Antibody engineering for increased potency, breadth and half-life

Sievers, Stuart A.; Scharf, Louise; West, Anthony P.; Bjorkman, Pamela J.

A similar effort to improve a bNAb is represented by development of a more potent variant of a VRC01-class antibody that was isolated from the VRC01/NIH45-46 donor [54 && ]. First, 454 pyrosequencing of B cell antibody gene transcripts allowed identification of a heavy chain clonal variant that was closely related to NIH45-46 and contained the same four-residue CDRH3 insert. When combined with the VRC01 light chain, the new bNAb, VRC07, was about two-fold more potent than VRC01. Although not antibody engineering per se, this result illustrates that deeper searching of bNAb donor repertoires can identify more potent clonal relatives; other examples include variants of PGT121 [47, 57, 58] , PGT141 [59] and 8ANC195 [26] . Next, all 20 amino acids were evaluated at VRC07 heavy chain position 54. Similar to results for NIH45-46 Gly54 HC mutants [55] , substitution of VRC07 Gly54 to larger residues, including Trp, Tyr, Phe and His, increased neutralization potency and breadth, but substitutions for large aromatic residues resulted in polyreactive recognition of non-HIV-1 antigens [54 && ]. A Gly54His substitution showed the least increase in polyreactivity while still increasing potency. The antibody light chain was also shortened to address observations that the V5 loop of gp120 interferes with the binding of VRC01-like antibodies [60] . On the basis of results from a screen of light chain truncations and modifications, a tworesidue N-terminal truncation as well as a Val3Ser LC mutation was selected [54 && ]. For in vivo experiments, mutations in the Fc region to improve plasma half-life through enhanced binding to FcRn (M428L/N434S, the 'LS' mutant [61] ) were introduced. Additional framework mutation reversions and solubilityenhancing mutations were investigated, but the antibody selected after in vivo half-life determinations, VRC07-523-LS, did not contain these mutations. VRC07-523 and VRC07-523-LS were five-to eight-fold more potent than VRC01 and neutralized 96% of tested HIV-1 strains in vitro. Viral challenge experiments in macaques verified that the increased in vitro potency correlated with improved protection from infection in vivo [54 && ]. Taken together, the NIH45- 46 and VRC07 examples illustrate the potential to improve the activity of VRC01-class bNAbs by filling the hydrophobic Phe43 CD4 pocket on gp120.