Denintuzumab Mafodotin Stimulates Immune Responses and Synergizes with CD20 Antibodies to Heighten Anti-Tumor Activity in Preclinical Models of Non-Hodgkin Lymphoma

Denintuzumab Mafodotin (SGN-CD19A) is an antibody-drug conjugate (ADC) composed of an anti-CD19 antibody attached to a synthetic cytotoxic agent, monomethyl auristatin F (MMAF). The ADC binds CD19, internalizes, and releases cys-mcMMAF, which ultimately results in G2-M growth arrest and induction of apoptosis in targeted cells (Law et. al. AACR 2011). SGN-CD19A has shown convincing antitumor activity in relapsed/refractory non-Hodgkin lymphoma patients, resulting in a significant number of objective responses, including complete responses with prolonged durability (Moskowitz et. al. Blood 2015). To further characterize the anti-tumor activity of SGN-CD19A, we evaluated whether immune-mediated cell killing and immunogenic cell death (ICD) act in addition to the previously described apoptotic cell death. We then examined whether these mechanisms of cell death are augmented by clinically approved CD20 antibodies.

Our results show that SGN-CD19A supports immune-mediated killing of lymphoma cell lines in vitro through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Assessment of disseminated and subcutaneous tumor xenografts showed that SGN-CD19A activity is reduced when the wild type antibody is replaced with IgG variants that lack effector function. We also found depletion of macrophage or NK cells eliminates the improved survival conferred by the parental antibody of SGN-CD19A. Together these data provide strong evidence that effector function contributes to activity of SGN-CD19A in vivo.

Next, we implemented an in vitro system pairing lymphoblastoid cell lines (LCLs) with autologous PBMC to investigate the effect of SGN-CD19A-mediated cell death on immune activation. Treatment of LCL with SGN-CD19A drove hallmarks of ICD as indicated by activation of the ER stress response along with upregulation of surface calreticulin. In vitro co-culture of SGN-CD19A treated LCL with autologous PBMC resulted in heightened monocyte activation and expansion of LCL-reactive cytotoxic T cells supporting a role for SGN-CD19A in augmenting anti-tumor immune responses.

Finally, we evaluated the anti-tumor activity of SGN-CD19A in the presence and absence of clinically relevant CD20 antibodies. Results showed that CD20-targeted antibodies impact internalization of CD19 antigen leading to improved drug delivery of SGN-CD19A. Blocking B cell signaling inhibits the effect of CD20 antibodies on CD19 and SGN-CD19A. The improved anti-tumor activity observed with SGN-CD19A plus CD20 therapeutics was confirmed in vivo using rituximab. In conclusion, our results demonstrate SGN-CD19A is a multifunctional ADC capable of eliciting cell killing through immune-mediated mechanisms as well as delivery of the cytotoxic payload, cys-mcMMAF. Importantly, the described mechanisms of SGN-CD19A activity are further augmented by CD20 antibodies. These studies support the ongoing clinical evaluation of SGN-CD19A, which is currently being tested in randomized Phase II clinical trials in combination with R-ICE (NCT02592876) as well as R-CHOP (NCT02855359).