A fusion protein containing IL13

Abstract

The present invention relates to a fusion protein comprising interleukin (13) and a regulatory cytokine, wherein the regulatory cytokine is, for example, an interleukin selected from interleukin (4), interleukin (10), interleukin (27), interleukin (33), transforming growth factor β (1), transforming growth factor β (2) and interleukin 13; to a nucleic acid molecule encoding such a fusion protein, a vector comprising such a nucleic acid molecule, and a host cell comprising such a nucleic acid molecule or such a vector. The present invention further relates to a method for producing such a fusion protein. The fusion protein or a gene therapy vector encoding the fusion protein can be used to prevent or treat a disease characterized by pathological pain, chronic pain, neuroinflammation and / or neurodegeneration.

Description

[0001] Cross-references

[0002] This application claims priority from Netherlands Patent Application No. 2022982 and Netherlands Patent Application No. 2022984, each of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention belongs to the field of neuroimmunology and pharmacology, and is particularly useful for treating chronic pain, neuroinflammatory and neurodegenerative diseases, and inflammatory diseases. The present invention particularly relates to novel fusion proteins comprising interleukin 13 (IL13) and regulatory cytokines (e.g., but not limited to, selected from the group consisting of interleukins: interleukin 4 (IL4), interleukin 10 (IL10), interleukin 27 (IL27), interleukin 33 (IL33), transforming growth factor β1 (TGFβ1), transforming growth factor β2 (TGFβ2), and IL13 itself) that are not physically fused together or physically fused together via a linker sequence. In particular, the present invention provides an IL4 / IL13, IL10 / IL13, IL27 / IL13, IL33 / IL13, TGFβ1 / IL13, TGFβ2 / IL13, or IL13 / IL13 fusion protein that has superior analgesic, neuroprotective, and anti-inflammatory activity compared to a combination of individual cytokines, or compared to a fusion protein of IL4 and IL10. The present invention also provides nucleic acid sequences encoding fusion proteins, such as IL4 / IL13 fusion protein, IL10 / IL13 fusion protein, IL27 / IL13 fusion protein, IL33 / IL13 fusion protein, TGFβ1 / IL13 fusion protein, TGFβ2 / IL13 fusion protein or IL13 / IL13 fusion protein, expression vectors comprising such nucleic acid sequences, host cells or host organisms altered to comprise nucleic acid sequences encoding IL4 / IL13 fusion protein, IL10 / IL13 fusion protein, IL27 / IL13 fusion protein, IL33 / IL13 fusion protein, TGFβ1 / IL13 fusion protein, TGFβ2 / IL13 fusion protein, IL / 13 / IL13 fusion protein, and the fusion proteins themselves. The present 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. Transgenic organisms containing the nucleic acid sequences of the present invention are also provided. The present invention also relates to pharmaceutical compositions containing, for example, IL4 / IL13, IL10 / IL13, IL27 / IL13, IL33 / IL13, TGFβ1 / IL13, TGFβ2 / IL13, or IL13 / IL13 fusion proteins. Finally, the present invention teaches the use of IL4 / IL13 or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13 fusion proteins as drugs, in particular for the prevention and / or treatment of chronic pain and conditions characterized by neuroinflammation, neurodegeneration or inflammation. Background Art

[0004] Chronic pain affects millions of people and constitutes the largest unmet need in modern medicine 1-4 In 2016, an estimated 20.4% of U.S. adults (50 million) had chronic pain, and 8.0% of U.S. adults (19.6 million) had high-impact chronic pain. 5 Opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the main classes of drugs used to combat pain. However, these analgesics ("painkillers") are often ineffective and have serious side effects (addiction, gastrointestinal bleeding, cardiovascular, etc.). It is estimated that approximately 50% of people with chronic pain (approximately 5%-10% of the general population) do not receive adequate pain relief. 6 .

[0005] People with chronic pain experience spontaneous pain, hyperalgesia (an increased experience of pain in response to noxious stimuli), and allodynia (pain evoked by normally non-painful stimuli). Pain has multiple causes and arises from biological processes at different anatomical levels. 7-10 The three main mechanisms of pain management are: the generation of stimuli that trigger sensory nerve endings in peripheral nerve ending areas; the stimulation, sensitization, and dysfunction of peripheral sensory neurons that transmit action potentials to the spinal cord; neurons and glial cells in the dorsal horn of the spinal cord, where the action potentials of peripheral neurons are transmitted to spinal pain neurons through synapses; and finally, the central mechanisms in the brain. All of these processes contribute differently to different types of chronic pain. Based on this contribution, pain is divided into several types, including nociceptive pain, peripheral and central neuropathic pain, and mixed pain. Analgesics used clinically only target one level of pain: NSAIDs reduce the generation of noxious stimuli in peripheral nerve ending areas, while opioids inhibit central mechanisms. Coupled with their notorious side effects, this explains the limited clinical efficacy of these analgesics.

[0006] Over the past decade, it has become increasingly clear that pain signals are not transmitted directly to the brain, but are mediated by neuroinflammatory processes involving glial cells and sensory neurons in the spinal cord. Cytokines are known to coordinate immune and inflammatory processes and are also crucial in controlling pain. Proinflammatory cytokines enhance inflammation and promote pain, while regulatory (e.g., anti-inflammatory) cytokines suppress inflammation. The balance between proinflammatory and regulatory cytokines determines the outcome of inflammatory responses in the body. 11 Although the pain-promoting effects of pro-inflammatory cytokines are well known 12 However, knowledge about the role of regulatory cytokines in pain is limited. 13 Blocking regulatory cytokines such as TGFβ, IL10, IL4, and IL13 significantly impairs transient inflammatory hyperalgesia 14 and chemotherapy-induced allodynia 15This effect of cytokines goes far beyond reducing inflammation and is consistent with anti-inflammatory drugs such as corticosteroids, which have only limited analgesic effects. 16,17 It is noteworthy that not only are glial cells regulated by cytokines, but sensory neurons themselves can also respond directly to cytokines. In fact, sensory neurons express receptors for all regulatory cytokines, although expression varies between neuronal subpopulations. 18 Pain caused by peripheral inflammation or nerve damage is associated with activation of spinal microglia and astrocytes, which promote pain by enhancing spinal pain signaling. 19-22 .

[0007] Given the role of neuroinflammation in chronic pain, regulatory cytokines may target pain at multiple levels. Indeed, they inhibit 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 proinflammatory mediators by glial cells. 21,22 However, the analgesic effect of treatment with independent regulatory cytokines (such as IL10 or IL13) or anti-inflammatory cytokines (IL1 receptor antagonists) is limited. 19 , probably because optimal analgesic activity requires the synergistic action of various regulatory cytokines and because their bioavailability is relatively poor due to rapid clearance by the kidneys. Therefore, a new strategy for the resolution of chronic pain with regulatory cytokines using fusion proteins of IL4 and IL10 has been proposed. 19,23 Intrathecal administration of analgesics is a common practice in pain management because it allows for lower doses of analgesics and reduces their toxicity. 24 Intrathecal injection of IL4 / IL10 fusion protein alleviates pain in mouse models of various types of pain 19 Notably, three repeated intrathecal administrations of the IL4 / IL10 fusion protein resulted in sustained relief of pain induced by paw inflammation (e.g., complete and permanent resolution of chronic pain, such as nociceptive pain), without modulating peripheral inflammation per se. 19 Interestingly, the IL4 / IL10 fusion protein was more effective than either the individual wild-type cytokines or even the combination of these cytokines. 19 .

[0008] In neuropathic pain 19 and osteoarthritis pain models 25Pain relief by IL4 / IL10 fusion proteins has also been observed in patients with neuropathic and osteoarthritis pain. However, the effects of IL4 / IL10 fusion proteins on neuropathic and osteoarthritis pain can be transient, even after multiple injections. Therefore, there is a need to provide molecules with long-term analgesic effects for the prevention or treatment of neuropathic and osteoarthritis pain. Summary of the Invention

[0009] The present disclosure provides a single molecule that targets neuroinflammation and neurodegeneration and has a lasting effect on chronic neuropathic pain. This molecule can be used to treat a variety of diseases or disorders with different etiologies in which chronic pain, neuroinflammation, and / or neurodegeneration play a role. The present disclosure provides a fusion protein comprising interleukin 13 (IL13) directly or indirectly linked to a regulatory cytokine.

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

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

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

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

[0014] In some aspects, disclosed herein is the use of a fusion protein comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence for promoting neuroprotection in a subject in need thereof.

[0015] In some aspects, disclosed herein are uses of fusion proteins comprising an interleukin 13 (IL13) amino acid sequence and a regulatory cytokine amino acid sequence for modulating the activity of a signaling pathway in cells of the nervous system.

[0016] In some embodiments, the regulatory cytokine is selected from interleukin 4 (IL4), interleukin 10 (IL10), interleukin 33 (IL33), transforming growth factor beta 1 (TGFβ1), transforming growth factor beta 2 (TGFβ2), and another 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 another IL13. In some embodiments, IL13 comprises wild-type IL13. In some embodiments, IL13 is mammalian IL13. In some embodiments, IL13 is human IL13. In some embodiments, the regulatory cytokine comprises wild-type regulatory cytokine. In some embodiments, the regulatory cytokine is mammalian regulatory cytokine. In some embodiments, the regulatory cytokine is a human regulatory cytokine. In some embodiments, interleukin 27 comprises interleukin 27 alpha (IL27A). In some embodiments, IL27A comprises an L134C substitution relative to SEQ ID NO: 36. In some embodiments, IL13 binds to interleukin 13 receptor alpha 1 (IL-13Rα1) with an affinity that is less than two times greater and less than two times less than wild-type IL13. In some embodiments, IL13 binds to interleukin 13 receptor alpha 2 (IL-13Rα2) with an affinity that is less than two times greater and less than two times less than wild-type IL13. In some embodiments, IL13 binds to interleukin 4 receptor alpha (IL-4Rα) with an affinity that is less than two times greater and less than two times less than wild-type IL13. In some embodiments, the regulatory cytokine amino acid sequence is a derivative sequence that binds to all subunits of the regulatory cytokine receptor with an affinity comparable to that of the wild-type regulatory cytokine. In some embodiments, the regulatory cytokine amino acid sequence is a derivative sequence of a natural receptor that activates a regulatory cytokine. In some embodiments, IL13 comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from SEQ ID NO: 2 and any one of SEQ ID NO: 9-15. In some embodiments, IL13 comprises an amino acid sequence selected from SEQ ID NO: 2 and any one of SEQ ID NO: 9-15. In some embodiments, IL13 comprises an amino acid sequence having 1 to 10 amino acid deletions, insertions, substitutions, or a combination thereof relative to a sequence selected from SEQ ID NO: 2 and any one of SEQ ID NO: 9-15.In some embodiments, the regulatory cytokine comprises an amino acid sequence having at least 90% sequence identity 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 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 to 10 amino acid deletions, insertions, substitutions, or combinations thereof relative to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 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, IL13 and the regulatory cytokine are covalently linked. In some embodiments, IL13 and the regulatory cytokine are connected via a linker. In some embodiments, the C-terminus of IL13 is connected to the N-terminus of the cytokine, optionally via a linker. In some embodiments, the N-terminus of IL13 is connected 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 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, postoperative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-related neuropathy, chemotherapy-induced neuropathy, polyneuropathy, mononeuropathy, multiple mononeuropathy, cranial neuropathy, primary motor neuropathy, primary sensory neuropathy, sensorimotor neuropathy, autonomic neuropathy, idiopathic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, pudendal neuralgia, atypical trigeminal neuralgia, sciatica, brachial plexopathy, or intercostal neuralgia. In some embodiments, the neuropathy is associated with pain, numbness, weakness, burning, atrophy, tingling, twitching, 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-orthopedic surgical pain, musculoskeletal pain, chemotherapy-related pain, chemotherapy-induced allodynia, pain after spinal cord injury, pain after stroke, 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, postoperative 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 associated with Alpers disease, arachnoiditis, arthrofibrosis, ataxia cerebral palsy, autoimmune atrophic gastritis, amyloidosis, hATTR amyloidosis, avascular necrosis, back pain, Batten disease, Behçet's disease (syndrome), breakthrough pain, burning mouth syndrome, bursitis, central autosomal dominant arteriopathy with subcortical infarcts 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-Tooth disease (CMT), chronic functional abdominal pain (CFAP), chronic pancreatitis, collapsed lung (pneumothorax), corticobasal degeneration, crush injury, corneal neuropathic pain, compression Syndrome, degenerative disc disease, dermatomyositis, dementia, dystonia, Ehlers-Danlos syndrome (EDS), endometriosis, eosinophilic myalgia syndrome (EMS), erythromelalgia, failed back surgery syndrome (FBSS), fibromyalgia, Friedreich's ataxia, frontotemporal dementia, glossopharyngeal neuralgia, growing pains, herniated disc, hydrocephalus, intercostal neuralgia, interstitial cystitis, juvenile dermatitis, knee injury, leg pain, Lewy body dementia, lumbago-hematuria syndrome, Lyme disease, meralgia paresthesia, mitochondrial disease, mixed dementia, motor neuron disease (MND), monomelic muscular dystrophy, multiple system atrophy (MSA), myositis, neck pain, occipital neuralgia, osteoporosis, rhabdomyolysis, Paget's disease, Parsonage-Turner syndrome Turner Syndrome), pelvic pain, peripheral neuropathy, phantom limb pain, nerve pinch, plantar fasciitis, polymyalgia rheumatica, polymyositis, post-herniorrhaphy pain syndrome, post-mastectomy pain syndrome, post-stroke pain, post-thoracotomy pain syndrome, post-polio syndrome, primary lateral sclerosis, psoriatic arthritis, pudendal neuralgia, radiculopathy, restless legs syndrome, rheumatoid arthritis (RA), sacroiliac joint dysfunction, sarcoidosis, Scheuemann's Kyphosis Disease, sciatica, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), herpes zoster, spasmodic torticollis, sphincter of Oddi dysfunction, spinal cord injury, spinal stenosis, syringomyelia, Tarlov cyst Cysts), tethered cord syndrome, thoracic outlet syndrome (TOS), TMJ disorder, transverse myelitis, traumatic brain injury, vascular pain, vulvodynia, cervical hyperextension, 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, inflammation includes chronic inflammation. In some embodiments, inflammation includes local inflammation or systemic inflammation. In some embodiments, inflammation is related to: inflammatory bowel disease, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, glomerulonephritis, sepsis, adult respiratory distress syndrome, dermatitis, sarcoidosis, allergic inflammation, psoriasis, ankylosing spondylarthritis, systemic lupus erythematosus, vasculitis, gout, allografts, xenografts, autoimmune diseases, Sjogren's disease (Sjogren's disease), burns, trauma, stroke, myocardial infarction, atherosclerosis, diabetes, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, cytokines or other therapeutic monoclonal antibodies administered in vivo toxicity induced. In some embodiments, nerve fiber degeneration is reduced. In some embodiments, nerve fiber loss is reduced. In some embodiments, the maintenance of nerve fiber density is promoted. In some embodiments, nerve fiber regrowth is promoted. In some embodiments, neuroprotection of the central nervous system is promoted. In some embodiments, neuroprotection of the peripheral nervous system is promoted. In some embodiments, the loss of nerve fibers in the epidermis is reduced. In some embodiments, neuronal dysfunction is reduced. In some embodiments, the fusion protein triggers a therapeutic effect that is greater than an equal amount of IL13, regulatory cytokines, or a combination thereof. In some embodiments, the fusion protein triggers a therapeutic effect that lasts longer than an equal amount of IL13, regulatory cytokines, or a combination thereof. In some embodiments, the fusion protein is present in a pharmaceutical composition comprising the fusion protein and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical 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 / mL to about 100 mg / mL. In some embodiments, the fusion protein is formulated for administration at a dose between about 0.5 μg / kg body weight and about 1 mg / kg body weight. In some embodiments, the fusion protein is formulated for administration with a controlled release formulation. In some embodiments, the fusion protein is formulated for administration by parenteral, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, 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 somatic sensory 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 cells are glial cells. In some embodiments, the nervous system cells are microglial cells. In some embodiments, the nervous system cells are infiltrating cells. In some embodiments, the nervous system cells are infiltrating macrophages. In some embodiments, the signaling pathway is modulated in the presence of pro-inflammatory mediators.

[0017] In some aspects, disclosed herein is a method of treating a neuropathy in a subject in need thereof, 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 pain in a subject in need thereof, 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 neurodegeneration or neuroinflammation in a subject in need thereof, 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 treating inflammation in a subject in need thereof, 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 promoting neuroprotection in a subject in need thereof, 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.

[0022] In some aspects, disclosed herein is a method of modulating signaling pathway activity 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.

[0023] In some embodiments, the regulatory cytokine is selected from interleukin 4 (IL4), interleukin 10 (IL10), interleukin 33 (IL33), transforming growth factor beta 1 (TGFβ1), transforming growth factor beta 2 (TGFβ2), and another 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, IL13 comprises wild-type IL13. In some embodiments, IL13 is mammalian IL13. In some embodiments, IL13 is human IL13. In some embodiments, the regulatory cytokine comprises a 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, interleukin 27 comprises interleukin 27 alpha (IL27A). In some embodiments, IL27A comprises an L134C substitution relative to SEQ ID NO: 36. In some embodiments, IL13 binds to interleukin 13 receptor alpha 1 (IL-13Rα1) with an affinity that is less than two times greater and less than two times less than wild-type IL13. In some embodiments, IL13 binds to interleukin 13 receptor alpha 2 (IL-13Rα2) with an affinity that is less than two times greater and less than two times less than wild-type IL13. In some embodiments, IL13 binds to interleukin 4 receptor alpha (IL-4Rα) with an affinity that is less than two times greater and less than two times less than wild-type IL13. In some embodiments, the regulatory cytokine amino acid sequence is a derivative sequence that binds to all subunits of the regulatory cytokine receptor with an affinity comparable to that of the wild-type regulatory cytokine. In some embodiments, the regulatory cytokine amino acid sequence is a derivative sequence of a natural receptor that activates a regulatory cytokine. In some embodiments, IL13 comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from SEQ ID NO: 2 and any one of SEQ ID NO: 9-15. In some embodiments, IL13 comprises an amino acid sequence selected from SEQ ID NO: 2 and any one of SEQ ID NO: 9-15. In some embodiments, IL13 comprises an amino acid sequence having 1 to 10 amino acid deletions, insertions, substitutions, or a combination thereof relative to a sequence selected from SEQ ID NO: 2 and any one of SEQ ID NO: 9-15.In some embodiments, the regulatory cytokine comprises an amino acid sequence having at least 90% sequence identity 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 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 to 10 amino acid deletions, insertions, substitutions, or combinations thereof relative to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 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, IL13 and the regulatory cytokine are covalently linked. In some embodiments, IL13 and the regulatory cytokine are connected via a linker. In some embodiments, the C-terminus of IL13 is connected to the N-terminus of the cytokine, optionally via a linker. In some embodiments, the N-terminus of IL13 is connected 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 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, postoperative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-related neuropathy, chemotherapy-induced neuropathy, polyneuropathy, mononeuropathy, multiple mononeuropathy, cranial neuropathy, primary motor neuropathy, primary sensory neuropathy, sensorimotor neuropathy, autonomic neuropathy, idiopathic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, pudendal neuralgia, atypical trigeminal neuralgia, sciatica, brachial plexopathy, or intercostal neuralgia. In some embodiments, the neuropathy is associated with pain, numbness, weakness, burning, atrophy, tingling, twitching, 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-orthopedic surgical pain, musculoskeletal pain, chemotherapy-related pain, chemotherapy-induced allodynia, pain after spinal cord injury, pain after stroke, 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, postoperative 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 associated with Alpers disease, arachnoiditis, arthrofibrosis, ataxia cerebral palsy, autoimmune atrophic gastritis, amyloidosis, hATTR amyloidosis, avascular necrosis, back pain, Batten disease, Behçet's disease (syndrome), breakthrough pain, burning mouth syndrome, bursitis, central autosomal dominant arteriopathy with subcortical infarcts 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-Tooth disease (CMT), chronic functional abdominal pain (CFAP), chronic pancreatitis, collapsed lung (pneumothorax), corticobasal degeneration, crush injury, corneal neuropathic pain, compression Syndrome, degenerative disc disease, dermatomyositis, dementia, dystonia, Ehlers-Danlos syndrome (EDS), endometriosis, eosinophilic myalgia syndrome (EMS), erythromelalgia, failed back surgery syndrome (FBSS), fibromyalgia, Friedreich's ataxia, frontotemporal dementia, glossopharyngeal neuralgia, growing pains, herniated disc, hydrocephalus, intercostal neuralgia, interstitial cystitis, juvenile dermatitis, knee injury, leg pain, Lewy body dementia, lumbago-hematuria syndrome, Lyme disease, meralgia paresthesia, mitochondrial disease, mixed dementia, motor neuron disease (MND), monomelic muscular dystrophy, multiple system atrophy (MSA), myositis, neck pain, occipital neuralgia, osteoporosis, rhabdomyolysis, Paget's disease, Parsonage-Turner syndrome Turner Syndrome), pelvic pain, peripheral neuropathy, phantom limb pain, nerve pinch, plantar fasciitis, polymyalgia rheumatica, polymyositis, post-herniorrhaphy pain syndrome, post-mastectomy pain syndrome, post-stroke pain, post-thoracotomy pain syndrome, post-polio syndrome, primary lateral sclerosis, psoriatic arthritis, pudendal neuralgia, radiculopathy, restless legs syndrome, rheumatoid arthritis (RA), sacroiliac joint dysfunction, sarcoidosis, Scheuemann's Kyphosis Disease, sciatica, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), herpes zoster, spasmodic torticollis, sphincter of Oddi dysfunction, spinal cord injury, spinal stenosis, syringomyelia, Tarlov cyst Cysts), tethered cord syndrome, thoracic outlet syndrome (TOS), TMJ disorder, transverse myelitis, traumatic brain injury, vascular pain, vulvodynia, cervical hyperextension, 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, inflammation includes chronic inflammation. In some embodiments, inflammation includes local inflammation or systemic inflammation. In some embodiments, inflammation is related to: inflammatory bowel disease, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, glomerulonephritis, sepsis, adult respiratory distress syndrome, dermatitis, sarcoidosis, allergic inflammation, psoriasis, ankylosing spondylarthritis, systemic lupus erythematosus, vasculitis, gout, allografts, xenografts, autoimmune diseases, Sjogren's disease (Sjogren's disease), burns, trauma, stroke, myocardial infarction, atherosclerosis, diabetes, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, cytokines or other therapeutic monoclonal antibodies induced by in vivo administration toxicity. 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 the 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, administration of the fusion protein triggers a greater degree of therapeutic effect than administration of an equal amount of IL13, regulatory cytokines, or a combination thereof. In some embodiments, administration of the fusion protein triggers a therapeutic effect that lasts longer than administration of an equal amount of IL13, regulatory cytokines, or a combination thereof. In some embodiments, administration results in a greater degree of pain relief compared to administration of an equivalent amount of IL13 and regulatory cytokines, alone or in combination, as measured by mechanical sensitivity of von Frey hairs in a paclitaxel-induced neuropathy mouse model. In some embodiments, the fusion protein is present in a pharmaceutical composition comprising the fusion protein and one or more pharmaceutically 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, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intraventricular, intracisternal, or intraparenchymal pathways. In some embodiments, the nervous system cells are neurons.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 somatic sensory 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 microglia. 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.

[0024] In some aspects, disclosed herein is a fusion protein comprising an interleukin 13 (IL13) amino acid sequence that is a wild-type IL13 sequence and a regulatory cytokine amino acid 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 that is a wild-type sequence.

[0026] 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.

[0027] 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 regulatory cytokine amino acid sequence is a derivative sequence that binds to all receptor subunits to which the wild-type regulatory cytokine binds with an affinity comparable to that of the wild-type regulatory cytokine.

[0028] In some embodiments, the regulatory cytokine is selected from interleukin 4 (IL4), interleukin 10 (IL10), interleukin 33 (IL33), transforming growth factor beta 1 (TGFβ1), transforming growth factor beta 2 (TGFβ2), and another 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 another interleukin 13 (aIL13). In some embodiments, IL13 is mammalian IL13. In some embodiments, IL13 is human IL13. In some embodiments, IL13 comprises wild-type IL13. In some embodiments, the regulatory cytokine comprises wild-type regulatory cytokine. In some embodiments, regulatory cytokines are mammalian regulatory cytokines. In some embodiments, regulatory cytokines are human regulatory cytokines. In some embodiments, interleukin 27 comprises interleukin 27 alpha (IL27A). In some embodiments, IL27A comprises an L134C substitution relative to SEQ ID NO: 36. In some embodiments, IL13 binds to interleukin 13 receptor alpha 1 (IL-13Rα1) with an affinity that is less than two times greater and less than two times less than wild-type IL13. In some embodiments, IL13 binds to interleukin 13 receptor alpha 2 (IL-13Rα2) with an affinity that is less than two times greater and less than two times less than wild-type IL13. In some embodiments, IL13 binds to interleukin 4 receptor alpha (IL-4Rα) with an affinity that is less than two times greater and less than two times less than wild-type IL13. In some embodiments, the regulatory cytokine amino acid sequence is a derivative sequence that binds to all subunits of the regulatory cytokine receptor with an affinity comparable to that of the wild-type regulatory cytokine. In some embodiments, the regulatory cytokine amino acid sequence is a derivative sequence of a natural receptor that activates the regulatory cytokine. In some embodiments, IL13 comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from SEQ ID NO: 2 and any one of SEQ ID NO: 9-15. In some embodiments, IL13 comprises an amino acid sequence selected from SEQ ID NO: 2 and any one of SEQ ID NO: 9-15. In some embodiments, IL13 comprises an amino acid sequence having 1 to 10 amino acid deletions, insertions, substitutions, or a combination thereof relative to a sequence selected from SEQ ID NO: 2 and any one of SEQ ID NO: 9-15.In some embodiments, the regulatory cytokine comprises an amino acid sequence having at least 90% sequence identity 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 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 to 10 amino acid deletions, insertions, substitutions, or combinations thereof relative to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 26-28, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 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, IL13 and the regulatory cytokine are covalently linked. In some embodiments, IL13 and the regulatory cytokine are connected via a linker. In some embodiments, the C-terminus of IL13 is connected to the N-terminus of the cytokine, optionally via a linker. In some embodiments, the N-terminus of IL13 is connected 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 glycosylation, fucosylation, sialylation, and pegylation. In some embodiments, the protein construct comprises an affinity tag. In some embodiments, a nucleic acid molecule encoding a 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 the nucleic acid. In some embodiments, the fusion protein is present in a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient.In some embodiments, the nucleic acid vector is present in a pharmaceutical composition that also includes a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in unit dosage form. In some embodiments, the fusion protein is present in a pharmaceutical composition in an amount of about 50 μg to about 100 mg / mL. In some embodiments, the fusion protein is formulated for administration at a dosage of about 0.5 μg to 1 mg / kg 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, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intraventricular, intracerebral cistern or intra-organ parenchymal pathway. In some embodiments, an effective amount of the pharmaceutical composition is administered to a subject in need. In some embodiments, the fusion protein is produced by culturing cells under conditions that allow the production of the fusion protein, wherein the cells comprise a polynucleotide sequence. In some embodiments, the fusion protein is harvested. In some embodiments, the fusion protein is purified from the harvested culture medium. In some embodiments, the fusion protein is used to crosslink the interleukin 13 receptor to a regulatory cytokine receptor.

[0029] 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 β1, transforming growth factor β2, and interleukin 13.

[0030] In some embodiments, interleukin 13 and the interleukin selected from interleukin 4, interleukin 10, interleukin 33, transforming growth factor β1, transforming growth factor β2 and interleukin 13 are connected 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 β1, transforming growth factor β2 and interleukin 13. In some embodiments, an interleukin selected from interleukin 4, interleukin 10, interleukin 33, transforming growth factor β1, transforming growth factor β2 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 modification is selected from glycosylation, fucosylation, sialylation and pegylation. In some embodiments, interleukin 13 is human interleukin 13. In some embodiments, 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 β1 is human transforming growth factor β1, and / or the transforming growth factor β2 is human transforming growth factor β2. 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 prepared by a method comprising the steps of culturing the host cell under conditions that allow the production of the fusion protein, and optionally purifying the fusion protein from the conditioned medium. In some embodiments, the fusion protein is present in a pharmaceutical composition that also includes a pharmaceutically acceptable carrier. In some embodiments, the fusion protein is used as a medicament. In some embodiments, the fusion protein is used to prevent or treat a condition 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 post-orthopedic surgical pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-surgical peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-related pain, chemotherapy-induced allodynia, complex regional pain syndrome, pain after spinal cord injury, pain after stroke, 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 fusion protein is used to prevent or treat a clinical condition in a mammal, such as a human, for which interleukin 13 is indicated. In some embodiments, the fusion protein is used to prevent or treat a clinical condition in a mammal, such as a human, for which interleukin 4 and / or interleukin 10 and / or interleukin 33 and / or interleukin 27 and / or transforming growth factor β1 and / or transforming growth factor β2 is indicated.

[0031] In certain aspects, disclosed herein is a gene therapy vector comprising one or more nucleotide sequences encoding interleukin 13 and an interleukin selected from interleukin 4, interleukin 10, interleukin 27, interleukin 33, transforming growth factor β1, transforming growth factor β2, and interleukin 13, for preventing or treating a condition characterized by chronic pain, neuroinflammation and / or neurodegeneration.

[0032] 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 post-orthopedic surgical pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-surgical peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-related pain, chemotherapy-induced allodynia, complex regional pain syndrome, pain after spinal cord injury, pain after stroke, 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.

[0033] In some aspects, the present invention relates to a fusion protein comprising at least 2, 3, 4, preferably 2 regulatory (e.g., anti-inflammatory) interleukins selected from interleukin 13 (IL13), interleukin 4 (IL4), interleukin 10 (IL10), interleukin 27 (IL27), interleukin 33 (IL33), transforming growth factor β1 (TGFβ1) and transforming growth factor β2 (TGFβ2).

[0034] Preferably, the present invention relates to a fusion protein comprising IL13 and another, ie a second, interleukin / cytokine, preferably selected from the group consisting of IL4, IL10, IL27, IL33, TGFβ1, TGFβ2 and IL13 itself.

[0035] In one embodiment, said IL13 and said interleukin selected from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2 or IL13 itself are linked via a linker.

[0036] In one embodiment, an interleukin selected from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2 or IL13 itself is fused to the N-terminus of IL13.

[0037] In another embodiment, IL13 is fused to the N-terminus of an interleukin selected from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2, or IL13 itself.

[0038] In one embodiment, the fusion protein further comprises one or more chemical modifications. The chemical modifications may be selected from:

[0039] Glycosylation, fucosylation, sialylation, and PEGylation.

[0040] In one embodiment, the IL13 is human IL13.

[0041] In one embodiment, the IL4 is human IL4.

[0042] In one embodiment, the IL10 is human IL10.

[0043] In one embodiment, the IL27 is human IL27.

[0044] In one embodiment, the IL33 is human IL33.

[0045] In one embodiment, the TGFβ1 is human TGFβ1.

[0046] In one embodiment, the TGFβ2 is human TGFβ2

[0047] In a second aspect, the present invention relates to a nucleic acid molecule comprising a polynucleotide encoding a fusion protein taught herein.

[0048] In another aspect, the present invention relates to a vector comprising a nucleic acid molecule taught herein.

[0049] In one aspect, the invention relates to a host cell comprising a nucleic acid molecule taught herein or a vector taught herein.

[0050] In one aspect, the invention provides a method of producing a fusion protein as taught herein, the method comprising the steps of: culturing a host cell as taught herein under conditions that allow production of the fusion protein as taught herein; and optionally, recovering the fusion protein.

[0051] In another aspect, the present invention provides a pharmaceutical composition comprising a fusion protein as taught herein and a pharmaceutically acceptable carrier or excipient.

[0052] The present invention also relates to the use of the fusion proteins as taught herein as medicaments, for example, for preventing or treating conditions characterized by pathological pain, chronic pain, neuroinflammation, neurodegeneration and / or local or systemic inflammation. In this context, "chronic" can be considered to last 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 condition is 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 can be selected from post-orthopedic surgical pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-surgical peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-related pain, chemotherapy-induced allodynia, chemotherapy-induced peripheral neuropathy, complex regional pain syndrome, pain after spinal cord injury, pain after stroke, multiple sclerosis, low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, chronic widespread pain, myofascial pain syndrome, Alzheimer's disease and Parkinson's disease, Huntington's disease and / or amyotrophic lateral sclerosis or multiple sclerosis.

[0053] In one aspect, the present invention relates to the use of a fusion protein as taught herein for preventing or treating a clinical condition in a mammal, such as a human, wherein IL4 or IL13 is indicated for the condition.

[0054] The present invention also relates to the use of a fusion protein as taught herein for preventing or treating a clinical condition in a mammal, such as a human, wherein IL10 or IL27 or IL33 or TGFβ1 or TGFβ2 is indicated for the condition.

[0055] Finally, the present invention provides vectors for use in the prevention or treatment of conditions characterized by chronic pain, neuroinflammation, neurodegeneration, and / or local or systemic inflammation. Said conditions are 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 selected from the group consisting of post-orthopedic pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-surgical peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-related pain, complex regional pain syndrome, pain after spinal cord injury, pain after stroke, multiple sclerosis, low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's and Parkinson's diseases, Huntington's disease, and / or amyotrophic lateral sclerosis or multiple sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The features of the present disclosure are particularly set forth in the appended claims. The features and advantages of the present disclosure may be better understood by reference to the following detailed description of illustrative embodiments in which the principles of the present disclosure are utilized, and the accompanying drawings, which are:

[0057] Figure 1 .Regulatory (e.g., anti-inflammatory) cytokines are required to subside chemotherapy-induced pain. Mice were injected intraperitoneally with 2 mg / kg paclitaxel on days 0 and 2 to induce transient painful chemotherapy-induced polyneuropathy. From day 6 onwards, mice received daily intrathecal injections of neutralizing antibodies against endogenous IL4 (n=4; hollow downward triangles) or IL13 (n=4; solid upward triangles) for 5 days (5 μg antibody per injection). As a control, isotype IgG was injected intrathecally into mice treated with paclitaxel (n=3; dotted line with hollow circles). As another control, mice were pretreated with vehicle instead of paclitaxel and control IgG (n=3; solid line with solid circles). Pain-like behavior was tracked over time by measuring mechanical sensitivity to touch using von Frey hair. Note that a lower 50% threshold indicates increased sensitivity. The data represent the mean and standard error of the mean. Statistical differences between mice treated with anti-IL13 IgG and control IgG are indicated as *p<0.05, **p<0.01, ***p<0.001. Statistical differences between mice treated with anti-IL4 IgG and control IgG are indicated as xp<0.05, xxp<0.01.

[0058] Figure 2.IL13 weakens paclitaxel-induced neuronal damage. Primary sensory neurons were cultured and treated overnight with paclitaxel (1 μM) to induce neuronal damage, which was quantified by measuring the neurite length after β3-tubulin staining. During treatment with chemotherapy drugs, vehicle (-) or a single cytokine (50 ng / ml) was added and the average length of neurons was measured. Data represent the mean and standard error of the mean of neurite length in micrometers for >10 cells measured in at least 2 experiments.

[0059] Figure 3 .Characterization of recombinant IL4 / IL13 fusion protein. His-tagged IL4 / IL13 fusion protein was expressed in HEK293 cells, purified using HIS-Select nickel affinity chromatography, and analyzed using high-pressure size exclusion chromatography (HP-SEC). The HP-SEC profile indicated 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 from 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.

[0060] Figure 4 .IL4 / IL13 fusion protein alleviates paclitaxel-induced persistent mechanical allodynia. Paclitaxel (8 mg / kg) was injected intraperitoneally into C57BL / 6 mice on days 0, 2, 4, and 6 (grey symbols on the X-axis) to induce persistent chemotherapy-induced polyneuropathy. IL4 / IL13 fusion protein (0.3 [open circles], 1 [open triangles], or 3 μg / mouse [open squares]; n=4 / group) or vehicle (n=4) was administered intrathecally on day 8, and the course of mechanical allodynia was tracked over time using von Frey hairs. Data are expressed as mean ± SEM. Statistics of the data were analyzed using a two-way ANOVA followed by a Tukey's multiple comparison test. 0.3 μg IL4 / IL13 fusion protein vs. vehicle, *, **, *** = p < 0.05, p < 0.01 and 0.001, respectively. 3 μg IL4 / IL13 fusion protein vs. vehicle, &, = p < 0.05. 1 μg IL4 / IL13 fusion protein vs. vehicle, x = p < 0.05.

[0061] Figure 5.IL4 / IL13 fusion protein has a more sustained effect on painful paclitaxel-induced neuropathy in mice than IL4 / IL10 fusion protein. Mice received four intraperitoneal injections of 8 mg / kg paclitaxel every other day (grey symbols on the X-axis) to induce persistent, painful, chemotherapy-induced polyneuropathy. On day 8, mice received 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 (closed circles, n=4). Pain-like behaviors were tested using von Frey hairs (see Figure 1 ). Note that a single administration of the IL4 / IL13 fusion protein resulted in sustained relief of pain (eg, permanent resolution of pain), whereas the IL4 / IL10 fusion protein had a temporary effect that lasted 2 days in this experiment.

[0062] Figure 6 .IL4 / IL13 fusion protein protects mice from paclitaxel-induced nerve damage. Mice received 4 intraperitoneal injections of 8mg / kg paclitaxel every other day to induce persistent, painful, chemotherapy-induced polyneuropathy. On the 8th day, they were treated with a single intrathecal injection of IL4 / IL13 fusion protein (0.7 μg) or vehicle. On the 15th day, the length of intraepidermal nerve fibers in the paw skin was determined by immunofluorescence visualization using the neuronal marker PGP9.5. Data for mice that were not treated with chemotherapy drugs (black columns; n=4) or injected with paclitaxel and subsequently treated with vehicle (-; n=6) or IL4 / IL3 fusion protein (IL4-13; n=4) are shown.

[0063] Figure 7 .IL4 / IL13 fusion protein better protects cultured neurons 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 the neurite length after β3-tubulin staining. During incubation with chemotherapy drugs, 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 were compared (black columns).

[0064] Figure 8.IL4 / IL13 cures oxaliplatin-induced polyneuropathy in mice, while IL4 or IL13 only has a partial temporary effect. Oxaliplatin (3 mg / kg) was injected intraperitoneally into mice every day for 5 days, followed by 5 days without treatment, and another 5-day oxaliplatin treatment cycle (gray symbols on the X-axis). On the first day after the last oxaliplatin injection, animals received intrathecal injections of IL4 / IL13 fusion protein (0.3 μg; open circles, n=4) or wild-type cytokines (0.15 μg; n=4, rectangle for IL4, triangle for IL13); or vehicle only (filled circles). Pain was measured with the von Frey test.

[0065] 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 overnight with oxaliplatin (5 μg / ml). Neuronal damage was then quantified by measuring the neurite length after β3-tubulin staining. During incubation with chemotherapy drugs, 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 were compared (black columns).

[0066] Figure 10 .The cytokine receptor subunits of IL10, IL4, IL13, IL33, IL27, TGFβ1, and TGFβ2 are expressed in the dorsal root ganglia of humans and mice. In order to evaluate whether the cytokine receptors targeted by the fusion protein of the present invention are expressed by the sensory system, Ray et al. (Pain 2018; 159: 1325-1345) extracted RNAseq data of cytokine receptor subunits of IL10, IL4, IL13, IL33, IL27, TGFβ1, and TGFβ2 in the dorsal root ganglia and spinal cord from the database, which can be obtained at https: / / www.utdallas.edu / bbs / painneurosciencelab / sensoryomics / drgtxome / ?go. RNA sequencing data are expressed as transcripts per million. For comparison, data on receptor expression in whole blood are also given.

[0067] Figure 11 Characterization of fusion proteins. Under non-reducing and reducing conditions, 4-12% gradient NuPage TM The purified fusion protein was analyzed on polyacrylamide gel and the bands were visualized by Coomassie staining.

[0068] Figure 12The two IL4 / IL13 fusion proteins disclosed herein protect 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 an equimolar dose of IL13 or a combination of unlinked cytokines. Inhibition of paclitaxel-induced reduction in neurite length was calculated. The fusion protein labeled IL4 / IL13 comprises SEQ ID NO: 4. The fusion protein labeled IL4 / IL13 comprises SEQ ID NO: 4. SKP The fusion protein comprises SEQ ID NO: 16. Data are shown as mean ±

[0069] SEM. Data were analyzed using two-way ANOVA mixed effects analysis followed by Tukey's multiple comparison test.

[0070] 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 an equimolar dose of IL13 or a combination of unconnected cytokines. The inhibition of paclitaxel-induced neurite length reduction was calculated. Data are shown as mean ± SEM. Data were analyzed using a two-way ANOVA mixed effects analysis followed by a Tukey multiple comparison test.

[0071] Figure 14 Protective effects of IL13 / IL13 and IL27 / IL13 on 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 an equimolar dose of IL13. Inhibition of paclitaxel-induced reduction in neurite length was calculated. Data are shown as mean ± SEM. Data were analyzed using a two-way ANOVA mixed-effects analysis followed by a Tukey multiple comparison test.

[0072] Figure 15Compared to the combination of unconnected cytokines, the fusion proteins containing IL13 disclosed herein elicit different kinase activity profiles in dorsal root ganglion (DRG) cells. PamGene kinase activity analysis was performed to assess global protein tyrosine kinase (PTK) activity in lumbar DRG homogenates isolated from mice with persistent paclitaxel-induced peripheral neuropathy after administration of IL4 / IL13 fusion protein, IL4+IL13 (combination of unconnected cytokines), and vehicle. Kinaseomic profiles were assessed 60 minutes after intrathecal administration of IL4 / IL13 fusion protein, cytokine combination, or vehicle (PBS). Peptides differentially phosphorylated based on one-way ANOVA analysis between mice treated with IL4 / IL13, IL4+IL13, and vehicle and naive mice (untreated; no paclitaxel, no intrathecal injection) are shown. Black indicates no significant change, while color indicates reduced phosphorylation.

[0073] Figure 16 Altered kinase activity in dorsal root ganglion (DRG) cells of female mice treated with IL4 / IL13 compared to the unlinked cytokine combination. A PamGene kinase activity assay was performed to assess global protein tyrosine kinase (PTK) activity in lumbar DRG homogenates isolated from female mice with persistent paclitaxel-induced CIPN following administration of an IL4 / IL13 fusion protein or IL4 + IL13 (unlinked cytokine combination). The figure shows predicted upstream kinases inferred from differentially phosphorylated peptide substrates identified by unpaired t-test comparisons between samples from females treated with IL4 / IL13 fusion protein and IL4 + IL13 (n = 3 animals per group). The figure is sorted with the highest specificity score at the top and the lowest specificity score at the bottom.

[0074] Figure 17 Altered kinase activity in dorsal root ganglion (DRG) cells of male mice treated with IL4 / IL13 compared to the unlinked cytokine combination. A PamGene kinase activity assay was performed to assess global protein tyrosine kinase (PTK) activity in lumbar DRG homogenates isolated from male mice with persistent paclitaxel-induced CIPN following administration of an IL4 / IL13 fusion protein or IL4 + IL13 (unlinked cytokine combination). The figure shows predicted upstream kinases inferred from differentially phosphorylated peptide substrates identified by unpaired t-test comparisons between samples from males treated with the IL4 / IL13 fusion protein and males treated with IL4 + IL13 (n = 3 animals per group). The figure is sorted with the highest specificity score at the top and the lowest specificity score at the bottom.

[0075] Figures 18A-18DSize 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

[0076] General Definition

[0077] The term "nucleic acid molecule" (or "nucleic acid sequence," "polynucleotide," or "nucleotide sequence") refers to a DNA or RNA molecule in single-stranded or double-stranded form, particularly a DNA encoding a protein of the present 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, such as a nucleic acid sequence in a bacterial host cell or in a plant nuclear or plastid genome.

[0078] The terms "protein" and "polypeptide" are used interchangeably to refer to a molecule composed of one or more chains of amino acids, without regard to a specific mode of action, size, three-dimensional structure, or origin. The term "isolated protein" is used to refer to a protein that is no longer in its natural environment, for example, in vitro or in a recombinant mammalian, bacterial, or plant host cell.

[0079] 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 connecting region or amino acid linker between the amino acid portions of the isolated proteins. A fusion protein also refers to a molecule having an amino acid sequence from or derived from two or more proteins, the proteins being non-covalently bound or linked (e.g., covalently linked) by a chemical cross-linker, with or without a spacer. A fusion protein may be a polypeptide construct.

[0080] The terms IL4, IL10, IL13, IL27, IL33, TGFβ1 and TGFβ2 preferably refer to the wild-type sequences of these respective cytokines, and / or mutant variants thereof that are capable of binding to at least one of their respective cytokine receptors or receptor subunits (e.g., preferably at least two).

[0081] The terms "TGFβ", "TGFβ1 / 2" and "TGFβ(1 or 2)" are used to refer to TGFβ1 and / or TGFβ2.

[0082] The term "IL4 / IL13 fusion protein" refers to a fusion polypeptide comprising at least IL4 and IL13, optionally coupled to each other via a linker. The fusion protein may comprise additional polypeptide sequences, such as a signal sequence, a His tag, one or more targeting sequences, an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0083] The term "IL10 / IL13 fusion protein" refers to a fusion polypeptide comprising at least IL10 and IL13, optionally coupled to each other via a linker. The fusion protein may comprise additional polypeptide sequences, such as a signal sequence, a His tag, one or more targeting sequences or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0084] The term "IL33 / IL13 fusion protein" refers to a fusion polypeptide comprising at least IL33 and IL13, optionally coupled to each other via a linker. The fusion protein may comprise additional polypeptide sequences, such as a signal sequence, a His tag, one or more targeting sequences or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0085] The term "TGFβ1 / IL13 fusion protein" refers to a fusion polypeptide comprising at least TGFβ1 and IL13, optionally coupled to each other via a linker. The fusion protein may comprise additional polypeptide sequences, such as a signal sequence, a His tag, one or more targeting sequences or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0086] The term "TGFβ2 / IL13 fusion protein" refers to a fusion polypeptide comprising at least TGFβ2 and IL13, optionally coupled to each other via a linker. The fusion protein may comprise additional polypeptide sequences, such as a signal sequence, a His tag, one or more targeting sequences or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0087] The term "IL13 / IL13 fusion protein" refers to a fusion polypeptide comprising at least two IL13 molecules, optionally coupled to each other via a linker. The fusion protein may comprise additional polypeptide sequences, such as a signal sequence, a His tag, one or more targeting sequences or an antibody Fc fragment, an extracellular matrix binding polypeptide, or any combination thereof.

[0088] As used herein, "linker" means a polypeptide used to couple two proteins or polypeptides, i.e., 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 of up to 100 amino acids in length, e.g., from 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.

[0089] As used herein, "interleukin-13" (IL13) preferably refers to any mammalian IL13, such as human IL13, mouse IL13, or an active species or variant (eg, allelic variant), (functional) fragment or derivative thereof.

[0090] As used herein, "interleukin-4" (IL4) preferably refers to any mammalian IL4, such as human IL4, mouse IL4, or an active species or variant (eg, allelic variant), (functional) fragment or derivative thereof.

[0091] As used herein, "interleukin-10" (IL10) preferably refers to any mammalian IL10, such as human IL10, mouse IL10, or an active species or variant (eg, allelic variant), (functional) fragment or derivative thereof.

[0092] As used herein, "interleukin-27" (IL27) may refer to any mammalian IL27, such as human IL27, mouse IL27, or an active species or variant (e.g., allelic variant), (functional) fragment, or derivative thereof. In some cases, IL27 refers to IL27 subunit α (IL27A), such as human IL27A. In some cases, IL27 refers to IL27 subunit β (IL27B), such as human IL27B. In some cases, IL27 refers to IL27A and IL27B.

[0093] As used herein, "interleukin-33" (IL33) preferably refers to any mammalian IL33, such as human IL33, mouse IL33, or an active species or variant (eg, allelic variant), (functional) fragment or derivative thereof.

[0094] As used herein, "transforming growth factor β1" (TGFβ1) preferably refers to any mammalian TGFβ1, such as human TGFβ1, mouse TGFβ1, or an active species or variant (such as an allelic variant), (functional) fragment or derivative thereof.

[0095] As used herein, "transforming growth factor β2" (TGFβ2) preferably refers to any mammalian TGFβ2, such as human TGFβ2, mouse TGFβ2, or an active species or variant (such as an allelic variant), (functional) fragment or derivative thereof.

[0096] When describing cytokines, the term "wild type" refers to a cytokine with an amino acid sequence that is naturally occurring and encoded by the germline genome of a given species. Species can have a wild type sequence, or two or more wild type sequences (e.g., having a standard wild type sequence and one or more non-standard wild type sequences). A wild type cytokine sequence can include a sequence that is truncated at the N and / or C-terminal end relative to a sequence encoded by an open reading frame. A wild type cytokine sequence can be a cytokine in a mature form that has been processed to remove N-terminal and / or C-terminal residues. A wild type cytokine can lack a signal peptide or can include a signal peptide (e.g., a signal peptide can be added to the N-terminal end of a wild type cytokine).

[0097] 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, containing one or more amino acid insertions, deletions, or substitutions relative to the wild-type sequence. A cytokine derivative binds to at least one subunit of the corresponding native receptor of the wild-type cytokine and induces signaling and / or cytokine activity. The binding affinity, signaling, and / or cytokine activity of a cytokine derivative may be the same as or different from that of the corresponding wild-type cytokine.

[0098] "Functionality" in relation to the fusion protein of the present invention (or its variant or fragment) refers to the ability to exhibit IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2 or other regulatory cytokines, such as anti-inflammatory cytokines) and IL13 functionality, for example, the ability to bind to at least one receptor subunit that binds to the wild-type cytokine.

[0099] Assays for evaluating the functional activity of these cytokines are well known to those skilled in the art. For example, functional assays for IL4 and IL10 are performed in the presence of anti-IL10 antibodies. 26 The release of TNF in whole blood was induced by lipopolysaccharide (LPS). The functional assay of IL13 was performed on TF1 human erythroleukemia cells. 27 The functional assay for IL33 is the production of IL6 by the mast cell line MC / 9. The assay for TGFβ1 or TGFβ2 is the inhibition of IL4-dependent growth of the mouse T cell line HT-2. The functional assay for IL27 can include the production of IL6 by LPS-stimulated THP-1 macrophages.

[0100] The term "gene" refers to a DNA sequence comprising a region (transcribed region) operably linked to a suitable regulatory region (e.g., a promoter) that is transcribed into an RNA molecule (e.g., mRNA) in a cell. Thus, a gene may comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising, for example, sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA), introns, and a 3' untranslated sequence comprising, for example, a transcription termination site.

[0101] "Gene expression" refers to the process by which a DNA region operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into biologically active RNA, i.e., RNA that can be translated into a biologically active protein or peptide (or active peptide fragment). "Polypeptide expression" further refers to the process by which mRNA is translated into a protein product, which may or may not be secreted.

[0102] As used herein, the term "promoter" refers to a nucleic acid sequence whose function is to control the transcription of one or more genes, located upstream of the gene transcription start site in the direction of transcription, and structurally identified by the presence of a binding site for RNA polymerase that depends on DNA, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art that directly or indirectly regulates the transcription amount of the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically (e.g., by external application of certain compounds) or developmentally regulated. A "tissue-specific" promoter is active only in a specific type of tissue or cell.

[0103] As used herein, the term "operably linked" refers to the connection of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a promoter or transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the linked DNA sequences are generally contiguous.

[0104] "Nucleic acid construct" or "vector" is understood herein to mean an artificial nucleic acid molecule produced using recombinant DNA technology that is used to deliver exogenous DNA into a host cell. Vectors typically contain further genetic elements to facilitate their use in molecular cloning, such as selectable markers or multiple cloning sites (see below).

[0105] " sequence identity " and " sequence similarity " can be determined by comparing two peptides or two nucleotide sequences using a global or local alignment algorithm. When sequences (when performing optimal comparison using default parameters by, for example, programs GAP or BESTFIT) share at least a certain minimum percentage of sequence identity (as defined below), they can be referred to as "substantially identical" or "substantially similar". GAP uses the Needleman and Wunsch global alignment algorithm to compare over the entire length of two sequences, maximizing the number of matches and minimizing the number of gaps. Typically, using GAP default parameters, gap creation penalty=50 (nucleotide) / 8 (protein) and gap extension penalty=3 (nucleotide) / 2 (protein). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89,915-919). Sequence alignments and percentage scores of sequence identity 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 percentages can be determined by searching databases using algorithms such as FASTA, BLAST, etc. Preferably, sequence identity refers to sequence identity over the entire length of the sequence.

[0106] "Host cell" or "recombinant host cell" or "transformed cell" refers to a new individual cell (or organism) produced by at least one nucleic acid molecule, especially a nucleic acid molecule comprising 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 present invention as an extrachromosomal (episomal) replicating molecule, or more preferably, includes the nucleic acid molecule or vector of the present invention integrated into the host cell genome.

[0107] The term "selectable marker" is a term familiar to those of ordinary skill in the art and is used herein to describe any genetic entity that, when expressed, can be used to select one or more cells containing the selectable marker. The selectable marker gene product confers, for example, antibiotic resistance or nutritional requirements.

[0108] The term "nervous system cell" refers to a cell found in the central nervous system or peripheral nervous system. A nervous system cell can be a neuron, a central nervous system cell, a peripheral nervous system cell, a glial cell, a microglia, an astrocyte, a Schwann cell, a satellite glial cell, an oligodendrocyte, an infiltrating cell, an infiltrating immune cell, an infiltrating bone marrow cell, an infiltrating lymphocyte, an infiltrating macrophage, an infiltrating neutrophil, an infiltrating lymphocyte, an infiltrating T cell, an infiltrating B cell, or an infiltrating natural killer cell. A neuron can be, for example, a sensory neuron, a somatosensory neuron, a visceral sensory neuron, a nociceptor, and / or an autonomic neuron.

[0109] In this document and its claims, the verb "to comprise" and its conjugations are used in its non-restrictive sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. It also covers the more restrictive verb "to consist of". Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements are present, unless the context clearly requires the presence of one and only one element. Thus, the indefinite article "a" or "an" usually means "at least one". It is further understood that when a "sequence" is referred to herein, this is usually referring to an actual physical molecule having a specific sequence of subunits (e.g., amino acids).

[0110] Proteins, nucleic acid sequences, vectors and host cells of the present invention

[0111] The present inventors provide a fusion protein comprising an IL13 protein and a regulatory cytokine, optionally physically fused together by a linker, such as a protein selected from IL4, IL10, IL27, IL33, TGFβ1, TGFβ2 or IL13 itself. The regulatory cytokines disclosed herein include, but are not limited to, IL4, IL10, IL13, IL27, IL33, TGFβ1 and TGFβ2. In particular, it was found that the fusion protein of the present invention has activity that is superior to its respective counterparts, i.e., individual regulatory cytokines (e.g., IL4 or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13, in treating conditions disclosed herein (e.g., neuropathic pain). Specifically, it was found that upon intrathecal administration, the fusion protein of the present invention has a lasting analgesic effect on neuropathic pain and eliminates the abnormal pain associated with chemotherapy-induced neuropathy. The inventors unexpectedly observed that this analgesic effect of the fusion protein of the present invention is better than that of the previously described IL4 and IL10. 19,23,26The analgesic effect of the fusion protein of the present invention lasts longer. Surprisingly, the fusion protein of the present invention also improves the neurodegeneration associated with chemotherapy. After incubation with chemotherapeutic drugs, it can prevent the shortening of neurites in vitro, and is more effective 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 unexpected discovery shows that the fusion protein has a unique effect compared to its single cytokine or the combination of these cytokines. In vivo, the fusion protein of the present invention weakened the reduction of nerve fibers in the epidermis after administration of chemotherapeutic drugs.

[0112] In one embodiment of the present invention, the nucleic acid sequence and amino acid sequence of 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, or an IL13 / IL13 fusion protein (including variants, derivatives, and fragments thereof) are provided. The IL4 / IL13 fusion protein, the IL10 / IL13 fusion protein, the IL27 / IL13 fusion protein, or the 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.

[0113] In some cases, the fusion proteins disclosed herein comprise two cytokines, C1 and C2, and are referred to in the format of C1 / C2 or C1-C2. The order in which the cytokines appear is not limiting and does not necessarily infer the orientation of the cytokines. For example, C1 / C2 or C1-C2 can include cytokine C1 on the C-terminal side of C2 or on the N-terminal side of C2. Similarly, unless otherwise indicated, a cytokine referred to as C1 / C2 can be the same as a cytokine referred to as C2 / C1.

[0114] In one aspect, a fusion protein comprising IL4 (or IL10 or IL13 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13 is provided.

[0115] Interleukin-13

[0116] The fusion proteins disclosed herein may comprise an IL13 protein or a variant, derivative or fragment thereof operably linked or fused directly or indirectly to a regulatory cytokine or a variant or derivative thereof. The IL13 protein is preferably a mammalian IL13 protein, such as human IL13 or mouse IL13. Non-limiting examples of amino acid sequences representing human IL13 are set forth in SEQ ID NO: 2 and SEQ ID NO: 9-15. Variants of IL13 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to any one of SEQ ID NO: 2 or SEQ ID NO: 9-15 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 as defined above. Variants, derivatives and fragments thereof also include proteins having IL13 activity that are derived from a polypeptide having an amino acid sequence of SEQ ID NO: 2 or any one of SEQ ID NOs: 9 to 15 by one or more amino acid substitutions, deletions or insertions. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

[0117] 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, at least, or at least 50 amino acid substitutions, deletions, or insertions relative to an IL13 sequence disclosed herein (e.g., a wild-type IL13 sequence).

[0118] In some embodiments, an IL13 of the present disclosure (e.g., an IL13 variant, derivative, or fragment thereof) may include at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL13 sequence disclosed herein (e.g., a wild-type IL13 sequence).

[0119] In some embodiments, the IL13 sequences disclosed herein (e.g., IL13 variants, derivatives, or fragments thereof) may comprise 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, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2- 20, 25, 30, 35, 36, 37, 38, 39, 40, 50, 50, 50, 60, 70, 80, 90, 100, 150, 150, 200, 250, 300, 400, 80, 90, 10 ...

[0120] In some embodiments, the IL13 sequences disclosed herein (e.g., IL13 variants, derivatives, or fragments 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 relative to the IL13 sequences disclosed herein (e.g., wild-type IL13 sequences). 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, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions may be continuous, non-continuous, or a combination thereof.

[0121] The IL13 of the present disclosure may comprise a wild-type IL13 sequence. Non-limiting examples of wild-type IL13 sequences include SEQ ID NOs: 2 and 9-15. SEQ ID NO: 12 may be the canonical wild-type IL13 sequence of the present disclosure.

[0122] The IL13 of the present disclosure may comprise an IL13 variant, derivative, or fragment thereof having one or more amino acid substitutions. For example, the IL13 variant, derivative, or fragment thereof may comprise an amino acid 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, N113, or a combination thereof, of SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative, or fragment thereof may comprise an amino acid substitution relative to position 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 a combination thereof, of SEQ ID NO: 2 or SEQ ID NO: 12. NO:12 contains the following substitutions: 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; 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 a combination thereof. In some embodiments, the IL13 variant, derivative, or fragment thereof comprises the substitutions L10H, R86T, D87G, T88R, and R108K relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative 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 relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative, or fragment thereof comprises substitutions V18I, R86T, D87G, T88S, L101Y, K104R, and K105A relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative, or fragment thereof comprises substitutions R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, and F107M relative 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 relative 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 relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative, or fragment thereof comprises the substitutions E12K and R111D relative to SEQ ID NO: 2 or SEQ ID NO: 12.In some embodiments, the IL13 variant, derivative, or fragment thereof comprises the substitutions E12Y and R65D relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative, or fragment thereof comprises the substitutions E12Y and S68D relative 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, and S68D relative 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 relative 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 relative to SEQ ID NO: 2 or SEQ ID NO: 12.

[0123] In some embodiments, the IL13 variant, derivative, or fragment thereof does not comprise 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 relative to SEQ ID NO: 2 or SEQ ID NO: 12. In some embodiments, the IL13 variant, derivative, or fragment thereof does not comprise 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; L10 1Y; 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 or N113D substitutions.

[0124] In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure bind to the IL13 receptor subunit with an affinity approximately comparable to that of the wild-type IL13 sequence. Comparable affinity can be an increase in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type IL13 sequence. Comparable affinity can be a decrease in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type IL13 sequence.

[0125] For example, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can 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 comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 with an affinity approximately comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα2 with an affinity approximately comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-4Rα with an affinity approximately comparable to that of the wild-type IL13 sequence. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 and IL-13Rα2 with an affinity approximately comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 and IL-4Rα with an affinity approximately comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα2 and IL-4Rα with an affinity approximately comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1, IL-13Rα2, and IL-4Rα with an affinity approximately comparable to that of wild-type IL13.

[0126] In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to an IL13 receptor subunit with an affinity at least comparable to that of wild-type IL13. For example, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can 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 comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 with an affinity at least comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα2 with an affinity at least comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof disclosed herein can bind to IL-4Rα with an affinity at least comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof disclosed herein can bind to IL-13Rα1 and IL-13Rα2 with an affinity at least comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof disclosed herein can bind to IL-13Rα1 and IL-4Rα with an affinity at least comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof disclosed herein can bind to IL-13Rα2 and IL-4Rα with an affinity at least comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof disclosed herein can bind to IL-13Rα1, IL-13Rα2, and IL-4Rα with an affinity at least comparable to that of wild-type IL13.

[0127] In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to an IL13 receptor subunit with an affinity that is at most comparable to that of wild-type IL13. For example, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can 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 that is at most comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 with an affinity that is at most comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα2 with an affinity that is at most comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-4Rα with an affinity that is at most comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 and IL-13Rα2 with an affinity that is at most comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 and IL-4Rα with an affinity that is at most comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα2 and IL-4Rα with an affinity that is at most comparable to that of wild-type IL13. In some embodiments, the IL13 or IL13 variant, derivative, or fragment thereof of the present disclosure can bind to IL-13Rα1, IL-13Rα2, and IL-4Rα with an affinity that is at most comparable to wild-type IL13.

[0128] In some embodiments, IL13 or an IL13 variant, derivative, or fragment thereof can bind to IL-13Rα1 with an affinity that is increased by 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 relative to the wild-type IL13 sequence. In some embodiments, IL13 or an IL13 variant, derivative, or fragment thereof can bind to IL-13Rα1 with an affinity that is decreased by 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 relative to the wild-type IL13 sequence.

[0129] In some embodiments, IL13 or an IL13 variant, derivative, or fragment thereof can bind to IL-13Rα2 with an affinity that is increased by 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 relative to the wild-type IL13 sequence. In some embodiments, IL13 or an IL13 variant, derivative, or fragment thereof can bind to IL-13Rα2 with an affinity that is decreased by 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 relative to the wild-type IL13 sequence.

[0130] In some embodiments, IL13 or an IL13 variant, derivative, or fragment thereof can bind to IL-4Rα with an affinity that is increased by 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 relative to the wild-type IL13 sequence. In some embodiments, IL13 or an IL13 variant, derivative, or fragment thereof can bind to IL-4Rα with an affinity that is decreased by 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 relative to the wild-type IL13 sequence.

[0131] In some embodiments, the IL13 or IL13 variant, derivative, or fragment thereof disclosed herein can activate a native IL13 receptor. The native IL13 receptor can be, for example, a receptor comprising an IL-13Rα1 subunit and an IL-4Rα subunit.

[0132] Interleukin-4

[0133] The fusion proteins disclosed herein may comprise an IL4 protein or a variant, derivative, or fragment thereof operably linked or fused directly or indirectly to an interleukin-13 or variant or derivative thereof. The IL4 protein is preferably a mammalian IL4 protein, such as human IL4 or mouse IL4. Non-limiting examples of the amino acid sequence of IL4 are set forth in SEQ ID NO: 1 and SEQ ID NOs: 26-28. Variants of IL4 include, for example, proteins that preferably have at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to any one of SEQ ID NO: 1 or SEQ ID NOs: 26-28 over their entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and the GAP default parameters as defined above. Variants also include proteins having IL4 activity that are derived from a polypeptide having an amino acid sequence of SEQ ID NO: 1 or any one of SEQ ID NOs: 26 to 28 by one or more amino acid substitutions, deletions, or insertions. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions, or insertions. 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, at least, or at least 50 amino acid substitutions, deletions, or insertions relative to an IL4 sequence disclosed herein (e.g., a wild-type IL4 sequence).

[0134] In some embodiments, an IL4 of the present disclosure (e.g., an IL4 variant, derivative, or fragment thereof) may include at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL4 sequence disclosed herein (e.g., a wild-type IL4 sequence).

[0135] In some embodiments, the IL4 sequences disclosed herein (e.g., IL4 variants, derivatives, or fragments thereof) may comprise 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, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20 or 20-25 amino acid substitutions, deletions or insertions.

[0136] In some embodiments, the IL4 sequence of the present disclosure (e.g., an IL4 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 relative to the IL4 sequence disclosed herein (e.g., a wild-type IL4 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, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions may be continuous, non-continuous, or a combination thereof.

[0137] The IL4 of the present disclosure may comprise a wild-type IL4 sequence. Non-limiting examples of wild-type IL4 sequences include SEQ ID NOs: 1 and 26-28. The canonical wild-type IL4 sequence of the present disclosure may be SEQ ID NO: 1.

[0138] The IL4 of the present disclosure may include IL4 variants, derivatives, or fragments thereof having one or more amino acid substitutions. For example, the IL4 variant, derivative, or fragment thereof may include an amino acid substitution at position K117, T118, R121, E122, Y124, S125, S128, S129, or a combination thereof, of SEQ ID NO: 1. In some embodiments, the IL4 variant, derivative, or fragment thereof comprises the following substitutions relative to SEQ ID NO: 1: K117R, T118V, R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F, Y124D, S125F, S128G, S129A, S125R, or a combination thereof. In some embodiments, the IL4 variant, derivative, or fragment thereof comprises substitutions K117R, T118V, R121Q, E122S, Y124W, S125F, S128G, and S129A relative to SEQ ID NO: 1. In some embodiments, the IL4 variant, derivative, or fragment thereof comprises substitutions R121D and Y124D relative to SEQ ID NO: 1.

[0139] In some embodiments, the IL4 variant, derivative, or fragment thereof does not comprise a substitution at position K117, T118, R121, E122, Y124, S125, S128, or S129 relative to SEQ ID NO: 1. In some embodiments, the IL4 variant, derivative, or fragment thereof does not comprise a K117R, T118V, R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F, Y124D, S125F, S128G, S125R, or S129A substitution.

[0140] In some embodiments, the IL4 or IL4 variants, derivatives or fragments of the present disclosure bind to the IL4 receptor subunit with an affinity approximately comparable to that of the wild-type IL4 sequence. Comparable affinity can be an affinity increase, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4 times, less than about 1.3 times, less than about 1.2 times or less than about 1.1 times, compared to the wild-type IL4 sequence. Comparable affinity can be an affinity decrease, for example, less than about 10, less than about 5, less than about 2, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4 times, less than about 1.3 times, less than about 1.2 times or less than about 1.1 times, compared to the wild-type IL4 sequence.

[0141] For example, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to interleukin 13 receptor alpha 1 (IL-13Rα1), common gamma chain, interleukin 4 receptor alpha (IL-4Rα), or a combination thereof, for example, with an affinity that is approximately comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 with an affinity that is approximately comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to the common gamma chain with an affinity that is approximately comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-4Rα with an affinity that is approximately comparable to that of the wild-type IL4 sequence. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 and the common gamma chain with an affinity that is approximately comparable to that of wild-type IL4. In some embodiments, IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can bind to IL-13Rα1 and IL-4Rα with an affinity approximately comparable to that of wild-type IL4. In some embodiments, IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can bind to the common gamma chain and IL-4Rα with an affinity approximately comparable to that of wild-type IL4. In some embodiments, IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can bind to IL-13Rα1, the common gamma chain, and IL-4Rα with an affinity approximately comparable to that of wild-type IL4.

[0142] In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure bind to the IL4 receptor subunit with an affinity that is at least comparable to that of the wild-type IL4 sequence. For example, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can 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 that is at least comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 with an affinity that is at least comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to the common gamma chain with an affinity that is at least comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-4Rα with an affinity that is at least comparable to that of the wild-type IL4 sequence. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can bind to IL-13Rα1 and the common gamma chain with an affinity at least comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can bind to IL-13Rα1 and IL-4Rα with an affinity at least comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can bind to the common gamma chain and IL-4Rα with an affinity at least comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can bind to IL-13Rα1, the common gamma chain, and IL-4Rα with an affinity at least comparable to that of wild-type IL4.

[0143] In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure bind to the IL4 receptor subunit with an affinity that is at most comparable to that of the wild-type IL4 sequence. For example, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can 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 that is at most comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-13Rα1 with an affinity that is at most comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to the common gamma chain with an affinity that is at most comparable to that of wild-type IL4. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof of the present disclosure can bind to IL-4Rα with an affinity that is at most comparable 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 the common gamma chain with an affinity that is at most comparable 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 an affinity that is at most comparable 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 the common gamma chain and IL-4Rα with an affinity that is at most comparable 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, the common gamma chain, and IL-4Rα with an affinity that is at most comparable to that of wild-type IL4.

[0144] In some embodiments, IL4 or an IL4 variant, derivative, or fragment thereof can bind to IL-13Rα1 with an affinity that is increased by 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 relative to the wild-type IL4 sequence. In some embodiments, IL4 or an IL4 variant, derivative, or fragment thereof can bind to IL-13Rα1 with an affinity that is decreased by 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 relative to the wild-type IL4 sequence.

[0145] In some embodiments, IL4 or IL4 variants, derivatives, or fragments thereof can bind to the common gamma chain with an affinity that is increased by 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 relative to the wild-type IL4 sequence. In some embodiments, IL4 or IL4 variants, derivatives, or fragments thereof can bind to the common gamma chain with an affinity that is decreased by 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 relative to the wild-type IL4 sequence.

[0146] In some embodiments, IL4 or an IL4 variant, derivative, or fragment thereof can bind to IL-4Rα with an affinity that is increased by 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 relative to the wild-type IL4 sequence. In some embodiments, IL4 or an IL4 variant, derivative, or fragment thereof can bind to IL-4Rα with an affinity that is decreased by 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 relative to the wild-type IL4 sequence.

[0147] In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can activate a native IL4 receptor. A native IL4 receptor can be, for example, a receptor comprising an IL-13Rα1 subunit and an IL-4Rα subunit or a common γ chain subunit and an IL-4Rα subunit. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can activate a native IL4 receptor when present in a fusion protein. In some embodiments, the IL4 or IL4 variants, derivatives, or fragments thereof disclosed herein can activate a native IL4 receptor when present as a polypeptide that is not part of a fusion protein, but do not activate a native IL4 receptor when present in a fusion protein.

[0148] In some embodiments, the polypeptides of the present disclosure do not comprise IL4. In some embodiments, the polypeptides of the present disclosure do not comprise SEQ ID NO: 1.

[0149] Interleukin-10

[0150] The fusion protein may comprise an IL10 protein, or a variant, derivative, or fragment thereof, operably linked or fused directly or indirectly to interleukin-10, or a variant or derivative thereof. The IL10 protein is preferably a mammalian IL10 protein, such as human IL10 or mouse IL10. An amino acid sequence representative of IL10 is set forth in SEQ ID NO:5. Variants of IL10 include, for example, proteins that preferably have at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or more, e.g., 100%, amino acid sequence identity to SEQ ID NO:5 over its entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and the default GAP parameters as defined above. Variants, derivatives, and fragments thereof also include proteins having IL10 activity that are derived from a polypeptide having the amino acid sequence of SEQ ID NO:5 by one or more amino acid substitutions, deletions, or insertions. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

[0151] In some embodiments, an 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, at least, or at least 50 amino acid substitutions, deletions, or insertions relative to an IL10 sequence disclosed herein (e.g., a wild-type IL10 sequence).

[0152] In some embodiments, an IL10 of the present disclosure (e.g., an IL10 variant, derivative, or fragment thereof) may include at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL10 sequence disclosed herein (e.g., a wild-type IL10 sequence).

[0153] In some embodiments, the IL10 sequences disclosed herein (e.g., IL10 variants, derivatives, or fragments thereof) may comprise 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, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2- 20, 25, 30, 35, 36, 37, 38, 39, 40, 50, 50, 50, 60, 70, 80, 90, 100, 150, 150, 200, 250, 300, 400, 80, 90, 10 ...

[0154] In some embodiments, the IL10 sequences disclosed herein (e.g., IL10 variants, derivatives, or fragments 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 relative to the IL10 sequences disclosed herein (e.g., wild-type IL10 sequences). 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, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions may be continuous, non-continuous, or a combination thereof.

[0155] The IL10 of the present disclosure may comprise 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 canonical wild-type IL10 sequence of the present disclosure.

[0156] The IL10 of the present disclosure may include IL10 variants, derivatives, or fragments thereof having one or more amino acid substitutions. For example, the IL10 variant, derivative, or fragment thereof may include an amino acid substitution at position I87, A89, H109, ​​R110, F111, Y153, M156, or a combination thereof, of SEQ ID NO: 5. In some embodiments, the IL10 variant, derivative, or fragment thereof comprises the following substitutions relative to SEQ ID NO: 5: M156, F111S, I87A, I87G, A89D, H109D, R110D, Y153D, M156D, A89D, H109E, R110E, Y153E, M156E, or a combination thereof.

[0157] In some embodiments, the IL10 variant, derivative, or fragment thereof does not comprise a substitution at position I87, A89, H109, ​​R110, F111, Y153, or M156 relative to SEQ ID NO: 5. In some embodiments, the IL10 variant, derivative, or fragment thereof does not comprise an M156, F111S, I87A, I87G, A89D, H109D, R110D, Y153D, M156D, A89D, H109E, R110E, Y153E, or M156E substitution.

[0158] In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein bind to the IL10 receptor subunit with an affinity that is approximately comparable to that of the wild-type IL10 sequence. Comparable affinity can be an increase in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type IL10 sequence. Comparable affinity can be a decrease in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type IL10 sequence.

[0159] For example, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to interleukin 10 receptor 1 (IL-10R1), interleukin 10 receptor 2 (IL-10R2), or a combination thereof with an affinity approximately comparable to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to IL-10R1 with an affinity approximately comparable to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to IL-10R2 with an affinity approximately comparable to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to both IL-10R1 and IL-10R2 with an affinity approximately comparable to that of wild-type IL10.

[0160] In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to an IL10 receptor subunit with an affinity at least comparable to that of wild-type IL10. For example, the IL10 or IL10 variants, derivatives, or fragments disclosed herein can bind to IL-10R1, IL-10R2, or a combination thereof with an affinity at least comparable to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments disclosed herein can bind to IL-10R1 with an affinity at least comparable to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments disclosed herein can bind to IL-10R2 with an affinity at least comparable to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments disclosed herein can bind to both IL-10R1 and IL-10R2 with an affinity at least comparable to that of wild-type IL10.

[0161] In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to an IL10 receptor subunit with an affinity that is at most comparable to that of wild-type IL10. For example, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to IL-10R1, IL-10R2, or a combination thereof with an affinity that is at most comparable to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to IL-10R1 with an affinity that is at most comparable to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to IL-10R2 with an affinity that is at most comparable to that of wild-type IL10. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can bind to both IL-10R1 and IL-10R2 with an affinity that is at most comparable to that of wild-type IL10.

[0162] In some embodiments, IL10 or an IL10 variant, derivative, or fragment thereof can bind to IL-10R1 with an affinity that is increased by 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 relative to the wild-type IL10 sequence. In some embodiments, IL10 or an IL10 variant, derivative, or fragment thereof can bind to IL-10R1 with an affinity that is decreased by 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 relative to the wild-type IL10 sequence.

[0163] In some embodiments, IL10 or an IL10 variant, derivative, or fragment thereof can bind to IL-10R2 with an affinity that is increased by 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 relative to the wild-type IL10 sequence. In some embodiments, IL10 or an IL10 variant, derivative, or fragment thereof can bind to IL-10R2 with an affinity that is decreased by 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 relative to the wild-type IL10 sequence.

[0164] In some embodiments, the IL10 or IL10 variants, derivatives, or fragments thereof disclosed herein can activate a native IL10 receptor. A native IL10 receptor can be, for example, a receptor comprising an IL-10R1 subunit and an IL-10R2 subunit. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments disclosed herein can activate a native IL10 receptor when present in a fusion protein. In some embodiments, the IL10 or IL10 variants, derivatives, or fragments disclosed herein can activate a native IL10 receptor when present as a polypeptide that is not part of the fusion protein, but does not activate a native IL10 receptor when present in a fusion protein.

[0165] In some embodiments, the polypeptides of the present disclosure do not comprise IL10.In some embodiments, the polypeptides of the present disclosure do not comprise SEQ ID NO:5.

[0166] Interleukin-33

[0167] The fusion protein may comprise an IL33 protein, or a variant, derivative, or fragment thereof, operably linked or fused directly or indirectly to interleukin-13, or a variant or derivative thereof. The IL33 protein is preferably a mammalian IL33 protein, such as 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, for example, proteins that preferably have at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, e.g., 100%, amino acid sequence identity to any of SEQ ID NO: 6 or SEQ ID NOs: 29-34 over their entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and the default GAP parameters as defined above. Variants, derivatives, and fragments thereof also include proteins having IL33 activity that are derived from a polypeptide having an 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 comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions, or insertions.

[0168] In some embodiments, an IL33 of the present disclosure (e.g., an IL33 variant, derivative, or fragment thereof) may comprise 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, at least, or at least 50 amino acid substitutions, deletions, or insertions relative to an IL33 sequence disclosed herein (e.g., a wild-type IL33 sequence).

[0169] In some embodiments, an IL33 of the present disclosure (e.g., an IL33 variant, derivative, or fragment thereof) may include at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL33 sequence disclosed herein (e.g., a wild-type IL33 sequence).

[0170] In some embodiments, the IL33 sequences disclosed herein (e.g., IL33 variants, derivatives, or fragments thereof) can comprise 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, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2- 20, 25, 30, 35, 36, 37, 38, 39, 40, 50, 50, 50, 60, 70, 80, 90, 100, 150, 150, 200, 250, 300, 400, 80, 90, 10 ...

[0171] In some embodiments, the IL33 sequences disclosed herein (e.g., IL33 variants, derivatives, or fragments 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 relative to the IL33 sequences disclosed herein (e.g., wild-type IL33 sequences). 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, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions may be continuous, non-continuous, or a combination thereof.

[0172] The IL33 of the present disclosure may comprise a wild-type IL33 sequence. Non-limiting examples of wild-type IL33 sequences include SEQ ID NOs: 6 and 29-34. SEQ ID NO: 6 may be a canonical wild-type IL33 sequence.

[0173] The IL33 of the present disclosure may include IL33 variants, derivatives, or fragments thereof having one or more amino acid substitutions. For example, an IL33 variant, derivative, or fragment thereof may include an amino acid substitution at position 1263 of SEQ ID NO: 6. In some embodiments, the IL33 variant, derivative, or fragment thereof comprises a substitution of 1263M relative to SEQ ID NO: 6.

[0174] In some embodiments, the IL33 variant, derivative, or fragment thereof does not comprise a substitution at position 1263 relative to SEQ ID NO: 6. In some embodiments, the IL33 variant, derivative, or fragment thereof does not comprise an I263M substitution.

[0175] In some embodiments, the IL33 or IL33 variant, derivative, or fragment thereof disclosed herein binds to the IL33 receptor subunit with an affinity approximately comparable to that of the wild-type IL33 sequence. Comparable affinity can be an increase in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type IL33 sequence. Comparable affinity can be a decrease in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type IL33 sequence.

[0176] For example, IL33 or IL33 variants, derivatives, or fragments thereof disclosed herein can bind to ST2 (IL1RL1), IL1RAP, or a combination thereof with an affinity approximately comparable to that of wild-type IL33. In some embodiments, IL33 or IL33 variants, derivatives, or fragments thereof disclosed herein can bind to ST2 with an affinity approximately comparable to that of wild-type IL33. In some embodiments, IL33 or IL33 variants, derivatives, or fragments thereof disclosed herein can bind to IL1RAP with an affinity approximately comparable to that of wild-type IL33. In some embodiments, IL33 or IL33 variants, derivatives, or fragments thereof disclosed herein can bind to ST2 and IL1RAP with an affinity approximately comparable to that of wild-type IL33.

[0177] In some embodiments, the IL33 or IL33 variants, derivatives, or fragments thereof disclosed herein can bind to an IL33 receptor subunit with an affinity at least comparable to that of wild-type IL33. For example, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can bind to ST2, IL1RAP, or a combination thereof with an affinity at least comparable to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can bind to ST2 with an affinity at least comparable to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can bind to IL1RAP with an affinity at least comparable to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can bind to ST2 and IL1RAP with an affinity at least comparable to that of wild-type IL33.

[0178] In some embodiments, the IL33 or IL33 variants, derivatives, or fragments thereof disclosed herein can bind to an IL33 receptor subunit with an affinity that is at most comparable to that of wild-type IL33. For example, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can bind to ST2, IL1RAP, or a combination thereof with an affinity that is at most comparable to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can bind to ST2 with an affinity that is at most comparable to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can bind to IL1RAP with an affinity that is at most comparable to that of wild-type IL33. In some embodiments, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can bind to ST2 and IL1RAP with an affinity that is at most comparable to that of wild-type IL33.

[0179] In some embodiments, IL33 or an IL33 variant, derivative, or fragment thereof can bind to ST2 with an affinity that is increased by 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 relative to the wild-type IL33 sequence. In some embodiments, IL33 or an IL33 variant, derivative, or fragment thereof can bind to ST2 with an affinity that is decreased by 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 relative to the wild-type IL33 sequence.

[0180] In some embodiments, IL33 or an IL33 variant, derivative, or fragment thereof can bind to IL1RAP with an affinity that is increased by 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 relative to the wild-type IL33 sequence. In some embodiments, IL33 or an IL33 variant, derivative, or fragment thereof can bind to IL1RAP with an affinity that is decreased by 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 relative to the wild-type IL33 sequence.

[0181] In some embodiments, the IL33 or IL33 variants, derivatives, or fragments thereof disclosed herein can activate a native IL33 receptor. A native IL33 receptor can be, for example, a receptor comprising an ST2 subunit and an IL1RAP subunit. In some embodiments, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can activate a native IL33 receptor when present in a fusion protein. In some embodiments, the IL33 or IL33 variants, derivatives, or fragments disclosed herein can activate a native IL33 receptor when present as a polypeptide that is not part of the fusion protein, but does not activate the native IL33 receptor when present in a fusion protein.

[0182] Transforming growth factor β1

[0183] The fusion protein may comprise a TGFβ1 protein or a variant, derivative or fragment thereof operably linked or fused directly or indirectly to interleukin-13 or a variant or derivative thereof. The TGFβ1 protein is preferably a mammalian TGFβ1 protein, such as 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 more, such as 100%, amino acid sequence identity to SEQ ID NO: 7 (the entire or underlined portion) 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 as defined above. Variants, derivatives or fragments thereof also include proteins having TGFβ1 activity that are derived from a polypeptide having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 21 by one or more amino acid substitutions, deletions or insertions. Preferably, such proteins contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

[0184] 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, at least or at least 50 amino acid substitutions, deletions, or insertions relative to the TGFβ1 sequence disclosed herein (e.g., a wild-type TGFβ1 sequence).

[0185] In some embodiments, the TGFβ1 of the present disclosure (e.g., a TGFβ1 variant, derivative, or fragment thereof) may include at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to the TGFβ1 sequence disclosed herein (e.g., a wild-type TGFβ1 sequence).

[0186] In some embodiments, the TGFβ1 sequence disclosed herein (e.g., a TGFβ1 variant, derivative, or fragment thereof) may comprise 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, 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, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20 or 20-25 amino acid substitutions, deletions or insertions.

[0187] In some embodiments, the TGFβ1 sequence of the present disclosure (e.g., a TGFβ1 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 relative to the TGFβ1 sequence disclosed herein (e.g., a wild-type TGFβ1 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, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions may be continuous, non-continuous, or a combination thereof.

[0188] The TGFβ1 of the present disclosure may comprise a wild-type TGFβ1 sequence. Non-limiting examples of wild-type TGFβ1 sequences include SEQ ID NOs: 7 and 21. The canonical TGFβ1 sequence may be SEQ ID NO: 21.

[0189] In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure bind to the TGFβ1 receptor subunit with an affinity approximately comparable to that of the wild-type TGFβ1 sequence. Comparable affinity can be an increase in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type TGFβ1 sequence. Comparable affinity can be a decrease in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type TGFβ1 sequence.

[0190] For example, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure can 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 approximately comparable to that of wild-type TGFβ1. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure can bind to TGFβR1 with an affinity approximately comparable to that of wild-type TGFβ1. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure can bind to TGFβR2 with an affinity approximately comparable to that of wild-type TGFβ1. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure can bind to ALK-1 with an affinity approximately comparable to that of the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof disclosed herein can bind to ALK-2 with an affinity approximately comparable to that of the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof disclosed herein can bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with an affinity approximately comparable to that of the wild-type TGFβ1 sequence.

[0191] In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure 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 comparable to that of wild-type TGFβ1. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure may bind to TGFβR1 with an affinity at least comparable to that of wild-type TGFβ1. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure may bind to TGFβR2 with an affinity at least comparable to that of wild-type TGFβ1. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure may bind to ALK-1 with an affinity at least comparable to that of the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof disclosed herein can bind to ALK-2 with an affinity at least comparable to that of the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof disclosed herein can bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with an affinity at least comparable to that of the wild-type TGFβ1 sequence.

[0192] In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure 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 that is at most comparable to wild-type TGFβ1. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure may bind to TGFβR1 with an affinity that is at most comparable to wild-type TGFβ1. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure may bind to TGFβR2 with an affinity that is at most comparable to wild-type TGFβ1. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure may bind to ALK-1 with an affinity that is at most comparable to the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof disclosed herein can bind to ALK-2 with an affinity that is at most comparable to the wild-type TGFβ1 sequence. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof disclosed herein can bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with an affinity that is at most comparable to the wild-type TGFβ1 sequence.

[0193] In some embodiments, TGFβ1 or a TGFβ1 variant, derivative, or fragment thereof can bind to TGFβR1, TGFβR2, ALK-1, or ALK-2 with an affinity that is increased by 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 relative to the wild-type TGFβ1 sequence. In some embodiments, TGFβ1 or a TGFβ1 variant, derivative, or fragment thereof can bind to TGFβR1, TGFβR2, ALK-1, or ALK-2 with an affinity that is decreased by 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 relative to the wild-type TGFβ1 sequence.

[0194] In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure can activate a native TGFβ1 receptor. A native TGFβ1 receptor can be, for example, a receptor comprising a TGFβR1 subunit and a TGFβR2 subunit. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure can activate a native TGFβ1 receptor when present in a fusion protein. In some embodiments, the TGFβ1 or TGFβ1 variants, derivatives, or fragments thereof of the present disclosure can activate a native TGFβ1 receptor when present as a polypeptide that is not part of a fusion protein, but do not activate a native TGFβ1 receptor when present in a fusion protein.

[0195] Transforming growth factor β2

[0196] The fusion protein may comprise a TGFβ2 protein or a variant, derivative or fragment thereof operably linked or fused directly or indirectly to interleukin-13 or a variant or derivative thereof. The TGFβ2 protein is preferably a mammalian TGFβ2 protein, such as 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 more, such as 100%, amino acid sequence identity to SEQ ID NO: 8 (the entire or underlined portion), SEQ ID NO: 22, or SEQ ID NO: 35, 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 as defined above. Variants, derivatives and fragments thereof also include proteins having TGFβ2 activity that are derived from a polypeptide having an amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 22 or SEQ ID NO: 35 by one or more amino acid substitutions, deletions or insertions. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

[0197] In some embodiments, the 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, at least or at least 50 amino acid substitutions, deletions, or insertions relative to the TGFβ2 sequence disclosed herein (e.g., a wild-type TGFβ2 sequence).

[0198] In some embodiments, the TGFβ2 of the present disclosure (e.g., a TGFβ2 variant, derivative, or fragment thereof) may include at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to a TGFβ2 sequence disclosed herein (e.g., a wild-type TGFβ2 sequence).

[0199] In some embodiments, the TGFβ2 sequences disclosed herein (e.g., TGFβ2 variants, derivatives, or fragments thereof) may comprise 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 ... , 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20 or 20-25 amino acid substitutions, deletions or insertions.

[0200] In some embodiments, the TGFβ2 sequences disclosed herein (e.g., TGFβ2 variants, derivatives, or fragments 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 relative to the TGFβ2 sequences disclosed herein (e.g., wild-type TGFβ2 sequences). 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, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions may be continuous, non-continuous, or a combination thereof.

[0201] The TGFβ2 of the present disclosure may comprise 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.

[0202] The TGFβ2 of the present disclosure may include TGFβ2 variants, derivatives, or fragments thereof having one or more amino acid substitutions. For example, the TGFβ2 variant, derivative, or fragment thereof may include an amino acid substitution at position R18, P36, or a combination thereof of SEQ ID NO: 22. In some embodiments, the TGFβ2 variant, derivative, or fragment thereof comprises a substitution of R18C, P36H, or a combination thereof relative to SEQ ID NO: 22. In some embodiments, the TGFβ2, fragment, or derivative thereof comprises a substitution of R18C and P36H relative to SEQ ID NO: 22.

[0203] In some embodiments, the TGFβ2 variant, derivative, or fragment thereof 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 thereof does not comprise an R18C or P36H substitution.

[0204] 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 comparable to that of the wild-type TGFβ2 sequence. Comparable affinity can be an increase in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type TGFβ2 sequence. Comparable affinity can be a decrease in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, 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, compared to the wild-type TGFβ2 sequence.

[0205] For example, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the present disclosure 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 approximately comparable to that of wild-type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the present disclosure may bind to TGFβR1 with an affinity approximately comparable to that of wild-type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the present disclosure may bind to TGFβR2 with an affinity approximately comparable to that of wild-type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the present disclosure may bind to ALK-1 with an affinity approximately comparable to that of the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof disclosed herein can bind to ALK-2 with an affinity approximately comparable to that of the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof disclosed herein can bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with an affinity approximately comparable to that of the wild-type TGFβ2 sequence.

[0206] In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure 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 comparable to that of wild-type TGFβ2. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure may bind to TGFβR1 with an affinity at least comparable to that of wild-type TGFβ2. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure may bind to TGFβR2 with an affinity at least comparable to that of wild-type TGFβ2. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure may bind to ALK-1 with an affinity at least comparable to that of the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof disclosed herein can bind to ALK-2 with an affinity at least comparable to that of the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof disclosed herein can bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with an affinity at least comparable to that of the wild-type TGFβ2 sequence.

[0207] In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure 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 that is at most comparable to wild-type TGFβ2. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure may bind to TGFβR1 with an affinity that is at most comparable to wild-type TGFβ2. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure may bind to TGFβR2 with an affinity that is at most comparable to wild-type TGFβ2. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure may bind to ALK-1 with an affinity that is at most comparable to the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof disclosed herein can bind to ALK-2 with an affinity that is at most comparable to that of the wild-type TGFβ2 sequence. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof disclosed herein can bind to TGFβR1, TGFβR2, ALK-1, and ALK-2 with an affinity that is at most comparable to that of the wild-type TGFβ2 sequence.

[0208] In some embodiments, TGFβ2 or a TGFβ2 variant, derivative, or fragment thereof can bind to TGFβR1, TGFβR2, ALK-1, or ALK-2 with an affinity that is increased by 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 relative to the wild-type TGFβ2 sequence. In some embodiments, TGFβ2 or a TGFβ2 variant, derivative, or fragment thereof can bind to TGFβR1, TGFβR2, ALK-1, or ALK-2 with an affinity that is decreased by 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 relative to the wild-type TGFβ2 sequence.

[0209] In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure can activate a native TGFβ2 receptor. A native TGFβ2 receptor can be, for example, a receptor comprising a TGFβR1 subunit and a TGFβR2 subunit. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure can activate a native TGFβ2 receptor when present in a fusion protein. In some embodiments, the TGFβ2 or TGFβ2 variants, derivatives, or fragments thereof of the present disclosure can activate a native TGFβ2 receptor when present as a polypeptide that is not part of a fusion protein, but do not activate a native TGFβ2 receptor when present in a fusion protein.

[0210] Interleukin-27

[0211] The fusion protein may comprise an IL27 protein or its variant, derivative or fragment operably linked or directly or indirectly fused to interleukin 13 or its variant or derivative. The IL27 protein is preferably a mammalian IL27 protein, such as human IL27 or mouse IL27, or its variant, derivative or fragment. The IL27 or IL27 variant, derivative or fragment of the present disclosure 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 provided in SEQ ID NO: 18). In some embodiments, the IL27 of the present disclosure comprises an IL27B subunit.

[0212] 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 that preferably have at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, e.g., 100%, amino acid sequence identity to SEQ ID NO:36 or SEQ ID NO:45 over their entire length. Amino acid sequence identity is preferably determined by pairwise alignment using the Needleman and Wunsch algorithm and the default GAP parameters as defined above. Variants, derivatives, and fragments thereof also include proteins having IL27 activity that are derived from a polypeptide having the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:45 by one or more amino acid substitutions, deletions, or insertions. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

[0213] In some embodiments, the 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, at least or at least 50 amino acid substitutions, deletions, or insertions relative to an IL27 sequence disclosed herein (e.g., a wild-type IL27 sequence).

[0214] In some embodiments, an IL27 of the present disclosure (e.g., an IL27 variant, derivative, or fragment thereof) may include at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL27 sequence disclosed herein (e.g., a wild-type IL27 sequence).

[0215] In some embodiments, the IL27 sequences disclosed herein (e.g., IL27 variants, derivatives, or fragments thereof) can comprise 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, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2- 20, 25, 30, 35, 36, 37, 38, 39, 40, 50, 50, 50, 60, 70, 80, 90, 100, 150, 150, 200, 250, 300, 400, 80, 90, 10 ...

[0216] In some embodiments, the IL27 sequences disclosed herein (e.g., IL27 variants, derivatives, or fragments 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 relative to the IL27 sequences disclosed herein (e.g., wild-type IL27 sequences). 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, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions may be continuous, non-continuous, or a combination thereof.

[0217] 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 a canonical wild-type IL27 sequence. In some embodiments, the IL27 sequence of the present disclosure comprises a substitution relative to the wild-type IL27 sequence.

[0218] Examples of IL27 variants, derivatives, or fragments thereof disclosed herein are IL27 variant sequences that can be secreted as functional immunomodulatory monomeric proteins, for example, an IL27A variant, derivative, or fragment thereof that can be secreted and function as a functional immunomodulatory monomeric protein without being bound to 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 thereof disclosed herein that comprises an amino acid substitution, L134C, relative to SEQ ID NO: 36 (which is L162C in the sequence containing the signal peptide) and can be secreted as a functional immunomodulatory monomeric protein.

[0219] The IL27 of the present disclosure may include IL27 variants, derivatives, or fragments thereof having one or more amino acid substitutions. For example, the IL27 variants, derivatives, or fragments thereof may include amino acid substitutions at positions F132, N132, L134, P135, E136, E137, L152, L153, ​​P154, or a combination thereof, of SEQ ID NO: 36. In some embodiments, the IL27 variants, derivatives, or fragments thereof are SEQ ID NO: 36. NO:36 contains the following substitutions: F132C, N132C, L134C, P135C, E136C, E137C, L152C, L153C, P154C, F132D, N132D, L134D, P135D, E136D, E137D, L152D, L153D, P154D, F132E, N132E, L134E, P135E, E136C E, 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.

[0220] In some embodiments, the IL27 variant, derivative, or fragment thereof does not comprise a substitution at position 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 thereof does not comprise F132C, N132C, L134C, P135C, E136C, E137C, L152C, L153C, P154C, F132D, N132D, L134D, P135D, E136D, E137D, L152D, L153D, P154D, F132E, N132E, L134E, P135C, E136C, E137C, L152C, L153C, P154C K, L152K, L153K, P154K, S31A, or L91P substitutions.

[0221] In some embodiments, the IL27 of the present disclosure or its IL27 variant, derivative or fragment binds to the IL27 receptor subunit with an affinity approximately equivalent to that of the wild-type IL27 sequence. A comparable affinity can be an increase in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4 times, less than about 1.3 times, less than about 1.2 times, or less than about 1.1 times, compared to the wild-type IL27 sequence. A comparable affinity can be a decrease in affinity, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4 times, less than about 1.3 times, less than about 1.2 times, or less than about 1.1 times, compared to the wild-type IL27 sequence.

[0222] For example, the IL27 or IL27 variants, derivatives, or fragments thereof disclosed herein can bind to interleukin 27 receptor alpha (IL-27RA), gpl30, or a combination thereof with an affinity approximately comparable to that of wild-type IL27. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments thereof disclosed herein can bind to IL-27RA with an affinity approximately comparable to that of wild-type IL27. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments thereof disclosed herein can bind to gpl30 with an affinity approximately comparable to that of wild-type IL27. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments thereof disclosed herein can bind to IL-27RA and gpl30 with an affinity approximately comparable to that of wild-type IL27.

[0223] In some embodiments, the IL27 or IL27 variants, derivatives, or fragments thereof disclosed herein can bind to an IL27 receptor subunit with an affinity at least comparable to that of wild-type IL27. For example, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can bind to IL-27RA, gp130, or a combination thereof with an affinity at least comparable to that of wild-type IL27. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can bind to IL-27RA with an affinity at least comparable to that of wild-type IL27. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can bind to gp130 with an affinity at least comparable to that of wild-type IL27. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can bind to both IL-27RA and gp130 with an affinity at least comparable to that of wild-type IL27.

[0224] In some embodiments, the IL27 or IL27 variants, derivatives, or fragments thereof disclosed herein can bind to an IL27 receptor subunit with an affinity that is at most comparable to that of wild-type IL27. For example, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can bind to IL-27RA, gpl30, or a combination thereof with an affinity that is at most comparable to that of wild-type IL27. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can bind to IL-27RA with an affinity that is at most comparable to that of wild-type IL27. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can bind to gpl30 with an affinity that is at most comparable to that of wild-type IL27. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can bind to IL-27RA and gpl30 with an affinity that is at most comparable to that of wild-type IL27.

[0225] In some embodiments, IL27 or an IL27 variant, derivative, or fragment thereof can bind to IL-27RA with an affinity that is increased by 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 relative to the wild-type IL27 sequence. In some embodiments, IL27 or an IL27 variant, derivative, or fragment thereof can bind to IL-27RA with an affinity that is decreased by 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 relative to the wild-type IL27 sequence.

[0226] In some embodiments, IL27 or an IL27 variant, derivative, or fragment thereof can bind to gp130 A with an affinity that is increased by 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 relative to the wild-type IL27 sequence. In some embodiments, IL27 or an IL27 variant, derivative, or fragment thereof can bind to gp130 with an affinity that is decreased by 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 relative to the wild-type IL27 sequence.

[0227] In some embodiments, the IL27 or IL27 variants, derivatives, or fragments thereof disclosed herein can activate a native IL27 receptor. A native IL27 receptor can be, for example, a receptor comprising an IL-27RA subunit and a gp130 subunit. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can activate a native IL27 receptor when present in a fusion protein. In some embodiments, the IL27 or IL27 variants, derivatives, or fragments disclosed herein can activate a native IL27 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate a native IL27 receptor when present in a fusion protein.

[0228] Fusion protein

[0229] The present disclosure provides fusion proteins comprising interleukin 13 (IL13) linked directly or indirectly to a regulatory cytokine, such as IL4, IL10, IL27, IL33, TGFβ1, TGFβ2, or another IL13.

[0230] In some embodiments, the fusion protein of the present disclosure can bind to a receptor present on the cell surface and form a complex with 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 (e.g., polypeptide chains). In some embodiments, the fusion protein of the present disclosure can bind to a receptor present on the cell surface and form a complex with 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 (e.g., polypeptide chains). In some embodiments, the fusion protein of the present disclosure can bind to a receptor present on the cell surface and form a complex with at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11 or at most 12 receptor subunits (e.g., polypeptide chains).

[0231] IL4 (or IL10 or IL13 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13 in the fusion protein may or may not be connected via a linker, such as a linker sequence, or via a chemical spacer.

[0232] The linker can be a peptide. The linker can comprise a linker sequence, such as a linker peptide sequence. The length of 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.

[0233] Joints as described herein can include flexible joints or rigid joints. Flexible joints can have sequences comprising glycine and serine residue segments. The small size of glycine and serine residues provides flexibility and allows the functional domains connected to move. The incorporation of serine or threonine can maintain the stability of the joint in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing unfavorable interactions between the joint and the protein portion. Flexible joints can also include other amino acids (such as threonine and alanine) to maintain flexibility, and polar amino acids (such as lysine and glutamine) to improve solubility.

[0234] The flexible linker may comprise SEQ ID NO: 3. The flexible linker may comprise a repeat of SEQ ID NO: 37 (GGGS), such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeats of SEQ ID NO: 37. The flexible linker may comprise a repeat of SEQ ID NO: 38 (GGGGS), such as 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, including SEQ ID NO: 40 (KESGSVSSEQLAQFRSLD) and SEQ ID NO: 41 (EGKSSGSGSESKST). SEQ ID NO: 42 (GSAGSAAGSGEF) linkers may also be used, in which large hydrophobic residues are minimized to maintain good solubility in aqueous solution. The length of the flexible linker may be adjusted to allow for proper folding or to achieve optimal biological activity of the fusion protein.

[0235] Rigid linkers can have, for example, an α-helical structure. α-Helical rigid linkers can be used as spacer sequences between protein domains. Rigid linkers can comprise a repeat of SEQ ID NO: 43 (EAAAK), such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeats of SEQ ID NO: 43. Rigid linkers can comprise a repeat of SEQ ID NO: 44 (EAAAR), such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeats of SEQ ID NO: 44. Rigid linkers can have a proline-rich sequence (XP) n, where X represents alanine, lysine, glutamine or any amino acid. The presence of proline in a non-helical linker can increase rigidity and allow for efficient separation of protein domains.

[0236] Joints of the present disclosure can include non-peptide joints, such as chemical joints. For example, two amino acid sequences of the present disclosure can be connected by chemical joints. Each chemical joint of the present disclosure can be an alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene or heteroarylene, any one of which is optionally substituted. In some embodiments, chemical joints of the present disclosure can be esters, ethers, amides, thioethers or polyethylene glycol (PEG). In some embodiments, the joint can reverse the order of the amino acid sequence in the compound, for example, so that the amino acid sequence connected by the joint is head to head, rather than head to tail. The limiting examples of this type of joint include the diester of dicarboxylic acid, such as oxalyl diester, malonyl diester, succinyl diester, glutaryl diester, adipoyl diester, pimeloyl diester, fumaryl diester, maleic diester, phthaloyl diester, isophthaloyl diester and terephthaloyl diester. Non-limiting examples of such linkers include diamides of dicarboxylic acids, such as oxalyl diamide, malonyl diamide, succinyl diamide, glutaryl diamide, adipoyl diamide, pimeloyl diamide, fumaryl diamide, maleic diamide, phthalyl diamide, isophthalyl diamide, and terephthalyl diamide. Non-limiting examples of such linkers include diamides of diamino linkers, such as ethylenediamine, 1,2-bis(methylamino)ethane, 1,3-diaminopropane, 1,3-bis(methylamino)propane, 1,4-bis(methylamino)butane, 1,5-bis(methylamino)pentane, 1,6-bis(methylamino)hexane, and piperyzine. Non-limiting examples of optional substituents include hydroxy, thiol, halogen, amino, nitro, nitroso, cyano, azido, sulfoxide, sulfone, sulfonamide, carboxyl, carboxaldehyde, imine, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, alkoxy, aryl, aryloxy, aralkyl, arylalkoxy, heterocyclyl, acyl, acyloxy, carbamate, amide, urea, epoxy, and ester.

[0237] In some embodiments, the N-terminus of a polypeptide sequence can be linked to the N-terminus of another polypeptide. In some embodiments, the C-terminus of a polypeptide sequence can be linked to the C-terminus of another polypeptide.

[0238] In some embodiments, the fusion proteins of the present disclosure do not comprise a linker.

[0239] Amino acids can include both genetically encoded and non-genetically encoded amino acids. Amino acids can include naturally occurring and non-naturally occurring amino acids. Amino acids can be L- or D-type. Substitutions can include conservative and / or non-conservative amino acid substitutions. A conservative amino acid substitution can be one amino acid substituted for another amino acid with similar biochemical properties (e.g., charge, size, and / or hydrophobicity). A non-conservative amino acid substitution can be one amino acid substituted for another amino acid with different biochemical properties (e.g., charge, size, and / or hydrophobicity). A conservative amino acid change can be, for example, a substitution that minimizes the effect on the secondary or tertiary structure of a polypeptide. A conservative amino acid change can be an amino acid change from one hydrophilic amino acid to another. Hydrophilic amino acids can include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K), and Arg (R). A conservative amino acid change can be an amino acid change 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 amino acid changes 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 amino acid changes from one basic amino acid to another basic amino acid. Basic amino acids may include His (H), Arg (R), and Lys (K). Conservative amino acid changes may be amino acid changes from one polar amino acid to another polar amino acid. Polar amino acids may include Asn (N), Gln (Q), Ser (S), and Thr (T). Conservative amino acid changes may be amino acid changes 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 can be an amino acid change from one aromatic amino acid to another aromatic amino acid. Aromatic amino acids can include Phe (F), Tyr (Y), and Trp (W). A conservative amino acid change can be an amino acid change from one aliphatic amino acid to another aliphatic amino acid. Aliphatic amino acids can 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.

[0240] Additional amino acid sequences may be present in the N-terminus and / or C-terminus of the fusion protein of the present invention, for example, affinity tags that promote purification. For example, a polyhistidine tag, a GST tag, a FLAG tag, a CBP tag, an HA tag or a Myc tag may be present in the C-terminus or N-terminus to promote purification. In some embodiments, for example, after purification, the affinity tag is removed from the fusion protein of the present invention. In some embodiments, the fusion protein of the present invention does not comprise an affinity tag (for example, the fusion protein can be purified by other methods). Additionally or alternatively, the fusion protein of the present invention may optionally comprise additional protein moieties, for example, moieties capable of targeting, for example, protein moieties 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.

[0241] IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) may be located at the N-terminus of IL13, or may be located 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.

[0242] 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 connected via a linker sequence.

[0243] 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 following staining for β3-tubulin.

[0244] In suitable embodiments, the fusion protein of the present invention exists in monomeric form, is purified into monomeric form and / or is used in monomeric form. In one embodiment, it has a molecular weight of 30 to 37 kDa. In some embodiments, the fusion protein of the present disclosure exists in multimeric form, is purified into multimeric form and / or is used in multimeric form, such as dimeric form or tetrameric form. In some embodiments, the fusion protein of the present disclosure exists in monomeric form, is purified into multimeric form and / or is used in multimeric form, such as dimeric form or tetrameric form. In some embodiments, the fusion protein of the present disclosure exists in monomeric form, is purified into monomeric form, dimer, trimer, tetramer, multimer or its arbitrary combination and / or is used as monomeric form, dimer, trimer, tetramer, multimer or its arbitrary combination. Dimer, trimer, tetramer or multimer can comprise covalent or non-covalently bound subunits.

[0245] In some embodiments, the IL4 / IL13 fusion protein of the present disclosure exists as a monomer, is purified into a monomer, and / or is used as a monomer. In some embodiments, the IL4 / IL13 fusion protein of the present disclosure exists as a dimer, is purified into a dimer, and / or is used as a dimer. In some embodiments, the IL4 / IL13 fusion protein of the present disclosure exists as a trimer, is purified into a trimer, and / or is used as a trimer. In some embodiments, the IL4 / IL13 fusion protein of the present disclosure exists as a tetramer, is purified into a tetramer, and / or is used as a tetramer. In some embodiments, the IL4 / IL13 fusion protein of the present disclosure exists as a multimer, is purified into a multimer, and / or is used as a multimer. In some embodiments, the IL4 / IL13 fusion protein of the present disclosure exists as a monomer and a dimer, is purified into a monomer and a dimer, and / or is used as a monomer and a dimer. In some embodiments, the IL4 / IL13 fusion protein exists as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, is purified as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, and / or is used as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof.

[0246] In some embodiments, the IL10 / IL13 fusion protein of the present disclosure exists as a monomer, is purified into a monomer, and / or is used as a monomer. In some embodiments, the IL10 / IL13 fusion protein of the present disclosure exists as a dimer, is purified into a dimer, and / or is used as a dimer. In some embodiments, the IL10 / IL13 fusion protein of the present disclosure exists as a trimer, is purified into a trimer, and / or is used as a trimer. In some embodiments, the IL10 / IL13 fusion protein of the present disclosure exists as a tetramer, is purified into a tetramer, and / or is used as a tetramer. In some embodiments, the IL10 / IL13 fusion protein of the present disclosure exists as a multimer, is purified into a multimer, and / or is used as a multimer. In some embodiments, the IL10 / IL13 fusion protein of the present disclosure exists as a monomer and a dimer, is purified into a monomer and a dimer, and / or is used as a monomer and a dimer. In some embodiments, the IL10 / IL13 fusion protein exists as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, is purified as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, and / or is used as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof.

[0247] In some embodiments, the IL13 / IL13 fusion protein of the present disclosure exists as a monomer, is purified into a monomer, and / or is used as a monomer. In some embodiments, the IL13 / IL13 fusion protein of the present disclosure exists as a dimer, is purified into a dimer, and / or is used as a dimer. In some embodiments, the IL13 / IL13 fusion protein of the present disclosure exists as a trimer, is purified into a trimer, and / or is used as a trimer. In some embodiments, the IL13 / IL13 fusion protein of the present disclosure exists as a tetramer, is purified into a tetramer, and / or is used as a tetramer. In some embodiments, the IL13 / IL13 fusion protein of the present disclosure exists as a multimer, is purified into a multimer, and / or is used as a multimer. In some embodiments, the IL10 / IL13 fusion protein exists as a monomer and a dimer, is purified into a monomer and a dimer, and / or is used as a monomer and a dimer. In some embodiments, the IL10 / IL13 fusion protein exists as, is purified as, and / or is used as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof.

[0248] In some embodiments, the IL27 / IL13 fusion protein of the present disclosure exists as a monomer, is purified into a monomer, and / or is used as a monomer. In some embodiments, the IL27 / IL13 fusion protein of the present disclosure exists as a dimer, is purified into a dimer, and / or is used as a dimer. In some embodiments, the IL27 / IL13 fusion protein of the present disclosure exists as a trimer, is purified into a trimer, and / or is used as a trimer. In some embodiments, the IL27 / IL13 fusion protein of the present disclosure exists as a tetramer, is purified into a tetramer, and / or is used as a tetramer. In some embodiments, the IL27 / IL13 fusion protein of the present disclosure exists as a multimer, is purified into a multimer, and / or is used as a multimer. In some embodiments, the IL27 / IL13 fusion protein of the present disclosure exists as a monomer and a dimer, is purified into a monomer and a dimer, and / or is used as a monomer and a dimer. In some embodiments, the IL27 / IL13 fusion protein exists as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, is purified as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, and / or is used as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof.

[0249] In some embodiments, the IL33 / IL13 fusion protein of the present disclosure exists as a monomer, is purified into a monomer, and / or is used as a monomer. In some embodiments, the IL33 / IL13 fusion protein of the present disclosure exists as a dimer, is purified into a dimer, and / or is used as a dimer. In some embodiments, the IL33 / IL13 fusion protein of the present disclosure exists as a trimer, is purified into a trimer, and / or is used as a trimer. In some embodiments, the IL33 / IL13 fusion protein of the present disclosure exists as a tetramer, is purified into a tetramer, and / or is used as a tetramer. In some embodiments, the IL33 / IL13 fusion protein of the present disclosure exists as a multimer, is purified into a multimer, and / or is used as a multimer. In some embodiments, the IL33 / IL13 fusion protein of the present disclosure exists as a monomer and a dimer, is purified into a monomer and a dimer, and / or is used as a monomer and a dimer. In some embodiments, the IL33 / IL13 fusion protein exists as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, is purified as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, and / or is used as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof.

[0250] In some embodiments, the TGFβ1 / IL13 fusion protein of the present disclosure exists as a monomer, is purified into a monomer, and / or is used as a monomer. In some embodiments, the TGFβ1 / IL13 fusion protein of the present disclosure exists as a dimer, is purified into a dimer, and / or is used as a dimer. In some embodiments, the TGFβ1 / IL13 fusion protein of the present disclosure exists as a trimer, is purified into a trimer, and / or is used as a trimer. In some embodiments, the TGFβ1 / IL13 fusion protein of the present disclosure exists as a tetramer, is purified into a tetramer, and / or is used as a tetramer. In some embodiments, the TGFβ1 / IL13 fusion protein of the present disclosure exists as a multimer, is purified into a multimer, and / or is used as a multimer. In some embodiments, the TGFβ1 / IL13 fusion protein of the present disclosure exists as a monomer and a dimer, is purified into a monomer and a dimer, and / or is used as a monomer and a dimer. In some embodiments, the TGFβ1 / IL13 fusion protein exists as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, is purified into a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, and / or is used as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof.

[0251] In some embodiments, the TGFβ2 / IL13 fusion protein of the present disclosure exists as a monomer, is purified into a monomer, and / or is used as a monomer. In some embodiments, the TGFβ2 / IL13 fusion protein of the present disclosure exists as a dimer, is purified into a dimer, and / or is used as a dimer. In some embodiments, the TGFβ2 / IL13 fusion protein of the present disclosure exists as a trimer, is purified into a trimer, and / or is used as a trimer. In some embodiments, the TGFβ2 / IL13 fusion protein of the present disclosure exists as a tetramer, is purified into a tetramer, and / or is used as a tetramer. In some embodiments, the TGFβ2 / IL13 fusion protein of the present disclosure exists as a multimer, is purified into a multimer, and / or is used as a multimer. In some embodiments, the TGFβ2 / IL13 fusion protein of the present disclosure exists as a monomer and a dimer, is purified into a monomer and a dimer, and / or is used as a monomer and a dimer. In some embodiments, the TGFβ2 / IL13 fusion protein exists as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, is purified as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof, and / or is used as a monomer, dimer, trimer, tetramer, multimer, or any combination thereof.

[0252] Preparation method

[0253] The fusion protein of the present invention can be prepared by conventional techniques of the skilled artisan. For example, it can be prepared using techniques for producing recombinant fusion proteins through cultured continuous cell lines. For example, a combination of recombinant DNA technology and gene transfection methods can be used to produce the fusion protein of the present invention in a host cell transfectoma.

[0254] For example, to express the fusion protein of the present invention, nucleic acid molecules encoding the fusion protein of the present invention can be prepared by standard molecular biology techniques. The nucleic acid molecules of the present invention are preferably operably linked to transcriptional control sequences such as a promoter and an optional 3' untranslated region. The nucleic acid molecules of the present invention can be inserted into a vector, such as an expression vector, so that the gene is operably linked to transcriptional and translational control sequences. An expression vector and transcriptional control sequences compatible with the host cell used for expression are selected. The nucleic acid molecules encoding the fusion protein of the present invention can be inserted into an expression vector by conventional methods. The nucleic acid molecules or vectors of the present invention may further include a nucleotide sequence encoding a signal peptide, which can promote secretion of the fusion protein from the host cell. The nucleotide sequence encoding the signal peptide can be operably linked to the nucleic acid molecule of the present invention. Preferably, the signal peptide is located at the amino terminus of the fusion protein of the present invention. Therefore, the nucleotide sequence encoding the signal peptide can be located 5' of the nucleic acid molecule encoding the fusion protein of the present invention. The signal peptide can be a cytokine signal peptide or a signal peptide from a non-cytokine protein. The signal peptide may not be present on the mature form of the fusion protein. The promoter can be constitutive or inducible. The vector can contain a selectable marker for selecting host cells carrying the vector. When the vector is a replicable vector, the vector may contain an origin of replication.

[0255] The fusion protein of the present invention can be synthesized de novo by chemical synthesis (using, for example, a peptide synthesizer provided by Applied Biosystems) or can be produced by recombinant host cells by expressing a nucleic acid sequence encoding the fusion protein, fragment derivative or variant. Variants and fragments are preferably functional, that is, they 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) and / or IL13 activity, preferably IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13 activity.

[0256] The functional activity of IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13, as well as the functional activity of the fusion protein comprising IL4 (or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2) and IL13 can be determined using conventional methods known to those skilled in the art. For example, a suitable assay for the functionality of IL4 and a fusion protein comprising IL4 (or IL10) and IL13 is lipopolysaccharide (LPS)-induced cytokine release (IL1, IL6, IL8, TNFα) in whole blood, optionally in the presence of an anti-IL10 antibody. Functional activity can also be determined by assessing the activation of intracellular signaling pathways when target cells are incubated with the fusion protein in the presence or absence of blocking antibodies to the cytokine portion of the fusion protein or its receptor.

[0257] On the other hand, there is provided an isolated nucleic acid sequence encoding any of the above-mentioned fusion proteins, such as cDNA, genomic DNA and RNA sequences. Due to the degeneracy of the genetic code, different nucleic acid sequences can encode the same amino acid sequence. Any nucleic acid sequence encoding the fusion protein of the present invention is referred to herein as "IL4 / IL13 (or IL10 / IL13, IL27 / IL13, IL33 / IL13, TGFβ1 / IL13, TGFβ2 / IL13, IL13 / IL13) encoding nucleic acid sequence". The nucleic acid sequence provided includes recombinant, artificial or synthetic nucleic acid sequences. It will be understood that when a sequence is described as a DNA sequence and RNA is mentioned, the actual base sequence of the RNA molecule is identical, with the difference being that thymine (T) is replaced by uracil (U). The nucleic acid sequences of the present invention are particularly suitable 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 producing these proteins, or for gene therapy purposes.

[0258] The nucleic acid 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, in particular the DNA sequence, can be inserted into an expression vector that produces (e.g., a large amount of) 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.

[0259] Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma virus. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter, may be used.

[0260] In addition to the nucleic acid molecules and regulatory sequences encoding IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion proteins, the recombinant expression vectors of the present invention can also carry additional sequences, such as sequences that regulate the replication of the vector in the host cell (e.g., origin of replication) and selectable marker genes. Selectable marker genes can help select host cells into which the vector has been introduced (see, for example, US 4,399,216, US 4,634,665, and US 5,179,017, Axel et al.). For example, selectable marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate on host cells into which the vector has been introduced. Preferred selectable marker genes include dihydrofolate reductase (DHFR) genes (for dhfr- host cells with methotrexate selection / amplification) and neo genes (for G418 selection). Finally, in addition to the nucleic acid sequence encoding the fusion protein of the present invention, the recombinant expression vector may also contain a gene encoding a glycosyltransferase.

[0261] In another aspect, the present invention relates to a host cell comprising a nucleic acid sequence of the present invention, or 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 prokaryotic cells and eukaryotic cells. The host cell can also be a cell line, such as a prokaryotic cell line or a eukaryotic cell line. The host cell is preferably an animal cell or cell line, such as a mammalian cell or cell line.

[0262] In one embodiment, the fusion protein of the present invention is expressed in eukaryotic cells, such as mammalian host cells. Preferred mammalian host cells for expressing the fusion protein of the present invention include CHO cells (including dhfr-CHO cells, described in (Urlaub et al., 1980)) used with the 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 comprising a nucleic acid sequence encoding the fusion protein of the present invention is introduced into a mammalian host cell, the fusion protein of the present invention can be produced by culturing the host cell for a period sufficient to allow the fusion protein to be expressed in the host cell, or more preferably for a period of time in which the fusion protein is secreted into the host cell culture medium in which the host cell is grown. Standard protein purification methods can be used to recover the fusion protein of the present invention from the culture medium.

[0263] Alternatively, the nucleic acid sequence encoding the fusion protein of the present invention can be expressed in other expression systems, including prokaryotes, such as microorganisms, such as Escherichia coli, or algae expression systems, insect cell expression systems, or cell-free protein synthesis systems. In addition, the fusion protein of the present invention can be produced in transgenic non-human animals, such as milk from sheep and rabbits or eggs from hens, or in transgenic plants.

[0264] The nucleic acid sequence of the present invention can be introduced into host cells by any standard technique known in the art. In order to express the fusion protein of the present invention, one or more expression vectors encoding the fusion protein can be transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass the various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, lipofectamine transfection, and lyophilization transfection, etc. The cell line that secretes the fusion protein of the present invention can be identified by measuring the presence of the fusion protein in the culture supernatant. The preferred screening procedure includes two consecutive steps, the first being to identify the cell line that secretes the fusion protein, and the second being to determine the quality of the fusion protein (e.g., the ability of the fusion protein to inhibit the production of cytokines by blood cells stimulated by LPS or other Toll-like receptor agonists), glycosylation pattern, etc.

[0265] For optimal expression in host cells, the fusion proteins encoding the present disclosure (e.g., IL4 / IL13 or IL10 / IL13 or IL27 / IL13 or IL33 / 13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13 fusion proteins) can be codon-optimized by adapting codon usage to the most preferred codon usage in host cell genes. Several techniques for modifying codon usage to that preferred by host cells can be found in patent and scientific literature. The exact method of codon usage modification is not important to the present invention.

[0266] In another embodiment of the present invention, PCR primers and / or probes and kits 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 are provided. Degenerate or specific PCR primer pairs for amplifying DNA encoding IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion proteins from a sample can be synthesized (see Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson et al. (2000) PCR-Basics: From Background to Bench, 1st edition, Springer Verlag, Germany). For example, any segment of 9, 10, 11, 12, 13, 14, 15, 16, 18 or more consecutive nucleotides of the nucleic acid sequence encoding the IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein (or its complementary chain) can be used as a primer or probe. Similarly, a DNA fragment of the nucleic acid sequence encoding the IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein can be used as a hybridization probe.The detection kit for the nucleic acid sequence encoding the IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein may comprise specific primers for the nucleic acid sequence 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 / or specific primers for the nucleic acid sequence encoding the IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein. 13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein encoding nucleic acid sequence specific probe, and related protocols for using primers or probes to specifically detect IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein encoding nucleic acid sequence in a sample. For example, such a detection kit can be used to determine whether a host cell has been transformed with a specific IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein encoding nucleic acid sequence of the present invention. Due to the degeneracy of the genetic code, certain amino acid codons can be replaced by other codons without changing the amino acid sequence of the protein.

[0267] In one aspect, the present invention relates to a method for producing a fusion protein of the present invention (e.g., IL4 / IL13 or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13 fusion protein), the method comprising the steps of: culturing a host cell of the present invention under conditions that allow production of the fusion protein (e.g., IL4 / IL13 or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or TGFβ2 / IL13 or IL13 / IL13 fusion protein); and optionally, recovering the fusion protein. A skilled person will routinely be able to select conditions that allow production of an IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein. In addition, those skilled in the art will be able to reclaim the fusion protein produced using conventional methods, including, but not limited to, chromatographic methods (including, but not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, metal binding, etc.), immunoprecipitation, HPLC, high speed centrifugation, precipitation, and differential dissolution and extraction. As mentioned above, the recovery or purification of the fusion protein can be promoted by adding, for example, a His tag to the fusion protein.

[0268] Pharmaceutical composition

[0269] In one aspect, the present invention relates to a pharmaceutical composition comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier.

[0270] Pharmaceutical compositions can be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional techniques (e.g., as described in Remington: The Science and Practice of Pharmacy, 19th ed., Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995).

[0271] The term "pharmaceutically acceptable carrier" refers to a carrier or excipient that is non-toxic and non-therapeutic in nature. Examples of such excipients are, but are not limited to, saline, Ringer's solution, dextrose solution, and Hank's solution.

[0272] The pharmaceutical composition can be administered by any suitable route and mode. It will be appreciated by those skilled in the art that the route and / or mode of administration will vary depending on the desired results.

[0273] The pharmaceutical composition according to the present invention can be formulated according to conventional procedures for administration by any route, preferably parenteral administration. The composition can be in the form of a liquid preparation, such as an oral or sterile parenteral solution or suspension.

[0274] The pharmaceutical compositions of the present invention include any form of pharmaceutical compositions suitable for parenteral administration.

[0275] In one embodiment, the pharmaceutical composition is administered parenterally.

[0276] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intraventricular, intracisternal, and intraparenchymal administration, e.g., injection and infusion.

[0277] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0278] In one embodiment, the fusion protein of the invention is administered in crystalline form by subcutaneous injection.

[0279] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, absorption delaying agents, and the like that are physiologically compatible.

[0280] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, they may be considered for use in the pharmaceutical compositions of the present invention. Preferably, the carrier is suitable for parenteral administration, such as intravenous or subcutaneous injection or infusion.

[0281] Pharmaceutical compositions must be sterile and stable under production and storage conditions generally. Compositions can be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. Examples of suitable aqueous and non-aqueous carriers that can be used for pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils, such as olive oil and injectable organic esters, such as ethyl acetate. Appropriate mobility can be maintained, for example, by using coating materials (such as lecithin), by maintaining the required particle size in the case of dispersions, and by using surfactants to maintain.

[0282] Sterile injectable solutions can be prepared by incorporating the active compound into a suitable solvent in the desired amount, for example, with one or a combination of the ingredients listed above, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients, such as those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.

[0283] Depending on the route of administration, the active compound, i.e., IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13 or TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein, can be coated with a material to protect it from acid and other natural conditions that may inactivate the compound. For example, the compound can be administered to a subject in a suitable carrier, such as a liposome. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., 1984).

[0284] The fusion protein of the present invention can also be prepared with a carrier that will prevent the fusion protein from releasing rapidly, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such preparations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0285] The pharmaceutical compositions of the present disclosure can be any pharmaceutical compound described herein in combination with other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. Pharmaceutical compositions facilitate administration of a compound to an organism.

[0286] The pharmaceutical preparation for administration can include an aqueous solution of the active compound in a water-soluble form. The suspension of the active compound can be prepared into 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 can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. The suspension can also include a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a high concentration solution. The active ingredient can be in powder form to be prepared with a suitable carrier, such as sterile pyrogen-free water, before use.

[0287] In practicing the methods of treatment or uses provided herein, a therapeutically effective amount of a compound described herein is administered as a pharmaceutical composition to a subject suffering from the 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 efficacy of the compound used, and other factors.

[0288] Pharmaceutical compositions can be formulated using one or more pharmaceutically acceptable carriers (including excipients and adjuvants that facilitate processing of the active compounds into pharmaceutically usable formulations). The formulations can be modified according to the chosen route of administration. Pharmaceutical compositions containing the compounds described herein can be prepared, for example, by mixing, dissolving, emulsifying, encapsulating, embedding, or compressing processes.

[0289] The pharmaceutical composition may include at least one pharmaceutically acceptable carrier, diluent or excipient and a compound described herein in the form of a free base or a pharmaceutically acceptable salt. The pharmaceutical composition may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0290] Methods for preparing compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form solid, semisolid, or liquid compositions. Solid compositions include, for example, powders, dispersible granules, and cachets. Liquid compositions include, for example, solutions in which the compound is dissolved, emulsions containing the compound, or solutions containing liposomes, micelles, or nanoparticles containing the compounds disclosed herein. Semisolid compositions include, for example, gels, suspensions, and creams. The compositions may be liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to use, or as emulsions. These compositions may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, and other pharmaceutically acceptable additives.

[0291] Non-limiting examples of dosage forms suitable for use with the present disclosure include liquids, powders, gels, nanosuspensions, nanoparticles, microgels, aqueous or oily suspensions, emulsions, and any combination thereof.

[0292] Non-limiting examples of pharmaceutically 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, emulsifiers, antimicrobial agents, spheronization agents, and any combination thereof.

[0293] Non-limiting examples of pharmaceutically acceptable carriers include saline, Ringer's solution, and dextrose solution. In some embodiments, the pH of the solution may be from about 5 to about 8, and may be from about 7 to about 7.5. Other carriers include sustained-release formulations, such as semipermeable matrices of solid hydrophobic polymers containing the compound. The matrix may be in the form of a shaped article, such as a film, liposome, microparticle, or microcapsule.

[0294] The pH of the disclosed compositions can be in the range of about 3 to about 12. The pH of the composition can 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 can 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 can 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.

[0295] Preparations of the present disclosure may comprise sugar, alcohol, antioxidant, buffer, antibacterial agent, solute or suspending agent or thickening agent that makes preparation and the blood of expected recipient isotonic. These compositions may also comprise preservative, wetting agent, emulsifying agent and dispersant. By adding various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid etc., the effect of microorganisms on the subject compound can be prevented. It is also desirable to include isotonic agents, such as sugar, sodium chloride etc. in the composition. In addition, the absorption of the injectable drug form can be extended by comprising an agent that delays absorption, such as aluminum monostearate and gelatin. If necessary, the preparation may be diluted with, for example, isotonic saline solution or glucose solution before use.

[0296] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a compound (e.g., a fusion protein) mixed with a pharmaceutically acceptable carrier and / or excipient, such as saline, phosphate-buffered saline, phosphate and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary agents include octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextran, 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 ethylene glycol.

[0297] In some embodiments, the pharmaceutical formulations disclosed herein may comprise: (i) a compound or fusion protein disclosed herein; (ii) a buffer; (iii) a nonionic detergent; (iv) a tonicity agent; and (v) a stabilizer. In some embodiments, the pharmaceutical formulations disclosed herein are stable liquid pharmaceutical formulations.

[0298] For solid compositions, solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, and magnesium carbonate.

[0299] The pharmaceutical carrier or excipient can be a solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, etc., which is physiologically compatible. The carrier can be suitable for administration by the routes disclosed herein (eg, parenteral).

[0300] The composition of the present invention can be, for example, an immediate release form or a controlled release formulation. Immediate release formulations can be formulated to allow compounds to act quickly. Non-limiting examples of immediate release formulations include readily soluble formulations. Controlled release formulations can be a pharmaceutical formulation that is adjusted so that the release rate and release profile of the active agent can be matched with physiological and time treatment requirements, or alternatively, has been formulated to achieve the release of the active agent at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., synthetic or naturally derived), other gelling agents (e.g., dietary fiber forming a gel), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed therein), granules in the matrix, polymer mixtures, and granule clusters.

[0301] In some embodiments, the formulations of the present disclosure include a thermal 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.

[0302] Non-limiting examples of pharmaceutically acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Edition (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 Edition (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[0303] The pharmaceutical composition can be applied topically, for example, by direct injection of the compound into an organ, optionally in the form of a reservoir or a sustained-release formulation or an implant. The pharmaceutical composition can be provided in the form of a quick-release formulation, in the form of an extended-release formulation, or in the form of a medium-release formulation. Quick-release forms can provide immediate release. Extended-release formulations can provide controlled-release or sustained-release.

[0304] In some embodiments, a pump can be used to deliver a pharmaceutical composition. In some embodiments, a pen delivery device can be used, for example, to deliver a composition of the present disclosure subcutaneously. Such pen delivery devices can be reusable or disposable. A reusable pen delivery device can use a replaceable cartridge containing a pharmaceutical composition disclosed herein. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. A disposable pen does not have a replaceable cartridge. In contrast, a disposable pen delivery device comes pre-loaded with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition within the reservoir is emptied, the entire device is discarded.

[0305] The pharmaceutical compositions described herein can be unit dosage forms suitable for single administration of precise dosages. In a unit dosage form, the formulation can be divided into unit doses containing an appropriate amount of one or more compounds, fusion proteins and / or therapeutic agents. A unit dose can be a packaged form of a formulation containing discrete amounts. Non-limiting examples are packaged injections, vials and ampoules. The aqueous suspension compositions disclosed herein can be packaged in single-dose, non-resealable containers. Multi-dose resealable containers can be used, for example, with or without preservatives. The injection formulations disclosed herein can be present in unit dosage form, for example, in ampoules or in multi-dose containers with preservatives.

[0306] In some embodiments, pharmaceutical preparation disclosed herein is a liquid preparation that can include about 50 μ g / mL to about 100 mg / mL fusion protein.Preparation can include, for example, at least 50 μ g / mL, at least 100 μ g / mL, at least 200 μ g / mL, at least 300 μ g / mL, at least 400 μ g / mL, at least 500 μ g / mL, at least 600 μ g / mL, at least 700 μ g / mL, at least 800 μ g / mL, at least 900 μ g / 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 at most 100 μg / mL, at most 200 μg / mL, at most 300 μg / mL, at most 400 μg / mL, at most 500 μg / mL, at most 600 μg / mL, at most 700 μg / mL, at most 800 μg / mL, at most 900 μg / mL, at most 1 mg / mL, at most 10 mg / mL, at most 20 mg / mL, or at most 50 mg / mL.

[0307] The dosage regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, as indicated by the exigencies of the therapeutic situation, a single bolus can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniform dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect associated with the required pharmaceutical carrier. The specifications of the dosage unit form of the present invention are indicated by and directly dependent on the following: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such an active compound for individual therapeutic sensitivity.

[0308] Actual dosage levels of the IL4 / IL13 (or IL10 / IL13 or IL27 / IL13 or IL33 / IL13TGFβ1 / IL13 or TGFβ2 / IL13 or IL13 / IL13) fusion protein in the pharmaceutical compositions of the present invention can 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 that is effective ("effective amount") to achieve the desired therapeutic response for a particular subject (e.g., patient), composition, and mode of administration, without being toxic to the subject (e.g., patient). The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and / or materials in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts.

[0309] 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 can be administered as intravenous injection or short-term infusion, in another embodiment, they are administered by slow continuous infusion over a long period of time (e.g., more than 24 hours) to reduce toxic side effects.

[0310] 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 protein of the invention can be administered as maintenance therapy, e.g., once a week for 6 months or longer.

[0311] 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 proteins of the present invention can be administered as intrathecal injections. And in yet another embodiment, they are administered via intrathecal or intraspinal drug delivery systems that allow continuous or repeated administration.

[0312] The therapeutic agents described herein can be administered before, during, or after the onset of a disease or condition, and the time for administering a composition containing a therapeutic agent can vary. For example, the composition can be used as a prophylactic and can be continuously administered to a subject with a condition or disease tendency to reduce the likelihood of the disease or condition occurring. The composition can be administered to the subject as soon as possible during or after the onset of symptoms. The initial administration can be by any feasible route, such as by any route described herein using any formulation described herein. After the onset of a disease or condition is detected or suspected, the therapeutic agent can be administered as soon as practicable and for the duration of time required to treat the disease, such as from about 1 month to about 3 months. The duration of treatment for each subject may be different.

[0313] The dosage can be based on the amount of fusion protein / kg body weight of the subject. The dosage of the fusion protein can 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. g / kg, 500μg / kg, 525μg / kg, 550μg / kg, 575μg / kg, 600μg / kg, 625μg / kg, 650μg / kg, 675μg / kg, 700μg / kg, 725μg / kg, 7 50μg / kg, 775μg / kg, 800μg / kg, 825μg / kg, 850μg / kg, 875μg / kg, 900μg / kg, 925μg / kg, 950μg / kg, 975μg / kg or 1mg / kg. The dosage of the fusion protein can be up to about 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 , 500μg / kg, 525μg / kg, 550μg / kg, 575μg / kg, 600μg / kg, 625μg / kg, 650μg / kg, 675μg / kg, 700μg / kg, 725μg / kg, 750 μg / kg, 775μg / kg, 800μg / kg, 825μg / kg, 850μg / kg, 875μg / kg, 900μg / kg, 925μg / kg, 950μg / kg, 975μg / kg or 1mg / kg.

[0314] 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 at most about 1 ng, at most 10 ng, at most 100 ng, at most 500 ng, at most 1 μg, at most 5 μg, at most 10 μg, at most 50 μg, at most 100 μg, at most 500 μg, at most at most 1 mg, at most 5 mg, at most 10 mg, at most 50 mg, or at most 100 mg.

[0315] The dosage can be determined with reference to the plasma concentration or local concentration of the fusion protein. The target plasma concentration or local concentration of the fusion protein can be 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 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 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 at most about 1 nM, at most about 10 nM, at most about 100 nM, at most about 1 μM, at most about 10 μM, at most about 100 μM, or at most about 1 mM.

[0316] As long as clinical needs, the use of fusion protein can continue.In some embodiments, fusion protein of the present disclosure can be used 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 present 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.

[0317] In some embodiments, the fusion protein can 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 during a treatment cycle. In some embodiments, a treatment cycle is 7 days, 14 days, 21 days, 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.

[0318] In some embodiments, the fusion protein is administered to the subject once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0319] Therapeutic uses

[0320] In another aspect, the present invention relates to the use of the fusion protein of the present invention or the nucleic acid encoding the same as a medicine.

[0321] In one aspect, the present invention relates to the use of fusion proteins or nucleic acids encoding them for treating pain, such as chronic pain. "Chronic" may mean that the pain persists for more than 2 weeks or for more than 1, 3, 6, 12 months, or even for more than 1, 2, 4, or 6 years. The pain may 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 types of pain.

[0322] Neuropathy can cause pain. In some embodiments, the fusion proteins of the present disclosure can be used to treat neuropathy. Neuropathy can be associated with pain, numbness, weakness, or a combination thereof.

[0323] In particular, it was found that the fusion protein of the present invention has a lasting analgesic effect on the 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 it can be used for preventing and treating neuropathic pain. Therefore, the fusion protein may be particularly suitable for treating neuropathy (such as neuropathy caused by chemotherapy) and other forms of peripheral neuropathic pain, such as postherpetic neuralgia, trigeminal neuralgia, post-traumatic 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, etc. In some embodiments, the fusion protein of the present invention can be used to treat postoperative cognitive dysfunction.

[0324] In some embodiments, the fusion proteins of the present disclosure can be used to treat conditions associated with cancer or chemotherapy, such as chemotherapy-induced neuropathy, chemotherapy-related pain, chemo brain, cancer-related cognitive impairment, cancer-related cognitive dysfunction. In some embodiments, the condition is associated with chemotherapy used to treat acute leukemia, astrocytoma, bile duct cancer (cholangiocarcinoma), bone cancer, breast cancer, brain stem glioma, bronchioalveolar lung cancer, adrenal cancer, cancer of the anal region, bladder cancer, endocrine system cancer, esophageal cancer, head and neck cancer, kidney cancer, parathyroid cancer, penile cancer, pleural / peritoneal cancer, salivary gland cancer, small intestine cancer, thyroid cancer, ureteral cancer, urethral cancer, cervical cancer, endometrial cancer, fallopian tube cancer, renal pelvis cancer, vaginal cancer, vulvar cancer, cervical cancer, chronic leukemia, colon cancer, colorectal cancer, skin melanoma, ependymoma, epidermoid tumor, Ewing's sarcoma, gastric cancer, glioblastoma, multiple sclerosis, leukemia ...cervic glioblastoma, glioma, hematologic malignancies, hepatocellular carcinoma (liver cancer), hepatoma, Hodgkin's disease, intraocular melanoma, Kaposi's sarcoma, lung cancer, lymphoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, muscle cancer, central nervous system (CNS) tumors, neuronal cancer, small cell lung cancer, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pediatric malignancies, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, soft tissue sarcoma, schwannoma, skin cancer, spinal axis tumors, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, uterine cancer, or tumors and metastases thereof, including refractory versions of any of the foregoing cancers or any combination thereof.

[0325] In further embodiments, the fusion proteins of the present invention or nucleic acids encoding the same can be used to treat pain and neurodegeneration in central nervous system disorders (including spinal cord injury, post-stroke pain, and multiple sclerosis). In some embodiments, the fusion proteins of the present disclosure can be used to treat postoperative cognitive dysfunction.

[0326] On the other hand, the present invention relates to the use of the fusion protein of the present invention or the nucleic acid encoding the same for treating chronic mixed nociceptive pain and neuropathic pain, such as low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, etc.

[0327] In another aspect, the fusion protein of the present invention or the nucleic acid encoding the same can be used to treat post-orthopedic pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, and the like.

[0328] In one embodiment, the disorder treated with the fusion protein of the invention is characterized by pain and can be selected from nociceptive pain, neuropathic pain, or mixed nociceptive-neuropathic pain.

[0329] In another embodiment, the fusion protein of the present invention or the nucleic acid encoding the same can be used to prevent chronic pain. In particular, the fusion protein can be used to prevent neuropathic pain and neurodegeneration in cancer patients receiving chemotherapy.

[0330] In another aspect, the present invention relates to use of the fusion protein of the present invention for preventing or treating a clinical disorder in a mammal, such as a human, wherein interleukin 13 is indicated for the disorder.

[0331] In another aspect, the present invention relates to the use of the fusion protein of the present invention for preventing or treating clinical disorders in mammals, such as humans, wherein interleukin-4, interleukin-10, or 27, IL33, TGFβ1, or TGFβ2 is suitable for the disorder.

[0332] According to one embodiment, the fusion proteins taught herein can be used to inhibit neuroinflammation due to activation of glial and neuronal cells in the central nervous system, infiltrating immune cells, or any combination thereof.

[0333] Therefore, the fusion protein of the present invention can be used to prepare a drug for alleviating neuroinflammatory response by inhibiting the activation of glial cells and neuronal cells in vivo.

[0334] In one embodiment, the fusion protein of the present invention or the nucleic acid encoding it can be used as an independent drug. In another embodiment, they are used in combination with other drugs. In some embodiments, the fusion protein of the present invention is used in combination with an analgesic such as acetaminophen / paracetamol, nonsteroidal anti-inflammatory drugs (NSAIDs) or opioids. In some embodiments, the fusion protein of the present invention can be used in combination with NSAIDs such as aspirin, ibuprofen, naproxen, celecoxib, ketorolac or diclofenac. In some embodiments, the fusion protein of the present invention can be used in combination with specific COX-2 inhibitors such as celecoxib. Rofecoxib or etoricoxib are used in combination. In some embodiments, the fusion protein of the present disclosure can be used in combination with corticosteroids such as dexamethasone or glycosteroids (e.g., hydrocortisone and prednisone). In some embodiments, the fusion protein of the present disclosure is administered in combination with an antagonist of a proinflammatory cytokine (e.g., an antibody derivative or other molecules thereof that bind to TNF-α (e.g., adalimumab, etanercept), other molecules that bind to IL-17 (e.g., secukinumab), other molecules that bind to IL-23 (e.g., guselkumab, ildrakizumab), or other molecules that bind to IL-12 and 11-23 (e.g., ustekinumab). In some embodiments, the fusion protein is administered in combination with hyaluronic acid. The multiple therapeutic agents can be administered in any order or simultaneously. In some embodiments, the compound of the present invention is administered in combination with, before, or after another drug.

[0335] In some embodiments, the fusion proteins of the present disclosure or nucleic acids encoding the same can be used to treat Alpers disease, arachnoiditis, arthrofibrosis, ataxia cerebral palsy, autoimmune atrophic gastritis, amyloidosis, hATTR amyloidosis, avascular necrosis, back pain, Batten disease, Behçet disease (syndrome), breakthrough pain, burning mouth syndrome, bursitis, central autosomal dominant arteriopathy with subcortical infarcts 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-Tooth disease (CMT) disease, chronic functional abdominal pain (CFAP), chronic pancreatitis, collapsed lung (pneumothorax), corticobasal degeneration, crush injury, corneal nerve Sexual pain, crush syndrome, degenerative disc disease, dermatomyositis, dementia, dystonia, Ehlers-Danlos syndrome (EDS), endometriosis, eosinophilic myalgia syndrome (EMS), erythromelalgia, failed back surgery syndrome (FBSS), fibromyalgia, Friedreich's ataxia, frontotemporal dementia, glossopharyngeal neuralgia, growing pains, herniated disc, hydrocephalus, intercostal neuralgia, interstitial cystitis, juvenile dermatitis, knee injury, leg pain, Lewy body dementia, lumbago-hematuria syndrome, Lyme disease, meralgia paresthesia, mitochondrial disease, mixed dementia, motor neuron disease (MND), monomelic muscular dystrophy, multiple system atrophy (MSA), myositis, neck pain, occipital neuralgia, osteoporosis, rhabdomyolysis, Paget's disease, Parsonage-Turner syndrome Turner Syndrome), pelvic pain, peripheral neuropathy, phantom limb pain, nerve pinch, plantar fasciitis, polymyalgia rheumatica, polymyositis, post-herniorrhaphy pain syndrome, post-mastectomy pain syndrome, post-stroke pain, post-thoracotomy pain syndrome, post-polio syndrome, primary lateral sclerosis, psoriatic arthritis, pudendal neuralgia, radiculopathy, restless legs syndrome, rheumatoid arthritis (RA), sacroiliac joint dysfunction, sarcoidosis, Scheuemann's Kyphosis Disease, sciatica, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), herpes zoster, spasmodic torticollis, sphincter of Oddi dysfunction, spinal cord injury, spinal stenosis, syringomyelia, Tarlov cyst Cysts), tethered cord syndrome, thoracic outlet syndrome (TOS), TMJ disorder, transverse myelitis, traumatic brain injury, vascular pain, vulvodynia, cervical hyperextension, or a combination thereof.

[0336] The fusion proteins of the present disclosure or nucleic acids encoding them can be used to treat neuropathy. Non-limiting examples of neuropathy include post-traumatic peripheral neuropathy, postoperative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-related neuropathy, chemotherapy-induced neuropathy, polyneuropathy, mononeuropathy, multiple mononeuropathy, cranial neuropathy, primary motor neuropathy, primary sensory neuropathy, sensorimotor neuropathy, autonomic neuropathy, idiopathic neuropathy, postherpetic neuralgia, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, pudendal neuralgia, atypical trigeminal neuralgia, sciatica, brachial plexopathy, or intercostal neuralgia. Neuropathy can be associated with, for example, pain, numbness, weakness, burning, atrophy, tingling, convulsions, or a combination thereof.

[0337] In some embodiments, the fusion proteins of the present disclosure or nucleic acids encoding the same can be used to treat autoimmune diseases. Non-limiting examples of autoimmune diseases include acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, obesity, adult-onset Still's disease, alopecia areata, ankylosing spondylitis, anti-glomerular basement membrane nephritis, antineutrophil cytoplasmic antibody-associated vasculitis, anti-N-methyl-D-aspartate receptor encephalitis, antiphospholipid syndrome, antisynthetase syndrome, aplastic anemia, autoimmune angioedema, autoimmune encephalitis, autoimmune bowel disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune neutrophil encephalitis, Granulocytopenia, autoimmune 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, Barlow's concentric sclerosis, Behçet's disease, Bickerstaff's encephalitis encephalitis), bullous pemphigoid, celiac disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy, allergic granulomatosis with polyangiitis, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complex regional pain syndrome, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes mellitus, discoid lupus erythematosus, endometriosis, myotitis, myotitis-associated arthritis, eosinophilic esophagitis, eosinophilic fasciitis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, Felty syndrome syndrome), fibromyalgia, gastritis, pemphigoid gestationis, 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 myasthenic syndromesyndrome), leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease, lupus nephritis, lupus vasculitis, Lyme disease, Meniere's syndrome, microscopic colitis, microscopic polyangiitis, mixed connective tissue disease, corneal erosion ulcer, morphea, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myocarditis, myositis, neuromyelitis optica, neuromyotonia, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, palindromic rheumatism, subacute cerebellar degeneration, Parry-Romberg syndrome Romberg syndrome), Pastor-Turner syndrome, streptococcal autoimmune neuropsychiatric disorders, pemphigus vulgaris, pernicious anemia, acute pityriasis lichenoides vulgaris, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary immunodeficiency, primary sclerosing cholangitis, progressive inflammatory 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, rheumatoid vasculitis, sarcoidosis, Schnitzler syndrome, scleroderma, Sjögren's syndrome, stiff-person syndrome, subacute bacterial endocarditis, Susac's syndrome syndrome, Sydenham 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.

[0338] In some embodiments, the fusion protein of the present disclosure or the nucleic acid encoding it can be used to treat inflammation. In some embodiments, inflammation is chronic inflammation. In some embodiments, the fusion protein of the present disclosure can be used to treat inflammation related to: inflammatory bowel disease, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, glomerulonephritis, sepsis, adult respiratory distress syndrome, dermatitis, sarcoidosis, allergic inflammation, psoriasis, ankylosing spondylarthritis, systemic lupus erythematosus, vasculitis, gout, allografts, xenografts, autoimmune diseases, Sjogren's disease, burns, trauma, stroke, myocardial infarction, atherosclerosis, diabetes, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, cytokines or other therapeutic monoclonal antibodies administered in vivo toxicity induced. In some embodiments, inflammation is chronic inflammation. In some embodiments, the fusion proteins of the present disclosure can 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 spondylarthritis, systemic lupus erythematosus, vasculitis, gout, allografts, xenografts, autoimmune diseases, Sjogren's disease, burns, trauma, stroke, myocardial infarction, atherosclerosis, diabetes, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injury, cytokine or other therapeutic monoclonal antibody-induced toxicity in vivo.

[0339] In some embodiments, the fusion protein is used to treat a disorder, wherein the disorder is not cancer.

[0340] Treatment (preventive or therapeutic) is generally composed of parenteral, preferably intrathecal, intra-articular, intravenous, intramuscular or subcutaneous administration of the fusion protein of the present invention or the nucleic acid encoding it. Dosage and administration regimen will depend on the degree of inhibition of the neuroinflammation and neurodegeneration targeted. Generally, the amount of the given fusion protein will be in the range of 0.5 μg to 1 mg / kg body weight. The dosage can be determined or adjusted by measuring the cytokine levels (IL13, IL4 or IL10 or IL27 or IL33 or TGFβ1 or TGFβ2, or a combination thereof) in the body compartment targeted after administration. Dosage can also be determined by measuring the patient's neuroinflammation, for example, by positron emission tomography (PET) imaging of microglia.

[0341] For parenteral administration, the fusion protein or nucleic acid encoding the fusion protein is preferably formulated into an injectable form in combination with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art, and examples include saline, dextrose solution, Ringer's solution, and solutions containing a small amount of human serum albumin.

[0342] Typically, the fusion proteins of the present invention can be formulated in such vehicles at a concentration of about 50 μg to about 100 mg / ml.

[0343] Gene therapy

[0344] The nucleic acid construct or vector of the present invention can be used as a gene therapy agent for treating the above-mentioned diseases.

[0345] Thus, in one aspect, the present invention relates to a vector as described above for use in the prevention or treatment of a condition characterized by visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain or mixed nociceptive-neuropathic pain, neuroinflammation and / or neurodegeneration, preferably wherein the condition can be selected from post-orthopedic surgical pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-surgical peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-related pain, complex regional pain syndrome, pain after spinal cord injury, pain after stroke, 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 may be Alpers disease, arachnoiditis, arthrofibrosis, ataxia cerebral palsy, autoimmune atrophic gastritis, amyloidosis, hATTR amyloidosis, avascular necrosis, back pain, Batten disease, Behçet's disease (syndrome), breakthrough pain, burning mouth syndrome, bursitis, central autosomal dominant arteriopathy with subcortical infarcts 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-Tooth disease (CMT), chronic functional abdominal pain (CFAP), chronic pancreatitis, collapsed lung (pneumothorax), corticobasal degeneration, crush injury, corneal neuropathic pain, crush syndrome Syndrome, degenerative disc disease, dermatomyositis, dementia, dystonia, Ehlers-Danlos syndrome (EDS), endometriosis, eosinophilic myalgia syndrome (EMS), erythromelalgia, failed back surgery syndrome (FBSS), fibromyalgia, Friedreich's ataxia, frontotemporal dementia, glossopharyngeal neuralgia, growing pains, herniated disc, hydrocephalus, intercostal neuralgia, interstitial cystitis, juvenile dermatitis, knee injury, leg pain, Lewy body dementia, lumbago-hematuria syndrome, Lyme disease, meralgia paresthesia, mitochondrial disease, mixed dementia, motor neuron disease (MND), monomelic muscular dystrophy, multiple system atrophy (MSA), myositis, neck pain, occipital neuralgia, osteoporosis, rhabdomyolysis, Paget's disease, Parsonage-Turner syndrome Turner Syndrome), pelvic pain, peripheral neuropathy, phantom limb pain, nerve pinch, plantar fasciitis, polymyalgia rheumatica, polymyositis, post-herniorrhaphy pain syndrome, post-mastectomy pain syndrome, post-stroke pain, post-thoracotomy pain syndrome, post-polio syndrome, primary lateral sclerosis, psoriatic arthritis, pudendal neuralgia, radiculopathy, restless legs syndrome, rheumatoid arthritis (RA), sacroiliac joint dysfunction, sarcoidosis, Scheuemann's Kyphosis Disease, sciatica, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), herpes zoster, spasmodic torticollis, sphincter of Oddi dysfunction, spinal cord injury, spinal stenosis, syringomyelia, Tarlov cyst Cysts), tethered cord syndrome, thoracic outlet syndrome (TOS), TMJ disorder, transverse myelitis, traumatic brain injury, vascular pain, vulvodynia, acute disseminated encephalomyelitis, acute optic neuritis, transverse myelitis, neuromyelitis optica, or cervical hyperextension.

[0346] As described earlier herein, the present invention relates to a fusion protein comprising at least 2, 3, 4, preferably 2 regulatory (e.g., anti-inflammatory) interleukins selected from 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 the present disclosure, it can be replaced by transforming growth factor beta 1 (TGFβ1) or transforming growth factor beta 2 (TGFβ2). Similarly, when "IL4" is mentioned in the present disclosure, this can be replaced by transforming growth factor beta 1 (TGFβ1) or transforming growth factor beta 2 (TGFβ2). The present disclosure therefore also includes fusion proteins of IL13 / TGFβ1 or IL13 / TGFβ2. In addition, the present disclosure includes fusion proteins of IL4 / TGFβ1 or IL4 / TGFβ2. Furthermore, the present disclosure includes fusion proteins of IL10 / TGFβ1 or IL10 / TGFβ2.

[0347] The invention will now be described with reference to the following examples, which illustrate particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and should not be construed as limiting the invention in any way.

[0348] Example

[0349] animal

[0350] All animal experiments were performed in accordance with international guidelines and were approved in advance by the Experimental Animal Committee of the University Medical Centre Utrecht. Male and female C57BL / 6 mice aged 8 to 16 weeks were used. Observers performing behavioral experiments were blinded to the treatments.

[0351] Materials and Methods

[0352] Expression and purification of IL4 / IL13 fusion protein.L4 / 13 fusion protein HEK293 cells were transiently transfected with a vector containing a transgene according to standard procedures (Y Derocher et al., Nucleic Acids Research 2002, Vol. 30, no2, e9). Briefly, a synthetic cDNA encoding the IL4 / IL13 fusion protein sequence of the present invention (GeneArt, ThermoFisherScientific) (see SEQ ID NO: 4, a 6-His tag was inserted into the N-terminus of the sequence) was cloned into an expression vector containing a cystatin signal sequence. HEK293E cells were then transfected with the expression vector and co-transfected with a vector carrying a transgene for β-galactoside α-2,3-sialyltransferase 5 (SIAT9; Homo sapiens) to optimize the capping of glycans by sialic acid. As previously described, cells were cultured in FreeStyle medium (Invitrogen) containing 0.9% primate and approximately 0.04% v / v fetal bovine serum. 19,26 On day 4 after transfection, the cell suspension was collected and centrifuged at 435 x g for 5 minutes. The supernatant was passed through 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 (PBS), pH 7.4.

[0353] Protein determination. Bradford (Bio-Rad), BCA (Thermo Scientific) and Qubit 1.0 (Thermo Scientific) were used to determine the protein content in the eluted and dialyzed fractions. All protein determinations were performed according to the manufacturer's protocol.

[0354] ELISA. The amount of fusion protein was determined based on the amount of individual cytokines and measured using 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.

[0355] SDS-PAGE and Western blotting. Fractions of the HIS-Select nickel affinity chromatography purification (load, flow-through, wash, elution / dialysis) were separated on 12% polyacrylamide SDS-Page gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes. The membranes were stained with IL4 antibody (Santa Cruz, SC-13555).

[0356] High performance size exclusion chromatography (HP-SEC).To determine its molecular weight and homogeneity, the IL4 / IL13 fusion protein was analyzed using high performance size exclusion chromatography (HP-SEC). Gel filtration (BioSuite 125 4 μm UHR SEC column; Waters; Cat# 186002161) was performed on a high performance liquid chromatography system (Shimadzu) using 50 mM phosphate buffer containing 0.5 M NaCl as the mobile phase. The column was calibrated with a protein mixture of thyroglobulin, bovine serum albumin, carbonic anhydrase, myosin, and ribonuclease before the run. 50 μl of 20 μg / ml of the purified IL4 / IL13 fusion protein was injected and separated on the column at a flow rate of 0.35 ml / min and at a pressure of 35 bar.

[0357] In vitro evaluation of chemotherapy-induced neurotoxicity. Dorsal root ganglion (DRG) neurons were cultured as previously described. 27 Briefly, adult mouse DRG neurons were dissected and then cultured in a Ca-free medium. 2+ and Mg 2+Cells were digested for 1 hour in an enzyme mixture of 5% HBSS, 5 mM HEPES, 10 mM glucose, type XI collagenase (5 mg / ml), and dispase (10 mg / ml), followed by mechanical trituration in DMEM containing 10% v / v heat-inactivated fetal bovine serum. 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), and 1% glutamax, and incubated in 24-well plates for 24 hours in the presence of paclitaxel (1 uM) or oxaliplatin (5 ug / ml) 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 along with the chemotherapeutic agents. As controls, cells were cultured in the absence of chemotherapeutic drugs or cytokines, as well as in the presence of chemotherapeutic drugs alone. After fixation with 4% paraformaldehyde, cells were stained with rabbit anti-mouse βIII-tubulin (ab18207, 1:1000; Abcam). Neurites were visualized using a Zeiss Axio Lab A1 microscope (Zeiss–Oberkochen, Germany), and at least 10 images were obtained per slide at 10x magnification using random sampling. Neurite length was measured using the ImageJ plugin Simple Neurite Tracer76. For three separate primary sensory cultures, the mean of neurite length per neuron of at least five neurons per condition was compared between groups.

[0358] Evaluation 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), with a 5-day treatment-free interval.

[0359] Noxious mechanical sensitivity in the hind paw was measured using von Frey hairs (Stoelting, Wood Dale, USA). Results were expressed as 50% paw withdrawal threshold using the up-down method.29 In some experiments, the length of intraepidermal nerve fibers in paw skin was determined by immunofluorescence staining of skin biopsies with the neuronal marker PGP9.5 at day 15. All experiments were performed in a blinded manner.

[0360] Statistical analysis. Unless otherwise stated, all data are expressed as mean ± SEM. The statistical significance of the data was analyzed by one-way or two-way ANOVA (with repeated measures if appropriate) followed by appropriate post hoc tests. A p value of p < 0.05 was considered significant.

[0361] Example 1. Endogenous IL4 and IL13 are required for normal resolution of paclitaxel-induced transient hyperalgesia.

[0362] To investigate the possible role of regulatory (e.g., anti-inflammatory) cytokines in the recovery of chemotherapy-induced polyneuropathy, a transient pain model of paclitaxel-induced pain was used. 15 On days 0 and 2, mice received two injections of low-dose (2 mg / kg) paclitaxel. From day 6 to day 10 after the start of chemotherapy treatment, mice received daily intrathecal injections of neutralizing antibodies against IL4, IL13, or control IgG antibodies (5 μg of antibody per injection; Figure 1 Mice receiving paclitaxel developed mechanical hyperalgesia starting on the first day of chemotherapy and resolved spontaneously one week after treatment was terminated ( Figure 1 In animals injected intrathecally with neutralizing antibodies against IL4 or IL13, resolution of hyperalgesia was delayed and persisted for at least 2 weeks. These data suggest that endogenously produced IL4 and IL13 are required for normal pain resolution after chemotherapy treatment ( Figure 1 ).

[0363] Example 2. Neuroprotective effects of IL4, IL10 and IL13.

[0364] To assess whether regulatory (e.g., anti-inflammatory) cytokines have neuroprotective properties, sensory neurons isolated from dorsal root ganglia were cultured overnight with paclitaxel together with IL4, IL10, or IL13 as described in the methods. Neurotoxicity was assessed by measuring neurite length using βIII-tubulin staining. Paclitaxel (1 μM) reduced neurite length, with approximately 50% of sensory neurons showing paclitaxel damage. Addition of IL13 during incubation with paclitaxel prevented the negative effects of paclitaxel on neurite length, whereas IL10 and IL4 did not ( Figure 2 ).

[0365] Example 3. Characterization of recombinant IL4 / 13 fusion protein.

[0366] Human IL4 / IL13 fusion protein with an N-terminal 6His tag (see SEQ ID NO: 4) was produced by transient transfection of HEK293 cells and purified as described in the methods. On 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 on SDS-PAGE and Coomassie staining. A homogeneous preparation ( Figure 3 (illustration in ).

[0367] Example 4. IL4 / IL13 fusion protein cures paclitaxel-induced persistent polyneuropathy.

[0368] The potential of an IL4 / IL13 fusion protein to inhibit chemotherapy-induced hyperalgesia was evaluated in a model of persistent paclitaxel-induced painful neuropathy 30 From day 0 to day 6, mice received 4 injections of paclitaxel (8 mg / kg) every other day. Paclitaxel-induced mechanical hyperalgesia began on the first day after the first injection and lasted for at least 3 weeks after cessation of chemotherapy treatment. Two days after the last paclitaxel injection, mice were intrathecally injected with 3 different doses of IL4 / IL13 fusion protein containing SEQ ID NO: 4 (0.3, 1 and 3 μg / mouse) ( Figure 4 All three doses of the IL4 / IL13 fusion protein significantly reduced paclitaxel-induced polyneuropathy. Importantly, the near normalization of mechanical hyperalgesia persisted for at least one week, demonstrating the potential of the IL4 / IL13 fusion protein for long-term resolution of chemotherapy-induced polyneuropathy.

[0369] Example 5. IL4 / IL13 fusion protein is more effective than IL4 / IL10 fusion protein or IL4 and IL13 combination therapy to cure chemotherapy-induced polyneuropathy.

[0370] Next, we evaluated whether the IL4 / IL13 fusion protein inhibited paclitaxel-induced polyneuropathy better than the IL4 / IL10 fusion protein or the combination of IL4 and IL13. After four injections of paclitaxel (8 mg / kg) every other day from day 0 to day 6, mice developed paclitaxel-induced painful polyneuropathy. The IL4 / IL13 fusion protein (comprising SEQ ID NO: 4) inhibited paclitaxel-induced mechanical hypersensitivity for at least 1 week, while the combination of wild-type IL4 and IL13 or the IL4 / IL10 fusion protein only inhibited paclitaxel-induced mechanical hypersensitivity for 1-2 days ( Figure 5The inhibition of paclitaxel-induced persistent allodynia by IL4 / IL13 fusion protein was associated with a reduction in paclitaxel-induced intraepidermal nerve fiber loss in paw skin ( Figure 6 ).

[0371] To assess whether the IL4 / IL13 fusion protein protects against paclitaxel-induced neurotoxicity in vitro, we measured neurite length in mouse sensory neurons cultured in the presence of paclitaxel with or without the fusion protein. Paclitaxel had a significant negative effect on neurite length compared to controls ( Figure 7 The presence of either IL4 / IL10 fusion protein or the combination of IL4 and IL13 had a moderate beneficial effect on neurite length. However, the presence of IL4 / IL13 fusion protein in the culture significantly prevented paclitaxel-induced neurotoxicity ( Figure 7 ). These data collectively demonstrate that the IL4 / IL13 fusion protein is unexpectedly superior to the IL4 / IL10 fusion protein or the combination of IL4 and IL13 in protecting neurons from the toxic effects of the chemotherapy drug paclitaxel. The superiority of the fusion protein over the combination is particularly surprising because the in vitro system is not affected by differential clearance of proteins from their sites of action. Instead, the data point to a unique role for the IL4 / IL13 fusion protein in neuroprotection.

[0372] Example 6. IL4 / IL13 fusion protein also cures oxaliplatin-induced polyneuropathy.

[0373] We investigated whether the neuroprotective effects of IL4 / IL13 fusion proteins are unique to paclitaxel-induced neurotoxicity or whether the neuroprotective effects are specific to a broader range of chemotherapy-induced polyneuropathy. Oxaliplatin, a platinum-based chemotherapy, was used to induce toxic neuropathy in mice. Mechanical allodynia was induced by two cycles of oxaliplatin injections five times daily, separated by 5 days without intraperitoneal injection, for at least 3 weeks. Figure 8 Intrathecal injection of IL4 / IL13 fusion protein (comprising SEQ ID NO: 4) on the day after the last oxaliplatin injection significantly reduced mechanical allodynia for 4 days ( Figure 8 Intrathecal injection of wild-type IL4 or wild-type IL13 temporarily inhibited oxaliplatin-induced mechanical allodynia for approximately 1 day, which was significantly shorter than the effect of the IL4 / IL13 fusion protein. Similarly, in vitro 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 IL4 / IL13 fusion protein protects against chemotherapy-induced neurotoxicity and chemotherapy-induced polyneuropathy.

[0374] Example 7. IL10 / IL13 and IL33 / IL13 fusion proteins

[0375] In addition, it was considered whether IL10 / IL13 fusion proteins or IL33 / IL13 fusion proteins had therapeutic effects in the medical indications disclosed herein. Surprisingly, cross-linking of the IL10 receptor and the IL13 receptor by administering an IL10 / IL13 fusion protein, or cross-linking of the IL33 receptor and the IL13 receptor by administering an IL33 / IL13 fusion protein, resulted in a stronger and more sustained therapeutic effect than the administration of an IL4 / IL10 fusion protein, particularly in a neuropathic pain model.

[0376] Example 8. Expression of cytokine receptors in dorsal root ganglia and spinal cord

[0377] The question of whether the cytokine receptors targeted by the fusion proteins of the present invention are expressed by the sensory system has been addressed. To analyze this, Ray et al. (Pain 2018; 159: 1325-1345) extracted RNAseq data of IL10, IL4, IL13, IL27, TGFβ1, and TGFβ2 receptors in dorsal root ganglia and spinal cord from a database available at https: / / www.utdallas.edu / bbs / painneurosciencelab / sensoryomics / drgtxome / ?go. RNA sequencing revealed the expression of receptor chains for IL10, IL4, IL13, IL27, TGFβ1, and TGFβ2 in dorsal root ganglia and spinal cord of humans and mice ( Figure 10 ; Data are expressed as transcripts per million).

[0378] Example 9. Fusion protein of the present disclosure

[0379] This example demonstrates the design and production of non-limiting examples of IL13-containing fusion proteins disclosed herein.

[0380] The IL4 / IL13 fusion protein of the present disclosure comprising SEQ ID NO: 1 and SEQ ID NO: 14 was designed. SEQ ID NO: 1 was linked to SEQ ID NO: 14 using a SEQ ID NO: 3 linker to produce SEQ ID NO: 16. A hexahistidine tag was added to the N-terminus and the fusion protein was produced by transient transfection of HEK293E cells as disclosed below. The fusion protein comprising SEQ ID NO: 16 has IL4 at the N-terminus and Figure 11 、 Figure 12 and Figure 18A Marked as IL4 / IL13 SKP .

[0381] An IL10 / IL13 fusion protein comprising SEQ ID NO:5 and SEQ ID NO:14 was designed. SEQ ID NO:5 was linked to SEQ ID NO:14 using a SEQ ID NO:3 linker to produce SEQ ID NO:17. A hexahistidine tag was added to the N-terminus and the fusion protein was produced by transient transfection of HEK293E cells as described below. The fusion protein comprising SEQ ID NO:17 contained IL13 at the N-terminus and Figure 11 、 Figure 13 and Figure 18D The cells are labeled as IL13 / IL10.

[0382] An IL27 / IL13 fusion protein comprising SEQ ID NO: 18 and SEQ ID NO: 14 was designed. SEQ ID NO: 18 was linked to SEQ ID NO: 14 using a SEQ ID NO: 3 linker to produce SEQ ID NO: 19. A hexahistidine tag was added to the N-terminus and the fusion protein was produced by transient transfection of HEK293E cells as described below. The fusion protein comprising SEQ ID NO: 19 contained IL13 at the N-terminus and Figure 11 、 Figure 14 and Figure 18C The cells are labeled as IL13 / IL27-A.

[0383] An IL13 / IL13 fusion protein of the present disclosure was designed containing two copies of SEQ ID NO: 14. SEQ ID NO: 14 was linked to the second copy of SEQ ID NO: 14 using a SEQ ID NO: 3 linker, generating SEQ ID NO: 20. A hexahistidine tag was added to the N-terminus, and the fusion protein was produced by transient transfection of HEK293E cells as disclosed below.

[0384] IL4 / IL13, IL10 / IL13, IL27 / IL13 and IL13 / IL13 fusion proteins were purified as described below. Size exclusion chromatography showed that the IL4 / IL13 fusion protein containing SEQ ID NO: 16 migrated as a single peak ( Figure 18A ), IL13 / IL13 fusion protein (containing SEQ ID NO: 20) migrated as a single peak ( Figure 18B ).

[0385] For IL27 / IL13 (IL13 / IL27-A), two peaks of recombinant protein were identified and they were pooled separately after gel filtration ( Figure 18C). Pool 1 is the higher molecular weight pool. Pool 2 has a lower molecular weight and is selected 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).

[0386] For IL10 / IL13 (IL13 / IL10), two peaks of recombinant protein were identified, which were pooled separately after gel filtration ( Figure 18D ). Pool 1 represents the high molecular weight pool and pool 2 represents the low molecular weight pool. Without wishing to be bound by any particular theory, IL10 / IL13 pool 1 may contain dimeric forms of the molecules and IL10 / IL13 pool 2 may contain monomeric forms of IL10 / IL13.

[0387] like Figure 11 As shown, under reducing and non-reducing conditions, 4-12% gradient polyacrylamide NuPage TM The purified proteins were analyzed on gel and the bands were visualized by Coomassie protein staining.

[0388] Expression and purification of IL13-containing fusion proteins The IL13-containing fusion proteins disclosed herein were produced by transient transfection of HEK293E cells. The cells were transfected with a pUPE expression vector containing a transgene encoding one of the IL13-containing fusion protein sequences. For purification, a hexahistidine affinity tag was cloned at the N-terminus of each IL13-containing protein. Six days after transfection, the conditioned medium containing the recombinant protein was harvested by low-speed centrifugation (10 minutes, 1000 x g) followed by high-speed centrifugation (10 minutes, 4000 x g).

[0389] The protein was purified by immobilized metal affinity chromatography (IMAC) via the His tag. Briefly, the recombinant protein was mixed with 0.5 ml Nickel excel binds at 20°C. Nickel containing bound protein is harvested by centrifugation excel and transferred to a gravity flow column. Non-specifically bound proteins were removed by washing the column with IMAC buffer (500 mM NaCl, 25 mM Tris, pH = 8.2) containing 0 and 10 mM imidazole. Proteins were eluted with IMAC buffer containing 500 mM imidazole. 2.5 ml fractions were collected and the fractions containing recombinant protein were combined. Conditioned medium and unbound IMAC components were separated by Analysis was performed by 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, 18,000 x g, 4°C).

[0390] The protein was further purified by gel filtration using a Superdex200 16 / 600 column equilibrated in PBS. Protein-containing fractions were analyzed by capillary electrophoresis and fractions containing recombinant protein 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.

[0391] Protein determination: Protein concentration was determined spectrophotometrically by measuring absorbance at 280 nm (DropSense 16, Trinean) and using the BCA (ThermoScientific) protein assay. All protein determinations were performed according to the manufacturer's protocol.

[0392] SDS-Page : Under reducing and non-reducing conditions, 4-12% polyacrylamide NuPage TM The purified proteins were analyzed on polyacrylamide gels and protein bands were visualized by Coomassie staining.

[0393] Example 10. Neuroprotective Effect of IL13-Containing Fusion Protein on Paclitaxel-Induced Sensory Neuron Injury

[0394] To elucidate the potential neuroprotective effects of various fusion proteins in which IL13 is linked to other regulatory cytokines (such as IL4, IL10, IL13, or IL27), the fusion proteins were tested in an in vitro paclitaxel-induced sensory neuron injury assay. Primary sensory neurons from mice were cultured for 24 hours in the presence of paclitaxel (1 μM) and various concentrations of each fusion protein or an equimolar dose of IL13 or an unlinked cytokine combination.

[0395] Culture of DRG neurons DRGs were cultured as previously described (Nat. Commun. 4, 1682 (2013)). Briefly, DRGs were dissected and placed in ice-cold dissection medium (HBSS w / o Ca 2+ and Mg 2+ After dissection, the axons were cut and the cells were washed with filtered enzyme mixture (HBSS w / o Ca 2+ and Mg 2+, 5mM HEPES, 10mM glucose, 5mg / ml collagenase type XI (Sigma) and 10mg / ml dispase (Gibco)) instead of dissection medium. DRG is incubated in enzyme mixture at 37 ℃ and 5% CO2 for 30 minutes. Subsequently, the enzyme mixture is inactivated with heat-inactivated fetal bovine serum (FBS, Sigma). Cells are cultured in glass coverslips coated with Dulbecco's modified Eagle medium (Gibco) in a 5% CO2 incubator at 37 ℃. The culture medium contains 10% FBS (Gibco), 2mmol / L glutamine (Gibco), 10,000IU / ml penicillin-streptomycin (Gibco) and laminin (0.02mg / ml, Sigma) on poly-L-lysine (0.01mg / ml, Sigma). Cells are used in the next 1-2 days.

[0396] Stimulation for neurite length measurement : After 24 hours of culture, DRG neurons were treated with paclitaxel (1 μM) alone (n=11) or in the presence of different concentrations of IL13 fusion protein (0.12nM, n=6; 0.6nM or 3nM, n=11) or equimolar doses of individual cytokines (n=6) for 24 hours (n values ​​represent the number of animals tested for each condition). Neurites were visualized using β3-tubulin staining, while 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 photos were taken per well. For each photo, the neurite length of each sensory neuron was determined and averaged as a single value for each animal and condition. The percentage inhibition of paclitaxel-induced neurite length loss (μ) for each mouse culture of 1 mouse was calculated according to the following formula 对照 -μ 紫杉醇 )-(μ 对照 -X 细胞因子 )) / (μ 对照 -μ 紫杉醇 )*100 (where μ is the neurite length of each neuron averaged across all samples and X is the neurite length of each individual sample).

[0397] β3-tubulin and NeuN staining: Cells were fixed in 4% PFA for 10 minutes, permeabilized with PBS containing 0.05% Tween-20, and then incubated in blocking buffer (1% BSA and 5% normal donkey serum in PBS containing 0.05% Tween-20 and 0.01% triton) for 1 hour. Cells were incubated 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) overnight at 4°C, then washed and incubated with AF488-conjugated donkey anti-rabbit and 568-conjugated donkey anti-mouse secondary antibodies (Thermofisher, 1:500) followed by DAPI (1:5000, Sigma) staining, and sections were mounted on slides using FluorSave reagent (Millipore).

[0398] Images were captured using an Olympus IX83 microscope (Olympus). Images 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 CellSens software. Neurite length was determined using the NeuralNetric macro in ImageJ (Fiji).

[0399] result

[0400] Paclitaxel reduced neurite length by approximately 67% compared to control cultured sensory neurons.

[0401] Figure 12 The comparison of the neuroprotective effects of two IL4 / IL13 fusion proteins compared to IL13 alone or a combination of IL4 and IL13 is shown. IL13 dose-dependently inhibited paclitaxel-induced neurite length reduction, with a maximum of approximately 9% ( Figure 12 The combination of IL4 and IL13 did not significantly inhibit paclitaxel-induced neurite length at any concentration tested.

[0402] The fusion protein labeled IL4 / IL13 comprises SEQ ID NO: 4. SKP The fusion protein comprises SEQ ID NO: 16. IL13 is located at the C-terminus of these constructs. IL4 / IL13 dose-dependently inhibited paclitaxel-induced neurite length reduction, with a maximum effect of 35% at 3 nM ( Figure 12 IL4 / IL13 inhibited paclitaxel-induced neurite length reduction significantly better than the combination of IL4 and IL13 or IL13 alone, with the degree of inhibition being approximately 4-fold higher at a concentration of 3 nM compared to IL13 ( Figure 12 ). Alternative IL4 / IL13 fusion protein IL4 / IL13 SKP The extent of inhibition of paclitaxel-induced neurite length reduction was similar to that of IL4 / IL13 at all concentrations tested ( Figure 12 ). IL4 / IL13 SKP The IL13 protein sequence (SEQ ID NO: 14) used in the fusion protein of IL4 (SEQ ID NO: 16) contains an additional serine at the N-terminus and an arginine near the C-terminus instead of a glutamine (position 112 in SEQ ID NO: 14). Despite these protein sequence differences, both fusion proteins containing IL4 and IL13 showed superior neuroprotection compared to IL4 and IL13 alone, as well as the combination of IL4 + IL13. Data are shown as mean ± SEM. Data were analyzed using a two-way ANOVA mixed-effects analysis followed by a Tukey's multiple comparison test.

[0403] Figure 13 The neuroprotective effects of two IL10 / IL13 fusion protein pools compared to equimolar concentrations of IL13 alone or a combination of IL10 and IL13 are shown. The IL10 / IL13 fusion protein comprises SEQ ID NO: 17, wherein IL13 is located at the N-terminus. The pool of this fusion protein is referred to as IL13 / IL10 in the figure. 池1 and IL13 / IL10 池2 At all doses tested, IL13 / IL10 池1 Significantly inhibited paclitaxel-induced neurite length reduction, ranging from 15% at 0.12 nM to 28% at 3 nM ( Figure 13 ). IL13 / IL10 池2 It also protects neurons from paclitaxel-induced neurite length reduction in a concentration-dependent manner. Importantly, IL13 / IL10 池1 and IL13 / IL10 池2 IL10 and IL13 combined at 3 nM significantly better protected against paclitaxel-induced neuronal damage ( Figure 13Data are shown as mean ± SEM. Data were analyzed using two-way ANOVA mixed effects analysis followed by Tukey's multiple comparison test.

[0404] Figure 14 A comparison of the neuroprotective effects of IL13 / IL13 fusion protein and IL27 / IL13 fusion protein compared to IL13 alone is provided. The IL13 / IL13 fusion protein comprises SEQ ID NO: 20. The IL13 / IL13 fusion protein inhibited paclitaxel-induced neurite length reduction by approximately 10% at 0.12 nM, which is significantly better than IL13 at this concentration. However, as the concentration increased, IL13 / IL13 did not provide more neuroprotection ( Figure 14 ).

[0405] The IL27 / IL13 fusion protein comprises SEQ ID NO: 19 (wherein IL13 is located at the N-terminus of the protein), Figure 14 IL13 / IL27-A 池2 . This fusion protein includes secreted mutant proteins of IL13 and IL27A (Müller et al., 2019). Pool 2 of IL27 / IL13 generated as described above was tested. At 0.6 nM, IL27 / IL13 inhibited paclitaxel-induced neuronal damage, with a maximum inhibition of approximately 21%. At this concentration, IL27 / IL13 performed better than IL13. At 3 nM, there was no significant difference in the neuroprotective effects of IL13 and IL27 / IL13. Data are shown as mean ± SEM. Data were analyzed using two-way ANOVA mixed effects analysis followed by Tukey's multiple comparison test.

[0406] These data suggest that multiple fusion proteins containing IL13 and regulatory cytokines can effectively reduce neurotoxicity, including fusion proteins containing different IL13 sequences, different regulatory cytokines, and IL13 at the N-terminus or C-terminus of the fusion protein. Multiple fusion proteins demonstrate superiority compared to IL13 alone, ...

Claims

1. A fusion protein consisting of a first cytokine which is IL13, a linker, and a second cytokine which is IL4, IL10 or IL13, wherein: (a) the second cytokine is IL4, and the fusion protein consists of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 4; (b) the second cytokine is IL10, and the fusion protein consists of the amino acid sequence of SEQ ID NO: 17; or (c) the second cytokine is IL13, and the fusion protein consists of the amino acid sequence of SEQ ID NO:

20.

2. The fusion protein according to claim 1, wherein the second cytokine is IL4 and the fusion protein consists of the amino acid sequence of SEQ ID NO:

16.

3. The fusion protein according to claim 1, wherein the second cytokine is IL4, and the fusion protein consists of the amino acid sequence of SEQ ID NO:

4.

4. The fusion protein according to claim 1, wherein the second cytokine is IL10, and the fusion protein consists of the amino acid sequence of SEQ ID NO:

17.

5. The fusion protein according to claim 1, wherein the first cytokine is IL13 and the fusion protein consists of the amino acid sequence of SEQ ID NO:

20. The fusion protein of claim 1 , wherein the fusion protein performs better than the combination of the first cytokine and the second cytokine in treating chemotherapy-induced peripheral neuropathy.

7. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is in unit dosage form.

9. A nucleic acid molecule comprising a polynucleotide sequence encoding the fusion protein according to any one of claims 1 to 6.

10. A vector comprising the nucleic acid molecule according to claim 9.

11. Use of the fusion protein according to any one of claims 1 to 5 in the preparation of a medicament for treating peripheral neuropathy in a subject in need thereof.

12. The use according to claim 11, wherein the peripheral neuropathy is chemotherapy-induced peripheral neuropathy.

13. The use according to claim 12, wherein the peripheral neuropathy is paclitaxel-induced peripheral neuropathy.

14. The use according to claim 12, wherein the peripheral neuropathy is oxaliplatin-induced peripheral neuropathy.

Images