Antibody engineering for increased potency, breadth and half-life

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

An early example of a bispecific anti-HIV-1 reagent fused the gp120-binding domains of CD4 to an scFv version of a CD4i antibody. CD4i antibodies recognize the conserved coreceptor-binding site on gp120 after it is exposed as a result of CD4 binding [101] . As IgGs, CD4i antibodies exhibit limited neutralization potencies because of steric constraints when gp120 is bound to CD4 on the target cell [7] . The broadly neutralizing reagent sCD4-17b was created by fusing CD4 to the variable regions of a CD4i antibody, 17b, thereby solving the steric problem because the epitope was exposed by CD4 binding when the virion was not bound to the target cell (Fig. 2c) [101] . The CD4-17b reagent was recently expressed in a chimeric antigen receptor (CAR) format on T cells to target and kill HIV-1infected cells [102 & ]. Later versions of CD4-CD4i reagents used an IgG architecture by N-terminally fusing CD4 to the Fabs of CD4i antibodies [103] . Despite containing only two CD4 moieties, the IgGbased CD4-CD4i reagents showed increased potencies compared with CD4-IgG2 (PRO542), a tetravalent CD4-Fc fusion protein [104] . A more recent reagent of this class combined a CD4 variant with m36, a CD4i single variable domain selected against different HIV-1 envelope proteins (Fig. 2d ) 106] . Bispecific IgGs containing variable regions from two different IgGs have been produced using the crossMab platform in which heavy chain heterodimerization occurs using a 'knob-into-hole' Fc architecture and V H and V L domains are swapped in one half of the reagent to ensure proper heavy chainlight chain pairing [107] (Fig. 2e) . Other architectures, such as dual variable domain constructs, attach a second Ig variable domain N-terminal to an IgG (Fig. 2f) . A promising reagent of this class was created from bNAbs against gp120 and gp41 [108] . Another potent chimeric reagent was constructed as an Fc fusion with CD4-and coreceptor-mimetic peptides attached to the Fc, thus targeting both the receptor and coreceptor sites on HIV-1 Env (Fig. 2g) [109 & ]. Bispecific reagents generally function by binding of one or the other Env-targeting component at a time. Simultaneous binding of both components of bispecific reagents to single Env trimers was recently shown to result in synergistic in vitro neutralization improvements of two to three orders of magnitude [13 & ], suggesting a promising strategy for achieving potency, breadth and resistance to viral mutations. Synergistic neutralization resulting in extremely high in vitro potencies was also observed for reagents combining anti-Env and antihost specificities (see Ho and Markowitz, this issue). For example, attaching the scFv of PG9 or PG16 to the N-terminus of ibalizumab (anti-CD4) yielded synergistic reagents, PG9-iMab and PG16-iMab, of exceptional potency that neutralized 100% of a 118-virus panel, with the majority of IC50s less than 0.01 mg/ml (Fig. 2h) [110 && ]. Another broad and potent bispecific reagent, iMabm36, joined m36 with ibalizumab [111] . This reagent neutralized 96% of a 118-member multiclade pseudovirus panel at a concentration of 10 mg/ml.