Development of mbIL-21 expanded Natural Killer cells for CLL immunotherapy
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Date
2021-04
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Abstract
Background & Purpose: Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia. While CLL treatment outcomes have improved significantly in recent years, CLL is still incurable and efforts to utilize curative cellular therapy has been limited by the intrinsic immunosuppressive nature of this disease. Natural killer (NK) cells are innate immune cells that function to destroy cancer or virus-infected cells. While NK cells can have potent anti-cancer effects, previous efforts to use them to treat patients with CLL have been constrained by poor NK cell growth and anti-leukemia activity against the NK-suppressive effects of CLL. Stimulating NK cells ex vivo with K562-based feeder cells expressing membrane-bound IL-21 (mbIL-21) results in significantly improved expansion and activation that leads to potent cytotoxicity against other cancer cells (Denman et al. PLoS ONE 2012). Collectively, these findings suggest that this expansion method may overcome the limitations seen with prior techniques for NK cell therapies for CLL. Our goal with this project was to test the in vitro and in vivo efficacy of mbIl-21-expanded NK cells as a potential novel cell therapy for CLL.
Methods: We isolated NK cells from peripheral blood of normal donors or CLL patients and expanded them in vitro using mbIL-21 expressing feeder cells and IL-2. Calcein release assays measured both direct cytotoxicity (without antibody) and antibody-dependent cell-mediated cytotoxicity (ADCC) against allogeneic and autologous patient CLL leukemia cells. To understand the mechanisms of NK targeting of CLL cancer cells, we used conjugation assays, death ligand blockade, and a degranulation inhibitor. Finally, we tested efficacy of expanded NK cells in vivo using a CLL cell line xenograft mouse model.
Results: Healthy donor-derived NK cells expand vigorously when stimulated with mbIL-21 expressing feeder cells (average 179-fold over 14 days, n=9) and show superior potency against CLL patient leukemia samples compared to unstimulated NK cells (e.g. at 2.5:1 NK:CLL ratio using obinutuzumab ADCC, 69% vs. 47% cytotoxicity, n=13 expanded/6 unstimulated, p=.004). In contrast to previous expansion strategies, this technique is able to overcome the existing NK suppression seen in CLL patients and expand CLL patient-derived NK cells equally effectively as normal donor-derived NKs (average 360-fold over 14 days, n=5). These patient-derived NKs are equally potent to normal donor-derived NKs, including against both unrelated allogeneic CLL and the patient's own autologous leukemia cells (obinutuzumab ADCC at 2.5:1 NK:CLL ratio, 68% and 70% killing, n=21 allogeneic/7 autologous). This may enable for the first time the effective use of a CLL patient's own NK cells as a therapeutic agent, lessening the risks of off-target immune reactions or NK cell rejection associated with allogeneic cell therapy. Primary CLL tumor cells are not susceptible to direct cytotoxicity (without targeting antibody) but are very vulnerable to ADCC using CLL-targeting antibodies rituximab or obinutuzumab (at 2.5:1 NK:CLL ratio, 10%, 52%, and 67% killing, respectively; n=10, p<.0001). This lack of direct cytotoxicity is correlated to low NK:CLL quantitative conjugate formation, suggesting a lack of tumor target recognition (21% binding untreated, 42% with rituximab, 50% with obinutuzumab, n=6, p=.001). Blocking antibodies to FasL, TNFα, and TRAIL show that only TRAIL contributes to anti-CLL direct cytotoxicity (at 2.5:1 NK:CLL, 29% killing untreated, 12% with TRAIL blockade, n=5, p<.0001). Application of folimycin (a degranulation inhibitor) eliminates killing activity in the presence or absence of targeting antibodies (1% killing at 2.5:1 ratio for both conditions, n=4-8), suggesting that perforin/granzyme release is the major mechanism of cytotoxicity. In vivo, treatment with expanded NK cells plus obinutuzumab prolongs survival after OSU-CLL xenograft in mice (median survival 49 days untreated vs. >6 months with NK+Obinutuzumab, n=7/group).
Conclusion & Implications: IL-21 expanded NK cells are a promising new therapy for CLL. NK cells stimulated with this technique show promising efficacy against human CLL cells both in vitro and in vivo. This includes both allogeneic and autologous tumor directed cytotoxicity. Our additional studies demonstrate a reliance on antibody targeting for CLL recognition by NK cells and emphasize the role of perforin/granzyme release in anti-leukemia activity, with some contribution by TRAIL. Based on these data, we are currently in the early stages of planning a clinical trial to test this therapy for patients with CLL.
Description
Health Sciences: 2nd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)
Keywords
leukemia, natural killer, NK cell, immunotherapy, CLL