Development of CD38 CAR Engineered Human Placental Hematopoietic Stem Cell Derived Natural Killer Cells (PNK-CAR38) As Allogeneic Cancer Immunotherapy
Authors: Somanchi Srinivas , Guo Xuan , He Shuyang , Mathur Rohit , Difiglia Andrea , Rana Hemlata , Ling Weifang , Edinger James , Kaufmann Gunnar F. , Zeldis Jerome B. , Ji Henry , Hariri Robert , Zhang Xiaokui 
Published: November 2019

Background: Natural killer (NK) cells exhibit innate anti-tumor activity owing to the expression of a multitude of activating receptors. Celularity has developed a GMP process for generating off-the-shelf and allogeneic human Placental Hematopoietic Stem Cell derived Natural Killer (PNK-007) cells. To develop a tumor targeted approach, we have engineered PNK cells to express Chimeric Antigen Receptor (CAR) against tumor antigen CD38. PNK cells expressing CD38 CAR (PNK-CAR38) have enhanced anti-tumor function against CD38+ lymphoma and multiple myeloma (MM) cell lines in pre-clinical studies and do not exhibit on-target off-tumor cytotoxicity against healthy CD38+ cells. Here we report the development of a CD38 CAR specific NK cell therapy.
Methods: PNK-CAR38 cells were generated through transduction of human placental CD34+ cells with a retroviral vector carrying second generation anti-CD38 chimeric antigen receptor (CAR2-anti-CD38 A2; CD38scFv-CD28CD3ζ) followed by expansion and differentiation of transduced CD34+ cells into NK cells in presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2. The anti-tumor activity of untransduced PNK and PNK-CAR38 cells was assessed against Lymphoma (Daudi and Raji) and Multiple Myeloma (Molp8, LP1 and OPM2) cell lines at various effector to target (E:T) ratios. PNK-CAR38 in vivo anti-tumor activity was assessed in a disseminated lymphoma xenograft model in NSG mice. Briefly, luciferase-expressing Daudi cells (3×106) were intravenously (IV) injected at Day0, followed by IV injection of PNK-CAR38 cells (20×106) at Day1 and Day3. Tumor burden was assessed weekly by Bioluminescence Imaging (BLI). Differences between the groups were calculated using two-tailed Student t-test.
Results: Placental CD34+ cells were efficiently gene-modified using a retroviral vector and achieved a median of 64% (range 38% to 87%; n=11 donors) CD38 CAR expression in PNK cells at the end of 35-day culture. The median PNK-CAR38 cell yield achieved was 29,000-fold (range 7,800-48,000 fold; n=8) with NK purity >90% measured by CD3-CD56+ population. In vitro an overall improvement in anti-tumor response by PNK-CAR38 over PNK cells was observed. In a NK resistant lymphoma cell line model Daudi, PNK-CAR38 cells from various donors (n=8) elicited 72%±10% lysis versus 6%±3.5% lysis by PNK alone (p<0.01) at 10:1 E:T ratio. PNK-CAR38 elicited significant cytotoxicity 27%±9% versus 13%±5% by PNK cells (p<0.01) against Raji cells depending on NK cell donor. Using MM lines, at 10:1 E:T ratio, PNK-CAR38 cells elicited 33%±16% lysis versus 21%±12% by PNK (p<0.01) against LP1; 44%±6% versus 28%±3% by PNK (p<0.01) against OPM2; and 32%±6% versus 2%±0.5% by PNK (p<0.01) against MOLP8. Since CD38 is also expressed on normal immune and progenitor cells, PNK-CAR38 cells were co-cultured with healthy donor-derived activated T cells and CD34+CD38+ progenitor cells. No on-target off-tumor cytotoxicity against these cells was observed. In the disseminated Daudi xenograft NSG model, PNK-CAR38 significantly reduced tumor burden (approximately 50% BLI reduction) compared to vehicle control 10 days post treatment.
Conclusions: The novel PNK-CAR38 demonstrated enhanced in vitro cytotoxic potential against lymphoma and MM lines and showed no on-target off-tumor cytotoxicity against healthy donor-derived cells in vitro. In vivo anti-tumor activity of PNK-CAR38 was shown in a lymphoma disseminated NSG model. Further development of PNK-CAR38 for hematological malignancies is warranted.

Disclosures
Somanchi: Celularity Inc: Employment. Guo:Celularity, Inc.: Employment. He:Celularity Inc: Employment. Mathur:Celularity Inc: Employment. Difiglia:Celularity Inc: Employment. Rana:Celularity Inc: Employment. Ling:Celularity Inc: Employment. Edinger:Celularity Inc: Employment. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.