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Abstract
Background AK117 is a humanized monoclonal antibody targeting CD47 which widely expresses on innate immune cells, such as macrophages, and functions as a regulator of phagocytosis. CD47 serves as the ligand for a receptor on these innate immune cells, SIRPα, which in turn delivers an inhibitory signal for phagocytosis. Hematology toxicity is the major concern of an anti-CD47 antibody. As an agent targeting CD47 being investigated as an anti-tumor therapeutic, AK117 is engineered on a human IgG4 scaffold to minimize recruitment of Fc-dependent effector functions, as well as identified with favorable hematology safety profile and robust pro-phagocytosis activity.
Methods Activity of AK117 binding to CD47 to block the interaction between CD47 and SIRPα were determined by FACS, and binding of AK117 to human RBC was also evaluated. Raji cells, HT-29 cells, and HL-60 cells which highly express CD47 were used as target cells to evaluate a pro-phagocytic activity of AK117 as a monotherapy or in combination with anti-EGFR antibody, anti-CD20 antibody or azacitidine. In in-vivo pharmacology studies, anti-tumor activity of AK117 was investigated in SCID/beige mouse Raji tumor model. Effects of AK117 on hemagglutination of human RBC at was tested. Hemoglobin (HGB) and hematocrit (HCT) was evaluated after single dose of 10 mg/kg AK117 or Hu-5F9 in male and female cynomolgus monkeys (n=1/gender).
Results AK117 could effectively binds to CD47, and competes with SIRPα for binding to the antigen on Raji cells (figure 1). AK117 alone or combines with anti-EGFR antibody, anti-CD20 antibody and azacitidine shows potent phagocytosis of tumor cells in a dose-dependent manner (figure 2). AK117 significantly inhibited tumor growth in these tumor models (figure 3). Favorable hematology safety profile of AK117 was observed. A significant weaker binding to human RBC of AK117 was identified (figure 4), and AK117 does not induce hemagglutination of human RBC up to a concentration of 1050 μg/mL, while Hu-5F9 triggers hemagglutination even at a low concentration of 1.44 μg/mL (figure 5). AK117 has minimal anemia effect in monkey studies compared to hu5F9-G4 after single dose in cynomolgus monkeys (figure 6). AK117 showed a rather superior safety profile to Hu5F9-G4 as a shorter duration of anemia.
Binding and Competition activity of AK117 to CD47. (A) FACS binding curves of AK117 and Hu5F9-G4 to CD47 on raji cells. (B) FACS competitive binding curve of AK117 and Hu5F9-G4 with SIRPαECD-mFc to CD47 on raji cells.
The pro-phagocytic activity against tumor cells. (A) The phagocytic index of raji cells by macrophages with AK117. (B) The phagocytic index of HL-60 cells by macrophages with AK117 and azacitidine. (C) The phagocytic index of HT-29 cells by macrophages with AK117 and cetuximab. (D) The phagocytic index of raji cells by macrophages with AK117 and rituximab.
Anti-tumor activity in raji tumor mouse model. The (A) Tumor growth curves and (B) Body weight curves of different groups in SCID/Beige mice with subcutaneous raji tumor.
Binding activity of AK117 to human RBCs. Binding Curves of Hu5F9-G4 and AK117 to CD47 on human RBCs
Hemagglutination effect on human erythrocytes. Hemagglutination effect of AK117 on human erythrocytes
HGB and HCT in cynomolgus monkeys. The curves of (A) hemoglobin and (B) Hematocrit at different times in cynomolgus monkeys.
Conclusions With pre-clinical pharmacology activities comparable to Hu5F9-G4 as well as superior safety properties demonstrated in non-clinical pharmacodynamics studies, AK117 has emerged as a promising new treatment for solid tumor.