Engineered natural ligand-based car: directed evolution of the stress-receptor nkp30

A method of producing a variant of a receptor, optionally a variant NK receptor, further optionally an NKp30 variant that interacts with a ligand preferentially expressed or expressed at higher levels on diseased cells than normal cells, optionally cancer or virally infected cells, comprising introducing one or mutations in a receptor, optionally a NK receptor polypeptide, further optionally a NKp30 polypeptide or a nucleic acid encoding an NK receptor polypeptide, further optionally a NKp30 polypeptide, optionally by directed evolution, further optionally using yeast or phage display, and determining in one or more screens whether said variant receptor, optionally a variant NK receptor, further optionally a NKp30 variant or a CAR, bispecific T cell engager or fusion protein containing said variant receptor, variant NK receptor, or variant NKp30 polypeptide or a nucleic acid encoding any of the foregoing when expressed exhibits one or more of the following:

• (i) binds to one or more of its endogenous ligands which are preferentially expressed or more highly expressed on diseased cells, e.g., cancerous or infected cells, e.g., B7H6 expressing cells, with higher affinity compared to the native receptor, e.g., NK receptor or NKp30;
• (ii) retains its fast association and dissociation profile to ligand expressing cells, e.g., NK ligand expressing cells or B7H6 expressing cells, compared to the native receptor, e.g., an NK receptor polypeptide, optionally NKp30;
• (iii) elicits a different cytokine profile than the native receptor, e.g., NK receptor polypeptide, optionally NKp30 or than an scFv that binds to the preferentially expressed ligand, optionally NK ligand such as B7H6 or TZ47, optionally against one or more different types of tumors which preferentially express the ligand bound by the receptor,
• (iv) exhibits improved in vitro or in vivo tumor cell killing of cells which express the ligand relative to the native receptor, optionally a NK receptor, optionally against tumors or other diseased cells which express low levels of the ligand, e.g., NKp30 or compared to a scFv that binds the NK ligand, optionally TZ47 and/or
• (v) binds to tumor cells or other diseased cells expressing low levels of the NK ligand, optionally B7H6 better than TZ47 and or the native receptor, optionally a NK receptor, further optionally NKp30.