The IL-15 superagonist ALT-803 enhances anti-CD20 antibody-directed NK cell ADCC and in vivo clearance of B cell lymphomas

Abstract

Anti-CD20 monoclonal antibodies (mAbs) have provided an important therapeutic passive immunotherapy approach for B cell malignancies. Typically anti-CD20 mAbs are combined with traditional chemotherapy and/or radiation therapy that have the potential for serious long term adverse complications. Novel approaches are needed to improve anti-CD20 mAb anti-lymphoma efficacy with agents that do not result in long term toxicity. ALT-803 is a super agonist IL-15 variant bound to an IL-15Ralpha - Fc fusion and represents a novel immunostimulatory agent for NK and T cells. Notably, ALT-803 exhibits extended in vivo pharmacokinetics and altered biodistribution compared to recombinant human IL-15. We hypothesized that ALT-803 would enhance anti-CD20-mAb-(rituximab) directed NK cell antibody dependent cellular cytotoxicity (ADCC) against B cell lymphomas. ALT-803 at concentrations of 0.35-35 ng/mL potentiated rituximab-triggered NK cell ADCC against the Raji (27% vs. 74%, E:T 25:1, P<0.01) and Daudi (39% vs. 84%, E:T 2:1, P<0.05) B cell lymphoma lines in vitro. Moreover, the activation of NK cells with ALT-803 significantly increased ADCC against primary human follicular lymphoma cells in vitro (11% vs. 33% at a 2.5:1 E:T ratio, P<0.001, N=5 primary follicular lymphomas, N=15 NK cell donors). One mechanism whereby ALT-803 may be modulating human NK cells is via the induction of cytotoxic effector molecules. After 24-48 hour in vitro activation with ALT-803 (0.35-350 ng/mL) an increased expression of granzyme B and perforin were observed in primary human NK cells (P<0.05). The effectiveness of ALT-803 enhancement of NK cell ADCC against B cell lymphoma cell lines was assessed with two in vivo mouse models. First, Daudi cells were engrafted into SCID mice (that have an intact NK cell compartment), and groups were treated with vehicle, rituximab (10 mg/kg), ALT-803 (0.2 mg/kg), or ALT-803+rituximab at day 15 and 18, and assessed for lymphoma percentages in the bone marrow at day 22. Mice treated with ALT-803+rituximab had significantly reduced Daudi B cell burden, compared to rituximab, ALT-803, or vehicle treatment (vehicle versus ALT-803+rituximab, 38% vs. 5%, P<0.01). At doses of 0.2-0.02 mg/kg the effect of ALT-803 on rituximab mediated lymphoma clearance was demonstrated (P<0.02 compared to vehicle treatment). Further, the survival of mice treated with ALT-803+rituximab was significantly longer compared vehicle control, rituximab alone, and ALT-803 alone groups (see KM survival curves, Figure, P<0.05). In our second in vivo model, malignant Raji B cells expressing luciferase were injected into immunodeficient NOD-SCID-gamma-c-/- (NSG) mice (100,000/mouse, day 0) followed by primary human NK cells (4 million / mouse, day 3), and treated (initiated on day 3) with vehicle, ALT-803 (0.05 mg/kg q3-4 days), rituximab (10 mg/kg day 3), or ALT-803+rituximab. At day 16, ALT-803+rituximab exhibited a significant reduction in Raji signal compared to the control groups (P<0.05). These results were confirmed in a second approach where an increased Raji cell numbers were engrafted (1 million / mouse, day 0), primary human NK cells (4 million / mouse) were infused on day 3, and groups of mice were treated with rituximab (5 mg/kg) or ALT-803 (0.2 mg/kg q3-4 days)+rituximab (mean photons per second at day 34, 3.8x109 versus 3.1x108, respectively, P<0.05). ALT-803 was well tolerated at all of the administered dose levels in combination with rituximab. Thus, ALT-803 represents an effective immunostimulatory agent that augments NK cell cytotoxic potential and ADCC against malignant follicular lymphoma and B cell lines in vitro, and significantly increases rituximab-directed clearance of B cell lymphoma by NK cells in two in vivo models. Based on these findings, a phase 1/2 clinical trial of ALT-803 plus rituximab is planned for patients with relapsed/refractory indolent non-Hodgkin lymphomas. (Figure Presented).