ABSTRACT

T-cell bispecific antibodies (TCBs) are a novel class of engineered immunoglobulins that unite monovalent binding to the T-cell receptor (TCR) CD3e chain and bivalent binding to tumor-associated antigens in order to recruit and activate T-cells for tumor cell killing. In vivo, T-cell activation is usually initiated via the interaction of the TCR with the peptide-HLA complex formed by the human leukocyte antigen (HLA) and peptides derived from intracellular proteins. TCR-like antibodies (TCRLs) that recognize pHLA-epitopes extend the target space of TCBs to peptides derived from intracellular proteins, such as those overexpressed during oncogenesis or created via mutations found in cancer. One challenge during lead identification of TCRL-TCBs is to identify TCRLs that specifically, and ideally exclusively, recognize the desired pHLA, but not unrelated pHLAs. In order to identify TCRLs suitable for TCRL-TCBs, large numbers of TCRLs have to be tested in the TCB format. Here, we propose a novel approach using chimeric antigen receptors (CARs) to facilitate the identification of highly selective TCRLs. In this new so-called TCRL-CAR-J approach, TCRL-candidates are transduced as CARs into Jurkat reporter-cells, and subsequently assessed for their specificity profile. This work demonstrates that the CAR-J reporter-cell assay can be applied to predict the profile of TCRL-TCBs without the need to produce each candidate in the final TCB format. It is therefore useful in streamlining the identification of TCRL-TCBs.

 抽象的

T 细胞双特异性抗体 (TCB) 是一类新型工程免疫球蛋白，它将与 T 细胞受体 (TCR) CD3e 链的单价结合和与肿瘤相关抗原的二价结合结合起来，以招募和激活肿瘤细胞的 T 细胞杀戮。在体内，T 细胞激活通常是通过 TCR 与人类白细胞抗原 (HLA) 和源自细胞内蛋白的肽形成的肽-HLA 复合物的相互作用来启动。识别 pHLA 表位的 TCR 样抗体 (TCRL) 将 TCB 的目标空间扩展到源自细胞内蛋白质的肽，例如在肿瘤发生过程中过度表达或通过癌症中发现的突变产生的肽。 TCRL-TCB 的先导物鉴定过程中的一项挑战是确定 TCRL 能够特异性地（理想情况下是排他地）识别所需的 pHLA，但不能识别不相关的 pHLA。为了识别适合TCRL-TCB的TCRL，必须以TCB格式测试大量TCRL。在这里，我们提出了一种使用嵌合抗原受体（CAR）的新方法来促进高选择性 TCRL 的鉴定。在这种新的所谓 TCRL-CAR-J 方法中，TCRL 候选物作为 CAR 转导到 Jurkat 报告细胞中，随后评估其特异性。这项工作表明，CAR-J 报告细胞测定可用于预测 TCRL-TCB 的概况，而无需以最终 TCB 格式生成每个候选物。因此，它对于简化 TCRL-TCB 的识别非常有用。