CD82 Is Sufficient to Uniquely Identify Pluripotent Stem Cell-Derived Hemogenic Endothelium with the Hematopoietic Lineage Potency to Give Rise to Bona Fide Lymphocytes

Chimeric antigen receptor (CAR)-T and natural killer (NK) cells represent compelling opportunities for the treatment of hematological malignancies and solid tumors. However, autologous and donor-derived allogeneic CAR-T and NK cell therapies are limited based on the quantity and quality of the starting engineered cells. We previously described our off-the-shelf induced pluripotent stem cell (iPSC)-derived CAR-T (CAR-iT) and CAR-NK (CAR-iNK) cell platforms that eliminate some of the hurdles associated with patient-derived cells by creating a homogenous cell product that is derived from a renewable bank of iPSC master cell lines. A major challenge in the manufacturing of iPSC derived immune cells is the derivation of hemogenic endothelium (HE), which is the rare and transient direct precursor of hematopoietic cells, making its in vitro specification from iPSCs inefficient and challenging. Furthermore, there is not a reliable marker or set of markers for the identification of HE in culture. HE identification has traditionally relied on detection of the transcription factor RUNX1 and the establishment of reporter cell lines, neither of which are suitable for the manufacture of HE cells destined to become therapeutic immune cells.

We have established a cytokine driven protocol for the differentiation of iPSCs that improves the efficiency of the derivation of RUNX1⁺ HE cells. To identify candidate HE surface markers, we compared iPSC-derived cells containing a high percentage of HE, based on RUNX1 expression, to control differentiated cells containing a low percentage of HE, via a cell surface antibody array. Through the array we identified several surface markers that were expressed by a higher percentage of CD34⁺ endothelial cells compared to control CD34⁺ cells. Several of the candidates identified, including CD44, CD61, CD226 and CD143 have been previously identified by other groups as candidate HE markers, thereby validating our screening method. We identified CD82 as a novel candidate HE marker, with expression uniquely restricted to the CD34⁺ endothelial population and positively correlating with RUNX1 expression (Fig. 1A). Furthermore, we demonstrated via limiting dilution assays that HE is enriched in the CD34⁺CD43^-CD82⁺ population (HE frequency, 1/38) compared to the CD34⁺CD43^-CD82^- population(HE frequency, 1/736) (Fig. 1B). Even more strikingly, isolation of cells based on CD82 alone was sufficient to enrich for HE cells (HE frequency, 1/44) and outcompeted CD34 enrichment alone, suggestive of its unique ability to identify the HE population (Fig. 1B). To determine if CD82⁺ HE cells could give rise to lymphocytes we carried them forward into T cell differentiation and showed thatthey efficiently gave rise to CAR-iT cells (i.e. >95% CD7+ T lymphocytes). The identification of CD82 for the enrichment of iPSC derived HE is a critical step to the development of definitive hematopoiesis with ability to give rise to all lineages of the hematopoietic compartment and facilitates the scaled and consistent manufacture of high-quality T and NK cells for off-the-shelf therapeutic applications.

Yzaguirre:Fate Therapeutics Inc.: Current Employment. Dege:Fate Therapeutics Inc.: Current Employment. Sivalingam:Fate Therapeutics Inc.: Current Employment. Schnellmann:Fate Therapeutics Inc.: Current Employment. Witty:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics: Current Employment.