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360 Targeting CD123 In Leukemic Stem Cells Using Dual Affinity Re-Targeting Molecules (DARTs®)

Program: Oral and Poster Abstracts
Type: Oral
Session: 615. Acute Myeloid Leukemia: Therapy, excluding Transplantation: Antibody-Based Targeted Therapy
Monday, December 9, 2013: 11:45 AM
La Nouvelle Ballroom C (Ernest N. Morial Convention Center)

Muneera H. AL Hussaini, MBBS1*, Julie Ritchey1*, Michael P. Rettig, PhD1, Linda Eissenberg, PhD1*, Geoffrey L. Uy, MD1, Gurunadh Chichili, PhD2*, Paul A Moore, PhD2*, Syd Johnson, PhD2*, Lynne Collins3*, Ezio Bonvini2* and John F. DiPersio, MD, PhD1

1Siteman Cancer Center, Washington University School of Medicine, Saint Louis, MO
2MacroGenics, Inc., Rockville, MD
3Molecular Imaging Center, Washington University School of Medicine, Saint Louis, MO

T-cell directed killing of tumor cells using bispecific reagents is a promising approach for the treatment of hematologic malignancies.  In contrast to B-cell malignancies, this approach has been limited in AML by the lack of tumor-specific antigens/targets.  CD123 (IL3RA) is highly and differentially expressed in AML blasts compared to normal hematopoietic stem and progenitor cells and is a potential target for immunotherapy.  We investigated the ability of a DART constructed from an antibody to CD123 (7G3) and a MacroGenics’ proprietary CD3 antibody to redirect T cells against CD123+ AML blasts.  DARTs consist of 2 independent polypeptides, each comprising the VH of one antibody in tandem with the VL of the other antibody.  The resultant heterodimer is stabilized by a disulfide bond at the carboxyl terminal domains of the 2 VH regions.  This construct binds to both the N-terminal extracellular domain of human CD123 and to the extracellular domain of CD3 in the human T-cell receptor complex.  Our in vitro studies demonstrate that the CD3xCD123 DART induces specific aggregation of TCR/CD3+ Jurkat or human T cells and human CD123-transduced K562 (K562CD123/GFP) cells compared to a control DART lacking specificity for one of the antigens (16±3.2% vs. 1.6±0.2%, p=0.0074) or when compared to CD3xCD123 DART incubated with control GFP-transduced K562 (K562GFP) cells.  Incubation of human T cells (1:1 ratio) with K562CD123/GFP and CD3xCD123 DART vs. control DARTS (10 ng/ml) for 5 days in vitro resulted in profound T-cell activation (CD25 expression, 88.8±2.7% vs.1.2±0.2%; p=0.0009), T-cell proliferation (VPD-450 proliferation assay; 98.2 ± 0.4% vs. 2.27± 0.4%, p=0.0001), expansion of the central memory T cell compartment (TCM, 62.6±1.5% vs. 5±1.3%, p<0.0001), and killing of K562CD123/GFP targets as measured by 7-AAD FACS (97±0.9% apoptosis relative to the control; p<0.0001) and chromium release assays (28.5% vs.3.1%; p=0.0002).  As expected, aggregation, T-cell activation and target cell killing was negligible when T cells and CD3xCD123 DART were incubated with control K562GFP cells.  Similar results were seen when A20 targets (BALB-C/H-2d B lymphoma cell line) overexpressing CD123 were used (7-AAD+ after 18 hours, 91.1±2.01% vs. 28.1±0.76%, p=0.0012).  In spite of very low E:T ratios (0.009:1-0.071:1), when primary frozen/thawed AML peripheral blood specimens (n=6; CD123+ blasts ranging from 34.7 to 87.2%) were used, there was a profound and universal CD3xCD123 DART-specific activation and expansion of the few human T cells present in these AML samples (ranging between 0.9 to 6.6% of cells).  After 6 days of in vitro incubation (37°C w/o exogenous IL-2) of each AML sample (1x106/ml) with CD3xCD123 DART (0.1 ng/ml) or control DARTs, there was a CD3xCD123 DART-specific increase in both T cell numbers (median: 8 fold, range:1.7-15 fold vs. 0.6-1 fold for control DARTs), and in the percentage of T cells expressing CD25 (median: 69.4 fold, range: 21.4-152.8 fold vs. 0.2-6.5 fold for control DARTs). This was accompanied by a dose-dependent reduction in blasts by 35±25% at 0.1 ng/ml DART and 99.6±0.05% at 10 ng/ml DART (p=0.0063).  In addition, AML colony-forming units (L-CFU) were also inhibited by 94 ±0.6% while CFU-GEMM, CFU-GM and BFU-E from cord blood and (G-CSF)-mobilized peripheral blood from normal donors were not affected by either CD3xCD123 DART or control DARTs (0.1-10 ng/ml), suggesting limited impact on normal human hematopoietic progenitors in vitro.  Bioluminescence imaging of irradiated NSG mice (n= 5/group; 300cGy) performed on days 3, 12, 19, and 28 after the infusion of 1.5 x 106 Click Beetle Red (CBR) luciferase+- transduced K562CD123/GFP cells on day 0 and both CD3xCD123 DART (0.5 mg/kg IV) and human T cells (3 x 106) on day 3 revealed no expansion of tumor cells in sharp contrast to NSG mice receiving either control DARTs and/or no human T cells (1415- fold expansion; p<0.0001).  Of interest is that at 6 weeks post-infusion of primary AML xenografts into NSG mice (E:T=0.009:1), there was near- complete elimination (>97%) of AML blasts from the peripheral blood even in the absence of exogenously added human T cells.  Clearing of primary human AML blasts from the spleen and bone marrow was also significant (40-77.8%) but less than that seen in the blood.  These results provide the basis for the CD3xCD123 DART as a novel reagent for the treatment of patients with CD123+ AML.   

Disclosures: Chichili: Macrogenics. Inc: Employment. Moore: Macrogenics. Inc.: Employment. Johnson: Macrogenics. Inc.: Employment. Bonvini: Macrogenics. Inc.: Employment.

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