| Background Prostate-specific membrane antigen(PSMA)is a type II tra nsmembrane protein composed of the extracellular C-terminus,the helical transmembrane region and the cytoplasmic N-terminus.Since the extracellular domain of PSMA can be recognized by antibodies,peptides,RNA aptamers and small molecules,it has become an ideal target molecule for targeted therapy.PSMA was originally found to be specifically expressed in normal prostate secretory epithelium,later it was found to be highly expressed in prostate cancer(PCa),and its expression was closely associated with disease progression.Recently,studies have reported that PSMA was also expressed in various solid tumors such as gastric cancer,pancreatic cancer,breast cancer,lung cancer,etc.,but it was expressed in neovascular of tumors rather than tumor cells.Therefore,besides being a therapeutic target for prostate cancer,PSMA can also be used as an anti-vascular therapeutic target for many tumors.Antibody-drug conjugate(ADC)is a new strategy for tumor-targeted therapy,which is composed of antibody and cytotoxic drugs.ADC combines the high targeting of antibodies with the potent killing of cytotoxic drugs,which has the function of specifically killing target cells,and has a broad application prospect.Previously,we have engineered a human single-chain antibody against PSMA obtained from a yeast display antibody library and have reconstructed it into a full-antibody PSMAb.We have confirmed that PSMAb can specifically bind to and internalize into PSMA-positive cells,haveing a good prospect for the development into ADCs.Objective 1)To investigate PSMA expression in urinary tumors.2)To investigate PSMA expression in other tumors.3)To study the therapeutic effect of PSMA-based antibody-drug conjugate on prostate cancer.Methods 1)Collect samples of prostatitis,benign prostatic hyperplasia and prostate cancer,performed immunohistochemical staining on PSMA,and analyzed PSMA expression in prostate cancer.Collect specimens of bladder cancer and renal cell carcinoma and perfor m immunohistochemical staining on CD31 and PSMA.Collect the information of bladder cancer patients and analyzed the association between vascular PSMA expression and clinicopathological features and prognosis.2)Collect specimens of hepatocellular carcinoma,breast cancer and ovarian cancer and performed immunohistochemical staining on CD31 and PSMA.Collect the information of hepatocellular carcinoma patients and analyze the association between vascular PSMA expression and clinicopathological features and prognosis.3)Amplify the PSMAb expression plasmid and transfected it into eukaryotic cells for expression,and harvested supernatants for purification.4)Examine the binding abilities of PSMAb to human PSMA and mouse PSMA by ELISA.5)Generate antibody drug conjugate PSMAb-MMAE throught the conjugation of PSMAb with microtubule inhibitor MMAE.6)Examine the binding and proapoptotic ability of PSMAb-MMAE to prostate cancer PC3 and C4-2 cells by flow cytometry.Examine the binding affinity of PSMAb-MMAE by cellular ELISA.Examine the internalization activity and microtubule inhibition of PSMAb-MMAE by indirect immunofluorescence staining.Analyze IC50 concentration of PSMAb-MMAE using Alamar Blue assay.7)Evaluate anti-tumor effect of PSMAb-MMAE on PSMA~+tumors in vivo using nude mice xenograft model.8)Evaluate the safety of PSMAb-MMAE preliminary through the monitor on mice bod y weight,liver and kidney function examination,and HE staining of important organs after treatment.Results 1)In urinary tumors,the expression of PSMA in prostate cancer is significantly higher than that of prostatitis and benign prostatic hyperplasia,with a positive rate of 93%.PSMA was expressed in tumor-associated vasculature of bladder cancer and renal cell carcinoma,and the positive rate of bladder cancer vascular was 60%.Vascular PSMA expression was closely associated with the clinicopathological features in patients with bladder cancer,such as tumor differentiation,tumor stage,lymph node metastasis,TNM stage,and Ki67 index,and patients with high vascular PSMA expression had shorter survival than patients with low vascular PSMA expression.The high vascular PSMA expression was closely related to the poor prognosis of patients with bladder cancer.2)In other tumors,PSMA was expressed in tumor-associated vasculature of hepatocellular carcinoma,breast cancer and ovarian cancer,and the positive rate of vascular PSMA expression in hepatocellular carcinoma is 76%.Vascular PSMA expression was closely associated with the clinicopathological features in patients with hepatocellular carcinoma,such as tumor differentiation,tumor stage,lymph node metastasis,TNM stage,and Ki67index,and patients with high vascular PSMA expression have shorter survival than patients with low vascular PSMA expression.The high vascular PSMA expression is closely related to the poor prognosis of patients with hepatocellular carcinoma.3)Obtained PSMAb with high purity.PSMAb can bind to human PSMA but not mouse PSMA.Successfully generated antibody-drug conjugate PSMAb-MMAE.PSMAb-MMAE can specifically bind with and internalize into PSMA~+C4-2 cells with a Kd value of 0.6nM.PSMAb-MMAE has a proapoptotic effect on C4-2 cells with an IC50value of 0.59nM.PSMAb-MMAE can inhibit the assembly of microtubules in C4-2 cells and can inhibit the growth of PSMA~+tumors in vivo.PSMAb-MMAE had no significant effect on body weight,liver and kidney function and tissue structure of important organs after its application in vivo.Conclusion PSMA was highly expressed in prostate cancer.PSMA was also expressed in tumor-associated vasculature in bladder cancer,renal cell carcinoma,hepatocellular carcinoma,breast cancer and ovarian cancer.PSMA expression is closely associated with the clinicopathological features and prognosis of patients with bladder cancer or hepatocellular carcinoma.PSMAb-MMAE can specifically kill PSMA positive cells in vitro,and has a significant therapeutic effect on prostate cancer xenograft model and has biological safety in vivo. |
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