Application of substance specifically binding to ICAM1 in preparation of tongue cancer treatment product

Abstract

The invention relates to the field of medicine, in particular to application of a substance specifically bound with ICAM1 in preparation of a tongue cancer treatment product, the substance specifically bound with ICAM1 is selected from antibody conjugate drugs or pharmaceutically acceptable salts or solvent compounds thereof, and the antibody conjugate is such as ICAM1-MC-VC-PAB-MMAE or ICAM1-Maleimide GGFG peptide-Dxd. The tongue cancer is selected from tongue squamous carcinoma or adenocarcinoma. The invention finds that tongue squamous cell carcinoma tissues mostly present ICAM1 high expression which is significantly different from normal tissues, and verifies that the ICAM1 or the coding gene thereof can be used as a target in preparation of tongue cancer treatment or diagnosis products, thereby providing a powerful theoretical basis for clinical treatment of tongue cancer.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the use of a substance specifically binding to ICAM1 in the preparation of a tongue cancer treatment product. Background Art

[0002] Tongue squamous cell carcinoma (TSCC) is the most common oral malignancy in the world and a type of head and neck squamous cell carcinoma. For patients with tongue cancer, surgery and chemoradiotherapy are often used clinically. The most commonly used and effective chemotherapy drug is 5-fluorouracil (5-FU). However, the prognosis of patients with tongue cancer is generally poor, with a five-year survival rate of only 40-50%. The main reason is the emergence of chemotherapy resistance after continuous use of 5-FU. Due to the unclear molecular mechanism, there is a serious lack of effective targeted drugs in clinical treatment. Therefore, it is urgent to select tumor markers that are highly expressed in tongue cancer tissue, develop a drug that can accurately target tongue cancer tissue, verify the feasibility of the drug, and evaluate its efficacy in vivo and in vitro.

[0003] The concept of antibody-drug conjugate (ADC) was proposed in the 1980s. Its main structure is a humanized antibody (antibody), which is coupled to a small molecule toxic warhead (payload) through a linker. The linker is usually a cleavable peptide, and the toxic warhead is mostly a small molecule with high activity but strong toxicity. It cannot be directly used for tumor chemotherapy due to its large side effects. The main mechanism of action of ADC is that after the drug solution enters the blood circulation through intravenous injection, it is captured by the target protein highly expressed on the cell surface when passing through the tumor tissue, enters the cell through endocytosis, and cleaves the cleavable linker or antibody under the action of lysosomes, releasing toxic small molecules, hindering the formation of microtubules or destroying the DNA structure, causing cell mitosis to arrest, thereby inhibiting tumor growth. Some lipophilic toxic small molecules can also pass through the cell membrane into adjacent cells, exerting a bystander killing effect. Over the past forty years, with the unremitting efforts of many researchers, the ADC drugs approved for marketing have been continuously updated and iterated. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a use of a substance that specifically binds to ICAM1 in the preparation of a tongue cancer treatment product, so as to solve the problems in the prior art.

[0005] To achieve the above objectives and other related objectives, the present invention provides the use of a substance that specifically binds to ICAM1 in the preparation of a product for treating or diagnosing tongue cancer.

[0006] Preferably, the substance that specifically binds to ICAM1 is selected from antibody-drug conjugates or pharmaceutically acceptable salts or solvent compounds thereof.

[0007] Preferably, the structure of the antibody-drug conjugate is as follows: Ab-(LD) n , wherein Ab is an anti-ICAM1 antibody, L is a linker, D is a cytotoxic agent, and n is an integer of 1-40.

[0008] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-ICAM1 antibody are shown as SEQ ID NOs. 1 and 2, respectively.

[0009] Preferably, the antibody-drug conjugate is ICAM1-MC-VC-PAB-MMAE or ICAM1-MaleimideGGFGpeptide-Dxd, the ICAM1 is an anti-ICAM1 antibody, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-ICAM1 antibody are shown in SEQ ID NOs. 1 and 2, respectively.

[0010] Preferably, the tongue cancer is selected from tongue squamous cell carcinoma or adenocarcinoma.

[0011] As described above, the use of the substance that specifically binds to ICAM1 in the preparation of a tongue cancer treatment product of the present invention has the following beneficial effects: for tongue squamous cell carcinoma (TSCC), whose molecular mechanism is currently extremely unclear and clinically lacks targeted drugs, the tumor marker ICAM1, which is highly expressed in its tissues, is used as a target, and ADC drugs are found to be useful for treating TSCC patients with highly expressed ICAM1, so as to fill the gap in targeted drugs in the field of tongue cancer. The experimental results show that the tongue squamous cell carcinoma tissues derived from patients mostly show high expression of ICAM1, which is significantly different from normal tissues; the information in the database also shows the same trend. At the same time, with the progression of the course of TSCC, the expression of ICAM1 shows a synergistic upward trend, and the upward trend is particularly obvious in the patient group who has received radiotherapy and chemotherapy after surgery. The research results of the present invention provide a strong theoretical basis for the subsequent animal experiments and clinical trials, and ICAM1-ADC shows great clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shown are the intergroup difference analysis results of different groups of TSCC samples and adjacent cancer tissue samples in the TCGA database of the present invention.

[0013] Figure 2 Shown are the intergroup differences in survival, immune score and matrix score of patients with different expression levels of ICAM1 in the TCGA database of the present invention.

[0014] Figure 3Shown is the intergroup difference analysis of TEM of tissue samples with different expression levels of ICAM1 in the TCGA database.

[0015] Figure 4 The figure shows antigen screening of two tongue cancer cell lines, SAS and CAL-27, and a control normal cell line, 293T, using flow cytometry.

[0016] Figure 5 Shown are pictures of cells incubated with anti-ICAM1-PE taken under a single-photon confocal microscope. The cells in the experimental group showed obvious ICAM1 expression and were located on the membrane surface.

[0017] Figure 6 Shown are HE staining and immunohistochemistry (IHC) staining of tongue cancer tissues from patients, and the statistical analysis of ICAM1 expression scores in cancer tissues and adjacent tissues of 26 patients is shown in the figure. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.001; no significant difference ns).

[0018] Figure 7 Shown is a schematic diagram of the ICAM1-ADC molecular structure.

[0019] Figure 8 The figure shows the flow cytometry combined with confocal microscopy analysis of the internalization capacity of ICAM1 in tongue cancer cell lines.

[0020] Fig. 9 The CCK-8 method is used to detect and compare the killing of ADC drugs and first-line chemotherapy drugs on cell lines, and the IC 50 value.

[0021] Fig.10 The flow chart of the distribution experiment of ICAM1-cy3 and ICAM1-MMAE-cy3 in nude mice is shown. The ex vivo organs and tumors of nude mice were photographed using IVIS, and the obtained data were analyzed and processed by GraphPad. The results are shown in the figure (*p<0.05; **p<0.01; ***p<0.001; no significant difference ns)

[0022] Fig.11 The figure shows the schematic diagram of PDX modeling and treatment process. The figure shows the solid photos of in vitro tumors and the changes in body weight and tumor volume of nude mice in each group at different time points. The ICAM1-DXd group showed significant differences compared with the PBS group, and the body weight of nude mice in each group did not show significant changes during the treatment (*p<0.05; **p<0.01; ***p<0.001; no significant difference ns).

[0023] Fig.12The figure shows the schematic diagram of subcutaneous tumor modeling and treatment process. The figure shows the solid photos of the in vitro tumors, and the changes in the body weight and tumor volume of each group of nude mice recorded at different time points. There were significant differences between the ICAM1-DXd and ICAM1-MMAE treatment groups compared with the PBS group, and the body weight of the nude mice in each group did not show significant changes during the treatment (*p<0.05; **p<0.01; ***p<0.001; no significant difference ns). DETAILED DESCRIPTION

[0024] Based on the ADC design principle, the present invention first determines an antigen epitope highly expressed in tongue cancer tissue as a target, which is a protein that is not expressed or low expressed in normal human tissue, so that the drug molecules can be accumulated in the tumor tissue as much as possible, avoiding or reducing the toxic side effects of the drug due to off-target. At the same time, after indiscriminate screening, the present invention discovered a tumor marker, Intercellular Cell Adhesion Molecule-1 (ICAM1), which is highly expressed in various cell lines (taking tongue squamous cell carcinoma cells SAS, SCC-4, CAL-27 and self-built cell line tongue 003#1 as examples) and not expressed in the control group (human embryonic kidney cell 293T). ICAM1 is a member of the cell adhesion molecule family. Its interaction with β2 integrins CD11a / CD18 (LFA-1) and CD11b / CD18 (MAC-1) on the surface of leukocytes is a crucial step in the migration of leukocytes across endothelial cells. Therefore, ICAM1 is often highly expressed at inflammatory sites. Previous studies have shown that high expression of ICAM1 promotes the invasion and migration of cancer cells. Therefore, this study intends to use ADC drugs to target ICAM1 for postoperative treatment or post-drug resistance treatment of patients with tongue cancer, in order to fill the current gap in targeted drugs in this field in clinical practice and improve the well-being of patients.

[0025] The present invention first provides the use of ICAM1 or its encoding gene as a target in preparing a tongue cancer treatment or diagnosis product.

[0026] The use of ICAM1 or its encoding gene as a target in the preparation of tongue cancer treatment or diagnosis products specifically refers to: using ICAM1 protein or its encoding gene as a recognition object, a substance that can reduce ICAM1 levels or kill tongue cancer cells.

[0027] The present invention also provides use of a substance that specifically binds to ICAM1 in preparing a product for treating or diagnosing tongue cancer.

[0028] In one embodiment, the substance that specifically binds to ICAM1 is selected from an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof.

[0029] In one embodiment, the structure of the antibody-drug conjugate is as follows: Ab-(LD)n, wherein Ab is an anti-ICAM1 antibody, L is a linker, D is a cytotoxic agent (or warhead), and n is an integer of 1-40.

[0030] In the present invention, the anti-ICAM1 antibody is selected from monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), multispecific antibodies (eg, bispecific antibodies) and antibody fragments (eg, Fab, F(ab')2, Fv, scFv). The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0031] The anti-ICAM1 antibody can be selected from any antibody or antigen-binding fragment thereof that can bind to ICAM1 in the prior art, such as the ICAM1 antibody of MabPlex.

[0032] In one embodiment, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-ICAM1 antibody are shown in SEQ ID NO. 1 and 2, respectively:

[0033] QVQLQQSGPELVRPGVSVKISCKGSGYTFIDYAIHWVKESHAKSLEWIGVISAYSGDTNYNQKFKGKATMTVDKSSNTAYLELARLTSEDSAIYYCARGGWLLLSFDYWGQGTTLTVSSA(SEQ ID NO.1)

[0034] DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNNYLHWYLQKSGQAPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGGGTKLEIKR(SEQ ID NO.2)

[0035] The anti-ICAM1 antibody is coupled to the cytotoxic agent via a linker. Linkers are divided into two categories: non-cleavable linkers and cleavable linkers. Cleavable linkers can be cleaved in the target cell and release the drug toxic agent. Cleavable linkers can be divided into two main categories: chemically unstable linkers and enzyme-unstable linkers. Chemically unstable linkers can be selectively cleaved due to the different properties of plasma and cytoplasm. Such properties include pH value, glutathione concentration, etc. Linkers sensitive to pH value are usually called acid-cleavable linkers. Such linkers are relatively stable in the neutral environment of blood (pH7.3-7.5), but will be hydrolyzed in weakly acidic endosomes (pH5.0-6.5) and lysosomes (pH4.5-5.0). Linkers sensitive to glutathione are also called disulfide bond linkers. Enzyme-unstable linkers, such as peptide linkers, can better control drug release. Peptide linkers can be effectively cut by lysosomal proteases, such as cathepsin B or plasmin (the content of such enzymes is increased in some tumor tissues). This peptide connection is very stable in the plasma circulation because the proteases are usually inactive due to the unfavorable pH value outside the cell and serum protease inhibitors. In view of the high plasma stability and good intracellular cleavage selectivity and effectiveness, enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical enzyme-labile linkers include Val-Cit (vc), Phe-Lys, Gly-Gly-Phe-Gly, etc.

[0036] The linker can include one or more linker components. Exemplary linker components include 6-maleimidocaproyl, maleimidopropionyl-valine-citrulline, alanine-phenylalanine, p-aminobenzoyloxycarboxyl, N-succinimidyl 4-(2-pyridylthio) pentanoate, N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate and N-succinimidyl (4-iodo-acetyl) aminobenzoate. Other exemplary linker components can also be linkers that include amino acid units to allow protease cleavage, thereby facilitating release of cytotoxic agents from antibody-drug conjugates after 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.

[0037] The cytotoxic agent is selected from a toxin, a chemotherapeutic drug, an antibiotic, a radioisotope, a growth inhibitory agent, a nanobody or a polypeptide.

[0038] Exemplary cytotoxic agents include: maytansine; maytansine-like substances; topoisomerase I inhibitors (such as camptothecin derivatives: DX-8951 derivative Dxd); tubulin inhibitors (such as monomethyl auristatin peptide E (MMAE) and monomethyl auristatin peptide F (MMAF); calicheamicins (such as calicheamicin); doxorubicin (such as doxorubicin); benzodipyrrole antibiotics (such as duocarmycins, CC-1065, etc.) and other cyclopropylpyrroloindole-4-one (cyclopropapyrroloind-4-one, CPI) derivatives, such as cyclopropylbenzidindole-4-one analogs, and pyrrolobenzodiazepines (PBD) or PBD dimers.

[0039] 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.

[0040] In one embodiment, 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).

[0041] The DAR value of the ICAM1-MMAE is 4.

[0042] The DAR value of the ICAM1-Dxd is 8.

[0043] The maleimide group in the linker is covalently coupled to the cysteine ​​in the anti-ICAM1 antibody.

[0044] In another embodiment, the substance that specifically binds to ICAM1 is selected from ICAM1 inhibitors.

[0045] ICAM1 inhibitors refer to molecules that have an inhibitory effect on ICAM1. Inhibitory effects on ICAM1 include, but are not limited to, inhibiting the level or activity of ICAM1.

[0046] Inhibiting ICAM1 activity means reducing the activity of ICAM1. Preferably, compared with before inhibition, the activity of ICAM1 is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0047] Inhibiting the level of ICAM1 may be by inhibiting the transcription or translation of the ICAM1 gene. Specifically, it may mean: preventing the transcription of the ICAM1 gene, or reducing the transcription activity of the ICAM1 gene, or preventing the translation of the ICAM1 gene, or reducing the translation level of the ICAM1 gene.

[0048] Those skilled in the art can use conventional methods to regulate ICAM1 gene expression, such as gene knockout, homologous recombination, interfering RNA, etc.

[0049] Preferably, compared with the wild type, ICAM1 gene expression is reduced by at least 10%, more preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, even more preferably by at least 90%, and most preferably, ICAM1 gene is not expressed at all.

[0050] The ICAM1 inhibitor includes, but is not limited to, nucleic acid molecules, small molecule chemical drugs, antibody drugs, polypeptides, proteins, interfering lentiviruses or adeno-associated viruses. The nucleic acid includes, but is not limited to, antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNAs or short hairpin RNAs (shRNAs) prepared by endoribonuclease III. The protein includes, for example, monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), multispecific antibodies (e.g., bispecific antibodies), nanobodies, and antibody fragments (e.g., Fab, F(ab')2, Fv, scFv).

[0051] The tongue cancer treatment or diagnosis product must include a substance that specifically binds to ICAM1, and uses the substance that specifically binds to ICAM1 as an effective ingredient.

[0052] The tongue cancer treatment or diagnosis product may be a single-component substance or a multi-component substance.

[0053] The form of the tongue cancer treatment or diagnosis product is not particularly limited and may be in the form of solid, liquid, gel, semi-fluid, aerosol or other material forms.

[0054] The tongue cancer treatment or diagnosis product is mainly targeted at mammals. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0055] The tongue cancer treatment or diagnosis products include but are not limited to drugs, health products, foods, etc.

[0056] The tongue cancer treatment product is selected from: targeted ICAM1 antibodies or antigen-binding fragments, exosomes, liposomes, SLP nanoparticles, CAR-T cells, oncolytic viruses, ADCs, small molecule drug conjugates, bispecific antibodies, nucleic acid molecules, small molecule chemical drugs, peptides, proteins, interfering lentiviruses or adeno-associated viruses, etc.;

[0057] The tongue cancer diagnostic product is selected from: exosomes, CTC, CT DNA, blood / urine protein or MRI / PET / ultrasound imaging probes, etc.

[0058] The substance that specifically binds to ICAM1 can be used in combination with other substances to prepare tongue cancer diagnosis products.

[0059] The tongue cancer is selected from squamous cell carcinoma or adenocarcinoma.

[0060] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0061] Before further describing the 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 terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0062] When the embodiments give a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by technicians in this field. In addition to the specific methods, equipment, and materials used in the embodiments, according to the technical personnel in this field's grasp of the prior art and the description of the present invention, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention. Reagents, equipment, and cell culture used in the embodiments

[0063] Experimental reagents:

[0064] Antibodies: Anti-IgG1-PE (biolegend), Anti-ICAM1-PE (biolegend), Anti-ICAM1antibody (Sigma-Aldrich). 1×PBS buffer (pH7.2-7.4, pH8.6, Solarbio), South American fetal bovine serum (ExCell Bio); double antibody (penicillin / streptomycin, Biosharp); New Zealand fetal bovine serum (Gbico); DMEM, DMEM / F12 complete medium (Gbico); bovine serum albumin (BSA, Sigma-Aldrich); Hoechst 33342 staining solution (Biosharp); 4% paraformaldehyde / universal tissue fixative (Biosharp); Cell Counting Kit-8 cell counting reagent (CCK8) (Uningwei); 0.25% trypsin + 0.02% EDTA solution (Cienry); BL203B serum-free cell freezing solution (Biosharp); flow cytometry magnetic beads (Bangs Laboratories); sodium pyruvate (Biosharp); non-essential amino acids (NEAA, MEM) hydrocortisone sodium succinate (Aladdin); 5-fluorouracil (Meilunbio);

[0065] ADC drugs: ICAM1-MMAE (DAR=4), ICAM1-DXd (Mabplex, DAR=8), the preparation method is shown in Example 3 of patent application number 2022103321761, and the structural schematic diagrams of the two ADC drugs are shown in Figure 7 As shown, ICAM1antibody is a human-mouse chimeric IgG1 antibody targeting CD54 protein, which is produced by a third-party company through CHO cell line expression, and the heavy chain and light chain amino acid sequences are shown in SEQ ID NO.3 and 4, respectively. The two toxic warheads are synthetic derivatives of sea dolitoxin 10 (Monomethyl auristatin E, MMAE) and camptothecin derivatives, which are topoisomerase I inhibitors. MMAE is a mitotic inhibitor that can inhibit tubulin polymerization and is widely used in ADC drug design; DXd is a toxic small molecule that inhibits cell growth by destroying the spatial structure of DNA and causing DNA damage. Due to the differences in the properties of the molecules themselves and the binding sites, the combination of different linkers and toxic small molecules has different drug resistance ratios (DARs). The DAR values ​​determined by the present invention using different linkers and toxic warheads are as follows: mc-vc-MMAE (DAR=4), GGFG-DXd (DAR=8). Both linkers are coupled to the exposed cysteine ​​thiol group at the hinge site of the antibody through their own maleimide group, so that the linker-toxic warhead is bound to the antibody.

[0066] QVQLQQSGPELVRPGVSVKISCKGSGYTFIDYAIHWVKESHAKSLEWIGVISAYSGDTNYNQKFKGKATMTVDKSSNTAYLELARLTSEDSAIYYCARGGWLLLSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:3)

[0067] DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNNYLHWYLQKSGQAPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:4).

[0068] Cell culture:

[0069] Human tongue squamous cell carcinoma cell line CAL-27 purchased from ATCC, SAS and SCC-4 purchased from Nanjing Kebai Biotechnology Co., Ltd., and primary cells (tongue 003#1) were selected as experimental groups, and human embryonic kidney cell line 293T (ATCC) was used as negative control. 293T and CAL-27 were cultured using DMEM medium with 10% South American fetal bovine serum and 1% double antibody; SAS cells were cultured using DMEM / F12 medium with 10% South American fetal bovine serum and 1% double antibody; SCC-4 was cultured using DMEM / F12 medium with 10% fetal bovine serum, 1% double antibody, which also contained 0.5mM sodium pyruvate and 400ng / mL hydrocortisone; tongue 003#1 was cultured using DMEM / F12 medium with 10% New Zealand fetal bovine serum and 1% double antibody, and 1% NEAA. The cells were placed in a humidified incubator containing 95% air and 5% CO2, and the constant temperature was adjusted to 37°C. All cells were passaged at least three times after recovery before the experiment was carried out. When the cell morphology changed during the passage, the cells were discarded and revived.

[0070] The steps for constructing the TSCC patient primary cell line Tongue 003#1 are as follows:

[0071] The TSCC tissue samples of the patients were frozen in advance, and completely thawed in a 37°C water bath. The samples were rinsed once with resuscitation solution, and the tissue precipitate was collected by low-speed centrifugation. The samples were then digested with digestion solution, and the cell suspension was collected by filtration and centrifugation to collect the cell precipitate. Red blood cell lysis solution was added, and the samples were mixed and lysed by inversion at room temperature. The cell precipitate was collected by centrifugation. The samples were inoculated into cell culture bottles for culture to obtain the primary cell line Tongue 003#1.

[0072] The contents of the embodiments are briefly described as follows:

[0073] Methods: In the experiment of screening antigen epitopes, three human tongue squamous cell carcinoma cell lines (SCC-4, SAS and CAL-27) and a self-established cell line (tongue 003#1) were selected as the experimental group, and the human embryonic kidney cell line 293T was selected as the negative control. For the selected cell lines, flow cytometry was first used to screen the overexpressed antigen epitopes unbiasedly, and the single-photon confocal microscope was used to perform imaging experiments on the behavior of cell antigen epitopes binding to the target antibody to verify the flow results. Immunohistochemical staining was performed on tumor tissue and adjacent tissue sections to confirm the expression of ICAM1 in patient tissues. In order to verify the behavior of cell endocytosis of antibodies, confocal imaging and flow cytometry were used to observe the endocytosis of the target antibody by the selected cell lines, and appropriate time nodes were selected for quantitative analysis. In order to verify the killing effect of ADC drugs (ICAM1-ADC) with ICAM1 as a molecular target at the cellular level, the CCK8 method was used to compare the efficacy and toxicity of the designed ICAM1-ADC with 5-fluorouracil, a first-line chemotherapy drug currently used in the clinical treatment of tongue cancer. In the in vivo validation part, the small animal in vivo optical imaging system was used to verify the distribution and enrichment of drug molecules in tumor-bearing nude mice of the BALB / c strain; finally, nude mice of the BALB / c strain were also selected, and patient tumor tissues were subcutaneously transplanted to construct PDX models. After tumor formation, PBS, ICAM1 naked antibody, 5-fluorouracil and ICAM1-ADC drugs were given respectively, and the tumor growth of nude mice in the experimental group and the control group was measured, recorded and compared; in addition, CDX models were constructed by subcutaneous injection of tongue cancer cells, and the treatment and measurement schemes were the same as above.

[0074] Results: Flow cytometry and confocal microscopy showed that ICAM1 was highly expressed on the cell membrane surface of the experimental group selected by the institute; immunohistochemistry showed that ICAM1 staining was overexpressed in human tongue squamous cell carcinoma tissue sections, while it was not expressed in adjacent tissues. The gradual internalization of antibodies by cells can be observed at different time points, and the antibody internalization rate is about 40% in four hours. CCK8 results showed that ICAM1-ADC drugs were less toxic to cells than chemotherapy drugs; while for normal cells, ICAM1-ADC had no obvious killing effect. In vivo distribution experiments showed that antibodies were highly enriched in tumor sites. After administration, the tumor volume of nude mice in the experimental group was significantly smaller than that in the control group, the overall tumor growth rate was significantly reduced, and the tumor-free survival period was significantly increased.

[0075] Conclusion: ADC drugs targeting ICAM1 have good anti-tumor effects and have extremely high potential for clinical transformation.

[0076] Example 1 Data Analysis

[0077] Using the TCGA database, 153 TSCC samples and 15 adjacent tissue samples were included in the study, and ICAM1 gene expression information and clinical information were extracted. Afterwards, the relationship between ICAM1 expression and clinical characteristics was analyzed according to tissue type, clinical stage, and gender. In addition, the correlation analysis between ICAM1 expression and survival was performed with the help of the above sample information. Finally, the relationship between ICAM1 and the tumor microenvironment was analyzed: ESTIMATE was used to perform matrix scoring and immune scoring on RNA expression data, and their differences were compared. CIBERSORT was used to compare the composition of immune cells between ICAM1 high and low expression groups, that is, the differences in immune cell infiltration levels.

[0078] The results showed that the expression of ICAM1 mRNA in tumor tissues was significantly higher than that in normal tissues. In addition, the expression level of ICAM1 was closely related to the progression of the disease and gradually increased with the progression of the disease. Moreover, the level of ICAM1 was significantly increased in the cancer tissues of TSCC patients who had previously received chemoradiotherapy. At the same time, there was no gender difference in the expression of ICAM1 ( Figure 1 ).

[0079] Next, the 153 TSCC samples were divided into ICAM1 low (n=76) and ICAM1 high (n=76) Figure 2 When evaluating the stromal and immune scores of RNA expression data using ESTIMATE, ICAM1 was observed high The scores of the group were significantly higher than those of ICAM1 low group, indicating that low ICAM1 expression was associated with poor prognosis (P<0.001).

[0080] Further analysis using CIBERSORT revealed that ICAM1 high CAM1 low There were differences in the level of immune cell infiltration between the groups. Upregulation of ICAM1 was associated with an increase in memory B cells, M1 macrophages, resting NK cells, and eosinophils in the tumor microenvironment, as well as the recruitment of neutrophils ( Figure 3 .).

[0081] Example 2 Target Verification

[0082] Flow cytometry: 69 cell membrane proteins were screened unbiasedly using two human TSCC cell lines (SAS and CAL-27) and human normal kidney cell line 293T by flow cytometry. 4 × 10 6Cells were blocked with PBS containing 1% bovine serum albumin (BSA) for 30 minutes under ice bath, and resuspended with 800 μL blocking solution, and divided into three groups. Each group was added with 2 μL PBS, IgG1-PE or ICAM1-PE, and then placed on a shaker in the dark for 45 minutes. After washing twice, the cells were detected by flow cytometry (CytoFLEX LX, Beckman Coulter). By comparing the mean fluorescence intensity (MFI) of ICAM1 and the control group, the expression of ICAM1 on the cell membrane can be roughly known. In order to obtain more accurate quantification, at the same time as the above experiment, the antibody bound to the cell was quantitatively analyzed using a magnetic bead quantitative kit: after shaking the magnetic beads, one drop was taken from each of the three groups, and the above operation was followed to conduct parallel experiments and then put on the computer, and imported into GraphPad to draw a standard curve for quantitative analysis of fluorescent antibodies.

[0083] Confocal localization: A single-photon confocal microscope (A1 HD25, NIKON) was used to take pictures to visually demonstrate the behavior of cell binding to antibodies. 1×10 6 After the cells are well attached, the culture medium is removed and the cells are rinsed once with PBS. Under ice bath conditions, the cells are blocked with PBS containing 1% BSA for 30 minutes. At 37°C, 2μL PBS, IgG1-PE or ICAM1-PE are added to each group respectively. After incubation, the cells are washed 1-2 times with PBS and fixed with 500μL paraformaldehyde for 20 minutes. Wash, add 1mL Hoechst dye solution, and stain at 37°C for 5-7 minutes. Wash 1-2 times with PBS, and finally add 1mL PBS to keep the cells moist and take pictures.

[0084] Immunohistochemistry (IHC)

[0085] Tongue cancer tissue samples were selected, rinsed with xylene (three times) for 10 minutes, immersed in alcohol gradient for 3 minutes, and then rinsed with double distilled water for 3 minutes. Then, antigen repair was performed with antigen repair solution (3g sodium citrate, 0.4g citric acid, 1000ml double distilled water). A preliminary experiment was designed to screen out the optimal antibody concentration, and the incubation steps were repeated at the optimal concentration. Diaminobenzidine (DAB) color development solution was prepared, and one drop of DAB was added to each piece. After color development for 1 minute, water was added to terminate. After washing with water 3 times, it was soaked in hematoxylin for 2 minutes, washed with water 3 times; DAPI staining was performed for 2 minutes, and washed with water 3 times. After that, it was immersed in gradient alcohol, dried naturally, and scanned on a machine (VS200, Olympus) after sealing.

[0086] The results are as follows Figures 4 to 6 As shown. 8 proteins are overexpressed on the surface of TSCC cells ( Figure 4), except CD73 and CD274, the other six proteins were overexpressed in the three cell lines and were therefore excluded. CD73 and CD274 were overexpressed in SAS cells, but their expression levels were 10.3 and 13.9 times higher than those in 293T cells, respectively; however, in CAL-27, their overexpression was limited, only 9.4 times (CD73) and 6.66 times (CD274) higher than those in 293T cells. The binding of SAS and CAL-27 cells to ICAM1 was 25.8 and 45.7 times higher than that of 293T cells, respectively, and the binding of 293T cells to ICAM1 was almost similar to that of IgG (293T CD54 / 293T IgG =1.128), confirming that ICAM1 is highly overexpressed in TSCC cells and lowly expressed in normal cells. The present invention also found that ICAM1 is highly overexpressed in TSCC cells and lowly expressed in normal cells in two other TSCC cell lines: SCC-4 (SCC-4 CD54 / SCC-4 IgG =7.47) and the self-established TSCC patient primary cell line Tongue 003#1 (Tongue 003#1 CD54 / tongue003#1 IgG =15.7) further confirmed the widespread overexpression of ICAM1 in TSCC cells. The average density of ICAM1 antibodies on the surface of each cell was further quantified by magnetic beads: 29125 (SAS), 179385 (CAL-27), 26963 (SCC-4), 75154 (Tongue 003#1) and 1764 (293T). The results observed by confocal single-photon microscopy were consistent with those of flow cytometry. At the same time, the results showed that the expression of ICAM1 was localized on the cell membrane surface, providing theoretical support for the binding of ADC to cells and subsequent internalization ( Figure 5 , the scale bar in the figure is 25 μm).

[0087] Based on the above theoretical support, the present invention uses patient-derived tongue cancer tissue and adjacent tissue for immunohistochemical staining ( Figure 6 ), the results showed that ICAM1 was highly expressed in cancer tissue sections of 26 patients, while adjacent tissues showed no staining, confirming the assumption that ICAM1 is highly expressed in tongue cancer tissues.

[0088] Example 3 demonstrates the behavior of antigen epitope-mediated endocytosis of antibodies

[0089] Flow cytometry quantification:

[0090] Cell collection: Trypsin digestion, collect 7×10 6Cells were centrifuged and the supernatant was removed; 6-8 mL of PBS was added to suspend and rinse once, centrifuged and the supernatant was removed. After adding 1.5 mL of PBS and suspending, the tubes were divided into 6 groups at 200 μL / tube. 2 μL of anti-ICAM1 was added to each tube, mixed and incubated on ice for 30 minutes. PBS was washed once and resuspended in 200 μL PBS. The endocytosis time of the six groups was 0, 15, 30, 60, 120 and 240 minutes respectively. After washing, the tubes were resuspended in 200 μL PBS, and 2 μL of anti-ICAM1 antibody-PE was added to each of the 6 tubes and incubated on ice for 30 minutes. PBS was washed twice and then put into the machine. Substitute the antibody quantity-MFI standard curve obtained by the quantitative method to obtain the average number of bound antibodies, calculate the endocytosis efficiency, and fit the endocytosis efficiency curve using GraphPad.

[0091] Confocal microscopy to verify endocytosis: 1×10 6 cells, add 1mL of culture medium, and incubate at 37℃ overnight. After good adhesion, remove the culture medium and rinse once with PBS. In an ice bath, add 1mL PBS and 2μL IgG-PE, and stain on ice for 45 minutes. Aspirate PBS, rinse once with pre-cooled PBS, add 1mL PBS, and place at 37℃ for internalization for 0, 15, 30, 60, 120, and 240 minutes. After each group of cells are fixed with paraformaldehyde for 20 minutes, wash with PBS, add 1mL Hoechst dye, stain at 37℃ for 20-30 minutes, and wash 1-2 times with PBS. Finally, add 1mL PBS to keep the cells moist and take pictures.

[0092] The results are as follows Figure 8 As shown, the results showed that after 4 hours, the internalization rate of ICAM1 antibody by SAS and CAL-27 reached about 40%, and confocal microscopy also showed a similar phenomenon: the fluorescent antibody gradually entered the cytoplasm over time.

[0093] Example 4 In vitro cytotoxicity experiment

[0094] The cytotoxicity of the two ICAM1-ADCs was evaluated in three human TSCC cell lines (SAS, SCC-4, CAL-27) and normal 293T cells and compared with the standard chemotherapeutic drug 5-FU.

[0095] CCK-8 experimental steps: 1×10 4 / 100μL suspension was inoculated into cells and pre-cultured in an incubator for 24h. Drugs were diluted with corresponding culture medium in a 10-fold concentration gradient (10-10 -6μg / mL), aspirate the culture medium in the 96-well plate, add 100μL of drug-containing culture medium to each column according to different concentration gradients (6 replicates for each concentration), and place it in the incubator for 72h. Aspirate the original culture medium and add 100μL CCK-8 working solution to each well. Incubate the culture plate in the incubator for 1-4 hours (the duration depends on the condition of each cell, and the OD value of the control group is optimal in the range of 0.8-1.2). Use a microplate reader (SPARK, TECAN) to measure the absorbance at a wavelength of 450nm, and then use EXCEL to process the obtained OD value to obtain the cell growth rate corresponding to each drug concentration. Finally, use GraphPad's 4-parameter nonlinear regression model to process the obtained data, fit the inhibition rate curve, and obtain the IC of the drug to the cell line. 50 value.

[0096] The results are as follows Fig. 9 As shown, the results showed that ICAM1-MMAE had strong anti-cancer activity against SAS (IC50: 91.4nM), CAL-27 (IC50: 267nM) and SCC-4 (IC50: 76nM) cells. ICAM1-DXd has a less toxic DNA topoisomerase inhibitor load, and the IC50 for TSCC killing is: SAS (41.6nM) and SCC-4 (545nM).

[0097] In addition, the two ICAM1-ADCs showed no significant toxicity against normal 293T cells that do not express ICAM1. Compared with the two ICAM1-ADCs, 5-FU showed exponentially lower cytotoxicity against TSCC cells of SAS (IC50: 32.9 μM), CAL-27 (IC50: 1.03 μM), and SCC-4 (IC50: 106 μM). Due to the lack of tumor specificity, 5-FU nonspecifically killed normal 293T cells (IC50: 1.66 μM) and TSCC cells. In addition, the naked ICAM1 antibody did not show any cytotoxicity at the cellular level. Therefore, the two ICAM1-ADCs showed a potent effect on human TSCC cells in vitro and deserve further study in the in vivo model of TSCC.

[0098] Example 5 In vivo drug molecule organ distribution experiment

[0099] IVIS imaging was used to determine the tumor specificity and biodistribution of ICAM1 antibodies and ICAM1-ADCs. ICAM1 antibodies and ICAM1-MMAE conjugated with fluorescent dye Cy3 were intravenously injected into nude mice implanted with TSCC tumors. Fifteen male BALB / c nude mice were subcutaneously injected with 3×10 SAS cells of the tongue squamous cell carcinoma cells. 6 / , when the mean tumor volume reaches 150mm 3When the concentration of ICAM1 and ICAM1-MMAE was about 1:5, the antibody-fluorescent conjugate was synthesized based on the principle that the free amino group of the monoclonal antibody can form a stable amide bond with cy3: ICAM1 and ICAM1-MMAE were added to 0.5 ml of PBS at pH 8.6 at a molar mass of 1:5, and incubated at room temperature for 4 hours. The mixture was passed through a desalting column, and the eluent was PBS. The antibodies with larger molecular weight were washed out first. According to the instructions of the desalting column, the second tube of liquid flowing out after the sample was added was collected to obtain the coupled ICAM1-cy3 and ICAM1-MMAE-cy3. At the same time, IgG-cy3 was synthesized under the same conditions as an isotype control. Afterwards, the absorbance of the antibody-fluorescent conjugate at 562 nm was determined by an enzyme-labeled instrument according to the BCA method. The concentration was calculated according to the standard curve. The mice bearing tumors (n=5 in each group) were given a dose of 2 mg / kg via the tail vein. The mice were killed 48 hours later, and the organs were removed. The small animal in vivo imaging system (IVIS Lumina Series III, PerkinElmer) was used to image each organ at a wavelength of 520-570 nm, and the average fluorescence intensity of each organ was quantitatively analyzed.

[0100] Organ brightness Fig.10 As shown in the figure, it can be clearly seen that in the ICAM1-PE group and the ICAM1-MMAE-PE group, the brightness of the tumor site is the highest, and the brightness of the liver is second, which is about half of the brightness of the tumor. In the IgG group, the brightness of the liver and tumor is almost the same, and the ROI is almost similar. After two-way analysis of variance, the ROI values ​​of the tumors among the experimental groups were significantly different. The mean ROI of the ICAM1 tumor was 2.32 times that of the IgG group, and the ICAM1-MMAE group was 1.86 times that of the IgG group. These in vivo tumor accumulation data are consistent with the in vitro near-infrared images of the resected tumors. These data confirm in vivo that ICAM1 has a more accurate tumor cell recognition ability compared with the isotype control.

[0101] Example 6 In vivo efficacy verification

[0102] In order to verify the in vivo targeted ablation effect of ICAM1-ADC on tumor tissue, we established nude mouse PDX and CDX models. The patient's surgical specimen (number Tongue 003#1) was selected for the experiment and implanted subcutaneously into the right shoulder of nude mice (5-6 weeks old, female) after resuscitation. There were 6 mice in each treatment group and control group. When the average tumor volume reached about 200mm 3 The treatment group was given 5-FU, ICAM1 naked antibody, ICAM1-DXd and ICAM1-MMAE, respectively, by tail vein injection, with a dose of 5 mg / kg; the control group was given PBS, 0.1 mL per mouse, and the drug was administered once a week according to the above scheme. From the first administration, the weight and tumor volume of each nude mouse were measured and recorded every other day until the tumor volume of the nude mice in the control group reached 1000mm 3, the experiment was stopped, the mice were killed, and each tumor mass was taken out for weighing and photographing. In the CDX experiment, SAS cells were selected and injected subcutaneously into the right abdomen of nude mice (6-7 weeks old, male) at 5×10 6 / mouse, with an average tumor volume of about 150mm 3 At the same time, the two drug-treated groups were given ICAM1-DXd and ICAM1-MMAE, respectively, and the control group was given PBS, with 8-9 mice in each group. The drug administration and measurement scheme were the same as above.

[0103] like Fig.11 As shown, 5 mg / kg was administered weekly through the tail vein, and the body weight and tumor volume of nude mice were recorded every two days. After an 11-day treatment period, the treatment was terminated, the tumors were removed and photographed. Although the efficacy of ICAM1-MMAE in the PDX model was not significantly better than that of other groups, this difference was attributed to the heterogeneity of the tissues used to establish the PDX model, but ICAM1-MMAE still had a tendency to inhibit tumors compared with the control group (P=0.2066). The tumor growth inhibition value (Tumor Growth Inhibition value, TGI) of ICAM1-DXd was 54.0%. At the end of treatment, the tumor volume (P=0.0147) and weight (P=0.0239) of the ICAM1-DXd group were significantly reduced compared with the PBS control group. The tumor weight of each group was recorded as the average value: 0.308g (ICAM1-DXd), 0.455g (ICAM1-MMAE), 0.548g (5-FU), 0.495g (ICAM1 naked antibody), 0.670g (PBS). Compared with ICAM1-MMAE, ICAM1-DXd had a stronger tumor inhibitory effect (P=0.0204), and the body weight of mice did not show significant fluctuations during the entire treatment period.

[0104] Considering that no significant tumor inhibitory effect of naked ICAM1 antibody and 5-FU was found in the more representative PDX model, from the perspective of animal welfare and following the 3R principle, only the ICAM1-ADCs group and the PBS control group were set up in the CDX treatment model ( Fig.12). After 11 days of treatment and measurement, the mice were killed, the tumors were removed and photographed, weighed and statistically analyzed. At the CDX model endpoint, both the ICAM1-DXd group (P=0.0004) and the ICAM1-MMAE group (P=0.0086) showed significant tumor inhibition compared with the PBS group. The tumor weight of each group was recorded as the average value: 0.080g (ICAM1-DXd), 0.212g (ICAM1-MMAE), 0.458g (PBS). The tumor weight of the experimental group was significantly lower than that of the control group (ICAM1-DXd: P<0.0001, ICAM1-MMAE: P=0.0086). The TGI of ICAM1-DXd and ICAM1-MMAE were 82.5% and 53.6%, respectively. At the same time, the tumor inhibition effect of ICAM1-DXd was slightly better than that of ICAM1-MMAE (P=0.3086), but there was no significant difference between the two. Similarly, there was no obvious fluctuation in the weight of the mice.

[0105] These findings suggest that ICAM1-ADCs, especially ICAM1-DXd, have the potential to serve as effective targeted therapeutics for TSCC cells and warrant further investigation in both preclinical and clinical settings.

[0106] In summary, the present invention found that intercellular adhesion molecule-1 (ICAM1) is highly overexpressed in tumor tissues of patients with tongue squamous cell carcinoma and is a potential drug target. It is also determined that an antibody-drug conjugate (ADC) targeting ICAM1 can provide an effective targeted drug with clinical transformation potential for patients with tongue squamous cell carcinoma.

[0107] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present 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 the present invention.

Claims

1. Use of a substance that specifically binds to ICAM1 in the preparation of a product for treating or diagnosing tongue cancer.

2. The use according to claim 1, characterized in that The substance that specifically binds to ICAM1 is selected from antibody-drug conjugates or pharmaceutically acceptable salts thereof.

3. The use according to claim 2, characterized in that: The structure of the antibody-drug conjugate is shown below: Ab-(LD) n , wherein Ab is an anti-ICAM1 antibody, L is a linker, D is a cytotoxic agent, and n is an integer of 1-40.

4. The use according to claim 3, characterized in that The anti-ICAM1 antibody is selected from a full-length antibody or an antibody fragment; preferably, the antibody fragment is selected from Fab, F(ab')2, Fv or scFv.

5. The use according to claim 3, characterized in that: The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-ICAM1 antibody are shown in SEQ ID NOs. 1 and 2, respectively; and / or, The linker is selected from a non-cleavable linker or a cleavable linker; preferably, the cleavable linker is selected from a chemically unstable linker or an enzyme-labile linker.

6. The use according to claim 3, characterized in that: The linker comprises one or more linker components, and the linker components are selected from any one or more of maleimidopropionyl, maleimidocaproyl, p-aminobenzoyloxycarboxyl, valine-citrulline, alanine-phenylalanine, glycine-glycine-glycine, glycine-valine-citrulline, glycine-glycine-phenylalanine-glycine; preferably, the linker is selected from maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarboxyl or maleimide-GGFG tetrapeptide.

7. The use according to claim 3, characterized in that: The cytotoxic agent is selected from toxins, chemotherapeutic drugs, antibiotics, radioactive isotopes, small molecule inhibitors, nanobodies or polypeptides; preferably, the cytotoxic agent is selected from any one or more of the following: maytansine, maytansine-like substances, topoisomerase I inhibitors, microtubule inhibitors, calicheamicin and its derivatives, doxorubicin and its derivatives; preferably, the cytotoxic agent is selected from Dxd, MMAE or MMAF.

8. The use according to claim 3, characterized in that: The antibody-drug conjugate is ICAM1-MC-VC-PAB-MMAE or ICAM1-Maleimide GGFG peptide-Dxd, the ICAM1 is an anti-ICAM1 antibody, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-ICAM1 antibody are shown in SEQ ID NO.1 and 2, respectively.

9. The use according to claim 1, characterized in that The substance that specifically binds to ICAM1 is an ICAM1 inhibitor; preferably, the ICAM1 inhibitor is selected from nucleic acid, small molecule chemical drugs, polypeptides, proteins, interfering lentivirus or adeno-associated virus.

10. The use according to claim 1, characterized in that The tongue cancer is selected from squamous cell carcinoma or adenocarcinoma.

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