Fusion proteins containing IL13

Abstract

The present invention relates to fusion proteins comprising interleukin (13) and a regulatory cytokine, e.g., an interleukin selected from interleukin (4), interleukin (10), interleukin (27), interleukin (33), transforming growth factor beta (1), transforming growth factor beta (2), and interleukin (13), nucleic acid molecules encoding such fusion proteins, vectors containing such nucleic acid molecules, and host cells containing such nucleic acid molecules or such vectors. The present invention further relates to methods for producing such fusion proteins. The fusion proteins, or gene therapy vectors encoding the fusion proteins, can be used in the prevention or treatment of conditions characterized by pathological pain, chronic pain, neuroinflammation, and / or neurodegeneration.

Description

[Technical field]

[0001] cross reference This application claims priority to Dutch Patent Applications Nos. 2022982 and 2022984, each of which is incorporated herein by reference in its entirety.

[0002] The present invention is in the field of neuroimmunology and pharmacology, in particular the treatment of chronic pain, neuroinflammatory and neurodegenerative diseases and inflammatory disorders.The present invention particularly relates to novel fusion proteins comprising interleukin 13 (IL13) and a regulatory cytokine, such as, but not limited to, an interleukin selected from interleukin 4 (IL4), interleukin 10 (IL10), interleukin 27 (IL27), interleukin 33 (IL33), transforming growth factor (TGF) beta 1 (TGFβ1), transforming growth factor beta 2 (TGFβ2) and IL13 itself, either physically fused or not fused through a linker sequence. In particular, the present invention provides IL4 / IL13, IL10 / IL13, IL27 / IL13, IL33 / IL13, TGFβ1 / IL13, TGFβ2 / IL13 or IL13 / IL13 fusion proteins endowed with superior analgesic, neuroprotective and anti-inflammatory activity than the individual cytokine combinations or than fusion proteins of IL4 and IL10. The invention also provides nucleic acid sequences encoding fusion proteins, such as an IL4 / IL13 fusion protein, an IL10 / IL13 fusion protein, an IL27 / IL13 fusion protein, an IL33 / IL13 fusion protein, a TGFβ1 / IL13 fusion protein, a TGFβ2 / IL13 fusion protein or an IL13 / IL13 fusion protein; expression vectors comprising such nucleic acid sequences; host cells or host organisms modified to carry a nucleic acid sequence encoding an IL4 / IL13 fusion protein, an IL10 / IL13 fusion protein, an IL27 / IL13 fusion protein, an IL33 / IL13 fusion protein, a TGFβ1 / IL13 fusion protein, a TGFβ2 / IL13 fusion protein, an IL13 / IL13 fusion protein; and the fusion proteins themselves. The invention further provides methods for producing IL4 / IL13, IL10 / IL13, IL27 / IL13, IL33 / IL13, TGFβ1 / IL13, TGFβ2 / IL13 or IL13 / IL13 fusion proteins using cells or organisms containing such nucleic acid sequences. Also provided are transgenic organisms comprising the nucleic acid sequences of the invention.The invention also relates to pharmaceutical compositions comprising, for example, IL4 / IL13 or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13 fusion proteins.Finally, the pharmaceutical use of IL4 / IL13 or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13 fusion proteins is taught herein, in particular for the prevention and / or treatment of chronic pain and conditions characterized by neuroinflammation, neurodegeneration or inflammation. [Background technology]

[0003] Chronic pain affects millions of people and constitutes the greatest unmet need in modern medicine. 1~4 In 2016, it was estimated that 20.4% of U.S. adults (50 million people) had chronic pain, and 8.0% of U.S. adults (19.6 million people) had high-impact chronic pain. 5 Opioids and non-steroidal anti-inflammatory drugs (NSAIDs) constitute the major classes of drugs that combat pain. However, these analgesics ("pain-killers") are often ineffective and have severe side effects (addictive, gastrointestinal bleeding, cardiovascular, and others). It is estimated that approximately 50% of chronic pain patients (approximately 5-10% of the total population) do not receive adequate pain relief. 6 .

[0004] People with chronic pain suffer from spontaneous pain, hyperalgesia (enhanced experience of pain in response to noxious stimuli) and allodynia (pain elicited by normally non-painful stimuli). Pain has multiple causes and arises from biological processes at various anatomical levels. 7~10Pain is caused by a number of different processes: generation of stimuli that trigger sensory nerve endings in the periphery; stimulation, sensitization and dysfunction of peripheral sensory neurons that transmit action potentials to the spinal cord; neurons and glial cells in the spinal dorsal horn, where action potentials from peripheral neurons are transmitted via synapses to spinal pain neurons; and finally central mechanisms in the brain. The contribution of all these processes to the various types of chronic pain varies. Depending on this contribution, pain is differentiated into several types, including nociceptive pain, peripheral central neuropathic pain and mixed pain. Analgesics used in the clinic target pain at only one level: NSAIDs reduce the generation of nociceptive stimuli in the periphery, while opioids inhibit central mechanisms. This explains the limited clinical effectiveness of these analgesics, along with their notorious toxic side effects.

[0005] In the past decade, it has become increasingly clear that pain signals do not go straight to the brain, but rather are modulated by neuroinflammatory processes involving glial cells and sensory neurons in the spinal cord. Cytokines are known to orchestrate immune and inflammatory processes and are also crucial in the control of pain. Proinflammatory cytokines enhance inflammation and promote pain, whereas regulatory (e.g., anti-inflammatory) cytokines attenuate inflammation. The balance between proinflammatory and regulatory cytokines determines the outcome of the inflammatory response in vivo. 11 The pain-promoting effects of proinflammatory cytokines are well known. 12 However, knowledge about the role of regulatory cytokines in pain is limited. 13 Blockade of regulatory cytokines, such as TGFβ, IL10, IL4, and IL13, reduced transient inflammatory hyperalgesia. 14 and chemotherapy-induced allodynia 15 This severely impairs the resolution of pain, demonstrating an important role for endogenous regulatory cytokines in pain resolution. The role of this cytokine goes far beyond reducing inflammation and does not include the role of anti-inflammatory drugs, such as corticosteroids, which only have limited analgesic effects. 16、17Notably, not only are glial cells modulated by cytokines, but sensory neurons themselves can also respond directly to cytokines. Indeed, sensory neurons express receptors for all regulatory cytokines, although expression differs between neuronal subsets. 18 Pain resulting from peripheral inflammation or nerve injury is associated with activation of spinal microglia and astrocytes, which promote pain by enhancing spinal pain signaling. 19~22 .

[0006] Given the role of neuroinflammation in chronic pain, regulatory cytokines could potentially target pain at multiple levels: indeed, they attenuate stimulation of nociceptors by reducing inflammation, they inhibit sensitization and dysfunction of sensory neurons, and they prevent activation of pain pathways in the spinal cord by attenuating the production of pro-inflammatory mediators by glial cells. 21、22 However, the analgesic effect of treatment with independent regulatory cytokines (e.g., IL10 or IL13) or anti-inflammatory cytokines (IL1 receptor antagonists) is limited. 19 This is probably because optimal analgesic activity requires synergistic action of various regulatory cytokines and because of their relatively poor bioavailability due to rapid clearance by the kidney. Therefore, a new strategy for resolving chronic pain with regulatory cytokines was proposed using a fusion protein of IL4 and IL10. 19、23 Intrathecal administration of analgesics is a common practice in pain management because it reduces the dose of analgesics and decreases the toxicity of the analgesics. 24 Intrathecal injection of IL4 / IL10 fusion protein reduces pain in a variety of different mouse models of pain. 19Remarkably, three repeated intrathecal administrations of IL4 / IL10 fusion protein resulted in sustained relief of pain induced by inflammation in the paw (i.e., complete and permanent resolution of chronic pain, e.g., nociceptive pain), without modulating peripheral inflammation per se. 19 Interestingly, the efficacy of the IL4 / IL10 fusion protein is superior to that of the individual wild-type cytokines and to that of the combination of these cytokines. 19 .

[0007] In addition, pain relief by IL4 / IL10 fusion protein was observed in a neuropathic pain model. 19 and osteoarthritis pain models 25 It has been observed in. However, the effect of IL4 / IL10 fusion protein on neuropathic and osteoarthritic pain may be transient even after multiple injections. Therefore, there is a need to provide a molecule for the prevention or treatment of neuropathic and osteoarthritic pain with a long-lasting analgesic effect. Summary of the Invention [Means for solving the problem]

[0008] The present disclosure provides a single molecule that targets neuroinflammation and neurodegeneration and has a long-lasting effect on chronic neuropathic pain.This molecule can be used for the treatment of various diseases or disorders with various etiologies in which chronic pain, neuroinflammation and / or neurodegeneration play a role.The present disclosure provides a fusion protein that includes interleukin 13 (IL13) directly or indirectly linked to a regulatory cytokine.

[0009] In some aspects, disclosed herein is a fusion protein comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence for use in treating a neuropathy in a subject in need thereof.

[0010] In some aspects, disclosed herein is a fusion protein comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence for use in treating pain in a subject in need thereof.

[0011] In some aspects, disclosed herein is a fusion protein comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence for use in treating neurodegeneration or neuroinflammation in a subject in need thereof.

[0012] In some aspects, disclosed herein is a fusion protein comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence for use in treating inflammation in a subject in need thereof.

[0013] In some aspects, disclosed herein is a fusion protein comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence for use in promoting neuroprotection in a subject in need thereof.

[0014] In some aspects, disclosed herein is a fusion protein comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence for use in modulating the activity of a signaling pathway in a nervous system cell.

[0015] In some embodiments, the regulatory cytokine is selected from the group consisting of interleukin 4 (IL4), interleukin 10 (IL10), interleukin 33 (IL33), transforming growth factor beta 1 (TGFβ1), transforming growth factor beta 2 (TGFβ2), and additional interleukin 13 (IL13). In some embodiments, the regulatory cytokine is IL4. In some embodiments, the regulatory cytokine is IL10. In some embodiments, the regulatory cytokine is IL33. In some embodiments, the regulatory cytokine is interleukin 27 (IL27). In some embodiments, the regulatory cytokine is TGFβ1. In some embodiments, the regulatory cytokine is TGFβ2. In some embodiments, the regulatory cytokine is additional IL13. In some embodiments, the IL13 comprises wild-type IL13. In some embodiments, the IL13 is mammalian IL13. In some embodiments, the IL13 is human IL13. In some embodiments, the regulatory cytokine comprises wild-type regulatory cytokine. In some embodiments, the regulatory cytokine is a mammalian regulatory cytokine. In some embodiments, the regulatory cytokine is a human regulatory cytokine. In some embodiments, the interleukin 27 comprises interleukin 27 alpha (IL27A). In some embodiments, IL27A comprises a L134C substitution compared to SEQ ID NO:36. In some embodiments, IL13 binds to interleukin 13 receptor alpha 1 (IL-13Rα1) with less than two-fold and more than half-fold lower affinity compared to wild-type IL13. In some embodiments, IL13 binds to interleukin 13 receptor alpha 2 (IL-13Rα2) with less than two-fold and more than half-fold lower affinity compared to wild-type IL13. In some embodiments, IL13 binds to interleukin 4 receptor alpha (IL-4Rα) with less than two-fold and more than half-fold lower affinity compared to wild-type IL13. In some embodiments, the regulatory cytokine amino acid sequence is a derived sequence that binds to all subunits of the receptor of the regulatory cytokine with an affinity comparable to that of the wild-type regulatory cytokine. In some embodiments, the regulatory cytokine amino acid sequence isA derived sequence that activates the native receptor of a regulatory cytokine. In some embodiments, IL13 comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs:9-15. In some embodiments, IL13 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs:9-15. In some embodiments, IL13 comprises an amino acid sequence having 1-10 amino acid deletions, insertions, substitutions or combinations thereof compared to a sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs:9-15. In some embodiments, the regulatory cytokine comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO:1, any one of SEQ ID NOs:26-28, SEQ ID NO:5, SEQ ID NO:6, any one of SEQ ID NOs:29-34, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:36 and SEQ ID NO:45. In some embodiments, the regulatory cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, any one of SEQ ID NOs:26-28, SEQ ID NO:5, SEQ ID NO:6, any one of SEQ ID NOs:29-34, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:36, and SEQ ID NO:45. In some embodiments, the regulatory cytokine comprises an amino acid sequence having 1-10 amino acid deletions, insertions, substitutions, or combinations thereof compared to a sequence selected from the group consisting of SEQ ID NO:1, any one of SEQ ID NOs:26-28, SEQ ID NO:5, SEQ ID NO:6, any one of SEQ ID NOs:29-34, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:36, and SEQ ID NO:45. In some embodiments, the IL13 and the regulatory cytokine are covalently linked. In some embodiments, the IL13 and the regulatory cytokine are linked by a linker. In some embodiments, the C-terminus of IL13 is linked to the N-terminus of the cytokine, optionally via a linker. In some embodiments, the N-terminus of IL13 is linked to the C-terminus of the cytokine, optionally via a linker.The protein construct further comprises one or more chemical modifications. In some embodiments, the one or more chemical modifications are selected from the group consisting of glycosylation, fucosylation, sialylation, and pegylation. In some embodiments, the protein construct comprises an affinity tag. In some embodiments, the neuropathy is post-traumatic peripheral neuropathy, post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, chemotherapy-induced neuropathy, polyneuropathy, mononeuropathy, multiple mononeuropathy, cranial neuropathy, predominantly motor neuropathy, predominantly sensory neuropathy, sensorimotor neuropathy, autonomic neuropathy, idiopathic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, pudenal neuralgia, variant trigeminal neuralgia, sciatica, brachial plexopathy, or intercostal neuralgia. In some embodiments, the neuropathy is accompanied by pain, numbness, weakness, burning sensation, atrophy, tingling, spasm, or a combination thereof. In some embodiments, the pain is chronic pain. In some embodiments, the pain is pathological pain, inflammatory pain, neuropathic pain, nociceptive pain or mixed nociceptive-neuropathic pain.In some embodiments, the pain is visceral nociceptive pain, non-visceral nociceptive pain, peripheral neuropathic pain, central neuropathic pain or a combination thereof.In some embodiments, the pain is post-operative orthopedic surgery pain, musculoskeletal pain, chemotherapy-related pain, chemotherapy-induced allodynia, post-spinal cord injury pain, post-stroke pain, low back pain, cancer pain or chronic visceral pain. In some embodiments, the pain is associated with irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic peripheral neuropathy, post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, peripheral neuropathy, nerve entrapment syndrome, chemotherapy-induced neuropathy, multiple sclerosis, chemotherapy-induced neurodegeneration, complex regional pain syndrome, osteoarthritis, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, polyneuropathy, or amyotrophic lateral sclerosis. In some embodiments, the pain is due to Alpers disease, arachnoiditis, fibrous joint disease, ataxic cerebral palsy, autoimmune atrophic gastritis, amyloidosis, hATTR amyloidosis, avascular necrosis, back pain, Batten disease, Behcet's disease (syndrome), breakthrough pain, burning mouth syndrome, bursitis,Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (Cadasil), cerebral ischemia, cerebro-oculo-facio-skeletal syndrome (COFS), carpal tunnel syndrome, cauda equina syndrome, central pain syndrome, cerebral palsy, cerebrospinal fluid (CSF) leakage, cervical stenosis, Charcot-Marie-Tooth (CMT) disease, chronic functional abdominal pain (CFAP), Pain), chronic pancreatitis, collapsed lung (pneumothorax), corticobasal degeneration, compression injury, corneal neuropathic pain, crush syndrome, degenerative disc disease, dermatomyositis, dementia, dystonia, Ehlers-Danlos syndrome (EDS), endometriosis, Eosinophilia-Myalgia Syndrome (EMS), erythromelalgia, failed back surgery syndrome (FBSS) Syndrome), fibromyalgia, Friedreich's ataxia, frontotemporal dementia, glossopharyngeal neuralgia, growing pains, herniated disc, hydrocephalus, intercostal neuralgia (Neuraligia), interstitial cystitis, juvenile dermatomyositis, knee injury, lower limb pain, Lewy body dementia, low back pain and hematuria syndrome, Lyme disease, dyssensory thigh pain, mitochondrial disease, mixed dementia, motor neurone disease (MND), Hirayama disease, multiple system atrophy (MSA), atrophy), myositis, neck pain, occipital neuralgia, osteoporosis, rhabdomyolysis, Paget's disease, Parsonage-Turner syndrome, pelvic pain, peripheral neuropathy, phantom limb pain, nerve compression, plantar fasciitis, rheumatica polymyalgia, polymyositis, post-herniorraphy pain syndrome, post-mastectomy pain syndrome, post-stroke pain, post-thorocotomy pain syndrome, post-polio syndrome, primary lateral sclerosis, psoriatic arthritis, pudendal neuralgia,Radiculopathy, restless legs syndrome, It may be associated with rheumatoid arthritis (RA), sacroiliac joint dysfunction, sarcoidosis, Scheuemann's kyphosis disease, sciatica, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), shingles, spasmodic torticollis, sphincter of Oddi dysfunction, spinal cord injury, spinal stenosis, syringomyelia, Tarlov's cyst, tethered spinal cord syndrome, thoracic outlet syndrome (TOS), temporomandibular joint disorders, transverse myelitis, brain trauma, vascular pain, vulvodynia, whiplash injury, or a combination thereof. In some embodiments, the neurodegeneration or neuroinflammation comprises Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, spinocerebellar ataxia or spinal muscular atrophy. In some embodiments, the inflammation comprises chronic inflammation. In some embodiments, the inflammation comprises local inflammation or systemic inflammation. In some embodiments, the inflammation is associated with inflammatory bowel disease, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, glomerulonephritis, sepsis, adult respiratory distress syndrome, dermatitis, sarcoidosis, allergic inflammation, psoriasis, ankylosing spondyloarthritis, systemic lupus erythematosus, vasculitis, gout, allografts, xenografts, autoimmune diseases, Sjogren's syndrome, burns, trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, and toxicity induced by in vivo administration of cytokines or other therapeutic monoclonal antibodies. In some embodiments, nerve fiber degeneration is reduced. In some embodiments, nerve fiber loss is reduced. In some embodiments, maintenance of nerve fiber density is promoted. In some embodiments, nerve fiber re-growth is promoted. In some embodiments, neuroprotection in the central nervous system is promoted. In some embodiments, neuroprotection in the peripheral nervous system is promoted. In some embodiments, intraepidermal nerve fiber loss is reduced. In some embodiments, neuronal dysfunction is reduced. In some embodiments, the fusion protein induces a therapeutic effect of greater magnitude than an equivalent amount of IL13, a regulatory cytokine, or a combination thereof.In some embodiments, the fusion protein induces a longer duration of therapeutic effect than an equivalent amount of IL13, a regulatory cytokine, or a combination thereof. In some embodiments, the fusion protein is present in a pharmaceutical composition comprising the fusion protein and one or more pharma- ceutically acceptable excipients. In some embodiments, the pharmaceutical composition is present in a unit dosage form. In some embodiments, the fusion protein is present in the pharmaceutical composition at a concentration of about 50 μg / mL to about 100 mg / mL. In some embodiments, the fusion protein is formulated for administration at a dose of about 0.5 μg / kg to about 1 mg / kg of body weight. In some embodiments, the fusion protein is formulated for administration in a controlled release formulation. In some embodiments, the fusion protein is formulated for administration by parenteral, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intraventricular, intracisternal, or intraparenchymal routes. In some embodiments, the nervous system cell is a neuron. In some embodiments, the nervous system cell is a central nervous system cell. In some embodiments, the nervous system cell is a peripheral nervous system cell. In some embodiments, the neuron is a sensory neuron. In some embodiments, the neuron is a somatosensory neuron. In some embodiments, the neuron is a visceral sensory neuron. In some embodiments, the neuron is a nociceptor. In some embodiments, the neuron is an autonomic neuron. In some embodiments, the nervous system cell is a glial cell. In some embodiments, the nervous system cell is a microglial cell. In some embodiments, the nervous system cell is an infiltrating cell. In some embodiments, the nervous system cell is an infiltrating macrophage. In some embodiments, the signaling pathway is modulated in the presence of a pro-inflammatory mediator.

[0016] In some aspects, disclosed herein is a method of treating a neuropathy in a subject in need thereof, the method comprising administering to the subject an effective amount of a fusion protein comprising an interleukin-13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence.

[0017] In some aspects, disclosed herein is a method of treating pain in a subject in need thereof, the method comprising administering to the subject an effective amount of a fusion protein comprising an interleukin-13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence.

[0018] In some aspects, disclosed herein is a method of treating neurodegeneration or neuroinflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of a fusion protein comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence.

[0019] In some aspects, disclosed herein is a method of treating inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of a fusion protein comprising an interleukin-13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence.

[0020] In some aspects, disclosed herein is a method of promoting neuroprotection in a subject in need thereof, the method comprising administering to the subject an effective amount of a fusion protein comprising an interleukin-13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence.

[0021] In some aspects, disclosed herein is a method of modulating the activity of a signaling pathway in a nervous system cell, the method comprising contacting the nervous system cell with a fusion protein comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence.

[0022] In some embodiments, the regulatory cytokine is selected from the group consisting of interleukin 4 (IL4), interleukin 10 (IL10), interleukin 33 (IL33), transforming growth factor beta 1 (TGFβ1), transforming growth factor beta 2 (TGFβ2), and additional interleukin 13 (aIL13). In some embodiments, the regulatory cytokine is IL4. In some embodiments, the regulatory cytokine is IL10. In some embodiments, the regulatory cytokine is IL33. In some embodiments, the regulatory cytokine is interleukin 27 (IL27). In some embodiments, the regulatory cytokine is TGFβ1. In some embodiments, the regulatory cytokine is TGFβ2. In some embodiments, the regulatory cytokine is aIL13. In some embodiments, the IL13 comprises wild-type IL13. In some embodiments, the IL13 is mammalian IL13. In some embodiments, the IL13 is human IL13. In some embodiments, the regulatory cytokine comprises wild-type regulatory cytokine. In some embodiments, the regulatory cytokine is a mammalian regulatory cytokine. In some embodiments, the regulatory cytokine is a human regulatory cytokine. In some embodiments, the interleukin 27 comprises interleukin 27 alpha (IL27A). In some embodiments, IL27A comprises a L134C substitution compared to SEQ ID NO:36. In some embodiments, IL13 binds to interleukin 13 receptor alpha 1 (IL-13Rα1) with less than two-fold and more than half-fold lower affinity compared to wild-type IL13. In some embodiments, IL13 binds to interleukin 13 receptor alpha 2 (IL-13Rα2) with less than two-fold and more than half-fold lower affinity compared to wild-type IL13. In some embodiments, IL13 binds to interleukin 4 receptor alpha (IL-4Rα) with less than two-fold and more than half-fold lower affinity compared to wild-type IL13. In some embodiments, the regulatory cytokine amino acid sequence is a derived sequence that binds to all subunits of the receptor of the regulatory cytokine with an affinity comparable to that of the wild-type regulatory cytokine.In some embodiments, the regulatory cytokine amino acid sequence is a derived sequence that activates the native receptor of the regulatory cytokine. In some embodiments, IL13 comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs:9-15. In some embodiments, IL13 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs:9-15. In some embodiments, IL13 comprises an amino acid sequence having 1-10 amino acid deletions, insertions, substitutions or combinations thereof compared to a sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs:9-15. In some embodiments, the regulatory cytokine comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO:1, any one of SEQ ID NOs:26-28, SEQ ID NO:5, SEQ ID NO:6, any one of SEQ ID NOs:29-34, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:36 and SEQ ID NO:45. In some embodiments, the regulatory cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, any one of SEQ ID NOs:26-28, SEQ ID NO:5, SEQ ID NO:6, any one of SEQ ID NOs:29-34, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:36, and SEQ ID NO:45. In some embodiments, the regulatory cytokine comprises an amino acid sequence having 1-10 amino acid deletions, insertions, substitutions, or combinations thereof compared to a sequence selected from the group consisting of SEQ ID NO:1, any one of SEQ ID NOs:26-28, SEQ ID NO:5, SEQ ID NO:6, any one of SEQ ID NOs:29-34, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:36, and SEQ ID NO:45. In some embodiments, the IL13 and the regulatory cytokine are covalently linked. In some embodiments, the IL13 and the regulatory cytokine are linked by a linker. In some embodiments, the C-terminus of IL13 is linked to the N-terminus of the cytokine, optionally via a linker.In some embodiments, the N-terminus of IL13 is linked to the C-terminus of the cytokine, optionally via a linker. In some embodiments, the fusion protein further comprises one or more chemical modifications. In some embodiments, the one or more chemical modifications are selected from the group consisting of glycosylation, fucosylation, sialylation and pegylation. In some embodiments, the protein construct comprises an affinity tag. In some embodiments, the neuropathy is post-traumatic peripheral neuropathy, post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, chemotherapy-induced neuropathy, polyneuropathy, mononeuropathy, multiple mononeuropathy, cranial neuropathy, predominantly motor neuropathy, predominantly sensory neuropathy, sensorimotor neuropathy, autonomic neuropathy, idiopathic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, pudendal neuralgia, variant trigeminal neuralgia, sciatica, brachial plexopathy or intercostal neuralgia. In some embodiments, the neuropathy is accompanied by pain, numbness, weakness, burning sensation, atrophy, tingling, spasms, or a combination thereof. In some embodiments, the pain is chronic pain. In some embodiments, the pain is neuropathic pain, nociceptive pain, or mixed nociceptive-neuropathic pain. In some embodiments, the pain is visceral nociceptive pain, non-visceral nociceptive pain, peripheral neuropathic pain, central neuropathic pain, or a combination thereof. In some embodiments, the pain is post-operative orthopedic surgery pain, musculoskeletal pain, chemotherapy-related pain, chemotherapy-induced allodynia, post-spinal cord injury pain, post-stroke pain, low back pain, cancer pain, or chronic visceral pain. In some embodiments, the pain is associated with irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic peripheral neuropathy, post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, peripheral neuropathy, nerve entrapment syndrome, chemotherapy-induced neuropathy, multiple sclerosis, chemotherapy-induced neurodegeneration, complex regional pain syndrome, osteoarthritis, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, polyneuropathy, or amyotrophic lateral sclerosis.In some embodiments, the pain is due to conditions including, but not limited to, Alpers disease, arachnoiditis, fibrotic joint disease, ataxic cerebral palsy, autoimmune atrophic gastritis, amyloidosis, hATTR amyloidosis, ischemic necrosis, back pain, Batten disease, Behcet's disease (syndrome), breakthrough pain, burning mouth syndrome, bursitis, cerebral autosomal dominant arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL), cerebral ischemia, cerebro-oculofacial-skeletal syndrome (COFS), carpal tunnel syndrome, cauda equina syndrome, central pain syndrome, cerebral palsy, cerebrospinal fluid (CSF) leak, cervical stenosis, Charcot-Marie's syndrome, cerebral palsy, cerebrospinal fluid (CSF) leak, cervical stenosis, Charcot-Marie's syndrome, cerebral palsy, cerebrospinal fluid (CSF) leak, cerebral stenosis, cerebral palsy ... CMT disease, chronic functional abdominal pain (CFAP), chronic pancreatitis, collapsed lung (pneumothorax), corticobasal degeneration, compression injury, corneal neuropathic pain, crush syndrome, degenerative disc disease, dermatomyositis, dementia, dystonia, Ehlers-Danlos syndrome (EDS), endometriosis, eosinophilic myalgia syndrome (EMS), erythromelalgia, lumbar postoperative pain syndrome (FBSS), fibromyalgia, Friedreich's ataxia, frontotemporal dementia, glossopharyngeal neuralgia, growing pains, herniated disc, hydrocephalus, intercostal neuralgia, interstitial cystitis, juvenile dermatomyositis, knee Injury, leg pain, dementia with Lewy bodies, low back pain and hematuria syndrome, Lyme disease, dyssensory thigh pain, mitochondrial disease, mixed dementia, motor neuron disease (MND), Hirayama disease, multiple system atrophy (MSA), myositis, neck pain, occipital neuralgia, osteoporosis, rhabdomyolysis, Paget's disease, Parsonage-Turner syndrome, pelvic pain, peripheral neuropathy, phantom limb pain, nerve compression, plantar fasciitis, polymyalgia rheumatica, polymyositis, postherniorrhaphy pain syndrome, postmastectomy pain syndrome, poststroke pain, postthoracotomy pain syndrome, postpolio syndrome, primary lateral sclerosis The neurodegeneration or neuroinflammation is associated with: sclerosis, psoriatic arthritis, pudendal neuralgia, radiculopathy, restless legs syndrome, rheumatoid arthritis (RA), sacroiliac joint dysfunction, sarcoidosis, Scheuermann's kyphosis, sciatica, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), herpes zoster, spasmodic torticollis, sphincter of Oddi dysfunction, spinal cord injury, spinal stenosis, syringomyelia, Tarlov's cyst, tethered spinal cord syndrome, thoracic outlet syndrome (TOS), temporomandibular joint disorders, transverse myelitis, brain trauma, vascular pain, vulvodynia, whiplash injury, or combinations thereof. In some embodiments, the neurodegeneration or neuroinflammation comprises Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, spinocerebellar ataxia, or spinal muscular atrophy.In some embodiments, the inflammation comprises chronic inflammation. In some embodiments, the inflammation comprises local or systemic inflammation. In some embodiments, the inflammation is associated with inflammatory bowel disease, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, glomerulonephritis, sepsis, adult respiratory distress syndrome, dermatitis, sarcoidosis, allergic inflammation, psoriasis, ankylosing spondyloarthritis, systemic lupus erythematosus, vasculitis, gout, allografts, xenografts, autoimmune diseases, Sjogren's syndrome, burns, trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, and toxicity induced by in vivo administration of cytokines or other therapeutic monoclonal antibodies. In some embodiments, IL13 treatment is indicated. In some embodiments, IL4, IL10, IL27, IL33, TGFβ1 or TGFβ2 treatment is indicated. In some embodiments, the method reduces nerve fiber degeneration. In some embodiments, the method reduces nerve fiber loss. In some embodiments, the method promotes maintenance of nerve fiber density. In some embodiments, the method promotes nerve fiber regrowth. In some embodiments, the method promotes neuroprotection in the central nervous system. In some embodiments, the method promotes neuroprotection in the peripheral nervous system. In some embodiments, the method reduces intraepidermal nerve fiber loss. In some embodiments, the method reduces neuronal dysfunction. In some embodiments, administering the fusion protein induces a therapeutic effect of greater magnitude than administering an equivalent amount of IL13, a regulatory cytokine, or a combination thereof. In some embodiments, administering the fusion protein induces a therapeutic effect of greater duration than administering an equivalent amount of IL13, a regulatory cytokine, or a combination thereof. In some embodiments, administering results in greater magnitude of pain relief compared to equivalent amounts of IL13 and regulatory cytokines administered individually or in combination, as measured by mechanical sensitivity to von Frey hairs in a paclitaxel-induced mouse neuropathy model. In some embodiments, the fusion protein is present in a pharmaceutical composition comprising the fusion protein and one or more pharma- ceutically acceptable excipients.In some embodiments, the composition is in unit dosage form. In some embodiments, the fusion protein is present in the pharmaceutical composition at a concentration of about 50 μg to about 100 mg / mL. In some embodiments, the fusion protein is administered at a dose of about 0.5 μg to 1 mg / kg body weight. In some embodiments. The fusion protein is administered in a controlled release formulation. In some embodiments, the fusion protein is administered by parenteral, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intraventricular, intracisternal, or intraparenchymal routes. In some embodiments, the nervous system cell is a neuron. In some embodiments, the nervous system cell is a central nervous system cell. In some embodiments, the nervous system cell is a peripheral nervous system cell. In some embodiments, the neuron is a sensory neuron. In some embodiments, the neuron is a somatosensory neuron. In some embodiments, the neuron is a visceral sensory neuron. In some embodiments, the neuron is a nociceptor. In some embodiments, the neuron is an autonomic neuron. In some embodiments, the nervous system cell is a glial cell. In some embodiments, the nervous system cell is a microglial cell. In some embodiments, the nervous system cell is an infiltrating cell. In some embodiments, the nervous system cell is an infiltrating macrophage. In some embodiments, the signaling pathway is modulated in the presence of a pro-inflammatory mediator.

[0023] Disclosed herein, in some aspects, is a fusion protein that includes an interleukin-13 (IL13) amino acid sequence that is a wild-type IL13 sequence and a regulatory cytokine amino acid sequence.

[0024] In some aspects, disclosed herein is a fusion protein that includes an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence that is a wild-type sequence. In some aspects, disclosed herein is a fusion protein comprising an interleukin-13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence, wherein the IL13 amino acid sequence is an IL13-derived sequence that binds to interleukin-13 receptor alpha 1 (IL-13Rα1), interleukin-13 receptor alpha 2 (IL-13Rα2), and interleukin-4 receptor alpha (IL-4Rα) with an affinity comparable to that of the wild-type interleukin-13 sequence.

[0025] In some aspects, disclosed herein is a fusion protein comprising an interleukin-13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence, where the regulatory cytokine amino acid sequence is a derived sequence that binds to all receptor subunits to which the wild-type version of the regulatory cytokine binds with an affinity equivalent to that of the wild-type regulatory cytokine.

[0026] In some embodiments, the regulatory cytokine is selected from the group consisting of interleukin 4 (IL4), interleukin 10 (IL10), interleukin 33 (IL33), transforming growth factor beta 1 (TGFβ1), transforming growth factor beta 2 (TGFβ2), and additional interleukin 13 (aIL13). In some embodiments, the regulatory cytokine is IL4. In some embodiments, the regulatory cytokine is IL10. In some embodiments, the regulatory cytokine is IL33. In some embodiments, the regulatory cytokine is interleukin 27 (IL27). In some embodiments, the regulatory cytokine is TGFβ1. In some embodiments, the regulatory cytokine is TGFβ2. In some embodiments, the regulatory cytokine is additional interleukin 13 (aIL13). In some embodiments, the IL13 is mammalian IL13. In some embodiments, the IL13 is human IL13. In some embodiments, the IL13 comprises wild-type IL13. In some embodiments, the regulatory cytokine comprises wild-type regulatory cytokine. In some embodiments, the regulatory cytokine is a mammalian regulatory cytokine. In some embodiments, the regulatory cytokine is a human regulatory cytokine. In some embodiments, the interleukin 27 comprises interleukin 27 alpha (IL27A). In some embodiments, IL27A comprises a L134C substitution compared to SEQ ID NO:36. In some embodiments, IL13 binds to interleukin 13 receptor alpha 1 (IL-13Rα1) with less than two-fold and more than half-fold lower affinity compared to wild-type IL13. In some embodiments, IL13 binds to interleukin 13 receptor alpha 2 (IL-13Rα2) with less than two-fold and more than half-fold lower affinity compared to wild-type IL13. In some embodiments, IL13 binds to interleukin 4 receptor alpha (IL-4Rα) with less than two-fold and more than half-fold lower affinity compared to wild-type IL13. In some embodiments, the regulatory cytokine amino acid sequence is a derived sequence that binds to all subunits of the receptor of the regulatory cytokine with an affinity comparable to that of the wild-type regulatory cytokine.In some embodiments, the regulatory cytokine amino acid sequence is a derived sequence that activates the native receptor of the regulatory cytokine. In some embodiments, IL13 comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs:9-15. In some embodiments, IL13 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs:9-15. In some embodiments, IL13 comprises an amino acid sequence having 1-10 amino acid deletions, insertions, substitutions or combinations thereof compared to a sequence selected from the group consisting of SEQ ID NO:2 and any one of SEQ ID NOs:9-15. In some embodiments, the regulatory cytokine comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO:1, any one of SEQ ID NOs:26-28, SEQ ID NO:5, SEQ ID NO:6, any one of SEQ ID NOs:29-34, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:36 and SEQ ID NO:45. In some embodiments, the regulatory cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, any one of SEQ ID NOs:26-28, SEQ ID NO:5, SEQ ID NO:6, any one of SEQ ID NOs:29-34, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:36, and SEQ ID NO:45. In some embodiments, the regulatory cytokine comprises an amino acid sequence having 1-10 amino acid deletions, insertions, substitutions, or combinations thereof compared to a sequence selected from the group consisting of SEQ ID NO:1, any one of SEQ ID NOs:26-28, SEQ ID NO:5, SEQ ID NO:6, any one of SEQ ID NOs:29-34, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:18, SEQ ID NO:36, and SEQ ID NO:45. In some embodiments, the IL13 and the regulatory cytokine are covalently linked. In some embodiments, the IL13 and the regulatory cytokine are linked by a linker. In some embodiments, the C-terminus of IL13 is linked to the N-terminus of the cytokine, optionally via a linker.In some embodiments, the N-terminus of IL13 is linked to the C-terminus of the cytokine, optionally via a linker. In some embodiments, the fusion protein further comprises one or more chemical modifications. In some embodiments, the one or more chemical modifications are selected from the group consisting of glycosylation, fucosylation, sialylation, and pegylation. In some embodiments, the protein construct comprises an affinity tag. In some embodiments, a nucleic acid molecule encoding the fusion protein is provided. In some embodiments, the nucleic acid molecule is codon-optimized for expression in a cell. In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the cell comprises a nucleic acid. In some embodiments, the fusion protein is present in a pharmaceutical composition that also comprises a pharma- ceutically acceptable excipient. In some embodiments, the nucleic acid vector is present in a pharmaceutical composition that also comprises a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition is present in a unit dosage form. In some embodiments, the fusion protein is present in the pharmaceutical composition at about 50 μg to about 100 mg / mL. In some embodiments, the fusion protein is formulated for administration as a dose of about 0.5 μg to 1 mg / kg of body weight. In some embodiments, the fusion protein is formulated for administration as a controlled release formulation. In some embodiments, the pharmaceutical composition is formulated for administration by parenteral, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intraventricular, intracisternal or intraparenchymal routes. In some embodiments, an effective amount of the pharmaceutical composition is administered to a subject in need thereof. In some embodiments, the fusion protein is produced by comprising culturing cells under conditions allowing for production of the fusion protein, the cells comprising the polynucleotide sequence. In some embodiments, the fusion protein is recovered. In some embodiments, the fusion protein is purified from the recovered culture medium. In some embodiments, the fusion protein is used to crosslink an interleukin-13 receptor with a regulatory cytokine receptor.

[0027] In some aspects, disclosed herein is a fusion protein comprising 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.

[0028] In some embodiments, 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. In some embodiments, interleukin-13 is fused to the N-terminus of an interleukin selected from interleukin-4, interleukin-10, interleukin-33, transforming growth factor beta 1, transforming growth factor beta 2, and interleukin-13. In some embodiments, an interleukin selected from interleukin-4, interleukin-10, interleukin-33, transforming growth factor beta 1, transforming growth factor beta 2, and interleukin-13 is fused to the N-terminus of interleukin-13. In some embodiments, the fusion protein further comprises one or more chemical modifications. In some embodiments, the chemical modifications are selected from the group consisting of glycosylation, fucosylation, sialylation, and pegylation. In some embodiments, the interleukin-13 is human interleukin-13. In some embodiments, the interleukin 4 is human interleukin 4, and / or the interleukin 10 is human interleukin 10, and / or the 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. In some embodiments, the fusion protein is encoded by a polynucleotide present in a nucleic acid molecule. In some embodiments, the nucleic acid molecule is present in a vector. In some embodiments, the nucleic acid molecule is present in a host cell. In some embodiments, the fusion protein is made by a method comprising culturing a host cell under conditions that allow for the production of the fusion protein, and optionally purifying the fusion protein from the conditioned culture medium. In some embodiments, the fusion protein is present in a pharmaceutical composition that also includes a pharma- ceutically acceptable carrier. In some embodiments, the fusion protein is used as a medicament.In some embodiments, the fusion protein is used in the prevention or treatment of conditions characterized by chronic pain, neuroinflammation or neurodegeneration. In some embodiments, 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. In some embodiments, 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 cord 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. In some embodiments, the fusion protein is used in the prevention or treatment of a clinical condition in which interleukin-13 is indicated in a mammal, e.g., a human. In some embodiments, the fusion protein is used in the prevention or treatment of a clinical condition in a mammal, e.g., a human, for which interleukin 4 and / or interleukin 10 and / or interleukin 33 and / or interleukin 27 and / or transforming growth factor beta 1 and / or transforming growth factor beta 2 is indicated.

[0029] In some aspects, disclosed herein are gene therapy vectors containing nucleotide sequences encoding interleukin 13 and an interleukin selected from interleukin 4, interleukin 10, interleukin 27, interleukin 33, transforming growth factor beta 1, transforming growth factor beta 2, and interleukin 13 for use in the prevention or treatment of conditions characterized by chronic pain, neuroinflammation and / or neurodegeneration.

[0030] In some embodiments, 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. In some embodiments, 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 cord 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.

[0031] In some aspects, the present invention relates to a fusion protein comprising at least two, three, four, preferably two regulatory (e.g., anti-inflammatory) interleukins selected from the group consisting of interleukin 13 (IL13), interleukin 4 (IL4), interleukin 10 (IL10), interleukin 27 (IL27), interleukin 33 (IL33), transforming growth factor beta 1 (TGFβ1) and transforming growth factor beta 2 (TGFβ2).

[0032] Preferably, the present invention relates to a fusion protein comprising IL13 and another, i.e. a second, interleukin / cytokine, preferably selected from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2 and IL13 itself.

[0033] In one embodiment, said IL13 and said interleukin selected from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2 or IL13 itself are connected by a linker.

[0034] In one embodiment, an interleukin selected from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2 or IL13 itself is fused to the N-terminus of IL13.

[0035] In another embodiment, IL13 is fused to the N-terminus of an interleukin selected from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2, or IL13 itself.

[0036] In one embodiment, the fusion protein further comprises one or more chemical modifications, which may be selected from the group consisting of glycosylation, fucosylation, sialylation and pegylation.

[0037] In one embodiment, said IL13 is human IL13. In one embodiment, said IL4 is human IL4. In one embodiment, said IL10 is human IL10.

[0038] In one embodiment, said IL27 is human IL27. In one embodiment, said IL33 is human IL33. In one embodiment, said TGFβ1 is human TGFβ1.

[0039] In one embodiment, said TGFβ2 is human TGFβ2. In a second aspect, the present invention relates to a nucleic acid molecule comprising a polynucleotide encoding a fusion protein as taught herein.

[0040] In another aspect, the present invention is directed to a vector comprising the nucleic acid molecules taught herein. In one aspect, the invention pertains to a host cell comprising a nucleic acid molecule taught herein or a vector taught herein.

[0041] In one aspect, the invention provides a method for producing a fusion protein as taught herein, the method comprising culturing a host cell as taught herein under conditions that allow for production of a fusion protein as taught herein; and, optionally, recovering the fusion protein.

[0042] In yet another aspect, the invention provides a pharmaceutical composition comprising a fusion protein as taught herein and a pharma- ceutically acceptable carrier or excipient. The invention also relates to the fusion proteins taught herein for use as medicines, e.g., for use in the prevention or treatment of conditions characterized by pathological pain, chronic pain, neuroinflammation, neurodegeneration, and / or local or systemic inflammation. In this regard, "chronic" may be considered as lasting for at least 1, 2, 3, 4, 5, 6, 10 or 12 months, or even at least 1, 2, 3, 4 or 5 years. The conditions may be characterized by visceral or non-visceral inflammatory pain, visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, mixed nociceptive-neuropathic pain, neuroinflammation, and / or neurodegeneration, and / or may be characterized by 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 The pain may be selected from the group consisting of: associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-related pain, chemotherapy-induced allodynia, chemotherapy-induced peripheral neuropathy, complex regional pain syndrome, post-spinal cord injury pain, post-stroke pain, multiple sclerosis, low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, chronic widespread pain, myofascial pain syndrome, Alzheimer's and Parkinson's disease, Huntington's disease, and / or amyotrophic lateral sclerosis or multiple sclerosis.

[0043] In one 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, e.g., a human, where IL4 or IL13 is indicated.

[0044] The present invention also relates to the fusion proteins taught herein for use in the prevention or treatment in a mammal, e.g., a human, of a clinical condition in which IL10 or IL27 or IL33 or TGFβ1 or TGFβ2 is indicated.

[0045] Finally, the present invention teaches vectors for use in the prevention or treatment of conditions characterized by chronic pain, neuroinflammation, neurodegeneration, and / or local or systemic inflammation, which may be characterized by visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, or mixed nociceptive-neuropathic pain, neuroinflammation, and / or neurodegeneration, and / or may be characterized by 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 The pain may be selected from the group consisting of peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, complex regional pain syndrome, post-spinal cord 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.

[0046] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure may be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief description of the drawings]

[0047] [Figure 1]Regulatory (e.g., anti-inflammatory) cytokines are required for the resolution of chemotherapy-induced pain. Mice were injected intraperitoneally with 2 mg / kg paclitaxel on days 0 and 2 to induce a transient, painful chemotherapy-induced polyneuropathy. From day 6 onwards, mice were injected intrathecally daily (5 μg antibody per injection) with neutralizing antibodies against endogenous IL4 (n=4; open downward triangles) or IL3 (n=4; filled upward triangles) for 5 days. As a control, mice treated with paclitaxel were injected intrathecally with isotype IgG (n=3; dotted line with open circle). As another control, mice were pretreated with vehicle instead of paclitaxel and control IgG (n=3; solid line with filled circle). Pain-like behavior was followed over time by measuring mechanical sensitivity to touch using von Frey hairs. Note that below the 50% threshold indicates high sensitivity. Data represent the 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-treated and control IgG-treated mice; xp<0.05, xx p<0.01 between anti-IL4 IgG-treated and control IgG-treated mice. [Diagram 2] IL13 attenuates paclitaxel-induced damage to neurons. Primary sensory neurons were cultured and treated overnight with paclitaxel (1 μM) to induce neuronal damage, which was quantified by measuring neurite length after β3-tubulin staining. During chemotherapy treatment, vehicle (-) or individual cytokines (50 ng / ml) were added and the average length of neurons was measured. Data represent the mean and standard error of the mean of neurite length (microns) of >10 cells measured in at least two experiments. [Diagram 3]Characterization of recombinant IL4 / IL13 fusion proteins. His-tagged IL4 / IL13 fusion proteins were expressed in HEK393 cells, purified using HIS-Select Nickel affinity chromatography, and analyzed by high pressure size exclusion chromatography (HP-SEC). The HP-SEC profile shows a homogeneous monomeric preparation. The inset shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the starting material (load), flow-through (FT), wash buffer (wash), and eluate of the His-tag purification column. The gel was stained with Coomassie Blue. Note that the IL4 / IL13 fusion protein migrates as a smear at 37 kDa. [Figure 4] IL4 / IL13 fusion protein reduces paclitaxel-induced persistent mechanical allodynia. C57BL / 6 mice were intraperitoneally administered paclitaxel (8 mg / kg) on ​​days 0, 2, 4, and 6 (gray symbols on the x-axis) to induce persistent chemotherapy-induced polyneuropathy. On day 8, IL4 / IL13 fusion protein (0.3 [open circle], 1 [open triangle], or 3 μg / mouse [open square]; n = 4 / group) or vehicle (n = 4) was administered intrathecally, and the course of mechanical allodynia was followed over time using von Frey hairs. Data are presented as mean ± SEM. Data statistics were analyzed by two-way ANOVA followed by Tukey's multiple comparison test. *, **, ***=p<0.05, p<0.01, and 0.001, respectively, 0.3 μg IL4 / IL13 fusion protein vs. vehicle. &=p<0.05, 3 μg IL4 / IL13 fusion protein vs. vehicle. x=p<0.05, 1 μg IL4 / IL13 fusion protein vs. vehicle. [Diagram 5]IL4 / IL13 fusion protein has a longer lasting effect on painful paclitaxel-induced neuropathy in mice compared to IL4 / IL10 fusion protein. Mice were intraperitoneally injected with 8 mg / kg paclitaxel four times every other day (gray symbols on the x-axis) to induce persistent painful chemotherapy-induced polyneuropathy. On day 8, mice were given a single intrathecal injection of IL4 / IL13 fusion protein (0.7 μg; open circles, n=4), IL4 / IL10 fusion protein (0.7 μg; open triangles, n=3), a combination of wild-type IL4 and IL13 (0.35 μg / cytokine; open squares, n=4), or vehicle (filled 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 results in sustained relief of pain (e.g., permanent relief of pain), whereas IL4 / IL10 fusion protein has a transient effect that lasts for 2 days in this experiment. [Figure 6] IL4 / IL13 fusion protein protects against paclitaxel-induced nerve damage in mice. Mice were injected intraperitoneally with 8 mg / kg paclitaxel four times every other day to induce persistent painful chemotherapy-induced polyneuropathy. On day 8, mice were treated with a single intrathecal injection of IL4 / IL13 fusion protein (0.7 μg) or vehicle. On day 15, the length of intraepidermal nerve fibers in the paw skin was determined by immunofluorescence visualization with the neuronal marker PGP9.5. Data are shown for mice not treated with chemotherapy (black bars; n=4) or mice injected with paclitaxel and subsequently treated with vehicle (-; n=6) or IL4 / IL13 fusion protein (IL4-13; n=4). [Figure 7]IL4 / IL13 fusion protein protects cultured neurons better from chemotherapy-induced damage than IL4 / IL10 fusion protein or a combination of IL4 and IL13. Primary sensory neurons were cultured overnight in the presence of paclitaxel (1 μM) to induce neuronal damage, which was quantified by measuring neurite length with β3-tubulin staining. During incubation with chemotherapy, vehicle (-), or IL4 / IL13 fusion protein (IL4-13), IL4 / IL10 fusion protein (IL4-10), or a combination of IL4 and IL13 (IL4+Il13) were added at equimolar concentrations. Neurons cultured in the absence of paclitaxel and cytokines are shown for comparison (black bars). [Figure 8] IL4 / IL13 cures oxaliplatin-induced polyneuropathy in mice, whereas IL4 alone or IL13 alone has a partial, temporary effect. Mice were intraperitoneally injected with oxaliplatin (3 mg / kg) daily for 5 days, followed by 5 days of no treatment and an additional 5 days of oxaliplatin treatment cycles (gray symbols on the x-axis). The day after the last oxaliplatin injection, animals were intrathecally injected with IL4 / IL13 fusion protein (0.3 μg; open circles, n=4), or wild-type cytokine (0.15 μg; n=4, rectangles for IL4 and triangles for IL13); or vehicle alone (filled circles). Pain was measured with the von Frey test. [Figure 9] IL4 / IL13 fusion protein, but not the combination of IL4 and IL13, protects cultured neurons from oxaliplatin-induced damage. Primary sensory neurons were cultured and treated with oxaliplatin (5 μg / ml) overnight. Neuronal damage was then quantified by measuring neurite length by β3-tubulin staining. During incubation with chemotherapeutic agents, vehicle (-), or IL4 / IL13 fusion protein, or a combination of Il4 and IL13 (IL4+IL13) was added at equimolar concentrations. Neurons cultured in the absence of oxaliplatin and cytokines are shown for comparison (black bars). [Figure 10] In human and mouse dorsal root ganglia, cytokine receptor subunits IL10, IL4, IL13, IL33, IL27, TGFβ1, and TGFβ2 are expressed. To evaluate whether the cytokine receptor targeted by the fusion protein of the present invention is expressed by the sensory system, RNAseq data of cytokine receptor subunits IL10, Il4, IL13, IL33, IL27, TGFβ1, and TGFβ2 in dorsal root ganglia and spinal cord were extracted from the database by Ray et al. (Pain 2018;159:1325-1345) available at https: / / www.utdallas.edu / bbs / painneurosciencelab / sensoryomics / drgtxome / ?go. RNA sequencing data is expressed in transcripts per million. For comparison, data on the expression of this receptor in whole blood is also shown. [Figure 11] Characterization of fusion proteins. Purified fusion proteins were analyzed on 4-12% gradient NuPage™ polyacrylamide gels under non-reducing and reducing conditions, and bands were visualized by Coomassie protein staining. [Figure 12] The two IL4 / IL13 fusion proteins of the present disclosure protect neurons from chemotherapy-induced neuronal damage. Primary sensory neurons were cultured in the presence of paclitaxel (1 μM) and various concentrations of each fusion protein, or equimolar doses of IL13 or unlinked cytokine combinations, for 24 hours. Inhibition of paclitaxel-induced reduction in neurite length was calculated. The fusion protein labeled IL4 / IL13 contains SEQ ID NO: 4. The fusion protein labeled IL4 / IL13SKP contains SEQ ID NO: 16. Data are presented as mean ± SEM. Data were analyzed by two-way ANOVA mixed-effects analysis followed by Tukey's multiple comparison test. [Figure 13]IL10 / IL13 protects neurons from chemotherapy-induced neuronal damage. Primary sensory neurons were cultured for 24 hours in the presence of paclitaxel (1 μM) and various concentrations of each fusion protein, or equimolar doses of IL13 or unlinked cytokine combinations. Inhibition of paclitaxel-induced reduction in neurite length was calculated. Data are presented as mean ± SEM. Data were analyzed by two-way ANOVA mixed-effects analysis followed by Tukey's multiple comparison test. [Figure 14] Protective effects of IL13 / IL13 and IL27 / IL13 against chemotherapy-induced neuronal damage. Primary sensory neurons were cultured in the presence of paclitaxel (1 μM) and various concentrations of each fusion protein or equimolar doses of IL13 for 24 h. Inhibition of paclitaxel-induced reduction in neurite length was calculated. Data are presented as mean ± SEM. Data were analyzed by two-way ANOVA mixed-effects analysis followed by Tukey's multiple comparison test. [Figure 15] The IL13-containing fusion proteins of the present disclosure induce distinct kinase activity profiles in dorsal root ganglion (DRG) cells compared to unlinked cytokine combinations. PamGene kinase activity profiling was performed to assess global protein tyrosine kinase (PTK) activity in homogenates of lumbar DRG isolated from mice with persistent paclitaxel-induced peripheral neuropathy following administration of IL4 / IL13 fusion proteins, IL4+IL13 (unlinked cytokine combinations), and vehicle. Kinomic profiles were assessed 60 minutes after intrathecal administration of IL4 / IL13 fusion proteins, cytokine combinations, or vehicle (PBS). Differentially phosphorylated peptides are shown based on one-way ANOVA analysis between mice treated with IL4 / IL13, IL4+IL13, and vehicle compared to naive mice (untreated; no paclitaxel, no intrathecal injection). Black indicates no significant change, and shades indicate reduced phosphorylation. [Figure 16]Changes in kinase activity in dorsal root ganglion (DRG) cells of female mice treated with IL4 / IL13 compared to unlinked cytokine combination. PamGene kinase activity profiling was performed to assess global protein tyrosine kinase (PTK) activity in homogenates of lumbar DRG isolated from female mice with persistent paclitaxel-induced CIPN after administration of IL4 / IL13 fusion protein or IL4+IL13 (unlinked cytokine combination). Graph shows predicted upstream kinases inferred from differentially phosphorylated peptide substrates identified by unpaired t-test comparison between samples from females treated with IL4 / IL13 fusion protein and those treated with IL4+IL13 (n=3 animals per group). Graph is sorted with highest specificity score at the top and lowest specificity score at the bottom. [Figure 17] Changes in kinase activity in dorsal root ganglion (DRG) cells of male mice treated with IL4 / IL13 compared to unlinked cytokine combination. PamGene kinase activity profiling was performed to assess global protein tyrosine kinase (PTK) activity in homogenates of lumbar DRG isolated from male mice with persistent paclitaxel-induced CIPN after administration of IL4 / IL13 fusion protein or IL4+IL13 (unlinked cytokine combination). Graph shows predicted upstream kinases inferred from differentially phosphorylated peptide substrates identified by unpaired t-test comparison between samples from males treated with IL4 / IL13 fusion protein and those treated with IL4+IL13 (n=3 animals per group). Graph is sorted with highest specificity score at the top and lowest specificity score at the bottom. [Figure 18A]Size exclusion chromatography of IL4 / IL13, IL10 / IL13, IL27 / IL13, and IL13 / IL13 fusion proteins. Size exclusion chromatography was performed on the IL4 / IL13, IL10 / IL13, IL27 / IL13, and IL13 / IL13 fusion proteins of the present disclosure. Figure 18A: Size exclusion chromatography of IL4 / IL13 fusion protein comprising SEQ ID NO: 16. Figure 18B: Size exclusion chromatography of IL13 / IL13 fusion protein comprising SEQ ID NO: 20. Figure 18C: Size exclusion chromatography of IL27 / IL13 fusion protein comprising SEQ ID NO: 19. Figure 18D: Size exclusion chromatography of IL10 / IL13 fusion protein comprising SEQ ID NO: 17. [Figure 18B] Size exclusion chromatography of IL4 / IL13, IL10 / IL13, IL27 / IL13, and IL13 / IL13 fusion proteins. Size exclusion chromatography was performed on the IL4 / IL13, IL10 / IL13, IL27 / IL13, and IL13 / IL13 fusion proteins of the present disclosure. Figure 18A: Size exclusion chromatography of IL4 / IL13 fusion protein comprising SEQ ID NO: 16. Figure 18B: Size exclusion chromatography of IL13 / IL13 fusion protein comprising SEQ ID NO: 20. Figure 18C: Size exclusion chromatography of IL27 / IL13 fusion protein comprising SEQ ID NO: 19. Figure 18D: Size exclusion chromatography of IL10 / IL13 fusion protein comprising SEQ ID NO: 17. [Figure 18C]Size exclusion chromatography of IL4 / IL13, IL10 / IL13, IL27 / IL13, and IL13 / IL13 fusion proteins. Size exclusion chromatography was performed on the IL4 / IL13, IL10 / IL13, IL27 / IL13, and IL13 / IL13 fusion proteins of the present disclosure. Figure 18A: Size exclusion chromatography of IL4 / IL13 fusion protein comprising SEQ ID NO: 16. Figure 18B: Size exclusion chromatography of IL13 / IL13 fusion protein comprising SEQ ID NO: 20. Figure 18C: Size exclusion chromatography of IL27 / IL13 fusion protein comprising SEQ ID NO: 19. Figure 18D: Size exclusion chromatography of IL10 / IL13 fusion protein comprising SEQ ID NO: 17. [Figure 18D] Size exclusion chromatography of IL4 / IL13, IL10 / IL13, IL27 / IL13, and IL13 / IL13 fusion proteins. Size exclusion chromatography was performed on the IL4 / IL13, IL10 / IL13, IL27 / IL13, and IL13 / IL13 fusion proteins of the present disclosure. Figure 18A: Size exclusion chromatography of IL4 / IL13 fusion protein comprising SEQ ID NO: 16. Figure 18B: Size exclusion chromatography of IL13 / IL13 fusion protein comprising SEQ ID NO: 20. Figure 18C: Size exclusion chromatography of IL27 / IL13 fusion protein comprising SEQ ID NO: 19. Figure 18D: Size exclusion chromatography of IL10 / IL13 fusion protein comprising SEQ ID NO: 17. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] General definition 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 DNA encoding a protein according to the invention. An "isolated nucleic acid sequence" refers to a nucleic acid sequence that is no longer in the natural environment from which it was isolated, e.g., a nucleic acid sequence within a bacterial host cell or a plant nuclear or plastid genome.

[0049] The terms "protein" or "polypeptide" are used interchangeably and refer to a molecule consisting of one or several chains of amino acids, regardless of a specific mode of action, size, three-dimensional structure, or origin. An "isolated protein" is used to refer to a protein that is no longer in its natural environment, e.g., in vitro, or within a recombinant mammalian, bacterial, or plant host cell.

[0050] The term "fusion protein" refers to a protein or polypeptide having an amino acid sequence from or derived from two or more proteins. A fusion protein may also include a linking region or linker of amino acids between amino acid moieties from or derived from separate proteins. A fusion protein also refers to a molecule having an amino acid sequence from or derived from two or more proteins that are non-covalently linked, or connected (e.g., covalently linked), with or without a spacer, or via a chemical crosslinking linker. A fusion protein may be a polypeptide construct.

[0051] The terms IL4, IL10, IL13, IL27, IL33, TGFβ1 and TGFβ2 preferably refer to the wild-type sequences of their respective cytokines and / or mutant forms thereof capable of binding to at least one (e.g., preferably at least two) of their respective cytokine receptors or receptor subunits.

[0052] The terms "TGFβ," "TGFβ1 / 2," and "TGFβ(1 or 2)" are used to refer to TGFβ1 and / or TGFβ2. The term "IL4 / IL13 fusion protein" refers to a fusion polypeptide comprising at least IL4 and IL13, optionally linked together via a linker. The fusion protein may contain additional polypeptide sequences, such as a signal sequence, a His tag, a targeting sequence, an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0053] The term "IL10 / IL13 fusion protein" refers to a fusion polypeptide comprising at least IL10 and IL13, optionally linked together via a linker. The fusion protein may contain additional polypeptide sequences, such as a signal sequence, a His tag, a targeting sequence or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0054] The term "IL33 / IL13 fusion protein" refers to a fusion polypeptide comprising at least IL33 and IL13, optionally linked together via a linker. The fusion protein may contain additional polypeptide sequences, such as a signal sequence, a His tag, a targeting sequence or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0055] The term "TGFβ1 / IL13 fusion protein" refers to a fusion polypeptide comprising at least TGFβ1 and IL13, optionally linked together via a linker. The fusion protein may contain additional polypeptide sequences, such as a signal sequence, a His tag, a targeting sequence or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0056] The term "TGFβ2 / IL13 fusion protein" refers to a fusion polypeptide comprising at least TGFβ2 and IL13, optionally linked together via a linker. The fusion protein may contain additional polypeptide sequences, such as a signal sequence, a His tag, a targeting sequence or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0057] The term "IL13 / IL13 fusion protein" refers to a fusion polypeptide comprising at least two IL13 molecules, optionally linked together via a linker. The fusion protein may contain additional polypeptide sequences, such as a signal sequence, a His tag, a targeting sequence or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0058] As used herein, "linker" refers to a polypeptide used to link two proteins or polypeptides, IL4 (or IL10 or IL27 or IL33 or TGFβ or IL13) and IL13. The linker is typically a stretch of amino acids, e.g., predominantly glycine and / or serine. In one embodiment, the linker is a stretch of amino acids having a length of up to 100 amino acids, e.g., about 2, 5, 7, 10, 15 amino acids, up to about 15, 20, 25, 30, 35, 50, 75 or 100 amino acids, preferably comprising predominantly serine and glycine residues.

[0059] As used herein, "interleukin-13" (IL13) preferably refers to any mammalian IL13, such as human IL13, murine IL13 or active species or variants (e.g. allelic variants), (functional) fragments or derivatives thereof.

[0060] As used herein, "interleukin-4" (IL4) preferably refers to any mammalian IL4, such as human IL4, murine IL4 or active species or variants (e.g. allelic variants), (functional) fragments or derivatives thereof.

[0061] As used herein, "interleukin-10" (IL10) preferably refers to any mammalian IL10, such as human IL10, murine IL10, or active species or variants (e.g. allelic variants), (functional) fragments or derivatives thereof.

[0062] As used herein, "interleukin-27" (IL27) may refer to any mammalian IL27, e.g., human IL27, mouse IL27, or active species or variants (e.g., allelic variants), (functional) fragments, or derivatives thereof. In some cases, IL27 refers to IL27 subunit alpha (IL27A), e.g., human IL27A. In some cases, IL27 refers to IL27 subunit beta (IL27B), e.g., human IL27B. In some cases, IL27 refers to IL27A and IL27B.

[0063] As used herein, "interleukin-33" (IL33) preferably refers to any mammalian IL33, such as human IL33, murine IL33 or an active species or variant (e.g. allelic variant), (functional) fragment or derivative thereof.

[0064] As used herein, "transforming growth factor β1" (TGFβ1) preferably refers to any mammalian TGFβ1, e.g., human TGFβ1, mouse TGFβ1 or active species or variants (e.g. allelic variants), (functional) fragments or derivatives thereof.

[0065] As used herein, "transforming growth factor β2" (TGFβ2) preferably refers to any mammalian TGFβ2, e.g., human TGFβ2, mouse TGFβ2, or active species or variants (e.g. allelic variants), (functional) fragments or derivatives thereof.

[0066] When describing a cytokine, the term "wild type" refers to a cytokine that has an amino acid sequence that is naturally occurring and encoded by the germline genome of a given species. A species may have one wild type sequence, or two or more wild type sequences (e.g., one reference wild type sequence and one or more non-reference wild type sequences). A wild type cytokine sequence may include a sequence that is truncated at the N- and / or C-terminus compared to the sequence encoded by the open reading frame. A wild type cytokine sequence may be a mature form of a cytokine that has been processed to remove N- and / or C-terminal residues. A wild type cytokine may lack a signal peptide or may include a signal peptide (e.g., a signal peptide may be added to the N-terminus of a wild type cytokine).

[0067] When describing a cytokine, the term "derivative" refers to a cytokine having an amino acid sequence that differs from the wild-type sequence by one or more amino acids, for example, contains one or more amino acid insertions, deletions or substitutions compared to the wild-type sequence. A cytokine derivative binds to at least one subunit of the corresponding native receptor for the wild-type cytokine and induces signal transduction and / or cytokine activity. The binding affinity, signal transduction and / or cytokine activity of a cytokine derivative may be the same as or different from that of the corresponding wild-type cytokine.

[0068] With respect to the fusion proteins of the invention (or variants or fragments thereof), "functional" refers to the ability to display both IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2 or other regulatory cytokines, e.g., anti-inflammatory cytokines) and IL13 functionality, e.g., the ability to bind to at least one receptor subunit to which a wild-type version of the cytokine binds.

[0069] Assays to assess the functional activity of these cytokines are well known to those of skill in the art. For example, a functional assay for IL4 and IL10 is lipopolysaccharide (LPS)-induced TNF release in whole blood in the presence of anti-IL10 antibodies. 26 The functional assay for IL13 is the proliferation of human TF1 erythroleukocytes. 27 An assay for IL33 function is IL6 production by the mast cell line MC / 9. An assay for TGFβ1 or TGFβ2 is inhibition of IL4-dependent proliferation of the murine T cell line HT-2. A functional assay for IL27 may include IL6 production by LPS-stimulated THP-1 macrophages.

[0070] The term "gene" refers to a DNA sequence comprising a region that is transcribed in a cell into an RNA molecule (e.g., mRNA) (transcribed region), operably linked to suitable regulatory sequences (e.g., promoters). Thus, a gene may comprise several operably linked sequences, e.g., a promoter, a 5' leader sequence including, e.g., sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA), introns, and a 3' untranslated sequence including, e.g., a transcription termination site.

[0071] "Expression of a gene" refers to the process by which a DNA region operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into RNA that is biologically active, i.e., capable of being translated into a biologically active protein or peptide (or active peptide fragment). "Expression of a polypeptide" further refers to the process by which mRNA is translated into a protein product that may or may not be secreted.

[0072] 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 in the direction of transcription of the transcription start site of a gene, and is structurally specified by the presence of a binding site for DNA-dependent RNA polymerase; a transcription start site; and any other DNA sequence, including but not limited to, a transcription factor binding site, a repressor and activator protein binding site, and any other nucleotide sequence known to those skilled 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 a certain compound) or developmentally regulated. A "tissue-specific" promoter is only active in a specific type of tissue or cell.

[0073] As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter, or rather a transcriptional regulatory sequence, is operably linked to a coding sequence when it affects the transcription of the coding sequence. Operatively linked means that the DNA sequences that are linked are typically contiguous.

[0074] "Nucleic acid construct" or "vector" is understood herein to mean an artificial nucleic acid molecule that results from the use of recombinant DNA technology and is used to deliver foreign DNA into a host cell. Vectors usually contain additional genetic elements that facilitate their use in molecular cloning, such as selectable markers or multiple cloning sites (see below).

[0075] "Sequence identity" and "sequence similarity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences can then be referred to as "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (defined below) when optimally aligned (e.g., by the programs GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, GAP default parameters are used, with gap creation penalty = 50 (nucleotides) / 8 (proteins), and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff and Henikoff, 1992, PNAS 89, pp. 915-919). Sequence alignment and sequence identity percentage scores can 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, similarity or identity percentage may be determined by searching against a database using algorithms such as FASTA, BLAST, etc. Preferably, sequence identity refers to sequence identity over the entire length of the sequence.

[0076] "Host cell" or "recombinant host cell" or "transformed cell" are terms that refer to new individual cells (or organisms) resulting from the expression of at least one nucleic acid molecule, including, inter alia, a nucleic acid molecule encoding a desired protein. The host cell is preferably a mammalian cell, a plant cell or a bacterial cell. The host cell may contain the nucleic acid molecule or vector of the invention as an extrachromosomal (episomal) replicating molecule, or more preferably, the nucleic acid molecule or vector of the invention integrated into the genome of the host cell.

[0077] The term "selectable marker" is a term known to those of skill in the art and is used herein to describe any genetic entity that, when expressed, can be used to select a cell(s) that contains the selectable marker. The selectable marker gene product confers, for example, antibiotic resistance or an auxotrophy.

[0078] The term "nervous system cell" refers to a cell found in the central nervous system or peripheral nervous system. Nervous system cells can be neurons, central nervous system cells, peripheral nervous system cells, glial cells, microglial cells, astrocytes, Schwann cells, satellite glial cells, oligodendrocytes, infiltrating cells, infiltrating immune cells, infiltrating myeloid cells, infiltrating lymphoid cells, infiltrating macrophages, infiltrating neutrophils, infiltrating lymphocytes, infiltrating T cells, infiltrating B cells, or infiltrating natural killer cells. Neurons can be, for example, sensory neurons, somatosensory neurons, visceral sensory neurons, nociceptors, and / or autonomic neurons.

[0079] In this document and its claims, the verb "comprises" and its conjugations are used in their open-ended sense to mean that the items following the word are included, but not that items not specifically mentioned are excluded. It also encompasses the more restrictive verb "consists 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 element is present, unless the context clearly requires that one and only one of the element is present. Thus, the indefinite article "a" or "an" usually means "at least one." It is further understood that when referring to a "sequence" herein, reference is generally made to an actual physical molecule having a particular sequence of subunits (e.g., amino acids).

[0080] Proteins, nucleic acid sequences, vectors and host cells of the invention The present inventors provide a fusion protein comprising an IL13 protein and a regulatory cytokine, such as a protein selected from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2, or IL13 itself, optionally physically fused together via a linker. The regulatory cytokines of the present disclosure include, but are not limited to, IL4, IL10, IL13, IL27, IL33, TGFβ1, and TGFβ2. In particular, the fusion protein of the present invention has been found to have superior activity in treating conditions disclosed herein (e.g., neuropathic pain) than its individual counterparts, i.e., separate regulatory cytokines (e.g., IL4 or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13. In particular, upon intrathecal administration, the fusion protein of the present invention has been found to have a long-lasting analgesic effect on neuropathic pain and suppress allodynia associated with chemotherapy-induced neuropathy. The present inventors believe that this analgesic effect of the fusion protein of the present invention is consistent with the previously described analgesic effect of the fusion protein of IL4 and IL10. 19、23、26We unexpectedly observed that the effect of the fusion protein of the present invention lasted longer than that of the fusion protein of the present invention. Surprisingly, the fusion protein of the present invention also ameliorated chemotherapy-related neurodegeneration. It prevented neurite shortening in vitro upon incubation with chemotherapy drugs, and was more potent in this effect than the fusion protein of IL4 and IL10, or the combination of IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13. The latter surprising finding indicates the unique effect of the fusion protein over individual cytokines or their combinations. In vivo, the fusion protein of the present invention attenuated the loss of intraepidermal nerve fibers upon administration of chemotherapy drugs.

[0081] In one embodiment of the invention, the nucleic acid and amino acid sequences of IL4 / IL13 fusion protein or IL10 / 1L13 fusion protein or IL27 / IL13 fusion protein or IL33 / IL13 fusion protein or TGFβ1 / IL13 fusion protein or TGFβ2 / IL13 or IL13 / IL13 fusion protein are provided, including variants, derivatives and fragments thereof. IL4 / IL13 fusion protein or IL10 / 1L13 fusion protein or IL27 / IL13 fusion protein or IL33 / IL13 fusion protein, as well as derivatives, functional fragments and variants thereof, exhibit IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) activity and IL13 activity.

[0082] In some cases, the fusion proteins disclosed herein contain two cytokines, C1 and C2, and are referred to in the form of C1 / C2 or C1-C2. The order in which the cytokines are listed is not limiting and does not necessarily indicate the orientation of the cytokines. For example, C1 / C2 or C1-C2 may contain C1 at the C-terminal side of the cytokine C2, or at the N-terminal side of C2. A cytokine similarly referred to as C1 / C2 may be the same as a cytokine referred to as C2 / C1, unless otherwise specified.

[0083] In one aspect, a fusion protein is provided comprising IL4 (or IL10 or IL13 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13. Interleukin 13 The fusion proteins disclosed herein may comprise an IL13 protein, or a variant, derivative or fragment thereof, operably linked to, or directly or indirectly fused to, a regulatory cytokine or a variant or derivative thereof. The IL13 protein is preferably a mammalian IL13 protein, e.g., human IL13, or mouse IL13. Non-limiting examples of amino acid sequences representing human IL13 are shown in SEQ ID NO:2 and SEQ ID NOs:9-15. Variants of IL13 include proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher, e.g., 100%, amino acid sequence identity, preferably over the entire length, with any one of SEQ ID NO:2 or SEQ ID NOs:9-15. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman-Wunsch algorithm and the GAP default parameters defined above. Also included are variants, derivatives and fragments thereof that have IL13 activity and are derived by one or more amino acid substitutions, deletions or insertions from a polypeptide having the amino acid sequence of SEQ ID NO:2 or any one of SEQ ID NOs:9 to 15. Preferably, such proteins contain 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.

[0084] In some embodiments, an IL13 of the present disclosure (e.g., an IL13 variant, derivative or fragment thereof) may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions, deletions or insertions compared to an IL13 sequence disclosed herein (e.g., a wild-type IL13 sequence).

[0085] In some embodiments, an IL13 of the disclosure (e.g., an IL13 variant, derivative or fragment thereof) may comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid substitutions, deletions or insertions compared to an IL13 sequence disclosed herein (e.g., a wild-type IL13 sequence).

[0086] In some embodiments, an IL13 sequence of the disclosure (e.g., an IL13 variant, derivative or fragment thereof) has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-46, 2-47, 2-48, 2-49, 2-50, 2-51, 2-52, 2-53, 2-54, 2-55, 2-56, 2-57, 2-58, 2-59, 2-60, 2-61, 2-62, 2-63, 2-64, 2-65, 2-66, 2-67, 2-68, 2-69, 2-70, 2-71, 2 , 2 to 9, 2 to 10, 2 to 15, 2 to 20, 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20 or 20 to 25 amino acid substitutions, deletions or insertions.

[0087] In some embodiments, the IL13 sequence of the present disclosure (e.g., an IL13 variant, derivative or fragment thereof) may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions, deletions or insertions compared to the IL13 sequence disclosed herein (e.g., a wild-type IL13 sequence). The amino acid substitutions may be conservative or non-conservative substitutions. One or more amino acid substitutions, deletions or insertions may be at the N-terminus, C-terminus, or combinations thereof within the amino acid sequence. The amino acid substitutions, deletions or insertions may be contiguous, non-contiguous, or combinations thereof.

[0088] The IL13 of the present disclosure may include a wild-type IL13 sequence. Non-limiting examples of wild-type IL13 sequences include SEQ ID NOs: 2 and 9 to 15. SEQ ID NO: 12 may be the reference wild-type IL13 sequence of the present disclosure.

[0089] The IL13 of the present disclosure may include IL13 variants, derivatives or fragments thereof having one or more amino acid substitutions. For example, the IL13 variants, derivatives or fragments thereof may include amino acid substitutions at positions L10, E12, R11, I14, E15, E16, V18, R65, S68, R86, D87, T88, K89, D98, L101, L103, K104, K105, L106, F107, R108, R111, F114, N113 or combinations thereof of SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof is L10F;L10I;L10V;L10A;L10D;L10T;L10H;R11S;R11N;R11H;R11L;R11I;I14L;I14F;I14V;I14M;V18L;V18F;V18I;E12A;R65D;R86K;R86T;R86M;D87E;D87K;D87R;D87G;D87S;T88S,T88I;T88K;T88R;K89R;K89T;K89M;L101F;L101 I;L101Y;L101H;L101N;K104R;K104T;K104M;K105T;K105A;K105R;K105E;F107L;F107I;F107V;F107M;R108K;R108T;R108M;E12K, E12I, E12C, E12S, E12R, E12Y, E12D, E15K, E16K, R65D, S68D, D98K, L101A, L103A, K104D, K105D, L106A, F107Y, R108D, R111D, F114D, N113D or combinations thereof. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10H, R86T, D87G, T88R and R108K compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10A, V18F, R86K, D87K, K89R, L101I, K104R and R108K compared to SEQ ID NO:2 or SEQ ID NO:12.In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R and K105T compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10V, K89R, L101N, K105E and R108T compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10D, R11I, V18I, R86K, D87K, K89R and R108K compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A and R108K compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10V, K89R, L101N, K105E and R108T compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions R11S, I14M, T88S, L101N, K105A and R108K compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T and R108K compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10H, R86T, D87G, T88R and R108K compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10A, V18F, R86K, D87K, K89R, L101I, K104R and R108K compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10T or L10D; R11I; V18I; R86K; D87K or D87G; T88S; K89R; L101Y; K104R; K105T; and R108K compared to SEQ ID NO:2 or SEQ ID NO:12.In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10A or L10V; R86T; D87G; T88K; K89R; L101N; K104R; K105A or K105E; and R108K or R108T compared to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10V, V18I, D87S, D88S, L101F, K104R and K105T compared to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R and K105T compared to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10V, V18I, D87S, T88S, L101F, K104R and K105T compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10V or L10I;D87S;T88S;K89R;L101H or L101F;K104R; and K105T compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10I;V18I;R86T;D87G;T88S;K89R;L101Y, L101H;K104R; and K105A compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions L10V; V18I; D87S; T88S; L101F; K104R; and K105T compared to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions V18I, R86T, D87G, T88S, L101Y, K104R and K105A compared to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A and F107M compared to SEQ ID NO: 2 or SEQ ID NO: 12.In some embodiments, the IL13 variant, derivative or fragment thereof comprises substitutions E12K and S68D compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises substitutions E12K and R108D compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises substitutions E12K and R111D compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises substitutions E12Y and R65D compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises substitutions E12Y and S68D compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises substitutions E12K, R65D and S68D compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions E12Y, R65D and S68D compared to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative or fragment thereof comprises the substitutions E12K, R65D, S68D and R111D compared to SEQ ID NO: 2 or SEQ ID NO: 12.

[0090] In some embodiments, the IL13 variant, derivative or fragment thereof does not contain a substitution at position L10, E12, R11, I14, E15, E16, V18, R65, S68, R86, D87, T88, K89, D98, L101, L103, K104, K105, L106, F107, R108, R111, F114 or N113 compared to SEQ ID NO:2 or SEQ ID NO:12. In some embodiments, the IL13 variant, derivative or fragment thereof is L10F;L10I;L10V;L10A;L10D;L10T;L10H;R11S;R11N;R11H;R11L;R11I;I14L;I14F;I14V;I14M;V18L;V18F;V18I;E12A;R65D;R86K;R86T;R86M;D87E;D87K;D87R;D87G;D87S;T88S, T88I;T88K;T88R;K89R;K89T;K89M;L101F;L101I;L101 Y;L101H;L101N;K104R;K104T;K104M;K105T;K105A;K105R;K105E;F107L;F107I;F107V;F107M;R108K;R108T;R108M;does not contain E12K, E12I, E12C, E12S, E12R, E12Y, E12D, E15K, E16K, R65D, S68D, D98K, L101A, L103A, K104D, K105D, L106A, F107Y, R108D, R111D, F114D or N113D substitutions.

[0091] In some embodiments, the disclosed IL13 or IL13 variants, derivatives or fragments thereof bind to the IL13 receptor subunit with an affinity approximately equivalent to that of the wild-type IL13 sequence. The equivalent affinity can be, for example, less than about 10-fold, less than about 5-fold, less than about 2-fold, less than about 1.9-fold, less than about 1.8-fold, less than about 1.7-fold, less than about 1.6-fold, less than about 1.5-fold, less than about 1.4-fold, less than about 1.3-fold, less than about 1.2-fold or less than about 1.1-fold affinity compared to the wild-type IL13 sequence. Comparable affinity can be, for example, greater than about 10-fold, greater than about 5-fold, greater than about 2-fold, greater than about 1.9-fold, greater than about 1.8-fold, greater than about 1.7-fold, greater than about 1.6-fold, greater than about 1.5-fold, greater than about 1.4-fold, greater than about 1.3-fold, greater than about 1.2-fold or greater than about 1.1-fold affinity compared to the wild-type IL13 sequence.

[0092] For example, the IL13 or IL13 variants, derivatives or fragments thereof of the present disclosure may bind to interleukin 13 receptor alpha 1 (IL-13Rα1), interleukin 13 receptor alpha 2 (IL-13Rα2), interleukin 4 receptor alpha (IL-4Rα), or a combination thereof with an affinity approximately equivalent to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments thereof of the present disclosure may bind to IL-13Rα1 with an affinity approximately equivalent to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments thereof of the present disclosure may bind to IL-13Rα2 with an affinity approximately equivalent to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments thereof of the present disclosure may bind to IL-4Rα with an affinity approximately equivalent to that of the wild-type IL13 sequence. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and IL-13Rα2 with approximately the same affinity as wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and IL-4Rα with approximately the same affinity as wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα2 and IL-4Rα with approximately the same affinity as wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1, IL-13Rα2 and IL-4Rα with approximately the same affinity as wild-type IL13.

[0093] In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to the IL13 receptor subunit with an affinity at least equal to that of wild-type IL13. For example, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to interleukin-13 receptor alpha 1 (IL-13Rα1), interleukin-13 receptor alpha 2 (IL-13Rα2), interleukin-4 receptor alpha (IL-4Rα), or a combination thereof with an affinity at least equal to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 with an affinity at least equal to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα2 with an affinity at least equal to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-4Rα with an affinity at least equivalent to that of the wild-type IL13 sequence. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and IL-13Rα2 with an affinity at least equivalent to that of the wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and IL-4Rα with an affinity at least equivalent to that of the wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα2 and IL-4Rα with an affinity at least equivalent to that of the wild-type IL13. In some embodiments, the disclosed IL13 or IL13 variants, derivatives or fragments thereof may bind to IL-13Rα1, IL-13Rα2 and IL-4Rα with an affinity at least equivalent to wild-type IL13.

[0094] In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to the IL13 receptor subunit with an affinity equal to or less than that of wild-type IL13. For example, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to interleukin-13 receptor alpha 1 (IL-13Rα1), interleukin-13 receptor alpha 2 (IL-13Rα2), interleukin-4 receptor alpha (IL-4Rα), or a combination thereof with an affinity equal to or less than that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 with an affinity equal to or less than that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα2 with an affinity equal to or less than that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-4Rα with an affinity equal to or less than the wild-type IL13 sequence. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and IL-13Rα2 with an affinity equal to or less than the wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and IL-4Rα with an affinity equal to or less than the wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα2 and IL-4Rα with an affinity equal to or less than the wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1, IL-13Rα2 and IL-4Rα with an affinity equal to or less than the wild-type IL13.

[0095] In some embodiments, IL13 or IL13 variants, derivatives or fragments thereof may bind to IL-13Rα1 with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL13 sequence. In some embodiments, IL13 or IL13 variants, derivatives or fragments thereof may bind to IL-13Rα1 with no more than about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL13 sequence.

[0096] In some embodiments, IL13 or IL13 variants, derivatives or fragments thereof may bind to IL-13Rα2 with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL13 sequence. In some embodiments, IL13 or IL13 variants, derivatives or fragments thereof may bind to IL-13Rα2 with about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold less affinity than the wild-type IL13 sequence.

[0097] In some embodiments, IL13 or IL13 variants, derivatives or fragments thereof may bind to IL-4Rα with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL13 sequence. In some embodiments, IL13 or IL13 variants, derivatives or fragments thereof may bind to IL-4Rα with no more than about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL13 sequence.

[0098] In some embodiments, the disclosed IL13 or IL13 variants, derivatives or fragments thereof can activate a native IL13 receptor. The native IL13 receptor can be, for example, a receptor that includes an IL-13Rα1 subunit and an IL-4Rα subunit.

[0099] Interleukin 4 The fusion proteins disclosed herein may comprise an IL4 protein, or a variant, derivative or fragment thereof, operably linked to, or directly or indirectly fused to, an interleukin-13 or variant or derivative thereof. The IL4 protein is preferably a mammalian IL4 protein, e.g., human IL4, or murine IL4. Non-limiting examples of amino acid sequences of IL4 are shown in SEQ ID NO:1 and SEQ ID NOs:26-28. Variants of IL4 include proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher, e.g., 100%, amino acid sequence identity, preferably over the entire length, with any one of SEQ ID NO:1 or SEQ ID NOs:26-28. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman-Wunsch algorithm and the GAP default parameters defined above. Variants also include proteins having IL4 activity that are derived by one or more amino acid substitutions, deletions or insertions from a polypeptide having the amino acid sequence of SEQ ID NO:1 or any one of SEQ ID NOs:26 to 28. Preferably, such proteins contain 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. In some embodiments, an IL4 of the present disclosure (e.g., an IL4 variant, derivative or fragment thereof) may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions, deletions or insertions compared to an IL4 sequence disclosed herein (e.g., a wild-type IL4 sequence).

[0100] In some embodiments, an IL4 of the present disclosure (e.g., an IL4 variant, derivative or fragment thereof) may include no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid substitutions, deletions or insertions compared to an IL4 sequence disclosed herein (e.g., a wild-type IL4 sequence).

[0101] In some embodiments, the IL4 sequence of the disclosure (e.g., an IL4 variant, derivative or fragment thereof) has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-21, 3-22, 3-23, 3-24, 3-25, 3-26, 3-27, 3-28, 3-29, 3-30, 3-31, 3-32, 3-33, 3-34, 3-35, 3-36, 3-37, 3-38, 3-39, 3-40, 3-41, 3-42, 3-43, 3-44, 3-45, 3-46, 3-47, 3-48, 3-49, 3-50, 3-51, 3-52, 3-53, 3-54, 3-55, 3-56, 3-57, 3-58, 3-59, 3-60, 3-61, 3-62, 3-63, 3-64, 3-65, 3-66, 3-67, 3-68, 3-69, 3-70, 3-71, 3-7 It may comprise from 2 to 9, 2 to 10, 2 to 15, 2 to 20, 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20 or 20 to 25 amino acid substitutions, deletions or insertions.

[0102] In some embodiments, the IL4 sequence of the present disclosure (e.g., an IL4 variant, derivative or fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions, deletions or insertions compared to the IL4 sequence disclosed herein (e.g., a wild-type IL4 sequence). The amino acid substitutions may be conservative or non-conservative substitutions. One or more amino acid substitutions, deletions or insertions may be at the N-terminus, C-terminus, or combinations thereof within the amino acid sequence. The amino acid substitutions, deletions or insertions may be contiguous, non-contiguous, or combinations thereof.

[0103] The IL4 of the present disclosure may include a wild-type IL4 sequence. Non-limiting examples of wild-type IL4 sequences include SEQ ID NOs: 1 and 26 to 28. The reference wild-type IL4 sequence of the present disclosure may be SEQ ID NO: 1.

[0104] The IL4 of the present disclosure may include IL4 variants, derivatives or fragments thereof with one or more amino acid substitutions.For example, the IL4 variants, derivatives or fragments thereof may include amino acid substitutions at positions K117, T118, R121, E122, Y124, S125, S128, S129 or combinations thereof in SEQ ID NO:1.In some embodiments, the IL4 variants, derivatives or fragments thereof include substitutions that are K117R, T118V, R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F, Y124D, S125F, S128G, S125R, S129A or combinations thereof compared to SEQ ID NO:1. In some embodiments, the IL4 variant, derivative or fragment thereof comprises the substitutions K117R, T118V, R121Q, E122S, Y124W, S125F, S128G and S129A compared to SEQ ID NO: 1. In some embodiments, the IL4 variant, derivative or fragment thereof comprises the substitutions R121D and Y124D compared to SEQ ID NO: 1.

[0105] In some embodiments, the IL4 variant, derivative or fragment thereof does not contain a substitution at position K117, T118, R121, E122, Y124, S125, S128 or S129 compared to SEQ ID NO: 1. In some embodiments, the IL4 variant, derivative or fragment thereof does not contain a substitution at position K117R, T118V, R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F, Y124D, S125F, S128G, S125R or S129A.

[0106] In some embodiments, the IL4 or IL4 variants, derivatives or fragments thereof of the present disclosure bind to the IL4 receptor subunit with approximately the same affinity as the wild-type IL4 sequence. The same affinity can be, for example, about 10 times less, about 5 times less, about 2 times less, about 1.9 times less, about 1.8 times less, about 1.7 times less, about 1.6 times less, about 1.5 times less, about 1.4 times less, about 1.3 times less, about 1.2 times less, or about 1.1 times less affinity than the wild-type IL4 sequence. The same affinity can be, for example, more than about 10 times, more than about 5 times, more than about 2 times, more than about 1.9 times, more than about 1.8 times, more than about 1.7 times, more than about 1.6 times, more than about 1.5 times, more than about 1.4 times, more than about 1.3 times, more than about 1.2 times, or more than about 1.1 times less affinity than the wild-type IL4 sequence.

[0107] For example, the IL4 or IL4 variants, derivatives or fragments thereof of the present disclosure may bind to, for example, interleukin 13 receptor alpha 1 (IL-13Rα1), common gamma chain, interleukin 4 receptor alpha (IL-4Rα), or a combination thereof with an affinity approximately equivalent to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments thereof of the present disclosure may bind to IL-13Rα1 with an affinity approximately equivalent to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments thereof of the present disclosure may bind to IL-4Rα with an affinity approximately equivalent to that of the wild-type IL4 sequence. In some embodiments, the IL4 or IL4 variants, derivatives or fragments thereof of the present disclosure may bind to IL-13Rα1 and common gamma chain with an affinity approximately equivalent to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments thereof of the present disclosure may bind to IL-13Rα1 and IL-4Rα with approximately the same affinity as wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments thereof of the present disclosure may bind to common gamma chain and IL-4Rα with approximately the same affinity as wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments thereof of the present disclosure may bind to IL-13Rα1, common gamma chain and IL-4Rα with approximately the same affinity as wild-type IL4.

[0108] In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure bind to the IL4 receptor subunit with an affinity at least equal to that of the wild-type IL4 sequence. For example, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to the interleukin 13 receptor alpha 1 (IL-13Rα1), common gamma chain, interleukin 4 receptor alpha (IL-4Rα), or a combination thereof with an affinity at least equal to that of the wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to the IL-13Rα1 with an affinity at least equal to that of the wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to the common gamma chain with an affinity at least equal to that of the wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to the IL-4Rα with an affinity at least equal to that of the wild-type IL4 sequence. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and common gamma chain with an affinity at least equal to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and IL-4Rα with an affinity at least equal to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to common gamma chain and IL-4Rα with an affinity at least equal to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1, common gamma chain and IL-4Rα with an affinity at least equal to that of wild-type IL4.

[0109] In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure bind to the IL4 receptor subunit with an affinity equal to or less than the wild-type IL4 sequence. For example, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to interleukin-13 receptor alpha 1 (IL-13Rα1), common gamma chain, interleukin-4 receptor alpha (IL-4Rα), or a combination thereof with an affinity equal to or less than the wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 with an affinity equal to or less than the wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to common gamma chain with an affinity equal to or less than the wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to IL-4Rα with an affinity equal to or less than the wild-type IL4 sequence. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and common gamma chain with an affinity equal to or less than that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1 and IL-4Rα with an affinity equal to or less than that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to common gamma chain and IL-4Rα with an affinity equal to or less than that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may bind to IL-13Rα1, common gamma chain and IL-4Rα with an affinity equal to or less than that of wild-type IL4.

[0110] In some embodiments, IL4 or IL4 variants, derivatives or fragments thereof may bind to IL-13Rα1 with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL4 sequence. In some embodiments, IL4 or IL4 variants, derivatives or fragments thereof may bind to IL-13Rα1 with no more than about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL4 sequence.

[0111] In some embodiments, the IL4 or IL4 variant, derivative or fragment thereof may bind to the common gamma chain with at least about 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1000 or 10,000 times greater affinity than the wild-type IL4 sequence. In some embodiments, the IL4 or IL4 variant, derivative or fragment thereof may bind to the common gamma chain with about 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1000 or 10,000 times less affinity than the wild-type IL4 sequence.

[0112] In some embodiments, the IL4 or IL4 variant, derivative or fragment thereof may bind to IL-4Rα with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL4 sequence. In some embodiments, the IL4 or IL4 variant, derivative or fragment thereof may bind to IL-4Rα with no more than about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL4 sequence.

[0113] In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may activate the native IL4 receptor. The native IL4 receptor may be, for example, a receptor that includes an IL-13Rα1 subunit and an IL-4Rα subunit, or a common gamma chain subunit and an IL-4Rα subunit. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may activate the native IL4 receptor when present in a fusion protein. In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure may activate the native IL4 receptor when present as a polypeptide that is not part of a fusion protein, but do not activate the native IL4 receptor when present in a fusion protein.

[0114] In some embodiments, the polypeptides of the present disclosure do not contain IL4. In some embodiments, the polypeptides of the present disclosure do not contain SEQ ID NO:1. Interleukin 10 The fusion protein may comprise an IL10 protein, or a variant, derivative or fragment thereof, operably linked to, or directly or indirectly fused with, an interleukin-10 or variant or derivative thereof. The IL10 protein is preferably a mammalian IL10 protein, e.g., human IL10, or murine IL10. One amino acid sequence representing IL10 is shown in SEQ ID NO:5. Variants of IL10 include proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher, e.g., 100%, amino acid sequence identity, preferably over the entire length, with SEQ ID NO:5. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman-Wunsch algorithm and the GAP default parameters as defined above. Variants, derivatives and fragments thereof also include proteins having IL10 activity derived by one or more amino acid substitutions, deletions or insertions from a polypeptide having the amino acid sequence of SEQ ID NO: 5. Preferably, such proteins contain 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.

[0115] In some embodiments, the IL10 of the present disclosure (e.g., an IL10 variant, derivative or fragment thereof) may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions, deletions or insertions compared to an IL10 sequence disclosed herein (e.g., a wild-type IL10 sequence).

[0116] In some embodiments, an IL10 of the present disclosure (e.g., an IL10 variant, derivative or fragment thereof) may include no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid substitutions, deletions or insertions compared to an IL10 sequence disclosed herein (e.g., a wild-type IL10 sequence).

[0117] In some embodiments, the IL10 sequences of the disclosure (e.g., IL10 variants, derivatives or fragments thereof) have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-21, 3-22, 3-23, 3-24, 3-25, 3-26, 3-27, 3-28, 3-29, 3-30, 3-31, 3-32, 3-33, 3-34, 3-35, 3-36, 3-37, 3-38, 3-39, 3-40, 3-41, 3-42, 3-43, 3-44, 3-45, 3-46, 3-47, 3-48, 3-49, 3-50, 3-51, 3-52, 3-53, 3-54, 3-55, 3-56, 3-57, 3-58, 3-59, 3-60, 3-61, 3-62, 3-63, 3-64, 3-65, 3-66, 3-67, 3-68, 3-69, 3-70, 3-71, , 2 to 9, 2 to 10, 2 to 15, 2 to 20, 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20 or 20 to 25 amino acid substitutions, deletions or insertions.

[0118] In some embodiments, the IL10 sequence of the present disclosure (e.g., an IL10 variant, derivative or fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions, deletions or insertions compared to an IL10 sequence disclosed herein (e.g., a wild-type IL10 sequence). The amino acid substitutions may be conservative or non-conservative substitutions. One or more amino acid substitutions, deletions or insertions may be at the N-terminus, C-terminus, or combinations thereof within the amino acid sequence. The amino acid substitutions, deletions or insertions may be contiguous, non-contiguous, or combinations thereof.

[0119] The IL10 of the present disclosure may include a wild-type IL10 sequence. A non-limiting example of a wild-type IL10 sequence is SEQ ID NO: 5. SEQ ID NO: 5 may be the reference wild-type IL10 sequence of the present disclosure.

[0120] The IL10 of the present disclosure may include IL10 variants, derivatives or fragments thereof with one or more amino acid substitutions. For example, the IL10 variants, derivatives or fragments thereof may include amino acid substitutions at positions I87, A89, H109, ​​R110, F111, Y153, M156 or combinations thereof in SEQ ID NO:5. In some embodiments, the IL10 variants, derivatives or fragments thereof include substitutions that are M156, F111S, I87A, I87G, A89D, Hl09D, R110D, Yl53D, Ml56D, A89D, Hl09E, R110E, Yl53E, Ml56E or combinations thereof compared to SEQ ID NO:5.

[0121] In some embodiments, the IL10 variant, derivative or fragment thereof does not contain a substitution at position I87, A89, H109, ​​R110, F111, Y153 or M156 compared to SEQ ID NO: 5. In some embodiments, the IL10 variant, derivative or fragment thereof does not contain a M156, F111S, I87A, I87G, A89D, Hl09D, R110D, Yl53D, Ml56D, A89D, Hl09E, R110E, Yl53E or Ml56E substitution.

[0122] In some embodiments, the disclosed IL10 or IL10 variants, derivatives or fragments thereof bind to the IL10 receptor subunit with an affinity approximately equivalent to that of the wild-type IL10 sequence. Equivalent affinity can be, for example, less than about 10-fold, less than about 5-fold, less than about 2-fold, less than about 1.9-fold, less than about 1.8-fold, less than about 1.7-fold, less than about 1.6-fold, less than about 1.5-fold, less than about 1.4-fold, less than about 1.3-fold, less than about 1.2-fold or less than about 1.1-fold affinity compared to the wild-type IL10 sequence. Comparable affinity can be, for example, greater than about 10-fold, greater than about 5-fold, greater than about 2-fold, greater than about 1.9-fold, greater than about 1.8-fold, greater than about 1.7-fold, greater than about 1.6-fold, greater than about 1.5-fold, greater than about 1.4-fold, greater than about 1.3-fold, greater than about 1.2-fold, or greater than about 1.1-fold affinity compared to the wild-type IL10 sequence.

[0123] For example, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to interleukin 10 receptor 1 (IL-10R1), interleukin 10 receptor 2 (IL-10R2), or a combination thereof with an affinity approximately equivalent to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R1 with an affinity approximately equivalent to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R2 with an affinity approximately equivalent to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R1 and IL-10R2 with an affinity approximately equivalent to that of wild-type IL10.

[0124] In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to the IL10 receptor subunit with at least the same affinity as wild-type IL10. For example, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R1, IL-10R2 or a combination thereof with at least the same affinity as wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R1 with at least the same affinity as wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R2 with at least the same affinity as wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R1 and IL-10R2 with at least the same affinity as wild-type IL10.

[0125] In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to the IL10 receptor subunit with an affinity equal to or less than that of wild-type IL10. For example, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R1, IL-10R2 or a combination thereof with an affinity equal to or less than that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R1 with an affinity equal to or less than that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R2 with an affinity equal to or less than that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may bind to IL-10R1 and IL-10R2 with an affinity equal to or less than that of wild-type IL10.

[0126] In some embodiments, IL10 or IL10 variants, derivatives or fragments thereof may bind to IL-10R1 with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL10 sequence. In some embodiments, IL10 or IL10 variants, derivatives or fragments thereof may bind to IL-10R1 with no more than about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL10 sequence.

[0127] In some embodiments, IL10 or IL10 variants, derivatives or fragments thereof may bind to IL-10R2 with at least about 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1000 or 10,000 times greater affinity than the wild-type IL10 sequence. In some embodiments, IL10 or IL10 variants, derivatives or fragments thereof may bind to IL-10R2 with about 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1000 or 10,000 times less affinity than the wild-type IL10 sequence.

[0128] In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may activate native IL10 receptor. Native IL10 receptor may be, for example, a receptor that includes IL-10R1 and IL-10R2 subunits. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may activate native IL10 receptor when present in a fusion protein. In some embodiments, the IL10 or IL10 variants, derivatives or fragments of the present disclosure may activate native IL10 receptor when present as a polypeptide that is not part of a fusion protein, but do not activate native IL10 receptor when present in a fusion protein.

[0129] In some embodiments, the polypeptides of the present disclosure do not contain IL10. In some embodiments, the polypeptides of the present disclosure do not contain SEQ ID NO:5. Interleukin 33 The fusion protein may comprise an IL33 protein, or a variant, derivative or fragment thereof, operably linked to, or directly or indirectly fused to, an interleukin-13 or variant or derivative thereof. The IL33 protein is preferably a mammalian IL33 protein, e.g., human IL33, or mouse IL33. Non-limiting examples of amino acid sequences representing IL33 include SEQ ID NO:6 and SEQ ID NOs:29-34. Variants of IL33 include proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher, e.g., 100%, amino acid sequence identity, preferably over the entire length, with any one of SEQ ID NO:6 or SEQ ID NOs:29-34. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman-Wunsch algorithm and the GAP default parameters defined above. Also included are variants, derivatives and fragments thereof, which have IL33 activity and are derived from a polypeptide having the amino acid sequence of SEQ ID NO:6 or any one of SEQ ID NOs:29 to 34 by one or more amino acid substitutions, deletions or insertions. Preferably, such proteins contain 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.

[0130] In some embodiments, an IL33 of the disclosure (e.g., an IL33 variant, derivative or fragment thereof) may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions, deletions or insertions compared to an IL33 sequence disclosed herein (e.g., a wild-type IL33 sequence).

[0131] In some embodiments, an IL33 of the disclosure (e.g., an IL33 variant, derivative or fragment thereof) may include no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid substitutions, deletions or insertions compared to an IL33 sequence disclosed herein (e.g., a wild-type IL33 sequence).

[0132] In some embodiments, an IL33 sequence of the disclosure (e.g., an IL33 variant, derivative or fragment thereof) has 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 30, 1 to 40, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, or more amino acids relative to an IL33 sequence disclosed herein (e.g., a wild-type IL33 sequence). , 2 to 9, 2 to 10, 2 to 15, 2 to 20, 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20 or 20 to 25 amino acid substitutions, deletions or insertions.

[0133] In some embodiments, the IL33 sequence of the present disclosure (e.g., an IL33 variant, derivative or fragment thereof) may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions, deletions or insertions compared to the IL33 sequence disclosed herein (e.g., a wild-type IL33 sequence). The amino acid substitutions may be conservative or non-conservative substitutions. One or more amino acid substitutions, deletions or insertions may be at the N-terminus, C-terminus, or combinations thereof within the amino acid sequence. The amino acid substitutions, deletions or insertions may be contiguous, non-contiguous, or combinations thereof.

[0134] The IL33 of the present disclosure may include a wild-type IL33 sequence. Non-limiting examples of wild-type IL33 sequences include SEQ ID NOs: 6 and 29 to 34. SEQ ID NO: 6 may be a reference wild-type IL33 sequence.

[0135] The IL33 of the present disclosure may include IL33 variants, derivatives or fragments thereof with one or more amino acid substitutions.For example, IL33 variants, derivatives or fragments thereof may include an amino acid substitution at position I263 of SEQ ID NO:6.In some embodiments, IL33 variants, derivatives or fragments thereof include a substitution of I263M compared to SEQ ID NO:6.

[0136] In some embodiments, the IL33 variant, derivative or fragment thereof does not contain a substitution at position I263 compared to SEQ ID NO: 6. In some embodiments, the IL33 variant, derivative or fragment thereof does not contain a I263M substitution.

[0137] In some embodiments, the disclosed IL33 or IL33 variants, derivatives or fragments thereof bind to the IL33 receptor subunit with an affinity approximately equivalent to that of the wild-type IL33 sequence. The equivalent affinity can be, for example, less than about 10-fold, less than about 5-fold, less than about 2-fold, less than about 1.9-fold, less than about 1.8-fold, less than about 1.7-fold, less than about 1.6-fold, less than about 1.5-fold, less than about 1.4-fold, less than about 1.3-fold, less than about 1.2-fold, or less than about 1.1-fold affinity compared to the wild-type IL33 sequence. Comparable affinity can be, for example, greater than about 10-fold, greater than about 5-fold, greater than about 2-fold, greater than about 1.9-fold, greater than about 1.8-fold, greater than about 1.7-fold, greater than about 1.6-fold, greater than about 1.5-fold, greater than about 1.4-fold, greater than about 1.3-fold, greater than about 1.2-fold, or greater than about 1.1-fold affinity compared to a wild-type IL33 sequence.

[0138] For example, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to ST2 (IL1RL1), IL1RAP or a combination thereof with an affinity approximately equivalent to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to ST2 with an affinity approximately equivalent to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to IL1RAP with an affinity approximately equivalent to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to ST2 and IL1RAP with an affinity approximately equivalent to that of wild-type IL33.

[0139] In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to the IL33 receptor subunit with an affinity at least equal to that of wild-type IL33. For example, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to ST2, IL1RAP or a combination thereof with an affinity at least equal to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to ST2 with an affinity at least equal to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to IL1RAP with an affinity at least equal to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to ST2 and IL1RAP with an affinity at least equal to that of wild-type IL33.

[0140] In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to the IL33 receptor subunit with an affinity equal to or less than that of wild-type IL33. For example, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to ST2, IL1RAP or a combination thereof with an affinity equal to or less than that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to ST2 with an affinity equal to or less than that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to IL1RAP with an affinity equal to or less than that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may bind to ST2 and IL1RAP with an affinity equal to or less than that of wild-type IL33.

[0141] In some embodiments, IL33 or IL33 variants, derivatives or fragments thereof may bind to ST2 with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL33 sequence. In some embodiments, IL33 or IL33 variants, derivatives or fragments thereof may bind to ST2 with about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold less affinity than the wild-type IL33 sequence.

[0142] In some embodiments, IL33 or IL33 variants, derivatives or fragments thereof may bind to IL1RAP with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold greater affinity than the wild-type IL33 sequence. In some embodiments, IL33 or IL33 variants, derivatives or fragments thereof may bind to IL1RAP with about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or 10,000-fold less affinity than the wild-type IL33 sequence.

[0143] In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may activate native IL33 receptor. Native IL33 receptor may be, for example, a receptor that includes ST2 subunit and IL1RAP subunit. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may activate native IL33 receptor when present in a fusion protein. In some embodiments, the IL33 or IL33 variants, derivatives or fragments of the present disclosure may activate native IL33 receptor when present as a polypeptide that is not part of a fusion protein, but do not activate native IL33 receptor when present in a fusion protein.

[0144] Transforming growth factor beta 1 The fusion protein may comprise a TGFβ1 protein, or a variant, derivative, or fragment thereof, operably linked to or directly or indirectly fused with interleukin 13 or a variant or derivative thereof. The TGFβ1 protein is preferably a mammalian TGFβ1 protein (e.g., human TGFβ1 or mouse TGFβ1). Non-limiting examples of amino acid sequences representing TGFβ1 include SEQ ID NO:7 and SEQ ID NO:21. Variants of TGFβ1 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher (e.g., 100%) amino acid sequence identity with SEQ ID NO:7 (entire or underlined) or SEQ ID NO:21, preferably over the entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and the GAP default parameters defined above. The variants, derivatives or fragments also include proteins having TGFβ1 activity that are derived by one or more amino acid substitutions, deletions or insertions from a polypeptide having the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:21. Preferably, such proteins contain 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.

[0145] In some embodiments, the TGFβ1 of the present disclosure (e.g., a TGFβ1 variant, derivative, or fragment thereof) may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions, deletions, or insertions relative to a TGFβ1 sequence disclosed herein (e.g., a wild-type TGFβ1 sequence).

[0146] In some embodiments, the TGFβ1 of the present disclosure (e.g., a TGFβ1 variant, derivative, or fragment thereof) may include no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid substitutions, deletions, or insertions relative to a TGFβ1 sequence disclosed herein (e.g., a wild-type TGFβ1 sequence).

[0147] In some embodiments, a TGFβ1 sequence of the disclosure (e.g., a TGFβ1 variant, derivative, or fragment thereof) may be 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 3-10, 3-15 ... The amino acid sequence may include substitutions, deletions, or insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 51 ...5, 56, 57, 58, 59, 51, 51, 52,

[0148] In some embodiments, the TGFβ1 sequence of the present disclosure (e.g., its TGFβ1 variant, derivative, or fragment) may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to the TGFβ1 sequence disclosed herein (e.g., wild-type TGFβ1 sequence). The amino acid substitutions may be conservative or non-conservative. The substitutions, deletions, or insertions of one or more amino acids may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions, deletions, or insertions of amino acids may be consecutive, non-consecutive, or a combination thereof.

[0149] The TGFβ1 of the present disclosure may include a wild-type TGFβ1 sequence. Non-limiting examples of wild-type TGFβ1 sequences include SEQ ID NO: 7 and 21. The canonical TGFβ1 sequence may be SEQ ID NO: 21.

[0150] In some embodiments, the TGFβ1 of the present disclosure, or its TGFβ1 variants, derivatives, or fragments, binds to the TGFβ1 receptor subunit with an affinity similar to that of the wild-type TGFβ1 sequence. Similar affinity can be, for example, less than about 10-fold, less than about 5-fold, less than about 2-fold, less than about 1.9-fold, less than about 1.8-fold, less than about 1.7-fold, less than about 1.6-fold, less than about 1.5-fold, less than about 1.4-fold, less than about 1.3-fold, less than about 1.2-fold, or less than about 1.1-fold affinity compared to the wild-type TGFβ1 sequence. Equivalent affinity can be, for example, greater than about 10-fold, greater than about 5-fold, greater than about 2-fold, greater than about 1.9-fold, greater than about 1.8-fold, greater than about 1.7-fold, greater than about 1.6-fold, greater than about 1.5-fold, greater than about 1.4-fold, greater than about 1.3-fold, greater than about 1.2-fold, or greater than about 1.1-fold affinity compared to the wild-type TGFβ1 sequence.

[0151] For example, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to transforming growth factor beta receptor 1 (TGFβR1), transforming growth factor beta receptor 2 (TGFβ2), activin receptor-like kinase 1 (ALK-1), activin receptor-like kinase 2 (ALK-2), or a combination thereof, with an affinity similar to that of wild-type TGFβ1. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR1 with an affinity similar to that of wild-type TGFβ1. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR2 with an affinity similar to that of wild-type TGFβ1. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to ALK-1 with an affinity similar to that of the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to ALK-2 with approximately the same affinity as the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with approximately the same affinity as the wild-type TGFβ1 sequence.

[0152] In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to transforming growth factor beta receptor 1 (TGFβR1), transforming growth factor beta receptor 2 (TGFβ2), activin receptor-like kinase 1 (ALK-1), activin receptor-like kinase 2 (ALK-2), or a combination thereof, with an affinity at least equivalent to that of wild-type TGFβ1. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR1 with an affinity at least equivalent to that of wild-type TGFβ1. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR2 with an affinity at least equivalent to that of wild-type TGFβ1. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to ALK-1 with an affinity at least equivalent to that of the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to ALK-2 with an affinity at least equivalent to that of the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβ2, ALK-1, and ALK-2 with an affinity at least equivalent to that of the wild-type TGFβ1 sequence.

[0153] In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to transforming growth factor beta receptor 1 (TGFβR1), transforming growth factor beta receptor 2 (TGFβ2), activin receptor-like kinase 1 (ALK-1), activin receptor-like kinase 2 (ALK-2), or a combination thereof with an affinity equal to or less than that of wild-type TGFβ1. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR1 with an affinity equal to or less than that of wild-type TGFβ1. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR2 with an affinity equal to or less than that of wild-type TGFβ1. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to ALK-1 with an affinity equal to or less than that of the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to ALK-2 with an affinity equal to or less than the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 of the present disclosure, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with an affinity equal to or less than the wild-type TGFβ1 sequence.

[0154] In some embodiments, TGFβ1, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβR2, ALK-1, or ALK-2 with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold greater affinity than the wild-type TGFβ1 sequence. In some embodiments, TGFβ1, or a TGFβ1 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβR2, ALK-1, or ALK-2 with an affinity that is about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold less than the wild-type TGFβ1 sequence.

[0155] In some embodiments, the TGFβ1 of the present disclosure, or its TGFβ1 variant, derivative, or fragment, can activate native TGFβ1 receptor. The native TGFβ1 receptor can be, for example, a receptor that includes a TGFβR1 subunit and a TGFβR2 subunit. In some embodiments, the TGFβ1 of the present disclosure, or its TGFβ1 variant, derivative, or fragment, can activate native TGFβ1 receptor when present in a fusion protein. In some embodiments, the TGFβ1 of the present disclosure, or its TGFβ1 variant, derivative, or fragment, can activate native TGFβ1 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate native TGFβ1 receptor when present in a fusion protein.

[0156] Transforming growth factor beta 2 The fusion protein may comprise a TGFβ2 protein, or a variant, derivative, or fragment thereof, operably linked or directly or indirectly fused to interleukin 13 or a variant or derivative thereof. The TGFβ2 protein is preferably a mammalian TGFβ2 protein (e.g., human TGFβ2 or mouse TGFβ2). Non-limiting examples of amino acid sequences representing TGFβ2 include SEQ ID NO:8, SEQ ID NO:22, and SEQ ID NO:35. Variants of TGFβ2 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or higher (e.g., 100%) amino acid sequence identity, preferably over the entire length, with SEQ ID NO:8 (all or underlined), SEQ ID NO:22, or SEQ ID NO:35. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and the GAP default parameters defined above. The variants, derivatives and fragments also include proteins having TGFβ2 activity that are derived by one or more amino acid substitutions, deletions or insertions from a polypeptide having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:22, or SEQ ID NO:35. Preferably, such proteins contain 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.

[0157] In some embodiments, a TGFβ2 of the present disclosure (e.g., a TGFβ2 variant, derivative, or fragment thereof) may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions, deletions, or insertions relative to a TGFβ2 sequence disclosed herein (e.g., a wild-type TGFβ2 sequence).

[0158] In some embodiments, a TGFβ2 of the present disclosure (e.g., a TGFβ2 variant, derivative, or fragment thereof) may include no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid substitutions, deletions, or insertions relative to a TGFβ2 sequence disclosed herein (e.g., a wild-type TGFβ2 sequence).

[0159] In some embodiments, a TGFβ2 sequence of the disclosure (e.g., a TGFβ2 variant, derivative, or fragment thereof) may be 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 3-10, 3-15 ... The amino acid sequence may include substitutions, deletions, or insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 51 ...5, 56, 57, 58, 59, 51, 51, 52,

[0160] In some embodiments, the TGFβ2 of the present disclosure (e.g., TGFβ2 variants, derivatives, or fragments thereof) may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to the TGFβ2 sequence disclosed herein (e.g., wild-type TGFβ2 sequence). The amino acid substitutions may be conservative or non-conservative. The substitutions, deletions, or insertions of one or more amino acids may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions, deletions, or insertions of amino acids may be consecutive, non-consecutive, or a combination thereof.

[0161] The TGFβ2 of the present disclosure may include a wild-type TGFβ2 sequence. Non-limiting examples of wild-type TGFβ2 sequences include SEQ ID NOs: 8, 22, and 35. SEQ ID NO: 22 may be the canonical wild-type TGFβ2 sequence of the present disclosure.

[0162] The TGFβ2 of the present disclosure may include a TGFβ2 variant, derivative, or fragment thereof, which has one or more amino acid substitutions.For example, the TGFβ2 variant, derivative, or fragment may include an amino acid substitution at position R18, position P36, or a combination thereof of SEQ ID NO:22.In some embodiments, the TGFβ2 variant, derivative, or fragment includes a substitution that is R18C, P36H, or a combination thereof with respect to SEQ ID NO:22.In some embodiments, the TGFβ2, fragment, or derivative thereof includes substitutions R18C and P36H with respect to SEQ ID NO:22.

[0163] In some embodiments, the TGFβ2 variant, derivative, or fragment does not comprise a substitution at position R18 or P36 relative to SEQ ID NO: 22. In some embodiments, the TGFβ2 variant, derivative, or fragment does not comprise an R18C substitution or a P36H substitution.

[0164] In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure bind to the TGFβ2 receptor subunit with an affinity approximately equivalent to that of the wild-type TGFβ2 sequence. Equivalent affinity can be, for example, less than about 10-fold, less than about 5-fold, less than about 2-fold, less than about 1.9-fold, less than about 1.8-fold, less than about 1.7-fold, less than about 1.6-fold, less than about 1.5-fold, less than about 1.4-fold, less than about 1.3-fold, less than about 1.2-fold, or less than about 1.1-fold affinity compared to the wild-type TGFβ2 sequence. Comparable affinity can be, for example, greater than about 10-fold, greater than about 5-fold, greater than about 2-fold, greater than about 1.9-fold, greater than about 1.8-fold, greater than about 1.7-fold, greater than about 1.6-fold, greater than about 1.5-fold, greater than about 1.4-fold, greater than about 1.3-fold, greater than about 1.2-fold, or greater than about 1.1-fold affinity compared to the wild-type TGFβ2 sequence.

[0165] For example, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to transforming growth factor beta receptor 1 (TGFβR1), transforming growth factor beta receptor 2 (TGFβ2), activin receptor-like kinase 1 (ALK-1), activin receptor-like kinase 2 (ALK-2), or a combination thereof, with approximately the same affinity as wild-type TGFβ2. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR1 with approximately the same affinity as wild-type TGFβ2. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR2 with approximately the same affinity as wild-type TGFβ2. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to ALK-1 with approximately the same affinity as wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to ALK-2 with approximately the same affinity as the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with approximately the same affinity as the wild-type TGFβ2 sequence.

[0166] In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to transforming growth factor beta receptor 1 (TGFβR1), transforming growth factor beta receptor 2 (TGFβR2), activin receptor-like kinase 1 (ALK-1), activin receptor-like kinase 2 (ALK-2), or a combination thereof, with an affinity at least equivalent to that of wild-type TGFβ2. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR1 with an affinity at least equivalent to that of wild-type TGFβ2. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR2 with an affinity at least equivalent to that of wild-type TGFβ2. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to ALK-1 with an affinity at least equivalent to that of the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to ALK-2 with an affinity at least equivalent to that of the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with an affinity at least equivalent to that of the wild-type TGFβ2 sequence.

[0167] In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to transforming growth factor beta receptor 1 (TGFβR1), transforming growth factor beta receptor 2 (TGFβR2), activin receptor-like kinase 1 (ALK-1), activin receptor-like kinase 2 (ALK-2), or a combination thereof with an affinity equal to or less than that of wild-type TGFβ2. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR1 with an affinity equal to or less than that of wild-type TGFβ2. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR2 with an affinity equal to or less than that of wild-type TGFβ2. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to ALK-1 with an affinity equal to or less than that of the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to ALK-2 with an affinity equal to or less than the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 of the present disclosure, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with an affinity equal to or less than the wild-type TGFβ2 sequence.

[0168] In some embodiments, TGFβ2, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβR2, ALK-1, or ALK-2 with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold greater affinity than the wild-type TGFβ2 sequence. In some embodiments, TGFβ2, or a TGFβ2 variant, derivative, or fragment thereof, may bind to TGFβR1, TGFβR2, ALK-1, or ALK-2 with an affinity that is about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold less than the wild-type TGFβ2 sequence.

[0169] In some embodiments, the TGFβ2 of the present disclosure, or its TGFβ2 variant, derivative, or fragment, can activate native TGFβ2 receptor. The native TGFβ2 receptor can be, for example, a receptor that includes a TGFβR1 subunit and a TGFβR2 subunit. In some embodiments, the TGFβ2 of the present disclosure, or its TGFβ2 variant, derivative, or fragment, can activate native TGFβ2 receptor when present in a fusion protein. In some embodiments, the TGFβ2 of the present disclosure, or its TGFβ2 variant, derivative, or fragment, can activate native TGFβ2 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate native TGFβ2 receptor when present in a fusion protein.

[0170] Interleukin 27 The fusion protein may comprise an IL27 protein or a variant, derivative, or fragment thereof operably linked to or directly or indirectly fused with interleukin-13 or a variant or derivative thereof. The IL27 protein is preferably a mammalian IL27 protein (e.g., human IL27 or mouse IL27, or a variant, derivative, or fragment thereof). The IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may comprise an IL27A subunit, an IL27B (EBI3) subunit, or a combination thereof. In some embodiments, the IL27 of the present disclosure comprises an IL27A subunit. In some embodiments, the IL27 of the present disclosure comprises a variant IL27A subunit disclosed below (e.g., as set forth in SEQ ID NO: 18). In some embodiments, the IL27 of the present disclosure comprises an IL27B subunit.

[0171] An example of an amino acid sequence representing IL27A is set forth in SEQ ID NO: 36. An example of an amino acid sequence representing IL27B is set forth in SEQ ID NO: 45. Variants of IL27 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher (e.g., 100%) amino acid sequence identity, preferably over the entire length, with SEQ ID NO: 36 or SEQ ID NO: 45. The amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and the GAP default parameters defined above. The variants, derivatives, or fragments also include proteins having IL27 activity that are derived from the polypeptide having the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 45 by substitution, deletion, or insertion of one or more amino acids. Preferably, such proteins contain 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.

[0172] In some embodiments, an IL27 of the present disclosure (e.g., an IL27 variant, derivative, or fragment thereof) may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions, deletions, or insertions relative to an IL27 sequence disclosed herein (e.g., a wild-type IL27 sequence).

[0173] In some embodiments, an IL27 of the disclosure (e.g., an IL27 variant, derivative, or fragment thereof) may include no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid substitutions, deletions, or insertions relative to an IL27 sequence disclosed herein (e.g., a wild-type IL27 sequence).

[0174] In some embodiments, an IL27 sequence of the disclosure (e.g., an IL27 variant, derivative, or fragment thereof) may be 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-15, 3-20, 3-20, 3-30, 3-40, 3-30, 3-40, 3-40, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 3-30, 3-40, 3-40, 3-40, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-3 ...40, 3-40, 3- It may include substitutions, deletions, or insertions of 2 to 10, 2 to 15, 2 to 20, 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20, or 20 to 25 amino acids.

[0175] In some embodiments, the IL27 sequence of the present disclosure (e.g., an IL27 variant, derivative, or fragment thereof) may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to the IL27 sequence disclosed herein (e.g., a wild-type IL27 sequence). The amino acid substitutions may be conservative or non-conservative. The one or more amino acid substitutions, deletions, or insertions may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions may be consecutive, non-consecutive, or a combination thereof.

[0176] The IL27 of the present disclosure may comprise a wild-type IL27 sequence. Non-limiting examples of wild-type IL27 sequences include SEQ ID NO: 36 (IL27A) and SEQ ID NO: 45 (IL27B). SEQ ID NO: 36 may be the canonical wild-type IL27 sequence. In some embodiments, the IL27 sequence of the present disclosure comprises one substitution relative to the wild-type IL27 sequence.

[0177] An example of an IL27 variant, derivative, or fragment of the present disclosure is an IL27 variant sequence that can be secreted as a functional immunomodulatory monomeric protein, for example, an IL27A variant, derivative, or fragment that can be secreted and function as a functional immunomodulatory monomeric protein without the need for association with the IL27B (EBI3) subunit. One or more amino acid substitutions, deletions, or insertions can be introduced to generate such molecules. SEQ ID NO: 18 is an example of an IL27 variant, derivative, or fragment of the present disclosure that contains one amino acid substitution L134C (which is L162C in the sequence including the signal peptide) relative to SEQ ID NO: 36 and can be secreted as a functional immunomodulatory monomeric protein.

[0178] The IL27 of the present disclosure may include IL27 variants, derivatives, or fragments thereof, which have one or more amino acid substitutions. For example, the IL27 variants, derivatives, or fragments may include amino acid substitutions at positions F132, N132, L134, P135, E136, E137, L152, L153, ​​P154, or combinations thereof of SEQ ID NO:36. In some embodiments, the IL27 variant, derivative, or fragment is selected from the group consisting of F132C, N132C, L134C, P135C, E136C, E137C, L152C, L153C, P154C, F132D, N132D, L134D, P135D, E136D, E137D, L152D, L153D, P154D, F132E, N132E, L134E, P1 35E, E136E, E137E, L152E, L153E, P154E, F132R, N132R, L134R, P135R, E136R, E137R, L152R, L153R, P154R, F132K, N132K, L134K, P135K, E136K, E137K, L152K, L153K, P154K, S31A, L91P, or a combination thereof.

[0179] In some embodiments, the IL27 variant, derivative, or fragment does not comprise a substitution at positions F132, N132, L134, P135, E136, E137, L152, L153, ​​or P154 relative to SEQ ID NO: 36. In some embodiments, the IL27 variant, derivative, or fragment does not comprise a substitution at positions F132, N132, L134, P135, E136, E137, L152, L153, ​​or P154 relative to SEQ ID NO: 36. does not contain 5E, E136E, E137E, L152E, L153E, P154E, F132R, N132R, L134R, P135R, E136R, E137R, L152R, L153R, P154R, F132K, N132K, L134K, P135K, E136K, E137K, L152K, L153K, P154K, S31A, or L91P substitutions.

[0180] In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, binds to the IL27 receptor subunit with an affinity that is about equivalent to the wild-type IL27 sequence. The equivalent affinity can be, for example, less than about 10-fold, less than about 5-fold, less than about 2-fold, less than about 1.9-fold, less than about 1.8-fold, less than about 1.7-fold, less than about 1.6-fold, less than about 1.5-fold, less than about 1.4-fold, less than about 1.3-fold, less than about 1.2-fold, or less than about 1.1-fold affinity compared to the wild-type IL27 sequence. Comparable affinity can be, for example, greater than about 10-fold, greater than about 5-fold, greater than about 2-fold, greater than about 1.9-fold, greater than about 1.8-fold, greater than about 1.7-fold, greater than about 1.6-fold, greater than about 1.5-fold, greater than about 1.4-fold, greater than about 1.3-fold, greater than about 1.2-fold, or greater than about 1.1-fold affinity compared to the wild-type IL27 sequence.

[0181] For example, the IL27 of the present disclosure, or an IL271 variant, derivative, or fragment thereof, may bind to interleukin-27 receptor alpha (IL-27RA), gp130, or a combination thereof with approximately the same affinity as wild-type IL27. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27RA with approximately the same affinity as wild-type IL27. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to gp130 with approximately the same affinity as wild-type IL27. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27RA and gp130 with approximately the same affinity as wild-type IL27.

[0182] In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to the IL27 receptor subunit with an affinity at least equal to that of wild-type IL27. For example, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27RA, gp130, or a combination thereof with an affinity at least equal to that of wild-type IL27. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27RA with an affinity at least equal to that of wild-type IL27. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to gp130 with an affinity at least equal to that of wild-type IL27. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27RA and gp130 with an affinity at least equal to that of wild-type IL27.

[0183] In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to the IL27 receptor subunit with an affinity equal to or less than that of wild-type IL27. For example, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27RA, gp130, or a combination thereof with an affinity equal to or less than that of wild-type IL27. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27RA with an affinity equal to or less than that of wild-type IL27. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to gp130 with an affinity equal to or less than that of wild-type IL27. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27RA and gp130 with an affinity equal to or less than that of wild-type IL27.

[0184] In some embodiments, IL27, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27A with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold greater affinity than the wild-type IL27 sequence. In some embodiments, IL27, or an IL27 variant, derivative, or fragment thereof, may bind to IL-27RA with about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold less affinity than the wild-type IL27 sequence.

[0185] In some embodiments, IL27, or an IL27 variant, derivative, or fragment thereof, may bind to gp130A with at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold greater affinity than the wild-type IL27 sequence. In some embodiments, IL27, or an IL27 variant, derivative, or fragment thereof, may bind to gp130 with about 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold less affinity than the wild-type IL27 sequence.

[0186] In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, can activate the native IL27 receptor. The native IL27 receptor can be, for example, a receptor that includes an IL-27RA subunit and a gp130 subunit. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, can activate the native IL27 receptor when present in a fusion protein. In some embodiments, the IL27 of the present disclosure, or an IL27 variant, derivative, or fragment thereof, can activate the native IL27 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate the native IL27 receptor when present in a fusion protein.

[0187] Fusion proteins The present disclosure provides fusion proteins that include interleukin 13 (IL13) directly or indirectly linked to a regulatory cytokine (e.g., IL4, IL10, IL27, IL33, TGFβ1, TGFβ2, or another IL13).

[0188] In some embodiments, the fusion proteins of the present disclosure may bind to a receptor present on the surface of a cell to form a complex (e.g., a polypeptide chain) having about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 receptor subunits. In some embodiments, the fusion proteins of the present disclosure may bind to a receptor present on the surface of a cell to form a complex (e.g., a polypeptide chain) having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 receptor subunits. In some embodiments, the fusion proteins of the present disclosure may bind to a receptor present on the surface of a cell to form a complex (e.g., a polypeptide chain) having 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, or 12 or less receptor subunits.

[0189] In this fusion protein, IL4 (or IL10, or IL13, or IL27, or IL33, or TGFβ1, or TGFβ2) and IL13 may or may not be connected by a linker (e.g., a linker sequence) or a chemical spacer.

[0190] The linker can be a peptide. The linker can include a linker sequence, for example a linker peptide sequence. The linker sequence can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 amino acid residues in length.

[0191] The linker described herein may include flexible linkers or rigid linkers. Flexible linkers may have sequences that include stretches of glycine and serine residues. The small size of glycine and serine residues provides flexibility, allowing the movement of the connected functional domain. The incorporation of serine or threonine may form hydrogen bonds with water molecules, thereby reducing undesirable interactions between the linker and protein moieties and maintaining the stability of the linker in aqueous solution. Flexible linkers may also include additional amino acids (e.g., threonine and alanine) to maintain flexibility, and polar amino acids (e.g., lysine and glutamine) to improve solubility.

[0192] The flexible linker may comprise SEQ ID NO:3. The flexible linker may comprise repeats of SEQ ID NO:37 (GGGS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO:37. The flexible linker may comprise repeats of SEQ ID NO:38 (GGGGS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO:38. Several other types of flexible linkers may also be used, such as SEQ ID NO:40 (KESGSVSSEQLAQFRSLD) and SEQ ID NO:41 (EGKSSGSGSESKST). The SEQ ID NO:42 (GSAGSAAGSGEF) linker may also be used, in which large hydrophobic residues are minimized and good solubility in aqueous solution is maintained. The length of the flexible linker may be adjusted to allow proper folding or to achieve optimal biological activity of the fusion protein.

[0193] The rigid linker may have, for example, an alpha-helical structure. The alpha-helical rigid linker may act as a spacer between protein domains. The rigid linker may comprise a repeat of SEQ ID NO: 43 (EAAAK), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 43. The rigid linker may comprise a repeat of SEQ ID NO: 44 (EAAAR), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 44. The rigid linker may have a proline-rich sequence (XP)n, where X represents alanine, lysine, glutamine, or any amino acid. The presence of proline in the non-helical linker may increase rigidity and allow efficient separation of protein domains.

[0194] The linker of the present disclosure may include a non-peptide linker (e.g., a chemical linker). For example, two amino acid sequences of the present disclosure may be connected by a chemical linker. Each chemical linker of the present disclosure may be an alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, any of which may be optionally substituted. In some embodiments, the chemical linker of the present disclosure may be an ester, ether, amide, thioether, or polyethylene glycol (PEG). In some embodiments, the linker may reverse the order of the amino acid sequence in the compound, for example, so that the amino acid sequence connected by the linker is head-to-head instead of head-to-tail. Non-limiting examples of such linkers include diesters of dicarboxylic acids, such as oxalyl diesters, malonyl diesters, succinyl diesters, glutaryl diesters, adipyl diesters, pimetyl diesters, fumaryl diesters, maleyl diesters, phthalyl diesters, isophthalyl diesters, and terephthalyl diesters. Non-limiting examples of such linkers include diamides of dicarboxylic acids, such as oxalyl diamides, malonyl diamides, succinyl diamides, glutaryl diamides, adipyl diamides, pimetyl diamides, fumaryl diamides, maleyl diamides, phthalyl diamides, isophthalyl diamides, and terephthalyl diamides. Non-limiting examples of such linkers include: diamides of diamino linkers, such as ethylenediamine, 1,2-di(methylamino)ethane, 1,3-diaminopropane, 1,3-di(methylamino)propane, 1,4-di(methylamino)butane, 1,5-di(methylamino)pentane, 1,6-di(methylamino)hexane, and pipyrizine.Non-limiting examples of optional substituents include hydroxyl, sulfhydryl, halogen, amino, nitro, nitroso, cyano, azide, sulfoxide, sulfone, sulfonamide, carboxyl, carboxaldehyde, imine, alkyl, halo-alkyl, alkenyl, halo-alkenyl, alkynyl, halo-alkynyl, alkoxy, aryl, aryloxy, aralkyl, arylalkoxy, heterocyclyl, acyl, acyloxy, carbamate, amide, ureido, epoxy, and ester groups.

[0195] In some embodiments, the N-terminus of a polypeptide sequence may be linked to the N-terminus of another polypeptide. In some embodiments, the C-terminus of a polypeptide sequence may be linked to the C-terminus of another polypeptide.

[0196] In some embodiments, the fusion proteins of the present disclosure do not include a linker. The amino acids may include genetically encoded and non-genetically encoded amino acids. The amino acids may include naturally occurring and non-naturally occurring amino acids. The amino acids may be in the L- or D-form. The substitutions may include conservative and / or non-conservative amino acid substitutions. A conservative amino acid substitution may be a substitution of one amino acid with another amino acid with similar biochemical properties (e.g., charge, size, and / or hydrophobicity). A non-conservative amino acid substitution may be a substitution of one amino acid with another amino acid with different biochemical properties (e.g., charge, size, and / or hydrophobicity). A conservative amino acid change may be, for example, a substitution that has minimal effect on the secondary or tertiary structure of a polypeptide. A conservative amino acid change may be an amino acid change from one hydrophilic amino acid to another hydrophilic amino acid. Hydrophilic amino acids may include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K), and Arg (R). Conservative amino acid changes may be from one hydrophobic amino acid to another hydrophilic amino acid. Hydrophobic amino acids may include Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), and Pro (P). Conservative amino acid changes may be from one acidic amino acid to another acidic amino acid. Acidic amino acids may include Glu (E) and Asp (D). Conservative amino acid changes may be from one basic amino acid to another basic amino acid. Basic amino acids may include His (H), Arg (R), and Lys (K). A conservative amino acid change may be an amino acid change from one polar amino acid to another polar amino acid. Polar amino acids may include Asn (N), Gln (Q), Ser (S), and Thr (T). A conservative amino acid change may be an amino acid change from one non-polar amino acid to another non-polar amino acid. Non-polar amino acids may include Leu (L), Val (V), Ile (I), Met (M), Gly (G), and Ala (A).A conservative amino acid change may be an amino acid change from one aromatic amino acid to another aromatic amino acid. Aromatic amino acids may include Phe (F), Tyr (Y), and Trp (W). A conservative amino acid change may be an amino acid change from one aliphatic amino acid to another aliphatic amino acid. Aliphatic amino acids may include Ala (A), Val (V), Leu (L), and Ile (I). In some embodiments, a conservative amino acid substitution is an amino acid change from one amino acid to another within one of the following groups: Group I: ala, pro, gly, gln, asn, ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met, ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.

[0197] At the N-terminus and / or C-terminus of the fusion protein of the present invention, there may be an additional amino acid sequence (e.g., an affinity tag to facilitate purification). For example, at the C-terminus or N-terminus, there may be a polyhistidine-tag, a GST-tag, a FLAG-tag, a CBP-tag, an HA-tag, or a Myc-tag to facilitate purification. In some embodiments, the affinity tag is removed from the fusion protein of the present disclosure, e.g., after purification. In some embodiments, the fusion protein of the present disclosure does not include an affinity tag (e.g., the fusion protein can be purified by other methods). Additionally or alternatively, the fusion protein of the present invention may optionally include additional protein moieties (e.g., a targetable moiety, e.g., a protein moiety comprising one or more antibody Fc regions). In some embodiments, the fusion protein comprises an antibody Fc region. In some embodiments, the fusion protein comprises an extracellular matrix binding polypeptide.

[0198] IL4 (or IL10, or IL27, or IL33, or TGFβ1, or TGFβ2) may be located at the N-terminus of IL13 or at the C-terminus of IL13. In a preferred embodiment, the IL4 (or IL10, or IL27, or IL33, or TGFβ1, or TGFβ2) molecule is located at the N-terminus of the IL13 molecule.

[0199] In one embodiment, the fusion protein of the invention consists essentially of IL4 (or IL10, or IL27, or IL33, or TGFβ1, or TGFβ2) and IL13, optionally linked by a linker sequence.

[0200] In one embodiment, the fusion proteins of the invention prevent or reduce neuronal damage to primary sensory neurons cultured overnight in the presence of oxaliplatin or paclitaxel, as quantified by measuring neurite length after β3-tubulin staining.

[0201] In a preferred embodiment, the fusion proteins of the present disclosure are present in, purified into, and / or used in monomeric form. In one embodiment, the fusion proteins have a molecular weight of 30-37 kDa. In some embodiments, the fusion proteins of the present disclosure are present in, purified into, and / or used in multimeric form (e.g., dimeric or tetrameric). In some embodiments, the fusion proteins of the present disclosure are present in, purified into, and / or used as monomers, dimers, trimers, tetramers, multimers, or any combination thereof. The dimers, trimers, tetramers, or multimers may include covalently or non-covalently linked subunits.

[0202] In some embodiments, the IL4 / IL13 fusion proteins of the present disclosure exist as monomers, are purified into monomers, and / or are used as monomers. In some embodiments, the IL4 / IL13 fusion proteins of the present disclosure exist as dimers, are purified into dimers, and / or are used as dimers. In some embodiments, the IL4 / IL13 fusion proteins of the present disclosure exist as trimers, are purified into trimers, and / or are used as trimers. In some embodiments, the IL4 / IL13 fusion proteins of the present disclosure exist as tetramers, are purified into tetramers, and / or are used as tetramers. In some embodiments, the IL4 / IL13 fusion proteins of the present disclosure exist as multimers, are purified into multimers, and / or are used as multimers. In some embodiments, the IL4 / IL13 fusion proteins exist as monomers and dimers, are purified into monomers and dimers, and / or are used as monomers and dimers. In some embodiments, the IL4 / IL13 fusion protein exists as, is purified into, and / or is used as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof.

[0203] In some embodiments, the IL10 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as monomers. In some embodiments, the IL10 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as dimers. In some embodiments, the IL10 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as trimers. In some embodiments, the IL10 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as tetramers. In some embodiments, the IL10 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as multimers. In some embodiments, the IL10 / IL13 fusion protein exists as, is purified into, and / or is used as monomers and dimers. In some embodiments, the IL10 / IL13 fusion protein exists as, is purified into, and / or is used as monomers, dimers, trimers, tetramers, multimers, or any combination thereof.

[0204] In some embodiments, the IL13 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as monomers. In some embodiments, the IL13 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as dimers. In some embodiments, the IL13 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as trimers. In some embodiments, the IL13 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as tetramers. In some embodiments, the IL13 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as multimers. In some embodiments, the IL10 / IL13 fusion protein exists as, is purified into, and / or is used as monomers and dimers. In some embodiments, the IL10 / IL13 fusion protein exists as, is purified into, and / or is used as monomers, dimers, trimers, tetramers, multimers, or any combination thereof.

[0205] In some embodiments, the IL27 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as monomers. In some embodiments, the IL27 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as dimers. In some embodiments, the IL27 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as trimers. In some embodiments, the IL27 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as tetramers. In some embodiments, the IL27 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as multimers. In some embodiments, the IL27 / IL13 fusion protein exists as, is purified into, and / or is used as monomers and dimers. In some embodiments, the IL27 / IL13 fusion protein exists as, is purified into, and / or is used as monomers, dimers, trimers, tetramers, multimers, or any combination thereof.

[0206] In some embodiments, the IL33 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as monomers. In some embodiments, the IL33 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as dimers. In some embodiments, the IL33 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as trimers. In some embodiments, the IL33 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as tetramers. In some embodiments, the IL33 / IL13 fusion proteins of the present disclosure exist as, are purified into, and / or are used as multimers. In some embodiments, the IL33 / IL13 fusion protein exists as, is purified into, and / or is used as monomers and dimers. In some embodiments, the IL33 / IL13 fusion protein exists as, is purified into, and / or is used as monomers, dimers, trimers, tetramers, multimers, or any combination thereof.

[0207] In some embodiments, the TGFβ1 / IL13 fusion proteins of the present disclosure are present as monomers, purified into monomers, and / or used as monomers. In some embodiments, the TGFβ1 / IL13 fusion proteins of the present disclosure are present as dimers, purified into dimers, and / or used as dimers. In some embodiments, the TGFβ1 / IL13 fusion proteins of the present disclosure are present as trimers, purified into trimers, and / or used as trimers. In some embodiments, the TGFβ1 / IL13 fusion proteins of the present disclosure are present as tetramers, purified into tetramers, and / or used as tetramers. In some embodiments, the TGFβ1 / IL13 fusion proteins of the present disclosure are present as multimers, purified into multimers, and / or used as multimers. In some embodiments, the TGFβ1 / IL13 fusion protein exists as, is purified into, and / or is used as a monomer and a dimer. In some embodiments, the TGFβ1 / IL13 fusion protein exists as, is purified into, and / or is used as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof.

[0208] In some embodiments, the TGFβ2 / IL13 fusion proteins of the present disclosure are present as monomers, purified into monomers, and / or used as monomers. In some embodiments, the TGFβ2 / IL13 fusion proteins of the present disclosure are present as dimers, purified into dimers, and / or used as dimers. In some embodiments, the TGFβ2 / IL13 fusion proteins of the present disclosure are present as trimers, purified into trimers, and / or used as trimers. In some embodiments, the TGFβ2 / IL13 fusion proteins of the present disclosure are present as tetramers, purified into tetramers, and / or used as tetramers. In some embodiments, the TGFβ2 / IL13 fusion proteins of the present disclosure are present as multimers, purified into multimers, and / or used as multimers. In some embodiments, the TGFβ2 / IL13 fusion protein exists as, is purified into, and / or is used as monomers and dimers. In some embodiments, the TGFβ2 / IL13 fusion protein exists as, is purified into, and / or is used as monomers, dimers, trimers, tetramers, multimers, or any combination thereof.

[0209] Manufacturing method The fusion protein of the present invention can be prepared by techniques that are routine for those skilled in the art.For example, the fusion protein can be prepared using techniques that allow the production of recombinant fusion protein by continuous cell lines in culture.For example, the fusion protein of the present invention can be produced in host cell transfectomas using a combination of recombinant DNA technology and gene transfection methods.

[0210] For example, to express the fusion protein of the invention, a nucleic acid molecule encoding the fusion protein of the invention can be prepared by standard molecular biology techniques. The nucleic acid molecule of the invention is preferably operably linked to a transcriptional regulatory sequence, such as a promoter, and optionally to a 3' untranslated region. The nucleic acid molecule of the invention can be inserted into a vector, such as an expression vector, such that the gene is operably linked to a transcriptional and translational regulatory sequence. The expression vector and the transcriptional regulatory sequence are selected to be compatible with the expression host cell used. The nucleic acid molecule encoding the fusion protein of the invention can be inserted into an expression vector by routine methods. The nucleic acid molecule or vector of the invention can further comprise a nucleotide sequence encoding a signal peptide, which can facilitate secretion of the fusion protein from a host cell. The nucleotide sequence encoding the signal peptide can be operably linked to the nucleic acid molecule of the invention. Preferably, the signal peptide is located at the amino terminus of the fusion protein of the invention, and therefore the nucleotide sequence encoding the signal peptide can be located 5' of the nucleic acid molecule encoding the fusion protein of the invention. The signal peptide can be a cytokine signal peptide or a signal peptide from a non-cytokine protein. The signal peptide may not be present in the mature form of the fusion protein. The promoter may be constitutive or inducible. The vector may contain a selectable marker for selection of vector-carrying host cells. The vector may contain an origin of replication if the vector is replicable.

[0211] The fusion proteins of the present invention may be synthesized de novo by chemical synthesis (e.g., using a peptide synthesizer provided by Applied Biosystems) or may be produced by a recombinant host cell by expressing a nucleic acid sequence encoding the fusion protein, fragment derivative, or variant. The variants and fragments are preferably functional, i.e., bind to at least one, two, three, four, or all of the corresponding membrane receptors or receptor subunits, and have IL4 (or IL10, or IL27, or IL33, or TGFβ1, or TGFβ2) activity and / or IL13 activity, preferably IL4 (or IL10, or IL27, or IL33, or TGFβ1, or TGFβ2) activity and IL13 activity.

[0212] The functional activity of IL4 (or IL10, or IL27, or IL33, or TGFβ1, or TGFβ2) and IL13, as well as the IL4 (or IL10, or IL27, or IL33, or TGFβ1, or TGFβ2) activity and IL13 constituting the fusion protein, can be determined using routine methods known to those skilled in the art. For example, a suitable assay for the functionality of IL4, and IL14 (or IL10) and IL13 constituting the fusion protein is lipopolysaccharide (LPS)-induced cytokine release (IL1, IL6, IL8, TNFα) in whole blood, optionally in the presence of anti-IL10 antibodies. Functional activity can also be determined by evaluating the activity of intracellular signaling pathways upon incubation of target cells with the fusion protein in the presence or absence of blocking antibodies against either the cytokine portion of the fusion protein or its receptor.

[0213] In another aspect, isolated nucleic acid sequences (e.g., cDNA sequences, genomic DNA sequences, and RNA sequences) are provided that code for any of the above fusion proteins. Due to the degeneracy of the genetic code, various nucleic acid sequences may code for the same amino acid sequence. Any nucleic acid sequence that codes for the fusion protein of the present invention is referred to herein as an "IL4 / IL13 (or IL10 / IL13, IL27 / IL13, IL33 / IL13, TGFβ1 / IL13, TGFβ2 / IL13, IL13 / IL13)-encoding nucleic acid sequence." The nucleic acid sequences provided include recombinant, artificial, or synthetic nucleic acid sequences. When a sequence is depicted as a DNA sequence and at the same time refers to RNA, it is understood that the actual base sequence of the RNA molecule is the same with the difference that thymine (T) is replaced by uracil (U). The nucleic acid sequences of the present invention are particularly useful for expressing the IL4 / IL13 (or IL10 / IL13, IL27 / IL13, IL33 / IL13, TGFβ1 / IL13, TGFβ2 / IL13, IL13 / IL13) fusion proteins of the present invention, for the production of any of these proteins, or for gene therapy purposes.

[0214] The nucleic acid sequence, particularly the DNA sequence, encoding the IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / 13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein of the present invention can be inserted into an expression vector to produce (e.g., large amounts of) the IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / 13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein. Suitable vectors include, but are not limited to, linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and the like. Non-limiting examples of viral vectors include retroviruses, adenoviruses, and adeno-associated viruses.

[0215] Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter, can be used.

[0216] In addition to the nucleic acid molecule encoding the IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein and the regulatory sequence, the recombinant expression vector of the present invention may carry additional sequences, such as sequences that regulate the replication of the vector in a host cell (e.g., an origin of replication), and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, a typical selectable marker gene confers resistance to drugs (e.g., G418, hygromycin, or methotrexate) on the 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, recombinant expression vectors can contain genes encoding glycosyltransferases, in addition to nucleic acid sequences encoding the fusion proteins of the invention.

[0217] In another aspect, the present invention relates to a host cell comprising a nucleic acid sequence of the present invention or comprising a nucleic acid construct or vector comprising a nucleic acid sequence of the present invention. The host cell can be any host cell. The host cell can be selected from a prokaryotic cell and a eukaryotic cell. The host cell can also be a cell line, such as a prokaryotic or eukaryotic cell line. The host cell is preferably an animal cell or an animal cell line, such as a mammalian cell or a mammalian cell line.

[0218] In one embodiment, the fusion protein of the invention is expressed in eukaryotic cells, such as mammalian host cells. Preferred mammalian host cells for expressing the fusion protein of the invention include CHO cells (e.g., dhfr-CHO cells, as 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 a recombinant expression vector containing a nucleic acid sequence encoding the fusion protein of the invention is introduced into a mammalian host cell, the fusion protein of the invention can be produced by culturing the host cell for a period of time sufficient to allow expression of the fusion protein in the host cell, or more preferably, for a period of time sufficient to allow secretion of the fusion protein into the culture medium in which the host cell is grown. The fusion protein of the invention can be recovered from the culture medium using standard protein purification methods.

[0219] Alternatively, nucleic acid sequences encoding the fusion proteins of the invention can be expressed in other expression systems, such as prokaryotic cells (e.g., microorganisms such as E. coli), or algal expression systems, insect cell expression systems, or cell-free protein synthesis systems. Additionally, the fusion proteins of the invention can be produced in transgenic non-human animals (e.g., sheep and rabbit milk, or chicken eggs) or transgenic plants.

[0220] The introduction of the nucleic acid sequence of the present invention into a host cell can be carried out by any standard technique known in the art. For the expression of the fusion protein of the present invention, an expression vector encoding the fusion protein can be 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 prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, lipofectamine transfection, and freeze-drying transfection, and the like. The cell line secreting the fusion protein of the present invention can be identified by assaying the culture supernatant for the presence of the fusion protein. The preferred screening procedure includes two consecutive steps: the first step is the identification of the cell line secreting the fusion protein, and the second step is the determination of the quality of the fusion protein (e.g., the ability of the fusion protein to inhibit cytokine production by blood cells stimulated by LPS or other Toll-like receptor agonists, glycosylation pattern, and the like).

[0221] For optimal expression in a host cell, the DNA sequence encoding the fusion protein of the present disclosure (e.g., an IL4 / IL13 fusion protein, or an IL10 / IL13 fusion protein, or an IL27 / IL13 fusion protein, or an IL33 / 13 fusion protein, or a TGFβ1 / IL13 fusion protein, or a TGFβ2 / IL13 fusion protein, or an IL13 / IL13 fusion protein) may be codon optimized by matching the codon usage to that most preferred in the host cell genes. Several techniques for altering codon usage to that preferred by the host cell can be found in the patent and scientific literature. The exact method of altering the codon usage is not critical to the present invention.

[0222] In another embodiment of the invention, PCR primers and / or probes and kits are provided for detecting DNA or RNA sequences encoding IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion proteins. Degenerate or specific PCR primer pairs can be synthesized to amplify DNA encoding an IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein from a sample (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 a nucleic acid sequence (or complementary strand) encoding an IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein may be used as a primer or probe. Similarly, a DNA fragment of a nucleic acid sequence encoding an IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein may be used as a hybridization probe.A kit for detecting a nucleic acid sequence encoding an IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein may comprise a primer specific for a nucleic acid sequence encoding an IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein, and / or a primer specific for a nucleic acid sequence encoding an IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein. The detection kit may comprise a probe specific for a nucleic acid sequence encoding a IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein and an associated protocol for using the primers or probes to specifically detect a nucleic acid sequence encoding an IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein in a sample. Such a detection kit may be used, for example, to determine whether a host cell has been transformed with a nucleic acid sequence encoding a particular IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein of the invention. Because of the degeneracy of the genetic code, some amino acid codons can be substituted for others without altering the amino acid sequence of the protein.

[0223] In one aspect, the invention relates to a method of producing a fusion protein of the disclosure (e.g., an IL4 / IL13 fusion protein, or an IL10 / IL13 fusion protein, or an IL27 / IL13 fusion protein, or an IL33 / IL13 fusion protein, or a TGFβ1 / IL13 fusion protein, or a TGFβ2 / IL13 fusion protein, or an IL13 / IL13 fusion protein), comprising culturing a host cell of the invention under conditions that allow for the production of the fusion protein (e.g., an IL4 / IL13 fusion protein, or an IL10 / IL13 fusion protein, or an IL27 / IL13 fusion protein, or an IL33 / IL13 fusion protein, or a TGFβ1 / IL13 fusion protein, or a TGFβ2 / IL13 fusion protein, or an IL13 / IL13 fusion protein), and optionally recovering the fusion protein. The skilled artisan will be able to routinely select conditions that allow the production of IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion proteins. In addition, the skilled artisan will be able to recover the produced fusion proteins using routine methods, including but not limited to chromatographic methods (such as but not limited to size-exclusion 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 mentioned above, recovery or purification of the fusion protein can be easily performed, for example by adding a His tag to the fusion protein.

[0224] Pharmaceutical Compositions In one aspect, the invention relates to a pharmaceutical composition comprising a fusion protein of the invention and a pharma- ceutically acceptable carrier.

[0225] The pharmaceutical composition can be prepared by the method described in, for example, Remington: The Science and Practice of Pharmacy, 1999. th The compositions may be formulated with pharma- ceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, according to conventional techniques (e.g., as described in U.S. Pat. No. 6,313,316, 1995).

[0226] The term "pharmaceutically acceptable carrier" refers to a carrier or excipient that is essentially non-toxic and non-therapeutic. Examples of such excipients include, but are not limited to, saline, Ringer's solution, dextrose solution, and Hank's solution.

[0227] The pharmaceutical compositions may be administered by any suitable route and mode. As will be appreciated by those of skill in the art, the route and / or mode of administration will vary depending on 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, and may be in the form of a liquid preparation, such as an oral or sterile parenteral solution or suspension.

[0228] Pharmaceutical compositions of the present invention include those suitable for any form of parenteral administration. In certain embodiments, the pharmaceutical composition is administered parenterally. The phrases "parenteral administration" and "administering parenterally", as used herein, refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, intradural substernal, intracerebral, intraocular, intralesional, intraventricular, intracapsular, intraparenchymal, e.g., injection and infusion.

[0229] In certain embodiments, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion. In one embodiment, the fusion proteins of the invention are administered in crystalline form by subcutaneous injection.

[0230] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and antioxidants, and absorption delaying agents, and the like that are physiologically compatible.

[0231] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Unless any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical composition of the present invention is contemplated.Preferably, this carrier is suitable for parenteral administration, for example, suitable for intravenous or subcutaneous injection or infusion.

[0232] Pharmaceutical compositions must typically 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 for high drug concentration.The examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include: water, ethanol, polyol (e.g., 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 maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0233] Sterile injection solution can be prepared by placing the required amount of active compound in a suitable solvent, for example with one or combination of the above-listed ingredients as necessary, and then carrying out microfiltration.Generally, dispersion is prepared by placing active compound in a sterile vehicle that contains a basic dispersion medium and other necessary ingredients (for example, other necessary ingredients from the above-listed ones).In the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which can obtain powder of active ingredient and any additional desired ingredients from the solution that has already been sterile-filtered.

[0234] Depending on the route of administration, the active compound (i.e., the present IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein) may be coated with a material to protect the compound 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 a suitable carrier, such as a liposome. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al., 1984).

[0235] The fusion protein of the present invention can also be prepared with a carrier that protects the fusion protein against rapid release (e.g., controlled release formulations, such as implants, transdermal patches, and microencapsulated delivery systems). Biodegradable and biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) can be used. Methods for preparing such formulations are generally known to those skilled in the art. For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0236] A pharmaceutical composition of the present disclosure can be a combination of any pharmaceutical compound described herein with other chemical components (e.g., carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients) that facilitate administration of the compound to an organism.

[0237] The pharmaceutical preparation for administration may include an aqueous solution of the active compound in water-soluble form. The suspension of the active compound may be prepared as an oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. The suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, to allow the preparation of highly concentrated solutions. The active ingredient may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0238] In carrying out the methods of treatment or use provided herein, a therapeutically effective amount of the compound described herein in a pharmaceutical composition is administered to a subject having a disease or condition to be treated.In some embodiments, the subject is a mammal, such as a human.The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors.

[0239] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, including excipients and auxiliary agents, which facilitates the processing of active compounds into preparations that can be used medicaments.Depending on the selected route of administration, formulation can be changed.The pharmaceutical compositions that contain the compounds described herein can be prepared, for example, by mixing, dissolving, emulsifying, encapsulating, encapsulating or compressing process.

[0240] The pharmaceutical composition may include at least one pharma- ceutically acceptable carrier, diluent, or excipient, and the compound described herein in the form of a free base or a pharma- ceutically acceptable salt.The pharmaceutical composition may include a solubilizer, a stabilizer, an osmolality enhancer, a buffer, and a preservative.

[0241] Methods for preparing compositions containing the compounds described herein include formulating the compounds with one or more inert, pharma- ceutically acceptable excipients or carriers to form solid, semi-solid, or liquid compositions. Solid compositions include, for example, powders, dispersible granules, and cachets. Liquid compositions include, for example, solutions in which the compounds are dissolved, emulsions containing the compounds, or solutions containing liposomes, micelles, or nanoparticles containing the compounds disclosed herein. Semi-solid compositions include, for example, gels, suspensions, and creams. The compositions can be in liquid solution or suspension, solid forms suitable for solution or suspension in liquid prior to use, or emulsions. The compositions can also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, and other pharma- ceutical acceptable additives.

[0242] Non-limiting examples of dosage forms suitable for use in the present disclosure include liquids, powders, gels, nanosuspensions, nanoparticles, microgels, aqueous or oily suspensions, emulsions, and any combination thereof.

[0243] Non-limiting examples of pharma- ceutically acceptable excipients suitable for use in the present disclosure include binders, disintegrants, anti-adherents, antistatic agents, surfactants, antioxidants, coating agents, colorants, plasticizers, preservatives, suspending agents, emulsifying agents, antibacterial agents, spheronizing agents, and any combination thereof.

[0244] Non-limiting examples of pharma-ceutically acceptable carriers include saline, Ringer's solution, and dextrose solution. In some embodiments, the pH of the solution can be about 5 to about 8, and can be about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the compound. The matrices can be in the form of shaped articles (e.g., films, liposomes, microparticles, or microcapsules).

[0245] The pH of the composition of the present disclosure may range from about 3 to about 12. The pH of the composition may be, for example, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 11, or about 11 to about 12 pH units. The pH of the composition may be, for example, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 pH units. The pH of the composition may be, for example, at least 3, at least 4, at least 5, at least 6, at least 6.2, at least 6.4, at least 6.6, at least 6.8, at least 7, at least 7.2, at least 7.4, at least 7.6, at least 7.8, at least 8, at least 9, at least 10, at least 11, or at least 12 pH units. The pH of the composition can be, for example, at most 3, at most 4, at most 5, at most 6, at most 6.2, at most 6.4, at most 6.6, at most 6.8, at most 7, at most 7.2, at most 7.4, at most 7.6, at most 7.8, at most 8, at most 9, at most 10, at most 11, or at most 12 pH units. The pharmaceutical formulations disclosed herein can have a pH of about 5.5 to about 8.5.

[0246] The formulations of the present disclosure may contain sugars, alcohol, antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. The compositions may also contain preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms on the subject compound may be achieved by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It is also desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. In addition, the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin, may prolong the absorption of injectable pharmaceutical forms. If desired, the formulation may be diluted, for example, with isotonic saline or dextrose solution, before use.

[0247] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a compound (e.g., a fusion protein) in admixture with a pharma- ceutically acceptable carrier and / or excipient (e.g., saline, phosphate buffered saline, phosphates and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins). Exemplary agents include: octylphenoxypolyethoxyethanol compounds, monostearate polyethylene glycol compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol.

[0248] In some embodiments, the pharmaceutical formulations disclosed herein may include (i) a compound or fusion protein disclosed herein; (ii) a buffer; (iii) a non-ionic surfactant; (iv) a tonicity agent; and (v) a stabilizer. In some embodiments, the pharmaceutical formulations disclosed herein are stable liquid pharmaceutical formulations.

[0249] For solid compositions, solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.

[0250] Pharmaceutical carriers or excipients can be physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The carrier can be suitable for administration by the routes disclosed herein (e.g., parenterally).

[0251] The composition of the present invention may be, for example, an immediate release form or a controlled release formulation. An immediate release formulation may be formulated to allow the compound to act quickly. Non-limiting examples of immediate release formulations include formulations that are easily soluble. A controlled release formulation may be a pharmaceutical formulation that is adapted to match the release rate and release profile of active agent with physiological requirements and chronotherapeutic requirements, or may be a pharmaceutical formulation that is formulated to release active agent at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., synthetic or natural origin), other gelling agents (e.g., gel-forming dietary fibers), matrix-based formulations (e.g., formulations that include polymeric materials in which at least one active ingredient is dispersed), granules in a matrix, polymer mixtures, and granular masses.

[0252] In some embodiments, the formulations of the present disclosure include a heat stabilizer, such as a sugar or sugar alcohol, such as sucrose, sorbitol, glycerol, trehalose, or mannitol, or any combination thereof. In some embodiments, the stabilizer is a sugar. In some embodiments, the sugar is sucrose, mannitol, or trehalose.

[0253] Non-limiting examples of pharma- ceutically acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[0254] The pharmaceutical composition can be administered locally, for example by direct injection of compound into organ, optionally in depot or sustained release formulation or implant.The pharmaceutical composition can be provided in the form of a fast release formulation, a sustained release formulation, or an intermediate release formulation.Fast release forms can provide immediate release.Sustained release formulations can provide controlled release or sustained delayed release.

[0255] In some embodiments, a pump may be used to deliver the pharmaceutical composition. In some embodiments, for example, a pen delivery device may be used for subcutaneous delivery of the disclosed composition. Such a pen delivery device may be reusable or disposable. A reusable pen delivery device may use a replaceable cartridge that contains the pharmaceutical composition disclosed herein. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. A disposable pen does not have a replaceable cartridge. Rather, a disposable pen delivery device is pre-filled with the pharmaceutical composition that is held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0256] The pharmaceutical compositions described herein may be in unit dosage form suitable for single administration of precise dosage. In unit dosage form, the formulation may be divided into unit doses containing appropriate amounts of one or more compounds, fusion proteins, and / or therapeutic agents. The unit dosage may be in the form of a package containing individual amounts of the formulation. Non-limiting examples are packaged injection solutions, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single-dose non-reclosable containers. Multi-dose reclosable containers may be used, for example, with or without preservatives. The formulations for injection disclosed herein may be in unit dosage form, for example, in ampoules or in multi-dose containers with preservatives.

[0257] In some embodiments, the pharmaceutical formulations disclosed herein are liquid formulations that may contain about 50 μg / mL to about 100 mg / mL of the fusion protein. The formulations may contain, for example, at least 50 μg / mL, at least 100 μg / mL, at least 200 μg / mL, at least 300 μg / mL, at least 400 μg / mL, at least 500 μg / mL, at least 600 μg / mL, at least 700 μg / mL, at least 800 μg / mL, at least 900 μg / mL, at least 1 mg / mL, at least 10 mg / mL, at least 20 mg / mL, or at least 50 mg / mL. In some embodiments, the formulation may contain 100 μg / mL or less, 200 μg / mL or less, 300 μg / mL or less, 400 μg / mL or less, 500 μg / mL or less, 600 μg / mL or less, 700 μg / mL or less, 800 μg / mL or less, 900 μg / mL or less, 1 mg / mL or less, 10 mg / mL or less, 20 mg / mL or less, or 50 mg / mL or less.

[0258] Dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. Unit dosage form, as used herein, refers to a physically discrete unit suitable as a single dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to provide the desired therapeutic effect together with the necessary pharmaceutical carrier. The specifications of the unit dosage form of the present invention are determined by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the technology of compounding such active compound for the treatment of individual susceptibility.

[0259] The actual dosage level of the IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13 TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein in the pharmaceutical compositions of the present invention may be varied to obtain an amount of IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein effective to achieve the desired therapeutic response for a particular subject (e.g., patient), composition, and mode of administration without exhibiting toxicity to the subject (e.g., patient). The selected dosage level will depend on various pharmacokinetic factors, such as the activity of the particular composition of the invention being employed, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition being employed, the age, sex, weight, condition, general health, and past medical history of the patient being treated, and similar factors known in the medical arts.

[0260] In one embodiment, the IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion proteins of the present invention may be administered as an intravenous injection or short-term infusion, or in another embodiment, administered as a slow continuous infusion over an extended period of time, such as greater than 24 hours, to reduce toxic side effects.

[0261] In yet another embodiment, the IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion proteins of the present invention may be administered as a maintenance therapy (e.g., once weekly for a period of six months or more).

[0262] In another embodiment, the IL4 / IL13 (or IL10 / IL13, or IL27 / IL13, or IL33 / IL13, or TGFβ1 / IL13, or TGFβ2 / IL13, or IL13 / IL13) fusion protein of the present invention may be administered as an intrathecal injection. In yet another embodiment, the fusion protein is administered via an intrathecal or intraspinal drug delivery system that allows for continuous or repeated administration.

[0263] The therapeutic agent described herein may be administered before, during, or after the onset of a disease or condition, and the timing of administering the composition containing the therapeutic agent may vary. For example, the composition may be used as a prophylactic and may be administered continuously to a subject prone to a condition or disease to reduce the likelihood of the onset of the disease or condition. The composition may be administered to a subject during or as soon as possible after the onset of symptoms. The initial administration may be by any route practical, for example, by any route described herein using any formulation described herein. The therapeutic agent may be administered as soon as possible after the onset of a disease or condition is detected or suspected, and for the period of time required to treat the disease (e.g., about 1 month to about 3 months). The length of treatment may vary from subject to subject.

[0264] Dosages may be based on the amount of the fusion protein per kilogram of subject body weight. Doses of fusion protein may be at least about 0.5 μg / kg, 1 μg / kg, 25 μg / kg, 50 μg / kg, 75 μg / kg, 100 μg / kg, 125 μg / kg, 150 μg / kg, 175 μg / kg, 200 μg / kg, 225 μg / kg, 250 μg / kg, 275 μg / kg, 300 μg / kg, 325 μg / kg, 350 μg / kg, 375 μg / kg, 400 μg / kg, 425 μg / kg, 450 μg / kg, 475 μg / kg, / kg, 500μg / kg, 525μg / kg, 550μg / kg, 575μg / kg, 600μg / kg, 625μg / kg, 650μg / kg, 675μg / kg, 700μg / kg, 725μg / kg, 750μg / kg, 775 μg / kg, 800 μg / kg, 825 μg / kg, 850 μg / kg, 875 μg / kg, 900 μg / kg, 925 μg / kg, 950 μg / kg, 975 μg / kg, or 1 mg / kg. The dosage of the fusion protein is approximately 1μg / kg, 25μg / kg, 50μg / kg, 75μg / kg, 100μg / kg, 125μg / kg, 150μg / kg, 175μg / kg, 200μg / kg, 225μg / k g, 250μg / kg, 275μg / kg, 300μg / kg, 325μg / kg, 350μg / kg, 375μg / kg, 400μg / kg, 425μg / kg, 450μg / kg, 475μg / kg, 500μ g / kg, 525μg / kg, 550μg / kg, 575μg / kg, 600μg / kg, 625μg / kg, 650μg / kg, 675μg / kg, 700μg / kg, 725μg / kg, 750μg / kg, 7 It can be 75 μg / kg, 800 μg / kg, 825 μg / kg, 850 μg / kg, 875 μg / kg, 900 μg / kg, 925 μg / kg, 950 μg / kg, 975 μg / kg, or 1 mg / kg or less.

[0265] In some embodiments, the dose can be at least about 1 ng, at least 10 ng, at least 100 ng, at least 500 ng, at least 1 μg, at least 5 μg, at least 10 μg, at least 50 μg, at least 100 μg, at least 500 μg, at least 1 mg, at least 5 mg, at least 10 mg, at least 50 mg, or at least 100 mg. The dose can be about 1 ng or less, 10 ng or less, 100 ng or less, 500 ng or less, 1 μg or less, 5 μg or less, 10 μg or less, 50 μg or less, 100 μg or less, 500 μg or less, 1 mg or less, 5 mg or less, 10 mg or less, 50 mg or less, or 100 mg or less.

[0266] Dosages may be determined with reference to the plasma or local concentration of the fusion protein. The target plasma or local concentration of the induced protein is at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 10 nM, at least about 15 ... M, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 10 μM, or at least about 100 μM. The target plasma or local concentration of the fusion protein can be about 1 nM or less, about 10 nM or less, about 100 nM or less, about 1 μM or less, about 10 μM or less, about 100 μM or less, or about 1 mM or less.

[0267] The administration of the fusion protein can continue as long as clinically necessary. In some embodiments, the fusion protein of the present disclosure can be administered for more than 1 day, more than 1 week, more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, more than 12 months, more than 13 months, more than 14 months, more than 15 months, more than 16 months, more than 17 months, more than 18 months, more than 19 months, more than 20 months, more than 21 months, more than 22 months, more than 23 months, or more than 24 months. In some embodiments, the fusion proteins of the disclosure are administered for less than 1 week, less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 7 months, less than 8 months, less than 9 months, less than 10 months, less than 11 months, less than 12 months, less than 13 months, less than 14 months, less than 15 months, less than 16 months, less than 17 months, less than 18 months, less than 19 months, less than 20 months, less than 21 months, less than 22 months, less than 23 months, or less than 24 months.

[0268] In some embodiments, the fusion protein may be administered to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times over a treatment cycle. In some embodiments, the treatment cycle is 7, 14, 21, or 28 days long. In some embodiments, the treatment cycle is 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 11 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 ​​months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months.

[0269] In some embodiments, the fusion protein is administered to a subject once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. therapeutic use In a further aspect, the present invention relates to the fusion protein of the invention or a nucleic acid encoding said fusion protein, for use as a medicament.

[0270] In one embodiment, the present invention relates to a fusion protein or a nucleic acid encoding the fusion protein for use in treating pain (e.g., chronic pain). "Chronic" can mean that the pain continues / has continued for more than 2 weeks, or more than 1, 3, 6, 12 months, or even more than 1, 2, 4, 6 years. The pain can include pain mediated by the central nervous system, the peripheral nervous system, or a combination thereof. Non-limiting types of pain include nociceptive pain, peripheral neuropathic pain, central neuropathic pain, and mixed pain.

[0271] Neuropathy can contribute to pain. In some embodiments, the fusion proteins of the present disclosure can be used to treat neuropathies. Neuropathy can be accompanied by pain, numbness, weakness, or a combination thereof.

[0272] Specifically, the fusion protein of the present invention has been found to have a long-lasting analgesic effect on allodynia associated with chemotherapy-related neuropathy and neurodegeneration in mice.In addition, the fusion protein prevents chemotherapy-induced neurodegeneration in vitro and in vivo.Therefore, the fusion protein of the present invention or the nucleic acid encoding the fusion protein can be used to prevent and treat neuropathic pain.Therefore, the fusion protein can be particularly useful for treating neuropathy (e.g., chemotherapy-induced neuropathy) and other forms of peripheral neuropathic pain (e.g., postherpetic neuralgia, trigeminal neuralgia, posttraumatic or postoperative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-related neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-related pain, complex regional pain syndrome, and the like).In some embodiments, the fusion protein of the present disclosure can be used to treat postoperative cognitive dysfunction.

[0273] In some embodiments, the fusion proteins of the present disclosure may be used to treat conditions associated with cancer or chemotherapy (e.g., chemotherapy-induced neuropathy, chemotherapy-associated pain, chemo brain, cancer-associated cognitive impairment, cancer-associated cognitive dysfunction). In some embodiments, the condition is associated with chemotherapy being used to treat: acute leukemia, astrocytoma, biliary tract cancer (cholangiocarcinoma), bone cancer, breast cancer, brain stem glioma, bronchioloalveolar cell lung cancer, cancer of the adrenal gland, cancer of the anal region, cancer of the bladder, cancer of the endocrine system, cancer of the esophagus, cancer of the head or neck, cancer of the kidney, cancer of the parathyroid gland, cancer of the penis, cancer of the pleura / peritoneum, cancer of the salivary gland, cancer of the small intestine, cancer of the thyroid gland, cancer of the ureter, cancer of the urethra, carcinoma of the cervix, carcinoma of the endometrium, carcinoma of the fallopian tube, carcinoma of the renal pelvis, carcinoma of the vagina, carcinoma of the vulva, cervical cancer, chronic leukemia, colon cancer, colorectal cancer, cutaneous melanoma, ependymoma, epidermoid tumor, Ewing's tumor tumors, gastric cancer, glioblastoma, glioblastoma multiforme, glioma, hematologic tumors, hepatocellular (liver) carcinoma, hepatoma, Hodgkin's disease, intraocular melanoma, Kaposi's sarcoma, lung cancer, lymphoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, muscle cancer, neoplasms of the central nervous system (CNS), neuronal carcinoma, small cell lung cancer, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, childhood malignancies, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, sarcoma of soft tissue, Schwannoma, skin cancer, tumors of the spinal axis, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular tumor, uterine cancer, or tumors and metastases thereof, such as refractory versions of any of the above cancers, or any combination thereof.

[0274] In further embodiments, the fusion protein of the present invention or the nucleic acid encoding the fusion protein can be used to treat pain and neurodegeneration in central nervous disorders (e.g., spinal cord injury, post-stroke pain, and multiple sclerosis). In some embodiments, the fusion protein of the present disclosure can be used to treat post-operative cognitive dysfunction.

[0275] In a further aspect, the invention relates to a fusion protein of the invention or a nucleic acid encoding the fusion protein for use in the treatment of chronic mixed nociceptive-neuropathic pain (e.g., low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, and the like).

[0276] In still further embodiments, the fusion proteins of the invention or nucleic acids encoding the fusion proteins may be used to treat post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, and the like.

[0277] In certain embodiments, the condition treated with the fusion proteins of the invention is characterized by pain, which may be selected from nociceptive pain, neuropathic pain, or mixed nociceptive-neuropathic pain.

[0278] In another embodiment, the fusion protein of the present invention or a nucleic acid encoding the fusion protein can be used to prevent chronic pain. In particular, the fusion protein can be used to prevent neuropathic pain and neurodegeneration in cancer patients treated with chemotherapy.

[0279] In another aspect, the present invention is directed to a fusion protein of the invention for use in the prevention or treatment of a clinical condition in a mammal (e.g., a human) in which interleukin-13 is indicated. In a further aspect, the present invention is directed to the fusion protein of the invention for use in the prevention or treatment of a clinical condition in a mammal (e.g. a human) in which interleukin 4, or interleukin 10, or 27, or IL33, or TGFβ1, or TGFβ2 are indicated.

[0280] According to one embodiment, the fusion proteins taught herein may be used to inhibit neuroinflammation resulting from activation of glial cells and neurons in the central nervous system, infiltrating immune cells, or any combination thereof.

[0281] As a result, the fusion proteins of the present invention may be used for the preparation of a medicament for attenuating neuroinflammatory responses by inhibiting the activation of glial cells and neurons in vivo. In some embodiments, the fusion proteins of the present disclosure or the nucleic acids encoding the fusion proteins may be used as independent drugs. In other embodiments, they are used in combination with other drugs. In some embodiments, the fusion proteins of the present disclosure are administered in combination with analgesics (e.g., acetaminophen / paracetamol, nonsteroidal anti-inflammatory drugs (NASIDs), or opioids). In some embodiments, the fusion proteins of the present disclosure may be used in combination with NSAIDs (e.g., aspirin, ibuprofen, naproxen, celecoxib, ketorolac, or diclofenac). In some embodiments, the fusion proteins of the present disclosure may be used in combination with certain COX-2 inhibitors (e.g., celecoxib (Celebrex®), rofecoxib, or etoricoxib). In some embodiments, the fusion proteins of the present disclosure may be used in combination with corticosteroids (e.g., dexamethasone or glucosteroids (e.g., hydrocortisone and prednisone)). In some embodiments, the fusion proteins of the present disclosure are administered in combination with an antagonist of a proinflammatory cytokine (e.g., an antibody derivative or other molecule that binds TNF-α (e.g., adalimumab, etanercept), IL-17 (e.g., secukinumab), IL-23 (e.g., guselkumab, ildrakizumab), or IL-12 and IL-23 (e.g., ustekinumab)). In some embodiments, the fusion protein is administered in combination with hyaluronic acid. The multiple therapeutic agents may be administered in any order or simultaneously. In some embodiments, the compounds of the present disclosure are administered in combination with, before, or after another drug.

[0282] In some embodiments, the fusion proteins of the present disclosure or nucleic acids encoding the fusion proteins may be used to treat: Alpers' disease, arachnoiditis, fibrotic joint disease, ataxic cerebral palsy, autoimmune atrophic gastritis, amyloidosis, hATTR amyloidosis, ischemic necrosis, back pain, Batten's disease, Behcet's disease (syndrome), breakthrough pain, burning mouth syndrome, bursitis, cerebral autosomal dominant arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL), cerebral ischemia, cerebro-oculo-facial-skeletal syndrome (COFS), carpal tunnel syndrome, cauda equina syndrome, central pain syndrome. group, cerebral palsy, cerebrospinal fluid (CSF) leakage, cervical stenosis, Charcot-Marie-Tooth (CMT) disease, chronic functional abdominal pain (CFAP), chronic pancreatitis, collapsed lung (pneumothorax), corticobasal degeneration, compression injury, corneal neuropathic pain, crush syndrome, degenerative disc disease, dermatomyositis, dementia, dystonia, Ehlers-Danlos syndrome (EDS), endometriosis, eosinophilic myalgia syndrome (EMS), erythromelalgia, lumbar postoperative pain syndrome (FBSS), fibromyalgia, Friedreich's ataxia, frontotemporal dementia, glossopharyngeal neuralgia, growing pains, herniated disc, hydrocephalus syndrome, intercostal neuralgia, interstitial cystitis, juvenile dermatomyositis, knee injury, leg pain, Lewy body dementia, low back pain and hematuria syndrome, Lyme disease, dyssensory thigh pain, mitochondrial disease, mixed dementia, motor neuron disease (MND), Hirayama disease, multiple system atrophy (MSA), myositis, neck pain, occipital neuralgia, osteoporosis, rhabdomyolysis, Paget's disease, Parsonage-Turner syndrome, pelvic pain, peripheral neuropathy, phantom limb pain, nerve compression, plantar fasciitis, polymyalgia rheumatica, polymyositis, postherniorrhaphy pain syndrome, postmastectomy pain syndrome, poststroke pain, postthoracotomy pain syndrome syndrome, post-polio syndrome, primary lateral sclerosis, psoriatic arthritis, pudendal neuralgia, radiculopathy, restless legs syndrome, rheumatoid arthritis (RA), sacroiliac joint dysfunction, sarcoidosis, Scheuermann's kyphosis, sciatica, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), herpes zoster, spasmodic torticollis, sphincter of Oddi dysfunction, spinal cord injury, spinal stenosis, syringomyelia, Tarlov's cyst, tethered spinal cord syndrome, thoracic outlet syndrome (TOS), temporomandibular joint disorder, transverse myelitis, brain trauma, vascular pain, vulvodynia, whiplash injury, or a combination thereof.

[0283] The fusion protein of the present disclosure or the nucleic acid encoding this fusion protein can be used to treat neuropathy.Non-limiting examples of neuropathy include: post-traumatic peripheral neuropathy, post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-related neuropathy, chemotherapy-induced neuropathy, polyneuropathy, mononeuropathy, multiple mononeuropathy, cranial neuropathy, mainly motor neuropathy, mainly sensory neuropathy, sensorimotor neuropathy, autonomic neuropathy, idiopathic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, pudendal neuralgia, variant trigeminal neuralgia, sciatica, brachial plexopathy, or intercostal neuralgia.Neuropathy can be accompanied by, for example, pain, numbness, weakness, burning sensation, atrophy, tingling, spasm, or combinations thereof.

[0284] In some embodiments, the fusion proteins of the present disclosure or nucleic acids encoding the fusion proteins may be used to treat autoimmune diseases. Non-limiting examples of autoimmune diseases include acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, adiposity, adult Still's disease, alopecia areata, ankylosing spondylitis, antiglomerular basement membrane nephritis, antineutrophil cytoplasmic antibody-associated vasculitis, anti-NMDA receptor encephalitis, antiphospholipid syndrome, antisynthetase syndrome, aplastic anemia, autoimmune angioedema, autoimmune encephalitis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune neutropenia, autoimmune inflammatory bowel disease ... epigenetic oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune polyendocrine syndrome type 2, autoimmune polyendocrine syndrome type 3, autoimmune progesterone dermatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, autoimmune urticaria, autoimmune uveitis, Baro concentric sclerosis, Behçet's disease, Bickerstaff encephalitis, bullous pemphigoid, celiac disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, -cancer syndrome, cold agglutinin disease, complex regional pain syndrome, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, discoid lupus erythematosus, endometriosis, enthesitis, enthesitis-associated arthritis, eosinophilic esophagitis, eosinophilic fasciitis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, Felty syndrome, fibromyalgia, gastritis, pemphigoid of pregnancy, giant cell arteritis, Goodpasture's syndrome, Graves' disease, Graves' ophthalmopathy, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, Henoch-Schönlein purpura, hidradenitis suppurativa, idiopathic dilated cardiomyopathy, idiopathic inflammatory demyelinating disease, IgA nephropathy, IgG4-related systemic disease, inclusion body myositis, inflammatory bowel disease (IBD), intermediate uveitis, interstitial cystitis, juvenile arthritis, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease, lupus nephritis, lupus vasculitis, Lyme disease, Meniere's disease, microscopic colitis, microscopic polyangiitis, mixed connective tissue disease, Mooren's ulcer, morphea, Mucha-Habermann disease,Multiple sclerosis, myasthenia gravis, myocarditis, myositis, neuromyelitis optica, neuromyotonia, opsoclonus-myoclonus ataxia, optic neuritis, Ord's thyroiditis, relapsing rheumatism, paraneoplastic cerebellar degeneration, Parry-Romberg syndrome, Parsonage-Turner syndrome, streptococcal-associated childhood autoimmune neuropsychiatric disorders, pemphigus vulgaris, pernicious anemia, acute pityriasis lichenoides, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary immunodeficiency, primary sclerosing cholangitis, progressive inflammation Neuropathy, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, rheumatic vasculitis, sarcoidosis, Schnitzler's syndrome, scleroderma, Sjögren's syndrome, stiff-body syndrome, subacute bacterial endocarditis, Southac syndrome, Sydenham's chorea, sympathetic ophthalmia, systemic lupus erythematosus, systemic scleroderma, thrombocytopenia, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, urticaria, urticarial vasculitis, vasculitis, and vitiligo.

[0285] In some embodiments, the fusion protein of the present disclosure or the nucleic acid encoding the fusion protein may be used to treat inflammation. In some embodiments, the inflammation is chronic inflammation. In some embodiments, the fusion protein of the present disclosure may be used to treat inflammation associated with: inflammatory bowel disease, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, glomerulonephritis, sepsis, adult respiratory distress syndrome, dermatitis, sarcoidosis, allergic inflammation, psoriasis, ankylosing spondyloarthritis, systemic lupus erythematosus, vasculitis, gout, allografts, xenografts, autoimmune diseases, Sjogren's syndrome, burns, trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, and toxicity induced by in vivo administration of cytokines or other therapeutic monoclonal antibodies. In some embodiments, the inflammation is chronic inflammation. In some embodiments, the fusion proteins of the present disclosure may be used to treat inflammatory bowel disease, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, glomerulonephritis, sepsis, adult respiratory distress syndrome, dermatitis, sarcoidosis, allergic inflammation, psoriasis, ankylosing spondyloarthritis, systemic lupus erythematosus, vasculitis, gout, allografts, xenografts, autoimmune diseases, Sjogren's syndrome, burns, trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, and toxicity induced by in vivo administration of cytokines or other therapeutic monoclonal antibodies.

[0286] In some embodiments, the fusion proteins are used to treat non-cancer conditions. Treatment (prophylactic or therapeutic) generally consists of parenterally (preferably intrathecally, intraarticularly, intravenously, intramuscularly, or subcutaneously) administering the fusion protein of the invention or a nucleic acid encoding the fusion protein. The dose and administration regimen will depend on the degree of inhibition of neuroinflammation and neurodegeneration desired. Typically, the amount of fusion protein administered ranges from 0.5 μg to 1 mg per kg of body weight. Dosage may be determined or adjusted by measuring cytokine levels (IL13, IL4, or IL10, or IL27, or IL33, or TGFβ1, or TGFβ2, or a combination thereof) in the targeted body compartment at the time of administration. Dosage may also be determined by measuring neuroinflammation in the patient, for example, by positron emission tomography (PET) of microglia.

[0287] For parenteral administration, the fusion protein or a nucleic acid encoding the fusion protein is preferably formulated in an injectable form in combination with a pharma- ceutically acceptable parenteral vehicle, such vehicles being known in the art and including, for example, saline, dextrose solution, Ringer's solution, and solutions containing small amounts of human serum albumin.

[0288] Typically, the fusion proteins of the invention can be formulated in such vehicles at a concentration of about 50 μg to about 100 mg per ml. Gene therapy The nucleic acid construct or vector of the present invention may be used as a gene therapy agent for the treatment of the above-mentioned conditions.Thus, in one aspect, the present invention is directed to the vector described above for use in the prevention or treatment of conditions characterized by visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, or mixed nociceptive-neuropathic pain, neuroinflammation, and / or neurodegeneration, preferably, said conditions are 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 The pain may be selected from the group consisting of 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 cord 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.In some embodiments, the condition is Alpers disease, arachnoiditis, fibrous joint disease, ataxic cerebral palsy, autoimmune atrophic gastritis, amyloidosis, hATTR amyloidosis, ischemic necrosis, back pain, Batten disease, Behcet's disease (syndrome), breakthrough pain, burning mouth syndrome, bursitis, cerebral autosomal dominant arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL), cerebral ischemia, cerebro-oculofacial-skeletal syndrome (COFS), carpal tunnel syndrome, cauda equina syndrome, central pain syndrome, cerebral palsy, cerebrospinal fluid (CSF) leak, cervical stenosis, Charcot-Marie-Tu CMT disease, chronic functional abdominal pain (CFAP), chronic pancreatitis, collapsed lung (pneumothorax), corticobasal degeneration, compression injury, corneal neuropathic pain, crush syndrome, degenerative disc disease, dermatomyositis, dementia, dystonia, Ehlers-Danlos syndrome (EDS), endometriosis, eosinophilic myalgia syndrome (EMS), erythromelalgia, lumbar postoperative pain syndrome (FBSS), fibromyalgia, Friedreich's ataxia, frontotemporal dementia, glossopharyngeal neuralgia, growing pains, herniated disc, hydrocephalus, intercostal neuralgia, interstitial cystitis, juvenile dermatomyositis, knee injury, lower limb pain, Lewy Dementia with small bodies, Low back pain and hematuria syndrome, Lyme disease, Dysensory thigh pain, Mitochondrial disease, Mixed dementia, Motor neuron disease (MND), Hirayama disease, Multiple system atrophy (MSA), Myositis, Neck pain, Occipital neuralgia, Osteoporosis, Rhabdomyolysis, Paget's disease, Parsonage-Turner syndrome, Pelvic pain, Peripheral neuropathy, Phantom limb pain, Nerve compression, Plantar fasciitis, Polymyalgia rheumatica, Polymyositis, Postherniorrhaphy pain syndrome, Postmastectomy pain syndrome, Poststroke pain, Postthoracotomy pain syndrome, Postpolio syndrome, Primary lateral sclerosis, Psoriatic arthritis, Vulvar nerve pain It may be menorrhagia, radiculopathy, restless legs syndrome, rheumatoid arthritis (RA), sacroiliac joint dysfunction, sarcoidosis, Scheuermann's kyphosis, sciatica, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), shingles, spasmodic torticollis, sphincter of Oddi dysfunction, spinal cord injury, spinal stenosis, syringomyelia, Tarlov's cyst, tethered spinal cord syndrome, thoracic outlet syndrome (TOS), temporomandibular joint disorders, transverse myelitis, brain trauma, vascular pain, vulvodynia, acute disseminated encephalomyelitis, acute optic neuritis, transverse myelitis, neuromyelitis optica, or whiplash injury.

[0289] As previously described herein, the present invention relates to a fusion protein comprising at least two, three, four (preferably two) regulatory (e.g., anti-inflammatory) interleukins selected from the group consisting of interleukin 13 (IL13), interleukin 4 (IL4), interleukin 10 (IL10), interleukin 27 (IL27), interleukin 33 (IL33), transforming growth factor beta 1 (TGFβ1), and transforming growth factor beta 2 (TGFβ2). Thus, when "IL13" is mentioned in this disclosure, it can be replaced with transforming growth factor beta 1 (TGFβ1) or transforming growth factor beta 2 (TGFβ2). Similarly, when "IL4" is mentioned in this disclosure, it can be replaced with transforming growth factor beta 1 (TGFβ1) or transforming growth factor beta 2 (TGFβ2). Thus, the present disclosure also encompasses IL13 / TGFβ1 or IL13 / TGFβ2 fusion proteins. Further, the present disclosure encompasses fusion proteins of IL4 / TGFβ1 or IL4 / TGFβ2. Additionally, the present disclosure encompasses fusion proteins of IL10 / TGFβ1 or IL10 / TGFβ2.

[0290] The present invention will now be described with reference to the following examples which show particularly advantageous embodiments, however it should be noted that the embodiments are illustrative and should not be construed as limiting the invention in any way. EXAMPLES

[0291] animal All animal experiments were performed according to international guidelines and with prior approval by the University Medical Centre Utrecht Laboratory Animal Committee. Experiments were performed using both male and female C57BL / 6 mice aged 8-16 weeks. Observers performing behavioral experiments were blinded to the treatments.

[0292] material and method Expression and purification of IL4 / IL13 fusion proteins. IL4 / IL13 fusion proteins HEK293 cells were transiently transfected with vectors containing the transgenes according to standard procedures (Y Derocher et al., Nucleic Acids Research 2002, vol 30, no 2, e9). Briefly, a synthetic cDNA (GeneArt, ThermoFisher Scientific) encoding the IL4 / IL13 fusion protein sequence of the present invention (see SEQ ID NO: 4; this sequence has a 6-His tag inserted at its N-terminus) was cloned into an expression vector containing a cystatin signal sequence. HEK293E cells were then transfected with this expression vector and co-transfected with a vector carrying a transgene for beta-galactoside alpha-2,3-sialyltransferase 5 (SIAT9: Homo sapiens) to optimize capping of glycans with sialic acid. Cells were transfected as previously described. 19,26 The cells were cultured in FreeStyle medium (Invitrogen) containing 0.9% primatone and approximately 0.04% v / v fetal bovine serum. Four days after transfection, the cell suspension was harvested and centrifuged at 435×g for 5 min. The supernatant was passed through a HIS-Select Nickel Affinity gel (Sigma-Aldrich) to purify the recombinant IL4 / IL13 fusion protein. The eluted fraction was dialyzed overnight at 4° C. against phosphate-buffered saline pH 7.4 (PBS).

[0293] Protein assays. Protein amounts in eluted and dialyzed fractions were determined using Bradford (Bio-Rad), BCA (Thermo Scientific), and Qubit 1.0 (Thermo Scientific). All protein assays were performed according to the manufacturer's protocols.

[0294] ELISA. The amount of fusion protein was determined based on the amount of individual cytokines measured by ELISA (IL4 Pelipair ELISA kit, Sanquin; IL13, DuoSet ELISA, R&D Systems). ELISA was 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.

[0295] SDS-Page and Western Blot. Fractions (load, flow-through, wash, elution / dialysis) from the HIS-Select Nickel affinity chromatography purification were separated on 12% polyacrylamide SDS-Page gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes. The membranes were stained with IL4 antibody (Santa Cruz, SC-13555).

[0296] High performance size exclusion chromatography (HP-SEC). To determine molecular weight and homogeneity, IL4 / IL13 fusion protein was analyzed by high performance size exclusion chromatography (HP-SEC). Gel filtration (BioSuite 125 4 μm UHR SEC column; water; catalog number 186002161) was performed on a high performance liquid chromatography system (Shimadzu) using 50 mM phosphate buffer with 0.5 M NaCl as the mobile phase. The column was calibrated prior to the run using a protein mixture of thyroglobulin, bovine serum albumin, carbonic anhydrase, myoglobulin, and ribonuclease. 50 μl of 20 μg / ml 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.

[0297] Assessment of chemotherapy-induced neurotoxicity in vitro. As previously described 27 In this study, dorsal root ganglion (DRG) neurons were cultured. Briefly, adult mouse DRG neurons were isolated and then treated with Ca 2+ and Mg 2+After digestion with an enzyme mixture containing HBSS-free, 5 mM HEPES, 10 mM glucose, collagenase type XI (5 mg / ml), and dispase (10 mg / ml), cells were mechanically disrupted in DMEM containing 10% heat-inactivated fetal bovine serum (v / v). Cells were centrifuged at 800 rpm for 5 min, resuspended in DMEM containing 4.5 g / L glucose, 4 mM L-glutamine, 110 mg / L sodium pyruvate, 10% fetal bovine serum, 1% penicillin-streptomycin (10,000 IU / ml), 1% glutamax, and incubated in 24-well plates in the presence of paclitaxel (1 uM) or oxaliplatin (5 ug / ml) for 24 hours to induce neurotoxicity. IL4 / IL10 fusion protein (100 ng / mL), IL4 / IL13 fusion protein (100 ng / mL), IL4 and IL13 (50 ng / mL each), IL4 (50 ng / mL), IL10 (50 ng / mL), and IL13 (50 ng / mL) were added together with the chemotherapeutic agents. As controls, cells were cultured in the absence of chemotherapeutic agents or cytokines and in the presence of chemotherapeutic agents alone. After fixation with 4% paraformaldehyde, cells were stained with rabbit anti-mouse βIII-tubulin (ab18207, 1:1000; Abcam). Neurites were visualized with a Zeiss Axio Lab A1 microscope (Zeiss-Oberkochen, Germany) using a random sampling method, and at least 10 images were made per slide at 10x magnification. Neurites length was measured with the ImageJ plugin Simple Neurite Tracer76. Mean neurite length per neuron for a minimum of five neurons per condition was compared between groups for three separate primary sensory cultures.

[0298] Assessment of chemotherapy-induced polyneuropathy and allodynia. To induce transient chemotherapy-induced polyneuropathy (CIPN), paclitaxel (2 mg / kg, Cayman Chemical Company) was injected intraperitoneally on days 0 and 2. To induce persistent paclitaxel-induced CIPN, paclitaxel (8 mg / kg, Cayman Chemical Company) was injected intraperitoneally on days 0, 2, 4, and 6. To induce persistent oxaliplatin-induced polyneuropathy, mice received two treatment cycles, each consisting of five daily intraperitoneal injections of 3 mg / kg oxaliplatin (Tocris) and a 5-day treatment-free interval.

[0299] Noxious mechanical sensitivity was measured in the hind paws using von Frey hairs (Stoelting, Wood Dale, USA). Results were analyzed using the up-down method. 29 The 50% paw-withdrawal threshold was expressed using the 50% paw-withdrawal threshold. In some experiments, the length of intraepidermal nerve fibers in the paw skin at day 15 was determined by immunofluorescence staining of skin biopsies with the neuronal marker PGP9.5. All experiments were performed in a blinded manner.

[0300] Statistical analysis. All data are expressed as mean ± SEM unless otherwise indicated. Data were analyzed for statistical significance by one-way or two-way ANOVA (with repeated measures as appropriate) followed by appropriate post-hoc tests. A p-value of p<0.05 was considered significant.

[0301] Example 1 Endogenous IL4 and IL13 are required for normal resolution of paclitaxel-induced transient hyperalgesia A transient pain model of paclitaxel-induced pain to investigate the role of regulatory (e.g., anti-inflammatory) cytokines in recovery from chemotherapy-induced polyneuropathy 15was used. Mice were injected twice with a low dose (2 mg / kg) of paclitaxel on days 0 and 2. Neutralizing antibodies against IL4, IL13, or control IgG antibodies were injected intrathecally daily (5 μg of antibody per injection; Figure 1) from days 6 to 10 after the start of chemotherapy treatment. Mice receiving paclitaxel developed mechanical hyperalgesia starting from the first day of chemotherapy, which resolved spontaneously 1 week after treatment was completed (Figure 1). In animals that were injected intrathecally 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 required for normal pain resolution after chemotherapy treatment (Figure 1).

[0302] Example 2 Neuroprotective Effects of IL4, IL10, and IL13 To evaluate whether regulatory (e.g., anti-inflammatory) cytokines have neuroprotective properties, sensory neurons isolated from dorsal root ganglia were cultured overnight with paclitaxel in combination with IL4, IL10, or IL13, as described in the methods above. Neurotoxicity was assessed by measuring neurite length using βIII-tubulin staining. Paclitaxel (1 μM) reduced neurite length by approximately 50%, indicating that paclitaxel damages sensory neurons. Addition of IL13 in culture with paclitaxel, but not IL10 and IL4, prevented the paclitaxel-induced adverse effects on neurite length (Figure 2).

[0303] Example 3. Characterization of recombinant IL4 / 13 fusion proteins Human IL4 / IL13 fusion protein with an N-terminal 6His-tag (see SEQ ID NO: 4) was produced by transient transfection into HEK293 cells and purified as described in the methods above. In HP-SEC, the purified IL4 / IL13 fusion protein migrated as a single peak with an apparent mass of 40 kDa (Figure 3). The preparation was also analyzed by SDS-PAGE and Coomassie staining. A homogenous preparation was detected migrating as a smear with a molecular weight of approximately 37 kDa (Inset 3).

[0304] Example 4 IL4 / IL13 fusion protein cures paclitaxel-induced persistent polyneuropathy The potential of IL4 / IL13 fusion protein to inhibit chemotherapy-induced hyperalgesia was examined in a model of persistent paclitaxel-induced painful neuropathy. 30 Paclitaxel (8 mg / kg) was injected into mice 4 times every other day on days 0-6. Paclitaxel induced mechanical hyperalgesia that began 1 day after the first injection and persisted for at least 3 weeks after cessation of chemotherapy treatment. Two days after the last paclitaxel injection, mice were intrathecally injected with three different doses (0.3, 1, and 3 μg / mouse) of IL4 / IL13 fusion protein containing SEQ ID NO: 4 (Figure 4). All three doses of IL4 / IL13 fusion protein significantly reduced paclitaxel-induced polyneuropathy. Importantly, the near-normalization of mechanical hyperalgesia persisted for at least 1 week, demonstrating the potential of IL4 / IL13 fusion protein for long-term resolution of chemotherapy-induced polyneuropathy.

[0305] Example 5 The efficacy of IL4 / IL13 fusion protein is superior to that of IL4 / IL10 fusion protein or combination therapy of IL4 and IL13 in curing chemotherapy-induced polyneuropathy We next assessed whether IL4 / IL13 fusion protein inhibited paclitaxel-induced painful polyneuropathy better than IL4 / IL10 fusion protein or the combination of IL4 and IL13. Mice developed paclitaxel-induced painful polyneuropathy after four injections of paclitaxel (8 mg / kg) every other day on days 0-6. IL4 / IL13 fusion protein (comprising SEQ ID NO: 4) inhibited paclitaxel-induced mechanical hypersensitivity for at least 1 week, whereas the combination of wild-type IL4 and IL13, or IL4 / IL10 fusion protein only inhibited paclitaxel-induced mechanical hypersensitivity for 1-2 days (Figure 5). Inhibition of paclitaxel-induced tonic allodynia by IL4 / IL13 fusion protein was associated with a reduction in paclitaxel-induced intraepidermal nerve fiber loss in paw skin (Figure 6).

[0306] To investigate in vitro whether IL4 / IL13 fusion protein protects against paclitaxel-induced neurotoxicity, neurite length was measured in mouse sensory neurons cultured in the presence of paclitaxel with or without fusion protein. Paclitaxel had a significant negative effect on neurite length when compared to the control group (Figure 7). The simultaneous presence of IL4 / IL10 fusion protein or a combination of IL4 and IL13 produced a moderate beneficial effect on neurite length. However, the presence of IL4 / IL13 fusion protein in the cultures significantly prevented paclitaxel-induced neurotoxicity (Figure 7). Thus, taken together, these data demonstrated that IL4 / IL13 fusion protein has an unexpectedly superior effect of protecting neurons against the toxic effects of the chemotherapeutic agent paclitaxel over IL4 / IL10 fusion protein or a combination of IL4 and IL13. The superiority of this fusion protein over the combination was surprising, especially since the in vitro system is not subject to differences in clearance of the proteins from the site of action. Rather, the data demonstrated a unique effect of the IL4 / IL13 fusion protein on neuroprotection.

[0307] Example 6 IL4 / IL13 fusion protein also cures oxaliplatin-induced polyneuropathy We investigated whether the neuroprotective effect of the IL4 / IL13 fusion protein was specific to paclitaxel-induced neurotoxicity or whether the neuroprotective effect was against a broader chemotherapy-induced polyneuropathy. The platinum-based chemotherapy drug oxaliplatin was used to induce toxic neuropathy in mice. Two cycles of five daily injections of oxaliplatin, separated by five days without intraperitoneal injection, induced mechanical allodynia that persisted for at least three weeks (Figure 8). Intrathecal injection of the IL4 / IL13 fusion protein (comprising SEQ ID NO: 4) two days after the last oxaliplatin injection significantly reduced mechanical allodynia for four days (Figure 8). Intrathecal injection of wild-type IL4 or wild-type IL13 transiently inhibited oxaliplatin-induced mechanical allodynia for approximately one day, and this inhibition was significantly shorter than the effect of the IL4 / IL13 fusion protein. Similarly, in vitro, the IL4 / IL13 fusion protein protected against oxaliplatin-induced neurotoxicity, whereas the combination of IL4 and IL13 did not (Figure 9). Thus, these data indicate that the IL4 / IL13 fusion protein protects against chemotherapy-induced neurotoxicity and chemotherapy-induced polyneuropathy.

[0308] Example 7 IL10 / IL13 and IL33 / IL13 Fusion Proteins Furthermore, it was investigated whether IL10 / IL13 fusion protein or IL33 / IL13 fusion protein has a therapeutic effect in medical applications as disclosed herein. Surprisingly, cross-linking of IL10 receptor and IL13 receptor by administration of IL10 / IL13 fusion protein or cross-linking of IL33 receptor and IL13 receptor by administration of IL33 / IL13 fusion protein can result in a stronger and longer therapeutic effect compared to administration of IL4 / IL10 fusion protein, especially in neuropathic pain models.

[0309] Example 8 Expression of cytokine receptors in the dorsal root ganglion and spinal cord We addressed whether the cytokine receptors targeted by the fusion proteins of the present invention are expressed by the sensory system. To analyze this, RNAseq data for IL10, IL4, IL13, IL27, TGFβ1, and TGFβ2 receptors in the dorsal root ganglion and spinal cord were extracted from the database by Ray et al. (Pain 2018;159:1325-1345), available at https: / / www.utdallas.edu / bbs / painneurosciencelab / sensoryomics / drgtxome / ?go. RNA sequencing revealed expression of IL10, IL4, IL13, IL27, TGFβ1, and TGFβ2 receptor chains in human and mouse dorsal root ganglion and spinal cord (Figure 10; data expressed in transcripts per million).

[0310] Example 9 Fusion Proteins of the Disclosure This example illustrates the design and production of non-limiting examples of IL13-containing fusion proteins of the present disclosure.

[0311] An IL4 / IL13 fusion protein of the present disclosure was designed comprising SEQ ID NO:1 and SEQ ID NO:14. SEQ ID NO:1 was linked to SEQ ID NO:14 using the linker of SEQ ID NO:3, resulting in SEQ ID NO:16. A hexahistidine tag was added to the N-terminus, and the fusion protein was produced by transient transfection into HEK293E cells as disclosed below. The fusion protein comprising SEQ ID NO:16 has IL4 located at the N-terminus and is shown in Figures 11, 12, and 18A as IL4 / IL13. SKP It is labeled as follows.

[0312] An IL10 / IL13 fusion protein of the present disclosure was designed comprising SEQ ID NO:5 and SEQ ID NO:14. SEQ ID NO:5 was linked to SEQ ID NO:14 using the linker of SEQ ID NO:3, resulting in SEQ ID NO:17. A hexahistidine tag was added to the N-terminus and the fusion protein was produced by transient transfection into HEK293E cells as disclosed below. The fusion protein comprising SEQ ID NO:17 contains IL13 at the N-terminus and is labeled IL13 / IL10 in Figures 11, 13, and 18D.

[0313] An IL27 / IL13 fusion protein of the present disclosure was designed comprising SEQ ID NO: 18 and SEQ ID NO: 14. SEQ ID NO: 18 was linked to SEQ ID NO: 14 using the linker of SEQ ID NO: 3, resulting in SEQ ID NO: 19. A hexahistidine tag was added to the N-terminus and the fusion protein was produced by transient transfection into HEK293E cells as disclosed below. The fusion protein comprising SEQ ID NO: 19 contains IL13 at the N-terminus and is labeled IL13 / IL27-A in Figures 11, 14, and 18C.

[0314] An IL13 / IL13 fusion protein of the present disclosure was designed that contains two copies of SEQ ID NO: 14 linked to another copy of SEQ ID NO: 14 using the linker of SEQ ID NO: 3, resulting in SEQ ID NO: 20. A hexahistidine tag was added to the N-terminus and the fusion protein was produced by transient transfection into HEK293E cells as disclosed below.

[0315] IL4 / IL13, IL10 / IL13, IL27 / IL13, and IL13 / IL13 fusion proteins were purified as disclosed below. Size exclusion chromatography showed that the IL4 / IL13 fusion protein containing SEQ ID NO: 16 migrated as a single peak (FIG. 18A), and the IL13 / IL13 fusion protein (containing SEQ ID NO: 20) migrated as a single peak (FIG. 18B).

[0316] In the case of IL27 / IL13 (IL13 / IL27-A), two peaks of recombinant protein were identified and these peaks were pooled separately after gel filtration (FIG. 18C). Pool 1 was the higher molecular weight pool. Pool 2 was the lower molecular weight and was chosen for further evaluation. Without wishing to be bound by any particular theory, pool 1 may contain multimerized and / or aggregated forms of the protein, while pool 2 may contain pure protein (e.g., monomer).

[0317] In the case of IL10 / IL13 (IL13 / IL10), two peaks of recombinant protein were identified and these peaks were pooled separately after gel filtration (FIG. 18D). Pool 1 represents the higher molecular weight pool and pool 2 represents the lower molecular weight pool. Without wishing to be bound by any particular theory, IL10 / IL13 pool 1 may contain a dimeric version of the molecule and IL10 / IL13 pool 2 may contain a monomeric version of IL10 / IL13.

[0318] As shown in FIG. 11, purified proteins were analyzed on 4-12% gradient polyacrylamide NuPage™ gels under reducing and non-reducing conditions and bands were visualized by Coomassie protein staining.

[0319] Expression and purification of IL13-containing fusion proteins. The IL13-containing fusion proteins of the present disclosure were produced by transient transfection into HEK293E cells. The cells were transfected with a pUPE expression vector containing a transgene encoding one of the IL13-containing fusion protein sequences. A hexahistidine affinity tag was cloned at the N-terminus of each IL13-containing protein to allow purification. Six days after transfection, conditioned medium containing recombinant proteins was collected by low speed centrifugation (10 min, 1000×g) followed by high speed centrifugation (10 min, 4000×g). Proteins were purified by His tag by immobilized metal affinity chromatography (IMAC). Briefly, recombinant proteins were bound to 0.5 ml of Nickel Sepharose® excel at 20° C. The Nickel Sepharose® excel containing bound proteins was collected by centrifugation and transferred into a gravity flow column. Non-specifically bound proteins were removed by washing the column with IMAC buffer (500 mM sodium chloride, 25 mM Tris, pH=8.2) containing 0 and 10 mM imidazole. Proteins were eluted with IMAC buffer containing 500 mM imidazole. Fractions of 2.5 ml were collected and recombinant protein-containing fractions were pooled. Conditioned medium and unbound IMAC fractions were analyzed by LabChip® capillary electrophoresis. The IMAC pool was concentrated to 2-4 ml using an Amicon 10 kDa spin filter. Aggregates were removed by centrifugation (10 min, 18000×g, 4°C).

[0320] The protein was further purified by gel filtration using a Superdex200 16 / 600 column equilibrated with PBS. Protein-containing fractions were analyzed by LabChip® capillary electrophoresis, and recombinant protein-containing fractions were pooled. The protein pool was sterilized by filtration through a 0.22 μm syringe filter, and the product was stored in 1 ml vials at -80°C.

[0321] Protein assay: Protein concentrations were determined spectrophotometrically by measuring absorbance at 280 nm (DropSense16, Trinean) and by using the BCA (Thermo Scientific) protein assay. All protein assays were performed according to the manufacturer's protocol.

[0322] SDS-Page: Purified proteins were analyzed on 4-12% polyacrylamide NuPage™ polyacrylamide gels under reducing and non-reducing conditions. Protein bands were visualized by Coomassie protein staining.

[0323] Example 10 Neuroprotective effect of IL13-containing fusion protein on paclitaxel-induced sensory neuron injury To determine the potential neuroprotective effects of various fusion proteins in which IL13 is linked to other regulatory cytokines (e.g., IL4, IL10, IL13, or IL27), these fusion proteins were tested in an in vitro paclitaxel-induced sensory neuron injury assay. Primary sensory neurons from mice were cultured for 24 h in the presence of paclitaxel (1 μM) and various concentrations of each fusion protein, or equimolar doses of IL13, or combinations of unlinked cytokines.

[0324] Culture of DRG neurons: DRGs were cultured as previously described (Nat. Commun. 4, 1682 (2013)). Briefly, DRGs were dissected and cultured in ice-cold dissection medium (HBSS w / o Ca). 2+ and Mg 2+ , 5 mM HEPES, and 10 mM glucose). After dissection, the axons were cut and the dissection medium was replaced with filtered enzyme mix (HBSS w / o Ca 2+ and Mg 2+ The DRGs were incubated at 37°C and 5% CO for 1 h at 4°C for 2 h, then replaced with 10 mM HEPES, 10 mM glucose, 5 mg / ml type XI collagenase (Sigma), and 10 mg / ml dispase (Gibco). 2The cells were incubated in the enzyme mix for 30 min at 37 °C. The enzyme mix was then inactivated with heat-inactivated fetal bovine serum (FBS, Sigma). The cells were incubated at 37 °C in 5% CO 2 Cells were cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% FBS (Gibco), 2 mmol / L glutamine (Gibco), and 10,000 IU / ml penicillin-streptomycin (Gibco) on glass coverslips coated with poly-L-lysine (0.01 mg / ml, Sigma) and laminin (0.02 mg / ml, Sigma) in an incubator. Cells were used for the next 1–2 days.

[0325] Stimulation for neurite length measurements: After 24 h of culture, DRG neurons were treated for 24 h with paclitaxel (1 μM) alone (n=11), or with various concentrations of IL13 fusion protein (0.12 nM, n=6; 0.6 nM or 3 nM, n=11), or in the presence of equimolar doses of individual cytokines (n=6) (n values ​​represent the number of animals tested for each condition). Neurites were visualized using β3-tubulin staining, and the number of sensory neurons was determined using NeuN staining. For each animal, 2-3 wells were evaluated (cytokines: 2 wells per concentration; paclitaxel or control: 3 wells), and 5 pictures were taken per well. For each picture, the neurite length per sensory neuron was determined and averaged into a single value per animal and condition. The percentage inhibition of paclitaxel-induced neurite length reduction per mouse culture per mouse was calculated according to the following formula (μ 対照 -μ パクリタキセル )-(μ 対照 -X サイトカイン ) / (μ 対照 -μ パクリタキセル ) × 100 (where μ is the neurite length per neuron averaged across all samples and X is the neurite length for each individual sample).

[0326] β3-Tubulin and NeuN staining: Cells were fixed with 4% PFA for 10 min, permeabilized with PBS containing 0.05% Tween-20, and subsequently incubated in blocking buffer (1% BSA and 5% normal donkey serum in PBS containing 0.05% Tween-20 and 0.01% Triton) for 1 h. Cells were incubated overnight at 4°C with rabbit anti-β3 tubulin (anti-βIII tubulin antibody-Neuronal Marker, ab18207, 1:1500, Abcam, Cambridge, UK) and NeuN (anti-NeuN antibody clone A60, MAB377, 1:500) Sigma Aldrich (Merck), Darmstadt, Germany), followed by washing and incubation with AF488-conjugated donkey anti-rabbit and 568-conjugated donkey anti-mouse secondary antibodies (Thermofisher, 1:500), followed by DAPI (1:5000, Sigma) staining, after which sections were mounted on slides with FluorSave reagent (Millipore).

[0327] Images were acquired using an Olympus IX83 microscope (Olympus). Pictures were analyzed using CellSens software (Olympus) and ImageJ (NIH) using the NeuralNetrics macro (Pani G, et al. MorphoNeuroNet: An automated method for dense neurite network analysis. Cytom Part A. 2014 Feb;85(2):188-99). Other plugins used were the Olympus Viewer plugin (Olympus). The number of neurons was automatically counted based on NeuN staining using Cell sense software. Neurite length was determined using the NeuralNetric macro in ImageJ (Fiji).

[0328] result Paclitaxel reduced neurite length by approximately 67% compared to control cultured sensory neurons.

[0329] Figure 12 shows the neuroprotective effect of two IL4 / IL13 fusion proteins compared with IL13 alone or the combination of IL4 and IL13. IL13 dose-dependently inhibited the paclitaxel-induced decrease in neurite length, up to about 9% at a concentration of 3 nM (Figure 12). The combination of IL4 and IL13 did not significantly inhibit paclitaxel-induced neurite length at any of the concentrations tested.

[0330] The fusion protein labeled IL4 / IL13 comprises SEQ ID NO: 4. IL4 / IL13 SKP The fusion proteins labeled as IL4 / IL13 contain SEQ ID NO: 16. IL13 is located at the C-terminus of these constructs. IL4 / IL13 inhibited paclitaxel-induced decrease in neurite length in a dose-dependent manner, with a maximal effect of 35% at 3 nM (FIG. 12). IL4 / IL13 inhibited paclitaxel-induced decrease in neurite length significantly better than the combination of IL4 and IL13 or IL13 alone, inhibiting approximately 4-fold at a concentration of 3 nM compared to IL13 (FIG. 12). An alternative IL4 / IL13 fusion protein, IL4 / IL13 SKP At all concentrations tested, IL4 / IL13 inhibited the paclitaxel-induced decrease in neurite length to a similar extent as IL4 / IL13 (Figure 12). SKP The IL...

Claims

1. (a) a first cytokine that is IL13 and comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or any one of SEQ ID NOs:9-15; and (b) a second cytokine, (i) a second cytokine which is IL4 and comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1 or any one of SEQ ID NOs:26-28; (ii) a second cytokine that is IL10 and comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5; (iii) a second cytokine that is IL13 and comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or any one of SEQ ID NOs:9-15; or (iv) a second cytokine that is IL33 and comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:6 or any one of SEQ ID NOs:29-34; and (c) a linker, a linker, wherein the C-terminus of the first cytokine is linked to the N-terminus of the linker, and the C-terminus of the linker is linked to the N-terminus of the second cytokine; or The C-terminus of the second cytokine is linked to the N-terminus of the linker, and the C-terminus of the linker is linked to the N-terminus of the first cytokine. A fusion protein comprising:

2. The fusion protein of claim 1 , wherein the second cytokine is IL4.

3. The fusion protein of claim 1 , wherein the second cytokine is IL10.

4. The fusion protein of claim 1 , wherein the second cytokine is IL13.

5. The fusion protein of claim 1 , wherein the second cytokine is IL33.

6. The fusion protein of any one of claims 1 to 5, wherein the first cytokine comprises a wild-type IL13 amino acid sequence.

7. The fusion protein of any one of claims 1 to 6, wherein the second cytokine comprises a wild-type IL4, IL10, IL13 or IL33 amino acid sequence.

8. The fusion protein of any one of claims 1 to 7, wherein the first cytokine is human IL13 and the second cytokine is human IL4, IL10, IL13 or IL33.

9. The fusion protein of claim 1, wherein the second cytokine is IL4 and comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:1 or any one of SEQ ID NOs:26-28.

10. The fusion protein of claim 1, wherein the second cytokine is IL4 and comprises an amino acid sequence that is SEQ ID NO:1 or any one of SEQ ID NOs:26-28.

11. 10. The fusion protein of claim 1 or 6, wherein the second cytokine is (a) is IL10 and comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5; (b) is IL13 and comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or any one of SEQ ID NOs:9-15; or (c) A fusion protein which is IL33 and comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:6 or any one of SEQ ID NOs:29-34.

12. 10. The fusion protein of claim 1 or 6, wherein the second cytokine is (a) is IL10 and comprises the amino acid sequence of SEQ ID NO:5; (b) is IL13 and comprises an amino acid sequence of SEQ ID NO:2 or any one of SEQ ID NOs:9-15; or (c) A fusion protein which is IL33 and comprises an amino acid sequence which is SEQ ID NO:6 or any one of SEQ ID NOs:29-34.

13. The fusion protein of any one of claims 1 to 5 and 9 to 12, wherein the first cytokine, which is IL13, comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or any one of SEQ ID NOs:9 to 15.

14. The fusion protein of any one of claims 1 to 5 and 9 to 12, wherein the first cytokine, which is IL13, comprises an amino acid sequence which is SEQ ID NO: 2 or any one of SEQ ID NOs: 9 to 15.

15. The fusion protein of any one of claims 1 to 14, wherein the C-terminus of the first cytokine is linked to the N-terminus of the linker and the C-terminus of the linker is linked to the N-terminus of the second cytokine.

16. 15. The fusion protein of any one of claims 1 to 14, wherein the C-terminus of the second cytokine is linked to the N-terminus of the linker and the C-terminus of the linker is linked to the N-terminus of the first cytokine.

17. The fusion protein according to any one of claims 1 to 16, wherein the linker comprises the amino acid sequence of SEQ ID NO:

3.

18. A fusion protein comprising the amino acid sequence of SEQ ID NO:

16.

19. The fusion protein of any one of claims 1 to 18, further comprising one or more chemical modifications.

20. 20. The fusion protein of claim 19, wherein the one or more chemical modifications are glycosylation, fucosylation, sialylation or pegylation.

21. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 20 and a pharma- ceutically acceptable excipient.

22. 22. The pharmaceutical composition of claim 21, in unit dosage form.

23. A nucleic acid molecule comprising a polynucleotide sequence encoding the fusion protein of any one of claims 1 to 20.

24. A vector comprising the nucleic acid molecule of claim 23.

25. 25. A composition for use in a method of treating a condition in a subject in need thereof, the composition comprising a fusion protein according to any one of claims 1 to 20, a pharmaceutical composition according to claim 21 or claim 22, a nucleic acid molecule according to claim 23, or a vector according to claim 24, the method comprising administering to the subject an effective amount of the composition.

26. 1. A fusion protein comprising a first cytokine and a second cytokine for use in a method of treating pain, inflammation, neuropathy or neurodegeneration in a subject in need thereof, the method comprising administering to the subject an effective amount of the fusion protein; (a) the first cytokine is IL13 and comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or any one of SEQ ID NOs:9-15; and (b) the second cytokine is (i) IL4 and comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1 or any one of SEQ ID NOs:26-28; (ii) is IL10 and comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5; (iii) is IL13 and comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or any one of SEQ ID NOs:9-15; or (iv) A fusion protein which is IL33 and comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:6 or any one of SEQ ID NOs:29-34.

27. 27. The fusion protein of claim 26, wherein the second cytokine is IL4.

28. 27. The fusion protein of claim 26, wherein the second cytokine is IL10.

29. 27. The fusion protein of claim 26, wherein the second cytokine is IL13.

30. 27. The fusion protein of claim 26, wherein the second cytokine is IL33.

31. The fusion protein of any one of claims 26 to 30, for treating a neurological disorder in a subject.

32. 32. The fusion protein of claim 31, wherein the neuropathy is chemotherapy-induced neuropathy.

33. 33. The fusion protein of claim 31 or 32, wherein the neuropathy is a peripheral neuropathy.

34. The fusion protein of any one of claims 26 to 30, for treating neurodegeneration in a subject.

35. The fusion protein of any one of claims 26 to 34, wherein nerve fiber degeneration is reduced or prevented in a subject.

36. The fusion protein of any one of claims 26 to 30, for treating pain in a subject.

37. The fusion protein of any one of claims 26 to 30, for treating inflammation in a subject.

38. 26. The composition of claim 25, wherein the condition comprises pain, inflammation, neuropathy or neurodegeneration.

39. 26. The composition of claim 25, wherein the condition is a neurological disorder.

40. 26. The composition of claim 25, wherein the neuropathy is chemotherapy-induced neuropathy.

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