CD3-epsilon/delta heterodimer recombinant protein induced diverse antibodies binding to Jurkat cells and primary T cells in mice 2260655

Abstract Introduction The T cell receptor (TCR)/CD3 complex is assembled by pairwise interactions involving the formation of dimers of CD3 epsilon with either CD3 delta or CD3 gamma. CD3-epsilon/delta (CD3ε/γ) and CD3-epsilon/gamma (CD3ε/γ) complexes are essential for T-cell activation and signaling. These molecules are key targets for biotherapeutic drugs such as T-cell engagers and bispecific antibodies, to treat cancers by linking T cells to tumor cells for targeted killing. To mimic the dimeric conformation of CD3ε/γ, we engineered a novel human CD3ε/γ cis-heterodimer recombinant protein. Methods To engineer the CD3ε/γ heterodimer, the ECDs of CD3 epsilon and delta subunits were fused with a heterodimer motif at the C-terminus of each chain. The recombinant CD3ε/γ heterodimer protein was expressed/purified from HEK293 cells. The CD3ε/γ protein were characterized by binding of CD3 specific antibodies. To evaluate the immunogenicity, mice were immunized with CD3ε/γ protein. The CD3ε/γ specific antibody titers in mice were measured by ELISA and antibody binding to Jurkat cells and primary T cells were measured by flow cytometry. Results The recombinant CD3ε/γ heterodimer protein could potently bind to the CD3-specific antibodies including OKT3 and UCHT1. The immunogenicity study in mice demonstrated that the CD3ε/γ heterodimer protein induced high titers of CD3-specific antibody responses. More importantly, the antibodies elicited by CD3ε/γ heterodimer protein demonstrated very good binding abilities to the T cell line Jurkat cells and primary human T cells as evaluated by flow cytometry. Conclusion The engineered CD3ε/γ cis-heterodimer protein could mimic the native conformation for in vitro antibody assessment. It can generate high levels of CD3-specific antibodies binding to T cell lines and primary T cells by immunization. This novel CD3ε/γ cis-heterodimer recombinant protein can be a very useful tool for basic immunology research and drug discovery. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)