CD8-specific designed ankyrin repeat proteins improve selective gene delivery into human and primate T lymphocytes

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 in vitro and in vivo. This approach might facilitate the genetic engineering of patient's own T lymphocytes possibly even shifting this concept from personalized medicine to an off-the shelf therapy in future. We describe here 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 non-human 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 lentiviral vectors (CD8-LVs) were then generated which delivered genes exclusively and specifically to human and NHP T-lymphocytes using the same targeting domain. These CD8-LVs were as specific for human T lymphocytes as their scFv-based counterpart, but could be produced to higher titers. Moreover, they were superior in transducing cytotoxic T cells 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 like non-human primates without the need for modifications in vector design