PO.IM01.14 · 免疫学
泛alphabeta-T细胞反应性抗TCR VHH双特异性抗体通过新型恒定区靶向显示出强效细胞毒性
Pan-alphabeta-T-cell reactive anti-TCR VHH bispecifics demonstrate potent cytotoxicity through novel constant region targeting
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：所有获批的T细胞衔接器均靶向CD3ε，但TCR复合物上的替代衔接位点可能产生差异化的功能特性。我们开发了基于VHH的T细胞衔接（TCE）臂，其结合一个先前未开发的、在TCRalpha/beta恒定区界面形成的四级表位。这些结合剂表现出泛alphabeta-T细胞反应性，并具有固有的人-食蟹猴交叉反应性。
方法：使用基于酵母的选择，针对重组人和食蟹猴TCRalpha/beta异二聚体鉴定抗TCR VHHs，采用合成文库和美洲驼免疫两种方法。VHHs的亲和力优化维持了跨物种识别。使用易错PCR生成的TCR恒定区突变体文库进行表位定位。解析了VHH-TCR复合物的结构。将掺入抗肿瘤相关抗原（TAA）结构域的双特异性抗体在跨肿瘤模型的T细胞依赖性细胞毒性（TDCC）测定中进行评估，并与临床CD3对照进行基准比较。
结果：分离出多个识别TCRalpha/beta恒定区界面保守四级表位的VHH谱系，提供TRBC1/C2覆盖和人-食蟹猴交叉反应性。结构分析确立了结合模式。尽管单价亲和力中等，抗TCR双特异性抗体仍表现出与基准CD3双特异性抗体和临床活性对照相当或更强的TDCC活性。在人和食蟹猴测定系统中均证实了功能活性，包括初步的细胞因子读数。
结论：靶向TCR恒定区内四级表位的基于VHH的双特异性抗体构成了一类新兴的T细胞衔接器，具有强效的细胞毒性活性和广泛的alphabeta-T细胞覆盖。TRBC1/C2包容性、结构定义以及人-食蟹猴交叉反应性的结合，使TCR恒定区靶向成为下一代T细胞重定向疗法有前景的替代方案。
查看英文原文 English abstract
Introduction: All approved T-cell engagers target CD3ε, but alternative engagement sites on the TCR complex may yield differentiated functional properties. We developed VHH-based T-cell-engaging (TCE) arms that bind a previously unexploited quaternary epitope formed at the TCRalpha/beta constant-region interface. These binders exhibit pan-alphabeta-T-cell reactivity with inherent human-cynomolgus cross-reactivity.
Methods: Anti-TCR VHHs were identified using yeast-based selections against recombinant human and cynomolgus TCRalpha/beta heterodimers, employing both synthetic libraries and llama immunization. Affinity optimization of VHHs maintained cross-species recognition. Epitope mapping was performed using TCR constant-region mutant libraries generated by error-prone PCR. Structures of VHH-TCR complexes were solved. Bispecifics incorporating anti-tumor associated antigen (TAA) domains were assessed in T-cell-dependent cytotoxicity (TDCC) assays across tumor models and benchmarked against clinical CD3 controls.
Results: Multiple VHH lineages that recognize a conserved quaternary epitope at the TCRalpha/beta constant-region interface were isolated, providing TRBC1/C2 coverage and human-cynomolgus cross-reactivity. Structural analyses established the binding mode. Despite moderate monovalent affinities, anti-TCR bispecifics demonstrated potent TDCC activity comparable to or exceeding benchmark CD3 bispecifics and clinically active controls. Functional activity was confirmed in both human and cynomolgus assay systems, including preliminary cytokine readouts.
Conclusions: VHH-based bispecifics that target a quaternary epitope within the TCR constant region constitute an emerging class of T-cell-engagers with potent cytotoxic activity and broad alphabeta-T-cell coverage. The combination of TRBC1/C2 inclusivity, structural definition, and human-cynomolgus cross-reactivity positions TCR constant-region targeting as a promising alternative for next-generation T-cell-redirecting therapeutics.
利益披露 Disclosure
M. B. Battles, None.