Preclinical characterization of FLEX-NKTM tetravalent NKp46 engager directed against GPC3 ( CYT-303) alone or in combination with iPSC derived Natural Killer cells (iNKs) against hepatocellular carcinoma (HCC)

Abstract Glypican-3 (GPC3) is highly expressed in multiple solid tumors including HCC while it is hardly expressed in adult normal tissues except placenta. GPC3 promotes Wnt-dependent cell proliferation, and its expression is correlated with poor prognosis in HCC. NK cells exhibit innate anti-tumor activity owing to the expression of multiple activating receptors, such as NKp46. NKp46 is expressed in all NK cells including tumor-infiltrating NK cells. FLEX-NKTrademark is a proprietary platform for production of tetravalent IgG1-like multifunctional antibody NK engagers with a novel FLEX-linker to allow for simultaneous binding of both the targeted cancer cells and NK cells. A novel humanized NKp46 binder that does not induce NKp46 internalization and a humanized GPC3 binder that targets the membrane-proximal lobe of the GPC3 were combined on the novel FLEX-NKTrademark scaffold to create the NK engager CYT-303. CYT-303 has higher affinity for GPC3 compared to NKp46 to skew binding preferentially to GPC3 expressing tumor cells prior to binding NK cells expressing NKp46 to optimize targeted killing. CYT-303 showed significantly higher dose dependent peripheral blood NK cell redirected cytotoxicity and degranulation against GPC3 expressing Hep3B tumor cells compared to GPC3 or NKp46 monoclonal antibodies alone suggesting that co-engagement of NKp46 and GPC3 via an immunological synapse is required for optimal tumor killing by CYT-303. Low NK cell numbers or suppression of NK cell function in the tumor microenvironment may limit the clinical activity of FLEX-NKTrademark engagers. iNK cells derived from iPSC, a uniform starting material with unlimited self-renewal capabilities, can be expanded to produce a universal off-the-shelf allogeneic therapy that can be used in combination with FLEX-NKTrademark engagers. We studied the efficacy of the combination of a FLEX-NKTrademark antibody and iNKs. The iNK cells express high levels of multiple activation receptors including NKp46 and showed good cytotoxic activity against HCC cell line Hep3B. The iNKs also showed anti-tumor activity in NSG-hIL15 mice bearing HCC subcutaneous tumors as demonstrated by the presence of CD56+CD3-, NKp46+, NKG2D+ iNKs in the tumors at day 21 post-intratumoral injection of the iNKs. CYT-303 greatly enhanced the cytotoxic activity of iNKs and cytolysis of Hep3B tumor cells in-vitro. CYT-303 and iNK cells, alone or in combination, demonstrate anti-tumor activity against HCC that warrants clinical development. Citation Format: Antonio Arulanandam, Liang Lin, Hao-Ming Chang, David Zou, Melissa Triggiano, Nejmi Dilmac, Yinan Wang, Shira Kahlon, Stanley Frankel, Jean Kadouche, Daniel Teper, Ofer Mandelboim, Wei Li. Preclinical characterization of FLEX-NK࣪ tetravalent NKp46 engager directed against GPC3 (CYT-303) alone or in combination with iPSC derived Natural Killer cells (iNKs) against hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2752.