Fusion protein

Abstract

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

Description

[Technical Field]

[0001] This invention relates to the p75NTR neurotrophic factor-binding protein (NBP)-Fc fusion protein. We describe the affinity and in vivo kinetics of such molecules, as well as their efficacy in pain treatment in animal models. The p75NTR(NBP)-Fc fusion protein is used in the treatment of pain and other neurotrophic factor-related 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 factors 4 / 5 (NT-4 / 5), act via 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 has been reported to bind to and activate all four neurotrophic factors, and to function independently of the other receptors. However, the Trk receptors are activated more selectively; namely, 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 co-expressed, they form a complex, which alters the signaling of both receptors (Huang and Reichardt, 2003, Annu Rev Biochem. 72:609-42). In fact, it has been suggested that p75NTR promotes the selectivity of each neurotrophic factor for each Trk receptor.

[0003] p75NTR belongs to the tumor necrosis factor receptor superfamily (TNFR-SF) and was the first member of this superfamily to be fully characterized. 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 are no sequence motifs 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-bonded p75NTR dimer formed via cysteine ​​residues within the transmembrane domain. This disulfide bond is required for effective neurotrophic factor-dependent signaling by p75NTR and plays a crucial role in the formation of intracellular and extracellular domains (Vilar et al., 2009 Neuron 62:72-83). Neurotrophic factors exist physiologically as non-covalently associated dimers (Bothwell and Shooter, 1977 J Biol Chem. 252(23):8532-6.) and have a distribution half-life of approximately 5 minutes (Tria et al., 1994 Exp Neurol. 127(2):178-83). Neurotrophic factor-dependent p75NTR activation involves the association of neurotrophic factor dimers with CRD2-4 of the two extracellular domains of the p75NTR dimer (He and Garcia, 2004 Science 304:870-875). Recent studies support a model in which neurotrophic factor binding causes the two extracellular domains of the p75NTR dimer to move closer to each other, thereby allowing the intracellular domain to extend outward around the disulfide bond in a snail-tongue-like motion, enabling it to associate with the signaling adapter 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 the transmembrane domain, as found in p75NTR, have not been previously reported in other TNFR-SF family members or any other membrane proteins.

[0005] p75NTR undergoes sequential protein cleavage by α-secretase and γ-secretase activity, as well as 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 proteins (Jung et al., 2003 J Biol Chem 278:42161-42169; Kanning et al., 2003 J Neurosci 23:5425-5436). This release of the p75NTR ICD into the cytoplasm promotes signaling by associated NRIF (Kenchappa et al., 2006 Neuron 50:219-232). The role of the extracellular domain of p75NTR following protein cleavage by α-secretase and γ-secretase activity and MMPs is not yet fully understood.

[0006] The literature has reported that NGF and other neurotrophic factors (BDNF, NT-3, and NT-4 / 5) play important roles in pain associated with pathological conditions such as osteoarthritis, pancreatitis, rheumatoid arthritis, psoriasis, pruritus, and 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 any neurotrophic factor: NGF or BDNF, NT-3, and NT-4 / 5 have been shown to significantly reduce pain. Furthermore, antibodies directed against 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 against pain-related endpoints following treatment with an anti-p75NTR antibody in a pain model (mechanical allodynia after sciatic nerve contusion). This study concluded that treatment with a p75NTR inhibitory antibody reduces CGRP and p75NTR expression, resulting in a significant reduction in pain. [Overview of the Initiative]

[0007] According to a first aspect of the present invention, a p75NTR neurotrophic factor-binding protein (NBP)-Fc fusion protein is provided, which includes: (a) the p75NTR(NBP) portion selected from any of sequence numbers 1 to 19; and (b) Immunoglobulin Fc moiety selected from any of sequence numbers 20-24.

[0008] Here, the p75NTR(NBP) portion and the Fc portion are connected via a linker, and the linker is of type G x It contains the peptide, where x is 1, 2, 3, 4, 5, or 6.

[0009] In a preferred embodiment, the linker does not include the sequence GGGGS and does not consist of.

[0010] In a further embodiment, the linker is GGG.

[0011] In a particularly preferred embodiment of the p75NTR(NBP)-Fc fusion protein according to the present invention, the Fc region is an Fc suitable for human use.

[0012] In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 1. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 2. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 3. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 4. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 5. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 6. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 7. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 8. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 9. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 10. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 11. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 12. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 14. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 15. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 16. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 17. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 18. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 19.

[0013] In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 20. In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 21. In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 22. In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 23. In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 24.

[0014] In a preferred embodiment, the p75NTR(NBP)-Fc fusion protein according to the present invention has a binding affinity (K) to any of NGF, BDNF, NT3, or NT4 / 5, measured by surface plasmon resonance at 20°C, between approximately 0.01 nM and approximately 50 nM. d ) and then join them.

[0015] In a second aspect of the present invention, a p75NTR(NBP)-Fc fusion protein described according to any of the other aspects of the present invention is provided for use in the treatment of pain.

[0016] In a third aspect of the present invention, a nucleic acid molecule encoding the p75NTR(NBP)-Fc fusion protein described in the first or second aspect of the present invention is provided, which may further comprise encoding a signal sequence.

[0017] In a fourth aspect of the present invention, a replicable expression vector for transfecting cells (optionally mammalian cells) is provided, said vector comprising the nucleic acid molecule described in the third aspect of the present invention.

[0018] Preferably, said replicable expression vector is a viral vector.

[0019] In a fifth aspect of the present invention, a host cell harbouring the nucleic acid molecule of the third aspect of the present invention is provided.

[0020] In a sixth aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, or the vector described in the fourth aspect of the present invention, is for use in the treatment of pain.

[0021] 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 toothache; and back pain. Treatment of pain includes, but is not limited to: prevention, amelioration, control, reduction in incidence, or delay of onset or progression of pain.

[0022] In a seventh aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, or the vector described in the fourth aspect of the present invention, is for use in the treatment of osteoarthritis.

[0023] In an eighth aspect, there is provided the p75NTR(NBP)-Fc fusion protein described in the first or second aspect, or the nucleic acid or vector described in the third or fourth aspect, wherein the p75NTR(NBP)-Fc fusion protein, or the nucleic acid molecule or vector, is for use in combination with, separately, sequentially, or simultaneously with a second pharmacologically active compound.

[0024] In a ninth aspect, the present invention provides a pharmaceutical composition comprising the p75NTR(NBP)-Fc fusion protein described in any aspect of the present invention, or a nucleic acid molecule or vector described in any aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0025] Preferably, the pharmaceutical composition is for use in any one or more of the prevention, improvement, control, reduction in the incidence, or delay in the onset or progression of osteoarthritis in mammals.

[0026] In one embodiment, the mammal is a human.

[0027] In another embodiment, the mammal is an animal, preferably a dog, cat, elephant, or horse. In a particularly preferred embodiment, the animal is a dog.

[0028] In a further aspect of the present invention, there is provided a kit comprising: (a) the p75NTR(NBP)-Fc fusion protein described in any aspect of the present invention, or a nucleic acid molecule or vector described in any aspect of the present invention, or the pharmaceutical composition described in 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 the prevention or treatment of pain, or the improvement, control, reduction in the incidence, or delay in the onset or progression of pain.

[0029] In yet another embodiment of the present invention, a method for treating and / or preventing pain in an animal is provided, the method comprising the step of administering to the individual a therapeutically effective amount of the p75NTR(NBP)-Fc fusion protein described in any aspect of the present invention, or a nucleic acid molecule or vector described in any aspect of the present invention, which may further comprise a pharmaceutically acceptable carrier or a pharmaceutical composition described in eighth aspect of the present invention. [Brief explanation of the drawing]

[0030] [Figure 1] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 1). [Figure 2] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 2). [Figure 3] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 3). [Figure 4] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 4). [Figure 5] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 5). [Figure 6] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 6). [Figure 7] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 7). [Figure 8] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 8). [Figure 9] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 9). [Figure 10] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 10). [Figure 11]The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 11). [Figure 12] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 12). [Figure 13] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 13). [Figure 14] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 14). [Figure 15] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 15). [Figure 16] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 16). [Figure 17] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 17). [Figure 18] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 18). [Figure 19] The amino acid sequence of the p75NTR(NBP) portion of the fusion protein according to the present invention (SEQ ID NO: 19). [Figure 20] The amino acid sequence of the appropriate human Fc portion of the fusion protein according to the present invention (SEQ ID NO: 20). [Figure 21] The amino acid sequence of the feline IgG-Fc portion of the fusion protein according to the present invention (SEQ ID NO: 21). [Figure 22] The amino acid sequence of the horse IgG-Fc portion of the fusion protein according to the present invention (SEQ ID NO: 22). [Figure 23] The amino acid sequence of the feline IgG-Fc portion of the fusion protein according to the present invention (SEQ ID NO: 23). [Figure 24] The amino acid sequence of the feline IgG-Fc portion of the fusion protein according to the present invention (SEQ ID NO: 24). [Modes for carrying out the invention]

[0031] According to a first aspect of the present invention, a p75NTR neurotrophic factor-binding protein (NBP)-Fc fusion protein is provided, which includes: (a) the p75NTR(NBP) portion selected from any of sequence numbers 1 to 19; and (b) Immunoglobulin Fc moiety selected from any of sequence numbers 20-24.

[0032] Here, the p75NTR(NBP) portion and the Fc portion are connected via a linker, and the linker is of type G x It contains the peptide, where x is 1, 2, 3, 4, 5, or 6.

[0033] G stands for glycine.

[0034] In a preferred embodiment, the linker does not include the sequence GGGGS and does not consist of.

[0035] In a further embodiment, the linker is GGG.

[0036] In a particularly preferred embodiment of the p75NTR(NBP)-Fc fusion protein according to the present invention, the Fc region is an Fc suitable for human use.

[0037] In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 1. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 2. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 3. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 4. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 5. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 6. In yet another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention includes or comprises the amino acid sequence shown in SEQ ID NO: 7. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 8. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 9. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 10. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 11. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 12. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 14. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 15. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 16. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 17. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 18. In another preferred embodiment, the p75NTR(NBP) portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 19.

[0038] In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 20. In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 21. In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 22. In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 23. In another preferred embodiment, the Fc portion of the fusion protein of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 24.

[0039] Preferably, the p75NTR neurotrophic factor-binding protein, i.e., p75NTR(NBP), is PEGylated, and more preferably glycosylated.

[0040] In addition, those skilled in the art will understand that the therapeutic potential of the molecules of the present invention can be enhanced by introducing specified mutations, such as YTE(M252Y / S254T / T256E) and LS(M428L / N434S), into the crystallizable fragment (Fc) domain. Such techniques are well known to those skilled in the art. The effect of introducing such mutations typically results in molecules with increased half-life and extended duration of action. The effect of introducing mutations is not limited to increases in half-life and duration of action.

[0041] The p75NTR(NBP)-Fc fusion protein of the present invention preferably has a binding affinity (K) between approximately 0.01 nM and approximately 50 nM to one or more of NGF, BDNF, NT3, or NT4 / 5. d ) binds. In some preferred embodiments, binding affinity (K d The binding affinity (K) is between approximately 0.01 nM and any of approximately 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, and is measured in an in vitro binding assay to NGF, BDNF, NT3, or NT4 / 5 as described herein, preferably by surface plasmon resonance at 20°C. In some further preferred embodiments, the binding affinity (K) is measured in an in vitro binding assay to NGF, BDNF, NT3, or NT4 / 5 as described herein, preferably by surface plasmon resonance at 20°C. d The binding affinity (K) is less than or equal to approximately 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, and is measured in an in vitro binding assay between the p75NTR(NBP)-Fc fusion protein and neurotrophic factors as described herein, preferably by surface plasmon resonance at 20°C. In a further, more preferred embodiment, the binding affinity (K) dThe ) is approximately 0.3 nM or approximately 1 nM and is measured in an in vitro binding assay of p75NTR(NBP)-Fc fusion protein with neurotrophic factors, as described herein, preferably by surface plasmon resonance at 20°C.

[0042] Preferably, the p75NTR(NBP)-Fc fusion protein of the present invention is intended for use in the treatment of pain. While we do not wish to be bound by any particular theory, the inventors believe that the p75NTR(NBP)-Fc fusion protein achieves efficacy in pain treatment by acting on (defining as modulating, or up-or down-regulating, the functional activity of the aforementioned neurotrophic factors, namely NGF, BDNF, NT3, or NT4 / 5, for example, the functional activity of the aforementioned neurotrophic factors resulting from their interaction with their respective receptors.

[0043] Preferably, the p75NTR(NBP)-Fc fusion protein acts on the functional activity of BDNF, which is evaluated by a functional assay for one of the following: neuronal and synaptic growth and differentiation, survival and differentiation in neuronal cell culture, Trk signaling, or stimulation of axonal elongation in vitro or in vivo.

[0044] Preferably, the p75NTR(NBP)-Fc fusion protein acts on the functional activity of NGF, which is assessed by measuring the binding of NGF to TrkA and its activation, and is demonstrated in a classical neuronal survival assay (e.g., Cowan et al., Annu. Rev. Neurosci. 2001;24:551-600).

[0045] Preferably, the p75NTR(NBP)-Fc fusion protein acts on the functional activity of NT3, which is assessed by measuring its binding to and activation of the endogenous Trk receptor activity of NT3, and is demonstrated in Trk receptor phosphorylation, mitogen-activated protein kinase phosphorylation reporter assays, or cell survival and neurite outgrowth assays.

[0046] Preferably, the p75NTR(NBP)-Fc fusion protein acts on the functional activity of NT4 / 5, which can be evaluated 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 a Matrigel angiogenesis assay for vascular endothelial growth factor (VEGF) / basic fibroblast growth factor-induced angiogenesis in vivo.

[0047] Preferably, the p75NTR(NBP)-Fc fusion protein binds to one or more contact residues among the neurotrophic factors NGF, NT3, BDNF, and NT4 / 5 (as shown in He and Garcia (2001) Science, 301, pages 870-805).

[0048] Preferably, the p75NTR(NBP)-Fc fusion protein is soluble, preferably soluble in aqueous solutions, and preferably soluble in biological fluids such as serum, plasma, and blood.

[0049] As used herein, the terms “Fc,” “immunoglobulin Fc,” or “Ig Fc” are understood to mean the carboxyl terminal portion or part thereof of the constant region of an immunoglobulin chain (preferably the constant region of an immunoglobulin heavy chain). Preferably, immunoglobulin Fc comprises: 1) CH1 domain, CH2 domain, and CH3 domain, optionally having an immunoglobulin hinge region; 2) CH1 domain and CH2 domain, optionally having an immunoglobulin hinge region; 3) CH1 domain and CH3 domain, optionally having an immunoglobulin hinge region; 4) CH2 domain and CH3 domain, optionally having an immunoglobulin hinge region; or 5) a combination of two or more domains selected from, but not limited to, CH1, CH2, and CH3, optionally combined with an immunoglobulin hinge region.

[0050] According to the present invention, the p75NTR(NBP)-Fc fusion protein preferably exhibits advantageous biological properties such as improved solubility of p75NTR(NBP), and / or improved stability of p75NTR(NBP), and / or improved serum half-life of p75NTR(NBP). Improved solubility is desirable to maximize the bioavailability of p75NTR(NBP) at administration, enabling accurate dosage determination and implementation. Improved solubility is advantageous in overcoming the problem of undesirable aggregates that can cause pain and lead to potential inflammation during in vivo delivery. Improved serum half-life has the advantage of facilitating a reduction in the dose level or frequency of administration required at therapeutic use to achieve equivalent or maintained therapeutic effects with delivered p75NTR(NBP). The extended half-life and higher stability in blood or serum have the advantage of enabling less frequent and / or lower dose levels of administration 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 circulation. The resulting less frequent or lower doses are advantageous in minimizing any potential toxic effects 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 of the molecule being well retained in the bloodstream when administered intravenously, reducing the risk of penetration to undesirable sites (e.g., the central nervous system), and making the molecule suitable for retention or concentration within targeted tissues.

[0051] Preferably, the p75NTR(NBP)-Fc fusion protein exhibits improved solubility and / or improved stability and / or improved serum half-life compared to p75NTR(NBP) alone. Preferably, the improved solubility is solubility in aqueous solutions such as water (preferably containing excipients such as buffers and / or salts, preferably at a physiological pH, preferably between pH 5 and pH 8, and preferably about pH 7), or solubility in biological fluids such as serum or blood. Preferably, the improved stability is the stability of the activity or structural integrity of the p75NTR(NBP) protein over a period of time, during storage, or after freeze-thaw cycles, due to the effects of denaturation, oxidation, fragmentation, or aggregation. Structural stability can be determined by standard measurements of denaturation, oxidation, aggregation, or flocculation, activity stability can be measured by binding or functional assays disclosed herein, and methods for measuring the serum half-life of the protein are known.

[0052] Preferably, the p75NTR(NBP)-Fc fusion protein can be expressed at high levels from diverse mammalian host cells to provide a single type, and can be efficiently purified by affinity chromatography (e.g., by binding to Staphylococcus aureus protein A). Preferably, the p75NTR(NBP)-Fc fusion protein can be dimerized, and preferably the dimer has increased affinity for neurotrophic factors NGF, BDNF, NT3, or NT4 / 5 compared to p75NTR(NBP) alone. Stronger binding has the advantage of higher potency and higher therapeutic effect, which are determined by the effect of p75NTR(NBP) and determined, for example, by neurotrophic factor function assays disclosed herein. Higher potency has the advantage of reducing potential toxicity or side effects in vivo, as the p75NTR(NBP)-Fc fusion protein can achieve equivalent therapeutic effect with lower doses.

[0053] Preferably, the p75NTR(NBP)-Fc fusion protein of the present invention has an in vivo half-life of one or more 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 preferably the p75NTR(NBP)-Fc fusion protein of the present invention has an in vivo half-life of about 24 hours or more.

[0054] More preferably, the p75NTR(NBP)-Fc fusion protein of the present invention has an in vitro half-life of one or more 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, more preferably the p75NTR(NBP)-Fc fusion protein of the present invention has an in vitro half-life of about 6 days or more. Preferably, stability is measured in a buffered aqueous solution near the physiological pH, preferably at 20°C or 37°C.

[0055] According to the preferred embodiments described above, the in vivo half-life is preferably the rat half-life or the human half-life, more preferably the human half-life. Preferably, the half-life is determined by measuring the serum level of the p75NTR(NBP)-Fc fusion protein of the present invention after in vivo administration (e.g., by intravenous or subcutaneous injection).

[0056] The p75NTR(NBP) portion and the immunoglobulin Fc portion of the p75NTR(NBP)-Fc fusion protein are linked by a linker. The linker preferably comprises or consists of one or more amino acids, or comprises or consists of a polypeptide sequence of amino acids, preferably about 1 to about 25 amino acids, preferably one of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, more preferably one of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids, and most preferably 13 amino acids.

[0057] Preferably, the linker is an α-helix, a β-strand, 3 10 The linker region comprises or consists of an amino acid polypeptide sequence that lacks any stable secondary structures, such as helices, π-helices, polyproline helices, or α-sheets. Preferably, the linker region comprises or consists of an amino acid polypeptide sequence that defines a flexible, dynamic, or unstructured polypeptide, such as a flexible loop, random coil, or flexible turn. Such unstructured polypeptides are often found in regions that link secondary structures in large protein molecules.

[0058] Preferably, the linker is an amino acid polypeptide sequence containing more than 50% or about 50% glycine and / or alanine and / or serine in the p75NTR(NBP), more preferably more than or near 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% glycine and / or alanine and / or serine in the p75NTR(NBP). Preferably, the linker region contains an amino acid polypeptide sequence containing both glycine and serine, or consists of, preferably having a higher proportion of glycine than serine, and preferably the linker region contains a flexible linker, or consists of.

[0059] Although not wishing to be bound by any particular theory, the inventors believe that flexible linkers overcome or prevent steric hindrance that may interfere with the aforementioned neurotrophic factor binding ability or biological activity of the p75NTR(NBP)-Fc fusion protein when compared to p75NTR(NBP) alone. Thus, the linker region preferably allows flexibility between the p75NTR(NBP) portion and the immunoglobulin Fc portion, and allows retention or improvement of the aforementioned biological activity of the p75NTR(NBP)-Fc fusion protein compared to free or native p75NTR(NBP) alone, which is determined by binding to a neurotrophic factor using a binding assay as described herein.

[0060] More preferably, the linker is immunologically inert, thus not causing complement-mediated lysis, not stimulating antibody-dependent cell-mediated cytotoxicity (ADCC), and not activating microglia or T cells. Preferably, the linker region has reduced one or more of these activities.

[0061] Even more preferably, the linker is known or predicted to be a flexible or dynamic or unstructured polypeptide from structural analysis or prediction, or comprises or consists of a polypeptide lacking a stable secondary structure.

[0062] The linker comprises a peptide of the formula G x where x is 1, 2, 3, 4, 5, or 6.

[0063] In preferred embodiments, the linker does not contain and does not consist of the sequence GGGGS.

[0064] In a further embodiment, the linker is GGG.

[0065] The p75NTR(NBP)-Fc fusion protein of the present invention may also contain protein cleavage sites, which may optionally be inserted between the p75NTR(NBP) portion and the immunoglobulin Fc portion. The protein cleavage sites may be located within the linker or at the junction between the linker and either the p75NTR(NBP) portion or / or the immunoglobulin Fc portion. The p75NTR(NBP) may optionally be cleaved from the immunoglobulin Fc portion before therapeutic formulation and / or administration.

[0066] Preferably, the linker and / or immunoglobulin Fc portion does not impair, or significantly impair, the p75NTR(NBP) portion: (a) Effects on the functional activity of neurotrophic factors NGF, BDNF, NT3, or NT4 / 5 (defined as modulating, or up-regulating, the functional activity of neurotrophic factors), (b) Binding affinity to any of NGF, BDNF, NT3, or NT4 / 5 having a binding affinity between approximately 0.1 nM and approximately 50 nM, (c) Ability to bind to each of the neurotrophic factors NGF, NT3, BDNF, and NT4 / 5 (preferably human NGF, NT3, BDNF, and NT4 / 5).

[0067] According to another aspect of the present invention, a nucleic acid molecule encoding the p75NTR(NBP)-Fc fusion protein described in the first or second aspect is provided. Preferably, the nucleic acid molecule is for use in the treatment of pain.

[0068] According to a preferred embodiment of the present invention, the nucleic acid molecule may further include a region encoding a signal sequence (preferably a p75NTR signal sequence, such as a DNA or RNA sequence).

[0069] According to another aspect of the present invention, a replicable expression vector for transfecting cells is provided, the vector comprising a nucleic acid molecule according to a third aspect, preferably the vector being a viral vector. Preferably the vector is for use in the treatment of pain.

[0070] Furthermore, according to the above aspects of the present invention, a method is provided for expressing a nucleic acid molecule or vector of the present invention to produce or secrete a p75NTR(NBP)-Fc fusion protein. Preferably, 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. Preferably, the nucleic acid molecule or vector is introduced into the cell in vitro or in vivo. Preferably, the expressed p75NTR(NBP)-Fc fusion protein is expressed in vitro and optionally further isolated and purified, or preferably, the expressed p75NTR(NBP)-Fc fusion protein is expressed in vivo, preferably, in vivo expression constitutes gene therapy. Preferably, the vector is a replicable expression vector and may be for transfecting mammalian cells, preferably, the vector is a viral vector.

[0071] According to another aspect of the present invention, a host cell is provided that holds a nucleic acid molecule or vector of any third or fourth aspect, preferably a mammalian cell.

[0072] According to another aspect of the present invention, a p75NTR(NBP)-Fc fusion protein, or a nucleic acid or vector, for use in the treatment of pain is provided. Pain may include, but is not limited to, the following: (a) Acute pain and / or spontaneous pain, (b) Chronic pain and / or progressive pain, (c) Inflammatory pain (including pain caused by arthritis, pain resulting from osteoarthritis or rheumatoid arthritis, inflammatory bowel disease, psoriasis, and eczema), (d) Nociceptive pain, (e) Neuropathic pain (including pain associated with diabetic neuropathy or postherpetic neuralgia), (f) Hyperalgesia, (g) Allodynia, (h) Central pain, central post-stroke pain, pain resulting from multiple sclerosis, pain resulting from spinal cord injury, or pain resulting from Parkinson's disease or epilepsy, (i) cancer pain, (j) Postoperative pain; (k) Visceral pain (including gastrointestinal and non-gastrointestinal visceral pain, pain resulting from gastrointestinal (GI) disorders, pain resulting from functional bowel disorders (FBD), pain resulting from inflammatory bowel disease (IBD), pain resulting from dysmenorrhea, pelvic pain, cystitis, interstitial cystitis, or pancreatitis), (l) Musculoskeletal pain, myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatic) arthritis, non-rheumatic arthritis, dystrophinopathy, glycogenolysis, polymyositis, suppurative myositis, (m) Cardiac or vascular pain, angina pectoris, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, edematous sclerosis (scleredoma), pain resulting from edematous sclerosis, or skeletal muscle ischemia. (n) Headache (including migraine, migraine with aura, migraine without aura, cluster headache, and tension headache), (o) Orofacial pain (including toothache, temporomandibular joint myofascial pain, or tinnitus), (p) Back pain, bursitis, menstrual cramps, migraines, referred pain, trigeminal neuralgia, hypersensitization, spinal cord injury and / or pain resulting from degeneration or stroke.

[0073] Treatment of pain includes, but is not limited to, prevention, improvement, control, reduction of incidence, or delay of onset or progression of pain.

[0074] In other aspects of the present invention, 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, is provided, where the p75NTR(NBP)-Fc fusion protein, or the nucleic acid molecule or vector, is intended to be used separately, sequentially, or simultaneously in combination with a second pharmacologically active compound. Preferably, the second pharmacologically active compound in the combination may include, but is not limited to, the following: Opioid analgesics, such as morphine, heroin, hydromorphone, oxymorphone, levorphanol, revallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyfene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbufine, or pentazocine; Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, diclofenac, diflusal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimeslide, nitroflurbiprofen, orsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin, or zomepirac; Barbiturate sedatives, such as amobarbital, aprobarbital, butabarbital, butarbital, mefobarbital, metalbital, methhexital, pentobarbital, phenobartital, secobarbital, tarbutal, theamylal, or thiopental; • Sedative benzodiazepines, such as chlordiazepoxide, chlorazepic acid, diazepam, flurazepam, lorazepam, oxazepam, temazepam, or triazolam; • Sedative H1 antagonists, such as diphenhydramine, pyriramine, promethazine, chlorpheniramine, or chlorcyclidine; • Sedatives, such as glutethimid, meprobamate, methacaron, or dichlorphenazone; • 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 dextorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid, budipine, EN-3231 (MorphiDex®, a combination preparation of morphine and dextromethorphan), topiramate, neramexane, or perzinhotel, which include 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 drugs, such as doxazosin, tamsulosin, clonidine, guanfacine, dexmedetomidine, modafinil, or 4-amino-6,7-dimethoxy-2-(5-methanesulfonamide-1,2,3,4-tetrahydroisoquinoline-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, especially NK-3, NK-2, or NK-1 antagonists, e.g., (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazosino[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-triazole-3-one (MK-869), aprepitant, ranepitant, dapitant, or 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine (2S,3S); • Muscarinic antagonists, such as oxybutynin, tolterodine, propiverine, tropsium chloride, dalifenacin, solifenacin, temiverine, and ipratropium; • COX-2 selective inhibitors, such as celecoxib, rofecoxib, parecoxib, valdecoxib, delacoxib, etoricoxib, or lumiracoxib; • Coal tar-based analgesics, especially paracetamol; • Neuroleptics, such as droperidol, chlorpromazine, haloperidol, perphenazine, thioridazine, mesolidazine, trifluoperazine, fluphenazine, clozapine, olanzapine, risperidone, ziprasidone, quetiapine, certindol, aripiprazole, sonepiprazole, blonanserin, iloperidone, perospirone, lacloprid, zotepine, bifepurnox, asenapine, lurasidone, amisulpride, balaperidone, palindore, eprivanserin, osanetant, rimonabant, meclinertan, Miraxion®, or salizotan; • Vanilloid receptor agonists (e.g., resiniferatoxin) or antagonists (e.g., capsazepine); • Beta-adrenergic drugs, such as propranolol; • Local anesthetics, such as mexiletine; • Corticosteroids, such as 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-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (MDL-100907); • Cholinergic (nicotinic) analgesics, e.g., ispronicline (TC-1734), (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403), (R)-5-(2-azetidinyl methoxy)-2-chloropyridine (ABT-594), or nicotine; • Tramador (registered trademark); • PDEV inhibitors, e.g., 5-[2-ethoxy-5-(4-methyl-1-piperazinyl-sulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidine-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 1,4-dione (IC-351 or tadalafil), 2-[2-ethoxy-5-(4-ethyl-piperazine-1-yl-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazine-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]pyrimidine-7-one, 5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidine-7-one, 5-[2-ethoxy-5-(4-ethylpiperazine-1-ylsulfonyl)pyridine-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidine-7 -ON, 4-[(3-chloro-4-methoxybenzyl)amino]-2-[(2S)-2-(hydroxymethyl)pyrrolidine-1-yl]-N-(pyrimidine-2-ylmethyl)pyrimidine-5-carboxamide, 3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-5-yl)-N-[2-(1-methylpyrrolidine-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, cyanodothiopine, ritoxetine, dapoxetine, nefazodone, sericlamine, and trazodone; • Norepinephrine reuptake inhibitors, such as maprotiline, lofepramine, mirtazapine, oxaprotiline, fezolamine, tomoxetine, mianserin, bupropion, bupropion metabolite hydroxybupropion, nomifensin, and biloxazine (Vivalan®), particularly selective norepinephrine reuptake inhibitors, such as reboxetine, specifically (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, e.g., 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-pyridinecarbonitride; 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-pyridinecarbonitride, 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, e.g., N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridine-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzenesulfonamide, or 4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridine-3-yl]carbonyl}amino)ethyl]benzoic acid; • Leukotriene B4 antagonists; for example, 1-(3-biphenyl-4-ylmethyl-4-hydroxychroman-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, e.g., zileuton, 6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl])phenoxymethyl]-1-methyl-2-quinolone (ZD-2138), or 2,3,5-trimethyl-6-(3-pyridylmethyl),1,4-benzoquinone (CV-6504); • Sodium channel blockers, e.g., lidocaine; or • 5-HT3 antagonists, e.g., ondansetron; and their pharmaceutically acceptable salts and solvates.

[0075] A further aspect of the present invention provides a method for treating, preventing, improving, controlling, reducing the incidence of, or delaying the onset or progression of pain or any of the aforementioned pains in an individual, the method comprising administering to the individual an effective amount of the p75NTR(NBP)-Fc fusion protein described in the first or second aspect or a preferred embodiment thereof, or a nucleic acid molecule or vector described in the third and fourth aspects.

[0076] In another preferred embodiment, the p75NTR(NBP)-Fc fusion protein of the present invention is suitable for the treatment of osteoarthritis. Specifically, it is suitable for stopping, delaying, and reversing the progression of the disease. In a specific embodiment, the protein of the present invention provides a cure for osteoarthritis.

[0077] In one embodiment of the present invention, it is suitable for the treatment of osteoarthritis in humans.

[0078] In one embodiment of the present invention, it is suitable for the treatment of osteoarthritis in the field of veterinary medicine. Preferably, the individual is a mammal, such as a companion animal like a horse, cat, or dog, or a domestic animal like a sheep, cow, or pig. Most preferably, the animal is a dog.

[0079] According to an eighth aspect of the present invention, a pharmaceutical composition is provided for treating, preventing, improving, controlling, reducing the incidence of, or delaying the onset or progression of pain or any of the aforementioned pains, the pharmaceutical composition comprising 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, and a pharmaceutically acceptable carrier and / or excipient.

[0080] Preferably, the p75NTR(NBP)-Fc fusion protein described in the first or second embodiment or their preferred embodiments, or the nucleic acid molecule or vector described in the third and fourth embodiments, or the pharmaceutically acceptable agent of the eighth embodiment, is prepared for or suitable for: oral, sublingual, buccal, topical, rectal, inhalation, percutaneous, subcutaneous, intravenous, intra-arterial, intramuscular, intracardiac, intramedullary, intradermal, intraperitoneal, transmucosal, vaginal, intravitreous, intra-articular, peri-articular, topical, or cutaneous administration.

[0081] Preferably, the p75NTR(NBP)-Fc fusion protein described in the first or second embodiment or its preferred embodiments, or the nucleic acid molecule or vector described in the third and fourth embodiments, or the pharmaceutical composition of the eighth embodiment, are prepared or suitable for administration before and / or during and / or after the onset of pain, or for such use.

[0082] Preferably, the p75NTR(NBP)-Fc described in the first or second embodiment or its preferred embodiments, or the nucleic acid molecule or vector described in the third and fourth embodiments, or the pharmaceutical composition of the eighth embodiment, is intended for or prepared for administration between 1 and 7 times per week, more preferably between 1 and 4 times per month, more preferably between 1 and 6 times per 6-month period, and more preferably between 1 and 12 times per year. Preferably, the pharmaceutical is administered peripherally or prepared for peripheral administration at administration intervals including, but not limited to, the following: once daily, once every 2, 3, 4, 5, or 6 days, once per week, once every 2 weeks, once every 3 weeks, once per month, 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 per year.

[0083] More preferably, the p75NTR(NBP)-Fc fusion protein described in the first or second embodiment or its preferred embodiments, or the nucleic acid molecule or vector described in the third and fourth embodiments, or the pharmaceutical composition of the eighth embodiment, is administered peripherally or prepared for peripheral administration via one or more of the following routes: oral, sublingual, buccal, topical, rectal, inhalation, transdermal, subcutaneous, intravenous, intra-arterial, or intramuscular, intracardiac, intramedullary, intradermal, intraperitoneal, transmucosal, vaginal, intravitreous, supercutaneous, intra-articular, peri-articular, or topical.

[0084] Preferably, the p75NTR(NBP)-Fc fusion protein described in the first or second aspect or their preferred embodiments, or the nucleic acid molecule or vector described in the third and fourth aspects, or the pharmaceutical composition of the eighth aspect, is intended for administration at a concentration of about 0.05 to about 200 mg / ml, or is prepared for such administration; preferably any of 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 with an error of about ±10%, most preferably about 3 mg / ml for veterinary use and about 0.1 for humans.

[0085] Preferably, the p75NTR(NBP)-Fc fusion protein described in the first or second aspect or their preferred embodiments, or the nucleic acid molecule or vector described in the third and fourth aspects, or the pharmaceutical composition of the eighth aspect, is intended for administration at a concentration of about 0.1 to about 200 mg / kg (body weight), or is prepared for that purpose; preferably any of ± about 10% error between 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), most preferably about 10 mg / kg for veterinary use and 0.3 for humans.

[0086] According to a ninth aspect of the present invention, a kit is provided which includes the following: (a) a p75NTR(NBP)-Fc fusion protein as described in the first or second aspect or a preferred embodiment thereof, or a nucleic acid molecule or vector as described in the third and fourth aspects, or a pharmaceutical composition of 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 individual for the prevention or treatment of pain, or for the improvement, control, reduction of incidence of pain, or delay of onset or progression of pain.

[0087] 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 an individual suitable for treatment based on identifying whether the individual has pain or symptoms of pain, or is at risk of developing such a condition. The instructions for administering the pharmaceutical composition may include information regarding the dosage, administration schedule, and route of administration with respect to the intended treatment.

[0088] According to yet another aspect of the present invention, 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, or a pharmaceutical composition of the eighth aspect is provided, which are used for the prevention or treatment of, or for the improvement, control, reduction of incidence, or delay of onset or progression thereof of a condition or symptoms of a condition related to one or more of the neurotrophic factors NGF, BDNF, NT-3, NT-4 / 5.

[0089] NGF (nerve growth factor) binds to at least two receptor classes: p75NTR and TrkA (transmembrane tyrosine kinase), and is involved in axonal growth, branching, and elongation. Conditions and symptoms associated with NGF 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 in multiple sclerosis. Decreased plasma levels of NGF (and BDNF as well) are associated with acute coronary syndrome and metabolic syndrome. NGF is also associated with various mental disorders, such as dementia, depression, schizophrenia, autism, Rett syndrome, anorexia nervosa, and bulimia nervosa, and is also thought to be involved in the development of Alzheimer's disease and neurodegenerative diseases. NGF has also been shown to accelerate wound healing and there is evidence suggesting its potential usefulness in treating skin and corneal ulcers. Furthermore, in rats, it has been shown to reduce neurodegeneration and promote peripheral nerve regeneration.

[0090] - BDNF (brain-derived neurotrophic factor) is a neurotrophic factor that supports the survival and growth of neurons during the development of the nervous system [protein sequences NP_001137277.1, NP_001041604]. BDNF binds to cell surface receptors TrkB and p75NTR and also modulates the activity of α7 nicotinic receptors. Conditions and symptoms associated with BDNF are known. BDNF has been shown to play a crucial role in the transmission of physiological and pathological pain, specifically in models of acute pain, inflammatory pain, and neuropathic pain, where BDNF synthesis is found to be significantly increased; BDNF has also been shown to be upregulated in chronic pain conditions, as well as in further conditions such as eczema and psoriasis. Downregulation of BDNF is 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.

[0091] Neurotrophic factor-4 (NT-4) (also known as neurotrophic factor-5 (NT-5)) is a neurotrophic factor that primarily signals via the p75NTR and TrkB receptors, promoting the survival of peripheral sensory and sympathetic neurons. The mature peptide of this protein is identical in all mammals 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 by neurons in the paravertebral and prevertebral sympathetic ganglia, spinal cord dorsal horn and anterior horn, and 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. NT-4 / 5 deficiency is associated with susceptibility to primary open-angle glaucoma. Neurotrophic factor 4 has also been shown to contribute to the survival of breast cancer cells and is a target that inhibits tumor growth. NT-4 / 5 is known to be involved in pain signaling pathways, including nociceptive pain, and upregulation of NT-4 / 5 is also observed in chronic inflammatory conditions of the skin, such as dermatitis, eczema, and pruritic lesions of atopic dermatitis. Downregulation of NT-4 / 5 is observed in Alzheimer's disease and Huntington's disease.

[0092] Neurotrophic factor-3 (NT-3) is a structurally related neurotrophic factor to β-NGF, BDNF, and NT-4. It controls 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 the embryo. NT3-related conditions and symptoms are known. NTF3-deficient mice, created by gene targeting, exhibit severe motor impairment of the limbs. NT-3 signals through the Trk receptor and promotes the growth and survival of nerve and glial cells [protein sequences NP_001096124.1 and NP_032768]. The amino acid sequences of NT-3 are identical in humans, mice, and rats. NT-3 and its corresponding receptor, tyrosine kinase C (TrkC), are known to modulate neuropathic and nociceptive pain, and the mechanisms of nociception and proprioception. For example, NT3 expression is increased in small DRG cells of neuropathic animals. NT3 expression is also associated with neuropathy, such as diabetic polyneuropathy and HIV-associated neuropathy, as well as large-diameter fiber neuropathy including atrophy. Furthermore, it is involved in the development of hyperalgesia (a decrease in the threshold for normal noxious stimuli), allodynia (non-noxious stimuli becoming noxious), and spontaneous pain (pain in the absence of obvious stimuli), and is a known regulator of myalgia.

[0093] The present invention will be described based on the following examples, which are illustrative and not limiting. [Examples]

[0094] [Design of p75-NTR Fc fusion protein] The specific allotype of the Fc portion of the p75-NTR Fc fusion protein was IgGza, an IgG pCon vector (see above).

[0095] The design of the p75-NTR Fc fusion protein was carried out in multiple stages: • The precise 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 to 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, which must be removed from the sequence. This is because cleavage reduces the in vivo bioactivity and PK profile of the p75-Fc product.

[0096] In the p75-NTR Fc fusion protein, suitable empirical linkers for use in linking the extracellular p75-NTR domain to Fc were identified. Linker sequences containing sites potentially involved in post-translational modification (PTM) were excluded.

[0097] Multiple variants of the p75-NTR Fc fusion protein were constructed in silico using a defined p75-NTR construct and appropriate portions 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 candidate linkers were attempted.

[0098] [Structural Modeling] The structural models of the proposed p75-NTR Fc fusion protein were generated using Lonza's modeling platform. Candidate structural template fragments of the p75-NTR and Fc portions were scored, ranked, and selected from both the in-house antibody database and the Protein Databank (PDB) based on their sequence identity and qualitative crystallographic indicators of the template structure, such as resolution (in angstroms (Å)).

[0099] Sequence alignments of structural template fragments for the p75-NTR Fc fusion protein were generated. The template fragments, along with the sequence alignments, were processed using MODELLER (Sali et al. 1993 J. Mol. Biol 234, 779-815). This protocol creates conformational constraints derived from a set of aligned structural templates. An ensemble of structures satisfying these constraints is generated by conjugated gradient and simulated annealing optimization procedures. One or more model structures are selected from this ensemble based on protein structure scores and energy scores derived from conformational constraint satisfaction. The models were refined, and side chains at locations with differences between the target and template were optimized and energy-minimized using a side-chain optimization algorithm. A set of visualization and computational tools were used to assess conformational variability of the structures, as well as to evaluate domain core and local packing to select one or more preferred models.

[0100] [Design of p75-NTR Fc fusion protein] The primary requirements for a linker selected for a variant are to allow flexibility in the fusion partner in the Fc fusion protein, to avoid introducing any residues that could potentially undergo PTM, and to maintain a low risk of immunogenicity.

[0101] Moving on to the process of protein expression, the methods for expressing proteins are well known to those skilled in the art.

[0102] The process began with codon optimization of the DNA sequence for efficient expression in the CHO cell line. This cell line has a signal sequence at the N-terminus that leads the protein to secretory expression, a suitable restriction enzyme site to enable cloning into a glutamine synthase (GS) vector, and a Kozak sequence between the 5′ restriction enzyme site and the "ATG" start codon. These sequences were submitted to Life Technologies, synthesized by the company, and provided in the cloning vector. Other vectors have different secretory sequences and different selection markers (e.g., dihydrofolate reductase (DHFR) instead of GS). A 1405 base pair (bp) DNA fragment encoding the protein was transferred from the cloning vector to the GS expression vector pXC-17 via digestion with HindIII and EcoRI. The digested products and vectors were ligated, and the ligated products were used to transform chemically competent Escherichia coli (E. coli) cells (e.g., TOP10 cells). A single colony was analyzed for the correct insert, one positive clone was selected, plasmid DNA was prepared, and sequenced (forward and reverse) to confirm the presence of the correct sequence. Sufficient linear DNA for the cell line construction process was created by linearizing the plasmid with the restriction enzyme PvuI.

[0103] Lonza Biologics' mammalian-derived Chinese hamster ovary (CHO) K1SV glutamine synthase knockout (GS-KO) expression system was used to produce the protein. The CHOK1SV GS-KO host cell line is a derivative of the CHOK1SV host cell line in which the endogenous GS gene is "knocked out". The host cell line was derived from Lonza Biologics' CHOK1SV GS-KO host working cell bank.

[0104] [Affinity of p75NTR-Fc in sequences 1-19 (sequence numbers 1-19) for NGF] Biacore tips were prepared in experiments involving amine coupling of protein A to flow cells 1 and 2. The single-cycle dynamics of NGF binding to captured p75-Fc were measured.

[0105] Bonding ability of the chip surface (R max ) depends on the level of ligand (fusion protein) immobilization. For kinetic analysis experiments, 50-100 RU R max This is recommended. By using the molecular weights of p75-Fc and NGF, the desired immobilization level for the fusion protein can be calculated.

[0106] R max = (NGF molecular weight / fusion protein molecular weight) × immobilization level × stoichiometric ratio: 50 = (13,500 / 102,000) × fixed level × 1

[0107] Therefore, the required immobilization level = (102,000 / 13,500) × 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 Protein A chips prior to single-cycle kinetic analysis.

[0108] Using manual execution, p75-Fc was captured on flow cell 2 of the Protein A tip until a desired level of approximately 380 RU was achieved. This was done with injection at a flow rate of 10 μl / min for 22 seconds and a p75-Fc concentration of 10 μg / ml, resulting in the capture of 418 RU of fusion protein on the Protein A surface.

[0109] First, NGF concentrations of 10, 5, 2.5, 1.25, and 0.625 nM were tested. These concentrations were tested for the K of the fusion protein. D However, this was because it was estimated to be within the range of NGF concentration.

[0110] The single-cycle dynamics analysis method included the following: - Step 1: Inject 0.625 nM NGF onto the captured p75-Fc at a rate of 30 μl / min for 120 seconds. - This process was then repeated with the injection 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 phase was performed by flowing a running buffer (HBS-EP) onto the chip.

[0111] After completion, the chip was regenerated on the protein A surface by injecting 10 mM glycine HCl (pH 2) at a rate of 30 μl / min for 60 seconds.

[0112] p75-Fc was then captured on the tip by injection at a concentration of 10 μg / ml at a flow rate of 10 μl / min for 38 seconds. This achieved the desired level of 430 RU. The single-cycle dynamic analysis procedure described above was then repeated.

[0113] [Data Analysis] Fusion protein-NGF binding data were analyzed using Biacore T200 evaluation software v1 in the following manner: - Data is recorded regarding the binding of NGF to the fusion protein on flow cell 2 (Fc=2) and the NGF flowing on 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" combined data. - 2-1 fused data for 0nM (HBS-EP running buffer only) injection are then subtracted from all subsequent 2-1 fused data to correct for baseline variability throughout the experiment. - Finally, this data was then fitted to a 1:1 binding model to determine the association rate (ka), dissociation rate (kd), and affinity (K). D Calculate the coupling properties including ).

[0114] These sequences showed appropriate affinity for NGF.

[0115] [P75NTR-Fc is analgesic.] The purpose of this study was to investigate the effect of chronic exposure to p75NTR-Fc on analgesic effects in rats with monosodium iodoacetate (MIA)-induced osteoarthritis (OA).

[0116] Previously, we showed that spontaneous pain can be assessed by measuring static load using an incapacitance tester, and that this correlates with knee histopathology. Preclinical studies using novel therapies for pain have been criticized for the potential to introduce bias into the data. To address this, both left and right knees were randomly selected for OA induction, and all operators in the daily in vivo work were blinded for the condition of each knee. While OA induction is typically performed only in the right knee in the literature, previous studies have not found consistent differences between OA induction in the left and right knees, regardless of the time or dose of MIA used.

[0117] [Preparation of MIA] MIA was prepared in 0.3 mg / 50 μl ETF-PBS (the 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 of ETF-PBS. The MIA was prepared the day before and stored in the dark at 4°C until needed.

[0118] [animal] 185 male Wistar rats (from Charles River, UK, weighing 110–130g upon arrival) were used in this study. Each animal was checked upon arrival and appeared healthy. They were randomly assigned to cages of two, and each rat was given a unique identification number by a tail tattoo. The animals were acclimatized to the animal facility for at least 10 days before the start of the experiment on day 0. After the rats had acclimatized to their environment, they were moved to a stock / treatment room where all in vivo procedures were performed. The animals were kept under fluorescent lighting, set to a 12-hour light-dark cycle (on 07:00, off 19:00), as recommended by the UK 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.

[0119] The 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. The nesting boxes and cages were autoclaved, and each cage was individually ventilated (IVC system).

[0120] [Experimental Design] The study design divided the animals into 21 groups: a control human antibody group (n=6), 19 groups of p75NTR-Fc tested with 3 mg / kg p75NTR-Fc (each linked to the Fc region, SEQ ID NO: 1-19, using a GGG linker), and a 3 mg / kg PG-007 group (Pfizer's tanezumab biosimilar anti-NGF antibody).

[0121] The antibody and p75NTR-Fc were administered by subcutaneous injection every 5 days for 25 days.

[0122] Body weight was measured, and baseline blood samples were collected from the tail vein on the morning of day 2. Baseline static load was measured at approximately the same time on day 1. On day 0, again at approximately the same time, all rats were treated with their respective antibodies or p75NTR-Fc fusion protein. Three hours later, all animals were injected with 0.3 mg of MIA into one knee joint (ETF-PBS was injected into the opposite knee).

[0123] [Randomization of treatment] Before the start of the experiment, the rats were weighed, and each cage containing two rats was randomly assigned to a treatment group to ensure that the average weight of the animals in each group was approximately the same. In addition to assigning each rat to a specific treatment group, further randomization was performed to ensure that MIA was injected into either the left or right knee of each rat (ETFPBS was injected into the opposite knee of each rat). The treatment group and the assignment of which knee to treat for each rat were generated using the random number generator in Microsoft Excel for Mac (version 14.1.1). Personnel who did not have contact with the animals performed the randomization procedure and assignments.

[0124] Two 7 ml polypropylene vials were labeled for each animal to indicate either the left or right knee (88 vials in total). Two researchers (one to record and verify against the master randomization sheet, and the other to dispense the intra-articular injection solution) prepared the 88 vials. Dispensing was performed sequentially, with the MIA vials being filled first, followed by the remaining vials filled with ETF-PBS (these were the vials for the opposite knee of each animal). Throughout the study, the in vivo researchers were blinded to the treatment status of all animals.

[0125] [Animal experimentation] [Intra-articular injection of the knee joint] All rats were anesthetized by isoflurane inhalation using a Boyles device. The hair on both knees of each animal was shaved, and the knees were wiped with ethanol. 50 μl containing either 0.3 mg of MIA in ETF-PBS or ETF-PBS alone was injected into each knee subpatellarly using a 0.5 ml sterile Becton Dickinson Micro-Fine insulin syringe fitted with a 27G needle.

[0126] [Assessment of spontaneous pain] Spontaneous pain was determined for each animal by measuring the load on the left and right hind limbs using an incapacitance tester (Linton Instruments, UK). Rats were placed in appropriately sized Perspex animal boxes on the incapacitance tester so that their hind legs were placed on separate sensors. The size of the boxes allowed the rats to sit comfortably without being cramped, but not to allow enough space to turn around. Once the rats were stable and calm, the load on each limb was recorded for 5 seconds, and the average force (in grams) applied by both hind limbs was recorded. The load distribution on the hind limbs was measured 5 times for each rat at each time point (the validity of which we have previously demonstrated), and the average of the 5 readings was calculated. Individual load data were converted to load distribution by dividing the load on the right limb by the total load on both hind limbs.

[0127] [Measurement of spontaneous pain after MIA-induced osteoarthritis] Spontaneous pain was assessed using an incapacitance tester to measure load distribution through the hind limb. Assessments were performed at baseline and 3 weeks after treatment for MIA.

[0128] These studies demonstrate that p75NTR-Fc is analgesic in a rat model of OA with MIA. The analgesic effect of p75NTR-Fc was greater than that observed with an anti-NGF antibody (PG-007: a biosimilar of the anti-NGF antibody tanezumab from Pfizer) at a similar dose (3 mg / kg subcutaneous administration).

Claims

1. p75NTR is a neurotrophic factor-binding protein (NBP)-Fc fusion protein, and: (a) A p75NTR(NBP) portion selected from any of sequence numbers 1 to 19; and (b) Immunoglobulin Fc moiety selected from any of sequence numbers 20-24; Includes, The p75NTR(NBP) portion and the Fc portion are connected via a linker, and the linker is of type G ｘ It contains the peptide, where x is 1, 2, 3, 4, 5, or 6. p75NTR is a neurotrophic factor-binding protein (NBP)-Fc fusion protein.

2. The p75NTR(NBP)-Fc fusion protein described in claim 1, The linker is GGG. p75NTR(NBP)-Fc fusion protein.

3. A p75NTR(NBP)-Fc fusion protein according to claim 1 or 2, Here, the Fc region is Fc suitable for human use. p75NTR(NBP)-Fc fusion protein.

4. The use of the p75NTR(NBP)-Fc fusion protein described in claims 1 to 3, For the treatment of pain, use.

5. A nucleic acid molecule encoding the p75NTR(NBP)-Fc fusion protein described in claims 1 to 3.

6. A replicable expression vector for transfecting cells, The vector comprises the nucleic acid molecule described in claim 5. A replicable expression vector.

7. The use of the nucleic acid molecule described in claim 5, or the vector described in claim 6, For the treatment of pain, use.

8. The use of the p75NTR(NBP)-Fc fusion protein described in claims 1 to 3, the nucleic acid molecule described in claim 5, or the vector described in claim 6, For use in the treatment of osteoarthritis, use.

9. A p75NTR(NBP)-Fc fusion protein according to claims 1 to 3, a nucleic acid molecule according to claim 5, or a vector according to claim 6, and a pharmaceutically acceptable carrier and / or excipient, Pharmaceutical composition.

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