Abstract 32: Engineering Cord Blood CD34+ Cells for the Generation of Allogeneic NK or Myeloid Cell Immunotherapies

Introduction Immune cells such as natural killer (NK) cells and myeloid cells are gaining attention as an alternative cellular immunotherapy to traditional chimeric antigen receptor (CAR)-T due to their vital functions including immune regulation, cytotoxicity, and phagocytosis of tumor cells, respectively. One major challenge to the generation of CAR-NK or CAR-myeloid cells is the ability to effectively engineer these mature immune cells. To address this, we have developed an ex vivo platform to engineer cord blood-derived CD34+ cells to express CARs early in the culture process. These engineered cells can then be expanded and differentiated into NK cells or monocytes and macrophages to target different cancers. Objectives We aim to develop engineered NK cell or monocyte and macrophage cell products derived from cord blood for off-the-shelf treatment of hematologic and solid malignancies. Methods We designed CARs of interest and cloned the constructs into γ-retroviral or lentiviral backbones to generate viral vectors. To generate engineered immune cells, we first isolated and pooled cord blood-derived CD34+ cells, which are expanded in an ex vivo cell culture process using a proprietary Notch ligand. Early in the process, the CD34+ cells are engineered by viral delivery. These engineered cells then follow additional phase(s) of culturing to differentiate into NK or myeloid cells. The cell cultures are assessed throughout the process for CAR expression and phenotyping is performed on differentiated cells according to the respective cell type. Results Engineering of cord blood-derived CD34+ cells to express CARs followed by differentiation resulted in engineered NK or myeloid cells that maintain viability and phenotyping comparable to non-engineered cells. The engineered immune cells can be cryopreserved and thawed while retaining CAR expression and CAR-specific anti-tumor function in vitro. Discussion The versatility of this platform is demonstrated by the ability to generate CAR-NK or CAR-myeloid cells from the same CD34+ cells that are engineered early, followed by directed differentiation. Not only is this approach significantly more cost-effective, it creates the potential to address more targets to drive anti-tumor immune responses. We are currently optimizing the engineering process, exploring non-viral engineering strategies, and designing payloads that are unique for each immune cell type. Overview of the generation of engineered NK or myeloid cell products from cord blood-derived CD34+ cells