Fusion protein and uses thereof

Abstract

Disclosed herein are methods of treatment of pain associated with osteoarthritis, pain associated with osteonecrosis, and pain associated with one or more subchondral insufficiency fractures; methods of improving physical function in a human subject with osteoarthritis, osteonecrosis, or one or more subchondral insufficiency fractures; and methods of treating osteoarthritis, or delaying or slowing the progression of osteoarthritis, or relieving a symptom of osteoarthritis, in humans with these diseases, comprising administering a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein.

Description

Attorney Docket No. LEVI0008-401-PC FUSION PROTEIN AND USES THEREOF

[0001] This application claims the benefit of priority of Great Britain patent application no.2402758.3, filed February 27, 2024, Great Britain patent application no. 2402762.5, filed February 27, 2024, Great Britain patent application no.2404695.5, filed April 02, 2024, Great Britain patent application no.2404697.1, filed April 02, 2024, Great Britain patent application no.2407860.0, filed June 03, 2024, Great Britain patent application no. 2408403.0, filed June 12, 2024, Great Britain patent application no.2416136.6, filed November 01, 2024, Great Britain patent application no.2416137.4, filed November 01, 2024, United States provisional application no.63 / 719,597, filed November 12, 2024, Great Britain patent application no.2417278.5, filed November 25, 2024, and Great Britain patent application no.2417279.3, filed November 25, 2024, the contents of which are incorporated by reference as if written herein in their entirety.

[0002] Pain, particularly chronic pain, is a major public health problem reported by approximately 20% of adults in the Western world. Despite slightly different definitions, chronic pain can be defined as a condition in which an individual experiences pain on most days or every day for more than three months (Zelaya et al., 2020). An estimated 116 million Americans are burdened by chronic pain, resulting in an annual cost of between $500–600 billion (Control and Prevention, 2001). Treatment of chronic pain continues to be one of the major challenges in clinical medicine (Schnitzer and Marks, 2015).

[0003] Pain is the cardinal manifestation of osteoarthritis (OA), and yet the least understood. Globally, OA is a leading cause of chronic pain and disability affecting millions of adults who are middle–aged and older adults worldwide (Wong et al., 2023). Despite the availability of a wide spectrum of non–pharmacologic and pharmacologic options, for many people with OA, chronic pain is not adequately controlled and leads to reduced activity and a markedly diminished quality of life (McNeil and Binette, 2001).

[0004] Osteoarthritis is a group of conditions in which the joints are degraded, leading to pain, tenderness, stiffness and locking of joints. It is the most common form of arthritis, affecting millions of people around the world. Treatment of osteoarthritis is limited to management of the condition, with no curative treatment options available. Management options include physiotherapy, administration of pain killers, and / or administration of anti– inflammatory drugs and, in some cases, joint replacement via surgery.

[0005] Nerve growth factor (NGF), is a member of the neurotrophin family of molecules and is critical for normal development of sympathetic neurons and sensory neuronsAttorney Docket No. LEVI0008-401-PC responsible for nociception and temperature sensation. In adults, withdrawal of NGF results in decreased sensitivity of peripheral nociceptors and a reduction in neuropeptide levels. NGF is known to bind two receptors on peripheral nociceptors, trkA and p75, which then activate a signaling pathway of intracellular kinases that eventually lead to neurite outgrowth and sensitization of these neurons (Hefti et al., 2006).

[0006] Neurotrophin–3 (NT–3), is a neurotrophin that is structurally related to beta–NGF, BDNF, and NT–4, and that controls survival and differentiation of mammalian neurons and the maintenance of the adult nervous system and may affect development of neurons in the embryo when it is expressed in human placenta. Conditions and symptoms associated with NT3 gene expression are known. NT3–deficient mice generated by gene targeting, display severe movement defects of the limbs. NT–3 signals through the Trk receptors and promotes the growth and survival of nerve and glial cells (see, e.g., Protein Sequence NP_001096124.1 and NP_032768).

[0007] The amino acid sequences of human and murine NT–3 are identical. NT–3 and its cognate receptor, tyrosine kinase C (TrkC), modulate neuropathic pain and nociceptive pain and the mechanism of nociception and proprioception. For example, NT3 expression is increased in the small DRG cells of neuropathic animals. NT–3 expression is also associated with neuropathies such as diabetic polyneuropathy and HIV–related neuropathy, large fiber neuropathy including atrophy, it is further involved in the development of hyperalgesia (a decrease in the threshold of a normally noxious stimuli), allodynia (a non–noxious stimulus becomes noxious), and spontaneous pain (pain in the apparent absence stimuli) and is a known modulator of muscle pain.

[0008] Excess neurotrophins (NTs) are implicated in OA and other painful conditions. Previous OA therapies selectively targeting NGF provided analgesia but were placed on clinical hold by the U.S. Food and Drug Administration in 2010 due to the emergence of significant joint pathologies. Reports of serious joint adverse events (AEs) due to osteonecrosis were noted during clinical trials of the anti–NGF antibodies, Tanezumab (Pfizer / Lilly) and Fasinumab (Regeneron / Sanofi) (Schnitzer, supra).

[0009] Other drugs for the treatment of chronic pain include non–steroidal anti– inflammatory drugs, anticonvulsant drugs, and opioids. However, these drugs have been found to have limited efficacy in clinical applications and severe side effects to patient health. For example, besides limited pharmacological efficacy, non–steroidal anti–inflammatory drugs’ side effects include gastrointestinal hemorrhage and nephrotoxicity, while the side effect of opioids is addiction (Hochberg, 2015).Attorney Docket No. LEVI0008-401-PC

[0010] The development of non–surgical interventions that are more effective and associated with fewer untoward effects than existing therapies remains a priority (Humphreys et al., 2022). Thus, there is an unmet clinical need for non–opioid, non–toxic and not–easy– abuse new drugs for treatment of acute and chronic pain. SUMMARY

[0011] Disclosed herein are methods of treatment of pain associated with osteoarthritis, pain associated with osteonecrosis, and pain associated with one or more subchondral insufficiency fractures; methods of improving physical function in a human subject with osteoarthritis, osteonecrosis, or one or more subchondral insufficiency fractures; and methods of treating osteoarthritis, or delaying or slowing the progression of osteoarthritis, or relieving a symptom of osteoarthritis, in humans with these diseases, comprising administering a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein. Compound E3 is such a novel p75NTR–Fc fusion protein and neurotrophin–3 inhibitor, which supplements endogenous p75NTR, thereby modulating neurotrophin levels, and inhibiting NT3. The structure of Compound E3 and its alternatives and glycosylated variants are detailed herein.

[0012] Provided herein are the following non-limiting embodiments. Other embodiments are disclosed throughout the specification.

[0013] In some embodiments, the present disclosure provides a method of treating pain associated with osteoarthritis, comprising administering to a human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions wherein after treatment with the composition, the subject is a responder to treatment by at least one measure of clinical response.

[0014] In some embodiments, the present disclosure provides a method of treating pain associated with osteonecrosis, comprising administering to a human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising:Attorney Docket No. LEVI0008-401-PC a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions.

[0015] In some embodiments, the present disclosure provides a method of treating pain associated with one or more subchondral insufficiency fractures, comprising administering to a human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, where x is 1, 2, 3, 4, 5 or 6 connecting the p75NTR(NBP) and Fc portions.

[0016] In some embodiments, the present disclosure provides a method of improving physical function in a human subject with osteoarthritis, osteonecrosis, or one or more subchondral insufficiency fractures, comprising administering to the human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions.

[0017] In some embodiments, the present disclosure provides a method of treating osteoarthritis, or delaying or slowing the progression of osteoarthritis, or relieving a symptom of osteoarthritis, in a human subject, comprising administering to the human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; andAttorney Docket No. LEVI0008-401-PC a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions.

[0018] Also provided herein are equivalent: Compositions comprising the fusion proteins disclosed above and herein for use in the indications and diseases disclosed in the methods recited above; Uses of the compositions comprising the fusion proteins disclosed above and herein in the treatment and effecting of changes in the indications and diseases disclosed in the methods recited above; and Uses of the compositions comprising the fusion proteins disclosed above and herein in the manufacture of medicaments for the treatment and effecting of changes in the indications and diseases disclosed in the methods recited above; including any doses and other limitations. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG.1A is a graph of the efficacy endpoints where the downward–facing arrow indicates the primary endpoint. Statistical significance was met for the primary and secondary efficacy endpoints at wk5 and wk17 where the mean change from baseline for patient– reported pain (y–axis) was plotted against time in weeks (x–axis).

[0020] FIG.1B is a graph of the efficacy endpoints. Statistical significance was met for the primary and secondary efficacy endpoints at wk5 and wk17 where the mean change from baseline for patient–reported physical function (y–axis) was plotted against time in weeks (x– axis).

[0021] FIG.1C is a graph of the efficacy endpoints. Statistical significance was met for the primary and secondary efficacy endpoints at wk5 and wk17 where the mean change from baseline for patient global assessment for OA (PGA–OA) (y–axis) was plotted against time in weeks (x–axis).

[0022] FIG.2 is an overview of the Study Schema showing a schedule of events.

[0023] FIG.3 describes the forms of glycosylation and the accepted notation for describing them. Some common glycans in the Fc and binding domain regions of protein, which are abbreviated as G0F, G1F, G2FS2, G3F and G2FS1 to indicate differences in monosaccharide composition, according to nomenclature provided by the Consortium for Functional Glycomics (CFG).

[0024] FIG.4 shows an example from the Symbol Nomenclature for Glycans (SNFG), another standard for depicting glycans but using colored shapes and text.Attorney Docket No. LEVI0008-401-PC

[0025] FIG.5 shows SEQ ID NO:1 and sites of O-Linked and N-Linked glycosylation.

[0026] FIG.6 shows a table indicating O-linked and N-linked glycosylation in SEQ ID NO:1. DETAILED DESCRIPTION

[0027] Compound E3(p75NTR–Fc) is a chimeric fusion protein that combines the Fc fragment of human immunoglobulin G1 (IgG1) with the p75 neurotrophin receptor (p75 NTR).

[0028] In some embodiments of the disclosure, Compound E3 acts as a neurotrophin–3 (NT–3) inhibitor that restores neurotrophin homeostasis and provides profound analgesia without use–limiting side effects, including rapid progression of osteoarthritis observed with anti–NGF antibodies. Without being bound by theory, it is hypothesized that Compound E3 provides analgesia via inhibition of NT–3 activity and returns neurotrophin homeostasis by supplementing the endogenous p75NTR binding protein to scavenge excess neurotrophins present in chronic pain states. Compound E3 retains the important trophic effects of the neurotrophins, including joint re–modelling.

[0029] After a protein is expressed in the cell it goes through a process called post translational modification (PTM). Molecules of sugars and carbohydrates attach themselves to the protein (a process called glycosylation). Example PTMs include phosphorylation, sulfation, sialic acid acetylation, and / or addition of LacdiNAc repeats. Protein molecules that are synthesized and secreted via the ER-Golgi pathway can be subjected to ER modifications such as O-fucosylation and O-mannosylation, with important effects on facilitating proper folding in the ER lumen. A major fraction of such ER-synthesized proteins are also modified by N-linked glycans at Asn-X-Ser / Thr sequons, and the attached large, hydrophilic sugar chains contribute to proper folding of nascent polypeptides emerging into the lumen of the ER. For example, preventing N-linked glycosylation using the inhibitor tunicamycin can have negative effects on the initial folding of such proteins. Heavily glycosylated proteins are protected from protease cleavage by glycans, by steric hindrance or negative charge. Glycosylation at single sites can regulate specific cleavage events with large impacts on protein activity.

[0030] Proteins tend to fold themselves into a particular conformation. Glycosylation can influence the way the protein folds, or act as a steric block, changing the ability of the protein to bind to other molecules in the body. Accordingly, glycosylation can have a great effect on the ability of a protein to be used for a particular indication. There can be variation in theAttorney Docket No. LEVI0008-401-PC glycosylated protein depending on the process by which the peptide has been expressed. Factors can include the cell from which the protein is expressed and the environmental conditions such as nutrients, oxygen levels and lysis. The choice of a particular cell to culture and the specific fermentation conditions can result in a protein that is more appropriate for use. Various forms of glycosylation and the accepted notation for describing them is shown in FIG.2, FIG.3 and FIG.4.

[0031] Due to the branching of the chains and post– translational modifications, their structures are very complex and difficult to characterize. The glycosylated protein will likely be a mixture due to differences in the amount of glycosylation; both in terms of the length of the various glycosylation chain and variation in the sites at which glycosylation can take place.

[0032] It is the convention to number the constituent amino acids of a protein from the N terminus to the COOH terminus. Particularly preferred embodiments include glycosylated proteins of SEQ ID NO:1, further characterized by glycosylation occurring at specific amino acid sites. Typically, glycosylation occurs on the side chain of an amino acid constituent of the protein. Sugars bind to either the N or O containing side chains (or both), such as the CH2C(O)NH2 side chain which defines asparagine, the CH2OH side chain which defines serine, and / or CH(OH)CH3 side chain which defines threonine. Within this specification, the N or O atom on the side chain will be referred to by the position of the constituent amino acid within the protein. Thus, the nitrogen-containing moiety on the side chain of the 32nd amino acid from the NH2 terminus of the constituent protein, will be referenced as ‘N32’.

[0033] In this context, N refers to an nitrogen containing moiety on the amino side chain and does not refer to a single letter amino acid code. In this context, specific amino acids will be referred to by three letter amino acid codes.

[0034] Some common glycans in mAb Fc are abbreviated as G0F, G1F, G2FS2, G3F & G2FS1 to indicate differences in monosaccharide composition.

[0035] The neutral N glycosylation profile in which sialic acid residues have been removed from the molecule ahead of analysis includes G0F (representing 19–32%, in some embodiments 20–25%, particularly In some embodiments 22–24% of the total neutral N– glycan population), G1F (representing 21–31%, in some embodiments 26–28% of the total neutral N–glycan population), G3F (representing 5–10%, in some embodiments 7–8% of the total neutral N–glycan population), G2FS1 representing 2–8%, in some embodiments 4–6% of the total neutral glycan population; and G2FS2 representing 10–20%, in some embodiments 14–17% of the total neutral glycan population.Attorney Docket No. LEVI0008-401-PC

[0036] In one embodiment, the protein is distinguished by glycosylation at both the N moieties, ‘N32’ and ‘N294’.

[0037] In some embodiments, the glycosylated protein is glycosylated at ASN32.

[0038] In some embodiments, the glycosylated protein is glycosylated at ASN294.

[0039] In some embodiments, the predominant glycan structure at ASN32 is G2FS2.

[0040] In some embodiments, the glycan structure at ASN294 is G1F.

[0041] In some embodiments, the glycan structure at ASN294 is G0F.

[0042] In some embodiments, the glycosylated protein is a mixture of glycosylation where the glycan structure at ASN294 are G0F and G1F.

[0043] In further embodiments the glycan’s at the N32 site have a MW of 1565or 3857 Da. The glycan’s at the N294 site have a molecular weight of 1419 or 2471 Da.

[0044] There are multiple constituent amino acids of the protein of SEQ ID NO:1, which possess a hydroxyl group on the side chain. In further embodiments, the peptide is characterized by glycosylation at between 1 and 12 of the OH containing amino acid side chains.

[0045] In another embodiment the N glycosylated protein also has O–linked glycosylation at 1 to 6 sites on the protein, in particular serine and threonine. In further embodiments, the peptide is characterized by O-linked glycosylation at between 1 and 12 of the OH containing amino acid side chains, in particular sides chains of serine and / or threonine residues.

[0046] In another embodiment wherein the protein is N-glycosylated, in particular as described in suitable embodiments described hereinabove, the N glycosylated protein also has O-linked glycosylation at 1 to 6 sites on the glycosylated protein, in particular at between 1 and 6 of the OH containing amino acid side chains, in particular sides chains of serine and / or threonine residues.

[0047] In another embodiment, there is O-linked glycosylation at 3 sites on the glycosylated protein.

[0048] In another embodiment, O linked glycosylation occurs at specific amino acids. Numbering the amino acids from N-terminus of the protein, with K being amino acid residue 1, O- linked glycosylation occurs at O44, O169, O171, O172, O179, O180, O183, O184, O198, O205, O206, ie the oxygen containing moiety on the side chain of amino acid residues 44, 169, 171, 172, 179, 180, 183, 184, 198, 205, 206, in particular the OH containing amino acid side chains, particularly sides chains of serine and / or threonine residues.

[0049] In a further embodiment, O-linked glycosylation occurs at, at least 1 site, in some embodiments 3 sites, in some embodiments 6 sites, in some embodiments all of these sites.

[0050] In a yet further embodiment, O-linked glycosylation occurs at position 169. This is independent of glycosylation at any other sites.Attorney Docket No. LEVI0008-401-PC

[0051] One of the glycans that can be found at an O–linked position is HexNAc1Neu5NAc1Hex1.

[0052] Another of the glycans that can be found at an O–linked position is HexNAc1Neu5NAc2Hex1.

[0053] The O-linked glycans can be sialylated with 1-2 sialic acid molecules.

[0054] In suitable embodiments, the peptide of the present disclosure is characterized by glycosylation at the N containing sidechain on the amino acids at N32, in particular the side chain of an asparagine (Asn) amino acid residue, and N294 in particular the side chain of an asparagine (Asn) amino acid residue and at between 1 and 12 of the OH containing amino acid sidechains, in particular the side chain of a serine (Ser) amino acid residue and / or threonine (Thr) residue.

[0055] In particular, the present disclosure is characterized by peptide of Seq No ID 1 with glycosylation at the N32 site, Asn32,, the N294 site, Asn294, and between 1 and 6 of the OH containing amino acid side chains, in particular the side chain of a serine (Ser) amino acid residue and / or threonine (Thr) residue.

[0056] In suitable embodiments the peptide of Seq ID No 1 with glycosylation at the Asn32 site, the Asn294 site and the between 1 and 3 of the OH containing amino acid side chains, in particular the side chain of a serine (Ser) amino acid residue and / or threonine (Thr) residue..

[0057] In suitable embodiments the peptide of Seq ID No1 with glycosylation at the Asn32 site, the Asn294 site and 3 of the OH containing amino acid side chains, in particular the side chain of a serine (Ser) amino acid residue and / or threonine (Thr) residue.

[0058] In an embodiment the disclosure provides a glycosylated protein wherein 90% of the glycosylated protein has a MW of 109kDa, the glycosylated protein is glycosylated at Asn294 where the glycan structures at Asn294 are G0F and G1F; and glycosylated at Asn32 where the predominant glycan structure is G2FS1 and G2FS2.

[0059] In another embodiment the disclosure provides a glycosylated protein wherein 90% of the glycosylated protein has a MW of 109kDa, the glycosylated protein is glycosylated at Asn294 where the glycan structures at Asn294 are G0F and G1F; and glycosylated at ASN32 where the predominant glycan structure is G2FS1 and there is O- linked glycosylation at 3 sites on the glycosylated protein, in particular at 3 of the OH containing amino acid side chains, in particular sides chains of serine and / or threonine residues.Attorney Docket No. LEVI0008-401-PC

[0060] In another embodiment the disclosure provides a glycosylated protein wherein 90% of the glycosylated protein has a MW of 109kDa, the glycosylated protein is glycosylated at

[0061] Asn294 where the glycan structures at Asn294 are G0F and G1F; and glycosylated at Asn32 where the predominant glycan structure is G2FS1 and there is O-linked glycosylation at 3 sites on the glycosylated protein, in particular at 3 of the OH containing amino acid side chains, in particular sides chains of serine and / or threonine residues; wherein the O-linked glycans comprise: HexNAc1Neu5NAc1Hex1 and HexNAc1Neu5NAc2Hex1.

[0062] The present invention provides for a molecule of Seq ID no 1, with both N and O linked glycosylation. Fig 2 and below, show Seq ID no 1, with N linked glycosylation sites shown in bold and O linked glycosylation sites shown underlined and in italics:with sequence ID No1 with glycosylation at one or more of positions 32, 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, 222 & 294.

[0064] The present invention further provides for a molecule, having 95% or greater homology with of sequence ID 3 with glycosylation at one or more of positions 32, 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, 222 & 294.

[0065] The present invention provides for a molecule of sequence ID 3 with glycosylation at one or more of positions 32, 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, 222 & 294

[0066] The present invention provides for a molecule as described above, with glycosylation at one of more of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206

[0067] In some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 & 16 of the positions.Attorney Docket No. LEVI0008-401-PC

[0068] In some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 & 11 of the positions.

[0069] The present invention provides for a molecule as described above wherein there is glycosylation at 1-3 of the positions

[0070] The present invention provides for a molecule as described above wherein there is glycosylation at 1-6 of the positions

[0071] The present invention provides for a molecule as described above wherein there is glycosylation at 3-6 of the positions

[0072] The present invention provides for a molecule as described above wherein there is glycosylation at 1-9 of the positions

[0073] The present invention provides for a molecule as described above wherein there is glycosylation at 6-9 of the positions

[0074] The present invention provides for a molecule as described above wherein there is glycosylation at 6-12 of the positions

[0075] The present invention provides for a molecule as described above wherein there is glycosylation at 1-12 of the positions

[0076] The present invention provides for a molecule as described above wherein there is glycosylation at 1-16 of the positions

[0077] The present invention provides for a molecule as described above wherein there is glycosylation at 6-16 of the positions.

[0078] A molecule as described above wherein:

[0079] The N glycan at position 32 is selected from

[0080] F(6)A2G(4)2S(3)2, F(6)A2G(4)2S(3)1

[0081] In some embodiments glycan is F(6)A2G(4)2S(3)2

[0082] The Glycan at position 169 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0083] In some embodiments the glycan is HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1) (HexNAc(1)Hex (1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0084] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(2)

[0085] The Glycan at position 171 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0086] In some embodiments the glycan is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0087] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(2)Attorney Docket No. LEVI0008-401-PC

[0088] The Glycan at position 172 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0089] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(2)

[0090] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(1)

[0091] The Glycan at position 179 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0092] In some embodiments the glycan is selected from HexNAc(1)Hex(1) or HexNAc(1)Hex(1)NeuAc(2)

[0093] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(2)

[0094] The Glycan at position 180 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0095] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)NeuAc(1) (N1S1), HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0096] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(2)

[0097] The Glycan at position 183 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0098] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0099] In some embodiments the glycan is selected from HexNAc(1) HexNAc(1)Hex(1) and HexNAc(1)Hex(1)NeuAc(2)

[0100] The Glycan at position 184 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0101] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0102] In some embodiments the glycan is selected from HexNAc(1) , H1N1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0103] The Glycan at position 185 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0104] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0105] In some embodiments the glycan is selected from HexNAc(1) , H1N1, HexNAc(1)Hex(1)NeuAC(1) , HexNAc(1)Hex(1)NeuAc(2)Attorney Docket No. LEVI0008-401-PC

[0106] The Glycan at position 198 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0107] In some embodiments the glycan is selected from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0108] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0109] The Glycan at position 199 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0110] In some embodiments the glycan is selected from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0111] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0112] The Glycan at position 205 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0113] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0114] The Glycan at position 206 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0115] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0116] The Glycan at position 216 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0117] In some embodiments the glycan is selected from HexNAc(1) or HexNAc(1)Hex(1)

[0118] In some embodiments the glycan is HexNAc(1)

[0119] The Glycan at position 217 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0120] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0121] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) ,

[0122] The Glycan at position 222 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)Attorney Docket No. LEVI0008-401-PC

[0123] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0124] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(2)

[0125] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1)

[0126] In some embodiments the glycan is selected from HexNAc(1) , HexNA(1)Hex(1) ,

[0127] The N glycan at position 294 is selected from

[0128] F(6)A2, F(6)A2[6}G(4)1 & F(6)A2[3}G(4)1, F(6)A2G(4)2

[0129] In some embodiments the glycan is F(6)A2

[0130] The present invention describes molecules with every combination of the disclosed glycans at 1-16 of the described glycan sites.

[0131] In one form of the invention is described a molecule having 90% or greater homology with sequence ID No1, with glycosylation at position 32, 294 and at 1-11 of each of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206.

[0132] In one form of the invention is described a molecule having 95% or greater homology with sequence ID No1, with glycosylation at position 32, 294 and at 1-11 of each of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206.

[0133] In one form of the invention is described a molecule of sequence ID No1, with glycosylation at position 32, 294 and at 1-11 of each of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206.

[0134] In some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 & 11 of the positions.

[0135] In some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 1-6 of the positions

[0136] In some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 3-6 of the positions

[0137] In some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 1-9 of the positions

[0138] In some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 6-9 of the positions

[0139] In some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 7-10 of the positionsAttorney Docket No. LEVI0008-401-PC

[0140] In some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 9-9 of the positionsIn some embodiments the present invention provides for a molecule as described above wherein there is glycosylation at 6-11 of the positions

[0141] In one form of the invention is described a molecule having 90% or greater homology with sequence ID No1, with glycosylation at each of positions 169, 171, 172, 177, 179, 180, 183, 184, 198, 199, 205, 206.

[0142] In one form of the invention is described a molecule having 95% or greater homology with sequence ID No1, with glycosylation at each of positions 169, 171, 172, 177, 179, 180, 183, 184, 198, 199, 205, 206.

[0143] In one form of the invention is described a molecule of sequence ID No1, with glycosylation at each of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206

[0144] In a further form of the invention, is described a molecule having 90% or greater homology with sequence ID No1, with glycosylation at each of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, wherein

[0145] The Glycan at position 169 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0146] In some embodiments the glycan is HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1) (HexNAc(1)Hex (1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0147] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(2)

[0148] The Glycan at position 171 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0149] In some embodiments the glycan is selected from HexNAc(1), HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(2)

[0150] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(2)

[0151] The Glycan at position 172 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0152] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0153] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(1)

[0154] The Glycan at position 179 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0155] In some embodiments the glycan is selected from HexNAc(1)Hex(1) or HexNAc(1)Hex(1)NeuAc(2)Attorney Docket No. LEVI0008-401-PC

[0156] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(2)

[0157] The Glycan at position 180 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0158] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)NeuAc(1) (N1S1), HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0159] In some embodiments the glycan is HexNAc(1)Hex(1)NeuAc(2)

[0160] The Glycan at position 183 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0161] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0162] In some embodiments the glycan is selected from HexNAc(1) HexNAc(1)Hex(1) and HexNAc(1)Hex(1)NeuAc(2)

[0163] The Glycan at position 184 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0164] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0165] In some embodiments the glycan is selected from HexNAc(1) , H1N1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0166] The Glycan at position 185 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0167] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0168] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAC(1) , HexNAc(1)Hex(1)NeuAc(2)

[0169] The Glycan at position 198 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0170] In some embodiments the glycan is selected from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0171] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0172] The Glycan at position 199 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0173] In some embodiments the glycan is selected from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)Attorney Docket No. LEVI0008-401-PC

[0174] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0175] The Glycan at position 205 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0176] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0177] The Glycan at position 206 is selected from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2)

[0178] In some embodiments the glycan is selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0179] In some embodiments the molecule has 95% homology with Seq ID No.1

[0180] In some embodiments the molecule is Seq ID No 1

[0181] In a further form of the invention, is described a molecule having 90% or greater homology with sequence ID No1, with glycosylation at each of positions 169, 171, 172, 177, 179, 180, 183, 184, 198, 199, 205, 206, wherein

[0182] The Glycan at position 169 is HexNAc(1)Hex(1)NeuAc(2)

[0183] The Glycan at position 171 is HexNAc(1)Hex(1)NeuAc(2)

[0184] The Glycan at position 172 is HexNAc(1)Hex(1)NeuAc(1)

[0185]

[0186] The Glycan at position 179 is HexNAc(1)Hex(1)NeuAc(2)

[0187] The Glycan at position 180 is selected from HexNAc(1)Hex(1)NeuAc(2)

[0188] The Glycans at position 183 are selected from

[0189] HexNAc(1) HexNAc(1)Hex(1) and HexNAc(1)Hex(1)NeuAc(2)

[0190] The Glycans at position 184 are selected from HexNAc(1) , H1N1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0191] The Glycans at position 185 is selected from

[0192] HexNAc(1) , HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAC(1) , HexNAc(1)Hex(1)NeuAc(2)

[0193] The Glycans at position 198 are selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0194] The Glycans at position 199 are selected from HexNAc(1) , HexNAc(1)Hex(1) , HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0195] The Glycans at position 205 are selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)Attorney Docket No. LEVI0008-401-PC

[0196] The Glycans at position 206 are selected from HexNAc(1) , HexNAc(1)Hex(1) , N1S1, HexNAc(1)Hex(1)NeuAc(1) , HexNAc(1)Hex(1)NeuAc(2)

[0197] In some embodiments the molecule has 95% homology with Seq ID No.1

[0198] In some embodiments the molecule is Seq ID No 1

[0199] It’s understood that the molecule is expressed and that thus will result in molecules with the same amino acid backbone, but that there will be variation in the glycosylation seen, with the end product a mixture of different glycosylated forms of the invention.

[0200] The molecule may also be found as a dimer.

[0201] In another embodiment, the invention provides a glycosylated protein wherein 90% of the glycosylated protein has a MW of 109kDa, the protein is glycosylated at ASN294 where the glycan structures at ASN294 are G0F and G1F; and glycosylated at ASN32 where the predominant glycan structure is G2FS1 and there is O–linked glycosylation at 3 sites on the protein.

[0202] In another embodiment, the invention provides a glycosylated protein wherein 90% of the glycosylated protein has a MW of 109kDa, the protein is glycosylated at ASN294 where the glycan structures at ASN294 are G0F and G1F; and glycosylated at ASN32 where the predominant glycan structure is G2FS1 and there is O–linked glycosylation at 3 sites on the protein where the O–linked glycans comprise HexNAc1Neu5NAc1Hex1 and HexNAc1Neu5NAc2Hex1.

[0203] Glycosylation affects glycosylated protein binding, solubility, stability, pharmacokinetics, and pharmacodynamics (PK / PD), bioactivity and safety (e.g., immunogenicity). Importantly, Fc glycoforms impact antibody structure and effector functions. Thus, the glycosylation pattern of a therapeutic mAb is a critical quality attribute (CQA) that is frequently discussed and reviewed. Overall, glycan structural analysis and heterogeneity control are critical for the quality of all glycosylated therapeutic proteins and are especially important for biosimilar products, where the chemical similarity to the reference product can eliminate or decrease the scope of clinical studies needed for marketing approval.

[0204] The glycans on the molecule are covalently bound to sialic acid molecules. One of the effects of sialic acids is to add a charge to the molecule. The effect of charge on a molecule is twofold. First, in influences the folding of the molecule and its arrangement in space. The structure a molecule takes is key to its ability to bind, both in terms of how strongly it binds to a site and its selectivity for a site over other binding positions. Second, theAttorney Docket No. LEVI0008-401-PC charge itself has an effect on binding and can be a key determinant of a molecules half–life within the body.

[0205] There may be 16–17 sialic acid molecules on each glycosylated monomer of SEQ ID NO:1. In some embodiments, there are 16.3–16.9 sialic acid molecules on each glycosylated monomer of SEQ ID NO:1. In another embodiment there are 16.55–16.65 sialic acid molecules on each glycosylated monomer of SEQ ID NO:1. There may also be variations in the sialic acids bound to the glycan. The predominant sialic acid is N- Acetylneuraminic acid (Neu5Ac) and the minor version is N-glycolyl neuraminic acid (Neu5Gc). Neu5Gc is not expressed in humans and therefore there is an immunogenicity risk associated with this sialic acid. Having a low level is an important quality attribute for a therapeutic glycosylated protein. ABBREVIATIONS ACRAmerican College of Rheumatology Accurate Pain ReportingAPRAttorney Docket No. LEVI0008-401-PC SAPStatistical Analysis Plan SIFSubchondral Insufficiency Fractures

[0206] Allodynia is defined as pain that occurs in response to a stimulus that normally would not cause pain.

[0207] “Average daily NRS pain” refers to the calculated average pain level, as measured on a Numerical Rating Scale (NRS), across a day, typically determined by taking the mean of multiple pain ratings recorded throughout the day on a 0–10 scale where 0 represents “no pain” and 10 represents “worst possible pain.” (Walker et al., 2019).

[0208] A “clinically meaningful within–patient change threshold” for osteoarthritis refers to the smallest amount of improvement in a patient’s symptoms, as measured by a specific assessment tool (like the WOMAC scale), that is considered significant enough to represent a real, noticeable difference in their condition, usually determined by comparing their baseline score to their post–treatment score and considering factors like patient perception of improvement (Conaghan et al., 2022).

[0209] A Baseline NRS Pain score is derived from the Average Daily NRS Pain score collected on the last seven days of the Diary Run–In Period (Day 10 – Day 14) and at least one knee was required to meet the criteria as described in the inclusion criteria.

[0210] Hyperalgesia is defined as a condition that causes increased sensitivity to pain, or an extreme response to pain.

[0211] Patient–reported outcome measurements (PROMs) are standardized questionnaires that allow patients to self–report on their health status, quality of life, symptoms, and functional abilities. PROMs provide insights into a patient’s perception of their health and well–being, and are used to assess the impact of treatments, e.g., pain or from the patient’s perspective.

[0212] The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) is a patient–reported outcome measure (PROM) that assesses pain, stiffness, and functional impairment in patients with osteoarthritis (OA). WOMAC is regularly used in clinical trialsAttorney Docket No. LEVI0008-401-PC in order to facilitate the interpretation of OA clinical trial endpoints. Thus, WOMAC is a self–reported questionnaire that takes about 12 minutes to complete. It consists of 24 questions that are scored on a scale of 0–4, with higher scores indicating worse pain, stiffness, and functional limitations. WOMAC is often used to measure improvement after knee arthroplasty (KA) for OA or rheumatoid arthritis. It can be used to evaluate the effectiveness of various interventions to help improve rehabilitation. WOMAC has three subscales: pain (0–20 points), stiffness (0–8 points), and physical function (0–68 points). The scores for each subscale are added together to calculate a total WOMAC score. The WOMAC can be completed on paper, over the telephone, or on a computer.

[0213] A “WOMAC domain” refers to one of the three categories of questions on the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaire, which are: pain, stiffness, and physical function. These are the three areas of joint health that the WOMAC assesses in patients with osteoarthritis.

[0214] A ‘patient global assessment of OA’ (PGA–OA) is a scale that measures the impression of disease severity regularly used in OA clinical trials in combination with WOMAC.

[0215] To aid the interpretation of clinical trials in patients with OA of the hip or knee, a meaningful response to treatment has been defined for the individual patient level, for the patient population defined by patients with moderate–to–severe OA of the hip or knee. Clinically important differences in WOMAC scores have been estimated to be between 0.3– 1.3 for patients with OA of the hip and knee, between 0.5–3.4 for those with OA of the knee, and between 0.3–3.6 for those with OA of the hip, depending on the domain assessed (where the original standardized 0–100 scale was converted to 0–10 scale).

[0216] A StEPP pain intensity score is a numerical rating of a patient’s pain level on a scale from 0 to 10, where 0 represents “no pain” and 10 represents “worst possible pain,” typically used to assess pain severity within the StEPP (Stepped Care for Pain Management) framework (Anekar et al., 2023).

[0217] The terms ‘minimal clinically important difference’ (MCID) and ‘minimum important difference’ (MID) are defined as the smallest amount of change in a patient’s condition that is considered meaningful and beneficial enough to be considered clinically significant. MCID thus represents the smallest improvement a patient perceives as worthwhile and would lead to a change in their management (McGlothlin and Lewis, 2014).

[0218] The term ‘meaningful within–patient change’ (MWPC) refers to the threshold on a clinical outcome measure that represents a clinically significant change in a patient’sAttorney Docket No. LEVI0008-401-PC condition, signifying a noticeable improvement or worsening compared to their baseline state, as determined by considering their individual situation and not just comparing them to a group average.

[0219] MWPCs for WOMAC domains in patients with moderate–to–severe OA of the hip or knee have been estimated. It has been established that a 2–category change on PGA– OA corresponds to an estimated 25.0–32.5% change in WOMAC domains. Thus, an approximately 30% reduction in WOMAC is considered to be moderately clinically meaningful. An estimated 12.5–16.2% change in WOMAC domains corresponds to a 1– category change on PGA–OA, suggesting that these improvements may be minimally clinically meaningful. Thus, the estimated MWPC for the 3 WOMAC domains have been established from 0.84–1.16 (0–10 numerical rating scale) and from 12.50–16.23%, depending on study and domain, that corresponded to a 1–category change on PGA–OA. For a 2– category change those values were from 1.68–2.31 and from 25.01–32.46%, respectively.

[0220] The least squares (“LS”) mean change from baseline is a statistical estimate of the change in a mean, as well as the initial mean level and the change point (Hawkins et al., 1986).

[0221] Patient Global Assessment (PGA) refers to a unidimensional measure of the participant’s overall assessment of disease impact on a given day. A single question was asked of the participant, and they were to select a number on a 11–point scale from 0 (“Very good”) to 10 (“Very bad”).

[0222] An “oligosaccharide” is a glycan containing more than 1 monosaccharide with a defined length, typically up to 30-40 residues.

[0223] The term “pain” as used herein may include: (a) acute pain and / or spontaneous pain; (b) chronic pain and or on-going pain; (c) inflammatory pain including any one of arthritic pain, pain resulting from osteoarthritis or the like; (d) nociceptive pain; (e) neuropathic pain, including painful diabetic neuropathy or pain associated with post-herpetic neuralgia; (f) hyperalgesia; (g) allodynia; (h) central pain, pain resulting from injury or fractures; and other types of pain. Treatment of pain includes, but is not limited to, preventing, ameliorating, controlling, reducing incidence of, or delaying the development or progression of pain and / or a symptom of pain.

[0224] A “polysaccharide” is usually reserved for glycans containing ≥ 30 monosaccharides lacking defined length and having a repeating structure.Attorney Docket No. LEVI0008-401-PC

[0225] A “glycan” is a general term denoting all kinds of saccharides linked to each other or to an aglycone (non-carbohydrate). A glycan may be composed of, for example, hexoses such as glucose, fucose, mannose, fructose, and galactose; N-acetylglucosamine; N- acetylgalactosamine; neuraminic acid; and other monosaccharides. As used herein, a “glycan,” “glycan structure,” or “glycoform” is equivalent to “glycosylation” at a given position.

[0226] A “sequon” is a sequence of consecutive amino acids in a protein that can serve as the attachment site to a polysaccharide. An example sequon is the tripeptide sequence Asn- X-Ser / Thr, which is the recognition signal for N-glycosylation, where X can be any amino acid except proline.

[0227] An “O-Linked glycan” is attached post-translationally to proteins in the Golgi apparatus by single step additions of sugar from nucleotide sugar donors. O-linked glycans are attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains of a peptide chain. O-glycans can be on adjacent Ser / Thr residues. The placement of a proline residue at either -1 or +3 relative to the serine or threonine is favorable for O-linked glycosylation.

[0228] An “N-Linked glycan” is attached co-translationally to proteins in the endoplasmic reticulum (ER) of cells using pre-assembled lipid-linked oligosaccharide donors. An N-Linked glycan is attached to the amide nitrogen (N) in the side chain of asparagine (Asn) or arginine (Arg) side-chains in a sequon. It is not normally possible for adjacent Asn residues to be N-glycosylated, although they can be near each other in sequence. A common consensus sequence for N-glycosylation is –Asn–X–Ser / Thr–. Some glycoproteins have 1 or more N-glycans and lack O-glycans, and vice versa; some glycoproteins have numerous N- and O-glycans. N-linked glycosylation involves participation of a special lipid called dolichol phosphate.

[0229] The C–terminal portion of an immunoglobulin is referred to as the crystallizable fragment of an antibody following papain digestion and comprises the CH2 and CH3 domains and is thus referred to as the “Fc” domain. The Fc domain is the portion of the lg recognized by cell receptors, such as the FcR, and to which the complement–activating protein, Clq, binds. Although the boundaries of the Fc domain may vary, the human IgG heavy chain Fc domain, as defined herein, comprises residue E216 to its carboxyl–terminus of the CH3 domain (or the CH4 domain for lgM and lgE antibodies), wherein the numbering is in the EU format as set forth in Edelman. The term “Fc domain” may refer to this sequenceAttorney Docket No. LEVI0008-401-PC in isolation, or this sequence in the context of an antibody, antibody fragment, or Fc fusion protein.

[0230] As used herein, the term, “Fc” or “immunoglobulin Fc” or “Ig Fc” is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, in particular an immunoglobulin heavy chain constant region, or a portion thereof. Particularly the immunoglobulin Fc comprises 1) a CH1 domain, a CH2 domain, and a CH3 domain, optionally with an immunoglobulin hinge region, 2) a CH1 domain and a CH2 domain, optionally with an immunoglobulin hinge region, 3) a CH1 domain and a CH3 domain, optionally with an immunoglobulin hinge region, 4) a CH2 domain and a CH3 domain, optionally with an immunoglobulin hinge region or 5) a combination of two or more domains selected from but not limited to CH1, CH2 and CH3 optionally combined with an immunoglobulin hinge region. Particularly the immunoglobulin Fc comprises at least an immunoglobulin hinge region, a CH2 domain and a CH3 domain, and optionally a CH1 domain. Particularly the immunoglobulin Fc comprises or consists of an Fc or a portion of an Fc of an immunoglobulin of isotype including but not limited to IgG, IgM, IgA, IgD, IgE, more particularly, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, sIgA, more particularly IgG1, igG2 or IgG4, most particularly IgG1. Optionally the immunoglobulin Fc also comprises amino acid mutations, deletions, substitutions or chemical modifications which serve to minimize complement fixation or antibody-dependent cellular cytotoxicity or which improve affinity of binding to the Fc receptor.

[0231] Further particularly the immunoglobulin Fc comprises or consists of any of: (a) a CH2 domain or portion thereof and a CH3 domain or portion thereof, (b) a CH2 domain or portion thereof, or (c) a CH3 domain or portion thereof, wherein the immunoglobulin Fc or portion thereof is of isotype including but not limited to IgG, IgM, IgA, IgD, igE, further particularly, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, sIgA, more particularly, IgG, IgG2 or IgG4, most particularly IgG1.

[0232] Particularly the immunoglobulin Fc comprises or consists of the carboxy terminal region of an immunoglobulin heavy chain and may comprise the CH2 and / or CH3 domains, or parts thereof, from IgG, igA or IgD antibody isotypes, or the CH2 and / or CH3 and / or CH4 domains, or parts thereof from IgM or IgE. Particularly the immunoglobulin Fc comprises or consists of a fragment of the Fc, comprising mainly CH3 and a small portion of CH2, as is derivable by pepsin digestion of the immunoglobulin. Particularly the immunoglobulin Fc comprises or consists of the full Fc region, comprising CH2 and CH3, additionally connected to the hinge region which is a short segment of heavy chain connecting the CH1 and CH2 regions in the intact immunoglobulin, as may be produced by papain digestion of the immunoglobulin. Particularly the immunoglobulin hinge region comprises or consists of a hinge region or part of a hinge region derived from an IgG particularly human IgG, more particularly selected from but not limited to IgG1, IgG2, IgG3, or IgG4, most particularly IgG1 or is alternatively a species or allelic variant of the foregoing hinge region embodiments. The hingeAttorney Docket No. LEVI0008-401-PC region or a part of an immunoglobulin hinge region can be located at the C or N-terminal end of the Fc region, particularly at the N-terminal end.

[0233] In addition, the skilled person would understand that the therapeutic potential of the molecule of the present disclosure may be enhanced by the introduction of defined mutations in the crystallizable fragment (Fc) domains eg YTE (M252Y / S254T / T256E) and LS (M428L / N434S). Such techniques are well known to one skilled in the art. The effect of introducing such a mutation typically results in a molecule with increased half-life and prolonged duration of action. The effects of introducing the mutation are not limited to increased half life and duration of action.

[0234] The term “EU format as set forth in Edelman” refers to the residue numbering of a human IgGl EU antibody as described in (Edelman, 2004). The human IgG2 and human IgG4 residue numbering is also in the EU format (Dillon et al., 2008); (Aalberse and Schuurman, 2002); (Schlothauer et al., 2016). The EU numbering of residues may be determined by aligning an antibody at regions of homology to the sequence of the antibody with a “standard” EU numbered sequence. Unless indicated to the contrary, all residue numbering of constant regions herein will be according to EU numbering.

[0235] The amino acid sequence of a non–naturally occurring Fc domain (also referred to herein as a “variant Fc domain”) may comprise one or more amino acid modifications. For example, polymorphisms have been observed at a number of Fc domain positions, including but not limited to positions 270, 272, 312, 315, 356, and 358, according to EU numbering, and thus slight differences between the presented sequence and sequences in the prior art may exist. Mutations may also be introduced into the Fc domain. Thus, an Fc region may be a naturally occurring Fc region, or a naturally occurring Fc region in which one or more amino acids have been substituted, added or deleted, provided that the Fc region has the desired biological properties. A desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain. In some embodiments of this disclosure, there may be first and second Fc domains that comprise mutations, to promote heterodimer formation, for example.

[0236] The human constant region of the fusion protein set forth herein may comprise a heavy chain constant region chosen from lgG1, lgG2, lgG3, lgG4, igM, igA, igE, and any allelic variation thereof known in the art. Any one or more of such sequences may be used in the present disclosure. In some embodiments, the heavy chain constant region is chosen from lgG1 and allelic variations thereof.

[0237] As used herein, the term “host cell proteins” (HCP) includes proteins derived from a host cell and can be unrelated to the desired protein of interest. Host cell proteins can be aAttorney Docket No. LEVI0008-401-PC process–related impurity which can be derived from the manufacturing process and can include three major categories: cell substrate–derived, cell culture–derived and downstream derived. Cell substrate–derived impurities include, but are not limited to, proteins derived from a host organism and nucleic acid (host cell genomic, vector, or total DNA). Cell culture–derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream–derived impurities include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, nonmetallic ion), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables.

[0238] As used herein, the term “liquid chromatography” refers to a process in which a biological / chemical mixture carried by a liquid can be separated into components as a result of differential distribution of the components as they flow through (or into) a stationary liquid or solid phase. Non–limiting examples of liquid chromatography include reverse phase liquid chromatography, ion–exchange chromatography, size exclusion chromatography, affinity chromatography, mixed–mode chromatography or hydrophobic chromatography.

[0239] As used herein, “affinity chromatography” can include separations including any method by which two substances are separated based upon their affinity to a chromatographic material. It can comprise subjecting the substances to a column comprising a suitable affinity chromatographic media. Non–limiting examples of such chromatographic media include, but are not limited to, Protein A resin, Protein G resin, affinity supports comprising an antigen against which a binding molecule (e.g., antibody) was produced, protein capable of binding to a protein of interest and affinity supports comprising an Fc binding protein. In one aspect, an affinity column can be equilibrated with a suitable buffer prior to sample loading. An example of a suitable buffer can be a Tris / NaCl buffer, pH around 7.0 to 8.0. A skilled artisan can develop a suitable buffer without undue burden. Following this equilibration, a sample can be loaded onto the column. Following the loading of the column, the column can be washed one or multiple times using, for example, the equilibrating buffer. Other washes, including washes employing different buffers, can be used before eluting the column. The affinity column can then be eluted using an appropriate elution buffer. An example of a suitable elution buffer can be an acetic acid / NaCl buffer, pH around 2.0 to 3.5. Again, the skilled artisan can develop an appropriate elution buffer without undue burden. The eluate can be monitored using techniques well known to those skilled in the art, including UV. For example, the absorbance at 280 nm can be employed, especially if the sample of interest comprises aromatic rings (e.g., proteins having aromatic amino acids like tryptophan).Attorney Docket No. LEVI0008-401-PC

[0240] As used herein, “ion exchange chromatography” can refer to separations including any method by which two substances are separated based on differences in their respective ionic charges, either on the molecule of interest and / or chromatographic material as a whole or locally on specific regions of the molecule of interest and / or chromatographic material, and thus can employ either cationic exchange material or anionic exchange material. Ion exchange chromatography separates molecules based on differences between the local charges of the molecules of interest and the local charges of the chromatographic material. A packed ion–exchange chromatography column or an ion–exchange membrane device can be operated in a bind–elute mode, a flowthrough mode, or a hybrid mode. After washing the column or the membrane device with an equilibration buffer or another buffer, product recovery can be achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute can be another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution). Anionic or cationic substituents may be attached to matrices in order to form anionic or cationic supports for chromatography. Non–limiting examples of anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups.

[0241] As used herein, the term “recombinant protein” refers to a protein produced as the result of the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein can be a fusion protein. In a particular aspect, the recombinant protein is the expression product of SEQ ID NO:1. In certain exemplary embodiments, the recombinant protein can be a fusion protein comprising the constant domain of an antibody of an isotype selected from group consisting of: IgG, igM, igA1, igA2, igD, or IgE. In certain exemplary embodiments the constant domain is derived from a full–length antibody (e.g., an IgG1) or alternatively the antibody can be a fragment (e.g., an Fc fragment or a Fab fragment).

[0242] As used herein, the term “sequence variant” of a polypeptide (e.g., of a p75NTR– Fc fusion protein) refers to a polypeptide comprising an amino acid sequence that is at least about 70–99.9% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to SEQID NO:1. A sequence comparison can be performed by, for example, a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respectiveAttorney Docket No. LEVI0008-401-PC sequences over the entire length of the respective reference sequences (e.g., expect threshold: 10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment). Sequence variants of a polypeptide (e.g., of a p75NTR–Fc fusion) may also refer to a polypeptide comprising SEQ ID NO:1 except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for example, missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions.

[0243] Some variants may also be covalent modifications that polypeptides undergo, either during (co–translational modification) or after (post–translational modification “PTM”) their ribosomal synthesis. PTMs are generally introduced by specific enzymes or enzyme pathways. Many occur at the site of a specific characteristic protein sequence (e.g., signature sequence) within the protein backbone. Several hundred PTMs have been recorded, and these modifications invariably influence some aspect of a protein's structure or function (Walsh, G. “Proteins” (2014) second edition, published by Wiley and Sons, Ltd., iSBN: 9780470669853, the entire teachings of which are herein incorporated). In certain exemplary embodiments, a protein composition can comprise more than one type of protein variant of a protein of interest.

[0244] With respect to Compound 3 (and proteins sharing structural characteristics therewith) deamidation variants can comprise deamidation of asparagine residue at Asn84 and / or Asn99 (or equivalent residue positions on proteins sharing certain structural characteristics of Compound 3). In this respect, protein variants can include both acidic species and basic species. Acidic species are typically the variants that elute earlier than the main peak from CEX or later than the main peak from AEX, while basic species are the variants that elute later than the main peak from CEX or earlier than the main peak from AEX.

[0245] As used herein, the terms “acidic species,” “AS,” “acidic region,” and “AR,” refer to the variants of a protein which are characterized by an overall acidic charge. For example, in recombinant protein preparations such acidic species can be detected by various methods, such as ion exchange.

[0246] As used herein, the terms “oxidative species,” “OS,” or “oxidation variant” refer to the variants of a protein formed by oxidation. Such oxidative species can also be detected by various methods, such as ion exchange.

[0247] As used herein, the terms “basic species,” “basic region,” and “BR,” refer to the variants of a protein, which are characterized by an overall basic charge, relative to theAttorney Docket No. LEVI0008-401-PC primary charge variant species present within the protein. For example, in recombinant protein preparations, such basic species can be detected by various methods, such as ion exchange.

[0248] It is understood that the molecules of the present disclosure may have additional conservative or non–essential amino acid substitutions, which do not have a substantial effect on their functions. Table 1 shows exemplary conservative amino acid substitutions. In some embodiments, the antibody constant domain can comprise an amino acid sequence at least 90% identical to amino acids 234–332 of a human IgG1 antibody and differ by one or more substitution(s). Exemplary polypeptide chains of heavy chain constant regions of the IgG1, igG2, and IgG4 subclasses are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8 in Table 2. An example of an IgG1 mutated sequence has the amino acid sequence set forth in SEQ ID NO:6.Attorney Docket No. LEVI0008-401-PC Table 1. Conservative amino acid substitutions Original amino acid residue One and three–letter codes Conservative substitutionAttorney Docket No. LEVI0008-401-PC Table 2. Exemplary Constant and Fc Domains Identifier Description Sequence SEQ ID NO:4 IgG2 heavy chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS D T E I V V W S D D R T A N L D S N K T I S Q S N K T I S QAttorney Docket No. LEVI0008-401-PC Enumerated Embodiments

[0249] Provided herein are the following non-limiting embodiments. Other embodiments are disclosed throughout the specification.

[0250] Embodiment 1. A method of treating pain associated with osteoarthritis, comprising administering to a human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions wherein after treatment with the composition, the subject is a responder to treatment by at least one measure of clinical response.

[0251] Embodiment 2. A method of treating pain associated with osteonecrosis, comprising administering to a human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions.

[0252] Embodiment 3. A method of treating pain associated with one or more subchondral insufficiency fractures, comprising administering to a human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, where x is 1, 2, 3, 4, 5 or 6 connecting the p75NTR(NBP) and Fc portions.

[0253] Embodiment 4. A method of improving physical function in a human subject with osteoarthritis, osteonecrosis, or one or more subchondral insufficiency fractures, comprising administering to the human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising:Attorney Docket No. LEVI0008-401-PC a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions.

[0254] Embodiment 5. A method of treating osteoarthritis, or delaying or slowing the progression of osteoarthritis, or relieving a symptom of osteoarthritis, in a human subject, comprising administering to the human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions.

[0255] Embodiment 6. The method of any of embodiments 1-5, wherein the linker is GGG and the Fc is a human Fc.

[0256] Embodiment 7. The method of any of embodiments 1-6, wherein the p75NTR(NBP) portion has at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:3.

[0257] Embodiment 8. The method of embodiment 7, wherein the p75NTR(NBP) portion has SEQ ID NO:3.

[0258] Embodiment 9. The method of any of embodiments 1-9, wherein the fusion protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:1.

[0259] Embodiment 10. The method of embodiment 9, wherein the fusion protein has SEQ ID NO:1.

[0260] Embodiment 11. The method of any of embodiments 1-11, wherein the fusion protein has glycosylation at one or more of positions chosen from positions 32, 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, 222, and 294.

[0261] Embodiment 12. The method of any of embodiments 1-11, wherein the fusion protein has glycosylation at one or more of positions chosen from positions 169, 171, 172,179, 180, 183, 184, 198, 199, 205, and 206.

[0262] Embodiment 13. The method of any of embodiments 11-12, wherein the fusion protein has glycosylation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the positions.Attorney Docket No. LEVI0008-401-PC

[0263] Embodiment 14. The method of any of embodiments 11-12, wherein the fusion protein has glycosylation at 1-3, 1-6, 1-9, 1-12, 1-16, 3-6, 3-9, 3-12, 3-16, 6-9, 6-12, 6-16, 7- 9, 7-10, 8-9, 9-12, or 9-16 of the positions.

[0264] Embodiment 15. The method of embodiment 14, wherein the fusion protein has glycosylation at 6-12 of the positions.

[0265] Embodiment 16. The method of embodiment 14, wherein the fusion protein has glycosylation at 7-10 of the positions.

[0266] Embodiment 17. The method of embodiment 14, wherein the fusion protein has glycosylation at positions 32, 294, and 5-8 of the positions chosen from positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, and 222.

[0267] Embodiment 18. The method of embodiment 17, wherein the fusion protein has glycosylation at positions 32, 294, and 7 of the positions chosen from positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, and 222.

[0268] Embodiment 19. The method of embodiment 14, wherein the fusion protein has glycosylation at positions 32, 294, and 5-8 of the positions chosen from positions 169, 171, 172,179, 180, 183, 184, 198, 199, 205, and 206.

[0269] Embodiment 20. The method of embodiment 19, wherein the fusion protein has glycosylation at positions 32, 294, and 7 of the positions chosen from positions 169, 171, 172,179, 180, 183, 184, 198, 199, 205, and 206.

[0270] Embodiment 21. The method of any of embodiments 1-20, wherein the fusion protein comprises N-glycosylation (i.e., is an N-glycan variant).

[0271] Embodiment 22. The method of any of embodiments 1-21, wherein the fusion protein composition of embodiment 2, having has at least one glycan chosen from G0F, G1F, G3F, G2FS1 and G2FS2.

[0272] Embodiment 23. The method of embodiment 22, wherein a. G0F represents about 19% - about 32% of the total neutral N-glycan population, b. G1F represents about 21% - about 31%, of the total neutral N-glycan population, c. G3F represents about 5% - about 10% of the total neutral N-glycan population, d. G2FS1 represents about 2% - about 8% of the total neutral glycan population; and e. G2FS2 represents about 10% - about 20% of the total neutral glycan population.

[0273] Embodiment 24. The method of embodiment 23, wherein a. G0F represents about 20% - about 25% of the total neutral N-glycan population, b. G1F represents about 26% - about 28%, of the total neutral N-glycan population, c. G3F represents about 7% - about 8% of the total neutral N-glycan population,Attorney Docket No. LEVI0008-401-PC d. G2FS1 represents about 4% - about 6% of the total neutral glycan population; and e. G2FS2 represents about 14% - about 17% of the total neutral glycan population.

[0274] Embodiment 25. The method of embodiment 24, wherein a. G0F represents about 22% - about 24% of the total neutral N-glycan population, b. G1F represents about 26% - about 28%, of the total neutral N-glycan population, c. G3F represents about 7% - about 8% of the total neutral N-glycan population, d. G2FS1 represents about 4% - about 6% of the total neutral glycan population; and e. G2FS2 represents about 14% - about 17% of the total neutral glycan population.

[0275] Embodiment 26. The method of any of embodiments 1-25, wherein SEQ ID NO:1 is glycosylated at Asn32.

[0276] Embodiment 27. The method of embodiment 26, wherein the N-glycan at Asn32 is chosen from F(6)A2G(4)2S(3)2 and F(6)A2G(4)2S(3)1.

[0277] Embodiment 28. The method of embodiment 26, wherein the predominant glycan at Asn32 is G2FS2.

[0278] Embodiment 29. The method of embodiment 26 wherein the N glycan at Asn32 is F(6)A2G(4)2S(3)2.

[0279] Embodiment 30. The method of any of embodiments 1-29, wherein SEQ ID NO:1 is glycosylated at Asn294.

[0280] Embodiment 31. The method of any of embodiments 1-29, wherein SEQ ID NO:1 is glycosylated at Asn32 and Asn294.

[0281] Embodiment 32. The method of any of embodiments 30-31, wherein the glycan at Asn294 is G1F.

[0282] Embodiment 33. The method of any of embodiments 30-31, wherein the glycan at Asn294 is G0F.

[0283] Embodiment 34. The method of any of embodiments 30-31, wherein the glycan at Asn294 is chosen from F(6)A2, F(6)A2[6}G(4)1, and F(6)A2[3}G(4)1.

[0284] Embodiment 35. The method of any of embodiments 30-31, wherein the glycan at Asn294 is F(6)A2.

[0285] Embodiment 36. The method of any of embodiments 30-31, wherein the glycosylated fusion protein is a mixture of glycosylation, and wherein the glycan at Asn294 is chosen from G0F and G1F, or a mixture thereof.

[0286] Embodiment 37. The method of any of embodiments 1-36, wherein the glycans at any one or more of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216,Attorney Docket No. LEVI0008-401-PC 217, and 222 are chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0287] Embodiment 38. The method of any of embodiments 1-36, wherein the glycans at each of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, and 222, if present, are chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0288] Embodiment 39. The method of embodiment 38, wherein the glycan at position 169 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0289] Embodiment 40. The method of embodiment 38, wherein the glycan at position 169 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0290] Embodiment 41. The method of embodiment 38, wherein the glycan at position 169 is chosen from HexNAc(1) and HexNAc(1)Hex(1).

[0291] Embodiment 42. The method of embodiment 38, wherein the glycan at position 169 is HexNAc(1)Hex(1)NeuAc(2).

[0292] Embodiment 43. The method of any of embodiments 1-36, wherein the glycan at position 171 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0293] Embodiment 44. The method of embodiment 43, wherein the glycan at position 171 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0294] Embodiment 45. The method of embodiment 43, wherein the glycan at position 171 is chosen from HexNAc(1) and HexNAc(1)Hex(1).

[0295] Embodiment 46. The method of embodiment 43, wherein the glycan at position 171 is HexNAc(1)Hex(1).

[0296] Embodiment 47. The method of embodiment 43, wherein the glycan at position 171 is HexNAc(1)Hex(1)NeuAc(2).

[0297] Embodiment 48. The method of any of embodiments 1-36, wherein the glycan at position 172 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0298] Embodiment 49. The method of embodiment 48, wherein the glycan at position 172 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).Attorney Docket No. LEVI0008-401-PC

[0299] Embodiment 50. The method of embodiment 48, wherein the glycan at position 172 is chosen from HexNAc(1) and HexNAc(1)Hex(1).

[0300] Embodiment 51. The method of embodiment 48, wherein the glycan at position 172 is HexNAc(1)Hex(1).

[0301] Embodiment 52. The method of embodiment 48, wherein the glycan at position 172 is HexNAc(1)Hex(1)NeuAc(1).

[0302] Embodiment 53. The method of any of embodiments 1-36, wherein the glycan at position 179 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0303] Embodiment 54. The method of embodiment 53, wherein the glycan at position 179 is chosen from HexNAc(1)Hex(1) and HexNAc(1)Hex(1)NeuAc(2).

[0304] Embodiment 55. The method of embodiment 53, wherein the glycan at position 179 is HexNAc(1)Hex(1).

[0305] Embodiment 56. The method of embodiment 53, wherein the glycan at position 179 is HexNAc(1)Hex(1)NeuAc(2).

[0306] Embodiment 57. The method of any of embodiments 1-36, wherein the glycan at position 180 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0307] Embodiment 58. The method of embodiment 57, wherein the glycan at position 180 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2) .

[0308] Embodiment 59. The method of embodiment 57, wherein the glycan at position 180 is HexNAc(1)Hex(1)NeuAc(2).

[0309] Embodiment 60. The method of any of embodiments 1-36, wherein the glycan at position 183 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0310] Embodiment 61. The method of embodiment 60, wherein the glycan at position 183 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0311] Embodiment 62. The method of embodiment 60, wherein the glycan at position 183 is chosen from HexNAc(1), HexNAc(1)Hex(1), and HexNAc(1)Hex(1)NeuAc(2).

[0312] Embodiment 63. The method of embodiment 60, wherein the glycan at position 183 is chosen from HexNAc(1) and HexNAc(1)Hex(1).Attorney Docket No. LEVI0008-401-PC

[0313] Embodiment 64. The method of any of embodiments 1-36, wherein the glycan at position 184 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0314] Embodiment 65. The method of embodiment 65, wherein the glycan at position 184 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2) .

[0315] Embodiment 66. The method of embodiment 65, wherein the glycan at position 184 is chosen from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0316] Embodiment 67. The method of any of embodiments 1-36, wherein the glycan at position 185 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0317] Embodiment 68. The method of embodiment 67, wherein the glycan at position 185 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2) .

[0318] Embodiment 69. The method of embodiment 67, wherein the glycan at position 185 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0319] Embodiment 70. The method of any of embodiments 1-36, wherein the glycan at position 198 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0320] Embodiment 71. The method of embodiment 70, wherein the glycan at position 198 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0321] Embodiment 72. The method of embodiment 70, wherein the glycan at position 198 is chosen from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2) .

[0322] Embodiment 73. The method of embodiment 70, wherein the glycan at position 198 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0323] Embodiment 74. The method of any of embodiments 1-36, wherein the glycan at position 199 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).Attorney Docket No. LEVI0008-401-PC

[0324] Embodiment 75. The method of embodiment 74 wherein the glycan at position 199 is chosen from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0325] Embodiment 76. The method of embodiment 74, wherein the glycan at position 199 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0326] Embodiment 77. The method of any of embodiments 1-36, wherein the glycan at position 205 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0327] Embodiment 78. The method of embodiment 77, wherein the glycan at position 205 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0328] Embodiment 79. The method of any of embodiments 1-36, wherein the glycan at position 206 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0329] Embodiment 80. The method of embodiment 79, wherein the glycan at position 206 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0330] Embodiment 81. The method of any of embodiments 1-36, wherein the glycan at position 216 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0331] Embodiment 82. The method of embodiment 81, wherein the glycan at position 216 is chosen from HexNAc(1) and HexNAc(1)Hex(1).

[0332] Embodiment 83. The method of embodiment 81, wherein the glycan at position 216 is HexNAc(1).

[0333] Embodiment 84. The method of any of embodiments 1-36, wherein the glycan at position 217 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0334] Embodiment 85. The method of embodiment 84, wherein the glycan at position 217 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0335] Embodiment 86. The method of embodiment 84, wherein the glycan at position 217 is chosen from HexNAc(1), HexNAc(1)Hex(1), and HexNAc(1)Hex(1)NeuAc(1).Attorney Docket No. LEVI0008-401-PC

[0336] Embodiment 87. The method of any of embodiments 1-36, wherein the glycan at position 222 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0337] Embodiment 88. The method of embodiment 87, wherein the glycan is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

[0338] Embodiment 89. The method of embodiment 87, wherein the glycan at position 222 is chosen from HexNAc(1), HexNAc(1)Hex(1), and HexNAc(1)Hex(1)NeuAc(2).

[0339] Embodiment 90. The method of embodiment 87, wherein the glycan at position 222 is chosen from HexNAc(1), HexNAc(1)Hex(1), and HexNAc(1)Hex(1)NeuAc(1).

[0340] Embodiment 91. The method of embodiment 87, wherein the glycan at position 222 is chosen from HexNAc(1) and HexNA(1)Hex(1).

[0341] Embodiment 92. The method of any of embodiments 1-91, wherein the fusion protein comprises O-glycosylation (i.e., is an O-glycan variant).

[0342] Embodiment 93. The method of embodiment 92, wherein there is O-linked glycosylation at 6-12, 7-10, 5-8 or 7 sites on SEQ ID NO:1 or SEQ ID NO:3.

[0343] Embodiment 94. The method of embodiment 92, wherein an O-linked glycan is HexNAc1Neu5NAc1Hex1.

[0344] Embodiment 95. The method of embodiment 92, wherein an O linked glycan is HexNAc1Neu5NAc2Hex1.

[0345] Embodiment 96. The method of any of embodiments 92-95, wherein an O- linked glycan is sialylated with 1-2 sialic acid molecules.

[0346] Embodiment 97. The method of any of embodiments 92-96, containing 16-17 moles of sialic acid molecules to every mole of the monomer of SEQ ID NO:1 or SEQ ID NO:3.

[0347] Embodiment 98. The method of any of embodiments 92-96, wherein there are 16.55-16.65 sialic acid molecules on each glycosylated monomer of SEQ ID NO:1 or SEQ ID NO:3.

[0348] Embodiment 99. The method of any of embodiments 96-98, wherein the sialic acids are chosen from N-Acetylneuraminic acid (Neu5Ac) and N-glycolyl neuraminic acid (Neu5Gc).

[0349] Embodiment 100. The method of embodiment 99, wherein there are 16.1-16.35 molecules of Neu5Ac for each monomer of SEQ ID NO:1 or SEQ ID NO:3.Attorney Docket No. LEVI0008-401-PC

[0350] Embodiment 101. The method of embodiment 99, wherein there are 0.255-0.265 molecules of Neu5G for each monomer of SEQ ID NO:1 or SEQ ID NO:3.

[0351] Embodiment 102. The method of any one of embodiments 1-101, wherein the composition is administered according to a dosing regimen comprising one or more dosing cycles.

[0352] Embodiment 103. The method of embodiment 102, wherein the dosing cycle is a 4-week dosing cycle comprising a single administration.

[0353] Embodiment 104. The method of embodiment 102, wherein the dosing regimen comprises 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, or 48 weeks of treatment.

[0354] Embodiment 105. The method of embodiment 102, the dosing regimen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ,11, or 12 dosing cycles.

[0355] Embodiment 106. The method of any one of embodiments 102-105, wherein administration comprises at least about 0.1 mg / kg, at least about 0.3 mg / kg, at least about 1.0 mg / kg, or at least about 2.0 mg / kg of the composition.

[0356] Embodiment 107. The method of any one of embodiments 102-105, wherein administration comprises about 0.1 mg / kg to about 2.0 mg / kg, about 0.1 mg / kg to about 1.0 mg / kg, or about 0.1 mg / kg to about 0.3 mg / kg of the composition.

[0357] Embodiment 108. The method of any one of embodiments 102-105, wherein administration comprises about 0.1 mg / kg, about 0.3 mg / kg, about 1.0 mg / kg, or about 2.0 mg / kg of the composition.

[0358] Embodiment 109. The method of any of embodiments 1-108 wherein the administration is by intravenous injection or infusion.

[0359] Embodiment 110. The method of any of embodiments 1-109, wherein the glycosylated protein binds to any of NGF, BDNF, NT3 or NT4 / 5 with a binding affinity (Kd) of between about 0.001 nM to about 50 nM.

[0360] Embodiment 111. The method of any one of embodiments 1-110, wherein after treatment with the composition, the subject is a responder to treatment by at least one measure of clinical response.

[0361] Embodiment 112. The method of embodiment 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean WOMAC pain scores compared to placebo based on patient-reported outcomes measurements (PROMs).

[0362] Embodiment 113. The method of embodiment 112, wherein the reduction in mean WOMAC pain scores occurs after 1 week of treatment, 2 weeks of treatment, 3 weeksAttorney Docket No. LEVI0008-401-PC of treatment, 4 weeks of treatment, 5 weeks of treatment, 6 weeks of treatment, 7 weeks of treatment, 8 weeks of treatment, 9 weeks of treatment, 10 weeks of treatment, 11 weeks of treatment, 12 weeks of treatment, 13 weeks of treatment, 14 weeks of treatment, 15 weeks of treatment, 16 weeks of treatment, or 17 weeks of treatment.

[0363] Embodiment 114. The method of embodiment 111, wherein the at least one measure of clinical response is at least about a ≥30% reduction in mean WOMAC pain scores compared to placebo based on patient-reported outcomes measurements (PROMs) by week 5.

[0364] Embodiment 115. The method of embodiment 111, wherein the at least one measure of clinical response is at least about a ≥50% reduction in mean WOMAC pain scores compared to placebo based on patient-reported outcomes measurements (PROMs) by week 17.

[0365] Embodiment 116. The method of embodiment 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean WOMAC physical functionality scores compared to placebo based on patient-reported outcomes measurements (PROMs).

[0366] Embodiment 117. The method of embodiment 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean WOMAC stiffness scores compared to placebo based on patient-reported outcomes measurements (PROMs).

[0367] Embodiment 118. The method of embodiment 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean StEPP pain intensity scores compared to placebo based on patient-reported outcomes measurements (PROMs).

[0368] Embodiment 119. The method of embodiment 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean PGA scores compared to placebo based on patient-reported outcomes measurements (PROMs).

[0369] Embodiment 120. The method of embodiment 111, wherein the at least one measure of clinical response is at least about a ≥30% reduction in mean WOMAC physical functionality scores compared to placebo based on patient-reported outcomes measurements (PROMs) by week 5.

[0370] Embodiment 121. The method of embodiment 111, wherein the at least one measure of clinical response is at least about a ≥30% reduction in mean WOMAC stiffnessAttorney Docket No. LEVI0008-401-PC scores compared to placebo based on patient-reported outcomes measurements (PROMs) by week 5.

[0371] Embodiment 122. The method of embodiment 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) or clinically meaningful reduction in mean NRS scores compared to placebo from week 5 to week 20.

[0372] Embodiment 123. The method of any of embodiments 1-122, wherein the at least one measure of clinical response is a statistically significant (p<0.05) or clinically meaningful reduction in the average daily NRS area under the curve (AUC) results compared to placebo.

[0373] Embodiment 124. The method of any of embodiments 1-3 and 6-115, 118, 119, 122, and 123, wherein reducing pain comprises at least a 1-point difference, at least a 2-point difference, at least a 3-point difference, or at least a 4-point difference in mean change from a baseline WOMAC pain score.

[0374] Embodiment 125. The method of any of embodiments 1-3 and 6-115, 118, 119, 122, and 123, wherein reducing pain comprises a 1-point to 4-point difference, a 1-point to 3-point difference, a 1-point to 2-point difference, a 2-point to 4-point difference, a 3-point to 4-point difference, a 4-point difference, a 3-point difference, a 2-point difference, or a 4- point difference in mean change from a baseline WOMAC pain score.

[0375] Embodiment 126. The method of any of embodiments 1-125 comprising administering the composition as a 4-weekly dose, wherein the method is capable of achieving a statistically significant or clinically meaningful reduction in pain as evaluated by WOMAC, StEPP or NRS as compared to placebo by week 17.

[0376] Embodiment 127. The method of any of embodiments 1 and 4-126, wherein the osteoarthritis is osteoarthritis of the knee, hip, shoulder, hands, neck, and / or lower back

[0377] Embodiment 128. The method of embodiment 127, wherein the osteoarthritis is osteoarthritis of the knee.

[0378] Embodiment 129. The method of any of embodiments 1-128, wherein the composition is formulated for intravenous administration.

[0379] Embodiment 130. The method of any of embodiments 1-129, wherein the composition is administered to the subject separately, sequentially, or simultaneously in combination with a second pharmacologically active compound.

[0380] Embodiment 131. The method of any of embodiments 1 and 4-130, wherein relief from the symptoms of osteoarthritis comprises one or more of reduction in pain,Attorney Docket No. LEVI0008-401-PC inflammation, swelling, tenderness, joint stiffness, and increase in joint mobility, or any combination thereof.

[0381] Embodiment 132. The method of any of embodiments 1-131, wherein the composition comprises a glycosylated variant of SEQ ID NO:1 or SEQ ID NO:3, and the glycosylated variant shows selectivity for NT3 over NGF.

[0382] Embodiment 133. The method of embodiment 132, wherein the composition comprises a glycosylated variant of SEQ ID NO:1 or SEQ ID NO:3, and the glycosylated variant shows selectivity for NT3 over NGF on the order of 50-1000x over the NGF KD.

[0383] Embodiment 134. The method of any of embodiments 1-133, wherein the composition has increased inhibition of NT3 and decreased inhibition of NGF. EXAMPLES Example 1 − Primary Structure of p75NTR–Fc Fusion Protein

[0384] In an embodiment of the disclosure, Compound E3 comprises the extracellular domain of human p75 neurotrophin receptor (p75NTR) coupled to the constant fragment of human immunoglobulin G1 (IgG1) and may be referred to as a p75NTR neurotrophin binding protein (NBP)–Fc fusion protein.

[0385] In some embodiments, Compound E3 comprises the primary amino acid sequence represented by SEQ ID NO:1, having the following primary structure: >SEQ ID NO:1 KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMS APCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPC LPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVV TTVMGGGGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG p75NTR portion is aa 1–209 of SEQ ID NO:1 (underlined) Linker is aa 210–212 of SEQ ID NO:1 IgG1–Fc portion is aa 213–443 of SEQ ID NO:1 (bold)

[0386] In some embodiments, Compound E3 comprises a sequence variant of a polypeptide (e.g., of a p75NTR–Fc fusion protein). A sequence variant p75NTR–Fc fusion protein comprises an amino acid sequence that is at least about 70–99.9% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to SEQ ID NO:1. Another aspect of the present disclosure provides a glycosylated protein having 5% or more homology with the protein ofAttorney Docket No. LEVI0008-401-PC SEQ ID NO:1. A p75NTR neurotrophin binding protein (NBP)–Fc fusion protein may also comprise a p75NTR(NBP) portion, having 5% or more homology with SEQ ID NO:3, and an immunoglobulin Fc portion, wherein, the p75NTR(NBP) and Fc portions are connected via a linker, the linker comprising a peptide of formula Gx, where x is 1, 2, 3, 4, 5 or 6 and wherein the linker does not comprise or consist of the sequence GGGGS. >SEQ ID NO:3 KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMS APCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPC LPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVV TTVMG

[0387] The amino acid sequence represented by SEQ ID NO:3 corresponds to amino acid residues 1–209 of SEQ ID NO:1, which comprises the extracellular binding domain of p75NTR. The italicized residues correspond to the stalk region, which is highly glycosylated and exists between the extracellular ligand-binding domains and the cell membrane in the native protein. Primary Structure Determination

[0388] Primary structure was assessed by RP HPLC–UV reduced tryptic peptide mapping and RP HPLC–MS and MS / MS reduced tryptic peptide mapping.

[0389] Tryptic peptide mapping with ultraviolet (UV) detection showed a well resolved profile at both 210 and 280 nm.

[0390] RP HPLC–MS reduced tryptic peptide mapping confirmed 100% of the detected sequence as the expected sequence. MS / MS fragmentation analysis confirmed the identity of peptides representing 66% of the expected sequence.

[0391] The N–terminal tryptic peptides obtained were detected as the expected sequence. The first 17 residues of SEQ ID NO:1 were confirmed by MS / MS analysis.

[0392] C–terminal tryptic peptides were detected as the expected sequence.7 terminal residues were confirmed by MS analysis.

[0393] Low levels of potential deamidated variants were detected for tryptic peptides T7 and T31 (each at 1% relative % deamidation). An oxidized variant was detected for tryptic peptide T15 (3% relative % oxidation) and was confirmed by MS / MS analysis to be due to oxidation of a methionine residue in that peptide.

[0394] 100% occupancy of the N–linked glycosylation sites on Asparagine32 (Asn32) and (Asn294) was detected. Occupancy was determined from the ratio of glycosylated trypticAttorney Docket No. LEVI0008-401-PC peptide to aglycosylated tryptic peptide. Samples were digested individually with trypsin, endoproteinase Glu–C (Glu–C) and chymotrypsin in order to provide representation of the glycopeptides for SEQ ID NO:1.

[0395] Following trypsin digestion, G2F+NeuAc was the predominant glycan structure detected at the Asn32 N–linked glycosylation site and G1F was the predominant glycan structure at the Asn294 N–linked glycosylation site. Following Glu–C digestion, only the glycans at Asn294 were detected, with the predominant glycan being G0F.

[0396] At least three O–linked glycosylation sites were identified from the combined trypsin, Glu–C and chymotrypsin digest data.

[0397] Mass Isoform Heterogeneity

[0398] The molecular weight and mass isoform profile were determined by ESI–MS. SEQ ID NO:1 was denatured and reduced, followed by treatment with peptide–N– glycosidase F (PNGaseF) and O–glycanase to remove N–linked and O–linked glycans respectively. ESI–MS detected a predominant isoform at 48,310 Da, which is consistent with the theoretical deglycosylated single chain (reduced) mass of 48,312 Da. Two further single chain species were detected with masses of 48,675 Da and 49,042 Da. These were likely to represent single chain species that were incompletely deglycosylated.

[0399] Isoforms were also detected within the mass range of 96,621 Da to 97,352 Da. These species were comparable in mass to the monomeric deglycosylated product (theoretical mass 96,564 Da) and were likely to represent incompletely reduced and / or incompletely deglycosylated species. The range of isoforms detected in reduced and non–reduced samples suggested O–linked glycans were present after O–glycanase treatment. Example 2 − Secondary Structure of p75NTR–Fc Fusion Protein

[0400] In some embodiments, the present disclosure also provides a glycosylated protein of SEQ ID NO:1 and may be referred to as a glycosylated p75NTR neurotrophin binding protein (NBP)–Fc fusion protein. A glycosylated p75NTR neurotrophin binding protein (NBP)–Fc fusion protein may comprise a p75NTR(NBP) portion, having 5% or more homology with SEQ ID NO:1 or SEQ ID NO:3, and an immunoglobulin Fc portion, wherein, the p75NTR(NBP) and Fc portions are connected via a linker, the linker comprising a peptide of formula Gx, where x is 1, 2, 3, 4, 5 or 6 and wherein the linker does not comprise or consist of the sequence GGGGS. Secondary Structure DeterminationAttorney Docket No. LEVI0008-401-PC

[0401] Secondary structure was assessed by Fourier transform infra–red spectroscopy (FTIR). Results produced spectra that consisted predominantly of β–sheet (35.8%) with α– helix detected at 10.5%. Free Thiol

[0402] Free thiol (sulphydryl group) concentration, under native and denatured conditions, was determined. The free thiol content detected under native and denatured conditions was less than the limit of quantitation (LOQ) (194.02 mmol / mol). Mass Heterogeneity

[0403] Mass heterogeneity was determined by size exclusion chromatography with light scatter detection (SEC–LS). The main peak had an apparent apex molecular weight of 109 kDa and represented 99.4% relative peak area. This was consistent with monomeric, glycosylated SEQ ID NO:1. A high molecular weight species was detected with an apparent apex molecular weight of 221 kDa (0.6% relative peak area), consistent with a dimer of SEQ ID NO:1. Example 3 − Manufacturing Process

[0404] The active ingredient of Compound E3 is manufactured according to current Good Manufacturing Practice (GMP) standards and formulated in 25mM histidine, 50mM sodium chloride, 200mM mannitol, pH6.5. CHO cells and fermentation medium were obtained from Lonza. The materials necessary for MabSelect SuReTMAffinity Chromatography were obtained from Sigma Aldrich / GE Lifesciences. The materials necessary for POROS XS Cation Exchange Chromatography were obtained from ThermoFisher Scientific. Vector Construction and Development of Candidate Cell Lines

[0405] The process starts with codon optimization of the DNA sequence to allow efficient expression in CHO cell lines with a signal sequence at the N–terminus that directs the protein for secreted expression and appropriate restriction enzyme sites to enable cloning into cloning into the glutamine synthetase (GS) vectors and a Kozak sequence between the 5’ restriction enzyme site and the ‘ATG’ start codon. These sequences were submitted to and synthesized by Life Technologies and provided within a cloning vector. Other vectors with different secretion sequences and a different selectable marker (example dihydrofolate reductase (DHFR) instead of GS). The 1405 base pair (bp) DNA fragment encoding SEQ ID NO:1 was removed from the cloning vector into the GS expression vector pXC–17 via HindIII and EcoR1 digestion. The digested products (SEQ ID NO:1 DNA fragment and vector were ligated and the ligated products used to transform chemically competent Escherichia Coli (E.Attorney Docket No. LEVI0008-401-PC coli) cells (for example TOP10 cells). Single colonies were analyzed for the correct insert and one positive clone was selected and plasmid DNA prepared and sequenced (in forward reverse orientation) to ensure the correct sequence was present. The plasmid was renamed p SEQ ID NO:1 / SGV. Sufficient linearized DNA for the cell line construction process was generated by linearization of p SEQ ID NO:1 / SGV with the restriction enzyme PvuI.

[0406] Lonza Biologics’ mammalian Chinese Hamster Ovary (CHO) K1SV Glutamine Synthetase – Knock Out (GS–KO) expression system was used to produce SEQ ID NO:1. The CHOK1SV GS–KO host cell line is a derivative of the CHOK1SV host cell line with the endogenous gene for GS ‘knocked out’. The host cell line was derived from Lonza Biologics’ CHOK1SV GS–KO host working cell bank designated the code 760–W (prepared from the master host cell bank 760–M).

[0407] Other exemplary CHO Cell lines include for example CHO–DGB with a DHFR selectable marker on the expression vector. There are also other GS–CHO knock out cell lines available that could be used (for example CHOSOURCE GS KO (Horizon discovery) and CHOZN (MilliporeSigma). There are also other mammalian systems available (for example PER.C6® human cells (Crucell).

[0408] Transfection by electroporation was performed via a single pulse using linearized plasmid DNA to generate stable CHOK1SV GS–KO transfectant minipools expressing SEQ ID NO:1. CaPO4 or lipid–based reagents (Lipofectamine, Fugene, Transfectin) can also be used to transfect cells.

[0409] A number of transfections was performed and split into a larger number of minipools. The day after transfection, selective medium containing methionine sulphoximine (MSX) was added to each transfectant minipool. The addition of MSX, which inhibits GS, to the growth medium increases the selective pressure in the transfectant minipools; following cloning this selective pressure is no longer required. If one used the same vector with a CHOK1SV cell line, the selective pressure would need to be applied throughout the cell line development and included in the early growth steps (before the inoculation of the fermenter).

[0410] After ~14 days of incubation, >500 transfectant minipools are expanded to suspension culture in wells of shaken 96 deep well plates.3 days after transfer to suspension culture, samples from >500 wells were assayed for protein production using the Octet® method and the product concentration data generated were used to identify the overall highest producer transfectant minipools. Multiple high producing transfectant minipools were combined to generate >4 enriched transfectant pools.Attorney Docket No. LEVI0008-401-PC

[0411] The enriched transfectant pools were single cell sorted using a fluorescence activated cell sorter (FACS). Following incubation and imaging, supernatant samples from wells identified as containing a single colony were screened for SEQ ID NO:1 production.

[0412] >500 clonal cell lines were subsequently selected for further evaluation in an abridged fed–batch suspension culture productivity screen and transferred to suspension culture in shaken 96 deep well plates. Other methods can be used to derive monoclonal cell lines (for example a two–step dilution cloning in 96–well plates or using semi–solid media plates.

[0413] >500 clonal cell lines were successfully adapted to suspension culture and were screened for productivity in a fed batch media. Following this screen, 20 clonal cell lines were selected for further evaluation, based upon their productivity ranking and the parental transfectant pool from which they were derived; these were expanded to culture in shake– flasks and their growth during routine subculture assessed.9 lead candidate cell lines were selected for progression and cryopreservation of associated research cell banks (RCB). Selection was based on the expression levels of SEQ ID NO:1, acceptable growth characteristics and image evidence that each cell line arose from a single colony.

[0414] A 70–generation cell line stability study was initiated with all 9 lead candidate cell lines in which 2 independently subcultured lineages of each cell line were then established. The growth and productivity characteristics for each lineage were then evaluated at two points (~5 and ~35 generations beyond that of the associated RCBs) in fed–batch miniature bioreactor culture screens designed to mimic the cGMP manufacturing bioreactor culture process. Product from cultures of each of the 9 cell lines evaluated at ~5 generations was also assessed for product quality using a range of assays.

[0415] After a review of all available data, six lead candidate cell lines were selected for continuation in the 70–generation cell line stability study. Once the 6 cell lines had all accrued greater than seventy generations beyond the associated RCB, a final fed–batch miniature bioreactor culture screen was undertaken in which early and late generation cultures were evaluated concurrently.

[0416] Following evaluation of the consistency of the growth, productivity and product characteristics data across the 70–generation study, the lead cell line was chosen. A 200 vial GMP master cell bank was manufactured from a single vial of the relevant RCB and tested in accordance with current regulatory requirements. When sufficient cells were obtained, cells were aliquoted in cryopreservation medium (92.5 % CM66 / 7.5% DMSO) into polypropylene vials (each containing approximately 1.5 x 107 viable cells) and cryopreservedAttorney Docket No. LEVI0008-401-PC in a controlled manner to -100.0°C. Vials are stored in a vapor phase liquid nitrogen autofill dewar in a good manufacturing practice (GMP) controlled area. Inoculum Expansion

[0417] The molecule of the present disclosure may be expressed from Chinese Hamster Ovary cell lines, in particular the cell line CHOK1SV GS–KO. In a particular embodiment, cells were grown in medium CM16 (UKSL–7212). The inoculum was grown, and then sequentially transferred via a series of containers of increasing volume: a) 5–30ml b) 30–50ml c) 50–100ml d) 100–200ml e) 200–400ml

[0418] Containers were kept on a shaker platform and maintained within a temperature range of 34–38ºC. Fermentation

[0419] The grown inoculum material was transferred to a 100L cell bag, in CN68 (UKSL–8689). The material was maintained within a temperature range of 34–38ºC. Harvest / Clarification

[0420] The production fermenter was harvested, and the cells and cell debris were removed by depth filtration. The clarified supernatant was aseptically filtered via 0.22 μm filters into sterile containers. The filled containers were transferred to a 5 ± 3°C product cold room pending further processing for a maximum hold time of 14 days prior to completion of first purification step. Purification by MabSelect SuReTMAffinity Chromatography

[0421] MabSelect SuReTMresin was used for the purification and was dedicated to bulk protein product and for multiple cycles.

[0422] The processed harvest supernatant was removed from the 5 ± 3°C cold room storage and allowed to equilibrate to ambient temperature (15 to 25°C) and divided into aliquots and loaded onto the MabSelect SuReTMcolumn in successive cycles. It may be processed individually or as a run consisting of multiple cycles. An aliquot contains sufficient product to load the column at up to 6 g protein / liter of packed matrix.

[0423] The chromatography column was packed using 50mM Sodium Phosphate / 250 mM sodium chloride pH 7.0. The column was Height Equivalent to a Theoretical Plate (HETP) tested and the peak asymmetry was calculated.Attorney Docket No. LEVI0008-401-PC

[0424] Each cycle proceeded as follows: • The column is cleaned with 0.1M Sodium Hydroxide before use and after each cycle. • The chromatography column was equilibrated with 50 mM Sodium Phosphate / 250 mM sodium chloride, pH 7.0 buffer. • Unbound contaminants were removed by washing the column in three stages: o 2CV of 50 mM Sodium Phosphate / 250 mM sodium chloride, pH 7.0 buffer. o 4 CV of 50 mM Sodium Phosphate / 2M sodium chloride, pH 7.0 buffer o 2CV of 50 mM Sodium Phosphate / 250 mM sodium chloride, pH 7.0 buffer. • Bound protein was eluted from the column as a single fraction using 100 mM Sodium Citrate pH 3.6. • The column was washed post elution using 100 mM Citric Acid pH 2.1 buffer.

[0425] The MabSelect SuReTMeluate was pH adjusted with 2M Acetic Acid or 2.0 M Tris Base and held at pH 3.50 ± 0.05 for between 60 and 75 minutes as a viral inactivation step, before adjusting the pH to 7.0 ± 0.10 with 2.0 M Tris Base. Concentration and Diafiltration

[0426] Once the MabSelect SuReTMchromatograms meet the defined acceptance criteria for comparability as detailed in the associated batch record documentation, the protein purified from all cycles was pooled and concentrated to 10.0 g / liter ± 1.0 g / liter using an ultrafiltration unit containing 50 kDa molecular weight cut off cassettes which were dedicated to this product and this process step. The product was then diafiltered into 40mM Sodium Acetate / 10mM Sodium Chloride pH 5.5 in preparation for the next column step. Step 6: Purification by POROS XS Cation Exchange Chromatography

[0427] POROS XS matrix was used for the purification of one product batch. Up to three cycles were performed and the resin was then discarded. The chromatography column was packed using 0.1 M sodium chloride solution. The column was HETP tested, and the peak asymmetry was calculated.

[0428] The concentrated / diafiltered MabSelect SuReTMeluates were removed from the 5 ± 3°C cold room storage and allowed to equilibrate to ambient temperature (15 to 25°C).

[0429] Each cycle proceeded as follows: • The column was cleaned with 3 CV 0.5M sodium hydroxide.Attorney Docket No. LEVI0008-401-PC • The chromatography column was pre–equilibrated with 250mM Sodium acetate pH5.5 (3CV) and then equilibrated with 3 CV 40 mM Sodium Acetate / 10mM Sodium Chloride pH5.5. • The concentrated / diafiltered Mabselect SuReTMeluate was loaded onto the column at up to 20 g / L of resin. • The unbound product was washed through the column and collected from the flow– through as a single fraction using a 40 mM Sodium Acetate / 10mM Sodium Chloride pH5.5. • Impurities bound to the column were removed by a post elution wash with 2.0M Sodium Chloride pH7.0. • The resin was subjected to a clean with 0.5M Sodium hydroxide (3 CV) and were then prepared for the next load by equilibrating with ≥5 CVs 40 mM Sodium Acetate / 10mM Sodium Chloride pH5.5. • Samples were compared for similarity before pooling ahead of the next step. POROS HQ Anion Exchange Chromatography

[0430] POROS HQ matrix was used for the purification of one product batch. Up to three cycles were performed and the resin was then discarded. The resin bound the product, allowing impurities to flow through the packed column. The bound product was eluted from the resin by increasing the conductivity of the buffer. Aggregated product was retained until eluted by a further increase in buffer conductivity.

[0431] The chromatography column was packed using 0.1 M sodium chloride solution. The column is HETP (Height Equivalent to a Theoretical Plate) tested, and the peak asymmetry was calculated.

[0432] The POROS XS eluates were removed from the 5 ± 3°C cold room storage and allowed to equilibrate to ambient temperature (15 to 25°C)

[0433] Each cycle proceeded as follows: 1. The column was cleaned with 3 CV 0.5M sodium hydroxide. 2. The chromatography column was pre–equilibrated with 200mM Sodium Phosphate pH7.0 (3CV) and then equilibrated with ≥5 CV 10mM Sodium Phosphate / 40mM Sodium Chloride pH7.0. 3. The POROS XS eluate was loaded onto the column at up to 20 g / L of resin. 4. The column was washed with 3 column volumes 10mM Sodium Phosphate / 40mM Sodium Chloride pH7.0 5. Product was eluted from the column with ≥3 CV 10mM Sodium Phosphate / 350mM Sodium Chloride pH7.0.Attorney Docket No. LEVI0008-401-PC 6. Impurities bound to the column were removed by a post elution wash with 3CV 2.0 M Sodium Chloride. 7. The resin was subjected to a clean with 0.5M Sodium hydroxide (3 CV). 8. 200mM Sodium Phosphate pH7.0 was then prepared for the next load by pre– equilibration with 3 CVs 200mM Sodium Phosphate pH7.0 followed by equilibration with 10mM Sodium Phosphate / 40mM Sodium Chloride pH7.0 9. Samples were compared for similarity. No pooling was necessary as stage 8 allows for sequential loading of the virus reduction filter. Virus Reduction Filtration

[0434] The POROS HQ eluate was passed through a 0.1μm pre–filter followed by a Planova™ 20N virus reduction filter up to a specified maximum pressure and volume of product per filter. The virus reduction filter was then flushed in 10mM Sodium Phosphate / 350mM Sodium Chloride pH7.0. Concentration and Diafiltration

[0435] Following the virus reduction filtration step, the product was concentrated to a maximum product concentration of 20 mg / mL using an ultrafiltration unit containing 30kDa molecular weight cut off cassettes which are dedicated to this product and process step. The product was then diafiltered into 25mM Histidine / 50mM Sodium Chloride / 200mM Mannitol pH6.5. The protein concentration was determined by A280nm. The final product concentration specified was 10.0 mg / L ± 0.8 mg / mL.

[0436] Step 10: Bulk filtration and Dispensing

[0437] The product was filtered through a 0.5 / 0.2 µm pre–filter followed by a 0.22 µm final filter into sterile high–density polyethylene (HDPE) containers. After use the filter was tested for integrity. The product was labelled and stored in quarantine at both 5 ± 3°C and ≤65°C pending batch disposition. Example 4 − Characterization of Glycosylated Forms of Protein

[0438] It is understood that the glycosylated protein will be a mixture of glycosylated proteins. Glycosylation heterogeneity was assessed by neutral and charged Nitrogen–linked (N–linked) oligosaccharide profiling, monosaccharide composition and sialic acid content. Putative glycan structures were further confirmed by mass spectrometry (MS) of the released N–linked glycans and from protease digestions analyzed by RP HPLC–MS and MS / MS reduced tryptic peptide mapping.Attorney Docket No. LEVI0008-401-PC

[0439] Overall, glycan analysis indicated the presence of both N–linked and Oxygen– linked (O–linked) glycans, which were present at multiple sites on the protein chain. The glycans detected were predominantly G0F, G1F, G2F, G3F, G4F, and G4F+GN+Hex.

[0440] As part of reduced neutral N–linked oligosaccharide profiling the detected glycans were predominantly G0F (representing 22.4% of the total glycan population), G1F (representing 21.8% of the total glycan population), G2F (representing 30.0% of the total glycan population), G3F (representing 7.6% of the total glycan population), G4F (representing 6.5% of the total glycan population) and G4F+GN+Hex (representing 3.8% of the total glycan population).

[0441] The charged N–linked oligosaccharide profile showed predominantly neutral glycans, but also mono–sialylated, di–sialylated, and tri–sialylated glycans

[0442] Neutral glycans were detected at a total of 55.1% mol / mol total glycan. Mono– sialylated glycans were detected at a total of 9.5% mol / mol total glycan. Di–sialylated glycans were detected at a total of 25.0% mol / mol total glycan. Tri–sialylated glycans were detected at a total of 10.4% mol / mol total glycan).

[0443] O–linked glycan composition (from material generated by sodium borohydride reductive release) was measured using a hydrophobic interaction chromatography–HPLC with charged aerosol detection (HILIC–CAD). The abundance was measured as % peak intensity. The reference standard was shown to contain two main glycan structures: NeuNAc α(2–3) Gal β(1–3) [NeuNAc α(2–6)] GalNAc (48.8%) and NeuNAcα(2–3)Galβ(1– 3)GalNAc (36.26%). The remaining O–glycans were as follows: NeuNAcα(2–3)Gal (5.92%) NeuNAc α(2–3) Gal β(1–3) [NeuGc α(2–6)] GalNAc (4.02%), NeuGcα(2–3)Galβ(1– 3)GalNAc (2.74%) NeuNAcα(2–6)GalNAc (–H2O) (2.22%), Gal β(1–3) NeuGc α(2– 3) GalNAc (0.47%) and Gal β(1–3) NeuNAc α(2–3) GalNAc (0.22%).

[0444] Monosaccharide composition analysis was consistent with the presence of N– linked glycans and O–linked glycans. The monosaccharides detected were mannose, glucosamine, galactose, galactosamine and fucose. Total percentage glycosylation (w / w) was 9.3%.

[0445] The molar content of the N–acetylneuraminic acid (Neu5Ac) form of sialic acid and the N–glycolylneuraminic acid (Neu5Gc) form of sialic acid was assessed.

[0446] The analysis showed:Attorney Docket No. LEVI0008-401-PC mol total mol mol sialic Neu5Ac / mol Neu5Gc / mol acid / mol Batch protein protein protein 1 33.294 0.45 33.744 2 32.692 0.522 33.214 3 32.622 0.521 33.143 Mean 32.87 0.50 33.37 Stddev 0.37 0.04 0.33 CV 1.12 8.30 0.98 Mass spectrometry of Immunomodulating metalloprotease (IMPa) and trypsin digested compound E3 to detect sites of O-glycosylation Prepare two samples for each experiment.

[0447] Prepare denaturation buffer by adding 1mL 1M Tris pH8.0 to a 10mL volumetric flask and make up to 10mL with 8M guanidine HCl. Add 100uL of denaturation buffer to an Eppendorf tube Add 200ug Compound E3 (i.e., 20µL of 10mg / mL Compound E3) to the tube containing the denaturation solution.

[0448] Add 1µL of 0.5M tris (2-carboxyethyl) phosphine (TCEP) reducing agent to tube. Close cap and vortex for a few seconds. Spin briefly in a microfuge to collect solution from tube wall. Incubate for 30 minutes at 37°C.

[0449] Add 2uL of 0.5M iodoacetamide alkylating agent to reduced sample. Close cap and vortex for a few seconds. Spin briefly in a microfuge to collect solution from tube wall. Incubate for 30 minutes at 37°C in the dark.

[0450] Prepare spin filters (ZebaTM Spin Desalting Columns, 7K MWCO (Thermo Fisher Scientific, Part No.89882) before use: • Remove the bottom of the filters (cap closed) and place in tube. • Loosen the cap but do not take off completely • Centrifuge 1500g for 1 minute at ambient temperature to remove the storage solution / Attorney Docket No. LEVI0008-401-PC • Add 300µL 0.1M Tris pH8.0 and then centrifuge for 1 minute Centrifuge 1500g for 1 minute at ambient temperature. • Perform step above twice to ensure column is equilibrated.

[0451] Add 120µL denatured, reduced and alkylated Compound E3 to the equilibrated spin filter in a clean tube. Centrifuge 1500g for 2 minutes at ambient temperature and collect the flow through for next stage. Digestion / O-Glycoprotease treatment Tube 1:

[0452] Add 5µL of O-Glycoprotease solution (IMPa) and incubate at 37°C for 5h. After incubation has completed add 20µL of 0.5µg / µL trypsin solution and incubate at 37°C for a further 16-18 h.

[0453] Stop the reaction by addition of 2.8µL of 10% trifluoroacetic acid (TFA) and then transfer to Waters polypropylene vials for analysis. Tube 2:

[0454] Add 20µL of 0.5µg / µL trypsin solution and incubate at 37°C for 16-18 hours. After incubation has completed add 5µL of O-Glycoprotease solution (IMPa) and incubate at 37°C for a further 5h.

[0455] Stop the reaction by addition of 2.8µL of 10% TFA and then transfer to Waters polypropylene vials for analysis. Chromatographic and MS acquisition parameter Chromatographic separation method for peptide mapping analysis UHPLC system Agilent 1290 Infinity II UHPLC with column heater and on- line UV detector Mobile phase A (MPA): 0.1% formic acid (FA), in water (v:v) Mobile phase B (MPB): 0.1% FA, in acetonitrile (v:v) Run / stop time: 98 min Injection volume: 10 µL Column temperature: set to 60 °C Autosampler temperature: set to 5 °C Column: Waters Acquity UPLC Peptide BEH 300 C18, 1.7 µm, 2.1 × 150 mm (Part No.186003687)Attorney Docket No. LEVI0008-401-PC Chromatographic gradient for peptide mapping setup. Flow Rate Time [min] Eluent A [%] Eluent B [%] [mL / min]Attorney Docket No. LEVI0008-401-PC MS acquisition parameters for peptide mapping analysis Mass spectrometer: Thermo Q-Exactive plus Orbitrap MS equipped with ESI (Electro Spray Ionization) Ion Source. Ionization: Electrospray Ionization (ESI) Detection mode: Heated-ESI Positive Spray voltage: 3.5 kV Capillary temperature: 350 °C Sheath gas: 35 units Auxiliary gas: 10 units Probe heater 350 °C Full MS Full scan m / z 200 – 3000; Resolution 140’000; 1 Microscan Data dependent MS2Top 5 signals from Full MS; Resolution 17’500; 1 Microscan (TopN): Divert valve: 0.0 min to waste, 1.8 min to source, 80.0 min to waste MS run time 98.0 minutes ^ Data acquisition was carried out on Chromeleon 7.2 software (Thermo Fisher Scientific). ^ The 5 most intense ions from the Full-MS scan were selected for MS / MS by using a data dependent scheme and dynamic exclusion. Data evaluation

[0456] Chromatogram and isotope extraction were performed in Chromeleon 7.2 (Thermo Fisher Scientific). The O-glycosylation site evaluation as performed using BioPharma Finder v3.2. Parameters for the databased protein search in the software Peaks v8.5. Reference sequence: Compound E3Attorney Docket No. LEVI0008-401-PC Mass spectrometry of O-endoprotease digested Compound E3 co-treated with PNGaseF and IdeS to detect site specific O-glycosylation

[0457] Sample preparation, LC-MS / MS analysis and data processing were based on Riley, N. M., & Bertozzi, C. R. (2022). Deciphering O-glycoprotease substrate preferences with O-Pair Search. Mol Omics, 18(10), 908-922. doi:10.1039 / d2mo00244b Sample

[0458] Twousing different O-glycoproteases were tested (with sample prep triplicates for each), either using OpeRATOR® enzyme (G2-OP1-020, Genovis) combined with pan sialidase (SialEXO®, G1-SM1-020, Genovis) or using ImpaRATOR™ enzyme(G1-IR1-020, Genovis). All enzymes were reconstituted according to manufacturer recommendations. Digestion Mix

[0459] A digestion mix in 20 mM Tris-HCl buffer (pH 7.5) and containing all required enzymes was prepared for each condition from stock solutions, to reach a final concentration of 2.5 U / µL of O-glycoproteases enzymes (OpeRATOR or ImpaRATOR), pan sialidase (in combination with OpeRATOR only) and IdeS (FabRICATOR®, A0-FR1-020, Genovis), and final concentration of 5 U / µL of PNGaseF (from a 100 U / µL solution in water, P0708S, New England Biolabs). Digestion

[0460] For each condition, add 50 µg (5 µL of 10mg / mL) Compound E3 to 20 µL of digestion mix, for a final 1:1 protease unit : protein weight ratio (2:1 for PNGaseF) as directed by the manufacturer. Incubate at 37°C for 5h under gentle agitation (500 rpm). Denaturation, Alkylation and sample preparation

[0461] After digestion, add 23 µL of a 4.2 M guanidine-HCl,10.4 mM DTT solution to the digest (final concentration of 2 M guanidine-HCl, 5 mM DTT) and incubate samples at room temperature for 1h under gentle agitation (500 rpm). Then, perform alkylation by adding 2 µL of 250 mM iodoacetamide (final concentration of 10 mM iodoacetamide) to the reduced samples, followed by incubation at room temperature for 1h in the dark under gentle agitation (500 rpm). Buffer exchange alkylated samples to 100 mM ammonium acetate using Biospin-6 columns (732-6227, Bio-Rad) according to manufacturer’s instructions. Vacuum-dry samples and resuspend pellets in 25 µL (final sample concentration of 2 µg / µL) of 99:1 water : acetonitrile with 0.2% formic acid prior to injection.Attorney Docket No. LEVI0008-401-PC LC-MS / MS analysis

[0462] Data were acquired using product-dependent triggering of electron- transfer / higher-energy collision dissociation (EThcD) scans as described in Riley, N. M., & Bertozzi, C. R. (2022). Deciphering O-glycoprotease substrate preferences with O-Pair Search. Mol Omics, 18(10), 908-922. doi:10.1039 / d2mo00244b.

[0463] O-glycopeptides separation was performed on a Vanquish Flex ultrahigh pressure liquid chromatography (UHPLC) instrument consisting of a Quaternary Pump F (VF-P20-A), Split Sampler FT with 25 μL autosampler loop (VF-A10-A), and a Column Compartment H (VH-C10-A) coupled to an Orbitrap Eclipse Tribrid mass spectrometer. Instrument modules were controlled with Thermo Scientific Xcalibur software version 4.7.

[0464] Inject approximately 3 µg of O-glycopeptides on a Hypersil Gold™ C18 column (100 × 1.0 mm, 3 μm, 175 Å, Thermo Fisher Scientific) heated to 40°C. Perform separation at 100 µL / min using the following gradient of mobile phases A (0.2% FA in water) and B (0.2% FA in acetonitrile): 5% B was held for the first 4 min, followed by an increase from 5% to 45% B from 4 to 64 min, an increase from 45% to 95% B from 64 to 74 min, isocratic flow at 95% B for 5 min and a re-equilibration at 5% B for 10 min. Eluted O-glycopeptides were analyzed on the mass spectrometer in peptide mode using a standard flow Ion MAX Source containing a heated electrospray ionisation (H-ESI) probe (Thermo Fisher Scientific, San Jose, CA, USA). Spray voltage was set to 3.5 kV, ion transfer tube and vaporizer temperatures to 275°C and 200°C, respectively, and sheath and auxiliary gas to 15 and 5 a.u., respectively. Survey scans of peptide precursors were acquired in the Orbitrap in the 400- 1,800 m / z range using a 60,000 (at m / z 200) resolution, a maximum injection time of 50 ms and a normalized automatic gain control (AGC) target of 100% (400,000 charges). Monoisotopic precursor selection was enabled for peptide isotopic distributions, precursors of z = 2 to 8 were selected for data-dependent MS / MS scans for 3 s of cycle time, and dynamic exclusion was enabled with a repeat count of 2, repeat duration of 20 s, exclusion duration of 20 s, and mass tolerance of 10 ppm. Priority filters were set to favour highest precursor charge states and lowest precursor m / z values. An isolation window of 2 m / z was used to select precursor ions with the quadrupole. EThcD scans were collected in product-dependent fashion, where the presence of oxonium ions (m / z 126.055, 138.0549, 144.0655, 168.0654, 186.076, 204.0865, 274.0921, 292.1027, and 366.1395) in a ‘‘scouting’’ high-energy collisional dissociation (HCD) MS / MS scan triggered acquisition of a second MS / MS scan. The ‘‘scout HCD’’ scan used an automated scan range determination and a first mass of 100Attorney Docket No. LEVI0008-401-PC Th, a normalized collision energy of 36, a normalized AGC target value of 100% (50000 charges), a maximum injection time setting of Auto (54 ms), and a 30,000 (at m / z 200) resolution. If at least four of the nine listed oxonium ions were present in the scout HCD scan within a ±15 ppm tolerance and were among the 20 most intense peaks, an EThcD MS / MS scan was triggered that used calibrated charge dependent parameters for calculating reagent AGC targets and ion–ion reaction times, a supplemental collision energy of 25, a scan range of m / z 120 to 4,000, a maximum injection time of 400 ms, a normalized AGC target of 200% (100,000 charges), and a resolution of 60,000 (at m / z 200). Data processing

[0465] All raw data were searched using O-Pair Search implemented in MetaMorpheus v.1.0.5, which is available at www.github.com / smith-chem-wisc / MetaMorpheus (Lu, L., Riley, N. M., Shortreed, M. R., Bertozzi, C. R., & Smith, L. M. (2020). O-Pair Search with MetaMorpheus for O-glycopeptide characterization. Nat Methods, 17(11), 1133-1138. doi:10.1038 / s41592-020-00985-5). A customized O-glycans database was created containing the following core 1 O-glycans: HexNAc(1) (203.0794 Da), HexNAc(1)Hex(1) (365.1322 Da), HexNAc(1)NeuAc(1) (494.1748 Da), HexNAc(1)Hex(1)NeuAc(1) (656.2276 Da) and HexNAc(1)Hex(1)NeuAc(2) (947.3230 Da).

[0466] The Glyco Search was performed using the customized O-glycan database with a maximum of 4 O-glycans allowed, top N candidates was set to 50, dissociation type set to HCD, child scan dissociation to EThcD, and oxonium ion fit was enabled. For in-silico digestion parameters of the FASTA file containing the sample sequence, decoy proteins were generated using reversed decoys, initiator methionine was set as variable, protease was set to non-specific with a peptide length from 3 to 125, 124 max missed cleavages and a maximum of 5 modifications per peptides with cysteine set as fixed modification. For fragment ion search parameters, mass tolerance was set to 10 ppm and 20 ppm for precursors and products, respectively, and the minimum score allowed was 3. All other parameters were kept as default. Each replicate was searched individually.

[0467] Additional data filtering was operated after O-Pair Search. Briefly, all decoy peptides were removed, and only level 1 glycopeptides (location probability > 0.75) with a Q-value higher than 0.01 that were identified in 2 over 3 replicates of one condition were kept. Finally, all identified glycopeptides were manually checked to confirm cleavage sites from the enzymes, with first amino acid of the peptide bearing a O-glycan and last amino acid corresponding to a potential cleavage site.Attorney Docket No. LEVI0008-401-PC Mass Spectrometry of de-sialylated and PNGaseF treated Compound E3 to measure O-glycan occupancy Sample preparation

[0468] 100 µg of material was brought to a final concentration of 1 mg / mL with 20 mM TRIS HCl pH 6.8. Enzymatic treatment was started by addition of 2.5 µL of sialidases (SialEXO, Genovis) and 1 µL of PNGase F (CarboClip, Asparia Glycomics). Samples were incubated overnight at 37°C under agitation. Following digestion, samples were transferred to HPLC vial and directly injected. LC-MS analysis

[0469] Inject 20 µg of sample on a NativePac column (2.1 x 50 mm, Thermo Scientific). Isocratic gradient was delivered by a Thermo Scientific Vanquish Flex uHPLC at a flow rate of 0.2 mL / min. Buffer was 50 mM ammonium acetate and column temperature was kept at 25°C. Column was connected to a Thermo Scientific Orbitrap Exploris MX mass detector. Mass spectrometer was operated at Intact Protein mode, with High Pressure settings for HCD gas trapping. Tune settings were as follows: sheath and auxiliary gasses were set at 28 and 10 arbitrary units respectively, ion transfer and vaporizer temperatures were set at 275 and 250°C, respectively. Orbitrap resolution was kept at 30,000 (at 200 m / z) and scan range was set between 2,500 and 8,000 m / z. In-source fragmentation was on and kept at 130 eV, while acquisition gain control was set to 100%, maximum injection time was 250 ms and microscan number was 10. Full method duration was 5 minutes. Data analysis

[0470] For intact analysis, raw data were deconvoluted using BioPharma Finder software v.5.2. Elution peak (0.5-1.0 RT) was selected through the average over selected time range tool and deconvoluted using the ReSpect algorithm. Briefly, model and output mass range were 90,000 and 110,000 Da, charge state range was between 10 and 50, deconvolution mass tolerance was set to 20 ppm and the minimum number of adjacent charge states was set to 4. Identification was achieved with a mass accuracy tolerance of 30 ppm. Site Specific N-linked glycan analysis of Compound E3 FabRICATOR Digestion

[0471] 20µl Compound E3 (10mg / mL) added to 80 µl 50 mM Tris HCL pH 7.9 (final conc.2mg / mL). Take 50µL (100µg) of this solution and add to 100units of FabRICATOR (Genovis AO-FR1-008). Mix contents via pipette action and incubate at 37 °C for 3 hours. Isolate the glycan peaks using charged variant analysis.Attorney Docket No. LEVI0008-401-PC Charged Variant Analysis Sample preparation: 10 µg Injection volume: 10 μL Column: Thermo Scientific ProPac 3R SCX column (2.0x50 mm) Column temperature: 25 ºC System: Thermo Scientific Vanquish UHPLC Software: XCalibur (Thermo Scientific) Solvent A: 25mM Ammonium Bicarbonate, 30 mM Acetic Acid Solvent B: 10mM ammonium Hydroxide Gradient: 30 minute linear gradient with a flow rate of 0.2 mL / min: Starting at 20 % Solvent B, increasing to 100 % Solvent B over 15.0 minutes, flowing at 100% Solvent B for 4.0 minutes, reducing to 0 % Solvent B for 3 minutes, returning to 20% Solvent B and equilibrating for 8.0 minutes. Wavelengths: 280 nm. Sample Temperature: 5 ºC Fraction collection: N32 glycan site elutes between 0-1.5 minutes (1stpeak). Peak collected in 1.5 mL tube. N294 glycan site elutes between 7.0-8.2 minutes (2ndpeak). Peak collected in 1.5 mL tube. In-solution release of N-glycans with PNGase F

[0472] Add each sample fraction onto separate Nanosep® 10 K MWCO filters (PALL) filter and spin @13,000 rpm until all protein is loaded onto the filter. Once all protein is on the filter add 100µl 20mM NaHCO3 to each filter and spin at 13,000rpm. Repeat this wash step 3 times before continuing to reduction step.

[0473] Add 90 μL of 20mM NaHCO3; 10 μL of 1 % Rapigest solution in 20mM NaHCO3; 2 μL 400 mM DTT. Mix the contents by pipette action and incubate on the filter at 65 °C for 15 minutes.

[0474] Alkylate samples by adding 2 μL of 80 mM iodoacetamide and incubate in the dark at room temperature for 30 min.Attorney Docket No. LEVI0008-401-PC

[0475] De-N-glycosylate samples by adding 2 μL PNGase F (NEB, P0709L). Mix via pipette action and incubate on the filter at 37 °C overnight.

[0476] Remove deglycosylated material by centrifugation @13,000 rpm and collect filtrate. Wash the filter twice with 100uL distilled H2O to wash all the released glycans through the filter. Dry samples in a vacuum centrifuge before adding 20 μL of 1 % formic acid and incubate at room temperature for 20 minutes.

[0477] Dry samples in a vacuum centrifuge prior to further processing. 2-AB Glycan labelling and clean-up Samples were labelled by adding 5 μL of 2-AB labelling solution (LudgerTag 2-AB labelling kit, Ludger, Abingdon, UK) to dried pellet and mix by pipette action. Incubate for 2 h at 65 °C. Remove excess 2-AB using amide resin Phytips from Phynexus. Dry sample in vacuum centrifuge. HILIC UPLC N-Glycan Method

[0478] The UPLC system was calibrated by running an external standard of 2-AB dextran ladder (2-AB labeled glucose homopolymer) alongside the sample runs. A fifth-order polynomial distribution curve was fitted to the dextran ladder and used to allocate glucose unit (GU) values from retention times, using Empower software (Waters).

[0479] Take the dried sample at the end of the previous step (2-AB Glycan labelling and clean-up) and resuspend pellet in 12.5µL 75% acetonitrile. Sample preparation: 70 % acetonitrile Injection volume: 10 μL Column: 1.7 µm BEH Glycan column (2.1 X 150 mm) Column temperature: 40ºC System: Waters Acquity UPLC equipped with a fluorescence detector Software: Empower 3 (Waters) Solvent A: 50 mM ammonium formate pH 4.4 Solvent B: Acetonitrile Gradient: 30 minute linear gradient with a flow rate of 0.561 mL / min (except for wash step): 30 % Solvent A for 1.47 minutes, increasing to 47 % Solvent A over 23.34 minutes, increasing to 70 % Solvent A over 0.69 minutes; 70 % Solvent A for 0.75 minutes and then for a further 0.3Attorney Docket No. LEVI0008-401-PC minutes at a reduced flowrate of 0.4 mL / min, returning to 30 % Solvent A over 0.3 minutes at a flow rate of 0.4 mL / min, then equilibrating with 30 % Solvent A for 1.95 minutes with the flow rate returned to 0.561 mL / min. Wavelengths: Excitation 330 nm and emission 420 nm. Data rate: 20 pts / sec and PMT gain: 20. Weak Wash: 80 % Acetonitrile Strong Wash: 20 % Acetonitrile Sample Temperature: 5 ºC Quantification of N-glycans

[0480] Released 2-AB labelled N-glycans for each sample are loaded in triplicate and analyzed as per HILIC UPLC method described. Resulting chromatograms are integrated using Waters Empower software. The relative percent area is used to express the abundance of each peak as a percentage of the total chromatographic area. Relative % area is taken as an average across the triplicate release. Liquid chromatography-mass spectrometry (LC-MS)

[0481] LC-MS analysis is performed for orthogonal confirmation of the N-glycan structural assignments. Compound E3 is prepared as described above (from FabRICATOR digestion through to end of 2-AB Glycan labelling and clean-up). Sample is analyzed as per LC-MS method described below and resulting MS1 data integrated using XCalibur software.

[0482] The dried sample at the end of the previous step (2-AB Glycan labelling and clean-up) was taken and resuspend pellet in 12.5µL 75% acetonitrile. LC-FLD-mass spectrometry Sample Temperature: 5 ºC Sample preparation: 75 % acetonitrile Injection volume: 10 μL Column: 1.7 μm Waters BEH Glycan column (1.0 x 150 mm). Column temperature: 60 ºC System: Thermo Scientific Q Exactive PlusAttorney Docket No. LEVI0008-401-PC Software: XCalibur (Thermo Scientific) Solvent A: 50 mM ammonium formate pH 4.4 Solvent B: Acetonitrile Gradient: 40 minute linear gradient with a flow rate of 0.15 mL / min: 28 % Solvent A for 1.0 minute, increasing to 43 % Solvent A over 30.0 minutes, increasing to 45 % Solvent A over 1 minute; returning to 28 % Solvent A over 4.0 minutes, then equilibrating with 30 % Solvent A for 4.0 minutes. Wavelengths: Excitation 320 nm and emission 420 nm. MS: Negative mode, spray voltage 3.40kV, Capillary temperature 320 °C, Aux gas heater temperature 300 °C, Sheath and sweep gas flow rate 30 and 10 L / h respectively, Scan range 450 to 2,500 m / z, Resolution 70,000. Example 5 − NT3 Affinity

[0483] p75NTR–Fc for NT3

[0484] In another aspect, the present disclosure provides a glycosylated protein as described herein wherein the glycosylated protein binds to NT3 with a binding affinity (KD) of between about 0.001 nM to about 50 nM.

[0485] The p75NTR(NBP)–Fc fusion protein of the present invention in some embodiments binds NT3 with a binding affinity (KD) of between about 0.001 nM to about 50 nM. In some preferred embodiments, the binding affinity (KD) is between about 0.001 nM and any of about 0.01nM, 0.02 nM, 0.05 nM, 1 nM, 1.5 nM, 2 nM, 2.5 nM, 3 nM, 3.5 nM, 4 nM, 4.5 nM, 5 nM, 5.5 nM, 6 nM, 6.5 nM, 7 nM, 7.5 nM, 8 nM, 8.5 nM, 9 nM, 9.5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM or 50 nM as measured in an in vitro binding assay for NGF, BDNF, NT3 or NT4 / 5 such as described herein in some embodiments as measured by surface plasmon resonance at 20 °C. In some further preferred embodiments, binding affinity (KD) is or is less than any of about 1 pM 10pM 25 pM, 50 pM 100 pM 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 950 pM or 1 nM as measured in an in vitro binding assay for p75NTR(NBP)–Fc fusion protein with the neurotrophins such as described herein, in some embodiments as measured by surface plasmon resonance at 20 °C. In a further preferred embodiment, the binding affinity (KD) is about 10 pM or about 1 nM, as measured in an in vitro binding assay for p75NTR(NBP)–Fc fusion protein with the neurotrophin. suchAttorney Docket No. LEVI0008-401-PC as described herein, in some embodiments as measured by surface plasmon resonance at 20 °C.

[0486] In some embodiments the p75NTR(NBP)-Fc fusion protein of the invention is for in the treatment of pain. Without wishing to be bound by any particular theory, the inventors believe that the p75NTR(NBP)-Fc fusion protein achieves efficacy in the treatment of pain by effecting the functional activity of the aforementioned neurotrophins, (defined as modulating or up or down regulating the functional activity of the neurotrophins, NT3 ,for example the functional activity of the aforementioned neurotrophin resulting from the interaction with the respective receptor. By inhibiting NT3, the molecule can provide pain relief. By not suppressing NGF levels the molecule does not interfere with the body’s natural joint regeneration process. This avoids the side effects seen with anti-NGF drugs such as Tanezumab and can lead to regeneration of the joint, that is disease modifying treatment of osteoarthritis.

[0487] In some embodiments, the p75NTR(NBP)–Fc fusion protein is selective for NT3 over NGF.

[0488] In some embodiments, the fusion protein is 25 times more selective for NT3 over NGF.

[0489] In some embodiments, the fusion protein is 50 times more selective for NT3 over NGF.

[0490] In some embodiments, the fusion protein is 500 times more selective for NT3 over NGF.

[0491] In some embodiments, the fusion protein is 1000 times more selective for NT3 over NGF.

[0492] In some embodiments, the fusion protein binding to NT3 (Kd) is 1–20pm and binding to NGF is 500–1000pM.

[0493] Binding may be assessed by measuring NGF binding to and activation of TrkA, as demonstrated in classical neuron survival assays (such as provided in Cohen et al., 2011). BINDING LEVELS

[0494] In some embodiments, the p75NTR(NBP)–Fc fusion protein effects the functional activity of NT3 as assessed by measuring NT3 binding to and activation of endogenous Trk receptor activity, as demonstrated in Trk receptor phosphorylation, mitogen–activated protein kinase phosphorylation reporter assays or cell survival and neurite extension assays.Attorney Docket No. LEVI0008-401-PC

[0495] In some embodiments, the NT3 Kd for a molecule of the present invention is generated using the Biacore binding assay as described herein.

[0496] The p75NTR(NBP)–Fc fusion protein of the invention may also comprise a proteolytic cleavage site, optionally interposed between the p75NTR(NBP) portion and the immunoglobin Fc portion. The proteolytic cleavage site may be located in the linker or at the junction of the linker with either the p75NTR(NBP) portion or / and the immunoglobin Fc portion. The p75NTR(NBP) may optionally be cleaved from the immunoglobin Fc portion prior to formulation and or administration for therapeutic purposes.

[0497] The glycosylated proteins of the present disclosure have been found to have numerous, surprising advantages when compared to close analogues.

[0498] Without being bound by theory, its believed that glycosylation significantly influences the stereochemistry of the molecule. This leads to several advantages including enhanced stability; increased and advantageous selectivity for key receptors; greater activity at key receptors; and / or stronger and selective binding to key targets; and / or disease modification in osteoarthritis patients is seen only with the glycosylated protein.

[0499] In a some embodiments, treatment of osteoarthritis includes slowing or arresting of disease progression and / or reduction in cartilage loss. Treatment of osteoarthritis can include reversal of disease progression, regrowth of cartilage and / or curative treatment. Disease progression may be determined by the rate of cartilage loss or regrowth. In some embodiments, disease progression may be monitored by determining the number of chondrocytes present in a joint. In some embodiments, the treatment of osteoarthritis may include relief from the symptoms of osteoarthritis, for example as measured by reduction in pain, inflammation, swelling, tenderness, or joint stiffness, or increase in joint mobility, or any combination of these.

[0500] In some embodiments, disease modification in osteoarthritis patients is seen only with the glycosylated protein.

[0501] In some embodiments, glycosylation increases the ability of the molecule to inhibit NT3 and decreases the ability to inhibit NGF.

[0502] In some embodiments, the glycosylated molecule has affinity for NT3 with a binding affinity of between about 1 pM to about 500 pM.

[0503] In some embodiments, the glycosylated molecule shows selectivity for NT3 over NGF on the order of 50–1000x over NGF KD.

[0504] A Biacore chip was prepared in an experiment in which Protein A was amine coupled to flow cells 1 and 2. Single cycle kinetics of NT3 binding to captured p75–Fc were measured.Attorney Docket No. LEVI0008-401-PC

[0505] The binding capacity (Rmax) of a chip surface depends on the immobilized level of the ligand (fusion protein). For a kinetics study an Rmax of 50–100 RU is advised. By using the molecular weights of the p75–Fc and NT–3, a desired immobilisation level for the fusion protein can be calculated.

[0506] Rmax = (NT3 molecular weight / fusion protein molecular weight) x immobilization level x stoichiometric ratio: 50 = (13,500 / 102,000) x immobilization level x 1.

[0507] Hence, the immobilization level required = (102,000 / 13,500) x 50 = 378 RU) SEQ ID NO:1 and SEQ ID NO:3 were immobilized onto the Protein A chip prior to single cycle kinetics.

[0508] Using a manual run, p75–Fc was captured onto flow cell 2 of the Protein A chip until the desired level of approx.380 RU was achieved. This was performed with a 22 second injection at a flow rate of 10 μl / min and p75–Fc concentration 10 μg / ml which resulted in 418 RU of the fusion protein captured onto the protein A surface.

[0509] In the first instance NT–3 concentrations of 10, 52.5, 1.25 and 0.625 nM were tested. These concentrations were tested as the KD for the fusion protein was approximated to be within this range of NT–3 concentrations.

[0510] The single cycle kinetics method involved:

[0511] – injecting 0.625 nM of NT–3 onto the captured p75–Fc for 120 seconds at 30 μl / min.

[0512] – this process was then repeated with an injection of NT–3 at 1.25 nM, followed by 2.5, 5 and 10 nM.

[0513] – after the final concentration of NT–3 had been injected a 600 second dissociation phase was performed by flowing the running buffer (HBS–EP) over the chip.

[0514] Once completed the chip was regenerated back to its Protein A surface by injecting 10 mM Glycine HCl, pH 2 for 60 seconds at 30 μl / min.

[0515] p75–Fc was then captured onto the chip by performing a 38 second injection at a flow rate of 10μl / min at a concentration of 10 μg / ml. This achieved the desired level of 430 RU. The single cycle kinetics procedure described above was then repeated. Data analysis

[0516] The fusion protein–NT–3 binding data was analyzed in the following manner using the Biacore T200 evaluation software v1: – Data is recorded for the binding of NT–3 to the fusion protein on flow cell 2 (Fc=2) and for NT–3 flowing over the control flow cell 1 (Fc=1; protein A alone).Attorney Docket No. LEVI0008-401-PC – The data from Fc=1 is then subtracted from Fc=2 to give “2–1” binding data. – The 2–1 binding data for an injection of 0 nM (HBS–EP running buffer alone) is then subtracted from all the 2–1 binding data to control for any drifts in baseline throughout the experiment. – Finally, this data is then fitted to a 1:1 binding model to calculate binding characteristics including association rates (ka), dissociation rates (kd) and affinities (KD).

[0517] The sequence showed suitable affinity for NT–3. Example 6 − Comparison with molecule lacking glycosylation.

[0518] A closely related peptide to the one of the present disclosure was created, varying at several positions to create a protein that would not possess the glycosylation of the present disclosure. The protein, disclosed in WO2016 / 146841 and in SEQ ID NO:2, has the following sequence: KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECV GLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSLGLVFSCQDKQNTVCEECPD GTYDEANHVDPCLPCTVCEDTERQLFECTRWADAECEEIPGGGGEPKSDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCVMHEALHNHYTQKSLLSLSPG

[0519] With respect to histological outcomes, the new molecule was not associated with efficacy at any dose studied. Given the variation in pathology severity scores within each group, any data trends are likely to be the result of the additive effects of variable group size (due to sample rejections) and variation in sample blocking (section depth and orientation). However, efficacy profiles with the compound of the present disclosure in this model showed a differential efficacy profile (cartilage and bone pathology inhibition and potentiation of mesenchyme cell expansion and homing) which would have overcome these artefacts. It is unlikely that significant efficacy has been masked.

[0520] Example 7 − Pharmaceutical Preparations and Kits

[0521] In some embodiments the glycosylated protein of the present disclosure is prepared for oral, sublingual, buccal, topical, rectal, inhalation, transdermal, subcutaneous, intravenous, intra–arterial, intramuscular, intracardiac, intraosseous, intradermal,Attorney Docket No. LEVI0008-401-PC intraperitoneal, transmucosal, vaginal, intravitreal, intra–articular, peri–articular, local or epicutaneous administration.

[0522] In some embodiments the glycosylated protein of the present disclosure or the pharmaceutical composition thereof is for, or prepared for administration between once to 7 times per week, further In some embodiments between once to four times per month, further In some embodiments between once to six times per 6 month period, further In some embodiments once to twelve times per year. In some embodiments the medicament is to be or prepared to be peripherally administered in a period including but not limited to: once daily, once every two, three, four, five or six days, weekly, once every two weeks, once every three weeks, monthly, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months or yearly.

[0523] Further in some embodiments the glycosylated protein or the pharmaceutical composition of this aspect is to be or prepared to be peripherally administered via a route including but not limited to one or more of; orally, sublingually, buccally, topically, rectally, via inhalation, transdermally, subcutaneously, intravenously, intra–arterially or intramuscularly, via intracardiac administration, intraosseously, intradermally, intraperitoneally, transmucosally, vaginally, intravitreally, epicutaneously, intra–articularly, peri–articularly or locally.

[0524] In some embodiments the glycosylated protein or the pharmaceutical composition is for, or is prepared for, administration at a concentration of between about 0.05 to about 200 mg / ml; In some embodiments at any one of about 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg / ml + / – about 10% error, most in some embodiments at about 3 mg / ml in veterinary applications and 0.1 in humans.

[0525] In some embodiments the glycosylated protein or the pharmaceutical composition is for, or is prepared for, administration at a concentration of between about 0.1 to about 200 mg / kg of body weight; in some embodiments at any one of about 0.5, 1, 5, 10,1520, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 mg / kg of body weight + / – about 10% error, most in some embodiments at about 10 mg / kg in veterinary applications and 0.3 in humans.

[0526] Pharmaceutical compositions of this disclosure are suitable for parenteral administration comprise one or more compounds of the disclosure in combination with one or more pharmaceutically–acceptable sterile isotonic aqueous or nonaqueous solutions,Attorney Docket No. LEVI0008-401-PC dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0527] Examples of aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0528] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0529] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally–administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0530] Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide–polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled.

[0531] Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

[0532] An exemplary formulation for injection, including transdermally, subcutaneously, intravenously, intra–arterially or intramuscularly administration would include a saline or oilAttorney Docket No. LEVI0008-401-PC solution, in some embodiments buffered. A specific example would include: 25 mM Histidine, 50 mM NaCl, 200 mM Mannitol and the compound or mixture of compounds of the present disclosure, buffered to pH 6.5.

[0533] According to another aspect of the present disclosure there is provided a method and a kit comprising: (a) the glycosylated protein or the pharmaceutical composition; and (b) instructions for the administration of an effective amount of said glycosylated protein, or pharmaceutical composition to an individual for any one or more of the prevention or treatment of eczema, psoriasis, dermatitis, prurigo lesions of atopic dermatitis, obsessive– compulsive disorder, depression, schizophrenia, anorexia nervosa, bulimia nervosa, cardiovascular diseases, coronary atherosclerosis, acute coronary syndromes, obesity, type 2 diabetes, metabolic syndrome, multiple sclerosis, accelerating wound healing, treatment of skin ulcers and corneal ulcers, neurodegeneration, neurodevelopmental or neurological conditions, Huntington's disease, Rett syndrome, dementia, Alzheimer's disease, autism, development neurodegenerative disorders, primary open angle glaucoma, to reduce neural degeneration and to promote peripheral nerve regeneration, cancer, breast cancer, rheumatoid arthritis, osteoarthritis, cystitis and endometriosis.

[0534] The kit may include one or more containers containing the glycosylated protein or pharmaceutical composition described herein and instructions for use in accordance with any of the methods and uses of the disclosure. The kit may further comprise a description of selecting an individual suitable for treatment. The instructions for the administration of the pharmaceutical composition may include information as to dosage, dosing schedule and routes of administration for the intended treatment. Example 8 − Clinical Trial of Compound E3

[0535] A pivotal, multiple–arm, multicenter, prospective, randomized, double–blind, placebo–controlled, Phase II study of Compound E3 monthly intravenous infusion for the treatment of pain due to osteoarthritis (OA) of the knee is described.

[0536] The trial recruited more than 518 patients and was designed to evaluate the efficacy, safety, and tolerability of weekly and monthly intravenous infusions of Compound E3 as compared to placebo in participants with radiographic and symptomatic knee osteoarthritis. Compound E3 was shown to substantially improve pain and / or hyperalgesia and anodynia and function without deleterious effects on joint structure.Attorney Docket No. LEVI0008-401-PC TRIAL DESIGN

[0537] The use of the WOMAC pain subscale is a U.S. Food and Drug Administration (FDA) recommended clinical trial efficacy endpoint for OA and considered a reliable, validated outcome measure. The study duration of 30 weeks included 14 weeks of follow–up via phone from the last dose administered and was considered sufficient to meet study objectives and ensure sufficient oversight of participant safety and follow–up of adverse events.

[0538] Radiographs of major joints bilaterally (knee–, hip–, and shoulder joints) and MRI of both knees during screening were included to determine eligibility. Only the Target Knee had an MRI completed at FU / EOT (follow–up / end–of–trial) visit (week 20). Safety assessments throughout the trial included physical examination, vital signs, clinical laboratory values, ECG, and AEs. Primary Objective

[0539] The primary objective was evaluating the efficacy of Compound E3 (multiple doses) compared to placebo in reducing pain due to knee OA. Secondary Objectives • Evaluating the efficacy of Compound E3 (multiple doses) compared to placebo in improving physical function. • Evaluating the efficacy of Compound E3 (multiple doses) compared to placebo in improving joint stiffness. • Evaluating the efficacy of Compound E3 (multiple doses) compared to placebo in a Patient Global Assessment (PGA). • Evaluating the proportion of responders based on various levels of reduced pain in participants receiving Compound E3 (multiple doses) compared to placebo. • Evaluating rescue medication use in the Compound E3 group (multiple doses) compared to placebo. Inclusion Criteria • Male or female participants between ≥40 and ≤80 years of age • BMI ≤40 kg / m2 • History of knee pain on most days for at least 3 months prior to screening • Confirmation of OA of the Target kneeAttorney Docket No. LEVI0008-401-PC • Radiographs of both knees with a Posterior–Anterior, Fixed–flexion view taken during the Screening Period

[0540] American College of Rheumatology (ACR) clinical and radiographic diagnostic criteria. Evidence of Target knee OA with a KL grade ≥2, determined through central reading.

[0541] Target Knee must have a score of ≥20 out of 50 on the WOMAC NRS 3.1 pain subscale during screening and at randomization.

[0542] The Baseline NRS Pain score were derived from the last seven days of the Diary Run–In Period and must meet following criteria:

[0543] Completion of Average Daily NRS Pain score on at least 6 of the 7 days.

[0544] Mean Average Daily NRS Pain score must be ≥4.0 and ≤9.0.

[0545] Mean Average Daily NRS Pain variability must be ≤1.5.

[0546] Willing to withdraw from any medication for OA including, but not limited to, Opioids (including semisynthetic opioids), Non–Steroidal Anti–inflammatories (NSAIDs), COX–2 inhibitors, Topical medication, and Serotonin and Noradrenaline Reuptake Inhibitors (SNRIs e.g., Duloxetine). Exclusion Criteria

[0547] Presence of OA of other major joints (including but not limited to nontarget knee) that could interfere with assessment of pain due to OA of the target knee, in the opinion of the investigator.

[0548] Current comorbid condition, other than OA, affecting target knee or systemic illness known to be significantly associated with arthritis or joint pathology affecting any joint, including but not necessarily limited to endocrinopathies or autoimmune disease with significant joint involvement (e.g., Rheumatoid Arthritis); Seronegative Spondyloarthropathies (e.g. Ankylosing Spondylitis, Psoriasis arthritis, Reactive arthritis) .

[0549] Pathological conditions significantly affecting joint and bone health, in the opinion of the Investigator should be excluded (including but not necessarily limited to following findings on x–rays and / or MRI): • Known presence of Rapidly Progressive Osteoarthritis (RPOA) (any joint) • Osteonecrosis (including avascular necrosis) (any joint) • Subchondral insufficiency fractures (SIF) (any major joint) Fractures or stress reactions with radiographic signs of ongoing healing processes (including but not limited to osteoporotic and pathological fractures) (any major joint) • Excessive malalignment of the knee (anatomical axis angle greater than 10 degrees) (target knee only)Attorney Docket No. LEVI0008-401-PC • Complete tear of the posterior meniscal root (partial tears and / or anterior meniscal tears not excluded) (either knee) • Large or extensive subchondral cysts (either knee) • Anserine or patellar bursitis (target knee only) • Significant articular bone loss (any joint) • Articular bone fragmentation or collapse (any joint) • Primary or metastatic tumor (any joint) • Joint infection (any joint) • Paget’s disease (any joint) • Osteochondritis dissecans (any joint)

[0550] Hip dislocation and congenital hip dysplasia with degenerative joint disease were excluded. Treatment Regimen

[0551] Participants were randomized to one of three active treatment arms and one placebo arm in 1:1:1:1 ratio. Treatment arms • 0.3 mg / kg dose intravenous infusion of Compound E3 • 1.0 mg / kg dose intravenous infusion of Compound E3 • 2.0 mg / kg dose intravenous infusion of Compound E3 • Placebo dose intravenous infusion Rescue Medication

[0552] Paracetamol, up to a maximum dose of 4000 mg / day, were dispensed by site at Visit 2 (Diary Run–In Visit). Prohibited medications included opioid medication and any other medications for treatment of pain due to OA of the Target Knee including, but not limited to, NSAIDs, topical medication, and SNRIs (e.g., Duloxetine). TRIAL METHODOLOGY

[0553] Participants were randomized to baseline then 4–weekly IV placebo or 0.3, 0.1 or 2 mg / kg Compound E3 through wk16, with safety follow–up to wk30. The primary endpoint was change in WOMAC pain to wk17, with secondary outcomes including function, Patient Global Assessment and >50% pain responders. X–rays of 6 large joints and MRI of kneesAttorney Docket No. LEVI0008-401-PC were utilized for inclusion / exclusion criteria at baseline, and safety evaluation at wk20. ITT analyses were performed using an ANCOVA model (covariates were baseline WOMAC score and site), with Dunnett’s step–down testing procedure comparing the 3 active arms versus placebo. Dosage, Administration and Post–infusion monitoring

[0554] Eligible participants were randomized in the ratio 1:1:1:1 to one of three parallel treatment arms or a placebo control arm according to the following: • Compound E3 intravenous infusion dose 0.3 mg / kg • Compound E3 intravenous infusion dose 1.0 mg / kg • Compound E3 intravenous infusion dose 2.0 mg / kg • Placebo intravenous infusion

[0555] There were five doses administered during the trial: one at each of the following visits: • Randomization (Visit 3, Day 1), 1st dose. • Visit 5 (Week 4), 2nd dose. • Visit 7 (Week 8), 3rd dose. • Visit 8 (Week 12), 4th dose. • Visit 9 (Week 16), 5th and last dose. Pain Assessments

[0556] As an essential part of the eligibility determination process, participants were evaluated based on following pain instruments: The WOMAC Pain subscale, and Average Daily NRS Pain.

[0557] The WOMAC Pain subscale NRS 3.1 (WOPAIN NRS) was completed for both knees during screening prior to starting the Diary Run–In Period, with the requirement that the participant be treatment–naïve or has not used analgesics during the 48 hours prior to completion and must be ≥20 out of 50.

[0558] Participants at Visit 1 who had taken analgesics within the 48 hours prior to Visit 1 had WOPAIN NRS completed prior to or at Visit 2 following the 48–hour analgesic washout. Average Daily Numeric Rating Scale (NRS) Pain ScoreAttorney Docket No. LEVI0008-401-PC

[0559] The Average Daily NRS Pain score measure uses the single 11–point numeric rating scale on which a participant selects a number between 0 (“No pain”) and 10 (“Worst possible pain”) that best reflects their knee pain severity over the past 24–hours. Boston Carpal Tunnel Questionnaire

[0560] The Boston Carpal Tunnel Questionnaire (BCTQ) is a measure of self–reported severity of symptoms and functional status associated with carpal tunnel syndrome. The questionnaire consists of a Symptom Severity scale (11 items) assessing severity of wrist or hand pain, change of sensation, and weakness in hand or wrist the past two weeks and a Functional Status scale (8 items) assessing ability to perform various hand / wrist related tasks. Each item is scored on a 5–point Likert scale from 1 (no symptoms) to 5 (most severe symptoms). The score for the Symptom Severity scale is the mean score of 11 items and the score for the Functional Status scale was the mean score of 8 items. Exclusion criteria is based on the Symptom Severity scale only mean score >3. Survey for Autonomic Symptoms

[0561] The Survey of Autonomic Symptoms consists of two columns with two separate scores: A “Number of symptoms score” and a “Total symptom impact score”. The first score refers to the total score from column Q1a where the participant answers Y / N to if he / she has had the specific symptom during the last 6 months. Yes counts as 1 point, No counts as 0 and total score for the Number of symptoms score ranges from 0–11 (males) and 0–12 (females). The “Total symptom impact score” is the total score from the second column, Q1b and was scored on impact of the symptoms the participant answered yes to having, ranging from 1 (“not bothered by this symptom”) to 5 (“bothered a lot”) (total score range 0–60). The Number of symptoms score was used to determine eligibility, with exclusion of participants with a score of > 3 (4 or more). Radiographic Assessments

[0562] For evaluation of relevant inclusion and exclusion criteria, radiographs of both knees and both shoulders and hips were taken during the screening period and at the end of the study. Knee radiographs acquired within three months prior to screening may be submitted for central reading, provided they meet the study requirements for acquisition and quality. Participants with partial or total joint replacements of any minor and major joints were not excluded from the trial, if the participant is otherwise considered eligible and central radiographic responsible personnel consider the prothesis compatible and safe upon imagingAttorney Docket No. LEVI0008-401-PC with results at sufficient quality. Conversely, any partial or total knee replacement of the target knee was exclusionary.

[0563] Participants with non–target knee joint replacements were considered ineligible but radiographic assessments provided additional information on joint and bone health related to the joint replacement, which could render the participant suitable for the study. MRI Assessments

[0564] Participants had MRI of both knees conducted prior to randomization and read centrally for confirmation of eligibility and selection of the Target Knee. A follow–up MRI of the Target Knee was only performed at FU / EOT. Other Physical Assessments Staircase Evoked Pain Procedure (StEPP)

[0565] The StEPP is a stress test and performance–based outcome measure developed to improve the sensitivity to detecting analgesic effect in clinical trials of knee OA. The test consists of stepping fully up onto a 20–cm high platform with one foot, then the other foot and back down (alternating lead leg at each up / down cycle), for a total of 24 times over a 5– minute period. Pain intensity assessments were performed for the Target Knee, immediately before and after the exercise on a scale of 0 to 10, with 0 representing “No pain” and 10 representing “Worst possible pain”. Musculoskeletal Exam

[0566] The musculoskeletal exam was completed as part of the physical exam at Visit 3, Visit 5, Visit 7, Visit 8, Visit 9 and Visit 11. The Musculoskeletal Exam includes assessments of shoulder–, hip– and knee joints. Each shoulder was assessed for range of motion, abduction, internal and external rotation. Both hip joints were examined for range of motion, internal and external rotation of groin or buttocks. Both knees were inspected for deformities, varus / valgus alignment and palpated for effusion, crepitus, and tenderness. Laboratory Tests Blood samples

[0567] All efficacy measures were completed prior to blood draws to limit any potential influence of the procedure on the general perception of pain. Laboratory values for an analyte that was outside normal range per the central laboratory were identified and repeated at the Investigator’s discretion. Serum Sampling for PharmacodynamicsAttorney Docket No. LEVI0008-401-PC

[0568] Blood samples for measurement of the pharmacokinetics (PK) of Compound E3 were collected at two timepoints during dosing visits (Visit 3, 5, 7, 8 and 9). Prior to administration of Compound E3, a pre–dose PK sample was taken, and following administration, a post–dose PK sample was taken. An additional 2mL of blood was taken at selected timepoints for the testing of biomarkers. Blood samples for pharmacodynamic biomarkers were collected prior to administration of Compound E3 and at Randomization (Visit 3) and Visit 5 (week 4) and at Visit 11. The PD biomarkers included biomarkers for inflammation and neurotrophin engagement associated with the mode of action of NGF and Neurotrophin–3 (NT3). Target Knee Selection

[0569] Target Knee were selected following completion of Radiographs, Diary Run–In Period and MRI visit and must meet following criteria (See Selection of Trial Population): • Confirmed OA based on ACR clinical and radiographic diagnostic criteria • Knee pain on most days for at least 3 months prior to Screening • KL grade of 2–4 (both inclusive) • WOMAC Pain subscale score of ≥20 out of 50 at Screening (Visit 1 or 2) and Randomization (Visit 3) • Baseline NRS Pain score derived from the last seven days of the Diary Run–In Period (Day 10– 14) which must meet following criteria: − Completion of Average Daily NRS Pain score on at least 6 of the 7 days − Mean Average Daily NRS Pain score between ≥4.0 and ≤9.0 − Mean Average Daily NRS Pain score variability ≤1.5 − No presence of pathology on Radiographs or MRI that would render the participant unsuitable for the study − Pain in nontarget knee due to OA, was required as per exclusion criterion #1 to not interfere with assessment of the Target Knee pain − If both knees meet all criteria, the dominant knee as determined by the Investigator was selected as the Target Knee − The Target Knee remained unchanged throughout the trial Diary Run–In Period

[0570] Participants were required to daily, in the evenings, enter their Average Daily NRS Pain for each knee and any use of Rescue Medication in the eDiary. After completion of the Diary Run–In Period and confirmation of eligibility, participants continued to report theirAttorney Docket No. LEVI0008-401-PC Average Daily NRS Pain scores for each knee in the eDiary until the Target Knee was selected at Visit 3 (Randomization). RESULTS

[0571] 518 people with knee OA were enrolled, balanced across groups (mean age 63.1– 65.4 years, mean BMI 29.3–30.3, female participants 51.5–61.5%). Statistical significance was met for the primary and secondary efficacy endpoints (FIG.1A, FIG.1B and FIG.1C) at wk5 and wk17 (p<0.05 vs placebo, all doses). More than 50% of the Compound E3–treated patients reported ≥50% reduction in pain and >25% reported ≥75% reduction at weeks 5 and 17. Compound E3 was well tolerated, with no increased incidence of SAEs, TEAEs and joint pathologies including rapidly progressive OA (Tables 2 and 3) compared to placebo. Incidence of anti–drug antibodies was low: 9 participants tested positive pre–dosing, 6 at week 5 or week 20, all at the lowest limit of detection.

[0572] Compound E3 demonstrated significant differences as compared to placebo at Week 17 for all primary and secondary endpoints for all doses evaluated: • The primary endpoint was WOMAC pain assessment (change from baseline at Week 17). The mean reduction in WOMAC pain score from baseline was greater than 50% for all three doses of Compound E3 and all statistically different to placebo. • Secondary endpoints were WOMAC subscales of function and joint stiffness, patient global assessment and daily pain scores and all statistically different to placebo.

[0573] Standard safety monitoring plus peripheral nervous system assessments showed Compound E3 to be well tolerated. There was no increase in incidence of rapidly progressive osteoarthritis (RPOA) as measured via detailed, closely examined, radiographic analysis. Efficacy

[0574] Statistically significant (p<0.05) reductions in mean WOMAC pain scores were observed with all Compound E3 dose groups compared to placebo at the Week 17 timepoint (primary efficacy endpoint). Overall, significant improvements with Compound E3 were also observed in the primary and secondary endpoints including WOMAC pain (interim timepoints), WOMAC physical function scores, WOMAC stiffness scores, WOMAC total scores, and StEPP pain intensity scores. In addition, significant improvements with Compound E3 were also observed in NRS and PGA scores.

[0575] Thus, Compound E3 demonstrated significant and clinically meaningful improvement in pain, function and other outcomes. Compound E3 was well tolerated at allAttorney Docket No. LEVI0008-401-PC doses studied, supporting the concept of supplementing endogenous p75NTR as a treatment for OA and other pain conditions. Primary Endpoint

[0576] The primary endpoint was a change in the WOMAC Pain subscale from Randomization (Visit 3) to Visit 10 (week 17). A statistically significant (p<0.05) reduction in mean WOMAC pain scores was observed with all Compound E3 dose groups compared to placebo at the Week 17 timepoint. Secondary Endpoints

[0577] A secondary endpoint was a change in the WOMAC Pain subscale from Randomization (Visit 3) to Visit 6 (week 5).

[0578] A statistically significant (p<0.05) reduction in mean WOMAC pain scores was observed with all Compound E3 dose groups compared to placebo at the Week 5 and Week 17 timepoints.

[0579] A secondary endpoint was a change in the WOMAC Physical function subscale from Randomization (Visit 3) to Visit 6 (week 5) and Visit 10 (week 17).

[0580] A statistically significant (p<0.05) reduction in mean WOMAC physical function scores was observed with all Compound E3 dose groups compared to placebo at the Week 5 and Week 17 timepoints.

[0581] A secondary endpoint was a change in the WOMAC Stiffness subscale from Randomization (Visit 3) to Visit 6 (week 5) and Visit 10 (week 17).

[0582] A statistically significant (p<0.05) reduction in mean WOMAC stiffness scores was observed with all Compound E3 dose groups compared to placebo at the Week 5 and Week 17 timepoints.

[0583] A secondary endpoint was a change in the StEPP from Randomization (Visit 3) to Visit 6 (week 5) and Visit 10 (week 17).

[0584] A statistically significant (p<0.05) reduction in mean StEPP pain intensity scores was observed with all Compound E3 dose groups compared to placebo at the Week 5 and Week 17 timepoints.

[0585] A secondary endpoint was a change in PGA from Visit 6 (week 5) to Visit 10 (week 17).

[0586] A statistically significant (p<0.05) reduction in mean PGA scores was observed with all Compound E3 dose groups compared to placebo at the Week 5 and Week 17 timepoints.Attorney Docket No. LEVI0008-401-PC

[0587] A secondary endpoint was participants achieving 30% and 50% reduction in WOMAC Pain subscale at week 5 and week 17 using a cumulative distribution function.

[0588] A statistically significant (p<0.05) proportion of participants had a ≥30% reduction in mean WOMAC pain scores with all Compound E3 dose groups compared to placebo at Week 5.

[0589] A statistically significant (p<0.05) proportion of participants had a ≥50% reduction in mean WOMAC pain scores with all Compound E3 dose groups compared to placebo at Week 5 and 17 (with the exception of the 0.3 mg / kg Compound E3 dose group at Week 17).

[0590] A secondary endpoint was reducing rescue medication usage during the trial.

[0591] Numerical reductions in the average daily rescue medication use were observed with all Compound E3 dose groups but no statistically significant differences were observed compared to placebo.

[0592] A secondary endpoint was a change in average weekly NRS Pain score from Baseline to Visit 6 (week 5) and Visit 10 (week 17).

[0593] Numerical reductions in NRS scores were observed in all Compound E3 dose groups compared to placebo beginning at Week 1 with statistical significance observed with the 0.3 and 2.0 mg / kg Compound E3 dose groups beginning at Week 2. A consistent statistically significant (p<0.05) reduction in mean NRS scores was observed with the 2.0 mg / kg Compound E3 group compared to placebo from Week 5 to Week 20.

[0594] A secondary endpoint was a reduction in area under the curve (AUC) of Average Daily NRS Pain score from Baseline to Visit 11 (week 20).

[0595] A statistically significant (p<0.05) reduction in the average daily NRS area under the curve (AUC) result was observed with the 2.0 mg / kg Compound E3 group compared to placebo. Table 3. Serious Adverse Events and Treatment Emergent Adverse EventsAttorney Docket No. LEVI0008-401-PC 0.3 mg / kg 1.0 mg / kg 2.0 mg / kg Placebo Event versus Dose (N=129) (N=130) (N=129) (N=129) 4) 1) 8) 1) 8) 1) 4)Attorney Docket No. LEVI0008-401-PC Table 4. Incidence of Rapidly Progressive OA Type 1 & 2 (R1, R2), Subchondral Insufficiency Fracture (SIF), Osteonecrosis (ON), and Total Joint Replacements (TJR), n (%) All joint events Kellgren–Lawrenceall TJRs Kellgren–Lawrence grade 4 † on entry *Elective surgery; OA of the shoulder present at baselineAttorney Docket No. LEVI0008-401-PC REFERENCE LIST AALBERSE, R. C. & SCHUURMAN, J.2002. IgG4 breaking the rules. Immunology, 105, 9–19. ANEKAR, A. A., HENDRIX, J. M. & CASCELLA, M.2023. WHO analgesic ladder. StatPearls [Internet]. StatPearls Publishing. COHEN, T. J., GUO, J. L., HURTADO, D. E., KWONG, L. K., MILLS, i. P., TROJANOWSKI, J. Q. & LEE, V. M.2011. The acetylation of tau inhibits its function and promotes pathological tau aggregation. Nature communications, 2, 252. CONAGHAN, P. G., DWORKIN, R. H., SCHNITZER, T. J., BERENBAUM, F., BUSHMAKIN, A. G., CAPPELLERI, J. C., VIKTRUP, L. & ABRAHAM, L.2022. WOMAC meaningful within–patient change: results from 3 studies of tanezumab in patients with moderate–to–severe osteoarthritis of the hip or knee. The Journal of Rheumatology, 49, 615–621. CONTROL, C. F. D. & PREVENTION 2001. Prevalence of disabilities and associated health conditions among adults––United States, 1999. MMWR. Morbidity and mortality weekly report, 50, 120–125. DILLON, T. M., RICCI, M. S., VEZINA, C., FLYNN, G. C., LIU, Y. D., REHDER, D. S., PLANT, M., HENKLE, B., LI, Y. & DEECHONGKIT, S.2008. Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. Journal of Biological Chemistry, 283, 16206–16215. EDELMAN, G. M.2004. Biochemistry and the sciences of recognition. Journal of Biological Chemistry, 279, 7361–7369. HEFTI, F. F., ROSENTHAL, A., WALICKE, P. A., WYATT, S., VERGARA, G., SHELTON, D. L. & DAVIES, A. M.2006. Novel class of pain drugs based on antagonism of NGF. Trends in pharmacological sciences, 27, 85–91. HOCHBERG, M.2015. Serious joint–related adverse events in randomized controlled trials of anti–nerve growth factor monoclonal antibodies. Osteoarthritis and cartilage, 23, S18–S21. HUMPHREYS, K., SHOVER, C. L., ANDREWS, C. M., BOHNERT, A. S., BRANDEAU, M. L., CAULKINS, J. P., CHEN, J. H., CUÉLLAR, M.–F., HURD, Y. L. & JUURLINK, D. N.2022. Responding to the opioid crisis in North America and beyond: recommendations of the Stanford–Lancet Commission. The Lancet, 399, 555–604. HUNTER, D. J., MCDOUGALL, J. J. & KEEFE, F. J.2008. The symptoms of osteoarthritis and the genesis of pain. Rheumatic Disease Clinics of North America, 34, 623–643. MCGLOTHLIN, A. E. & LEWIS, R. J.2014. Minimal clinically important difference: defining what really matters to patients. Jama, 312, 1342–1343. MCNEIL, J. & BINETTE, J.2001. Prevalence of Disabilities and Associated Health Conditions Among Adults––United States, 1999. JAMA: Journal of the American Medical Association, 285.Attorney Docket No. LEVI0008-401-PC SCHLOTHAUER, T., HERTER, S., KOLLER, C. F., GRAU–RICHARDS, S., STEINHART, V., SPICK, C., KUBBIES, M., KLEIN, C., UMAÑA, P. & MÖSSNER, E.2016. Novel human IgG1 and IgG4 Fc–engineered antibodies with completely abolished immune effector functions. Protein Engineering, Design and Selection, 29, 457–466. SCHNITZER, T. & MARKS, J.2015. A systematic review of the efficacy and general safety of antibodies to NGF in the treatment of OA of the hip or knee. Osteoarthritis and cartilage, 23, S8–S17. WALKER, B. J., POLANER, D. M. & BERDE, C. B.2019. Acute pain. A practice of anesthesia for infants and children. Elsevier. WONG, A. Y., SAMARTZIS, D. & MAHER, C.2023. The global burden of osteoarthritis: past and future perspectives. The Lancet Rheumatology, 5, e496–e497. ZELAYA, C. E., DAHLHAMER, J. M., LUCAS, J. W. & CONNOR, E. M.2020. Chronic pain and high–impact chronic pain among US adults, 2019.

[0596] All references, patents or applications, U.S. or foreign, cited in the application are hereby incorporated by reference as if written herein in their entireties. Where any inconsistencies arise, material literally disclosed herein controls.

[0597] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

Attorney Docket No. LEVI0008-401-PC CLAIMS What is claimed is:

1. A method of treating pain associated with osteoarthritis, comprising administering to a human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions wherein after treatment with the composition, the subject is a responder to treatment by at least one measure of clinical response.

2. A method of treating pain associated with osteonecrosis, comprising administering to a human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions.

3. A method of treating pain associated with one or more subchondral insufficiency fractures, comprising administering to a human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, where x is 1, 2, 3, 4, 5 or 6 connecting the p75NTR(NBP) and Fc portions.

4. A method of improving physical function in a human subject with osteoarthritis, osteonecrosis, or one or more subchondral insufficiency fractures, comprising administering to the human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising:Attorney Docket No. LEVI0008-401-PC a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions.

5. A method of treating osteoarthritis, or delaying or slowing the progression of osteoarthritis, or relieving a symptom of osteoarthritis, in a human subject, comprising administering to the human subject in need thereof one or more doses of a therapeutically effective amount of a composition comprising a glycosylated p75NTR neurotrophin binding protein (NBP)-Fc fusion protein, comprising: a p75NTR(NBP) portion, having at least 85% sequence identity with SEQ ID NO:3; an immunoglobulin Fc portion; and a linker of formula Gx, wherein x is 1, 2, 3, 4, 5 or 6, connecting the p75NTR(NBP) and Fc portions.

6. The method of any of claims 1-5, wherein the linker is GGG and the Fc is a human Fc.

7. The method of any of claims 1-6, wherein the p75NTR(NBP) portion has at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:

3.

8. The method of claim 7, wherein the p75NTR(NBP) portion has SEQ ID NO:

3.

9. The method of any of claims 1-9, wherein the fusion protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:

1.

10. The method of claim 9, wherein the fusion protein has SEQ ID NO:

1.

11. The method of any of claims 1-11, wherein the fusion protein has glycosylation at one or more of positions chosen from positions 32, 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, 222, and 294.

12. The method of any of claims 1-11, wherein the fusion protein has glycosylation at one or more of positions chosen from positions 169, 171, 172,179, 180, 183, 184, 198, 199, 205, and 206.

13. The method of any of claims 11-12, wherein the fusion protein has glycosylation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the positions.

14. The method of any of claims 11-12, wherein the fusion protein has glycosylation at 1-3, 1-6, 1-9, 1-12, 1-16, 3-6, 3-9, 3-12, 3-16, 6-9, 6-12, 6-16, 7-9, 7-10, 8-9, 9-12, or 9-16 of the positions.

15. The method of claim 14, wherein the fusion protein has glycosylation at 6-12 of the positions.Attorney Docket No. LEVI0008-401-PC 16. The method of claim 14, wherein the fusion protein has glycosylation at 7-10 of the positions.

17. The method of claim 14, wherein the fusion protein has glycosylation at positions 32, 294, and 5-8 of the positions chosen from positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, and 222.

18. The method of claim 17, wherein the fusion protein has glycosylation at positions 32, 294, and 7 of the positions chosen from positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, and 222.

19. The method of claim 14, wherein the fusion protein has glycosylation at positions 32, 294, and 5-8 of the positions chosen from positions 169, 171, 172,179, 180, 183, 184, 198, 199, 205, and 206.

20. The method of claim 19, wherein the fusion protein has glycosylation at positions 32, 294, and 7 of the positions chosen from positions 169, 171, 172,179, 180, 183, 184, 198, 199, 205, and 206.

21. The method of any of claims 1-20, wherein the fusion protein comprises N- glycosylation (i.e., is an N-glycan variant).

22. The method of any of claims 1-21, wherein the fusion protein composition of claim 2, having has at least one glycan chosen from G0F, G1F, G3F, G2FS1 and G2FS2.

23. The method of claim 22, wherein a. G0F represents about 19% - about 32% of the total neutral N-glycan population, b. G1F represents about 21% - about 31%, of the total neutral N-glycan population, c. G3F represents about 5% - about 10% of the total neutral N-glycan population, d. G2FS1 represents about 2% - about 8% of the total neutral glycan population; and e. G2FS2 represents about 10% - about 20% of the total neutral glycan population.

24. The method of claim 23, wherein a. G0F represents about 20% - about 25% of the total neutral N-glycan population, b. G1F represents about 26% - about 28%, of the total neutral N-glycan population, c. G3F represents about 7% - about 8% of the total neutral N-glycan population, d. G2FS1 represents about 4% - about 6% of the total neutral glycan population; and e. G2FS2 represents about 14% - about 17% of the total neutral glycan population.

25. The method of claim 24, wherein a. G0F represents about 22% - about 24% of the total neutral N-glycan population, b. G1F represents about 26% - about 28%, of the total neutral N-glycan population, c. G3F represents about 7% - about 8% of the total neutral N-glycan population,Attorney Docket No. LEVI0008-401-PC d. G2FS1 represents about 4% - about 6% of the total neutral glycan population; and e. G2FS2 represents about 14% - about 17% of the total neutral glycan population.

26. The method of any of claims 1-25, wherein SEQ ID NO:1 is glycosylated at Asn32.

27. The method of claim 26, wherein the N-glycan at Asn32 is chosen from F(6)A2G(4)2S(3)2 and F(6)A2G(4)2S(3)1.

28. The method of claim 26, wherein the predominant glycan at Asn32 is G2FS2.

29. The method of claim 26 wherein the N glycan at Asn32 is F(6)A2G(4)2S(3)2.

30. The method of any of claims 1-29, wherein SEQ ID NO:1 is glycosylated at Asn294.

31. The method of any of claims 1-29, wherein SEQ ID NO:1 is glycosylated at Asn32 and Asn294.

32. The method of any of claims 30-31, wherein the glycan at Asn294 is G1F.

33. The method of any of claims 30-31, wherein the glycan at Asn294 is G0F.

34. The method of any of claims 30-31, wherein the glycan at Asn294 is chosen from F(6)A2, F(6)A2[6}G(4)1, and F(6)A2[3}G(4)1.

35. The method of any of claims 30-31, wherein the glycan at Asn294 is F(6)A2.

36. The method of any of claims 30-31, wherein the glycosylated fusion protein is a mixture of glycosylation, and wherein the glycan at Asn294 is chosen from G0F and G1F, or a mixture thereof.

37. The method of any of claims 1-36, wherein the glycans at any one or more of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, and 222 are chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

38. The method of any of claims 1-36, wherein the glycans at each of positions 169, 171, 172, 179, 180, 183, 184, 198, 199, 205, 206, 216, 217, and 222, if present, are chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

39. The method of claim 38, wherein the glycan at position 169 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

40. The method of claim 38, wherein the glycan at position 169 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

41. The method of claim 38, wherein the glycan at position 169 is chosen from HexNAc(1) and HexNAc(1)Hex(1).Attorney Docket No. LEVI0008-401-PC 42. The method of claim 38, wherein the glycan at position 169 is HexNAc(1)Hex(1)NeuAc(2).

43. The method of any of claims 1-36, wherein the glycan at position 171 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

44. The method of claim 43, wherein the glycan at position 171 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

45. The method of claim 43, wherein the glycan at position 171 is chosen from HexNAc(1) and HexNAc(1)Hex(1).

46. The method of claim 43, wherein the glycan at position 171 is HexNAc(1)Hex(1).

47. The method of claim 43, wherein the glycan at position 171 is HexNAc(1)Hex(1)NeuAc(2).

48. The method of any of claims 1-36, wherein the glycan at position 172 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

49. The method of claim 48, wherein the glycan at position 172 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

50. The method of claim 48, wherein the glycan at position 172 is chosen from HexNAc(1) and HexNAc(1)Hex(1).

51. The method of claim 48, wherein the glycan at position 172 is HexNAc(1)Hex(1).

52. The method of claim 48, wherein the glycan at position 172 is HexNAc(1)Hex(1)NeuAc(1).

53. The method of any of claims 1-36, wherein the glycan at position 179 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

54. The method of claim 53, wherein the glycan at position 179 is chosen from HexNAc(1)Hex(1) and HexNAc(1)Hex(1)NeuAc(2).

55. The method of claim 53, wherein the glycan at position 179 is HexNAc(1)Hex(1).

56. The method of claim 53, wherein the glycan at position 179 is HexNAc(1)Hex(1)NeuAc(2).

57. The method of any of claims 1-36, wherein the glycan at position 180 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).Attorney Docket No. LEVI0008-401-PC 58. The method of claim 57, wherein the glycan at position 180 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2) .

59. The method of claim 57, wherein the glycan at position 180 is HexNAc(1)Hex(1)NeuAc(2).

60. The method of any of claims 1-36, wherein the glycan at position 183 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

61. The method of claim 60, wherein the glycan at position 183 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

62. The method of claim 60, wherein the glycan at position 183 is chosen from HexNAc(1), HexNAc(1)Hex(1), and HexNAc(1)Hex(1)NeuAc(2).

63. The method of claim 60, wherein the glycan at position 183 is chosen from HexNAc(1) and HexNAc(1)Hex(1).

64. The method of any of claims 1-36, wherein the glycan at position 184 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

65. The method of claim 65, wherein the glycan at position 184 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2) .

66. The method of claim 65, wherein the glycan at position 184 is chosen from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

67. The method of any of claims 1-36, wherein the glycan at position 185 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

68. The method of claim 67, wherein the glycan at position 185 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2) .

69. The method of claim 67, wherein the glycan at position 185 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

70. The method of any of claims 1-36, wherein the glycan at position 198 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

71. The method of claim 70, wherein the glycan at position 198 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

72. The method of claim 70, wherein the glycan at position 198 is chosen from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2) .Attorney Docket No. LEVI0008-401-PC 73. The method of claim 70, wherein the glycan at position 198 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

74. The method of any of claims 1-36, wherein the glycan at position 199 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

75. The method of claim 74 wherein the glycan at position 199 is chosen from HexNAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

76. The method of claim 74, wherein the glycan at position 199 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

77. The method of any of claims 1-36, wherein the glycan at position 205 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

78. The method of claim 77, wherein the glycan at position 205 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

79. The method of any of claims 1-36, wherein the glycan at position 206 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

80. The method of claim 79, wherein the glycan at position 206 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

81. The method of any of claims 1-36, wherein the glycan at position 216 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

82. The method of claim 81, wherein the glycan at position 216 is chosen from HexNAc(1) and HexNAc(1)Hex(1).

83. The method of claim 81, wherein the glycan at position 216 is HexNAc(1).

84. The method of any of claims 1-36, wherein the glycan at position 217 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

85. The method of claim 84, wherein the glycan at position 217 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

86. The method of claim 84, wherein the glycan at position 217 is chosen from HexNAc(1), HexNAc(1)Hex(1), and HexNAc(1)Hex(1)NeuAc(1).Attorney Docket No. LEVI0008-401-PC 87. The method of any of claims 1-36, wherein the glycan at position 222 is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

88. The method of claim 87, wherein the glycan is chosen from HexNAc(1), HexNAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2).

89. The method of claim 87, wherein the glycan at position 222 is chosen from HexNAc(1), HexNAc(1)Hex(1), and HexNAc(1)Hex(1)NeuAc(2).

90. The method of claim 87, wherein the glycan at position 222 is chosen from HexNAc(1), HexNAc(1)Hex(1), and HexNAc(1)Hex(1)NeuAc(1).

91. The method of claim 87, wherein the glycan at position 222 is chosen from HexNAc(1) and HexNA(1)Hex(1).

92. The method of any of claims 1-91, wherein the fusion protein comprises O- glycosylation (i.e., is an O-glycan variant).

93. The method of claim 92, wherein there is O-linked glycosylation at 6-12, 7-10, 5-8 or 7 sites on SEQ ID NO:1 or SEQ ID NO:

3.

94. The method of claim 92, wherein an O-linked glycan is HexNAc1Neu5NAc1Hex1.

95. The method of claim 92, wherein an O linked glycan is HexNAc1Neu5NAc2Hex1.

96. The method of any of claims 92-95, wherein an O-linked glycan is sialylated with 1-2 sialic acid molecules.

97. The method of any of claims 92-96, containing 16-17 moles of sialic acid molecules to every mole of the monomer of SEQ ID NO:1 or SEQ ID NO:

3.

98. The method of any of claims 92-96, wherein there are 16.55-16.65 sialic acid molecules on each glycosylated monomer of SEQ ID NO:1 or SEQ ID NO:

3.

99. The method of any of claims 96-98, wherein the sialic acids are chosen from N- Acetylneuraminic acid (Neu5Ac) and N-glycolyl neuraminic acid (Neu5Gc).

100. The method of claim 99, wherein there are 16.1-16.35 molecules of Neu5Ac for each monomer of SEQ ID NO:1 or SEQ ID NO:

3.

101. The method of claim 99, wherein there are 0.255-0.265 molecules of Neu5G for each monomer of SEQ ID NO:1 or SEQ ID NO:

3.

102. The method of any one of claims 1-101, wherein the composition is administered according to a dosing regimen comprising one or more dosing cycles.

103. The method of claim 102, wherein the dosing cycle is a 4-week dosing cycle comprising a single administration.Attorney Docket No. LEVI0008-401-PC 104. The method of claim 102, wherein the dosing regimen comprises 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, or 48 weeks of treatment.

105. The method of claim 102, the dosing regimen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ,11, or 12 dosing cycles.

106. The method of any one of claims 102-105, wherein administration comprises at least about 0.1 mg / kg, at least about 0.3 mg / kg, at least about 1.0 mg / kg, or at least about 2.0 mg / kg of the composition.

107. The method of any one of claims 102-105, wherein administration comprises about 0.1 mg / kg to about 2.0 mg / kg, about 0.1 mg / kg to about 1.0 mg / kg, or about 0.1 mg / kg to about 0.3 mg / kg of the composition.

108. The method of any one of claims 102-105, wherein administration comprises about 0.1 mg / kg, about 0.3 mg / kg, about 1.0 mg / kg, or about 2.0 mg / kg of the composition.

109. The method of any of claims 1-108 wherein the administration is by intravenous injection or infusion.

110. The method of any of claims 1-109, wherein the glycosylated protein binds to any of NGF, BDNF, NT3 or NT4 / 5 with a binding affinity (Kd) of between about 0.001 nM to about 50 nM.

111. The method of any one of claims 1-110, wherein after treatment with the composition, the subject is a responder to treatment by at least one measure of clinical response.

112. The method of claim 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean WOMAC pain scores compared to placebo based on patient-reported outcomes measurements (PROMs).

113. The method of claim 112, wherein the reduction in mean WOMAC pain scores occurs after 1 week of treatment, 2 weeks of treatment, 3 weeks of treatment, 4 weeks of treatment, 5 weeks of treatment, 6 weeks of treatment, 7 weeks of treatment, 8 weeks of treatment, 9 weeks of treatment, 10 weeks of treatment, 11 weeks of treatment, 12 weeks of treatment, 13 weeks of treatment, 14 weeks of treatment, 15 weeks of treatment, 16 weeks of treatment, or 17 weeks of treatment.

114. The method of claim 111, wherein the at least one measure of clinical response is at least about a ≥30% reduction in mean WOMAC pain scores compared to placebo based on patient-reported outcomes measurements (PROMs) by week 5.

115. The method of claim 111, wherein the at least one measure of clinical response is at least about a ≥50% reduction in mean WOMAC pain scores compared to placebo based on patient-reported outcomes measurements (PROMs) by week 17.Attorney Docket No. LEVI0008-401-PC 116. The method of claim 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean WOMAC physical functionality scores compared to placebo based on patient-reported outcomes measurements (PROMs).

117. The method of claim 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean WOMAC stiffness scores compared to placebo based on patient-reported outcomes measurements (PROMs).

118. The method of claim 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean StEPP pain intensity scores compared to placebo based on patient-reported outcomes measurements (PROMs).

119. The method of claim 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) reduction in mean PGA scores compared to placebo based on patient-reported outcomes measurements (PROMs).

120. The method of claim 111, wherein the at least one measure of clinical response is at least about a ≥30% reduction in mean WOMAC physical functionality scores compared to placebo based on patient-reported outcomes measurements (PROMs) by week 5.

121. The method of claim 111, wherein the at least one measure of clinical response is at least about a ≥30% reduction in mean WOMAC stiffness scores compared to placebo based on patient-reported outcomes measurements (PROMs) by week 5.

122. The method of claim 111, wherein the at least one measure of clinical response is a statistically significant (p<0.05) or clinically meaningful reduction in mean NRS scores compared to placebo from week 5 to week 20.

123. The method of any of claims 1-122, wherein the at least one measure of clinical response is a statistically significant (p<0.05) or clinically meaningful reduction in the average daily NRS area under the curve (AUC) results compared to placebo.

124. The method of any of claims 1-3 and 6-115, 118, 119, 122, and 123, wherein reducing pain comprises at least a 1-point difference, at least a 2-point difference, at least a 3- point difference, or at least a 4-point difference in mean change from a baseline WOMAC pain score.

125. The method of any of claims 1-3 and 6-115, 118, 119, 122, and 123, wherein reducing pain comprises a 1-point to 4-point difference, a 1-point to 3-point difference, a 1-point to 2-point difference, a 2-point to 4-point difference, a 3-point to 4-point difference, a 4- point difference, a 3-point difference, a 2-point difference, or a 4-point difference in mean change from a baseline WOMAC pain score.Attorney Docket No. LEVI0008-401-PC 126. The method of any of claims 1-125 comprising administering the composition as a 4- weekly dose, wherein the method is capable of achieving a statistically significant or clinically meaningful reduction in pain as evaluated by WOMAC, StEPP or NRS as compared to placebo by week 17.

127. The method of any of claims 1 and 4-126, wherein the osteoarthritis is osteoarthritis of the knee, hip, shoulder, hands, neck, and / or lower back 128. The method of claim 127, wherein the osteoarthritis is osteoarthritis of the knee.

129. The method of any of claims 1-128, wherein the composition is formulated for intravenous administration.

130. The method of any of claims 1-129, wherein the composition is administered to the subject separately, sequentially, or simultaneously in combination with a second pharmacologically active compound.

131. The method of any of claims 1 and 4-130, wherein relief from the symptoms of osteoarthritis comprises one or more of reduction in pain, inflammation, swelling, tenderness, joint stiffness, and increase in joint mobility, or any combination thereof.

132. The method of any of claims 1-131, wherein the composition comprises a glycosylated variant of SEQ ID NO:1 or SEQ ID NO:3, and the glycosylated variant shows selectivity for NT3 over NGF.

133. The method of claim 132, wherein the composition comprises a glycosylated variant of SEQ ID NO:1 or SEQ ID NO:3, and the glycosylated variant shows selectivity for NT3 over NGF on the order of 50-1000x over the NGF KD.

134. The method of any of claims 1-133, wherein the composition has increased inhibition of NT3 and decreased inhibition of NGF.

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