Structural features of bovine colostral immunoglobulin that confer proteolytic stability in a simulated intestinal fluid.

Bovine colostral antibodies, purified from cow's milk produced immediately after calving, have enhanced resistance to degradation by intestinal proteases relative to antibodies from human or bovine serum, making them of particular interest as orally administered therapeutic agents. However, the basis of this resistance is not well defined. We evaluated the stability of AVX-470, a bovine colostral anti-tumor necrosis factor (TNF) polyclonal antibody used in early clinical studies for treatment of ulcerative colitis, using conditions that mimic the human small intestine. AVX-470 was degraded ∼3 times more slowly than human IgG antibodies or infliximab (a monoclonal mouse-human chimeric IgG). Bovine IgG1 antibodies, the primary component of AVX-470, were slowly cleaved to F(ab')2 fragments. In contrast, bovine IgG2 and human IgG1 antibodies were cleaved rapidly into Fab and smaller fragments, pointing to specific regions where additional stability might be gained. Infliximab was modified to incorporate the sequences from these regions, including the bovine IgG1 hinge region and a predicted disulfide bonding motif linking the upper hinge region, the CH1 domain, and the light chain. This infliximab-bovine IgG1 chimera (bovinized infliximab) retained the antigen binding and neutralization activity of the WT sequence but was degraded 9-fold more slowly than the unmodified infliximab. This remarkable increase in stability with as few as 18 amino acid substitutions suggests that this bovinization process is a means to enable oral delivery of proven therapeutic antibodies as well as novel antibodies to targets that have been previously inaccessible to therapies delivered by injection.

Effects of AVX-470, an Oral, Locally Acting Anti-Tumour Necrosis Factor Antibody, on Tissue Biomarkers in Patients with Active Ulcerative Colitis.

BACKGROUND AND AIMS: AVX-470 is an orally administered, bovine-derived, anti-tumour necrosis factor (TNF) antibody with local activity in the gastrointestinal tract. In the first-in-human clinical trial of AVX-470 in active ulcerative colitis, we evaluated inflammatory biomarkers in colon tissue as measures of disease activity and early response to treatment. METHODS: Thirty-six patients received active drug (AVX-470 at 0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Colon biopsy samples were collected from 5 regions of colon at baseline and week 4. Tissue inflammatory biomarkers were evaluated by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), epithelial cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and bovine immunoglobulin by immunohistochemistry and mass spectrometry. Endoscopic activity (Ulcerative Colitis Endoscopic Index of Severity [UCEIS]) at colonoscopy was assessed in each colonic region by a central reader. RESULTS: Bovine immunoglobulin was observed in mucosal tissue before and after dosing in lamina propria and submucosal layers of biopsy tissue. Baseline levels of TNF, myeloperoxidase (MPO), CD68 and interleukin (IL)-1ß and, to a lesser extent, IL-6 mRNA were 2- to 3-fold higher in distal vs proximal colon tissue, corresponding to the 2- to 3-fold differences in baseline severities of endoscopic scores. Reductions of >10-fold in TNF and, to lesser extents, in MPO and epithelial cell apoptosis were observed in proximal and distal colon biopsies after 4 weeks of AVX-470 3.5g/day treatment. Reductions in TNF scores were correlated with changes in MPO and CD3 immunohistochemistry scores. CONCLUSIONS: These results are consistent with anti-TNF activity of orally administered AVX-470 in colon mucosal tissue in ulcerative colitis patients and demonstrate the utility of tissue biomarkers in assessing disease and treatment response in early clinical studies. CLINICAL TRIAL REGISTRATION NUMBER: This trial was registered with Clinicaltrials.gov as study NCT01759056 and with EudraCT as study 2012-004859-27.

AVX-470: a novel oral anti-TNF antibody with therapeutic potential in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, which is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF). We developed and characterized AVX-470, a novel polyclonal antibody specific for human TNF. We evaluated the oral activity of AVX-470m, a surrogate antibody specific for murine TNF, in several well-accepted mouse models of IBD. METHODS: AVX-470 and AVX-470m were isolated from the colostrum of dairy cows that had been immunized with TNF. The potency, specificity, and affinity of both AVX-470 and AVX-470m were evaluated in vitro and compared with infliximab. AVX-470m was orally administered to mice either before or after induction of colitis, and activity was measured by endoscopy, histopathology, immunohistochemistry, and quantitative measurement of messenger RNA levels. Colitis was induced using either 2,4,6-trinitrobenzene sulfonate or dextran sodium sulfate. RESULTS: AVX-470 and AVX-470m were shown to be functionally comparable in vitro. Moreover, the specificity, neutralizing potency, and affinity of AVX-470 were comparable with infliximab. Orally administered AVX-470m effectively reduced disease severity in several mouse models of IBD. Activity was comparable with that of oral prednisolone or parenteral etanercept. The antibody penetrated the colonic mucosa and inhibited TNF-driven mucosal inflammation with minimal systemic exposure. CONCLUSIONS: AVX-470 is a novel polyclonal anti-TNF antibody with an in vitro activity profile comparable to that of infliximab. Oral administration of a surrogate antibody specific for mouse TNF is effective in treating mouse models of IBD, delivering the anti-TNF to the site of inflammation with minimal systemic exposure.

Suppression of CD4+ T cell activation by a novel inhibitor of Src family kinases.

The Src family kinases Lck and Fyn play an important role in T cell development and function. We have synthesized a novel small molecule, A-420983, which inhibits Lck and Fyn, as well as other Src family kinases, but has selectivity with respect to non-Src family kinases. A-420983 completely inhibited antigen-stimulated production of IFN-gamma and IL-4 by mouse Th1 and Th2 cells, respectively. Antigen-induced T cell proliferation was also blocked by treatment with A-420983. In contrast, IL-15-induced proliferation was unaffected by A-420983, suggesting that TCR-independent pathways of T cell activation were not impaired. When mice were dosed orally, A-420983 inhibited TCR-mediated c-jun and ZAP-70 phosphorylation in CD4+ T cells and suppressed the disease course of established EAE. Treatment with A-420983 for 7 days resulted in a block in thymocyte development at the CD4- CD8- stage, consistent with inhibition of Lck and Fyn in vivo. These results demonstrate that a small molecule inhibitor of Lck and Fyn can block TCR-induced T cell activation in vitro and in vivo. Furthermore, CNS demyelination mediated by activated encephalitogenic CD4+ T cells is dependent upon the kinase activity of these Src family members. We conclude that inhibition of Src family kinases may represent a promising strategy for the treatment of T cell-mediated disorders.