(C) Peritoneal infiltration of neutrophils in mice (n = 5) after injection of PBS only or monosodium urate crystals (MSU) followed by administration of PBS, anakinra 30 mg/kg, isotype human antibody 15 mg/kg, P2D7KK 5 mg/kg or P2D7KK 15 mg/kg. and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1 monoclonal antibody is usually a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal. Keywords: interleukin 1, antibody, immunotherapy, drug discovery, animal models Introduction Interleukin (IL)-1 is usually a potent cytokine that drives both the acute and chronic phases of the inflammatory response and plays an essential role in innate immune Laniquidar response.1-4 IL-1 activation and release arises from the activation of inflammasomes, which are large protein complexes constituting users of the NOD-like receptor (NLRs) or PYHIN protein families.5 Upon sensing microbial or danger-associated molecules, these intracellular receptors recruit the adaptor protein ASC, which engages and activates caspase-1. Activated caspase-1 in turn processes the IL-1 precursor into the active and secreted IL-1 cytokine. Physiologically, inflammasomes activation serves as a natural means to defend against pathogens by initiating innate immune responses. However, several endogenous brokers of nonpathogenic origins released upon tissue damage are known to activate the inflammasomes, leading to pathological outcomes.5 For example, the inflammasome activators monosodium urate (MSU) crystals and islet amyloid polypeptides can induce gout disease and Type 2 diabetes, respectively.6,7 Furthermore, genetic defects have also been linked to inflammasome activation-related diseases.8 For example, cryopyrin-associated periodic syndromes (CAPS) arise from a mutation in the CIAS1 gene encoding for cryopyrin/NLRP3, a component of the inflammasome complex that responds to danger signals, resulting in increased inflammasome activity and consequently enhanced IL-1 release. High levels of IL-1 have also been implicated in more common inflammatory and autoimmune diseases, including but not limited to gout, rheumatoid arthritis and diabetes.9,10 As such, IL-1 has been actively pursued as a target for therapeutic antibody development. To date, three recombinant protein drugs targeting IL-1 signaling have been approved for clinical use. Anakinra, marketed as Kineret?, is usually a recombinant IL-1 receptor antagonist (IL-1ra) produced in periplasm. Of the 62 clones analyzed, all showed slower dissociation than clone 2H, with the dissociation rates 1.5 to 30 occasions slower than the parent antibody (representative clones shown in Fig.?3). The off-rate improvements observed from this semi-quantitative analysis using crude Fab samples were confirmed by analysis of 15 purified Fabs (data not shown). DNA analysis of these 62 clones revealed 56 unique sequences, with only slight variations Laniquidar between clones. Out of 9 Laniquidar possible residues at each mutated position, only 2C3 amino acids predominated. Interestingly, while other positions can accommodate mutant residues of various chemical natures, we found the glutamic acid at position 8 of this CDR was completely conserved among all selected clones (Table 1). The conservation of this invariant residue may likely be attributed to a strongly favored electrostatic conversation with a positively charged residue at the antigenic epitope. Four clones, of which the dissociation rates spread across a range of improvement, were chosen to study the affinity-neutralization potency relationship. Affinity measurements by SPR indicated an increase of 21 to 43-fold and 9 to 27-fold for human and mouse IL-1, respectively, compared with the parent clone 2H (Table 1). Neutralization potency of these clones was decided using MRC5 cell-based assays. They inhibited human and mouse IL-1 6 to 36-fold (Fig.?2A) and 1.5 to 12-fold (Fig.?2B), more potently than 2H, respectively. The improvement in affinity Laniquidar and neutralization showed a parallel pattern. Table 2. CDR3L mutagenesis library design assessments: *< 0.05, **< 0.01, Rabbit polyclonal to ZNF268 ***< 0.001. (C) Peritoneal infiltration of neutrophils in mice (n = 5) after injection of PBS only or monosodium urate crystals (MSU) followed by administration of PBS, anakinra 30 mg/kg, isotype human antibody 15 mg/kg, P2D7KK 5 mg/kg or P2D7KK 15 mg/kg. Shown are mean sem on 3 impartial experiments. Data analyzed by ANOVA followed by a multiple test with Bonferronis correction: ****< 0.0001. (D) Survival of mice (n = 10) after inoculation of human myeloma cells. Survival curves were analyzed with a log-rank (Mantel-Cox). P2D7KK efficacy was also tested in a mouse model of peritonitis induced by MSU crystals, which trigger quick and strong neutrophils recruitment in the Laniquidar peritoneal cavity. This model has been widely used to mimic the acute inflammatory response common of gout in humans.23 P2D7KK effectively reduced neutrophils recruitment in the peritoneum after injection of MSU crystals compared with that in mice receiving the isotype control (Fig.?5C). P2D7KK treatment prevented myeloma cell-induced death in vivo IL-6 is the central survival and proliferation factor for multiple myeloma and IL-1 appears to be its major inducer in the bone marrow.24,25 Blockade of the IL-1 signaling by the receptor antagonist IL-1ra has.
(C) Peritoneal infiltration of neutrophils in mice (n = 5) after injection of PBS only or monosodium urate crystals (MSU) followed by administration of PBS, anakinra 30 mg/kg, isotype human antibody 15 mg/kg, P2D7KK 5 mg/kg or P2D7KK 15 mg/kg