Targeting CD123 in Leukemic Stem Cells Using Dual Affinity Re-Targeting Molecules (DARTs )

Transcription

1 Targeting CD123 in Leukemic Stem Cells Using Dual Affinity Re-Targeting Molecules (DARTs ) Muneera AL Hussaini 1, Julie Ritchey 1, Linda Eissenberg 1, Geoff Uy 1, Mike Rettig 1, Matthew Holt 1, Gurunadh Chichili 3, Paul Moore 3, Syd Johnson 3, Ezio Bonvini 3, Lynne Collins 2, John DiPersio 1 1 Division of Oncology and 2 Department of Radiology, Washington University School of Medicine, St. Louis, MO, and 3 MacroGenics, Inc., Rockville, MD, USA DiPersio Lab DiPersioLab.org

2 Introduction 50% of AML patients relapse or have refractory disease Newer chemotherapies have not improved outcomes. New immunotherapies have shown promise for the treatment of other cancers including mabs, chimeric antigen receptor T cells (CARs) and bispecific agents. The use of bispecific agents in AML has been limited by the lack of suitable AML-specific tumor-associated antigens/targets.

3 CD123 IL-3 receptor α: Low affinity ligand binding subunit of the IL-3 receptor Binds IL-3 and heterodimerizes with the common β subunit of the GM-IL-5-IL-3 receptor complex to induce proliferative and anti-apoptotic signaling. Differentially overexpressed in 93% of AML patients. A marker of quiescent leukemic stem cells (Jordan, et al., Leukemia, 2000) and for AML MRD detection.

4 VL VH VH VL Terminology Anti-target Anti-CD3 -S-S- scfv 1 scfv 2 BiTE= Bispecific T cell Engager V H and V L from one antigen recognition site are combined in a single chain variable fragment (scfv). Two scfv are linked in tandem to make a BiTE. DART = Dual Affinity Re-Targeting V H from one antibody is combined with V L of another. Two such chains become stably associated due to a covalent disulfide bridge between the two V H subunits. Advantage: Improved stability and manufacturability.

5 DARTs Used in These Studies Parent antibodies DARTs + CD3 x CD123 (Active DART) -S-S- Anti-CD123 Anti-CD3 Anti-CD123 + Anti-Fluorescein FITC x CD123 (Inactive DART) -S-S- Anti-CD3 + Anti-Fluorescein CD3 x FITC (Inactive DART) -S-S-

6 Structure of CD3 x CD123 DART (Dual Affinity Re-Targeting) VH VH VH VH VL VL VL VL -S-S- Anti-CD3 (MacroGenics) Killing CD123 + Target Anti-CD123 (7G3) CD123 + Target Aggregation T cell CD3 x CD123 DART T cell activation and proliferation T cell T cell T cell T cell T T cell cell T cell T cell

7 Does CD3 x CD123 DART Mediate Effector-Target Cell Aggregation in Vitro? K562 GFP-CD123 K562 No DART CD3 XFITC FITC x CD % 1.35% 1.26% Aggregated Cells GFP Jurkat VPD450 K562 GFP CD3 x CD123 DART K562 GFP-CD123 CD3 x CD123 DART 1.54% 17.2% 1.54% vs 17.2% GFP VPD450

8 CD3 x CD123 DART Induces Human T Cell-target Aggregation, T-cell Activation and Proliferation in Vitro B T Cell Activation 1.1% C T Cell Proliferation 1.8% Control DART CD3 CD123 DART 98.5% 98.4% CD3 CD25 VPD450

9 Effect of CD3 x CD123 DART on T cell Phenotype HuT cells incubated with K562 CD123/GFP (1:1 ratio) and CD3xCD123 vs. control DARTS (10 ng/ml) for 5 days

10 Does CD3 x CD123 DART Mediates Killing of CD123- overexpressing K562 by T Cells? K562 GFP-CD123 K562 GFP 4-Hour Killing Curve

11 CD3 x CD123 DART Induces T-cell Proliferation and Re-directed Killing of Primary AML Blasts No DART CD3 X FITC FITC x CD123 CD3 CD123 DART Blasts Side Scatter Lymphocytes CD45 Six-day incubation

12 CD3 x CD123 DART Induces T-cell Activation and Proliferation in AML Patient Samples Activation P<0.001 Proliferation P<0.001 Six-day incubation

13 CD3 x CD123 DART Induces a Dose-dependent Elimination of Primary AML Blasts in Vitro E:T= 1:111 CD123%= 69.2% Six-day incubation

14 K562 GFP-CD123-luciferase+ Leukemia NSG Xenograft Model K562 CD123+LuCBR+ egfp+ (1.5X10e6 cells/mouse) N=5/group E:T=2:1 D -1 D 0 D 3 D 4 D 5-8 D 12 D 19 D28 Irradiation (300 cgy) Baseline BLI Pan T cells (3X10e6/mouse) + DARTs (0.5mg/kg) or PBS DARTs (0.5mg/kg) or PBS BLI Monitoring Groups A. T cells only B. K562 + T cells + PBS C. K562 + T cells + CD3 x FITC D. K562 + T cells + FITC x CD123 E. K562 + T cells + CD3 x CD123 DART

15 CD3 x CD123 DART Inhibits CD123+ K562 Leukemia Growth in Vivo T Cells Only PBS CD3 CD123 CD3xCD123 Day 3 CD3 x CD123 DART Day 12 Day 19 CD3 x CD123 DART p < (PBS vs. CD3 x CD123)

16 Primary AML NSG Xenograft Model Day 0 Sublethal Irradiation (300 Gy) Day 3 Intravenous Injection of Patient sample No.10 5X10e6 Cells/mouse Day 3-D7 Daily Intravenous DART Injection (0.5mg/kg/mouse) CD3 x FITC (Control) CD3 x CD123 DART 6 wks Sacrifice+ Harvest Bone marrow, spleen, Peripheral Blood +Flow analysis N=3/group Baseline E:T= 1:111 (AML #10)

17 CD3 x CD123 DART Eliminates Primary AML Blasts in Vivo PB HuCD45 Hu CD123 CD3 DART BM Murine CD45 HuCD33 HuCD45 hucd123 CD3 X CD123 DART Spleen MuCD45 HuCD33

18 Does CD3 x CD123 DART Affect Normal Progenitor Survival and Proliferation? Dose of DART Umbilical cord blood cells were incubated with DARTs for 4 hours and then cultured in methylcellulose for 7 days.

19 Conclusions CD3 x CD123 DART binds both human CD3 and CD123 to mediate targeteffector cell aggregation, T-cell activation, proliferation and re-directed killing of CD123+ K562 and primary human AML blasts in vitro and in vivo CD3 x CD123 DART induces in vitro skewing of both CD4 and CD8 subsets toward increased central memory and early effectors and decreases naïve T cells. CD3 x CD123 DART does not affect colony formation from umbilical cord blood. Effects of CD3 x CD123 DART on normal human hematopoiesis will need to be further explored including on those normal cells which are known to express CD123 (plasmacytoid dendritic cells, mast cells, macrophages, etc). A phase 1 study of CD3 x CD123 DART (MGD006) in relapsed and refractory AML will open at Washington University in the 1st half of 2014.

20 Acknowledgements DiPersio Lab John DiPersio Julie Ritchey Linda Eissenberg Geoffrey Uy Mike Rettig Matthew Holt MacroGenics Ezio Bonvini Gurunadh Chichili Paul Moore Syd Johnson DiPersio Lab DiPersioLab.org