SUMMARYThe type 2 helper T cell (T H 2) cytokine interleukin (IL)-4 is thought to play a central role in the early stages of asthma. In an effort to develop an antibody treatment for asthma that neutralizes the effects of IL-4, a murine monoclonal antibody, 3B9, was generated with specificity for human IL-4. In vitro studies demonstrated that 3B9 inhibited IL-4-dependent events including IL-5 synthesis, T H 2 cell activation and up-regulation of immunoglobulin E expression. 3B9 was then humanized (pascolizumab, SB 240683) to reduce immunogenicity in humans. SB 240683 demonstrated species specificity for both monkey and human IL-4 with no reactivity to mouse, rat, cow, goat or horse IL-4. Pascolizumab inhibited the response of human and monkey T cells to monkey IL-4 and effectively neutralized IL-4 bioactivity when tested against several IL-4-responsive human cell lines. Affinity studies demonstrated rapid IL-4 binding by pascolizumab with a slow dissociation rate. In vivo pharmacokinetic and chronic safety testing in cynomolgus monkeys demonstrated that pascolizumab was well tolerated, and no adverse clinical responses occurred after up to 9 months of treatment. Three monkeys developed an anti-idiotypic response that resulted in rapid pascolizumab clearance. However, in the chronic dosing study the antibody response was transient and not associated with clinical events. In conclusion, pascolizumab is a humanized anti-IL-4 monoclonal antibody that can inhibit upstream and downstream events associated with asthma, including T H 2 cell activation and immunoglobulin E production. Clinical trials are under way to test the clinical efficacy of pascolizumab for asthma.

The recruitment of inflammatory cells into sites of inflammation is a normal physiological response designed to fight infection, remove damaged cells, and stimulate healing. However, the excessive recruitment of such cells often exacerbates tissue damage, slows healing, and in some cases leads to host death. Therefore, inhibition of inflammatory cell recruitment may be an appropriate therapeutic strategy in a number of inflammatory diseases, such as asthma, reperfusion injury, arthritis, and inflammatory bowel disease.Chemokines are a superfamily of approximately 30 distinct small secreted proteins, and additional members continue to be identified (1, 2). They are classified into two major groups, CXC and CC, based on the position of the first two of their four invariant cysteines (3). The actions of chemokines are mediated via interactions with 7-TM 1 G protein-coupled receptors on the surface of immune and inflammatory cells. To date, 18 unique chemokine receptors, including 11 CC chemokine receptors, have been cloned (4, 5).The properties of the chemokines suggest that they are essential for leukocyte trafficking and inflammatory processes and thus are important components in a number of disease states (6, 7). Eosinophils are proinflammatory granulocytes that play a major role in allergic diseases, such as bronchial asthma (8), allergic rhinitis (9), atopic dermatitis (10), and eosinophilic gastroenteritis (11). Upon activation, eosinophils release lipid mediators, cytotoxic proteins, oxygen metabolites, and cytokines, all of which have the potential to produce pathophysiology. Recent studies have clearly demonstrated the presence of eosinophils or eosinophil-specific products in inflamed lung biopsy tissues in human asthma (10).Although the molecular mechanism responsible for the selective infiltration of eosinophils into inflamed tissue has not been elucidated, recently the CC chemokine eotaxin was identified in guinea pig lung following antigen challenge in sensitized guinea pigs (12, 13). Furthermore, neutralizing antibodies to eotaxin in a mouse model of allergy demonstrated inhibition of eosinophil recruitment when administered before the antigen challenge (14). Five CCR3 ligands have been shown to induce eosinophil transendothelial migration using human umbilical vein endothelial cells. This migration is inhibited by pretreatment with anti-CCR3. In addition, a human lung epithelial cell line (BEAS-2B), stimulated with proinflammatory cytokines, has been shown to produce eotaxin and 16). In humans, biopsies obtained from asthmatic lung have shown increased levels of CCR3 and its ligands, eotaxin, eotaxin-2, RANTES, and MCP-4, both at the mRNA and pro-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence should be addressed: Dept. of Immunology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of ...

Wild type human (h) interleukin 5 (wt IL5) Human interleukin 5 (hIL5)1 is a T cell-derived cytokine which plays an important role in the differentiation, proliferation, and activation of eosinophils (Sanderson et al., 1992;Bentley et al., 1992). Natural hIL5 is a disulfide-linked, homodimeric glycoprotein with 115 residues per chain. The high resolution crystal structures of both Escherichia coli-expressed (Milburn et al., 1993) and Drosophila-expressed hIL5 (Johanson et al., 1995) have revealed a core of two four-helix bundles. Each four-helix bundle resembles the four-helix bundle seen in IL2 (Bazan and McKay, 1992), IL4 (Smith et al., 1992), and GM-CSF (Diederichs et al., 1991). However, the bundle organization in IL5 is unique in that helix D of one monomer combines with helices A, B, and C of the second monomer, and vice versa. In the two-bundle structure, the A and D helices form one face, the B and C helices a second. The organization of structural features on each face is palindromic, as observed for restriction sites in DNA.Human IL5 receptor is composed of two different chains, denoted ␣ and ␤ (Tavernier et al., 1991). The ␣ chain is specific for IL5 (Murata et al., 1992) and has a K d in the 0.1-1 nM range depending on the assays and receptor forms examined (Li et al., 1996). In contrast, the ␤ chain of hIL5R is not cytokine-specific but is shared with the receptors for IL3 and GM-CSF (Tavernier et al., 1991) and appears needed for signal transduction. A soluble form of hIL5 receptor ␣ chain (shIL5R␣), which has only the extracellular domain of IL5R␣, also has been described. This binds to hIL5 with nanomolar affinity (Tavernier et al., 1991;Johanson et al., 1995). Despite the dimeric nature of IL5, a 1:1 binding stoichiometry between shIL5R␣ and hIL5 has been reported (Devos et al., 1993;Johanson et al., 1995).New insights into the binding mechanism of IL5 to its receptor are emerging with the availability of high resolution structure and mutagenesis techniques. Data from hybrid constructs of mouse/human IL5 suggest that the carboxyl-terminal 36 residues of IL5 interact directly with the IL5R␣ and confer species specificity (McKenzie et al., 1991). By Ala-scanning mutagenesis of the carboxyl-terminal region of hIL5, we found previously that Glu-110 and Trp-111 contribute significantly to receptor binding . In addition, mutation of residues (Glu-89 and Arg-91) in the loop between helices C and D have been found to affect binding to IL5R␣ (Tavernier et al., 1995;Graber et al., 1995). All of these residues cluster around the interface between the two 4-helix bundles of hIL5 and appear to constitute a central patch for binding to a single molecule of hIL5R␣ . In contrast, residue Glu-13, which is at the distal ends of the IL5 dimer away from the helix bundle interface, was suggested to interact with the ␤ chain of IL5R, since mutation at this position resulted in loss of biological activity but did not affect the binding affinity to the ␣ chain (Tavernier et al., 1995;Graber et al., 1995).While emerging...

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