Engineering Novel Antibody-Like Reagents to Improve Neutralization of HIV

The HIV pandemic continues to burden more than 30 million individuals worldwide despite over 30 years of intensive global research. Today, more than 70% of all HIV infection takes place in Sub-Saharan Africa, the same countries where severe AIDS and AIDS-related opportunistic infections prevail. While antiretroviral therapy (ARV) has proven to be very effective in controlling disease in much of the Western world, these African countries lack both the economic and clinical resources necessary to successfully deliver drugs to all the patients in need. In the past decade, clinical screens of a variety of elite controller cohorts have identified hundreds of potent antibodies from the blood of rare patients that demonstrate potential in passive immunotherapy, gene therapy and vector prophylaxis. In general, these antibodies are produced in the IgG form, which is a dimer containing two identical antigen-binding fragments (Fabs) whose recognition sites are separated by a distance of approximately 15nm. This distance is relatively conserved from one IgG to another, and is in general inflexible within a single antibody. This design however is not optimized for HIV binding due to two reasons: 1. The much larger separation distance between epitope sites on the gp160 trimer; 2. The scarcity of HIV epitopes on the surface of the virus. Both contribute to the hypothesis that IgGs cannot achieve avidity. Due to the wide applicability of BNAs in gene therapy and passive immunotherapy, as well as their potential in acting as a scaffold for guiding future vaccine design, this project serves to engineer novel antibody-like reagents using a variety of existing antigen recognition fragments of BNAs as the epitope-binding domain for improved structures. We hypothesize that by altering the separation distance of individually linked Fab fragments, and by combining non-canonical combinations of existing Fabs into a single construct and thus generating new fusion proteins, we can generate multivalent reagents that ultimately will have greater neutralization of HIV. Over 60 new antibody-like reagents have been synthesized. Neutralization assays have demonstrated improved neutralization potential for various constructs against a standard panel of HIV strains. One promising bi-specific protein has an IC50 of 33nM, compared to 97nM and infinite nanomolar IC50s of the individual Fabs when not linked together. Another bi-specific construct has an IC50 of 0.56nM, compared to 2.1nM and 14.4nM of the individual Fabs, a 4 to 25-fold improvement. This project overall has validated the use of a structural linker in the design of improved HIV-neutralizing antibodies.