Production Of Multivalent Anti-PSMA Nanobodies And The Toxicity Evaluation Of Nanobody-drug Conjugate

Prostate cancer is one of the cancers with the highest morbidity and mortality in males.Prostate-specific membrane antigen（PSMA）is highly expressed in prostate cancer,and its expression is closely related to the progression of the disease,which makes it an ideal target in the treatment of prostate cancer.Antibody-drug conjugates（ADCs）are composed of antibodies and small molecule toxic drugs.Due to their novel drug-delivery capability,ADCs have become an effective mean in the clinical treatment of cancers.However,there are some shortcomings of the monoclonal antibody molecules in the traditional ADCs,such as poor tissue penetration,high production and engineering costs,and certain immunogenic risks.Derived from the variable regions of camelid heavy chain antibodies,nanobodies are the smallest antibody fragments known to have complete antigen-binding capacity.Nanobodies have the advantages of deep tissue penetration,easy production and engineering,and low immunogenicity,and have been developed and applied to the diagnosis and treatment of various cancers.In this dissertation,based on PSMA-targeting nanobody,prokaryotic expression systems were constructed to achieve efficient soluble expression of monovalent and multivalent nanobodies.Next,the PSMA binding affinity and endocytosis capacity of monovalent and multivalent nanobodies were evaluated.Finally,a multivalent nanobody with better drugability was selected to construct a nanobody-drug conjugate,which was confirmed to have the ability to selectively kill PSMA-positive tumor cells.The main results are as follows:（1）Efficient soluble expression of monovalent and multivalent nanobodies in E.coli was achieved.Based on PSMA-targeting nanobody JVZ-007,four nanobody expression plasmids22b-jvz007₁,22b-jvz007₂,22b-jvz007₃,and 22b-jvz007₄ were constructed by isocaudamer strategy and were transformed into E.coli Rosetta（DE3）.All four kinds of nanobodies,JVZ-007₁,JVZ-007₂,JVZ-007₃ and JVZ-007₄ were efficiently expressed as soluble form at the shake flask level,with yields of 151.5,324.4,278.7 and 209.5 mg·L^-1,respectively.（2）The ability of nanobodies to recognize and bind to PSMA-positive prostate cancer cells was verified.It was proved by immunofluorescence and flow cytometry that monovalent nanobody and three kinds of multivalent nanobodies can specifically bind to PSMA-positive cells.Then,the binding EC50 values of nanobodies to PSMA were evaluated by cell-based ELISA.Compared with the binding affinity to PSMA-positive cells of JVZ-007₁,those of multivalent nanobodies JVZ-007₂,JVZ-007₃,JVZ-007₄ were increased 22,16 and 18 times,respectively.（3）The endocytosis ability of monovalent and multivalent nanobodies was evaluated.It was proved by immunofluorescence that monovalent and multivalent nanobodies can be successfully endocytosed by PSMA-positive cells and can be degraded through the lysosomal pathway.Then,the endocytosis half-life and total endocytosis rate of monovalent and multivalent nanobodies were evaluated by flow cytometry.It was proved that multivalent nanobodies have faster endocytosis（half-life<4 minutes）and higher overall endocytosis rate（>80%）.（4）The nanobody-drug conjugate was constructed and its selective cancer killing ability of PSMA-positive cells was evaluated.The bivalent nanobody JVZ-007₂ with the best comprehensive performance was selected as the model multivalent nanobody,and the cytotoxic drug MMAE was modified at the C-terminal of JVZ-007₂ using the enhanced transpeptidase e Srt A.In vitro cytotoxicity experiments showed that the constructed nanobody-drug conjugate JVZ-007₂-VC-PAB-MMAE can specifically kill PSMA-positive prostate cancer cells,with an IC50 value of 88.28 nmol·L^-1,while it has very low toxicity to PSMA-negative cells.