# In vivo affinity maturation of the HIV-1 Env-binding domain of CD4

## TL;DR

Scientists improved a human protein used to fight HIV by letting mice B-cells naturally enhance its effectiveness in the body.

## Contribution

A novel in vivo method for affinity maturation of non-antibody protein biologics is demonstrated.

## Key findings

- Engineered murine B cells successfully affinity matured the CD4-Ig-v0 protein in vivo.
- Somatic hypermutations increased CD4-Ig-v0's binding affinity and neutralization potency tenfold.
- Pharmacokinetic properties of CD4-Ig-v0 remained unaffected after in vivo maturation.

## Abstract

Many human proteins have been repurposed as biologics for clinical use. These proteins have been engineered with in vitro techniques that improve affinity for their ligands. However, these approaches do not select against properties that impair efficacy such as protease sensitivity or self-reactivity. Here we engineer the B-cell receptor of primary murine B cells to express a human protein biologic without disrupting their ability to affinity mature. Specifically, CD4 domains 1 and 2 (D1D2) of a half-life enhanced-HIV-1 entry inhibitor CD4-Ig (CD4-Ig-v0) were introduced into the heavy-chain loci of murine B cells, which were then adoptively transferred to wild-type mice. After immunization, transferred B cells proliferated, class switched, affinity matured, and efficiently produced D1D2-presenting antibodies. Somatic hypermutations found in the D1D2-encoding region of engrafted B cells improved binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein (Env) and the neutralization potency of CD4-Ig-v0 by more than ten-fold across a global panel of HIV-1 isolates, without impairing its pharmacokinetic properties. Thus, affinity maturation of non-antibody protein biologics in vivo can guide development of more effective therapeutics.