Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escape.

INTRODUCTION

Amid the persistent threat of future pandemics, the continuous evolution of SARS-CoV-2 exposed critical challenges for vaccine efficacy and therapeutic interventions, highlighting the need for rapid and adaptable approaches to respond to immune escape variants.

METHODS

Here, we report the use of immortalized B cell libraries from human peripheral blood mononuclear cells (PBMCs) and tonsil tissues to uncover B cell clones exhibiting cross-reactive neutralization against various SARS-CoV-2 variants and perform directed evolution of immortalized B cell clones to produce antibodies with improved binding and neutralization against emerging SARS-CoV-2 variants.

RESULTS

Immortalization of PBMC and tonsil-derived human B cells was achieved through transduction with retroviral vectors encoding apoptosis inhibitors, yielding transduction efficiencies of 67.5% for PBMCs and 50.2% for tonsil-derived cells. Analysis revealed that immortalized B cell libraries produced with this method retain diverse immunoglobulin isotype representations. Through high-throughput functional screening of approximately 40,000 B cells per library, we identified 12 unique clones with neutralization activity for SARS-CoV-2, leading to selection of monoclonal antibodies with robust neutralization activity against Delta and BA.5 variants. We applied our directed evolution approach to libraries generated by ex vivo AID-induced somatic hypermutation (SHM) of immortalized B cell clones to enhance the affinity and cross-reactivity, resulting in improved binding and neutralization potency to escape variants such as EG.5.1 and JN.1. Furthermore, we engineered a bi-paratopic antibody combining KBA2401, a broadly neutralizing antibody binding to highly conserved epitope on Spike-RBD, and KBA2402, a broadly binding non-neutralizing antibody, resulting in enhanced potency against SARS-CoV-2 variant JN.1 and KP.3.

DISCUSSION: Our findings illustrate the use of immortalized B cell libraries for development of therapeutics that adapt to viral evolution and highlight the application of ex vivo directed evolution in refining antibody responses against emerging immune escape SARS-CoV-2 variants. The approach here described offers a promising pathway for rapid therapeutic development in the face of evolving viral threats.