Glypican-3 (GPC3) is an oncofetal antigen that is highly expressed in multiple solid tumors, including hepatocellular carcinoma, and is barely expressed in adult normal tissues except the placenta. NKp46 activation receptor is expressed in all-natural killer (NK) cells, including tumor-infiltrating NK cells. FLEX-NK^TM is a platform for the production of tetravalent multifunctional antibody NK cell engagers (NKE). CYT-303 was designed using the FLEX-NK scaffold, incorporating a novel humanized NKp46 binder that does not induce NKp46 internalization and a humanized GPC3 binder that targets the membrane-proximal lobe to mediate NK cell-redirected killing of HCC tumors. CYT-303 shows sub-nanomolar binding affinities to both GPC3 and NKp46. CYT-303 was highly potent and effective in mediating NK cell-redirected cytotoxicity against multiple HCC tumor cell lines and tumor spheroids. More interestingly, it can reverse the dysfunction induced in NK cells following repeated rounds of serial killing of tumors. It also mediated antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity against GPC3-expressing HCC tumors. In vivo, CYT-303 showed no toxicity or cytokine release in cynomolgus monkeys up to the highest dose (60 mg/kg), administered weekly by intravenous infusion for 28 days. These results demonstrate the potential of CYT-303 to be a safe and effective therapy against HCC.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC₅₀ in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.