Preclinical Evaluation of CSL362/JNJ-56022473 Single Agent in in Vitro Assays
Authors: Syed Khaja , Pietsch Christine , Axel Amy , Forslund Ann , Sasser Kate , Salvati Mark 
Published: November 2019

Acute myeloid leukemia (AML) is a hematologic malignancy that affects the normal production of neutrophils, red blood cells, and platelets. AML blasts and leukemic stem cells, but not normal hematopoietic progenitor cells, show elevated expression of CD123 (IL-3Rα) (Jordon et al Leukemia 2000), providing a potential therapeutic target for AML. CSL362 is an anti-CD123 monoclonal antibody engineered to have high affinity CD16 binding leading to enhanced, antibody-dependent cell-mediated cytotoxicity (ADCC) (Busfield et al Leukemia 2014) and antibody-dependent cell-mediated phagocytosis (ADCP) of CD123-positive AML cells through the activation of natural killer cells and macrophages. A CSL362 variant, JNJ-56022473, was generated from a new cell line and shows comparable biological activity to CSL362. In this study, we evaluated CD123 receptor expression in various AML cell lines and assessed CSL362 and JNJ-56022473-mediated cytotoxicity in AML cells.

CD123 receptor expression was determined in 14 AML cell lines by fluorescence activated cell sorting utilizing three clones of anti-CD123 antibody (7G3, 6H6, and 9F5). CD123 receptors were detected in all cell lines examined, and expression ranged from ~80 to ~16,000 receptors/cell. Using human peripheral blood mononuclear cells as effector cells and CSL362 (10µg/ml) in an ADCC assay, we found that cell lines with high (~16,000 receptors/cell) to intermediate (~2,500 receptors/cell) CD123 expression were susceptible to CSL362-mediated ADCC (68.5%-32.8% lysis). In the assay conditions used here, the threshold for ADCC activity was ~1,800-2,500 CD123 receptors/cell where 32.8% lysis was observed at threshold compared to 0.9% lysis with control (no CSL362 antibody). The level of CD123 expression correlated with CSL362-mediated ADCC activity.

CSL362 also induced ADCP of MV4-11 cells at 0.1 and 1.0µg/ml compared to PBS control (3.8 and 2.6 fold, respectively). JNJ-56022473 induced ADCP of MV4-11 (3.0 and 2.2 fold, respectively) and KG-1 (1.8 and 1.7 fold, respectively) cells when tested at 0.1 and 1.0µg/ml, but not at 10µg/ml compared to PBS control. In TF-1 cells, no ADCP activity was observed with either CSL362 or JNJ-56022473. The greater ADCP activity observed in MV4-1 (~9,700 receptors/cell) and KG-1 (~4,800 receptors/cells) cells correlated with higher levels of CD123 expression compared to TF-1 cells (~2,500 receptors/cell).

To determine if CSL362 and JNJ-56022473 could elicit complement-mediated lysis, complement-dependent cytotoxicity (CDC) assays were performed in three AML cell lines. CSL362 and JNJ-56022473 (0.1-10µg/ml) failed to mediate CDC in OCl-AML5 (~16,000 receptors/cell), MV4-11 (~9,700 receptors/cell), and KG-1 (~4,800 receptors/cell) cells.

In summary, CD123 receptors were expressed in all 14 AML cells lines examined, with expression levels ranging from ~80 to ~16,000 receptors/cell. The level of CD123 expression correlated with CSL362-induced ADCC activity; higher expression levels exhibited greater ADCC activity. Both CSL362 and JNJ-56022473-mediated ADCP were significantly enhanced at 0.1 and 1.0µg/ml compared to control. Both antibodies failed to elicit a CDC response.

Disclosures

Syed: Janssen Research & Development at Johnson & Johnson: Employment. Pietsch:Janssen Research & Development at Johnson & Johnson: Employment. Axel:Janssen: Employment. Forslund:Janssen Research & Development at Johnson & Johnson: Employment. Sasser:Janssen Pharmaceuticals: Employment. Salvati:Janssen Research & Development at Johnson & Johnson: Employment.