Adoptive T cell immunotherapy in combination with gene therapy is a promising treatment concept for chronic infections and cancer. Recently, receptor-targeted lentiviral vectors (LVs) were shown to enable selective gene transfer into particular types of lymphocytes both in vitro and in vivo. This approach might facilitate the genetic engineering of a patient's own T lymphocytes, possibly even shifting this concept from personalized medicine to an off-the shelf therapy in future. Here, we describe novel high-affinity binders for CD8 consisting of designed ankyrin repeat proteins (DARPins), which were selected to bind to the CD8 receptor of human and nonhuman primate (NHP) cells. These binders were identified by ribosome display screening of DARPin libraries using recombinant human CD8 followed by receptor binding analysis on primary lymphocytes. CD8-targeted LVs (CD8-LVs) were then generated that delivered genes exclusively and specifically to human and NHP T lymphocytes by using the same targeting domain. These CD8-LVs were as specific for human T lymphocytes as their single-chain variable fragment-based counterpart, but they could be produced to higher titers. Moreover, they were superior in transducing cytotoxic T cells both in vitro and in vivo when equal particle numbers were applied. Since the here described CD8-LVs transduced primary T lymphocytes from NHP and human donors equally well, they offer the opportunity for preclinical studies in different animal models including large animals such as NHPs without the need for modifications in vector design.

中文翻译：

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CD8 特异性设计的锚蛋白重复蛋白改善了人类和灵长类 T 淋巴细胞的选择性基因传递。

过继 T 细胞免疫疗法与基因疗法相结合是治疗慢性感染和癌症的一种很有前景的概念。最近，受体靶向慢病毒载体 (LVs) 被证明能够在体外和体内选择性地将基因转移到特定类型的淋巴细胞中. 这种方法可能会促进患者自身 T 淋巴细胞的基因工程，甚至可能在未来将这一概念从个性化医疗转变为现成的治疗。在这里，我们描述了由设计的锚蛋白重复蛋白 (DARPins) 组成的新型高亲和力 CD8 结合剂，这些蛋白被选择用于与人类和非人类灵长类动物 (NHP) 细胞的 CD8 受体结合。这些结合物是通过使用重组人 CD8 对 DARPin 文库进行核糖体展示筛选，然后对原代淋巴细胞进行受体结合分析来鉴定的。然后生成 CD8 靶向 LVs (CD8-LVs)，通过使用相同的靶向域将基因专门和特异性地传递给人类和 NHP T 淋巴细胞。这些 CD8-LV 对人 T 淋巴细胞具有特异性，就像它们基于单链可变片段的对应物一样，但它们可以产生更高的滴度。此外，它们在转导细胞毒性 T 细胞方面具有优势当应用相同的粒子数时，体外和体内。由于这里描述的 CD8-LVs 转导来自 NHP 和人类供体的原代 T 淋巴细胞同样好，它们为不同动物模型的临床前研究提供了机会，包括大型动物，如 NHPs，无需修改载体设计。