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Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes.

ABSTRACT: Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.

SUBMITTER: Strokappe NM

PROVIDER: S-EPMC6640723 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes.

Strokappe Nika M NM Hock Miriam M Rutten Lucy L Mccoy Laura E LE Back Jaap W JW Caillat Christophe C Haffke Matthias M Weiss Robin A RA Weissenhorn Winfried W Verrips Theo T

Antibodies (Basel, Switzerland) 20190618 2

Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown ...[more]

PMID: 31544844

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Project description:UnlabelledThe potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 μg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans.ImportanceTo prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different antibody binding arms could potentially display neutralization characteristics better than those of any single parental antibody. Here we show that bispecific antibodies contain the binding specificities of the two parental antibodies and that a single bispecific antibody can neutralize 97% of viral strains with a high overall potency. These findings support the use of bispecific antibodies for the prevention or treatment of HIV-1 infection.

Project description:BackgroundThe discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%-30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003).MethodsTo determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further.ResultsPM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo.ConclusionsA unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

Dataset Information

Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes.

Publications

Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes.

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