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Acute and chronic leukemias, including CD34(+) CML cells, demonstrate increased expression of the Wilms tumor gene 1 product (WT1), making WT1 an attractive therapeutic target. However, WT1 is a currently undruggable, intracellular protein. ESKM is a human IgG1 T-cell receptor mimic monoclonal antibody directed to a 9-amino acid sequence of WT1 in the context of cell surface HLA-A*02. ESKM was therapeutically effective, alone and in combination with tyrosine kinase inhibitors (TKIs), against Philadelphia chromosome-positive acute leukemia in murine models, including a leukemia with the most common, pan-TKI, gatekeeper resistance mutation, T315I. ESKM was superior to the first-generation TKI, imatinib. Combination therapy with ESKM and TKIs was superior to either drug alone, capable of curing mice. ESKM showed no toxicity to human HLA-A*02:01(+) stem cells under the conditions of this murine model. These features of ESKM make it a promising nontoxic therapeutic agent for sensitive and resistant Ph(+) leukemias.

Preferentially expressed antigen in melanoma (PRAME) is a well-validated target for T cell-based immunotherapy in leukemias and solid tumors. PRAME is a retinoic acid receptor binding protein that prevents retinoic acid-mediated differentiation, proliferation arrest, and apoptosis. As a cancer-testis antigen, PRAME has limited expression in healthy adult tissue that is restricted to the testes, ovaries, and endometrium. However, PRAME is over-expressed in multiple cancers including ALL, AML, melanomas, and breast cancers, making it a specific and highly attractive therapeutic target. PRAME is an intracellular protein making it impossible to target using traditional antibodies and it is not currently druggable. After proteasomal processing, the PRAME300-309 peptide is presented on the cell surface in the context of HLA*A02:01 molecules, for recognition by CD8 T cells. We therefore hypothesized that a TCR-mimic (TCRm) monoclonal antibody that recognizes surface PRAME300-309 presented by HLA*A02:01 could have therapeutic activity. Here, we describe Pr20, a therapeutic TCRm antibody, specific for the PRAME300-309 peptide in complex with HLA*A02:01, identified through a human scFv phage display library screen. Pr20 was engineered into full length human IgG1. Pr20 exhibited specific binding to PRAME300-309 -pulsed TAP-deficient T2 cells and bound PRAME+/ HLA*A02:01+ Ph+ ALL and AML, demonstrating that endogenously presented PRAME300-309 could be recognized by Pr20. Pr20 was determined to have 4 nM binding affinity by scatchard plot analysis. The specific epitope was mapped using alanine substitutions of non-anchor residues in the PRAME300-309 peptide and determined to primarily require the C-terminal residues. Pr20M, an afucosylated form of the antibody with enhanced Fc binding, mediated antibody-dependent cellular cytotoxicity (ADCC) in-vitro in a PRAME+/ HLA*A02:01+ restricted manner. Pharmacokinetic studies in C57BL/6 mice indicated that Pr20M was stable in-vivo and biodistribution studies in HLA*A02:01 transgenic mice suggested that there was no significant antibody sink. Pr20M was therapeutically active in established xenograft leukemia models in NSG mice (T, B, and NK-deficient). Interestingly, Pr20 binding to PRAME+/HLA*A02:01+ melanomas was minimally detectable, but was dramatically increased upon treatment with IFNγ, which also led to an increased sensitivity to ADCC. The data provide rationale for developing TCRm antibodies against intracellular oncoproteins as therapeutics. Disclosures No relevant conflicts of interest to declare.

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.