CD19 Targeting of Lymphoid Malignancies by Novel Fc-Domain Engineered Monoclonal Antibody.

Abstract 3725

Poster Board III-661

CD19 is a lineage-specific B-cell antigen, expressed at a high density on CLL cells, that contributes to B-cell receptor signaling but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified Fc-domain designed to enhance binding of FcγRIIIa that is predominately expressed on Natural Killer (NK)-cells. Utilizing freshly isolated chronic lymphocytic leukemia (CLL) patient B-cells we demonstrate that XmAb5574 lacks significant internalization seen with other anti-CD19 antibodies [maximum internalization for XmAb5574 was only 27.9% at 30-minutes (95%CI 14.5%, 41.4%)], thereby enhancing its ability to induce potent antibody-dependent cellular cytotoxicity (ADCC). Annexin V/PI flow cytometry analysis revealed that XmAb5574 mediates modest direct cytotoxicity not significantly different from Rituximab (0.6% increase, 95%CI -10.5%, 11.7%, p=0.91), and no complement mediated cytotoxicity (CDC) against primary CLL B-cells. Multi-color flow cytometry and monocyte derived macrophages (MDM) were used to assess XmAb5574 antibody dependent cellular phagocytosis (ADCP) against CLL cells and revealed no significant impact of the Fc-domain modification on MDM induced ADCP against CLL cells as compared to the wild type parental anti-CD19 antibody (12.37% vs. 10.51%, p=0.58). Interestingly, utilizing NK-cells and CLL cells isolated from normal donors and CLL patients, and employing autologous and allogeneic effector-target (E:T) conditions, XmAb5574 was found to mediate significantly higher ADCC when compared to the control humanized anti-CD19 non-engineered antibody (26.9% higher at E:T 25:1, p=0.0004 for allogeneic conditions, and 23.6% higher, p=0.004 for autologous conditions). ADCC mediated by XmAb5574 was also significantly higher as compared to Rituximab (33.5% higher at E:T 25:1, p<0.0001 for allogeneic conditions and 27.1% higher, p=0.004 for autologous conditions), a therapeutic antibody widely utilized in the treatment of CLL, hence confirming the functional in vitro efficacy and utility of the Fc-domain modification. By using inhibitor studies we further provide mechanistic insight into the XmAb5574–dependent ADCC mediated by NK-cells through a Granzyme B dependent mechanism. XmAb5574 also enhanced NK-cell activation as exhibited by an increased phosphorylation of Erk1/2 downstream of Fcγ receptor. The enhancement of subsequent cytolytic and secretory function was shown by the measurement CD107a up regulation on the surface of NK-cells (19.4% increase, p=0.005, as compared to wild type anti-CD19 antibody), and interferon-gamma release as measured by ELISA assays (6.4 times higher, p=0.007, as compared to wild type anti-CD19 antibody). Notably, enhanced NK-cell mediated ADCC observed with XmAb5574 against primary CLL B-cells could be augmented further by treatment with Lenalidomide (17.9% higher, p=0.04). These findings provide strong pre-clinical evidence for further clinical development of XmAb5574, both as monotherapy and in combination with Lenalidomide, for the therapy of CLL and related CD19⁺ B-cell malignancies. We also provide mechanistic insight into the utility and feasibility of Fc-domain engineering of specific antibodies, which will enhance their efficacy through an increased ability to recruit the innate immune system to more effectively control tumor progression.