For the in vivo pharmacokinetic study in Figure 6B, data points represent the mean 10E8V2.0/iMab plasma concentrations from seven mice at each time point indicated, and the error bars represent the standard deviation. 118 HIV-1 pseudotyped viruses tested with a mean 50% inhibitory concentration (IC50) of 0.002 g/mL. 10E8V2.0/iMab also potently neutralized 99% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ~50% of new infections worldwide. Importantly, 10E8V2.0/iMab reduced virus load substantially in HIV-1-infected humanized mice, and also provided complete protection when administered prior to virus challenge. These bispecific antibodies hold promise as novel prophylactic and/or therapeutic agents in the fight against HIV-1. Graphical Abstract INTRODUCTION The field of HIV-1 neutralizing antibodies has progressed rapidly in recent years (Mascola and Haynes, 2013). Numerous potent and broad neutralizing mAbs have been isolated from infected humans since 2009 (Blattner et al., 2014; Doria-Rose et al., 2014; Huang et al., 2012; Mouquet et al., 2012a; Rudicell et al., 2014; Scheid et al., 2011; Sok et al., 2014; Walker et al., 2011; Walker et al., 2009; Wu et al., 2010). Impressive anti-HIV-1 activity has been noted with select mAb combinations in vitro (Klein et al., 2012; Kong et al., 2015; Sok et al., 2014) and in vivo (Halper-Stromberg et al., 2014; Klein et al., 2012). Structure-based modifications of antibodies have also resulted in further improvements in anti-HIV-1 activity (Diskin et al., 2011). We (Pace et al., 2013b; Sun et al., 2014) and others (Gardner et al., 2015) have engineered antibodies with dual specificity that showed marked enhancement of virus-neutralization breadth and potency. It should be noted, however, that the aforementioned anti-HIV-1 bispecific antibodies or antibody-like molecules deviate from the normal antibody structure, thereby raising concerns about their potential immunogenicity, unfavorable pharmacokinetic properties, and manufacturing challenges. One approach to construct bispecific antibodies with normal architecture utilizes BF-168 the so-called CrossMAb technology (Schaefer et al., 2011). In this study, we used this technology to generate a library of bispecific antibodies, which were then characterized for their activities against HIV-1. Two bispecific antibodies, 10E8V2.0/iMab and 10E8V1.1/P140, emerged that potently neutralized the majority of circulating HIV-1 strains tested in vitro. 10E8V2.0/iMab was advanced into proof-of-concept in vivo studies, demonstrating potent activity as a single agent in humanized mouse models of HIV-1 treatment and prevention. RESULTS HIV CrossMAbs possess potent and broad antiviral activity against HIV-1 As schematically shown in Fig. 1A, the creation of a knob in one H-chain and a hole in the other H-chain favors the formation of H-chain heterodimers, while the crossover of CL and CH1 sequences in one arm of the antibody favors correct H-L-chain pairings in both arms. Each bispecific antibody was engineered, as shown in Fig. 1A, so that one arm targeted either the human CD4 receptor via the Fab of ibalizumab (iMab) (Burkly et al., 1992; Jacobson et al., 2009; Pace et al., 2013a; Reimann et al., 1997; Song et al., 2010; Toma et al., 2011) or the human CCR5 co-receptor via the Fab of mAb PRO140 (P140) (Tenorio, 2011; Trkola et al., 2001). The other arm targeted one of the five neutralizing epitope clusters on the viral envelope glycoproteins using one of the recently isolated HIV-1-neutralizing mAbs (Blattner et al., 2014; Huang et al., 2012; Scheid et al., 2011; Walker et al., 2011). In this fashion, we created a library of 20 bispecific antibodies, including the specific examples in Fig. 1A. Open in a separate window Figure 1 HIV CrossMAbs possess potent and broad antiviral activity against a diverse panel of 118 Tier-2 HIV-1 Env pseudoviruses(A) Schematic of an HIV CrossMAb and list of examples of parental antibodies from which each CrossMAb was derived. (B) IC50 (top panel) and maximum percent inhibition (MPI, bottom panel) comparison of select HIV CrossMAbs and Rabbit Polyclonal to Histone H2A their parental mAbs. Asterisks refer to data obtained from other sources (3BNC117 MPI data from personal communication with Michel Nussenzweig and PGT151 IC50 and MPI data from Blattner et al., 2014). Error bars indicate median interquartile range. (C) Percent of viruses neutralized (based on IC50 values) by 10E8/P140 and 10E8/iMab, and their BF-168 parental mAbs. Neutralization by penta-mix is included as a reference (Klein et al., 2012). To confirm the dual specificity of the HIV CrossMAb format, we characterized the binding activity of 3BNC117/iMab, for example, by surface plasmon resonance and ELISA. This bispecific CrossMAb bound both soluble human being CD4 BF-168 and HIV-1 gp120 monomer with affinities that are similar to those of the parental antibodies (Fig. S1A and data not shown). Moreover, 3BNC117/iMab successively bound soluble human being CD4 and monomeric gp120 (Fig. S1B), again confirming its dual specificity. Each bispecific antibody in the library, along with its parental mAbs, was then tested for HIV-1 neutralization in vitro against a panel of 118 HIV-1 pseudotyped viruses representing varied clades.