Application Of Antibody-Drug Conjugates Based On Phage Display Technology In The Treatment Of Prostate Cancer

BackgroundProstate cancer is one of the most common malignancies in males worldwide.Early prostate cancer can be cured by surgery and radiotherapy.When patients progress to intermediate and advanced prostate cancer,androgen deprivation therapy(ADT)is the most important treatment,and then almost all patients will develop castration-resistant prostate cancer,which is the main cause of death in prostate cancer patients.Chemotherapy drugs are used in second-line therapy and are indicated for symptomatic prostate cancer,but adverse effects and drug resistance limit their use in prostate cancer.Therefore,how to achieve accurate and efficient removal of cancer cells is a key point in the development of anti-cancer drugs.ObjectiveSpecific prostate cancer targets suitable for antibody-drug conjugate(ADC)were screened,antibody libraries were established through phage display technology,fully human antibodies were constructed,novel antibody conjugates were prepared,and the biological functions of novel ADC in vivo and in vitro were studied in prostate cancer.MethodsThe membrane protein data and the differential genes of bulk transcriptome sequencing were analyzed,and the membrane proteins that were highly expressed in prostate cancer and lowly expressed in normal tissues were screened out by bulk transcriptome sequencing and single-cell transcriptome sequencing.The specific expression of target antigens in prostate cancer was verified,and finally the target antigens for the preparation of ADCs were screened.The peripheral blood of prostate cancer patients was collected to construct a single-chain bacteriophage antibody library;single-chain antibodies with high affinity were screened out through cell enrichment;full-length antibodies were constructed by eukaryotic expression;and antibodies were conjugated with monomethyl auristatin E(MMAE)to prepare ADCs.The biological activity of ADCs on prostate cancer cell lines was verified by cytotoxicity detection experiments.Detection of the effect of ADCs on the cell cycle by flow cytometry.The subcutaneous tumor model was established and administered by mouse tail vein to verify the inhibition of tumor growth and evaluate the safety of ADCs in vivo.The subcutaneous tumor tissue was sequenced by bulk transcriptomics to explore the possibility of combining drugs.ResultsFinally,three genes were screened for high expression in prostate cancer and low expression in normal tissues,and these three genes were used as candidate target antigens,screened again by single-cell transcriptome analysis,and finally selected prostate specific membrane antigen(PSMA)encoded by the FOLH1 gene as the target of ADCs.Establish a phage single-chain antibody bank from the peripheral blood of prostate cancer patients;the antibody library has sufficient storage capacity;and the insertion rate,diversity,and display rate of the antibody library show that the antibody library can carry out the next step of enrichment.Through three rounds of cell panning,enough positive clones were obtained,and a full-length fully human antibody was constructed after sequencing.The affinity and internalization activity of the antibody were verified by flow cytometry,and then the ADC was prepared by conjugation with MMAE,named 15873-MMAE.15873-MMAE can kill PSMA-positive cell lines,but not PSMA-negative cell lines,and the effect of 15873-MMAE is weakened after knocking down PSMA expression in PSMA-positive cell lines.Flow cytometry found that 15873-MMAE is dose-dependent in inducing cell cycle arrest in the G2/M phase,which can inhibit tumor growth in vivo and has drug safety.Subsequent bulk transcriptome sequencing analysis found that the mitosis damage pathway of tumor tissue after administration was regulated,and it affected the metabolic mode of tumor cells.ConclusionsPSMA can be used as an ideal target antigen for the development of ADCs in prostate cancer.15873-MMAE,which targets PSMA and was developed by phage display technology,specifically kills PSMA-expressing cells and subcutaneous transplant tumors in vivo and in vitro and is safe.Subsequent transcriptome sequencing verified the killing mechanism of 15873-MMAE and considered its combination with inhibitors of metabolic pathways.