Therapeutic molecules containing variants of the extracellular domain of the p75 neurotrophin receptor (p75NTR)

Abstract

The present invention relates to effective therapies for pain, inflammation, and osteoarthritis, particularly in companion animals such as dogs. The present invention provides isolated polypeptides comprising a fusion protein containing a variant p75 neurotrophin receptor (p75NTR) extracellular domain, or a portion thereof, to an Fc domain. Nucleic acids encoding the protein and methods of using the same are also included in the present invention.

Description

Technical Field

[0001] Related Applications and Incorporation by Reference This application claims the benefit of priority from UK Application No. 2301838.5 (filed on February 9, 2023), UK Application No. 2301839.3 (filed on February 9, 2023), US Application No. 18 / 187,377 (filed on March 21, 2023) and US Application No. 18 / 187,414 (filed on March 21, 2023).

[0002] All documents cited or referenced in this specification (the "documents cited in this specification"), and all documents cited or referenced in the documents cited in this specification, and any manufacturer's instructions, descriptions, product specifications, and product sheets regarding any product mentioned in this specification, or any document incorporated by reference in this specification, are incorporated herein by reference and may be employed in the practice of the present invention. More specifically, all documents referenced are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference.

[0003] Declaration of Sequence This application includes a sequence listing submitted electronically, which is incorporated herein by reference in its entirety. The XML copy was created on February 1, 2024, named P44829WO1 SL.xml, and is 194,555 bytes in size.

[0004] The present invention relates to effective pain therapies in humans and companion animals.

Background Art

[0005] The need for therapies for pain relief is very great. Further, effective pain treatment with minimal side effects is required.

[0006] Pain relief treatments currently include non-steroidal anti-inflammatory drugs (NSAIDs), several of which help control pain and inflammation associated with osteoarthritis. Some NSAIDs are approved by the FDA. However, there is a need for more effective pain treatments with minimal side effects.

[0007] Neurotrophins are a family of proteins involved in the proliferation, maintenance, and survival of neurons. Since the discovery of nerve growth factor (NGF) in the 1950s, many biological processes involving NGF have been identified. NGF is crucial for neuronal proliferation and maintenance and also plays a role in the inflammation and maintenance of pancreatic beta cells. NGF binds to at least two receptors, including tropomyosin receptor kinase A (TrkA) and the low-affinity NGF receptor (LNGFR / p75NTR). The neurotrophin family also includes structurally related brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) (also known as NT-5 or NT-4 / 5). BDNF and NT-4 function primarily via tropomyosin receptor kinase B (TrkB). NT-3 binds to tropomyosin receptor kinase C (TrkC) with high affinity, but can also signal via TrkB. All four neurotrophins bind to p75NTR with low affinity.

[0008] NGF induces peripheral sensitization both in vitro and in vivo, as indicated by increased responses of DRG neurons to temperature or capsaicin in its presence. NGF also induces transcriptional regulation after retrograde axonal transport, as shown by immunohistochemistry demonstrating upregulation of BDNF after subarachnoid NGF treatment. Furthermore, NGF may induce the sprouting of peripheral afferent nerves into diseased joints and cancerous tissues (Denk et al, Annual Review of Neuroscience, Vol.40:307-325, 2017).

[0009] NGF is expressed at low levels in adulthood, but injury, inflammation, or NGF release triggers the activation of inflammatory cells. These cells then produce and secrete NGF, resulting in both short-term and long-term effects. While NGF has a well-known multifunctional role in nociceptive processes, the precise downstream signaling pathways of NGF receptor activation that mediate nociception are complex and not fully understood. The role of NGF in nociception, as well as in the development and / or maintenance of chronic pain, has made it an attractive target for pain therapy for the treatment of chronic pain conditions (Barker et al, Journal of Pain Research, 2020:13 1223-1241).

[0010] Very low doses of monoclonal antibodies (mAbs) against NGF can alleviate chronic pain. However, during clinical trials in humans, a small number of patients treated with mAbs against NGF developed rapidly progressive joint degeneration due to interactions with NSAID treatment. Complete NGF elimination has been shown to cause impaired bone and cartilage repair (Denk et al, Annual Review of Neuroscience, Vol.40:307-325, 2017). Therapies based on species-specific mAbs targeting NGF are currently under development for use in dogs and cats for the management of osteoarthritis (OA)-related pain (Enomoto et al, Vet Rec. 2019 Jan 5;184(1):23 and WO2019177690). However, given the side effects observed in clinical trials in humans, there is a need to develop alternative therapies targeting NGF.

[0011] The reference or identification of any document in this application does not constitute an admission that such document is available as prior art for the present invention. [Overview of the Initiative]

[0012] There is a need for effective pain therapy in humans and companion animals. This invention provides a pain therapy with reduced side effects compared to anti-NGF antibody therapy. The protein of this invention binds to NGF, which is elevated in pain states, and therefore binds to excess NGF without completely blocking NGF signaling, restoring normal NGF levels. While we do not wish to be constrained by theory, the inventors believe this ensures the level of NGF signaling necessary for healthy function. Furthermore, the fusion protein of this invention is considered highly effective even at very low doses.

[0013] The inventors used an analgesic strategy to reduce circulating NGF, but not to completely deplete it. For this purpose, they used the extracellular domain (ECD) of the p75 neurotrophin receptor (p75NTR) and fused it to Fc to increase its half-life. p75NTR binds NGF, proNGF, and other brain-derived neurotrophic factors (BDNF, NT3, NT4), mediating different cellular activities. The inventors further used a structure-induced mutation strategy to generate p75NTR variants with advantageous biochemical properties. Point mutations were introduced into the extracellular domain of p75NTR, which was used to modulate the binding affinity of p75NTR to its ligand, NGF. These variant p75NTR molecules have altered binding properties compared to wild-type p75NTR and may enable efficient NGF binding without complete removal of NGF, which has been shown to cause negative side effects such as joint aggravation.

[0014] In a first embodiment, the present invention relates to an isolated polypeptide comprising a p75 neurotrophin receptor (p75NTR) extracellular domain, wherein the p75NTR extracellular domain comprises one or more variant amino acids at positions 75, 109, 133, and / or 134.

[0015] The present invention also relates to an isolated nucleic acid encoding an isolated polypeptide comprising a p75NTR extracellular domain, wherein the p75NTR extracellular domain comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134.

[0016] The present invention also relates to a vector comprising the nucleic acid described above.

[0017] The present invention also relates to a host cell comprising the nucleic acid or vector described above.

[0018] One aspect of the present invention relates to a fusion protein comprising a p75NTR extracellular domain, wherein the p75NTR extracellular domain comprises one or more variant amino acids at positions 75, 109, 133, and / or 134, and comprises a half-life extension portion.

[0019] The present invention also relates to nucleic acids encoding the aforementioned fusion protein.

[0020] The present invention also relates to a vector comprising a nucleic acid encoding the aforementioned fusion protein.

[0021] The present invention also relates to a host cell containing nucleic acid encoding the aforementioned fusion protein, or a vector containing nucleic acid encoding the aforementioned fusion protein.

[0022] One aspect of the present invention relates to a pharmaceutical composition comprising an isolated polypeptide or a fusion protein as described above and herein.

[0023] One aspect of the present invention relates to a method for treating NGF-related disorders in a subject, comprising administering an isolated p75NTR protein, a fusion protein, or a pharmaceutical composition as described above and herein.

[0024] One aspect of the present invention relates to the use of the isolated p75NTR protein described above and herein, the fusion protein described above and herein, or the pharmaceutical composition described above and herein in the treatment of NGF-related disorders in a subject.

[0025] One aspect of the present invention relates to the isolated p75NTR protein described above and herein, or the fusion protein described above and herein for use in the treatment of NGF-related disorders in a subject.

[0026] One aspect of the present invention is a method of inhibiting NGF activity in a subject, the method comprising administering the isolated p75NTR protein described above and herein, the fusion protein described above and herein, or the pharmaceutical composition described above and herein.

[0027] One aspect of the present invention relates to a kit comprising the isolated p75NTR protein described above and herein, the fusion protein described above and herein, or the pharmaceutical composition described above and herein, and optionally instructions for use.

[0028] In certain embodiments, the p75NTR is of human origin. In certain embodiments, the p75NTR is of non-human primate origin. In certain embodiments, the p75NTR is of animal origin. In certain embodiments, the p75NTR is of companion animal origin, including but not limited to dog, cat, horse, cow, sheep, or camel.

[0029] In certain embodiments, the isolated p75NTR protein of the present invention may be used to treat or inhibit NGF activity in humans. In certain embodiments, the isolated p75NTR protein of the present invention may be used to treat or inhibit NGF activity in non-human primates. In certain embodiments, the isolated p75NTR protein of the present invention may be used to treat or inhibit NGF activity in animals. In certain embodiments, the isolated p75NTR protein of the present invention may be used to treat or inhibit NGF activity in companion animals.

[0030] One aspect of the present invention relates to the treatment of osteoarthritis using an isolated p75NTR protein. In certain embodiments, the isolated p75NTR protein described herein is used to treat or prevent osteoarthritis in humans. In certain embodiments, the isolated p75NTR protein described herein is used to treat or prevent osteoarthritis in non-human primates. In certain embodiments, the isolated p75NTR protein described herein may be used to treat or prevent osteoarthritis in animals. In certain embodiments, the isolated p75NTR protein described herein may be used to treat or prevent osteoarthritis in companion animals. In certain embodiments, the method may include slowing or stopping disease progression, reversing disease progression, cartilage regrowth, and / or curative treatment. In certain embodiments, the method may be determined by the rate of cartilage loss or regrowth. In certain embodiments, the treatment of osteoarthritis may include slowing or stopping disease progression, reversing disease progression, cartilage regrowth, and / or curative treatment. In certain embodiments, treatment may be determined by the rate of cartilage loss or regrowth. In certain embodiments, treatment of osteoarthritis may include slowing or halting disease progression, reversing disease progression, cartilage regrowth, and / or curative treatment. In certain embodiments, treatment may be determined by the rate of cartilage loss or regrowth.

[0031] Therefore, the object of the present invention is not to include any previously known product, process for manufacturing a product, or method for using a product in the present invention, so that the applicant reserves the right to disclose any previously known product, process, or method for disavowing any previously known product, process, or method. The present invention is not intended to include any product, process, or method for manufacturing a product or using a product that does not satisfy the written description and enablement requirements of the USPTO (Section 112, paragraph 1) or the EPO (Section 83 of the EPC), and as a result, the applicant should be further noted to reserve the right to disclose herein any previously described product, process for manufacturing a product, or method for using a product. In practice of the present invention, it may be advantageous to comply with Section 53(c) of the EPC and Sections 28(b) and (c) of the EPC Rules. All rights to expressly disavow any embodiment that is the subject matter of any granted patent(s) of the applicant in the lineage of this application, or in any other lineage, or in any prior application of any third party are expressly reserved. Nothing in this specification should be construed as a warranty.

[0032] In this disclosure, particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” may have the meanings assigned to them in U.S. patent law, for example, they may mean “includes,” “included,” and “including,” and terms such as “consisting essentially of” and “consists essentially of” may have the meanings assigned to them in U.S. patent law, for example, they may allow elements not expressly described but exclude elements found in the prior art or elements that affect the basic or novel features of the present invention.

[0033] These and other embodiments are disclosed or evident from the embodiments for carrying out the following inventions and are encompassed by those embodiments.

[0034] The present invention is illustrated in the following non-limiting figures. [Brief explanation of the drawing]

[0035] [Figure 1] Sequence alignments of the p75NTR extracellular domains of human (SEQ ID NO: 73), dog (SEQ ID NO: 7), cat (SEQ ID NO: 38), horse (SEQ ID NO: 5), camel (SEQ ID NO: 101), cattle (SEQ ID NO: 36), and pig (SEQ ID NO: 110). Amino acids 1-160 are shown. The stalk region and alpha and gamma-secretase cleavage sites are not shown. Sequence alignments performed using EXPASY Clustal Omega. Mutant residues according to the present invention are indicated by boxes. [Figure 2] A 3D model of canine p75NTR-ECD in a complex with a canine NGF (shown as an illustration) base generated using homology modeling for human pdb 1SG1 (shown as an illustration). Residues of p75NTR-ECD important for binding to NGF are circled. [Figure 3-1] Binding affinity of the p75NTR variant. Each panel represents a representative SPR sensorogram (raw data shown as dots, fitted data using 1:1 Langmuir fitting shown as lines) containing five different concentrations of PetML119wt, the variant, and bezin-vetomab. Table 1 shows the kinetic values ​​obtained from the fitting. [Figure 3-2]Binding affinity of the p75NTR variant. Each panel represents a representative SPR sensorogram (raw data shown as dots, fitted data using 1:1 Langmuir fitting shown as lines) containing five different concentrations of PetML119wt, the variant, and bezin-vetomab. Table 1 shows the kinetic values ​​obtained from the fitting. [Figure 4] Thermal stability analysis of the p75NTR variant. The intrinsic fluorescence measurements obtained from each molecule are shown in the graph. The calculated Tm for these molecules are shown in the summary table in Figure 4. [Figure 5] TF-1 cell proliferation assay. The Y-axis shows the normalized mean fluorescence intensity value relative to the control, while the X-axis shows the concentration of PetML119wt, variant, or bezin-vetomab used. A fitting curve using a sigmoid function and the resulting IC50 and ICMAX values ​​are shown for each molecule tested. [Figure 6] TrkA activation assay. The Y-axis shows the normalized luminescence AU value relative to the control, while the X-axis shows the concentration of PetML119wt, variant, or bezinbetomab used. A fitting curve using a sigmoid function and the resulting IC50 and ICMAX values ​​are shown for each molecule tested. [Figure 7] Schematic diagram of an in vivo trial. [Figure 8] Determination of nerve growth factor (h-rNGF) binding affinity in humans and rats. Each panel represents a representative SPR sensorogram (raw data shown as dots, fitted data using 1:1 Langmuir fitting shown as lines) containing five different concentrations of PetML308, PetML309, and PetML319. Tables 6 and 7 show the kinetic values ​​obtained from the fitting. [Figure 9] Thermal denaturation by UnCle. The intrinsic fluorescence measurements obtained from each molecule are shown in the graph for each molecule tested. Table 8 shows the melting temperature for each molecule. [Figure 10] TrkA activation assay. The Y-axis shows the normalized luminescence AU value relative to the control, while the X-axis shows the concentration of one of the PetML308, PetML309, or PetML319 used. Fitting curve using a sigmoid function, and the resulting IC50 and ICMAX values ​​for each molecule tested. [Figure 11-1] In vivo analgesic effect. Dynamic weight-bearing analysis in rats using a monosodium iodoacetate (MIA) osteoarthritis model. Figure 11A: Day 3. Figure 11B: Day 6. [Figure 11-2] In vivo analgesic effect. Dynamic weight-bearing analysis in rats using monosodium iodoacetate (MIA) osteoarthritis model. Figure 11C Day 14. Figure 11D Day 21. [Figure 12] pK analysis of rats using a MIA osteoarthritis model. Figure 12A: Plasma p75-Fc pK. Figure 12B: Plasma NGF. [Figure 13-1] Joint diameter measurement using an MIA osteoarthritis model. [Figure 13-2] Joint diameter measurement using an MIA osteoarthritis model. [Figure 14-1] Investigation of the anti-inflammatory effects of the p75NTR-Fc molecule in the DH82 cell line. [Figure 14-2] Investigation of the anti-inflammatory effects of the p75NTR-Fc molecule in the DH82 cell line. [Figure 14-3] Investigation of the anti-inflammatory effects of the p75NTR-Fc molecule in the DH82 cell line. [Figure 15] Disease-modifying osteoarthritis drugs (DMOADs) in rats. [Modes for carrying out the invention]

[0036] Next, the present invention will be described further. The following paragraphs will define different aspects of the present invention in more detail. Each of the aspects thus defined may be combined with any other aspect or more of the aspects unless otherwise expressly indicated. In particular, any feature shown to be preferred or advantageous may be combined with any one or more other features shown to be preferred or advantageous.

[0037] In general, the nomenclature and techniques used in relation to cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and the chemistry and hybridization of proteins and nucleic acids described herein are well known and commonly used in the art. The methods and techniques described herein are generally carried out in accordance with conventional methods well known in the art, and unless otherwise indicated, are carried out as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009), and Antibody Engineering, 2nd Ed., Vols. 1 and 2, Ontermann and Duebel, eds., Springer-Verlag, Heidelberg (2010).

[0038] Enzyme reactions and purification techniques are carried out in accordance with the manufacturer's specifications, either as commonly achieved in the art or as described herein. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as their laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as in patient treatment.

[0039] The present invention provides a biotherapeutic agent for human and veterinary use, comprising a p75NTR fusion protein for use in the treatment of humans or companion animals such as dogs, cats, cattle, horses, pigs, or camels.

[0040] P75NTR Variant In a first aspect, the present invention relates to an isolated polypeptide comprising the extracellular domain of the p75 neurotrophin receptor (p75NTR), wherein the isolated polypeptide comprises one or more variant amino acids at positions 75, 109, 133, and / or 134 within the p75NTR.

[0041] As used herein, the term p75NTR protein refers to a p75NTR protein that binds NGF, proNGF, and / or other neurotrophins (BDNF, NT-3, and / or NT-4 / 5). As used herein, this means that a p75NTR protein can bind to NGF and inhibit NGF biological activity and / or downstream pathways mediated by NGF signaling. NGF-binding proteins reduce NGF biological activity, including downstream pathways mediated by NGF signaling, and / or reduce the amount of NGF that is circulating and can bind to its receptors trkA and NGFR (p75NTR). In certain embodiments, the term p75NTR protein refers to a p75NTR protein that binds proNGF, i.e., an NGF precursor molecule. As used herein, this means that a p75NTR protein can bind to proNGF and inhibit proNGF biological activity and / or downstream pathways mediated by NGF signaling. ProNGF-binding proteins reduce NGF biological activity, including downstream pathways mediated by NGF signaling, and / or reduce the amount of NGF circulating that can bind to its receptors, trkA and NGFR (p75NTR). The p75NTR protein of the present invention can bind to proNGF and / or NGF. The p75NTR protein of the present invention may exhibit preferential binding to NGF or proNGF. NGF promotes neuronal survival and differentiation. NGF is first synthesized as proNGF, a precursor which is the dominant form in the central nervous system. NGF and proNGF bind to TrkA / p75NTR to mediate cell survival and to sorbitol / p75NTR to promote apoptosis.

[0042] As used herein, the term companion animal refers to a dog, cat, or horse. In one embodiment, the companion animal is a dog. In one embodiment, the companion animal is a cat. In another embodiment, the animal being treated may be a cow or a pig. In another embodiment, the animal being treated may be a camel.

[0043] The term “isolated” molecule, protein, or polypeptide refers to a molecule, protein, or polypeptide that substantially does not contain other proteins or polypeptides having different antigen specificities. Furthermore, the protein or polypeptide may substantially not contain other cellular material and / or chemical substances. Accordingly, the proteins, nucleic acids, and polypeptides described herein are preferably isolated. Thus, as used herein, “isolated” protein or polypeptide means a protein or polypeptide identified, isolated, and / or recovered from components of its natural cell culture environment. Contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the protein or polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0044] The terms “polypeptide” and “protein” are used interchangeably and refer to polymers of amino acid residues, not limited to a minimum length. Peptides, oligopeptides, dimers, polymers, etc., are also included in this definition, whether they are composed of linearly arranged amino acids linked by peptide bonds, whether produced biologically, recombinantly, or synthetically, and whether they are composed of naturally occurring or non-naturally occurring amino acids. Both full-length proteins and their fragments are included in the definition. These terms also include co-translational and post-translational modifications of polypeptides, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, and proteolytic cleavage (e.g., cleavage by furin or metalloproteinases and prohormone-converting enzymes (PCs)). Furthermore, for the purposes of this invention, “polypeptide” includes proteins that have undergone modifications to their natural sequence, such as deletions, additions, substitutions, and post-translational modifications (which are generally essentially conserved, as is known to those skilled in the art), as long as the protein maintains the desired activity. These modifications can be intentional, such as through site-directed mutagenesis, or accidental, such as mutations in the host producing the protein, or errors in PCR amplification or other recombinant DNA methods. Polypeptides or proteins are composed of amino acids arranged in a linear chain linked by peptide bonds, but in contrast to peptides, they have a clearly defined conformation.

[0045] In contrast to peptides, proteins generally consist of chains of 50 or more amino acids. For the purposes of this invention, the term "peptide" as used herein typically refers to a sequence of amino acids consisting of a single chain of D- or L-amino acids, or a mixture of D- and L-amino acids linked by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length.

[0046] This invention relates to a molecule comprising a variant of the p75NTR extracellular domain. The variant p75NTR may be, but is not limited to, a human p75NTR variant, a canine p75NTR variant, a feline p75NTR variant, a horse p75TR variant, a bovine p75NTR variant, or a camel p75NTR variant. The canine, feline, bovine, horse, and camel p75NTR proteins all have a very high degree of sequence similarity, as shown in Figure 1. The variant extracellular domain of p75NTR contains different amino acids at one or more positions in the polypeptide chain compared to that of wild-type p75NTR. The variant amino acids may be present at one or more of positions 75, 109, 133, and / or 134 in the extracellular domain of p75NTR. Positions 75, 109, 133, and 134 are highlighted in Figure 1. The position numbering is based on the amino acid sequences of canine, feline, horse, bovine, and human p75NTR shown in SEQ ID NOs: 1, 3, 5, 36, and 71, respectively. The position numbering is based on the amino acid sequences of human p75NTR ECD (SEQ ID NO: 73), canine p75NTR ECD (SEQ ID NO: 7), feline p75NTR ECD (SEQ ID NO: 38), horse p75NTR ECD (SEQ ID NO: 5), camel p75NTR ECD (SEQ ID NO: 101), bovine p75NTR ECD (SEQ ID NO: 36), and pig p75NTR ECD (SEQ ID NO: 110). See Figure 1.

[0047] The term “variant amino acid,” as used herein, refers to any amino acid that is not present in the wild-type amino acid sequence (in this case, the wild-type p75NTR sequence). The wild-type sequence may be the wild-type mammalian p75NTR sequence. The wild-type sequence may be the wild-type human p75NTR sequence. The wild-type sequence may be the wild-type dog p75NTR sequence, wild-type cat p75NTR sequence, wild-type horse p75NTR sequence, wild-type cattle p75NTR sequence, or wild-type camel sequence disclosed herein. The wild-type dog, cat, horse, cattle, and human p75NTR sequences are shown in Figure 1, and in Sequence IDs 1, 3, 5, 36, and 71. When referring to the wild-type p75NTR sequence, this may refer to the entire sequence or a portion of it, for example, the extracellular domain.

[0048] Variant amino acids may be the result of substituting, replacing, or modifying the original (e.g., wild-type or germline) amino acids in a protein sequence with different amino acids. The process of amino acid substitution or replacement can be carried out using standard techniques available to those skilled in the art, such as recombinant DNA techniques. Amino acid modification may also be carried out post-translation, and various chemical or bioconjugation methods can be used to modify such amino acids. The amino acids are altered compared to the natural (wild-type / germline) sequence as found in nature in the wild-type (WT). In this specification, “wild-type,” “WT,” or “natural” means the naturally occurring amino acid or nucleotide sequence, including allelic mutations. Wild-type proteins or polypeptides have an amino acid or nucleotide sequence that is not intentionally modified.

[0049] As used herein, the term “amino acid” refers to one of the 20 naturally occurring (canonical) amino acids or any non-natural analogue (non-canonical amino acid) that may be present at a specific defined position within a peptide sequence. “Amino acid” encompasses both naturally occurring and synthetic amino acids. However, in most cases, only naturally occurring amino acids are used when proteins are recombinantly produced. Variant amino acids may include one of the 20 canonical amino acids. Variant amino acids may include non-canonical amino acids (also known as non-natural amino acids), such as hydroxyproline, hydroxylysine, phosphoserine, phosphothreonine, phosphotyrosine, N-acetyllysine, and methyllysine.

[0050] The variant amino acid at position 75 may be an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The variant amino acid at position 109 may be selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, and valine. The variant amino acid at position 133 may be selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and tyrosine. The variant amino acid at position 134 may be selected from alanine, arginine, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0051] The variant amino acid at position 75 may be an amino acid containing a polar side chain. For example, the variant amino acid at position 75 may be selected from serine, threonine, tyrosine, tryptophan, asparagine, glutamine, or cysteine. Preferably, the variant amino acid at position 75 contains a small polar side chain, such as serine or threonine. In one embodiment, the variant amino acid at position 75 is threonine.

[0052] The variant amino acid at position 109 may be an amino acid containing an aromatic side chain. For example, the variant amino acid at position 109 may be selected from histidine, tyrosine, phenylalanine, or tryptophan. In one embodiment, the variant amino acid at position 109 is histidine. In one embodiment, the variant amino acid at position 109 is tyrosine.

[0053] The variant amino acid at position 133 may be an amino acid containing a charged side chain. For example, the variant amino acid at position 133 may be selected from arginine, histidine, lysine, aspartic acid, or glutamic acid. In one embodiment, the variant amino acid at position 133 may be an amino acid containing a negatively charged side chain. For example, the variant amino acid at position 133 may be selected from arginine, histidine, or lysine. In one embodiment, the variant amino acid at position 133 is arginine.

[0054] The variant amino acid at position 134 may be an amino acid containing a hydrophobic side chain. For example, the variant amino acid at position 134 may be selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one embodiment, the variant amino acid at position 134 may be an amino acid containing a non-aromatic hydrophobic side chain. For example, the variant amino acid at position 134 may be selected from alanine, valine, isoleucine, leucine, or methionine. In one embodiment, the variant amino acid at position 134 is leucine or isoleucine. In one embodiment, the variant amino acid is leucine.

[0055] Amino acid modifications generally refer to and include substitutions, insertions, and deletions, with the former often preferred. The variant extracellular domain of the p75NTR of the present invention as described herein comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134, and may contain additional variant amino acids within the polypeptide sequence. These additional variant amino acids may include any number of further modifications, as long as the function of the protein described herein remains. It will be apparent to those skilled in the art that additional mutations may occur spontaneously within the amino acid sequence, or that additional mutations may be genetically engineered to, for example, increase stability or decrease glycosylation. In one embodiment, since the goal is often to alter function with a minimum number of modifications, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications are commonly used. The variant polypeptide sequence will preferably have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with respect to the wild-type or parent sequence. It should be noted that the percentage of identity will depend on the number of amino acids, depending on the size of the sequence.

[0056] In this specification, “protein variant” or “variant protein” means a protein that differs from the wild-type protein in that it has at least one amino acid modification. The isolated polypeptide according to the present invention contains the extracellular domain of the p75 neurotrophin receptor (p75NTR), which contains a variant amino acid at one or more of the positions 75, 109, 133, and / or 134 within the p75NTR, but those skilled in the art will understand that further variant amino acids may be present within the p75NTR compared to the wild-type p75NTR. The positions referred to herein are those of the p75NTR extracellular domain. The parent polypeptide may be naturally occurring, or may be a wild-type (WT) polypeptide, or may be a modified version of a WT polypeptide. A variant polypeptide may refer to the polypeptide itself, a composition containing the polypeptide, or the amino sequence encoding it. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g., about 1 to about 10 amino acid modifications compared to the parent, preferably about 1 to about 5 amino acid modifications. The variant polypeptide sequences described herein preferably have at least about 80% identity with the parent polypeptide sequence, most preferably at least about 90% identity, and more preferably at least about 95% identity. The variants do not contain human sequences.

[0057] As used herein, “parent polypeptide” and “parent protein” mean an unmodified polypeptide that is subsequently modified to produce a variant. Such parent polypeptide may be a naturally occurring polypeptide, or a variant or manipulated version of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition containing the parent polypeptide, or the amino acid sequence encoding it.

[0058] The native form of the p75 neurotrophin receptor, p75NTR, exists as a transmembrane glycoprotein. Family members are characterized by multiple cysteine-rich domains for ligand binding, a single transmembrane extracellular domain (ECD), and a non-catalytic cytoplasmic domain. As used herein, a portion of p75NTR or a portion of the ECD of p75NTR contains at least one neurotrophin-binding domain.

[0059] The endogenous soluble ECD of p75NTR is produced by proteolysis regulated by α-secretases and γ-secretases that cleave the protein near the membrane junction of the ECD. This cleavage leads to the release of the cytoplasmic domain, which freely binds NGF as a natural antagonist to NGF signaling.

[0060] In one embodiment, the extracellular domain of p75NTR according to the present invention comprises a full-length extracellular domain (ECD) or a portion thereof. The portion of the ECD of p75NTR may comprise various cleavages of the full-length ECD. Cleavages of the ECD of p75NTR can be produced using techniques known in the art, such as recombinant DNA technology. In one embodiment, the extracellular domain of p75NTR comprises or consists of a full-length ECD. In one embodiment, the extracellular domain of p75NTR comprises the extracellular domain (ECD) of the p75NTR protein adjacent to the ECD and additional C-terminal amino acids. For example, a portion of the p75NTR protein may have ECD and at least 1-5 or 5-10 amino acids of the p75NTR protein adjacent to ECD, e.g., C-terminal amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or the C-terminal amino acids of the companion animal p75NTR protein adjacent to ECD may have ECD and at least 10-20 amino acids, or ECD and at least 20-30 amino acids, or ECD and at least 30-40 amino acids, or ECD and at least 40-50 amino acids, or ECD and at least 50-60 amino acids, or ECD and at least 60-70 amino acids. Alternatively, it may contain ECD and at least 70-80 amino acids, or ECD and at least 80-90 amino acids, or ECD and at least 90-100 amino acids, or ECD and at least 100-110 amino acids, or ECD and at least 110-120 amino acids, or ECD and at least 120-130 amino acids, or ECD and at least 130-140 amino acids, or ECD and at least 140-150 amino acids, or ECD and at least 150-160 amino acids, or ECD and at least 160-170 amino acids, or ECD and at least 170-180 amino acids.

[0061] In one embodiment, the α-secretase and γ-secretase cleavage sites within the ECD are removed. In one embodiment, all or part of the stalk region is removed. As used herein, the stalk region refers to the amino acids downstream from the conserved EEIP sequence at positions 161-164. In one embodiment, the stalk region within the ECD, as well as the α-secretase and γ-secretase cleavage sites, are removed. In one embodiment, a portion of the N-terminus of the mature p75NTR protein, for example, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more at the N-terminus, is removed. In one embodiment, a portion of the amino acids immediately upstream of the stalk region, for example, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more consecutive amino acids, is removed.

[0062] In one embodiment, the p75NTR extracellular domain is canine and includes or consists of SEQ ID NO: 7 or a variant thereof or a part thereof. The variant according to the present invention includes a variant amino acid at one or more of positions 75, 109, 133, and / or 134, for example, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58. In one embodiment, an isolated polypeptide comprising a companion animal p75 neurotrophin receptor (p75NTR) extracellular domain, wherein the p75NTR includes a variant amino acid at one or more of positions 75, 109, 133, and / or 134, includes or consists of SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58.

[0063] In another embodiment, the isolated companion animal p75NTR extracellular domain is cat and comprises or consists of SEQ ID NO: 38 or a variant thereof or a portion thereof. The variant according to the present invention comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134, e.g., SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69. In one embodiment, an isolated polypeptide comprising a companion animal p75 neurotrophin receptor (p75NTR) extracellular domain, wherein the p75NTR comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134, comprises or consists of SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69.

[0064] In another embodiment, the isolated p75NTR extracellular domain is a human p75NTR extracellular domain and includes or consists of SEQ ID NO: 71 or a variant thereof or a portion thereof. The variant according to the present invention includes a variant amino acid at one or more of positions 75, 109, 133, and / or 134, e.g., SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, or SEQ ID NO: 96. In one embodiment, an isolated polypeptide comprising a human p75 neurotrophin receptor (p75NTR) extracellular domain, wherein the p75NTR includes a variant amino acid at one or more of positions 75, 109, 133, and / or 134, includes or consists of SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, or SEQ ID NO: 96.

[0065] In another embodiment, the isolated p75NTR extracellular domain is a bovine p75NTR extracellular domain and includes or consists of SEQ ID NO: 36 or a variant thereof or a portion thereof. The variant according to the present invention includes variant amino acids at one or more of positions 75, 109, 133, and / or 134, e.g., SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131.

[0066] In another embodiment, the isolated p75NTR extracellular domain is a camel p75NTR extracellular domain and includes or consists of SEQ ID NO: 99 or a variant thereof or a portion thereof. The variant according to the present invention includes variant amino acids at one or more of positions 75, 109, 133, and / or 134, e.g., SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107.

[0067] In another embodiment, the isolated p75NTR extracellular domain is a porcine p75NTR extracellular domain and includes or consists of SEQ ID NO: 108 or a variant thereof or a portion thereof. The variant according to the present invention includes variant amino acids at one or more of positions 75, 109, 133, and / or 134, e.g., SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116.

[0068] In another embodiment, the isolated p75NTR extracellular domain is a horse p75NTR extracellular domain and includes or consists of SEQ ID NO: 5 or a variant thereof or a portion thereof. The variant according to the present invention includes variant amino acids at one or more of positions 75, 109, 133, and / or 134, e.g., SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124.

[0069] The ECD of p75NTR has a stalk region that is prone to O-glycosylation (e.g., SEQ ID NO: 9, the inu-stalk region). Glycosylation in proteins can cause manufacturing difficulties. Therefore, in one embodiment, the isolated ECD may contain deletions within the stalk region to reduce the number of O-glycosylation sites within the stalk region, for example, to form a cleaved stalk region. The cleaved stalk region may contain any number of amino acids from the stalk region. For example, the stalk region may contain 1-10, 1-20, or 1-30 amino acids. The stalk region may be removed in the embodiments described herein. Point mutations can be introduced into the stalk region to generate a stalk region with a reduced number of O-glycosylation sites. For example, point mutations can be used to introduce variant amino acids into the O-glycosylation sites to prevent glycosylation occurring at these sites. Therefore, as used herein, a portion of the ECD may be an ECD that does not have a stalk region and 3' sequence α-secretase and γ-secretase cleavage sites (e.g., SEQ ID NO: 34).

[0070] Therefore, in one embodiment, the ECD of p75NTR is a cleaved protein from which the O-glycosylated stalk region has been removed.

[0071] The p75NTR molecules described herein can bind to proNGF and / or NGF. ProNGF and NGF have different activities in vivo; proNGF exhibits pro-inflammatory, catabolic, and apoptotic effects, while NGF has anti-inflammatory, anabolic, and proliferative effects. In one embodiment, an isolated polypeptide containing a p75NTR ECD, wherein the p75NTR contains a variant amino acid at one or more of positions 75, 109, 133, and / or 134, can bind to NGF and inhibit downstream pathways(s) mediated by NGF biological activity and / or NGF signaling. An isolated polypeptide containing a p75NTR ECD may also bind to proNGF and inhibit downstream pathways(s) mediated by proNGF biological activity and / or proNGF signaling. An isolated polypeptide containing a p75NTR ECD may preferentially bind to and inhibit NGF over proNGF. Isolated polypeptides containing p75NTR ECD may preferentially bind to and inhibit pro-NGF rather than NGF. For example, in certain embodiments where an anti-inflammatory effect is desired, having a p75NTR molecule that preferentially binds to pro-NGF rather than NGF may be advantageous.

[0072] An isolated polypeptide comprising a p75NTR ECD, wherein the p75NTR comprises one or more variant amino acids at positions 75, 109, 133, and / or 134, has altered binding affinity to its target compared to a wild-type p75NTR ECD. The p75NTR ECD of the present invention may have altered binding affinity to one or more or NGF, proNGF BDNF, NT3, NT4 compared to a wild-type p75NTR ECD. In some embodiments, the binding affinity of the variant p75NTR ECD of the present invention is increased to one or more or NGF, proNGF, BDNF, NT3, NT4 compared to a wild-type p75NTR ECD. In some embodiments, the binding affinity of the variant p75NTR ECD of the present invention is decreased to one or more or NGF, proNGF, BDNF, NT3, NT4 compared to a wild-type p75NTR ECD.

[0073] In one embodiment, an isolated polypeptide comprising a p75NTR ECD, wherein the p75NTR comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134, exhibits altered binding affinity to NGF compared to a wild-type p75NTR ECD. In one embodiment, the p75NTR ECD of the present invention exhibits altered binding affinity to NGF compared to a wild-type p75NTR ECD, but retains similar binding affinity to BDNF, NT3, and / or NT4. In one embodiment, the binding affinity of the variant p75NTR ECD is increased to NGF compared to a wild-type p75NTR ECD. In one embodiment, the binding affinity of the variant p75NTR ECD of the present invention is decreased to NGF compared to a wild-type p75NTR ECD. In one embodiment, an isolated polypeptide comprising a p75NTR ECD, wherein the p75NTR comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134, exhibits altered binding affinity to proNGF compared to a wild-type p75NTR ECD. In one embodiment, the p75NTR ECD of the present invention exhibits altered binding affinity to proNGF compared to a wild-type p75NTR ECD, but retains similar binding affinity to BDNF, NT3, and / or NT4. In one embodiment, the binding affinity of the variant p75NTR ECD is increased to proNGF compared to a wild-type p75NTR ECD. In one embodiment, the binding affinity of the variant p75NTR ECD of the present invention is decreased to proNGF compared to a wild-type p75NTR ECD.

[0074] nucleic acid In another embodiment, the present invention relates to an isolated nucleic acid encoding an isolated polypeptide comprising a p75 neurotrophin receptor (p75NTR) extracellular domain, wherein the p75NTR comprises one or more variant amino acids at positions 75, 109, 133, and / or 134 within the p75NTR.

[0075] In one embodiment, p75NTR is of human origin, and the isolated nucleic acid encodes SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, or SEQ ID NO: 96. In one embodiment, the companion animal is a dog, and the isolated nucleic acid encodes SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58. In one embodiment, the companion animal is a cat, and the isolated nucleic acid encodes SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69.

[0076] "Isolated nucleic acid molecules" means genomes, mRNA, cDNA, or synthetic DNA or RNA, or some combination thereof, in which the isolated polynucleotides are not associated with all or some of the polynucleotides found in nature, or the isolated polynucleotides are linked to polynucleotides that are not linked in nature.

[0077] In another embodiment, the present invention relates to a vector, plasmid, transcription, expression cassette, or nucleic acid construct comprising a p75NTR ECD, for example, a nucleic acid encoding the ECD or a portion thereof as described herein.

[0078] The construct may include a suitable leader sequence. The term "leader sequence" is used interchangeably with "signal sequence." Therefore, in some embodiments, a nucleic acid sequence / nucleic acid construct encoding a fusion protein may also include a leader sequence. The leader sequence is constructed as part of a protein and cleaved when the protein is secreted. Any suitable leader sequence may be used, including endogenous p75 leaders of the relevant species (e.g., human, dog, horse, cat, cow, camel), endogenous p75 leaders of different species, e.g., a mouse IgG leader, or another leader sequence known in the art, e.g., a Campath leader sequence (see US8,362,208B2), or a natural immunoglobulin germline leader sequence such as an artificial sequence. Such leader sequences can help enhance protein expression.

[0079] In another embodiment, the present invention relates to human, companion animal, or other p75NTR ECDs, such as nucleic acids encoding the aforementioned ECDs, or host cells comprising vectors, plasmids, vectors, transcriptions, expression cassettes, or constructs.

[0080] The expression vector used in this invention can be constructed from a starting vector, such as a commercially available vector. After the vector is constructed and nucleic acid molecules are inserted into the appropriate sites within the vector, the completed vector can be inserted into a suitable host cell for amplification and / or polypeptide expression.

[0081] The term "vector" refers to a construct that can deliver one or more genes or sequences of interest to a host cell and, in some embodiments, can be expressed. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensers, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells such as producer cells.

[0082] The present invention also relates to isolated recombinant host cells comprising one or more nucleic acid molecule plasmids, vectors, transcription or expression cassettes as described above. Transformation of selected host cells with an expression vector can be achieved by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method of selection is, in part, a function of the type of host cell used.

[0083] The host cell may be a eukaryote or prokaryote, such as a bacterium, virus, plant, fungus, mammal, or other suitable host cell. In one embodiment, the cell is an E. coli cell. In another embodiment, the cell is a yeast cell. In yet another embodiment, the cell is a Chinese hamster ovary (CHO) cell, a HeLa cell, or other cell that would be apparent to those skilled in the art. Mammalian cell lines available as hosts for expression include, but are not limited to, immortalized cell lines known in the art and available from the American Type Culture Collection (ATCC), and any cell line used in expression systems known in the art can be used to produce the recombinant polypeptide of the present invention.

[0084] Generally, host cells are transformed with recombinant expression vectors containing protein-coding DNA. Potential host cells include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include COS-7 cells, L cells, CI27 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, or their derivatives, and related cell lines grown in serum-free medium, such as HeLa cells, BHK cell lines, CVIIEBNA cell lines, human fetal kidney cells such as 293, 293EBNA, or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues, exoplants, HL-60, U937, HaK, or Jurkat cells. Selectively, mammalian cell lines such as HepG2 / 3B, KB, NIH 3T3, or S49 can be used for polypeptide expression, for example, when it is desirable to use polypeptides in various signaling or reporter assays.

[0085] Other suitable host cells include insect cells using expression systems such as baculoviruses in insect cells, plant cells, transgenic plants and transgenic animals, and viruses and nucleic acid vectors.

[0086] Alternatively, polypeptides can be produced in fungal cell lines and lower eukaryotes such as yeast, or in prokaryotes such as bacteria. Suitable yeasts include S. cerevisiae, S. pombe, Kluyveromyces strain, Pichia pastoris, Candida, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include E. coli, B. subtilis, S. typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. When proteins are produced in yeast or bacteria, it may be desirable to modify the product produced therein, for example, by phosphorylation or glycosylation at the appropriate site, in order to obtain a functional product. Such covalent bonding can be achieved using known chemical or enzymatic methods.

[0087] Host cells, when cultured under appropriate conditions, can be used to express proteins, which can then be collected from the culture medium (if the host cells secrete the protein into the medium) or directly from the producing host cells (if the protein is not secreted). The selection of appropriate host cells depends on various factors, including the desired expression level, polypeptide modifications desirable or required for activity (such as glycosylation or phosphorylation), and the ease of folding into biologically active molecules.

[0088] In another embodiment, the present invention also relates to the use of the isolated p75NTR protein or a portion thereof in a fusion protein having another portion (e.g., a half-life extension portion as described in more detail below). Thus, the p75NTR protein or a portion thereof can be provided covalently or coupled to the half-life extension portion. Alternatively, it can be provided incorporated into a liposome. The present invention further relates to the isolated p75NTR protein or a portion thereof for use in therapy. Furthermore, the isolated p75NTR protein or a portion thereof for use in the treatment of pain-related diseases, such diseases are described in more detail below.

[0089] In some embodiments, the half-life of the p75NTR protein is extended to improve its pharmacokinetic (PK) properties.

[0090] Fusion protein Accordingly, in another embodiment, the present invention relates to a fusion protein comprising the extracellular domain of the p75 neurotrophin receptor (p75NTR), wherein the p75NTR comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134, and comprises another moiety. The fusion protein may comprise the extracellular domain of the p75 neurotrophin receptor (p75NTR), wherein the p75NTR comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134, and comprises another moiety.

[0091] For example, this other portion could be a half-life extension portion. Thus, the p75NTR protein or a portion thereof (e.g., the extracellular domain) is coupled to the half-life extension portion. As described above, the p75NTR protein or a portion thereof may be a human, dog, cat, bovine, horse, or camel p75NTR protein or a portion thereof. Thus, the p75NTR protein or a portion thereof used in the fusion protein may include, or consist of, a sequence, a portion thereof, or a variant selected from SEQ ID NOs: 1, 3, 5, 7, 34, 36, 38, 71, or 73, in particular, the variant may include a variant amino acid at one or more of positions 75, 109, 133, and / or 134. In certain embodiments, all or a portion of the stalk region of the p75NTR is removed. In certain embodiments of the canine p75NTR protein or a portion thereof, the stalk region (e.g., SEQ ID NO: 9) is removed. In certain embodiments of the feline p75NTR protein or a portion thereof, the stalk region is removed. In certain embodiments of the human p75NTR protein or a portion thereof, the stalk region (e.g., SEQ ID NO: 25) is removed.

[0092] The half-life extension portion is described. For example, the half-life extension portion may be selected from the following non-restrictive list: human immunoglobulin Fc domains, companion animal immunoglobulin Fc domains, polyethylene glycol (PEG), PEG derivatives, simple lipids, lipid dicarboxylic acids, lipids with additional portions, human or companion animal serum albumin-binding factors, e.g., small molecule binding factors, or antibodies / antibody fragments that bind human or companion animal serum albumin, companion animal serum albumin, or albumin-binding domains (ABDs) of streptococcal protein G. Examples of lipids include glucagon-like peptide 1 (GLP-1), its analog GLP-1 liraglutide, and semaglutide or cholesterol. Advantageously, the use of immunoglobulin Fc domains facilitates protein purification. In particular, Fc binding to protein A can be used in purification procedures. The presence of immunoglobulin Fc domains can also stabilize the overall folding of fusion proteins and extend their half-life.

[0093] In certain embodiments, when the half-life extension portion is an Fc domain, a serum albumin-binding factor, or serum albumin, the extracellular domain and half-life extension portion of p75NTR are of the same species origin / specific. For example, in one embodiment, the half-life extension portion is the companion animal Fc domain of the corresponding companion animal. For example, if the extracellular domain of p75NTR is from a dog, the Fc domain is a canine Fc domain. If the extracellular domain of p75NTR is from a cat, the Fc domain is a feline Fc domain. If the extracellular domain of p75NTR is from a horse, the Fc domain is a horse Fc domain. If the extracellular domain of p75NTR is from a cow, the Fc domain is a cow Fc domain.

[0094] In one embodiment, when the half-life extension portion is an Fc domain, human serum albumin-binding factor, or human serum albumin, both the extracellular domain and the half-life extension portion of p75NTR are derived from humans.

[0095] Human serum albumin-binding factors, for example, antibodies to fragments thereof, can be entirely human or can be humanized. Human serum albumin-binding factors bind to human serum albumin.

[0096] However, given the high sequence similarity between p75 proteins, in certain embodiments, if the half-life extension portion is a companion animal Fc domain, a companion animal serum albumin-binding factor, or companion animal serum albumin, then the p75NTR protein or a portion thereof and the half-life extension portion are not derived from / specific to the same companion animal. For example, in one embodiment, the half-life extension portion is the companion animal Fc domain of the corresponding companion animal, but the p75 protein or a portion thereof is from a different companion animal. For example, for the treatment of dogs, if the Fc domain is a canine Fc domain, then the p75NTR protein or a portion thereof, such as the extracellular domain, may be derived from a different animal, e.g., a cat, a cow, a horse, a pig, or a camel. For example, if the Fc domain is a feline Fc domain for the treatment of cats, the p75NTR protein or a portion thereof, such as the extracellular domain, may be derived from a different animal, such as a dog, cow, pig, horse, or camel. For example, if the Fc domain is a horse Fc domain for the treatment of cats, the p75NTR protein or a portion thereof, such as the extracellular domain, may be derived from a different animal, such as a cat, cow, dog, pig, or camel. In yet another embodiment, a human p75 or a portion thereof fused to a companion animal Fc can be used.

[0097] Companion animal serum albumin-binding factors, such as antibodies or fragments thereof, may be canine or canine-modified, catine or feline-modified, horseine or horseine-modified, cattle or catine-modified, cameline or camelidine, or pigine or pigine-modified. Companion animal serum albumin-binding factors may bind to canine, catine, cattle, horseine, or camelid serum albumin.

[0098] In one embodiment, the half-life extension portion is a wild-type or variant Fc domain. The term variant is as defined above. For example, an Fc domain variant may have a modified half-life compared to a wild-type Fc domain. In one embodiment, the Fc domain is an Fc domain, i.e., a wild-type domain or a variant thereof. Variant Fc domains are described, for example, in WO2020 / 142625.

[0099] As used herein, “Fc” or “Fc region” or “Fc domain” means a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain (CH1), and possibly a portion of the hinge. In one embodiment, the Fc domain comprises the constant region immunoglobulin domains CH2, CH3, and the hinge region between CH1 and CH2, or a portion of the hinge region.

[0100] The proteolytic digestion of antibodies releases different fragments, referred to as Fv (variable fragment), Fab (antigen-binding fragment), and Fc (crystallized fragment). The Fc fragment contains the carboxyl-terminal portions of both H chains, which are held together by a disulfide. The constant domain of the Fc fragment is involved in mediating the effector function of the antibody.

[0101] In dogs, there are four IgG heavy chains designated A, B, C, and D. These heavy chains represent four distinct subclasses of canine IgG, designated IgG-A, IgG-B, IgG-C, and IgG-D. The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. (Vet.Immunol.Immunopathol.80:259-270(2001)). Exemplary amino acid and DNA sequences for these heavy chains are also available from the GenBank database (IgGA: accession number AAL35301.1, IgGB: accession number AAL35302.1, IgGC: accession number AAL35303.1, IgGD: accession number AAL35304.1). The amino acid sequences of IgG-A, IgG-B, IgG-C, and IgG-D used by the inventors, and according to aspects and embodiments of the invention, are provided as Sequence IDs 15, 16, 17, and 18.

[0102] In humans, Fc refers to the last two constant-region immunoglobulin domains of IgA, IgD, and IgG, the last three constant-region immunoglobulin domains of IgE and IgM, and the mobile hinge N-terminus to these domains. In the case of IgA and IgM, Fc may include the J chain. In the case of IgG, the Fc domain includes the immunoglobulin domains CH2 and CH3, as well as the lower hinge region between CH1 and CH2. While the boundaries of the Fc region may differ, the human IgG heavy chain Fc region is typically defined as containing residues C226 or P230 at its carboxyl terminus, and its numbering follows the EU index, as in Kabat.

[0103] As used herein, Fc may refer to an Fc region on its own or to this region in relation to an Fc fusion ("fusion composition" or "fusion construct"), as described herein. An Fc domain comprises all or part of an Fc region, i.e., the N or C-terminal sequence may be removed from a wild-type or variant Fc domain as long as it does not affect its function.

[0104] In short, IgG function is generally achieved, sometimes on effector cells, through the interaction between the Fc region of Ig and the Fcγ receptor (FcγR) or another binding molecule. This can induce effector cells to kill target cells to which antibodies bind through their variable (V) region. Antibodies against soluble antigens can also form immune complexes that target FcγR, resulting in the uptake (opsonization) of immune complexes, or the induction of effector cells and the release of cytokines.

[0105] As used herein, “Fc gamma receptor,” “FcγR,” or “Fc gamma R” means any member of the protein family that binds to the Fc region of an IgG antibody and is encoded by the FcγR gene.

[0106] In humans, three classes of FcγR have been characterized, but the situation is further complicated by the emergence of multiple receptor morphologies. The three classes are as follows:

[0107] (i) FcγRI(CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc, bind monomeric IgG with high affinity and are expressed on macrophages, monocytes, and possibly neutrophils and eosinophils.

[0108] (ii) FcγRII(CD32) binds to complexed IgG with medium to low affinity and is widely expressed. These receptors can be classified into two important types: FcγRIIa and FcγRIIb. The "a" form of the receptor is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process, arising as two alternative alleles. The "b" form appears to play a role in the inhibitory process and is found on B cells, macrophages, as well as mast cells and eosinophils. On B cells, the "b" form appears to function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, the b form acts to inhibit phagocytosis, such as that mediated via FcγRIIa. On eosinophils and mast cells, the b form may help suppress the activation of these cells by IgE binding to its distinct receptor.

[0109] (iii) FcγRIII(CD16) binds to IgG with moderate to low affinity and exists in two forms. FcγRIIIa is found on NK cells, macrophages, eosinophils, as well as some monocytes and T cells, and mediates ADCC.

[0110] FcγRIIIb is highly expressed on neutrophils. Both types have different homogeneous morphologies.

[0111] The canine Fc receptor is described in Bergeron et al. (LMBergeron et al.; Veterinary Immunology and Immunopathology 157(2014)31-41). Dogs possess RI, RIIb, and RIII receptors, but not Riia.

[0112] Beyond binding to FcγR, IgG antibodies can activate complement, which can also trigger cell lysis, opsonization, or cytokine release and inflammation. The Fc region also mediates properties such as the transport of IgG to the neonatal region (via so-called "FcRn"), an increased half-life (possibly mediated via FcRn-type receptors), and autoaggregation. The Fc region is also responsible for interactions with protein A and protein G (this interaction appears to be analogous to FcRn binding).

[0113] As used herein, “effector function” refers to a biochemical event resulting from the interaction between an antibody Fc region and an Fc receptor or ligand. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0114] In one embodiment, the ECD and other parts of p75NTR are linked to the linker portion, or otherwise conjugated, connected, or covalently or noncovalently bonded. Suitable linkers are known to those skilled in the art. For example, the linker is a peptide linker such as a glycine and / or alanine and / or threonine and / or serine-rich linker, e.g., a glycine-serine linker such as (G4S)n, where n is 1 to 4.

[0115] In another embodiment, the linker may be cleavable. The linker may be an acid-unstable linker, a proteolytically cleavable linker, an enzymatically cleavable linker, or an oxidation-sensitive linker.

[0116] In one embodiment of the fusion protein, the companion animal p75NTR protein or a portion thereof includes or consists of a variant canine p75NTR ECD or a portion thereof. In one embodiment, the ECD is a canine ECD and includes or consists of a sequence selected from SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58. In one embodiment, the ECD is a feline ECD and includes or consists of a sequence selected from SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69. In one embodiment, the ECD is human and includes or consists of a sequence selected from SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, or SEQ ID NO: 96.

[0117] Accordingly, in one embodiment, the present invention relates to a fusion protein comprising a canine p75NTR ECD linked to a canine Fc domain. In one embodiment, the ECD comprises or consists of a sequence selected from SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58, or a variant of said sequence operably linked to the canine Fc domain. In one embodiment, the present invention relates to a fusion protein comprising a feline p75NTR ECD linked to a feline Fc domain. In one embodiment, the ECD comprises or consists of a sequence selected from SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69, or a variant of said sequence operably linked to the feline Fc domain. In one embodiment, the present invention relates to a fusion protein comprising a human p75NTR ECD linked to a human Fc domain. In one embodiment, the ECD comprises or consists of a sequence selected from SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, or SEQ ID NO: 96, or a variant of said sequence operably linked to the human Fc domain.

[0118] In one embodiment, the fusion protein of the present invention preferably binds to one or more of NGF, BDNF, NT3, or NT4 / 5 with a binding affinity (Kd) of about 1 pM to about 100 nM. In some preferred embodiments, the binding affinity (Kd), when measured in an in vitro binding assay for NGF, proNGF, BDNF, NT3, or NT4 / 5 as described herein, is between about 5 pM and any of the following: about 10 pM, 20 pM, 40 pM, 50 pM, 100 pM, 0.2 nM, 0.5 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, 50 nM, or 100 nM. A range of less than nanomoles is preferred.

[0119] In one embodiment, the fusion protein comprises SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58, or a variant thereof. The fusion protein according to the present invention may comprise a variant p75NTR ECD having a variant amino acid at one or more of positions 75, 109, 133, and / or 134, operably linked to a canine Fc domain. The Fc domain in the fusion protein construct may be a wild-type canine Fc domain, such as SEQ ID NO: 20. The Fc domain in the fusion protein construct may be a variant canine Fc domain modified to increase half-life, such as SEQ ID NO: 21. In this domain, the mutant YTE is introduced at residues Y252-T254 of the wild-type Fc domain using EU numbering. The fusion protein can be produced by combining a variant p75NTR ECD, such as SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58, with a canine Fc domain, such as SEQ ID NO: 20 or 21. The fusion protein may contain, or consist of, a sequence selected from SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 59.

[0120] In one embodiment, the fusion protein comprises a wild-type feline p75NTR ECD operably linked to a feline Fc domain. In another embodiment, the fusion protein comprises or consists of Sequence ID No. 39, for example, the fusion protein comprises a wild-type feline p75 ECD operably linked to a feline Fc domain. The Fc domain in the construct of Sequence ID No. 39 is a wild-type feline IgG2 Fc domain. In one embodiment, the fusion protein comprises or consists of Sequence ID No. 42, for example, the fusion protein comprises a feline p75 ECD operably linked to a feline Fc domain. The Fc domain in the construct of Sequence ID No. 42 is a wild-type feline IgG1 Fc domain. In yet another embodiment, the fusion protein comprises or consists of Sequence ID No. 44, for example, the fusion protein comprises a feline p75 ECD operably linked to a feline Fc domain. The Fc domain in the construct of Sequence ID No. 44 is a wild-type feline IgG3 Fc domain.

[0121] In one embodiment, the fusion protein comprises SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69, or a variant thereof. The fusion protein according to the present invention may comprise a variant feline p75NTR ECD having a variant amino acid at one or more of positions 75, 109, 133, and / or 134 operably linked to a feline Fc domain. The Fc domain in the fusion protein construct may be a wild-type feline Fc domain such as SEQ ID NO: 24, 25, 26, or 41. For example, the fusion protein may be produced by combining a variant p75NTR ECD such as SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69 with a feline Fc domain such as SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 41. The Fc domain in the fusion protein construct may be a variant feline Fc domain modified to increase half-life, and those skilled in the art will be able to determine a preferred half-life-extending variant. The fusion protein may contain, or consist of, a sequence selected from SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 70.

[0122] A modified companion animal Fc domain (e.g., a dog, cat, or horse Fc domain) containing one or more half-life extension mutations can be used in the fusion protein of the present invention. Those skilled in the art will know that any other known mutations that increase half-life can also be introduced into the Fc domain.

[0123] In one embodiment, the fusion protein includes or comprises SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, or SEQ ID NO: 97, or variants thereof. The fusion protein according to the present invention may include a variant p75NTR ECD having a variant amino acid at one or more of positions 75, 109, 133, and / or 134 operably linked to a human Fc domain. The Fc domain in the fusion protein construct may be a wild-type human Fc domain such as SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80. The Fc domain in the fusion protein construct may be a variant human Fc domain modified to increase its half-life. Suitable modifications for increasing the half-life of a human Fc domain are known in the art. The fusion protein may be produced by combining a variant p75NTR ECD such as SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, or SEQ ID NO: 96 with a human Fc domain such as SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80. The fusion protein may contain, or consist of, a sequence selected from SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, or SEQ ID NO: 97.

[0124] According to the present invention, the fusion protein exhibits advantageous biological properties, including improved solubility, stability, and / or improved serum half-life. These examples demonstrate that the described molecules are highly stable under both temperature and chemical stress, exhibiting unfolding only when incubated at temperatures above 70°C, with Tm1 not accumulating from around 67°C up to 95°C. The improved half-life allows for a reduced administration frequency (single dose compared to existing treatments requiring daily administration). This effect is demonstrated while exhibiting a potent analgesic effect. In certain embodiments, the fusion protein of the present invention is approximately 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 , 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, or longer in vivo, and more preferably, the p75NTR(NBP)-Fc fusion protein of the present invention has an in vivo half-life of about 24 hours or longer.

[0125] In another embodiment, the fusion protein of the present invention is approximately 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, 13 The fusion protein has an in vitro half-life of one or longer of 4, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 62, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, or 210 days ± 1 day, and furthermore, the fusion protein has an in vitro half-life of about 6 days or longer than 1 month. In one embodiment, the half-life is 14 days.

[0126] According to the preferred embodiment described above, the in vivo half-life may be the half-life in a rat or a corresponding companion animal, such as a dog or cat or a horse or a pig or a camel, or in a human.

[0127] The fusion protein of the present invention can act at very low doses, yet is highly effective.

[0128] According to the present invention, the fusion protein exhibits a favorable safety profile. This is, for example, when the subject maintains normal body weight and hematological parameters and does not produce anti-drug antibodies after administration of the fusion protein.

[0129] In another embodiment, the present invention relates to an isolated nucleic acid encoding the above-mentioned fusion protein, for example, a nucleic acid encoding a fusion protein such as SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56 or SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94 or SEQ ID NO: 97. In one embodiment, the nucleic acid includes or consists of a sequence selected from SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, or SEQ ID NO: 60. In one embodiment, the nucleic acid includes or consists of a sequence selected from SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 95, or SEQ ID NO: 98.

[0130] In another embodiment, the present invention relates to vectors, plasmids, vectors, transcriptions, expression cassettes or constructs comprising the nucleic acids described above.

[0131] In another embodiment, the present invention relates to a host cell comprising the nucleic acid vector, plasmid, vector, transcription, expression cassette, or construct described above. Suitable host cells are described elsewhere in this specification.

[0132] In another embodiment, the P75NTR protein, a portion thereof, or a fusion protein is labeled with a detectable or functional label. The label may include, but is not limited to, fluorophores, phosphors, radiolabels, enzymes, chemiluminescents, nuclear magnetic resonance activity labels, or photosensitizers, and may be any molecule that produces or induces the production of a signal. Thus, binding may be detected and / or measured by detecting fluorescence or emission, radioactivity, enzyme activity, or photoabsorbance.

[0133] Pharmaceutical composition In another embodiment, a pharmaceutical composition or fusion protein of the present invention is provided, comprising an isolated polypeptide containing the extracellular domain of the p75 neurotrophin receptor (p75NTR), wherein one or more variant amino acids are present at positions 75, 109, 133, and / or 134 within the p75NTR. The fusion proteins or pharmaceutical compositions described herein can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, nasal, pulmonary, intradermal, intravitreous, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, inhalation, or topical, particularly to the ear, nose, eye, or skin, or by inhalation. In another embodiment, delivery is the delivery of a nucleic acid encoding a drug, for example, a nucleic acid encoding the molecule of the present invention is delivered.

[0134] Parenteral administration includes, for example, intravenous, intramuscular, intra-arterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, intra-articular, or subcutaneous administration. Preferably, the composition is administered parenterally.

[0135] A pharmaceutically acceptable carrier or vehicle may be particulate, such as the composition being in tablet or powder form. The term "carrier" refers to a diluent, adjuvant, or excipient administered with the drug-antibody conjugate of the present invention. Such pharmaceutically acceptable carriers may be liquids, such as water and oils, and may be of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Carriers may include saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants may be used. In one embodiment, when administered to a subject, the polypeptide or composition and the pharmaceutically acceptable carrier of the present invention are sterile. Water is a preferred carrier when the drug-antibody conjugate of the present invention is administered intravenously. Saline solutions, as well as aqueous dextrose and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers.

[0136] Pharmaceutical compositions may be in liquid form, such as solutions, syrups, emulsions, or suspensions. Liquids may be useful for oral administration, or for injection, infusion (e.g., IV infusion), or subcutaneous delivery.

[0137] When intended for oral administration, the composition may be in solid or liquid form, and semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered as either solid or liquid in this specification.

[0138] As a solid composition for oral administration, the composition can be formulated in the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, or corn starch; lubricants such as magnesium stearate; flow enhancers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavor, and coloring agents. If the composition is in the form of a capsule (e.g., a gelatin capsule), the composition may contain a liquid carrier such as polyethylene glycol, cyclodextrin, or fatty oil, in addition to the types of materials described above.

[0139] When intended for oral administration, the composition may contain one or more of the following: sweeteners, preservatives, dyes / colorants, and flavor enhancers. For administration by injection, the composition may also contain one or more of the following: surfactants, preservatives, humectants, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0140] The composition can take the form of one or more medication units.

[0141] In certain embodiments, it may be desirable to administer the composition topically to the area requiring treatment, or by intravenous injection or infusion.

[0142] The amount of polypeptides, fusion proteins, or pharmaceutical compositions described herein that are effective / active in treating a specific disease or condition depends on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may be optionally used to help identify the optimal dosage range. The exact dose used in a composition also depends on the route of administration and the disease or severity of the disease described above, and should be determined according to the physician's judgment and the circumstances of each patient. Factors such as age, weight, sex, diet, administration time, excretion rate, host condition, drug combination, response sensitivity, and disease severity should be taken into consideration.

[0143] Typically, this amount is at least about 0.01% of the polypeptide of the present invention relative to the mass of the composition. When intended for oral administration, this amount can be varied in the range of about 0.1% to about 80% by mass of the composition. A preferred oral composition may contain about 4% to about 50% of the polypeptide of the present invention relative to the mass of the composition.

[0144] The composition can be prepared such that the parenteral administration unit contains approximately 0.01% to approximately 2% by mass of the polypeptide of the present invention.

[0145] For therapeutic use, the composition is available in concentrations of approximately 0.01 mg / kg to 250 mg / kg, 0.05 mg / kg to 250 mg / kg, 0.1 mg / kg to 250 mg / kg, 0.5 mg / kg to 250 mg / kg, 1 mg / kg to 250 mg / kg, 0.01 mg / kg to 100 mg / kg, 0.05 mg / kg to 100 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.5 mg / kg to 100 mg / kg, and 1 mg / kg. The composition may contain approximately 0.1 mg / kg to 250 mg / kg of the subject's body weight, preferably approximately 0.1 mg / kg to 20 mg / kg, more preferably approximately 1 mg / kg to 10 mg / kg of the subject's body weight. In one embodiment, the composition is administered in doses of about 1 to 30 mg / kg, for example, about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The administration schedule can vary, for example, from once a week to once every two, three, or four weeks or more.

[0146] Treatment can be administered, for example, once a month or once every two months. This is advantageous over daily administration because it improves compliance and minimizes stress on the patient.

[0147] As used herein, “to treat,” “to treat,” or “treatment” means to inhibit or alleviate a disease or condition. For example, treatment may include delaying the onset of a disease or symptoms associated with a disease, and / or reducing the severity of such symptoms that would or are expected to occur with the disease. These terms include improvement of existing symptoms, prevention of additional symptoms, and improvement or prevention of the underlying causes of such symptoms. Thus, these terms indicate that a beneficial outcome is conferred on at least some of those being treated. Many medical treatments are effective for some patients receiving treatment, but not for all patients.

[0148] The terms “subject” or “patient” refer to a human or animal, preferably a human or companion animal (e.g., a dog or cat), or an animal such as a horse, cow, pig, or camel, that is the subject of treatment, observation, or experimentation.

[0149] therapeutic use In another embodiment, the present invention relates to the use of an isolated polypeptide, fusion protein, or pharmaceutical composition comprising the p75NTR protein or a portion thereof as described herein in the treatment or prevention of a disease. In another embodiment, the present disclosure relates to the use of the polypeptide, fusion protein, or pharmaceutical composition as described herein in the manufacture of a medicament for the treatment or prevention of a disease enumerated herein. The present invention further relates to a method for treating a disease in a subject, comprising an effective amount of the polypeptide, fusion protein, or pharmaceutical composition as described herein to the subject. The present invention further relates to an isolated polypeptide comprising the p75NTR protein as described herein, or a fusion protein as described herein, for use in the treatment or prevention of a disease enumerated herein.

[0150] For example, the disease is an NGF-related disorder. For example, the disease is a pro-NGF-related disorder.

[0151] In one embodiment, NGF-related disorders are selected from the group consisting of cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. In one embodiment, NGF-related disorders include pain. In one embodiment, the pharmaceutical composition is used to treat pain. In one embodiment, the pharmaceutical composition is used for the treatment of pain, and the type of pain is selected from osteoarthritis pain, rheumatoid arthritis pain, surgical and postoperative pain, incision pain, systemic inflammatory pain, cancer pain, traumatic pain, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain. In one embodiment, pain includes osteoarthritis pain. In one embodiment, pain includes surgical and postoperative pain. In one embodiment, pain includes cancer pain.

[0152] In one or more embodiments, an isolated polypeptide comprising the extracellular domain of the p75 neurotrophin receptor (p75NTR) of the present invention, comprising one or more variant amino acids at positions 75, 109, 133, and / or 134 within the p75NTR, is intended for use in humans, dogs, cats, horses, cattle, or camels. In one or more embodiments, the p75NTR protein or a portion thereof, a fusion protein, or a pharmaceutical composition of the present invention is intended for use in dogs. In one or more embodiments, the p75NTR protein or a portion thereof, a fusion protein, or a pharmaceutical composition of the present invention is intended for use in cats. In one or more embodiments, the variant p75NTR protein or a portion thereof, a fusion protein, or a pharmaceutical composition of the present invention is intended for use in horses. In one or more embodiments, the p75NTR protein or a portion thereof, a fusion protein, or a pharmaceutical composition of the present invention is intended for use in cattle. In one or more embodiments, the p75NTR protein or a portion thereof, a fusion protein or pharmaceutical composition of the present invention is for use in camels. In one or more embodiments, the p75NTR protein or a portion thereof, a fusion protein or pharmaceutical composition of the present invention is for use in humans.

[0153] In one embodiment, an isolated polypeptide, fusion protein, or pharmaceutical composition comprising the extracellular domain of the p75 neurotrophin receptor (p75NTR) of the present invention, wherein the isolated polypeptide comprises one or more variant amino acids at positions 75, 109, 133, and / or 134 within the p75NTR, is administered together with one or more therapeutic agents, for example, a therapeutic agent for treating pain.

[0154] The p75NTR protein of the present invention is administered optionally in combination with one or more active agents, including other analgesics. Such active agents include analgesics, antihistamines, antipyretics, anti-inflammatory drugs, antibiotics, antivirals, and anticytokine agents. Examples of active agents include TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, hepatocyte growth factor (HGF), hepcidin agonists, antagonists, and modifiers (including antibodies reactive to any of the above), and antibodies reactive to any of their receptors.Active agents include, but are not limited to, 2-arylpropionic acid, aceclofenac, acemetacin, acetylsalicylic acid (aspirin), alclofenac, aluminoprofen, amoxiprine, ampylon, arylalkanoates, azapropazon, benorylate / benorilate, benoxaprofen, bromfenac, carprofen, celecoxib, magnesium choline salicylate, clofezon, COX-2 inhibitors, dexibprofen, dexketoprofen, diclofenac, diflunisal, droxicam, ethenzamide, etodolac, etoricoxib, faislamine, fenamic acid, fenbufen, fenoprofen, flufenamic acid, flunoxaprofen, flurbiprofen, ibuprofen, ibu Examples include proxam, indomethacin, indoprofen, kebzon, ketoprofen, ketrolac, romoxicam, loxoprofen, lumiracoxib, magnesium salicylate, meclofenamic acid, mefenamic acid, meloxicam, metamizole, methyl salicylate, mofebutazone, nabumetone, naproxen, n-arylanthranilic acid, oxamethacin, oxaprozin, oxicam, oxyfenbutazone, parecoxib, phenazone, phenylbutazone, phenylbutazone, piroxicam, pirprofen, profen, proglummetacin, pyrazolidine derivatives, lofecoxib, salicylate salicylate, salicylamide, salicylate, sulfinpyrazone, sulindac, suprofen, tenoxicam, tiaprofenic acid, tolfenamic acid, tolmetin, and valdecoxib.

[0155] Antihistamines can be any compound that counteracts the action of histamine or its release from cells (e.g., mast cells). Examples of antihistamines include, but are not limited to, acribastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclidine, cetirizine, cetirizine analogs, chlorpheniramine, clemastine, CS 560, cyproheptadine, desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine, loratidine, mescopolamine, mizolastine, nolastemizole, phenindamine, promethazine, pyriramine, terfenadine, and tranilast.

[0156] Antibiotics include, but are not limited to, amikacin, aminoglycosides, amoxicillin, ampicillin, ansamycin, aruphenamine, azithromycin, azurocillin, aztreonam, bacitracin, carbasephalm, carbapenem, carbenicillin, cefaclor, cefadroxil, cephalexin, cephalothin, cephalothin, cephamandol, cefazolin, cefdinir, cefditoren, cefepime, cefixime, cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprodil, ceftazidime, and ceftibutene. Ceftizoxime, ceftoviprol, ceftriaxone, cefuroxime, cephalosporin, chloramphenicol, cilastatin, ciprofloxacin, clarithromycin, clindamycin, cloxacillin, colistin, cotrimoxazole, dalfopristin, demeclocycline, dicloxacillin, zilithromycin, doripenem, doxycycline, enoxacin, ertapenem, erythromycin, ethambutol, flucloxacillin, fosfomycin, furazolidone, fusidic acid, gatifloxacin, geldanamycin, gentama Icin, glycopeptide, harbimycin, imipenem, isoniazid, kanamycin, levofloxacin, lincomycin, linezolid, lomefloxacin, loracalbef, macrolide, mafenide, meropenem, methicillin, metronidazole, mezlocillin, minocycline, monobactam, moxifloxacin, mupirocin, nafcillin, neomycin, netylmycin, nitrofurantoin, norfloxacin, ofloxacin, oxacillin, oxytetracycline, paromomycin, penicillin, penicillins, piperacillin, platen Simycin, polymyxin B, polypeptide, prontosil, pyrazinamide, quinolone, quinupristin, rifampicin, rifampin, roxithromycin, spectinomycin, streptomycin, sulfacetamide, sulfamethizol, sulfanilimide, sulfasalazine, sulfisoxazole, sulfonamide, teicoplanin, telithromycin, tetracycline, tetracyclines, ticarcillin, tinidazole, tobramycin, trimethoprim, trimethoprim-sulfamethoxazole, troleandomycin,Examples include trovafloxacin and vancomycin.

[0157] Other active agents include aldosterone, beclomethasone, betamethasone, corticosteroids, cortisol, cortisone acetate, deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate, glucocorticoids, hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids, and triamcinolone. Any preferred combination of these active agents is also considered.

[0158] The most common form of current treatment for osteoarthritis (OA) and associated pain is NSAIDs (which are also pain relievers). NSAIDs are not always sufficiently effective, typically need to be administered daily, and are not approved for long-term use in cats in the United States. In addition, there are safety and tolerability concerns regarding the use of NSAIDs in both dogs and cats, especially in long-term treatment. Long-term concomitant administration of NSAIDs with anti-NGF mAbs is not recommended.

[0159] In certain embodiments, treatment involves the concomitant administration of dietary supplements containing omega-3 fatty acids, microlactin, and / or glucosamine / chondroitin to support joint health. Adecane (polysulfated glycosaminoglycan) is an FDA-approved disease-modifying agent that inhibits cartilage loss and may also be administered concomitantly.

[0160] Isolated polypeptides, fusion proteins, or pharmaceutical compositions comprising the extracellular domain of the p75 neurotrophin receptor (p75NTR), wherein one or more variant amino acids are present at positions 75, 109, 133, and / or 134 within the p75NTR, can be administered concurrently with or at different times to other therapies or therapeutic compounds or therapies, for example, simultaneously, separately, or sequentially.

[0161] In another embodiment, the present invention relates to the use of an isolated polypeptide, fusion protein, or a pharmaceutical composition comprising the p75NTR protein or a portion thereof, in the treatment or prevention of osteoarthritis. In another embodiment, the present disclosure relates to an isolated polypeptide, fusion protein, or a pharmaceutical composition comprising the p75NTR protein or a portion thereof, in the manufacture of a medicament for the treatment or prevention of osteoarthritis. The present invention further relates to a method for treating osteoarthritis in a subject, comprising administering to the patient an effective amount of an isolated polypeptide, fusion protein, or a pharmaceutical composition comprising the isolated polypeptide or fusion protein. The present invention further relates to an isolated polypeptide, fusion protein, or a pharmaceutical composition comprising the p75NTR protein or a portion thereof, for use in the treatment or prevention of osteoarthritis.

[0162] In methods, uses, or recipients of the p75NTR protein or portion thereof, or compositions containing such p75NTR protein or portion thereof, “subject” or “patient” refers to a human or animal, preferably a human or companion animal such as a dog or cat, that is the subject of treatment, observation, or experimentation. Other animals that may be suitable for treatment include, but are not limited to, cattle, pigs, horses, and camels.

[0163] The p75NTR protein or portion thereof refers to a p75NTR protein or portion thereof suitable for administration to the subject. Preferably, the p75NTR protein or portion thereof is obtained from the same species as the subject that accepts the p75NTR protein or portion thereof, or a composition containing said p75NTR protein or portion thereof.

[0164] In one embodiment, the p75NTR protein or a portion thereof is for the treatment of animals such as cats, dogs, pigs, cattle, horses, or camels. In one embodiment, the companion animal is a dog. In one embodiment, the companion animal is a cat. In one embodiment, the p75NTR protein or a portion thereof or a pharmaceutical composition for use in the treatment of osteoarthritis comprises a canine p75NTR protein such as SEQ ID NO: 1. In one embodiment, the p75NTR protein or a portion thereof or a pharmaceutical composition for use in the treatment of osteoarthritis comprises a feline p75NTR protein such as SEQ ID NO: 3. In one embodiment, the p75NTR protein or a portion thereof or a pharmaceutical composition for use in the treatment of osteoarthritis comprises a bovine p75NTR protein such as SEQ ID NO: 36. In one embodiment, the p75NTR protein or a portion thereof or a pharmaceutical composition for use in the treatment of osteoarthritis comprises a camel p75NTR protein such as SEQ ID NO: 99. In one embodiment, the p75NTR protein or a portion thereof or a pharmaceutical composition for use in the treatment of osteoarthritis comprises a porcine p75NTR protein such as SEQ ID NO: 108. In one embodiment, the p75NTR protein or a portion thereof or a pharmaceutical composition for use in the treatment of osteoarthritis comprises a horse p75NTR protein such as SEQ ID NO: 5. In one embodiment, the p75NTR protein or a portion thereof is for human treatment, and the p75NTR protein or a portion thereof or a pharmaceutical composition for use in the treatment of osteoarthritis comprises a human p75NTR protein such as SEQ ID NO: 71.

[0165] In one embodiment, the p75NTR protein or a portion thereof for use in the treatment of osteoarthritis includes an extracellular domain or a portion thereof. In one embodiment, the treatment is for dogs, and the p75NTR extracellular domain includes SEQ ID NO: 7. A portion of the extracellular domain, for example, SEQ ID NO: 34, may be used as described herein. In one embodiment, the treatment is for cats, and the p75NTR extracellular domain includes SEQ ID NO: 38. In one embodiment, the treatment is for humans, and the p75NTR extracellular domain includes SEQ ID NO: 73. In one embodiment, the p75NTR extracellular domain includes a camel p75NTR extracellular domain such as SEQ ID NO: 101. In one embodiment, the p75NTR extracellular domain includes a pig p75NTR extracellular domain such as SEQ ID NO: 110. In one embodiment, the p75NTR extracellular domain includes a horse p75NTR extracellular domain such as SEQ ID NO: 118. In one embodiment, the p75NTR extracellular domain includes a bovine p75NTR extracellular domain such as SEQ ID NO: 125. In one embodiment, the p75NTR protein or a portion thereof may comprise one of the variant p75NTR proteins, fusion proteins, or pharmaceutical compositions described herein above.

[0166] The p75NTR protein or a portion thereof for use in the treatment of osteoarthritis may be provided as a fusion protein. The fusion protein comprises an isolated companion animal p75NTR extracellular domain or a portion thereof and a half-life extension portion. For example, the p75NTR extracellular domain of SEQ ID NOs. 7, 38, 73, 101, 110, 118, or 125 may be ligated to the half-life extension portion. Suitable half-life extension portions are described herein and may be used in fusion proteins, e.g., Fc domains, serum albumin-binding factors, or PEG.

[0167] In embodiments in which a fusion protein containing an Fc domain is used to treat osteoarthritis, the Fc domain and the p75NTR extracellular domain or a portion thereof are linked by a linker. In one embodiment, the linker is a peptide linker, for example, (GGGG)n or (G4S)n, where n is 1 to 4.

[0168] In one embodiment, the fusion protein for use in the treatment of osteoarthritis comprises a canine extracellular domain (e.g., SEQ ID NO: 7) or a portion thereof (e.g., SEQ ID NO: 34). The portion of the canine extracellular domain in SEQ ID NO: 34 comprises a canine ECD that does not contain a stalk and does not have alpha and gamma-secretase cleavage at the 3' of the stalk region.

[0169] The Fc domain present in the fusion protein may be a canine Fc domain. In some embodiments, the fusion protein for use in the treatment of osteoarthritis comprises SEQ ID NO: 11 or SEQ ID NO: 13. In some embodiments, the fusion protein for use in the treatment of osteoarthritis comprises SEQ ID NO: 11. In some embodiments, the fusion protein for use in the treatment of osteoarthritis comprises a variant canine p75NTR ECD containing a variant amino acid at one or more of positions 75, 109, 133, and / or 134, and as described herein, for example, the fusion protein may contain or consist of SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 59. In some embodiments, the fusion protein may contain or consist of SEQ ID NO: 47 or SEQ ID NO: 59.

[0170] The Fc domain present in the fusion protein may be a feline Fc domain. In exemplary embodiments, the fusion protein for use in the treatment of osteoarthritis comprises SEQ ID NO: 39, SEQ ID NO: 42, or SEQ ID NO: 44. In some embodiments, the fusion protein for use in the treatment of osteoarthritis comprises SEQ ID NO: 42. In some embodiments, the fusion protein for use in the treatment of osteoarthritis comprises a variant feline p75NTR ECD containing one or more variant amino acids at positions 75, 109, 133, and / or 134, and as described herein, for example, the fusion protein may contain or consist of SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 70. In some embodiments, the fusion protein may contain or consist of SEQ ID NO: 62 or SEQ ID NO: 70.

[0171] The Fc domain present in the fusion protein may be a human Fc domain. In exemplary embodiments, the fusion protein for use in the treatment of osteoarthritis comprises SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. In some embodiments, the fusion protein for use in the treatment of osteoarthritis comprises a variant cat p75NTR ECD containing one or more variant amino acids at positions 75, 109, 133, and / or 134, and as described herein, for example, the fusion protein may comprise or consist of SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, or SEQ ID NO: 97. In some embodiments, the fusion protein may comprise or consist of SEQ ID NO: 85 or SEQ ID NO: 97.

[0172] The p75NTR molecule for use in the treatment of osteoarthritis can bind proNGF and / or NGF. In the treatment of osteoarthritis, it may be advantageous to use a molecule that preferentially binds to proNGF rather than NGF. As shown in the examples herein, p75NTR containing a variant amino acid at position 75 may preferentially bind to proNGF rather than NGF. In one embodiment, an isolated polypeptide, a fusion protein, containing the p75NTR protein or a portion thereof for use in the treatment of osteoarthritis, contains a variant amino acid at position 75.

[0173] The treatment of osteoarthritis may be measured by several factors, for example, and may include slowing or stopping disease progression, reversing disease progression, cartilage regrowth, and / or curative treatment. In some embodiments, the treatment of osteoarthritis is determined by the rate of cartilage loss or regrowth.

[0174] In one embodiment, treatment for osteoarthritis includes prophylactic treatment. Prophylactic treatment may include treatment to prevent the onset of the disease. Such treatment may be provided to the subject before the onset of symptoms.

[0175] In one embodiment, treatment for osteoarthritis comprises the administration of a second compound. Suitable additional therapeutic compounds are described above herein.

[0176] In one embodiment, the treatment for osteoarthritis further includes the treatment of one or more NGF-related disorders in companion animals. In one embodiment, administration of the p75NTR protein, a portion thereof, or a fusion protein results in the treatment of both osteoarthritis and one or more NGF-related disorders. Advantageously, the molecules described herein have been shown to possess both disease-modifying osteoarthritis activity and anti-inflammatory activity. In one embodiment, one or more NGF-related disorders are cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. In one embodiment, the NGF-related disorder is a pain-related disorder. In one embodiment, pain is selected from osteoarthritis pain, rheumatoid arthritis pain, surgical and postoperative pain, incision pain, systemic inflammatory pain, cancer pain, traumatic pain, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain.

[0177] Therefore, the molecules of the present invention can be advantageously used to treat osteoarthritis and provide analgesic effects. Similarly, the molecules of the present invention can be advantageously used to treat inflammation and provide analgesic effects. This is advantageous because subjects with osteoarthritis or inflammation may also suffer from pain. Suitable molecules include SEQ ID NO: 11, as demonstrated in Examples 11 and 12, and lower affinity molecules such as SEQ ID NO: 47 or SEQ ID NO: 59 for the treatment of dogs. Similarly, suitable molecules include SEQ ID NO: 42, and lower affinity molecules such as SEQ ID NO: 62 or SEQ ID NO: 70 for the treatment of cats.

[0178] In one embodiment, the treatment of osteoarthritis also includes the treatment of NGF-related disorders such as inflammation. In one embodiment, inflammation is an inflammatory disease, condition, or symptom. In one embodiment, inflammatory diseases, conditions, or symptoms are selected from the group consisting of single or multiple organ failure or dysfunction, sepsis, cytokine storm, fever, neurological dysfunction or injury, loss of taste or smell, cardiac dysfunction, pulmonary dysfunction, hepatic dysfunction, acute or chronic respiratory dysfunction, graft-versus-host disease (GVHD), cardiomyopathy, vasculitis, fibrosis, ocular inflammation, skin inflammation, gastroenteritis, tenosynovitis, allergies, asthma, glomerulonephritis, pancreatitis, hepatitis, non-alcoholic steatohepatitis (NASH), inflammatory arthritis, gout, multiple sclerosis, psoriasis, acute respiratory distress syndrome (ARDS), diabetic ulcers, non-healing wounds, lupus, autoimmune diseases associated with acute or chronic inflammation, and acute or chronic inflammation associated with viral, bacterial or fungal infections, swelling, tenderness, joint contracture or decreased joint mobility. In one embodiment, the inflammation is systemic. In one embodiment, the inflammation is localized.

[0179] Further methods and kits The present invention also provides in vitro, ex vivo, or in vivo methods for inhibiting NGF activity in a subject, comprising administering an isolated polypeptide, fusion protein, or pharmaceutical composition comprising the extracellular domain of the p75 neurotrophin receptor (p75NTR) of the present invention, wherein the isolated polypeptide comprises one or more variant amino acids at positions 75, 109, 133, and / or 134 within the p75NTR. In one or more embodiments, the present invention provides a method for producing the fusion protein of the present invention by culturing host cells of the present invention under conditions that result in the production of the fusion protein, and then isolating the fusion protein from the host cells or the culture medium of the host cells.

[0180] In another embodiment, the present invention provides a kit for the treatment or prevention, diagnosis, prognosis, or monitoring of a disease, comprising an isolated polypeptide comprising the extracellular domain of the p75 neurotrophin receptor (p75NTR) of the present invention, wherein the isolated polypeptide comprises one or more variant amino acids at positions 75, 109, 133, and / or 134 within the p75NTR, the isolated polypeptide, fusion protein, or pharmaceutical composition. Such a kit may include other components, packaging, and / or instructions.

[0181] In another embodiment, the present invention provides isolated polypeptides, fusion proteins, or pharmaceutical compositions comprising the extracellular domain of the p75 neurotrophin receptor (p75NTR) of the present invention, packaged in a lyophilized form or in an aqueous medium, wherein the isolated polypeptide comprises one or more variant amino acids at positions 75, 109, 133, and / or 134 within the p75NTR.

[0182] In another embodiment, an isolated polypeptide, fusion protein, or pharmaceutical composition comprising the p75 neurotrophin receptor (p75NTR) extracellular domain of the present invention as described herein, wherein the isolated polypeptide comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134 within the p75NTR, for use in non-therapeutic purposes such as diagnostic tests and assays. Accordingly, the present invention also provides the above-mentioned p75NTR protein and fusion protein for use in diagnostic methods for detecting NGF in subjects known to have or suspected to have NGF-related disorders, but not limited to human, canine, or feline subjects. A method for detecting NGF in subjects known to have or suspected to have NGF-related disorders may include exposing a sample from the subject to a labeled protein of the present invention and detecting the labeled protein. The diagnostic method may be used to quantitatively or qualitatively detect NGF in a sample or to detect the presence of cells expressing NGF.

[0183] Further aspects and embodiments of the present invention will be apparent to those skilled in the art upon consideration of this disclosure, including the following experimental examples.

[0184] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. The foregoing disclosure provides a general description of the subject matter encompassed within the scope of the invention, including methods for constructing and using the invention, and its best mode; however, the following examples are provided to enable those skilled in the art to practice the invention and to provide a further complete written description thereof. However, those skilled in the art will understand that the details of these examples should not be read as limiting the invention. The scope of the invention should be understood from the claims and their equivalents attached to this disclosure. Various further aspects and embodiments of the invention will be apparent to those skilled in the art in consideration of this disclosure.

[0185] All documents referenced herein, including any references to gene accession numbers and references to patent publications, are incorporated herein in their entirety.

[0186] When used herein, “and / or” should be interpreted as a specific disclosure of each of two particular features or components, with or without the other. For example, “A and / or B” should be interpreted as each of the specific disclosures of (i) A, (ii) B, and (iii) A and B, as each of which is described separately herein. Unless the context otherwise indicates, the above descriptions and definitions of features are not limited to any particular aspect or embodiment of the Invention, but apply equally to all aspects and embodiments described herein.

[0187] Numbered clauses 1. An isolated polypeptide comprising a companion animal p75 neurotrophin receptor (p75NTR) extracellular domain, wherein the p75NTR contains a variant amino acid at one or more of the positions 75, 109, 133, and / or 134. 2. The isolated polypeptide according to Clause 1, wherein the variant amino acid at position 75 of p75NTR includes a polar side chain. 3. An isolated polypeptide according to any preceding clause, wherein the variant amino acid at position 75 of p75NTR is selected from serine, threonine, tyrosine, tryptophan, asparagine, glutamine, or cysteine, preferably threonine. 4. An isolated polypeptide according to any preceding clause, wherein the variant amino acid at position 109 of p75NTR includes an aromatic side chain. 5. An isolated polypeptide according to any preceding clause, wherein the variant amino acid at position 109 of p75NTR is selected from histidine, tyrosine, phenylalanine, or tryptophan. 6. An isolated polypeptide according to any preceding clause, wherein the variant amino acid at position 133 of p75NTR includes a charged side chain. 7. An isolated polypeptide according to any prior clause, wherein the variant amino acid at position 133 of p75NTR is selected from arginine, histidine, lysine, aspartic acid, or glutamic acid. 8. An isolated polypeptide according to any one of the clauses 1 to 6, wherein the variant amino acid at position 133 of p75NTR includes a negatively charged side chain. 9. The isolated polypeptide according to Clause 8, wherein the variant amino acid at position 133 of p75NTR is selected from arginine, histidine, or lysine, and is preferably arginine. 10. An isolated polypeptide according to any preceding clause, wherein the variant amino acid at position 134 of p75NTR includes a hydrophobic side chain. 11. An isolated polypeptide according to any preceding clause, wherein the variant amino acid at position 134 of p75NTR is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. 12. An isolated polypeptide according to any one of the clauses 1 to 10, wherein the variant amino acid at position 134 of p75NTR includes a non-aromatic hydrophobic side chain. 13. The isolated polypeptide according to Clause 12, wherein the variant amino acid at position 134 of p75NTR is selected from alanine, valine, isoleucine, leucine, or methionine, and is preferably leucine. 14. An isolated polypeptide as described in any preceding clause, wherein the companion animal is a cat, dog, pig, cow, horse, or camel. 15. An isolated polypeptide according to any preceding clause, wherein the companion animal is a dog, and p75NTR contains or consists of a sequence selected from SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58. 16. An isolated polypeptide according to any one of Clauses 1 to 14, wherein the companion animal is a cat, and p75NTR contains or consists of a sequence selected from SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69. 17. An isolated polypeptide according to any one of Clauses 1 to 14, wherein the companion animal is a pig and p75NTR contains or consists of a sequence selected from SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116; the companion animal is a horse and p75NTR contains or consists of a sequence selected from SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, or SEQ ID NO: 124; the companion animal is a cow and p75NTR contains or consists of a sequence selected from SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131; or the companion animal is a camel and p75NTR contains or consists of a sequence selected from SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107. 18. The isolated polypeptide described in the preceding clause, in which the p75NTR extracellular domain is cleaved. 19. Isolated nucleic acid encoding an isolated polypeptide as described in the preceding clause. 20. A vector containing nucleic acids as described in Clause 19. 21. Host cells containing nucleic acids as described in Clause 19 or vectors as described in Clause 20. 22. A fusion protein comprising a companion animal p75NTR extracellular domain, wherein the p75NTR comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134, and comprises a half-life extension portion. 23. The fusion protein described in Clause 22, wherein the half-life extension portion is selected from an Fc domain, a serum albumin-binding factor, or PEG. 24. The fusion protein described in Clause 22, wherein the half-life extension portion is a wild-type or mutant Fc domain. 25. A fusion protein according to any one of clauses 22 to 24, wherein the half-life extension portion is an Fc domain, and the p75NTR extracellular domain or a portion thereof is linked to the Fc domain by a linker. 26. The fusion protein described in Clause 25, wherein the linker is a peptide linker. 27. Peptide linker, (G4S) n The fusion protein described in Clause 26, wherein n is 1 to 4 in the formula. 28. A fusion protein as described in any one of clauses 22-27, wherein the companion animal is a cat, dog, pig, cow, horse, or camel. 29. A fusion protein according to any one of clauses 22 to 28, wherein the Fc domain is a canine Fc domain. 30. The fusion protein according to Clause 29, wherein the fusion protein includes or consists of a sequence selected from SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 59. 31. A fusion protein according to any one of clauses 22 to 28, wherein the Fc domain is a feline Fc domain. 32. The fusion protein according to Clause 31, wherein the fusion protein includes or consists of a sequence selected from SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 70. 33. A nucleic acid encoding a fusion protein as described in any one of clauses 22 to 32. 34. A vector containing nucleic acids as described in Clause 33. 35. A host cell containing the nucleic acid described in Clause 33 or the vector described in Clause 34. 36. A pharmaceutical composition comprising an isolated polypeptide as described in any one of Clauses 1 to 18, or a fusion protein as described in any one of Clauses 22 to 32. 37. A method for treating NGF-related disorders in companion animals, comprising administering an isolated companion animal p75NTR protein as described in any one of Clauses 1 to 18, a fusion protein as described in any one of Clauses 22 to 32, or a pharmaceutical composition as described in Clause 36. 38. Use of an isolated companion animal p75NTR protein according to any one of Embodiments 1 to 18, a fusion protein according to any one of Clauses 22 to 32, or a pharmaceutical composition according to Embodiment 36 in the manufacture of a pharmaceutical for the treatment of NGF-related disorders in companion animals. 39. An isolated companion animal p75NTR protein according to any one of Embodiments 1 to 18, or a fusion protein according to any one of Clauses 22 to 32, for use in the treatment of NGF-related disorders in companion animals. 40. Isolated companion animal p75NTR protein or fusion protein for use in the manner described in Clause 37, or the use described in Clause 38, or the use described in Clause 39, for NGF-related disorders being cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. 41. NGF-related disorders are pain-related disorders, as described in, used by, or for use by isolated companion animal p75NTR protein as described in Clause 40. 42. The method, use, or use of isolated companion animal p75NTR protein as described in Clause 41, wherein the pain is selected from osteoarthritis pain, rheumatoid arthritis pain, surgical and postoperative pain, incision pain, systemic inflammatory pain, cancer pain, traumatic pain, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain. 43. NGF-related disorder is inflammation, inflammation is an inflammatory disease, condition, or symptom, and optionally, inflammatory disease, condition, or symptom is single or multiple organ failure or dysfunction, sepsis, cytokine storm, fever, neurological dysfunction or injury, loss of taste or smell, cardiac dysfunction, pulmonary dysfunction, hepatic dysfunction, acute or chronic respiratory failure, graft-versus-host disease (GVHD), cardiomyopathy, vasculitis, fibrosis, ocular inflammation, skin inflammation, gastroenteritis, tenosynovitis, allergy, asthma, glomerulonephritis , a companion animal p75NTR protein isolated for use or use in the manner described in Clause 40, selected from the group consisting of pancreatitis, hepatitis, non-alcoholic steatohepatitis (NASH), inflammatory arthritis, gout, multiple sclerosis, psoriasis, acute respiratory distress syndrome (ARDS), diabetic ulcers, non-healing wounds, lupus, autoimmune diseases associated with acute or chronic inflammation, and acute or chronic inflammation associated with viral, bacterial or fungal infections, swelling, tenderness, joint contracture or decreased joint mobility. 44. A method for inhibiting NGF activity in a companion animal, comprising administering an isolated companion animal p75NTR protein as described in any one of Clauses 1 to 18, a fusion protein as described in any one of Clauses 22 to 32, or a pharmaceutical composition as described in Clause 36. 45. The method, use, or isolated companion animal p75NTR protein described in any one of the clauses 41-43, or the method described in clause 44, including the administration of a second compound for treating pain. 46. ​​A kit comprising an isolated companion animal p75NTR protein as described in any one of Clauses 1 to 18, a fusion protein as described in any one of Clauses 22 to 32, or a pharmaceutical composition as described in Clause 36, and optionally, instructions for use. 47. A method for treating osteoarthritis in a companion animal, comprising administering an effective amount of isolated companion animal p75NTR protein or a portion thereof, or a pharmaceutical composition comprising said isolated companion animal p75NTR protein or a portion thereof. 48. Use of an isolated companion animal p75NTR protein or a portion thereof, or a pharmaceutical composition containing the isolated companion animal p75NTR protein or a portion thereof, for the manufacture of a medicament for the treatment of osteoarthritis in companion animals. 49. Isolated companion animal p75NTR protein or a portion thereof, or a pharmaceutical composition comprising said isolated companion animal p75NTR protein or a portion thereof, for use in the treatment of osteoarthritis in companion animals. 50. Isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use according to the method described in Clause 47, or the use described in Clause 48, or the use described in Clause 49, wherein the companion animal is a cat, dog, pig, cow, horse, or camel. 51. The method, use, or use of isolated companion animal p75NTR protein or portion thereof, or a pharmaceutical composition thereof, as described in Clause 50, wherein the isolated companion animal p75NTR protein or portion thereof comprises one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 36, SEQ ID NO: 99, or SEQ ID NO: 108. 52. A companion animal p75NTR protein or a portion thereof, or a pharmaceutical composition for use, by any one of the methods described in any one of the clauses 47 to 51, comprising an extracellular domain or a portion thereof, wherein the p75NTR protein or a portion thereof comprises an extracellular domain or a portion thereof. 53. An isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use as described in Clause 52, or for use for use, wherein the p75NTR extracellular domain comprises one of SEQ ID NO: 7, SEQ ID NO: 38, SEQ ID NO: 101, SEQ ID NO: 110, SEQ ID NO: 118, or SEQ ID NO: 125. 54. The method, use, or use of an isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use as described in any one of the items 47 to 53, wherein the p75NTR comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134. 55. A variant amino acid at position 75 of p75NTR, comprising a polar side chain, isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use, as described in Clause 54, or for use. 56. The variant amino acid at position 75 of p75NTR is selected from serine, threonine, tyrosine, tryptophan, asparagine, glutamine, or cysteine, preferably threonine, as described in any one of clauses 54 to 55, or an isolated companion animal p75NTR protein or a portion thereof or a pharmaceutically acceptable composition for use. 57. A variant amino acid at position 109 of p75NTR comprising an aromatic side chain, the method, use, or use of an isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use as described in any one of the items 54 to 56. 58. The variant amino acid at position 109 of p75NTR is selected from histidine, tyrosine, phenylalanine, or tryptophan, wherein the method, use, or use of isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use as described in any one of clauses 54 to 57. 59. A variant amino acid at position 133 of p75NTR comprising a charged side chain, the method, use, or use of an isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use as described in any one of the clauses 54 to 58. 60. The variant amino acid at position 133 of p75NTR is selected from arginine, histidine, lysine, aspartic acid, or glutamic acid, wherein the method, use, or use of isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use as described in any one of clauses 54 to 59. 61. A variant amino acid at position 133 of p75NTR comprising a negatively charged side chain, the method, use, or use of an isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use as described in any one of the clauses 54 to 60. 62. The variant amino acid at position 133 of p75NTR is selected from arginine, histidine, or lysine, preferably arginine, wherein the isolated companion animal p75NTR protein or a portion thereof or a pharmaceutically acceptable composition for use, as described in Clause 61. 63. A variant amino acid at position 134 of p75NTR comprising a hydrophobic side chain, the method, use, or use of an isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use as described in any one of clauses 54 to 62. 64. The variant amino acid at position 134 of p75NTR is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, wherein the method, use, or use of isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition thereof is described in any one of clauses 54 to 63. 65. A variant amino acid at position 134 of p75NTR comprising a non-aromatic hydrophobic side chain, the method, use, or use of an isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use as described in any one of clauses 54 to 64. 66. The variant amino acid at position 134 of p75NTR is selected from alanine, valine, isoleucine, leucine, or methionine, preferably leucine, wherein the isolated companion animal p75NTR protein or a portion thereof or a pharmaceutically acceptable composition for use as described in Clause 65. 67. The companion animal is a cat, dog, pig, cow, horse, or camel, the method, use, or isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use as described in any one of the clauses 54 to 66. 68. The method, use, or isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use according to any one of the clauses 54 to 67, wherein the companion animal is a dog, and p75NTR comprises or consists of a sequence selected from SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58. 69. The method, use, or isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use according to any one of the claims 54 to 67, wherein the companion animal is a cat, and p75NTR comprises or consists of a sequence selected from SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, or SEQ ID NO: 69. 70. The extracellular domain of p75NTR is cleaved in an isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use as described in any one of clauses 54 to 69. 71. The isolated companion animal p75NTR protein is provided as a fusion protein comprising the isolated companion animal p75NTR extracellular domain or portion thereof and a half-life extension portion, as described in any one of Clauses 47 to 70, or an isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use. 72. Isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use, use, or application as described in Clause 71, wherein the half-life extension portion is selected from the Fc domain, serum albumin-binding factor, or PEG. 73. Isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use, use, or application as described in Clause 72, wherein the half-life extension portion is the wild-type or mutant Fc domain. 74. The half-life extension portion is an Fc domain, and the Fc domain is linked to the p75NTR extracellular domain or a portion thereof by a linker, an isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use as described in any one of clauses 71 to 73. 75. Isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use, use, or otherwise for use as described in Clause 74, wherein the linker is a peptide linker. 76. Companion animal p75NTR protein or a portion thereof or a pharmaceutical composition as described in, used, or isolated in the manner of Clause 75, wherein the peptide linker is (GGGG)n or (G4S)n, where n is 1 to 4. 77. The method, use, or use of isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use as described in any one of the clauses 71 to 76, wherein the fusion protein comprises one of SEQ ID NOs: 11, 13, 39, 42, 47, 50, 53, 56, 59, 62, 64, 66, 68, or 70. 78. The treatment of the osteoarthritis thereof includes delaying or halting disease progression, reversing disease progression, cartilage regrowth, and / or curative treatment, as described in any one of Clauses 47 to 77, or isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition for use. 79. The treatment as described in Clause 78, or a portion thereof or a pharmaceutically acceptable composition for use, where the treatment is determined by the rate of cartilage loss or regrowth. 80. The method, use, or use of isolated companion animal p75NTR protein or portion thereof or pharmaceutically active composition for use, as described in any one of the paragraphs 47 to 79, wherein the treatment of the osteoarthritis further comprises the treatment of one or more NGF-related disorders in a companion animal. 81. One or more NGF-related disorders are cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation, as described in Clause 80, or isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use. 82. NGF-related disorders are pain-related disorders, as described in, used by, or for use by isolated companion animal p75NTR protein or portion thereof or a pharmaceutical composition as described in Clause 81. 83. The method, use, or use of isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use, as described in Clause 82, wherein the pain is selected from osteoarthritis pain, rheumatoid arthritis pain, surgical and postoperative pain, incision pain, systemic inflammatory pain, cancer pain, traumatic pain, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain. 84. If NGF-related disorder is inflammation, and inflammation is an inflammatory disease, condition, or symptom, then the method, use, or use of isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition as described in Clause 81. 85. The method, use, or isolated companion animal p75NTR protein or portion thereof or pharmaceutical composition for use of an inflammatory disease, condition, or symptom selected from the group consisting of single or multiple organ failure or dysfunction, sepsis, cytokine storm, fever, neurological dysfunction or injury, loss of taste or smell, cardiac dysfunction, pulmonary dysfunction, hepatic dysfunction, acute or chronic respiratory dysfunction, graft-versus-host disease (GVHD), cardiomyopathy, vasculitis, fibrosis, ocular inflammation, skin inflammation, gastroenteritis, tenosynovitis, allergy, asthma, glomerulonephritis, pancreatitis, hepatitis, non-alcoholic steatohepatitis (NASH), inflammatory arthritis, gout, multiple sclerosis, psoriasis, acute respiratory distress syndrome (ARDS), diabetic ulcer, non-healing wound, lupus, autoimmune diseases associated with acute or chronic inflammation, and acute or chronic inflammation associated with viral, bacterial or fungal infection, swelling, tenderness, joint contracture or decreased joint mobility. 86. The treatment includes prophylactic treatment, the method, use, or isolated companion animal p75NTR protein or portion thereof or pharmaceutical composition for use as described in any one of the clauses 47 to 85. 87. The method, use, or use of isolated companion animal p75NTR protein or a portion thereof or a pharmaceutical composition for use, as described in any one of the clauses 47 to 86, comprising administering a second compound.

[0188] Numbered Embodiments 1. An isolated polypeptide comprising the extracellular domain of the human p75 neurotrophin receptor (p75NTR), wherein the p75NTR comprises one or more variant amino acids at positions 75, 109, 133, and / or 134. 2. The isolated polypeptide according to Embodiment 1, wherein the variant amino acid at position 75 of p75NTR includes a polar side chain. 3. An isolated polypeptide according to any prior embodiment, wherein the variant amino acid at position 75 of p75NTR is selected from serine, threonine, tyrosine, tryptophan, asparagine, glutamine, or cysteine, preferably threonine. 4. An isolated polypeptide according to any prior embodiment, wherein the variant amino acid at position 109 of p75NTR includes an aromatic side chain. 5. An isolated polypeptide according to any prior embodiment, wherein the variant amino acid at position 109 of p75NTR is selected from histidine, tyrosine, phenylalanine, or tryptophan. 6. An isolated polypeptide according to any prior embodiment, wherein the variant amino acid at position 133 of p75NTR includes a charged side chain. 7. An isolated polypeptide according to any prior embodiment, wherein the variant amino acid at position 133 of p75NTR is selected from arginine, histidine, lysine, aspartic acid, or glutamic acid. 8. An isolated polypeptide according to any one of Embodiments 1 to 6, wherein the variant amino acid at position 133 of p75NTR includes a negatively charged side chain. 9. The isolated polypeptide according to Embodiment 8, wherein the variant amino acid at position 133 of p75NTR is selected from arginine, histidine, or lysine, and is preferably arginine. 10. An isolated polypeptide according to any prior embodiment, wherein the variant amino acid at position 134 of p75NTR includes a hydrophobic side chain. 11. An isolated polypeptide according to any prior embodiment, wherein the variant amino acid at position 134 of p75NTR is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. 12. An isolated polypeptide according to any one of Embodiments 1 to 10, wherein the variant amino acid at position 134 of p75NTR includes a non-aromatic hydrophobic side chain. 13. The isolated polypeptide according to Embodiment 12, wherein the variant amino acid at position 134 of p75NTR is selected from alanine, valine, isoleucine, leucine, or methionine, and is preferably leucine. 14. An isolated polypeptide according to any prior embodiment, wherein p75NTR contains or consists of a sequence selected from SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, or SEQ ID NO: 96. 15. The isolated polypeptide described in the prior embodiment, in which the p75NTR extracellular domain is cleaved. 16. Isolated nucleic acid encoding an isolated polypeptide as described in a prior embodiment. 17. A vector comprising the nucleic acid described in Embodiment 16. 18. A host cell comprising the nucleic acid described in Embodiment 16 or the vector described in Embodiment 17. 19. A fusion protein comprising a human p75NTR extracellular domain, wherein the p75NTR comprises one or more variant amino acids at positions 75, 109, 133, and / or 134, and comprises a half-life extension portion. 20. The fusion protein according to Embodiment 19, wherein the half-life extension portion is selected from an Fc domain, a serum albumin-binding factor, or PEG. 21. The fusion protein according to Embodiment 19, wherein the half-life extension portion is a wild-type or mutant Fc domain. 22. A fusion protein according to any one of embodiments 19 to 21, wherein the half-life extension portion is an Fc domain, and the p75NTR extracellular domain or a portion thereof is linked to the Fc domain by a linker. 23. The fusion protein according to Embodiment 22, wherein the linker is a peptide linker. 24. The peptide linker is (G4S) n The fusion protein according to Embodiment 23, wherein n is 1 to 4 in the formula. 25. A fusion protein according to any one of Embodiments 19 to 24, wherein the Fc domain is a human Fc domain. 26. The fusion protein according to Embodiment 25, wherein the fusion protein includes or consists of a sequence selected from SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, or SEQ ID NO: 97. 27. A nucleic acid encoding a fusion protein as described in any of embodiments 19 to 26. 28. A vector comprising the nucleic acid described in Embodiment 27. 29. A host cell comprising the nucleic acid described in Embodiment 27 or the vector described in Embodiment 28. 30. A pharmaceutical composition comprising an isolated polypeptide according to any of Embodiments 1 to 15, or a fusion protein according to any of Embodiments 19 to 26. 31. A method for treating NGF-related disorders in a subject, comprising administering a human p75NTR protein according to any of Embodiments 1 to 15, a fusion protein according to any of Embodiments 19 to 26, or a pharmaceutical composition according to Embodiment 30. 32. Use of an isolated human p75NTR protein according to any of Embodiments 1 to 15, a fusion protein according to any of Embodiments 19 to 26, or a pharmaceutical composition according to Embodiment 30 in the manufacture of a pharmaceutical for the treatment of NGF-related disorders in a subject. 33. An isolated human p75NTR protein according to any of Embodiments 1 to 15, a fusion protein according to any of Embodiments 19 to 26, or a pharmaceutical composition according to Embodiment 30, for use in the treatment of NGF-related disorders in subjects. 33. Use according to Embodiment 31 or Embodiment 32, wherein the NGF-related disorder is cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. 34. The method or use of Embodiment 33, wherein the NGF-related disorder is a pain-related disorder. 35. The method or use of Embodiment 34, wherein the pain is selected from osteoarthritis pain, rheumatoid arthritis pain, surgical and postoperative pain, incision pain, systemic inflammatory pain, cancer pain, traumatic pain, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain. 36. A method for inhibiting NGF activity in a subject, comprising administering an isolated human p75NTR protein according to any of Embodiments 1 to 15, a fusion protein according to any of Embodiments 19 to 26, or a pharmaceutical composition according to Embodiment 30. 37. The method or use of Embodiment 34 or Embodiment 35, or the method of Embodiment 36, comprising the administration of a second compound for treating pain. 38. A kit comprising an isolated human p75NTR protein according to any of Embodiments 1 to 15, a fusion protein according to any of Embodiments 19 to 26, or a pharmaceutical composition according to Embodiment 30, and optionally, instructions for use. 39. A method for treating osteoarthritis in humans, comprising administering an effective amount of isolated human p75NTR protein or a portion thereof, or a pharmaceutical composition comprising said isolated human p75NTR protein or a portion thereof. 40. Use of isolated human p75NTR protein or a portion thereof, or a pharmaceutical composition containing said isolated human p75NTR protein or a portion thereof, for the manufacture of a pharmaceutical for the treatment of osteoarthritis in humans. 41. Isolated human p75NTR protein or a portion thereof, or a pharmaceutical composition comprising said isolated human p75NTR protein or a portion thereof, for use in the treatment of osteoarthritis in humans. 42. Isolated human p75NTR protein or a portion thereof, or a pharmaceutical composition for use according to Embodiment 39, the use described in Embodiment 40, or the use described in Embodiment 41, wherein isolated human p75NTR protein or a portion thereof comprises SEQ ID NO: 71. 43. A p75NTR protein or a portion thereof comprising an extracellular domain or a portion thereof, isolated human p75NTR protein or a portion thereof, or a pharmaceutically acceptable composition for use, according to any of Embodiments 39 to 42, or for use. 44. An isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition for use, use, or for use as described in Embodiment 43, wherein the p75NTR extracellular domain comprises SEQ ID NO: 73. 45. The method, use, or use of an isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use according to any of Embodiments 39 to 44, wherein the human p75NTR comprises a variant amino acid at one or more of positions 75, 109, 133, and / or 134. 46. ​​The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use according to Embodiment 45, wherein the variant amino acid at position 75 of p75NTR includes a polar side chain. 47. The variant amino acid at position 75 of p75NTR is selected from serine, threonine, tyrosine, tryptophan, asparagine, glutamine, or cysteine, preferably threonine, as described in any of Embodiments 45 to 46, or an isolated human p75NTR protein or portion thereof or a pharmaceutically acceptable composition for use. 48. A variant amino acid at position 109 of p75NTR comprising an aromatic side chain, the method, use, or use of an isolated human p75NTR protein or portion thereof or a pharmaceutically acceptable composition as described in any of Embodiments 45-47. 49. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition for use as described in any of Embodiments 45-48, wherein the variant amino acid at position 109 of p75NTR is selected from histidine, tyrosine, phenylalanine, or tryptophan. 50. A variant amino acid at position 133 of p75NTR comprising a charged side chain, the method, use, or use of an isolated human p75NTR protein or portion thereof or a pharmaceutical composition for use as described in any of Embodiments 45 to 49. 51. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use according to any of Embodiments 45-50, wherein the variant amino acid at position 133 of p75NTR is selected from arginine, histidine, lysine, aspartic acid, or glutamic acid. 52. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use according to any of Embodiments 45 to 51, wherein the variant amino acid at position 133 of p75NTR comprises a negatively charged side chain. 53. The variant amino acid at position 133 of p75NTR is selected from arginine, histidine, or lysine, preferably arginine, wherein the method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition is described in Embodiment 52. 54. The variant amino acid at position 134 of p75NTR comprises a hydrophobic side chain, the method, use, or use of isolated human p75NTR protein or portion thereof or a pharmaceutical composition for use as described in any of Embodiments 45 to 53. 55. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use as described in any of Embodiments 45 to 54, wherein the variant amino acid at position 134 of p75NTR is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. 56. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use according to any of Embodiments 45 to 55, wherein the variant amino acid at position 134 of p75NTR comprises a non-aromatic hydrophobic side chain. 57. The variant amino acid at position 134 of p75NTR is selected from alanine, valine, isoleucine, leucine, or methionine, preferably leucine, wherein the method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition is described in Embodiment 56. 58. A method, use, or isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use according to any of Embodiments 45 to 57, wherein p75NTR comprises or consists of a sequence selected from SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 93, or SEQ ID NO: 96. 59. Isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use, by any of the methods, uses, or for use described in any of Embodiments 45 to 58, wherein the extracellular domain of p75NTR is cleaved. 60. The method, use, or use of an isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition according to any one of Embodiments 39 to 59, wherein an isolated human p75NTR protein is provided as a fusion protein comprising the isolated human p75NTR extracellular domain or a portion thereof and a half-life extension portion. 61. Isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use, by the method of Embodiment 60, or for use, wherein the half-life extension portion is selected from the Fc domain, serum albumin-binding factor, or PEG. 62. Isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use, by the method of Embodiment 61, or for use, wherein the half-life extension portion is a wild-type or mutant Fc domain. 63. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use according to any of Embodiments 60 to 62, wherein the half-life extension portion is an Fc domain, and the p75NTR extracellular domain or a portion thereof is linked to the Fc domain by a linker. 64. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition according to Embodiment 63, wherein the linker is a peptide linker. 65. A human p75NTR protein or a portion thereof or a pharmaceutical composition isolated by the method, use, or method described in Embodiment 64, wherein the peptide linker is (GGGG)n or (G4S)n, where n is 1 to 4. 66. A human p75NTR protein or a portion thereof isolated by any of embodiments 60 to 65, or a pharmaceutical composition, wherein the fusion protein comprises one of SEQ ID NOs: 81, 82, 83, 85, 88, 91, 94, or 97. 67. The method, use, or isolated human p75NTR protein or portion thereof or a pharmaceutically acceptable composition for use according to any one of Embodiments 39 to 66, wherein the treatment of the osteoarthritis includes slowing or halting disease progression, reversing disease progression, cartilage regrowth, and / or curative treatment. 68. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutical composition for use as described in Embodiment 67, wherein the treatment is determined by the rate of cartilage loss or regrowth. 69. The method, use, or use of isolated human p75NTR protein or portion thereof or a pharmaceutically acceptable composition for use according to any one of Embodiments 39 to 68, wherein the treatment of the osteoarthritis further comprises the treatment of one or more NGF-related disorders in humans. 70. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition according to Embodiment 69, wherein one or more NGF-related disorders are cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation. 71. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition according to Embodiment 70, wherein the NGF-related disorder is a pain-related disorder. 72. The method, use, or isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition for use according to Embodiment 71, wherein the pain is selected from osteoarthritis pain, rheumatoid arthritis pain, surgical and postoperative pain, incision pain, systemic inflammatory pain, cancer pain, traumatic pain, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain. 73. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition for use according to Embodiment 70, wherein the NGF-related disorder is inflammation, and the inflammation is an inflammatory disease, condition, or symptom. 74. The method, use, or isolated human p75NTR protein or portion thereof or a pharmaceutical composition for use according to Embodiment 73, wherein the inflammatory disease, condition, or symptom is selected from the group consisting of single or multiple organ failure or dysfunction, sepsis, cytokine storm, fever, neurological dysfunction or injury, loss of taste or smell, cardiac dysfunction, pulmonary dysfunction, hepatic dysfunction, acute or chronic respiratory dysfunction, graft-versus-host disease (GVHD), cardiomyopathy, vasculitis, fibrosis, ocular inflammation, skin inflammation, gastroenteritis, tenosynovitis, allergy, asthma, glomerulonephritis, pancreatitis, hepatitis, non-alcoholic steatohepatitis (NASH), inflammatory arthritis, gout, multiple sclerosis, psoriasis, acute respiratory distress syndrome (ARDS), diabetic ulcer, non-healing wound, lupus, autoimmune diseases associated with acute or chronic inflammation, and acute or chronic inflammation associated with viral, bacterial or fungal infection, swelling, tenderness, joint contracture or decreased joint mobility. 75. The treatment, use, or use of an isolated human p75NTR protein or portion thereof or a pharmaceutical composition for use according to any one of embodiments 39 to 74, including prophylactic treatment. 76. The method, use, or use of isolated human p75NTR protein or a portion thereof or a pharmaceutically acceptable composition for use according to any one of embodiments 39 to 75, comprising administering a second compound. The present invention is further described by non-limiting embodiments. [Examples]

[0189] Example 1 - Structural analysis of the p75NTR-NGF interaction Models of the p75NTR extracellular domain and NGF were generated using an alpha-folding server (using the human crystal structure - pdb code 3BUK as a template) (Figure 2). Interaction residues between p75NTR and NGF were identified using distance and geometric cutoffs. Mutations were identified to improve / decrease binding and improve expression.

[0190] Figure 1 shows the alignment of p75NTR sequences from dogs, cats, cattle, and horses, demonstrating high sequence identity among the species. Dog vs. Cat: 1 / 164 (over 99% identical) Dogs vs. Horses: 4 / 164 (97.6% identical). Cat vs. Horse: 5 / 164 (97% identical). Dogs vs. Cows: 2 / 164 (98.8% identical).

[0191] Example 2 - Protein construct and CHO-s transfection / expression The amino acid sequence of the PetML119 variant is shown in the following sequence section.

[0192] For protein production, DNA constructs were generated to encode a chimeric Fc fusion protein containing a selected canine IgG constant region (between the hinge and the C-terminus) fused to the extracellular domain of canine p75 lacking the predicted O-glycosylation and γ-secretase sites.

[0193] Both the canine IgG-B Fc domain and the p75 extracellular domain (res 31-194 from UniProtKB-J9PAM0) were synthesized. Both genes were PCR amplified using Q5 high-fidelity DNA polymerase (specific primers containing overlapping regions were used to enable assembly) and assembled into the mammalian expression vector PetML119var using NEBuilder HIFI DNA Assembly (New England Biolabs). In the expression vector, the fusion protein chain and antibiotic resistance gene expression units are flanked by DNA transposon piggyBac terminal inversion repeats, mediating stable integration into host cells in the presence of piggyBac transposase. The expression vector was then transfected with PiggyBac transposase into suitable mammalian cell lines such as CHO cells to obtain stable expression. 1 × 10⁶ units were used for fusion protein production. 6 Selected CHO cells at a concentration of / mL were seeded in 800mL of culture medium (F17 + 4mM l-Gln + 0.3% P188 + 1:500 ACA) and incubated at 32°C and 8% CO2 with shaking at 130 rpm. 2% HyClone Cell Boost 7a supplement + 0.2% HyClone Cell Boost 7b supplement and 2mM glucose were added to the medium daily from day 4 of overproduction. The culture supernatant was collected on day 10, and protein concentrations were determined using surface plasmon resonance (Biacore 8K, Cytiva Life Sciences) with a Protein A tip.

[0194] Typically, PetML119vars showed peak expression on day 10 of production, reaching 20–150 mg / L. High-affinity molecules (PetML119-S109Y, PetML119-S109H, PetML119-V133R) showed a 2-fold improvement compared to the WT molecule (SEQ ID NO: 11), while lower-affinity molecules (PetML119-E75T, PetML119-D134L) showed slightly lower titers. PetML119: 60 mg / L PetML119-E75T: 20mg / L PetML119-S109Y: 130 mg / L PetML119-S109H: 150 mg / L PetML119-V133R: 120 mg / L PetML119-D134L: 30 mg / L

[0195] Example 3 - Purification of PetML119var Cell suspensions derived from stable transfect clones of PetML119var, cultured for at least 7 days as described, were incubated for 10 minutes in Sartoclear Dynamics® Lab V (SDLV-0500-20C-E) and then filtered using a 0.22 μm filter. The clear supernatant was loaded onto a 20 mL Mabselect sure LX pre-packed column (17547402) pre-equilibrated with PBS. The column was washed with 40 mL of PBS (2 CV), and the fusion protein was then eluted using a 0.1 M glycine pH 2.7 gradient (0–100% in 2 CV). The fractions containing the fusion protein were pooled together and neutralized with 100 mM TRIS pH 8 (final concentration).

[0196] The neutralized fusion protein pool fraction was concentrated to 5 mL and loaded onto PBS pre-equilibriumized HiLoad 16 / 600 Superdex 200 pg (28989335) as the second step of purification. The monomer fraction was pooled (based on the retention time of previously analyzed protein standards), and the protein concentration was assessed using NanoDrop®One (Thermo Scientific®).

[0197] Following the protocol described above, purified products with a concentration of approximately 10–60 mg / L were obtained. The variants were purified to a concentration comparable to that of the waste product (WT). The yield of the variants did not change during the purification process compared to the WT. PetML119: After two-step purification, 30 mg / L PetML119-E75T: After two-step purification, 10 mg / L PetML119-S109Y: After two-step purification, 60 mg / L PetML119-S109H: After two-step purification, 63.3 mg / L PetML119-V133R: After two-step purification, 56.6 mg / L PetML119-D134L: After two-step purification, 13.3 mg / L

[0198] Example 4 - HPLC Analytical Chromatography The purity of the purified material was evaluated using both size exclusion chromatography (SEC) for oligomerization analysis and cation exchange chromatography (SCX) for charge variant analysis.

[0199] HPLC-SEC chromatography (column: BioResolve SEC mAb 200A, 2.5um column, WATERS) was performed using a WATERS ACQUITY H-class Bio with PBS as the mobile phase, having an isocratic flow rate of 0.575 mL / min.

[0200] HPLC-SCX chromatography (column: BioResolve SCX mAb column, 3 μm, 4.6 mm × 100 mm) was performed using a WATERS ACQUITY H-class Bio with MES pH5 as the mobile phase, and a salt gradient was used to separate charge variants at a flow rate of 0.9 mL / min.

[0201] 10 μL of each sample was injected into both H-SEC and H-SCX using the protocol described above. For each molecule, the monomer species and the percentage of area (indicating protein concentration) were determined.

[0202] Both PetML119wt and var exhibited very high purity (over 99%) due to HSEC, and hardly any charge variants (presumably corresponding to different glycoforms) were observed due to HSCX, confirming that the p75 variant does not affect the final purity and homogeneity of the product.

[0203] Example 5 - Verification of Protein A Binding Affinity The purified fusion protein in PBS was concentrated to 5 mg / mL using a centrifugal concentrator (Sartorious-VS02H22). The protein concentration was evaluated using NanoDrop®One (Thermo Scientific®) with UV absorbance at 280 nm.

[0204] The binding affinity of the fusion protein to protein A was evaluated using Biacore 8K (Cytiva).

[0205] In short, Sensor Chip Protein A (Cytiva) was docked to a Biacore 8K chip, equilibrated at room temperature for 30 minutes, and then electrophoresis buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween20) was applied to the SPR chip surface.

[0206] Six fusion protein dilutions (1:3 dilution) of PetML119var were prepared in electrophoresis buffer by diluting them from 1 μM to 4 nM, and their kinetics were evaluated using a single-cycle kinetics method (Biacore Assay Handbook, Cytiva). Kinetics and / or affinity quantification were performed using Biacore Insight according to standard analytical methods.

[0207] The results indicate that none of the p75NTR mutations induced changes in protein A binding.

[0208] Example 6 - Determination of nerve growth factor (h-rNGF) and other neuronal root binding affinities in humans and rats The purified fusion protein in PBS was concentrated to 5 mg / mL using a centrifugal concentrator (Sartorious-VS02H22). The protein concentration was evaluated using NanoDrop®One (Thermo Scientific®) with UV absorbance at 280 nm.

[0209] The binding affinity of fusion proteins to human and rat NGF was evaluated using Biacore 8K (Cytiva). Briefly, a Protein A Sensor Chip (Cytiva) was docked to Biacore 8K, equilibrated at room temperature for 30 minutes, and then electrophoresis buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween20) was applied to the SPR chip surface.

[0210] PetML119var was diluted in electrophoresis buffer to a concentration of 6 nM. These were immobilized using a capture step of 10 μL / min for 90 seconds, followed by injection of electrophoresis buffer to remove unbound products.

[0211] Human NGF (from Bio-Techne Ltd-556-NG / CF / 256-GF-100 / CF), human BDNF (11166-BD), human NT-3 (267-N3-025 / CF), human NT-4 (268-N4-025 / CF), and proNGF were diluted to 100 nM in electrophoresis buffer, and then further diluted to 4.68 nM by a 1:2 ratio. The kinetics were evaluated using multi-cycle kinetics with a capture step (30 seconds of association - 300 seconds of dissociation), followed by a regeneration step (0.1 M glycine, pH 2.2, contact time 60 seconds, FR 30 μL / min). Kinetic quantification was performed using Biacore Insight according to standard analytical methods.

[0212] The results showed subnanomolar KDs for both human NGFs with the improved PetML119wt variant (Figure 3 and Table 1). Variants with lower affinity for NGF showed 1 / 20 and 1 / 40 reduced affinities compared to those observed with PetML119wt. Both PetML119wt and the variant showed dissociation from NGF over time, indicating that the binding is reversible. This is in contrast to what was observed with an anti-NGF mAb (bezinevetomab) where the binding remained constant over time.

Table 1-1

[0213]

Table 1-2

[0214]

Table 1-3

[0215] Using the same approach, the binding of the same molecule to three other neurotrophins (BDNF, NT-3 and NT-4) was also tested. The results showed that a rational mutation to the p75NTR ECD selectively changes NGF binding without affecting other mature NTs (only NT-4 showed a decrease in binding affinity with E75T and D134L, which is probably due to some local atomic similarity between these two NTs).

[0216] The binding affinities of PetML119 and bedin-vetomab to proNGF were tested using the same approach to confirm that p75NTR-Fc can bind to this form with higher affinity than the mature form. PetML119 showed a 171-fold higher affinity for the propeptide (0.0019 nM proNGF vs. 0.309 nM NGF). Bedin-vetomab, an antibody produced against the mature NGF protein, showed similar results for both proNGF and mature NGF.

[0217] Example 7 - Determination of unfolding and oligomerization The purified fusion protein in PBS was concentrated to 5 mg / mL using a centrifugal concentrator (Sartorious-VS02H22). The protein concentration was evaluated using NanoDrop®One (Thermo Scientific®) with UV absorbance at 280 nm.

[0218] Tm and Tag analyses were performed on UnCle from Unchained Labs using standard protocols.

[0219] In short, we used 10 μL of fusion protein to determine unfolding and aggregation events during temperature increase (from 25°C to 95°C).

[0220] As expected from previous results, PetML119 did not exhibit aggregation up to 95°C, with a Tm1 of approximately 78°C. Similarly, all mutants did not exhibit aggregation up to 95°C, with a Tm1 of approximately 76°C (Figure 4).

[0221] Example 8 - In vitro NGF inhibition assay To evaluate the biological activity of the p75 fusion protein, we used NGF-dependent cell lines. Our fusion protein exhibits superior NGF sequester, resulting in slower growth compared to controls.

[0222] The TF-1 cell line was purchased from ATCC (CRL-2003) and maintained in culture using a standard sterile method with complete RPMI (10% FBS + 2 mM l-Gln + 10 ng / mL hNGF).

[0223] Two million TF-1 cells were labeled with 2.5 μM CFSE cell trace (Invitrogen-C34554) in 1 mL of RPMI at room temperature in the dark for only 30 minutes. The cells were then washed twice in complete RPMI medium, counted again, and seeded into 24-well plates at 10,000 cells / mL (total volume 1 mL per well).

[0224] Dilutions of PetML119wt and variants or controls (bedinbetomab-anti-NGF IgG control, and RPMI without NGF as a negative control) were diluted in 100 μL of RPMI medium from a concentration of 3 μM to 91.25 nM. 100 μL of the protein dilution was added to each well.

[0225] Plates were analyzed after 3 days. Briefly, 1 mL of cell suspension was centrifuged at 300 g at room temperature for 5 minutes, washed twice with FACS buffer (PBS + 3% FBS + 3 mM EDTA), and finally resuspended in 100 μL of FACS buffer. Cells were acquired using a CytoFLEX Flow Cytometer with the following parameters (FSC: 20; SSC: 50; FITC: 1; threshold: 1313131). Cells were gated based on FITC fluorescence (more fluorescence, less proliferation), and the percentage of growth inhibition was calculated considering 100% inhibited TF-1 cells cultured in RPMI without NGF and 0% inhibited cells cultured in complete RPMI medium. IC50 values ​​were calculated using Graphpad.

[0226] PetML119wt, variants, and bedin-vetomab were able to inhibit TF-1 proliferation in a dose-dependent manner (Figure 5, Table 2). Incubation with bedin-vetomab showed complete inhibition, while incubation with PetML119wt had an ICMAX of approximately 60–70%. Lower affinity NGF-binding molecules showed reduced inhibitory capacity compared to PetML119wt (particularly the D134L variant). High affinity NGF-binding molecules, as with bedin-vetomab, showed improved inhibitory capacity compared to PetML119wt. Importantly, the ICMAX of all variants did not reach 100, indicating partial inhibition. This is in contrast to anti-NGF bedin-vetomab, which showed an ICMAX of 100, indicating complete inhibition. [Table 2]

[0227] Example 9 - PathHunter® eXpress Receptor Tyrosine Kinase Functional Assay Kit To further demonstrate that our molecule can inhibit NGF pathway activation in vitro, we used the PathHunter® eXpress receptor tyrosine kinase function assay kit. This included cell lines engineered to fuse the TrkA intracellular domain with a small complementary fragment of β-Gal.

[0228] A larger portion of β-gal, called EA of "enzyme acceptor", was fused to a protein containing a phospho-tyrosine binding domain. Ligand-induced activation of the receptor causes either homodimerization or heterodimerization of the receptor, which causes cross-phosphorylation. Subsequently, the SH2-EA fusion protein specifically binds to the phosphorylated receptor, resulting in complementation of two fragments of β-gal and formation of a functional enzyme. Then, β-gal activity is detected quantitatively using the chemiluminescent substrate of the PathHunter detection kit. Briefly, PetML119wt, var and the positive control anti-NGF mAb (Bevacizumab) at concentrations ranging from 3 μM to 91.25 nM were used, while NGF was used at 2 nM.

[0229] The luminescence readings showed that the anti-NGF mAb had a distinct sigmoid-shaped inhibition curve reaching 100% TrkA inhibition, similar to that seen in the TF-1 assay (Figure 6). In contrast to anti-NGF, and consistent with the TF-1 assay, the PetML119 molecules showed a shallower inhibition curve that did not reach 100% inhibition. Also, the high-affinity NGF PetML119 molecules showed increased maximal inhibition (although not reaching 100% inhibition) compared to WT, as well as decreased IC50, while the lower-affinity NGF PetML119 molecules showed decreased maximal inhibition and increased IC50 compared to WT, which was also consistent with the TF-1 inhibition assay (Table 3).

[0230] Collectively, these results suggest that the anti-NGF mAb Bevacizumab differs from the PetML119 molecules in both inhibition concentration and amplitude of inhibition. The inventors' new molecules of rationally engineered PetML119 variants may approach Bevacizumab-level NGF inhibition or further reduce PetML119wt NGF inhibition. Importantly, this is achieved without achieving 100% inhibition of NGF, which provides a mechanistic basis underlying its expected DMOAD activity in vivo.

Table 3

[0231] Example 10 - MIA-induced OA in rats (efficacy, pK, and joint diameter measurements) The efficacy and half-life of PetML119 and its variants were analyzed using a rat model of OA. The detailed protocol is shown below. The model is shown in Figure 7.

[0232] Induction of arthritis Osteoarthritis was chemically induced in rats under isoflurane anesthesia by intra-articular (IA) injection of 3 mg (in 25 μL of physiological saline) of monosodium iodoacetate (MIA) into the right hindlimb knee joint. Ophthalmic ointment was applied to both eyes under anesthesia. The day of the IA injection of MIA was counted as day 0.

[0233] Assignment to treatment group Baseline dynamic weight-bearing (DWB) was measured for all rats. Body weight (BW) was also measured simultaneously. Rats were anesthetized, and MIA was injected into the right knee joint via the medial aspect of the patellar tendon, nearly perpendicular to the tibia (intra-articular (IA)). The dose level of MIA for IA injection was selected based on previous literature reports in rodents (Bove et al.: Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis. Osteoarthritis Cartilage. 2003 Nov;11(11):821-830). Animals showing a significant weight-bearing difference between the MIA-injected limb (right) and the healthy limb (left) were assigned to the study. Randomization was performed based on both baseline DWB and BW (randomization of two variables).

[0234] Evaluation of Dynamic Weight Load (DWB) Dynamic weight loading was assessed using the BioSeb® automated DWB system, according to the manufacturer's manual. A 2-minute recording was made for each rat. Dynamic weight loading data analysis was performed offline using BioSeb® software. The system automatically calculated the weight load for each limb and tail. Weight was measured for each rat immediately before DWB and at each time point in the study. DWB measurements were taken at different points in time according to the study design schedule. The total distance traveled was also recorded during DWB data analysis.

[0235] Administration of test items Rats in groups 1-2 received intravenous (IV) injections of the vehicle, while rats in groups 3-8 received IV injections of the test item at a predetermined dose once on day 3, as shown in the table below. Study on monosodium iodoacetate (MIA)-induced rats research design [Table 4-1] [Table 4-2]

[0236] During the study, treated animals were observed for all clinical signs. No adverse signs were observed. DWB was analyzed on days 3, 6, 14, and 21. Joint diameter was measured using a caliper on the right knee joint (medial) on days 3, 6, 14, and 21.

[0237] In short, PetML119, PetML119-S109Y, and PetML119-E75T (single dose) were tested for their analgesic effects (Figure 11). All molecules tested showed good analgesic effects, reaching their maximum effect on day 6. The analgesic effect was dose-dependent. As expected, PetML119-E75T, a lower affinity p75NTR variant, showed decreased efficacy in pain relief over the long term (i.e., after day 14), while WT and PetML119-S109Y remained effective until the end of the study. Similarly, measurements of joint diameter showed dose-dependent knee hypertrophy from day 3 to the end of the study (WT and S109Y only, E75T only up to day 6) (Figure 13). This is usually associated with increased inflammation.

[0238] PK blood samples were collected from 5 rats per group every other day using a standard procedure.

[0239] serum pk analysis The sandwich ELISA for quantifying the serum levels of the inventors' fusion protein was configured as follows. 30 μL of 2 μg / mL capture antibody (mouse anti-canine p75 Ab-->MAB367-SP (Novus Bio)) diluted with PBS + 0.1 M sodium bicarbonate was fixed overnight at 4°C on a half-area ELISA plate (microplate, 96 wells, PS, half-area, clear, catalog number: 675061). The plate was washed twice with 200 μL of blocking solution (PBS + 5% DNFM + 0.2% Tween20), and blocking was performed at room temperature for 3 hours with 150 μL of blocking solution across all wells. Serum from different time points / groups of rat studies was diluted 100-fold in blocking solution (2 μL serum + 198 μL blocking solution), and 30 μL was added to the relevant wells. Standards from PetML119 were prepared by diluting the fusion protein in rat serum at a 1:5 ratio from 100 μg / mL to 1 ng / mL. The standard material was then diluted 100-fold in blocking solution, and 30 μL was added to the relevant wells. The serum was incubated at room temperature for 1 hour while shaking at 450 rpm. The plate was washed twice with 200 μL of blocking solution, the detection antibody (SA5-10309 (ThermoFisher)) was diluted 1:40000 in blocking solution, 30 μL was added to each well, and the mixture was left at room temperature for 30 minutes while shaking at 450 rpm. The plate was washed twice with 200 μL of blocking solution, the HRP conjugate antibody under development (A16035 (ThermoFisher)) was diluted 1:10000 in the blocking solution, 30 μL was added to each well, and the mixture was left at room temperature for 30 minutes while shaking at 450 rpm. The plate was washed twice with 200 μL of blocking solution, then twice with 200 μL of PBS + 0.2% Tween 20, and finally 50 μL of TMB (TMB Chromogen Solution (for ELISA) --> 002023) was added to each well. After 10 minutes, when the standard curve showed saturation at the first two points, the reaction was stopped by adding 50 μL of 1 M sulfuric acid. We read all wells using CLARIOstar Plus (BMG LABTECH) with endpoint absorbance at 650nm and 450nm. The values ​​were imported into Graph Pad, and single-phase decay fitting was applied to estimate their half-lives.

[0240] PetML119var showed a similar plasma half-life (Figure 12A, Table 5). [Table 5]

[0241] NGF is generally undetectable (or present at very low levels) in plasma. Systemic administration of NGF-binding molecules (i.e., mAbs or Fc fusion proteins) results in increased plasma levels of NGF. Rats treated with PetML119-E75T experienced a faster NGF peak (24 hours) than those treated with the WT molecule PetML119 (144 hours). Conversely, rats treated with PetML119-S109Y experienced a later NGF peak (336 hours). In this study, we demonstrate that plasma NGF levels were detectable upon induction of MIA and administration of our p75-Fc molecule. In addition, p75NTR variants exhibited distinct peak levels of NGF correlated with their affinity for this molecule. This is consistent with the prolonged analgesic effects observed with PetML119 and PetML119-S109Y during the 6-day loss of activity of PetML119-E75T. Observed differences in NGF inhibition can lead to in vivo anti-inflammatory / DMOAD effects, as observed in joint diameter measurements.

[0242] The variant molecule is expected to exhibit the same safety properties as demonstrated in previous in vivo studies in dogs. This is shown, for example, in Example 12 of WO2023 / 067358, in which the animals maintained normal body weight and hematological parameters and did not produce anti-drug antibodies after administration of the fusion protein. Maintenance of normal body weight after administration is also shown, for example, in Example 11 of WO2023 / 067358. This is in contrast to the dexamethasone administration in which body weight decreased over time.

[0243] Example 11 - Disease-modifying osteoarthritis drug (DMOAD) in rats We optimized a model to evaluate the therapeutic effect of the p75NTR-Fc molecule in OA rats. Briefly, osteoarthritis was induced by injecting a low dose of MIA (0.3 mg) into either the right or left knee of a rat. After 4 weeks, when signs of arthritis were observed, the animals were treated IV twice at 14-day intervals (days 28 and 42) with either the p75NTR-Fc molecule (3 doses: 0.5 mg / kg, 0.2 mg / kg, and 0.05 mg / kg) or a vehicle, as a control. Then, on day 56, each individual in each group was euthanized. IHC (H&E, Saffron-O staining) was performed on the affected knees, and the Mankin scoring system was used for the medial, lateral, medial, and lateral femoral regions to score for structural changes, clonalization, chondrocyte loss, SOFG stain loss, osteophytes, and composite scores (sum of all previous scores). Serum was collected at different time points to assess systemic levels of active compounds, NGF, and other biomarkers.

[0244] The results shown in Figure 15 demonstrate a dose-dependent effect of p75NTR-Fc, with the lowest concentration exhibiting the most severe DMOAD effect for all osteoarthritis phenotypes analyzed.

[0245] Example 12 - Investigation of the anti-inflammatory effects of the p75NTR-Fc molecule in the DH82 cell line ProNGF and NGF have different activities in vivo; the former exhibits pro-inflammatory, catabolic, and apoptotic effects, while the latter has anti-inflammatory, anabolic, and proliferative effects. To evaluate whether the preferential binding of proNGF (and less than NGF) to the p75NTR-Fc molecule affects inflammation, an in vitro assay using DH82 cell lines (canine macrophages such as CRL-3590™ ATCC) has been established.

[0246] In short, cells were seeded at a density of 1 million / mL in 6-well plates using EMEM + 15% thermally inactivated FBS and incubated in a static incubator at 37°C and 5% CO2 for 16 hours. Pro-inflammatory effects were induced (or used as controls) by adding 100 ng / mL of LPS, 2 ng / mL of IL1β, or culture medium alone to the cells. Two concentrations of PetML119 (625 nM or 1 nM) or culture medium alone were added to the cells simultaneously. After 24 hours, the supernatant was collected and used for quantification of pro-inflammatory cytokines.

[0247] TNFα and IL-6 were used in accordance with the provider's specifications, along with NGF (detecting both pro-NGF and mature NGF) ELISAs (ab193687, ab193760 from AbCam, and CA6000 from R&D Systems).

[0248] The results showed that both LPS and IL1β induced the secretion of pro-inflammatory cytokines and total NGF compared to the control (Figure 14). Treatment with PetML119 showed a dose-response suppression of total NGF levels (Figure 14). Interestingly, PetML119 had different activity against TNFα and IL-6 depending on the concentration used. At high concentrations, PetML119 induced higher secretion of cytokines, while at low concentrations, it showed a decrease in their levels.

[0249] This intriguing finding correlates with the preferential binding of PetML119 to proNGF, in contrast to NGF. At high concentrations, the p75NTR-Fc molecule can reduce both proNGF and mature NGF, preventing the anti-inflammatory activity of the mature form. At low concentrations, PetML119 can preferentially block proNGF (due to its lower affinity for mature NGF), thus reducing its pro-inflammatory effects, while free mature NGF can function as an anti-inflammatory agent, thus showing a reduction in TNFα and IL-6. Similarly, variant molecules with lower affinity are expected to preferentially block proNGF. Lower affinity variant molecules, such as E75T, are expected to have anti-inflammatory effects at higher concentrations.

[0250] Example 13 - Human p75NTR ECD Stork Modified Protein Construct and CHO-s Transfection / Expression The amino acid sequences of the protein constructs are listed below. The amino acid sequences are provided in the sequence section. PetML308-Human p75NTR ECD Whole Stork IgG1 (SEQ ID NO: 81) PetML309-Human p75NTR ECD Partial Stork IgG1 (SEQ ID NO: 82) PetML319-Human p75NTR ECD without stalk (SEQ ID NO: 83).

[0251] DNA constructs were generated to encode a chimeric Fc fusion protein containing a selected human IgG constant region (between the hinge and the C-terminus) fused to the extracellular domain of human p75, either containing the predicted γ-secretase site (PetML308 - whole stalk), lacking it (PetML309 - partial stalk), or lacking both O-glycosylation and the γ-secretase site (PetML319 - no stalk).

[0252] Both the human IgG1 Fc domain and the p75 extracellular domain (res 31-194 from UniProtKB) were synthesized. Both genes were PCR amplified using Q5 high-fidelity DNA polymerase (specific primers containing overlapping regions were used to enable assembly) and assembled into the mammalian expression vector PetML319var using NEBuilder HIFI DNA Assembly (New England Biolabs). In the expression vector, the fusion protein chain and antibiotic resistance gene expression units are flanked by DNA transposon piggyBac terminal inversion repeats, mediating stable integration into host cells in the presence of piggyBac transposase. The expression vector was then transfected with PiggyBac transposase into suitable mammalian cell lines such as CHO cells to obtain stable expression. 1 × 10⁶ units were used for fusion protein production. 6 Selected CHO cells at a concentration of / mL were seeded in 800mL of culture medium (F17 + 4mM l-Gln + 0.3% P188 + 1:500 ACA) and incubated at 32°C and 8% CO2 with shaking at 130 rpm. 2% HyClone Cell Boost 7a supplement + 0.2% HyClone Cell Boost 7b supplement and 2mM glucose were added to the medium daily from day 4 of overproduction. The culture supernatant was collected on day 10, and protein concentrations were determined using surface plasmon resonance (Biacore 8K, Cytiva Life Sciences) with a Protein A tip.

[0253] Typically, the human p75-Fc molecule shows peak expression on day 10 of production, reaching 20–60 mg / L, exhibiting best performance without stalk molecules, followed by partial and complete stalks. PetML308: 20 mg / L PetML309: 30 mg / L PetML319: 60 mg / L

[0254] Example 14 - Purification of Human p75-Fc Molecules Cell suspensions derived from PetML319, PetML308, or a stable transfect clone of PetML309, cultured for at least 7 days as described, were incubated for 10 minutes in Sartoclear Dynamics® Lab V (SDLV-0500-20C-E) and then filtered using a 0.22 μm filter. The clear supernatant was loaded onto a 20 mL Mabselect sure LX pre-packed column (17547402) pre-equalized with PBS. The column was washed with 40 mL of PBS (2 CV), and then the fusion protein was eluted using a 0.1 M glycine pH 2.7 gradient (0–100% in 2 CV). The fractions containing the fusion protein were pooled together and neutralized with 100 mM TRIS pH 8 (final concentration).

[0255] The neutralized fusion protein pool fraction was concentrated to 5 mL and loaded onto PBS pre-equilibriumized HiLoad 16 / 600 Superdex 200 pg (28989335) as the second step of purification. The monomer fraction was pooled (based on the retention time of previously analyzed protein standards), and the protein concentration was assessed using NanoDrop®One (Thermo Scientific®).

[0256] Following the protocol described above, purified products with concentrations of approximately 10–30 mg / L were obtained. No differences in terms of purification and recovery were reported between wt and variants, and this difference is attributed to different titers. PetML308: After two-step purification, 10 mg / L PetML309: After two-step purification, 12 mg / L PetML319: After two-step purification, 30 mg / L

[0257] Example 15 - HPLC Analytical Chromatography The purity of the purified material was evaluated using both size exclusion chromatography (SEC) for oligomerization analysis and cation exchange chromatography (SCX) for charge variant analysis.

[0258] HPLC-SEC chromatography (column: BioResolve SEC mAb 200A, 2.5um column, WATERS) was performed using a WATERS ACQUITY H-class Bio with PBS as the mobile phase, having an isocratic flow rate of 0.575 mL / min.

[0259] HPLC-SCX chromatography (column: BioResolve SCX mAb column, 3 μm, 4.6 mm × 100 mm) was performed using a WATERS ACQUITY H-class Bio with MES pH5 as the mobile phase, and charge variants were separated using a salt gradient at a flow rate of 0.9 mL / min.

[0260] 10 μL of each sample was injected into both H-SEC / H-SCX using the protocol described above. For each molecule, the monomer species and the percentage of area (indicating protein concentration) were determined.

[0261] PetML319 showed very high purity (over 99%) by HSEC, and little charge variant (presumably corresponding to different glycoforms) was observed by HSCX. However, PetML309 and 308 showed the presence of aggregates, leading to the idea that this stalk region may negatively affect the development of these molecules.

[0262] Example 16 - Verification of Protein A Binding Affinity The purified fusion protein in PBS was concentrated to 5 mg / mL using a centrifugal concentrator (Sartorious-VS02H22). The protein concentration was evaluated using NanoDrop®One (Thermo Scientific®) with UV absorbance at 280 nm.

[0263] The binding affinity of the fusion protein to protein A was evaluated using Biacore 8K (Cytiva).

[0264] In short, Sensor Chip Protein A (Cytiva) was docked to a Biacore 8K chip, equilibrated at room temperature for 30 minutes, and then electrophoresis buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween20) was applied to the SPR chip surface.

[0265] PetML319, PetML308, or PetML309 were prepared in electrophoresis buffer in fusion protein dilutions ranging from 1 μM to 4 nM (6 concentrations at a 1:3 dilution), and their kinetics were evaluated using a single-cycle kinetics method (Biacore Assay Handbook, Cytiva). Kinetics and / or affinity quantification were performed using Biacore Insight according to standard analytical methods.

[0266] The results indicate that all molecules possess excellent protein A binding.

[0267] Example 17 - Determination of nerve growth factor (h-rNGF) binding affinity in humans and rats The purified fusion protein in PBS was concentrated to 5 mg / mL using a centrifugal concentrator (Sartorious-VS02H22). The protein concentration was evaluated using NanoDrop®One (Thermo Scientific®) with UV absorbance at 280 nm.

[0268] We used Biacore 8K (Cytiva) to evaluate the binding affinity of fusion proteins to human and rat NGF.

[0269] In short, a Protein A Sensor Chip (Cytiva) was docked to a Biacore 8K, equilibrated at room temperature for 30 minutes, and then electrophoresis buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween20) was applied to the surface of the SPR chip.

[0270] PetML319, PetML308, or PetML309 were diluted in electrophoresis buffer to a concentration of 6 nM. These were immobilized using a capture step of 10 μL / min for 90 seconds, followed by injection of electrophoresis buffer to remove unbound products.

[0271] Human and rat NGF (Bio-Techne Ltd-556-NG / CF / 256-GF-100 / CF) were diluted to 100 nM in electrophoresis buffer and then further diluted 1:2 to 4.68 nM. The kinetics were evaluated using multi-cycle kinetics with a capture step (30 seconds association - 300 seconds dissociation) followed by a regeneration step (0.1 M glycine, pH 2.2, contact time 60 seconds, FR 30 μL / min). Kinetic quantification was performed using Biacore Insight according to standard analytical methods (Table 6).

[0272] [Table 6]

[0273] The results showed sub-nanomolecular KD for both human NGF and rat NGF containing PetML319, but PetML309 and 308 showed slightly lower affinity, respectively (Table 7). [Table 7]

[0274] Example 18 - Determination of unfolding and oligomerization The purified fusion protein in PBS was concentrated to 3 mg / mL using a centrifugal concentrator (Sartorious-VS02H22). The protein concentration was evaluated using NanoDrop®One (Thermo Scientific®) with UV absorbance at 280 nm.

[0275] Tm and Tag analyses were performed on UnCle from Unchained Labs using standard protocols (Figure 9).

[0276] In short, we used 10 μL of fusion protein to determine unfolding and aggregation events during temperature increase (from 25°C to 95°C). All molecules did not aggregate up to 95°C, with Tm1 being approximately 76°C. (Table 8) [Table 8]

[0277] Example 19 - PathHunter® eXpress Receptor Tyrosine Kinase Functional Assay Kit To further demonstrate that our molecule can inhibit NGF pathway activation in vitro, we used the PathHunter eXpress assay and engineered cell lines to have a TrkA intracellular domain fused with a smaller complementary fragment of β-Gal. The larger portion of β-gal, called EA of the "enzyme acceptor," is fused to a protein containing a phosphotyrosine-binding domain. Ligand-induced activation of the receptor leads to either homodimerization or heterodimerization of the receptor, resulting in cross-phosphorylation. The SH2-EA fusion protein then specifically binds to the phosphorylated receptor, leading to the complementation of the two β-gal fragments and the formation of a functional enzyme. β-gal activity is then quantitatively detected using a chemiluminescent substrate from the PathHunter detection kit.

[0278] In short, PetML119 wt, var, and a positive control anti-NGF mAB (bezin betomab) were used at concentrations ranging from 3 μM to 91.25 nM, while NGF was used at 2 nM. Luminescence readings showed that the anti-NGF mAb had a distinct sigmoid-shaped inhibition curve reaching 100% TrkA inhibition, similar to what was observed in the TF-1 assay. In contrast to anti-NGF, and also consistent with the TF-1 assay, the PetML119 molecule did not reach 100% inhibition. Furthermore, the high-affinity NGF PetML119 molecule showed increased maximum inhibition (still not 100%) and decreased IC50, while the lower-affinity NGF PetML119 molecule showed little to no inhibition, also consistent with the TF-1 inhibition assay.

[0279] Taken together, these results suggest that the anti-NGF mAb bedinbetomab differs from the human p75 molecule in both inhibitory concentration and inhibition amplitude. Furthermore, as mentioned above, no stalk molecule showed better performance than the partial or complete stalk molecules, likely due to reduced stability and binding ability. [Table 9]

[0280] array Canine p75NTR protein (SEQ ID NO: 1) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQ DRQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPSEDSDSTAPSTEEPELPPDQEIIASTMADVVTTVMGSSQPVVTRGTADN LIPVYCSILAAVVVGLVAYIAFKRWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGISVDSQSLHDQQPHTQTAAGQALKGDGGLYSSLPPAKREEVEKLLNGSAGDTWRHLAGELGYQPEHIDSFTHEACPARALLASWAAQDSATLDALLAALRRIQRADIVESLCSESTATSPV ECD is underlined. Canine p75NTR nucleic acid sequence (SEQ ID NO: 2) The leader row in JPEG2026511340000015.jpg118152 is underlined.

[0281] Feline p75NTR protein (SEQ ID NO: 3) The text JPEG2026511340000016.jpg36152ECD is underlined. Cat p75NTR nucleic acid sequence (SEQ ID NO: 4) The leader row in JPEG2026511340000017.jpg118153 is underlined.

[0282] Horse p75NTR protein (SEQ ID NO: 5) The text JPEG2026511340000018.jpg36153ECD is underlined. Horse p75NTR nucleic acid sequence (SEQ ID NO: 6) The leader row in JPEG2026511340000019.jpg118152 is underlined.

[0283] Canine p75NTR protein ECD (SEQ ID NO: 7) The ECD region in JPEG2026511340000020.jpg20152wt includes the stalk region (underlined), and alpha and gamma-secretase cleavage (bold) at 3' of the stalk region. Canine p75NTR ECD nucleic acid sequence (SEQ ID NO: 8) AAGGAGGCATGTCCCACTGGCCTGTACACCCACAGCGGCGAGTGCTGCAAAGCCTGCAATCTGGGTGAGGGGGTGGCCCAGCCTTGCGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACCTTCTCGGACGTGGTGAGCGCCACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTGCAGAGCATGTCGGCGCCGTGCGTGGAGGCGGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGACGAGACGACGGGCCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGGCTCGTGTTCTCGTGCCAGGACAGGCAGAACACCGTGTGCGAGGAGTGTCCCGACGGCACGTACTCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTGCGCGAGTGCACGCGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACCCGGTCCACACCCTCAGAGGACTCGGACAGCACCGCCCCCAGCACAGAGGAGCCAGAGCTACCTCCAGATCAAGAAATCATAGCCAGCACCATGGCAGATGTGGTGACCACAGTGATGGGCAGCTCTCAGCCTGTAGTGACCCGAGGAACCGCTGACAAC Canine ECD of the p75NTR stalk region protein (SEQ ID NO: 9) WITRSTPSEDSDSTAPSTEEPELPPDQEIIASTMADVVTTVM Canine ECD of the p75NTR stalk region nucleic acid sequence (SEQ ID NO: 10) TGGATTACCCGGTCCACACCCTCAGAGGACTCGGACAGCACCGCCCCCAGCACAGAGGAGCCAGAGCTACCTCCAGATCAAGAAATCATAGCCAGCACCATGGCAGATGTGGTGACCACAGTGATG

[0284] Canine p75NTR ECD-Canine IgGB wt Fc protein fusion (SEQ ID NO: 11) JPEG2026511340000021.jpg41153 Signal peptide-canine p75-ECD-linkerGGGG-canine Fc-B-wt Canine p75NTR ECD-Canine IgGB wt Fc nucleic acid sequence (SEQ ID NO: 12) ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGTCCGTGACCACCGGCGTGCACTCCAAAGAGGCTTGTCCTACCGGCCTGTACACCCACTCTGGCGAGTGTTGCAAGGCCTGTAATCTCGGCGAAGGCGTGGCACAACCTTGTGGCGCTAATCAGACAGTGTGCGAGCCTTGCCTGGACTCCGTGACCTTCTCTGATGTGGTGTCTGCCACCGAGCCATGCAAGCCTTGTACCGAGTGTGTGGGCCTGCAGTCCATGTCTGCCCCTTGTGTGGAAGCCGACGACG CCGTGTGTAGATGTGCCTACGGCTACTACCAGGACGAGACAACCGGAAGATGCGAGGCCTGCAGAGTGTGTGAAGCTGGCTCTGGACTGGTGTTCTCCTGCCAAGACAGACAG AACACCGTGTGCGAGGAATGCCCTGACGGCACCTACTCTGATGAGGCCAATCACGTGGACCCCTGCCTGCCTTGTACTGTGTGCGAAGATACCGAGCGGCAGCTGCGCGAGTGTACCAGATGGGCTGATGCCGAGTGCGAAGAGATCCCTGGAGGTGGCGGACGCGAGAATGGCAGAGTGCCTAGACCTCCTGACTGCCCTAAGTGCCCTGCTCCTGAAATGCTCGGCGGACCCTCCGTGTTCATCTTCCCACCTAAGCCTAAGGACACCCTGCTGATCGCTCGGACCCCTGAAGTGACATGCGTGGTGGTGGATCTGGACCCCGAGGATCCTGAGGTGCAGATCAGTTGGTTCGTGGACGGCAAGCAGATGCAGACCGCTAAGACCCAGCCTAGAGAGGAACAGTTCAACGGCACCTACAGAGTGGTGTCTGTGCTGCCTATCGGCCACCAGGATTGGCTGAAGGGCAAGCAGTTTACCTGCAAAGTGAACAACAAGGCCCTGCCTTCTCCAATCGAGCGGACCATCTCTAAGGCCAGAGGCCAGGCTCATCAGCCTTCCGTGTATGTCCTGCCACCTAGCCGCGAGGAACTGTCCAAGAACACCGTGTCTCTGACCTGCCTGATCAAGGACTTCTTCCCTCCTGACATCGACGTGGAATGGCAGTCCAACGGCCAGCAAGAGCCCGAGTCTAAGTACCGGACAACCCCTCCACA GCTGGACGAGGACGGCTCCTACTTCCTGTACTCCAAGCTGTCCGTGGACAAGTCTCGGTGGCAGAGAGGCGACACCTTCATCTGTGCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCACTCCCCTGGCAAGTGA The leader row is underlined.

[0285] Canine p75NTR ECD-Canine Fc YTE protein fusion (SEQ ID NO: 13) JPEG2026511340000022.jpg42154 Signal peptide-canine p75-ECD-linkerGGGG-canine Fc-B-YTE Canine p75NTR ECD-Fc YTE nucleic acid sequence (SEQ ID NO: 14) ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGTCCGTGACCACCGGCGTGCACTCCAAAGAGGCTTGTCCTACCGGCCTGTACACCCACTCTGGCGAGTGTTGCAAGGCCTGTAATCTCGGCGAAGGCGTGGCACAACCTTGTGGCGCTAATCAGACAGTGTGCGAGCCTTGCCTGGACTCCGTGACCTTCTCTGATGTGGTGTCTGCCACCGAGCCATGCAAGCCTTGTACCGAGTGTGTGGGCCTGCAGTCCATGTCTGCCCCTTGTGTGGAAGCCGACGACGCCGTGTGTAGATGTGCCTACGGCTACTACCAGGACGAGACAACCGGAAGATGCGAGGCCTGCAGAGTGTGTGAAGCTGGCTCTGGACTGGTGTTCTCCTGCCAAGACAGACAGAACACCGTGTGCGAGGAATGCCCTGACGGCACCTACTCTGATGAGGCCAATCACGTGGACCCCTGCCTGCCTTGTACTGTGTGCGAAGATACCGAGCGGCAGCTGCGCGAGTGTACCAGATGGGCTGATGCCGAGTGCGAAGAGATCCCTGGAGGTGGCGGACGCGAGAATGGCAGAGTGCCTAGACCTCCTGACTGCCCTAAGTGCCCTGCTCCTGAAATGCTCGGCGGACCCTCCGTGTTCATCTTCCCACCTAAGCCTAAGGACACCCTGTATATCACTCGGGAACCTGAAGTGACATGCGTGGTGGTGGATCTGGACCCCGAGGATCCTGAGGTGCAGATCAGTTGGTTCGTGGACGGCAAGCAGATGCAGACCGCTAAGACCCAGCCTAGAGAGGAACAGTTCAACGGCACCTACAGAGTGGTGTCTGTGCTGCCTATCGGCCACCAGGATTGGCTGAAGGGCAAGCAGTTTACCTGCAAAGTGAACAACAAGGCCCTGCCTTCTCCAATCGAGCGGACCATCTCTAAGGCCAGAGGCCAGGCTCATCAGCCTTCCGTGTATGTCCTGCCACCTAGCCGCGAGGAACTGTCCAAGAACACCGTGTCTCTGACCTGCCTGATCAAGGACTTCTTCCCTCCTGACATCGACGTGGAA TGGCAGTCCAACGGCCAGCAAGAGCCCGAGTCTAAGTACCGGACAACCCCTCCACAGCTGGACGAGGACGGCTCCTACTTCCTGTACTCCAAGCTGTCCGTGGACAAGTCTCGGTGGCAGAGAGGCGACACCTTCATCTGTGCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCACTCCCCTGGCAAGTGA The leader row is underlined.

[0286] IgG-A (SEQ ID NO: 15) MEFVLGWVFLVAILQGVQGEVQLVESGGDLVKPAGSLRLSCVASGFTFSNNAMNWVRQAPGKGLQWVAGINSGGSTASADAVKGRFTISRDNAKNTVYLQMNSLTAEDTAVYYCAKV IGNWIATSDLDYWGQGTLVIVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVVHPASNTKVD KPVFNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPPSPI ERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK IgG-B(SEQ ID NO:16) MEFVLGWVFLVAILQGVQGEVQLVESGGDLVKPAGSLRLSCVASGFTFSNNAMNWVRQAPKGGLQWVAGINSGGSTASADAVKGRFTISRDNAKNTVYLQMSLTAEDTAVYCAKVIGNWIATSDLDYWGQGTLVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTGSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETTCNVAHPASTKVDKP VPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDIVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQKSLSHPGK IgG-C(SEQ ID NO:17) MEFVLGWVFLVAILQGVQGEVQLVESGGDLVKPAGSLRLSCVASGFTFSNNAMNWVRQAPKGGLQWVAGINSGGSTASADAVKGRFTISRDNAKNTVYLQMSLTAEDTAVYCAKVIGNWIATSDLDYWGQGTLVISSASTTAPSVFPLAPSCGSQSGSTVALACLVSGYIPEPVTVSWNSGSLTSGVHTFPSILQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPATNTKVDK PVVKECECCKCNCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQKSLSHPGK

[0287] IgG-D(SEQ ID NO:18) MEFVLGWVFLVAILQGVQGEVQLVESGGDLVKPAGSLRLSCVASGFTFSNNAMNWVRQAPGKGLQWVAGINSGGSTASADAVKGRFTISRDNAKNTVYLQMNSLTAEDTAVYYCAKV IGNWIATSDLDYWGQGTLVIVSSASSTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLKSSGLYSLSSMVTVPSSRLPSETFTCNVVHPATNTKVD KPVPKESTCKCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPI ERTISKARGQAHQPGVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHEALQNHYTDLSLSHSPGK Canine A steady-state region (SEQ ID NO: 19) FNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIE RTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK Canine B steady-state region (SEQ ID NO: 20) RENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSP IERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQKSLSHSPGK Canine B-YTE constant region (SEQ ID NO: 21) RENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLYITREPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSP IERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQKSLSHSPGK

[0288] Canine C constant region (SEQ ID NO: 22) ECECKCNCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPI EEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQKSLSHSPGK Canine D steady-state region (SEQ ID NO: 23) ESTCKCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPPSPIER TISKARGQAHQPGVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHEALQNHYTDLSLSHSPGK Cat_IGG1V1 steady-state region (SEQ ID NO: 24) TDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSP IERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK Cat_IGG1V2 steady-state region (SEQ ID NO: 25) TDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSP IERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK Cat_IGG2 steady-state region (SEQ ID NO: 26) KTASTIESKTGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSA MERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK

[0289] Horse_IGHG1 steady-state region (SEQ ID NO: 27) VIKECNGGCPAECLQVGPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFPDVQFNWYVDGVETHTATTEPKQEQFNSTYRVVSVLPIQHKDWLSGKEFKCKVNNKALPVER TISKPTGQPREPQVYVLAPHRDELSKNKVSVTCLVKDFYPTDIDIEWKSNGQPEPETKYSTTPAQLDSDGSYFLYSKLTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKSPGK Horse_IGHG2 steady-state region (SEQ ID NO: 28) CVLSAEGVIPIPSVPKPQCPPYTHSKFLGGPSVFIFPPNPKDALMISRTPVVTCVVVNLSDQYPDVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTNVGV PQPISRAISRGKGPSRVPQVYVLPPHPDELAKSKVSVTCLVKDFYPPDISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNHFTKTDISESLGK Horse_IGHG3 steady-state region (SEQ ID NO: 29) TTPPCPCECPKCPAPELLGGPSVFIFPPKDVLMITRTPEVTCLVVDVSHDSSDVLFTWYVDGTEVKTAKTMPNEEQNNSTYRVVSVLRIQHQDWLNGKKFKCKVNNQALPAPVE RTISKATGQTRVPQVYVLAPHPDELSKNKVSVTCLVKDFLPTDITVEWQSNEHPEPEGKYRTTEAQKDSDGSYFLYSKLTVETDRWQQGTTFTCVVMHEALHNHVMQKNVSHSPGK

[0290] Horse_IGHG4 steady-state region (SEQ ID NO: 30) VIKECNGGCPAECLQVGPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFPDVQFNWYVDGVETHTATTEPKQEQFNSTYRVVSVLPIQHKDWLSGKEFKCKVNNKALPVER TISKPTGQPREPQVYVLAPHRDELSKNKVSVTCLVKDFYPTDIDIEWKSNGQPEPETKYSTTPAQLDSDGSYFLYSKLTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKSPGK Horse_IGHG5 steady-state region (SEQ ID NO: 31) VVKGSPCPKCPAPELPGGPSVFIFPPKPKDVLKISRKPEVTCVVVDLGHDDPDVQFTWFVDGVETHTATTEPKEEQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTNKALPVER TTSKAKGQLRVPQVYVLAPHPDELAKNTVSVTCLVKDFYPPEIDVEWQSNEHPEPEGKYSTTPAQLNSDGSYFLYSKLSVETSRWKQGESFTCGVMHEAVENHYTQKNVSHSPGK Horse_IGHG6 steady-state region (SEQ ID NO: 32) KEPCCCPKCPGRPSVFIFPPNPKDTLMISRTPEVTCVVVDVSQENPDVKFNWYVDGVEAHTATTKAKEKQDNSTYRVVSVLPIQHQDWRRGKEFKCKVNNRALPAPVERTIT KAKGELQDPKVYILAPHREEVTKNTVSVTCLVKDFYPPDINVEWQSNEEPEPEVKYSTTPAQLDGDGSYFLYSKLTVETDRWEQGESFTCVVMHEAIRHTYRQKSITNFPGK

[0291] Uma_IGHG7 steady-state region (SEQ ID NO: 33) VIKECGGCPTCPECLSVGPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFPDVQFNWYVDGVETHTATTEPKQEQNNSTYRVVSILAIQHKDWLSGKEFKCKVNNQALPAPVQK TISKPTGQPREPQVYVLAPHRDELSKNKVSVTCLVKDFYPTDIDIEWKSNGQPEPETKYSTTPAQLDSDGSYFLYSKLTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKSPGK

[0292] A portion of canine ECD (excluding the stalk and not containing alpha and gamma-secretase cleavage at 3' of the stalk region) that can be used in fusion constructs having the mutations described herein (SEQ ID NO: 34) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDRQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIP A tandem repeat of the canine ECD portion (without stalks and without alpha and gamma-secretase cleavage at 3' of the stalk region) that can be used in fusion constructs having the mutations described herein (SEQ ID NO: 117) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDRQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIP KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDRQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIP

[0293] Canine p75NTR ECD nucleic acid sequence used in the fusion construct (encoding only the first two stalk amino acids GR and not containing alpha and gamma secretase cleavage at 3' of the stalk region) (SEQ ID NO: 35) AAGGAGGCATGTCCCACTGGCCTGTACACCCACAGCGGCGAGTGCTGCAAAGCCTGCAATCTGGGTGAGGGGGTGGCCCAGCCTTGCGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACA GCGTGACCTTCTCGGACGTGGTGAGCGCCACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTGCAGAGCATGTCGCGCCGTGCGTGGAGGCGGACGACGCCGTGTGCCGCTGCGCCTAC GGCTACTACCAGGACGAGACGACGGGCCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGGCTCGTGTTCTCGTGCCAGGACAGGCAGAACACCGTGTGCGAGGAGTGTCCCGACGGCA CGTACTCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTGCGCGAGTGCACGCGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGT Bovine p75 NTR protein (SEQ ID NO: 36) The text "JPEG2026511340000023.jpg36153ECD" is underlined.

[0294] Bovine p75 NTR nucleic acid (SEQ ID NO: 37) Feline p75NTR protein ECD (SEQ ID NO: 38) KEACPTGLFTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQ DRQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPSEGSDSTAPSTEEPEVPPEQDLIASTVADVVTTVMGSSQPVVTRGTADN

[0295] Feline p75NTR ECD-Feline IgG2 wt Fc protein fusion (SEQ ID NO: 39) JPEG2026511340000024.jpg41152 Signal peptide-feline p75-ECD-linkerGGGG-feline Fc-IgG2 Feline p75NTR ECD-Feline IgG2 wt Fc nucleic acid sequence (SEQ ID NO: 40) ATGGATGGACCTAGACCTCTGCTGCTGCTCCTGCCTCTGCTGTTGGGAGTTTCTCTCGGCGGAGCCAAAGAGGCTTGTCCTACCGGCCTGTTTACCCACTCTGGCGAGTGTTGCAAGGCCTGTAATCTCGGCGAAGGCGTGGCACAACCTTGTGGCGCTAATCAGACAGTGTGCGAGCCTTGCCTGGACTCCGTGACCTTCTCTGATGTGGTGTCTGCCACCGAGCCATGCAAGCCTTGTACCGAGTGTGTGGGCCTGCAGTCCATGTCTGCCCCTTGTGTGGAAGCCGACGACGCCGTGTGTAGATGTGCCTACGGCTACTACCAGGACGAGACAACCGGAAGATGCGAGGCCTGCAGAGTGTGTGAAGCTGGCTCTGGACTGGTGTTCTCCTGCCAAGACAGACAGAACACCGTGTGCGAGGAATGCCCTGACGGCACCTACTCTGATGAGGCCAATCACGTGGACCCCTGCCTGCCTTGTACTGTGTGCGAAGATACCGAGCGGCAGCTGCGCGAGTGTACCAGATGGGCTGATGCCGAGTGCGAAGAGATCCCTGGAAGAGGCGGAGGCGGAGTGCCTAAGACCGCTTCTACCATCGAGTCCAAGACCTGCGACTGCCCTAAGTGCCCTGTGCCTGAAATTCCTGGCGCTCCCTCCGTGTTCATCTTCCCACCTAAGCCTAAGGACACCCTGTCCATCTCTCGGACCCCTGAAGTGACCTGCCTGGTGGTTGATCTGGGCCCTGACGACTCCAACGTGCAGATCACTTGGTTTGTGGACAACACCGAGATGCACACCGCCAAGACCAGACCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGCCCATCCTGCACCAGGATTGGCTGAAGGGCAAAGAATTCAAGTGCAAAGTGAACTCCAAGAGCCTGCCTTCCGCCATGGAACGGACCATCTCTAAGGCTAAGGGCCAGCCTCATGAGCCCCAGGTGTACGTTCTGCCTCCTACACAAGAGGAACTGTCCGAGAACAAAGTGTCCGTGACATGCCTGATCAAGGGCTTTCACCCTCCTGATATCGCCGTGGAATGGGAGATCACCGGACAGCCTGAGCCTGAGAACAACTACCAGACCACACCTCCTCAGCTGGACAGCGACGGAACCTACTTCCTGTACTCCCGGCTGTCCGTGGACAGATCCCATTGGCAGAGAGGCAACACCTACACCTGTTCCGTGTCTCACGAGGCCCTGCACTCTCATCACACCCAGAAGTCCCTGACACAGTCCCCTGGCAAG Cat IgG3 (SEQ ID NO: 41) LPPCKCPKCPVPEIPGGPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPIVHQDWLTGKEFKCKVNSKALPSAIERTISKAKGQPHEPQVYVLPPAQEELSENKVCVTCLIKGFYPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSRLSMDRSRWQSGNTYTCSVSHEALHSHHTQKSLTQSPGK

[0296] Cat p75NTR ECD-Cat IgG1 wt Fc protein fusion (SEQ ID NO: 42) JPEG2026511340000025.jpg41153 Signal peptide-feline p75-ECD-linkerGGGG-feline Fc-IgG1 Feline p75NTR ECD-Feline Ig1 wt Fc nucleic acid sequence (SEQ ID NO: 43) ATGGATGGCCCTAGACCTCTGCTGCTGCTGTTGCCTCTGCTCCTGGGAGTTTCTCTCGGCGGAGCCAAAGAGGCTTGTCCTACCGGCCTGTTTACCCACTCTGGCGAGTGTTGCAAGGCCTGTAATCTCGGCGAAGGCGTGGCACAACCTTGTGGCGCTAATCAGACAGTGTGCGAGCCTTGCCTGGACTCCGTGACCTTCTCTGATGTGGTGTCTGCCACCGAGCCATGCAAGCCTTGTACCGAGTGTGTGGGCCTGCAGTCCATGTCTGCCCCTTGTGTGGAA GCCGACGACGCCGTGTGTAGATGTGCCTACGGCTACTACCAGGACGAGACAACCGGAAGATGCGAGGCCTGCAGAGTGTGTGAAGCTGGCTCTGGACTGGTGTTCTCCTGCCAAGACAGACAGAACACCGTGTGCGAGGAATGCCCTGACGGCACCTACTCTGATGAGGCCAATCACGTGGACCCCTGCCTGCCTTGTACTGTGTGCGAAGATACCGAGCGGCAGCTGCGCGAGTGTACCAGATGGGCTGATGCCGAGTGCGAAGAGATTCCTGGCGGAGGCGGAGTGCGCAAGACAGATCATCCTCCTGGACCTAAGCCTTGCGACTGCCCTAAGTGTCCCGCTCCTGAAATGCTCGGCGGACCCAGCATCTTCATCTTCCCACCTAAGCCAAAGGACACCCTGTCCATCTCTCGGACCCCTGAAGTGACCTGCCTGGTGGTTGATCTGGGCCCTGACGATTCCGACGTGCAGATCACTTGGTTTGTGGACAACACCCAGGTGTACACAGCCAAGACCTCTCCAAGAGAGGAACAGTTCAACTCCAC CTACAGAGTGGTGTCCGTGCTGCCCATCCTGCACCAGGATTGGCTGAAGGGCAAAGAATTCAAGTGCAAAGTGAACTCCAAGAGCCTGCCTTCTCCAATCGAGCGGACCATCTCCAAGGCTAAGGGCCAGCCTCATGAGCCTCAGGTGTACG TTCTGCCTCCTGCTCAAGAGGAACTGTCCCGGAACAAAGTGTCTGTGACCTGTCTGATCAAGAGCTTTCACCCTCCTGATATCGCCGTGGAATGGGAGATCACCGGACAGCCTGAGCCTGAGAACAACTACCGGACCACACCTCCTCAGCTG GACAGCGACGGCACATACTTCGTGTACTCCAAGCTGTCCGTGGACAGATCCCACTGGCAGCGGGGCAATACCTACACCTGTTCCGTGTCTCACGAGGCCCTGCACTCTCATCACACCCAGAAGTCCCTGACACAGTCCCCTGGAAAGTGATGA

[0297] Feline p75NTR ECD-Feline IgG3 wt Fc protein fusion (SEQ ID NO: 44) JPEG2026511340000026.jpg42152 Signal peptide-feline p75-ECD-linkerGGGG-feline Fc-IgG3 Feline p75NTR ECD-Feline IgG3 wt Fc nucleic acid sequence (SEQ ID NO: 45) ATGGATGGCCCTAGACCTCTGCTGCTGCTGTTGCCTCTGCTCCTGGGAGTTTCTCTCGGCGGAGCCAAAGAGGCTTGTCCTACCGGCCTGTTTACCCACTCTGGCGAGTGTTGCAAGGCCTGTAATCTCGGCGAAGGCGTGGCACAACCTTGTGGCGCTAATCAGACAGTGTGCGAGCCTTGCCTGGACTCCGTGACCTTCTCTGATGTGGTGTCTGCCACCGAGCCATGCAAGCCTTGTACCGAGTGTGTGGGCCTGCAGTCCATGTCTGCCCCTTGTGTGGAAGCCGACGACGCCGTGTGTAGATGTGCCTACGGCTACTACCAGGACGAGACAACCGGAAGATGCGAGGCCTGCAGAGTGTGTGAAGCTGGCTCTGGACTGGTGTTCTCCTGCCAAGACAGACAGAACACCGTGTGCGAGGAATGCCCTGACGGCACCTACTCTGATGAGGCCAATCACGTGGACCCCTGCCTGCCTTGTACTGTGTGCGAAGATACCGAGCGGCAGCTGCGCGAGTGTACCAGATGGGCTGATGCCGAGTGCGAAGAGATTCCTGGCGGAGGCGGAGTTCTGCCTCCTTGCAAGTGTCCTAAGTGCCCCGTGCCTGAAATCCCTGGCGGCCCTTCCGTGTTCATCTTCCCACCTAAGCCTAAGGACACCCTGTCCATCTCTCGGACCCCTGAAGTGACCTGCCTGGTGGTTGATCTGGGCCCTGACGACTCCAACGTGCAGATCACTTGGTTTGTGGACAACACCGAGATGCACACCGCCAAGACCAGACCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGCCCATCGTGCACCAGGATTGGCTGACCGGCAAAGAATTCAAGTGCAAAGTGAACAGCAAGGCCCTGCCTTCCGCCATCGAGCGGACAATCTCTAAGGCTAAGGGCCAGCCTCACGAGCCCCAGGTTTACGTTTTGCCTCCTGCTCAAGAGGAACTGTCCGAGAACAAAGTGTGCGTGACCTGTCTGATCAAGGGCTTCTACCCTCCTGATATCGCCGTGGAATGGGAGATCACCGGACAGCCTGAGCCTGAGAACAACTACCGGACCACACCTCCTCAGCTGGATTCCGACGGCACATACTTCGTGTACTCCCGGCTGAGCATGGACAGATCCAGATGGCAGTCCGGCAACACCTACACCTGTTCCGTGTCTCACGAGGCCCTGCACTCTCATCACACCCAGAAGTCCCTGACACAGTCCCCTGGCAAG variant Inu p75NTR ECD E75T (Sequence ID 46) JPEG2026511340000027.jpg15152 Canine p75NTR ECD E75T-Canine IgGB wt Fc (Sequence ID 47) JPEG2026511340000028.jpg42153 Signal peptide-canine p75-ECD-linkerGGGG-canine Fc-B wt Canine p75NTR ECD E75T-canine IgGB wt Fc nucleic acid sequence (SEQ ID NO: 48) ATGGAATGGAGCTGGGTGTTTCTGTTTTTTCTGAGCGTGACCACCGGCGTGCATAGCAAAGAAGCGTGCCCGACCGGCCTGTATACCCATAGCGGCGAATGCTGCAAAGCGTGCAACCTGGGCGAAGGCGTGGCGCAGCCGTGCGGCGCGAACCAGACCGTGTGCGAACCGTGCCTGGATAGCGTGACCTTTAGCGATGTGGTGAGCGCGACCGAACCGTGCAAACCGTGCACCGAATGCGTGGGCCTGCAGAGCATGAGCGCGCCGTGCGTGGAAGCGACCGATGCGGTGTGCCGCTGCGCGTATGGCTATTATCAGGATGAAACCACCGGCCGCTGCGAAGCGTGCCGCGTGTGCGAAGCGGGCAGCGGCCTGGTGTTTAGCTGCCAGGATCGCCAGAACACCGTGTGCGAAGAATGCCCGGATGGCACCTATAGCGATGAAGCGAACCATGTGGATCCGTGCCTGCCGTGCACCGTGTGCGAAGATACCGAACGCCAGCTGCGCGAATGCACCCGCTGGGCGGATGCGGAATGCGAAGAAATTCCGGGCCGCGGCGGCGGCGGCCGCGAAAACGGCCGCGTGCCGCGCCCGCCGGATTGCCCGAAATGCCCGGCGCCGGAAATGCTGGGCGGCCCGAGCGTGTTTATTTTTCCGCCGAAACCGAAAGATACCCTGCTGATTGCGCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATCTGGATCCGGAAGATCCGGAAGTGCAGATTAGCTGGTTTGTGGATGGCAAACAGATGCAGACCGCGAAAACCCAGCCGCGCGAAGAACAGTTTAACGGCACCTATCGCGTGGTGAGCGTGCTGCCGATTGGCCATCAGGATTGGCTGAAAGGCAAACAGTTTACCTGCAAAGTGAACAACAAAGCGCTGCCGAGCCCGATTGAACGCACCATTAGCAAAGCGCGCGGCCAGGCGCATCAGCCGAGCGTGTATGTGCTGCCGCCGAGCCGCGAAGAACTGAGCAAAAACACCGTGAGCCTGACCTGCCTGATTAAAGATTTTTTTCCGCCGGATATTGATGTGGAATGGCAGAGCAACGGCCAGCAGGAACCGGAAAGCAAATATCGCACCACCCCGCCGCAGCTGGATGAAGATGGCAGCTATTTTCTGTATAGCAAACTGAGCGTGGATAAAAGCCGCTGGCAGCGCGGCGATACCTTTATTTGCGCGGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGCCATAGCCCGGGCAAA

[0298] Canine p75NTR ECD S109Y (SEQ ID NO: 49) JPEG2026511340000029.jpg15152 Canine p75NTR ECD S109Y - Canine IgGB wt Fc (SEQ ID NO: 50) JPEG2026511340000030.jpg41153 Signal Peptide - Canine p75 - ECD - Linker GGGG - Canine Fc - B wt

[0299] Canine p75NTR ECD S109Y-canine IgGB wt Fc nucleic acid sequence (SEQ ID NO: 51) ATGGAATGGAGCTGGGTGTTTCTGTTTTTTCTGAGCGTGACCACCGGCGTGCATAGCAAAGAAGCGTGCCCGACCGGCCTGTATACCCATAGCGGCGAATGCTGCAAAGCGTGCAACCTGGGCGAAGGCGTGGCGCAGCCGTGCGGCGCGAACCAGACCGTGTGCGAACCGTGCCTGGATAGCGTGACCTTTAGCGATGTGGTGAGCGCGACCGAACCGTGCAAACCGTGCACCGAATGCGTGGGCCTGCAGAGCATGAGCGCGCCGTGCGTGGAAGCGGATGATGCGGTGTGCCGCTGCGCGTATGGCTATTATCAGGATGAAACCACCGGCCGCTGCGAAGCGTGCCGCGTGTGCGAAGCGGGCAGCGGCCTGGTGTTTTATTGCCAGGATCGCCAGAACACCGTGTGCGAAGAATGCCCGGATGGCACCTATAGCGATGAAGCGAACCATGTGGATCCGTGCCTGCCGTGCACCGTGTGCGAAGATACCGAACGCCAGCTGCGCGAATGCACCCGCTGGGCGGATGCGGAATGCGAAGAAATTCCGGGCCGCGGCGGCGGCGGCCGCGAAAACGGCCGCGTGCCGCGCCCGCCGGATTGCCCGAAATGCCCGGCGCCGGAAATGCTGGGCGGCCCGAGCGTGTTTATTTTTCCGCCGAAACCGAAAGATACCCTGCTGATTGCGCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATCTGGATCCGGAAGATCCGGAAGTGCAGATTAGCTGGTTTGTGGATGGCAAACAGATGCAGACCGCGAAAACCCAGCCGCGCGAAGAACAGTTTAACGGCACCTATCGCGTGGTGAGCGTGCTGCCGATTGGCCATCAGGATTGGCTGAAAGGCAAACAGTTTACCTGCAAAGTGAACAACAAAGCGCTGCCGAGCCCGATTGAACGCACCATTAGCAAAGCGCGCGGCCAGGCGCATCAGCCGAGCGTGTATGTGCTGCCGCCGAGCCGCGAAGAACTGAGCAAAAACACCGTGAGCCTGACCTGCCTGATTAAAGATTTTTTTCCGCCGGATATTGA TGTGGAATGGCAGAGCAACGGCCAGCAGGAACCGGAAAGCAAATATCGCACCACCCCGCCGCAGCTGGATGAAGATGGCAGCTATTTTCTGTATAGCAAACTGAGCGTGGATAAAAGCCGCTGGCAGCGCGGCGATACCTTTATTTGCGCGGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGCCATAGCCCGGGCAAA Inu p75NTR ECD S109H (Sequence ID 52) JPEG2026511340000031.jpg15152

[0300] Canine p75NTR ECD S109H-Canine IgGB wt Fc (Sequence ID 53) JPEG2026511340000032.jpg42153 Signal peptide-canine p75-ECD-linkerGGGG-canine Fc-B wt Canine p75NTR ECD S109H-canine IgGB wt Fc nucleic acid sequence (SEQ ID NO: 54) ATGGAATGGAGCTGGGTGTTTCTGTTTTTTCTGAGCGTGACCACCGGCGTGCATAGCAAAGAAGCGTGCCCGACCGGCCTGTATACCCATAGCGGCGAATGCTGCAAAGCGTGCAACCTGGGCGAAGGCGTGGCGCAGCCGTGCGGCGCGAACCAGACCGTGTGCGAACCGTGCCTGGATAGCGTGACCTTTAGCGATGTGGTGAGCGCGACCGAACCGTGCAAACCGTGCACCGAATGCGTGGGCCTGCAGAGCATGAGCGCGCCGTGCGTGGAAGCGGATGATGCGGTGTGCCGCTGCGCGTATGGCTATTATCAGGATGAAACCACCGGCCGCTGCGAAGCGTGCCGCGTGTGCGAAGCGGGCAGCGGCCTGGTGTTTCATTGCCAGGATCGCCAGAACACCGTGTGCGAAGAATGCCCGGATGGCACCTATAGCGATGAAGCGAACCATGTGGATCCGTGCCTGCCGTGCACCGTGTGCGAAGATACCGAACGCCAGCTGCGCGAATGCACCCGCTGGGCGGATGCGGAATGCGAAGAAATTCCGGGCCGCGGCGGCGGCGGCCGCGAAAACGGCCGCGTGCCGCGCCCGCCGGATTGCCCGAAATGCCCGGCGCCGGAAATGCTGGGCGGCCCGAGCGTGTTTATTTTTCCGCCGAAACCGAAAGATACCCTGCTGATTGCGCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATCTGGATCCGGAAGATCCGGAAGTGCAGATTAGCTGGTTTGTGGATGGCAAACAGATGCAGACCGCGAAAACCCAGCCGCGCGAAGAACAGTTTAACGGCACCTATCGCGTGGTGAGCGTGCTGCCGATTGGCCATCAGGATTGGCTGAAAGGCAAACAGTTTACCTGCAAAGTGAACAACAAAGCGCTGCCGAGCCCGATTGAACGCACCATTAGCAAAGCGCGCGGCCAGGCGCATCAGCCGAGCGTGTATGTGCTGCCGCCGAGCCGCGAAGAACTGAGCAAAAACACCGTGAGCCTGACCTGCCTGATTAAAGATTTTTTTCCGCCGGATATTGATGTGGAATGGCAGAGCAACGGCCAGCAGGAACCGGAAAGCAAATATCGCACCACCCCGCCGCAGCTGGATGAAGATGGCAGCTATTTTCTGTATAGCAAACTGAGCGTGGATAAAAGCCGCTGGCAGCGCGGCGATACCTTTATTTGCGCGGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGCCATAGCCCGGGCAAA

[0301] Canine p75NTR ECD V133R (SEQ ID NO: 55) JPEG2026511340000033.jpg16152 Canine p75NTR ECD V133R - Canine IgGB wt Fc (SEQ ID NO: 56) JPEG2026511340000034.jpg41153 Signal peptide - Canine p75 - ECD - linker GGGG - Canine Fc - B wt

[0302] Canine p75NTR ECD V133R-Canine IgGB wt Fc nucleic acid sequence (SEQ ID NO: 57) Inu p75NTR ECD D134L (Sequence ID 58) JPEG2026511340000035.jpg15152 Canine p75NTR ECD D134L-Canine IgGB wt Fc (Sequence ID 59) JPEG2026511340000036.jpg41153 Signal peptide-canine p75-ECD-linkerGGGG-canine Fc-B wt Canine p75NTR ECD D134L-canine IgGB wt Fc nucleic acid sequence (SEQ ID NO: 60) ATGGAATGGAGCTGGGTGTTTCTGTTTTTTCTGAGCGTGACCACCGGCGTGCATAGCAAAGAAGCGTGCCCGACCGGCCTGTATACCCATAGCGGCGAATGCTGCAAAGCGTGCAACCTGGGCGAAGGCGTGGCGCAGCCGTGCGGCGCGAACCAGACCGTGTGCGAACCGTGCCTGGATAGCGTGACCTTTAGCGATGTGGTGAGCGCGACCGAACCGTGCAAACCGTGCACCGAATCGTGGGCCTGCAGAGCATGAGCGCGCCGTGCGTGGAAGCGGATGATGCGGTGTGCCGCTGCGCGTATGGCTATTATCAGGATGAAACCACCGGCCGCTGCGAAGCGTGCCGCGTGTGCGAAGCGGGCAGCGGCCTGGTGTTTAGCTGCCAGGATCGCCAGAACACCGTGTGCGAAGAATGCCCGGATGGCACCTATAGCGATGAAGCGAACCATGTGCTGCCGTGCCTGCCGTGCACCGTGTGCGAAGATACCGAACGCCAGCTGCGCGAATGCACCCGCTGGGCGGATGCGGAATGCGAAGAAATTCCGGGCCGCGGCGGCGGCGGCCGCGAAAACGGCCGCGTGCCGCGCCCGCCGGATTGCCCGAAATGCCCGGCGCCGGAAATGCTGGGCGGCCCGAGCGTGTTTATTTTTCCGCCGAAACCGAAAGATACCCTGCTGATTGCGCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATCTGGATCCGGAAGATCCGGAAGTGCAGATTAGCTGGTTTGTGGATGGCAAACAGATGCAGACCGCGAAAACCCAGCCGCGCGAAGAACAGTTTAACGGCACCTATCGCGTGGTGAGCGTGCTGCCGATTGGCCATCAGGATTGGCTGAAAGGCAAACAGTTTACCTGCAAAGTGAACAACAAAGCGCTGCCGAGCCCGATTGAACGCACCATTAGCAAAGCGCGCGGCCAGGCGCATCAGCCGAGCGTGTATGTGCTGCCGCCGAGCCGCGAAGAACTGAGCAAAAACACCGTGAGCCTGACCTGCCTGATTAAAGATTTTTTTCCGCCGGATATTGATGTGGAATGGCAGAGCAACGGCCAGCAGGAACCGGAAAGCAAATATCGCACCACCCCGCCGCAGCTGGATGAAGATGGCAGCTATTTTCTGTATAGCAAACTGAGCGTGGATAAAAGCCGCTGGCAGCGCGGCGATACCTTTATTTGCGCGGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGCCATAGCCCGGGCAAA

[0303] Cat p75NTR ECD E75T (Sequence ID 61) JPEG2026511340000037.jpg15152 Feline p75NTR ECD E75T-Feline IgG1 (SEQ ID NO: 62) JPEG2026511340000038.jpg41153 Signal peptide-feline p75-ECD-linkerGGGG-feline Fc-IgG1

[0304] Cat p75NTR ECD S109Y (Sequence ID 63) JPEG2026511340000039.jpg15152 Feline p75NTR ECD S109Y-Feline Fc IgG1 (Sequence ID 64) JPEG2026511340000040.jpg41153 Signal peptide-feline p75-ECD-linkerGGGG-feline Fc-IgG1

[0305] Cat p75NTR ECD S109H (Sequence ID 65) JPEG2026511340000041.jpg15152 Feline p75NTR ECD S109H-Feline Fc IgG1 (Sequence ID 66) JPEG2026511340000042.jpg67168 Signal peptide-feline p75-ECD-linkerGGGG-feline Fc-IgG1

[0306] Cat p75NTR ECD V133R (Sequence ID 67) JPEG2026511340000043.jpg15152 Feline p75NTR ECD V133R-Feline Fc IgG1 (SEQ ID NO: 68) JPEG2026511340000044.jpg41153 Signal peptide-feline p75-ECD-linkerGGGG-feline Fc-IgG1

[0307] Cat p75NTR ECD D134L (Sequence ID 69) JPEG2026511340000045.jpg15152 Feline p75NTR ECD D134L-Feline Fc IgG1 (Sequence ID 70) JPEG2026511340000046.jpg67156 Signal peptide-feline p75-ECD-linkerGGGG-feline Fc-IgG1

[0308] Human p75NTR protein (SEQ ID NO: 71) >sp|P08138|TNR16_Human Tumor Necrosis Factor Receptor Superfamily Member 16 OS=Homo sapiens OX=9606 GN=NGFR PE=1 SV=1 MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQ EPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGISVDSQSLHDQQPH TQTASGQALKGDGGLYSSLPPAKREEVEKLLNGSAGDTWRHLAGELGYQPEHIDSFTHEACPVRALLASWATQDSATLDALLAALRRIQRADLVESLCSESTATSPV The complete p75NTR aa sequence including signal peptide, ECD, transmembrane and intracellular domains. Human p75NTR nucleic acid sequence (SEQ ID NO: 72) Human p75NTR protein ECD (SEQ ID NO: 73) with a stalk region JPEG2026511340000047.jpg36161 The WT ECD region includes the stalk region (underlined), as well as alpha and gamma-secretase cleavage (bold) at the 3' of the stalk region. Human p75NTR ECD nucleic acid sequence (SEQ ID NO: 74) AAGGAGGCCTGCCCCACCGGCCTGTACACCCACAGCGGCGAGTGCTGCAAGGCCTGCAACCTGGGCGAGGGCGTGGCCCAGCCCTGCGGCGCCAACCAGACCGTGTGCGAGCCCTGCCTGGACAGCGTGACCTTCAGCGACGTGGTGAGCGCCACC GAGCCCTGCAAGCCCTGCACCGAGTGCGTGGGCCTGCAGAGCATGAGCCCCCTGCGTGGAGGCCGACGACGCCGTGTGCAGGTGCGCCTACGGCTACTACCAGGACGAGACCACCGGCAGGTGCGAGGCCTGCAGGGTGTGCGAGGCCGGCAGC GGCCTGGTGTTCAGCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACCTACAGCGACGAGGCCAACCACGTGGACCCCTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGAGGCAGCTGAGGGAGTGCACCAGGTGGCC GACGCCGAGTGCGAGGAGATCCCCGGCAGGTGGATCACCAGGAGCACCCCCCCCGAGGGCAGCGACAGCACCGCCCCCAGCACCCAGGAGCCCGAGGCCCCCCGAGCAGGACCTGATCGCCAGCACCGTGGCCGGCGTGGTGACCACCACCGTGATG

[0309] Human ECD of p75NTR stalk region protein (SEQ ID NO: 75) JPEG2026511340000048.jpg16159 WT ECD stalk region (underlined), and alpha and gamma secretase cleavage at 3' of the stalk region (bold). Human ECD (SEQ ID NO: 76) of the p75NTR stalk region nucleic acid sequence. GGCAGGTGGATCACCAGGAGCACCCCCCCCGAGGGCAGCGACAGCACCGCCCCAGCACCCAGGAGCCCGAGGCCCCCCGAGCAGGACCTGATCGCCAGCACCGTGGCCGGCGTGGTGACCACCGTGATG Human IgG1 (SEQ ID NO: 77) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 (SEQ ID NO: 78) ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG3 (SEQ ID NO: 79) ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK Human IgG4 (SEQ ID NO: 80) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK PetML308_Human p75NTR ECD Whole Stork IgG1 (SEQ ID NO: 81) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQ DKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGGGGEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0310] PetML309_Human p75NTR ECD partial Stork IgG1 (SEQ ID NO: 82) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGRWITRSTPPEGGGGEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PetML319_Human p75NTR ECD Stork-free IgG1 (SEQ ID NO: 83) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human p75NTR ECD E75T (Sequence ID 84) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEATDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIP Human p75NTR ECD E75T-Human IgG1 Fc (SEQ ID NO: 85) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEATDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0311] Human p75NTR ECD E75T-Human IgG1 Fc nucleic acid sequence (SEQ ID NO: 86) AAGGAGGCCTCCCACCCGGCCGTTACACCCACAGCGGCGAGTGCTGCAAGGCCTGCAACCTGGGCGAGGGCGTGGCCAGCCCTGCGGCGCCAACCAGACCGTGTGCGGACCCTGCCTGGACAGCGGTGACCTTCAGCGACGGTGGTGAGCGCCACCACCGAGCCCTGTGCGGTGCGCTACGGCCTACTACCAGGACGACACCAGCACCGGCAGGTCGAGGCTGCGAGGTCGAGGTCGAGGTCGAGGTCGAGGTCCA GCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG

[0312] Human p75NTR ECD S109Y (SEQ ID NO: 87) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFYCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIP Human p75NTR ECD S109Y - Human IgG1 Fc (SEQ ID NO: 88) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFYCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0313] Human p75NTR ECD S109Y-Human IgG1 Fc nucleic acid sequence (SEQ ID NO: 89) Human p75NTR ECD S109H (SEQ ID NO: 90) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFHCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIP

[0314] Human p75NTR ECD S109H-Human IgG1 Fc (SEQ ID NO: 91) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFHCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLECTRWADAECEEIPGGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human p75NTR ECD S109H-Human IgG1 Fc nucleic acid sequence (SEQ ID NO: 92)

[0315] Human p75NTR ECD V133R (SEQ ID NO: 93) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHRDPCLPCTVCEDTERQLRECTRWADAECEEIP Human p75NTR ECD V133R-Human IgG1 Fc (SEQ ID NO: 94) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHRDPCLPCTVCEDTERQLECTRWADAECEEIPGGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human p75NTR ECD V133R-Human IgG1 Fc nucleic acid sequence (SEQ ID NO: 95)

[0316] Human p75NTR ECD D134L (Sequence ID 96) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVLPCLPCTVCEDTERQLRECTRWADAECEEIP Human p75NTR ECD D134L-Human IgG1 Fc (SEQ ID NO: 97) KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVLPCLPCTVCEDTERQLECTRWADAECEEIPGGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0317] Human p75NTR ECD D134L-Human IgG1 Fc nucleic acid sequence (SEQ ID NO: 98)

[0318] Examples of the p75NTR protein of the present invention derived from camels and pigs are incorporated into the sequence listing XML as follows: Camel p75NTR protein (SEQ ID NO: 99) Camel p75NTR nucleic acid sequence (SEQ ID NO: 100) Camel p75NTR protein ECD (SEQ ID NO: 101) Camel p75NTR protein ECD nucleic acid sequence (SEQ ID NO: 102) Camel p75NTR ECD E75T (Sequence ID 103) Camel p75NTR ECD S109Y (Sequence ID 104) Camel p75NTR ECD S109H (Sequence ID 105) Camel p75NTR ECD V133R (Sequence ID 106) Camel p75NTR ECD D134L (Sequence ID 107) Porcine p75NTR protein (SEQ ID NO: 108) Pig p75NTR nucleic acid sequence (SEQ ID NO: 109) Porcine p75NTR protein ECD (SEQ ID NO: 110) Porcine p75NTR protein ECD nucleic acid sequence (SEQ ID NO: 111) Pig p75NTR ECD E75T (Sequence ID 112) Pig p75NTR ECD S109Y (Sequence ID 113) Pig p75NTR ECD S109H (Sequence ID 114) Pig p75NTR ECD V133R (Sequence ID 115) Pig p75NTR ECD D134L (Sequence ID 116) Horse p75NTR protein ECD (SEQ ID NO: 118) Horse p75NTR protein ECD nucleic acid sequence (SEQ ID NO: 119) Uma p75NTR ECD E75T (Sequence ID 120) Uma p75NTR ECD S109Y (Sequence ID 121) Uma p75NTR ECD S109H (Sequence ID 122) Uma p75NTR ECD V133R (Sequence ID 123) Uma p75NTR ECD D134L (Sequence ID 124) Bovine p75NTR protein ECD (SEQ ID NO: 125) Bovine p75NTR protein ECD nucleic acid sequence (SEQ ID NO: 126) Cow p75NTR ECD E75T (Sequence ID 127) Cow p75NTR ECD S109Y (Sequence ID 128) Cow p75NTR ECD S109H (Sequence ID 129) Cow p75NTR ECD V133R (Sequence ID 130) Cow p75NTR ECD D134L (Sequence ID 131)

Claims

1. An isolated polypeptide comprising a companion animal p75 neurotrophin receptor (p75NTR) extracellular domain, wherein the p75NTR comprises one or more variant amino acids at positions 75, 109, 133, and / or 134.

2. The variant amino acid at position 75 of p75NTR includes a polar side chain, and optionally, The isolated polypeptide according to claim 1, wherein the variant amino acid is selected from serine, threonine, tyrosine, tryptophan, asparagine, glutamine, or cysteine, and preferably threonine.

3. The variant amino acid at position 109 of p75NTR includes an aromatic side chain, optionally, The isolated polypeptide according to claim 1 or 2, wherein the variant amino acid is selected from histidine, tyrosine, phenylalanine, or tryptophan.

4. The isolated polypeptide according to any one of claims 1 to 3, wherein the variant amino acid at position 133 of p75NTR includes a charged side chain.

5. The isolated polypeptide according to any one of claims 1 to 4, wherein the variant amino acid at position 133 of p75NTR is selected from arginine, histidine, lysine, aspartic acid, or glutamic acid.

6. The variant amino acid at position 133 of p75NTR includes a negatively charged side chain, optionally, The isolated polypeptide according to any one of claims 1 to 4, wherein the variant amino acid is selected from arginine, histidine, or lysine, and is preferably arginine.

7. The isolated polypeptide according to any one of claims 1 to 6, wherein the variant amino acid at position 134 of p75NTR includes a hydrophobic side chain.

8. The isolated polypeptide according to any one of claims 1 to 7, wherein the variant amino acid at position 134 of p75NTR is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

9. The isolated polypeptide according to any one of claims 1 to 7, wherein the variant amino acid at position 134 of p75NTR comprises a non-aromatic hydrophobic side chain, and optionally the variant amino acid is selected from alanine, valine, isoleucine, leucine, or methionine, preferably leucine.

10. The isolated polypeptide according to any one of claims 1 to 9, wherein the companion animal is a cat, a dog, a pig, a cow, a horse, or a camel.

11. If the companion animal is a dog and the p75NTR contains or consists of a sequence selected from sequence number 46, sequence number 49, sequence number 52, sequence number 55, or sequence number 58, or if the companion animal is a cat and the p75NTR contains or consists of a sequence selected from sequence number 61, sequence number 63, sequence number 65, sequence number 67, or sequence number 69, or if the companion animal is a pig and the p75NTR contains or consists of a sequence selected from sequence number 112, sequence number 113, sequence number 114, sequence number 115, or sequence number 116, or if the companion animal is a horse The isolated polypeptide according to any one of claims 1 to 10, wherein the p75NTR includes or consists of a sequence selected from SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, or SEQ ID NO: 124, or the companion animal is a cow and the p75NTR includes or consists of a sequence selected from SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131, or the companion animal is a camel and the p75NTR includes or consists of a sequence selected from SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO:

107.

12. The isolated polypeptide according to any one of claims 1 to 11, wherein the p75NTR extracellular domain is cleaved.

13. An isolated nucleic acid encoding an isolated polypeptide according to any one of claims 1 to 12.

14. A vector comprising the nucleic acid described in claim 13.

15. A host cell comprising the nucleic acid described in claim 13 or the vector described in claim 14.

16. A fusion protein comprising a companion animal p75NTR extracellular domain, wherein the p75NTR comprises one or more variant amino acids at positions 75, 109, 133, and / or 134, and comprises a half-life extension portion.

17. The fusion protein according to claim 16, wherein the half-life extension portion is selected from an Fc domain, a serum albumin-binding factor, or PEG, and optionally the half-life extension portion is a wild-type or mutant Fc domain.

18. The aforementioned half-life extension portion is an Fc domain, and the p75NTR extracellular domain or a portion thereof and the Fc domain are linked by a linker, and optionally, The linker is a peptide linker, and further optionally, The peptide linker is (GGGG)n or (G 4 S) n The fusion protein according to claim 16 or 17, wherein n is 1 to 4 in the formula.

19. The fusion protein according to any one of claims 16 to 18, wherein the companion animal is a cat, dog, pig, cow, horse, or camel.

20. The Fc domain is a canine Fc domain, and the fusion protein contains or consists of a sequence selected from SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO:

59. The fusion protein according to any one of claims 16 to 19, wherein the Fc domain is a feline Fc domain, and the fusion protein includes or consists of a sequence selected from SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO:

70.

21. A nucleic acid encoding a fusion protein according to any one of claims 16 to 20.

22. A vector comprising the nucleic acid described in claim 21.

23. A host cell comprising the nucleic acid described in claim 21 or the vector described in claim 22.

24. A pharmaceutical composition comprising an isolated polypeptide according to any one of claims 1 to 12, or a fusion protein according to any one of claims 16 to 20.

25. A method for treating an NGF-related disorder in a companion animal, comprising administering an effective amount of an isolated companion animal p75NTR protein according to any one of claims 1 to 12, a fusion protein according to any one of claims 16 to 20, or a pharmaceutical composition according to claim 24.

26. An isolated companion animal p75NTR protein according to any one of claims 1 to 12, or a fusion protein according to any one of claims 16 to 20, or a pharmaceutical composition according to claim 24, for use in the treatment of one or more NGF-related disorders in companion animals.

27. The method according to claim 25, or an isolated companion animal p75NTR protein, fusion protein, or pharmaceutical composition for use according to claim 26, wherein the one or more NGF-related disorders are cardiovascular disease, atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, pain, and inflammation.

28. The aforementioned NGF-related disorder is a pain-related disorder, and is selected as: The method according to claim 27, or an isolated companion animal p75NTR protein, fusion protein, or pharmaceutical composition for use, wherein the pain is selected from osteoarthritis pain, rheumatoid arthritis pain, surgical and postoperative pain, incision pain, systemic inflammatory pain, cancer pain, traumatic pain, neuropathic pain, neuralgia, diabetic neuropathy pain, pain associated with rheumatic diseases, pain associated with musculoskeletal diseases, visceral pain, and gastrointestinal pain.

29. The NGF-related disorder is inflammation, and the inflammation is an inflammatory disease, condition, or symptom, and optionally, the inflammatory disease, condition, or symptom is one or more organ failure or dysfunction, sepsis, cytokine storm, fever, neurological dysfunction or injury, loss of taste or smell, cardiac dysfunction, pulmonary dysfunction, hepatic dysfunction, acute or chronic respiratory failure, graft-versus-host disease (GVHD), cardiomyopathy, vasculitis, fibrosis, ocular inflammation, skin inflammation, gastroenteritis, tenosynovitis, allergy, asthma, glomerulonephritis, pancreatitis, hepatitis A companion animal p75NTR protein, fusion protein, or pharmaceutical composition for use according to claim 28, selected from the group consisting of non-alcoholic steatohepatitis (NASH), inflammatory arthritis, gout, multiple sclerosis, psoriasis, acute respiratory distress syndrome (ARDS), diabetic ulcers, non-healing wounds, lupus, autoimmune diseases associated with acute or chronic inflammation, and acute or chronic inflammation associated with viral, bacterial, or fungal infections, swelling, tenderness, joint contracture, or decreased joint mobility.

30. A method for inhibiting NGF activity and / or pro-NGF activity in a companion animal, comprising administering an isolated companion animal p75NTR protein according to any one of claims 1 to 12, a fusion protein according to any one of claims 16 to 20, or a pharmaceutical composition according to claim 24.

31. An isolated companion animal p75NTR protein, fusion protein, or pharmaceutical composition for use according to any one of claims 25 to 30, comprising administering a second compound.

32. A kit comprising an isolated companion animal p75NTR protein according to any one of claims 1 to 12, a fusion protein according to any one of claims 16 to 20, or a pharmaceutical composition according to claim 24, and optionally, instructions for use.

33. A method for treating osteoarthritis in a companion animal, comprising administering an effective amount of isolated companion animal p75NTR protein or a portion thereof, or a pharmaceutical composition comprising the isolated companion animal p75NTR protein or a portion thereof.

34. A pharmaceutically acceptable composition comprising an isolated companion animal p75NTR protein or a portion thereof, or the isolated companion animal p75NTR protein or a portion thereof, for use in the treatment of osteoarthritis in companion animals.

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