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.

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.

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.

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).

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.

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.

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.

Evidence for therapeutic drug monitoring of targeted anticancer therapies.

Therapeutic drug monitoring (TDM) provides valuable guidance for dose adjustment of antibiotics, immunosuppressives, antiepileptics, and other drugs, but its use for traditional anticancer therapies has been limited. Perhaps the most important obstacle is the impractical requirement of multiple blood samples to adequately define systemic exposure of drugs that have a short elimination half-life and are given by intermittent intravenous injections. However, the newer targeted anticancer therapies have different pharmacokinetic (PK) and dosing characteristics compared with traditional cytotoxic drugs, making it possible to estimate the steady-state drug exposure with a single trough-level measurement. Recent evidence indicates that certain PK parameters, including trough levels, are correlated with clinical outcomes for many of these agents, including imatinib, sunitinib, rituximab, and cetuximab. Although the current evidence is insufficient to mandate TDM in routine practice, a concerted investigation should be encouraged to determine whether the steady-state trough measurements of targeted agents will have a practical place in the clinical care of patients with cancer.

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.

(99m)Tc-labeled rituximab for imaging B lymphocyte infiltration in inflammatory autoimmune disease patients.

PURPOSE: The rationale of the present study was to radiolabel rituximab with 99m-technetium and to image B lymphocytes infiltration in the affected tissues of patients with chronic inflammatory autoimmune diseases, in particular, the candidates to be treated with unlabelled rituximab, in order to provide a rationale for 'evidence-based' therapy. PROCEDURES: Rituximab was labelled with (99m)Tc via 2-ME reduction method. In vitro quality controls of (99m)Tc-rituximab included stability assay, cysteine challenge, SDS-PAGE, immunoreactive fraction assay and competitive binding assay on CD20+ve Burkitt lymphoma-derived cells. For the human pilot study, 350-370 MBq (100 µg) of (99m)Tc-rituximab were injected in 20 patients with different chronic inflammatory autoimmune diseases. Whole body anteroposterior planar scintigraphic images were acquired 6 and 20 h p.i. RESULTS: Rituximab was labelled to a high labelling efficiency (>98%) and specific activity (3515-3700 MBq/mg) with retained biochemical integrity, stability and biological activity. Scintigraphy with (99m)Tc-rituximab in patients showed a rapid and persistent spleen uptake, and the kidney appeared to be a prominent source for the excretion of radioactivity. Inflamed joints showed a variable degree of uptake at 6 h p.i. in patients with rheumatoid arthritis indicating patient variability; similarly, the salivary and lacrimal glands showed variable uptake in patients with Sjögren's syndrome, Behçet's disease and sarcoidosis. Inflammatory disease with particular characteristics showed specific uptake in inflammatory lesions, such as, dermatopolymyositis patients showed moderate to high skin uptake, a sarcoidosis patient showed moderate lung uptake, a Behçet's disease patient showed high oral mucosa uptake and a polychondritis patient showed moderate uptake in neck cartilages. In one patient with systemic lupus erythematosus, we did not find any non-physiological uptake. CONCLUSION: Rituximab can be efficiently labelled with (99m)Tc with high labelling efficiency. The results suggest that this technique might be used to assess B lymphocyte infiltration in affected organs in patients with autoimmune diseases; this may provide a rationale for anti-CD20 therapies.

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.

Preclinical studies on the mechanism of action and the anti-lymphoma activity of the novel anti-CD20 antibody GA101.

GA101 is a novel glycoengineered Type II CD20 monoclonal antibody. When compared with rituximab, it mediates less complement-dependent cytotoxicity (CDC). As expected for a Type II antibody, GA101 appears not to act through CDC and is more potent than the Type I antibody rituximab in inducing cell death via nonclassical induction of apoptosis cytotoxicity, with more direct cytotoxicity and more antibody-dependent cell-mediated cytotoxicity. We evaluated the antitumor activity of GA101 against the human-transformed follicular lymphoma RL model in vivo in severe combined immunodeficient mice (SCID) mice. GA101 induced stronger inhibition of tumor growth than rituximab. Combination of GA101 with cyclophosphamide in vivo confirmed the superiority of GA101 over rituximab. Neutralizing the complement system with cobra venom factor partially impaired the antitumor activity of rituximab, but had no impact on the efficacy of GA101. In vitro GA101 more potently induced cell death of RL cells than rituximab. The expression of a limited number of genes was found to be induced by both antibodies after exposure in vitro. Among these, early growth response 1 and activation transcription factor 3 were confirmed to be increased at the protein level, suggesting a possible role of these proteins in the apoptotic signalling of anti-CD20 antibodies. These data imply that GA101 is superior to rituximab not only as a single agent, but also in combination with chemotherapy. These data suggest the presence of novel signalization pathways activated after exposure to anti-CD20 antibodies.

Interindividual variability of response to rituximab: from biological origins to individualized therapies.

Rituximab has markedly changed the treatment of B-cell malignancies. Despite its widespread use, however, its precise mode of action and the impact of host- and tumor-related factors on rituximab-activated biological pathways were only recently clarified. Biological mechanisms resulting in complete resistance to rituximab may exist at both the cellular and subcellular level; however, their frequency and their impact on clinical response are unclear. The identification of Fcγ receptor polymorphisms that can influence anti-CD20 antibody activity has resulted in the development of third-generation anti-CD20 antibodies. However, it is also now appreciated that pharmacokinetic variability is a major factor affecting clinical response to anti-CD20 antibodies. The concept of antigenic mass, which takes into account the total tumor load and the expression levels of the target antigen CD20, is able to explain the correlation between rituximab plasma concentrations and treatment responses. Thus, it can be hypothesized that dosing regimens that take this information into account will help to improve response rates.

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.

Radiolabeling of rituximab with (188)Re and (99m)Tc using the tricarbonyl technology.

INTRODUCTION: The most successful clinical studies of immunotherapy in patients with non-Hodgkin's lymphoma (NHL) use the antibody rituximab (RTX) targeting CD20(+) B-cell tumors. Rituximab radiolabeled with ß(-) emitters could potentiate the therapeutic efficacy of the antibody by virtue of the particle radiation. Here, we report on a direct radiolabeling approach of rituximab with the (99m)Tc- and (188)Re-tricarbonyl core (IsoLink technology). METHODS: The native format of the antibody (RTX(wt)) as well as a reduced form (RTX(red)) was labeled with (99m)Tc/(188)Re(CO)(3). The partial reduction of the disulfide bonds to produce free sulfhydryl groups (-SH) was achieved with 2-mercaptoethanol. Radiolabeling efficiency, in vitro human plasma stability as well as transchelation toward cysteine and histidine was investigated. The immunoreactivity and binding affinity were determined on Ramos and/or Raji cells expressing CD20. Biodistribution was performed in mice bearing subcutaneous Ramos lymphoma xenografts. RESULTS: The radiolabeling efficiency and kinetics of RTX(red) were superior to that of RTX(wt) ((99m)Tc: 98% after 3 h for RTX(red) vs. 70% after 24 h for RTX(wt)). (99m)Tc(CO)(3)-RTX(red) was used without purification for in vitro and in vivo studies whereas (188)Re(CO)(3)-RTX(red) was purified to eliminate free (188)Re-precursor. Both radioimmunoconjugates were stable in human plasma for 24 h at 37 °C. In contrast, displacement experiments with excess cysteine/histidine showed significant transchelation in the case of (99m)Tc(CO)(3)-RTX(red) but not with pre-purified (188)Re(CO)(3)-RTX(red). Both conjugates revealed high binding affinity to the CD20 antigen (K(d) = 5-6 nM). Tumor uptake of (188)Re(CO)(3)-RTX(red) was 2.5 %ID/g and 0.8 %ID/g for (99m)Tc(CO)(3)-RTX(red) 48 h after injection. The values for other organs and tissues were similar for both compounds, for example the tumor-to-blood and tumor-to-liver ratios were 0.4 and 0.3 for (99m)Tc(CO)(3)-RTX(red) and for (188)Re(CO)(3)-RTX(red) 0.5 and 0.5 (24 h pi). CONCLUSION: Rituximab could be directly and stably labeled with the matched pair (99m)Tc/(188)Re using the IsoLink technology under retention of the biological activity. Labeling kinetics and yields need further improvement for potential routine application in radioimmunodiagnosis and therapy.

Efficacy of rituximab monotherapy for an elderly hemodialysis patient with primary cardiac lymphoma.

We report a case of primary cardiac lymphoma (PCL) occurring in a 76-year-old man during maintenance hemodialysis. Chest computed tomography (CT) revealed a tumor with pericardial effusion in the left ventricular posterior wall. Cytological examination of the pericardial fluid revealed monotonous lymphoid cells positive for B-cell markers, and clonal immunoglobulin heavy chain gene rearrangement was detected, indicating B-cell lymphoma. Rituximab monotherapy was administered biweekly at the therapeutic level on hemodialysis. The follow-up chest CT showed tumor disappearance with pericardial fluid after two courses of therapy. Rituximab monotherapy was effective for an elderly hemodialysis patient with PCL.