Identification Of CD47 Monoclonal Antibody And Study On The Structural Biology Of Its Complex

Immunotherapy is a novel method of cancer therapy following surgical resection,radiotherapy,and chemotherapy,which refers to the recognition and killing of tumor cells by activating the autoimmune system.As an immune regulatory signal,immune checkpoints participate in maintaining the balance of the human immune system and play an important role in suppressing the excessive activation of immunity.As a result,immune checkpoints as an essential role by inhibiting the activity of tumor infiltrating immune cells in the process of tumor growth and development.Current research is mainly focus on activating T lymphocytes in tumors to treat cancer through targeting immune checkpoints,such as CTLA-4 and PD-1,PD-L1,etc.As an important part of the innate immune system,macrophages play a vital role in the anti-tumor response.Under normal circumstances,CD47 forms a signal complex with SIRPαon the surface of macrophages and transmits signals to inhibit the excessive activation of macrophages.Most of cancer cells express CD47 to inhibit tumor phagocytosis mediated by macrophages and maintain the growth and progression of tumors.Anti-CD47 therapy has become a new type of tumor treatment attribute to increasing of the phagocytosis of tumor cells by macrophages.At the same time,targeting CD47 also promote the anti-tumor T cell immune response,which has gradually become a hot target for cancer treatment.Recently,CD47 has come to fore of immunotherapy research,many CD47antibodies have been developed.In order to further to understand the interaction mechanism of CD47 and its antibody,the current study verified the effectiveness of CD47 antibody TDC-4,and analyzed the crystal structure of its complex with CD47.We obtained the mammalian expression vector expressing the CD47 antibody TDC-4 from the group of Professor Lin Jizhen of Fujian Medical University and constructed the vector expressing the specific sequence of CD47（aa 19-135）.We isolated and purified the human CD47（aa 19-135）protein fragment and the TDC-4 antibody upon transfection into Expi293F^TM cells.We studied the effects of multiple glycosylation sites in the extracellular region of CD47 protein on the antibody binding affinity.The purified CD47（aa19-135）and TDC-4 were used to prepare the complex of CD47 and TDC-4 by molecular sieve.Through multiple exploration and optimization of crystal growth conditions,a crystal with good growth potential was obtained and we did the analysis of its composite structure.We also used ELISA,flow cytometry to identify the affinity of the antibody,and verify the cytotoxic effect of the antibody on lung cancer cell lines.In summary,we accomplished the verifying of the affinity between the CD47antigen and antibody,and the efficacy evaluation and toxicity verification of the antibody.Subsequently,we analyzed the crystal structure of the CD47 antigen-antibody complex for exploring and discussing the interaction sites and modes of action.These experimental results provide a scientific basis for exploring the role of TDC-4monoclonal antibody in tumor therapy and combination therapy.