Potentiation of Anti-Myeloma Activity of Daratumumab with Combination of Cyclophosphamide, Lenalidomide or Bortezomib Via a Tumor Secretory Response That Greatly Augments Macrophage-Induced ADCP

Introduction

Daratumumab (Dara) is the first CD38-directed monoclonal antibody (moAb) approved anywhere in the world as well as the first moAb approved for the treatment of Multiple Myeloma (MM). While currently approved as monotherapy, recent results from randomized trials have shown dramatic activity in relapsed MM patients in combination with both lenalidomide (Len) and bortezomib (Bor) and Dara is likely to become an intrinsic component of MM regimens in the future. Based on pre-clinical data Dara is thought to have pleiotropic mechanisms of activity, killing myeloma cells via a direct anti-CD38 effect, induction of complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Therefore, we focused our attention on potential mechanisms of enhancing macrophage (Mφ)-mediated ADCP. Previously, Pallasch reported that resistance to Mφ-mediated killing in lymphoid malignancy can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, Cyclophosphamide (Cy) was shown to induce an acute secretory activating phenotype from treated tumor cells, which led to Mφ infiltration and phagocytic activity in the bone marrow. Given this background we sought to establish the existence of this phenomenon in MM in the context of drugs likely to be used in combination with Dara, i.e. Cy, Len and Bor, respectively.

Materials and Methods:

MM1S MM cells were conditioned with low dose single agents, Cy, Len and Bor for 24 hours. Media containing single agent chemotherapy was removed and replaced with fresh media for 24 hours (TCM). THP-1 Mφ were conditioned with TCM for up to 48 hours and then incubated at 2:1 effector target ratio with CFSE labelled MM1S cells for 18 hours in the presence or absence of Dara or isotype control antibody. The % of Dara-specific tumour cell clearance was calculated as the number of live CFSE labelled tumour cells in Dara-treated cells/Isotype-treated cells. Additionally, Mφ were pre-incubated with Cytochalasin D (CytoD), an inhibitor of actin polymerization, to determine if the mechanism of tumour cell clearance is mediated by Mφ mediated ADCP. MM1S expression of CD47, as well as Mφ expression of FC receptor expression, CD32 and CD64 was analysed by flow cytometry. Mφ migration was assessed using transwell assays assessment of fluorescently labelled Mφ migration to Cy, Len and /or Bor TCM.

Results:

Exposure of Mφ to TCM from Cy, Len and Bor treated MM1S cells significantly enhanced % Dara specific clearance of MM1S cells (p<0.01). The effect was also maintained when combinations of the Cy, Len and Bor were used. This effect was partially reversed by incubation of Mφ co-cultures with CytoD (p<0.01), indicating ADCP as one of the likely mechanisms of tumour cell clearance. We observed reduced expression of the "don't eat me" antigen, CD47, on chemotherapy treated MMIS cells after 24 hours, which could enhance phagocytosis. In addition, we detected induction of CD32 and CD64 Fc gamma receptor expression on conditioned Mφ as well as altered migratory capacity, which was more evident in Cy and Bor TCM conditioned Mφ.

Conclusions:

Exposure of MM cells to both low doses of Cy, Len and Bor leads to a secretory response, which, along with downregulation of CD47, greatly augments Mφ induced ADCP of Dara coated MM cells. This effect may explain in part the dramatic synergy observed between Len, Bor and Dara in clinical trials and provides a rationale for combining Cy with Dara in the clinic. Future research, including use of patient samples, will focus on understanding the mechanisms responsible for this anti-tumour effect.

Reference:

Pallasch CP et al. Sensitizing protective tumor microenvironments to antibody-mediated therapy. Cell. 2014 Jan 30;156(3):590-602.