3074 Background: SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40. It is derived from dacetuzumab, a humanized IgG1 previously developed for B-lineage malignancies. Antibody glycosylation is essential for Fc receptor-mediated activity and nonfucosylated antibodies may show improved efficacy via increased binding to FcγRIIIa (CD16). Methods: Enhanced functionality of SEA-CD40 was determined through FcγRIIIa binding, immune activation, and induction of antigen-specific T-cells Results: While SEA-CD40 and the parent antibody dacetuzumab bind to CD40 with similar affinity, the non-fucosylated SEA-CD40 has a higher affinity for both low (158F) and high (158V) affinity FcγRIIIa. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent ADCC activity and improved agonistic signaling to antigen presenting cells (APCs). SEA-CD40 treatment of human PBMCs elicits a robust immune response consisting of proinflammatory cytokine production, APC maturation and up-regulation of co-stimulatory receptors on APCs with activity at antibody concentrations as low as 10 ng/ml. Utilizing a surrogate antibody against mouse CD40, the immune stimulatory properties of nonfucosylated anti-CD40 were confirmed in vivo in syngeneic tumor models. SEA-CD40 induction of antigen specific T-cells was assessed using human peripheral blood mononuclear cells (PBMCs) exposed to the M1 influenza antigen. SEA-CD40 stimulated the expansion of influenza specific T-cells and elevated their production of IFNγ. Likewise, SEA-CD40 also stimulated T-cell proliferation and IFNγ production in PBMCs from melanoma, pancreatic, or breast cancer patients in response to a mixture of the tumor-associated antigens MAGE-A1/A3. Interestingly, antigen-specific T-cell responses to both the influenza and tumor antigens were enhanced in the presence of blocking antibodies to CTLA4 or PD1. Conclusions: These observations demonstrate the potential of combining the non-fucosylated agonistic SEA-CD40 with immune check point inhibitors to generate more effective adaptive antitumor immune responses.
Leukemias with MLL gene rearrangement are associated with a poor prognosis. Natural killer (NK) cell therapy is a potential treatment, but leukemia cells may be resistant. Here, we sought to determine the susceptibility of MLL-rearranged leukemia cells to NK cell lysis and to develop a novel immunotherapeutic approach to optimize NK cell therapy, including the use of an antibody against leukemia-associated antigen and the elimination of killer-cell immunoglobulin-like receptor (KIR)-mediated inhibition.Three MLL-rearranged leukemia cell lines (RS4;11, SEM, and MV4-11) and primary leukemia blasts were assessed for surface phenotype and susceptibility to NK cell lysis with or without antibodies against CD19 (XmAb5574), CD33 (lintuzumab), or KIR ligands.All three cell lines were resistant to NK cell lysis, had some inhibitory KIR ligands and protease inhibitor-9, and expressed low levels of NKG2D activating ligands and adhesion molecules. After treatment with XmAb5574 or lintuzumab, MLL-rearranged leukemia cells were efficiently killed by NK cells. The addition of pan-major histocompatibility complex class I antibody, which blocked inhibitory KIR-HLA interaction, further augmented degranulation in all three KIR2DL1, KIR2DL2/3, and KIR3DL1 subsets of NK cells based on the rule of missing-self recognition. A mouse model showed a decreased rate of leukemia progression in vivo as monitored by bioluminescence imaging and longer survival after antibody treatment.Our data support the use of a triple immunotherapy approach, including an antibody directed against tumor-associated antigen, KIR-mismatched NK cell transplantation, and inhibitory KIR blockade, for the treatment of NK cell-resistant MLL-rearranged leukemias.
