Human Placental CD34+-Derived Natural Killer Cells with High Affinity and Cleavage Resistant CD16 (CYNK-101) for ADCC Mediated Cancer Immunotherapy
Authors: Kang Lin , Raitman Irene , Rotondo Salvatore , Gleason Joseph , He Shuyang , Somanchi Srinivas , Rousseva Valentina , Guo Xuan , Rana Hemlata , DiFiglia Andrea , Ye Qian , Mahlakõiv Tanel , Van Der Touw William , Hariri Robert , Zhang Xiaokui 
Published: November 2020

CYNK-101, an allogeneic off-the-shelf human placental CD34+-derived natural killer (NK) cell product, is genetically modified to express a variant of CD16 (FcγRIII) via lentiviral vector transduction. When targeting tumors with monoclonal antibodies, NK cells are key effectors of antibody dependent cellular cytotoxicity (ADCC) following recognition of antibody Fc by CD16. CYNK-101 expresses the CD16 variant (CD16VP) which has a high-affinity due to a Valine at amino acid position 158 along with proteolytic cleavage resistance imparted by Proline at amino acid position 1971,2,3,4. We hypothesize that expressing CD16VP enhances anti-tumor activity of CYNK-101 in combination with monoclonal antibody therapy. Reported here are the in vitro and ex vivo results of evaluating CYNK-101 cytotoxicity against human epidermal growth factor receptor 2 (HER2)+ solid tumors in combination with Trastuzumab, an anti-HER2 monoclonal antibody.
Human placental CD34+ cells were transduced with lentivirus expressing CD16VP, and cultured in the presence of cytokines, including TPO, SCF, Flt3L, IL-7, IL-15, and IL-2, to generate CYNK-101 cells. Lentiviral transduction with CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells (n=7) expanded at 12041 ± 6394-fold and differentiated into CYNK-101 being &gt;90% CD56+CD3-. CYNK-101 expressed 74.1 ± 5.6% (n=7) of CD16 at the end of cultivation. Proteolytic cleavage resistance conferred by CD16VP in CYNK-101 was evaluated by activating NK cells with PMA/ionomycin (PMAi) to induce CD16 shedding. While 4h PMAi treatment resulted in &gt;89% CD16 cleavage on non-transduced NK cells, &lt;11% cleavage was observed on CYNK-101 cells, demonstrating CD16 shedding resistance.
The in vitro anti-tumor activity of CYNK-101 against HER2+ solid tumor cell lines was assessed in combination with Trastuzumab. At effector to target (E:T) ratio of 0.6:1, CYNK-101 (n=7 donors) showed enhanced lysis of NCI-N87 gastric cancer cells in the presence of Trastuzumab compared to that of IgG control, 41.2 ± 11.1% vs. 5.3 ± 4.7% at 4h, 91.3 ± 15.0% vs. 7.5 ± 9.3% at 24h, respectively (p&lt;0.005). Increased production of GM-CSF, IFN-γ, and TNF-α was shown in CYNK-101 plus Trastuzumab against NCI-N87 at 24h compared to that of IgG control. Overall, the enhanced ADCC activity of CYNK-101 in combination with Trastuzumab has been demonstrated against both HER2+ gastric cancer cell lines, NCI-N87 and OE-19; and HER2+ breast cancer cell lines, AU565, BT-474, HCC-1954, SKBR-3, and ZR-75-30.
To further evaluate ex vivo ADCC activity of CYNK-101, CYNK-101 cells were intravenously (IV) injected at 2x107 into busulfan-pretreated NOD-scid IL2Rγnull immunodeficient (NSG) mice at Day 0. Recombinant human IL-15 was intraperitoneally injected at Days 0, 2, 4, 6, 8, 10 and 12. At Day 13, CYNK-101 cells were isolated from mouse livers (ex vivo-CYNK-101) and assessed for phenotype and ADCC activity. Ex vivo-CYNK-101 exhibited enhanced cytotoxicity against NCI-N87 in combination with Trastuzumab compared to that of IgG control (30.9% vs. 6.6% at 4h, 90.7% vs. 9.1% at 24h) at the E:T ratio of 0.5:1, as well as increased cytokine productions of GM-CSF, IFN-γ, and TNF-α at 24h. Compared to pre-infusion CYNK-101, ex vivo-CYNK-101 showed not only a higher ADCC activity against NCI-N87, but also a more matured NK cell phenotype with increased expression of CD16, KIR, NKG2D, CD94, and CD11a. Besides, these ex vivo-CYNK-101 cells demonstrated CD16 shedding resistance post PMAi stimulation.
In summary, our results demonstrated enhanced in vitro and ex vivo ADCC activities of CYNK-101 in combination with Trastuzumab against HER2+ solid tumors. CYNK-101 exhibited high resistance to CD16 shedding in vitro and ex vivo. Further maturation of CYNK-101 cells post IV infusion was accompanied by ADCC activity enhancement. Evaluation of in vivo ADCC activity of CYNK-101 in combination with Trastuzumab will include both subcutaneous and orthotopic gastric cancer models.

Disclosures
Kang: Celularity Inc.: Current Employment. Raitman:Celularity Inc.: Current Employment. Rotondo:Celularity Inc.: Current Employment. Gleason:Celularity Inc.: Current Employment, Current equity holder in private company. He:Celularity Inc.: Current Employment, Current equity holder in private company. Somanchi:Celularity Inc.: Current Employment, Current equity holder in private company. Rousseva:Celularity Inc.: Current Employment. Guo:Celularity Inc.: Current Employment. Rana:Celularity Inc.: Current Employment. DiFiglia:Celularity Inc.: Current Employment. Ye:Celularity Inc.: Current Employment, Patents & Royalties. Mahlakõiv:Celularity Inc.: Current Employment. Van Der Touw:Celularity Inc.: Current Employment. Hariri:Celularity Inc.: Current Employment, Current equity holder in private company. Zhang:Celularity Inc.: Current Employment, Current equity holder in private company.