fusion proteins

Abstract

The present invention relates to a p75NTR neurotrophic factor-binding protein (NBP)-Fc fusion protein comprising a p75NTR (NBP) portion and an immunoglobulin portion. In one embodiment, the p75NTR (NBP)-Fc fusion protein is for use in treating pain and / or painful symptoms.

Description

[Technical Field]

[0001] The present invention relates to p75NTR neurotrophin-binding protein (NBP)-Fc fusion proteins. We describe the affinity and in vivo kinetics of such molecules, as well as their efficacy in treating pain in animal models. p75NTR(NBP)-Fc fusion proteins are used to treat pain and other neurotrophin-associated conditions, such as psoriasis, eczema, rheumatoid arthritis, cystitis, endometriosis, and osteoarthritis. [Background technology]

[0002] Neurotrophic factors, namely, neurotrophic growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), and neurotrophic factor 4 / 5 (NT-4 / 5), act through four receptors: the low-affinity p75 neurotrophic factor receptor (p75NTR) and the high-affinity tyrosine kinase receptors TrkA, TrkB, and TrkC. The low-affinity receptor p75NTR is activated by binding to all four neurotrophic factors and functions independently of the other receptors. However, Trk receptors are activated more selectively; NGF is a selective ligand for TrkA, BDNF is a ligand for TrkB, and NT-3, 4 / 5 are ligands for TrkC. In addition, it has been reported that when p75NTR and Trk proteins are coexpressed, they form a complex, which alters the signaling of both receptors (Huang and Reichardt, 2003, Annu Rev Biochem. 72:609-42). Indeed, it has been suggested that p75NTR promotes the selectivity of each neurotrophic factor for its respective Trk receptor.

[0003] p75NTR belongs to the tumor necrosis factor receptor superfamily (TNFR-SF) and was the first fully characterized member of this superfamily. This superfamily (encoded by approximately 30 genes in humans) is defined by a ligand-binding domain consisting of one or more (typically four) repeats of a 40-amino acid cysteine-rich domain (CRD), which was first identified in p75NTR (Johnson et al., 1986 Cell 47:545-554; Radeke et al., 1987 Nature 325:593-597). In contrast, there is no sequence motif common to the intracellular domains of all TNFR-SF family members. As a result, the signaling mechanisms of TNFR-SF proteins are remarkably diverse.

[0004] A unique feature of the p75NTR structure is the presence of a disulfide-linked p75NTR dimer formed through cysteine ​​residues within the transmembrane domain. This disulfide bond is required for effective neurotrophin-dependent signaling by p75NTR and plays a key role in the formation of the intracellular and extracellular domains (Vilar et al., 2009 Neuron 62:72-83). Physiologically, neurotrophins exist as noncovalently associated dimers (Bothwell and Shooter, 1977 J Biol Chem. 252(23):8532-6), with a distribution half-life of approximately 5 minutes (Tria et al., 1994 Exp Neurol. 127(2):178-83). Neurotrophin-dependent p75NTR activation involves the association of neurotrophin dimers with CRDs 2–4 of the two extracellular domains of the p75NTR dimer (He and Garcia, 2004 Science 304:870–875). Recent studies support the model that neurotrophin binding brings the two extracellular domains of the p75NTR dimer into close proximity with each other, allowing the intracellular domain to spread outward in a snail-tongue-like manner around a disulfide bond, allowing it to associate with the signaling adaptor proteins NRIF and TRAF6 (Vilar et al., 2009 J Cell Sci 122:3351–3357; Vilar et al., 2009 Neuron 62:72–83). Disulfide bonds within transmembrane domains such as those present in p75NTR have not previously been reported in other TNFR-SF family members or any other membrane proteins.

[0005] p75NTR undergoes sequential proteolytic cleavage by α- and γ-secretase activity and matrix metalloproteinases (MMPs), releasing its intracellular domain (ICD) into the cytoplasm in a manner similar to the cleavage-dependent signaling pathways of Notch and β-amyloid precursor protein (Jung et al., 2003 J Biol Chem 278:42161-42169; Kanning et al., 2003 J Neurosci 23:5425-5436). Release of the p75NTR ICD into the cytoplasm via this pathway promotes signal transduction by associated NRIFs (Kenchappa et al., 2006 Neuron 50:219-232). The role of the extracellular domain of p75NTR following proteolytic cleavage by α- and γ-secretase activity and MMPs remains unclear.

[0006] It has been reported in the literature that NGF and other neurotrophic factors (BDNF, NT-3, and NT-4 / 5) play important roles in pathological conditions such as osteoarthritis, pancreatitis, rheumatoid arthritis, psoriasis, pruritus, and pain caused by multiple sclerosis (Watanabe et al., 2008 J Neurosci Res. 86(16):3566-74; Raychaudhuri et al., 2011 Arthritis Rheum. 63(11):3243-52; Barthel et al., 2009 Arthritis Res Ther. 11(3):R82; Truzzi et al., 2011 Cell Death Differ.18:948-58; McDonald et al., 2011 Curr Med Chem. 18:234-44; Yamaoka et al., 2007 J Dermatol Sci. 46(1):41-51). Selective antibodies against either neurotrophic factor: NGF or BDNF, NT-3 and NT-4 / 5 have been shown to significantly reduce pain. Furthermore, antibodies directed against the neurotrophic factor receptor p75NTR, TrkA, TrkB, or TrkC have also been shown to be effective in pain models (Orita S et al., 2010 J Orthop Res. 28:1614-20; Svensson P et al., 2010 Pain. 148:473-80; Iwakura et al., 2010 J Hand Surg Am. 35:267-73; Cirilio et al., 2010 Cell Mol Neurobiol. 30:51-62; Pezet et al., 2010 Pain. 90:113-25; Hayashi et al., 2011 J Pain. 12:1059-68; Chu et al., 2011 Pain. 152:1832-7; Ueda et al., 2010 J Pharmacol Sci. 112:438-43; Ghilardi et al., 2010 Bone. 48:389-98; Fukui et al., 2010 J Orthop Res. 28:279-83).Fukui et al. (2010) demonstrated significant efficacy in pain-related endpoints after treatment with an anti-p75NTR antibody in a pain model (mechanical allodynia after sciatic nerve crush). This study concluded that treatment with a p75NTR inhibitor antibody reduced CGRP and p75NTR expression, resulting in a significant reduction in pain. Summary of the Invention

[0007] According to a first aspect of the present invention, there is provided a p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein comprising: (a) a portion of p75NTR(NBP) having an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 1 and 2; and (b) an immunoglobulin Fc portion having an amino acid sequence having at least 90% identity to a sequence selected from any one of SEQ ID NOs: 3 to 6; wherein the p75NTR(NBP) portion and the Fc portion are linked via a linker, and the linker has the formula G x where x is 1, 2, 3, 4, 5, or 6.

[0008] In one embodiment, the linker does not comprise and consist of the sequence GGGGS.

[0009] In a further embodiment, the linker is GGG.

[0010] In a further embodiment of the p75NTR(NBP)-Fc fusion protein according to the invention, the p75NTR(NBP) is canine p75NTR(NBP). In another embodiment of the p75NTR(NBP)-Fc fusion protein according to the invention, the p75NTR(NBP) is SEQ ID NO: 1. In another embodiment of the p75NTR(NBP)-Fc fusion protein according to the invention, the Fc region is canine Fc.

[0011] In yet another embodiment, the p75NTR(NBP) portion of the fusion protein of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the p75NTR(NBP) portion of the fusion protein of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the Fc portion of the fusion protein of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the Fc portion of the fusion protein of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, the Fc portion of the fusion protein of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5. In another embodiment, the Fc portion of the fusion protein of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6.

[0012] In one embodiment, the p75NTR(NBP)-Fc fusion protein of the present invention has a binding affinity (K) of between about 0.01 nM and about 50 nM to either NGF, BDNF, NT3, or NT4 / 5, as measured by surface plasmon resonance at 20°C. d ) to join them.

[0013] In a second aspect of the invention, there is provided a p75NTR(NBP)-Fc fusion protein as described according to any of the other aspects of the invention for use in the treatment of pain.

[0014] In a third aspect of the present invention, there is provided a nucleic acid molecule encoding a p75NTR(NBP)-Fc fusion protein as described in the first or second aspect of the invention, and optionally further comprising encoding a signal sequence.

[0015] In a fourth aspect of the invention, there is provided a replicable expression vector for transfecting a cell (optionally a mammalian cell), said vector comprising a nucleic acid molecule as described in the third aspect of the invention.

[0016] Suitably, the replicable expression vector is a viral vector.

[0017] In a fifth aspect of the invention, there is provided a host cell harboring a nucleic acid molecule of the third aspect of the invention.

[0018] In a sixth aspect of the invention, the nucleic acid molecule according to the third aspect of the invention, or the vector according to the fourth aspect of the invention, is for use in the treatment of pain.

[0019] Pain includes, but is not limited to, acute pain, chronic pain, inflammatory pain, nociceptive pain, neuropathic pain, hyperalgesia, allodynia, central pain, cancer pain, postoperative pain, visceral pain, musculoskeletal pain, cardiac or vascular pain, headache, including migraine, orofacial pain, including dental pain, and back pain. Treatment of pain includes, but is not limited to, preventing, ameliorating, controlling, reducing the incidence, or delaying the onset or progression of pain.

[0020] In a seventh aspect of the invention, the nucleic acid molecule according to the third aspect of the invention, or the vector according to the fourth aspect of the invention, is for use in the treatment of osteoarthritis.

[0021] In an eighth aspect, there is provided a p75NTR(NBP)-Fc fusion protein according to the first or second aspect, or a nucleic acid or vector according to the third or fourth aspect, wherein said p75NTR(NBP)-Fc fusion protein, or nucleic acid molecule or vector is for separate, sequential or simultaneous use in combination with a second pharmacologically active compound.

[0022] In a ninth aspect, the present invention provides a pharmaceutical composition comprising a p75NTR(NBP)-Fc fusion protein as described in any aspect of the invention, or a nucleic acid molecule or vector as described in any aspect of the invention, and a pharmaceutically acceptable carrier and / or excipient.

[0023] Suitably, the pharmaceutical composition is for use in any one or more of preventing, ameliorating, controlling, reducing the incidence or delaying the onset or progression of pain in an animal, for example a mammal such as a dog, cat, elephant or horse, hi one embodiment, the animal is a dog.

[0024] In a further aspect of the invention, a kit is provided comprising: (a) a p75NTR(NBP)-Fc fusion protein according to any aspect of the present invention, or a nucleic acid molecule or vector according to any aspect of the present invention, or a pharmaceutical composition according to the eighth aspect; and (b) Instructions for administering an effective amount of the p75NTR(NBP)-Fc fusion protein, nucleic acid molecule, vector, or pharmaceutical composition to an animal for any one or more of preventing or treating pain, or ameliorating, controlling, reducing the incidence, or delaying the onset or progression of pain.

[0025] In yet another aspect of the present invention, there is provided a method for treating and / or preventing pain in an animal, said method comprising the step of administering to said individual a therapeutically effective amount of a p75NTR(NBP)-Fc fusion protein as described in any aspect of the present invention, or a nucleic acid molecule or vector as described in any aspect of the present invention, which may further comprise a pharmaceutically acceptable carrier, or which may further comprise a pharmaceutical composition as described in the eighth aspect of the present invention. [Brief explanation of the drawings]

[0026] [Figure 1] Amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 1). [Figure 2] Amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the invention (SEQ ID NO: 2). Canine P75NTR. [Figure 3] Amino acid sequence of the feline IgG-Fc portion of the fusion protein according to the invention (SEQ ID NO: 3). [Figure 4]Amino acid sequence of the horse IgG-Fc portion of a fusion protein according to the invention (SEQ ID NO: 4). [Figure 5] Amino acid sequence of the feline IgG-Fc portion of the fusion protein according to the invention (SEQ ID NO: 5). [Figure 6] Amino acid sequence of the feline IgG-Fc portion of the fusion protein according to the invention (SEQ ID NO: 6). DETAILED DESCRIPTION OF THE INVENTION

[0027] According to a first aspect of the present invention, there is provided a p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein comprising: (a) a portion of p75NTR(NBP) having an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 1 and 2; and (b) an immunoglobulin Fc portion having an amino acid sequence having at least 90% identity to a sequence selected from any one of SEQ ID NOs: 3 to 6; wherein the p75NTR(NBP) portion and the Fc portion are linked via a linker, and the linker has the formula G x where x is 1, 2, 3, 4, 5, or 6.

[0028] In one embodiment, the linker does not comprise and consist of the sequence GGGGS.

[0029] In a further embodiment, the linker is GGG.

[0030] In a suitable embodiment, the p75NTR(NBP) portion is an amino acid sequence having at least 95% identity to a sequence selected from any of SEQ ID NOs: 1 and 2.

[0031] In a suitable embodiment, the p75NTR(NBP) portion is selected from either SEQ ID NO:1 and SEQ ID NO:2.

[0032] In a suitable embodiment, the immunoglobulin Fc portion is an amino acid sequence having at least 95% identity to a sequence selected from any of SEQ ID NOs: 3-6.

[0033] In a suitable embodiment, the immunoglobulin Fc portion is an amino acid sequence selected from any of SEQ ID NOs: 3-6.

[0034] In one embodiment of the p75NTR(NBP)-Fc fusion protein according to the invention, the p75NTR(NBP) is canine p75NTR(NBP). In another particularly preferred embodiment of the p75NTR(NBP)-Fc fusion protein according to the invention, the p75NTR(NBP) is SEQ ID NO: 1. In another embodiment of the p75NTR(NBP)-Fc fusion protein according to the invention, the Fc region is canine Fc.

[0035] In yet another embodiment, the p75NTR(NBP) portion of the fusion protein according to the invention comprises or consists of the amino acid sequence shown in (SEQ ID NO: 1).

[0036] In yet another embodiment, the p75NTR(NBP) portion of the fusion protein according to the invention comprises or consists of the amino acid sequence set forth in (SEQ ID NO: 2): Canine P75NTR.

[0037] In yet another embodiment, the Fc portion of a fusion protein according to the invention comprises or consists of the amino acid sequence shown in (SEQ ID NO: 3).

[0038] In yet another embodiment, the Fc portion of a fusion protein according to the invention comprises or consists of the amino acid sequence shown in (SEQ ID NO: 4).

[0039] In yet another embodiment, the Fc portion of a fusion protein according to the invention comprises or consists of the amino acid sequence shown in (SEQ ID NO: 5).

[0040] In yet another embodiment, the Fc portion of a fusion protein according to the invention comprises or consists of the amino acid sequence shown in (SEQ ID NO: 6).

[0041] In a suitable embodiment of the p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein, the p75NTR (NBP) portion is SEQ ID NO:2 and the immunoglobulin Fc portion is SEQ ID NO:3.

[0042] In a suitable embodiment of the p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein, the p75NTR (NBP) portion is SEQ ID NO:2 and the immunoglobulin Fc portion is SEQ ID NO:4.

[0043] In a suitable embodiment of the p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein, the p75NTR (NBP) portion is SEQ ID NO:2 and the immunoglobulin Fc portion is SEQ ID NO:5.

[0044] In a suitable embodiment of the p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein, the p75NTR (NBP) portion is SEQ ID NO:2 and the immunoglobulin Fc portion is SEQ ID NO:6.

[0045] In another embodiment of the p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein described above, the linker moiety is GGG.

[0046] Suitably, the p75NTR neurotrophic factor binding protein, ie p75NTR(NBP), is PEGylated and, more preferably, glycosylated.

[0047] The p75NTR(NBP)-Fc fusion protein of the present invention has a binding affinity (K ) of about 0.01 nM to about 50 nM to any one or more of NGF, BDNF, NT3, or NT4 / 5. d In some embodiments, the binding affinity (K d) is between about 0.01 nM and any of about 0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 1.5 nM, 2 nM, 2.5 nM, 3 nM, 3.5 nM, 4 nM, 4.5 nM, 5 nM, 5.5 nM, 6 nM, 6.5 nM, 7 nM, 7.5 nM, 8 nM, 8.5 nM, 9 nM, 9.5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, or 50 nM, as measured in an in vitro binding assay for NGF, BDNF, NT3, or NT4 / 5 as described herein, preferably as measured by surface plasmon resonance at 20° C. d ) is less than or equal to about any of 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 950 pM, or 1 nM, as measured in an in vitro binding assay of p75NTR(NBP)-Fc fusion protein and neurotrophic factor as described herein, preferably as measured by surface plasmon resonance at 20°C. In a further embodiment, the binding affinity (K d ) is about 0.3 nM or about 1 nM, as measured in an in vitro binding assay of p75NTR(NBP)-Fc fusion protein with neurotrophic factors as described herein, preferably measured by surface plasmon resonance at 20°C.

[0048] Suitably, the p75NTR(NBP)-Fc fusion protein of the present invention is for use in the treatment of pain. Without wishing to be bound by a particular theory, the inventors believe that the p75NTR(NBP)-Fc fusion protein achieves its effectiveness in the treatment of pain by acting on (defined as modulating, or up- or down-regulating) the functional activity of the aforementioned neurotrophic factors, i.e., NGF, BDNF, NT3, or NT4 / 5, for example, the functional activity of the aforementioned neurotrophic factors resulting from interaction with their respective receptors.

[0049] Suitably, the p75NTR(NBP)-Fc fusion protein affects the functional activity of BDNF, as assessed by any of the following functional assays: neuronal and synaptic growth and differentiation, survival and differentiation in neuronal cell culture, Trk signaling, and stimulation of axonal outgrowth in vitro or in vivo.

[0050] Suitably, the p75NTR(NBP)-Fc fusion protein affects the functional activity of NGF, which is assessed by measuring NGF binding to and activation of TrkA, as demonstrated in a classical neuronal survival assay (e.g., as described in Cowan et al., Annu. Rev. Neurosci. 2001;24:551-600).

[0051] Suitably, the p75NTR(NBP)-Fc fusion protein affects the functional activity of NT3, as assessed by measuring the binding of NT3 to and activation of endogenous Trk receptor activity, as shown in Trk receptor phosphorylation, mitogen-activated protein kinase phosphorylation reporter assays, or cell survival and neurite outgrowth assays.

[0052] Suitably, the p75NTR(NBP)-Fc fusion protein affects the functional activity of NT4 / 5, as assessed by measuring NT4 / 5 in in vitro or in vivo phosphorylation and activation assays, for example, in a myelin basic protein (MBP) phosphorylation assay, or in an in vivo Matrigel angiogenesis assay of vascular endothelial growth factor (VEGF) / basic fibroblast growth factor-induced angiogenesis.

[0053] Suitably, the p75NTR(NBP)-Fc fusion protein binds to one or more contact residues of the neurotrophic factors NGF, NT3, BDNF, and NT4 / 5 (as shown in He and Garcia (2001) Science, 301, pages 870-805).

[0054] Suitably, the p75NTR(NBP)-Fc fusion protein is soluble, for example soluble in aqueous solution, and may further be soluble in biological fluids such as serum, plasma, blood and the like.

[0055] As used herein, the term "Fc" or "immunoglobulin Fc" or "Ig Fc" is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region (preferably an immunoglobulin heavy chain constant region), or a portion thereof. Preferably, the immunoglobulin Fc comprises: 1) a CH1 domain, a CH2 domain, and a CH3 domain, optionally with an immunoglobulin hinge region, 2) a CH1 domain and a CH2 domain, optionally with an immunoglobulin hinge region, 3) a CH1 domain and a CH3 domain, optionally with an immunoglobulin hinge region, 4) a CH2 domain and a CH3 domain, optionally with an immunoglobulin hinge region, or 5) a combination of two or more domains selected from, but not limited to, a CH1, a CH2, and a CH3, optionally combined with an immunoglobulin hinge region.

[0056] According to the present invention, p75NTR(NBP)-Fc fusion proteins may exhibit advantageous biological properties, such as improved solubility of p75NTR(NBP), stability of p75NTR(NBP), and / or improved serum half-life of p75NTR(NBP). Improved solubility is desirable for maximizing the bioavailability of p75NTR(NBP) upon administration and enabling accurate dosing of p75NTR(NBP). Improved solubility is advantageous in overcoming the problem of undesirable aggregation, which can cause pain and potentially lead to inflammation during in vivo delivery. Improved serum half-life has the advantage of facilitating reduced dosage levels or less frequent administration required during therapeutic use to achieve comparable or sustained therapeutic effects of the delivered p75NTR(NBP). The extended half-life and greater stability in blood or serum have the advantage of allowing for less frequent and / or lower dosage regimens, thus reducing potential toxicity or side effects in vivo. In this case, the p75NTR(NBP)-Fc fusion protein is more potent in its therapeutic effect and / or more stable in the circulation. The resulting lower or less frequent administration is advantageous in minimizing any potential toxic or side effects potentially associated with p75NTR(NBP) administration. The molecular weight of the p75NTR(NBP)-Fc fusion protein is also increased compared to p75NTR(NBP) alone, which has the advantage that the molecule is better retained in the circulation when administered intravenously, reducing the risk of penetration into undesirable sites (e.g., the central nervous system) and making the molecule more suitable for retention or concentration in targeted tissues.

[0057] Suitably, the p75NTR(NBP)-Fc fusion protein exhibits improved solubility of p75NTR(NBP), improved stability of p75NTR(NBP), and / or improved serum half-life compared to p75NTR(NBP) alone. Suitably, the improved solubility is solubility in an aqueous solution such as water (which may contain excipients such as buffers and / or salts, and which may be at physiological pH, between pH 5 and pH 8, or about pH 7), or in a biological fluid such as serum or blood. Suitably, the improved stability is stability of the activity or structural integrity of the p75NTR(NBP) protein over a period of time, during storage, or after freeze-thawing due to the effects of denaturation, oxidation, fragmentation, or aggregation. Structural stability can be determined by standard measurements of denaturation, oxidation, aggregation, or aggregation, and activity stability can be measured by binding or functional assays disclosed herein, and methods for measuring serum half-life of proteins are known.

[0058] Suitably, the p75NTR(NBP)-Fc fusion protein can be expressed at high levels from a variety of mammalian host cells to provide a single species and can be efficiently purified by affinity chromatography (e.g., by binding to Staphylococcus aureus protein A). Suitably, the p75NTR(NBP)-Fc fusion protein can dimerize, and preferably the dimer has increased affinity for the neurotrophic factors NGF, BDNF, NT3, or NT4 / 5 compared to p75NTR(NBP) alone. Stronger binding has the advantage of higher potency and greater therapeutic efficacy, as determined by the effects of p75NTR(NBP), e.g., by the neurotrophic factor functional assays disclosed herein. Higher potency has the advantage that the p75NTR(NBP)-Fc fusion protein can achieve equivalent therapeutic efficacy at lower doses, thereby reducing potential toxicity or side effects in vivo.

[0059] Suitably, the p75NTR(NBP)-Fc fusion proteins of the invention have an in vivo half-life of near or greater than any one of the following: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 , 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 62, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, or 210 hours ± 1 hour, more suitably, the p75NTR(NBP)-Fc fusion proteins of the present invention have an in vivo half-life of about 24 hours or more.

[0060] Further optionally, the p75NTR(NBP)-Fc fusion proteins of the invention have an in vitro half-life of near or greater than any one of the following: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118 , 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 62, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, or 210 days ± 1 day, and optionally, the p75NTR(NBP)-Fc fusion proteins of the present invention have an in vitro half-life of about 6 days or more. Suitably, stability is measured in a buffered aqueous solution at near physiological pH, for example at 20°C or 37°C.

[0061] According to the foregoing embodiments, the in vivo half-life may be the half-life in rats or the half-life in humans. Suitably, the half-life is determined from measuring serum levels of the p75NTR(NBP)-Fc fusion protein of the present invention after in vivo administration (e.g., by intravenous or subcutaneous injection).

[0062] The p75NTR(NBP) portion and the immunoglobulin Fc portion of the p75NTR(NBP)-Fc fusion protein are linked by a linker that may comprise or consist of one or more amino acids, or may comprise or consist of a polypeptide sequence of amino acids, and may be about 1 to about 25 amino acids, or any one of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, or even any one of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids, such as 13 amino acids.

[0063] Suitably, the linker may be an alpha helix, a beta strand, a three 10 The linker region may comprise or consist of a polypeptide sequence of amino acids that lacks any stable secondary structure, such as a helix, a π-helix, a polyproline helix, an α-sheet, etc. Suitably, the linker region may comprise or consist of a polypeptide sequence of amino acids that defines a flexible or dynamic or unstructured polypeptide, such as a flexible loop, a random coil, or a flexible turn. Such unstructured polypeptides are often found in regions that connect secondary structures in large protein molecules.

[0064] Suitably, the linker is a polypeptide sequence of amino acids that comprises greater than or about 50% glycine and / or alanine and / or serine in p75NTR(NBP), and may comprise greater than or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% glycine and / or alanine and / or serine in p75NTR(NBP). Suitably, the linker region comprises or consists of a polypeptide sequence of amino acids that comprises both glycine and serine, preferably having a higher proportion of glycine than serine, and the linker region may comprise or consist of a flexible linker.

[0065] Without wishing to be bound by any particular theory, the inventors believe that the flexible linker overcomes or prevents steric hindrance that may interfere with the aforementioned neurotrophic factor binding ability or biological activity of the p75NTR(NBP)-Fc fusion protein compared to p75NTR(NBP) alone. Thus, the linker region allows flexibility between the p75NTR(NBP) portion and the immunoglobulin Fc portion, allowing the aforementioned biological activity of the p75NTR(NBP)-Fc fusion protein to be retained or improved compared to free or native p75NTR(NBP) alone, as determined by binding to neurotrophic factors using binding assays such as those described herein.

[0066] Further optionally, the linker is immunologically inert, such that it does not trigger complement-mediated lysis, stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or activate microglia or T cells. Suitably, the linker region has reduced one or more of these activities.

[0067] Further optionally, the linker comprises or consists of a polypeptide that is known or predicted from structural analysis or structural prediction to be a flexible or dynamic or unstructured polypeptide, or to lack stable secondary structure.

[0068] The linker has the formula G x where x is 1, 2, 3, 4, 5, or 6.

[0069] In one embodiment, the linker does not comprise and consist of the sequence GGGGS.

[0070] In a further embodiment, the linker is GGG.

[0071] The p75NTR(NBP)-Fc fusion proteins of the present invention may also include a proteolytic cleavage site, optionally inserted between the p75NTR(NBP) portion and the immunoglobulin Fc portion. The proteolytic cleavage site may be located within the linker or at the junction of the linker with either the p75NTR(NBP) portion or / and the immunoglobulin Fc portion. The p75NTR(NBP) may optionally be cleaved from the immunoglobulin Fc portion prior to formulation and / or administration for therapeutic purposes.

[0072] Suitably, the linker and / or immunoglobulin Fc portion does not impair or significantly impair the p75NTR(NBP) portion: (a) an effect on the functional activity of the neurotrophic factors NGF, BDNF, NT3, or NT4 / 5 (defined as modulating, or up- or down-regulating, the functional activity of a neurotrophic factor); (b) a binding affinity for either NGF, BDNF, NT3, or NT4 / 5, with a binding affinity between about 0.1 nM and about 50 nM; (c) the ability to bind to each of the neurotrophic factors NGF, NT3, BDNF, and NT4 / 5 (preferably human NGF, NT3, BDNF, and NT4 / 5).

[0073] According to another aspect of the present invention there is provided a nucleic acid molecule encoding a p75NTR(NBP)-Fc fusion protein according to the first or second aspect, suitably for use in the treatment of pain.

[0074] According to one embodiment of the present invention, the nucleic acid molecule may further comprise a region encoding a signal sequence (preferably a p75NTR signal sequence, eg, a DNA or RNA sequence).

[0075] According to another aspect of the present invention there is provided a replicable expression vector for transfecting a cell, said vector comprising the nucleic acid molecule of the third aspect, said vector may be a viral vector. Suitably said vector is for use in the treatment of pain.

[0076] Further, according to the above aspect of the present invention, there is provided a method for expressing a nucleic acid molecule or vector of the present invention to produce or secrete a p75NTR(NBP)-Fc fusion protein. Suitably, the method comprises introducing the nucleic acid molecule or vector into a cell, expressing the nucleic acid therein, and producing or secreting the p75NTR(NBP)-Fc fusion protein. Suitably, the nucleic acid molecule or vector is introduced into the cell in vitro or in vivo. Suitably, the expressed p75NTR(NBP)-Fc fusion protein is expressed in vitro and optionally further isolated and purified, or optionally, the expressed p75NTR(NBP)-Fc fusion protein may be expressed in vivo, optionally constituting gene therapy. Suitably, the vector is a replicable expression vector and may be for transfecting mammalian cells, suitably the vector is a viral vector.

[0077] According to another aspect of the present invention there is provided a host cell harboring the nucleic acid molecule or vector of either the third or fourth aspect, which may be a mammalian cell.

[0078] According to another aspect of the present invention, there is provided a p75NTR(NBP)-Fc fusion protein, or a nucleic acid or vector for use in treating pain, which may include, but is not limited to: (a) acute pain and / or spontaneous pain; (b) chronic and / or progressive pain; (c) inflammatory pain (including any of arthritic pain, pain caused by osteoarthritis or rheumatoid arthritis, pain caused by inflammatory bowel disease, psoriasis, and eczema); (d) nociceptive pain; (e) neuropathic pain (including pain associated with painful diabetic neuropathy or postherpetic neuralgia); (f) hyperalgesia, (g) allodynia, (h) central pain, central post-stroke pain, pain due to multiple sclerosis, pain due to spinal cord injury, or pain due to Parkinson's disease or epilepsy; (i) cancer pain, (j) Postoperative pain; (k) visceral pain (including gastrointestinal visceral pain and non-digestive visceral pain, pain due to gastrointestinal (GI) disorders, pain due to functional bowel disorders (FBD), pain due to inflammatory bowel disease (IBD), pain due to dysmenorrhea, pelvic pain, cystitis, interstitial cystitis, or pancreatitis); (l) Musculoskeletal pain, myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatic) arthropathy, non-articular rheumatism, dystrophinopathy, glycogenolysis, polymyositis, pyomyositis, (m) cardiac or vascular pain, pain resulting from angina, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma, or skeletal muscle ischemia; (n) headache (including migraine, migraine with aura, migraine without aura, cluster headache, and tension-type headache); (o) orofacial pain (including toothache, temporomandibular myofascial pain, or tinnitus), or (p) Back pain, bursitis, menstrual pain, migraine, referred pain, trigeminal neuralgia, hypersensitisation, pain due to spinal cord trauma and / or degeneration or stroke.

[0079] Treating pain includes, but is not limited to: preventing, ameliorating, controlling, reducing the incidence, or delaying the onset or progression of pain.

[0080] According to another aspect of the present invention, there is provided a p75NTR(NBP)-Fc fusion protein as described in the first or second aspect or preferred embodiments thereof, or a nucleic acid molecule or vector as described in the third and fourth aspects, wherein said p75NTR(NBP)-Fc fusion protein or said nucleic acid molecule or vector is for separate, sequential or simultaneous use in combination with a second pharmacologically active compound. Suitably, said second pharmacologically active compound of said combination may include, but is not limited to: · Opioid analgesics, such as morphine, heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, or pentazocine; nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin, or zomepirac; barbiturates, such as amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobartital, secobarbital, talbutal, theamylal, or thiopental; · Sedating benzodiazepines, such as chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, or triazolam; Sedating H1 antagonists, such as diphenhydramine, pyrilamine, promethazine, chlorpheniramine, or chlorcyclizine; · Sedatives, such as glutethimide, meprobamate, methaqualone, or dichloralphenazone; Skeletal muscle relaxants, such as baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol, or orphrenadine; NMDA receptor antagonists, such as dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextromethorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid, budipine, EN-3231 (MorphiDex®, a combination formulation of morphine and dextromethorphan), topiramate, neramexane, or perzinfotel, including NR2B antagonists, such as ifenprodil, traxoprodil, or (-)-(R)-6-{2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl-3,4-dihydro-2(1H)-quinolinone; alpha-adrenergic (receptor antagonist) drugs, such as doxazosin, tamsulosin, clonidine, guanfacine, dexmetatomidine, modafinil, or 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline; Tricyclic antidepressants, such as desipramine, imipramine, amitriptyline, or nortriptyline; anticonvulsants, such as carbamazepine, lamotrigine, topiramate, or valproic acid; Tachykinin (NK) antagonists, particularly NK-3, NK-2, or NK-1 antagonists, such as (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione (TAK-637), 5-[[(2R,3S)-2-[(1R -1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one (MK-869), aprepitant, lanepitant, dapitant, or 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine(2S,3S); Muscarinic antagonists, such as oxybutynin, tolterodine, propiverine, tropsium chloride, darifenacin, solifenacin, temiverine, and ipratropium; COX-2 selective inhibitors, such as celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib; · Coal tar-based painkillers, especially paracetamol; neuroleptics, such as droperidol, chlorpromazine, haloperidol, perphenazine, thioridazine, mesoridazine, trifluoperazine, fluphenazine, clozapine, olanzapine, risperidone, ziprasidone, quetiapine, sertindole, aripiprazole, sonepiprazole, blonanserin, iloperidone, perospirone, raclopride, zotepine, bifeprunox, asenapine, lurasidone, amisulpride, balaperidone, palindore, eplivanserin, osanetant, rimonabant, meclineltant, Miraxion®, or sarizotan; Vanilloid receptor agonists (e.g., resiniferatoxin) or antagonists (e.g., capsazepine); beta-adrenergic (receptor antagonist) drugs, e.g., propranolol; Local anesthetics, e.g., mexiletine; Corticosteroids, e.g., dexamethasone; 5-HT receptor agonists or antagonists, especially 5-HT 1B / 1D agonists, such as eletriptan, sumatriptan, naratriptan, zolmitriptan, or rizatriptan; 5-HT 2A Receptor antagonists, for example, R(+)-α-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (MDL-100907); Cholinergic (nicotinic) analgesics, such as ispronicline (TC-1734), (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403), (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594), or nicotine; Tramadol (registered trademark); PDEV inhibitors, such as 5-[2-ethoxy-5-(4-methyl-1-piperazinyl-sulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil), (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]-pyrido[3,4-b]indo 2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one (vardenafil), 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3 -d]pyrimidin-7-one, 5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulfonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidine-7-one -one, 4-[(3-chloro-4-methoxybenzyl)amino]-2-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide, 3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide; · Cannabinoids; · Metabotropic glutamate subtype 1 receptor (mGluR1) antagonists; Serotonin reuptake inhibitors, such as sertraline, sertraline metabolite demethylsertraline, fluoxetine, norfluoxetine (fluoxetine demethyl metabolite), fluvoxamine, paroxetine, citalopram, citalopram metabolite desmethylcitalopram, escitalopram, d,l-fenfluramine, femoxetine, ifoxetine, cyanodothiepin, ritoxetine, dapoxetine, nefazodone, cericlamine, and trazodone; noradrenaline (norepinephrine) reuptake inhibitors, such as maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine, tomoxetine, mianserin, bupropion, the bupropion metabolite hydroxybupropion, nomifensine, and viloxazine (Vivalan®), especially selective noradrenaline reuptake inhibitors, such as reboxetine, in particular (S,S)-reboxetine; Serotonin-norepinephrine dual reuptake inhibitors, such as venlafaxine, venlafaxine metabolite O-desmethylvenlafaxine, clomipramine, clomipramine metabolite desmethylclomipramine, duloxetine, milnacipran, and imipramine; Inducible nitric oxide synthase (iNOS) inhibitors, such as S-[2-[(1-Iminoethyl)amino]ethyl]-L-homocysteine, S-[2-[(1-Iminoethyl)amino]ethyl]-4,4-dioxo-L-cysteine, S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, (2S,5Z)-2-amino-2-methyl-7-[(1-Iminoethyl)amino]-5-heptenoic acid, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)-butyl]thio]-5-chloro-3-pyridinecarbonitrile; 2-[[(1R,3S)-3-amino-4-hydroxy-1-( 5-thiazolyl)butyl]thio]-4-chlorobenzonitrile, (2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolebutanol, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluoromethyl)-3-pyridinecarbonitrile, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile, N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine, or guanidinoethyl disulfide; Acetylcholinesterase inhibitors, e.g., donepezil; Prostaglandin E2 subtype 4 (EP4) antagonists, such as N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzenesulfonamide, or 4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]benzoic acid; Leukotriene B4 antagonists; for example, 1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylic acid (CP-105696), 5-[2-(2-carboxyethyl)-3-[6-(4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]-valeric acid (ONO-4057), or DPC-11870, 5-lipoxygenase inhibitors, such as zileuton, 6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl])phenoxy-methyl]-1-methyl-2-quinolone (ZD-2138), or 2,3,5-trimethyl-6-(3-pyridylmethyl),1,4-benzoquinone (CV-6504); sodium channel blockers, such as lidocaine; or 5-HT3 antagonists, e.g., ondansetron; and pharmaceutically acceptable salts and solvates thereof.

[0081] According to a further aspect of the present invention there is provided a method of treating, preventing, ameliorating, controlling, reducing the incidence of or delaying the onset or progression of pain, or any of the aforementioned pain, in an individual, said method comprising administering to said individual an effective amount of a p75NTR(NBP)-Fc fusion protein as described in the first or second aspect or preferred embodiments thereof, or a nucleic acid molecule or vector as described in the third and fourth aspects.

[0082] In another embodiment, the p75NTR(NBP)-Fc fusion protein of the present invention is suitable for treating osteoarthritis, particularly for halting, slowing, and reversing the progression of the disease. In a specific embodiment, the protein of the present invention provides a cure for osteoarthritis.

[0083] Suitable embodiments of the present invention provide a p75NTR(NBP)-Fc fusion protein of the present invention for the treatment of osteoarthritis, wherein treatment includes reversing disease progression, regenerating cartilage, and / or curative treatment.

[0084] In a suitable embodiment, disease progression is determined by the rate of cartilage loss or regeneration.

[0085] In further suitable embodiments, wherein osteoarthritis includes prophylactic treatment.

[0086] The present invention is applicable in the veterinary field. Suitably, the individual is a mammal, suitably a companion animal such as a horse, cat or dog, or a livestock animal such as a sheep, cow or pig. Other suitable animals include elephants. Most suitably, the animal is a dog.

[0087] Without limiting the described invention, one skilled in the art will appreciate that certain IgG sequences may be particularly suitable as components of fusion proteins for particular animals, for example, in a fusion protein for use in dogs, canine IgG sequences may be particularly suitable.

[0088] According to an eighth aspect of the present invention there is provided a pharmaceutical composition for the treatment, prevention, amelioration, control, reduction of incidence or delay of onset or progression of pain, or any one or more of the aforementioned pains, said pharmaceutical composition comprising a p75NTR(NBP)-Fc fusion protein as described in the first or second aspect or embodiments thereof, or a nucleic acid molecule or vector as described in the third and fourth aspects, and a pharmaceutically acceptable carrier and / or excipient.

[0089] Suitably, the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or an embodiment thereof, or the nucleic acid molecule or vector according to the third and fourth aspects, or the medicament of the eighth aspect is prepared for or is suitable for oral, sublingual, buccal, topical, rectal, inhalation, transdermal, subcutaneous, intravenous, intraarterial, intramuscular, intracardiac, intramedullary, intradermal, intraperitoneal, transmucosal, intravaginal, intravitreal, intra-articular, peri-articular, topical or epicutaneous administration.

[0090] Suitably, the p75NTR(NBP)-Fc fusion protein described in the first or second aspect or an embodiment thereof, or the nucleic acid molecule or vector described in the third and fourth aspects, or the pharmaceutical composition of the eighth aspect, is prepared for or suitable for administration before and / or during and / or after the onset of pain, or for such use.

[0091] Suitably, the p75NTR(NBP)-Fc according to the first or second aspect or embodiments thereof, or the nucleic acid molecule or vector according to the third and fourth aspects, or the pharmaceutical composition of the eighth aspect, is for or is prepared for administration between 1 and 7 times per week, further optionally between 1 and 4 times per month, further optionally between 1 and 6 times per 6 month period, further optionally between 1 and 12 times per year. Suitably, the medicament is or is prepared to be administered peripherally at dosing intervals including, but not limited to, once daily, once every 2, 3, 4, 5, or 6 days, once weekly, once every 2 weeks, once every 3 weeks, once monthly, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once yearly.

[0092] Further optionally, the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or preferred embodiments thereof, or the nucleic acid molecule or vector according to the third and fourth aspects, or the pharmaceutical composition according to the eighth aspect, is or is prepared to be administered peripherally, via one or more of the following routes, including but not limited to: oral, sublingual, buccal, topical, rectal, inhalation, transdermal, subcutaneous, intravenous, intraarterial, or intramuscular, intracardiac, intraosseous, intradermal, intraperitoneal, transmucosal, intravaginal, intravitreal, epicutaneous, intra-articular, peri-articular, or topical.

[0093] Suitably, the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or embodiments thereof, or the nucleic acid molecule or vector according to the third and fourth aspects, or the pharmaceutical composition of the eighth aspect, is for administration at, or is prepared for, a concentration of about 0.05 to about 200 mg / ml; optionally about 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / ml ± about 10% error, for example about 3 mg / ml for veterinary use and about 0.1 mg / ml for humans.

[0094] Suitably, the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or embodiments thereof, or the nucleic acid molecule or vector according to the third and fourth aspects, or the pharmaceutical composition of the eighth aspect, is for or is prepared for administration at a concentration of about 0.1 to about 200 mg / kg body weight; optionally about 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or about 200 mg / kg body weight, plus or minus about 10%, for example about 10 mg / kg for veterinary use and 0.3 for humans.

[0095] According to a ninth aspect of the present invention there is provided a kit comprising: (a) a p75NTR(NBP)-Fc fusion protein according to the first or second aspect or an embodiment thereof, or a nucleic acid molecule or vector according to the third and fourth aspects, or a pharmaceutical composition according to the eighth aspect; and (b) Instructions for administering to an individual an effective amount of the p75NTR(NBP)-Fc fusion protein, nucleic acid molecule, vector, or pharmaceutical composition for any one or more of preventing or treating pain, or ameliorating, controlling, reducing the incidence, or delaying the onset or progression of pain.

[0096] The kit may include one or more containers containing the p75NTR(NBP)-Fc fusion protein, nucleic acid, vector, or pharmaceutical composition described herein, and instructions for use according to any of the methods and uses of the present invention. The kit may further include instructions for selecting individuals suitable for treatment based on identifying whether the individual has pain or a pain symptom or is at risk for such a condition. Instructions for administering the pharmaceutical composition may include information regarding the dosage, administration schedule, and route of administration for the intended treatment.

[0097] According to yet another aspect of the present invention there is provided a p75NTR(NBP)-Fc fusion protein as defined in the first or second aspect or embodiments thereof, or a nucleic acid molecule or vector as defined in the third and fourth aspects, or a pharmaceutical composition of the eighth aspect, for use in preventing or treating, or ameliorating, controlling, reducing the incidence of, or delaying the onset or progression of, a condition or a symptom of a condition associated with any one or more of the neurotrophic factors NGF, BDNF, NT-3, NT-4 / 5.

[0098] Nerve growth factor (NGF) binds to at least two receptor classes: p75NTR and TrkA (transmembrane tyrosine kinase) and is involved in axon growth, branching, and elongation. NGF-related conditions and symptoms are known. NGF is expressed in and associated with inflammatory conditions and pain [protein sequences NP_002497.2, NP_038637]. NGF has also been shown to play a role in numerous cardiovascular diseases, such as coronary atherosclerosis, obesity, type 2 diabetes, and metabolic syndrome, as well as multiple sclerosis. Decreased plasma levels of NGF (and BDNF) are associated with acute coronary syndrome and metabolic syndrome. NGF is also associated with various psychiatric disorders, such as dementia, depression, schizophrenia, autism, Rett syndrome, anorexia nervosa, and bulimia nervosa, and has also been implicated in the development of Alzheimer's disease and neurodegenerative disorders. NGF has also been shown to accelerate wound healing, and there is evidence that it may be useful in treating skin and corneal ulcers. It has also been shown to reduce nerve degeneration and promote peripheral nerve regeneration in rats.

[0099] Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor that supports the survival and growth of neurons during nervous system development [protein sequences NP_001137277.1, NP_001041604]. BDNF binds to the cell surface receptors TrkB and p75NTR and regulates the activity of the α7 nicotinic receptor. BDNF-associated conditions and symptoms are known. BDNF has been shown to play an important role in physiological and pathological pain transmission, specifically in models of acute, inflammatory, and neuropathic pain, where BDNF synthesis is found to be significantly increased; BDNF has also been shown to be upregulated in chronic pain states and in additional conditions such as eczema and psoriasis. BDNF downregulation has been observed in depression, schizophrenia, obsessive-compulsive disorder, Alzheimer's disease, Huntington's disease, Rett syndrome, and dementia, as well as anorexia nervosa and bulimia nervosa.

[0100] Neurotrophin-4 (NT-4), also known as neurotrophin-5 (NT-5), is a neurotrophic factor that signals primarily through p75NTR and TrkB receptors and promotes the survival of peripheral sensory and sympathetic neurons. The mature peptide of this protein is identical in all mammalian species studied, including humans, pigs, rats, and mice. [Protein sequences NP_006170, NP_937833]. NT-4 is synthesized by most neurons in the dorsal root ganglia (DRG), as well as neurons in the paravertebral and prevertebral sympathetic ganglia, dorsal and ventral horns of the spinal cord. It is found to be expressed in many tissues, including the prostate, thymus, placenta, and skeletal muscle. Conditions and symptoms associated with NT-4 / 5 are known. Deficiency of NT-4 / 5 is associated with susceptibility to primary open-angle glaucoma. Neurotrophin-4 has also been shown to contribute to breast cancer cell survival and be a target for inhibiting tumor growth. NT-4 / 5 are known to be involved in pain signaling, including nociceptive pain. Upregulation of NT-4 / 5 is also observed in chronic inflammatory conditions of the skin, such as dermatitis, eczema, and prurigo lesions of atopic dermatitis. Downregulation of NT-4 / 5 is observed in Alzheimer's disease and Huntington's disease.

[0101] Neurotrophic factor-3 (NT-3) is a neurotrophic factor structurally related to β-NGF, BDNF, and NT-4. It regulates the survival and differentiation of mammalian neurons and the maintenance of the adult nervous system, and when expressed in the human placenta, it can affect neuronal development in embryos. Conditions and symptoms associated with NT3 are known. NTF3-deficient mice generated by gene targeting exhibit severe limb movement disorders. NT-3 signals through Trk receptors and promotes the growth and survival of neural and glial cells [protein sequences NP_001096124.1 and NP_032768]. The amino acid sequences of human, mouse, and rat NT-3 are identical. NT-3 and its corresponding receptor, tyrosine kinase C (TrkC), are known to regulate neuropathic and nociceptive pain, as well as nociception and proprioception. For example, NT3 expression is increased in small DRG cells of neuropathic animals. NT3 expression is also associated with neuropathies such as diabetic polyneuropathy and HIV-associated neuropathies, large fiber neuropathies including atrophy, and is involved in the development of hyperalgesia (a decrease in the threshold for normally noxious stimuli), allodynia (non-noxious stimuli becoming noxious), and spontaneous pain (pain in the absence of any apparent stimulus), and is a known regulator of muscle pain.

[0102] The invention will now be described with reference to the following examples, which are intended to illustrate but not limit the invention. [Example]

[0103] [Design of p75-NTR Fc fusion protein] The specific allotype of the Fc portion of the p75-NTR Fc fusion protein was IgG pCon vector IgGza (see above).

[0104] The design of the p75-NTR Fc fusion protein proceeded in multiple steps: The exact construct of the p75-NTR sequence used in the Fc fusion protein was defined. Several factors were considered, including: The p75-NTR Fc fusion must be able to bind multiple neurotrophic factors, including at least NGF, BDNF, NT-3, and NT-4; flexibility must be maintained in the p75-NTR Fc fusion protein. The extracellular domain of p75-NTR-Fc (SEQ ID NO: 1) contains unnecessary α-secretase cleavage sites that must be removed from the sequence because they are subject to cleavage, thereby reducing the in vivo biological activity and PK profile of the p75-Fc product.

[0105] Suitable empirical linkers suitable for use in linking the extracellular p75-NTR domain to Fc in a p75-NTR Fc fusion protein were identified. Linker sequences containing sites potentially involved in post-translational modifications (PTMs) were excluded.

[0106] Multiple variants of the p75-NTR Fc fusion protein were constructed in silico using a defined p75-NTR construct and the appropriate portion of the Fc region, with different candidate linker sequences. Structural modeling and analysis of the C-terminus of the p75-NTR extracellular domain, the Fc hinge region, and the candidate linkers were undertaken.

[0107] [Structural Modeling] A structural model of the proposed p75-NTR Fc fusion protein was generated using Lonza's modeling platform. Candidate structural template fragments of the p75-NTR and Fc portions were scored, ranked, and selected from both an in-house antibody database and the Protein Data Bank (PDB) based on their sequence identity and qualitative crystallographic indices of the template structure, such as resolution (in angstroms (Å)).

[0108] A sequence alignment of the structural template fragments to the p75-NTR Fc fusion protein was generated. The template fragments, along with the sequence alignment, were processed by MODELLER (Sali et al. 1993 J. Mol. Biol 234, 779-815). This protocol generates conformational constraints derived from a set of aligned structural templates. An ensemble of structures satisfying the constraints is generated using conjugate gradient and simulated annealing optimization procedures. One or more model structures are selected from this ensemble based on the protein structure score and energy scores derived from the degree of conformational constraint satisfaction. The models were refined, and side chains at positions that differed between the target and template were optimized and energy minimized using a side chain optimization algorithm. A suite of visualization and computational tools was used to evaluate the conformational variability of the structures and to evaluate the core and local packing of domains to select one or more preferred models.

[0109] [Design of p75-NTR Fc fusion protein] The main requirements for the linker selected for the variant are to allow flexibility of the fusion partner in the Fc fusion protein, to avoid the introduction of any residues that may undergo PTM, and to maintain a low immunogenicity risk.

[0110] [Affinity of p75NTR-Fc of Sequence 1 (SEQ ID NO: 1) and Sequence 2 (SEQ ID NO: 2) for NGF] Biacore chips were prepared in experiments where Protein A was amine coupled to flow cells 1 and 2. Single-cycle kinetics of NGF binding to captured p75-Fc were measured.

[0111] The binding capacity of the chip surface (R max ) depends on the immobilization level of the ligand (fusion protein). For kinetic analysis experiments, an R of 50-100 RU is recommended. maxUsing the molecular weights of p75-Fc and NGF, the desired immobilization level for the fusion protein can be calculated.

[0112] R max = (molecular weight of NGF / molecular weight of fusion protein) × immobilization level × stoichiometry: 50 = (13,500 / 102,000) × Fixation Level × 1

[0113] Therefore, the required immobilization level = (102,000 / 13,500) x 50 = 378 RU. p75NTR-Fc and NTR-Fc of sequence 1 (SEQ ID NO: 1) and sequence 3 (SEQ ID NO: 3) were immobilized on a Protein A chip prior to single-cycle kinetic analysis.

[0114] Using a manual run, p75-Fc was captured onto flow cell 2 of a Protein A chip until the desired level of approximately 380 RU was achieved. This was done using a 22 second injection at a flow rate of 10 μl / min and a p75-Fc concentration of 10 μg / ml, resulting in 418 RU of fusion protein being captured on the Protein A surface.

[0115] Initially, NGF concentrations of 10, 5, 2.5, 1.25, and 0.625 nM were tested. These concentrations were tested because the K D However, this was estimated to be within the range of NGF concentrations.

[0116] Single cycle kinetic analysis methods included: - injecting 0.625 nM NGF over the captured p75-Fc at 30 μl / min for 120 seconds; - This treatment was then repeated with injections of 1.25 nM NGF, followed by 2.5, 5, and 10 nM After the final concentration of NGF was injected, a 600-second dissociation step was performed by flowing running buffer (HBS-EP) over the chip.

[0117] Upon completion, the chip was regenerated with a Protein A surface by injecting 10 mM glycine HCl (pH 2) at 30 μl / min for 60 seconds.

[0118] p75-Fc was then captured onto the chip at a concentration of 10 μg / ml using a 38-second injection at a flow rate of 10 μl / min, achieving the desired level of 430 RU. The single-cycle kinetic analysis procedure described above was then repeated.

[0119] [Data Analysis] Fusion protein-NGF binding data were analyzed using Biacore T200 evaluation software v1 in the following manner: - Data are recorded for binding of NGF to the fusion protein on flow cell 2 (Fc=2) and for NGF flowing over control flow cell 1 (Fc=1; Protein A only). - The data from Fc=1 is then subtracted from Fc=2 to obtain the "2-1" binding data. - The 2-1 binding data for the 0 nM (HBS-EP running buffer only) injection is then subtracted from all 2-1 binding data to correct for baseline fluctuations throughout the experiment. - Finally, this data is then fitted to a 1:1 binding model to obtain the association rate (ka), dissociation rate (kd), and affinity (K D ) to calculate binding properties.

[0120] Both sequences showed appropriate affinity for NGF.

[0121] [P75NTR-Fc is analgesic.] The aim of this study was to investigate the effect of chronic exposure to p75NTR-Fc on the analgesic effect in monosodium iodoacetate (MIA)-induced osteoarthritis (OA) in rats.

[0122] We previously demonstrated that spontaneous pain assessment can be performed by measuring static load using an incapacitance tester and that this correlates with knee histopathology. Preclinical studies using novel pain treatments have been criticized for the potential for bias in the data. To address this, both left and right knees were randomly selected for OA induction, and all operators of daily in vivo work were blinded to the condition of each knee. While the literature typically dictates that OA induction is performed only in the right knee, previous studies have not found consistent differences between OA induction in the left and right knees, regardless of the time point or dose of MIA used.

[0123] [Preparation of MIA] MIA was prepared at 0.3 mg / 50 μl ETF-PBS (volume used for each intra-articular injection), which corresponds to a 6 mg / ml stock solution. 302 mg of MIA was weighed and dissolved in 50.3 ml ETF-PBS. MIA was prepared the day before and stored in the dark at 4°C until needed.

[0124] [animal] Forty-four male Wistar rats (from Charles River, UK, weighing 110–130 g upon arrival) were used in this study. Each animal was identified upon arrival and appeared healthy. They were randomly assigned to cages of two, and each rat was assigned a unique identification number via a tail tattoo. Animals were acclimated to the animal facility for at least 10 days before the start of the study on day 0. After the rats had acclimated to their environment, they were transferred to the stock / treatment room, where all in vivo procedures were performed. Animals were kept under fluorescent lighting with a 12-h light / dark cycle (lights on 07:00, lights off 19:00), as recommended by the Home Office Animals (Scientific Procedures) Act 1986. The room was air-conditioned, and air temperature (21°C ± 2°C) and relative humidity were measured daily.

[0125] Rats were fed irradiated feed (Scientific Animal Food and Engineering, Augy, France) and had free access to autoclaved water. Each batch of feed was regularly checked and tested for composition and contaminants. Nest boxes and cages were autoclaved, and each cage was individually ventilated (IVC system).

[0126] [Experimental design] The study design divided animals into five groups: a control human antibody group (n=6), a 0.3 mg / kg p75NTR-Fc group, a 1 mg / kg p75NTR-Fc group, a 3 mg / kg p75NTR-Fc group, and a 3 mg / kg PG-007 group (a biosimilar anti-NGF antibody to Pfizer's tanezumab).

[0127] Antibodies and p75NTR-Fc were administered by subcutaneous injection every 5 days for 25 days.

[0128] Body weights were measured, and baseline blood samples were collected from the tail vein on the morning of day -2. Baseline static load measurements were taken at approximately the same time on day -1. On day 0, all rats were treated with the respective antibodies or p75NTR-Fc fusion protein, again at approximately the same time. Three hours later, all animals were injected intra-articularly with 0.3 mg of MIA on one side (ETF-PBS was injected into the contralateral knee).

[0129] Randomization of Treatment Prior to the start of the study, rats were weighed and each cage of two rats was randomly assigned to a treatment group, ensuring that the average weight of animals in each group was approximately equal. In addition to assigning each rat to a specific treatment group, further randomization was performed so that each rat received an injection of MIA into either its left or right knee (each rat's contralateral knee was injected with ETFPBS). The assignment of each rat to treatment group and which knee received treatment was generated using a random number generator in Microsoft Excel for Mac (version 14.1.1). Personnel with no contact with the animals performed the randomization procedure and assignment.

[0130] Two 7 ml polypropylene vials were labeled for each animal to indicate the left or right knee (88 vials total). Two individuals (one recorded and verified against the master randomization sheet, and the other dispensed the intra-articular injection solution) prepared the 88 vials. Dispensing was performed sequentially, with the MIA vials filled first, followed by the remaining vials with ETF-PBS (this was the vial for each animal's contralateral knee). Throughout the study, the in vivo investigator was blinded to the treatment status of all animals.

[0131] [Animal experiments] [Intra-articular injection of the knee] All rats were anesthetized with isoflurane inhalation using a Boyles apparatus. Both knees of each animal were clipped and wiped with ethanol. Each knee was injected under the patellar ligament with 50 μl of either 0.3 mg of MIA in ETF-PBS or ETF-PBS alone using a 0.5 ml sterile Becton Dickinson Micro-Fine insulin syringe fitted with a 27G needle.

[0132] [Evaluation of spontaneous pain] Spontaneous pain was assessed for each animal by measuring the load of the left and right hind paws using an incapacitance tester (Linton Instruments, UK). Rats were placed in an appropriately sized Perspex animal box on the incapacitance tester, with each hind paw resting on a separate sensor. The box size was such that the rat could sit comfortably without feeling cramped, but did not allow enough space for it to turn around. Once the rat was stable and calm, the load of each paw was recorded for 5 seconds, and the average force (in grams) exerted by both hind paws was recorded. Hind paw load distribution was measured five times for each rat at each time point (the validity of which has been previously demonstrated), and the average of the five readings was calculated. Individual load data were converted to load distribution by dividing the load of the right paw by the combined load of both hind paws.

[0133] [Measurement of spontaneous pain after MIA-induced OA] Spontaneous pain was assessed using an incapacitance tester to measure load distribution through the hind paw. Assessments were performed at baseline and after 3 weeks of treatment with MIA.

[0134] These studies demonstrate that p75NTR-Fc is analgesic in the MIA rat model of OA. The analgesic effect of p75NTR-Fc was greater than that observed with an anti-NGF antibody (PG-007, a biosimilar of the Pfizer anti-NGF antibody tanezumab) at a similar dose (3 mg / kg subcutaneously).

Claims

1. A p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein comprising: (a) a portion of p75NTR(NBP) having an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 1 and 2; and (b) an immunoglobulin Fc portion having an amino acid sequence having at least 90% identity to a sequence selected from any of SEQ ID NOs: 3-6; Including, The p75NTR(NBP) portion and the Fc portion are linked via a linker, the linker having the formula G ｘ wherein x is 1, 2, 3, 4, 5, or 6; p75NTR neurotrophin-binding protein (NBP)-Fc fusion protein.

2. 2. The p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein of claim 1, The p75NTR(NBP) portion is SEQ ID NO:

2. p75NTR neurotrophin-binding protein (NBP)-Fc fusion protein.

3. 3. The p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein of claim 1 or 2, the immunoglobulin Fc portion is SEQ ID NO: 3; p75NTR neurotrophin-binding protein (NBP)-Fc fusion protein.

4. 3. The p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein of claim 1 or 2, the immunoglobulin Fc portion is SEQ ID NO: 4; p75NTR neurotrophin-binding protein (NBP)-Fc fusion protein.

5. 3. The p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein of claim 1 or 2, the immunoglobulin Fc portion is SEQ ID NO: 5; p75NTR neurotrophin-binding protein (NBP)-Fc fusion protein.

6. 3. The p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein of claim 1 or 2, the immunoglobulin Fc portion is SEQ ID NO: 6; p75NTR neurotrophin-binding protein (NBP)-Fc fusion protein.

7. A p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein according to any one of claims 1 to 6, The linker moiety is GGG. p75NTR neurotrophin-binding protein (NBP)-Fc fusion protein.

8. A p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein according to any one of claims 1 to 6, For use in the treatment of pain, p75NTR neurotrophin-binding protein (NBP)-Fc fusion protein.

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