Generation and Screening of Various CD70 CAR NK Cells Identify the Most Effective Construct Against Hematologic Malignancies

The ability to re-direct the intrinsic power of the immune system against cancer has resulted in unprecedented outcomes in certain patients with otherwise incurable diseases. Natural killer (NK) cells, similar to T cells, are powerful immune effectors that possess intrinsic anti-tumor properties and are capable of generating a strong cytotoxic response upon engaging tumor cells. However, unlike T cells, NK cells do not identify specific antigens and do so in a way that is HLA-independent, posing minimal risk of GvHD. Thus, NK cells have are being explored as a possible allogeneic source for over-the-counter cellular immunotherapy. CD70, the ligand for the CD27 receptor, is an attractive "pan-cancer antigen", since in addition to being expressed in hematologic malignancies such as acute myeloid leukemia (AML) and lymphoma, it is also expressed on many solid tumors, including bladder, lung, triple negative breast cancer, renal cell carcinoma, pancreatic cancer, and melanoma. Moreover, CD70 is only transiently found on activated T and B lymphocytes and on dendritic cells. To target CD70-expressing cancers, we designed and tested a number of different chimeric antigen receptors (CAR). Briefly, we used the natural interaction between CD70 and CD27 to make a CAR construct that incorporated the extracellular domain of human CD27, which is the natural receptor for CD70. We generated various second-generation CAR constructs that differed in their transmembrane domains (CD27 vs. CD28) and costimulatory domains. We included co-stimulatory domains that are NK-centric (DAP10 and DAP12), T-cell-centric (CD28), or bi-centric (4-1BB). The CD3ζ chain was used as a signaling endodomain, and all the CAR constructs also included IL-15 to enhance in vivo proliferation and persistence. Cord blood (CB)-derived NK cells were efficiently retrovirally transduced with the different CAR constructs and subjected to an in vitro functional screen against various cancer cells to evaluate the best constructs for in vivo validation (Figure 1). The top constructs from our in vitro screen were further evaluated in multiple in vivo models, including a Raji lymphoma model and a THP-1 AML model. The best CAR construct in terms of superior tumor control and prolongation of survival was selected for GMP manufacturing. Based on these preclinical findings, a Phase I/II clinical study is now being conducted at our center to assess the safety and effectiveness of CD70 CAR NK cells against CD70-expressing hematologic malignancies (NCT05092451).

Figure 1: Flow chart showing the screening process to identify the most effective CAR construct.