Engineering Structurally-Novel Antibody-Like Reagents to Improve Neutralization of HIV

Abstract

Human immunodeficiency virus (HIV) infection is a worldwide epidemic affecting more than 30 million individuals today. Since its initial outbreak, an estimated 20 million deaths have resulted and nearly 3 million new cases arise annually in adults and children. Combinational antiretroviral therapy (ART) has been highly effective at controlling infection to those fortunate enough to have access, but no drug is able to clear infection and thus treatment must be given lifelong. This is particularly challenging given that nearly 70% of infected individuals live in Sub-Saharan Africa and in regions where adequate delivery infrastructure may not exist. Thus, only about 25% of infected individuals in Africa currently receive treatment. Clinical screens of HIV-resistant individuals have identified a class of broadly-neutralizing antibodies (BNAs) that have significant potential for passive immunotherapy. This project serves to engineer and construct new antibody-like reagents inspired from BNAs with improved HIV neutralization efficiencies. When expressed using adeno-associated virus, a long-term supply of these reagents could be generated to provide a potent alternative to costly conventional drug therapy to individuals all over the world. By utilizing concepts of molecular biology and structural biology, over 60 new antibody-like reagents have been synthesized. These reagents utilize the powerful binding affinities of some of best natural antibody fragments to date, while at the same time, are designed to be better suited to binding to HIV virions compared to the immunoglobulin G (IgG) form of native antibodies. One key modification is the removal of the constant fragment region of the IgGs through molecular cloning and the addition of a rigid, stable, and length-variable protein linker joining two antigen-binding fragments (Fabs) at either end. Secondly, non-canonical combinations of different Fabs are coupled at the ends of the linker, generating bi-specific and completely new proteins. 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 HIV-neutralizing antibodies.