Derek Thomas Brown

Mechanism of action of certolizumab pegol (CDP870): In vitro comparison with other anti-tumor necrosis factor α agents

Background: Inhibitors of tumor necrosis factor &agr; (TNF&agr;) have demonstrated significant efficacy in chronic inflammatory diseases, including Crohns disease (CD). To further elucidate the mechanisms of action of these agents, we compared the anti‐TNF&agr; agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems. Methods: The ability of each anti‐TNF&agr; agent to neutralize soluble and membrane‐bound TNF&agr;; mediate cytotoxicity, affect apoptosis of activated human peripheral blood lymphocytes and monocytes; induce degranulation of human peripheral blood granulocytes, and modulate lipopolysaccharide (LPS)‐induced interleukin (IL)‐1&bgr; production by human monocytes was measured in vitro. Results: All 4 agents neutralized soluble TNF&agr; and bound to and neutralized membrane TNF&agr;. Infliximab and adalimumab were comparable in their ability to mediate complement‐dependent cytotoxicity and antibody‐dependent cell‐mediated cytotoxicity, and to increase the proportion of cells undergoing apoptosis and the level of granulocyte degranulation. Etanercept generally mediated these effects to a lesser degree, while certolizumab pegol gave similar results to the control reagents. LPS‐induced IL‐1&bgr; production was inhibited by certolizumab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept. Conclusions: In contrast to the other anti‐TNF&agr; agents tested, certolizumab pegol did not mediate increased levels of apoptosis in any of the in vitro assays used, suggesting that these mechanisms are not essential for the efficacy of anti‐TNF&agr; agents in CD. As certolizumab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS‐induced IL‐1&bgr; release from monocytes, inhibition of cytokine production may be important for efficacy of anti‐TNF&agr; agents in CD.

High-Throughput Screening for High Affinity Antibodies

UCB Selected Lymphocyte Antibody Method (SLAM) is a rapid and efficient process for the generation of high-quality monoclonal antibodies, in which variable region gene sequences are recovered directly from specific, single B cells. Monoclonal antibody generation has been limited in the past by the relatively low efficiency of the hybridoma process. UCB SLAM process is well suited to high-throughput screening and has been extensively automated at UCB. If necessary, in excess of 1 times 10 9 B cells can be screened in a campaign, to discover a rare therapeutic antibody candidate, which meets the stringent selection criteria. Primary screening for antigen binders, on purified or cell expressed antigen, is performed using a homogeneous fluorescence assay format. Supernatants from positive wells are consolidated to allow further secondary screening and selection of antibodies with desired characteristics. Individual, specific B cells are identified using a fluorescence based method and isolated using a micromanipulator. The antibody variable region genes are cloned from DNA extracted from the single B cell. The genes are sequenced then prepared for transient expression to confirm activity. Antibodies with affinities (K D) in the sub 10 pM range against a range of therapeutic targets are routinely recovered using this process.

An Antibody That Promotes Adhesion Events Mediated by Both LFA-1 and CR3

Members of the Leu-CAM (β 2 integrin) family of adhesion molecules are widely distributed on circulating lecukocytes, where they play an important role in many cell-cell interactions (reviewed in 1). This family forms a subset of the integrin supergene family and its members show the typical integrin noncovalently associated α/β heterodimer structure (2). There are three members of the family; LFA-1 (CD11a/CD18), CR3 (CD11b/ CD18), and p150.95 (CD11c/CD18) with α-chain molecular weights of 170, 165, and 150 kDa, respectively (2). These glycoproteins exist on the surface of leukocytes as noncovalently linked heterodimers; all share a common β chain (CD18) with a molecular weight of 95 kDa. The different α chains which characterize the individual members of the group share extensive homology (3).