Design of an Avelumab Nanobody and Investigation of Its Binding to the PD-L۱ Antigen

PD-L۱ is expressed on a variety of normal cells, including epithelial and antigen-presenting cells (APCs). PD-L۱'s natural ligand is PD-۱, which is expressed on the surface of T and B lymphocytes and myeloid cells. After PD-۱/PD-L۱ binding, the immune-related cell receives a negative modulation signal that prevents activation, maintaining systemic self-tolerance. Cancer cells overexpress PD-L۱ molecules on their membranes, leading to T-cell depletion and immune escape. Therefore, PD-L۱ promotes the proliferation and survival of cancer cells by binding to its receptor. Currently, avelumab, an anti-PD-L۱ monoclonal antibody, has been approved by the FDA. Avelumab blocks the PD-۱/PD-L۱ binding. This blockade reinvigorates T cells and enhances the cytotoxic activity of T cells against tumor cells, shifting the immune system from a passive to an active anti-tumor state. Nanobodies consist only of the heavy chain, and they are more effective than antibodies due to having several advantages. CDR grafting is a novel method used in this project to design nanobodies. We used this method to replace the CDRs of avelumab monoclonal antibody with those of caplacizumab and utilized the framework region (FWR) of caplacizumab, which is a VHH approved by the FDA. Method: In this study, we utilized the SabDab server to determine the CDR sequence of the antibody. Then, the CDRs of avelumab were integrated into the VHH structure of caplacizumab, and a nanobody sequence was designed. The Swiss Model server was employed to model the three-dimensional structure of the nanobody. The ۳D structure of the PD-L۱ antigen was retrieved from the PDB server with the code ۳BIS. The HADDOCK ۲.۴ server was used to perform molecular docking, and the best docking result was selected based on the scores obtained from HADDOCK. The type of CDR amino acids is very important in binding antigens to antibodies, and through targeted mutagenesis, amino acids can be replaced to strengthen the binding. Therefore, the PROT-ON server was used to perform targeted mutagenesis. The mutagenesis process was carried out using PyMOL software. Furthermore, to predict the binding affinity between the nanobody and the antigen, and to calculate the binding free energy (ΔG), the PRODIGY and I-Mutant servers were used. Results: The PROT-ON report states that replacing arginine at position ۳۲ with serine results in an increase in the number of bonds between the antigen and the nanobody. The results from Prodigy showed a ΔG of -۹.۲ kcal/mol and a Kd of ۱.۹e-۰۷. The I-Mutant ۲.۰ indicated that the stability of the nanobody's three-dimensional structure increased after mutagenesis. Conclusion: Effective binding between antigens and therapeutic nanobodies is essential for optimal functionality. One approach to enhance this binding is through site-directed mutagenesis, which allows for targeted modifications. In this study, we used site-directed mutagenesis to increase the binding strength between the Avelumab nanobody and the PD-L۱ antigen.

کلیدواژه ها:

Avelumab ، molecular docking ، PROT-ON server ، Nanobody ، PD-L۱