Abstract 646: Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1
Authors: Herrmann Amanda , Im Jin , Lu Sijie , Molldrem Jeffrey 
Published: May 2015

Acute (AML) and chronic (CML) myeloid leukemia together account for approximately 50% of newly diagnosed leukemias. Both AML and advanced CML have dismal survival outcomes but are susceptible to immunotherapy. We discovered PR1 (VLQELNVTV) as an effective human leukocyte antigen (HLA)-A2 restricted peptide and have targeted it in clinical trials using PR1 peptide vaccine. PR1 is derived from the serine proteases proteinase-3 and neutrophil elastase which are highly expressed in myeloid leukemia blasts. PR1 was shown to be immunogenic in myeloid malignancies, and we are currently developing several PR1 targeting therapies. We have completed the development and humanization of a T-cell receptor-like monoclonal antibody (h8F4) that targets PR1/HLA-A2 and eliminates primary human AML xenografts in NSG mice. To improve the efficacy of h8F4, we have developed a bi-specific T-cell engaging (BiTE) antibody that targets leukemia PR1/HLA-A2 and the T-cell receptor CD3. This would enhance the anti-leukemia immune response by bringing the T-cell in close proximity to the leukemia cell, and by further activating the T-cells to eliminate the target leukemia. Here we demonstrate successful construction and expression of the h8F4 BiTE in CHO cells. The antibody was purified from cell culture supernatant via Ni-NTA column chromatography, and identity was confirmed using western blot analysis. Next, flow cytometry was used to test the binding specificities of each targeting region of the BiTE antibody. The h8F4 BiTE antibody showed increased binding to T2 cells pulsed with PR1 peptide compared with T2 pulsed with irrelevant CMV peptide pp65 (MFI=627 and 72.4, respectively). In addition, the h8F4 BiTE demonstrated 6.8 fold higher binding to wild type Jurkat cells, which express CD3, in comparison with the CD3-lacking mutant Jurkat cell line J.RT3-T3.5. These results indicate that both regions of the h8F4 BiTE can efficiently recognize their intended targets. Next, we investigated whether h8F4 BiTE engagement with both PR1/HLA-A2 on T2 cells and CD3 on healthy donor PBMC could activate T-cells. We show with flow cytometry that upon 24h incubation in the presence of h8F4 BiTE and T2 cells pulsed with PR1 peptide, both CD4 and CD8 T-cells increase their surface expression of CD69 (21 fold in CD4; 10.3 fold in CD8), CD25 (35 fold in CD4; 3.5 fold in CD8) and CD71 (26.6 fold in CD4; 640 fold in CD8) compared to co-incubation in the absence of BiTE protein. In conclusion, this study shows that we have successfully developed a h8F4 BiTE which recognizes both the CD3 on T cells and PR1/HLA-A2 complexes on myeloid leukemia. Furthermore, the h8F4 BiTE can activate multiple T-cell subsets from healthy donor PBMC as early as 24h following co-incubation with PR1-pulsed T2 cells. This novel BiTE antibody could provide a safer and more potent treatment option for patients with aggressive myeloid leukemias that will improve upon currently available highly toxic standard therapies.

Citation Format: Amanda Herrmann, Jin Im, Sijie Lu, Jeffrey Molldrem. Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2014-646