In Vitro Pharmacology of AVX-470, an Oral Anti-TNF Polyclonal Antibody for the Treatment of IBD

Abstract

AVX-470 is a bovine polyclonal anti-TNF antibody that is being developed as an oral therapeutic for the treatment of IBD. The antibody is designed to work locally within the inflamed GI tract, minimizing systemic exposure and the resulting systemic immunosuppression. Oral delivery of a surrogate antibody specific for murine TNF (AVX-470 m) has previously been shown to effectively treat DSS- and TNBS-induced colitis in mice. The goal of this study is to determine the in vitro pharmacological parameters of AVX-470, and to compare some of these parameters to the monoclonal antibody infliximab. AVX-470 was generated by immunization of pregnant dairy cows with recombinant human TNF. Colostrum was collected and the immunoglobulin fraction was enriched, with a majority of the polyclonal antibody of bovine isotype IgG1. AVX-470 binding to TNF was quantified by direct ELISA on TNF-coated plates. Neutralization of soluble TNF by AVX-470 was determined using the cellbased L929 assay. Sandwich ELISAs were used to determine AVX-470 cross-reactivity with other cytokines. The TNF-specific component of AVX-470 was isolated on a TNF-affinity column and the activity of the affinity-purified and parent materials were compared in both a TNF-specific binding ELISA and in the L929 TNF neutralization assay. Affinity purified AVX-470 (AVX-470A) was used to measure the binding affinity to human TNF by both Surface Plasmon Resonance (SPR) and by competitive ELISA. Flow cytometric analysis was used to assess the ability of AVX-470 to signal through transmembrane TNF and induce apoptosis in PMA and Ionomycin activated human PBMC, as measured by annexin V and propidium iodide uptake. AVX-470 bound to human TNF in a solid phase ELISA with a high titer, and robustly neutralized soluble human TNF in a cell-based assay. While AVX-470 bound strongly to human TNF, it bound less well to non-human primate TNF, and bound weakly to other species (human>cynomolgus monkey/rhesus macaque>dog>>rodent and cow). AVX-470 demonstrated no cross reactivity to TNFrelated cytokines. By both SPR and competitive ELISA, AVX-470A showed an affinity to TNF in the high picomolar range, comparable to that of infliximab. The monoclonal antibody infliximab displayed a very sharp inhibition curve while the polyclonal AVX-470A displayed a shallower inhibition curve, indicative of a broad range of antibodies directed against multiple epitopes on TNF. AVX-470 induced apoptosis by reverse signaling in activated human PBMC, similar to that of infliximab, while control bovine immunoglobulin showed no effect on apoptosis. AVX-470 is a potent, specific, high affinity anti-TNF antibody which binds to TNF in a solid phase binding assay, neutralizes soluble TNF in the L929 cell-based assay, and induces reverse signaling through membrane TNF as measured by induction of apoptosis. In affinity measurements, TNF neutralization, and apoptosis studies, AVX-470 was comparable to infliximab. AVX-470 has some reactivity to non-human primate TNF, but little to no reactivity to other species tested. This finding has enabled a toxicology study in cynomolgus monkeys. Taken together, these in vitro data support the use of AVX-470 as a therapeutic agent for the treatment of IBD.