The IL15 Superagonist Complex ALT-803 Potently Enhances the Proliferative Capacity and Activity of in Vitro Expanded Natural Killer Cells

Introduction

We previously reported on the treatment of 8 relapsing high-risk multiple myeloma (HRMM) patients with ex vivo activated/expanded NK cells (ENKs). We demonstrated that interleukin-2 (IL2) provided suboptimal cytokine support post adoptive transfer of ENKs. Aiming to improve upon prior data we hypothesize that the novel IL15 superagonist complex ALT-803 will provide superior support compared to IL2 and enhance ENK activity, proliferation and persistence.

Methods

To compare the activity of IL2 and ALT-803 on ENKs, we performed long-term in vitro culture studies. Analogous to the 8-day clinical production method, healthy donor (HD) peripheral blood mononuclear cells (PBMCs) were first co-cultured with the leukemic cell line K562 modified to express both membrane-bound IL15 and the costimulatory molecule 4-1BBL, and high-dose IL2. We deliberately chose to maintain IL2 in the expansion method rather than substituting ALT-803 because our FDA-approved GMP-grade expansion was validated using IL2. On expansion Day 8 (the day clinical ENK products are infused), the ENKs were either maintained at high-dose (300U/mL) IL2, or otherwise cultured at low-dose (20U/mL) IL2 or 35ng/mL ALT-803, in all conditions without K562 stimulator cells. Proliferation (fold-change from baseline) was measured on Days 14, 21, and 28, and expression of key activating and inhibitory markers was assessed via flow cytometry. Relative cytolytic ability was determined via 4-hour chromium (Cr)-release assays against K562 and the MM cell lines OPM2, JJN3, and U266.

Results

Proliferation of ALT-803-ENKs was superior to both IL2-ENK conditions at all assessed time points; on Day 28, fold-expansion of ALT-803-ENKs was 753, versus 156- and 557-fold for ENKs maintained in 20U or 300U/mL IL2, respectively, Figure 1A. Immunophenotyping studies demonstrated significant differences between ENKs cultured in 20U/mL IL2 versus ALT-803-treated ENKs, including activation (CD26, CD69, DNAM1, NKp30) and functional (CD16, perforin, granzyme, TRAIL) molecules. ALT-803-treated ENKs maintained an activated phenotype, whereas IL2 ENKs (20U/mL) exhibited reduced expression of several markers by Day 14, with further decreases noted on Days 21 and 28. Representative examples are depicted in Figure 1B. Morphologically, IL2-ENKs acquired apoptotic features after extended culture, whereas ALT-803-ENKs did not. Though cytolytic ability was variable on Days 14 and 21, the activity of ALT-803-ENKs exceeded that of IL2-ENKs (20U/mL) against K562, OPM2, JJN3, and U266 cell lines on Day 28. High-dose IL2-ENKs exhibited a similar phenotype and cytolytic ability compared to ALT-803-ENKs.

Conclusions

We compared clinically-relevant doses of IL2 and ALT-803 to determine whether ALT-803 is superior at maintaining ENK activity and proliferative capacity. ALT-803 ENKs exhibited robust proliferation through Day 28 and sustained expression of key activation and functional markers, highlighting the superiority of ALT-803 in this setting. After four weeks of continuous culture, ALT-803-ENKs maintained greater cytolytic ability against MM cell lines. Though high-dose IL2 is capable of sustaining ENK activation state in vitro, this dose is not achievable in humans. Unlike IL2, ALT-803 does not induce expansion of T regulatory cells, and has a more favorable toxicity profile. Importantly, ALT-803 stimulated and maintained expression of CD16, indicating that it would be well-suited to pair with monoclonal antibodies for the support of antibody-dependent cellular cytotoxicity. Further studies are in progress to study the effects of ALT-803 on ENKs in the presence of suppressive cytokines/cells to determine if ALT-803 is able to overcome the effects of the tumor microenvironment.

Figure 1

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Figure 1

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