Furthermore, super-resolution microscopy studies of intact virions have revealed that this affinity of these mutated antibodies for native Env increased (Carravilla et al., 2019). site-selective chemical modification to optimize the function of antibodies that target membrane-proximal epitopes. == Graphical Abstract == == In Brief == Rujas et al. describe the site-selective chemical modification of antibodies to improve the molecular recognition of epitopes at LX-1031 membrane surfaces. The modification using aromatic compounds dramatically enhanced the virus neutralization potency and native antigen binding efficiency of HIV-1 antibodies directed against the membrane-embedded MPER epitope. == INTRODUCTION == Chemical modification of proteins is a method widely used to engineer proteins and to elucidate their function in the cell (Isenegger and Davis, 2019;Krall et al., 2016;Sakamoto and Hamachi, 2019). In antibodies (Abs), chemical modifications are generally introduced to link the protein to a second molecule to generate functionality, such as in Ab-drug conjugates, or to label the protein for analytical purposes. In addition, site-selective chemical modification provides a potential route to optimize Ab function beyond the limits LX-1031 imposed by the collection of natural amino acids (Isenegger and Davis, 2019;Krall et al., 2016;Sakamoto and Hamachi, 2019). Here, we sought to improve Ab recognition of integral membrane antigens by site-specific chemical conjugation of synthetic aromatic compounds. Integral membrane proteins represent one of the largest fraction of antibody-based therapeutic targets under clinical evaluation, including tumor-associated antigens such as the tetraspanin CD20, or members of several receptor families such as the human epidermal growth factor receptor tyrosine kinase and the tumor necrosis factor-related apoptosis-inducing ligand receptors (Hendriks et al., 2017), diverse families of ion-channels (Hutchings et al., 2019), G-protein-coupled receptors (Hutchings et al., 2017), and viral glycoproteins from relevant human pathogens, such as the Ebola virus or the human immunodeficiency virus type-1 (HIV-1) (Walker and Burton, 2018). Many Abs targeting these integral membrane-antigens reportedly bind to membrane-proximal regions, i.e., epitopes that are uncovered close to, or lying around the membrane surface (Flyak et al., 2018;Hutchings et al., 2017;Klein et al., 2013;Lee et al., 2020;Pahuja et al., 2018;Xu et al., 2005). The membrane-proximal epitope MPER, existing in the HIV-1 envelope glycoprotein Env, epitomizes this class of antigen determinant (Huang et al., 2012;Krebs et al., 2019;Pinto et al., 2019;Rantalainen et al., 2020;Stiegler et al., 2001;Williams et al., 2017;Zhang et al., 2019). One Ab that binds to this region, 10E8, has been extensively studied both structurally and functionally (Huang et al., 2012;Irimia et al., 2017;Lee et al., 2016;Rantalainen et al., 2020;Rujas et al., 2016) and thus is a relevant candidate for rational optimization by protein engineering. Effective binding of 10E8 to Env translates into viral neutralization, hence, higher affinity of this Ab for the antigen would result in greater capacity to block cell contamination (Carravilla et al., 2019). The evidence accumulated so far suggests that engagement of Env by 10E8 requires not only the recognition of the proper protein epitope, but also additional contacts to HDAC3 adjust the Ab surface to the viral membrane interface (Irimia et al., 2016,2017;Lee et al., 2016;Rantalainen et al., 2020;Rujas et al., 2016). Recent studies reported by us and LX-1031 others have demonstrated that this neutralization potency of this Ab can be improved 10-fold by mutating residues located at the contact interface with the viral membrane (Kwon et al., 2018;Rujas et al., 2018). Furthermore, super-resolution microscopy studies of intact virions have revealed that this affinity of these mutated antibodies for native Env increased (Carravilla et al., 2019). In other words, optimal accommodation of the viral membrane onto the Ab surface is a critical aspect for efficient viral neutralization. Here, we have examined the hypothesis that 10E8-like Abs can be rationally optimized by grafting synthetic aromatic compounds at sites that, while remote from the epitope-binding site, may facilitate its conversation with the viral membrane. The antibody was specifically modified with iodoacetamide derivatives at specific places predicted to improve the conversation of 10E8 with the viral membrane. As a result, the biological function of the modified antibody increased dramatically using various benchmark methodologies and biological assays. Following the same strategy, mutants of 10E8 of reduced efficacy and a less potent Ab arising from a different lineage were also modified achieving much greater potency. Collectively, our data provide a proof-of-principle to support site-selective conjugation with aromatic compounds as a rational approach to improve Ab recognition of epitopes that are located at membrane interfaces. == RESULTS == == Strategy to Optimize Antibody 10E8 by Chemical Modification with Aromatic Compounds == Our rational approach seeks the promotion of interfacial affinity of the surface of the anti-HIV Ab 10E8 that comes into contact with.