Fusion proteins comprising interleukin 10 and interleukin 4

Abstract

The present invention relates to a fusion protein comprising interleukin 10 and interleukin 4, a nucleic acid molecule encoding such a fusion protein, a vector comprising such a nucleic acid molecule, and a host cell comprising such a nucleic acid molecule or such a vector. The present invention further relates to a method for producing such a fusion protein. The fusion protein or a gene therapy vector encoding the fusion protein can be used for the prevention and / or treatment of osteoarthritis, chronic pain, a condition characterized by local or systemic inflammation, immune activation and / or lymphoid tissue proliferation.

Description

[0001] Case Analysis

[0002] This application is a divisional application of patent application number 201280066029.9, entitled "Fusion protein including interleukin 10 and interleukin 4". Technical Field

[0003] This invention belongs to the fields of immunology and pharmacology, and particularly relates to the treatment of osteoarthritis, chronic pain, inflammatory diseases or conditions, and related diseases and conditions. This invention particularly relates to novel fusion proteins comprising interleukin-4 (IL-4) and interleukin-10 (IL-10) selectively fused together via a linker. Specifically, this invention provides an IL4-IL10 fusion protein exhibiting stronger biological activity than a combination of individual cytokines. This invention also provides a nucleic acid sequence encoding the IL4-IL10 fusion protein, an expression vector containing such a nucleic acid sequence, a host cell or host organism altered to carry the nucleic acid sequence encoding the IL4-IL10 fusion protein, and the IL4-IL10 fusion protein itself. This invention further provides a method for producing the IL4-IL10 fusion protein using cells or organisms carrying such a nucleic acid sequence. Transgenic organisms containing the nucleic acid sequence of this invention are also provided. This invention also relates to pharmaceutical compositions containing the IL4-IL10 fusion protein. Finally, this article teaches the use of the IL4-IL10 fusion protein in medicines specifically for the prevention and / or treatment of osteoarthritis and / or conditions characterized by local or systemic inflammation, immune activation, lymphoid tissue hyperplasia, and / or chronic pain. Background Technology

[0004] Inflammatory diseases and related conditions, such as local or systemic inflammation, immune activation and / or lymphoproliferation, are selected from the group consisting of sepsis, adult respiratory distress syndrome, allogeneic and xenograft, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylarthitis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerulonephritis, immune complex-induced and other forms of vasculitis, multiple sclerosis, Sjögren's disease, gout, lymphoproliferative disorders of non-Hodgkin's lymphoma and B-cell chronic lymphocytic leukemia, burns, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, toxicity induced by in vivo administration of cytokines or therapeutic monoclonal antibodies, chronic pain syndromes and neurogenic and / or inflammatory pain. These are the most common debilitating conditions observed in clinical practice. Inflammation is an important process in these medical conditions, in which pro-inflammatory and anti-inflammatory cytokines play an important role as mediators of the inflammatory process.

[0005] Pro-inflammatory cytokines promote local and systemic inflammation. Among the numerous pro-inflammatory cytokines, two are particularly important: tumor necrosis factor (TNFα) and interleukin-1 (IL1β). Much effort has been devoted to developing therapeutic strategies targeting the inhibition of TNFα and IL1β. In particular, the reduction of the biological activity of TNFα and IL1β has been achieved through several strategies, such as neutralizing antibodies, soluble receptors, receptor antagonists, and inhibitors of proteases that convert inactive precursors into active molecules. For example, TNFα inhibitors include... (anti-TNFα antibody) (fully human anti-TNFα antibody) (TNF-receptor-Fc fusion protein), and anakinase ( IL-1 receptor antagonists (IL-1ra) have been tested in clinical trials. While blocking TNFα and / or IL-1β has been successful in many patients with rheumatoid arthritis and inflammatory bowel disease, not all patients respond well to this type of treatment intervention. Therefore, there remains a pressing need for effective alternative treatment strategies for inflammatory diseases, chronic pain, and related conditions.

[0006] Osteoarthritis (OA) is the most common joint condition, with radiographic and / or clinical evidence of OA present in the majority of individuals over 65 years of age. The most frequently affected sites are the hands, knees, hips, and spine. Importantly, symptoms are usually associated with significant functional impairment, as well as signs and symptoms of inflammation, including pain, stiffness, and loss of motor function. Characteristic structural changes in OA include progressive loss of articular cartilage, increased thickness of the subchondral plate, formation of new bone (osteophytes) at the joint margins, and the development of subchondral bone cysts. Additionally, at the junction of the articular cartilage and adjacent subchondral bone, at the so-called tide mark, remnants of calcified cartilage are present. As OA progresses, there is evidence of vascular infiltration and calcified cartilage entering this area of ​​the articular cartilage, further contributing to a reduction in articular cartilage thickness. These structural changes in the articular cartilage and surrounding bone can lead to alterations in the contours of adjacent articular articular surfaces. These changes, along with corresponding alterations in subchondral bone remodeling and bone modulus, can further contribute to the development of an unfavorable biomechanical environment and enhance the progression of articular cartilage degeneration. Multiple factors have been shown to influence the development of OA, including the presence of multiple joint disorders, increasing age, associated intra-articular crystal deposition, obesity, joint instability and / or dislocation, muscle weakness, and peripheral neuropathy. These factors can be categorized into several types, including genetic influences, mechanical factors, and age-related effects. Only a very limited range of interventional treatments have been proposed for OA, with the few available interventions targeting pain control rather than functional impairment.

[0007] Therefore, there is a need to provide a single molecule that is clinically feasible and can be used for the prevention or treatment of OA, as well as chronic pain, and for the prevention or treatment of conditions characterized by local or systemic inflammation, immune activation, and / or lymphoid tissue hyperplasia. This would enable the clinical development of a single molecule for a wide range of diseases or conditions with very different etiologies. Summary of the Invention

[0008] In a first aspect, the present invention relates to a fusion protein comprising interleukin 4 (IL4) and interleukin 10 (IL10).

[0009] In one embodiment, the IL4 and the IL10 are connected by a connector.

[0010] In one implementation, IL4 is fused to the N-terminus of IL10.

[0011] In one implementation, IL10 is fused to the N-terminus of IL4.

[0012] In one embodiment, the fusion protein further includes one or more chemical modifications. The chemical modifications may be selected from the group consisting of glycosylation, fucosylation, sialylation, and polyethylene glycolation.

[0013] In one implementation, the IL10 is human IL10.

[0014] In one implementation, the IL4 is human IL4.

[0015] In a second aspect, the present invention relates to nucleic acid molecules, said nucleic acid molecules comprising polynucleotides encoding fusion proteins taught herein.

[0016] In another aspect, the present invention relates to carriers comprising nucleic acid molecules taught herein.

[0017] In one aspect, the present invention relates to host cells including nucleic acid molecules or vectors taught herein.

[0018] In one aspect, the present invention provides a method for producing a fusion protein as taught herein, the method comprising the steps of: culturing host cells as taught herein in a manner that allows for the production of a fusion protein as taught herein; and optionally, recovering the fusion protein.

[0019] In another aspect, the present invention provides pharmaceutical compositions comprising a fusion protein as taught herein and a pharmaceutically acceptable carrier.

[0020] The present invention also relates to fusion proteins as taught herein, for use as medicines, for example for the prevention or treatment of conditions characterized by local or systemic inflammation, immune activation, lymphoid tissue proliferation and / or pain.

[0021] The condition characterized by local or systemic inflammation, immune activation, lymphoid hyperplasia, and / or chronic pain may be selected from the group consisting of: sepsis, adult respiratory distress syndrome, allogeneic and xenograft transplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, osteoarthritis, ankylosing spondylitis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerulonephritis, immune complex-induced and other forms of vasculitis, multiple sclerosis, Sjögren's disease, gout, lymphoid hyperplasia diseases such as non-Hodgkin's lymphoma and B-cell chronic lymphocytic leukemia, burns, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, peripheral neuropathy, toxicity induced by in vivo administration of cytokines or therapeutic monoclonal antibodies, chronic pain syndromes, and neurogenic and / or inflammatory pain.

[0022] In one aspect, the present invention relates to fusion proteins as taught herein for the prevention or treatment of clinical conditions in mammals, such as humans, where IL10 is an indication.

[0023] The present invention also relates to fusion proteins as taught herein for the prevention or treatment of clinical conditions in mammals, such as humans, where IL4 is an indicator.

[0024] Finally, the present invention teaches, as described herein, a carrier for the prevention or treatment of conditions characterized by local or systemic inflammation, immune activation, lymphoproliferation, and / or chronic pain, wherein said conditions may be selected from: sepsis, adult respiratory distress syndrome, allogeneic and xenograft transplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, osteoarthritis, ankylosing spondylitis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerulonephritis, immune complex-induced and other forms of vasculitis, multiple sclerosis, Sjögren's disease, gout, lymphoproliferative disorders such as non-Hodgkin's lymphoma and B-cell chronic lymphocytic leukemia, burns, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, toxicity induced by in vivo administration of cytokines or therapeutic monoclonal antibodies, chronic pain syndromes, and neurogenic and / or inflammatory pain. Detailed Implementation

[0025] General definition

[0026] The term "nucleic acid molecule" (or "nucleic acid sequence," "polynucleotide," or "nucleotide sequence") refers to a DNA or RNA molecule in single-stranded or double-stranded form, particularly DNA encoding proteins according to the present invention. "Isolated nucleic acid sequence" refers to a nucleic acid sequence that is no longer located in its natural environment from which it has been isolated, such as a nucleic acid sequence in a bacterial host cell or in a plant nuclear genome or plastid genome.

[0027] The terms “protein” and “polypeptide” are used interchangeably and refer to a molecule composed of chains of amino acids, without specifying a particular mode of action, size, 3D structure, or origin. “Isolated protein” is used to refer to a protein that is no longer present in its natural environment, such as in vitro or in recombinant bacterial or plant host cells.

[0028] The term "fusion protein" refers to a protein or polypeptide having an amino acid sequence derived from two or more proteins. Fusion proteins may also include linker regions or amino acid junctions derived from individual proteins between amino acid motifs.

[0029] The term "IL4-IL10 fusion protein" refers to a fusion polypeptide containing at least IL4 and IL10 optionally coupled to each other via a linker. Fusion proteins may include additional polypeptide sequences, such as signal sequences, histidine tags, antibody Fc fragments, etc.

[0030] As used herein, "linker" refers to a polypeptide used to couple two proteins or polypeptides such as IL4 and IL10. Linkers are typically extensions of amino acids, primarily glycine and / or serine. In embodiments, linkers are extensions of amino acids having a length up to 100 nucleotides, for example, about 2, 5, 7, 10, 15 amino acids, up to about 15, 20, 25, 30, 35, 50, 75, or 100 amino acids, preferably primarily comprising serine and glycine residues.

[0031] As used in this article, "interleukin-10" (IL10) refers to IL10 of any mammal, such as human IL10, mouse IL10, or its active species or allele variants, fragments or derivatives.

[0032] As used in this article, "interleukin-4" (IL4) refers to IL4 in any mammal, such as human IL4, mouse IL4, or its active species or allele variants, fragments or derivatives.

[0033] As used herein, the term "dimer" refers to a molecule in which two polypeptides are stably bound together by covalent or non-covalent interactions. The term "homogeneous dimer" refers to a molecule in which two identical polypeptides are stably bound together by covalent or non-covalent interactions. In suitable embodiments, they are stably bound together by non-covalent interactions.

[0034] The term "functional" in relation to the fusion protein (or its variants or fragments) of the present invention refers to the ability to simultaneously exhibit the functionality of IL4 and IL10. The functional assays for IL4 and IL10 are based on the release of lipopolysaccharide (LPS)-induced cytokines (e.g., IL1, IL6, IL8, TNFα) from whole blood.

[0035] The term "gene" refers to a DNA sequence containing a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell, operatively linked to a suitable regulatory region (e.g., a promoter). Thus, a gene can include several operatively linked sequences, such as a promoter, a 5' leader sequence containing, for example, a sequence involved in translation initiation, a (protein) coding region (cDNA or genomic DNA), an intron, and a 3' untranslated sequence containing, for example, a transcription termination site.

[0036] A “3'UTR” or “3' untranslated sequence” (often also called a “3' untranslated region” or “3' end”) refers to a nucleic acid sequence located downstream of the coding sequence of a gene, which includes, for example, a transcription termination site and (in most, but not all, eukaryotic mRNAs) a polyadenylation signal (e.g., AAUAAA or its variants). After transcription termination, the mRNA transcript downstream of the polyadenylation signal can be cleaved and a polyadenylated tail can be added, which is involved in the transport of the mRNA to the cytoplasm (where translation takes place).

[0037] "Gene expression" refers to the process in which a DNA region operatively linked to a suitable regulatory region (particularly a promoter) is transcribed into biologically active RNA, which can be translated into biologically active proteins or peptides (or active peptide fragments). Furthermore, "peptide expression" refers to the process in which mRNA is translated into a protein product that may or may not be secreted.

[0038] “Transcriptional regulatory sequences” are defined herein as nucleic acid sequences capable of regulating the transcription rate of a (coding) sequence operatively linked to said transcriptional regulatory sequence. Therefore, a transcriptional regulatory sequence as defined herein will include all sequence elements necessary to initiate transcription (promoter elements), maintain and regulate transcription (including, for example, attenuators or enhancers). Although primarily referring to upstream (5') transcriptional regulatory sequences of the coding sequence, this definition also includes regulatory sequences present downstream (3') of the coding sequence.

[0039] As used herein, the term "promoter" refers to a nucleic acid segment that controls the transcription of one or more genes, located upstream in the transcriptional direction from the transcription start site of said gene, and characterized by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art to directly or indirectly regulate the amount of transcription from that promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically regulated (e.g., by external application of certain compounds) or developmentally regulated. A "tissue-specific" promoter is active only in specific types of tissues or cells.

[0040] As used herein, the term "operably linked" refers to the connection of polynucleotide elements in terms of their functional relationship. A nucleic acid is "operably linked" when it has a functional relationship with another nucleic acid sequence. For example, if promoters, more precisely transcriptional regulatory sequences, affect the transcription of a coding sequence, then they are operably linked to the coding sequence. Operablely linked also means that the linked DNA sequences are typically contiguous.

[0041] In this article, "nucleic acid construct" or "vector" can be understood as an artificial nucleic acid molecule produced using recombinant DNA technology and used to deliver exogenous DNA into host cells. Vectors typically include other genetic elements that facilitate their use in molecular cloning, such as selectable markers, multiple cloning sites, etc. (see below).

[0042] "Strict hybridization conditions" can be used to identify nucleotide sequences that are substantially identical to a specified nucleotide sequence. Strict conditions are sequence-dependent and vary across different environments. Typically, the thermal melting point (T0) of the specific sequence is chosen at a defined ionic strength and pH. m Strict conditions requiring a temperature approximately 5°C lower. m This is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (at a defined ionic strength and pH). Choose typical stringent conditions, where the salt concentration is approximately 0.02 mol at pH 7 and the temperature is at least 60 °C. Decreasing the salt concentration and / or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridization (RNA blotting using, for example, a 100 nt probe) include, for example, washing at 63 °C in 0.2 × SSC for 20 minutes at least once, or equivalent conditions. Stringent conditions for DNA-DNA hybridization (DNA blotting using, for example, a 100 nt probe) include, for example, washing at 50 °C (typically about 55 °C) in 0.2 × SSC for 20 minutes at least once (typically twice), or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

[0043] Sequence identity and sequence similarity can be determined by aligning two peptide or nucleotide sequences using global or local alignment algorithms. Sequences are then described as "substantially identical" or "substantially similar" when they share at least a minimum percentage of sequence identity (as defined below) when optimally aligned using, for example, programs like GAP or BESTFIT with default parameters. GAP performs a full-length alignment of two sequences using the Needleman and Wunsch global alignment algorithms, maximizing the number of matches and minimizing the number of gaps. Typically, GAP's default parameters are used, with a gap penalty of 50 (nucleotides) / 8 (protein) and a gap extension penalty of 3 (nucleotides) / 2 (protein). For nucleotides, the default scoring matrix used is nwsgapdna, while for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignment and sequence identity percentage scores can be determined using computer programs such as GCG WisconsinPackage (version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA) or EmbossWin version 2.10.0 (using the program "needle"). Alternatively, similarity or identity percentages can be determined by searching a database using algorithms such as FASTA or BLAST. Preferably, sequence identity refers to sequence identity across the entire sequence length.

[0044] The terms "host cell," "recombinant host cell," or "transformed cell" refer to a new individual cell (or organism) resulting from the introduction of at least one nucleic acid molecule (particularly a nucleic acid molecule containing an encoding desired protein). The host cell is preferably a plant cell or a bacterial cell. The host cell may contain the nucleic acid molecule or vector of the present invention as an extrachromosomal (epitaxy) replication molecule, or more preferably, the nucleic acid molecule or vector of the present invention integrated into the genome of the host cell.

[0045] The term "selectable marker" is a term familiar to those skilled in the art and is used herein to describe any genetic entity that, when expressed, can be used to select one or more cells containing the selected marker. Selectable marker gene products confer, for example, antibiotic resistance or nutritional requirements.

[0046] The term “biological half-life” refers to the time required for the amount of a substance (e.g., protein) in a biological system (e.g., the circulatory system in an animal or human body) to decrease to half its value due to biological processes (e.g., kidney clearance and degradation).

[0047] In this document and its claims, the verb "comprising" and its inflections are used in their non-limiting sense to mean including items that follow the word, but not excluding items not explicitly mentioned. It also covers the more restrictive verb "consisting of." Furthermore, when an element is referred to with the indefinite article "an" or "a," the possibility of more than one of the said elements is not excluded, unless the context clearly requires that there be exactly one of the element. Therefore, the indefinite article "an" or "a" generally means "at least one." It should also be understood that when "sequence" is referred to herein, it generally refers to an actual physical molecule having a sequence of a certain subunit (e.g., an amino acid).

[0048] The protein, nucleic acid sequences, vectors, and host cells of this invention

[0049] The inventors have now devised a novel “single-molecule therapy.” Specifically, they provide a fusion protein comprising IL4 and IL10 proteins selectively fused together via a linker. In particular, the fusion protein of the present invention has been found to exhibit superior biological activity (e.g., inhibition of TNFα and IL1β) compared to its individual counterparts, i.e., IL4 and IL10, respectively. Furthermore, the fusion protein of the present invention has been found to be significantly larger (~35 kD) than the individual cytokines (both <20 kD). The inventors have also unexpectedly discovered that the fusion protein itself forms a dimer (through the IL10 portion of the fusion protein), thus providing a fusion protein with even larger molecular weights (~70 kD). This increased molecular weight and the resulting increase in molecular radius, compared to the individual cytokines, not only significantly prolongs the biological half-life of the IL4-IL10 fusion protein in circulation but also enhances its bioavailability at inflammatory sites to unprecedented levels. The fusion protein of this invention also significantly prolongs the therapeutic window for the synergistic effect between IL4 and IL10 because it delivers both cytokines to the inflammatory site, where they can exert their effects in equal amounts over time when they are present with each other. Moreover, it has been unexpectedly found that the fusion protein of this invention also exhibits dual therapeutic effects. In particular, when administered systemically, the IL4-IL10 fusion protein displays anti-inflammatory activity, while when administered intrathecally, it acts as an anti-hypnotic agent.

[0050] In one embodiment of the invention, the nucleic acid sequence and amino acid sequence (including its variants and fragments) of the IL4-IL10 fusion protein are provided. The IL4-IL10 fusion protein, as well as its functional fragments and variants, exhibit IL4 activity and IL10 activity.

[0051] In one aspect, a fusion protein comprising IL4 and IL10 is provided. The fusion protein comprises the IL4 protein, or a variant or fragment thereof. The IL4 protein is preferably a mammalian IL4 protein, such as human IL4 or mouse IL4. One amino acid sequence of IL4 is described in SEQ ID NO:1. Variants of IL4 include proteins having, for example, at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or greater than 100% amino acid sequence identity with SEQ ID NO:1, preferably in full length. Amino acid sequence identity is preferably determined by pairing using the Needleman and Wunsch algorithm as defined above and the GAP default parameter. Variants also include proteins having IL4 activity derived by substitution, deletion, or insertion of one or more amino acids having the amino acid sequence SEQ ID NO:1. Preferably, such proteins include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

[0052] The fusion protein further comprises IL10 protein, or variants or fragments thereof. The IL10 protein is preferably a mammalian IL10 protein, such as human IL10 or mouse IL10. An amino acid sequence representing IL10 is described in SEQ ID NO:2. Variants of IL10 include proteins having, for example, at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater, such as 100%, amino acid sequence identity with SEQ ID NO:2, preferably in full length. Amino acid sequence identity is preferably determined by pairing using the Needleman and Wunsch algorithms as defined above and the GAP default parameter. Variants also include proteins having IL10 activity derived by substitution, deletion, or insertion of one or more amino acids having the amino acid sequence SEQ ID NO:2. Preferably, such proteins include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

[0053] IL4 and IL10 in the fusion protein may or may not be linked by a linker. Additional amino acid sequences may be present at the N- and / or C-terminus of the fusion protein of the present invention, for example, to facilitate purification. For example, a histidine tag may be present at the C- or N-terminus for purification purposes. Alternatively, the IL4-IL10 fusion protein of the present invention may optionally include additional protein moieties, such as targetable portions, for example, protein moieties comprising one or more antibody Fc regions.

[0054] IL4 can be located at the N-terminus of IL10, or it can be located at the C-terminus of IL10. In a preferred embodiment, the IL4 molecule is located at the N-terminus of the IL10 molecule. The subsequent fusion protein was found to exhibit higher specific activity (data not shown) than the IL4-IL10 fusion protein in which the IL4 molecule is located at the C-terminus of the IL10 molecule.

[0055] IL4-IL10 fusion proteins can exist in their monomeric form, in which case they have a kDa of approximately 35 kDa, or they can be dimeric IL4-IL10 fusion proteins, in which case two IL4-IL10 fusion proteins can bind nonvalently to each other, in which case they have a kDa of approximately 70 kDa.

[0056] In embodiments, the fusion protein of the present invention is substantially composed of IL4 and IL10 selectively linked via a linker.

[0057] In embodiments, both the IL4 and IL10 portions of the fusion protein of the present invention are active and capable of signaling cells to inhibit the production of at least one inflammatory cytokine or mediator, such as IL1β, IL6, IL8, or TNFα. Preferably, at least TNFα, IL6, and IL8 are inhibited.

[0058] In this embodiment, the fusion protein inhibits the production of cytokines (e.g., TNFα, IL1β, IL6, IL8 and other inflammatory cytokines) while inducing the secretion of inhibitory molecules, such as IL1 receptor antagonists and soluble TNF receptors (through inflammation) and other cells stimulated by endotoxins, other Toll-like receptor (TLR) agonists or other stimulants.

[0059] In embodiments, the fusion protein of the present invention inhibits the expression of adhesion molecules in inflammatory cells, endothelial cells, and other cells stimulated by endotoxins, other TLRs, or other stimuli.

[0060] In one embodiment, the fusion protein inhibits the expression of tissue factor in endothelial cells, inflammatory cells, and other cells stimulated by endotoxins, other TLR agonists, or other stimuli.

[0061] In embodiments, the fusion protein of the present invention inhibits the generation of oxygen free radicals in inflammatory cells and other cells stimulated by endotoxins, other TLR agonists, or other stimuli.

[0062] In the embodiments, in the presence or absence of autoantigens or non-autoantigens, or superantigen-stimulated antigen-presenting cells, the fusion protein inhibits the activity of IFNγ- and IL17-secreting Th1 and Th17 cells, and induces or maintains the in vitro expression of generated FoxP3-secreting T cells (CD25+), TGFβ-secreting Th2, Tr1, and Th3 cells. The superantigen includes staphylococcal enterotoxin B (SEB) or mitogens (e.g., including CD3, CD28, phytohemagglutinin (PHA)) or phorbol myristate acetate (PMA).

[0063] In one implementation, the fusion proteins inhibit the expression of activated Fcγ receptors while simultaneously inducing the expression of inhibitory Fcγ receptors, preventing cells (e.g., monocytes, macrophages, and dendritic cells) from being activated by immune complexes containing IgG.

[0064] In suitable embodiments, the fusion protein of the present invention exists in the form of a homodimer.

[0065] In one embodiment, it has a molecular weight greater than 60 kDa.

[0066] The fusion protein of the present invention can be prepared using techniques conventional to those skilled in the art. For example, it can be prepared using techniques provided for generating recombinant fusion proteins from passaged cell lines in a culture medium. For example, the fusion protein of the present invention can be generated in host cell transfected tumors using a combination of recombinant DNA technology and gene transfection methods.

[0067] For example, to express the fusion protein of the present invention, a nucleic acid molecule encoding the fusion protein of the present invention can be prepared using standard molecular biology techniques. The nucleic acid molecule of the present invention preferably has a transcriptional regulatory sequence, such as a promoter, and an optional 3' untranslated region operably linked. The nucleic acid molecule of the present invention can be inserted into a vector (e.g., an expression vector) such that the gene is operably linked to transcriptional and translational control sequences. The expression vector and transcriptional regulatory sequence are selected to match the expression host cell used. The nucleic acid molecule encoding the fusion protein of the present invention can be inserted into the expression vector using conventional methods. The nucleic acid molecule or vector of the present invention may further include a nucleotide sequence encoding a signal peptide that can promote the secretion of the fusion protein by the host cell. The nucleotide sequence encoding the signal peptide can be operably linked to the nucleic acid molecule of the present invention. Preferably, the signal peptide is operably linked to the amino terminus of the fusion protein of the present invention, so that the nucleotide sequence encoding the signal peptide can be located at the 5' of the nucleic acid molecule encoding the fusion protein of the present invention. The signal peptide can be a cytokine signal peptide or a signal peptide derived from a non-cytokine protein. The promoter can be constitutive or inducible. The vector may include a selectable marker for selecting a host cell carrying the vector. When the vector is a reproducible vector, the vector may include an origin of replication.

[0068] The fusion protein according to the invention can be synthesized de novo by chemical synthesis (using, for example, a peptide synthesizer provided by AppliedBiosystems), or it can be produced by recombinant host cells by expressing a nucleic acid sequence encoding the fusion protein, fragment, or variant. The variants and fragments are preferably functional, i.e., possessing IL4 and / or IL10 activity, preferably IL4 and IL10 activity.

[0069] The anti-inflammatory activity and thus functionality of IL4 and IL10, as well as the IL4-IL10 fusion protein, can be determined using conventional methods. For example, a suitable assay for the functionality of IL4 and IL10, as well as the IL4-IL10 fusion protein, is the release of lipopolysaccharide (LPS)-induced cytokines (IL1β, IL6, IL8, TNFα) in whole blood, as illustrated in Example 5, for example.

[0070] In another aspect, isolated nucleic acid sequences, such as cDNA, genomic DNA, and RNA sequences, encoding any of the aforementioned fusion proteins are provided. Due to the degeneracy of the genetic code, multiple nucleic acid sequences can encode the same amino acid sequence. Any nucleic acid sequence encoding the fusion protein of the present invention is referred to herein as the "IL4-IL10 fusion protein encoding nucleic acid sequence." The provided nucleic acid sequences include recombinant, artificial, or synthetic nucleic acid sequences. It should be understood that when RNA is referred to and the sequence is described as a DNA sequence, the actual base sequence of the RNA molecule is the same, the difference being that thymine (T) is replaced by uracil (U). The nucleic acid sequences of the present invention are specifically used for expressing the IL4-IL10 fusion protein of the present invention for the production of these proteins or for gene therapy purposes.

[0071] The nucleic acid sequence, particularly the DNA sequence, encoding the IL4-IL10 fusion protein of the present invention can be inserted into an expression vector to produce (high amounts) of the IL4-IL10 fusion protein. Suitable vectors include, but are not limited to, straight-chain nucleotides, plasmids, phage particles, granules, RNA vectors, viral vectors, etc. Non-limiting examples of viral vectors include retroviruses, adenoviruses, and adenovirus-associated viruses.

[0072] Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and multitumor promoters. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters, may be used.

[0073] In addition to the nucleic acid molecule encoding the IL4-IL10 fusion protein and the regulatory sequence, the recombinant expression vector of the present invention may carry additional sequences, such as those regulating vector replication in host cells (e.g., origin of replication), and selectable marker genes. These selectable marker genes facilitate selection of host cells in which the vector has been introduced (see, for example, US 4,399,216, US 4,634,665, and US 5,179,017 by Axel et al.). For example, selectable marker genes typically confer resistance to drugs (e.g., G418, hygromycin, or methotrexate) in host cells in which the vector has been introduced. Preferred selectable marker genes include a dihydrofolate reductase (DHFR) gene (for selection / amplification with methotrexate in DHFR-host cells) and a neo gene (for G418 selection). Finally, in addition to the nucleic acid sequence encoding the fusion protein of the present invention, the recombinant expression vector may contain a gene encoding a glycosyltransferase.

[0074] In another aspect, the present invention relates to host cells comprising the nucleic acid sequences of the present invention, or nucleic acid constructs or vectors comprising the nucleic acid sequences of the present invention. The host cell can be any host cell. The host cell can be selected from prokaryotic and eukaryotic cells. The host cell can also be a cell line, such as a prokaryotic or eukaryotic cell line. The host cell is preferably an animal cell or cell line, such as a mammalian cell or cell line.

[0075] In one embodiment, the fusion protein of the present invention is expressed in eukaryotic cells (e.g., mammalian host cells). Preferred mammalian host cells for expressing the recombinant IL4-IL10 fusion protein of the present invention include CHO cells (including dhfr-CHO cells, described in (Urlaub et al., 1980), selectively labeled with DHFR), NS / 0 myeloma cells, COS cells, HEK293 cells, and SP2.0 cells. When a recombinant expression vector containing the nucleic acid sequence encoding the IL4-IL10 fusion protein is introduced into a mammalian host cell, the fusion protein of the present invention can be generated by culturing the host cells for a sufficiently long period of time to allow the fusion protein to be expressed in the host cells, or more preferably to be secreted into the culture medium in which the host cells are grown. The fusion protein of the present invention can be recovered from the culture medium in which the host cells are grown, or can be purified from the culture medium using standard protein purification methods.

[0076] Alternatively, the nucleic acid sequence encoding the fusion protein of the present invention can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g., Escherichia coli, algae, and insect cells. Furthermore, the fusion protein of the present invention can be produced in transgenic non-human animals (e.g., from the milk of sheep and rabbits or from the eggs of hens) or transgenic plants.

[0077] Introducing the nucleic acid sequence of the present invention into host cells can be performed using any standard techniques known in the art. For the expression of the fusion protein of the present invention, the expression vector encoding the fusion protein can be transfected into host cells using standard techniques. The term "transfection" in various forms is intended to encompass the various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, liposome transfection, and lyophilization. Cell lines secreting the fusion protein of the present invention can be identified by measuring the presence of the fusion protein in the culture supernatant. A preferred screening process includes two sequential steps: first, identifying cell lines secreting the fusion protein; and second, determining the properties of the fusion protein, such as its ability to inhibit LPS or other Toll-like receptor agonists, glycosylation patterns, etc., stimulating blood cells to produce cytokines.

[0078] For optimal expression in host cells, the DNA sequence encoding the IL4-IL10 fusion protein can be codon-optimized by adjusting the most preferred codon usage in the host cell gene. Some techniques for modifying the host cell-preferred codon usage can be found in patents and scientific literature. The precise method of codon usage modification is not critical to this invention.

[0079] In another embodiment of the invention, PCR primers and / or probes and a kit for detecting DNA or RNA sequences encoded by the IL4-IL10 fusion protein are provided. The degenerate or specific PCR primer pairs used to amplify the IL4-IL10 fusion protein-encoded DNA from the sample can be synthetic (see Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany). For example, any extension of 9, 10, 11, 12, 13, 14, 15, 16, 18 or more adjacent nucleotides of the IL4-IL10 fusion protein-encoded nucleic acid sequence (or complementary strand) can be used as a primer or probe. Similarly, DNA fragments encoding the IL4-IL10 fusion protein-encoded nucleic acid sequence can be used as hybridization probes. The detection kit for the IL4-IL10 fusion protein-encoding nucleic acid sequence may include primers specific to the IL4-IL10 fusion protein-encoding nucleic acid sequence, and / or probes specific to the IL4-IL10 fusion protein-encoding nucleic acid sequence, and an assay design for specifically detecting the IL4-IL10 fusion protein-encoding nucleic acid sequence in a sample using said primers or probes. Such a detection kit can, for example, be used to determine whether host cells have been transformed by the specific IL4-IL10 fusion protein-encoding nucleic acid sequence of the present invention. Due to the degeneracy of the genetic code, certain amino acid codons can be substituted by other amino acid codons without altering the amino acid sequence of the protein.

[0080] In one aspect, the present invention relates to a method for producing an IL4-IL10 fusion protein, the method comprising the steps of: culturing host cells of the invention under conditions allowing for the production of the IL4-IL10 fusion protein; and optionally, recovering the fusion protein. Those skilled in the art can conventionally select the conditions allowing for the production of the IL4-IL10 fusion protein. Furthermore, those skilled in the art can recover the produced fusion protein using conventional methods, including but not limited to chromatographic methods (including but not limited to size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, metal binding, etc.), immunoprecipitation reactions, HPLC, ultracentrifugation, precipitation and differential dissolution, and extraction. As mentioned above, the recovery or purification of the fusion protein can be facilitated by, for example, adding a histidine tag to the fusion protein.

[0081] Pharmaceutical Composition

[0082] In one aspect, the present invention relates to pharmaceutical compositions comprising the fusion protein of the present invention and a pharmaceutically receptive carrier.

[0083] The pharmaceutical composition can be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients using conventional techniques (e.g., as described in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995).

[0084] The term "pharmaceutically acceptable carrier" refers to a natural, non-toxic, and non-therapeutic carrier or excipient. Examples of such excipients are, but are not limited to, saline, Ringer's solution, glucose solution, and Hank's solution. Non-aqueous excipients, such as non-volatile oils and ethyl oleate, may also be used. A preferred excipient is a 5% aqueous solution of glucose saline. The excipient may contain small amounts of additives, such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.

[0085] The pharmaceutical composition may be administered via any suitable route and manner. As those skilled in the art will understand, the route and / or manner of administration will vary depending on the desired outcome.

[0086] The pharmaceutical compositions according to the invention can be formulated according to conventional methods for administration via any route, such as oral, topical, parenteral, sublingual, transdermal, or inhalation. The compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams, or liquid formulations (e.g., in the form of oral or sterile parenteral solutions or suspensions, or in the form of sprays, aerosols, or other conventional inhalation methods).

[0087] The pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical (including sublingual and sublingual), rectal, vaginal and / or parenteral administration.

[0088] In one embodiment, the pharmaceutical composition is administered parenterally into the intestine.

[0089] As used herein, the terms “parental administration” and “prescription” refer to administration methods that differ from enteral and local administration, typically via injection, including but not limited to intravenous injection, intramuscular injection, intra-arterial injection, intrathecal injection, intracapsular injection, intraorbital injection, intracardiac injection, intradermal injection, intraperitoneal injection, tracheal injection, subcutaneous injection, subepidermal injection, intra-articular injection, subcapsular injection, subarachnoid injection, spinal injection, epidural and intrasternal injection, and infusion.

[0090] In this embodiment, the pharmaceutical composition is administered via intravenous or subcutaneous injection or infusion.

[0091] In one embodiment, the fusion protein of the present invention is administered in crystalline form via subcutaneous injection.

[0092] As used in this article, “pharmaceuticalally acceptable carriers” include any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, antioxidants, and absorption delay agents.

[0093] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. Such media and reagents are well known in the art for use with pharmaceutically acceptable active substances. Their use in the pharmaceutical compositions of the present invention is contemplated unless any conventional media or reagent is incompatible with the active compound. Preferably, the carrier is suitable for parenteral administration, such as intravenous or subcutaneous injection or infusion.

[0094] Pharmaceutical compositions must generally be sterile and stable under preparation and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of this invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). For example, suitable flowability can be maintained by using coating materials (such as lecithin), maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0095] Prolonged absorption of injectable drugs can be achieved by including delayed absorption agents (such as monostearate and gelatin) in the composition.

[0096] A sterile injectable solution can be prepared by adding the required amount of the active compound to a suitable solvent having, for example, one or more of the components listed above (as needed), followed by filtration and sterilization. Typically, a dispersion is prepared by introducing the active compound into a sterile carrier containing an alkaline dispersion medium and other desired components (e.g., from those listed above). In the case of sterile powders used to formulate sterile injectable solutions, a preferred preparation method is vacuum drying and freeze-drying (lyophilization), which yields a powder of the active ingredient and any other desired components from its previously sterile filtered solution.

[0097] Depending on the route of administration, the active compound, namely the IL4-IL10 fusion protein, can be coated onto a material to protect it from acids and other natural conditions that could inactivate the compound. For example, the compound can be administered to the subject in a suitable carrier (e.g., liposomes). Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al., 1984).

[0098] The fusion protein of the present invention can also be formulated with a carrier that prevents its rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for formulating such formulations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0099] Dosing regimens can be adjusted to provide the best desired response (e.g., therapeutic effect). For example, a single bolus can be administered, fractional doses can be administered over time, or the dose can be reduced or increased accordingly, as indicated by the urgency of the treatment situation. For ease of administration and uniform dosing, it is particularly advantageous to formulate parenteral compositions in unit dosage form. As used herein, unit dosage form refers to physically separate units suitable for single-dose administration to the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the desired pharmaceutical carrier. The specifications of the unit dosage form of the present invention are determined by and directly depend on the following: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art of formulating such active compounds for individual sensitivity treatment.

[0100] To obtain an effective and non-toxic amount (“effective amount”) of IL4-IL10 fusion protein in the pharmaceutical compositions of the present invention to achieve the desired therapeutic response for a specific patient, composition, and route of administration, the actual dose level of the IL4-IL10 fusion protein can be varied. The selected dose level will depend on various pharmacokinetic factors, including the activity of the specific composition administered according to the present invention, route of administration, time of administration, excretion rate, duration of treatment, other pharmaceutical compounds and / or substances used in conjunction with the specific composition administered, the age, sex, weight, condition, general health status and medical history of the patient to be treated, and similar factors known in the medical field.

[0101] In one embodiment, the IL4-IL10 fusion proteins of the present invention can be administered by intravenous injection or short-term infusion. In another embodiment, they are administered by long-term, slow, continuous infusion, for example, over 24 hours, in order to reduce toxic side effects.

[0102] In another embodiment, the IL4-IL10 fusion protein of the present invention can be administered as maintenance therapy, for example, once a week for 6 months or longer.

[0103] Therapeutic uses

[0104] In a further aspect, the present invention relates to the fusion protein of the present invention used as a drug or a pharmaceutical composition comprising the fusion protein.

[0105] In one aspect, the present invention relates to a fusion protein of the present invention or a pharmaceutical composition comprising the fusion protein for the prevention or treatment of osteoarthritis. In particular, the fusion protein of the present invention has been found to have chondroprotective activity. Therefore, the fusion protein can be used to prevent and treat cartilage damage, particularly in osteoarthritis. Furthermore, it has been found that in canine models of osteoarthritis, dogs given the fusion protein of the present invention experienced significantly less pain compared to dogs not given the fusion protein of the present invention. Thus, the fusion protein of the present invention can be particularly used for the prevention or treatment of osteoarthritis (prevention or treatment of cartilage degeneration) and its associated chronic pain.

[0106] In a further aspect, the present invention relates to the fusion protein of the present invention or pharmaceutical compositions comprising the fusion protein for the prevention or treatment of osteoarthritis, chronic pain, conditions characterized by local or systemic inflammation, immune activation and / or lymphocyte proliferation.

[0107] In the embodiments, the condition characterized by local or systemic inflammation, immune activation, and / or lymphoid hyperplasia is selected from the group consisting of: sepsis, adult respiratory distress syndrome, allogeneic and xenograft transplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylitis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerulonephritis, immune complex-induced and other forms of vasculitis, multiple sclerosis, Sjögren's disease, gout, lymphoid hyperplasia diseases such as non-Hodgkin's lymphoma and B-cell chronic lymphocytic leukemia, burns, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, toxicity induced by in vivo administration of cytokines or therapeutic monoclonal antibodies, chronic pain syndromes, and neurogenic and / or inflammatory pain.

[0108] In an implementation, the condition is characterized by pain, and may be selected from inflammatory pain and neuropathic pain.

[0109] In another aspect, the present invention relates to the fusion protein of the present invention for the prevention or treatment of clinical conditions in which IL10 is indicated in mammals such as humans.

[0110] In a further aspect, the present invention relates to the fusion protein of the present invention for the prevention or treatment of clinical conditions in mammals, such as humans, indicating IL4.

[0111] In embodiments of the present invention, the fusion polypeptide of the present invention is used to prevent or treat diseases or conditions in which inhibiting the production of pro-inflammatory cytokines and other inflammatory mediators is beneficial.

[0112] In another aspect, the present invention relates to a method for preventing or treating a disease or condition in which the inhibition of the production of pro-inflammatory cytokines and other inflammatory mediators has a beneficial effect, comprising the step of administering an effective amount of the fusion protein of the invention to a subject in need of it to treat or prevent said disease or condition.

[0113] In one implementation, such a disease or condition is an inflammatory disease mediated by the production of pro-inflammatory cytokines (e.g., TNF, IL1β, IL6), chemokines (e.g., IL8), and other inflammatory mediators.

[0114] According to one embodiment, the fusion proteins taught herein can be used to inhibit the production and release of cytokines and other inflammatory mediators by cells (e.g., macrophages, monocytes, T-lymphocytes, and other cells). Therefore, the fusion proteins of the present invention can be used to formulate drugs that attenuate inflammatory responses by inhibiting the release of cytokines and other inflammatory mediators from these cells in vivo. The fusion proteins of the present invention can be used as a drug on their own or in combination with other drugs.

[0115] Treatment (prophylactic or therapeutic) may be administered via parenteral, preferably intravenous, intramuscular, intrathecal, epidural, intraspinal, or subcutaneous routes. However, for pharmaceutical compositions comprising the fusion proteins listed above, other routes of administration as described above may also be used. Dosage and administration regimen may depend on the degree of inhibition of the production and release of the targeted inflammatory cytokines. Typically, the amount of fusion protein administered ranges from 0.5 μg to 1 mg / kg body weight. When administered as a biological sample, the dosage can be determined or adjusted by measuring the levels of circulating cytokines (IL10, IL4).

[0116] For parenteral administration, the fusion protein is preferably formulated into an injectable form by mixing it with a pharmaceutically acceptable parenteral carrier. Such carriers are well known in the art, and examples include saline, glucose solution, Ringer's solution, and solutions containing a small amount of human serum albumin.

[0117] Typically, the fusion protein of the present invention can be formulated in such a carrier at a concentration of about 50 μg to about 100 mg / ml. In one embodiment of the invention, the fusion protein is administered by intravenous injection.

[0118] Other details regarding dosing are set forth in the above section concerning pharmaceutical compositions including the fusion protein of the present invention.

[0119] Gene therapy

[0120] The nucleic acid constructs or vectors of the present invention can be used as gene therapy agents for treating the diseases listed above. In one embodiment of the present invention, adenovirus-associated virus is used as a gene therapy vector.

[0121] Therefore, one aspect of the present invention relates to a gene therapy vector as described above for the prevention or treatment of osteoarthritis, chronic pain, and conditions characterized by local or systemic inflammation, immune activation, and / or lymphoid tissue hyperplasia, preferably wherein the conditions characterized by local or systemic inflammation, immune activation, and / or lymphoid tissue hyperplasia are selected from: sepsis, adult respiratory distress syndrome, allogeneic and xenograft transplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylitis, such as systemic lupus erythematosus and rheumatoid arthritis. The group includes autoimmune diseases of arthritis, glomerulonephritis, immune complex-induced and other forms of vasculitis, multiple sclerosis, Sjögren's disease, gout, lymphoproliferative disorders such as non-Hodgkin's lymphoma and B-cell chronic lymphocytic leukemia, burns, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, and toxicity induced by in vivo administration of cytokines or therapeutic monoclonal antibodies, chronic pain syndromes, and neuropathic and / or inflammatory pain.

[0122] The present invention will now be described with reference to the following embodiments, which provide particularly preferred embodiments. However, it should be noted that these embodiments are exemplary and should not be construed as limiting the invention in any way.

[0123] sequence list

[0124] amino acid sequence of SEQ ID NO:1–IL4

[0125] HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS

[0126] amino acid sequence of SEQ ID NO:2–IL10

[0127] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0128] SEQ ID NO:3 – Amino acid sequence of the linker

[0129] GSGGGGSGT

[0130] The amino acid sequence of the SEQ ID NO:4–IL4-IL10 fusion protein (the linker sequence is underlined; IL4 is located at...). (N-terminus of IL10)

[0131] HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS GSGGGGSGT SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN Attached Figure Description

[0132] Figure 1 This displays the levels of the IL4-IL10 fusion protein obtained from HEK293 cells transfected with the IL4-IL10 fusion protein. IL4 (A) and IL10 (B) in the supernatant of HEK293 cells transfected with the pUPE expression vector carrying the IL4-IL10 fusion protein were detected in a sandwich ELISA assay according to the manufacturer's instructions. Results are expressed as optical density (OD) versus dilution of the culture supernatant.

[0133] Figure 2 Immunochemical identification of the IL4-IL10 fusion protein was demonstrated using a cross-ELISA. In the cross-ELISA, the supernatant of HEK293 cells transfected with a pUPE expression vector carrying the IL4-IL10 fusion protein was detected using either anti-IL4 (A) or anti-IL10 monoclonal antibody (used as a capture antibody) (B) and biotinylated anti-IL10 (A) or anti-IL4 (B) monoclonal antibody (used as a detection antibody). Recombinant IL10 was used as a negative control. The results showed that the IL4-IL10 protein could be detected by both its IL10 fraction (Figure A) and its IL4 fraction (Figure B).

[0134] Figure 3 Western blotting was used to identify the IL4-IL10 fusion protein in the supernatant of HEK293 cells. Electrophoretic analysis of 10 μL of supernatant obtained from HEK293 cells transfected with cDNA encoding the IL4-IL10 fusion protein was performed on SDS-PAGE. The blots were developed with labeled anti-IL4 antibody (A) or labeled anti-IL10 antibody (B). Recombinant IL4 and IL10 were used as controls. Lane 1 = molecular marker, Lane 2 = untreated IL4-IL10 fusion protein, Lane 3 = deglycosylated IL4-IL10 fusion protein, Lane 4 = IL4, and Lane 5 = IL10.

[0135] Figure 4Results of high-performance size exclusion chromatography (HPLC) assays identifying the polymerization of the IL4-IL10 fusion protein are shown. Samples of the IL4-IL10 fusion protein, recombinant IL4, and recombinant IL10 were run on an HPLC system. Columns were calibrated using a mixture of proteins including thyroglobulin, bovine serum albumin (66 kDa, first dashed line on the left), carbonic anhydrase (30 kDa, second dashed line on the left), myosin (18 kDa, third dashed line on the left), and ribonuclease (13.7 kDa, fourth dashed line on the left). Using these markers, the molecular weight of the protein in each fraction was estimated by comparing retention times. The concentrations of IL4 and IL10 in each run fraction were measured by ELISA and expressed as ng / ml. IL4 eluted as a distinct monomer, IL10 as a distinct dimer, and the elution pattern of the IL4-IL10 fusion protein showed a distinct dimer.

[0136] Figure 5 The IL4-IL10 fusion protein inhibits LPS-induced cytokine release in whole blood cultures. Heparinized blood from healthy volunteers was diluted 1:10 in culture medium and cultured with LPS at a concentration of 10 ng / mL in the presence of various concentrations of the IL4-IL10 fusion protein. TNFα in the supernatant was measured by ELISA. Figure A shows the ability of the IL4-IL10 fusion protein to inhibit TNF production (results are expressed as inhibition %). Note that no inhibition of TNF production was observed when a similar volume of medium without the IL4-IL10 fusion protein was tested. Figure B shows the inhibition of LPS-induced TNFα (left), IL6 (middle), and IL8 (right) production (concentrations of TNFα, IL6, and IL8 measured from the supernatant are given on the Y-axis). The IL4-IL10 fusion protein was detected at 100 ng / mL and its effect was compared with that of a mixture of recombinant IL4 and IL10 at a final concentration of 50 ng / mL for each. Figure C shows that, compared to culture medium alone (without antibodies), the addition of both anti-IL4 receptor (anti-IL4R) and anti-IL10 receptor (anti-IL10R) receptor-blocking antibodies completely eliminated the effect of the IL4-IL10 fusion protein on LPS-induced IL6 production. Note that the addition of either anti-IL4 receptor (anti-IL4R) or anti-IL10 receptor (anti-IL10R) receptor-blocking antibodies (rather than both) only partially eliminated the effect of the IL4-IL10 fusion protein.

[0137] Figure 6The strong inhibitory effect of the IL4-IL10 fusion protein on the production of Th1 and Th17 cytokines was shown to be correlated with the percentage of (FoxP3+) T-cells persistently regulated in monocyte cultures activated by the superantigen Staphylococcus enterotoxin B (SEB). Activation of bone marrow cells and B cells is critically dependent on the balance between pro-inflammatory Th1 and Th17 cytokines and CD4 T cells regulating FoxP3- expression. To evaluate the effect of the IL4-IL10 fusion protein on T cell activation, monocytes were isolated from peripheral blood (PBMCs) of healthy donors. The percentage of (FoxP3+) T-cells was determined by the presence or absence of SEB (1 ng / mL) and / or the IL4-IL10 fusion protein in PBMCs (5.10 ng / mL). 5 Treatment with IL4-IL10 for 3 days induced T cell activation. After proliferation during this culture period (Fig. A), FoxP3 expression and pro-inflammatory T-cell cytokine production were measured (Fig. B). SEB induced significant proliferation associated with upregulation of FoxP3 expression (Fig. B) (Fig. A). Proliferation (Fig. A) and FoxP3 expression (Fig. B) were almost entirely unaffected by the IL4-IL10 fusion protein. Conversely, Th1 and Th17 cytokines IFNγ (left, Fig. C), IL17 (middle, Fig. C), and TNFα (right, Fig. C) were all strongly reduced by the IL4-IL10 fusion protein. In conclusion, this confirms that the IL4-IL10 fusion protein strongly alters the balance between inhibitory CD4 T cells and pro-inflammatory Th1 and Th17 cells.

[0138] Figure 7 This study demonstrates the stabilizing effect of the IL4-IL10 fusion protein on IgG receptor expression on monocytes in staphylococcal enterotoxin B (SEB)-activated monocyte cultures. The balance between activating and repressive receptors for IgG (FcγRs) plays a crucial role in immune complex-mediated activation of bone marrow cells and lymphocytes. To evaluate the effect of the IL4-IL10 fusion protein on FcγR expression on monocytes, peripheral blood monocytes (PBMCs) were isolated from healthy donors. PBMCs were treated with the superantigen SEB (0.1 ng / mL) and / or the IL4-IL10 fusion protein (5.1 ng / mL) in or without the superantigen SEB (0.1 ng / mL). 5T-cell-dependent monocyte activation was induced for 2 days ( / ml). FcγRs expression was measured after this culture period. IL10 upregulated the expression of FcγRI, FcγRIIa, and Fcγriii. IL4 alone showed a slight decrease in the expression of these activated FcγRs compared to cells cultured in the absence of cytokines. The combination of IL4 and IL10 with the IL4-IL10 fusion protein normalized the expression of FcγRI and FcRγIIa. A small increase in FcγRIII expression was measured in the combination of IL4 and IL10 or the IL4-IL10 fusion protein, although negligible compared to the upregulation of this receptor induced by IL10 alone. IL4 alone showed a slight decrease in FcγRIIb expression. The combination of IL4 and IL10 and the IL4-IL10 fusion protein did not alter the expression of inhibitory FcγRIIb. Meanwhile, the results confirm that the IL4-IL10 fusion protein stably activates the expression of FcγRs, which in turn inhibits immune complex-induced immune activation.

[0139] Figure 8 This study illustrates the dose-response effect of the IL4-IL10 fusion protein on chondrocyte proteoglycan turnover. Cartilage grafts from five donors were exposed to 50% v / v blood from five donors (n=5) for 4 days. During blood exposure, the IL4-IL10 fusion protein was added at concentrations ranging from 0.0001 to 100 ng / mL. After a 12-day recovery period, the rate of proteoglycan synthesis (A), release (B), and content (C) were measured. Compared to control cartilage, the rate of proteoglycan synthesis and content were significantly reduced due to blood exposure, while proteoglycan release was increased (indicated by an asterisk; p<0.05). Hash labels indicate statistically significant differences compared to 50% v / v blood (p<0.05), while dashed lines in A and B highlight the sigmoid appearance of the dose-response. The mean ± quartile range is described. Adding the IL4-IL10 fusion protein to cartilage cultures resulted in a dose-dependent restoration of the proteoglycan synthesis rate; normalization of proteoglycan release; and inhibition of the decrease in content.

[0140] Figure 9This study demonstrates the prevention of blood-induced cartilage damage by the IL4-IL10 fusion protein. Cartilage grafts from eight donors were exposed to 50% v / v blood from eight donors for four days. During blood exposure, the IL4-IL10 fusion protein, as well as IL4, IL10, and combinations of IL4 and IL10 (all at 10 ng / mL), were added. Blood exposure reduced the rate of proteoglycan synthesis in the cartilage grafts by 76% (A, p = 0.012). Compared with blood exposure alone, the IL4-IL10 fusion protein increased the rate of proteoglycan synthesis by 241%. Blood-exposed cartilage showed a 59% increase in proteoglycan release (B, p = 0.017). The addition of the IL4-IL10 fusion protein reduced blood-induced proteoglycan release (p = 0.012) back to control levels. Compared with control, blood-exposed cartilage showed a 10% decrease in proteoglycan content (C, p = 0.012). Compared with blood exposure alone, the IL4-IL10 fusion protein significantly increased proteoglycan content (p = 0.012). * indicates statistical significance compared to control, and # indicates statistical significance compared to 50% v / v blood (p < 0.05). Mean ± quartile range is described.

[0141] Figure 10 The IL4-IL10 fusion protein was shown to reduce pain in a canine Groove model of osteoarthritis. Osteoarthritis (OA) was induced in four dogs, and 1 ml of the IL4-IL10 fusion protein was administered intra-articularly at 5 weeks (1 μg / ml) and 7 weeks (10 μg / ml) after OA induction (see arrow). Force plate analysis (FPA) was performed every 2 weeks from week 3 before induction to week 8 after induction, and daily after the IL4-IL10 fusion protein injection. The peak signal of standing force indicated that the loading of the OA joint (test joint vs. contralateral joint) was almost normalized compared to pre-injection levels. This effect of load on pain relief was obtained over several days after the load decreased again. After the second injection at week 7, the unloading changed from 7% (compared to pre-OA load) to 2% compared to reaching the pre-OA load. Therefore, the positive and almost complete normalization of the loading pattern of the affected OA joints was again confirmed by the IL4-IL10 fusion protein. The asterisk (*) indicates p = 0.05 compared to the pre-injection value, while the # indicates p < 0.05 compared to the baseline value. A curved dashed line indicates the natural progression of OA pain (unloading) without treatment.

[0142] Figure 11This study illustrates the time course of carrageenan-induced thermal hyperalgesia in mice treated with the IL4-IL10 fusion protein. The latency of the heat-shrink paw response was determined using a Hargreaves assay. Mice received intrapedicular injections of carrageenan (see the first arrow on the left), and the reduction in the latency of the heat-shrink paw response was measured. Intrathecal injection of the IL4-IL10 fusion protein (see the second arrow on the left) significantly reduced the hyperalgesic response to intrapedicular carrageenan. Although the reduction in the latency of the heat-shrink paw response shown in mice treated with the IL4-IL10 fusion protein remained significantly smaller than that in saline-treated mice after 48 hours, the effect of the IL4-IL10 fusion protein was clearly less pronounced after 2 days. Data are presented as mean ± SEM. * indicates p < 0.05; ** indicates p < 0.001.

[0143] Figure 12 The Hargreaves assay was used to determine the latency of the heat-shrink paw response. Mice received intrapedicular injections of carrageenan, and the reduction in the latency of the heat-shrink paw response was measured. Six days after induction of hyperalgesia, intrathecal injections (see arrow) of IL4 or IL10 (A) or the IL4-IL10 fusion protein (B) were administered. Both IL4 and IL10 slightly reduced the hyperalgesic response to carrageenan pain in the paw, but the combined effect of IL4 and IL10 was negligible compared to the effect of the IL4-IL10 fusion protein. The effects of IL4 or IL10 alone lasted for 1 day, while the effects of the IL4-IL10 fusion protein lasted for a much longer period, up to 4 days. Data are presented as mean ± SEM. * indicates p < 0.05; ** indicates p < 0.001.

[0144] Figure 13 The IL4-IL10 fusion protein inhibited LPS-induced cytokine release in whole blood cultures. Heparinized blood from healthy volunteers was diluted 1:10 in culture medium and cultured with 10 ng / mL LPS in the presence of multiple pools containing different concentrations of the IL4-IL10 fusion protein construct. TNFα in the supernatant was measured by ELISA. Results are expressed as % inhibition. TNF production was 0% inhibited in the absence of the IL4-IL10 fusion protein. Compared to pools 1 and 3, where the IL10 c-terminus is linked to the n-terminus of IL4, pools 2 and 4, where the IL4 c-terminus is linked to the n-terminus of IL10, showed the highest inhibition of TNFα production at similar concentrations. This indicates that the functionality of the IL4-10 fusion protein depends on the way individual cytokines are linked within the IL4-IL10 fusion protein.

[0145] Example

[0146] Example 1. Transfection of HEK293 cells

[0147] Methods: HEK293 cells were transiently transfected with a transgenic vector according to standard methods (Y Derocher et al., Nucleic Acids Research 2002, Vol. 30, no. 2, e9). Briefly, the IL4-IL10 fusion protein insert was cloned into a pUPE expression vector containing a serum cystatin signal sequence. HEK293E cells were then transfected with the pUPE expression vector containing the IL4-IL10 fusion protein of this invention. Simultaneously, cells were co-transfected with a vector carrying a transgene for human β-galactosyltransferase 5 (SIAT 9) to optimize sialic acid-based capping of the glycan. Cells were cultured in FreeStyle medium (Invitrogen) containing 0.9% primatone and ~0.04%, v / v fetal bovine serum. Five days after transfection, the conditioned medium was collected by low-speed centrifugation, then concentrated using a 10 kDa QuixStand hollow fiber column (GE Healthcare), and dialyzed with phosphate-buffered saline (PBS).

[0148] Example 2. ELISA assay for immunochemical detection of IL4-IL10 fusion protein, IL4, and IL10.

[0149] methodAccording to the manufacturer's instructions, the amounts of IL4 and IL10 in culture supernatant or chromatographic fractions are measured using an ELISA (IL4 PeliPair ELISA Kit; Sanquin, Amsterdam, the Netherlands; Cat#M9314 or IL10 PeliPair ELISA Kit; Sanquin; Cat#M9310). In short, catching antibodies against IL4 or IL10 are diluted 1:200 in phosphate-buffered saline (pH 7.4 (PBS)) and plated overnight on an ELISA plate. All subsequent steps are performed in PBS supplemented with 0.1% w / v, Tween-20 (PBS-T). Dose-response curves are detected based on serial dilutions to obtain recombinant IL4 or IL10 from 100 to 2 pg / ml. The binding antibody was detected using streptavidin-poly-HRP (Sanquin), followed by incubation with TMB (3,3',5,5"-tetramethylbenzidine; Invitrogen, Carlsbad, CA, USA; Cat#SB02). The reaction was terminated with 1M sulfuric acid (Chem Lab; Cat#_CL05-2658-1000). The ELISA results were compared with control curves using recombinant IL4 and IL10 dilutions provided by the manufacturer.

[0150] A cross-ELISA for the specific detection of the IL4-IL10 fusion protein was performed using an anti-IL4-coated plate and a biotinylated anti-IL10 monoclonal antibody. The cross-ELISA was performed with the same precision as the IL10 ELISA, except that an anti-IL4-coated plate was used instead of an anti-IL10-coated plate. The anti-IL4-coated plate was prepared precisely as described for the IL4 ELISA. Since there are no standards for this assay, the results are given as OD. In this IL4-L10 fusion protein-specific cross-ELISA, a fixed amount of supernatant equivalent to 75 pg / ml of control recombinant IL10 and the IL4-IL10 fusion protein was detected.

[0151] result Detection of IL4-IL10 fusion protein. Two ELISA assays (see...). Figure 1 and Figure 2 Dose-response curves were obtained from the culture supernatant of transfected HEK293 cells, indicating the presence of IL4 and IL10 proteins with a structural conformation recognizable by the monoclonal antibodies used in ELISA. Figure 1Then, a modified ELISA (cross-ELISA) was used to specifically measure the IL4-IL10 fusion protein without measuring recombinant wild-type cytokine molecules (see [link to ELISA]). Figure 2 (Figures A and B). When the equivalent of 75 pg / ml of recombinant IL10 and IL4-IL10 fusion protein was measured in this cross-ELISA, only the IL4-IL10 fusion protein emitted a signal, while IL10 did not. Figure 2 Figures A and B) are not present. Therefore, these results confirm that only the supernatant obtained from HEK293 cells (transfected with the sequence of the IL4-IL10 fusion protein of the present invention) contains a protein in which the IL4 and IL10 sequences are linked together.

[0152] Example 3. SDS-Page and Western Blotting

[0153] method Samples were diluted 1:1 in sample buffer containing 710 mM 2-mercaptoethanol (Tris-HCl pH 6.8, 25%, w / v, glycerol, 2%, w / v, SDS, 0.01%, w / v, bromophenol blue; BioRad, Richmond, VA, USA, Cat#161-0737) and incubated at 100 °C for 10 min. Next, samples were loaded onto 7.5%, w / v, polyacrylamide Tris / polyacrylamide gels (SDS-free Mini-PROTEAN TGX Precast Gels; BioRad, Cat#456-1023). Molecular weight markers (Western C Standard, 250-10 kD; BioRad; Cat#161-0376) were run in separate lanes. Electrophoresis was performed under reducing conditions using Tris / glycine / SDS buffer (BioRad; Cat#_161-0732). To identify the IL4-IL10 fusion protein, Western blotting was performed with anti-IL4 or anti-IL10 antibodies. Proteins were separated on SDS-PAGE as described above, and then transferred to a PVDF-membrane (BioRad; Cat#_161-0277) at 100V for 1 hour using a protein blot in Tris / glycine buffer (BioRad; Cat#_161-0734).

[0154] Following the blot, the membrane was incubated in PBS-T containing 4%, w / v milk powder (Elk milk powder; Campina, Zaltbommel, Netherlands) to block any remaining binding sites. The PVDF membrane was then washed 3× in PBS-T and incubated for 1 hour with primary antibodies (mouse IgG1 anti-human IL4; Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat#SC80093 or mouse IgG1 anti-human IL10; Santa Cruz; Cat#SC32815) in 1% w / v milk (dissolved in PBST). After another washing step, the blot was incubated for 1 hour with secondary antibodies (horse-horseradish peroxidase (HRP)-bound goat anti-mouse IgG; Santa Cruz; Cat SC2005) and Western C-labeled detection antibody (StrepTactin-HRP; BioRad; Cat#161-0382) in PBST-1% milk. ECL solution (GE Healthcare, Diegem, Belgium, Cat#RPN2132) was added to the washed membrane, which was then transferred to a cartridge and developed using a Kodak Imager for 15 minutes. To further characterize the IL4-IL10 fusion protein, the culture supernatant was also treated with PNGase F (Sigma Aldrich, cat#_G5166) according to the manufacturer's instructions to deglycosylate the IL4-IL10 fusion protein, followed by analysis on SDS-PAGE and Western blotting as described above.

[0155] result Both wild-type IL4 and IL10 migrate, exhibiting relative migration (Mr) consistent with MW < 20kD. Figure 3 Lanes 4 and 5 of the blot are shown in A and B. Based on the supernatant obtained from transfected HEK293, the IL4-IL10 fusion protein migrated as a double band with Mr consistent with MW ~30~35kD. Both bands were resisted by anti-IL4 (…). Figure 3 A) and anti-IL10 monoclonal antibody ( Figure 3 B) is identifiable, therefore both represent variants of the IL4-IL10 fusion protein, presumably of different glycoforms. Notably, no glycoforms corresponding to either recombinant wild-type IL4 or IL10 were detected. Figure 3 The bands of Mr were observed in lanes 2) of A and 3B. These results confirmed that only the IL4-IL10 fusion protein was detected in the supernatant of transfected HEK293 cells, without the individual cytokines (i.e., IL4 and IL10). To confirm this, Figure 3The double bands described in lanes 2 of swim bladders A and 3B are glycosylated. The supernatant containing the IL4-IL10 fusion protein was treated with PNGase F for deglycosylation, and the supernatant was compared with the untreated supernatant by Western blotting. The results showed that only one band was detected after deglycosylation (see [link to original text]). Figure 3 Lane 3 in the two imprints shown in A and 3B confirms the double bands seen in lane 2 of both imprints. Figure 2 A and B) are actually the IL4-IL10 fusion protein of the present invention, but in glycosylated form.

[0156] Example 4. Gel filtration-size exclusion chromatography (SEC) of the IL4-IL10 fusion protein

[0157] method To determine the molecular weight of the IL4-IL10 fusion protein, high-performance size exclusion chromatography (HP-SEC) was performed. Gel filtration was performed on a Shimadzu HPLC system (BioSuite 125 4μm UHRSEC Column; Waters; Cat#186002161) with a mobile phase of 20mM phosphate buffer containing 0.5M NaCl. The column was calibrated prior to run using thyroglobulin, bovine serum albumin, carbonic anhydrase, myosin, and ribonuclease. The IL4-IL10 fusion protein was purified by cation exchange by pooling and concentrating the fraction with the highest IL4-IL10 fusion protein content (2 ml pooled fraction was concentrated to 100 μl, containing 2 μg of IL4-IL10 fusion protein). 50 μl of the 20 μg / ml pooled IL4-IL10 fusion protein was injected and passed through the column at a flow rate of 0.35 ml / min and a pressure of 35 bar. A 175 μl fraction was collected, and IL4 and IL10 levels were measured in the IL4 and IL10 ELISA (1 / 500 dilution) described above. Similar runs were performed using recombinant human IL4 (Sigma, Cat#_I4269) and recombinant human IL10 (Sigma, Cat#_I9276) to compare the molecular size of the IL4-IL10 fusion protein with that of wild-type cytokines.

[0158] result The HP-SEC elution pattern of IL4 indicated that IL4 exists as a monomer (~15 kDa). The elution pattern of IL10 showed that the cytokine exists in a dimer form (~40 kDa), which is the naturally occurring form of IL10 (Zdanov et al., Stucture 1995, 3:591-601). The pattern of the IL4-IL10 fusion protein indicated that it exists primarily in a dimer form (~70 kDa). Figure 4 ).

[0159] Example 5. Assay for measuring pro-inflammatory cytokines

[0160] method TNFα production was measured using a commercially available ELISA kit (TNF-α Pelipair ELISA Kit; Sanquin, Amsterdam, the Netherlands; Cat#M9323) according to the manufacturer's instructions. In short, each well was plated with anti-TNFα capture antibody diluted 1:150 in carbonate / dicarbonate buffer (pH 9.6), washed three times with PBS-T, and incubated with 2.5% w / v bovine serum albumin (BSA; Roche Applied Science, Mannheim, Germany, Cat#10735108001) in PBS to block any remaining binding sites on the plate. After another washing step, each well was incubated with the sample diluted in PBS-T. A standard curve of recombinant TNF-α at concentrations of 200–1.56 pg / ml in PBS-T was used for reference. Recombinant TNF-α was provided with the kit. Finally, TNF-α binding was detected by incubation in biotinylated anti-TNF-α and streptavidin-poly-HRP (Sanquin; Cat#_2032) in PBS-T. Bound HRP was visualized using TMB (3,3'5,5"-tetramethylbenzidine; Invitrogen; Cat#SB02). For ELISA, 1M sulfuric acid (Chem Lab; Cat#_CL05-2658-1000) was added. Results were compared with the standard curve of recombinant TNF-α and expressed as pg / ml. IL-6 and IL-8 (purchased from Sanquin) and IFNγ and IL-17 (purchased from Biosource) were measured using a similar ELISA method.

[0161] Example 6. Assay for measuring the activity of IL4-IL10 fusion protein, IL4, and IL10.

[0162] methodThe release of cytokines (IL6, IL8, TNFα) induced by lipopolysaccharide (LPS) in whole blood was used as a functional assay for IL4 and IL10. Heparinized human blood was obtained from healthy volunteers and diluted 1:10 in RPMI medium (Glutamax; Invitrogen, Cat#_61870010) supplemented with Pen / Strep (PAA Laboratories, Pasching, Austria; Cat#P11-013). LPS (lipopolysaccharide; Sigma; Cat#_L4391) was added to obtain a final concentration of 10 ng / mL. The IL4-IL10 fusion protein was added to a final concentration of 100 ng / mL. Recombinant human IL4 (Sigma, cat#_i4269) and recombinant human IL10 (Sigma, Cat#_I9276) were added to a final concentration of 50 ng / mL each as controls. To confirm the activities of IL10 and IL4, receptor blocking antibodies against human IL4-receptor (a-hIL4-R; R&D Systems; Minneapolis, MN, USA, Cat#MAB230) and human IL10-receptor (a-hIL4-R; R&D Systems; Minneapolis, MN, USA, Cat#MAB230) were added at final concentrations of 10 μg / ml and 20 μg / ml, respectively. Whole blood cultures were then cultured at 37°C for 18 hours, with the supernatant collected and stored at -80°C until cytokine detection was performed.

[0163] result The level of TNFα present in the supernatant was measured using ELISA and expressed as the % inhibition of TNFα. Figure 5 A). The results showed that in the absence of the IL4-IL10 fusion protein, TNFα production was inhibited by 0% ( Figure 5 A). When testing similar volumes of buffer lacking the IL4-IL10 fusion protein, no inhibition of TNFα was observed. Figure 5 A).

[0164] The inhibition of LPS-induced TNFα, IL6, and IL8 production by the IL4-IL10 fusion protein or the combination of IL4 and IL10 was also measured using ELISA. Figure 5B). The concentrations of TNFα, IL6, and IL8 present in the supernatant are given on the Y-axis. The IL4-IL10 fusion protein was detected at 50 ng / mL and compared with the effects of a mixture of recombinant human IL4 and IL10 at a final concentration of 25 ng / mL each. The results showed that, relative to the control (i.e., cytokine-free medium), both the IL4-IL10 fusion protein and the combination of IL4 and IL10 significantly inhibited LPS-induced TNF, IL6, and IL8 production. The effect of the IL4-IL10 fusion protein on LPS-induced IL6 production was completely eliminated relative to the control (e.g., medium only). However, compared to the control case (e.g., culture medium only), when one of the two parts (i.e., IL4 or IL10) is blocked by a receptor blocking antibody against human IL4-receptor (a-hIL4-R) or human IL-10-receptor (a-IL10-R), the effect of the IL4-IL10 fusion protein on LPS-induced IL6 production is partially eliminated.

[0165] Example 7. The role of the IL4-IL10 fusion protein in balancing pro-inflammatory responses (Th1 and Th17 cytokine-expressing cells) and T-cell activity (FoxP3-expressing CD4 T cells).

[0166] method The activation of bone marrow cells and B cells depends critically on the pro-inflammatory Th1 and Th17 responses and the T-cell homeostasis of FoxP3-expressing CD4 T cells. To evaluate the effect of the IL4-IL10 fusion protein on T-cell cytokine production, monocytes from peripheral blood (PBMCs) were isolated from healthy donors. Briefly, blood was diluted 1:1 with RPMI 1640 medium containing penicillin (100 U / ml, Yamanouchi, Leiderdorp, The Netherlands), streptomycin (100 mg / ml, Fisiopharma, Milan, Italy), and glutamine (2 mM, Gibco BRL). PBMCs were isolated by Ficoll-Paque density gradient centrifugation (Pharmacia, Uppsala, Sweden). PBMCs (5.10) were then centrifuged at 37°C. 5PBMCs were cultured for 3 days in RPMI / glutamax (Gibco BRL) supplemented with penicillin (100 U / ml), streptomycin (100 mg / ml), and 10% pooled fetal bovine serum (FCS, Gibco BRL). PBMCs were cultured with or without the superantigen staphylococcal enterotoxin B (SEB, 1 ng / mL) and / or IL4-IL10 fusion protein. After this culture period, the supernatant was collected, cell-free, and stored at -80°C until IFNγ, IL17, and TNFα were detected by ELISA. Cell proliferation was also measured by 3H-thymidine binding. During the last 18 hours of the 3-day cell culture, 3H-thymidine (5 mCi / ml, NEN LifeScience Products, Amsterdam, The Netherlands) was added to each well. After this culture period, cells were collected, and 3H-thymidine binding was measured by liquid scintillation counting, expressed as counts per minute (CPM). Furthermore, intracellular FoxP3 staining was performed using an APC-conjugated rat anti-human FoxP3 staining apparatus (eBioscience, San Diego, USA). For intracellular staining, an APC-labeled rat isotype control antibody (eBioscience) was used. The percentage of positive / negative cells was determined based on the markers used in the isotype control assay. Cells were collected using a FACScan flow cytometer, and data were analyzed using FlowJo software, version 7.5 (Tree Star Inc., Oregon, USA).

[0167] The synergistic effect of the IL4-IL10 fusion protein on the expression of the inhibitory Fcγ receptor (FcγRIIb) on monocytes was also examined. This was compared with the regulation of activation of FcγRI and FcγRIII. PBMCs (1.10 μL) were cultured at 37 °C in RPMI / glutamax (Gibco BRL) supplemented with penicillin (100 U / ml), streptomycin (100 mg / ml), and 10% pooled human AB serum (Gibco BRL). 6PBMCs were cultured for 4 days with or without the superantigen SEB (1 ng / mL) and / or the IL4-IL10 fusion protein. Following this culture period, FcγR expression was evaluated by FACS analysis. For FACS analysis, monocytes were cultured with APC-labeled CD14 mAb, FITC-labeled anti-FcγRI and anti-FcgRIII mabs (Pharmingen), and FITC-labeled anti-FcgRIIb mab (2B6, Genmab, Utrecht, Netherlands). Cells were collected using FACScan flow cytometry, and data were analyzed using FlowJo software, version 7.5 (Tree Star Inc., Oregon, USA).

[0168] result SEB induces significant proliferation, which is associated with the upregulation of FoxP3 expression. Both proliferation and FoxP3 expression are almost unaffected by the IL4-IL10 fusion protein (which is respectively...). Figure 6 (Figures A and B). Conversely, Th1 and Th17 cytokines IFNγ (left), IL-17 (middle), and TNFα (right) were all strongly reduced by the IL4-IL10 fusion protein and the combination of IL4 and IL10. Figure 6 C). In summary, this confirms that the IL4-10 fusion protein strongly alters the balance between inhibitory regulatory CD4 T cells and pro-inflammatory Th1 and Th17 cells.

[0169] Example 8. Effects of the IL4-IL10 fusion protein on the balance between activated Fcγ receptors I and III and inhibitory FcγRIIb.

[0170] method The balance between activated and inhibitory receptors of IgG (FcγRs) plays a crucial role in immune complex-mediated activation of bone marrow cells and lymphocytes. To evaluate the effect of the IL4-IL10 fusion protein on FcγR expression on monocytes, monocytes derived from peripheral blood (PBMCs) were isolated from healthy donors. PBMCs (5.1 ng / mL) were treated with the superantigen staphylococcal enterotoxin B (SEB) (0.1 ng / mL) and / or the IL4-IL10 fusion protein. 5 T-cell-dependent monocyte activation was induced by inducing FcγR expression for 2 days ( / ml). FcγR expression was measured after this culture period.

[0171] result IL10 upregulates the expression of FcγRI, FcγRIIa and FcγRIII. Figure 7Under these conditions, IL4 showed a slight decrease in the expression of these activated FcγRs compared to cells cultured in the absence of cytokines. The combination of IL4 and IL10 with the IL4-IL10 fusion protein normalized the expression of FcγRI and FcγIIa. Although a slight increase in FcγRIII was observed with the combination of IL4 and IL10 with the IL4-IL10 fusion protein, this was negligible compared to the upregulation of this receptor induced by IL10 alone. IL10 alone showed a slight increase in the expression of inhibitory FcγRIIb, while IL4 alone showed a slight decrease in the expression of this receptor. The combination of IL4 and IL10 with the IL4-IL10 fusion protein did not alter the expression of inhibitory FcγRIIb. Figure 7 In summary, this confirms that the IL4-IL10 fusion protein of the present invention stably activates the expression of FcγRs, thereby inhibiting immune complex-induced immune activation.

[0172] Example 9. Cartilage culture for blood-induced cartilage injury

[0173] method With the approval of the medical ethics board of University Medical Centre Utrecht, articular cartilage tissue from healthy individuals was obtained from humeral head autopsies within 24 hours of donor death. Donors (n=8; mean age 69.8±8.7 years, 3 males, 5 females) had no known history of joint disease. Full-thickness cartilage sections were aseptically excised from the humeral head (excluding the basal bone) and preserved in phosphate-buffered saline (PBS, pH 7.4). Within 1 hour of autopsy, the sections were aseptically cut into small full-thickness cubic explants and weighed (range 5–15 mg, accuracy ±0.1 mg). The explants were cultured individually in 96-well round-bottom microtiter plates (at 5% CO2 in air, pH 7.4, 37°C, and 95% humidity). The culture medium consisted of glutamine (2 mM), penicillin (100 IU / mL), streptomycin sulfate (100 μg / mL; PAA), ascorbic acid (85 μM; Sigma), and 10% heat-inactivated monosodium glutamate (MSG). + The serum (Gemini Bioproducts) consists of Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen).

[0174] For each experiment, fresh blood was drawn from healthy human donors (n=8, mean age 28.0 ± 5.0 years, 2 males and 6 females) into collection tubes (nr. 367895; Becton Dickinson). To simulate human joint bleeding, cartilage was exposed to 50% v / v whole blood for 4 days, which was considered the time for blood to drain naturally from the joint cavity. After blood exposure, the cartilage grafts were washed twice under culture conditions for 45 minutes to remove all additives and then cultured for another 12 days. The culture medium was refreshed every 4 days. In the first experimental setup using cartilage and blood from five of eight donors, dose-response profiles of the IL4-IL10 fusion protein were prepared by adding concentrations of 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 ng / mL (n=5) of the IL4-IL10 fusion protein during blood exposure. In separate experiments, the optimal concentration of 10 ng / mL of the IL4-IL10 fusion protein was compared with similar concentrations of IL10 plus IL4 combinations and individual components (each 10 ng / mL, n=8).

[0175] As a measure of proteoglycan synthesis rate, proteoglycans are one of the main cartilage matrix components. The sulfate rate of added glycosaminoglycans (GAGs) was determined. At the end of each experiment, 74 kBq of Na2 was added to each well. 35 SO4 (NEX-041-H without carrier; DuPont). Four hours after pulse labeling of newly formed sulfurized GAG, cartilage samples were washed twice in cold PBS and stored at -20°C. Thawed samples were digested with 2% papain (Sigma) at 65°C for 2 hours. The rate of proteoglycan synthesis was determined by precipitating GAG with 0.3M cetylpyridine chloride monohydrate (CPC; Sigma) in 0.2M NaCl. The precipitate was dissolved in 3M NaCl, and the amount of radioactivity was measured by liquid scintillation analysis. Radioactivity counts were normalized to the specific activity of the culture medium, labeling time, and wet weight of the cartilage. Results are expressed as nanomoles of sulfate added per gram of wet cartilage tissue (nmol / h*g).

[0176] The proteoglycan content and release of proteoglycans into the culture medium of each cartilage graft were determined by staining and by applying papain digest to the cartilage samples and Alcian Blue (Sigma) precipitation of GAG in the culture medium, respectively. Chondroitin sulfate (Sigma) was used as a reference, and staining was quantified by absorbance measurement at 620 nm. For content and release, the results are expressed as mg GAG / wet weight of cartilage tissue (mg / g) and mg GAG / wet weight of tissue released over 4 days (mg / g), respectively. Due to focal differences in cartilage composition and bioactivity, proteoglycan recycling parameters were measured in 10 cartilage grafts from the same donor, obtained randomly and processed separately. The average value of these 10 samples was used as the representative value for the cartilage donor.

[0177] result Cartilage exposure to 50% v / v blood for 4 days significantly reduced the rate of proteoglycan synthesis by 74%. Figure 8 A; p = 0.043). The addition of the IL4-IL10 fusion protein resulted in a dose-dependent recovery of the proteoglycan synthesis rate above 0.1 ng / mL of the IL4-IL10 fusion protein (onwards), and a significant increase in the proteoglycan synthesis rate was observed compared to blood exposure without the IL4-IL10 fusion protein (all p = 0.043). Figure 8 A). By adding IL4-IL10 fusion protein at a concentration of 0.1 ng / mL or higher, the 62% increase in proteoglycan release due to blood exposure was statistically significant and reversible (returning to control levels). Figure 8 B, all p = 0.043 except for 1 ng / mL, p = 0.080). This will lead to normalization of proteoglycan release, meaning that different forms of control cultures will no longer be different. When cartilage grafts are exposed to blood, the total proteoglycan content decreases by 11% ( Figure 8 C; p = 0.043). The decrease in blood exposure was inhibited by adding IL4-IL10 fusion protein at concentrations above 0.3 ng / mL (all p = 0.043, except for 1 and 100 ng / mL, both of which had p = 0.080).

[0178] To compare the effects of the IL4-IL10 fusion protein on the combined and individual components of cytokines, the IL4-IL10 fusion protein, IL4, IL10, and combinations thereof (10 ng / mL each) were measured in the same assay. Due to blood exposure, the rate of proteoglycan synthesis decreased by 76%. Figure 9A; p = 0.012). Both IL10 and IL4 significantly increased the rate of proteoglycan synthesis compared to blood (p = 0.017 and p = 0.012, respectively). Use of the IL4-IL10 fusion protein at the same concentration also increased the rate of proteoglycan synthesis, thus proving equally effective as the combination of the two individual cytokines (241% and 245%, respectively, compared to blood exposure). Furthermore, the effect of the IL4-IL10 fusion protein was statistically significantly better than that of IL10 alone (p = 0.025). There was no significant difference in the rate of proteoglycan synthesis between cultures using the IL4-IL10 fusion protein, the combination of the two cytokines, and IL4 alone compared to the control. Complete recovery, or normalization, of the blood-induced inhibition of proteoglycan synthesis was achieved. Blood exposure to cartilage increased proteoglycan release by 59% (…). Figure 9 B; p = 0.017). The addition of IL10 reduced this enhanced release (p = 0.012 compared to blood). When compared to blood exposure, IL-4, the combination of IL-4 and IL-10, and the IL4-IL10 fusion protein greatly reduced the release (all p = 0.012), and were equally effective with combinations of individual cytokines (p = 0.611).

[0179] Cartilage exposed to blood showed a 10% decrease in proteoglycan content. Figure 9 C; p = 0.012). The cytokines IL4 and IL10 alone did not prevent this decrease (p = 0.093 and p = 0.327, respectively, compared to blood exposure). However, the IL4-IL10 fusion protein significantly increased proteoglycan content compared to blood exposure without the additive (p = 0.012).

[0180] Example 10. Canine Groove Model for Pain and Osteoarthritis

[0181] methodThe effects of the IL4-IL10 fusion protein on pain and functional capacity in the canine Groove model of osteoarthritis. The Groove model's characteristics reflect those of human osteoarthritis (OA), making it a suitable model for studying human OA. The Groove model is unique because degenerative cartilage changes are progressive, eventually leading to osteoarthritis (OA), while synovial inflammation decreases over time. Therefore, the evaluation of the direct effects of drugs on cartilage degeneration and pain can be less influenced by the possible indirect effects on inflammation. Furthermore, this model is unique because there are no long-term triggers for joint damage, making it more sensitive to treatment. Long-term triggers for joint damage, such as joint instability used in other OA (canine) models, would inhibit the potential beneficial effects of treatment. In conclusion, the Groove model is suitable for detecting the therapeutic effects of the IL4-IL10 fusion protein on cartilage damage and pain-induced disability via OA. Pain and functional capacity are considered very important parameters in clinical osteoarthritis research because these parameters, rather than structural changes, compel patients to seek medical care. In canine models, changes in braking, uprighting, and ground reaction forces (FRAP) indicators of pain and functional capacity can be evaluated using force-plate analysis (FPA). Joint loading is influenced by pain and functional capacity, depending on the stage of joint degeneration. A significant reduction in unloading was observed in the first two weeks following OA induction, likely due to surgery-related pain. However, after three weeks, more stable unloading of the affected limb was observed as a result of OA-related pain.

[0182] Osteoarthritis (OA) was induced in the right knee of four Mongrels dogs (hybrid, skeletally mature) using the Groove model. Ten longitudinal and diagonal grooves, 0.5 mm deep, were created on the weight-bearing portion of the femoral ankle. Hemorrhage and soft tissue injury were minimized to prevent the inflammatory component from dominating, as in inflammatory-driven models and specific arthritis models. Following surgery, the synovium, fascia, and skin were sutured. The unoperated contralateral knee served as a control. Five weeks after OA induction, an intra-articular injection of 1 ml of IL4-IL10 fusion protein (1 μg / ml) was administered (see first arrow on the left). Two weeks (7 weeks) after the first injection, a second injection of a higher dose (10 μg / ml) was administered (see second arrow on the left).

[0183] Ground gait pattern, as a measure of pain and functional ability, was evaluated using force-plate analysis (FPA). In canine models, longitudinal variations in braking, erection, and throbbing reaction forces (GRFs) for each leg were assessed using FPA. The force-plate (FP) was embedded on the surface of the peak GRF at a sampling (100 Hz) level of 11 m channels. Forces were normalized by weight and time and expressed in N / kg. A single trainer guided the dog on the FP at a constant speed (1 ± 0.2 m / s) using a leash. A successful run was defined as consecutive, distinct paw strikes of the right foreleg and right hind paw, or left foreleg and left hind paw, respectively. Ten correct runs were collected from each side of the dog, and the GRFs for each of the four legs were averaged. FPA was performed every two weeks from 3 weeks before induction to 8 weeks after induction. Additional daily FPA was performed after infusion of the IL4-IL10 fusion protein (weeks 5 and 7).

[0184] Results: The results showed that after the first injection of the IL4-IL10 fusion protein, as indicated by the peak signal of standing force, the load of the OA joint (experimental joint relative to the contralateral control joint) was almost normalized (inhibited by 2% compared with the pre-OA load) compared to the pre-injection level (9% inhibition compared with the pre-OA load), as indicated by the peak signal of standing force. Figure 10 Several days after the load was reduced again, this effect of load reduction was observed, showing pain relief. Following the second injection at week 7, a positive effect of the IL4-IL10 fusion protein was also seen on the loading pattern of the affected OA joints. This was demonstrated by the reduction in unloading from -7% (compared to pre-OA load) to -2%, almost completely normalizing the condition again. Therefore, in this canine model of OA, the IL4-IL10 fusion protein was able to reduce pain.

[0185] Example 11. Canine Groove Model for Pain and Osteoarthritis

[0186] methodOsteoarthritis (OA) was induced in the right knee of Mongrels dogs (hybrid, skeletally mature) using the Groove model. Ten longitudinal and diagonal grooves, 0.5 mm deep, were created on the weight-bearing portion of the femoral ankle. Hemorrhage and soft tissue injury were minimized to prevent the inflammatory component from dominating, as in control models of inflammation-driven and specific arthritis. Following surgery, the synovium, fascia, and skin were sutured. The unoperated contralateral knee served as a control. Dogs were divided into two groups. Group 1 received an intra-articular injection of IL4-IL10 fusion protein 5 weeks after OA induction. Group 2 received an intra-articular injection of both IL4 and IL10, but in free form, 5 weeks after OA induction. Two weeks after the first injection (week 7), a second injection was given, with higher doses than those given to Group 1 (10 μg / ml IL4-IL10 fusion protein) and Group 2 (5 μg / ml IL4 and 5 μg / ml IL10).

[0187] Ground gait patterns, as a measure of pain and functional ability, were evaluated using force-plate analysis (FPA). In a canine model, longitudinal variations in braking, erection, and throbbing reaction forces (GRFs) for each leg were assessed using FPA. The force-plate (FP) was embedded on the surface of the peak GRF at a sampling (100 Hz) frequency of 11 m channels. Forces were normalized by weight and time and expressed in N / kg. A single trainer guided the dog on the FP at a constant speed (1 ± 0.2 m / s) using a leash. A successful run was defined as consecutive, distinct paw strikes of the right foreleg and right hind paw, or left foreleg and left hind paw, respectively. Ten successful runs were collected from each side of the dog, and the GRFs for each of the four legs were averaged. FPA was performed every two weeks from 3 weeks before induction to 8 weeks after cessation. In both groups, additional daily FPA was performed at weeks 5 and 7 following injection of the IL4-IL10 fusion protein (Group 1) and injection of free forms of IL4 and IL10 (Group 2).

[0188] result The results showed that after two injections (weeks 5 and 7), treatment with either the IL4-IL10 fusion protein (Group 1) or the combination of free IL4 and IL10 (Group 2) had a positive effect on the loading pattern of the affected OA joints. However, at weeks 5 and 7, treatment with the IL4-IL10 fusion protein showed a significantly greater improvement in the loading pattern of the affected OA joints compared to treatment with the combination of free IL4 and IL10. The effects of treatment with the IL4-IL10 fusion protein were also observed to be more durable over time compared to treatment with the combination of free IL4 and IL10. Therefore, the IL4-IL10 fusion protein of this invention significantly reduces pain in a canine model of OA compared to the effects obtained with the combination of free IL4 and IL10.

[0189] Example 12. Carrageenan-induced hyperalgesia model in rodents.

[0190] method On day 0, hyperalgesia was induced in female C57BL / 6 mice by intrapedicular injection of 5 μl λ-carrageenan diluted in saline (2% w / v; Sigma-Aldrich, St. Louis, MO, USA) in the hind paw. Intrapedicular injection of saline alone did not induce detectable hyperalgesia. Response to infrared thermal stimulation was measured as the paw withdrawal latency using the Hargreaves test (IITC Life Science Woodland Hills, CA). The beam intensity was selected to induce a baseline paw withdrawal latency of approximately 8 seconds. The baseline paw withdrawal latency was measured over three consecutive days. Hyperalgesia was confirmed by a sustained decrease in paw withdrawal latency for at least 10 days following carrageenan injection. On day 6, mice received a single intrathecal injection of IL4 (100 ng), IL10 (100 ng), or the IL4-IL10 fusion protein (40, 100, and 200 ng), or a carrier (saline) (see [link to relevant documentation]). Figure 11 and 12 (The arrow in the image).

[0191] result The decreased latency of the withdrawal response indicates that mice exhibit hyperalgesia up to 6 days after carrageenan injection. Figure 11 On day 6, mice received a single intrathecal injection of 100 ng of the IL4-IL10 fusion protein (n=4) or saline as a control (n=4). Following injection of the IL4-IL10 fusion protein, inhibition of hyperalgesia was confirmed by a decrease in the claw withdrawal latency value returning to baseline. Figure 11 The effect of a single dose of the IL4-IL10 fusion protein lasted up to 2 days. After 48 hours, the effect decreased, but the reduction in latency was still significantly different from that in control saline-treated mice. Figure 11 ).

[0192] In supplemental experiments using the same methods described above, carrageenan-induced thermal hyperalgesia was also evaluated in mice treated with IL4, IL10, or the IL4-IL10 fusion protein. Specifically, on day 6 following the induction of hyperalgesia, intrathecal injection of IL4 or IL10 (A) or the IL4-IL10 fusion protein (B) was administered. Figure 12 Although both IL4 and IL10 slightly reduced the hyperalgesic response to carrageenan pain in the sole of the foot, this effect was negligible compared to the effect of the IL4-IL10 fusion protein. Figure 12The effects of IL4 or IL10 alone last for 1 day, while the effects of the IL4-IL10 fusion protein last for a much longer period, up to 4 days. Figure 12 B).

[0193] Example 13: Carrageenan-induced hyperalgesia model in mice

[0194] Methods: On day 0, hyperalgesia was induced in female C57BL / 6 mice by intrapedicular injection of 5 μl λ-carrageenan diluted in saline (2% w / v; Sigma-Aldrich, St. Louis, MO, USA) in the hind paw. Intrapedicular injection of saline alone did not induce detectable hyperalgesia. Response to infrared thermal stimulation was measured using the Hargreaves test (IITC Life Science Woodland Hills, CA), as the latency of paw withdrawal. The beam intensity was selected to induce a baseline paw withdrawal response latency of approximately 8 seconds. The baseline paw withdrawal response latency was measured over three consecutive days. Hyperalgesia was confirmed by a sustained decrease in paw withdrawal response latency for at least 10 days following carrageenan injection. On day 6, mice received a single intrathecal injection of IL4-IL10 fusion protein (40, 100 and 200 ng), or a solution containing a combination of free IL4 (100 ng) and IL10 (100 ng), or a carrier (saline).

[0195] result The results showed that, compared to the control (vector treatment), treatment with the IL4-IL10 fusion protein or the combination of free IL4 and IL10 significantly reduced hyperalgesia. However, treatment with the IL4-IL10 fusion protein exhibited a significantly greater inhibitory effect on hyperalgesia compared to treatment based on the combination of free IL4 and IL10. Furthermore, the effect of a single dose of the IL4-IL10 fusion protein on hyperalgesia tolerance was observed to be significantly longer over time than the effect of equivalent doses of individual cytokines (i.e., the combination of free IL4 and IL10).

[0196] Example 14. Activity of IL4-IL10 fusion protein on LPS-induced TNF production in whole blood cultures

[0197] methodThe activity of the IL4-IL10 fusion protein was functionally determined using lipopolysaccharide (LPS)-induced cytokine release (TNFα) from whole blood. Heparinized human blood was obtained from healthy volunteers and diluted 1:10 in RPMI 1640 medium (Glutamax; Invitrogen; Cat#_61870010) supplemented with Pen / Strep (Pasching, Australia, PAA Laboratories; Cat#_P11-013). LPS (lipopolysaccharide; Sigma; Cat#_L4391) was added to a final concentration of 10 ng / ml. Four different pools containing different IL4-IL10 fusion protein constructs were analyzed. The differences between the pools lay in the conjugation of IL4 and IL10 (e.g., C-terminus of IL4 conjugated to N-terminus of IL10, or vice versa). Different constructs were added at final concentrations of 2, 10, 20, 30, 40, and 50 ng / ml. The whole blood cultures were then incubated at 37°C for 18 hours, with the supernatant collected and stored at -80°C until TNFα levels were measured. TNFα levels in the supernatant were measured using an ELISA assay (as described in Examples 2 and 5 above).

[0198] result The results showed an inhibition of TNFα production. No inhibition of TNFα production was observed in the absence of the IL4-IL10 fusion protein. Pools 2 and 4, where the IL4 c-terminus is linked to the n-terminus of IL4 (used at the same concentration), showed the highest inhibition of TNFα production compared to pools 1 and 3, where the IL10 c-terminus is linked to the n-terminus of IL10. These results suggest that the functionality of the IL4-10 fusion protein depends on how individual cytokines are linked within the IL4-IL10 fusion protein.

[0199]

[0200]

[0201]

[0202] sequence list <110> UMC Utrecht Holding BV <120> A fusion protein including interleukin-10 and interleukin-4 <130> IP40-171074 <141> 2012-11-08 <150> US 61 / 556,843 <151> 2011-11-08 <150> US 61 / 691,816 <151> 2012-08-22 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 129 <212> PRT <213> homo sapiens <400> 1 His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser 1 5 10 15 Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile 20 25 30 Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala 35 40 45 Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg 50 55 60 Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile 65 70 75 80 Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 85 90 95 Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe 100 105 110 Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser 115 120 125 Ser <210> 2 <211> 160 <212> PRT <213> homo sapiens <400> 2 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80 Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 145 150 155 160 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <400> 3 Gly Ser Gly Gly Gly Gly Ser Gly Thr 1 5 <210> 4 <211> 298 <212> PRT <213> Artificial sequence <400> 4 His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser 1 5 10 15 Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile 20 25 30 Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala 35 40 45 Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg 50 55 60 Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile 65 70 75 80 Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 85 90 95 Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe 100 105 110 Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser 115 120 125 Ser Gly Ser Gly Gly Gly Gly Ser Gly Thr Ser Pro Gly Gln Gly Thr 130 135 140 Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met 145 150 155 160 Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln 165 170 175 Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu 180 185 190 Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln 195 200 205 Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp 210 215 220 Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg 225 230 235 240 Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser 245 250 255 Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys 260 265 270 Gly With Tyr Lys Also Served With Glu Phe Asp And Phe With Asn Tyr Ile 275 280 285 Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 290,295

Claims

1. Use of a fusion protein in the preparation of a medicament for treating a condition in an individual with such need, wherein the condition is inflammatory pain or osteoarthritis; wherein the fusion protein is a functional fusion protein and is composed of interleukin-4, a linker, and interleukin-10 from the N-terminus to the C-terminus, or is composed of interleukin-10, a linker, and interleukin-4 from the N-terminus to the C-terminus, wherein the amino acid sequence of interleukin-4 is as shown in SEQ ID NO: 1 and the amino acid sequence of interleukin-10 is as shown in SEQ ID NO:

2.

2. The use as described in claim 1, wherein the condition is osteoarthritis.

3. The use as described in claim 1, wherein the condition is inflammatory pain.

4. The use as described in claim 1, wherein, The fusion protein consists of interleukin 4, the linker, and interleukin 10 from the N-terminus to the C-terminus.

5. The use as described in claim 1, wherein, The fusion protein is composed of interleukin 10, the linker, and interleukin 4 from the N-terminus to the C-terminus.

6. The use as described in any one of claims 1 to 5, wherein the fusion protein further comprises one or more chemical modifications.

7. The use as described in claim 6, wherein, The chemical modification is selected from the group consisting of glycosylation, sialylation, and polyethylene glycolation.

8. The use as described in claim 6, wherein, The chemical modification is fucosylation.

9. The use according to any one of claims 1 to 5, wherein the amino acid sequence of the connector comprises the amino acid sequence of SEQ ID NO:

3.

10. The use according to any one of claims 1 to 5, wherein the amino acid sequence of the connector consists of the amino acid sequence of SEQ ID NO:

3.

11. The use according to any one of claims 1 to 5, wherein the amino acid sequence of the fusion protein comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:

4.

12. The use according to any one of claims 1 to 5, wherein the amino acid sequence of the fusion protein comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

4.

13. The use according to any one of claims 1 to 5, wherein the amino acid sequence of the fusion protein comprises the amino acid sequence of SEQ ID NO:

4.

14. The use according to any one of claims 1 to 5, wherein the amino acid sequence of the fusion protein consists of the amino acid sequence of SEQ ID NO:

4.

15. A pharmaceutical composition comprising a fusion protein and a pharmaceutically acceptable excipient or carrier, wherein the fusion protein is a functional fusion protein and is composed of interleukin-4, a linker, and interleukin-10 from the N-terminus to the C-terminus, or is composed of interleukin-10, a linker, and interleukin-4 from the N-terminus to the C-terminus, wherein the amino acid sequence of interleukin-4 is as shown in SEQ ID NO: 1 and the amino acid sequence of interleukin-10 is as shown in SEQ ID NO:

2.

16. The pharmaceutical composition of claim 15, wherein the excipient is a diluent.

17. The pharmaceutical composition of claim 15, wherein the concentration of the fusion protein is between 50 μg / mL and 100 mg / mL.

18. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is in unit dose form.

19. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is formulated for intrathecal administration.

20. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is formulated for intra-articular administration.

21. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is formulated for subcutaneous, intracapsular, intravenous, subarachnoid, or epidural administration.

22. The pharmaceutical composition of claim 15, wherein, The fusion protein consists of interleukin 4, the linker, and interleukin 10 from the N-terminus to the C-terminus.

23. The pharmaceutical composition of claim 15, wherein, The fusion protein is composed of interleukin 10, the linker, and interleukin 4 from the N-terminus to the C-terminus.

24. The pharmaceutical composition of any one of claims 15 to 23, wherein the amino acid sequence of the linker comprises the amino acid sequence of SEQ ID NO:

3.

25. The pharmaceutical composition of any one of claims 15 to 23, wherein the amino acid sequence of the linker consists of the amino acid sequence of SEQ ID NO:

3.

26. The pharmaceutical composition of any one of claims 15 to 23, wherein the amino acid sequence of the fusion protein comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:

4.

27. The pharmaceutical composition of any one of claims 15 to 23, wherein the amino acid sequence of the fusion protein comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

4.

28. The pharmaceutical composition of any one of claims 15 to 23, wherein the amino acid sequence of the fusion protein comprises the amino acid sequence of SEQ ID NO:

4.

29. The pharmaceutical composition of any one of claims 15 to 23, wherein the amino acid sequence of the fusion protein comprises the amino acid sequence of SEQ ID NO:

4.

30. A fusion protein comprising, from the N-terminus to the C-terminus, interleukin 4, a linker, and interleukin 10, or from the N-terminus to the C-terminus, interleukin 10, a linker, and interleukin 4, wherein the amino acid sequence of interleukin 4 is as shown in SEQ ID NO: 1, and the amino acid sequence of interleukin 10 is as shown in SEQ ID NO:

2.

31. A fusion protein whose amino acid sequence consists of the amino acid sequence of SEQ ID NO:

4.

32. Use of a gene therapy vector in the preparation of a medicament for treating a condition in an individual with such need, said condition being inflammatory pain or osteoarthritis, said gene therapy vector comprising a polynucleotide encoding a fusion protein, said fusion protein being a functional fusion protein and consisting of interleukin-4, a linker, and interleukin-10 from the N-terminus to the C-terminus, or consisting of interleukin-10, a linker, and interleukin-4 from the N-terminus to the C-terminus, said amino acid sequence of interleukin-4 being as shown in SEQ ID NO: 1 and said amino acid sequence of interleukin-10 being as shown in SEQ ID NO: 2.

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