Biological Characterization and Differential Gene Expression Analysis of CYT-338 NK Cell Engager (NKE) Against CD38 Expressing Tumors Including Multiple Myeloma (MM)

Introduction: CYT-338 is a NK cell engager antibody designed using Cytovia's proprietary FLEX-NK^TM platform containing a novel FLEX-linker to redirect NK cells expressing NKp46 activation receptor to kill CD38 expressing tumors including multiple myeloma (MM). The human IgG1 backbone provides additional Fc effector functionality to mediate antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC) against CD38 positive tumors. Here, we further characterized CYT-338 tumor cytotoxicity and show potent killing of MM tumor spheroids and serial killing activity of MM tumors in the presence of Cytovia's iPSC derived NK (iNK) or peripheral blood NK cells (PBNKs). Finally, we compared anti-tumor effects of CYT-338 and daratumumab in primary MM patient samples ex-vivo via multi-omic profiling and subsequent differential gene expression analysis to elucidate potential mechanisms contributing to the increased NK cell cytotoxicity observed with CYT-338.

Methods: MM1S tumor spheroids were established in ultra-low attachment U-bottom plates and killing assays were conducted with iNKs or PBNKs using the Incucyte ^TM Live Cell Analysis System. MM1S tumor cell serial killing assays were conducted by repeatedly adding the same iNK or PBNKs alone or in combination with CYT-338 to fresh tumor cells following each round of tumor killing. CYT-338 Fc effector function against MM1S tumors was evaluated for antibody dependent cellular phagocytosis (ADCP) using human macrophages differentiated from purified monocytes isolated from peripheral blood and for complement dependent cytotoxicity (CDC) in the presence of rabbit complement. The cytotoxicity of CYT-338 and daratumumab was evaluated using newly diagnosed MM patient CD138+ tumor cells and autologous NK cells purified from bone marrow and peripheral blood and co-cultured in a customized microfluidic platform for 24 hrs. Differential gene expression using mRNA isolated from these cultures was measured using Nanostring's nCounter® using the Pan cancer IO360^TM gene panel. Gene expression pathway analysis was conducted using Principal component analysis.

RESULTS: CYT-338 increased the ability of iNK and PBNK to kill MM1S tumor spheroids that peaked 2-3 days following of initiation of killing. Serial killing activity of iNKs and PBNKs against MM1S tumors declined over additional rounds of killing but the combination with CYT-338 maintained serial killing at original levels over multiple rounds of killing suggesting the ability of CYT-338 to overcome NK cell exhaustion. In the ADCP assay using purified human macrophages CYT-338 showed potent dose dependent killing of MM1S tumors indicating an additional effector cell pathway deployed by CYT-338. In addition, CYT-338 showed dose dependent CDC against MM1S tumors invoking the complement fixation pathway for MM killing. Autologous patient NK cell and MM co-cultures treated with CYT-338 showed increased cytotoxicity compared to daratumumab. Gene ontology enrichment analysis showed similar and dissimilar gene expression profiles with CYT-338 and daratumumab. CYT-338 and daratumumab showed increased expression of innate immunity and cytokine signaling pathway related genes (eg. IFI35, GZMK, JAK/STAT) and increased upregulation and down regulation of inhibitory receptors respectively (TIGIT and KIR3DL2). In contrast CYT-338 treatment showed highest enrichment for lymphocyte and leukocyte activation receptor related genes (eg. CD96, CD244, and CD2) and daratumumab showed highest enrichment for leukocyte migration and chemotaxis genes (eg. PECAM-1, CCL8, CXCL1). These differentially expressed genes may partly explain the increased cytotoxicity observed with CYT-338.

Conclusions: CYT-338 mediates potent anti- tumor activity against MM cell lines and patient tumors in the presence of effector cells (PBNK, iNK or MM patient NK cells). Evidence for additional CYT-338 effector function invoking macrophages and complement mediated MM tumor killing was obtained. Differential gene expression analysis provides mechanistic clues for a distinct cytotoxicity profile for CYT-338 from daratumumab that supports further evaluation of this potent anti-CD38 antibody.

Lin:Cytovia Therapeutics: Current Employment. Chang:Cytovia Therapeutics: Current Employment, Patents & Royalties: Compositions and Methods Relating to Genomic Modifications in Avian Primordial Germ Cells [US 10,577,612 - issued 03MAR2020]. Wu:LynxBio: Current Employment. Titz:LynxBio: Current Employment, Patents & Royalties: Inventor on patents filed by WARF. Chen:LynxBio: Current Employment. Chao Ma:Cytovia Therapeutics: Current Employment. Ashok:Cytovia Therapeutics: Current Employment. Zou:Cytovia Therapeutics: Current Employment. Dilmac:Cytovia Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bensussan:Inserm: Current Employment, Patents & Royalties. Kadouche:Cytovia Therapeutics: Current Employment, Current equity holder in publicly-traded company. Pak:LynxBio: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Frankel:Cytovia Therapeutics: Current Employment, Current equity holder in publicly-traded company. Teper:Cytovia Therapeutics: Current Employment. Li:Cytovia Therapeutics: Current Employment. Arulanandam:Cytovia Therapeutics: Current Employment.