Potent Immunotherapy Of Human Placental Cd34+-Derived Natural Killer Cells With High Affinity And Cleavage Resistant Cd16 (Cynk-101) Plus Trastuzumab For Her2+Gastric Cancer.

Abstract Introduction: Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in ~20% of gastric cancer cases and correlated with poor outcome. Celularity is developing CYNK-101, an allogeneic, off-the-shelf human placental CD34+-derived NK cell product with genetically modified CD16 variant (CD16VP) for cancer treatment. CYNK-101 is designed to enhance the anti-tumor antibody dependent cellular cytotoxicity (ADCC) activity by expressing high-affinity and cleavage resistant CD16VP. Here we report the preclinical efficacy data of CYNK-101 plus Trastuzumab, an anti-HER2 monoclonal antibody, against HER2+ gastric cancer cells. Methods: Human placental CD34+ cells were transduced with lentivirus expressing CD16VP and cultured with cytokines to generate CYNK-101. ADCC activity of CYNK-101 plus Trastuzumab against HER2+ gastric cancer cells was measured by real-time xCELLigence, and cytokine secretion was quantified by Luminex. Ex vivo-CYNK-101 was isolated from NOD-scid IL2Rγnull immunodeficient (NSG) mice 13 days post CYNK-101 infusion. A subcutaneous NCI-N87 xenograft NSG-hIL15 transgenic mouse model was used for in vivo study. Results and conclusion: CYNK-101 was generated from multiple placental CD34+ donors (n=7) with >90% CD56+CD3-and 74.1 ± 5.6% CD16 expression. While 4h PMAi treatment resulted in >89% CD16 cleavage on non-transduced NK cells, <11% cleavage from CYNK-101 demonstrated CD16 shedding resistance. At an effector to target (E:T) ratio of 1:1, CYNK-101 (n=7) showed enhanced lysis of NCI-N87 cells in the presence of Trastuzumab compared to IgG control, 59.6 ± 13.7% vs. 9.8 ± 3.2% at 4h, 97.1 ± 7.1% vs. 26.2 ± 16.1% at 24h, respectively (p<0.001), and secreted increased GM-CSF, IFN-γ, and TNF-α at 24h. Ex vivo-CYNK-101 plus Trastuzumab exhibited enhanced cytotoxicity against NCI-N87 compared to IgG control (30.9% vs. 6.6% at 4h, 90.7% vs. 9.1% at 24h) at the E:T of 0.5:1, as well as increased production of GM-CSF, IFN-γ, and TNF-α at 24h. Compared to CYNK-101 pre-infusion, ex vivo-CYNK-101 showed not only a higher ADCC activity against NCI-N87, but also a more matured NK phenotype with increased expression of CD16, KIR, NKG2D, and CD94. In addition, CD16 shedding resistance was demonstrated on ex vivo-CYNK-101. CYNK-101 in vivo anti-tumor ADCC activity was assessed in a subcutaneous xenograft NCI-N87 mouse model. Significant tumor reduction was shown in the animals treated with CYNK-101 plus Trastuzumab compared to vehicle control, Trastuzumab or CYNK-101 alone (P<0.0001, n=20 mice per group). In summary, our results demonstrated synergistic anti-tumor ADCC activities of CYNK-101 plus Trastuzumab against HER2+ gastric cancer cells in vitro, ex vivo and in vivo. Further development of CYNK-101 in combination with Trastuzumab for HER2+ gastric cancer immunotherapy is underway. Citation Format: Lin Kang, Shuyang He, Irene Raitman, Salvatore Rotondo, Joseph Gleason, Valentina Rousseva, Xuan Guo, Hemlata Rana, Qian Ye, Robert Hariri, Xiaokui Zhang. Potent immunotherapy of human placental CD34+-derived natural killer cells with high affinity and cleavage resistant CD16 (CYNK-101) plus Trastuzumab for HER2+ gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 58.