NL2022982B1

NL2022982B1 - A fusion protein comprising IL13

Info

Publication number: NL2022982B1
Application number: NL2022982A
Authority: NL
Inventors: Eijkelkamp Niels; Erik Hack Cornelis; Prado Sanchez Judith; Popov- Čeleketić Jelena; Versteeg Sabine
Original assignee: Synerkine Pharma B V
Priority date: 2019-04-19
Filing date: 2019-04-19
Publication date: 2020-10-27

Abstract

The invention is concerned with a fusion protein comprising interleukin 13 and an interleukin chosen from interleukin 4, interleukin 10, interleukin 33, transforming growth factor beta 1, transforming growth factor beta 2, and interleukin 13, a nucleic acid molecule encoding such fusion protein, a vector comprising such nucleic acid molecule, and a host cell comprising 5 such nucleic acid molecule or such vector. The invention further pertains to a method for producing such fusion protein. The fusion protein or a gene therapy vector encoding the fusion protein may be used in the prevention or treatment of a condition characterized by chronic pain, neuro-inflammation and or neurodegeneration.

Description

P33747NL0O0O Title: A fusion protein comprising IL13 Field of the invention The present invention is in the field of neuro-immunology and pharmacology, particularly for treatment of chronic pain, neuro-inflammatory and neurodegenerative diseases, and inflammatory disorders. The invention particularly relates to a novel fusion protein comprising interleukin 13 (IL13) and an interleukin chosen from interleukin 4 (IL4), interleukin 10 (IL10), interleukin 33 (IL33), transforming growth factor beta 1 (TGFB1), transforming growth factor beta 2 (TGFB2), and IL13 itself, either or not physically fused together through a linker sequence. Particularly, the present invention provides an IL4/IL13, IL10/IL13, IL33/IL13, IL13- TGFB1/2, IL13/IL13 fusion protein endowed with a superior analgesic activity over a combination of the individual cytokines, or over a fusion protein of IL4 and IL10. The present invention also provides nucleic acid sequences encoding an IL4/IL13 fusion protein, IL10/1L13 fusion protein, IL33/IL13, IL13-TGFB1/2 or IL13/IL13 fusion protein, expression vectors comprising such nucleic acid sequences, host cells or host organisms altered to harbour the nucleic acid sequence encoding the IL4/IL13 fusion protein, IL10/IL13 fusion protein, or IL33/IL13 fusion protein, IL13-TGFB1/2 fusion protein, and the fusion protein itself. The invention further provides methods for producing an IL4/IL13, IL10/IL13, IL33/IL13, IL13- TGFB1/2 or IL13/IL13 fusion protein using a cell or organism harbouring such nucleic acid sequences. Transgenic organisms comprising the nucleic acid sequence of the invention are also provided. The present invention also relates to pharmaceutical compositions comprising the IL4/IL13 or IL10/IL13 or IL33/IL13 or IL13-TGFR1/2 or IL13/IL13fusion protein. Finally, the use of the IL4/IL13 or IL10/IL13 or IL33/IL13 or IL13-TGFR1/2 or IL13/IL13 fusion protein as a medicament, in particular for the prevention and/or treatment of chronic pain and of conditions characterized by neuro-inflammation, neuro-degeneration, or inflammation is taught herein. Background of the invention Chronic pain affects millions of people and constitutes the largest unmet need of modern medicine’. In 2016, an estimated 20.4% of U.S. adults (50.0 million) had chronic pain and

8.0% of U.S. adults (19.6 million) had high-impact chronic pain’. Opioids and non-steroidal anti- inflammatory drugs (NSAIDs) constitute the main classes of drugs to combat pain. However, these analgesics (“pain-killers”) are often ineffective and have severe side effects (addiction, gastrointestinal bleeding, cardiovascular, other). It is estimated that ~50% of chronic pain patients (~5-10% of the total population) do not receive adequate pain relief®.

22. People with chronic pain suffer from spontaneous pain, hyperalgesia (a heightened experience of pain to a noxious stimulus) and allodynia (pain caused by a normally non-painful stimulus). Pain has multiple causes and results from biological processes at various anatomic levels”:1°: the generation of stimuli that trigger sensory nerve endings in the periphery; the stimulation, sensitization, and dysfunction of peripheral sensory neurons that transmit action potentials to the spinal cord; neurons and glial cells in the dorsal horn of the spinal cord where action potentials from peripheral neurons are transmitted to spinal pain neurons via synapses, and finally central mechanisms in the brain. The contribution of all of these processes to different types of chronic pain varies. Depending on this contribution pain is discriminated in several types, including nociceptive pain, peripheral and central neuropathic pain, and mixed types of pain. Analgesic drugs used in the clinic, target pain at only one level: NSAIDs reduce the generation of nociceptive stimuli in the periphery, whereas opioids inhibit central mechanisms. Together with their notorious toxic side effects, this explains the limited clinical efficacy of these analgesics.

Last decade it has become increasingly clear that pain signals are not straightforward transferred to the brain but rather are modulated by neuro-inflammatory processes involving glial cells in the spinal cord as well as sensory neurons. Cytokines are well-known to orchestrate immune and inflammatory processes, and also are crucial in the control of pain. Pro- inflammatory cytokines enhance inflammation and promote pain whilst regulatory (anti- inflammatory) cytokines dampen inflammation. The balance between proinflammatory and regulatory cytokines determines the outcome of inflammatory reactions in vivo''. Although pain promoting effects of pro-inflammatory cytokines are well-known, knowledge on the role of regulatory cytokines in pain is limited. Blocking of regulatory cytokines such as TGF, IL10, IL4, and IL13, severely impairs the resolution of transient inflammatory hyperalgesia’* and chemotherapy-induced allodynia'®, demonstrating a critical role for endogenous regulatory cytokines in pain resolution. This role of cytokines goes far beyond reducing inflammation and is fundamentally different from that of anti-inflammatory drugs such as corticosteroids which only have limited analgesic effects’. Notably, not only glial cells are modulated by cytokines, but also sensory neurons themselves can directly respond to cytokines. Indeed, sensory neurons express receptors for all regulatory cytokines, though expression differs among neuronal subsets'®. Pain resulting from peripheral inflammation or nerve damage is associated with the activation of spinal microglia and astrocytes that promote pain by enhancing spinal pain signal transmission1®22.

Considering the role of neuro-inflammation in chronic pain, regulatory cytokines potentially can target pain at multiple levels. Indeed, they dampen stimulation of nociceptors by reducing inflammation, they suppress sensitization and dysfunction of sensory neurons, and they prevent activation of pain pathways in the spinal cord by attenuating the production of pro-

23. inflammatory mediators by glial cells222. However, the analgesic effects of therapy with stand- alone regulatory {e.g., IL10 or IL13) or anti-inflammatory cytokines (IL1-receptor antagonist) are limited'®, presumably because optimal analgesic activity requires synergy of various regulatory cytokines, and because of their relatively poor bioavailability due to rapid clearance by the kidney. Therefore, a new strategy to resolve chronic pain with regulatory cytokines has been proposed using a fusion-protein of IL4 and IL10°323. Intrathecal administration of analgesics is common practice in pain treatment as this reduces the dose and decreases toxicity of an analgesic drug?*. Intrathecal injection of IL4/IL10 fusion protein reduces pain in mouse models for a variety of different types of pain’, Remarkably, three repeated intrathecal administrations of IL4/IL10 fusion protein completely and permanently resolves chronic nociceptive pain induced by inflammation in the paw, without modulating peripheral inflammation itself’. Interestingly, the efficacy of IL4/IL10 fusion protein is superior to that of stand-alone wild-type cytokines, and even to that of the combination of these cytokines1?. Resolution of pain by IL4/IL10 fusion protein was also observed in neuropathic'® and osteoarthritis pain models?®. However, the effect of IL4/IL10 fusion protein on neuropathic and osteoarthritis pain is transient, even after multiple injections. Therefore, there is a need for providing a molecule for prevention or treatment of neuropathic pain and osteoarthritis pain that has a long-lasting analgesic effect.

Summary of the invention The present invention discloses such a molecule. It describes a single molecule that targets neuro-inflammation and -degeneration and has a long-lasting effect on chronic neuropathic pain. This molecule can be used for the treatment of various diseases or disorders with different etiology, in which chronic pain, neuro-inflammation and/or neuro-degeneration play a role. In a first aspect, the present invention relates to a fusion protein comprising at least 2, 3, 4, preferably 2 (anti-inflammatory) interleukins chosen from the group consisting of interleukin 13 {IL13), interleukin 4 (IL4), interleukin 10 (IL10), interleukin 33 (IL33), transforming growth factor beta 1 (TGFB1), and transforming growth factor beta 2 (TGFB2). Preferably, the present invention relates to a fusion protein comprising an IL13 and another, i.e. a second, interleukin/cytokine, preferably chosen from IL4, IL10, IL33, TGFB1, TGFB2, and IL13 itself. In an embodiment, said IL13 and said interleukin chosen from IL4, IL10, IL33, TGFB1, TGFB2, or IL13 itself are connected by a linker. In an embodiment, the interleukin chosen from IL4, IL10, IL33, TGFB1, TGFB2, or IL13 itself is fused N-terminal of the IL13.

-4- In another embodiment, the IL13 is fused N-terminal of the interleukin chosen from IL4, IL10, IL33, TGFB1, TGFB2, or IL13 itself. In an embodiment, said fusion protein further comprises one or more chemical modifications. Said chemical modifications may be selected from the group consisting of glycosylation, fucosylation, sialylation, and pegylation. In an embodiment, said IL13 is human IL13. In an embodiment said IL4 is human IL4. In an embodiment, said IL10 is human IL10. In an embodiment, said IL33 is human IL33. In an embodiment, said TGFB1 is human TGFB1. In an embodiment, said TGFB2 is human TGFp2 In a second aspect, the present invention pertains to a nucleic acid molecule comprising a polynucleotide encoding the fusion protein taught herein. In another aspect, the present invention is directed to a vector comprising the nucleic acid molecule taught herein.

In an aspect, the present invention is concerned with a host cell comprising the nucleic acid molecule taught herein or the vector taught herein.

In an aspect, the present invention provides a method for producing a fusion protein as taught herein, said method comprising the steps of: culturing a host cell as taught herein under conditions permitting the production of the fusion protein as taught herein; and optionally, recovering the fusion protein.

In yet another aspect, the present invention provides for a pharmaceutical composition comprising the fusion protein as taught herein, and a pharmaceutically acceptable carrier. The invention also pertains to a fusion protein as taught herein for use as a medicament, such as for use in the prevention or treatment of a condition characterized by chronic pain, neurcinflammation, neurodegeneration, and/or local or systemic inflammation. In this regard, “chronic” can be regarded as persisting at least 1, 2, 3, 4, 5, 6, 10, 12 months, or even at least 1, 2, 3, 4, 5 years. Said condition may be characterized by visceral or non- visceral nociceptive pain, peripheral or central neuropathic pain, mixed nociceptive- neuropathic pain, neuro-inflammation, and/or neuro-degeneration, and/or may be selected from the group consisting of post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, chemotherapy-induced allodynia, complex regional pain syndrome, past-spinal injury pain, post-stroke pain, multiple sclerosis, low back pain,

-5. osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, chronic widespread pain, myofascial pain syndrome, Alzheimer’s disease and Parkinson’s disease, Huntington’s disease, and/or amyotrophic lateral sclerosis, or multiple sclerosis.

In an aspect, the invention relates to a fusion protein as taught herein for use in the prevention or treatment of a clinical condition in a mammal, such as a human, for which IL4 or IL13 is indicated.

The invention is also concerned with a fusion protein as taught herein for use in the prevention or treatment of a clinical condition in a mammal, such as a human, for which IL10 or IL33 or TGFB1 or TGFB2 is indicated.

Finally, the invention teaches a vector for use in the prevention or treatment of a condition characterized by chronic pain, neuroinflammation, neurodegeneration, and/or local or systemic inflammation. Said condition may be characterized by visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, or mixed nociceptive-neuropathic pain, neuro-inflammation, and/or neuro-degeneration, and/or may be selected from the group consisting of post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post- herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV- associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, complex regional pain syndrome, post-spinal injury pain, post-stroke pain, multiple sclerosis, low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndromes, Alzheimer's disease and Parkinson’s disease, Huntington's disease, and/or amyotrophic lateral sclerosis, or multiple sclerosis.

Detailed description of the invention

GENERAL DEFINITONS The term “nucleic acid molecule” (or “nucleic acid sequence”, “polynucleotide”, or “nucleotide sequence”) refers to a DNA or RNA molecule in single or double stranded form, particularly a DNA encoding a protein according to the invention. An “isolated nucleic acid sequence” refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g., the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.

The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of one or several chains of amino acids, without reference to a specific mode of action, size, three-dimensional structure or origin. An “isolated protein” is used to refer to a protein which is

-6- no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

The term “fusion protein” refers to a protein or polypeptide that has an amino acid sequence derived from two or more proteins. The fusion protein may also include linking regions or a linker of amino acids between amino acid portions derived from separate proteins. The fusion protein also refers to a molecule that has an amino acid sequence derived from two or more proteins which are connected via chemical crosslinkers with or without a spacer.

The terms IL4, IL10, IL13, IL33, TGFB1, and TGFB2 preferably refer to the wild-type sequences of these respective cytokines, and/or to mutated variants thereof capable of binding to at least one, preferably at least two, of their respective cytokine receptors.

The term TGFB is used to refer to TGFB1 and/or TGFB2.

The term “IL4/IL13 fusion protein” refers to a fusion polypeptide comprising at least (human) IL4 and IL13, optionally coupled to one another via a linker. The fusion protein may comprise additional polypeptide sequences, e.g., a signal sequence, a His-tag, targeting sequence(s), or an antibody Fc fragment.

The term “IL10/IL13 fusion protein” refers to a fusion polypeptide comprising at least (human) IL10 and IL13, optionally coupled to one another via a linker. The fusion protein may comprise additional polypeptide sequences, e.g., a signal sequence, a His-tag, targeting sequence(s) or an antibody Fc fragment.

The term “IL33/IL13 fusion protein” refers to a fusion polypeptide comprising at least (human) IL33 and IL13, optionally coupled to one another via a linker. The fusion protein may comprise additional polypeptide sequences, e.g., a signal sequence, a His-tag, targeting sequence(s) or an antibody Fc fragment.

The term “TGFB1/IL13 fusion protein” refers to a fusion polypeptide comprising at least (human) TGFB1 and IL13, optionally coupled to one another via a linker. The fusion protein may comprise additional polypeptide sequences, e.g., a signal sequence, a His-tag, targeting sequence(s) or an antibody Fc fragment.

The term “TGFB2/IL13 fusion protein” refers to a fusion polypeptide comprising at least (human) TGFB2 and IL13, optionally coupled to one another via a linker. The fusion protein may comprise additional polypeptide sequences, e.g., a signal sequence, a His-tag, targeting sequence(s) or an antibody Fc fragment.

The term “IL13/IL13 fusion protein” refers to a fusion polypeptide comprising at least two (human) IL13 molecules, optionally coupled to one another via a linker. The fusion protein may comprise additional polypeptide sequences, e.g., a signal sequence, a His-tag, targeting sequence(s) or an antibody Fc fragment.

-7- As used herein, a “linker” means a polypeptide used to couple two proteins or polypeptides, in casu IL4 (or IL10 or IL33 or TGFB) and IL13. The linker typically is a stretch of amino acids, e.g., predominantly glycine and/or serine. In an embodiment, the linker is a stretch of amino acids having a length of up to 100 amino acids, such as from about 2, 5, 7, 10, 15 amino acids uptoabout 15, 20, 25, 30, 35, 50, 75, or 100 amino acids, preferably comprising predominantly serine and glycine residues.

As used herein, “interleukin-13” (IL13) preferably refers to any mammalian IL13, such as human IL13, mouse IL13, or an active species or allelic variant, (functional) fragment or derivative thereof.

As used herein, “interleukin-4” (IL4) preferably refers to any mammalian IL4, such as human IL4, mouse IL4, or an active species or allelic variant, (functional) fragment or derivative thereof. As used herein, “interleukin-10” (IL10) preferably refers to any mammalian IL10, such as human IL10, mouse IL10, or an active species or allelic variant, (functional) fragment or derivative thereof.

As used herein, “interleukin-33” (IL33) preferably refers to any mammalian IL33, such as human IL33, mouse IL33, or an active species or allelic variant, (functional) fragment or derivative thereof.

As used herein, “transforming growth factor B1” (TGFB1) preferably refers to any mammalian TGFB1, such as human TGFB1, mouse TGFB1, or an active species or allelic variant, (functional) fragment or derivative thereof.

As used herein, “transforming growth factor 82" (TGFB2) preferably refers to any mammalian TGFB2, such as human TGFB2, mouse TGFB2, or an active species or allelic variant, (functional) fragment or derivative thereof.

“Functional”, in relation to the fusion proteins of the present invention (or variants or fragments thereof), refers to the capability to display both IL4 (or IL10 or IL33 or TGFB1/2) and IL13 functionality. Assays to assess the functional activity of these cytokines are well known to those skilled in the art. For example, a functional assay for IL4 and IL10 is the lipopolysaccharide (LPS) induced TNF release in whole blood in presence of anti-IL10 antibody?®. A functional assay for IL13 is the proliferation of TF1 human erythroleukemic cells?7. An assay for IL33 function is IL6 production by the mast cell line MC/9. An assay for TGF B1/2 is inhibition of IL4- dependent growth of mouse T-cell line HT-2.

The term “gene” means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. a mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5’ leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA), introns, and a 3’ non-translated sequence comprising e.g. transcription termination sites.

-8- “Expression of a gene” refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment). “Expression of a polypeptide” additionally refers to a process wherein an mRNA is translated into a protein product, which may or may not be secreted.

As used herein, the term "promoter" refers to a nucleic acid sequence that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the 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 (e.g. by external application of certain compounds) or developmentally regulated.

A "tissue specific’ promoter is only active in specific types of tissues or cells.

As used herein, the term "operably linked" refers to a linkage of polynuclectide elements in a functional relationship.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.

For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence.

Operably linked means that the DNA sequences being linked are typically contiguous.

A “nucleic acid construct” or “vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and which is used to deliver exogenous DNA into a host cell.

Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, or multiple cloning sites (see below). “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms.

Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps.

Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring

-9- matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum82 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, 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, percent similarity or identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc. Preferably, the sequence identity refers to the sequence identity over the entire length of the sequence.

A "host cell" or a "recombinant host cell" or “transformed cell” are terms referring to a new individual cell (or organism) arising as a result of at least one nucleic acid molecule, especially comprising a nucleic acid molecule encoding a desired protein. The host cell is preferably a mammalian cell, plant cell or a bacterial cell. The host cell may contain the nucleic acid molecule or vector of the present invention as an extra-chromosomally (episomal) replicating molecule, or more preferably, comprises the nucleic acid molecule or vector of the present invention integrated in the genome of the host cell.

The term "selectable marker" is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. Selectable marker gene products confer for example antibiotic resistance or nutritional requirements.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb “to consist of”. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to.

Proteins, nucleic acid sequences, vectors and host cells of the invention The present inventors provide a fusion protein comprising an IL13 protein and a protein chosen from IL4, IL10, IL33, TGF, or IL13 itself, optionally physically fused together via a linker. Particularly, the fusion protein of the present invention was found to have a superior activity in a treatment for neuropathic pain over its individual counterparts, i.e., IL4 (or IL10 or IL33 or TGFB1/2) and IL13 separately. Specifically, it was found that, upon intrathecal administration, the fusion protein of the present invention has a long-lasting analgesic effect on neuropathic pain, and abrogates allodynia associated with chemotherapy-induced neuropathy.

-10 - The inventors unexpectedly observed that this analgesic effect of the fusion protein of the present invention lasted longer than that of a previously described fusion protein of IL4 and IL1019232 Surprisingly, the fusion protein of the present invention also improved chemotherapy-associated neuro-degeneration. It prevented the shortening of neurites in vitro upon incubation with chemotherapeutic drugs, and was more potent regarding this effect than a fusion protein of IL4 and IL10, or the combination of IL4 (or IL10 or IL33 or TGFB) and IL13. The latter surprising finding indicates a unique effect of the fusion protein over its individual cytokines or the combination of these. In vivo, the fusion protein of the present invention attenuated the decrease of intraepidermal nerve fibers upon administration of chemotherapeutic drugs.

In one embodiment of the invention nucleic acid sequences and amino acid sequences of IL4/IL13 fusion proteins or IL10/IL13 fusion proteins or IL33/IL13 fusion proteins or TGFB1/L13 fusion proteins or TGFB2/IL13 fusion proteins are provided (including variants and fragments thereof). The IL4/IL13 fusion proteins or IL10/IL13 fusion proteins or IL33/IL13 fusion proteins, as well as functional fragments and variants thereof, display IL4 {or IL10 or IL33 or TGFB1/2) activity as well as IL13 activity.

In one aspect, a fusion protein comprising IL4 (or IL10 or IL33) and IL13 is provided. The fusion protein may comprise an IL4 protein, or a variant or fragment thereof. The IL4 protein is preferably a mammalian IL4 protein, such as a human IL4, or mouse IL4. One amino acid sequence of IL4 is set forth in SEQ ID NO:1. Variants of IL4 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO:1, preferably over the entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as defined above. Variants also include proteins having IL4 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO: 1. Preferably, such proteins comprise from 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

The fusion protein further comprises an IL13 protein, or a variant or fragment thereof. The IL13 protein is preferably a mammalian IL13 protein, such as a human IL13, or mouse IL13. One amino acid sequence representing IL13 is set forth in SEQ ID NO:2. Variants of IL13 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO:2, preferably over the entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as defined above. Variants also include proteins having IL13 activity, which have been derived,

-11 - by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO:2. Preferably, such proteins comprise from 1, 2,3,4,5,6,7,8,9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

The fusion protein may comprises an IL10 protein, or a variant or fragment thereof.

The IL10 protein is preferably a mammalian IL10 protein, such as a human IL10, or mouse IL10. One amino acid sequence representing IL10 is set forth in SEQ ID NO:5. Variants of IL10 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO:5, preferably over the entire length.

Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as defined above.

Variants also include proteins having IL10 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO:5. Preferably, such proteins comprise from 1, 2,3,4,5,6, 7,8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

The fusion protein may comprises an IL33 protein, or a variant or fragment thereof.

The IL33 protein is preferably a mammalian IL33 protein, such as a human IL33, or mouse IL33. One amino acid sequence representing IL33 is set forth in SEQ ID NQ:6. Variants of IL33 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO:6, preferably over the entire length.

Variants also include proteins having IL33 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO:8. Preferably, such proteins comprise from 1, 2,3,4,5,8, 7,8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

The fusion protein may comprises a TGFB1 protein, or a variant or fragment thereof.

The TGFB1 protein is preferably a mammalian TGFB1 protein, such as a human TGFB1, or mouse TGFB1. One amino acid sequence representing TGFB1 is set forth in SEQ ID NO:7. Variants of TGFB1 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO:7 (whole or underlined part), preferably over the entire length.

Variants also include proteins having TGFB1 activity, which have been derived, by way of one or more amino acid substitutions,

-12- deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO:7. Preferably, such proteins comprise from 1, 2, 3,4, 5,6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions. The fusion protein may comprises a TGFB2 protein, or a variant or fragment thereof. The TGFB2 protein is preferably a mammalian TGFB2 protein, such as a human TGFB2, or mouse TGFB2. One amino acid sequence representing TGFB2 is set forth in SEQ ID NO:8. Variants of TGFB2 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO:8 (whole or underlined part), preferably over the entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as defined above. Variants also include proteins having TGFB2 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO:8. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

The IL4 (or IL10 or IL33 or TGFB1/2) and IL13 in the fusion protein may or may not be connected by a linker sequence or by a chemical spacer. Additional amino acid sequences may present at the N- and/or C-terminus of the fusion protein of the present invention, e.g., to facilitate purification. For example, a histidine-tag may be present at the C-or N-terminus to facilitate purification. Alternatively, the fusion protein of the invention may optionally comprise additional protein moieties, such as moieties capable of targeting, e.g., a protein moiety comprising one or more antibody Fc regions.

The IL4 (or IL10 or IL33 or TGFB1/2) may be located N-terminal of the IL13, or may be located C-terminal of the IL13. In a preferred embodiment, the IL4 (or IL10 or IL33 or TGFR1/2) molecule is located N-terminal of the IL13 molecule.

In an embodiment, the fusion protein of the invention consists essentially of IL4 {or IL10 or IL33 or TGFB1/2) and IL13, optionally linked by a linker sequence.

In an embodiment, the fusion protein of the present invention prevents neuronal damage to primary sensory neurons cultured overnight in presence of paclitaxel as quantified by measuring the neurite length after f3-tubulin staining.

In a suitable embodiment, the fusion protein of the present invention is present in a monomeric form. In one embodiment, it has a molecular weight of 30 to 37 kDa.

The fusion protein of the present invention may be prepared by techniques which are routine to the skilled person. For example, it may be prepared using a technique which provides for the production of recombinant fusion proteins by continuous cell lines in culture. For example, fusion proteins of the present invention can be produced in a host cell transfectoma

-13- using a combination of recombinant DNA techniques and gene transfection methods.

For example, to express the fusion proteins of the present invention, a nucleic acid molecule encoding the fusion proteins of the present invention can be prepared by standard molecular biology techniques. The nucleic acid molecule of the invention is preferably operably linked to transcription regulatory sequences such as a promoter, and optionally a 3 untranslated region. The nucleic acid molecule of the present invention may be inserted into a vector, such as an expression vector, such that the genes are operatively linked to transcriptional and translational control sequences. The expression vector and transcription regulatory sequences are selected to be compatible with the expression host cell used. The nucleic acid molecule encoding a fusion protein of the present invention may be inserted into the expression vector by routine methods. The nucleic acid molecule or vector of the present invention may further include a nucleotide sequence encoding a signal peptide, which may facilitate secretion of the fusion protein from the host cell. Said nucleotide sequence encoding a signal peptide may be operably linked to the nucleic acid molecule of the present invention.

Preferably, said signal peptide is located at the amino terminus of the fusion protein of the present invention, and as such, the nucleotide sequence encoding said signal peptide may be located 5’ of the nucleic acid molecule encoding the fusion protein of the present invention. The signal peptide may be a cytokine signal peptide or a signal peptide from a non-cytokine protein. The promoter may be constitutive or inducible. The vector may comprise a selectable marker for selection of a vector-carrying host cell. The vector may comprise an origin of replication when the vector is a replicable vector.

The fusion protein according to the invention may be synthesized de novo by chemical synthesis (using e.g. a peptide synthesizer such as supplied by Applied Biosystems) or may be produced by recombinant host cells by expressing the nucleic acid sequence encoding the fusion protein, fragment or variant. Variants and fragments are preferably functional, i.e., they bind at least to one, preferably two of the corresponding membrane-receptor(s) and have IL4 (or IL10 or IL33 or TGFB1/2) and/or IL13 activity, preferably IL4 (or IL10 or IL33 or TGF(1/2) and IL13 activity.

The functional activity of IL4 (or IL10 or IL33 or TGFB1/2) and IL13, as well as the IL4 (or IL10 or IL33 or TGFB1/2) and IL13 comprising fusion protein can be determined using routine methods known for those skilled in the art. For example, a suitable assay for functionality of IL4, as well as the IL4 (or IL10) and IL13 comprising fusion protein, is the lipopolysaccharide (LPS) induced cytokine release (IL1, IL6, IL8, TNFa) in whole blood, optionally in the presence of anti-IL10 antibody. Functional activity may also be determined by assessing the activation of intracellular signalling pathways upon incubation of target cells with the fusion protein in presence or absence of blocking antibody against either cytokine moiety of the fusion protein or their receptors.

-14 - In another aspect, isolated nucleic acid sequences encoding any of the above fusion proteins are provided, such as cDNA, genomic DNA and RNA sequences. Due to the degeneracy of the genetic code various nucleic acid sequences may encode the same amino acid sequence. Any nucleic acid sequence encoding the fusion proteins of the invention are herein referred to as “IL4/IL13 (or IL10/IL13, IL33/IL13, TGFB(1 or 2)/IL13, IL13/IL13) encoding nucleic acid sequences”. The nucleic acid sequences provided include recombinant, artificial or synthetic nucleic acid sequences. It is understood that when sequences are depicted as DNA sequences while RNA is referred to, the actual base sequence of the RNA molecule is identical with the difference that thymine (T) is replaced by uracil (U). The nucleic acid sequences of the invention are particularly useful for expression of the [L4/IL13 (or IL10/IL13, IL13/IL33, TGFB(1 or 2)/1L13, IL13/IL13) fusion protein of the invention, for either the production of these proteins or for gene therapy purposes.

The nucleic acid sequence, particularly DNA sequence, encoding the IL4/IL13 (or IL10/IL13 or IL33/13 or TGFB{1 or 2)/IL13, or IL13/IL13) fusion protein of this invention can be inserted in expression vectors to produce (high amounts of) IL4/IL10 (or IL10/IL13 or IL33/13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein. Suitable vectors include, without limitation, linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector include a retrovirus, an adenovirus, and an adeno- associated virus.

Preferred regulatory sequences for mammalian host cell expression 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}, adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter.

In addition to the nucleic acid molecules encoding IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion proteins and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., US 4,399,216, US 4,634,665 and US 5,179,017, all by Axel et al). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). Finally, the recombinant expression vector may contain a gene that codes for a glycosyl transferase in addition to the nucleic acid sequence encoding the fusion proteins of the present invention.

In another aspect, the present invention relates to a host cell comprising a nucleic acid

-15- sequence of the present invention, or a nucleic acid construct or vector comprising a nucleic acid sequence of the present invention. The host cell may be any host cell. The host cell may be selected from prokaryotic and eukaryotic cells. The host cell may 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.

In one embodiment the fusion proteins of the present invention are expressed in eukaryotic cells, such as mammalian host cells. Preferred mammalian hast cells for expressing the recombinant synerkines of the invention include CHO cells (including dhfr-CHO cells, described in (Urlaub et al., 1980), used with a DHFR selectable marker, NS/0 myeloma cells, COS cells, HEK293 cells, PER.C6 cells,SP2.0 cells, or other cells. When recombinant expression vectors comprising nucleic acid sequences encoding a fusion protein according to the present invention are introduced into mammalian host cells, the fusion proteins of the present invention may be produced by culturing the host cells for a period of time sufficient to allow for expression of the fusion proteins in the host cells or, more preferably, secretion of the fusion proteins into the culture medium in which the host cells are grown. The fusion proteins of the present invention can be recovered from the culture medium using standard protein purification methods.

Alternatively, the nucleic acid sequences encoding the fusion proteins of the invention can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g. E. coli, algi, as well as insect cells. Furthermore, the fusion proteins of the present invention can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or eggs from hens, or in transgenic plants.

Introduction of the nucleic acid sequence of the present invention into a host cell may be carried out by any standard technique known in the art. For expression of the fusion proteins of the present invention, the expression vector(s) encoding the fusion protein may transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium- phosphate precipitation, DEAE-dextran transfection, lipofectamine transfection and freeze-dry method transfection, and the like. Cell lines that secrete fusion proteins of the present invention can be identified by assaying culture supernatants for the presence of the fusion protein. The preferred screening procedure comprises two sequential steps, the first being identification of cell lines that secrete the fusion protein, the second being determination of the quality of the fusion protein such as the ability of the fusion protein to inhibit cytokine production by blood cells stimulated with LPS or other Toll-like receptor agonists, glycosylation patterns, and other.

For optimal expression in a host cell, the IL4/IL13 (or IL10/IL13 or IL33/13 or TGFB{1 or 2)/IL13 or IL13/IL13) fusion protein encoding DNA sequences can be codon-optimized by

-16 - adapting the codon usage to that most preferred in host cell genes. Several techniques for modifying the codon usage to that preferred by the host cells can be found in patent and scientific literature. The exact method of codon usage modification is not critical for this invention.

In another embodiment of the invention PCR primers and/or probes and kits for detecting the IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein encoding DNA or RNA sequences are provided. Degenerate or specific PCR primer pairs to amplify IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFR(1 or 2)/IL13 or IL13/IL13) fusion protein encoding DNA from samples can be synthesized (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 stretch of 9, 10, 11, 12, 13, 14, 15, 16, 18 or more contiguous nucleotides of an IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein encoding nucleic acid sequence (or the complement strand) may be used as primer or probe.

Likewise, DNA fragments of an IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/1L13) fusion protein encoding nucleic acid sequence can be used as hybridization probes.

A detection kit for an IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein encoding nucleic acid sequence may comprise primers specific for an IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein encoding nucleic acid sequence and/or probes specific for an IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein encoding nucleic acid sequences, and an associated protocol to use the primers or probe to detect specifically IL4/IL13 {or IL10/IL13 or IL33-IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein encoding nucleic acid sequence in a sample.

Such a detection kit may, for example, be used to determine, whether a host cell has been transformed with a specific an IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein encoding nucleic acid sequence of the invention. Because of the degeneracy of the genetic code, some amino acid codons can be replaced by others without changing the amino acid sequence of the protein.

In an aspect, the present invention is concerned with a method for producing an 1L4/1L13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein, said method comprising the steps of: culturing a host cell of the present invention under conditions permitting the production of the IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein; and optionally, recovering the fusion protein. The skilled person will be capable of routinely selecting conditions permitting production of the IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein. Additionally, a person skilled in the art will be capable of recovering the fusion protein produced using routine methods, which include, without limitation, chromatographic methods (including, without limitation, size exclusion

-17 - chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, metal binding, and the like), immunoprecipitation, HPLC, ultracentrifugation, precipitation and differential solubilisation, and extraction. As said above, recovery or purification of the fusion protein may be facilitated by adding, for example, a His-tag to the fusion protein. Pharmaceutical composition In an aspect, the invention relates to a pharmaceutical composition comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques (e.g., as described in Remington: The Science and Practice of Pharmacy, 19 Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995).

The term “pharmaceutically acceptable carrier” relates to carriers or excipients, which are inherently nontoxic and nontherapeutic. Examples of such excipients are, but are not limited to, saline, Ringer's solution, dextrose solution and Hank's solution.

The pharmaceutical composition may be administered by any suitable route and mode. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

The pharmaceutical compositions according to the invention may be formulated in accordance with routine procedures for administration by any route, preferably parenteral. The compositions may be in the form of liquid preparations, such as oral or sterile parenteral solutions or suspensions.

The pharmaceutical compositions of the present invention include those suitable for any form of parenteral administration.

In an embodiment, the pharmaceutical composition is administered parenterally.

The phrases "parenteral administration” and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

In an embodiment the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

In an embodiment the fusion proteins of the invention are administered in crystalline form by subcutaneous injection.

As used herein, “pharmaceutically acceptable carrier’ includes any and all solvents,

-18- dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical composition of the invention is contemplated. Preferably, the carrier is suitable for parenteral administration, e.g. intravenous or subcutaneous injection or infusion.

Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Depending on the route of administration, the active compound, i.e, the IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB{1 or 2)/IL13 or IL13/IL13) fusion proteins, may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate the compound. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., 1984).

The fusion proteins of the present invention may also be prepared with carriers that will protect it against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,

-19- collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

Actual dosage levels of the IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB{1 or 2)/IL13 or IL13/IL13) fusion proteins in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGFB(1 or 2)/IL13 or IL13/IL13) fusion protein which is effective (“effective amount”) to achieve the desired therapeutic response for a particular patient, compasition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

In one embodiment the IL#IL13 (or IL10/IL13 or IL33/IL13 or TGFB{1 or 2)/IL13 or IL13/1L13) fusion proteins of the present invention can be given as intravenous injection or a short infusion, in another embodiment, they are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.

In yet another embodiment, the IL4/IL 13 (or IL10/IL13 or IL33/IL13 or TGF (1 or 2)/IL13 or IL13/IL13} fusion proteins of the present invention can be administered as maintenance therapy, such as, e.g., once a week for a period of 6 months or more.

In another embodiment, the IL4/IL13 (or IL10/IL13 or IL33/IL13 or TGF (1 or 2)/IL13 or IL13/IL13) fusion proteins of the present invention can be administered as an intrathecal injection. And in yet another embodiment they are administered through an intrathecal or intraspinal drug delivery system which allows continuous or repeated administration.

-20 - Therapeutic Uses In a further aspect, the present invention relates to the fusion protein of the present invention for use as a medicament.

In an aspect, the present invention pertains to the fusion protein for use in treating chronic pain. “Chronic” may mean that the pains persists/persisted over more than 2 weeks or more than 1, 3, 6, 12 months, or even more than 1, 2, 4, 6 years. Particularly, it was found that the fusion protein of the present invention has a long-lasting analgesic effect against allodynia associated with chemotherapy-associated neuropathy and neurodegeneration in mice. In addition, the fusion protein prevents neurodegeneration induced by chemotherapy in vitro and in vivo. Therefore, the fusion protein of the invention may be used for prevention and treatment neuropathic pain. As such, the fusion protein may be particularly useful for the treatment of chemotherapy induced neuropathy, and other forms of peripheral neuropathic pain such as post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV- associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, complex regional pain syndrome, and other.

In a further embodiment, the fusion protein of the present invention may be used to treat pain and neurodegeneration of central neuropathic disorders including spinal cord injury, post- stroke pain and multiple sclerosis.

In a further aspect, the present invention pertains to a fusion protein of the present invention for use in treatment of chronic mixed nociceptive and neuropathic pain such as low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndromes, and other.

In yet a further aspect, the fusion protein of the present invention may be used to treat post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, and other.

In an embodiment, the condition treated with the fusion protein of the present invention is characterized by pain and may be selected from nociceptive pain, neuropathic pain, or mixed nociceptive-neuropathic pain.

In another embodiment, the fusion protein of the present invention may be used to prevent chronic pain. Particularly, the fusion protein may be used to prevent neuropathic pain and neurodegeneration in cancer patients treated with chemotherapy.

In another aspect, the invention is directed to a fusion protein of the present invention for use in the prevention or treatment of a clinical condition in a mammal, such as a human, for which interleukin 13 is indicated.

-21 - In a further aspect, the invention is directed to a fusion protein of the present invention for use in the prevention or treatment of a clinical condition in a mammal, such as a human, for which interleukin 4 or interleukin 10 or IL33 or TGFB1 or TGFB2 is indicated.

According to one embodiment the fusion proteins taught herein can be used for inhibiting neuro-inflammation due to activation of glial and neuronal cells in the central nerve system.

As a result, the fusion proteins of the present invention can be used for the preparation of a medicament to attenuate neuro-inflammatory reactions by inhibiting the activation of glial cells and neuronal cells in vivo.

In an embodiment the fusion proteins of the present invention can be used as stand- alone drug. In another embodiment they are used in combination with other drugs.

Treatment (prophylactic or therapeutic) will generally consist of administering the fusion protein of the present invention parenterally, preferably intrathecally, intraarticularly, intravenously, intramuscularly or subcutaneously. The dose and administration regimen will depend on the extent of inhibition of neuroinflammation and neurodegeneration aimed at. Typically, the amount of the fusion protein given will be in the range of 0.5 ug to 1 mg per kg of body weight. The dosage can be determined or adjusted by measuring cytokine levels (IL13, IL4 or IL10 or IL33 or TGFB1 or TGFB2) in the body compartment targeted upon administration. The dose can also be determined by measuring neuro-inflammation in a patient for example by positron emission tomography (PET) imaging of microglia.

For parenteral administration, the fusion protein is preferably formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well- known in the art and examples include saline, dextrose solution, Ringer's solution and solutions containing small amounts of human serum albumin.

Typically, the fusion proteins of the present invention may be formulated in such vehicles at a concentration of from about 50 ug to about 100 mg per ml.

Gene therapy The nucleic acid constructs or vectors of the present invention may be used as gene therapy agents for treatment of the conditions set forth above.

As such, in an aspect the invention is directed to a vector as described above for use in the prevention or treatment of a condition characterized by visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, or mixed nociceptive-neuropathic pain, neuroinflammation and/or neurodegeneration, preferably wherein said condition may be selected from the group consisting of post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-operative

22.

peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, complex regional pain syndrome, post-spinal injury pain, post-stroke pain, multiple sclerosis, low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's disease and Parkinson’s disease, Huntington's disease, and/or amyotrophic lateral sclerosis, or multiple sclerosis.

As described earlier herein, the present invention relates to a fusion protein comprising atleast 2, 3, 4, preferably 2 (anti-inflammatory) interleukins chosen from the group consisting of interleukin 13 (IL13), interleukin 4 (IL4), interleukin 10 (IL10), interleukin 33 {IL33), transforming growth factor beta 1 (TGFB1), and transforming growth factor beta 2 (TGFB2). Accordingly, where reference is made in the present disclosure to “IL13”, this may be replaced by transforming growth factor beta 1 (TGFB1), or transforming growth factor beta 2 (TGFB2).

Similarly, where reference is made in the present disclosure to “IL4”, this may be replaced by transforming growth factor beta 1 (TGFB1), or transforming growth factor beta 2 (TGFB2). The present disclosure thus also encompasses a fusion protein of IL13/ TGFB1 or IL13/ TGFB2. Further, the present disclosure encompasses a fusion protein of IL4/ TGFB1 or IL4/ TGFB2. Additionally, the present disclosure encompasses a fusion protein of IL10/ TGFB1 or IL10/ TGFp2.

The present invention will now be illustrated with reference to the following examples, which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be construed as restricting the invention in any way.

SEQUENCE LISTING SEQ ID NO:1 — Amino acid sequence of human IL4

HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRC LGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKY SKC

SS SEQ ID NQ:2 — Amino acid sequence of human IL13

PGRYPPSTALRELIEELVNITONGKAPLCNGSMVYWSINLTAGMYCAALESLINVSGUSAIEKT

QRMLSGECPHKVSAGOFSSLHVRDTKIEVAGEVKDLLLHLKKLEREGGFEN SEQ ID NO:3 — Amino acid sequence of suitable linker

- 23.

GSGGGGSGT SEQ ID NO:4 — Amino acid sequence of an IL4-IL13 fusion protein (the linker sequence is underlined)

HKCDITLGEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRC LGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKC SSGSGGGGSGTPSPVPPSTALRELIEELVYNITONQKAPLONGSMVWSINLTAGMYCAALESL

INVSGCOSAIEKTQORMLSGECPHKVSAGQFSSLHVRDTKIEVAGEVEDLLLHLKKLEREGOEN SEQ ID NO:5 — Amino acid sequence of human IL10

SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLG CQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQ

VKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO:6 — Amino acid sequence of human 1L33

MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFR RETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGISPITEYLA SLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTL SPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHL

ALIKVDSSENLCTENILFKLSET SEQ ID NO:7 — Amino acid sequence of human immature and mature (underlined) TGFB1:

MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPS QGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFK QSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLL APSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLA TIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWK WIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVG

RKPKVEQLSNMIVRSCKCS SEQ ID NO:8 — Amino acid sequence of human immature and mature (underlined) TGFB2:

MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVP PEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSETVCPVVTTPSGS VGSLCSRQSQVLCGYLDAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLGNPKARV PEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISL HCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPS YRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAG

-24-

ACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCK

CS Brief description of the figures related to the invention Figure 1. Anti-inflammatory cytokines are required for resolution of chemotherapy- induced pain. Mice were intraperitoneally injected with 2 mg/kg paclitaxel at day 0 and 2 to induce transient painful chemotherapy-induced polyneuropathy. From day 6 on, mice received daily intrathecal injections of neutralizing antibodies to endogenous IL4 (n=4; open downward triangles), or IL13 (n=4; closed upward triangles) for 5 days (5 ug antibody per injection). As a control, isotype IgG was injected intrathecal in mice treated with paclitaxel (n=3; dotted line with open circles). As another control, mice were pretreated with vehicle instead of paclitaxel and control IgG (n=3; closed line with closed circles). Pain-like behavior was followed over time by measuring mechanical sensitivity to touch using von Frey hairs. Note that a lower 50% threshold indicates increased sensitivity. Data represent mean and standard error of the mean. Statistical differences are indicated as * p<0.05, ** p<0.01, *** p<0.001 between anti-IL13 IgG versus control IgG treated mice. x p<0.05, xx p<0.01, *** p<0.001 between anti-IL13 IgG versus control IgG treated mice.

Figure 2. IL13 attenuates paclitaxel-induced damage to neurons. Primary sensory neurons were cultured and treated overnight with Paclitaxel (1 uM} to induce neuronal damage that was quantified by measuring the neurite length after B3-tubulin staining. Vehicle (-) or individual cytokines (50 ng/ml) were added during treatment with the chemotherapeutic drug, and the average length of neurons was measured. Data represent mean and standard error of the mean of the neurite length in microns of >10 cells measured in at least 2 experiments.

Figure 3. Characterization of recombinant IL4/IL13 fusion protein. His-tagged IL4/IL13 fusion protein (IL4-13 FP) was expressed in HEK293 cells, purified using HIS-Select Nickel Affinity chromatography, and analyzed with High Pressure Size Exclusion Chromatography (HP-SEC). The HP_SEC profile indicates a homogenous monomeric preparation. Insert shows Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of starting material (load), flow through (FT), washing buffer (wash) and eluate of the His-tag purification column. The gel was stained with Coomassie Blue. Note that IL4/IL13 fusion protein migrates as a smear at 37 kDa.

Figure 4. IL4/IL13 fusion protein relieves paclitaxel-induced persistent mechanical allodynia. Paclitaxel (8 mg/kg) was administered intraperitoneally to C57BL/6 mice on days 0, 2, 4 and 6 (grey symbols on the X-axis) to induce persistent chemotherapy-induced polyneuropathy. IL4/IL13 fusion protein (0.3 [open circle], 1 [open triangle] or 3 pg/mouse [open square]; n=4/group) or vehicle (n=4) was administered intrathecally at day 8, and the course of

- 95. mechanical allodynia was followed over time using von Frey hairs. Data is represented as mean t SEM. Statistics of the data were analysed with two-way ANOVA followed by Tukey's multiple comparisons test. *, **, *** = p<0.05, p<0.01, and 0.001, 0.3 ug IL4/IL13 fusion protein versus vehicle respectively. &, && = p<0.05, p<0.01 respectively, 3 ug IL4/IL13 fusion protein versus vehicle. x = p<0.05, 1 ug IL4/IL13 fusion protein versus vehicle. Figure 5. IL4/IL13 fusion protein has a longer lasting effect on painful paclitaxel-induced neuropathy in mice than IL4/IL10 fusion protein. Mice received 4 intraperitoneal injections of 8 mg/kg paclitaxel every other day (grey symbols on X-axis) to induce persisting painful chemotherapy-induced polyneuropathy. At day 8, mice received a single intrathecal injection of IL4/IL13 fusion protein (0.7 ug; open circles, n=4), IL4/IL10 fusion protein (0.7 ug; open triangles, n=3), the combination of wildtype IL4 and IL13 (0.35 ug/cytokine; open squares, n=4) or vehicle (closed circles, n=4). Pain-like behavior was tested with von Frey hairs (see figure 1). Note that a single administration of IL4/IL13 fusion protein permanently resolves pain, whereas IL4/IL10 fusion protein has a temporary effect lasting 2 days.

Figure 6. IL4/IL13 fusion protein protects against paclitaxel-induced nerve damage in mice. Mice received 4 intraperitoneal injections of 8 mg/kg paclitaxel every other day to induce persisting painful chemotherapy-induced polyneuropathy. On day 8, they were treated with a single intrathecal injection of IL4/IL13 fusion protein (0.7 ug), or vehicle. On day 15, the length of intraepidermal nerve fibers in the paw skin was determined upon immunofluorescent visualization with the neuronal marker PGP9.5. The data of mice not treated with chemotherapeutic drug (black bar; n=4), or injected with paclitaxel and subsequently treated with vehicle (- ; n=6) or IL4/IL3 fusion protein (IL4-13; n=4), are shown.

Figure 7. IL4IL13 fusion protein protects cultured neurons against chemotherapy induced damage better than IL4/IL10 fusion protein or the combination of IL4 and IL13.

Primary sensory neurons were cultured overnight in presence of paclitaxel (1 pM) to induce neuronal damage that was quantified by measuring the neurite length upon B3-tubulin staining. Vehicle (-) or IL4/IL13 fusion protein (IL4-13), IL4IL10 fusion protein (IL4-10) or the combination of IL4 and IL13 (IL4+IL13) were added at equimolar concentrations during incubation with the chemotherapeutic drug. Neurons cultured in absence of paclitaxel and cytokines are shown for comparison (black bar).

Figure 8. IL4/IL13 cures oxaliplatin-induced polyneuropathy in mice whereas IL4 or IL13 only have a partial transient effect. Oxaliplatin (3 mg/kg) was daily injected intraperitoneally in mice for 5 days followed by 5 days no treatment and another 5 days of an oxaliplatin treatment cycle (grey symbols on X-axis). On the day after the last oxaliplatin injection animals received an intrathecal injection of IL4/IL13 fusion protein (0.3 kg; open circles, n=4) or the wild-type cytokines {0.15 ug; n=4, rectangles for IL4 and triangles for IL13}; or vehicle only (closed circles). Pain was measured with von Frey test.

- 96 - Figure 9. IL4/IL13 fusion protein, but not the combination of IL4 and IL13, protects cultured neurons against oxaliplatin-induced damage. Primary sensory neurons were cultured and treated overnight with oxaliplatin (5 pg/ml). Neuronal damage was then quantified by measuring the neurite length upon B3-tubulin staining. Vehicle (-) or IL4/IL13 fusion protein or the combination of IL4 and IL13 (IL4+IL13) were added at equimolar concentrations during incubation with the chemotherapeutic drug. Neurons cultured in absence of oxaliplatin and cytokines are shown for comparison (black bar).

Figure 10. Cytokine-receptors of IL-10, IL-4, IL-13, and TGFB1/2 are expressed in the dorsal root ganglia of human and mouse. To evaluate whether cytokine receptors targeted by fusion proteins of the present invention are expressed by the sensory system, RNAseq data of receptors for IL-10, IL-4, IL-13, and TGFB1/2 in the dorsal root ganglia and spinal cord were extracted from the data base by Ray et al. (Pain 2018;159:1325-1345) as available on hitps www. utdallas edu/bbs/painneurosciencelab/sensoryomics/drgtxome/?qo. RNA sequencing data are expressed as transcripts per million. For comparison, data for expression of the receptors in whole blood are also given.

EXAMPLES Animals.

All animal experiments were performed in agreement with international guidelines and with prior approval by the University Medical Centre Utrecht experimental animal committee. Experiments were conducted using both male and female C57BL/6 mice aged between 8 and 16 weeks. Observers carrying out behavioural experiments were blinded to treatment.

Material and methods IL4/IL13 fusion protein. 1L4-13 fusion protein HEK293 cells were transiently transfected according to standard procedures with a vector containing a transgene (Y Derocher et al., Nucleic Acids Research 2002, vol 30, no 2, e9). Briefly, synthetic cDNA (GeneArt, ThermoFisher Scientific) coding for the IL4-IL13 fusion protein sequence of the present invention (see SEQ ID no.4, N-terminal of this sequence a 6-His tag was inserted) was cloned in a pUPE expression vector, containing a cystatin signal sequence. HEK293E cells were then transfected with the pUPE expression vector, and co-transfected with a vector carrying the transgene for beta-galactoside alpha-2,3-sialyltransferase 5 (SIAT 9; homo sapiens) to optimize capping of the glycans with sialic acid. Cells were cultured in FreeStyle medium (Invitrogen) with 0.9% primatone and ~ 0.04 %, v/v, fetal calf serum as described before'®?. Cell suspension was collected on day 4 after transfection, and centrifuged at 435xg for 5 minutes. The supernatant was passed through a HIS-Select Nickel Affinity gel (Sigma-Aldrich)

-27- to purify the recombinant IL4/IL13 fusion protein.

Elution fraction was dialysed overnight at 4°C against phosphate buffered saline, pH 7.4 (PBS). Protein assays.

Bradford (Bio-Rad), BCA (Thermo Scientific), and Qubit 1.0 (Thermo Scientific) were used to determine the amount of protein in the eluded & dialysed fractions.

All protein assays were performed according to the manufacturer's protocols.

ELISAs.

The amount of fusion protein was determined based on the amount of the individual cytokines, measured with ELISA (IL4 Pelipair ELISA kit, Sanquin; IL13, DuoSet ELISA, R&D Systems) ELISAs were performed according to the manufacturer's instructions.

Concentrations were calculated based on the theoretical molecular weight of the fusion protein compared to the individual cytokines.

SDS-Page and Western Blot.

Fractions of the HIS-Select Nickel affinity chromatography purification (load, flow-through, wash, elution/dialysis) were separated on 12% polyacrylamide SDS-Page gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes.

Membranes were stained with IL4 antibody (Santa Cruz, SC-13555). High Performance Size Exclusion Chromatography (HP-SEC). To determine its molecular weight and homogeneity, IL4/IL13 fusion protein was analyzed with High Performance Size Exclusion Chromatography (HP-SEC). The gel filtration (BioSuite 125 4 um UHR SEC Column; Waters; Cat# 186002161) was performed on a High-Performance Liquid Chromatography System (Shimadzu) with 50 mM phosphate buffer containing 0.5 M NaCl as mobile phase.

The column was calibrated prior to the run using a protein mix of thyroglobulin, bovine serum albumen, carbonic anhydrase, myoglobulin, and ribonuclease.

Fifty ul of 20 pg/ml of purified IL4/IL13 fusion protein was injected and separated on the column at a flow rate of 0.35 ml/min and under a pressure of 35 bar.

Evaluation of chemotherapy-induced neurotoxicity in vitro.

Dorsal root ganglion neurons were cultured as described previously?’. Briefly, adult mice DRG neurons were dissected out and subsequently digested in an enzyme mixture containing Ca?- and Mg?’-free HBSS, 5 mM HERES, 10 mM glucose, collagenase type XI (Emg/ml) and dispase (10 mg/ml) for 1 hour before mechanical trituration in DMEM containing 10%, v/v, heat-inactivated felal calf serum.

Celis were centrifuged for 5 min at 800 rpm, resuspended in DMEM containing 4.5 g/l. glucose, 4 mM L-glutamine, 110 mg/L sodium pyruvate, 10% fetal calf serum, 1% penicillin- streptomycin (10,000 IU/mi}, 1% glutamax, 125 ng/ml nerve growth factor, and incubated in 24 wells plates for 24 hours in presence of paclitaxel (1 uM) or oxaliplatin (5 ug/ml) to induce

- 28 - neurotoxicity. 1L4/IL10 fusion protein {100 ng/mL), IL4/IL13 fusion protein (100 ng/mL), IL4 an IL13 (50 ng/mL each), IL4 (50 ng/mL), IL10 (50 ng/mL), and IL13 {50 ng/mL) were added together with the chemotherapeutic agent. As controls cells were also cultured in absence of chemotherapeutic drugs or cytokines, and in presence of chemotherapeutic drugs only. After fixation with 4% paraformaldehyde, cells were stained with rabbit anti-mouse Blll-tubulin (ab18207, 1:1000; Abcam). Neurites were visualized with a Zeiss Axio Lab A1 microscope (Zeiss — Oberkochen, Germany) and using a random sampling method, at least 10 images per glass slide were made at a magnification of 10x. The length of neurites was measured with the ImageJ plugin Simple Neurite Tracer78. The averages of neurite length per neuron for a minimum of five neurons per condition were compared between groups for the three individual primary sensory cultures.

Chemotherapy-induced polyneuropathy and assessment of allodynia. To induce transient chemotherapy-induced polyneuropathy (CIPN), paclitaxel (2 mg/kg, Cayman Chemical Company) was injected intraperitoneally on days O and 2. To induce persistent paclitaxel- induced CIPN paclitaxel (8 mg/kg, Cayman Chemical Company) was injected intraperitoneal on day 0, 2, 4 and 6. To induce persistent oxaliplatin-induced polyneuropathy, mice received two treatment cycles, each consisting of 5 daily intraperitoneal injections of 3 mg/kg oxaliplatin (Tocris) with a 5 days free interval.

Noxious mechanical sensitivity in the hind paws was measured using von Frey hairs (Stoelting, Wood Dale, USA). Results were expressed as the 50% paw-withdrawal threshold using the up-and-down method23. In some experiments the length of intraepidermal nerve fibers in the paw skin at day 15 was determined by immunofluorescent staining of skin biopsies with the neuronal marker PGP9.5. All experiments were performed in a blinded manner.

Statistical analysis. Unless indicated otherwise, all data are expressed as mean + SEM. Data were analysed for statistical significance by one-way or two-way ANOVA (with repeated measures if appropriate) followed by the appropriate post-hoc test. A p value of p<0.05 was considered significant.

Example 1. Endogenous IL4 and IL13 are necessary for normal resolution of paclitaxel- induced transient hyperalgesia.

To investigate the possible role of anti-inflammatory cytokines on recovery from chemotherapy-induced polyneuropathy, the transient pain model of paclitaxel-induced pains was used. Mice received 2 injections of low dose (2 mg/kg) of paclitaxel on days 0 and 2. From days 6 to 10 after start of chemotherapy treatment, mice received a daily intrathecal injection of neutralizing antibodies against IL4, IL13 or control IgG antibodies (5 pg antibody per

- 29. injection; Fig. 1). Mice that received paclitaxel, developed mechanical hyperalgesia starting on the first day of chemotherapy, which resolved spontaneously after one week of treatment termination (Fig. 1). In animals intrathecally injected with neutralizing antibodies against IL4 or IL13, resolution of hyperalgesia was delayed and persisted for at least 2 weeks. These data indicate that endogenously produced IL4 and IL13 are necessary for normal pain resolution after chemotherapy treatment (Fig. 1). Example 2. Neuroprotective effects of IL4, IL10 and IL13. To assess whether anti-inflammatory cytokines possess neuroprotective properties sensory neurons isolated from the dorsal root ganglion were cultured overnight with paclitaxel in combination of either IL4, IL10 or IL13 as described in methods. Neurotoxicity was evaluated by measuring neurite length using BIII-tubulin staining. Paclitaxel (1 uM) reduced neurite length with ~50% indicating paclitaxel damaged sensory neurons. Addition of IL13 during the culture with paclitaxel prevented the paclitaxel-induced negative effect on neurite length, whilst IL10 and IL4 did not (Fig. 2). Example 3. Characterization of recombinant IL4/13 fusion protein. Human IL4/IL13 fusion protein (see SEQ ID no 4) with an N-terminal 6 His-tag was produced by transient transfection of HEK293 cells and purified as described in methods. On HP-SEC the purified IL4/IL13 fusion protein migrated as a single peak with an apparent mass of 40 kDa (Fig.3). The preparation was also analyzed on SDS-PAGE and Coomassie staining. A homogenous preparation migrating as a smear with a molecular mass of ~37 kDa was detected (insert Fig.3). Example 4. IL4/IL13 fusion protein cures paclitaxel-induced persistent polyneuropathy. The potential of IL4-13 fusion protein to inhibit chemotherapy-induced hyperalgesia was evaluated in model of persistent paclitaxel-induced painful neuropathy®°. Mice received 4 injections of paclitaxel (8 mg/kg) every other day from day 0 to 6. Paclitaxel induced mechanical hyperalgesia that started on the first day after the first injection and that persisted at least 3 weeks after chemotherapy-treatment was stopped. Two days after the last paclitaxel injection, mice were injected intrathecally with 3 different doses of 1L4/IL13 fusion protein (0.3, 1 and 3 ug/mouse) (Fig. 4). All three doses of IL4/IL13 fusion protein markedly reduced paclitaxel- induced polyneuropathy. Importantly, the almost normalization of mechanical hyperalgesia lasted for at least a week, demonstrating the potential of the 1L4-13 fusion protein for long- lasting resolution of chemotherapy-induced polyneuropathy.

Example 5. Potency of IL4/IL13 fusion protein is superior over IL4/IL10 fusion protein or IL4 and IL13 combination therapy to cure chemotherapy-induced polyneuropathy.

-30- Next it was assessed whether IL4/IL13 fusion protein inhibits paclitaxel-induced polyneuropathy better than IL4/IL10 fusion protein, or the combination of IL4 and IL13. Mice developed paclitaxel-induced painful polyneuropathy after 4 injections of paclitaxel (8 mg/kg) every other day from day 0 to 6. IL4/IL13 fusion protein inhibited paclitaxel-induced mechanical hypersensitivity for at least 1 week, whilst the combination of wildtype IL4 and IL13, or IL4/IL10 fusion protein only inhibited paclitaxel-induced mechanical hypersensitivity for 1-2 days (Fig. 5). The inhibition of paclitaxel-induced persistent allodynia by IL4IL13 fusion protein was associated with reduced paclitaxel-induced intra-epidermal nerve fibre loss in the paw skin (Fig. 6).

To evaluate whether IL4/IL13 fusion protein protects against neurotoxicity induced by paclitaxel in vitro we measured neurite length of mouse sensory neurons cultured in presence of paclitaxel with or without fusion protein. Paclitaxel had a significant negative effect on neurite length when compared to the control group (Fig. 7). Simultaneous presence of IL4/IL10 fusion protein or the combination of IL4 and IL13 had a moderate beneficial effect on neurite length.

However, presence of IL4-IL13 fusion protein in the culture markedly prevented paclitaxel- induced neurotoxicity (Fig. 7). Thus, these data together demonstrated an unexpected superior effect of IL4/IL13 fusion protein over IL4/IL10 fusion protein or the combination of IL4 and IL13 to protect neurons against toxic effects of the chemotherapeutic drug paclitaxel. In particular the superiority of the fusion protein over the combination was surprising as the in vitro system is not affected by different clearance of the proteins from the site of action. Rather the data pointed to a unique effect of IL4/IL13 fusion protein regarding neuroprotection.

Example 6. IL4/IL13 fusion protein also cures polyneuropathy induced by oxaliplatin.

It was investigated whether neuroprotective effects of IL4/IL13 fusion protein are unique for paclitaxel-induced neurotoxicity or whether neuroprotective effects are against a broader spectrum of chemotherapy-induced polyneuropathy. Toxic neuropathy was induced in mice using a platinum-based chemotherapeutic drug, oxaliplatin. Two cycles of 5 times a daily injection of oxaliplatin, separated by 5 days without intraperitoneal injection, induced mechanical allodynia that persisted for at least 3 weeks (Fig. 8). Intrathecal injection of IL4/IL13 fusion protein on the second day after the last oxaliplatin injection reduced mechanical allodynia significantly for 4 days (Fig. 8). Intrathecal injection of either wild-type IL4 or wild-type IL13 transiently inhibited oxaliplatin-induced mechanical allodynia for ~1 day, which effect was significantly shorter than IL4-13. Similarly, in vitro IL4/IL13 fusion protein protected against oxaliplatin-induced neurotoxicity, whilst the combination of IL4 and IL13 did not (Fig. 9). Thus, these data indicate that IL4IL13 fusion protein protects against chemotherapy-induced neurotoxicity and chemotherapy-induced polyneuropathy.

-31- Example 7. IL10/IL13 fusion protein Further, it was considered whether an IL10/L13 fusion protein or IL33/IL13 fusion protein would have a therapeutic effect in the medical indications as disclosed herein. Surprisingly, cross-linking of the IL10 receptor and the IL13 receptor by administration of an IL10/L13 fusion protein, or the cross-linking of the IL33 receptor and the IL13 receptor by administration of an IL33/IL13 fusion protein, can lead to a stronger and prolonged therapeutic effect, in comparison to administration of an IL4/IL10 fusion protein, and in particular in the neuropathic pain model. Example 8 Finally, it was addressed whether cytokine receptors targeted by fusion proteins of the present invention are expressed by the sensory system. To analyze this, RNAseq data of receptors for IL-10, IL-4, IL-13, and TGFB1/2 in the dorsal root ganglia and spinal cord were extracted from the data base by Ray et al. (Pain 2018:159:1325-1345) as available on niips-//www.utdallas sdu/bbs/painneurosciencelab/sensoryomics/drgikome/?go. RNA sequencing revealed expression of receptors for IL-10, IL-4, IL-13, TGFB1 and TGFB2 in the dorsal root ganglia and spinal cord of human and mouse (Figure 10; data are expressed as transcripts per million). References

1. Breivik H, et al. Survey of chronic pain in Europe: prevalence, impact on daily life, and treatment. Eur J Pain 2006;10:287-333.

2. Johannes CB, et al. The prevalence of chronic pain in United States adults: results of an Internet-based survey. J Pain 2010;11:1230-9.

3. Langley PC. The prevalence, correlates and treatment of pain in the European Union. Curr Med Res Opin 2011;27:483-80.

4. van Hecke O, et al. Chronic pain epidemiology and its clinical relevance. Br J Anaesth 2013;111:13-8

5. Dahlhammer J, et al. Prevalence of Chronic Pain and High-Impact Chronic Pain Among Adults — United States, 2018. Morbidity and Mortality Weekly Report (MMWR) 2018;67:1001-6

6. Breivik H, et al. The individual and societal burden of chronic pain in Europe: the case for strategic prioritisation and action to improve knowledge and availability of appropriate care. BMC Public Health 2013;13:1229

7. Scholz J, et al. The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 2007;10:1361-8

8. Woolf CJ, et al. Neuronal plasticity: increasing the gain in pain. Science 2000;288:1765-9

-32.

9. Ji R-R, et al. Pain regulation by non-neuronal cells and inflammation. Science. 2016;354:572-7

10. Chen G, et al. Microglia in Pain: Detrimental and Protective Roles in Pathogenesis and Resolution of Pain. Neuron 2018;100:1292-311

11. Serhan CN, et al. Resolution of inflammation: state of the art, definitions and terms. FASEB J 2007;21:325-32

12. Schaible HG. Nociceptive neurons detect cytokines in arthritis. Arthritis Res Ther 2014;16:470

13. Raoof R, et al. Divergent roles of immune cells and their mediators in pain. Rheumatology (Oxford) 2018;57:429-40

14. Willemen HL, et al. Monocytes/Macrophages control resolution of transient inflammatory pain. J Pain 2014;15:496-506

15. Krukowski K, et al. CD8+ T Cells and Endogenous IL10 Are Required for Resolution of Chemotherapy-Induced Neuropathic Pain. J Neurosci 2016;36:11074-83

18. Rijsdijk M, et al. The effects of glucocorticoids on neuropathic pain: a review with emphasis on intrathecal methylprednisolone acetate delivery. Anesth Analg 2014;118:1097-

112.

17. Rijsdijk M, et al. No beneficial effect of intrathecal methylprednisolone acetate in postherpetic neuralgia patients. Eur J Pain 2013;17:7 14-23.

18. Usoskin D, et al. Unbiased classification of sensory neuron types by large-scale single- cell RNA sequencing. Nat Neurosci 2015;18:145-53

19. Eijkelkamp N, et al. IL4-10 Fusion Protein Is a Novel Drug to Treat Persistent Inflammatory Pain. J Neurosci 2016;36:7353-63.

20. Old EA, et al. The role of glia in the spinal cord in neuropathic and inflammatory pain. Handb Exp Pharmacol 2015;227:145-70

21. JiRR, et al. Glia and pain: is chronic pain a gliopathy? Pain 2013;154 Suppl 1:510-28.

22. Grace PM, et al. Pathological pain and the neuroimmune interface. Nat Rev Immunol 2014;14:217-31.

23. WO 2013/070076 A1 Fusion protein comprising interleukin 4 and interleukin 10. JAG van Roon, SAY Hartgring, CE Hack, C Louws, FPJG Lafeber (inventors).

24. Bottros MM, et al. Current perspectives on intrathecal drug delivery. J Pain Res 2014;7:615-26

25. Steen-Louws C, et al. IL4-10 fusion protein has chondroprotective, anti-inflammatory and potentially analgesic effects in the treatment of osteoarthritis. Osteoarthritis Cartilage 2018;26:1127-35

26. Steen-Louws C, et al. IL4-10 fusion protein: a novel immunoregulatory drug combining activities of interleukin 4 and interleukin 10. Clin Exp Immunol 2019;195:1-9.

-33.

27. Thom G, et al. Probing a protein—protein interaction by in vitro evolution. PNAS 2006;103:7619-24

28. Eijkelkamp N, et al. A role for Piezo2 in EPAC1-dependent mechanical allodynia. Nat Commun 2013;4:1682

29. Chaplan SR, et al. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994;53:55-63

30. Toma W, et al. Effects of paclitaxel on the development of neuropathy and affective behaviors in the mouse. Neuropharmacology 2017;117:305-15

SEQLTXT

SEQUENCE LISTING <110> UMC Utrecht Holding B.V. <120> A fusion protein comprising IL13 <130> P33747NL00 <160> 8 <170> PatentIn version 3.5 <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 Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala 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 Pagina 1

SEQLTXT <210> 2 <211> 113 <212> PRT <213> Homo sapiens <400> 2 Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Glu Leu Ile Glu Glu 1 5 10 15 Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly Ser

Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala Leu 40 45 Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr Gln 50 55 60 Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln Phe 65 70 75 80 Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe Val 85 90 95 Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Gln Phe 100 105 110 Asn <210> 3 <211> 9 <212> PRT <213> Homo sapiens <400> 3 Gly Ser Gly Gly Gly Gly Ser Gly Thr 1 5 Pagina 2

SEQLTXT <210> 4 <211> 251 <212> PRT <213> Homo sapiens <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

Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala 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 Pro Gly Pro Val Pro Pro 130 135 140 Ser Thr Ala Leu Arg Glu Leu Ile Glu Glu Leu Val Asn Ile Thr Gln 145 150 155 160 Asn Gln Lys Ala Pro Leu Cys Asn Gly Ser Met Val Trp Ser Ile Asn 165 170 175 Pagina 3

SEQLTXT Leu Thr Ala Gly Met Tyr Cys Ala Ala Leu Glu Ser Leu Ile Asn Val 180 185 190 Ser Gly Cys Ser Ala Ile Glu Lys Thr Gln Arg Met Leu Ser Gly Phe 195 200 205 Cys Pro His Lys Val Ser Ala Gly Gln Phe Ser Ser Leu His Val Arg 210 215 220 Asp Thr Lys Ile Glu Val Ala Gln Phe Val Lys Asp Leu Leu Leu His 225 230 235 240 Leu Lys Lys Leu Phe Arg Glu Gly Gln Phe Asn 245 250 <210> 5 <211> 160 <212> PRT <213> Homo sapiens <400> 5 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

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 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 Pagina 4

SEQLTXT 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> 6 <211> 270 <212> PRT <213> Homo sapiens <400> 6 Met Lys Pro Lys Met Lys Tyr Ser Thr Asn Lys Ile Ser Thr Ala Lys 1 5 10 15 Trp Lys Asn Thr Ala Ser Lys Ala Leu Cys Phe Lys Leu Gly Lys Ser

Gln Gln Lys Ala Lys Glu Val Cys Pro Met Tyr Phe Met Lys Leu Arg 40 45 Ser Gly Leu Met Ile Lys Lys Glu Ala Cys Tyr Phe Arg Arg Glu Thr 50 55 60 Thr Lys Arg Pro Ser Leu Lys Thr Gly Arg Lys His Lys Arg His Leu 65 70 75 80 Val Leu Ala Ala Cys Gln Gln Gln Ser Thr Val Glu Cys Phe Ala Phe 85 90 95 Gly Ile Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp Ser Ser 100 105 110 Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu Ser Thr 115 120 125 Pagina 5

SEQLTXT Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser Tyr Glu 130 135 140 Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys Val Leu 145 150 155 160 Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly Asp Gly 165 170 175 Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp Phe 180 185 190 Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys Cys 195 200 205 Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met His 210 215 220 Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val Phe Ile 225 230 235 240 Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser Ser Glu 245 250 255 Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr 260 265 270 <210> 7 <211> 390 <212> PRT <213> Homo sapiens <400> 7 Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Leu 1 5 10 15 Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr

Pagina 6

SEQLTXT Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala 40 45 Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser 50 55 60 Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu 65 70 75 80 Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu 85 90 95 Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu 100 105 110 Met Val Glu Thr His Asn Glu Ile Tyr Asp Lys Phe Lys Gln Ser Thr 115 120 125 His Ser Ile Tyr Met Phe Phe Asn Thr Ser Glu Leu Arg Glu Ala Val 130 135 140 Pro Glu Pro Val Leu Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Leu 145 150 155 160 Lys Leu Lys Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn 165 170 175 Asn Ser Trp Arg Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp Ser 180 185 190 Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln Trp Leu 195 200 205 Ser Arg Gly Gly Glu Ile Glu Gly Phe Arg Leu Ser Ala His Cys Ser 210 215 220 Cys Asp Ser Arg Asp Asn Thr Leu Gln Val Asp Ile Asn Gly Phe Thr 225 230 235 240 Pagina 7

SEQLTXT Thr Gly Arg Arg Gly Asp Leu Ala Thr Ile His Gly Met Asn Arg Pro 245 250 255 Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu Gln 260 265 270 Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser 275 280 285 Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys 290 295 300 Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala Asn 305 310 315 320 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr 325 330 335 Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala 340 345 350 Ala Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr 355 360 365 Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val 370 375 380 Arg Ser Cys Lys Cys Ser 385 390 <210> 8 <211> 442 <212> PRT <213> Homo sapiens <400> 8 Met His Tyr Cys Val Leu Ser Ala Phe Leu Ile Leu His Leu Val Thr 1 5 10 15 Pagina 8

SEQLTXT Val Ala Leu Ser Leu Ser Thr Cys Ser Thr Leu Asp Met Asp Gln Phe

Met Arg Lys Arg Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu 40 45 Lys Leu Thr Ser Pro Pro Glu Asp Tyr Pro Glu Pro Glu Glu Val Pro 50 55 60 Pro Glu Val Ile Ser Ile Tyr Asn Ser Thr Arg Asp Leu Leu Gln Glu 65 70 75 80 Lys Ala Ser Arg Arg Ala Ala Ala Cys Glu Arg Glu Arg Ser Asp Glu 85 90 95 Glu Tyr Tyr Ala Lys Glu Val Tyr Lys Ile Asp Met Pro Pro Phe Phe 100 105 110 Pro Ser Glu Thr Val Cys Pro Val Val Thr Thr Pro Ser Gly Ser Val 115 120 125 Gly Ser Leu Cys Ser Arg Gln Ser Gln Val Leu Cys Gly Tyr Leu Asp 130 135 140 Ala Ile Pro Pro Thr Phe Tyr Arg Pro Tyr Phe Arg Ile Val Arg Phe 145 150 155 160 Asp Val Ser Ala Met Glu Lys Asn Ala Ser Asn Leu Val Lys Ala Glu 165 170 175 Phe Arg Val Phe Arg Leu Gln Asn Pro Lys Ala Arg Val Pro Glu Gln 180 185 190 Arg Ile Glu Leu Tyr Gln Ile Leu Lys Ser Lys Asp Leu Thr Ser Pro 195 200 205 Thr Gln Arg Tyr Ile Asp Ser Lys Val Val Lys Thr Arg Ala Glu Gly 210 215 220 Pagina 9

SEQLTXT Glu Trp Leu Ser Phe Asp Val Thr Asp Ala Val His Glu Trp Leu His 225 230 235 240 His Lys Asp Arg Asn Leu Gly Phe Lys Ile Ser Leu His Cys Pro Cys 245 250 255 Cys Thr Phe Val Pro Ser Asn Asn Tyr Ile Ile Pro Asn Lys Ser Glu 260 265 270 Glu Leu Glu Ala Arg Phe Ala Gly Ile Asp Gly Thr Ser Thr Tyr Thr 275 280 285 Ser Gly Asp Gln Lys Thr Ile Lys Ser Thr Arg Lys Lys Asn Ser Gly 290 295 300 Lys Thr Pro His Leu Leu Leu Met Leu Leu Pro Ser Tyr Arg Leu Glu 305 310 315 320 Ser Gln Gln Thr Asn Arg Arg Lys Lys Arg Ala Leu Asp Ala Ala Tyr 325 330 335 Cys Phe Arg Asn Val Gln Asp Asn Cys Cys Leu Arg Pro Leu Tyr Ile 340 345 350 Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly 355 360 365 Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys Pro Tyr Leu Trp Ser Ser 370 375 380 Asp Thr Gln His Ser Arg Val Leu Ser Leu Tyr Asn Thr Ile Asn Pro 385 390 395 400 Glu Ala Ser Ala Ser Pro Cys Cys Val Ser Gln Asp Leu Glu Pro Leu 405 410 415 Thr Ile Leu Tyr Tyr Ile Gly Lys Thr Pro Lys Ile Glu Gln Leu Ser 420 425 430 Pagina 10

SEQLTXT Asn Met Ile Val Lys Ser Cys Lys Cys Ser 435 440 Pagina 11

Claims

-34 - CONCLUSIONS

A fusion protein comprising an interleukin 13 and an interleukin selected from interleukin 4, interleukin 10, interleukin 33, transforming growth factor beta 1, transforming growth factor beta 2 and interleukin 13.

The fusion protein of claim 1, wherein said interleukin 13 and said interleukin selected from interleukin 4, interleukin 10, interleukin 33, transforming growth factor beta 1, transforming growth factor beta 2 and interleukin 13 are linked by a linker sequence.

The fusion protein of any of claims 1 to 2, wherein the interleukin 13 is N-terminally fused from the interleukin selected from interleukin 4, interleukin 10, interleukin 33, transforming growth factor beta 1, transforming growth factor beta 2, and interleukin 13.

The fusion protein of any one of claims 1 to 2, wherein the interleukin selected from interleukin 4, interleukin 10, interleukin 33, transforming growth factor beta 1, transforming growth factor beta 2 and interleukin 13, is N-terminally fused of the interleukin 13.

The fusion protein of any one of the preceding claims, further comprising one or more chemical modification (s).

The fusion protein of claim 5, wherein the chemical modification is selected from the group consisting of glycosylation, fucosylation, sialylation and pegylation.

The fusion protein of any one of the preceding claims, wherein the interleukin 13 is human interleukin 13.

The fusion protein of any preceding claim, wherein said interleukin 4 is human interleukin 4 and / or said interleukin 10 is human interleukin 10 and / or said interleukin 33 is human interleukin 33 and / or the transforming growth factor beta 1 is human transforming growth factor beta 1 and / or the transforming growth factor beta 2 is human transforming growth factor beta 2.

A nucleic acid molecule comprising a polynucleotide encoding the fusion protein of any one of the preceding claims.

-35.

A vector comprising the nucleic acid molecule of claim 9.

A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

A method for producing a fusion protein according to any one of claims 1-8, said method comprising the steps of: - culturing a host cell according to claim 11 under conditions that allow the production of the fusion protein according to enable any of claims 1-8; - optionally, purifying the fusion protein from the conditioned culture medium.

A pharmaceutical composition comprising the fusion protein of any of claims 1 to 8 and a pharmaceutically acceptable carrier.

A fusion protein according to any of claims 1-8 for use as a medicament.

A fusion protein according to any of claims 1 to 8 for use in the prevention or treatment of a condition characterized by chronic pain, neuroinflammation or neurodegeneration.

The fusion protein for use according to claim 15, wherein the condition is further characterized by visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, or mixed nociceptive-neuropathic pain, neuroinflammation and / or neurodegeneration.

Fusion protein for use according to claims 15 or 18, wherein the condition is selected from the group consisting of post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, chemotherapy-induced allodynia, complex regional pain syndrome, post-spinal injury pain, post-stroke pain, multiple sclerosis, low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's and Parkinson's disease, Huntington's disease and / or amyotrophic lateral sclerosis or multiple sclerosis.

- 36 -

The fusion protein of any one of claims 1 to 8 for use in the prevention or treatment of a clinical condition in a mammal, such as a human, for which interleukin 13 is indicated.

A fusion protein according to any of claims 1 to 8 for use in the prevention or treatment of a clinical condition in a mammal, such as a human, for which interleukin 4 and / or interleukin 10 and / or interleukin 33 and / or transforming growth factor beta 1 and / or transforming growth factor beta 2 is indicated.

20. Gene therapy vector containing nucleotide sequence (s) encoding interleukin 13 and an interleukin selected from interleukin 4, interleukin 10, interleukin 33, transforming growth factor beta 1, transforming growth factor beta 2 and interleukin 13, for use in the prevention or treatment of a condition which is characterized by chronic pain, neuroinflammation and / or neurodegeneration.

The gene therapy vector of claim 20, wherein said condition is further characterized by visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, or mixed nociceptive-neuropathic pain, neuroinflammation and / or neurodegeneration.

The gene therapy vector of claims 20 or 21, wherein said condition is selected from the group consisting of post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic neuralgia, post-traumatic operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, chemotherapy-induced allodynia, complex regional pain syndrome, post-spinal injury pain, post-stroke pain, low, multiple sclerosis back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's and Parkinson's disease, Huntington's disease and / or amyotrophic lateral sclerosis or multiple sclerosis.