CYT-338 NK Cell Engager Mutispecific Antibody Engagement of NKp46 Activation Receptor Overcomes Anti-CD38 Mab Mediated Fratricide of NK Cells and Accords Enhanced Cancer Immunotherapy

Background and Significance: Despite the clinical success of the first anti-CD38 targeted monoclonal antibody, daratumumab, approved for the treatment of multiple myeloma (MM), significant challenges remain such as CD38 antigen loss/internalization and/or natural killer (NK) cell fratricide resulting in resistance to treatment over time.

Study Design and Methods: To overcome the above-mentioned challenges, Cytovia has utilized its proprietary multifunctional tetravalent Flex Engager ^TM bispecific antibody IgG scaffold platform that contains a flexible linker for simultaneous engagement of effector immune cells and tumors targets. Through this, we generated CYT-338, an NK cell engager mutispecific antibody that targets CD38 on MM tumors and NKp46 and CD16 activation receptors on NK cells to mediate potent NK cell redirected killing of MM tumor cells more efficaciously than daratumumab. In serial killing assays against MM tumors, CYT-338 reversed dysfunctional NK cells to its original activation state and prevented exhaustion of killing activity of NK cells over time. In addition, we have also previously reported that CYT-338 has a higher potency over daratumumab for antibody dependent cellular phagocytosis (ADCP) of MM tumors. Preclinically,CYT-338 shows significantly reduced NK cell fratricide compared to daratumumab but the reason for this was unknown. We hypothesized that CYT-338 could overcome anti-CD38 mAb mediated fratricide of NK cells by engaging NKp46 activation receptor. To this end we incubated peripheral blood NK cells (PBNK) with either anti-CD38 mAb, daratumumab, CYT-338 or human IgG1 isotype control either in the presence or absence of blocking anti-NKp46 mAb (IgG1) for 24 hrs at 37 ^oC and stained with Annexin V apoptosis and Zombie NIR viability dye and analyzed NK fratricide by flow cytometry. We also evaluated the ability of anti-NKp46 mAb to inhibit daratumumab-mediated fratricide of NK cells under the same conditions.

Current Status: Through functional preclinical investigations, we have been able to deconvolute the novel molecular mechanisms whereby NKp46 and CD16 co-engagement signals by CYT-338 on NK cells are able to overcome the CD38 apoptotic fratricide signals induced by the single anti-CD38 mAb. Specifically, we have found that blockade of CYT-338 binding to NKp46 by anti-NKp46 IgG1 antibody reversed the fratricide protection provided by CYT-338, indicating that the fratricide protection was mediated by NKp46 activation signals. In addition, the single anti-NKp46 mAb was able to inhibit daratumumab-mediated NK cell fratricide providing direct evidence that daratumumabmediated fratricide can also be reversed by engagement of NKp46 activation receptor. Conclusions: The unique NKp46 activation mechanism provided by CYT-338 for reducing NK cell fratricide and maintaining NK cell levels together with the enhanced potency including reversal of NK cell dysfunction makes it an attractive best-in-class anti-CD38 therapeutic against MM compared to daratumumab. These results support the development of CYT-338 in both monotherapy and combination with other complementary immunotherapy agents being developed or approved for MM. A first-in-human Phase 1 study targeting patients with relapsed/refractory MM is in development in the U.S.