ALBERT

Description: Abstract 2599 The Wilms' tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in leukemias and a wide range of cancers. WT1 may be expressed in leukemia stem cells. RMFPNAPYL, “RMF”, a WT1-derived CD8 T cell epitope presented by HLA-A0201, is a validated target for T cell-based immunotherapy, used in multiple clinical trials of vaccines and cellular therapies. Therefore, we hypothesized that a “TCR-like antibody” specific for the WT1 peptide/HLA-A0201 complex might be an effective therapeutic agent. Using phage display technology, we generated 2 lead, high avidity (Kd 〈 0.2nM), fully human monoclonal antibodies (mAb) specific for the WT1 RMF peptide/HLA-A0201 complex. One version, ESK1, is a native human IgG1. A second version, ESKM, with enhanced antibody dependent human effector cell cytotoxicity (ADCC) function due to altered Fc glycolsylation was also prepared. ESK mAb bind to leukemia lines and other cancer cell lines, as well as primary leukemia cells that are both WT1+ and HLA-A0201+. In vitro, both ESK mAb mediated ADCC against CML cells, at concentrations below 3 ug/ml, but ESKM was about 8–10 fold more potent and could kill targets with far fewer peptide/MHC complexes expressed on the cell surface. At therapeutic doses of ESKM, there was no difference in biodistribution between the 2 mAb in C57BL6 mice or in mice that were transgenic for HLA-A0201. Low doses of ESK (25–100ug twice) effectively treated an established disseminated, ALL or human bcr/abl + lymphoid leukemias in a NSG mouse model (T, B and NK deficient) and prolonged survival. In mice, ESKM was slightly more effective than ESK1. An F(Ab')2 version of the antibody had no anti-tumor effect, indicating that an Fc-mediated mechanism plays the major role in therapy. When combined with a single infusion of human CD34-, CD3-, human NK and monocyte effectors in the NSG mice, therapeutic effects of the mAb were more pronounced and more durable. There was no therapeutic effect of either mAb on WT1 low/A0201 negative disseminated Daudi ALL in mice. There was no observed toxicity in HLA-A0201 transgenic mice at the therapeutic mAb dose and schedule. ESK mAb are potential therapeutic agents for ALL, CML, other leukemias and cancers over-expressing the WT1 oncoprotein. Its expression in early leukemia cells may allow for elimination of the progenitors. The data also provide proof of concept for developing therapeutic mAb targeting important intracellular oncogenic proteins. Disclosures: Yan: Eureka: Employment, Equity Ownership. Liu:Eureka: Employment, Equity Ownership.

Description: Acute and chronic leukemias, including CD34+ CML stem cells, overexpress the Wilms tumor gene 1 (WT1) protein, making WT1 an attractive therapeutic target. ESKM is a fully human IgG1 antibody that targets a 9 amino acid sequence (RMF) of the protein WT1 in the context of HLA-A0201, allowing it to target an undruggable, widely expressed, intracellular oncogene product. BV173 is an HLA-A0201+, human Ph+ ALL cell line that expresses WT1, and tagged by our lab with luciferase. We engineered a tyrosine kinase inhibitor (TKI) resistant BV173-R cell line by transducing BV173 with the resistant T315I Bcr-Abl plasmid. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated in vitro by chromium release assay, utilizing human PBMC effectors. Tumor growth in vivo was assessed in NOD/SCID gamma (NSG) mice with bioluminescence imaging (BLI). RT-PCR was used to evaluate minimal residual disease in mice with negative BLI signal at the end of therapy. Imatinib, dasatinib, and ponatinib were used at up to maximally tolerated doses, given IP once daily. ESKM was administered at 100 µg twice weekly IP. ESKM mediated ADCC against both BV173 and BV173-R cell lines in vitro. In a BV173 engrafted human leukemia xenograft model, ESKM was more potent than imatinib, with median tumor growth reduction of 78% vs 52%. Combination of imatinib and ESKM therapy resulted in a 94% reduction in leukemic growth. High dose dasatinib (40 mg/kg daily) was more potent than ESKM, but discontinuation of therapy due to dasatinib toxicity resulted in relapse. Combination with ESKM therapy with dasatinib resulted in cure in 75% of mice, confirmed by bone marrow RT-PCR three weeks after termination of therapy. For mice cytoreduced with dasatinib followed by consolidation therapy with ESKM, delayed relapse was observed, but no cures. ESKM was highly superior to imatinib and dasatinib against the T315I BV173-R leukemia in vivo. Cures were not achieved with combination therapy of ESKM and either first or second generation TKIs against resistant T315I leukemia. Ponatinib at 10 mg/kg had higher efficacy than ESKM alone against BV173-R, but mice treated with combination of ESKM and ponatinib had superior tumor reduction. CONCLUSION: ESKM is an effective therapeutic antibody for sensitive and T315I Ph+ ALL. Resistant T315I Ph+ leukemic growth is inhibited more effectively by ESKM therapy compared to imatinib and dasatinib, and combination therapy with ESKM is superior to ponatinib. Supported by the Leukemia and Lymphoma Society, NIH R01CA55349, P01 23766 and T32CA62948-18. Disclosures: Yan: Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership.

Description: The Wilms’ tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8 T cell HLA-A0201epitope, is a validated target for T cell-based immunotherapy. We generated a high affinity, fully human IgG1 mAb specific for the RMF/HLA-A0201 complex. The mAb shows potent anti-leukemia activity both in vitro and in vivo in mouse models. Bi-specific T cell engaging antibodies (BiTE) have been used effectively to target cell surface proteins and kill cancers. We have developed a new, potent form of the ESK mAb that is a bi-specific T cell engaging antibody (BiTE) specific for tumor cells coexpressing the intracellular oncoprotein, WT-1 and HLA A0201. ESK-BiTE and an irrelevant control BiTE were constructed with ESK1 scFv or irrelevant ScFv on one arm, and anti-CD3 ScFv fragment as the other arm. The BiTE constructs were expressed in CHO cells. Both the ESK-BiTE and the control BiTE bind human CD3 T cells. The ESK-BiTE selectively bind to leukemia cells that express both WT1 and HLA-A0201. The ESK-BiTE activated human resting T cells and EBV-specific T cells retargeting potent cytotoxicity against WT-1+ HLA A0201+ human leukemia cells in vitro. In an NSG mouse xenograft model, injection of ESK-BiTE (20 ug/ml) twice a week, following I.V. administration of 2x107 human EBV-specific T cells, once a week, significantly inhibited the growth of a previously established disseminated HLA A0201+, WT-1+ human Ph+ ALL, BV173 expressing luciferase, as measured by bioluminescence imaging. In a second NSG mouse model mice injected I.V. with an aggressive human AML, SET-2, on day 0, were treated on day 4 with ESK-Bite for 6 days consecutively at 20 ug/day together with EBV-specific T cells given twice a week. In this setting, the ESK-BiTE and T cells resulted in undetectable leukemic growth for 14 days post-leukemia inoculation, with a minimal tumor burden detected by day 18, while all control groups showed massive increases in leukemia burden by day 14. Mice bearing SET-2 leukemia, that received ESK-BiTE and T cells also showed longer survival and delayed limb paralysis. As expected, the human T cells, which were EBV-specific, did not induce signs of GVHD in mice. Our data provide evidence that ESK-BiTE is a potent and specific therapeutic agent against aggressive human leukemias expressing WT1 and HLA-A0201. This is the first study showing efficacy of a TCR-like BiTE antibody targeting an intracellular tumor antigen expressed at low density. Supported by the Leukemia and Lymphoma Society, NIH R01CA55349 and P01 23766. Disclosures: No relevant conflicts of interest to declare.