Application of substance specifically binding to ICAM1 in combination with RET inhibitor in preparation of product for treating cancer caused by RET variation

Abstract

The invention relates to the field of medicine, in particular to application of a substance specifically binding to ICAM1 in combination with an RET inhibitor in preparation of a product for treating cancers caused by RET variation, and the cancers caused by RET gene variation are solid tumors resistant to small-molecule inhibitors. The substance which is specifically bound with the ICAM1 is selected from an antibody coupling medicine, and the antibody coupling medicine is selected from anti-ICAM1mAb-MC-VC-PAB-MMAE or anti-ICAM1mAb-MC-GGFG-Dxd, and the substance which is specifically bound with the ICAM1 is selected from a substance which is specifically bound with the ICAM1, a substance which is specifically bound with the ICAM1 and a substance which is specifically bound with the ICAM1. According to the invention, a generally highly expressed antigen epitope ICAM1 is found in a solid tumor with RET gene variation, and the precise targeting effect and the efficient inhibition capability of ICAM1-MMAE and ICAM1-DXd on tumor cells with RET mutation are evaluated on the in-vivo and in-vitro aspects; the ICAM1-ADCs and the obtained specific RET inhibitor BLU-667 are combined for use, and the combined synergistic capability of the ICAM1-ADCs and the obtained specific RET inhibitor BLU-667 is evaluated.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the use of a substance that specifically binds to ICAM1 in combination with a RET inhibitor in the preparation of a product for treating cancers caused by RET mutations. Background Technology

[0002] The RET gene, discovered in 1985, is a proto-oncogene located on chromosome 10q11.2. It encodes a transmembrane glycoprotein receptor tyrosine kinase, which is activated upon interaction with ligands of the glial-derived neurotrophic factor (GDNF) family, triggering downstream signaling and activating the MAPK, JAK-STAT, and PI3K-AKT-mTOR proliferation pathways, thus affecting cell proliferation or cell lifespan.

[0003] RET gene mutations can be broadly categorized into two types: mutations and fusions. Point mutations in the extracellular domain enriched with cysteine ​​at the N-terminus of the RET protein, its hydrophobic transmembrane domain, and its intracellular tyrosine kinase domain lead to the loss of ligand dependence, meaning that phosphorylation occurs without ligands or monomeric pairing. Conversely, RET gene fusion rearrangements cause the RET protein to lose its extracellular domain, similarly enabling activation without ligands. Therefore, RET gene alterations can lead to various cancers or cause certain developmental disorders, including thyroid cancer, non-small cell lung cancer (NSCLC), multiple endocrine neoplasia type 2, and megacolon. Fusions of RET with fusion partners such as KIF5B, ROS, and ALK can be detected in samples from patients with lung adenocarcinoma and colorectal cancer. Point mutations in the gene encoding the extracellular region of the RET protein can lead to hereditary MEN2A syndrome. Furthermore, RET alterations are detected in almost all patients with medullary thyroid carcinoma (MTC), with the most common mutation being the M918T mutation. In addition, evidence suggests that the RET signaling pathway plays a crucial role in drug resistance, making it an effective therapeutic target for developing selective inhibitors to treat cancer. The recently marketed small molecule inhibitor pralatinib (BLU-667) is a RET-specific receptor tyrosinase inhibitor (TKI) approved for clinical use in the treatment of RET fusion-positive papillary thyroid carcinoma and non-small cell lung cancer, as well as mutant medullary thyroid carcinoma. Phase I clinical trials showed that the objective response rate (ORR) for RET fusion-positive non-small cell lung cancer patients who had previously received platinum-based therapy and those who had not received prior systemic therapy was 61% and 70%, respectively, while the ORR for RET fusion-positive thyroid cancer patients was 89%. Moreover, pralatinib demonstrated significant efficacy against all solid tumors with RET gene fusions, regardless of tumor type or fusion partner. However, patients treated with pralatinib have a higher probability of developing resistance. For example, one study focused on patients who had previously received selpercatinib (LOXO-292), including one MTC patient with a RET mutation and one NSCLC patient with CCDC6-RET fusion. The results showed that the MTC patient had a RETG810C / S mutation in the solvent region and a RETY806C / N mutation in the hinge region; the NSCLC patient also showed cross-resistance to BLU-667 due to the RETG810C / S mutation. Another related study, after biopsies of tissues and blood from 18 RET fusion NSCLC patients who had received LOXO-292 / BLU-667 treatment, found two samples with acquired RET mutations, both affecting the RET residues at the kinase solvent front; three resistant samples showed MET amplification, and one sample showed KRAS amplification. Drug resistance is mainly due to the fact that after RET is inhibited, tumor cells maintain their original proliferative activity by activating the MET pathway.

[0004] Clinical treatment of lung and thyroid cancer often employs radiotherapy, chemotherapy, and surgery. However, small molecule inhibitors are prone to drug resistance, and their availability is rapidly evolving, resulting in a scarcity of sustainable drugs. Therefore, more targeted antibody-drug conjugates (ADCs) have emerged. The concept of ADCs was proposed by researchers in the 1980s and has been around for over 30 years. ADCs use chemical and biological methods to conjugate cytotoxic small molecules (payloads) and monoclonal antibodies via linkers. The antibody specifically recognizes tumor cell surface antigens, delivering the cytotoxic target molecule directly to the tumor. This allows the drug to accumulate at the lesion site, increasing drug concentration, improving treatment efficacy, and simultaneously reducing the drug's toxic side effects on normal tissues.

[0005] After intravenous injection, ADC drugs are transported to tumor tissue via the bloodstream. Their monoclonal antibodies specifically bind to tumor cell surface antigens, and endocytosis brings the drug into the cell. Intracellular proteases cleave linkers (or antibodies), releasing toxic small molecules that act on the DNA in the cell nucleus, cutting the DNA chain or acting on microtubules to block cell division, thereby killing tumor cells. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the object of the present invention is to provide the use of a substance that specifically binds to ICAM1 in combination with a RET inhibitor in the preparation of a product for treating cancers caused by RET mutations, thereby solving the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention first provides the use of ICAM1 or its encoding gene as a target in the preparation of products for treating cancers caused by RET gene mutations.

[0008] In this invention, the RET gene variant is selected from RET gene mutation or RET gene fusion.

[0009] In this invention, the cancer caused by the RET gene mutation is selected from thyroid cancer, non-small cell lung cancer (NSCLC), multiple endocrine tumor type 2, esophageal cancer, breast cancer, melanoma, leukemia, or colorectal cancer. The thyroid cancer is selected from medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC). The non-small cell lung cancer is lung adenocarcinoma.

[0010] Preferably, the cancer caused by the RET gene mutation is a solid tumor.

[0011] Preferably, the cancer caused by the RET gene mutation is a cancer resistant to small molecule inhibitors.

[0012] The small molecule inhibitor is a RET receptor tyrosine kinase inhibitor. Preferably, the small molecule inhibitor is pralatinib (BLU-667) or serpatinib.

[0013] The present invention also provides the use of substances that specifically bind to ICAM1 in the preparation of products for treating cancers caused by RET gene mutations.

[0014] The present invention also provides the use of substances that specifically bind to ICAM1 in combination with substances that specifically bind to RET in the preparation of products for treating cancers caused by RET gene mutations.

[0015] Preferably, the substance that specifically binds to ICAM1 is selected from antibody-drug conjugates, and the antibody-drug conjugate is selected from anti-ICAM1 mAb-MC-VC-PAB-MMAE or anti-ICAM1 mAb-MC-GGFG-Dxd.

[0016] The substance that specifically binds to RET is a RET inhibitor. The RET inhibitor is a small molecule inhibitor; preferably, the small molecule inhibitor is a RET receptor tyrosine kinase inhibitor; preferably, the small molecule inhibitor is pralatinib or serpatinib.

[0017] As described above, the use of the ICAM1-specific substance of the present invention in combination with a RET inhibitor in the preparation of products for treating cancers caused by RET mutations has the following beneficial effects: The present invention discovered a universally highly expressed antigenic epitope, ICAM1, in solid tumors with RET gene mutations (taking MTC and NSCLC as examples), and explored the potential regulatory relationship between RET protein activation and high ICAM1 expression. It was proposed for the first time that RET phosphorylation may indirectly upregulate ICAM1 expression levels by activating NF-κB. Subsequently, the precise targeting and efficient inhibitory effects of ICAM1-MMAE and ICAM1-DXd on RET-mutant MTC, NSCLC, and RET-fused NSCLC cells were evaluated in vitro and in vivo. Furthermore, ICAM1-ADCs were combined with the already approved specific RET inhibitor BLU-667 to evaluate the synergistic effect of the two and to further explore the combination mechanism. Attached Figure Description

[0018] Figure 1-1 The graph shows the expression levels of 73 tumor-specific antigens in LC-2 / ad, TT, and 293T.

[0019] Figure 1-2 The expression levels of ICAM1 are shown in LC-2 / ad, TT, H23, H2009, and 293T.

[0020] Figure 1-3 The image shows ICAM1 as a cell membrane localization using immunofluorescence.

[0021] Figure 1-4 The image shows the expression of ICAM1 in MTC tumor tissue and normal tissue.

[0022] Figure 1-5 The image shows the expression of ICAM1 in NSCLC tumor tissue and normal tissue.

[0023] Figure 1-6 The data shows the changes in ICAM1 expression levels after 0-168 hours of LC-2 / ad inhibition by BLU-667, as detected by flow cytometry and Western blotting.

[0024] Figure 1-7 The data shows the changes in ICAM1 expression levels after H23 was inhibited by BLU-667 for 0-168 hours, as detected by flow cytometry and Western blotting.

[0025] Figure 1-8 The image shows the changes in ICAM1 expression levels detected by flow cytometry and Western blotting 0-168 hours after TT administration.

[0026] Figure 1-9 The results show the efficiency of H23 and LC-2 / ad internalization of ICAM1 antibodies within 0-4 hours as measured by flow cytometry.

[0027] Figure 1-10 This is shown as the process by which fluorescent antibodies gradually enter the cytoplasm of TT, H23, and LC-2 / ad cells over time, captured using confocal imaging.

[0028] Figure 2-1 The diagram shows the drug structures of ICAM1-MMAE and ICAM1-DXd.

[0029] Figure 2-2 The results show the cytotoxicity of BLU-667, ICAM1-MMAE, ICAM1-DXd, ICAM1 antibody, and cisplatin / doxorubicin in various cell lines as determined by the CCK-8 assay.

[0030] Figure 2-3 The biodistribution of ICAM1-MMAE and IgG in tumor-bearing nude mice is shown.

[0031] Figure 2-4 The tumor-suppressive effects of ICAM1-DXd, ICAM1-MMAE, ICAM1 antibody, and cisplatin in tumor-bearing nude mice are shown (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0032] Figure 3-1The results show the in vitro toxicity test and synergy index calculation of H23 after the combination of ICAM1-MMAE and BLU-667.

[0033] Figure 3-2 The results show the in vitro toxicity test and synergy index calculation of H23 after the combination of ICAM1-DXd and BLU-667.

[0034] Figure 3-3 The results show the in vitro toxicity test and synergy index calculation of H2009 after the combination of ICAM1-MMAE and BLU-667.

[0035] Figure 3-4 The results show the in vitro toxicity test and synergy index calculation of H2009 after the combination of ICAM1-DXd and BLU-667.

[0036] Figure 3-5 The results show the in vitro toxicity test and synergy index calculation of LC-2 / ad after the combination of ICAM1-MMAE and BLU-667.

[0037] Figure 3-6 The results show the in vitro toxicity test and synergy index calculation of LC-2 / ad after the combination of ICAM1-DXd and BLU-667.

[0038] Figure 3-7 The results show the in vitro toxicity test and synergistic index calculation of TT after the combination of ICAM1-MMAE and BLU-667.

[0039] Figure 3-8 The results show the in vitro toxicity test and synergistic index calculation of TT after the combination of ICAM1-DXd and BLU-667.

[0040] Figure 3-9 The display shows that ICAM1-MMAE will intercept LC-2 / ad during the G2 / M period.

[0041] Figure 3-10 The display shows that ICAM1-DXd will intercept LC-2 / ad during the S phase.

[0042] Figure 3-11 The display shows that BLU-667 will intercept LC-2 / ad in the G0 / G1 phase.

[0043] Figure 3-12 The display shows that ICAM1-MMAE will intercept H23 during the G2 / M period.

[0044] Figure 3-13 The display shows that ICAM1-DXd will intercept H23 in the S phase.

[0045] Figure 3-14The display shows that BLU-667 will intercept H23 in the G0 / G1 phase.

[0046] Figure 3-15 The display shows that ICAM1-MMAE will intercept TT during the G2-M period.

[0047] Figure 3-16 The display shows that ICAM1-DXd will intercept TT during the S phase.

[0048] Figure 3-17 The results showed no significant effect of BLU-667 on the TT cell cycle. Detailed Implementation

[0049] This invention utilizes ICAM1 as a target to develop a tumor-targeting drug for patients with solid tumors (NSCLC and MTC) with RET gene mutations (taking NSCLC and MTC as examples) that can be used in combination with BLU-667. At the same time, in vivo and in vitro validation experiments were designed to explore the relationship between ICAM1 expression and RET phosphorylation level, verify the tumor-suppressive effect of ICAM1-ADCs, and further verify the combined synergistic effect of the two and the different sites of action of the drugs.

[0050] This invention first provides the use of ICAM1 or its encoding gene as a target in the preparation of products for treating cancers caused by RET gene mutations.

[0051] In this invention, the RET gene variant is selected from RET gene mutation or RET gene fusion.

[0052] In this invention, the cancer caused by the RET gene mutation is selected from thyroid cancer, non-small cell lung cancer (NSCLC), multiple endocrine tumor type 2, esophageal cancer, breast cancer, melanoma, leukemia, or colorectal cancer. The thyroid cancer is selected from medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC). The non-small cell lung cancer is lung adenocarcinoma.

[0053] In some embodiments of the present invention, the cancer caused by the RET gene mutation is a solid tumor.

[0054] In some embodiments of the present invention, the cancer caused by the RET gene mutation is a cancer resistant to small molecule inhibitors.

[0055] Small molecule inhibitors are a class of organic compound molecules that can target specific proteins, reducing their activity or inhibiting biochemical reactions. The small molecule inhibitor is a RET receptor tyrosine kinase inhibitor. In some embodiments of the present invention, the small molecule inhibitor is pralatinib (BLU-667) or serpatinib.

[0056] The present invention also provides the use of substances that specifically bind to ICAM1 in the preparation of products for treating cancers caused by RET gene mutations.

[0057] Substances that specifically bind to ICAM1 can reverse resistance to small molecule inhibitors in cancers caused by RET gene mutations.

[0058] The present invention also provides the use of substances that specifically bind to ICAM1 in combination with substances that specifically bind to RET in the preparation of products for treating cancers caused by RET gene mutations.

[0059] In one embodiment, the substance that specifically binds to ICAM1 is selected from antibody-drug conjugates or pharmaceutically acceptable salts or solvent compounds thereof.

[0060] In one embodiment, 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] QVQLQQSGPELVRPGVSVKISCKGSGYTFIDYAIHWVKESHAKSLEWIGVISAYSGDTN YNQKFKGKATMTVDKSSNTAYLELARLTSEDSAIYYCARGGWLLLSFDYWGQGTTLTVSS A(SEQ ID NO: 1)

[0065] DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNNYLHWYLQKSGQAPKLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGGGTKLEIKR (SEQ ID NO: 2)

[0066] In one embodiment, the amino acid sequences of the heavy and light chains of the anti-ICAM1 antibody are shown as SEQ ID NO.3 and 4 respectively.

[0067] MGWSCIILFLVATATGVHSQVQLQQSGPELVRPGVSVKISCKGSGYTFIDYAIHWVKESHAKSLEWIGVISAYSGDTNYNQKFKGKATMTVDKSSNTAYLELARLTSEDSAIYYCARGGWLLLSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:3)

[0068] MGWSCIILFLVATATGVHSDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNNYLHWYLQKSGQAPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:4)

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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).

[0074] The DAR value of the ICAM1-MMAE is 4.

[0075] The DAR value of the ICAM1-DXD is 8.

[0076] The maleimide group in the linker is covalently coupled to the cysteine ​​residue in the anti-ICAM1 antibody.

[0077] In some embodiments of the present invention, the ICAM1-MMAE and ICAM1-DXD are the ICAM1-MMAE and ICAM1-DXD in patent application number 2022103321761.

[0078] In some embodiments of the present invention, the substance that specifically binds to RET is a RET inhibitor. A RET inhibitor refers to a molecule that has an inhibitory effect on RET. An inhibitory effect on RET includes, but is not limited to, inhibiting the level or activity of RET.

[0079] Inhibiting RET activity means reducing RET activity.

[0080] Suppressing RET levels can be achieved by inhibiting the transcription or translation of the RET gene. Specifically, this can mean preventing the RET gene from being transcribed, reducing the transcriptional activity of the RET gene, preventing the RET gene from being translated, or reducing the translation level of the RET gene.

[0081] Those skilled in the art can use conventional methods to regulate RET gene expression, such as gene knockout, homologous recombination, and interfering RNA.

[0082] The RET 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.

[0083] In some embodiments of the present invention, the RET inhibitor is a small molecule chemical drug inhibitor. The small molecule chemical drug inhibitor is a RET receptor tyrosine kinase inhibitor. In some embodiments of the present invention, the small molecule chemical drug inhibitor is pralatinib (BLU-667) or serpatinib.

[0084] Substances that specifically bind to ICAM1 and substances that specifically bind to RET exhibit synergistic effects when used in combination. This invention has demonstrated that, under different test subjects (i.e., different ex vivo cells) and with different substances that specifically bind to ICAM1 (ICAM1-MMAE, ICAM1-DXd) and substances that specifically bind to RET, both substances exhibit synergistic effects, differing only in the concentration at which the synergistic effect is achieved. For example, in the NCI-H23 cell embodiment of this invention, the substance that specifically binds to RET is BLU-667 at a final concentration of 0.25–1.00 μg / mL, and the substance that specifically binds to ICAM1 is ICAM1-MMAE at a final concentration of 0.1 μg / mL–1 μg / mL; or, the substance that specifically binds to RET is BLU-667 at a final concentration of 0.1–2 μg / mL, and the substance that specifically binds to ICAM1 is ICAM1-Dxd at a final concentration of 0.01 μg / mL–10 μg / mL. For example, in the NCI-H2009 cell embodiment of the present invention, the substance specifically binding RET is BLU-667 at a final concentration of 1-2 μg / mL, and the substance specifically binding ICAM1 is ICAM1-MMAE at a final concentration of 0.1 μg / mL-1 μg / mL; or, the substance specifically binding RET is BLU-667 at a final concentration of 0.25-0.5 μg / mL, and the substance specifically binding ICAM1 is ICAM1-MMAE at a final concentration of 1 μg / mL; or, the substance specifically binding RET is BLU-667 at a final concentration of 1-2 μg / mL, and the substance specifically binding ICAM1 is ICAM1-Dxd at a final concentration of 10 μg / mL. For example, in the LC-2 / ad cell embodiment of the present invention, the substance specifically binding RET is at a final concentration of 10 μg / mL. -3 ~10 -4 BLU-667 at a final concentration of 1 μg / mL, wherein the substance specifically binding to ICAM1 is ICAM1-MMAE at a final concentration of 1 μg / mL; or, the substance specifically binding to RET at a final concentration of 5 × 10 μg / mL. -4 BLU-667 at a final concentration of μg / mL, wherein the substance specifically binding to ICAM1 is ICAM1-Dxd at a final concentration of 0.01 μg / mL to 1 μg / mL. For example, in the TT cell embodiment of the present invention, the substance specifically binding to RET is ICAM1-Dxd at a final concentration of 10 μg / mL. -3 BLU-667 at a final concentration of μg / mL, wherein the substance specifically binding to ICAM1 is ICAM1-MMAE at a final concentration of 0.01–10 μg / mL; or, the substance specifically binding to RET is ICAM1-MMAE at a final concentration of 10 μg / mL. -3BLU-667 at a concentration of μg / mL, wherein the substance specifically binding to ICAM1 is ICAM1-Dxd at a final concentration of 1 μg / mL to 10 μg / mL. The aforementioned concentration range falls near the IC50 value; lower drug concentrations result in better inhibitory effects and exhibit synergistic effects.

[0085] When used in combination, the dosage of the substance that specifically binds to ICAM1 and the substance that specifically binds to RET can be calculated by referring to data from animal experiments, but the specific administration should be determined by the results of clinical trials and the clinician.

[0086] Substances that specifically bind to ICAM1, in combination with substances that specifically bind to RET, achieve therapeutic effects by arresting the cell cycle of tumor cells.

[0087] This invention relates to a "combination" as a route of administration, which refers to administering at least one dose of a substance specifically binding to ICAM1 and at least one dose of a substance specifically binding to RET within a certain time period, wherein both substances exhibit pharmacological effects. The time period can be within a dosing cycle, such as within 4 weeks, 3 weeks, 2 weeks, 1 week, or within 24 hours, more preferably within 12 hours. The substance specifically binding to ICAM1 and the substance specifically binding to RET can be administered simultaneously or sequentially. The combined route of administration described in this invention is selected from simultaneous administration, independently prepared and co-administered administration, or independently prepared and sequentially administered administration.

[0088] The present invention also provides a pharmaceutical composition comprising a substance that specifically binds to ICAM1 and a substance that specifically binds to RET.

[0089] The substances that specifically bind to ICAM1 and those that specifically bind to RET are the same as previously described and will not be repeated here.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] Example 1 Target Screening

[0094] The approach of this embodiment is as follows:

[0095] 1) The primary problem addressed in this section is the indiscriminate screening of highly expressed tumor-specific antigens in RET solid tumor cells (using NSCLC cell lines NCI-H23, NCI-H2009, LC-2 / ad, and MTC cell line TT as examples), while these antigens exhibit low expression in normal tissues (using normal human embryonic kidney cells 293T as an example); among which

[0096] LC-2 / ad was found to have a CCDC6-RET fusion, TT was found to carry a RET C634W mutation, H23 was a RET missense mutation, and H2009 was a silent mutation. The next step was to verify whether this antigen was truly expressed in patient tissues and located on the cell membrane surface.

[0097] Due to the presence of RET gene mutations in the experiment, the potential relationship between the sustained activation of RET protein caused by RET gene mutations and the high expression of this antigen was further explored from the perspective of signaling pathways.

[0098] 2) Further validate the universality of the antigens obtained from screening in the RET variant solid tumor patient population.

[0099] 3) Based on the ADC mechanism, it is also necessary to quantify the binding force between the antibody and the antigen, and to confirm that the antibody-antigen complex after the antigen binds to its antibody will be stably internalized by the target cells.

[0100] Based on the above objectives, this section designs methods such as flow cytometry, immunohistochemical staining, Western blotting, and confocal single-photon microscopy to screen and verify specific antigenic epitopes.

[0101] Experimental steps

[0102] Cell culture

[0103] Human MTC cell line TT (purchased from Zhejiang Meisen Cell Technology Co., Ltd.), human non-small cell lung adenocarcinoma cell lines LC-2 / ad, NCI-H2009, and NCI-H23 (purchased from Wuhan Huana Biotechnology Co., Ltd.), and human embryonic kidney cell line 293T (purchased from ATCC) were selected as negative controls. 293T was cultured in DMEM medium, H2009 in DMEM / F12 1:1 medium, H23 in RPMI 1640 medium, LC-2 / ad in 1640 / F12 1:1 medium, and TT in F12K medium. All media were supplemented with 10% New Zealand fetal bovine serum and 1% penicillin / streptomycin antibiotics. Cells were cultured in a humidified incubator containing 95% air and 5% CO2 at a constant temperature of 37°C. Each cell line was accompanied by STR identification upon purchase and was negative for mycoplasma. All cells were passaged at least three times after thawing before experiments. Cells were discarded and rethawed when morphological changes occurred.

[0104] Target screening

[0105] Flow cytometry was used to screen for highly expressed antigenic epitopes in the experimental group of tumor cells. TT, LC-2 / ad, and 293T cells were collected by trypsin digestion, and the cells were counted at 8 × 10⁸ cells per cell using a cell counter (Countess 3FL, ThermoFisher). 7Cells were placed in an ice bath and their non-specific antigenic epitopes were blocked with PBS containing 1% bovine serum albumin (BSA) for 30 minutes. The cells were then resuspended in 8 mL of blocking buffer and divided into 75 groups, each containing 100 μL of the solution. Each group was further supplemented with 2 μL of PBS, IgG, anti-human CD9, anti-human CD166, anti-human CD29, anti-human CD142, anti-human CD47, anti-human CD71, anti-human CD49b, anti-human CD44, anti-human CD54, anti-human CD227 (MUC-1), anti-human EGFR, anti-human CD324 (E-Cadherin), anti-human CD51 / 61, anti-human EphA2, anti-human CD141 (Thrombomodulin), anti-human CD340 (erbB2 / HER-2), anti-human CD276 (B7-H3), and anti-human... CD66a / c / e, anti-human CD46, anti-human CD266 (Fn14, TWEAKReceptor), anti-human CD274 (B7-H1, anti-human PD-L1), anti-human CD49a, anti-human CD107a (LAMP-1), anti-human SSEA-5, anti-human erbB3 / HER-3, anti-humanSSEA-4, anti-human CD73(Ecto-5'-nucleotidase), anti-mouse / human CD15(SSEA-1), anti-human Notch, anti-human / mouseCD49f, anti-human CD271(NGFR), anti-humanROR1, anti-human CD184(CXCR4), anti-human CD105, anti-human CD87, anti-humanCD140b(PDGFRβ), anti-human PSMA(FOLH1), anti-human CD70, anti-human / mouseCD317(BST2, PDCA-1), anti-human CD152(CTLA-4), anti-human CD325(N-Cadherin),anti-human / mouseSSEA-3, anti-human CD49e, anti-human CD192(CCR2), anti-human CD146, anti-human CD304(Neuropilin-1), anti-human CD221(IGF-1R), anti-human CD254(TRANCE, RANKL), anti-human CD24, anti-human CD309(VEGFR2), anti-human CD31, anti-human CD202b (Tie2 / Tek), anti-human CD144 (VE-Cadherin), anti-human CD197 (CCR7), anti-human CD203c (E-NPP3), anti-human CD126 (IL-6Rα), anti-human CD62E, anti-human CD181 (CXCR1), anti-human CD56 (NCAM), anti-human CD140a(PDGFRα), anti-humanCD104, anti-human CD90(Thy1), anti-human CD371(CLEC12A), anti-human CD50(ICAM-3), anti-human VEGFR-3(FLT-4), anti-human CD171(L1CAM), anti-human CD62P(P-Selectin), anti-human CD117(c-kit), anti-human TM4SF20, anti-human CD257(BAFF, BLYS), anti-human anti-FOLR1(Folate Binding Protein), anti-human CD106, anti-human CD195(CCR5)Phospho(Ser349)1, all of the above antibodies were commercially available antibodies labeled with PE fluorescence. All flow cytometry tubes were then incubated on a shaker (150 rpm) in the dark for 45 minutes. After washing twice, the samples were analyzed using a flow cytometer (CytoFLEXLX, Beckman Coulter). Samples from each group were collected at 1×10⁻⁶ after the gate. 4The average fluorescence intensity (MFI) of each tumor marker protein was obtained by analyzing individual cells in the three cell types. To obtain more accurate quantification, the antibodies bound to the cells were quantitatively analyzed simultaneously using a magnetic bead quantitative assay kit: after shaking the magnetic bead suspension well, one drop was taken from each of the three groups, and parallel experiments were performed following the same procedure. The data were then imported into GraphPad to plot a standard curve for the quantitative analysis of fluorescent antibodies. The MFI of each tumor marker was substituted into the standard curve to obtain its expression level, which was then imported into GraphPad and processed to obtain a heatmap of tumor marker expression levels.

[0106] Target validation

[0107] (1) Flow cytometry analysis

[0108] ICAM1 is known to be highly expressed in tumor cells and lowly expressed in 293T normal cells. Therefore, two additional experimental groups, H23 and H2009, were added to verify the expression of ICAM1 on its cell membrane. 4 × 10⁴ H23 cells were collected by trypsin digestion. 6 Cells were isolated and incubated in an ice bath. Non-specific antigenic epitopes were blocked in PBS containing 1% bovine serum albumin (BSA) for 30 minutes. The cells were then resuspended in 800 μL of blocking buffer and divided into three groups. Each group was incubated with 2 μL of PBS, IgG-PE, and ICAM1-PE, respectively, and then incubated in a shaker (150 rpm) in the dark for 45 minutes. After washing twice with PBS, the cells were analyzed by flow cytometry. Comparing the mean fluorescence intensity (MFI) of ICAM1 with the control group provides a rough estimate of ICAM1 expression on the cell membrane. Substituting the antibody quantity versus MFI standard curve obtained using the magnetic bead quantification method described above yields the number of ICAM1 units expressed.

[0109] (2) Confocal positioning

[0110] Using a single-photon confocal microscope (A1 HD25, NIKON), photographs were taken to visually demonstrate the antibody binding behavior of cells. 1 × 10⁶ cells were seeded per glass-bottomed culture dish. 6 After the cells adhered well to the culture dish, the culture medium was removed, and the cells were washed once with PBS. In an ice bath, each culture dish was blocked for 30 minutes with 1 mL of PBS containing 1% BSA. Then, the blocking solution was removed from the culture dishes, and 1 mL of freshly prepared blocking solution was added. 2 μL of PBS, IgG-PE, or ICAM1-PE were added to each group, respectively. The cells were incubated in an ice bath on a shaker (150 rpm) in the dark for 45 minutes. After incubation, the cells were washed 1-2 times with pre-cooled PBS, and fixed with 500 μL of paraformaldehyde for 20 minutes. After washing, 1 mL of Hoechst staining solution was added, and staining was performed at 37°C for 5-7 minutes. The cells were washed 1-2 times with PBS, and finally, 1 mL of PBS was added to keep the cells moist before imaging.

[0111] (3) Immunohistochemistry (IHC)

[0112] MTC tissue samples obtained from Zhejiang Cancer Hospital were used in the experiment. Sections were prepared using a paraffin microtome. The prepared cationic slides were dried at 72°C for 20 minutes, and immediately immersed in xylene (three times, 10 minutes each time). Afterwards, the slides were rinsed in a gradient alcohol solution (100%-100%-100%-95%-80%) for 3 minutes each time. After rinsing with water for 3 minutes, antigen retrieval was performed. Antigen retrieval solution (3g sodium citrate, 0.4g citric acid, and 1000mL double-distilled water) was added to an autoclave and brought to a boil. The rinsed cationic slides were then placed in the autoclave. Once the pressure valve started blowing, the timer was started for 2.5 minutes, and then the autoclave was turned off. After natural cooling, the lid was opened, and the cationic slides were removed and rinsed with water three times, 3 minutes each time. After rinsing, the cationic slides were soaked in PBS for 5 minutes, and then immersed in 3% hydrogen peroxide for 10 minutes. After rinsing, wash three times with water for two minutes each time, and then soak in PBS for five minutes. After soaking, remove the cationic slide, shake off the PBS on the slide, draw circles with an oil pen (to prevent antibody loss), and then add the prepared ICAM1 antibody solution to the tissue. Incubate at 37°C for 1 hour. After incubation, remove the cationic slide, wash with water, and then soak in PBS for 5 minutes. After shaking off the PBS, add enhancement solution, incubate at room temperature for 20 minutes, then wash three times with water for two minutes each time, and soak in PBS for 5 minutes. After shaking off the PBS, add secondary antibody (mouse / rabbit), incubate at room temperature for 20 minutes, then wash three times with water for two minutes each time, and soak in PBS for 5 minutes. Prepare DAB chromogenic solution (1 drop of stock solution in 1 mL), add one drop of DAB to each slide (mainly to cover the tissue), time for 3 minutes, and stop with water. Wash three times with water for two minutes each time. The slides were then immersed in hematoxylin for 2 minutes, washed three times with water for 2 minutes each time; the samples were then treated with a self-prepared blue-inducing solution for 2 minutes, washed three times with water for 2 minutes each time; finally, they were rinsed with a gradient of alcohols, air-dried, mounted, and scanned using a VS200 (Olympus) scanner. For the IHC staining experiment of lung cancer tissue, this study used tissue microarrays purchased from Shanghai Chipchao Biotechnology Co., Ltd., and the staining experimental procedures were the same as above.

[0113] RET protein phosphorylation and ICAM1 expression

[0114] (1) Streaming Validation

[0115] To verify the changes in ICAM1 expression levels in tumor cells after administration of the small molecule inhibitor BLU-667, a flow cytometry experiment was designed to examine the changes in ICAM1 expression levels over time. Using TT cells as an example, a 12-well plate was used, with a cell seeding density of 2 × 10⁶ cells per well. 5 After pre-culturing at 37℃ for 24 hours, the culture medium was aspirated and replaced with fresh culture medium containing the drug (BLU-667). The drug concentration was 1×10⁻⁶.-3 μg / mL (IC50 value of BLU-667 against TT cells) was used in each experimental group for 1.5 hours, 6 hours, 12 hours, 24 hours, 72 hours, and 168 hours, with untreated cells serving as a control group. During the experiment, at 72 hours after administration in the 168-hour group, the medium was changed at the original concentration in each well to prevent the accumulation of cell metabolites in the culture medium, which could affect cell state or even lead to cell death. Immediately after culture, the original culture medium was aspirated, and the cells were washed once with pre-chilled PBS. Samples from each group were digested with trypsin, collected, centrifuged, and resuspended in 500 μL of freshly prepared 1% BSA blocking buffer. Cells were blocked on ice for 30 minutes, centrifuged again, and the supernatant was discarded. Cells were resuspended in 200 μL of blocking buffer, and 2 μL of ICAM1-PE fluorescent antibody was added to each group. The cells were incubated at 150 rpm on a shaker in an ice bath in the dark for 45 minutes. After the incubation, the cells were washed twice with PBS before analysis. After gate selection using CytExpert software, a collection rate of 1×10⁶ cells / well was set. 4 One analysis sample.

[0116] Data processing: The obtained data were gated using FlowJo_V10 software to obtain the MFI values ​​of each sample. These values ​​were then substituted into the standard curve obtained by the magnetic bead quantification method to obtain the ICAM1 expression level per unit cell. The data were then imported into GraphPad to fit the curve of ICAM1 expression level per unit cell as a function of BLU-667 inhibition time.

[0117] For the H23 experiment, the drug concentration was increased to 1 μg / mL (this mutant is not sensitive to BLU-667), and the remaining procedures were the same as above; the LC-2 / ad experiment was exactly the same as above.

[0118] (2) BCA protein quantification experiment

[0119] Experimental Procedure: Dilute bovine serum albumin (BSA) standard to obtain a protein standard of 0.5 mg / mL. Perform serial dilutions according to the manufacturer's instructions, adding each diluted concentration of protein standard and the protein sample to be tested to a 96-well plate (three replicates per sample). Then prepare the BCA working solution according to the manufacturer's instructions (solution A:solution B = 1:50). Add the BCA working solution to each well and mix well (the time interval between adding the BCA working solution to each well should be as short as possible). After sealing, incubate at 37°C for 30 min. Cool to room temperature, using the blank as a control, and measure the absorbance of the sample at 562 nm or near that wavelength. Subtract the average absorbance of the blank standard at 562 nm from the absorbance values ​​of each standard and the protein sample to be tested at 562 nm.

[0120] Data Processing: Enter the corresponding standard curve values ​​and protein sample values ​​into an Excel worksheet. Select the OD value of the standard curve and the concentration of the standard protein. Insert an "XY scatter plot" based on the obtained data and add a trend curve. Then set the trend line format and check "Show standard curve formula" and R. 2 Value. To ensure the accuracy of protein concentration measurements based on sample OD values ​​derived from the standard curve, R... 2 The theoretical value should be infinitely close to 1 (the actual value is above 0.99). Substituting the sample OD value into the formula of the standard curve, the concentration of the protein in the sample to be tested can be derived.

[0121] (3) Western Blot Validation

[0122] To determine the changes in the levels of various proteins in cells after treatment with the small molecule inhibitor BLU-667, a Western blotting experiment was designed for further verification.

[0123] Preparation of solutions required for the experiment:

[0124] 1.10× Protein Electrophoresis Buffer (SDS Running Buffer): Tris 30g, Glycine 144g, SDS 10g, add deionized water to a final volume of 1L. When using, take 100mL of 10× Running Buffer and add it to 900mL of deionized water.

[0125] 2.10× Transfer Buffer: Glycine 29g, Tris 58g, SDS 3.7g, add deionized water to a final volume of 1L. When using, take 100mL of 10× Transfer Buffer, add 200mL of methanol solution, and add 700mL of deionized water.

[0126] 3. TBST washing solution: 50 mL Tris-HCl (1 M, pH 7.5), 8 g NaCl, 0.2 g KCl, 5 mL Tween 20, and deionized water to a final volume of 1 L.

[0127] 4. Formulation of primary and secondary antibody stripping solution: Tris 0.76g, SDS 2g and β-mercaptoethanol 700μL, add deionized water to make up to 100mL, and adjust the pH to 6.8 with HCl.

[0128] Taking the incubation of p-RET antibody in TT cells as an example, a 12-well plate was used, with a cell seeding density of 2 × 10⁶ cells per well. 5 After pre-culturing at 37℃ for 24 hours, the culture medium was aspirated and replaced with fresh culture medium containing the drug (BLU-667). The drug concentration was 1×10⁻⁶.-3 μg / mL (IC50 value of BLU-667 against TT cells) was used at 24, 72, and 168 hours after drug administration, with untreated cells serving as a control group. During the experiment, at 72 hours after drug administration in the 168-hour group, the medium for each well was changed at the original concentration to prevent the accumulation of cell metabolites in the culture medium over time, which could affect cell state or even lead to cell death. After the culture was completed at all time points, the 24-well plates were immediately placed on ice, and the liquid in each well was aspirated. Each well was washed twice with pre-chilled PBS. Then, 100 μL of pre-chilled cell lysis buffer (containing protease inhibitor at a 1:50 ratio, phosphorylated protease inhibitor at a 1:100 ratio, and 1 μmol / L sodium orthovanadate) was added to each well. After lysing on ice for 30 minutes, the solution from each well was collected and centrifuged at 1×10⁻⁶ °C. 5 Centrifuge for 15 minutes. Immediately after centrifugation, aspirate the supernatant and determine the total protein content of each sample using the BCA method. Then, add 25 μL of SDS (sodium dodecyl sulfate) loading buffer to each sample, mix well, and then boil in a preheated 95°C metal bath for 10 minutes. After cooling, the samples are ready for loading.

[0129] Electrophoresis: The sample loading amount was 40 μg of total protein per well, and the constant voltage was set to 80 V. Electrophoresis was performed for 120 minutes.

[0130] Transfer: After electrophoresis, clamp the electrophoresis gel and cellulose membrane together using the "sandwich method", place them in the transfer tank, and transfer the membrane at a constant current of 100mA for 80 minutes in an ice bath.

[0131] Sealing: After completion, soak the cellulose membrane in the sealing solution and place it on a shaker at 150 rpm for 60 minutes.

[0132] Primary antibody incubation: Incubate p-RET antibody (rabbit antibody, dilution 1:1000) overnight at 4°C. After incubation, wash the cellulose membrane three times with TBST washing buffer for 10 minutes each time.

[0133] Secondary antibody incubation: Incubate the secondary antibody (goat anti-rabbit, dilution ratio 1:10000) at room temperature for 60 minutes. After incubation, wash the cellulose membrane 3 times, 10 minutes each time.

[0134] Development: Prepare the developing solution, immediately immerse the cellulose membrane in the developing solution for 2-3 minutes, then expose and develop it on the machine. Adjust the contrast to obtain the corresponding bands.

[0135] Internal control incubation: Then, at room temperature, wash the cellulose membrane with primary and secondary antibody elution buffer for 20 minutes, and re-incubate with β-actin (internal control, primary antibody dilution ratio 1:30000). Repeat the experiment under the above primary and secondary antibody incubation conditions to obtain the target band.

[0136] When performing this experiment on other cell types, the H23 concentration was 1 μg / mL, and all other conditions were the same as above; the loading buffer for LC-2 / ad was replaced with 30 μL LDS (lithium dodecyl sulfate), and 5% β-mercaptoethanol preheated to 95°C was added, with all other conditions being the same as above.

[0137] Data processing: After obtaining the corresponding bands of each protein, the corresponding gray values ​​of each band were obtained by processing them with the "Gels" module of ImageJ software; the ratio of each band to the gray value of β-actin on the same membrane was calculated, and the curves of the change of each protein amount with the drug administration time were obtained by processing with GraphPad.

[0138] Demonstrating the behavior of intracellular antibodies

[0139] (1) Flow cytometry to determine the amount of intracellular antibodies

[0140] Pancreatic enzyme digestion, 7×10 6 H23 cells were centrifuged and the supernatant was discarded. The cells were resuspended in 6-8 mL of PBS and washed once, then centrifuged again and the supernatant was discarded. After resuspending in 1.5 mL of PBS, the cells were divided into 6 equal groups of 200 μL each. 2 μL of ICAM1 antibody was added to each group, mixed well, and incubated on ice at 150 rpm for 30 minutes. The cells were washed once with PBS and resuspended in 200 μL of PBS. The endocytosis durations for the six groups were 0, 15, 30, 60, 120, and 240 minutes, respectively. After the endocytosis, each group was washed twice with 500 μL of PBS, resuspended in 200 μL of PBS, and 2 μL of PE fluorescent secondary antibody (anti-ICAM1 antibody) was added to each of the 6 tubes. The tubes were incubated on ice at 150 rpm for 30 minutes, washed twice with PBS, and then loaded onto the cytometer. The average number of bound antibodies was obtained by substituting the antibody quantity obtained by the quantitative method into the MFI standard curve, and the efficiency of antibody internalization was calculated. The internalization efficiency curve was then fitted using GraphPad. The procedure for the LC-2 / ad cell flow cytometry quantitative internalization experiment is the same as above.

[0141] (2) Confocal verification of intracellular antibody phenomenon

[0142] Inoculate 1×10⁶ cells per glass-bottomed culture dish. 6Add 1 mL of culture medium to each TT cell, and incubate overnight at 37°C. After good adhesion, remove the culture medium and wash once with PBS. Add 1 mL of PBS and 2 μL of ICAM1-PE antibody to each culture dish, place on a shaker (150 rpm), and incubate on ice for 45 minutes. Aspirate the liquid from the culture dish, wash once with pre-chilled PBS, add another 1 mL of PBS, and incubate at 37°C for endocytosis at 0, 15, 30, 60, 120, and 240 minutes. After each time point, fix the cells with paraformaldehyde for 20 minutes, wash with PBS, add 1 mL of Hoechst staining solution, stain at 37°C for 5-7 minutes, and wash twice with PBS. Finally, add 1 mL of PBS to keep the cells moist and image. The confocal internalization experiment for LC-2 / ad and H23 cells is performed in the same manner.

[0143] Results and Discussion

[0144] 1. By incubating TT and LC-2 / ad cells with fluorescent antibodies against 73 tumor-specific proteins, flow cytometry was used to unbiasedly screen for the antigenic epitope CD54, which was highly expressed on the cell membranes of TT and LC-2 / ad cells and not expressed or expressed at low levels in 293T cells. Figure 1-1 ICAM1, also known as ICAM1, was identified through screening. Based on these results, experiments were designed to further validate the expression of ICAM1 in other cell lines of MTC and NSCLC, as well as in patient tissues.

[0145] Subsequently, the expression of ICAM1 in two other solid tumor cell lines carrying RET gene mutations (human non-small cell lung adenocarcinoma cell lines H2009 and H23) was validated. Flow cytometry analysis showed that both H23 and H2009 cells exhibited high ICAM1 expression. Specifically, the average binding amount of ICAM1 antibody on TT cells was 845,474 per cell, H23 cells were 2,084,625 per cell, H2009 cells were 201,656 per cell, and LC-2 / ad cells were 111,848 per cell, which were approximately 479, 1181, 114, and 63 times that of 293T cells, respectively. Figure 1-2 ).

[0146] Images captured by confocal single-photon microscopy also showed that ICAM1 was highly expressed in TT, LC-2 / ad, H23, and H2009 cells, and extensively localized on the cell membrane surface. These results suggest that ICAM1 expression levels are generally abnormally elevated in solid tumors carrying RET gene mutations. Figure 1-3 ).

[0147] Based on the above theoretical support, we performed immunohistochemical staining and scoring on MTC tissues from 30 patients. The results showed that ICAM1 staining was positive in cancer tissue sections from 24 patients, with a positive rate of 80.0%, and ICAM1 expression was observed in adjacent tissues from 7 patients, accounting for 23.3%. Figure 1-4 In addition, immunohistochemical staining was performed using a microarray containing cancerous tissue and normal tissue controls from 30 lung adenocarcinoma patients. Two tissues detached during staining. The ICAM1 expression of 28 pairs of tissues was ultimately summarized. Of these, 19 patients' lung adenocarcinoma tissues showed ICAM1 positivity (67.9%), and 6 patients' normal tissues showed ICAM1 expression (21.4%). Figure 1-5 ).

[0148] 2. RET protein phosphorylation and ICAM1 expression

[0149] Based on previous work, this invention proposes a synergistic relationship between abnormally elevated ICAM1 expression levels and hyperphosphorylation of RET protein. To verify this hypothesis, flow cytometry was first used in experiments, employing 1×10⁻⁶ flow cytometry. -3 After 1.5 hours of inhibition of LC-2 / ad by the small molecule inhibitor BLU-667 at a concentration of μg / mL, the expression level of ICAM1 showed a decreasing trend, which is consistent with the trend of p-RET expression level changes after LC-2 / ad treatment with BLU-667 shown in previous studies. This decreasing trend reached an extremely low value 72 hours after administration. Finally, after 168 hours, the expression level of ICAM1 rebounded. Statistical analysis of three parallel experiments showed that the expression level of ICAM1 in the 72-hour treatment group was significantly lower than that in the control group (p = 0.0108), while the ICAM1 expression level in the 168-hour treatment group was significantly higher than that in the control group (p = 0.0081). Figure 1-6 ).

[0150] In experiments with H23 cells, the expression level of ICAM1 significantly decreased 24 hours after drug administration (p = 0.0053), reaching its lowest value at 72 hours (p < 0.0001). However, at 168 hours, the ICAM1 expression level significantly rebounded, reaching 3 times that of the control group (p < 0.0001) and 10 times that of the 72-hour drug administration group (p < 0.0001). Figure 1-7 ).

[0151] ICAM1 expression showed an upward trend 72 hours after drug administration, but did not show a significant upward trend after 168 hours, which may be related to the abnormally slow proliferation of TT. Figure 1-8 ).

[0152] In the Western blot experiment, the LC-2 / ad band was lighter than the control group after 24 hours of inhibition with BLU-667, indicating a significant decrease in RET phosphorylation level. The band reached an extremely low value after 72 hours of inhibition, showing a recovery trend at 168 hours, but not exceeding the phosphorylation level of the control group. Correspondingly, NF-κB, downstream of RET, also showed a decreasing trend at the lowest point of RET phosphorylation, reaching a maximum value 168 hours after administration. The trend of ICAM1 was roughly the same as that of NF-κB, with its expression level reaching its highest point 168 hours after administration. Figure 1-6 For H23 cells, a similar trend of change was observed. Figure 1-7 ).

[0153] For TT cells, RET phosphorylation levels reached extremely low levels 72 hours after drug administration and slightly increased at 168 hours. However, ICAM1 levels reached extremely low levels 168 hours after drug administration, indicating that the inhibition of ICAM1 was delayed compared to RET phosphorylation. ICAM1 expression may be regulated by the aforementioned signaling pathways. Figure 1-8 ).

[0154] In summary, hyperphosphorylation of RET protein can lead to increased ICAM1 expression levels to some extent. This provides further theoretical support for designing therapeutics targeting ICAM1 to treat solid tumors with RET gene mutations.

[0155] 3. Demonstrate the behavior of antigenic epitopes mediating antibody endocytosis within cells.

[0156] Based on the mechanism of action of ADCs, they must be stably internalized by tumor cells in order to release toxic small molecules intracellularly and exert tumor-suppressive effects. Therefore, flow cytometry was used to measure the molecular weight fraction (MFI) of H23 and LC-2 / ad cells at different time points. The MFI was then substituted into the MFI-antibody count standard curve obtained by magnetic bead quantification to obtain the amount of ICAM1 antibody internalized by cells at different time points. The internalization rate was further calculated. The results showed that after 4 hours, the internalization rate of ICAM1 antibody in both H23 and LC-2 / ad cells reached 70-80%. Figure 1-9 ).

[0157] This phenomenon was visually demonstrated using confocal microscopy: the fluorescent antibody gradually entered the cytoplasm over time, and the red fluorescence in the cytoplasm reached its peak after 4 hours of internalization. Figure 1-10 ).

[0158] Example 2 Characterization of ICAM1-ADC

[0159] The approach of this embodiment is as follows: Experiments were designed both in vitro and in vivo to verify the safety, targeting, and tumor-suppressive effects of two ADCs, ICAM1-MMAE and ICAM1-DXd. First, the inhibition rates of ICAM1-ADCs against H23, H2009, LC-2 / ad, and TT were quantified using the CCK-8 assay, and the half-maximal inhibitory concentration (IC50) was measured. Next, an in vivo distribution experiment was designed to verify the targeting effect of ICAM1-ADCs in animals. Finally, a nude mouse subcutaneous tumor model was established using H23 cells, and groups treated with the first-line clinical chemotherapy drugs cisplatin, ICAM1-naked antibody, and ICAM1-MMAE and ICAM1-DXd were set up to explore the tumor-suppressive effect of ICAM1-ADCs in animals.

[0160] In vitro experiments of ADC drugs

[0161] ICAM1 antibody is a human-mouse chimeric IgG antibody targeting the CD54 protein, prepared through expression in CHO cell lines. The two toxic agents used in this study were: a synthetic derivative of sarsaparilla toxin 10 (Monomethyl auristatin E, MMAE), a mitotic inhibitor that inhibits microtubule polymerization and can arrest cell growth and division by disrupting spindle formation and cytoskeleton structure during mitosis; and a topoisomerase I inhibitor, a camptothecin derivative (DXd), a small toxic molecule that inhibits cell mitosis and tumor growth by disrupting DNA spatial structure, causing DNA damage, and arresting DNA replication. Both of these toxic agents are widely used in ADC drug design. In vitro cell killing experiments were conducted.

[0162] To quantify the inhibitory effects of first-line clinical chemotherapy drugs—cisplatin (lung cancer) / doxorubicin (thyroid cancer), BLU-667, ICAM1 naked antibody, ICAM1-MMAE, and ICAM1-DXd—on tumor cell growth in vitro, a cell killing assay was designed. A preliminary experiment was conducted before the formal experiment to determine the appropriate cell seeding rate and culture time after drug administration. Cells were seeded at 3 × 10⁻⁶ cells / year. 3 / 100μL, 1×10 4 / 100μL and 5×10 4 TT cells were seeded at a concentration of 100 μL per well; six replicates were set up for each cell concentration, and three replicates were prepared under the same conditions. After incubation for 48 h, 72 h, and 96 h, the original culture medium was aspirated, and 100 μL of CCK-8 working solution (CCK-8 stock solution: corresponding culture medium = 1:9) was added to each well. The culture plates were incubated at 37°C. The optimal seeding density for the cells was determined when the OD value reached the range of 0.8-1.2 within 1-4 hours. The final seeding density for TT cells was determined to be 5 × 10⁶ cells / well.4 / 100μL, the seeding density of LC-2 / ad cells was 2×10⁶. 4 / 100μL, the inoculation density for both H23 and H2009 was 1×10⁻⁶. 4 / 100μL; the culture time after drug administration was set to 96h.

[0163] Taking TT cells as an example, in a 96-well plate, 5 × 10⁶ cells per well were used. 4 TT cells were seeded with 100 μL suspension and incubated statically in an incubator for 24 h to allow them to adhere. The drug was then serially diluted 10-fold with the appropriate culture medium (10⁻¹⁰ μL / mL). -6 For each well in a 96-well plate, 100 μL of drug-containing medium (6 replicates per concentration) was added to each column according to different concentration gradients. The plates were incubated for 72 h. After incubation, the original medium was removed, and 100 μL of CCK-8 working solution was added to each well. The plates were incubated until the OD value of the control group was in the range of 0.8-1.2. The absorbance of each well in the experimental group was measured at 450 nm using a microplate reader (SPARK, TECAN). The OD values ​​were then processed using EXCEL to obtain the cell growth rate corresponding to each drug concentration. Finally, the data were processed using a 4-parameter nonlinear regression model in GraphPad to fit the inhibition rate curve and obtain the IC50 value of the drug against TT cells. The cell killing experiments of LC-2 / ad, H23, and H2009 were basically performed in the same manner, but the incubation time after adding CCK-8 working solution was slightly different due to varying cell viability.

[0164] In vivo ADC drug molecular organ distribution experiment

[0165] To verify the actual distribution of the ICAM1-ADCs developed in this study in animals and their targeting effect on tumors, an organ distribution experiment was designed. All experimental animals were purchased from Shanghai Silex Animal Co., Ltd., and the animal experiments were conducted at the animal platform of the Hangzhou Institute of Medical Sciences, Chinese Academy of Sciences. This experiment used 15 female BALB / c nude mice (5-6 weeks old). NSCLC cell line H23 (8 × 10⁸ g / L) was subcutaneously injected into the right axilla. 6 One cell / animal (mixed with standard concentration of matrix gel at a 1:1 ratio), until the average tumor volume reaches 100 mm. 3Around 4 hours later, antibody-fluorescent dye was synthesized: ICAM1-MMAE and ATTO647 dye were mixed at a molar ratio of 1:5 and incubated at room temperature in a liquid environment with pH=8.6 for 4 hours. After passing through a desalting column, the conjugated ICAM1-MMAE-ATTO647 was collected. Simultaneously, IgG-ATTO647 was synthesized under the same conditions as an isotype control. After quantifying BCA protein using an ELISA reader, tumor-bearing nude mice (n=5 per group) were immediately injected via tail vein at a dose of 2 mg / kg. After 48 hours, the mice were sacrificed, and organs were removed. Each organ was imaged at a wavelength of 660-710 nm using a small animal in vivo imaging system (IVIS Lumina Series III, PerkinElmer). The average fluorescence intensity of each group of organs was quantitatively analyzed using the Living Image software accompanying the small animal in vivo imaging system. The fluorescence intensity values ​​were then imported into GraphPad, and statistical analysis was performed using the Ordinary one-way ANOVA function.

[0166] In vivo efficacy verification

[0167] The experiment used the H23 NSCLC cell line carrying the RET gene mutation and highly expressing ICAM1. The cells were implanted into the right axilla of nude mice (5-6 weeks old, female). Six mice were placed in each of the treatment and control groups, with 8 × 10⁸ cells implanted. 6 One cell per tumor (mixed with standard concentration of matrix gel at a 1:1 ratio). Wait until the average tumor volume reaches approximately 100 mm. 3 Drug administration began. The treatment group received cisplatin, ICAM1 naked antibody, ICAM1-DXd, and ICAM1-MMAE via tail vein injection at a dose of 5 mg / kg, respectively. The control group received 0.1 mL of PBS per mouse per administration. Administering drugs was done weekly according to the above regimen. From the first administration, the body weight and tumor volume of each nude mouse were measured and recorded every other day until the maximum tumor volume in the control group reached 500 mm². 3 The experiment was stopped, the animals were euthanized, and each tumor mass was removed, weighed, and photographed for record-keeping. The drugs were administered twice during the experiment.

[0168] Data processing: The body weight, tumor volume, and weight values ​​of nude mice were imported into GraphPad. Ordinary one-way ANOVA was used for statistical analysis of tumor weight, and two-way ANOVA was used for statistical analysis of changes in body weight and tumor volume. Tumor growth inhibition rate (TGI) was calculated as follows: TGI = (1 - tumor weight in treatment group / tumor weight in control group) * 100%.

[0169] Results and Discussion

[0170] Determination of ICAM1-ADCs structure and DAR value

[0171] Due to differences in molecular properties and binding sites, different combinations of linkers and toxic small molecules exhibit different DAR values ​​after conjugation and purification with ICAM1 antibodies. We used Mc-vc-MMAE (DAR = 4) and Mc-GGFG-DXd (DAR = 8), which were named ICAM1-MMAE and ICAM1-DXd, respectively. Both linker-toxic warheads are coupled to the antibody hinge region via their maleimide groups, whereby the linker-toxic warhead binds to the antibody. Figure 2-1 ).

[0172] Cytotoxicity assay

[0173] At the cellular level, the CCK8 method was used to gradient-dose cell growth inhibition to calculate the IC50 values ​​of various drugs in different cell lines. The results showed that ICAM1-MMAE had a strong killing effect on NCI-H23 (326 pM), LC-2 / ad (974 pM), NCI-H2009 (14.3 nM), and TT (1.71 μM). ICAM1-DXd showed strong toxicity to some experimental groups of cells: NCI-H23 (129 nM), LC-2 / ad (1.71 nM), and TT (9.92 nM) and H2009 (120 nM). Moreover, ICAM1-ADCs had no significant killing effect on the control group 293T cells that did not express ICAM1. BLU-667 showed strong killing effects against NCI-H23 (1.82 μM), NCI-H2009 (2.14 μM), LC-2 / ad (17.6 nM), and TT (2.79 nM), and also had some killing effect on 293T (36.8 nM). However, for chemotherapy drugs, cisplatin only had a killing effect on NCI-H23 tumor cells (45.5 μM), considering that other cell lines have become resistant to cisplatin; doxorubicin (ADR) showed a killing effect on the MTC cell line TT (57.7 nM), but the toxicity of chemotherapy drugs to 293T (cisplatin: 37.6 μM, doxorubicin: 49.8 nM) was almost consistent with that to tumor cells. In addition, naked ICAM1 antibody did not show any killing effect at the cellular level. Therefore, the targeted killing effect of ADC is significantly better than that of traditional first-line chemotherapy drugs. Figure 2-2 ).

[0174] In vivo targeted experiments

[0175] When the average tumor volume in tumor-bearing nude mice reached 100 mm... 3In an alkaline liquid environment, synthesized fluorescent antibodies IgG-ATTO647 and ICAM1-MMAE-ATTO647 were used. Uncoupled antibodies and fluorescent molecules were removed using a desalting column. After quantification by BCA, the fluorescent antibodies (2 mg / kg) were immediately injected into tumor-bearing nude mice via the tail vein. After 48 hours, the mice were sacrificed, and organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumor tissue, were dissected. The fluorescence intensity of each organ and tumor was recorded using IVIS. The average fluorescence intensity of each organ in each group is shown below. Figure 2-3 As shown, it can be visually observed that the fluorescence intensity at the tumor site in the experimental group (injected with ICAM1-MMAE-ATTO647) was significantly higher than that in other organs; among the normal organs, the liver showed the highest fluorescence intensity, proving that the liver tissue exhibited the highest concentration of drug accumulation, consistent with pharmacokinetic theory. Subsequently, Living Image was used to calculate the average fluorescence intensity of each tissue, and the calculated fluorescence values ​​were imported into GraphPad. Two-way ANOVA was used to statistically analyze the average fluorescence intensity of each site. The fluorescence intensity deposited at the tumor site in the experimental group was significantly higher than that in other organs; while in the control group, IgG-ATTO647 did not show the same tumor targeting, and the average fluorescence intensity at the tumor site was approximately the same as that at the liver site. Figure 2-3 ).

[0176] CDX treatment results

[0177] H23 cells were subcutaneously implanted into the right shoulder of nude mice until the tumor volume reached 100 mm. 3 Afterwards, the mice were divided into groups of six, and each group received PBS, cisplatin, BLU-667, ICAM1-MMAE, and ICAM1-DXd via tail vein, respectively. After 14 days of treatment (administered twice), tumor volume and body weight were recorded every other day for each group. The excised tumors obtained at the end of the experiment are shown in the figure. The body weight and tumor volume of the mice in each group were imported into GraphPad, and two-way ANOVA analysis yielded the results shown in the figure. Significant differences were found between ICAM1-MMAE and ICAM1-DXd and the PBS control group. Furthermore, the tumor growth rate in the ICAM1-DXd group was significantly slower than in other groups in the later stages, indicating tumor growth inhibition. After measuring the weight of the excised tumors, t-test analysis was performed in GraphPad. The tumor weight in the ICAM1-DXd treatment group was significantly lower than that in the PBS group. The calculated tumor growth inhibition rates (TGI) for each treatment group were: cisplatin 106.5%, ICAM1 antibody 74.6%, ICAM1-MMAE 60.4%, and ICAM1-DXd 5.9%. In summary, ICAM1-DXd exhibited the strongest tumor inhibition rate, and the body weight of nude mice in each group did not fluctuate significantly throughout the treatment process, demonstrating that ICAM1-ADCs have no significant organ toxicity. Figure 2-4 ).

[0178] Example 3: Drug Combination Regimen

[0179] The approach of this embodiment is as follows: First, CCK-8 assays were designed to combine BLU-667 with ICAM1-MMAE and ICAM1-DXd, respectively, to verify the in vitro cell-killing effects of these two combinations and to calculate the synergistic index of each drug combination and concentration. After confirming the synergistic effect after combination, cell cycle assays were designed to determine the sites of action of BLU-667, ICAM1-MMAE, and ICAM1-DXd during the cell division cycle at the phenotypic level. Finally, RNA-seq assays were designed to explore whether there are different mechanisms of action between single drugs and drug combinations at the cellular transcriptome level.

[0180] Verification of the in vitro killing effect of combined drugs

[0181] To demonstrate that the combined use of ICAM1 and BLU-667 results in a stronger inhibitory effect at lower concentrations, a cell-killing assay using this drug combination was designed. Using TT cells as an example, 100 μL of the drug was added to each well of a 96-well plate at a concentration of 5 × 10⁶ cells / well. 4 TT cells were seeded at a concentration of [number] cells and cultured statically in an incubator for 24 hours to allow them to adhere. A gradient of ICAM1-MMAE combined with different concentrations of BLU-667 was established. Based on the cell killing results of the single-drug experiments described above, the concentration gradient of BLU-667 was set near the IC50 value of TT cells (1×10⁻⁶). -5 5×10 -5 1×10 -4 and 1×10 -3 (μg / mL), with an ICAM1-MMAE monotherapy group serving as a control. ICAM1-MMAE was serially diluted 10-fold (10-10 μg / mL) using appropriate culture media. -6 For each 96-well plate, 100 μL of drug-containing medium (6 replicates per concentration) was added to each column according to different concentration gradients. The plates were incubated for 72 h. After incubation, the original medium was removed, and 100 μL of CCK-8 working solution was added to each well. The plates were incubated until the OD value of the control group was in the range of 0.8-1.2. The absorbance of each experimental group's wells at 450 nm was measured using a microplate reader (SPARK, TECAN). The OD values ​​were then processed using EXCEL to obtain the cell growth rate corresponding to each drug concentration. Finally, the data were processed using a 4-parameter nonlinear regression model in GraphPad to fit the inhibition rate curve. The BLU-667 concentration in the LC-2 / ad experimental group was 1 × 10⁻⁶. -4 μg / mL, 5×10-4 μg / mL and 1×10 -3 μg / mL; the BLU-667 concentrations in the H23 and H2009 experimental groups were 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively. The experimental procedures for using BLU-667 in combination with ICAM1-DXd and the concentration settings of BLU-667 were the same as above.

[0182] Data processing:

[0183] Simplified CI value calculation: The classic Chou-Talalay method in existing technology calculates the CI value by analyzing the drug dose-response curve. The CI value reflects the synergistic effect when drugs are used in combination. A CI value less than 1 indicates a synergistic effect, equal to 1 indicates an additive effect, and greater than 1 indicates an antagonistic effect.

[0184] The combined experimental data (cell viability for each concentration pair) were input into R Studio and converted into cell inhibition rates using the R package developed with SynergyFinder (a standalone web application for interactive analysis and visualization of response data from multiple drug dose combinations). The output was a cell growth inhibition rate matrix ("12×12" or "10×10") with BLU-667 concentration as the ordinate and ICAM1-ADCs concentration as the abscissa. Subsequently, the study used the HSA model and the Loewe additivity model to calculate the synergistic effects of each drug combination.

[0185] For two-drug combinations, the HSA model reflects a single-drug combination (E AB The effect produced is greater than that of a single-component drug A (E). A ) and B(E B The extent of the maximum effect produced, corresponding to the synergy index of a single joint trial, can be calculated as follows:

[0186]

[0187] By definition, the combined effect is better when the HSA score is greater than 10.

[0188] In the Loewe additivity model for calculating the expected response, it is assumed that the two drugs have the same effect (a basic additive effect exists). Therefore, at any given cell inhibition rate, a slice of the response surface should show a linear relationship between the doses of the two drugs. In mathematical terms, if a dose a' of drug A produces the same effect as a dose b' of drug B, then for any dose combination (a, b), a / a' + b / b' = 1, meaning it must produce the same effect as a' and b'. The synergy index can be calculated as follows:

[0189]

[0190] Therefore, according to the above definition, all combinations of drug concentrations with a score higher than zero indicate a synergistic effect.

[0191] Next, the experimental data were further processed. The cell viability values ​​of each concentration combination were averaged, integrated, and the "ReshapeData" function in "synergyfinder" was used to obtain the drug's inhibition rate on cells. Then, the "CalculateSynergy" function was used to calculate the drug synergy coefficient. After the above processing, the "Plot2DrugHeatmap" command was used to output heatmaps of the inhibition rate and synergy index of each concentration combination on cells. The "Plot2DrugContour" command was used to obtain a 2D distribution map of the synergy index of each drug combination, and the data was visualized.

[0192] Drug site of action verification

[0193] To investigate the mechanism of drug synergistic effects, a cell cycle experiment was designed to verify the location of each drug action within the cell cycle and whether the duration of drug action affects the site of action. The experiment included three different drug concentrations and two different drug administration times.

[0194] Cell treatment: Taking TT cells as an example, the cells were treated at a rate of 5 × 10⁶ cells / year. 5 The culture medium was seeded into 6-well plates at a specific density and pre-cultured at 37°C for 24 hours. Afterward, the culture medium was aspirated. The drug concentrations of ICAM1-MMAE and ICAM1-DXd were 0.1 μg / mL, 1 μg / mL, and 10 μg / mL, respectively, while the drug concentration of BLU-667 was 1 × 10⁻⁶. -5 1×10 -4 and 1×10 -3 μg / mL; two administration time points were set at 48 hours and 72 hours, respectively, as well as a blank control group. Immediately after the experiment, the culture medium was aspirated, washed once with pre-cooled PBS, digested with trypsin, and the cells were collected and centrifuged.

[0195] Cell cycle assay: Collected cells were washed twice with pre-chilled PBS, and the supernatant was discarded (leaving 50 μL in the tube). The bottom of the tube was gently tapped to disperse the cells. Pre-chilled 70% ethanol was prepared, and the bottom of the tube was gently shaken using a vortex mixer while adding 70% ethanol dropwise. A total of 500 μL of pre-chilled ethanol was added to each flow cytometry tube. After all the ethanol was added, the tube was transferred to a 4°C freezer for overnight fixation. After 12 hours, the tube was centrifuged, the supernatant was discarded, and the cells were washed once with pre-chilled PBS. Propidium iodide staining solution was prepared (propidium iodide staining solution was diluted 1:20 in staining buffer, and the ratio of RNase A to buffer was 1:50). 500 μL of staining solution was added to each sample tube, and the cell pellet was slowly and thoroughly resuspended. The cells were incubated at 37°C in the dark for 30 minutes. Immediately after incubation, the cells were analyzed using flow cytometry at an excitation wavelength of 488 nm for red fluorescence.

[0196] Data Processing: The collected sample data were processed using FlowJo_V10 software. A pulse width-pulse area plot was used as a gate to select a single cell population. This gate was then applied to the scatter plot to exclude obvious cell debris. A "Cell Cycle" analysis was added under the selected cell population to fit the cell proportions for each cell cycle. To ensure accuracy, the peak width at 2N (G0 / G1 phase) or 4N (G2 / M phase) was manually determined. The software automatically determined the positions and fluorescence intensities of other peaks based on the DNA content of each cycle. The theoretical basis for manually determining the peak width is to control the CV value below 10% to ensure data reliability. CV (coefficient of variation) is a measure of data dispersion, defined as the standard deviation (SD) divided by the mean (M), and expressed as a percentage (SD / M × 100%). The final cell cycle data are presented as the percentage of cells in G0 / G1, S, and G2 / M phases.

[0197] The cell percentages of each experimental group were imported into GraphPad for further visualization and statistical analysis, and bar charts were created.

[0198] The procedure for LC-2 / ad cell cycle experiments is the same as above.

[0199] Results and Discussion

[0200] Verification of the in vitro killing effect of combined drugs

[0201] The experiment selected lung adenocarcinoma cell lines NCI-H2009 and NCI-H23 containing RET gene point mutations. Gradual concentrations of ICAM1-MMAE combined with a specific dose of BLU-667 were administered, along with an ICAM1-MMAE monotherapy group. CCK-8 assays were performed, and the CI values ​​for each combination therapy group on each cell line were calculated. If synergistic drug concentration combinations were found, the synergistic index for each concentration was fitted using R language based on the wet-scale experimental data to generate an inhibition rate matrix, and a heatmap of the synergistic index was output. The effects of drug combinations are discussed separately for each cell type.

[0202] (1) H23 cells: The half-maximal inhibitory concentration (IC50) of BLU-667 against H23 cells was approximately 0.97 μg / mL. Therefore, the concentration gradient of BLU-667 used in combination was set around 1 μg / mL (2, 1, 0.5, and 0.25 μg / mL). For ICAM1-MMAE, the IC50 value was 326 pM (4.9 × 10⁻⁶ pM). -2 When used in combination with BLU-667 at a concentration of 0.5 μg / mL, to achieve the same half-maximal inhibitory effect, the concentration of ICAM1-MMAE was 183 pM (2.7 × 10⁻⁶ μg / mL). -2 At this concentration (μg / mL), the CI value of the drug combination was 1.05, failing to achieve the expected synergistic effect; while when BLU-667 was used in combination at a concentration of 0.25 μg / mL, to achieve the same half-maximal inhibitory effect, the concentration of ICAM1-MMAE was 176 pM (2.6 × 10 μg / mL). -2 At this concentration (μg / mL), the CI value of the drug combination was 0.82, indicating a synergistic effect when used in combination. This suggests that BLU-667 and ICAM1-MMAE have the potential for synergistic enhancement in inhibiting the growth of H23 cells.

[0203] like Figure 3-1 As shown, the synergistic index calculation results indicate that ICAM1-MMAE exhibits synergistic effects when used in combination with 1.00 μg / mL BLU-667 at various concentration gradients. Even at ICAM1-MMAE concentrations of 1×10⁻⁶, the synergistic effect is further enhanced. -3The highest synergistic index was observed at a concentration of μg / mL, with an inhibition rate of 62.31% against H23 at this concentration. Analysis of other data showed that the synergistic index was high in the 2×3 matrix for BLU-667 (0.25-1 μg / mL) and ICAM1-MMAE (0.1-1 μg / mL), with inhibition rates against H23 ranging from 75.48% to 98.12%. Note: The synergistic index in this invention provides both HSA and Loewe scores. Using the HSA score, drug combinations with a score higher than 10 are considered to have good synergistic effects; using the Loewe score, a score higher than 0 indicates synergistic enhancement. In case of conflict between the two scoring methods, the Loewe score prevails, and the HSA score is only used as a reference.

[0204] For ICAM1-DXd, the IC50 value was 129.2 nM (19.4 μg / mL). When combined with BLU-667 at a concentration of 0.5 μg / mL, to achieve the same half-maximal inhibitory effect, the concentration of ICAM1-DXd was 6.79 nM (1.02 μg / mL), with a CI of 0.57, indicating a synergistic effect. When combined with BLU-667 at a concentration of 0.25 μg / mL, to achieve the same half-maximal inhibitory effect, the concentration of ICAM1-DXd was 28.7 nM (4.31 μg / mL), with a CI of 0.48, also indicating a synergistic effect. Based on these simplified calculations, it is evident that the combination of BLU-667 and ICAM1-DXd also possesses reasonable synergistic potential for inhibiting H23 growth. Figure 3-2 ).

[0205] The synergy index calculation results showed that, for H23 cells, except for a BLU-667 concentration of 10 μg / mL, no synergy was observed with ICAM-DXd at any concentration. Furthermore, within the BLU-667 concentration range of 0.5–1 μg / mL, ICAM-DXd showed synergy with ICAM-DXd at concentrations of 1 × 10⁻⁶ μg / mL. -6 -1×10 -3 Except for concentrations in the μg / mL range that did not exhibit synergistic effects, all other concentration pairs showed synergistic effects when used in combination. When the ICAM-DXd concentration was around 10 μg / mL, the inhibition rate of H23 was high; only 1×10⁻⁶ μg / mL was added. -6 BLU-667 at a concentration of μg / mL can inhibit more than 60% of H23 cells; BLU-667 concentrations of 1×10 -3 μg / mL showed the highest synergistic index, and the inhibition rate reached 72.29% at this concentration.

[0206] 2) H2009 cells: The half-maximal killing concentration of BLU-667 is approximately 1.14 μg / mL, so the concentration gradient of BLU-667 used in combination was set around 1 μg / mL (2, 1, 0.5 and 0.25 μg / mL).

[0207] For ICAM1-MMAE, the IC50 value was 14.35 nM (2.15 μg / mL). When combined with BLU-667 at a concentration of 0.5 μg / mL, to achieve the same half-maximal inhibitory effect, the concentration of ICAM1-MMAE was 8.24 nM (1.24 μg / mL). At this concentration, the IC50 value of this drug combination was 1.02, failing to achieve the expected synergistic effect. Furthermore, when combined with BLU-667 at a concentration of 1 μg / mL, to achieve the same half-maximal inhibitory effect, the concentration of ICAM1-MMAE was 0.84 pM (1.25 × 10⁻⁶ pM). -4 At this concentration (μg / mL), the CI value of the drug combination was 0.88, indicating a synergistic effect when used in combination. Based on the above simplified calculations, it is known that BLU-667 and ICAM1-MMAE have synergistic potential for inhibiting the growth of H2009 cells within a reasonable combined concentration range. Figure 3-3 ).

[0208] The results showed that ICAM1-MMAE exhibited good synergistic effects when used in combination with 1.00 μg / mL BLU-667 at various concentration gradients. The highest synergistic index was observed at the same ICAM1-MMAE concentration of 1.00 μg / mL, at which point the inhibition rate of H2009 reached 86.34%. Analysis of other data indicated that even when both drugs were at concentrations below 0.01 μg / mL, the synergistic effect was superior to that of either drug alone.

[0209] For ICAM1-DXd, the IC50 value was 120 nM (18.0 μg / mL). When combined with BLU-667 at a concentration of 0.5 μg / mL, to achieve the same half-maximal inhibitory effect, the concentration of ICAM1-DXd was 52.4 nM (7.87 μg / mL), with a CI of 0.88, indicating a synergistic effect. However, when combined with BLU-667 at a concentration of 1 μg / mL, to achieve the same half-maximal inhibitory effect, the concentration of ICAM1-DXd was 16.9 nM (2.54 μg / mL), with a CI of 1.02, failing to achieve the expected synergistic effect. Based on the above simplified calculations, it is evident that the combination of BLU-667 and ICAM1-DXd also possesses reasonable synergistic potential for inhibiting the growth of H2009. Figure 3-4 ).

[0210] The synergistic index calculation results showed that when ICAM1-DXd concentrations of 1-10 μg / mL were used in combination with 0.25-0.5 μg / mL BLU-667, it had reasonable synergistic potential and a high inhibition rate against H2009. In addition, when the ICAM1-DXd concentration reached 10 μg / mL, the synergistic index with BLU-667 was high, and the inhibition rate against H2009 was over 60%.

[0211] (3) LC-2 / ad cells: The IC50 value of BLU-667 was 9.40 × 10⁻⁶. -3 μg / mL. The concentration of BLU-667 used in combination was 1×10 μg / mL. -3 μg / mL, 5×10 -4 μg / mL, 1×10 -4 g / mL.

[0212] For ICAM1-MMAE, the IC50 value was 0.974 nM (0.146 μg / mL), when the concentration of BLU-667 used in combination was 10. -3 To achieve the same half-maximal inhibitory effect, the concentration of ICAM1-MMAE was 23.8 pM (3.57 × 10⁻⁶ μg / mL). -3 When the concentration of the drug combination was 5 × 10 μg / mL, the CI value was 0.131; while when the concentration of BLU-667 used in combination was 5 × 10 μg / mL, the CI value was 0.131. -4 To achieve the same half-maximal inhibitory effect, the concentration of ICAM1-MMAE was 797 pM (0.120 μg / mL). At this concentration, the CI of the drug combination was 0.87, indicating a synergistic effect when the two concentrations were used together. Based on the above simplified calculations, it is known that BLU-667 and ICAM1-MMAE have the potential for synergistic enhancement in inhibiting the growth of LC-2 / ad cells within a reasonable combined concentration range. Figure 3-5 ).

[0213] The calculation results of the synergy index show that ICAM1-MMAE is at 1×10 -6 -1 μg / mL concentration range and 1×10 -3 When used in combination with BLU-667 at a concentration of μg / mL, good synergistic effects were observed. The highest synergistic index was observed at an ICAM1-MMAE concentration of 1.00 μg / mL, at which point the inhibition rate against LC-2 / ad reached 94.19%. Analysis of other data showed that when the BLU-667 concentration was less than or equal to 1×10⁻⁶ μg / mL, the synergistic effect was better. -5At a concentration of μg / mL, there is a synergistic effect that is superior to that of single drugs, but the inhibition rate on cell growth is too low at this point; while the inhibitory effect of BLU-667 at a concentration of ≥1 μg / mL mainly comes from BLU-667 alone. Neither of these situations is considered a reasonable combination of drug concentrations.

[0214] For ICAM1-DXd, the IC50 value was 1.71 nM (0.257 μg / mL), when the concentration of BLU-667 used in combination was 1 × 10⁻⁶. -3 To achieve the same half-maximal inhibitory effect, the concentration of ICAM1-DXd was 3.03 nM (0.455 μg / mL). At this concentration, the CI of the drug combination was 1.82, indicating no synergistic effect. However, when combined with BLU-667 at a concentration of 10 μg / mL... -4 To achieve the same half-maximal inhibitory effect, the concentration of ICAM1-DXd is 1.60 nM (0.239 μg / mL). At this concentration, the CI of the drug combination is 0.94, indicating a synergistic inhibitory effect. Based on the above simplified calculations, it is evident that the combined use of BLU-667 and ICAM1-DXd also has reasonable synergistic potential for inhibiting the growth of LC-2 / ad.

[0215] The synergistic index calculation results show that ICAM1-DXd concentrations between 0.1 μg / mL and 1 μg / mL are significantly higher than those with concentrations less than 5 × 10⁻⁶. -4 When used in combination with μg / mL BLU-667, it exhibits reasonable synergistic potential; furthermore, when the ICAM1-DXd concentration reaches 1 μg / mL, the highest synergistic index is observed when used in combination with BLU-667, and the inhibition rate against LC-2 / ad is above 90%. Figure 3-6 ).

[0216] (4) TT cells: The IC50 value of BLU-667 was 1.49 × 10⁻⁶. -3 μg / mL. The concentration of BLU-667 used in combination was 10 μg / mL. -3 μg / mL, 10 -4 μg / mL, 5×10 -5 μg / mL, 1×10 -5 μg / mL;

[0217] For ICAM1-MMAE, the IC50 value was 1.72 μM (257.8 μg / mL). At various BLU-667 concentrations used in combination, to achieve the same half-maximal inhibitory effect, the concentration of ICAM1-MMAE was significantly lower than the half-maximal inhibitory concentration (MCC) of the single drug. This was achieved at a BLU-667 spiking level of 1×10⁻⁶. -3 μg / mL, 10 -4 μg / mL, 5×10 -5μg / mL, 1×10 -5 At μg / mL, the CI values ​​were (0.67, 0.73, 0.74, and 0.71), all showing a synergistic effect. Figure 3-7 ).

[0218] The synergistic index calculation results show that higher concentrations of ICAM1-MMAE (approximately 10 μg / mL) and lower concentrations below 1×10⁻⁶ are associated with lower concentrations of ICAM1-MMAE. -3 When used in combination with μg / mL BLU-667, both showed good synergistic effects. Furthermore, 1×10 -3 The highest cell inhibition rate was achieved at a concentration of μg / mL, reaching 94.36%.

[0219] For ICAM1-DXd, the IC50 value was 9.92 nM (1.49 μg / mL), when the concentration of BLU-667 used in combination was 1×10⁻⁶. -3 To achieve the same half-maximal inhibitory effect, the concentration of ICAM1-DXd was 843 nM (0.13 μg / mL). At this concentration, the CI of the drug combination was 0.76, indicating a synergistic effect. This suggests that the drug combination has a more significant inhibitory effect on TT than the single drug. Figure 3-8 ).

[0220] However, the synergistic index calculation results only show that the ICAM1-DXd concentration at 10 μg / mL is similar to that at 1×10 -3 μg / mL BLU-667 exhibits a significant synergistic effect.

[0221] In summary, although the synergistic effects of ICAM1-MMAE and ICAM1-DXd with BLU-667 varied at different combined concentrations and in different cell lines, there were synergistic drug concentration pairs in each experimental group, which can provide guidance for their combined application.

[0222] Cell cycle experiment

[0223] To determine the sites of action of each drug, cell cycle experiments were designed.

[0224] In the LC-2 / ad experimental group, after 48 hours of inhibition at a concentration of 10 μg / mL, cells in the ICAM1-MMAE group were significantly intercepted in the G2-M phase. Figure 3-9 After 48 hours of inhibition at a concentration of 10 μg / mL, the ICAM1-DXd group showed significant cell interception in the S phase. Figure 3-10 ); while BLU-667 group 1×10 -3 After inhibition at a concentration of μg / mL for 48 hours, the number of G0-G1 phase cells significantly increased. Figure 3-11 ).

[0225] In the H23 experimental group, after inhibition at a concentration of 10 μg / mL for 48 hours, cells in the ICAM1-MMAE group were significantly intercepted in the G2-M phase. Figure 3-12 After 48 hours of inhibition at a concentration of 10 μg / mL, the ICAM1-DXd group showed significant cell interception in the S phase. Figure 3-13 ); while BLU-667 group 1×10 -3 After inhibition at a concentration of μg / mL for 48 hours, the number of G0-G1 phase cells significantly increased. Figure 3-14 ).

[0226] In the TT experimental group, after inhibition at a concentration of 10 μg / mL for 48 hours, cells in the ICAM1-MMAE group were significantly intercepted in the G2-M phase. Figure 3-15 After 48 hours of inhibition at a concentration of 10 μg / mL, the number of S-phase cells in the ICAM1-DXd group increased ( Figure 3-16 In the BLU-667-treated group, the proportion of cells in each cell cycle did not change significantly, making it impossible to determine the site of drug action. Figure 3-17 ).

[0227] In addition, intergroup comparisons showed that ICAM1-MMAE, ICAM1-DXd, and BLU-667 all acted on different cell cycles, consistent with theoretical expectations; intragroup comparisons showed that the drug effects were dose-dependent, and the duration of administration had little effect on the inhibitory effect.

[0228] in conclusion

[0229] This study identified a highly expressed antigenic epitope, ICAM1, in solid tumors with RET gene mutations (MTC and NSCLC as examples) and explored the potential regulatory relationship between RET protein activation and ICAM1 high expression. It was the first time that RET phosphorylation may indirectly upregulate ICAM1 expression levels by activating NF-κB. Subsequently, the precise targeting and efficient inhibition of ICAM1-MMAE and ICAM1-DXd on RET-mutant MTC, NSCLC, and RET-fusion NSCLC cells were evaluated in vitro and in vivo. Furthermore, ICAM1-ADCs were combined with the approved specific RET inhibitor BLU-667 to assess their synergistic effect and further explore the combined mechanism.

[0230] The RET gene is a proto-oncogene encoding a receptor tyrosine kinase. Mutations in RET proteins lead to binding phosphorylation and persistent activation of downstream signaling pathways, resulting in abnormal cell proliferation. Previous studies have shown that RET fusions are present in 1%-2% of NSCLC, and RET and RAS mutations are present in 90% of MTC patients. Although the number of gene fusion partners and mutation sites identified that are associated with cancer development is limited, and most remaining RET mutations are missense mutations with unknown biological significance, studies have found that missense mutations in the extracellular domain of RET lead to ligand-independent dimerization, while mutations in the cytoplasmic catalytic site are closely related to substrate-specific autophosphorylation.

[0231] Treatment for solid tumors carrying RET mutations currently relies primarily on surgery, radiotherapy, chemotherapy, and oral kinase inhibitors. In recent years, immunotherapy has also been applied to treat these tumors. However, these methods still fall short of clinical needs. Firstly, 10% of MTC patients already have distant metastases at diagnosis, and this proportion increases during follow-up. For NSCLC patients, late-stage metastasis is the leading cause of death; therefore, surgery and local radiotherapy are largely ineffective for patients with metastases. Secondly, the use of kinase inhibitors can lead to acquired mutations in tumor cells, allowing them to escape drug inhibition by activating parapathic pathways. Although specific RET kinase inhibitors are constantly being updated, many studies have found cross-resistance with new TKIs in patients who have previously used TKIs and developed resistance, meaning the drug's activity typically only lasts for a limited time. Furthermore, because RET gene fusions lead to primary resistance to immunotherapy, immune checkpoint inhibitors are ineffective for NSCLC patients carrying RET fusions (ORR 0%-7%, median PFS 2.2-3.4 months).

[0232] In summary, this study addresses the current lack of targeted drugs for treating RET variant solid tumors by designing an ICAM1-targeting ADC. The expression level of ICAM1 on the surface of MTC and NSCLC cells is 63-1181 times higher than that of normal 293T cells. This significant difference in expression levels ensures low off-target toxicity. Furthermore, NSCLC and MTC cells exhibit a high internalization rate of the ICAM1 antibody (80% internalized after 4 hours), providing a prerequisite for the efficient release of the toxic small molecule. ICAM1-MMAE showed strong killing effects on tumor cells in vitro (326 pM-1.71 μM), while ICAM1-DXd also showed killing effects of 1.71 nM-129 nM. In vivo, ICAM1-ADCs showed good targeting, accumulating at tumor cell sites approximately four times that of the liver. Additionally, in the CDX treatment model, the tumor growth rate in the ICAM1-DXd group was significantly lower than that in the control group.

[0233] Furthermore, flow cytometry and Western blotting results validated the hypothesis that RET protein phosphorylation upregulates ICAM1 expression levels through NF-κB activation. In each experimental group, ICAM1 expression levels decreased and then increased after BLU-667 inhibition, indicating that reduced RET phosphorylation inhibits some ICAM1 expression on the cell membrane. However, after continuous drug administration, ICAM1 expression levels rebounded to varying degrees, possibly due to the activation of a protective mechanism that upregulates other pathways that regulate ICAM1. This potential drug resistance mechanism provides more binding targets for combination therapy with ICAM1-ADCs. Simultaneously, Western blotting experiments confirmed that the trend of NF-κB activation levels was consistent with that of p-RET and ICAM1, suggesting that NF-κB, as an important member of the signaling pathway, participates in the regulation of ICAM1 by RET protein.

[0234] In the combination of ICAM1-ADCs and BLU-667, although each experimental group showed varying degrees of synergistic effect, the combination of ICAM1-MMAE and ICAM1-DXd with BLU-667 showed a more significant synergistic effect in the BLU-667-insensitive cell line H23. This suggests that the combination regimen may provide a stronger inhibitory effect for individuals that are insensitive or resistant to BLU-667.

[0235] Previous studies have shown that acquired MET amplification is common in patients resistant to specific TKIs; and MET has been shown to promote the transition of cells from G1 to S phase through multiple mechanisms. MET amplification is a potential mechanism for drug resistance in RET fusion-type lung cancer patients. BLU-667 inhibits cells in the G0-G1 phase, hindering normal cell division and inducing apoptosis. When MET amplification occurs, mitosis breaks through G1 phase inhibition and enters the next stage. At this time, using ICAM1-DXd, which targets the S phase, or ICAM1-MMAE, which targets the G2 / M phase, will produce a stronger tumor-suppressive effect.

[0236] In summary, this study developed rational ICAM1-ADCs for the clinical treatment of RET-mutant solid tumors. The combination of BLU-667 and ICAM1-ADCs showed encouraging tumor cell inhibition in in vitro experiments, and it is expected to serve as an alternative treatment option for patients with advanced / metastatic RET solid tumors and patients resistant to TKIs.

[0237] 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.

Claims

1. Use of ICAM1 or its encoding gene as a target in the preparation of products for treating cancers caused by RET gene mutations.

2. Use of substances that specifically bind to ICAM1 in the preparation of products for treating cancers caused by RET gene mutations.

3. Use of substances that specifically bind to ICAM1 in combination with substances that specifically bind to RET in the preparation of products for treating cancers caused by RET gene mutations.

4. The use according to any one of claims 1 to 3, characterized in that, RET gene variants are selected from RET gene mutations or RET gene fusions.

5. The use according to any one of claims 1 to 3, characterized in that, The cancer caused by the RET gene mutation is a cancer resistant to small molecule inhibitors.

6. The use according to claim 5, characterized in that, The small molecule inhibitor is a RET receptor tyrosine kinase inhibitor; preferably, the small molecule inhibitor is pralatinib or serpatinib.

7. The use according to any one of claims 1 to 3, characterized in that, The cancer caused by the RET gene mutation is a solid tumor; preferably, the cancer caused by the RET gene mutation is selected from thyroid cancer, non-small cell lung cancer, multiple endocrine tumor type 2, esophageal cancer, breast cancer, melanoma or colorectal cancer; preferably, the thyroid cancer is selected from medullary thyroid carcinoma or papillary thyroid carcinoma; preferably, the non-small cell lung cancer is lung adenocarcinoma.

8. The use according to any one of claims 1 to 3, characterized in that, The substance that specifically binds to ICAM1 is selected from antibody-drug conjugates or their pharmaceutically acceptable salts or solvent compounds, and 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.

9. The use according to claim 8, characterized in that, The antibody-drug conjugate further includes one or more of the following features: 1) 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 NO.1 and 2, respectively; 2) The linker is selected from non-breakable linkers or breakable linkers; preferably, the breakable linker is selected from chemically unstable linkers or enzyme-unstable linkers; 3) The linker comprises one or more linker components selected from maleimide propionyl, maleimide hexanoyl, p-aminobenzoyloxycarboxyl, valine-citrulline, alanine-phenylalanine, glycine-glycine-glycine, glycine-valine-citrulline, glycine-glycine-phenylalanine-glycine; preferably, the linker is selected from maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarboxyl or maleimide-GGFG tetrapeptide; 4) The cytotoxic agent is selected from toxins, chemotherapy drugs, antibiotics, radioactive isotopes, or growth inhibitors; 5) The cytotoxic agent is selected from any one or more of the following: maytansin, maytansin-like substances, topoisomerase I inhibitors, tubulin inhibitors, kazimidic acid and its derivatives, doxorubicin and its derivatives; preferably, the cytotoxic agent is selected from DX-8951 derivative Dxd, MMAE or MMAF.

10. The use according to claim 8, characterized in that, The antibody-drug conjugate is selected from anti-ICAM1mAb-MC-VC-PAB-MMAE or anti-ICAM1 mAb-MC-GGFG-Dxd.

11. The use according to claim 3, characterized in that, The substance that specifically binds to RET is a RET inhibitor; preferably, the RET inhibitor is a small molecule inhibitor; preferably, the small molecule inhibitor is pralatinib or serpatinib.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a substance that specifically binds to ICAM1 and a substance that specifically binds to RET. Preferably, the substance that specifically binds to ICAM1 is an antibody-drug conjugate as described in claim 9, and / or, the substance that specifically binds to RET is a small molecule RET inhibitor.

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