Preparation And Functional Analysis Of Anti-CD3ε Nanobody

CD3 is a cluster of differentiation antigens(CD)on T cell membrane,and forms a CD3-TCR complex with T cell receptor(TCR),which plays an important role in antigen recognition and immune signal transmission.CD3ε is a key component of the CD3 complex,and the binding of CD3ε by antibodies induces significant biological effects on T cells.Thus,CD3ε becomes an attractive therapeutic target for antibody drugs.CD3ε antibodies have been developed for the treatment of autoimmune diseases such as type 1 diabetes and organ transplant rejection.In addition,CD3ε bispecific antibodies are also used for tumor immunotherapy by recruiting T lymphocytes to kill tumor cells.However,conventional anti-CD3ε antibodies,especially single-chain antibodies,have many disadvantages such as large molecular weight,weak stability,difficulty in obtaining,and high preparation cost.Therefore,this research proposes to develop CD3ε nanobodies,which have small molecular weight,high stability,high solubility,and low production cost as new potential drugs for autoimmune diseases and tumor immunotherapy.In this study,a CD3ε nanobody phage library was established after immunizing camel with the extracellular domain of CD3ε.Two candidate nanobodies(CD3ε-Nb1、CD3ε-Nb57)that can recognize the CD3ε antigen were obtained by phage display technology and were identified by routine ELISA.Western Blot was used to identify the specificity of two nanobodies for the detection of target antigen CD3ε.The binding of CD3ε nanobodes to T cell were also investigated by immunofluorescence assay,and the specificity of anti-CD3ε-Nb57 for endogenous antigen recognition was preliminarily identified.In this study,we screened the CD3ε nanobodies and its related research work provide an experimental basis for further studies on the biological functions of CD3εnanobodies and the development of biomedical methods based on CD3ε nanobodies.