A novel approach to evaluate the pharmacokinetic biocomparability of a monoclonal antibody derived from two different cell lines using simultaneous crossover design.

A parallel study design with a large number of subjects has been a typical path for pharmacokinetic (PK) biocomparability assessment of biotherapeutics with long half-lives and immunogenic propensity, for example, monoclonal antibodies (mAb). A recently published innovative bioanalytical method that can quantify mAb produced from two different cell lines in the same sample opened an avenue to exploring a simultaneous crossover study design for PK biocomparability assessment of biotherapeutics. Siltuximab, a chimeric IgG1 mAb-targeting interleukin-6, was studied as an example. The pharmacokinetic biocomparability of siltuximab derived from mouse myeloma (Sp2/0) cells and Chinese hamster ovary cells was previously assessed and demonstrated in a clinical PK biocomparability study that enrolled more than 140 healthy subjects using a parallel trial design. The biocomparability was successfully shown in six cynomolgus monkeys in a preclinical proof-of-concept study using the new crossover study design supported by the analytical method. The impact of antidrug antibodies on the assessment of biocomparability was minimal. This novel approach opened up a new arena for the evaluation of PK biocomparability of biotherapeutics with unique molecular signatures such as a mAb derived from different cell lines.

A novel approach for the simultaneous quantification of a therapeutic monoclonal antibody in serum produced from two distinct host cell lines.

Therapeutic monoclonal antibodies (mAbs) possess a high degree of heterogeneity associated with the cell expression system employed in manufacturing, most notably glycosylation. Traditional immunoassay formats used to quantify therapeutic mAbs are unable to discriminate between different glycosylation patterns that may exist on the same protein amino acid sequence. Mass spectrometry provides a technique to distinguish specific glycosylation patterns of the therapeutic antibody within the same sample, thereby allowing for simultaneous quantification of the same mAb with different glycosylation patterns. Here we demonstrate a two-step approach to successfully differentiate and quantify serum mixtures of a recombinant therapeutic mAb produced in two different host cell lines (CHO vs. Sp2/0) with distinct glycosylation profiles. Glycosylation analysis of the therapeutic mAb, CNTO 328 (siltuximab), was accomplished through sample pretreatment consisting of immunoaffinity purification (IAP) and enrichment, followed by liquid chromatography (LC) and mass spectrometry (MS). LC-MS analysis was used to determine the percentage of CNTO 328 in the sample derived from either cell line based on the N-linked G1F oligosaccharide on the mAb. The relative amount of G1F derived from each cell line was compared with ratios of CNTO 328 reference standards prepared in buffer. Glycoform ratios were converted to concentrations using an immunoassay measuring total CNTO 328 that does not distinguish between the different glycoforms. Validation of the IAP/LC-MS method included intra-run and inter-run variability, method sensitivity and freeze-thaw stability. The method was accurate (%bias range = -7.30-13.68%) and reproducible (%CV range = 1.49-10.81%) with a LOQ of 2.5 µg/mL.

Therapeutic targeting of the IL-12/23 pathways: generation and characterization of ustekinumab.

Preclinical and clinical studies conducted in the mid-1990s reported strong association and causality between the T-cell helper (T(H)) 1 inductor cytokine interleukin (IL)-12 and numerous immune-mediated disorders, which spurred the development of therapeutic agents targeting IL-12 function. One of the first to enter the clinic, ustekinumab, is a human monoclonal antibody (mAb) that binds to the p40 subunit of IL-12. Subsequent to the generation of ustekinumab, it was discovered that IL-23 also contains the p40 subunit. Thus, although ustekinumab was designed to target IL-12, it also modulates IL-23, a cytokine important to the development and/or maintenance of T(H)17 cells. Clinical observations established that IL-12/23p40 is integral to the pathologies of psoriasis, psoriatic arthritis and Crohn's disease. The molecular and cellular evaluations conducted in ustekinumab clinical programs have provided numerous insights into the pathologic processes of these disorders, illustrating how a novel molecular entity can contribute to our understanding of disease. The individual contributions of these cytokines to specific pathologies require investigation and clinical evaluation of the role of IL-12- and IL-23-specific inhibitors.

Positive treatment effects of ustekinumab in psoriasis: analysis of lesional and systemic parameters.

Ustekinumab, a human anti-interleukin (IL)-12/IL-23p40 monoclonal antibody has demonstrated significant efficacy in patients with moderate-to-severe psoriasis. Skin lesion biopsies, cell surface markers on peripheral blood lymphocytes, and ex vivo T-helper (Th)1/Th2 cytokine responses from peripheral blood mononuclear cells (PBMC) from patients receiving ustekinumab 45 or 90 mg, or placebo were evaluated at baseline and week 12. Inflammatory serum protein levels were measured at baseline, week 2 and week 12. At week 12, median epidermal thickness decreased from 312.1 to 132.7 microm, and median levels of cellular proliferation (Ki67) and T-cell infiltration (CD3) decreased by 84.3% and 70.7%, respectively, in the combined ustekinumab group (all P < or = 0.002). Serum levels of tumor necrosis factor (TNF)-alpha, C-C motif ligand 27 (CCL27) and other inflammatory cytokines remained unchanged. Minimal variation in the percentage of T cells expressing cutaneous lymphocyte antigen (CLA) was observed following ustekinumab treatment, with no significant variation in the percentage of cells expressing CD45RA, CD45RO, CD25, human leukocyte antigen-DR (HLA-DR), and C-X-C motif receptor 3 (CXCR3). No apparent effect on the magnitude of Th1/Th2 responses to external stimuli in PBMC was observed following placebo or ustekinumab treatment. Ustekinumab improves histological psoriasis measures, with minimal impact on the systemic immune system.

Multiple-dose pharmacokinetics and safety of an ibuprofen-pseudoephedrine cold suspension in children.

Two studies were conducted to characterize multiple-dose pharmacokinetics and potential drug interactions of ibuprofen and pseudoephedrine combined in a suspension and to evaluate safety of this combination in children with common cold, flu, or sinusitis. In the pharmacokinetic study, 24 healthy children aged 4-11 years were administered ibuprofen -pseudoephedrine suspension at 7.5 and 1.125 mg/kg, respectively, every 6 hours for 5 doses. Serial blood samples were drawn over 6 hours after final dose for assessment of steady-state pharmacokinetics. In the open-label, multicenter safety study, more than 100 children aged 2-11 years experiencing symptomatic rhinitis were enrolled. Ibuprofen -pseudoephedrine suspension was administered as needed at similar mg/kg doses every 6-8 hours for up to 3 days. Subjects enrolled in the pharmacokinetic study showed no accumulation of either drug; their weight-adjusted clearances were independent of age, and results were comparable with those from previous single-ingredient studies. For ibuprofen, oral clearance (Cl/F) was 77.5 + or - 16.4 mL/kg/h and volume of distribution (Vd/F) was 0.147 + or - 0.037 L/kg. For pseudoephedrine, Cl/F was 12.3 + or - 2.2 mL/kg/min and Vd/F was 2.52 + or - 0.47 L/kg. In the safety study, adverse events were reported for 18.4% of subjects; most were mild to moderate intensity. There was little difference in incidence of adverse events among different age and weight groups. In conclusion, administration of combined ibuprofen and pseudoephedrine in children demonstrated similar pharmacokinetics when compared with reports of the pharmacokinetics for the single-ingredient products, consistent with no apparent drug interactions. The combination suspension was generally well tolerated.

Population pharmacokinetics of golimumab, an anti-tumor necrosis factor-alpha human monoclonal antibody, in patients with psoriatic arthritis.

The population pharmacokinetics of subcutaneously administered golimumab (50 mg or 100 mg every 4 weeks) were characterized in patients with active psoriatic arthritis (PsA) in GO-REVEAL, a randomized, double-blind, placebo-controlled, phase 3 study. A total of 2029 serum golimumab concentrations from 337 patients were analyzed using NONMEM. A 1-compartment pharmacokinetic model with first-order absorption and elimination was chosen to describe the observed concentration-time data. For a patient of standard weight (70 kg), the population estimates (typical value +/- standard error) for golimumab pharmacokinetic parameters were as follows: apparent clearance = 1.38 +/- 0.04 L/d, apparent volume of distribution = 24.9 +/- 1.04 L, and absorption rate constant = 0.908 +/- 0.121 per day. The between-subject variability was 37.6% in apparent clearance and 37.9% in apparent volume of distribution. Body weight, antibody-to-golimumab status, baseline C-reactive protein level, and smoking status were identified as significant covariates on apparent clearance. Body weight was also a significant covariate on apparent volume of distribution. None of the concomitant medications examined (methotrexate, corticosteroids, and nonsteroidal anti-inflammatory drugs) were significant covariates on apparent clearance, although the median trough golimumab concentration in patients receiving methotrexate was higher than for those not receiving methotrexate. These significant covariates account for part of the variability in systemic exposure to golimumab observed in patients with PsA.

Modulation of CLA, IL-12R, CD40L, and IL-2Ralpha expression and inhibition of IL-12- and IL-23-induced cytokine secretion by CNTO 1275.

Cytokines interleukin (IL)-12 and IL-23 are implicated in the pathogenesis of psoriasis. IL-12 causes differentiation of CD4+ T cells to interferon-gamma (IFN-gamma)-producing T helper 1 (Th1) cells, while IL-23 induces differentiation to IL-17-producing pathogenic Th17 cells. The effects of the monoclonal antibody to IL-12/23 p40 subunit (CNTO 1275) on IL-12 receptor (IL-12R) expression, markers associated with skin homing, activation, and cytokine secretion were investigated in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors. PBMCs were activated in the presence or absence of recombinant human (rh) IL-12 or rhIL-23, with or without CNTO 1275. CNTO 1275 inhibited upregulation of CLA, IL-12R, IL-2Ralpha and CD40L expression and also inhibited IL-12- and IL-23-induced IFN-gamma, IL-17A, tumor necrosis factor (TNF)-alpha, IL-2, and IL-10 secretion. Thus, the therapeutic effect of CNTO 1275 may be attributed to the IL-12/23 neutralization, resulting in decreased expression of skin homing and activation markers, and IL-12- and IL-23-induced cytokine secretion.

Changes in biomarkers of inflammation and bone turnover and associations with clinical efficacy following infliximab plus methotrexate therapy in patients with early rheumatoid arthritis.

OBJECTIVE: To determine if changes in biomarkers of inflammation and bone turnover in response to treatment with infliximab plus methotrexate (MTX) versus MTX alone are associated with improvement in clinical measures of signs, symptoms, and structural damage in early rheumatoid arthritis. METHODS: Sera were collected from patients in the ASPIRE study who received 3 mg/kg (n = 48) or 6 mg/kg infliximab plus MTX (n = 55), or MTX alone (n = 41). Several baseline biomarker levels correlated with changes in median percentage of American College of Rheumatology improvement (ACR-N), 50% improvement in ACR response (ACR50), and van der Heijde-modified Sharp score (vdHSS) at Week 54. RESULTS: Infliximab plus MTX treatment resulted in more rapid decreases in levels of matrix metalloproteinase-3 (MMP-3), intercellular cell adhesion molecule-1, interleukin 8 (IL-8), and tumor necrosis factor-a than treatment with MTX alone. Baseline levels and decreases from baseline to Weeks 6 and 54 in MMP-3 correlated with improvement in ACR-N response at Week 54. An increase in IL-8 levels from baseline to Week 54 correlated with worsening in vdHSS at Week 54 in the MTX-alone group. Regression analysis of markers at baseline showed that MMP-3 was the only variable associated with ACR50 response and less worsening in vdHSS at Week 54. CONCLUSION: Treatment with infliximab plus MTX resulted in a rapid decrease in inflammation markers. MMP-3 levels at different timepoints were consistently associated with clinical improvements at Week 54 in the infliximab plus MTX group, while increases in IL-8 levels correlated with a worsening in vdHSS at Week 54 in the MTX-alone group.

A phase 1, double-blind, placebo-controlled study evaluating single subcutaneous administrations of a human interleukin-12/23 monoclonal antibody in subjects with plaque psoriasis.

OBJECTIVE: To evaluate safety, pharmacokinetics, pharmacodynamics, and clinical response of single subcutaneous (s.c.) administrations of a human monoclonal antibody against the p40 subunit of IL-12/23 (IL-12/23 mAb) in subjects with moderate-to-severe psoriasis. METHODS: Twenty-one subjects were enrolled sequentially into 4 dose cohorts (0.27, 0.675, 1.35, and 2.7 mg/kg) and randomized to IL-12/23 mAb or placebo in a 4:1 ratio. Laboratory/clinical parameters and pharmacokinetics were evaluated through Week 24; mRNA cytokine expression was measured in psoriatic plaques at Week 1. RESULTS: Mostly mild adverse events and no serious adverse events were reported. The pharmacokinetics (Cmax and AUC) of IL-12/23 mAb increased in an approximately dose-proportional manner. Of the 17 subjects who received IL-12/23 mAb, 13 achieved PASI 75 (compared with no placebo subjects). mRNA expression of IL-8, IL-18, and IFN-gamma in psoriatic plaques decreased in subjects with sustained Psoriasis Area and Severity Index (PASI) improvement. LIMITATIONS: Interpretation of results is limited due to the small sample size in each dose cohort. CONCLUSION: A single s.c. administration of IL-12/23 mAb was well tolerated and showed clinical response in subjects with moderate-to-severe psoriasis.

A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs.

Bioanalytical assessments of anti-drug antibodies (ADAs) provide an understanding of the immunogenicity of biological drug molecules. The potential to induce ADAs after treatment with biologics is a safety issue that has become an important consideration in the development of biologics and a critical aspect of regulatory filings. US and European regulatory agencies are recommending that sponsors study immunogenicity using a risk-based approach, encouraging sponsors to formulate and implement their own risk management plans and to conduct discussions with the agencies when necessary. It follows from this that the greater the safety risks of ADAs, the more diligently one should clarify the immunogenicity of the product. Here we propose a general strategy to broadly assign immunogenicity risk levels to biological drug products, and present risk level-based 'fit-for-purpose' bioanalytical schemes for the investigations of treatment-related ADAs in clinical and nonclinical studies.

Pharmacokinetics and safety of golimumab, a fully human anti-TNF-alpha monoclonal antibody, in subjects with rheumatoid arthritis.

Golimumab is a fully human antitumor necrosis factor alpha (TNF-alpha) monoclonal antibody that is being developed for intravenous and subcutaneous administration. To assess the pharmacokinetics and safety of the intravenous formulation of golimumab, 36 adult subjects with rheumatoid arthritis were randomly assigned to receive a single infusion of placebo or golimumab (0.1, 0.3, 1, 3, 6, or 10 mg/kg). Serum concentrations of golimumab were determined using a validated enzyme-linked immunosorbent assay method. In addition to the noncompartmental analysis and compartmental modeling, a population pharmacokinetics analysis using NONMEM was also conducted. Both the maximum serum concentration and the area under the serum concentrationtime curve appeared to increase in a dose-proportional manner. The median half-life ranged from 7 to 20 days. A 2-compartment population pharmacokinetic model adequately described the pharmacokinetics of golimumab. The following pharmacokinetic parameters (typical value [% coefficient of variation]) were estimated from the population pharmacokinetic model: clearance (CL: 0.40 [10.1%] L/d), volume of distribution in the central compartment (V(c): 3.07 [6.4%] L), intercompartmental clearance (Q: 0.42 [15.5%] L/d), and volume of distribution in the peripheral compartment (V(p): 3.68 [11.8%] L). Interindividual variability of the pharmacokinetic parameters was quantified for CL (44.3%), V(c) (25.5%), Q (44.6%), and V(p) (44.6%). Residual variability was estimated to be 15.0%. Body weight was found to be an important covariate on V(c). Golimumab was generally well tolerated. The pharmacokinetics of golimumab appeared to be linear over the dose range evaluated in this study.

An anti-IL-12p40 antibody down-regulates type 1 cytokines, chemokines, and IL-12/IL-23 in psoriasis.

Psoriasis is characterized by activation of T cells with a type 1 cytokine profile. IL-12 and IL-23 produced by APCs are essential for inducing Th1 effector cells. Promising clinical results of administration of an Ab specific for the p40 subunit of IL-12 and IL-23 (anti-IL-12p40) have been reported recently. This study evaluated histological changes and mRNA expression of relevant cytokines and chemokines in psoriatic skin lesions following a single administration of anti-IL-12p40, using immunohistochemistry and real-time RT-PCR. Expression levels of type 1 cytokine (IFN-gamma) and chemokines (IL-8, IFN-gamma-inducible protein-10, and MCP-1) were significantly reduced at 2 wk posttreatment. The rapid decrease of these expression levels preceded clinical response and histologic changes. Interestingly, the level of an anti-inflammatory cytokine, IL-10, was also significantly reduced. Significant reductions in TNF-alpha levels and infiltrating T cells were observed in high responders (improvement in clinical score, > or =75% at 16 wk), but not in low responders. Of importance, the levels of APC cytokines, IL-12p40 and IL-23p19, were significantly decreased in both responder populations, with larger decreases in high responders. In addition, baseline levels of TNF-alpha significantly correlated with the clinical improvement at 16 wk, suggesting that these levels may predict therapeutic responsiveness to anti-IL-12p40. Thus, in a human Th1-mediated disease, blockade of APC cytokines by anti-IL-12p40 down-regulates expression of type 1 cytokines and chemokines that are downstream of IL-12/IL-23, and also IL-12/IL-23 themselves, with a pattern indicative of coordinated deactivation of APCs and Th1 cells.

A phase I study evaluating the safety, pharmacokinetics, and clinical response of a human IL-12 p40 antibody in subjects with plaque psoriasis.

The potential therapeutic activity of a human monoclonal antibody to the human interleukin-12 p40 subunit (anti-IL-12p40) has been established both in vitro and in vivo, warranting a first-in-human investigation in psoriasis. This phase I, first-in-human, non-randomized, open-label study evaluated the short-term safety, pharmacokinetics, and clinical response of single, ascending, intravenous (IV) doses of anti-IL-12p40 in subjects with moderate-to-severe psoriasis vulgaris. Eighteen subjects with at least 3% body surface area involvement were enrolled in four dose groups (0.1, 0.3, 1.0, and 5.0 mg per kg). Safety, pharmacokinetics, and clinical response (e.g., Psoriasis Area and Severity Index (PASI)) were monitored at baseline and at specific time points over a 16-wk follow-up period. Anti-IL-12p40 was generally well tolerated. No related serious adverse events or infusion reactions were reported, and most adverse events were mild. IV anti-IL-12p40 yielded linear pharmacokinetics, with a mean terminal half-life of approximately 24 d. Dose-dependent associations with both the rate and extent of clinical response were observed across the four dose groups. Twelve of 18 subjects (67%) achieved at least a 75% improvement in PASI between 8 and 16 wk after study agent administration. Significant and sustained concentration-dependent improvements in psoriatic lesions were observed in most subjects.

Validation and comparative analysis of a multiplexed assay for the simultaneous quantitative measurement of Th1/Th2 cytokines in human serum and human peripheral blood mononuclear cell culture supernatants.

There is increasing evidence suggesting a relationship between cytokine levels and disease pathogenesis, which has led to interest in analyzing multiple cytokines in biological fluids and culture supernatants for various research and clinical studies. The introduction of methodologies allowing simultaneous measurement of interrelated biomarkers/cytokines has further revolutionized this process. In contrast to tissue culture supernatant, the measurement of cytokines in serum has proven to be difficult to characterize in multiplexed formats because of the presence of large dynamic concentration ranges of proteins and other interfering factors that are present in this matrix. In the present study, we have used the microsphere-based multiplex method to simultaneously quantitate and compare six analytes, encompassing a representation of the Th1/Th2 cytokine panel (interleukin (IL)-2, IL-4, IL-5, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and IL-10), in both serum and culture supernatants from peripheral blood mononuclear cells (PBMCs). A detailed validation procedure for these determinations is described along with a comparative analysis of the performance of the multiplexed assay in serum and culture supernatant matrices. Our results indicate that precision of the multiplexed assay is comparable in both culture supernatant and serum. However, the accuracy of quantification of cytokines in the serum matrix but not in culture supernatant may be compromised depending upon the cytokine being analyzed. Therefore, one must use caution when interpreting data from such complex matrices. Nevertheless, this assay format is appropriate to profile cytokines in clinical trial samples.

Immunogenicity of therapeutic monoclonal antibodies.

There are currently 13 monoclonal antibodies or antibody constructs approved for therapeutic use in the US and at least another 400 products are in clinical testing or undergoing regulatory review. Despite widespread use and development of therapeutic monoclonal antibodies, limited information exists regarding the incidence and consequence of immune antibodies generated against many of these products. In this review, analytical methodology and recent data on the immunogenicity of approved therapeutic monoclonal antibodies will be presented. The implications of these findings in relation to potential concerns for repeated or chronic treatment with these powerful and specific therapeutic agents will be discussed.