Computational-driven epitope verification and affinity maturation of TLR4-targeting antibodies

Bilal Ahmad ¹, Maria Batool^1,2, Moon Suk Kim¹ and Sangdun Choi ^1,2,*

¹Department of Molecular Science and Technology, Ajou University, Suwon, 16499, Korea; bilalpharma77@gmail.com (B.A.); mariabatool.28@gmail.com (M.B.); moonskim@ajou.ac.kr (M.S.K.)

²S&K Therapeutics, Woncheon Hall 135, Ajou University, Suwon, 16499, Korea

*Correspondence: ; Fax: +82-31-219-1615; Tel: +82-31-219-2600

4.3. Epitope prediction and CDR annotations

The RCSB PDB database was used to extract the 3D coordinates of TLR4 (PDB ID: 3FXI) and TLR4-specific monoclonal antibodies (mAbs) Hu 15C1 (PDB ID: 4R7D) and C2E3 (PDB ID: 4R7N).The TLR4–MD2 complex was used to create a monomeric TLR4 structure, which was then optimized in the 2019 molecular operating environment (MOE). After removing the water of crystallization, hydrogen atoms and partial charges were added, incomplete and broken side chains were redesigned, and hybridization events were regulated in MOE at default structure preparation conditions. To predict the conformational epitope, the prepared TLR4 and mAb structure was subjected to Epipred tool of the SAbPred [1,2]web server for the prediction of conformational epitopes. Antigen epitopes are classified according to the combined conformational matching of antibody-antigen structures through geometric fitting and knowledge-based asymmetric antibody-antigen score. An epitope's score is determined by:

where T_ab and T_ag are the amino acid types of the antibody and antigen residues that correspond to node n, respectively. The Ramachandran plot was used to validate the stereochemistry and geometry of chosen epitopes using the MOE's inbuilt functionality. The residues forming the conformational epitope can be visualized in MOE by selecting the b-factor the epitopic residues will be highlighted.

Standardized numbering methods are required to precisely define complementary determining regions (CDR), frameworks, and light- and heavy-chain residues that affect the binding affinity and/or specificity of the antibody-antigen interaction, which is a prerequisite for antibody humanization. The most widely used schemes, which are now universally acknowledged by immunologists, were developed by Kabat and Chothia. The other antibody numbering schemes are Martin [3], Gelfand [4-6], IMGT [7], and Honeger (Aho) [8]. Chothia and Lesk's [9,10] numbering technique was used to annotate CDRs of antibodies acquired from the PDB database. CDRs of mAbs were used as ligand sites in docking and simulations instead of the whole Fab region.

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