US 201701. 89384A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0189384 A1 Penny et al. (43) Pub. Date: Jul. 6, 2017

(54) REVERSING INTESTINAL INFLAMMATION (60) Provisional application No. 61/526,574, filed on Aug. BY INHIBITING RETNOIC ACID 23, 2011. METABOLISM Publication Classification (71) Applicant: THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNOR (51) Int. Cl. UNIVERSITY, STANFORD, CA (US) A6II 3/428 (2006.01) A6IR 9/00 (2006.01) (72) Inventors: Hweixian Leong Penny, Singapore A63L/484 (2006.01) (SG); Edgar George Engleman, (52) U.S. Cl. Atherton, CA (US); Nupur CPC ...... A6 IK3I/428 (2013.01); A61K 31/4184 Bhattacharya, Palo Alto, CA (US) (2013.01); A61K 9/0053 (2013.01) (21) Appl. No.: 15/464,010 (57) ABSTRACT (22) Filed: Mar. 20, 2017 An agent that increases local concentration of retinoic acid (RA) in the intestine through modifying enzymatic pathways Related U.S. Application Data involved in RA metabolism is administered in a dose effec (63) Continuation of application No. 14/962.782, filed on tive to inhibit or reverse production of inflammatory media Dec. 8, 2015, now Pat. No. 9,623,013, which is a tors by intestinal dendritic cells and thereby reduce intestinal continuation of application No. 13/592,180, filed on inflammation and tumor growth associated with intestinal Aug. 22, 2012, now Pat. No. 9.248,120. inflammation. Patent Application Publication Jul. 6, 2017. Sheet 1 of 16 US 2017/O189384 A1

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US 2017/O 189384 A1 Jul. 6, 2017

REVERSING INTESTINAL INFLAMMATION differentiation. In addition to its ability to support the BY INHIBITING RETNOIC ACID development of T17 cells, IL-23 induces the secretion of METABOLISM IL-17 by non-T-cells in an inflammatory environment, and both T cells and monocytes serve as sources of increased CROSS REFERENCE expression in the mucosa of IBD patients. 0001. This application claims benefit and is a Continua 0007 An understanding and manipulation of inflamma tion of application Ser. No. 14/962,782 filed Dec. 8, 2015, tory cells in the gut is of great interest. The present invention which claims the benefit and is a Continuation of application addresses this issue. Ser. No. 13/592,180, filed Aug. 22, 2012, now U.S. Pat. No. 9,248,120, issued Feb. 2, 2016, which claims benefit of U.S. SUMMARY OF THE INVENTION Provisional Patent Application No. 61/526,574, filed Aug. 0008 Methods are provided for reducing intestinal 23, 2011, which applications are incorporated herein by inflammation, particularly chronic inflammation, and tumor reference in their entirety. growth precipitated by intestinal inflammation. In the meth ods of the invention, an effective dose of an agent is FEDERALLY SPONSORED RESEARCH AND provided to the individual, where the agent increases local DEVELOPMENT concentration of retinoic acid (RA) in the intestine through 0002 This invention was made with Government support modifying enzymatic pathways involved in RA metabolism. under contract CA141468 awarded by the National Institutes 0009. In some embodiments, an inhibitor of CYP26A1 is of Health. The Government has certain rights in the inven administered in a dose effective form to neutralize a pre tion. existing inflammatory environment, prevent the develop ment of inflammation, or maintain the tolerogenic functions BACKGROUND OF THE INVENTION of intestinal dendritic cells that maintain intestinal tolerance by inducing Treg formation. Alternatively, an agent that 0003 Pathological inflammation is emerging as an under increases activity of retinaldehyde dehydrogenase or retinol lying mechanism for numerous diseases. For example, the dehydrogenase may be administered in a dose effective to major forms of idiopathic IBD, ulcerative colitis and neutralize or prevent inflammation in the intestine. The Crohn's disease are chronic inflammatory disorders of the appropriate dose may be determined by evaluating the effect gastrointestinal tract. Animal studies have shown that of the agent on functions of intestinal dendritic cells, for chronic intestinal inflammation precipitates as well as propa example by monitoring synthesis of cytokines such as IL-23 gates tumor growth. and IL-17, or by overall analysis of intestinal inflammation. 0004. A number of sentinel cell populations in the intes The methods of the invention exclude administration of tinal mucosa continuously monitor luminal microbes and retinoic acid directly. other antigens. Among the Subsets of antigen-presenting 0010 Dendritic cells are shown to be reprogrammed due cells, myeloid-derived dendritic cells are a dominant sub to local loss of the vitamin A metabolite, retinoic acid (RA), type in the intestinal lamina propria and show considerable which occurs as a result of insufficient retinaldehyde dehy functional plasticity depending on the location, state of drogenase and an overabundance of the transcriptional co maturation, and stage of inflammation. Dendritic cells form repressor, Ctbp1. The reprogrammed dendritic cells secrete an extensive network beneath the intestinal epithelium and mainly pro-inflammatory cytokines and induce Th17 forma project long processes through the interstices of epithelial tion. Treatment by the methods of the invention replenish cells to sample luminal antigens. In response to TLR ligands, RA and neutralize the inflammatory phenotype of the den immature dendritic cells produce IL-23, which contributes dritic cells. to development of intestinal inflammation in murine models 0011. Other aspects and features will be readily apparent of colitis and intestinal inflammation. The remarkable to the ordinarily skilled artisan upon reading the present capacity of dendritic cells to orchestrate distinct immune disclosure. responses is aided by a panoply of environmental cues, which condition the cells to adopt specific phenotypes in different settings. DCs in gut-associated lymphoid tissue are BRIEF DESCRIPTION OF THE DRAWINGS of particular interest because they maintain tolerance to 0012. The invention is best understood from the follow commensal flora as well as mount protective inflammatory ing detailed description when read in conjunction with the responses in the face of pathogen incursion. accompanying drawings. The patent or application file con 0005 Migration of innate immune cells such as neutro tains at least one drawing executed in color. Copies of this phils, macrophages, and dendritic cells into target mucosal patent or patent application publication with color drawing tissues depends on the expression of cytokines, chemokines (s) will be provided by the Office upon request and payment and adhesion molecules. Recruitment of activated neutro of the necessary fee. It is emphasized that, according to phils, dendritic cells and macrophages into the lamina pro common practice, the various features of the drawings are pria in general amplifies the local immune response, not to-scale. On the contrary, the dimensions of the various whereas activated natural killer cell recruitment seems to features are arbitrarily expanded or reduced for clarity. enhance antimicrobial factors, leading to attenuation of Included in the drawings are the following figures. inflammation. 0013 The invention is best understood from the follow 0006. The CD4 T-cell lineage (T,17) is characterized by ing detailed description of exemplary embodiments when the production of IL-17. Its development is promoted by read in conjunction with the accompanying drawings. It is IL-23, in addition to IL-6 and TGFbeta. Evidence indicates emphasized that, according to common practice, the various that RORct (retinoic acid-related orphan nuclear hormone features of the drawings are not necessarily to-scale. On the receptor gamma-t) is necessary for T17 commitment and contrary, the dimensions of the various features are arbi US 2017/O 189384 A1 Jul. 6, 2017

trarily expanded or reduced for clarity. Included in the antagonist LE540 was added to whole SI-LPDC-T cell drawings are the following figures: co-culture wells, in addition to TGF-31. Cells were re 0014 FIG. 1A-1E. APC' SI-LPDCs accumulate over stimulated on day 4 with plate-bound anti-CD3 and anti time and secrete more pro-inflammatory cytokines com CD28 (1 lug/ml each) for 18 hours. Supernatants were pared to WT SI-LPDCs upon TLR stimulation. DCs were collected on day 5 to assay for cytokine production by defined as PI- EpCAM-CD45+ DX5- CD3e- CD19 standard ELISA. Representative bar graphs show the mean CD11c MHCII+ in all the tissues. O. WT: ... APC' production (with SEM) of IL10 (FIG. 2E), IL17A (FIG.2F) (FIG. 1A) A representative contour plot depicting how DCs and IL6 (FIG. 2G). Data in this figure were obtained at late are gated by CD11c and MHC II expression. (FIG. 1B) Abar stage disease, and are representative of 4 independent graph of the mean frequency (with SEM) of CD11c-- experiments, with DCs pooled from at least 5 WT and at MHCII+ DCs as a percentage of PI- Lineage-cells. (FIG. least 3 APC" mice per experiment. An unpaired student's 1C) Representative contour plots of SPL, mLN/PP and t test with 95% confidence interval was performed. P-O. SI-LP DCs divided by CD11 b and CD103 expression. 05=*; p<0.001=**; p<0.0001=***. Below each FACS plot are bar graphs showing the mean 0016 FIG. 3A-3C. APC'* SI-LP DCs and IECs have frequency (with SEM) of each subset in that tissue as a lower expression of Raldhs than WT, resulting in loss of RA percentage of total CD11c-- MHCII+ DCs. (FIG. 1D) Mean in the tumor milieu. O WT: ... APC'". Total RNA frequency (with SEM) of cells expressing the co-stimulatory from FACS-purified SI-LPDCs, splenic DCs (FIG. 3A) and molecules CD40, CD80, CD83, CD86 and PDL1 as a epithelial cells (IECs) (FIG. 3B) was extracted and assayed percentage of the different CD11 b CD103 subsets. Data for in triplicate by qPCR for the genes Raldh1a1, Raldh1a2. (FIG. 1A-1D) were obtained at intermediate stage disease. Raldhla3 and Ctbp1 (FIG. 3C). Each sample was normal Inset values in the contour plots are the mean frequencies of ized to ubiquitin b expression. Representative bar graphs each population pooled using at least 4 mice per strain (WT show mean relative expression (with SEM) of triplicate and APC") per experiment, from 5 independent experi samples. Data are representative of 3 independent qPCR ments. (FIG. 1E) 5x10 FACS-purified WT and APC' experiments, with DCs and IECs pooled from 3 sorts per SI-LPDCs were stimulated with a panel of 6 different TLR timepoint, using at least 5 WT and at least 3 APC" mice agonists—Pam3Csk4 (1 lug/ml), Poly I:C (10 ug/ml), LPS per sort. An unpaired student's t test with 95% confidence (10 ug/ml), flagellin (1 lug/ml), R848 (10 ug/ml), CpG 2336 interval was performed. P-0.05=*; p<0.001=**; p<0. (10 ug/ml). Supernatants were collected after 48 hours. 0001=3; 33. Representative bar graphs show mean production (with 10017 FIG. 4A-4F. FAP adenoma tissue exhibits IL-17 SEM) of the cytokines IL-6, TNFC. and IL-2p40 as measured driven inflammation and loss of RA. Normal colon, FAP by standard ELISA. Data in (FIG. 1E) was obtained at late adenoma and sessile serrated polyp (SSP) sections were stage of disease. Data are representative of 4 independent stained by immunohistochemistry for IL-17A (FIG. 4A) or experiments, with DCs pooled from 8 mice per strain (WT co-stained by immunofluorescence with DC-SIGN and and APC") per experiment. An unpaired student's t test |B-catenin (FIG. 4B), Ctbp1 (FIG. 4C), Raldh1a1 (FIG. 4D), with 95% confidence interval was performed. P<0.05=*: Raldhla2 (FIG. 4E), CYP26A1 (FIG. 4F). Shown are p-0.001=**; p<0.0001=***. samples from one representative normal colon, two repre 0.015 FIG. 2A-2G. APC' SI-LPDCs have a reduced sentative FAPadenoma and one representative SSP patient. capacity to induce Foxp3+T and IL-10 producing CD4+ Images for FAP P1 show serial sections of the same polyp T cells, but instead generate Th17 cells, in an RA-dependent with underlying adjacent normal grossly uninvolved tissue. manner. 2x10 of sorted CD11c MHCII DCs were co Overall, samples from a total of 11 normal colon, 8 FAP cultured for 5 days with 1x10 MACS-enriched CD4+ adenoma, 2 FAPadenocarcinoma and 4 SSP patients were CD62L + Foxp3- naive T cells from the spleen and lymph analyzed. All images were captured using the same exposure nodes of OT-II TCR-transgenic mice, along with OVA time and the same brightfield or fluorescence settings for peptide and 10 ng/ml recombinant human TGF-B. 5 ng/ml of each protein of interest. White magnification bars in (FIG. recombinant human IL-2 was added to the cultures every 4A-4F) are 100 uM. other day beginning on day 2. O. WT: ... APC' (FIG. 0018 FIG. 5A-5I. A vitamin A-deficient diet exacerbates 2A) DCs used in these cultures were purified from SPL, disease in APC' mice (FIG. 5A-5D) 10 week old WT mLN/PP and SI-LP, and stimulated with 5 nM OVAs. (open circles) and APC" (filled triangles) mice were Representative bar graph shows the mean frequency (with placed on 1500 ppm Celecoxib (: , s ); 100 ppm Rosigli

SEM) of Foxp3 induction from 9 independent experiments, taZone (« , »); a vitamin A deficient diet (0 IU/g of with DCs pooled from at least 5 WT and at least 3 APC' Vitamin A) ( x); or a base diet (4 IU/g of vitamin A) ( mice per experiment. (FIG. 2B) SI-LPDCs were used in * , x) for 10 consecutive weeks. Mice were monitored for these co-cultures, and stimulated with 200 nM, 1 uM and 5 disease progression on the basis of hematocrit and body uM OVAsso. Representative bar graph shows the mean weight change. Line graphs show Kaplan-Meier Survival frequency (with SEM) of Foxp3 induction from 4 indepen curves (FIG. 5A), mean change in percentage body weight dent experiments, with DCs pooled from at least 5 WT and over original body weight (FIG. 5B) and hematocrits (FIG. at least 3 APC" mice per experiment. (FIG. 2C) CD11c+ 5C), with SEMs. 5 mice were used per strain for each diet. MHCII+ SI-LP DCs were further sorted into the CD103+ Data are representative of 2 independent experiments. Liaro and CD103- Subsets and cultured with naive CD4 T cells as zole, a CYP26A1 inhibitor, ameliorates disease in APC'" before. Bar graph shows the mean frequency (with SEM) of mice by increasing local RA which reverses the SI-LPDC Foxp3 induction in a representative experiment of 2 inde pro-inflammatory phenotype. 8 week old WT and APC'" pendent experiments, with DCs pooled from at least 5 WT mice were placed on 40 ppm of Liarozole for 6 consecutive and at least 3 APC' mice per experiment. (FIG. 2D) In weeks. APC'* Base diet :: APC'* - Liarozole : these experiments, 10 nM all-trans RA or 1 uM of the RAR WT Liarozole « . Disease was monitored as described in US 2017/O 189384 A1 Jul. 6, 2017

(FIG. 5A-5D). In these experiments, small intestinal polyps Representative dot plots of Foxp3 versus CFSE in Thy 1.2+ were enumerated at 14 weeks, the point of euthanasia. A CD4+ cells. The inset values of the bordered population scatter plot shows the mean number of polyps (with SEM) show the mean frequency of Foxp3+ cells as a percentage of (FIG.5H) between APC' mice on Liarozole compared to Thy 1.2+ CD4+ cells. (FIG. 8A) DCs used in these cultures base diet. 5 mice were used per strain in each diet. Data are were purified from SPL, mLN/PP and SI-LP (FIG. 8B) representative of 2 independent experiments. Significance in SI-LPDCs were incubated with 200 nM, 1 uM and 5 uM disease indicators of the different diets was calculated by OVA. (FIG. 8C) SI-LPDCs were isolated from early, comparing values to those obtained on the base diet. For intermediate and late stage disease. calculating significance in DAI, a Wilcoxon-rank-Sum test (0022 FIG. 9A-9B. ADH class I and II expression in was used. (FIG. 5I) All-trans retinoic acid (RA) was APC" IECs and SI-LPDCs increased at intermediate extracted from the duodenum, jejunum, ileum, colon and stage and decreased at Subsequent late stage compared to eye and quantified by tandem mass spectrometry. WT base WT counterparts. RT-qPCR on FACS-purified WT and diet C : APC'* Base diet S : APC' Liarozole APC'* SI-LP DCs, splenic DCs (FIG.9A) and epithelial S. Bar graphs show mean amounts of retinoic acid per cells (IECs) (FIG. 9B) was performed as described in FIG. gram tissue (with SEM) among mice of the same strain. 5 3. C. WT II : APC' Representative bar graphs show WT, 5 APC' and 4 Liarozole-treated APC' mice mean relative expression (with SEM) of triplicate samples. were used in this experiment. For data in this figure, an Data are representative of 3 independent qPCR experiments, unpaired student's t test with 95% confidence interval was with DCs and IECs pooled from 3 sorts per time point, using performed unless otherwise stated. P-0.05=*; p<0.001=**: at least 5 WT and at least 3 APC' mice per sort. Shown p<0.0001=***. 8 week-old WT and APC' mice were is the ADH class I and II expression in DCs (a) and in IECs placed on 40 ppm of Liarozole for 6 consecutive weeks as (FIG.9B). described in FIGS.5A-5I. WT base diet C : APC' (0023 FIG. 10A-10B. Quantification of retinyl esters and retinol in vivo. Retinyl esters (RE) and all-trans retinol Base diet : APC' Liarozole . (FIG. 5A) At the (ROL) were extracted from the duodenum, jejunum, ileum, 14 week time point of euthanasia, 2x10 FACS-purified WT colon and eye and quantified by HPLC. Bar graphs show and APC" SI-LPDCs were stimulated with Pam3Csk4 (1 mean amounts of retinoid per gram tissue (with SEM) ug/ml), LPS (10 ug/ml) and R848 (10 ug/ml). Supernatants among mice of the same strain. 5 WT, 5 APC" and 4 were collected after 48 hours. Representative bar graphs Liarozole-treated APC" mice were used in this experi show mean production (with SEM) of the cytokines IL-6, ment. An unpaired student's t test with 95% confidence TNFC. and IL-2p40 as measured by standard ELISA. Data interval was performed. P-0.05=*; p<0.001=**; p<0. are representative of 2 independent experiments, with at 0001=3; 33. least 2 mice per strain. (FIG. 5B) AT induction assay was 0024 FIG. 11. Isotype control for immunohistochemistry performed as described in FIG. 2 on SI-LPDCs sorted from and immunofluorescence. Normal colon sections were these mice. Representative bar graph shows the mean fre stained with an isotype control (Rabbit IgG) to the rabbit quency (with SEM) of Foxp3 induction from 2 independent primaries used against B-catenin, Ctbp1, Raldhlal. experiments, with DCs pooled from at least 2 mice per Raldh1a2 and CYP26A1. strain. (FIG. 5C) T cells in the SI-LPDC-T cell co-culture 0025 FIG. 12. Wildtype mice on base, rosiglitazone, was re-stimulated as described in FIG. 2 and assayed for celecoxib and vitamin A-deficient diets gain in weight IL10, IL17A and IL6 by standard ELISA as before. For data comparably. Wildtype mice on the various diets 10 week-old in this figure, an unpaired student's t test with 95% confi WT mice were placed on 1500 ppm Celecoxib, 100 ppm dence interval was performed. P-0.05=*; p<0.001=**; p<0. Rosiglitazone, a vitamin A deficient diet (0 IU/g of vitamin 0001=3; 33. A), or on a base diet (4 IU/g of vitamin A), for 10 0.019 FIG. 6. Proposed Model: In the APC' mouse, consecutive weeks. Line graph show mean change in per the immune state of the intestine, departs from homeostatic centage body weight over original body weight. Data are tolerance to chronic inflammation. Truncated APC protein representative of 2 independent experiments. leads to an accumulation of the transcription factor Ctbp1. 0026 FIG. 13. APC' SI-LPDCs display higher which in turns Suppresses retinol dehydrogenases in the expression of Cox2 compared to WTSI-LPDCs. RT-qPCR epithelium and may lead to a reduction in local concentra on FACS-purified WT and APC' SI-LP DCs was per tions of RA. Upregulation of Cox2 in the inflamed tissue formed as described in FIG. 3. C. WT: APC'''“ leads to greater production of PGE, which has been Representative bar graph shows mean relative expression reported to suppress Raldh1a2 in DCs and likely does so to (with SEM) of triplicate samples. Data is from 1 indepen the surrounding epithelium as well. Lack of RA in the dent qPCR experiment, with DCs and pooled from 3, using intestinal milieu conditions or reprograms. SI-LPDCs to a at least 5 WT and at least 3 APC' mice per sort. pro-inflammatory phenotype, in which IL6 production com (0027 FIG. 14A-14F. Direct supplementation of RA does bined with TGFB found locally, contributes to a deleterious not affect tumor frequency or intestinal RA levels in APC Th17 response that promotes tumor growth. '* mice, but Liarozole, a CYP26A1 inhibitor, improves 0020 FIG. 7. Morphology of sorted SI-LPDCs from WT all tested disease parameters. Groups of 8 week-old APC and APC' mice. Sorted PI-EpCAM- CD45+ DX5 '"" mice were injected i.p. with or without 200 g/mouse CD3e-CD19- CD11c" MHCII+ LPDCs were stained with RA in sunflower oil injected twice weekly while on a base May Grumwald-Giemsa. diet, or placed on 40 ppm of Liarozole for 6 consecutive 0021 FIG. 8A-8C. APC' SI-LPDCs have a reduced weeks. APC' on Base diet?: APC' on Liarozole?; capacity to induce Foxp3+T in a tissue-specific, dose APC" given RA i.p./; WT on Liarozole. (FIG. 14A) dependent and time-dependent manner. A T induction Small intestine tumors were enumerated at 14 weeks. Scatter assay was performed as described in FIG. 2. (FIG. 8A-8C) plot shows the mean number of tumors (with SEM) in US 2017/O 189384 A1 Jul. 6, 2017

APC" mice on base, Liarozole or RA i.p. (a). Line graphs either, neither or both limits are included in the smaller show mean change in percentage body weight (FIG. 14B) ranges is also encompassed within the invention, Subject to and hematocrit (FIG. 14C). For (FIG. 14A-14C), data for any specifically excluded limit in the stated range. Where the mice on base diet and LiaroZole are aggregated from 4 stated range includes one or both of the limits, ranges independent experiments, with at least 4 mice per group, excluding either or both of those included limits are also while data for RA i.p.-injected mice are aggregated from 2 included in the invention. independent experiments, with at least 3 mice per group. 0032 Unless defined otherwise, all technical and scien (FIG. 14D) Concomitant to experiments performed in FIG. tific terms used herein have the same meaning as commonly 4D, RA was quantified by LC/MS in 5 Liarozole treated and understood by one of ordinary skill in the art to which this 5 RA i.p.-injected APC" mice. Shown are mean RA invention belongs. Although any methods and materials levels (with SEM) in each tissue isolated at the 14 week time similar or equivalent to those described herein can be used point. (FIG. 14E) Dot plots show the mean frequency of in the practice or testing of the present invention, exemplary IL-17A- and IFNY-producing CD4 T cells in freshly-iso methods and materials are now described. All publications lated SI-LP from WT. APC" and Liarozole-treated APC mentioned herein are incorporated herein by reference to "" mice. Also shown are bar graphs depicting mean disclose and describe the methods and/or materials in con frequency (with SEM) of IL-17A- and IFNY-producing nection with which the publications are cited. It is under CD4+ T cells from these mice. (FIG. 14F) CD103- SI stood that the present disclosure Supercedes any disclosure LPDCs from WT, APC''' and Liarozole-treated APC'''“ of an incorporated publication to the extent there is a mice were co-cultured in the T cell differentiation assays contradiction. performed as in FIG. 1A-1E. Inset values on the represen 0033. It must be noted that as used herein and in the tative dot plots show the mean frequency of IL-10- and appended claims, the singular forms “a”, “an', and “the IL-17A- producing CD4 T cells induced. In (ef) IL-17A, include plural referents unless the context clearly dictates IFNY', or IL-10" cells are shown as a percentage of CD4 T otherwise. Thus, for example, reference to “a cell includes cells. Data in (FIG. 14E-14F) are from 2 independent a plurality of such cells and reference to “the polypeptide' experiments, with at least 4 mice per group. DCs obtained in includes reference to one or more polypeptides and equiva (FIG. 14F) were pooled from all mice in the same group in lents thereof known to those skilled in the art, and so forth. each experiment. P-0.05=*; p<0.001=**; p<0.0001=***. 0034. It is further noted that the claims may be drafted to 0028 FIG. 15. Talarozole, a highly specific inhibitor of exclude any element which may be optional. As such, this CYP26A1, ameliorates disease in APC" mice. 8 week statement is intended to serve as antecedent basis for use of old APC' mice were placed on 8 ppm of Talarozole for such exclusive terminology as “solely”, “only' and the like 6 consecutive weeks. APC' Base diet: APC' in connection with the recitation of claim elements, or the Talarozole. At 14 weeks, tumors in the Small and large use of a “negative limitation. intestine were enumerated. Results shown are aggregated 0035. The publications discussed herein are provided from 2 independent experiments, with 5 mice per group. solely for their disclosure prior to the filing date of the 0029 FIG. 16. Paraffin embedded normal, inflamed, dys present application. Nothing herein is to be construed as an plastic or carcinoma colon sections were stained by immu admission that the present invention is not entitled to ante nofluorescence for CYP26A1 (in red) and DC-SIGN (in date such publication by virtue of prior invention. Further, green). Shown are samples from 2 different patients. Patient the dates of publication provided may be different from the 1: Normal, Dysplastic and Carcinoma sections from a collec actual publication dates which may need to be independently tomy sample and Patient 2 Inflamed and dysplastic regions confirmed. from a collectomy sample. All images were captured using 0036. The term “effective dose” or “effective dosage” is the same exposure time and the same brightfield or fluores defined as an amount Sufficient to achieve or at least partially cence settings for each protein of interest. Similar results achieve the desired effect. The term “therapeutically effec were obtained from staining with Raldh1a1 and Raldh1a2. tive dose' is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a DETAILED DESCRIPTION OF THE patient already suffering from the disease. Amounts effective EMBODIMENTS for this use will depend upon the severity of the disorder 0030. It is to be understood that the invention is not being treated and the general state of the patients own limited to particular embodiments described, as such may, of immune system. course, vary. It is also to be understood that the terminology 0037 “Polypeptide' and “protein’ as used interchange used herein is for the purpose of describing particular ably herein, can encompass peptides and oligopeptides. embodiments only, and is not intended to be limiting, since Where “polypeptide' is recited herein to refer to an amino the scope of the present invention will be limited only by the acid sequence of a naturally-occurring protein molecule, appended claims. "polypeptide' and like terms are not necessarily limited to 0031 Where a range of values is provided, it is under the amino acid sequence to the complete, native amino acid stood that each intervening value, to the tenth of the unit of sequence associated with the recited protein molecule, but the lower limit unless the context clearly dictates otherwise, instead can encompass biologically active variants or frag between the upper and lower limits of that range is also ments, including polypeptides having Substantial sequence specifically disclosed. Each Smaller range between any similarity or sequence identify relative to the amino acid stated value or intervening value in a stated range and any sequences provided herein. In general, fragments or variants other stated or intervening value in that stated range is retain a biological activity of the parent polypeptide from encompassed within the invention. The upper and lower which their sequence is derived. limits of these Smaller ranges may independently be 0038. As used herein, “polypeptide refers to an amino included or excluded in the range, and each range where acid sequence of a recombinant or non-recombinant poly US 2017/O 189384 A1 Jul. 6, 2017 peptide having an amino acid sequence of i) a native excipient, dileuent, carrier and adjuvant as used in the polypeptide, ii) a biologically active fragment of an poly specification and claims includes both one and more than peptide, or iii) a biologically active variant of an polypep one such excipient, dileuent, carrier, and adjuvant. tide. Polypeptides suitable for use can be obtained from any 0045. As used herein, a “pharmaceutical composition' is species, e.g., mammalian or non-mammalian (e.g., reptiles, meant to encompass a composition Suitable for administra amphibians, avian (e.g., chicken)), particularly mammalian, tion to a Subject, Such as a mammal, especially a human. In including human, rodenti (e.g., murine or rat), bovine, Ovine, general a “pharmaceutical composition' is sterile, and is porcine, murine, or equine, particularly rat or human, from usually free of contaminants that are capable of eliciting an any source whether natural, synthetic, semi-synthetic or undesirable response within the Subject (e.g., the compound recombinant. In general, polypeptides comprising a (s) in the pharmaceutical composition is pharmaceutical sequence of a human polypeptide are of particular interest. grade). Pharmaceutical compositions can be designed for 0039. The term “derived from indicates molecule that is administration to Subjects or patients in need thereof via a obtained directly from the indicated source (e.g., when a number of different routes of administration including oral, protein directly purified from a cell, the protein is "derived buccal, rectal, parenteral, intraperitoneal, intradermal, intra from the cell) or information is obtained from the source, cheal and the like. e.g. nucleotide or amino acid sequence, from which the 0046 T helper 17 cells (Th17) are a subset of T helper molecule can be synthesized from materials other than the cells, characterized by their production of interleukin 17 Source of information. (IL-17). They are considered developmentally distinct from 0040. The term "isolated indicates that the recited mate Th1 and Th2 cells and excessive amounts of the cell are rial (e.g., polypeptide, nucleic acid, etc.) is substantially thought to play a key role in autoimmune disease. In separated from, or enriched relative to, other materials with humans, a combination of TGF-B, IL-1B and IL-23 induces which it occurs in nature (e.g., in a cell). A material (e.g., Th17 differentiation from naive T cells. Both interferon polypeptide, nucleic acid, etc.) that is isolated constitutes at gamma (IFNY) and IL-4, the main stimulators of Th1 and least about 0.1%, at least about 0.5%, at least about 1% or Th2 differentiation respectively, negatively regulate Th17 at least about 5% by weight of the total material of the same differentiation. type (e.g., total protein, total nucleic acid) in a given sample. 0047. T helper 1 cells (Th1). Proliferating helper T cells 0041. The terms “subject' and “patient” are used inter that develop into effector T cells differentiate into two major changeably herein to mean a member or members of any subtypes of cells known as Th1 and Th2 cells. Th1 cells mammalian or non-mammalian species that may have a primarily produce IFN-Y and TNF-B cytokines. IFN-y need for the pharmaceutical methods, compositions and increases the production of interleukin-12 by dendritic cells treatments described herein. Subjects and patients thus and macrophages, and via positive feedback, IL-12 stimu include, without limitation, primate (including humans), lates the production of IFN-Y in helper T cells, thereby canine, feline, ungulate (e.g., equine, bovine, Swine (e.g., promoting the Th1 profile. IFN-Y also inhibits the production pig)), avian, and other Subjects. Humans and non-human of cytokines Such as IL-4. Conditions that polarize to the animals having commercial importance (e.g., livestock and Tl type include antigen presenting cells and IL-12. domesticated animals) are of particular interest. As will be 0048 Interleukin-17 (IL-17) refers to a group of cytok evidence from the context in which the term is used, subject ines called the IL-17 family. IL-17 shows high homology to and patient refer to a Subject or patient Susceptible to viral IL-17 encoded by an open reading frame of the T infection by a Flaviviridae virus, particularly HCV. lymphotropic rhadinovirus Herpesvirus saimiri. To elicit its 0042 “Mammal’ means a member or members of any functions, IL-17 binds to a type I cell surface receptor called mammalian species, and includes, by way of example, IL-17R of which there are at least three variants IL17RA, canines; felines; equines; bovines; Ovines; rodentia, etc. and IL17RB, and IL17RC. Members of the IL-17 family include primates, particularly humans. Non-human animal models, IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and particularly mammals, e.g. primate, murine, lagomorpha, IL-17F. All members of the IL-17 family have a similar etc. may be used for experimental investigations. protein structure, with four highly conserved cysteine resi 0043. The term “unit dosage form,” as used herein, refers dues critical to their 3-dimensional shape, although with no to physically discrete units suitable as unitary dosages for sequence similarity to any other known cytokines. Numer human and animal Subjects, each unit containing a prede ous immune regulatory functions have been reported for the termined quantity of compounds calculated in an amount IL-17 family. sufficient to produce the desired effect in association with a 0049. IL-23 alpha subunit is a protein encoded by the pharmaceutically acceptable diluent, carrier or vehicle. The IL23A gene (see Oppmann et al. (2001) Immunity 13 (5): specifications for the novel unit dosage forms depend on the 715-25). This gene encodes the p19 subunit of the heterodi particular compound employed and the effect to be achieved, meric cytokine interleukin 23 (IL23). IL23 is composed of and the pharmacodynamics associated with each compound this protein and the p40 subunit of interleukin 12. The in the host. receptor of IL23 is formed by the beta 1 subunit of IL12 0044. A “pharmaceutically acceptable excipient,” “phar (IL12RB1) and an IL23 specific subunit, IL23R. Both IL23 maceutically acceptable diluent,” “pharmaceutically accept and IL12 can activate the transcription activator STAT4, and able carrier, and “pharmaceutically acceptable adjuvant” stimulate the production of interferon-gamma (IFNG). In means an excipient, diluent, carrier, and adjuvant that are contrast to IL12, which acts mainly on naive CD4(+) T cells, useful in preparing a pharmaceutical composition that are IL23 preferentially acts on memory CD4(+) T cells. generally safe, non-toxic and neither biologically nor oth 0050. Dendritic cell. As used herein, the term refers to erwise undesirable, and include an excipient, diluent, carrier, any member of a diverse population of morphologically and adjuvant that are acceptable for veterinary use as well as similar cell types found in lymphoid or non-lymphoid tis human pharmaceutical use. “A pharmaceutically acceptable Sues. Dendritic cells are a class of “professional antigen US 2017/O 189384 A1 Jul. 6, 2017

presenting cells, and have a high capacity for sensitizing Sandell et al. (2007) Genes Dev. 21: 1113-1124; Wu et al. MHC-restricted T cells. Dendritic cells may be recognized (2002) Invest. Ophthal. Vis. Sci. 43: 3365-3372. Agonists of by function, or by phenotype, particularly by cell Surface RDH10, or other agents that increase activity of RDH10 are phenotype. These cells are characterized by their distinctive of interest for the methods of the invention. morphology, intermediate to high levels of surface MHC 0056. The term colorectal cancer includes all cancers of class II expression and ability to present antigen to T cells, the colon and/or rectum, but particularly adenocarcinoma of particularly to naive T cells (Steinman et al. (1991) Ann. the colon (e.g., mucinous (colloid) adenocarcinoma or signet Rev. Immunol. 9:271; incorporated herein by reference for ring adenocarcinoma). Other types of colorectal cancer its description of Such cells). included by the term include the following varieties of colon 0051. The vitamin A metabolite all-trans-retinoic acid cancer: neuroendocrine, lymphoma, melanoma, squamous (RA) is an essential signaling molecule in embryonic devel cell, sarcoma and carcinoid. The term colorectal cancer also opment and throughout life; a potent regulator of cell includes all stages of colorectal cancer; for example, under differentiation, proliferation, and apoptosis in various cell the Modified Duke Staging System or TNM system (Tumor, types. RA acts through specific RA nuclear receptors Node, Metastasis). The stages associated with these systems (RARC, B, and Y) and their heterodimeric counterparts, the are well known by practitioners of ordinary skill in the art. retinoid-X-receptors (C., B, and Y) to positively or negatively 0057. In the methods of the invention, the agent(s) may regulate expression of RA target genes by binding to their be administered to a subject to treat an inflammatory con respective response elements. Vitamin A deficiency has been dition, which inflammation may predispose to colorectal linked to increased susceptibility to carcinogenesis in animal cancer, and may include individuals having familial models. Although essentially all cell types express nuclear adenomatous polyposis (FAP), hereditary nonpolyposis retinoic acid receptors, cellular responsiveness is determined colon cancer (HNPCC) (i.e., Lynch I Syndrome or Lynch II by RA bioavailability regulated by the coordinated balance Syndrome), inflammatory bowel disease, Such as chronic between vitamin A nutritional status and RA biosynthesis ulcerative colitis (UC) or Crohn's disease, other family and catabolism. The RA-metabolizing cytochrome P450s cancer syndromes (e.g., Peutz-Jegher Syndromem and CYP26A1, B1, and C1 convert RA into rapidly excreted Familial Juvenile Polyposis), adenomatous polyps (e.g., oxoderivatives (4-OH RA, 4-oxo RA, 18-OH RA), while sessile (flat with a broad base and no stalk); tubular (com retinaldehyde dehydrogenase generates RA. Inhibitors of the posed of tubular glands extending downward from the outer CYP26 gene family are of interest for use in the methods of Surface of the polyp); Villous (composed of fingerlike epi the invention, including without limitation CYP26A1. thelial projections extending outward from the surface of the 0052 CYP26A1 is a member of the cytochrome P450 bowel mucosa); pedunculated (attached by a narrow base superfamily of enzymes. The cytochrome P450 proteins are and a long stalk), and sporadic forms of colon cancer. monooxygenases. This endoplasmic reticulum protein acts 0.058 Familial adenomatous polyposis (FAP) is an inher on retinoids, including all-trans-retinoic acid (RA), with ited condition in which numerous polyps form mainly in the both 4-hydroxylation and 18-hydroxylation activities. Two epithelium of the large intestine. In general, while these alternatively spliced transcript variants of this gene, which polyps start out benign, malignant transformation into colon encode the distinct isoforms, have been reported. cancer occurs when not treated. Familial juvenile polyposis 0053. Inhibitors of CYP26A1 can increase local concen (FJP) is an autosomal dominant condition characterized by trations of RA. For example, an orally administered inhibi multiple juvenile polyps of the gastrointestinal (GI) tract. tor, particularly a formulation that provides for enteric Kindreds have been described in which there is involvement delivery, can raise the RA concentration in intestinal tissues. of the colon only, the upper GI tract or both upper and lower Inhibitors of CYP26A1 are known in the art, and include, GI tracts. FJP is a hamartomatous polyposis syndrome. without limitation, talarozole, ketoconazole; liarozole; S Adenomatous polyps (adenomas) of the colon and rectum (R*,R)—N-4-2-(dimethylamino)-1-(1H-imidazole-1-yl) may be benign (noncancerous) growths that may be precur propyl-phenyl 2-benzothiazolamine (R116010); (R) N Sor lesions to colorectal cancer. In general, polyps greater 4-2-ethyl-1-(1H-1, 2,4-triazol-1-yl)butylphenyl-2- than one centimeter in diameter are associated with a greater benzothiazolamine (R115866); the retinoic acid receptor risk of cancer. If polyps are not removed, they typically (RAR)Yagonist CD1530; the pan-RAR agonist 4-(E)-2-(5. continue to grow and can become cancerous. 6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-pro 0059 Crohn's Disease (Regional Enteritis; Granuloma penylbenzoic acid; peroxisome proliferator-activated tous Ileitis or Ileocolitis) is a chronic transmural inflamma receptor ligands rosiglitaZone and pioglitaZone; etc. tory disease that usually affects the distal ileum and colon 0054 Aldehyde dehydrogenase 1 (retinal dehydrogenase, but may occur in any part of the GI tract. Symptoms include RALDH1) is a liver cytosolic isoform of acetaldehyde diarrhea and abdominal pain. Abscesses, internal and exter dehydrogenase. Retinaldehyde is generated by ADH1 from nal fistulas, and bowel obstruction may arise. Extraintestinal retinol, and its concentration is determined in large part by symptoms, particularly arthritis, may occur. Diagnosis is by its subsequent catabolism by RALDH1 to retinoic acid, see colonoscopy and barium contrast studies. The most common Hempel et al. (1984) Europ. J. Biochem. 141: 21-35; Hsu et initial presentation is chronic diarrhea with abdominal pain, al. (1985) Proc. Nat. Acad. Sci. 82: 3771-3775. Agonists of fever, anorexia, and weight loss. The abdomen is tender, and RALDH1, or other agents that increase activity of RALDH1 a mass or fullness may be palpable. Gross rectal bleeding is are of interest for the methods of the invention. unusual except in isolated colonic disease, which may 0055 Retinol dehydrogenase 10 (RDH10) generates all manifest similarly to ulcerative colitis. Some patients pres trans retinal from all-trans retinol and may plan an important ent with an acute abdomen that simulates acute appendicitis role in the photic visual cycle. The first oxidative step of or intestinal obstruction. About 33% of patients have peria vitamin A metabolism is catalyzed in large part by RDH10 nal disease (especially fissures and fistulas), which is some and is critical for spatiotemporal synthesis of retinoic acid, times the most prominent or even initial complaint. In US 2017/O 189384 A1 Jul. 6, 2017

children, extraintestinal manifestations frequently predomi tered in a dose effective to maintain the tolerogenic functions nate over GI symptoms; arthritis, fever of unknown origin, of dendritic cells. The appropriate dose may be determined anemia, or growth retardation may be a presenting symptom, by evaluating the effect of the agent on functions of dendritic whereas abdominal pain or diarrhea may be absent. cells, or by overall analysis of intestinal inflammation. The 0060 Established Crohn's disease is rarely cured but is methods of the invention exclude administration of retinoic characterized by intermittent exacerbations and remissions. acid directly. The administration of inhibitors of CYP26A1, Some patients suffer severe disease with frequent, debilitat including without limitation liarozole and talarozole, are of ing periods of pain. However, with judicious medical particular interest. therapy and, where appropriate, Surgical therapy, most 0065. The term “therapeutically effective amount” or patients function well and adapt Successfully. Disease-re “therapeutically effective dosage' may mean that amount or lated mortality is very low. GI cancer, including cancer of dosage of an agent or combination thereof of the invention the colon and Small bowel, is the leading cause of excess or composition thereof that will elicit a biological or medical Crohn's disease-related mortality. response of a tissue, system, patient, Subject or host that is 0061 Irritable bowel syndrome consists of recurring being sought by the administrator (Such as a researcher, upper and lower GI symptoms, including variable degrees of doctor or veterinarian) which includes any measurable alle abdominal pain, constipation or diarrhea, and abdominal viation of the signs, symptoms and/or clinical indicia of bloating. Diagnosis is clinical. Treatment is generally symp intestinal inflammation, including colorectal cancer (e.g., tomatic, consisting of dietary management and drugs, tumor growth and/or metastasis) including the prevention, including anticholinergics and agents active at serotonin slowing or halting of progression of the inflammation to any receptors. There are no consistent motility abnormalities. degree whatsoever. Some patients have an abnormal gastro-colonic reflex, with 0.066 Dosage regimens may be adjusted to provide the delayed, prolonged colonic activity. There may be reduced optimum desired response (e.g., a therapeutic response). For gastric emptying or disordered jejunal motility. Some example, a single dose may be administered or several patients have no demonstrable abnormalities, and in those divided doses may be administered over time or the dose that do, the abnormalities may not correlate with Symptoms. may be proportionally reduced or increased as indicated by Small-bowel transit varies: sometimes the proximal small the exigencies or the particular circumstances or require bowel appears to be hyperreactive to food or parasympath ments of the therapeutic situation. For example, dosage may omimetic drugs. Intraluminal pressure Studies of the sigmoid be determined or adjusted, by a practitioner of ordinary skill show that functional constipation can occur with hyperre in the art (e.g., physician or Veterinarian) according to the active haustral segmentation (ie, increased frequency and patient’s age, weight, height, past medical history, present amplitude of contractions). In contrast, diarrhea is associated medications and the potential for cross-reaction, allergies, with diminished motor function. Thus strong contractions sensitivities and adverse side-effects. For example, the phy can, at times, accelerate or delay transit. sician or veterinarian could start doses of the agent at levels 0062 Diagnosis is based on characteristic bowel patterns, lower than that required in order to achieve the desired time and character of pain, and exclusion of other disease therapeutic effect and gradually increase the dosage until the processes through physical examination and routine diag desired effect is achieved. The effectiveness of a given dose nostic tests. Diagnostic testing should be more intensive or treatment regimen of an agent of the invention can be when “red flags' are present: older age, weight loss, rectal determined, for example, by determining the concentration bleeding, vomiting. Proctosigmoidoscopy with a flexible of pro-inflammatory cytokines such as IL-23; whether a fiberoptic instrument should be performed. Introduction of tumor being treated in the Subject shrinks or ceases to grow. the sigmoidoscope and air insufflation frequently trigger The size and progress of a tumor can be easily determined, bowel spasm and pain. The mucosal and vascular patterns in for example, by X-ray, magnetic resonance imaging (MRI) IBS usually appear normal. or visually in a Surgical procedure. In general, tumor size 0063 Colonoscopy is preferred for patients >40 with a and proliferation can be measured by use of a thymidine change in bowel habits, particularly those with no previous PET scan (see e.g., Wells et al., Clin. Oncol. 8: 7-14 (1996)). IBS symptoms, to exclude colonic polyps and tumors. In Generally, the thymidine PET scan includes the injection of patients with chronic diarrhea, particularly older women, a radioactive tracer, such as 2-'C-thymidine, followed by mucosal biopsy can rule out possible microscopic colitis. a PET scan of the patient’s body (Vander Borght et al., Gastroenterology 101: 794-799, 1991; Vander Borght et al., Methods of the Invention J. Radiat. Appl. Instrum. Part A, 42: 103-104 (1991)). Other 0064 Methods are provided for reducing intestinal tracers that can be used include 'FI-FDG (18-fluorode inflammation, particularly chronic inflammation, and tumor oxyglucose), 'IIIUdR (5-'Iliodo-2'-deoxyuridine), growth precipitated by intestinal inflammation. In the meth 7Br BrdUrd (Bromodeoxyuridine), 'FIFLT (3'-deoxy-3' ods of the invention, an effective dose of an agent is fluorothymidine) or 'CIFMAU (2'-fluoro-5-methyl-1-3- provided to the individual, where the agent increases local D-arabinofuranosyluracil). concentration of retinoic acid (RA) in the intestine through 0067 Methods for treating or preventing intestinal modifying enzymatic pathways involved in RA metabolism. inflammation, including inflammation leading to colorectal In particular, an inhibitor of a CYP26, enzyme, for example cancer by administering a pharmaceutical composition com CYP26A1, may be administered in a dose effective to prising an agent that increases RA levels by altering enzy neutralize an inflammatory environment and/or maintain the matic activity involved in RA metabolism, in association tolerogenic functions of intestinal dendritic cells that main with a pharmaceutically acceptable carrier are also within tain intestinal tolerance by inducing Treg formation. Alter the scope of the present invention (e.g., in a single compo natively, an agent that increases activity of retinaldehyde sition or separately in a kit) as are combinations and com dehydrogenase or retinol dehydrogenase may be adminis positions including Such pharmaceutical compositions. The US 2017/O 189384 A1 Jul. 6, 2017 pharmaceutical compositions may be prepared by any meth and polyvinylpyrrolidone. Emulsifying agents include Poly ods well known in the art of pharmacy; see, e.g., Gilman, et sorbate 80 (TWEEN-80). A sequestering or chelating agent al., (eds.) (1990), The Pharmacological Bases of Therapeu of metal ions includes EDTA (ethylenediaminetetraacetic tics, 8th Ed., Pergamon Press; A. Gennaro (ed.), Reming acid) or EGTA (ethylene glycol tetraacetic acid). Pharma ton's Pharmaceutical Sciences, 18th Edition, (1990), Mack ceutical carriers may also include ethyl alcohol, polyethyl Publishing Co., Easton, Pa.; Avis, et al., (eds.) (1993) ene glycol and propylene glycol for water miscible vehicles; Pharmaceutical Dosage Forms: Parenteral Medications and Sodium hydroxide, hydrochloric acid, citric acid or Dekker, N.Y.: Lieberman, et al., (eds.) (1990) Pharmaceu lactic acid for pH adjustment. tical Dosage Forms: Tablets Dekker, N.Y.; and Lieberman, 0073. In an embodiment of the invention, preparations et al., (eds.) (1990), Pharmaceutical Dosage Forms: Disperse for parenteral administration can include Sterile solutions Systems Dekker, N.Y. ready for injection, sterile dry soluble products, such as 0068 A pharmaceutical composition can be prepared lyophilized powders, ready to be combined with a solvent using conventional pharmaceutically acceptable excipients just prior to use, including hypodermic tablets, sterile Sus and additives and conventional techniques. Such pharma pensions ready for injection, Sterile dry insoluble products ceutically acceptable excipients and additives include non ready to be combined with a vehicle just prior to use and toxic compatible fillers, binders, disintegrants, buffers, pre sterile emulsions. The Solutions may be either aqueous or servatives, anti-oxidants, lubricants, flavorings, thickeners, nonaqueous. coloring agents, emulsifiers and the like. All routes of 0074 The concentration of the agent of the invention, administration are contemplated including, but not limited which is optionally in association with a further chemothera to, parenteral (e.g., Subcutaneous, intravenous, intraperito peutic agent, can be adjusted so that a dose provides an neal, intramuscular, topical, intra-peritoneal, inhalation, effective amount to produce the desired pharmacological intra-cranial) and non-parenteral (e.g., oral, transdermal, effect. As discussed herein, the exact dose depends, in part, intranasal, intraocular, Sublingual, rectal and topical). on the age, weight and condition of the patient or animal as 0069 Oral administration is of interest, including enteric is known in the art. formulations, which may include acid stable agents that 0075 Implantation of a slow-release or sustained-release maintain activity under gastrointestinal conditions, enteric system, Such that a constant level of dosage is maintained, coatings of pills, and the like, where there is a significant is also contemplated herein. Briefly, an active agent is activity of the agent in intestinal tissues. dispersed in a solid inner matrix, e.g., polymethylmethacry 0070 Injectables can be prepared in conventional forms, late, polybutylmethacrylate, plasticized or unplasticized either as liquid Solutions or Suspensions, Solid forms suitable polyvinylchloride, plasticized nylon, plasticized polyethyl for solution or Suspension in liquid prior to injection, or as eneterephthalate, natural rubber, polyisoprene, polyisobuty emulsions. The injectables, Solutions and emulsions can also lene, polybutadiene, polyethylene, ethylene-vinylacetate contain one or more excipients. Excipients include, for copolymers, silicone rubbers, polydimethylsiloxanes, sili example, water, Saline, dextrose, glycerol or ethanol. In cone carbonate copolymers, hydrophilic polymers such as addition, if desired, the pharmaceutical compositions to be hydrogels of esters of acrylic and methacrylic acid, collagen, administered may also contain minor amounts of non-toxic cross-linked polyvinylalcohol and cross-linked partially auxiliary Substances Such as wetting or emulsifying agents, hydrolyzed polyvinyl acetate, that is surrounded by an outer pH buffering agents, stabilizers, solubility enhancers, and polymeric membrane, e.g., polyethylene, polypropylene, other such agents, such as for example, Sodium acetate, ethylene/propylene copolymers, ethylene/ethyl acrylate Sorbitan monolaurate, triethanolamine oleate and cyclodex copolymers, ethylene/vinylacetate copolymers, silicone rub trins. bers, polydimethyl siloxanes, neoprene rubber, chlorinated 0071. In an embodiment of the invention, pharmaceuti polyethylene, polyvinylchloride, vinylchloride copolymers cally acceptable carriers used in parenteral preparations with vinyl acetate, vinylidene chloride, ethylene and pro include aqueous vehicles, nonaqueous vehicles, antimicro pylene, ionomer polyethylene terephthalate, butyl rubber bial agents, isotonic agents, buffers, antioxidants, local anes epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, thetics, Suspending and dispersing agents, emulsifying ethylene/vinyl acetate/vinyl alcohol terpolymer, or ethylene/ agents, sequestering or chelating agents and other pharma vinyloxyethanol copolymer, that is insoluble in body fluids. ceutically acceptable Substances. The antibody or fragment diffuses through the outer poly 0072 Examples of aqueous vehicles include sodium meric membrane in a release rate controlling step. The chloride injection, Ringers Injection, isotonic dextrose percentage of active agent contained in Such parenteral Injection, sterile water injection, dextrose and lactated Ring compositions is highly dependent on the specific nature ers Injection. Nonacqueous parenteral vehicles include fixed thereof, as well as the activity of the antibody or antigen oils of vegetable origin, cottonseed oil, corn oil, Sesame oil binding fragment, which is optionally in association with a and peanut oil. Antimicrobial agents in bacteriostatic or further chemotherapeutic agent, and the needs of the Subject. fungistatic concentrations may be added to parenteral prepa 0076 Agents set forth herein can be formulated into a rations packaged in multiple-dose containers which include Sustained release formulation including liposomal formula phenols or cresols, mercurials, benzyl alcohol, chlorobuta tions such as unilamellar vesicular (ULV) and multilamellar nol, methyl and propyl p-hydroxybenzoic acid esters, thime vesicular (MLV) liposomes. rosal, benzalkonium chloride and benzethonium chloride. Example 1 Isotonic agents include Sodium chloride and dextrose. Buf fers include phosphate and citrate. Antioxidants include 0077. The present invention is intended to exemplify the Sodium bisulfate. Local anesthetics include procaine hydro present invention and not to be a limitation thereof. Methods chloride. Suspending and dispersing agents include Sodium and compositions disclosed below fall within the scope of carboxymethylcellulose, hydroxypropyl methylcellulose the present invention. US 2017/O 189384 A1 Jul. 6, 2017

0078. In the majority of sporadic colorectal cancers and for generating the altered CD4 T cell response in the gut in FAP. tumorigenesis is initiated from mutations that arise demonstrated in previous studies. No significant changes in in the APC tumor suppressor gene. In the APC" murine the expression of costimulatory molecules (CD40, CD80, model, numerous adenomas develop in the intestines, CD83, CD86 or PDL1) were seen on these cells (FIG. 1d). closely resembling FAP APC'" homozygotes die in Nonetheless, when stimulated with a panel of TLR agonists, utero, and APC" heterozygotes spontaneously lose their purified SI-LPDCs from APC' mice, especially those wildtype allele during puberty and start developing polyps with late-stage disease, secreted much larger amounts of the by week 10. Although these mice do not develop invasive pro-inflammatory cytokines TNFO, IL-6 and IL-12p40 com metastases, they eventually succumb to a fatal anemia pared to SI-LPDCs from WT mice (FIG. 1e). through excessive intestinal bleeding. 0.083 APC' SI-LPDCs are impaired in their ability to 0079 Prior studies in APC" mice show that chronic induce T and instead promote Th17 formation. To intestinal inflammation precipitates as well as propagates address whether APC' SI-LPDCs are able to induce tumor growth and that Th17 cells are the responsible Foxp3". To de novo, we cultured CFSE-labeled naive immune effector cells. Since chronic inflammation in the CD4"CD62LFoxp3 T cells from OT-II TCR transgenic intestine predisposes to colorectal cancer, it is Surprising that mice with purified DCs in the presence of TGFB and the the role of local DCs has yet to be examined in models such ovalbuming peptide. After 5 days of co-culture, the as APC'". The remarkable capacity of these cells to frequency of Foxp3"CD4 T cells was determined. The orchestrate distinct immune responses is aided by a panoply results show that splenic DCs from both genotypes induced of environmental cues, which condition the cells to adopt Foxp3" cells weakly, while mLN/PPDCs were more potent specific phenotypes in different settings. DCs in gut-associ inducers of Foxp3" cells, consistent with previous studies. ated lymphoid tissue are of particular interest because they Interestingly, APC' SI-LPDCs were impaired in their maintain tolerance to commensal flora as well as mount capacity to induce Foxp3 cells compared to WT controls protective inflammatory responses in the face of pathogen (FIG. 2a, FIG. 7a). This impairment was observed begin incursion. ning at intermediate stage and became more apparent at late 0080. The experiments described in this report indicate stage (FIG. 7c). Similar results were obtained across a 25 that small intestine LPDCs (SI-LPDCs) play a critical role in fold range of peptide concentration (FIG. 2b, FIG. 7b). the inflammatory process that underlies tumor progression in I0084. When we measured the levels of key immuno the APC" mouse. Whereas SI-LPDCs in healthy control modulatory cytokines in the supernatants of these DC-T cell mice induce the formation of Tre APC SI-LPDCs are co-cultures, the results revealed a striking six-fold reduction reprogrammed to induce Th17 formation. A marked reduc in IL-10 generated in APC" LPDC co-cultures compared tion in the intestine of the vitamin A metabolite, retinoic acid to the WT control cultures (FIG.2e). Moreover, there was a (RA), which under homeostatic conditions maintains the concomitant and similarly dramatic increase in IL-17A tolerogenic functions of LPDCs, explains the reprogram (FIG. 20, consistent with the documented role of Th17 cells ming of these cells. Since we find identical defects in RA in adenoma development. Intracellular staining of the T cells metabolism in FAP tissue, the same mechanism that drives in the APC" co-cultures confirmed the presence of inflammation in APC" mice contributes to tumor forma IL-17A producing cells and a reduction of IL-10 producing tion in FAP. cells. 0081 Inflammatory DCs accumulate in the SI-LP of I0085. As CD103* SI-LPDCs are the main cells respon APC' mice. We initially compared the frequency of DCs sible for generating immune tolerance in the intestinal in various tissues in APC" mice and their WT littermate environment, we sorted SI-LPDCs into CD103" and controls. DCs were identified as EpCAMCD45"Lin CD103 subsets to determine which subset accounted for the MHCII"CD11c" and analyzed by flow cytometry at 10, 14 observed impairment in Foxp3 induction. APC" CD103" and 18 weeks of age, time points that correspond to minimal SI-LPDCs were four-fold less able to induce Foxp3" T cells (<30 polyps, <2 mm diameter), intermediate (30-60 polyps, compared to their WT counterparts (FIG. 2c). In contrast, 0.5-4 mm diameter) and late stage (>60 polyps, 1-6 mm CD103. SI-LPDCs from both genotypes were equally poor diameter) disease, respectively. As shown in FIG. 1a-b, DCs in inducing Foxp3 expression (FIG. 2c), consistent with accumulated in the SI-LP as disease progressed. In the previous studies. In addition, co-cultures of APC'" steady state. DCs in the gut environment are comprised of CD103* SI-LPDCs and T cells, but not CD103 LPDC-T three phenotypically distinct populations CD103"CD11b, cell co-cultures, contained large amounts of IL-17A. These CD103*CD11b and CD103CD11b DCs. CD103*CD11 results show that the CD103 Subset of SI-LPDCs in APC b. DCs are enriched in the Peyer's patches, whereas CD103" '" mice is not only responsible for defective induction of CD11b" and CD103CD11b" DCs are found mainly in the T, but also for the generation of Th17 cells. LP. Although there were some differences in the frequency I0086 Retinoic acid (RA) reverses the inflammatory phe of splenic DC subsets between APC" and WT control notype of APC" LPDCs. The unique capacity of the mice, no significant differences in the percentages of the CD103* SI-LPDC subset to store and metabolize vitamin A three key subsets were observed in the mesenteric lymph into RA explains the specialized role of these cells in nodes (mLN), Peyer's Patches (PP) and most importantly, generating and maintaining intestinal tolerance. We hypoth the SI-LP (FIG. 1c). These analyses were performed at esized that the cytokine profile of the pro-inflammatory intermediate stage, as the APC" mice lose their Peyer's APC" SI-LPDCs could be modulated by changes in the Patches by late stage disease, accompanying other develop local RA concentration. To address this possibility we added mental abnormalities such as early thymic involution, sple RA or the RA receptor antagonist, LE540, to the LPDC-T nomegaly and lymphodepletion. cell co-cultures and assessed the induction of Foxp3' T cells. I0082) We considered the possibility that the DCs accu LE540 abrogated Foxp3 induction in both WT and APC'" mulating in the SI-LP of APC" mice may be responsible SI-LPDC co-cultures (FIG. 2d), whereas RA enhanced US 2017/O 189384 A1 Jul. 6, 2017

Foxp3 induction in the APC" SI-LPDC co-cultures to the were shown to express abnormally high levels of Ctbp1, levels seen in WT co-cultures (FIG. 2d). Addition of RA also which correlated with low levels of the intestinal retinol strongly inhibited IL17A production in the APC" co dehydrogenase Rdh11 and intestinal differentiation defects. cultures. Since Th17 differentiation requires IL-6 in con Moreover, reintroduction of the APC protein into human junction with TGFB, and since no exogenous IL-6 was colon carcinoma cell lines led to a proteasome-dependent added to our cultures, we postulated that the APC'" destruction of Ctbp1, concomitant with increased expression LPDC co-culture supernatants may contain IL-6. This was of the retinol dehydrogenase, DHRS9. To determine if the confirmed (FIG. 2g). RA also potently inhibited IL-10 loss of RA we observed in the APC" gut may be due to production in both WT and APC' co-cultures, consistent upregulation of Ctbp1, we assessed Ctbp1 transcript levels with a previous study. in SI-LPDCs and IECs. A significant elevation of Ctbp 1 was 0087 Loss of RA is linked to both reduced expression of seen in APC'* IECs compared to WTIECs beginning at retinaldehyde dehydrogenases (Raldh) and defective regu intermediate stage disease (FIG. 3c). Surprisingly, there was lation of the transcriptional co-repressor C-terminal binding a parallel accumulation of Ctbp 1 in the APC' SI-LPDCs protein 1 (Ctbp1). Vitamin A, or retinol, when absorbed in as well. These findings suggest that along with loss of Raldh the intestine, is either converted to a storage form or expression, elevated levels in Ctbp 1 in both cell types may metabolized to RA. In the latter, retinol is catalyzed to retinal Suppress intestinal retinol dehydrogenase expression, by two families of enzymes, the alcohol dehydrogenases thereby contributing to the loss of RA observed in the tumor (ADHS) and the short-chain dehydrogenase reductases milieu. (RDH), and subsequently oxidized to bioactive RA by the 0091 FAP adenomas exhibit the same inflammatory Raldh enzymes. To ascertain whether APC" LPDCs are changes and abnormalities in RA metabolism as seen in able to produce RA, we used RT-qPCR to measure the APC" mice. To assess whether our findings in the expression of several key Raldh enzymes in these cells. In APC" model are predictive of the human condition, we line with previous studies, Raldh expression in the SI-LP of used immunohistochemistry and immunofluorescence to both WT and APC" mice was consistently higher than analyze the colonic polyps of FAP patients. For comparison, control splenic DCs (FIG. 3b). At early stage, APC'" we analyzed biopsies of normal colon as well as sessile SI-LPDCs expressed equal or higher levels of Raldh1a1 and serrated polyps (SSP), the latter serving as a source of Raldh1a2 compared to WT SI-LPDCs (FIG. 3a). However, non-APC mutated adenomas. IL17A expression was strik as the disease progressed in APC" mice, expression of ingly elevated in FAP adenomas compared to adjacent these enzymes, especially Raldhlal, declined markedly. uninvolved colon, normal colon or SSP (FIG. 4a). Staining 0088 Recent studies have shown that RA in the gut of the same tissues with an isotype control antibody is shown induces Raldh expression. Since IECs constitutively express in FIG. 11. These findings indicate that the inflammatory these retinol-metabolizing enzymes and can efficiently gen environment in FAP polyps is similar to that seen in the erate RA, we postulated that defective RA production in the APC' mouse model. IECs might help explain the loss of Raldh expression 0092. We next examined the tissues for expression of key observed in the APC' LPDCs. As shown in FIG. 3b, Vitamin A metabolizing enzymes and the proteins that modu Raldh1a1 and Raldh1a2 expression in sorted CD45TEp late them. Consistent with published reports, cytoplasmic CAM IECs did not change significantly through early and B-catenin was expressed at higher levels in FAPadenomas intermediate stage disease. At late stage, however, APC'" compared to adjacent uninvolved tissue and to SSP. Inter IECs exhibited approximately five- and two-fold reduction estingly, however, B-catenin was not expressed by cells in the expression of Raldh1a1 and Raldh1a2, respectively, expressing dendritic cell-specific Intercellular Adhesion compared to WTIECs. Of note, compared to WTIEC, ADH Molecule-3-Grabbing Non-integrin (DC-SIGN) in the tis class I and II expression in APC" IEC increased at sues tested (FIG. 4b). Ctbp1, the transcription factor that intermediate stage and decreased at Subsequent late stage negatively regulates intestinal RDHS, was present at much (FIG.9). Taken together, these data show that both APC'" higher concentrationin FAPIECs than in the IECs of normal SI-LPDCs and IECs lose expression of the critical Raldh1a1 or SSP (FIG. 5c), consistent with previous reports, Remark and Raldhla2 enzymes by late stage disease, and have a ably, Raldh1a1 and Raldh1a2 expression was almost com reduced RA-production capacity compared to their WT pletely abolished in IECs of FAP adenoma tissue and not counterparts. SSP (FIG. 5d-e), validating our findings in the APC'" 0089. To confirm that the changes observed at the tran mouse. Finally, the RA catabolizing enzyme CYP26A1 was script level of these enzymes correlated with a loss of RA in markedly upregulated in the epithelia (FIG. 5f) as has been the local tumor milieu, we utilized tandem mass spectrom reported. Since FAPadenomas contain little RALDH activ etry to quantitatively profile endogenous retinoids in late ity (possibly due to excess Ctbp 1), but excessive CYP26A1, stage APC" duodenum, jejunum, ileum, colon and eye. the net result is likely a dearth of RA in the local tumor As shown in FIG. 5i, RA levels in APC" tissues were milieu. diminished compared to WT controls, with the difference 0093 Polyposis is exacerbated by a vitamin A deficient reaching statistical significance in the ileum, where there is (VAD) diet and ameliorated by Liarozole, a CYP26A1 the highest frequency of polyps, and in the colon. Although inhibitor. Mice fed a vitamin A-deficient diet have dramati retinol was reduced in the APC" ileum, tissues from both cally reduced DC Raldh expression. Given the likelihood genotypes had comparable amounts retinyl esters, indicating that diminished RA explains the pro-inflammatory profile of that the deficit in RA is not due to a lack of substrate (FIG. SI-LPDCs in APC' mice, we postulated that prolonged 10). vitamin A-deficiency would intensify Raldh loss, drive 0090 Previous studies have demonstrated that the APC inflammation and accelerate polyp development. To test this protein can directly bind to Ctbp 1 in both Drosophila hypothesis, groups of WT and APC' mice were placed melanogaster and human cell lines. APC-mutant Zebrafish on a diet containing no vitamin A for 10 weeks, beginning US 2017/O 189384 A1 Jul. 6, 2017

at 8-10 weeks of age, after which they were returned to a indicate that as disease progresses, there is an accumulation normal rodent chow diet. Groups of mice treated with of pro-inflammatory DCs in the SI-LP that induce the Celecoxib (a cyclooxygenase-2 inhibitor) and RosiglitaZone formation of Th17 cells. This stands in dramatic contrast to (a peroxisome proliferator-activated receptor-Y agonist), the SI-LPDCs of healthy mice, which do not promote were included as positive and negative controls, respec inflammation but instead maintain immune tolerance tively. These compounds were added to “base' mouse chow through the generation of T. CD103+ LPDCs, believed which contained 4 IU Vitamin Afg. Disease development and to be responsible for tolerance induction in the intestine, severity were monitored on the basis of Survival, changes in were present in greater numbers in APC" than healthy body weight, hematocrit and polyp count at the point of control mice, but secreted pro-inflammatory cytokines and euthanasia. induced the formation of Th17 cells rather than T. The 0094 WT mice on each diet gained weight comparably induction of Th17 cells by APC' SI-LPDCs likely rep (FIG. 12) and did not develop polyps. APC' mice on the resents a critical control point in shaping the inflammatory base diet survived throughout the 10-week period of disease milieu that drives tumor growth, and explains the predomi monitoring, but all succumbed by week 24 (FIG. 5a). nance of IL-17 in the inflamed APC" intestine. Compared to mice on the base diet APC" mice treated 0098. Since SI-LPDCs play such an important role in with Celecoxib had markedly prolonged survival, while tumor-promoting inflammation, identifying the mechanism mice that received RosiglitaZone had reduced Survival con that induces their unusual phenotype in APC" mice is sistent with previous reports (FIG. 5a). Interestingly, VAD key to understanding how the inflammatory cascade is diet-fed APC" mice had particularly aggressive disease triggered. We hypothesized that these cells could have and Succumbed even more rapidly than mice on Rosiglita undergone reprogramming in response to one or more fac Zone (FIG. 5c-d). The body weights of APC' mice tors in the tumor microenvironment. Our efforts focused on placed on base or VAD diets, or treated with Rosiglitazone, RA because of the well documented role of this molecule in decreased comparably, while those treated with Celecoxib maintaining a tolerant state in the intestine. Addition of RA gained weight steadily (FIG. 5b). to the T induction cultures not only enhanced the capacity 0095 Since decreasing RA in the intestinal environment of APC' SI-LPDCs to induce Foxp3+ T cells but also exacerbates disease in APC" mice, we wanted to test the prevented the induction of Th17 cells. Conversely, addition hypothesis that increasing intestinal RA would ameliorate of the RAR antagonist, LE540, resulted in further diminu disease. Oral administration of RA was not feasible, since it tion of Foxp3 induction and augmented the production of is known to stimulate further polyp growth in APC" mice IL17A by more than five-fold. due to the induction of CYP26A1 and consequent loss of 0099. The absolute amount of RA in the intestines of RA. Instead, we sought to increase intestinal RA by pre mice with APC mutations has not been reported previously, venting its breakdown with a CYP26A1 inhibitor, Liarozole, likely due to the technical difficulty of carrying out such which was added to the base diet of 8 week old mice for a measurements. By preventing exposure of tissues to white period of 10 weeks, as above. Remarkably, the Liarozole light, which rapidly degrades retinoids, and utilizing mass treated mice exhibited a striking reduction in the number of spectroscopy to measure RA, we could achieve accurate RA polyps as well as a significant increase in body weight, quantitation. Our results revealed highly significant reduc although their hematocrits deteriorated to the same extent as tions of RA in the ileum and colon of APC" mice, but not untreated controls (FIG. 6a-d). To confirm that Liarozole in other sites. Although some RA remained in the intestines, mediated its effects by increasing local RA concentration in the reductions seen are nonetheless noteworthy, as retinol the intestine, we measured retinoid levels in the treated bound to its specific retinol-binding protein (RBP) is strictly mice. As shown in FIG. 6e, RA levels returned to normal in regulated and maintained in plasma at about 2 uM despite LiaroZole treated mice, especially in the ilium. daily fluctuations in dietary intake of vitamin A. Only in 0096. To assess whether the Liarozole mediated increase situations of severe, prolonged vitamin A-deficiency, where in intestinal RA influenced immune outcome by re-repro stores of retinyl esters in the liver are depleted, is there a gramming the pro-inflammatory APC'". SI-LPDCs, SI drop in plasma concentration of retinol-RBP, and by asso LPDCs from Liarozole-treated mice were isolated and tested ciation, RA. functionally. The results show that APC'". SI-LPDCs 0100. The loss of RA in the tumor milieu appears to be from Liarozole-treated miceno longer produced substantial due to both diminished synthesis and excessive breakdown amountsof pro-inflammatory cytokines when stimulated of this molecule. We found that the critical Raldh enzymes with TLR agonists (FIG. 60, and instead of inducing Th17 that control RA production decline as disease progresses, formation, promoted the formation of IL-10 secreting and this this is likely explained, in part, by the overexpres Foxp3+Tregs (FIG. 6g-h) at levels similar to wildtype mice. sion of Ctbp 1, which suppresses Rdh. Ctbp1 is normally Taken together, these findings show that whereas exacerbat degraded by APC, but accumulates in the absence of func ing RA deficiency in the intestine accelerates disease, restor tional APC. Our immunofluorescence data confirmed a ing RA reverses the reprogramming of SI-LPDCs, thereby marked accumulation of Ctbp1 in FAPIECs. Inactivation of preventing deleterious Th17 responses and ameliorating the APC gene also results in constitutive expression of disease. f3-catenin, which upregulates the major RA catabolic 0097. The role of intestinal inflammation in the develop enzyme, CYP26A1. Indeed, the CYP26A1 transcript has ment of adenomas in the APC" model of spontaneous been found to be upregulated in whole tissue isolated from neoplasia has been well documented. However, the few both APC" and FAPadenomas, sporadic colorectal car immunological studies performed on APC" mice and cinomas, and the intestine of APC-mutant Zebrafish related APC mutation models have focused almost exclu embryos. Here we validate these observations, specifically sively on altered T cell responses, with no attention directed identifying epithelial cells as one of perhaps several cell at the underlying cause of these changes. Our findings types in FAP colon displaying CYP26A1 upregulation. US 2017/O 189384 A1 Jul. 6, 2017

Finally, prostaglandin E2 (PGE2), which has been reported against CD45.2, CD49b, CD3e, CD19, CD11c, MHC II, to inhibit Raldh1a2 expression in DCs, may contribute to the EpCAM and propidium iodide (PI), and sorted using a loss of RA. It is well established that FAP and APC'" FACS Aria (BD Biosciences). In some cases, DCs were epithelium exhibit constitutively high Cox2 expression. In additionally sorted using anti-CD103. addition, we found that the Cox2 transcript is overexpressed in late stage APC'* SI-LPDCs compared to their WT 0106 Flow Cytometric analysis. Isolated small intestine counterparts (FIG. 13). This finding is consistent with the lamina propria (SI-LP) cells, splenocytes and mesenteric possibility that Cox2-overexpression and abundance of lymph node cells were resuspended in 1% BSA in PBS PGE2 in the FAP colon may lead to suppression of Raldh1a2 (FACS buffer). After Fc blockade with anti-FcyRIII/II (BD in both DCs and epithelia. Taken together, our results point Biosciences), cells were stained with Live/Dead Blue (Invit to at least 3 mechanisms that cooperate to Suppress the RA rogen) or PI, and monoclonal antibodies (all Biolegend) levels in the tumor milieu of APC" mice. Moreover, against CD40, CD80, CD83, CD86, PDL1, Thy 1.2 and these data indicate that the APC mutation underlying malig CD4. Intracellular Foxp3 and cytokines were stained using nant transformation of intestinal epithelia is directly linked antibodies against Foxp3, IL 10, IL6, IL17A (all eBiosci to the immune defect driving tumor growth. We summarize ence) per manufacturers instructions. Flow cytometric our key findings and others in an illustration depicted in FIG. analysis was performed on a LSRII flow cytometer (BD 6d. Biosciences). 0101 Importantly, human FAPadenoma tissue exhibited several of the same defects that we found in the APCMini+ (0.107 T cell differentiation assay. 2x10 sorted CD11c' mouse intestine. Not only was there a marked elevation in MHCII+ DCs were co-cultured with 1x10 MACS-enriched IL17 expression, signifying Th17 driven inflammation, but CD4+CD62L+Foxp3- naive T cells from the spleen and in addition we observed concomitant Raldh downregulation lymph nodes of OT-II TCR-transgenic mice, along with and CYP26A1 upregulation in colonic epithelial cells. OVA peptide (New England Peptide) and 10 ng/ml Defective RA production combined with enhanced RA recombinant human TGF-31 (Peprotech). On day 5, cells breakdown would produce a deficit of similar magnitude to were harvested and analyzed for intracellular Foxp3 or that seen in APC" mice. intracellular cytokines. In some experiments, cells were 0102) To investigate the role of RA in disease develop restimulated on day 4 with 1 g/ml each of plate-bound ment and progression, we sought to alter RA concentration anti-CD3 and anti-CD28 (BD Biosciences), with or without in the intestine in vivo, by removing or administering Brefeldin A for 18 hr. Where indicated, 10 nM all-trans RA molecules in the diet that are known to affect RA production (Sigma), or 1 uM LE540 (Wako Chemicals) was added to or breakdown. Our results revealed that a diet deficient in culture wells. Vitamin A exacerbates disease, while the CYP26A1 inhibi tor, Liarozole, ameliorates disease as indicated by a dramatic 01.08 ELISA. Purified SI-LPDCs were stimulated for 48 reduction in the number of intestinal as well as steady weight hr with Toll-like receptor agonists—1 ug/ml Pam3Csk4, 10 ga1n. ug/ml Poly I:C, 10 ug/ml LPS, 1 lug/ml flagellin, 10 g/ml 0103 Since increasing intestinal RA proved highly effi R848, 10 ug/ml CpG 2336 (Invivogen). Supernatants from cacious in APC" mice, doing so in patients with APC these experiments were assayed for IL-6, TNFO, IL12-p40 mutation associated bowel disease may be initiated. Small and those from were DC-T cell co-cultures were assayed for molecule agonists of Rdhs and Raldhs, or inhibitors of IL-6, IL-10 and IL17A, performed according to manufac CYP26 such as Liarozole, are logical candidates. Indeed, turers instructions (eBioScience). Liarozole has been evaluated in clinical trials for diseases unrelated to colorectal cancer and is apparently well-toler 0109 Quantitation of gene expression using real-time ated. Maintaining sufficient RA in the intestinal environment PCR. Total RNA from purified LP DCs, splenic DCs and could therefore, reverse inflammation and reduce tumor epithelial cells was extracted using RNAeasy kits (Qiagen), burden, as we observed in APC" mice. and the DNase-treated total RNA was reverse-transcribed using High-Capacity Reverse Transcription Kit (Applied Materials and Methods Biosystems) according to manufacturers instructions. Gene 0104 Mice. Breeding pairs of APC' male and WT expression of ADH class 1-III, Raldh1a1-3, Ctbp1, and C57BL/6 female mice were purchased from The Jackson Cox2 was determined by quantitative PCR with Power Laboratory and bred on-site. OT-II TCR transgenic Rag' SYBR Green PCR Master Mix (Applied Biosystems) per mice were purchased from Taconic. TNF^' mice were manufacturers instructions using a 7900HT real-time PCR kindly provided by Dr. Frank Jirik of the University of instrument (Applied Biosystems). Ubiquitin levels were Calgary, Canada. All mice were housed in an American measured in a separate reaction and used to normalize the Association for the Accreditation of Laboratory Animal data. Careaccredited animal facility, maintained in pathogen-free 0110 Quantitation of tissue retinoids. Retinyl esters conditions on a standard rodent chow ad libitum unless (RE), all-trans retinol, and all-trans retinoic acid (RA) were otherwise stated. extracted from the duodenum, jejunum, ileum, colon and 0105 Isolation of DCs. Epithelial cells from the small eye using procedures to those described previously. RA was intestine were washed in PBS with vigorous stirring and the quantified by LC/MS/MS with atmospheric pressure chemi remaining intestinal pieces digested twice with Type VIII cal ionization. RE and retinol were quantified by HPLC. Collagenase (Sigma-C2139) and DNase I (Sigma). Spleen Tissues were harvested and retinoids handled under yellow and mesenteric lymph nodes were digested with collagenase light using only glass laboratory equipment. Results were Type IV (Worthington, 200 U/ml). For purification of DCs. normalized to per gram tissue weight, or to control groups cells were stained with monoclonal antibodies (Biolegend) as fold-change. US 2017/O 189384 A1 Jul. 6, 2017

Histology. colon of TNF^' and AOM/DSS mice, corresponding to the very locations where inflammation and carcinoma was 0111. Immunohistochemistry. occurring respectively. 0112 Formalin-fixed, paraffin-embedded, 5 M-thick, I0120 Taken together, an RA deficiency can occur in human tissue sections were stained with the primary anti generalized settings of chronic intestinal inflammation and body rabbit IL17A (Protein Tech Group (PTG) 13082-1- in colorectal cancer, Suggesting that the beneficial effects of AP), and the secondary rabbit-HRP polymer (Dako Envi modulation of local intestinal RA levels extends to gener sion). Antigen retrieval was performed using Human Diva alized chronic inflammation in the gut. Decloaker citrate buffer (Biocare Medical). I0121. As with many other hormones and vitamins that 0113 Immunofluorescence. can be dysregulated in disease, reinstating RA to the appro 0114 Primary antibodies, all from PTG and rabbit anti priate dynamic range may be key. RA has a short elimination human unless otherwise noted, were mouse DC-SIGN (Den half-life in vivo and in cultured cells just 6-7 hours. Retinol dritics DDX0202), B-catenin (51067-2-AP), Ctbp1 (Human bound to its specific retinol-binding protein in plasma is Protein Atlas (HPA) HPAO18987), Raldh1a1 (15910-1-AP), strictly regulated and maintained at about 2 LM despite daily Raldh1a2 (HPAO10022), CYP26A1 (HPAC6498). Chicken fluctuations from dietary intake of vitamin A. Only in secondary antibodies include anti-rabbit Alexa647 and anti situations of severe vitamin A-deficiency where stores of mouse Alexa488 (Invitrogen). 11 normal colon, 8 FAP retinyl esters in the liver are depleted, is there a drop in adenoma, 2 FAP adenocarcinoma and 4 sessile serrated plasma concentration of retinol-RBP. Corresponding RA polyp patients were analyzed. Images were collected using levels in plasma are in the range of 5-10 nM, more than two a Leica DM2500 confocal laser scanning microscope, and orders of magnitude lower than that of its substrate retinol, analyzed using the LAS AF software. reinforcing the notion that retinol metabolism is tightly regulated and often restricted to certain settings. Local RA 0115 Drug Treatment. concentrations in the APC" ileum and colon may be 0116) 1500 ppm of Celecoxib (Celebrex, Pfizer), 100 reduced to a point where tolerance is broken, but not to the ppm of Rosiglitazone (Avandia, Glaxo Smith Kline), or 40 point where compensatory mechanisms that maintain toler ppm of Liarozole (Tocris Pharmaceuticals) was incorporated ance in severe RA deficiency starts to kick in; or to the point into a base diet (4 IU/g of vitamin A) by Research Diets. A that Th17 cells cannot induce inflammation anymore. vitamin A deficient (0 IU/g) diet (VAD) was also studied. 0.122 We measured RA levels in Liarozole-treated APC Hematocrits were measured every 2 weeks, while weights '"" mice and observed a return to wildtype levels of RA in and DAI were measured every week. At the point of the ileum, so we validated that Liarozole did indeed increase euthanasia, polyps were enumerated and measured using a local concentrations of RA to homeostatic levels, and not to Stereomicroscope at 10x magnification, from the gastro Supraphysiological levels. duodenual junction to the ceco-colic junction. I0123. Since decreasing RA in the intestinal environment 0117 Statistics. exacerbates disease in APC" mice, we tested the recip 0118. An unpaired student's t test (2-tailed) with a 95% rocal hypothesis that increasing intestinal RA would ame confidence interval was performed in Prism (Graphpad) in liorate disease. RA was administered intraperitoneally (i.p.) all experiments unless otherwise stated. Kaplan-Meier sur twice weekly for 6 weeks to APCMin/+ mice, using the vival curves were analyzed with the log-rank test. Differ same protocol that has been reported to attenuate ileitis in a ences of DAI in the drug treatment studies were analyzed mouse model of Crohn's disease. Surprisingly, compared to using the Wilcoxon Rank-Sum test in R. Where needed, controls, this regimen did not improve disease outcome in meant-SEM was represented on graphs. PK0.05=*; p<0. terms of tumor frequency, body weight or hematocrit. Con 001=**, p<0.0001=***. sistent with these findings, SI-LPDCs isolated from APC '"" mice that received RA i.p. retained their proinflamma Example 2 tory phenotype and ability to induce the formation of Th17 cells. A likely explanation for this apparent paradox is that Drug Treatment of Inflammatory Bowel Disease RA i.p. did not increase RA levels in the ileum, due to persistent breakdown of RA from upregulated CYP26A1 in 0119 Inflammatory DCs that infiltrate the GALT during APC" tissue. Indeed, direct RA supplementation via the autoimmune colitis appear to amplify Th17 responses that diet has been shown to stimulate tumor formation in the exacerbate intestinal pathology, similar to our observations APC model. in the APC' model of intestinal cancer. To address 0.124 Given these results, we decided to try an alternative whether the loss of local RA as observed in our studies strategy to increase intestinal RA by targeting the upregu extended beyond APC-associated GI cancers, we quantified lated CYP26A1 with Liarozole, an inhibitor of this enzyme. endogenous tissue retinoids as before in TNF^' and Compared to untreated and RA i.p.-treated mice, Liarozole azoxymethane/dextran-sodium-Sulphate (AOM/DSS)- treated mice exhibited a striking reduction in tumor number treated mice. A model of Crohn's disease that exhibit in the jejunum and ileum (FIG. 14a), as well as a substantial chronic ileitis, TNF^' mice provide a model to examine increase in body weight (FIG. 14b), and hematocrit (FIG. whether there was a similar RA deficiency in cases of 14c). Importantly, Th17 cells were reduced to WT levels generalized chronic intestinal inflammation. The AOM/DSS (FIG. 14e), and SI-LPDCs from these mice failed to induce carcinogen and mucosal injury-induced model of colorectal Th17 cells, instead promoting the formation of IL-10-se carcinogenesis provided another model of colorectal cancer, creting CD4+ T cells (FIG. 14f). Moreover, RA in the ileum which is chemically-induced instead of spontaneously-aris of Liarozole-treated mice was restored to WT levels (FIG. ing, like the APC'". Intriguingly, compared to WT tissue, 145d), likely explaining the therapeutic effect. Consistent we observed a marked reduction in RA in both the ileum and with this interpretation, treatment of APC" mice with US 2017/O 189384 A1 Jul. 6, 2017

Talarozole, a more potent and more specific CYP26A1 CD103T subsets to assess the role of each subset in the inhibitor than Liarozole38, also ameliorated disease (FIG. observed impairment in Foxp3 induction. APC' CD103" 15). SI-LPDCs were four-fold less efficient at inducing Foxp3"T 0.125 Taken together, these findings indicate that whereas cells compared to their WT counterparts. In contrast, increasing the RA deficit in the intestine exacerbates disease, cD103. SI-LPDCs from both genotypes were equally poor restoring RA to normal levels reverses the pro-inflammatory at inducing Foxp3 expression. Both CD103 and CD103" phenotype of SI-LPDCs, attenuates Th17-driven inflamma APC" SI-LPDCs induced more Th17 cells compared to tion and ameliorates disease. the WT SILPDC Subsets. These results show that the CD103* subset of SILPDCs in APC' mice is responsible Example 3 for the observed defect in TReg induction, and that both CD103 and CD103* SI-LPDCs likely contribute to the Inflammatory DCs Accumulate in the SI-LP of Th17-skewed inflammation observed in these mice. APC'" Mice and Promote Th17 Formation and 0.130. To directly assess the role of DCs in tumor pro Tumor Growth gression, we generated bone marrow (BM) chimeras to 0126 The frequency of DCs in various tissues was com selectively and constitutively ablate DCs using BM donor pared in APC" mice and their WT littermate controls. cells in which Diphtheria Toxin A (DTA) is activated in DCs (PI-EpCAMCD45"LinCD11'MHCII) were ana CD11c expressing cells. Chimeras generated with WT and lyzed at 10, 14 and 18 weeks of age, which correspond to APC' BM were used for comparison. Depletion of DCs early- (<30 adenomas, <2 mm diameter), intermediate-(30 resulted in a significant decrease in total tumor number, due 60 adenomas, 0.5-4 mm diameter) and late-stage (>60 mainly to a marked tumor reduction in the ileum where the adenomas, 1-6 mm diameter) disease, respectively. DCs incidence of tumors in APC" mice is highest, providing accumulated in the SI-LP as disease progressed and by strong evidence that DCs are a key driver of tumor devel intermediate stage the frequency of SI-LPDCs was more opment. In contrast, APC' mice reconstituted with WT than 3 times greater in APC'" than WT mice. BM had similar numbers of tumors compared to control 0127. In the steady state, DCs in the gut consist of three APC' BM-reconstituted APC' mice, suggesting that phenotypically distinct populations: CD103"CD11b, the intestinal environment of the APC' host overrides any CD103*CD11b and CD103CD11b DCs. Although there potential benefit afforded by a reconstituted WT immune were some differences in the frequencies of splenic DC system. subsets between APC" and WT control mice, no signifi Example 4 cant differences in the percentages of the three main DC subsets were observed in the mesenteric lymph nodes Tissue Histology (mLN)/Peyer's Patches (PP) or SI-LP. These analyses were performed at intermediate-stage, as APC" mice lose their 0131 Immunohistochemical and immunofluorescence Peyer's Patches by late-stage disease. Further studies of staining of intestinal biopsies from FAP and APC" mice SI-LPDCs from APC" mice revealed that, although their shows the presence of identical markers of inflammation as expression of costimulatory molecules was similar to that of well as abnormal RA metabolism, including Raldh1a1, WT SI-LPDCs, they secreted much higher levels of the Raldh1a2 and CYP26A1. These findings support the view pro-inflammatory cytokines TNFO, IL-6 and IL-12p40 that the underlying pathology in APC" disease is similar to under basal conditions and in response to a panel of Toll-like that of FAP. Tumors in the APC" model are primarily in the receptor agonists. Moreover, APC" SI-LPDCs induced small intestine, whereas the tumors in FAP are primarily in fewer Foxp3" TRegs in a conventional TReg induction the colon. One reason for this difference is that the mouse assay. This impairment was observed at intermediate-stage colon has very few DCs, while DCs are abundant in the and became even more apparent at late-stage disease. Simi human colon. Drugs that work in APC" mice also work in lar results were obtained across a 25-fold range of peptide FAP providing additional evidence that the pathogenesis of concentrations. Splenic DCs from both genotypes weakly disease is the same in the two species. induced Foxp3" T cells, while mLN/PP DCs were more 0.132. Patients with ulcerative colitis, a type of inflam potent inducers of Foxp3' T cells, consistent with previous matory bowel disease, are at greatly increased risk for studies. developing colon cancer due to the presence of chronic I0128 Supernatants obtained from APC" SI-LPDC-T inflammation. Once regions of microscopic dysplasia are cell co-cultures contained a striking six-fold reduction in identified in the colons of Such patients, the colons are IL-10 compared to WT SI-LPDC co-cultures. Moreover, typically removed because dysplasia almost always there was a concomitant and similarly dramatic increase in becomes cancer. IL-17A, a key cytokine involved in adenoma development. 0.133 Based on staining data, the same abnormalities in Since Th17 differentiation requires IL-6 in addition to TGFB retinoic acid metabolism that were found in APC" mice, and no exogenous IL-6 was added to our cultures, we patients with FAP and mice with experimentally induced measured IL-6 in co-culture Supernatants and, as expected, colitis and Crohn's disease were present in the dysplastic there were larger amounts in APC'". SI-LPDC co-cul areas of patients with ulcerative colitis. tures. Consistent with these findings, IL-23, known to be 1-12. (canceled) essential for the maintenance of Th17 cells, though not 13. A method of reducing polyp development and/or detectable by ELISA, was expressed at higher levels in tumor burden in an individual who has a mutation in the APC' SI-LPDCs compared to WT cells in situ. adenomatous polyposis coli (APC) gene, the method com 0129. As CD103* SI-LPDCs are the main cells respon prising: sible for generating immune tolerance in the intestinal administering to the individual an effective dose of an environment, we sorted SI-LPDCs into CD103* and agent to reduce polyp development and/or tumor bur US 2017/O 189384 A1 Jul. 6, 2017

den in said individual, wherein the agent increases local 22. The method of claim 13, wherein the agent is Liaro concentration of retinoic acid (RA). Zole or Talarozole. 14. The method of claim 13, wherein the agent is not R.A. 23. The method of claim 13, wherein said administering 15. The method of claim 14, wherein the agent increases comprises oral administration. local concentration of RA through modifying enzymatic 24. The method of claim 13, wherein the individual has pathways involved in RA metabolism. familial adenomatous polyposis (FAP). 16. The method of claim 15, wherein the agent increases 25. The method of claim 15, wherein said administering activity of retinaldehyde dehydrogenase or retinol dehydro comprises oral administration. genase in intestinal tissues. 26. The method of claim 15, wherein the individual has 17. The method of claim 16, wherein the agent inhibits familial adenomatous polyposis (FAP). activity of a retinoic acid catabolizing enzyme. 27. The method of claim 18, wherein said administering 18. The method of claim 17, wherein the retinoic acid comprises oral administration. catabolizing enzyme is a protein of the CYP26 family. 28. The method of claim 18, wherein the individual has 19. The method of claim 18, wherein the retinoic acid familial adenomatous polyposis (FAP). catabolizing enzyme is CYP26A1 29. The method of claim 20, wherein said administering 20. The method of claim 19, wherein the agent comprises comprises oral administration. one or more of liarozole, talarozole, ketoconazole, S 30. The method of claim 20, wherein the individual has (R*,R)—N-4-2-(dimethylamino)-1-(1H-imidazole-1-yl) familial adenomatous polyposis (FAP). propyl-phenyl-2-benzothiazolamine (R116010), and (R)— 31. The method of claim 22, wherein said administering N-4-2-ethyl-1-(1H-1,2,4-triazol-1-yl)butylphenyl-2- comprises oral administration. benzothiazolamine (R115866). 32. The method of claim 22, wherein the individual has 21. The method of claim 20, wherein the agent is Liaro familial adenomatous polyposis (FAP). Zole or Talarozole. k k k k k