Characterization of the robust humoral immune response to GSK2618960, a humanized anti-IL-7 receptor monoclonal antibody, observed in healthy subjects in a Phase 1 study.

Interleukin-7 (IL-7) signaling modulates T cell activity and is implicated in numerous autoimmune diseases. An anti-IL-7 receptor monoclonal antibody (GSK2618960) biotherapeutic was evaluated in healthy subjects for safety, pharmacokinetics (PK), pharmacodynamics (PD) and immunogenicity in a single-dose escalation phase I study. We found that antibodies against GSK2618960 (i.e., anti-drug antibodies or ADA) developed in 83% and 100% of GSK2618960-treated subjects in the 0.6 and 2.0 mg/kg dose cohorts, respectively. Of the ADA positive subjects, 64% (7 of 11) had detectable neutralizing activity. Further investigation revealed the presence of GSK2618960-specific memory B cells, indicating the development of immunological memory for the ADAs. Ex vivo stimulation of peripheral blood mononuclear cell (PBMC) samples demonstrated a relatively strong CD4+ T cell proliferation response to GSK2618960 as compared to the control anti-RSV antibody (which is known to have only low immunogenic potential), confirming the high immunogenic potential of GSK2618960. Furthermore, GSK2618960 was found to bind in vitro monocyte-derived dendritic cells (DCs). GSK2618960 treatment of PBMCs increased the proportion of DC cells showing an increase in expression of CD83, CD86 and CD209, which indicated enhanced DC differentiation and activation relative to the isotype control anti-ß amyloid antibody. Collectively, the evidence supports that the high incidence of observed clinical immunogenicity was likely related to the receptor-mediated activity by GSK2618960.

Anti-IL-7 receptor α monoclonal antibody (GSK2618960) in healthy subjects - a randomized, double-blind, placebo-controlled study.

AIM: Interleukin (IL)-7 signalling modulates T cell activity and is implicated in numerous autoimmune diseases. The present study investigated the safety, pharmacokinetics, target engagement, pharmacodynamics and immunogenicity of GSK2618960, an IL-7 receptor-α subunit (CD127) monoclonal antibody. METHODS: A double-blind (sponsor-unblind) study of a single intravenous infusion of either GSK2618960 (0.6 mg kg-1 or 2.0 mg kg-1 ) or placebo was carried out in 18 healthy subjects over 24 weeks. RESULTS: GSK2618960 was well tolerated; there were no serious or significant adverse events. The observed half-life was 5 (±1) days (2.0 mg kg-1 ), with nonlinear pharmacokinetics. Full receptor occupancy (>95%) was observed until day 8 (0.6 mg kg-1 ) and day 22 (2.0 mg kg-1 ). Maximal inhibition of IL-7-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation was observed in 5/6 subjects until day 22 (2.0 mg kg-1 ). Mean circulating IL-7 and soluble receptor (CD127) levels were increased above baseline during days 2 and 15 (0.6 mg kg-1 ) and days 2 and 22 (2.0 mg kg-1 ). No meaningful changes were observed in absolute numbers or proportions of immune cell populations or inflammatory cytokine profiles (IL-6, tumour necrosis factor-α, interferon-γ, IL-2). Persistent antidrug antibodies (ADAs) were detected in 5/6 subjects administered a dose of 0.6 mg kg-1 (neutralizing in 2/6) and in 6/6 subjects administered 2.0 mg kg-1 (neutralizing in 5/6). CONCLUSION: GSK2618960 was well tolerated and blocked IL-7 receptor signalling upon full target engagement. Although there was no discernible impact on peripheral T cell subsets in healthy subjects, GSK2618960 may effectively modulate the autoinflammatory activity of pathogenic T cells in diseased tissue. A relatively short half-life is likely the result of target-mediated rather than ADA-mediated clearance.

Low Percentage of Signal Regulatory Protein α/ß+ Memory B Cells in Blood Predicts Development of Anti-drug Antibodies (ADA) in Adalimumab-Treated Rheumatoid Arthritis Patients.

An important goal for personalized treatment is predicting response to a particular therapeutic. A drawback of biological treatment is immunogenicity and the development of antibodies directed against the drug [anti-drug antibodies (ADA)], which are associated with a poorer clinical outcome. Here we set out to identify a predictive biomarker that discriminates rheumatoid arthritis (RA) patients who are more likely to develop ADA in response to adalimumab, a human monoclonal antibody against tumor necrosis factor (TNF)α. By taking advantage of an immune-phenotyping platform, LEGENDScreen™, we measured the expression of 332 cell surface markers on B and T cells in a cross-sectional adalimumab-treated RA patient cohort with a defined ADA response. The analysis revealed seven differentially expressed markers (DEMs) between the ADA+ and ADA- patients. Validation of the DEMs in an independent prospective European cohort of adalimumab treated RA patients, revealed a significant and consistent reduced frequency of signal regulatory protein (SIRP)α/ß-expressing memory B cells in ADA+ vs. ADA- RA patients. We also assessed the predictive value of SIRPα/ß expression in a longitudinal RA cohort prior to the initiation of adalimumab treatment. We show that a frequency of < 9.4% of SIRPα/ß-expressing memory B cells predicts patients that will develop ADA, and consequentially fail to respond to treatment, with a receiver operating characteristic (ROC) area under the curve (AUC) score of 0.92. Thus, measuring the frequency of SIRPα/ß-expressing memory B cells in patients prior to adalimumab treatment may be clinically useful to identify a subgroup of active RA subjects who are going to develop an ADA response and not gain substantial clinical benefit from this treatment.

A Microdose PET Study of the Safety, Immunogenicity, Biodistribution, and Radiation Dosimetry of 18F-FB-A20FMDV2 for Imaging the Integrin αvß6.

The αvß6 integrin is involved in the pathogenesis of cancer and fibrosis. A radiolabeled 20-amino-acid αvß6-binding peptide, derived from the foot and mouth virus (NAVPNLRGDLQVLAQKVART [A20FMDV2]), has been developed to image αvß6 levels preclinically. This study was designed to translate these findings into a clinical PET imaging protocol to measure the expression of αvß6 in humans. Methods: Preclinical toxicology was undertaken, and a direct immunoassay was developed for 4-fluorobenzamide (FB)-A20FMDV2. Four healthy human subjects (2 male and 2 female) received a single microdose of 18F-FB-A20FMDV2 followed by a multibed PET scan of the whole body over more than 3 h. Results: There were no findings in the preclinical toxicology assessments, and no anti-A20FMDV2 antibodies were detected before or after dosing with the PET ligand. The mean and SD of the administered mass of 18F-FB-A20FMDV2 was 8.7 ± 4.4 µg (range, 2.7-13.0 µg). The mean administered activity was 124 ± 20 MBq (range, 98-145 MBq). There were no adverse or clinically detectable pharmacologic effects in any of the subjects. No significant changes in vital signs, laboratory study results, or electrocardiography results were observed. Uptake of radioactivity was observed in the thyroid, salivary glands, liver, stomach wall, spleen, kidneys, ureters, and bladder. Time-activity curves indicated that the highest activity was in the bladder content, followed by the kidneys, small intestine, stomach, liver, spleen, thyroid, and gallbladder. The largest component of the residence times was the voided urine, followed by muscle, bladder, and liver. Using the mean residence time over all subjects as input to OLINDA/EXM, the effective dose was determined to be 0.0217 mSv/MBq; using residence times from single subjects gave an SD of 0.0020 mSv/MBq from the mean. The critical organ was the urinary bladder, with an absorbed dose of 0.18 mGy/MBq. Conclusion:18F-FB-A20FMDV2 successfully passed toxicology criteria, showed no adverse effects in this first-in-humans study, and has an effective dose that enables multiple scans in a single subject.

False-positive immunogenicity responses are caused by CD20+ B cell membrane fragments in an anti-ofatumumab antibody bridging assay.

An electrochemiluminescent (ECL) bridging assay to detect anti-ofatumumab antibodies (ADA) in human serum samples was developed and validated. Using this assay format, clinical samples were first screened to identify potential ADA positive samples, which were then further tested by adding excess drug, confirming the positive signals as drug specific. However, when the method was implemented into clinical studies for ADA testing, a high positive rate was observed in the pre-dose samples collected from patients with chronic lymphocytic leukemia (CLL). Since the positive signals were not associated with ofatumumab (Ofa) treatment, and diminished after treatment, it was suspected that matrix interference might be responsible, resulting in false-positive responses. We performed a series of experimental investigations to identify, characterize, minimize or eliminate the possible false-positive responses. One possible source was identified to be CD20 (the target of Ofa) present on cell membrane fragments (CMFs). The false-positive responses caused by CD20(+) CMFs could be reduced by solid-phase immunodepletion, ultracentrifugation, or inhibited by adding another anti-CD20 antibody (rituximab). As a consequence, the ADA method was modified to minimize the matrix interference caused by CD20(+) CMFs and, then, validated for sample testing.

Development and validation of a cell-based SEAP reporter assay for the detection of neutralizing antibodies against an anti-IL-13 therapeutic antibody.

A cell-based bioassay capable of detecting neutralizing antibodies (NAb) specific to a therapeutic anti-IL-13 monoclonal antibody was developed, validated and used to analyze normal human and asthma serum samples. At the time of this study, a neutralizing assay was unavailable for anti-IL-13 antibody therapeutics with sufficient rigor for validation. Thus, we describe here a method and considerations for validation. The assay used IL-13 responsive HEK293 cells transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cells were plated at 5.4×10(4) per assay well due to 90% confluence on the subsequent day. Optimal IL-13 and anti-IL-13 concentrations were determined to be 600 pg/mL and 900 ng/mL respectively. We demonstrated the assay's cut point, sensitivity, specificity/cross reactivity, selectivity/matrix interference, and precision. Also, we demonstrated how the drug inhibitory concentration (IC(50), IC(75), and IC(90)) can affect sensitivity and dynamic range/assay window. We characterized the differences in assay response between serum samples of normal population and asthma population. Asthma samples demonstrated an elevated OD ratio in average compared to normal samples. Thus, separate cut points were needed and calculated to be 1.78 and 2.43 for normal and asthma serum, respectively. The assay sensitivity was 670 ng/mL with the positive control (affinity purified rabbit anti-drug polyclonal antibodies). Potential false positives resulting from endogenous serum cytokines including IL-13, IL-4, and Interferon alpha (INF-α) were evaluated and the results indicated that the interfering concentrations for these cytokines are much higher than the respective physiological concentrations. Based on these data, the risk of false positive by endogenous cytokines was considered to be low. In addition, irrelevant anti-drug positive control antibodies were evaluated for assay specificity and did not demonstrate neutralizing capability. Further, no matrix interference in the intended patient population was found when using a final assay serum concentration of 16.7%. The validated assay had acceptable intra- and inter- assay precision in that all %CVs were ≤25%. Overall, this assay successfully proceeded through validation and was used to determine NAb responses within serum samples.

Production of ex vivo lipopolysaccharide-induced tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 is suppressed by trans-resveratrol in a concentration-dependent manner.

Trans-resveratrol is a biologically active compound present in certain foods that has anti-inflammatory and anticancer properties. These beneficial effects are derived from both the immune system and cytokines. The purpose of this study was to determine the immunomodulatory effect of trans-resveratrol on the ex vivo production of inflammatory and anti-inflammatory cytokines stimulated by lipopolysaccharides (LPS). Trans-resveratrol (0, 0.01, 0.1, 1, and 10 microM) was added to blood samples from male Sprague-Dawley rats (n = 6) along with 100 U of LPS (Escherichia coli serotype, 055B5). The samples were then incubated for 4 h at 37 degrees C and centrifuged. Finally, concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in the plasma were analyzed using an enzyme-linked immunosorbent assay (ELISA). The production of inflammatory (TNF-alpha and IL-1beta) and anti-inflammatory (IL-6) cytokines was suppressed by trans-resveratrol in a concentration-dependent manner. These results support the hypothesis that the immunomodulatory effect of trans-resveratrol plays an important role in disease conditions that involve an overproduction of inflammatory cytokines.

Peripheral lymph node stromal cells can promote growth and tumorigenicity of breast carcinoma cells through the release of IGF-I and EGF.

The regional lymph nodes draining primary breast carcinomas are generally the first site to be invaded by disseminating tumor cells. The extent of lymph node involvement remains the most reliable indicator for staging and prognosis of breast cancer. We have investigated host-tumor interactions between breast carcinoma cells and the lymph node stroma, which may control the outcome of lymph node infiltration. In a previous study, we identified integrin-mediated cell adhesion as a correlate of the metastatic potential of human and rat carcinoma cells. Our present objective was to determine whether lymphatic stromal cells can affect cancer cell growth through the elaboration of growth-modulating factors. Two lymphatic stromal cell lines, ST-A4 and ST-B12, were established from normal rat lymph node stromal cell cultures. SFM conditioned by these cells increased the proliferation of human (Hs578T and MCF-7) and rat (TMT-081) breast carcinoma cells by up to 7-fold and augmented their ability to form colonies in semisolid agar by up to 41-fold. This effect was specific as normal, diploid human breast epithelial cells (Hs578Bst), a nontumorigenic, immortalized human breast epithelial cell line (MCF-10A) and a nonmetastatic rat mammary carcinoma cell line (MT-W9B) had either no or reduced responses. RT-PCR analysis revealed that both lymph node stromal cell lines expressed mRNA transcripts for multiple growth factors, including IGF-I, EGF, HGF and PDGF-alpha, and produced detectable levels of IGF-I, EGF and PDGF-alpha, as assessed by Western blotting. Antibody-mediated depletion assays identified IGF-I and EGF as the major mitogenic factors in the CM. The identification of these cells raises the possibility that the lymph node microenvironment may contribute actively to the process of cancer cell dissemination.