Fusion protein

Abstract

This disclosure relates to neurotrophins, fusion proteins comprising monomeric Ig Fc domains or fragments thereof, and methods for producing them.

Description

[Technical Field]

[0001] Related application data This application claims priority from Australian Patent Application No. 2023 / 902574, filed on 14 August 2023, entitled “A Fusion Protein.” The entire contents of this application are incorporated herein by reference.

[0002] Sequence List This application is filed together with an electronic sequence listing. The entire contents of the sequence listing are incorporated herein by reference.

[0003] This disclosure relates to neurotrophins, fusion proteins comprising monomeric Ig Fc domains or fragments thereof, and methods for producing them. [Background technology]

[0004] Neurotrophins are a family of four structurally and functionally related proteins that regulate the growth, maintenance, and apoptosis of neurons and injured neurons in the developing nervous system. In mammals, there are four neurotrophins: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4), the latter also known as neurotrophin 5 (NT-4 / 5).

[0005] Many neurotrophins are currently undergoing clinical trials and hold potential for treating various diseases and disorders. However, due to their low concentrations in mammalian tissues, isolating neurotrophins from natural sources is technically difficult, expensive, and impractical. Recently, attempts have focused on developing efficient methods for neurotrophin production.

[0006] Mammalian expression systems produce neurotrophins in low yields, are labor- and reagent-intensive, and consequently are not practical for scale-up. Escherichia coli expression systems lack the ability to produce the disulfide bonds present in neurotrophins. Therefore, neurotrophins often fold incorrectly and tend to aggregate into inactive complexes, requiring extensive downstream processing to achieve biologically active dimers. Yeast can offer a high degree of initial purity, but yields are low, and expressed neurotrophins may be partially inactive.

[0007] Therefore, it will be clear to those skilled in the art that there is a need for improved methods for producing neurotrophins in the art. [Overview of the project]

[0008] In the process leading to the present invention, the inventors attempted to provide an improved method for producing neurotrophins such that the neurotrophins (i) have improved expression levels (e.g., high yield), and / or (ii) can form homodimers, and / or (iii) have improved biological activity, and / or (iv) have increased receptor binding affinity. To this end, the inventors have surprisingly identified a fusion protein that enables the production of neurotrophins with improved expression levels and high yield in mammalian cells. Advantageously, the fusion protein can form homodimers with improved biological activity and / or improved binding affinity.

[0009] Based on the foregoing, the present disclosure provides a fusion protein comprising (i) a neurotrophin and (ii) a monomer immunoglobulin (Ig) crystallizable fragment (Fc) domain or fragment thereof.

[0010] This disclosure is, (i) neurotrophins, and (ii) Provide a fusion protein comprising a monomer immunoglobulin (Ig) crystallizable fragment (Fc) domain or a fragment thereof.

[0011] In one example, neurotrophins form homodimers.

[0012] In one example, the fusion protein exhibits improved expression levels compared to wild-type or unmodified neurotrophins.

[0013] In one example, the fusion protein is characterized by increased activation of the tyrosine receptor kinase (Trk) receptor compared to the wild-type neurotrophin.

[0014] In one example, a Trk receptor can be a TrkA receptor, a TrkB receptor, a TrkC receptor, or a combination thereof. For example, the Trk receptor is a TrkA receptor. In another example, the Trk receptor is a TrkB receptor. In yet another example, the Trk receptor is a TrkC receptor.

[0015] In one example, the fusion protein is characterized by increased activation of the TrkA receptor compared to the wild-type neurotrophin. In another example, the fusion protein is characterized by increased activation of the TrkB receptor compared to the wild-type neurotrophin. In yet another example, the fusion protein is characterized by increased activation of the TrkC receptor compared to the wild-type neurotrophin.

[0016] For example, the fusion protein has an affinity constant (K) of 400-800 nM. D ) and binds to the TrkA receptor at neutral pH.

[0017] For example, the fusion protein has an affinity constant (K) of 20-60 nM. D ) and binds to the TrkB receptor at neutral pH.

[0018] For example, the fusion protein has an affinity constant (K) of 0-5 nM. D ) and binds to the TrkC receptor at neutral pH.

[0019] In one example, the monomeric Ig Fc domain or a fragment thereof contains one or more amino acid substitutions.

[0020] In one example, a monomeric Ig Fc domain or fragment thereof contains one or more amino acid substitutions that reduce dimerization with another monomeric Ig Fc domain or fragment thereof. In another example, a monomeric Ig Fc domain or fragment thereof contains one or more amino acid substitutions that inhibit dimerization with another monomeric Ig Fc domain or fragment thereof.

[0021] In some examples, the monomeric Ig Fc domain or a fragment thereof contains substitutions at one or more positions selected from the group consisting of 234, 235, 237, and 329 relative to the sequence shown in Sequence ID No. 10 according to the EU numbering system.

[0022] In one example, the monomeric Ig Fc domain or fragment includes substitutions at position 234 and position 235 relative to the sequence shown in sequence number 10 according to the EU numbering system. In another example, the monomeric Ig Fc domain or fragment includes substitution at position 237 relative to the sequence shown in sequence number 10 according to the EU numbering system. In yet another example, the monomeric Ig Fc domain or fragment includes substitution at position 329 relative to the sequence shown in sequence number 10 according to the EU numbering system. In several examples, the monomeric Ig Fc domain or fragment includes substitutions at position 234, position 235, position 237, and position 329 relative to the sequence shown in sequence number 10 according to the EU numbering system.

[0023] In some examples, the monomeric Ig Fc domain or a fragment thereof contains at least one substitution selected from the group consisting of L234A, L235A, G237A, and P329A relative to the sequence shown in Sequence ID No. 10 according to the EU numbering system.

[0024] In one example, the monomeric Ig Fc domain or a fragment thereof, (i) Leucine substituted with alanine at the position corresponding to amino acid 234 of Sequence ID No. 10 according to the EU numbering system, (ii) Leucine substituted with alanine at the position corresponding to amino acid 235 of Sequence ID No. 10 according to the EU numbering system, (iii) Glycine substituted with alanine at the position corresponding to amino acid 237 of Sequence ID No. 10 according to the EU numbering system, and (iv) comprising one or more amino acid substitutions selected from the group consisting of proline substituted with glycine at the position corresponding to amino acid 329 of Sequence ID No. 10 according to the EU numbering system.

[0025] In one example, the monomeric Ig Fc domain or fragment contains leucine substituted with alanine at the position corresponding to amino acid 234 of SEQ ID NO: 10 according to the EU numbering system, and leucine substituted with alanine at the position corresponding to amino acid 235 of SEQ ID NO: 10. In another example, the monomeric Ig Fc domain or fragment contains glycine substituted with alanine at the position corresponding to amino acid 237 of SEQ ID NO: 10 according to the EU numbering system. In yet another example, the monomeric Ig Fc domain or fragment contains proline substituted with glycine at the position corresponding to amino acid 329 of SEQ ID NO: 10 according to the EU numbering system.

[0026] In one example, the monomeric Ig Fc domain or a fragment thereof, (i) Leucine substituted with alanine at the position corresponding to amino acid 234 of Sequence ID No. 10 according to the EU numbering system, (ii) Leucine substituted with alanine at the position corresponding to amino acid 235 of Sequence ID No. 10 according to the EU numbering system, (iii) Glycine substituted with alanine at the position corresponding to amino acid 237 of Sequence ID No. 10 according to the EU numbering system, and (iv) Contains proline substituted with glycine at the position corresponding to amino acid 329 of Sequence ID No. 10 according to the EU numbering system.

[0027] In one example, a monomeric Ig Fc domain or fragment thereof includes at least one N-glycosylation site that reduces dimerization with another monomeric Ig Fc domain or fragment thereof. In another example, a monomeric Ig Fc domain or fragment thereof includes at least one N-glycosylation site that inhibits dimerization with another monomeric Ig Fc domain or fragment thereof.

[0028] In one example, a monomeric Ig Fc domain or fragment thereof contains one or more amino acid substitutions and at least one N-glycosylation site that reduces dimerization with another monomeric Ig Fc domain or fragment thereof. In another example, a monomeric Ig Fc domain or fragment thereof contains one or more amino acid substitutions and at least one N-glycosylation site that inhibits dimerization with another monomeric Ig Fc domain or fragment thereof.

[0029] In one example, the monomeric Ig Fc domain or a fragment thereof contains at least one N-glycosylation site within the CH3 domain.

[0030] In one example, the monomeric Ig Fc domain or a fragment thereof, (i) at the position corresponding to amino acid 364 of Sequence ID No. 10 according to the EU numbering system, or (ii) Contains at least one N-glycosylation site at the position corresponding to amino acid 407 of Sequence ID No. 10 according to the EU numbering system.

[0031] In one example, the monomeric Ig Fc domain or a fragment thereof, (i) at the position corresponding to amino acid 364 of Sequence ID No. 10 according to the EU numbering system, and (ii) The system includes at least two N-glycosylation sites at the position corresponding to amino acid 407 of Sequence ID No. 10 according to the EU numbering system.

[0032] In one example, the N-terminus of the monomeric Ig Fc domain or a fragment thereof is linked to the C-terminus of the neurotrophin. In another example, the C-terminus of the monomeric Ig Fc domain or a fragment thereof is linked to the N-terminus of the neurotrophin. In yet another example, the N-terminus of the monomeric Ig Fc domain or a fragment thereof is linked to the C-terminus of the neurotrophin by a linker. In yet another example, the C-terminus of the monomeric Ig Fc domain or a fragment thereof is linked to the N-terminus of the neurotrophin by a linker.

[0033] For example, the linker contains serine.

[0034] In one example, the monomeric Ig Fc domain or fragment thereof includes deletions at positions corresponding to amino acids 1-233 of SEQ ID NO: 10 according to the EU numbering system, leucine substituted with alanine at the position corresponding to amino acid 234 of SEQ ID NO: 10 according to the EU numbering system, leucine substituted with alanine at the position corresponding to amino acid 235 of SEQ ID NO: 10 according to the EU numbering system, and serine inserted between the positions corresponding to amino acids 235 and 236 of SEQ ID NO: 10.

[0035] For example, the monomeric Ig Fc domain or fragment thereof includes deletions at positions corresponding to amino acids 1-233 of SEQ ID NO: 10 according to the EU numbering system, leucine substituted with alanine at the position corresponding to amino acid 234 of SEQ ID NO: 10 according to the EU numbering system, leucine substituted with alanine at the position corresponding to amino acid 235 of SEQ ID NO: 10 according to the EU numbering system, serine inserted between the positions corresponding to amino acids 235 and 236 of SEQ ID NO: 10, and proline substituted with glycine at the position corresponding to amino acid 329 of SEQ ID NO: 10 according to the EU numbering system.

[0036] In one example, the monomeric Ig Fc domain or a fragment thereof contains a deletion at a position corresponding to amino acids 1-235 of Sequence ID No. 10 according to the EU numbering system.

[0037] In one example, the monomeric Ig Fc domain or a fragment thereof contains deletions at positions corresponding to amino acids 1-235 of SEQ ID NO: 10 according to the EU numbering system, and glycine substituted with alanine at the position corresponding to amino acid 237 of SEQ ID NO: 10 according to the EU numbering system.

[0038] In one example, the monomeric Ig Fc domain or a fragment thereof contains deletions at positions corresponding to amino acids 1-235 of SEQ ID NO: 10 according to the EU numbering system, and a proline substituted with glycine at the position corresponding to amino acid 329 of SEQ ID NO: 10 according to the EU numbering system.

[0039] For example, neurotrophins are selected from a group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4).

[0040] In one example, the neurotrophin is NT-3. For example, NT-3 is human NT-3. In one example, human NT-3 contains the sequence shown in Sequence ID No. 1.

[0041] For example, neurotrophin 4 is NT-4. For instance, neurotrophin 4 is human NT-4. For example, human NT-4 contains the sequence shown in sequence number 4.

[0042] For example, neurotrophin is BDNF. For example, BDNF is human BDNF. For example, human BDNF contains the sequence shown in SEQ ID NO: 6.

[0043] For example, neurotrophin is NGF. For example, NGF is human NGF. For example, human NGF contains the sequence shown in Sequence ID No. 8.

[0044] In one example, the monomeric Ig Fc domain or a fragment thereof contains the sequence shown in Sequence ID No. 2.

[0045] In one example, the monomeric Ig Fc domain or a fragment thereof contains the sequence shown in SEQ ID NO: 35. In another example, the monomeric Ig Fc domain or a fragment thereof contains the sequence shown in SEQ ID NO: 36. In another example, the monomeric Ig Fc domain or a fragment thereof contains the sequence shown in SEQ ID NO: 37. In another example, the monomeric Ig Fc domain or a fragment thereof contains the sequence shown in SEQ ID NO: 38. In another example, the monomeric Ig Fc domain or a fragment thereof contains a consensus sequence that includes the sequence shown in SEQ ID NO: 39.

[0046] In one example, the fusion protein contains the neurotrophin and the sequence shown in SEQ ID NO: 35. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 36. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 37. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 38. In yet another example, the fusion protein contains a consensus sequence that includes the sequence shown in SEQ ID NO: 39.

[0047] In one example, the fusion protein contains the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 6 and the sequence shown in SEQ ID NO: 2. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 8 and the sequence shown in SEQ ID NO: 2. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 4 and the sequence shown in SEQ ID NO: 2.

[0048] In one example, the fusion protein contains the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 35. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 6 and the sequence shown in SEQ ID NO: 35. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 8 and the sequence shown in SEQ ID NO: 35. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 4 and the sequence shown in SEQ ID NO: 35.

[0049] In one example, the fusion protein contains the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 36. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 6 and the sequence shown in SEQ ID NO: 36. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 8 and the sequence shown in SEQ ID NO: 36. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 4 and the sequence shown in SEQ ID NO: 36.

[0050] In one example, the fusion protein contains the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 37. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 6 and the sequence shown in SEQ ID NO: 37. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 8 and the sequence shown in SEQ ID NO: 37. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 4 and the sequence shown in SEQ ID NO: 37.

[0051] In one example, the fusion protein contains the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 38. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 6 and the sequence shown in SEQ ID NO: 38. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 8 and the sequence shown in SEQ ID NO: 38. In yet another example, the fusion protein contains the sequence shown in SEQ ID NO: 4 and the sequence shown in SEQ ID NO: 38.

[0052] In one example, the fusion protein includes a consensus sequence containing the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 39. In another example, the fusion protein includes a consensus sequence containing the sequence shown in SEQ ID NO: 6 and the sequence shown in SEQ ID NO: 39. In yet another example, the fusion protein includes a consensus sequence containing the sequence shown in SEQ ID NO: 8 and the sequence shown in SEQ ID NO: 39. In yet another example, the fusion protein includes a consensus sequence containing the sequence shown in SEQ ID NO: 4 and the sequence shown in SEQ ID NO: 39.

[0053] In one example, the fusion protein contains the sequence shown in SEQ ID NO: 21. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 23. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 24. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 25. In another example, the fusion protein contains the sequence shown in SEQ ID NO: 26. In another example, the fusion protein has a consensus sequence that includes the sequence shown in SEQ ID NO: 28.

[0054] This disclosure also provides nucleic acids that encode or express the fusion proteins disclosed herein.

[0055] In one example, the nucleic acid encoding the fusion protein is a DNA sequence containing the sequence shown in SEQ ID NO: 15. In another example, the nucleic acid encoding the fusion protein is a DNA sequence containing the sequence shown in SEQ ID NO: 17.

[0056] This disclosure also provides proneurotrophins and nucleic acids that encode or express monomeric immunoglobulin (Ig) crystallizable fragment (Fc) domains or fragments thereof.

[0057] This disclosure also provides expression constructs comprising nucleic acids disclosed herein.

[0058] In one example, the expression construct contains the sequence shown in SEQ ID NO: 18. In another example, the expression construct contains the sequence shown in SEQ ID NO: 20.

[0059] In one example, the expression construct includes the sequence shown in SEQ ID NO: 12. In another example, the expression construct includes the sequence shown in SEQ ID NO: 14.

[0060] This disclosure also provides host cells that express a fusion protein disclosed herein, or a nucleic acid disclosed herein, or an expression construct disclosed herein.

[0061] For example, the host cell is a mammalian cell. Mammalian cells are selected from the group consisting of HEK cells, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, 211A cells, and their derivatives.

[0062] For example, mammalian cells are HEK cells. For instance, mammalian cells are HEK293 cells.

[0063] In another example, the mammalian cell is a CHO cell. For example, the mammalian cell is an ExpiCHO cell. In yet another example, the mammalian cell is a CHOK1 cell.

[0064] This disclosure also relates to a method for producing the fusion protein disclosed herein, (a) A step of culturing a cell line containing nucleic acid encoding a fusion protein, (b) A method is provided comprising the step of isolating the fusion protein from a cell line.

[0065] This disclosure also provides compositions comprising superparticles, the superparticles comprising fusion proteins disclosed herein.

[0066] This disclosure provides a method for promoting the survival of spiral ganglion neurons in a target ear, comprising administering a fusion protein, composition, nucleic acid, or expression vector described herein.

[0067] This disclosure also provides a method for treating hearing impairment in a subject, comprising administering a fusion protein, composition, nucleic acid, or expression vector described herein.

[0068] This disclosure also provides the use of the fusion proteins, compositions, nucleic acids, or expression vectors described herein in the manufacture of pharmaceuticals for promoting the survival of spiral ganglion neurons in the ear of a subject.

[0069] This disclosure further provides the use of the fusion proteins, compositions, nucleic acids, or expression vectors described herein in the manufacture of pharmaceuticals for the treatment of hearing impairment.

[0070] This disclosure also provides fusion proteins, compositions, nucleic acids, or expression vectors described herein for use in promoting the survival of spiral ganglion neurons in the ear of interest.

[0071] This disclosure also provides fusion proteins, compositions, nucleic acids, or expression vectors described herein for use in the treatment of hearing impairment. [Brief explanation of the drawing]

[0072] [Figure 1] This is a graphical representation of a protein purification chromatogram showing the elution profile of NT3-Fc from a 5 mL HiTrap PrismA column. (A) Complete affinity purification run. (B) Magnified view of the eluted NT3-Fc peak. Traces of the chromatogram represent protein absorbance at 280 nM (mAU, solid line), conductivity of the purified solution (mS / cm, dashed line), and concentration of the elution buffer (%), dotted line. The protein fraction marked in gray was pooled. [Figure 2] This is a graphical representation of the size exclusion chromatography (SEC) elution profile for NT3-Fc in 1×PBS. (A) Complete chromatogram. (B) Magnified view of the elution. [Figure 3]This is a graphical representation of the buffer exchange elution profile for NT3-Fc to 1 / 4×PBS on a HiPrep 26 / 10 desalting column (Cytiva#17508701). The protein showed a broad elution profile due to protein overload. 1×PBS was favorably removed, as indicated by elution peaks (smaller peaks = conductivity traces) in the 80–130 mL range. The protein fraction marked in gray was pooled. [Figure 4] (A) Graph representation of affinity-purified NT3-Fc from HEK293F cells and (B) Coomassie-stained SDS-PAGE from ExpiCHO-S cells. Samples were incubated with or without a reducing agent (DTT, dithiothreitol) as shown. NuPAGE BIS-TRIS 4-12%, 200V, 40 min. [Figure 5] (A) is the total ion chromatogram of the NT3-Fc sample, and (B) is a graphical representation of the deconvoluted spectrum of the labeled peak. The 20-fold diluted sample produced a clean chromatographic peak at 21.2 min containing a mass of approximately 42,288 Da, separated by masses consistent with multiple hexoses and HexNAc variants. [Figure 6A] (A) Total ion chromatogram of the deglycosylated NT3-Fc sample, (B) Deconvoluted MS spectrum of the labeled peaks, and (C) Graph representation of the enlarged section. [Figure 6B] (A) Total ion chromatogram of the deglycosylated NT3-Fc sample, (B) Deconvoluted MS spectrum of the labeled peaks, and (C) Graph representation of the enlarged section. [Figure 7] This is a pictorial representation of a chromatogram showing the elution profile of NT3-Fc from a 5 mL HiTrap PrismA column. The areas marked in gray are pooled. [Figure 8]This is a graphical representation of the size exclusion chromatography elution profile for NT3-Fc in 1×PBS on a HiLoad 26 / 600 Superdex 200 pg column. NT3-Fc elution shows a uniform profile indicated by a single symmetrical peak. The gray-marked region is pooled. To assess the molecular weight of the eluted NT3-Fc homodimer, Bio-Rad protein standards (bovine thyroglobulin [670kDa], bovine γ-globulin [158kDa], chicken ovalbumin [44kDa], horse myoglobin [17kDa], and vitamin B12 [1.35kDa]) were analyzed on the same column. The elution volumes of these standards are indicated by arrows. [Figure 9A] This is a graphical representation of fully processed SPR binding data showing the interaction between injected NT3-Fc and immobilized Trk receptors (TrkA: top row; TrkB: middle row; and TrkC: bottom row). Three sets of experimental datasets are shown in columns A-C (n=1 (A), n=2 (B), and n=3 (C)). (D) shows the fitting of the binding response at equilibrium (t=80-85 sec, plotted against analyte concentration) to a simple (1:1) binding isotherm. [Figure 9B] This is a graphical representation of fully processed SPR binding data showing the interaction between injected NT3-Fc and immobilized Trk receptors (TrkA: top row; TrkB: middle row; and TrkC: bottom row). Three sets of experimental datasets are shown in columns A-C (n=1 (A), n=2 (B), and n=3 (C)). (D) shows the fitting of the binding response at equilibrium (t=80-85 sec, plotted against analyte concentration) to a simple (1:1) binding isotherm. [Figure 10](A) A graph representation of a fully processed SPR binding sensogram (solid line) demonstrating that the NT3-Fc protein produced in a CHO cell host (ExpiCHO-S) following transient transfection has the same binding kinetics to the TrkC receptor as (B) purified NT3-Fc following transfection of HEK293 Freestyle cells. In this case, NT3-Fc was immobilized on a solid support. Binding affinity was determined in an experiment consisting of five sequential injections of sequentially diluted TrkC (3x, from 243 nM to 3 nM). The superimposed dotted line represents the global fitting of the data to a 1:1 interaction model. (C) Binding interaction parameters extracted from the sensograms shown in A and B. [Figure 11] This is a graphical representation of the CellSensor TrkC assay, showing (A) representative dose-response curves for fresh NT3 and NT3-Fc, and (B) EC50 values ​​of NT3 and NT3-Fc based on the dose-response curves. Data are presented as mean ± standard error of the mean. [Figure 12] (A) Graph representation of the NT3-Fc loading capacity of ultraparticles over 84 days. Each data point represents the loading capacity of the ultraparticle at x days after manufacturing. Approximately 4 μg of NT3-Fc can be loaded into one ultraparticle. (B) Elution of NT3-Fc from loaded ultraparticles. Data are presented as mean ± standard error of the mean. [Figure 13A] Graph representations of cat cochlear sections after treatment with NT3-Fc-loaded ultrafine particles (SP) on a round window membrane (RWM), stained with anti-human IgG-HRP and DAB. (A) and (B) Cats with normal hearing, bilaterally implanted with NT3-Fc-loaded ultrafine particles on the RWM for 48 hours. (C) Negative control, a cat with normal hearing without implantation. (D) Positive control, a cat with normal hearing, with NT3-Fc-loaded ultrafine particles implanted inside the cochlea. [Figure 13B]Graph representations of cat cochlear sections after treatment with NT3-Fc-loaded ultrafine particles (SP) on a round window membrane (RWM), stained with anti-human IgG-HRP and DAB. (A) and (B) Cats with normal hearing, bilaterally implanted with NT3-Fc-loaded ultrafine particles on the RWM for 48 hours. (C) Negative control, a cat with normal hearing without implantation. (D) Positive control, a cat with normal hearing, with NT3-Fc-loaded ultrafine particles implanted inside the cochlea. [Figure 14A] (A) Graph representation of scanning electron images of superparticles. (B) NT3-SP implanted on the RWM of a cat. (C) Image of the cochlea and hair cells of a cat with a schematic representation of superparticles on the RWM. Arrows indicate localized regions (32 kHz) where significant synaptic repair was observed. (D) Cochlear synapses in NT3-SP treated ears and control ears for the cochlear region at 32 kHz (the region closest to the circular window membrane as measured). The Y axis is synapse / inner hair cell, the x axis is the treatment condition, NT is NT3.Fc, and Norm is normal (undamaged). Significantly higher synaptic density in surviving inner hair cells was observed in NT3-Fc treated cochlea compared to controls (n=8 cats, ANCOVA, p=0.002). [Figure 14B] (A) Graph representation of scanning electron images of superparticles. (B) NT3-SP implanted on the RWM of a cat. (C) Image of the cochlea and hair cells of a cat with a schematic representation of superparticles on the RWM. Arrows indicate localized regions (32 kHz) where significant synaptic repair was observed. (D) Cochlear synapses in NT3-SP treated ears and control ears for the cochlear region at 32 kHz (the region closest to the circular window membrane as measured). The Y axis is synapse / inner hair cell, the x axis is the treatment condition, NT is NT3.Fc, and Norm is normal (undamaged). Significantly higher synaptic density in surviving inner hair cells was observed in NT3-Fc treated cochlea compared to controls (n=8 cats, ANCOVA, p=0.002). [Figure 15] This is a schematic representation of a modified pCAGGS mammalian expression construct. [Figure 16]This is a graphical representation of a chromatogram showing the elution profile of BDNF-Fc from a 5 mL HiTrap PrismA column. The gray-marked region is pooled. [Figure 17] This is a graphical representation of the preparative size exclusion chromatography elution profile (solid line) of BDNF-Fc. Bio-Rad protein standards (dotted line) were analyzed on the same column, and the molecular weight of the eluted protein peaks was evaluated. The molecular weights of these standards are indicated by arrows. [Figure 18] This is a pictorial representation of the Coomassie-stained SDS-PAGE analysis of purified BDNF-Fc samples. Concentrated samples were incubated at 95°C for 5 minutes before loading onto the gel. The gel used was NuPAGE Bis-Tris 4-12% in MES-SDS buffer, run at 180V for 50 minutes. Samples were analyzed under reducing and non-reducing conditions. [Figure 19] This graph shows the size exclusion chromatography results for an analyzed purified BDNF-Fc dimer sample (approximately 50 μg, solid line). A Bio-Rad protein standard (dotted line) was analyzed on the same column, and the molecular weight of the eluted protein was evaluated. The molecular weight of the standard is indicated by the arrow. [Figure 20] This is a graphical representation (solid line sensorogram) of processed SPR binding data for recombinant TrkB binding to immobilized BDNF-Fc. The experiment consisted of eight sequential injections of continuously diluted TrkB (2x from 320 nM to 2.5 nM). The superimposed dashed line represents the global fitting of the data to a 1:1 interaction model. [Figure 21] This is a graphical representation of the CellSensor TrkB assay, showing (A) representative dose curves for BDNF and BDNF-Fc, and (B) EC50 values ​​of BDNF and BDNF-Fc based on the dose curves. Data are presented as mean ± standard error of the mean. [Figure 22]This is a graphical representation of the purification profile of NT3.diFc (PrismA, solid line). (A) Two protein peaks representing recombinant NT3.diFc were sequentially eluted with 0.1M sodium acetate at pH 4.0 (999.8 mins) and pH 3.5 (1024 mins). (B) Magnified view of the peaks eluted at pH 4.0 and pH 3.5. [Figure 23] This graph shows the size exclusion chromatography results for eluting NT3-diFc at pH 4.0 (solid line) and pH 3.5 (dashed line). For comparison, a previously purified NT3-Fc sample (dotted line) is included in this figure. The elution volume and MW of Bio-Rad protein standards analyzed on the same column are highlighted with black arrows. [Figure 24] This is a graphical representation of Coomassie-stained SDS-PAGE of NT3-diFc samples collected during purification. Concentrated samples were incubated at 95°C for 5 minutes before loading onto the gel. The gel used was NuPAGE Bis-Tris 4-12% in MES-SDS buffer, run at 180V for 50 minutes. Samples were analyzed under reducing (lanes 2-5) and non-reducing (lanes 6-10) conditions. [Figure 25] This is a graphical representation of processed SPR binding data (colored lines in the sensorogram) for a graphical representation of processed SPR binding kinetics (solid line) for recombinant TrkB binding to immobilized NT3-Fc samples (captured on a protein A SPR chip). (A) NT3-Fc, (B) NT3-diFc sample eluted at pH 4.0. Both binding experiments consisted of five sequential injections (colored sensorograms) of sequentially diluted TrkB (3-fold serial dilutions from 243 nM to 3 nM). The superimposed dashed lines represent the global fitting of the data to a 1:1 interaction model. [Figure 26A] (A) A pictorial representation of sequence alignments for the matured and processed amino acid sequences of five mono-Fc constructs, and (B) a dimerized Fc construct. Hinge regions, extra serine insertions, mono-Fc mutations involved in preventing dimerization, and LALA mutations are all shaded. [Figure 26B](A) A pictorial representation of sequence alignments for the matured and processed amino acid sequences of five mono-Fc constructs, and (B) a dimerized Fc construct. Hinge regions, extra serine insertions, mono-Fc mutations involved in preventing dimerization, and LALA mutations are all shaded. [Figure 27] (A) A graph showing the number of cochlear synapses per inner hair cell in mouse explants. Three experimental conditions were investigated: normal, KA control, and KA=NT3-Fc treatment. Treatment of explants with kainic acid (KA) damaged synapses, resulting in a reduction in their number per inner hair cell. Treatment of explants with NT3-Fc (at a concentration of 1 nM) after KA damage resulted in synaptic recovery, with more synapses observed in cochlear hair cells. Normal explants were untreated. Representative images of cochlear explants are shown for each condition in (B), showing inner hair cells (gray) containing cochlear synapses (white dots). [Table 1] [Modes for carrying out the invention]

[0073] General Throughout this specification, unless otherwise specifically stated or the context requires, any reference to a single step, composition, group of steps, or group of compositions shall be construed as encompassing one or more (i.e., one or more) of those steps, compositions, groups of steps, or groups of compositions.

[0074] Those skilled in the art will understand that this disclosure is susceptible to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or indicated herein, as well as any and all combinations or any two or more of such steps or features.

[0075] This disclosure is not limited in scope by the specific examples described herein, which are intended to be illustrative only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0076] Any example in this disclosure shall apply mutatis mutandis to any other example in this disclosure unless otherwise specified. In other words, any particular example in this disclosure may be combined with any other particular example in this disclosure (except where mutually exclusive).

[0077] Any example in this disclosure disclosing a particular feature or group of features or a method or step in a method shall be construed as providing express support for excluding a particular feature or group of features or a method or step in a method.

[0078] Unless otherwise specifically defined, all technical and scientific terms used herein shall be construed to have the same meaning as that commonly understood by those skilled in the art (for example, in cell culture, recombinant proteins, immunology, protein chemistry, and biochemistry).

[0079] Unless otherwise specified, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. This is described and explained through literature from sources such as al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0080] The descriptions and definitions of variable regions and their parts, immunoglobulins, antibodies and their fragments as used herein can be further clarified by the discussions in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991; Bork et al., J Mol. Biol. 242, 309-320, 1994; Chothia and Lesk J. Mol Biol. 196:901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0081] The terms "and / or," for example, "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be construed as providing explicit support for both meanings or either meaning.

[0082] Throughout this specification, the word “comprise,” or variations thereof such as “comprises” or “comprising,” is understood to mean including any specified element, component, or step, or group of elements, components, or steps, but not to mean excluding any other element, component, or step, or group of elements, components, or steps.

[0083] Selection Definition As used herein, the terms “polynucleotide,” “nucleotide sequence,” or “nucleic acid” are understood to mean a sequence of consecutive nucleotides (or bases) covalently bonded to a phosphodiester backbone. Conventionally, sequences are presented from the 5' end to the 3' end unless otherwise specified. For example, a nucleic acid is a DNA sequence. In one example, a nucleic acid is an RNA sequence. In another example, a nucleic acid is an mRNA sequence. In one example, mRNA may be conventional mRNA (cRNA) or self-amplified RNA (sa-mRNA).

[0084] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches, taking into account the degree to which sequences are identical across a comparison window, considering proper alignment using standard algorithms. Thus, the “percentage of sequence identity” is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, U) or amino acid residues occur in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “percentage of matches” calculated by the DNASIS computer program (version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA).

[0085] As used herein, the terms “encode,” “encodes,” or “encoding” refer to a region of nucleic acid that can be transcribed or translated to produce RNA, polypeptides, or proteins. For example, the terms “encode,” “encodes,” or “encoding” refer to a region of RNA (e.g., mRNA) that can be transcribed to a polypeptide or protein, or, as used in the context of DNA, a region of DNA that can be transcribed to produce mRNA that can be translated into a polypeptide or protein.

[0086] The terms “polypeptide” or “polypeptide chain” will be understood to mean a sequence of amino acids linked by peptide bonds. For example, a protein will be interpreted as containing a single polypeptide chain, i.e., a sequence of amino acids linked by peptide bonds, or a sequence of polypeptide chains (i.e., polypeptide complexes) covalently or non-covalently linked to one another. A sequence of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0087] The term “protein” should be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. In some examples, a protein is a fusion protein. As used herein, a “fusion protein” is a protein comprising at least two domains, at least two of which are translated as a single unit and joined together to produce a single protein.

[0088] The terms “isolated protein” or “isolated polypeptide” refer to a protein or polypeptide that, by its origin or source of derivative, is not associated with any naturally associated components in its natural state and substantially contains no other proteins from the same source. A protein can be substantially free of naturally associated components or substantially purified by isolation using protein purification techniques known in the art. “Substantially purified” means that the protein is substantially free of contaminants, for example, free of contaminants in a percentage of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0089] As used herein, the term “variant” refers to a protein that has undergone one or more amino acid substitutions using well-known techniques for site-directed mutagenesis or any other conventional method.

[0090] As used herein, “amino acid substitution” refers to the replacement of an amino acid with another amino acid at a specific position within a polypeptide sequence.

[0091] For example, a substitution is a conservative substitution. A conservative substitution means the substitution of one or more amino acids with alternative amino acids that share similar properties. Those skilled in the art recognize that various amino acids have similar properties and are therefore "conservative." One or more such amino acids in a protein, polypeptide, or peptide can often be substituted by one or more other such amino acids without eliminating the desired activity of that protein, polypeptide, or peptide. Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine can often be substituted for each other (e.g., amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferred to be used to substitute for each other (because they have relatively short side chains), and valine, leucine, and isoleucine are preferred to be used to substitute for each other (because they have larger aliphatic side chains that are hydrophobic). Other amino acids that can often be substituted for each other include phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains). It will be understood that amino acid substitutions within the scope of this disclosure can be made using naturally occurring or non-naturally occurring amino acids. For example, in this specification, a methyl group on alanine may be replaced with an ethyl group, and / or minor modifications may be made to the peptide skeleton. Whether natural or synthetic amino acids are used, it is preferable that only L-amino acids are present.

[0092] Those skilled in the art will know that an "antibody" is a variable region consisting of multiple polypeptide chains, for example, V L polypeptides and V HIt will be recognized that antibodies are generally considered to be proteins containing polypeptides. Antibodies also generally contain constant domains, some of which, in the case of heavy chains, can be located within constant regions containing constant fragments or crystallizable fragments (Fc). H and V L These interact to form an Fv containing an antigen-binding region that can specifically bind to one or more closely related antigens. Generally, the light chain from mammals is either a κ light chain or a λ light chain, and the heavy chain from mammals is α, δ, ε, γ, or μ. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass. The term “antibody” also encompasses humanized antibodies, primate-derived antibodies, human antibodies, and chimeric antibodies.

[0093] The terms "full-length antibody," "intact antibody," or "whole antibody" are used synonymously to refer to an antibody in a substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, whole antibodies include those that possess both heavy and light chains.

[0094] As used herein, the term “binding” in relation to interactions with tyrosine receptor kinase (Trk) receptor fusion proteins means that the interaction depends on the presence of specific structures on the Trk receptor and the fusion protein. For example, fusion proteins generally recognize and bind to specific structures on the Trk receptor, rather than the receptor itself.

[0095] As used herein, the term “activate” in relation to interaction with a tyrosine receptor kinase (Trk) receptor fusion protein means that the interaction stimulates the Trk receptor and results in a flow-on effect.

[0096] For the purpose of clarification, and as will be obvious to those skilled in the art based on the subject matter illustrated herein, references to “affinity” herein refer to the interaction, binding, or association of a fusion protein with a Trk receptor.

[0097] For the purpose of clarification, and as will be obvious to those skilled in the art based on the descriptions herein, the reference to “at least about affinity” is understood to mean that affinity is equal to or greater than the listed values ​​(i.e., lower than the values ​​listed as affinity), i.e., an affinity of 2nM is greater than an affinity of 3nM. In other words, the term could mean “affinity less than or equal to X,” where X is one of the values ​​listed herein.

[0098] The term "soluble" is used in the context of this disclosure to refer to a fusion protein expressed in a soluble form. For example, aggregation of foreign proteins into insoluble inclusions is a limiting factor for various recombinant expression systems. In some cases, the soluble fusion proteins of this disclosure do not aggregate into insoluble inclusions following expression in a suitable expression system disclosed herein. In some cases, a fusion protein expressed in a "soluble form" can be purified, extracted, or obtained from solution as a complete tertiary structure. In certain specific cases, the fusion proteins of this disclosure may have improved solubility. The term "solubility" is used herein to refer to the degree of solubility relative to insoluble expression of the fusion proteins disclosed herein. In some cases, the fusion proteins of this disclosure may have improved solubility relative to wild-type neurotrophins.

[0099] In some cases, the fusion proteins of this disclosure may have improved translation for wild-type neurotrophins. In other cases, the fusion proteins of this disclosure may have improved stability for wild-type neurotrophins.

[0100] As used herein, the terms “to treat,” “to cure,” or “to treat” include administering any of the fusion proteins or compositions described herein to reduce or eliminate at least one symptom of a particular disease or condition, or to slow the progression of a disease or condition.

[0101] As used herein, the terms “prevent,” “prevent,” or “prevent” include providing prevention of the onset or recurrence of hearing impairment or symptoms of hearing impairment in a subject. The individual may have a predisposition or risk of developing the disease or disease recurrence, but has not yet been diagnosed with the disease or recurrence.

[0102] The “effective dose” refers to the minimum effective amount in the required dosage and duration to achieve the desired outcome. For example, the desired outcome may be a therapeutic or prophylactic outcome. The effective dose may be provided in one or more doses. In some examples of this disclosure, the term “effective dose” means the amount required to treat a disease or condition previously described herein. In some examples of this disclosure, the term “effective dose” means the amount required to bring about a change in factors related to a disease or condition previously described herein. The effective dose may vary depending on the disease or condition being treated or the factors being modified, and also depending on body weight, age, racial background, sex, health and / or physical condition, and other factors related to the mammal being treated. Typically, the effective dose falls within a relatively broad range (e.g., “dosage” range) that can be determined through routine trial and error by healthcare professionals. Therefore, this term should not be construed as limiting this disclosure to a specific amount. The effective dose may be administered in a single dose or in doses repeated once or several times over a period of treatment.

[0103] The “therapeutic dose” is the minimum concentration required to produce at least a measurable improvement in a particular disease or condition. The therapeutic dose as used herein may vary depending on factors such as the patient’s disease state, age, sex, and weight, as well as the ability of the fusion protein or composition to induce the desired response in the individual. The therapeutic dose is also defined as the therapeutically beneficial effect outweighing any toxic or adverse effects of the fusion protein or composition. For example, the therapeutic dose is interpreted as a sufficient amount of the fusion protein or composition to reduce or inhibit one or more symptoms of hearing impairment or its complications.

[0104] As used herein, the term “preventive effective dose” shall be interpreted as meaning a sufficient amount of fusion protein or composition to prevent, inhibit, or delay the onset of one or more detectable symptoms of a condition.

[0105] As used herein, the term “subject” shall be interpreted as meaning any animal, including humans and, for example, mammals. Illustrative subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0106] Fusion protein This disclosure provides (i) a neurotrophin and (ii) a fusion protein comprising a monomer immunoglobulin (Ig) crystallizable fragment (Fc) domain or a fragment thereof.

[0107] In the context of this disclosure, the term "fusion protein" is used to refer to a protein produced by the conjugation of two or more originally separate polypeptides, or by the expression of a nucleic acid(s) encoding it in a format suitable for providing a fusion protein.

[0108] Fusion proteins can be formed by various methods known in the art. For example, a fusion protein can be formed by joining two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. In another example, a fusion protein can be formed by linking one polypeptide to another through a reaction between amino acid side chains (e.g., a disulfide bond between cysteine ​​residues on each polypeptide). In yet another example, a fusion protein can be formed by the chemical coupling of two or more polypeptides, or it can be expressed as a single polypeptide from a single consecutive nucleic acid sequence encoding the fusion protein. In yet another example, a fusion protein can be prepared using conventional recombination techniques in molecular biology to join two genes in-frame into a single nucleic acid sequence, and then express the nucleic acid in a suitable host cell under conditions in which the fusion protein is produced. In other words, the methods for producing fusion proteins as contained herein are not particularly limited, as long as the resulting fusion protein contains (i) a neurotrophin and (ii) a monomeric immunoglobulin (Ig) crystallizable fragment (Fc) domain or fragment thereof.

[0109] The fusion proteins of this disclosure may be provided in host cells or tissues. Alternatively, in another example, the fusion proteins may be provided in a purified or partially purified composition. Thus, in certain examples, the fusion proteins of this disclosure may be “purified” or “isolated.” These terms are used in the context of this disclosure to generally refer to proteins that have been isolated from lipids, nucleic acids, other peptides and proteins, and other contaminating molecules relevant in their expression system. Preferably, the purified protein contains at least 60%, more preferably at least 75%, and more preferably at least 90% of the other components in the expression system. Methods for producing the fusion proteins of this disclosure and the nucleic acids encoding them are discussed further below.

[0110] The fusion proteins of this disclosure may have improved expression levels. In one example, the fusion protein improves the expression level of the conjugated neurotrophin compared to the wild-type or unmodified neurotrophin. In several examples, the fusion protein improves the expression level of the conjugated neurotrophin in mammalian cell lines compared to the wild-type or unmodified neurotrophin in mammalian cell lines.

[0111] In one example, the fusion protein can form a homodimer. Therefore, in some examples, the fusion protein of this disclosure may contain a neurotrophin dimer. In some examples, the dimer is provided via dimerization with another neurotrophin of the same type. For example, the fusion protein of this disclosure may contain a neurotrophin-3 dimer. Another fusion protein of this disclosure may contain a BDNF dimer.

[0112] Neurotrophin This disclosure provides a fusion protein comprising the neurotrophin of this disclosure.

[0113] Neurotrophins are growth factors expressed in the brain and peripheral tissues that control many aspects of neuronal function, including the proliferation of neural progenitor molecules, neuronal morphology, synaptic plasticity, and even cell death following injury. In fact, one cause of spiral ganglion neuron degeneration is the loss of endogenous neurotrophin supply. Neurotrophins mediate these actions by activating two distinct classes of receptors: the tyrosine receptor kinase (Trk) family of receptor tyrosine kinases and p75NTR, a member of the TNF receptor superfamily. Specifically, nerve growth factor (NGF) binds to TrkA with high affinity, brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT-4) bind to TrkB with high affinity, and neurotrophin 3 (NT-3) binds to TrkC with high affinity. Apart from the activation of TrkC, NT-3 also activates TrkA and TrkB, albeit with lower affinity. Therefore, in certain cases, neurotrophins contained herein may be characterized based on their ability to activate Trk receptors. For example, neurotrophin variants relating to the Sequence IDs described herein may be characterized based on their ability to activate and / or bind to the relevant Trk receptors at a level corresponding to their corresponding neurotrophins having the amino acid sequence disclosed herein.

[0114] Neurotrophins share several structural and chemical properties, including over 50% sequence homology in their primary structure, nearly identical molecular weights, three disulfide bonds forming a cysteine ​​knot, and isoelectric points ranging from 9 to 10. "Wild-type" or "unmodified" neurotrophins originate from mammals in natural conditions without additional modifications or mutations.

[0115] Those skilled in the art will understand that neurotrophins are initially synthesized as larger precursor molecules containing signal peptides, propeptides, and peptides. For example, precursors may be "proneurotrophin 3," "proneurotrophin 4," "pro-BDNF," or "pro-NGF," which undergo proteolytic cleavage to yield mature neurotrophins. Proneurotrophins are precursor forms of neurotrophins and can function as high-affinity apoptotic ligands for selected neuronal populations.

[0116] For example, this disclosure also encompasses precursor forms of the fusion proteins described herein. For instance, a precursor fusion protein may comprise a proneurotrophin as described herein and a monomeric Ig Fc domain or a fragment thereof. For example, a precursor fusion protein comprises proneurotrophin 3 as described herein and a monomeric Ig Fc domain or a fragment thereof. For example, a precursor fusion protein comprises proneurotrophin 4 as described herein and a monomeric Ig Fc domain or a fragment thereof. For example, a precursor fusion protein comprises proBDNF as described herein and a monomeric Ig Fc domain or a fragment thereof. For example, a precursor fusion protein comprises proNGF as described herein and a monomeric Ig Fc domain or a fragment thereof.

[0117] Various neurotrophins are known in this field, and in certain cases, they are described based on their respective amino acid sequences. Examples of neurotrophins include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4).

[0118] Therefore, in one example, the neurotrophins are NGF, BDNF, and NT-3. In another example, the neurotrophins are NGF, BDNF, and NT-4. In yet another example, the neurotrophins are BDNF, NT-3, and NT-4. In yet another example, the neurotrophins are NGF and BDNF. In yet another example, the neurotrophins are NGF, NT-3, and NT-4. In yet another example, the neurotrophins are NGF and BDNF. In yet another example, the neurotrophins are NGF and NT-3. In yet another example, the neurotrophins are NGF and NT-4. In yet another example, the neurotrophins are BDNF and NT-3. In yet another example, the neurotrophins are BDNF and NT-4. In yet another example, the neurotrophins are NT-3 and NT-4.

[0119] In one example, the neurotrophin is neurotrophin 3 (NT-3). For example, the neurotrophin can be mammalian NT-3. In one example, the neurotrophin is human NT-3. In one example, NT-3 contains the sequence shown in SEQ ID NO: 1. In one example, NT-3 as incorporated in this disclosure is a variant of SEQ ID NO: 1. For example, NT-3 may contain a sequence that is at least 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 1. In one example, the variant is identified based on its sequence identity to SEQ ID NO: 1 and its ability to bind / activate Trk at a level corresponding to a neurotrophin containing the amino acid sequence shown in SEQ ID NO: 1.

[0120] In one example, the neurotrophin is neurotrophin 4 (NT-4). For example, the neurotrophin can be mammalian NT-4. In one example, the neurotrophin is human NT-4. In one example, NT-4 contains the sequence shown in SEQ ID NO: 4. In one example, NT-4 as incorporated in this disclosure is a variant of SEQ ID NO: 4. For example, NT-4 may contain a sequence that is at least 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 4. In one example, the variant is identified based on its sequence identity to SEQ ID NO: 4 and its ability to bind / activate Trk at a level corresponding to a neurotrophin containing the amino acid sequence shown in SEQ ID NO: 4.

[0121] In one example, neurotrophin is nerve growth factor (NGF). For example, neurotrophin can be mammalian NGF. In another example, neurotrophin is human NGF. In one example, NGF contains the sequence shown in Sequence ID No. 8. In one example, NGF contained herein is a variant of Sequence ID No. 8. For example, NGF may contain a sequence that is at least 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to Sequence ID No. 8. In one example, a variant is identified based on its sequence identity to Sequence ID No. 8 and its ability to bind to / activate Trk at a level corresponding to neurotrophin containing the amino acid sequence shown in Sequence ID No. 8.

[0122] In one example, neurotrophin is brain-derived neurotrophic factor (BDNF). For example, neurotrophin can be mammalian BDNF. In another example, neurotrophin is human BDNF. In one example, BDNF contains the sequence shown in SEQ ID NO: 6. In another example, BDNF as contained herein is a variant of SEQ ID NO: 6. For example, BDNF may contain a sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In one example, a variant is identified based on its sequence identity to SEQ ID NO: 6 and its ability to bind to / activate Trk at a level corresponding to neurotrophin containing the amino acid sequence shown in SEQ ID NO: 6.

[0123] In one example, a fusion protein can dimerize with another neurotrophin or fusion protein. In another example, a fusion protein forms a homodimer. In yet another example, a neurotrophin, when provided herein as a disclosure of a fusion protein, forms a homodimer. In such an example, the neurotrophin component of the fusion protein disclosed herein mediates the dimerization.

[0124] As used herein, the term “dimer” refers to a protein complex comprising at least two polypeptides. At least two polypeptides may associate with each other via one or both of covalent and non-covalent (e.g., electrostatic, π-effect, van der Waals force, and hydrophobic effect) interactions. The two polypeptides may have the same amino acid sequence or they may be different. If the two polypeptides are identical, the dimer is called a homodimer. If the two polypeptides are different, the dimer is called a heterodimer.

[0125] In one example, the neurotrophin component of the fusion protein disclosed herein is provided as a homodimer. For example, the homodimer of this disclosure comprises an NT-3 molecule dimerized with another NT-3 molecule. In another example, the homodimer is an NT-4 molecule dimerized with another NT-4 molecule. In yet another example, the homodimer is a BDNF molecule dimerized with another BDNF molecule. In one example, the homodimer is an NGF molecule dimerized with another NGF molecule.

[0126] In another example, the neurotrophins of this disclosure are heterodimers. For example, an NT-3 molecule dimerizes with an NT-4, BDNF, or NGF molecule. In yet another example, an NT-4 molecule dimerizes with an NT-3, BDNF, or NGF molecule. In one example, a BDNF molecule dimerizes with an NT-3, NT-4, or NGF molecule. In another example, an NGF molecule dimerizes with an NT-3, NT-4, or BDNF molecule.

[0127] Crystallizable fragments (Fc) The terms “crystallizable fragment” or “Fc” are used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes Fc domain variants and Fc domain fragments.

[0128] Those skilled in the art will be familiar with the human Fc domain region.

[0129] For example, the human IgG heavy chain Fc domain region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the Fc domain produced by host cells may undergo post-translational cleavage of one or more amino acids, particularly one or two, from the C-terminus of the heavy chain. Thus, the Fc domain may contain cleaved variants of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the EU index). Therefore, the C-terminal lysine (K447), or C-terminal glycine (G446) and lysine (K447), may or may not be present in the Fc domain region.

[0130] Where applicable, the numbering of amino acid residues in the Fc domain region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0131] The terms "Kabat's EU numbering system" or "EU numbering system" are understood to mean that the numbering of immunoglobulin heavy chains is based on the EU index, as taught in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of human IgG1 EU.

[0132] Wild-type Fc is naturally homodimerized, a characteristic resulting from strong high-affinity interactions between the two CH3 domains and the two disulfide bonds within the hinge region.

[0133] The fusion proteins of this disclosure include a “monomer Ig Fc domain” or a fragment thereof. Therefore, the Fc components of the disclosed fusion proteins are distinguishable from their wild-type counterparts, at least in terms of their monomeric structure. In the context of this disclosure, the term “monomer Ig Fc domain” is used to refer to a monomeric polypeptide containing, for example, a single CH2 domain and a single CH3 domain, a sequence of amino acids corresponding to the Fc portion of a heavy chain. Therefore, a monomeric Ig Fc domain does not contain a hinge region. Such monomeric polypeptides generally contain amino acid substitutions (may be multiple) relative to the wild-type Fc, which point towards a monomeric structure. For example, a monomeric Ig Fc domain contains an N-glycosylation site (may be multiple) within a CH3 domain that inhibits dimerization with another CH3 domain.

[0134] Various examples of monomeric Fc are known in the field of this technology and will be discussed further below.

[0135] Any Ig Fc domain region can be modified to produce the monomeric Ig Fc domain or fragments thereof of this disclosure. As considered herein, generally speaking, Ig Fc domains are derived from human Ig. However, Ig Fc domains may also be derived from Ig of any other mammalian species, including, for example, camelid species, rodents (e.g., mice, rats, rabbits, guinea pigs), or non-human primates (e.g., chimpanzees, macaques). Furthermore, Ig Fc regions may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, as well as any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In certain examples, the Fc domain is an IgG Fc domain (e.g., a human IgG region). In certain examples, the Ig Fc domain is an IgG1 Fc domain (e.g., human IgG1). In certain cases, an Ig Fc domain is a chimeric Ig Fc domain containing parts of several different Ig Fc domains. Therefore, it will be understood that the scope of this disclosure encompasses alleles, variants, and mutations of Ig Fc domains that provide monomeric Ig Fc domains.

[0136] In one example, the monomeric Ig Fc domain or a fragment thereof is derived from IgG. For example, the monomeric Ig Fc domain or a fragment thereof is derived from human IgG. In another example, the monomeric Ig Fc domain or a fragment thereof is derived from IgG1. In yet another example, the monomeric Ig Fc domain or a fragment thereof is derived from human IgG1. In yet another example, the monomeric Ig Fc domain or a fragment thereof is derived from IgG4. In yet another example, the monomeric Ig Fc domain or a fragment thereof is derived from human IgG4.

[0137] In one example, the monomeric Ig Fc domain or a fragment thereof enables dimerization of the fusion protein. In such an example, the fusion protein described herein forms a homodimer. In such an example, the fusion protein described herein maintains the biological activity of the neurotrophin.

[0138] In one example, a monomeric Ig Fc domain or fragment does not induce an immune or cellular event. For example, a monomeric Ig Fc domain or fragment does not induce antibody-dependent cytotoxicity (ADCC). In another example, a monomeric Ig Fc domain or fragment does not induce complement-dependent cytotoxicity (CDC). In one example, a monomeric Ig Fc domain or fragment does not induce phagocytosis. In another example, a monomeric Ig Fc domain or fragment does not induce opsonization.

[0139] In one example, a monomeric Ig Fc domain or a fragment thereof facilitates improved expression levels of the fusion protein.

[0140] For example, the monomeric Ig Fc domain or a fragment thereof contains one or more amino acid substitutions relative to the sequence shown in SEQ ID NO: 10.

[0141] For example, when considering the positioning of substitutions within the Ig Fc domain of a fusion protein, the position numbering is relative to the sequence shown in Sequence ID No. 10, not to the entire fusion protein. Therefore, when a neurotrophin is fused to the N-terminus of the Ig domain, the first residue when considering mutations within the Fc domain is the first residue in the sequence shown in Sequence ID No. 10.

[0142] The Ig Fc domain may be further cleavable or may contain substitutions to prevent dimerization and / or reduce ADCC and / or CDC. The ability of the Ig Fc domain fragment to dimerize can be determined using any assay recognized in the art, such as gel filtration or size exclusion chromatography.

[0143] In one example, a monomeric Ig Fc domain or fragment thereof includes at least one N-glycosylation site that reduces dimerization with another monomeric Ig Fc domain or fragment thereof. In another example, a monomeric Ig Fc domain or fragment thereof includes at least one N-glycosylation site that inhibits dimerization with another monomeric Ig Fc domain or fragment thereof. In yet another example, a monomeric Ig Fc domain or fragment thereof includes at least one N-glycosylation site that prevents dimerization with another monomeric Ig Fc domain or fragment thereof. Several exemplary sets of N-glycosylation sites are described in EP2494061, which is incorporated herein by reference.

[0144] In some cases, the monomer Ig Fc domain or a fragment thereof contains at least one N-glycosylation site. For example, the monomer Ig Fc domain or a fragment thereof contains at least one N-glycosylation site within the CH3 domain.

[0145] In some cases, the monomeric Ig Fc domain or a fragment thereof corresponds to the sequence shown in Sequence ID No. 10 by the EU numbering system: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 3 It contains at least one N-glycosylation site at one or more positions selected from the group consisting of 09, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439.

[0146] In one example, the monomeric Ig Fc domain or a fragment thereof, (i) at the position corresponding to amino acid 364 of Sequence ID No. 10 according to the EU numbering system, or (ii) Contains at least one N-glycosylation site at the position corresponding to amino acid 407 of Sequence ID No. 10 according to the EU numbering system.

[0147] In one example, the monomeric Ig Fc domain or a fragment thereof, (i) at the position corresponding to amino acid 364 of Sequence ID No. 10 according to the EU numbering system, and (ii) The system includes at least two N-glycosylation sites at the position corresponding to amino acid 407 of Sequence ID No. 10 according to the EU numbering system.

[0148] In some cases, the monomeric Ig Fc domain or a fragment thereof corresponds to the sequence shown in Sequence ID No. 10 according to the EU numbering system: 237, 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303 This includes substitutions at one or more positions selected from the group consisting of 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. Several exemplary substitutions are described in US7335742, Ishino et al. 2013, J of Biological Chemistry 288:23:16529-16537, Ying et al. 2012, J of Biological Chemistry 287:23:19399-19408, Wang et al. 2017, Frontiers in Immunology 8:1545, and Wikipedianson et al. 2013, mAbs 5:3, each of which is incorporated herein by reference.

[0149] In some examples, the monomeric Ig Fc domain or a fragment thereof contains substitutions at one or more positions selected from the group consisting of 234, 235, 237, and 329 relative to the sequence shown in Sequence ID No. 10 according to the EU numbering system.

[0150] In some examples, the monomeric Ig Fc domain or a fragment thereof contains at least one substitution selected from the group consisting of G237A, S298A, E333A, K334A, P329G, L351S, T366R, L368H, P395K, F405E, Y407N, and K409T, relative to the sequence shown in Sequence ID No. 10 according to the EU numbering system.

[0151] In some examples, the monomeric Ig Fc domain or a fragment thereof contains at least one substitution selected from the group consisting of L234A, L235A, G237A, and P329A relative to the sequence shown in Sequence ID No. 10 according to the EU numbering system.

[0152] In one example, the monomeric Ig Fc domain or a fragment thereof, (i) Serine substituted with alanine at the position corresponding to amino acid 298 of Sequence ID No. 10 according to the EU numbering system, (ii) Glutamic acid substituted with alanine at the position corresponding to amino acid 333 of Sequence ID No. 10 according to the EU numbering system, (iii) Lysine substituted with alanine at the position corresponding to amino acid 334 of Sequence ID No. 10 according to the EU numbering system, (iv) Leucine substituted with serine at the position corresponding to amino acid 351 of Sequence ID No. 10 according to the EU numbering system, (v) Threonine substituted with arginine at the position corresponding to amino acid 366 of Sequence ID No. 10 according to the EU numbering system, (vi) Leucine substituted with histidine at the position corresponding to amino acid 368 of Sequence ID No. 10 according to the EU numbering system, (vii) Proline substituted with lysine at the position corresponding to amino acid 395 of Sequence ID No. 10 according to the EU numbering system, (viii) Phenylalanine substituted with glutamic acid at the position corresponding to amino acid 405 of Sequence ID No. 10 according to the EU numbering system, (ix) Tyrosine substituted with asparagine at the position corresponding to amino acid 407 of Sequence ID No. 10 according to the EU numbering system, (x) Lysine substituted with threonine at the position corresponding to amino acid 409 of Sequence ID No. 10 according to the EU numbering system, and comprising one or more amino acid substitutions selected from the group consisting of (x) lysine substituted with threonine at the position corresponding to amino acid 409 of Sequence ID No. 10, and

[0153] In one example, the monomeric Ig Fc domain or a fragment thereof, (i) Serine substituted with alanine at the position corresponding to amino acid 298 of Sequence ID No. 10 according to the EU numbering system, (ii) Glutamic acid substituted with alanine at the position corresponding to amino acid 333 of Sequence ID No. 10 according to the EU numbering system, (iii) Lysine substituted with alanine at the position corresponding to amino acid 334 of Sequence ID No. 10 according to the EU numbering system, (iv) Leucine substituted with serine at the position corresponding to amino acid 351 of Sequence ID No. 10 according to the EU numbering system, (v) Threonine substituted with arginine at the position corresponding to amino acid 366 of Sequence ID No. 10 according to the EU numbering system, (vi) Leucine substituted with histidine at the position corresponding to amino acid 368 of Sequence ID No. 10 according to the EU numbering system, (vii) Proline substituted with lysine at the position corresponding to amino acid 395 of Sequence ID No. 10 according to the EU numbering system, (viii) Phenylalanine substituted with glutamic acid at the position corresponding to amino acid 405 of Sequence ID No. 10 according to the EU numbering system, (ix) Tyrosine substituted with asparagine at the position corresponding to amino acid 407 of Sequence ID No. 10 according to the EU numbering system, and (x) Consists of one or more amino acid substitutions selected from the group consisting of lysine substituted with threonine at the position corresponding to amino acid 409 of Sequence ID No. 10 according to the EU numbering system.

[0154] In one example, the monomeric Ig Fc domain or a fragment thereof has the following amino acid substitutions: (xi) Tyrosine substituted with asparagine at the position corresponding to amino acid 407 of SEQ ID NO: 10 according to the EU numbering system, and (xii) Contains lysine substituted with threonine at the position corresponding to amino acid 409 of SEQ ID NO: 10 according to the EU numbering system.

[0155] In one example, the monomeric Ig Fc domain or a fragment thereof, (i) Leucine substituted with alanine at the position corresponding to amino acid 234 of Sequence ID No. 10 according to the EU numbering system, (ii) Leucine substituted with alanine at the position corresponding to amino acid 235 of Sequence ID No. 10 according to the EU numbering system, (iii) Glycine substituted with alanine at the position corresponding to amino acid 237 of Sequence ID No. 10 according to the EU numbering system, and (iv) comprising one or more amino acid substitutions selected from the group consisting of proline substituted with glycine at the position corresponding to amino acid 329 of Sequence ID No. 10 according to the EU numbering system.

[0156] In some cases, the constituent Fc domain regions of a monomeric Ig Fc domain or its fragments are linked together by a linker.

[0157] Methods for producing single-chain Fc domain regions are known in the art (see, for example, US2009 / 0252729 and US2011 / 0081345).

[0158] To enhance the manufacturability of the fusion proteins disclosed herein, it may be desirable that the monomeric Ig Fc domain does not contain any non-disulfide-bonded cysteine ​​residues. Therefore, in certain examples, the monomeric Ig Fc domain does not contain any free cysteine ​​residues.

[0159] Linker In some examples, the components of the fusion protein of this disclosure are linked by a linker.

[0160] In some examples, the linker is a polypeptide linker. For example, the linker is a serine residue. In other examples, the linker contains alanine, alanine, and serine.

[0161] The polypeptide linkers of this disclosure are at least one amino acid long and can be of varying lengths. In some examples, the polypeptide linkers of this disclosure are about 1 to about 50 amino acids long. In other examples, the polypeptide linkers of this disclosure are about 5 to about 10 amino acids long. In other examples, the polypeptide linkers of this disclosure are about 10 to about 20 amino acids long. In other examples, the polypeptide linkers of this disclosure are about 15 to about 50 amino acids long.

[0162] In some examples, the linker contains or is a chemical linker. In some examples, the linker is one or more ethylene glycol (EG) units, e.g., two or more EG units (i.e., polyethylene glycol (PEG)). In some examples, the linker contains or consists of a polyethylene glycol (PEG) linker. Polyethylene glycol or PEG refers to a chemical compound composed of repeating ethylene glycol units. An exemplary “PEG linker” contains a compound of the formula: H-(0-CH2-CH2)n-OH, where n is a positive integer (e.g., 1, 10, 20, 50, 100, 200, 300, 400, 500, 600). In some examples, the PEG linker is PEG1000. In some examples, the PEG linker is PEG2000. In some examples, the PEG linker is PEG3000.

[0163] In some examples, the fusion protein contains monomeric Fc domains or fragments linked to serine, which are sequentially linked to neurotrophins by linkers. In some examples, the fusion protein contains monomeric Fc domains or fragments linked by ala-ala-ser, which are sequentially linked to neurotrophins by linkers.

[0164] In other examples, polypeptide linkers include or consist of gly-ser linkers. As used herein, the term “gly-ser linker” refers to a peptide consisting of glycine and serine residues. An example gly / ser linker is given by the formula (Gly4Ser) n The formula contains the amino acid sequence, where n is a positive integer (e.g., 1, 2, 3, 4, or 5). In certain examples, the gly / ser linker is (Gly4Ser)1. In some examples, the gly / ser linker is (Gly4Ser)2. In some examples, the gly / ser linker is (Gly4Ser)3 or (Gly4Ser)4.

[0165] Other linkers suitable for use in the fusion proteins of this disclosure are known in the art, for example, the serine-rich linker disclosed in US5525491, the helix-forming peptide linker (e.g., A(EAAAK)nA(n=2~5)) disclosed in Arai et al, Protein Eng 2001;14:529-32, or the stable linker disclosed in Chen et al, Mol Pharm 2011;8:457-65.

[0166] Other linkers include the GS linker (i.e., (GS)n), the GGSG linker (i.e., (GGSG)n), the GSAT linker, the SEG linker, and the GGS linker (i.e., (GGSGGS)n), where n is a positive integer (e.g., 1, 2, 3, 4, or 5).

[0167] In one example, the N-terminus of a monomeric Ig Fc domain or a fragment thereof is linked to the C-terminus of a neurotrophin by a linker. For example, the N-terminus of a monomeric Ig Fc domain or a fragment thereof is linked to the C-terminus of a neurotrophin by serine.

[0168] In another example, the C-terminus of a monomeric Ig Fc domain or a fragment thereof is linked to the N-terminus of a neurotrophin by a linker.

[0169] Fusion protein activity In certain specific examples, the present disclosure provides a fusion protein having improved biological activity and / or increased receptor binding affinity.

[0170] As used herein, "biologically active" or "biological activity" refers to the ability of a fusion protein to affect the normal biological processes of a conjugated neurotrophin, for example, such as tyrosine receptor kinase (Trk) receptor activation. For example, improved biological activity refers to an increase in the ability of a fusion protein to affect normal biological processes relative to wild-type or unmodified neurotrophin.

[0171] Those skilled in the art will understand that neurotrophins are biologically active as non-covalently bound homodimers.

[0172] The fusion proteins of the present disclosure can be readily screened for biological activity and / or binding affinity, for example, as described below.

[0173] In one example, the biological activity of the fusion protein can be assessed in vivo, for example, in an animal model. In such examples, the fusion protein can be placed in the cochlea of a subject to determine whether the fusion protein directs synaptic repair.

[0174] Affinity measurements can be determined by standard methodologies, for example, immunoassays, surface plasmon resonance (SPR; e.g., using Biacore™ technology [Cytiva], Rich and Myszka Curr. Opin. Biotechnol 11:54, 2000, Englebienne Analyst. 123:1599, 1998), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. For example, the dissociation rate constant (k d ) or association rate constant (k a ) or affinity constant (K D ) can be determined for the fusion proteins described herein and appropriate controls (e.g., wild-type).

[0175] In some cases, the fusion protein has a similar K to the tyrosine receptor kinase (Trk) receptor compared to an unmodified neurotrophin (e.g., a neurotrophin that does not have a monomeric Ig Fc domain or fragment thereof). D or improved K D (That is, a lower K) D It has a value.

[0176] In one example, the fusion protein binds to the Trk receptor with increased affinity compared to the unmodified neurotrophin.

[0177] In one example, the fusion protein is characterized by increased activation of the Trk receptor compared to the unmodified neurotrophin.

[0178] The binding affinity of the fusion protein can also be determined non-quantitatively using flow cytometry. For example, the fusion protein is added to CHO cells stably expressing the Trk receptor, and then stained with a marker (to detect target binding) and another marker (to detect binding) at acidic (pH 5.5) and neutral (pH 7.4) pH, and analyzed by flow cytometry. Relative binding to the Trk receptor is determined, for example, by calculating the average fluorescence intensity relative to unmodified neurotrophins.

[0179] In one example, the Trk receptor is the TrkA receptor. In another example, the Trk receptor is the TrkB receptor. In yet another example, the Trk receptor is the TrkC receptor.

[0180] For example, the fusion protein has an affinity constant (K) less than 800 nM. D ) binds to the TrkA receptor at neutral pH, the TrkA receptor is immobilized on a solid support, and the binding of the fusion protein to the TrkA receptor is determined using surface plasmon resonance (SPR). In one example, the fusion protein has a K700 nM DThe fusion protein binds to the TrkA receptor at a neutral pH, and the TrkA receptor is immobilized on a solid support. The binding of the fusion protein to the TrkA receptor is determined using SPR. In one example, the fusion protein is 400 nM to 800 nM K D The fusion protein binds to the TrkA receptor at a neutral pH, and the TrkA receptor is immobilized on a solid support. The binding of the fusion protein to the TrkA receptor is determined using SPR. In one example, the fusion protein is subjected to a K2-500nM to 700nM K2-5 D The fusion protein binds to the TrkA receptor at a neutral pH, and the TrkA receptor is immobilized on a solid support. The binding of the fusion protein to the TrkA receptor is determined using SPR. In one example, the fusion protein is subjected to a K2-600nM-700nM K2-600nM K2-600nM K2-7 D The fusion protein binds to the TrkA receptor at a neutral pH, and the TrkA receptor is immobilized on a solid support. The binding of the fusion protein to the TrkA receptor is determined using SPR.

[0181] For example, a fusion protein containing NT-3 has an affinity constant (K) of less than 800 nM. D ) binds to the TrkA receptor at neutral pH, and the TrkA receptor is immobilized on a solid support, and the binding of the fusion protein to the TrkA receptor is determined using surface plasmon resonance (SPR). In one example, the fusion protein containing NT-3 has a K700 nM D The fusion protein binds to the TrkA receptor at a neutral pH, and the TrkA receptor is immobilized on a solid support. The binding of the fusion protein to the TrkA receptor is determined using SPR. In one example, the fusion protein containing NT-3 is bound at 400 nM to 800 nM K D The fusion protein binds to the TrkA receptor at a neutral pH, and the TrkA receptor is immobilized on a solid support. The binding of the fusion protein to the TrkA receptor is determined using SPR. In one example, the fusion protein containing NT-3 is bound at 500 nM to 700 nM K DThe fusion protein binds to the TrkA receptor at a neutral pH, and the TrkA receptor is immobilized on a solid support. The binding of the fusion protein to the TrkA receptor is determined using SPR. In one example, the fusion protein containing NT-3 is bound at 600 nM to 700 nM K D The fusion protein binds to the TrkA receptor at a neutral pH, and the TrkA receptor is immobilized on a solid support. The binding of the fusion protein to the TrkA receptor is determined using SPR.

[0182] For example, the fusion protein has an affinity constant (K) less than 60 nM. D ) binds to the TrkB receptor at neutral pH, and the TrkB receptor is immobilized on a solid support, and the binding of the fusion protein to the TrkB receptor is determined using surface plasmon resonance (SPR). In one example, the fusion protein has a K content of less than 50 nM. D The fusion protein binds to the TrkB receptor at a neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR. In one example, the fusion protein has a K2-20nM to 60nM K2-20nM D The fusion protein binds to the TrkB receptor at neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR. In one example, the fusion protein has a K2-20nM to 60nM K2-20nM D The fusion protein binds to the TrkB receptor at a neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR.

[0183] For example, a fusion protein containing NT-3 has an affinity constant (K) of less than 60 nM. D ) binds to the TrkB receptor at neutral pH, and the TrkB receptor is immobilized on a solid support, and the binding of the fusion protein to the TrkB receptor is determined using surface plasmon resonance (SPR). In one example, the fusion protein containing NT-3 has at least 50 nM K DThe fusion protein binds to the TrkB receptor at a neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR. In one example, the fusion protein containing NT-3 is bound to a K2-20nM to 60nM K2-2 D The fusion protein binds to the TrkB receptor at neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR. In one example, the fusion protein containing NT-3 has a K2-30K2-50K2K2 ratio. D The fusion protein binds to the TrkB receptor at a neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR. In one example, the fusion protein containing NT-3 is bound to a K2-40nM (40nM-50nM) D The fusion protein binds to the TrkB receptor at a neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR.

[0184] For example, a fusion protein containing BDNF has an affinity constant (K) of less than 50 nM. D ) binds to the TrkB receptor at neutral pH, and the TrkB receptor is immobilized on a solid support, and the binding of the fusion protein to the TrkB receptor is determined using surface plasmon resonance (SPR). In one example, the fusion protein containing BDNF has a K content of less than 40 nM. D The fusion protein binds to the TrkB receptor at a neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR. In one example, the fusion protein containing BDNF has a K2-20nM to 60nM K2-20nM D The fusion protein binds to the TrkB receptor at a neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR. In one example, the fusion protein containing BDNF has a K2-30nM concentration of 30nM-50nM. DThe fusion protein binds to the TrkB receptor at a neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR. In one example, the fusion protein containing BDNF has a K2-40nM concentration. D The fusion protein binds to the TrkB receptor at a neutral pH, and the TrkB receptor is immobilized on a solid support. The binding of the fusion protein to the TrkB receptor is determined using SPR.

[0185] For example, the fusion protein has an affinity constant (K) less than 10 nM. D ) binds to the TrkC receptor at neutral pH, and the TrkC receptor is immobilized on a solid support, and the binding of the fusion protein to the TrkC receptor is determined using surface plasmon resonance (SPR). In one example, the fusion protein has a K content of less than 5 nM. D The fusion protein binds to the TrkC receptor at a neutral pH, and the TrkC receptor is immobilized on a solid support. The binding of the fusion protein to the TrkC receptor is determined using SPR.

[0186] For example, the fusion protein has a K content of 0.1 nM to 5 nM. D The fusion protein binds to the TrkC receptor at neutral pH, and the TrkC receptor is immobilized on a solid support. Binding of the fusion protein to the TrkC receptor is determined using SPR. In one example, the fusion protein has a K2-0.5nM to 2nM K2-0. D The fusion protein binds to the TrkC receptor at neutral pH, and the TrkC receptor is immobilized on a solid support. The binding of the fusion protein to the TrkC receptor is determined using SPR. In one example, the fusion protein has a K content of 1nM to 2nM. D The fusion protein binds to the TrkC receptor at a neutral pH, and the TrkC receptor is immobilized on a solid support. The binding of the fusion protein to the TrkC receptor is determined using SPR.

[0187] For example, a fusion protein containing NT-3 has an affinity constant (K) of less than 10 nM. D) binds to the TrkC receptor at neutral pH, and the TrkC receptor is immobilized on a solid support, and the binding of the fusion protein to the TrkC receptor is determined using surface plasmon resonance (SPR). In one example, the fusion protein containing NT-3 has a K content of less than 5 nM. D The fusion protein binds to the TrkC receptor at a neutral pH, and the TrkC receptor is immobilized on a solid support. The binding of the fusion protein to the TrkC receptor is determined using SPR.

[0188] For example, a fusion protein containing NT-3 has a K content of 0.1 nM to 5 nM. D The fusion protein binds to the TrkC receptor at a neutral pH, and the TrkC receptor is immobilized on a solid support. The binding of the fusion protein to the TrkC receptor is determined using SPR. In one example, the fusion protein containing NT-3 has a K2-0.5nM to 2nM K2-0. D The fusion protein binds to the TrkC receptor at neutral pH, and the TrkC receptor is immobilized on a solid support. The binding of the fusion protein to the TrkC receptor is determined using SPR. In one example, the fusion protein containing NT-3 has a K2-12K2 ratio of 1nM to 2nM. D The fusion protein binds to the TrkC receptor at a neutral pH, and the TrkC receptor is immobilized on a solid support. The binding of the fusion protein to the TrkC receptor is determined using SPR.

[0189] In one example, the biological activity of a fusion protein is evaluated. Methods for evaluating biological activity are known in the art and / or illustrated herein. For example, the fusion protein is evaluated in vitro and / or ex vivo. One example is a cell culture assay such as CellSensor® (https: / / www.thermofisher.com / order / catalog / product / K1491, Gunewardene et al. J. Control Release 342:295-307, 2022).

[0190] In one example, the fusion protein exhibits improved biological activity (e.g., enhanced biological activity) compared to an unmodified neurotrophin.

[0191] For example, the fusion protein has increased biological activity compared to the unmodified neurotrophin. In one example, the biological activity is (EC 50 This is determined based on the following: For example, a fusion protein with increased biological activity will have an EC of less than 100 pM. 50 It has. In one example, a fusion protein with increased biological activity has an EC of less than 70 pM. 50 It has. In one example, a fusion protein with increased biological activity has an EC of less than 60 pM. 50 It has. For example, the fusion protein has an EC of 20 pM to 100 pM. 50 It has. For example, the fusion protein has an EC of 30 pM to 60 pM. 50 It has. For example, the fusion protein has an EC of 50 pM to 60 pM. 50 It holds.

[0192] In one example, the fusion protein has improved solubility relative to the wild-type neurotrophin. Methods for determining the solubility of the fusion protein of this disclosure are known in the art and / or described herein. In some examples, solubility is determined using gel filtration or size exclusion chromatography. As will be understood by those skilled in the art, if a compound aggregates, the mass of the aggregate will be higher than the mass of the compound itself. Therefore, if the mass of the fusion protein is similar to the mass of the wild-type neurotrophin, it can be determined that the fusion protein is expressed primarily in a soluble form.

[0193] In another example, the fusion protein has improved expression levels compared to wild-type neurotrophins. Methods for determining the expression levels of the fusion proteins of this disclosure are known in the art and / or described herein. In one example, the expression levels of the fusion proteins are determined using SDS-PAGE.

[0194] Method for producing fusion proteins This disclosure provides nucleic acids, expression constructs, and host cells encoding the fusion proteins disclosed herein. Methods for producing the fusion proteins, including expressing these polynucleotides, are also provided.

[0195] As those skilled in the art will understand, the production of neurotrophins is partly difficult and time-consuming due to low yields and the need to refold the neurotrophins isolated from E. coli to achieve correct disulfide bond formation. Advantageously, the fusion proteins described herein are synthesized (synethesised) as monomers that subsequently dimerize. Thus, in certain examples, the fusion proteins of this disclosure facilitate high yields and prospects for large-scale production.

[0196] In one example, this disclosure encompasses nucleic acids encoding fusion proteins disclosed herein. In one example, the nucleic acid encodes a proneurotrophin and a monomeric Ig Fc domain or a fragment thereof. In a particular example, the nucleic acid encodes a proneurotrophin and a monomeric Ig Fc domain or a fragment thereof, with a linker or spacer between the proneurotrophin and the monomeric Ig Fc domain or a fragment thereof. In one example, the nucleic acid is provided in a host cell suitable for nucleic acid expression.

[0197] For example, proneurotrophin is proneurotrophin 3 (proNT-3). For example, proneurotrophin can be mammalian proNT-3. In one example, proneurotrophin is human proNT-3. In one example, proNT-3 contains the sequence shown in SEQ ID NO: 3. In one example, proNT-3 as incorporated herein is a variant of SEQ ID NO: 3. For example, proNT-3 may contain a sequence that is at least 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 3.

[0198] In one example, the proneurotrophin is proneurotrophin 4 (pro-NT-4). For example, the proneurotrophin can be mammalian pro-NT-4. In one example, the proneurotrophin is human pro-NT-4. In one example, pro-NT-4 contains the sequence shown in Sequence ID No. 5. In one example, pro-NT-4 as incorporated herein is a variant of Sequence ID No. 5. For example, pro-NT-4 may contain a sequence that is at least 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to Sequence ID No. 5.

[0199] In one example, proneurotrophin is nerve growth factor (proNGF). For example, proneurotrophin can be mammalian proNGF. In another example, proneurotrophin is human proNGF. In one example, proNGF contains the sequence shown in Sequence ID No. 9. In one example, proNGF is a variant of Sequence ID No. 9. For example, proNGF may contain a sequence that is at least 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to Sequence ID No. 9.

[0200] In one example, proneurotrophin is brain-derived neurotrophic factor (pro-BDNF). For example, proneurotrophin can be mammalian pro-BDNF. In another example, proneurotrophin is human pro-BDNF. In one example, pro-BDNF contains the sequence shown in SEQ ID NO: 7. In another example, pro-BDNF is a variant of SEQ ID NO: 7. For example, pro-BDNF may contain a sequence that is at least 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to SEQ ID NO: 7.

[0201] For example, the fusion protein includes a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No. 1, and a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No. 2.

[0202] For example, the fusion protein includes a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No. 6, and a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No. 2.

[0203] For example, the fusion protein contains a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 21. For example, the fusion protein contains a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 23. For example, the fusion protein contains a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 24. For example, the fusion protein contains a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 25. For example, the fusion protein contains a sequence that has at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 26.

[0204] In some cases, the fusion protein is expressed in a soluble form by the host cell.

[0205] For example, the nucleic acid encoding the fusion protein is a DNA sequence containing a sequence that has at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No. 15. For example, the nucleic acid encoding the fusion protein is a DNA sequence containing a sequence that has at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in Sequence ID No. 17.

[0206] For example, the expression construct includes a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 18. For example, the expression construct includes a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 20.

[0207] In one example, the expression construct encodes a sequence that has at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 12. In another example, the expression construct encodes a sequence that has at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 14.

[0208] The nucleic acids encoding the fusion proteins disclosed herein are typically inserted into an expression construct or vector for introduction into a host cell which can be used to produce a desired amount of the claimed fusion protein. Thus, in some examples, this disclosure provides expression constructs or vectors comprising the nucleic acids disclosed herein, as well as host cells comprising these constructs or vectors and the nucleic acids.

[0209] One of the advantages of this approach is that the signal peptide and prodomain are cleaved during synthesis and secretion, providing neurotrophins that align more closely with their naturally occurring counterparts.

[0210] For example, this disclosure provides nucleic acids that encode or express the fusion proteins described herein.

[0211] In another example, the disclosure provides proneurotrophins and nucleic acids that encode or express monomeric immunoglobulin (Ig) crystallizable fragment (Fc) domains or fragments thereof. For example, the proneurotrophin is proneurotrophin 3. In one example, the proneurotrophin is proneurotrophin 4. In one example, the proneurotrophin is pro-BDNF. In one example, the proneurotrophin is pro-NGF.

[0212] In some cases, the constituent Fc domain regions of a monomeric Ig Fc domain or its fragments are linked together by a linker.

[0213] Numerous expression constructs or vector systems may be used for the purposes of this disclosure, for example, by any preferred method including cloning of a suitable sequence, or by the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphorumidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; or by using an automated synthesis apparatus such as that described in Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984, Beaucage & DNA can be prepared by direct chemical synthesis, such as the solid-phase phosphoramidite triester method described by Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, and the solid support method described in U.S. Patent No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with complementary sequences or by polymerization with DNA polymerase using the single strand as a template. Those skilled in the art will recognize that while the chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences can be obtained by ligation of shorter sequences.

[0214] Exemplary nucleic acid sequences encoding monomeric Ig Fc domains can be prepared by cloning techniques. Examples of instructions sufficient to guide those skilled in the art through appropriate cloning and sequencing techniques, as well as numerous cloning exercises, can be found in Sambrook et al., (see above), Berger and Kimmel (eds.), (see above), and Ausubel, (see above). Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, Calif.), and Applied Biosystems (Foster City, Calif.), Thermo Fisher Scientific, as well as many other commercial suppliers known to those skilled in the art.

[0215] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustaining sequence replication systems (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to those skilled in the art.

[0216] The terms “vector,” “expression vector,” or “expression construct” are used synonymously herein for the purposes of this specification and the claims, meaning a vector used in accordance with this disclosure as a vehicle for introducing and expressing a desired gene in a cell. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses.

[0217] More generally, once a vector or nucleic acid sequence encoding a fusion protein is prepared, the expression vector may be introduced into a suitable host cell. That is, the host cell can be transfected. The introduction of the expression vector into the host cell can be achieved by various techniques well known to those skilled in the art. These include, but are not limited to, transfection, electroporation, protoplast fusion, cell fusion with enveloped DNA, microinjection, and infection with an intact virus. See Ridgway, AAG, "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472; and Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Most preferably, the introduction of the vector into the host cell is via transfection (e.g., calcium phosphate precipitation, FuGENE, Lipofectamine (trade name), and polyethyleneimine (PEI)). The transformed host cells are grown under conditions suitable for the production of the fusion protein, and the expression of the fusion protein is assayed. The nucleic acid sequences encoding the fusion proteins of this disclosure can be transiently transfected into host cells. Cell lines may also be produced by stably integrating the nucleic acid sequences encoding the fusion proteins together with sequences encoding selectable genetic markers, such as glutamine synthetase (GS) or dihydrofolate reductase (DHFR), to enable the use of drug selection for inducing cell lines that produce the protein of interest. Exemplary assay techniques include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), or fluorescence-activated cell sorting and analysis (FACS), and immunohistochemistry.

[0218] In certain specific examples, in vitro production can be "scaled up" to obtain a large amount of the desired fusion protein. Techniques for mammalian cell culture under tissue culture conditions are known in the art, and include, for example, homogeneous suspension culture in an airlift reactor or continuous stirred tank reactor, or immobilized or entrapped cell culture, for example, in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatography methods, for example, gel filtration, ion exchange chromatography, chromatography on DEAE cellulose, and / or (immuno)affinity chromatography.

[0219] Isolation and purification of the expressed fusion protein can be performed by conventional means using, for example, preparative chromatography and immunological separation. Once expressed, the fusion protein can be purified by standard procedures in the art including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see generally R. Scopes, Protein Purification, Springer-Verlag, N.Y., 1982, and Janson, Jan-Christer, Protein Purification: Principles, High Resolution Methods, and Applications, 3rd Edition, Wiley Series in Methods of Biochemical Analysis). Substantially pure compositions with at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or higher homogeneity can be used for pharmaceutical purposes. Once purified partially or to the desired homogeneity, when used therapeutically, the fusion protein should be substantially free of endotoxin.

[0220] Non-limiting examples of suitable mammalian cells include HEK cells, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, 211 A cells, and derivatives thereof.

[0221] In one example, the host cell is a HEK cell. For example, the HEK cell is a HEK293 cell. In one example, the HEK cell is a Freestyle HEK293-F cell. In another example, the HEK cell is a HEK293T cell. In one example, the host cell is a CHO cell. In one example, the CHO cell is an ExpiCHO cell. In one example, the CHO cell is a CHOK1 cell. In one example, the host cell is a BHK cell. In one example, the host cell is an MDCK cell.

[0222] Superparticle The present disclosure provides a composition comprising a superparticle, wherein the superparticle comprises the fusion protein described herein. The present disclosure also provides a composition comprising a superparticle, wherein the superparticle comprises the nucleic acid or expression vector described herein.

[0223] The term "superparticle" is used in the context of the present disclosure to refer to an agglomerated particle comprising a network of pores. The network of pores provides the superparticle with a large pore volume and surface area for carrying a payload, for example, a fusion protein. A large pore volume and surface area is advantageous because it can enhance the amount of payload that can be carried by the superparticle. In certain examples, the superparticle is an agglomerated nanoparticle.

[0224] In one example, the superparticle contains at least 1.5 μg of fusion protein. In another example, the superparticle contains at least 2.0 μg of fusion protein. In yet another example, the superparticle contains at least 3.0 μg of fusion protein. In yet another example, the superparticle contains at least 4.0 μg of fusion protein. In yet another example, the superparticle contains at least 5.0 μg of fusion protein. In yet another example, the superparticle contains at least 6.0 μg of fusion protein. In yet another example, the superparticle contains at least 7.0 μg of fusion protein. In yet another example, the superparticle contains at least 8.0 μg of fusion protein. In yet another example, the superparticle contains at least 9.0 μg of fusion protein. In yet another example, the superparticle contains at least 10 μg of fusion protein. In yet another example, the superparticle contains at least 11 μg of fusion protein. In yet another example, the superparticle contains at least 12 μg of fusion protein. In yet another example, the superparticle contains at least 15 μg of fusion protein. In another example, the superparticle contains at least 20 μg of fusion protein.

[0225] In one example, the superparticle contains at least 1.5 μg of nucleic acid or expression vector. In another example, the superparticle contains at least 2.0 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 3.0 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 4.0 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 5.0 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 6.0 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 7.0 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 8.0 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 9.0 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 10 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 11 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 12 μg of nucleic acid or expression vector. In another example, the superparticle contains at least 15 μg of nucleic acid or expression vector. In yet another example, the superparticle contains at least 20 μg of nucleic acid or expression vector.

[0226] Therapeutic efficacy may be improved by administering a superparticle containing multiple different fusion proteins. Therefore, in some examples, a superparticle may contain at least two, at least three, at least four, or at least five different fusion proteins. For example, a superparticle may contain a fusion protein containing NGF and a fusion protein containing NT-3. In another example, a superparticle may contain a fusion protein containing NT-4 and a fusion protein containing NT-3. In yet another example, a superparticle may contain a fusion protein containing BDNF and a fusion protein containing NT-3.

[0227] Therapeutic efficacy can be improved by administering a superparticle containing multiple different nucleic acids or expression vectors. Therefore, in some examples, a superparticle may contain at least two, at least three, at least four, or at least five different nucleic acids or expression vectors. For example, a superparticle may contain a fusion protein containing NGF and a fusion protein containing NT-3. In another example, a superparticle may contain a fusion protein containing NT-4 and a fusion protein containing NT-3. In yet another example, a superparticle may contain a fusion protein containing BDNF and a fusion protein containing NT-3.

[0228] In one example, a superparticle contains at least two payloads, one of which is a fusion protein and the other is any drug useful for treating a disorder. In another example, a superparticle contains at least two payloads, one of which is a nucleic acid or expression vector and the other is any drug useful for treating a disorder. Examples of drugs include polynucleotides, antibodies, monoclonal antibodies, antibody fragments, antibody-drug conjugates, proteins, biologically active proteins, fusion proteins, recombinant proteins, peptides, polypeptides, synthetic polypeptides, vaccines, therapeutic serums, viruses, polynucleotides, cells such as stem cells or parts thereof, and biological products such as small molecules. Exemplary drugs include neurotrophic factors, steroids, or antioxidants. Examples of neurotrophic factors include the agents discussed above that have known therapeutic efficacy in directly or indirectly enhancing cell survival from the auditory system and / or their synaptic connections. Some exemplary neurotrophic factors include members of the cilioneurotrophic factor (CNTF) family, e.g., CNTF, leukemia inhibitory factor (LIF), interleukin-6 (IL-6), glial maturation factor (GMF), insulin-like growth factor-1 (IGF-1), neuregulin 1, neuregulin 2, neuregulin 3, etc. Examples include neuregulin 4, vascular endothelial growth factor (VEGF), members of the glial cell-derived neurotrophic factor (GDNF) family, such as GDNF, neuruturin (NRTN), artemin (ARTN), and percefin (PSPN), ephrins, such as A1, A2, A3, A4, A5, B1, B2, and B3, interleukins, such as IL-11, antibodies or other binding proteins, such as anti-tropomyosin receptor kinase (Trk) B, anti-Trk C, or binding proteins that interact with the p75 neurotrophin receptor, such as p75 neurotrophin receptor antagonists. In another example, neurotrophic factors include nucleic acids. For example, neurotrophic factors can include gene therapies, silencing RNAs such as siRNA or miRNA, and expression constructs such as DNA plasmids containing the nucleic acid of interest.In some cases, neurotrophic factors are expression constructs containing nucleic acids that encode opsins. In other cases, superparticles may include antineoplastic agents such as cisplatin or related compounds, antibiotics such as aminoglycosides or related compounds such as tobrahmycin, loop diuretics such as furosemide, antimetabolites such as methotrexate, salicylates such as aspirin, or radioactive moieties, and neurotrophins.

[0229] Various ear interventions, such as surgical procedures and the implantation of hearing devices, can result in side effects in the middle and inner ear, including tissue damage, inflammation, and / or infection. The resulting biological responses to such side effects may indirectly affect the growth or viability of cells from the auditory system and / or their synaptic connections. Therefore, in some cases, the fusion proteins of this disclosure may aid in tissue repair, reduction of inflammation, and / or reduction of infection.

[0230] In one example, the superparticles of this disclosure are made from nanoparticles having a diameter of approximately 1 nm to 100 nm. In another example, the superparticles are made from microparticles having a diameter of approximately 0.1 μm to 100 μm. In yet another example, the superparticles are made from nanoparticles and microparticles.

[0231] Exemplary particles forming the superparticles of this disclosure include organic particles, inorganic particles, metallic particles, or combinations thereof. Exemplary organic particles include polymer particles such as polyglycolic acid (PGA), polylactic acid (PLA), poly(methacrylic acid), poly(ethacrylic acid), polyacrylic acid (PAA), poly(N-isopropylacrylamide), poly(N,N-dimethylacrylamide), polyamide, poly-2-hydroxybutyrate (PHB), gelatin, polycaprolactone (PCL), and poly(lactic acid-coglycolic acid) (PLGA). Exemplary inorganic particles include mineral fillers such as heavy fillers or high-density fillers, pigments, clays, and other synthetic particles. Other exemplary inorganic particles include high-density minerals such as barite, hematite, and magnesium oxide, and inorganic oxides including titanium dioxide, calcium oxide, zinc oxide, magnesium oxide, cerium oxide, zirconium dioxide, and silicon dioxide. In one example, the material is silicon dioxide (i.e., silica). Therefore, in one example, the superparticles can be called silica superparticles. Exemplary metallic particles include gold, silver, and copper. In one example, the superparticles may consist of the same particles. For example, the superparticles may consist substantially of silica particles. In another example, the superparticles may consist of different particles, such as silica and clay particles. In yet another example, the superparticles may contain at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different particles.

[0232] In some examples, the superparticles include polyelectrolytes or polyelectrolyte materials. Examples of such superparticles are disclosed in WO2006 / 037160. In these examples, the polyelectrolyte may be a positively charged polyelectrolyte (or capable of being positively charged) or a negatively charged polyelectrolyte (or capable of being negatively charged), or have a net charge of zero.

[0233] The superparticles of this disclosure can have a variety of shapes. For example, a superparticle can have a spherical shape. Exemplary spherical shapes include spheres and ovals. In another example, a superparticle can have a non-spherical shape. Exemplary non-spherical shapes include dumbbells, hemispheres, disks, tetrahedra, fibers, spherical cylinders, and irregular shapes. In some examples, a superparticle can have a regular structure. For example, a superparticle can include a regular array.

[0234] The spherical superparticles of this disclosure may be characterized by their diameter. For example, the superparticles of this disclosure have a diameter greater than 100 μm. For example, the superparticles of this disclosure can have diameters of at least about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, and about 1000 μm. For example, a superparticle can have a diameter of about 550 μm. This diameter of the superparticle is advantageous because it allows for high drug loading while facilitating inner ear delivery via cannula. In other examples, the superparticles can have diameters of approximately 150 μm to 1000 μm, 200 μm to 900 μm, and 300 μm to 800 μm. In yet another example, the superparticles can have diameters of approximately 400 μm to 600 μm. In yet another example, the superparticles can have diameters of approximately 450 μm to 550 μm. In yet another example, the superparticles can have diameters of approximately 520 μm to 580 μm. In yet another example, the superparticles can have diameters of approximately 460 μm to 540 μm. In yet another example, the superparticles can have diameters of approximately 470 μm to 530 μm. In yet another example, the superparticles can have diameters of approximately 480 μm to 520 μm. In yet another example, the superparticles can have diameters of approximately 490 μm to 510 μm. In other examples, superparticles can be characterized by a width spanning the widest point of their three-dimensional structure. For example, a superparticle may have a width that coincides with the diameter exemplified above.

[0235] In some cases, superparticles can be produced from compositions comprising nanoparticles and alginic acid or its polysaccharide derivatives. Various examples of nanoparticles are provided above. In some cases, the nanoparticles have a bimodal pore structure. Nanoparticles can be prepared using various methods. One such method is described in Cui et al. 2015, ACS Nano, 9, 1571-1580.

[0236] In another example, the superparticles according to this disclosure are produced by electrospraying. An example of electrospraying is outlined in Jaworek A., 2007 Powder Technology 1, 18-35. Another example of electrospraying is illustrated below. In one example, this disclosure encompasses a method for producing superparticles, comprising electrospraying a composition comprising nanoparticles and alginic acid or a polysaccharide derivative thereof into a dicationic aqueous solution.

[0237] Those skilled in the art will understand that electrospray parameters can be optimized based on the type of solution used for electrospraying. For example, voltage and flow rate can be optimized to provide ultraparticles of a desired size. In one example, the flow rate is approximately 6–10 mL / h -1 In another example, the flow rate is approximately 7-9 mL / h. -1 In another example, the flow rate is approximately 8 mL / h. -1 In one example, the voltage is approximately 10-25kV. In another example, the voltage is approximately 11-20kV. In yet another example, the voltage is approximately 12-14kV. In yet another example, the voltage is approximately 13kV.

[0238] In one example, the ultraparticles are produced by electrospraying a nanoparticle solution. In another example, the concentration of nanoparticles in the solution is approximately 20 mg / mL. In yet another example, the concentration of nanoparticles in the solution is approximately 30 mg / mL. In yet another example, the concentration of nanoparticles in the solution is approximately 40 mg / mL. In yet another example, the concentration of nanoparticles in the solution is approximately 50 mg / mL. In yet another example, the concentration of nanoparticles in the solution is approximately 60 mg / mL.

[0239] In another example, the superparticles are produced by electrospraying a nanoparticle solution containing alginate or a derivative thereof. In one example, the nanoparticle solution is 5 mg mL -1 It is prepared from an alginate solution. In one example, the nanoparticle solution is 10 mg mL -1 It is prepared from an alginate solution. In one example, the nanoparticle solution is 20 mg mL -1 It is prepared from an alginate solution. In one example, the nanoparticle solution is 30 mg mL -1 It is prepared from an alginate solution. In another example, the nanoparticle solution is 5 mg mL -1 ~30mg mL -1 It is prepared from an alginate solution. In another example, the nanoparticle solution is 10 mg mL -1 ~30mg mL -1 It is prepared from an alginate solution. In another example, the nanoparticle solution is 20 mg mL -1 ~30mg mL -1 It is prepared from an alginate solution.

[0240] In one example, the nanoparticle solution was 5 mg mL in water. -1 It is prepared from alginic acid. In one example, the nanoparticle solution is prepared in 10 mg mL of water. -1 It is prepared from alginic acid. In one example, the nanoparticle solution is prepared in 20 mg mL of water. -1 It is prepared from alginic acid. In one example, the nanoparticle solution is prepared in 30 mg mL of water. -1 It is prepared from alginic acid. In another example, the nanoparticle solution is prepared in 5 mg mL of water. -1 ~30mg mL -1is prepared from alginic acid. In another example, the nanoparticle solution is 10 mg mL in water -1 ~30 mg mL -1 is prepared from alginic acid. In another example, the nanoparticle solution is 20 mg mL in water -1 ~30 mg mL -1 is prepared from alginic acid.

[0241] In another example, supraparticles are produced by electrospraying a nanoparticle solution containing alginic acid.

[0242] Alginic acid derivatives are not particularly limited as long as they form a gel at a defined temperature. In one example, the alginic acid derivative is a polysaccharide derivative. Alginic acid derivatives include various alginate forms. Examples include sodium alginate, potassium alginate, and calcium alginate. Other examples include barium alginate and strontium alginate. In one example, the alginic acid is sodium alginate. In another example, supraparticles are produced by electrospraying a nanoparticle solution containing alginic acid.

[0243] Alginates of various viscosities may be used to produce supraparticles according to the present disclosure, depending on the desired size and shape of the supraparticles. For example, an alginate having a viscosity of about 20 to 300 mPa*s can be used. In another example, the alginate has a viscosity of about 20 to 200 mPa*s. In one example, the alginate has a viscosity of 20 mPa*s. In another example, the alginate has a viscosity of 100 mPa*s. In another example, the alginate has a viscosity of 200 mPa*s.

[0244] In one example, supraparticles are produced by electrospraying a composition comprising nanoparticles into an aqueous solution. In one example, this is a dicationic aqueous solution. Exemplary dicationic components include Ca 2+ and Ba 2+Examples include the following. For example, the aqueous solution may comprise calcium chloride. In another example, the aqueous solution comprises barium chloride.

[0245] In one example, the superparticles are produced by electrospraying a composition, wherein the concentration of alginate in the composition is 5 mg mL -1 to 30 mg mL -1 and the concentration of nanoparticles in the composition is 20 mg mL -1 to 50 mg mL -1 and the voltage is 10 kV to 25 kV. In another example, the superparticles are produced by electrospraying a composition, wherein the concentration of alginate in the composition is 10 mg mL -1 to 30 mg mL -1 and the concentration of nanoparticles in the composition is 30 mg mL -1 to 50 mg mL -1 and the voltage is 11 kV to 21 kV. In another example, the superparticles are produced by electrospraying a composition, wherein the concentration of alginate in the composition is 20 mg mL -1 to 30 mg mL -1 and the concentration of nanoparticles in the composition is 35 mg mL -1 to 45 mg mL -1 and the voltage is 12 kV to 14 kV. In these examples, the flow rate can be 8 mL h -1 .

[0246] In another example, the superparticles are produced by electrospraying a composition, wherein the concentration of alginate in the composition is 30 mg mL -1 and the concentration of nanoparticles in the composition is 40 mg mL -1 the voltage is 13 kV, and the flow rate is 8 mL h -1 .

[0247] In one example, the ultrafine particles produced using the method defined herein are subjected to calcination to remove alginate or a derivative thereof. In one example, the calcination is carried out at approximately 500°C. In another example, the calcination is carried out at approximately 550°C. In yet another example, the calcination is carried out at approximately 600°C. In yet another example, the calcination is carried out at approximately 650°C. In yet another example, the calcination is carried out at approximately 700°C. In one example, the calcination is carried out for about 6 to 30 hours. In yet another example, the calcination is carried out for about 10 hours. In yet another example, the calcination is carried out for about 20 hours. In yet another example, the calcination is carried out for about 30 hours.

[0248] The method for preparing fusion protein-loaded superparticles is not particularly limited, as long as the resulting superparticles can be loaded with at least 1.5 μg of fusion protein. Preferably, the resulting superparticles can deliver a payload to the ear of a target. Exemplary loading methods are outlined in Wang et al. (2009) J.Mater.Chem. 19, 6451 and include encapsulation and inclusion of the fusion protein. In one non-limiting example, superparticles may be loaded by contacting them with an aqueous solution of fusion protein, followed by incubation for a period of time. The fusion protein solution may contain an amount of fusion protein greater than the amount to be loaded into the superparticle, and the incubation can be carried out at room temperature. The loading of the fusion protein may be enhanced by stirring the solution containing the superparticles and fusion protein.

[0249] Those skilled in the art will understand that the required level of fusion protein is likely to be influenced by the fusion protein itself and the indications treated by this disclosure.

[0250] The superparticles may have pore sizes selected from the examples considered below. In some examples, the superparticles are microporous. The term "microporous" is used in the context of this disclosure to refer to particles having a pore size of less than about 2 nm. For example, microporous superparticles may have pore sizes of about 0.5 nm to about 2 nm. In other examples, microporous superparticles may have pore sizes of about 1 nm to about 2 nm and about 1.5 nm to about 2 nm. In yet another example, the superparticles are mesoporous. The term "mesoporous" is used in the context of this disclosure to refer to particles having pores with a diameter of about 2 nm to about 50 nm. For example, mesoporous superparticles may have pore sizes of about 2 nm to about 50 nm. In other examples, mesoporous superparticles may have pore sizes of about 2 nm to about 40 nm and about 2 nm to about 30 nm. In yet another example, the superparticles are macroporous. In the context of this disclosure, the term "macroporous" is used to refer to particles having a pore size greater than approximately 50 nm. For example, macroporous hyperparticles have a pore size of approximately 50 nm to approximately 500 nm. In other examples, macroporous hyperparticles have pore sizes of approximately 50 nm to approximately 250 nm, approximately 50 nm to approximately 150 nm, and approximately 50 nm to approximately 100 nm.

[0251] In one example, the superparticles are composed of microporous nanoparticles. In another example, the superparticles are composed of nanoparticles with pore sizes of approximately 0.5 nm to 2 nm. In yet another example, the superparticles are composed of nanoparticles with pore sizes of approximately 1 nm to 2 nm and approximately 1.5 nm to 2 nm. In yet another example, the superparticles are composed of mesoporous nanoparticles. In one example, the superparticles are composed of nanoparticles with pore sizes of approximately 2 nm to 50 nm. In yet another example, the superparticles are composed of nanoparticles with pore sizes of approximately 2 nm to 40 nm and approximately 2 nm to 30 nm. In yet another example, the superparticles are composed of macroporous nanoparticles. In one example, the superparticles are composed of nanoparticles with pore sizes of approximately 50 nm to 95 nm. In yet another example, the superparticles are composed of nanoparticles with pore sizes of approximately 50 nm to 85 nm, approximately 50 nm to 75 nm and approximately 50 nm to 65 nm.

[0252] In other examples, superparticles include pore sizes in the range of 1 nm to 200 nm. It will be understood by those skilled in the art that superparticles can include a range of pore sizes. For example, a single superparticle can be microporous, mesoporous, and macroporous.

[0253] Those skilled in the art will understand that the pore size of superparticles can be measured, for example, by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray computed tomography. Those skilled in the art can identify superparticles having the pore sizes exemplified above by measuring the width across the widest point of their three-dimensional structure. In some examples, the widest point or pore may be on the surface of the superparticle.

[0254] Use of fusion proteins This disclosure provides a method for promoting the survival of spiral ganglion neurons in the ear of a subject, comprising administering a fusion protein or composition described herein. This disclosure also provides a method for treating hearing impairment in a subject, comprising administering a fusion protein or composition described herein. This disclosure provides a method for promoting the survival of spiral ganglion neurons in the ear of a subject, comprising administering a nucleic acid or expression vector described herein. This disclosure also provides a method for treating hearing impairment in a subject, comprising administering a nucleic acid or expression vector described herein.

[0255] This disclosure also provides the use of the fusion proteins or compositions described herein in the manufacture of a pharmaceutical product for promoting the survival of spiral ganglion neurons in a target ear. This disclosure further provides the use of the fusion proteins or compositions described herein in the manufacture of a pharmaceutical product for the treatment of hearing impairment. This disclosure also provides the use of the nucleic acids or expression vectors described herein in the manufacture of a pharmaceutical product for promoting the survival of spiral ganglion neurons in a target ear. This disclosure further provides the use of the nucleic acids or expression vectors described herein in the manufacture of a pharmaceutical product for the treatment of hearing impairment.

[0256] This disclosure also provides a fusion protein or composition described herein for use in promoting the survival of spiral ganglion neurons in the ear of a subject. This disclosure also provides a fusion protein or composition described herein for use in the treatment of hearing impairment. This disclosure also provides a nucleic acid or expression vector described herein for use in promoting the survival of spiral ganglion neurons in the ear of a subject. This disclosure also provides a nucleic acid or expression vector described herein for use in the treatment of hearing impairment.

[0257] Therefore, in one example, the fusion protein or composition according to this disclosure is administered to a subject in an amount effective to treat a disease or disorder in the subject. In another example, the nucleic acid or expression vector according to this disclosure is administered to a subject in an amount effective to treat a disease or disorder in the subject. In one example, the disease or disorder is hearing loss. The term “hearing loss” is used in the context of this disclosure to refer to any reduction in the subject’s ability to detect or process sound. Therefore, references to hearing loss include partial hearing loss or complete inability to hear.

[0258] In some cases, fusion proteins treat hearing loss. In some cases, the compositions, nucleic acids, and expression vectors described herein treat hearing loss. In such cases, the treatment repairs synaptic connections in the cochlea of ​​the subject.

[0259] In some cases, hearing loss is characterized as sensory neuronal hearing loss (SNHL). In the context of this disclosure, SNHL is used to refer to hearing loss resulting from damage to delicate sensory hair cells in the cochlea, or loss of their synaptic connections with spiral ganglion neurons (SGNs), or dysfunction of cochlear Schwann cells or glial cells. In some cases, hearing loss is characterized as age-related hearing loss. In other cases, hearing loss is noise-induced. In other cases, hearing loss is disease-induced or genetic. In other cases, hearing loss is induced by exposure to otoxins, such as aminoglycosides.

[0260] In some cases, the subject is a mammal. In other cases, the subject is a human. For example, a human subject may be an adult. In some cases, a human subject may be a child. Other exemplary mammalian subjects include companion animals such as dogs or cats, or domestic animals such as horses or cattle. The terms “subject,” “patient,” or “individual” are terms that can be used synonymously in this disclosure depending on the context.

[0261] In one example, the fusion protein or composition according to this disclosure is administered intraperitoneally. The disclosure also encompasses a method for delivering a fusion protein or composition to cells, tissues, or organs in a subject via the ear, comprising administering the fusion protein or composition according to this disclosure to the ear of the subject. In this example, the method may deliver the fusion protein or composition to cells from the inner ear, middle ear, and / or vestibular system of the subject. In another example, the method delivers the fusion protein or composition to nerve cells, nerve tissue, or the brain of the subject. In one example, the nucleic acid or expression vector according to this disclosure is administered intraperitoneally. The disclosure also encompasses a method for delivering a nucleic acid or expression vector to cells, tissues, or organs in a subject via the ear, comprising administering the nucleic acid or expression vector according to this disclosure to the ear of the subject. In this example, the method may deliver the nucleic acid or expression vector to cells from the inner ear, middle ear, and / or vestibular system of the subject. In another example, the method delivers the nucleic acid or expression vector to nerve cells, nerve tissue, or the brain of the subject. In one example, the fusion protein or composition is administered onto the eardrum. In this example, the fusion protein or composition may be formulated for topical administration (e.g., drops, gels, foams, sprays). In one example, the nucleic acid or expression vector is administered onto the tympanic membrane. In this example, the nucleic acid or expression vector may be formulated for topical administration (e.g., drops, gels, foams, sprays).

[0262] In another example, the compositions of this disclosure are administered into the “middle ear” cavity. In another example, the fusion proteins, nucleic acids, or expression vectors of this disclosure are administered into the “middle ear” cavity. In the context of this disclosure, the term “middle ear” is used to refer to the space between the tympanic membrane and the inner ear. Thus, the middle ear is outside all inner ear tissue. For example, a fusion protein or composition may be administered into the middle ear by injection through the tympanic membrane. In this example, the fusion protein or composition may be administered as an accumulation injection. In another example, an opening in the tympanic membrane may be made by the treating clinician to facilitate access to the middle ear for the fusion protein or composition. When a fusion protein or composition is administered into the middle ear, it may be administered over a circular window and / or oval window(s). For example, nucleic acids or expression vectors may be administered into the middle ear by injection through the tympanic membrane. In this example, nucleic acids or expression vectors may be administered as an accumulation injection. In another example, an opening in the tympanic membrane may be made by the treating clinician to facilitate access to the middle ear for the nucleic acid or expression vector. When administering nucleic acids or expression vectors into the middle ear, the nucleic acids or expression vectors may be administered over a circular window and / or oval window(s).

[0263] In another example, a fusion protein or composition is administered to the inner ear. For example, a fusion protein or composition can be administered to the cochlea. In one example, a fusion protein or composition can be administered to the basal gyrus of the cochlea. In yet another example, a nucleic acid or expression vector is administered to the inner ear. For example, a nucleic acid or expression vector can be administered to the cochlea. In one example, a nucleic acid or expression vector can be administered to the basal gyrus of the cochlea. Surgical techniques for obtaining access to the cochlea or other structures of the inner ear are known in the art. Exemplary techniques for surgically accessing the human cochlea are described, for example, in Clark GM, et al., "Surgery for an improved multiple-channel cochlear implant," Ann Otol Rhinol Laryngol 93:204-7, 1984, and Clark GM, et al., "Surgical and safety considerations of multichannel cochlear implants in children," Ear and Hearing Suppl. 12:15S-24S, 1991.

[0264] Various ear interventions, such as surgical procedures and the implantation of hearing devices, can result in side effects in the middle and inner ear, including tissue damage, inflammation, and / or infection. The resulting biological responses to such side effects can indirectly affect cell growth or viability from the auditory system and / or their synaptic connections. Therefore, in some cases, neurotrophins may aid in tissue repair, reduction of inflammation, and / or reduction of infection.

[0265] The combination of fusion proteins or compositions with ear interventions is discussed above. In these examples, the ear intervention may be performed concurrently with the administration of the fusion protein. For example, a cochlear device can be implanted at the same time as the fusion protein. However, increased survival of spiral ganglion neurons can improve the usefulness of the cochlear implant. Therefore, it may be desirable to implant the cochlear device after the administration of the fusion protein. For example, the cochlear device can be implanted approximately one month, two months, three months, or six months after the administration of the fusion protein. In these examples, additional fusion proteins may be administered together with the cochlear device.

[0266] In one example, a first dose of the fusion protein or composition is administered to the cochlea of ​​the subject, and at least a second dose of the fusion protein or composition is administered to the circular and / or oval window(s) of the subject.

[0267] The combination of ear interventions with nucleic acids or expression vectors has been discussed above. In these examples, the ear intervention may be performed concurrently with the administration of nucleic acids or expression vectors. For example, a cochlear device can be implanted simultaneously with the nucleic acid or expression vector. However, increased survival of spiral ganglion neurons can improve the usefulness of the cochlear implant. Therefore, it may be desirable to implant the cochlear device after the administration of nucleic acids or expression vectors. For example, the cochlear device can be implanted approximately 1 month, 2 months, 3 months, or 6 months after the administration of nucleic acids or expression vectors. In these examples, additional fusion proteins may be administered together with the cochlear device.

[0268] In one example, a first dose of the nucleic acid or expression vector is administered to the cochlea of ​​the subject, and at least a second dose of the nucleic acid or expression vector is administered to the subject's circular window and / or oval window(s).

[0269] In one example, a therapeutically effective amount of fusion protein or composition is administered to the target ear. In another example, multiple fusion proteins or compositions are administered to the target ear. For example, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 fusion proteins or compositions can be administered to the target ear. In yet another example, approximately 1 to 10 fusion proteins or compositions are administered. In yet another example, approximately 2 to 9, approximately 3 to 8, approximately 4 to 7, or approximately 5 to 6 fusion proteins or compositions are administered to the target ear. In one example, a therapeutically effective amount of nucleic acid or expression vector is administered to the target ear. In one example, multiple nucleic acids or expression vectors are administered to the target ear. For example, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 nucleic acids or expression vectors can be administered to the target ear. In yet another example, approximately 1 to 10 nucleic acids or expression vectors are administered. In other cases, approximately 2-9, 3-8, 4-7, or 5-6 nucleic acids or expression vectors are administered to the target ear.

[0270] Those skilled in the art can determine, through routine experiments, the effective and non-toxic amount of a fusion protein, composition, nucleic acid, or expression vector for the purpose of treating hearing impairment. For example, the therapeutically active amount of a fusion protein, composition, nucleic acid, or expression vector may vary depending on factors such as the disease stage and body weight of the subject, as well as the ability of the fusion protein to induce the desired response in the subject. The drug regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, as indicated by the urgency of the treatment situation. However, generally, the effective dose is expected to be in the range of about 1–200 mg / kg body weight.

[0271] Kit and other compositions of the substance Another example of the present disclosure is a kit comprising a fusion protein, composition, nucleic acid, or expression vector of the present disclosure useful for doing so in the ear of an object where the promotion of spiral ganglion neuron survival is required. (i) at least one fusion protein, composition, nucleic acid, or expression vector of the Disclosure (ii) Instructions for using the kit to promote the survival of spiral ganglion neurons in the ear of the subject, and (iii) Provide a kit that optionally includes at least one additional therapy.

[0272] This disclosure also relates to a kit for treating hearing impairment in subjects requiring treatment for hearing impairment, (i) at least one fusion protein, composition, nucleic acid, or expression vector of the Disclosure (ii) Instructions for using the kit to promote the survival of spiral ganglion neurons in the ear of the subject, and (iii) Provide a kit that optionally includes at least one additional therapy.

[0273] In this example of the Disclosure, the instructions (or accompanying information) are on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. The label or accompanying information indicates that the fusion protein, composition, nucleic acid, or expression vector is to be used to treat a therapeutically eligible subject, e.g., a subject who has or is predisposed to developing one of the conditions described herein, and provides specific guidance regarding the dosage and interval of the fusion protein, composition, nucleic acid, or expression vector and any other medicinal products. The kit may further include additional containers containing pharmaceutically acceptable diluent buffers, e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0274] The kit optionally further comprises a container containing a second medicament, wherein the fusion protein, composition, nucleic acid, or expression vector is a first medicament, and the article further comprises instructions on a package insert for treating a subject with an effective amount of the second medicament. The second medicament may be the therapeutic protein set forth above.

[0275] The present disclosure includes the following non-limiting examples.

[0276] Example 1: Production and Purification of Neurotrophin 3 Fusion Protein in HEK293 Freestyle Cells Cell Culture and Transfection Codon-optimized synthetic DNA fragments were synthesized by GenScript and subcloned into a modified pCAGGS mammalian expression vector via SacI-XhoI restriction enzyme sites (Miyazaki et.al., Gene 79:269-277, 1989) (Figure 15). HEK293T cells were grown as adherent cultures in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 10% v / v fetal bovine serum (Serana) and 1×GlutaMAX™ supplement (Thermo Fisher Scientific) in a humidified incubator at 37°C and 5% CO2. For both transient transfections, 4×10 5 cells were seeded into 6-well tissue culture plates the day before transfection. Prior to transfection, the culture volume was reduced to 1 mL, and transfection was performed using 1 μg of plasmid DNA and 4 μL of FuGENE® Transfection Reagent (Promega) according to the recommended protocol. After 24 hours, the culture medium was replaced with 1 mL of serum-free Freestyle 293 Expression Medium (Thermo Fisher Scientific), harvested after an additional 48 hours of culture, clarified by brief centrifugation, and stored at -20°C.

[0277] Freestyle HEK293-F cells (Thermo Fisher Scientific) were grown as suspension cultures in Freestyle293 expression medium using a humidified shaking incubator (37°C, 5% CO2, 130 rpm). Cells were maintained and 2.1 × 10⁶ cells were grown to set up a transfection experiment using polyethyleneimine (PEI, Polysciences) at a ratio of 1:3 [DNA:PEI] with 1 μg of plasmid per 1 mL of culture. 6 The solution was scaled up to 1,800 mL in an Erlenmeyer shaker flask (Corning) with a viable cell density (ViCD) of 100 cells / mL. The transfection mix was prepared by adding the plasmid DNA solution to warm (37°C) PBS, then adding the PEI solution, followed by gentle mixing, and incubated at room temperature for 15 minutes.

[0278] Protein production was carried out according to a standard protocol developed by CSIRO. The cultures were maintained in a humidified incubator (37°C, 5% CO2, 130 rpm) for the duration of the production period (9 days). At the end of production, the cells were removed by centrifugation at 4°C for 20 minutes at 4000 rpm, filtered through a 0.2 μm filter (Nalgene Rapid Flow Filter), and the protein supernatant was collected before freezing at -80°C.

[0279] Affinity chromatography Prior to affinity chromatography, the system was cleaned / disinfected with 1M NaOH + 2M NaCl, including a 30-minute incubation. The alkali-resistant column was also treated with 1M NaOH and disinfected for a period of 30 minutes. A chromatogram showing the elution profile of NT3-Fc is shown in Figure 1. Elution occurred as a single elution peak with a small shoulder. The UV detector was saturated. The shaded area was pooled and prepared for loading onto a Superdex 200 pg 26 / 600 column. [Table 2] [Table 3]

[0280] Preparative size exclusion chromatography (SEC) Column calibration was performed to meet the expected specifications. The total protein volume of 32 mL was divided into three parts, with each part being approximately 10 mL for loading onto a Superdex 200 pg 26 / 600 column. A total of three SEC runs were performed. The elution profiles for each SEC run were identical in 1×PBS. For NT3-Fc, there was a major peak eluting at the expected volume, with a small aggregate peak eluting at the void volume and as a shoulder before the major peak (Figure 2). [Table 4]

[0281] Fractions from the main peak were pooled and combined over three SEC runs. The concentration of the pooled fraction (approximately 80 mL) was measured to approximately 0.8 mg / mL. The sample was concentrated to a volume of approximately 25 mL on a 10 kDa MWCO spin filter and then aseptically filtered through a 0.2 μm filter. The final concentrated sample concentration was 2.19 mg / mL. An estimated loss of approximately 10 mg occurred during concentration, which is expected in this step.

[0282] 1 / 4 desalting exchange with PBS The purpose of the final desalting column was buffer exchange from 1×PBS to 1 / 4×PBS (approximately 2.5 mM phosphate and 35 mM NaCl). Broad elution and indistinct peak shapes were due to protein overload (Figure 3). The protein was favorably buffer-exchanged (smaller peak = conductivity trace), as indicated by the lower conductivity trace of the NT3-Fc elution peak in 1 / 4×PBS compared to 1×PBS which eluted at 80–130 mL. [Table 5]

[0283] SDS-PAGE analysis All samples were incubated at 95°C for 5 minutes before loading onto the gel. The gel was prepared in NuPAGE BIS-TRIS 4-12% and MES-SDS buffer and run at 200V for 40 minutes. Six samples equivalent to 5 μg each (affinity loaded, flow-through, and eluate in both 1×PBS and 1 / 4×PBS) were collected and treated either with a reducing agent (DTT) (R) or without a reducing agent (DTT) (NR).

[0284] A faint band corresponding to approximately 40 kDa may indicate the presence of NT3-Fc in the culture supernatant (affinity loading, Figure 4A). Comparing the affinity loading and affinity flow-through, there was a difference in the protein band intensity of the indicated NT3-Fc(NR) (approximately 40 kDa, see lanes 1 vs 2), indicating complete affinity capture. The elution profile was as expected. The affinity eluate in the non-reduced form showed slight band laddering at higher molecular weights (lane 4), which was not visible in the reduced form (lanes 5 and 6). A faint band corresponding to approximately 75 kDa in the affinity eluate likely indicated the dimerized form of NT3-Fc. Despite the band laddering, the NT3-Fc sample was pure, without other contaminating proteins.

[0285] mass spectrometry MS analysis of the untreated sample yielded multiple masses of approximately 42289 Da, separated by hexose and N-acetylhexose masses (Figure 5). The sample was then deglycosylated with PNGaseF and analyzed by rpHPLC-MS (reverse-phase liquid chromatography coupled to electrospray mass spectrometry). Both reduced and unreduced samples were analyzed.

[0286] The deglycosylated sample yielded a mass of 37604 Da (Figure 6), approximately 128 Da less than the expected mass of 37731.55 Da. This apparent difference was explained by the loss of C-terminal lysine, a common modification that occurs in antibodies or Fc fusions.

[0287] Endotoxin test Pooled and filtered protein samples were pre-tested for endotoxin levels and diluted 1:10 with fresh, sterile MilliQ (MQ) water that passed the test. Endotoxin levels were measured using the Endosafe® PTS assay. Cartridge sensitivity: 5–0.05 EU / mL [Table 6]

[0288] Details and dispensing of the final product Approximately 54 mg of NT3-Fc protein was captured by a three-step purification regime: protein A (PrismA), followed by preparative SEC (Superdex 200 pg 26 / 600), and final desalting in 1 / 4×PBS buffer. Due to the high concentration of the sample, multiple preparative SEC steps were performed. All preparative SEC runs were pooled. The pooled sample showed a uniform profile when analyzed by analytical SEC. The final PBS buffer was diluted to 1 / 4×PBS (2.5 mM phosphate, 35 mM NaCl). Intact MS showed that the NT3-Fc was of the expected size, considering terminal lysine loss. The final sample was filtered through a 0.2 μm filter into 50, 100, and 500 μL aliquots and stored at 4°C. The final sample passed endotoxin testing.

[0289] Example 2: Production and purification of neurotrophin 3 fusion protein in ExpiCHO cells Cell culture and transfection Codon-optimized synthetic DNA fragments were synthesized using GenScript and subcloned into a modified pCAGGS mammalian expression vector via a SacI-XhoI restriction enzyme site (Miyazaki et.al., Gene 79:269-277, 1989) (Figure 15).

[0290] ExpiCHO cells Transient transfection of suspension-adapted cultures of ExpiCHO cells (ThermoFisher Scientific) was performed using the Thermo Fisher Scientific ExpiCHO-S® expression system (catalog number A29129). Cell culture, growth parameters, transient expression, and nutritional supplementation followed the manufacturer's recommendations. In short, vials of ExpiCHO-S® cells were thawed in ExpiCHO expression medium (catalog number A2910002) in a 125 mL shaking flask and cultured in a humidified and shaking CO2 incubator (37°C, 5% CO2, 130 rpm). The cultures were periodically subcultured and expanded every 3-4 days to reach the required transfection volume. On the day of transfection, the cell density was 6.0 × 10⁶. 6 The cells were adjusted to 100 mL total cell volume and transferred to a 500 mL shaking flask to prepare for transfection. Transfection was performed using plasmid DNA (final concentration of 1 mg of DNA per liter of culture) according to the manufacturer's recommendations. Protein production was carried out using a standard nutrition regime according to the manufacturer's protocol. The culture supernatant was collected 9 days after transfection by centrifugation at 4000 rpm for 30 minutes at 4°C under low endotoxin conditions, followed by sterile filtration through 0.2 μm and stored at 4°C.

[0291] Affinity chromatography NT3-Fc purification from ExpiCHO_S culture supernatant was performed using the same methodology as described in Example 1.

[0292] Protein elution from PrismA occurred as a single elution peak, which appeared jagged due to UV detector saturation (Figure 7). [Table 7] [Table 8]

[0293] Preparative size exclusion chromatography (SEC) The pooled eluate from affinity chromatography escaped with the expected molecular weight in preparative SEC (Figure 8). [Table 9] [Table 10]

[0294] Protein concentration The concentration of the pooled fraction (approximately 32 mL) was measured at 0.55 mg / mL. The sample was concentrated to a volume of approximately 8.3 mL on a 10 kDa MWCO spin filter and then filtered through a 0.2 μm filter. The final concentrated sample had a concentration of 1.90 mg / mL. An estimated loss of approximately 2 mg occurred during the concentration process.

[0295] SDS-PAGE analysis All samples were treated with iodoacetamide and then incubated in sample loading buffer at 95°C for 5 minutes before loading onto the gel. The gel was prepared using NuPAGE BIS-TRIS 4-12% and MES-SDS buffer and run at 180V for 45 minutes. At approximately 40 kDa, there was a major band corresponding to NT3-Fc (lanes 3 and 4, Figure 4B).

[0296] Endotoxin test Pooled and filtered protein samples were pre-tested for endotoxin levels and diluted 1:10 with fresh, sterile MilliQ (MQ) water that passed the test. Endotoxin levels were measured using the Endosafe® PTS assay. Cartridge sensitivity: 5–0.05 EU / mL [Table 11]

[0297] Product details and dispensing Approximately 15.77 mg of NT3-Fc protein was captured by a two-step purification regime: affinity HiTrap MabSelect PrismA column followed by preparative SEC (S200pg 26 / 60). The final sample was filtered through a 0.2 μm filter into 5 × 1 mL, 5 × 0.5 mL, and 8 × 0.1 mL aliquots and stored at 4°C.

[0298] Example 3: Characterization of Neurotrophin 3 fusion protein Surface plasmon resonance (SPR) for NT3-Fc binding to immobilized Trk receptors SPR measurements were performed using a Biacore T200 instrument (Cytiva). First, a multi-cycle dynamics approach was used to demonstrate the binding interaction between the injected NT3-Fc protein and the immobilized Trk receptor on the chip surface. Pilot capture experiments were performed using Trk proteins at different concentrations to ensure that approximately equal amounts of each Trk could be captured during subsequent binding experiments. All SPR binding experiments were performed at 25°C using 1×HBS-EP+ as the instrument's running buffer. Trk receptors were diluted to 1 μg / mL (TrkC, TrkB) and 5 μg / mL (TrkA) in SPR binding buffer and captured on the antihistidine mAb chip surface at the start of each binding cycle. Following capture, NT3-Fc was injected onto the Trk receptor surface at 30 μL / min for 90 seconds, and then dissociated in the running buffer for 300 seconds. The antihistidine mAb surface was regenerated by a single 60-second injection of 10 mM glycine at pH 1.5. This binding cycle (1. capture, 2. analyte injection, and 3. regeneration) was repeated several times with different analyte (NT3-Fc) concentrations (3-fold dilution) injected. The experimental design for this assay is outlined in Table 11. [Table 12]

[0299] SPR binding assay for TrkC binding to immobilized NT3-Fc SPR experiments were performed at 25°C using 1×HBS-EP+ / N+ (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% (v / v) Tween 20) as the running buffer for the instrument (Biacore T200). A multi-cycle dynamics approach was used to determine the binding affinity to recombinant human TrkC interacting with the immobilized NT3-Fc sample.

[0300] In short, purified protein samples were diluted in the instrument's running buffer and captured onto the SPR chip surface containing amine-coupling protein A (Cytiva, catalog number: 29127556) at the start of each binding cycle. Typically, these captures were performed for 60 seconds at a constant flow rate of 10 μL / min. Pilot experiments were conducted to determine the appropriate dilution of NT3-Fc to achieve a consistent capture level of 300 RU (1 RU = 1 pg protein / mm2). Following capture, TrkC protein (Sino Biological, catalog number 10048-H08H) was injected at 30 μL / min for 90 seconds, and then dissociated in the running buffer for 180 seconds. The Protein A chip surface was regenerated between each binding by injecting 10 mM glycine at 30 μL / min for 30 seconds.

[0301] The final SPR binding data and a summary of binding interactions derived from these experiments are shown in Figure 10, Figure 25, and Table 22. Estimated affinity (K) for TrkC binding to immobilized NT3-Fc samples. D The value was estimated to be approximately 50-150 nM.

[0302] Processing and analysis of SPR data SPR experimental data were processed and analyzed using Scrubber software (www.biologic.com.au). Flow cell 1 and "zero-buffer-blank" injection were used to reference the binding signal. Affinity parameter (K D This was derived by fitting each set of experimental data in equilibrium to a Langmuir 1:1 coupled model.

[0303] Affinity determination Figure 9 illustrates binding data showing the interaction between immobilized Trk receptors and injected NT3-Fc produced from HEK cells. The assay configuration for these initial SPR experiments involved injecting dimerized NT3-Fc in solution onto immobilized Trk receptors. Affinity parameters were extracted using the Langmuir 1:1 binding model. [Table 13]

[0304] In SPR experiments, the polymeric state of the injected reagent is important. If the injected species (in solution) is a monomer (TrkC), the estimated binding parameter represents the true affinity. On the other hand, if the injected species is a dimer, avidity (total affinity) is measured. See Table 12, K. D Because the model used to extract the values ​​was a 1:1 model, these estimates represent only apparent affinity. Nevertheless, this assay approach is useful because it allows for a direct comparison of NT3-Fc interacting with different Trk receptors in the same assay. Furthermore, it is also relevant in the biological context where NT3 homodimers interact with immobilized Trk receptors.

[0305] Figure 10 shows SPR binding data and a summary of binding interactions for monomeric TrkC interaction-immobilized NT3-Fc. In this case, true affinity estimates are generated. Importantly, these results demonstrate that purified NT3-Fc produced in a CHO cell host (ExpiCHO) following transient transfection has the same TrkC receptor binding kinetics as purified NT3-Fc following transfection of HEK293Freestyle cells.

[0306] In vitro bioactivity CHO K1 cell lines containing the CellSensor construct (TrkC-NFAT-bla) were cultured in growth medium (DMEM containing GlutaMAX) supplemented with 10% dialysis FBS, 1×MEM NEAA (Minimum Essential Medium Non-Essential Amino Acids) solution, 25 mM Hepes, blastosidine (5 μg / mL), and zeosin (200 μg / mL). Cells were then cultured in a medium without blastosidine and zeosin, at a rate of 1.0 × 10⁶ per well. 4 Cells were seeded overnight in 96-well plates. The following day, the cells were treated with the fusion protein for 5 hours. Subsequently, the wells were loaded with the β-lactamase LiveBLAzer-FRET B / G substrate for 2 hours. Fluorescence at 450 and 510 nm was recorded using a plate reader. Figure 11 illustrates the biological activity of NT3 and NT3-Fc.

[0307] Example 4: Characterization of neurotrophin 3 fusion protein loaded onto ultraparticles Load Each ultraparticle (SP) was loaded in a solution containing 5.5 μg of fusion protein per particle and loaded at room temperature for 3 days. First, the SPs were sterilized with 100 μL of ethanol (80% v / v) at room temperature (approximately 22°C) for 4 hours. Next, the SPs were washed 6 times with sterile Milli-Q water. Then, a phosphate buffer solution containing the fusion protein was added to the SPs at a ratio of 5.5 μg of fusion protein per particle. After 3 days, the supernatant was collected for analysis. The concentration of fusion protein in the supernatant was measured using Nanodrop and MicroBCA assays. The amount of fusion protein loaded into the SPs was calculated by subtracting the amount of protein in the supernatant from the amount of protein in the original stock solution (i.e., solution before loading minus solution after loading). Figure 12A illustrates that the loading volume of NT3-Fc remained consistent over 84 days.

[0308] Elution The loaded particles were individually separated and transferred to 100 μL of phosphate-buffered solution (PBS) containing 0.2% BSA and 0.05% sodium azide. The particle elution profiles were analyzed for 63 days. At specific time points (days 3, 7, 14, 21, 28, 35, 42, 49, 56, and 63), the PBS solution was collected as an elution sample. The protein concentration in each elution sample was determined by ELISA. Fresh PBS was replenished after each collection. Figure 12B illustrates the elution profile of NT3-Fc from the loaded ultraparticles.

[0309] Bioactivity of the eluted fusion protein CHO K1 cell lines containing the CellSensor construct (TrkC-NFAT-bla) were cultured in growth medium (DMEM containing GlutaMAX) supplemented with 10% dialysis FBS, 1×MEM NEAA (Minimum Essential Medium Non-Essential Amino Acids) solution, 25 mM Hepes, blastosidine (5 μg / mL), and zeosin (200 μg / mL). Cells were then cultured in a medium without blastosidine and zeosin, at a rate of 1.0 × 10⁶ per well. 4 Cells were seeded overnight in a 96-well plate. The following day, the cells were treated with eluted fusion protein at a concentration of 50 pM for 5 hours. Subsequently, the wells were loaded with β-lactamase LiveBLAzer-FRET B / G substrate for 2 hours. Fluorescence at 450 and 510 nm was recorded using a plate reader.

[0310] In vivo pharmacokinetic studies NT3-Fc-loaded superparticles (SPs) were implanted bilaterally in cats with normal hearing via a circular window approach. Aseptic surgical techniques were used to expose the circular window membrane. The SPs (n=30) were positioned on the circular window membrane using a 21-gauge polyurethane catheter, a fibrin sealant was placed on top of the particles, and then a Kwik seal was applied over the cured fibrin sealant to maintain particle placement. The surrounding muscle layer was sutured, and the skin incision was closed with staples. The treatment period lasted 48 hours.

[0311] At the completion of the treatment period, the animals were stopped and intracardiac perfused with 0.9% NaCl (37°C), followed by 10% neutral buffered formalin (10% NBF; 4°C). The tympanic bulla was removed from the temporal bone and the cochlea was dissected. The cochlea was then post-fixed in 10% NBF and then transferred to 10% ethylenediaminetetraacetic acid (EDTA) in PBS at room temperature for decalcification. The cochlea was further sectioned using a cryostat. The sectioned cochlear tissue was blocked for endogenous peroxidase with EPB solution (3% H2O2) for 20 minutes, followed by treatment in 0.1% Tween-20 PBS for 20 minutes. The tissue was then treated with goat anti-human IgG (Fc-specific) antibody (1:500 in 0.1% Tween-20 PBS) for 4 hours, washed with PBS, and then treated with DAB solution for 1 minute. The tissue was rinsed with water, dehydrated with ethanol, and then imaged.

[0312] Cats with normal hearing and no implants were used as negative controls, and cats with normal hearing and NT3-Fc-loaded superparticles implanted via an intracochlear approach were used as positive controls. Figure 13 shows cat cochlear sections stained with anti-human IgG-HRP and DAB after 48 hours of treatment with NT3-Fc-loaded SPs on a round window membrane (RWM).

[0313] In vivo efficacy study This study investigated the efficacy of NT3-Fc SP delivered to the circular window membrane in noise-exposed cats. Cats (n=8) were bilaterally deafened three days prior to implantation surgery (124 dB 16 kHz pure tone for 100 minutes under anesthesia). NT3-SP (n=25) was unilaterally implanted on the circular window membrane using the same surgical approach as above, achieving a total NT3 dosage of approximately 105 ug per ear over a two-month treatment period. No implantation was performed on the contralateral ear. The cochlea was prepared for surface preparation, and cochlear hair cells and synapses were quantified in the NT3-SP treated ear and the unimplanted control ear. Analysis of cochlear synapses at the highest measured frequency (32 kHz cochlear region - closest to the circular window membrane) showed a significant effect of NT3-SP treatment on cochlear synapse repair (ANCOVA p=0.002). Figure 14 illustrates the significantly higher synaptic density in the 32 kHz cochlear region in NT3-Fc-treated cochleas compared to controls, and in NT3-Fc SPs implanted on the RWM of cats (n=8 cats).

[0314] Example 5: Production and purification of BDNF fusion protein in ExpiCHO cells Cell culture and transfection A gene construct encoding the human BDNF gene, fused to a non-dimerized version of the human antibody Fc gene fragment, was synthesized using GenScript and subcloned to modified pCAGGS mammalian expression via a SacI-XhoI restriction site (Figure 15).

[0315] ExpiCHO cells Transient transfection of suspension-adapted cultures of ExpiCHO cells (ThermoFisher Scientific) was performed using the Thermo Fisher Scientific ExpiCHO-S® expression system (catalog number A29129). Cell culture, growth parameters, transient expression, and nutritional supplementation followed the manufacturer's recommendations. In short, vials of ExpiCHO-S® cells were thawed in ExpiCHO expression medium (catalog number A2910002) in a 125 mL shaking flask and cultured in a humidified and shaking CO2 incubator (37°C, 5% CO2, 130 rpm). The cultures were periodically subcultured and expanded every 3-4 days to reach the required transfection volume. On the day of transfection, the cell density was 6.0 × 10⁶. 6 The cells were adjusted to 100 mL / mL and transferred to a 500 mL shaking flask with a total cell volume of 100 mL or 150 mL, and prepared for transfection. Transfection was performed using plasmid DNA (final concentration of 1 μg of DNA per liter of culture) according to the manufacturer's recommendations.

[0316] Protein yield monitoring BDNF-Fc production was induced using a standard nutrition regime according to the manufacturer's protocol. Cultures were collected 7 days post-transfection (PF23-11-12, viability: 94.9%, ViCD: 8.52 × 10⁶ cells / mL) by centrifugation at 4000 rpm / 30 min / 4°C under low endotoxin conditioning, followed by 0.2 μm sterile filtration. Cell counts were measured using trypan blue staining.

[0317] Production yield was monitored using SPR. The Fc portion allows the fusion protein to bind to the Protein A SPR chip docked inside the SPR instrument (Biacore 8K, 8-channel instrument, Cytiva). Specifically, the amount of secreted BDNF-Fc protein was monitored using a calibration-dependent concentration assay (CDCA). This approach establishes a standard curve (SPR binding response vs. injected concentration) using a reference sample. The results of these SPR experiments are shown in Table 13. [Table 14]

[0318] Summary of the purification process: BDNF-Fc was purified using a two-step chromatography method: 1) PrismA affinity chromatography column and 2) size exclusion chromatography column. The final protein sample was concentrated to 0.8 mg / mL and analyzed by SDS-PAGE, SEC for analysis, SPR, and mass spectrometry.

[0319] Affinity chromatography on a PrismA column Elution occurred as a single elution peak (Figure 16). [Table 15] [Table 16]

[0320] Preparative SEC The column calibration was in accordance with the expected specifications. The main protein peak eluted at molecular weight is expected to be the BDNF-Fc dimer (approximately 85 kDa). [Table 17]

[0321] Fractions collected from preparative SECs (Figure 17) spanning the main 85 kDa peak were combined and buffer-exchanged by two rounds of concentration and dilution in 1× PBS using an Amicon® Ultra centrifuge filter (10 kDa MWCO; Merck). A summary of this process is outlined in Table 17. [Table 18]

[0322] SDS-PAGE All three samples were analyzed by SDS-PAGE under reducing and non-reducing conditions (Table 18). The proteins moved at approximately 45 kDa in SDS-PAGE (Figure 18). No significant differences in migration were observed between reducing and non-reducing conditions for these proteins. This observation is consistent with BDNF-Fc, a properly processed mature non-covalent dimer that dissociates. [Table 19]

[0323] Size exclusion chromatography Analytical size exclusion chromatography was first performed in a high-salt buffer (1 × PBS + 350 mM NaCl). The protein sample eluted at a predicted molecular weight of approximately 85 kDa, consistent with the molecular weight of the BDNF-Fc dimer (Figure 19). [Table 20]

[0324] Final processing The BDNF-Fc samples were filtered through a 0.2 μm Nanosep filter (Cytiva) and stored at 4°C.

[0325] Example 6: Characterization of BDNF fusion protein SPR binding assay All SPR experiments were performed at 25°C using 1×HBS-EP+ / N+ (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% (v / v) Tween 20) as the running buffer for the instrument (Biacore 8K). A multi-cycle dynamics approach was used to determine the binding affinity to recombinant human TrkB interacting with the immobilized BDNF-Fc sample.

[0326] In short, purified protein samples were diluted in the instrument's running buffer and captured onto the SPR chip surface containing amine-coupling protein A (Cytiva, catalog number: 29127556) at the start of each binding cycle. Typically, these captures were performed for 60 seconds at a constant flow rate of 10 μL / min. Pilot experiments were conducted to determine the appropriate dilution of BDNF-Fc to achieve a consistent capture level of 300 RU (1 RU = 1 pg protein / mm2). Following capture, TrkB protein (Sino Biological, catalog number 10047-H08H) was injected at 30 μL / min for 90 seconds, and then dissociated in the running buffer for 180 seconds. The Protein A chip surface was regenerated between each binding by injecting 10 mM glycine at 30 μL / min for 30 seconds.

[0327] The final SPR binding data and a summary of binding interactions derived from these experiments are shown in Figure 20 and Table 20. Estimated affinity (K) for TrkB binding to immobilized BDNF-Fc samples. D The value was estimated to be 32 nM. [Table 21]

[0328] Endotoxin test Endotoxin levels were measured using the Endosafe® PTS assay (cartridge sensitivity 5-0.05 EU / mL), with BDNF-Fc endotoxin levels <0.500 EU / mL.

[0329] In vitro bioactivity of BDNF-Fc CHO K1 cell lines containing the CellSensor construct (TrkB-NFAT-bla) were cultured in growth medium (DMEM containing GlutaMAX) supplemented with 10% dialysis FBS, 0.1 mM NEAA (non-essential amino acid) solution, 25 mM HEPES, penicillin (100 U / mL) / streptomycin (100 μg / mL), blastosidine (5 μg / mL), and zeosin (200 μg / mL). Cells were then cultured in medium without blastosidine and zeosin at a rate of 1.0 × 10⁶ cells per well. 4 Cells were seeded overnight in a 96-well plate. The following day, the cells were treated with BDNF and BDNF-Fc for 5 hours. Subsequently, the wells were loaded with LiveBLAzer-FRET B / G substrate for 2 hours. Fluorescence at 450 and 510 nm was recorded using a plate reader. Figure 21 illustrates the biological activity of BDNF and BDNF-Fc.

[0330] Example 7: Purification and analysis of dimeric neurotrophic fusion protein To demonstrate the importance of fusion proteins containing monomeric Ig Fc domains in improving expression levels (e.g., high yield) and / or improving biological activity and / or increasing receptor binding, human NT3 was fused to a dimerized human Fc protein fragment (hereinafter, "NT3-diFc") in transient ExpiCHO cell culture.

[0331] Compared to the original NT3-Fc, the production of active NT3-diFc is significantly lower, which is likely due to the complexity of forming a properly assembled molecule that dimerizes through both the Fc and NT3 moieties.

[0332] The NT3-diFc material was purified by affinity HiTrap MabSelect PrismA chromatography (Figure 22).

[0333] Size exclusion chromatography NT3-diFc and NT3-Fc samples were analyzed using analytical SEC. NT3-diFc (PrismA pH 4.0) samples were eluted from a Superdex 200 10 / 300 SEC column at three main peaks with estimated molecular weights of >670 kDa (9.5 mL), approximately 80 kDa (14.9 mL), and approximately 50 kDa (15.7 mL). NT3-diFc (PrismA pH 3.5 sample) was eluted primarily as high molecular weight aggregates (>670 kDa). In comparison, NT3-Fc was eluted on the same column under identical conditions at 14.0 mL (Figure 23).

[0334] SDS-PAGE NT3-diFc migrated as the dominant band in SDS-PAGE (indicated by arrows in Figure 24 and Table 21) at approximately 40 kDa under reduction and 80 kDa under non-reducing conditions, which is consistent with properly processed mature NT3-diFc (expected Mw of approximately 39 kDa for reduced monomers and approximately 78 kDa for non-reducing dimers). Non-reducing samples contained many protein bands migrating at higher molecular weights, indicating a high tendency for aggregation for this construct. [Table 22]

[0335] SPR assay SPR binding experiments using recombinant TrkC (receptor) confirmed that NT3-diFc was active because it bound to TrkC with an affinity equivalent to that of NT3-Fc (Figure 25 and Table 22). The SPR binding activity of NT3-diFc was localized to a peak at 14.9 mL (80 kDa). [Table 23]

[0336] Example 8: Purification and analysis of alternative neurotrophic fusion proteins The NT3-Fc constructs in Table 23 were designed to further investigate the production and purification of NT3-Fc. Figure 26 shows the sequence alignment of these constructs. Table 24 provides a summary of the expression and purification results. [Table 24] [Table 25]

[0337] Example 9: Mouse cochlear explant synaptic degeneration assay material and method Mouse offspring were selected at 3–5 days postnatology, and cochleas were isolated from the temporal bones. Explants were cultured overnight (37°C, 5% CO2) in culture medium (Dulbecc's modified Eagle medium supplemented with 1% FBS, 1% N-2, ampicillin, and amphotericin B). Cochlear synaptic injury (synaptic degeneration) was induced using kainic acid (KA) (0.5 mM), and the explants were then cultured for 3 days in NT3-Fc diluted in culture medium. After the 3-day culture period, the explants were fixed in PFA and stored in PBS at 4°C.

[0338] To detect and image cochlear hair cells and their synapses (presynaptic and postsynaptic dots), explants were incubated overnight at 4°C in the following primary antibodies: PSD-95 (postsynaptic dots), CtBP2 (presynaptic dots), and Myo7A (inner hair cells, IHC). Secondary antibodies that fluoresce under a confocal microscope at a given wavelength include 405 anti-rabbit IgG (blue), 488 anti-mouse IgG2a (green), and 568 anti-mouse IgG1 (red). Slides containing the explants were covered with coverslips before imaging.

[0339] The explants were first imaged at 10x magnification using a fluorescence microscope. The length of the cochlea was measured using ImageJ, and the central cochlear region (50% along the length of the cochlea) was identified. This region was then imaged at 63x magnification using a Stellaris 5 Leica confocal microscope. The same region of the cochlea was analyzed across all samples.

[0340] High-magnification images were analyzed using specialized software (Imaris). IHCs and synapse / IHC counts were performed. Cochlear synapses were defined as the count of presynaptic and postsynaptic points within 1 micron of each other, and quantified as the number of synapses per inner hair cell.

[0341] result Treatment of explants with NT3-Fc (at a concentration of 1 nM) after KA injury resulted in synaptic restoration, with more synapses observed in cochlear hair cells (Figure 27).

Claims

1. (i) Neurotrophins, and (ii) A fusion protein comprising a monomer immunoglobulin (Ig) crystallizable fragment (Fc) domain or fragment thereof.

2. The fusion protein according to claim 1, wherein the neurotrophin forms a homodimer.

3. The fusion protein according to claim 1 or 2, wherein the fusion protein has improved expression levels in mammalian cells compared to wild-type or unmodified neurotrophins.

4. The fusion protein according to any one of claims 1 to 3, characterized in that the fusion protein exhibits increased activation of the tyrosine receptor kinase (Trk) receptor compared to wild-type neurotrophin.

5. The fusion protein according to claim 4, wherein the Trk receptor is a TrkA receptor, a TrkB receptor, a TrkC receptor, or any combination thereof.

6. The aforementioned fusion protein has an affinity constant (K) of 400-800 nM. Ｄ The fusion protein according to claim 5, wherein it binds to the TrkA receptor at a neutral pH.

7. The aforementioned fusion protein has an affinity constant (K) of 20-60 nM. Ｄ The fusion protein according to claim 5 or 6, wherein it binds to the TrkB receptor at a neutral pH.

8. The aforementioned fusion protein has an affinity constant (K) of 0 to 5 nM. Ｄ A fusion protein according to any one of claims 5 to 7, wherein the protein binds to the TrkC receptor at a neutral pH.

9. The fusion protein according to any one of claims 1 to 8, wherein the monomeric IgFc domain or fragment thereof comprises one or more amino acid substitutions and / or at least one N-glycosylation site that reduce and / or inhibit dimerization with another monomeric IgFc domain or fragment thereof.

10. The monomer IgFc domain or a fragment thereof (i) Leucine substituted with alanine at the position corresponding to amino acid 234 of Sequence ID No. 10 according to the EU numbering system, (ii) Leucine substituted with alanine at the position corresponding to amino acid 235 of Sequence ID No. 10 according to the EU numbering system, (iii) Glycine substituted with alanine at the position corresponding to amino acid 237 of Sequence ID No. 10 according to the EU numbering system, and / or (iv) A fusion protein according to any one of claims 1 to 9, comprising one or more amino acid substitutions selected from the group consisting of proline substituted with glycine at the position corresponding to amino acid 329 of SEQ ID NO: 10 according to the EU numbering system.

11. The monomer IgFc domain or a fragment thereof (i) Leucine substituted with alanine at the position corresponding to amino acid 234 of Sequence ID No. 10 according to the EU numbering system, (ii) Leucine substituted with alanine at the position corresponding to amino acid 235 of Sequence ID No. 10 according to the EU numbering system, (iii) Glycine substituted with alanine at the position corresponding to amino acid 237 of Sequence ID No. 10 according to the EU numbering system, and (iv) The fusion protein according to any one of claims 1 to 9, comprising a proline substituted with glycine at the position corresponding to amino acid 329 of SEQ ID NO: 10 according to the EU numbering system.

12. The fusion protein according to any one of claims 1 to 11, wherein the monomer Ig Fc domain or a fragment thereof comprises at least one N-glycosylation site within the CH3 domain.

13. The monomer IgFc domain or a fragment thereof (i) at the position corresponding to amino acid 364 of sequence number 10 in the EU numbering system, or (ii) The fusion protein according to any one of claims 1 to 12, comprising at least one N-glycosylation site at the position corresponding to amino acid 407 of Sequence ID No. 10 according to the EU numbering system.

14. The monomer IgFc domain or a fragment thereof (i) at the position corresponding to amino acid 364 of sequence number 10 in the EU numbering system, and (ii) The fusion protein according to any one of claims 1 to 13, comprising at least two N-glycosylation sites at the position corresponding to amino acid 407 of Sequence ID No. 10 according to the EU numbering system.

15. The fusion protein according to any one of claims 1 to 14, wherein the N-terminus of the monomer IgFc domain or a fragment thereof is linked to the C-terminus of the neurotrophin by a linker.

16. The fusion protein according to any one of claims 1 to 14, wherein the C-terminus of the monomer IgFc domain or a fragment thereof is linked to the N-terminus of the neurotrophin by a linker.

17. The fusion protein according to claim 15 or 16, wherein the linker contains serine.

18. The fusion protein according to any one of claims 1 to 17, wherein the neurotrophin is selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4).

19. The fusion protein according to claim 18, wherein the neurotrophin is NT-3.

20. The fusion protein according to claim 19, wherein the NT-3 is human NT-3.

21. The fusion protein according to claim 20, wherein the human NT-3 is shown in Sequence ID No.

1.

22. The fusion protein according to claim 18, wherein the neurotrophin is BDNF.

23. The fusion protein according to claim 22, wherein the BDNF is human BDNF.

24. The fusion protein according to claim 23, wherein the human BDNF is shown in Sequence ID No.

6.

25. The fusion protein according to any one of claims 1 to 24, wherein the monomer Ig Fc domain or a fragment thereof is shown in Sequence ID No.

2.

26. A fusion protein according to any one of claims 1 to 14, comprising the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO:

2.

27. A fusion protein according to any one of claims 1 to 14, comprising the sequence shown in SEQ ID NO: 6 and the sequence shown in SEQ ID NO:

2.

28. A fusion protein according to any one of claims 1 to 14, comprising the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO:

2.

29. A nucleic acid that encodes or expresses the fusion protein according to any one of claims 1 to 28.

30. Nucleic acids encoding or expressing a proneurotrophin and a monomeric immunoglobulin (Ig) crystallizable fragment (Fc) domain or fragment thereof.

31. The nucleic acid according to claim 30, wherein the nucleic acid sequence is a DNA sequence containing the sequence shown in Sequence ID No.

15.

32. The nucleic acid according to claim 30, wherein the nucleic acid sequence is a DNA sequence containing the sequence shown in Sequence ID No.

17.

33. An expression construct comprising the nucleic acid described in any one of claims 29 to 32.

34. The expression construct according to claim 33, comprising the sequence shown in sequence number 18.

35. The expression construct according to claim 33, comprising the sequence shown in Sequence ID No.

20.

36. The expression construct according to claim 33, comprising the sequence shown in sequence number 12.

37. The expression construct according to claim 33, comprising the sequence shown in sequence number 14.

38. A host cell that expresses a fusion protein according to any one of claims 1 to 28, or a nucleic acid according to any one of claims 29 to 32, or an expression construct according to any one of claims 33 to 37.

39. The host cell according to claim 38, wherein the host cell is a mammalian cell.

40. The host cell according to claim 39, wherein the mammalian cell is selected from the group consisting of HEK cells, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC 5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH 3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, 211A cells, and derivatives thereof.

41. The host cell according to claim 39 or 40, wherein the mammalian cell is a HEK cell, preferably a HEK293 cell.

42. The host cell according to claim 39 or 40, wherein the mammalian cell is a CHO cell, preferably a CHOK1 cell.

43. A method for producing a fusion protein according to any one of claims 1 to 28, (a) A step of culturing a cell line containing nucleic acid encoding the fusion protein, (b) A method comprising the step of isolating the fusion protein from the cell line.

44. A composition comprising ultraparticles, wherein the ultraparticles comprise a fusion protein according to any one of claims 1 to 28.

Images