Ex Vivo Activity Profile of the CD123xCD3 Duobody® Antibody JNJ-63709178 Against Primary Acute Myeloid Leukemia Bone Marrow Samples
Authors: Forslund Ann , Syed Khaja , Axel Amy , McDaid Ronan , Li Yingzhe , Ballesteros Juan , Gaudet Francois , Attar Ricardo M. , Salvati Mark , Huang Fei , Sasser Kate 
Published: October 2019

Background/Aims:Acute myeloid leukemia (AML) is a hematologic malignancy that affects the normal production of neutrophils, red blood cells, and platelets. CD123 (IL-3 receptor alpha) is over-expressed on AML stem cells and blasts, compared with normal hematopoietic progenitor and stem cells, and represents a promising target for the development of antibody-based AML therapies (Jordan et al., Leukemia 2000;14:1777-1784).

The outcome for patients with AML remains poor, and effective therapeutics are needed in this patient population. JNJ-63709178 is a promising new bispecific antibody that recruits T cells to tumor cells through a tumor-specific antigen-binding arm, CD123, and a T-cell-specific CD3 arm. JNJ-63709178 is a humanized antibody on an IgG4 scaffold with potent in vitro and in vivo T-cell-mediated killing of CD123-positive leukemic cells. The Fc domain of JNJ-63709178 was also engineered to diminish binding to Fcγ receptors (to reduce the potential for nonselective T-cell activation), yet preserve binding to human FcRn to maintain a long serum half-life. JNJ-63709178 is currently being explored in a Phase 1 study in patients with relapsed or refractory AML.

Methods: To evaluate the ex vivo effects of this class of bispecific antibodies on primary samples from patients with AML (15 newly diagnosed, 1 previously treated and 1 unknown), patient bone marrow samples were collected and received within 24-48 hours at Vivia Biotech and analyzed by Vivia's proprietary ExviTech flow cytometry system. This platform preserves the whole bone marrow native environment by retaining all cell types, matrix, and proteins important for long-term growth of primary human bone marrow and blood. CNTO 9958, a close analog of JNJ-63709178 with similar affinities for CD123 and CD3 and functionally similar to JNJ-63709178, was utilized for these studies. Patient samples were evaluated for blast count, CD123 quantification on blast cells (QuantiBRITE PE beads, Beckton Dickinson), T-cell count and activation markers (CD25, ICOS, and PD1), and effector-to-target (E:T) ratios were determined. Following 4 to 120 hours of culturing with CNTO 9958, the bone marrow aspirate was extensively profiled by an automated multi-parameter flow cytometry system at Vivia Biotech. Growth and depletion of leukemic blasts and T-cell subsets were monitored upon CNTO 9958 antibody treatment and expansion and activation of CD4+ and CD8+T-cells were measured at multiple time points.

Results: CD123 expression was detected in 16 of 17 primary AML bone marrow samples examined, and expression ranged from ~1300 to ~24000 receptors/cell on AML blasts. Blast cell depletion was measured in the same 17 primary AML bone marrow samples across different time points and after incubating with varied concentrations of CNTO 9958. CNTO 9958 was able to effectively reduce blasts levels with EC50 <0.5 µg/mL (median EC50: 0.11 µg/mL, range: 0.001-500 µg/mL) at 72 hours' incubation in 14 of 16 samples. Measurement of T-cell subtypes showed proliferation of both CD4+ and CD8+T cells as well as increased activation (CD25+ and ICOS+) on effector T cells. There was a correlation between proliferation of cytotoxic T cells and blast depletion. There was no correlation between effect and CD123 level. E:T ratios as low as 1:54.3 (range 1:2.6-1:54.3) resulted in efficient depletion of leukemic blasts with CNTO 9958. Baseline activation of T cells (CD8+CD25+) was observed to correlate with the efficacy of CNTO 9958 on blast depletion.

Conclusions: Vivia Biotech's ExviTech flow cytometry system provides a biologically relevant platform to evaluate patient bone marrow samples ex vivo, preserving the tumor microenvironment of the bone marrow sample, including blasts and white blood cells. Using this system to evaluate AML patient samples, CD123 was expressed in 16 of 17 primary AML bone marrow samples, with expression densities ranging from ~1,300 to ~24,000 receptors/cell. The CD123xCD3 bispecific antibody CNTO 9958 was able to recruit and activate endogenous patient T cells to efficiently kill CD123-positive AML cells in a time- and dose-dependent manner, even at the very low E:T ratios observed and irrespective of CD123 receptor density in AML patient samples. These results support clinical evaluation of JNJ-63709178 in patients with AML.

Disclosures

Forslund: Janssen Pharmaceuticals Research and Development: Employment. Syed:Janssen Pharmaceuticals Research and Development: Employment. Axel:Janssen Pharmaceuticals Research and Development: Employment. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Ballesteros:Vivia Biotech: Employment, Equity Ownership. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Attar:Janssen: Employment. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Huang:Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Sasser:Johnson & Johnson: Equity Ownership; Janssen Pharmaceuticals R&D: Employment.