Abstract
Abstract
CD40 is highly expressed in B-cell malignancies and its activation is a growth and survival signal for these cells. We have generated a novel, highly potent, fully human anti-CD40 IgG1 monoclonal antibody, CHIR-12.12 using XenoMouse® mice (Abgenix, Inc), a strain of transgenic mice expressing fully human IgG antibodies. CHIR-12.12 has at least two mechanisms of cytotoxicity in vitro: it blocks CD40-ligand mediated CD40 activation and mediates B-cell killing by ADCC. In-vitro crossreactivity studies were performed and the antibody showed cross reactivity to non-human primate but not to rodent tissue. To investigate the potential long-term toxicity of the antibody and to observe the recovery of the induced B-cell depletion, a single-dose toxicity study was conducted in cynomolgus monkeys. Three animals per sex per group (two animals per sex in control group) were given a single intravenous dose of 0, 10, or 100 mg/kg CHIR-12.12 and were monitored for 23 weeks post dosing. In addition to a detailed physical examination, samples for clinical chemistry, hematology and coagulation parameters as well as urinalysis were taken every 2 weeks. FACS analyses were performed every two weeks to monitor changes in B-cell, T-cell subpopulation and NK-cell numbers. In addition, CD40 expression and antigen saturation were measured on the remaining B-cell populations over 23 weeks. To investigate T-cell activation and T-cell function after CHIR-12.12 dosing, CD25 and CD40 expression on T-cells were monitored and at Week 23 a Mixed Lymphocyte Reaction (MLR) test was performed. The results of the study showed that a single dose of CHIR-12.12 was well tolerated and did not elicit any adverse clinical signs or effect on body weights, clinical pathology and hematology including differential blood counts. Immunophenotyping analysis showed a dramatic reduction of B-cells after dosing. The remaining B-cells mostly consist of the CD20high CD40low population, which is a population uniquely found in cynomolgus monkey blood but not in human blood (Vugmeyster et al.; Cytometry. 2003 Apr;52A). Both treatment groups showed a recovery of B-cells to normal pre-dose values in the majority of animals 19 weeks after dosing. In contrast, the average CHIR-12.12 antigen saturation level returned to pre-dose values in the low dose group after 11 weeks and in the high dose group after 19 weeks. T-cell counts (CD3, CD4 and CD8 subpopulation) showed no treatment related changes in absolute cell numbers throughout the study. No changes in T-cell activation measured by CD25 and CD40 expression were detected. T-cell responsiveness was not altered as measured by MLR. The pharmacokinetic analysis showed that, consistent with other human IgG antibodies, CHIR-12.12 had a small volume of distribution, limited to the blood volume, and was slowly eliminated from the circulation. Dose-dependency was observed with the area under serum concentration-time curve (AUC) increasing in a greater than dose-proportional fashion. Likewise, the half-life also increased with dose, ranging from 10.3 days at 10 mg/kg to 16.8 days at 100 mg/kg. In conclusion, all animals remained healthy and no evidence of immune impairment other then the expected reversible B-cell depletion was detected with a single dose of CHIR-12.12 up to 100 mg/kg. These results support the clinical development of CHIR-12.12 antibody for treatment of B-cell malignancies.