CD66c-targeted antibody coupling medicine and application thereof in preparation of medicine for treating gastroesophageal junction cancer

Abstract

The invention relates to the field of biological medicine, in particular to a CD66c-targeted antibody coupling medicine and application thereof in preparation of a medicine for treating gastroesophageal junction cancer, the structure of the antibody coupling medicine is shown as follows: Ab-(L-D) n, Ab is an anti-CD66c antibody, the anti-CD66c antibody is selected from a full-length antibody or an antibody fragment, L is a connexon, n is a positive integer, and n is a positive integer. The linker is selected from a non-breakable linker or a breakable linker; d is a cell toxic agent, the cell toxic agent is selected from toxins, chemotherapeutics, antibiotics, radioisotopes or growth inhibitors, and n is an integer from 1 to 40. The CD66c is found to be a novel gastro-esophageal junction cancer ADC target spot, meanwhile, the synthesized CD66c-DXd ADC is proved to have a remarkable killing effect on the gastro-esophageal junction cancer at the in-vivo and in-vitro levels, and a promising targeted therapy candidate drug is provided for targeted therapy of the gastro-esophageal junction cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an antibody-drug conjugate targeting CD66c and its use in preparing a drug for treating gastroesophageal junction cancer. Background Art

[0002] Gastroesophageal junction (GEJ) cancer refers to adenocarcinoma arising within 5 cm in either direction of the esophagogastric junction. While the incidence of distal gastric cancer has been declining globally in recent years, the incidence of GEJ cancer has been on a significant upward trend, with over 1.5 million patients diagnosed with GEJ cancer annually, and the incidence continues to rise. Currently, combined treatment options, including surgical resection, chemotherapy, and immunotherapy, are the most effective treatments for GEJ cancer. However, most GEJ cancer patients present with locally advanced disease or distant metastases at diagnosis, with a two-year survival rate of less than 20%. Targeted therapies are limited to patients with advanced, metastatic HER2-positive tumors, benefiting a very limited population. With the use of immune checkpoint inhibitors, immunotherapy for GEJ cancer has made progress. However, due to the heterogeneity and complexity of the immune microenvironment in GEJ cancer, the application of immunotherapy in GEJ cancer still faces numerous challenges, such as hyperprogression. On the other hand, due to its anatomical proximity to the stomach, patients with GEJ cancer are typically enrolled in clinical trials targeting gastric cancer, and there is a lack of clinical research specifically targeting GEJ cancer. Furthermore, the risk factors for GEJ and gastric cancers differ significantly, thus creating a need to better understand and identify potential prognostic indicators and drug targets for GEJ cancer. From a clinicopathological perspective, GEJ cancer exhibits higher aggressiveness, later TNM stage, younger age, higher recurrence rate, and lower survival rate compared to gastric cancer.

[0003] Antibody-drug conjugates (ADCs) are a new class of anti-tumor drugs consisting of monoclonal antibodies that specifically bind to tumor-associated antigens, linked to small molecule cytotoxins via various linkers. The structural composition of ADCs consists of three parts: a monoclonal antibody that specifically binds to the target antigen on the tumor surface, a stable chemical linker, and a cytotoxic small molecule drug. Based on the characteristics of specific binding to the targeted antigen, ADC drugs can selectively kill tumors and significantly reduce side effects (off-target effects) on normal organs, exhibiting specificity and efficacy that traditional anti-tumor drugs cannot achieve. In recent years, ADC drugs have made rapid progress in clinical and preclinical research. In 2020, the New England Journal of Medicine published a Phase III clinical study report on the clinical trial of Trastuzumab Deruxtecan (DS-8201, an ADC targeting HER2) for the treatment of HER2-positive gastric cancer patients. The objective response rate of gastric cancer subjects was 51%, significantly better than the control chemotherapy group (14%). It is worth noting that 11 patients receiving ADC treatment achieved complete remission. Subsequently, the interim analysis results of the DESTINY-PanTumor02 study of trastuzumab deruxteca in the treatment of HER2-expressing pan-tumor types were released at the 2023 American Society of Clinical Oncology meeting. A total of 267 patients were enrolled in the study, with an overall objective response rate of 37.1%, a complete response rate of 5.6%, and a disease control rate of 83.2%, significantly superior to previous chemotherapy data. In summary, ADCs are revolutionizing oncology clinical practice by leveraging the high targeting properties of monoclonal antibodies and the high antitumor activity of small molecule toxins.

[0004] Patients with GEJ cancer typically have a poor prognosis, a situation exacerbated by the lack of GEJ cancer-specific clinical trial data evaluating therapeutic agents in the broader gastric cancer category. ADCs are a promising cancer treatment strategy that leverages the specificity of antibodies to deliver potent cytotoxic drugs directly to tumor cells, thereby improving efficacy and reducing systemic toxicity. Continued research and development in the ADC field holds great promise for the future of precision cancer medicine. A major obstacle to developing ADC drugs targeting GEJ cancer is finding appropriate membrane targets that can effectively distinguish malignant GEJ cancer cells from normal tissue. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an antibody-drug conjugate targeting CD66c and its use in the preparation of a drug for treating gastroesophageal junction cancer, so as to solve the problems in the prior art.

[0006] To achieve the above objectives and other related objectives, the present invention provides an antibody-drug conjugate having the following structure: Ab-(LD)n, wherein Ab is an anti-CD66c antibody selected from a full-length antibody or an antibody fragment, L is a linker selected from a non-cleavable linker or a cleavable linker; D is a cytotoxic agent selected from a toxin, a chemotherapeutic drug, an antibiotic, a radioactive isotope or a growth inhibitory agent, and n is an integer from 1 to 40.

[0007] The present invention also provides use of CD66c or its encoding gene as a target in preparing a product for treating or diagnosing gastroesophageal junction cancer.

[0008] The present invention also provides use of a substance that specifically binds to CD66c in preparing a product for treating or diagnosing gastroesophageal junction cancer.

[0009] As described above, the CD66c-targeting antibody-drug conjugate of the present invention and its use in the preparation of a drug for treating gastroesophageal junction cancer have the following beneficial effects: CD66c is demonstrated to be a novel ADC target for gastroesophageal junction cancer, and CD66c-DXd is demonstrated to have a significant killing effect on gastroesophageal junction cancer both in vivo and in vitro, providing a promising targeted therapy candidate for the targeted treatment of gastroesophageal junction cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shown is CD66c overexpression in GEJ cancer. (A) Heat map of the top 20 DEPs in AEG tumor samples and paired NAT samples; (B) Venn diagram of the top 20 DEPs and membrane proteins in the cancer surface group atlas; (C) Heat map of the normal tissue expression index for eight candidate targets; (D) Bar chart of the expression levels of established ADC targets (HER2, TROP2, and CLDN18) and CD66c in tumor and precancerous tissues of each GEJ cancer patient; (E) Heat map of clinicopathological characteristics of different CD66c expression levels; (F) Histogram comparing CD66c protein expression in adjacent normal tissues and different TNM stages; (G) Representative images of CD66c IHC staining in human normal gastric tissues, primary GEJ cancer, and metastatic lymph nodes; (H) H-score of CD66c in adjacent normal tissues and different TNM stages. ***p<0.001; **p<0.01; *p<0.05..

[0011] Figure 2CD66c is shown to be a potential ADC target for the treatment of gastroesophageal junction cancer. (A) Flow cytometry (PE-labeled antibody) shows the expression of CD66c on the surface of human gastroesophageal junction cancer cells and normal GES-1 cells; (B) Representative flow cytometry images show the expression of CD66a, CD66c, and CD66e in OE-19 and SK-GT-4 cells; (C) IF staining of CD66c in OE-19 and SK-GT-4 cells.

[0012] Figure 3 PE-conjugated CD66c antibody is clearly internalized by GEJ cancer cells. (A) Representative images of IF staining of PE-CD66c antibody internalization in OE19 and SK-GT-4 cells at different time points (0 min, 30 min, 60 min, 120 min, and 240 min); (B) Flow cytometric quantification of PE-CD66c antibody internalization in OE19 and SK-GT-4 cells.

[0013] Figure 4 Shown are the synthesis and in vitro efficacy of CD66c-DXd. (A) Volcano plot of 252 potential GEJ drug candidates; (B) Schematic diagram and chemical structure of the CD66c-DXd linker and warhead; (C) MADLI-TOF / TOF mass spectrometry results of CD66c antibody and CD66c-DXd; (D) In vitro cell growth inhibitory activity in OE19, SK-GT-4, and GES-1 cells.

[0014] Figure 5 Shown is the in vivo therapeutic potential of CD66c-DXd for GEJ treatment. (A) Schematic diagram of the gastroesophageal cancer model. Model mice were injected with OE19 cells and then injected with PBS, paclitaxel, or CD66c-DXd via the tail vein at a dose of 5 mg / kg / week. (B) Images of subcutaneous tumors excised from mice treated with PBS, paclitaxel, and CD66c-DXd (n = 6 per group). (C) Growth curves of OE19 tumors. (D) Tumor mass at the endpoint of OE19 tumors. (E) Body weight of mice bearing subcutaneous OE19 tumors. (F) Representative Ki67 IHC staining images of tumors in the PBS, paclitaxel, and CD66c-DXd groups. Scale bar 20 μm. (G) Quantification of the proportion of Ki67-positive cells in the PBS, paclitaxel, and CD66c-DXd groups. (H) HE staining of major organs, including heart, lung, liver, spleen, and kidney, in the PBS, paclitaxel, and CD66c-DXd groups. (I) Blood biochemical indicators (liver function: ALT, AST, and TB; renal function: Cre and BUN) in the PBS, paclitaxel, and CD66c-DXd groups. *p<0.05; **p<0.01; ***p<0.001. DETAILED DESCRIPTION

[0015] The applicant previously conducted multi-omics analysis of proteomics, phosphorylation proteomics, whole exome sequencing and transcriptome RNA sequencing on 103 pairs of gastroesophageal junction cancer tumor tissues and paired adjacent cancer tissues, and found that CD66c is a new drug target for gastroesophageal junction cancer. Therefore, this study synthesized CD66c-ADCs to verify the characteristic expression of this new target in gastroesophageal junction cancer and explore the potential of CD66c-ADCs for the treatment of gastroesophageal junction cancer. The results of the present invention show that CD66c-DXd has significant anti-tumor efficacy in the gastroesophageal junction cancer model with limited toxicity. The expression level and tumor specificity of CD66c in gastroesophageal junction cancer are significantly higher than other established ADC targets, including HER2, CLDN18.2 and TROP2. The specificity of the efficacy was also determined in the treatment experiment of CD66c-negative cell line (GES1). This study provides a paradigm for identifying specific membrane targets and synthesizing ADCs with customized payloads for specific cancers, advancing the development of targeted cancer therapies.

[0016] The present invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof. The structure of the antibody-drug conjugate is as follows: Ab-(LD)n, wherein Ab is an anti-CD66c antibody, L is a linker, D is a cytotoxic agent, and n is an integer of 1-40.

[0017] CD66c is highly overexpressed in tumors and can effectively internalize cells. CD66c, also known as CEACAM6, is a multifunctional glycoprotein located at the 19q13.2 site in the human genome, mediating interactions with integrin receptors.

[0018] In the present invention, the anti-CD66c antibody is selected from monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody fragments (eg, Fab, F(ab')2, Fv, scFv). The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0019] The anti-CD66c antibody can be selected from any antibody or antigen-binding fragment thereof in the prior art that can bind to CD66c, such as Bayer's Bay1834942 CD66c antibody.

[0020] In one embodiment, the heavy chain variable region of the anti-CD66c antibody includes CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.2, and CDR-H3 as shown in SEQ ID NO.3, and the light chain variable region of the anti-CD66c antibody includes CDR-L1 as shown in SEQ ID NO.4, CDR-L2 as shown in SEQ ID NO.5, and CDR-L3 as shown in SEQ ID NO.6.

[0021] TYGIGVG (SEQ ID NO. 1)

[0022] HIWWNDNKYYSTSLKT (SEQ ID NO. 2)

[0023] ISLPYFDY (SEQ ID NO. 3)

[0024] KASQNVGTAVA (SEQ ID NO. 4)

[0025] SASNRYT (SEQ ID NO. 5)

[0026] QQYSSYPLT (SEQ ID NO. 6)

[0027] The amino acid sequences of the heavy and light chains of the anti-CD66c antibody are shown in SEQ ID NO. 7 and 8, respectively: QVTLRESGPALVKPTOTLTLTCTFSGFSLSTYGIGVGWIROPPGKALEWLAHIWWNDNKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARISLPYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVOFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHODWLNGKEYKCKVSNKGLPAPIEKTI SKTKGOPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO.7)

[0028] DIQLTQSPSFLSASVGDRVTITCKASQNVGTAVAWYQQKPGKAPKLLIYSASNRYTGVPSR

[0029] FSGSGSGTEFTLTISSLQPEDFATYYCQQYSSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEOL

[0030] KSGTASVVCLLNNFYPREAKVOWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHOGLSSPVTKSFNRGEC(SEQ ID NO.8)

[0031] The anti-CD66c antibody is coupled to the cytotoxic agent via a linker. Linkers are divided into two categories: non-cleavable linkers and cleavable linkers. Cleavable linkers can be cleaved within the target cell and release the drug agent. Cleavable linkers can be divided into two main categories: chemically labile linkers and enzyme-labile linkers. Chemically labile linkers can be selectively cleaved due to differences in the properties of plasma and cytoplasm. Such properties include pH value, glutathione concentration, etc. pH-sensitive linkers, commonly referred to as acid-cleavable linkers, are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be hydrolyzed in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Glutathione-sensitive linkers are also called disulfide bond linkers. Enzyme-labile linkers, such as peptide linkers, can better control drug release. Peptide linkers can be effectively cleaved by lysosomal proteases such as cathepsin B or plasmin (the levels of which are elevated in some tumor tissues). These peptide linkages are highly stable in the plasma circulation because proteases are generally inactive due to the unfavorable extracellular pH and serum protease inhibitors. Due to their high plasma stability and good intracellular cleavage selectivity and efficiency, enzyme-labile linkers are widely used as cleavable linkers in antibody-drug conjugates. Typical enzyme-labile linkers include Val-Cit (vc), Phe-Lys, and Gly-Gly-Phe-Gly.

[0032] The linker can include one or more linker components. Exemplary linker components include 6-maleimidocaproyl, maleimidopropionyl-valine-citrulline, alanine-phenylalanine, p-aminobenzoyloxycarboxyl, N-succinimidyl 4-(2-pyridylthio) pentanoate, N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate and N-succinimidyl (4-iodo-acetyl) aminobenzoate. Other exemplary linker components can also be linkers that include amino acid units to allow protease cleavage, thereby facilitating the release of cytotoxic agents from antibody-drug conjugates after exposure to intracellular proteases (such as lysosomal enzymes). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides and pentapeptides. Exemplary dipeptides include: valine-citrulline; alanine-phenylalanine; phenylalanine-lysine; or N-methyl-valine-citrulline. Exemplary tripeptides include: glycine-valine-citrulline or glycine-glycine-glycine. Exemplary tetrapeptides include: glycine-glycine-phenylalanine-glycine (abbreviated as GGFG).

[0033] The cytotoxic agent is selected from the group consisting of toxins, chemotherapeutic drugs, antibiotics, radioisotopes, and growth inhibitory agents.

[0034] Exemplary cytotoxic agents include: maytansine; maytansine-like substances; topoisomerase I inhibitors (such as camptothecin derivatives: DX-8951 derivative Dxd); tubulin inhibitors (such as monomethyl auristatin peptide E (MMAE) and monomethyl auristatin peptide F (MMAF); calicheamicins (such as calicheamicin); doxorubicin (such as doxorubicin); benzodipyrrole antibiotics (such as duocarmycins, CC-1065, etc.) and other cyclopropylpyrroloindole-4-one (cyclopropapyrroloind-4-one, CPI) derivatives, such as cyclopropylbenzidindole-4-one analogs, and pyrrolobenzodiazepines (PBD) or PBD dimers.

[0035] 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 one of the following: 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40.

[0036] In one embodiment, the antibody-drug conjugate is anti-CD66c mAb-GGFG-Dxd (abbreviated as CD66c-Dxd).

[0037] In one embodiment, the DAR value of the CD66c-DXD is 3.6.

[0038] The maleimide group in the linker is covalently coupled to the cysteine in the anti-CD66c antibody.

[0039] The present invention also provides a method for preparing the antibody-drug conjugate, comprising the following steps:

[0040] 1) mixing a disulfide bond reducing agent with the anti-CD66c antibody to reduce at least a portion of the disulfide bonds in the cysteine residues of the anti-CD66c antibody to sulfhydryl groups;

[0041] 2) Mixing the linker-cytotoxic agent with the product of step 1) to couple the anti-CD66c antibody to the linker to obtain the antibody-drug conjugate.

[0042] In step 1), the disulfide bond reducing agent is selected from DTT, tris(2-carboxyethyl)phosphine or their salts. In one embodiment, the disulfide bond reducing agent is selected from tris(2-carboxyethyl)phosphine hydrochloride.

[0043] In certain embodiments of the present invention, the reactions of steps 1) and 2) are carried out in a solvent. Those skilled in the art can select the appropriate type and amount of solvent to allow the reactants to be fully dispersed in the reaction system. The solvent can be a buffer. More specifically, the solvent can be selected from any one or more of a borate buffer and a phosphate buffer.

[0044] In certain embodiments of the present invention, the reaction temperature of step 1) or step 2) is 0-37° C. In certain embodiments of the present invention, the reaction temperature is 10-35° C. Preferably, the reaction temperature is 15-30° C.

[0045] In step 1), the amount of the disulfide bond reducing agent is generally equal to or in excess of the anti-CD66c antibody on a molar basis. In certain embodiments of the present invention, the molar ratio of the disulfide bond reducing agent to the anti-CD66c antibody is 1-50:1. In a preferred embodiment, the molar ratio of the disulfide bond reducing agent to the anti-CD66c antibody is 10-30:1. In a more preferred embodiment, the molar ratio of the disulfide bond reducing agent to the anti-CD66c antibody is 10-20:1. The linker and the cytotoxic agent can be a commercially available reagent that has been successfully linked, i.e., a linker-cytotoxic agent; the linker and the cytotoxic agent can also be homemade.

[0046] In certain embodiments of the present invention, the linker and the cytotoxic agent have already been successfully linked to form a linker-cytotoxic agent. The amounts of different linker-cytotoxic agents used in step 2) may vary, but the amount of linker-cytotoxic agent is generally equal to or in excess of the product of step 1) on a molar basis. In certain embodiments of the present invention, the molar ratio of linker-cytotoxic agent to the product of step 1) is 10-50:1. In a preferred embodiment, the molar ratio of linker-cytotoxic agent to the product of step 1) is 10-40:1. In a more preferred embodiment, the molar ratio of linker-cytotoxic agent to the product of step 1) is 15-25:1.

[0047] In certain embodiments of the present invention, step 2) further comprises removing unreacted linkers and cytotoxic agents.

[0048] The present invention also provides use of CD66c or its encoding gene as a target in preparing a product for treating or diagnosing gastroesophageal junction cancer.

[0049] The use of CD66c or its encoding gene as a target in the preparation of gastroesophageal junction cancer treatment or diagnosis products specifically refers to: using CD66c protein or its encoding gene as an identification object, and a substance that can reduce CD66c levels or kill gastroesophageal junction cancer cells.

[0050] In the present invention, the gastroesophageal junction cancer refers to a tumor occurring at the junction of the stomach and esophagus. This area usually refers to the connecting area between the lower esophagus and the upper stomach. The tumor in this area may manifest as a tumor in the lower esophagus extending into the stomach, or a tumor in the upper stomach extending into the esophagus.

[0051] The gastroesophageal junction cancer is selected from Siewert type I, Siewert type II or Siewert type III.

[0052] In Siewert type I, the tumor is centered in the lower esophagus and extends toward the stomach. In Siewert type II, the tumor is centered at the gastroesophageal junction. In Siewert type III, the tumor is centered in the upper stomach and extends toward the esophagus.

[0053] The present invention also provides use of a substance that specifically binds to CD66c in preparing a product for treating or diagnosing gastroesophageal junction cancer.

[0054] In one embodiment, the substance that specifically binds to CD66c is selected from an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof.

[0055] In one embodiment, the structure of the antibody-drug conjugate is as follows: Ab-(LD)n, wherein Ab is an anti-CD66c antibody, L is a linker, D is a cytotoxic agent, and n is an integer of 1-40.

[0056] In one embodiment, the antibody-drug conjugate is CD66c-Dxd.

[0057] In another embodiment, the substance that specifically binds to CD66c is selected from CD66c inhibitors.

[0058] A CD66c inhibitor refers to a molecule that has an inhibitory effect on CD66c. Inhibitory effects on CD66c include, but are not limited to, inhibiting the level or activity of CD66c.

[0059] Inhibiting CD66c activity means reducing CD66c activity. Preferably, compared to before inhibition, CD66c activity is reduced by at least 10%, more preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0060] Inhibiting the CD66c level may be by inhibiting the transcription or translation of the CD66c gene. Specifically, it may mean preventing the CD66c gene from being transcribed, or reducing the transcriptional activity of the CD66c gene, or preventing the CD66c gene from being translated, or reducing the translation level of the CD66c gene.

[0061] Those skilled in the art can use conventional methods to regulate CD66c gene expression, such as gene knockout, homologous recombination, interfering RNA, etc.

[0062] Preferably, compared with the wild type, CD66c gene expression is reduced by at least 10%, more preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, even more preferably by at least 90%, and most preferably, CD66c gene is not expressed at all.

[0063] The CD66c inhibitors include, but are not limited to, nucleic acid molecules, carbohydrates, lipids, small molecule drugs, 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 prepared by endoribonuclease III or short hairpin RNA (shRNA).

[0064] The gastroesophageal junction cancer treatment or diagnosis product must include a substance that specifically binds to CD66c and use the substance that specifically binds to CD66c as an active ingredient.

[0065] The gastroesophageal junction cancer treatment or diagnosis product may be a single-component substance or a multi-component substance.

[0066] The form of the gastroesophageal junction cancer treatment or diagnosis product is not particularly limited and may be in the form of solid, liquid, gel, semi-fluid, aerosol, or other substances.

[0067] The gastroesophageal junction cancer treatment or diagnosis product is primarily intended for mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes, or humans.

[0068] The gastroesophageal junction cancer treatment or diagnosis product is a drug.

[0069] The gastroesophageal junction cancer treatment product is selected from: targeted CD66c antibodies or antigen-binding fragments, exosomes, liposomes, SLP nanoparticles, CAR-T cells, oncolytic viruses, small molecule drug conjugates, bispecific antibodies, nucleic acid molecules, small molecule chemical drugs, peptides, proteins, interfering lentiviruses or adeno-associated viruses, etc.; the gastroesophageal junction cancer diagnosis product is selected from: exosomes, CTC, CT DNA, blood / urine protein or MRI / PET / ultrasound imaging probes, etc.

[0070] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0071] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0072] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0073] Brief description of the embodiment:

[0074] Methods: First, multi-omics data analysis of a large sample of GEJ cancer cells identified CD66c as a potential target for GEJ cancer. Immunohistochemistry (IHC) was used to further validate the differential expression of CD66c in GEJ cancer and adjacent tissues. Immunofluorescence (IF) staining, flow cytometry, and single-photon confocal microscopy were used to determine the expression level and subcellular location of the novel target CD66c in two human GEJ cancer cell lines (OE19 and SK-GT-4) and one normal gastric epithelial cell line (GES-1). The endocytosis of anti-CD66c antibodies in GEJ cancer cells was assessed by confocal imaging, and the endocytosis efficiency was analyzed by flow cytometry. Furthermore, a candidate ADC (CD66c-DXd) was designed, prepared, and characterized. The inhibitory activity of the ADC candidate in GEJ cancer cells and normal gastric epithelial cells was evaluated in vitro using a CCK8 assay. The antitumor activity of CD66c-DXd was further evaluated in an in vivo model.

[0075] Results: Multi-omics data from a large sample of GEJ cancer cells identified CD66c as a potential target. In vitro chromatin immunohistochemistry (IHC) analysis of clinical specimens confirmed that the expression level of the novel target CD66c was significantly higher in GEJ cancer tissues than in adjacent adjacent tissues. Two GEJ cancer cell lines overexpressed the CD66c target protein, while normal gastric epithelial cells did not express CD66c. The cell membrane localization of the CD66c target protein was determined. GEJ cancer cells overexpressing CD66c displayed significant endocytosis activity in response to an anti-CD66c antibody. The half-maximal inhibitory concentration (IC50) of the ADC candidate, CD66c-DXd, was determined to be 22.8 nM and 28.4 nM in OE19 and SK-GT-4 tumor cells, respectively, demonstrating promising antitumor activity. Furthermore, CD66c-DXd demonstrated effective and durable tumor regression in an OE19 GEJ cancer xenograft model with an excellent safety profile.

[0076] Conclusion: CD66c was identified as a novel ADC target for the treatment of GEJ cancer. Based on this, an ADC drug (CD66c-DXd) was designed, prepared, and characterized. Its in vivo and in vitro antitumor activity and biosafety were determined, providing a potential targeted therapy candidate for GEJ cancer.

[0077] Materials and experimental methods used in the examples

[0078] 1.1 Clinical samples, cell lines, and main reagents

[0079] Human gastroesophageal junction cancer cell lines (OE19 and SK-GT-4) and normal gastric epithelial cells (GES-1) were purchased from Tongpai (Shanghai) Biotechnology Co., Ltd. DMEM, RPMI-1640, and fetal bovine serum were purchased from Gibco. The CCK8 kit (catalog number BS350B) was purchased from BioshRP; PE anti-mouse IgG1 (catalog number 406608) was purchased from Biolegend; and PE-CD66c antibody (catalog number ab275676) was purchased from Abcam.

[0080] 1.2 Experimental methods

[0081] 1.2.1 Immunohistochemistry (IHC) Immunohistochemistry (IHC) was used to detect the differences in CD66c expression levels in 95 human gastroesophageal junction cancer tissues, 49 adjacent cancer tissues, and 48 metastatic lymph nodes.

[0082] 1.2.2 Cell Culture and Passaging GEJ cancer cells and human gastric epithelial cells were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM) or RMPI-1640 medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin in a 37°C, 5% CO2 incubator. The medium was changed every 2-3 days. When the cell density reached approximately 90% and the cells were in the logarithmic growth phase, they were trypsinized for passaging and subsequent experiments.

[0083] 1.2.3 Flow cytometry (FACS) Flow cytometry was used to detect the expression levels of CD66c in two human gastroesophageal junction cancer cell lines OE19 and SK-GT-4 and one normal gastric epithelial cell line GES-1 cultured in vitro. 1×10 6 The cells were washed twice with PBS. The obtained cells were blocked with 1% bovine serum albumin (BSA) in PBS for 30 minutes in an ice bath. After BSA blocking, the cells were incubated with phycoerythrin (PE)-conjugated CD66c antibodies for 1 hour at room temperature. PE-conjugated IgG was used as a control. The cells were washed three times with 1% BSA in PBS, resuspended in PBS, and the expression intensity of CD66c in each cell line was assessed using flow cytometry.

[0084] 1.2.4 Immunofluorescence staining (IF) The subcellular location of the target protein in two gastroesophageal junction cancer cell lines, OE19 and SK-GT-4, was observed under a single-photon confocal microscope. 6Cells were seeded into three confocal dishes, each containing 1 mL of culture medium, and cultured overnight at 37°C. The culture medium was removed and the cells were washed once with PBS. Block the cells with 1 mL of PBS containing 1% BSA for 15-30 minutes on ice. After blocking, the liquid was aspirated and the cells were incubated with antibodies at 37°C in three groups: Group 1: Incubate the cells with 1 mL of PBS for 1 hour; Group 2: Incubate the cells with 2 μL of IgG-PE and 1 mL of PBS for 1 hour; Group 3: Incubate the cells with 2 μL of CD66c-PE and 1 mL of PBS for 1 hour; wash once with 1 mL of PBS; add 1 mL of Hoechst nuclear stain and stain at 37°C for 20-30 minutes; wash once or twice with 1 mL of PBS and observe under a single-photon confocal microscope.

[0085] 1.2.5 Imaging flow cytometry Imaging flow cytometry was used to determine whether CD66c antibodies could selectively enter GEJ cancer cells and be rapidly transported to intracellular lysosomes. 6 Cells were seeded onto confocal microplates, with 5 plates plated. Each dish was incubated with 1 ml of culture medium at 37°C overnight. The culture medium was removed, and 2 μl of CD66c-PE in 1 ml of PBS was added on ice. After staining on ice for 30 minutes, the cells were washed once with cold PBS. The cells were then added with 1 ml of PBS and allowed to internalize at 37°C for 0, 30, 60, 120, and 240 minutes. At the end of each time period, the cells were washed 1-2 times with PBS, fixed with 4% paraformaldehyde for 10 minutes, and washed 1-2 times with PBS. The PBS was aspirated, and 1 ml of Hoechst staining solution was added. The cells were stained at 37°C for 20-30 minutes, the stain was aspirated, and the cells were washed 1-2 times with PBS. Finally, the cells were photographed, analyzed, and stored using a confocal microscope.

[0086] 1.2.6 ADC Drug Design, Preparation, and Characterization The structural composition of ADC drugs determines their high targeting and tumor cell killing properties. Therefore, based on the identification of the target protein CD66c, we selected the appropriate antibody, linker, and warhead, determined the drug-antibody ratio (DAR), and designed CD66c-DXd, which was then prepared and characterized.

[0087] Preparation steps of ADC drugs:

[0088] 1) Prepare the CD66c antibody at a concentration of 10 mg / mL. Add this solution (1.00 mL) to a 2.0 mL polypropylene test tube, and add a 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0667 mL; 10 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.050 mL). After confirming that the pH of this solution is 7.4 ± 0.1, reduce the disulfide bonds in the hinge region of the antibody by incubating at 37°C for 1 hour.

[0089] 2) Conjugation of the antibody to the linker-drug warhead: After incubating the above solution in a 22°C water bath for 10 minutes, 10 mM GGFG-DXd (molar ratio to antibody: 10:1) was added and incubated in a 22°C water bath for 40 minutes to conjugate the drug linker to the antibody;

[0090] 3) Purification: A NAP-25 column was equilibrated with 10 mM acetate buffer (pH 5.5) containing 5% sorbitol. The antibody-drug conjugate reaction aqueous solution (approximately 2.5 mL) was loaded onto the NAP-25 column and eluted with the manufacturer's specified amount of buffer to collect the antibody fraction. The collected fraction was reloaded onto the NAP-25 column and eluted with buffer. The gel filtration purification process was repeated 2 to 3 times to obtain the purified antibody-drug conjugate.

[0091] 1.2.7 In vitro cytotoxicity assay - CCK8 assay. The IC50 values of two human gastroesophageal junction cancer cell lines, OE19 and SK-GT-4, and one normal gastric epithelial cell line, GES-1, were determined using the CCK8 assay. Cells were seeded at a density of 10,000 cells per well in 96-well culture dishes and cultured overnight. After adherence, CD66c-DXd was added to the plates at concentrations ranging from 0 to 10 μg / mL, using 10-fold serial dilutions for a total of eight concentrations in triplicate wells. After 72 hours of incubation with the drugs, the culture medium was discarded, and the CCK8 assay reagent was diluted 10-fold with fresh cell culture medium. 100 μL / well was added to the 96-well plates and incubated at 37°C. The absorbance (OD) was read at a wavelength of 450 nm using a microplate reader. Cell viability was determined by comparing the absorbance of drug-treated cells with that of untreated control cells.

[0092] 1.2.8 Statistical Methods GraphPad Prism 9.0 and FlowJo V10 were used to analyze the experimental data. Variable data are expressed as mean ± variance (SD). Differences between groups were analyzed using the t-test, with P < 0.05 considered statistically significant. For survival analysis, the Mantel-Cox test in GraphPad Prism 9.0 was used, with significance levels categorized as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. GraphPad Prism 9.0 was used to plot the experimental data.

[0093] Example 1CD66c is a potential ADC target for advanced gastroesophageal junction cancer.

[0094] Our previous study reported proteomic and phosphoproteomic analyses of 103 gastric cancer tumors and paired normal adjacent tissues (NATs), as well as RNA sequencing of 83 tumor-NAT pairs. Figure 1 A shows the top 20 differentially expressed proteins (DEPs) based on 103 AEG tumor proteomics data. Next, we cross-referenced the Cancer Surfaceome Atlas and found that 8 of the top 20 DEPs were localized to the cell membrane ( Figure 1 B). The expression of these eight proteins in normal human tissues was compared using the Human Protein Atlas (HPA). To quantify and compare the expression of different targets in normal tissues, a normal tissue expression index was defined, which is the sum of the 41 tissue expression scores in the HPA, with high expression scored as 3, moderate expression as 2, low expression as 1, and no expression as 0. CD66c was compared with other established ADC targets for GEJ cancer (HER2, TROP2, and CLDN18.2). Figure 1 D is a histogram of the logarithmic fold change of CD66c in tumor tissue and precancerous tissue of each gastroesophageal junction cancer patient, indicating that the tumor expression specificity of CD66c is the highest among the candidate targets. Figure 1 E shows the heat map relationship between clinical characteristics and CD66c expression levels of 103 gastric cancer patients, indicating that CD66c expression increases significantly with the progression of TNM stage ( Figure 1 F). To further validate the proteomic findings, IHC staining of CD66c was performed in tissue microarrays of the GEJ cancer cohort ( Figure 1 G). IHC results showed that CD66c immunostaining was mainly membranous, and CD66c was not expressed or was low in normal gastric tissue samples ( Figure 1 G). Figure 1As shown in Figure H, the IHC H score in patients with stage III-IV GEJ cancer was significantly higher than that in patients with stage I-II and normal gastric tissue. Together, these data support CD66c as a potential ADC target for advanced GEJ cancer. Therefore, developing an ADC for patients with CD66c-positive GEJ cancer is crucial for improving the poor clinical prognosis of GEJ cancer.

[0095] Example 2 CD66c is overexpressed in gastroesophageal junction tumor cell lines and is significantly internalized

[0096] Flow cytometry was used to investigate the expression of CD66c on the cell membrane of two human gastroesophageal junction cancer cell lines (OE19 and SK-GT-4) and a normal gastric epithelial cell line (GES-1). Figure 2 As shown in A, OE19 cells showed high levels of CD66c expression, while SK-GT-4 cells showed moderate levels of expression. In contrast, CD66c expression was undetectable in the normal human epithelial cell line GES1. In addition, we compared the surface density of various CEACAM family members, including CD66a (CEACAM1), CD66c (CEACAM6), and CD66e (CEACAM5), in gastroesophageal junction cancer cell lines. Among them, CD66c was expressed at the highest levels in OE19 and SK-GT-4 cells ( Figure 2 B). Immunofluorescence (IF) staining further confirmed the presence of CD66c on the cytoplasmic membrane of gastroesophageal junction cancer cells ( Figure 2 C). Simultaneously, the endocytic activity of CD66c antibodies in GEJ cancer cells was quantitatively assessed using two independent methods. IF staining showed that PE-conjugated CD66c antibodies initially bound to the plasma membrane of OE19 and SK-GT-4 cells and were subsequently internalized into endosomes and lysosomes via antigen-mediated endocytosis over time ( Figure 3 A). Flow cytometry analysis quantified the internalization kinetics of CD66c antibody, showing that after 4 hours of culture, the internalization rates of OE19 cells and SK-GT-4 cells were 46.7% and 30.5%, respectively ( Figure 3 B) This demonstrates the high specificity and internalization of the CD66c target, and provides the possibility for the successful development of an ADC drug targeting CD66c.

[0097] Example 3 Design, preparation and characterization of ADC drugs

[0098] To identify therapeutic payloads for advanced GEJ cancer, an unbiased screening of potential drug candidates was performed based on GEJ cancer proteomics. First, a search was conducted for DEPs identified by GEJ cancer proteomics using genomics of cancer drug sensitivity (GDSC) and other approaches. This comprehensive approach identified 252 DEPs that are targets of US FDA-approved drugs or drug candidates currently in clinical trials. Figure 4 A shows the volcano plot of these 252 DEPs. Based on the p-value of the false discovery rate (FDR), the top 10 upregulated DEPs for potential ADC payloads are NNMT, CTSB, TYMP, NAMPT, MMP14, GLS, COL1A1, TOP1, CDK1, and PLAUR. Considering the accessibility of ADC payloads, the potent TOP1 inhibitor DXd and its corresponding linker (GGFG) were selected as the optimized ADC formulation. CD66c-GGFG-DXd (CD66c-DXd) was subsequently synthesized via cysteine-maleimide conjugation ( Figure 4 B). The drug-antibody ratio (DAR) of CD66c-DXd was 3.6 ( Figure 4 C). Next, the in vitro toxicity of CD66c-DXd was determined by quantifying its half-maximal inhibitory concentrations (IC50s) in GEJ cancer cell lines ( Figure 4 D) The IC50 of CD66c-DXd against OE-19 and SK-GT-4 cells were 22.8 nM and 28.4 nM, respectively.

[0099] Example 4 In vivo therapeutic efficacy of CD66c-DXd

[0100] The in vivo efficacy of CD66c-DXd was evaluated using a subcutaneous gastroesophageal xenograft mouse model of OE19 cells. Figure 5 A). First, CD66c-DXd was injected via the tail vein at a dose of 5 mg / kg / week. The control group was treated with PBS (sham) or paclitaxel at the same dose and schedule as the tumor-bearing mice. Figure 5 As shown in Figure BC, the CD66c-DXd group showed significant and sustained tumor regression throughout the experiment. The difference in tumor size between the CD66c-DXd group and the control group was statistically significant (PBS vs. CD66c-DXd, adjusted p = 0.004; paclitaxel vs. CD66c-DXd, adjusted p = 0.011; Figure 5 C). The average tumor weights were 1.26 g (PBS), 0.59 g (paclitaxel), and 0.21 g (CD66c-DXd). Figure 5 D). Importantly, no significant differences in body weight were observed among the three treatment groups ( Figure 5E), indicating that CD66c-DXd has limited toxicity and is well tolerated by mice. Immunohistochemical staining of Ki67, a cell proliferation marker, was performed on subcutaneous xenografts to assess tumor cell proliferation in the different treatment groups. The PBS-treated group showed a large number of Ki67-positive cells, indicating a high level of cell proliferation. In contrast, the CD66c-DXd-treated group showed a significant decrease in Ki67-positive cells, indicating that CD66c-DXd has a strong inhibitory effect on tumor growth ( Figure 5 F). Statistical analysis confirmed the antiproliferative effect of CD66c-DXd (PBS vs. CD66c-DXd, adjusted p < 0.001; paclitaxel vs. CD66c-DXd, adjusted p = 0.025; Figure 5 G). Treatment-related toxicity in major organs (heart, lung, liver, spleen, and kidney) was assessed by histological analysis using HE staining. HE staining showed no obvious drug-related necrosis or degenerative changes in these organs in any treatment group ( Figure 5 H). In addition, serum biochemical indicators were measured to evaluate the organ function and potential toxicity of ADC. There were no significant differences in liver function indicators (ALT, AST, and TB) and renal function indicators (Cre and BUN) among the three groups, indicating that there were no obvious off-target effects in vivo ( Figure 5 I). These results demonstrate the efficacy and safety of CD66c-DXd in targeting GEJ cancer tumors and are well tolerated.

[0101] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. An antibody-drug conjugate, characterized in that: The structure of the antibody-drug conjugate is as follows: Ab-(LD)n, wherein Ab is an anti-CD66c antibody, the anti-CD66c antibody is selected from a full-length antibody or an antibody fragment, L is a linker, and the linker is selected from a non-cleavable linker or a cleavable linker; D is a cytotoxic agent, and the cytotoxic agent is selected from a toxin, a chemotherapeutic drug, an antibiotic, a radioactive isotope or a growth inhibitor, and n is an integer of 1-40.

2. The antibody-drug conjugate according to claim 1, characterized in that: The heavy chain variable region of the anti-CD66c antibody includes CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.2 and CDR-H3 as shown in SEQ ID NO.3, and the light chain variable region of the anti-CD66c antibody includes CDR-L1 as shown in SEQ ID NO.4, CDR-L2 as shown in SEQ ID NO.5 and CDR-L3 as shown in SEQ ID NO.

6.

3. The antibody-drug conjugate according to claim 1, characterized in that: The linker comprises one or more linker components, and the linker components are selected from any one or more of maleimidopropionyl, maleimidocaproyl, p-aminobenzoyloxycarboxyl, valine-citrulline, alanine-phenylalanine, glycine-glycine-glycine, glycine-valine-citrulline, and glycine-glycine-phenylalanine-glycine.

4. The antibody-drug conjugate according to claim 1, characterized in that: The cytotoxic agent is selected from any one or more of the following: maytansine, maytansine-like agents, topoisomerase I inhibitors, tubulin inhibitors, calicheamicin and its derivatives, doxorubicin and its derivatives; preferably, the cytotoxic agent is selected from DX-8951 derivative Dxd, MMAE or MMAF.

5. The method for preparing the antibody-drug conjugate according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: 1) mixing a disulfide bond reducing agent with the anti-CD66c antibody to reduce at least part of the disulfide bonds in the cysteine ​​of the anti-CD66c antibody to sulfhydryl groups; 2) Mixing the linker, the cytotoxic agent and the product of step 1) to couple the anti-CD66c antibody to the linker to obtain the antibody-coupled drug.

6. The preparation method according to claim 5, characterized in that: Also includes one or more of the following features: A. the disulfide bond reducing agent in step 1) is selected from DTT, tris(2-carboxyethyl)phosphine or their salts; B. The reaction of steps 1) and 2) is carried out in a solvent; preferably, the solvent is a buffer; more preferably, the solvent is selected from any one or more of a borate buffer and a phosphate buffer; C. The reaction temperature of step 1) or step 2) is 0-37°C; D. In step 1), the molar ratio of the disulfide bond reducing agent to the anti-CD66c antibody is 1-50:

1.

7. Use of CD66c or its encoding gene as a target in the preparation of therapeutic or diagnostic products for gastroesophageal junction cancer.

8. Use of a substance that specifically binds to CD66c in the preparation of a product for treating or diagnosing gastroesophageal junction cancer.

9. The use according to claim 8, characterized in that The substance that specifically binds to CD66c is selected from an antibody-drug conjugate or a pharmaceutically acceptable salt thereof; preferably, the antibody-drug conjugate is selected from the antibody-drug conjugate according to any one of claims 1-4.

10. The use according to claim 9, characterized in that The substance that specifically binds to CD66c is a CD66c inhibitor; preferably, the CD66c inhibitor is selected from nucleic acids, small molecule chemical drugs, polypeptides, proteins, interfering lentiviruses, adeno-associated viruses, nanoparticles, liposomes, extracellular vesicles or cells.

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