Pre-clinical and translational pharmacology of a human interleukin-22 IgG fusion protein for potential treatment of infectious or inflammatory diseases.

Interleukin (IL)-22 plays protective roles in infections and in inflammatory diseases that have been linked to its meditation of innate immunity via multiple mechanisms. IL-22 binds specifically to its heterodimeric receptor, which is expressed on a variety of epithelial tissues. UTTR1147A is a recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of human immunoglobulin (Ig) G4. Here, we report extensive in vitro and in vivo nonclinical studies that were conducted to characterize the pharmacological activity of UTTR1147A. The in vitro activity and potency of UTTR1147A were analyzed using primary human hepatocytes and human colonic epithelial cell lines. Assessment of in vivo efficacy was performed in a mouse colitis model and by measuring relevant pharmacodynamic biomarkers, including antimicrobial peptides REG3A/ß, serum amyloid protein A (SAA) and lipopolysaccharide binding protein (LBP). The pharmacokinetic and pharmacodynamic characteristics of UTTR1147A were assessed in healthy mice, rats and cynomolgus monkeys. UTTR1147A induced STAT3 activation through binding to IL-22 receptor expressed in primary human hepatocytes and human colon cell lines. In both, activation occurred in a concentration-dependent manner with similar potencies. In the mouse colitis model, murine IL-22Fc- (muIL-22Fc) treated groups at doses of 1.25 µg and above had statistically lower average histologic colitis scores compared to the control treated group. Administration of muIL-22Fc or UTTR1147A was associated with a dose-dependent induction of PD markers REG3ß and SAA in rodents as well as REG3A, SAA and LBP in cynomolgus monkeys. The combined data confirm pharmacological activity of IL-22Fc and support potential regenerative and protective mechanisms in epithelial tissues.

Improving therapeutic efficacy of a complement receptor by structure-based affinity maturation.

CRIg is a recently discovered complement C3 receptor expressed on a subpopulation of tissue-resident macrophages. The extracellular IgV domain of CRIg (CRIg-ECD) holds considerable promise as a potential therapeutic because it selectively inhibits the alternative pathway of complement by binding to C3b and inhibiting proteolytic activation of C3 and C5. However, CRIg binds weakly to the convertase subunit C3b (K(D) = 1.1 microm), and thus a relatively high concentration of protein is required to reach nearly complete complement inhibition. To improve therapeutic efficacy while minimizing risk of immunogenicity, we devised a phage display strategy to evolve a high affinity CRIg-ECD variant with a minimal number of mutations. Using the crystal structure of CRIg in complex with C3b as a guide for library design, we isolated a CRIg-ECD double mutant (Q64R/M86Y, CRIg-v27) that showed increased binding affinity and improved complement inhibitory activity relative to CRIg-ECD. In a mouse model of arthritis, treatment with a Fc fusion of CRIg-v27 resulted in a significant reduction in clinical scores compared with treatment with an Fc fusion of CRIg-ECD. This study clearly illustrates how phage display technology and structural information can be combined to generate proteins with nearly natural sequences that act as potent complement inhibitors with greatly improved therapeutic efficacy.

Analysis of germline GLI1 variation implicates hedgehog signalling in the regulation of intestinal inflammatory pathways.

BACKGROUND: Ulcerative colitis (UC) and Crohn's disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis. METHODS AND FINDINGS: Using a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C-->G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09-1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1(+/lacZ) mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1(+/lacZ) mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model. CONCLUSIONS: HH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.

Quantification of cortical bone loss and repair for therapeutic evaluation in collagen-induced arthritis, by micro-computed tomography and automated image analysis.

OBJECTIVE: Ex vivo and in vivo micro-computed tomography (micro-CT) combined with a novel image analysis algorithm were used to quantify cortical bone loss and periosteal new bone formation for therapeutic evaluation in a murine model of collagen-induced arthritis. METHODS: An automated algorithm was created to locate 5 metatarsophalangeal and 3 metacarpophalangeal joints in 3-dimensional micro-CT images of mouse paws for evaluation of joint cortical bone volume (JCBV) within close proximity of the joints as well as cortical bone mineral density and periosteal new bone formation within the paws. For validation, automated estimates of JCBV were compared with radiographic visual scores (RVS) in 4 treatment groups (n = 9 per group): rat anti-mouse CD11a monoclonal antibody, methotrexate (MTX), anti-CD11a plus MTX, and saline only. In a separate study, serial images of hind limbs were evaluated in 2 treatment groups: murine tumor necrosis factor receptor II-Fc fusion protein (mTNFRII; n = 10) and control antibody (n = 7). RESULTS: Automated estimates of the JCBV were significantly correlated with the RVS (hind paws R = -0.94, front paws R = -0.81, combined R = -0.87). The anti-CD11a group had significantly higher JCBV compared with controls. In the serial study, the automated estimate of JCBV detected significant treatment effects in the mTNFRII-Fc group compared with controls. Cortical bone mineral density was significantly higher in all treatment groups compared with controls. CONCLUSION: Micro-CT combined with a novel image analysis technique (estimation of JCBV) provides a fully automated means to quantify bone destruction in a mouse model of rheumatoid arthritis.