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Preclinical Support for Combination Therapy in the Treatment of Autoimmunity with Atacicept1 ZymoGenetics, Inc., Seattle, WA 98102, USA Correspondence: Dennis Miller, ZymoGenetics, Inc., 1201 Eastlake Ave. E., Seattle, WA 98102, USA; phone: (206) 442-6600; fax: (206) 442-6608; e-mail:info{at}zgi.com.
Atacicept, a fully human recombinant fusion protein that blocks the activity of BLyS (B-Lymphocyte Stimulator) and APRIL (a proliferation-inducing ligand), is undergoing clinical evaluation in B-cell-mediated diseases, including autoimmune disorders. Nonclinical studies in mice and cynomolgus monkeys demonstrate dose-dependent, reversible decreases in circulating Ig concentrations and reductions in mature B cells in the peripheral blood and lymphoid tissues. However, the combination of atacicept with purine synthesis inhibitors (e.g., mycophenolate mofetil [MMF]) and anti-B-cell monoclonal therapy (e.g., rituximab) has not been evaluated. Atacicept does not augment hematological toxicities associated with MMF, including anemia or thrombocytopenia. Combination of atacicept with MMF or rituximab reduced B cells in the periphery (MMF) or tissues (MMF and rituximab) further than did monotherapy, as was the case with atacicept–MMF combination therapy and serum Ig concentrations. Overall, atacicept appears to augment the pharmacologic activity toward B cells of current immunosuppressive therapies without increasing the hematological toxicities associated with MMF. Enhanced reduction in B cells and Ig concentrations associated with atacicept combination therapy may increase therapeutic activity or allow dose reduction in autoimmune patients. Findings from nonclinical safety studies support clinical evaluation of atacicept combination therapies.
Key Words: autoimmunity • biotechnology-derived therapeutics • nonclinical safety assessment Abbreviations: ANOVA, analysis of variance • APRIL, a proliferation-inducing ligand • BLyS, B-lymphocyte stimulator • Ig, immunoglobulin • MMF, mycophenolate mofetil • SLE, systemic lupus erythematosus • TACI, transmembrane activator and calcium modulator and cyclophilin-ligand interactor
The mechanisms underlying the initiation and progression of autoimmune conditions such as rheumatoid arthritis and systemic lupus erythematosus (SLE) are an active area of experimental and epidemiological research. Several factors are associated with the development of autoimmunity, including individual genetics, environmental factors, and alterations in the regulatory proteins involved in maintaining tolerance (Atassi and Casali 2008). Because the majority of B cells produced in the bone marrow are autoreactive, a number of checkpoints have evolved to ensure tolerance. Central tolerance in the bone marrow removes high-avidity B-cell clones through receptor editing or apoptosis, whereas autoreactive B-cell clones that reach the periphery are removed through various mechanisms, such as competition for survival factors (Cornall, Goodnow, and Cyster 1995; Wardemann et al. 2003). A failure of peripheral tolerance mechanisms designed to eliminate autoreactive B cells may underlie many forms of autoimmunity (Shlomchik, Craft, and Mamula 2001). B-lymphocyte stimulator (BLyS, CD257) and a proliferation-inducing ligand (APRIL, CD256) are members of the tumor necrosis factor ligand superfamily and appear to be key cytokines involved in the proliferation, maturation, and survival of B cells (Moore et al. 1999; Dillon et al. 2006; Mackay, Silveira, and Brink 2007). Transgenic mice that overexpress BLyS develop features of autoimmunity, including anti-double-stranded DNA antibodies, proteinuria, and glomerulonephritis, consistent with an SLE-like disorder (Mackay et al. 1999; Gross et al. 2000; Khare et al. 2000). In contrast, BLyS knockout mice have reduced mature B cells and serum immunoglobulin (Ig) levels (Gross et al. 2001). Transgenic mice that overexpress APRIL exhibit increased B cells in peripheral lymph nodes and elevated serum IgM concentrations (Stein et al. 2002), whereas APRIL knockout mice have impaired IgA class switching (Castigli et al. 2004). Moreover, the survival of bone marrow plasma cells, which are highly efficient at producing high-affinity, class-switched antibodies, is dependent on the presence of BLyS or APRIL, and a decrease in both BLyS and APRIL appears to be required to reduce the numbers of bone marrow and plasma cells (Benson et al. 2008). These and other data suggest that competition for these ligands may play a role in establishing peripheral tolerance, and aberrant expression may be associated with autoimmunity (reviewed in Rolink and Melchers 2002; Mackay et al. 2003). Elevated serum concentrations of BLyS and APRIL have been reported in patients with SLE (Stohl 2004; Koyama et al. 2005) and in the sera and synovial fluid of patients with rheumatoid arthritis (Cheema et al. 2001; Tan et al. 2003; Nagatani et al. 2007). Thus, the available mechanistic and epidemiological data suggest that BLyS and APRIL may be useful therapeutic targets in the treatment of B-cell disorders, including autoimmunity. Atacicept is a recombinant Fc fusion protein containing the extracellular, ligand-binding portion of the human transmembrane activator and calcium modulator and cyclophilin-ligand interactor (TACI) receptor and a modified Fc portion of human IgG1. Atacicept binds to, and neutralizes, the activity of BLyS and APRIL and is currently in clinical evaluation for the treatment of rheumatoid arthritis, SLE, multiple sclerosis, and B-cell malignancies. Nonclinical safety studies have been conducted in mice and cynomolgus monkeys, including those involving acute and chronic toxicity, safety pharmacology, and host resistance (Roque et al. 2006; Carbonatto et al. 2008). These studies demonstrate that repeated subcutaneous atacicept administration was well tolerated when administered every other day to mice or twice weekly to monkeys at up to 80 mg/kg for four weeks in both species, or 10 mg/kg for twenty-six weeks in mice or thirty-nine weeks in monkeys. Clinical pathological and histological evaluations revealed changes consistent with the proposed mechanism of action of this molecule, including decreased serum Ig concentrations, decreased mature B-cell concentrations in peripheral blood, and reductions in the follicular marginal zone of the spleen and the mantle surrounding germinal centers of the lymph nodes. Host-resistance studies have demonstrated no effect on pathogen clearance or survival of mice treated with atacicept and subsequently challenged with either influenza virus (Roque et al. 2006) or Streptococcus bacteria (Heffernan et al. 2008). Many aspects of the pharmacological activity of atacicept, including general safety, are comparable to that reported for other recombinant therapeutic agents designed to neutralize BLyS activity, such as belimumab (Halpern et al. 2006) and BR3-Fc (Vugmeyster et al. 2006). Similar to findings with atacicept, treatment with either agent was associated with decreased B cells in the peripheral blood and lymphoid tissues and with reduced marginal zones in the spleen and lymph nodes. However, under the conditions tested, atacicept had a more pronounced effect on serum total Ig concentrations relative to that reported for belimumab (data not reported for BR3-Fc; Halpern et al. 2006). These data suggest that the reduction of both BLyS and APRIL may lead to a relatively greater effect on antibody levels compared with a reduction in BLyS alone. Although clinical data will be needed to validate these ligands as therapeutic targets, it is evident that these new therapeutic approaches along with anti-B-cell monoclonal antibodies (e.g., rituximab, targeting CD20+ B cells) will shed important light on the role of discrete B-cell subsets in the treatment of autoimmunity. The activity of atacicept is unique compared with that of other available immunosuppressive therapies used to reduce autoimmune disease severity, such as corticosteroids (e.g., prednisone), purine synthesis inhibitors (e.g., mycophenolate mofetil [MMF] and azathioprine), cyclophosphamide, or rituximab. The combination of atacicept with these or other immunomodulatory agents has the potential to increase the therapeutic activity directed toward autoimmunity or to allow dose reduction to minimize adverse effects without loss of efficacy. We summarize the results from several nonclinical safety studies conducted to evaluate the safety and pharmacodynamic effects of atacicept combined with MMF or rituximab. Overall, results from these studies demonstrate that atacicept does not augment the hematological toxicities associated with purine synthesis inhibitors. Combination therapy of atacicept with either MMF or rituximab leads to greater reductions in B cells in the periphery (MMF) or tissues (MMF and rituximab) compared with monotherapy. In addition, combination treatment of atacicept with MMF is associated with a greater reduction in serum Ig concentrations compared with monotherapy.
Test Items and Formulation The manufacturing and characterization of atacicept have been previously described (Roque et al. 2006; Carbonatto et al. 2008). MMF (CellCept® oral suspension) and rituximab (MabThera®) were supplied by F. Hoffmann-LaRoche, Ltd., Basel, Switzerland. These agents were stored and prepared per the manufacturer’s instructions and used within the labeled expiry dates.
Animal Care and Use, and Regulatory Compliance
Combination-Study Designs
The safety of atacicept and MMF was evaluated in mice because of demonstrated activity of both agents in this species. The study evaluated the safety and tolerability of atacicept alone, MMF alone, and concurrent administration during up to a thirteen-week dosing period and thirteen-week recovery period (Table 2). The dose route, dose-level selection, and dosing schedule were designed to mimic the proposed clinical dosing regimen.
General Toxicology Clinical and anatomic pathology assessments of mice and cynomolgus monkeys were conducted using methods similar to those previously described, including assessments of mortality, physical appearance, behavior, body weight, food consumption (estimated in monkeys), hematology, serum chemistry, coagulation (cynomolgus monkeys only), organ weight, gross necropsy, microscopic pathology, toxicokinetics, anti-atacicept antibodies, flow cytometric immunophenotyping, and total serum Ig concentration (Carbonatto et al. 2008). For brevity, clarification is provided only for those methods that differ substantially from those described previously and to clarify study-specific sampling schedules. Hematology and serum chemistry analyses were conducted on blood collected from four male and four female mice on days 34 (week 5), 90 (week 13), 124 (week 18), and 180 (week 26). Hematology, serum chemistry, and coagulation analyses were conducted using blood collected from all available monkeys during predosing (days–2 or –3) and weeks 4, 8, 13, 17, 23, 27, and 31. A comprehensive gross necropsy and microscopic pathology evaluation was conducted on all cynomolgus monkeys (Table 1). Similarly, a comprehensive gross necropsy and microscopic pathology evaluation was conducted on four male and four female mice at the end of five or thirteen weeks of treatment and five or thirteen weeks of recovery (Table 2). In addition to standard hematoxylin and eosin analyses, immunohistochemical analysis for B cells (CD20) and T cells (CD3) was performed on the spleen and mesenteric lymph nodes of cynomolgus monkeys.
Pharmacodynamics Flow cytometric analyses of whole blood were conducted using whole blood samples anticoagulated with ethylenediaminetetraacetic acid, collected on days 34 (week 5), 90 (week 13), 124 (week 18), and 180 (week 26) from four male and four female mice, and during weeks–1, 4, 8, 13, 17, 23, 27, and 31 from all available cynomolgus monkeys. In addition to flow cytometric immunophenotyping of peripheral blood in cynomolgus monkeys, sections of spleen were collected at the scheduled necropsy of all animals for analyses. The immunophenotyping panels used for analyses of peripheral blood and spleen samples from cynomolgus monkeys are summarized in Table 3.
Pharmacokinetics Serum atacicept concentration analyses were performed using blood collected from two male and two female mice from atacicept-treated groups (groups 2 and 4) at two, six, and forty-eight hours after treatment on days 1, 34 (week 5), and 90 (week 13). In addition, sera were analyzed from one male and one female mouse from non-atacicept-treated groups (groups 1 and 3) two hours after dosing on days 1, 34 (week 5), and 90 (week 13) to confirm that they had not been incorrectly dosed with atacicept. Serum atacicept concentration analyses were performed using blood collected from all cynomolgus monkeys at baseline (week–1) and during weeks 1, 4, 8, 13, 17, 23, 27, and 31 at two, six, twenty-four, forty-eight, and seventy-two hours after treatment. In addition, sera collected from animals at baseline and during weeks 7, 23, 27, and 31 were evaluated for the presence of anti-atacicept antibodies.
Statistical Analyses
Atacicept and Rituximab in Cynomolgus Monkeys Administration of atacicept and rituximab alone or in combination was well tolerated by cynomolgus monkeys. No animals died during the study and no meaningful clinical signs or changes in body weight, food consumption, hematological, or coagulation parameters were seen among treated animals during the treatment and the recovery periods. Atacicept pharmacokinetics appeared to be unaffected by rituximab as ascertained by periodic serum atacicept concentration analyses throughout the study duration. For example, analyses of atacicept exposure (as AUC0–72 h) revealed no remarkable differences across atacicept treated animals in groups 3 to 5 during weeks 5 and 13 (Table 4). Decreased average atacicept exposure observed among group 5 animals at week 13 is attributable to decreased exposure in two of eight remaining animals, both females, who also demonstrated decreased exposure as early as week 5. However, it was not possible to test sera from these animals for the presence of anti-atacicept antibodies, so it could not be confirmed if the decreased exposure was attributable to the development of anti-atacicept antibodies. Evaluation of sera for anti-atacicept antibodies demonstrated at least one positive antibody titer among animals in groups 3 only (3 of 8 animals) but not animals in groups 4 (0 of 8 animals) or 5 (0 of 4 animals). Sera were not evaluated to establish rituximab pharmacokinetics during concurrent atacicept administration.
Treatment with atacicept alone resulted in marked decreased serum Ig concentrations consistent with previously reported findings (Carbonatto et al. 2008). No notable effect on serum Ig concentrations was observed upon treatment with rituximab alone, and neither combination treatment enhanced reduction of serum Ig concentrations relative to atacicept alone. Results are presented depicting the effects on serum total IgM concentrations of monotherapy and combination therapy (Figure 1). Although results are not shown for serum total IgG and IgA concentrations, the profiles of response are similar to those depicted for IgM. Treatment with atacicept (groups 3–5) was associated with a decrease of ~50% in the mean predose baseline IgA and IgG concentrations, whereas IgM concentrations decreased >95% during the dosing period; serum concentrations recovered to predose levels during the recovery period (Table 5).
Treatment-related changes in B-cell subsets (but not T cells) were observed in animals treated with rituximab and atacicept, whether administered alone or in combination. A modest decrease (–20%) in B cells (CD20+/CD40+) was observed in the peripheral blood in animals treated with atacicept only (group 3) during the treatment period (weeks 13 and 17) compared with the predose baseline. In contrast, a marked decrease of B cells in the blood (CD40+/CD20+), with almost complete loss of cells expressing CD20 and a consequent effect on all B-cell subsets (>–98%), was observed during the rituximab treatment period (week 4) in the peripheral blood of animals of groups 2, 4, and 5. Normalization of B cells to within 20% of baseline was observed after thirteen weeks of recovery among animals treated with atacicept only (group 3) or concurrent atacicept and rituximab (group 5). In contrast, B cells remained decreased among animals treated with rituximab alone (group 2) or sequential atacicept and rituximab (group 4). For example, CD40+/CD20+ cells remained decreased by > 70% after a thirteen-week dose-free period, compared with the predose baseline concentration among animals in groups 2 and 4. The combination of rituximab and atacicept induced a greater pharmacological effect on B lymphocytes in spleen compared with treatment with the single agents alone and appeared to prolong the time to B-cell recovery upon termination of treatment (Figure 2). This was most pronounced in flow cytometric enumeration of spleen for total (CD40+) and CD40+/CD20+ B cells. For example, analysis of isolated splenocytes from group 5 animals (concurrent rituximab and atacicept) revealed a near-complete depletion of detectable CD40+ cells (0.7 ± 0.8%, n = 4) at week 5 relative to vehicle-treated group 1 animals (45.3 ± 7.2%, n = 2) or to group 2 animals treated with rituximab alone (21.3 ± 14.4%, n = 4). Normalization in the abundance of CD40+ splenocytes was seen by the end of the recovery period (week 27).
A reduction of B lymphocytes in other compartments of the lymphoid system was also observed upon histological evaluation of lymphoid tissues from animals treated with rituximab, atacicept, or both. Histological effects in the spleen were characterized by a decreased number and size of the follicles and germinal centers in the white pulp and by the reduced size of the marginal zone surrounding the follicles and of the periarteriolar lymphoid sheets. Similarly, a decreased number and size of germinal centers was observed in the lymph nodes of treated animals compared with control animals. Among animals treated with rituximab or atacicept, the effects in spleen and lymph nodes were generally characterized as moderate to severe, with greater severity among animals dosed with either combination. Immunohistochemical staining evaluations using anti-CD20 antibodies confirmed that these effects were attributable to a decreased B-lymphocyte population (Figure 3), particularly among animals treated with both atacicept and rituximab. A clear trend to recovery was noted by week 27 in all treatment groups, with only minimal and occasional B-cell reduction in a few animals receiving only rituximab or atacicept and with a moderate decrease among animals receiving both drugs (data not shown).
Minimal-to-slight changes were noted at the injection site in animals treated subcutaneously in the hind limbs with atacicept and in the tails of animals injected with rituximab, compared with animals receiving the vehicle. These changes were characterized histologically as vasculitis/perivasculitis and fibroplasia in the subcutis or dermis. Minimal changes were still present in single animals from different groups after the recovery period.
Atacicept and MMF in CD-1 Mice
Sera from animals collected during the dosing phase of the study were uniformly negative for the presence of anti-atacicept antibodies during the dosing phase (i.e., weeks 5 and 13, n = 6), with the exception of 1/6 positive group 4 animals (atacicept + MMF) at week 5. In contrast, four of six animals each from groups 2 (atacicept only) and 4 were antibody positive after five weeks of recovery. Because serum samples were taken from different mice, individual antibody profiles could not be followed over time or correlated with alterations in pharmacokinetics. Overall, no differences were observed in the incidence rate of anti-atacicept-binding antibodies following administration of atacicept alone or in combination with MMF. In the group treated with MMF alone, one female was thin and had skin pallor and piloerection from week 7 up to the end of the treatment period. Piloerection, together with depression in body weight growth, was observed in one male of the same dose group at the end of the treatment period. Skin sores were present in the MMF group at a higher frequency (5/40 animals) compared with controls (3/40), animals treated with atacicept (2/40), and the combination treatment group (1/40). The development of sores has been observed among controls in other studies, and the time of appearance of sores was comparable in treated animals and concurrent controls. Microscopic evaluation of these lesions revealed acute/subacute inflammation and epidermal hyperplasia. The pathogenesis of the sores was not further evaluated or characterized. Given the similar incidence rate and development of sores across treatment groups, no attribution to treatment was made. A trend toward decreased body weight growth gain (~5% vs. controls) was noted in males given MMF alone, starting from week 6 of dosing up to the end of recovery. A slight decrease in food intake was observed in both sexes of the same group during the treatment and withdrawal periods. Body weights and food consumption of animals treated with atacicept alone or in combination with MMF were comparable with controls. Hematology analyses demonstrated a decreased red cell mass (~20% decrease vs. controls in erythrocyte count, hematocrit, and hemoglobin concentration) upon treatment with MMF alone and with combination therapy at the end of the five- and thirteen-week treatment periods. At the same times, an increase in platelets was also observed in both sexes, peaking in females given MMF (~66% increase vs. controls) at the end of the treatment period. These findings resolved by five weeks of withdrawal. No treatment-related changes were seen in animals treated with atacicept alone, and atacicept did not appear to augment the hematological alterations associated with MMF administration. Consistent with previously reported effects, evaluation of total circulating Ig concentrations revealed a marked effect of atacicept on IgG, IgA, and IgM levels relative to controls (Table 7). By day 35 of treatment, animals treated with atacicept alone exhibited a reduction in IgG concentration of approximately 50% compared with concurrent controls (261 ± 307 µg/ml vs. 520 ± 394 µg/ml). In contrast, an elevated IgG concentration (940 ± 654 µg/ml) on day 35 was observed among animals treated with MMF alone compared with controls. Treatment with atacicept and MMF in combination was associated with a slightly greater decrease in IgG levels (149 ± 119 µg/ml) compared with atacicept alone. Measurement of serum IgA also revealed a slightly greater decrease in levels among animals treated with the combination relative to animals treated with atacicept alone by day 91. Atacicept treatment alone was sufficient to nearly deplete serum IgM concentrations by day 35 (Figure 4). Recovery in serum Ig concentrations was generally observed by the end of the dose-free observation period (day 182).
Treatment with atacicept or MMF, alone or in combination, led to a decrease in peripheral B-cell concentrations, particularly mature B cells (Table 7). Treatment with atacicept alone reduced mature B cells by week 5, to approximately 29% of the level observed in concurrent controls. Treatment with MMF alone was also associated with a decrease in mature B cells (63% of the level of concurrent controls). The combination of atacicept with MMF reduced peripheral B-cell concentration to a greater extent than atacicept alone (reduced to 8% of the concentration observed for concurrent controls). At the end of treatment (week 13), animals treated with MMF alone had the greatest overall reduction in the B-cell populations. There was a reduced effect of atacicept or atacicept and MMF on B cells at the end of treatment compared with week 5. Inspection of individual animal data demonstrated a high degree of variability in response by week 13. Whether this may be attributed to the development of antidrug antibodies and/or alterations in atacicept exposure is uncertain, as sera from these mice were not available for antibody and drug concentration assessments. The B-cell populations were similar to controls by the end of the recovery period, although animals previously treated with MMF had a persistently lower B-cell concentration. Microscopic evaluation of a comprehensive panel of tissues revealed treatment-related findings in the spleen, lymph nodes (mesenteric and mandibular), and injection site of animals of the various treated groups at weeks 5 (interim sacrifice) and 13 (end of dosing). In the spleen, a decrease in lymphoid follicle structures (involving germinal centers and/or marginal zone) was seen in almost all males and females given atacicept alone or in combination with MMF. In the mesenteric and mandibular lymph nodes, a decrease in lymphoid follicle structures (involving germinal centers and/or marginal zone) was seen in all animals treated with atacicept alone or in combination with MMF. Treatment with MMF was associated with extramedullary hematopoiesis in the spleen. At the injection site, subcutaneous subacute inflammation and/or infiltration of eosinophils were observed in females given atacicept alone or in combination with MMF and in males given atacicept alone. All these effects resolved during the recovery period.
The nonclinical safety of atacicept in combination with MMF or rituximab was evaluated in thirteen-week studies conducted in mice or cynomolgus monkeys, respectively. These studies were designed according to recommendations for combination safety testing published by the FDA (2006), which encompasses adjunctive combination therapy wherein two therapeutic agents are administered concomitantly (although not necessarily at the same time or under a fixed dose) and there is intent to obtain a marketing label for concomitant use. The FDA guidance also encompasses fixed-dose combinations, wherein the two therapeutic agents are combined in a single-dose form, and can thus be compared against EMEA guidance supporting fixed-dose combinations (EMEA 2008). These two guidance documents are highly similar in their recommendations for the development of therapeutic combinations. Under the FDA guidance, combinations intended for chronic clinical administration wherein one agent is currently approved and the other is new, one should conduct a comprehensive safety program for the novel single agent (according to appropriate standards for drugs [i.e., International Conference on Harmonisation [ICH] M3] or biologic agents [i.e., ICH S6]) and conduct a bridging study of up to ninety days’ duration to evaluate combination treatment. We have completed a comprehensive nonclinical development program for atacicept monotherapy consistent with ICH S6 guidance (Carbonatto et al. 2008). In addition, the clinical and marketing development goals for atacicept include the potential for approval as adjunctive therapy with standard-of-care immunomodulators. Thus, we conducted the nonclinical testing of atacicept combination in accordance with the FDA guidance document to include evaluation periods of thirteen weeks to support chronic clinical dosing. In designing these studies, we used an allometrically scaled human equivalent dose and regimen for MMF and rituximab in combination with an exaggerated human-equivalent dose and regimen for atacicept. Given our previous experience with atacicept, we did not expect substantial interference in exposure or pharmacological activity associated with antibody formation. Moreover, we relied on published data and pilot studies to plan the studies reported here with the goals of assay development, evaluation of unexpected pharmacodynamic or toxicological interactions, and assessment of the allometrically scaled doses. We found the conduct of pilot studies to be invaluable to develop experience with the combination and inform the design of the definitive study. Overall, the combination of rituximab and atacicept was well tolerated and induced greater pharmacological reduction of B lymphocytes in lymphoid tissues compared with treatment with the single agents alone. Similarly, the combination of atacicept and MMF was well tolerated and demonstrated enhanced reduction of serum Ig levels throughout the dosing period and of B-cell concentrations in the peripheral blood through week 5 of treatment. Moreover, atacicept treatment did not augment MMF-induced hematological effects on red blood cells or platelets. These data suggest that combination treatment with atacicept may augment the effects of B-cell-targeted therapies without incurring novel or untoward toxicity. However, these nonclinical studies, which were conducted over short exposure periods in healthy animals, do not address the possible risk of infection associated with combination therapy. Because these combinations appear to be highly effective in modulating specific B-cell populations, these combinations offer the promise of selective reduction of discrete B-cell populations in the treatment of autoimmunity. Further study of these combinations is warranted to establish the safety profile of these therapeutic regimens and whether the enhanced pharmacological activity seen in these nonclinical studies will translate into improved clinical outcomes.
The work presented here was the product of a joint collaboration between Merck Serono International, an affiliate of Merck KGaA, Darmstadt, Germany, and ZymoGenetics, Inc. The author gratefully acknowledges the contributions of Laura Fava, Manuela Onidi, Sergio Peano, Alberto Renoldi, Simona Riva, and Enrico Vigna (Merck Serono); and Ken Bannink, Stacey Dillon, Alisa Littau, Jane Gross, Jane Heffernan, Julie Hill, Steve Hughes, Richard Roque, and Kirk Van Ness (ZymoGenetics). The nonclinical study of atacicept and rituximab was conducted at Nerviano Medical Sciences and involved the contributions of Ugo Bonfanti, Giovanni Buzzi, Paolo Colombo, Anna Maria Giusti, Monica Longo, and Miriam Magistrelli.
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This version was published on January
1, 2009 Toxicologic Pathology, Vol. 37, No. 1,
89-99 (2009)
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Abstract
Introduction
.05) were indicated by the ANOVA, a test for differences between the control and the treatment groups was conducted using Dunnett’s test. When data had nonhomogeneous variance, a Kruskal–Wallis nonparametric ANOVA analysis was conducted, and a Mann–Whitney U test was conducted if warranted. 
