WO2020222109A1

WO2020222109A1 - Csf-1/csf-1r gene set

Info

Publication number: WO2020222109A1
Application number: PCT/IB2020/053951
Authority: WO
Inventors: Carl L. Manthey; Stanley M. Belkowski; Matthew J. LOZA; Beverley A. Moore
Original assignee: Janssen Biotech, Inc.
Priority date: 2019-05-02
Filing date: 2020-04-27
Publication date: 2020-11-05

Abstract

The disclosure is related to a CSF-1/CSF-1R gene set and methods for using the same in disease treatment and diagnostics.

Description

CSF-1/CSF-1R GENE SET

FIELD

The disclosure is related to a CSF-1/CSF-1R gene set and uses of the same in disease treatment and diagnosis.

BACKGROUND

Cells of the macrophage lineage are present constituently in all bodily tissues where they mediate multiple processes in both health and disease. In general,“resident” tissue macrophages function as sentinels to defend against foreign bodies and pathogens, and they contribute to tissue homeostasis. However, during inflammatory events, macrophage numbers can increase 3- to 4-fold. Secreted mediator profiling indicates these macrophages exhibit a pro-inflammatory phenotype, producing tumor necrosis factor alpha (TNFa), IL-1, IL-6, and IL-23, and are capable of activating T-cells (MacDonald TT, Monteleone I, Fantini MC, et al. Regulation of homeostasis and inflammation in the intestine. Gastroenterology 2011 ; 140: 1768-75.) These cytokines and T cell activation are validated targets in human disease.

Although many ligands/receptors regulate macrophage-lineage proliferation and differentiation, among these, the colony stimulating factor 1 receptor (CSF-1R) and its ligands, CSF-1 (also known as macrophage colony-stimulating factor (M-CSF)) and interleukin-34, are prominent. In mice, genetic deletion of CSF-1R results in marked to moderate reductions in macrophage-lineage cells in most tissues examined, together with marked reduction in

Langerhans cells and partial reductions in some dendritic cell populations (Dai XM, Ryan GR, Hapel AJ, et al. Blood 2002;99: 111-20.). A similar, albeit milder, phenotype occurs in mice with CSF-1 -deficiency, while IL-34-deficient mice exhibit selective reductions in Langerhans and microglial cells (Wang Y, Szretter KJ, Vermi W, et al. Nat Immunol 2012;13:753-60.).

Although CSF-1 is expressed at homeostatic levels in healthy tissues, expression by stromal cells may be induced by TNFa and IL-1 (Hamilton JA, Filonzi EL, Ianches G. Growth Factors 1993;9: 157-65.). Moreover, some T cells may also express CSF-1 upon antigen stimulation (Fontana MF, de Melo GL, Anidi C, et al. PLoS Pathog 2016;12:el006046.). These data are consistent with observations that CSF-1 mRNA was one of a subset of cytokine mRNAs elevated in both Crohn’s and UC mucosa (West NR, Hegazy AN, Owens BMJ, et al. Nat Med

2017;23:579-589.). There remains a need to identify and characterize genetic surrogates of the biological pathways activated in CSF-1 R mediated diseases or disorders that may be used as targets for drug development and for patient stratification.

SUMMARY

The disclosure provides, at least in part, a gene set representative of CSF-1/CSF-1R activation and its use in the treatment, prognosis, and diagnosis of multiple diseases. Several of the various features of the invention will be described hereinafter. It is to be understood that the invention is not limited in its application to the details set forth in the following embodiments, claims, description and figures. The invention is capable of other embodiments and of being practiced or carried out in numerous other ways.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 : A schematic of GS-MISS.

FIG. 2: Colon mucosa biopsy microarray dataset (GSE16879) was submitted to GSVA using a CSF-1 induced gene set (A) and a TNFa induced gene set (B). The colon mucosal biopsies were from healthy controls (clear boxes) or from subjects with CD before (cross-hatched boxes) and after (grey boxes) infliximab therapy.

FIG. 3: GS-MISS of CSF-1/CSF-1R gene set in CD responders and non-responders before and after treatment with infliximab and compared to healthy control.

FIG. 4: Clinical and histopathology scores for T cell transfer colitis studies comparing CSF1R-I to vehicle. CNTO5048 (anti-TNFa) served as a positive control. (A) change in body weight; (B) colon weight/length ratio; (C) Sum of histological scores. (D) Estimated fraction of cellular infiltrate that were neutrophils. Naive control group, n=4 (Study #1) or n=8 (Study #2). PBS group, n=8. All remaining groups, n=10. *^#P<0.05 relative to naive or vehicle control, respectively. #, p<0.5; ##, p<0.01; *** or ###, p<0.001; **** or ####, p<0.0001.

FIG. 5: Individual Components of Sum of Scores

FIG. 6: Colonic mucosa from mice with T cell transfer-induced colitis was stained for F4/80 (A) and CD3 (B) antigens. Data are positive pixels determined by digital imaging of three sections per mouse per colonic region. Naive control group, n=6. All remaining groups, n=10. *P<0.05 relative to respective vehicle control for either proximal or distal colon.

FIG. 7: CSF-1/CSF-1R gene set showed excellent directional concordance between the TCT model and the human Crohn’s dataset.

FIG. 8: Gene set variation analyses (GSVA) of T cell transfer colitis mucosal transcriptome using mouse homologues of the human CSF-1 (A) and TNFa (B) induced gene sets. Naive and isotype controls, n=6. All remaining groups, n=8.

FIG. 9: Gene set variation analyses (GSVA) of T cell transfer colitis mucosal gene set using gene sets enriched in M1 macrophages (A), M0 resident macrophages (B) and activated Th1 T cells (C). Naive and isotype controls, n=6. All remaining groups, n=8.

FIG. 10: M1 inflammatory macrophage gene set.

FIG. 11 : M0 macrophage gene set.

FIG. 12: Th1 lymphocyte gene set.

FIG. 13: IPA pathway analysis (2018) of CD and T cell transfer colitis datasets: Values are Z scores. Pathways with P £0.01 and Z>2.7 in any one of the four comparisons were included in the table.

FIG. 14: CSF-1/CSF-1R gene set GS-MISS identifies ulcerative colitis (UC) as a disease with increased CSF-1 /CSF-1 R pathway activity and in need of CSF-1 R inhibition.

FIG. 15: CSF-1/CSF-1R gene set GS-MISS correlates with response to infliximab in UC and CD.

DETAIL D DESCRIPTION

Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Generally, nomenclatures used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those known and commonly used in the art. Enzymatic reactions, protein expression and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects. That the disclosure may be more readily understood, select terms are defined below.

Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A," (alone) and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

"About" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result or embodiment, "about" means within one standard deviation from the mean per the practice in the art, or a range of up to 10%, whichever is larger. About may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The term“antibody” includes, but is not limited to a protein, or polypeptide sequence, traditionally derived from an immunoglobulin molecule, and which specifically binds with an antigen. Antibodies may be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources.

Antibodies may be tetramers of immunoglobulin molecules. Also included are, for example, domain antibodies (dAbs), diabodies, camelid antibodies and engineered camelid antibodies, asymmetric IgG-like antibodies (e.g., triomab/quadroma, Trion Pharma/Fresenius Biotech; knobs-into-holes, Genentech; Cross MAbs, Roche; electrostatically matched antibodies, AMGEN; LUZ-Y, Genentech; strand exchange engineered domain (SEED) body, EMD Serono; biolonic, Merus; and Fab-exchanged antibodies, Genmab), symmetric IgG-like antibodies (e.g., dual targeting (DT)-Ig, GSK/Domantis; two-in-one antibody, Genentech; crosslinked MAbs, karmanos cancer center; mAb², F-star; and Cov X-body, Cov X/Pfizer), IgG fusions (e.g., dual variable domain (DVD)-Ig, Abbott; IgG-like bispecific antibodies, Eli Lilly; Ts2Ab,

Medimmune/AZ; BsAb, ZymoGenetics; HERCULES, Biogen Idee; TvAb, Roche) Fc fusions (e.g., ScFv/Fc fusions, Academic Institution; SCORPION, Emergent BioSolutions/Trubion, ZymoGenetics/BMS; dual affinity retargeting technology (Fc-DART), MacroGenics; dual (ScFv)₂-Fab, National Research Center for Antibody Medicine) Fab fusions (e.g., F(ab)2,

Medar ex/ AMGEN; dual-action or Bis-Fab, Genentech; Dock-and-Lock (DNL), ImmunoMedics; bivalent bispecific, Biotechnol; and Fab-Fv, UCB-Celltech), ScFv- and diabody-based antibodies (e.g., bispecific T cell engagers (BiTEs), Micromet; tandem diabodies (Tandab), Affimed;

DARTs, MacroGenics; Single-chain diabody, Academic; TCR-like antibodies, AIT, Receptor Logics; human serum albumin ScFv fusion, Merrimack; and COMBODIES, Epigen Biotech), IgG/non-IgG fusions (e.g., immunocytokins, EMDSerono, Philogen, ImmunGene,

ImmunoMedics; superantigen fusion protein, Active Biotech; and immune mobilising mTCR Against Cancer, ImmTAC) and oligoclonal antibodies (e.g., Symphogen and Merus). Any of these types of antibodies may be used to make anti-CSF-1 or anti-CSF-1R antibodies to be used in the methods disclosed herein.

An "antigen” (e.g., CSF-1) refers to a compound, composition, or substance, e.g., lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid, that can stimulate the production of antibodies or a T cell response in an animal, including compositions (such as one that includes a tumor-specific protein) that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited, but excludes prior art embodiments.

The“CSF-1/CSF-1 R gene set” encompasses the following genes: ARHGAP18, ARHGAP22, ATF3, BCAT1, CASP1, CCL8, CCR2, CD14, CD163, CLEC5A, COL7A1, CSF1R, CTSB, DSE, EPB41L3, FBP1, FCN1, FCRLA, FLJ42418, FMNL3, FPR3, FUCA1, fflVEP3, HLA-DPA1, HLA-DRA, HNMT, IFNB1, IQSEC2, KYNU, LAP3, LRPl, MAFB, MARCH 1, MARCKSLl, MERTK, MRAS, MS4A14, MS4A6A, MS4A7, MYOF, NAPSB, OAS1, OLR1, RIN2, SCD, SDS, SEMA6B, SIGLEC1, SIGLEC16, STARD4, TFEC, TGM2, THBS1, TMEM180, VCAN, VSIG4, and ZNF330.

As used herein, the terms "determining", "assessing", "assaying", "measuring" and "detecting" refer to both quantitative and qualitative determinations, and as such, the term "determining" is used interchangeably herein with "assaying," "measuring," and the like. Where a quantitative determination is intended, the phrases "determining an amount" of an analyte and the like may be used. Where a qualitative and/or quantitative determination is intended, the phrase "determining a level" of an analyte or "detecting" an analyte is used.

Any range disclosed herein is intended to encompass the endpoints of that range unless stated otherwise. Ranges provided herein are understood to be shorthand for all the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

42, 43, 44, 45, 46, 47, 48, 49, or 50.

By "reference" on“control” is meant a standard of comparison. The standard may be an established method in the art. A control reference method is a reference method in which all of the parameters are identical to those of the method being compared with exception of the variable being tested. It may also be the average value for the parameter being measured from what is typically used or known in the art.

It should be noted that the "responsiveness" of a subject to a CSF-1, CSF-1R, or TNF- alpha inhibitor refers to the success or failure of treatment of the subject with the CSF-1, CSF- 1R, or TNF-alpha inhibitor.

The term“response/respond” to a CSF-1, CSF-1R, or TNF-alpha inhibitor refers to an improvement following treatment with the CSF-1, CSF-1R, and/or TNF-alpha inhibitor in at least one relevant clinical parameter as compared to an untreated subject diagnosed with the same pathology ( e.g the same type, stage, degree and/or classification of the pathology), or as compared to the clinical parameters of the same subject prior to treatment with the TNF-alpha inhibitor. Hence, improvement of clinical symptom(s) following treatment implicates that the subject is a "responder" to the treatment. On the other hand, a negative response to the treatment with the CSF-1, CSF-1 R, or TNF-alpha inhibitor means that the subject has no sufficient improvement in clinical symptoms, or has a complete lack of improvement of clinical symptoms, or has a worsening of clinical symptoms characterizing the pathology (the IBD condition), with or without appearance of antibodies ( e.g ., antibody against infliximab) which neutralize the CSF- 1, CSF-1R, or TNF-alpha inhibitor. Such a subject is a "non-responder" to the treatment.

The term“subject” as used herein includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles.

As used herein, the terms "treat," "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and/or duration of a disorder, e.g., a proliferative disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of the disorder resulting from the administration of one or more therapies. In some embodiments, the wherein the one or more symptoms ameliorated are selected from the group consisting of: weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, distended or painful abdomen, bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination. In specific embodiments, the terms "treat," "treatment" and "treating" refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In other embodiments the terms "treat", "treatment" and "treating" refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g. , stabilization of a physical parameter, or both. In other embodiments the terms "treat", "treatment" and "treating" refer to the reduction or stabilization of tumor size or cancerous cell count.

IDENTIFICATION OF A CSF-1 INDUCIBLE GENE SET

In one embodiment, to enable estimation of CSF-1R-driven biology in disease datasets, a CSF-1 -inducible gene set was derived consisting of fifty-seven genes induced >2-fold by ex vivo treatment of whole human blood cultures with CSF-1. In one embodiment, the disclosure provides a newly discovered CSF-1 -inducible gene set, which is also referred to herein as a CSF-1 gene set. In one embodiment, the members of this gene set are considered to encode mRNAs whose expression is related to the activation of CSF-1 R by CSF-1. In one embodiment, the gene set as a group represents an aspect of a cell’s biology that characterizes the cellular transcriptional component specific to CSF-1R activation by CSF-1. In one embodiment, the expression pattern of the gene set represents engagement of CSF-1/CSF-1R signaling.

In one embodiment, the CSF-1 gene set is comprised of 57 genes (ARHGAP18,

ARHGAP22, ATF3, BCAT1, CASP1, CCL8, CCR2, CD14, CD163, CLEC5A, COL7A1, CSF1R, CTSB, DSE, EPB41L3, FBP1, FCN1, FCRLA, FLJ42418, FMNL3, FPR3, FUCA1, HIVEP3, HLA-DPA1, HLA-DRA, HNMT, IFNB1, IQSEC2, KYNU, LAP3, LRP1, MAFB, MARCH 1, MARCKSLl, MERTK, MRAS, MS4A14, MS4A6A, MS4A7, MYOF, NAPSB, OAS1, OLR1, RIN2, SCD, SDS, SEMA6B, SIGLEC1, SIGLEC16, STARD4, TFEC, TGM2, THBS1, TMEM180, VCAN, VSIG4, ZNF330) whose expression has a geometric means >2-fold higher relative to unstimulated control conditions (p<0.05) at either 4 or 24 hours post treatment with CSF-1. In one embodiment, the CSF-1 gene set consists essentially of the recited 57 genes. The 57 gene CSF-1-induced gene set described herein is novel and includes genes not previously known to be regulated by CSF-1 [e.g., DSE, KYNU, MS4A6A, MS4A7, and MYOF.

In one embodiment, this gene set is referred to as the CSF-1-CSF-1R gene set or CSF-1 gene set. As explained above, the disclosure provides that this gene set is a surrogate for activation of CSF-1-driven biology, or activation of CSF-1/CSF-1R signaling.

In one embodiment, using gene set variation analysis (GSVA), the CSF-1-induced gene set was determined to be highly enriched in a previously published microarray dataset generated from human Crohn’s Disease mucosal biopsies. See, e.g, Arijs I, De Hertogh G, Lemaire K, et al. Mucosal gene expression of antimicrobial peptides in inflammatory bowel disease before and after first infliximab treatment. PLoS One 2009;4:e7984. In one embodiment, the same results were obtained with GS-MISS analysis. In one embodiment, Ingenuity Pathway Analysis identified“activation of phagocytes” as the top enriched disease and function category (p = 4x10- ¹⁰) in the gene set. In one embodiment, the disclosure provides a gene set that represents activation of CSF1- induced biology. In one embodiment, the disclosure provides a gene set that represents “activation of phagocytes.” too.

METHODS OF IDENTIFYING QUANTITATIVE AND QUALITATIVE CHANGES IN THE EXPRESSION OF THE CSF-1/CSF-1R GENE SET

In one embodiment, the disclosure provides for methods of measuring the expression level of the gene set in vitro. The gene expression level may be determined by any method known by the skilled person. In one embodiment, the expression levels are measured at the mRNA level. Methods for determining the quantity of mRNA are well known in the art and include, but are not limited to, quantitative or semi-quantitative RT-PCR, real time quantitative or semi-quantitative RT-PCR, Nanostring technology, microarray and sequencing based approaches

In one embodiment, the nucleic acids contained in a sample ( e.g ., cells or tissue prepared from the patient) may be first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. These nucleic acids may be frozen to be stored before use. The extracted messenger RNA (mRNA) may be then detected by hybridization and/or amplification (e.g., RT-PCR). Quantitative or semi-quantitative RT-PCR is preferred. Other methods of mRNA amplification include, but are not limited to, ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA). In one embodiment, the quantity of mRNA is measured by quantitative RT-PCR.

In some embodiments, the quantity of mRNA in a sample may also be measured using the Nanostring's NCOUNTER™ Digital Gene Expression System (Geiss et al. 2008 Nat.

Biotechnol. 26:317-325) which captures and counts individual mRNA transcripts by a molecular bar-coding technology and is commercialized by Nanostring Technologies, or the QuantiGene® Plex 2.0 Assay (Affymetrix).

In a preferred embodiment, the quantity of mRNA may be determined using approaches based on high-throughput sequencing technology such as RNA-Seq (Wang et al. Nat Rev Genet. 2009 January; 10(1): 57-63) or sequencing technologies using microfluidic systems. In another embodiment, the expression level of a gene may also be determined by measuring the quantity of mRNA by transcriptome approaches, in particular by using DNA microarrays. To determine the expression level of a gene, the sample, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and may be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art. Examples of DNA biochips suitable to measure the expression level of the genes of interest include, but are not limited to, Human Genome U133 Plus 2.0 array (Affymetrix).Next Generation Sequencing methods (NGS) may also be used.

GENE EXPRESSION SCORES

In one embodiment, an expression score for the CSF-1/CSF-1R gene set is calculated by Gene set median individual scoring system (GS-MISS). In one embodiment, an expression score for the CSF-1/CSF-1R gene set is calculated by a gene set variation analysis (GSVA).

Gene set median individual scoring system (GS-MISSj

GS-MISS is a method that assigns to each individual sample a score based on the median relative expression (or change in expression) of a set of genes in a specified gene signature. The GS-MISS scores could be calculated for evaluation of either levels of a gene signature pre- treatment (e.g., for prediction of response to a specific treatment) or pharmacodynamic changes in the gene signature after an intervention (e.g., to determine whether the treatment is biologically effective in suppressing the target of the treatment). The GS-MISS is amenable to qPCR-based approach, not requiring genome-wide data (e.g., microarray, RNAseq) for calculation of enrichment scores of the gene signature relative to all other genes. A schematic illustrating how GS-MISS can be applied to evaluate pharmacodynamic changes is presented in FIG. 1.

GS-MISS for CSF1R-inhibitor pre-treatment evaluations.

In one embodiment, for evaluation of pre-interventional levels of the CSF-1/CSF-1R signature, the GS-MISS score may be calculated for each patient- visit sample by: Calculating the ratio of baseline gene expression in the patient sample over the

(previously established constant for) geometric mean of gene expression in a healthy control population, for each gene in the signature for a given patient sample;

Scoring each individual sample with log₂-transform of median of gene expression ratios among the genes in the signature.

In one embodiment, for evaluation of levels of the CSF-1/CSF-1R gene signature in clinical studies or in clinical settings before initiating treatment with a CSF1R- inhibitor, the 57 genes in the gene set, or a minimal subset of genes in the gene set, may be evaluated by qPCR- based approaches or RNAseq to establish the expression levels of the genes. The constant for geometric mean of gene expression in a healthy control population would be previously established.

In another embodiment, a pooled set of RNA samples from healthy control population may be run as a standard during the qPCR (or RNAseq) evaluation of the patient sample, with the expression value used as the geometric mean of gene expression in a healthy control population.

In one embodiment, when evaluating the score before treatment and the GS-MISS score is at least 0.265 points above the established healthy control constant (2^GS-MISS>1.20; i.e., >20% higher), then this would suggest that CSF-1 activity is elevated compared to that observed in a healthy population. In one embodiment this result may indicate that a CSF-1 R inhibitor may be warranted for treatment. The exact criteria may be set at different thresholds depending on experience, e.g., the extent of dysregulation of the gene signature in the specific disease of interest compared to demographically-matched healthy control subjects. In other embodiments, the GS-MISS score is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 300% above the established healthy control constant, relative to the value corresponding to the geometric mean observed for a healthy control set of samples.

In one embodiment, when evaluating the score after treatment with a TNF inhibitor, if the GS-MISS score remains at least 0.265 points above the established healthy control constant (2^GS-MISS> 1.20; i.e., >20% higher), then this would suggest that CSF-1 activity remained despite treatment and that a CSF-1 R inhibitor may be warranted for next line treatment. The exact criteria may be set at different thresholds depending on experience, e.g., the extent of dysregulation of the gene signature in the specific disease of interest compared to demographically-matched healthy control subjects.

GS-MISS for pharmacodynamic evaluations

If the molecular fingerprint of a therapeutic target (here, represented as the CSF-1/CSF- 1R gene set) at a relevant site of disease pathology is not normalized (i.e., returned from a dysregulated state towards normal expression levels) upon treatment with e.g., a CSF-1Ri or a TNFi, then it may be that there has either not been biologically sufficient engagement of target; or engagement of the target is not sufficient per se to modify disease. In one embodiment, normalization of fingerprint may translate to positive clinical efficacy. One of the utilities of this approach is to triage assets that are less likely to work, while retaining those that pass for evaluation in proof-of-concept trials.

In another embodiment, gene set median individual scoring system (GS-MISS) is a method that assigns to each individual sample a score based on the median relative expression (or change in expression) of a set of genes in a specified gene signature. In one embodiment, for evaluation of pharmacodynamics, the GS-MISS score is calculated for each subject-visit sample by:

Calculating the ratio of week X/baseline gene expression for each gene in the signature for a given subject, for each subject-visit sample;

Score each individual sample with log₂-transform of median of gene expression ratios among the genes in the signature.

In one embodiment, this approach is used in clinical trial setting to evaluate whether a CSF-1R inhibitor has effectively modulated CSF-1R biological activity. In one embodiment, statistical comparisons are performed between active and placebo treatment groups using GS- MISS scores as the outcome using the appropriate statistical test. Alternatively, if comparison to a placebo group is not to be performed, a one-sample t-test vs. 0 could be performed to evaluate significance of treatment effect. In one embodiment, general minimal passing criteria would be p<0.05 and GS-MISS>0.26 (2^GS-MISS>= 1.2, i.e., 20% difference), though criteria may be set at different thresholds depending on experience, e.g., extent dysregulation of the gene signature in the disease of interest compared to demographically-matched healthy control subjects. OTHER METHODS FOR CALCULATING GENE EXPRESSION SCORES

Any bioinformatic method known in the art may be used to analyze the gene expression data for the disclosed CSF-1 gene set. In one embodiment, the method comprises gene set enrichment (GSE) analysis, where a set of genes may be characterized between two groups. In one embodiment, the GSE comprises Gene Set Variation Analysis (GSVA). Gene Set Variation Analysis (GSVA) is a non-parametric, unsupervised method that estimates the relative enrichment of a gene set of interest across a sample population. Hence, it allows one to observe the variation in the activity of a set of genes such as a pathway or a gene signature,

corresponding to a particular biological condition, within an entire gene expression set, which may be defined a priori. It produces a value, termed enrichment score (ES), per sample and gene set, which may be examined for associations with clinical features of interest. Hanzelmann S, Castelo R, Guinney, JBMC Bioinformatics. 2013 Jan 16;14:7. doi: 10.1186/1471-2105-14-7, incorporated herein by reference.

Non-limiting examples of other methods that can used to assess CSF-1/CSF-1R gene set/transcriptome expression levels include, without limitation, FuncAssociate, PANTHER, ErmineJ, Pathway-Express, GoMiner, Enrichr, WebGestalt, DAVID, ConceptGen, FIDEA, Ingenuity Pathway Analysis (IPA), Reactome enrichment analysis, PAGE, and LRpath.

METHODS OF:

Measuring Increased Activation or Engagement of CSF-1/CSF-IR Signaling in a Tissue;

and

Use of the CSF-1/CSF-1R Induced Gene Set To Identify Diseases Where CSF-1 Driven

Biology Is Strong

In one embodiment, the disclosure provides a method of measuring increased activation or engagement of CSF-1 /CSF-1R signaling in a tissue comprising:

processing mRNA from a tissue sample through a gene expression system measuring the expression of a set of genes comprising, consisting, or consisting essentially of ARHGAP18, ARHGAP22, ATF3, BCAT1 CASP1 , CCL8, CCR2, CD14, CD163, CLEC5A, COL7A1 , CSF1 R, CTSB, DSE, EPB41 L3, FBP1, FCN1, FCRLA, FLJ42418, FMNL3, FPR3, FUCA1 , HIVEP3, HLA-DPA1, HLA-DRA, HNMT, IFNB1, IQSEC2, KYNU LAP3, LRP1, MAFB MARCH1, MARCKSL1 , MERTK, MRAS, MS4A14, MS4A6A, MS4A7 MYOF, NAPSB, OAS 1 , OLR1 , RIN2, SCD, SDS, SEMA6B, SIGLEC1 , SIGLEC16, STARD4, TFEC, TGM2, THBS1 , TMEM1 80, VCAN, VSIG4, and ZNF330; and

calculating a gene expression score, wherein an increase in the expression score in the tissue sample relative to the expression score in a control tissue, or a control score, indicates increased activation or engagement of CSF-1/CSF-1R signaling in the tissue.

In one embodiment of this method, the expression level of all genes in the gene set (57 genes described below) is combined into an enrichment score by a gene set median individual scoring system (GS-MISS). In one embodiment of this, the expression level of all genes in the gene set is combined into a gene set variation analysis (GSVA) expression score. In one embodiment of this method, the enrichment score is increased by at least 0.265 (20% increase), at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 300%, relative to the value corresponding to the geometric mean observed for a healthy control set of samples.

The“CSF-1/CSF-1R gene set" encompasses the following genes:

ARHGAP18, ARHGAP22, ATF3, BCAT1, CASP1, CCL8, CCR2, CD14, CD163, CLEC5A, COL7A1, CSF1R, CTSB, DSE, EPB41L3, FBP1, FCN1, FCRLA, FLJ42418, FMNL3, FPR3, FUCA1, fflVEP3, HLA-DPA1, HLA-DRA, HNMT, IFNB1, IQSEC2, KYNU, LAP3, LRP1, MAFB, MARCH 1, MARCKSL1, MERTK, MRAS, MS4A14, MS4A6A, MS4A7, MYOF, NAPSB, OAS1, OLR1, RIN2, SCD, SDS, SEMA6B, SIGLEC1, SIGLEC16, STARD4, TFEC, TGM2, THBS1, TMEM180, VC AN, VSIG4, and ZNF330. In one embodiment, the measured genes consist of, or consist essentially of, ARHGAP18, ARHGAP22, ATF3, BCAT1, CASP1, CCL8, CCR2, CD14, CD163, CLEC5A, COL7A1, CSF1R, CTSB, DSE, EPB41L3, FBP1, FCN1, FCRLA, FLJ42418, FMNL3, FPR3, FUCA1, HIVEP3, HLA-DPA1, HLA-DRA, HNMT, IFNBl, IQSEC2, KYNU, LAP3, LRP1, MAFB, MARCH 1, MARCKSL1, MERTK, MRAS, MS4A14, MS4A6A, MS4A7, MYOF, NAPSB, OAS1, OLR1, RIN2, SCD, SDS, SEMA6B, SIGLEC1, SIGLEC16, STARD4, TFEC, TGM2, THBSl, TMEM180, VC AN, VSIG4, and ZNF330.

In one embodiment of this method, wherein the control tissue is obtained from non- lesional tissue matching lesional tissue from the same individual. In one embodiment of this method, wherein the control is healthy skin for a skin test sample or healthy mucosa for a colon mucosa test sample from the same individual. In one embodiment of this method, the controls are a collecti on of healthy tissues or a historic reference from that tissue from a population of healthy individuals or pooled samples of healthy tissue from a population of healthy individuals, the same tissue before treatment, and the like.

In one embodiment of this method the tissue comprises colon tissue, preferably colonic mucosa. In one embodiment of this method the indi vidual is a mammal, preferably a human.

In one embodiment, the invention provides a method of assessing activation or engagement of CSF-1/CSF-1R-driven biology, activation of CSF-1/CSF-1R-induced signaling, or enrichment in CSF-1/CSF-1R-response genes, in a disease or disorder by measuring the expression of the CSF-1 gene set genes in a sample comprising diseased tissue and a sample of a corresponding healthy tissue. All of these assessments are examples of the functional significance of the CSF-1/CSF-1R gene set, and these terms are used interchangeable throughout this specification. In one embodiment, diseases in which there is“activation of the gene set” (e.g. detection of a relative increase in the gene set signal relative to a reference) are diseases in which CSF1 -driven biology is strong or significantly engaged. In one embodiment, identification of this biology in a particular disease is helpful to the development of new drug targets for treatment. In one embodiment, the terms“activation of the gene set,”“increase/decrease in gene set expression,” and enrichment, elevation, increased expression of, increase in enrichment score, all mean that the gene set enrichment score (e.g., Gene set median individual scoring system (GS- MISS) score, or

GSVA) is increased or decreased relative to the score in a reference sample. In one embodiment,“activation of the gene set” means at least a 0.2 unit increase in the expression score for the gene set in the tissue, relative to a control, baseline level understood to represent baseline CSF-1 signaling. In other embodiments, there is at least a 0.4, at least a 0.5, at least a 0.6, at least a 0.7, at least a 0.8, at least a 0.9, at least a 1, at least a 5, at least a 10, or at least a 100 unit increase.

The disclosure provides that the term“CSF-1/CSF-1R biology is involved or is strong” in a tissue means that cellular processes and pathways involving CSF-1/CSF-1R signaling are more active or engaged in the tissue than in a reference control. The CSF-1/CSF-1R gene set represents a particular subset of genes from the cellular transcriptome whose expression is increased at least two-fold by CSF-1 stimulation of healthy whole blood. The disclosure provides that, if these particular genes are responsive to CSF-1 stimulation, they likely are involved in CSF-1 signaling or CSF-1 related biology.

In one embodiment, the measured genes comprise a combination of any two or more of ARHGAP18, ARHGAP22, ATF3, BCAT1, CASP1, CCL8, CCR2, CD14, CD163, CLEC5A, COL7A1, CSF1R, CTSB, DSE, EPB41L3, FBP1, FCN1, FCRLA, FLJ42418, FMNL3, FPR3, FUCA1, HIVEP3, HLA-DPA1, HLA-DRA, HNMT, IFNB1, IQSEC2, KYNU, LAP3, LRP1, MAFB, MARCH 1, MARCKSLl, MERTK, MRAS, MS4A14, MS4A6A, MS4A7, MYOF, NAPSB, OAS1, OLR1, RIN2, SCD, SDS, SEMA6B, SIGLEC1, SIGLEC16, STARD4, TFEC, TGM2, THBS1, TMEM180, VC AN, VSIG4, and ZNF330. In one embodiment, the measured genes consist of, or consist essentially of, ARHGAP18, ARHGAP22, ATF3, BCAT1, CASP1, CCL8, CCR2, CD 14, CD163, CLEC5A, COL7A1, CSF1R, CTSB, DSE, EPB41L3, FBP1, FCN1, FCRLA, FLJ42418, FMNL3, FPR3, FUCA1, HIVEP3, HLA-DPA1, HLA-DRA, HNMT, IFNB1, IQSEC2, KYNU, LAP3, LRP1, MAFB, MARCH 1, MARCKSLl, MERTK, MRAS, MS4A14, MS4A6A, MS4A7, MYOF, NAPSB, OAS1, OLR1, RIN2, SCD, SDS, SEMA6B, SIGLEC1, SIGLEC16, STARD4, TFEC, TGM2, THBS1, TMEM180, VCAN, VSIG4, and ZNF330. In this context, the term“consist essentially of’ means that other genes may be measured but they do not need to be considered for purposes of assessing the status of the CSF- 1/CSF-1R gene set and do not affect the result or conclusion.

In one embodiment, a greater than two-fold increase in the gene expression level of at least two genes of the CSF-1 gene set in a tissue sample relative to that expression level in a reference control ( e.g ., normal tissue) indicates increased activation of CSF-1/CSF-1R-driven biology or signaling in the sample tissue. In one embodiment, a greater than two-fold increase in the gene expression level of all 57 genes of the CSF-1 gene set in a tissue sample relative to that expression level in a reference control (e.g., normal tissue) indicates increased activation of CSF- 1/CSF-1R-driven biology or signaling in the sample tissue.

In one embodiment, the gene expression measurements are subject to gene set enrichment (GSE) analysis. In one embodiment, the GSE comprises Gene Set Variation Analysis (GSVA).

In one embodiment, the expression levels of the genes in the CSF-1 gene set are converted into a combined score (e.g., an expression score or enrichment score).

In one embodiment, the expression score of the CSF-1/CSF-1R gene set (or

representative subset) is calculated by gene set median individual scoring system (GS-MISS). In one embodiment, an increase in the expression score or enrichment score of a first sample relative to that of a second sample indicates higher activation of CSF-1 induced biology or biological response in the first sample. In one embodiment, the first sample is a colon sample from a subject suspected of having an inflammatory bowel disorder and the second sample is a colon sample from a healthy subject, or an average value for a number of colon samples from healthy subjects.

In one embodiment, the disclosure provides strong evidence of robust activation of CSF- 1/CSF1R- induced biology or activation in Crohn’s disease. CSF-1 -inducible genes from the gene set were similarly enriched in a T cell transfer murine model of colitis and known gene sets representative of cells of the macrophage lineage were enriched in both the model and in human disease.

Ingenuity Pathway Analysis (IPA) analysis of the CSF-1/CSF-1R gene set was done, as was IPA of a TCT colitis disease signature, and a previously described Crohn’s disease signature. In one embodiment, IPA identified“activation of phagocytes” as enriched in the CSF- 1/CSF-1R gene set and strong pathway level concordance between the mouse model and human disease (FIG. 13).

In one embodiment, the activation of the CSF-1/CSF-1R gene set in a tissue sample from a subject may be used to identify the subject as having an above normal/average number of macrophages in the tissue sample. In one embodiment, the activation of the CSF-1/CSF-1R gene set in a tissue sample from a subject may be used to identify the subject as having a macrophage- related disorder. In one embodiment, the activation of the CSF-1/CSF-1R gene set in a tissue sample from a subject may be used to identify the subject as having an inflammatory disease in the tissue from which the sample was obtained. Non-limiting examples of inflammatory and otherwise macrophage-related disorders that may be used together with the disclosed methods are provided below.

In one embodiment, the presence of circulating cells in the blood wherein there is detectable activation of the CSF-1/CSF-1R gene set may be an indicator of inflammation elsewhere in the body. In another embodiment, activation of the CSF-1 /CSF-1R gene set in a tissue sample is an indicator that CSF-1 signaling is or has been stimulated in the tissue. In another embodiment, activation of the CSF-1/CSF-1R gene set in a tissue sample is an indicator that CSF-1 -mediated biology is or has been activated in the tissue. In one embodiment, the tissue sample comprises colon tissue. In one embodiment, the tissue sample comprises colon mucosa. In one embodiment, the tissue sample comprises a blood sample. Virtually any tissue/cell sample comprising mRNA may be used to detect increased activation of the CSF-1/CSF-1R gene set. Additional non-limiting embodiments of samples that may be used to measure increased activation of the CSF-1/CSF-1R gene set include a sample/biopsy from any tissue where inflammation or otherwise another situation of macrophage accumulation is suspected, a blood sample, a sample obtained by blood draw; a sample obtained by finger-stick/prick or heel-prick; a CNS fluid sample; a synovial fluid sample, cells isolated from the subject ( e.g immune cells, cells isolated from cheeks or gums, hair roots); and a sample that is not directly from a subject but is derived from or comprises cells grown and/or processed in vitro.

MARKER OF RESPONSE TO TREATMENT

In one embodiment, changes in the expression/signal of the CSF-1/CSF-1R gene set may be used to monitor a subject’s disease or disorder response to treatment. IPA analysis and functional curation of the gene set revealed the gene set to be highly enriched for genes significant to’’activation of phagocytes”. Consequently, in one embodiment, diseases in which macrophage activation plays a role may be diseases in which monitoring the CSF-1/CSF-1R gene set signal is a means for monitoring treatment response. Non-limiting examples of diseases whose treatment may be monitored by the disclosed methods may be found below at

“METHODS OF TREATMENT WITH CSF-1/CSF-1R INHIBITORS.”

As disclosed above, the CSF-1/CSF-1R gene set of the disclosure is enriched/activated in inflammatory bowel disorders such as colitis and Crohn’s disease. In a mouse model of colitis/Crohn’s disease, a CSF-1R inhibitor (CSF1R-I) effectively restored both the CSF-1/CSF- 1R gene set and macrophage gene sets signals (see EXAMPLES) to baseline. This suppression of the CSF-1/CSF-1R gene set in mice treated with CSF1R-I, further validated the CSF-1/CSF- 1R gene set as a strong indicator of CSF-1 -activated biology.

The reduction in CSF-1/CSF-1R gene set and macrophage signal strength was accompanied by preclinical efficacy in the form of prevented or reversed weight loss, reduced gross pathology (colon weight to length ratios) and reduced histopathology scores based on inflammation and on mucosa hyperplasia, and damage. F4/80 immunohistochemistry was concordant with diminished macrophage gene signal strength. Association with reduced F4/80⁺ macrophages, CD3 staining was reduced as was the enrichment of Th1 T-cell associated gene sets (described in EXAMPLES).

Further, at the individual gene level, the CSF-1/CSF-1R gene set showed excellent directional concordance between the TCT model and the human Crohn’s dataset (FIG. 7). The disclosure provides that the data not only highlight the strength of CSF-1 biology in Crohn’s disease but also the translational value of the CSF-1/CSF-1R gene set for clinical and preclinical pharmacodynamic assessment of CSF-1R inhibitors. In one embodiment, the disclosure provides that although initially blood-derived, the CSF-1-induced gene set may be particularly useful for assessment of pharmacodynamic effect within any other tissues for which such methods were not previously available.

In one embodiment, the level of activation of the CSF-1 /CSF-1 R gene set may be used as a surrogate for disease parameters such reduced histological disease scores, and reduced macrophages and CD3+ lymphocytes. In one embodiment, the alteration of the expression level of the CSF-1/CSF-1R gene set/activation of the gene set in a tissue sample from a subject being treated measured prior to (baseline) and after treatment may be used as a pharmacodynamic parameter for response to the treatment. In one embodiment, the change in expression level of the CSF1 gene set is a surrogate for preclinical efficacy. In one embodiment, the change in expression level of CSF1 gene set is a surrogate for histological disease scores. In one embodiment, the change in expression level is a surrogate for the presence of

macrophages/mononuclear cells and CD3+ lymphocytes.

In one embodiment, the subject is or has been treated with a CSF-1R tyrosine kinase inhibitor and a post- treatment decrease in CSF/CSF-1R gene set activation relative to baseline levels indicates significant attenuation of one or more disease parameters. In one embodiment, the treatment comprises a CSF-1 inhibitor. In on embodiment, the CSF-1R inhibitor is one of the compounds disclosed in US Publication No. 2009/0105296. In one embodiment, the treatment comprises JNJ-40346527.

GSVA performed on colonic mucosa collected from Crohn’s disease patients undergoing Infliximab therapy demonstrated that CSF-1 driven pathways were indeed expressed in human Crohn’s disease, and that the CSF-1 /CSF-1R gene set was resolved in those that showed good clinical response to therapy. A more robust enrichment of the CSF-1/CSF-1R gene set was observed in clinical non-responders prior to treatment with Infliximab, but the signature failed to resolve with treatment. In the mouse colonic mucosa, GSVA analyses indicated that the CSF- 1/CSF-1R gene set was present in the preclinical model of T cell dependent colitis. The gene set was not significantly impacted by anti-TNFa therapy but was normalized following treatment with CSF1R-I along with concomitant amelioration of disease. The disclosure thus provides evidence that multiple mechanisms contribute to disease pathology in inflammatory bowel diseases (IBD), and that enhanced CSF-1/CSF-1R signaling may represent a differential disease mechanism within select IBD patient subsets that may be used for patient stratification. Anti- TNFa therapy reversed those biological process that were altered in disease in both species, and the effect of CSF1R-I in mice was similar to that achieved by anti-TNFa therapy in humans.

The disclosure provides that CSF-1-driven biology is elevated in Crohn’s disease mucosa, even more so in infliximab non-responders. The disclosure also provides that CSF-1 is responsible, at least in part, for increased macrophage numbers and macrophage-driven pathology in tissues.

In one embodiment, the alteration of the expression of the CSF1 gene set in a tissue sample from a subject being treated measured prior to and after anti-TNFa treatment may be used as a pharmacodynamic parameter for response to the treatment.

In one embodiment, the subject is or has been treated with an anti-TNFa treatment and a post-treatment decrease in CSF-1/CSF-1R gene set activation relative to baseline levels indicates significant attenuation of one or more disease parameters. In one embodiment, the decrease is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, reduction in enrichment score.

In one embodiment, the disease parameters comprise reduced histological disease scores, and reduced macrophages and CD3+ lymphocytes. In one embodiment, the CSF-1/CSF-1R gene set is a surrogate for histological disease scores. In one embodiment, the CSF1 gene set is a surrogate for macrophages and CD3+ lymphocytes.

In one embodiment, the disease is an inflammatory bowel disease. In one embodiment, the disease is colitis. In one embodiment, the disease is Crohn’s Disease.

In one embodiment, activation of CSF-1/CSF-1R gene set with a GS-MISS enrichment score above 0.93 (90% increase) relative to the value corresponding to the geometric mean observed for a healthy control set of samples indicates that the subject is likely to be non- responsive to anti-TNF-alpha treatment.

In one embodiment, activation of CSF-1/CSF-1R gene set prior to treatment and a lack of decrease in that activation after anti-TNF-alpha treatment indicates that the IBD/CD is resistant to treatment with anti-TNF-alpha. In one embodiment, the decrease is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, reduction in enrichment score. In one embodiment the GS-MISS enrichment score is above 0.9, above 1, above 5, above 10, or above 100.

In one embodiment, the anti-TNF-alpha treatment consists of treatment with infliximab. Non-limiting examples of other anti-TNF-alpha inhibitors that may be used in the methods of the disclosure are described elsewhere in this specification.

Additional non-limiting examples of diseases in which the CSF-1/CSF-1R gene set might be activated are described below. In non-limiting embodiments, these methods, as well as all the other methods provided in this disclosure, may be used in the context of any of these diseases.

PATIENT STRATIFICATION IN METHODS OF TREATMENT

In one embodiment, the disclosure provides improvements in disease treatment through responsive patient selection guided by the relative strength of the CSF-1/CSF-1R signal. This patient selection may be followed by, optionally, treatment with an appropriate drug. In one embodiment, disclosure provides support for the use of modulators of the CSF-1/CSF-1R gene set in the treatment of subjects where the gene set is elevated in the pertinent tissue. In one embodiment, the disclosure provides the use of modulators of CSF-1/CSF-1R signaling in the treatment of subjects where the CSF-1/CSF-1R gene set is elevated in the pertinent tissue, preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, above reference. In one embodiment, the disclosure provides for the use of CSF1R tyrosine kinase inhibitors in the treatment of subjects where the CSF-1/CSF-1R gene set is elevated in the pertinent tissue, preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, above reference. In one embodiment, the disclosure provides for the use of JNJ-40346527 in the treatment of subjects where the CSF-1/CSF-1R gene set is elevated in the pertinent tissue, for example, colonic mucosa in Crohn’s disease, preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%,.

In one embodiment, the disclosure provides a method of identifying a subject in need of treatment as responsive to anti-CSF-1/CSF-1R treatment and, optionally, treating the subject’s disease, comprising:

processing mRNA from a baseline/non-treated tissue sample from the subject through a gene expression system measuring the expression of the CSF1-CSF1R gene set represented by an expression score;

obtain the expression score in the sample, wherein an increase in the CSF1-CSF1R gene set expression score in the sample relative to the expression score in normal tissue indicates that the subject is responsive to anti-CSF-1/CSF-1R treatment; and, optionally,

treating the subject with an anti-CSF-1/CSF-1R treatment; wherein the tissue sample is harvested from a suspected diseased tissue.

In one embodiment the increase is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, relative to the appropriate reference.

In one embodiment, the disclosure provides a method of identifying a Crohn’s Disease subject in need of treatment as non-responsive to anti-TNF-alpha treatment and, optionally, treating the subject’s disease, comprising:

processing mRNA from a baseline/non-treated colon tissue sample from the subject through a gene expression system measuring the expression of at least the genes in Table I;

measuring the expression level of at least those genes in the sample, wherein a gene expression pattern in the sample relative to the expression levels in normal colon as that represented in the table indicates that the subject is non-responsive to anti-TNF-alpha treatment; and, optionally,

treating the subject with a drug other than anti-TNF-alpha treatment.

processing mRNA from a baseline/non-treated colon tissue sample from the subject through a gene expression system measuring the expression of at least the genes in Table I; measuring the expression level of at least those genes in the sample, wherein a gene expression pattern in the sample relative to the expression levels in normal colon exhibiting >90% of the changes indicated in the table indicates that the subject is non- responsive to anti- TNF-alpha treatment; and, optionally,

treating the subject with a drug other than anti-TNF-alpha treatment.

TABLE I

In one embodiment, the term“expression pattern” refers to the pattern of increased and decreased expression levels of the listed genes in the sample.

In one embodiment, the anti-TNF-alpha treatment comprises/consists of infliximab. In one embodiment, the anti-TNF-alpha treatment comprises an anti-TNF-alpha antibody or a small molecule TNF-alpha inhibitor, including, without limitation, those exemplified below.

In one embodiment, the drug is a CSF-1/CSF-1R inhibitor. In one embodiment, the drug is JNJ-40346527. In one embodiment, the drug is a c-fms kinase inhibitor among those described in U.S. Patent Publication No. 2014/0045789A1. In one embodiment, the drug is a CSF-1 or a CSF-1R inhibitor including, without limitation, those exemplified below.

In one embodiment, the disclosure provides a method of identifying a Crohn’s Disease subject in need of treatment as responsive to anti-TNF-alpha treatment, and, optionally, further treating the subject’s disease, comprising:

processing mRNA from a baseline/non-treated colon tissue sample through a gene expression system measuring the expression of at least the genes in Table II;

measuring the expression level of at least those genes in the sample, wherein a gene expression pattern in the baseline sample, relative to expression levels in normal colon as that represented in the table, indicates that the subject is responsive to anti-TNF-alpha treatment; and, optionally,

treating the subject with an anti-TNF1alpha treatment.

TABLE II

In one embodiment, the anti-TNF-alpha treatment comprises/consists of infliximab. In one embodiment, the anti-TNF-alpha treatment comprises an anti-TNF-alpha antibody or a small molecule TNFalpha inhibitor, including, without limitation, those exemplified below.

In one embodiment, the disclosure provides a method of identifying a Crohn’s Disease subject in need of treatment as responsive to anti-TNF-alpha treatment and, optionally, treating the subject’s disease, comprising:

processing mRNA from a baseline/non- treated colon tissue sample from the subject through a gene expression system measuring the expression of at least the genes in Table II; measuring the expression level of at least those genes in the sample, wherein a gene expression pattern in the sample relative to the expression levels in normal colon exhibiting >80% of the changes indicated in Table II but less than 90% of the changes indicated in Table II indicates that the subject is responsive to anti-TNF-alpha treatment; and,

treating the subject with an anti-TNF-alpha treatment.

In one embodiment, the disclosure provides a method of identifying a Crohn’s Disease subject in need of treatment as having responded or being responding to anti-TNF-alpha treatment, and, optionally, treating the subject’s disease, comprising:

processing mRNA from a post-anti-TNF-alpha treatment colon tissue sample from the subject through a gene expression system measuring the expression of at least the genes in Table III;

measuring the expression level of at least those genes in the sample, wherein a gene expression pattern in the sample, relative to expression levels in a baseline/pre-treatment from the same subject, as that represented in the table indicates that the subject has responded or is responding to anti-TNF-alpha treatment; and optionally;

treating the subject with anti-TNF-alpha treatment.

TABLE III

Any anti-TNF-alpha treatment may be used in the methods of the disclosure. In one embodiment, the anti-TNF-alpha treatment comprises, consists, or consists essentially of a TNFalpha inhibitor. In one embodiment, the inhibitor is infliximab (e.g., marketed as

REMICADE™, REMSEVIA™, INFLECTRA™). In certain embodiments, the TNF-alpha inhibitor is adalimumab (e.g., marketed as HUMIRA™ and EXEMPTIA), Certolizumab pegol (e.g., CDP870, marketed as CEVIZIA™), etanercept, golimumab, or combinations thereof, as well as small molecule TNFa-inhibitors (e.g., those described in Richmond et al, Small

Molecules as Anti-TNF Drugs Curr Med Chem. 2015;22(25):2920-42).In certain embodiments, the anti-TNF-alpha inhibitor is adalimumab. In certain embodiments, the TNF-alpha inhibitor is certolizumab. In certain embodiments, the TNF-alpha inhibitor is etanercept. In certain embodiments, the TNF-alpha inhibitor is golimumab. In some embodiments, the TNF-alpha inhibitor is used in combination with other medications such as prednisone, methotrexate, hydroxychloroquine, leflunomide, or sulfasalazine.

processing mRNA from a baseline/non- treated colon tissue sample from the subject through a gene expression system measuring the expression of at least the genes in Table III; measuring the expression level of at least those genes in the sample, wherein a gene expression pattern in the sample relative to the expression levels in normal colon exhibiting >80% of the changes indicated in Table III indicates that the subject is responding to anti-TNF- alpha treatment; and, optionally

treating the subject with an anti-TNF-alpha treatment.

METHODS OF TREATMENT WITH CSF-1/CSF-1R INHIBITORS

In one embodiment, the disclosure provides for the treatment (e.g., inhibiting, reducing, ameliorating, or preventing) of a disease in which the CSF-1/CSF-1R gene set is

elevated/activated in a diseased tissue sample relative to a reference sample/level, with an effective amount of a CSF-1/CSF-1R inhibitor. In one embodiment, the reference sample level is the median level of the CSF-1/CSF-1R gene set in a control sample of the same tissue from a reference population. A non-limiting example of a reference population includes healthy humans, or a control healthy tissue. More details on the appropriate controls are provided elsewhere in this application.

In one embodiment, the disclosure provides that any disease or disorder in which the CSF-1/CSF-1R gene set is increasingly activated may be treated with a CSF-1 inhibitor. In one embodiment, the disclosure provides that inflammatory bowel disease, Chron’s Disease, and Colitis may be treated with administration to the subject of one or more CSF1 inhibitors. In one embodiment, the disclosure provides a method for treatment or prophylaxis of an inflammatory bowel disease in a subject with a CSF1 inhibitor. In one embodiment, the disclosure provides a method for treatment or prophylaxis of Chron’s disease in a subject with a CSF1 inhibitor. In one embodiment, the disclosure provides a method for treatment or prophylaxis of colitis in a subject with a CSF1 inhibitor.

In one non-limiting embodiment, the CSF1 inhibitor is selected from antisense oligonucleotides or small interfering RNAs, and anti-CSFl antibodies such as those sold by Roche (e.g, RG7155), Pfizer (PD-0360324), and Novartis (MCS110).

In one embodiment, the disclosure provides that any disease or disorder in which the CSF-1/CSF-1R gene set is activated may be treated with a CSF-1R inhibitor. In one

embodiment, the disclosure provides that inflammatory bowel disease, Chron’s Disease, and Colitis may be treated with administration to the subject of one or more CSF-1R inhibitors. In one embodiment, the disclosure provides a method for treatment or prophylaxis of an

inflammatory bowel disease in a subject with a CSF-1R inhibitor. In one embodiment, the disclosure provides a method for treatment or prophylaxis of Chron’s disease in a subject with a CSF-1 R inhibitor. In one embodiment, the disclosure provides a method for treatment or prophylaxis of colitis in a subject with a CSF-1R inhibitor.

In one non-limiting embodiment, the CSF-1R inhibitor is selected from an anti-CSFIR antibody (e.g. Emactuzumab is also known as RG7155 or RO5509554; FPA008 is a humanized mAb; AMG820; IMC-CS4 (LY3022855)); a small molecule inhibitor, ( e.g., JNJ-40346527; JNJ- 28312141, heteroaryl amides, quinolinone series, pyrido-pyrimide series (all developed by Johnson and Johnson); BLZ945 (Novartis), PLX7486, ARRY-382, a chimeric antigen receptor, Hodgkin's lymphoma, anaplastic large cell lymphoma, Pexidrtinib is also known as PLX3397 or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-06-(trifluoromethyl)pyridin-3- yl)methyl)pyridin-2-amine; GW 2580 (CAS 870483-87-7), KΪ20227 (CAS 623142-96-1),

AC708 by Ambit Siosciences, or any CSF1R inhibitor listed in Cannarile et al. Journal for ImmunoTherapy of Cancer (2017) 5:53 and US20180371093, incorporated herein by reference for the inhibitors they discloses. In one embodiment, the CSF-1R inhibitor is described in US Publication No. 2009/0105296.

The disclosure provides several diseases in which CSF-1/CSF1R signaling may be increasingly activated relative to a control level. Treatment of those diseases may be monitored by use of the CSF-1/CSF-1R gene set disclosed herein. The gene set levels may be measured before, during, and after treatment to monitor progression of the disease. In one embodiment, the gene set is used to diagnose the subject as having one of these diseases.

In some embodiments, the disease or condition to be treated, diagnosed, or monitored according to the methods of the disclosure is a cancer, an autoimmune disease, and any otherwise inflammatory disease.

In some embodiments, the disease is an autoimmune disease including but not limited to Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, inflammatory bowed disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, acute disseminated encephalomyelitis, Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hepatitis, Bullous pemphigoid, dermatomyositis, diabetes mellitus type 1, Goodpasture's syndrome, Guillain-Barre syndrome, Idiopathic thrombocytopenic purpura, Lupus erythematosus, Mixed Connective Tissue Disease, multiple sclerosis, myasthenia gravis, narcolepsy, Pemphigus vulgaris, Pernicious anemia, Polymyositis, Primary biliary cirrhosis, Sjogren's syndrome, Temporal arteritis, Ulcerative Colitis, Vasculitis, or Wegener's granulomatosis.

In one embodiment, the disease is an inflammatory disease or macrophage-related disease selected from rheumatoid arthritis, conjunctivitis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, bronchitis, tuberculosis, chronic cholecystitis, acute pancreatitis, sepsis, asthma, chronic obstructive pulmonary disease, ankylosing spindilitis, hindradentis suppurativa, lichen planus, psoriatic arthritis, non-infectious uveitis, erythema multiforme, Stevens-Johnson syndrome, dermal inflammatory disorders such as psoriasis, atopic dermatitis, and toxic epidermal necrosis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), inflammatory pustular skin disease, cancer-associated inflammation, reduction of tumor-associated angiogenesis, diabetes, graft versus host disease and associated tissue rejection inflammatory responses, inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, a delayed-type hypersensitivity, immune-mediated and inflammatory elements of CNS disease; e.g., Alzheimer disease, Parkinson disease, multiple sclerosis,

Systemic Juvenile Idiopathic Arthritis, immune-mediated diseases, including but not limited to multiple sclerosis, lupus nephritis, rheumatoid arthritis, and to treat neurological diseases, including but not limited to amyotrophic lateral sclerosis (ALS) and Huntington's disease, inhibition of cell proliferation and survival in CSF-1R ligand-dependent and CSF-1 ligand- independent CSF-1R expressing tumor cells, monocytes, and infiltrating macrophages:

neovascular ocular disease.

In another embodiment, the disease is an inflammatory disease or a macrophage-related disease selected from cancer, autoimmune diseases, macrophage activation syndrome, atherosclerosis, diabetes mellitus, Kawasaki disease, asthma, hemophagocytic

lymphohistiocytosis, sarcoidosis, periodontitis, Whipple's disease, pulmonary alveolar proteinosis, macrophage related pulmonary disease, Leishmaniasis, obesity complications, hemodialysis related inflammation, microbial infection, viral infection, inflammation, and complications thereof.

In some embodiments, the disease is a cancer including but not limited to adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, Castleman's Disease, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g., Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors,

gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, Non- Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (adult soft tissue cancer), melanoma skin cancer, non-melanoma skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer ( e.g ., uterine sarcoma), vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia.

In some embodiments, the disease is an inflammatory disease, a cardiovascular disease, a metabolic disease, or a neurodegenerative disease

In some embodiments, the CSF-1/CSF-1R inhibitor is a small molecule and is administered at a dose between 50 mg and 1500 mg, e.g., between 75 mg and 1000 mg, between 100 mg and 900 mg, between 200 mg and 800 mg, between 300 mg and 700 mg, between 400 mg and 600 mg, between 100 mg and 700 mg, between 100 mg and 500 mg, between 100 mg and 300 mg, between 700 mg and 900 mg, between 500 mg and 900 mg, between 300 mg and 900 mg, between 75 mg and 150 mg, between 100 mg and 200 mg, between 200 mg and 400 mg, between 500 mg and 700 mg, or between 800 mg and 1000 mg, e.g., at a dose of 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg.

In one embodiment, the therapeutically effective amount of the antibody administered may be in the range of about 0.1 to about 50 mg/kg of patent body weight whether by one or more administrations, with a typical range of antibody used being about 0.3 to about 20 mg/kg, more preferably about 0.3 to about 15 mg/kg, administered daily, for example. However, other dosage regimens may be useful. The progress of this therapy may be monitored by conventional techniques or by the methods of the disclosure described above.

In one embodiment, treatment and/or prophylaxis require the administration of an effective amount of CSF-1/CSF-1R inhibitor to the diseased subject. "Effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit.

The appropriate dosage (“therapeutically effective amount”) of the CSF-1/CSF-1R inhibitor will depend, for example, on the condition to be treated, the severity and course of the condition, whether the inhibitor is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the inhibitor, the type of CSF-1/CSF-1R inhibitor used, and the discretion of the attending physician. The CSF-1/CSF-1R inhibitor is suitably administered to the patent at one time or over a series of treatments and may be administered to the patent at any time from diagnosis onwards. The CSF-1/CSF-1R inhibitor may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, mode of

administration, and composition, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compositions of the disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. The compositions may be administered with medical devices known in the art. Non-limiting embodiments include a needle, a needleless hypodermic injection device, a variable flow implantable infusion apparatus for continuous drug delivery, an osmotic drug delivery system having multi-chamber compartments, and the like.

If desired, the effective daily dose of therapeutic compositions may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present disclosure to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

COMBINATIONS

In some embodiments, the anti-CSF1-/CSF-1R inhibitor or anti-TNF1alpha inhibitor treatments may be used in combination with each other, and with other therapies. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

As non-limiting examples, the combination therapy may include a composition of the anti-CSF1-/CSF-1R inhibitors and/or anti-TNF1alpha inhibitors of the present disclosure co- formulated with, and/or co-administered with, one or more additional therapeutic agents, e.g., one or more anti-cancer agents, cytotoxic or cytostatic agents, hormone treatment, vaccines, checkpoint inhibitors (e.g. anti-PD/PDL1 antibodies nivolumab, pembrolizumab, avelumab, durvalumab, PDR001, atezolizumab, keytruda), and/or other immunotherapies (RG7876);

androgen-deprivation therapy plus external radiotherapy, paclitaxel, eribulin, temozolomide plus external radiotherapy, vemurafenib, PLX9486, paclitaxel plus bevacizumab, carboplatin plus gemcitabine, cyclophosphamide. Additional suitable cytotoxic agents are described in

REMINGTON'S PHARMACEUTICAL SCIENCES, 22nd Ed. (Mack Publishing Co. 2012), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS , 13th Ed. (MacMillan Publishing Co. 2017), as well as all other editions of these publications. Other non-limiting examples of drugs that may be used as combination treatments in the disclosed methods may be also be found in PCT publication WO2018237173A1, incorporated herein by reference for those examples.

ADMINISTRATION OF THE ANTI-CSF-1/CSF-1R AND ANTI-TNF-ALPHA COMPOSITIONS

In one embodiment, the compositions disclosed herein may be administered to a subject in need of treatment with anti-CSF-1/CSF-1R and/or anti-TNF-alpha treatment, preferably a human, in accord with any known methods. Non-limiting methods include intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes, and the like. In some preferred embodiments, the composition is administered to the subject by intravenous administration. For such purposes, the composition may be injected using a syringe or via an IV line, for example. In addition, the composition may be incorporated into sustained release matrices such as biodegradable polymers, the polymers being implanted in the vicinity of where delivery is desired, for example, the colonic mucosa. The method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and sustained administration for a predetermined period of time.

Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail may be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein.

EXAMPLES

BRIEF SUMMARY OF THE EXAMPLES DESCRIBED BELOW

Macrophage-lineage cells have been linked to the pathogenesis of Crohn’s disease. Colony stimulating factor- 1 (CSF-1), a mediator of macrophage differentiation and survival, is reportedly elevated in Crohn’s mucosa, but a role in disease has not be determined. To assess if CSF-1 may play a role in inflammatory bowel disease, the inventors examined transcriptional data from Crohn’s mucosa for evidence of CSF-1 pathway activation and tested CSF1R-I, a small molecule inhibitor of CSF-1 receptor kinase (CSF-1R), for ability to inhibit inflammation and disease indices in mouse T-cell transfer colitis.

A CSF-1/CSF-1R gene set was created from microarray data of human whole blood cultured ex vivo with CSF-1, and gene set variation analysis was performed using existing Crohn’s mucosa microarray data. Colitis was induced in C.B-17 SCID mice by i.p. injection of CD45+RBhigh T cells. Commencing day 14 or day 21, mice were treated PO QD until study termination (day 42) with vehicle or CSF1R-I. Endpoints included colon weight/length ratios and histopathology scores, and macrophage and T cells were assessed by F4/80 and CD3 immunohistochemistry. Mucosal gene expression was investigated using RNAseq.

CSF-1/CSF-1R gene set enrichment scores were elevated in the mucosal gene sets of Crohn’s disease and in mouse T cell transfer colitis. In the mouse model, CSF1R-I inhibited the increase in colon weight/length ratio by ~50%, reduced histological disease scores by ~60%, and reduced F4/80+ mononuclear cells and CD3+ lymphocytes. RNAseq analysis confirmed CSF-1/CSF-1R gene set was sharply reduced in mice treated with CSF1R-I as were gene sets enriched in“M1” and“M0” macrophages and in activated T cells. Ingenuity pathway analysis demonstrated significant concordance for disease associated biological pathways between Crohn’s disease and the mouse model (FIG. 13).

CSF-1 biology is activated in Crohn’s disease and in mouse T cell transfer colitis.

Inhibition of CSF-1R by CSF1R-I was associated with significantly attenuated clinical disease scores and reduced inflammatory gene expression in murine colitis.

JNJ-40346527, 4-cyano-N-[2-(4,4-dimethylcyclohex-1-en-1-yl)-6-(2,2,6,6-tetramethyl- tetrahydro-2H-pyran-4-yl) pyridin-3-yl]-1H-imidazole-2-carboxamide (abbreviated JNJ527), was prepared at Janssen as described (Kolodziejczyk K SA, Teleha CA, Weerts KJH. Process for the preparation of C-FMS kinase inhibitor. U.S. Patent Publication No. US 2014/0045789A1). For oral (PO) dose administration at 10 ml/kg, CSF1R-I was prepared fresh daily in vehicle (0.5% w/v METHOCEL*F4M Premium hydroxypropyl methylcellulose in deionized water).

CNTO5048, a neutralizing anti-mouse TNFa monoclonal antibody and an isotype control antibody, CNTO6601, were also prepared at Janssen. For intraperitoneal (IP) administration at 10 ml/kg, the antibodies were formulated at 0.3 mg/ml in sterile phosphate buffered saline.

EXAMPLE 1

Generation of human CSF-1 and TNFa-induced gene sets To identify CSF-1 induced genes, whole blood from three healthy donors was drawn into EDTA vacutainer tubes. Two aliquots of 1 ml were transferred to sterile 2 ml Eppendorf tubes. Recombinant human CSF-1 (Peprotech) was added to one of the two replicate tubes to a final concentration of 50 ng/ml, with the second replicate tube retained as an unstimulated control sample. Tubes were affixed to a tube rotator and incubated at 37°C, 5% CO₂. Aliquots (100 mL) were collected from all tubes at 4 and 24 hr, and RNA isolated using a MagMAX total RNA isolation kit (Thermo Fisher Scientific) according to the manufactures protocol. cDNA was generated and aliquoted onto GeneChip Human Transcriptome Array 2.0 (ThermoFisher) and analyzed via the manufacturer’s online proprietary software. The CSF-1/CSF-1R gene set was defined as comprising or consisting essentially of 57 genes (ARHGAP18, ARHGAP22, ATF3, BCAT1, CASP1, CCL8, CCR2, CD14, CD163, CLEC5A, COL7A1, CSF1R, CTSB, DSE, EPB41L3, FBP1, FCN1, FCRLA, FLJ42418, FMNL3, FPR3, FUCA1, fflVEP3, HLA-DPA1, HLA-DRA, HNMT, IFNB1, IQSEC2, KYNU, LAP3, LRPl, MAFB, MARCH 1, MARCKSLl, MERTK, MRAS, MS4A14, MS4A6A, MS4A7, MYOF, NAPSB, OAS1, OLR1, RIN2, SCD, SDS, SEMA6B, SIGLEC1, SIGLEC16, STARD4, TFEC, TGM2, THBS1, TMEM180, VCAN, VSIG4, ZNF330), all of which have geometric means >2-fold relative to unstimulated control condition (p<0.05) at either 4 or 24 hours of stimulation of whole blood cells with CSF-1.

To create a TNFa-induced gene set, triplicate cultures of A549, HT-29, HEK293, and primary human keratinocytes were cultured without (unstimulated control) or with 10 ng/ml human recombinant TNFa (Peprotech) for 4-to-24-h and gene expression analyzed by

Affymetrix GeneChip(R) HG-U133+2 array or HG-U133+PM array plate. TNFa-induced gene set was comprised of 25 genes (ATXN1, BID, BIRC3, C3, CCL20, CD83, CLDN1, CXCL1, CXCL2, CXCL5, CYLD, DRAM1, IFNGR1, IKBKE, IL32, IL8, KIF3C, KLKIO, LAMC2, NFKB2, NFKBIA, OPTN, SOD2, TNFAIP3, TNIP1) with geometric means ³1.5-fold for TNF- stimulated vs. unstimulated control samples (p<0.05) in at least two cell types.

To assess variations in the expression of the CSF-1/CSF-1R gene set gene set, GSVA analysis was conducted and GSVA expression scores calculated.

EXAMPLE 2

Gene set variation analysis (GSVA) analysis of human Crohn’s mucosa

Transcriptomic profiles of individual RNA samples from Crohn’s disease and healthy control mucosal biopsies have been previously published (NCBI/NIH GEO DataSet GSE16879) (Arijs I, De Hertogh G, Lemaire K, Quintens R et al. PLoS One 2009 Nov 24;4(11):e7984.).

Raw cel files from Affymetrix GeneChip(R) HG-U133+2 arrays were subjected to robust multi- array average normalization, and paired analysis was conducted for before and after treatment samples from the same patient for differences in gene expression due to drug treatment. GLM (general linear model) was used to obtain gene expression differences between diseased and healthy controls. Gene set variation analysis (GSVA) was performed as described by

Hanzelmann (Hanzelmann S, Castelo R, Guinney J. BMC Bioinformatics 2013;14:7.), mapping the CSF-1 and TNFa-induced gene sets onto the GSE16879 Crohn’s disease and healthy control normalized dataset.

EXAMPLE 3

T Cell Transfer Model of Colitis

The in-life portion of the murine colitis model studies reported herein were performed by Bolder BioPATH, Boulder Colorado. Female Balb/C mice (12 weeks of age) and Fox Chase C.B-17 SCID mice (5-6 weeks of age) were obtained from Harlan, Inc., Indianapolis, Indiana. Animals were maintained under a 12 hr light/dark cycle and were provided with standard rodent chow and water ad libitum. Mice were acclimated to animal facility housing for a minimum of 5 days prior to initiation of the study. All animal handling and housing procedures were conducted in accordance with Institutional Animal Care and Use Committee guidelines

Colitis was induced as described previously (Leach, MW, Andrew GD, et al. Amer J Pathol 1996; 148(5): 1503-1515.). Single cell suspensions were prepared from the spleens of BALBc mice by trituration and passed through 40 pm cell separator mesh (BD Falcon). Red cells were lysed with RBC lysis buffer, and T cells were collected using a MACS CD4+ separator kit (Miltenyi Biotech). Naive T cells were then isolated by FACS sorting using a specific antibody against CD45RB (Dynal® Mouse CD4 Negative Isolation kits, Invitrogen,

CA). Female Fox Chase C.B-17 SCID which lack B and T cells were inoculated

intraperitoneally (IP) with a minimum of 4× 105 CD4+CD45RBhigh naive T cells (100 mL/mouse injections). Animals so treated develop progressive and unrelenting inflammatory colitis. Two dosing paradigms were employed to determine the efficacy of CSF1R-I:

“Prophylactic” dosing was initiated on day 14, a time when naive T cells are expected to have fully engrafted, but before clinical signs of disease are manifest. “Therapeutic” dosing commenced on day 21 when mice exhibit clinical signs of disease. Treatment groups are summarized in Table IV.

Study 1 - Dose response. Prophylactic Dosing: On study day 0, SCID recipient mice were inoculated with naive T cells as described above. On study day 14, mice were randomized by body weight into treatment groups receiving vehicle, or 5, 10, or 20 mg/kg CSF1R-I. Dosing was administered orally (PO) commencing day 14 post transfer and continued daily (QID) through day 41.

Study 2 - Prophylactic vs. Therapeutic Dosing: SCID mice underwent T cell transfer on day 0. On study day 14, all mice were randomized by body weight into treatment groups, and prophylactic treatment was initiated with 15 mg/kg CSF1R-I or vehicle. A third group of mice received vehicle until day 21 at which time therapeutic dosing of compound was initiated.

Dosing was administered PO, QID through day 41. Other groups of mice were treated intraperitoneally (IP), every third day (Q3D) commencing day 21 with either PBS, CNTO5048 (anti-mouse TNFa), or an isotype control. Study 2 included additional parallel groups of mice designated for RNAseq analysis. Specifically, additional SCID mice with adoptive CD45RB^hi T cell transfer were dosed therapeutically with vehicle or with JNJ527, CNTO5048 or isotype control. Additional SCID mice without T cell transfer and a group of immunocompetent Balb/c mice were included as disease- free controls.

EXAMPLE 4

Clinical and Histological Assessment of T Cell Transfer Colitis

All mice were weighed on study day 0 and then every other day commencing on day 14. Final body weights were determined on day 41 with subsequent tissue harvest on day 42 following an overnight fast. Percent change in body weight relative to day 14 (Study 1) or day 0 (Study 2) was reported. At necropsy, the entire colon was harvested from each animal, inspected visually, measured for length, and weighed. Results were reported as weight/length ratios. Colons were fixed in 10% neutral buffered formalin (NBF) for 48 hr, transferred to 70% ethanol and embedded in paraffin within 1 week of fixation. Two paraffin blocks were prepared from each animal: 1 block containing 3 pieces of proximal colon, and 1 block containing 3 pieces of distal colon (~1 cm long each). Paraffin embedded blocks were sectioned, processed for H&E and scored for pathological changes by a trained pathologist (Bolder BioPATH. Individual components of the pathology score are provided in FIG. 5.

Immunohistochemical staining for F4/80 and CD3 was performed at HistoTox Labs, Boulder, CO. Digital images from three proximal and three distal colon tissue sections were captured at 40X using Aperio ScanScope XT (Leica Biosystems, Danvers, MA). Staining was quantified using Aperio GENIE pattern recognition software which detects the number and intensity of positively stained pixels and the total tissue area. Results were reported as total positive pixels normalized to tissue area (FIG. 6).

EXAMPLE 5

Analyses of gene expression in the mouse T cell transfer colitis model

On study day 42, mice were euthanized and mucosa from the middle 2/3 of the colon was harvested by separating the mucosa from the muscle layer. Mucosa were collected in Matrix Lysis-D tubes, immediately snap frozen in liquid nitrogen and stored at -80 C for subsequent RNA extraction. Mucosal homogenates were prepared in 1 ml RLT buffer with the aid of a Bead Ruptor bead mill (Omni International) and total RNA was isolated using a Qiagen RNeasy Mini kit according to the manufacturer’s protocol. RNAseq was performed by BGI Americas Corporation (Cambridge, MA). FASTQ files were obtained from BGI and processed using ArrayStudio (OmicSoft/QIAGene, Research Triangle Park, NC). QC passed FASTQ files were mapped to mouse mmlO reference genome using gene model ENSEMBLE.R72 to obtain gene expression quantifications. Deseq2 and GLM (General Linear Model) methods implemented in Arraystudio were used to compare gene expression differences between treatment groups using the original RNA sequence read count data at individual gene level. Ingenuity Pathway Analysis indicated strong pathway level concordance between the mouse model and human disease (FIG 13). Furthermore, treatment of mice with CSF1R-I effectively and broadly countered disease pathway activation.

GSVA was performed using the CSF-1 and TNFa -induced gene lists after translation to mouse homologues (FIG. 8A and 8B). GSVA was also performed using gene lists enriched for M0 and M1 macrophages and activated Th1 T cells (FIGs. 10-12). The cell-type lists were derived from a matrix of genes reported to have low/limited expression outside the

hematopoietic system but to have high differential expression in 24 subsets of lymphoid and myeloid cells (Chen Z, Huang A, Sun J, et al. Sci Rep 2017;7:40508.). For use herein, gene sets were derived by sorting the top 30 genes by signal intensity for the indicated cell type (lists are provided in FIG. 10-12). EXAMPLE 6

Gene Set Variation Analysis ( GSVA ) supports a role for

CSF-1 in Crohn 's disease

To enable estimation of CSF-1R-driven biology in disease datasets, a CSF-1/CSF-1R gene set was derived consisting of fifty-seven genes induced >2-fold by CSF-1 in ex vivo whole human blood cultures (see EXAMPLE 1 for full list). Consistent with the role of CSF-1 as a macrophage regulatory factor, Ingenuity Pathway Analysis identified“activation of phagocytes” as the top enriched disease and function category (p = 4×10^-10).

A transcriptomic analysis of Crohn’s disease colonic mucosa biopsies before and after patient treatment with Infliximab was published previously (Arijs I, De Hertogh G, Lemaire K, Quintens R et al. PLoS One 2009 Nov 24;4(11):e7984. PMID: 19956723). Using this Crohn’s disease dataset and the CSF-1/CSF-1R gene set, gene set variation analysis (GSVA) was performed to estimate the relative strength of CSF-1 biology within Crohn’s disease colonic mucosa. Patients were classified into those with good clinical response to Infliximab vs. non- responding patients. Response was assessed by endoscopy and histology in CD patients 4 to 6 weeks after the first infliximab treatment and responders were defined as having complete mucosal healing.

When compared to the healthy control group, the CSF-1/CSF-1R gene set enrichment scores were robustly elevated prior to infliximab treatment in CD patients who did not achieve response criteria (FIG. 2). Enrichment of the CSF-1 -induced gene set was not reduced following treatment, consistent with non-response. The CSF-1 pathway signal was also significantly increased at baseline in patients who subsequently achieve the response criteria, although to a lesser extent than observed in non-responders. This signal was lowered to the level of healthy mucosa following clinically effective treatment. Further, the expression of CSF-1R mRNA was consistent with increased CSF-1R pathway activity, viz., at baseline relative to healthy controls, expression of CSF-1R was significantly 1.92-fold higher in responders and 2.78-fold in non- responders and was restored to healthy levels with treatment only in responders. FIG. 7 provides a summary of the CSF-1/CSF-1R gene set in this Crohn’s cohort at the individual gene level. Collectively, the data supported a hypothesis that CSF-1 driven biology is elevated in Crohn’s disease mucosa, even more so in infliximab non-responders. For comparative purposes, GSVA was similarly performed using a TNFa-induced gene set consisting of twenty-five genes significantly up-regulated by TNFa in nonhematopoietic cells (see METHODS for full list). Similar to the CSF-1 gene set, the TNFa gene set was enriched in Crohn’s mucosa, was highest in infliximab non- responders, and restored toward healthy levels only in the responders (FIG. B). The CSF-1 and the TNFa gene sets did not intersect at the gene level, i.e., there were no common genes.

EXAMPLE 7

The CSF-1R kinase inhibitor , CSF1R-I, reduced severity of murine colitis when dosed prophylacticaUy or therapeutically

Transfer of naive T cells into severe combined immunodeficiency (SCID) mice provides a highly reproducible and easily manipulated model of colitis that has characteristics similar to chronic, idiopathic inflammatory bowel disease (IBD) in humans (Leach, MW, Andrew GD, et al. Amer J Pathol 1996; 148(5): 1503-1515.). To assess the net role of CSF-1 in experimental colitis we employed the T cell transfer colitis model together with a selective small molecule inhibitor of CSF-1 R kinase, CSF1R-I, described previously (Genovese MC, Hsia E, Belkowski SM, et al. J Rheumatol 2015;42: 1752-60.). The model is induced when naive T cells, are introduced into a T cell deficient, syngeneic recipient. The T cells are taken up by the gut associated lymphoid tissue where they are exposed to gut associated antigens presented by dendritic cells. Thus primed, the T cells undergo clonal expansion and enter the lymphatic circulation, express gut homing markers, and traffic back to the gastrointestinal tract where they become colitogenic effector cells upon re-exposure to cognate antigens, triggering chronic inflammation in the colon.

In an initial study (Study #1), three dose levels of CSF1R-I were administered prophylacticaUy starting on day 14 post- injection of T cells, a point where pathogenic T cells have expanded and trafficked back to the GI tract, but before clinical signs of disease are manifest. In a second, confirmatory study (Study #2), a single dose level was administered starting on day 14 or on day 21, the latter considered to model therapeutic dosing. In both studies, dosing continued once per day until study termination on day 42. The second study included a group administered (i.p) CNTO5048, a neutralizing mAb to murine TNFa, to serve as a positive control. Additional groups were administered an isotype mAb control or PBS alone. Body weight loss is a common feature of experimental models of colitis, and weight losses averaging 10-15% occurred by day 40 in the oral vehicle- treated or isotype control-treated disease groups while the naive, disease- free group experienced 5-10% weight gain (FIG. 4A).

Treatment with CSF1R-I significantly prevented disease-induced body weight loss at all three dose levels in the first study and in the follow-on study body weight loss was prevented and reversed by both prophylactic and therapeutic dosing, an effect similar to CNTO5048.

Unexpectedly, the i.p. PBS-treated group in the second study did not show weight loss in this study, although subsequent indices of disease were evident. This may have been the result of the smaller group size for this group and chance accumulation of mice with mild disease in this group.

In this model, inflammation results in mucosal hyperplasia and edema reflected as an increase in the colon weight/length (W/L) ratio. FIG. 4B shows that the disease-associated increase in W/L ratio in vehicle-treated mice was significantly reduced by treatment with CSF1R-I. The therapeutic dosing regimen was found to be equally effective in reducing disease parameters. Histological assessment of disease is shown in FIG. 4C as the sum of four scoring parameters, i.e., inflammation, glandular loss, hyperplasia, and erosions. Sum of scores was markedly increased in animals with disease and significantly reduced by treatment with CSF1R-I (results for individual components are provided in FIG. 5). Further, CSF1R-I significantly reduced the disease associated neutrophil infiltrate (FIG. 4D). Overall, CSF1R-I showed a shallow dose-dependency with numerically maximal effect at 20 mg/kg.

EXAMPLE 8

CSF1R-I reduced recruitment of F4/80+ macrophages and CD3+ T cells during

TCT-colitis

The disclosure provides that CSF1R inhibition by CSF1R-I may lead directly to reduced macrophage numbers and have a secondary impact on T cells. To examine the effect of CSF1R- I on these cells, mucosal F4/80⁺ macrophages and CD3⁺ T cells were assessed by

immunohistochemistry (FIG. 6B and FIG. 6D). F4/80⁺ macrophages in the mucosa were increased in the proximal and distal colon of mice with TCT-colitis as compared to naive SCID mice. Staining was principally in the lamina propria. CSF1R-I, administered prophylactically or therapeutically, blocked the disease-related increase F4/80⁺ cells. CD3⁺ staining was, as expected, low/absent in the naive control SCID mice but CD3⁺ cells were abundant and distributed across all tissue levels in vehicle-treated mice with TCT-colitis. The T cell infiltrate was also significantly reduced in animals treated with CSF1R-I. EXAMPLE 9

Tramcriptomic analysis confirms increased strength of CSF-1 -driven biology in T cell colitis that is inhibited by CSF1R-I resulting in reduced macrophage and T cell inflammatory gene sets

To probe further the therapeutic mechanisms of CSF1R-I in TCT colitis, Study #2 included additional parallel cohorts of mice designated for RNAseq of colonic mucosa. An overall summary of differential gene expression is provided in Table V. T-cell transfer colitis was associated with >2000 genes differentially expressed >2-fold when compared to naive mice. The number of genes reaching significance was reduced 94% in the group treated with CSF1R-I, an effect similar to CNTO5048. Conversely, when compared to vehicle-treated mice with disease, CSF1R-I and CNTO5048 significantly impacted 1091 and 1280 genes, respectively.

Table V. Differential gene expression statistics from RNAseq of TCT coliti

study #2

To assess the translatability of the T cell transfer colitis model to human disease, IPA pathway analysis was performed using the genes differentially expressed in the aforementioned Crohn’s disease dataset (GSE16879) and in the mouse dataset described herein. Pathway comparisons were based on those impacted in human Crohn’s disease. At the pathway level, there was strong concordance between the mouse model and human disease (FIG. 13). Further, treatment of mice with CSF1R-I effectively and broadly countered disease pathway activation.

Next, GSVA was employed to assess CSF-1 pathway strength in the model. Mild elevation in CSF-1 pathway strength was apparent in naive SCID mice compared to Balb/c, suggesting a compensatory increase in innate pathways in this T and B cell deficient strain. However, similar to human Crohn’s disease, expression of the CSF-1/CSF-1R gene set was further enriched in mice with TCT colitis (FIG. 8A and FIG. 8B), and good concordance at the individual gene level existed between Crohn’s disease and the mouse model. The CSF-1/CSF- 1R gene set also provided an opportunity to assess directly the pharmacodynamics of CSF1R-I in disease tissue. Consistent with its mechanism as a CSF-1R inhibitor, CSF1R-I caused a sharp reduction in CSF-1-pathway strength to a level that was similar to that in Balb/c mice. In contrast, CNTO5048 had a weak suppressive net effect on the CSF-1/CSF-1R gene set that did not reach significance, although several individual genes were suppressed concordant with CSF1R-I. Conversely, CNTO5048 had a marked suppressive impact on the TNFa-induced gene set which was otherwise sharply elevated in the vehicle-treated group, while CSF1R-I had a moderate suppressive net effect on the TNFa gene set.

Although the loss of F4/80⁺ cells suggested a reduction in macrophages, it may also have reflected reduced expression of the F4/80 antigen as CSF-1 is reported to induce F4/80 antigen expression (Waddell LA, Lefevre L, Bush SJ, et al.. Front Immunol 2018;9:2246). To examine further the impact of CSF1R-I on the complement of mucosal macrophages and to confirm the impact on T cell activation contributing to colitis, we employed further gene set analysis. We made use of a matrix of genes reported to have low/limited expression outside the hematopoietic system but to have high and differential expression within subsets of lymphoid and myeloid cells (Chen Z, Huang A, Sun J, et al. Sci Rep 2017;7:40508). From this gene matrix, gene sets were derived enriched in inflammatory“M1” macrophages, resident-like“M0” macrophages and activated Th1 lymphocytes. Consistent with F4/80 immunohistochemistry, GSVA indicated the M1 and resident macrophage gene sets were enriched in the inflamed mucosa of vehicle-treated mice, while CSF1R-I treatment was associated with sharp reductions in both gene sets (FIG. 9 A, FIG. 9B). Similarly, genes enriched in activated Th1 lymphocytes were markedly increased in the inflamed mucosa, and significantly reduced with CSF1R-I treatment, consistent with the overall anti-inflammatory activity of the compound in the model (FIG. 9C). The full gene sets used herein and expression data at the individual gene level are provided in FIG. 10 through 12.

The disclosure shows enrichment of CSF-1 -inducible genes in Crohn’s colonic mucosa. Enrichment in CD was mirrored by enrichment of CSF-1 -inducible genes in the murine TCT model of colitis, wherein gene sets for cells of the macrophage lineage were also enriched. In TCT colitis, the CSF-1 R inhibitor, CSF1R-I, effectively restored the CSF-1 and macrophage gene sets to baseline. Reduction in CSF-1 and macrophage signal strength correlated with improved disease activity and histological parameters and in reduced the enrichment of a Thl T- cell associated gene set and reduced CD3⁺ T cells staining. IPA analysis of the TCT-colitis and CD gene signatures indicated good concordance at the level of biological pathways.

CSF1R-I restored those pathways in TCT colitis toward normal similar to pathway restoration in CD patients responding to anti-TNF-a therapy. Overall, the results support evaluation of CSF1R-I in CD.

Using GSVA analysis, the disclosure demonstrated the utility of a novel gene set induced by CSF-1 in human blood. While the gene set includes genes not previously known to be regulated by CSF-1, IPA functional curation confirmed it to be highly enriched for genes significant to macrophage biology. Robust suppression of the gene set in mice treated with CSF- 1R inhibitor further supported the use of the gene set as an indicator of CSF-1R mediated biology. Using the gene set with GSVA provided strong, contextual evidence of CSF-1R- mediated biology in CD, consistent with prior data indicating increased expression of CSF-1R and its ligands in Crohn’s. At the individual gene level, the CSF-1 -induced gene set showed excellent directional concordance between the TCT-colitis model and CD (FIG. 7). Taken together, the disclosure highlights the signature of CSF-1 biology in CD and the translational value of the gene set for clinical and preclinical pharmacodynamic assessment of CSF-1R inhibitors. Although blood-derived, the CSF-1 -induced gene set may be particularly useful for assessment of pharmacodynamic effect within tissues for which methods were not previously available.

It is known that macrophage numbers increase in Crohn’s disease colonic mucosa and contribute to disease processes. Transcriptomic analysis of the TCT model indicated a powerful suppressive effect of CSF1R-I on the total population of macrophage-lineage cells. IPA analysis of the TCT colitis disease signature and the Crohn’s disease signature revealed striking concordance at the pathway level. In CD anti-TNF therapy reversed the disease-associated pathways and the effect of CSF1R-I in mice was similar to that achieved by anti-TNF therapy in humans. GSVA performed on colonic mucosa collected from Crohn’s disease patients undergoing infliximab therapy demonstrated that CSF-1 driven pathways were indeed expressed in human, and that the signature was resolved in those that showed good clinical response to therapy. A more robust enrichment of the CSF-1 signature was observed in clinical non- responders prior to treatment with infliximab, but the signature failed to resolve with treatment.

Application of the CSF-1 and TNFa gene sets to the murine TCT model revealed differences in the mechanistic impact of CSF1R-I and anti-TNFa. GSVA indicated the CSF- 1/CSF-1R gene set was enriched with disease, only weakly and nonsignificantly impacted by anti-TNFa therapy, but robustly normalized following treatment with CSF1R-I along with concomitant amelioration of disease. Together, these observations provide evidence that multiple mechanisms may drive disease pathology in IBD, and that enhanced CSF-1/CSF-1R signaling could represent a differential disease mechanism within select IBD patient subsets.

Collectively, the disclosure provides that CSF-1 driven biology is elevated in Crohn’s disease mucosa, even more so in infliximab non-responders, and that CSF-1 is responsible, at least in part, for increased macrophage-driven pathology. The disclosure also provides that inhibition of CSF-1/CSF-1R signaling by CSF1R-I may be effective in managing inflammatory bowel disease. CSF-1 and TNFa may mediate pathogenic processes that exhibit different degrees of dominance in some patient subsets, and this compound may be of particular benefit for patients who fail anti-TNFa therapy.

EXAMPLE 10

CSF-1/CSF-1R gene set GS-MISS identifies CD patients in need of CSF-1R inhibition , identifies CD patients having responded to anti~TNF~alpha therapy , and identifies CD patients unlikely to respond to anti-TNF -alpha therapy

Relative to healthy control subjects, 17 of 18 CD patients had GS-MISS scores > 0.265 points above healthy control levels identifying these patients as in need of a CSF-1R inhibitor and identifying CD in general as a disease with increased CSF-1/CSF-1R pathway activity (FIG. 3). Following infliximab therapy, GS-MISS scores were reduced in the CD patients responding to infliximab (8 of 11 subjects with GS-MISS scores <0.265 points above heathy controls while all non-responders remained >0.265 points above healthy controls. Further, relative to responders, nonresponders had high GS-MISS scores prior to infliximab therapy, i.e., all non- responders had a GS-MISS score >0.93 above healthy controls while none of the responders reached this level.

EXAMPLE 11

CSF-1/CSF-1R gene set GS-MISS identifies ulcer stive colitis (UC) as a disease with increased CSF-1/CSF-1R pathway activity and in need of CSF-1R inhibition .

A transcriptomic analysis of UC colonic mucosa biopsies before and after patient treatment with Infliximab was published previously (Arijs I, De Hertogh G, Lemaire K, Quintens R et al. PLoS One 2009 Nov 24;4(11):e7984.). Using this UC disease dataset and the CSF- 1/CSF-1R gene set, GS-MISS scores were calculated for each subject before and after infliximab therapy (FIG. 14). Relative to healthy control subjects, 23 of 24 UC patients had GS-MISS scores > 0.265 points above healthy control levels identifying these patients as in need of a CSF- lR.

EXAMPLE 12

CSF-1/CSF-1R gene set GS-MISS correlates with response to infliximab in UC and

CD

CSF-1/CSF-1R gene set GS-MISS was used for pharmacodynamic evaluation of infliximab treatment in UC and CD patients (FIG. 15). For this purpose, for each subject, the ratio of gene expression for each gene was calculated after treatment relative to before treatment. As groups, CD and UC patients responding to infliximab demonstrated good pharmacodynamic response to therapy, i.e., UC and CD responders achieved group average reductions of ³0.263 points while the non-responder groups did not.

INCORPORATION BY REFERENCE

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

EQUIVALENTS

The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

Claims

CLAIMS We claim:

1. A method of measuring increased activation or engagement of CSF1/CSF-1R signaling in a tissue comprising:

a. processing mRNA from a tissue sample through a gene expression system measuring the expression of a set of genes comprising, consisting, or consisting essentially of ARHGAP18, ARHGAP22, ATF3, BCAT1, CASP1, CCL8, CCR2, CD 14, CD163, CLEC5A, COL7A1, CSF1R, CTSB, DSE, EPB41L3, FBP1, FCN1, FCRLA, FLJ42418, FMNL3, FPR3, FUCA1, fflVEP3, HLA-DPA1, HLA-DRA, HNMT, IFNB1, IQSEC2, KYNU, LAP3, LRP1, MAFB, MARCH1, MARCKSLl, MERTK, MRAS, MS4A14, MS4A6A, MS4A7, MYOF, NAPSB, OAS1, OLR1, RIN2, SCD, SDS, SEMA6B, SIGLEC1, SIGLEC16, STARD4, TFEC, TGM2, THBS1, TMEM180, VCAN, VSIG4, and ZNF330, also known as the CSF-1/CSF-1R gene set; and

b. calculating a gene expression score, wherein an increase in the expression score in the tissue sample relative to the expression score in a control tissue, or to a control score, indicates increased activation or engagement of CSF1/CSF-1R signaling in the tissue.

2. The method of claim 1, wherein the expression level of all genes in the gene set is

combined into an enrichment score by a gene set median individual scoring system (GS- MISS).

3. The method of claim 1, wherein the set of genes is referred to as the CSF-1/CSF-1R gene set, and the expression level of all genes in the gene set is combined into a gene set variation analysis (GSVA) expression score.

4. The method of claim 2, wherein the enrichment score is increased by at least 0.265 (20% increase), at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 300%, relative to the value corresponding to the geometric mean observed for a healthy control set of samples.

5. A method of diagnosing and, optionally, treating a subject in need thereof, as having a disease or disorder in which CSF-1/CSF-1R biology is engaged/involved or is strong, comprising: measuring increased activation of CSF-1/CSF-1R signaling in a diseased tissue sample from a subject by a method according to claim 1; wherein increased activation of CSF-1/CSF-1R signaling in the diseased tissue sample indicates that subject has a disease or disorder in which CSF-1/CSF-1R biology is engaged/involved or is strong.

6. The method of claim 5, further comprising treating the subject with a CSF-1 inhibitor or a CSF-1R inhibitor.

7. The method of claim 5, further comprising treating the subject with anti-TNF-alpha

treatment/TNF-alpha inhibitor.

8. The method of claim 5, wherein the disease is an inflammatory bowel disease, preferably Crohn’s Disease.

9. The method of claim 5, wherein the disease is an autoimmune disease, cancer,

cardiovascular disease, metabolic disease, or neurodegenerative disease.

10. A method of identifying a Crohn’s Disease subject in need of treatment as non- responsive to anti-TNF-alpha treatment comprising:

a. processing mRNA from a baseline/non-treated diseased colon tissue sample from the subject through a gene expression system as described in claim 1 and obtaining an expression score;

b. wherein a gene expression GS-MISS enrichment score above 0.93 (90% increase) relative to the value corresponding to the geometric mean observed for a healthy control set of samples indicates that the subject is likely to be non-responsive to anti-TNF-alpha treatment.

11. The method of claim 10, wherein the GS-MISS enrichment score is above 1, above 5, above 10, or above 100.

12. The method of claim 11, further comprising administering a CSF-1or CSF-1R inhibitor to the subject.

13. The method of claim 11, wherein the anti-TNF-alpha treatment consists essentially of treatment with infliximab.