Minimal physiologically-based pharmacokinetic (mPBPK) model for a monoclonal antibody against interleukin-6 in mice with collagen-induced arthritis.

Therapeutic monoclonal antibodies (mAb) targeting soluble inflammatory cytokines exert their pharmacological effects in rheumatoid arthritis through binding and neutralizing free cytokines in target tissue sites. Therefore suppression of free cytokines in such sites directly relates to the magnitude of therapeutic response. Although the interrelationships between mAb and cytokines have been examined in the systemic circulation, less is known about the interaction of mAb and cytokines in inflamed joints. In the present study, the interplay between the mAb, CNTO 345, and its target IL-6 in serum as well as ankle joint synovial fluid were characterized in collagen-induced arthritic mice. A minimal physiologically-based pharmacokinetic model with target-mediated drug disposition (TMDD) features in serum and ankle joint synovial fluid was developed for the assessment of the TMDD dynamics of CNTO 345 and IL-6. Our model indicates that TMDD kinetics in ankle joints differ greatly from that in serum. The differences can be attributed to the limited tissue distribution of CNTO 345 in ankle joint synovial fluid, the significant rise of the IL-6 baseline in ankle joint synovial fluid in comparison with serum, and the relative time-scales of elimination rates between CNTO 345, free IL-6 and CNTO 345-IL-6 complex in serum and ankle joint synovial fluid.

Ofatumumab is more efficient than rituximab in lysing B chronic lymphocytic leukemia cells in whole blood and in combination with chemotherapy.

Ofatumumab (OFA) is a human anti-CD20 Ab approved for treatment of fludarabine-refractory B chronic lymphocytic leukemia (B-CLL). The efficacy of different immunotherapeutic strategies is best investigated in conditions that are as physiologic as possible. We have therefore compared the activity OFA and rituximab (RTX), alone or in combination with chemotherapeutic agents in unmanipulated whole blood assays, using flow cytometry. OFA (10-100 µg/ml) lysed B-CLL targets in whole blood more efficiently and with faster kinetics than RTX, with a mean 56% lysis at 24 h compared with 16%. This activity of OFA was fully complement dependent, as shown by >99% inhibition by anti-C5 Ab eculizumab and a lack of NK cell activation in whole blood. OFA-mediated NK cell activation was blocked by complement. OFA-mediated lysis could be increased an additional 15% by blocking CD55 and CD59 complement inhibitors. Interestingly, OFA-mediated lysis correlated significantly with CD20 expression levels (r(2) = 0.79). OFA showed overlapping dose response curves similar to those for RTX in phagocytosis assays using either human macrophages or neutrophils. However, phagocytosis was inhibited in the presence of serum or whole blood. Finally, combined treatment with mafosfamide and fludarabine showed that these therapeutic drugs are synergistic in B-CLL whole blood assays and show superior activity when combined with OFA compared with RTX. These results confirm in B-CLL samples and in physiologic conditions the superior complement mediated cytotoxicity induced by OFA alone compared with RTX, the lack of NK cell activation, and phagocytosis in these conditions and suggest effective chemoimmunotherapy strategies using this new generation anti-CD20 Ab.

Effects of hypertonic buffer composition on lymph node uptake and bioavailability of rituximab, after subcutaneous administration.

The subcutaneous administration of biologics is highly desirable; however, incomplete bioavailability after s.c. administration remains a major challenge. In this work we investigated the effects of excipient dependent hyperosmolarity on lymphatic uptake and plasma exposure of rituximab as a model protein. Using Swiss Webster (SW) mice as the animal model, we compared the effects of NaCl, mannitol and O-phospho-L-serine (OPLS) on the plasma concentration of rituximab over 5 days after s.c. administration. An increase was observed in plasma concentrations in animals administered rituximab in hypertonic buffer solutions, compared with isotonic buffer. Bioavailability, as estimated by our pharmacokinetic model, increased from 29% in isotonic buffer to 54% in hypertonic buffer containing NaCl, to almost complete bioavailability in hypertonic buffers containing high dose OPLS or mannitol. This improvement in plasma exposure is due to the improved lymphatic trafficking as evident from the increase in the fraction of dose trafficked through the lymph nodes in the presence of hypertonic buffers. The fraction of the dose trafficked through the lymphatics, as estimated by the model, increased from 0.05% in isotonic buffer to 13% in hypertonic buffer containing NaCl to about 30% for hypertonic buffers containing high dose OPLS and mannitol. The data suggest that hypertonic solutions may be a viable option for improving s.c. bioavailability.

Optimization of rituximab for the treatment of DLBCL (I): dose-dense rituximab in the DENSE-R-CHOP-14 trial of the DSHNHL.

BACKGROUND: To improve outcome of elderly patients with diffuse large B-cell lymphoma, dose-dense rituximab was evaluated in the prospective DENSE-R-CHOP-14 trial. PATIENTS AND METHODS: Rituximab (375 mg/m(2)) was given on days 0, 1, 4, 8, 15, 22, 29, 43, 57, 71, 85, and 99 together with six CHOP-14 cycles. Results were to be compared with patients who had received the same chemotherapy in combination with eight 2-week applications of rituximab in RICOVER-60. RESULTS: One hundred twenty-four patients are assessable. Dose-dense rituximab resulted in considerably higher serum levels during the first 50 days of treatment, but rituximab exposure time was not prolonged. Grade 3 and 4 infections were exceptionally high in the first 20 patients without anti-infective prophylaxis, but decreased after introduction of prophylaxis with aciclovir and cotrimoxazole in the remaining 104 patients (from 13% to 6% per cycle and from 35% to 18% per patient; P = 0.007 and P = 0.125, respectively). Patients with international prognostic index = 3-5 had higher complete response/complete response unconfirmed rates (82% versus 68%; P = 0.033) than in the respective RICOVER-60 population, but this did not translate into better long-term outcome, even though male hazard was decreased (event-free survival: from 1.5 to 1.1; progression-free survival: from 1.7 to 1.1; overall survival: from 1.4 to 1.0). CONCLUSIONS: Dose-dense rituximab achieved higher rituximab serum levels, but was not more effective than eight 2-week applications in the historical control population, even though minor improvements in poor-prognosis and male patients cannot be excluded. The increased, though manageable toxicity, precludes its use in routine practice. Our results strongly support anti-infective prophylaxis with aciclovir and cotrimoxazole for all patients receiving R-CHOP.

Rituximab pharmacokinetics in children and adolescents with de novo intermediate and advanced mature B-cell lymphoma/leukaemia: a Children's Oncology Group report.

The ANHL01P1 trial was undertaken to determine pharmacokinetics and safety following the addition of rituximab to French-American-British/Lymphome Malins de Burkitt (FAB/LMB96) chemotherapy in 41 children and adolescents with Stage III/IV mature B-cell lymphoma/leukaemia. Patients received rituximab (375 mg/m(2) ) days -2 and 0 of two induction cycles and day 0 of two consolidation cycles. Highest peak levels were achieved following the second dose of each induction cycle [299 ± 19 and 384 ± 25 µg/ml (Group-B); 245 ± 31 and 321 ± 32 µg/ml (Group-C)] with sustained troughs and t½ of 26-29 d. Rituximab can be safely added to FAB chemotherapy with high early rituximab peak/trough levels and a long t½.

Progression of structural damage is not related to rituximab serum levels in rheumatoid arthritis patients.

OBJECTIVE: The most cost-effective dosing regimen for rituximab treatment in RA is currently unknown. The objective of this study is to determine whether low rituximab serum levels are associated with progression of structural damage in RA patients. METHODS: Sixty-two RA patients were treated with rituximab in three different centres. Structural damage was assessed on radiographs of hands and feet before and 1 year after therapy using the Sharp-van der Heijde scoring method (SHS). Patients were divided into progressors vs non-progressors based on different cut-off values. Rituximab serum levels were measured by sandwich ELISA after 4 and 12 weeks (Leiden University Medical Center and University Medical Centre, Utrecht cohorts) or 4 and 16 weeks (Academic Medical Center/University of Amsterdam cohort). RESULTS: There was no difference in rituximab levels between progressors and non-progressors 4 weeks and 12 or 16 weeks after initiation of treatment in the different cohorts. There was also no correlation between rituximab levels at week 4 or week 12 or 16 and change in SHS score after 1 year. CONCLUSION: Low rituximab serum levels are not associated with progression of structural damage in RA patients. The results do not support the use of dosages higher than 2 × 1000 mg rituximab to inhibit progression of joint destruction.

Rapid clearance of rituximab may contribute to the continued high incidence of autoimmune hematologic complications of chemoimmunotherapy for chronic lymphocytic leukemia.

Rituximab is an effective treatment for autoimmune cytopenias associated with chronic lymphocytic leukemia. Despite the incorporation of rituximab into fludarabine-based chemotherapy regimens, the incidence of autoimmune cytopenias has remained high. Inadequate rituximab exposure due to rapid antibody clearance may be a contributing factor. To test this hypothesis, we measured serum rituximab levels in patients treated with fludarabine and rituximab (375 mg/m(2)). All patients had undetectable rituximab trough levels by the end of cycle 1, and one-third had undetectable levels already on Day 6 of cycle 1. Although rituximab trough levels increased progressively with each cycle, only by cycle 4 did the median trough level exceed 10 ug/mL. The median half-life of rituximab during cycle 1 was 27 hours, compared to 199 hours during cycle 4 (P<0.0001). There was a significant inverse correlation between the rituximab half-life in cycle 1 and the degree of tumor burden (P=0.02). Two patients who were identified as having subclinical autoimmune hemolysis prior to therapy were given additional doses of rituximab during the initial cycles of therapy and did not develop clinically significant hemolysis. One patient who developed clinically significant hemolysis during therapy was given additional rituximab doses during cycles 3-5 and was able to successfully complete his treatment. In conclusion, rituximab is cleared so rapidly during the initial cycles of therapy for chronic lymphocytic leukemia that most patients have only transient serum levels. More frequent dosing of rituximab may be required to prevent autoimmune complications in at-risk patients (clinicaltrials.gov identifier:00001586).

Mechanism of action of type II, glycoengineered, anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic leukemia whole blood assays in comparison with rituximab and alemtuzumab.

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1-100 µg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.

Expression of apoptosis- and vitamin D pathway-related genes in hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and therapeutic options are scarce. As they might represent future targets for cancer therapy, the expression of apoptosis-related genes in HCC is of particular interest. In this pilot study, we further examined apoptosis-related genes in human HCC and also focused on vitamin D signaling as this might be a regulator of HCC cell apoptosis. METHODS: We employed tumor tissue and serum samples from 62 HCC patients as well as 62 healthy controls for these studies. Tissue and serum specimens were analyzed by quantitative RT-PCR, immunohistochemistry and ELISA. RESULTS: In HCC patients the apoptosis marker M30 was found to be elevated and several pro-apoptotic (TRAIL, FasL and FasR) as well as anti-apoptotic genes (Mcl-1 and Bcl-2) were simultaneously upregulated in tumor tissue and especially tumor-surrounding tissue as compared to healthy control livers. Moreover, vitamin D serum levels were decreased in HCC patients whereas vitamin D receptor mRNA expression was increased in tumor tissue and tumor-surrounding tissue as compared to healthy livers. CONCLUSIONS: In human HCC, M30 serum levels are elevated indicating an increased cell turnover. Modulation of the vitamin D pathway might be a supportive, pro-apoptotic HCC therapy.

Chemokine/cytokine profiling after rituximab: reciprocal expression of BCA-1/CXCL13 and BAFF in childhood OMS.

The aim of the study was to test the hypothesis that B-cell repopulation following rituximab (anti-CD20) therapy is orchestrated by chemokines and non-chemokine cytokines. Twenty-five children with opsoclonus-myoclonus syndrome (OMS) received rituximab with or without conventional agents. A comprehensive panel of 40 chemokines and other cytokines were measured in serum by ELISA and multiplexed fluorescent bead-based immunoassay. Serum BAFF concentration changed dramatically (even after first infusion) and inversely with B-cell depletion/repopulation and CXCL13 concentration at 1, 3, and 6 months. Negative correlations were found for BAFF concentration vs blood B cell percentage and serum CXCL13 concentration; positive correlations with serum rituximab concentrations. Six months after initiation of therapy, no significant difference in the levels of APRIL, CXCL10, IL-6, or 17 other cytokines/chemokines were detected. These data reveal a major role for BAFF in peripheral B cell repopulation following rituximab-induced B-cell depletion, and novel changes in CXCL13. ClinicalTrials.gov NCT0024436.

Misleading hepatitis B test results due to intravenous immunoglobulin administration: implications for a clinical trial of rituximab in immune thrombocytopenia.

BACKGROUND: Rituximab may cause reactivation of hepatitis B virus (HBV) even in patients with remote HBV infection. Thus, the presence of hepatitis B core antibodies (anti-HBc) was an exclusion criterion for a randomized trial of rituximab for patients with immune thrombocytopenia. A high seroprevalence of anti-HBc observed among patients screened for the trial prompted this substudy to investigate for an association between anti-HBc seropositivity and exposure to intravenous immunoglobulin (IVIG). STUDY DESIGN AND METHODS: This was a retrospective case-control study that was a substudy of a randomized controlled trial. RESULTS: Of 24 trial participants screened at one center, 11 (45.8%) were anti-HBc positive and of those, 10 (90.0%) had received IVIG in the preceding 4 weeks. Of 13 seronegative patients screened, five (38.5%) had received IVIG (odds ratio, 16; 95% confidence interval, 1.5-166.1). Seven (70%) of 10 seropositive participants subsequently reverted to negative upon repeat testing. Serial testing before and after IVIG (n = 2) demonstrated transient anti-HBc that lasted for up to 11 weeks after the last dose of IVIG. Samples from three of five different IVIG products were found to contain anti-HBc. CONCLUSIONS: Passive transfer of anti-HBc from certain IVIG products may lead to misinterpretation of hepatitis test results with implications for treatment and clinical trial eligibility. To avoid misleading test results, anti-HBc should be measured before or 3 months after IVIG administration; alternatively an IVIG product known to be free of anti-HBc should be used.

Application of high-resolution MS in the quantification of a therapeutic monoclonal antibody in human plasma.

BACKGROUND: Monoclonal antibodies are the fastest growing class of protein therapeutics. Ligand-binding assays have been the technique of choice for the quantification of these large proteins; however, LC-MS and more recently LC-HRMS have been gaining momentum as robust alternatives for the bioanalysis of antibodies in biological matrices. RESULTS: Optimization of sample preparation and LC-HRMS analysis in MRM(HR) mode has allowed us to develop a highly specific dual-peptide targeted assay for the quantification of Rituximab, in human plasma. The linearity of the assay was established from 1.0 to 200 µg/ml for both light and heavy chain surrogate peptides, with accuracy and precision within 15%. CONCLUSION: LC-HRMS can be an effective tool for the quantification of monoclonal antibodies in regulated bioanalysis.

A quantitative flow cytometric assay for determining binding characteristics of chimeric, humanized and human antibodies in whole blood: proof of principle with rituximab and ofatumumab.

Clinical successes of antibody-based drugs has led to extensive (pre-) clinical development of human(ized) monoclonal antibodies in a great number of diseases. The high specificity of targeted therapy with antibodies makes it ideally suited for personalized medicine approaches in which treatments needs are tailored to individual patients. One aspect of patient stratification pertains to the accurate determination of target occupancy and target expression to determine individual pharmacodynamic properties as well as the therapeutic window. The availability of reliable tools to measure target occupancy and expression on diseased and normal cells is therefore essential. Here, we evaluate a novel human antibody detection assay (Human-IgG Calibrator assay), which allows the flow cytometric quantification of therapeutic antibodies bound to the surface of cells circulating in whole blood. This assay not only permits the determination of the number of specific antibody bound per cell (sABC), but, when combined with quantification of exogenously added mouse antibody, also provides information on binding kinetics and antigen modulation. Our data indicate that the calibrator assay has all properties required for a pharmacodynamic tool to quantify target occupancy of chimeric, humanized and human therapeutic antibodies during therapy, as well as to collect valuable information on both antibody and antigen kinetics.

Validation of a Gyrolab™ assay for quantification of rituximab in human serum.

INTRODUCTION: Gyrolab™ technology presents a technology breakthrough for large molecule bioanalysis to support biologic drug development. The advantages of this innovative platform include fully automated nanoscale immunoassay capability, better assay reproducibility and data quality, small reagent and sample volumes, and rapid assay development and validation as a result of reduced run time. Although Gyrolab has been increasingly used in method development in discovery environment, few fully validated Gyrolab assays have been reported. Here we report a method validation of a Gyrolab assay to determine rituximab levels in human serum. METHODS: Rituximab is captured on a Bioaffy™ CD by a biotinylated rat anti-idiotypic monoclonal antibody against rituximab and detected by an Alexa Fluor®-labeled anti-human IgG antibody. Assay conditions were optimized to give required sensitivity and dynamic range. The assay validation was conducted according to the current industry standards for GLP-regulated immunoassays. RESULTS: The intrabatch precision and accuracy for the assay were determined using spiked human serum samples and shown to have a coefficient of variation (CV) of <11% with a mean bias <20%. The interbatch precision (CV) and absolute mean bias were both <12% with the total error <25%. Adequate spike recovery was demonstrated in serum samples of healthy individuals and solid tumor patients. The dilutional linearity test showed that the determined concentrations adjusted with various dilution factors had a linear relationship with the expected concentrations and that there was no hook effect. The method has been validated for the quantification of rituximab in human serum from 90 to 60,000 ng/mL with a minimum required dilution of 30. DISCUSSION: The Gyrolab assay was proved to be accurate, precise and selective, with a comparable sensitivity to the ELISA method, but provided an automated nanoscale assay with a significantly wider assay dynamic range for the determination of rituximab in human serum during pharmacokinetics/toxicokinetics studies.

The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters: Case studies.

To interpret pharmacokinetic (PK) data of biotherapeutics, it is critical to understand which drug species is being measured by the PK assay. For therapeutic antibodies, it is generally accepted that "free" circulating antibodies are the pharmacologically active form needed to determine the PK/pharmacodynamic (PD) relationship, safety margin calculations, and dose projections from animals to humans and the eventual characterization of the exposure in the clinic. However, "total" drug may be important in evaluating the dynamic interaction between the drug and the target, as well as the total drug exposure. In the absence of or with low amounts of soluble ligand/shed receptor, total and free drug species are often equivalent and their detection is less sensitive to assay formats or reagent choices. In contrast, in the presence of a significant amount of ligand, assay design and characterization of assay reagents are critical to understanding the PK profiles. Here, we present case studies where different assay formats affected measured PK profiles and data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters.

Application of knockout mouse models to investigate the influence of FcγR on the tissue distribution and elimination of 8C2, a murine IgG1 monoclonal antibody.

The current work examines the role of Fcγ-receptors on the elimination and tissue distribution of 8C2, a model murine IgG1 monoclonal antibody. The plasma pharmacokinetics of (125)Iodine-labeled 8C2 were investigated in C57BL/6 control mice, FcγRI/RIII knockout mice, and FcγRIIb knockout mice, following intravenous doses of 0.04, 0.1 and 0.4 mg/kg. Plasma samples were collected and radioactivity was counted. Concentration data were analyzed with a population pharmacokinetic model. Additionally, the tissue disposition of 8C2 was investigated using whole body autoradioluminography (WBAL) and via counting excised tissues. Areas under the plasma concentration vs. time curves AUC(0-10 days) ±SD (nM×days) were: 12.3±0.3, 12.5±1.3 and 15.1±1.2 at 0.04 mg/kg; 39.3±2.0, 28.9±2.7 and 42.0±9.4 at 0.1 mg/kg; and 225±19, 158±19 and 204±26 at 0.4 mg/kg in C57BL/6, FcγRI/RIII(-/-) and FcγRIIb(-/-) mice. Strain was not a statistically significant predictor for any of the parameters of the population model. 8C2 plasma clearance, distribution clearance, and central compartment volume were 0.00543 L/days/kg, 0.0598 L/days/kg, and 0.057 L/kg. No substantial differences in 8C2 tissue uptake were identified by analysis of excised tissues or by WBAL. In conclusion, FcγR knockout is associated with only minor effects on the plasma and tissue disposition of 8C2, a model murine IgG1 mAb.

Prediction of human plasma concentration-time profiles of monoclonal antibodies from monkey data by a species-invariant time method.

We have previously reported that human total body clearance (CL) and steady-state volume of distribution (Vss) of monoclonal antibodies (mAbs) could be predicted reasonably well from monkey data alone using simple allometry with scaling exponents of 0.79 and 1.12 (for soluble targets), and 0.96 and 1.00 (for membrane-bound targets). In the present study, to predict the plasma concentration-time profiles of mAbs in humans, we employed simple dose-normalization and species-invariant time methods (elementary Dedrick plot and complex Dedrick plot), based on the monkey data and the scaling exponents we previously determined. The results demonstrated that the species-invariant time methods were able to provide higher accuracy of prediction than simple dose-normalization, regardless of the type of target antigens (soluble or membrane-bound). The accuracy between elementary Dedrick plot and complex Dedrick plot was nearly equivalent. The predicted human CL and Vss using species-invariant time methods were within mostly 2-fold differences from the observed values. The prediction not only of pharmacokinetic (PK) parameters but also of the plasma concentration-time profile in humans can serve as guidelines for better planning of clinical studies on mAbs.