Interleukin-31 monoclonal antibody
A cell-based assay for assessing the functional activity of a canine or feline IL-31 inhibitor candidate is provided. Such a canine or feline IL-31 inhibitor candidate can be an isolated antibody that specifically binds to at least one of canine Interleukin-31 (IL-31) or feline IL-31. Such antibodies can be in the form of diagnostic and/or veterinary compositions useful for treating a pruritic and/or allergic condition in dogs or cats.
1. A cell-based assay for assessing the functional activity of a canine or feline IL-31 inhibitor candidate, comprising:
providing cells that endogenously express receptors for and are responsive to canine or feline IL-31;
incubating canine or feline IL-31 in the presence of a composition comprising an IL-31 inhibitor candidate or a vehicle control;
treating the cells with the incubated canine or feline IL-31;
measuring the direct phosphorylation of a protein induced by the canine or feline IL-31 treatment; and
determining whether the composition comprising the canine or feline IL-31 inhibitor candidate inhibited the canine or feline IL-31 induced direct protein phosphorylation relative to the vehicle control.
2. The cell-based assay of claim 1 , wherein the measuring step comprises measuring direct phosphorylation of a STAT protein.
3. The cell-based assay of claim 1 , wherein the cells are monocyte cells.
4. The cell-based assay of claim 3 , wherein the monocyte cells are canine DH82 cells.
5. The cell-based assay of claim 1 , wherein the direct protein phosphorylation induced by the canine or feline IL-31 treatment results in an increase in signal.
6. The cell-based assay of claim 1 , wherein inhibition of the direct protein phosphorylation induced by the canine or feline IL-31 inhibitor candidate results in a decrease in signal.
7. The cell-based assay of claim 1 , wherein the canine or feline IL-31 inhibitor candidate is a recombinant canine or feline anti-IL-31 antibody.
8. The cell-based assay of claim 7 , wherein the recombinant canine or feline anti-IL-31 antibody is contained in a hybridoma culture supernatant.
9. The cell-based assay of claim 1 , wherein the canine or feline IL-31 is co-incubated with the canine or feline IL-31 inhibitor candidate prior to treating the cells with the canine or feline IL-31.
10. The cell-based assay of claim 1 , wherein the cells are preincubated with a gamma interferon prior to the canine or feline IL-31 treatment for a period of time sufficient to increase IL-31 receptor expression.
11. The cell-based assay of claim 10 , wherein the cells are preincubated with canine gamma interferon.
12. The cell-based assay of claim 11 , wherein the cells are subsequently serum starved prior to the canine or feline IL-31 treatment.
13. The cell-based assay of claim 1 , wherein a canine or feline IL-31 inhibitor candidate that inhibits greater than 50% of the direct protein phosphorylation relative to the vehicle control is selected for further purification and/or characterization.
14. The cell-based assay of claim 13 , further comprising identifying the 1050 values of the canine or feline IL-31 inhibitor candidate via the cell-based assay.
15. The cell-based assay of claim 1 , wherein inhibition of the canine or feline IL-31 induced direct protein phosphorylation observed in the assay correlates with inhibition of IL-31-mediated pruritus in dogs or cats.
16. A cell-based assay for assessing the functional activity of a canine or feline IL-31 inhibitor candidate,
comprising:
providing canine DH82 monocyte cells that endogenously express receptors for and are responsive to canine or feline IL-31;
incubating canine or feline IL-31 in the presence of a composition comprising an IL-31 inhibitor candidate or a vehicle control;
treating the DH82 monocyte cells with the incubated canine or feline IL-31;
measuring a biological activity in the DH82 monocyte cells induced by the canine or feline IL-31 treatment; and
determining whether the composition comprising the canine or feline IL-31 inhibitor candidate inhibited the canine or feline IL-31 induced biological activity relative to the vehicle control.
17. The cell-based assay of claim 16 , wherein the biological activity is direct phosphorylation of a protein induced by the canine or feline IL-31 treatment.
18. The cell-based assay of claim 17 , wherein the protein is a STAT protein.
19. The cell-based assay of claim 17 , wherein the direct protein phosphorylation induced by canine or feline IL-31 treatment results in an increase in signal.
20. The cell-based assay of claim 17 , wherein inhibition of the direct protein phosphorylation induced by the canine or feline IL-31 inhibitor candidate results in a decrease in signal.
21. The cell-based assay of claim 16 , wherein the canine or feline IL-31 inhibitor candidate is a recombinant canine or feline anti-IL-31 antibody.
22. The cell-based assay of claim 21 , wherein the recombinant canine or feline anti-IL-31 antibody is contained in a hybridoma culture supernatant.
23. The cell-based assay of claim 16 , wherein the canine or feline IL-31 is co-incubated with the canine or feline IL-31 inhibitor candidate prior to treating the DH82 monocyte cells with the canine or feline IL-31.
24. The cell-based assay of claim 16 , wherein the cells are preincubated with a gamma interferon prior to the canine or feline IL-31 treatment for a period of time sufficient to increase IL-31 receptor expression.
25. The cell-based assay of claim 24 , wherein the cells are preincubated with canine gamma interferon.
26. The cell-based assay of claim 25 , wherein the cells are subsequently serum starved prior to the canine or feline IL-31 treatment.
27. The cell-based assay of claim 17 , wherein a canine or feline IL-31 inhibitor candidate that inhibits greater than 50% of the direct protein phosphorylation relative to the vehicle control is selected for further purification and/or characterization.
28. The cell-based assay of claim 27 , further comprising identifying the 1050 values of the canine or feline IL-31 inhibitor candidate via the cell-based assay.
29. The cell-based assay of claim 17 , wherein inhibition of the canine or feline IL-31 induced direct protein phosphorylation observed in the assay correlates with inhibition of IL-31-mediated pruritus in dogs or cats.