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

> **NIH NIH R21** · SCRIPPS RESEARCH INSTITUTE, THE · 2021 · $221,875

## Abstract

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.

## Key facts

- **NIH application ID:** 10115604
- **Project number:** 5R21AI150346-02
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** James Even Voss
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $221,875
- **Award type:** 5
- **Project period:** 2020-03-01 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10115604

## Citation

> US National Institutes of Health, RePORTER application 10115604, Directed Evolution of HIV Broadly Neutralizing Antibodies Using a Novel CRISPR-Engineered B cell in Vitro Affinity Maturation Platform (5R21AI150346-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10115604. Licensed CC0.

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