Marsman Casper, Heinen Jurgen, Clerico Mosina Vanessa, Maijoor Kelly, Bakker Arjen Q, Koslowski Michael, Villa Alessandra, Gullà Stefano
Kling Biotherapeutics, Amsterdam, Netherlands.
Front Immunol. 2025 Aug 18;16:1648717. doi: 10.3389/fimmu.2025.1648717. eCollection 2025.
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.
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.
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.
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.
在未来大流行持续存在的威胁下，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的不断演变给疫苗效力和治疗干预带来了严峻挑战，凸显了需要快速且适应性强的方法来应对免疫逃逸变体。
在此，我们报告了利用来自人外周血单个核细胞（PBMC）和扁桃体组织的永生化B细胞文库，以发现对各种SARS-CoV-2变体表现出交叉反应中和作用的B细胞克隆，并对永生化B细胞克隆进行定向进化，以产生对新出现的SARS-CoV-2变体具有更强结合力和中和作用的抗体。
通过用编码凋亡抑制剂的逆转录病毒载体转导，实现了PBMC和扁桃体来源的人B细胞的永生化，PBMC的转导效率为67.5%，扁桃体来源细胞的转导效率为50.2%。分析表明，用该方法产生的永生化B细胞文库保留了多样化的免疫球蛋白同种型表现。通过对每个文库约40000个B细胞进行高通量功能筛选，我们鉴定出12个对SARS-CoV-2具有中和活性的独特克隆，从而筛选出对德尔塔和BA.5变体具有强大中和活性的单克隆抗体。我们将定向进化方法应用于通过体外活化诱导胞嘧啶脱氨酶（AID）诱导永生化B细胞克隆的体细胞高频突变（SHM）产生的文库，以增强亲和力和交叉反应性，从而提高对EG.5.1和JN.1等逃逸变体的结合力和中和效力。此外，我们构建了一种双特异性抗体，将结合刺突蛋白受体结合域（Spike-RBD）上高度保守表位的广谱中和抗体KBA2401和具有广泛结合能力的非中和抗体KBA2402结合在一起，从而增强了对SARS-CoV-2变体JN.1和KP.3的效力。
我们的研究结果说明了利用永生化B细胞文库开发适应病毒进化的治疗方法，并突出了体外定向进化在优化针对新出现的免疫逃逸SARS-CoV-2变体的抗体反应中的应用。这里描述的方法为面对不断演变的病毒威胁进行快速治疗开发提供了一条有前景的途径。
