The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors.

针对细胞表面抗原 CD19 的过继性 T 细胞疗法的效力已在造血系统癌症中得到证实。在非造血肿瘤中确定适当的靶点一直很困难，但一类已显示出希望的抗原是异常 O 糖蛋白表位。人们早就知道，O-连接（苏氨酸或丝氨酸）糖的合成失调发生在许多癌症中，并且这可能导致含有由单个N-乙酰半乳糖胺（GalNAc，已知）组成的 O-聚糖的细胞表面蛋白的表达作为 Tn 抗原）而不是通常延伸的碳水化合物。此前，我们使用抗体 237 的 scFv 片段作为嵌合抗原受体 (CAR) 来介导对小鼠肿瘤细胞的识别，这些肿瘤细胞在 podoplanin（也称为 OTS8）中具有同源 Tn 糖肽表位。在 237 Fab:Tn-OTS8-糖肽复合物的结构指导下，我们进行了深度突变扫描，显示 Tn-聚糖侧翼的残基对相互作用贡献了显着的结合能。在酵母展示系统中设计 237-scFv 文库使我们能够分离出对 Tn-OTS8 具有更高亲和力的 scFv 变体。使用非同源人类抗原 Tn-MUC1 进行选择，产生了与多种 Tn 糖蛋白广泛反应的 scFv 变体。当配置为 CAR 时，表达这些 scFv 变体的工程化 T 细胞对 O-连接糖基化缺陷的小鼠和人类癌细胞系表现出更高的活性。该策略为 CAR 提供了 Tn 肽特异性，全部基于单一 scFv 支架，允许测试相同 CAR 在小鼠中的毒性以及对小鼠和人类肿瘤的功效。