HK1240500A1 - Methods of using interleukin-10 for treating diseases and disorders - Google Patents

Description

Methods of treating diseases and disorders using interleukin-10

The application is a divisional application of an invention application with the application date of 2014, 04, 15, China application number of 201480024021.5 and the invention name of 'a method for treating diseases and symptoms by using interleukin-10'.

Cross Reference to Related Applications

This application claims the benefit of priority from U.S. provisional application serial No. 61/813,563 (which is incorporated herein by reference in its entirety) filed on 18/4/2013.

Technical Field

The present application relates to methods of using IL-10 and related agents for treating or preventing a variety of diseases and disorders.

Introduction to the design reside in

The cytokine interleukin-10 (IL-10) is a pleiotropic cytokine that regulates multiple immune responses by acting on T cells, B cells, macrophages, and Antigen Presenting Cells (APCs). IL-10 can inhibit immune responses by inhibiting the expression of IL-1 α, IL-1 β, IL-6, IL-8, TNF- α, GM-CSF and G-CSF of activated monocytes and activated macrophages, and it also inhibits IFN- γ production by NK cells. Although IL-10 is expressed primarily in macrophages, expression has also been detected in activated T cells, B cells, mast cells and monocytes. In addition to suppressing immune responses, IL-10 also exhibits immunostimulatory properties, including stimulation of proliferation of IL-2 and IL-4 treated thymocytes, enhancement of B cell viability, and stimulation of MHC class II expression.

Human IL-10 is a homodimer that becomes biologically inactive after the non-covalent interaction between two monomer subunits is disrupted. Data from the published crystal structure of IL-10 indicate that the functional dimer exhibits some similarity to IFN- γ (ZDanov et al, (1995) Structure (Lond)3: 591-one 601).

As a result of its pleiotropic activity, IL-10 has been associated with a wide variety of diseases, disorders and conditions, including inflammatory conditions, immune-related disorders, fibrotic disorders and cancer. Clinical and preclinical assessments with IL-10 for many such diseases, disorders and conditions have consolidated their therapeutic potential. In addition, PEGylated IL-10 has been shown to be more effective in certain therapeutic contexts than non-PEGylated IL-10.

Given the prevalence and severity of IL-10 associated diseases, disorders and conditions, novel dosing regimens and parameters that optimize efficacy, patient tolerance, and the like would be of great value in enhancing the therapeutic usefulness of IL-10 and pegylated IL-10 and agents related thereto.

SUMMARY

The present disclosure encompasses methods of treating and/or preventing various diseases, disorders and conditions, and/or symptoms thereof, using IL-10, modified (e.g., pegylated) IL-10, and related agents described herein, and compositions thereof. More specifically, the present disclosure relates to optimized dosing parameters that achieve and maintain efficacy in the treatment and/or prevention of various diseases, disorders, and conditions in a subject, while minimizing adverse effects associated therewith. As shown in detail below, optimization of such dosing parameters includes, for example, assessment of pharmacokinetic and pharmacodynamic parameters associated with absorption, distribution, metabolism and excretion ("ADME") taking into account route of administration and other factors. It will be understood that terms related to ADME and other parameters are intended to have their generally accepted meanings in the relevant scientific field, unless otherwise indicated herein. For example, the term "serum half-life" orRefers to the elimination half-life (i.e., the time at which the serum concentration of the agent has reached half its initial or maximum value).

According to the methods described herein, the disease, disorder or condition and/or symptoms thereof may be a proliferative disorder, such as cancer or a cancer-related disorder, or a fibrotic disorder, such as cirrhosis of the liver, NASH and NAFLD. Although not limited to a particular cancer, the cancer may be a solid tumor, including tumors associated with colon cancer, melanoma, and squamous cell carcinoma, or it may be a hematological disorder.

In other embodiments, the disease, disorder, or condition is a viral disorder, including, but not limited to, human immunodeficiency virus, hepatitis b or c virus, or cytomegalovirus. In other embodiments, the disease, disorder, or condition is an immune disorder or an inflammatory disorder, which may be acute or chronic. Examples of immune and inflammatory disorders include inflammatory bowel disease, psoriasis, rheumatoid arthritis, multiple sclerosis, and Alzheimer's disease.

In particular embodiments, the disease, disorder, or condition is a cardiovascular disorder, including atherosclerosis. A subject with a cardiovascular disorder may have elevated cholesterol.

In other embodiments, the disease, disorder, or condition is thrombosis or a thrombotic condition.

As discussed further below, human IL-10 is a homodimer and each monomer comprises 178 amino acids, the first 18 of which comprise a signal peptide. Particular embodiments of the disclosure comprise a mature human IL-10 polypeptide lacking a signal peptide (see, e.g., U.S. patent No. 6,217,857), or a mature human PEG-IL-10. In other specific embodiments, the IL-10 agent is a variant of mature human IL-10. The variant may exhibit less than, comparable to, or greater than the activity of mature human IL-10; in certain embodiments, the activity is comparable to or greater than the activity of mature human IL-10.

Certain embodiments of the present disclosure encompass modification of IL-10 to enhance one or more properties (e.g., pharmacokinetic parameters, efficacy, etc.). In particular embodiments, IL-10 is modified by, for example, pegylation, glycosylation, albumin (e.g., Human Serum Albumin (HSA)) conjugation, and hydroxyethyl starch (hesylation). In further embodiments, modification of IL-10 does not result in therapeutically relevant deleterious effects on immunogenicity, and in still further embodiments, the modified IL-10 is less immunogenic than unmodified IL-10. The terms "IL-10", "IL-10 polypeptide", "agent", and the like are intended to be broadly construed and include, for example, human and non-human IL-10 related polypeptides, including homologs, variants (including muteins) and fragments thereof, as well as IL-10 polypeptides having, for example, a leader sequence (e.g., a signal peptide), as well as modified forms of the foregoing. In other particular embodiments, the terms "IL-10", "IL-10 polypeptide", "agent" are agonists. Particular embodiments relate to pegylated IL-10, which is also referred to herein as "PEG-IL-10". The present disclosure also encompasses nucleic acid molecules encoding the aforementioned polypeptides.

Particular embodiments of the present disclosure relate to methods of treating or preventing a disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an IL-10 agent, wherein the amount is sufficient to obtain a mean IL-10 serum trough concentration of at least 0.1 ng/mL. The method of treatment or prevention may be mediated by CD8+ T cells.

Other embodiments relate to methods of treating or preventing a disease, disorder, or condition in a subject (e.g., a human), comprising administering to the subject a therapeutically effective amount of an IL-10 agent, wherein the amount is sufficient to maintain a mean IL-10 serum trough concentration over a period of time, wherein the mean IL-10 serum trough concentration is at least 0.1ng/mL, and wherein the mean IL-10 serum trough concentration is maintained for at least 90% of the period of time. In particular embodiments of the present disclosure, the mean IL-10 serum trough concentration is at least 0.2ng/mL, at least 0.3ng/mL, and at least 0.4ng/mL, at least 0.5ng/mL, at least 0.6ng/mL, at least 0.7ng/mL, at least 0.8ng/mL, at least 0.9ng/mL, at least 1ng/mL, at least 1.2ng/mL, at least 1.25ng/mL, at least 1.3ng/mL, at least 1.4ng/mL, at least 1.5ng/mL, at least 1.6ng/mL, at least 1.7ng/mL, at least 1.8ng/mL, at least 1.85ng/mL, at least 1.9ng/mL, at least 1.95ng/mL, at least 1.97ng/mL, and at least 1.98ng/mL, at least 1.99ng/mL, at least 2.0ng/mL, or greater than 2 ng/mL.

In further embodiments, the period of time is at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, or greater than 3 months.

In particular embodiments of the present disclosure, the mean IL-10 serum trough concentration is maintained for the period of time or for at least 85% of the period of time, at least 90%, at least 95%, at least 98%, at least 99% or 100% of the period of time.

It is expected that a dosing regimen sufficient to maintain a desired steady-state serum trough concentration (e.g., 0.1ng/mL or 2ng/mL) will result in an initial serum trough concentration that is greater than the desired steady-state serum trough concentration. Due to the pharmacokinetic and pharmacodynamic characteristics of IL-10 in mammalian subjects, the initial trough concentration (e.g., obtained by administering one or more loading doses followed by a series of maintenance doses) gradually decreases over a period of time, even when the dosing parameters (amount and frequency) are kept constant. After this period, gradual, constant reduction ceased and steady-state serum trough concentrations were maintained.

For example, parenteral administration (e.g., SC and IV) of 0.1 mg/kg/day of an IL-10 agent (e.g., mIL-10) to mice (e.g., C57BL/6 mice) is required to maintain a steady state serum trough concentration of, for example, 2.0 ng/mL. However, this steady state serum trough concentration was not obtained until about 30 days after the initial administration at 0.1 mg/kg/day (and also after any initial dose). Conversely, after an initial serum trough concentration (e.g., 2.5ng/mL) has been reached, the concentration is gradually and continuously decreased over a period of time, e.g., about 30 days, after which the desired steady-state serum trough concentration (e.g., 2.0ng/mL) is maintained. One of ordinary skill in the art will be able to determine the dose required to maintain the desired steady state trough concentration using, for example, ADME and patient-specific parameters.

The present disclosure encompasses methods wherein an IL-10 agent may comprise at least one modification to form a modified IL-10 agent, wherein the modification does not alter the amino acid sequence of the IL-10 agent. In some embodiments, the modified IL-10 agent is a PEG-IL-10 agent. The PEG-IL-10 agent may comprise at least one PEG molecule covalently attached to at least one amino acid residue of at least one subunit of IL-10, or in other embodiments, a mixture of mono-PEGylated and di-PEGylated IL-10. The PEG component of the PEG-IL-10 agent may have a molecular mass greater than about 5kDa, greater than about 10kDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 50 kDa. In some embodiments, the molecular mass is about 5kDa to about 10kDa, about 5kDa to about 15kDa, about 5kDa to about 20kDa, about 10kDa to about 15kDa, about 10kDa to about 20kDa, about 10kDa to about 25kDa, or about 10kDa to about 30 kDa.

In some embodiments, the modified IL-10 agent comprises at least one Fc fusion molecule, at least one serum albumin (e.g., HSA or BSA), an HSA fusion molecule, or an albumin conjugate. In additional embodiments, the modified IL-10 agent is glycosylated, hydroxyethylated or comprises at least one albumin binding domain. Some modified IL-10 agents may comprise more than one type of modification. In particular embodiments, the modification is site-specific. Some embodiments comprise a linker. Modified IL-10 agents are discussed in detail below.

The present disclosure also encompasses the use of gene therapy in conjunction with the teachings herein. For use and methods of gene therapy, cells of a subject can be transformed in vivo with a nucleic acid encoding an IL-10 related polypeptide as set forth herein. Alternatively, the treatment can be effected by transforming cells in vitro with the transgene or polynucleotide, followed by transplantation of the cells into the tissue of the subject. In addition, primary cell isolates or established cell lines can be transformed with transgenes or polynucleotides encoding IL-10-related polypeptides, and then optionally transplanted into a tissue of a subject.

The present disclosure encompasses methods wherein an IL-10 agent is administered to a subject at least 2 times per day, at least 1 time per 48 hours, at least 1 time per 72 hours, at least 1 time per week, at least 1 time per 2 weeks, at least 1 time per month, at least 1 time per 2 months, or at least 1 time per 3 months. Some embodiments further comprise administering the IL-10 agent with at least one additional prophylactic or therapeutic agent, examples of which are set forth below.

The IL-10 agent may be administered by any effective route. In some embodiments, it is administered by parenteral injection (including subcutaneous injection).

Particular embodiments of the present disclosure relate to pharmaceutical compositions comprising an amount of an IL-10 agent (e.g., a therapeutically effective amount), including those agents described above, in combination with one or more pharmaceutically acceptable diluents, carriers, or excipients (e.g., isotonic injection fluids). The pharmaceutical composition is typically a pharmaceutical composition suitable for human administration. Furthermore, in some embodiments, the pharmaceutical composition comprises at least one additional prophylactic or therapeutic agent.

Certain embodiments of the present disclosure encompass sterile containers containing one of the above-described pharmaceutical compositions and optionally one or more additional components. For example, but not limiting of, the sterile container may be a syringe. In other embodiments, the sterile container is a component of a kit; the kit can also contain, for example, a second sterile container containing at least one prophylactic or therapeutic agent.

Brief Description of Drawings

FIG. 1 depicts the amino acid sequences of human and mouse IL-10.

FIG. 2A depicts the concentration of MCP-1 (pg/mL) in PBMCs at increasing concentrations of IL-10. IL-10 increased MCP-1 secretion at concentrations of 1ng/mL and above.

FIG. 2B depicts the concentration of MCP-1 (pg/mL) in PBMCs stimulated with LPS at increasing concentrations of IL-10. IL-10 is an inhibitor of LPS-mediated PBMC activation, and IL-10 was added at a concentration of 1ng/mL or more to significantly inhibit MCP-1 secretion.

Detailed description of the invention

Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments shown herein, and further that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the upper and lower limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," etc. in connection with the recitation of claim elements, or use of a "negative" limitation.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. In addition, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

SUMMARY

The present disclosure encompasses the use of the agents described herein and compositions thereof for the treatment and/or prevention of various diseases, disorders, and conditions, and/or symptoms thereof. In certain aspects of the disclosure, such treatment or prevention is achieved through the use of specific dosing parameters. In some embodiments, the agent is administered to achieve an optimized serum trough concentration for treating, for example, inflammatory and immune-related disorders, fibrotic disorders, cancer and cancer-related disorders or cardiovascular disorders (e.g., atherosclerosis).

In some embodiments of the disclosure, an IL-10 agent is administered to a subject having or at risk of developing a disease or disorder treatable with an IL-10 agent (e.g., an IL-10 polypeptide) in an amount sufficient to obtain a serum trough concentration of greater than about 0.1ng/mL, in certain embodiments a serum trough concentration of greater than about 1ng/mL, while in other embodiments a serum trough concentration of greater than about 2 ng/mL.

It should be noted that any reference to the polypeptides and nucleic acid molecules of the present disclosure used in conjunction with "human" is not intended to be limiting as to the manner or source in which the polypeptide or nucleic acid is obtained, and rather such reference order may correspond to the sequence of a naturally occurring human polypeptide or nucleic acid molecule. In addition to human polypeptides and nucleic acid molecules encoding them, the present disclosure also encompasses IL-10 related polypeptides and corresponding nucleic acid molecules from other species.

Definition of

Unless otherwise indicated, the following terms are intended to have the meanings indicated below. Other terms are defined elsewhere throughout the specification.

The terms "patient" or "subject" are used interchangeably to refer to a human or non-human animal (e.g., a mammal).

The terms "administration", "administering", and the like, when used, for example, in a subject, cell, tissue, organ, or biological fluid, refer to, for example, contacting IL-10 or PEG-IL-10, a nucleic acid (e.g., a nucleic acid encoding native human IL-10), a pharmaceutical composition comprising the foregoing, or a diagnostic agent, with the subject, cell, tissue, organ, or biological fluid. In the context of cells, administration includes contacting the agent with the cell (e.g., in vitro or ex vivo), as well as contacting the agent with a fluid, wherein the fluid is in contact with the cell.

The term "treating/managing" or the like refers to a process of action, such as administration of IL-10 or a pharmaceutical composition comprising IL-10, initiated after a disease, disorder or condition or symptoms thereof have been diagnosed, observed, or the like, to temporarily or permanently eliminate, reduce, inhibit, alleviate, or ameliorate at least one of the underlying causes of the disease, disorder or condition afflicting the subject, or at least one of the symptoms associated with the disease, disorder or condition afflicting the subject. Thus, treatment includes inhibiting (e.g., arresting the development or further development of the disease, disorder or condition or clinical symptoms associated therewith) active disease. The term may also be used in other contexts, such as where IL-10 or PEG-IL-10 is in contact with an IL-10 receptor, for example, in a mobile or colloidal phase.

As used herein, the term "in need of treatment" refers to a judgment by a physician or other caregiver that a subject is in need of treatment or will benefit from treatment. This determination is made based on a number of factors that are within the realm of the expertise of the physician or caregiver.

The terms "prevent," "preventing," "prevention," and the like refer to a process that initiates an action (such as administration of IL-10 or a pharmaceutical composition comprising IL-10) in a manner (e.g., prior to onset of a disease, disorder, condition, or symptom thereof) that temporarily or permanently arrests, suppresses, inhibits, or reduces the risk of developing a disease, disorder, condition, or the like, or delays onset thereof, in a background of a subject generally predisposed to the particular disease, disorder, or condition (as by, for example, absence of clinical symptoms). In certain instances, the term also refers to slowing the progression of a disease, disorder, or condition or inhibiting its progression to a deleterious or otherwise undesirable state.

As used herein, the term "in need of prevention" refers to a judgment made by a doctor or other caregiver that a subject needs or will benefit from preventive care. This determination is made based on a number of factors that exist within the expertise of the physician or caregiver.

The phrase "therapeutically effective amount" refers to an amount of an agent that, when administered to a subject, is capable of having any detectable positive effect on any symptom, aspect, or feature of a disease, disorder, or condition, either alone or as part of a pharmaceutical composition, as well as administered to the subject in a single dose or as part of a series of doses. A therapeutically effective amount may be determined by measuring the relevant physiological effects, and may be adjusted in conjunction with dosing regimens and diagnostic analysis of the subject's condition, among others. For example, measurement of the amount of inflammatory cytokine produced after administration can indicate whether a therapeutically effective amount has been used.

The phrase "in an amount sufficient to produce a change" means that there is a detectable difference between the level of the indicator measured before (e.g., the baseline level) and after administration of the particular therapy. Indicators include any objective parameter (e.g., serum concentration of IL-10) or subjective parameter (e.g., the subject's health well-being).

The term "small molecule" refers to a compound having a molecular weight of less than about 10kDa, less than about 2kDa, or less than about 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, and synthetic molecules. Therapeutically, small molecules are more permeable to cells, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

The term "ligand" refers to a peptide, polypeptide, membrane-associated or membrane-bound molecule or complex thereof, which may, for example, act as an agonist or antagonist of a receptor. "ligands" include natural and synthetic ligands such as cytokines, cytokine variants, analogs, muteins, and binding compositions derived from antibodies. "ligands" also include small molecules, such as peptidomimetics of cytokines and peptidomimetics of antibodies. The term also includes agents that are neither agonists nor antagonists but bind to the receptor without significantly affecting its biological properties such as signal transduction or adhesion. Furthermore, the term includes membrane-bound ligands that have been altered in soluble form, e.g., by chemical or recombinant means. The ligand or receptor may be entirely intracellular, i.e., it may be present in the cytosol, nucleus or some other intracellular compartment. The complex of a ligand and a receptor is referred to as a "ligand-receptor complex".

The terms "inhibitor" and "antagonist" or "activator" and "agonist" refer to, for example, inhibitory or activating molecules, respectively, for activation of a ligand, receptor, cofactor, gene, cell, tissue, or organ. An inhibitor is a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes, or down regulates, for example, a gene, protein, ligand, receptor, or cell. An activator is a molecule that increases, activates, facilitates, enhances activation, sensitizes, or up regulates, for example, a gene, protein, ligand, receptor, or cell. An inhibitor may also be defined as a molecule that reduces, blocks or inactivates constitutive activity. An "agonist" is a molecule that interacts with a target to cause or promote enhanced activation of the target. An "antagonist" is a molecule that resists the action of an agonist. Antagonists prevent, reduce, inhibit or neutralize the activity of an agonist, and antagonists may also prevent, inhibit or reduce the constitutive activity of a target, e.g., a target receptor, even when the identified agonist is not present.

The terms "modulate", "modulate" and the like refer to the ability of a molecule (e.g., activator or inhibitor) to directly or indirectly enhance or reduce the function or activity of an IL-10 agent (or nucleic acid molecule encoding same); or enhancing the ability of the molecule to produce an effect comparable to that of an IL-10 agent. The term "modulator" broadly means a molecule that can achieve the above-described activity. For example, a modulator of a gene, receptor, ligand or cell, for example, is a molecule that alters the activity of the gene, receptor, ligand or cell, where the activity can be activated, inhibited or altered in its regulatory properties. The modulator may act alone, or it may use a cofactor, such as a protein, metal ion, or small molecule. The term "modulator" includes agents that act by the same mechanism of action as IL-10 (i.e., agents that modulate the same signal transduction pathway as IL-10 in a manner similar thereto) and are capable of eliciting a biological response comparable to (or greater than) that elicited by IL-10.

Examples of modulators include small molecule compounds and other bio-organic molecules. Libraries (e.g., combinatorial libraries) of many small molecule compounds are commercially available and can be used as a starting point to identify modulators. One skilled in the art can develop one or more assays (e.g., biochemical or cell-based assays) in which libraries of such compounds can be screened to identify one or more compounds having a desired property; subsequently, the skilled pharmaceutical chemist can optimize such compound or compounds by, for example, synthesizing and evaluating analogs and derivatives thereof. Synthetic and/or molecular modeling studies can also be used for the identification of activators.

"activity" of a molecule may describe or refer to the binding of the molecule to a ligand or receptor; catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; (ii) antigenic activity; modulation of the activity of other molecules, and the like. The term may also refer to activity that modulates or maintains interactions (e.g., adhesion) between cells, or maintains structure (e.g., cell membrane) of cells. "activity" may also refer to specific activity, e.g., [ catalytic activity ]/[ mg protein ], or [ immunological activity ]/[ mg protein ], concentration in a biological compartment, and the like. The term "proliferative activity" includes activities which are necessary for or specifically associated with the performance of said biological process, for example, to promote normal cell division as well as cancer, tumors, dysplasia, cell transformation, metastasis and angiogenesis.

As used herein, "equivalent," "equivalent activity," "activity equivalent to … …," "equivalent effect," "effect equivalent to … …," and the like are relative terms that can be observed quantitatively and/or qualitatively. The meaning of the terms generally depends on the context in which they are used. For example, two agents that both activate a receptor may be considered to have comparable effects from a qualitative standpoint, but if one agent is only able to achieve 20% of the activity of the other agent (as measured in art-accepted assays (e.g., dose-response assays) or in art-accepted animal models), then two agents may be considered to lack comparable effects from a quantitative standpoint. "equivalent" when comparing one result to another (e.g., one result versus a reference standard) generally means that one result deviates from the reference standard by less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In particular embodiments, a result is comparable to a reference standard if it deviates from the reference standard by less than 15%, less than 10%, or less than 5%. For example, but not limited to, activity or effect may refer to efficacy, stability, solubility, or immunogenicity.

The term "response" of, for example, a cell, tissue, organ or organism, includes biochemical or physiological behavior, e.g., changes in concentration, density, adhesion or migration within a biological compartment, gene expression rate or differentiation state, wherein the changes are associated with activation, stimulation or treatment or with intrinsic mechanisms such as genetic programming. In certain contexts, the terms "activation," "stimulation," and the like refer to activation of a cell, e.g., by internal mechanisms, as well as by external or environmental factors; however, the terms "inhibit", "down-regulate" and the like refer to the opposite effect.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins having heterologous amino acid sequences; a fusion protein having heterologous and homologous leader sequences; a fusion protein with or without an N-terminal methionine residue; fusion proteins with immunologically labeled proteins, and the like.

It is understood that throughout this disclosure reference is made to amino acids according to the single or three letter code. For the convenience of the reader, the single and three letter amino acid codes are provided below:

as used herein, the term "variant" includes naturally occurring variants and non-naturally occurring variants. Naturally occurring variants include homologs (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, between species) and allelic variants (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, between individuals within a species). Non-naturally occurring variants include polypeptides and nucleic acids comprising changes in the amino acid or nucleotide sequence, respectively, where changes in the sequence are artificially introduced (e.g., muteins); for example, changes are made in the laboratory by human intervention ("human manual"). Thus, in this context, "mutant protein" broadly refers to a recombinant protein that typically has single or multiple amino acid substitutions and is typically derived from a cloned gene that has undergone site-directed or random mutagenesis, or a mutation from a completely synthetic gene.

The terms "DNA," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length (deoxyribonucleotides or ribonucleotides or analogs thereof). Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger rna (mrna), complementary dna (cdna), recombinant polynucleotides, vectors, probes, primers, and the like.

As used herein, in the context of the structure of a polypeptide, "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxy terminus") refer to the last amino and carboxy termini, respectively, of the polypeptide, while the terms "N-terminus" and "C-terminus" refer to the relative positions in the amino acid sequence of the polypeptide toward the N-terminus and C-terminus, respectively, and may include residues on the N-terminus and C-terminus, respectively. "N-terminal" or "C-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue, wherein the first and second amino acid residues are covalently joined to provide a contiguous amino acid sequence.

"derived from" in the context of an amino acid sequence or polynucleotide sequence (e.g., "derived from" the amino acid sequence of an IL-10 polypeptide) means that the polypeptide or nucleic acid has a sequence based on the sequence of a reference polypeptide or nucleic acid (e.g., a naturally occurring IL-10 polypeptide or a nucleic acid encoding IL-10), and is not meant to be limiting with respect to the source or method in which the protein or nucleic acid is produced. For example, the term "derived from" includes homologues or variants of the reference amino acid or DNA sequence.

In the context of polypeptides, the term "isolated" refers to a polypeptide of interest that, if found in nature, is present in an environment other than the environment in which it may naturally occur. "isolated" is intended to include polypeptides within a sample that are substantially enriched for a polypeptide of interest and/or wherein the polypeptide of interest is partially or substantially purified. When a polypeptide does not naturally occur, "isolated" means that the polypeptide has been separated from the environment in which it was produced by synthetic or recombinant means.

By "enriched" is meant that the sample is non-naturally manipulated (e.g., by a scientist) such that the polypeptide of interest is a) present at a higher concentration (e.g., at least 3-fold more, at least 4-fold more, at least 8-fold more, at least 64-fold more, or more) than the concentration of the polypeptide in the starting sample, such as a biological sample (e.g., a sample in which the polypeptide is naturally occurring or in which it is present after administration), or b) is present at a higher concentration than the environment in which the polypeptide is produced (e.g., as in a bacterial cell).

By "substantially pure" is meant that the component (e.g., polypeptide) constitutes greater than about 50% of the total composition content, typically greater than about 60% of the total polypeptide content. More generally, "substantially pure" refers to a composition in which at least 75%, at least 85%, at least 90%, or more of the total composition is the target component. In some cases, the polypeptide will constitute greater than about 90%, or greater than about 95%, of the total composition content.

The term "specific binding" or "selective binding," when referring to a ligand/receptor, antibody/antigen or other binding pair, refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics. Thus, under specified conditions, a specified ligand binds to a particular receptor and does not bind in significant amounts to other proteins present in the sample. The antibody or binding component derived from the antigen binding site of the antibody of contemplated methods binds its antigen or a variant or a mutation thereof with an affinity of at least 2-fold more, at least 10-fold more, at least 20-fold more or at least 100-fold more for any other antibody or binding component derived therefromAnd (3) changing protein. In particular embodiments, the antibody will have a size greater than about 10⁹The affinity in liters/mol, as determined by, for example, Scatchard analysis (Munsen, et al 1980Analyt. biochem.107: 220-.

IL-10 and PEG-IL-10

The anti-inflammatory cytokine IL-10, also known as human Cytokine Synthesis Inhibitory Factor (CSIF), is classified as a type 2 cytokine, a group of cytokines including IL-19, IL-20, IL-22, IL-24(Mda-7) and IL-26, interferons (IFN-. alpha., -gamma., -kappa., -omega. and-tau), and interferon-like molecules (limitin, IL-28A, IL-28B and IL-29).

IL-10 is a cytokine with pleiotropic effects in immune regulation and inflammation. It is produced by mast cells, counteracting the inflammatory effects of these cells at the site of the allergic reaction. Although it is capable of inhibiting the synthesis of proinflammatory cytokines such as IFN- γ, IL-2, IL-3, TNF α, and GM-CSF, IL-10 is also stimulatory for certain T cells and mast cells and stimulates B cell maturation, proliferation, and antibody production. IL-10 blocks NF-. kappa.B activity and is involved in the regulation of the JAK-STAT signal transduction pathway. It also induces the cytotoxic activity of CD8+ T cells and antibody production by B cells, and it inhibits macrophage activity and pro-tumor inflammation. Modulation of CD8+ T cells is dose-dependent, with higher doses inducing stronger cytotoxic responses.

Human IL-10 is a homodimer with a molecular mass of 37kDa, where each 18.5kDa monomer contains 178 amino acids (the first 18 amino acids of which comprise the signal peptide) and two pairs of cysteine residues that form intramolecular disulfide bonds. IL-10 dimer becomes biologically inactive after the noncovalent interaction between two monomer subunits is disrupted.

The present disclosure encompasses human IL-10 and murine IL-10(IL-10 exhibits 80% homology) and uses thereof. In addition, the scope of the present disclosure includes IL-10 orthologs and modified forms thereof from other mammalian species, including rat (accession NP-036986.2; GI 148747382); cattle (accession NP-776513.1; GI 41386772); sheep (accession NP-001009327.1; GI 57164347); dog (accession ABY 86619.1; GI 166244598); and rabbits (accession AAC 23839.1; GI 3242896).

As alluded to above, the terms "IL-10", "IL-10 polypeptide", "IL-10 agent", and the like, are intended to be broadly construed and include, for example, human and non-human IL-10 related polypeptides, including homologs, variants (including muteins) and fragments thereof, as well as IL-10 polypeptides having, for example, a leader sequence (e.g., a signal peptide) and modified forms of the foregoing. In other particular embodiments, the IL-10, IL-10 polypeptide, and IL-10 agent are agonists.

The IL-10 receptor, the type II cytokine receptor, is composed of alpha and beta subunits, also known as R1 and R2, respectively. Receptor activation requires binding to both alpha and beta. One homodimer of an IL-10 polypeptide binds to alpha and another homodimer of the same IL-10 polypeptide binds to beta.

The utility of recombinant human IL-10 is usually limited by its relatively short serum half-life, which can be attributed to such as renal clearance, proteolytic degradation and blood stream in monomer. Thus, various approaches have been developed to improve the pharmacokinetic profile of IL-10 without disrupting its dimeric structure (thereby adversely affecting its activity). PEGylation of IL-10 results in improvement of certain pharmacokinetic parameters (e.g., serum half-life) and/or enhancement of activity. For example, particular embodiments of the present disclosure include methods of optimizing treatment of proliferative disorders (e.g., cancer) using PEG-IL-10.

As previously noted, the present disclosure also encompasses the use of gene therapy in conjunction with the teachings herein. Gene therapy is achieved by delivering genetic material (typically packaged in a vector) to endogenous cells within a subject to introduce novel genes, to introduce additional copies of preexisting genes, to impair the function of existing genes, or to repair existing but non-functioning genes. Once inside the cell, the nucleic acid is expressed by the cellular machinery, resulting in the production of the protein of interest. In the context of the present disclosure, gene therapy is used as a therapeutic agent to deliver a nucleic acid encoding an IL-10 agent for the treatment or prevention of the diseases, disorders, or conditions described herein.

As alluded to above, for the use and methods of gene therapy, cells of a subject may be transformed in vivo with a nucleic acid encoding an IL-10-related polypeptide as set forth herein. In addition, cells are transformed in vitro with the transgene or polynucleotide and subsequently transplanted into a tissue of a subject to effect treatment. In addition, primary cell isolates or established cell lines can be transformed with transgenes or polynucleotides encoding IL-10-related polypeptides, and then optionally transplanted into a tissue of a subject.

As used herein, the terms "PEGylated IL-10" and "PEG-IL-10" refer to IL-10 molecules having one or more polyethylene glycol molecules covalently attached (typically by a linker, such that the attachment is stable) to at least one amino acid residue of the IL-10 protein. The terms "mono-pegylated IL-10" and "mono-PEG-IL-10" refer to a single amino acid residue of a polyethylene glycol molecule covalently attached (typically through a linker) to one subunit of the IL-10 dimer. In certain embodiments, the PEG-IL-10 used in the present disclosure is a mono PEG-IL-10 in which 1 to 9 PEG molecules are covalently attached by a linker to the alpha amino group of the amino acid residue at the N-terminus of one subunit of the IL-10 dimer. Mono-PEGylation on one IL-10 subunit typically results in a heterogeneous mixture of non-PEGylated, mono-PEGylated, and di-PEGylated IL-10 due to subunit shuffling. Furthermore, allowing the pegylation reaction to proceed to completion will typically result in non-specific and multi-pegylated IL-10, thereby reducing its biological activity. Thus, particular embodiments of the present disclosure include administering a mixture of mono-and di-pegylated IL-10 produced by the methods described herein (e.g., experimental section).

In particular embodiments, the average molecular weight of the PEG moiety is from about 5kDa to about 50 kDa. While the method or site of attachment of PEG to IL-10 is not critical, in certain embodiments pegylation does not alter, or only minimally alters, the activity of the IL-10 agent. In certain embodiments, the increase in half-life is greater than any decrease in biological activity. The biological activity of PEG-IL-10 is typically measured by assessing the levels of inflammatory cytokines (e.g., TNF-a or IFN- γ) in the serum of subjects challenged with bacterial antigens (lipopolysaccharide (LPS)) and treated with PEG-IL-10, as described in U.S. patent No. 7,052,686.

IL-10 variants can be prepared for a variety of purposes under consideration, including increasing serum half-life, reducing immune responses to IL-10, facilitating purification or preparation, reducing the conversion of IL-10 to its monomeric subunit, improving efficacy and reducing the severity or occurrence of side effects during therapeutic use. Amino acid sequence variants are typically predetermined variants not found in nature, although some variants may be post-translational variants, such as glycosylation variants. Any variant of IL-10 may be used, so long as it retains the appropriate level of IL-10 activity. In a tumor context, suitable IL-10 activity includes, for example, infiltration of CD8+ T cells into the tumor site, expression of inflammatory cytokines such as IFN- γ, IL-4, IL-6, IL-10, and RANK-L from these infiltrating cells, and elevated levels of IFN- γ in a biological sample.

The phrase "conservative amino acid substitution" refers to a substitution that preserves the activity of a protein by replacing an amino acid in the protein with an amino acid having a side chain of similar acidity, basicity, charge, polarity, or side chain size. Conservative amino acid substitutions typically require the substitution of amino acid residues within the following groups: 1) l, I, M, V, F, respectively; 2) r, K, respectively; 3) f, Y, H, W, R, respectively; 4) g, A, T, S, respectively; 5) q, N, respectively; and 6) D, E. Guidance for substitutions, insertions or deletions may be based on alignment of the amino acid sequences of different variant proteins or proteins from different species. Thus, in addition to any naturally occurring IL-10 polypeptide, the present disclosure encompasses polypeptides having 1,2,3,4, 5,6, 7,8, 9, or 10, typically no more than 20, 10, or 5 amino acid substitutions, wherein the substitutions are typically conservative amino acid substitutions.

The present disclosure also encompasses active fragments (e.g., subsequences) of mature IL-10 that contain contiguous amino acid residues derived from mature IL-10. The length of contiguous amino acid residues of a peptide or polypeptide subsequence varies depending on the particular naturally occurring amino acid sequence from which the subsequence is derived. Generally, peptides and polypeptides may be from about 20 amino acids to about 40 amino acids, from about 40 amino acids to about 60 amino acids, from about 60 amino acids to about 80 amino acids, from about 80 amino acids to about 100 amino acids, from about 100 amino acids to about 120 amino acids, from about 120 amino acids to about 140 amino acids, from about 140 amino acids to about 150 amino acids, from about 150 amino acids to about 155 amino acids, from about 155 amino acids up to full length peptides or polypeptides.

In addition, an IL-10 polypeptide can have a defined sequence identity over a defined length of contiguous amino acids (e.g., a "comparison window") as compared to a reference sequence. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, adv.appl.Math.2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol.biol.48:443(1970), by the similarity search method of Pearson & Lipman, Proc.Nat' l.Acad.Sci.USA 85:2444(1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al, eds., 1995 suppl.).

As an example, a suitable IL-10 polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to about 20 amino acids to about 40 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 120 amino acids, about 120 amino acids to about 140 amino acids, about 140 amino acids to about 150 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids up to a contiguous segment of a full-length peptide or polypeptide.

As discussed further below, IL-10 polypeptides may be isolated from a natural source (e.g., an environment other than that in which they naturally occur) and may also be recombinantly produced (e.g., in a genetically modified host cell, such as a bacterium, yeast, pichia, insect cell, etc.), wherein the genetically modified host cell is modified with a nucleic acid comprising a nucleotide sequence encoding the polypeptide. IL-10 polypeptides can also be produced synthetically (e.g., by cell-free chemical synthesis).

The present disclosure encompasses nucleic acid molecules encoding IL-10 agents, including naturally occurring and non-naturally occurring isoforms, allelic and splice variants thereof. The disclosure also includes nucleic acid sequences that differ from naturally occurring DNA sequences in one or more bases but which, due to the degeneracy of the genetic code, are still translated into an amino acid sequence corresponding to an IL-10 polypeptide.

IL-10 serum concentration

Plasma levels of IL-10 in the methods described herein can be characterized in several ways, including: (1) a mean IL-10 serum trough concentration above a certain specified level or within a certain range of levels; (2) a mean IL-10 serum trough concentration above a specified level for an amount of time; (3) steady state IL-10 serum concentration levels above or below a specified level or within a range of levels; or (4) C of a concentration profile above or below a specified level or within a range of levels_max. As shown herein, mean serum trough IL-10 concentrations have been found to be particularly important for efficacy in certain indications.

In some embodiments of the present disclosure, plasma level concentration profiles that can be produced include greater than about 0.1ng/mL, greater than about 0.15ng/mL, greater than about 0.2ng/mL, greater than about 0.25ng/mL, greater than about 0.3ng/mL, greater than about 0.35ng/mL, greater than about 0.4ng/mL, greater than about 0.45ng/mL, greater than about 0.5ng/mL, greater than about 0.55ng/mL, greater than about 0.6ng/mL, greater than about 0.65ng/mL, greater than about 0.7ng/mL, greater than about 0.75ng/mL, greater than about 0.8ng/mL, greater than about 0.85ng/mL, greater than about 0.9ng/mL, greater than about 0.95ng/mL, greater than about 1.0ng/mL, greater than about 1.1ng/mL, greater than about 1.2ng/mL, greater than about 1.3ng/mL, greater than about 1.4ng/mL, An average IL-10 serum trough concentration of greater than about 1.5ng/mL, greater than about 1.6ng/mL, greater than about 1.7ng/mL, greater than about 1.8ng/mL, greater than about 1.9ng/mL, greater than about 2.0ng/mL, greater than about 2.1ng/mL, greater than about 2.2ng/mL, greater than about 2.3ng/mL, greater than about 2.4ng/mL, greater than about 2.5ng/mL, greater than about 2.75ng/mL, or greater than about 3.0 ng/mL.

In particular embodiments directed to treating or preventing a cancer-associated disease, disorder or condition is treated by achieving a dose of greater than about 0.5ng/mL, greater than about 0.55ng/mL, greater than about 0.6ng/mL, greater than about 0.65ng/mL, greater than about 0.7ng/mL, greater than about 0.75ng/mL, greater than about 0.8ng/mL, greater than about 0.85ng/mL, greater than about 0.9ng/mL, greater than about 0.95ng/mL, greater than about 1.0ng/mL, greater than about 1.1ng/mL, greater than about 1.2ng/mL, greater than about 1.3ng/mL, greater than about 1.4ng/mL, greater than about 1.5ng/mL, greater than about 1.6ng/mL, greater than about 1.7ng/mL, greater than about 1.8ng/mL, greater than about 1.9ng/mL, greater than about 2.0ng/mL, greater than about 2.1ng/mL, A mean IL-10 serum trough concentration of greater than about 2.3ng/mL, greater than about 2.4ng/mL, greater than about 2.5ng/mL, greater than about 2.75ng/mL, or greater than about 3.0ng/mL to optimize therapy.

Particular embodiments of the present disclosure comprise mean IL-10 serum trough concentrations within the following ranges: about 0.1ng/mL to about 1.0ng/mL, about 0.1ng/mL to about 0.9ng/mL, about 0.1ng/mL to about 0.8ng/mL, about 0.1ng/mL to about 0.7ng/mL, about 0.1ng/mL to about 0.6ng/mL, about of 0.1ng/mL to about 0.5ng/mL, about 0.2ng/mL to about 1.0ng/mL, about 0.2ng/mL to about 0.9ng/mL, about 0.2ng/mL to about 0.8ng/mL, about 0.2ng/mL to about 0.7ng/mL, about 0.2ng/mL to about 0.6ng/mL, about 0.2ng/mL to about 0.5ng/mL, about 0.3ng/mL to about 1.0ng/mL, about 0.3ng/mL to about 0.9ng/mL, about 0.3ng/mL to about 0ng/mL, about 0.7ng/mL, about 0.3ng/mL to about 0.7ng/mL, about 0ng/mL to about 0.7ng/mL, about 0ng/mL, about, About 0.3ng/mL to about 0.6ng/mL, about 0.3ng/mL to about 0.5ng/mL, about 0.3ng/mL to about 0.4ng/mL, about 0.4ng/mL to about 1.0ng/mL, about 0.4ng/mL to about 0.9ng/mL, about 0.4ng/mL to about 0.8ng/mL, about 0.4ng/mL to about 0.7ng/mL, about 0.4ng/mL to about 0.6ng/mL, about 0.4ng/mL to about 0.5ng/mL, about 0.5ng/mL to about 1.0ng/mL, about 0.5ng/mL to about 0.9ng/mL, about 0.5ng/mL to about 0.8ng/mL, about 0.5ng/mL to about 0.7ng/mL, about 0.5ng/mL to about 0.6ng/mL, about 0.5ng/mL to about 0.8ng/mL, about 0.5ng/mL to about 2ng/mL, about 2ng/mL to about 0.7ng/mL, about 0.5ng/mL, about 0.6ng/mL, about 2ng/mL, about 0.9ng/mL, About 1.0ng/mL to about 2.1ng/mL, about 1.0ng/mL to about 2.0ng/mL, about 1.0ng/mL to about 1.9ng/mL, about 1.0ng/mL to about 1.8ng/mL, about 1.0ng/mL to about 1.7ng/mL, about 1.0ng/mL to about 1.6ng/mL, about 1.0ng/mL to about 1.5ng/mL, about 1.9ng/mL to greater than about 2.5ng/mL, about 1.9ng/mL to about 2.4ng/mL, about 1.9ng/mL to about 2.3ng/mL, about 1.9ng/mL to about 2.2ng/mL, or about 1.9ng/mL to about 2.1 ng/mL.

In particular embodiments directed to the treatment or prevention of an anti-inflammatory disease, disorder or condition, by obtaining 0.1ng/mL to 1.0ng/mL, 0.1ng/mL to 0.9ng/mL, 0.1ng/mL to 0.8ng/mL, 0.1ng/mL to 0.7ng/mL, 0.1ng/mL to 0.6ng/mL, 0.1ng/mL to 0.5ng/mL, 0.2ng/mL to 1.0ng/mL, 0.2ng/mL to 0.9ng/mL, 0.2ng/mL to 0.8ng/mL, 0.2ng/mL to 0.7ng/mL, 0.2ng/mL to 0.6ng/mL, 0.2ng/mL to 0.5ng/mL, 0.3ng/mL to 1.0ng/mL, 0.3ng/mL to 0.9ng/mL, 0.8ng/mL to 0.6ng/mL, 0.2ng/mL to 0.5ng/mL, 0.3ng/mL, 0ng/mL to 0.6ng/mL, 0.7ng/mL, 0.6ng/mL, or a, An average IL-10 trough concentration of 0.3ng/mL to 0.5ng/mL, 0.3ng/mL to 0.4ng/mL, 0.4ng/mL to 1.0ng/mL, 0.4ng/mL to 0.9ng/mL, 0.4ng/mL to 0.8ng/mL, 0.4ng/mL to 0.7ng/mL, 0.4ng/mL to 0.6ng/mL, 0.4ng/mL to 0.5ng/mL, 0.5ng/mL to 1.0ng/mL, 0.5ng/mL to 0.9ng/mL, 0.5ng/mL to 0.8ng/mL, 0.5ng/mL to 0.7ng/mL, or 0.5ng/mL to 0.6ng/mL to optimize therapy.

The experimental section describes the evaluation of the therapeutic efficacy of mIL-10 and PEG-mIL-10 in PDV6 squamous cell carcinoma and CT-26 colon carcinoma, where the mIL-10 and mPEG-IL-10 dosing parameters (amount and frequency of administration) are sufficient to achieve an IL-10 serum trough concentration of 1-2ng/mL on average. As described in the experimental section, PEG-IL-10 treatment resulted in complete response, whereas IL-10 treatment showed anti-tumor function but not complete response.

The efficacy of IL-10 treatment of hepatitis C can also be assessed. A mouse model with a functional immune system susceptible to hepatitis C virus (see Dorner, M. (09.6.2011) Nature 474: 208-. Using the teachings presented herein and the basis of knowledge of one of ordinary skill in the art, the effects of mIL-10 and PEG-mIL-10 administered to achieve mean IL-10 serum trough concentrations of about 0.1ng/mL, about 0.5ng/mL, about 1.0ng/mL, about 1.5ng/mL, and about 2ng/mL can be evaluated.

Although not prevalent at therapeutic doses in most patient populations, administration of higher doses of IL-10 has caused adverse effects (e.g., headache, anemia, and effects on the liver) in a limited number of subjects. Fortunately, such adverse effects are not prevalent when mean IL-10 serum concentrations of 0.1-2ng/mL are maintained over the duration of treatment. Yet another embodiment of the present disclosure provides a method for monitoring a subject receiving IL-10 therapy to predict an adverse effect, thereby potentially avoiding the adverse effect, the method comprising: (1) measuring the peak concentration of IL-10 in the subject; (2) measuring a trough concentration of IL-10 in the subject; (3) calculating peak-to-valley fluctuations; and (4) using the calculated peak-to-valley fluctuations to predict potential adverse effects in the subject. Smaller peak-to-valley fluctuations indicate a lower likelihood that the subject will experience IL-10 related adverse effects. In certain embodiments, specific peak-to-trough fluctuations of treatment of a particular disease, disorder, and condition using a particular dosing parameter are determined, and those fluctuations are used as a reference standard.

In addition to the IL-10 dosing-related parameters described above, volume of distribution considerations are also relevant. For most drugs, plasma drug concentrations decline in a multi-exponential manner. Immediately after intravenous administration, the drug rapidly distributes throughout the initial space (minimally defined as the plasma volume) followed by a slower equilibrium distribution to the extravascular space (e.g., certain tissues). Intravenous IL-10 administration was combined with such a two-compartment kinetic model (see Rachmawai, H. et al (2004) pharm. Res.21(11): 2072-78). The pharmacokinetics of subcutaneous recombinant hIL-10 have also been studied (Radwanski, E. et al (1998) pharm. Res.15(12): 1895-1901). In addition, IL-10 modifications have been introduced in an attempt to target cytokines to specific cell types (see rachmawaii, H. (5.2007) Drug met.dist.35(5): 814-21).

As described further below, the anti-tumor efficacy of IL-10 and PEG-IL-10 observed in mice results from the induction of cytotoxic enzymes in CD8+ T cells, leading to the killing of tumor cells. Many anti-cancer compounds (including, but not limited to, apoptosis-inducing agents) are administered in cycles. Commonly, a single dose or series of doses near the Maximum Tolerated Dose (MTD) is administered, including a single application or series of high doses near the Maximum Tolerated Dose (MTD), followed by discontinuation of the dosing ("drug holiday") to allow normal physiological recovery of the patient. For example, the dosing strategy is used for cytotoxic chemotherapeutic antibody therapies, such as anti-vegf (avastin), and for short-acting biological agents, such as PROLEUKIN (IL-2).

Murine studies were performed to generate data that help understand the pharmacokinetic parameters of IL-10 therapy and help optimize human tumor treatment regimens. As described in the experimental section, although mice receiving the same amount of drug administered in a single dose or multiple doses over the course of one week did have similar total exposure, mice receiving daily doses showed the greatest reduction in tumor size (table 15). In addition, treatment regimens that result in maintenance of serum trough concentrations greater than about 1ng/mL (e.g., 1.1-2.1ng/mL) exhibited the greatest reduction in tumor size and weight (table 16).

The present disclosure encompasses administration of any dose that results in maintenance of a serum trough concentration of greater than about 0.1ng/mL (e.g., 0.1-2ng/mL, 0.1-1ng/mL, 0.5-1.5ng/mL, or 1.1-2.1 ng/mL). For example, when the subject is a human, the non-PEGylated hIL-10 can be administered at a dose of greater than 15 μ g/kg/day, greater than 18 μ g/kg/day, greater than 20 μ g/kg/day, greater than 21 μ g/kg/day, greater than 22 μ g/kg/day, greater than 23 μ g/kg/day, greater than 24 μ g/kg/day, or greater than 25 μ g/kg/day. When the subject is a human, a PEG-hIL-10 comprising a relatively small PEG (e.g., 5kDa mono-di-PEG-hIL 10) can be administered at a dose of greater than 2.0. mu.g/kg/day, greater than 2.3. mu.g/kg/day, greater than 2.5. mu.g/kg/day, greater than 2.6. mu.g/kg/day, greater than 2.7. mu.g/kg/day, greater than 2.8. mu.g/kg/day, greater than 2.9. mu.g/kg/day, greater than 3.0. mu.g/kg/day, greater than 3.1. mu.g/kg/day, greater than 3.2. mu.g/kg/day, greater than 3.3. mu.g/kg/day, greater than 3.4. mu.g/kg/day, or greater than 3.5. mu.g/kg/day.

Role of CD8+ T cells in IL-10 function

CD8 (differentiation group 8) is a transmembrane glycoprotein that acts as a co-receptor for the T Cell Receptor (TCR). The CD8 co-receptor is expressed primarily on the surface of Cytotoxic T Lymphocytes (CTLs), but it is also found on other cell types, including natural killer cells (NK). Like the TCR, CD8 binds to Major Histocompatibility Complex (MHC) molecules, but is specific for MHC class I proteins.

CD8 function requires the formation of a dimer comprising a pair of CD8 chains. The two isoforms α and β of CD8 exist, and the most common form of CD8 comprises the CD8- α and CD8- β chains, two members of the immunoglobulin superfamily. CD 8-a interacts with MHC class I molecules and this interaction holds the T cell receptor of cytotoxic T cells in close association with the target cell during antigen-specific activation. Cytotoxic T cells with CD8 surface protein are referred to as "CD 8+ T cells". CD8+ T cells (CTLs and NK cells) recognize antigens of specific infected target cells (cell surface peptides or proteins typically resulting from infection by intracellular pathogens), and if those antigens differ from the normal antigen profile of the subject ("autoimmunity"), CD8+ T cells are activated and induce apoptosis of the target cells.

There are several situations in which the antigen profile is different. For example, when a pathogen (e.g., a virus) invades a cell, the cell produces "non-self" cell surface antigens, and CD8+ T cells initiate an immune response in an attempt to eradicate the infected cell. Another scenario occurs, where some cellular proteins are modified due to mutations at the nucleic acid and/or amino acid level. Cancer cells typically have many mutations and are recognized as 'distinct' by CD8+ T cells. The presence of CD8+ T cells in human cancers is associated with longer survival.

In both of the above scenarios, activated CD8+ T cells produce IFN γ, perforin and granzyme B. IFN gamma is important for further up-regulation of antigen "presentation" on target cells, the antigen being present on MHC class I proteins. Perforin and granzyme B mediate killing of target cells (e.g., viruses and cancer).

Perforin, a cytolysin found in the granules of CTLs and NK, inserts itself into the plasma membrane of the target cell after degranulation. Perforin has structural and functional similarities to complement component 9(C9), and like C9, perforin produces a transmembrane tubule and is capable of non-specifically lysing a variety of target cells. Perforin is a crucial effector molecule for T-cell and NK-cell mediated cell lysis.

As suggested above, granzyme B is a serine protease expressed by Cytotoxic T Lymphocytes (CTL) and Natural Killer (NK) cells. CTL and NK cells recognize a specific infected target cell population and induce apoptosis of cells that have 'non-self' antigens (peptides or proteins usually produced by intracellular pathogen infection) on their surface. Granzyme B is crucial for the rapid induction of apoptosis of target cells produced by CTLs in a cell-mediated immune response.

IL-10 plays multiple roles in the activation of CD8+ T cells. For example, IL-10 induces effector molecules (IFN γ, perforin and granzyme B) in memory CD8+ T cells (which have been produced during previous infection or vaccination). Such memory CD8+ T cells are the cells responsible for providing long-term antiviral protection to a subject. Although memory CD8+ T cell production and expansion can occur when IL-10 is absent (Vicari, A. and Trinchieri, G. (2004) immunity. Rev.202: 223-236), the fact that IL-10 directly activates such cells provides a unique and alternative therapeutic approach. Although chronic viral infection has been associated with CD8+ T cells (Virgin, h. et al (2009) Cell 138, page 30), treatment of subjects (e.g., mice) with non-pegylated IL-10 or pegylated IL-10 has not been described.

In view of the above, embodiments of the present disclosure are based on the association between CD8+ T cells and cancer and viral infection. Thus, certain methods of treating and/or preventing cancer-related diseases, disorders and conditions, such as maintaining a mean IL-10 serum concentration of, for example ≧ 0.5ng/mL, ≧ 1ng/mL, or ≧ 2ng/mL, should also be applicable for the treatment of virus-related diseases, disorders and conditions.

In contrast to other cytokines, IL-10 can be considered to be a highly potent immunomodulatory and immunosuppressive factor. The role of CD8+ T cells in chronic inflammation has not yet been fully elucidated. However, because the involvement of IFN γ in cancer and virus-related disorders is mediated at least in part by CD8+ T cells, and because the IL-10-T cell pathway involved in the control of inflammation-related disorders (through the down-regulation of inflammatory cytokines) is also involved in IFN γ, CD8+ T cells also play a critical role in inflammation. Thus, IL-10 may prove to be an important therapeutic agent in the current stable anti-inflammatory agents.

Method for producing IL-10

The polypeptides of the disclosure can be produced by any suitable method, including non-recombinant (e.g., chemical synthesis) and recombinant methods.

A.Chemical synthesis

When the polypeptide is chemically synthesized, the synthesis may be carried out by a liquid phase or a solid phase. Solid Phase Peptide Synthesis (SPPS) allows for the incorporation of unnatural amino acids and/or peptide/protein backbone modifications. Various forms of SPPS, such as 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc), can be used to synthesize the disclosed polypeptides. Details of chemical synthesis are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. chem.6: 3-10; and Camarero J.A. et al, (2005) ProteinPept Lett.12: 723-8).

Solid phase peptide synthesis can be performed as described below. The alpha functional group (na) and any reactive side chains are protected with acid-or base-labile groups. The protecting group is stable under the conditions used to attach the amide bond but can be easily cleaved without damaging the peptide chain already formed. Suitable protecting groups for the alpha-amino functional group include, but are not limited to, the following: boc, benzyloxycarbonyl (Z), O-chlorobenzyloxycarbonyl, di-phenylisopropyloxycarbonyl, tert-pentyloxycarbonyl (Amoc), α -dimethyl-3, 5-dimethoxy-benzyloxycarbonyl, O-nitrothio, 2-cyano-tert-butyloxycarbonyl, Fmoc, 1- (4, 4-dimethyl-2, 6-dioxocyclohexyl-1-ylidene) ethyl (Dde, etc.

Suitable side chain protecting groups include, but are not limited to: acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl), benzyloxycarbonyl (Z), tert-butoxycarbonyl (Boc), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, tert-butyl (tBu), tert-butyldimethylsilyl, 2-chlorobenzyl, 2-chlorobenzyloxycarbonyl, 2, 6-dichlorobenzyl, cyclohexyl, cyclopentyl, 1- (4, 4-dimethyl-2, 6-dioxocyclohexyl-1-ylidene) ethyl (Dde), isopropyl, 4-methoxy-2, 3-6-trimethylbenzylsulfonyl (Mtr), 2,3,5,7, 8-pentamethylchroman-6-sulfonyl (Pmc), pivaloyl, tetrahydropyran-2-yl, tosyl (Tos), 2,4, 6-trimethoxybenzyl, trimethylsilyl and trityl (Trt).

In solid phase synthesis, the C-terminal amino acid is coupled to a suitable support material. Suitable support materials are those which are inert to the reagents and reaction conditions used for the step-wise polycondensation and cleavage reaction of the synthesis and which are insoluble in the reaction medium to be used. Examples of commercially available support materials include styrene/divinylbenzene copolymers which have been modified with reactive groups and/or polyethylene glycol; chloromethylated styrene/divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers and the like. When it is desired to prepare the peptide acids, polystyrene (1%) -divinylbenzene or derivatized with 4-benzyloxybenzyl-alcohol (Wang-Anchor) or 2-chlorotrityl chloride may be usedIn the case of peptide amides, polystyrene (1%) divinylbenzene or derived from 5- (4' -aminomethyl) -3',5' -dimethoxyphenoxy) pentanoic acid (PAL-anchor) or p- (2, 4-dimethoxyphenyl-aminomethyl) -phenoxy (Rink-amide-anchor) may be used

The linkage to the polymeric support may be achieved by reacting the C-terminal Fmoc-protected amino acid with the support material by adding an activating agent in ethanol, acetonitrile, N-Dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N-methylpyrrolidone or similar solvents at room temperature or elevated temperature (e.g., between 40 ℃ and 60 ℃) and utilizing a reaction time of, for example, 2 to 72 hours.

The coupling of N α -protected amino acids (e.g., Fmoc amino acids) to PAL, Wang or Rink anchors may be carried out, for example, with the aid of coupling reagents such as N, N '-Dicyclohexylcarbodiimide (DCC), N' -Diisopropylcarbodiimide (DIC) or other carbodiimides, 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU) or other uronium salts, O-acyl-urea, benzotriazol-1-yl-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) or other phosphonium salts, N-hydroxysuccinimide, other N-hydroxyimides or oximes, in the presence or absence of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole, for example by means of TBTU with or without addition of a base such as, for example, Diisopropylethylamine (DIEA), triethylamine or N-methylmorpholine, for example diisopropylethylamine, using a reaction time of 2 to 72 hours (for example, in a 1.5 to 3 fold excess (for example a2 fold excess) of the amino acid and coupling reagent, and at a temperature of about 10 ℃ to 50 ℃, for example 25 ℃, in a solvent such as dimethylformamide, N-methylpyrrolidone or dichloromethane, for example dimethylformamide, for 3 hours).

It is also possible to use active esters (e.g., pentafluorophenyl, p-nitrophenyl, etc.), symmetrical anhydrides of N α -Fmoc-amino acids, acid chlorides or acid fluorides thereof, instead of coupling reagents, under the above-described conditions.

The N α -protected amino acid (e.g., Fmoc amino acid) can be coupled to the 2-chlorotrityl resin in dichloromethane by adding DIEA and using a reaction time of 10 to 120 minutes, e.g., 20 minutes, but not limited to using the solvent and the base.

The sequential coupling of protected amino acids can be carried out according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer. After cleavage of the na-Fmoc protecting group of the coupled amino acid on the solid phase by treatment with, for example, piperidine in dimethylformamide (10% to 50%) for 5 to 20 minutes, for example, piperidine in 50% DMF for 2x2 minutes, and piperidine in 20% DMF for 1x15 minutes, a 3 to 10 fold excess (e.g., a 10 fold excess) of the next protected amino acid is coupled to the previous amino acid in an inert, non-aqueous polar solvent such as dichloromethane, DMF, or a mixture of both, and at a temperature between about 10 ℃ and 50 ℃, for example, at 25 ℃. The previously mentioned reagents for coupling the first na-Fmoc amino acid to the PAL, Wang or Rink anchor are suitable as coupling reagents. Active esters of protected amino acids or their chlorides or fluorides or symmetrical anhydrides may also be used as replacements.

At the end of the solid phase synthesis, the peptide is cleaved from the support material, with simultaneous cleavage of the side chain protecting groups. Cleavage can be carried out with trifluoroacetic acid or other strongly acidic medium in 0.5 to 3 hours (e.g., 2 hours) with the addition of 5% -20% V/V of a scavenger such as dimethyl sulfide, ethyl methyl sulfide, thioanisole, thiocresol, m-cresol, anisole, ethanedithiol, phenol, or water, e.g., 15% V/V dimethyl sulfide/ethanedithiol/m-cresol 1:1: 1. Peptides with fully protected side chains were obtained by cleavage of the 2-chlorotrityl anchor with glacial acetic acid/trifluoroethanol/dichloromethane 2:2: 6. The protected peptide can be purified by chromatography on silica gel. If the peptide is attached to the solid phase by a Wang anchor and if a peptide with C-terminal alkanoylation is to be obtained, cleavage can be carried out by aminolysis using an alkylamine or a fluoroalkylamine. The aminolysis is carried out at a temperature of about-10 ℃ to 50 ℃ (e.g., about 25 ℃) and a reaction time of about 12 to 24 hours (e.g., about 18 hours). In addition, the peptide can be cleaved from the carrier by re-esterification, for example using methanol.

The acidic solution obtained can be mixed with 3 to 20 fold amount of cold diethyl ether or n-hexane, e.g. 10 fold excess diethyl ether, to precipitate the peptide, thereby separating the scavenger and cleaved protecting groups remaining in the ether. Further purification can be performed by reprecipitating the peptide several times from glacial acetic acid. The precipitate obtained can be treated in water or tert-butanol or a mixture of the two solvents, for example a tert-butanol/water 1:1 mixture, and subsequently freeze-dried.

The peptides obtained can be purified by various chromatographic methods including ion exchange in a weakly basic resin in the form of acetate, ion exchange in a non-derivatized polystyrene/divinylbenzene copolymer (e.g.,XAD), adsorption chromatography on silica gel, ion exchange chromatography, e.g. on carboxymethyl cellulose, e.g. onPartition chromatography, countercurrent distribution chromatography or High Pressure Liquid Chromatography (HPLC) carried out on G-25, for example, reversed phase HPLC carried out on an octyl or octadecyl silane bonded silicon (ODS) phase.

B.Recombinant production

Methods describing the production of human and mouse IL-10 can be found, for example, in U.S. Pat. No. 5,231,012, which teaches methods for producing proteins having IL-10 activity, including recombinant and other synthetic techniques. IL-10 may be of viral origin, and the cloning and expression of viral IL-10(BCRF1 protein) from EB virus is disclosed in Moore et al, (1990) Science 248: 1230. IL-10 is obtained in a variety of ways using standard techniques known in the art, such as those described herein. Recombinant human IL-10 is also commercially available, for example, from PeproTech, Inc., Rocky Hill, N.J.

When producing a polypeptide using recombinant techniques, the polypeptide is produced as an intracellular or secreted protein using any suitable construct and any suitable host cell, which may be prokaryotic or eukaryotic, such as a bacterial (e.g., e.coli) or yeast host cell, respectively. Other examples of eukaryotic cells that can be used as host cells include insect cells, mammalian cells, and/or plant cells. When mammalian host cells are used, they may include human cells (e.g., HeLa, 293, H9, and Jurkat cells); mouse cells (e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos 7, and CV 1); and hamster cells (e.g., Chinese Hamster Ovary (CHO) cells).

A variety of host-vector systems suitable for polypeptide expression can be used according to standard methods known in the art. See, e.g., Sambrook et al, 1989 Current Protocols in Molecular Biology Cold spring harbor Press, New York; and Ausubel et al 1995 Current Protocols in Molecular Biology, Wiley and Sons. Methods for introducing genetic material into a host cell include, for example, transformation, electroporation, conjugation, calcium phosphate methods, and the like. The method for transfer may be selected to provide for stable expression of the introduced nucleic acid encoding the polypeptide. The nucleic acid encoding the polypeptide may be provided as an inheritable episomal element (e.g., plasmid) or may be integrated into the genome. A variety of suitable vectors for the production of the polypeptide of interest are commercially available.

The vector may provide for extrachromosomal maintenance in the host cell or may provide for integration into the host cell genome. Expression vectors provide transcriptional and translational control sequences, and may provide inducible or constitutive expression, wherein the coding regions are operably linked under the transcriptional control of a transcriptional initiation region, and a transcriptional and translational termination region. Generally, transcriptional and translational regulatory sequences can include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, and enhancer or activation sequences. The promoter may be constitutive or inducible, and may be a strong constitutive promoter (e.g., T7).

Expression constructs typically have convenient restriction sites located near the promoter sequence to provide for insertion of the nucleic acid sequence encoding the protein of interest. Selectable markers effective in the expression host may be provided to aid in the selection of cells containing the vector. In addition, the expression construct may also include additional elements. For example, an expression vector may have one or two replication systems, allowing it to be maintained in an organism, such as a mammalian or insect cell (for expression) and a prokaryotic host (for cloning and amplification). In addition, the expression construct may contain a selectable marker gene to allow selection of transformed host cells. Alternative genes are well known in the art and may vary depending on the host cell used.

Isolation and purification of the protein can be achieved according to methods known in the art. For example, proteins can be isolated from lysates of cells genetically modified to constitutively and/or after induction, or from synthesis reaction mixtures by immunoaffinity purification, which typically involves contacting the sample with anti-protein antibodies, washing to remove non-specifically bound material, and eluting the specifically bound proteins. The isolated protein is further purified by dialysis and other methods commonly used for protein purification. In one embodiment, the protein is isolated using metal chelate chromatography. The protein may contain modifications to facilitate separation.

The polypeptide may be prepared in substantially pure or isolated form (e.g., free of other polypeptides). The polypeptide can be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). For example, a purified polypeptide can be provided such that the polypeptide is present in a composition that is substantially free (e.g., less than about 90%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1%) of other expressed proteins.

Recombinant techniques are used to manipulate various IL-10 related nucleic acids known in the art to provide constructs capable of encoding IL-10 polypeptides to produce IL-10 polypeptides. It is understood that when a particular amino acid sequence is provided, one of ordinary skill in the art will recognize a variety of different nucleic acid molecules encoding such amino acid sequences in view of her background and experience in, for example, molecular biology.

Amide bond substitution

In some cases, IL-10 includes one or more linkages other than peptide bonds, e.g., at least 2 adjacent amino acids are linked by a linkage other than an amide bond. For example, one or more amide linkages within the backbone of IL-10 may be substituted in order to reduce or eliminate undesirable proteolytic or other degradation, and/or to enhance serum stability, and/or to limit or increase conformational flexibility.

In another example, a linkage that can be used as an isostere of an amide linkage such as-CH₂NH-、-CH₂S-、-CH₂CH₂-, -CH-CH- (cis and trans) -, -COCH₂-、-CH(OH)CH₂-or-CH₂SO-replaces one or more amide bonds (-CO-NH-) in IL-10. One or more amide linkages in IL-10 may also be replaced by, for example, a reduced isosteric pseudopeptide bond. See Couder et al (1993) int.J.peptide Protein Res.41: 181-184. Such alternatives and how to implement them are known to those of ordinary skill in the art.

Amino acid substitutions

One or more amino acid substitutions may be made in the IL-10 polypeptide. The following are non-limiting examples:

a) alkyl-substituted hydrophobic amino acids, including alanine, leucine, isoleucine, valine, norleucine, (S) -2-aminobutyric acid, (S) -cyclohexylalanine or substituted with a group derived from C₁-C₁₀Other simple α -amino acid substitutions with aliphatic side chain substitutions of carbon (including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions);

b) aromatic-substituted hydrophobic amino acids, including phenylalanine, tryptophan, tyrosine, sulfotyrosine, diphenylalanine, 1-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, including amino, alkylamino, dialkylamino, aza, halo (fluoro, chloro, bromo, or iodo), or alkoxy (C) groups of the aromatic amino acids listed above₁To C₄) Substituted forms, illustrative examples of which are 2-, 3-or 4-aminophenylalanineAcid, 2-, 3-or 4-chlorophenylalanine, 2-, 3-or 4-methylphenylalanine, 2-, 3-or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl-or 5-methoxyphenylalanine, 2' -,3' -or 4' -amino-, 2' -,3' -or 4' -chloro-, 2,3 or 4-diphenylalanine, 2' -,3' -, or 4' -methyl-, 2-, 3-or 4-diphenylalanine, and 2-or 3-pyridylalanine;

c) substitution of amino acids containing basic side chains, including arginine, lysine, histidine, ornithine, 2, 3-diaminopropionic acid, homoarginine, including alkyl, alkenyl or aryl-substitution of previous amino acids (from C)₁-C₁₀Branched, linear or cyclic) derivatives, whether the substituent is on a heteroatom in, for example, the pro-R position (such as α nitrogen, or the distal nitrogen or nitrogens) or on the α carbon compounds used as illustrative examples include N-isopropyl-lysine, 3- (4-tetrahydropyridyl) -glycine, 3- (4-tetrahydropyridyl) -alanine, N- γ, γ' -diethyl-homoarginine, compounds such as α -methyl-arginine, α -methyl-2, 3-diaminopropionic acid, α -methyl-histidine, α -methyl-ornithine, wherein the alkyl group occupies the pro-R position at α -carbon, also included are amides formed from alkyl, aromatic acids, heteroaromatic acids (wherein the heteroaromatic group has one or more nitrogen, oxygen or sulfur atoms, singly or in combination), carboxylic acids or many well known activated derivatives such as acyl chlorides, activated esters, activated cyclic amides and related derivatives, and any of lysine, ornithine or 2, 3-diaminopropionic acid;

d) substitution of acidic amino acids, including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, 2, 4-diaminopropionic acid, ornithine or lysine, and the alkyl, aryl, aralkyl, heteroaryl sulfonamides of tetrazole-substituted alkyl amino acids;

e) substitution of side chain amide residues, including asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine; and

f) substitutions of hydroxyl-containing amino acids, including serine, threonine, homoserine, 2, 3-diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine or threonine.

In some cases, IL-10 contains one or more of naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids, or D-enantiomers of amino acids. For example, IL-10 may comprise only D-amino acids. For example, an IL-10 polypeptide may comprise one or more of the following residues: hydroxyproline, beta-alanine, o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2, 3-diaminopropionic acid, alpha-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, naphthylalanine, pyridylalanine 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, beta-2-thienylalanine, beta-aminobenzoic acid, p-aminotoluic acid, 2, 3-diaminopropionic acid, alpha-aminoisobutyric acid, alpha-methylglycine, N-methylglycine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2, 4-diaminobutyric acid, rho-aminophenylalanine, N-methylvaline, homocysteine, homoserine, -aminocaproic acid, ω -aminoheptanoic acid, ω -aminocaprylic acid, ω -aminotetradecanoic acid, cyclohexylalanine, α, γ -diaminobutyric acid, α, β -diaminopropionic acid, -aminopentanoic acid and 2, 3-diaminobutyric acid.

Additional modifications

Cysteine residues or cysteine analogs can be introduced into the IL-10 polypeptide to provide linkage to another peptide via disulfide linkage or to provide cyclization of the IL-10 polypeptide. Methods of introducing cysteine or cysteine analogs are known in the art; see, for example, U.S. patent No. 8,067,532.

Cyclizing the IL-10 polypeptide. One or more cysteines or cysteine analogs can be introduced into the IL-10 polypeptide, wherein an introduced cysteine or cysteine analog can form a disulfide bond with a further introduced cysteine or cysteine analog. Other methods of cyclization include the introduction of oxime or lanthionine linkers(ii) a See, for example, U.S. patent No. 8,044,175. Any combination of amino acids (or non-amino acid moieties) that can form a cyclized bond can be used and/or introduced. The cyclic bond may be formed using an amino acid (or using an amino acid and- (CH2)_n-CO-or- (CH2)_n-C₆H₄-CO-) with any combination of functional groups allowing the introduction of bridges. Some examples are disulfides, disulfide moieties such as- (CH2)_nCarbonyl bridges (carba bridges), thioacetals, thioether bridges (cystathionine or lanthionine), and also bridged esters and ethers. In these examples, n can be any integer, but is typically less than 10.

Other modifications include, for example, N-alkyl (or aryl) substitutions (psi [ CONR ]), or backbone crosslinks that create lactams and other cyclic structures. Other derivatives include C-terminal hydroxymethyl derivatives, ortho-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminal modified derivatives, including substituted amides such as alkyl amides and hydrazides.

In some cases, one or more L-amino acids are replaced with one or more D-amino acids in the IL-10 polypeptide.

In some cases, the IL-10 polypeptide is a retro-inverso (retroverso) analog (see, e.g., Sela and Zisman (1997) FASEB J.11: 449). Retro-inverso peptide analogs are isomers of linear polypeptides in which the orientation of the amino acid sequence is reversed (inverted) and in which the chiral D or L form of one or more amino acids is inverted (modified), e.g., using D-amino acids rather than L-amino acids. [ see, e.g., Jameson et al (1994) Nature 368: 744; and Brady et al (1994) Nature 368:692 ].

The IL-10 polypeptide may include a "protein transduction domain" (PTD), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic molecule that facilitates passage across a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. PTDs attached to another molecule facilitate the passage of the molecule across the membrane, for example from the extracellular space into the intracellular space or from the cytosol into an organelle. In some embodiments, the PTD is covalently attached to the amino terminus of the IL-10 polypeptide, while in other embodiments, the PTD is covalently attached to the carboxy terminus of the IL-10 polypeptide. Exemplary protein transduction domains include, but are not limited to, the minimal undecapeptide protein transduction domain (according to residues 47-57 of HIV-1TAT comprising YGRKKRRQRRR; SEQ ID NO://); a poly-arginine sequence comprising a number of arginine residues sufficient to direct entry into a cell (e.g., 3,4, 5,6, 7,8, 9, 10, or 10-50 arginines); the VP22 domain (Zender et al (2002) Cancer Gene ther.9(6): 489-96); drosophila antennal protein transduction domains (Noguchi et al (2003) Diabetes 52(7): 1732-1737); truncated human calcitonin peptide (Trehin et al (2004) pharm. research 21: 1248-1256); polylysine (Wender et al (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLMKR (SEQ ID NO: /); transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: /); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: /); and RQIKIWFQNRRMKWKK (SEQ ID NO: v). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: /), RKKRRQRRR (SEQ ID NO: /); an arginine homopolymer of 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following sequences: YGRKKRRQRRR (SEQ ID NO://); RKKRRQRR (SEQ ID NO://); YARAAARQARA (SEQ ID NO://); THRLPRRRRRR (SEQ ID NO://) and GGRRARRRRRR (SEQ ID NO://).

Carboxyl group COR of amino acid at C-terminal of IL-10 polypeptide₃Can be in free form (R)₃OH) or in the form of physiologically tolerated alkali metal or alkaline earth metal salts such as sodium, potassium or calcium salts. The carboxyl groups can also be replaced by primary, secondary or tertiary alcohols, such as, for example, methanol, branched or unbranched C₁-C₆-alkanols, such as ethanol or tert-butanol. The carboxyl group can also be replaced by a primary or secondary amine such as ammonia, branched or unbranched C₁-C₆Alkyl amines or C₁-C₆A di-alkylamine such as methylamine or dimethylamine.

Amino acid NR at the N-terminus of IL-10 Polypeptides₁R₂The amino group of (A) may be in free form (R)₁H and R₂H) or in physiologically tolerated salts such as, for example, chlorinationIn the form of substance or acetate. The amino group may also be acetylated with an acid, such that R₁Is H and R₂Acetyl, trifluoroacetyl or adamantyl. The amino group may be present in the form of an amino protecting group conventionally used in peptide chemistry, such as the amino protecting groups described above (e.g., Fmoc, benzyloxy-carbonyl (Z), Boc, and Alloc). The amino group may be N-alkylated, wherein R₁And/or R₂＝C₁-C₆Alkyl or C₂-C₈Alkenyl or C₇-C₉An aralkyl group. The alkyl residue may be linear, branched or cyclic (e.g., ethyl, isopropyl and cyclohexyl, respectively).

Specific modifications that enhance and/or mimic IL-10 function

It is often beneficial, and sometimes urgently desirable, to improve one of the physical properties of the treatment modalities disclosed herein (e.g., IL-10) and/or the manner in which they are administered. Improvements in physical properties include, for example, modulation of immunogenicity; methods of increasing water solubility, bioavailability, serum half-life, and/or therapeutic half-life; and/or modulating biological activity. Certain modifications may also be useful, for example, to improve antibodies (e.g., epitope tags) for detection assays and to facilitate protein purification. It is generally necessary to confer such improvements without adversely affecting the biological activity of the treatment modality and/or increasing its immunogenicity.

PEGylation of IL-10 is one specific modification encompassed by the present disclosure, however other modifications include, but are not limited to, glycosylation (N-and O-linked); polysialylation; albumin fusion molecules comprising serum albumin (e.g., Human Serum Albumin (HSA), cynomolgus monkey (cyno) serum albumin, or Bovine Serum Albumin (BSA)); albumin binding through, for example, a conjugated fatty acid chain (acylation); and an Fc fusion protein.

PEGylation:clinical efficacy of protein therapeutics is often limited by short plasma half-life and susceptibility to protease degradation. Studies of various therapeutic proteins (e.g., filgrastim) have shown that such difficulties can be overcome by various modificationsTo overcome this, it involves conjugating or linking the polypeptide sequence to any of a variety of non-proteinaceous polymers such as polyethylene glycol (PEG), polypropylene glycol or polyalkylene oxide. This is typically achieved by covalent binding of a linking moiety to both proteinaceous and non-proteinaceous polymers such as PEG. Such PEG conjugated biomolecules have been shown to have clinically useful properties, including better physical and thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulating half-life and reduced clearance, reduced immunogenicity and antigenicity, and reduced toxicity.

In addition to the beneficial effect of pegylation on pharmacokinetic parameters, pegylation itself may enhance activity. For example, PEG-IL-10 has been shown to be more effective against certain cancers than non-PEGylated IL-10 (see, e.g., EP 206636A 2).

PEGs suitable for conjugation to a polypeptide sequence are generally soluble in water at room temperature and have the general formula R (O-CH)₂-CH₂)_nO-R, wherein R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it typically has from 1 to 8 carbons. PEG conjugated to polypeptide sequences may be linear or branched. The present disclosure encompasses branched PEG derivatives, "star-PEG" and multi-arm PEG. The molecular weight of the PEG used in the present disclosure is not limited to any particular range, and examples are shown elsewhere herein; for example, certain embodiments have a molecular weight of 5kDa to 20kDa, whereas other embodiments have a molecular weight of 4kDa to 10 kDa.

The present disclosure also encompasses compositions of conjugates in which the PEG has different values of n, such that various different PEGs are present in a particular ratio. For example, some compositions comprise a mixture of conjugates, wherein n ═ 1,2,3, and 4. In some compositions, the percentage of conjugates where n-1 is 18-25%, the percentage of conjugates where n-2 is 50-66%, the percentage of conjugates where n-3 is 12-16%, and the percentage of conjugates where n-4 is up to 5%. Such compositions can be produced using reaction conditions and purification methods known in the art. Exemplary reaction conditions are described throughout the specification. Cation exchange chromatography can be used to separate the conjugates, followed by identification of fractions containing purified (conjugates without unmodified protein sequences and with other numbers of attached PEGs) conjugates with, for example, the desired number of attached PEGs.

Pegylation most commonly occurs on the alpha amino group on the N-terminus of the polypeptide, the amino group on the side chain of a lysine residue, and the imidazole group on the side chain of a histidine residue. Since most recombinant polypeptides have a single alpha as well as multiple amino and imidazole groups, many positional isomers can be produced depending on the linker chemistry. General pegylation strategies known in the art can be used herein. PEG can be conjugated to a polypeptide of the present disclosure through a terminal reactive group ("spacer") that mediates a bond between one or more of the free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. PEGs having spacers that can be conjugated to free amino groups include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide. Another activated polyethylene glycol that can be bound to free amino groups is 2, 4-bis (O-methoxypolyethylene glycol) -6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride. Activated polyethylene glycols bound to free carboxyl groups include polyoxyethylenediamine.

Conjugation of one or more of the polypeptide sequences of the present disclosure to PEG with a spacer can be performed by various conventional methods. For example, the conjugation reaction can be carried out at a temperature of 4 ℃ to room temperature, in solution at a pH of 5 to 10, using a molar ratio of reagent to protein of 4:1 to 30:1, for 30 minutes to 20 hours. The reaction conditions may be selected to direct the reaction toward producing predominantly the desired degree of substitution. Generally, low temperatures, low pH (e.g., pH ≧ 5) and short reaction times tend to reduce the number of attached PEGs, whereas high temperatures, neutral to high pH (e.g., pH ≧ 7) and longer reaction times tend to increase the number of attached PEGs. Various methods known in the art can be used to terminate the reaction. In some embodiments, the reaction is terminated by acidifying the reaction mixture and freezing at, for example, -20 ℃. Pegylation of various molecules is discussed, for example, in U.S. Pat. nos. 5,252,714, 5,643,575, 5,919,455, 5,932,462, and 5,985,263. PEG-IL-10 is described, for example, in U.S. Pat. No. 7,052,686. Specific reaction conditions contemplated for use herein are shown in the experimental section.

The present disclosure also encompasses the use of PEG mimetics. Recombinant PEG mimetics have been developed that retain the attributes of PEG (e.g., extended serum half-life) while imparting several additional advantageous properties. For example, simple polypeptide chains (including, e.g., Ala, Glu, Gly, Pro, Ser, and Thr) that have been fused to a peptide or protein drug of interest (e.g., Amunix's XTEN technology; Mountain View, CA) can be recombinantly produced that are capable of forming extended conformations similar to PEG. This eliminates the need for an additional conjugation step in the manufacturing process. Furthermore, established molecular biology techniques enable control of the side chain composition of polypeptide chains, thereby allowing optimization of immunogenicity and manufacturing performance.

Glycosylation: for the purposes of this disclosure, "glycosylation" is intended to broadly refer to the enzymatic process of attaching glycans to proteins, lipids, or other organic molecules. The use of the term "glycosylation" in connection with the present disclosure is generally intended to refer to the addition or elimination of one or more sugar moieties (by removal of potential glycosylation sites or by deletion of glycosylation using chemical and/or enzymatic methods), and/or the addition of one or more glycosylation sites that may or may not be present in the native sequence. In addition, the phrase includes qualitative changes in the glycosylation of the native protein, including changes in the nature and proportions of the various sugar moieties present.

Glycosylation can significantly affect the physical properties (e.g., solubility) of polypeptides such as IL-10 and can also be important in protein stability, secretion, and subcellular localization. Glycosylated polypeptides may also exhibit enhanced stability or may improve one or more pharmacokinetic properties, such as half-life. In addition, the improvement in solubility may, for example, enable the production of formulations that are more suitable for pharmaceutical administration than formulations comprising non-glycosylated polypeptides.

Proper glycosylation may be necessary for biological activity. Indeed, some genes from eukaryotes, when expressed in bacteria (e.g., E.coli) that lack the cellular processes for glycosylating proteins, produce recovered proteins that are nearly inactive or inactive because they lack glycosylation.

The addition of glycosylation sites can be achieved by altering the amino acid sequence. Changes to the polypeptide can be made, for example, by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites) with the one or more amino acid residues. The structure of the N-linked or O-linked oligosaccharides and sugar residues found in each type may be different. One type of sugar commonly found in both is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acids are usually the terminal residues of both N-linked and O-linked oligosaccharides and, due to their negative charge, may confer acidic properties to the glycoprotein. Particular embodiments of the present disclosure include the generation and use of N-glycosylation variants.

The polypeptide sequences of the present disclosure may optionally be altered at the nucleic acid level, particularly by mutating the nucleic acid encoding the polypeptide at preselected bases (so as to create codons that will translate into the desired amino acids). Another method of increasing the number of sugar moieties on a polypeptide is by chemically or enzymatically coupling a glycoside to the polypeptide. Removal of the carbohydrate may be achieved chemically or enzymatically, or by substitution of codons encoding amino acid residues that are glycosylated. Chemical deglycosylation techniques are known, and enzymatic cleavage of the sugar moiety on a polypeptide can be achieved by using a variety of endo-or exo-glycosidases.

Dihydrofolate reductase (DHFR) -deficient Chinese Hamster Ovary (CHO) cells are common host cells for the production of recombinant glycoproteins. These cells do not express the enzyme β -galactoside α -2, 6-sialyltransferase and thus do not add sialic acid in the α -2,6 linked form to the N-linked oligosaccharides of the glycoproteins produced in these cells.

PolysialylationThe present disclosure also encompasses the use of polysialylation, conjugation of polypeptides to naturally occurring biodegradable α - (2 → 8) linked polysialic acid ("PSA"), to improve the stability and pharmacokinetics in vivo, PSA is a biodegradable, non-toxic, highly hydrophilic natural polymer that prolongs its serum half-life in view of its high apparent molecular weight in blood, in addition, polysialylation of many peptide and protein therapeutics has resulted in significantly reduced proteolysis, retention of in vivo activity, and reduction of immunogenicity and antigenicity (see, e.g., g., g.gregoriadis et al, int.j.pharmaceutics 300(1-2): 125-30.) as well as modification with other conjugates (e.g., PEG), various techniques for site-specific polysialylation are available (see, e.g., t.linddhout et al, (18) PNAS108(18) 7397-7402).

Albumin fusion: additional suitable components and molecules for conjugation include albumins such as Human Serum Albumin (HSA), cynomolgus serum albumin, and Bovine Serum Albumin (BSA).

Mature HSA (a 585 amino acid polypeptide (-67 kDa) with a serum half-life of-20 days) is primarily responsible for maintenance of colloid osmotic blood pressure, blood pH, and transport and distribution of many endogenous and exogenous ligands. The protein has 3 structurally homologous domains (domains I, II and III), exists almost completely in an alpha-helical conformation, and is highly stable via 17 disulfide bridges. The 3 major drug binding regions of albumin are located on each of the 3 domains IB, IIA and IIIA within the subdomain.

Albumin synthesis occurs in the liver, which produces a transient primary product, preproprotein. Thus, full-length HSA has a signal peptide of 18 amino acids (MKWVTFISLLFLFSSAYS; SEQ ID NO://), followed by a 6 amino acid prodomain (RGVFRR; SEQ ID NO: /); this 24 amino acid residue peptide may be referred to as a prepro domain. HSA can be expressed and secreted using its endogenous signal peptide as the prepro domain. Alternatively, HSA can be expressed and secreted using an IgK signal peptide fused to the mature construct. The prepro-albumin is rapidly co-translationally cleaved at its amino terminus in the lumen of the endoplasmic reticulum to produce the stable 609 amino acid precursor polypeptide, pre-albumin. The preprotein is then passed through the golgi apparatus where it is converted to a 585 amino acid mature albumin by furin-dependent amino-terminal cleavage.

The primary amino acid sequence, structure and function of albumin are highly conserved across species, as are the processes of albumin synthesis and secretion. Albumin serum proteins compared to HSA are present in e.g. cynomolgus monkeys, cows, dogs, rabbits and rats. Bovine Serum Albumin (BSA) is most structurally similar to HSA in non-human species (see, e.g., Kosa et al, J Pharm Sci.96(11):3117-24, 11.2007). The present disclosure encompasses the use of albumin from non-human species, including, but not limited to, those shown above, in processes such as drug development.

In accordance with the present disclosure, albumin can be conjugated to drug molecules (e.g., polypeptides described herein) at the carboxy-terminus, the amino-terminus, the carboxy-terminus and the amino-terminus, as well as internally (see, e.g., USP 5,876,969 and USP 7,056,701).

In the HSA-drug molecule conjugates encompassed by the present disclosure, various forms of albumin can be used, such as albumin secretion pre-sequences and variants thereof, fragments and variants thereof, and HSA variants. Such forms typically have one or more desired albumin activities. In additional embodiments, the disclosure includes fusion proteins comprising a polypeptide drug molecule fused directly or indirectly to albumin, albumin fragments, albumin variants, and the like, wherein the fusion protein has greater plasma stability than the unfused drug molecule and/or the fusion protein retains the therapeutic activity of the unfused drug molecule. In some embodiments, indirect fusion is achieved through a linker, such as a peptide linker or a modified form thereof.

Intracellular cleavage can be performed enzymatically by, for example, furin or caspase. The cells express low levels of these endogenous enzymes, which are capable of cleaving parts of the fusion molecule intramolecularly; thus, some polypeptides are secreted from the cell without being conjugated to HSA, whereas some polypeptides are secreted in the form of fusion molecules comprising HSA. Embodiments of the present disclosure encompass the use of various furin fusion constructs. For example, a construct comprising the sequence RGRR, RKRKKR, RKKR or RRRKKR can be designed.

The present disclosure also encompasses extracellular lysis (i.e., ex vivo lysis) by which the fusion molecule is secreted from the cell, subjected to purification, and subsequently lysed. It is understood that excision can separate the intact HSA-linker complex or less intact HSA-linker complex from mature IL-10.

As alluded to above, fusion of albumin to one or more polypeptides of the present disclosure may be achieved, for example, by genetic manipulation (so as to link a nucleic acid encoding HSA or a fragment thereof to a nucleic acid encoding one or more polypeptide sequences). Subsequently, a suitable host is transformed or transfected with the fused nucleotide sequence, for example in the form of a suitable plasmid, to express the fusion polypeptide. Expression can be achieved in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo from, for example, transgenic organisms. In some embodiments of the disclosure, expression of the fusion protein is performed in a mammalian cell line, such as a CHO cell line. Transformation is used broadly herein to refer to genetic alteration of a cell resulting from direct uptake through the cell membrane, integration and expression of exogenous genetic material (exogenous nucleic acid). Transformation occurs naturally in some species of bacteria, but it can also be achieved in other cells by artificial means.

In addition, albumin itself may be modified to extend its circulating half-life. Fusion of the modified albumin to IL-10 can be achieved by gene manipulation techniques described above or by chemical conjugation; the resulting fusion molecule has a half-life that exceeds the half-life of the fusion with unmodified albumin (see WO 2011/051489).

Alternative albumin binding strategies:several albumin binding strategies have been developed as alternatives to direct fusion, including albumin junctions through conjugated fatty acid chains (acylation)And (6) mixing. Because serum albumin is a transport protein for fatty acids, these natural ligands with albumin binding activity have been used to extend the half-life of small protein therapeutics. For example, insulin detemir (LEVEMIR), an approved product for diabetes, comprises a myristyl chain conjugated to a genetically modified insulin, resulting in a long acting insulin analog.

The present disclosure also encompasses fusion proteins comprising an Albumin Binding Domain (ABD) polypeptide sequence and a sequence of one or more of the polypeptides described herein. Any ABD polypeptide sequence described in the literature may be a component of a fusion protein. The components of the fusion protein may optionally be covalently bonded through linkers such as those described herein. In some embodiments of the disclosure, the fusion protein comprises the ABD polypeptide sequence as the N-terminal portion and the polypeptide described herein as the C-terminal portion.

The present disclosure also encompasses fusion proteins comprising a fragment of an albumin binding polypeptide, which fragment substantially retains albumin binding; or a multimer of albumin binding polypeptides or fragments thereof comprising at least two albumin binding polypeptides or fragments thereof (as monomer units). For a general discussion of ABD and related technology, see WO 2012/050923, WO 2012/050930, WO 2012/004384 and WO 2009/016043.

Conjugation to other molecules: additional suitable components and molecules for conjugation include, for example, thyroglobulin; tetanus toxoid; diphtheria toxoid; polyamino acids such as poly (D-lysine: D-glutamic acid); VP6 polypeptide of rotavirus; influenza virus hemagglutinin, influenza virus nucleoprotein; keyhole Limpet Hemocyanin (KLH); and hepatitis b virus core protein and surface antigens; or any combination of the foregoing.

Thus, the present disclosure encompasses the conjugation of one or more additional components or molecules at the N and/or C terminus of a polypeptide sequence, such as another polypeptide (e.g., a polypeptide having an amino acid sequence heterologous to the subject polypeptide) or a carrier molecule. Thus, exemplary polypeptide sequences can be provided as conjugates with another component or molecule.

Conjugate modifications can result in polypeptide sequences that retain activity, with additional or complementary functions or activities derived from yet another molecule. For example, polypeptide sequences may be conjugated to molecules, e.g., to increase solubility, storage, in vivo or shelf half-life or stability, reduction in immunogenicity, delayed or controlled in vivo release, and the like. Other functions or activities include conjugates that reduce toxicity relative to the unconjugated polypeptide sequence, conjugates that target one type of cell or organ more efficiently than the unconjugated polypeptide sequence, or drugs that further counteract the etiology or effect associated with a disease, disorder, or condition (e.g., cancer) as set forth herein.

IL-10 polypeptides can also be conjugated to large slowly metabolized macromolecules, such as proteins; polysaccharides such as sepharose, agarose, cellulose or cellulose beads; polymeric amino acids such as polyglutamic acid, or polylysine; an amino acid copolymer; inactivated virus particles; inactivated bacterial toxins such as toxoid or leukotoxin molecules from diphtheria, tetanus, cholera; inactivated bacteria; and dendritic cells. Such conjugated forms can be used to generate antibodies to the polypeptides of the disclosure, if desired.

Additional candidate components and molecules for conjugation include those suitable for isolation or purification. Specific non-limiting examples include binding molecules such as biotin (biotin-avidin protein specific binding pair), antibodies, receptors, ligands, lectins, or molecules comprising a solid support, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.

Purification methods such as cation exchange chromatography can be used to separate the conjugates by charge differences, which effectively separates the conjugates into their various molecular weights. For example, a cation exchange column can be loaded, followed by washing with-20 mM sodium acetate pH 4, followed by elution with a linear (0M to 0.5M) NaCl gradient buffered at a pH of about 3 to 5.5, e.g., at a pH of 4.5. The content of the fractions obtained by cation exchange chromatography can be identified by molecular weight using conventional methods (e.g., mass spectrometry, SDS-PAGE, or other known methods for separating molecular entities according to molecular weight).

Fc-fusion molecules: in certain embodiments, the amino-or carboxy-terminus of a polypeptide sequence of the present disclosure can be fused to an immunoglobulin Fc region (e.g., a human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, so biopharmaceutical products may require less frequent administration. Fc binds to nascent Fc receptors (FcRn) in endothelial cells lining the blood vessels, and after binding, the Fc fusion molecule is protected from degradation and is released back into the circulation, thereby allowing the molecule to remain in the circulation for a longer period of time. This Fc binding is believed to be the mechanism by which endogenous IgG retains its long plasma half-life. Recently, Fc-fusion technology has linked a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to traditional Fc-fusion conjugates. Examples of other Fc-related techniques suitable for use with the polypeptides disclosed herein are described in WO 2013/113008.

Other modifications: the present disclosure contemplates the use of other modifications to IL-10, now known or later developed, to improve one or more properties. One such method for extending the circulating half-life, enhancing the stability, reducing the clearance rate, or altering the immunogenicity or allergenicity of a polypeptide of the present disclosure includes modifying the polypeptide sequence by hydroxyethyl starch modification using hydroxyethyl starch derivatives linked to other molecules to modify the characteristics of the polypeptide sequence. Various aspects of hydroxyethylstarch are described, for example, in U.S. patent application nos. 2007/0134197 and 2006/0258607.

The present disclosure also encompasses fusion molecules comprising SUMO (as a fusion tag) (LifeSensors, inc.; Malvern, PA). The fusion of the polypeptides described herein to SUMO may exhibit several beneficial effects, including increased expression, improved solubility, and/or assistance in the development of purification methods. SUMO proteases recognize the tertiary structure of SUMO and cleave the fusion protein at the C-terminus of SUMO, thereby releasing the polypeptide with the desired N-terminal amino acid described herein.

Joint: linkers and their use have been described above. Any of the foregoing components and molecules used to modify the polypeptide sequences of the present disclosure may optionally be conjugated through a linker. Suitable linkers include "flexible linkers," which are generally of sufficient length to allow some movement between the modified polypeptide sequence and the linked components and molecules. Linker molecules are typically about 6-50 atoms in length. The linker molecule can also be, for example, an aryl acetylene, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof. Suitable linkers can be readily selected and can be of any suitable length, such as1 amino acid (e.g., Gly), 2,3,4, 5,6, 7,8, 9, 10-20, 20-30, 30-50, or more than 50 amino acids.

Exemplary flexible linkers include glycine polymers (G)_nGlycine-serine polymers (e.g., (GS)_n、GSGGS_n、GGGS_n、(G_mS_o)_n、(G_mS_oG_m)_n、(G_mS_oG_mS_oG_m)_n、(GSGGS_m)_n、(GSGS_mG)_nAnd (GGGS)_m)_nAnd combinations thereof, wherein m and o are each independently selected from integers of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and thus can serve as neutral tethers between components. Exemplary flexible linkers include, but are not limited to, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, and GSSSG.

Therapeutic and prophylactic uses

The present disclosure encompasses the use of IL-10 polypeptides (e.g., PEG-IL-10) described herein in the treatment or prevention of a wide range of diseases, disorders or conditions and/or symptoms thereof. Indeed, the teachings of the present disclosure are intended for use in any disease, disorder, or condition for which obtaining or maintaining the above-described IL-10 mean serum trough concentration parameters may be beneficial. Although specific uses are described in detail below, it should be understood that the present disclosure is not so limited. Furthermore, while general classifications of particular diseases, disorders, and conditions are shown below, some diseases, disorders, and conditions may be members of more than one classification (e.g., cancer-related disorders and fibrosis-related disorders), and other diseases may not be members of any of the disclosed classifications.

Fibrotic disorders and cancer.According to the present disclosure, IL-10 (e.g., PEG-IL-10) may be used to treat or prevent proliferative conditions or disorders, including cancer (e.g., cancer of the uterus, cervix, breast, prostate, testis, gastrointestinal tract (e.g., cancer of the esophagus, oropharynx, stomach, small or large intestine, colon, or rectum), kidney cells, bladder, bone marrow, skin, head or neck, skin, liver, gall bladder, heart, lung, pancreas, salivary gland, adrenal gland, thyroid, brain (e.g., glioma), ganglia, Central Nervous System (CNS), and Peripheral Nervous System (PNS), and cancers of the hematopoietic and immune systems (e.g., spleen or thymus). The present disclosure also provides methods of treating or preventing other cancer-related diseases, disorders or conditions, including, for example, immunogenic tumors, non-immunogenic tumors, dormant tumors, virus-induced cancers (e.g., epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, and papilloma virus), adenocarcinomas, lymphomas, carcinomas, melanomas, leukemias, myelomas, sarcomas, teratomas, chemically-induced cancers, metastases, and angiogenesis. The present disclosure contemplates, for example, reducing tolerance to tumor or cancer cell antigens by modulating the activity of regulatory T cells and/or CD8+ T cells (see, e.g., Ramirez-Montagut, et al (2003) Oncogene22: 3180-. In particular embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, or leukemia. The use of the term cancer-related diseases, disorders and conditions is intended to be broadly construedRefers to conditions directly or indirectly associated with cancer and includes, for example, angiogenesis and precancerous conditions such as dysplasia.

In some embodiments, the present disclosure provides methods of treating a proliferative condition, a cancer, a tumor, or a precancerous condition with an IL-10 polypeptide (e.g., PEG-IL-10) and at least one additional therapeutic or diagnostic agent, examples of which are elsewhere herein.

The present disclosure also provides methods of treating or preventing fibrotic diseases, disorders and conditions. As used herein, the phrase "fibrotic diseases, disorders and conditions" and similar terms (e.g., "fibrotic disorders") and phrases are to be construed broadly so that it includes any condition that can lead to the formation of fibrotic or scar tissue (e.g., fibrosis in one or more tissues). For example, injuries (e.g., wounds) that can produce scar tissue include wounds to the skin, eye, lung, kidney, liver, central nervous system, and cardiovascular system. The phrase also includes scar tissue formation resulting from stroke and tissue adhesions, for example, as a result of injury or surgery.

As used herein, the term "fibrosis" refers to the formation of fibrous tissue as a repair or reaction process rather than as a normal component of an organ or tissue. Fibrosis is characterized by the accumulation of fibroblasts in any particular tissue and collagen deposition beyond that normally deposited.

Fibrotic disorders include, but are not limited to, fibrosis resulting from wound healing, systemic and local scleroderma, atherosclerosis, restenosis, inflammation and fibrosis of the lung, idiopathic pulmonary fibrosis, interstitial lung disease, cirrhosis of the liver, fibrosis due to chronic hepatitis b or c virus infection, kidney disease (e.g., glomerulonephritis), heart disease due to scar tissue, keloids and hypertrophic scars, and ocular diseases such as macular degeneration as well as retinal and vitreoretinopathy. Other fibrotic diseases include chemotherapy drug-induced fibrosis, radiation-induced fibrosis, and injuries and burns.

Fibrotic disorders are often liver-related, and there is frequently a link between such disorders and inappropriate accumulation of hepatic cholesterol and triglycerides within hepatocytes. This accumulation appears to cause a pro-inflammatory response that leads to liver fibrosis and cirrhosis. Liver disorders with fibrotic components include nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH).

Cardiovascular diseases.The present disclosure also encompasses the use of IL-10 polypeptides (e.g., PEG-IL-10) described herein for the treatment and/or prevention of certain cardiovascular-related diseases and or associated metabolic-related diseases, disorders, and conditions, and disorders related thereto.

As used herein, the terms "cardiovascular disease", "heart disease" and the like refer to any disease affecting the cardiovascular system, primarily heart disease, vascular disease of the brain and kidneys, and peripheral arterial disease. Cardiovascular diseases are a range of diseases including coronary heart disease (i.e., ischemic heart disease or coronary artery disease), atherosclerosis, cardiomyopathy, hypertension, hypertensive heart disease, pulmonary heart disease, heart rhythm disorders, endocarditis, cerebrovascular disease, and peripheral artery disease. Cardiovascular disease is a leading cause of death in the world, and although it usually affects the elderly, the antecedent cause of cardiovascular disease, particularly atherosclerosis, begins early in life.

Particular embodiments of the present disclosure relate to the use of IL-10 polypeptides for the treatment and/or prevention of atherosclerosis, a chronic condition in which the arterial wall thickens to form plaques as a result of the accumulation of fatty substances such as cholesterol and triglycerides. Atherosclerosis generally involves a chronic inflammatory reaction in the arterial wall, which is mainly caused by the accumulation of macrophages and promoted by Low Density Lipoproteins (LDL) (failure to adequately remove fat and cholesterol from macrophages by functional high density lipoproteins). A chronically enlarged atherosclerotic lesion may cause complete occlusion of the lumen, which may only be manifested when the lumen is so narrow that there is insufficient blood supply to downstream tissues, resulting in ischemia.

IL-10 polypeptides, which may be associated with, for example, cardiovascular disease (e.g., atherosclerosis), cerebrovascular disease (e.g., stroke), and peripheral vascular disease, may be particularly advantageous in the treatment and/or prevention of cholesterol-related disorders. For example, but not limited to, IL-10 polypeptides can be used to reduce blood cholesterol levels in a subject. In determining whether a subject has hypercholesterolemia, there is no clear boundary between normal and abnormal cholesterol levels, and interpretation of values associated with other health conditions and risk factors is required. Nevertheless, the following guidelines are generally used in the united states: total cholesterol <200mg/dL is desirable, 200-239mg/dL is critically high, and ≧ 240mg/dL is high. Higher total cholesterol levels increase the risk of cardiovascular disease, and levels of LDL or non-HDL cholesterol are both predictive of future coronary heart disease. When assessing hypercholesterolemia, it is often useful to measure all lipoprotein subfractions (VLDL, IDL, LDL and HDL). A particular therapeutic goal is to reduce LDL while maintaining or increasing HDL.

Thrombosis and thrombotic conditions.

Thrombosis, the formation of a thrombus (blood clot) within a blood vessel that results in the obstruction of the flow of blood through the circulatory system, can be caused by abnormalities in one or more of the following aspects (Virchow's tripad): hypercoagulability or increased blood clotting, vascular endothelial cell damage or disturbed blood flow (stasis, turbulence).

Thrombosis is generally classified as venous or arterial thrombosis, each of which may assume several subtypes. Venous thrombosis includes Deep Vein Thrombosis (DVT), portal vein thrombosis, renal vein thrombosis, internal jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, and cerebral sinus thrombosis. Arterial thrombosis includes stroke and myocardial infarction.

The present disclosure encompasses other diseases, disorders, and conditions, including atrial thrombosis and polycythemia vera (also known as erythema, essential polycythemia, and polycythemia vera), myeloproliferative blood disorders in which the bone marrow produces too many RBCs, WBCs, and/or platelets.

Immune disorders and inflammatory conditions.As used herein, terms such as "immune disease," "immune condition," "immune disorder," "inflammatory disease," "inflammatory condition," "inflammatory disorder," and the like, are intended to broadly include any immune or inflammatory-related condition (e.g., pathological inflammation and autoimmune disease). Such conditions are often inevitably interwoven with other diseases, disorders and conditions. For example, an "immune condition" may refer to proliferative conditions such as cancer, tumors, and angiogenesis, including infections (acute and chronic), tumors, and cancers that resist eradication by the immune system.

A non-limiting list of immune and inflammatory-related diseases, disorders, and conditions that may be caused, for example, by inflammatory cytokines includes arthritis, renal failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergy, fibrosis, surgical complications (e.g., where inflammatory cytokines prevent healing), anemia, and fibromyalgia. Other diseases and conditions that may be associated with chronic inflammation include alzheimer's disease, congestive heart failure, stroke, aortic valve stenosis, atherosclerosis, osteoporosis, Parkinson's disease, infections, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), allergic contact dermatitis and other eczemas, systemic sclerosis, transplantation, and multiple sclerosis.

Some of the above-described diseases, disorders, and conditions for which IL-10 (e.g., PEG-IL-10) may be particularly effective (due to, for example, limitations of current therapies) are described in more detail below.

The IL-10 polypeptides of the present disclosure may be particularly effective in the treatment and prevention of Inflammatory Bowel Disease (IBD). IBD includes Crohn's Disease (CD) and Ulcerative Colitis (UC), both of which are chronic idiopathic diseases that can affect any part of the gastrointestinal tract and are associated with many adverse reactions, and patients with long-term UC are at increased risk of developing colon cancer. Current treatment of IBD is aimed at controlling inflammatory symptoms, and while certain agents (e.g., corticosteroids, aminosalicylates, and standard immunosuppressive agents (e.g., cyclosporine, azathioprine, and methotrexate)) have met with limited success, long-term treatment can cause liver damage (e.g., fibrosis or cirrhosis) and bone marrow suppression, and patients often become noncompliant with such treatment.

Psoriasis (a common series of immune-mediated chronic skin diseases) affects over 450 million people in the united states, 150 of which are believed to have moderate to severe forms of the disease. In addition, over 10% of psoriasis patients develop psoriatic arthritis, which damages the connective tissue around bones and joints. Improved understanding of the underlying physiology of psoriasis has led to the introduction of agents that target, for example, the activity of T lymphocytes and cytokines responsible for the inflammatory properties of the disease. Such agents include TNF-alpha inhibitors (also used in the treatment of Rheumatoid Arthritis (RA)), including ENBREL (etanercept), REMICADE (infliximab) and homira (adalimumab), and T-cell inhibitors such as AMEVIVE (alefacept) and raptvia (efletuzumab). While several of these agents are effective to some extent in certain patient populations, they have not been shown to be effective in treating all patients.

Rheumatoid Arthritis (RA), which is generally characterized by chronic inflammation of the membrane lining (synovium) of the joint, affects about 1% of the us population, or 210 million people in the united states. Further understanding of the role of cytokines, including TNF- α and IL-1, in inflammatory processes has enabled the development and introduction of a new class of disease modifying antirheumatic drugs (DMARDs). Agents, some of which overlap with the treatment modalities of RA, include ENBREL (etanercept), REMICADE (infliximab), HUMIRA (adalimumab), and KINERET (anakinra). Although some of these agents alleviate symptoms, inhibit the progression of structural damage, and improve physical function in a particular patient population, there remains a need for alternative agents with improved efficacy, complementary mechanisms of action, and fewer/less severe adverse effects.

Subjects with Multiple Sclerosis (MS), a severely debilitating autoimmune disease that includes multiple areas of inflammation and myelin scarring in the brain and spinal cord, can be helped specifically with the IL-10 polypeptides described herein, as current treatments only alleviate symptoms or delay progression of disability.

Similarly, IL-10 polypeptides are particularly advantageous for subjects suffering from neurodegenerative disorders such as Alzheimer's Disease (AD), brain disorders that severely impair a patient's mind, memory, and speech processing, and Parkinson's Disease (PD), a progressive disorder of the CNS characterized by, for example, abnormal movement, stiffness, and tremor. These conditions are progressive and debilitating and no therapeutic drug is available.

Viral diseases.There is increasing interest in the role of IL-10 in viral diseases. IL-10 has been postulated to produce stimulatory and inhibitory effects, depending on its receptor binding activity.

For example, the effect of inhibiting IL-10 function to enhance antiviral immunity and vaccine efficacy has been considered (see Wilson, E., (2011) Curr Top Microbiol Immunol.350: 39-65). In addition, the role of IL-10 in Human Immunodeficiency Virus (HIV) function has been studied. In addition to inhibition of human immunodeficiency virus type 1 (HIV-1) replication, IL-10 may also promote viral retention by inactivation of effector immune mechanisms (Naicker, D., et al, (2009) J InfectDis.200.3: 448-452). Another study has identified a subset of IL-10 producing B cells capable of modulating T cell immunity in chronic Hepatitis B Virus (HBV) infection.

Although the above studies indicate that IL-10 inhibition may be beneficial, specific viral infections comprising a CD8+ T cell component may be candidates for treatment and/or prevention by administration of IL-10. This is supported by the positive role that IL-10 plays in certain cancers through the regulation of regulatory T cells and/or CD8+ T cells.

The present disclosure encompasses the use of IL-10 polypeptides in the treatment and/or prevention of any viral disease, disorder or condition for which treatment with IL-10 may be beneficial. Examples of viral diseases, disorders, and conditions contemplated include hepatitis b, hepatitis c, HIV, herpes virus, and Cytomegalovirus (CMV).

Pharmaceutical composition

The IL-10 polypeptides of the present disclosure can be in the form of a composition suitable for administration to a subject. Typically, such compositions are "pharmaceutical compositions" comprising IL-10 and one or more pharmaceutically or physiologically acceptable diluents, carriers or excipients. In certain embodiments, the IL-10 polypeptide is present in a therapeutically acceptable amount. Pharmaceutical compositions may be used in the methods of the present disclosure; thus, for example, pharmaceutical compositions can be administered to a subject ex vivo or in vivo to practice the therapeutic and prophylactic methods and uses described herein.

The pharmaceutical compositions of the present disclosure may be formulated to be compatible with the desired method or route of administration; exemplary routes of administration are shown herein. In addition, the pharmaceutical compositions can be used in combination with other therapeutically active agents or compounds described herein to treat or prevent diseases, disorders, and conditions, as encompassed by the present disclosure.

The pharmaceutical compositions generally comprise a therapeutically effective amount of an IL-10 polypeptide encompassed by the present disclosure and one or more pharmaceutically and physiologically acceptable formulating agents. Suitable pharmaceutically or physiologically acceptable diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl paraben, ethyl paraben, or n-propyl paraben), emulsifiers, suspending agents, dispersants, solvents, fillers, extenders, detergents, buffers, vehicles, diluents, and/or adjuvants. For example, a suitable vehicle may be a physiological saline solution or citrate buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. One of ordinary skill in the art will readily recognize a variety of buffers that may be used in the pharmaceutical compositions and dosage forms contemplated herein. Typical buffering agents include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. As an example, the buffer component may be a water-soluble substance such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Acceptable buffers include, for example, Tris buffer, N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), 2- (N-morpholino) ethanesulfonic acid (MES), 2- (N-morpholino) ethanesulfonic acid sodium salt (MES), 3- (N-morpholino) propanesulfonic acid (MOPS), and N-Tris [ hydroxymethyl ] methyl-3-aminopropanesulfonic acid (TAPS).

After the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored in ready-to-use form, in lyophilized form requiring reconstitution prior to use, in liquid form requiring dilution prior to use, or in other acceptable forms. In some embodiments, the container is a disposable container (e.g., a disposable vial, ampoule, syringe, or auto-injector (similarly, e.g.,) While in other embodiments multiple use containers (e.g., multiple use vials) are provided. Any drug delivery device may be used to deliver IL-10, including implants (e.g., implantable pumps) and catheter systems, slow syringe pumps, and devices, all of which are well known to those of ordinary skill in the art. Depot injections, usually administered subcutaneously or intramuscularly, may also be used to release the polypeptides disclosed herein over a defined period of time. Depot injectable formulations are typically solid or oil-based and typically comprise at least one of the formulation components set forth herein. One of ordinary skill in the art is familiar with the possible formulations and uses of depot injections.

Pharmaceutical compositionCan be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which are mentioned herein. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable diluents, solvents and dispersion media which can be employed include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL^TM) (BASF, Parsippany, NJ) or Phosphate Buffered Saline (PBS), ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Prolonged absorption of a particular injectable formulation can be brought about by the inclusion of an agent that delays absorption (e.g., aluminum monostearate or gelatin).

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, capsules, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups, solutions, microbeads or elixirs. Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the preparation of pharmaceutical compositions, and such compositions may contain one or more agents such as, for example, sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets, capsules and the like contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.

Tablets, capsules and the like suitable for oral administration may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by techniques known in the art to form osmotic therapeutic tablets for controlled release. Additional agents include biodegradable or biocompatible particles or polymeric substances, such as polyesters, polyamino acids, hydrogels, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene-vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylene vinyl acetate copolymers, to control the delivery of the administered composition. For example, oral agents may be encapsulated in microcapsules prepared by coacervation techniques or by interfacial polymerization, or in colloidal drug delivery systems, by using hydroxymethylcellulose or gelatin-microcapsules or poly (methylmethacylate) microcapsules, respectively. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Methods for preparing the above formulations will be apparent to those of ordinary skill in the art.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin, or microcrystalline cellulose, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture thereof. Such excipients may be suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as naturally occurring phosphatides (e.g. lecithin), or condensation products of an alkylene oxide with fatty acids (e.g. polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g. for heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g. polyoxyethylene sorbitan monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g. polysorbitol monooleate). The aqueous suspensions may also contain one or more preservatives

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

The disclosed pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin, or a mixture of these oils. Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth; naturally occurring phospholipids, such as soy, lecithin and esters or partial esters derived from fatty acids; hexitol anhydrides, for example, sorbitol monooleate; and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitol monooleate.

The formulations may also include carriers to protect the composition from rapid degradation or elimination from the body, such as controlled release formulations, including implants, liposomes, hydrogels, prodrugs and microencapsulated delivery systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate, alone or in combination with a wax, may be used.

The present disclosure encompasses administration of IL-10 polypeptides in the form of suppositories for rectal administration. Suppositories can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycols.

The IL-10 polypeptides encompassed by the present disclosure are in the form of any other suitable pharmaceutical composition now known or later developed (e.g., sprays for nasal or inhalation use).

The concentration of the polypeptide or fragment thereof in the formulation can vary widely (e.g., from less than about 0.1%, typically about 2% or at least about 2% up to 20% to 50% or more by weight) and is typically selected, for example, according to the particular mode of administration selected, based primarily on fluid volume, viscosity, and subject-based factors.

Route of administration

The present disclosure encompasses the administration of IL-10 and compositions thereof in any suitable manner. Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intraparenchymal), and intracerebroventricular), oral, nasal, vaginal, sublingual, ocular, rectal, topical (e.g., transdermal), sublingual, and inhalation. Depot injections, typically administered subcutaneously or intramuscularly, may also be used to release the IL-10 polypeptides disclosed herein over a defined period of time.

Particular embodiments of the present disclosure encompass parenteral administration, and in other particular embodiments, parenteral administration is subcutaneous administration.

Combination therapy

The present disclosure encompasses the use of IL-10 (e.g., PEG-IL-10) in combination with one or more active therapeutic agents (e.g., cytokines) or other prophylactic or therapeutic modalities (e.g., irradiation). In such combination therapies, the various active agents often have different mechanisms of action. Such combination therapy may be particularly advantageous because it allows for a reduction in the dosage of the one or more agents, thereby reducing or eliminating the adverse effects associated with the one or more agents; moreover, such combination therapies may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder or condition.

As used herein, "combination" is intended to include therapies that can be administered alone, e.g., separately formulated for separate administration (e.g., as may be provided in a kit), as well as therapies that can be administered together in a single formulation (i.e., "co-formulation").

In certain embodiments, the IL-10 polypeptide is administered or applied sequentially, e.g., wherein one agent is administered prior to one or more other agents. In other embodiments, the IL-10 polypeptide is administered simultaneously, e.g., wherein two or more agents are administered at the same time or at about the same time; the two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Whether two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

The IL-10 polypeptides of the present disclosure may be used in combination with one or more other (active) agents in any suitable manner under circumstances. In one embodiment, treatment with at least one active agent of the present disclosure and at least one IL-10 polypeptide is maintained for a period of time. In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., when the subject is stable) while treatment with an IL-10 polypeptide of the disclosure is maintained at a constant dosing regimen. In further embodiments, treatment with at least one active agent is reduced or discontinued (e.g., when the subject is stable), while treatment with an IL-10 polypeptide of the disclosure is reduced (e.g., lower dose, less frequent dosing, or shorter treatment regimen). In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., when the subject is stable) and treatment with an IL-10 polypeptide of the disclosure is increased (e.g., higher dose, more frequent dosing or longer treatment regimen). In another embodiment, treatment with at least one active agent is maintained and treatment with an IL-10 polypeptide of the disclosure is reduced or discontinued (e.g., lower dose, less frequent dosing or shorter treatment regimen). In another embodiment, treatment with at least one active agent and treatment with an IL-10 polypeptide of the disclosure is reduced or discontinued (e.g., lower dose, less frequent dosing or shorter treatment regimen).

Fibrotic disorders and cancer.The present disclosure provides methods of treating and/or preventing a proliferative condition, cancer, tumor, or pre-cancerous disease, disorder, or condition using an IL-10 polypeptide (e.g., PEG-IL-10) and at least one additional therapeutic or diagnostic agent.

Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzotepa, carboquone, metoclopramide, and uretepa; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; nitrogen mustards such as chlorambucil, napthalamine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine oxide hydrochloride, melphalan, neomechlorethamine, cholesterol chlorambucil, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorourethrin, fotemustine, lomustine, nimustine, ramustine; antibiotics such as acrifamycin, actinomycin, amtriptan, azaserine, bleomycin, actinomycin C, calicheamicin, carrubicin, carminomycin, carubicin, chromamycin, chromomycin, dactinomycin, daunorubicin, ditobicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, isorubicin, idarubicin, sisomicin, mitomycin, mycophenolic acid, norramycin, olivomycin, pelomycin, porphyrinomycin, puromycin, trirubicin, roxobicin, streptonigrin, streptozocin, tubercidin, ubenimex, setastin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as carpoterone, drotandrosterone propionic acid, epitioandrostanol, meiandrane, testolactone; anti-adrenal classes such as luminal, mitotane, trostane; folic acid replenisher such as folinic acid; acetic acid glucurolactone; an aldehydic phosphoramide glycoside; (ii) aminolevulinic acid; amsacrine; (ii) a hundred-pad (bestrabucil); bisantrene; edatrexate (edatraxate), dexfofane (dexfamine); decarburized colchicine (demecolcine); diazaquinone; eformin (elformithine); ammonium etiolate; etoglut; gallium nitrate; a hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidanol; nitraminopropanine (nitrarine); pentostatin; methionine; pirarubicin; podophyllinic acid; 2-ethyl hydrazide; (ii) procarbazine; lezoxan; a texaphyrin; spiro germanium; alternarionic acid; a tri-imine quinone; 2,2' -trichlorotriethylamine; uratan; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulcitol; pipobroman; adding cytosine (cytosine); cytarabine (Ara-C); cyclophosphamide; thiotepa; taxanes such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum complexes such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; novier; noxiatrone (novantrone); (ii) teniposide; daunorubicin; aminopterin; (ii) Hirodad; ibandronate sodium; CPT 11; a topoisomerase inhibitor; difluoromethyl ornithine (DMFO); tretinoin; epothilones (esperamicins); capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Chemotherapeutic agents also include anti-hormonal agents used to modulate or inhibit the effects of hormones on tumors, such as anti-estrogen agents, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5) -imidazole, 4-hydroxy tamoxifen, trovaxifene, raloxifene, onapristone, and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and a pharmaceutically acceptable salt, acid or derivative of any of the above. In certain embodiments, the combination therapy comprises administration of a hormone or related hormonal agent.

Additional therapeutic modalities that can be used in combination with an IL-10 polypeptide include cytokines or cytokine antagonists such as IL-12, INF α or anti-epidermal growth factor receptor, radiation therapy, monoclonal antibodies to another tumor antigen, complexes of monoclonal antibodies with toxins, T cell adjuvants, bone marrow transplantation, or antigen presenting cells (e.g., dendritic cell therapy). Vaccines (e.g., in the form of a soluble protein or in the form of a nucleic acid encoding a protein) are also provided herein.

Cardiovascular diseases.The present disclosure provides methods of treating and/or preventing certain cardiovascular and/or metabolic-related diseases, disorders, and conditions, and disorders related thereto, using an IL-10 polypeptide (e.g., PEG-IL-10) and at least one additional therapeutic or diagnostic agent.

Examples of therapeutic agents useful in combination therapy for the treatment of hypercholesterolemia (and thus atherosclerosis generally) include statins that inhibit enzymatic synthesis of cholesterol (e.g., CRESTOR, LESCOL, LIPITOR, MEVACOR, pravacrol, and ZOCOR); bile acid resins that sequester cholesterol and prevent its absorption (e.g., COLESTID, LO-CHOLEST, PREVALITE, QUESTRAN, and WELCHOL); ezetimibe (ZETIA) which blocks cholesterol absorption; fibric acids that lower triglycerides and may slightly increase HDL (e.g., TRICOR); niacin (e.g., NIACOR) for moderately lowering LDL cholesterol and triglycerides; and/or combinations of the foregoing (e.g., VYTORIN (ezetimibe and simvastatin). alternative cholesterol treatments that may be candidates for use in combination with the IL-10 polypeptides described herein include various supplements and herbs (e.g., garlic, policosanol (policosanol), and myrrh (guggul)).

Immune disorders and inflammatory disorders.The present disclosure provides methods for treating and/or preventing immune and/or inflammatory-related diseases, disorders, and conditions, and disorders related thereto, utilizing an IL-10 polypeptide (e.g., PEG-IL-10) and at least one additional therapeutic or diagnostic agent.

Examples of therapeutic agents for use in combination therapy include, but are not limited to, the following: non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen and other propionic acid derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fencloc acid, fenclorac, fenti, furofenac, ibufenac, xofenac, obenzoic acid (oxpinac), sulindac, thiopinac, tolmetin, zidometacin, and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid, and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenican), oxicams (oxicams), Piroxicam, sudoxicam and tenoxicam), salicylic acids (acetylsalicylic acid, sulfasalazine) and pyrazolones (azapropazone, beripipret (bezpiperylon), feprazone, mofebuzone, oxybuprazone, phenylbutazone). Other combinations include cyclooxygenase-2 (COX-2) inhibitors.

Other active agents for use in combination include steroids such as prednisolone, prednisone, methylprednisolone, betamethasone, dexamethasone, or hydrocortisone. Such combinations may be particularly advantageous because one or more side effects of steroids may be reduced or even eliminated by gradually reducing the required steroid dose when treating a patient in combination with an IL-10 polypeptide of the invention.

Additional examples of active agents for use in combination therapy, such as rheumatoid arthritis, include cytokine inhibitory anti-inflammatory drugs (CSAIDs); antibodies or antagonists against other human cytokines or growth factors such as TNF, LT, IL-1 β, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF or PDGF.

Specific combinations of active agents can interfere at different points in the autoimmune and subsequent inflammatory cascades, and include TNF antagonists such as chimeric, humanized or human TNF antibodies, REMICADE, anti-TNF antibody fragments (e.g., CDP870) and soluble p55 or p75TNF receptor, derivatives thereof, p75TNFRIgG (ENBREL) or p55TNFR1gG (LENERCEPT), soluble IL-13 receptor (sIL-13), and also TNF α converting enzyme (TACE) inhibitors; similarly, an IL-1 inhibitor (e.g., an interleukin-1 converting enzyme inhibitor) may be effective. Other combinations include interleukin 11, anti-P7, and P-selectin glycoprotein ligand (PSGL). Other examples of agents for use in combination with the IL-10 polypeptides described herein include interferon beta 1a (avonex); interferon β 1b (betaseron); (ii) copaxone; high pressure oxygen; intravenous injection of immunoglobulin; cladribine (clavibine); and antibodies or antagonists against other human cytokines or growth factors (e.g., antibodies against CD40 ligand and CD 80).

The present disclosure includes pharmaceutically acceptable salts, acids or derivatives of any of the above agents.

Viral diseases.The present disclosure provides methods for treating and/or preventing viral diseases, disorders, and conditions, and disorders related thereto, utilizing an IL-10 polypeptide (e.g., PEG-IL-10) with at least one additional therapeutic or diagnostic agent (e.g., one or more additional antiviral agents and/or one or more additional non-viral agents).

Such combination therapies include antiviral agents that target various viral life cycle stages and have different mechanisms of action, including, but not limited to, the following: inhibitors of viral uncoating (e.g., amantadine and rimantadine); reverse transcriptase inhibitors (e.g., acyclovir, zidovudine, and lamivudine); agents targeting integrase; agents that prevent the attachment of transcription factors to viral DNA; agents that affect translation (e.g., antisense molecules) (e.g., fomivirsen); agents that modulate translation/ribozyme function; a protease inhibitor; a viral assembly modulator (e.g., rifampin); and agents that prevent release of the viral particles (e.g., zanamivir and oseltamivir). The treatment and/or prevention of certain viral infections (e.g., HIV) often requires a panel ("cocktail") of antiviral agents.

Other antiviral agents contemplated for use in combination with the IL-10 polypeptide include, but are not limited to, the following: abacavir, adefovir, amantadine, amprenavir, abidol, atazanavir, ritriptan (atripla), boceprevirrette, cidofovir, cobivir (combivir), darunavir, delavirdine, didanosine, icosandiol, edexuridine, efavirenz, emtricitabine, emfuvirdi, entecavir, famciclovir, fosamprenavir, foscarnet sodium, ganciclovir, ibacitabine, isoprinosine (imuvir), idoxuridine, imiquimod, indinavir, inosine, various interferons (e.g., peginterferon alpha-2 a), lopinavir, lovenamine, malavir, morpholine, metsaxazone, nelfinavir, nevirapine, bud, sarsa (nevirar), penciclovir, lamivuvir, letavir, letivir, letamivir, picavir, vallisinovir, vallisoprid, vallisinovir, valacilin, foscarnet, valacilin, foscamitabine, valaciclovir, Pyrimidine, saquinavir, stavudine, telaprevir, tenofovir, tiravir, trifluridine (trifluridine), triazivir, acetamantane, teluvada, valacyclovir, valganciclovir, vickviro (vicriviroc), vidarabine, talivirine (viramidine) and zalcitabine.

The present disclosure includes pharmaceutically acceptable salts, acids or derivatives of the above agents.

Administration of drugs

The IL-10 polypeptides of the disclosure can be administered to a subject in an amount that depends on, for example, the purpose of the administration (e.g., the degree of regression desired); the age, weight, sex, and health and physical condition of the subject to whom the formulation is to be administered; the route of administration; and the nature of the disease, disorder, condition, or symptom thereof. The dosing regimen may also take into account the presence, nature and extent of any adverse effects associated with the agent to be administered. The amount of an effective dose and dosage regimen can be readily determined from, for example, safety and dose-escalation assays, in vivo studies (e.g., animal models), and other methods known to those of ordinary skill in the art.

The present disclosure encompasses administration of IL-10 to achieve a certain serum trough concentration and/or to maintain a certain mean serum trough concentration. Methods specific for IL-10 are described elsewhere herein and in this section below.

Generally, the dosing parameters specify that the dose amount is less than the amount that would have irreversible toxicity to the subject (i.e., the maximum tolerated dose, "MTD") and is not less than the amount required to produce a measurable effect on the subject. Such amounts are determined by, for example, pharmacokinetic and pharmacodynamic parameters associated with ADME, by consideration of the route of administration and other factors.

An Effective Dose (ED) is the dose or amount of an agent that produces a therapeutic response or desired therapeutic effect in a portion of the subject to whom it is administered. The "half effective dose" or ED50 of an agent is the dose or amount of the agent that produces a therapeutic response or desired therapeutic effect in 50% of the population to which the agent is administered. While ED50 is often used as a reasonably expected measure of the therapeutic efficacy of an agent, it is not necessarily the dose that a clinician might consider appropriate given all relevant factors. Thus, in some cases, the effective amount is above the calculated ED50, in other cases the effective amount is below the calculated ED50, and in other cases the effective amount is the same as the calculated ED 50.

In addition, an effective dose of an IL-10 polypeptide of the disclosure can be an amount that, when administered to a subject in one or more doses, produces a desired result relative to a healthy subject. For example, for a subject experiencing a particular disorder, an effective dose can be a dose that improves a diagnostic parameter, metric, marker, etc. of the disorder by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, where 100% is defined as the diagnostic parameter, metric, marker, etc. exhibited by normal subjects.

The amount of PEG-IL-10 necessary to treat the diseases, disorders, or conditions described herein is based on the IL-10 activity of the conjugated protein, which can be determined by IL-10 activity assays known in the art. For example, in a tumor context, suitable IL-10 activity includes, for example, infiltration of CD8+ T cells into the tumor site, expression of inflammatory cytokines such as IFN- γ, IL-4, IL-6, IL-10, and RANK-L from these infiltrating cells, and elevated levels of IFN- γ in a biological sample.

A therapeutically effective amount of an IL-10 agent may range from about 0.01 to about 100 μ g protein/kg body weight/day, from about 0.1 to 20 μ g protein/kg body weight/day, from about 0.5 to 10 μ g protein/kg body weight/day, or from about 1 to 4 μ g protein/kg body weight/day. In some embodiments, the IL-10 agent is administered by continuous infusion to deliver about 50 to 800 μ g protein/kg body weight/day (e.g., about 1 to 16 μ g protein/kg body weight/day of the IL-10 agent). The rate of infusion may vary depending on, for example, adverse effects and assessment of blood counts.

For administration as an oral dosage, the composition may be provided in the form of tablets, capsules, and the like containing 1.0 to 1000 milligrams of the active ingredient, specifically 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient.

Specific dosing regimens (e.g., frequency of dosing) for IL-10 polypeptides are described elsewhere herein.

In certain embodiments, the doses of the disclosed IL-10 polypeptides are contained in "unit dosage forms". The phrase "unit dosage form" refers to physically discrete units, each unit containing a predetermined amount of an IL-10 polypeptide of the present disclosure, alone or in combination with one or more additional pharmaceutical agents, sufficient to produce the desired therapeutic effect. It will be appreciated that the parameters of the unit dosage form will depend on the particular agent and the therapeutic effect to be achieved.

Medicine box

The present disclosure also encompasses kits comprising IL-10 and pharmaceutical compositions thereof. The kit is generally in the form of a physical structure containing the various components as described below, and can be used, for example, to practice the methods described above (e.g., administration of an IL-10 polypeptide to a subject in need of restoration of cholesterol homeostasis).

The kit can include one or more of the IL-10 polypeptides disclosed herein (provided, e.g., in a sterile container), which can be in the form of a pharmaceutical composition suitable for administration to a subject. The IL-10 polypeptide can be in a ready-to-use form or in a form that requires, e.g., reconstitution or dilution prior to administration. Where the IL-10 polypeptide is in a form that requires reconstitution by a user, the kit may further include buffers, pharmaceutically acceptable excipients, and the like packaged together with or separately from the IL-10 polypeptide. When combination therapy is contemplated, the kit may contain several agents separately or they may have been combined in a kit. Each component of the kit may be packaged in a single container, and all of the various containers may be in a single package. Kits of the present disclosure can be designed to properly maintain the conditions necessary for the components contained therein (e.g., refrigeration or freezing).

The kit may contain a label or package insert including identifying information about the components therein and instructions for their use (e.g., dosage parameters for the active ingredient, clinical pharmacology, including mechanism of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.). The label or insert may include manufacturer information such as lot number and expiration date. The label or package insert can be, for example, integrated within the physical structure containing the components, contained separately within the physical structure, or affixed to a component of the kit (e.g., an ampoule, tube, or vial).

The label or insert may further comprise or be incorporated in a computer readable medium such as a magnetic disk (e.g., hard disk, card, memory disc), an optical disk such as CD-or DVD-ROM/RAM, DVD, MP3, a magnetic tape, or an electronic storage medium such as RAM and ROM, or a hybrid of such media such as magnetic/optical storage media, FLASH media, or memory type cards. In some embodiments, the actual instructions are not present in the kit, but provide a means for obtaining the instructions from a remote source, e.g., via the internet.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments that may be performed. It is to be understood that the exemplary description, which is written now, need not be made, but rather the description can be made to produce the data and the like described herein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius (° c), and pressure is at or near atmospheric. Standard abbreviations are used, including the following: bp is base pair; kb is kilobase; pl to picoliter; s or sec is seconds; min is minutes; h or hr-hr; aa ═ amino acids; kb is kilobase; nt-nucleotide; ng equals nanogram; μ g to μ g; mg ═ mg; g is gram; kg is kg; dL or dL-deciliter; μ L or μ L ═ microliter; mL or mL ═ mL; l or L ═ liter; μ M to micromolar; mM ═ millimole; m is mole; kDa ═ kilodaltons; i.m. intramuscularly (di); i.p. ═ intraperitoneally (di); i.v. or IV ═ intravenously (di); s.c or SC subcutaneous (earth); QD is once daily; BID twice daily; once per week QW; QM is once a month; HPLC ═ high performance liquid chromatography; BW is body weight; u is a unit; ns is not statistically significant; PBS ═ phosphate buffered saline; PCR ═ polymerase chain reaction; NHS ═ N-hydroxysuccinimide; DMEM (Dulbeco's Modification of Eagle's Medium); GC-genomic copy; EDTA is ethylenediaminetetraacetic acid.

Materials and methods

The following general materials and methods may be used in the examples below:

standard methods in Molecular Biology are described (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Ausubel, et al (2001) Current Protocols in Molecular Biology, Vol.1-4, John Wiley and Sons, Inc. New York, N.Y., which describe Cloning and DNA mutagenesis in bacterial cells (Vol.1), Cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol.3), and bioinformatics (Vol.4)).

The scientific literature describes methods for Protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins and glycosylation of proteins (see, e.g., Coligan, et al (2000) Current Protocols in Protein Science, Vol.1-2, John Wiley and Sons, Inc., NY).

The generation, purification and fragmentation of polyclonal and monoclonal Antibodies is described (e.g., Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan et al (2001) Current Protocols in Immunology, vol 4, John Wiley, inc., NY); methods for flow Cytometry, including Fluorescence Activated Cell Sorting (FACS), are available (see, e.g., Shapiro (2003) practical flow Cytometry, John Wiley and Sons, Hoboken, NJ); and fluorescent reagents suitable for modifying nucleic acids (including nucleic acid primers and Probes), polypeptides and antibodies (for use as, for example, diagnostic reagents) are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see, e.g., Louis et al (2002) basic Histology: Text and Atlas, McGraw-Hill, New York, N.Y.).

Immune cell (CD 4)⁺And CD8⁺T cells) can be affected by antibody-mediated elimination. For example, 250 μ g of CD 4-or CD 8-specific antibody was injected weekly and cell depletion was verified using FACS and IHC analysis.

Software packages and databases for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments are available (see, e.g., the GCG Wisconsin software package (Accelrys, Inc., San Diego, Calif.); and DeCypher;)^TM(TimeLogic Corp.,Crystal Bay,NV)。

Immunocompetent Balb/C or B cell deficient Balb/C mice were obtained from The Jackson Lab., Bar Harbor, ME and used according to standard methods (see, e.g., Martin et al (2001) feed. Immun.,69(11):7067-73 and Compton et al (2004) Comp. Med.54(6): 681-89). Other mouse strains suitable for experimental work encompassed by The present disclosure are known to those of ordinary skill in The art and are generally available from The Jackson Lab.

Unless otherwise indicated, PDV6 squamous cell carcinoma of skin was used in the experiments described herein (see, e.g., Langowski et al (2006) Nature442: 461-465). Other oncology-related models and cell lines may be used, such as the Ep2 breast cancer, CT26 colon cancer, and 4T1 breast cancer models (see, e.g., Langowski et al (2006) Nature442:461-465), and are known to those of ordinary skill in the art. Non-oncology related models and cell lines (e.g., models of inflammation) can also be used and are known to those of ordinary skill in the art.

Serum IL-10 concentration levels and exposure levels can be determined by standard methods used in the art. For example, IL-10 exposure levels can be determined by collecting whole blood (. about.50. mu.l/mouse) from mouse tail snips into flat capillaries, separating serum from blood cells by centrifugation, and by standard ELISA kits and techniques. Additional methods for determining IL-10 serum concentrations are described below.

Production of PEGylated IL-10

The present disclosure encompasses the synthesis of pegylated IL-10 by any method known to one of ordinary skill in the art. The following description of several alternative synthesis schemes for generating mixtures of mono-PEG-IL-10 and mono/di-PEG-IL-10 is intended to be illustrative only. While mixtures of mono-PEG-IL-10 and mono/di-PEG-IL-10 have many comparable properties, mixtures of selectively pegylated mono-and di-PEG-IL-10 improve the yield of the final pegylated product (see, e.g., U.S. patent No. 7,052,686 and U.S. patent publication No. 2011/0250163).

In addition to utilizing her own skills to produce and use PEGs (and other drug delivery technologies) suitable for use in practicing the present disclosure, one of ordinary skill in the art is also familiar with many commercial suppliers of PEG-related technologies (and other drug delivery technologies). For example, NOF America Corp (Irvine, CA) provides monofunctional linear PEG, bifunctional PEG, multi-arm PES, branched PEG, heterofunctional PEG, branched PEG, and releasable PEG; and Parchem (New Rochelle, NY) is a global distributor of PEG products and other specialty raw materials.

Exemplary PEG-IL-10 synthetic scheme No. 1.IL-10 can be at pH 7.0,100mM NaCl against 10mM sodium phosphate dialysis. Dialyzed IL-10 can then be diluted 3.2-fold to a concentration of 4mg/mL using dialysis buffer. Prior to addition of linker SC-PEG-12K (Delmar Scientific Labs; Maywood, IL), 1 volume of 100mM sodium tetraborate pH9.1 may be added to 9 volumes of diluted IL-10 to raise the pH of the IL-10 solution to 8.6. The SC-PEG-12K linker can be dissolved in dialysis buffer and an appropriate volume can be addedLinker solution (1.8 to 3.6 moles linker per mole IL-10) was added to the diluted IL-10 solution to initiate the pegylation reaction. The reaction can be carried out at5 ℃ to control the reaction rate. The reaction solution may be gently stirred during the pegylation reaction. When the mono-PEG-IL-10 yield (as determined by size exclusion HPLC (SE-HPLC)) approaches 40%, the reaction was stopped by adding 1M glycine solution to a final concentration of 30 mM. The pH of the reaction solution was slowly adjusted to 7.0 using HCl solution, then the reaction solution was filtered using a 0.2 micron filter and stored at-80 ℃.

Exemplary PEG-IL-10 synthetic scheme No. 2.MonoPEG-IL-10 was prepared using methoxy-PEG-aldehyde (PALD-PEG) as a linker (Inhale Therapeutic Systems Inc., Huntsville, AL; also available from NOF America Corp (Irvine, Calif.)). The PALD-PEG may have a molecular weight of 5kDa, 12kDa or 20 kDa. IL-10 was dialyzed and diluted as described above except that the pH of the reaction buffer was 6.3 to 7.5. The activated PALD-PEG linker was added to the reaction buffer at a molar ratio of 1: 1. Aqueous cyanoborohydride is added to the reaction mixture to achieve a final concentration of 0.5 to 0.75 mM. The reaction was carried out at room temperature (18-25 ℃) by gentle stirring for 15-20 hours. The reaction was quenched with 1M glycine. The yield was analyzed by SE-HPLC. Single PEG-IL-10 was separated from unreacted IL-10, PEG linker and di-PEG-IL-10 by gel filtration chromatography and characterized by RP-HPLC and bioassay (e.g., stimulation of IL-10-reactive cells or cell lines).

Exemplary PEG-IL-10 synthetic scheme No. 3.IL-10 (e.g., rodent or primate) was dialyzed against 50mM sodium phosphate, 100mM sodium chloride (pH in the range of 5-7.4). 5K PEG-propionaldehyde was reacted with IL-10 at a concentration of 1-12mg/mL in a molar ratio of 1:1-1:7 in the presence of 0.75-30mM sodium cyanoborohydride. Alternatively, the reaction can be activated with picoline borane in a similar manner. The reaction was incubated at 5-30 ℃ for 3-24 hours.

The pH of the PEGylation reaction was adjusted to 6.3, and 7.5mg/mL of hIL-10 was reacted with PEG such that the ratio of IL-10 to PEG linker was 1: 3.5. The final concentration of cyanoborohydride was-25 mM, and the reaction was carried out at 15 ℃ for 12-15 hours. Mono-and di-PEG IL-10 are the most reaction products, with concentrations of each at the end of the run of-45-50%. The reaction may be quenched using an amino acid such as glycine or lysine or alternatively a Tris buffer. A variety of purification methods, such as gel filtration, anion and cation exchange chromatography, and size exclusion HPLC (SE-HPLC), can be used to isolate the desired PEGylated IL-10 molecules.

Exemplary PEG-IL-10 synthetic scheme No. 4.IL-10 was dialyzed against 10mM sodium phosphate pH 7.0,100mM NaCl. Dialyzed IL-10 was diluted 3.2-fold to a concentration of about 0.5 to 12mg/mL using dialysis buffer. Prior to the addition of linker SC-PEG-12K (Delmar Scientific Laboratories, Maywood, Ill.), 1 volume of 100mM sodium tetraborate, pH9.1, was added to 9 volumes of diluted IL-10 to raise the pH of the IL-10 solution to 8.6. The SC-PEG-12K linker was dissolved in dialysis buffer and an appropriate volume of linker solution (1.8 to 3.6 moles linker per mole IL-10) was added to the diluted IL-10 solution to initiate the pegylation reaction. The reaction was carried out at5 ℃ to control the reaction rate, and the reaction solution was gently stirred. When the mono-PEG-IL-10 yield (as determined by size exclusion HPLC (SE-HPLC)) approaches 40%, the reaction was stopped by adding 1M glycine solution to a final concentration of 30 mM. The pH of the reaction solution was slowly adjusted to 7.0 using HCl solution and the reaction was filtered using a 0.2 micron filter and stored at-80 ℃.

The materials shown below, including table 15 and the descriptions thereof, are essentially abstracted from U.S. patent publication No. 2011/00911419 (the co-inventor of U.S. patent publication No. 2011/00911419 is also the inventor of the present application), and the teachings therein and variations thereof have broad applicability and can be used in and/or modified in a number of different contexts. Similarly, the teachings of other publications in the related and/or technical fields (see, e.g., USP nos. 6,387,364 and 7,052,684, and PCT publication No. WO 2006/075138), together with the general knowledge of one of ordinary skill in the art, can form the basis for additional experimental work.

Tumor model and tumor analysis

Any field accepted tumor models, assays, etc. can be used to assess the effects of IL-10 and PEG-IL-10 on various tumors. The tumor models and tumor analyses described below are representative of those that can be utilized and used to generate and evaluate the data shown in tables 1-15.

At 10⁴、10⁵Or 10⁶Individual cell/tumor inoculation tumor cells from syngeneic mice were injected subcutaneously or intradermally. Ep2 breast cancer, CT26 colon cancer, PDV6 squamous cell carcinoma of the skin, and 4T1 breast cancer models can be used (see, e.g., Langowski et al (2006) Nature442: 461-465). Immunocompetent Balb/C or B-cell deficient Balb/C mice may be used. PEG-mIL-10 can be administered to immunocompetent mice, however PEG-hIL-10 treatment can be used in B cell deficient mice. Make the tumor reach 100-250mm³Then the process is started. IL-10, PEG-mIL-10, PEG-hIL-10 or buffer control were administered subcutaneously at sites distant from tumor implantation. Tumor growth is typically monitored 2 times per week using electronic calipers.

Tumor tissue and lymphoid organs were harvested at different endpoints to measure mRNA expression of a number of inflammatory markers and immunohistochemistry for several inflammatory cell markers was performed. Tissues were snap frozen in liquid nitrogen and stored at-80 ℃. Primary tumor growth is typically monitored 2 times per week using electronic calipers. Can use the formula (Wide)²x long/2) the tumor volume was calculated, where length is the longer dimension. Make the tumor reach 90-250mm³Then the process is started.

Administration of IL-10 and/or PEG-IL-10

The tumor models and tumor analysis methods described above were used to generate the data shown below. However, as alluded to above, these same models and methodologies may be used in other experimental settings.

To immune activity of mice were administered mouse IL-10(mIL-10) or PEG-mIL-10, however, the PEG-hIL-10 treatment for B cell deficient mice. Murine IL-10, PEG-mIL-10, PEG-hIL-10, or vehicle control were administered subcutaneously at sites distant from tumor implantation. The SC-PEG-12K linker was used to prepare the PEG-mIL-10 used in these studies. The biological activity of mIL-10 and PEG-mIL-10 was assessed by using a short-term proliferation bioassay using MC/9, a mouse mast cell line expressing the endogenous mIL-10 receptor (R1 and R2). MC/9 cells proliferate in response to co-stimulation with mIL-4 and mIL-10 (MC/9 cells do not proliferate using mIL-4 or mIL-10 alone). Proliferation was measured by colorimetry using Alamar Blue (Alamar Blue), a growth indicator dye based on the detection of metabolic activity. The biological activity of recombinant or PEG-mIL-10 was assessed by EC50 values, or the concentration of protein at which half maximal stimulation was observed in the dose-response curve (table 1).

TABLE 1

As indicated in Table 1, the specific activity of PEG-mIL-10 used in the experiments was approximately 1/7 that is the activity of mIL-10, based on the MC/9 bioassay.

PEG-mIL-10 was also administered every other day to Ep2 breast cancer bearing mice. The treatment is effective in reducing tumor size and inducing tumor rejection.

TABLE 2

Treatment with PEG-mIL-10 was also effective in reducing tumor size in the tumor models of PDV6, CT-26, and 4T1 syngeneic immunocompetent mice (see tables 3,4, and 5).

TABLE 3

TABLE 4

TABLE 5

Dose titration study

In dose titration studies, tail vein bleeds were collected from representative mice of each group at times corresponding to expected peak and trough dose levels. Harvested sera were assayed for mIL-10 concentration using a Meso Scale Discovery platform (combination of electrochemiluminescence detection and patterned array) based on multi-array technology. Two-tailed unpaired student's t-test was used to compare the mean tumor volume of mIL-10 or PEG-mIL-10 treated mice, grouped by serum mIL-10 concentration, to their corresponding vehicle control group. Welch correction was used when two groups had unequal variance (t-test with p < 0.05).

Dose titration of PEG-mIL-10 and mIL-10 in mice with 4T1 breast cancer showed that control of primary tumors and lung metastases was dose titratable with both mIL-10 and PEG-mIL-10. As shown in Table 6, PEG-mIL-10 is more effective than mIL-10 at any given dose. Twice daily treatment was started on day 17 after implantation, at which time the mean tumor volume was 84-90mm³. The treatment group consisted of 14 mice per group, while the control group had 8 mice per group. Tris and Hepes buffers are controls for mIL-10 and PEG mIL-10, respectively.

TABLE 6

Dose titration of PEG-mIL-10 and mIL-10 in mice with PDV6 squamous cell carcinoma indicated that control of primary tumors was dose titratable with both mIL-10 and PEG-mIL-10, although PEG-MIL-10 was more effective than mIL-10 at any given dose (Table 7). High dose PEG-mIL-10 treatment resulted in near 100% tumor regression and subsequent resistance to rechallenge (table 8). Treatment 2 times daily was started on day 23 post-implantation, at which time the mean tumor volume was 107-109mm³And for all mIL10 treatment groups and 0.01mg/kg PEG mIL-10 treatment group continued until day 55. 0.1mg/kg PEG-mIL-10 treatment was terminated on day 48, at which time 100% tumor regression was seen, whereas the remaining groups were treated until day 51. The treatment group consisted of 10 mice per group, while each vehicle control contained 6 mice. Tris buffer and Hepes buffer are vehicle controls for mIL-10 and PEG mIL-10, respectively. Reimplantation was performed 85 days after the initial implantation and 4 weeks after the last PEG-mIL10 treatment. Each group contained 10 mice.

TABLE 7

TABLE 8

Lung metastasis study

Post-lung resection (table 9) by visual inspection or as described in Current Protocols in Immunology (section 20.2.4) John Wiley and Sons, inc, New York; lung metastases in the 4T1 breast cancer model were quantified by counting lung metastatic colonies after culture (table 10) as described in Harlow and Lane (1999). Briefly, lungs harvested from 4T1 tumor-bearing mice were minced and digested with collagenase/elastase, then cultured in media containing 6-thioguanine in a limiting dilution assay. Only 4T1 cells were 6-thioguanine resistant and were passable at 10Quantification was done by counting colonies after 14 days of culture. Twice daily treatment was started on day 17 after implantation, at which time the mean tumor volume was 84-90mm³. Tris and Hepes buffers are controls for mIL-10 and PEGmIL-10, respectively. Lung metastases were measured as the number of metastatic colonies cultured per lung.

TABLE 9

Watch 10

Administration of PEG-mIL-10 or IL-10 to mice with 4T1 breast cancer reduced the rate of metastasis and increased CD8+ T cell infiltration and expression of immunostimulatory cytokines as measured by quantitative RT-PCR (tables 11 and 12). The number of infiltrating CD8+ T cells was counted from representative sections of several tumors stained by immunohistochemistry for CD8 surface marker and confirmed by staining with anti-CD 3 and anti-TCR α β antibody.

TABLE 11

PEG-mIL-10 is more potent than IL-10 in the induction of inflammatory cytokines. Total RNA was extracted from the homogenized tumor sample and reverse transcribed as previously described (see, e.g., Homey, et al (2000) J. Immunol.164: 3465-3470). Complementary DNA was quantitatively analyzed for cytokine expression by the fluorogenic 5' -nuclease PCR assay (see, e.g., Holland, et al (1991) Proc. Natl. Acad. Sci.88: 7276-7280). Specific PCR products were measured continuously in 40 cycles by ABI PRISM 7700 sequence detection System (Applied Biosystems). Values were normalized to ubiquitin proteins. The logarithmically transformed data were subjected to Kruskal-Wallis statistical analysis (median method). The expression level (log-transformed) corresponds to the amount of inflammatory cytokine expressed in the tumor sample, such that the higher the expression level (log-transformed), the greater the amount of inflammatory cytokine expressed in the tumor sample.

TABLE 12

Depletion of immune cells

CD4+ and CD8+ T cells were depleted by antibody-mediated depletion. For this purpose 250. mu.g of CD 4-or CD 8-specific antibody were injected weekly. Cell depletion was verified using FACS and IHC analysis.

Depletion of B cell deficient BALB/c mice (C.129-Igh-6) Using CD4 antibody^tmlCgn) The CD4+ T cells in (C) inhibited the function of PEG-hIL-10 on tumors (Table 13).

Watch 13

Depletion of CD8+ T cells completely inhibited the effect of PEG mIL-10 on syngeneic tumor growth (table 14).

TABLE 14

Dosing frequency and serum trough concentration of IL-10

Murine studies were designed and conducted to enhance understanding of pharmacokinetic parameters of IL-10 therapy and to generate data in mice for optimizing tumor treatment regimens for recombinant human IL-10(rhIL-10) in human subjects.

Mice were inoculated with PDV6 tumor cells and tumors were grown for 2.5 weeks to reach 100mm³. Groups of tumor-bearing mice (n 10/group) were subsequently treated with the same weekly dose (0.7 mg/kg/week) with a) one bolus SC injection once weekly, or b) several SC injections in divided doses throughout the week, including 2 (0.35mg/kg) weekly, every other day (-0.25 mg/kg, so that the total weekly dose is 0.7mg/kg) and daily (0.1 mg/kg/day), administered with 5kDa mono-di-pegmigl-10. Similar total exposure (area under the curve, AUC) was observed when all mice received the same amount of drug over the course of one week. Peak exposure was highest in mice dosed once a week, whereas the minimum drug exposure (trough) was highest in mice receiving smaller daily doses. Surprisingly, as indicated in table 15, the mice dosed daily exhibited the highest antitumor efficacy, indicating that serum trough exposure is very important for antitumor function, whereas peak exposure is not crucial for antitumor function.

Watch 15

Dosing regimens
		Control	813.9522
Daily	43.196
		Every other day	170.186
Every 2 weeks	347.315
		Every week	425.572

In two tumor models: the required serum trough concentrations were further probed in PDV6 tumor in C57BL/6 mice and CT26 colon cancer cells in Balb/C mice. Mice were grown to 100mm using standard methods³Treatment was then initiated by administration of 5kDa mono-di PEGmIL-10 for 4 weeks. Subsequently, serum trough concentrations of IL-10 were measured in tumor-bearing mice that received different treatment protocols. IL-10 serum trough concentrations were then correlated with the resulting tumor size. As indicated in table 16, mice with serum trough IL-10 above 1ng/mL had persistent small tumors and rejected their tumors.

TABLE 16

To confirm critical trough concentrations in human cells, hIL-10 was added to cultures of human Peripheral Blood Mononuclear Cells (PBMCs) at increasing concentrations. PBMC cultures were not treated or cultures were stimulated with Lipopolysaccharide (LPS). IL-10 is known to inhibit LPS-mediated activation of PBMCs. The activity was measured as secretion of the chemokine MCP-1. Both LPS and IL-10 induced secretion of MCP-1, but inhibited each other's chemokine-inducing activity. IL-10 increased MCP-1 secretion in the absence of LPS at concentrations of 1ng/mL and above (FIG. 2A). In contrast, in PBMC stimulated with LPS, the addition of IL-10 at a concentration of 1ng/mL significantly inhibited MCP-1 secretion (FIG. 2B). This confirms the biological activity of IL-10 to induce and inhibit the respective biological processes.

Effect of IL-10 on cytokines and cholesterol in human subjects

Determination of serum IL-10 concentration in human subjects.Application to human volunteersAn amount of rhIL-10SC or IV, and drawing the whole blood sample into the heparin anticoagulant-containing tube at the desired post-administration time. Serum rhIL-10 or PEG-rhIL-10 concentrations were determined using standard sandwich enzyme-linked immunosorbent assay (ELISA) kits. Typically, the ELISA assay is determined to be selective, linear and reproducible over a concentration range of 0.1 to 10ng/mL, and the limit of quantitation (LOO) is 0.1 ng/mL. Serum samples were also analyzed by ELISA for the presence of antibodies that bind to hIL-10. In addition, selected serum samples were analyzed using a validated bioassay (containing the mouse mast cell line MC 9); the cell line proliferates in response to IL-10. This bioassay is used to measure the bioactivity of GMP-produced rHuIL-10 and PEG-rHuIL-10 and to measure the bioactivity of IL-10 in patient sera. Typically, ELISA and bioassay measurements of IL-10 concentration and activity show corresponding values.

Determination of TNF α and IL-1 β concentrations in human subjects.IL-10 has anti-inflammatory function in patients with chronic inflammatory disease, and TNF α and IL-1 β represent key inflammatory cytokines released in such disease.A concentration of TNF α and IL-1 β in a blood sample obtained from a human subject is determined.typically, 3mL of venous blood are aseptically collected at about 0 hours when the SC or IV is administered rhIL-10 and at 0.5, 2,3,4, 6, 8, 12, 16, 24, 48, 72, and 96 hours post-dose.the sample is subjected to a cytokine release assay in the presence of LPS and an anticoagulant, and TNF α and IL-1 β concentrations are measured using an ELISA assay.LPS stimulates the release of TNF α and IL-1 β from blood cells.

In samples collected 0.5-12 hours after IV administration of subject with rHuIL-10, the release of TNF α and IL-1 β was inhibited. In samples collected from subjects dosed with rHuIL-10SC, TNF α and IL-1 β release was inhibited from 0.5 hours to 24 hours. Serum concentrations of rHuIL-10 in these human subjects were determined by ELISA. The inhibition of TNF α and IL-1 β correlated with the serum concentration of rHuIL-10. Serum concentrations of rHuIL-10 increased and remained elevated for 48 hours post-dose. However, TNF α and IL-1 β release was inhibited only at concentrations of rHuIL-10 of 0.2ng/mL or above 0.2 ng/mL; when the concentration is less than 0.1ng/mL, the release of TNF α and IL-1 β is not inhibited. After 12 hours post IV administration of rHuIL-10 and 24 hours post SC administration, serum concentrations dropped below 0.2ng/mL and release of TNF α and IL-1 β was observed. These data indicate that in order to observe anti-inflammatory function in patients with chronic inflammatory disease, a serum trough concentration of IL-10 of 0.2ng/mL or higher than 0.2ng/mL must be achieved.

Assay of PEG-IL-10 modulation of INF γ and cholesterol in human cancer patients.IL-10 induces IFN γ in CD8+ T cells, and IFN γ induction is essential for IL-10 mediated tumor rejection in mice. When treated with PEG-rmIL-10 at concentrations that induce tumor regression in control mice, IFN γ -deficient mice failed to reject their tumors (data not shown). Thus, IFN γ was measured in serum from patients treated with PEG-rhIL-10.

Cancer patients had been SC injected with different doses of PEG-rhIL-10 daily after education with appropriate administration techniques. Serum IL-10 concentrations were determined using a sandwich ELISA as described previously. Serum samples taken before the first dose or after 28 days of dosing were measured for IFN γ using the Luminex bead assay (Luminex corp.; Austin, TX).

As indicated in Table 17, patients receiving 1. mu.g/kg PEG-IL-10 had serum trough levels of IL-10 from 0.4 to 1.1ng/mL, whereas patients receiving a 2.5. mu.g/kg PEG-IL-10 dose had serum trough levels of IL-10 from 0.4 to 2.6 ng/mL.

IFN γ is primarily signaled via the Jak-Stat pathway. Jak-Stat signaling involves the recruitment of a cascade of receptors and activation of members of the Janus kinase family (Jak: Jak 1-3 and Tyk2) and Stat kinase family (Stat 1-6, including Stat5a and Stat5b) to control transcription of target genes by specific response elements. Because this signaling mechanism is characteristic of many members of the cytokine receptor superfamily, IFN γ -induced Jak-Stat signaling is an example of current class II cytokine receptor signaling. As indicated in table 17, patients with serum trough levels of 1ng/mL or greater showed induction of IFN γ in serum, whereas patients with serum trough levels below 1ng/mL failed to show induction of IFN γ. Referring to table 17, IFN γ induction was defined as a value greater than 1.

TABLE 17

These data indicate that in order to observe a therapeutic effect in a cancer/tumor setting, a serum trough concentration of IL-10 of 1ng/mL or above 1ng/mL must be achieved. Importantly, serum trough concentrations rather than dose levels are determinants of IFN γ induction.

Cholesterol in serum samples drawn from cancer patients was measured before administration of PEG-rhIL-10 or after 1 week of daily SC dosing (1 μ g/kg; 2.5 μ g/kg; or 5 μ g/kg; n ═ 3-4 patients/dose). Referring to Table 18, patients receiving 1 μ g/kg achieved a mean daily serum cholesterol concentration of 0.4ng/mL and had a reduction in cholesterol of 7.8%; patients receiving 2.5 μ g/kg achieved a mean daily serum cholesterol concentration of 1ng/mL and had a 19% reduction in cholesterol; and patients receiving 5 μ g/kg achieved a mean serum cholesterol trough concentration of 2ng/mL and had a 38% reduction in cholesterol. Thus, each dosing regimen resulted in a treatment-related reduction in serum cholesterol, indicating that a mean IL-10 serum trough concentration of about 0.2ng/mL to 0.4ng/mL was effective.

Watch 18

Dosage form	1ug/kg	2.5	5
				n	4	4	3
Mean serum valley (day 15)	0.4	1.8	3.6
				Mean cholesterol reduction (1 week)	7.8％	20％	37％

Specific embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the disclosed embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description, and it is contemplated that such variations may be utilized as appropriate by those of ordinary skill in the art. Accordingly, it is intended that the invention be practiced otherwise than as specifically described herein, and that the invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described components in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers and other references cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

The present application relates to the following embodiments.

1. A method of treating or preventing a disease, disorder, or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an IL-10 agent, wherein the amount is sufficient to obtain a mean IL-10 serum trough concentration of at least 0.1 ng/mL.

2. A method of treating or preventing a disease, disorder, or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an IL-10 agent, wherein the amount is sufficient to maintain a mean IL-10 serum trough concentration over a period of time;

wherein the mean IL-10 serum trough concentration is at least 0.1ng/mL, and

wherein the mean IL-10 serum trough concentration is maintained for at least 90% of the period of time.

3. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 1.5 ng/mL.

4. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 1.85 ng/mL.

5. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 2.0 ng/mL.

6. The method of any of embodiments 2-5 or 69-75 wherein the period of time is at least 12 hours.

7. The method of embodiment 6, wherein the period of time is at least 24 hours.

8. The method of embodiment 7, wherein the period of time is at least 48 hours.

9. The method of embodiment 8, wherein the period of time is at least 72 hours.

10. The method of embodiment 9, wherein the period of time is at least 1 week.

11. The method of embodiment 10, wherein the period of time is at least 2 weeks.

12. The method of embodiment 11, wherein the period of time is at least 1 month.

13. The method of any one of embodiments 2-12 or 69-75, wherein the mean IL-10 serum trough concentration is maintained for at least 95% of the period of time.

14. The method of embodiment 13, wherein the mean IL-10 serum trough concentration is maintained for at least 98% of the period of time.

15. The method of embodiment 14, wherein the mean IL-10 serum trough concentration is maintained for 100% of the period of time.

16. The method according to any one of embodiments 1-15, wherein the IL-10 agent is mature human IL-10.

17. The method according to any one of embodiments 1-15, wherein the IL-10 agent is a variant of mature human IL-10, and wherein the variant exhibits activity comparable to the activity of mature human IL-10.

18. The method according to any one of embodiments 1-17, wherein the disease, disorder, or condition is a proliferative disorder.

19. The method of embodiment 18, wherein the proliferative disorder is cancer.

20. The method of embodiment 19, wherein the cancer is a solid tumor or a hematological disorder.

21. The method according to any one of embodiments 1-17, wherein the disease, disorder or condition is an immune disorder or an inflammatory disorder.

22. The method of embodiment 21, wherein the immune or inflammatory disorder is selected from the group consisting of: inflammatory bowel disease, psoriasis, rheumatoid arthritis, multiple sclerosis and alzheimer's disease.

23. The method according to any one of embodiments 1-17, wherein the disease, disorder or condition is thrombosis or a thrombotic condition.

24. The method according to any one of embodiments 1-17, wherein the disease, disorder or condition is a fibrotic disorder.

25. The method of any one of embodiments 1-17, wherein the disease, disorder, or condition is a viral disorder.

26. The method of embodiment 25, wherein the viral disorder is selected from the group consisting of: human immunodeficiency virus, hepatitis b virus, hepatitis c virus and cytomegalovirus.

27. The method according to any one of embodiments 1-17, wherein the disease, disorder, or condition is a cardiovascular disorder.

28. The method of embodiment 27, wherein the cardiovascular disorder is atherosclerosis.

29. The method of embodiment 27 or 28, wherein the subject has elevated cholesterol.

30. The method of any one of embodiments 1-29, wherein the IL-10 agent comprises at least one modification to form a modified IL-10 agent, wherein the modification does not alter the amino acid sequence of the IL-10 agent.

31. The method of embodiment 30, wherein the modified IL-10 agent is a PEG-IL-10 agent.

32. The method of embodiment 31, wherein the PEG-IL-10 agent comprises at least one PEG molecule covalently attached to at least one amino acid residue of at least one subunit of IL-10.

33. The method of embodiment 31 or 32, wherein the PEG-IL-10 agent comprises a mixture of mono-pegylated and di-pegylated IL-10.

34. The method according to any one of embodiments 31-33, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of about 5kDa to about 20 kDa.

35. The method of any one of embodiments 31-33, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of greater than about 20 kDa.

36. The method of any one of embodiments 31-33, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of at least about 30 kD.

37. The method of embodiment 30, wherein the modified IL-10 agent is an Fc fusion molecule.

38. The method of embodiment 30, wherein the modified IL-10 agent comprises serum albumin.

39. The method of embodiment 38, wherein the serum albumin is Human Serum Albumin (HSA).

40. The method of embodiment 39, wherein the modified IL-10 agent is an HSA fusion molecule or an albumin conjugate.

41. The method of embodiment 30, wherein the modified IL-10 agent is glycosylated.

42. The method of embodiment 30, wherein the modified IL-10 agent is hydroxyethylated precipitated.

43. The method of embodiment 30, wherein the modified IL-10 agent comprises an Albumin Binding Domain (ABD).

44. The method of any one of embodiments 30-43, wherein the modification is site-specific.

45. The method of any one of embodiments 30-36, 38, and 39, wherein the modification comprises a linker.

46. The method according to any one of embodiments 1-45, wherein the subject is administered the IL-10 agent at least twice daily.

47. The method according to any one of embodiments 1-45, wherein the subject is administered the IL-10 agent at least once daily.

48. The method according to any one of embodiments 1-45, wherein the IL-10 agent is administered to the subject at least once every 72 hours.

49. The method according to any one of embodiments 1-45, wherein the subject is administered the IL-10 agent at least once weekly.

50. The method according to any one of embodiments 1-45, wherein the subject is administered the IL-10 agent at least once every 2 weeks.

51. The method according to any one of embodiments 1-45, wherein the subject is administered the IL-10 agent at least once a month.

52. The method of any one of embodiments 1-51, further comprising administering at least one additional prophylactic or therapeutic agent.

53. The method according to any one of embodiments 1-52, wherein the subject is a human.

54. The method according to any one of embodiments 1-53, wherein said administering is by parenteral injection.

55. The method according to embodiment 54, wherein the parenteral injection is subcutaneous injection.

56. The method according to any one of embodiments 1-55, wherein said treatment or prevention is mediated by CD8+ T cells.

57. A pharmaceutical composition comprising an amount of an IL-10 agent according to any one of embodiments 1 to 56 and a pharmaceutically acceptable diluent, carrier or excipient.

58. The pharmaceutical composition of embodiment 57, wherein the excipient is an isotonic injection solution.

59. The pharmaceutical composition of embodiment 57, wherein the composition is suitable for human administration.

60. The pharmaceutical composition according to any one of embodiments 57-59, further comprising at least one additional prophylactic or therapeutic agent.

61. A sterile container comprising the pharmaceutical composition of any one of embodiments 57-60.

62. The sterile container of embodiment 61, wherein said sterile container is a syringe.

63. A kit comprising the sterile container of embodiment 61 or embodiment 62.

64. The kit of embodiment 63, further comprising a second sterile container comprising at least one additional prophylactic or therapeutic agent.

65. The method of embodiment 1, wherein the amount is sufficient to obtain a mean IL-10 serum trough concentration of at least 0.5 ng/mL.

66. The method of embodiment 1, wherein the amount is sufficient to obtain a mean IL-10 serum trough concentration of at least 1.0 ng/mL.

67. The method of embodiment 1, wherein the amount is sufficient to obtain a mean IL-10 serum trough concentration of at least 1.5 ng/mL.

68. The method of embodiment 1, wherein the amount is sufficient to obtain a mean IL-10 serum trough concentration of at least 2.0 ng/mL.

69. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 0.2 ng/mL.

70. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 0.4 ng/mL.

71. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 0.6 ng/mL.

72. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 0.8 ng/mL.

73. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 1.0 ng/mL.

74. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 1.25 ng/mL.

75. The method of embodiment 2, wherein the mean IL-10 serum trough concentration is at least 1.75 ng/mL.

The present application also relates to the following embodiments.

1. A composition for use in a method of treating cancer in a human subject, wherein the method comprises administering to the human subject by injection a therapeutically effective amount of a PEG-IL-10 agent, wherein the amount is sufficient to maintain an IL-10 serum trough concentration in the human subject at or above 0.2ng/mL for a period of at least 24 hours, wherein the human subject is also treated with at least one therapeutic agent.

2. The composition for use according to embodiment 1, wherein the at least one therapeutic agent comprises 5-fluorouracil (5-FU).

3. The composition for use of embodiment 1, wherein the at least one therapeutic agent comprises a platinum complex.

4. The composition for use according to embodiment 1, wherein the at least one therapeutic agent comprises a folic acid analog.

5. The composition for use of embodiment 4, wherein the at least one therapeutic agent further comprises a platinum complex.

6. The composition for use according to embodiment 5, wherein the at least one therapeutic agent further comprises 5-fluorouracil (5-FU).

7. The composition for use according to any one of embodiments 1-6, wherein the PEG-IL-10 agent is administered to maintain an IL-10 serum trough concentration at or above 0.2ng/mL for a period of at least 2 weeks.

8. The composition for use according to any one of embodiments 1-6, wherein the PEG-IL-10 agent is administered to maintain an IL-10 serum trough concentration at or above 0.2ng/mL for a period of at least 1 month.

9. The composition for use according to any one of embodiments 1-6, wherein said PEG-IL-10 agent comprises mature human IL-10.

10. The composition for use according to any one of embodiments 1-6, wherein said PEG-IL-10 agent comprises a variant of mature human IL-10, and wherein said variant exhibits activity comparable to the activity of mature human IL-10.

11. The composition for use according to any one of embodiments 1-6, wherein the PEG-IL-10 agent comprises at least one PEG molecule covalently attached to at least one amino acid residue of at least one subunit of mature human IL-10.

12. The composition for use of embodiment 11, wherein the PEG-IL-10 agent comprises a mixture of mono-pegylated and di-pegylated IL-10.

13. The composition for use according to embodiment 12, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of about 5kDa to about 20 kDa.

14. The composition for use according to embodiment 13, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of about 5kDa to about 10 kDa.

15. The composition for use according to embodiment 13, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of about 5 kDa.

16. The composition for use according to any one of embodiments 1-6, wherein the PEG-IL-10 agent is administered to the subject once daily.

17. The composition for use of embodiment 16, wherein said administration is by subcutaneous injection.

18. The composition for use according to any one of embodiments 1-17, wherein the cancer is a cancer of the pancreas, kidney cells, or colon.

Use of a PEG-IL-10 agent in the manufacture of a medicament for use in a method of treating cancer in a human subject, wherein the method comprises administering to the human subject a therapeutically effective amount of a PEG-IL-10 agent, wherein the amount is sufficient to maintain IL-10 serum trough concentrations in the human subject at or above 0.2ng/mL for a period of at least 24 hours, wherein the human subject is also treated with at least one therapeutic agent.

20. The use of embodiment 19, wherein the at least one therapeutic agent comprises 5-fluorouracil (5-FU).

21. The use of embodiment 19, wherein the at least one therapeutic agent comprises a platinum complex.

22. The use according to embodiment 19, wherein the at least one therapeutic agent comprises a folic acid analog.

23. The use of embodiment 22, wherein the at least one therapeutic agent further comprises 5-fluorouracil (5-FU).

24. The use of embodiment 23, wherein the at least one therapeutic agent further comprises a platinum complex.

25. The use of embodiment 19, wherein the PEG-IL-10 agent is administered to maintain IL-10 serum trough concentrations at or above 0.2ng/mL for a period of at least 2 weeks.

26. The use of embodiment 19, wherein the PEG-IL-10 agent is administered to maintain IL-10 serum trough concentrations at or above 0.2ng/mL for a period of at least 1 month.

27. The use of embodiment 19, wherein the PEG-IL-10 agent comprises mature human IL-10.

28. The use of embodiment 19, wherein the PEG-IL-10 agent comprises a variant of mature human IL-10, and wherein said variant exhibits activity comparable to the activity of mature human IL-10.

29. The use of any one of embodiments 19-28, wherein the PEG-IL-10 agent comprises at least one PEG molecule covalently attached to at least one amino acid residue of at least one subunit of mature human IL-10.

30. The use of embodiment 29, wherein the PEG-IL-10 agent comprises a mixture of mono-pegylated and di-pegylated IL-10.

31. The use of embodiment 30, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of about 5kDa to about 20 kDa.

32. The use of embodiment 30, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of about 5kDa to about 10 kDa.

33. The use of embodiment 30, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of about 5 kDa.

34. The use of embodiment 30, wherein the PEG-IL-10 agent is administered to the subject once daily.

35. The use of embodiment 34, wherein the administration is by subcutaneous injection.

36. The use according to any one of embodiments 19-35, wherein the cancer is a cancer of the pancreas, kidney cells, or colon.

Claims (8)

1. A method of treating or preventing a disease, disorder, or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an IL-10 agent, wherein the amount is sufficient to obtain a mean IL-10 serum trough concentration of at least 0.1 ng/mL.

2. A method of treating or preventing a disease, disorder, or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an IL-10 agent, wherein the amount is sufficient to maintain a mean IL-10 serum trough concentration over a period of time;

wherein the mean IL-10 serum trough concentration is at least 0.1ng/mL, and

wherein the mean IL-10 serum trough concentration is maintained for at least 90% of the period of time.

3. The method of claim 1 or 2, further comprising administering at least one additional prophylactic or therapeutic agent.

4. A pharmaceutical composition comprising an amount of an IL-10 agent of any one of claims 1-3 and a pharmaceutically acceptable diluent, carrier or excipient.

5. The pharmaceutical composition of claim 4, further comprising at least one additional prophylactic or therapeutic agent.

6. A sterile container comprising the pharmaceutical composition of claim 4 or 5.

7. A kit comprising the sterile container of claim 6.

8. The kit of claim 7, further comprising a second sterile container comprising at least one additional prophylactic or therapeutic agent.