Engineering bispecific antibodies targeting HIV-1 subtype C

Abstract

The introduction of antiretroviral therapy has revolutionised the management of HIV-1 infection, effectively reducing morbidity and restoring life expectancy of HIV-1 patients. Continual improvements into the tolerability, increasing the genetic resistance barrier and reduction in antiretroviral drug dosing frequency have improved patient adherence and outcomes, but nonetheless require life-long therapy. By contrast, the development of an effective HIV-1 prophylactic vaccine or practical cure strategy remains elusive. Ongoing development of novel therapies and preventative strategies will be crucial in bringing an end to the global HIV-1 pandemic. Several immune-based therapeutic and preventative strategies using combinations of isolated HIV-1 broadly neutralising antibodies are currently undergoing clinical development. However, the high cost of combination immunotherapy/immunoprophylaxis in comparison to antiretroviral drugs remains a major obstacle in wide-spread clinical implementation, especially in resource-limited settings like Africa. Specifically engineered antibodies comprising dual- (bispecific) or tri-paratopes (trispecific) have been developed as an alternative to combination-based therapy. This study aimed to design and evaluate novel bispecific neutralising antibodies that outperform (i.e., exhibit improved breadth and potency) combinations of antibodies in preventing HIV-1 infection in vitro. A range of bispecific constructs were designed and incorporated the knob-in-a-hole and CrossMab mutations to promote preferential assembly of heterologous heavy and light antibody polypeptide chains into a native-like IgG configuration. Antibody sequences used in the bispecific constructs included known broadly neutralizing antibodies against HIV-1 as well as xvi ibalizumab (iMab), which targets human CD4. The bispecific antibodies and parental monoclonal antibodies were produced by transient transfection of HEK293T cells and purified by protein-A affinity chromatography followed by size-exclusion chromatography. Purified antibodies were evaluated by SDS-PAGE under reducing and non-reducing conditions and confirmed heterologous assembly of the dual heavy and light chain constituents in the bispecific antibody configurations. The bispecificity of the antibodies was confirmed by binding ELISA to HIV-1 Env and two-domain/four- domain human CD4 immobilized proteins. A diverse panel of 21 pseudoviruses inclusive of the global HIV-1 panel, was used to evaluate the breadth and potency of the bispecific antibodies in an in vitro HIV-1 neutralization assay, and compared to their monoclonal parental antibodies, alone or in combination, and selected bispecific antibodies such as 10E08-iMab and PG9-iMab. We successfully engineered and characterised two novel bispecific antibodies, iMab- Cap256 (Chapter 2; Publication 1) and iMab-N6 (Chapter 3; Publication 2). Our data demonstrate that both antibodies exhibited greater breadth over their respective parental antibodies when evaluated individually and in combination. Impressively, iMab-Cap256 exhibited remarkable potency over its parental antibodies (on average 4× and 59× more potent than CAP25 and iMab, respectively). Although iMab-N6 showed no enhancement in potency over its parental antibodies, it is calculated to have an exceptional IC80 coverage at < 1 μg/ml of 90% of global circulating HIV-1 strains, versus 86% and 48% for the N6 and iMab parental antibodies, respectively. These findings are significant as an IC80 < 1μg/ml may predict real-world clinical outcomes in protecting against HIV-1 acquisition in humans. In summary, the described bispecific antibodies may be particularly important in the context of HIV-1 xvii subtype C infection, which predominates global infections, and requires uniquely tailored treatment and prevention strategies to overcome the resource-limited challenges facing these endemic regions. These novel bispecific antibodies show great potential for further clinical development given their breadth and potency, but understandably require further testing and development before entering human clinical trials.