USES OF ANTAGONIST, NON-DEPLETING OX40 ANTIBODIES

1 . A method of increasing the activity of regulatory T cells (Tregs) comprising contacting the Tregs with an antagonist, non-depleting antibody or antigen-binding fragment thereof that specifically binds to OX40.

2 . The method of claim 1 , wherein the Tregs are in a subject, optionally wherein the subject has an autoimmune or inflammatory disease or disorder.

3 . A method of increasing the activity of Tregs in a subject with an autoimmune or inflammatory disease or disorder comprising administering to the subject an antagonist, non-depleting antibody or antigen-binding fragment thereof that specifically binds to OX40.

4 . A method of preventing or treating an autoimmune or inflammatory disease or disorder in a subject comprising administering to the subject an antagonist, non-depleting antibody or antigen-binding fragment thereof that specifically binds to OX40, wherein the antibody or antigen-binding fragment increases Treg activity.

5 . The method of any one of claims 1-4 , wherein the antibody or antigen-binding fragment thereof increases stability of the Tregs.

6 . The method of any one of claims 1-5 , wherein the antibody or antigen-binding fragment thereof increases Foxp3 expression in Tregs, optionally wherein 1 μg/ml concentration of the antibody or antigen-binding fragment increases Foxp3 expression by at least 10%.

7 . The method of any one of claims 1-6 , wherein the antibody or antigen-binding fragment thereof increases Helios expression in Tregs, optionally wherein 1 μg/ml concentration of the antibody or antigen-binding fragment increases Helios expression by at least 10%.

8 . The method of any one of claims 1-7 , wherein the antibody or antigen-binding fragment thereof increases Treg proliferation, optionally wherein 1 μg/ml of the antibody or antigen-binding fragment increases Treg proliferation by at least 10%.

9 . The method of any one of claims 1-8 , wherein the antibody or antigen-binding fragment thereof inhibits binding of OX40-ligand (OX40L) to OX40 with an IC50 of no more than 100 nM.

10 . The method of any one of claims 1-9 , wherein the antibody or antigen-binding fragment thereof agonizes OX40, optionally wherein the antibody or antigen-binding fragment thereof agonizes OX40 by about 2-fold to about 5-fold at the IC50 concentration for inhibition of OX40L binding.

11 . The method of any one of claims 1-10 , wherein the antibody or antigen-binding fragment thereof does not bind to FcγR.

12 . The method of any one of claims 1-11 , wherein the antibody or antigen-binding fragment thereof is engineered to have decreased binding to FcγR.

13 . The method of any one of claims 1-12 , wherein the antibody or antigen-binding fragment thereof comprises an Fc region comprising the amino acid substitution (i) N297A, (ii) N297G, (iii) L234A and L235A, or (iv) a combination thereof, numbered according to the EU numbering system.

14 . The method of any one of claims 1-13 , wherein the antibody or antigen-binding fragment thereof is aglycosylated.

15 . The method of any one of claims 1-14 , wherein the antibody or antigen-binding fragment thereof is engineered to have decreased glycosylation or grown in a host cell to have decreased glycosylation.

16 . The method of any one of claims 1-10 , wherein the antibody or antigen-binding fragment thereof is an IgG4.

17 . The method of any one of claims 1-16 , wherein the antibody or antigen-binding fragment thereof binds to human OX40 and cynomolgus monkey ( Macaca fascicularis ) OX40.

18 . The method of any one of claims 1-17 , wherein the antibody or antigen-binding fragment is chimeric, humanized, or human.

19 . The method of any one of claims 1-18 , wherein the antibody or antigen-binding fragment is a full length antibody.

20 . The method of any one of claims 1-18 , wherein the antibody or antigen-binding fragment is an antigen binding fragment.

21 . The method of claim 20 , wherein the antigen binding fragment comprises a Fab, Fab′, F(ab′) 2 , single chain Fv (scFv), disulfide linked Fv, V-NAR domain, IgNar, IgGΔCH2, minibody, F(ab′) 3 , tetrabody, triabody, diabody, single-domain antibody, (scFv) 2 , or scFv-Fc.

22 . The method of any one of claims 1-21 , wherein the antibody or antigen-binding fragment is monoclonal.

23 . The method of any one of claims 1-22 , wherein the antibody or antigen-binding fragment is recombinant.

24 . The method of any one of claims 2-23 , wherein the autoimmune or inflammatory disease or disorder is selected from the group consisting of psoriasis, graft versus host disease, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, amyotrophic lateral sclerosis (ALS), multiple sclerosis, ulcerative colitis, Crohn's disease, HCV-related vasculitis, alopecia areata, ankylosing spondylitis, Sjögren's Syndrome, autoimmune hepatitis, inflammatory bowel disease (IBD), colitis, vasculitis, temporal arthritis, lupus, celiac disease, polymyalgia rheumatic, and arthritis.

25 . The method of any one of claims 2-23 , wherein the autoimmune or inflammatory disease or disorder is graft versus host disease.

26 . A method for selecting an anti-OX40 antibody or antigen-binding fragment thereof capable of increasing the activity of regulatory T cells (Tregs), the method comprising (i) providing an anti-OX40 antibody or antigen-binding fragment thereof, and (ii) assaying the ability of the antibody or antigen-binding fragment thereof to increase Foxp3 expression in the Tregs to determine whether the antibody or antigen-binding fragment thereof is capable of increasing Foxp3 expression in the Tregs by at least 10%.

27 . A method for selecting an anti-OX40 antibody or antigen-binding fragment thereof capable of increasing the activity of regulatory T cells (Tregs), the method comprising (i) providing a plurality of recombinant anti-OX40 antibodies or antigen-binding fragments thereof; and (ii) assaying the ability of the antibodies or antigen-binding fragments thereof to increase Foxp3 expression in the Tregs to determine whether the antibodies or antigen-binding fragments thereof are capable of increasing Foxp3 expression in the Tregs by at least 10%.

28 . A method for selecting an anti-OX40 antibody or antigen-binding fragment thereof capable of increasing the activity of regulatory T cells (Tregs), the method comprising (i) providing an anti-OX40 antibody or antigen-binding fragment thereof, and (ii) assaying the ability of the antibody or antigen-binding fragment thereof to increase Helios expression in the Tregs to determine whether the antibody or antigen-binding fragment thereof is capable of increasing Helios expression in the Tregs by at least 10%.

29 . A method for selecting an anti-OX40 antibody or antigen-binding fragment thereof capable of increasing the activity of regulatory T cells (Tregs), the method comprising (i) providing a plurality of recombinant anti-OX40 antibodies or antigen-binding fragments thereof; and (ii) assaying the ability of the antibodies or antigen-binding fragments thereof to increase Helios expression in the Tregs to determine whether the antibodies or antigen-binding fragments thereof are capable of increasing Helios expression by at least 10%.

30 . A method for selecting an anti-OX40 antibody or antigen-binding fragment thereof capable of increasing the activity of regulatory T cells (Tregs), the method comprising (i) providing an anti-OX40 antibody or antigen-binding fragment thereof, and (ii) assaying the ability of the antibody or antigen-binding fragment thereof to increase each of Foxp3 and Helios expression in Tregs to determine whether the antibody or antigen-binding fragment is capable of increasing each of Foxp3 and Helios expression by at least 10%.

31 . A method for selecting an anti-OX40 antibody or antigen-binding fragment thereof capable of increasing the activity of regulatory T cells (Tregs), the method comprising (i) providing a plurality of recombinant anti-OX40 antibodies or antigen-binding fragments thereof; and (ii) assaying the ability of the antibodies or antigen-binding fragments thereof to increase each of Foxp3 and Helios expression in Tregs to determine whether the antibodies or antigen-binding fragments thereof are capable of increasing each of Foxp3 and Helios expression by at least 10%.

32 . A method for selecting an anti-OX40 antibody or antigen-binding fragment thereof capable of increasing the activity of regulatory T cells (Tregs), the method comprising (i) providing an anti-OX40 antibody or antigen-binding fragment thereof, (ii) assaying the ability of the antibody or antigen-binding fragment thereof to inhibit binding of OX40-ligand (OX40L) to OX40 to determine whether the antibody or antigen-binding fragment thereof is capable of inhibiting binding of OX40L to OX40 with an IC50 of no more than 100 nM; and (iii) assaying the ability of the antibody or antigen-binding fragment thereof to induce signaling of OX40 to determine whether the antibody or antigen-binding fragment thereof is capable of inducing signaling of OX40 by about 2-fold to about 5-fold at the IC50 concentration for inhibition of OX40L binding; wherein (ii) and (iii) can be performed in any order.

33 . The method of claim 32 further comprising assaying the ability of the antibody or antigen-binding fragment thereof to increase Foxp3 expression in Tregs to determine whether the antibody or antigen-binding fragment thereof is capable of increasing Foxp3 expression in the Tregs by at least 10%.

34 . The method of claim 32 or 33 further comprising assaying the ability of the antibody or antigen-binding fragment thereof to increase Helios expression in Tregs to determine whether the antibody or antigen-binding fragment thereof is capable of increasing Helios expression in the Tregs by at least 10%.

35 . A method for selecting an anti-OX40 antibody or antigen-binding fragment thereof capable of increasing the activity of regulatory T cells (Tregs), the method comprising (i) providing a plurality of anti-OX40 antibodies or antigen-binding fragments thereof; (ii) assaying the ability of the antibodies or antigen-binding fragments thereof to inhibit binding of OX40-ligand (OX40L) to OX40 to determine whether the antibodies or antigen-binding fragments thereof are capable of inhibiting binding of OX40L to OX40 with an IC50 of no more than 100 nM; and (iii) assaying the ability of the antibodies or antigen-binding fragments thereof to induce signaling of OX40 to determine whether the antibodies or antigen-binding fragments thereof are capable of inducing signaling of OX40 by about 2-fold to about 5-fold at their IC50 concentration for inhibition of OX40L binding; wherein (ii) and (iii) can be performed in any order.

36 . The method of claim 35 further comprising assaying the ability of the antibodies or antigen-binding fragments thereof to increase Foxp3 expression in Tregs to determine whether the antibodies antigen-binding fragments thereof are capable of increasing Foxp3 expression in the Tregs by at least 10%.

37 . The method of claim 35 or 36 further comprising assaying the ability of the antibodies or antigen-binding fragments thereof to increase Helios expression in Tregs to determine whether the antibodies or antigen-binding fragments thereof are capable of increasing Helios expression in the Tregs by at least 10%.

38 . The method of any one of claims 26, 28, 30, and 32-34 , wherein the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment.

39 . The method of any one of claims 26-31, 33, 34, and 36-38 , wherein the assaying the ability to increase Foxp3 and/or Helios expression comprises contacting Tregs with the antibody or antigen-binding fragment thereof and measuring the change in expression of Foxp3 and/or Helios.

40 . The method of any one of claims 32-39 , wherein the assaying the ability to inhibit binding of OX40-ligand (OX40L) to OX40 comprises contacting cells expressing OX40 with OX40L in the presence and the absence of the antibody or antigen-binding fragment thereof and measuring the OX40 signaling in the cells, optionally wherein the measuring of the signaling comprises measuring activity of a NF-κB response element.

41 . The method of any one of claims 32-40 , wherein the assaying the ability to induce signaling of OX40 comprises contacting cells expressing OX40 with the antibody or antigen-binding fragment thereof and measuring the OX40 signaling in the cells, optionally wherein the measuring of the signaling comprises measuring activity of a NF-κB response element

42 . A method of making an anti-OX40 antibody or antigen-binding fragment thereof comprising culturing a host cell comprising one or more polynucleotides encoding an antibody or antigen-binding fragment thereof selected by the method of any one of claims 26-41 to be (a) capable of increasing Foxp3 expression in Tregs by at least 10%, (b) capable of increasing Helios expression in Tregs by at least 10%; and/or (c) capable of inhibiting binding of OX40L to OX40 with an IC50 of no more than 100 nM and capable of inducing signaling of OX40 by about 2-fold to about 5-fold at the IC50 concentration for inhibition of OX40L binding.

43 . The method of claim 42 , wherein the host cell comprises a first polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof and a second polynucleotide comprising a nucleic acid sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first polynucleotide and the second polynucleotide are in the same vector or are in different vectors.

44 . An anti-OX40 antibody or antigen-binding fragment thereof produced by the method of claim 42 or 43 .

45 . The anti-OX40 antibody or antigen-binding fragment thereof of claim 44 , wherein the antibody or antigen-binding fragment thereof is a non-depleting antibody, optionally wherein the antibody or antigen-binding fragment thereof is an IgG4 antibody or antigen-binding fragment thereof.

46 . A method of increasing the activity of a Treg comprising contacting the Treg with an antibody or antigen-binding fragment thereof selected by the method of any one of claims 26-41 to be (a) capable of increasing Foxp3 expression in Tregs by at least 10%, (b) capable of increasing Helios expression in Tregs by at least 10%; and/or (c) capable of inhibiting binding of OX40L to OX40 with an IC50 of no more than 100 nM and capable of inducing signaling of OX40 by about 2-fold to about 5-fold at the IC50 concentration for inhibition of OX40L binding.

47 . A method of increasing the activity of a Treg comprising contacting the Treg with the antibody or antigen-binding fragment thereof of claim 44 or 45 .

48 . The method of claim 46 or 47 , wherein the contacting is in vitro.

49 . The method of claim 46 or 47 , wherein the Treg is in a subject, optionally wherein the subject has an autoimmune or inflammatory disease or disorder.