Abstract 5612: Evaluating the cytotoxic effectiveness of a novel TCR-like bispecific T cell engager targeting the PR1/HLA-A2 leukemia antigen
Authors: Herrmann Amanda Cernosek , Im Jin Seon , He Hong , Anna Sergueeva , Lu Sijie , Molldrem Jeffrey 
Published: August 2018

Overall 5-year survival rates of myeloid leukemia patients remain low at &lt;30%, and new therapies for these patients are desperately needed. PR1 (VLQELNVTV) is a human leukocyte antigen (HLA)-A2 restricted peptide derived from serine proteases proteinase-3 and neutrophil elastase, which are aberrantly expressed in myeloid leukemia blasts, including acute myelogenous leukemia (AML). PR1 was shown to be immunogenic in myeloid malignancies, and we have conducted preclinical development and humanization of a T cell receptor-like monoclonal antibody (h8F4) that targets the leukemia-associated PR1/HLA-A2 complex and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC) following repeat dosing. To improve the potency of h8F4, we have developed a bispecific T cell-engaging antibody that targets PR1/HLA-A2 on leukemia and CD3 on neighboring T cells. Here we demonstrate successful production and purification of the h8F4 bispecific antibody. Utilizing flow cytometry, we confirm PR1/HLA-A2 and CD3-specific binding characteristics, T cell activation in the presence of PR1/HLA-A2, and importantly AML target cell cytotoxicity after h8F4 bispecific antibody engagement with healthy donor effector T cells. Cytotoxicity assays were performed with both AML cell lines and primary patient AML blasts serving as target cells and health donor PBMC as a source of effector T cells at an E:T ratio of 2:1. Results indicate up to 60% leukemia-specific lysis with 2nM h8F4 bispecific antibody after only 18 hours of incubation. In vivo data also confirm significant elimination of the leukemia cell line U937 (as evidenced by % leukemia cells detected in peripheral blood) in an NSG-U937 AML xenograft mouse model when compared to control groups treated with effector cells alone. Mice were infused with 5,000 leukemia cells followed by 3 million normal healthy donor PBMC. These infusions were followed by a week of daily h8F4 bispecific antibody injections (20ng/injection). Both bioluminescence and flow cytometry analysis two weeks following the final injection show a marked difference between the h8F4 bispecific antibody treatment group and controls, with a 1.5-fold reduction in circulating leukemia cells and an overall increase in survival. In conclusion, these studies demonstrate the therapeutic potential of a novel bispecific antibody targeting the PR1/HLA-A2 leukemia-associated antigen. This bispecific antibody appears to increase the potency of h8F4 with rapid elimination of AML compared to h8F4, and our studies justify potential development as a treatment option for patients with high-risk AML.

Citation Format: Amanda Cernosek Herrmann, Jin Seon Im, Hong He, Sergueeva Anna, Sijie Lu, Jeffrey Molldrem. Evaluating the cytotoxic effectiveness of a novel TCR-like bispecific T cell engager targeting the PR1/HLA-A2 leukemia antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5612.