Highly frequent anti-idiotype antibody in cynomolgus monkeys developed against mouse-derived regions of anti-Fas antibody humanized by complementarity determining region grafting

Abstract

Background and purpose: We investigated the immunogenicity of a humanized anti-human Fas monoclonal antibody, R-125224, in cynomolgus monkeys to estimate its efficacy, as well as its toxicity in clinical situations.

Experimental approach: R-125224 was intravenously administered to cynomolgus monkeys at single doses of 0.4, 1.2, 6 and 30 mg kg(-1), and the plasma concentrations of R-125224 and anti-R-125224 antibody (ARA) were measured. We conducted a competitive enzyme-linked immunosorbent assay to determine which part of R-125224 was recognized by ARA. We also examined the retention of radioactivity in mononuclear cells and granulocytes after the injection of [(125)I]-R-125224 to a collagen-induced arthritis monkey model.

Key results: After i.v. administration of R-125224, the elimination of the plasma R-125224 concentrations was accelerated at around 10 days post-dose, and 10 of 12 monkeys were ARA positive. From an epitope analysis of ARA, the ARA produced in monkeys recognized the mouse-derived regions located in complementarity determining regions, but could not recognize the human IgG. After the injection of [(125)I]-R-125224 to a collagen-induced arthritis monkey model, a significantly longer retention of the radioactivity in mononuclear cells compared to granulocytes was observed.

Conclusions and implications: In monkeys, the development of antibodies against R-125224 is rapid and highly frequent. Our hypothesis is that this highly frequent development of ARA might be due to the binding of R-125224 to immune cells, and its circulation in monkey blood might contribute to an increase in its chances of being recognized as an immunogen.

Figure 1

Plasma concentrations of R-125224 (A–D) and anti-R-125224 antibody (E–H) after i.v. administration to cynomolgus monkeys at a dose of 0.4 (A, E), 1.2 (B, F), 6 (C, G) and 30 mg·kg⁻¹ (D, H).

Figure 2

Relative binding of anti-R-125224 antibody and biotin-R-125224 in the presence of antigens (control human IgG, R-125224, Fab of R-125224 and m-HFE7A) at doses of 0.4 (A) and 1.2 mg·kg⁻¹ (B). IC₅₀ values (competitive antigen/biotin-R-125224) of R-125224, Fab of R-125224 and m-HFE7A were 0.496, 1.02, and 0.664 respectively.

Figure 3

Binding activity of R-125224 and Fas antigen in the presence of anti-R-125224 antibody.

Figure 5

Trichloroacetic acid (TCA)-precipitable ratios to total radioactivity in mononuclear cells, granulocytes and blood. Statistical analysis comparing the values at 24 and 168 h to that at 1 h in blood and immune cells was performed with Student's t-test; **P < 0.05.

Figure 4

Concentrations of radioactivity in granulocytes, mononuclear cells and blood after i.v. administration of [¹²⁵I]-R-125224 to cynomolgus monkeys at a dose of 0.4 mg·kg⁻¹.