The IL-2/IL-15 Mimetic NL-201 Prevents Myeloma Relapse after ASCT By Expanding Highly Cytolytic T Cells in the Bone Marrow That Are Resistant to Exhaustion
Authors: Minnie Simone A , Nemychenkov Nicole S , Takahashi Shuichiro , Schmidt Christine R , Legg Samuel RW , Ensbey Kathleen S , Hill Geoffrey R 
Published: November 2021

Abstract
Multiple myeloma (MM) is a bone marrow (BM) resident hematological malignancy that is becoming increasingly recognized as one amenable to immunotherapy, although no therapies have yet provided durable, long-term disease control. Autologous stem cell transplantation (ASCT), the standard of care in eligible patients, provides a window for intervention with immunotherapy due to the induction of inflammation in the context of lymphodepletion at a time where there is also minimal residual disease and a disrupted tumor microenvironment (TME). We have previously established that the addition of T cells to BM grafts results in enhanced long-term myeloma control post-transplant in mice. Novel approaches aimed at improving and/or expanding the endogenous T cell response early post-ASCT may therefore prove highly effective with the benefit of avoiding ex vivo processing associated with other cellular therapies. To explore this, we utilized the IL-2/IL-15 mimetic NL-201: a de novo cytokine mimetic that signals via the beta and gamma subunits of the IL-2 receptor without engaging IL-2Rα (CD25). In pre-clinical studies, NL-201 has demonstrated the ability to signal to effector CD4 and CD8 T cells while avoiding the toxicity usually associated with IL-2 signaling via IL-2Rα.
We hypothesized that NL-201 would enhance control of myeloma progression by stimulating T cell proliferation and activation early post-ASCT. We transplanted lethally irradiated Vk*MYC myeloma-bearing B6 recipients with BM and T cells graft from B6 donors and administered NL-201 from D+7 to week 6 (225 μg/kg weekly I.P). NL-201 promoted potent anti-myeloma immunity that was dependent on CD4 and CD8 T cells, but not NK cells (median survival was 68 days for control mice, unreached at &gt;120 days for NL-201 alone or with NK depletion, 86 days for NL-201 with CD8 depletion, and 74 days with CD4 depletion; PBS vs NL-201 p&lt;0.01; PBS vs NL-201 + αNK1.1 p&lt;0.01; NL-201 vs NL-201 + αCD4 or αCD8 p&lt;0.05). To further elucidate potential mechanisms of action we harvested BM from PBS and NL-201-treated mice 2 days after the last dose was administered and performed comprehensive immunophenotyping with high parameter flow cytometry. We grouped recipients based on whether they had controlled myeloma (MM-controlled) or had active disease progression at the time of harvest (MM-relapsed) to reveal immunological phenotypes that were dependent and independent of myeloma in the TME. In these experiments, all NL-201-treated recipients had controlled myeloma at time of harvest. Mechanistically, NL-201 significantly expanded the total number of CD8 T cells in the BM compared to PBS-treated mice with controlled or relapsed MM (PBS-treated mean CD8 T cell number was 1.0 x 10 5/femur vs 7.7 x 10 5/femur in NL-201-treated mice) but did not impact CD8 T cell number in peripheral blood. Memory CD8 T cells (CD44+CD62L+) were preferentially expanded, while the frequency of exhausted CD8 T cells (TOX +PD-1 +TIGIT +CD39 +; T EX) was reduced in NL-201-treated mice compared to both PBS-treated MM-relapsed and MM-controlled mice (75% T EX in PBS MM-relapsed, 15% PBS MM-controlled, 2% in NL-201; p&lt;0.001). Surprisingly, &gt;80% of the memory CD8 T cells in NL-201-treated mice produced granzyme B compared to &lt;10% in PBS-treated mice. Granzyme B production was also observed in conventional CD4 T cells in response to NL-201 treatment, and the frequency of regulatory T cells was reduced by 50% after NL-201 compared to PBS MM-controlled and MM-relapsed mice (p&lt;0.001).
NL-201 expanded bone marrow resident cytotoxic memory CD8 and CD4 T cells that are resistant to exhaustion, whilst reducing the frequency of regulatory T cells in the BM TME. Together, these data highlight the promising therapeutic potential of NL-201 in multiple myeloma and support testing NL-201 in clinical trials for the treatment of hematological malignancies.

Disclosures
Hill: NapaJen Pharma: Consultancy; Roche: Research Funding; Syndax Pharmaceuticals: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Applied Molecular Transport: Research Funding; Compass Therapeutics: Research Funding; NeoLeukin Therapeutics: Consultancy; Generon Corporation: Consultancy.