The existing repertoire of HIV-1 patient derived broadly neutralising antibodies (bNAbs) that target the HIV-1 envelope glycoprotein (Env) present numerous and exciting opportunities for immune-based therapeutic and preventative strategies against HIV-1. Combination antibody therapy is required to ensure greater neutralization coverage and limit Env mediated escape mutations following treatment pressure. Engineered bispecific bNAbs (bibNAbs) assimilate the advantages of combination therapy into a single antibody molecule with several configurations reporting potency enhancement as a result of the increased avidity and simultaneous engagement of targeted epitopes. We report the engineering of a novel bibNAb (iMab-CAP256) comprising the highly potent, CAP256.VRC26.25 bNAb with anticipated extension in neutralization coverage through pairing with the host directed, anti-CD4 antibody, ibalizumab (iMab). Recombinant expression of parental monoclonal antibodies and the iMab-CAP256 bibNAb was performed in HEK293T (Human embryonic kidney 293 T antigen) cells, purified to homogeneity by Protein-A affinity chromatography followed by size exclusion chromatography. Antibody assembly and binding functionality of Fab moieties was confirmed by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and ELISA, respectively. Breadth and potency were evaluated against a geographical diverse HIV-1 pseudovirus panel (n = 20). Overall, iMab-CAP256 demonstrated an expanded neutralizing coverage, neutralizing single, parental antibody resistant pseudovirus strains and an enhanced neutralization potency against all dual sensitive strains (average fold increase over the more potent parental antibody of 11.4 (range 2 to 31.8). Potency enhancement was not observed for the parental antibody combination treatment (iMab + CAP256) suggesting the presence of a synergistic relationship between the CAP256 and iMab paratope combination in this bibNAb configuration. In addition, iMab-CAP256 bibNAbs exhibited comparable efficacy to other bibNAbs PG9-iMab and 10E08-iMab previously reported in the literature. The enhanced neutralization coverage and potency of iMAb-CAP256 over the parental bNAbs should facilitate superior clinical performance as a therapeutic or preventative strategy against HIV-1.

中文翻译：

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设计和表征一种针对 HIV-1 的新型高效双特异性抗体 iMab-CAP256

现有的 HIV-1 患者来源的广泛中和抗体 (bNAb) 以 HIV-1 包膜糖蛋白 (Env) 为靶点，为基于免疫的 HIV-1 治疗和预防策略提供了大量令人兴奋的机会。需要联合抗体疗法以确保更大的中和覆盖范围并限制治疗压力后 Env 介导的逃逸突变。工程双特异性 bNAb (bibNAb) 将联合治疗的优势吸收到单个抗体分子中，并报告了由于增加的亲和力和同时结合靶向表位而导致的效力增强的几种配置。我们报告了一种新型 bibNAb (iMab-CAP256) 的工程，包括高效的 CAP256.VRC26。25 bNAb 通过与宿主导向的抗 CD4 抗体 ibalizumab (iMab) 配对，预计会扩大中和覆盖范围。亲本单克隆抗体和 iMab-CAP256 bibNAb 的重组表达在 HEK293T（人胚胎肾 293 T 抗原）细胞中进行，通过蛋白 A 亲和层析和尺寸排阻层析纯化至同质。Fab 部分的抗体组装和结合功能分别通过 SDS-PAGE（十二烷基硫酸钠聚丙烯酰胺凝胶电泳）和 ELISA 确认。针对不同地域的 HIV-1 假病毒面板（n = 20）评估了广度和效力。总体而言，iMab-CAP256 展示了扩大的中和覆盖范围，中和单一、