TW202330613A - Methods of treating crohn’s disease using integrin beta7 antagonists - Google Patents

Abstract

Methods of treating Crohn's disease are provided. Also provided are methods of administering and dosing integrin beta7 antagonists, such as anti-integrin beta7 antibodies. In addition, methods of administrating and dosing such integrin beta7 antagonists to maintain improvement of Crohn's disease, including clinical remission and/or endoscopic improvement, are provided.

Description

Treatment of Crohn's disease using integrin β7 antagonists

The present invention provides methods of treating Crohn's disease. Methods of administering and administering integrin beta7 antagonists, such as anti-integrin beta7 antibodies, are also provided. Additionally, methods of administering and administering such integrin beta7 antagonists to maintain improvement in Crohn's disease, including clinical remission and/or endoscopic improvement, are provided.

Inflammatory bowel disease (IBD) is a chronic inflammatory autoimmune disorder of the gastrointestinal (GI) tract that manifests clinically as ulcerative colitis (UC) or Crohn's disease (CD). CD is a chronic transmural inflammatory disease that may affect any part of the entire GI tract, whereas UC is inflammation of the mucosa of the colon. Both conditions are clinically characterized by frequent bowel movements, malnutrition and dehydration, along with interference with activities of daily living. The etiology of IBD is complex, and many aspects of its pathogenesis remain unclear.

CD is a chronic, relapsing form of IBD that can affect any part of the gastrointestinal tract, with 40%-50% of cases affecting the small intestine. CD is characterized by patchy, transmural inflammatory, ulcerative, and granulomatous lesions interspersed with healthy intestinal segments (skip lesions). The disease is progressive; uncontrolled inflammation develops into stricturing or penetrating complications such as prestenotic dilatation, obstruction (stenosis), and intra-abdominal or perianal fistulas and abscesses (penetrating). Clinical signs and symptoms include chronic diarrhea, abdominal pain, cachexia, abdominal mass or tenderness, and telltale signs of fistulas. The disease course is variable; patients may experience a severe initial episode followed by few symptoms for 10 years (43%) or chronic and persistent symptoms (19%) or relapsing-remitting symptoms (32%) (Baumgart DC, et al., Lancet 380:1590-605, 2012).

Annual incidence rates of CD reported in Europe, Asia and the Middle East, and North America are 12.7, 5.0, and 20.2/100,000 person-years, respectively (Molodecky NA, et al., Gastroenterology 142:46-54, 2012). The current prevalence in North America is reported to be 319/100,000 people ( ibid. ). Disease-related mortality in CD accounts for approximately 30% of deaths in this population and is due to clinical and/or surgical complications early in the disease course or bowel cancer later in the disease course. The global incidence of CD is expected to continue to increase substantially, affecting individuals during their most formative and productive years of life, resulting in long-term costs to patients, healthcare systems, and society (Duricova D. et al., Inflamm Bowel Dis 16: 347-53, 2010).

To date, CD has no cure. Therefore, the current goals of treatment for CD are to induce and maintain symptom improvement, induce mucosal healing, avoid surgery, and improve quality of life (Lichtenstein GR, et al., Am J Gastroenterol 104:465-83, 2009; Van Assche G, et al., J Crohns Colitis. 4:63-101, 2010).

Systemic corticosteroids (CS) have become the mainstay of treatment for induction of remission and are effective in approximately 80% of patients (Summers RW, et al., Gastroenterology 77:847-69, 1979; Malchow H, et al., Gastroenterology 86:249- 66, 1984). However, they are less effective as maintenance therapy, with only 28% of patients achieving a prolonged response after 1 year of treatment, and 32% becoming steroid dependent (Faubion WA, et al., Gastroenterology 121:255-60, 2001; Peyrin-Biroulet L, et al., Am J Gastroenterol 105:289–97, 2010). Even if a patient's symptoms improve, less than 30% of patients are expected to achieve endoscopic improvement with steroid therapy (Modigliani R, et al., Gastroenterology 98:811-8, 1990). Adverse effects of steroids are well documented and result in 50% of patients discontinuing treatment; long-term safety outcomes include osteoporosis, cataracts, and diabetes.

Immunosuppressants (IS) (eg, azathioprine [AZA], 6-mercaptopurine [6-MP], or methotrexate [MTX]) are often administered to induce remission in patients who are intolerant or refractory to steroids and maintaining remission in patients who achieved quiescent CD. Immunosuppressants were given with or without steroid bridging depending on the patient's symptoms during the 2-4 months after the onset of IS response. In patients with ileal or ascending colon disease, cortisol is a less toxic and better tolerated bridging agent because of its rapid first-pass metabolism resulting in low systemic bioavailability. Over the past 20 years, earlier initiation of IS has been advocated to modify the course of inflammatory disease. However, a decrease in bowel resection and complication rates has not been observed during this period (Cosnes J, et al., Gut 54:237-41, 2005). This may reflect poor uptake of this top-down treatment due to concerns about systemic toxicity, including leukopenia, thrombocytopenia, and increased risk of lymphoma associated with administration of AZA and 6-MP (Prefontaine E, et al., Cochrane Database Syst Rev (4):CD000545, 2009) and hepatotoxicity and alopecia associated with MTX administration (Hausmann J, et al., Inflamm Bowel Dis 16:1195-202, 2010).

The development of anti-tumor necrosis factor (TNF)-α monoclonal antibodies (mAbs) provides additional therapeutic options. Although anti-TNF is effective in a large proportion of patients, efficacy is suboptimal; response rates after 4 weeks of induction therapy are less than 35%, and among patients who respond to induction therapy, at the 20-30 week maintenance assessment, less than Fifty percent of patients achieve remission (Peyrin-Biroulet L, et al., Aliment Pharmacol Ther 33:870-9, 2011). Additionally, 30% of patients have been reported to be first-time non-responders to anti-TNF therapy when assessed 4 weeks after induction therapy (Targan SR, et al., N Engl J Med 337:1029-35, 1997; Sandborn WJ, et al. Human, Ann Intern Med 2007:19;146:829-38. Epub 2007 Apr 30), possibly because the underlying pathobiology is not TNF-α driven and may therefore benefit from drugs with different mechanisms. It is estimated that 30%-40% of patients will be secondary non-responders (i.e. initially respond) but lose response or become intolerant during the first year of treatment (Colombel JF, et al, Gastroenterology 132: 52-65, 2007). Secondary unresponsiveness has been attributed to the production of neutralizing antibodies, resulting in low drug serum levels, to accelerated drug clearance, or to biological escape mechanisms that may benefit from treatments with different pharmacological targets. Anti-TNF is also associated with significant side effects, including serious infections, opportunistic infections, lupus-like reactions, and an increased risk of lymphoma (Siegal CA, et al., Therap Adv Gastroenterol 2:245–51, 2009). Tolerability issues include infusion reactions (occurring in 9%-17% of patients treated with infliximab, see de Vries HS, et al., Br J Clin Pharmacol 71:7-19, 2011) and injection site Reaction (occurs in 10% of patients treated with adalimumab, see van der Heijde D, et al., Arthritis Rheum. 54:2136-46, 2006). Overall, the benefits and risks of this class of drugs are considered acceptable, but treatments with better benefit-risk profiles are still needed to reduce inflammation and clinical sequelae and improve the long-term prognosis of CD patients.

Integrins are α/β heterodimeric cell surface glycoprotein receptors that play roles in many cellular processes including leukocyte adhesion, signaling, proliferation and migration, and gene regulation (Hynes, RO, Cell, 1992, 69:11-25; and Hemler, ME, Annu. Rev. Immunol., 1990, 8:365-368). They are composed of two heterodimeric, non-covalently interacting α and β transmembrane subunits that specifically bind to different cell adhesion molecules (CAMs) on endothelial, epithelial and extracellular matrix proteins. In this way, integrins can function as tissue-specific cell adhesion receptors, helping to recruit leukocytes from the blood to nearly all tissue sites in a highly regulated manner, playing a role in homing leukocytes to normal tissues as well as to sites of inflammation. role (von Andrian et al., N Engl J Med 343:1020–34 (2000)). In the immune system, integrins are involved in leukocyte trafficking, adhesion, and infiltration during inflammatory processes (Nakajima, H. et al. , J. Exp. Med., 1994, 179:1145-1154). Different expressions of integrins can regulate the adhesive properties of cells, and different integrins participate in different inflammatory responses. (Butcher, EC et al. , Science, 1996, 272:60-66). β7-containing integrins ( i.e. α4β7 and αEβ7) are mainly expressed on monocytes, lymphocytes, eosinophils, basophils and macrophages, but not on neutrophils (Elices, MJ et al. , Cell, 1990, 60:577-584).

Antiintegrins are another class of biologics approved for the treatment of CD. Natalizumab is an antiintegrin approved in the United States only for the treatment of moderately to severely active CD. The use of natalizumab, which blocks both α4β1 and α4β7, has been criticized due to concerns that inhibition of α4β1/VCAM-1 binding increases the risk of progressive multipartite leukoencephalopathy (PML), a rare but serious CNS infection. limit. Vedolizumab is a gut-selective anti-integrin recently approved for CD, but it only targets the α4β7 integrin receptor (which inhibits the binding of T lymphocytes to the adhesion molecule MAdCAM-1) and is Administered by intravenous (IV) infusion. In the pivotal trial of vedolizumab, 31% of patients experienced a clinical response, defined as a > 100-point reduction in Crohn's Disease Activity Index [CDAI] score from baseline, after 6 weeks of induction therapy; up to 39% of patients with vedolizumab Lizumab responders achieved remission (defined as CDAI score < 150) after 46 weeks of maintenance therapy, compared with 22% of patients given placebo (Sandborn WJ, et al., N Engl J Med 369(8):711- 721, 2013; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013). Although vedolizumab holds promise as a new treatment for CD, there is still a need for a more convenient therapy that is gut-selective and achieves better response and remission rates.

α4β7 integrin (the target of vedolizumab) is a leukocyte homing receptor that plays a role in cell migration to the intestinal mucosa and associated lymphoid tissues (such as Peyer's patches in the small intestine and lymphoid follicles in the large intestine). and mesenteric lymph nodes) are important. In the intestine, leukocyte rolling and firm adhesion to the mucosal endothelium are initiated by chemokine signaling and mediated via mucosal addressin cell adhesion molecule (MAdCAM)-1-related sialic acid Lewis X. Chemokine signaling induces α4β7 integrin to undergo a change in MAdCAM‑1 binding affinity from low to high. Leukocytes then cease and begin the process of extravasation across the vascular endothelium into underlying tissues. This extravasation process is thought to occur both in normal immune cell recycling states and in inflammation (von Andrian et al., supra ). The number of α4β7 ⁺ cells in the infiltrate and the expression of the ligand MAdCAM‑1 are higher at sites of chronic inflammation, such as in the gut of patients with UC or CD (Briskin et al., Am J Pathol 151:97–110 (1997); Souza et al., Gut 45:856-63 (1999)). α4β7 preferentially binds to high endothelial venous and vascular cell adhesion molecule (VCAM)-1 expressing MAdCAM-1, as well as to the extracellular matrix molecule fibronectin fragment CS-1 (Chan et al., J Biol Chem 267:8366–70 ( 1992); Ruegg et al., J Cell Biol 17:179–89 (1992); Berlin et al., Cell 74:185–95 (1993)). Together with MAdCAM-1, which is constitutively expressed in intestinal mucosal vessels, α4β7 integrin plays a selective role in leukocyte intestinal tropism but does not appear to contribute to homing of leukocytes to peripheral tissues or the CNS. In contrast, peripheral lymphatic transport is related to the interaction of α4β1 with VCAM-1 (Yednock et al., Nature 356:63–6 (1992); Rice et al., Neurology 64:1336–42 (2005)).

Another member of the β7 integrin family that is expressed only on T lymphocytes and is associated with mucosal tissue is the αEβ7 integrin, also known as CD103. αEβ7 integrin selectively binds to E-cadherin on epithelial cells and has been proposed to play a role in T cell retention in mucosal tissue within the intraepithelial lymphocyte compartment (Cepek et al., J Immunol 150:3459 -70 (1993); Karecla et al. Eur J Immunol 25:852–6 (1995)). αEβ7 ⁺ cells in the lamina propria have been reported to exhibit cytotoxicity toward stressed or infected epithelial cells (Hadley et al., J Immunol 159:3748–56 (1997); Buri et al., J Pathol 206:178–85 (2005) )). Expression of αEβ7 is increased in CD (Elewaut et al., Acta Gastroenterol Belg 61:288–94 (1998); Oshitani et al., Int J Mol Med 12:715–9 (2003)), and anti-αEβ7 antibodies have been reported Treatment alleviates experimental colitis in mice, suggesting a role for αEβ7 ⁺ lymphocytes in experimental models of IBD (Ludviksson et al., J Immunol 162:4975–82 (1999)).

Humanized monoclonal antibodies directed against the β7 integrin subunit have been described previously. See, for example, International Patent Publication No. WO2006/026759. One such antibody, evolizumab (rhuMAb β7), is derived from the rat anti-mouse/human monoclonal antibody FIB504 (Andrew DP, et al., J Immunol 153:3847-61, 1994). Designed to contain human IgG1 heavy chain and κ1 light chain framework. International Patent Publication No. WO2006/026759.

Evolizumab (subcutaneously administered mAb) is a novel antiintegrin distinct from vedolizumab that targets α4β7 and αEβ7 receptors, respectively, that regulate the trafficking and retention of T cell subsets in the intestinal mucosa. Thus, evolizumab provides potential additive therapeutic effects in CD via a dual mechanism of action (MOA) without the need for systemic immunosuppression. Evolizumab binds with high affinity to α4β7 (Holzmann B, et al., Cell 56:37-46, 1989; Hu M, et al., Proc Natl Acad Sci USA 89:8254-8, 1992) and αEβ7 (Cepek KL, et al., J Immunol 150:3459-70, 1993). Through this mechanism, it blocks the homing and retention of leukocyte subsets in the intestinal mucosa via binding to cell adhesion molecules (MAdCAM-1) and E-cadherin, respectively. Therefore, it represents a novel intestinal mucosal-selective anti-delivery agent whose selectivity can abrogate systemic immunosuppression by preferentially targeting delivery to the intestine over other organs and tissues. Data from multiple nonclinical general toxicity studies lasting up to 6 months indicate that evolizumab had no adverse effects on any organ system. See, for example, Stefanich et al., British Journal of Pharmacology 162 : 1855–1870, 2011; International Patent Publication No. WO 2009/140684.

Of note, unlike natalizumab, evolizumab does not bind to α4β1 or inhibit the interaction of α4β1 with VCAM-1 and lymphocyte distribution and homing into the CNS and peripheral lymphoid tissues. Therefore, evolizumab is not expected to increase the risk of progressive multipartite leukoencephalopathy (PML). The safety of evolizumab has been evaluated in adult Phase 1 and 2 studies in which patients with moderately to severely active UC received single or multiple doses of IV or subcutaneous (SC) evolizumab. A total of 158 patients were treated with evolizumab, and there were no significant adverse safety signals, including any evidence of an increased incidence of serious or opportunistic infections associated with evolizumab treatment. It is recognized that clinical experience with evolizumab is limited and no events of PML have been reported in patients treated with evolizumab.

To date, the primary outcome measure in Crohn's disease clinical trials is the Crohn's Disease Activity Index (CDAI), which has served as the basis for the approval of several drug treatments, including, for example, vedolizumab and natalizumab. The CDAI was developed by returning clinicians' global assessment of disease activity on eighteen potential items representing patient-reported outcomes (PROs) (i.e., abdominal pain, pain that awakens the patient from sleep, appetite), physical signs (ie, mean daily temperature, abdominal mass), medication use (ie, loperamide or opioids for diarrhea), and laboratory tests (ie, hematocrit). Backward stepwise regression analysis identified eight independent predictors, which were the number of liquid or soft stools, severity of abdominal pain, overall sense of well-being, occurrence of extraintestinal symptoms, need for antidiarrheal medication, presence of abdominal mass, hematocrit and weight. The final score is a composite of these eight items, adjusted using regression coefficients and standardization to construct an overall CDAI score, ranging from 0 to 600, with higher scores indicating greater disease activity. Widely used benchmarks are: CDAI <150 defines clinical remission, 150 to 219 defines mildly active disease, 220 to 450 defines moderately active disease, and above 450 defines very severe disease (Best WR, et al., Gastroenterology 77:843-6, 1979). Vedolizumab and natalizumab have been approved based on proven clinical response, defined as CDAI < 150.

Although the CDAI has been used for more than 40 years and serves as the basis for drug approval, it has several limitations as an outcome measure in clinical trials. For example, most global scores come from patient diary card items (pain, number of liquid bowel movements, and general feeling of well-being), which are poorly defined and not standardized (Sandler et al., J. Clin. Epidemiol 41:451-8, 1988; Thia et al. Inflamm Bowel Dis 17:105-11, 2011). Additionally, pain was measured on a four-point scale rather than the newer seven-point scale. The remaining five index items play a small role in identifying efficacy signals and may be sources of measurement noise. Additionally, there are concerns about the poor criterion validity of the CDAI, the reported lack of correlation between the CDAI and endoscopic measures of inflammation (which may make the CDAI poor at discriminating active CD from irritable bowel syndrome), and the high reported placebo rate (Korzenik et al., N Engl J Med. 352:2193-201, 2005; Sandborn WJ, et al., N Engl J Med. 353:1912-25, 2005; Sandborn WJ, et al., Ann Intern 19;146:829 -38, 2007, Epub 30 April 2007; Kim et al., Gastroenterology 146: (5 supplement 1) S-368, 2014).

Therefore, there is general agreement that additional or alternative measures of CD symptoms are needed. In fact, the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) registration requirements for new drugs for Crohn's disease have recently been changing and focus on various patient reports in addition to objective assessment of inflammation, discussed further below. Outcomes (PRO) (see, e.g., Willet et al., Clin Gastroenterol Hepatol 2014, 12 (8):1246-1256.e1683; Khanna et al. Aliment Pharmacol Ther. 2015;41(1):77–86).

Another way to assess the extent and severity of Crohn's disease is endoscopy. Typical endoscopic lesions of Crohn's disease have been described in many studies and include, for example, aphthous ulcers, "perforated ulcers," cobblestone formation, and strictures. Endoscopic assessment of such lesions was used to develop the first validated endoscopic score, the Crohn's Disease Endoscopic Index of Severity (CDEIS) (Mary et al., Gut 39:983-9, 1989). Recently, because CDEIS is time-consuming, complex, and not suitable for routine use, the Simplified Endoscopic Activity Score (SES-CD) for Crohn's disease was developed and validated (Daperno et al., Gastrointest. Endosc. 60(4):505-12 , 2004). SES-CD consists of four endoscopic variables (ulcer size, proportion of surface covered by ulcer, proportion of surface with any other pathology (e.g., inflammation), and presence of narrowing [stenosis]), and is evaluated across five ileocolic segments. These variables are scored, with each variable or assessment rated from 0 to 3.

Despite these recent advances in the development and validation of new PRO measures and new endoscopic measures of CD disease extent and severity, there are no published prospective clinical studies to date that can be used to determine how to conduct prospective clinical trials in phase III clinical trials. These new measures are used in the design and conduct of studies (i.e., pivotal or registration studies) designed to evaluate the effectiveness of specific dosing regimens of therapeutic candidates (including, for example, evolizumab) in inducing remission and, importantly, Efficacy in maintaining remission and improving other CD disease signals.

The inventions described herein satisfy some of the above needs and provide other benefits.

All references (including patent applications and publications) cited herein are incorporated by reference in their entirety for any purpose.

The methods of the present invention are based, at least in part, on surprising and unexpected results from the maintenance phase of a clinical study of the safety and efficacy of evolizumab in patients with Crohn's disease. These results demonstrate that during the maintenance phase, patients treated with evolizumab achieved the co-primary endpoints of clinical response and endoscopic improvement compared with placebo. The results also showed that patients treated with evolizumab achieved the secondary endpoints of corticosteroid-free response and endoscopic response compared with placebo.

Accordingly, in one aspect, a method of treating a patient suffering from Crohn's disease is provided. In certain embodiments, the method comprises subcutaneously administering an integrin beta7 antagonist to the patient for a treatment period of at least 52 weeks or at least 66 weeks after initiation of induction therapy, wherein 14 weeks after initiation of induction therapy, the patient is determined to A reduction in Crohn's Disease Activity Index (CDAI) score of 70 points or more from baseline has been achieved, in which the patient maintains clinical remission during the treatment period.

In a further embodiment, the patient maintains endoscopic improvement in addition to maintaining clinical remission. In yet other embodiments, the patient further maintains endoscopic remission, corticosteroid-free clinical remission, or both endoscopic remission and corticosteroid-free clinical remission.

In other embodiments, the patient receives corticosteroid therapy in addition to induction therapy during an induction period, wherein the induction period is 14 weeks, and wherein at the end of the induction period, corticosteroid therapy is reduced over time until discontinued. In some embodiments, the corticosteroid therapy is (i) less than or equal to 20 mg of prednisone per day and wherein the corticosteroid therapy is reduced by 2.5 mg of prednisone per week until discontinued, or (ii) less than or equal to 20 mg of prednisone per day. or equal to 6 mg of oral budesonide with corticosteroid therapy reduced by 3 mg of oral budesonide every 2 weeks until discontinued.

In certain embodiments, the integrin β7 antagonist is a humanized monoclonal anti-integrin β7 antibody. In certain such embodiments, the anti-integrin β7 antibody comprises three light chain hypervariable regions (HVR): HVR-L1, HVR-L2, and HVR-L3, and three heavy chain HVRs: HVR-H1, HVR -H2 and HVR-H3, where: (i) HVR-L1 contains the amino acid sequence RASESVDDLLH (SEQ ID NO: 1); (ii) HVR-L2 contains the amino acid sequence KYASQSIS (SEQ ID NO: 2); (iii) HVR-L3 contains the amino acid sequence QQGNSLPNT (SEQ ID NO:3); (iv) HVR-H1 contains the amino acid sequence GFFITNNYWG (SEQ ID NO:4); (v) HVR-H2 contains the amino acid sequence GYISYSGSTSYNPSLKS (SEQ ID NO: 5; and (vi) HVR-H3 contains the amino acid sequence RTGSSGYFDF (SEQ ID NO:6); or the amino acid sequence ARTGSSGYFDF (SEQ ID NO:7).

In certain such embodiments, the anti-integrin β7 antibody further comprises: a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; and a heavy chain variable region comprising SEQ ID NO: 8 The amino acid sequence shown in NO: 9. In certain embodiments, an anti-integrin β7 antibody comprises: a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11 The amino acid sequence shown in SEQ ID NO: 12; or a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, an anti-integrin β7 antibody comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 Amino acid sequence is shown.

In certain of these embodiments, the anti-integrin β7 antibody further comprises: a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11 An amino acid sequence, or a heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 12. In certain such embodiments, the anti-integrin β7 antibody is evolizumab.

In another aspect, a method of treating a patient who has moderately to severely active Crohn's disease prior to administration of induction therapy is provided. In some embodiments, induction therapy includes one or more therapeutic agents selected from: 5-aminosalicylates, antibiotics, cortisol, systemic corticosteroids, thiopurines, methotrexate, anti-TNF agents , infliximab, adalimumab, pegylated certolizumab, vedolizumab, ustekinumab, natalizumab, efalizumab, evolizumab , Janus kinase (JAK) inhibitors, upadatinib and filgotinib.

In yet another aspect, the patient is determined to have (1) a CDAI score greater than or equal to 220 and less than or equal to 480 at any time during the seven days prior to the start of induction therapy and (2) an average of Daily fluid/soft stool frequency (SF) score greater than or equal to 6 or mean daily SF greater than 3 and mean daily abdominal pain (AP) score greater than 1. In some embodiments, the patient is determined to have active inflammation, wherein active inflammation is determined by a SES-CD score of greater than or equal to 7 as determined by ileocolonoscopy. In some embodiments, the patient has isolated ileitis or post ileocecal resection and wherein the patient is determined to have active inflammation, wherein the active inflammation is due to clones greater than or equal to 4 as determined by ileocolonoscopy The disease was determined based on the Simplified Endoscopic Index (SES-CD) score.

In another aspect, the patient is refractory to or intolerant to one or more therapies selected from immunosuppressive therapy, corticosteroid therapy, and anti-TNF therapy. In some embodiments, the patient responds poorly to anti-TNF therapy. In some embodiments, the immunosuppressant therapy is selected from the group consisting of 6-mercaptopurine, azathioprine, and methotrexate. In some embodiments, corticosteroid therapy is selected from the group consisting of prednisone and oral cortisol. In some embodiments, the anti-TNF therapy is selected from the group consisting of infliximab, adalimumab, and pegylated certolizumab.

In yet another aspect, the anti-integrin beta7 antibody is administered at a uniform dose of 105 mg every 4 weeks from week 14 to at least week 52 or to at least week 66 of induction therapy.

In yet another aspect, clinical remission is due to a fluid/soft stool frequency (SF) average daily score of less than or equal to three and an abdominal pain (AP) average daily score of less than or equal to three, averaged over a period of at least four days prior to assessment. Determined if there is no worsening of SF or AP compared to baseline. In some embodiments, the SF average daily score and the AP average daily score are averaged over the seven days preceding the assessment.

In some of the above embodiments, the endoscopic improvement is determined by the Simplified Endoscopic Index for Crohn's Disease (SES-CD) score, and the SES-CD score is compared to the SES-CD score determined at baseline. , a reduction of at least fifty percent.

In certain of the above embodiments in which the patient has discontinued corticosteroid treatment, the patient does not receive treatment with one or more corticosteroids for at least 24 consecutive weeks after discontinuing corticosteroid treatment.

In another aspect, the patient maintains endoscopic response, and endoscopic response is determined by SES-CD score and the SES-CD score is less than or equal to four. In some embodiments, the patient is an ileal patient and has an SES-CD score of less than or equal to two. In some embodiments, the SES-CD score does not include segments with subcategory scores greater than one. In some embodiments, the subcategories are selected from the size and extent of ulcer, affected surface, and narrowing.

In yet another aspect, a method of treating Crohn's disease according to any of the embodiments includes administering an integrin β7 antagonist using a prefilled syringe or a prefilled syringe in combination with an autoinjector.

Cross-references to related applications

This application claims the benefit of Provisional Application No. 63/278,891, filed on November 12, 2021, the contents of which are incorporated herein by reference in their entirety. sequence list

This application contains a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML replica was created on November 7, 2022, is named P37218-WO_SL.xml, and is 12,529 bytes in size.

Unless otherwise defined, 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. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992) provides general guidance to those skilled in the art regarding many of the terms used in this application. some definitions

For the purposes of interpreting this specification, the following definitions will apply and, wherever appropriate, terms used in the singular will also include the plural and vice versa. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

As used in this specification and the appended claims, the singular forms "a/an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "protein" includes a plurality of proteins; reference to "cells" includes mixtures of cells, and so on.

Ranges provided in this specification and accompanying claims include both endpoints and all points between the endpoints. So, for example, the range 2.0 to 3.0 includes 2.0, 3.0, and all points between 2.0 and 3.0.

"Treatment" or "treating" and its grammatical variations refers to a clinical intervention that attempts to alter the natural course of a disease in an individual or cell being treated, and may be performed prophylactically or during clinical pathology. The desired therapeutic effects include preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.

"Treatment regimen" means the combination of dosage, frequency, or duration of treatment, with or without the addition of a second drug.

"Effective treatment regimen" means a treatment regimen that will provide a beneficial response to the patient receiving the treatment.

"Modified treatment" means a change in treatment regimen, including changing the dose, frequency or duration of treatment, and/or adding a second drug.

As used herein, "clinical remission" means an average daily score of Fluid/Soft Stool Frequency (SF) of less than or equal to three and an average daily score of Abdominal Pain (AP) of less than or equal to 1 for either sub-score, averaged over a seven-day period. Scores did not worsen from baseline. The assessment of SF and AP is described in Example 1.

"Corticosteroid-free clinical remission" or "corticosteroid-free remission" means maintenance of clinical remission or maintenance of remission without the use of concomitant corticosteroid therapy.

The "Crohn's Disease Activity Index" score or "CDAI" score is measured by the number of liquid or soft stools, abdominal pain, general feeling of well-being, presence of comorbidities, use of LOMOTIL® or other opioids A composite of eight assessments consisting of diarrhea, presence of abdominal mass, hematocrit and percentage deviation from standard body weight. Further details are described in Example 1.

The "Simplified Endoscopic Score for Crohn's Disease" or "SES-CD" refers to a composite of four assessments, each rated from zero to three, including the size of the ulcer, the proportion of surface covered by the ulcer, and the presence of any other lesions. The proportions of the surface and the presence of narrowing (also called stenosis). Further details are described in Example 1.

As used herein, "baseline" refers to clinical (e.g., signs or symptoms) or laboratory values that describe a patient's condition as determined prior to administration of a certain therapeutic agent, such as an integrin beta7 antagonist (such as evolizumab). . Examples of clinical or laboratory values from which baseline values may be determined include, but are not limited to, hematology and clinical chemistry values (such as hemoglobin, hematocrit, platelet count, sodium, potassium, chloride, etc.), CDAI score, SES-CD score , fluid/soft stool frequency (SF) or abdominal pain (AP) scores, or other patient-reported outcomes (such as IBDQ or CD-PRO/SS).

"Endoscopic improvement" is defined as a decrease in baseline SES-CD score greater than or equal to fifty percent.

“Endoscopic response” is defined as a SES-CD score of four or less (two or less in patients with ileum), excluding subcategory scores greater than one (e.g., ulcer size and extent, affected surface, or narrowing) segment.

"CDAI-70 response" is defined as a decrease in CDAI score of at least seventy points from baseline.

As used herein, "induction therapy" refers to the use of a therapeutic agent or combination of therapeutic agents in an initial regimen to treat the signs and symptoms of Crohn's disease. Induction therapy is usually given for a period of time ranging from about six weeks to about fourteen weeks. Induction therapy may refer to the initial regimen for a patient after an initial diagnosis of Crohn's disease, or after intolerance or poor response to a previous regimen or initial regimen in a clinical trial of a treatment candidate. Exemplary therapeutic agents useful for induction therapy of Crohn's disease include, for example, but are not limited to: 5-aminosalicylates, antibiotics, cortisol, systemic corticosteroids, thiopurines, methotrexate, anti-TNF agents (such as infliximab, adalimumab, and pegylated certolizumab), and other biologics (including but not limited to vedolizumab, ustekinumab, natalizumab , efalizumab and evolizumab), and Janus kinase (JAK) inhibitors (including but not limited to upadatinib and filgotinib).

As used herein, "disease worsening" means that during the induction phase of treatment of a patient with an anti-β7 integrin antagonist (such as evolizumab), the CDAI score ten weeks after the start of treatment is greater than the patient's CDAI score at baseline or week zero. CDAI score.

As used herein, "clinical relapse" means meeting the following criteria at two consecutive visits with a healthcare provider during the maintenance phase of treatment of a patient with an anti-β7 integrin antagonist, such as evolizumab At least one of them and at least one of two consecutive CDAI scores is greater than or equal to 220: (1) CDAI score is greater than or equal to baseline or week zero score; (2) CDAI score is greater than fourteen weeks after the start of treatment has a score greater than or equal to one hundred points.

The term "sample" as used herein refers to a composition obtained or derived from a patient or individual of interest, which includes, for example, cells and/or other molecules that are characterized and/or identified based on physical, biochemical, chemical and/or physiological properties. entity. For example, the phrase "disease sample" and variations thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cellular and/or molecular entities to be characterized. Samples may be obtained from tissue of the individual of interest or from peripheral blood of the individual.

"β7 integrin antagonist" or "β7 antagonist" means any molecule that inhibits one or more biological activities or blocks the binding of β7 integrin to one or more of its related molecules. The antagonists of the present invention can be used to modulate one or more aspects of integrin β7-related effects, including but not limited to association with α4 integrin subunit, association with αE integrin subunit, α4β7 integrin subunit, Binding of nectin to MAdCAM, VCAM-1, or fibronectin and binding of αEβ7 integrin to E-cadherin. These effects may be mediated by any biologically relevant mechanism, including disruption of ligand binding to the β7 subunit or to α4β7 or αEβ7 dimeric integrins, and/or by disruption of the interaction between the α and β integrin subunits. association, thereby inhibiting the formation of dimeric integrins. In one embodiment of the invention, the β7 antagonist is an anti-β7 integrin antibody (or anti-β7 antibody). In one embodiment, the anti-β7 integrin antibody is a humanized anti-β7 integrin antibody, and more specifically a recombinant humanized monoclonal anti-β7 antibody (or rhuMAb β7). In some embodiments, the anti-β7 antibodies of the invention are anti-integrin β7 antagonist antibodies that inhibit or block the binding of the β7 subunit to the α4 integrin subunit, the association with the αE integrin subunit, Binding of α4β7 integrin to MAdCAM, VCAM-1, or fibronectin and αEβ7 integrin to E-cadherin.

"β7 subunit" or "β7 subunit" refers to the human β7 integrin subunit (Erle et al ., (1991) J. Biol. Chem. 266:11009-11016). The β7 subunit associates with the α4 integrin subunit (such as the human α4 subunit) (Kilger and Holzmann (1995) J. Mol. Biol. 73:347-354). α4β7 integrin has been reported to be expressed on most mature lymphocytes and on a smaller number of thymocytes, myeloid cells, and mast cells. (Kilshaw and Murant (1991) Eur. J. Immunol. 21:2591-2597; Gurish et al. , (1992) 149: 1964-1972; and Shaw, SK and Brenner, MB (1995) Semin. Immunol. 7:335 ). The β7 subunit also associates with the αE subunit, such as the human αE integrin subunit (Cepek, K. L, et al. (1993) J. Immunol. 150:3459). αEβ7 integrin is expressed on intestinal epithelial lymphocytes (iIEL) (Cepek, KL (1993) supra ).

"αE subunit" or "αE integrin subunit" or "αE subunit" or "αE integrin subunit" or "CD103" refers to a protein that has been found to be associated with β7 integrin on intraepithelial lymphocytes Integrin subunit, of which αEβ7 integrin mediates iEL binding to intestinal epithelium expressing E-cadherin (Cepek, KL et al. (1993) J. Immunol. 150:3459; Shaw, SK and Brenner, MB (1995) Semin. Immunol. 7:335).

"MAdCAM" or "MAdCAM-1" are used interchangeably in the context of the present invention and refer to the protein mucosal addressin cell adhesion molecule-1, which is a single-chain polypeptide consisting of a short cytoplasmic tail, a transmembrane region and three Extracellular sequence composed of immunoglobulin-like domains. The cDNA of mouse, human and macaque MAdCAM-1 has been cloned (Briskin, et al. , (1993) Nature, 363:461-464; Shyjan et al. , (1996) J. Immunol. 156:2851-2857).

"VCAM-1" or "Vascular Cell Adhesion Molecule-1" "CD106" refers to the ligand of α4β7 and α4β1, which is expressed on activated endothelium and plays a role in endothelial-leukocyte interactions such as leukocyte binding and migration during inflammation. Very important.

"Gastrointestinal inflammatory disorders" are a group of chronic disorders that cause inflammation and/or ulceration in the mucous membranes. Such conditions include, for example, inflammatory bowel diseases ( such as Crohn's disease, ulcerative colitis, indeterminate colitis and infectious colitis), mucositis ( such as oral mucositis, gastrointestinal mucositis, nasal mucositis and rectal mucositis). enteritis), necrotizing enterocolitis, and esophagitis.

"Inflammatory bowel disease" or "IBD" are used interchangeably herein and refer to bowel diseases that cause inflammation and/or ulceration, and include, but are not limited to, Crohn's disease and ulcerative colitis.

"Crohn's disease (CD)" and "ulcerative colitis (UC)" are chronic inflammatory bowel diseases of unknown etiology. Unlike ulcerative colitis, Crohn's disease can affect any part of the intestine. The most prominent feature of Crohn's disease is the granular, reddish-purple edematous thickening of the intestinal wall. As inflammation progresses, these granulomas often lose their outer borders and merge with surrounding tissue. Diarrhea and intestinal obstruction are the main clinical features. Like ulcerative colitis, the course of Crohn's disease can be continuous or relapsing, mild or severe, but unlike ulcerative colitis, Crohn's disease cannot be cured by resection of the affected bowel segment. Most people with Crohn's disease require surgery at some point, but then the disease usually relapses and often requires ongoing medical treatment.

Crohn's disease can involve any part of the digestive tract from the mouth to the anus, but it usually occurs in the ileocolic, small intestinal, or colo-anorectal areas. Histopathologically, the disease manifests as intermittent granulomas, crypt abscesses, dehiscences, and aphthous ulcers. The inflammatory infiltrate is a mixture of lymphocytes (T cells and B cells), plasma cells, macrophages, and neutrophils. There is a disproportionate increase in IgM- and IgG-secreting plasma cells, macrophages, and neutrophils.

The anti-inflammatory drugs sulfasalazine and 5-aminosalicylic acid (5-ASA) are used to treat mildly active colonic Crohn's disease and are often prescribed in an attempt to maintain disease remission. Metronidazole and ciprofloxacin are similar in potency to sulfasalazine and are specifically indicated for the treatment of perianal disease. In more severe cases, corticosteroids are prescribed to treat active exacerbations and sometimes maintain remission. Azathioprine and 6-mercaptopurine have also been used in patients requiring long-term corticosteroid administration. These drugs have been shown to be useful for long-term prevention. Unfortunately, there can be an extremely long delay (up to 6 months) before effect occurs in some patients. Antidiarrheal drugs may also provide symptomatic relief in some patients. Nutritional therapy or elemental diets may improve a patient's nutritional status and induce symptomatic improvement in acute illness, but they do not induce sustained clinical remission. Antibiotics are used to treat secondary small intestinal bacterial overgrowth and to treat purulent complications.

"Effective dose" means the amount effective to achieve the desired therapeutic or preventive outcome at the required dosage and time period.

As used herein, the term "patient" refers to any individual individual in need of treatment. In certain embodiments, the patient herein is a human.

The "individual" in this article is usually a human being. In certain embodiments, the subject is a non-human mammal. Exemplary non-human mammals include laboratory animals, domestic animals, pet animals, sporting animals, and livestock, such as mice, cats, dogs, horses, and cattle. Typically, individuals are eligible for treatment, such as treatment of inflammatory conditions of the gastrointestinal tract.

As used herein, an individual's "lifespan" refers to the individual's remaining lifespan after initiation of treatment.

The terms “poor response,” “intolerance,” and “refractory response” refer to persistent activity despite a history of treatment with one or more therapeutic agents (e.g., corticosteroids, immunosuppressants, and/or anti-TNF) Signs and/or symptoms of disease.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation in a form that permits the biological activity of the active ingredient contained therein to be effective and does not contain other substances that would be unacceptably toxic to the individual to whom the composition is to be administered. components.

"Pharmaceutically acceptable carrier" means an ingredient in a pharmaceutical composition or formulation other than the active ingredient that is not toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies ( e.g., bispecific Antibodies, provided they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). Antibodies can be human, humanized and/or affinity matured antibodies.

An "antibody fragment" encompasses only a portion of an intact antibody, wherein the portion preferably retains at least one of, typically most or all, the functions normally associated with that portion when present in an intact antibody. In one embodiment, the antibody fragment contains the antigen-binding site of an intact antibody and therefore retains the ability to bind antigen. In another embodiment, the antibody fragment, e.g., an antibody fragment comprising an Fc region, retains at least one of the biological functions normally associated with an Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC Function and complement fixation. In one embodiment, the antibody fragment is a monovalent antibody with an in vivo half-life that is substantially similar to that of the intact antibody. For example, such antibody fragments may comprise an antigen-binding arm associated with an Fc sequence capable of conferring stability to the fragment in vivo .

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of antibodies that is substantially homogeneous, that is , the individual antibodies making up the population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigen. Furthermore, each monoclonal antibody system is directed against a single epitope on the antigen, as opposed to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence of an antibody derived from a specific species or belonging to a specific antibody class or subclass, and the chain The remainder is identical or homologous to the corresponding sequences of antibodies and fragments of such antibodies derived from another species or belonging to another antibody class or subclass, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al. , Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

"Humanized" forms of non-human ( eg , murine) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Most humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable region of the recipient are modified by a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate (animal)) with residues in the hypervariable region that have the desired specificity, affinity, and ability. In some cases, human immunoglobulin framework region (FR) residues are substituted with corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not present in the recipient or donor antibodies. These modifications are made to further improve antibody potency. Typically, a humanized antibody will comprise substantially all of at least one (and usually two) variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and All FRs are those of human immunoglobulin lo sequence. The humanized antibody also optionally contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, see Jones et al ., Nature 321:522-525 (1986); Riechmann et al. , Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 ( 1992). See also the following review articles and references cited therein: Vaswani & Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

An "isolated" antibody is one that has been identified and separated and/or recovered from components of its natural environment. Contaminant components of its natural environment are substances that may interfere with the diagnostic or therapeutic use of antibodies and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody (1) is purified to greater than 95% by weight of the antibody, as determined by the Lowry method, and typically greater than 99% by weight; (2) is purified to Sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spin-cup sequencer; or (3) Purified to homogeneity by use of the N-terminal or internal amino acid sequence under reducing or non-reducing conditions. SDS-PAGE was performed using Coomassie blue or silver dye. Isolated antibodies (or constructs) include the antibody in situ within recombinant cells because at least one component of the antibody's natural environment will not be present. Typically, however, isolated antibodies will be prepared by at least one purification step.

As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain that is highly variable in sequence and/or forms structurally defined loops. Typically, antibodies contain six hypervariable regions; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Many descriptions of highly variable regions are in use and are covered herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al ., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In contrast, Chothia refers to the position of a structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariable region represents a compromise between the Kabat CDR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. "Contact" highly variable regions are based on analysis of available complex crystal structures. The residues of each of these HVRs are shown below. Ring Kabat ikB Chothia get in touch with L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat number) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia number) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

The hypervariable region may include the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 49-56 or 50-56 or 52-56 (L2) and 89-97 ( L3), and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al. , supra.

"Framework" or "FR" residues are those variable domain residues other than the highly variable region residues as defined herein.

The "human consensus skeleton" is a skeleton that represents the most common amino acid residues in a series of human immunoglobulin VL or VH skeleton sequences. Typically, human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences. Typically, a subgroup of sequences is as described by Kabat et al . In one embodiment, for VL, the subgroup is subgroup κ I as described by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III as described by Kabat et al .

An "affinity matured" antibody has one or more changes in one or more of its CDRs that result in improved affinity of the antibody for the antigen compared to the parent antibody without these changes. In certain embodiments, affinity matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are generated by procedures known in the industry. Marks et al. Bio/Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described in: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1996); Yelton et al. , J. Immunol. 155:1994-2004 (1995); Jackson et al. , J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889- 896 (1992).

As used herein, the phrase "substantially similar" or "substantially the same" means that two numerical values are similar enough that one skilled in the art would consider them to be identical when measured by those values ( e.g., Kd values) In the context of the biological properties being measured, the difference between the two values has little biological and/or statistical significance.

The term "variable" refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of an antibody. It is concentrated in three segments called hypervariable regions in the light and heavy chain variable domains. The highly conserved part of the variable domain is called the framework region (FR). The variable domains of the native heavy and light chains each contain four FRs, predominantly in a β-sheet configuration, connected by three highly variable regions, which form a loop connecting the β-sheet structure and in some cases forming a β-sheet structure. -Part of the folding structure. The hypervariable regions in each chain are tightly bound together by FR, and together with the hypervariable region of the other chain, help form the antigen-binding site of the antibody (see Kabat et al ., Sequences of Proteins of Immunological Interest , Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as antibodies participating in antibody-dependent cellular cytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-binding fragments, termed "Fab" fragments, each with a single antigen-binding site; and a residual "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment produces an F(ab') ₂ fragment that has two antigen-binding sites and is still capable of cross-linking with antigen.

"Fv" is the smallest antibody fragment that contains complete antigen recognition and antigen-binding sites. This region consists of a dimer of a heavy chain and a light chain variable domain that are tightly, non-covalently associated. In this configuration, the three hypervariable regions of each variable domain interact to define the antigen-binding site on the surface of the _VH - _VL dimer. Six highly variable regions collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or only half of an Fv containing three highly variable domains specific for the antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab= fragments differ from Fab fragments in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH refers to Fab' in which the cysteine residue of the constant domain carries at least one free thiol group. F(ab') ₂ antibody fragments are originally manufactured as pairs of Fab' fragments with a hinge cysteine between them. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies from any vertebrate species can be assigned to one of two distinct classes called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant domains.

Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subtypes (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different types of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and commonly described, for example, in Abbas et al., Cellular and Mol. Immunology , 4th edition (WB Saunders, Co., 2000). An antibody can be part of a larger fusion molecule formed by the covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody that is in its substantially complete form and is not an antibody fragment as defined below. These terms particularly refer to antibodies having a heavy chain containing an Fc region.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc region. Although the boundaries of the Fc region of immunoglobulin heavy chains may vary slightly, the Fc region of human IgG heavy chains is generally defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. For example, the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, compositions of intact antibodies can include populations of antibodies with all K447 residues removed, populations of antibodies with no K447 residues removed, and populations of antibodies with mixtures of antibodies that do and do not contain K447 residues.

Unless otherwise indicated, the numbering of residues in immunoglobulin heavy chains in this document is that of the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest , 5th ed., Public Health Service, National Institutes of Health ( National Institutes of Health), Bethesda, MD (1991), which is expressly incorporated by reference. "EU index such as in Kabat" refers to the residue number of the human IgG1 EU antibody.

"Functional Fc fragments" have the "effector function" of the native sequence Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors ( e.g., B cell receptor; BCR ), etc. Such utility functions typically require binding of the Fc region to a binding domain ( eg , an antibody variable domain) and can be assessed using various assays such as those disclosed herein.

"Native sequence Fc region" includes amino acid sequences that are identical to the amino acid sequences of Fc regions found in nature. Native sequence human Fc region includes native sequence human IgG1 Fc region (non-A and A allotype); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, and naturally occurring variants thereof .

"Variant Fc region" includes an amino acid sequence that differs from the native sequence Fc region due to at least one amino acid modification. In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, e.g. , about one amino acid substitution in the native sequence Fc region or the Fc region of the parent polypeptide. To about ten amino acid substitutions, and in certain embodiments from about one to about five amino acid substitutions. In certain embodiments, a variant Fc region herein is at least about 80% homologous to, or at least about 90% homologous to, a native sequence Fc region and/or an Fc region of a parent polypeptide, or It is at least about 95% homologous to it.

An "amino acid sequence variant" antibody as used herein is an antibody that has an amino acid sequence that is different from the primary species antibody. In certain embodiments, the amino acid sequence variant is at least about 70% homologous to a primary species antibody, or at least about 80% homologous, or at least about 90% homologous to a primary species antibody. Amino acid sequence variants have substitutions, deletions, and/or additions at certain positions in or near the amino acid sequence of the primary species antibody. Examples of amino acid sequence variants herein include acidic variants ( e.g. , desamidine antibody variants), basic variants, having an amine-terminal leader extension on one or both of their light chains ( e.g. , VHS-) Antibodies, antibodies with C-terminal lysine residues on one or both of their heavy chains, etc., and include combinations of variants of the amino acid sequences of the heavy chain and/or light chain. Antibody variants of particular interest herein are antibodies that contain an amine-terminal leader extension on one or both of their light chains, optionally further containing additional amino acid sequences and/or glycosylation differences relative to the primary species antibody.

The term "immunosuppressive agent" or "immunosuppressant" as used herein for adjunctive therapy refers to a substance that acts to suppress or mask the immune system of the individual being treated herein. This would include substances that inhibit cytokine production, downregulate or inhibit self-antigen presentation, or mask MHC antigens. Exemplary non-biological therapeutic agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs); ganciclovir; tacrolimus (tacrolimus); glucocorticoids (such as cortisol or aldosterone); anti-inflammatory agents (such as cyclooxygenase inhibitors); 5-lipoxygenase inhibitors; or leukotriene receptor antagonists; purine antagonists ( such as azathioprine or mycophenolate mofetil (MMF); alkylating agents (such as cyclophosphamide); bromocryptine; danazol; diamine; glutaraldehyde (which masks MHC antigens, as described in U.S. Patent No. 4,120,649); anti-idiotypic antibodies directed against MHC antigens and MHC fragments; cyclosporine; 6-mercaptopurine; steroids (such as corticosteroids or glucocorticoids or glucocorticoid analogs , such as prednisone, methylpenic cortisol, including SOLU-MEDROL.RTM. methylpenic cortisol succinate and dexamethasone); dihydrofolate reductase inhibitors (such as methotrexate (oral or subcutaneous)); antimalarial agents (such as chloroquine and hydroxychloroquine); sulfasalazine; leflunomide; streptokinase; streptococcal enzyme; FK506; RS-61443; chlorambufen acid; deoxyspergualin; rapamycin. Exemplary biologics include cytokine antibodies and cytokine receptor antibodies (e.g., antagonists, including anti-interferon-alpha, -beta, or -gamma antibodies), anti-tumor necrosis factor (TNF)-alpha antibodies (infliximab [e.g., REMICADE]; adalimumab [e.g., HUMIRA®]), anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibody, anti-interleukin-2 (IL-2) antibody and anti-IL-2 receptor antibodies, and interleukin-6 (IL-6) receptor antibodies and antagonists; anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; allogeneic anti-lymphocytes globulin; pan-T antibodies, anti-CD3 or anti-CD4/CD4a antibodies; anti-idiotypic antibodies against MHC antigens and MHC fragments; soluble peptides containing LFA-3 binding domains (WO 90/08187 Published July 26, 1990 ); transforming growth factor-beta (TGF-beta); RNA or DNA from the host; T cell receptor (Cohen et al. , U.S. Patent No. 5,114,721); T cell receptor fragment (Offner et al ., Science, 251: 430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO 91/01133); BAFF antagonists (such as BAFF or BR3 antibodies or immunoadhesins); interfere with T cell helper signaling Biologics, such as anti-CD40 receptor or anti-CD40 ligand (CD154), include blocking antibodies to CD40-CD40 ligand ( e.g. , Durie et al., Science, 261: 1328-30 (1993); Mohan et al ., J. Immunol., 154: 1470-80 (1995)) and CTLA4-Ig (Finck et al ., Science, 265: 1225-7 (1994)); and T cell receptor antibodies (EP 340,109), such as T10B9.

As used herein, "corticosteroid-free" refers to a patient (e.g., a patient with Crohn's disease) who does not use corticosteroids to treat the disease or symptoms of the disease during the patient's corticosteroid-free period. For example, a patient with Crohn's disease who had been corticosteroid-free for 12 months had not used corticosteroids for 12 months to treat symptoms of Crohn's disease.

As used herein, the term "ameliorates" or "amelioration" means the reduction, attenuation or elimination of a condition, disease, disorder or phenotype, including abnormalities or symptoms.

A "symptom" of a disease or condition (such as an inflammatory bowel disease, such as ulcerative colitis or Crohn's disease) means any morbid phenomenon or deviation from normal structure, function, or sensation experienced by an individual that is indicative of the disease.

The expression "therapeutically effective amount" means an amount effective in preventing, ameliorating, or treating a disease or condition (such as an inflammatory bowel disease, such as ulcerative colitis or Crohn's disease). For example, a "therapeutically effective amount" of an antibody refers to an amount of the antibody that is effective in preventing, ameliorating, or treating a particular disease or condition. Similarly, a "therapeutically effective amount" of a combination of an antibody and a second compound refers to an amount of the antibody and the second compound that is effective in combination to prevent, ameliorate, or treat a particular disease or condition.

It should be understood that the term "combination" of two compounds does not mean that these compounds must be administered mixed with each other. Thus, treatment with such combinations or use of such combinations includes mixtures of compounds or separate administration of compounds, and includes administration on the same day or on different days. Thus, the term "combination" refers to the use of two or more compounds alone or in admixture with each other for treatment. For example, when an antibody is administered to an individual in combination with a second compound, whether the antibody and second compound are administered to the individual individually or as a mixture, the antibody is present in the individual and the second compound is also present in the individual. In certain embodiments, a compound other than the antibody is administered before the antibody. In certain embodiments, the compound other than the antibody is administered after the antibody.

For the purposes of this article, "tumor necrosis factor-alpha (TNF-alpha)" means a protein containing a molecule as described in Pennica et al., Nature, 312:721 (1984) or Aggarwal et al., JBC, 260:2345 (1985) Amino acid sequence of human TNF-α molecule.

“TNF-α inhibitors” as used herein are agents that generally inhibit the biological function of TNF-α to a certain extent by binding to TNF-α and neutralizing its activity. Examples of TNF inhibitors specifically considered herein are etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), golimumab ( SIMPONI®) and pegylated certolizumab (CIMZIA®).

"Corticosteroid" refers to any of several synthetic or naturally occurring substances having the general chemical structure of a steroid that mimic or enhance the effects of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, penicillin cortisol (including methyl penicillin cortisol), dexamethasone cortisol, aldehyde cortisol, and bencortisol.

"Antagonist" means a molecule capable of neutralizing, blocking, inhibiting, preventing, reducing or interfering with the activity of a specific or designated protein, including in the case of a ligand its binding to one or more receptors or at the receptor's binding to one or more ligands. Antagonists include antibodies and their antigen-binding fragments, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcription and translation control Sequence etc. Antagonists also include small molecule inhibitors of proteins, as well as fusion proteins that specifically bind to proteins and thereby isolate them from binding to their targets, receptor molecules and derivatives, antagonist variants of proteins, antisense molecules against proteins, RNA adaptations body and protein-targeting ribozymes.

"Self-injection device" means a medical device used for self-administration of a therapeutic agent, such as by a patient or family caregiver. Self-injection devices include auto-injector devices and other devices designed for self-administration.

Various other terms are defined or otherwise characterized herein. Compositions and Methods A. Anti-Integrin β7 Antibodies

In one embodiment, the β7 integrin antagonist is an anti-integrin β7 antibody. 1. Monoclonal antibodies

Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al. , Nature, 256:495 (1975), or can be prepared by recombinant DNA methods (see, eg, U.S. Patent No. 4,816,567).

In the hybridoma approach, mice or other appropriate host animals (e.g., hamsters) are immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing protein. Alternatively, lymphocytes can be immunized ex vivo. After immunization, lymphocytes are isolated and fused to a myeloma cell line using an appropriate fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103) Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable medium that may contain one or more substances that inhibit the growth or survival of the unfused parental myeloma cells (also known as fusion partners). For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), selective media for hybridomas typically include hypoxanthine, aminopterin, and thymidine (HAT media). These substances prevent the growth of HGPRT-deficient cells.

In certain embodiments, the fusion protein partner myeloma cells are cells that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to selective media that select for unfused parental cells. In certain embodiments, the myeloma cell line is a murine myeloma line, such as murine myeloma derived from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA. lines, and SP-2 and derivatives, such as X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human heterologous myeloma cell lines for the production of human monoclonal antibodies have also been described (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al. , Monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, Inc., New York, 1987)).

The medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies against the antigen. In certain embodiments, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard assay described by Munson et al. , Anal. Biochem., 107:220 (1980). Once hybridoma cells producing antibodies with the desired specificity, affinity, and/or activity are identified, clones can be subselected by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies : Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 media. In addition, hybridoma cells can grow in vivo as ascites tumors in the animals, for example by intraperitoneally injecting the cells into mice. By conventional antibody purification methods, such as affinity chromatography ( for example , using protein A or protein G-Sepharose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc., the The monoclonal antibodies secreted by sub-selection are separated from the culture medium, ascites fluid or serum.

DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional methods ( eg , by using oligonucleotide probes capable of binding specifically to genes encoding murine antibody heavy and light chains). Hybridoma cells are used as the source of this DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells (such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody proteins) to Obtain the synthesis of monoclonal antibodies in recombinant host cells. Review articles on the recombinant expression of antibody-encoding DNA in bacteria include: Skerra et al ., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992) .

In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques such as those described by McCafferty et al. , Nature, 348:552-554 (1990). Clackson et al ., Nature, 352:624-628 (1991) and Marks et al ., J. Mol. Biol., 222:581-597 (1991) describe the use of phage libraries to isolate murine and human antibodies, respectively. Subsequent publications described the generation of high affinity (in the nM range) human antibodies by chain shuffling (Marks et al. , Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a means to construct very large Strategies for phage libraries (Waterhouse et al. , Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

The DNA encoding the antibody can be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy and light chain constant domain (CH and CL) sequences for homologous murine sequences (U.S. Patent No. 4,816,567; and Morrison, et al. , Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences may replace the constant domain of an antibody, or they may replace the variable domain of one antigen-binding site of an antibody to produce an antigen-binding site that contains one specific for an antigen and another specific for a different antigen. Chimeric bivalent antibodies with specific antigen-binding sites.

Exemplary anti-integrin beta7 antibodies are Fib504, Fib 21, 22, 27, 30 (Tidswell, M. J Immunol. 1997 Aug 1;159(3):1497-505) or humanized derivatives thereof . Humanized antibodies to Fib504 are disclosed in detail in U.S. Patent Publication No. 20060093601 (issued under U.S. Patent No. 7,528,236), the contents of which are incorporated by reference in their entirety (see also discussion below). 2. Humanized antibodies

The anti-integrin β7 antibodies of the invention may further comprise humanized antibodies. Humanized forms of non-human ( e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ , or other antigen-binding subsequences of an antibody) , which contains minimal sequences derived from non-human immunoglobulins. Humanized Antibodies Antibodies include human immunoglobulins (recipient antibodies) in which residues from the complementarity determining regions (CDRs) of the recipient have been derived from a non-human species (donor antibody) with the required specificity, affinity, and capacity , such as mouse, rat or rabbit or residue substitutions in the CDR. In some cases, Fv backbone residues of human immunoglobulins are substituted with corresponding non-human residues. Humanized antibodies can also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Typically, a humanized antibody will comprise substantially all of at least one (and usually two) variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR The region is the FR region of the human immunoglobulin consensus sequence. The humanized antibody optimally also contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al. , Nature, 321:522-525 (1986); Riechmann et al. , Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and they are typically taken from the "import" variable domain. Humanization can be performed essentially as described by Winter and colleagues [Jones et al ., Nature, 321:522-525 (1986); Riechmann et al. , Nature, 332:323-327 (1988); Verhoeyen et al. , Science , 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of human antibodies. Accordingly, such "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567) in which substantially less than the entire human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues are substituted with residues at similar positions in rodent antibodies. When the antibody is intended for human therapeutic use, the choice of human variable domains (light and heavy chains) used to prepare the humanized antibody is very important to reduce antigenicity and HAMA reactions (human anti-mouse antibodies). According to the so-called "best fit" method, the variable domain sequences of rodent antibodies are screened against the entire genetic library of known human variable domain sequences. Identify the human V domain sequence that is closest to the rodent V domain sequence and accept the human framework region (FR) as the humanized antibody (Sims et al ., J. Immunol. 151:2296 (1993); Chothia et al ., J . Mol. Biol., 196:901 (1987)). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. , Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al. , J. Immunol. 151:2623 (1993)) . More importantly, the antibodies are humanized to retain high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to certain embodiments, humanized antibodies are prepared by analyzing the parental sequence 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 can be used to illustrate and display possible three-dimensional configurations of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e. analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues from the receptor and import sequences can be selected and combined to achieve desired antibody properties, such as increased affinity for the target antigen. In general, the hypervariable region residues are directly and most substantially involved in affecting antigen binding.

Exemplary humanized anti-integrin β7 antibodies include, but are not limited to, rhuMAb β7, also known as ivolizumab, which is a humanized monoclonal antibody directed against integrin subunit β7 and is derived from rat anti- -Mouse/human monoclonal antibody FIB504 (Andrew et al. , 1994 J Immunol 1994;153:3847-61). It is engineered to contain the human immunoglobulin IgG1 heavy chain and kappa 1 light chain framework and is produced from Chinese hamster ovary cells. This antibody binds to two integrins, α4β7 (Holzmann et al. 1989 Cell, 1989;56:37-46; Hu et al. , 1992, Proc Natl Acad Sci USA 1992;89:8254-8) and αEβ7 (Cepek et al. Man , 1993 J Immunol 1993;150:3459-70), which regulate the transport and retention of lymphocyte subsets in the gastrointestinal tract and are involved in inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ( CD). rhuMAb β7 (also known as evolizumab) is an intercellular linker between α4β7 and its ligands (mucosal addressin cell adhesion molecule-1 [MAdCAM]-1, vascular cell adhesion molecule [VCAM]-1, and fibronectin) A potent in vitro blocker of the interaction between αEβ7 and its ligand (E-cadherin). rhuMAb β7 (also known as evolizumab) reversibly binds with similarly high affinity to β7 on lymphocytes from rabbit, cynomolgus monkey, and human. It also binds with high affinity to mouse β7. The amino acid sequence and preparation and use of rhuMAb β7 (also known as evolizumab) and its variants are described in detail, for example, in U.S. Patent Application Publication No. 20060093601 (issued under U.S. Patent No. 7,528,236), the contents of which The entire text is incorporated into this article.

The sequence listing in this document provides sequence information for ivolizumab. As shown in the sequence listing, the heavy chain may lack the C-terminal lysine (HC.v1, SEQ ID NO:11)) or may contain the C-terminal lysine (HC.v2, SEQ ID NO:12). As is well known in the art, the C-terminal amine residue can be trimmed during the manufacturing process. 3. Vectors, host cells and recombinant methods for antibody production

Also provided are isolated nucleic acids encoding anti-β7 antibodies or polypeptide agents described herein, vectors and host cells containing the nucleic acids, and recombinant techniques for producing the antibodies.

For recombinant production of an antibody, the nucleic acid encoding it can be isolated and inserted into a replicable vector for further selection (amplification of DNA) or expression. In another example, antibodies can be produced by homologous recombination, for example , as described in U.S. Patent No. 5,204,244, which is expressly incorporated herein by reference. DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional methods ( eg , by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains). Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a message sequence, an origin of replication sequence, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence, for example , as authorized on July 9, 1996 This is described in issued U.S. Patent No. 5,534,615, which is expressly incorporated herein by reference.

Suitable host cells for colonization or expression of DNA in vectors herein are prokaryotic, yeast or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria (such as Gram-negative or Gram-positive organisms, e.g. Enterobacteriaceae, such as E. coli spp., e.g. E. coli ), Enterobacteriaceae, Erwinia spp. (Erwinia), Klebsiella, Proteus, Salmonella ( e.g. , Salmonella typhimurium), Serratia ( e.g. , Serratia flavonoids), and Shiga Shigella, and Bacillus (such as Bacillus subtilis and Bacillus licheniformis) ( for example , Bacillus licheniformis 41P, disclosed in DD 266,710 on April 12, 1989), Pseudomonas (such as Pseudomonas aeruginosa) ) and Streptomyces spp. One selected host for E. coli is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative rather than restrictive.

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable propagation or expression hosts for vectors encoding anti-β7 integrin antibodies. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as, for example , K. lactis ), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), Vaal K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus (K. marxianus); Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as, for example , Neurospora, Penicillium, Campylobacter (Tolypocladium); and Aspergillus hosts, such as A. nidulans and A. niger.

Suitable host cells for expressing glycosylated anti-beta7 antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants have been identified and have been identified from species such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), black Hosts such as Drosophila melanogaster (Drosophila melanogaster) and Bombyx mori are correspondingly permissive insect host cells. Various viral strains for transfection are publicly available, for example , the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and these viruses can be used as viruses according to the present invention herein, particularly for transfection. Stained Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

However, the greatest interest has been in vertebrate cells, and their propagation in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are: monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) or secondary selection for growth in suspension culture293 cells, Graham et al. , J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al. , Proc. Natl. Acad Sci. USA 77:4216 (1980)); mouse testicular Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney Cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); bovine and mouse liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al. , Annals NYAcad. Sci. 383:44-68 ( 1982)); MRC 5 cells; FS4 cells; and human liver cancer cell line (Hep G2).

Host cells are transformed with expression or selection vectors described above for anti-β7 integrin antibody production and cultured in conventional nutrient media appropriately modified to induce promoters, select transformants, or amplify the desired coding Sequence of genes.

Host cells used to produce anti-β7 integrin antibodies of the invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, Ham et al ., Meth. Enz. 58:44 (1979), Barnes et al ., Anal. Biochem. 1 02:255 (1980), U.S. Patent No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430 ; WO 87/00195; or any culture medium described in US Pat. Re. 30,985 can be used as the host cell culture medium. Any of these media can be supplemented with hormones and/or other growth factors (such as insulin, transgenes, etc.) as needed. ferritin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN.TM. drugs) ), trace elements (defined as inorganic compounds typically present in final concentrations in the micromolar range) and glucose or equivalent energy sources. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture Conditions, such as temperature, pH, etc., are those previously used to select host cells for expression and will be clear and obvious to those skilled in the art.

When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, particulate debris (host cell or lysed fragments) is removed as a first step, for example by centrifugation or ultrafiltration. Carter et al ., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli . Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonate fluoride (PMSF) for approximately 30 min. Cell debris can be removed by centrifugation. When antibodies are secreted into the culture medium, supernatants from such expression systems are typically first concentrated using commercially available protein concentration filters (eg, Amicon or Millipore Pellicon ultrafiltration devices). Protease inhibitors (such as PMSF) can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of exogenous contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a typical purification technique. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ.1, γ.2, or γ.4 heavy chains (Lindmark et al ., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma.3 (Guss et al. , EMBO J. 5:15671575 (1986)). The matrix to which affinity ligands are attached is most commonly agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter preparation times compared to agarose. When the antibody contains the C.sub.H3 domain, Bakerbond ABX.TM.resin (JT Baker, Phillipsburg, NJ) can be used for purification. Depending on the antibody to be recovered, other techniques for protein purification can also be used (such as fractionation on ion exchange columns, ethanol precipitation, reversed phase HPLC, silica column chromatography, chromatography on heparin SEPHAROSE.TM, Chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatographic focusing, SDS-PAGE and ammonium sulfate precipitation). After any preliminary purification steps, the mixture containing the antibodies of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer with a pH between about 2.5-4.5, typically at low salt concentrations ( e.g. , from Approximately 0-0.25 M salt) is carried out. 4. Specific therapeutic agents

Provided herein are therapeutic agents for the treatment of Crohn's disease. In one embodiment, the therapeutic agent is an anti-integrin β7 antibody, also known as evolizumab. Evolizumab as an IgG1 antibody. In one embodiment, the anti-integrin β7 antibody comprises three heavy chain HVRs: HVR-H1 (SEQ ID NO:4), HVR-H2 (SEQ ID NO:5), and HVR-H3 (selected from SEQ ID NO: :6 and SEQ ID NO:7). In one embodiment, the anti-integrin β7 antibody comprises three light chain HVRs: HVR-L1 (SEQ ID NO:1), HVR-L2 (SEQ ID NO:2), and HVR-L3 (SEQ ID NO:3 ). In one embodiment, the anti-integrin β7 antibody includes three heavy chain HVRs and three light chain HVRs: HVR-H1 (SEQ ID NO:4), HVR-H2 (SEQ ID NO:5), and HVR-H3 (Selected from SEQ ID NO:6 and SEQ ID NO:7), HVR-L1 (SEQ ID NO:1), HVR-L2 (SEQ ID NO:2) and HVR-L3 (SEQ ID NO:3). In one embodiment, the anti-integrin β7 antibody comprises: a variable heavy chain region VH having the amino acid sequence of SEQ ID NO:9. In some embodiments, an anti-integrin β7 antibody comprises: a variable light chain region VL having the amino acid sequence of SEQ ID NO:8. In one embodiment, the anti-integrin β7 antibody comprises: a variable heavy chain region VH having the amino acid sequence of SEQ ID NO: 9; and a variable light chain region VL having the amino acid sequence of SEQ ID NO: 8 Amino acid sequence. In one embodiment, the anti-integrin β7 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. In one embodiment, the anti-integrin β7 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 10. In one embodiment, an anti-integrin β7 antibody comprises: a heavy chain having an amino acid sequence selected from SEQ ID NO: 11 and SEQ ID NO: 12; and a light chain having SEQ ID Amino acid sequence of NO:10. Anti-integrin β7 antibodies are further described in Intn’l Pub. No. 2006/026759.

In another aspect, an anti-integrin β7 antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, identical to the amino acid sequence of SEQ ID NO: 11 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical contains substitutions relative to the reference sequence (eg conservative substitutions), insertions or deletions, but anti-integrin β7 antibodies containing this sequence retain the ability to bind to human integrin β7. In certain embodiments, a total of 1 to 10 amino acids are substituted, altered, inserted and/or deleted in SEQ ID NO: 11. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (i.e., in the FR). The anti-integrin β7 antibody contains the VH sequence of SEQ ID NO:11 or SEQ ID NO:12, as appropriate, including post-translational modifications of this sequence.

In another aspect, an anti-integrin β7 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) that shares at least 90%, 91%, 92% with the amino acid sequence of SEQ ID NO: 10 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions relative to the reference sequence (eg conservative substitutions), insertions or deletions, but anti-integrin β7 antibodies containing this sequence retain the ability to bind to human integrin β7. In certain embodiments, in SEQ ID NO: 10, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (i.e., in the FR). Optionally, the anti-integrin β7 antibody contains the VL sequence of SEQ ID NO:10, including post-translational modifications of this sequence.

In yet another embodiment, the anti-integrin β7 antibody comprises: a VL domain that is at least 90%, 91%, 92%, 93%, 94%, 95%, identical to the amino acid sequence of SEQ ID NO: 10. 96%, 97%, 98%, 99% or 100% sequence identity; and a VH domain that has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO:11 , 95%, 96%, 97%, 98%, 99% or 100% sequence identity. B.Pharmaceutical preparations

Therapeutic formulations containing the therapeutic agents, antagonists or antibodies of the invention are prepared by mixing the antibody with the desired purity and optionally a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th ed., Osol, A. Ed. (1980)), in the form of aqueous solutions, lyophilized or other dry formulations. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations employed and include: buffering agents such as phosphates, citrates, histidine and other organic acids; antioxidants, Including ascorbic acid and methionine; preservatives (such as stearyldimethylbenzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; benzalkonium chloride; bensonine chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol ); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, gluten Amino acids, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose or Polyethylene glycol (PEG).

The formulations described herein may also contain more than one active compound as required for the particular indication being treated, typically such compounds having complementary active ingredients without adversely affecting each other. Such molecules are suitably present in combination in amounts effective for the intended purpose.

Preparations for in vivo administration must be sterile. This is easily accomplished through sterile membrane filtration. C. invest

Administration of the anti-integrin β7 antibody and any second therapeutic compound can be performed simultaneously, for example, as a single composition or as two or more different compositions using the same or different routes of administration. Alternatively or additionally, administration may be performed sequentially in any order. In certain embodiments, there may be intervals from minutes to days, to weeks to months, between administrations of two or more compositions. For example, an anti-integrin β7 antibody can be administered first, followed by a second therapeutic compound. However, administration of a second therapeutic compound concurrently with or prior to the anti-integrin β7 antibody is also contemplated.

The standard of care for individuals with moderately to severely active CD includes treatment with standard doses of: systemic corticosteroids, such as prednisone (or prednisone equivalent) or corticosteroids; immunosuppressants Such as azathioprine, 6-mercaptopurine or methotrexate; or tumor necrosis factor inhibitors (anti-TNF) such as infliximab, adalimumab or pegylated certolizumab. Other antiintegrin therapies have been approved for the treatment of CD, and they are natalizumab and vedolizumab.

In one embodiment, the present invention treats Crohn's disease (CD) in a human subject with CD comprising administering to the subject an effective amount of a therapeutic agent (such as an anti-β7 integrin antibody), and further comprising administering to the subject Administer an effective amount of a second drug, i.e., immunosuppressant, corticosteroid, anti-TNF, analgesic, antidiarrheal, antibiotic, or combination thereof.

In an exemplary embodiment, the second drug is selected from the group consisting of: 6-mercaptopurine, azathioprine, methotrexate, prednisone (or prednisone equivalent), cortisol alcohol, infliximab, adalimumab, and pegylated certolizumab.

All these second drugs can be used in combination with each other or alone with the first drug, so the expression "second drug" used herein does not mean that it is the only drug in addition to the first drug. Accordingly, the second drug need not be one drug but may constitute or contain more than one such drug.

Co-administration as used herein includes co-administration, use of separate formulations or single drug formulations, and sequential administration in any order, where there is usually a phase in which both (or all) active agents (drugs) exert their biological activity simultaneously. time period.

Combination administration of a second drug includes co-administration (simultaneous administration), use of separate formulations or single drug formulations, and sequential administration in any order, where usually one of the two (or all) active agents (drugs) is present The time period during which it exerts its biological activity at the same time. E. Design a treatment plan

Drug development is a complex and expensive process. The cost of bringing a new drug to market is estimated to be between $800 million and $1 billion. Less than 10% of drugs in Phase I clinical trials advance to approval. Two key reasons for late-stage drug failure are a lack of understanding of the relationship between dose-concentration response and unexpected safety events. Given this situation, it is important to have enabling tools that can help predict how drugs will work in vivo and aid the success of clinical treatment candidates (Lakshmi Kamath , Drug Discovery and Development; Modeling Success in PK/PD Testing Drug Discovery & Development (2006)).

Pharmacokinetics (PK) characterizes the absorption, distribution, metabolism, and elimination characteristics of a drug. Pharmacodynamics (PD) defines the physiological and biological responses to administered drugs. PK/PD modeling establishes mathematical and theoretical links between these two processes and helps to better predict drug effects. Integrated PK/PD modeling and computer-aided experimental design through simulation are being incorporated into many drug development programs and are having an increasing impact (Lakshmi Kamath , Drug Discovery and Development; Modeling Success in PK/PD Testing Drug Discovery & Development (2006)).

PK/PD testing is typically performed at every stage of the drug development process. As development becomes increasingly complex, time-consuming and cost-intensive, companies want to better leverage PK/PD profiles to eliminate flawed drug candidates at the outset and identify those most likely to achieve clinical success . (Lakshmi Kamath, see above ).

PK/PD modeling approaches have proven useful in determining relationships between biomarker responses, drug levels, and dosing regimens. The PK/PD profile of a drug candidate and the ability to predict patient response to it are critical to the success of clinical trials. Recent advances in molecular biology techniques and a better understanding of targets in various diseases have confirmed biomarkers as good clinical indicators of drug therapeutic efficacy. Biomarker assays help identify biological responses to drug candidates. Once a biomarker is clinically validated, trial simulations can be effectively modeled. Biomarkers have the potential to achieve surrogate status and may one day surrogate clinical outcomes in drug development. (Lakshmi Kamath , see above ).

The amount of biomarkers in the peripheral blood can be used to identify the biological response to integrin β7 antagonist treatment and therefore can serve as a good clinical indicator of the therapeutic efficacy of the candidate treatment.

Traditional PK/PD modeling in drug development defines parameters such as drug dose concentration, drug exposure effects, drug half-life, drug concentration over time, and drug effect over time. When used more broadly, quantitative techniques such as drug modeling, disease modeling, trial modeling and market modeling can support the entire development process, allowing better decisions to be made through explicit consideration of risk and better utilization of knowledge. Several PK/PD modeling tools are available to drug development researchers, such as WinNonlin and the Knowledgebase Server (PKS), developed by Pharsight, Inc., Mountain View, CA.

The foregoing written description and the following examples are believed to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and the following examples, and such modifications are within the scope of the appended claims. .

It will be understood that application of the teachings of the present invention to a particular problem or situation will be within the ability of one of ordinary skill in the art in light of the teachings contained herein.

Other details of the invention are illustrated by the following non-limiting examples. The disclosures of all cited documents in the specification are expressly incorporated herein by reference. Examples Example 1 A Phase III , randomized, double-blind, placebo-controlled, multicenter study to evaluate the efficacy and safety of evolizumab as induction and maintenance therapy in patients with moderately to severely active Crohn's disease Research reason for research

The purpose of this study is to evaluate the efficacy and safety of evolizumab, an anti-integrin with a unique mechanism of action (MOA) that has been shown to destroy α4β7/MAdCAM-1 and αEβ7/ E-cadherin binds, thereby inhibiting the trafficking and retention of inflammatory T cells in the intestinal mucosa.

Although evolizumab has not been studied as maintenance therapy in people with CD, the pharmacology of evolizumab on intestinal CD4+ and CD8+ T cells isolated from patients with UC and resected patients with CD Preliminary performance studies of the target (integrin beta7 receptor) indicate similar levels of performance between the two diseases. The reported efficacy of vedolizumab (anti-α4β7 mAb) in CD demonstrates the role of α4β7 in the pathobiology of the disease (Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013) and follows from It was concluded from these studies that evolizumab would be effective in CD. In fact, results from an induction study (cohort 1 described below) demonstrated that evolizumab treatment demonstrated clinically meaningful endoscopic improvements compared with placebo 14 weeks after treatment initiation. Evolizumab-related symptom relief was observed as early as week 6 and continued through week 14 (Sandborn et al., United Eur Gastroenterol J. 2017;5(8):1138–50). Furthermore, because αEβ7 + expression has been reported to be elevated in CD patients (Elewaut D, et al., Acta Gastroenterol Belg 61:288-94, 1998; Oshitani N, et al., Int J Mol Med 12:715-9, 2003 ) and observed increased manifestations from distal to proximal intestinal tract, the dual MOA of evolizumab improves response to disease without the need for systemic immunosuppression compared with existing anti-integrin and anti-TNF therapies. Efficacy of CD.

In a global phase II study in patients with UC, evolizumab was effective in the treatment of moderate to severe UC and was 105 mg every 4 weeks [Q4W] (100 mg nominal dose) 10 weeks after treatment initiation. ) achieved a placebo-adjusted clinical response rate of 20.5% (p = 0.058) and an endoscopic response rate of 10.3% (p = 0.004) in the all-comer population (Vermeire S, et al., Gastroenterology 144,S1: S-36, 2013; Vermeire S, et al., Lancet 384: 309–18, 2014; Lin et al., Gastroenterology 146:307–315, 2014). In addition, evolizumab had an acceptable safety profile, with no clinically significant safety signals observed.

Although the above phase II results in UC are encouraging, topline results from five phase III studies in UC have been mixed and disappointing (Roche press release, August 10, 2020, available at www(dot)roche( dot)com). Of note, evolizumab met its primary endpoint of induction of remission compared with placebo in only two of the three induction studies ( ibid. ). Additionally, evolizumab failed to meet its primary endpoint (remission) compared with placebo as maintenance treatment in patients with ulcerative colitis ( ibid. ). Therefore, the efficacy of evolizumab therapy in inducing remission in UC in some studies failed to predict the efficacy of evolizumab as maintenance therapy in UC.

Likewise, encouraging results were previously reported in a CD study comparing the efficacy of evolizumab versus placebo in inducing remission (as noted above (Sandborn et al., United Eur Gastroenterol J. 2017;5( 8):1138–50)) The efficacy of evolizumab treatment in maintaining remission, including maintaining remission for at least one year, cannot be predicted. Therefore, the study design and endpoints discussed below, as well as the planned analysis of clinical outcomes, were designed to understand the efficacy of evolizumab treatment as induction versus maintenance therapy. research design

This will be a multicenter, phase III, double-blind, placebo-controlled study to evaluate the efficacy, safety and efficacy of evolizumab compared with placebo during induction and maintenance treatment of moderately to severely active CD. tolerance. This study was conducted in accordance with the recommendations of the European Medicines Agency (EMA) Guidance on New Medicinal Products for CD (CPMP/EWP/2284/99 Rev. 1) and the U.S. Food and Drug Administration (FDA)-approved registration trials for CD therapies. Induction and maintenance therapy were combined in a rerandomization/withdrawal design to evaluate the efficacy and safety of evolizumab treatment in inducing and maintaining remission.

The study design will consist of 1) a screening period (up to 35 days) to determine whether patients are eligible to participate in the study, 2) an induction period (14 weeks), and then 3) a maintenance period (52 weeks) to target patients who exhibit symptoms at the end of the induction period. patients with a CDAI-70 response (defined as a reduction of at least 70 points from baseline CDAI score), and 4) the safety follow-up phase (12 weeks), after the last dose of study drug during the maintenance phase, open-label for those not participating in the study Expansion Part 1 of patients receiving evolizumab (see Figures 1 and 2). Upon completion of the safety follow-up phase, patients will be asked to enter a 92-week extended PML monitoring phase (open-label extension study). An independent Data Monitoring Committee (iDMC) will continue to monitor safety and study conduct.

Patients will have moderately to severely active CD, as defined during the screening phase by clinical signs and symptoms that result in a CDAI score between ≥ 220 and ≤ 480, which will be determined at randomization Calculated on the same day, electronic diary PRO information is required for at least 4 days in the 7 days before randomization; and (a) mean bowel movement frequency (SF) > 6 or (b) mean SF > 3 and mean abdominal pain (AP) > 1, use Electronic diary (e-diary) patient-reported outcome (PRO) data for the 7 days before randomization were calculated on the day of randomization. Active inflammation is also required, defined as a SES-CD score ≥ 7, or in the case of solitary ileitis or following ileocecal resection, a score ≥ 4, as determined by screening ileocolonoscopy using central reading model scoring. Check to confirm.

The study population will include patients refractory to or intolerant to one or more of the following therapies: 1) corticosteroids (CS), 2) immunosuppressants (IS), or 3) anti-tumor necrosis factor (TNF) ( or poor response to anti-tumor necrosis factor [TNF-IR]). Patients enrolled based on refractory or intolerance to CS and/or IS lines may or may not have previously received anti-TNF.

Approximately 1,150 patients will be randomized into the study from approximately 420 global study sites by joining one of three cohorts. Recruitment will be done sequentially, first into Cohort 1, then Cohort 2, and finally into Cohort 3. screening stage

Patients will be assessed for eligibility during the 35-day screening period (see Figures 1 and 2). The main eligibility criteria are as follows.

During the screening phase, patients receiving CS must have been taking a stable dose of ≤ 20 mg/day prednisone (or equivalent) or ≤ 6 mg/day oral cortisol for at least 2 weeks immediately before randomization. Likewise, eligible patients requiring background IS therapy (eg, azathioprine [AZA] or equivalent, 6-mercaptopurine [6-MP] or equivalent, or methotrexate [MTX]) must be treated immediately Receive a stable IS dosage regimen for at least 8 weeks before randomization. Patients who had received anti-TNF therapy had to discontinue that therapy for at least 8 weeks before randomization.

Ileocolonoscopy should be performed during the screening phase and at least 9 days before randomization to allow sufficient time for central reader scoring and determination of eligibility and to avoid the impact of ileocolonoscopy bowel preparation for determining baseline SF, AP and patient-reported outcomes by CDAI score (i.e., abdominal pain, overall sense of well-being, and bowel movement frequency). induction period

Eligible patients will sequentially join one of three cohorts for a 14-week induction period (see Figure 1).

Patients enrolled in Cohort 1 (double-blind, placebo-controlled, exploratory cohort; n = 300) will be randomized in a 1:2:2 ratio at weeks 0, 2, 4, 8, and 12 of the 14-week induction period. Receive placebo, evolizumab 105 mg SC Q4W (low dose), or evolizumab 210 mg SC (high dose) (note that patients randomized to low dose evolizumab will receive receive placebo injections - see below). Patients enrolled in Cohort 2 (evolizumab dose-blind, active treatment cohort; n = 350) will be randomized in a 1:1 ratio to receive a low-dose or high-dose regimen of evolizumab. Patients enrolled in Cohort 3 (double-blind, placebo-controlled, pivotal cohort; n = 500) will be randomized in a 2:3:3 ratio to receive placebo or low- or high-dose evolizumab. To maintain blinding because the low and high doses of evolizumab are in syringes of different sizes, patients in all three cohorts will receive two injections at weeks 0, 4, 8, and 12. Patients randomized to low-dose evolizumab will receive one injection of placebo (matched to the high-dose prefilled syringe) and one injection of low-dose evolizumab at each dose, except at Week 2, when they Will receive a placebo injection. Patients randomized to high-dose evolizumab will receive one injection of placebo and one high-dose evolizumab at each dose, except at Week 2, when they will receive a high-dose injection. Finally, patients randomized to placebo will receive two placebo injections at each dose, except at week 2, when they will receive one placebo injection.

Randomization in all cohorts will be based on concomitant oral CS treatment (yes vs. no), concomitant IS treatment (yes vs. no), baseline CDAI ≤ 330 (yes vs. no), and prior exposure to anti-TNF (yes vs. No) to layer. Enrollment will be managed to ensure that no more than approximately 60% of patients in Cohort 3 are exposed to TNF and no more than approximately 10% of patients in each cohort have a CDAI score between > 450 and ≤ 480 .

During the induction phase, patients in all cohorts must keep their CS and IS treatment doses stable (if CS/IS at baseline is required). Increasing the dose of these medications would be considered first aid therapy. Additionally, every effort should be made to keep antidiarrheal medications at a fixed dose, if necessary. The effect of titrating antidiarrheal drugs on placebo response rates in SF and AP has not been studied in the moderately to severely active CD population; dose adjustments may confound interpretation of the data. Any changes in concomitant medications must be documented on the electronic case report form (eCRF).

Between weeks 10 and 14 (inclusive), there is an option to move to the open-label extension (OLE) study (Part 1), in which patients can receive open-label AIDS. Rozizumab. It can only be performed if the patient experiences disease exacerbation, defined as a CDAI week 10 score that is higher than the patient's baseline (week 0) score.

At week 14, patients who achieve a CDAI-70 response without rescue therapy will continue into the maintenance phase until a sample size of approximately 480 patients is reached to join the maintenance phase. Patients who are not eligible for the maintenance phase may be eligible to participate in the Part 1 open-label extension (OLE) study. Patients who are in the induction phase after completion of enrollment into the maintenance phase may join Part 1 ( OLE) study (if eligible).

Patients requiring surgical intervention for CD at any time during the induction phase will have study treatment discontinued, enter the safety follow-up phase, and will be required to enter the Part 2 Safety Monitoring (SM) study for PML monitoring. Patients who spontaneously withdraw from the induction phase and do not meet eligibility criteria for OLE treatment will also enter the safety follow-up phase and will be required to enter the Part 2 (SM) study for PML monitoring. Concomitant CD therapy during induction phase

Concomitant treatments included any medications (e.g., prescription, over-the-counter, herbal or homeopathic remedies, nutritional supplements) used by the patient from 4 weeks before screening until the study completion/early termination visit. Antidiarrheal medications are allowed if used to control chronic diarrhea, but every effort should be made to keep the dose/regimen stable. Investigators and/or patients must document any changes in dose/regimen after baseline (Week 0). Occasional use of nonsteroidal anti-inflammatory drugs (NSAIDs) or acetaminophen for pain relief (e.g., in the setting of headache, arthritis, myalgia, etc.) is allowed. Prophylactic aspirin is also allowed, up to 325 mg/day. Patients should not take any medications for the treatment of ongoing signs and symptoms of CD that are contraindicated with evolizumab; these include, but are not limited to: anti-integrins, anti-adhesion molecules (e.g., anti-MAdCAM-1), drugs other than AZA and T- or B-cell depleting agents (or equivalents) other than 6-MP, TNF antagonists, IL-23 ± IL-12 antagonists (such as ustekinumab), antimetabolites, cyclosporine and tacrolimus.

CS enemas/suppositories and/or topical (rectal) 5-aminosalicylate (5-ASA) preparations are also prohibited. Patients taking these drugs will be considered non-responders for the purposes of any endoscopic analysis. These treatments must be discontinued, but patients may continue receiving evolizumab and may still be eligible to participate in the Part 1 (OLE) study. First aid therapy during the induction period

This was defined as medications prescribed for new or worsening CD symptoms and included any new CS or IS therapy for CD or any increase in the dose or regimen of baseline Crohn's medications. Antibiotics, 5-ASA, antidiarrheals, probiotics, herbal/ayurvedic, nutritional and homeopathic supplements are not considered first aid treatments. Patients requiring rescue medication during the induction phase will be considered non-responders to the primary analysis and will not be eligible for the maintenance phase. maintenance period

At the end of the induction period (week 14), patients will be assessed for CDAI score and will undergo a comprehensive endoscopy (ileocolonoscopy) with central readings to determine SES-CD score. Every effort must be made to schedule the ileocolonoscopy at the 14th week visit or no later than 5 calendar days after this visit; the procedure may not be scheduled before the 14th week. Electronic diary data on patient-reported outcomes (i.e., abdominal pain, overall comfort, and bowel movement frequency, recorded 7 days before bowel preparation) will be used to calculate SF, AP, and CDAI scores at week 14, thereby eliminating the impact of bowel preparation on these outcomes any impact.

Patients who receive placebo during the induction phase and achieve a CDAI-70 response will receive pseudorandomization to blinded placebo treatment during the maintenance phase. Patients who receive evolizumab and achieve a CDAI-70 response at Week 14 and are not using rescue therapy will be randomized to the maintenance phase to receive placebo or evolizumab 105 mg SC Q4W in a 1:1 ratio ( See Figure 2). This will continue until a sample size of approximately 480 patients is reached to join the maintenance phase.

Randomization calls may occur between Week 14 (the last visit of the induction phase) and Week 16 if the patient has been assessed as eligible for the maintenance phase. Randomization will be based on CDAI response at weeks 10 and 14 (yes vs. no), induction dose regimen (low dose vs. high dose), concomitant oral CS therapy (yes vs. no), and prior exposure to anti-TNF ( yes vs. no) for stratification. The first dose of the maintenance phase is administered at the week 16 clinic visit.

Patients should remain on stable doses of IS therapy throughout treatment unless dose reduction or discontinuation is required due to drug-related toxicity. CS dosage should be tapered starting at week 14. Patients who are unable to tolerate CS tapering without CD symptoms or recurrence of steroid discontinuation symptoms may have their CS dose increased, but should not exceed the dose administered at randomization. The dose tapering regimen must be restarted within 2 weeks.

During the maintenance phase, patients who experienced clinical relapse had the option to transfer to the Part 1 (OLE) study. Clinical relapse was defined as meeting at least one of the following criteria on two consecutive visits (which may include unscheduled visits) and at least one of two consecutive CDAI scores ≥220: CDAI score ≥ baseline (0 week) score or CDAI score is ≥100 points higher than the score in week 14.

All patients who complete their final maintenance visit at Week 66 are eligible for enrollment in the Part 1 (OLE) study. Patients not enrolled in Part 1 (OLE) will enter a 12-week safety follow-up phase, after which they will be asked to enroll in the 92-week extended PML monitoring phase (Part 2 [SM] study). Patients requiring surgical intervention for CD at any time during the maintenance phase will discontinue study treatment, enter the safety follow-up phase, and will be required to enter the Part 2 (SM) study for PML monitoring. Patients who self-discontinue from the maintenance phase and do not meet OLE treatment eligibility criteria will also enter the safety follow-up phase and will be required to enter the Part 2 (SM) study for PML monitoring. Patients who withdraw will complete the early withdrawal assessment listed in the assessment schedule; patients who withdraw will not be replaced. Management of concomitant CD therapy in the maintenance phase

During the maintenance phase, corticosteroid doses should be tapered starting at Week 14 according to the following schedule: ≤20 mg/day prednisone (or equivalent) with dose adjustments by 2.5 mg weekly dose reductions until discontinued; ≤6 mg/day oral cortisol, with dose adjustments by reducing the dose by 3 mg every 2 weeks until discontinued.

If necessary, patients may increase their corticosteroid dose to their baseline dose (i.e., dose at randomization), but the dose tapering regimen should be restarted within 2 weeks. Patients receiving concomitant IS therapy (AZA, 6-MP, MTX) must remain on stable doses throughout the study unless dose reduction or discontinuation is required due to drug-related toxicity. The dose of antidiarrheal medication should also be kept stable.

Accepted criteria for discontinuation of IS due to toxicity include, but are not limited to, acute pancreatitis, severe leukopenia, severe thrombocytopenia, or significant elevations from baseline in liver-related enzymes, particularly in the setting of elevated total bilirubin. The final decision to discontinue IS remains at the discretion of the investigator. maintenance first aid therapy

This is defined as medications prescribed for new or worsening CD symptoms and includes: any new CS or IS therapy for CD; increasing the dose of IS therapy above the baseline (week 0) dose; increasing the dose of CS therapy Increase dose above patient's baseline (week 0) dose (for patients requiring CS at baseline). Antibiotics, 5-ASA, antidiarrheals, probiotics, herbal/ayurvedic, nutritional and homeopathic supplements are not considered first aid treatments. Patients requiring rescue therapy during the maintenance phase may enter the Part 1 OLE study early (if eligible) and will be asked to complete an early exit visit assessment. Contraindicated therapies are as described in the induction phase. Study drug or placebo administration

Patients will receive evolizumab or placebo via SC injection according to their treatment assignment. For the entire study, patients and investigators were blinded to study drug assignment (or evolizumab dose assignment for induction cohort 2 patients). During the induction phase, study drug will be administered at the study site, and patients will be trained to self-administer. A healthcare professional (HCP) will administer the first two doses, and patients or their caregivers will administer subsequent doses under the supervision of the HCP. Upon completion of the maintenance period after Week 16, patients may elect to return to the study site or self-administer/have a caregiver administer their dose of Q4W at home within ±3 days of their scheduled dose, unless a PK blood draw is planned, at which time In this case, the dose must be administered on the day of the visit after the blood is drawn or within 3 days of the visit. outcome measure; efficacy target

Analysis of clinical response will be based on patient-reported fluid/soft stool frequency (SF) and abdominal pain (AP) values (derived from 7-day average scores) and centrally read, clinician-reported endoscopic improvement values, which are As assessed by the Simplified Endoscopic Score for Crohn's Disease (SES-CD), which is the sum of four endoscopic variables assessed in the five ileocolic segments of the intestine (Daperno et al., Gastrointest Endosc 2004;60: 505-12). Co-primary efficacy objective

The co-primary efficacy endpoints of this study are as follows: (1) Clinical remission, defined as the mean daily score of SF ≤3 and the mean daily score of AP ≤1, averaged over the 7-day period before the visit, any subscore compared with baseline compared with no worsening; and (2) endoscopic improvement, defined as a ≥50% decrease in SES-CD from baseline.

The primary efficacy objectives of this study will be analyzed separately for the induction and maintenance phases, as described below. Induction Phase: The efficacy of the evolizumab dose regimen in inducing clinical remission and endoscopic improvement compared with placebo was independently assessed at the end of the induction phase (week 14). Maintenance Phase: For patients who achieve a Crohn's Disease Activity Index (CDAI)-70 response (defined as a CDAI reduction of at least 70 points from baseline) at Week 14, Aiello is independently assessed at 1 year of maintenance therapy (Week 66) Lizumab achieved clinical remission and endoscopic improvement compared with placebo. secondary goals

The secondary objectives of the induction phase of this study are as follows: (1) to evaluate the efficacy of evolizumab compared with placebo in achieving clinical remission at week 6; (2) to evaluate the efficacy of evolizumab compared with placebo in achieving clinical remission at week 6; Efficacy achieved at Week 14 with SES-CD ≤4 (≤2 for ileal patients), excluding segments with subcategory scores >1 (i.e., for ulcer size and extent, affected surface, or narrowing); and ( 3) To assess the reduction in signs and symptoms of CD achieved by the evolizumab dosing regimen compared with placebo at week 14 as measured by Crohn's disease patient-reported outcome signs and symptoms (CD-PRO/SS ) indicator evaluation.

Unless otherwise stated, for patients who achieve CDAI-70 at Week 14, the secondary objectives of the maintenance phase of this study are as follows: (1) For patients who achieve clinical remission at Week 14, evaluate the efficacy of evolizumab versus Efficacy of evolizumab compared with placebo in maintaining clinical remission at Week 66; (2) Evaluate the efficacy of evolizumab compared with placebo in achieving corticosteroid-free clinical remission at Week 66 in patients receiving corticosteroids at baseline (3) For patients who achieve endoscopic improvement at week 14, evaluate the efficacy of evolizumab compared with placebo in maintaining endoscopic improvement at week 66; (4) Evaluate the efficacy of evolizumab in maintaining endoscopic improvement at week 66; (4) Lizumab versus placebo achieved SES-CD ≤4 (≤2 for ileal patients) at Week 66, excluding subcategory scores with >1 (i.e., for ulcer size and extent, affected surface, or narrow (5) Evaluate the efficacy of evolizumab compared with placebo in achieving durable clinical remission during 1 year of maintenance therapy (i.e., at weeks 24, 28, and 32 of the maintenance period , ≥ 4 of 6 in-clinic assessment visits at Weeks 44, 56, and 66); (6) among patients receiving corticosteroids at baseline, no corticosteroid clinical response at Week 66 (corticosteroids were discontinued for at least 24 weeks before week 66); and (7) changes from baseline to week 66 in CD signs and symptoms as assessed by the CD-PRO/SS index were assessed. exploratory goals

The exploratory efficacy objectives of this study were as follows: (1) to evaluate the efficacy of evolizumab compared with placebo in reducing fecal calprotectin levels at the end of the induction and maintenance phases; (2) to evaluate the efficacy of evolizumab compared with placebo Efficacy compared with placebo in reducing C-reactive protein (CRP) levels at the end of the induction and maintenance phases; (3) Assessing the time from week 14 to experiencing a major CD-related event (including hospitalization, bowel surgery, and non-study procedures) time; (4) evaluate the effect of evolizumab on drainage fistula closure compared with placebo; (5) evaluate the efficacy of evolizumab compared with placebo in achieving SES-CD 0 at week 66; (6) Determine the efficacy of evolizumab in achieving histological improvement at the end of the induction and maintenance phases compared with placebo; (7) Evaluate the efficacy of evolizumab in achieving CDAI at week 66 compared with placebo The efficacy of remission; (8) Assess changes from baseline to week 14 in patient-reported health-related quality of life (HRQOL) as assessed by the Inflammatory Bowel Disease Questionnaire (IBDQ); (9) Assess changes from baseline to week 66 Changes in patient-reported HRQOL as assessed by IBDQ; (10) Evaluate erolizumab vs. Placebo comparison.

In addition to the analysis targeting the above objectives, other analyzes of CDAI will be conducted. In addition, analysis of resource utilization and other patient-reported outcomes will be performed using tools including, but not limited to, the EuroQOL five-dimensional questionnaire (EQ-5D). security goals

The safety objectives of this study are to evaluate the overall safety and tolerability of evolizumab compared with placebo during the induction therapy period and maintenance therapy period; to evaluate the incidence and severity of infection-related adverse events; to evaluate malignant tumors to assess the incidence and severity of immunogenic reactions (antitherapeutic antibodies [ATA]); and to assess the incidence and severity of hypersensitivity events. pharmacokinetic targets

The pharmacokinetic objective of this study was to evaluate evolizumab serum concentrations at several predose time points during the induction phase (week 14) and at steady-state during the maintenance phase in patients rerandomized to evolizumab. ;Characterize inter-individual variability and potential covariate effects on evolizumab serum exposure;Study the relationship between serum exposure and clinical response and remission and endoscopic changes during induction and maintenance treatment;Characterize evolizumab PK profile and relationship between serum exposure to evolizumab and β7 receptor occupancy of evolizumab on peripheral blood T and B lymphocyte subsets in patients with CD (in the PK/PD substudy middle). study population; patients

The target group is refractory to or intolerant to CS and/or IS therapy, and has not previously received anti-TNF therapy (TNF-naive) or has been exposed to one or more anti-TNF therapies, and has previously responded to CS and/or IS therapy and/or poor response, refractory response, or intolerance to anti-TNF.

Patients who are refractory to CS therapy despite a history of at least one 4-week induction regimen (including a dose equivalent to ≥ 30 mg/day oral prednisone for 2 weeks or IV for 1 week or ≥ 9 mg/day oral cortisol) , still have signs/symptoms of ongoing active disease. Patients who are intolerant to CS therapy have a history of conditions including, but not limited to, Cushing’s syndrome, osteopenia/osteoporosis, hyperglycemia, insomnia, and infection.

Patients refractory to IS therapy despite at least 12 weeks of oral AZA (or equivalent) (≥ 1.5 mg/kg) or 6-MP (or equivalent) (≥ 0.75 mg/kg) or MTX (≥ 15 mg/week) regimen and still have signs/symptoms of persistent active disease. Patients who are intolerant to IS therapy have a history of intolerance to AZA (or equivalent), 6-MP (or equivalent), and/or MTX (including but not limited to infection, nausea/vomiting, abdominal pain, pancreatic inflammation, liver function test abnormalities, lymphopenia, and thiopurine methyltransferase genetic polymorphism).

An initial lack of response to anti-TNF therapy means that the patient has not responded (eg, by receiving ≥ 2 induction doses of infliximab [≥ 5 mg/kg] or adalimumab [160 mg/80 mg or 80 mg/40 mg] or pegylated certolizumab [≥ 400 mg] as evidenced by persistent CD-related signs/symptoms). Insufficient nonresponse secondary to anti-TNF therapy means that the patient initially responded to infliximab (≥ 5 mg/kg) or adalimumab (≥ 40 mg) or pegylated certolizumab (≥ 400 mg). ) respond to induction therapy but experience signs/symptoms associated with CD relapse during the maintenance phase. Intolerance to anti-TNF therapy means the patient experienced significant injection site reactions, congestive heart failure, infection, or any other condition that precluded continued use of anti-TNF therapy at any time. Patients who have not previously demonstrated poor response to or intolerance to one or more anti-TNF therapies are eligible for study participation if they are intolerant to or refractory to corticosteroid or immunosuppressive therapy. Inclusion criteria

Patients must meet the following study enrollment criteria: (1) able and willing to provide written informed consent; (2) 18 to 80 years of age; (3) Males and non-postmenopausal females: during treatment and after the last dose of study drug Use of effective contraception for at least 24 weeks; (4) diagnosis of CD based on clinical and endoscopic evidence established > 3 months prior to screening visit; (5) established during screening as defined in the study design section above Moderately to severely active disease; (6) ileum and/or colon involvement with at least four colon segments traversable by a pediatric endoscope, or three segments (colon and/or colon in patients undergoing intestinal resection for CD) or ileum); (7) Complete surveillance colonoscopy ≤ 12 months before screening if colon disease duration > 10 years, or ≤ 5 years before screening if the patient has any risk factors for bowel cancer (screening Monitoring can be carried out during this period); (8) Experience intolerance, refractory disease or non-response to at least one of CS therapy, IS therapy and/or anti-TNF therapy (as defined above) within 5 years since screening. Exclusion criteria

Patients who met any of the following criteria were excluded from the study. Exclusion criteria related to gastrointestinal health included: (1) subtotal colectomy with ileorectal anastomosis or total colectomy; (2) short bowel syndrome; (3) presence of ileostomy or colostomy ; (4) Evidence of fixed stricture or small bowel stenosis with prestenotic dilatation, precluding adequate endoscopic evaluation of the bowel; (5) Diagnosis of UC or indeterminate colitis; (6) Suspected ischemic Colitis, radiation colitis, or microscopic colitis; (7) evidence of abdominal or perianal abscess; (8) anticipated need for surgery for CD-related complications during the study period; (9) past or current adenomatous colon polyps; (10) Past or current disease-related colonic mucosal dysplasia (past age-related polyps are acceptable); (11) Sinus tracts with evidence of infection (e.g., purulent discharge) in the clinical judgment of the investigator. Fistulas associated with Crohn's disease were not excluded.

Exclusion criteria related to prior or concomitant therapy included: (1) receipt of any of adalimumab, pegylated certolizumab, or infliximab for CD within 8 weeks prior to randomization; One; (2) any prior treatment with ustekinumab within 14 weeks before randomization; (3) use of evolizumab or other anti-integrin agents (including vedolizumab, natalizumab (4) any prior treatment with anti-adhesion molecules (e.g., anti-MAdCAM-1); (5) use of T- or B-cell therapy ≤ 12 months before randomization Prior treatment with cell-depleting agents (e.g., rituximab, alemtuzumab, or velcilizumab), except AZA and 6-MP (or equivalent); (6) 12 weeks prior to study randomization or within the 5 half-lives of the investigational product (whichever is longer); (7) Received any investigational treatment, including investigational vaccines; (7) Neutralization of chimeric, human or humanized antibodies, fusion proteins or murine proteins History of mild or severe allergy or allergic/anaphylaxis, or to ivolizumab (active drug substance) or any of the excipients (L-histamine, L-arginine, succinic acid, polysorbate 20 ) history of hypersensitivity; (8) treatment with corticosteroid enemas/suppositories and/or topical (rectal) 5-aminosalicylate (5-ASA) preparations ≤ 2 weeks before randomization; (9) treatment with Patients who continue tube feeding, prescribed formula diet and/or parenteral nutrition/nutrition treatment for CD ≥ 3 weeks before treatment; (10) Patients who are expected to need tube feeding, prescribed formula diet and/or parenteral nutrition/nutrition treatment for CD during the study period patients; (11) received any live or attenuated vaccine ≤ 4 weeks before randomization; (12) used IV steroids during screening, except for a single IV steroid dose administered in the emergency department; (13) Use cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil ≤ 4 weeks before treatment; (14) long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs). Prophylactic aspirin, up to 325 mg/day, is allowed, as is occasional use of NSAIDs to treat conditions such as headache, arthritis, myalgia, and menstrual cramps; (15) If receiving oral CS, patients are excluded unless Dose stable at ≤ 20 mg/day prednisone (or equivalent) for ≥ 2 weeks prior to randomization; (16) if receiving ongoing treatment with oral 5-aminosalicylate (5-ASA), if immediately following Patients were excluded if their dosage was unstable ≥ 4 weeks before randomization; (17) if they were receiving probiotics (e.g., Culturelle, Saccharomyces boulardii ) or over-the-counter supplements (e.g., N-acetylglucosamine, curcumin) If on-going treatment with an IS (e.g., 6-MP, AZA, or MTX), patients were excluded if the dose was unstable ≥ 2 weeks immediately before randomization; (18) if on-going treatment with an IS (e.g., 6-MP, AZA, or MTX), patients would be excluded Patients were excluded if dose was unstable ≥ 8 weeks before randomization;(19) Patients were excluded if dose was unstable ≥ 2 weeks immediately before randomization if receiving ongoing antibiotic therapy for CD outer. Patients may continue to receive ongoing antidiarrheal medication (eg, loperamide or phenylephrine and atropine), preferably reaching a stable dose ≥ 2 weeks before randomization. However, if the patient and/or treating physician decide to change the dose or regimen of antidiarrheal medication at any time during the screening period, these patients will be allowed to participate in the study.

Exclusion criteria related to infection risk include: (1) congenital or acquired immunodeficiency; (2) positive HIV ELISA test result confirmed by Western blot; (3) positive hepatitis C virus (HCV) antibody test result, Patients who tested positive for HBsAg during the screening hepatitis B assessment were excluded from the study unless the patient had documented undetectable HCV RNA > 6 months after successfully completing a course of HCV antiviral therapy; (4) Patients who test positive for hepatitis core antibody (HBcAb) but negative for hepatitis B surface antigen (HBsAg) must confirm a negative hepatitis B virus (HBV) DNA test result to be eligible for study participation and are required to receive HBV during the study period Regular monitoring of DNA; (5) Positive stool egg or parasite test results or positive stool culture for pathogens during screening; (6) Clostridium difficile infection and/or Clostridium difficile infection within 8 weeks before the baseline visit Evidence of treatment or treatment of other enteric pathogens for Clostridium difficile infection; (7) history of active or latent tuberculosis confirmed by screening test or suspected active tuberculosis on chest X-ray taken within 3 months of randomization; (8) History of recurrent opportunistic infections and/or severe or disseminated viral infections; (9) Any serious opportunistic infection occurring ≤6 months before screening; (10) Any current or recent signs or symptoms of infection ( ≤8 weeks before screening); (11) any major infectious episode requiring hospitalization or treatment with intravenous antibiotics ≤8 weeks before screening or oral antibiotics ≤4 weeks before screening.

Exclusion criteria related to general safety include: (1) pregnancy or lactation; (2) lack of peripheral venous access; (3) hospitalization within 4 weeks before randomization; (4) inability to comply with the study protocol in the opinion of the investigator; (5) ) Severe uncontrolled comorbidities such as neurological, cardiac, pulmonary, renal, hepatic, endocrine, or GI disorders (other than CD); (6) Neurological conditions or diseases that may interfere with PML monitoring; (7) Screening neurological conditions Clinically significant abnormality on examination; (8) History of demyelination; (9) History of severe neurological disease, including stroke, MS, brain tumors, neurodegenerative disease, or poorly controlled epilepsy; (10) ≤ 6 before screening Months of alcohol, drug or chemical product abuse history; (11) Conditions other than CD that may require > 20 mg/day prednisone (or equivalent) treatment during the study; (12) Within 5 years before screening History of cancer, including hematologic malignancies, solid tumors, and carcinoma in situ; (13) Evidence of the presence of adenocarcinoma in situ (AIS), high-grade squamous intraepithelial lesion (HSIL), or cervical intraepithelial neoplasia (CIN) at any time ) History of cervical smear results > Grade 1; (14) History of organ transplantation or cell transplantation; (15) Magnetic resonance imaging (MRI) scans are considered unsafe due to the presence of metals in the body that may pose a hazard during any potential scan of patients. Materials and Methods Evolizumab and placebo; Treatment

Evolizumab will be provided by the trial sponsor as a one-time PFS containing 150 mg/mL evolizumab for SC administration. To maintain blinding to study drug allocation during the induction phase, all patients will receive injections from two PFS at weeks 0, 4, 8, and 12: 1-mL PFS with a 0.7-mL injection volume (providing placebo or 105 mg evolizumab); and 2.25-mL PFS with a 1.4-mL injection volume (offering placebo or 210 mg evolizumab). Depending on dose allocation, one PFS (patients assigned to active drug) or two PFS (patients assigned to placebo) will contain placebo. At Week 2, all patients will receive a 1.4-mL injection of study drug from a 2.25-mL PFS (delivering placebo or 210 mg evolizumab). The PFS will contain placebo for patients in the low-dose evolizumab and placebo arms and active drug for patients in the high-dose evolizumab arm. During the maintenance phase, patients will receive injections starting from the 1-mL PFS in a 0.7-mL injection volume (delivering placebo or 105 mg evolizumab).

Evolizumab was formulated at 150 mg/mL in 20 mM histidine, 0.2 M argininosuccinate, and 0.04% (wt/vol) polysorbate 20, pH 5.8. Each PFS is intended for single-dose parenteral administration and is preservative-free.

The composition of the placebo drug product was identical to the active drug product without evolizumab.

Study drugs must be stored according to the details on the product label. The storage temperature is indicated on the medication label. PFS of study drugs should be refrigerated at 2℃-8℃ (35.6℉-46.4℉) and protected from excessive light and heat. PFS should not be frozen, shaken, or stored at room temperature. PFS containing study drug is stable at room temperature (up to 30°C [86°F]) for no more than 8 hours. If the syringe has been left at room temperature for longer than this time, it should not be used. Crohn's disease activity assessment

For each patient, a detailed history of CD was collected, including date of diagnosis, disease severity, hospitalization, and extraintestinal manifestations at screening. As mentioned above, disease severity will be assessed using CDAI, SF, AP and SES-CD. Additional details for CDAI are provided in Table 1 below, and SF and AP are described below. SES-CD (see Table 2) is assessed endoscopically and is based on four factors (ulcer size, proportion of ulcer surface, proportion of surface affected by other lesions, and degree of stenosis) in up to five ileocolic segments. synthesis. CDAI, SF, and AP scores will be calculated at weeks 0, 10, 14, 24, 28, 32, 44, 56, and 66 (or early exit visit). SES-CD assessments will be conducted at screening and at weeks 14 and 66 (or early exit visit). Abdominal pain will be additionally assessed throughout the study by the Abdominal Pain Questionnaire (APQ) recorded daily in an electronic diary (range 0-10, with 0 indicating no pain and 10 indicating pain as severe as imaginable).

The CDAI quantifies the signs and symptoms of CD in patients. The CDAI consists of eight factors; each factor is summed after adjusting for a weighting factor (Best et al. 1979). Components of the CDAI include frequency of liquid or soft stools, abdominal pain, general feeling of well-being, presence of comorbidities, use of LOMOTIL® (feperidone/atropine) or other opioids for diarrhea, presence of abdominal mass, hematocrit, and Percent deviation from standard body weight. Of the eight factors in the CDAI, three are patient-reported (number of liquid or soft stools, abdominal pain, and general feeling of well-being) and four are based on physician assessment (presence of complications, use of LOMOTIL® or other opioids for diarrhea , presence of abdominal mass, and percent deviation from normal weight, based on the patient's weight at the time of visit), and one factor is based on a blood test (hemocrit). Patients will report daily in an electronic diary the severity of their abdominal pain, frequency of loose stools and overall feeling of well-being. A weighted sum of the 7-day average scores was calculated for the PRO portion of the CDAI score. Provide the patient with the Bristol Stool Scale as a reference for identifying loose stools (types 6 and 7 on the Bristol Stool Scale; see Figure 3). Because ileocolonoscopy preparation can interfere with the assessment of other clinical parameters, electronic log entries used to calculate the complete CDAI should not correspond to bowel preparation, the day of endoscopy, or the day after endoscopy.

Two patient-reported factors will be assessed: liquid or soft stool frequency (SF) and abdominal pain (AP). Scores were calculated using the unweighted average number of liquid/very soft stools and the average AP (0-3 scale) in the 7 days before the assessment visit. Patients will report daily in an electronic diary their frequency of loose stools (Bristol Stool Scale [Figure 3] will be provided) and severity of abdominal pain. As with the CDAI, SF and AP scores should not use electronic diary entries corresponding to bowel preparation, the day of endoscopy, or the day after endoscopy to avoid interference related to ileocolonoscopy. Additionally, the 0-10 Abdominal Pain Questionnaire (APQ) is an 11-point numeric rating scale used to assess the worst daily abdominal pain. Higher scores indicate more severe abdominal pain (0 = no pain; 10 = pain as severe as imaginable). The review norm for APQ is 24 hours. As with the CDAI, SF, and AP scores, the calculated APQ score should not use electronic log entries corresponding to bowel preparation, the day of endoscopy, or the day after endoscopy to avoid interference related to ileocolonoscopy .

Another assessment of disease activity is the patient-reported outcome tool called CD-PRO/SS. The development of this PRO is described in Higgins et al., Journal of Patient-Reported Outcomes (2018) 2:24, and the PRO module and user manual can be found at https(colon)(slash)(slash)www(dot)evidera(dot )com(slash)crohns-disease-patient-reported-outcomes-cd-pro. The 14-item questionnaire (some questions have supplementary questions on severity/frequency) contains two domains: CD signs and symptoms and systemic symptoms. CD-PRO/SS assesses the presence of signs and symptoms of CD and, in some cases, the severity or frequency of symptoms. The lookback specification for CD-PRO/SS metrics is 24 hours. Patients will complete CD-PRO/SS indicators for at least 9 consecutive days before and after each visit, following procedures in the electronic log. To allow for early or late visits within the visit window, patients may complete up to 12 days of CD-PRO/SS. Table 1. Crohn’s Disease Activity Index (CDAI) Category count initial total multiplication factor total Number of liquid/very soft stools Total number of liquid/very soft stools in 7 days (reported 7 days immediately before study visit) x 2 stomach ache Sum of daily abdominal pain scores over 7 days, 3-point scale: 0=none, 1=mild, 2=moderate, 3=severe (reported 7 days immediately before study visit) x 5 Overall sense of comfort Sum of daily general comfort scores over 7 days, 4-point scale: O = overall good, 1 = slightly below standard, 2 = poor, 3 = very poor, 4 = terrible (reported 7 days immediately before study visit) x 7 Extraintestinal Manifestations of Crohn's Disease Total number of checked boxes (select all matching items) x 20 Arthritis/joint pain Iritis/uveitis Erythema nodosum/pyoderma gangrenosum/aphthous stomatitis Anal fissure, fistula or abscess Other anal fistula Fever over 37.8°C in the past week Lomotil/Imodium/opioids for diarrhea yes=1 x30 No=0 abdominal mass None=0 x 10 Not sure=2 OK=5 Hematocrit (%) ^a Male: Subtract value from 47 x 6 Female: Subtract value from 42 Weight ^b (1 - (weight/standard weight)) x 100 x 1 final rating Add total: a If the hematocrit subtotal is <0, enter 0. b If the weight subtotal is <-10, enter -10. Adapted from: Best WR, Becktel JM, Singleton JW, Kern F, Jr. Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. Gastroenterology1976;70(3):439-44. Table 2. Definition of SES-CD (Daperno et al. , Gastrointest. Endosco. 60:505, 2004) Simplified endoscopic scoring for Crohn's disease variable 0 1 2 3 ulcer size without Aphthous ulcers (Ø 0.1 to 0.5 cm) Large ulcers (Ø 0.5 to 2 cm) Very large ulcers (Ø > 2 cm) ulcer surface without <10% 10-30% ＞30% affected surface unaffected segment <50% 50-75% ＞75% Narrow existence without Single, can pass Multiple, you can pass Unable to pass Ø, diameter. Rationale for primary and key secondary study endpoints

The key cohort of this study was designed to evaluate relief of signs and symptoms and endoscopic improvement as co-primary outcomes in CD. Clinical remission is defined as the average number of liquid/soft stools (SF) ≤3 in the 7 days before the evaluation visit and the average AP score (0-3 scale) (AP) ≤1 in the 7 days before the evaluation visit, for any subscore There was no deterioration compared to baseline. The primary clinical endpoint is based on previous FDA advice, the changing global regulatory environment (new EMA CD Guidance on New Medicinal Products released in July 2018; CHMP 2016), and results from the exploratory induction of Cohort 1 in this study . Due to the limitations of the index, both the FDA and the EMA in their new guidance discourage the use of the CDAI as the primary endpoint in CD registration studies. Instead, co-primary endpoints consisting of patient-reported signs/symptoms and endoscopic measurements are encouraged.

Use of SF and AP was consistent with FDA recommendations for use of SF and AP 7-day scores associated with CDAI response. Using unweighted indices of AP and SF to define clinical remission and response is easier to interpret by clinicians, patients, and caregivers, and is more likely to represent the symptoms and symptoms of patients with moderate to severe CD who experience varying degrees of SF and AP during an episode. Clinically meaningful improvement in disease activity.

Endoscopic improvement, defined as a ≥50% change in SES-CD score from baseline (Ferrante M, et al., Gastroenterology 145:978-86, 2013), was the co-primary endpoint. SES-CD consists of four endoscopic variables (ulcer, ulcerated surface, inflamed surface, and presence of narrowing), which are scored in five ileocolic segments. The SES-CD was prospectively developed and validated in patients with mild to severe CD (per CDAI) by Daperno et al., Gastrointest. Endosc. 60(4):505-12, 2004. This scoring system is recommended by the FDA, and physicians generally prefer it to other indicators because it quantifies ulcer size (rather than qualitatively assesses ulcer characteristics), determines the proportion of ulcers in a segment (rather than by visual analog scale), and better inter-rater reliability (the interclass correlation coefficients of SES-CD and Crohn's disease endoscopic index of severity (CDEIS) were 0.83 and 0.71, respectively; Khanna R, et al., Inflamm Bowel Dis 20:1850-61, 2014). Because scoring will not be adjusted for the number of visible segments, only segments visible at baseline will be included in the endpoint assessment. This means that any new narrowing due to inflammation (which renders the segment unassessable for study post-treatment scoring) will classify patients as not achieving endoscopic improvement in the secondary endpoint analysis. Similarly, any improvement in inflammation (which renders the segment evaluable for study post-treatment scoring) will not be reflected in the endpoint assessment. The statistical analysis plan (see below) will describe any sensitivity analyzes planned to assess the impact of these missing data on the endpoints. Given limited experience with endoscopic scoring systems in general gastroenterology practice, all ileocolonoscopies in the study will be recorded at the study site, but a central reader will determine SES-CD.

Published studies have used multiple endoscopic endpoints to assess efficacy, which appear to have been arbitrarily defined. Mucosal healing, defined qualitatively as the absence of mucosal ulceration, has been studied as a primary or secondary endpoint in a number of phase IV studies (Rutgeerts P, et al., Gastro 126:1593-610, 2004 [natalizumab]; Rutgeerts P, et al., Gastrointest Endosc 63:433-42, 2006 [infliximab]; Colombel JF, Sandborn WJ, Reinisch W, et al. Infliximab, azathioprine, or combination therapy for Crohn's disease. et al., N Engl J Med 362:1383-95, 2010 [ infliximab and azathioprine]; Hebuterne et al., Gut 62:201-8, 2013 [certolizumab]; Rutgeerts P, et al., Gastroenterology 142(5):1047-9, 2012 [adalimumab]). Some studies have defined endoscopic thresholds representing endoscopic response, but the clinical significance of these scores is unclear (Hebuterne et al., Gut 62:201-8, 2013 [certolizumab]; Rutgeerts P, et al., Gastroenterology 142( 5):1047-9, 2012). In this study, endoscopic improvement was defined as the proportion of patients demonstrating a ≥50% reduction in SES-CD compared with pretreatment baseline scores.

This endpoint will be measured at Week 14 for induction and at Week 66 for maintenance in patients who achieve a clinical response at Week 14. The endpoint was based on a recent post hoc analysis of the SONIC trial, which determined that a ≥50% reduction in SES-CD score was predictive of CS-free CDAI remission after 50 weeks of biologic therapy (Ferrante M, et al., Gastroenterology 145:978-86, 2013). This definition is also appropriate when considering the large variability in SES-CD changes at the end of the 6-week induction period measured in a sample of 24 placebo patients with moderately to severely active CD who participated One of two trials of new biologics (Ferrante et al. Gastroenterology 138, Issue 5, Supplement 1, S-358, 2010). The data set showed that 6 patients achieved a 50% reduction in both SES-CD or CDEIS scores and at least a 5-point reduction in SES-CD or CDEIS scores. Patient Population Rationale

Patients with uncontrolled moderately to severely active CD are at risk for developing stricturing or penetrating complications of inflammation, as well as symptoms that impact quality of life. The goals of treatment for CD are to induce and maintain symptom improvement, induce mucosal healing, and improve quality of life. However, for a large proportion of patients, current therapies fail to achieve these goals. Therefore, the study population will include patients refractory to one or more of the following therapies: 1) CS, 2) IS, or 3) anti-TNF (or TNF-IR). Patients enrolled based on refractory or intolerance to CS and/or IS lines may or may not have previously received anti-TNF. From previous studies using evolizumab in ulcerative colitis (Rutgeerts, PJ et al., Gut 62:1122-1130, 2013; Vermeire et al., Lancet 384:309-18, 2014) and vedolizumab Data from the anti-GEMINI 2 and 3 studies (Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013; Sands BE, et al., Gastroenterology 147:618-27, 2014) have confirmed the anti-α4β7 mechanism of action in these patients. Good efficacy in subgroups. Patients belonging to each subgroup will be identified based on the refractory and inappropriate response criteria described herein.

Patients aged 18 to 80 years with moderately to severely active CD will be studied. This age range is typical of patients enrolled in clinical trials of new investigational agents in CD and reflects the observation that adult CD can become or remain moderately to severely active disease at any age. Given that the primary clearance mechanism of evolizumab is neither renal clearance nor first-pass metabolism, the risk of accumulation in patients >65 years of age is considered low and may also be mitigated by laboratory exclusions associated with poor renal and hepatic function.

According to EMA guidelines, eligible patients must have had confirmed CD for at least 3 months and have moderately to severely active disease confirmed by endoscopic evidence of inflammation. This is expected to improve the specificity of CDAI, SF and AP assessment and allow assessment of endoscopic improvement endpoints. On the basis of constructing the SES-CD, patients with isolated ileitis or ileocecal resection would be expected to have lower baseline scores compared with patients with ileocolic disease, regardless of the same degree of inflammation and ulceration in the affected segments. Therefore, different SES-CD entry scores have been proposed for these patient subgroups. It’s no secret that there are no good treatment options for patients with fistula disease. This subgroup was included in this protocol to evaluate the effect of evolizumab on draining fistulas. Basic principles of induction and maintenance period design

Patients randomized to the induction phase will be based on disease activity based on a CDAI score of ≤ 330 or > 330, which predicts CDAI response and response rate to biologic therapy; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013; Sands BE, et al., Gastroenterology 147:618-27, 2014), CS use, IS use, and prior anti-TNF failure (all indicators of disease activity). These factors were considered sufficient to mitigate the risk of imbalances in disease severity across treatment groups.

Assessment of induction of clinical remission and endoscopic improvement at week 14 is justified by the observation that anti-integrin therapy has a slower onset of action compared with anti-TNF therapy (Sandborn WJ, et al., N Engl J Med 353:1912-25, 2005; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013), and clinical consensus that the onset of endoscopic improvement is typically observed 16-24 weeks after induction therapy, especially In treatment-refractory patients (Consensus is supported by observational data showing that Mayo Clinic score response with evolizumab takes up to 14 weeks in TNF-IR patients with UC (see Vermeire et al., Lancet 384 :309-18, 2014). A challenge of the 14-week induction period is the need to keep concomitant CD therapy stable over the duration to avoid confounding endpoint analyses. For patients who require rescue therapy to treat flare-ups and who are responding to therapy but are unable to taper This is problematic for patients whose CS dose. This study design addresses this issue by allowing for the use of rescue therapy in the event of disease exacerbation, in which case patients would be classified as non-responders to the primary analysis, and limit maximum baseline CS to ≤ 20 mg/day prednisone equivalent dose. Although treatment adjustments are not prohibited, increasing antidiarrheal dosages should be avoided. Patients should keep their antidiarrheal dosages as stable as possible because titrating antidiarrheal medications can The impact of placebo response rates on clinical remission is unknown.

The induction phase included an exploratory cohort (Cohort 1) that was large enough to assess the effect size and accuracy of the statistical planning assumptions for the new endpoint, the clinical validity of the dichotomous endpoint definition, and the efficacy of the endpoint in the key cohort (Cohort 3). Test hierarchy. The study also included an active treatment induction cohort (cohort 2) to generate a sufficient number of symptom responders for endpoint assessment in the maintenance phase. The final cohort (Cohort 3) is the key induction cohort and will generate data for the endpoint analysis of the induction study.

Patients who achieve a CDAI-70 response at week 14 and are not using rescue therapy will be randomized into the maintenance phase until a maintenance phase sample size of approximately 480 patients is reached. Patients who achieved clinical remission and endoscopic improvement at week 14 were expected to be a subpopulation within this group based on expert clinical opinion. Randomization into the maintenance phase will be stratified by the use of disease activity (as described for the induction phase, except that CDAI remission [score < 150] at weeks 10 and 14 will be used instead of CDAI ≤ 330 or > 330). Additionally, randomization will be stratified by assignment to low-dose or high-dose evolizumab (allowing assessment of the impact of low-dose or high-dose induction therapy on maintenance endpoints). Given the large number of strata, any potential imbalance in the proportion of endoscopic improvers at week 14 will be addressed using covariate-adjusted analysis of the endoscopic improvement endpoint at week 66.

The duration of the maintenance phase (52 weeks) is considered appropriate by the FDA and EMA to determine the benefit of long-term therapy. During the first 8 weeks of the maintenance phase, patients receiving CS during the induction phase should have weekly dose reductions consistent with current American College of Gastroenterology and European Crohn's Disease and Colitis Organization guidelines for CS tapering and following EMA advice to avoid rapid tapering. The tapering schedule will allow assessment of the key secondary outcome of achieving corticosteroid-free clinical remission at week 66 in patients entering the maintenance phase.

An analysis of data from a phase I study of patients with UC comparing induction therapy with evolizumab versus placebo (Vermeire S, et al., Gastroenterology 144, S1:S-36, 2013) showed that patients receiving concomitant IS or Infection rates were similar between evolizumab and placebo in the subgroup of patients who received CS therapy and in the subgroup who did not receive these concomitant therapies. Although this is a small data set with limited exposure duration, more data with longer exposure durations are available for vedolizumab. A recent analysis of the vedolizumab development program showed a modest increase in infection rates in patients taking IS regardless of whether they received vedolizumab or placebo. Additionally, infection and serious infection rates in the vedolizumab-treated group were similar in patients who also received IS versus those who did not (Colombel et al. Gut 2016;0:1-13).

The risk of PML is expected to be lower with evolizumab compared with other parenterally-selective biologics, and no PML events were reported in the UC phase II or OLE studies. However, the potential risks of PML and other serious infections have been carefully considered. Patient safety will be monitored throughout the study through vital sign assessments, safety laboratory assessments, neurological examinations, and ongoing review of adverse events. If any patient develops an allergic reaction, develops PML, certain malignancies, colonic mucosal dysplasia, or certain severe infections, they will permanently discontinue study treatment. During the maintenance phase of the study, patients who experience clinical relapse in CD status will have the option to participate in an open-label study of evolizumab.

Following the final visit of the treatment period, patients not participating in the OLE study will undergo an additional 12 weeks of safety assessment, beginning after the last dose of study drug. This period reflects a sufficient time frame for elution of evolizumab based on an estimated elimination half-life of 11 days. Rationale for evolizumab dosage, dose range, and schedule

Because evolizumab has been evaluated in patients with moderately to severely active UC in a phase II study (Vermeire et al., Lancet 384:309-18, 2014), it was administered at 105 mg (nominal dose 0.7 mL 150 mg/mL evolizumab formulation) administered every 4 weeks (Q4W) (Doses 0, 4, and 8) and 315 mg Q4W + loading dose (LD [420 mg Week 0, Week 0 315 mg Ld]) at 2, 4 and 8 weeks, achieved clinically meaningful disease remission induction without obvious safety issues, and a dosing regimen of evolizumab (105 mg SC Q4W) was also proposed as this One of the doses to be tested in the study. Additionally, during the induction phase, patients will be randomly assigned to receive placebo, low-dose evolizumab, or high-dose evolizumab.

Low-dose evolizumab (105 mg) was administered to Q4W SC (weeks 0, 4, 8, and 12). The Q4W 105 mg SC low-dose regimen was designated as the dose range for the induction phase based on the following considerations: (1) In the Phase II UC trial, the nominal dose of 100 mg (0.7 mL of 150 mg/mL solution via vial and syringe) , demonstrated clinically meaningful induction of remission in patients with UC at an actual dose of 105 mg, administered Q4W SC and had a favorable safety profile in a phase II trial (Vermeire et al., Lancet 384:309-18, 2014 ); (2) For all patients who provided evaluable samples in the Phase II trial, exposure to 105 mg Q4W SC administration was shown to be sufficient to achieve maximal beta7-receptor occupancy in blood and colon tissue, supra , (3 ) The population PK/PD model predicts that doses lower than the 105-mg SC Q4W regimen (e.g., 50 mg Q4W SC) will result in approximately 44% of patients losing maximal beta7 receptor occupancy during the Q4W dosing interval and that exposure is likely in the non-linear PK range.

In addition to the 105-mg Q4W dose, a higher dose regimen of 210 mg SC at weeks 0, 2, 4, 8, and 12 was designated as the dose range for the induction phase based on the following considerations: (1) Despite similar pathogenesis, However, compared with UC, CD exhibits more complex anatomic disease manifestations throughout the GI tract (i.e., transmural inflammation, patchy distribution, and stenosis). A positive exposure-response relationship with vedolizumab, a cognate anti-integrin antibody, was recently reported following the induction period of a phase III clinical trial in patients with CD. Results showed that the induction dose was sufficient to achieve complete receptor occupancy in all patients (Rosario M, et al., (Abstract) Crohn's and Colitis Foundation of America, Advances in Inflammatory Bowel Diseases, Abstract P-140, 2013), but in Increased clinical response/remission was observed in patients with higher drug concentrations (Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013; Rosario M, et al., (Abstract), European Crohn's and Colitis Organization Congress, Abstract P489, 2014). These observations from the vedolizumab studies suggest that higher evolizumab exposure may have the potential to provide additional clinical benefit in this patient population; (2) In addition to the 210 mg Q4W dose, the The additional 210 mg dose over 2 weeks is designed to preload evolizumab exposure so that exposure reaches steady state more quickly. Early loading doses of anti-TNF agents or anti-integrin antibodies (Rutgeerts P, et al., Gastro 126:1593-610, 2004) were found to be effective in inducing clinical remission (CDAI score < 150) and were implemented in this study. loading dose strategy; (3) for patients with CD, the recommended higher dose of 210 mg × 5 doses SC (at weeks 0, 2, 4, 8, and 12) would result in 2.5 times the total dose of dose separation and is expected to achieve exposure levels that are 30% lower than the 315 mg + 420 mg LD cohort studied in the UC Phase II trial, which had an acceptable safety/tolerability profile (see above ). In conclusion, given the favorable safety profile and positive clinical outcomes observed in the nominal 100-mg Q4W cohort (actual dose 105 mg) in the evolizumab UC Phase II study, the 105-mg Q4W dose was evaluated for CD induction therapy is appropriate. Furthermore, given the complex pathophysiology of CD, the positive exposure-efficacy relationship observed with vedolizumab treatment despite complete receptor saturation in the blood, the available safety margin and acceptable safety profile of evolizumab It is scientifically reasonable to evaluate the higher dose regimen of 210 mg (weeks 0, 2, 4, 8, and 12) to further understand the dose-response relationship in patients with CD during the induction phase. Rationale for maintenance dose regimens

Patients who respond to evolizumab during the induction phase (achieve at least a 70-point reduction in CDAI score from baseline, a “CDAI-70 response”) will be randomly assigned again during the maintenance phase to receive 105 mg of evolizumab or placebo. Q4W; Patients who respond to placebo during the induction phase will continue to receive placebo during the maintenance phase. The PK profile of mAbs (anti-TNF or anti-integrin) has been reported to be similar in UC and CD patient populations (Ternant et al., Ther Drug Monit 2008 30:523-9; Fasanamde et al., Eur J Clin Pharmacol 2009; 65:1211-28; Awni et al. abstract published in ECCO, 2013; Feagan et al., N Engl J Med 2013;369:699-710; Sandborn et al., Aliment Pharmacol Ther 2013;37:204-13). The Q4W 105 mg SC low-dose regimen was designated during the maintenance phase based on the following considerations: (1) The 105-mg Q4W SC dose planned for the CD phase III study (by population modeling) is expected to be consistent in >85% of patients Maintains complete β7-receptor occupancy. The 100 mg Q4W SC nominal dose (105 mg actual dose) administered in the UC Phase II study demonstrated acceptable safety (see above ); (2) the same anti-integrin vedolizumab was Remission was successfully maintained with an 8-week regimen that provided mean steady-state trough serum concentrations sufficient to maintain maximal receptor occupancy (Rosario M, et al., (Abstract) Crohn's and Colitis Foundation of America, Advances in Inflammatory Bowel Diseases , Abstract P-140, 2013; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013). Rationale for control groups

This study will use a placebo-treated control group to evaluate the efficacy, safety, and tolerability of patients receiving evolizumab and background CD compared with patients receiving placebo and background CD. Given the inherent variability in disease onset and the use of subjective assessments (such as PROs), the use of control groups is necessary. Patients in the control cohort will receive the same study evaluations as evolizumab-treated patients. A 1:1 ratio of evolizumab to placebo-treated patients was necessary to achieve statistical comparison of the primary endpoint between these treatment groups. Patient-reported outcome assessment

PROs (loose stool frequency, abdominal pain, and overall well-being components of the IBDQ, CD-PRO/SS, EQ-5D, APQ, and CDAI) will be collected to help characterize patient-reported clinical characteristics of evolizumab. These instruments will be translated into local languages as necessary. PRO data are collected electronically through the use of electronic PRO (ePRO) devices (i.e., e-logs and tablets). Research staff will provide patients with electronic diaries and instructions for completing PRO questionnaires electronically (for those PROs that need to be completed outside the clinic). Patients will also be instructed to contact the study site immediately if they have any questions about the use of electronic diaries at any time during screening or during the study. For example, when a PRO is to be completed in the clinic, the patient will fill it out on a tablet. At each clinic visit, electronic patient data collected since the last study visit were reviewed. ePRO data are collected and assessed at visits. During the screening process, patients will be instructed on the appropriate use and completion of questions in the electronic diary. Signs and symptoms of CD, particularly frequency of liquid or soft stools, abdominal pain, and general feeling of well-being, must be recorded daily throughout the study, including the screening period. To ensure instrument validity and data standards meet health authority requirements, PROs completed at the study site ( IBDQ and EQ-5D) are administered.

CDAI, SF, AP and CD-PRO/SS have been discussed above. The IBDQ assesses patients' health-related quality of life (HRQOL; Guyatt G, et al., J Clin Epidemiol 42(5):403-408, 1989; Irvine EJ, J Pediatr Gastroenterol Nutr 28:S23–7, 1999). The 32-item questionnaire contains four domains: intestinal symptoms (10 items), systemic symptoms (5 items), emotional functioning (12 items), and social functioning (5 items). The items are rated on a 7-point Likert scale, with higher scores indicating better HRQOL. Retrospective norms for the IBDQ are 2 weeks. Patients completed the IBDQ on a tablet at the study site at baseline, weeks 0, 14, 44, and 74, before other non-PRO assessments were completed and before patients received any disease status information or study medication during the visit.

The EuroQol Five-Dimensional Questionnaire (EQ-5D) is a generic, preference-based HRQOL that provides a single index value for health status (Rabin R, et al., Enn Med 33:337-43, 2001). The tool includes questions about mobility, self-care, daily activities, pain/discomfort, and anxiety/depression that can be used to synthesize a patient's health status. At weeks 0, 14, 44, and 74 (or early withdrawal visit), patients were at the study site on a tablet computer before other non-PRO assessments were completed and before patients received any disease status information or study drug during the visit. Complete the EQ-5D. Statistical considerations and analysis plan

For the purposes of statistical analysis, the induction and maintenance phases will be considered separate studies. Open-label cohort 2 served as a tributary cohort and provided sufficient sample size to sustain analysis. Induction data from Cohort 2 will also be considered exploratory data and may be analyzed to inform decision making and the development of a statistical analysis plan. Table 3 below summarizes the sample size for each cohort during the induction period. Table 3. Induction phase sample size for each cohort. Number of patients group total placebo Low dose (105 mg) High dose (210 mg) Exploratory induction group (group 1) ~300 ~60 ~120 ~120 Active treatment induction cohort (cohort 2) ~350 NA ~175 ~175 Key induction group (group 3) ~496 ~124 ~186 ~186 NA = Not applicable; No. = Quantity

The sample size for Cohort 1 (see Table 3) provided approximately 90% power to detect a ≥20% difference in PRO2 or CDAI response rates between each evolizumab arm and placebo (assuming placebo response rates ≤ 15%, similar to results reported in the GEMINI 2 trial of vedolizumab in patients with CD; Sandborn WJ, et al., Aliment Pharmacol Ther 37:204-13, 2013) and approximately 80% power to detect within 15% difference in microscopic response compared to placebo (assuming placebo response rate ≤ 10%) and two-sided chi-square test at 10% significance level.

Pivotal analyzes during the induction phase will be performed using only patients from cohort 3. In this pivotal induction cohort, patients will be randomized in a 2:3:3 ratio to receive placebo, evolizumab 105 mg, or evolizumab 210 mg. The co-primary endpoint will be tested using the Cochran–Mantel–Haenszel (CMH) test at the 5% significance level, where both clinical response and endoscopic improvement are required to be significant. The sample size for Cohort 3 is expected to provide ≥85% power to detect clinical response in each evolizumab group versus placebo (SF mean daily score ≤ 3 and AP mean daily score ≤ 1) Rate difference ≥ 15%, assuming placebo response rate ≤ 15% and two-sided test at 5% significance level. Additionally, Cohort 3 will provide ≥80% power to detect a 10% difference in proportion of endoscopic improvement in each evolizumab group versus placebo, assuming a placebo response rate of ≤5% and a 5% Two-sided test of significance level. The chi-square test was used to confirm the power calculation.

The size of cohort 2 was approximately 350 patients to obtain a sufficient number of patients for maintenance analysis. Table 4. Power estimates for primary and key secondary efficacy analyzes during the critical induction and maintenance phases. Research end point Check your strength Assumed reaction rate Sample size per group induce clinical remission ＞ 85% Placebo=15% Evolizumab=30% Placebo=124 Evolizumab 105 mg=186 Evolizumab 210 mg= 186 endoscopic improvements ＞ 80% Placebo=5% Erolizumab=15% Placebo = 124 Evolizumab 105 mg = 186 Evolizumab 210 mg = 186 maintain clinical remission ＞ 90% ^a Placebo < 20% Evolizumab < 35% Placebo=210 Evolizumab 105 mg=210 endoscopic improvements ~90% ^a Placebo < 30% Evolizumab = 45% Placebo=210 Evolizumab 105 mg=210 All analyzes used type I error support, α =5.0%. ^aAnalysis of all maintenance-phase patients randomized to receive evolizumab during the induction phase.

A total of approximately 480 patients who achieve a CDAI-70 response at Week 14 will be eligible to enter the maintenance phase. Co-primary endpoints will be used in the analysis of the maintenance phase: clinical response (SF mean daily score ≤3 and AP mean daily score ≤1) and endoscopic improvement at week 66. A maintenance co-primary analysis will be performed on all patients rerandomized to the maintenance phase who were randomized to evolizumab (105 mg or 210 mg) during the induction phase. The co-primary endpoint will be tested at the 5% significance level using the CMH test, where both clinical response and endoscopic improvement are required to be significant. Note that to maintain blinding, patients randomized to placebo during the induction phase will undergo pseudorandomization and receive placebo during the maintenance phase. These patients will not form part of the pivotal maintenance analysis.

Of the approximately 480 patients expected to enter the maintenance phase, approximately 420 patients will be rerandomized to receive placebo or evolizumab, and approximately 60 patients will be pseudorandomized to placebo. These projections are based on the assumption that 50% of patients receiving evolizumab during the induction phase and 40% of patients receiving placebo will be eligible for the maintenance phase.

Assuming a maintenance sample size of 210 patients per group, using a 1:1 allocation to receive placebo or evolizumab 105 mg, maintenance analyzes performed at the 5% significance level will provide the following power: (1) Week 66 Clinical remission (SF mean daily score ≤3 and AP mean daily score ≤1): Assuming a placebo response rate of up to 20% at week 66, at least 90% power to detect a 15% treatment difference; (2) Endoscopic improvement: Assuming a 30% placebo improvement rate at week 66, there is approximately 90% power to detect a 15% treatment difference.

In addition, the study was designed to provide adequate power for the following secondary analyses: (1) Among patients who achieved clinical remission at week 14, clinical remission was achieved at week 66: sample size N = 186 (i.e., 93 per group) would Provides 80% power to detect a 20% treatment difference, assuming a placebo rate of up to 30%. This assumes a Week 14 clinical response rate of ≥22% in evolizumab patients.

Demographic and baseline characteristics of all randomized patients, such as age, sex, race, region, use of corticosteroids and immunosuppressives, anti-TNF therapy, disease duration, disease, will be summarized by treatment group using descriptive statistics for the induction and maintenance phases degree, average daily SF, average daily AP, CDAI, SES-CD, fecal calprotectin and CRP. Study drug exposure (number of study treatments and treatment duration) will be summarized by treatment group. Efficacy analysis

For the purposes of statistical analysis, the induction and maintenance phases will be considered separate studies. Patients who were not evaluable for efficacy at a specific time point due to missing data were considered nonresponders for all categorical endpoints. In addition, CD requires emergency therapy and/or surgical intervention and/or administration of prohibited drugs (e.g., anti-integrins, T- or B-cell depleting agents, TNF antagonists, IL-23 ± IL-12 antagonists (e.g., ustekinumab), antimetabolites, cyclosporine, tacrolimus, immunosuppressants such as AZA (or equivalent), 6-MP, and MTX) will be considered non-responders for the analysis By. The following analyzes will be performed on the primary efficacy endpoint and key secondary efficacy endpoints: (1) Subgroup analysis to assess consistency of results between prespecified subgroups (including baseline anti-TNF status [naïve vs. IR] , baseline CS status [receiving CS or not], baseline IS status [receiving IS or not], age, sex, fecal calprotectin, C-reactive protein, region) and (2) sensitivity analysis to evaluate the results to the main analysis Robustness of methods (e.g., handling exits, adjusting cohorts). induction period

Efficacy analyzes during the induction phase will be performed separately for each cohort and will include all patients who are randomized and receive at least one dose of the study (adjusted intention-to-treat population [mITT]). Patients will be grouped according to the treatment assigned at randomization.

Cohort 1 : Exploratory analyzes were performed when all patients in Cohort 1 (n = 300) completed the Week 14 visit or were prematurely discontinued from the study. At this point, the trial sponsors were only informed of the patient-level data and treatment allocation of patients in the induction phase of Cohort 1. Patients, site monitors, and investigators remained blinded to patient-specific treatment assignment. Induction data from Cohort 1 were exploratory in nature and were evaluated prior to beginning the critical induction period in Cohort 3. Maintenance data from Cohort 1 patients will form part of the critical maintenance analysis and will remain blinded to all sponsors and study sites until the database is locked.

Cohort 2 : Cohort 2 was considered a "tributary" cohort to help achieve the sample size required to sustain the study. All primary and secondary efficacy parameters will be summarized descriptively for each treatment group. Descriptive statistics will be used to summarize demographic and baseline characteristics for each treatment group, such as age, sex, race, region, use of corticosteroids and immunosuppressants, disease duration, and CD activity score.

Cohort 3 : The co-primary endpoint analysis will compare the proportion of patients achieving clinical response or endoscopic improvement at Week 14 in each evolizumab dose group versus the placebo group.

Differences between each evolizumab group and the placebo group will be assessed using the CMH test statistic, stratified by the factors used at randomization. Absolute treatment differences will be provided along with 95% two-sided confidence interval (CI) estimates.

All categorical secondary endpoints will be analyzed using the same methodology as the primary endpoint. For all efficacy endpoints, descriptive summary statistics will be provided for each treatment group.

Continuous endpoints will be analyzed using analysis of covariance (ANCOVA) models with the stratification variables used at randomization and the baseline values of study measurements as covariates.

The co-primary endpoints will each be tested at the 5% significance level, and both need to be significant for the co-primary endpoint to be considered significant. The overall type I error rate will be maintained at 5% using a stratified model to test both evolizumab doses versus placebo and corresponding key secondary endpoints. The remaining secondary endpoints and all exploratory endpoints will be considered supportive information and will not be adjusted for multiple comparisons.

The co-primary efficacy endpoints for the induction phase were: (1) the proportion of patients in clinical remission at week 14 and (2) the proportion of patients achieving endoscopic improvement at week 14. maintenance period

Efficacy analyzes during the maintenance phase will include all evolizumab induction patients randomized into the maintenance phase who receive at least one dose of study drug (mITT population). Patients will be grouped according to the treatment assigned when they are randomized into the maintenance phase.

For all categorical endpoints, differences in proportions between the two treatment groups will be assessed using the CMH test. The CMH test will include important stratification factors, such as those used to randomly assign patients to the maintenance phase. Stratification factors for rerandomization into the maintenance phase included sustained CDAI response at weeks 10 and 14. Due to changes in endpoints introduced in version 6 of the protocol, this stratification factor in the analysis replaces clinical response at week 14 with clinical response at week 66, and endoscopic improvement at week 14 with week 66 Weekly analysis of endoscopic improvements. Sensitivity analyzes will be performed to include the ongoing CDAI stratification factor used at randomization. Tests will be performed at a two-sided 5% significance level. Absolute treatment differences will be provided along with 95% two-sided CI estimates. All categorical secondary endpoints will be analyzed using the same methodology as the primary endpoint.

Continuous endpoints will be analyzed using an ANCOVA model with the stratification variables used at randomization and the baseline values measured by the study as covariates.

If the primary endpoint is statistically significant, key secondary endpoints will be tested sequentially.

Patients who are not evaluable for efficacy at a specific time point (e.g., due to missing data or early enrollment in the OLE study) will be considered non-responders for all categorical endpoints.

The co-primary efficacy endpoints of the maintenance phase were: among patients who achieved a CDAI-70 response at Week 14, (1) the proportion of patients who achieved clinical remission at Week 66 and (2) the proportion of patients who achieved endoscopic improvement at Week 66. Results Patient Demographics: Induction and Maintenance Phase

As shown in Table 5 below, baseline characteristics during the induction phase were mostly balanced across treatment groups. Differences in distribution by gender and region were noted. Table 5. Induction phase demographics and baseline treatments. Placebo (N=97) Evolizumab 105mg (N=143) Evolizumab 210mg (N=145) Age, mean ± SD 37.4 + 13.7 38.3 + 13.4 36.5 + 13.1 gender, male 60.8% 51.7% 52.4% female 39.2% 48.3% 47.6% white race 83.5% 81.8% 88.3% Body weight (kg), mean ± SD 71.5 + 17.1 76.7 + 20.1 73.5 + 17.6 area Eastern/Central Europe 32.0% 37.1% 44.8% Western Europe/Northern Europe, Canada, Australia, New Zealand 35.1% 28.0% 23.4% USA 12.4% 23.8% 22.8% Asia 0.0% 1.4% 0.0% latin america 15.5% 5.6% 3.4% other 5.2% 4.2% 5.5% Previous oral CS use 38.1% 37.8% 36.6% Previous IS use (IxRS) 24.7% 27.3% 24.8% Previous TNF use Not received TNF 41.2% 46.9% 49.7% Refractory/loss of response 46.4% 49.0% 42.1% intolerance 9.3% 3.5% 6.9% unknown 3.1% 0.7% 1.4%

As shown in Table 6 below, baseline disease characteristics during the induction phase were mostly balanced across treatment groups. Differences in disease site and fecal caldefensin distribution were noted. Table 6. Baseline disease characteristics during the induction period. Placebo (N=97) Evolizumab 105mg (N=143) Evolizumab 210mg (N=145) Disease duration (years), median (Q1 - Q3) 7.9 (3.7-14.3) 6.1 (2.0-11.7) 6.9 (3.0-14.4) disease site ileum 23.7% 16.8% 17.2% colon 17.5% 19.6% 25.5% Colon + Ileum 58.8% 63.6% 57.2% CDAI, mean ± SD 329.4 ± 64.0 326.3 ± 60.4 328.0 ± 61.0 AP score, mean±SD 2.03 ± 0.56 1.98 ± 0.59 1.97 ± 0.56 SF score, mean ± SD 6.40 ± 2.30 6.59 ± 2.43 6.76 ± 2.75 CD-PRO/SS function, mean ± SD 6.61 ± 2.53 6.53 ± 2.32 6.58 ± 2.16 CD-PRO/SS intestinal, mean ± SD 8.67 ± 2.34 8.80 ± 2.23 8.98 ± 2.21 SES-CD score, mean ± SD 13.32 ± 7.51 14.36 ± 7.21 13.12 ± 7.66 Fecal calprotectin (ug/g) <250 28.4% 17.1% 24.6% 250-500 15.8% 25.0% 12.7% ＞ 500 55.8% 67.9% 62.7% CRP (mg/L), median (Q1 - Q3) ＜ 2.87 31.3% 22.4% 29.0% 2.87-10 25.0% 30.8% 31.0% >10 43.8% 46.9% 40.0%

As shown in Table 7 below, baseline characteristics during the maintenance phase were mostly balanced across treatment groups. Differences in distribution by gender and region were noted. Table 7. Maintenance phase demographics and baseline treatment. Evolizumab/placebo (N=217) Evolizumab/Evolizumab 105mg (N=217) Age, mean ± SD 37.9 + 12.6 38.8 + 12.9 gender, male 45.6% 54.8% female 54.4% 45.2% white race 88.9% 83.9% Body weight (kg), mean ± SD 72.8 + 18.6 75.7 + 19.6 area Eastern/Central Europe 38.2% 40.1% Western Europe/Northern Europe, Canada, Australia, New Zealand 36.9% 24.9% USA 17.1% 24.9% Asia 2.8% 3.7% latin america 3.2% 5.1% other 1.8% 1.4% Previous oral CS use 42.4% 41.9% Previous IS use 32.7% 30.9% Previous TNF use Not received TNF 40.6% 42.4% Refractory/loss of response 53.0% 51.6% intolerance 6.5% 4.1% unknown 0 1.8% induction dose 105mg 49.8% 50.2% 210mg 50.2% 49.8%

As shown in Table 8 below, baseline disease characteristics during the maintenance phase were mostly balanced across treatment groups. Table 8. Baseline disease characteristics during the maintenance phase. Evolizumab/placebo (N=217) Evolizumab/Evolizumab 105mg (N=217) Disease duration (years), median (Q1 - Q3) 7.8 (2.7-13.5) 6.6 (2.9-12.1) disease site ileum 19.8% 16.1% colon 22.1% 18.9% Colon + Ileum 58.1% 65.0% CDAI, mean ± SD 327.3 ± 65.2 322.3 ± 58.4 AP score, mean ± SD 1.93 ± 0.54 1.97 ± 0.53 SF score, mean ± SD 6.55 ± 2.87 6.38±2.94 CD-PRO/SS function, mean ± SD 6.61 ± 2.16 6.70 ± 2.21 CD-PRO/SS intestinal, mean ± SD 8.70 ± 2.28 8.36 ± 2.60 SES-CD score, mean ± SD 13.02 ± 7.03 13.63 ± 6.87 Fecal calprotectin (ug/g) <250 21.5% 19.1% 250-500 16.4% 15.3% ＞ 500 62.1% 65.6% CRP (mg/L), median (Q1 - Q3) ＜ 2.87 25.8% 28.1% 2.87-10 32.3% 30.0% >10 41.9% 41.9% Induction period results

As shown in Table 9 below, in the adjusted intention-to-treat (mITT) population, evolizumab 210 mg administered at weeks 0, 2, 4, 8, and 12 resulted in improved outcomes in patients with Crohn's disease compared with placebo. The co-primary endpoints of clinical response and endoscopic improvement were not reached at week 14. Table 9. Co-primary endpoint results during the induction period. induction period clinical remission endoscopic improvements Etro 210mg N=145 PBO N=96 Etro 210mg N=145 PBO N=96 Proportion: 33.1% 29.2% Proportion: 27.4% 21.6% Effect size (95%Cl): 3.8% (-8.3%, 15.3%) Effect size (95%Cl): 5.8% (-5.4%, 17.1%) p-value: 0.5235 p-value: 0.3170

Of the 12 secondary endpoints, none showed statistically significant and clinically meaningful results, and none showed nominally significant and clinically meaningful results.

Overall, evolizumab was safe and well tolerated during the induction phase, with no new safety signals. Maintenance period results

As shown in Table 10 below, in the adjusted intention-to-treat (mITT) population, 105 mg of evolizumab was administered every 4 weeks compared with placebo during the maintenance phase (i.e., after the end of the induction phase at Week 14). Patients with Crohn's disease achieved the co-primary endpoints of clinical remission and endoscopic improvement at week 66. Table 10. Maintenance period co-primary endpoint results. maintenance period clinical remission endoscopic improvements Etro/Etro 105mg N=217 Etro/PBO N=217 Etro/Etro 105mg N=217 Etro/PBO N=217 Proportion: 35.0% 24.0% Proportion: 23.6% 12.2% Effect size (95%Cl): 11.3% (2.7%, 19.7%) Effect size (95%Cl): 11.5% (4.1%, 18.8%) p-value: 0.0088 p-value: 0.0026

Of the seven secondary endpoints, two showed statistically significant and clinically meaningful results, as shown in Table 11 below (indicated by bold outlines). In addition, four of the secondary endpoints showed nominally significant and clinically meaningful results (indicated by light boxes). Table 11. Maintenance period secondary endpoint results. end point Evolizumab/placebo (N=217) Evolizumab/Evolizumab 105mg (N=217) Treatment difference (95% CI) Nominal p-value adjust p-value Key Secondary, Family 2 Week 66 CDAI remission 66 (30.4%) 83 (38.2%) 8.1 (-0.87, 16.83) 0.0728 0.0971 Endoscopic relief at week 66 13 (5.9%) 26 (12.1%) 6.2 (0.54, 11.93) 0.0240 0.0480 Key secondary, family 3 Clinical remission at week 66 for clinical responders at week 14 38/97 (39.2%) 61/108 (56.5%) 17.3 (3.52, 30.27) 0.0127 0.0677 Clinical remission without CS at week 66 in patients with baseline CS 10/91 (11.0%) 27/93 (29.0%) 18.6 (11.07, 25.96) 0.0013 0.0480 Key secondary, family 4 durable clinical remission 43 (19.8%) 67 (30.9%) 11.2 (3.04, 19.24) 0.0063 0.0677 Key secondary, family 5 Changes to CD-PRO/SS - n 180 180 CD-PRO/SS Feature Changes in Week 66 -1.4 (0.2) -1.7 (0.2) -0.3 (-0.9, 0.4) 0.4009 0.4009 CD-PRO/SS Intestinal changes at week 66 -1.7 (0.3) -2.0 (0.3) -0.3 (-1.0, 0.4) 0.3849 0.4009

Overall, evolizumab was safe and well tolerated during the maintenance phase, with no new safety signals. sequence list SEQ ID NO instruction sequence 1 Anti-integrin beta7 antibody HVR-L1 RASESVDDLLH 2 Anti-integrin beta7 antibody HVR-L2 KYASQSIS 3 Anti-integrin beta7 antibody HVR-L3 QQGNSLPNT 4 Anti-integrin beta7 antibody HVR-H1 GFFITNNYWG 5 Anti-integrin beta7 antibody HVR-H2 GYISYSGSTSYNPSLKS 6 Anti-integrin beta7 antibody HVR-H3.v1 RTGSSGYFDF 7 Anti-integrin beta7 antibody HVR-H3.v2 ARTGSSGYFDF 8 anti-integrin beta7 antibody VL DIQMTQSPSSSLSASVGDRVTITCRASESVD DLLHWYQQKPGKAPKLLIKYASQSISGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQ GNSLPNTFGQGTKVEIKR 9 Anti-integrin beta7 antibody VH EVQLVESGGGLVQPGGSLRLSCAASGFFIT NNYWGWVRQAPGKGLEWVGYISYSGSTSYN PSLKSRFTIS RDTSKNTFYLQMNSLRAEDT AVYYCARTGSSGYFDFWGQGTLVTVSS 10 anti-integrin beta7 antibody LC DIQMTQSPSS LSASVGDRVT ITCRASESVD DLLHWYQQKPGKAPKLLIKYASQSISGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ GNSLPNTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGL SSPVTKSFN RGEC 11 Anti-integrin beta7 antibody HC.v1 EVQLVESGGG LVQPGGSLRLSCAASGFFIT NNYWGWVRQAPGKGLEWVGYISYSGSTSYN PSLKSRFTIS RDTSKNTFYLQMNSLRAEDT AVYYCARTGSSGYFDFWGQG TLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTV DKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG 12 Anti-integrin beta7 antibody HC.v2 EVQLVESGGG LVQPGGSLRLSCAASGFFIT NNYWGWVRQAPGKGLEWVGYISYSGSTSYN PSLKSRFTIS RDTSKNTFYLQMNSLRAEDT AVYYCARTGSSGYFDFWGQG TLVTVSSAST KGPSVFPLAPSSKSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTV DKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

Figure 1 shows the study protocol for the induction phase of a Phase III clinical study as described in Example 1. Anti-TNF = anti-tumor necrosis factor; CD = Crohn's disease; ETRO = evolizumab; IS = immunosuppressant; SC = subcutaneous; Wk = weekly. Figure 2 shows the study protocol for the maintenance phase of the Phase III clinical study as described in Example 1. CS = corticosteroids; ETRO = evolizumab; OLE = open-label extension; PBO = placebo; q4w = every 4 weeks; Re-Rx = rerandomization; Wk = week. Figure 3 shows a medical aid called the Bristol Stool Scale as described in Example 1.

TW202330613A_111143114_SEQL.xml

Claims (33)

A method of treating a patient suffering from Crohn's disease, the method comprising subcutaneously administering an integrin beta7 antagonist to the patient after initiation of induction therapy for a treatment period of at least 52 weeks or at least 66 weeks, wherein after the initiation of induction therapy After 14 weeks, the patient is determined to have achieved a reduction in Crohn's Disease Activity Index (CDAI) score of 70 points or more from baseline, and the patient maintains clinical remission during the treatment period. The method of claim 1, wherein the patient further maintains endoscopic improvement. The method of Claim 1 or Claim 2, wherein the patient further maintains endoscopic remission, corticosteroid-free clinical remission, or both endoscopic remission and corticosteroid-free clinical remission. The method of any one of claims 1 to 3, wherein the patient receives corticosteroid therapy in addition to the induction therapy during an induction period, wherein the induction period is 14 weeks, and wherein at the end of the induction period, the corticosteroid therapy Steroid therapy is reduced over time until discontinuation. As claimed in claim 4, wherein the corticosteroid therapy is (i) less than or equal to 20 mg of prednisone per day and wherein the corticosteroid therapy is reduced by 2.5 mg of prednisone per week until discontinued, or (ii) ) less than or equal to 6 mg of oral budesonide per day and in which corticosteroid therapy is reduced by 3 mg of oral budesonide every 2 weeks until discontinued. The method of any one of claims 1 to 5, wherein the integrin β7 antagonist is a monoclonal anti-integrin β7 antibody. The method of claim 6, wherein the anti-integrin β7 antibody is a chimeric antibody or a humanized antibody. The method of claim 6 or claim 7, wherein the anti-integrin β7 antibody is an antibody fragment. The method of any one of claims 6 to 8, wherein the anti-integrin β7 antibody includes three light chain hypervariable regions (HVR): HVR-L1, HVR-L2 and HVR-L3, and three heavy chains HVR: HVR-H1, HVR-H2 and HVR-H3, among which: (i) HVR-L1 contains the amino acid sequence RASESVDDLLH (SEQ ID NO: 1); (ii) HVR-L2 contains the amino acid sequence KYASQSIS (SEQ ID NO: 2); (iii) HVR-L3 contains the amino acid sequence QQGNSLPNT (SEQ ID NO: 3); (iv) HVR-H1 contains the amino acid sequence GFFITNNYWG (SEQ ID NO: 4); (v) HVR-H2 contains the amino acid sequence GYISYSGSTSYNPSLKS (SEQ ID NO: 5); and (vi) HVR-H3 contains the amino acid sequence RTGSSGYFDF (SEQ ID NO: 6); or the amino acid sequence ARTGSSGYFDF (SEQ ID NO: 7). The method of claim 9, wherein the anti-integrin β7 antibody includes: a light chain variable region (variable region domain), which includes the amino acid sequence shown in SEQ ID NO: 8; and a heavy chain variable region , which contains the amino acid sequence shown in SEQ ID NO: 9. The method of claim 9, wherein the anti-integrin β7 antibody comprises: a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; and a heavy chain comprising SEQ ID NO: 11 The amino acid sequence shown in SEQ ID NO: 12; or a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 12. The method of claim 9, wherein the anti-integrin β7 antibody comprises: a light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a heavy chain variable region comprising SEQ ID NO: 9 The amino acid sequence shown in . The method of claim 9, wherein the anti-integrin β7 antibody comprises: a light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 11 An amino acid sequence, or a heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 12. The method of any one of claims 9 to 13, wherein the anti-integrin β7 antibody is etrolizumab. The method of any one of claims 1 to 14, wherein the patient already has moderately to severely active Crohn's disease prior to administration of the induction therapy. The method of any one of claims 1 to 15, wherein the induction therapy includes one or more therapeutic agents selected from the group consisting of: 5-aminosalicylate, antibiotics, corticosteroids, systemic corticosteroids , thiopurine, methotrexate, anti-TNF agent, infliximab, adalimumab, certolizumab pegol, Vedolizumab, ustekinumab, natalizumab, efalizumab, evolizumab, Janus kinase (JAK) inhibitors, Upadacitinib and filgotinib. The method of claim 15 or claim 16, wherein the patient is determined to have (1) a CDAI score of greater than or equal to 220 and less than or equal to 480 at any time during the seven days prior to the start of the induction therapy and (2) during the Mean daily fluid/soft stool frequency (SF) score greater than or equal to 6 or mean daily SF greater than 3 and mean daily abdominal pain (AP) score greater than 1 for seven consecutive days before induction therapy. Claim the method of any one of items 15 to 17, wherein the patient is determined to have active inflammation, wherein the active inflammation is due to a SES-CD of greater than or equal to 7 as determined by ileocolonoscopy Determined by rating. Claim the method of any one of items 15 to 18, wherein the patient suffers from solitary ileitis or is post-ileocecal resection and wherein the patient is determined to have active inflammation, wherein the activity Inflammation was determined by a Simplified Endoscopic Index for Crohn's disease (SES-CD) score of greater than or equal to 4 as determined by ileocolonoscopy. The method of any one of claims 15 to 19, wherein the patient is refractory to or intolerant to one or more therapies selected from the group consisting of immunosuppressive therapy, corticosteroid therapy, and anti-TNF therapy. The method of claim 15 to 19, wherein the patient responds poorly to anti-TNF therapy. The method of claim 20, wherein the immunosuppressive therapy is selected from the group consisting of mercaptopurine, azathioprine and methotrexate. The method of claim 20, wherein the corticosteroid therapy is selected from the group consisting of prednisone and oral cortisol. The method of claim 20 or claim 21, wherein the anti-TNF therapy is selected from the group consisting of infliximab, adalimumab and pegylated certolizumab. The method of any one of claims 1 to 24, wherein the anti-integrin β7 antibody is administered at a rate of 105 mg every 4 weeks from the 14th week to at least the 52nd week or to at least the 66th week after the initiation of the induction therapy. Administer a uniform dose. If the method of any one of claims 1 to 25, in which clinical remission is the cause, averaged over the four-day period preceding the assessment or averaged over the seven-day period preceding the assessment, the average daily score for Liquid/Loft Stool Frequency (SF) is less than or It was determined as equal to three and the mean daily abdominal pain (AP) score was less than or equal to one and there was no worsening of SF or AP compared to baseline. The method of claim 2 to 26, wherein endoscopic improvement is determined by a Simplified Endoscopic Index for Crohn's Disease (SES-CD) score, and wherein the SES-CD score is the same as that determined at baseline. The SES-CD score was reduced by at least fifty percent. The method of any one of claims 3 to 27, wherein the patient has discontinued corticosteroid therapy and wherein the patient has not received treatment with one or more corticosteroids for at least 24 consecutive weeks after discontinuing corticosteroid therapy. The method of any one of claims 3 to 28, wherein the endoscopic response is determined by a SES-CD score and wherein the SES-CD score is less than or equal to four. The method of claim 29, wherein the patient is an ileal patient and the SES-CD score is less than or equal to two. The method of request 29 or request 30, wherein the SES-CD score does not contain segments with subcategory scores greater than one. The method of claim 31, wherein the subcategory is selected from the group consisting of ulcer, affected surface, and size and degree of narrowing. The method of any one of the preceding claims, wherein the integrin β7 antagonist is administered using a prefilled syringe or a combination of a prefilled syringe and an automatic injector.