ANTI-CD25 MONOCLONAL ANTIBODY (MAB):AN IMMUNOMODULATING DRUG CANDIDATE FOR THE TREATMENT OF T-CELL MEDIATED DISEASES

INTRODUCTION AND BACKGROUND Anti-CD25 is a fully human IgG1 kappa monoclonal antibody (mAb) against the alpha subunit of the interleukin 2 receptor (IL2Rα, also known as CD25 or TAC antigen) of potential clinical interest in autoimmune diseases, including multiple sclerosis, uveitis, type 1 diabetes and psoriasis. Anti-CD25 acts mainly by inhibiting the proliferation of T cells and by consequence T cell clonal expansion and cytokine production. IL2 is a potent immunomodulator whose major function is the activation of various cells of the immune system, i.e. T cells (including CD4+ CD25+ regulatory T cells), B cells, NK cells and macrophages, which express CD25 upon antigen stimulation. Anti-CD25 is presented with a potential therapeutic application in T cell mediated diseases, including organ transplant rejection and autoimmune disease, such as multiple sclerosis (MS). The key to immunotherapeutic success with an anti-CD25 is to elicit the correct balance of effector and regulatory T cells. Nevertheless, the effects of an anti-CD25 antibody on the balance between pro-inflammatory T cells versus anti-inflammatory regulatory T cells are still unclear. The potential advantages of Anti-CD25 are the high affinity for human CD25 and the fact that it is a fully humanized mAb, potentially less immunogenic, leading to longer duration of therapeutic effect.It is difficult to foresee which subpopulation of CD25 cells will be the most inhibited by blocking the IL-2Rα as a consequence of in vivo administration of Anti-CD25 mAb. Such aspects were investigated in non-human primates. The ICH S6 guidance providing general principles for designing scientifically acceptable preclinical safety evaluation programs, to support clinical development and marketing authorization, was followed. The purpose of the thesis was to assess the potential adverse effects resulting from the repeated administration of Anti-CD25 mAb in Cynomolgus monkeys. Selection of the relevant animal species for preclinical safety studies was based first on interspecies amino-acid sequence homology of CD25 extracellular domain. The capacity of Anti-CD25 to bind monkey (Rhesus, Cynomolgus and marmoset), minipig, mouse, rat and rabbit CD25 receptor expressed on CD3+ T cells was evaluated using resting or activated Peripheral Blood Mononuclear Cells (PBMCs). The results obtained from Anti-CD25 cross-reactivity evaluation to PBMCs showed that Cynomolgus monkey was the only species where binding to CD25-bearing cells was shown at potentially relevant in vivo concentrations. Therefore the Cynomolgus monkey was considered the relevant species to be used in the preclinical development program of Anti-CD25. EXPERIMENTAL DESIGN The thesis will show data from a study where Anti-CD25 mAb was given by intravenous route (i.v.) at doses of 5, 25, and 125 mg/kg or subcutaneously (s.c.) at a dose of 75 mg/kg to 22 males and 22 females (4 animals/sex for groups 1, 2, and 3 and 5 animals/sex for groups 4 and 5) once a week for 4 consecutive weeks (total of 5 doses) as shown in the table below: Group Doses Volume of administr. Concentr. In vehicle No. of males No. of females Group (mg /kg/ week) (ml/Kg) (mg/ml) identification [admin.Route] --------------------------------------------------------------------------------------------------------------------------------------------------------------- 1 0 (vehicle) [IV] 2 0 4 4 white 2 5 [IV] 2 2.5 4 4 yellow 3 25 [IV] 2 12.5 4 4 green 4 125 [IV] 2 62.5 5 5 red 5 75 [SC] 2 37.5 5 5 blue During the study, general clinical observations, clinical pathology, CD25 expression, CD25 saturation and down-modulation on lymphocyte subsets, NK cell activity, cytokine release markers, toxicokinetics and host anti-drug Ab (ADA) were evaluated. One week after the last treatment 3 monkeys/sex/group were sacrificed for pathology investigations, while the remaining animals were subjected to a recovery period and then sacrificed. RESULTS General examinations: No animals died during the study. No clinical signs (general and local) or behavioral changes were observed in any monkey. No treatment-related hematological changes were seen in any animal treated at the various dosages by either intravenous or subcutaneous route at the end of the treatment or at any test point of the recovery period. No treatment-related changes and no systemic changes were noted at either sacrifice. Toxicokinetics evaluations demonstrated that serum levels increased between the first and the last treatment at all doses by both administration routes. A good compound bioavailability was detected when administered by subcutaneous route at 75 mg/kg. No gender difference was found. Male animals of all the treated groups (from group 2 to group 5) were found positive for the presence of binding anti-drug antibodies to Anti-CD25 whereas females were negative. Antibodies in males were revealed starting from week 6 of the recovery period with a mean titer of >100. They were detected till the last week of recovery. Leukocyte and immunophenotyping analysis showed no effects at any dose and by either administration route. Special examinations: 1. PD markers leukocyte and lymphocyte subsets; CD25 expression, saturation and down-modulation on lymphocyte subsets Immunophenotyping analysis and CD25 expression/saturation/down-modulation were performed on peripheral blood collected in EDTA and analyzed by flow cytometry.CD25 expression in healthy Cynomolgus monkeys was detected only on CD4+ T-cells and this expression was revealed in about 4% of this lymphocyte subpopulation. Anti-CD25 mAb administered at dosages of 5, 25 and 125 mg/kg by iv route, and 75 mg/kg by sc route was able to completely bind and saturate IL-2Rα expressed on CD4+ T-cells, as detected 3 and 6 hours after the treatment, confirming data obtained in previous experiments. CD25 saturation and CD25 down-modulation was observed in all dosed animals until the end of treatment (Day 28). CD25 desaturation was observed during recovery in a dose-dependent manner, and this effect was more evident in female animals. 2. Other biomarkers Natural killer (NK) activity was measured by the ability of effector cells (PBMCs) to kill the target cells (human K562 cell line). Heparinized blood samples were obtained from all animals at the following time point: pre-dose, at the end of treatment (on week 5) and at three timepoints during the recovery period (in weeks 13, 25 and 28). No toxicologically relevant variations were observed in NK activity, in any animal at any time point. CRS (cytokine release syndrome) MARKERS: TNFα, IL-6, IFNγ, IL-1β, IL-2, IL-4, IL-10, IL-8, MCP-1 Blood samples (plasma) were collected from animals at the following time points: day 0 (predose), day1 (2h, 6h, 24h) and day 29 (2h, 6h, 24h). Simultaneous detection of several cytokines/chemokines was performed with Luminex100 system. IL-1β, IL-2 and IFN-γ as well as TNF-α, IL-4 and IL-10 did not show any significant change over time. Nor was any effect seen on IL-8. MCP-1 showed a time effect but this was not statistically relevant. The modulation was similar both in the control group and in treated groups. IL-6 level showed a rise at 2h and 6h after administration of the two higher i.v. doses of Anti-CD25 mAb. This was observed both after the 1st and the 5th administration, with the female animals showing a slightly higher response. The increase in IL-6 was also clearly seen over time. 3. Functional test T-cell proliferation assay and Treg cells number evaluation The functional tests were set to evaluate the antiproliferative and immunomodulatory effect of Anti-CD25 mAb .T cell proliferation assay and FoxP3 staining were performed at the following time point: pre-dose, 24h after the last administration and at 3 recovery time-points (8 , 18, 25 weeks after the last treatment). There were no effects on the ex-vivo T-cell proliferation assay. Anti 25 mAb significantly decreased the percentage of T regulatory cells 24h after the last administration of 125 mg/kg i.v. and 75 mg/kg s.c. CONCLUSION Neither toxicologically relevant changes in clinical observations on hematology, blood chemistry and coagulation parameters nor in histology examinations were found. Toxicokinetics demonstrated the compound accumulated in serum upon prolonged dosing by both administration routes. A good s.c. bioavailability was shown. Anti-CD25 mAb was shown to bind and saturate CD25 (IL-2Rα) expressed on blood CD4 T-cells. This effect was observed at all doses and by both administration routes since the first dosing and was maintained through the dosing period. CD25 desaturation occurred dose-dependently during the recovery period. A trend towards down-modulation was observed at all doses. There was no elevation in the CRS (cytokine release) markers measured, except for IL-6 which showed transient increases at the two higher i.v. doses. This effect could indicate a possible CRS (cytokine release syndrome) in vivo. Regulatory T cells percentage was significantly decreased at the highest i.v. or s.c. doses. This effect seems to correlate with the CD25 expression down-modulation. In conclusion, Anti-CD25 mAb did not result in any adverse toxicological effect either on clinical observations or clinical and morphological pathology, when administered by intravenous or subcutaneous route. However, transient increases in serum IL-6 and reduction in the percentage of blood regulatory T-cells were observed. The results presented in this thesis support further investigations on the potential therapeutic value of the Anti-CD25 mAb evaluated in human diseases where autoimmunity may play a pathogenetic role.