Targeting the HIV-1 Spike and Coreceptor with Bi- and Trispecific Antibodies for Single-Component Broad Inhibition of Entry

Abstract

Protection against acquiring human immunodeficiency virus (HIV) infection may not require a vaccine in the conventional sense, because broadly neutralizing antibodies (bNAbs) alone prevent HIV infection in relevant animal challenge models. Additionally, bNAbs as therapeutics can effectively suppress HIV replication in infected humans and in animal models. Combinations of bNAbs are generally even more effective, and bNAb-derived multivalent antibody-like molecules also inhibit HIV replication both in vitro and in vivo To expand the available array of multispecific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAbs that recognize HIV Env exclusively, a bispecific CrossMAb targeting two epitopes on the major HIV coreceptor, CCR5, and bi- and trispecifics that cross-target both Env and CCR5. These newly designed molecules displayed exceptional breadth, neutralizing 98 to 100% of a 109-virus panel, as well as additivity and potency compared to those of the individual parental control IgGs. The bispecific molecules, designed as tandem single-chain variable fragments (scFvs) (10E8fv-N6fv and m36.4-PRO 140fv), displayed median 50% inhibitory concentration (IC₅₀s) of 0.0685 and 0.0131 μg/ml, respectively. A trispecific containing 10E8-PGT121-PGDM1400 Env-specific binding sites was equally potent (median IC₅₀ of 0.0135 μg/ml), while a trispecific molecule targeting Env and CCR5 simultaneously (10E8Fab-PGDM1400fv-PRO 140fv) demonstrated even greater potency, with a median IC₅₀ of 0.007 μg/ml. By design, some of these molecules lacked Fc-mediated effector function; therefore, we also constructed a trispecific prototype possessing reconstituted CH2-CH3 domains to restore Fc receptor binding capacity. The molecules developed here, along with those described previously, possess promise as prophylactic and therapeutic agents against HIV.IMPORTANCE Broadly neutralizing antibodies (bNAbs) prevent HIV infection in monkey challenge models and suppress HIV replication in infected humans. Combinations of bNAbs are more effective at suppression, and antibody-like molecules engineered to have two or three bNAb combining sites also inhibit HIV replication in monkeys and other animal models. To expand the available array of multispecific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAb binding sites that recognize the HIV envelope glycoprotein (Env) or the HIV coreceptor (CCR5) or that cross-target both Env and CCR5. Several of the bi- and trispecific molecules neutralized most viruses in a diverse cross-clade panel, with greater breadth and potency than those of the individual parental bNAbs. The molecules described here provide additional options for preventing or suppressing HIV infection.

Keywords: CrossMAb; HIV envelope; HIV-1; anti-CCR5; bispecific antibody; trispecific antibody.

FIG 1

Epitopes on HIV Env targeted by selected bNAbs. The Env trimer is shown as a space-filling model, and the epitopes targeted are highlighted clockwise from bottom left, as follows: the MPER epitope (10E8; sea green), the CD4bs (N6; light green), the V2 apex (PGDM1400; blue, with the N160 glycan highlighted), and the N332 glycan, with the V3 GDIR residues shown in dark pink (PGT121). The binding sites are enlarged in the flanking black boxes. Approximate distances between these epitopes are as follows: 10E8 to N6, 94 Å; N6 to PGDM1400, 33 Å; and PGDM1400 to PGT121, 54 Å.

FIG 2

Design and neutralization profile of the bispecific molecule 10E8fv-N6fv. (Top) The design of the tandem scFv molecule is shown as a linear box schematic along with a cartoon depiction of the bispecific. (Bottom) Neutralization potencies (IC₅₀s, in micrograms per milliliter) in the TZM-bl cell in vitro neutralization assay. In this and subsequent figures, “>40” indicates that the IC₅₀ was not detectable at the highest concentration used, 40 μg/ml. ND, not determined.

FIG 3

Design and neutralization profile of trispecific 10E8Fab-PGT121fv-PGDM1400fv variants. (A) Schematic representation of the trispecific design. Chains A and B (top) were combined to form the trispecific molecule (middle). scFv, single-chain variable fragment; VH, heavy chain variable region; VL, light chain variable region; IL-2 SS, interleukin-2 signal sequence; L, linker. The various orientations of the scFvs are shown at the bottom (H1 to H4 and L1 to L4), with different combinations used to make the eight variants tested (e.g., H4 + L1 was used to form the V8 trispecific). (B) Binding of 10E8Fab-PGT121fv-PGDM1400fv.V8 to NFL trimers compared to that of the parental IgGs by ELISA. (C) Neutralization profiles of the eight 10E8Fab-PGT121fv-PGDM1400fv variants, the individual parental IgGs (10E8, PGT121, and PGDM1400), and the three parental IgGs in combination against a 28-virus panel initially selected based on susceptibility or resistance to the parental IgGs. The three parental IgGs were combined at a concentration of 0.33 μg/ml each (total concentration, 1 μg/ml). IC₅₀ values (in micrograms per milliliter) are shown.

FIG 4

Neutralization profile of trispecific variant 10E8Fab-PGT121fv-PGDM1400fv.V8 against a 109-virus panel. Neutralization potencies of 10E8Fab-PGT121fv-PGDM1400fv.V8 (IC₅₀s, in micrograms per milliliter) in the TZM-bl cell neutralization assay are given. ND, not determined. Parental IgGs that formed the basis for the trispecific were also tested, and their IC₅₀s are shown.

FIG 5

Design and neutralization profiles of trispecific V8 variants. Neutralization profiles of the following trispecific 10E8Fab-PGT121fv-PGDM1400fv.V8 variants are given, with cartoon schematics shown above: V8Fc (Fc region incorporated for binding to FcRn) (A), V8LL (longer linker between the Fab and scFvs) (B), and V8.4DS (improved solubility) (C). IC₅₀ values (in micrograms per milliliter) are indicated. ND, not determined. (D) Binding of the V8LL and V8.4DS variants to NFL trimers compared to that of the parental trispecific 10E8Fab-PGT121fv-PGDM1400fv.V8 and PGDM1400 IgG by ELISA.

FIG 6

cMAb design and binding characteristics. (A) Crystal structure of CCR5 (PDB entry 4MBS), with the extracellular loop 2 (ECL2) region shown in red and the N-terminal (Nt) region (PDB entry 2L87) superimposed in blue. The orange lines represent the top and bottom layers of the plasma membrane, with the transmembrane region in between. The top view is shown on the right. (B) Cartoon representation of the cMAb, including the T366W change in the RoAb13 CH3 domain to give the knob (red) and the modification of three residues (T366S/L368A/Y407V) to create the hole (blue) in the CH3 domain of the other corresponding heavy chain on PRO 140. The key or knob-in-hole (KIH) design was used to ensure that the two different heavy chains would pair up. The constant domain of the light chain (CL) (gray) and constant domain 1 of the heavy chain (CH1) (black) on the PRO 140 side of the IgG molecule were swapped to ensure correct association of the light chains. The KIH design and the CL/CH1 swap on one of the IgG arms ensure that the IgG molecules are in the bispecific cMAb format, eliminating the undesirable monospecific IgG formats. On the right is an SDS-PAGE gel showing PRO 140 IgG (lanes 1), RoAb13 (IgG) (lanes 2), and the cMAb (lanes 3), run under nonreducing (NR) and reducing (R) conditions. (C) Human (PRO 140, RoAb13, and cMAb) and mouse (CTC8 and 45523) anti-CCR5 antibodies are represented as cartoons. PRO 140 and 45523 are ECL2 specific, while RoAb13 and CTC8 are Nt-specific antibodies. The cMAb is dually specific, recognizing both Nt and ECL2 epitopes. (D) Table summarizing the cross-competition results between the human antibodies (PRO 140, RoAb13, and cMAb) and the mouse antibodies (CTC8 and 45523) to confirm the binding specificity of the cMAb. MFI, mean fluorescence intensity; APC, allophycocyanin; FITC, fluorescein isothiocyanate.

FIG 7

Neutralization breadth of the RoAb13-PRO 140 cMAb. Cartoons of RoAb13, PRO 140, and the RoAb13-PRO 140 cMAb are shown at the top, with their neutralization potencies (IC₅₀s, in micrograms per milliliter) given below.

FIG 8

Neutralization by Env-CCR5 cross-targeting molecules. Neutralization profiles are given for the Env-CCR5-targeting molecules without (−) or with (+) TPST-1 coexpression for designs incorporating PGDM1400. IC₅₀ values (in micrograms per milliliter) are provided. ND, not determined.

FIG 8

Neutralization by Env-CCR5 cross-targeting molecules. Neutralization profiles are given for the Env-CCR5-targeting molecules without (−) or with (+) TPST-1 coexpression for designs incorporating PGDM1400. IC₅₀ values (in micrograms per milliliter) are provided. ND, not determined.