Introduction: The angiotensin-converting enzyme 2 (ACE2) is the effective primary receptor for SARS-CoV-2. The interaction between ACE2 and the spike protein of the virus is the crucial step for virus entry into the target cells. ACE2 receptor can be blocked by neutralizing antibodies (nAbs) such as CR3022 which targets the virus receptor-Binding site. Enhancing the Binding affinity between CR3022 and ACE2 would lead to a more efficient blockade of virus entry. Methods: In this regard, the amino acids with central roles in the Binding affinity of CR3022 antibody to spike protein were substituted. The best mutations to increase the affinity of antibodies were also selected based on protein-protein docking and molecular dynamics simulations. Result: The variants 45 (H: 30I/G, H: 55D/F, H: 103S/Y, L: 59T/F, L: 98Y/A), 60(H: 31T/D, H: 55D/E, H: 103S/Y, L: 59T/D, L: 98Y/F), 67(H: 30I/G, H: 55D/F, H: 103S/Y, L: 56 W/L, L: 59T/Y, L: 61E/G), 69(H: 31T/D, H: 55D/F, H: 103S/Y, L: 59T/F, L: 98Y/A), and 71(H: 31T/D, H: 55D/F, H: 103S/Y) with respective Binding affinities of-167. 3,-167. 5,-161. 6,-173. 0, and-169. 8 Kcal/mol had higher Binding affinities against the RBD of the SARS-CoV2 spike protein compared to the wild-type Ab. Conclusion: The engineered antibodies with higher Binding affinities against the target protein can improve specificity and sensitivity. Thus, a more successful blockade of the ACE2 is achieved, resulting in a better therapeutic outcome. In silico studies can pave the way for designing these engineered molecules avoiding the economic and ethical challenges.

Background: TIM-3 is an inhibitory receptor expressed in a variety of cells, including dendritic cells, T-helper 1 lymphocytes, and natural killer cells. Binding of this protein to its ligand, CEACAM1, causes T-cell exhaustion, a specific condition in which effector T cells lose their ability to proliferate and produce cytokines. Blocking this inhibitory receptor is known to be an effective strategy for treating cancer and other related diseases. Therefore, in this study, in order to block the inhibitory receptor of TIM-3, we designed and produced recombinantly a protein with a high Binding affinity to this receptor. Methods: The extracellular domain of CEACAM1 involved in Binding to TIM-3 was mutated using R script to obtain a variant with the increased Binding affinity to TIM-3. The Binding energy of the mutant protein was calculated using the FoldX module. Finally, after recombinant production of the most appropriate CEACAM1 variant (variant 39) in E. coli, its secondary structure was determined by CD spectroscopy. Results: The Binding free energy between variant 39 and TIM-3 decreased from-5. 63 to-14. 49 kcal/mol, indicating an increased Binding affinity to the receptor. Analysis of the secondary structure of this variant also showed that the mutation did not significantly alter the structure of the protein. Conclusion: Our findings suggest that variant 39 could bind to TIM-3 with a higher Binding affinity than the wild-type, making it a proper therapeutic candidate for blocking TIM-3.