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BackgroundDespite recent success of monoclonal antibodies (mAb) and T cell redirecting therapies, relapsed/refractory (RR) multiple myeloma (MM) patients remain in urgent need of new therapies with distinct mechanisms of action (MoA). Modakafusp alfa, previously known as TAK-573, is a novel immune-targeted attenuated cytokine specifically designed to deliver interferon-alpha to CD38-expressing cells via a CD38-targeting IgG4 mAb. Preliminary results of a first in human phase 1 trial with modakafusp alfa monotherapy (1.5mg/kg, every four weeks) in RRMM patients (90% anti-CD38 mAb refractory; 37% anti-BCMA agent exposed) indicate a promising 40% overall response with manageable toxicity (Kaufman et al. EHA 2022 abstract S181). Prior studies suggest a unique and multimodal MoA of modakafusp alfa, including anti-proliferative effects on tumor cells as well as stimulation of CD38-positive immune effector cells. Towards a thorough understanding of these immunomodulatory effects, specifically regarding the activation of innate immunity, we here studied the effects of modakafusp alfa on the anti-MM activity of NK cells, which highly express CD38.AimsWe assessed the ex vivo activity of modakafusp alfa on NK cell activation, degranulation, and antibody-independent cytotoxicity. In addition, we investigated the impact of modakafusp alfa on NK cell-mediated, antibody-dependent cellular cytotoxicity (ADCC) against MM cells using daratumumab (anti-CD38) or elotuzumab (anti-SLAMF7).MethodsNK cell activation was explored by flow cytometry after 24-hour incubation of peripheral blood mononuclear cells (PBMC) from healthy donors with modakafusp alfa or negative/specificity controls. These pre-incubated PBMCs were also used to examine NK cell degranulation after subsequent 4 hour stimulation with K562 or PMA/Ionomycin, or in a 16-hour cytotoxicity assay against K562. In addition, modakafusp alfa pre-incubated PBMCs or enriched NK cells were assessed for antibody-independent cytotoxicity or daratumumab/elotuzumab-mediated ADCC in 24-48 hour assays against MM cell lines RPMI-8226, MM.1S or UM9. Alternatively, modakafusp alfa was directly added to the assays without pre-incubation steps.ResultsWe found that incubation of PBMCs with modakafusp alfa for 24 hours resulted in upregulation of activation markers CD38 and CD69 on the surface of NK cells. In addition, modakafusp alfa pre-incubation significantly improved the degranulation of NK cells after 4-hour stimulation with K562 or PMA/Ionomycin. Importantly, overnight pre-incubation of PBMCs or enriched NK cells with modakafusp alfa showed enhanced antibody-independent cytotoxicity against K562 tumor cells and MM cell lines RPMI-8226, MM.1S or UM9. Consequently, this also resulted in augmented killing of MM cells in the presence of daratumumab or elotuzumab. We observed similar effects when modakafusp alfa was directly added to the cytotoxicity assays without pre-incubation of immune effector cells.ConclusionsModakafusp alfa rapidly activates NK cells, and improves their degranulation and anti-MM effectivity. This important MoA also results in improved efficacy of daratumumab or elotuzumab in short-term cytotoxicity assays. Our findings warrant clinical investigation of combining modakafusp alfa with NK cell-exploiting immunotherapies.DisclosuresCollins:Takeda Development Center Americas, Inc (TDCA): Current Employment, Current equity holder in publicly-traded company. Sampson:Takeda Development Center Americas, Inc (TDCA): Current Employment, Current equity holder in publicly-traded company. Van De Donk:Cellectis: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Zweegman:BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Mutis:Genmab: Research Funding; Janssen: Research Funding; Takeda: Research Funding.Author notes*Asterisk with author names denotes non-ASH members.

The authors note that Fig. 3 appeared incorrectly. "The authors would like to note that in Fig. 3B, the untreated (control) 031M organoids and those of the 081RF sup + a-IL-6 condition showed duplicate images at different exposures." The corrected figure and its legend appear below. The online version has been corrected.