| CTLA4/CD80 signaling is a vital pathway for retaining the homeostasis of immune system.The T cell receptor CTLA4 could competitively bind to CD80 on antigen presenting cells(APC)with CD28 due to its higher binding affinity,and transduce negative signaling that inhibit T cell activation.Several therapeutic agents targeting the CTLA4/CD80 signaling have been approved by FDA.They can be classified as CTLA4-Fc fusion proteins and anti-CTLA4 monoclonal antibodies.CTLA4-Fc fusion proteins bind to CD80 with high specificity and can act as CD80 antagonists by blocking the costimulatory molecule CD28 bind to CD80,thus inhibit T cell activation and avoid immune system over-activity.While anti-CTLA4 antibody can bind to CTLA4 and cause steric competition of CTLA4 interact with CD80,thus release the brake on T cell response and result in tumor killing.So far,the clinical blockades of CTLA4/CD80 signal pathway are mainly biological macromolecules,while few small molecules is reported.Given the shortcomings of macromolecular antibodies,such as expressed in eukaryotic system with high cost and poor tumor penetrating ability,researches on small molecular inhibitors showed potential prospect and high demands.Recombinant proteins derived from the tenth unit of human type Ⅲfibronectin(FN3)termed monobody were studied as CTLA4 analogs in this study.FN3 is a ubiquitous protein expressed in human body,whose structure highly resembles the variable domain of antibody.It is composed of 94 amino acids only.In this study,FN3 was used as a scaffold to display the critical domains which contribute to the CD80 binding,generating small molecular CTLA4 analogs.To select the grafted domains on CTLA4,a peptide EL 16 derived from CD80-binding domain(MYPPPY motif)in the complementarity determining region(CDR)3 of CTLA4 was synthesized and found to inhibit the interaction of CTLA4 with CD80 significantly.Afterward,the peptide EL 16 as well as the CDR1 of CTLA4 which is also critical for its binding to CD80 were grafted onto FN3 and obtained a novel CD80 binding monobody protein CFN13.CFN13 showed 80%binding affinity compared to CTLA4.In addition,to increase the half-life,CFN13 was fused to human IgG1 Fc to generate CFN13-Fc fusion protein.As expected,CFN13-Fc bound to CD80 in a dosage-dependent manner as CFN13 did,and displayed 41.0%and 31.4%inhibition on the interaction of CTLA4-Fc with CD80 at 200 μg/ml and 100 μg/ml respectively.Moreover,peptide EL 16 could inhibit CFN13-Fc binding to CD80 significantly,with the inhibition ratio of 64.3%and 52.8%at 100 and 50 μg/ml respectively,indicating that the peptide EL16 and CFN13-Fc shared the similar binding sites with CD80 and the CDR3 motif of CTLA4 contributed more than CDR1 in binding to CD80.The 3D structures of peptide EL16 and recombinant protein CFN13 were constructed by SWISS-MODEL.The binding modes of EL16/CD80 and CFN13/CD80 were simulated by ZDOCK.We hypothesized that the shared‘MYPPPY’ motifs of both peptide EL16 and CFN13 made hydrophobic contacts with the nonpolar surface of CD80 and contributed most to their binding with CD80.In summary,our study provides insights into small molecular analogs of CTLA4. |
