Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques

Abstract

The development of an effective AIDS vaccine has been challenging because of viral genetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs). We engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to those of human bnAbs, and conferred complete immunity against a mixture of simian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and they may be applicable for treatment of diverse diseases, including infections, cancer, and autoimmunity.

Fig. 1. CODV-Ig bispecific antibody design and neutralization titers of the VRC01/10E8 bispecific antibodies

(A) CODV-Ig bispecific antibody design with two different orientations of 10E8 and VRC01. (B) Neutralization titers (IC₅₀) in µg/ml of VRC01/10E8 bispecific Abs and parental Abs against a select panel of 19 previously circulating HIV-1 strains highlighted in red, yellow and grey indicating highest, medium and lowest potency respectively.

Fig. 2. Neutralization titers of VRC01/PGT121 and VRC01/PGT128 based bispecific antibodies

Neutralization titers (IC₅₀) in µg/ml of the VRC01/PGT121 (A) and VRC01/PGT128 (B) bispecific Abs against a select panel of 20 circulating HIV-1 strains, with highlights as in Fig.1.

Fig. 3. Neutralization titers of trispecific antibodies and broadly neutralizing antibodies, and sequential binding of alternative Env epitopes

(A) The neutralization titers (IC₅₀) of different bnAbs and trispecific Abs against a genetically diverse panel of 208 circulating HIV-1 strains. The solid line denotes the median IC₅₀ neutralization titer of sensitive viruses while the dotted line indicates median titers of all 208 viral strains. The percentage of resistant viruses are shown in the top line. (B) The breadth and potency of the trispecific Abs compared to other bnAbs. (C) Sequential binding of three antigens to the trispecific Ab, VRC01/PGDM1400-10E8v4 in the indicated order. The RSC3 (44) antigen represents monomeric gp120 optimized for the CD4 binding site ab VRC01. MPER peptide interacts with 10E8 (7), and gp140 trimer for PGDM1400 was derived from the gp140ΔN6 (BG505) protein.

Fig. 4. Serum antibody levels in rhesus macaques infused with parental and trispecific antibodies

The concentration of VRC01, N6 and the two trispecific Abs containing a Fc mutation to extend half-life, were measured in serum over the course of 14 days after intravenous administration of a single 10 mg/kg dose of each antibody. Each data point represents the mean +/− SEM of the values from 2–6 animals per group (VRC01, n=6; N6, n=4; each trispecific Ab, n=2) and determined in replicates from two independent experiments.

Fig. 5. Crystal structure of the CODV Fab and a structure model of the trispecific antibody

(A) Configuration of the trispecific antibody, color-coded by parental antibody. Dark shades (red or green) refer to heavy chain while pastels indicate light chain peptides. (B) Crystal structure of the PGDM1400-10E8v4 CODV Fab in side and top views. CDRH3s from the two Fvs are labeled to highlight the antigen binding region gp41 MPER was modeled in by superposing PDB 5IQ9 on to the 10E8v4 Fv. (C) VRC01/gp120 structure (PDB 4LST) and the CODV Fab were modeled onto the b12 structure (PDB 1HZH) by overlaying the CH1-CL domains. Color codes are matched in A, B, and C.

Fig. 6. Trispecific and broad neutralizing antibody sensitivity of SHIVs, plasma antibody levels and viremia in rhesus macaques

(A) The IC50 neutralizing titers (ug/ml) of VRC01, PGDM1400, and VRC01/10E8v4-PGDM1400 against replication competent SHIV BaLP4 or SHIV 325c. (B) Plasma levels of VRC01, PGDM1400 and VRC01/PGDM1400-10E8v4 in rhesus macaques (n=8 on each arm, done in two separate experiments with 4 animals each). All animals were administered 5 mg/kg of the indicated antibody intravenously. Each data point represents the mean +/− SEM of the values from all 8 animals per group. (C) Plasma viral loads in rhesus macaques (n=8 per group) challenged with a mixture of SHIV BaLP4 and SHIV 325c, 5 days after intravenous administration of either VRC01, PGDM1400 or VRC01/PGDM1400-10E8v4.