WO2010022227A1 - Methods for monitoring il-10 therapy - Google Patents

Abstract

The present invention provides biomarkers to monitor IL-10 therapy. In particular biomarkers are provided to assess target engagement and efficacy of tumor treatment.

Description

METHODS FOR MONITORING IL- 10 THERAPY

FIELD OF THE INVENTION

[0001] The present invention provides methods for monitoring IL- 10 therapy. In particular, the present invention provides biomarkers to assess IL-IO target engagement and efficacy of tumor treatment.

BACKGROUND OF THE INVENTION

[0002J Cancers and tumors can be controlled or eradicated by the immune system.

The immune system includes several types of lymphoid and myeloid cells, e.g., monocytes, macrophages, dendritic cells (DCs), eosinophils, T cells, B cells, and neutrophils. These lymphoid and myeloid cells produce secreted signaling proteins known as cytokines. The cytokines include, e.g., interleukin-10 (IL-IO), interferon-gamma (ϊFNγ), IL-12. and IL-23. Immune response includes inflammation, i.e., the accumulation of immune cells system ically or in a particular location of the body. In response to an infective agent or foreign substance, immune cells secrete cytokines which, in turn, modulate immune cell proliferation, development, differentiation, or migration. Excessive immune response can produce pathological consequences, such as autoimmune disorders, whereas impaired immune response may result in cancer. Anti-tumor response by the immune system includes innate immunity, e.g., as mediated by macrophages, NK cells, and neutrophils, and adaptive immunity, e.g., as mediated by antigen presenting cells (APCs). T cells, and B cells {see. e.g., Abbas, el al. (eds.) (2000) Cellular and Molecular Immunology, W. B. Saunders Co.. Philadelphia. PΛ: Oppenheim and Feidmann (eds ) (20O i ) Cytokine Reference, Academic Press, San Diego, CA; von Andrian and Mackay (200Oj New Engl. J MeJ, 343: 1020-1034; Davidson and Diamond (2001 ) .Vevv Engl, J Med. 345:340-350).

|0003] Initially known as cytokine synthesis inhibitor factor or CSIF, interieukin-10

(IL-10) is a potent immunomoduiator of hematopoietic cells, particularfy immune cells. Cells such as activated Th2 cells, B cells, keratinoc\tes. monocytes and macrophages produce IL- 10. See, e.g., Moore et a]., Annu Rev Immunol. 1 1 : 165 (1993). IL-IO inhibits activation and effector functions of a number of cells that include T ceils, monocytes and macrophages. in particular. IL-10 inhibits cjtokine synthesis, including that of IL-I , IFN-γ. and TNF. by cells such as Th I cells, natural killer ceils, monocytes, and macrophages. See, e g.. Florentine et al., (1989) ./ Exp. Med.. 170:2081-2095; Fiorentino et al., (1991 ) J Immunol. 146:3444; Hsu et al., (1992) Int. Immunol. 4:563; Hsu et al._s (1992) lnt Immunol, 4:563; D^f Andrea et a!., ( 1993) J. Exp. Med. 178 : 104 ϊ : de Waal Malefyt et al ., ( 1991 ) J Exp Med. 174:915; Fiorentirto et a!.. (199I) J Immunol. 147:3815.

[0004] Methods of modulating immune response have been used in the treatment of cancers, e.g., melanoma. These methods include treatment either w ith cytokines such as IL- 2, IL-IO, IL- 12, tumor necrosis factor-alpha (TNFalpha), IFNy, granulocyte macrophage- colony stimulating factor (GM-CSF), and transforming growth factor (TGF), or with cytokine antagonists (e.g., antibodies), lnterleukin-10 was first characterized as a cytokine synthesis inhibitory factor (CSIF; see, e.g., Fiorentino, et al (1989) J Exp. Med. 170:2081 -2095). IL- 10 is a pleiotropic cytokine produced by T cells. B cells, monocytes, that can function as both an immunosuppressant and irnmunostimulant (see, e.g., Groux, et al. (1998) J. Immunol. 160:3188-3193; and l iagenbaugh, et aϊ. (1997) J. Exp. Med 185:2101-21 10). [0005] Animal models suggest that IL-10 can induce NK-cell activation and facilitate target-cell destruction in a dose-dependent manner (see, e.g., Zheng, et al. (1996) ,/ Exp. Med. 184:579-584; Kundu, ct al. (1996) J. Natl Cancer Inst. 88:536-541). Further studies indicate that the presence of IL-IO in the tumor microenvironment correlates with better patient survival (see, e.g., Lu. et al. (2004) J. Clin. Oncol. 22:4575-4583). IL-10 induces cytotoxic activ ity of CDS T-ceils, antibody production of B-cell and suppresses macrophage activity and tumor promoting inflammation (see, Chen and Zlotnik (1991 } j. Immunol. 147:528-534; Groux. et al. (1999) J. Immunol. 162: 1723-1729; and Bergman, et al. (1996) J. Immunol. 15?'233 -238). The regulation of CDS ceils is dose dependent wherein higher doses induce stronger cytotoxic responses.

{0006] As the level of II -10 in the microenvironment is critical to induce cytotoxic activ ity, a need exists to monitor IL-10 levels (both nathe and engineered and/or linked IL-10 proteins) in the serum of patients. The present invention fulfills this need by providing bϊomarkers to assess IL-10 receptor engagement as well IL-10 efficacy levels, thereby iding information to monitor IL-10 therapies. SUMMARY OF THE INVENTION

[0007] The present invention is based, in part, upon the discovery of a set of biomarkers that have increased expression upon IL-I O target engagement, and a set if biomarkers that have increased expression correlating with tumor treatment efficacy, [0Θ08J The present invention provides a method for monitoring efficacy of an IL-10 therapeutic agent in treatment of a subject for a tumor or cancer, said method comprising: a) obtaining a baseline biological sample from said subject prior to administering a dose of the IL-IO therapeutic agent; b) measuring the level of at least one biomarker in the baseline biological sample by gene expression analysis or immunoassay, c) administering the IL-10 therapeutic agent to the subject; d) obtaining from the subject at least one subsequent biological sample; e) measuring the level of the biomarker in the subsequent sample by gene expression analysis or immunoassay; f) comparing the level of the biomarker in the subsequent sample with the level of the biomarker in the baseline biological sample; g) and correlating a change in the level of the biomarker with treatment efficacy, wherein an increase of at least one biomarker in the subsequent biological sample indicates an effective dose of the IL-10 therapeutic agent. In certain embodiments, the subject is human and the tumor or cancer is from an organ selected from the group consisting of skin, colon, thyroid, ovarian, lung, pancreas and brain. In yet a further embodiment the tumor is selected from the group consisting of: colon cancer, ovarian cancer, breast cancer, melanoma, Sung cancer, glioblastoma, and leukemia. In another embodiment, the IL- ! 0 therapeutic agent is selected from the group consisting of native IL-10. pegylated IL-I O (PEG-IL-10), and IL-IO conjugated to an Fc portion of an immunoglobulin (IL-10-ϊg). The biomarker is selected from the group consisting of IP-10 and MfG, In certain embodiments, the increase in IP-10 or MIG expression in the subsequent biological sample is at least 2 fold to at Seast 19 fold. In further embodiments, the gene expression analysis is selected from the group consisting of Northern blotting, quantitative PCR. SAGE, and DNA microarray. the immunassay selected from the group consisting of ElJSA, RlA, Western blot, luminescent immunoassay, flourescent immunoassay. The present invention also provides a method for monitoring target engagement following administration of an IL-IO therapeutic agent to treat a subject for a tumor or cancer, said method comprising^* a) obtaining a baseline biological sample from said subject prior to administering a dose of the IL-10 therapeutic agent; b) measuring the level of at least one biomarker in the baseline biological sample

gene expression or immunoassay, c) administering the IL-I O therapeutic agent to the subject; d) obtaining from the subject at least one subsequent bioiogical sample; e) measuring the level of the biomarker in the subsequent sample b> gene expression analysis or immunoassay; f) comparing the level of the biomarker in the subsequent sample with the level of the biomarker in the baseline biological sample; and g) correlating a change in the level of the biomarker target engagement, wherein an increase of at least one biomarker in the subsequent biological sample indicates target engagement. In certain embodiments, the subject is human. The tumor or cancer is from an organ selected from the group consisting of skin, colon, thyroid, ovarian, lung, pancreas, and brain. In further embodiments, the tumor is selected from the group consisting of; colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, and leukemia. 3n other embodiments, the IL-10 therapeutic agent is selected from the group consisting of native IL-10, pegylated IL- 10 (PEG-IL-10), and IL-I O conjugated to an Fc portion of an immunoglobulin (IL-IO-Ig). The biomarker is chosen from the group consisting of MCP-I and phosphorylation of STAT3. In further embodiments, the gene expression analysis is selected from the group consisting of Northern blotting, quantitative PCR, SAGE, and DNA microarray, the immunoassay is selected from the group consisting of ELΪSA, RIA. Western blot, luminescent immunoassay, flourescent immunoassay.

DETAILED DESCRIPTION

J0009J As used herein, including the appended claims, the singular forms of words such as "a,^" "an.^*' and ''the." include their corresponding plural references unless the context ciearh dictates otherwise.

[ΘOlOj All references cited herein arc incorporated by reference to the same extent as if each individual publication, patent application, or patent, was specifically and indiv idual!} indicated to be incorporated b> reference.

I. Definitions.

[0011] "Activity^"' of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor, to catalytic activity, to the ability to stimulate gene expression, to antigenic activity, to the modulation of activ ities of other molecules, and the like. "Activity" of a molecule ma} also refer to activity in modulating or maintaining ccll-to-cel! interactions, e.g., adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton. "Activity^"' may also mean specific activity, e.g., [catalytic activity]/[mg protein], or [immunological acti% ity]'[mg protein), or the like.

[0012] "Activation.'^" "stimulation.^"' and "treatment,^"' as it applies to celts or to receptors, may have the same meaning, e.g.. activation, stimulation, or treatment of a cell or receptor with a ligand, unless indicated otherwise by the context or explicitly. "Ligand^"' encompasses natural and synthetic ligands, e.g., cytokines, cytokine variants, analogues, muteins, and binding compositions derived from antibodies. "Ligand^'" also encompasses smail molecules, e.g.. peptide mtmetics of cytokines and peptide mimetics of antibodies. "Activation'^" can refer to cell activation as regulated by internal mechanisms as well as bv external or environmental factors. "Response.^"' e.g., of a cell, tissue, organ, or organism, encompasses a change in biochemical or physiological behav ior, e g., concentration, den-.it}. adhesion, or migration vuthin a biological compartment, rate of gene expression, or state of differentiation, where the change is correlated with actπ ation, stimulation, or treatment, or with internal mechanisms such as genetic programming.

[0013] "Activity^" of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor, to catalytic activity : to the ability to stimulate gene expression or eel! signaling, differentiaiioii. or maturation; to antigenic activ ity, to the modulation of activities of other molecules, and the like. "Activity^" of a molecule may also refer to activity in modulating or maintaining ceil-to-ceii interactions, e.g., adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton. "Activity^"' can also mean specific activity, e.g., [catalytic activity]/[mg protein], or [immunological activity ]/[mg proteinj. concentration in a biological compartment, or the like. "Proliferative activity^" encompasses an activity that promotes, that is necessary for. or that is specifically associated with, e.g., normal cell di\ ision, as well as cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.

[0014J "Administration" and "treatment,^" as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, compound, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration^" and "'treatment^" can refer, e.g., to therapeutic, placebo, pharmacokinetic, diagnostic, research, and experimental methods. "Treatment of a cell" encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell. "Treatment,^" as it applies to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications. "Treatment^"' as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses contact of PEG-IL-10 to a human or animal subject, a cell, tissue, physiological compartment, or physiological fluid. "Treatment of a cell^" also encompasses situations where PEG-ILTO contacts IL-IO receptor (heterodimer of IL-10R1 and IL-10R2) e.g.. in the fluid phase or colloidal phase, as well as situations where an IL- IO agonist or antagonist contacts a fluid, e.g., where the fluid is in contact w ith a cell or receptor, but where it has not been demonstrated that the agonist or antagonist directly contacts the eel! or receptor.

[0015] "Effective amount" encompasses an amount sufficient to ameliorate or prevent a s>mptom or sign of the medϊcaϊ condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects (see. e.g., U S. Pat No. 5,888,530 issued to Netti. ei at ) \n effecth e amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects. The effect will result in an improvement of a diagnostic measure or parameter by at least 5%. usually by at least 10%. more usually al least 20%, most usually at least 30%. preferably at least 40%, more preferably at least 50%, most preferably at feast 60%. ideally at least 70%. more ideally at least 80%. and most Ideally at least 90%, where 100% is defined as the diagnostic parameter shown by a normal subject (see, e.g., Maynard, el al, (1996) A Handbook of SOPs Jor Good Clinical Practice. Interpharm Press. Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice. Urcb Publ., London, UK). An effective amount of PEG-IL-10 would be an amount sufficient to reduce a tumor volume, inhibit tumor growth, prevent metastasis, or increase CD8+ T cell infiltration in to the tumor site.

[0016] "Exogenous" refers to substances that are produced outside an organism, cell, or human body, depending on the context. "'Endogenous" refers to substances that are produced within a cell, organism, or human body, depending on the context. [0017] "'Specifically" or '^"selectively" binds, when referring to a ligand/receptor. antibody/antigen, or other binding pair, indicates a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated conditions, a specified ligand binds to a particular receptor and does not bind in a significant amount to other proteins present in the sample. The antibody, or binding composition derived from the antigen-binding site of an antibody, of the contemplated method binds to its antigen, or a variant or mutein thereof, with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20- times greater, and most preferably at least 100-times greater than the affinity w ith any other antibody, or binding composition derived thereof. In a preferred embodiment the antibody will have an affinity that is greater than about ΪO^V Hters/rnol, as determined, e.g., by Scatchard analysis (Munsen, ei al (19&0) Λmihi Biochem. 107:220-239). jOOlSj "interleukiπ-iO^" or ''IL-I O^", as used herein, whether conjugated to a polyethy lene glycol or in a non-conjugated form, is a protein comprising two subunits noncovalently joined to form a homodimer. As used herein, unless otherwise indicated "mterleukin-Ϊ O'^" and "IL-IO^'' can refer to human or mouse IL- 10 (Gen batik Accession Nos. NP__000563; M37897: or US 6,217,857) which are also referred to as "hIL-10" or "mIL- i O^". [0019] ""Pegyiated !L-IO^" or "PEG-IL-I O^*' is an IL-IO molecule having one or more polyethylene glycol molecules covalently attached to one or more than one amino acid residue of the IL-10 protein via a linker, such that the attachment is stable. The terms "monopegylated IL-IO" and '"mono-PEG-lL-IO". mean that one polyethylene glycol molecule is covalently attached to a single amino acid residue on one subunit of the IL-10 dimer via a linker. The average molecular weight of the PEG moiety is preferably between about 5.000 and about 50.000 daltons. The method or site of PEG attachment to IL- 10 is not critical, but preferably the pegylation does not alter, or only minimally alters, the activity of the biologically active molecule. Preferably, the increase in half-life is greater than any decrease in biological activ ity . For PEG-IL- 10, biological activity is typically measured by assessing the levels of inflammatory cytokines (e.g., TN Fa, IFNγ) in the serum of subjects challenged vvith a bacterial antigen (lipopolysaccharide, LPS) and treated with PEG-IL-10, as described in US 7,052,686.

[0020) ''IL-I O therapeutic agent^"' as used herein, includes native IL- 10 or IL-10 conjugated to heterologous conjugates. Heterologous conjugates can be the Fc portion of an immunoglobulin molecule, serum albumin, or other known proteins or peptides that increase IL-10 serum half life (e.g., superoxide disrnutase (SOD)). Other conjugates include polyethylene glycol, (PEG), oligosaccharides, etc.

J0021J As used herein, "serum half-life", abbreviated "t _{1 2}", means elimination half- life, i.e., the time at which the serum concentration of an agent has reached one-half its initial or maximum value. The term "increased serum half-life" used herein in reference to a synthetic agent means that the synthetic agent is cleared at a slower rate than either the non- synthetic, endogenous agent or the recombinant!)- produced version thereof. [0022] The term "biomarker^"' as used in the present application refers generally to a molecule, including a gene, protein, carbohydrate structure, or glycolipid. the expression of which in biological sample can be detected by standard methods (or methods disclosed herein) and is indicative of IL-10 target engagement or I!,- IO efficacy in the treatment of tumors. Optionally, the expression of such a biomarker is determined to be higher than that observed for a control sample. Optionally, for example, the expression of such a biomarker w ill be determined using a gene expression microarray , quantitative PCR or immunohistochemistry (JHC) assay [0023] Bj "subject" or "patient" is meant any single subject for which therapy is desired, including humans. Also intended to be included as a subject are any subjects involved in clinical research trials not showing an> clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls. [0024] The term "mammal" as used herein refers to any mammal classified as a mammal, including humans, cows, horses, dogs and cats, in a preferred embodiment of the invention, the mammal is a human.

[0025] By "biological sample" is meant a collection of similar cells obtained from a tissue of a subject or patient. The source of the sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate: blood or any blood constituents (e.g., serum); bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The sample may also be primary or cultured cells or cell lines. Optionally, the sample is obtained from a primary or metastatic tumor. The tissue sample may contain compounds which are not naturallv intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. [0026] By "correlate^"' or ^"correlating" is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first anahsis or protocol to determine whether a second analvsis or protocol should be performed. With respect to various embodiments herein, one may use the results of an analytical assay such as mRNA expression or an immunoassa\ to determine whether a dosage of IL- 10 engages the receptor and/ or is efficacious in reducing the size of a tumor.

[0027] By "nucleic acid^" is meant to include an> DNA or RNA. For example, chromosomal, mitochondrial, viral and/or bacteπal nucleic acid present in tissue sample. The term "nucleic acid" encompasses either or both strands of a double stranded nueiete acid molecule and includes anv fragment or portion of an intact nucleic acid molecule. [0028J By "gene^" is meant any nucleic acid sequence or portion thereof w ith a functional role in encoding or transcribing a protein or regulating other gene expression. The gene ma\ consist of ali the nucleic acids responsible for encoding a functional protein or onh a portion of the nucleic acids responsible for encoding or expressing a protein The nucleic acid sequence may contain a genetic abnormality within exons, introns, initiation or termination regions, promoter sequences, other regulator) sequences or unique adjacent regions to the gene.

[0029] The term ""gene expression analysis^*' is the measurement of mRNA in a sample. Gene expression analysis can be performed by methodologies that include, but are not limited to, Northern blots, PCR, including real-time or quantitative PCR, serial analysis of gene expression (SAGE), DNA microarrays, etc.

[0030] The term "immunoassay^"' refers to assays combining immunological and chemical methods to detect protein levels in a sample, including biological samples. Immunoassays include ELISA, RIA. Western blots. luminescent assays, fluorescent assays, etc.

[0031] The word "iabel" when used herein refers to a compound or composition which is conjugated or fused directly or indirectly to a reagent such as a nucleic acid probe or an antibody and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g.. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

[0032] The term "antibody'^* herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies {e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0033] ""Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')i, and F\ fragments; diabodies; linear antibodies; singie-chain antibody molecules; and multispecific antibodies formed from antibody fragments. J0034] "Native antibodies^*" are usually heterotetrameric glycoproteins of about ϊ 50.000 daltoπs. composed of two identical light (L) chains and two identical heavy (S I) chains. Each light chain is linked to a

chain b> one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin tsotjpes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (Vu) followed b> a number of constant domains. Each light chain has a variable domain at one end (V_? ) and a constant domain at its other end: the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0035] The term "variable^" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable or complementary determining regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariabie regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC). [0036] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc^" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment >ie Ids an F(ab'); fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

|0037)

^" is the minimum antibod) fragment w hich contains a complete antigen- recognition and antigen-binding site. This region consists of a dimer of one heav> chain and one light chain variable domain in tight, non-covalent association, ft is Sn this configuration that the three hvpervaπable regions of each variable domain interact to define an antigen- binding site on the surface of the Vn-Vi dimer Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. [0038J The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the

chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0039] The "light chains^'' of antibodies (imniunoglobuiins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ> and lambda (λ). based on the amino acid sequences of their constant domains.

[0040] Depending on the amino acid sequence of the constant domain of their hea\y chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, JgD, IgE. IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl , lgG2. IgG3, IgG4, IgA. and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, λ, ε, γ. and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[004Ϊ] "Single-chain Fv" or "scFv" antibody fragments comprise the Vn and V^ domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the Vn and V_E domains which enables the scFv to form the desired structure for antigen binding. For a re\ ievi of scFv see Piueckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer- Vertag. New York, pp. 269-315 (1994). f0042j The term "diabodies^" refers to small antibody fragments with two antigen- binding sites, which fragments comprise a

variable domain (Vn) connected to a ϊight-chain variable domain (V; ) in the same polypeptide chain (V_H-V_I ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites. Diabodies are described more fully in, for example. EP 404,097: WO 93/ 1 1 161 ; and Hollinger et aL (1993) Proc. Natl. Acad ScL USA, 90:6444-6448. f0043] 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. Monoclonal antibodies are higblj specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibodv preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridorna culture, uncontaminated b> other immunoglobulins. The modifier ^"monoclonal^" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention maj be made by the hybridoma method first described by Kohler et aL (1975) Nature. 256:495, or may be made by recombinant DNA methods (see, e.g.. U.S. Pat. No. 4,816,567) The "monoclonal antibodies'^' may also be isolated from phage antibody libraries using the techniques described in Ciackson et al., ( 1991) Nature, 352:624-628 and Marks et aL, (1991) J. Mot. Biol, 222:581 -597. for example.

[0044] The monoclonal antibodies herein specifically include "chimeric^"' antibodies

(immunoglobulins) 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 antibodv class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies demed from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as ihe> exhibit the desired biological acik itj. (L^*. S Pat. No. 4,816,567; Morπson et aL ( 1984) Proc Natl Acad Sa USA, 81 :6851-6855), Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkev. such as baboon, rhesus or c\r!omolgus monkey) and human constant region sequences (U.S. Pat. No. 5.693.780) [0045] "Humanized^"' forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, 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 capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. Jn general, the humanized antibody will comprise substantially ail of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially ali of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., (1986) Mature 321 :522-525; Riechmann et al., (1988) Nature 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol. 2:593-596. [0046] The term "hvpervariable region'' when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "'complementarity determining region^*' or "CDR^" (e.g. residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (H l ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain: Kabat et al., Sequences of Proteins of Immunological Interest, 5th Hd. Public Health Service, National Institutes of Health. Bethesda, Md. (1991 )) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (Ll ). 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H l), 53-55 (H2) and 96-101 (H3) in the heavy chain vaπabic domain; Chothia and Lesk ,/ Mo! Biol. 196:901-917 ( 1987)) "Framework" or "FR^" residues are those v-aπabϊe domain residues other than the hypenariable region residues as herein defined. [0047] An antibody "v\ hich binds^"" an antigen of interest is one capable of binding that antigen w tth sufficient affinity and/ or av idity such that the antibodv is useful as a therapeutic or diagnostic agent for targeting a cell expressing the antigen (0048] For the purposes herein, "immunotherapy" will refer to a method of treating a mammal (preferably a human patient) with an antibody, wherein the antibody may be an unconjugated or "naked" antibody, or the

may be conjugated or fused with heterologous molecule(s) or agent(s), such as one or more cytotoxic agent(s), thereby generating an "immunoconjugate".

[0049] An "'isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural ens ironmcnt. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antϊbod}, and

include en/ymes. hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (I) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ w ithin recombinant ceils since at least one component of the antibody's natural environment will not be present. Ordinarily, isolated antibody wili be prepared by at least one purification step. [005Oj The expression "effective amount" refers to an amount of an agent (e.g. IL- 10) which is effective for preventing, ameliorating or treating the disease or condition in question.

[0051 j The terms "treating", "treatment'^" and "therapy" as used herein refer to curative therapy, prophylactic therapy, and preventative therapy. Consecutive treatment or administration refers to treatment on at least a daily basis without interruption in treatment by one or more

Intermittent treatment or administration, or treatment or administration in an intermittent fashion, refers to treatment that is not consecutive, but rather c\dic in nature. fOO52] The term "cytokine^" h a generic term for proteins released

one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokiπes. monokines, chemokines. and traditional polypeptide hormones, In particular, chemokines such as gamma-interferon-induced monokine (MlG: also known as CXCL9), interferon inducible cytokine, IP- 10 {also known as CXCL] O). and monocytes ehemoattractant protein- 1 (ΪVfCP-1 ), Table 1 : Biomarker Accession Numbers

[0053] "STAT3" or "signal transducer and activator of transcription 3" as used herein is a biomarker indicative of IL-I O target engagement or engagement with the IL-10R complex. STAT3 is a factor that is phosphorylated upon binding of IL- 10 to the IL- 1OR complex.

[0054] The term ''cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of ceils. The term is intended to include radioactive isotopes (e.g.. i^m, I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

[0055] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyciosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa. carboquone, meturedopa, and uredopa; ethyienimines and methylarnelamines including altretamine, triethylenemelamine, trietylenephosphoramide, trielhylenethtophosphoramide and trimethylolomelamine; acetogenins (especially bυllatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; caiiystatin; CC- 1065 {including its adozelesin, carzeiestn and bizelesiti synthetic analogues); cryptophycins (particularly cryptophycin I and cryptophyciri 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMϊj; eleutherobin; pancratistafin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine. cholophosphamtde, esirarnustme, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphaianu novernbichin, pheπesteriπe, prednimustine, trofbsfarnide, uracil mustard; niirosureas such as earmustøe. chlorozotoctn, fotemustine. lomυstine. nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamiein. especially calicheamicin gamma l ϊ and caiicheamicin phill, see, e.g., Agnew, Chem. ϊntl. Ed. Engl,, 33: 183-186 (1994): dvnemicin, including dvnemicin A: bisphosphonates, such as clodronate: an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores), aclacinomysins. actinomycin, authramycin. azaserine, bleomycins, cactinomycin, carabicin. caminomycin, carzinophiiin, chromomvcins. dactinomycin, daunorubicin. detorubicin, ό-diazo-S-oxo-L-norieucine. doxorubicin (Adriamycin™) (including morpholino-doxorubicin, cyanomorpholino-doxorabicin. 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcel lomycin, mitom>cins such as mitomycin C, mycophenolic acid, nogalamycin. olivomycins, peplomycine potfiromycin, puromycin. quelamycin, rodorubicin. streptonigrin, streptozocin, tuberctdin, ubenimex, zinostatin, zorubicin; anti-melabolites such as methotrexate and 5-fluorouracii (5-FU); folic acid analogues such as denopterin. methotrexate, pteropterin, trimetrexate: purine analogs such as fludarabine, ό-mercapto purine, thiamiprine, thioguanine; pyrimidine analogs such as ancttabine. azacitidine, 6-azauridine, carmofur, cylarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate. cpitiostanof, mepitiostane, lestolactone; anti-adrenals such as aminoglutethimide, mitotane. triSostane: folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene: edatraxate; defofamine; demecolctne; diaziquone; eiformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate: hydroxyurea; lentinan: lonidamine; maytansinoids such as maytansine and ansamitocins: mitoguazone: rmtoxantrone; mopidamol: nitracrine; pentostatin: phenamet; pirarubicin: losoxantrone; podophyllintc acid; 2-ethylhydrazide: procarbazine; PSK®; razoxane; rhizoxin; sizofuran; spirogermamum; tenuazonic acid: trta/iquone; 2. 2'.2"-trichIorϋtricth\ iamine; trichothccenes (especially T-2 toxin, verracurin A. roridsn A and anguϊdiπe). urethan: \ indesine; dacarbazine; mannomustme, mitobmnitoh mitolactoi; pipobroman; gacytosinc: arabinoside ("Ara-C^"); cyciophosphamide; thfotepa; taxoids. e.g. paclitaxel (TAXOLI:. Bristol-Myers Squibb Oncology, Princeton, NJ.) and doxetaxel (TAXOTHRHS, Rhόrte-Poulenc Rorer, Antony. France), chlorambucil: gemcitabine (Gemzar^1M); 6-thioguanine: mercaptopurine; methotrexate; platinum analogs such as cispiatϊn and carboplatin; \ inblastine; platinum: etoposidc (VP- 16); ifosfamlde; mitoxantrone; vincristine; vinorelbine (Navelbine™); novantrone: teniposide; edatrexatc; daunomjciti; aminopterin; xeloda: ibandrotiate; CPT-1 1 : topoisomerase inhibitor RFS 2000:

(DMfO); retinoids such as rctinoic acid: capecitabine; and pharmaceutically acceptable salts, acids or derivatives of airy of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor moduJators (SERMs). including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4- hydroxytamoxifen. trioxifene, keoxifene. LYl 17018. onapristone, and toremifene (Fareston); aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example. 4(5)-imidazoles, aminoglutethirnide. megestrol acetate (Megace™). exemestane. formestane, fadrozole, vorozole (Rivisor™). letrozole (Femara^τvl). and anastrozole (Arimidex™); and anti-androgens such as ffutamide. nϊlutamide, bicalutamide, leuproiide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0056] A "'growth inhibitory agent^* ' when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell over expressing any of the genes identified herein, either in vitro or in vivo. Thus, the growth inhibitor)- agent is one which significant!} reduces the percentage of cells over expressing such genes in S phase. Examples of growth inhibitor} agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine) taxol, and topo II inhibitors such as doxorubicin, eptrubtein, daunorubicin, etoposide. and bleomycin. Those agents that arrest Gl also spill over into S-ρhase arrest for example. DNA alkylating agents such as tamoxifen, prednisone, dacarba/ine, mechiorethamine. cisplatin, methotrexate. 5- fluorouracil, and ara-C. Further information can be found in The Molecular Basis υf Cancer.. Mendelsohn and Israel, eds.. Chapter 1. entitled "Cell

regulation, oncogens, and antineoplastic drugs"

Vlurakami et aS. (% B Saunders" Philadelphia. !995j, [0057] The terms "cancer^", "cancerous"^", or "malignant^" refer to or describe the physiological condition in mammals that is typically characterized by unregulated eel! growth. Examples of cancer include but are not limited to. carcinoma, lymphoma, leukemia, blastema, and sarcoma. More particular examples of such cancers include squamous eel! carcinoma myeloma, smalϊ-cell lung cancer, non-^maϋ ceil lung cancer, glioma, hodgkin s lymphoma, non-hod gkin's lymphoma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer.

II. General.

[0058] The present invention provides methods of monitoring target engagement and efficacy of IL-10 in the treatment of tumors. In particular, serum levels of MCP-I and STAT3 are biomarkers of IL-10 target engagement, while serum levels of MIG and IP-10 are biomarkers of efficacy .

A. Sample Collection and Preparation

[0059] The biomarkers can be measured in bioiogical samples from cancer subjects before and after exposure of the subject to a therapeutic drug. Accordingly, samples may be collected from a subject over a period of time. Furthermore, obtaining numerous samples from a subject over a period of time can be used to verify results from earlier detections and/or identify a differential expression as a result of exposure to a therapeutic drug. Generally, biological samples can be collected from a subject via biopsy, but may be collected using other known clinical methods, such as the collection of peripheral blood, including serum, or bone marrow for hematological malignancies, or the collection of peripheral blood mononuclear cells for surrogate assays of target inhibition, Tjiornarker measurement.

[G060J In one embodiment of the invention, the samples are analyzed without additional preparation and Or separation procedures. In another embodiment of the invention, sample preparation and-'or separation can invoKe. w ithout limitation, any of the following procedures., depending on the type of sample collected and/or types of biomarkers searched: removal of high abundance polypeptides: addition of preservatives and calibrants. desalting of sampler concentration of sample substances; protein digestions; and fraction collection. In yet another embodiment of the invention, sample preparation techniques concentrate information-rich biomarkers and deplete polypeptides or other substances that would carr> little or no information such as those that are bϊghlv abundant in or native to the tumor. [0061] In another embodiment of the invention, sample preparation takes piace in a manifold or preparation/separation device. Such a preparation/separation device may, for example, be a mtcrofluidics device. In yet another embodiment of the invention, the preparation/separation device interfaces directly or indirectl> with a detection device. Such a preparation/separation device ma>, for example, be a fluidics device. [0062J In another embodiment of the invention, the removal of undesired polypeptides (e.g., high abundance, uninformatne, or undetectable polypeptides) can be achieved using high affinity reagents, high molecular weight filters, column purification, ultracentrifugation and/or electrodialysis. High affinity reagents include antibodies that selectively bind to high abundance polypeptides or reagents that have a specific pH, ionic value, or detergent strength. High molecular weight filters include membranes that separate molecules on the basis of size and molecular weight. Such filters ma\ further

reverse osmosis, nanofiltration, ultrafiltration and micro filtration.

[0063] Lltracentrifugation constitutes another method for removing undesired polypeptides. Ultracentrifugation is the centrifugation of a sample at about 60.000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles. Finally, electrodialysis is an electromembrane process in which ions are transported through ion permeable membranes from one solution to another under the influence of a potential gradient. Since the membranes used in electrodiaiysis have the ability to selectively transporlions having positive or negative charge and reject ions of the opposite charge, electrodiaiysis is useful for concentration, removal, or separation of electrolytes. [0064] In another embodiment of the invention, the manifold or microfluidics device performs electrodsaijsis to remove high molecular weight polypeptides or undesired poh peptides. Electrodialysis can be used first to allow only molecules under approximate!) 30 kD to pass through into a second chamber. A second membrane with a ver\ small molecular weight (roughh 500 D) allows smaller molecules to egress the second chamber. [0065 j Upon preparation of the samples, biomarkers of interest may be separated in another embodiment of the invention. Separation can take place in the same location as the preparation or in another location. In one embodiment of the invention, separation occurs in the same mtcrofluidics device where preparation occurs, but in a different iocation on the device. Samples can be removed from an initial manifold location to a microfiuidics device using various means, including an electric field, In another embodiment of the invention, the samples are concentrated during their migration to the microfluidics dev ice using reverse phase beads and an organic solvent elution such as 50% methanol. This elutes the molecuies into a channel or a well on a separation

ice of a microfluidics device. [0066] Chromatography constitutes another method for separating subsets of substances. Chromatography is based on the differential absorption and elution of different substances. Liquid chromatography (LC), for example, involves the use of fluid carrier over a non-mobile phase. Conventional LC columns have an in inner diameter of roughly 4.6 mm and a flow rate of roughly 1 ml/min. Micro-LC has an inner diameter of roughly 1.0 mm and a flow rate of roughly 40 μl/rnin. Capillary LC utilizes a capillary with an inner diameter of roughly 300 μm and a flow rate of approximately 5 ul/min, Nano-LC is available with an inner diameter of 50 um-1 mm and flow rates of 200 nl/min. The sensitivity of nano-LC as compared to HPLC is approximately 3700 fold. Other types of chromatography contemplated for additional embodiments of the invention include, without limitation, thin-layer chromatography (TLC), reverse-phase chromatography, high-performance liquid chromatography (HPLC), and gas chromatography (GC).

[0067] In another embodiment of the invention, the samples are separated using capillary electrophoresis separation. This will separate the molecules based on their eletrophoretic mobility at a given pH (or hydrophobicity).

[0068] In another embodiment of the invention, sample preparation and separation are combined using microfluidics technology. A microfluidic device is a device that can transport liquids including \ arious reagents such as analytes and elutions between different locations using microchannel structures.

[0069] A biomarker can be modified before analysis to improve its resolution or to determine iis identity. For example, the biomarker may be subject to proteolytic digestion before analysis. Any protease can be used. Proteases, ^uch as trypsin, that are ϊikeh to cleave the biomarkers into a discrete number of fragments are particular!) useful. The fragments that result from digestion function as a fingerprint for the products, thereby enabling their detection indirectly. This is particularly useful where there are biomarkers w ith similar molecuϊar masses that might be confused for the product in question. Also, proteolytic fragmentation is usefuS for high molecular weight products, because smaller products are more easily resolved by mass spectrometry. In specific embodiments, the proteases occur or naturally exist in the tumor sample.

(0070] To improve detection resolution of the biomarkers, neuraminidase can, for instance, be used to remove terminal sialic acid residues from glycoproteins to improve binding to an anionic adsorbent (e.g., catSonic exchange ProteinChip® arrays) and to improve detection resolution. In another example, the biomarkers can be modified by the attachment of a tag of particular molecular weight that specifically bind to molecular markers, further distinguishing them. Optionally, after detecting such modified products, the identity of the products can be further determined by matching the physical and chemical characteristics of the modified products in a protein database (e.g., SwissProt).

[0071J It has been found that proteins frequently exist in a sample in a plurality of different forms characterized by a detectably different mass. These forms can result from either, or both, of pre- and post-translational modification. Pre-translational modified forms include allelic variants, slice variants and RNA editing forms. Post-translationally modified forms include forms resulting from proteolytic cleavage (e.g., fragments of a parent protein), glycosyiation, phosphorylation, lipidation, oxidation, methylation, cystinylation, sulphonation and acetylation. Modified forms of any biomarker of this invention also may be used, themselves, as biomarkers in the profiles. In certain cases, the modified forms may exhibit better discriminatory power in diagnosis than the specific forms set forth herein. (0072] For some of the method embodiments of the invention, it may be helpful to purify the biomarker whose differential presence has been detected by the methods disclosed herein prior to subsequent analysis. Nearly any means known to the art for the purification and separation of small molecular weight substances, e.g., anion or cation exchange chromatography, gas chromatography, liquid chromatography or high pressure liquid chromatography may be used. Methods of selecting suitable separation and purification techniques and means of carry ing them out are known in the art (see. e.g , Labadarious et. aL (1984) J Chromatography 310:223-231. and references cited therein, and Shahrokhin and Gehrke. ( 1968} ,/ Chromatography 36:31 -41 , and Niessen (1998) J. Chromatography 794:407-435). To the extent that it is desired to determine the differential presence of a nucleic acid biomarker, the biomarker may be purified using known methods including, slab or capillary gel electrophoresis [0073] In another embodiment of the method of the invention, purification of the hiomarker comprises fractioning a sample comprising one or more protein products b> size- exclusion chromatography and collecting a fraction that includes the one or more products; and⁷or fractioning a sample comprising the one or more products by anion exchange chromatography and collecting a fraction that includes the one or more products. Fractionation is monitored for purity on normal phase and immobilized nickel arrays. Generating data on immobilized biomarker fractions on an arrav is accomplished b> subjecting the array to laser ionization and detecting intensity of signal for mass/charge ratio; and transforming the data into computer readable form. Preferably, fractions are subjected to gel electrophoresis and correlated with data generated by mass spectrometry. In one aspect, gel bands representative of potential bϊomarkers are excised and subjected to enzymatic treatment and are applied to biochip arrav s for peptide mapping.

B. Detection and Quantitation of Biomarkers

[0074] Any suitable method can be used to detect (a differential presence of) one or more of the biomarkers described herein. Successful practice of the invention can be achieved with one or a combination of methods that can detect and, preferably, quantify the biomarkers. These methods include, without limitation, hybridization-based methods including those employed in biochip arrays, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, eilipsometry and atomic force microscopy. For nucleic acid biomarkers, methods for detection and quantitation include PCR, quantitative PCR, northern blot analysis, southern blot analysis, mass spectrometry and the like.

[0075] Methods ma> further include, by one or more of electrosprav ionization mass spectrometry (ESI-MS), ESI-MS/MS. ESI-MSz(MS)", matrix-assisted laser desorption ionization time-of- flight mass spectrometry (MA! -DKI OF-MS). surface-enhanced laser desorption ionization time-of-flight rna^s spectrometry (SELDI- IOI- -Vl S). desorption/ioniyation on silicon i DK)S). secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-IOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS). APCI-MS/MS, APCI-(MS)", atmospheric pressure photoionization mass spectrometry (APPI-MS). APP1-MS/MS. and APPI-(MS)_n. quadrupole mass spectrometry , fourier transform ma» spectrometry (ITVfS). and ion trap mass spectrometry. where n is an integer greater than zero. Spectroscopic methods for detecting and quantifying protein biomarkers are known in the art and are described, for example in L. S. Pat. Nos. 5,719,060; 6,225.047; 5,719,060; 6,124, 137 and PCT International Publication No. WO 03/64594.

[0076] fn another embodiment of the invention, the biomarkers of the invention are measured by a method other than mass spectrometry or other than methods that rely on a measurement of the mass of the biomarker. In one such embodiment that does not rel> on mass, the biomarkers of this invention are measured by immunoassay. Immunoassay requires bsospecifϊc capture reagents, such as antibodies, to capture the biomarkers. Antibodies, can be produced by methods well known in the art, e.g.. by immunizing animals with the biomarkers. Biomarkers can be isolated From samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.

[0077] This invention contemplates traditional immunoassays including, for example, sandwich immunoassays including F-XISA or fluorescence-based immunoassays, as well as other enzyme immunoassays. Nephelometry is an assay done in liquid phase, in which antibodies are in solution. Binding of the antigen to the antibody results in changes in absorbance, which is measured. In the SELDI-based immunoassay, a biospecific capture reagent for the biomarker is attached to the surface of an MS probe, such as a pre-activated ProteinChip array. The biomarker is then specifically captured on the biochip through this reagent, and the captured biomarker is detected by mass spectrometry. [0078] In one embodiment, the biomarker can be a nucleic acid, wherein the nucleic acid can be detected and/or quantified using methods known in the art. For example, nucleic acid biomarkers may be detected using PCR (disclosed in U.S. Pat. No. 4,683, 195. U.S. Pat. No. 4.683,202 and U.S. Pat. No. 4.965, 188 and others in detail). In one embodiment, a real time PCR method is used to enabϊe a convenient and reliable quantitative measurement of biomarker nucleic acids having a wide dynamic range. The real time PCR technique includes the method by using a TaqMan probe using AB1PRISM77G0. ™. (Applied Biosy stems) and the method by using LightCycler. ™. (Ropche Diagnostics). Particularly in the latter case, in a high rate reaction cycle in which a temperature cycle of PCR is completed for some 10 minutes, a change of an amplified amount of a DN' Λ synthesized for every cycle can be detected in a real time. DNA detection method of the real time PCR method includes 4 methods using a DMA-binding pigment (intercalator), a hybridization probe (kissing probe), TaqMan probe, or Sunrise Uniprimer (molecular beacon). On the other hand, the expression of a biomarker gene can be analyzed

using a DNA-binding pigment such as SYBR Greeπl. SYBR Greenl is a binding pigment specific to a double strand of the DNA and, when bound to a double strand, an inherent fluorescence intensity is reinforced. By adding SYBR Green! at the PCR reaction and measuring the fluorescence intensity at the end of each cycle of an elongation reaction, the increase in a PCR product can be detected. For detection of a biomarker gene, similar to normal PCR, a primer is designed by using a commercialized gene anal>sis software on the basis of a sequence of the biomarker gene. SYBR Greenl detects a nonspecific product and, thus, requires designing an optimal primer. Required designing standards are a length of an oligomer, a base composition of the sequence, a GC content, and a Tm value.

[0079] Detection methods may include use of a microarray/biochip array. Biochip arrays useful in the invention include protein and nucleic acid arrays. One or more biomarkers are captured on the biochip array and subjected to laser ionization to detect the molecular weight of the products. Analysis of the products is. for example, by molecular weight of the one or more biomarkers against a threshold intensity that is normalized against total ion current.

[0080] The biochip surfaces may, for example, be ionic, anionic, hydrophobic; comprised of immobilized nickel or copper ions, comprised of a mixture of positive and negative ions: and/or comprised of one or more antibodies, single or double stranded nucleic acids, proteins, peptides or fragments thereof, amino acid probes, or phage display libraries. Man> protein biochips are described in the art. These include, for example, protein biochips produced b\ Ciphergen Bios> stems (Fremont, Calif.), Packard BioScience Company (Meriden Conn.), Zyomyx (Hay ward. Calif.) and Ph\ ios (Lexington, Mass.). Examples, of such protein biochips arc described in the following patents or patent applications: U.S. Pai. No. 6,225,047 (Hutchens and Yip. "Use of retentate chromatography to generate difference maps,^" May I. 2001 ); International publication WO 99/51773 (Kuimelis and Wagner, "Addressable protein arrays." Oct. 14, 1999): U.S. Pat. No. 6,329.209 (Wagner et a!.. "Arrays of protein-capture agents and methods of use thereof,^'1 Dec, 1 1. 2001 ) and Internationa! publication WO 00/56934 (Englert et a!.. "Continuous porous matrix arravs.^" Sep. 28, 2000). [0081] Biomarkers may be captured with capture reagents immobilized to a solid support, such as a biochip, a multiwell microliter plate, a resin, or nitrocellulose membranes that are subsequently probed for the presence of proteins. Capture can be on a chromatographic surface or a biospecific surface. For example, a tumor sample containing the biomarkers may be placed on the active surface of a biochip for a sufficient time to allow binding. Then, unbound molecules are washed from the surface using a suitable eluant, such as phosphate buffered saline. In general, the more stringent the eluant, the more tightly the proteins must be bound to be retained after the wash.

[0082] Upon capture on a biochip, analvtes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method. Also of interest is the use of mass spectrometry, for example, SELDI. Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). Optical methods include microscopy (both confocai and non-confocal). imaging methods and non-imaging methods. Immunoassays in various formats (e.g., ELlSA) are popular methods for detection of analytes captured on a solid phase. Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy.

C. Qualification of Cancer Status

[0083] The biomarkers of the present invention have a number of uses. For example, identification of an anti-tumor response or monitoring the efficacy of a therapeutic drug in a cancer subject takes into account the amount of the biomarker(s) in a tumor sample before and after exposure of the subject to a therapeutic drug (up or down regulation of the bϊomarker(sj). The amounts are measured under the same or substantial!} similar experimental conditions but at different time periods preceding and follow ing treatment. The biomarkers of the invention can also be used to identify an agent useful in the treatment of cancer. f0084] The detection of a differential presence of a plurality of biornarkers in a tumor sample may improve the indication of therapeutic efficacy of a therapeutic drug in the treatment of a cancer.

[0085J The biomarkers of the present invention may also be used for determining efficacy and/or receptor engagement following therapy with and IL-I O therapeutic agent. The biomarkers can be used alone or in combination with other products. The biomarkers are differentially present in tumor samples of a cancer patient before and after exposure to a therapeutic drug. For example, some of the markers are expressed at an elevated level and/or are present at a higher frequency after treatment, while some of the products are expressed at a decreased level and/or are present at a lower frequency after treatment. Therefore, generating a biomarker profile for a subject would provide useful information regarding cancer status.

[0086] In certain embodiments of the methods of qualifying cancer status, the methods further comprise managing subject treatment based on the status. The invention also provides for such methods where the biomarkers (or specific combination of biomarkers) are measured again after such subject management. In these cases, the methods are used to monitor the status of the cancer, e.g., candidacj for treatment with IL-IO or a modified IL-I O protein, response to IL-I O treatment, remission of the disease or progression of the disease. [0087] The biomarkers of the present invention can be used to adjust the dosage of therapeutic IL-10 or PEG-IL-10 provided to a subject. For example, MCP-I serum protein level expression and phosphorylation of STAT3 is indicative of target engagement. To achieve maximal target engagement of JL-IO or PEG-IL-10, the clinician can adjust the dosage such that the expression of MCP-I and/or phosphorylation STAT3 is optimized. Similarly, the expression of MlG and/or IP-10 is indicative of tumor inhibition efficacj. Again, by optimizing the expression of MIG and/or IP-10 by varying IL-10 or PEG-IL-10 dosage, the clinician can achieve maximal tumor inhibition. Thus, bv changing the dosage level in a systematic manner, the optimal level of therapeutic IL-10 or PEG-IL- IO is determined for a subject. Amelioration of symptoms ma> be monitored concurrent! \. This method is used to find an optimal dosage level at

to treat a subject. (0088( In another embodiment of the invention, the output from a detection device can subsequently be processed, stored, and further analyzed or assajed using a bio- informatics ^\stem A bio-informatics system tτsa\ include one or more of the follow ing, without limitation: a computer; a plurality of computers connected to a network; a signal processing tool(s); a pattern recognition tooi(s): a tool(s) to control flow rate for sample preparation, separation, and detection.

[0089] The data processing utilizes mathematical foundations. In another embodiment of the invention, djnamic programming is used to align a separation axis with a standard separation profile. Intensities may be normalized, for example, by fitting roughly 90% of the intensity

into a standard spectrum. The data sets can then be fitted using wavelets designed for separation and mass spectrometer data. In yet another embodiment of lhe invention, data processing filters out some of the noise and reduces spectrum dimensionality, potentially allowing for pattern recognition.

[0090] Foliowing data processing, pattern recognition tools can be utilized to identify subtle differences between phenot>pic states. Pattern recognition tools are based on a combination of statistical and computer scientific approaches, which provide dimensionalitj reduction. Such tools are scalable. Data so obtained may be stored on a computer readable medium.

D. Kits

[0091] In one aspect, the invention provides kits for qualifying cancer status in a subject, wherein the kits can be used to detect the differential presence of the biomarkers described herein. For example, the kits can be used to detect a differential presence of any combination of the biomarkers in tumor samples of cancer subjects before and after exposure to a therapeutic drug. The kits of the invention have many applications. For example, the kits can be used to monitor efficacy of a therapeutic drug in a cancer subject. The kits can also be used to identify agents useful in the treatment of cancer.

[O092J In specific embodiments, kits of The invention contain a biomarker. which is optionally botopicaih or fluorescently labeled.

J0093] The kits of the invention ma> include instructions, reagents, testing equipment

(test tubes, reaction vessels, needles, s>ringes, etc.). standards tor calibration, and'or equipment. Reagents ma\ include acids, bases, oxidi/ing agents, and marker species, fhe instructions prov ided in a kit according to the invention ma> be directed to suitable operational parameters in the form of a label or a separate insert. [0094] The kits may also include an adsorbent, wherein the adsorbent retains one or more biomarkers described herein (polynucleotide or polypeptide), and written instructions for use of the kit for qualification of cancer status in a subject. Such a kit could, for example, comprise: (a) a substrate comprising an adsorbent thereon, wherein the adsorbent is suitable for binding a biomarker, and (b) instructions to detect the biomafker(s) by contacting a tumor sample v\ iih the adsorbent and detecting the ρroduct(s) retained by the adsorbent. Accordingly, the kit could comprise (a) a DNA probe that specifically binds to a biomarker: and (b) a detection reagent. Such a kit could further comprise an eluant (as an alternative or in combination with instructions) or instructions for making an eluant, wherein the combination of the adsorbent and the eluanl allows detection of the biomarker using, for example, gas phase ion spectrometry.

[0095] This invention is further illustrated by the following examples, which should not be construed as limiting. A skilled artisan should readily understand that other similar instruments with equivalent function/specification, either commercially available or user modified, are suitable for practicing the instant invention. Rather, the in\ention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.

EXAMPLES

ϊ. General methods.

[0100] Standard methods in molecular biology are described. Mamatis el al (1982)

Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY: Sam brook and Russell (2001) Molecular Cloning, 3^{rd '} ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA. Standard methods also appear in Ausbel et al, (2001) Current Protocols in Molecular Biology, VoIs.1-4, John Wiley and Sons. Inc. New York, NY. which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1). cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0101] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described. Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc.. New York, Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described. See, e.g., Coiigan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3. John Wiley and Sons, Inc., NY. NY. pp. 16.0.5-16.22.17; Sigma-Aldrtch, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ., pp. 384-391. Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protcoh in Immunology, Vol. /, John Wiley and Sons, Inc.. New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press. Cold Spring Harbor. NY; Harlow and Lane, supra. Standard techniques for characterizing iigand 'receptor interactions are available. See, e.g.. Coiigan et al. (2001 ) Current Protcoh in Immunology, VoI 4, John

Inc.. New York, [0102] Methods for flow cytometry, including fluorescence activated celϊ sorting detection s\ stems (FACS*'), are available. See, e g.. Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice. John Wiley and Sons. Hoboken, NJ; Gi\an (2001 ) Flow Cytometry, 2^nd ed.: Witey-Liss, Hoboken. NJ: Shapiro (2003) Practical Flow Cytometry. John Wiley and Sons. Hoboken, NJ Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g.. as diagnostic reagents, are available. Molecular Probes (2003) Catalogue, Molecular Probes, Inc.. Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis. MO. [0103] Standard methods of histolog> of the immune system are described. See, e.g ,

Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY: Hiatt, ei al. (2000) Color Atlas of Histology, Lippincott Williams, and Wiikins. Phila, PA; Louis, et al. (2002) Basic Histology-Text and Atlas, McGraw-Hill. New York, NY.

[0104J Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available. See, e.g., GenBank. Vector NTl* Suite (Informax, fnc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher* (TimeLogic Corp., Crystal Bay, Nevada); Menne et at (2000) Bioinprmatics 16; 741-742; Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren et al. (2002) Comput. Methods Programs B iomed. 68: 177-181 ; von Heijne (1983) Eur. J. Biochem. 133: 17-21 ; von Heijne (1986) Nucleic Acids Res. 14:4683-4690.

ΪI. Tumor implantation and blood sample collection.

(0105] Female C57/B6 mice approximately 8 to 10 weeks of age were used. The mouse squamous cell carcinoma line PDV6 was cultured in DMIiM with 10% FBS. 1 x10⁶ cells were injected subcutaneously in the right flank of each mouse and tumors were monitored regularly. Tumor staging and dosing of IL-10 or control was determined when average tumor size reached approximately 100-150 mm^J (e.g., at day S O to day 14 post- implant). Animals were then treated with PEG-IL-10 or vehicle control subcutaneously at designated dosing regimen. At given time points, animals were euthani/ed in accordance with IACL¹C recommendations and blood samples were collected.

IH. Serum sample collection and chemoklnes measurement.

[βløόj Using a serum separator tube to collect the mouse blood samples, the blood was allowed to clot for 30 minutes, then centrifuged for 15 minutes at approximately lOOOg to separate the serum. The serum sample was either snap frozen or assayed (e g.. ELISA) immediate]}. The serαm concentrations of MCP-5. MCP-K IP- 10 and MIO were quantified using the appropriate commercial ELISA kits (R&D Systems) according to the manufacturer's instructions.

[0107J Single dosing of administration of PEG-IL-IG (0.1 mg/kg) induces early elevation of mouse IV1CP-5 in the serum in the diseased or non-diseased animal, (see, e.g., tables 2 and 3).

Table 2: MCP-5 induction upon PEG-IL-10 treatment of tumor bearing mouse.

Table 3; Mouse MCP-5 induction upon PEG-IL-JO treatment of non-tumor bearing mouse.

[0108] Mouse MIG and IP-10 were induced later following PEG-IL-10 administration to tumor bearing mice (see, e.g., tables 4 and 5). Increased expression of MIG and IP-10 correlated with reduction of tumor volumes.

Table 4: Mouse MlG induction after administration of PEG-IL-10

IV. Human PBMC preparation, stimulation, and chemokities measurement.

[0109] Human peripheral blood mononuclear cells (PBMC) were prepared by

Histopaqυe (SϊgrnaI0771) density centrifugation and cultured with RPM1-1640 containing 10% of heat-inactivated fetal bovine serum.

[O110J To investigation of chemokine production, human PBMC were maintained in the presence or absence of human IL-10 or human PEG-IL- 10. After 24h and 48h, the supernatants were collected. The concentrations of MCP-I, IP-10 and MIG were quantified using commercial ELISA kits (R&D Systems) according to the manufacturer's instructions.

Table 6: Induction of human MCP-I from unprimed PBMC treated with varying concentrations of PEG-IL-10.

Table 7: Induction of human MIG from unprimed PBMC treated with varying concentrations of PEG-IL-] 0.

Table S: Induction of human IP-IO from unprimed PBMC with varying concentrations of PEG-IL- 10

From the above. PEG-! L- 10 induces mouse MCP- 5 in the sera early after administration to tumor bearing and naϊve mice. The human homolog of MCP-5, MCP-I (see. e.g., Sarafi et al. (1997) J. Exp. Med.l 85:99-109). is induced in a dose dependent manner, upon addition of PEG-FL-10 to unprimed PBMC cultures. Therefore MCP-] can be used as a marker for IL-10 target engagement.

[0100] Further induction of IP-] 0 and MlG appear to occur fater following administration of PEG-IL-10 to tumor bearing mice, and was correlated to tumor volume reduction. Therefore IP- 10 and MIG are markers for efficacy.

V. Human PBMC cell ϊysate preparation for STAT3 phosphorylation assay

[0101] To assess STATS phosphorylation levels, human PBMC were maintained in the presence or absence of human IL-10 or human PEG-IL- 10. After 24h and 48h, the cells were harvested by centrifugation at 500g at 4⁰C for 5 minutes. The pellet was washed once with coid PBS and subsequently incubated with complete lysis buffer (150 mM NaCl₅ 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100) containing protease (Protease Inhibitor Set III. EMD Biosciences. Cat. No. 539134) and phosphatase inhibitors (Phosphatase Inhibitors ! and IL Sigma. Cat. Nos. P2850 and P5726. respectively) on ice, for 20 minutes. After removal of cell debris by centrifugation, the cell lysatc was snap frozen for a STA T3 phosphorylation assay VI. STAT3 phosphorylation assay

[0102] The STAT3 phosphorylation assay employed standard ELISA format to assess

PEG-IL-10 engagement with its target receptor. 96 well flat bottom immunoplates were coated with I GO μl/well of STAT3 specific capture antibody (MAB 1799; R&D Systems) at a concentration of ] μg/ml. 80 μl of ceil iysate, described above, was added to each well of the HLlSA plate. Plates were incubated with shaking at room temperature. Plates were washed six times with IX wash buffer (50 mM Tris, pH 7.5, 0.15 M NaCL 0.02% Tween-20; Meso Scale Discovery, Cat. No. R61 TX-2). Detection antibody (CeIi Signaling, Cat. No. 9145) was diluted to a final concentration of 1 : 167, and 100 μi was added to each well. Plates were incubated with shaking at room temperature for 2 hours. Plates were again washed six time with IX wash buffer. A secondary antibody (anti-rabbit HRP conjugate, Pierce. Cat. No. 31460) was diluted 1 :5000, and 100 μl was added to each well. Plates were incubated and washed as above. Plates were read in an ELISA plate reader at an absorbance of 450-650 nm.

Table 9: STAT3 Phosphorylation with varying concentrations of PEG-IL-10

[0103] STAT3 phosphorylation is induced upon administration of PEG-IL-10. and iherefore can be used as a biomarker for target engagement. VII. Rapid modulations of MCP-5 and its receptor CCR2 mRNA expression by PEG-mIL-10 in tumor tissue.

[0104J 4Tl tumor-bearing mice received Imρk ρeg-1L- 10 subcuteously. Tumor tissue was harvested at 0.5, 1 , 2, 6. 12. 24 and 36 hours post dose. Total RNA was extracted and mRNA expression level was assessed by Taqmaπ real-time PCR. Table 10 shows the rnRNA expression of MCP-5 and its receptor CCR2 pooled sample of three individual animals at each time point. MCP-I was used as a negative control. Each sample was normalized to unbiquitin.

Table 10: mRNA expression of MCP-] , MCP-5 and CCR2 in tumor tissues of PEG-IL-10 treated mice

VlH. IL-10 Induced MCP-5 Production is Dependent on IL-I ORβ [0105] C57BL/6 mice or IL-I ORβ knockout mice were treated with 0.25 mg/kg of

PEG-mlL-S 0, and the sera were collected at 0, 4, and 8 hours post treatment. The serum concentration of MCP-5 (pg/^'mL) was assessed by using a commercial ELlSA kit for R&D System. The induction of MCP-5 was inhibited in the IL-I ORβ deficient mouse compared to that of in wild-type mouse. There were five mice in the wild-type group and seven mice in the IL-I ORβ knockout group.

Table 1 1 : MCP-5 protein expression in wild-type (WT) and ΪL-l ORβ KO mice following PHG-IL-10 treament (non-tumor bearing mice).

IX. MCP-5 Levels in PDV6-IL-I0Rβ Knockout Mice Treated with IL-IO Minicircle

DNA

(0106] C57BL/6 mice or IL-lORβ knockout mice were treated with όug of rnlL-IO minicircle DNA or control minicircle DlSIA (hATT) (see, e.g., US Patent Application 2004/0214329), at day 24 after PDV6 tumor implantation, and sera were collected 24 days after treatment. The serum concentration of MCP-5 was assessed by using a commercial ELlSA kit from R&D Systems using manufacturer's instructions. Table 12 shows that induction of MCP-5 was inhibited in the IL-lORβ deficient mouse compared to the wi id-type mouse.

Table 12: MCP- 5 serum levels following hydrodynamic delivery of IL-IO or control minicircle ("MC") DNA at day 24 post tumor implantation

Table 13 shows the relative tumor size at various time points following delivery of IL-10 or control minicircle ("MC^") DNA to IL-I ORβ KO or wild type mice.

X. IL-IO induced MlG elevation is dependent on T cell presence, but MCP-S induction is KOt dependent on T cell presence |0107| RAG2 knockout mice or wild type C57BL6 mice were treated with 6 μgs of mIL-10 MC or control hAAT MC at day 12 after PDV6 tumor implantation. Sera were collected 24 days after treatment. The serum concentrations of MCP-5 and MlG were assessed by using a commercial ELiSA kits from R&D Systems using manufacturer's instructions. Table 14 shows that MCP-5 concentration is elevated in the T cell deficient RAG mice treated with IL-IO MC DNA, Thus MCP-5 expression is not dependent on the presence of T cells.

Table 14: MCP-5 concentration at day 24 post HDD delivery of MC DNA

[0108] Table 15 shows that MEG expression is inhibited in RAG mice given IL-10

MC DNA, thus induction of MIG expression is dependent on the presence of T cells.

Table 15: MIG concentration at day 24 post HDD delivery of MC DNA

[Θ109] Table 1.6 shows that tumor growth is not dependent upon the presence of T cells.

Table 16: Average tumor size through day 40 post MC DNA delivery in RAG mice

Claims

WHAT IS CLAIMED IS:

1. A method for monitoring efficac> of an IL-I O therapeutic agent in treatment of a subject for a tumor or cancer, said method comprising: a) obtaining a baseline biological sample from the subject prior to administering a dose of the IL-10 therapeutic agent; b) measuring the level of at least one biomarker in the baseline biological sample b\ gene expression analysis or immunoassay. c) administering the IL- 10 therapeutic agent to the subject; d) obtaining from the subject at least one subsequent biological sample; e) measuring the level of the biomarker in the subsequent sample by gene expression analysis or immunoassay; f) comparing the level of at least one biomarker in the subsequent sample with the level of at least one biomarker in the baseline biological sample, wherein an increase of at least one biomarker in the subsequent biological sample indicates an effective dose of the IL- 10 therapeutic agent.

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

3. The method of claim 1. wherein the tumor or cancer is from an organ selected from the group consisting of skin, colon, thyroid, ovarian, lung, pancreas and brain.

4. The method of claim 1 wherein the tumor is selected from the group consisting of; colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, and leukemia,

5. The method of claim L therein the IL-IO therapeutic agent is selected from the group consisting of nathe IL-I O. pegylated IL-IO (PhG-IL-I O). and IL-HJ conjugated to an Fc portion of an immunoglobulin (ΪL~10-ϊg).

6. The method of claim 1. wherein the biomarker is selected from the group consisting of 3 PH O and MlG.

7. The method of claim 6, wherein the increase in IP-IO or MIG expression in the subsequent biological sample is at least 2 fold to at least 19 fold.

8. The method of claim 1, wherein the gene expression analysis is selected from the group consisting of Northern blotting, quantitative PCR, SAGE, and DNA microarray.

9. The method of claim 1 wherein the immunoassay is selected from the group consisting of ELISA. RIA, Western blot, luminescent immunoassay, fluorescent immunoassay.

10. A method for monitoring target engagement following administration of an IL-IO therapeutic agent to treat a subject for a tumor or cancer, said method comprising: a) obtaining a baseline biological sample from the subject prior to administering a dose of the IL-10 therapeutic agent; b) measuring the level of at least one biomarker in the baseline biological sample by gene expression analysis or immunoassay, c) administering the IL-IO therapeutic agent to the subject; d) obtaining from the subject at least one subsequent biological sample; e) measuring the level of the biomarker in the subsequent sample by gene expression analysis or immunoassay; f) comparing the level of at least one biomarker in the subsequent biological sample with the level of at least one biomarker in the baseline biological sample, wherein an increase of at least one biomarker in the subsequent biological sample indicates target engagement.

1 1. The method of claim 10. wherein the subject is human.

12. The method of claim 10, wherein the tumor or cancer is from an organ selected from the group consisting of skin, colon, thyroid, ovarian, lung, pancreas, and brain. i 3 The method of claim 10 wherein the tumor is selected from the group consisting of: colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, and leukemia.

14. The method of claim 10, wherein the IL-10 therapeutic agent is selected from the group consisting of native IL-10, pegvlated JL-IO (PEG-IL-I O), and IL-10 conjugated to an Fc portion of an immunoglobulin (IL-lO-lg),

15. The method of claim 10. wherein the biomarker Ls chosen from the group consisting of MCP-I and phosphorylation of STAT3.

16. The method of claim 10, wherein the gene expression analysis is selected from the group consisting of Northern blotting, quantitative PCR, SAGE, and DNA mϊcroarrav.

17. The method of claim 10. wherein the immunoassay is selected from the group consisting of ELISA, RIA. Western blot, luminescent immunoassay, fluorescent immunoassav.