Immunomodulatory Effects and Adaptive Immune Response to Daratumumab in Multiple Myeloma
Authors: Krejcik Jakub , Casneuf Tineke , Nijhof Inger , Verbist Bie , Bald Jaime , Plesner Torben , Liu Kevin , van de Donk Niels W.C.J. , Weiss Brendan , Ahmadi Tahamtan , Lokhorst Henk M. , Mutis Tuna , Sasser A. Kate 
Published: October 2019

Abstract
Introduction: Daratumumab (DARA) is a novel human monoclonal antibody that targets CD38, a protein that is highly expressed on multiple myeloma (MM) cells. DARA acts through multiple immune effector-mediated mechanisms, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis. In two clinical studies (NCT00574288 [GEN501] and NCT01985126 [Sirius]) of DARA monotherapy in patients with relapsed and refractory MM, overall response rates were 36% and 29%, respectively. CD38 is highly expressed in myeloma cells but also expressed in lymphocytes and other immune cell populations. Therefore, the effects of DARA on immune cell populations and adaptive immune response pathways were investigated.
Methods: The patient population investigated included treated subjects with MM that were relapsed after or were refractory to ≥2 prior therapies (GEN501) or had received ≥3 prior therapies, including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD), or were refractory to both a PI and an IMiD (Sirius). Patients assessed in this analysis were treated with 16 mg/kg DARA. When both studies were combined, median age (range) was 64 (31-84) years and median time from diagnosis was 5.12 (0.77-23.77) years. Seventy-six percent of patients had received >3 prior therapies and 91% were refractory to their last treatment. Clinical response was evaluated using IMWG consensus recommendations. Peripheral blood (PB) samples and bone marrow (BM) biopsies/aspirates were taken at prespecified time points and immunophenotyped by flow cytometry to enumerate various T-cell sub-types. T-cell clonality was measured by TCR sequencing. Antiviral T-cell response and regulatory T-cell (Treg) activity were analysed by functional in vitro assays. T-cell subpopulation counts were modelled over time with linear mixed modelling. Two group comparisons were performed using non-parametric Wilcoxon rank sum tests.
Results: Data from 148 patients receiving 16 mg/kg DARA in GEN501 (n = 42) and Sirius (n = 106) were analyzed for changes in immune response. In PB, robust mean increases in CD3+ (44%), CD4+ (32%) and CD8+ (62%) T-cell counts per 100 days were seen with DARA treatment. However, responding evaluable patients (n = 45) showed significantly greater increases from baseline than nonresponders (n = 93) in CD3+ (P = 0.00012), CD4+ (P = 0.00031), and CD8+ (P = 0.00018) T cells. In BM aspirates the number of CD3+, CD4+, and CD8+ T-cells increased during treatment compared to baseline (the median percent increases were 19.95%, 5.66%, and 26.99% [n = 58]). Additionally, CD8+: CD4+ T-cell ratios significantly increased compared to baseline in both PB (P = 0.00017), and BM (P = 0.00016). T cell clonality, assessed by TCR sequencing, increased after DARA treatment compared with pretreatment (P = 0.049), with greater sums of absolute expansion in the repertoire (P = 0.037), as well as greater maximum expansion of a single clone (P = 0.048) in responders compared to nonresponders. Increased antiviral T-cell responses were observed post-DARA treatment, particularly in responders. Interestingly, a novel subpopulation of regulatory T cells was identified that expressed high levels of CD38. These cells comprised ~10% of all Tregs and were depleted by one DARA infusion. In ex vivo analyses, CD38+ Tregs appeared to be highly immune suppressive compared to CD38-Tregs.
Conclusions: Robust T cell increases, increased CD8+: CD4+ ratios, increased antiviral responses, and increased T cell clonality were all observed after DARA treatment in a heavily pretreated, relapsed, and refractory patient population not expected to have strong immune responses. Improved clinical responses were associated with changes in these parameters. In addition, a sub-population of regulatory T cells expressing high CD38 levels was determined to be extremely immune suppressive and sensitive to DARA treatment. These data suggest a previously unknown immune modulatory role of DARA that may contribute to its efficacy, and a potential role for CD38 immune targeted therapies. We postulate that there are several distinct and complementary mechanisms that contribute to DARA's efficacy including increased antigen presentation through phagocytosis, targeting of immune suppressive Tregs, and increased adaptive immune responses.
JK and TC contributed equally to this work.

Disclosures
Casneuf: Janssen: Employment. Verbist:Janssen: Employment. Bald:Janssen: Employment. Plesner:Genmab: Membership on an entity's Board of Directors or advisory committees; Roche and Novartis: Research Funding; Janssen and Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Liu:Janssen: Employment. van de Donk:Janssen Pharmaceuticals: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Weiss:Janssen and Onclave: Research Funding; Janssen and Millennium: Consultancy. Ahmadi:Janssen: Employment. Lokhorst:Genmab: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria. Mutis:Janssen: Research Funding; Genmab: Research Funding.