WO2019160907A1 - Regulating cd4+ t cells to treat clostridium difficile infection - Google Patents

Abstract

Clostridium difficile infections (GDIs) are the number one cause of hospital acquired diarrhea in the United States. Several clinical studies have reported higher incidence and severity of GDI in patients with ulcerative colitis and/or Crohn's disease. In order to understand the factors underlying increased severity of GDI in IBD patients, we utilized a Dextran Sulfate Sodium (DSS) murine model of inflammatory colitis. In support of clinical observations, our data showed that mice treated with DSS and then infected with C. difficile developed a more severe C. difficile disease compared to untreated mice. Increased severity of disease was measured by increased mortality, weight loss and clinical scores. Importantly, comparison of C. difficile burden between mice with prior DSS colitis and untreated controls showed no differences in C. difficile colonization between the two groups, suggesting that increased severity of disease might be due to the host immune response to infection. Immunophenotyping of immune cells recruted to the colon at the peak of GDI revealed increased levels of CD4+ T cells at the site of infection in mice with prior DSS colitis. Furthermore, depletion of CD4+ T cells using a monoclonal antibody protected mice with prior DSS colitis from severe C. difficile disease. These data have identified a novel role for CD4+ T cells in contributing to increased severity of GDI. Our current studies are aimed at understanding the downstream mechanisms by which these T cells are acting to drive severe disease.

Description

REGULATING CD4+ T CELLS TO TREAT

CLOSTRIDIUM DIFFICILE INFECTION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No. 62/630,370 filed February 14, 2018, the disclosure of which is incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.

All 14734, and AI124214, awarded by The National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

The Gram positive anaerobe Clostridium difficile (C. difficile) causes mild to severe antibiotic associated diarrhea, pseudomembranous colitis, toxic megacolon, and death. Clostridium difficile infections (CDIs) are the number one cause of hospital- acquired diarrhea in the United States. Several clinical studies have reported higher incidence and severity of CDI in patients with one of the two major forms of inflammatory bowel disease (IBD): ulcerative colitis and Crohn’s disease.

Clostridium difficile is a spore-forming, Gram-positive, anaerobic bacterium that was identified as the cause of antibiotic-associated pseudomembranous colitis in 1978 (Bartlett et al., 1978). Despite an overall decline in infectious disease mortalities over the last 3.5 decades, mortality due to diarrheal diseases in the U.S. continues to rise (Bcheraoui et al., 2018). This trend is attributed to the spread of hypervirulent strains of C. difficile over the last decade (Bacci et al., 2011; Shuman and Malani, 2018), including the epidemic ribotype 027 strain used in our studies. The Center for Disease Control estimates that C. difficile causes almost 500,000 infections and 29,000 deaths annually in the United States alone (Lessa et al., 2015). These reports and others highlight the importance of studying C. difficile infection (CDI) and identifying novel targets for reducing disease severity.

Inflammatory Bowel Disease (IBD) is a set of conditions, including Crohn’s disease and ulcerative colitis, characterized by gastrointestinal tract inflammation due to dysregulation of the immune response to commensal bacteria (Baumgart and Carding, 2007). IBD is an independent risk factor for CDI (Issa, et al., 2007;

Nguyen et al., 2008, Cojocariu et al., 2015). Studies from the National Inpatient Sample, the largest short-stay hospital discharge database in the U.S., as well as single-center studies have reported a higher incidence of CDI in IBD patients compared to non-IBD patients (Rodemann et al., 2007; Issa, et al., 2007; Nguyen et al., 2008). Moreover, CDI in IBD patients is more severe as characterized by reduced times from admission to CDI, longer hospital stays, increased surgery and in-hospital mortality rates (Rodemann et al., 2007; Nguyen et al., 2008;

Ananthakrishnan et al., 2008). However, the cause of increased incidence and severity of CDI in IBD patients remains poorly understood.

There is a long felt need in the art for compositions and methods useful for preventing and treating CDIs, particularly those in subjects with colitis and inflammatory bowel disease. The present application satisfies these needs.

SUMMARY OF THE INVENTION

T-helper 17 (Thl7) cells are a unique CD4⁺ T-cell subset characterized by production of interleukin- 17 (IL- 17). The present application shows an increase in Thl7 cells is associated with increased severity of infection and that Thl7 cells in an inflammatory bowel disease model produce predominantly IL-17A, but little IFN-g or IL-22. The use of antibodies directed against IL-17A or against its receptor were protective in the early stages of C. difficile infection.

The present application further discloses that decreasing the number of CD4+ T cells (also referred to as CD4 cells and CD4+ cells) decreases severity of infection. It is also disclosed herein that in humans high levels of IL-6 or IL-23 in the serum of C. difficile subjects correlates with severe disease increased risk for mortality post infection. Therefore, the invention encompasses methods for measuring IL-6 and IL-23 and determining whether a subject is a risk for increased mortality post infection.

Based on the disclosure herein, the present invention provides for preventing or treating CDIs by inhibiting an increase in the number of CD4+ T cells or by decreasing the number of CD4+ cells. In one aspect, the CD4+ cells are Thl7 cells. Based on the disclosures provided herein, the present application encompasses preventing and treating C. difficile infection by targeting CD4+ cells and Thl7 cells. The targeting can include methods to kill the cells or to inhibit the cells from increasing in number. In one aspect, the CD4 protein on a CD4+ cell can be targeted with an inhibitor. Useful antibody inhibitors against human CD4 include, but are not limited to, OKT-4 and RPA-T4.

Based on the disclosure herein, the present invention includes compositions and methods for designing and implement treatments targeting, inter alia, CD4+ T cells, Thl7 cells, or upstream/downstream mechanisms (including, IL-23, IL-22, IL- 17A, IL-17F, IL-17RA, IL-6, and TGF ) to protect against severe C. difficile disease.

CDI is the number one hospital- acquired infection in the United States. CDI is more common and severe in inflammatory bowel disease patients. Disclosed herein are studies on the mechanism by which prior colitis exacerbates CDI. Mice were given Dextran Sulfate Sodium (DSS)-colitis, recovered for two weeks, and infected with C. difficile. Mortality and CDI severity were increased in DSS -treated mice compared to controls. Severe CDI was dependent on CD4+ T cells, highlighting the importance of studying their role in the pathogenesis of C. difficile. Adoptive transfer of Thl7 cells was sufficient to increase CDI-associated mortality. Finally, in humans, the Thl7 cytokines IL-6 and IL-23 are disclosed herein to be associated with severe CDI and patients with high serum IL-6 were 7.6 times more likely to die post-infection. These findings establish a central role for Thl7 cells in CDI pathogenesis following colitis and identify them as a potential target for preventing severe disease.

Without wishing to be bound by any particular hypothesis, it is hypothesized herein that in IBD patients, aberrant immune responses resulting in gut inflammation are the cause of increased CDI severity. Indeed, studies in mouse models as well as human patients have shown that the type of immune response mounted against CDI by the host can determine the severity and outcome of infection (Feghaly et ah,

2013; Buonomo et ah, 2016; Cowardin et ah, 2016). Here, Dextran Sulfate Sodium (DSS) induced colitis was used to model how prior gut inflammation due to IBD can influence the outcome of subsequent CDI by altering the host immune response.

The results show that colitis induces Thl7 cells that are still present in the mesenteric lymph nodes after full recovery from colitis. It is demonstrated herein using adoptive transfer that these Thl7 cells are sufficient to increase mortality after challenge with C. difficile. Prior to this study, previous reports showed similar CDI- associated mortality in WT and Ragl-/- mice and so a role for T cells during primary CDI has been overlooked (Abt et ah, 2015). Disclosed herein is an important and novel role for CD4+ T cells in general, and Thl7 cells in particular, during CDI and identify them as a potential therapeutic target for patients with IBD who are at risk for severe disease.

The present application provides for the use of inhibitors such as antibodies directed against cytokines such as IL-17A that bind with the cytokine and inhibit the cytokine from binding with its cognate receptor or inhibit it from eliciting its normal activity.

In one embodiment, an inhibitor of the invention includes, but is not limited to, a small molecule, drug, prodrug, or an antibody, or a biologically active fragment or homolog thereof.

The present application provides for the use of antibodies directed against cytokine receptors such as IL-17RA that inhibit the receptor from binding with its cognate ligand or from performing its functions.

In one aspect, the methods of the present application prevent an increase in cytokines, including, but not limited to, IL-6, IL-17, IL-22, and IL-23.

In one aspect, the methods of the invention inhibit CDI mortality. In one aspect, they inhibit increased CDI mortality in a subject.

The present application provides for the use of inhibitors, including antibodies, directed against the CD4 protein. In one aspect, an antibody of the invention is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, F(ab)2 fragments of a monoclonal antibody, a chimeric antibody, a single-chain antibody, a synthetic antibody, bi-specific antibody, and a humanized antibody, and active fragments and homologs thereof.

Antibodies of the invention can include their fragments that maintain the desired activity described herein, as well as those that have been humanized, are single chain, etc.

The present application provides compositions and methods useful for preventing or treating CDI in a subject in need of such a treatment. In one aspect, the method comprises administering to the subject a pharmaceutical composition comprising a pharmaceutical-acceptable carrier, optionally at least one additional therapeutic agent, and an effective amount of at least one inhibitor, wherein the useful inhibitors include, but are not limited to, an inhibitor of CD4+ T cells,

Thl7 cells, CD4, IL-17A, IL-17F, IL-17RA, IL-22, and IL-6. In one aspect, the inhibitor of IL-17A is monoclonal antibody clone 41809. In one aspect, the inhibitor of IL-17RA is monoclonal antibody clone 133617. In one aspect, the inhibitor of CD4 is the antibody OKT-4 or RPA-T4. In one aspect, two or more inhibitors are administered, wherein the two or more inhibitors are directed against different molecules. In one aspect, two inhibitors are administered. In another aspect, three or more inhibitors are administered, wherein the three or more inhibitors are directed against different molecules. In one aspect, when more than one inhibitor is administered each one is an antibody, or a biologically active fragment or homolog thereof.

Subjects with colitis are at a higher mortality risk when infected with C. difficile , therefore, in one embodiment, as a preventive measure a subject with colitis can be administered a pharmaceutical composition of the invention prior to being admitted to a hospital or at the time of admission, where the subject is being admitted for a procedure or due to some illness. In one aspect, the composition is administered before the subject is infected. In one aspect, the colitis is acute colitis. In one aspect, the subject has irritable bowel syndrome. In one aspect, the colitis is ulcerative colitis, Crohn’s colitis, diversion colitis, ischemic colitis, infectious colitis, fulminant colitis, collagenous colitis, chemical colitis, microscopic colitis, lymphocytic colitis, or atypical colitis.

In one embodiment, the subject has had at least one previous C. difficile infection.

In one embodiment, when a subject with colitis is diagnosed with a C.

difficile infection in its early stages, a pharmaceutical composition of the invention is administered to the subject.

In one embodiment, the pharmaceutical composition is administered within five days after the subject has been infected with C. difficile. In one aspect, the pharmaceutical composition is administered within three days after the subject has been infected with C. difficile. In another aspect, the pharmaceutical composition is administered within two days after the subject has been infected with C. difficile. In yet another aspect, the pharmaceutical composition is administered within one day after the subject has been infected with C. difficile. The present application further encompasses compositions and methods for determining if there is a CDI or the severity of a CDI infection in a subject with colitis and to determine the likelihood or risk of mortality from the infection. In one aspect, higher levels of IL-6 and IL-23 in a subject infected with C. difficile is an indication that it is a severe infection. In one aspect, the high levels of IL-6 and IL- 23 are in the ceca of the subject. By higher levels is meant higher than a standard or that the levels were measured before infection and one or more times during the course of infection in the subject. In one aspect, a subject with severe CDI is at a risk for increased mortality.

An antibody of the invention can be administered in any suitable fashion, including, but not limited to, intravenously, intraperitoneally, locally, and parenterally.

The dose of antibody to be administered, the number of doses to be delivered, and the time course of administration can be determined based on things such as the health and age of the subject and the severity of the cancer and the specific cancer being treated. In one aspect, a dose of an antibody of the invention can be from about 0.1 mg/kg body weight to about 25.0 mg/kg body weight, or about 1.0 to about 15, or about 5.0 to about 10 mg/kg body weight. In one aspect, a dose is selected from the group consisting of 0.1, 0.5, 0.75, 0.83, 1.0, 1.25, 1.5, 2.0,

2.5, 3.0, 3.5, 4.0, 4.5, 5,0, 5.5, 6.0, 6,5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,

11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, and 25.0 mg/kg body weight.

The number of doses to be administered can be, for example, one or more per day, per week, per month or per year. This may vary, for example, depending on, for example, the response of the cancer to the treatment. The number of doses to be administered can be varied as well for the same reasons. When more than one dose is administered, the doses can be administered, for example every day, every other day, every third day, every fourth day, weekly, twice weekly, three times weekly, monthly, or any regimen determined by the clinician treating the subject. In one aspect, the pharmaceutical composition is administered at least twice. In another aspect, it is administered at least five times. In yet another aspect, it is administered at least ten times. The present invention further provides a pharmaceutical composition comprising an effective amount of at least one antibody of the invention, a pharmaceutically-acceptable carrier, and optionally at least one additional therapeutic agent.

The present invention further provides a kit. The kit may comprise at least one antibody of the invention, a pharmaceutical composition, a pharmaceutically- acceptable carrier, an applicator, and an instructional material for the use thereof.

Various aspects and embodiments of the invention are described in further detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Figure 1 (comprising FIGS 1A to 1H): Increased CDI-associated mortality in mice with prior DSS colitis. 6-week old C57B1/6J mice were treated with 2% DSS or no treatment in the drinking water for 6 days, then allowed to recover for 2 weeks. Both groups were treated with antibiotics and infected with 1c10⁶-1c10⁷ CFU of C. difficile strain R20291. (A) Model for CDI after recovery from DSS colitis. (B) DSS-induced weight loss and recovery before C. difficile infection (h=18-19 per group). (C) FITC dextran detection assay in the serum was used to determine gut permeability in untreated mice, during acute DSS colitis, and after recovery from DSS colitis (n=8-l0 per group). After infection, survival (D), clinical scores: weight loss, coat appearance, eyes/nose discharge, activity, posture and diarrhea (E) and weight loss (F) of the two groups were assessed twice a day for 7 days (n=l6 per group). Day 0 post infection in (D-F) refers to Day 27 in the timeline in (A). (G) Ceca contents were collected from mice with/without prior DSS colitis on day 2. The samples were homogenized and plated anaerobically on C. difficile- selective agar plates (h=10-14 per group). (H) Toxins were detected within cecal contents using the C. difficile TOXA/B enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (n=l8 per group). Data represent mean ± SEM.*p<0.05, **r<0.01, ***r<0.001, ****p<0.000l by two- tailed t-test (B, F-H), one-way analysis of variance (C), Logrank statistical test (D) and Mann- Whitney test (E). These data are combined from two independent experiments. See also Figures 5 (comprising 5A and 5B) and 6 (comprising 6A to 6D) (also referred to as Sl and S2).

Figure 2 (comprising FIGS 2A to 2H): CD4+ T cells increase C. difficile disease severity in mice with prior DSS colitis. Mice were given DSS or regular water (as described before), recovered for 2 weeks and infected with C. difficile. Colon tissue was isolated, processed into single-cell suspension and stained for flow cytometry. (A) Representative flow plots depicting colonic CD4+ T cells (CD45+ TCRP+). (B) Quantification of colonic CD4+ T cells (n=10-12 per group). (C-F) Mice were injected i.p. with 400 pg a-CD4 or IgG isotype control on days -6, -3 and on the day of infection with C. difficile. (C) Representative flow plots showing depletion of colonic CD4 T cells. (D-F) Survival, weight loss and clinical scores were assessed twice daily for 6 days (n=6 per group). (G) Donor mice were given 2% DSS or regular water for 6 days. On day 7, CD4+ T cells were isolated from the colon and mesenteric lymph nodes using negative selection magnetic beads and the purity was checked by flow cytometry. (H) lxl()⁶ CD4+ T cells were transferred into naive, antibiotic-treated recipients. The following day, recipient mice were infected with C. difficile. Survival curves were compared using a Logrank statistical test. Weight loss and clinical scores were compared using a one-way analysis of variance and Kruskal Wallis test, respectively (n=10 per group per experiment).

Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, # p<0.05: DSS IgG compared to Untreated IgG. Data are combined from two independent experiments. See also Figure 7 (comprising 7A and 7B), also referred to as FIG. S3.

Figure 3 (comprising FIGS. 3A to 31): Increased Thl7 responses in mice with prior DSS colitis cause severe subsequent CDI. (A-D) Mice were given DSS or regular water (as described before), recovered for 2 weeks and infected with C. difficile. On day 2 of infection, colon and mesenteric lymph node tissue was isolated, processed into single-cell suspension and stained for flow cytometry. (A) Representative flow plots and quantification of Thl7 cells (CD45+ TCRP+ CD4+ ROR ;/t+ FOXP3-) (n=5-6 per group per experiment). (B) IL-17-GFP reporter mice were used to determine the number and frequency of IL-17A+ CD4 T cells during infection by flow cytometry (n=5 per group per experiment). (C) For protein data, cecal tissue was homogenized using a bead beater, data is normalized to total protein quantified using a Pierce BCA Assay (n=8-14 per group). (D) cecal IL-23 protein levels plotted against clinical scores. (E) Mice were injected i.p. with 125 pg a-IL- 17RA or IgG isotype control on days -1, 1 and 2 of infection (n=l6 per group). (F- I) Naive CD4+ T cells were isolated from the spleen of IL17A-GFP reporter mice and differentiated into Thl7 cells ex vivo in the presence of TGFP and IL-6. (F)

CD3e+CD4+IL-l7A+ and CD3e+CD4+IL-l7A- T cells were sorted and lxl0⁶cells were transferred i.p. to antibiotic-treated recipients. (G-I) One day following the T cell transfer, recipients were infected with C. difficile. Survival, weight loss and clinical scores were assessed twice daily (n= 10 per group). Data represent mean ± SEM.*p<0.05, **r<0.01, ***p<0.00l by a two-tailed student t-test (A-C), Pearson correlation statistical test (D), Logrank statistical test (E, G), Kruskal-Wallis test (H) and one-way analysis of variance (I). Data are representative of two independent experiments. See also Figures 7 (comprising 7A and 7B) and 8 (comprising 8A to 8C) (also referred to as Figs. S3 and S4).

Figure 4 (comprising FIGS. 4A to 4C): The Thl7 cytokines IL-6 and IL- 23 in the serum of C. difficile patients correlate with severe disease. IL-23 and IL-17A in the serum of C. difficile patients were quantified using high sensitivity ELIS As from R&D. IL-6 and IL-4 were quantified using a Luminex bead-based multiplex assay. (A) Kaplan-Meier survival curve post CDI diagnosis for patients with non-severe CDI (WBC<l5,000 per microliter, n=226) and severe CDI

(WBC³l5,000 per microliter, h=100). (B) serum IL-17A, IL-23, IL-6 and IL-4 for patients with non-severe and severe CDI, n=323, 323 and 362, 379 respectively. (C) Kaplan-Meier survival curve post CDI diagnosis for patients categorized into quartiles based on IL-6 serum levels, n=92-94 per quartile. Data represent mean ± SEM.*p<0.05, **r<0.01, ***p<0.00l using Logrank (A, C) and Mann- Whitney (B) statistical tests. See also Table Sl.

Figure 5 (comprising FIGS. 5A and 5B), (also referred to as Figure SI): Recovery from DSS colitis. Related to Figure 1. Mice were given untreated or DSS-treated water and cecal tissue was collected either during acute DSS or after recovery. (A) cecal sections were isolated and fixed in Bouin’s solution for 24 hours before paraffin embedding and H&E staining. Samples were then blindly scored by two independent scorers based on epithelial disruption, submucosal edema, inflammatory infiltrate, mucosal thickening and luminal exudates. (B) cecal tissue was homogenized using a bead beater, cytokine levels were determined using a Luminex MAGPIX bead-based multiplex analyzer and normalized to total protein quantified using a Pierce BCA Assay. Data represent mean ± SEM.*p<0.05, **r<0.01, ***p<0.00l using a one-way analysis of variance. Data are

representative of two independent experiments.

Figure 6 (comprising Figs. 6A to 6C), (also referred to as Figure S2): DSS-induced changes in the gut microbiota alone are not sufficient to increase CDI severity. Related to Figure 1. Mice were given untreated or DSS-treated water (A) Multidimensional scaling (MDS) plot of Bray-Curtis dissimilarity index comparing fecal communities from mice after recovery from DSS colitis vs untreated mice, before antibiotic treatment. (B) Survival curve of untreated and DSS-treated mice that were co-housed or housed separately for three weeks then infected with C. difficile. (C) MDS plot of Bray-Curtis dissimilarity index from cecal samples of DSS-treated and untreated mice post antibiotic treatment and before C. difficile infection. (D) Bar chart displaying relative abundance of family level OTUs in the cecal communities of DSS-treated and untreated mice post antibiotic treatment and before C. difficile infection. *p<0.05, statistical comparisons were made using permutational multivariate analysis of variance (PERMANOVA) (A,C) and a Logrank statistical test (B).

Figure 7 (comprising Fig. 7A and 7B), (also referred to as Figure S3): Characterization of immune responses to CDI in untreated and DSS-treated mice. Related to Figures 2 and 3. Mice were given DSS or regular water (as described before), recovered for 2 weeks and infected with C. difficile. On day 2 of infection, Colon tissue was isolated, processed into single-cell suspension and stained for flow cytometry. (A) Quantification of neutrophils (CD45+ CDllb-i- Ly6G+), eosinophils (CD45+ CDllb-i- SiglecF+), Ly6C^hl and Ly6C^lG monocytes (CD45+ CDllb-i- Ly6C+) (n=9-l0 per group per experiment). (B) Quantification of different (CD45+ TCRP+/CD3+) CD4+ T cell subsets, as well as CD8+ T cells (n=5-6 per group per experiment). Data represent mean ± SEM.*p<0.05, **p<0.0l by a two-tailed student t-test. Data are representative of at least two independent experiments.

Figure 8 (comprising Figs. 8A, 8B, and 8C) (also referred to as Figure S4): Pathogenic Thl7 cells in DSS mice are predominantly IFNy- and IL-22-.

Related to Figure 3. Mice were given DSS or regular water (as described before) and recovered for 2 weeks. On day 0 before infection and day 2 after infection, colon and mesenteric lymph node tissue was isolated, processed into single-cell suspension, stimulated with PMA/ionomycin and stained for flow cytometry. (A) Representative flow plots and quantification of Thl7 cells (CD45+ TCRP+ CD4+) on days 0 and 2 of infection, (n=6-10 per group per experiment). (B) On day 2 of infection, cecal tissue was homogenized using a bead beater, protein levels were measured by ELISA or a Luminex bead-based multiplex assay. Data is normalized to total protein quantified using a Pierce BCA assay (h=10-12 per group). (C) Survival analysis of mice injected i.p. with 125 pg a-IL-l7A or IgG isotype control on days -1, 1 and 2 of infection with C. difficile (n=8 per group). Data represent mean ± SEM.*p<0.05, **p<0.0l by a two-tailed student t-test or Mann-Whitney statistical test (A, B) and Logrank statistical test (C).

DETAILED DESCRIPTION

Abbreviations and Acronyms

Abx- antibiotics (also referred to as ABX)

aCDT- anti-CDT neutralizing nanobody

ANOVA- analysis of variance

aTLR2- anti-TLR2 neutralizing antibody

BMDC- bone marrow derived dendritic cells

C. difficile- Clostridium difficile

CD4- cluster of differentiation 4 protein

CDI- Clostridium difficile infection

CDT- C. difficile transferase

CFU- colony forming unit

DSS- Dextran Sulfate Sodium

EOP- eosinophil progenitor

FITC- Fluorescein isothiocyanate

H&E- hematoxylin and eosin

IBD- inflammatory bowel disease

IL- interleukin

IL-17RA- interleukin 17 receptor A

IL1R2- interleukin 1 receptor type 2

IL4R- interleukin 4 receptor

IL-25- Interleukin 25 IL-33- Interleukin 33

kg- kilogram

LSR- lipolysis stimulated lipoprotein receptor

mg- milligram

mg- microgram

PBS- phosphate buffered saline

PRR- Pattern Recognition Receptor

SEAP - Secreted Embryonic Alkaline Phosphatase

siRNA- small interfering RNA

TLR2- Toll- Like Receptor 2

T-reg- regulatory T cell

TSLP- thymic stromal lymphopoietin

WBC- white blood cell

YM-l- chitinase-like 3 (also referred to as Chil3)

Definitions

In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.

Unless defined otherwise, all 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section.

The articles“a” and“an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term "about,” as used herein, means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term“about.”

The terms“additional therapeutically active compound” or“additional therapeutic agent”, as used in the context of the present invention, refers to the use or administration of a compound for an additional therapeutic use for a particular injury, disease, or disorder being treated. Such a compound, for example, could include one being used to treat an unrelated disease or disorder, or a disease or disorder which may not be responsive to the primary treatment for the injury, disease or disorder being treated.

As use herein, the terms“administration of’ and or“administering” a compound should be understood to mean providing a compound of the invention or a prodrug of a compound of the invention to a subject in need of treatment.

An "agent" useful for treating a C. difficile infection, as used herein means any compound, molecule, or cell that can directly or indirectly be used to treat an infection. Cells can include, for example, eosinophils or one of more types of bacteria. Such an "agent" can also be referred to as a "useful agent".

As used herein, an“agonist” is a composition of matter which, when administered to a mammal such as a human, enhances or extends a biological activity attributable to the level or presence of a target compound or molecule of interest in the mammal.

As used herein,“alleviating a disease or disorder symptom,” means reducing the severity of the symptom or the frequency with which such a symptom is experienced by a patient, or both. A“therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

The term“alterations in peptide structure” as used herein refers to changes including, but not limited to, changes in sequence, and post-translational modification.

As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D

Glutamic Acid Glu E

Lysine Lys K

Arginine Arg R

Histidine His H

Tyrosine Tyr Y

Cysteine Cys C

Asparagine Asn N

Glutamine Gln Q

Serine Ser s

Threonine Thr T

Glycine Gly G

Alanine Ala A

Valine Val V

Leucine Leu L

Isoleucine He I

Methionine Met M

Proline Pro P

Phenylalanine Phe F

Tryptophan Trp W

The term“amino acid” is used interchangeably with“amino acid residue,” and may refer to a free amino acid and to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide. The expression“amino acid” as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids.“Standard amino acid” means any of the twenty standard L-amino acids commonly found in naturally occurring peptides.“Nonstandard amino acid residue” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein,

“synthetic amino acid” also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide’s circulating half-life without adversely affecting their activity.

Additionally, a disulfide linkage may be present or absent in the peptides of the invention.

Amino acids have the following general structure:

Amino acids may be classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxy lie (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.

The nomenclature used to describe the peptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified.

The term“basic” or“positively charged” amino acid, as used herein, refers to amino acids in which the R groups have a net positive charge at pH 7.0, and include, but are not limited to, the standard amino acids lysine, arginine, and histidine.

As used herein, an“analog”, or "analogue", of a chemical compound is a compound that, by way of example, resembles another in structure but is not necessarily an isomer (e.g., 5-fluorouracil is an analog of thymine).

An“antagonist” is a composition of matter which when administered to a mammal such as a human, inhibits a biological activity attributable to the level or presence of a compound or molecule of interest in the mammal.

The term“antibody,” as used herein, refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin subunit molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.

An“antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules.

An“antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules.

By the term“synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

As used herein, the term“secondary antibody” refers to an antibody that binds to the constant region of another antibody (the primary antibody).

The term“antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates.

The term "antigenic determinant" as used herein refers to that portion of an antigen that makes contact with a particular antibody (i.e., an epitope). When a protein or fragment of a protein, or chemical moiety is used to immunize a host animal, numerous regions of the antigen may induce the production of antibodies that bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the "immunogen" used to elicit the immune response) for binding to an antibody. The term“antimicrobial agents” as used herein refers to any naturally- occurring, synthetic, or semi-synthetic compound or composition or mixture thereof, which is safe for human or animal use as practiced in the methods of this invention, and is effective in killing or substantially inhibiting the growth of microbes.

“Antimicrobial” as used herein, includes antibacterial, antifungal, and antiviral agents.

The term "at least two antibiotics", as used herein, means at least two different antibiotics.

As used herein, the term“antisense oligonucleotide” or antisense nucleic acid means a nucleic acid polymer, at least a portion of which is complementary to a nucleic acid which is present in a normal cell or in an affected cell.“Antisense” refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences. The antisense oligonucleotides of the invention include, but are not limited to, phosphorothioate oligonucleotides and other modifications of oligonucleotides.

An“aptamer” is a compound that is selected in vitro to bind preferentially to another compound (for example, the identified proteins herein). Often, aptamers are nucleic acids or peptides because random sequences can be readily generated from nucleotides or amino acids (both naturally occurring or synthetically made) in large numbers but of course they need not be limited to these.

As used herein, the term "attach", or "attachment", or "attached", or

"attaching", used herein interchangeably with "bind", or "binding" or "binds' or "bound" refers to any physical relationship between molecules that results in forming a stable complex, such as a physical relationship between a ligand, such as a peptide or small molecule, with a "binding partner" or "receptor molecule." The relationship may be mediated by physicochemical interactions including, but not limited to, a selective noncovalent association, ionic attraction, hydrogen bonding, covalent bonding, Van der Waals forces or hydrophobic attraction.

As used herein, the term "avidity" refers to a total binding strength of a ligand with a receptor molecule, such that the strength of an interaction comprises multiple independent binding interactions between partners, which can be derived from multiple low affinity interactions or a small number of high affinity

interactions.

The term“binding” refers to the adherence of molecules to one another, such as, but not limited to, enzymes to substrates, ligands to receptors, antibodies to antigens, DNA binding domains of proteins to DNA, and DNA or RNA strands to complementary strands.

“Binding partner,” as used herein, refers to a molecule capable of binding to another molecule.

The term“biocompatible”, as used herein, refers to a material that does not elicit a substantial detrimental response in the host.

The term“biological sample,” as used herein, refers to samples obtained from a subject, including, but not limited to, skin, hair, tissue, blood, plasma, serum, cells, sweat, saliva, feces, tissue and/or urine.

As used herein, the term“biologically active fragments” or“bioactive fragment” of the polypeptides encompasses natural or synthetic portions of the full length protein that are capable of specific binding to their natural ligand or of performing the function of the protein. For example, a“functional” or“active” biological molecule is a biological molecule in a form in which it exhibits a property by which it is characterized. A functional enzyme, for example, is one which exhibits the characteristic catalytic activity by which the enzyme is characterized.

As used herein, the term "biopsy tissue" refers to a sample of tissue that is removed from a subject for the purpose of determining if the sample contains cancerous tissue. In some embodiment, biopsy tissue is obtained because a subject is suspected of having cancer. The biopsy tissue is then examined for the presence or absence of cancer.

"Blood components" refers to main/important components such as red cells, white cells, platelets, and plasma and to other components that can be derived such as serum. As used herein, the term "carrier molecule" refers to any molecule that is chemically conjugated to the antigen of interest that enables an immune response resulting in antibodies specific to the native antigen.

The term“cell surface protein” means a protein found where at least part of the protein is exposed at the outer aspect of the cell membrane. Examples include growth factor receptors.

As used herein, the term“chemically conjugated,” or“conjugating chemically” refers to linking the antigen to the carrier molecule. This linking can occur on the genetic level using recombinant technology, wherein a hybrid protein may be produced containing the amino acid sequences, or portions thereof, of both the antigen and the carrier molecule. This hybrid protein is produced by an oligonucleotide sequence encoding both the antigen and the carrier molecule, or portions thereof. This linking also includes covalent bonds created between the antigen and the carrier protein using other chemical reactions, such as, but not limited to glutaraldehyde reactions. Covalent bonds may also be created using a third molecule bridging the antigen to the carrier molecule. These cross-linkers are able to react with groups, such as but not limited to, primary amines, sulfhydryls, carbonyls, carbohydrates, or carboxylic acids, on the antigen and the carrier molecule. Chemical conjugation also includes non-covalent linkage between the antigen and the carrier molecule.

A“coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.

The term“competitive sequence” refers to a peptide or a modification, fragment, derivative, or homolog thereof that competes with another peptide for its cognate binding site.

“Complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs). Thus, it is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

“Co-administer” can include simultaneous and/or sequential administration of two or more agents.

A“compound,” as used herein, refers to any type of substance or agent that is can be considered a drug, or a candidate for use as a drug, as well as combinations and mixtures of the above.

The terms“comprises”,“comprising”, and the like can have the meaning ascribed to them in U.S. Patent Law and can mean“includes”,“including” and the like. As used herein,“including” or“includes” or the like means including, without limitation.

As used herein, the term“conservative amino acid substitution” is defined herein as an amino acid exchange within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

Asp, Asn, Glu, Gln;

III. Polar, positively charged residues:

His, Arg, Lys; IV. Large, aliphatic, nonpolar residues:

Met Leu, Ile, Val, Cys

V. Large, aromatic residues:

Phe, Tyr, Trp

A“control” cell is a cell having the same cell type as a test cell. The control cell may, for example, be examined at precisely or nearly the same time the test cell is examined. The control cell may also, for example, be examined at a time distant from the time at which the test cell is examined, and the results of the examination of the control cell may be recorded so that the recorded results may be compared with results obtained by examination of a test cell.

A“test” cell is a cell being examined.

“Cytokine,” as used herein, refers to intercellular signaling molecules, the best known of which are involved in the regulation of mammalian somatic cells. A number of families of cytokines, both growth promoting and growth inhibitory in their effects, have been characterized including, for example, interleukins, interferons, and transforming growth factors. A number of other cytokines are known to those of skill in the art. The sources, characteristics, targets and effector activities of these cytokines have been described.

The term“delivery vehicle” refers to any kind of device or material which can be used to deliver compounds in vivo or can be added to a composition comprising compounds administered to a plant or animal. This includes, but is not limited to, implantable devices, aggregates of cells, matrix materials, gels, etc.

As used herein, a“derivative” of a compound refers to a chemical compound that may be produced from another compound of similar structure in one or more steps, as in replacement of H by an alkyl, acyl, or amino group.

The use of the word“detect” and its grammatical variants refers to measurement of the species without quantification, whereas use of the word “determine” or“measure” with their grammatical variants are meant to refer to measurement of the species with quantification. The terms“detect” and“identify” are used interchangeably herein.

As used herein, a“detectable marker” or a“reporter molecule” is an atom or a molecule that permits the specific detection of a compound comprising the marker in the presence of similar compounds without a marker. Detectable markers or reporter molecules include, e.g., radioactive isotopes, antigenic determinants, enzymes, nucleic acids available for hybridization, chromophores, fluorophores, chemiluminescent molecules, electrochemically detectable molecules, and molecules that provide for altered fluorescence-polarization or altered

light-scattering.

The term "directed against " means that the compound being recited, whether a small molecule, drug, prodrug, or an antibody, or a biologically active fragment or homolog thereof, binds to the target and inhibits or stimulates its activity, or all of the above, according to the context in which the term is used.

A“disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a“disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

As used herein, the term“domain” refers to a part of a molecule or structure that shares common physicochemical features, such as, but not limited to, hydrophobic, polar, globular and helical domains or properties such as ligand binding, signal transduction, cell penetration and the like. Specific examples of binding domains include, but are not limited to, DNA binding domains and ATP binding domains.

An“effective amount” generally means an amount which provides the desired local or systemic effect, such as enhanced performance. For example, an effective dose is an amount sufficient to affect a beneficial or desired clinical result. The dose could be administered in one or more administrations and can include any preselected amount. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including size, age, injury or disease being treated and amount of time since the injury occurred or the disease began. One skilled in the art, particularly a physician, would be able to determine what would constitute an effective dose.

As used herein, the term“effector domain” refers to a domain capable of directly interacting with an effector molecule, chemical, or structure in the cytoplasm which is capable of regulating a biochemical pathway. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

An“enhancer” is a DNA regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.

The term“epitope” as used herein is defined as small chemical groups on the antigen molecule that can elicit and react with an antibody. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly five amino acids or sugars in size. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity.

As used herein, an“essentially pure” preparation of a particular protein or peptide is a preparation wherein at least about 95%, and preferably at least about 99%, by weight, of the protein or peptide in the preparation is the particular protein or peptide.

As used in the specification and the appended claims, the terms "for example," "for instance," "such as," "including" and the like are meant to introduce examples that further clarify more general subject matter. Unless otherwise specified, these examples are provided only as an aid for understanding the invention, and are not meant to be limiting in any fashion.

The terms“formula” and“structure” are used interchangeably herein.

As used herein,“Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. In one embodiment the bispecific antibodies of the invention comprise at least one Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Due to the exchange of either the variable regions or the constant regions, said Fab fragment is also referred to as“cross-Fab fragment” or “xFab fragment” or“crossover Fab fragment”. Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). This crossover Fab molecule is also referred to as CrossFab(VLVH). On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab molecule is also referred to as CrossFab(CLCHl).

A“fragment” or“segment” is a portion of an amino acid sequence, comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide. The terms“fragment” and“segment” are used interchangeably herein. As used herein, the term“fragment,” as applied to a protein or peptide, can ordinarily be at least about 3-15 amino acids in length, at least about 15-25 amino acids, at least about 25-50 amino acids in length, at least about 50-75 amino acids in length, at least about 75-100 amino acids in length, and greater than 100 amino acids in length.

As used herein, the term“fragment” as applied to a nucleic acid, may ordinarily be at least about 20 nucleotides in length, typically, at least about 50 nucleotides, more typically, from about 50 to about 100 nucleotides, at least about 100 to about 200 nucleotides, at least about 200 nucleotides to about 300

nucleotides, at least about 300 to about 350, at least about 350 nucleotides to about 500 nucleotides, at least about 500 to about 600, at least about 600 nucleotides to about 620 nucleotides, at least about 620 to about 650, and or the nucleic acid fragment will be greater than about 650 nucleotides in length. As used herein, a“functional” biological molecule is a biological molecule in a form in which it exhibits a property by which it is characterized. A functional enzyme, for example, is one which exhibits the characteristic catalytic activity by which the enzyme is characterized.

As used herein,“health care provider” includes either an individual or an institution that provides preventive, curative, promotional or rehabilitative health care services to a subject, such as a patient.

“Homologous” as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3 TTGCC5' and 3'TATGGC share 50% homology.

As used herein,“homology” is used synonymously with“identity.”

The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul [50; 1990]), modified as in Karlin and Altschul [51; 1993]. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. [52], and can be accessed, for example at the National Center for Biotechnology Information (NCBI) world wide web site. BLAST nucleotide searches can be performed with the NBLAST program (designated“blastn” at the NCBI web site), using the following parameters: gap penalty = 5; gap extension penalty = 2;

mismatch penalty = 3; match reward = 1; expectation value 10.0; and word size = 11 to obtain nucleotide sequences homologous to a nucleic acid described herein.

BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI“blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. [53]. Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

As used herein, the term“hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the length of the formed hybrid, and the G:C ratio within the nucleic acids.

By the term“immunizing a subject against an antigen” is meant

administering to the subject a composition, a protein complex, a DNA encoding a protein complex, an antibody or a DNA encoding an antibody, which elicits an immune response in the subject, and, for example, provides protection to the subject against a disease caused by the antigen or which prevents the function of the antigen.

The term“immunologically active fragments thereof’ will generally be understood in the art to refer to a fragment of a polypeptide antigen comprising at least an epitope, which means that the fragment at least comprises 4 contiguous amino acids from the sequence of the polypeptide antigen.

As used herein, the term“induction of apoptosis” means a process by which a cell is affected in such a way that it begins the process of programmed cell death, which is characterized by the fragmentation of the cell into membrane-bound particles that are subsequently eliminated by the process of phagocytosis.

The term“inhibit,” as used herein, refers to the ability of a compound, agent, or method to reduce or impede a described function, level, activity, rate, etc., based on the context in which the term“inhibit” is used. Preferably, inhibition is by at least 10%, more preferably by at least 25%, even more preferably by at least 50%, and most preferably, the function is inhibited by at least 75%. The term“inhibit” is used interchangeably with“neutralize”,“reduce” and“block” in the context of the definition of inhibit.

The term“inhibiting IL-17A” or any other cytokine or their respective receptors means to inhibit the activity, levels, or expression of IL-17A and is interpreted based on the context in which it is used. In one aspect, it refers to inhibiting its signaling activity by inhibiting it from binding with a ligand such as IL-17RA. The term“inhibition of IL-17A” when referring to a compound means that the compound is capable of“inhibiting IL-17A”. When using an antibody to inhibit, the term inhibiting also encompasses "blocking" or "neutralizing".

The term“inhibit a complex,” as used herein, refers to inhibiting the formation of a complex or interaction of two or more proteins, as well as inhibiting the function or activity of the complex. The term also encompasses disrupting a formed complex. However, the term does not imply that each and every one of these functions must be inhibited at the same time.

The term“inhibit a protein,” as used herein, refers to any method or technique which inhibits protein synthesis, levels, activity, or function, as well as methods of inhibiting the induction or stimulation of synthesis, levels, activity, or function of the protein of interest. The term also refers to any metabolic or regulatory pathway which can regulate the synthesis, levels, activity, or function of the protein of interest. The term includes binding with other molecules and complex formation. Therefore, the term“protein inhibitor” refers to any agent or compound, the application of which results in the inhibition of protein function or protein pathway function. However, the term does not imply that each and every one of these functions must be inhibited at the same time.

By the term "inhibits CDI mortality" is meant that the treatment being used reduces the amount of mortality relative to no treatment.

The term "inhibiting" a cell as used herein means to inhibit or decrease an increase in cell number of CD4+ cells or TH17 cells, or an increase in production of cytokines known to be associated with CDI, or to inhibit the activity of the cytokines or their receptors.

As used herein“injecting or applying” includes administration of a compound of the invention by any number of routes and means including, but not limited to, topical, oral, buccal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,

intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal means.

As used herein, an“instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the peptide or antibody of the invention in the kit for diagnosing or effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal.

The instructional material of the kit of the invention may, for example, be affixed to a container which contains the identified compound invention or be shipped together with a container which contains the identified compound. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

The terms "intestinal microbiota", "gut flora", and "gastrointestinal microbiota" are used interchangeably to refer to bacteria in the digestive tract.

The term“isolated” refers to a compound, including antibodies, nucleic acids or proteins/peptides, or cell that has been separated from at least one component which naturally accompanies it.

An“isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. A“ligand” is a compound that specifically binds to a target receptor.

A“receptor” is a compound that specifically binds to a ligand.

A ligand or a receptor (e.g., an antibody)“specifically binds to” or“is specifically immunoreactive with” a compound when the ligand or receptor functions in a binding reaction which is determinative of the presence of the compound in a sample of heterogeneous compounds. Thus, under designated assay (e.g., immunoassay) conditions, the ligand or receptor binds preferentially to a particular compound and does not bind in a significant amount to other compounds present in the sample. For example, a polynucleotide specifically binds under hybridization conditions to a compound polynucleotide comprising a

complementary sequence; an antibody specifically binds under immunoassay conditions to an antigen bearing an epitope against which the antibody was raised.

A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988, Antibodies, A

Laboratory Manual, Cold Spring Harbor Publications, New York) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity .

As used herein, the term“linkage” refers to a connection between two groups. The connection can be either covalent or non-covalent, including but not limited to ionic bonds, hydrogen bonding, and hydrophobic/hydrophilic interactions.

As used herein, the term“linker” refers to a molecule that joins two other molecules either covalently or noncovalently, e.g., through ionic or hydrogen bonds or van der Waals interactions, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at the 5’ end and to another complementary sequence at the 3’ end, thus joining two non-complementary sequences.

The term“measuring the level of expression” or“determining the level of expression” as used herein refers to any measure or assay which can be used to correlate the results of the assay with the level of expression of a gene or protein of interest. Such assays include measuring the level of mRNA, protein levels, etc. and can be performed by assays such as northern and western blot analyses, binding assays, immunoblots, etc. The level of expression can include rates of expression and can be measured in terms of the actual amount of an mRNA or protein present. Such assays are coupled with processes or systems to store and process information and to help quantify levels, signals, etc. and to digitize the information for use in comparing levels.

The term "microbiota" refers to an assemblage of microorganisms localized to a distinct environment.

The term“modulate”, as used herein, refers to changing the level of an activity, function, or process. The term“modulate” encompasses both inhibiting and stimulating an activity, function, or process.

The term“nucleic acid” typically refers to large polynucleotides. By “nucleic acid” is meant any nucleic acid, whether composed of

deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

As used herein, the term“nucleic acid” encompasses RNA as well as single and double-stranded DNA and cDNA. Furthermore, the terms,“nucleic acid,” “DNA,”“RNA” and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called“peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. By“nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of

phosphodiester linkages or modified linkages such as phosphotriester,

phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged

phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil). Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single- stranded polynucleotide sequence is the 5'-end; the left- hand direction of a double- stranded polynucleotide sequence is referred to as the 5'- direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the“coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as“downstream sequences.”

The term“nucleic acid construct,” as used herein, encompasses DNA and RNA sequences encoding the particular gene or gene fragment desired, whether obtained by genomic or synthetic methods.

Unless otherwise specified, a“nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

The term“oligonucleotide” typically refers to short polynucleotides, generally, no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence {i.e., A, U, G, C) in which“U” replaces“T.”

By describing two polynucleotides as“operably linked” is meant that a single- stranded or double- stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other. By way of example, a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region.

The term“otherwise identical sample”, as used herein, refers to a sample similar to a first sample, that is, it is obtained in the same manner from the same subject from the same tissue or fluid, or it refers a similar sample obtained from a different subject. The term“otherwise identical sample from an unaffected subject” refers to a sample obtained from a subject not known to have the disease or disorder being examined. The sample may of course be a standard sample. By analogy, the term“otherwise identical” can also be used regarding regions or tissues in a subject or in an unaffected subject. These can be used as controls, as can standard samples comprising known amounts of the target to be detected or measured.

As used herein,“parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

The term“peptide” typically refers to short polypeptides.

As used herein, the term "peptide ligand" (or the word "ligand" in reference to a peptide) refers to a peptide or fragment of a protein that specifically binds to a molecule, such as a protein, carbohydrate, and the like. A receptor or binding partner of the peptide ligand can be essentially any type of molecule such as polypeptide, nucleic acid, carbohydrate, lipid, or any organic derived compound.

The term“per application” as used herein refers to administration of a drug or compound to a subject.

The term“pharmaceutical composition” shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

As used herein, the term“pharmaceutically-acceptable carrier” means a chemical composition with which an appropriate compound or derivative can be combined and which, following the combination, can be used to administer the appropriate compound to a subject.

“Pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary application.

As used herein,“pharmaceutical compositions” include formulations for human and veterinary use. “Plurality” means at least two.

A“polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single- stranded or a double- stranded nucleic acid.

“Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.

“Synthetic peptides or polypeptides” means a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those of skill in the art.

As used herein, the term "prebiotic" refers to an agent that increases the number and/or activity of one or more desired bacteria. Non-limiting examples of prebiotics useful in the methods of the present invention include

fructooligosaccharides (e.g., oligofructose, inulin, inulin-type fructans),

galactooligosaccharides, amino acids, alcohols, and mixtures thereof.

By“presensitization” is meant pre-administration of at least one innate immune system stimulator prior to challenge with an agent. This is sometimes referred to as induction of tolerance.

The term“prevent,” as used herein, means to stop something from

happening, or taking advance measures against something possible or probable from happening. In the context of medicine,“prevention” generally refers to action taken to decrease the chance of getting a disease or condition.

A "preventive" or "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs, or exhibits only early signs, of a disease or disorder. A prophylactic or preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with developing the disease or disorder.

“Primer” refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single- stranded, but may be double- stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

As used herein, the term "probiotic" refers to a substantially pure bacteria (i.e., a single isolate), or a mixture of desired bacteria, and may also include any additional components that can be administered to a mammal for restoring microbiota. Such compositions are also referred to herein as a "bacterial inoculant." Probiotics or bacterial inoculant compositions of the invention are preferably administered with a buffering agent to allow the bacteria to survive in the acidic environment of the stomach, i.e., to resist low pH and to grow in the intestinal environment. Such buffering agents include sodium bicarbonate, milk, yogurt, infant formula, and other dairy products.

A“prodrug” refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug, or may demonstrate increased palatability or be easier to formulate.

As used herein, the term“promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

A“constitutive” promoter is a promoter which drives expression of a gene to which it is operably linked, in a constant manner in a cell. By way of example, promoters which drive expression of cellular housekeeping genes are considered to be constitutive promoters.

An“inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A“tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

A“prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

As used herein,“protecting group” with respect to a terminal amino group refers to a terminal amino group of a peptide, which terminal amino group is coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis. Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups, for example, tert-butoxycarbonyl or adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides, vol. 3, pp. 3- 88 (Academic Press, New York, 1981) for suitable protecting groups.

As used herein,“protecting group” with respect to a terminal carboxy group refers to a terminal carboxyl group of a peptide, which terminal carboxyl group is coupled with any of various carboxyl-terminal protecting groups. Such protecting groups include, for example, tert-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.

The term“protein” typically refers to large polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.

The term“protein regulatory pathway”, as used herein, refers to both the upstream regulatory pathway which regulates a protein, as well as the downstream events which that protein regulates. Such regulation includes, but is not limited to, transcription, translation, levels, activity, posttranslational modification, and function of the protein of interest, as well as the downstream events which the protein regulates.

The terms“protein pathway” and“protein regulatory pathway” are used interchangeably herein.

As used herein, the term“purified” and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment. The term“purified” does not necessarily indicate that complete purity of the particular molecule has been achieved during the process. A“highly purified” compound as used herein refers to a compound that is greater than 90% pure. In particular, purified sperm cell DNA refers to DNA that does not produce significant detectable levels of non-sperm cell DNA upon PCR amplification of the purified sperm cell DNA and subsequent analysis of that amplified DNA. A“significant detectable level” is an amount of contaminate that would be visible in the presented data and would need to be addressed/explained during analysis of the forensic evidence.

“Recombinant polynucleotide” refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.

A host cell that comprises a recombinant polynucleotide is referred to as a “recombinant host cell.” A gene which is expressed in a recombinant host cell wherein the gene comprises a recombinant polynucleotide, produces a“recombinant polypeptide.”

A“recombinant polypeptide” is one which is produced upon expression of a recombinant polynucleotide.

A“receptor” is a compound that specifically binds to a ligand.

A“ligand” is a compound that specifically binds to a target receptor.

A“recombinant cell” is a cell that comprises a transgene. Such a cell may be a eukaryotic or a prokaryotic cell. Also, the transgenic cell encompasses, but is not limited to, an embryonic stem cell comprising the transgene, a cell obtained from a chimeric mammal derived from a transgenic embryonic stem cell where the cell comprises the transgene, a cell obtained from a transgenic mammal, or fetal or placental tissue thereof, and a prokaryotic cell comprising the transgene.

The term "reduces recurrent infection" means that the number or percentage of subjects who get another C. difficile infection following a course of treatment for an initial C. difficile infection is lower compared to the number who had received standard doses or standard duration therapies.

The term“regulate” refers to either stimulating or inhibiting a function or activity of interest.

As used herein, the term“reporter gene” means a gene, the expression of which can be detected using a known method. By way of example, the Escherichia coli lacZ gene may be used as a reporter gene in a medium because expression of the lacZ gene can be detected using known methods by adding the chromogenic substrate o-nitrophenyl-P-galactoside to the medium (Gerhardt et ah, eds., 1994, Methods for General and Molecular Bacteriology, American Society for

Microbiology, Washington, DC, p. 574).

A“sample,” as used herein, refers preferably to a biological sample from a subject, including, but not limited to, normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, semen, tears, and urine. A sample can also be any other source of material obtained from a subject which contains cells, tissues, or fluid of interest. A sample can also be obtained from cell or tissue culture.

As used herein, the term“secondary antibody” refers to an antibody that binds to the constant region of another antibody (the primary antibody).

By the term“signal sequence” is meant a polynucleotide sequence which encodes a peptide that directs the path a polypeptide takes within a cell, i.e., it directs the cellular processing of a polypeptide in a cell, including, but not limited to, eventual secretion of a polypeptide from a cell. A signal sequence is a sequence of amino acids which are typically, but not exclusively, found at the amino terminus of a polypeptide which targets the synthesis of the polypeptide to the endoplasmic reticulum. In some instances, the signal peptide is proteolytically removed from the polypeptide and is thus absent from the mature protein.

By“small interfering RNAs (siRNAs)” is meant, inter alia, an isolated dsRNA molecule comprised of both a sense and an anti-sense strand. In one aspect, it is greater than 10 nucleotides in length. siRNA also refers to a single transcript which has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin. siRNA further includes any form of dsRNA (proteolytically cleaved products of larger dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA) as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides.

As used herein, the term“solid support” relates to a solvent insoluble substrate that is capable of forming linkages (preferably covalent bonds) with various compounds. The support can be either biological in nature, such as, without limitation, a cell or bacteriophage particle, or synthetic, such as, without limitation, an acrylamide derivative, agarose, cellulose, nylon, silica, or magnetized particles.

By the term“specifically binds to”, as used herein, is meant when a compound or ligand functions in a binding reaction or assay conditions which is determinative of the presence of the compound in a sample of heterogeneous compounds.

The term“standard,” as used herein, refers to something used for

comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function. Standard can also refer to an“internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in

determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured. Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker. Standard can also refer to a healthy individual.

A“subject” of analysis, diagnosis, or treatment is a vertebrate, including a mammal, such as a human. Mammals include, but are not limited to, humans, farm animals, sport animals, and pets.

As used herein, a“subject in need thereof’ is a patient, animal, mammal, or human, who will benefit from the method of this invention.

As used herein, a "substantially homologous amino acid sequences" includes those amino acid sequences which have at least about 95% homology, preferably at least about 96% homology, more preferably at least about 97% homology, even more preferably at least about 98% homology, and most preferably at least about 99% or more homology to an amino acid sequence of a reference antibody chain. Amino acid sequence similarity or identity can be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm. The default settings used for these programs are suitable for identifying substantially similar amino acid sequences for purposes of the present invention.

"Substantially homologous nucleic acid sequence" means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the

corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not significantly affecting the peptide function occur. Preferably, the substantially identical nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence. The percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more.

Substantial identity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm. Suitable nucleic acid hybridization conditions to determine if a nucleotide sequence is substantially similar to a reference nucleotide sequence are: 7% sodium dodecyl sulfate SDS, 0.5 M NaP0₄, 1 mM EDTA at 50°C with washing in 2X standard saline citrate (SSC), 0.1% SDS at 50°C; preferably in 7% (SDS), 0.5 M NaP0₄, 1 mM EDTA at 50°C. with washing in IX SSC, 0.1% SDS at 50°C; preferably 7% SDS, 0.5 M NaP0₄, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C; and more preferably in 7% SDS, 0.5 M NaP0₄, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, GCS program package (Devereux et al., 1984 Nucl. Acids Res. 12:387), and the BLASTN or FASTA programs (Altschul et al., 1990 Proc. Natl. Acad. Sci. USA. 1990 87:14:5509-13; Altschul et al., J. Mol. Biol. 1990 215:3:403-10; Altschul et al., 1997 Nucleic Acids Res. 25:3389-3402). The default settings provided with these programs are suitable for determining substantial similarity of nucleic acid sequences for purposes of the present invention. The term“substantially pure” describes a compound, e.g., a protein or polypeptide, cell or nucleic acid that has been separated from components which naturally accompany it. Typically, a compound is substantially pure when at least 10%, including at least 20%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95%, at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis, or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.

As used herein, a "substantially homologous amino acid sequences" or "substantially identical amino acid sequences" includes those amino acid sequences which have at least about 92%, or at least about 95% homology or identity, including at least about 96% homology or identity, including at least about 97% homology or identity, including at least about 98% homology or identity, and at least about 99% or more homology or identity to an amino acid sequence of a reference antibody chain. Amino acid sequence similarity or identity can be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm. The default settings used for these programs are suitable for identifying substantially similar amino acid sequences for purposes of the present invention.

"Substantially homologous nucleic acid sequence" or "substantially identical nucleic acid sequence" means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not significantly affecting the peptide function occur. In one embodiment, the substantially identical nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence. The percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is at least about 50%, 65%, 75%, 85%, 92%, 95%, 99% or more. Substantial identity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm.

Suitable nucleic acid hybridization conditions to determine if a nucleotide sequence is substantially similar to a reference nucleotide sequence are: 7% sodium dodecyl sulfate SDS, 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 2X standard saline citrate (SSC), 0.1% SDS at 50°C; preferably in 7% (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C. with washing in IX SSC, 0.1% SDS at 50°C; preferably 7% SDS, 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C; and more preferably in 7% SDS, 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, GCS program package. The default settings provided with these programs are suitable for determining substantial similarity of nucleic acid sequences for purposes of the present invention.

By the term "susceptible to C. difficile infection", as used herein, refers to a subject who, due to a prior disease state, treatment, or condition has now become more susceptible to such an infection than if they had not had the prior disease, treatment, or condition. Such susceptible subjects are described herein and others are also known in the art.

The term“symptom,” as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a“sign” is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse and other observers.

A“therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

A“therapeutically effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.

As used herein, the term“transgene” means an exogenous nucleic acid sequence comprising a nucleic acid which encodes a promoter/regulatory sequence operably linked to nucleic acid which encodes an amino acid sequence, which exogenous nucleic acid is encoded by a transgenic mammal. As used herein, the term“transgenic mammal” means a mammal, the germ cells of which comprise an exogenous nucleic acid.

As used herein, a“transgenic cell” is any cell that comprises a nucleic acid sequence that has been introduced into the cell in a manner that allows expression of a gene encoded by the introduced nucleic acid sequence.

The term to“treat,” as used herein, means reducing the frequency with which symptoms are experienced by a patient or subject or administering an agent or compound to reduce the frequency with which symptoms are experienced.

As used herein,“treat,”“treating”, or“treatment” includes treating, ameliorating, or inhibiting an injury or disease related condition or a symptom of an injury or disease related condition. In one embodiment the disease, injury or disease related condition or a symptom of an injury or disease related condition is prevented; while another embodiment provides prophylactic treatment of the injury or disease related condition or a symptom of an injury or disease related condition. The term“symptom,” as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a“sign” is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse and other observers.

By the term“vaccine,” as used herein, is meant a composition which when inoculated into a subject has the effect of stimulating an immune response in the subject, which serves to fully or partially protect the subject against a condition, disease or its symptoms. The term vaccine can encompass prophylactic as well as therapeutic vaccines. A combination vaccine is one which combines two or more vaccines, or two or more compounds or agents.

A“vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term“vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer or delivery of nucleic acid to cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, recombinant viral vectors, and the like. Examples of non- viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA and the like.

“Expression vector” refers to a vector comprising a recombinant

polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient ex acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.

Embodiments

The present invention provides compositions and methods for preventing and treating C. difficile infection. In one aspect, the subject suffers from colitis. In one aspect, the subject suffers from irritable bowel syndrome.

Colitis includes, but is not limited to, acute colitis, chronic colitis, inflammatory bowel disease, Ulcerative colitis, Crohn’s colitis, Diversion colitis, Ischemic colitis, Infectious colitis, Fulminant colitis, Collagenous colitis, Chemical colitis, Microscopic colitis, Lymphocytic colitis and Atypical colitis.

The present application discloses increased CDI-associated mortality with prior colitis and that an increase in the number of CD4+T cells is associated with increased disease severity of C. difficile infection in subjects with prior colitis. In one aspect, an increase in IL-17A+CD4+ T cells is associated with increased disease severity in C. difficile infection in subjects with prior colitis.

The present application further discloses increased Thl7 responses cause severe subsequent CDI in subjects with prior colitis.

It is disclosed herein that the Thl7 cytokines IL-6 and IL-23 are increased in the serum of subjects infected with C. difficile and that the increase correlates with severe disease (See Figs. 4A to 4C). It is also disclosed that IL-17A and IL-17F increase in colitis (DSS mouse model), that Thl7 cells are predominantly IL-22 negative (See Fig. S4A), and in cecal tissue IL-22 is significantly higher in DSS mice (See Fig. S4B; IL-22 can be produced by cell types other than Thl7 cells).

It is disclosed herein that depleting CD4+ cells reduces CDI severity. In one embodiment, inhibiting the increase in number of CD4+ cells upon infection or decreasing the number of CD4+ cells is useful for reducing CDI severity in a subject with colitis. In one aspect, the colitis is inflammatory bowel syndrome. In one aspect, the colitis is acute. In one aspect, the colitis is chronic.

The present application provides compositions and methods for preventing or treating Clostridium difficile (C. difficile) infection (CDI) in a subject in need thereof. In one aspect, the method comprises administering to the subject a pharmaceutical composition comprising a pharmaceutical-acceptable carrier, optionally at least one additional therapeutic agent, and an effective amount of at least one inhibitor selected from the group consisting of an inhibitor of CD4+ T cells, Thl7 cells, CD4, IL-17A, IL-17F, IL-17RA, and IL-6.

In one embodiment, a subject of the invention with prior colitis is administered a pharmaceutical composition of the invention as a preventative measure to reduce severity of a possible CDI infection. In another embodiment, a subject of the invention with colitis is administered a pharmaceutical composition of the invention as a preventative measure to reduce severity of a possible CDI infection. In yet another embodiment, a subject of the invention with prior colitis or present colitis, and who has been infected with C. difficile, is administered a pharmaceutical composition of the invention as a preventative measure to reduce severity of the CDI infection. In yet another embodiment, a subject of the invention with prior colitis or present colitis, and who has been infected with C. difficile, is administered a pharmaceutical composition of the invention to treat the CDI infection. In one aspect, the subject is treated shortly after infection.

In one aspect, at least two different inhibitors are administered. In one aspect, they are co-administered.

In one aspect, the method inhibits CDI mortality.

In one aspect, the method inhibits an increase in IL-6, IL-17, and IL-23 associated with CDI and with an increased risk of CDI mortality.

In one embodiment, the application provides compositions and methods for targeting Thl7 cells to prevent or treat CDI in subjects suffering from IBD.

In one embodiment, the application provides compositions and methods for targeting Thl7 cell effector cytokines to prevent or treat CDI in subjects suffering from IBD.

In one aspect, an inhibitor is directed against CD4. In one aspect, the inhibitor is the antibody OKT-4 or the antibody RPA-T4. A pharmaceutical composition of the invention can be administered as a preventative/prophylactic measure to a subject with colitis or who has had colitis previously. In one aspect, it can be administered in conjunction with a pending hospital stay or at the time of admission to a hospital.

A pharmaceutical composition of the invention can be administered to a subject with colitis or who has had colitis previously when they may have been exposed to C. difficile.

A pharmaceutical composition of the invention can be administered to a subject with colitis or who has had colitis previously when they may have been exposed to C. difficile or shortly after they have been infected. In one aspect, a subject is treated within one week of infection. In another aspect, a subject is treated within 5 days of infection. In one aspect, a subject is treated within 3 days of infection. In one aspect, a subjects is treated within 2 days of infection. In another aspect, a subject is treated within one day of being infected.

In one embodiment, the present application provides compositions and methods useful for targeting upstream/downstream mechanisms, including, IL-23, IL-17A, IL-17F, IL-6, and TGF , to protect against severe C. difficile disease.

The invention provides compositions and methods for treating a subject of the invention when the subject is found to have increased levels of IL-6 and IL-23.

Therefore, the present invention further encompasses development of additional anti-human specific antagonist (inhibitory) antibodies, including polyclonal antibodies, F(ab)2 fragments, monoclonal antibodies, chimeric antibodies, humanized monoclonal anti-human antibodies, synthetic antibodies, bi specific antibodies, and single chain antibodies, and useful homologs and fragments thereof, for use in the invention. The antibody(s), or biologically active fragments or homologs thereof, can be subjected to preclinical testing, for example in vitro with human or animal cells or in animal models, as well as clinical testing. The invention also encompasses the identification and development of other compounds which mimic the antagonistic effect of monoclonal antibodies on treating CDI as disclosed herein.

A dosage regimen for treatment with the active agents is based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary, but can be determined routinely by a physician using standard methods.

In one aspect, an antibody of the invention can be administered at a dose of about 0.01 mg/kg to about 100 mg/kg body weight. In another aspect, an antibody of the invention can be administered at a dose of about 0.1 mg/kg to about 50 mg/kg. In yet another aspect, an antibody of the invention can be administered at a dose of about 1.0 mg/kg to about 25 mg/kg body weight. In another aspect, an antibody of the invention can be administered at a dose of about 0.1, 0.5, 0.75, 0.833, 1.0, 1.25,

1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5,0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0,

18.5, 19.0, 19.5, and 20.0 mg/kg body weight. The invention further encompasses similar increments within each range of doses described herein.

In one embodiment, the agonist or additional therapeutic agent is

administered at a dose of about 1 pg/kg body weight to about lg/kg body weight.

The treatment regimen will vary depending on the disease being treated, based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. The treatment can include administration of a pharmaceutical composition of the invention once or more than once. Other therapeutic drugs and agents can be administered as well.

In one embodiment, a dose can be administered once a week. In another embodiment, a dose can be administered at least once a week. In one embodiment, a dose is administered two or more times a week. In another embodiment, a dose is administered three or more times a week. In another embodiment, a dose is administered ever third day. In one embodiment, the duration of treatment can be for up to one year, or up to six months, or up to three months.

In one embodiment, an antibody of the invention is purified.

In one embodiment, an antibody of the invention is substantially pure.

The invention also provides compositions and methods for determining the severity of the CDI and for determining the type and timing of treatment based on the diagnosis. For example, when a subject is found to have higher levels of IL-6 and IL-23, the subject is at increased risk for mortality and the physician can design a treatment regimen accordingly. The invention further provides for the use of the proteins or peptides where one or more conservative amino acid substitutions are made in the sequence and that the substitution has no effect on the desired biological activity, where such activity is desired. In one aspect, one conservative amino acid substitution is made. In one aspect, at least two conservative amino acid substitutions are made. When two or more substitutions are made, they do not have to be at adjacent amino acid residue positions.

Methods of generating antibodies (i.e., monoclonal and polyclonal) are well known in the art. Antibodies may be generated via any one of several methods known in the art, which methods can employ induction of in-vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi D. R. et ah, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter G. et ah, 1991. Nature 349:293-299) or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV) -hybridoma technique (Kohler G. et ah, 1975. Nature 256:495-497; Kozbor D. et ah, 1985. J. Immunol. Methods 81:31-42; Cote R J. et ah, 1983. Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030; Cole S P. et ah, 1984. Mol. Cell. Biol. 62:109-120).

It will be appreciated that for human therapy or diagnostics, humanized antibodies can be used. Humanized forms of nonhuman (e.g., murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having— preferably minimal— portions derived from nonhuman antibodies. Humanized antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a

complementarity determining region of a nonhuman species (donor antibody) such as mouse, rat, or rabbit having the desired functionality. In some instances, Fv framework residues of the human antibody are replaced by corresponding nonhuman residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a nonhuman antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence. Humanized antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988. Nature 332:323-329; and Presta, 1992. Curr. Op. Struct. Biol. 2:593-596).

Methods for preparing antibodies are described herein (see Embodiments and Examples) and are known in the art, including isolation and identification of single chain molecules produced by techniques such as phage or yeast display.

This invention encompasses methods of screening compounds to identify those compounds that act as agonists (stimulate) or antagonists (inhibit) of the protein interactions and pathways described herein. Screening assays for antagonist compound candidates are designed to identify compounds that bind or complex with the peptides described herein, or otherwise interfere with the interaction of the peptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, including protein- protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.

In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, one of the peptides of the complexes described herein, or the test compound or drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non- covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the peptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the peptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.

If the candidate compound interacts with, but does not bind to a particular peptide identified herein, its interaction with that peptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co purification through gradients or chromatographic columns. In addition, protein- protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London), 340:245- 246 (1989); Chien et ah, Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Complete kits for identifying protein-protein interactions between two specific proteins using the two-hybrid technique are available. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a peptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.

To assay for antagonists, the peptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the peptide indicates that the compound is an antagonist to the peptide. The peptide can be labeled, such as by radioactivity. Any convenient monoclonal antibody (mAb) may be utilized. Alternatively, any convenient polyclonal antibody against CD24 may be utilized. Methods of generating antibodies (i.e., monoclonal and polyclonal) are well known in the art. Antibodies may be generated via any one of several methods known in the art, which methods can employ induction of in-vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi D. R. et ah, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter G. et ah, 1991. Nature 349:293-299) or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV) -hybridoma technique (Kohler G. et ah, 1975. Nature 256:495-497; Kozbor D. et ah, 1985. J. Immunol. Methods 81:31-42; Cote R J. et ah, 1983. Proc. Natl. Acad. Sci. U.S.A. 80:2026- 2030; Cole S P. et ah, 1984. Mol. Cell. Biol. 62:109-120). Anti-CD24 antibodies, both polyclonal and monoclonal, suitable for use in the methods and compositions of the present invention are commercially available, for example, from Santa Cruz Biotechnology (Santa Cruz, Calif.), AbDSerotec (Kidlington, UK) and Life Span BioSciences, Inc (Seattle Wash.).

It will be appreciated that for human therapy or diagnostics, humanized antibodies are preferably used. Humanized forms of nonhuman (e.g., murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having— preferably minimal— portions derived from nonhuman antibodies.

Humanized antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementarity determining region of a nonhuman species (donor antibody) such as mouse, rat or rabbit having the desired functionality. In some instances, Fv framework residues of the human antibody are replaced by corresponding nonhuman residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a nonhuman antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence. Humanized antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988. Nature 332:323-329; and Presta, 1992. Curr. Op. Struct. Biol. 2:593-596).

Methods for humanizing nonhuman antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as imported residues which are typically taken from an imported variable domain. Humanization can be essentially performed as described (see, for example: Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988. Science 239:1534-1536; U.S. Pat. No.

4,816,567) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions. Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage or yeast display libraries [see, for example, Hoogenboom and Winter, 1991. J. Mol. Biol. 227:381; Marks et al., 1991. J. Mol. Biol. 222:581; Cole et al., "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, pp. 77 (1985); Boerner et al., 1991. J. Immunol. 147:86-95). Humanized antibodies can also be made by introducing sequences encoding human immunoglobulin loci into transgenic animals, e.g., into mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon antigenic challenge, human antibody production is observed in such animals which closely resembles that seen in humans in all respects, including gene rearrangement, chain assembly, and antibody repertoire. Ample guidance for practicing such an approach is provided in the literature of the art (for example, refer to: U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; Marks et al., 1992. Bio/Technology 10:779-783; Lonberg et al., 1994. Nature 368:856-859; Morrison, 1994. Nature 368:812-13; Fishwild et al., 1996. Nature Biotechnology 14:845-51; Neuberger, 1996. Nature Biotechnology 14:826; Lonberg and Huszar, 1995. Intern. Rev.

Immunol. 13:65-93).

Once antibodies are obtained, they may be tested for activity, for example via ELISA.

According to some aspects of the present invention, the method includes providing to the subject a therapeutic compound in combination with a

pharmaceutically acceptable carrier.

According to some aspects of the present invention, the antibody or combination can be provided using any one of a variety of delivery methods.

Delivery methods and suitable formulations are described herein below with respect to pharmaceutical compositions.

Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a subject's body.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more

physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

It will be appreciated, of course, that the proteins or peptides of the invention may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e.

chemical substituents suitable to protect and/or stabilize the N- and C-termini from “undesirable degradation”, a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C₁-C₅ branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N- terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH₂), and mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide's C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without effect on peptide activity.

Other modifications can also be incorporated without adversely affecting the activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the present invention are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms. Acid addition salts of the present invention are also contemplated as functional equivalents. Thus, a peptide in accordance with the present invention treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclic and the like, to provide a water soluble salt of the peptide is suitable for use in the invention.

Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or

deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phospho threonine.

Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.

Antibodies and their Preparation

Antibodies directed against proteins, polypeptides, or peptide fragments thereof of the invention may be generated using methods that are well known in the art. For instance, U.S. patent application no. 07/481,491, which is incorporated by reference herein in its entirety, discloses methods of raising antibodies to peptides. For the production of antibodies, various host animals, including but not limited to rabbits, mice, and rats, can be immunized by injection with a polypeptide or peptide fragment thereof. To increase the immunological response, various adjuvants may be used depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.

The antigenic fragments of the proteins of the invention may include, for example, peptide antigens that are at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 or up to about 200 amino acids in length. Of course, these are prepared based on the length of the starting protein or peptide. Also included are full-length unprocessed protein as well as mature processed protein. These various length antigenic fragments may be designed in tandem order of linear amino acid sequence of the immunogen of choice, such as SAS1R, or staggered in linear sequence of the protein. In addition, antibodies to three-dimensional epitopes, i.e., non-linear epitopes, can also be prepared, based on, e.g., crystallographic data of proteins.

Hosts may also be injected with peptides of different lengths encompassing a desired target sequence.

For the preparation of monoclonal antibodies, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be utilized. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4:72), and the EBV- hybridoma technique (Cole et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) may be employed to produce human monoclonal antibodies. In another embodiment, monoclonal antibodies are produced in germ-free animals.

In one embodiment, any new monoclonal antibody described herein, or made using the methods described herein, and the hybridomas making the antibodies, as well as those not described herein, will be deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209) and assigned Accession Numbers. The deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of

Microorganisms for the Purposes of Patent Procedure and made available for use under those terms. This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposits will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between the University of Virginia and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC section 122 and the Commissioner's rules pursuant thereto (including 37 CFR section 1.14 with particular reference to 886 OG 638). The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. Nucleic acid and amino acid sequences will be deposited with GenBank and made accessible to the public.

In accordance with the invention, human antibodies may be used and obtained by utilizing human hybridomas (Cote el al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,

Inc., pp. 77-96). Furthermore, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452- 454) by splicing the genes from a mouse antibody molecule specific for epitopes of SLLP polypeptides together with genes from a human antibody molecule of appropriate biological activity can be employed; such antibodies are within the scope of the present invention. Once specific monoclonal antibodies have been developed, the preparation of mutants and variants thereof by conventional techniques is also available.

Humanized (chimeric) antibodies are immunoglobulin molecules comprising a human and non-human portion. More specifically, the antigen combining region (or variable region) of a humanized chimeric antibody is derived from a non-human source (e.g., murine) and the constant region of the chimeric antibody (which confers biological effector function to the immunoglobulin) is derived from a human source. The humanized chimeric antibody should have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule. A large number of methods of generating chimeric antibodies are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and 4,975,369). Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S. Pat. No. 5,482,856.

In another embodiment, this invention provides for fully human antibodies. Human antibodies consist entirely of characteristically human polypeptide sequences. The human antibodies of this invention can be produced in using a wide variety of methods (see, e.g., U.S. Pat. No. 5,001,065, for review).

In one embodiment, techniques described for the production of single-chain antibodies (U.S. Patent No. 4,946,778, incorporated by reference herein in its entirety) are adapted to produce protein- specific single-chain antibodies. In another embodiment, the techniques described for the construction of Fab expression libraries (Huse et al, 1989, Science 246:1275-1281) are utilized to allow rapid and easy identification of monoclonal Fab fragments possessing the desired specificity for specific antigens, proteins, derivatives, or analogs of the invention.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragment; the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent; and Fv fragments.

The generation of polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies which specifically bind the antigen therefrom.

Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well-known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynski et al. (1988, Blood, 72:109-115). Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide.

Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.

A nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4): 125- 168) and the references cited therein. Further, the antibody of the invention may be“humanized” using the technology described in Wright et al., (supra) and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77(4):755-759).

To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY).

Bacteriophage which encode the desired antibody, may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophage which do not express the antibody will not bind to the cell. Such panning techniques are well known in the art.

Processes such as those described above, have been developed for the production of human antibodies using M13 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M13 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage which encode the Fab portion of an antibody molecule. However, the invention should not be construed to be limited solely to the generation of phage encoding Fab antibodies. Rather, phage which encode single chain antibodies (scFv/phage antibody libraries) are also included in the invention. Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CH1) of the heavy chain. Single chain antibody molecules comprise a single chain of protein comprising the Ig Fv fragment. An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein. Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al., 1991, J. Mol. Biol. 222:581-597. Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, Nature

Medicine 1:837-839; de Kruif et al. 1995, J. Mol. Biol.248:97-l05).

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., ELISA (enzyme-linked immunosorbent assay). Antibodies generated in accordance with the present invention may include, but are not limited to, polyclonal, monoclonal, chimeric (i.e., “humanized”), and single chain (recombinant) antibodies, Fab fragments, and fragments produced by a Fab expression library.

The peptides of the present invention may be readily prepared by standard, well-established techniques, such as solid-phase peptide synthesis (SPPS) as described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Illinois; and as described by Bodanszky and Bodanszky in The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York. At the outset, a suitably protected amino acid residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin.“Suitably protected” refers to the presence of protecting groups on both the a-amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions that will not affect the final peptide product. Stepwise synthesis of the oligopeptide is carried out by the removal of the N-protecting group from the initial amino acid, and couple thereto of the carboxyl end of the next amino acid in the sequence of the desired peptide. This amino acid is also suitably protected. The carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support-bound amino acid by formation into a reactive group such as formation into a carbodiimide, a symmetric acid anhydride or an“active ester” group such as hydroxybenzotriazole or pentafluorophenly esters.

Examples of solid phase peptide synthesis methods include the BOC method that utilized tert-butyloxcarbonyl as the a-amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protect the a-amino of the amino acid residues, both methods of which are well-known by those of skill in the art.

To ensure that the proteins or peptides obtained from either chemical or biological synthetic techniques is the desired peptide, analysis of the peptide composition should be conducted. Such amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively, or additionally, the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in order, may also be used to determine definitely the sequence of the peptide.

Prior to its use, the peptide can be purified to remove contaminants. In this regard, it will be appreciated that the peptide will be purified to meet the standards set out by the appropriate regulatory agencies. Any one of a number of a conventional purification procedures may be used to attain the required level of purity including, for example, reversed-phase high-pressure liquid chromatography (HPLC) using an alkylated silica column such as C₄ -,C₈- or C_l8- silica. A gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion-exchange chromatography can be also used to separate peptides based on their charge.

Substantially pure peptide obtained as described herein may be purified by following known procedures for protein purification, wherein an immunological, enzymatic or other assay is used to monitor purification at each stage in the procedure. Protein purification methods are well known in the art, and are described, for example in Deutscher et al. (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).

The invention further encompasses the use of aptamers. In one embodiment, an aptamer is a compound that is selected in vitro to bind preferentially to another compound (in this case the identified proteins). In one aspect, aptamers are nucleic acids or peptides, because random sequences can be readily generated from nucleotides or amino acids (both naturally occurring or synthetically made) in large numbers but of course they need not be limited to these. In another aspect, the nucleic acid aptamers are short strands of DNA that bind protein targets. In one aspect, the aptamers are oligonucleotide aptamers. Oligonucleotide aptamers are oligonucleotides which can bind to a specific protein sequence of interest. A general method of identifying aptamers is to start with partially degenerate oligonucleotides, and then simultaneously screen the many thousands of oligonucleotides for the ability to bind to a desired protein. The bound oligonucleotide can be eluted from the protein and sequenced to identify the specific recognition sequence. Transfer of large amounts of a chemically stabilized aptamer into cells can result in specific binding to a polypeptide of interest, thereby blocking its function. [For example, see the following publications describing in vitro selection of aptamers: Klug et ah, Mol. Biol. Reports 20:97-107 (1994); Wallis et ah, Chem. Biol. 2:543-552 (1995);

Ellington, Curr. Biol. 4:427-429 (1994); Lato et ah, Chem. Biol. 2:291-303 (1995); Conrad et ah, Mol. Div. 1:69-78 (1995); and Uphoff et ah, Curr. Opin. Struct. Biol. 6:281-287 (1996)]. Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers can be limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation.

The idea of using single- stranded nucleic acids (DNA and RNA aptamers) to target protein molecules is based on the ability of short sequences (20 mers to 80 mers) to fold into unique 3D conformations that enable them to bind targeted proteins with high affinity and specificity. RNA aptamers have been expressed successfully inside eukaryotic cells, such as yeast and multicellular organisms, and have been shown to have inhibitory effects on their targeted proteins in the cellular environment.

This invention encompasses methods of screening compounds to identify those compounds that act as agonists (stimulate) or antagonists (inhibit) of the protein interactions and pathways described herein. Screening assays for antagonist compound candidates are designed to identify compounds that bind or complex with the peptides described herein, or otherwise interfere with the interaction of the peptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, including protein- protein binding assays, biochemical screening assays, high-throughput assays, immunoassays, and cell-based assays, which are well characterized in the art.

All assays for antagonists are common in that they call for contacting the compound or drug candidate with a peptide identified herein under conditions and for a time sufficient to allow these two components to interact.

In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, one of the peptides of the complexes described herein, or the test compound or drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non- covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the peptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the peptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.

If the candidate compound interacts with, but does not bind to a particular peptide identified herein, its interaction with that peptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co purification through gradients or chromatographic columns. In addition, protein- protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London), 340:245- 246 (1989); Chien et ah, Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Complete kits for identifying protein-protein interactions between two specific proteins using the two-hybrid technique are available. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a peptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.

Other assays and libraries are encompassed within the invention, such as the use of phylomers® and reverse yeast two-hybrid assays (see Watt, 2006, Nature Biotechnology, 24:177; Watt, U.S. Pat. No. 6,994,982; Watt, U.S. Pat. Pub. No. 2005/0287580; Watt, U.S. Pat. No. 6,510,495; Barr et al., 2004, J. Biol. Chem., 279:41:43178-43189; the contents of each of these publications is hereby incorporated by reference herein in their entirety). Phylomers® are derived from sub domains of natural proteins, which makes them potentially more stable than conventional short random peptides. Phylomers® are sourced from biological genomes that are not human in origin. This feature significantly enhances the potency associated with Phylomers® against human protein targets. Phylogica’s current Phylomer® library has a complexity of 50 million clones, which is comparable with the numerical complexity of random peptide or antibody Fab fragment libraries. An Interacting Peptide Library, consisting of 63 million peptides fused to the B42 activation domain, can be used to isolate peptides capable of binding to a target protein in a forward yeast two hybrid screen. The second is a Blocking Peptide Library made up of over 2 million peptides that can be used to screen for peptides capable of disrupting a specific protein interaction using the reverse two-hybrid system.

The Phylomer® library consists of protein fragments, which have been sourced from a diverse range of bacterial genomes. The libraries are highly enriched for stable subdomains (15-50 amino acids long). This technology can be integrated with high throughput screening techniques such as phage display and reverse yeast two-hybrid traps.

The present application discloses compositions and methods for regulating the proteins described herein, and those not disclosed which are known in the art are encompassed within the invention. For example, various modulators/effectors are known, e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules, or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides.

The present invention also provides nucleic acids encoding peptides, proteins, and antibodies of the invention. By“nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged

phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

It is not intended that the present invention be limited by the nature of the nucleic acid employed. The target nucleic acid may be native or synthesized nucleic acid. The nucleic acid may be from a viral, bacterial, animal or plant source. The nucleic acid may be DNA or RNA and may exist in a double- stranded, single- stranded or partially double- stranded form. Furthermore, the nucleic acid may be found as part of a virus or other macromolecule. See, e.g., Fasbender et ah, 1996, J. Biol. Chem. 272:6479-89 (polylysine condensation of DNA in the form of adenovirus).

In some circumstances, as where increased nuclease stability is desired, nucleic acids having modified intemucleoside linkages may be preferred. Nucleic acids containing modified intemucleoside linkages may also be synthesized using reagents and methods that are well known in the art. For example, methods for synthesizing nucleic acids containing phosphonate phosphorothioate,

phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate, formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate, dimethylene- sulfide (- CH2-S-CH2), diinethylene-sulfoxide (-CH2-SO-CH2), dimethylene- sulfone (-CH2- S02-CH2), 2'-0-alkyl, and 2'-deoxy2'-fluoro phosphorothioate intemucleoside linkages are well known in the art (see Uhlmann et ah, 1990, Chem. Rev. 90:543- 584; Schneider et ah, 1990, Tetrahedron Lett. 31:335 and references cited therein).

The nucleic acids may be purified by any suitable means, as are well known in the art. For example, the nucleic: acids can be purified by reverse phase or ion exchange HPLC, size exclusion chromatography or gel electrophoresis. Of course, the skilled artisan will recognize that the method of purification will depend in part on the size of the DNA to be purified. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

The present invention also encompasses pharmaceutical and therapeutic compositions comprising the compounds of the present invention.

The present invention is also directed to pharmaceutical compositions comprising the compounds of the present invention. More particularly, such compounds can be formulated as pharmaceutical compositions using standard pharmaceutically acceptable carriers, fillers, solublizing agents and stabilizers known to those skilled in the art.

When used in vivo for therapy, the antibodies of the invention are administered to the subject in therapeutically effective amounts (i.e., amounts that have a desired therapeutic effect). In one aspect, they will be administered parenterally. The dose and dosage regimen will depend, for example, upon the degree of the anemia, the characteristics of the particular antibody or other compound used, e.g., its therapeutic index, the subject, and the subject's history. In one embodiment, at least one antibody or other agonist compound is administered once, or more than once, or even continuously over a period of 1-2 weeks.

Optionally, the administration is made during the course of adjunct therapy such as antimicrobial treatment, or administration of, for example, a cytokine(s), or other EPO or erythropoiesis regulatory agent.

For parenteral administration, an antibody can be formulated, for example, in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic, and non-therapeutic. Examples of such vehicle are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes can be used as carriers. The vehicle can contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibodies will typically be formulated in such vehicles at concentrations of about 1.0 mg/ml to about 10 mg/ml.

In one aspect, the invention provides for the use of IgM antibodies; however, IgG molecules by being smaller can be more able than IgM molecules to localize to certain types of infected cells. Therefore, in one aspect, IgG antibodies are useful in the practice of the invention. The antibody compositions used can be formulated and dosages established in a fashion consistent with good medical practice taking into account the condition or disorder to be treated, the condition of the individual patient, the site of delivery of the composition, the method of administration, and other factors known to practitioners. The antibody compositions are prepared for administration according to the description of preparation of polypeptides for administration, infra.

As is well understood in the art, biospecific capture reagents include antibodies, binding fragments of antibodies which bind to activated integrin receptors on metastatic cells (e.g., single chain antibodies, Fab' fragments, F(ab)'2 fragments, and scFv proteins and affibodies (Affibody, Teknikringen 30, floor 6,

Box 700 04, Stockholm SE-10044, Sweden; See U.S. Pat. No. 5,831,012, incorporated herein by reference in its entirety and for all purposes)). Depending on intended use, they also can include receptors and other proteins that specifically bind another biomolecule.

The hybrid antibodies and hybrid antibody fragments include complete antibody molecules having full length heavy and light chains, or any fragment thereof, such as Fab, Fab', F(ab')2, Fd, scFv, antibody light chains and antibody heavy chains. Chimeric antibodies which have variable regions as described herein and constant regions from various species are also suitable. See for example, U.S. Application No. 20030022244.

Initially, a predetermined target object is chosen to which an antibody can be raised. Techniques for generating monoclonal antibodies directed to target objects are well known to those skilled in the art. Examples of such techniques include, but are not limited to, those involving display libraries, xeno or humab mice, hybridomas, and the like. Target objects include any substance which is capable of exhibiting antigenicity and are usually proteins or protein polysaccharides.

Examples include receptors, enzymes, hormones, growth factors, peptides and the like. It should be understood that not only are naturally occurring antibodies suitable for use in accordance with the present disclosure, but engineered antibodies and antibody fragments which are directed to a predetermined object are also suitable.

The present invention is also directed to pharmaceutical compositions comprising the compounds of the present invention. More particularly, such compounds can be formulated as pharmaceutical compositions using standard pharmaceutically acceptable carriers, fillers, solublizing agents and stabilizers known to those skilled in the art.

In accordance with one embodiment, a method of treating a subject in need of treatment is provided. The method comprises administering a pharmaceutical composition comprising at least one compound of the present invention to a subject in need thereof. Compounds identified by the methods of the invention can be administered with known compounds or other medications as well.

The invention also encompasses the use of pharmaceutical compositions of an appropriate compound, and homologs, fragments, analogs, or derivatives thereof to practice the methods of the invention, the composition comprising at least one appropriate compound, and homolog, fragment, analog, or derivative thereof and a pharmaceutically-acceptable carrier.

The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day.

The invention encompasses the preparation and use of pharmaceutical compositions comprising a compound useful for treatment of the diseases disclosed herein as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

As used herein, the term“physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of

pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit. It will be understood by the skilled artisan that such pharmaceutical compositions are generally suitable for administration to animals of all sorts.

Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys. The invention is also contemplated for use in

contraception for nuisance animals such as rodents.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a“unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.

Controlled- or sustained-release formulations of a pharmaceutical

composition of the invention may be made using conventional technology.

As used herein,“additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other“additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

Typically, dosages of the compound of the invention which may be administered to an animal, preferably a human, range in amount from 1 pg to about 100 g per kilogram of body weight of the subject. While the precise dosage administered will vary depending upon any number of factors, including, but not limited to, the type of animal and type of disease state being treated, the age of the subject and the route of administration. In one aspect, the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the subject. In another aspect, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of the subject.

The compound may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the condition or disease being treated, the type and age of the subject, etc.

The invention is also directed to methods of administering the compounds of the invention to a subject. In one embodiment, the invention provides a method of treating a subject by administering compounds identified using the methods of the invention. Pharmaceutical compositions comprising the present compounds are administered to an individual in need thereof by any number of routes including, but not limited to, topical, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate, and the like. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

The invention also includes a kit comprising the composition of the invention and an instructional material which describes adventitially administering the composition to a cell or a tissue of a mammal. In another embodiment, this kit comprises a (preferably sterile) solvent suitable for dissolving or suspending the composition of the invention prior to administering the compound to the mammal.

As used herein, an“instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the peptide of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of alleviation the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the peptide of the invention or be shipped together with a container which contains the peptide. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

Other techniques known in the art may be used in the practice of the present invention, including those described in international patent application WO

2006/091535 (PCT/US 2006/005970), the entirety of which is incorporated by reference herein.

It will be appreciated, of course, that the proteins or peptides of the invention may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e.

chemical substituents suitable to protect and/or stabilize the N- and C-termini from “undesirable degradation”, a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C₁-C₅ branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N- terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH₂), and mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide's C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without effect on peptide activity.

Acid addition salts of the present invention are also contemplated as functional equivalents. Thus, a peptide in accordance with the present invention treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclic and the like, to provide a water soluble salt of the peptide is suitable for use in the invention. Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or

deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phospho threonine.

Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or non-standard synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.

The invention includes the use of beta-alanine (also referred to as b-alanine, b-Ala, bA, and bA, having the structure:

beta alanine

Sequences are provided herein which use the symbol“bA”, but in the Sequence Listing submitted herewith“bA” is provided as“Xaa” and reference in the text of the Sequence Listing indicates that Xaa is beta alanine.

Peptides useful in the present invention, such as standards, or modifications for analysis, may be readily prepared by standard, well-established techniques, such as solid-phase peptide synthesis (SPPS) as described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Illinois; and as described by Bodanszky and Bodanszky in The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York. At the outset, a suitably protected amino acid residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin. “Suitably protected” refers to the presence of protecting groups on both the a-amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product. Stepwise synthesis of the oligopeptide is carried out by the removal of the N-protecting group from the initial amino acid, and couple thereto of the carboxyl end of the next amino acid in the sequence of the desired peptide. This amino acid is also suitably protected. The carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support-bound amino acid by formation into a reactive group such as formation into a carbodiimide, a symmetric acid anhydride or an“active ester” group such as hydroxybenzotriazole or pentafluorophenly esters.

Examples of solid phase peptide synthesis methods include the BOC method which utilized tert-butyloxcarbonyl as the a-amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protect the a-amino of the amino acid residues, both methods of which are well-known by those of skill in the art.

Incorporation of N- and/or C- blocking groups can also be achieved using protocols conventional to solid phase peptide synthesis methods. For incorporation of C-terminal blocking groups, for example, synthesis of the desired peptide is typically performed using, as solid phase, a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal blocking group. To provide peptides in which the C-terminus bears a primary amino blocking group, for instance, synthesis is performed using a p-methylbenzhydrylamine (MB HA) resin so that, when peptide synthesis is completed, treatment with hydrofluoric acid releases the desired C-terminally amidated peptide. Similarly, incorporation of an N-methylamine blocking group at the C-terminus is achieved using N-methylaminoethyl-derivatized DVB, resin, which upon HF treatment releases a peptide bearing an N-methylamidated C- terminus. Blockage of the C-terminus by esterification can also be achieved using conventional procedures. This entails use of resin/blocking group combination that permits release of side-chain peptide from the resin, to allow for subsequent reaction with the desired alcohol, to form the ester function. FMOC protecting group, in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this purpose, with cleavage from the support being effected by TFA in dicholoromethane. Esterification of the suitably activated carboxyl function e.g. with DCC, can then proceed by addition of the desired alcohol, followed by deprotection and isolation of the esterified peptide product.

Incorporation of N-terminal blocking groups can be achieved while the synthesized peptide is still attached to the resin, for instance by treatment with a suitable anhydride and nitrile. To incorporate an acetyl blocking group at the N- terminus, for instance, the resin-coupled peptide can be treated with 20% acetic anhydride in acetonitrile. The N-blocked peptide product can then be cleaved from the resin, deprotected and subsequently isolated.

To ensure that the peptide obtained from either chemical or biological synthetic techniques is the desired peptide, analysis of the peptide composition should be conducted. Such amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively, or additionally, the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in order, may also be used to determine definitely the sequence of the peptide.

Prior to its use, the peptide may be purified to remove contaminants. In this regard, it will be appreciated that the peptide will be purified so as to meet the standards set out by the appropriate regulatory agencies. Any one of a number of a conventional purification procedures may be used to attain the required level of purity including, for example, reversed-phase high performance liquid

chromatography (HPLC) using an alkylated silica column such as C₄ -,C₈- or Cis- silica. A gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion-exchange chromatography can be also used to separate peptides based on their charge.

Substantially pure protein obtained as described herein may be purified by following known procedures for protein purification, wherein an immunological, enzymatic or other assay is used to monitor purification at each stage in the procedure. Protein purification methods are well known in the art, and are described, for example in Deutscher et al. (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).

As discussed, modifications or optimizations of peptide ligands of the invention are within the scope of the application. Modified or optimized peptides are included within the definition of peptide binding ligand. Specifically, a peptide sequence identified can be modified to optimize its potency, pharmacokinetic behavior, stability and/or other biological, physical and chemical properties.

Amino Acid Substitutions

In certain embodiments, the disclosed methods and compositions may involve preparing peptides with one or more substituted amino acid residues. In various embodiments, the structural, physical and/or therapeutic characteristics of peptide sequences may be optimized by replacing one or more amino acid residues.

Other modifications can also be incorporated without adversely affecting the activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the present invention are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.

The skilled artisan will be aware that, in general, amino acid substitutions in a peptide typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions). The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.

For example, one can make the following isosteric and/or conservative amino acid changes in the parent polypeptide sequence with the expectation that the resulting polypeptides would have a similar or improved profile of the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: including alanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid, S-cyclohexylalanine or other simple alpha-amino acids substituted by an aliphatic side chain from C1-10 carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions. Substitution of aromatic-substituted hydrophobic amino acids: including phenylalanine, tryptophan, tyrosine, biphenylalanine, l-naphthylalanine, 2- naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy-substituted forms of the previous listed aromatic amino acids, illustrative examples of which are: 2-, 3- or 4-aminophenylalanine, 2-, 3- or 4- chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or 4- methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxy tryptophan, 2'-, 3'-, or 4'-amino-, 2'-, 3'-, or 4'-chloro-, 2,3, or 4-biphenylalanine, 2', -3',- or 4'-methyl- 2, 3 or 4-biphenylalanine, and 2- or 3-pyridylalanine.

Substitution of amino acids containing basic functions: including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl, or aryl-substituted (from Ci-Cio branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms (such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro- R position for example. Compounds that serve as illustrative examples include: N- epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)- alanine, N,N-gamma, gamma'-diethyl-homoarginine. Included also are compounds such as alpha methyl arginine, alpha methyl 2,3-diaminopropionic acid, alpha methyl histidine, alpha methyl ornithine where alkyl group occupies the pro-R position of the alpha carbon. Also included are the amides formed from alkyl, aromatic, heteroaromatic (where the heteroaromatic group has one or more nitrogens, oxygens, or sulfur atoms singly or in combination) carboxylic acids or any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives) and lysine, ornithine, or 2,3- diaminopropionic acid.

Substitution of acidic amino acids: including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine or lysine and tetrazole-substituted alkyl amino acids.

Substitution of side chain amide residues: including asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine.

Substitution of hydroxyl containing amino acids: including serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine or threonine. It is also understood that the amino acids within each of the categories listed above can be substituted for another of the same group.

For example, the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making conservative substitutions, the use of amino acids can include various hydropathic indices. In one aspect, the hydropathic indices are within +/- 2, in another they are within +/- 1 , and in one aspect, they are within +/- 0.5.

Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4) In one aspect, the replacement of amino acids with others of similar hydrophilicity is provided by the invention.

Other considerations include the size of the amino acid side chain. For example, it would generally not be preferable to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine. The effect of various amino acid residues on protein secondary structure is also a consideration. Through empirical study, the effect of different amino acid residues on the tendency of protein domains to adopt an alpha-helical, beta- sheet or reverse turn secondary structure has been determined and is known in the art (see, e.g., Chou & Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables of conservative amino acid substitutions have been constructed and are known in the art. For example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R) gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed. For interior residues, conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp. (See, e.g., PROWL Rockefeller University website). For solvent exposed residues, conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have been constructed to assist in selection of amino acid substitutions, such as the PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in an encoded peptide sequence are well known and a matter of routine experimentation for the skilled artisan, for example by the technique of site-directed mutagenesis or by synthesis and assembly of oligonucleotides encoding an amino acid substitution and splicing into an expression vector construct.

Linkers

Additionally, modifications encompassed by the invention include introduction of linkers or spacers between the targeting sequence of the binding moiety or binding polypeptide and a detectable label or therapeutic agent. For example, use of such linkers/spacers can improve the relevant properties of the binding peptides (e.g., increase serum stability, etc.). These linkers can include, but are not restricted to, substituted or unsubstituted alkyl chains, polyethylene glycol derivatives, amino acid spacers, sugars, or aliphatic or aromatic spacers common in the art.

In other embodiments, therapeutic agents, including, but not limited to, cytotoxic agents, anti- angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes or other agents may be used as adjunct therapies when using the antibody/peptide ligand complexes described herein.

Nucleic acids useful in the present invention include, by way of example and not limitation, oligonucleotides and polynucleotides such as antisense DNAs and/or RNAs; ribozymes; DNA for gene therapy; viral fragments including viral DNA and/or RNA; DNA and/or RNA chimeras; mRNA; plasmids; cosmids; genomic DNA; cDNA; gene fragments; various structural forms of DNA including single- stranded DNA, double- stranded DNA, supercoiled DNA and/or triple-helical DNA; Z-DNA; and the like. The nucleic acids may be prepared by any conventional means typically used to prepare nucleic acids in large quantity. For example, DNAs and RNAs may be chemically synthesized using commercially available reagents and synthesizers by methods that are well-known in the art (see, e.g., Gait, 1985, OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press, Oxford, England)). RNAs may be produce in high yield via in vitro transcription using plasmids such as SP65 (Promega Corporation, Madison, WI).

The invention further provides cells transfected with the nucleic acid containing an enhancer/promoter combination of the invention.

Promoters may be coupled with other regulatory sequences/elements which, when bound to appropriate intracellular regulatory factors, enhance ("enhancers") or repress ("repressors") promoter-dependent transcription. A promoter, enhancer, or repressor, is said to be "operably linked" to a transgene when such element(s) control(s) or affect(s) transgene transcription rate or efficiency. For example, a promoter sequence located proximally to the 5' end of a transgene coding sequence is usually operably linked with the transgene. As used herein, term "regulatory elements" is used interchangeably with "regulatory sequences" and refers to promoters, enhancers, and other expression control elements, or any combination of such elements.

Promoters are positioned 5' (upstream) to the genes that they control. Many eukaryotic promoters contain two types of recognition sequences: TATA box and the upstream promoter elements. The TATA box, located 25-30 bp upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase II to begin RNA synthesis as the correct site. In contrast, the upstream promoter elements determine the rate at which transcription is initiated. These elements can act regardless of their orientation, but they must be located within 100 to 200 bp upstream of the TATA box.

Enhancer elements can stimulate transcription up to lOOO-fold from linked homologous or heterologous promoters. Enhancer elements often remain active even if their orientation is reversed (Li et ah, J. Bio. Chem. 1990, 266: 6562-6570). Furthermore, unlike promoter elements, enhancers can be active when placed downstream from the transcription initiation site, e.g., within an intron, or even at a considerable distance from the promoter (Yutzey et ah, Mol. and Cell. Bio. 1989, 9:1397-1405).

It is known in the art that some variation in this distance can be

accommodated without loss of promoter function. Similarly, the positioning of regulatory elements with respect to the transgene may vary significantly without loss of function. Multiple copies of regulatory elements can act in concert. Typically, an expression vector comprises one or more enhancer sequences followed by, in the 5' to 3' direction, a promoter sequence, all operably linked to a transgene followed by a polyadenylation sequence.

The present invention further relies on the fact that many enhancers of cellular genes work exclusively in a particular tissue or cell type. In addition, some enhancers become active only under specific conditions that are generated by the presence of an inducer such as a hormone or metal ion. Because of these differences in the specificities of cellular enhancers, the choice of promoter and enhancer elements to be incorporated into a eukaryotic expression vector is determined by the cell type(s) in which the recombinant gene is to be expressed.

In one aspect, the regulatory elements of the invention may be heterologous with regard to each other or to a transgene, that is, they may be from different species. Furthermore, they may be from species other than the host, or they also may be derived from the same species but from different genes, or they may be derived from a single gene.

The present invention further encompasses kits.

Compositions of the present invention may be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the therapeutic compound as described herein.

In some embodiments, the kit may include a therapeutic compound (as described herein), metal or plastic foil, such as a blister pack, a dispenser device or an applicator, tubes, buffers, and instructions for administration. The various reagent components of the kits may be present in separate containers, or some or all of them may be pre-combined into a reagent mixture in a single container, as desired. The dispenser device or applicator may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

In some cases, the kit includes at least one dose of monoclonal antibody (mAb) to CD24.

In some cases, the kit includes at least one dose of a fragment of monoclonal antibody (mAb).

In some cases, the kit includes at least one dose of an expression vector comprising a nucleic acid sequence encoding the full length or segments of the protein of interest.

The invention is now described with reference to the following Examples and Embodiments. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, are provided for the purpose of illustration only and specifically point out some embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Therefore, the examples should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Examples

Experimental Model and Subject Details- Mice

C57BL6 and IL17-GFP reporter mice were ordered from Jackson Laboratories. All mice were housed under specific pathogen-free conditions at the University of Virginia’s animal facility. All experiments were performed according to provisions of the Animal Welfare Act of 1996 and were approved by the University of Virginia Institutional Animal Care and Use Committee. Littermates were randomly assigned to experimental groups. Male mice were used that were 6 weeks old at the start of each experiment. During the infection, mice were weighed and scored daily and euthanized if they lost more than 25% of the starting body weight, or had a clinical score of 14 or above.

Human subjects

Information on the sex and sample size of the human subjects in each experimental group is listed in Table Sl. Ages ranged from 12-97 years, with a mean of 60 years and a median of 63 years. Cox proportional hazards model was used to adjust for age among other variables including sex, race, Charlson comorbidity index and ICU admission. Patient data collection and analysis were approved by the UVA Institutional Review Board (IRB-HSR #16926).

DSS treatment and C. difficile infection

For DSS treatment, mice were given 2% Dextran Sulfate Sodium (DSS, Affymetrix) in the drinking water for 6 days, then switched to regular drinking water and allowed to recover. During DSS colitis, weight loss was monitored and mice were euthanized in the rare instance that they developed severe DSS colitis. For C. difficile infections, age and sex-matched mice were given 45 mg/L vancomycin (Mylan), 35 mg/L colistin (Sigma), 35 mg/L gentamicin (Sigma) and 215 mg/L metronidazole (Hospira) ad libitum for 3 days. Then, mice were switched to regular drinking water for two days. On the day before infection, mice were injected intraperitoneally with 0.016 mg/g of clindamycin (Hospira). To prepare the inoculum, C. difficile strain R20291 was grown from a frozen glycerol stock onto Brain Heart Infusion (BHI) agar plates supplemented with cycloserine and cefoxitin (C. difficile supplement, Sigma) overnight at 37°C in an anaerobic chamber (Shel Labs). A single colony was then transferred into a tube containing BHI liquid media and grown overnight at 37°C anaerobically. The next day, the liquid culture was centrifuged and the pellet was washed twice with PBS, the optical density determined using a spectrophotometer and the inoculum was diluted to 5xl0⁷ CFU/ml using sterile, anaerobic PBS. The inoculum was loaded into sterile syringes and transported in sealed biohazard bags to the animal facility. The concentration of each inoculum was confirmed by counting CFUs after plating on BHI plates supplemented with 1% sodium taurocholate and grown overnight at 37°C anaerobically. Mice were orally gavaged with 5xl0⁶ CFU/mouse in randomized order. During the infection, mice were weighed and scored daily and euthanized if they developed severe disease based on the scoring criteria. Clinical scores are based on weight loss, coat appearance, eyes/nose discharge, activity, posture and diarrhea. To quantify C. difficile burden during infection, cecal contents were suspended using sterile, anaerobic PBS, serially diluted and plated on BHI plates supplemented with cycloserine and cefoxitin (C. difficile supplement, Sigma). After an overnight incubation at 37°C in an anaerobic chamber, CFUs of C. difficile were counted and normalized to stool weight. Toxins A/B were quantified using the ELISA C. difficile TOX A/B II kit from Techlab according to the manufacturer’s instructions and normalized to stool weight.

FITC dextran Assay

Mice were gavaged with 40 mg/ 100 g body weight Fluorescein

isothiocyanate (FITC)-dextran solution (Sigma). 4 hours later, mice were sacrificed and a spectrophotometer was used to detect FITC in the serum at 485/530 nm.

LP Isolation and Flow Cytometry

To isolate the lamina propria, colons were removed, cut longitudinally and rinsed thoroughly in Hank’s balanced salt solution (HBSS) supplemented with 5% FBS and 25 mM HEPES. Epithelial cells were removed with two 20-minute incubations in pre- warmed HBSS with 15 mM HEPES, 5 mM EDTA, 10% FBS and 1 mM dithiothreitol in a 37°C shaking incubator. The colons were then cut into small sections and incubated in pre-warmed RPMI media containing 0.17 mg/ml liberase TL (Sigma) and 30 mg/ml DNase (Sigma) in a 37°C shaking incubator. Following the digestion step, the tissue was passed through 40 and 100 mM cell strainers, respectively, counted and re-suspended to a concentration of lxlO⁷ cells/ml in fluorescence-activated cell sorting (FACS) buffer (PBS with 2% FBS). lxlO⁶ cells were plated in a 96-well plate and stained for flow cytometry. After an Fc blocking step (anti-mouse CD16/32 TruStain, BioLegend) and staining with a fixable viability dye (Zombie Aqua, Biolegend), the following antibodies were used for staining: CDl lb-APC (Ml/70), CD45-APC-Cy7 (30-F11), CDl lc-BV42l (N418), CD3e-APC (500A2), CD4-PE (RM4-4), CD45-PE/Cy7 (30-F11), T-bet- BV421 (4B10), TCRp-PerCp/Cy5.5 (H57-597), CD4-FITC (GK1.5), CD3e-FITC (500A2),CD45-BV42l (30-F11), CD4-PE/Cy7 (GK1.5), GATA3-AF647

(16E10A23), TCRp-FITC (H57-597), IL-17A-PE (TC11-18H10.1), IFNy-APC (XMG1.2), Ly6G-PeCy7 (1A8) , Ly6C-FITC (HK1.4) (BioLegend); SiglecF-PE (E50-2440) (BD Biosciences); FOXP3-APC (FJK-l6s), RORyt-PE (B2D), IL-22- PerCP-eFluor 710 (IL22JOP) (Thermo Fisher Scientific).

For intracellular transcription factor staining, a FOXP3/Transcription Factor Staining Buffer Set (eBioscience, catalog# 00-5523-00) was used according to the manufacturer’s instructions. For intracellular cytokine staining, isolated colonic cells were re-suspended in 5 ml 10% percoll (Sigma, catalog# P1644) and layered over 80% percoll, centrifuged at lOOOxg for 15 minutes at RT with maximum

acceleration and no brakes. Lymphocytes were collected at the interphase, washed with RPMI, plated at lxlO^{5 6} cells per well. Cells were stimulated in T cell culture media (RPMI Media 1640, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 5 mM 2-P-mercaptoethanol, BD GolgiPlug containing

Brefeldin A (BD Cytofix/Cytoperm kit cat#555028), 50 ng/ml PMA, 750 ng/ml ionomycin) for 4 hours at 37°C with 5% C0₂. Following the stimulation, cells were stained using Cytofix/Cytoperm™ Plus kit (BD, catalog# 555028) according to the manufacturer’s protocol. Flow cytometry data was acquired using an LSR Fortessa cytometer (BD Biosciences) and data analysis was performed via FlowJo.

Tissue protein and cytokine analysis

Cecal tissue was isolated and rinsed gently with PBS, then homogenized by bead-beating in 400 pl of a buffer containing lx HALT protease inhibitor (Pierce) and 5 mM HEPES. The homogenate was incubated on ice for 30 minutes after the addition of 400 ul of another buffer containing lx HALT protease inhibitor (Pierce), 5 mM HEPES and 2% Triton X-100. Finally, the lysates were spun at l3,000g for 5 minutes and the supernatants were transferred to a new tube and used for protein analysis. Mouse tissue IL-23, IL-17A, IL-17F and IL-22 ELISAs were done according to the manufacturer’s instructions (DuoSet, R&D). Protein levels were normalized to the total protein concentration determined using a Pierce BCA Protein Assay (Thermo Fisher Scientific). For TNFa, IFNy, KC, IL-6, IL-la, IL- 1 b,

GMCSF and IFNy cecal tissue was processed using the same protocol and total protein was quantified using a Pierce BCA Protein Assay. Cytokine levels were determined using a Luminex MAGPIX bead-based multiplex analyzer through the University of Virginia’s Flow Cytometry Core Facility. Human serum IL-23 and IL- 17A were measured using the Human IL-23 Quantikine ELISA Kit and Human IL- 17 Quantikine HS ELISA Kit from R&D, respectively, according to the

manufacturer’s instructions. Human serum IL-6 and IL-4 were measured using a Luminex bead-based multiplex assay from R&D according to the manufacturer’s instructions.

Cell depletion and antibody neutralization

For CD4 cell depletion: DSS-treated and untreated mice were injected i.p. with 400 pg a-CD4 (BioXcell, BE0003-1, clone GK1.5) or IgG isotype control (BioXcell, BE0090) on days -6, -3 and on the day of infection with C. difficile as previously reported (Moynihan et ah, 2016). Flow cytometry was used to confirm depletion of CD4-expressing T cells from the colon with 90% efficiency. Human anti-CD24 antibodies with cell depletion activity include, for example, MAb anti human CD4, clone OKT-4 (BioXcell, BE0003-2; ThermoFisher/eBio science #14- 0049082 (RPA-T4)). The OKT-4 and the RPA-T4 monoclonal antibodies react with human CD4. The CD4 antigen is a 55 kDa cell surface type I membrane

glycoprotein belonging to the immunoglobulin superfamily. CD4 is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes,

macrophages, and dendritic cells. CD4 acts as a co-receptor which in cooperation with the T cell receptor (TCR) interacts with class II MHC molecules displayed by antigen presenting cells (APC). CD4 is expressed by the majority of thymocytes, most helper T cells, a subset of NK-T cells and weakly by dendritic cells and macrophages. CD4 plays an important role in the development of T cells and is required for mature T cells to function optimally. For IL-17RA blockade and IL- 17A neutralization: DSS-treated and untreated mice were injected i.p. with 125 pg _(X-IL-17RA (R&D Systems, catalog# MAB4481), 125 pg a-IL-l7A (R&D Systems, catalog# MAB421) or IgG isotype control (R&D Systems, MAB006) on days -1, 1 and 2 of C. difficile infection.

T cell transfers For the adoptive transfer of CD4+ T cells, mice were either treated with DSS or regular drinking water. On day 7 after DSS treatment, colons and mesenteric lymph nodes were removed and processed into single-cell suspensions as described above. Then, CD4+ T cells were isolated using a negative selection CD4+ T cell isolation kit from Miltenyi Biotec. The purity of the cells isolated was checked by flow cytometry. lxlO⁶ cells were transferred i.p. into naive recipients on the day before infection with C. difficile.

For the adoptive transfer of Thl7 cells, splenocytes were isolated from IL17A-GFP reporter mice and cultured ex vivo using a CellXVivo Mouse Thl7 Cell Differentiation Kit (R&D Systems) according to the manufacturer’s instructions. After differentiation, the cells were stained for CD3e and CD4 expression and viability was determined using 7-AAD Viability Staining Solution (BioLegend). IL- 17A+ and IL-17A- CD4+ T cells were sorted using an Influx Cell Sorter (BD Biosciences) and lxlO⁶ cells were transferred into naive recipients on the day before C. difficile infection.

16S rRNA Sequencing

Fecal pellets were collected from mice given untreated or DSS -treated water after a two-week recovery period and before antibiotics treatment, on day 21 in Figure 1A. Cecal contents were collected from untreated or DSS-treated mice after antibiotics treatment, on day 27 in Figure 1A, and diluted 1:2 with sterile PBS.

DNA was extracted using Qiagen MagAttract PowerMicrobiome kit DNA/RNA kit (Qiagen, catalog no. 27500-4-EP). The V4 region of the 16S rRNA gene was amplified from each sample using the dual indexing sequencing strategy as described previously (Kozich et ah, 2013). Sequencing was done on the Illumina MiSeq platform, using a MiSeq Reagent Kit V2 500 cycles (Illumina cat# MS 102- 2003), according to the manufacturer’s instructions with modifications found in the Schloss SOP at the Schloss Lab/MiSeq WetLab website. The mock community produced ZymoBIOMICS Microbial Community DNA Standard (Zymo Research cat# D6306) was sequenced to monitor sequencing error. The overall error rate was 0.019%.

Sequence Curation and Analysis

Raw sequences were curated using the software package mothur version 1.39.5 (Schloss et ah, 2009) following the Illumina MiSeq standard operating procedure. Briefly, paired end reads were assembled into contigs and aligned to the V4 region using the SLIVA 16S rRNA sequence database (Quast et al., 2013) , sequences that failed to align or were flagged as possible chimeras were removed.

A naive Bayesian classifier using the Ribosomal Database Project (Wang et al., 2007) classified sequences. Operational Taxonomic Units (OTUs) using a 97% similarity cutoff were generated using the Opticlust clustering algorithm (Westcott et al., 2017).

The number of sequences in each sample was then rarefied to 25,000 sequences to minimize bias due to uneven sampling. Following curation in mothur, further data analysis and figure generation was carried out in R (v 3.4.1) using the package vegan (Oksanen et al, 2018). This includes determining the axes for the multidimensional scaling (MDS) plots using Bray-Curtis dissimilarity calculated from sequence abundance. Additionally, vegan was used to determine significance between groups using PERMANOVA. The sequences associated with analysis were deposited to the SRA under the bioproject PRJNA475161. Final figures were modified and arranged in Adobe Illustrator CC.

Histology

Cecal tissue was isolated and fixed in Bouin’s solution for 24 hours then transferred to 70% ethanol, paraffin embedded and stained with haematoxylin and eosin (H&E). The tissue was sectioned onto slides and scored by two independent, blinded observers. Each sample was given a score of 0-3 for each of the following parameters: epithelial disruption, submucosal edema, inflammatory infiltrate, mucosal thickening and luminal exudates as described previously (Pawlowski et al., 2010).

QUANTIFICATION AND STATISTICAL ANALYSIS

For mouse data, survival curves were compared using a Log-Rank (Mantel- Cox) statistical test. Comparisons between two groups were assessed using a student t-test or Mann- Whitney test depending on whether the data were normally distributed. Statistical significance between multiple groups was tested using a one way multiple analysis of variance (ANOVA) or Kruskal-Wallis. For human data, patients were categorized into quartiles based on IL-6 serum levels. Survival curves were compared using a Kaplan-Meyer test, then further evaluated by a Cox proportional hazards model to adjust for age, sex, race, Charlson comorbidity index and ICU admission. Statistical significance between severe and non-severe patients was assessed using a Mann- Whitney test. Data are presented as mean ± SEM. *p<0.05, **r<0.01, ***p<0.00l,

****p<0.000.

DATA AND SOFTWARE AVAILABILITY

The sequences associated with the 16S microbiota analysis were deposited to the SRA under the bioproject PRJNA475161.

C. difficile infections are the number one cause of hospital-acquired diarrhea in the United States. Several clinical studies have reported higher incidence and severity of CDI in patients with one of the two major forms of inflammatory bowel disease (IBD): ulcerative colitis and Crohn’s disease. In order to understand the factors underlying increased severity of CDI in IBD patients, we utilized a Dextran Sulfate Sodium (DSS) murine model of inflammatory colitis. In support of clinical observations, our data showed that mice treated with DSS and then infected with C. difficile developed a more severe C. difficile disease compared to untreated mice. Increased severity of disease was measured by increased mortality, weight loss and clinical scores. Importantly, comparison of C. difficile burden between mice with prior DSS colitis and untreated controls showed no differences in C. difficile colonization between the two groups, suggesting that increased severity of disease might be due to the host immune response to infection. Immunophenotyping of immune cells recruited to the colon at the peak of CDI revealed increased levels of CD4+ T cells at the site of infection in mice with prior DSS colitis (please see Appendix). Furthermore, depletion of CD4+ T cells using a monoclonal antibody protected mice with prior DSS colitis from severe C. difficile disease (please see Appendix).

This invention has identified a novel role that CD4+ T cells play in exacerbating the severity of C. difficile infection. We have shown that depleting these cells protects from severe C. difficile disease. It is also shown in the Appendix that an increase in Thl7 cells in mice with prior DSS suggesting that these cells are causing increased severity of C. difficile disease (please see Appendix). These findings can be used to design therapeutics targeting CD4+ T cells, Thl7 cells, or upstream/downstream mechanisms (IL-23, IL-22, IL-17A, IL-6, TGFb) to protect against severe C. difficile disease.

Sequences IL-17RA (human)- see GenBank Accession Nos. NP_055154.3 (866 a.a.) and NP_00l276834.l (832 a.a.)

IL-17 (human)- see GenBank: AAC50341.1, 155 a.a.

IL-17A (human) precursor- see GenBank NP_002l8l, 155 a.a.

Inhibitory Antibodies

Antibodies are available that are inhibitory against the human cytokines and their receptors. Some of the useful antibodies are:

Anti-Human IL-17 RA/IL-17 R Antibody. R&D Systems # MAB 177, Monoclonal Mouse IgGl Clone # 133617.

Anti-Human IL-17 RA/IL-17 R Antibody. R&D # AF177, Polyclonal Goat IgG. Anti-Human/Primate IL-17/IL-17A Antibody. R&D # MAB317, Monoclonal Mouse IgG2B Clone # 41809.

Anti-Human IL-17/IL-17A Antibody. R&D # AF-317-NA, Polyclonal Goat IgG.

Results-

(FIGS. 5-8 are also referred to as Supplemental Figs. 1-4 or Figs. SI to S4)

Increased CDI-associated mortality in mice with prior DSS colitis

To determine whether prior gut inflammation can predispose the host to severe subsequent CDI, we designed a mouse model where we treated mice with 2% DSS for 6 days to induce gut inflammation. After a two-week recovery period, mice were challenged with C. difficile (Figure 1 A). The recovery period was sufficient to reverse the DSS-induced weight loss, as well as restore gut barrier integrity as measured by permeability to FITC dextran (Figure 1, B-C). Examination of hematoxylin and eosin (H&E) stained histopathological sections of the ceca revealed that although DSS treatment caused an acute disruption of the epithelial barrier, the recovery period was sufficient to restore epithelial integrity (Figure SI A).

Furthermore, several studies have shown that DSS treatment causes the upregulation of several inflammatory cytokines, including TNFa, IFNy, KC, IL-6, IL-la and IL- 1b (Alex et ah, 2009; Arai et ah, 1998 and Jeengar et ah, 2017). Our data showed that although these cytokines were elevated during acute DSS colitis, the levels of these inflammatory cytokines were comparable between the untreated and DSS- treated groups after the recovery period (Figure SI B). Despite the resolution of acute colitis, mice previously treated with DSS had higher CDI-associated mortality when compared to previously untreated controls (Figure 1 D). Mice with prior DSS colitis also had more severe CDI when scored for: weight loss, coat appearance, eyes/nose discharge, activity, posture and diarrhea (Figure 1, E-F). We observed that DSS mice developed severe disease early starting at day 2 of infection.

Interestingly, when we measured C. difficile burden at the peak of infection on day 2, no differences were found between DSS mice and untreated mice (Figure 1 G). Additionally, C. difficile toxin A/B levels during infection were comparable between the two groups (Figure 1 H). Next, we tested whether increased severity of disease in the DSS-treated mice was due to DSS-induced changes in the gut microbiota. To characterize changes in the bacterial communities in the gut, we utilized 16S rRNA gene sequencing of the V4 region. We found that after two weeks of recovery, the DSS-treated mice had a significantly different fecal microbiota from the untreated mice (Figure S2 A). However, when we analyzed the cecal communities after antibiotic treatment, we found that the two groups were no longer significantly different suggesting that antibiotic treatment normalizes the changes in the gut microbiota caused by DSS (Figure S2 C-D). Finally, to test the role of the microbiota in this model, we co-housed DSS-treated mice with untreated mice before C. difficile infection. We found that co-housing was not sufficient to protect DSS mice from severe CDI or to worsen disease severity for untreated mice (Figure S2 B). Given the similarities in C. difficile colonization and toxin production and the lack of protection after co-housing, we hypothesized that increased severity of CDI in DSS mice is due to differences in the host immune response to infection.

CD4+ T cells increase CDI severity in mice with prior DSS colitis

To understand the differences in the host immune response to CDI between DSS and untreated mice that might influence disease severity, we used flow cytometry to characterize immune cell recruitment to the colon on day 2, the peak of infection. We found no significant difference in the numbers of neutrophils, eosinophils, Ly6C^hl and Ly6C^lG monocytes between the two groups (Figure S3 A). Surprisingly, CD4+ T cell numbers were consistently and significantly higher in DSS mice compared to untreated mice during CDI, both as a total number and as a percentage of TCRP+ T cells (Figure 2, A-B). Given the robust increase in CD4+ T cells, we hypothesized that these cells are the cause of increased severity of CDI in DSS mice. To test this hypothesis, first we depleted CD4-expressing cells using an anti-CD4 depleting antibody. We used flow cytometry to confirm successful depletion of CD4+ T cells from the colon lamina propria using the anti-CD4 antibody (Figure 2 C). Interestingly, depletion of CD4-expressing cells from DSS mice prior to CDI protected them from increased severity of disease as measured by reduced mortality, weight loss and clinical scores (Figure 2, D-F). Since CD4 can also be expressed by NK T cells, splenic DCs and lymphoid tissue inducer (LTi) cells in mice, we tested directly whether DSS-induced CD4+ T cells are sufficient to exacerbate CDI severity. To do this, we isolated CD4+ T cells from the colon lamina propria and mesenteric lymph nodes of both DSS-treated and untreated mice using a negative selection microbeads isolation kit. We used flow cytometry to confirm that the isolated cells were CD3+ CD4+ T cells (with a purity of 98.8%) (Figure 2 G) and transferred them into naive recipients that were subsequently infected with C. difficile. Mice receiving CD4+ T cells from DSS-treated donors had significantly lower survival rates post-infection compared to mice receiving CD4+ T cells from untreated donors and mice that did not receive a T cell transfer (Figure 2 H). Taken together, these studies are the first to show that in mice with prior gut inflammation, CD4+ T cells are necessary and sufficient to increase CDI-associated mortality. Increased Thl7 responses in mice with prior DSS colitis during CDI

Given our discovery that CD4+ T cells play an essential role during CDI in mice with prior gut inflammation, we aimed to further characterize the T cell subset that might exacerbate CDI severity. Using flow cytometry, we found a significant increase in Thl7 cells both in absolute numbers and as a percentage of CD4+ T cells in DSS mice during CDI (Figure 3 A). Similarly, using IL-17A-GFP reporter mice we found higher numbers of IL-l7A-producing CD4+ T cells during CDI in DSS mice compared to untreated controls (Figure 3 B). Furthermore, when we quantified colonic Thl, Th2, and T_reg cells, we did not find a compensatory increase in these cell subsets. Instead, our data showed a disruption in the homeostatic Thl7/T_reg balance and a significant skew towards Thl7 responses in DSS mice (Figure S3 B). Therefore, we focused our studies on understanding the role of Thl7 cells during CDI.

Thl7 cells express the IL-23 receptor, and the presence of IL-23 during Thl7 differentiation has been shown to lead to a pathogenic Thl 7 phenotype that has been implicated in autoimmunity, inflammation and infection (Langrish et ah, 2005; Lee et al. 2012). Furthermore, the IL-23 axis upstream of Thl7 cells has long been thought to play a role in the pathogenesis of IBD (McGovern and Powrie, 2007; Ahern et al., 2008). Finally, studies in our lab and others have shown that blocking IL-23 signaling during CDI protects from severe disease (Buonomo et al., 2013). Therefore, we investigated whether mice with DSS colitis have higher IL-23 levels before and after CDI. Indeed, we found higher levels of IL-23 protein in the ceca of DSS mice on days 0 and 2 of infection. Additionally, we found higher levels of the Thl7 cytokine IL-17A in the ceca of DSS mice during infection (Figure 3 C). Finally, our data showed a direct correlation between IL-23 levels in the cecum during infection and CDI severity (Figure 3 D). Taken together, these findings indicated that a colitis-induced skew towards Thl7 responses was associated with worse disease outcome during CDI.

In addition to the production of IL-17A, Thl7 cells can produce other cytokines including IL-17F, IL-22, IFNy and GMCSF. In order to determine which of these cytokines are important for the Thl7-mediated exacerbation of CDI severity, we used flow cytometry and ELISA assays to compare the levels of these cytokines between DSS and untreated mice. We found that the majority of IL-17A+ T cells do not co-express IFNy or IL-22. Our analysis showed that on Day 0 before infection, DSS mice had elevated levels of IL-17A+ IFNy- and IL-17A+ IL-22- T cells in the mesenteric lymph nodes but not in the colon. On Day 2 during infection, however, we found significantly increased numbers of these cells in the mesenteric lymph nodes as well as the colon (Figure S4 A). Additionally, we found an increase in the levels of IL-17F and IL-22 cecal proteins in DSS mice during infection and no difference in GMCSF and IFNy (Figure S4 B).

Because of the significant increase in IL-17A and IL-17F protein levels in the tissue and because TH17 cells in DSS mice were predominantly negative for IFNy and IL-22, we hypothesized that IL-17 signaling is the mechanism by which Thl7 cells cause increased severity of disease. To test this hypothesis, we used an antibody directed against the IL-17RA, which binds to IL-17A homodimers and with less affinity to IL-17A IL-17F heterodimers and IL-17F homodimers, and effectively neutralizes the activity of the receptor. Our data showed that blocking IL-17RA protected DSS mice from severe disease early during infection (Figure 3 E). Similarly, neutralizing IL-17A provided significant protection early during infection (Figure S4 C). Together, these data suggest that IL-17 signaling is important for Thl7-mediated exacerbation of CDI severity in DSS mice.

Adoptive transfer of Thl7 cells is sufficient to increase CDI severity

Given the significant Thl7 skew in DSS mice and our discovery that CD4+

T cells are necessary and sufficient to cause severe CDI in these mice, we wanted to directly test whether Thl7 cells alone can exacerbate CDI severity. To do this, we isolated naive CD4+ T cells from the spleens of IL-17-GFP reporter mice and differentiated them ex vivo into Thl7 cells. On the day before infection, we sorted CD3+ CD4+ T cells into IL-17A+ and IL-17A- cells (Figure 3 F). The sorted cells were transferred into naive recipient mice that were subsequently infected with C. difficile. After infection, mice that received IL-17A+ CD4+ T cell transfer had higher CDI-associated mortality rates, weight loss and clinical scores compared to mice that either received IL-17A- CD4+ T cells or no T cell transfer (Figure 3, G- I). It is important to note that while recipients of the IL-17A- CD4+ T cells had similar survival and clinical scores to the PBS controls, they lost more weight post infection. This modest effect is likely due to some IL-17A- CD4 T cells gaining IL- 17A expression post-transfer. Indeed, when we looked by flow cytometry after infection, we found some GFP+ cells in the colons of these mice. Taken together, these results establish a central role for Thl7 cells in determining the severity and outcome of CDI following colitis.

Serum IL-6 and IL-23 in C. difficile patients correlate with severe disease

In light of our data establishing a role for Thl7 cells during CDI in a mouse model, we wished to explore whether Thl7 responses are also important during human CDI. Serum samples from C. difficile patients were analyzed for IL-6, IL- 23, IL-17A and IL-4 protein levels. Severe CDI was defined with a white blood cell (WBC) count >15,000 per microliter as previously described (Shivashankar et al. 2013; Yu et al. 2017). We used a Kaplan-Meier survival analysis to confirm that the severe CDI group had significantly lower survival probability (Figure 4 A). When patients were categorized based on disease severity, those with severe CDI had significantly higher serum IL-6 and IL-23 levels compared to those with non-severe CDI. We found no difference in serum IL-17A levels between the two groups

(Figure 4 B). In addition to its role in Thl7 differentiation, IL-6 can promote IL-4 production by Th2 cells and lead to enhanced Th2 differentiation through an IL-4 feedback loop (Dienz and Rincon, 2009). However, when we measured the levels of IL-4 in the human serum samples, we actually found a decrease in IL-4 levels in patients with severe CDI suggesting no enhanced Th2 responses downstream of IL-6 (Figure 4 B). Strikingly, when we divided the patients into four quartiles based on IL-6 levels in the serum, patients with higher IL-6 (second, third and fourth quartiles) were significantly less likely to survive post-infection than patients in the lowest quartile (Figure 4 C). After adjustment for age, sex, race, comorbidities and ICU admission, patients in the highest quartile for serum IL-6 were 7.6 times less likely to survive than those in the lowest quartile (p=0.0009) (Table SI). A survival analysis based on serum IL-23 levels could not be completed due to the high number of samples under the assay’s limit of detection, which limited the statistical power of the survival studies for this cytokine. Taken together, these data suggest that serum IL-6 and IL-23, two cytokines upstream of pathogenic Thl7 cells, likely play a role in increasing CDI- associated mortality in human patients.

Table SI: mortality risk of C. difficile patients based on IL-6 serum levels.

Related to Figure 4.

a Correlation between IL-6 levels and survival assessed via a Cox proportional hazards model. M= male, F= female.

b Mortality risk (hazard ratio) adjusted for age, sex, race, Charlson comorbidity index and ICU admission

Discussion

The present application provides the first demonstration that in a colitis setting, Thl7 cells are an important source of IL-17A and these cells alone can increase the risk for severe CDI. The findings disclosed herein are particularly important in light of new evidence showing that IBD patients have higher numbers of Thl7 cells in the colon and circulation (Hegazy et ah, 2017), which might explain their increased risk for severe C. difficile colitis. In addition to C. difficile infection, Salmonella typhimurium infections have also been linked to IBD (Gradel et ah, 2009); Hegazy et al. found more S. typhimurium- specific Thl7 cells in PBMCs from patients with IBD compared to controls. There is also evidence that suppressing T cell responses during S. typhimurium infection can protect the host from severe disease by preventing the downstream neutrophil recruitment and off-target tissue pathology (Godinez, 2008). This suggests that aberrant Thl7 responses in IBD patients may generally put them at risk for severe gastrointestinal infections.

Previous studies have found that targeting IL-23 and IL-17A can protect mice from C. difficile -associated epithelial damage and mortality (Buonomo et ah, 2013; McDermott et ah, 2016; Tateda et ah, 2016). Here, it was found that neutralizing IL-17A and blocking the IL-17RA receptor protected DSS mice from severe disease early on during infection. Blocking the receptor was more robust at providing protection suggesting that IL-17F, also elevated in DSS mice, might play a role in exacerbating CDI severity. Blocking IL-17 signaling did not provide protection later during infection, suggesting that perhaps some IL-17 signaling is required to control the infection. Indeed, other studies have shown that although increased neutrophilia downstream of IL-17A is associated with severe disease, complete depletion of neutrophils does not protect mice from disease because of their essential role in clearing the infection (Jarchum et ah, 2012 and Feghaly et ah, 2013).

Interestingly, in a T cell transfer model of colitis, Wedebye et al. 2013 found that blocking IL-17A and IL-17F simultaneously was more successful at ameliorating colitis than blocking either cytokine individually. Furthermore, targeting Thl7 cells with pharmacologic inhibitors of RORyt was successful in reducing gut inflammation in two different murine models of IBD (Withers et al., 2016). The findings of our study suggest that specifically targeting Thl7 cells and their effector cytokines in IBD patients may also protect them from acquiring severe CDI.

The limitations of this study include the use of DSS colitis as a way to induce gut inflammation prior to CDI. DSS colitis does not perfectly model human IBD because it is chemically-induced colitis. However, for the purposes of this study, DSS colitis did cause a skew towards Thl7 responses that persisted beyond the resolution of acute colitis and allowed us to study the effect of the Thl7 axis on subsequent CDI. Therefore, the model we developed can be used for future studies to identify the downstream mechanisms that are required for the Thl7-mediated exacerbation of CDI. Another strength of the DSS model for our studies is that it is an acute form of colitis, which allows for full recovery before CDI. Recently, Zhou et al. (2018) showed that mice with acute DSS colitis are more susceptible to C. difficile infection. However, the cause of this increased susceptibility is unclear and could be attributed to the physical damage to the epithelial barrier during acute DSS colitis. In patients, time-to-CDI diagnosis data suggest that a high percentage of IBD patients acquire CDI during remission and not during an IBD flare (Rodemann et al., 2007). For this reason, we designed a model where mice are recovered from acute colitis before CDI and we suggest that this model might better reflect what happens in IBD patients.

Despite a previous report by Yu et al. (2017) suggesting that serum IL-17A was associated with severe CDI, here, no difference was found in IL-17A between the two patient groups in the present study. The two studies were done on two different cohorts and our study included 323 patients, compared to 36 in the Yu et al. report. While differences in IL-6 and IL-23 systemically in the serum are disclosed herein, perhaps the effects on IL-17A production are localized to colonic Thl7 cells and evaluation of tissue IL-17A levels might reveal differences between the two patient groups.

Future experimentation is needed to understand how Thl7 cells exacerbate CDI severity. Thl7 cells produce IL-17A, F and IL-22 that can act on epithelial cells, which in turn produce several chemokines that ultimately lead to neutrophil recruitment (Ouyang et al., 2008). While neutrophils are important for defense against bacterial infections, they often can have off-target damaging effects on host tissue. The results for day 2 herein did not show enhanced neutrophil recruitment in DSS mice, but future studies can further characterize the effects of Thl7 cells on neutrophil activation and function during CDI.

The incidence of C. difficile over the last two decades has continued to rise and the CDC identifies C. difficile as an urgent threat. Although antibiotic treatment is the first line of defense against CDI, the antibiotic-induced disruption of the gut microbiota contributes to disease relapse in 1 of 5 patients. Therefore, discovering new therapies is essential for treating this life-threatening infection. Previous studies have reported a role for CD4 T cells in protection against CDI via a TLR4- dependent recognition of C. difficile surface layer proteins (Ryan et al. 2011).

However, role for these cells during infection remains underexplored. Using adoptive transfer studies, we have shown for the first time that Thl7 cells are sufficient to increase CDI severity. Furthermore, by analyzing serum samples from C. difficile patients, we found that IL-23 and IL-6 correlate with disease severity and that patients with high IL-6 levels are significantly more likely to die after infection. This is supported by a previous study that described higher levels of IL-6 in 8 patients with severe CDI (Rao et al. 2014). These mouse studies and their human correlate suggest than in patients with increased pathogenic Thl7 responses, such as IBD, targeting these cells may not only ameliorate IBD symptoms but also reduce their risk of developing severe CDI.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference herein in their entirety.

Headings are included herein for reference and to aid in locating certain sections. These headings are not intended to limit the scope of the concepts described therein under, and these concepts may have applicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specific

embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention.

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Claims

CLAIMS What is claimed is:

1. A method for preventing or treating Clostridium difficile (C. difficile) infection (CDI) in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutical-acceptable carrier, optionally at least one additional therapeutic agent, and an effective amount of at least one inhibitor selected from the group consisting of an inhibitor of CD4+ T cells, Thl7 cells, CD4, IL-17A, IL-17F, IL-17RA, IL-22, and IL-6.

2. The method of claim 1, wherein the subject suffers from colitis.

3. The method of claim 1, wherein the subject suffers from irritable bowel syndrome.

4. The method of claim 1, wherein the method inhibits CDI mortality.

5. The method of claim 3, wherein the method inhibits CDI mortality.

6. The method of claim 1, wherein the method inhibits an increase in IL-6, IL-17, and IL-23.

7. The method of claim 1, wherein the pharmaceutical composition is

administered before the subject is infected with C. difficile.

8. The method of claim 1, wherein the pharmaceutical composition is

administered shortly after the subject is infected with C. difficile.

9. The method of claim 1, where the inhibitor is a small molecule, drug, prodrug, or an antibody, or a biologically active fragment or homolog thereof.

10. The method of claim 9, wherein the inhibitor is an antibody.

11. The method of claim 10, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, F(ab)2 fragments of a monoclonal antibody, a chimeric antibody, a single-chain antibody, a synthetic antibody, bi- specific antibody, and a humanized antibody, and active fragments and homologs thereof.

12. The method of claim 11, wherein said fragment is an F(ab)2 fragment.

13. The method of claim 11, wherein the antibody is a monoclonal antibody

14. The method of claim 1, wherein one inhibitor is administered.

15. The method of claim 1, wherein at least two inhibitors are administered.

16. The method of claim 14, where the inhibitor is directed against IL-17A.

17. The method of claim 14, wherein the inhibitor is directed against IL-17RA.

18. The method of claim 14, wherein the inhibitor is directed against IL-6.

19. The method of claim 14, wherein the inhibitor is directed against IL-17F.

20. The method of claim 14, wherein the inhibitor is directed against CD4.

21. The method of claim 15, wherein the pharmaceutical composition comprises an inhibitor directed against IL-17A and an inhibitor directed against IL-17F.

22. The method of claim 21, wherein each inhibitor is an antibody.

23. The method of claim 20, wherein the inhibitor an antibody.

24. The method of claim 23, wherein the antibody is OKT-4 or RPA-T4.

25. The method of claim 1, wherein the method inhibits an increase in CD4+ T cells.

26. The method of claim 1, wherein the method inhibits an increase in TH17 cells.

27. The method of claim 26, wherein the method decreases the number of CD4+ T cells.

28. The method of claim 27, wherein the method kills CD4+ T cells.

29. The method of claim 1, wherein the method decreases the number of Thl7 cells.

30. The method of claim 1, wherein the composition is administered at least twice.

31. The method of claim 30, wherein the composition is administered at least five times.

32. The method of claim 31, wherein the composition is administered at least 10 times.

33. The method of claim 1, wherein the composition is administered at least once per day.

34. The method of claim 33, wherein the composition is administered at least once per week.

35. The method of claim 33, wherein the composition is administered at least twice per week.

36. The method of claim 33, wherein the composition is administered at least once per month.

37. The method of claim 36, wherein the composition is administered at least twice per month.

38. The method of claim 23, wherein the antibody is administered at a dose ranging from about 0.1 mg/kg to about 25.0 mg/kg body weight.

39. The method of claim 38, wherein the antibody is administered at a dose ranging from about 1.0 mg/kg to about 15.0 mg/kg body weight.

40. The method of claim 39, wherein the antibody is administered at a dose ranging from about 5.0 mg/kg to about 10.0 mg/kg body weight.

41. The method of claim 1, wherein the subject is human.

42. The method of claim 2, wherein the colitis is selected from the group consisting of acute colitis, chronic colitis, inflammatory bowel disease, ulcerative colitis, Crohn’s colitis, diversion colitis, ischemic colitis, infectious colitis, fulminant colitis, collagenous colitis, chemical colitis, microscopic colitis, lymphocytic colitis and atypical colitis.

43. The method of claim 1, wherein the pharmaceutical composition is administered within five days after the subject has been infected with C. difficile.

44. The method of claim 43, wherein the pharmaceutical composition is administered within 3 days after the subject has been infected with C. difficile.

45. The method of claim 44, wherein the pharmaceutical composition is administered within 1 day after the subject has been infected with C. difficile.

46. A method for preventing C. difficile infection in a subject with colitis, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically-acceptable carrier, optionally at least one additional therapeutic agent, and an effective amount of at least one inhibitor selected from the group consisting of an inhibitor of CD4+ T cells, Thl7 cells, CD4, IL-17A, IL-17F, IL-17RA, IL-22, and IL-6.

47. A method for determining whether a subject with colitis is infected with C. difficile , the method comprising measuring the levels of IL-6 and IL-23 in a sample obtained from the subject and comparing the levels to standard levels, wherein an increase in the levels of IL-6 and IL-23 is an indication the subject is infected with C. difficile.

48. The method of claim 47, wherein when the levels of IL-6 and IL-23 are determined to be increased in the subject, the subject is at a risk for increased mortality.

49. The method of claim 48, wherein when subject is determined to be at risk for increased mortality, a pharmaceutical composition comprising a pharmaceutical- acceptable carrier, optionally at least one additional therapeutic agent, and an effective amount of at least one inhibitor selected from the group consisting of an inhibitor of CD4+ T cells, Thl7 cells, CD4, IL-17A, IL-17F, IL-17RA, and IL-6 is administered to the subject.

50. The method of claim 49, wherein the inhibitor of IL-17A is monoclonal antibody clone 41809.

51. The method of claim 49, wherein the inhibitor of IL- 17RA is monoclonal antibody clone 133617.

52. A kit for preventing of treating CDI in a subject with colitis or who has had colitis, the kit comprising an effective amount of one or more inhibitors selected from the group consisting of an inhibitor of CD4+ T cells, Thl7 cells, CD4, IL-17A, IL-17F, IL-17RA, and IL-6, a pharmaceutically-acceptable carrier, an applicator, and an instructional material for the use thereof.