Human Placental CD34 +-Derived Natural Killer Cells with High Affinity and Cleavage Resistant CD16 (CYNK-101) in Combination with Daratumumab for Immunotherapy Against CD38 Expressing Hematological Malignancies
Authors: Raitman Irene , Gleason Joseph , Rotondo Salvatore , He Shuyang , Rousseva Valentina , Guo Xuan , Rana Hemlata , van der Touw William , Ye Qian , Kang Lin , Hariri Robert , Zhang Xiaokui 
Published: November 2021

Abstract
Natural Killer (NK) cells are key mediators of antibody dependent cellular cytotoxicity (ADCC) via the CD16 Fc receptor. NK cellular therapies can effectively be targeted to tumor antigens when combined with tumor specific antibodies. Celularity Inc. is developing human placental CD34 +-derived, cryopreserved, off-the-shelf, allogenic NK cells (CYNK-101) with a high IgG binding affinity and proteinase cleavage resistant CD16 variant (CD16VP) for cancer treatment. We hypothesize that expressing CD16VP augments anti-tumor ADCC activity. Reported here are the in vitro and in vivo results of evaluating CYNK-101 cytotoxicity against CD38 expressing multiple myeloma (MM) and lymphoma tumor cell lines when in combination with daratumumab, an anti-CD38 monoclonal antibody.
Human placental CD34 + cells were transduced with lentivirus expressing CD16VP and cultured in the presence of cytokines to generate CYNK-101 cells. The in vitro cytotoxic activity of CYNK-101 against CD38 + MM (MOLP-8, LP-1, MM.1S) and lymphoma (Daudi) tumor cell lines, and normal B-cells was assessed in combination with daratumumab via flow cytometry based ADCC assays and cytokine secretion was assessed via multiplex Luminex analysis. In vivo ADCC activity of CYNK-101 was assessed using a disseminated B-cell lymphoma xenograft model in B-NDG-hIL15 mice. B-NDG-hIL15 mice lack T, B, and NK cells and are transgenic for human IL-15 to support CYNK-101 persistence and maturation. Luciferase expressing Daudi cells (3×10 6) were intravenously (IV) injected on Day 0 three days after preconditioning with a myeloablative dose of busulfan to allow for better tumor cell engraftment. CYNK-101 cells (1x10 7) and/or daratumumab (0.05 mg/kg) were IV injected on Days 7, 14 and 21. Tumor burden was assessed weekly by bioluminescence imaging (BLI) and the mice were followed for assessment of their survival (n=5 mice per group).
In vitro ADCC studies indicate enhanced cytolysis of CYNK-101 in combination with daratumumab against both MM and lymphoma tumor cells compared to that of IgG control. At 24h at the effector to target (E:T) ratio of 5:1, CYNK-101 (n=5 donors) demonstrated a cytolysis of 87.6 ± 6.3% with daratumumab vs. 37.3 ± 9.5% with IgG control against MOLP-8 (p&lt;0.001), 73.9 ± 2.5% vs. 32.1 ± 7.2% against LP-1 (p&lt;0.001), 77.2 ± 11.5% vs. 67.4 ± 10.7% against MM.1S (p&lt;0.001), and 54.7 ± 24.0% vs. 4.3 ± 2.6% against Daudi (p&lt;0.01) tumor cells. Secretion of GM-CSF, IFN-γ, and TNF-α was increased in CYNK-101 co-cultured with the tumor cell lines in the presence of daratumumab for 24h (n=5 donors, p&lt;0.05). When cocultured with mixed LP-1 and CD38 + normal B-cells, CYNK-101 in combination with daratumumab displayed specific cytotoxicity against LP-1, while sparing CD38 + normal B-cells even at an E:T ratio up to 100:1, demonstrating that CYNK-101 can distinguish CD38 + tumor cells from CD38 + normal cells. Additionally, despite expression of CD38 on CYNK-101 there was no NK fratricide observed when CYNK-101 were in combination with daratumumab.
In vivo studies in the lymphoma xenograft model revealed a significant decrease in tumor burden as evidenced from bioluminescence imaging at day 28 (1 week after last CYNK-101 injection) for mice that received CYNK-101 in combination with daratumumab compared to vehicle control (p&lt;0.001), CYNK-101 (p&lt;0.05) and daratumumab (p&lt;0.05). Furthermore, CYNK-101 in combination with daratumumab demonstrated an enhanced survival benefit with a median survival of 35 days versus a median survival of 28 days for the vehicle treated group (p&lt;0.005).
In summary, our results demonstrate enhanced in vitro and in vivo ADCC activities of CYNK-101 in combination with daratumumab against CD38 + hematological tumors and warrant further development of this combination therapy for these cancers.

Disclosures
Raitman: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Gleason: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rotondo: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. He: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rousseva: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Guo: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Rana: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. van der Touw: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Ye: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Kang: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Hariri: Celularity Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang: Celularity Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months.