ALBERT

Description: IL-21 is a cytokine produced by activated CD4+ T cells that augments the growth, survival, and function of NK, T and phagocytic cells. IL-21 also plays an important role in regulating B cell proliferation and differentiation. In a previous study of cynomolgus monkeys treated with recombinant human IL-21, we demonstrated increased numbers of NK cells and phagocytic cells expressing Fcγ RI and III, enhancement of ex vivo antibody dependent cellular cytotoxicity (ADCC) activity, altered lymphocyte trafficking, and improved B cell depletion with subclinical doses of rituximab. The current study aimed to extend these findings to a clinically relevant rituximab dose regimen, and to evaluate the safety and tolerability of IL-21 administration in this setting. Three groups of cynomolgus monkeys (n=8) were treated by IV injection once weekly for four weeks with rituximab (10 mg/kg), concurrently with vehicle or IL-21 (0.3 or 1.5 mg/kg). Standard toxicology data were collected. The number and activation state of peripheral blood leukocyte subsets were determined by flow cytometry. Effects on B cells within spleen and lymph nodes were evaluated by immunohistochemistry. Co-administration of rituximab and IL-21 was well tolerated at both dose levels of IL-21 tested. The primary toxicological responses to the combination treatment were moderate anemia and thrombocytopenia, which were IL-21 dose dependent and similar in magnitude to those observed in previous IL-21 toxicology studies. In animals treated with rituximab alone, circulating B cells were depleted to a nadir 0.3–3 % of baseline on Day 11 of the study (Figure 1). In contrast, significantly lower B cell nadirs were observed following the first treatment with rituximab and IL-21 combination therapy (p〈 0.01, ANOVA). In animals receiving the 1.5 mg/kg dose of IL-21, circulating B cells were depleted to a nadir 0 −1 % of baseline on Day 4, followed by partial recovery and further depletion to 0.3 – 2% of baseline on Day 11. More importantly, B cell recovery was significantly delayed in the group treated with rituximab and 1.5 mg/kg IL-21, compared to rituximab treatment alone (p〈 0.05, ANOVA/PLSD). Thirty days following the fourth and final treatment, B cell numbers had recovered to only 6% of baseline, compared to 22% in the rituximab monotherapy treated group. Additionally, coadministration of IL-21 with rituximab increased circulating activated monocytes, granulocytes and NK cells compared with rituximab monotherapy. In conclusion, addition of IL-21 to a standard weekly, 4-dose regimen of IV rituximab was well-tolerated in cynomolgus monkeys and resulted in more complete, rapid and durable depletion of circulating CD20-expressing target cells. As depletion of B cells in this model serves as a surrogate for clearance of CD20-expressing malignant cells, these data support the evaluation of IL-21 and rituximab combination therapy in patients with advanced CD20 positive malignancies. Figure Figure

Description: 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. To select an appropriate animal species to conduct toxicology studies, a variety of animal species including mouse, rat, rabbit, cynomolgus monkey, rhesus monkey and marmoset monkey were screened for CHIR-12.12 cross-reactivity. The antibody showed binding to rabbit and non-human primate CD40, but not to rodent CD40. Based on functional assays, the cynomolgus monkey was then identified as the appropriate species for toxicity studies: Similar to its activity in human lymphocytes, CHIR-12.12 binds to cynomolgus cell surface CD40 and inhibits CD40-ligand induced proliferation of cynomolgus lymphocytes. A range-finding toxicology study was conducted with CHIR-12.12 doses of 0, 1, 3, 10 and 30 mg/kg once a week for 3 weeks in two cynomolgus monkeys per group per sex. One week after the last dose (Day 22), the animals were humanely euthanized for pathological and histopathological evaluation of all tissues. During the study flow cytometric analysis were performed to monitor changes in B-cell, T-cell and NK-cell numbers. In addition, the antigen saturation on B-cells was monitored over time. Samples for clinical pathology, hematology and urinalysis were collected as well as samples for pharmacokinetics and anti-human antibody determination. The results of the study showed that 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. The expected reduction in B-cell counts was observed as early as 4 hours after the first dosing and at all subsequent time points throughout the study. T-cell counts (CD3+, CD4+ and CD8+ cells) showed no changes. Dose dependent antigen saturation was observed through the whole study. Necropsy and histopathology showed minimal findings in the pancreas of single animals. No significant treatment related adverse events were seen in any organs. The pharmacokinetics of CHIR-12.12 was characterized by small volume of distribution limited to blood volume and slow clearance. The elimination half-life after single dose increased with dose and tended to increase further after multiple dose administration. The half-life ranged between 1.8 days at the 1mg/kg dose to about 7.5 days in the 30 mg/kg group. In conclusion, there were no dose-limiting toxicities in any organ following 3 weekly doses of CHIR-12.12 up to 30 mg/kg. The observed B-cell reduction is the therapeutic target of CHIR-12.12 and an indication for the bioactivity of CHIR-12.12 and is therefore not considered adverse. These results support the clinical development of CHIR-12.12 antibody for treatment of B-cell malignancies.

Description: 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.