Methods of treating inflammatory bowel disease with combination therapy of antibodies to IL-23 and TNF α

文档序号：473852 发布日期：2021-12-31 浏览：21次中文

阅读说明：本技术 用针对IL-23和TNFα的抗体的联合疗法治疗炎性肠病的方法 (Methods of treating inflammatory bowel disease with combination therapy of antibodies to IL-23 and TNF α ) 是由 M·杰米纳罗 C·奥布莱恩 J·佩里古于 2020-05-21 设计创作，主要内容包括：本发明公开了一种治疗炎性肠病诸如溃疡性结肠炎的方法,该方法包括施用IL-23抑制剂诸如抗IL-23p19抗体(例如,古塞库单抗)和TNF-α抑制剂诸如抗TNF-α抗体(例如,戈利木单抗)。(A method of treating inflammatory bowel disease, such as ulcerative colitis, comprising administering an IL-23 inhibitor, such as an anti-IL-23 p19 antibody (e.g., Gustauzumab) and a TNF-a inhibitor, such as an anti-TNF-a antibody (e.g., golimumab).)

1. A method of treating an inflammatory disease in a patient, the method comprising:

a) administering a first synergistically therapeutically effective amount of an inhibitor of IL-23; and

b) administering a second synergistically therapeutically effective amount of a TNF- α inhibitor, wherein said method is effective to treat said inflammatory disease and said patient exhibits a clinical response.

2. The method of claim 1, wherein the inflammatory disease is inflammatory bowel disease and the patient exhibits a clinical response based on a clinical endpoint selected from the group consisting of: mayo score, partial Mayo score, ulcerative colitis endoscopic severity index (UCEIS), marker CRP and/or fecal calprotectin, and patient reported outcomes and symptom measurements.

3. The method of claim 2, wherein the inhibitor of IL-23 comprises an anti-IL-23 p19 antibody or antigen-binding fragment thereof, and the TNF-a inhibitor comprises an anti-TNF-a antibody or antigen-binding fragment thereof.

4. The method of claim 3, wherein the inflammatory bowel disease is Crohn's disease.

5. The method of claim 3, wherein the inflammatory bowel disease is Ulcerative Colitis (UC) or indeterminate colitis.

6. The method of claim 5, wherein the inflammatory bowel disease is moderate to severe active Ulcerative Colitis (UC).

7. The method of claim 6, wherein the patient was previously treated with a TNF-a inhibitor alone, and wherein the UC has not experienced remission following the previous treatment.

8. The method of claim 6, wherein the patient was previously treated with an inhibitor of IL-23 alone, and wherein the UC has not experienced remission following the previous treatment.

9. The method of claim 6, wherein the anti-IL-23 p19 antibody comprises: a) 1-3 and 4-6; b) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or c) the heavy chain amino acid sequence of SEQ ID NO 9 and the light chain amino acid sequence of SEQ ID NO 10.

10. The method of claim 6, wherein the anti-TNF α antibody comprises: a) 11-13 and 14-16; b) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or c) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

11. The method of claim 6, wherein the anti-IL-23 p19 antibody comprises: a) 1-3 and 4-6; b) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or c) the heavy chain amino acid sequence of SEQ ID NO 9 and the light chain amino acid sequence of SEQ ID NO 10, and the anti-TNF α antibody comprises: a) 11-13 and 14-16; b) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or c) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

12. A method of treating ulcerative colitis in a patient, the method comprising:

a) administering a first synergistically therapeutically effective amount of an anti-IL-23 p19 antibody comprising (i) heavy chain Complementarity Determining Region (CDR) amino acid sequences of SEQ ID NOs: 1-3 and light chain CDR amino acid sequences of SEQ ID NOs: 4-6, (ii) heavy chain variable region amino acid sequence of SEQ ID NO:7 and light chain variable region amino acid sequence of SEQ ID NO:8, or (iii) heavy chain amino acid sequence of SEQ ID NO:9 and light chain amino acid sequence of SEQ ID NO: 10; and

b) administering a second synergistically therapeutically effective amount of an anti-TNF- α antibody comprising (i) the heavy chain CDR amino acid sequences of SEQ ID NOS: 11-13 and the light chain CDR amino acid sequences of SEQ ID NOS: 14-16, (ii) the heavy chain variable region amino acid sequence of SEQ ID NO:17 and the light chain variable region amino acid sequence of SEQ ID NO:18, or (iii) the heavy chain amino acid sequence of SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:20, wherein the method is effective to treat ulcerative colitis and the patient exhibits a clinical response based on a clinical endpoint selected from the group consisting of: mayo score, partial Mayo score, ulcerative colitis endoscopic severity index (UCEIS), marker CRP and/or fecal calprotectin, and patient reported outcomes and symptom measurements.

13. The method of claim 12, wherein the anti-TNF α antibody and the anti-IL-23 p19 antibody are administered at a ratio of 1:2 to 2:1 (w/w).

14. The method of claim 12, wherein the anti-TNF α antibody and the anti-IL-23 p19 antibody are administered at a ratio of 15:1 to 400:1 (w/w).

15. The method of claim 12, wherein the anti-IL-23 p19 antibody and the anti-TNF-a antibody are administered simultaneously.

16. The method of claim 12, wherein the anti-IL-23 p19 antibody and the anti-TNF-a antibody are administered sequentially.

17. The method of claim 12, wherein the anti-IL-23 p19 antibody and the anti-TNF-a antibody are administered within one day of each other.

18. The method of claim 12, wherein the anti-IL-23 p19 antibody is administered at an initial intravenous dose of 200mg, an intravenous dose of 200mg at weeks 4 and 8 and a subsequent subcutaneous dose of 100mg once every 8 weeks, and the anti-TNF-a antibody is administered at an initial subcutaneous dose of 200mg and a subsequent subcutaneous dose of 100mg at weeks 2, 6 and 10.

19. The method of claim 18, wherein the patient exhibits clinical remission based on a clinical endpoint selected from the group consisting of: mayo score, partial Mayo score, ulcerative colitis endoscopic severity index (UCEIS), marker CRP and/or fecal calprotectin, and patient reported outcomes and symptom measurements.

20. The method of claim 19, wherein the clinical endpoint is measured at about 12 weeks after initial treatment.

21. The method of claim 19, wherein the clinical endpoint is based on the Mayo score.

22. A method of reducing inflammation of the colon of a patient having inflammatory bowel disease, the method comprising:

a) administering a first synergistically therapeutically effective amount of an anti-IL-23 p19 antibody or antigen-binding fragment thereof; and

b) administering a second synergistically therapeutically effective amount of an anti-TNF-alpha antibody, or antigen-binding fragment thereof, wherein the method is effective to reduce inflammation of the colon of the patient to a level comparable to the colon of a normal subject.

23. The method of claim 22, wherein the inflammation in a tissue sample from the colon of the patient is minimal or normal following administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-a antibody or antigen-binding fragment thereof.

24. The method of claim 22, wherein gland loss in a tissue sample from the colon of the patient is minimal or normal following administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-a antibody or antigen-binding fragment thereof.

25. The method of claim 22, wherein erosion is minimal or normal in a tissue sample from the colon of the patient after administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-a antibody or antigen-binding fragment thereof.

26. The method of claim 22, wherein mucosal thickness and hyperplasia in a tissue sample of the colon from the patient are each minimal or normal following administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-a antibody or antigen-binding fragment thereof.

27. The method of claim 22, wherein the histopathology of the colon after administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-a antibody or antigen-binding fragment thereof is the same as the histopathology of normal tissue.

28. The method of claim 22, wherein the anti-IL-23 p19 antibody or antigen-binding fragment thereof comprises: a) 1-3 and 4-6; b) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or c) the heavy chain amino acid sequence of SEQ ID NO 9 and the light chain amino acid sequence of SEQ ID NO 10; and the anti-TNF- α antibody or antigen-binding fragment thereof comprises d) the heavy chain CDR amino acid sequences of SEQ ID NOS: 11-13 and the light chain CDR amino acid sequences of SEQ ID NOS: 14-16; e) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or f) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

29. The method of claim 28, wherein the anti-TNF α antibody, or antigen-binding fragment thereof, and the anti-IL-23 p19 antibody, or antigen-binding fragment thereof, are administered at a ratio of 1:2 to 2:1 (w/w).

30. The method of claim 28, wherein the anti-TNF α antibody, or antigen-binding fragment thereof, and the anti-IL-23 p19 antibody, or antigen-binding fragment thereof, are administered at a ratio of 15:1 to 400:1 (w/w).

31. The method of claim 28, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered simultaneously.

32. The method of claim 28, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered sequentially.

33. The method of claim 28, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered within one day of each other.

34. A method of treating inflammatory bowel disease in a patient and reducing weight loss in the patient, the method comprising:

a) administering a first synergistic treatment and a weight loss reducing effective amount of an anti-IL-23 p19 antibody or antigen-binding fragment thereof; and

b) administering a second synergistic treatment and a weight loss reducing effective amount of an anti-TNF-alpha antibody, or antigen-binding fragment thereof.

35. The method of claim 34, wherein the anti-TNF α antibody, or antigen-binding fragment thereof, and the anti-IL-23 p19 antibody, or antigen-binding fragment thereof, are administered at a ratio of 15:1 to 400:1 (w/w).

36. The method of claim 34, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered simultaneously.

37. The method of claim 34, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered sequentially.

38. The method of claim 34, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered within one day of each other.

39. The method of claim 34, wherein the anti-IL-23 p19 antibody or antigen-binding fragment thereof comprises: a) 1-3 and 4-6; b) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or c) the heavy chain amino acid sequence of SEQ ID NO 9 and the light chain amino acid sequence of SEQ ID NO 10; and the anti-TNF- α antibody or antigen-binding fragment thereof comprises d) the heavy chain CDR amino acid sequences of SEQ ID NOS: 11-13 and the light chain CDR amino acid sequences of SEQ ID NOS: 14-16; e) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or f) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

40. A method of treating moderate to severe active ulcerative colitis in a human patient, the method comprising:

a) administering from 0.0005mg/kg to 0.002mg/kg of an anti-IL-23 p19 antibody or antigen-binding fragment thereof, said anti-IL-23 p19 antibody or antigen-binding fragment thereof comprising the sequence: (i) 1-3 and 4-6; (ii) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or (iii) the heavy chain amino acid sequence of SEQ ID NO 9 and the light chain amino acid sequence of SEQ ID NO 10, and

b) administering from 0.020mg/kg to 0.125mg/kg of an anti-TNF-alpha antibody, or antigen-binding fragment thereof, comprising the sequence: (iv) 11-13 and 14-16; (v) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or (vi) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

41. The method of claim 40, wherein the method is effective to treat the ulcerative colitis.

42. The method of claim 41, wherein the patient exhibits clinical remission based on a clinical endpoint selected from the group consisting of: mayo score, partial Mayo score, ulcerative colitis endoscopic severity index (UCEIS), marker CRP and/or fecal calprotectin, and patient reported outcomes and symptom measurements.

43. The method of claim 40, wherein the following are present in an aqueous solution of the pharmaceutical composition: 100mg/mL of the anti-IL-23 p19 antibody; 7.9% (w/v) sucrose, 4.0mM histidine, 6.9mM L-histidine monohydrochloride monohydrate; polysorbate 80 at 0.053% (w/v) of the composition, and the following are present in an aqueous solution of the pharmaceutical composition: 100mg/mL of the anti-TNF- α antibody; 4.1% (w/v) sorbitol, 5.6mM L-histidine and L-histidine monohydrochloride monohydrate; polysorbate 80 at 0.015% (w/v) of the composition.

Background

Inflammatory Bowel Disease (IBD), including Crohn's Disease (CD) and Ulcerative Colitis (UC), is characterized by idiopathic intestinal inflammation, disruption of the epithelial barrier, and microbial dysbiosis. While the use of biologies, such as anti-TNF α antibody therapy, has changed the clinical management of IBD, many patients do not achieve the clinical response of using induction therapy, and the short-term remission rate of biological therapy used as monotherapy is < 20% (2).

The role of IL-23 in promoting intestinal inflammation has been demonstrated in several mouse models, in which reduced colitis was exhibited in mice treated with neutralizing anti-IL-23 p19 antibody or in mice with gene deletion of the p19 subunit of IL-23 (1, 3-5). Genome-wide association studies (GWAS) have identified polymorphisms in the IL-23 receptor gene (IL23R) that are associated with both risk and protection of IBD (6). In patients with moderate to severe Crohn's disease, phase 2 results of two anti-IL-23 agents, Riseduzumab (BI 655066) and Brazimab (MEDI2070, AMG-139), were recently reported to show efficacy. Although anti-IL-23 therapy may have a role in the treatment of IBD, it is expected that a population of patients may not respond fully to IL-23 alone as observed with anti-TNF α therapy.

There is a need for improved treatment of IBD, particularly in patients who are non-responsive to therapy based on anti-TNF α antibodies or anti-IL-23 antibodies alone.

Disclosure of Invention

One aspect of the invention is a method of treating inflammatory bowel disease in a patient (subject). The method comprises administering a first synergistically therapeutically effective amount of an IL-23 inhibitor and administering a second synergistically therapeutically effective amount of a TNF- α inhibitor. The method is effective to treat inflammatory bowel disease and the first and second synergistically effective amounts are the same or different.

In some embodiments, the inflammatory bowel disease is Ulcerative Colitis (UC). In some embodiments, the inflammatory bowel disease is crohn's disease. In some embodiments, the inflammatory bowel disease is indeterminate colitis. In some embodiments, the subject was previously treated with a TNF- α inhibitor alone, and the inflammatory bowel disease has not experienced remission after the previous treatment. In some embodiments, the subject was previously treated with an IL-23 inhibitor alone, and the inflammatory bowel disease has not experienced remission after the previous treatment.

In various embodiments, the IL-23 inhibitor comprises a pharmaceutical composition of an anti-IL-23 p19 antibody (also referred to herein as anti-p 19 or anti-IL-23) or antigen-binding fragment thereof. In various embodiments, the TNF- α inhibitor comprises a pharmaceutical composition of an anti-TNF- α antibody, or antigen-binding fragment thereof. In some embodiments, the anti-IL-23 p19 antibody comprises a human antibody or a humanized antibody. In some embodiments, the anti-TNF α antibody comprises a human antibody or a humanized antibody.

In some embodiments, the inhibitor of IL-23 comprises: 100mg/mL of Gusseuzumab antibody (also known as CNTO1959) (manufactured by Janssen Biotech, Inc. and incorporated)Commercially available) or an antigen-binding fragment thereof, the gucesacumab antibody or antigen-binding fragment thereof comprising the following gucesacumab CDR sequences: (i) 1 heavy chain CDR amino acid sequence (CDRH1) of SEQ ID NO, SEQ IDThe heavy chain CDR amino acid sequence of NO:2 (CDRH2) and the heavy chain CDR amino acid sequence of SEQ ID NO:3 (CDRH 3); and (ii) the light chain CDR amino acid sequence of SEQ ID NO. 4 (CDRL1), the light chain CDR amino acid sequence of SEQ ID NO. 5 (CDRL2), and the light chain CDR amino acid sequence of SEQ ID NO. 6 (CDRL 3); 7.9% (w/v) sucrose, 4.0mM histidine, 6.9mM L-histidine monohydrochloride monohydrate; polysorbate 80 at 0.053% (w/v) of the pharmaceutical composition; wherein the diluent is water under standard conditions.

Another aspect of the methods of the invention comprises administering a pharmaceutical composition comprising: 100mg/mL of an isolated anti-IL-23 specific antibody having the amino acid sequence of the Guseneca single-anti heavy chain variable region of SEQ ID NO. 7 and the amino acid sequence of the Gusenecab light chain variable region of SEQ ID NO. 8; 7.9% (w/v) sucrose, 4.0mM histidine, 6.9mM L-histidine monohydrochloride monohydrate; polysorbate 80 at 0.053% (w/v) of the pharmaceutical composition; wherein the diluent is water under standard conditions.

Another aspect of the methods of the invention comprises administering a pharmaceutical composition comprising: 100mg/mL of an isolated anti-IL-23 specific antibody having the amino acid sequence of the Guseluzumab heavy chain of SEQ ID NO. 9 and the amino acid sequence of the Guseluzumab light chain of SEQ ID NO. 10; 7.9% (w/v) sucrose, 4.0mM histidine, 6.9mM L-histidine monohydrochloride monohydrate; polysorbate 80 at 0.053% (w/v) of the pharmaceutical composition; wherein the diluent is water under standard conditions.

The sequence of the Gusaikumab is as follows:

in various embodiments, the TNF- α inhibitor comprises golimumab (manufactured by Janssen Biotech, incCommercially available) or an antigen-binding fragment thereof, the golimumab or the antigen-binding fragment thereof comprising the sequence shown in SEQ ID NO:

exemplary anti-TNF-alpha antibody sequences (golimumab)

CDRs determined by Kabat

Amino acid sequence of anti-TNF- α antibody complementarity determining region heavy chain 1 (CDRH 1): (SEQ ID NO:11)

SYAMH

Amino acid sequence of anti-TNF- α antibody complementarity determining region heavy chain 2 (CDRH 2): (SEQ ID NO:12)

FMSYDGSNKKYADSVKG

Amino acid sequence of anti-TNF- α antibody complementarity determining region heavy chain 3 (CDRH 3): (SEQ ID NO:13)

DRGIAAGGNYYYYGMDV

Amino acid sequence of anti-TNF- α antibody complementarity determining region light chain 1 (CDRL 1): (SEQ ID NO:14)

RASQSVYSYLA

Amino acid sequence of anti-TNF- α antibody complementarity determining region light chain 2 (CDRL 2): (SEQ ID NO:15)

DASNRAT

Amino acid sequence of anti-TNF- α antibody complementarity determining region light chain 3 (CDRL 3): (SEQ ID NO:16)

QQRSNWPPFT

Amino acid sequence of anti-TNF-alpha antibody heavy chain variable region (CDR underlined): (SEQ ID NO:17)

Amino acid sequence of anti-TNF-alpha antibody light chain variable region (CDR underlined): (SEQ ID NO:18)

Amino acid sequence of the heavy chain (CDR underlined) of the anti-TNF- α antibody: (SEQ ID NO:19)

Amino acid sequence of the light chain (CDR underlined) of the anti-TNF- α antibody: (SEQ ID NO:20)

In some embodiments, the anti-TNF α antibody and the anti-IL-23 p19 antibody are administered at a ratio of 1:2 to 2:1 (w/w). In some embodiments, the anti-TNF α antibody and the anti-IL-23 p19 antibody are administered at a ratio ranging from 15:1 to 400:1(w/w) or from 2:1 to 14: 1.

In some embodiments, the anti-IL-23 p19 antibody and the anti-TNF α antibody are administered simultaneously or on the same day for an initial dose, and the administration of both antibodies is staggered by two or more weeks between administration for subsequent doses. In some embodiments, the anti-IL-23 p19 antibody and the anti-TNF α antibody are administered sequentially. In some embodiments, the anti-IL-23 p19 antibody and the anti-TNF α antibody are administered within one day of each other.

In another aspect, a method of reducing inflammation of the colon of a subject having an inflammatory bowel disease is provided. The method comprises administering a first synergistic inflammation reducing effective amount of an anti-IL-23 p19 antibody, and administering a second synergistic inflammation reducing effective amount of an anti-TNF α antibody. The method is effective in reducing inflammation of the colon of the subject to a level comparable to the colon of a normal patient. The first synergistic inflammation reducing effective amount and the second synergistic inflammation reducing effective amount are the same or different.

In some embodiments, following administration of the anti-IL-23 p19 antibody and the anti-TNF α antibody, inflammation is minimal or normal in a tissue sample from the colon of the subject. In some embodiments, gland loss in a tissue sample from the colon of the subject is minimal or normal following administration of the anti-IL-23 p19 antibody and the anti-TNF α antibody. In some embodiments, erosion in a tissue sample from the colon of the subject is minimal or normal following administration of the anti-IL-23 p19 antibody and the anti-TNF α antibody. In some embodiments, mucosal thickness and hyperplasia in a tissue sample from the colon of the subject are each minimal or normal following administration of the anti-IL-23 p19 antibody and the anti-TNF α antibody. In some embodiments, the histopathology of the colon following administration of the anti-IL-23 p19 antibody and the anti-TNF α antibody is about the same (or the same) as the histopathology of normal tissue.

In another aspect, a method of treating inflammatory bowel disease and reducing weight loss in a subject is provided. The method comprises the following steps: (a) administering a first synergistic treatment and a weight loss reducing effective amount of an anti-IL-23 p19 antibody or antigen-binding fragment thereof; and (b) administering a second synergistic treatment and weight loss reducing effective amount of an anti-TNF- α antibody, or antigen-binding fragment thereof; wherein the first synergistic treatment and weight loss effective amount is the same or different than the second synergistic treatment and weight loss effective amount.

In various embodiments, the method is effective in treating inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is ulcerative colitis. In some embodiments, the inflammatory bowel disease is crohn's disease. In some embodiments, the inflammatory bowel disease is indeterminate colitis. In some embodiments, the method is effective to inhibit weight loss (e.g., weight loss associated with inflammatory bowel disease).

In another aspect, there is provided a method of preventing inflammation of the colon in a subject having an inflammatory bowel disease, the method comprising: (a) administering a first synergistic inflammation reducing effective amount of an IL-23 inhibitor; and (b) administering a second synergistic inflammation reducing effective amount of a TNF- α inhibitor. The method is effective in reducing inflammation of the colon of the subject to a level comparable to the colon of a normal patient. The first synergistic inflammation reducing effective amount and the second synergistic inflammation reducing effective amount are the same or different.

In one embodiment, gucegurumab is administered to a UC patient at an initial intravenous dose of 200mg, an intravenous dose of 200mg at weeks 4 and 8, and a subsequent subcutaneous dose of 100mg once every 8 weeks; golimumab was administered at an initial subcutaneous dose of 200mg and subsequent subcutaneous doses of 100mg at weeks 2, 6 and 10. UC patients will be evaluated by the Mayo score to determine clinical response or remission. The clinical response measured at week 12 was defined as a decrease in the Mayo score of ≧ 30% and ≧ 3 points from baseline, and a decrease in the rectal bleeding sub-score (RBS) of ≧ 1 or 0 or 1 of RBS. Clinical remission measured at week 12 was defined as a Mayo score ≦ 2, and no separate sub-score >1. Additional measures of clinical response are used within the scope of the invention.

Drawings

FIGS. 1A and 1B show the results of a weight loss assay performed on mice after treatment with low (FIG. 1A, 50. mu.g) and high (FIG. 1B, 500. mu.g) doses of anti-TNF-. alpha.and anti-IL-23 p19 antibodies, alone or in combination. Each line represents the group mean of error bars with standard error (n-9 antibody treatments; n-5 PBS controls; n-3 naive controls) and is shown as percent change from day-1 (dashed line). Some error bars are within the size of the symbol and are not shown. Disease was induced by administration of anti-CD 40 antibody (BioXCell, catalog No. BE0016-2, agonist CD40 Ab clone FGK4.55, lot No. 5345/0515).

FIGS. 2A and 2B show the results of histopathological studies of the colon of mice treated with low dose (FIG. 2B, 50. mu.g/mouse) anti-TNF-. alpha.and/or anti-IL-23 p19 antibody and high dose (FIG. 2B, 500. mu.g/mouse) anti-TNF-. alpha.and/or anti-IL-23 p19 antibody, respectively. Disease is induced by administration of anti-CD 40 antibodies.

Figure 3A shows the humanization treatment signature of anti-TNF α or anti-IL-23 p19 monotherapy from an anti-CD 40 model of murine colitis projected onto the crohn's disease response assessment (CERTIFI) human IBD gene expression network for induced ustlizumab anti-interleukin-12/23. FIG. 3A shows the overlap between the genes present in the anti-TNF α and anti-IL-23 p19 subnetworks, as shown in the Venn diagram. FIG. 3B shows the largest connecting component of the shared anti-TNF α and anti-IL-23 p19 subnetworks.

FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D show the intraperitoneal injection of female RAG2 administered to an anti-IL-23 p19 antibody (FIG. 4B) with an isotype control antibody (FIG. 4A), or an anti-TNF α antibody (FIG. 4C) at 50, 15, 5, 1.5, 0.5, 0.15 μ g/mouse^-/-Results of weight loss analysis performed on mice. Disease is induced by administration of anti-CD 40 antibodies. As shown in fig. 4D, statistics comparing each group to isotype controls were generated.

FIGS. 5A, 5B, and 5C show the intraperitoneal injection of female RAG2 administered to an anti-IL-23 p19 antibody (FIG. 5B) at 50, 15, 5, 1.5, 0.5, 0.15 μ g/mouse, or an anti-TNF α antibody (FIG. 5C) at 150 and 15 μ g/mouse^-/-Results of histopathological studies performed on the colon of mice. Disease is induced by administration of anti-CD 40 antibodies.

FIGS. 6A, 6B, 6C and 6D show the results of weight loss assays performed on mice dosed with control antibody (FIG. 6A), 500 μ g/mouse anti-TNF α antibody alone (FIG. 6B), 1.5, 5 or 25 μ g/mouse anti-IL-23 p19 antibody alone (FIG. 6C), or a combination of 500 μ g/mouse anti-TNF α antibody and 1.5, 5 or 25 μ g/mouse anti-IL-23 p19 antibody (FIG. 6D). Disease is induced by administration of anti-CD 40 antibodies. FIG. 6E illustrates compilation of data from different groups.

FIGS. 7A, 7B and 7C show the results of histopathological studies of the colon of mice dosed with 500 μ g/mouse anti-TNF α antibody alone, mouse anti-IL-23 p19 antibody alone, or a combination of 500 μ g/mouse anti-TNF α antibody and anti-IL 23p19 antibody at a concentration of 1.5 μ g (FIG. 7A), 5 μ g (FIG. 7B) or 25 μ g (FIG. 7C) of mouse anti-IL-23 p19 antibody. Disease is induced by administration of anti-CD 40 antibodies.

FIG. 8 shows the results of a network analysis of humanized colon gene expression signatures based on anti-TNF α (500 μ g) or high dose anti-IL-23 p19(25 μ g) monotherapy intersecting with the gene expression signature from combination therapy (500 μ g anti-TNF α and 1.5 μ g anti-IL-23 p 19). This analysis was performed to determine whether the molecular response of anti-TNF α and low dose anti-IL-23 p19 antibody combined treatment was additive or unique compared to either therapy alone. Identifying a unique sub-network of about 200 genes; this subnetwork is rich in fibroblasts and extracellular matrix tissue, cell types and pathways involved in wound repair and mucosal healing.

Detailed Description

Defining:

unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

"about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, "about" means within one standard deviation, or up to a range of 5% (whichever is greater), according to common practice in the art, unless otherwise explicitly stated in the examples or elsewhere in the specification.

"administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treatment of cells encompasses contact of the reagent with the cells, and contact of the reagent with a fluid, wherein the fluid is in contact with the cells. "administration" and "treatment" also refer to, for example, in vitro and ex vivo treatment of a cell, by a reagent, a diagnostic, a binding composition, or by another cell. When applied to a human, veterinary or research subject, "treatment" refers to therapeutic treatment, prophylactic (preventative) measures, research and diagnostic applications. When applied to a human, veterinary or research subject, or a cell, tissue or organ, "treatment" encompasses contact of the agent with the animal subject, cell, tissue, physiological compartment or physiological fluid. "treatment of a cell" also encompasses situations where an agent contacts a target such as an IL-23 receptor (e.g., in the fluid or colloidal phase), as well as situations where an agonist or antagonist does not contact the cell or receptor.

"treating (Treat or treating)" may also refer to the administration of a therapeutic agent, such as a composition described herein, either internally or externally to a patient in need of the therapeutic agent. Typically, the agent is administered in an amount effective to prevent or ameliorate one or more symptoms of the disease or one or more adverse effects treated with a different therapeutic agent, whether by preventing development of such symptoms or adverse effects, inducing regression of such symptoms or adverse effects, or inhibiting progression of such symptoms or adverse effects to any clinically measurable degree. The amount of therapeutic agent that is effective to alleviate the symptoms or adverse effects of any particular disease (also referred to as a "therapeutically effective amount") may vary depending on factors such as: the disease state, the age and weight of the patient, the ability of the therapeutic agent to elicit a desired response in the patient, the general health of the patient, the method of administration, the route and dosage, and the severity of the side effects.

As used herein, an "inhibitor" is any agent that reduces the activity of a target molecule. In particular, antagonists of IL-23 or TNF- α are agents that decrease the biological activity of IL-23 or TNF- α, for example, by blocking the binding of IL-23 or TNF- α to its receptor or otherwise decreasing its activity (e.g., as measured in a bioassay).

As used herein, "anti-IL-23 specific antibody," "anti-IL-23 antibody," "antibody portion" or "antibody fragment" and/or "antibody variant" and the like include any protein or peptide comprising: the molecule comprises at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one Complementarity Determining Region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof, or a portion of at least one of an IL-23 receptor or a binding protein that can be incorporated into an antibody of the invention. Such antibodies optionally also affect specific ligands, such as, but not limited to, in the context of such antibodies modulating, decreasing, increasing, antagonizing, agonizing, moderating, alleviating, blocking, inhibiting, abrogating, and/or interfering with at least one IL-23 activity or binding, or IL-23 receptor activity or binding, in vitro, in situ, and/or in vivo. As a non-limiting example, a suitable anti-IL-23 antibody, specified portion or variant of the invention can bind to at least one IL-23 molecule, or specified portion, variant or domain thereof. Suitable anti-IL-23 antibodies, specified portions or variants can also optionally affect at least one of IL-23 activity or function, such as but not limited to RNA, DNA or protein synthesis, IL-23 release, IL-23 receptor signaling, membrane IL-23 cleavage, IL-23 activity, IL-23 production and/or synthesis.

The term "antibody" is also intended to encompass antibodies, digested fragments, specified portions and variants thereof, including antibody mimetics or antibody portions that comprise structures and/or functions that mimic an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. Functional fragments include antigen binding fragments that bind to mammalian IL-23. For example, antibody fragments capable of binding IL-23 or a portion thereof include, but are not limited to, Fab fragments (e.g., obtained by papain digestion), Fab ' fragments (e.g., obtained by pepsin digestion and partial reduction), and F (ab ') 2 fragments (e.g., obtained by pepsin digestion), facb fragments (e.g., obtained by plasmin digestion), pFc ' fragments (e.g., obtained by pepsin or plasmin digestion), Fd fragments (e.g., obtained by pepsin digestion, partial reduction, and reaggregation), Fv or scFv fragments (e.g., obtained by molecular biology techniques).

Such fragments may be produced by enzymatic cleavage, synthetic or recombinant techniques, as are well known in the art and/or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural termination site. For example, a combinatorial gene encoding a F (ab') 2 heavy chain portion can be designed to include a DNA sequence encoding the CH1 domain and/or hinge region of the heavy chain. The various portions of the antibody can be chemically linked together by conventional techniques or can be prepared as a continuous protein using genetic engineering techniques.

"humanized antibody" refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. Humanized antibodies may comprise substitutions in the framework such that the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

"human antibody" refers to an antibody having a heavy chain variable region and a light chain variable region, wherein both the framework and the antigen-binding site are derived from sequences of human origin. If the antibody comprises a constant region or a part of a constant region, the constant region is also derived from a sequence of human origin.

"subject" or "patient" used interchangeably includes any human or non-human animal. "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.

"tumor necrosis factor", "TNF" or "TNF- α" refers to the well-known human tumor necrosis factor- α (TNF- α), which is a multifunctional pro-inflammatory cytokine. TNF- α triggers proinflammatory pathways that lead to tissue damage such as degradation of cartilage and bone, induces adhesion molecules, induces procoagulant activity on vascular endothelial cells, increases adhesion of neutrophils and lymphocytes, and stimulates the release of platelet activating factor from macrophages, neutrophils, and vascular endothelial cells.

TNF- α exists as a soluble protein and as a precursor form, called transmembrane TNF- α, which is expressed as a cell surface type II polypeptide. Transmembrane TNF- α is processed by metalloproteases such as TNF- α converting enzyme (TACE) between residues Ala76 and Va177, resulting in the release of a soluble form of TNF- α of 157 amino acid residues. Soluble TNF-alpha is a homotrimer of 17-kDa cleaved monomers. Transmembrane TNF- α also exists as a homotrimer of 26-kD uncleaved monomers.

In a first aspect, a method of treating inflammatory bowel disease in a subject is provided. The method comprises administering a first synergistically therapeutically effective amount of an IL-23 inhibitor and administering a second synergistically therapeutically effective amount of a TNF- α inhibitor. The method is effective to treat inflammatory bowel disease and the first and second synergistically effective amounts are the same or different.

The combination of anti-TNF α antibodies and anti-IL-23 p19 antibodies can provide systemic effects as well as local effects on the intestine or colon. This combination may provide a greater systemic effect than treatment with either anti-TNF α antibody or anti-IL-23 p19 antibody alone. The combination may provide excellent anti-inflammatory activity in the treatment of human IBD. anti-IL-23 p19 antibodies are highly effective in blocking the development of IBD (e.g., colitis and crohn's disease) but do not block anti-CD 40-induced weight loss, whereas anti-TNF α antibodies can provide significant protection against anti-CD 40-induced weight loss and a degree of protection against IBD. Each antibody and the combination may provide a differential effect on local inflammation and systemic inflammation.

Various anti-IL-23 antibodies may be used, such as any of the anti-IL-23 antibodies described in U.S. patent 7,491,391 published 2-17 days 2009 and U.S. patent publication 2018/0094052 published 4-5 days 2018, both of which are incorporated herein by reference.

Various anti-TNF α antibodies can be used. For example, any of the anti-IL-23 antibodies described in U.S. patent 7,250,165 published on month 31 of 2007 and U.S. patent publication 2017/0218092 published on month 3 of 2017, both of which are incorporated herein by reference, may be used.

Various host animals can be used to produce anti-TNF- α antibodies. For example, Balb/c mice can be used to generate mouse anti-human TNF- α antibodies. Antibodies prepared from Balb/c mice and other non-human animals can be humanized using various techniques to produce more human-like sequences.

Anti IL-23 antibodies can optionally through the IL-23 high affinity binding to characterization, and optionally with low toxicity. anti-TNF α antibodies can optionally be characterized by high affinity binding to TNF α, and optionally have low toxicity. In particular, the antibodies, designated fragments or variants of antibodies may be used in situations where the individual components such as the variable regions, constant regions and frameworks are individually and/or collectively, optionally and preferably of low immunogenicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties, may help achieve a therapeutic result. "Low immunogenicity" is defined herein as producing a significant HAHA, HACA or HAMA response in less than about 75%, or preferably less than about 50%, of treated patients and/or producing low titers (less than about 300, preferably less than about 100, as measured by a dual antigen enzyme immunoassay) in treated patients (Elliott et al, Lancet 344:1125-1127(1994), which is incorporated herein by reference in its entirety). For an anti-IL-23 antibody, "low immunogenicity" can also be defined as the incidence of titratable levels of antibody against an IL-23 antibody in patients treated with an anti-IL-23 antibody during a treatment period occurring in less than 25% of patients treated with the recommended dose of the recommended course of therapy, preferably in less than 10% of patients treated with the recommended dose of the recommended course of therapy. For an anti-TNF α antibody, "low immunogenicity" may also be defined as the incidence of titratable levels of antibody against anti-TNF α antibody in patients treated with the anti-TNF α antibody during a treatment period occurring in less than 25% of patients treated with the recommended dose of the recommended course of therapy, preferably in less than 10% of patients treated with the recommended dose of the recommended course of therapy.

As is well known in the art, at least one of the anti-IL-23 antibody and the anti-TNF α antibody used in the methods described herein can be prepared from a cell line, a mixed cell line, immortalized cells, or a clonal population of immortalized cells. See, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY (1987-2001); sambrook et al, Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor, N.Y. (1989); harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); edited by Colligan et al, Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); colligan et al, Current Protocols in Protein Science, John Wiley & Sons, NY (1997-2001), each of which is incorporated herein by reference in its entirety.

anti-IL-23 antibodies and/or anti-TNF-alpha antibodies can also be generated by immunizing a transgenic animal (e.g., mouse, rat, hamster, non-human primate, etc.) capable of producing a full complement of human antibodies, as described herein and/or as known in the art. Cells producing human anti-IL-23 antibodies can be isolated from such animals and immortalized using suitable methods, such as those described herein.

anti-IL-23 antibodies for use in the methods described herein can also be prepared using at least one anti-IL-23 antibody-encoding nucleic acid to provide transgenic animals or mammals (such as goats, cattle, horses, sheep, rabbits, and the like) that produce such antibodies in their milk. anti-TNF- α antibodies for use in the methods described herein can also be prepared using at least one anti-TNF- α antibody-encoding nucleic acid to provide transgenic animals or mammals (such as goats, cows, horses, sheep, rabbits, etc.) that produce such antibodies in their milk. Such animals may be provided using known methods. See, for example, but not limited to, U.S. patents 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, 5,304,489, etc., each of which is incorporated herein by reference in its entirety.

anti-IL-23 antibodies can have a range of affinities (Ks)_D) Binds to human IL-23. In a preferred embodiment, the human mAb can optionally bind to human IL-23 with high affinity. Example (b)For example, a human mAb can be equal to or less than about 10^-7M, such as, but not limited to, 0.1 to 9.9 (or any range or value therein). times.10^-7、10^-8、10^-9、10^-10、10^-11、10^-12、10^-13Or any range or value of K therein_DBinds to human IL-23.

anti-TNF-alpha antibodies can have a range of affinities (Ks)_D) Binds human TNF-alpha. In a preferred embodiment, the human mAb can optionally bind to human TNF- α with high affinity. For example, a human mAb can be equal to or less than about 10^-7M, such as, but not limited to, 0.1 to 9.9 (or any range or value therein). times.10^-7、10^-8、10^-9、10^-10、10^-11、10^-12、10^-13Or any range or value of K therein_DBinds human TNF-alpha.

The anti-IL-23 antibody may be of IgG1, IgG2, IgG3, or IgG4 isotype. The anti-TNF-alpha antibody may be of IgG1, IgG2, IgG3 or IgG4 isotype.

Without being bound by theory, the beneficial effects of combining an anti-IL-23 p19 antibody with an anti-TNF α antibody may result from the different gene expression changes induced by each antibody. As described in example 1 and at least fig. 2A and 2B, at doses where each antibody provided similar protection against colonic inflammation (fig. 2, 50 μ g anti-IL-23 p19 and 500 μ g anti-TNF α), different changes in intestinal gene expression were observed in mice when IL-23p19 was blocked compared to TNF α. These changes in gene expression may also be useful in human diseases. Integration of the "humanized" murine anti-TNF α and anti-IL-23 p19 gene signatures with the human intestinal biopsy gene network may allow for the focus of only genes expressed and altered in human intestinal tissue. Additional context for the potential molecular impact of each antibody on human IBD can be obtained by generating a therapeutic subnetwork that includes genes in the network that are removed one step (i.e., strongly correlated) from the genes within each tag. Each of the anti-TNF α and anti-IL-23 p19 subnetworks showed unique single antibody gene signatures, allowing insight into the biology of the target by two mechanisms.

The therapeutic effect according to the methods described herein can be determined, for example, by assessing the degree of weight loss, nutrient absorption, and histopathological studies of tissue samples. Histopathological studies may include measuring one or more of submucosal edema, inflammation, gland loss, erosion, mucosal thickness, and hyperplasia. Submucosal edema can be quantified by measuring the thickness from the muscularis mucosae to the inner boundary of the outer muscle layer (e.g., in the non-tangential region believed to be most representative of the severity of the change). The inflammation score may reflect the degree of infiltration of macrophages, lymphocytes and neutrophils into the colon. Glandular loss of crypt epithelium and remaining glandular epithelium can be quantified by assessing the percentage of affected mucosa. Erosion reflects loss of surface epithelium and can be scored by assessing the percentage of mucosa affected (e.g., by mucosal bleeding). Mucosal thickness can be assessed by measuring the non-tangential regions of the slice that best represent the overall mucosal thickness. The increased thickness reflects gland elongation and mucosal hyperplasia.

The overall histopathological score may be obtained by measuring one or more of submucosal edema, inflammation, gland loss, erosion, mucosal thickness, and hyperplasia. An exemplary scoring system for mice is described in example 1. Similar systems are useful for human and other mammalian subjects.

In some embodiments, the inflammatory bowel disease is colitis, e.g., ulcerative colitis. Colitis may involve irritation, swelling and other signs of inflammation of the colon. Ulcers (Sores and ulcers) are present in ulcerative colitis.

In some embodiments, the inflammatory bowel disease is crohn's disease. Crohn's disease may be confined to the colon, but may also be present in other tissues such as the small intestine. Crohn's disease may involve inflammation of the colon and small intestine. There may even be inflammation of the mouth, anus, skin, eyes, joints and/or liver.

In some embodiments, the subject was previously treated with a TNF- α inhibitor alone, and the inflammatory bowel disease has not experienced remission after the previous treatment. In some embodiments, the subject was previously treated with an IL-23 inhibitor alone, and the inflammatory bowel disease has not experienced remission after the previous treatment. The methods described herein may be beneficial for subjects that are non-responsive to monotherapy treatment with a TNF-alpha inhibitor (e.g., an anti-TNF-alpha antibody) or an IL-23 inhibitor (e.g., an anti-IL-23 p19 antibody). Based on the results described herein, subjects can respond much better to a combination of a TNF-a inhibitor (e.g., anti-IL-23 p19 antibody) and an IL-23 inhibitor (e.g., anti-IL-23 p19 antibody) when both an anti-TNF-a antibody and an anti-IL-23 p19 antibody are administered, showing a significant improvement in the histopathology of the colon (as compared to either antibody alone).

In various embodiments, the inhibitor of IL-23 comprises an anti-IL-23 p19 antibody or antigen-binding fragment thereof. These can bind to the p19 subunit of IL-23.

In various embodiments, the TNF- α inhibitor comprises an anti-TNF- α antibody, or antigen-binding fragment thereof. In some embodiments, the anti-IL-23 p19 antibody comprises a human antibody or a humanized antibody. In some embodiments, the anti-TNF α antibody comprises a human antibody or a humanized antibody.

anti-IL-23 antibodies and/or anti-TNF α antibodies may also be humanized or prepared as human antibodies engineered to retain high affinity for the antigen and other favorable biological properties. Humanized (or human) antibodies can also optionally be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. These displayed assays enable analysis of the likely role of residues in the functional performance of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, Framework (FR) residues can be selected and combined from consensus and import sequences to enable desired antibody characteristics, such as increased affinity for a target antigen.

Humanization or engineering of the antibodies of the invention may be performed using any known method, such as, but not limited to, those described in Winter (Jones et al, Nature 321:522 (1986); Riechmann et al, Nature 332:323 (1988); Verhoeyen et al, Science 239:1534 (1988)); sims et al, J.Immunol.151:2296 (1993); chothia and Lesk, J.mol.biol.196:901 (1987); carter et al, Proc.Natl.Acad.Sci.U.S.A.89:4285 (1992); presta et al, J.Immunol.151:2623(1993), and U.S. patents: 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, 4,816,567, each of which is incorporated herein by reference in its entirety.

In another aspect, a method of reducing inflammation of the colon of a subject having an inflammatory bowel disease is provided. The method comprises administering a first synergistic inflammation reducing effective amount of an IL-23 inhibitor, and administering a second synergistic inflammation reducing effective amount of a TNF-alpha inhibitor. The method is effective in reducing inflammation of the colon of the subject to a level comparable to the colon of a normal patient. The first synergistic inflammation reducing effective amount and the second synergistic inflammation reducing effective amount are the same or different. Prevention or reduction of inflammation can be measured by histopathological analysis, the degree of weight loss, and the degree of inflammation.

In some embodiments, the inflammation score is very low or normal in a histopathological study of a tissue sample from the colon of the subject following administration of the IL-23 inhibitor and the TNF- α inhibitor. Minimal inflammation may reflect the presence of only one or two small foci, where mononuclear inflammatory cells (MNIC) may be background mucosal lymphoid aggregates.

In some embodiments, the gland loss score is very low or normal in a histopathological study of a tissue sample from the colon of the subject following administration of the IL-23 inhibitor and the TNF-a inhibitor. Minimal gland loss may involve only one or two small gland loss focal regions.

In some embodiments, the erosion score is very low or normal in a histopathological study of a tissue sample from the colon of the subject following administration of the IL-23 inhibitor and the TNF-a inhibitor. Minimal erosion may involve only one or two small focal areas of mucosal erosion.

In some embodiments, the mucosal thickness and proliferation score are each very low or normal in histopathological studies of tissue samples from the colon of a subject following administration of an IL-23 inhibitor and a TNF- α inhibitor. A very small mucosal thickness may involve an increase in mucosal thickness of less than 25% compared to the thickness of normal mucosal tissue.

In some embodiments, the histopathology of the colon following administration of the IL-23 inhibitor and the TNF- α inhibitor is about the same (or the same) as the histopathology of normal tissue. Histopathology can be assessed by measuring one or more of submucosal edema, inflammation, gland loss, erosion, mucosal thickness, and hyperplasia. Any or all of these parameters may be measured and scored. An exemplary scoring system is described in example 1.

In various embodiments, the IL-23 inhibitor is an anti-IL-23 p19 antibody or antigen-binding fragment thereof. Exemplary anti-IL-23 p19 antibodies and fragments are described in us patent 7,491,391 published 2.17.2009, and which is incorporated by reference herein in its entirety. In various embodiments, the TNF- α inhibitor is an anti-TNF- α antibody, or antigen-binding fragment thereof.

In some embodiments, the anti-TNF α antibody and the anti-IL-23 p19 antibody are administered at a ratio of 1:2 to 2:1 (w/w). The ratio can be calculated from the dose of one antibody in a patient (in mg/kg) and the dose of another antibody in the same patient (in mg/kg). In some embodiments, the anti-TNF α antibody and the anti-IL-23 p19 antibody are administered at a ratio of 15:1 to 400:1 (w/w). The ratio can be calculated from the dose of one antibody in a patient (in mg/kg) and the dose of another antibody in the same patient (in mg/kg).

Administration of an anti-TNF α antibody and an anti-IL-23 p19 antibody to a subject (e.g., a human patient) at a ratio of 1:2 to 2:1(w/w) can provide enhanced treatment of IBD (e.g., colitis and crohn's disease) in the subject. In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:2 to 1:1.8 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1.9 to 1:1.7 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1.8 to 1:1.6 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1.7 to 1:1.5 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1.6 to 1:1.4 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1.5 to 1:1.3 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1.4 to 1:1.2 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1.3 to 1:1.1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1.2 to 1:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1.1 to 1.1:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1:1 to 1.2:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1.1:1 to 1.3:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1.2:1 to 1.4:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1.3:1 to 1.5:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1.4:1 to 1.6:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1.5:1 to 1.7:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1.6:1 to 1.8:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1.7:1 to 1.9:1 (w/w). In some embodiments, the ratio of anti-TNF α antibody to anti-IL-23 p19 antibody is 1.8:1 to 2:1 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is about 1:2, 1:1.8, 1:1.5, 1:1.2, 1:1, 1.2:1, 1.5:1, 1.8:1, or 2:1 (w/w).

The lowest active dose of anti-IL-23 p19 antibody can be administered to a subject (e.g., a human patient) with a larger dose of anti-TNF α antibody to prevent the development of inflammatory bowel disease (e.g., colitis and crohn's disease). The ratio of the lowest active dose of anti-IL-23 p19 antibody to the larger dose of anti-TNF α antibody may range from 1:400 to 1:15 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:400 to 1:350 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:370 to 1:320 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:350 to 1:300 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:300 to 1:250 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:280 to 1:230 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:250 to 1:200 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:220 to 1:170 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:170 to 1:120 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:150 to 1:100 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:120 to 1:80 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:100 to 1:60 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:80 to 1:40 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:60 to 1:30 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:50 to 1:25 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:40 to 1:20 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:35 to 1:15 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is about 1:400, 1:300, 1:200, 1:150, 1:100, 1:75, 1:50, 1:25, or 1:15 (w/w).

In some embodiments, the anti-TNF α antibody and the anti-IL-23 p19 antibody are administered at a ratio of 15:1 to 400:1 (w/w). In some embodiments, a) the anti-IL-23 p19 antibody or antigen-binding fragment thereof and b) the anti-TNF- α antibody or antigen-binding fragment thereof are administered simultaneously. In some embodiments, a) the anti-IL-23 p19 antibody or antigen-binding fragment thereof and b) the anti-TNF- α antibody or antigen-binding fragment thereof are administered sequentially. a) The anti-IL-23 p19 antibody or antigen-binding fragment thereof and b) the anti-TNF-alpha antibody or antigen-binding fragment thereof can be administered within one hour, two hours, three hours, six hours, 12 hours, one day, two days, three days, or four days of each other.

In some embodiments, the combination of a) an anti-IL-23 p19 antibody or antigen-binding fragment thereof and b) an anti-TNF α antibody or antigen-binding fragment thereof is effective to treat a subject previously treated with the anti-TNF α antibody alone without significantly alleviating inflammatory bowel disease. In some embodiments, the combination of a) an anti-IL-23 p19 antibody or antigen-binding fragment thereof and b) an anti-TNF α antibody or antigen-binding fragment thereof is effective to treat a subject previously treated with an anti-IL-23 p19 antibody alone without significantly alleviating inflammatory bowel disease.

In another aspect, a method of treating inflammatory bowel disease in a human subject is provided. The method comprises the following steps: (a) administering 0.0005mg/kg to 0.002mg/kg of an anti-IL-23 p19 antibody or antigen-binding fragment thereof; and (b) administering 0.020mg/kg to 0.125mg/kg of an anti-TNF- α antibody, or antigen-binding fragment thereof. In various embodiments, the method is effective in treating inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is colitis. In some embodiments, the inflammatory bowel disease is crohn's disease. In some embodiments, the method is effective to inhibit weight loss (e.g., weight loss associated with inflammatory bowel disease).

(a) The anti-IL-23 p19 antibody or antigen-binding fragment thereof and (b) the anti-TNF-a antibody or antigen-binding fragment thereof can be administered simultaneously, sequentially or within one day of each other.

In various embodiments, administration of 0.020mg/kg to 0.125mg/kg of an anti-TNF α antibody and 0.020mg/kg to 0.125mg/kg of an anti-IL-23 p19 antibody to a subject (e.g., a human patient) can provide enhanced treatment of IBD (e.g., colitis and crohn's disease) in the subject. Initial results evaluated in mice with a combination of anti-TNF α and anti-IL-23 p19 at 50 μ g each showed that this combination provided enhanced protection against colitis relative to a single treatment at the same dose. See example 1. In some embodiments, 0.020mg/kg to 0.040mg/kg of the anti-TNF α antibody and 0.020mg/kg to 0.040mg/kg of the anti-IL-23 p19 antibody are administered to the human subject. In some embodiments, 0.030mg/kg to 0.050mg/kg of anti-TNF α antibody and 0.030mg/kg to 0.050mg/kg of anti-IL-23 p19 antibody are administered to a human subject. In some embodiments, 0.040mg/kg to 0.060mg/kg of the anti-TNF α antibody and 0.040mg/kg to 0.060mg/kg of the anti-IL-23 p19 antibody are administered to a human subject. In some embodiments, 0.050mg/kg to 0.070mg/kg of the anti-TNF α antibody and 0.050mg/kg to 0.070mg/kg of the anti-IL-23 p19 antibody are administered to the human subject. In some embodiments, 0.060mg/kg to 0.080mg/kg of anti-TNF α antibody and 0.060mg/kg to 0.080mg/kg of anti-IL-23 p19 antibody are administered to a human subject. In some embodiments, 0.070mg/kg to 0.090mg/kg of an anti-TNF α antibody and 0.070mg/kg to 0.090mg/kg of an anti-IL-23 p19 antibody are administered to a human subject. In some embodiments, 0.080 to 0.100mg/kg of anti-TNF α antibody and 0.080 to 0.100mg/kg of anti-IL-23 p19 antibody are administered to a human subject. In some embodiments, 0.090mg/kg to 0.110mg/kg of an anti-TNF α antibody and 0.090mg/kg to 0.110mg/kg of an anti-IL-23 p19 antibody are administered to a human subject. In some embodiments, 0.100mg/kg to 0.125mg/kg of an anti-TNF α antibody and 0.100mg/kg to 0.125mg/kg of an anti-IL-23 p19 antibody are administered to a human subject.

In various embodiments, the anti-IL-23 p19 antibody is administered to a subject (e.g., a human patient) daily, every two days, every three days, every four days, every five days, every six days, or once a week. In various embodiments, the anti-TNF α antibody is administered to the subject (e.g., a human patient) once a day, every two days, every three days, every four days, every five days, every six days, or weekly. In some embodiments, both the anti-IL-23 p19 antibody and the anti-TNF α antibody are administered once daily, every two days, every three days, every four days, every five days, every six days, or weekly.

The anti-IL-23 p19 antibody and the anti-TNF α antibody can be administered to a subject (e.g., a human patient) in combination. Alternatively, the anti-IL-23 p19 antibody and the anti-TNF α antibody can be administered separately to the subject. If administered separately, the antibodies can be administered within three hours, six hours, twelve hours, one day, two days, three days, or four days of each other.

In another aspect, a lowest active dose of an anti-IL-23 p19 antibody can be administered with a larger dose of an anti-TNF α antibody to prevent recurrence of inflammatory bowel disease (e.g., ulcerative colitis, indeterminate colitis, and/or crohn's disease) when the subject is in remission from the inflammatory bowel disease. The ratio of the lowest active dose of anti-IL-23 p19 antibody to the larger dose of anti-TNF α antibody may range from 1:400 to 1:15 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:400 to 1:350 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:370 to 1:320 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:350 to 1:300 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:300 to 1:250 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:280 to 1:230 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:250 to 1:200 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:220 to 1:170 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:170 to 1:120 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:150 to 1:100 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:120 to 1:80 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:100 to 1:60 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:80 to 1:40 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:60 to 1:30 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:50 to 1:25 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:40 to 1:20 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is 1:35 to 1:15 (w/w). In some embodiments, the ratio of anti-IL-23 p19 antibody to anti-TNF α antibody is about 1:400, 1:300, 1:200, 1:150, 1:100, 1:75, 1:50, 1:25, or 1:15 (w/w).

In various embodiments, the anti-IL-23 p19 antibody is administered daily, every two days, every three days, every four days, every five days, every six days, or once a week. In various embodiments, the anti-TNF α antibody is administered daily, every second day, every third day, every fourth day, every fifth day, every sixth day, or weekly. In some embodiments, both the anti-IL-23 p19 antibody and the anti-TNF α antibody are administered once daily, every two days, every three days, every four days, every five days, every six days, or weekly.

The anti-IL-23 p19 antibody and the anti-TNF α antibody can be administered in combination. Alternatively, the anti-IL-23 p19 antibody and the anti-TNF α antibody may be administered separately.

Combining anti-TNF α antibody (500 μ g/mouse) treatment with the lowest active dose of anti-IL-23 p19 antibody provides superior efficacy in preventing the development of colitis when compared to either of these doses of single antibody treatment. See, e.g., example 5. Colon gene signature analysis of this combination therapy versus anti-TNF α or anti-IL-23 p19 monotherapy identifies a unique set of genes modulated by combination therapies that are enriched for fibroblasts and extracellular matrix tissue, cell types and pathways involved in wound repair. This new finding suggests that combined treatment with antibodies directed against TNF α and IL-23p19 may provide superior efficacy in the treatment of colitis and inflammatory bowel syndrome. In addition, the combined treatment of antibodies against TNF α and IL-23p19 may have a synergistic effect due to the modulation of specific gene networks involved in mucosal healing.

The data in example 5 show that combined treatment with antibodies against TNF α and IL-23p19 can provide superior protection against colitis compared to treatment with either antibody as monotherapy. The colitis may be acute colitis. Without being bound by theory, transcriptomics and gene network analysis identified both overlapping and distinct molecular effects for each monotherapy and revealed a unique set of genes affected by a combination of treatments involving the wound repair process. Taken together, these findings indicate that combination therapy with anti-TNF α antibodies and anti-IL-23 p19 antibodies can provide a synergistic effect in reducing intestinal inflammation. Synergy may be elicited by targeting of common inflammatory pathways. Synergy may result from the treatment of different cell types involved in the onset of IBD and the effects on genes involved in tissue repair.

Preparation

Each of the anti-TNF α and anti-IL-23 (e.g., anti-IL-23 p19) antibodies can be present in a stable formulation. Stable formulations can include phosphate buffers with saline or selected salts, as well as preservative solutions and formulations containing preservatives, as well as multi-purpose preserved formulations suitable for pharmaceutical or veterinary use, comprising an anti-IL-23 (e.g., anti-IL-23 p19) antibody in a pharmaceutically acceptable formulation. The preservative formulation may comprise at least one known preservative or optionally selected from the group consisting of: at least one of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, a polymer, or a mixture thereof, dissolved in an aqueous diluent. Any suitable concentration or mixture may be used, such as about 0.0015%, or any range, value, or fraction therein. Non-limiting examples include: no preservative, about 0.1% -2% m-cresol (e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.9%, 1.0%), about 0.1% -3% benzyl alcohol (e.g., 0.5%, 0.9%, 1.1%, 1.5%, 1.9%, 2.0%, 2.5%), about 0.001% -0.5% thimerosal (e.g., 0.005%, 0.01%), about 0.001% -2.0% phenol (e.g., 0.05%, 0.25%, 0.28%, 0.5%, 0.9%, 1.0%), 0.0005% -1.0% alkyl parabens (e.g., 0.00075%, 0.0009%, 0.001%, 0.002%, 0.005%, 0.0075%, 0.009%, 0.01%, 0.02%, 0.05%, 0.075%, 0.09%, 0.1%, 0.2%, 0.3%, 0.5%, 0.0075%, 0.75%, etc.).

The aqueous diluent may also contain a pharmaceutically acceptable preservative. Preferred preservatives include those selected from the group consisting of: phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of preservative used in the formulation is a concentration sufficient to produce an antimicrobial effect. The concentration depends on the preservative selected and is readily determined by the skilled person.

Other excipients such as isotonic agents, buffers, antioxidants and preservative enhancers may be added to the diluent. Isotonic agents such as glycerol are often used in known concentrations. Physiologically tolerated buffers are preferably added to provide improved pH control. The formulation may cover a wide pH range, such as from about pH 4 to about pH 10, with a preferred range of from about pH 5 to about pH 9, and a most preferred range of from about 6.0 to about 8.0. Preferably the formulations of the present invention have a pH between about 6.8 and about 7.8. Preferred buffers include phosphate buffers, most preferably sodium phosphate, especially Phosphate Buffered Saline (PBS).

Other additives such as pharmaceutically acceptable solubilizers such as Tween20 (polyoxyethylene (20) sorbitan monolaurate), Tween40 (polyoxyethylene (20) sorbitan monopalmitate), Tween80 (polyoxyethylene (20) sorbitanSugar alcohol monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer) and PEG (polyethylene glycol) or non-ionic surfactants such as Polysorbate 20 or 80 or Poloxamer 184 or 188,Polyols, other block copolymers, and chelates such as EDTA and EGTA, may be added to the formulation or composition to reduce aggregation. These additives may be useful if the formulation is to be applied using a pump or plastic container. The presence of a pharmaceutically acceptable surfactant can reduce any tendency of the antibody to aggregate.

The formulations of the invention may be prepared by a method comprising mixing at least one anti-IL-23 antibody or anti-TNF α antibody with a selected buffer. The buffer may be a phosphate buffer comprising saline or a selected salt. At least one anti-IL-23 antibody and a buffer are mixed in an aqueous diluent using conventional dissolution and mixing procedures. For example, to prepare a suitable formulation, a measured amount of at least one antibody in water or a buffer is combined with a desired buffer in an amount of water sufficient to provide the protein and buffer at the desired concentrations. Variations of this method will be recognized by those of ordinary skill in the art. For example, the order of addition of the ingredients, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the concentration and mode of application used.

A stable or preserved formulation comprising one or both of an anti-IL-23 antibody and an anti-TNF α antibody may be provided to a patient as a clear solution or as a dual vial comprising one vial of lyophilized at least one antibody reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent. Either a single solution vial or a double vial requiring reconstitution can be reused multiple times and can satisfy a single or multiple cycles of patient treatment and thus provide a more convenient treatment regimen than currently available.

For parenteral administration, the anti-IL-23 antibody or anti-TNF α antibody may be formulated as a solution, suspension, emulsion, granule, powder, or lyophilized powder, provided in combination with or separate from a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, ringer's solution, dextrose solution, and about 1% -10% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation may be sterilized by known or suitable techniques.

Suitable Pharmaceutical carriers are described in the recent version of Remington's Pharmaceutical Sciences, a.osol (standard reference text in the art).

Many known and developed means can be used in accordance with the present invention to administer a pharmaceutically effective amount of at least one anti-IL-23 antibody or anti-TNF α antibody. Although pulmonary administration is used in the following description, other modes of administration may be used in accordance with the present invention with suitable results. The IL-23p19 antibodies of the invention may be delivered in a vehicle as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other means described herein or known in the art.

Formulations for parenteral administration may contain conventional excipients. Exemplary common excipients include, but are not limited to, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Aqueous or oily suspensions for injection may be formulated according to known methods using suitable emulsifying or wetting agents and suspending agents. The injectable agents may be non-toxic, parenterally-administrable diluents, such as aqueous solutions in solvents, sterile injectable solutions or suspensions. As a usable solvent or solvent, water, ringer's solution, isotonic saline, or the like is allowed to be used; as a common solvent or suspending solvent, sterile fixed oils may be used. For these purposes, any kind of non-volatile oils and fatty acids may be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semisynthetic mono-or diglycerides or triglycerides.

Formulations for oral administration may include co-administration of adjuvants (e.g., resorcinol and non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall, and co-administration of enzyme inhibitors (e.g., trypsin inhibitor, diisopropyl fluorophosphate (DFF) and aprotinin) to inhibit enzymatic degradation. Us patent 6,309,663 teaches formulations for delivering hydrophilic agents, including proteins and antibodies, and combinations of at least two surfactants intended for oral, buccal, mucosal, nasal, pulmonary, vaginal transmembrane or rectal administration. The active ingredient compounds in solid dosage forms for oral administration may be mixed with at least one additive selected from the group consisting of sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginates, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers and glycerides. These dosage forms may also contain other types of additives, for example inactive diluents, lubricants such as magnesium stearate, parabens, preservatives such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrants, binders, thickeners, buffering agents, sweeteners, flavoring agents, fragrances and the like.

It may be desirable to deliver a compound of the invention to a subject by a single administration over a prolonged period of time, for example, one week to one year. A variety of sustained release, depot or implant dosage forms may be utilized. For example, the dosage form may contain pharmaceutically acceptable, non-toxic salts of the compounds which have low solubility in body fluids, e.g., (a) acid addition salts with polybasic acids such as phosphoric, sulfuric, citric, tartaric, tannic, pamoic, alginic, polyglutamic, naphthalene monosulfonic or disulfonic acids, polygalacturonic acids, and the like; (b) salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, or the like, or salts with organic cations formed from, for example, N' -dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as a zinc tannate salt. In addition, the compounds of the present invention or preferably relatively insoluble salts such as those described above may be formulated in a gel suitable for injection, for example, in an aluminum monostearate gel with, for example, sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like.

Examples

The invention is further described and illustrated by the following examples. However, the use of these and other embodiments anywhere in this specification is merely illustrative and in no way limits the scope and meaning of the invention or any exemplary terms. Likewise, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention will be apparent to those skilled in the art upon reading this specification, and such variations may be made without departing from the spirit or scope of the invention. Accordingly, the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Example 1: mono-antibody directed to TNF alpha or IL-23p19 in a CD40 antibody-induced colitis model Dose ranging of a treatment and combination study

Three separate studies were performed. In all three studies, animals were randomized by weight, assigned to treatment groups, and each group was labeled with a specific number from 1 to 10. The previous day was treated with vehicle (PBS) and mAb as a single intraperitoneal (ip) injection (day-1), followed by induction of disease by intraperitoneal injection of 0.2mg of CD40 agonist antibody in 0.2ml of PBS into each animal (day 0).

Naive control mice were not treated and housed in individual cages until termination on day 7. Clinical signs of disease were observed daily. Body weight was measured and recorded daily from day-1 until termination on day 7. At study termination (day 7), excess CO was utilized₂Animals were euthanized and colon tissue was removed and processed accordingly for histological analysis.

After euthanasia, the colon (defined as the segment of the intestine between the cecum and rectum) was excised and rinsed with ice cold PBS to remove fecal contents. One centimeter of proximal colon was placed in a histological cassette and immersed in fixative (10% neutral buffered formalin, NBF). After 24 hours, the cassettes were removed from the fixative and transferred to 70% ethanol and stored refrigerated until processing. Dividing the remaining colon tissue into three equal portions; the first third was frozen in liquid nitrogen for PK analysis, the second third in liquid nitrogen for cytokine analysis, and the last third (distal, proximal to rectum) was stored on ice in 1ml RNAlater (Ambion)^TM) Until all animals were euthanized and the tissues removed accordingly, and then frozen for RNA extraction and gene expression analysis. All frozen samples were stored at-80 ℃ until further processing.

In all three studies, animals were randomized by weight, assigned to treatment groups, and each group was labeled with a specific number from 1 to 10. The previous day was treated with vehicle (PBS) and mAb as a single intraperitoneal (ip) injection (day-1), followed by induction of disease by intraperitoneal injection of 0.2mg of CD40 agonist antibody in 0.2ml of PBS into each animal (day 0). Naive control mice were not treated and housed in individual cages until termination on day 7. Clinical signs of disease were observed daily. Body weight was measured and recorded daily from day-1 until termination on day 7. On day 7, excess CO was utilized₂Animals were euthanized and colon tissue was removed and processed accordingly for histological analysis.

In the first study (study 1), anti-TNF α or anti-IL-23 p19 mAb was evaluated in a CD40 colitis model. These antibodies were evaluated individually at a dose of 500. mu.g or 50. mu.g per mouse, or in combination (i.e., 500. mu.g + 500. mu.g/mouse each or 50. mu.g + 50. mu.g/mouse each). The protocol is summarized in table 1 below.

Table 1: in CD40 colitis model/study 1, single antibody treatment against TNF α and IL-23p19 was compared to groups Combinations (at the same high and low doses)Evaluation of (2), ELN: immunopharmacology WC-2018-

CNTO 3723 is a murine anti-IL-23 p19 monoclonal antibody (neutralizing IL-23p19 mAb). CNTO5048 is a murine anti-TNF α monoclonal antibody (neutralizing TNF α mAb). CNTO6601 refers to the isotype control used throughout the experiment. CNTO6601 does not specifically bind to TNF α or IL-23p 19.

Anti-inflammatory activity of anti-TNF α and anti-IL-23 p19 antibody treatments, alone or in combination, were evaluated in an anti-CD 40 antibody-induced colitis model. Conjugation of the co-stimulatory receptor CD40 via agonist antibodies causes lymphopenia (T cell and B cell deficient) RAG2^-/-Acute innate systemic and colonic inflammatory responses in mice, where the inflammatory response in the colon peaked around day 7 before resolution (ELN Immunopharmacology WC-2015-. IL-23 drives local colonic inflammation in this model.

Although TNF α expression can control the manifestation of systemic diseases (e.g., weight loss), TNF α has only a modest effect on colitis development. (1) The inventors sought to investigate the different molecular effects of anti-TNF α versus anti-IL-23 p19 antibody treatment on intestinal gene expression and to determine whether the combined treatment of anti-TNF α and anti-IL-23 p19 showed enhanced efficacy over either monotherapy. On day-1, RAG2 was treated with 0.5mg or 0.05mg of anti-TNF α antibody (CNTO5048), 0.5mg or 0.05mg of anti-IL-23 p19 antibody (CNTO3732), a combination of both antibodies (0.5mg or 0.05mg each), 1.0mg of isotype control antibody (CNTO6601), or 10ml/kg PBS as RAG2^-/-Mice were dosed intraperitoneally once. (RAG 2 used in all the embodiments herein^-/-The mice are 8-10 week old female mice derived from Taconic Farms. ) One day later, on day 0, all animals were challenged intraperitoneally with anti-CD 40 antibody (0.2mg) to induce inflammation.

Weight loss analysis was performed after low dose (50. mu.g) and high dose (500. mu.g) antibody treatment. Body weight was monitored from day-1, at which time the mice were injected with antibody or PBS until termination on day 7.

The data are shown in fig. 1A and 1B. Each line represents the group mean of error bars with standard error (n-9 antibody treatments; n-5 PBS controls; n-3 naive controls) and is shown as percent change from day-1 (dashed line). Some error bars are within the size of the symbol and are not shown. FIG. 1A shows low dose (50. mu.g/mouse) and FIG. 1B shows high dose antibody treatment (500. mu.g/mouse). Statistical significance of the differences in weight loss between the antibody-treated and isotype control groups was analyzed by two-way anova using the Dunnett multiple comparison test, and the P-value for each time point is shown in the table. P-values indicating significance are highlighted in bold/italics. ELN: immunopharmacology WC-2018-.

The CD40 mAb-induced colitis model was characterized by a biphasic weight loss with an initial rapid weight loss within 24-48 hours after CD40 agonist antibody administration followed by a recovery and a second phase of weight loss on days 5-7. A single treatment with anti-IL-23 p19 antibody (0.5mg and 0.05mg) did not protect mice from initial rapid weight loss, but promoted faster recovery after day 2 with overall dose-dependent partial protection against weight loss during the second phase of the disease, as shown in fig. 1A and 1B.

In contrast, a single treatment with anti-TNF α antibodies (0.5mg and 0.05mg) completely protected mice from weight loss throughout the duration of the study at both doses. Similar to single antibody treatment against TNF α, the combined treatment resulted in complete protection from weight loss at both doses (fig. 1A and 1B). No adverse effects were observed for the low or high dose combination treatment of anti-TNF α/IL-23p 19.

At termination (day 7), colon histopathology scores were determined for the low and high dose antibody treated groups. Proximal colon sections were stained with H & E and examined for histopathological changes by a blinded pathologist using a severity score of 0-20 according to the following protocol.

For the proximal colon, two (2) pieces were cut and embedded in paraffin. Sections (5 μm) were excised and stained with hematoxylin and eosin (H & E). Histopathology of the two colon segments from each animal was evaluated separately and the mean value for each animal was used in the group analysis. For each H & E stained section, submucosal edema was quantified by measuring the thickness from the muscularis mucosae to the inner boundary of the outer muscle layer in the non-tangential region believed to be most representative of the severity of this change.

The inflammation score reflects the degree of macrophage, lymphocyte, and neutrophil (PMN) infiltration. Severity scores were assigned according to the following criteria:

0 is normal;

0.5 is very small; one or two small foci, mononuclear inflammatory cells (MNIC), may be background mucosal lymphoid aggregates. However, if the aggregates are Pan's patches, they are not scored as abnormal

1-small, with large focal areas or minimal spread of MNIC and neutrophils, no glandular segregation, and may be predominantly in the area of submucosal edema or mesentery

2-mild, mild diffuse or multifocal affects 11% -25% of the mucosa, with small or multifocal glandular segregation, with no segregation in most areas

3-moderate, 26% -50% of affected mucosa have little to mild focal or multifocal glandular segregation due to inflammatory cell infiltration, less in the remaining regions of mucosa, and some regions have no glandular segregation due to inflammation

Significant, 51-75% of affected mucosa have mild to moderate glandular segregation due to inflammatory cell infiltration, little to mild in the remaining areas of mucosa,

but all glands had some degree of separation due to infiltration

5-severe, 76% -100% of affected mucosa have moderate to marked glandular separation areas due to inflammatory cell infiltration, mild to moderate in the rest of the mucosa

Determining a gland loss score. Crypt epithelium and remaining glandular epithelium loss were scored based on approximate percentages of affected mucosa as follows:

0 ═ none

0.5-min, 1 or 2 small focal regions of gland depletion or mucosal erosion

1-10% of affected mucosa

2 mild, 11% -25% affected mucosa

3-moderate, 26% -50% of affected mucosa

Significant 4, 51% -75% of affected mucosa

5-100% of affected mucosa with severe degree

And determining the erosion score. The loss of surface epithelium is scored based on the approximate percentage of affected mucosa as follows. This is usually associated with mucosal bleeding (reflecting bleeding observed clinically and at necropsy):

0 ═ none

0.5-min, 1 or 2 small focal regions of gland depletion or mucosal erosion

1-10% of affected mucosa

2 mild, 11% -25% affected mucosa

3-moderate, 26% -50% of affected mucosa

Significant 4, 51% -75% of affected mucosa

5-100% of affected mucosa with severe degree

Mucosal thickness and proliferation score were determined. Mucosal thickness is measured in the non-tangential region of the slice that best represents the overall mucosal thickness. This parameter is indicative of gland elongation and mucosal hyperplasia. Proliferation scores were derived from the measurements as follows:

0-200 μm-normal

0.5-201 μm-250 μm-min

251 μm to 350 μm, very small

2-351 μm-450 μm mild

3-451-550 μm-medium

4-551 μm-650 μm is significant

5 >650 μm and severe

The histopathological score is the sum of inflammation, gland loss, erosion and hyperplasia scores. Ranging from 0 to 20. Histopathological scores are shown in fig. 2A and 2B. In these figures, each bar represents the group mean with standard error. No histopathological results were observed in naive animals. FIG. 2A shows the results for low doses of antibody (50. mu.g/mouse). FIG. 2B shows the results of the high dose treatment group (500. mu.g/mouse). The significance of the differences between the treated groups and the corresponding vehicle and isotype controls was analyzed by one-way anova and Sidak multiple comparison test. ELN: immunopharmacology WC-2018-.

In the proximal colon, treatment with isotype antibody (1000 μ g/mouse) showed a reduced tendency to histopathology when compared to disease control (PBS), but this did not reach statistical significance. Single treatment with anti-TNF α antibody significantly reduced colonic inflammation at the high dose (500 μ g, fig. 2B), but not at the low dose (50 μ g, fig. 2A), when compared to isotype control.

A single dose of anti-IL-23 p19 antibody was highly effective at high doses (500 μ g, fig. 2B), thereby completely preventing the development of colitis. At low doses (50 μ g, fig. 2A), single treatment significantly reduced histopathology compared to the isotype group, but did not completely prevent colitis. The high dose combination of the two antibodies (500. mu.g anti-TNF. alpha. + 500. mu.g anti-IL-23 p 19/mouse, FIG. 2B) completely prevented colitis in the disease model, similar to the high dose single anti-IL-23 p19 treatment.

The low dose combination treatment (50 μ g anti-TNF α +50 μ g anti-IL-23 p 19/mouse, fig. 2A) was significantly more effective than the single anti-TNF α treatment and showed a trend of improved protection compared to the single treatment against IL-23p19, indicating that this combination has potentially superior efficacy.

Example 2: anti-TNF alpha and anti-IL-23 p19 treatment affected unique genes in the intestine

The readings for systemic and local inflammation showed differential effects with anti-TNF α and anti-IL-23 p19 treatments. In this example, it was evaluated whether the treatment of example 1 above had a different molecular effect on intestinal gene expression. To generate the intestinal gene signature, mRNA is isolated from the distal colon and submitted for microarray analysis.

For RNA extraction, tissue samples were thawed on ice and transferred to a new tube containing 900. mu.l Qiazol (Qiagen) and one metal bead, followed by lysis using TissueLyser II for passage at 30S^-1Run for 1 minute to disrupt and homogenize the tissue. To each sample was added 180. mu.l chloroform, vortexed for 30 seconds, incubated at room temperature for two minutes, and centrifuged at 14,000rpm for 15 minutes at 4 ℃ to separate the mixture into an organic phase and an aqueous phase. Using RNeasy 96 well plate kit (Qiagen), 150 μ l of aqueous phase was used for RNA extraction, including an on-column DNase digestion step, all according to the manufacturer's protocol. The quality and quantity of the isolated RNA was determined by the Nanodrop at the Nanodrop 8000 instrument (Thermoscientific) and by the LabChip GX (DNA 5K/RNA/CZE chip used with GXTouch/GXII Touch HT) on the Caliper instrument (Life Science) according to the manufacturer's protocol. For Caliper analysis, an aliquot of colonic RNA was diluted 1:4 with molecular-scale water.

The following exclusion criteria were used to determine which samples were to be accepted for gene expression analysis by microarray. Nanodrop absorbance 260/280 (protein amount of nucleic acid) should be > 1.8. Nanodrop absorbance 260/230 (amount of nucleic acid salt) should be close to 2. If nanodrop absorbance 260/230 is less than 1.5, repurification is performed. The Caliper RIN (RNA integrity number) should be 5-10. If less than 5, the accuracy of microarray analysis may be affected. RNA was shipped to BioStorage Technologies (Indianapolis, IN) for microarray analysis.

Gene expression differential analysis was performed by comparing the effect of anti-TNF α or anti-IL-23 p19 with that of isotype control treatment. Since treatment with doses of 50 μ g anti-IL-23 p19 or 500 μ g anti-TNF α resulted in similar levels of reduction in histological inflammation (fig. 2), the inventors selected these colonic gene expression signatures for further evaluation to mitigate the potential confounding effects of differential cell infiltration gene expression.

Biological pathway overlap and enrichment for each treated murine gene signature was evaluated (Enrichr: http:// amp. pharm. mssm. edu/Enrichr /). The overlap of the individual gene signatures generated by anti-TNF α or anti-IL-23 p19 treatments was relatively small, with only 11% of the genes shared between the signatures, and did not show any specific pathway enrichment. The anti-TNF α treated gene signature (267 genes, FDR <0.05, FC >1.2) enriches metabolic pathways and cytokine-cytokine receptor interactions, while the anti-IL-23 p19 gene signature (765 genes, FDR <0.05, FC >1.2) enriches circadian rhythm and p53 signaling.

Example 3: single antibody treatment with anti-TNF alpha and anti-IL-23 p19 affects overlapping and different portions of the human IBD network

In cooperation with the mountain Sinai School of Medicine (New York, NY), a predictive Bayesian network model was generated for integrating transcriptional and genetic data from intestinal biopsy samples derived from CERTIFI clinical trials for Crohn's disease (847 IBD biopsies, 28 non-IBD control biopsies; 7,796 gene nodes) (7, 10). This type of molecular integration network provides a data-driven framework for studying gene-gene interactions in disease situations. To convert the anti-TNF α and anti-IL-23 p19 monotherapy gene signatures generated in murine colitis models to clinical disease, the murine gene signature was integrated with the human IBD patient gene network. As described above, based on the tissue inflammation similar effects were selected for 50 g IL-23p19 and 500 g anti TNF alpha dose for evaluation.

To bridge murine model data to the human IBD network, a "humanized" version of each treatment gene signature was first generated by mapping the murine genes to their human orthologous genes (767 genes for anti-IL-23 p19 and 274 genes for anti-TNF α). The NCBI isogene (https:// www.ncbi.nlm.nih.gov/homogene) database (Build 68,04/14/2014) was used to map murine genes to their human orthologous genes. Each NCBI gene Id of each murine gene profile was matched to all corresponding human members of the same cluster of putative orthologs.

A database entry is considered significant if its one-sided Fisher's exact test E value (Bonferroni corrected p value) is less than 0.05.

Hypergeometric tests were performed in Excel (hypgom. dist function) to determine the enrichment of IBD GWAS loci in the gene subnetwork. The list of genes for enrichment of the IBD GWAS locus is derived from Jostins et al, Nature 2012(8) and Liu et al, Nature Genetics 2015 (9).

Using these humanized gene tags, enrichment analysis of each treatment tag was extended to the human pathway. The anti-TNF α treated gene signature enriches cellular responses to stress and lipids, reactive oxygen species metabolism, inflammatory response genes, and up-regulated genes in patient biopsies. anti-IL-23 p19 treatment signatures enrich for cellular metabolism, regulation of proliferation, and gene downregulation in biopsies from IBD patients.

Next, these humanized gene signatures are mapped onto a CERTIFI bayesian network using a web-based network visualization tool and a processing sub-network is generated. The gene list is generated and imported as a text file partitioned by tabs. The gene list is applied to the T26 Pan-Intestine bayes network (CERTIFI network (7)), and the genes within the network and their first neighbors (genes within 1 step of the selected gene, either incoming or outgoing) are used to create sub-networks.

These sub-networks of treatment comprise genes modified in mouse models by anti-TNF α or anti-IL-23 p19 treatment, which are reflected in human IBD tissue and in the genes they are immediately adjacent in the network. Thus, an enriched analysis of these sub-networks can provide insight into the biological pathways targeted by each therapeutic agent in the case of human diseased tissue.

Figures 3A and 3B show humanization treatment signatures of anti-TNF α or anti-IL-23 p19 monotherapy from an anti-CD 40 model of murine colitis projected onto the CERTIFI human IBD gene expression network. First gene neighbors within a human IBD network are extracted to create a processing subnetwork. The overlap between the genes present in the anti-TNF α and anti-IL-23 p19 subnetworks is shown by the central Venn diagram. The largest connecting component of the shared subnetwork of anti-TNF α and anti-IL-23 p19 is shown in FIG. 3B.

Although specific biology was not enriched in the cross-analysis of the original gene signature, the largest connecting component of the network neighbor shared by both anti-TNF α and anti-IL-23 p19The focused analysis revealed an enrichment of dysregulated genes and IBD GWAS loci in IBD patient tissues, suggesting that the efficacy of these different mechanisms may be mediated in part by targeting a common core inflammatory pathway. The intersection of these two therapeutic subnetworks significantly enriched the IBD GWAS locus (p 0.001) and up-regulated genes (multiple tags; top tag E value 7.25E-27) in IBD patient tissues (figure 3). A unique portion of the anti-TNF subnetwork is highly enriched in neutrophils and CD11b⁺Macrophage gene signature (E values of 8.28E-10 and 2.41E-06, respectively), while a unique portion of the anti-IL-23 p19 subnetwork is highly enriched in colonic epithelial cells (E value of 1.27E-32), consistent with the role of IL-23 in promoting expression of cytokines that affect epithelial cell biology, such as IL-17A and IL-22. The relative enrichment of bone marrow cells and epithelial cells in the unique regions of the network, anti-TNF α and anti-IL-23 p19, respectively, raises the additional hypothesis that combination therapy using two antibodies can provide beneficial effects by targeting different cell types involved in the pathogenesis of IBD. Significantly similar results were observed when the same type of network analysis was performed in an orthogonal murine model of intestinal inflammation (T cell metastasis model of colitis) using gene signatures derived from anti-TNF α or anti-IL-23 p19 therapeutic treatments (ELN: jperrigo-2016-00002). Taken together, these network analyses suggest that the anti-TNF α and anti-IL-23 p19 mechanisms of action are distinct, but focus on the molecular driver of intestinal inflammation.

Example 4: escalating dose of anti-TNF alpha and anti-IL-23 p19 antibody treatment in anti-CD 40 antibody induced colitis Range analysis (study 2)

To enable further evaluation of the efficacy of the combination therapy, a escalating dose response study in a model of CD40 antibody-induced colitis was performed to determine the lowest effective dose of each antibody. Female RAG2 one day prior to disease induction with anti-CD 40 agonist antibodies^-/-Mice were dosed intraperitoneally with anti-IL-23 p19 antibodies (CNTO 3723, 50, 15, 5, 1.5, 0.5, 0.15 μ g/mouse), anti-TNF α antibodies (CNTO5048, 150 and 15 μ g/mouse) or isotype controls (50 μ g/mouse). The protocol is summarized in table 2 below.

Table 2: in CD40 colitis modelLower dose range for a Single antibody against TNF α and IL-23p19 in study 2 Evaluation of circumference, ELN: immunopharmacology WC-2016-

Test article	Pathway(s)	Dosage form	Number of animals
				Original time		Is free of	5
Solvent (PBS)	ip	10ml/kg, day-1	5
				CNTO 6601	ip	50 μ g/mouse, day-1	10
CNTO 3723	ip	50 μ g/mouse, day-1	10
				CNTO 3723	ip	15 μ g/mouse, day-1	10
CNTO 3723	ip	5 μ g/mouse, day-1	10
				CNTO 3723	ip	1.5. mu.g/mouse, day-1	10
CNTO 3723	ip	0.5. mu.g/mouse, day-1	10
				CNTO 3723	ip	0.15 μ g/mouse, day-1	10
CNTO 5048	ip	150 μ g/mouse, day-1	10
				CNTO 5048	ip	15 μ g/mouse, day-1	10

Body weight was monitored from day-1, at which time the mice were injected with antibody or PBS until termination on day 7. The data are shown in fig. 4A to 4D. Each line represents the mean of the group with standard error (n-10 antibody treatments; n-5 PBS controls; n-3 naive controls) and is shown as percent change from day-1 (dashed line). The significance of the differences between each treated group and the isotype control group was analyzed by two-way anova using the Dunnett multiple comparison test, and the resulting p-values for each study day are shown in the table. P-values indicating significant differences are highlighted in bold/italics. ELN: immunopharmacology WC-2016-.

A significant increase in the fraction of body weight loss was observed in the isotype control group when compared to vehicle control. From day 2 onwards, treatment with anti-IL-23 p19 antibody showed partial dose-dependent protection against weight loss at the two highest doses (15, 50 μ g/mouse). Only at the lowest dose of anti-IL-23 p19 antibody (0.15. mu.g/mouse) was protection from weight loss observed, as shown in FIG. 4B. Treatment with anti-TNF α antibody completely protected against weight loss at the higher dose (150 μ g/mouse), but only partial protection was noted at the lower dose (15 μ g/mouse). See fig. 4C.

Histopathological analysis of the proximal colon was performed as follows after single antibody treatment for dose ranging measurements. At termination (day 7), proximal colon sections were removed, washed, fixed, and then stained with H & E. The stained specimens were examined for histopathological changes using the protocol in example 1 above using a severity score of 0-20 by a blinded pathologist. The data are shown in fig. 5A to 5C. No histopathological results were observed in naive animals. The significance of the differences between the antibody-treated groups and the corresponding isotype controls was analyzed by a one-way anova-Sidak multiple comparison test. The line plots the group value. ELN: immunopharmacology WC-2016-.

Colon histopathology demonstrated dose-dependent protection of colitis by anti-IL-23 p19 antibody treatment, as shown in FIG. 5B. At a dose of 50. mu.g/mouse, anti-IL-23 p19 antibody treatment provided almost complete protection. Partial protection was detected at antibody doses of 15 μ g and 5 μ g, and no protection was observed at doses of 1.5 μ g and lower. In contrast, two dose levels of anti-TNF α antibody (150, 15 μ g) did not detect significant treatment efficacy for colon histopathology. See fig. 5C. These results demonstrate that blocking IL-23 signaling is highly effective for colitis in this model. Inhibition of TNF α, while effective on systemic inflammation (as measured by improvement in weight loss), provides only modest protection against colitis in this model.

Example 5: fixed doses of anti-TNF alpha antibody and different doses of anti-IL- Anti-inflammatory Activity of the combination of 23p19 antibodies (study 3)

A combination study was performed in the CD40 colitis model using a fixed dose of anti-TNF α antibody (500 μ g/mouse) in combination with different doses of anti-IL-23 p19 antibody (1.5, 5, 25 μ g/mouse). anti-IL-23 p19 antibody was also included in response to a single dose. The protocol is summarized in table 3 below.

Table 3: single high dose TNF α antibody treatment and Low dose IL + alone in CD40 colitis model/study 3 23p19 evaluation versus combination, ELN: immunopharmacology WC-2016-

The following high dose anti TNF alpha and low dose anti IL-23p19 antibody single and combined treatment after body weight loss determination.

Body weight was monitored from day-1, when mice were injected with antibody (isotype control: 525 μ g; anti-TNF α: 500 μ g; anti-IL-23 p 19: 25,5, 1.5 μ g) or PBS (10ml/kg) until termination on day 7. The data are shown in figure 6. Each line represents the group mean (n ═ 10 antibody treatments and vehicle; n ═ 5 naive controls) and is shown as the percent change from day-1 (dashed line). For each treatment group, the isotype control group was analyzed for significance of differences by two-way anova with Dunnett's multiple comparison test. P-values for each study day are shown in the table and highlighted in bold/italics if they indicate significance. ELN: immunopharmacology WC-2016-.

Consistent with previous studies, high doses of anti-TNF α antibodies completely protected against weight loss, as shown in fig. 6B. In contrast, a single treatment with all doses of anti-IL-23 p19 antibody provided partial protection against weight loss, particularly in the late stages of anti-CD 40 antibody-induced disease. See fig. 6C. The combination of anti-TNF α antibody and anti-IL-23 p19 antibody provided no additional detectable beneficial effect on the inhibition of weight loss compared to monotherapy (fig. 6D). Without being bound by theory, this effect may be due to the robust efficacy of anti-TNF α antibody monotherapy on this parameter.

Histopathological analysis of the proximal colon was performed after single and combined antibody treatment with high-dose anti-TNF α and low-dose anti-IL-23 p19 antibodies. At termination (day 7), proximal colon tissue samples were removed, washed, fixed, and then stained with H & E and examined for histopathological changes by a blinded pathologist using a severity score of 0-20, as described in example 1 above. The data are shown in fig. 7A to 7C. No histopathological results were observed in naive animals. The significance of the differences between the antibody-treated groups and the corresponding isotype controls was analyzed by a one-way anova-Sidak multiple comparison test. The line plots the group value. ELN: immunopharmacology WC-2016-.

As shown in fig. 7A-7C, anti-TNF α antibodies (500 μ g/mouse) did not provide significant protection against colon histopathology compared to isotype control. anti-IL-23 p19 antibody (1.5, 5 and 25. mu.g/mouse) treatment showed dose-dependent protection against colitis, with no protection observed at the lowest dose (1.5. mu.g/mouse). Partial protection against colitis was observed with two higher doses (5 and 25 μ g/mouse).

Due to the low amount of antibody used for anti-IL-23 p19, statistical significance of single anti-IL 23p19 treatment was calculated against vehicle control, but not against high dose (525 μ g/mouse) isotype control. All combined treatments showed significant protection against colon inflammation compared to the anti-TNF α treatment alone. See fig. 7A-7C. Notably, neither monotherapy treatment provided any protection of colon histopathology at the lowest combination dose evaluated (500 μ g/mouse TNF α +1.5 μ g/mouse anti-IL-23 p19), but showed significant improvement in histopathology when given in combination. See fig. 7A. Unexpectedly, a relatively small amount of anti-IL-23 p19 antibody in combination with an anti-TNF α antibody (e.g., at a ratio of 1:333(w/w)) provided this significant improvement in colon histopathology. It was also unexpected that the colon histopathological score observed in the group receiving 500. mu.g/mouse TNF α + 1.5. mu.g/mouse anti-IL-23 p19 was not statistically different from the colon histopathological score observed in the isotype control group. These results indicate that combined treatment with a fixed high dose of TNF α mAb and a suboptimal low dose of IL-23p19 provides superior protection against both cytokines compared to monotherapy.

Example 5: combined treatment of anti-TNF alpha and anti-IL-23 p19 affects a unique subnetwork that enriches the wound healing pathway

The molecular impact of combination therapy with anti-TNF α and anti-IL-23 p19 antibodies was determined relative to monotherapy. The humanized colon gene expression signature of either anti-TNF α (500 μ g) or high dose anti-IL-23 p19(25 μ g) monotherapy was crossed with the gene expression signature from the combination therapy (500 μ g anti-TNF α/1.5 μ g anti-IL-23 p19) to determine whether the molecular responses to the anti-TNF α and low dose anti-IL-23 p19 antibody combination treatment were additive or unique compared to either therapy alone.

The 25 μ g dose of anti-IL-23 p19 treatment was chosen for comparison in order to compare the effect of the combination treatment of anti-TNF α with suboptimal doses of anti-IL-23 p19 with that of a monotherapy dose of anti-IL-23 p19 with efficacy in this model.

As in study 1, humanized colon gene signatures were generated for each single and combination therapy treatment group for evaluation of signature overlap, generation of treatment sub-networks, and enrichment analysis performed. The data are shown in the left panel of fig. 8. It was found that twenty-two-hundred genes were uniquely differentiation regulated following combination therapy (500. mu.g anti-TNF. alpha./1.5. mu.g anti-IL-23 p19) relative to either monotherapy (500. mu.g anti-TNF. alpha. or 25. mu.g anti-IL-23 p 19). These genes are projected onto the CERTIFI intestinal Bayesian network. The resulting maximal ligation component inducing a 1-step network was subjected to enrichment analysis and the results are shown in the right panel of fig. 8. Network analysis of these 220 genes identified a unique sub-network (shown in figure 8) that enriched fibroblast and extracellular matrix tissues, a combined treatment of cell types and pathways involved in wound repair and mucosal healing. Thus, anti-TNF α and anti-IL-23 p19 therapies may provide additional beneficial effects when used in combination by targeting both common and unique disease-related pathways.

Example 6: clinical study of anti-TNF alpha and anti-IL-23 p19 treatment in UC

Phase 2a randomization, double-blind, active control, parallel cohort, multicenter, proof-of-concept clinical study to evaluate the efficacy and safety of combination therapy with both gucekumab and golimumab in participants with moderate to severe active ulcerative colitis

Gusaikumab (CNTO 1959 or CNTO1959)) Is a fully human immunoglobulin G1 λ monoclonal antibody (mAb) that binds with high specificity and affinity to the p19 subunit of human Interleukin (IL) -23. Binding of Gusenuzumab to IL-23 blocks binding of extracellular IL-23 to cell surface IL-23 receptors, inhibiting IL-23 specific intracellular signaling and subsequent activation and cytokine production. Ancient Securizumab is currently approved in the United states, European Union, Canada and several other countries for the treatment of moderate to severe plaque psoriasis. In addition, Gucekumab was also evaluated in psoriatic arthritis (PsA) and Crohn's disease worldwide.

Golimumab (CNTO 148 or) Is a fully human anti-tumor necrosis factor alpha (TNF α) mAb that binds TNF α with high affinity. This interaction prevents TNF α from binding to its receptor, thereby inhibiting the biological activity of TNF α. Golimumab is approved in 90 countries worldwide for the treatment of moderate to severe active Ulcerative Colitis (UC). In addition, golimumab is approved for 1 or more of the following indications worldwide: rheumatoid Arthritis (RA), PsA, Ankylosing Spondylitis (AS), nonradiative Axial spondyloarthritis (nr-Axial SpA) and polyarticular juvenile idiopathic arthritis (pJIA).

Target and endpoint

The study will consist of 2 different phases: a 12-week combination comparison phase followed by a 26-week monotherapy phase.

Target

Main object of

Joint comparison phase

Evaluation of clinical efficacy of combination therapy with both gucegurumab and golimumab in participants with moderate to severe active UC.

Evaluation of safety of combination therapy with both gucegurumab and golimumab in participants with moderate to severe active UC.

Secondary target

Joint comparison phase

Evaluating the effect of combination therapy using both gucecuriab and golimumab on the improvement of endoscopy.

Assessing the effect of combination therapy using both gucecurimab and golimumab on disease-specific health-related quality of life (HRQOL), including fatigue.

The efficacy of combination therapy with both gucegurumab and golimumab was assessed by the negative response signature status at baseline.

Evaluation of Pharmacokinetics (PK), immunogenicity, and Pharmacodynamics (PD) of combination therapy with gucekumab and golimumab, including changes in C-reactive protein (CRP), fecal calprotectin, and other PD biomarkers.

Monotherapy phases

Evaluation of the clinical efficacy of combination therapy followed by gusucumab monotherapy.

Evaluation of the safety of combination therapy followed by gusucumab monotherapy.

Evaluation of the effect of combination therapy followed by gusucizumab monotherapy on endoscopic improvement.

Evaluation of the effect of combination therapy followed by guceusumab monotherapy on disease-specific HRQOL including fatigue.

Efficacy of combination therapy followed by gusucumab monotherapy was assessed by negative response signature status at baseline.

Evaluation of the change in PK, immunogenicity and PD, including CRP, fecal calprotectin and other PD biomarkers, of combination therapy followed by gusucumab monotherapy.

Exploratory target

Explore the effect of combination therapy on Patient Reported Outcome (PRO) instruments (e.g., Bristol stool scale [ BSFS ] and patient impression of global change in UC severity [ PGIC ]).

Terminal point

Primary endpoint

The clinical response at week 12, defined as a decrease in Mayo score of ≧ 30% and ≧ 3 points from baseline, and a decrease in rectal bleeding sub-score (RBS) of ≧ 1 or RBS of 0 or 1.

Important secondary endpoint

Clinical remission at week 12, defined as a Mayo score ≦ 2, and no separate sub-score >1.

Note that: other mitigation definitions may be considered and will be fully described in Statistical Analysis Planning (SAP).

Hypothesis

Combination therapy with both guceuzumab and golimumab will result in a clinical response rate at week 12 that is better than both monotherapies.

Overall design

This is a phase 2a randomized, double-blind, active control, parallel cohort, multicenter, interventional proof of concept (POC) clinical study designed to evaluate the efficacy and safety of combination therapy with both gucekumab and golimumab in adults with moderate to severe active ulcerative colitis. The target population was 18 to 65 year old males or females with moderate to severe activity UC, as defined by a Mayo score of 6 to 12, inclusive, at baseline, including an endoscopic sub-score of ≧ 2 obtained during the central examination of the video endoscopy. The participants must be naive to the TNF antagonist and fail or are intolerant to conventional oral or Intravenous (IV) therapy with corticosteroids or immunomodulators (6-mercaptopurine [6-MP ] or azathioprine [ AZA ]).

Immunomodulators (6-MP, AZA, and methotrexate [ MTX ]) had to be discontinued for at least 2 weeks prior to study intervention at the first dose. For participants who received oral corticosteroids at baseline, the investigator had to gradually decrease the daily dose of corticosteroid starting at week 6. All participants will evaluate clinical exacerbations of UC throughout the study. Generally, the dose of concomitant therapy with UC should remain stable at week 38 (except for oral corticosteroids which decrease from week 6) and should not begin concomitant therapy with UC unless the investigator deems medically necessary. The initiation of the forbidden therapy will lead to an interruption of the study intervention.

Endoscopy with central readings is planned for screening/baseline, week 12 and week 38. The consenting participants will have additional endoscopy at week 4, which will also be assessed by the central reader. Efficacy, PK and PD parameters, biomarkers and safety will be evaluated according to the activity schedule (SoA). Pharmacogenomic blood samples will be collected from participants who agreed to this part of the protocol (where local regulations allow). Participation in pharmacogenomic studies is optional.

Temporal analysis is planned to inform future clinical development. Database locks (DBLs) were scheduled at weeks 12 and 38, and final DBLs were scheduled after all participants completed the security follow-up visit. An independent Data Monitoring Committee (DMC) will be commissioned to perform the study.

Number of participants

The study will recruit targets of 210 participants, 70 participants per intervention group plan.

Intervention group and duration

The study will consist of 2 different phases: a 12-week combination comparison phase followed by a 26-week monotherapy phase. At week 0, the targets of 210 participants will be randomized at a 1:1:1 ratio to combination therapy with both gutecumab and golimumab, gutecumab monotherapy or golimumab monotherapy layered by concomitant use of corticosteroids at baseline (Y/N). Participants randomized to combination therapy will receive a guseculizumab monotherapy after week 12. After week 12, participants randomized to the monotherapy group will continue on their initial randomized monotherapy. The combination therapy arm will employ the same dosage regimen of both gucecuriab and golimumab used in the respective monotherapy intervention groups to facilitate scientific interpretation of the results. The following is a description of the 3 intervention groups:

combination therapy: at week 0, 200mg of Gucekumab was injected intravenously, and 200mg of golimumab was injected Subcutaneously (SC); subcutaneous injection of 100mg of golimumab at weeks 2, 6 and 10; intravenous injection of 200mg Gusaikomab at weeks 4 and 8, followed by 100mg Gusaikomab subcutaneously every 8 weeks

Gusucumab monotherapy: 200mg of Gusaikomab intravenously at weeks 0, 4, and 8, followed by 100mg of Gusaikomab subcutaneously every 8 weeks

Golimumab monotherapy: subcutaneous injection of 200mg golimumab at week 0, followed by subcutaneous injection of 100mg golimumab at week 2, and then subcutaneous injection of 100mg golimumab every 4 weeks (q4w)

In addition, placebo administration (IV or SC) will be given appropriately to maintain blinding throughout the study.

The total participant duration will be up to 58 weeks in total (screening: up to 8 weeks; treatment duration: 38 weeks [ 12 weeks for the combination comparison phase; 26 weeks for the monotherapy phase ]; safety follow-up: approximately 16 weeks after the last study intervention administered at week 34). The end of the study will be defined as the time when the last participant completed his or her final security visit.

Evaluation of efficacy (end point)

Efficacy evaluations will include the following:

mayo score and partial Mayo score

Ulcerative colitis endoscopic severity index (UCEIS)

Inflammatory PD markers including CRP and fecal calprotectin

Patient report outcome measures for assessing HRQOL outcomes and fatigue (i.e., inflammatory bowel disease questionnaire [ IBDQ ], patient report outcome measurement information System [ PROMIS ] -29, and PROMIS fatigue 7-item profile [7a ])

Exploratory patient reports a measurement of symptoms, including PGIC for BSFS and UC severity

Other evaluation of efficacy (end-point)

Efficacy evaluations will include the following:

combined comparison phase (i.e., to week 12)

Endoscopic healing at week 12 (Mayo endoscopic score 0 or 1).

Normalization of mucosal endoscopic appearance (Mayo endoscopic sub-division 0).

Histological healing at week 12.

Mucosal healing at week 12 (complex Mayo endoscopic and histological healing).

Change in total scores of Inflammatory Bowel Disease Questionnaires (IBDQ) at weeks 6 and 12 from baseline.

Improvement in IBDQ score >20 at week 6 and week 12.

Changes in 7 domains of the Patient Report Outcome Measurement Information System (PROMIS) -29 from baseline and abdominal pain value rating scale at week 6 and week 12.

Fatigue responses at weeks 6 and 12 (based on PROMIS fatigue profile 7 a; as will be defined in SAP).

Clinical response, clinical remission and endoscopic healing at week 12 by negative response signature status at baseline.

Change in Mayo score from baseline at week 12.

Change in Mayo score from baseline by week 12.

Change in CRP from baseline by week 12.

Change in fecal calprotectin concentration by week 12 from baseline.

Participants with abnormal CRP concentrations at baseline were normalized for CRP concentration at week 12.

Normalization of fecal calprotectin concentration at week 12 for participants with abnormal fecal calprotectin concentration at baseline.

Ulcerative colitis severity endoscopic index (UCEIS) scores at week 0 and 12, obtained by the level of Mayo endoscopic score at the respective follow-up.

Change in the UCEIS score at week 12 from baseline.

The UCEIS score at week 12 is ≦ 4.

Visit, hospitalization and surgery by UC-related emergency department of week 12.

Monotherapy phase (i.e., after week 12)

Clinical remission at week 38.

Clinical response at week 38.

Participants who achieved clinical response at week 12 maintained clinical response at week 38.

Endoscopic healing at week 38.

Normalization of mucosal endoscopic appearance at week 38.

Histological healing at week 38.

Mucosal healing at week 38.

Clinical remission at week 38 and not concurrent corticosteroid receipt.

Participants who achieved clinical remission at week 12 maintained clinical remission at week 38.

Change in total IBDQ score from baseline at week 24 and week 38.

Improvement in IBDQ score >20 at week 24 and week 38.

Change in 7 domains of PROMIS-29 at week 24 and week 38 from baseline and abdominal pain value rating scale.

Fatigue responses at week 24 and week 38.

Clinical response, clinical remission and endoscopic healing at week 38 by negative response signature status at baseline.

Change in Mayo score from baseline at week 38.

Change in Mayo score from baseline by week 38.

Change in CRP from baseline by week 38.

Change in fecal calprotectin concentration by week 38 from baseline.

Participants with abnormal CRP concentration at baseline were normalized for CRP concentration at week 38.

Normalization of fecal calprotectin concentration at week 38 for participants with abnormal fecal calprotectin concentration at baseline.

A UCEIS score at week 38 by the level of Mayo endoscopic score at week 38.

Change in the UCEIS score from baseline at week 38.

The UCEIS score at week 38 is ≦ 4.

Visit, hospitalization and surgery by UC-related emergency department of week 38.

Exploratory endpoint

The score of BSFS over time.

The distribution of PGIC of UC severity over time.

Pharmacokinetic and immunogenicity assessment

Serum samples will be analyzed by or under the supervision of the sponsor using validated, specific and sensitive immunoassays to determine the concentrations of and detection of the anti-gusucumab and anti-golimumab antibodies, respectively.

Pharmacodynamic and biomarker evaluation

Biomarker assessments will be performed to examine the biological response to treatment and identify biomarkers associated with the gucekumab and/or golimumab in UC treatment. Assessment will include assessment of relevant biomarkers in serum, stool, whole blood and mucosal biopsy samples (RNA [ ribonucleic acid ], histology and single cell isolation).

Pharmacogenomic (DNA) evaluation

Approximately 5mL of pharmacogenomic whole blood samples (where permitted by local regulations) will be collected for the genetic analysis specified in the SoA. Whole blood deoxyribonucleic acid (DNA) samples were collected only by participants who signed consent for genetic evaluation. Participation in pharmacogenomic sub-studies is optional.

Security assessment

Safety assessments performed at each study visit will include assessment of adverse events (AE, visit and those occurring between assessment visits), Tuberculosis (TB) assessment and other infection assessments, clinical laboratory blood tests (hematology and chemistry), vital signs, suicide predisposition assessment, concomitant drug review, observation of injection site reactions, AEs related to infusion time, and/or hypersensitivity reactions.

Statistical method

Sample size determination

A sample size of 210 participants (70 per intervention group) was determined by the ability to detect significant differences in participant ratios in week 12 (primary endpoint) clinical responses between combination therapy and two monotherapies using a unilateral chi-square test, with 0.1 significance levels for each comparison. The study was sized so that the combination therapy had approximately 80% capacity based on the simulation to achieve both comparisons to the primary endpoint versus the monotherapy. The proportion of clinical response participants at week 12 of the combination therapy was assumed to be 75%, based on the additive effect of the two monotherapies (20% improvement of each monotherapy relative to the 35% historical placebo response).

Efficacy analysis

All randomized participants who received at least 1 dose of study intervention will be included in the efficacy analysis. Participants will be analyzed according to the treatment group they were randomized, regardless of the treatment they received.

For testing of the primary endpoint, the efficacy of the combination therapy will be compared against each monotherapy. For both statistical comparisons of the primary endpoints, the Cochran-Mantel-haenszel (cmh) chi-square test, stratified by simultaneous use of corticosteroids (Y/N) at baseline, will be used. For each comparison, the tests will be performed simultaneously at a unilateral 0.1 level of significance. The study will be considered positive if the combination treatment group differs significantly from the two monotherapy groups at the primary endpoint.

If both tests of the primary endpoint were positive, the efficacy of the combination therapy will be compared to each monotherapy for the primary secondary endpoint using the CMH chi-square test (one-sided test) stratified by simultaneous use of corticosteroids (Y/N) at baseline. For each comparison, the tests will be performed simultaneously at a unilateral 0.1 level of significance.

Analysis of other efficacy endpoints will be performed without adjustment for multiple comparisons and will provide nominal p-values.

Security analysis

Safety data will be summarized including, but not limited to, AE, Severe Adverse Events (SAE), infection, severe infection, changes in laboratory assessments, and changes in vital signs. AEs arising during treatment will be used to adjust the active (MedDRA) system organ class and preferred terminology summaries through the treatment group and medical dictionary.

Other analysis

Pharmacokinetic analysis

Serum gusucimumab and golimumab concentrations over time for each treatment group were summarized using descriptive statistics.

Population PK modeling may be performed at appropriate times. If these population PK analyses were performed, the results of these analyses would be presented in separate reports.

Immunogenicity assays

All participants who received at least 1 dose of both gucekumab or golimumab and had appropriate samples for testing the antibodies to both gucekumab and golimumab (i.e., participants who obtained at least 1 sample after the first administration of gucekumab or golimumab, respectively) will be pooled for the incidence of antibodies to both gucekumab and golimumab.

Pharmacokinetic/pharmacodynamic analysis

Where appropriate, the relationship between serum concentrations of both gucegurumab and golimumab and efficacy measurements and/or related biomarkers can be explored graphically. Additional analyses may be performed if necessary.

Biomarker analysis

The treatment groups will summarize changes in serum protein analytes, stool biomarkers and biopsies as well as whole blood RNA obtained over time. Correlations between baseline levels of the selected markers and changes from baseline and treatment response will be explored. Biomarker analyses will be summarized in separate technical reports.

Pharmacogenomic analysis

Genetic (DNA) analysis will only be performed in participants who signed consent for participation in the pharmacogenomic studies. These analyses are considered exploratory and will be summarized in separate technical reports.

This patent application describes various examples and embodiments of the present invention. It must be kept in mind, however, that various modifications may be made to the examples and implementations described without departing in principle from the scope and spirit of the invention. In this regard, other embodiments are included within the scope of the items listed below. Moreover, all numerical ranges recited herein include all sub-ranges subsumed therein, as well as any individual value within such ranges. All publications, patents and patent applications mentioned in this specification are herein incorporated by reference.

The invention may be described in conjunction with the following numbered embodiments：

1. An inhibitor of IL-23 and an inhibitor of TNF-a for use in treating an inflammatory disease in a patient, wherein the inhibitors are in synergistically therapeutically effective amounts and the patient exhibits a clinical response.

2. The IL-23 inhibitor and TNF-a inhibitor for use according to embodiment 1, wherein the inflammatory disease is inflammatory bowel disease and the patient exhibits a clinical response based on a clinical endpoint selected from the group consisting of: mayo score, partial Mayo score, ulcerative colitis endoscopic severity index (UCEIS), marker CRP and/or fecal calprotectin, and patient reported outcomes and symptom measurements.

3. The IL-23 inhibitor and TNF-a inhibitor for use according to any of the preceding embodiments, wherein the IL-23 inhibitor comprises an anti-IL-23 p19 antibody or antigen-binding fragment thereof, and the TNF-a inhibitor comprises an anti-TNF-a antibody or antigen-binding fragment thereof.

4. The IL-23 inhibitor and TNF-a inhibitor for use according to any of the preceding embodiments, wherein the inflammatory bowel disease is crohn's disease.

5. The IL-23 inhibitor and TNF-a inhibitor for use according to any of the preceding embodiments, wherein the inflammatory bowel disease is Ulcerative Colitis (UC) or indeterminate colitis.

6. The IL-23 inhibitor and TNF-a inhibitor for use according to any of the preceding embodiments, wherein the inflammatory bowel disease is moderate to severe active Ulcerative Colitis (UC).

7. The IL-23 inhibitor and TNF-a inhibitor for use according to any of the preceding embodiments, wherein the patient was previously treated with the TNF-a inhibitor alone, and wherein the UC has not experienced remission following the previous treatment.

8. The IL-23 inhibitor and TNF-a inhibitor for use according to any of the preceding embodiments, wherein the patient was previously treated with the IL-23 inhibitor alone, and wherein the UC has not experienced remission following the previous treatment.

9. The IL-23 inhibitor and TNF-a inhibitor for use according to any of the preceding embodiments, wherein the anti-IL-23 p19 antibody comprises: a) 1-3 and 4-6; b) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or c) the heavy chain amino acid sequence of SEQ ID NO 9 and the light chain amino acid sequence of SEQ ID NO 10.

10. The IL-23 inhibitor and TNF-a inhibitor for use according to any of the preceding embodiments, wherein the anti-TNF α antibody comprises: a) 11-13 and 14-16; b) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or c) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

11. The IL-23 inhibitor and TNF-a inhibitor for use according to any of the preceding embodiments, wherein the anti-IL-23 p19 antibody comprises: a) 1-3 and 4-6; b) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or c) the heavy chain amino acid sequence of SEQ ID NO 9 and the light chain amino acid sequence of SEQ ID NO 10, and the anti-TNF α antibody comprises: a) 11-13 and 14-16; b) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or c) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

12. An anti-IL-23 antibody or fragment thereof and an anti-TNF- α antibody or fragment thereof for use in treating ulcerative colitis in a patient, wherein the anti-IL-23 p19 antibody comprises (i) the heavy chain Complementarity Determining Region (CDR) amino acid sequences of SEQ ID NOs: 1-3 and the light chain CDR amino acid sequences of SEQ ID NOs: 4-6, (ii) the heavy chain variable region amino acid sequence of SEQ ID NO:7 and the light chain variable region amino acid sequence of SEQ ID NO:8, or (iii) the heavy chain amino acid sequence of SEQ ID NO:9 and the light chain amino acid sequence of SEQ ID NO: 10; and the anti-TNF- α antibody comprises (i) the heavy chain CDR amino acid sequences of SEQ ID NOS: 11-13 and the light chain CDR amino acid sequences of SEQ ID NOS: 14-16, (ii) the heavy chain variable region amino acid sequence of SEQ ID NO:17 and the light chain variable region amino acid sequence of SEQ ID NO:18, or (iii) the heavy chain amino acid sequence of SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:20, wherein the antibody is in a synergistically therapeutically effective amount and the use is effective to treat ulcerative colitis and the patient exhibits a clinical response based on a clinical endpoint selected from the group consisting of: mayo score, partial Mayo score, ulcerative colitis endoscopic severity index (UCEIS), marker CRP and/or fecal calprotectin, and patient reported outcomes and symptom measurements.

13. The anti-IL-23 antibody and anti-TNF-a antibody for use according to embodiment 12, wherein the anti-TNF-a antibody and the anti-IL-23 p19 antibody are administered at a ratio of 1:2 to 2:1 (w/w).

14. The anti-IL-23 and anti-TNF-a antibodies for use according to embodiment 12 or 13, wherein the anti-TNF-a and anti-IL-23 p19 antibodies are administered at a ratio of 15:1 to 400:1 (w/w).

15. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 12 to 14, wherein the anti-IL-23 p19 antibody and the anti-TNF-a antibody are administered simultaneously.

16. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 12 to 14, wherein the anti-IL-23 p19 antibody and the anti-TNF-a antibody are administered sequentially.

17. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 12 to 14 and 16, wherein the anti-IL-23 p19 antibody and the anti-TNF-a antibody are administered within one day of each other.

18. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 12 to 17, wherein the anti-IL-23 p19 antibody is administered at an initial intravenous dose of 200mg, an intravenous dose of 200mg at weeks 4 and 8 and a subsequent subcutaneous dose of 100mg once every 8 weeks, and the anti-TNF-a antibody is administered at an initial subcutaneous dose of 200mg and a subsequent subcutaneous dose of 100mg at weeks 2, 6 and 10.

19. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 12 to 18, wherein the patient exhibits clinical remission based on a clinical endpoint selected from the group consisting of: mayo score, partial Mayo score, ulcerative colitis endoscopic severity index (UCEIS), marker CRP and/or fecal calprotectin, and patient reported outcomes and symptom measurements.

20. The anti-IL-23 antibody and anti-TNF-a antibody for use according to embodiment 19, wherein the clinical endpoint is measured at about 12 weeks after the initial treatment.

21. The anti-IL-23 antibody and anti-TNF-a antibody for use according to embodiment 19 or 20, wherein the clinical endpoint is based on the Mayo score.

22. An anti-IL-23 antibody or fragment thereof and an anti-TNF-a antibody or fragment thereof for use in reducing inflammation of the colon of a patient having an inflammatory bowel disease, wherein said antibodies are in synergistically therapeutically effective amounts and said use is effective to reduce inflammation of the colon of the patient to a level comparable to the colon of a normal subject.

23. The anti-IL-23 antibody and anti-TNF-alpha antibody for use according to embodiment 22, wherein the inflammation in a tissue sample of the colon from the patient is minimal or normal following administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-alpha antibody or antigen-binding fragment thereof.

24. The anti-IL-23 antibody and anti-TNF-a antibody for use according to embodiment 22, wherein gland loss in a tissue sample of the colon from the subject is minimal or normal following administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-a antibody or antigen-binding fragment thereof.

25. The anti-IL-23 antibody and anti-TNF-a antibody for use according to embodiment 22, wherein erosion in a tissue sample of the colon from the subject is minimal or normal following administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-a antibody or antigen-binding fragment thereof.

26. The anti-IL-23 antibody and anti-TNF-alpha antibody for use according to embodiment 22, wherein mucosal thickness and hyperplasia in a tissue sample of the colon from the subject are each minimal or normal following administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-alpha antibody or antigen-binding fragment thereof.

27. The anti-IL-23 antibody and anti-TNF-alpha antibody for use according to embodiment 22, wherein the histopathology of the colon after administration of the anti-IL-23 p19 antibody or antigen-binding fragment thereof and the anti-TNF-alpha antibody or antigen-binding fragment thereof is the same as the histopathology of normal tissues.

28. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 22 to 27, wherein the anti-IL-23 p19 antibody or antigen-binding fragment thereof comprises: a) 1-3 and 4-6; b) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or c) the heavy chain amino acid sequence of SEQ ID NO 9 and the light chain amino acid sequence of SEQ ID NO 10; and the anti-TNF- α antibody or antigen-binding fragment thereof comprises d) the heavy chain CDR amino acid sequences of SEQ ID NOS: 11-13 and the light chain CDR amino acid sequences of SEQ ID NOS: 14-16; e) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or f) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

29. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 22 to 28, wherein the anti-TNF-a antibody or antigen-binding fragment thereof and the anti-IL-23 p19 antibody or antigen-binding fragment thereof are administered at a ratio of 1:2 to 2:1 (w/w).

30. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 22 to 28, wherein the anti-TNF-a antibody or antigen-binding fragment thereof and the anti-IL-23 p19 antibody or antigen-binding fragment thereof are administered at a ratio of 15:1 to 400:1 (w/w).

31. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 22 to 30, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered simultaneously.

32. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 22 to 30, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered sequentially.

33. The anti-IL-23 antibody and anti-TNF-a antibody for use according to any one of embodiments 22 to 30, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered within one day of each other.

34. An anti-IL-23 antibody or fragment thereof and an anti-TNF-a antibody or fragment thereof for use in treating an inflammatory bowel disease in a patient and reducing weight loss in said patient.

35. The anti-IL-23 antibody or antigen-binding fragment thereof and anti-TNF-a antibody or antigen-binding fragment thereof for use according to embodiment 34, wherein the anti-TNF a antibody or antigen-binding fragment thereof and the anti-IL-23 p19 antibody or antigen-binding fragment thereof are administered at a ratio of 15:1 to 400:1 (w/w).

36. The anti-IL-23 antibody or antigen-binding fragment thereof and anti-TNF-a antibody or antigen-binding fragment thereof for use according to embodiment 34 or 35, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered simultaneously.

37. The anti-IL-23 antibody or antigen-binding fragment thereof and anti-TNF-a antibody or antigen-binding fragment thereof for use according to embodiment 34 or 35, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered sequentially.

38. The anti-IL-23 antibody or antigen-binding fragment thereof and anti-TNF-a antibody or antigen-binding fragment thereof for use according to any one of embodiments 34, 35 or 37, wherein the a) anti-IL-23 p19 antibody or antigen-binding fragment thereof and the b) anti-TNF-a antibody or antigen-binding fragment thereof are administered within one day of each other.

39. The anti-IL-23 antibody or antigen-binding fragment thereof and anti-TNF-a antibody or antigen-binding fragment thereof for use according to any one of embodiments 34 to 38, wherein the anti-IL-23 p19 antibody or antigen-binding fragment thereof comprises: a) 1-3 and 4-6; b) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or c) the heavy chain amino acid sequence of SEQ ID NO 9 and the light chain amino acid sequence of SEQ ID NO 10; and the anti-TNF- α antibody or antigen-binding fragment thereof comprises d) the heavy chain CDR amino acid sequences of SEQ ID NOS: 11-13 and the light chain CDR amino acid sequences of SEQ ID NOS: 14-16; e) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or f) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

40. An anti-IL-23 antibody or fragment thereof and an anti-TNF-a antibody or fragment thereof for use in treating moderate to severe active ulcerative colitis in a human patient, wherein the anti-IL-23 p19 antibody or antigen binding fragment thereof is administered at 0.0005mg/kg to 0.002mg/kg and comprises the following sequences: (i) 1-3 and 4-6; (ii) the heavy chain variable region amino acid sequence of SEQ ID NO. 7 and the light chain variable region amino acid sequence of SEQ ID NO. 8; or (iii) the heavy chain amino acid sequence of SEQ ID NO:9 and the light chain amino acid sequence of SEQ ID NO:10, and the anti-TNF- α antibody or antigen-binding fragment thereof is administered at 0.020mg/kg to 0.125mg/kg and comprises the following sequences: (iv) 11-13 and 14-16; (v) the heavy chain variable region amino acid sequence of SEQ ID NO 17 and the light chain variable region amino acid sequence of SEQ ID NO 18; or (vi) the heavy chain amino acid sequence of SEQ ID NO 19 and the light chain amino acid sequence of SEQ ID NO 20.

41. The anti-IL-23 antibody or antigen-binding fragment thereof and anti-TNF-a antibody or antigen-binding fragment thereof for use according to embodiment 40, wherein the use is effective to treat the ulcerative colitis.

42. The anti-IL-23 antibody or antigen-binding fragment thereof and anti-TNF-a antibody or antigen-binding fragment thereof for use according to embodiment 40 or 41, wherein the patient exhibits clinical remission based on a clinical endpoint selected from the group consisting of: mayo score, partial Mayo score, ulcerative colitis endoscopic severity index (UCEIS), marker CRP and/or fecal calprotectin, and patient reported outcomes and symptom measurements.

43. The anti-IL-23 antibody or antigen-binding fragment thereof and anti-TNF-a antibody or antigen-binding fragment thereof for use according to any one of embodiments 40 to 42, wherein the following are present in an aqueous solution of the pharmaceutical composition: 100mg/mL of the anti-IL-23 p19 antibody; 7.9% (w/v) sucrose, 4.0mM histidine, 6.9mM L-histidine monohydrochloride monohydrate; polysorbate 80 at 0.053% (w/v) of the composition, and the following are present in an aqueous solution of the pharmaceutical composition: 100mg/mL of the anti-TNF- α antibody; 4.1% (w/v) sorbitol, 5.6mM L-histidine and L-histidine monohydrochloride monohydrate; polysorbate 80 at 0.015% (w/v) of the composition.

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Methods of treating inflammatory bowel disease with combination therapy of antibodies to IL-23 and TNF α

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