At week four, 3 x 106splenocytes containing > 50% blasts from untreated mice were transferred to secondary recipient mice. engraftment, supporting possible activity of 8F4 against the subset of AML with self-renewing potential. Our data provide evidence that 8F4 antibody is usually highly active in AML, including chemotherapy-resistant disease, supporting its potential use as a therapeutic agent in patients with AML. == INTRODUCTION == Monoclonal antibodies (mAbs) against tumor-specific or lineage-specific antigens are effective treatments for a growing number of cancers. Most of the mAbs used in the clinical setting target surface proteins that although expressed by normal cells, have unique expression patterns around the malignant cells. However, the majority of onco-mutated proteins and tumor-specific antigens are expressed within the tumor cell, in the nucleus or cytoplasm; targeting such proteins with mAbs has proven to be a difficult task. Nevertheless, intracellular proteins can be useful targets for immunotherapy. In acute myeloid leukemia (AML), a neoplasm largely resistant to standard therapies, the potential of allogeneic hematopoietic stem cell transplantation (HSCT), a proven potentially curative therapy, is due to its graft-versus-leukemia effect that is mediated by RWJ 50271 donor cytotoxic T lymphocytes (CTL).1Specifically, peptides from intracellular proteins within the AML blasts are processed and presented on cell surface major histocompatibility class I (MHC-I) antigens. These peptide/MHC-I complexes are recognized by the T cell receptor (TCR) on CD8+CTL, which in the appropriate tumor environment can eliminate the malignant cells.2,3TCR-like mAbs that target peptide/MHC-I around the tumor cell surface have been designed and are promising as novel cancer immunotherapies.47While the TCR binds to cognate peptide/MHC ligands with low affinity because of rapid off-rates,8,9TCR-like mAbs bind to surface peptide/MHC-I with several orders of magnitude higher affinity and therefore may have therapeutic advantages.4,5,1012Despite the technical challenges of developing mAbs with specificity for peptides in the context of MHC-I, a number of TCR-like mAbs targeting intracellular tumor-associated antigens have been investigated, and a few have shown encouraging activity against tumor cell lines,6,7,13including leukemia cell lines.14,15 PR1 is a human leukocyte antigen HLA-A2 restricted 9-mer peptide derived from the myeloid serine proteases proteinase 3 (P3) and neutrophil elastase (NE),10which are normally contained intracellularly within azurophilic granules in normal granulocytes. NE and P3 have been shown to be aberrantly expressed in AML and chronic myeloid leukemia (CML).2,16,17PR1-specific CTL have been shown to lyse malignant and dysplastic cells in AML, CML, and myelodysplastic syndrome (MDS), and were also have been shown to contribute to cytogenetic remission in CML.3,1820We developed a TCR-like mouse mAb, 8F4, which binds to the PR1/HLA-A2 complex on the surface of AML.218F4 mediates both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of AML. Importantly, 8F4 inhibits leukemia stem cells (i.e. LSC) but not normal hematopoietic progenitor cells inin vitrocolony forming assays.21However, thein vivoeffect of 8F4 on main leukemia cells has not been explored. Here, we analyzed thein vivoeffects of 8F4 in a patient-derived xenograft (PDX) model. Specifically, main cells from patients with a variety of AML subtypes were inoculated into NODscidIL2 receptor gamma-chain knock out (NSG) mice.22We show that treatment of established AML xenografts with 8F4 reduced human AML. In secondary transfer experiments, we found that 8F4 depleted AML, including cells with self-renewing potential. Taken together, our findings justify the further development of 8F4 as a potential therapeutic agent for patients with AML. RWJ 50271 == MATERIALS AND METHODS == == Patients and donors == Human AML samples were collected from patients treated at the University or RWJ 50271 college of Texas MD Anderson Malignancy Center (MDACC) after obtaining written informed consent under protocols approved by MDACC Institutional Review Table (IRB). The HLA status of the patients and other data, including previous treatments and end result, were obtained Rabbit Polyclonal to GFP tag from the patients’ electronic medical record. The HLA screening was conducted at the MDACC HLA typing Laboratory. Patients UPN14, UPN7 and UPN8 were molecularly typed as HLA-A02:01:01; patient UPN5 experienced serologic typing only and was identified as HLA-A2. Mononuclear cells were separated by gradient density centrifugation using histopaque 1077 (Sigma-Aldrich). == Assessment of PR1/HLA-A2 expression and susceptibility to 8F4-mediated cytotoxicity == 8F4 mAb was generated in BALB/c mice as previously explained.218F4 was affinity purified from hybridoma supernatant and directly conjugated to Alexa-647 fluorochrome (Invitrogen). To assess PR1/HLA-A2 expression, samples were stained in the presence of blocking antibody bb7.2, as described.21To account for variance in staining conditions performed on different days, 8F4 median fluorescence intensity (MFI) RWJ 50271 was normalized to the MFI of IgG-binding compensation beads (eBioscience), stained with 8F4 (maximum [Max] MFI); PR1/HLA-A2 expression was reported as % Maximum MFI. To study susceptibility of samples to 8F4-mediated killing circulation cytometry-based CDC assay was used as explained.21 == Mouse model == NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG), NOD.Cg-PrkdcscidTg(HLA-A2.1)1Enge/DvsJ (NOD scid/HLA-A2), NOD.CB17-Prkdcscid/J (NODscid), C57BL/6-Tg(HLA-A2.1)1Enge/J (B6/HLA-A2), and C57BL/6J (B6).