Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (KD values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent KD and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens

IFN-gamma Impairs Release of IL-8 by IL-1beta-stimulated A549 Lung Carcinoma Cells

Background Production of interferon (IFN)-gamma is key to efficient anti-tumor immunity. The present study was set out to investigate effects of IFNgamma on the release of the potent pro-angiogenic mediator IL-8 by human A549 lung carcinoma cells. Methods A549 cells were cultured and stimulated with interleukin (IL)-1beta alone or in combination with IFNgamma. IL-8 production by these cells was analyzed with enzyme linked immuno sorbent assay (ELISA). mRNA-expression was analyzed by real-time PCR and RNase protection assay (RPA), respectively. Expression of inhibitor-kappaBalpha, cellular IL-8, and cyclooxygenase-2 was analyzed by Western blot analysis. Results Here we demonstrate that IFNgamma efficiently reduced IL-8 secretion under the influence of IL-1beta. Surprisingly, real-time PCR analysis and RPA revealed that the inhibitory effect of IFNgamma on IL-8 was not associated with significant changes in mRNA levels. These observations concurred with lack of a modulatory activity of IFNgamma on IL-1beta-induced NF-kappaB activation as assessed by cellular IkappaB levels. Moreover, analysis of intracellular IL-8 suggests that IFNgamma modulated IL-8 secretion by action on the posttranslational level. In contrast to IL-8, IL-1beta-induced cyclooxygenase-2 expression and release of IL-6 were not affected by IFNgamma indicating that modulation of IL-1beta action by this cytokine displays specificity. Conclusions Data presented herein agree with an angiostatic role of IFNgamma as seen in rodent models of solid tumors and suggest that increasing T helper type 1 (Th1)-like functions in lung cancer patients e.g. by local delivery of IFNgamma may mediate therapeutic benefit via mechanisms that potentially include modulation of pro-angiogenic IL-8

Control of Cell Migration and Inflammatory Mediators Production by CORM-2 in Osteoarthritic Synoviocytes

BackgroundOsteoarthritis (OA) is the most widespread degenerative joint disease. Inflamed synovial cells contribute to the release of inflammatory and catabolic mediators during OA leading to destruction of articular tissues. We have shown previously that CO-releasing molecules exert anti-inflammatory effects in animal models and OA chondrocytes. We have studied the ability of CORM-2 to modify the migration of human OA synoviocytes and the production of chemokines and other mediators sustaining inflammatory and catabolic processes in the OA joint.Methodology/Principal FindingsOA synoviocytes were stimulated with interleukin(IL)-1β in the absence or presence of CORM-2. Migration assay was performed using transwell chambers. Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. CORM-2 reduced the proliferation and migration of OA synoviocytes, the expression of IL-8, CCL2, CCL20, matrix metalloproteinase(MMP)-1 and MMP-3, and the production of oxidative stress. We found that CORM-2 reduced the phosphorylation of extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2 and to a lesser extent p38. Our results also showed that CORM-2 significantly decreased the activation of nuclear factor-κB and activator protein-1 regulating the transcription of chemokines and MMPs in OA synoviocytes.Conclusion/SignificanceA number of synoviocyte functions relevant in OA synovitis and articular degradation can be down-regulated by CORM-2. These results support the interest of this class of agents for the development of novel therapeutic strategies in inflammatory and degenerative conditions

The preclinical pharmacology of the high affinity anti-IL-6R Nanobody (R) ALX-0061 supports its clinical development in rheumatoid arthritis

Introduction: The pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody (R) with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology. Methods: ALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control. Results: ALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration. Conclusions: ALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA

Chemokines in rheumatoid arthritis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46938/1/281_2004_Article_BF00832002.pd