Classifications

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  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

• ANTI-CXCL9, ANTI-CXCL10, ANTI-CXCL11, ANTI-CXCL13, ANTI-CXCR3 AND ANTI-CXCR5 AGENTS FOR INHIBITION OF INFLAMMATION
• This application generally relates to methods and compositions for inhibiting inflammation.
• the application relates to the use of anti-CXCL9, anti-CXCLlO, anti-CXCLl 1, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 agents, and/or other antiinflammatory agents for prevention and treatment of inflammatory diseases.
• Host cells have surface receptors that associate with ligands to signal and regulate host cell activities.
• Administration of anti-TNF-a antibody or soluble TNF-a receptor has been shown to inhibit inflammatory diseases.
• the side effects associated with this treatment can result in an increased risk of infections (e.g., tuberculosis) and other adverse reactions by mechanisms not fully understood.
• antibody therapies focused on membrane bound molecules like CD40 have a propensity for inhibiting inflammation and graft-host diseases. While other targeted host cell therapies to prevent inflammatory diseases are being developed, there is no known single surface or secreted factor that will stop all inflammatory diseases. Consequently, the development of therapies to exploit newly identified specific host cell targets is required.
• Chemokines represent a superfamily of small, cytokine-like proteins that are resistant to hydrolysis, promote neovascularization or endothelial cell growth inhibition, induce cytoskeletal rearrangement, activate or inactivate lymphocytes, and mediate chemotaxis through interactions with G-protein-coupled receptors. Chemokines can mediate the growth and migration of host cells that express their receptors. The cellular mechanisms responsible for the function of chemokines are often, but not entirely, Ca 2+ flux dependent and pertussis toxin-sensitive. However, the precise mechanisms for chemokine- mediated events are not known.
• the present invention relates to methods and compositions for treating or preventing inflammatory diseases or conditions.
• the method comprises the step of administering to a subject diagnosed with an inflammatory disease or condition an effective amount of an anti-inflammatory agent that (1) inhibits the expression of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5, or (2) inhibits the interaction between any one of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5, or (3) inhibits a biological activity of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5.
• the method comprises the step of administering to a subject diagnosed with an inflammatory disease or condition a therapeutically effective amount of an anti-CXCL9 antibody, an anti-CXCLlO antibody, an anti-CXCLl 1 antibody, an anti-CXCL13 antibody, and anti-CXCR3 antibody, an anti-CXCR5 antibody, or combination thereof.
• the agent or antibody is administered in a dosage range from about 10 ⁇ g/ g body weight/day to about 10 mg/kg body weight/day.
• the agent may comprise an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence, or agent-encoding expression vector.
• siRNA short interfering RNA
• a method for enhancing effect of anti-inflammatory therapy comprises administering to a subject who is receiving or has received anti-inflammatory therapy an effective amount of an anti-inflammatoryingthat (1) inhibits the expression of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5, or (2) inhibits the interaction between CXCR3 and CXCL9, CXCLIO or CXCLl 1, and the interaction between CXCR5 and CXCLl 3, or (3) inhibits a biological activity of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5, wherein the agent comprises an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence,
• an anti-inflammatorying that
• the subject is receiving anti-inflammatory therapy.
• the subject has received anti-inflammatory therapy and has exhibited anti-inflammatory drug-resistance to an anti-inflammatory agent.
• the present invention provides a pharmaceutical composition, comprising an anti-inflammatory agent capable of (1) inhibiting the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5; (2) inhibiting the interaction between any one of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, or (3) inhibiting a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, wherein the anti-inflammatory agent is an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence, or agent-encoding expression vector; and a pharmaceutically acceptable carrier.
• an anti-inflammatory agent capable of (1)
• FIG. 1 shows IFN- ⁇ , IP-10, MIG, I-TAC, and CXCR3 mRNA expression during murine colitis.
• FIG. 2 shows histological analysis of IBD in TCR ⁇ x ⁇ ⁇ " mice that received CD45RB HI or CXCR3 + CD4 + T cells by adoptive transfer.
• FIG. 3 shows SAA levels and the development of colitis in IL-10 "7" mice.
• SAA concentrations > 200 ⁇ g / ml were associated with the onset of asymptomatic colitis at week 0.
• FIG. 4 shows changes in body weight of IL-10 " _ mice.
• FIG. 5 shows association of serum IL-6 and SAA levels with murine colitis.
• FIG. 6 shows total fecal and serum Ab levels in IL-10 "7" mice.
• FIG. 7 shows serum IL-12, IFN- ⁇ , IL-2, TNF-a, IL-la, and IL- ⁇ levels in IL-10 " _ mice with IBD.
• FIG. 8 shows histological characteristics of colitis presented by IL-10 "7" mice.
• FIG. 9 shows that anti-CXCLlO antibody abrogates severe colitis.
• FIG. 10 shows Thl cytokine, CXCL10 and CXCR3 mRNA expression in mucosal tissue during severe colitis.
• FIG. 11 shows Thl and inflammatory cytokine levels in serum during severe colitis progression.
• FIG. 12 shows anti-CXCLlO antibody effects on colitis pathology.
• FIG. 13 shows histological and immunofluorescence localization of CXCL9, CXCLIO, CXCLl 1, and TNF-a in the colon of CD patients.
• FIG. 14 shows M. avium subsp. paratuberculosis (MAP)-specific serum Ab responses in IL-10 "7" mice during spontaneous colitis.
• MAP M. avium subsp. paratuberculosis
• FIG. 15 shows histological characteristics of IL-10 "7" mice challenged with M. avium subsp. paratuberculosis (MAP).
• FIG. 16 shows changes in body weight of IL-10 "7" mice after MAP challenge.
• FIG. 17 shows serum cytokine levels in IL-10 _/" mice after MAP challenge.
• FIG. 18 shows anti-peptide #25 Ag (from MPT59)-induced proliferation and IL-2 production by CD4 + T cells from IL-10 " " mice.
• FIG. 19 shows serum CXCR3 ligands and mycobacterial -specific Ab responses in IBD patients.
• FIG. 20 shows changes in SAA levels in IBD patients and in IL-10 7" mice after mycobacterial challenge.
• FIG. 21 shows intestinal histological characteristics of IL-10 7" mice challenged with Mycobacteria.
• FIG. 22 shows serum CXCL9, CXCLIO and CXCLl 1 concentrations in IC patients.
• FIG. 23 shows histological changes after CYP-induced cystitis.
• FIG. 24 shows CXCR3, CXCL9, CXCLIO, and CXCLl 1 mRNA expression in CYP-treated mice.
• FIG. 25 shows upregulated CXCLIO expression during active CD.
• FIG. 26 shows upregulated expression of CXCLl 1 and CXCL9 during active CD.
• FIG. 27 shows upregulated serum concentrations of serum amyloid A (SAA) and IL-6 in CD patients.
• FIG. 28 shows serum IL-12p40 and IFN- ⁇ levels correlate during CD.
• FIG. 29 shows inflammatory cytokine levels during active CD.
• FIG. 30 shows histological characteristics of colitis in normal and CD patients with high serum CXCR3 ligand concentrations.
• FIG. 31 shows CXCR3 ligands and TNFa expression in colons of normal and CD patients by histopathological examination.
• treat refers to a method of alleviating or abrogating a disorder and/or its attendant symptoms.
• prevent refers to a method of barring a subject from acquiring a disorder and/or its attendant symptoms.
• prevent refers to a method of reducing the risk of acquiring a disorder and/or its attendant symptoms.
• anti-inflammatory activity or "anti-inflammatory response” refer to a reduction or prevention of inflammation manifested in a change in cells, such as proliferation, activation, gene expression, and the like.
• a reduction in inflammation may include, for example, reducing the secretion or expression of inflammatory cytokines, chemokines, cytokine/chemokine receptors; adhesion molecules, proteases, and/or immunoglobulins; reducing chemotaxis or migration of cells; reducing the blood
• anti-inflammatory agent refers to a biologic agent which, upon binding to a protein reduces or prevents inflammatory activity or upon binding to a nucleic acid encoding an inflammatory protein product reduces or blocks expression of an mRNA or protein corresponding to the inflammatory protein product.
• Anti-inflammatory agents are to be distinguished from anti-inflammatory small molecule chemical compounds as further described herein.
• anti-inflammatory agents include antibodies, antibody fragments, short interfering RNAs (siRNAs), aptamers, synbodies, binding agents, peptides, aptamer-siRNA chimeras, single stranded antisense oligonucleotides, triplex forming oligonucleotides, ribozymes, external guide sequences, agent-encoding expression vectors, and the like.
• siRNAs short interfering RNAs
• aptamers aptamers
• synbodies binding agents
• binding agents peptides
• aptamer-siRNA chimeras single stranded antisense oligonucleotides
• triplex forming oligonucleotides triplex forming oligonucleotides
• ribozymes external guide sequences
• agent-encoding expression vectors and the like.
• antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site or epitope binding domain that specifically binds
• antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit specific binding to a target antigen.
• specifically bind or “immunoreacts with” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity with other polypeptides.
• antibody also includes antibody fragments that comprise a portion of a full length antibody, generally the antigen binding or variable region thereof.
• anti-inflammatory antibody refers to an antibody or antibody fragment agent.
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
• the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
• Humanized forms of non-human antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and/or capacity.
• donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and/or capacity.
• Bispecific antibodies are antibodies that have binding specificities for at least two different antigens.
• heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells. It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving the use of crosslinking agents.
• they may be prepared by fusing two antibodies or fragments thereof by recombinant DNA techniques known to those of skill in the art.
• nucleic acid refers to a polydeoxyribonucleotide (DNA or an analog thereof) or polyribonucleotide (RNA or an analog thereof) made up of at least two, and preferably ten or more bases linked by a backbone structure.
• nucleic acids may include base analogs ⁇ e.g., inosine) and abasic positions (i.e., a phosphodiester backbone that lacks a nucleotide at one or more positions).
• nucleic acids include single-stranded (ss), double-stranded (ds), or triple-stranded polynucleotides or oligonucleotides of DNA and RNA.
• polynucleotide refers to nucleic acids containing more than 10 nucleotides.
• oligonucleotide refers to a single stranded nucleic acid containing between about 15 to about 100 nucleotides.
• promoter is to be taken in its broadest context and includes transcriptional regulatory elements (TREs) from genomic genes or chimeric TREs therefrom, including the TATA box or initiator element for accurate transcription initiation, with or without additional TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) which regulate activation or repression of genes operably linked thereto in response to developmental and/or external stimuli, and trans-acting regulatory proteins or nucleic acids.
• the promoter may be constitutively active or it may be active in one or more tissues or cell types in a developmentally regulated manner.
• a promoter may contain a genomic fragment or it may contain a chimera of one or more TREs combined together.
• terapéuticaally effective amount of an anti-inflammatory antibody, agent or small molecule inhibitor, or combination thereof refers to an amount effective in the prevention or treatment of a disorder for the treatment of which the anti-inflammatory agent or combination thereof is effective.
• a “disorder” or “disease” is any inflammatory condition that would benefit from treatment with the antibody, agent or small molecule inhibitor.
• IBD inflammatory bowel disease
• Ulcerative colitis is a chronic, episodic, inflammatory disease of the large intestine and rectum characterized by bloody diarrhea. Ulcerative colitis is characterized by chronic inflammation in the colonic mucosa and can be categorized according to location: “proctitis” involves only the rectum, “proctosigmoiditis” affects the rectum and sigmoid colon, “left-sided colitis” encompasses the entire left side of the large intestine, “pancolitis” inflames the entire colon.
• Crohn's disease also called “regional enteritis,” is a chronic autoimmune disease that can affect any part of the gastrointestinal tract but most commonly occurs in the ileum (the area where the small and large intestine meet). Crohn's disease, in contrast to ulcerative colitis, is characterized by chronic inflammation extending through all layers of the intestinal wall and involving the mesentery as well as regional lymph nodes. Whether or not the small bowel or colon is involved, the basic pathologic process is the same.
• Ulcerative colitis and Crohn's disease can be distinguished from each other clinically, endoscopically, pathologically, and serologically in more than 90% of cases; the remainder are considered to be indeterminate IBD.
• mucosal tissue refers to any tissue in which mucosal cells are found, such tissues, include, for example, gastro-intestinal tissues (e.g., stomach, small intestine, large intestine, rectum), uro-genital tissue (e.g., vaginal tissue, penile tissue, urethra), nasal-larynx tissue (e.g., nasal tissue, larynx tissue), mouth (buccal tissue) to name a few.
• gastro-intestinal tissues e.g., stomach, small intestine, large intestine, rectum
• uro-genital tissue e.g., vaginal tissue, penile tissue, urethra
• nasal-larynx tissue e.g., nasal tissue, larynx tissue
• mouth a few.
• mucosal tissues are known and easily identifiable by one of skill in the art.
• inhibitors is a relative term, an agent inhibits a response or condition if the response or condition is quantitatively diminished following administration of the agent, or if it is diminished following administration of the agent, as compared to a reference agent.
• prevents does not necessarily mean that an agent completely eliminates the response or condition, so long as at least one characteristic of the response or condition is eliminated.
• compositions that reduces or prevents an inflammatory response can, but does not necessarily completely eliminate such a response, so long as the response is measurably diminished, for example, by at least about 50%, such as by at least about 70%, or about 80%, or even by about 90% of (that is to 10% or less than) the response in the absence of the agent, or in comparison to a reference agent.
• the term "increased level” refers to a level that is higher than a normal or control level customarily defined or used in the relevant art.
• an increased level of immunostaining in a tissue is a level of immunostaining that would be considered higher than the level of immunostaining in a control tissue by a person of ordinary skill in the art.
• biological sample refers to material of a biological origin, which may be a body fluid or body product such as blood, plasma, urine, saliva, spinal fluid, stool, sweat or breath.
• a biological sample may include tissue samples, cell samples, or combination thereof.
• Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
• CXCL9, CXCL10, and CXCL11 chemokines are ligands for the CXCR3 chemokine receptor.
• CXCL13 chemokine is the ligandsfor the CXCR5 chemokine receptor.
• Each of these chemokine ligands and their receptor are locally upregulated and play a role in various inflammatory diseases, including inflammatory bowel diseases.
• CXCL9, - CXCL10, CXCL11 and CXCL13 chemokines enhance inflammation both in vivo and in vitro.
• CXCR3 and CXCR3 are members of the chemokine receptor family of G protein coupled receptors (GPCRs). Interaction of CXCR3 with CXCL9, CXCL10, and CXCL11 and/or interaction of CXCR5 with CXCL13 activate inflammation.
• GPCRs G protein coupled receptors
• One aspect of the present application relates to methods for inhibiting inflammation using agents that inhibit the expression or activity of CXCL9, CXCL10, CXCL11 CXCL13, CXCR3 or CXCR5.
• Activity include, for example, transcription, translation, intracellular translocation, secretion, signal transduction, phosphorylation by kinases, cleavage by proteases, homophilic and heterophilic binding to other proteins, ubiquitination, and the like.
• a method for treating or preventing an inflammatory condition in a subject comprises administering to a subject diagnosed with an inflammatory disease an effective amount of an anti-inflammatory agent that: (1) inhibits the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, or (2) inhibits the interaction between CXCR3 and any one of CXCL9, CXCL10, and CXCL11 or interaction between CXCR5 and CXCL13, or (3) inhibits a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5.
• a therapeutically effective amount of at least one anti- CXCL9, anti-CXCLlO, anti-CXCLl 1, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is administered to a subject in need thereof as a sole anti-inflammatory agent.
• a therapeutically effective amount of at least one anti-CXCL9, anti- CXCLl 0, anti-CXCLl 1, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is administered to a subject in need thereof as a primary anti-inflammatory agent, in conjunction with the treatment of the subject beforehand, at the same time, or afterward with a therapeutically effective amount of a secondary anti-inflammatory agent.
• An anti-inflammatory agent is a biologic agent capable of reducing or preventing inflammation.
• exemplary anti-inflammatory agent include anti-inflammatory antibodies, short interfering R As (siRNAs), CXCL9-binding agents, CXCLlO-binding agents, CXCL11 -binding agents, CXCL 13 -binding agents, CXCR5 -binding agents and CXCR3 -binding agents, antisense oligonucleotides, ribozymes, triplex forming
• the method comprises administering to a subject diagnosed with an inflammatory disease a therapeutically effective amount of an anti-CXCL9 antibody, an anti-CXCLlO antibody, an anti-CXCLl 1 antibody, an anti-CXCR3 antibody, an anti-CXCL13 antibody, an anti-CXCR5 antibody or a combination thereof, resulting in reduced inflammation.
• Exemplary inflammatory diseases or conditions include, but are not limited to, anaphylaxis, septic shock, osteoarthritis, rheumatoid arthritis, psoriasis, asthma, allergies (e.g., drug, insect, plant, food), atherosclerosis, delayed type hypersensitivity, dermatitis, diabetes mellitus, juvenile onset diabetes, graft rejection, inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, enteritis, and interstitial cystitis; multiple sclerosis, myasthemia gravis, Grave's disease, Hashimoto's thyroiditis, pneumonitis, prostatitis, psoriasis, nephritis, pneumonitis, chronic obstructive pulmonary disease, chronic bronchitis rhinitis, spondyloarthropathies, scheroderma, systemic lupus erythematosus, and thyroiditis.
• anaphylaxis
• the subject is diagnosed with an inflammatory condition that results in elevated CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5 expression.
• the method of treatment further comprises the step of determining whether the level of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR53 expression is elevated in a tissue from the subject, and, if so, administering to the subject a therapeutically effective amount of an anti-inflammatory agent that: (1) inhibits the expression of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5, or (2) inhibits the interaction between CXCR3 and any one of CXCL9, CXCLIO, and CXCLl 1, or the interation between CXCR5 and CXCLl 3, or (3) inhibits a biological activity of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, C
• the therapeutically effective amount of an anti-CXCL9, anti-CXCLl 0, anti-CXCLl 1, anti-CXCLl 3, anti-CXCR3 and/or anti-CXCR5 antiinflammatory agent augments the effectiveness of one or more additional therapeutically effective agents or small molecule agents in inhibiting inflammation.
• the therapeutically effective amount of the anti-CXCL9, anti-CXCLl 0, anti- CXCLl 1, anti-CXCLl 3, anti-CXCR3 and/or anti-CXCR5 anti-inflammatory agent reduces the amount of the one or more additional therapeutically effective agents or small molecule agents required for inhibiting inflammation.
• treatment of a subject with an anti-CXCL9, anti- CXCL10, anti-CXCLl l, anti-CXCL13, anti-CXCR5 and/or anti-CXCR3 anti-inflammatory agent is carried out in conjunction with the treatment of the subject beforehand, at the same time, or afterward with a therapeutically effective amount of at least one secondary agent directed against a chemokine, cytokine, receptor thereof, or derivatives thereof, including soluble receptors and the like.
• a method for enhancing effect of anti-inflammatory therapy comprises administering to a subject who is receiving or has received antiinflammatory therapy an effective amount of an anti-inflammatory agent that (1) inhibits the expression of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5, or (2) inhibits the interaction between any one of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5 or (3) inhibits a biological activity of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5, wherein the agent comprises an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence, or agent
• an anti-inflammatory agent
• the subject is receiving anti-inflammatory therapy.
• the subject has received anti-inflammatory therapy, but has exhibited anti-inflammatory drug-resistance to an anti-inflammatory agent.
• the subject is administered an effective amount of an anti-CXCL9 antibody, an anti-CXCLlO antibody, an anti-CXCLl 1 antibody, an anti- CXCL13 antibody, anti-CXCR3 antibody, an anti-CXCR5 antibody, or combination thereof for .
• An anti-inflammatory agent may include any inhibitor of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5 activity and/or expression.
• exemplary antiinflammatory agents include antibodies, short interfering RNA (siRNA), aptamer-siRNA chimeras, single stranded antisense oligonucleotides, triplex forming oligonucleotides, ribozymes, external guide sequences, agent-encoding expression vectors, and combination thereof.
• An anti-inflammatory antibody may be an anti-chemokine antibody, an anti-chemokine receptor antibody, anti-cytokine antibody, an anti-cytokine receptor antibody, an anti-proinflammatory peptide antibody, or a combination thereof (e.g., bispecific antibody).
• a preferred anti-inflammatory antibody of the present application is one which binds to human CXCL9, CXCLIO, CXCLl 1 or CXCLl 3 and preferably blocks (partially or completely) the ability of CXCL9, CXCLIO, CXCLl 1 or CXCLl 3 to bind and/or activate the CXCR3 or CXCR5 receptor.
• Another preferred antibody of the present invention is one which binds to human CXCR3 or CXCR5 and preferably blocks (partially or completely) the ability of a cell carrying the receptor, such as an epithelial, endothelial or lymphoid cell, from binding to and/or being activated by CXCL9, CXCLIO CXCLl 1 and/or CXCLl 3.
• the anti-CXCL9, anti-CXCLlO, anti-CXCLl l, anti- CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is a monoclonal antibody.
• the anti-CXCL9, anti-CXCLlO, anti-CXCLl l, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is a humanized antibody.
• the anti- CXCL9, anti-CXCLlO, anti-CXCLl l, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is an antibody fragment.
• the anti-CXCL9, anti- CXCLlO, anti-CXCLl 1, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is a humanized antibody fragment.
• the anti-CXCL9, anti-CXCLl 0, anti-CXCLl 1, anti- CXCLl 3, anti-CXCR3 or anti-CXCR5 antibody binds to CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 or CXCR5, respectively, with a kd value in the range of 0.01 pM to 10 ⁇ , 0.01 pM to 1 ⁇ , 0.01 pM to 100 nM, 0.01 pM to 10 nM, 0.01 pM to 1 nM, 0.1 pM to 10 ⁇ , 0.1 pM to 1 ⁇ , 0.1 pM to 100 nM, 0.1 pM to 10 nM, 0.1 pM to 1 nM, 1 pM to 10 ⁇ , 1 pM to 1 ⁇ , 1 pM to 100 nM, 1 pM to 10 nM, 1 pM to 1 ⁇ , 1 pM to 100 nM, 1
• the anti-CXCL9, anti-CXCLlO, anti-CXCLl 1, anti-CXCLl 3, anti-CXCR3 or anti-CXCR5 antibody binds to non-target proteins with a kd value of greater than 100 nM.
• the anti-CXCL9, anti-CXCLlO, anti-CXCLl 1, anti-CXCL13, anti-CXCR3 or anti-CXCR5 antibody binds to the target protein (i.e., CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 or CXCR5, respectively, with a Kd value in the range of 0.01 pM to 100 nM or 0.01 pM to 10 nM, and binds to non-target proteins with a Kd value of greater than 100 nM.
• An anti-inflammatory antibody may be administered in any form suitable for neutralizing CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and/or CXCR5 activity.
• Exemplary antibody or antibody derived fragments may include any member of the group consisting of: IgG, antibody variable region; isolated CDR region; single chain Fv molecule (scFv) comprising a VH and VL domain linked by a peptide linker allowing for association between the two domains to form an antigen binding site; bispecific scFv dimer; minibody comprising a scFv joined to a CH3 domain; diabody (dAb) fragment; single chain dAb fragment consisting of a VH or a VL domain; Fab fragment consisting of VL, VH, CL and CHI domains; Fab' fragment, which differs from a Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region; Fab'-SH fragment, a Fab' fragment in which the cysteine residue(s) of the constant domains bear a free thiol group; F(ab') 2
• Fv, scFv, or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains.
• any or all of the targeting domains therein and/or Fc regions may be "humanized” using methodologies well known to those of skill in the art.
• the anti-inflammatory antibody is modified to remove the Fc region.
• an anti-CXCR3 antibody or antibody fragment thereof is conjugated to or fused to a second antibody or antibody binding fragment to enhance its binding to target cells carrying the CXCR3 receptor.
• an anti-inflammatory agent may be conjugated to one or more secondary anti-inflammatory agent(s), such as an anti-inflammatory small molecule(s) to provide a further level of anti-inflammatory activity.
• secondary anti-inflammatory agent(s) such as an anti-inflammatory small molecule(s)
• Short interfering RNAs fsiRNAs
• siRNA is a double-stranded RNA that can be engineered to induce sequence-specific post-transcriptional gene silencing of mRNAs corresponding to any one of the above-described chemokine, cytokine or receptors thereof.
• siRNAs exploit the mechanism of RNA interference (RNAi) for the purpose of "silencing” gene expression of targeted chemokine-, cytokine- or receptor genes. This "silencing” was originally observed in the context of transfecting double stranded RNA (dsRNA) into cells. Upon entry therein, the dsRNA was found to be cleaved by an RNase Ill-like enzyme, Dicer, into double stranded small interfering RNAs (siRNAs) 21-23 nucleotides in length containing 2 nucleotide overhangs on their 3' ends.
• RNAi RNA interference
• RISC RNAi induced silencing complex
• the anti-inflammatory agent comprises a synthetic siRNA.
• Synthetically produced siRNAs structurally mimic the types of siRNAs normally processed in cells by the enzyme Dicer.
• Synthetically produced siRNAs may incorporate any chemical modifications to the RNA structure that are known to enhance siRNA stability and functionality.
• the siRNAs may be synthesized as a locked nucleic acid (LNA)-modified siRNA.
• LNA locked nucleic acid
• An LNA is a nucleotide analogue that contains a methylene bridge connecting the 2'-oxygen of the ribose with the 4'carbon. The bicyclic structure locks the furanose ring of the LNA molecule in a 3'-endo conformation, thereby structurally mimicking the standard RNA monomers.
• the anti-inflammatory agent may comprise an expression vector engineered to transcribe a short double-stranded hairpin-like RNA
• shRNA that is processed into a targeted siRNA inside the cell.
• the shRNAs can be cloned in suitable expression vectors using kits, such as Ambion's SILENCER ® siRNA Construction Kit, Imgenex's GENESUPPRESSORTM Construction Kits, and Invitrogen's BLOCK-ITTM inducible RNAi plasmid and lentivirus vectors.
• Synthetic siRNAs and shRNAs may be designed using well known algorithms and synthesized using a conventional DNA/RNA synthesizer.
• a variety of chemokine-, cytokine- and receptor-targeted siRNAs may be commercially obtained from Origen
• the anti-inflammatory agent is a CXCL9-, CXCLl 0-, CXCLl 1-, CXCLl 3-, CXCR3- or CXCR5-binding agent.
• the binding agent may comprise any non-antibody protein, peptide, or synthetic binding molecule, such as an aptamer or synbody, which is capable of specifically binding directly or indirectly to CXCL9, CXCLl 0, CXCLl 1, CXCLl 3, CXCR3 or CXCR5 so as to inhibit the interaction and/or activation between CXCR3 and CXCL9, CXCL10 or CXCLl 1 ; or the interaction and/or activation between CXCR5 and CXCLl 3, or which inhibits a biological activity of CXCL9, CXCLl 0, CXCLl 1, CXCL13, CXCR3 and/or CXCR5, which is associated with reducing or preventing an inflammatory response.
• the CXCL9-, CXCLl 0-, CXCLl 1-, CXCLl 3, CXCR3 and/or CXCR5- binding agents may be produced by any conventional method for generating high-affinity binding ligands, including SELEX, phage display, and other methodologies, including combinatorial chemistry- and/or high throughput methods known to those of skill in the art.
• An aptamer is a nucleic acid version of an antibody that comprises a class of oligonucleotides that can form specific three dimensional structures exhibiting high affinity binding to a wide variety of cell surface molecules, proteins, and/or macromolecular structures.
• Aptamers are commonly identified by an in vitro method of selection sometimes referred to as Systematic Evolution of Ligands by Exponential enrichment or "SELEX". SELEX typically begins with a very large pool of randomized polynucleotides which is generally narrowed to one aptamer ligand per molecular target.
• aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
• An aptamer can be chemically linked or conjugated to the above described nucleic acid inhibitors to form targeted nucleic acid inhibitors, such as aptamer-siRNA chimeras.
• An aptamer-siRNA chimera contains a targeting moiety in the form of an aptamer which is linked to an siRNA.
• a cell internalizing aptamer When using an aptamer-siRNA chimera, it is preferable to use a cell internalizing aptamer. Upon binding to specific cell surface molecules, the aptamer can facilitate internalization into the cell where the nucleic acid inhibitor acts. In one
• both the aptamer and the siRNA comprises RNA.
• the aptamer and the siRNA may comprise any nucleotide modifications as further described herein.
• the aptamer comprises a targeting moiety specifically directed to binding cells expressing the chemokine-, cytokine- and/or receptor target genes, such as lymphoid, epithelial cell, and/or endothelial cells.
• Synbodies are synthetic antibodies produced from libraries comprised of strings of random peptides screened for binding to target proteins of interest. Synbodies are described in US 2011/0143953 and Diehnelt et al, PLoS One, 5(5):el0728 (2010).
• CXCL9-, CXCL10-, CXCL11-, CXCL13-, CXCR3-, CXCR5-binding agents can be engineered to bind target molecules very tightly with Kds between 10 "10 to 10 "12 M.
• the CXCL9-, CXCL10-, CXCL11-, CXCL13-, CXCR3-, CXCR5-binding agents can be engineered to bind target molecules very tightly with Kds between 10 "10 to 10 "12 M.
• CXCL11-, CXCL13-, CXCR3- or CXCR5-binding agent bind the target molecule with a Kd less than 10 ⁇ 6 , less than 10 "8 , less than 10 "9 , less than 10 "10 , or less than 10 "12 M.
• the anti-inflammatory inhibitor agent may comprise an antisense oligonucleotide or polynucleotide capable of inhibiting the expression of CXCL9, CXCLIO, CXCL11, CXCL13, CXCR3 and/or CXCR5.
• the antisense may comprise an antisense oligonucleotide or polynucleotide capable of inhibiting the expression of CXCL9, CXCLIO, CXCL11, CXCL13, CXCR3 and/or CXCR5.
• oligonucleotide or polynucleotide may comprise a DNA backbone, RNA backbone, or chemical derivative thereof.
• polynucleotide comprises a single stranded antisense oligonucleotide or polynucleotide targeting for degradation.
• the anti-inflammatory inhibitor agent comprises a single stranded antisense oligonucleotide complementary to a CXCL9, CXCLIO, CXCL11, CXCL12, CXCR3 or CXCR5 mRNA sequence.
• the single stranded antisense oligonucleotide or polynucleotide may be synthetically produced or it may be expressed from a suitable expression vector.
• the antisense nucleic acid is designed to bind via
• the antisense oligonucleotide is chemically or structurally modified to promote nuclease stability and/or increased binding.
• the antisense oligonucleotides are modified to produce oligonucleotides with nonconventional chemical or backbone additions or substitutions, including but not limited to peptide nucleic acids (PNAs), locked nucleic acids (LNAs), morpholino backboned nucleic acids, methylphosphonates, duplex stabilizing stilbene or pyrenyl caps, phosphorothioates, phosphoroamidates, phosphotriesters, and the like.
• PNAs peptide nucleic acids
• LNAs locked nucleic acids
• morpholino backboned nucleic acids methylphosphonates
• duplex stabilizing stilbene or pyrenyl caps phosphorothioates
• phosphoroamidates phosphotriesters, and the like.
• the modified oligonucleotides may incorporate or substitute one or more of the naturally occurring nucleotides with an analog; internucleotide modifications incorporating, for example, uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
• phosphorodithioates etc.
• modifications incorporating intercalators e.g., acridine, psoralen, etc.
• chelators e.g., metals, radioactive metals, boron, oxidative metals, etc.
• alkylators e.g., alpha anomeric nucleic acids, etc.
• the single stranded oligonucleotides are internally modified to include at least one neutral charge in its backbone.
• the single stranded oligonucleotides are internally modified to include at least one neutral charge in its backbone.
• oligonucleotide may include a methylphosphonate backbone or peptide nucleic acid (PNA) complementary to the target-specific sequence. These modifications have been found to prevent or reduce helicase-mediated unwinding.
• PNA peptide nucleic acid
• the use of uncharged probes may further increase the rate of hybridization to polynucleotide targets in a sample by alleviating the repulsion of negatively-charges nucleic acid strands in classical hybridization.
• PNA oligonucleotides are uncharged nucleic acid analogs for which the phosphodiester backbone has been replaced by a polyamide, which makes PNAs a polymer of 2-aminoethyl-glycine units bound together by an amide linkage.
• PNAs are synthesized using the same Boc or Fmoc chemistry as are use in standard peptide synthesis. Bases (adenine, guanine, cytosine and thymine) are linked to the backbone by a methylene carboxyl linkage. Thus, PNAs are acyclic, achiral, and neutral.
• Methylphosphonate-containing oligonucleotides are neutral DNA analogs containing a methyl group in place of one of the non-bonding phosphoryl oxygens.
• Oligonucleotides with methylphosphonate linkages were among the first reported to inhibit protein synthesis via anti-sense blockade of translation.
• the phosphate backbone in the oligonucleotides may contain phosphorothioate linkages or phosphoroamidates. Combinations of such
• oligonucleotide linkages are also within the scope of the present invention.
• the oligonucleotide may contain a backbone of modified sugars joined by phosphodiester intemucleotide linkages.
• the modified sugars may include furanose analogs, including but not limited to 2-deoxyribofuranosides, a-D- arabinofuranosides, a-2'-deoxyribofuranosides, and 2',3'-dideoxy-3'-aminoribofuranosides.
• the 2-deoxy-P-D-ribofuranose groups may be replaced with other sugars, for example, ⁇ -D-ribofuranose.
• ⁇ -D-ribofuranose may be present wherein the 2-OH of the ribose moiety is alkylated with a CI -6 alkyl group (2-(0 ⁇ Cl-6 alkyl) ribose) or with a C2-6 alkenyl group (2-(0 ⁇ C2-6 alkenyl) ribose), or is replaced by a fluoro group (2-fluororibose).
• LNA locked nucleic acids
• exemplary LNA oligonucleotides include modified bicyclic monomeric units with a 2'-0-4'-C methylene bridge, such as those described in U.S. Patent No. 6,268,490, the disclosures of which are incorporated by reference herein.
• Chemically modified oligonucleotides may also include, singly or in any combination, 2'-position sugar modifications, 5-position pyrimidine modifications (e.g, 5-(N- benzylcarboxyamide)-2'-deoxyuridine, 5-(N-isobutylcarboxyamide)-2'-deoxyuridine, 5-(N- [2-( 1 H-indole-3 yl)ethyl] carboxyamide)-2'-deoxyuridine, 5-(N-[ 1 -(3 -trimethylammonium) propyl]carboxyamide)-2'-deoxyuridine chloride, 5-(N-napthylcarboxyamide)-2'- deoxyuridine, and 5-(N-[l-(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine), 8- position purine modifications, modifications at exocyclic amines, substitution of 4- thiouridine, substitution of 5-bromo- or 5-iodo-
• Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahymena ribozymes.
• ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo.
• Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates, such as CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 or CXCR5 mRNAs.
• Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence.
• TFOs Triplex forming oligonucleotides
• TFOs Triplex forming oligonucleotides
• TFOs When TFOs interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. TFOs can bind target regions with high affinity and specificity.
• the triplex forming molecules bind the target molecule with a d less than 10 "6 , 10 "8 , 10 "10 , or 10 "12 .
• Exemplary TFOs for use in the present invention include PNAs, LNAs, and LNA modified PNAs, such as Zorro-LNAs.
• ETSs External guide sequences
• EGSs External guide sequences
• RNase P RNase P
• EGSs can be designed to specifically target an mRNA molecule of choice.
• RNAse P aids in processing transfer RNA (tRNA) within a cell.
• Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate.
• eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotic cells.
• a method for treating or preventing an inflammatory condition in a subject comprises administering to a subject diagnosed with an inflammatory disease an effective amount of an expression vector expressing an anti-CXCL9, anti- CXCL10, anti-CXCLl 1, anti-CXCL13 agent, anti-CXCR3 agent and/or anti-CXCR5 agent.
• the method comprises administering to a subject diagnosed with an inflammatory disease an effective amount of an expression vector expressing an an anti- CXCL9, anti-CXCLlO, anti-CXCLl l, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody.
• the method comprises administering to a subject diagnosed with an inflammatory disease an effective amount of an expression vector expressing an an anti-CXCL9, anti-CXCLlO, anti-CXCLl l, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 siRNA.
• the expression vector can be any expression vector capable of delivering and expressing a polynucleotide encoding an anti-CXCL9, anti-CXCLlO, anti- CXCL11, anti-CXCL13, anti-CXCR5 and/or anti-CXCR3 agent including antibodies, siRNAs, antisense oligonucleotides or polynucleotides, and the like.
• expression vector includes any nucleic acid capable of directing expression of a nucleic acid.
• Expression vectors may be delivered to cells using two primary delivery schemes: viral-based delivery systems using viral vectors and non- viral based delivery systems using, for example, plasmid vectors.
• viral-based delivery systems using viral vectors
• non- viral based delivery systems using, for example, plasmid vectors.
• plasmid vectors such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, these methods can be used to target certain diseases and cell populations by using the targeting characteristics inherent to the carrier or engineered into the carrier.
• the nucleic acids that are delivered to cells contain one or more transcriptional regulatory elements, including promoters and/or enhancers, for directing the expression of siRNAs.
• a promoter comprises a DNA sequence that functions to initiate transcription from a relatively fixed location in regard to the transcription start site.
• a promoter contains TRE elements required for basic interaction of RNA polymerase and transcription factors, and may operate in conjunction with other upstream elements and response elements.
• Preferred promoters are those capable of directing expression in a target cell of interest.
• the promoters may include constitutive promoters (e.g., HCMV, SV40, elongation factor- la (EF-la)) or those exhibiting preferential expression in a particular cell type of interest.
• Enhancers generally refer to DNA sequences that function away from the transcription start site and can be either 5' or 3' to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase and/or regulate transcription from nearby promoters.
• the promotor and/or enhancer may be specifically activated either by light or specific chemical inducing agents.
• inducible expression systems regulated by administration of tetracycline or dexamethasone, for example, may be used.
• gene expression may be enhanced by exposure to radiation, including gamma irradiation and external beam radiotherapy (EBRT), or alkylating chemotherapeutic drugs.
• EBRT gamma irradiation and external beam radiotherapy
• TREs Cell or tissue-specific transcriptional regulatory elements
• Expression vectors generally contain sequences for transcriptional termination, and may additionally contain one or more elements positively affecting mRNA stability.
• An expression vector may further include an internal ribosome entry site (IRES) between adjacent protein coding regions to facilitate expression two or more proteins from a common mRNA in an infected or transfected cell.
• the expression vectors may further include nucleic acid sequence encoding a marker product. This marker product may be used to determine if the gene has been delivered to the cell and is is being expressed.
• Preferred marker genes are the E. coli lacZ gene, which encodes ⁇ -galactosidase, and green fluorescent protein (GFP).
• the anti-CXCL9, anti- CXCL10, anti-CXCLl l, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5- antibody- or siRNA encoding sequences are delivered from viral-derived expression vectors.
• Exemplary viral vectors may include or be derived from adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, poxvirus, HIV virus, lentivirus, retrovirus, Sindbis and other RNA viruses, and the like. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors.
• Retroviruses include Murine Moloney Leukemia virus (MMLV), HIV and other lentivirus vectors.
• Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells.
• Poxviral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.
• Viral delivery systems typically utilize viral vectors having one or more genes removed and with and an exogenous gene and/or gene/promotor cassette being inserted into the viral genome in place of the removed viral DNA. The necessary functions of the removed gene(s) may be supplied by cell lines which have been engineered to express the gene products of the early genes in trans.
• Non-viral expression vectors are utilized for delivery of plasmid vectors or other bioactive non nucleic acid agents using lipid formulations comprising, for example, liposomes, such as cationic liposomes ⁇ e.g., DOTMA, DOPE, DC-cholesterol) and anionic liposomes.
• liposomes can be further conjugated to one or more proteins or peptides to facilitate targeting to a particular cell, if desired.
• Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract.
• an anti-inflammatory agent can be administered as a component of a microcapsule or nanoparticle that can be targeted to a cell type of interest using targeting moieties described herein or that can be designed for slow release of one or more anti-inflammatory agent (s) in accordance with a predetermined rate of release or dosage.
• the nucleic acids may be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Arlington, AZ).
• the nucleic acids may be in solution or suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to cells of interest), receptor mediated targeting of DNA through cell specific ligands or viral vectors targeting e.g., lymphoid, epithelial or endothelial cells. In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored
• the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of
• receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration.
• a therapeutically effective amount of at least one anti- CXCL9, anti-CXCLlO, anti-CXCLl 1, and/or anti-CXCR3 antibody is administered to a subject in need thereof in conjunction with a secondary anti-inflammatory agent.
• the a secondary anti-inflammatory agent may be given before, at the same time, or after the administration of the antibody or antibodies.
• the secondary anti-inflammatory agent is directed against a chemokine, cytokine, receptor thereof, or combination thereof.
• the secondary anti-inflammatory agent may comprise an anti-inflammatory antibody, short interfering RNA (siRNA), chemokine and chemokine receptor binding agents, antisense oligonucleotides, triplex forming oligonucleotides, ribozymes, external guide sequences, agent-encoding expression vectors or an anti-inflammatory small molecule chemical compound.
• the secondary anti-inflammatory agent comprise another an anti-inflammatory antibody directed to determinants on CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5.
• the secondary antiinflammatory agent comprises an antibody or an agent directed against a secondary chemokine, cytokine, or receptor thereof.
• the secondary anti-inflammatory agent is an antiinflammatory agent directed against a chemokine, cytokine or receptor thereof.
• chemokine or chemokine receptor targeted in accordance with the present invention including protein and cDNA sequences, respectively, from NIH-NCBI GenBank, are described in Table 1.
• the secondary anti-inflammatory agent binds specifically to a cytokine or cytokine receptor.
• cytokine or cytokine receptor targets and/or their reactive inhibitory products include, but are not limited to, interferon-a, - ⁇ , or - ⁇ ; tumor necrosis factor (TNF)-alpha, e.g., (infliximab (REMICADE ® ), adalimumab (HUMIRA ® ), D2E7 (BASF Pharma), and HUMICADE ® (Celltech)); soluble forms of the TNF receptor (etanercept (ENBREL ® )); CD20, including rituximab (RITUXAN ® ), humanized 2H7, 2F2 (Hu-Max-CD20), human CD20 antibody (Genmab), and humanized A20 antibody (Immunomedics); TNF-beta; interleukin-2 (IL-2), including daclizuma
• TNF tumor necrosis
• interleukin-1 receptor including IL-1 receptor agents, such as anakinra (KINERET ® ); LFA-1, including anti-CD 1 la, anti-CD 18 antibodies, and soluble peptides containing a LFA- 3 binding domain; anti-L3T4 antibodies; interleukin-1 ⁇ (IL- ⁇ ); interleukin-8 (IL-8);
• IL-1 receptor agents such as anakinra (KINERET ® )
• LFA-1 including anti-CD 1 la, anti-CD 18 antibodies, and soluble peptides containing a LFA- 3 binding domain
• anti-L3T4 antibodies interleukin-1 ⁇ (IL- ⁇ ); interleukin-8 (IL-8);
• IFN- ⁇ interferon- ⁇
• VEGF vascular endothelial growth factor
• LIF leukemia inhibitory factor
• CP-1 monocyte chemoattractant protein-1
• RANTES interleukin-10
• IL-12 interleukin-12
• MMP2 matrix metalloproteinase 2
• IP- 10 macrophage
• inflammatory protein la MlPla
• macrophage inflammatory protein 1 ⁇ ⁇
• pan-T including anti-CD3 or anti-CD4/CD4a antibodies
• BAFF zTNF4, BLyS
• BAFF receptor BR3
• anti-idiotypic antibodies for MHC antigens and MHC fragments CD40 receptor and anti-CD40 ligand (CD154); CTLA4-Ig
• T-cell receptor antibodies such as T10B9; heterologous anti-lymphocyte globulin; streptokinase; transforming growth factor-beta (TGF- beta); streptodomase; RNA or DNA from the host; chlorambucil; deoxyspergualin; T-cell receptor; and T-cell receptor fragments.
• Anti-inflammatory small molecule chemical compounds include, but are not are not limited to, small molecule compounds or medicaments selected from the group consisting of analgesics, such as aspirin or TYLENOL ® (Acetaminophen); 2- amino-6-aryl-5-substituted pyrimidines; nonsteroidal anti-inflammatory drugs (NSAIDs), such as acemetacin, amtolmetin, azapropazone, benorilate, benoxaprofen, benzydamine hydrochloride, bromfenal, bufexamac, butibufen, carprofen, celecoxib, choline salicylate, diclofenac dipyone, droxicam, etodolac, etofenamate, etoricoxib, felbinac, fentiazac, floctafenine, ibuprofen,
• analgesics such as aspirin or TYLENOL ® (Acet
• corticosteroids including oral glucocorticosteroids or glucocorticoid analogs, e.g., prednisone; methylprednisolone, including SOLU-MEDROL ® and methylprednisolone sodium succinate, triamcinolone, and betamethasone,
• dexamethasone aminosalicylate; azathioprine, calcineurin inhibitors, such as cyclosporine, tacrolimus (FK-506), and sirolimus (rapamycin); RS-61443 (mycophenolate mofetil);
• dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); anti-malarial agents, such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide;
• AZULFIDINE sulfasalazine
• PKAQUENIL hydroxychloroquine
• the primary anti-inflammatory agent and the secondary anti-inflammatory agent are directed against the same chemokine/chemokine receptor. In other embodiments, the primary anti-inflammatory agent and the secondary antiinflammatory agent are directed against different chemokine/chemokine receptor. Table 2 describes the association between inflammatory disease and certain chemikine and chemikine receptors.
• Atherosclerosis CXCL1, CXCL2, CXCL3, CXCL4, CXCR1, CXCR2
• the anti-inflammatory agents may be administered to the subject with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
• known methods such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
• the anti-inflammatory agent(s) may be administered directly to an infiammed tissue.
• mucosal tissue may be directly contacted with the anti-inflammatory agent(s).
• dermal tissue may be contacted directly with the anti-inflammatory agent(s) in a cream, lotion, or ointment.
• pulmonary tissue e.g., bronchoalveolar tissue may be contacted by inhalation of a liquid or powder aspirate.
• the anti-inflammatory agent may also be placed on a solid support such as a sponge or gauze for administration against the target chemokine to the affected tissues.
• the anti-inflammatory agents of the instant application can be administered in the usually accepted pharmaceutically acceptable carriers.
• Acceptable carriers include, but are not limited to, saline, buffered saline, and glucose in saline.
• Solid supports, liposomes, nanoparticles, microparticles, nanospheres or microspheres may also be used as carriers for administration of the anti-inflammatory agents.
• the appropriate dosage ("therapeutically effective amount") of the antiinflammatory agents will depend, for example, on the condition to be treated, the severity and course of the condition, the mode of administration, whether the antibody or agent is administered for preventive or therapeutic purposes, the bioavailability of the particular agent(s), previous therapy, the age and weight of the patient, the patient's clinical history and response to the antibody, the type of the anti-inflammatory agent used, discretion of the attending physician, etc.
• the anti-inflammatory agent is suitably administered to the patent at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards.
• the anti-inflammatory agent may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.
• the therapeutically effective amount of the antiinflammatory agent e.g., antibodies and/or anti-inflammatory small molecule compounds
• the therapeutically effective amount of the antiinflammatory agent will be in the range of about 1 ng/kg body weight/day to about 100 mg/kg body weight/day whether by one or more administrations.
• each antiinflammatory agent is administered in the range of from about 1 ng/kg body weight/day to about 10 mg/kg body weight/day, about 1 ng/kg body weight/day to about 1 mg/kg body weight/day, about 1 ng/kg body weight/day to about 100 ⁇ g/kg body weight/day, about 1 ng/kg body weight/day to about 10 ⁇ g/kg body weight/day, about 1 ng/kg body weight/day to about 1 ⁇ g/kg body weight/day, about 1 ng/kg body weight/day to about 100 ng/kg body weight/day, about 1 ng/kg body weight/day to about 10 ng kg body weight/day, about 10 ng/kg body weight/day to about 100 mg/kg body weight/day, about 10 ng/kg body weight/day to about 10 mg/kg body weight/day, about 10 ng/kg body weight/day to about 1 mg/kg body weight/day, about 10 ng/kg body weight/day to about 100 ⁇ g/kg body weight/day, about 10
• the anti-inflammatory agent e.g., antibodies and/or anti-inflammatory small molecule compounds
• the anti-inflammatory agent is administered at a dose of 500 ⁇ g to 20 g every three days, or 25 mg/kg body weight every three days.
• each anti-inflammatory agent is administered in the range of about 10 ng to about 100 ng per individual administration, about 10 ng to about 1 ⁇ g per individual administration, about 10 ng to about 10 ⁇ g per individual administration, about 10 ng to about 100 ⁇ g per individual administration, about 10 ng to about 1 mg per individual administration, about 10 ng to about 10 mg per individual administration, about 10 ng to about 100 mg per individual administration, about 10 ng to about 1000 mg per injection, about 10 ng to about 10,000 mg per individual administration, about 100 ng to about 1 ⁇ g per individual administration, about 100 ng to about 10 ⁇ g per individual administration, about 100 ng to about 100 ⁇ g per individual administration, about 100 ng to about 1 mg per individual administration, about 100 ng to about 10 mg per individual administration, about 100 ng to about 100 mg per individual administration, about 100 ng to about 1000 mg per injection, about 100 ng to about 10,000 mg per individual administration, about 1 ⁇ g to about 10 ⁇ g per individual administration, about 1 ⁇ g per individual administration, about 10 ng
• administration about 10 ⁇ g to about 100 ⁇ g per individual administration, about 10 ⁇ g to about 1 mg per individual administration, about 10 ⁇ g to about 10 mg per individual administration, about 10 ⁇ g to about 100 mg per individual administration, about 10 ⁇ g to about 1000 mg per injection, about 10 ⁇ g to about 10,000 mg per individual administration, about 100 ⁇ g to about 1 mg per individual administration, about 100 ⁇ g to about 10 mg per individual administration, about 100 ⁇ g to about 100 mg per individual administration, about 100 ⁇ g to about 1000 mg per injection, about 100 ⁇ g to about 10,000 mg per individual administration, about 1 mg to about 10 mg per individual administration, about 1 mg to about 100 mg per individual administration, about 1 mg to about 1000 mg per injection, about 1 mg to about 10,000 mg per individual administration, about 10 mg to about 100 mg per individual administration, about 10 mg to about 1000 mg per injection, about 10 mg to about 10,000 mg per individual administration, about 100 mg to about 1000 mg per injection, about 100 mg to about 10,000 mg per individual administration and about 1000 mg to about 10,000 mg per individual administration.
• the dosage will be dependant on the condition, size, age and condition of the patient.
• TNBS 2,4,6-trinitrobenesulfonic acid/ethanol
• DSS dextran sulfate sodium
• Another colitis model uses dextran sulfate sodium (DSS), which induces an acute colitis manifested by bloody diarrhea, weight loss, shortening of the colon and mucosal ulceration with neutrophil infiltration.
• DSS-induced colitis is characterized histologically by infiltration of inflammatory cells into the lamina intestinal, with lymphoid hyperplasia, focal crypt damage, and epithelial ulceration. These changes are thought to develop due to a toxic effect of DSS on the epithelium and by phagocytosis of lamina limba cells and production of TNF-alpha and IFN-gamma.
• DSS is regarded as a T cell-independent model because it is observed in T cell-deficient animals such as SCID mice.
• the administration of the anti-inflammatory agent of the present appliction can be evaluated in the TNBS or DSS models for amelioration of gastrointestinal disease.
• CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and CXCR5 are believed to play a role in the inflammatory response in inflammatory bowel disorders, including colitis, and the neutralization of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR3 and CXCR5 activity by administrating the anti-inflammatory agent of the present application can provide a potential therapeutic approach for gastrointestinal inflammatory diseases, including IBD.
• the particular chemokines which give rise to inflammatory diseases differ with the disease. They also differ among individuals. Hence, it is wise, when treating an individual, to identify the particular chemokines which are increased in the tissues of the patient. By exposing patient tissue samples to the particular antibodies against each of the chemokines and evaluating the amount of antibody/chemokine binding, it is possible to evaluate the level of expression for each chemokine to enable a determination of the appropriate type and amount of antibodies to administer for a given inflammatory disease.
• the antibody may be administered, as appropriate or indicated, a single dose as a bolus or by continuous infusion, or as multiple doses by bolus or by continuous infusion. Multiple doses may be administered, for example, multiple times per day, once daily, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6 weeks or monthly. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.
• compositions and Kits for Treating or Preventing Inflammatory Conditions are Compositions and Kits for Treating or Preventing Inflammatory Conditions
• compositions and kits for treating or preventing inflammatory conditions comprises an anti-inflammatory agent capable of (1) inhibiting the expression of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR5 and/or CXCR3; (2) inhibiting the interaction between any one of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR5 and/or CXCR3, or (3) inhibiting a biological activity of CXCL9, CXCLIO, CXCLl 1, CXCLl 3, CXCR5 and/or CXCR3, wherein the anti-inflammatory agent is an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence, agent-encoding expression
• siRNA short interfering RNA
• composition of the present invention may contain a single type of antibody directed against any one of CXCL9, CXCL10, CXCL11, CXCL13, CXCR5 and CXCR3, or two or more antibodies directed against the same chemokine or chemokine receptor, different chemokines or chemokine receptors, or combinations thereof as described above.
• the composition may also contain therapeutically effective amounts of other antiinflammatory agents as described above.
• the language "pharmaceutically acceptable carrier” is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, lubricants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
• the use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions.
• the pharmaceutically acceptable carrier comprises serum albumin.
• the pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
• routes of administration include parenteral, e.g., intrathecal, intra-arterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine; propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as
• ethylenediaminetetraacetic acid ethylenediaminetetraacetic acid
• buffers such as acetates, citrates or phosphates
• agents for the adjustment of tonicity such as sodium chloride or dextrose.
• pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
• the injectable composition should be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the requited particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens,
• chlorobutanol phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a neuregulin) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• the active compound e.g., a neuregulin
• dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
• Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
• the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
• a lubricant such as magnesium stearate or Stertes
• a glidant such as colloidal silicon dioxide
• the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• Systemic administration can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
• the pharmaceutical compositions are formulated into ointments, salves, gels, or creams as generally known in the art.
• the pharmaceutical composition is formulated for sustained or controlled release of the active ingredient.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from e.g. Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions
• liposomes targeted to infected cells with monoclonal antibodies to viral antigens can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
• Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
• Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
• Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
• the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
• IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
• Such information can be used to more accurately determine useful doses in humans.
• the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
• Primer Design Messenger RNA sequences for CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 were obtained from the NIH-NCBI gene bank database (Table 1). Primers were designed using the BeaconJ 2.0 computer program. Thermodynamic analysis of the primers was conducted using computer programs: Primer Premier J and MIT Primer 3. The resulting primer sets were compared against the entire human genome to confirm specificity.
• RNA contamination was removed from these samples by treatment with 10 U/ ⁇ of RNase free DNase (Invitrogen, San Diego, Calif.) for 15 minutes at 37° C. RNA was then precipitated and resuspended in RNA Secure (Ambion, Austin, Tex.). cDNA was generated by reverse transcribing approximately 2 ⁇ g of total RNA using Taqman7 reverse transcription reagents (AppliedBiosystems, Foster City, Calif.) according to manufacturers protocols.
• RNase free DNase Invitrogen, San Diego, Calif.
• cDNAs were amplified with specific human cDNA primers, to CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23- 1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2, using SYBR7 Green PCR master mix reagents (Applied Biosystems) according to manufacturers protocol.
• SYBR7 Green PCR master mix reagents (Applied Biosystems) according to manufacturers protocol.
• the level of copies of mRNA of these targets were evaluated by real-time PCR analysis using the BioRad Icycler and software (Hercules, Calif).
• Anti-sera preparation The 15 amino acid peptides from chemokines CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23- 1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 (Table 1) were synthesized (Sigma Genosys, The Woodlands, Tex.) and conjugated to hen egg lysozyme (Pierce, Rockford, 111.) to generate the antigens for subsequent immunizations for anti-sera preparation or monoclonal antibody generation.
• chemokine peptide conjugates were quantified by the chromogenic Limulus amebocyte lysate assay (Cape Cod, Inc., Falmouth, Miss.) and shown to be ⁇ 5 EU/mg.
• 100 ⁇ g of the antigen was used as the immunogen together with complete Freund's adjuvant Ribi Adjuvant system (RAS) for the first immunization in a final volume of 1.0 ml. This mixture was administered in 100 ml aliquots on two sites of the back of the rabbit subcutaneously and 400 ml intramuscularly in each hind leg muscle.
• Ribi Adjuvant system Ribi Adjuvant system
• chemokine peptide conjugates were quantified by the chromogenic Limulus amebocyte lysate assay (Cape Cod, Inc., Falmouth, Miss.) and shown to be ⁇ 5 EU/mg. 100 ⁇ g of the antigen was used as the immunogen together with complete Freund's adjuvant Ribi Adjuvant system (RAS) for the first immunization in a final volume of 200 ⁇ . This mixture was
• mice subcutaneously administered in 100 ⁇ aliquots at two sites of the back of a rat, mouse, or immunoglobulin-humanized mouse. Two weeks later, animals received 100 ⁇ g of the antigen in addition to incomplete Freund's adjuvant for 3 subsequent immunizations.
• Serum were collected and when anti -CXCL9, -CXCL10, -CXCL11, -CCRL1, -CCRL2, -CCR5, - CCL1, -CCL2, -CCL3, -CCL4, -CCL4L1, -CCL5, -CCL7, -CCL8, -CCL14-1, -CCL14-2, - CCL14-3, -CCL15-1, -CCL15-2, -CCL16, -CCL19, -CCL23-1, -CCL23-2, -CCL24, -CCL26, -CCR6, -CCL20, and -CCL25, -CCL25-1, -CCL25-2 antibody titers reached 1 :2,000,000, hosts were sacrificed and splenocytes were isolated for hybridoma generation.
• B cells from the spleen or lymph nodes of immunized hosts were fused with immortal myeloma cell lines ⁇ e.g., YB2/0).
• Hybridomas were next isolated after selective culturing conditions ⁇ i.e., HAT-supplemented media) and limiting dilution methods of hybridoma cloning.
• Cells that produce antibodies with the desired specificity were selected using ELIS A.
• Hybridomas from normal rats or mice were humanized with molecular biological techniques in common use. After cloning a high affinity and prolific hybridoma, antibodies were isolated from ascites or culture supernatants and adjusted to a titer of
• TMB Tetramethylbenzidine
• T helper cell derived cytokines, IL-la, IL- ⁇ , IL-2, IL-12, IFN- ⁇ , TNF-a, in serum were also determined by Beadlyte mouse multi-cytokine detection system kit provided by BioRad, following manufacturer instructions. Filter bottom plates were rinsed with 100 ⁇ of bio-plex assay buffer and removal using a Millipore Multiscreen Separation Vacuum Manifold System (Bedford, Mass.), set at 5 in Hg. IL-la, IL- ⁇ , IL-2; IL-12, IFN- ⁇ , TNF-a beads in assay buffer were added into wells.
• SAA Serum Amyloid Protein A
• the SAA levels were determined by ELISA using a kit supplied by Biosource International, (Camarillo, Calif). Briefly, 50 ⁇ of SAA-specific monoclonal antibody solution was used to coat micro-titer strips to capture SAA. Serum samples and standards were added to wells and incubated for 2 hours at RT. After washing in the assay buffer, the HRP-conjugated anti-SAA monoclonal antibody solution was added and incubated for 1 hour at 37° C. After washing, 100 ⁇ Tetramethylbenzidine (TMB) substrate solution was added and the reaction was stopped after incubation for 15 minutes at RT. After the stop solution was added, the plates were read at 450 nm.
• TMB Tetramethylbenzidine
• the disease score could range from 0 (no change in any segment) to a maximum of 12 with Grade 4 lesions of segments.
• RT-PCR products obtained using CXCL9-, CXCL10-, CXCL11-, CCRL1-, CCRL2-, CCR5-, CCL1-, CCL2-, CCL3-, CCL4-, CCL4L1-, CCL5-, CCL7-, CCL8-, CCL14-1-, CCL14-2-, CCL14-3- , CCL15-1-, CCL15-2-, CCL16-, CCL19-, CCL23-1-, CCL23-2-, CCL24-, CCL26-, CCR6-, CCL20-, and CCL25-, CCL25-1-, CCL25-2-specific primer sets did not cross react with other gene targets due to exclusion of primers that annealed to host sequences.
• the primers used produced different size amplicon products relative the polymorphisms that resulted in CCL4 versus CCL4L1, CCL14-1, CCL14-2, versus CCL14-3, CCL15-1 versus CCL15-2, CCL23-1 versus CCL23-2, and CCL25, CCL25-1, versus CCL25-2.
• dermatitis e.g., mellitus, juvenile onset
• graft rejection inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis, enteritis)
• multiple sclerosis myasthemia gravis
• pneumonitis psoriasis, nephritis, rhinitis
• chemokines used in the methods of the invention are known. Their accession numbers for the protein sequences are identified in Table 1.
• inflammatory diseases differ with the disease. They also differ among individuals. Hence, it is wise, when treating an individual, to identify the particular chemokines which are increased in the tissues of the patient. Using the antibodies produced against each of the chemokines and exposing the tissue samples from the patient to the particular antibodies, then evaluating the amount of antibody/chemokine binding, it is possible to evaluate the level of expression for each chemokine and to administer to the patient the particular antibodies that will bind the excessive chemokine.
• This tailored approach to treatment of inflammatory disease is novel, and a particularly valuable aspect of the invention.
• EXAMPLE 2 mRNA Expression of IFN- ⁇ , CXCL10. MIG. I-TAC. CXCR3 in Murine Colitis
• FIG. 1 shows mRNA expression of IFN- ⁇ , CXCL10, MIG, I-TAC, and CXCR3 during murine colitis.
• Laminar flow barriers were removed from the housing cages of IL-10 "7" mice, on C57BL/6 background, for the spontaneous development of colitis.
• the levels of IFN- ⁇ , IP- 10, MIG, I-TAC, and CXCR3 mRNA expression were ascertained after RT-PCR analysis that was capable of detecting > 20 copies of transcribed cDNA.
• the Log 10 copies of transcripts are expressed relative to actual copies of 18S rRNA.
• EXAMPLE 3 Histological Analysis of IBP in TCR ⁇ x ⁇ "7" Mice That Received CD45RB HI or CXCR3 + CD4 + T Cells by Adoptive Transfer
• FIG. 2 shows histological analysis of IBD in TCR ⁇ x ⁇ "7" mice that received CD45RB HI or CXCR3 + CD4 + T cells by adoptive transfer.
• Cross sections of intestines demonstrate the differences in wall thickness, enlargement of mucosal layer, crypt malformation, and leukocyte infiltration using hematoxylin-eosin staining of 6 ⁇ paraffin sections.
• FIG. 3 shows serum amyloid A (SAA) levels and the development of colitis in IL-10 " _ mice.
• SAA concentrations > 200 ⁇ g / ml were associated with the onset of asymptomatic colitis at week 0.
• Mice received 200 ⁇ of pre-immune- (open circles) or anti- mouse CXCL10 (closed circles) Ab solutions every 3 days.
• Sera were collected every 2 weeks and the data presented are the mean SAA concentrations ⁇ SEM.
• FIG. 4 shows changes in body weight of IL-10 "/_ mice.
• the wasting disease associated with murine CD was observed by monitoring the change in initial body mass at week 0.
• IL-10 _/" mice received 200 ⁇ of pre-immune- (open circles) or anti-mouse CXCL10 (closed circles) Ab solutions every 3 days.
• Body masses were recorded every 2 weeks and the change from initial body mass was expressed as a percentage: weight at week 0 minus weight at week 1 , 3, 5, 7, 9, or 11 divided by the weight at week 0.
• EXAMPLE 6 Association of Serum IL-6 and SAA Levels with Murine Colitis
• FIG. 5 shows association of serum IL-6 and SAA levels with murine colitis.
• IL-10 7" mice received 200 ⁇ of pre-immune- (open boxes) or anti-mouse CXCL10 (closed boxes) Ab solutions every 3 days.
• the data presented are the mean SAA or IL-6 concentrations ⁇ SEM.
• EXAMPLE 7 Total fecal and serum Ab levels in IL-10 'A mice
• FIG. 6 shows total fecal and serum Ab levels in IL-10 "7" mice.
• Groups of 5 IL- 10-/- mice received 200 ⁇ of either pre-immune- (open squares) or anti-mouse IP- 10- (closed squares) Ab solutions every 3 days.
• the data presented are the mean concentration of total Ig Abs (ng / ml) ⁇ SEM.
• Total IgA and IgG Abs in fecal extracts or IgM, IgGl, IgG2a, IgG2b, and IgG3 Abs in serum were collected at week 1 1 and levels determined by ELISA.
• Asterisk(s) indicate statistically significant differences, i.e.,/? ⁇ 0.05 (*), between the 2 groups.
• mice had similar levels of IgM, IgGl, IgG2b, and IgG3 Abs.
• total serum IgG2a levels were significantly higher in mice with active colitis, as compared with anti-CXCLlO Ab-treated mice (FIG. 6).
• the results indicate that blockade of CXCLIO attenuated total IgG2a levels and the excretion of IgG Abs during CD, consistent with the predicted imbalance of Thl»Th2 cytokine levels during CD.
• EXAMPLE 8 Serum IL-12, IFN- ⁇ . IL-2. TNF-q. IL-la, and IL- ⁇ ⁇ Levels in IL-10 7' Mice with IBP
• FIG. 7 shows serum IL-12, IFN- ⁇ , IL-2, TNF-a, IL-la, and IL- ⁇ levels in IL-10 "7" mice with IBD.
• IL-10 7" mice received 200 ⁇ of either pre-immune- (open squares) or anti-mouse IP- 10- (closed squares) Ab solutions every 3 days.
• Serum cytokines, at week 11, levels were determined by ELISA. The data presented are the mean cytokine
• Control groups showed moderately higher levels of serum IL-12 p40, compared with IP- 10 Ab-treated mice (FIG. 7).
• anti-CXCLlO Ab therapy dramatically decreased IFN- ⁇ levels in IL-10 7" mice, as well as the levels of IL-2, TNF-a, IL- la, and IL- ⁇ levels.
• Overproduction of IL-2, IL-12, TNF-a, IL-la, and IL- ⁇ during IBD has been well documented.
• the significant decreases in serum IL-2, TNF-a, IL-la, and IL- 1 ⁇ levels by CXCLIO blockade (FIG. 7) is consistent with the inflammatory state of the host with active colitis being significantly reduced by anti-CXCLlO Ab treatment.
• FIG. 8 shows histological characteristics of colitis presented by IL-10 _/" mice. Changes in mice that received 200 ⁇ of either pre-immune- (C or D) or anti-mouse IP- 10- (A or B) Ab solutions every 3 days. Following sacrifice at week 11, the intestines were fixed, sectioned at 6 ⁇ , and stained. Sections were examined microscopically at a magnification view of 40X (A and C) or 200X (B and D).
• FIG. 9 shows that anti-CXCLlO antibody abrogates severe colitis.
• IL-10 "/" mice received 200 ⁇ of control Ab (open circles) or anti -mouse CXCL10 Ab (closed circles) every 3 days starting 14 weeks after the onset of symptomatic colitis, when mice had lost about 10 to 15% of their initial body weight and attained a peak in SAA levels, and continued until the mice were sacrificed at week 26.
• the level of SAA ⁇ SEM and body weight of the IL-10 " mice were recorded every week, and the change from initial body weight was expressed as a percentage of the weight before the onset of colitis (week -2) minus the weight at subsequent weeks divided by the weight before the onset of colitis (+ SEM).
• Data represents the mean of three independent experiments involving 5 mice per groups. Asterisks indicate statistically significant differences (p ⁇ 0.01) between anti-CXCLlO Ab- and control Ab-treated groups.
• Chronic colitis in the IL-10 - ⁇ mice corresponded with an increase in SAA levels (>300 / g/mL)(FIG 9A) and with a 10%— 15% reduction in the body weight of the mice compared with their initial body weight (FIG. 9B).
• CXCL10 blockade in mice with chronic colitis alleviated weight loss when compared with the weight loss experienced by IL-10 ⁇ /_ mice with chronic colitis treated with control Ab.
• EXAMPLE 11 Thl Cytokine. CXCL10 and CXCR3 mRNA Expression in Mucosal Tissue During Severe Colitis
• FIG. 10 shows Thl cytokine, CXCL10 and CXCR3 mRNA expression in mucosal tissue during severe colitis.
• mice received 200 ⁇ of either control Ab (solid bars), or anti-CXCLlO Ab (hashed bars) or normal WT mice (open bars), every 3 days starting 14 weeks after the onset of symptomatic colitis, when mice had lost about 15% of their initial body weight.
• total RNA was isolated from the colons and mesenteric lymph nodes (MLNs) of mice treated with either control Ab, wild type or anti-CXCLlO Ab.
• the levels of IFN- ⁇ , CXCLIO, TNF-a, IL-12p40, and CXCR3 mRNA expression were ascertained by an RT-PCR analysis capable of detecting > 20 copies of transcribed cDNA. Logio copies of transcripts are expressed relative to actual copies of 18S rR A ⁇ SEM in FIG. 10. Data represents the mean of three independent experiments involving 5 mice per group. Asterisks indicate statistically significant differences (p ⁇ 0.01) between anti-CXCLlO and control Ab-treated groups.
• EXAMPLE 12 Thl and Inflammatory Cytokine Levels in Serum During Severe Colitis Progression
• FIG. 11 shows Thl and inflammatory cytokine levels in serum during severe colitis progression.
• IL-10 "7" mice received 200 ⁇ of either control Ab (open circles) or anti- CXCL10 Ab (closed circles) every 3 days, starting 14 weeks after the onset of symptomatic colitis, which continued through week 26.
• levels of serum cytokines at week 26 were determined by an ELISA capable of detecting > 10 pg/ml of IL-12p40, IL-2, TNF-a, IFN- ⁇ , IL-l , and IL- ⁇ .
• the data presented are the mean concentrations ⁇ SEM.
• Asterisk (s) indicate statistically significant differences, i.e., /? ⁇ 0.01 (*), between the two groups.
• Experimental groups consisted of 5 mice, and experiments were repeated 3 times. Data represents the mean of 3 independent experiments.
• FIG. 12 shows anti-CXCLlO antibody effects on colitis pathology.
• mice with chronic colitis that were treated with either control Ab, (Panels A and B) or anti-CXCLlO Ab (panels C-D) as before. Sections were examined by light microscopy. Experimental groups consisted of 5 mice and were repeated 3 times.
• mice that received anti-CXCLl O Ab showed a significant reduction in intestinal inflammation.
• An increase in leukocyte infiltrates (FIG. 12 A) and distortion of glandular architecture (FIG. 12B) were observed in the intestines during chronic colitis.
• Anti- CXCLlO Ab reduced the lymphocyte infiltration and partially restored the glandular and goblet cell architecture (FIG. 12C), which also coincided with lengthening of intestinal crypts FIG. 12D).
• the mean histologic scores of IL-10 _/ ⁇ mice with severe colitis that received control Ab were significantly higher than the scores of mice treated with anti- CXCL10 Ab (data not shown).
• SAA levels correlated with the severity of colitis as determined by histologic analysis.
• Pathologic changes included leukocyte infiltrates in the LP of the colon of control Ab-treated IL-10 ⁇ /_ mice, with the number of these infiltrates being reduced after CXCLI O blockade. Taken together, the results show a marked improvement in the characteristic intestinal inflammation associated with chronic colitis after CXCLIO blockade.
• EXAMPLE 14 Histological and Immunofluorescence Localization of CXCL9, CXCLIO, CXCL1 1. and TNF-a in the Colon of CD Patients
• FIG. 13 shows histological and immunofluorescence localization of CXCL9, CXCLIO, CXCL11 , and TNF-a in the colon of CD patients. Histopathology of colonic changes in the intestines of CD patients and normal control were fixed, sectioned at 6 ⁇ , and stained with hematoxylin and eosin or anti- CXCL9, CXCLIO, CXCL11 or TNF-a antibodies. Sections were examined at a magnification view of 13 OX. The inflamed colon demonstrates the differences in mucosal wall thickness, crypt malformation, leukocyte infiltration, and glandular elongation between normal and CD patients.
• EXAMPLE 15 MAP-Specific Serum Ab Responses in IL-10 -/ ⁇ Mice During Spontaneous Colitis
• FIG. 14 shows M. avium subsp. paratuberculosis (MAP)-specific serum Ab responses in IL-10 - " mice during spontaneous colitis.
• the data presented are the mean + SD concentration (ng/ml) of MAP-specific IgG subclasses from three separate experiments.
• Asterisks (*) indicate statistically significant differences, i.e.,p ⁇ 0.01, compared to controls.
• Mice experimental groups consisted of 15 mice. Assays were repeated 3 times.
• FIG. 14 shows that MAP-specific IgG2a Ab responses were significantly higher in mice with spontaneous colitis, kept under conventional housing, than in similar control mice without disease, which were housed under germfree conditions. This is consistent with the previously described imbalance of cytokine levels (Thl > Th2) during colitis, suggesting there is a Thl -biased humoral response associated with the progression of colitis.
• EXAMPLE 16 Histological characteristics of IL-10 " " mice challenged with MAP
• FIG. 15 shows histological characteristics of IL-10 "7" mice challenged with MAP. 14 weeks post challenge, histopathologies of colons from IL- 10 " ⁇ mice that received a single dose of 200 ⁇ of control vehicle (cream only), 10 4 CFU of live MAP in cream, or 10 4 CFU of heat-killed MAP in cream by gavage and maintained under otherwise germ-free conditions were fixed, sectioned at 6 ⁇ , and stained with hematoxylin and eosin. Mild (open triangles) and heavy (solid triangles) cellular infiltrates were noted in groups (i.e., live MAP » heat-killed MAP > controls).
• FIG. 15 shows that the intestinal tissues of mice challenged with live M. avium subsp. paratuberculosis exhibited increased levels of cellular infiltrates, which consisted of lymphocytes and, occasionally, polymorphonuclear cells.
• the colitis progression was more aggressive in mice that received live M. avium subsp. paratuberculosis, as noted by multifocal lesions, or aggregates of cellular infiltrates, in all regions of their large intestines.
• epithelial cells in mice challenged with live M. avium subsp. paratuberculosis were hypertrophied, the intestinal crypt length was decreased, and elongated glandular cells were also present in both the mucosa and the submucosa.
• EXAMPLE 17 Changes in Body Weight of IL-10 ⁇ Mice After MAP Challenge
• FIG. 16 shows changes in body weight of IL-10 7" mice after MAP challenge.
• the wasting disease associated with murine colitis was observed by monitoring the body weight during colitis progression.
• IL-10 7" mice on B6 background received a single dose of 200 ⁇ normal control (cream, open circles), 10 4 CFUs of live MAP in cream (solid circles) or 10 4 CFUs of pasteurized MAP in cream (triangles) and maintained under otherwise germ- free conditions. Percentage of initial body weight of IL-10 7" mice was recorded biweekly. The data presented are the mean + SD of 3 separate experiments. Asterisks (*) indicate statistically significant differences, i.e., p ⁇ 0.01, compared to controls. Experimental groups consisted of 15 mice and assays were repeated 3 times.
• FIG. 16 shows that mice challenged with avium subsp. paratuberculosis and housed under otherwise germfree conditions lost more body weight and experienced higher SAA levels than did similar mice challenged with heat-killed M. avium subsp.
• mice challenged with live M. avium subsp. paratuberculosis show rapid colitis progression associated with elevated SAA levels and reductions in body weight compared with the control group.
• EXAMPLE 18 Serum Cytokine Levels in IL-10 7" Mice after MAP Challenge
• FIG. 17 shows serum cytokine levels in IL-10 7" mice after MAP challenge.
• IL-10 7" mice on a B6 background, received a single dose of 200 ⁇ of the control vehicle (i.e., cream), 10 4 CFUs of live MAP in cream, or 10 4 CFUs heat-killed MAP in cream by gavage and maintained under otherwise germ-free conditions.
• the levels of serum TNF-a and IFN- ⁇ and CXCL9, CXCLIO, and CXCLl 1 14 weeks after challenge were determined by ELISA, capable of detecting > 10 pg/ml TNF-a, IFN- ⁇ or CXCR3 ligand.
• TNF-a TNF-a, CXCL10, and CXCL11.
• FIG. 18 shows anti-peptide #25 Ag (from MPT59)-induced proliferation and IL-2 production by CD4 + T cells from IL-10 "7" mice.
• IL-10 "7" mice on B6 background, received a single dose of 200 ⁇ of control vehicle (open bars, cream only), 10 4 CFUs of live MAP in cream (hatched bars), or 10 4 CFUs of heat-killed MAP in cream (solid bars) and maintained under otherwise germ-free conditions.
• CD4 + lymphocytes derived from the MLN, and PPs of the mice were purified and cultured at density of 5 x 10 6 cells/ml with peptide #25 (1 ⁇ g/ml) for 3 days with ⁇ -irradiated APCs (10 6 cells/ml). Cytokines present in culture supernatants were determined ELISA. Proliferation was measured by BrdU incorporation. The data presented are the mean OD 450 for proliferative responses or the mean of IL-2 secretion (pg /ml) ⁇ SD of quadruplicate cultures. Asterisks (*) indicate statistically significant differences, i.e,,p ⁇ 0.01, compared to controls. Experimental groups consisted of 15 mice and experiments were repeated three times.
• FIG. 18 shows that peptide 25-stimulated CD4 + T cells from the MLN and PP of mice previously challenged with either live or heat-killed M. avium subsp.
• paratuberculosis exhibited marked increases in BrdU incorporation compared with similar CD4 + T cells from mice challenged with cream alone. These results suggest that Ag restimulation after exposure to M. avium subsp. paratuberculosis enhances CD4 + T-cell proliferation.
• EXAMPLE 20 Serum CXCR3 Ligands and Mycobacterial-Specific Ab Responses in IBP Patients
• FIG. 19 shows serum CXCR3 ligands and mycobacterial -specific Ab responses in IBD patients.
• EXAMPLE 21 Changes in SAA Levels in IBD Patients and in IL-IO 7' Mice after
• FIG. 20 shows changes in SAA levels in IBD patients and in IL-IO 7" mice after mycobacterial challenge.
• IL-10 7" mice on B6 background received 200 ⁇ of cream milk alone (open circles; control) or cream milk containing 10 4 CFU of live (closed circles) or heat-killed (closed triangles) M. avium paratuberculosis.
• Mycobacteria-enhanced colitis as well as IBD patients and healthy donors were measured by ELISA.
• Experimental groups consisted of 5 mice, and experiments were repeated 3 times. The data presented are the mean ⁇ SEM concentration of SAA. Asterisks indicate statistically significant differences, i.e.,p ⁇ 0.01, between control and Mycobacteria-treated groups or healthy donors and IBD patients.
• FIG. 21 shows intestinal histological characteristics of IL-10 "7" mice challenged with Mycobacteria.
• IL-10 7" mice on B6 background received 200 ⁇ of cream milk alone ((open circles; control) or cream milk containing 10 4 CFU of live (closed circles) or heat-killed (closed triangles) M. avium paratuberculosis.
• intestines were fixed, sectioned at 6 ⁇ , and stained with hematoxylin and eosin. Sections were examined by light microscopy. Experimental groups consisted of 5 mice and experiments were repeated 3 times.
• mice challenged with Mycobacteria showed higher increases in leukocyte infiltrates, which consisted of lymphocytes and occasionally polymorphonuclear cells as well as a higher frequency of lymphoid follicles in live versus heat-killed Mycobacteria-c allenged groups (FIG. 21).
• colitis was more aggressive in mice that received live Mycobacteria, as noted by multi-focal lesions and aggregates of leukocyte infiltrates in the large intestines, than compared to control mice.
• EXAMPLE 23 Serum CXCL9, CXCL10 and CXCL11 Concentrations in IC Patients
• FIG. 22 shows serum CXCL9, CXCL10 and CXCL11 concentrations in IC patients.
• the data presented are the mean CXCL9, CXCL10, and CXCL11 of IC patient and normal healthy donors concentrations ⁇ SEM.
• Asterisks (“k) indicate statistically significant differences, i.e.,/? ⁇ 0.01, between the healthy donors and IC patients.
• Panel B Control or anti-CXCLlO Ab solutions were administered 2 days prior to CYP challenge and every 2 days thereafter. Five days after CYP administration, the serum levels of CXCL9, CXCL10, and CXCL11 were determined by ELISA. The data presented are the mean concentrations ⁇ SEM in each group. Asterisks (*) indicate statistically significant (p ⁇ 0.01) differences between unaffected and CYP-induced groups. Triangles indicate statistically significant (p ⁇ 0.01) differences between control Ab- and anti-CXCLlO Ab-treated groups administered CYP.
• the serum levels of CXCL9 and CXCL10 in IC patients were significantly higher than levels in unaffected healthy donors.
• the difference in serum levels between IC patients and healthy donors were greatest for CXCL9 (p ⁇ 0.001), followed by CXCL10 (p ⁇ 0.01) and CXCL11 (p > 0.1).
• CXCR3 ligand levels also correlated (although not statistically significant) with disease severity as manifested by pathological reports for each individual patient (data not shown). Further, these patients showed multiple pathological features of tissue damage that frequently included urothelium denudation, mucosal edema, and/or leukocyte infiltration.
• FIG. 23 shows histological changes after CYP-induced cystitis.
• Control or anti-mouse CXCLl 0 Ab solutions were administered 2 days prior to CYP treatment and every 2 days thereafter.
• Five days after CYP administration the urinary bladders of the mice were fixed, sectioned at 6 ⁇ , and stained with hematoxylin and eosin. The sections were examined microscopically at magnification views of 10X and 100X.
• Panels A and C show the magnified sections from control Ab-treated mice, while Panels B and D display similar sections from anti-CXCLlO Ab-treated mice given CYP to illustrate inflamed bladders and characterized differences in mucosal wall thickness, enlargement of mucosal layer, leukocyte infiltration, and glandular elongation.
• Control Ab-treated mice given CYP showed pathological signs of cystitis ⁇ i.e., urinary bladder inflammation, discontinuous uroepitheium).
• affected mice treated with anti-CXCLlO Ab displayed a reduction in cystitis, as noted by a decrease in urinary bladder leukocyte infiltrates (FIG. 23).
• Histological differences between control Ab- and anti-CXCLlO Ab-treated mice with CYP-induced cystitis were considered significant and showed that CXCL10 blockade significantly reduced CYP-induced cystitis.
• EXAMPLE 25 CXCR3, -9, -10, and -11 mRNA Expression in CYP-Treated Mice
• FIG. 24 shows CXCR3, CXCL9, CXCLl 0, and CXCLl 1 mRNA expression in CYP-treated mice.
• Control or anti-mouse CXCL10 Ab solutions were administered 2 days prior to CYP treatment and every 2 days thereafter.
• Five days after CYP administration total RNA was isolated from the spleen, iliac lymph nodes, or urinary bladder of the mice.
• Panel A RT-PCR analysis of CXCR3, CXCL9, CXCL10, or CXCLl 1 mRNA expression was performed.
• Panel B RT- PCR analysis of IFN- ⁇ , IL-12 p40, or TNF-a mRNA expression was performed.
• CYP-induced cystitis in mice led to substantial increases in the expression of CXCLl 0, CXCLl 1, and CXCR3 mRNA by urinary bladder leukocytes as well as modest increases in the expression of CXCL9 and CXCR3 transcripts by iliac lymph node lymphocytes than compared to normal, untreated mice.
• the expression of these IFN- ⁇ - and nuclear factor kappa B (NFicB)-inducible chemokines and CXCR3 mRNAs were significantly diminished in splenocytes from CYP-treated mice than compared to similar cells from control mice.
• NFicB nuclear factor kappa B
• Anti-CXCLIO Ab treatment significantly decreased the expression of CXCL9 and CXCR3 mRNAs by iliac lymph node leukocytes and reduced the production of CXCL9, CXCL10, CXCL11, and CXCR3 mRNAs by urinary bladder leukocytes.
• mice with CYP-induced cystitis exhibited increased IFN- ⁇ mRNA expression by iliac lymph node lymphocytes compared to similar cells from unaffected mice.
• the expression of IFN- ⁇ , IL-12p40, and TNF-a mRNAs by urinary bladder lymphocytes from mice with cystitis were significantly decreased following anti-CXCLlO Ab treatment than compared to similar cells from CYP-induced mice treated with control Ab.
• EXAMPLE 26 Serum CXCL10 Concentrations During Active Crohn's Disease (CD
• FIG. 25 shows upregulated CXCL10 expression during active CD.
• the levels of CXCL10 were determined by an ELISA assay capable of detecting > 20 pg/ml of CXCL10.
• the data presented are the mean CXCL10 concentrations ⁇ SEM in CD patients and healthy donors. Asterisk(s) indicate statistically significant differences, i.e., p ⁇ 0.05 (*), between the 2 groups.
• EXAMPLE 27 Serum CXCL11 and CXCL9 Concentrations During Active Crohn's Disease
• FIG. 26 shows upregulated expression of CXCL11 and CXCL9 during active CD.
• the levels of serum CXCL11 and CXCL9 were determined by ELISA that was capable of detecting > 20 pg/ml of each Thl cytokine.
• the data presented are mean CXCL11 (FIG. 26A) and CXCL9 (FIG. 26B) concentrations ⁇ SEM in CD patients and healthy donors.
• Asterisk(s) indicate statistically significant differences, i.e., p ⁇ 0.05 (*), between the 2 groups.
• EXAMPLE 28 Serum Amyloid Protein A (SAA) and IL-6 Concentrations During Active Crohn's Disease
• FIG. 27 shows upregulated serum concentrations of serum amyloid A (SAA) and IL-6 in CD patients.
• the data presented are the mean of SAA (FIG. 27A) and IL-6 (FIG. 27B) concentrations ⁇ SEM in CD patients and healthy donors.
• Asterisk(s) indicate statistically significant differences, i.e.,p ⁇ 0.05 (*), between the 2 groups. This data is consistent with elevated SAA and serum IL-6 levels corresponding with the severity of CD.
• EXAMPLE 29 Serum IL-12p40 and IFN-v Levels Correlate During Active Crohn's Disease
• FIG. 28 shows serum IL-12p40 and IFN- ⁇ levels correlate during CD.
• the data presented are the mean IL-12p40 (FIG. 28 A) and IFN- ⁇ (FIG. 28B) concentrations ⁇ SEM from the serum of CD patients and healthy donors.
• Asterisk(s) indicate statistically significant differences, i.e., p ⁇ 0.05 (*), between the 2 groups.
• EXAMPLE 30 Inflammatorv Cytokine Levels During Active Crohn's Disease
• FIG. 29 shows inflammatory cytokine levels during active CD.
• the levels of serum TNF-a and IL- ⁇ were determined by ELISA that was capable of detecting > 20 pg / ml of each cytokine.
• the data presented are the mean TNF-a (FIG. 29 A) and IL- ⁇ (FIG. 29B) concentrations ⁇ SEM from serum of CD patients and healthy donors.
• Asterisk(s) indicate statistically significant differences, i.e., p ⁇ 0.05 (*), between the 2 groups.
• FIG. 30 shows histological characteristics of colitis in normal and CD patients with high serum CXCR3 ligand concentrations. Histopathology of colonic biopsy from normal healthy donors and CD patients were fixed, sectioned at 6 ⁇ , and stained with hematoxylin and eosin. Sections were examined by microscopy.
• FIG. 30 shows that the colon in CD patients demonstrates differences in crypt malformation, leukocyte infiltration, glandular elongation/hyperplasia, and edema between normal and CD patients.
• EXAMPLE 32 CXCL9, CXCL10, CXCL1 1 and TNFa Expression in Colons of CD Patients
• FIG. 31 shows CXCR3 ligands and TNFa expression in colons of normal and CD patients by histopathological examination.
• the colons from normal and CD patients were frozen, fixed, sectioned at 6 ⁇ , and stained fluorescently for CXCL9-, CXCL10-, CXCL11- and TNFa-positive cells. Sections were examined by fluorescent con-focal microscopy.
• FIG. 31 shows that the colon from a CD patient shows increased leukocyte infiltration compared with a normal control patient.

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