Directed Evolution of HIV Broadly Neutralizing Antibodies Using a Novel CRISPR-Engineered B cell in Vitro Affinity Maturation Platform

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Abstract Passive transfer of HIV broadly neutralizing antibodies (bnAbs) is promising as an alternative to antiretroviral drugs because they are generally well tolerated, have long in vivo half-lives, and can activate the host immune system. Post-discovery improvement of bnAb breadth and potency should make their application as a medical intervention more practical. We have developed a novel antibody directed evolution platform we would like to employ for this purpose. This platform uses genome editing to replace immunoglobulin variable regions with those from specific monoclonal antibodies in a human B cell line. Endogenous activation-induced induced cytidine deaminase (AID) introduces mutations at these engineered loci generating antibody variants with altered antigen binding properties. The new antibody is expressed using endogenous constant genes as cell surface IgM, allowing for selection variants with desired binding properties using target antigen selection probes. We have successfully used this platform to generate and select variants of an antibody which show improved HIV neutralizing breadth and potency as a proof of concept. IgM secreted from engineered cells can be harvested from cell supernatants and characterized during the directed evolution of cell lines. Single cell sequencing of evolved cells can be performed to uncover the genetic basis for altered binding and for the generation of improved mAbs for expression as recombinant proteins. In this R21, we propose engineering the HIV bnAbs VRC01 and CAP256-VRC26.25 into our B cell line in order to evolve a CD4 binding site and V2-apex directed bnAbs with improved neutralizing breadth, potency and potentially biophysical properties, using a variety of different selection strategies. If successful, this platform would be a valuable resource for post-discovery improvement of not only HIV bnAbs, but for any monoclonal of therapeutic interest.