Fusion protein

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 certain embodiments, the p75NTR (NBP)-Fc fusion proteins are used to treat pain and / or symptoms of pain.

Description

Background of the Invention

[0002] Neurotrophic factors, including neurotrophic growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), and neurotrophic factor 4 / 5 (NT-4 / 5), exert their effects through four receptors: the low-affinity p75 neurotrophic receptor (p75NTR) and the high-affinity tyrosine kinase receptors; TrkA, TrkB, and TrkC. The low-affinity receptor p75NTR can bind to and be activated by all four neurotrophic factors, and its function has been reported to be independent of the other receptors. However, the Trk receptor exhibits stronger activation selectivity; that is, NGF is the selective ligand for TrkA, BDNF is the ligand for TrkB, and NT-3, 4 / 5 are the ligands for TrkC. Furthermore, it has been reported that when p75NTR and Trk proteins are co-expressed, they form a complex that alters the signal transduction of both receptors (Huang and Reichardt, 2003, Annu Rev Biochem. 72:609-42). In fact, studies have shown that p75NTR can promote the selectivity of various neurotrophic factors for their respective Trk receptors.

[0003] p75NTR is a member of the tumor necrosis factor receptor superfamily (TNFR-SF) and 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 (usually four) repeating 40-amino acid cysteine-rich domains (CRDs), which were initially discovered in p75NTR (Johnson et al., 1986 Cell 47:545–554; Radeke et al., 1987 Nature 325:593–597). In contrast, the intracellular domains of all TNFR-SF family members do not share any sequence motifs. Therefore, the signaling mechanisms of TNFR-SF proteins vary considerably.

[0004] An unusual feature of the p75NTR structure is the presence of a disulfide-linked p75NTR dimer formed via cysteine ​​residues within the transmembrane domain. This disulfide bond is required for efficient neurotrophic factor-dependent signaling by p75NTR and plays a crucial role in the formation of both intracellular and extracellular domains (Vilar et al., 2009 Neuron 62:72–83). Neurotrophic factors physiologically exist in a non-covalently bound dimer form (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). Neurotrophic factor-dependent p75NTR activation involves CRD2-4 binding of the neurotrophic factor dimer to both 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 approach each other, forcing the intracellular domain to open in a snail-tong-like motion centered on disulfide bonds, thereby enabling the intracellular domain to bind to the signal transduction adaptor proteins NRIF and TRAF6 (Vilar et al., 2009 J Cell Sci 122:3351–3357, Vilar et al., 2009 Neuron 62:72–83). Intra-membrane disulfide bonds (such as those present in p75NTR) have not been previously described in other TNFR-SF family members or any other membrane protein.

[0005] p75NTR undergoes sequential proteolytic cleavage by α-secretase and γ-secretase activity and matrix metalloproteinases (MMPs), releasing its intracellular domain (ICD) into the cytoplasm, similar to the cleavage-dependent signaling pathway of Notch and β-amyloid precursor (Jung et al., 2003 J Biol Chem 278:42161–42169; Kanning et al., 2003 J Neuro-sci 23:5425–5436). This intracellular release of p75NTR ICD promotes related NRIF signaling (Kenchappa et al., 2006 Neuron 50:219–232). The role of the p75NTR extracellular domain after α-secretase and γ-secretase activity and MMP proteolytic cleavage is not fully understood.

[0006] Records show that NGF and other neurotrophic factors (BDNF, NT-3 and NT-4 / 5) play important roles in pathology, such as pain caused by 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 CurrMed 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 against neurotrophic factor receptors p75NTR Trk A, Trk B, or Trk C have also been shown to be effective in pain models (Orita S et al., 2010 J Orthop Res. 28:1614-20;). et al., 2010 Pain. 148:473-80; Iwakura et al., 2010 J Hand Surg Am. 35:267-73; Cirilio et al., 2010 CellMol 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. 2010; 28:279-83). Fukui et al. (2010) demonstrated in a pain model (mechanical abnormal pain following sciatic nerve compression) that anti-p75NTR antibody treatment significantly improved pain-related endpoints. The study concluded that treatment with p75NTR inhibitory antibodies reduced the expression of CGRP and p75NTR, thereby significantly alleviating pain.

[0007] This invention relates to a p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein. We describe the affinity and in vivo kinetics of this molecule, as well as its efficacy in treating pain in animal models. The p75NTR(NBP)-Fc fusion protein may be used to treat pain and other neurotrophic factor-related diseases, such as psoriasis, eczema, rheumatoid arthritis, cystitis, endometriosis, and osteoarthritis.

[0008] Invention Summary

[0009] According to a first aspect of the present invention, a p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein is provided, comprising:

[0010] (a) The p75NTR (NBP) portion selected from one of Seq ID Nos. 1-19; and

[0011] (b) Immunoglobulin Fc fraction selected from one of Seq ID Nos. 20-24

[0012] The p75NTR (NBP) and Fc sections are connected via a connector, which includes type G. x The peptide, where x is 1, 2, 3, 4, 5 or 6.

[0013] In a preferred embodiment, the connector does not contain or consist of the sequence GGGGS.

[0014] In a further implementation, the connector is GGG.

[0015] In a particularly preferred embodiment of the p75NTR(NBP)-Fc fusion protein according to the invention, the Fc region is an Fc suitable for human use.

[0016] In yet 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. 1.

[0017] 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. 2.

[0018] 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. 3.

[0019] 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. 4.

[0020] 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. 5.

[0021] 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. 6.

[0022] 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. 7.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[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. 23.

[0039] 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.

[0040] In a preferred embodiment, the p75NTR(NBP)-Fc fusion protein according to the invention binds to any one of NGF, BDNF, NT3, or NT4 / 5, with a binding affinity (K... d The value ranges from approximately 0.01 nM to approximately 50 nM, as measured by surface plasmon resonance at 20 °C.

[0041] In a second aspect of the invention, a p75NTR(NBP)-Fc fusion protein according to any aspect of the invention is provided for the treatment of pain.

[0042] In a third aspect of the invention, a nucleic acid molecule encoding a p75NTR(NBP)-Fc fusion protein according to the first or second aspect of the invention is provided, optionally further comprising a coding signal sequence.

[0043] In a fourth aspect of the invention, a reproducible expression vector for transfecting cells (optionally mammalian cells) is provided, the vector comprising a nucleic acid molecule according to a third aspect of the invention.

[0044] Preferably, the reproducible expression vector is a viral vector.

[0045] In a fifth aspect of the invention, a host cell containing the nucleic acid molecule of the third aspect of the invention is provided.

[0046] In a sixth aspect of the invention, a nucleic acid molecule according to the third aspect of the invention or a carrier according to the fourth aspect of the invention is used for the treatment of pain.

[0047] Pain includes, but is not limited to: acute pain; chronic pain; inflammatory pain; nociceptive pain; neuropathic pain; hyperalgesia; anomalous pain; central pain; cancer pain; postoperative pain; visceral pain; musculoskeletal pain; cardiac or vascular pain; headache (including migraine); maxillofacial pain (including toothache); and back pain. Treatment of pain includes, but is not limited to, prevention, improvement, control, reduction of pain incidence, or delay of pain development or progression.

[0048] In a seventh aspect of the invention, a nucleic acid molecule according to the third aspect of the invention or a carrier according to the fourth aspect of the invention is used to treat osteoarthritis.

[0049] In the eighth aspect, 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, is provided, wherein the p75NTR(NBP)-Fc fusion protein or nucleic acid molecule or vector is used alone, sequentially or simultaneously in combination with a second pharmacologically active compound.

[0050] In a ninth aspect, the present invention provides a pharmaceutical composition comprising a p75NTR(NBP)-Fc fusion protein or a nucleic acid molecule or carrier according to any aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0051] Preferably, the pharmaceutical composition is used for one or more of the following purposes: preventing, improving, controlling, reducing the incidence of osteoarthritis in mammals, or delaying the development or progression of pain.

[0052] In one implementation, the mammal is a human.

[0053] In one embodiment, the mammal is preferably a dog, cat, elephant, or horse. In a particularly preferred embodiment, the animal is a dog.

[0054] In another aspect of the present invention, a kit is provided, comprising:

[0055] (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

[0056] (b) Instructions for use for administering an effective amount of the p75NTR(NBP)-Fc fusion protein, nucleic acid molecule, carrier or pharmaceutical composition to an animal for the prevention or treatment of pain, or for improving, controlling, reducing the incidence of pain, or delaying the development or progression of pain.

[0057] In another aspect of the invention, a method for treating and / or preventing pain in animals is provided, comprising administering to the individual a therapeutically effective amount of a p75NTR(NBP)-Fc fusion protein according to any aspect of the invention, or a nucleic acid molecule or carrier according to any aspect of the invention, optionally further comprising a pharmaceutically acceptable carrier, or a pharmaceutical composition according to an eighth aspect of the invention. Attached Figure Description

[0058] Figure 1 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 1).

[0059] Figure 2 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 2).

[0060] Figure 3 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 3).

[0061] Figure 4 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 4).

[0062] Figure 5 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 5).

[0063] Figure 6 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 6).

[0064] Figure 7 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 7).

[0065] Figure 8 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 8).

[0066] Figure 9 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 9).

[0067] Figure 10 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 10).

[0068] Figure 11 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 11).

[0069] Figure 12 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 12).

[0070] Figure 13 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 13).

[0071] Figure 14 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 14).

[0072] Figure 15 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 15).

[0073] Figure 16 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 16).

[0074] Figure 17 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 17).

[0075] Figure 18 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 18).

[0076] Figure 19 The amino acid sequence of the p75NTR (NBP) portion of the fusion protein according to the present invention (SEQ ID No. 19).

[0077] Figure 20 The amino acid sequence of the suitable human Fc portion of the fusion protein according to the present invention (SEQ ID No. 20).

[0078] Figure 21 The amino acid sequence of the cat IgG-Fc portion of the fusion protein according to the present invention (SEQ ID No. 21).

[0079] 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).

[0080] Figure 23 The amino acid sequence of the cat IgG-Fc portion of the fusion protein according to the present invention (SEQ ID No. 23).

[0081] Figure 24 The amino acid sequence of the cat IgG-Fc portion of the fusion protein according to the present invention (SEQ ID No. 24). Detailed Implementation

[0082] According to a first aspect of the present invention, a p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein is provided, comprising:

[0083] (a) The p75NTR (NBP) portion selected from one of Seq ID Nos. 1-19; and

[0084] (b) Selected from the Fc portion of immunoglobulin from one of Seq ID Nos. 20-24.

[0085] The p75NTR (NBP) and Fc sections are connected via a connector, which includes type G. x The peptide, where x is 1, 2, 3, 4, 5 or 6.

[0086] G stands for glycine.

[0087] In a preferred embodiment, the connector does not contain or consist of the sequence GGGGS.

[0088] In a further implementation, the connector is GGG.

[0089] In a particularly preferred embodiment of the p75NTR(NBP)-Fc fusion protein according to the invention, the Fc region is an Fc suitable for human use.

[0090] In yet 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. 1.

[0091] 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. 2.

[0092] 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. 3.

[0093] 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. 4.

[0094] 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. 5.

[0095] 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. 6.

[0096] 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. 7.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] Appropriately, p75NTR neurotrophic factor binding protein p75NTR (NBP) is polyethylene glycol-modified, more preferably glycosylated.

[0115] Furthermore, those skilled in the art will understand that the therapeutic potential of the molecules of the present invention can be enhanced by introducing defined mutations (e.g., YTE mutations (M252Y / S254T / T256E) and LS mutations (M428L / N434S)) into the crystallizable fragment (Fc) region. Such techniques are well known to those skilled in the art. The effect of introducing such mutations generally extends the half-life and duration of action of the molecule. However, the effects of mutation introduction are not limited to extending half-life and duration of action.

[0116] The p75NTR(NBP)-Fc fusion protein of the present invention preferably binds to any one or more of NGF, BDNF, NT3, or NT4 / 5, with a binding affinity (K). d The molecular weight (K) is approximately 0.01 nM to approximately 50 nM. In some preferred embodiments, the binding affinity (K) is... d The binding affinity (K) is between about 0.01 nM and 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 the in vitro binding assays of NGF, BDNF, NT3, or NT4 / 5 described herein, preferably as measured by surface plasmon resonance at 20°C. In some more preferred embodiments, the binding affinity (K) is... dThe binding affinity is or less than about 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 the p75NTR(NBP)-Fc fusion protein with the neurotrophic factor described herein, preferably as measured by surface plasmon resonance at 20°C. In a more preferred embodiment, the binding affinity (K0.05) is... d The value is approximately 0.3 nM or approximately 1 nM, as measured in an in vitro binding assay of the p75NTR(NBP)-Fc fusion protein with the neurotrophic factor described herein, preferably as measured by surface plasmon resonance at 20°C.

[0117] Preferably, the p75NTR(NBP)-Fc fusion protein of the present invention is used for the treatment of pain. Not wishing to be bound by any particular theory, the inventors believe that the p75NTR(NBP)-Fc fusion protein achieves its therapeutic effect on pain by influencing the aforementioned neurotrophic factors NGF, BDNF, NT3, or NT4 / 5 (e.g., the functional activity of the neurotrophic factors resulting from their interaction with their respective receptors) (defined as regulating or upregulating or downregulating the functional activity of the neurotrophic factors).

[0118] Preferably, the p75NTR(NBP)-Fc fusion protein affects the functional activity of BDNF, as assessed by functional assays of any one of the following: neuronal and synaptic growth and differentiation, neuronal cell survival and differentiation in culture, Trk signaling, and stimulation of axonal growth in vitro or in vivo.

[0119] Preferably, the p75NTR(NBP)-Fc fusion protein affects the functional activity of NGF, as assessed by measuring the binding and activation of NGF to TrkA, as demonstrated by classic neuronal survival assays (e.g., provided by Cowan et al., Annu. Rev. Neurosci. 2001;24:551–600).

[0120] Preferably, the p75NTR(NBP)-Fc fusion protein affects the functional activity of NT3, as assessed by measuring the binding and activation activity of NT3 with the endogenous Trk receptor, such as as confirmed by Trk receptor phosphorylation, mitogen-activated protein kinase phosphorylation reporter gene assays, or cell survival and neurite extension assays.

[0121] Preferably, the p75NTR(NBP)-Fc fusion protein affects the functional activity of NT4 / 5, as assessed by measuring NT4 / 5 phosphorylation and activation assays in vitro or in vivo, such as in myelin basic protein (MBP) phosphorylation assays or in in vivo vascular endothelial growth factor (VEGF) / basic fibroblast growth factor induced angiogenesis in a matrix gel angiogenesis assay.

[0122] Preferably, the p75NTR(NBP)-Fc fusion protein binds to one or more of the contact residues of neurotrophic factors NGF, NT3, BDNF and NT4 / 5, as shown in He and Garcia (2001) Science, 301, pp. 870-805.

[0123] Preferably, the p75NTR(NBP)-Fc fusion protein is soluble, preferably soluble in aqueous solution, and more preferably soluble in biological fluids such as serum, plasma, or blood.

[0124] As used herein, the term "Fc" or "immunoglobulin Fc" or "Ig Fc" refers to the carboxyl-terminal portion, or a portion thereof, of the constant region of the immunoglobulin chain (preferably the constant region of the immunoglobulin heavy chain). Preferably, 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, CH1, CH2, and CH3, optionally combined with an immunoglobulin hinge region.

[0125] According to the present invention, the p75NTR(NBP)-Fc fusion protein preferably exhibits superior biological properties, including improved p75NTR(NBP) solubility and / or p75NTR(NBP) stability and / or improved p75NTR(NBP) serum half-life. Improved solubility is necessary to maximize the bioavailability of p75NTR(NBP) at administration and to determine and administer an accurate dose of p75NTR(NBP). Improved solubility helps overcome the problem of aggregates, which are undesirable and can cause pain and potential inflammation during in vivo delivery. An improved serum half-life is advantageous because, during therapeutic use, it helps to reduce the level or frequency of dose requirements to achieve or maintain a therapeutic effect comparable to or maintained by the delivered p75NTR(NBP). The extended half-life and higher stability in blood or serum have the advantage of allowing for lower frequency dosing and / or lower dosing regimens, thereby reducing potential toxicity or side effects in vivo. In this context, the p75NTR(NBP)-Fc fusion protein exhibits stronger therapeutic efficacy and / or greater stability in blood circulation. The resulting lower dose or lower frequency of dosing helps minimize any potential toxicities or side effects potentially associated with p75NTR(NBP) administration. The increased molecular weight of the p75NTR(NBP)-Fc fusion protein compared to p75NTR(NBP) alone offers the advantage that, upon intravenous administration, the molecule remains well-retained in the bloodstream, reducing the risk of penetration into undesirable sites (e.g., the central nervous system) and ensuring appropriate retention or concentration of the molecule in the target tissue.

[0126] Preferably, the p75NTR(NBP)-Fc fusion protein exhibits improved p75NTR(NBP) solubility and / or improved p75NTR(NBP) stability and / or improved serum half-life compared to p75NTR(NBP) alone. Preferably, the improved solubility is the solubility in an aqueous solution (e.g., water, preferably containing excipients, such as buffers and / or salts), preferably at physiological pH, preferably between pH 5 and pH 8, preferably around pH 7, or in biological fluids (e.g., serum or blood). Preferably, the improved stability is the stability of the p75NTR(NBP) protein's activity or structural integrity over a period of time, during a period of storage, or after freeze-thaw cycles, due to 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, with methods for measuring the serum half-life of proteins known.

[0127] Preferably, 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, for example by binding to Staphylococcus aureus protein A. Preferably, the p75NTR(NBP)-Fc fusion protein can dimerize, and preferably, the dimer has a higher affinity for neurotrophic factors NGF, BDNF, NT3, or NT4 / 5 compared to p75NTR(NBP) alone. Tighter binding results in higher potency and therapeutic efficacy, which can be determined by the action of p75NTR(NBP), for example by the neurotrophic factor functional analysis disclosed herein. The benefit of higher potency is that the p75NTR(NBP)-Fc fusion protein can achieve the same therapeutic efficacy with lower doses, thereby reducing potential toxicity or side effects in vivo.

[0128] Preferably, the p75NTR(NBP)-Fc fusion protein of the present invention has an in vivo half-life of approximately or greater than any 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 approximately or more than 24 hours.

[0129] More preferably, the p75NTR(NBP)-Fc fusion protein of the present invention has an in vitro half-life of approximately or greater than any 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, 1 20, 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 in vitro half-life of the p75NTR(NBP)-Fc fusion protein of the present invention is about 6 days or more. Preferably, the stability is measured at approximately physiological pH in a buffered aqueous solution, preferably at 20°C or 37°C.

[0130] According to the aforementioned preferred embodiments, the in vivo half-life is preferably the half-life in rats or in humans, more preferably the half-life in humans. Preferably, the half-life is determined by measuring the level of the p75NTR(NBP)-Fc fusion protein of the present invention in serum after in vivo administration (e.g., intravenous or subcutaneous injection).

[0131] The p75NTR (NBP) and immunoglobulin Fc portions of the p75NTR (NBP)-Fc fusion protein are linked by a linker. Preferably, the linker optionally comprises or is composed of one or more amino acids, or a polypeptide sequence comprising or composed of amino acids, preferably about 1 to about 25 amino acids, preferably any one of 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, more preferably any 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.

[0132] Preferably, the joint comprises or is composed of any stable secondary structures (e.g., α-helices, β-chains, 3-chains). 10The linker region consists of amino acid polypeptide sequences that define flexible or dynamic or unstructured polypeptides (e.g., flexible loops, random coils, or flexible turns). Preferably, the linker region comprises or is composed of amino acid polypeptide sequences that define flexible or dynamic or unstructured polypeptides. Such unstructured polypeptides are commonly used to link secondary structural regions in large protein molecules.

[0133] Preferably, the linker is an amino acid polypeptide sequence in the p75NTR (NBP) containing greater than or about 50% glycine and / or alanine and / or serine; more preferably, the p75NTR (NBP) contains greater than or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% glycine and / or alanine and / or serine. Preferably, the linker region comprises or is composed of an amino acid polypeptide sequence containing glycine and serine; preferably, the proportion of glycine is greater than the proportion of serine; preferably, the linker region comprises or is composed of a flexible linker.

[0134] Without being bound by any particular theory, the inventors believe that a flexible linker can overcome or avoid steric hindrance, which can interfere with the aforementioned neurotrophic factor binding capacity or biological activity of the p75NTR(NBP)-Fc fusion protein compared to p75NTR(NBP) alone. Therefore, the linker region preferably allows flexibility between the p75NTR(NBP) moiety and the immunoglobulin Fc moiety, and allows for the maintenance or improvement of the aforementioned biological activity of the p75NTR(NBP)-Fc fusion protein compared to free or native p75NTR(NBP) alone, as determined by binding to neurotrophic factors using the binding assays described herein.

[0135] More preferably, the linker is immune inert, such that it does not trigger complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), and does not activate microglia or T cells. Preferably, one or more of these activities in the linker region are reduced.

[0136] More preferably, the linker comprises or is composed of polypeptides that are known or predicted, based on structural analysis or structural prediction, to be flexible, dynamic, or unstructured, or polypeptides lacking stable secondary structures.

[0137] Connector-included G x The peptide, where x is 1, 2, 3, 4, 5 or 6.

[0138] In a preferred embodiment, the connector does not contain the sequence GGGGS or is not composed of the sequence GGGGS.

[0139] In a further implementation, the connector is GGG.

[0140] The p75NTR(NBP)-Fc fusion protein of the present invention may further include a proteolytic cleavage site, which may optionally be inserted between the p75NTR(NBP) moiety and the immunoglobulin Fc moiety. This proteolytic cleavage site may be located in the linker or at the junction between the linker and the p75NTR(NBP) moiety and / or the immunoglobulin Fc moiety. The p75NTR(NBP) may optionally be cleaved from the immunoglobulin Fc moiety prior to formulation or administration for therapeutic purposes.

[0141] Preferably, the linker and / or immunoglobulin Fc partially do not impair or significantly impair the p75NTR (NBP) portion, with the following effects:

[0142] (a) Effects on the functional activity of neurotrophic factors NGF, BDNF, NT3, or NT4 / 5 (defined as regulation or upregulation or downregulation of the functional activity of neurotrophic factors).

[0143] (b) The binding affinity for any one of NGF, BDNF, NT3, or NT4 / 5 is between about 0.1 nM and about 50 nM.

[0144] (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).

[0145] According to another aspect of the invention, a nucleic acid molecule encoding a p75NTR(NBP)-Fc fusion protein according to a first or second aspect is provided. Preferably, the nucleic acid molecule is used for the treatment of pain.

[0146] According to a preferred embodiment of the invention, the nucleic acid molecule may also contain a region encoding a signal sequence, preferably a p75NTR signal sequence, such as a DNA or RNA sequence.

[0147] According to another aspect of the invention, a reproducible expression vector for transfecting cells is provided, the vector comprising the nucleic acid molecule described in the third aspect, preferably a viral vector. Preferably, the vector is used for treating pain.

[0148] According to the above aspects of the present invention, a method for expressing the nucleic acid molecule or vector of the present invention to produce or secrete the p75NTR(NBP)-Fc fusion protein is further provided. Preferably, the method comprises introducing the nucleic acid molecule or vector into cells and expressing the nucleic acid therein to produce or secrete the p75NTR(NBP)-Fc fusion protein. Preferably, the nucleic acid molecule or vector is introduced into cells in vitro or alternatively in vivo. Preferably, the expressed p75NTR(NBP)-Fc fusion protein is expressed in vitro, optionally further isolated and purified; or, preferably, the expressed p75NTR(NBP)-Fc fusion protein is expressed in vivo, preferably, the in vivo expression constituting gene therapy. Preferably, the vector is a reproducible expression vector, optionally used for transfecting mammalian cells; preferably, the vector is a viral vector.

[0149] According to another aspect of the invention, a host cell containing a nucleic acid molecule or vector of the third or fourth aspect is provided, preferably, the cell is a mammalian cell.

[0150] According to another aspect of the invention, a p75NTR(NBP)-Fc fusion protein for treating pain is provided, or a nucleic acid or vector for treating pain. Pain may include, but is not limited to:

[0151] (a) Acute pain and / or spontaneous pain,

[0152] (b) Chronic pain and / or persistent pain,

[0153] (c) Inflammatory pain includes any of the following: arthritis pain, pain caused by osteoarthritis or rheumatoid arthritis, pain caused by inflammatory bowel disease, psoriasis, and eczema.

[0154] (d) Nociceptive pain,

[0155] (e) Neuropathic pain, including pain associated with diabetic neuropathy or postherpetic neuralgia.

[0156] (f) Hyperalgesia,

[0157] (g) Abnormal pain,

[0158] (h) Central pain, central post-stroke pain, pain caused by multiple sclerosis, pain caused by spinal cord injury, or pain caused by Parkinson's disease or epilepsy.

[0159] (i) Cancer pain,

[0160] (j) Postoperative pain,

[0161] (k) Visceral pain, including digestive and non-digestive visceral pain, pain caused by gastrointestinal (GI) disorders, pain caused by functional bowel disorder (FBD), pain caused by inflammatory bowel disease (IBD), dysmenorrhea, pelvic pain, pain caused by cystitis, interstitial cystitis, or pancreatitis.

[0162] (l) Musculoskeletal pain, myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatoid) arthropathy, non-articular rheumatism, muscular dystrophy, glycogenolysis, polymyositis, pyogenic myositis,

[0163] (m) Pain in the heart or blood vessels, angina pectoris, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleromas, pain caused by scleromas or skeletal muscle ischemia.

[0164] (n) Headache, including migraine, migraine with aura, migraine without aura, cluster headache, tension headache.

[0165] (o) Oral and facial pain, including toothache, temporomandibular fascia pain, or tinnitus, or

[0166] (p) Back pain, bursitis, menstrual pain, migraine, referred pain, trigeminal neuralgia, hypersensitivity, pain caused by spinal trauma and / or degeneration or stroke.

[0167] Treatment of pain includes, but is not limited to, prevention, improvement, control, reduction of the incidence of pain, or delay of the development or progression of pain.

[0168] According to another aspect of the invention, a p75NTR(NBP)-Fc fusion protein according to a first aspect or a second aspect or a preferred embodiment thereof, or a nucleic acid molecule or carrier according to a third aspect and a fourth aspect, wherein the p75NTR(NBP)-Fc fusion protein or the nucleic acid molecule or carrier is used in a combination with a second pharmacologically active compound, used separately, sequentially or simultaneously. Preferably, the second pharmacologically active compound in the combination may include, but is not limited to:

[0169] • Opioid analgesics, such as morphine, heroin, hydromorphone, hydroxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, or pentazocine;

[0170] Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, and mefenamic acid. The following are listed: acid, meloxicam, nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin, or zomepirac;

[0171] • Barbiturate sedatives, such as amobarbital, aprobarbital, butabarbital, butabarbital, mephobarbital, metharbital, methoexital, pentobarbital, phenobartital, secobarbital, talbutal, thiamylal, or thiopental.

[0172] Benzodiazepines with sedative effects, such as chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, or triazolam;

[0173] • H1 antagonists with sedative effects, such as diphenhydramine, pyrilamine, promethazine, chlorpheniramine, or chlorcyclizine;

[0174] • Sedatives, such as glutethimide, meprobamate, methaqualone, or dichloralphenazone;

[0175] • Skeletal muscle relaxants, such as baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, mesocarbamol, or orphrenadine.

[0176] • NMDA receptor antagonists, such as dextromethorphan ((+)-3-hydroxy-N-methylmorphorphenanthranil) or its metabolite dextromethorphan ((+)-3-hydroxy-N-methylmorphorphenanthranil), ketamine, memantine, pyrroloquinoline quinine, cis-4-(phosphomethyl)-2-piperidinic acid, budipine, EN-3231 (MorphiDex®, a combination of morphine and dextromethorphan), topiramate, neramexane, or perzinfotel (including NR2B antagonists such as afenidil, traxoprodil, or (–)-(R)-6-{2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl-3,4-dihydro-2- (1H)-quinolinone);

[0177] • Alpha-adrenergic drugs, such as doxazosin, tamsulosin, clonidine, guanfacine, dexmetatomidine, modafinil, or 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4-tetrahydroisoquinoline-2-yl)-5-(2-pyridyl)quinazoline;

[0178] • Tricyclic antidepressants, such as desipramine, imipramine, amitriptyline, or nortriptyline;

[0179] • Anticonvulsants, such as carbamazepine, lamotrigine, topiratmate, or valproate;

[0180] • Tachykinin (NK) antagonists, especially 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]diazacyclooctano[2,1-g][1,7]-naphthyl-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);

[0181] Muscarinic antagonists, such as oxybutynin, tolterodine, propiverine, tropsium chloride, darifenacin, solifenacin, temiverine, and ipratropium;

[0182] • Selective COX-2 inhibitors, such as celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib.

[0183] • Coal tar analgesics, especially acetaminophen;

[0184] • Tranquilizers, such as droperidol, chlorpromazine, haloperidol, perphenazine, thioridazine, mesoridazine, trifluoperazine, fluphenazine, clozapine, olanzapine, risperidone, ziprasidone, quetiapine, sertindole, aripiprazole, and solanine. Sonepirazole, blonanserin, iloperidone, perospirone, raclopride, zotepine, bifeprunox, asenapine, lurasidone, amisulpride, balaperidone, palindore, epilisarserin, osanetant, rimonabant, meclinertant, Miraxion®, or sarizotan;

[0185] • Vanillin receptor agonists (e.g., resinferatoxin) or antagonists (e.g., capsazepine);

[0186] • Beta-adrenergic drugs, such as propranolol;

[0187] • Local anesthetics, such as mexiletine;

[0188] • Corticosteroids, such as dexamethasone;

[0189] • 5-HT receptor agonists or antagonists, especially 5-HT 1B / 1D Agonists, such as eletriptan, sumatriptan, naratriptan, zolmitriptan, or rizatriptan;

[0190] ·5-HT2A Receptor antagonists, such as R(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (MDL-100907);

[0191] • Cholinergic (nicotine) analgesics, such as ispronicline (TC-1734), (E)-N-methyl-4-(3-pyridyl)-3-buten-1-amine (RJR-2403), (R)-5-(2-azacyclobutylmethoxy)-2-chloropyridine (ABT-594) or nicotine;

[0192] Tramadol®;

[0193] • PDEV inhibitors, such as 5-[2-ethoxy-5-(4-methyl-1-piperazinylsulfonyl)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)-pyrazolo[2',1':6,1]pyrido[3,4-b]indole-1,4-dione (IC- 351 or tadalafil), 2-[2-ethoxy-5-(4-ethylpiperazin-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-pyridyl)-3-ethyl-2-(1-ethyl-3-azacyclobutyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 5-(5-acetyl-2-propoxy-3-pyridyl)-3-ethyl-2-(1-isopropyl-3-azacyclobutyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one Pyrimidine-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, 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;

[0194] ·Cannabinoids;

[0195] • Metabolic glutamate subtype 1 receptor (mGluR1) antagonists;

[0196] • Serotonin reuptake inhibitors, such as sertraline, sertraline metabolite demethylsertraline, fluoxetine, norfluoxetine (fluoxetine demethyl metabolite), fluvoxamine, paroxetine, citalopram, citalopram metabolite demethylcitalopram, escitalopram, d,l-fenfluramine, femoxetine, ifoxetine, cyanodothiepin, litoxetine, dapoxetine, nefazodone, cericlamine, and trazodone;

[0197] • Norepinephrine reuptake inhibitors, such as maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine, tomoxetine, mianserin, buproprion, hydroxybuproprion (a metabolite of buproprion), nomifenensine, and viloxazine (Vivalan®), especially selective norepinephrine reuptake inhibitors, such as reboxetine, particularly (S,S)-reboxetine;

[0198] • Dual serotonin-norepinephrine reuptake inhibitors, such as venlafaxine, venlafaxine metabolite O-desmethylvenlafaxine, clomipramine, clomipramine metabolite norclomipramine, duloxetine, milnacipran, and imipramine;

[0199] • 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-pyridinecarboxylate; 2-[[(1 R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-4-chlorobenzonitrile, (2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolylbutanol, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluoromethyl)-3-pyridinecarboxynitrile, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile, N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-formamidinium or guanidinylethyl disulfide;

[0200] • Acetylcholinesterase inhibitors, such as donepezil;

[0201] • 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;

[0202] • Leukotriene B4 antagonists; such as 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]valerate (ONO-4057) or DPC-11870,

[0203] • 5-Lipooxygenase 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);

[0204] • Sodium channel blockers, such as lidocaine; or

[0205] • 5-HT3 antagonists, such as ondansetron;

[0206] And its pharmaceutically acceptable salts and their solvates.

[0207] According to another aspect of the invention, a method is provided for treating, preventing, improving, controlling, reducing an individual's pain or the incidence of any of the aforementioned pain, or delaying its development or progression, comprising administering to the individual an effective amount of the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or a preferred embodiment thereof, or a nucleic acid molecule or carrier according to the third and fourth aspects.

[0208] In another preferred embodiment, the p75NTR(NBP)-FC fusion protein of the present invention is suitable for treating osteoarthritis. Specifically, it is used to prevent, slow, and reverse disease progression. In one specific embodiment, the protein of the present invention can be used to cure osteoarthritis.

[0209] In one embodiment of the invention, it is applicable to the treatment of osteoarthritis in humans.

[0210] In one embodiment of the invention, it is applicable to the treatment of osteoarthritis in the veterinary field. Preferably, the individual is a mammal, such as a companion animal like a horse, cat, or dog, or a farm animal like a sheep, cow, or pig. Most preferably, the animal is a dog.

[0211] According to an eighth aspect of the invention, a pharmaceutical composition is provided for treating, preventing, improving, controlling, reducing the incidence of pain or any of the aforementioned pain, or delaying its development or progression, comprising a p75NTR(NBP)-Fc fusion protein according to the first or second aspect or a preferred embodiment thereof, or a nucleic acid molecule or carrier according to the third and fourth aspects, and a pharmaceutically acceptable carrier and / or excipient.

[0212] Preferably, the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or its preferred embodiment, or the nucleic acid molecule or carrier according to the third and fourth aspects or the drug according to the eighth aspect, is prepared for or suitable for oral, sublingual, buccal, topical, rectal, inhalation, transdermal, subcutaneous, intravenous, intra-arterial, intramuscular, intracardiac, intraosseous, intradermal, intraperitoneal, transmucosal, vaginal, intravitreal, intra-articular, periarticular, topical or epipic administration.

[0213] Preferably, the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or its preferred embodiment, or the nucleic acid molecule or carrier according to the third and fourth aspects or the pharmaceutical composition according to 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.

[0214] Preferably, the p75NTR(NBP)-Fc according to the first or second aspect or its preferred embodiment, or the nucleic acid molecule or carrier according to the third and fourth aspects or the pharmaceutical composition of the eighth aspect, is used or prepared for application one to seven times per week, more preferably one to four times per month, more preferably one to six times every six months, and more preferably one to twelve times per year. Preferably, the drug is used or prepared for peripheral application during periods including but not limited to: once daily, once every two, three, four, five, or six days, once weekly, once every two weeks, once every three weeks, once per month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once per year.

[0215] More preferably, the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or its preferred embodiment, or the nucleic acid molecule or carrier according to the third and fourth aspects, or the pharmaceutical composition of the eighth aspect, will be or be prepared for peripheral administration via one or more of the following routes, including but not limited to: oral, sublingual, buccal, local, rectal, via inhalation, transdermal, subcutaneous, intravenous, intra-arterial or intramuscular, via intracardiac, intraosseous, intradermal, intraperitoneal, via mucosa, vagina, intravitreal, epidermal, intra-articular, periarticular or local.

[0216] Preferably, the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or its preferred embodiment, or the nucleic acid molecule or carrier according to the third and fourth aspects or the pharmaceutical composition of the eighth aspect, is used or prepared for administration at a concentration of about 0.05 to about 200 mg / ml; optionally, it is administered at any of the following concentrations: 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 in veterinary applications and 0.1 mg / ml in humans.

[0217] Preferably, the p75NTR(NBP)-Fc fusion protein according to the first or second aspect or its preferred embodiment, or the nucleic acid molecule or carrier according to the third and fourth aspects or the pharmaceutical composition of the eighth aspect, is used or prepared for administration at a concentration between about 0.1 and about 200 mg / kg body weight; preferably administered at any one of 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 + / - about 10% error, most preferably administered at about 10 mg / kg in veterinary applications and at 0.3 mg / kg in humans.

[0218] According to a ninth aspect of the present invention, a kit is provided comprising:

[0219] (a) a p75NTR(NBP)-Fc fusion protein according to the first or second aspect or its preferred embodiment, or a nucleic acid molecule or carrier according to the third and fourth aspects or a pharmaceutical composition according to the eighth aspect; and

[0220] (b) Instructions for use for administering an effective amount of the p75NTR(NBP)-Fc fusion protein, nucleic acid molecule, carrier or pharmaceutical composition to an individual for the prevention or treatment of pain, or for improving, controlling, reducing the incidence of pain, or delaying the development or progression of pain.

[0221] The kit may include one or more containers containing the p75NTR (NBP)-Fc fusion protein, nucleic acid, carrier, or pharmaceutical composition described herein, and instructions for use according to any method and purpose of the invention. The kit may also include instructions for selecting suitable individuals for treatment based on identifying whether an individual suffers from pain or pain symptoms, or is at risk for such pain. Instructions for use of the pharmaceutical composition may include information regarding the dosage, dosing regimen, and route of administration for the intended treatment.

[0222] According to another aspect of the invention, a p75NTR(NBP)-Fc fusion protein according to the first or second aspect or a preferred embodiment thereof, or a nucleic acid molecule or carrier according to the third and fourth aspects or a pharmaceutical composition according to the eighth aspect, is provided for any one or more of the following: prevention or treatment or for improving, controlling, reducing the incidence of or delaying the development or progression of a disease or its symptoms associated with any one or more of neurotrophic factors NGF, BDNF, NT-3, NT-4 / 5.

[0223] -NGF (nerve growth factor) binds to at least two types of receptors: p75NTR and TrkA, a transmembrane tyrosine kinase involved in axonal growth, branching, and elongation. Diseases and symptoms associated with NGF are known. NGF is expressed and associated with inflammatory diseases and pain [protein sequences NP_002497.2, NP_038637]. Furthermore, NGF has been shown to play a role in many cardiovascular diseases, such as coronary atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, and multiple sclerosis. Decreased plasma NGF (and BDNF) levels are associated with acute coronary syndrome and metabolic syndrome. NGF is also associated with a variety of mental illnesses, such as dementia, depression, schizophrenia, autism, Rett syndrome, anorexia nervosa, and bulimia nervosa, and is also associated with the development of Alzheimer's disease and neurodegenerative diseases. NGF has also been shown to accelerate wound healing, and there is evidence that it can be used to treat skin ulcers and corneal ulcers. It has also been shown to reduce neurodegeneration in rats and promote peripheral nerve regeneration.

[0224] BDNF (brain-derived neurotrophic factor) is a neurotrophic factor that supports neuronal survival and growth during nervous system development [protein sequences NP_001137277.1, NP_001041604]. BDNF binds to cell surface receptors TrkB and p75NTR and also regulates the activity of alpha-7 nicotinic receptors. Diseases and symptoms associated with BDNF are known. Studies have shown that BDNF plays an important role in the transmission of physiological and pathological pain, particularly in models of acute pain, inflammatory pain, and neuropathic pain, where BDNF synthesis is significantly increased; furthermore, BDNF has been shown to be upregulated in chronic pain as well as in other diseases such as eczema and psoriasis. Downregulation of BDNF 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.

[0225] Neurotrophic factor-4 (NT-4), also known as neurotrophic factor-5 (NT-5), is a neurotrophic factor that primarily signals via p75NTR and TrkB receptors and promotes the survival of peripheral sensory sympathetic neurons. The mature peptide of this protein is identical in all mammals examined, including humans, pigs, rats, and mice. [Protein sequence NP_006170, NP_937833]. NT-4 is synthesized by most neurons in the dorsal root ganglion (DRG), as well as neurons in the paravertebral and prevertebral sympathetic ganglia, the dorsal horn, and ventral horn of the spinal cord, and is expressed in a variety of tissues, including the prostate, thymus, placenta, and skeletal muscle. Diseases 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 breast cancer cell survival and is a target for inhibiting tumor growth. NT-4 / 5 is known to be involved in pain signaling systems, such as nociceptive pain. Upregulation of NT-4 / 5 is also seen in chronic inflammatory skin diseases, such as dermatitis, eczema, and prurigo lesions of atopic dermatitis. Downregulation of NT-4 / 5 is seen in Alzheimer's disease and Huntington's disease.

[0226] Neurotrophic factor-3 (NT-3) is a neurotrophic factor structurally associated with β-NGF, BDNF, and NT-4. It controls the survival and differentiation of mammalian neurons and the maintenance of the adult nervous system, and its expression in the human placenta may affect the development of embryonic neurons. Diseases and symptoms associated with NT-3 are known. NTF3-deficient mice induced by gene targeting technology exhibit severe limb motor deficits. NT-3 signals via the Trk receptor to promote the growth and survival of nerve cells 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 homologous receptor tyrosine kinase C (TrkC) are known to modulate neuropathic pain and nociceptive pain, as well as nociceptive and proprioceptive mechanisms; for example, increased NT-3 expression is observed in small DRG cells of neuropathic animals. NT3 expression is also associated with neuropathy, such as diabetic polyneuropathy and HIV-associated neuropathy, large fiber neuropathy (including atrophy), and it is also involved in the development of hyperalgesia (usually a lowered threshold for noxious stimuli), anomalous pain (non-noxious stimuli becoming noxious), and spontaneous pain (pain in the absence of a significant stimulus), and is a known modulator of muscle pain.

[0227] The present invention will now be described with reference to the following embodiments, which are for illustrative purposes but do not limit the invention.

[0228] Example

[0229] p75-NTR Fc fusion protein design

[0230] The specific allotype of the Fc portion of the p75-NTR Fc fusion protein is the IgG pCon vector IgGza (see above).

[0231] The design of the p75-NTR Fc fusion protein is carried out in several stages:

[0232] The specific construct for the p75-NTR sequence to be used in the Fc fusion protein was determined. Factors considered included:

[0233] The p75-NTR Fc fusion protein should be able to bind several neurotrophic factors, including at least NGF, BDNF, NT-3, and NT-4; the p75-NTR Fc fusion protein must retain its flexibility.

[0234] The extracellular domain of p75-NTR-Fc (SEQ ID No. 1) contains unwanted α-secretase cleavage sites, which must be removed from the sequence because they will be cleaved, thereby reducing the bioactivity and PK properties of the p75-Fc product in vivo.

[0235] Empirical adaptors suitable for connecting the extracellular p75-NTR domain and Fc in p75-NTR-Fc fusion proteins have been identified. Adaptor sequences containing sites that may be involved in post-translational modifications (PTMs) have been excluded.

[0236] Using different potential adaptor sequences, a defined p75-NTR construct, and appropriate portions of the Fc region, several variants of the p75-NTR Fc fusion protein were constructed on a computer. We attempted structural modeling and analysis of the C-terminus of the p75-NTR extracellular domain, the Fc hinge region, and the potential adaptors.

[0237] Structural modeling

[0238] Using the Lonza modeling platform, a structural model of the proposed p75-NTR Fc fusion protein was generated. Candidate structural template fragments for the p75-NTR and Fc regions were scored, ranked, and screened from internal antibody and protein databases (PDBs) based on sequence identity and qualitative crystallographic measures of the template structure (e.g., resolution in Å).

[0239] Sequence alignments were generated between the structural template fragment and the p75-NTR Fc fusion protein. The template fragment and sequence alignment were performed using MODELLER (Sali et al. 1993 J. Mol. Biol 234, 779-815). This approach creates conformational constraints based on the aligned structural template set. A set of structures satisfying the constraints is created using a conjugate gradient and simulated annealing optimization procedure. One or more model structures are selected from this set based on energy scores, derived from the protein structure score and the degree to which the conformational constraints are satisfied. The models are examined, and a sidechain optimization algorithm is used to optimize sidechains at different positions between the target and the template, minimizing energy. A suite of visualization and computational tools is used to evaluate the conformational variability of the structures and the core and local stacking of domains to select one or more preferred models.

[0240] p75-NTR Fc fusion protein design

[0241] A key requirement for selecting a linker for a variant is to allow for flexibility in the fusion partner in the Fc fusion protein, avoid introducing any residues that can carry PTM, and maintain a low risk of immunogenicity.

[0242] The process of protein expression is well known to those skilled in the art.

[0243] The process begins with codon optimization of the DNA sequence to achieve efficient expression in CHO cell lines, an N-terminal signal peptide sequence to guide the secretory expression of the protein, and appropriate restriction endonuclease sites to facilitate cloning into a glutamine synthase (GS) vector. A Kozak sequence is inserted between the 5' restriction endonuclease site and the "ATG" start codon. These sequences are submitted to Life Technologies for synthesis and are provided as cloning vectors. Vectors with different secretory sequences and different selection markers (e.g., dihydrofolate reductase (DHFR) instead of GS) are also available. The 1405-base-pair DNA fragment encoding the protein is transferred from the cloning vector to the GS expression vector pXC-17 via HindIII and EcoR1 digestion. The digestion product is ligated to the vector, and the ligation product is used to transform chemically competent *E. coli* cells (e.g., TOP10 cells). Correct insertion is confirmed by single-clone analysis; a positive clone is selected, and plasmid DNA is extracted and sequenced in both forward and reverse directions to verify the sequence presence. By linearizing the plasmid with the restriction endonuclease PvuI, sufficient linearized DNA was produced for the cell line construction process.

[0244] Protein production was achieved using Lonza Biologics' mammalian Chinese hamster ovary (CHO) K1SV glutamine synthase knockout (GS-KO) expression system. The CHOK1SV GS-KO host cell line is a derivative of the CHOK1SV host cell line, with its endogenous glutamine synthase gene knocked out. This host cell line is derived from Lonza Biologics' CHOK1SV GS-KO host working cell bank.

[0245] Affinity of p75NTR-Fc of sequences 1-19 (SEQ ID No. 1-19) to NGF

[0246] In the experiment, a Biacore chip was prepared in which protein A was coupled to flow cells 1 and 2 via amines. The single-cycle kinetics of NGF binding to the captured p75-Fc were measured.

[0247] Adhesion capability of chip surface (R max The effectiveness depends on the level of immobilization of the ligand (fusion protein). For kinetic studies, R is recommended. max The ideal immobilization level of the fusion protein can be calculated using the molecular weights of p75-Fc and NGF, ranging from 50 to 100 RU.

[0248] R max =(NGF molecular weight / fusion protein molecular weight) × immobilization level × stoichiometry: 50 =(13,500 / 102,000) × immobilization level × 1.

[0249] Therefore, the required fixation level = (102,000 / 13,500) x 50 = 378 RU. Sequence 1 (SEQ ID No. 1) and Sequence 3 (SEQ ID No. 3) p75NTR-Fc and NTR-Fc are fixed to the Protein A chip before single-cycle dynamics.

[0250] Using manual run, p75-Fc was captured onto flow cell 2 of the protein A chip until the desired level of approximately 380 RU was reached. The injection time was 22 seconds, at a flow rate of 10 μl / min and a p75-Fc concentration of 10 μg / ml, ultimately capturing 418 RU of the fusion protein onto the surface of protein A.

[0251] First, NGF concentrations of 10, 5, 2.5, 1.25, and 0.625 nM were tested. These concentrations were determined because the fusion protein's K+... D The values ​​are roughly within this NGF concentration range.

[0252] The single-cycle dynamics method involves:

[0253] - 0.625 nM NGF was injected into the captured p75-Fc at a rate of 30 μl / min for 120 seconds.

[0254] - Then repeat this process, using 1.25 nM NGF injections, followed by 2.5, 5, and 10 nM NGF injections.

[0255] - After the final concentration of NGF is injected, a 600-second dissociation phase is performed by flowing run buffer (HBS-EP) through the chip.

[0256] After completion, the chip was regenerated back to its protein A surface by injecting 10 mM glycine HCl at a rate of 30 μl / min, pH 2, for 60 seconds.

[0257] Then, p75-Fc was captured onto the chip by injection at a flow rate of 10 μl / min and a concentration of 10 μg / ml for 38 seconds. This achieved the expected level of 430 RU. The single-cycle kinetics procedure described above was then repeated.

[0258] Data Analysis

[0259] The fusion protein-NGF binding data were analyzed using Biacore T200 evaluation software v1 in the following manner:

[0260] - Record data on the binding of NGF to the fusion protein in flow cell 2 (Fc=2), and data on the flow of NGF through the control flow cell 1 (Fc=1; protein A alone).

[0261] Then subtract the data of Fc=1 from Fc=2 to get the combined data of "2-1".

[0262] - Then subtract the 2-1 binding data from the injection of 0 nM (HBS-EP run buffer alone) from all 2-1 binding data to control for any baseline drift throughout the experiment.

[0263] Finally, this data was fitted to a 1:1 binding model to calculate binding properties, including binding rate (ka), dissociation rate (kd), and affinity (K). D ).

[0264] The sequence exhibits an appropriate affinity for NGF.

[0265] P75NTR-Fc has analgesic effects.

[0266] The aim of this study was to investigate the effect of long-term exposure to p75NTR-Fc on the efficacy of treatment for pain in rats with iodoacetic acid (MIA)-induced osteoarthritis (OA).

[0267] Previously, we have shown that spontaneous pain can be assessed by measuring static weight-bearing using a disability assessment device, and that this is correlated with the histopathology of the knee. Preclinical studies using novel pain therapies have been criticized for their susceptibility to data bias. To address this issue, left and right knees were randomly selected to induce OA, and all operators performing routine in vivo tasks were unaware of the status of each knee. According to the literature, OA induction is typically performed only in the right knee, but in previous studies, we found no consistent difference in OA induction between the left and right knees regardless of the time point used or the dose of MIA.

[0268] Preparation of MIA

[0269] Prepare a stock solution of MIA at a concentration of 0.3 mg / 50 μl ETF-PBS (for each intra-articular injection), equivalent to 6 mg / ml. Weigh 302 mg of MIA and dissolve it in 50.3 ml of ETF-PBS. Prepare the MIA one day in advance and store it at 4°C protected from light until use.

[0270] animal

[0271] This study used 185 male Wistar rats (from Charles River, UK) weighing 110–130 grams upon arrival. Each animal was examined upon arrival and appeared healthy. They were randomly assigned to cages of two, each rat assigned a unique identification number by a tattoo on its tail. Animals were acclimatized in the animal room for at least 10 days prior to the start of Day 0 of the study. Once acclimatized, the rats were transferred to the storage / operation room, where all internal procedures were performed. Lighting was provided by fluorescent lamps with a 12-hour light-dark cycle (on at 07:00, off at 19:00), in accordance with the recommendations of the Home Office Animal (Scientific Procedures) Act 1986. The room was air-conditioned, and air temperature (21°C ± 2°C) and relative humidity were measured regularly.

[0272] Rats were fed irradiated feed (Scientific Animal Food and Engineering, Augy, France) and had free access to autoclaved water. Each batch of feed underwent routine ingredient and contaminant testing and screening. Nests and cages were autoclaved, and each cage had individual ventilation (IVC system).

[0273] Experimental Design

[0274] The study was designed with 21 animal groups: control human antibody (n=6), 19 groups of p75NTR-Fc tested at 0.3 mg / kg p75NTR-Fc (sequences 1-19, all with GGG linkers, linked to SEQ ID no 20 as the Fc region) and 3 mg / kg PG-007 (a biosimilar anti-NGF antibody of Pfizer Tanezumab).

[0275] The antibody and p75NTR-Fc were injected subcutaneously every 5 days for 25 days.

[0276] Body weight was measured and baseline blood samples were collected from the tail vein on the morning of Day -2. Baseline static weight-bearing was measured at approximately the same time on Day -1. On Day 0, also at approximately the same time, all rats received either their respective antibody or p75NTR-Fc fusion protein treatment. Three hours later, all animals received an intra-knee injection of 0.3 mg MIA (contralateral knee injection of ETF-PBS).

[0277] Randomized treatment

[0278] Before the study began, the rats were weighed and two rats per cage were randomly assigned to the treatment group to ensure that the average weight of the animals in each group was approximately equal. In addition to assigning each rat to a specific treatment group, further randomization was performed by injecting MIA into either the left or right knee of each rat (ETFPBS was injected into the contralateral knee of each rat). The treatment group assignment and the knee joint receiving treatment for each rat were determined using the random number generator in Microsoft Excel for Mac (version 14.1.1). Randomization and assignment were performed by staff who did not have contact with the animals.

[0279] Two 7 ml polypropylene vials were labeled for each animal, one for the left knee and one for the right knee (88 vials in total). Two people (one responsible for scoring and verifying the randomization master table, and the other responsible for dispensing the intra-articular injection solution) prepared the 88 vials. Dispensing was performed sequentially, first filling the MIA vials, then filling the remaining vials (the vials for the contralateral knee of each animal) with ETF-PBS. Throughout the in vivo study, the scientists were unaware of the treatment status of any of the animals.

[0280] animal experiments

[0281] Intra-articular injection of the knee

[0282] All rats were anesthetized using the Boyles device with inhaled isoflurane. Hair was clipped from both knees of each animal, and the knees were wiped with ethanol. 50 μl of 0.3 mg MIA in ETF-PBS solution or ETF-PBS solution alone was injected into each knee via the infrapatellar ligament using a 0.5 ml sterile Becton Dickinson Micro-Fine insulin injector (with a 27 G needle).

[0283] Assessment of spontaneous pain

[0284] To determine spontaneous pain, the weight-bearing capacity of each animal's left and right hind limbs was measured using a disability testing device (Linton Instruments, UK). Rats were placed in appropriately sized plexiglass animal boxes on the disability testing device, with their hind feet positioned on separate sensors. The boxes were sized to allow the rats to sit comfortably without being cramped, but also without sufficient space to turn around. After the rats had stabilized and calmed down, the weight-bearing capacity of each rat's hind limbs was recorded over 5 seconds, along with the average force exerted by both hind limbs (in grams). At each time point, the weight distribution of each rat's hind paws was measured five times (the validity of which has been previously demonstrated), and the average of the five readings was calculated. Individual weight-bearing data were converted into weight distribution by dividing the weight of the right limb by the total weight of both hind limbs.

[0285] Measurement of spontaneous pain after MIA-induced OA

[0286] Hind limb weight distribution was measured using a disability assessment device, and spontaneous pain was evaluated. Assessments were performed at baseline and 3 weeks after treatment for MIA.

[0287] These studies clearly demonstrate that p75NTR-Fc has an analgesic effect in a rat model of OA with MIA. At similar doses (3 mg / kg subcutaneous injection), p75NTR-Fc exhibits superior analgesic efficacy compared to the anti-NGF antibody (PG-007: a biosimilar of Pfizer's anti-NGF antibody Tanezumab).

Claims

1. A p75NTR neurotrophic factor binding protein (NBP)-Fc fusion protein, comprising: (a) The p75NTR (NBP) portion selected from one of Seq ID Nos. 1-19; and (b) Immunoglobulin Fc fraction selected from one of Seq ID Nos. 20-24; in, The p75NTR(NBP) and Fc sections are connected via a connector, which includes type G. x The peptide, where x is 1, 2, 3, 4, 5 or 6.

2. The p75NTR(NBP)-Fc fusion protein according to claim 1, wherein the linker is GGG.

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

4. Use of the p75NTR(NBP)-Fc fusion protein as described in any one of claims 1-3 for pain treatment.

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

6. A reproducible expression vector for transfecting cells, said vector comprising the nucleic acid molecule as described in claim 5.

7. Use of the nucleic acid molecule as described in claim 5 or the carrier as described in claim 6 for pain treatment.

8. Use of the p75NTR(NBP)-Fc fusion protein as described in any one of claims 1-3, the nucleic acid molecule as described in claim 5, or the vector as described in claim 6 for the treatment of osteoarthritis.

9. A pharmaceutical composition comprising the p75NTR(NBP)-Fc fusion protein as described in any one of claims 1-3, the nucleic acid molecule as described in claim 5, or the carrier as described in claim 6, and a pharmaceutically acceptable carrier and / or excipient.

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