144 RTX-240, an allogeneic engineered red blood cell expressing 4–1BBL and IL-15TP, promotes NK cell functionality in vitro and in vivo

Background Agonist antibodies and recombinant cytokines have had limited success in the clinic due to three factors: severe toxicity leading to a narrow therapeutic index, the diminished activity of an agonistic antibody compared with natural ligand, and the lack of multiple signals needed to effectively activate most cell types. To address these limitations, Rubius Therapeutics has developed RTX-240, an allogeneic cellular therapy using red blood cells genetically engineered to express 4-1BBL and IL-15/IL-15Ra fusion (IL-15TP) in their natural conformation on the cell surface. RTX-240 is designed to recapitulate human biology by broadly stimulating adaptive and innate immunity to generate an anti-tumor response and provide improved safety due to the restricted biodistribution of red blood cells to the vasculature. Here we demonstrate that RTX-240 is highly active in preclinical models. Methods PBMCs or NK cells were treated with RTX-240 in vitro. mRBC-240 was used for in vivo studies. Results Treatment of either PBMCs or isolated NK cells with RTX-240 induced a dose-dependent increase in NK cell activation, proliferation and functionality. These effects were further enhanced with increased 4-1BBL and IL-15TP expression on the surface of RTX-240. NK cell counts, NKp44 and Trail expression were increased 150, 4.6 and 6-fold over media control, respectively. Activation of NK cells with RTX-240, followed by incubation with K562 targets enhanced NK cell cytotoxicity (1.3-2.8 over control), that was accompanied by increased NK cell activation (CD69) and degranulation (CD107a) (3.1-fold and 1.9-fold, respectively). RTX-240-activated NK cells showed higher frequency of CD56dim/CD16+ NK cells, which have been reported to induce natural and ADCC-dependent cytotoxicity. Correspondingly, RTX-240 promoted enhanced ADCC-induced killing of Raji cells when combined with anti-CD20 mAb (1.4-fold over control). Intravenous administration of mRBC-240 to a B16F10 intravenous lung metastases model led to NK cell expansion on Day 4 (3.8-fold over control). These NK cells were cytotoxic (Granzyme B+) and highly proliferative (Ki67+) (1.4-fold and 18.8-fold over control, respectively). Treatment with mRBC-240 increased the frequency of terminally differentiated NK cells (NK1.1+/CD11b+/CD27-/KLRG1+) in the tumor (2.1-fold increase over control). Terminally differentiated NK cells are highly cytotoxic and their frequency in the tumor was strongly correlated with efficacy in this model (p=0.0001). Conclusions Taken together, these data indicate that RTX-240 promotes NK cell activity and functionality in preclinical models. RTX-240 has now entered a first-in-human Phase 1 trial for the treatment of patients with relapsed/refractory or locally advanced solid tumors, with a planned arm evaluating RTX-240 in relapsed/refractory acute myeloid leukemia.