Construction Of A Pan-Solid Tumor-Associated Antigen Atlas And Its Application To Antibody-Drug Conjugates And Chimeric Antigen Receptor T Cells

Cancer has become a major health hazard for humans,and according to the WHO's International Agency for Research on Cancer(IARC),in 2020,there will be approximately 19.3million new cases and 10.0 million cancer deaths in men and women combined worldwide,of which 18.0 million and 9.3 million will be from solid tumors.However,data from systematic evaluations of oncology treatments show that the majority of anticancer drugs approved by the European Medicines Agency in 2009-13 failed to produce significant improvements in overall patient quality of life.Therefore,there is a need to continue to improve the clinical efficacy of antineoplastic drugs.Antibody-Drug Conjugates(ADCs)and Chimeric Antigen Receptor(CAR)T-cell therapies are rapidly evolving tumor-targeted therapies that identify and ablate tumors by specifically recognizing target antigens that are differentially overexpressed between malignant cells and normal tissue cells.The key to the safety and efficacy of ADC and CAR-T lies in the tumor specificity of the target antigens they identify.It is therefore critical to identify the ideal target antigens.The ideal target antigens are expressed specifically by malignant cells,however,tumor-specific antigens are few and far between.The expression of tumor-associated antigens is elevated on tumor cells and restricted on normal cells,a property that allows them to also serve as effective target molecules for the localization of malignant tumors.Objective:Starting from the discovery of ideal tumor-associated antigens,we focus on building a new platform for tumor-associated antigens discovery,constructing a comprehensive pan-solid tumor tumor-associated antigens atlas,providing research convenience for cancer researchers and a wide range of scientific and pharmaceutical researchers.Method:1.Based on the the uniformly processed RNA sequencing data of TCGA and GTEx,the difference analysis of 20242 HUGO genes in 19 types of solid tumors was performed;by setting the threshold to Benjamini-Hochberg adjusted p value equal to 0.01 and log2 Fold Change equal to1.0,we kept those HUGO genes significantly overexpressed in tumour cell populations;we futher excluded those proteins without extracellular domains by using protein subcellular localization information;data sets integrating m RNA expression information of GTEx,HPA and FANTOM5 and protein abundance information of HPA and HPM were used to obtain protein molecules with restricted expression in normal tissues;by using the publicly available Blood Spot platform,we found those proteins that are restrictedly expressed in the bone marrow Lin-CD34+ CD38-CD90+ CD45RA-HSCs and Lin-CD34+ CD38-CD90-45RA-MPPs.2.Convert the read count data of RNA sequencing into TPM format,and use the TPM value of each gene in its paired indications and normal tissues as input data for differential expression analysis.The non-parametric Mann-Whitney U test was used to calculate and plot the differential expression profile of the expression of each candidate target molecule in a specific indication compared to its expression in each normal human tissue.3.Extract and sort out the cancer histological grade,pathological staging and TNM staging information in the pan-cancer phenotype data from TCGA;confirm the non-silent somatic mutations(SNP and INDEL)of target genes by mining MC3("multi-center and multi-cancer mutation identification")TCGA MAF("mutation annotation format")data.Combine m RNA expression data to compile a comprehensive dataset for the evaluation of heterogeneous expression patterns of target genes.4.Download and extract target genomic mutation data from TCGA,collect carcinogenic or functional genomic mutation information from Onco KB,annotate and count the mutation types and alteration frequency of target genes,and identify functional targets that carry clinically actionable alterations.5.CDH17-specific monoclonal antibodies are prepared by the hybridoma technology,and then flow cytometry,overexpression colocalization analysis,binding affinity detection,antibody sequencing,and variable region sequence evolution relationship analysis are used to multiplely characterize various indicators of the antibody.CDH17 targeting ADC are prepared by coupling mc-Val Cit PABC-MMAE.Combining the in vitro cytotoxicity experiments and cell internalization evaluations,complete the preliminary screening verification of effector molecules.6.By constructing CDX animal models of KM12 SM and COLO205 colon cancer cell lines,the in vivo anti-tumor activities of chi2E21-MMAE and chi2F12-MMAE are comprehensively evaluated.To evaluate the tissue toxicity of CDH17 targeted ADC in mice,we prepare ADC molecule that cross-reacts with human and mouse CDH17 proteins and perform HE staining on the heart/lung/liver/kidney/colon/rectum/spleen of the administered tumor-bearing mice.7.CD3+ T cells are infected by lentiviral vector to prepare LYPD3 targeting CAR-T cells.The ability of CAR-T cells to differentiate into effector cells is determined by detecting the differentiation of CAR-T cells.Through the in vitro cytotoxicity experiments and cytokine secretion detection,the specific killing effect of LYPD3 targeting CAR-T cells on tumor cells is studied.By counting CAR-T cells in the circulating blood of tumor-bearing mice and detecting the level of T cell infiltration in tumor tissues,the mechanism by which LYPD3-targeted CAR-T cells exert a long-lasting anti-tumor effect in mice is revealed.Result:1.Develop a specialized algorithm and compile a comprehensive data set that integrates large-scale transcriptome,proteome and genome information of pan-solid tumors and normal tissues.2.Systematically identify potential tumor-associated antigens.Through our tumor-associated antigens discovery platform,75 potential tumor-associated antigens are screened from 20242 HUGO genes.Especially BACE2,CDH17,CELSR1,CELSR2,COL17A1,COL23A1,CRIM1,DSC3,GPR87,LYPD3,MUC17,NOX1,PTPRN2,SCTR,TRPM8,VCAM1 et al.are worthy of further preclinical evaluation research.3.ADC molecules developed based on CDH17 protein can significantly ablate KM12 SM and COLO205 xenograft tumors in vivo,and show a certain tumor suppressive effect on the xenograft tumors derived from colon cancer patients with a high expression of CDH17 protein.chi2F12-MMAE exibited a high level of in vitro cytotoxicity to COLO205 with high drug sensitivity(IC50 = 60.9 p M),and chi2E21-MMAE and chi1A13-MMAE showed comparable levels of inhibitory effect to COLO205(IC50: 925.8 p M vs.998.3 p M).Twice monotherapy intravenous injections(Q2W)of chi2E21-MMAE or chi2F12-MMAE can inhibit the growth of KM12 SM in mice in a dose-dependent manner,but cannot completely eliminate tumors.In the COLO205 CDX model,chi2F12-MMAE is injected intravenously in the same way,and the first injection dose of 1.5 mg/kg can smooth the growth of xenograft tumors,and the second injection after two weeks can continue to have the oncolytic effect;the xenograft tumors of some mice in the 3 mg/kg middle-dose treatment group rebounded in the later stage;the 6 mg/kg high-dose treatment can cure xenograft tumors.4.CDH17 targeted ADC has relatively controllable tissue toxicity in mice.The ADC molecule677-MMAE,which can cross-react with human and mouse CDH17 protein,is prepared.It is observed that there is inflammation in the local tissues of the colon and rectum of the mice in the677-MMAE administration group,and a large number of lymphocytes accumulate;but in other tissues,there is no inflammatory response.In addition,during the observation period,chi2E21-MMAE,chi2F12-MMAE or 677-MMAE are well tolerated by mice in vivo,and no mice die and no weight loss of more than 10% is observed.5.LYPD3-targeted CAR-T cells exert long-lasting anti-tumor effects on PC9 xenograft tumors in mice.In addition,during the observation period,the mice tolerate LYPD3-CAR-T well,and no mice die and no weight loss of more than 10% is observed.Further investigation indicate that LYPD3-CAR-T cells not only exist in large amounts in the peripheral blood of tumor-bearing mice,but also infiltrate into the PC9 xenograft tumors that are highly responsive to LYPD3-CAR-T treatment.In addition,a large number of CD3+ T cells appear in the spleen of the mice in the LYPD3-CAR-T treatment group,but not in the control group.The memory phenotype of CAR-T cells is essential for stimulating a lasting immune response.The data shows that before the infusion of LYPD3-CAR-T cells,the proportion of Tn/Tscm cells and Tcm cells are as high as52.4% and 21.7%,respectively,and thus the LYPD3-CAR-T cells have a good potential to differentiate into effector cells.Conclusion:By developing a tumor-associated antigens recognition algorithm and integrating large-scale transcriptome,proteome and genome data from 19 types of solid tumors and normal tissues,we complete the exploration tumor-associated antigens for pan-solid tumors.By investigating the anti-tumor activity of CDH17-targeted ADC and LYPD3-targeted CAR-T cells in model mice,the feasibility of developing CDH17 and LYPD3 proteins as ADC and CAR-T targets,respectively,is demonstrated.