Mascola: BNAbs for Passive Immunization
The overall aim of this proposal is to develop an improved HIV neutralizing antibody that prevents HIV infection in adult and pediatric populations at risk for HIV-1 infection. Advances in the identification of broadly neutralizing antibodies to HIV have raised new opportunities to prevent infection through the administration of antibodies that neutralize diverse strains of the virus. Among these antibodies, an exceptionally broadly neutralizing antibody, VRC01, has been isolated by the Vaccine Research Center, National Institute of Allergy and Infection Diseases, NIH (VRC, NIAID, NIH). VRC01 neutralizes more than 90% of genetically diverse HIV strains by binding to the conserved CD4 binding site of the HIV-1 gp120 envelope. While this antibody is currently advancing into phase I clinical studies in human subjects, with the goal of evaluating its safety and tolerability in the target populations , a solution to the global HIV epidemic relies on improvements in antibody design that increase potency and circulating half-life, while maintaining the high breadth of neutralization. This increase in efficacy would reduce the amount of product and the frequency of dosing required to prevent HIV infection. Additionally, improvements in manufacturing to improve product yield are likely attainable. The cumulative effect of these improvements in potency, half-life and manufacturing efficiency could have a substantial impact on the cost and feasibility of an antibody product to prevent HIV infections.
This grant, led by Dr. Mascola, aims to develop second-generation antibody products to test the concept of passive immunization in adults. In the initial work, VRC01-like antibodies were optimized using several different approaches, including improved FcγIII binding to improve ADCC activity, and modifications to enhance binding to FcRn in order to prolong serum half-life and augment mucosal transport. The purpose of these modifications is to reduce the requirement for product while retaining the ability of the antibody to confer protection against infection by diverse strains. The research was performed by the VRC and extramural collaborators at the University of Minnesota and Brigham and Women’s Hospital with the goal of identifying a final candidate for Phase I clinical trial testing of the passive immunization concept of HIV prevention in adult and pediatric populations.
Significant progress has been achieved. VRC investigators identified VRC07, a clonal relative of VRC01 that is several times more potent. Rational structure-based and bioinformatics design were used to optimize VRC07 neutralization potential, minimize immunogenicity, and increase in vivo half-life. After screening more than 300 variants, four candidates were selected based on in vitro neutralization and autoreactivity analyses. A ‘LS’ Fc mutation (developed by Xencor) designed to extend half-life and increase concentrations in mucosal tissue was incorporated into the antibodies. Pharmacokinetic analyses in rhesus macaques demonstrated half-life values ranging from 7-10 days, compared to 5 days for VRC07 and 5-6 days for VRC01. After evaluation of all of the results, VRC07-523-LS was found to represent the best next-generation anti-CD4bs mAb, and it is currently being developed for clinical trials.
The grant is currently focusing on optimization of a second antibody to an independent site on the virus, in order to expand breadth of coverage and reduce likelihood of virus escape. The current lead candidate is 10E8, developed by Mark Connors at NIAID. This mAb targets the membrane proximal region (MPER) of HIV-1 gp41. The overall objective is to approach the challenge of improving 10E8 solubility in a rational, systematic manner. In the studies being conducted, novel 10E8 variants will be expressed, purified, and evaluated for improvement in solubility, as well as for effects on neutralization effectiveness. Methods being tested to improve solubility include “supercharging” the protein surface such that the overall charge of exposed antibody residues is increased in one direction (either positive or negative). Supercharging has been used for decreasing protein aggregation, isotype switching, mutation of exposed hydrophobic patches and glycosylation site additions.
Create and test modifications to improve solubility of the 10e8 antibody
1. Create modifications of a) surface charge, b) isotype switching, c) mutation of exposed hydrophobic patches, d) glycosylation site additions
2. In vitro analysis of mutants using turbidity measurements, dynamic light scattering, aggregation tests with Size Exclusion Chromatography (SEC), stability tests with DSC (Differential Scanning Calorimetry) and neutralization assays
3. In vivo pharmacokinetic (PK) half-life studies