Arna Andrews

Molecular cloning, expression and characterization of ovine TNFα

Tumour necrosis factor α (TNFα) is a cytokine with a wide range of effects on both lymphoid and non‐lymphoid cell types. By hybridization with a human TNFα cDNA probe the corresponding ovine cDNA was isolated from a lipopolysaccharide (LPS) stimulated alveolar macrophage cDNA library. The sequence of the cDNA clone showed that ovine TNFα encodes a polypeptide of 234 amino acids that, based on analysis of human TNFα is processed to a protein of 157 amino acids. The nucleotide and amino acid sequences showed a high degree of homology to the equivalent human and mouse molecules. In a mammalian COS cell expression system the ovine cDNA was found to encode a protein which was able to lyse actinomycin‐D treated WEHI‐164 cells and induce COS cells to produce and secrete interleukin 6 (IL‐6). Further experiments demonstrated the importance of sequences within the 3’untranslated region of the cDNA in determining the level of expression of ovine TNFα Northern blot analysis was used to analyse the kinetics of induction of ovine TNFα mRNA in alveolar macrophages stimulated with a variety of mitogens. Addition of LPS increased mRNA encoding TNFα at 1 hand 5 h but not 24 h post stimulation. In contrast, addition of phorbol myristic acid (PMA) led to increased TNFα mRNA at 5 h while the combination of PMA and ionomycin increased the level of specific mRNA detected at 1 h, 5 h and 24 h. From genomic analysis ovine TNFα appears to exist as a single copy.

Recombinant cytokines as immunological adjuvants

This paper describes the bacterial expression and purification of bioactive recombinant ovine interleukin‐2 (rovIL‐2), interleukin‐lα (rovlL‐lα) and tumour necrosis factor α. These purified proteins had specific activities in appropriate bioassays of 1 × 107, 1 times; 107 and 1 × 105 U/mg, respectively. Recombinant ovIL‐lα was assessed as an immunological adjuvant for the sheep response to the model protein avidin. When delivered either intradermally or intramuscularly in conjunction with avidin in aluminium hydroxide the rovIL‐1α significantly enhanced the secondary humoral response. Doses of 1, 10 or 100 μg per sheep enhanced the humoral response to a similar extent. Recombinant ovIL‐1β had similar adjuvant activity in that it was demonstrated to significantly enhance the sheep humoral response to an experimental H. contortus antigen. This increase in specific antibody, however, did not correlate with enhanced protection against infection with third stage H. contortus larvae. In addition incorporation of rovIL‐1 P into the formulation was shown not to alter the isotype profile of H. contortus antigen specific antibody.

Molecular cloning and characterization of a ruminant interleukin-6 cDNA

By hybridization with a human interleukin‐6 (IL‐6) cDNA fragment a corresponding ruminant (ovine) cDNA was isolated from a lipopolysaccharide (LPS)‐stimulated alveolar macrophage library. The nucleotide sequence of the cDNA and the predicted amino acid sequence of the protein showed significant homology to the human and murine molecules. Ovine IL‐6 cDNA encodes a polypeptide of 208 amino acids that, based on analysis of human IL‐6, is processed to a protein of 180 amino acids. Northern blot analysis and the 7TD1 bioassay were used to analyse regulatory aspects of IL‐6 production by primary ovine fibroblasts. Both LPS and recombinant ovine IL‐1α were shown to induce IL‐6 mRNA with peak levels occurring at 1 h post‐stimulation and declining thereafter. When fibroblasts were pretreated with cyclohexamide prior to stimulation the level of induction by LPS and IL‐1α increased dramatically and peak levels were observed at 5 h post‐stimulation. The level of secreted IL‐6 increased rapidly over the first 24 h and continued to increase over the next 48 h.

Therapeutic Targeting of the G-CSF Receptor Reduces Neutrophil Trafficking and Joint Inflammation in Antibody-Mediated Inflammatory Arthritis

G-CSF is a hemopoietic growth factor that has a role in steady state granulopoiesis, as well as in mature neutrophil activation and function. G-CSF– and G-CSF receptor–deficient mice are profoundly protected in several models of rheumatoid arthritis, and Ab blockade of G-CSF also protects against disease. To further investigate the actions of blocking G-CSF/G-CSF receptor signaling in inflammatory disease, and as a prelude to human studies of the same approach, we developed a neutralizing mAb to the murine G-CSF receptor, which potently antagonizes binding of murine G-CSF and thereby inhibits STAT3 phosphorylation and G-CSF receptor signaling. Anti–G-CSF receptor rapidly halted the progression of established disease in collagen Ab-induced arthritis in mice. Neutrophil accumulation in joints was inhibited, without rendering animals neutropenic, suggesting an effect of G-CSF receptor blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti–G-CSF receptor–treated mice showed alterations in cell adhesion receptors, with reduced CXCR2 and increased CD62L expression. Furthermore, blocking neutrophil trafficking with anti–G-CSF receptor suppressed local production of proinflammatory cytokines (IL-1β, IL-6) and chemokines (KC, MCP-1) known to drive tissue damage. Differential gene expression analysis of joint neutrophils showed a switch away from an inflammatory phenotype following anti–G-CSF receptor therapy in collagen Ab-induced arthritis. Importantly, G-CSF receptor blockade did not adversely affect viral clearance during influenza infection in mice. To our knowledge, we describe for the first time the effect of G-CSF receptor blockade in a therapeutic model of inflammatory joint disease and provide support for pursuing this therapeutic approach in treating neutrophil-associated inflammatory diseases.

Production of a human neutralizing monoclonal antibody and its crystal structure in complex with ectodomain 3 of the interleukin-13 receptor α1.

Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.

A neutralizing anti–G‐CSFR antibody blocks G‐CSF–induced neutrophilia without inducing neutropenia in nonhuman primates

Neutrophils are the most abundant WBCs and have an essential role in the clearance of pathogens. Tight regulation of neutrophil numbers and their recruitment to sites of inflammation is critical in maintaining a balanced immune response. In various inflammatory conditions, such as rheumatoid arthritis, vasculitis, cystic fibrosis, and inflammatory bowel disease, increased serum G‐CSF correlates with neutrophilia and enhanced neutrophil infiltration into inflamed tissues. We describe a fully human therapeutic anti–G‐CSFR antibody (CSL324) that is safe and well tolerated when administered via i.v. infusion to cynomolgus macaques. CSL324 was effective in controlling G‐CSF–mediated neutrophilia when administered either before or after G‐CSF. A single ascending‐dose study showed CSL324 did not alter steady‐state neutrophil numbers, even at doses sufficient to completely prevent G‐CSF–mediated neutrophilia. Weekly infusions of CSL324 (≤10 mg/kg) for 3 wk completely neutralized G‐CSF–mediated pSTAT3 phosphorylation without neutropenia. Moreover, repeat dosing up to 100 mg/kg for 12 wk did not result in neutropenia at any point, including the 12‐wk follow‐up after the last infusion. In addition, CSL324 had no observable effect on basic neutrophil functions, such as phagocytosis and oxidative burst. These data suggest that targeting G‐CSFR may provide a safe and effective means of controlling G‐CSF–mediated neutrophilia as observed in various inflammatory diseases.