Use of icam1 or a gene encoding same in the preparation of a therapeutic or diagnostic product for cervical cancer

Abstract

The present application relates to the field of medicine, in particular to the use of ICAM1 or its encoding gene in the preparation of a cervical cancer treatment or diagnosis product, ICAM1 or its encoding gene can be used as a molecular target of a cervical cancer treatment product or a cervical cancer diagnosis product, specifically, a substance specifically binding to ICAM1, such as an antibody conjugate drug or a pharmaceutically acceptable salt thereof, or an ICAM1 inhibitor can be used in the preparation of a cervical cancer treatment product, and an MRI molecular probe specifically binding to ICAM1 is used in the preparation of a molecular diagnosis product of ICAM1 positive cervical cancer or in the preparation of a product for predicting the treatment effect of an ICAM1 related drug for cervical cancer. The antibody conjugate drug prepared by ICAM1 or its encoding gene in the present application has a significant killing effect on cervical tumor cells without killing normal epithelial cells, which overcomes the shortcoming of traditional chemotherapy drugs in killing tumor cells while damaging normal tissues and cells.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the use of ICAM1 or its encoding gene in the preparation of products for the treatment or diagnosis of cervical cancer. Background Technology

[0002] Cervical cancer remains one of the most prevalent and deadliest malignant tumors among women. Current treatment methods for cervical cancer include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Currently used first-line chemotherapy and radiotherapy drugs indiscriminately kill both tumor cells and normal tissues, easily leading to drug resistance and chemotherapy-related complications. A large proportion of cervical cancer patients eventually progress to advanced stages (stage IV or recurrent disease). Due to the narrow therapeutic window and lack of targeted therapy of first-line chemotherapy drugs, the 5-year survival rate for patients with advanced cervical cancer is only 15%-20%.

[0003] In recent years, antibody-drug conjugates (ADCs), as a novel type of targeted therapy, have become a hot topic in anti-tumor research due to their unique advantages such as high targeting, high killing power, and suitable molecular size, showing broad research potential and prospects. They selectively kill tumor cells without damaging normal organs and tissues, demonstrating good efficacy in various solid tumors, including breast cancer and gastric cancer. Although the development of ADC drugs has always been a hot topic in anti-tumor drug research, existing ADC drugs still cannot meet clinical needs to date. For example, for patients with cervical cancer who relapse after first-line chemotherapy, there is a lack of available second-line drugs, especially targeted ADC drugs. Currently, the molecular subtyping of cervical cancer in clinical practice is relatively lacking, and research on related molecular markers is not mature enough to guide clinical medication. Therefore, screening for new tumor-specific targets or mutated antigens, researching and developing new ADC targeted drugs, conducting precision medicine research, reducing damage to normal tissues, improving prognosis, and increasing the five-year survival rate of cervical cancer patients are crucial.

[0004] Intercellular adhesion molecule-1 (ICAM-1), also known as CD54, is a member of the immunoglobulin superfamily (IGSF) and is an important adhesion molecule mediating adhesion reactions. ICAM-1 is a novel drug target for cervical cancer. This target is a cell surface glycoprotein that regulates intercellular adhesion during inflammatory damage, viral infection, and tumorigenesis. Satisfactory experimental results have been obtained in in vitro and in vivo studies of multiple myeloma and pancreatic cancer, and ADCs based on this new target have demonstrated significant and durable tumor regression effects in these solid tumor animal models. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide the use of ICAM1 or its encoding gene in the preparation of cervical cancer treatment or diagnostic products, in order to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides the use of ICAM1 or its encoding gene as a molecular recognition target in the preparation of cervical cancer diagnostic or therapeutic products.

[0007] The present invention also provides the use of substances that specifically bind to ICAM1 in the preparation of products for the treatment or diagnosis of cervical cancer.

[0008] Preferably, the substance that specifically binds to ICAM1 is selected from antibody-drug conjugates or their pharmaceutically acceptable salts or solvent compounds.

[0009] Preferably, the structure of the antibody-drug conjugate is as follows: Ab-(LD) n Where Ab is an anti-ICAM1 antibody, L is a linker, D is a cytotoxic agent, and n is an integer from 1 to 40.

[0010] Preferably, the heavy chain and light chain amino acid sequences of the anti-ICAM1 antibody are shown in SEQ ID NO.1 and 2, respectively.

[0011] Preferably, the antibody-drug conjugate is selected from anti-ICAM1 mAb-MC-VC-PAB-MMAE (abbreviated as ICAM1-MMAE) or anti-ICAM1 mAb-MC-GGFG-Dxd (abbreviated as ICAM1-Dxd).

[0012] As described above, the use of ICAM1 or its encoding gene as a molecular recognition target in the preparation of cervical cancer diagnostic or therapeutic products of the present invention has the following beneficial effects: It identifies ICAM1 as a novel target for the treatment of cervical cancer, and this target can serve as a target for ADCs (antibody-drug conjugates). Furthermore, the in vitro antitumor activity and biosafety of two ADC candidate drugs were determined, and IC1-MMAE, an optimal antibody-drug conjugate targeting the ICAM1 target protein, was identified, providing a potential targeted therapeutic candidate for cervical cancer. Attached Figure Description

[0013] Figure 1 IHC staining images of cervical cancer tissue and adjacent tissue (a), staining intensity of ICAM1+ and ICAM1- in tumor specimens (b), and survival curves (c).

[0014] Figure 2The image shows ICAM1 expression in cervical cancer cells SiHa (a), CaSki (b), and normal cervical cells HcerEpic (c).

[0015] Figure 3 The image shows IF images of cervical cancer cells SiHa (a) and CaSki (b).

[0016] Figure 4 Internalization images and efficiency curves of cervical cancer cells SiHa (a, c) and CaSki (b, d) are shown.

[0017] Figure 5 The chemical structures of the anti-ICAM1 antibody (a) and two sets of linker-cytotoxic agents are shown (b, c).

[0018] Figure 6 The IC50 values ​​show the in vitro inhibitory activity of ADC drugs and chemical drugs against cervical cancer cell lines and normal cervical cells. Detailed Implementation

[0019] This invention first provides the use of ICAM1 or its encoding gene as a target in the preparation of products for the treatment or diagnosis of cervical cancer.

[0020] The use of ICAM1 or its encoding gene as a target in the preparation of cervical cancer treatment or diagnostic products specifically refers to substances that can reduce ICAM1 levels or kill cervical cancer cells by using ICAM1 protein or its encoding gene as a recognition target.

[0021] The present invention also provides the use of substances that specifically bind to ICAM1 in the preparation of products for the treatment or diagnosis of cervical cancer.

[0022] In one embodiment, the substance that specifically binds to ICAM1 is selected from antibody-drug conjugates or pharmaceutically acceptable salts or solvent compounds thereof.

[0023] In one embodiment, the structure of the antibody-drug conjugate is shown below: Ab-(LD) n Where Ab is an anti-ICAM1 antibody, L is a linker, D is a cytotoxic agent, and n is an integer from 1 to 40.

[0024] In this invention, the anti-ICAM1 antibody is selected from monoclonal antibodies (including full-length antibodies having the Fc region of an immunoglobulin), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, F(ab')2, Fv, scFv). The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0025] The anti-ICAM1 antibody can be selected from any antibody or antigen-binding fragment of the prior art that can bind to ICAM1, such as the ICAM1 antibody from MabPlex.

[0026] In one embodiment, the heavy chain and light chain amino acid sequences of the anti-ICAM1 antibody are shown in SEQ ID NO. 1 and 2, respectively.

[0027] The anti-ICAM1 antibody is conjugated to a cytotoxic agent via a linker. Linkers are classified into two types: non-cleavable linkers and cleavable linkers. Cleavable linkers can cleave within the target cell, releasing the drug / toxin. Cleavable linkers can be further divided into two main categories: chemically unstable linkers and enzyme-unstable linkers. Chemically unstable linkers can selectively cleave due to differences in plasma and cytoplasmic properties. Such properties include pH value and glutathione concentration. pH-sensitive linkers are often called acid-cleavable linkers; these linkers are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be hydrolyzed in weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Glutathione-sensitive linkers are also called disulfide linkers. Enzyme-unstable linkers, such as peptide linkers, allow for better control of drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B or plasmin (these enzymes are present in increased amounts in some tumor tissues). These peptide links are highly stable in plasma circulation because unsuitable extracellular pH and serum protease inhibitors typically render the proteases inactive. Given their high plasma stability and good intracellular cleavage selectivity and efficiency, enzyme-unstable linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical enzyme-unstable linkers include Val-Cit(vc), Phe-Lys, and Gly-Gly-Phe-Gly.

[0028] Linkers may comprise one or more linker components. Exemplary linker components include 6-maleimide hexanoyl, maleimide propionyl-valine-citrulline, alanine-phenylalanine, p-aminobenzoyloxycarboxyl, N-succinimide-4-(2-pyridylthio)valerate, N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate, and N-succinimide-(4-iodo-acetyl)aminobenzoate. Other exemplary linker components may be linkers comprising amino acid units to allow protease cleavage, thereby facilitating the release of cytotoxic agents from antibody-drug conjugates upon exposure to intracellular proteases (such as lysosomal enzymes). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include: valine-citrulline; alanine-phenylalanine; phenylalanine-lysine; or N-methyl-valine-citrulline. Exemplary tripeptides include: glycine-valine-citrulline or glycine-glycine-glycine. Exemplary tetrapeptides include: glycine-glycine-phenylalanine-glycine.

[0029] The cytotoxic agent is selected from toxins, chemotherapy drugs, antibiotics, radioactive isotopes, and growth inhibitors.

[0030] Exemplary cytotoxic agents include: maytansin; maytansin-like agents; topoisomerase I inhibitors (such as camptothecin derivatives: DX-8951 derivative Dxd); tubulin inhibitors (such as monomethylostatin peptide E (MMAE) and monomethylostatin peptide F (MMAF); cazithromycins (such as cazithromycin); doxorubicins (such as doxorubicin); benzodipyrrole antibiotics (such as duocarmycins, CC-1065, etc.) and other cyclopropylpyrroloind-4-one (CPI) derivatives, such as cyclopropylbenzoind-4-one analogs, as well as pyrrolobenzodiazepines (PBD) or PBD dimers.

[0031] In the structure Ab-(LD)n of the antibody-drug conjugate, n is the drug-antibody ratio (DAR value), and the range of n is selected from any of the following: 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40.

[0032] In one embodiment, the antibody-drug conjugate is selected from anti-ICAM1 mAb-MC-VC-PAB-MMAE (hereinafter referred to as ICAM1-MMAE) or anti-ICAM1 mAb-MC-GGFG-Dxd (hereinafter referred to as ICAM1-Dxd).

[0033] The DAR value of the ICAM1-MMAE is 4.

[0034] The DAR value of the ICAM1-DXD is 8.

[0035] The maleimide group in the linker is covalently coupled to the cysteine ​​residue in the anti-ICAM1 antibody.

[0036] In one embodiment, the method for preparing the antibody-drug conjugate includes the following steps:

[0037] 1) Mix the disulfide bond reducing agent with the anti-ICAM1 antibody so that the disulfide bonds in the cysteine ​​of the anti-ICAM1 antibody are at least partially reduced to thiol groups;

[0038] 2) The linker, cytotoxic agent and product of step 1) are mixed to conjugate the anti-ICAM1 antibody with the linker to obtain the antibody-conjugated drug.

[0039] The disulfide bond reducing agent mentioned in step 1) is selected from DTT, tris(2-carboxyethyl)phosphine, or salts thereof. In one embodiment, the disulfide bond reducing agent is selected from tris(2-carboxyethyl)phosphine hydrochloride.

[0040] In some embodiments of the present invention, the reactions in steps 1) and 2) are carried out in a solvent. Those skilled in the art can select a suitable type and amount of solvent to ensure that the reactants are sufficiently dispersed in the reaction system. The solvent may be a buffer solution. More specifically, the solvent may be selected from one or more of borate buffer and phosphate buffer.

[0041] In some embodiments of the present invention, the reaction temperature of step 1) or step 2) is 0-37°C. In some embodiments of the present invention, the reaction temperature is 10-35°C. Preferably, the reaction temperature is 15-30°C.

[0042] In step 1), the amount of disulfide reducing agent is typically equal to or in excess of the anti-ICAM1 antibody in molar terms. In some embodiments of the invention, the molar ratio of disulfide reducing agent to anti-ICAM1 antibody is 1-50:1. In a preferred embodiment, the molar ratio is 1-30:1. In a more preferred embodiment, the molar ratio is 5-20:1.

[0043] Linkers and cytotoxic agents can be commercially available reagents that have already been successfully linked, i.e., linkers-cytotoxic agents; linkers and cytotoxic agents can also be prepared in-house.

[0044] In some embodiments of the present invention, the linker and the cytotoxic agent have been successfully linked to form a linker-cytotoxic agent. The amount of different linker-cytotoxic agents used in step 2) may vary, but the amount of linker-cytotoxic agent is generally equal to or excessive relative to the product of step 1) in molar terms. In some embodiments of the present invention, the molar ratio of linker-cytotoxic agent to the product of step 1) is 1-50:1. In a preferred embodiment, the molar ratio of linker-cytotoxic agent to the product of step 1) is 1-40:1. In a more preferred embodiment, the molar ratio of linker-cytotoxic agent to the product of step 1) is 5-25:1.

[0045] In some embodiments of the present invention, step 2) further includes the removal of unreacted linkers and cytotoxic agents.

[0046] In another embodiment, the substance that specifically binds to ICAM1 is selected from ICAM1 inhibitors.

[0047] ICAM1 inhibitors are molecules that inhibit ICAM1. Inhibition of ICAM1 includes, but is not limited to, inhibiting the level or activity of ICAM1.

[0048] Inhibiting ICAM1 activity means reducing ICAM1 activity. Preferably, compared to before inhibition, ICAM1 activity is reduced by at least 10%, more preferably by at least 30%, even more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0049] Inhibiting ICAM1 levels can be achieved by suppressing the transcription or translation of the ICAM1 gene. Specifically, this can mean preventing the ICAM1 gene from being transcribed, reducing its transcriptional activity, preventing its translation, or reducing its translation level.

[0050] Those skilled in the art can use conventional methods to regulate ICAM1 gene expression, such as gene knockout, homologous recombination, and interfering RNA.

[0051] Preferably, compared with the wild type, ICAM1 gene expression is reduced by at least 10%, more preferably by at least 30%, even more preferably by at least 50%, even better by at least 70%, even better by at least 90%, and most preferably by no expression of the ICAM1 gene at all.

[0052] The ICAM1 inhibitors include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, peptides, proteins, interfering lentiviruses, adeno-associated viruses, nanoparticles, liposomes, extracellular vesicles, or cells. The nucleic acids include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA or short hairpin RNA (shRNA) prepared by ribonuclease I or II.

[0053] The cervical cancer treatment or diagnostic product must include a substance that specifically binds to ICAM1, and use a substance that specifically binds to ICAM1 as the active ingredient.

[0054] The cervical cancer treatment or diagnostic product may be a single-component substance or a multi-component substance.

[0055] There are no special restrictions on the form of the cervical cancer treatment or diagnostic product; it can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0056] The cervical cancer treatment or diagnostic products are primarily targeted at mammals. Preferred mammals include rodents, even-toed ungulates, perissodactyls, lagomorphs, and primates. Preferred primates include monkeys, apes, or humans.

[0057] The cervical cancer treatment or diagnostic products include, but are not limited to, drugs, health products, and food.

[0058] The cervical cancer treatment products are selected from: targeted ICAM1 antibodies or antigen-binding fragments, exosomes, liposomes, SLP nanoparticles, CAR-T cells, oncolytic viruses, ADCs, small molecule conjugates, bispecific antibodies, nucleic acid molecules, small molecule chemicals, peptides, proteins, interfering lentiviruses or adeno-associated viruses, etc.; the cervical cancer diagnostic products are selected from: exosomes, CTCs, CT DNA, blood / urine proteins or imaging probes such as MRI / PET / ultrasound, etc.

[0059] The cervical cancer mentioned is selected from squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma.

[0060] Squamous cell carcinoma is classified into three grades according to histological differentiation: Grade I is well-differentiated squamous cell carcinoma, Grade II is moderately differentiated squamous cell carcinoma (non-keratinizing large cell type), and Grade III is poorly differentiated squamous cell carcinoma (small cell type), which is mostly undifferentiated small cell.

[0061] Adenocarcinoma accounts for 15% to 20% of cervical cancers, and there are two main histological types:

[0062] ① Mucinous adenocarcinoma: This is the most common type, originating from columnar mucinous cells in the cervical canal. Microscopically, it reveals glandular structures with multilayered proliferation of glandular epithelial cells, significant atypia, and mitotic figures. Cancer cells protrude into the glandular lumen in a papillary manner. It can be classified as well-, moderately, or poorly differentiated adenocarcinoma.

[0063] ② Malignant adenoma: also known as microadenocarcinoma, it is a well-differentiated cervical canal mucosal adenocarcinoma. The cancerous glands are numerous, varying in size and shape, and appear as punctate protrusions extending into the deep layer of the cervical stroma. The glandular epithelial cells do not show atypia, and lymph node metastasis is common.

[0064] Adenosquamous carcinoma accounts for 3% to 5% of cervical cancers. It is formed by the simultaneous differentiation and development of reserve cells into glandular cells and squamous cells. The cancerous tissue contains both adenocarcinoma and squamous cell carcinoma components.

[0065] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0066] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0067] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0068] The following examples first used immunohistochemistry (IHC) to detect the differential expression levels of the novel target protein ICAM1 in cervical cancer and adjacent normal tissues. Second, immunofluorescence (IF) staining, flow cytometry, and single-photon confocal microscopy were used to determine the location of the novel target ICAM1 in two types of human cervical cancer cells (Si-Ha and Ca-Ski) and one type of normal cervical cells (HcerEpic). Subsequently, confocal imaging was used to observe the endocytic capacity of cervical cancer cells against ICAM1 antibodies, and flow cytometry was used to analyze its endocytic efficiency. Finally, the CCK8 assay was used to evaluate the in vitro inhibitory activity of two ADC candidates with different linkers and cytotoxic agents, IC1-MMAE (ICAM1-MC-VC-PAB-MMAE) and IC1-Dxd (ICAM1-Maleimide GGFG peptide-Dxd), in cervical cancer cells and normal cervical cells. Clinical IHC studies showed that the expression level of the novel target ICAM1 in cervical cancer tissue was significantly higher than that in adjacent normal tissue. The positive rate of ICAM1 expression in cervical cancer tissue was 24.32% (27 / 111), while the positive rate in adjacent normal tissue was 0% (0 / 25). Two cervical cancer cell lines overexpressed the ICAM1 target protein; the ICAM1 expression in cervical cancer cells SiHa and CaSki was 80-fold and 106-fold higher than that in normal cervical cells HcerEpic, respectively. The location of the ICAM1 target protein on the cell membrane was determined. Cervical cancer cells with high ICAM1 expression showed significant endocytic activity against ICAM1 antibodies, with an internalization efficiency exceeding 30% within 1 hour. IC1-MMAE showed IC50 values ​​of 0.1198 ug / ml and 1.149 ug / ml in SiHa and CaSki tumor cells, respectively, demonstrating good antitumor activity. In summary, these examples identify ICAM1 as a novel ADC target for the treatment of cervical cancer. The in vitro antitumor activity and biosafety of two ADC candidates were determined, and IC1-MMAE, the optimal antibody-drug conjugate targeting the ICAM1 protein, was identified, providing a promising targeted therapy candidate for cervical cancer.

[0069] The main experimental materials used in this embodiment are as follows:

[0070] Cervical cancer tissue microarrays (array number: HUteS136Su01, batch number: XT17-039) were purchased from Shanghai Chimchao Biotechnology Co., Ltd. Human cervical cancer cell lines (Si-Ha and Ca-Ski) were stored in the cell banks of Zhejiang Cancer Hospital and Shanghai Cancer Hospital; normal cervical cells (HcerEpic) were purchased from Tongpai (Shanghai) Biotechnology Co., Ltd. MEM, RPMI-1640, and fetal bovine serum were purchased from Gibco, USA. The CCK8 kit (catalog number: BS350B) was purchased from Biospirp; PE anti-mouse IgG1 (catalog number: 406608) and Purified anti-human CD54 (catalog number: 322702) were both purchased from Biolegend, USA. The antibody-drug conjugates Anti-ICAM1 mAb-MC-VC-PAB-MMAE, Anti-ICAM1 mAb-MC-GGFG-Dxd, and Anti-ICAM1 mAb were prepared in-house by the applicant. Anti-ICAM1 mAb was synthesized by a third-party company. The heavy and light chain amino acid sequences of Anti-ICAM1 mAb are shown in SEQ ID NO. 1 and 2, respectively.

[0071] MGWSCIILFLVATATGVHSQVQLQQSGPELVRPGVSVKISCKGSGYTFIDYAIHWVKESHAKSLEWIGVISAYSGDTNYNQKFKGKATMTVDKSSNTAYLELARLTSEDSAIYYCARG GWLLLSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 1)MGWSCIILFLVATATGVHSDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNNYLHWYLQKSGQAPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHV PLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:2)

[0072] MC-VC-PAB-MMAE and MC-GGFG-Dxd were purchased from Taoshu Biotechnology.

[0073] The cell culture method in this example is as follows:

[0074] Cervical cancer cells and human cervical epithelial cells were cultured in MEM medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in a 5% CO2 incubator using standard methods. The culture medium was changed every 2-3 days. Cells that reached approximately 90% confluence and were in the logarithmic growth phase were digested with trypsin and then passaged for subsequent experiments.

[0075] The statistical methods used in this example are as follows:

[0076] Statistical analysis of the experimental data was performed using GraphPad Prism 8.0 and FlowJo V10. Quantitative data are expressed as mean ± variance (SD). Differences between two groups were analyzed using t-tests, with P < 0.05 considered statistically significant. For survival analysis, the Mantel-Cox test in GraphPad Prism 8.0 was used to determine significance levels as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. GraphPad Prism 8.0 was also used to create relevant images of the experimental data.

[0077] Example 1: Immunohistochemical staining (IHC) experiment of clinical specimens

[0078] Immunohistochemistry (IHC) was used to detect the differences in ICAM1 target protein expression levels in 25 groups of human cervical cancer tissues and adjacent normal tissues, as well as 86 cervical cancer tissues. TMA semi-quantitative analysis of ICAM1 expression in tissue microarrays was performed. Since no ICAM1-related scoring criteria for cervical cancer were found, following the suggestion of Professor Zhu Xiu from the Cancer Hospital of the University of Chinese Academy of Sciences, the staining intensity (1+, 2+, 3+) was multiplied by the percentage of positive tumor cells. For example, (2+, 50%) was quantified as 1.0. >0 was considered as expression (ICAM1+, positive), and =0 was considered as no expression (ICAM1-, negative).

[0079] A total of 111 surviving cervical tumor cases were included in the biochip study, of which 107 were squamous cell carcinomas and 4 were adenocarcinomas. The positive rate of the novel target ICAM1 in cervical cancer tissue was 24.32% (27 / 111). Among them, high-grade: 26.87% (18 / 67); low-grade: 21.05% (8 / 38); adenocarcinoma: 17.67% (1 / 6). The positive rate in adjacent normal tissue was 0 (0 / 25). The IHC staining results were quantified as ICAM1+ expression intensity. The staining intensity of ICAM1+ in tumor samples was statistically different from that of ICAM1- (P < 0.0001).

[0080] This indicates that the expression level of ICAM1 in human cervical tumor tissues is significantly higher than that in adjacent normal tissues, while adjacent normal tissues do not express the ICAM1 target protein. The ICAM1 target protein is specifically expressed in cervical cancer tissues and has the potential to serve as a target for ADC drugs. Typical immunohistochemical (IHC) images are shown below. Figure 1 a and Figure 1 As shown in b.

[0081] Kaplan-Meier statistical analysis of survival data from 111 patients using a biochip revealed that cervical cancer patients with high ICAM1 expression had a significantly worse prognosis (P < 0.05). Therefore, developing an adjuvant drug (ADC) for ICAM1-positive cervical cancer patients is of great significance for improving the poor clinical prognosis of cervical cancer.

[0082] Example 2: Flow cytometry (FACS) detection of ICAM1 expression intensity in cell line

[0083] The expression levels of ICAM1 in two human cervical cancer cell lines, SiHa and CaSki, and one normal cervical cell line, HcerEpic, were detected by flow cytometry. 1×10⁻⁶ cells were collected. 6 Cells were washed twice with PBS. The resulting cells were then blocked in ice bath with 1% bovine serum albumin (BSA) in PBS for 30 minutes. After BSA blocking, cells were incubated at room temperature for 1 hour with an ICAM1 antibody bound to phycoerythrin (PE). PE-bound IgG served as a control. Cells were washed three times with 1% BSA in PBS, resuspended in PBS, and the expression intensity of ICAM1 in each cell line was assessed using flow cytometry.

[0084] Flow cytometry results showed that the two cervical cancer cell lines, SiHa and CaSk, overexpressed the ICAM1 target protein. The ICAM1 peak (orange) and the non-specific IgG peak (blue) were significantly separated, while the ICAM1 and IgG peaks in the normal cervical cell line HcerEpic largely overlapped. The ICAM1 target protein expression levels in SiHa and CaSki were 80-fold and 106-fold higher than those in HcerEpic, respectively. Flow cytometry results indicate that ICAM1 is highly expressed in human cervical cancer cells SiHa and CaSki, but lowly expressed in normal cervical cells. This verifies the high specificity of the ICAM1 target and provides a possibility for the successful development of an ADC drug targeting the ICAM1 target protein. Flow cytometry results are shown below. Figure 2 .

[0085] Example 3: Immunofluorescence staining (IF) to verify the presence of ICAM1 target protein on the cell membrane.

[0086] The subcellular locations of target proteins in two cervical cancer cell lines, SiHa and CaSki, were observed using a single-photon confocal microscope. 1×10⁻⁶ cells were used. 6Cells were seeded in three confocal dishes, each with 1 mL of culture medium, and incubated overnight at 37°C. The culture medium was removed, and the cells were washed once with PBS. Under ice-bath conditions, 1 mL of PBS containing 1% BSA was used to block the cells for 15-30 min. After blocking, the liquid was aspirated, and the cells were incubated with antibodies in three groups at 37°C: Group ① cells were incubated with 1 mL of PBS for 1 h; Group ② cells were incubated with 2 μl of 1gG-PE + 1 mL of PBS for 1 h; Group ③ cells were incubated with 2 μl of ICAM1-PE + 1 mL of PBS for 1 h. Cells were washed once with 1 mL of PBS; 1 mL of Hoechst nuclear staining solution was added, and the cells were stained statically at 37°C for 20-30 min; the cells were washed 1-2 times with 1 mL of PBS and observed under a single-photon confocal microscope.

[0087] After treating two cervical cancer cell lines, SiHa and CaSki, with immunofluorescence (IF), the location of the ICAM1 target protein on the cells was observed under a single-photon confocal microscope. The red fluorescent circles represent ICAM1 antibodies with PE (polyethylene glycol), and the blue fluorescent patches represent cell nuclei stained with Hoechst stain. A ring of red fluorescence can be seen on the cell membrane of both SiHa and CaSki cervical cancer cell lines, which highly express ICAM1, confirming that the ICAM1 target protein is localized on the cell membrane. The immunofluorescence staining results are as follows: Figure 3 .

[0088] Example 4: Imaging flow cytometry to quantify the intracellularization efficiency of ICAM1 antibody

[0089] Imaging flow cytometry was used to determine whether ICAM1 ADCs could selectively enter cervical cancer cells and be rapidly transported to intracellular lysosomes. 1×10 6 Five cells were seeded in confocal microscopy dishes, each with 1 ml of culture medium, and incubated overnight at 37°C. The culture medium was removed, and the cells were placed on ice with 2 μl of ICAM-PE and 1 ml of PBS. After staining on ice for 30 min, the cells were washed once with cold PBS. 1 ml of PBS was added, and the cells were incubated at 37°C for 0, 30, 60, 120, and 240 mins for endocytosis. At the end of each incubation period, the cells were washed 1-2 times with PBS, followed by fixation with 4% paraformaldehyde for 10 min, and then washed 1-2 times with PBS. The PBS was removed, and 1 ml of Hoechst staining solution was added. The cells were stained at 37°C for 20-30 min, the staining solution was removed, and the cells were washed 1-2 times with PBS. Finally, the cells were photographed, analyzed, and stored using a confocal microscope.

[0090] Based on the targeting mechanism of ADCs, whether anti-ICAM1 antibodies can be internalized by antigen targets on the cell membrane is another important factor affecting the efficacy of ADCs. Therefore, this invention attempts to use imaging flow cytometry to determine whether ICAM1 ADCs can selectively enter cervical cancer cells and rapidly transport to intracellular lysosomes. Imaging flow cytometry was used to quantify the internalization rate of ICAM1 antibodies in two cervical cancer cell lines, SiHa and CaSki, and the normal cell line HcerEpic. ICAM1 target proteins were highly overexpressed in SiHa and CaSki. The two tumor cell lines, SiHa and CaSki, which highly expressed target proteins, showed a significant tendency to internalize anti-ICAM1 antibody conjugates, and over time, the target protein antibody conjugates migrated into the tumor cells, such as... Figure 4 a and Figure 4 b. Both observed cervical cancer cell lines achieved an internalization rate of 30% within 1 hour, such as Figure 4 c and Figure 4 d.

[0091] These results demonstrate the significant potential utility of ICAM1 as an ADC target for cervical cancer, enabling the design, preparation, and characterization of ICAM1 antibody-drug conjugates, and the determination of their in vitro and in vivo anti-cervical cancer activity. Confocal fluorescence images and endocytosis efficiency curves are shown below. Figure 4 As shown.

[0092] Example 5: Design, preparation, and characterization of ADC drugs

[0093] The structural composition of ADC drugs determines their high targeting and tumor cell killing efficacy. Whether the drug can be phagocytosed by tumor cells and effectively released intracellularly is a crucial factor in determining the effectiveness of ADCs. Therefore, after confirming that ADC drugs can be internalized through surface antigen-mediated processes, it is also necessary to determine whether the drug is transported intracellularly to lysosomes, where the linker is degraded, and the drug is released in a controlled manner to kill tumor cells. Therefore, based on the identification of the target protein ICAM1, suitable antibodies, linkers, and cytotoxic agents were selected, the drug / antibody ratio (DAR) was determined, several ICAM1-ADC candidate drugs were designed, and they were prepared and characterized.

[0094] A set of ICAMI ADCs was designed and constructed by conjugating monoclonal anti-ICAM1 chimeric antibodies to two different linkers (MC-VC-PAB, Maleimide GGFG peptide) and different drug agents (MMAE, Dxd), including ICAMI-MC-VC-PAB-MMAE (IC1-MMAE) and ICAMI-Maleimide GGFG peptide-Dxd (IC1-Dxd). Specific chemical structures are shown below. Figure 5The specific preparation process is as follows:

[0095] The ICAM1 antibody was reacted with tris(2-carboxyethyl)phosphonic acid hydrochloride in borate buffer at 25°C for 2 hours to partially reduce the disulfide bonds in cysteine ​​to thiol groups. Then, excess MC-VC-PAB-MMAE (molar ratio 10:1) or MC-GGFG-DXD (molar ratio 20:1) was added, and the thiol groups on the ICAM1 monoclonal antibody were reacted with the MC-VC-PAB-MMAE or MC-GGFG-DXD linkers in PBS buffer (pH 6.5) at 25°C for 2 hours to generate the final products ICAM1-MMAE or ICAM1-DXD. Unreacted MC-VC-PAB-MMAE or MC-GGFG-DXD was removed by ultrafiltration. The drug-antibody ratio (DAR) of ICAM1-MMAE was controlled to be 4, and the DAR of ICAM1-DXD was controlled to be 8, by controlling the feed ratio.

[0096] The DAR values ​​of the two ICAM1 ADCs were determined by hydrophobic interaction chromatography (HIC), and the DAR of IC1-MMAE was 4 and that of IC1-Dxd was 8.

[0097] Example 6 In vitro cytotoxicity experiment - CCK8 assay

[0098] The CCK8 assay was used to determine the IC50 values ​​of two human cervical cancer cell lines, Si-Ha and Ca-Ski, and one normal cervical cell line, HcerEpic. Cells were seeded at a density of 5000 cells per well in 96-well plates and cultured overnight. After adhesion, two different ICAM1 ADCs (IC1-MMAE and IC1-Dxd) were added at concentrations of 0-10 μg / mL, serially diluted 10-fold (8 concentrations in 3 parallel wells). After 72 hours of cell and drug culture, the original culture medium was discarded, and the CCK8 assay reagent was diluted 10-fold with fresh cell culture medium. 100 μL / well was added to each 96-well plate and incubated at 37°C. The absorbance (OD) was then read at 450 nm using a microplate reader. Cell viability was determined by comparing the absorbance of drug-treated cells with that of untreated control cells.

[0099] Comparing the in vitro inhibitory activity (IC50) of two ADC candidates in two cervical cancer cell lines (Si-Ha and Ca-Ski) and normal cervical cells (HcerEpic), it can be seen that IC1-MMAE and IC1-Dxd showed significantly greater killing effects in Si-Ha and Ca-Ski tumor cells than in normal cervical cells (HcerEpic), while IC1-Dxd had virtually no killing effect on normal cervical cells. In Si-Ha and Ca-Ski, IC1-MMAE showed better killing effects, with IC50 values ​​of 0.1198 μg / ml and 1.149 μg / ml, respectively; IC1-Dxd showed slightly weaker killing effects, with IC50 values ​​generally above 10 μg / ml, which is 10-100 times higher than that of IC1-MMAE.

[0100] Compared with cisplatin (Cis) and paclitaxel (Pac), the first-line standard chemotherapy drugs for cervical cancer treatment, Pac showed a significantly greater killing effect on cell lines than Cis, with an IC50 difference of 1000-10000 times. Cis showed no significant difference in killing effect between tumor cells and normal cervical cells, with IC50 values ​​above 100 μg / ml for both. Pac's IC50 was 0.1254 μg / ml for Si-Ha and 0.0036 μg / ml for Ca-Ski, but its killing power on normal cervical cells was even stronger, with an IC50 of 0.0016 μg / ml, representing a 2-100 times stronger killing effect, which may lead to more adverse reactions.

[0101] Therefore, considering all factors, IC1-MMAE was deemed the optimal ICAM1 ADC drug. This experiment determined the IC50 values ​​of two ADC drugs, IC1-MMAE and IC1-Dxd, and two first-line clinical chemotherapy drugs, Cis and Pac, in two human cervical cancer cell lines (Si-Ha and Ca-Ski) and one normal cervical cell line (HcerEpic). The results are as follows: Figure 6 .

[0102] This invention identifies ICAM1 as a novel ADC target for the treatment of cervical cancer, and designs, prepares, and characterizes two ADC candidate drugs, confirming their good in vitro antitumor activity and biosafety, providing a new direction for the development of a new class of ADC drugs for cervical cancer.

[0103] Based on the compositional characteristics of ADC drugs, this invention verified the overexpression level of the ICAM1 target protein on the surface of cervical cancer tissues and cell lines, and its low expression or absence in adjacent normal cervical cells. In conjunction with the targeting mechanism of ADCs, whether anti-ICAM1 antibodies can be internalized by antigen targets on the cell membrane is another important factor affecting the efficacy of ADCs. Interference related to internalization, transport, or recycling, antigen shedding, and lysosomal degradation defects of ADCs can all lead to reduced drug release, thus affecting the efficacy of ADCs. Therefore, the internalization efficiency of ICAM1 ADCs mediated by ICAM1+ cervical cancer cells was also measured, achieving a satisfactory internalization efficiency of 30% within 1 hour.

[0104] In designing the ADC candidate drugs IC1-MMAE and IC1-Dxd, by comparing commonly used monoclonal antibodies, an antibody targeting the IgG1 subtype, specifically an anti-ICAM1 antibody, was chosen. Cleavable linkers were selected, including the linker MC-Val-Cit-PAB (MC-VC-PAB), a protease-cleavable linker. The maleimide-GGFG peptide linker, which is more stable in vivo, was chosen, allowing for better control of drug release under the cleavage action of intracellular enzymes (such as proteases). The selected cytotoxic agents MMAE and Dxd are clinically used ADC agents, including monomethyl auristatin E (MMAE), a microtubule inhibitor that acts on cell proliferation and differentiation and inhibits cell mitosis, and deruxtecan (DXd), a highly effective derivative of exatecan, a water-soluble structural analog of camptothecin with a unique hexagonal structure, and a topoisomerase I inhibitor. The antibody / drug ratios (DARs) of 4 and 8 are within the appropriate range for clinically available ADCs, balancing efficacy and toxicity. The study revealed that both ADC candidates, IC1-MMAE and IC1-Dxd, have their advantages, demonstrating superior performance compared to Cis and Pac in specifically killing tumor cells. Compared to IC1-MMAE, IC1-Dxd exhibits virtually no killing effect on normal cervical cells, demonstrating good targeting specificity; IC1-MMAE shows better overall tumor cell killing efficacy, with an IC50 value 10 times higher than IC1-Dxd.

[0105] In summary, this study explored the possibility of developing novel ADCs targeting ICAM1 in cervical cancer, identified IC1-MMAE as an optimal drug for ICAM1-targeted ADCs, and provided a promising targeted therapy candidate for cervical cancer.

[0106] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention. sequence list <110> Zhejiang Cancer Hospital Institute of Basic Medical Sciences and Cancer, Chinese Academy of Sciences (in preparation) <120> Use of ICAM1 or its encoding gene in the preparation of products for the treatment or diagnosis of cervical cancer. <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 468 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg 20 25 30 Pro Gly Val Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Ile Asp Tyr Ala Ile His Trp Val Lys Glu Ser His Ala Lys Ser Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Ala Tyr Ser Gly Asp Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Asn 85 90 95 Thr Ala Tyr Leu Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Trp Leu Leu Leu Ser Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 <210> 2 <211> 238 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu 35 40 45 Val His Ser Asn Gly Asn Asn Tyr Leu His Trp Tyr Leu Gln Lys Ser 50 55 60 Gly Gln Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110 Ser Gln Ser Thr His Val Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235

Claims

1. Use of substances that specifically bind to ICAM1 in the preparation of cervical cancer treatment products; The cervical cancer is selected from squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma; The substance that specifically binds to ICAM1 is selected from antibody-drug conjugates or pharmaceutically acceptable salts thereof; The structure of the antibody-drug conjugate is as follows: Ab-(LD)n, where Ab is an anti-ICAM1 antibody, L is a linker, D is a cytotoxic agent, and n is an integer from 1 to 40.

2. The use according to claim 1, characterized in that, Anti-ICAM1 antibodies are selected from full-length antibodies or antibody fragments.

3. The use according to claim 2, characterized in that, The antibody fragment is selected from Fab, F(ab')2, Fv or scFv.

4. The use according to claim 1, characterized in that, The heavy chain and light chain amino acid sequences of the anti-ICAM1 antibody are shown in SEQ ID NO.1 and 2, respectively.

5. The use according to claim 1, characterized in that, The connector is selected from either unbreakable connectors or breakable connectors.

6. The use according to claim 5, characterized in that, The breakable linker is selected from chemically unstable linkers or enzyme-unstable linkers.

7. The use according to claim 1, characterized in that, The connector comprises one or more connector components selected from maleimide propionyl, maleimide hexanoyl, p-aminobenzoyloxycarboxyl, valine-citrulline, alanine-phenylalanine, glycine-glycine-glycine, glycine-valine-citrulline, and glycine-glycine-phenylalanine-glycine.

8. The use according to claim 1, characterized in that, The linker is selected from maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarboxyl or maleimide-GGFG tetrapeptide.

9. The use according to claim 1, characterized in that, The cytotoxic agent is selected from toxins, chemotherapy drugs, antibiotics, radioactive isotopes, or growth inhibitors.

10. The use according to claim 1, characterized in that, The cytotoxic agent is selected from one or more of the following: topoisomerase I inhibitors, tubulin inhibitors, kazimidic acid and its derivatives, doxorubicin and its derivatives.

11. The use according to claim 10, characterized in that, The microtubule inhibitor is selected from one or more of the following: maytansin, maytansin-like substances.

12. The use according to claim 9, characterized in that, The cytotoxic agent is selected from DX-8951 derivative Dxd, MMAE, or MMAF.

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