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Study Of Human Anti-Prostate Specific Membrane Antigen (PSMA) Antibody-Drug Conjugates

Posted on:2017-08-15Degree:MasterType:Thesis
Country:ChinaCandidate:J H WuFull Text:PDF
GTID:2334330503489217Subject:Immunology
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Background: Prostate cancer(PCa) is a common urinary tumor which is a serious threat to men's health. Prostate-specific membrane antigen(PSMA), which is specifically expressed in the surface of PCa cells, is an ideal target for the therapy of PCa. Antibody-drug conjugates(ADC) is composed of antibody, chemical drug and "Linker". It maintains the function of both antibody and drug, can specifically kill cancer cells, thus is a very promising antitumor drug.Objective:To construct the eukyryatic expression vector of anti-PSMA human antibody PSMAb, and to transfect eukaryotic cells for expression and purification, and to analyze its biofunction. To prepare antibody-drug conjugate using PSMAb and Maytansinoid(DM1) and to analyze the function of PSMAb-DM1 in vitro and in vivo.Methods: The sequence of the variable region of anti-PSMA heavy and light chain were obtained from an sc Fv which was screened from a yeast display sc Fv library. The genes of full heavy and light chain were obtained by linking the variable region sequence with corresponding heavy and light chain constant region sequence.The heavy and light chain sequences were optimized to ensure high expression in mammalian CHO cells before synthesization. The two genes were synthesized and cloned into eukaryotic expression vector pCDNA3, and the expressing vector contained light chain or heavy chain sequence was named pCDNA3-LC or pCDNA3-HC respectively. The pCDNA3-LC and pCDNA3-HC plasmids were transiently transfected into CHO-S cells at the ratio of 4:1 for expression. The cell culture medium was collected and the PSMAb antibody was purified using protein A affinity chromatography. Cellular ELISA, flow cytometry and indirect immunofluorescence staining were applied to evaluate the bio-function of PSMAb. The PSMAb antibody was also conjugated with the near infrared dye IRDye 800 CW and injected into xenografted nude mice to examine its distribution in vivo. The PSMAb antibody was conjugated with microtubule inhibitor DM1 to obtain the ADC PSMAb-DM1. The specific killing of PSMAb-DM1 on PSMA positive cancer cells was analyzed both in vitro and in vivo.Results: The sequences of heavy and light chain gene were correct as confirmed by gene sequencing. The light and heavy chain expressing plasmids were confirmed by restriction enzyme digestion analysis. The PSMAb antibody was successfully expressed after the plasmids were transiently transfected into CHO-S cells. PSMAb antibody with high purity was purified using protein A affinity chromatography.The PSMAb antibody was proved to have high binding affinity, can specifically bind with and internalized into PSMA positive cancer cells, using cellular ELISA, flow cytometry and indirect immun ofluorescence staining. In vivo study showed that the IRDye 800 CW conjugated PSMAb can specifically distribute in PSMA positive cancer tissues. After being conjugated with drug DM1, the PSMAb-DM1 maintained high binding ability with PSMA positive cancer cells, as shown by flow cytometry. And the PSMAb-DM1 still can internalize into PSMA positive cancer cells as detected by indirect immunofluorescent staining. The PSMAb-DM1 can effectively kill and induce apoptosis in PSMA positive cancer cells in vitro, as shown by apoptosis and Alamar Blue analysis. The IC50 of PSMAb-DM1 was calculated to be 0.12 n M. In vivo study showed that PSMAb-DM1 can effectively inhibit the growth of PSMA positive tumor.Conclusions: 1) The eukaryotic expressing plasmids for human anti-PSMA light and heavy chain were successfully constructed. 2) High purity PSMAb antibody was obtained using affinity chromatography, after the expressing plasmids were transiently transfected in CHO-S cells. 3) The PSMAb antibody was proven to have high binding affinity, can specifically bind with and internalize into PSMA positive cancer cells, detected by cellular ELISA, flow cytometry and indirect immun ofluorescence staining. 4) The antibody-drug conjugate PSMAb-DM1 was successfully prepared through conjugating PSMAb with DM1. 5) The PSMAb-DM1 can specifically kill and induce apoptosis in PSMA positive cancer cells in vitro, and can effectively inhibit the growth of PSMA positive cancer in vivo.
Keywords/Search Tags:PSMA, human antibody, antibody-drug conjugate, prostate cancer, target therapy of tumor
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