Long-acting nerve growth factor polypeptide and its uses

Abstract

The present invention relates to a sustained-release nerve growth factor (NGF) polypeptide comprising an NGF portion and an Fc portion from the N-terminus to the C-terminus, as well as a method for producing the same and uses thereof.

Description

cross reference

[0001] This application claims priority to International Patent Application No. PCT / CN2020 / 129925, filed November 19, 2020, the entire contents of which are incorporated herein by reference. (Submit sequence listing as an ASCII TEXT file)

[0002] The contents of the ASCII TEXT text file submitted below (File name: 202009094982_SEQLIST.txt, Recorded date: 2020.09.09, Size: 169KB) are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Format (CRF). [Technical field]

[0003] The present invention relates to a sustained-release nerve growth factor (NGF) polypeptide comprising an NGF portion and an Fc portion from the N-terminus to the C-terminus, and a production method and use thereof. [Background technology]

[0004] Neurotrophins are a family of highly homologous growth factors that are important for the survival and maintenance of neurons during the development and maturation of the vertebrate nervous system. Limited production of neurotrophins can lead to the degeneration or death of peripheral nervous system (PNS) or central nervous system (CNS) neurons.

[0005] Nerve growth factor (NGF) is the first member of the neurotrophic factor family and was first discovered in mouse sarcoma cells in 1953 by Italian scientist Levi-Montlcini. NGF is a nerve growth regulator with two biological functions: providing nutrients to neurons and promoting neurite outgrowth, and plays an important regulatory role in the development, differentiation, growth, regeneration and functional expression of central and peripheral nerve neurons. NGF contains three subunits, α, β and γ. The β subunit is the active region consisting of two single chains non-covalently bound.

[0006] NGF has been researched for decades, but due to limitations and problems in practical application, there are very few NGF products on the market, most of which are mainly used to treat ophthalmic diseases such as corneal ulcers, optic nerve contusion and visual impairment.

[0007] Like other proteins, the biological activity of NGF depends on its secondary and tertiary structure, and therefore it is particularly important to maintain its biological activity during preparation, purification, storage and administration.

[0008] In addition, during treatment, NGF can cause intolerable pain in some patients, limiting its use to some extent. Pain is classified into two types according to neurophysiological mechanisms: sensory pain and neuropathic pain. The former is directly caused by noxious stimuli and is associated with tissue damage or inflammatory responses, also called inflammatory pain. The latter is chronic pain directly caused by somatosensory nervous system damage or disease. NGF causes pain by influencing the release of inflammatory mediators, opening of ion channels, and promoting the growth of nerve fibers, and is involved in the pathophysiological process of pain. It is involved in the generation of pain by regulating ion channels and molecular signaling. Some scholars speculate that NGF may cause pain by promoting the expression of pain-inducing substances, and may also change the sprouting and regeneration of neurons after injury in organisms. Studies have shown that the maximum dose of NGF that does not cause hyperalgesia in humans was 0.03 μg / kg (Petty et al., Ann Neurol. 1994, 36(2):244-246). However, such low doses limit the application of NGF and limit the expansion of indications, such as to the central nervous system.

[0009] As a protein drug, NGF's active part in promoting nerve growth is mainly β-NGF. β-NGF has a sedimentation coefficient of 2.5S, a molecular weight of 13.5KDa, and is easily filtered by glomeruli during metabolism, resulting in a short half-life in the body. Research has shown that when β-NGF drug is injected intramuscularly into mice with a frequency of once a day, T1 / 2(β)=2.2h, Tmax=0.5h. NGF shows adverse pain response at the injection site or the injected lower limb during the injection period, so it is desirable to reduce the total number of administrations and the frequency of administration.

[0010] All publications, patents, patent applications, and the disclosures of the disclosed patent applications mentioned herein are hereby incorporated by reference in their entirety. Summary of the Invention

[0011] One aspect of the present invention relates to a sustained-release nerve growth factor (NGF) polypeptide comprising, from the N-terminus to the C-terminus, an NGF portion and an Fc portion, wherein the NGF portion comprises an amino acid sequence shown in any one of SEQ ID NOs: 1 to 4 (e.g., an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3), and the Fc portion is derived from IgG1 Fc or IgG4 Fc.

[0012] In some embodiments, any of the above sustained-release NFG polypeptides has an NGF portion fused to an Fc portion via a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence shown in any one of SEQ ID NOs: 68 to 99, for example, an amino acid sequence shown in any one of SEQ ID NOs: 68 to 72, or SEQ ID NOs: 68 or 69. In some embodiments, the peptide linker comprises an amino acid sequence (GGGGS)n (SEQ ID NO: 70, where n is 1, 2, 3, 4, 5, or 6).

[0013] In some embodiments, any of the above long-acting NGF polypeptides are derived from IgG1 Fc, in which the Fc portion comprises the amino acid sequence of SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises a mutation at one or more positions selected from E233, L234, L235, G236, G237, N297, A327, A330, and P331 relative to SEQ ID NO: 8. In some embodiments, the Fc portion comprises one or more mutations selected from E233P, L234V, L234A, L235A, L235E, G236del, G237A, N297A, A327G, A330S, and P331S relative to SEQ ID NO: 8. In some embodiments, the Fc portion further deletes the first five amino acids of SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises L234A, L235A, and P331S mutations relative to SEQ ID NO: 8. In some embodiments, the Fc portion comprises the amino acid sequence of SEQ ID NO: 11 or 12. In some embodiments, the long-acting NGF polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 62-64. In some embodiments, the Fc portion comprises E233P, L234V, L235A, G236del, A327G, A330S and P331S mutations relative to SEQ ID NO: 8. In some embodiments, the Fc portion comprises the amino acid sequence of SEQ ID NO: 15 or 16. In some embodiments, the long-acting NGF polypeptide comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the Fc portion comprises L234A, L235E, G237A, A330S and P331S mutations relative to SEQ ID NO: 8. In some embodiments, the Fc portion comprises the amino acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the long-acting NGF polypeptide comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, the Fc portion comprises N297A mutation relative to SEQ ID NO: 8. In some embodiments, the Fc portion comprises the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the sustained release NGF polypeptide comprises the amino acid sequence of SEQ ID NO:61.

[0014] In some embodiments, any of the above long-acting NGF polypeptides are derived from IgG4 Fc, wherein the Fc portion comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the Fc portion comprises mutations at one or more positions selected from S228, F234, and L235 relative to SEQ ID NO: 17. In some embodiments, the Fc portion comprises one or more mutations selected from S228P, F234A, and L235A relative to SEQ ID NO: 17. In some embodiments, the Fc portion comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the long-acting NGF polypeptide comprises the amino acid sequence of SEQ ID NO: 67.

[0015] In some embodiments, any of the above long-acting NGF polypeptides have a half-life of at least 10 hours (e.g., a half-life of at least one of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, and 100 hours) when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection.

[0016] In some embodiments, any of the above sustained-release NGF polypeptides cause less pain (e.g., reduce pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) compared to an NGF polypeptide comprising an NGF portion of the amino acid sequence of SEQ ID NO:4.

[0017] Other aspects of the invention also relate to isolated nucleic acids encoding any of the long-acting NGF polypeptides described herein, vectors comprising said nucleic acids, host cells (e.g., CHO cells, HEK293 cells, Hela cells or COS cells) comprising said nucleic acids or vectors, compositions (e.g., pharmaceutical compositions) or kits, and articles of manufacture comprising any of the long-acting NGF polypeptides described herein. Another aspect of the invention relates to methods of using any of the long-acting NGF polypeptides described herein or pharmaceutical compositions comprising same to treat an NGF-related disorder (e.g., a neurological disorder or a non-neurological disorder) in an individual (e.g., a human). [Brief description of the drawings]

[0018] [Figure 1A] FIG. 1A shows a sequence alignment of human wild type IgG1 Fc IGHG1*05 and the IgG1 Fc natural variant IGHG1*03 (FIG. 1A). [Figure 1B] FIG. 1B shows the sequence alignment of modified IgG1 Fc M1-5 with IGHG1*03 (FIG. 1B). [Figure 1C] FIG. 1C shows the sequence alignment of modified IgG1 Fc M3-5 with IGHG1*03 (FIG. 1C). [Figure 1D] FIG. 1D shows a sequence alignment of modified IgG1 Fc M5-5 and IGHG1*03 (FIG. 1D). [Figure 1E] FIG. 1E shows the sequence alignment of modified IgG1 Fc M7 with IGHG1*03 (FIG. 1E). [Figure 1F] FIG. 1F shows a sequence alignment of the modified IgG4 Fc with human wild-type IgG4 Fc (FIG. 1F). [Diagram 2] Figure 2 shows the preproNGF structure, including the signal peptide (SP), the propeptide, and mature NGF. Furin cleavage at the major cleavage site is responsible for processing proNGF to mature NGF. [Figure 3A]FIG. 3A shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (NGF-4-12PAA) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3B] FIG. 3B shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (FD-G4Fc) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3C] FIG. 3C shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (WM-G24Fc) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3D] FIG. 3D shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (2-118-L3G4-BM) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3E] FIG. 3E shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (NGF-1-15M7) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3F] FIG. 3F shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (2-1-15M7) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3G]FIG. 3G shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (NGF-L3Fc10M7) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3H] FIG. 3H shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (2-L3Fc10-M3-5) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3I] FIG. 3I shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (NGF-118-L3Fc10-M3-5) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3J] FIG. 3J shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (2-118-L3Fc10-M3-5) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3K] FIG. 3K shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (2-118-LFc10-M1-5) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3L] FIG. 3L shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (2-118-L3Fc10-M5-5) at different time points in a 40° C. accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 3M]Figure 3M shows the aggregate percentage, fragment content percentage, and monomer content percentage of mature NGF-Fc fusion protein (2-118-L3Fc1оM7-5) at different time points in a 40°C accelerated stability study as measured by size exclusion chromatography (SEC). [Figure 4A] FIG. 4A shows the detection of mature NGF-Fc fusion protein (NGF-4-12PAA) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 4B] FIG. 4B shows the detection of mature NGF-Fc fusion protein (FD-G4Fc) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS). [Figure 4C] FIG. 4C shows the detection of mature NGF-Fc fusion protein (WM-G24Fc) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS). [Figure 4D] FIG. 4D shows the detection of mature NGF-Fc fusion protein (2-118-L3G4-BM) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 4E] FIG. 4E shows the detection of mature NGF-Fc fusion protein (NGF-1-15M7) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS). [Figure 4F] FIG. 4F shows the detection of mature NGF-Fc fusion protein (2-1-15M7) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 4G]FIG. 4G shows the detection of mature NGF-Fc fusion protein (NGF-L3Fc10M7-5) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 4H] FIG. 4H shows the detection of mature NGF-Fc fusion protein (2-L3Fc10-M3-5) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 4I] FIG. 4I shows the detection of mature NGF-Fc fusion protein (NGF-118-L3Fc10-M3-5) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 4J] FIG. 4J shows the detection of mature NGF-Fc fusion protein (2-118-L3Fc10-M3-5) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 4K] FIG. 4K shows the detection of mature NGF-Fc fusion protein (2-118-L3Fc10-M1-5) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 4L] FIG. 4L shows the detection of mature NGF-Fc fusion protein (2-118-L3Fc10-M5-5) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 4M] FIG. 4M shows the detection of mature NGF-Fc fusion protein (2-118-L3Fc10M7-5) at different time points in a 40° C. accelerated stability study as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. [Figure 5A]5A is a graph showing the results of proliferation activity of TF-1 cells under treatment with each NGF-Fc fusion protein, except for SuTaiShengR mouse NGF, NGF variant 118aa (mNGF118), and recombinant human NGF (rhNGF) as controls. [Figure 5B] 5B is a graph showing the results of proliferation activity of TF-1 cells under treatment with each NGF-Fc fusion protein, except for SuTaiShengR mouse NGF, NGF variant 118aa (mNGF118), and recombinant human NGF (rhNGF) as controls. [Figure 6A] 6A is a graph showing the results of the biological activity of each NGF-Fc fusion protein on the growth of superior cervical ganglion (SCG) in rats, with SuTaiShengR mouse NGF and mNGF118 as controls, and PBS as a negative control. [Figure 6B] Figure 6B is a graph showing the results of the biological activity of each NGF-Fc fusion protein on the growth of superior cervical ganglion (SCG) in rats, with SuTaiShengR mouse NGF and mNGF118 as controls, and PBS as a negative control. [Figure 6C] Figure 6C is a graph showing the results of the biological activity of each NGF-Fc fusion protein on the growth of superior cervical ganglion (SCG) in rats, with SuTaiShengR mouse NGF and mNGF118 as controls, and PBS as a negative control. [Figure 6D] Figure 6D is a graph showing the results of the biological activity of each NGF-Fc fusion protein on the growth of superior cervical ganglion (SCG) in rats, with SuTaiShengR mouse NGF and mNGF118 as controls, and PBS as a negative control. [Figure 7A] FIG. 7A is a graph showing the pharmacokinetic (PK) curves of 2-118-L3Fc10-M3-5, 2-118-L3G4-BM and control mNGF118 (β-NGF118aa mutant without Fc fusion) in plasma. [Figure 7B] FIG. 7B shows the in vivo half-life of these (FIG. 7A) at an intramuscular dose of 235 μg / kg. [Figure 8] 8 is a graph showing the wound healing rate of diabetic mice treated with NGF (SuTaiShengR mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM), with PBS treatment serving as a negative control. [Figure 9A] FIG. 9A is a graph showing the proliferation rate of a human ovarian granulosa tumor cell line (KGN) treated with NGF (SuTaiShengR mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM). [Figure 9B] FIG. 9B is a graph showing secreted estrogen concentrations of human ovarian granulosa tumor cell line (KGN) treated with NGF (SuTaiShengR mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM). [Figure 9C] FIG. 9C is a graph showing the number of follicles at different levels in a rat premature ovarian failure animal disease model treated with NGF (SuTaiShengR mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM). [Figure 10A] FIG. 10A is a graph showing corneal sodium fluorescein staining scores in neurotrophic keratitis animal models treated with NGF (SuTaiShengR mouse NGF or mNGF118), NGF-Fc fusion proteins (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM), or 0.9% sodium chloride solution as a negative control. [Figure 10B] FIG. 10B is a graph showing the average corneal nerve length in an animal model of neurotrophic keratitis treated with NGF (SuTaiShengR mouse NGF or mNGF118), NGF-Fc fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM), or a negative control 0.9% sodium chloride solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention relates to a long-acting NGF polypeptide comprising an NGF portion and an Fc portion from the N-terminus to the C-terminus. The terms "long-acting NGF polypeptide", "long-acting NGF-Fc fusion protein" and "long-acting NGF construct" are used interchangeably.

[0020] NGF plays an important regulatory role in the development, differentiation, growth, regeneration and functional expression of central and peripheral nerve neurons. NGF has been used to treat nervous system growth abnormalities such as amblyopia, neuroma, various nerve injuries and nervous system diseases. However, NGF causes pain or has a short half-life in vivo, so low dose restrictions to avoid hyperalgesia, and frequent side effects such as nausea and vomiting limit the wide application of NGF. Fusing protein-based drugs to moieties with longer half-lives and / or larger molecular weights is one strategy to confer sustained activity to certain protein drugs. However, how to increase or maintain biological activity while extending the half-life of protein drugs remains a clinically challenging problem.

[0021] The sustained-release NGF polypeptide described herein has one or more of the following excellent effects. 1) The sustained-release NGF polypeptide according to the present invention has high biological activity both in vitro and in vivo (e.g., promotes the growth of superior cervical ganglion), and is superior to existing NGF-Fc fusion proteins or NGF drugs. 2) The sustained-release NGF polypeptide according to the present invention has a long half-life in vivo, which is not only much longer than NGF proteins without a fusion moiety, but also significantly longer than existing NGF-Fc fusion proteins, thereby reducing the frequency and total number of administrations, providing convenience to patients and reducing costs. 3) The sustained-release NGF polypeptide according to the present invention can reduce side effects such as pain, and even provide analgesia, thereby increasing the patient's tolerable dose, expanding the range of indications, and enabling application to the central nervous system. 4) The sustained-release NGF polypeptide according to the present invention reduces or minimizes antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), thereby avoiding undesirable immune responses during treatment. 5) The sustained-release NGF polypeptide according to the present invention has excellent thermal stability (e.g., high melting temperature (Tm) and / or high aggregation onset temperature (Tagg)). 6) The sustained-release NGF polypeptide according to the present invention exhibits excellent stability and maintains drug properties under accelerated stress (e.g., heating) with little or no fragmentation, aggregate formation and / or aggregate growth. 7) The sustained-release NGF polypeptide according to the present invention is highly effective in the in vivo treatment of NGF-related diseases, e.g., neurological diseases such as diabetic neuropathy, Alzheimer's disease and neurotrophic keratitis, and non-neurological diseases such as premature ovarian failure and spermatogenic disorders (e.g., oligozoospermia, asthenozoospermia, oligozoospermia), and has a therapeutic effect equal to or greater than that of an NGF portion not fused to Fc.

[0022] Thus, one aspect of the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion and an Fc portion from the N-terminus to the C-terminus, wherein the NGF portion comprises an amino acid sequence shown in any one of SEQ ID NOs: 1 to 4, and the Fc portion is derived from IgG1 Fc or IgG4 Fc.

[0023] Further aspects of the present invention relate to isolated nucleic acids encoding said sustained-release NGF polypeptides, vectors comprising said nucleic acids, host cells comprising said nucleic acids or vectors, methods for producing said sustained-release NGF polypeptides, pharmaceutical compositions, and products comprising said sustained-release NGF polypeptides, as well as methods of using said sustained-release NGF polypeptides or pharmaceutical compositions to treat diseases (e.g., diabetic neuropathy, nervous system diseases associated with neuronal degeneration or damage, such as Alzheimer's disease and neurotrophic keratitis, and non-nervous system diseases, such as premature ovarian failure and spermatogenic disorders). I. Definition

[0024] Unless the context clearly dictates otherwise, the practice of the present invention will employ conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of the art, as detailed below. The techniques are fully described in the literature, including Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0025] As used herein, "treatment" or "treating" is an approach to achieve beneficial or desired results, including clinical results. For purposes of the present invention, such beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease, preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improving the condition of the disease, relieving the disease (partially or fully), reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, improving the quality of survival and / or prolonging survival. "Treatment" also includes reducing the pathological consequences of the disease. The methods of the present invention are intended to take into account any one or more of these aspects of treatment. For example, a patient is considered to be successfully "treated" if one or more symptoms associated with the disease are alleviated or eliminated, including, but not limited to, alleviating symptoms caused by the disease, improving the quality of life of a patient suffering from the disease, reducing the dose of other medications required to treat the disease, and / or prolonging the individual's survival.

[0026] The term "prevention" and similar words such as "prevented," "preventing," and the like refer to a method of preventing, inhibiting, or reducing the likelihood of a disease or condition recurring. It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition before the disease or condition recurs.

[0027] As used herein, "delaying" disease progression means to prolong, inhibit, prolong, retard, stabilize and / or slow the progression of the disease. The amount of time delayed may vary depending on the history of the disease and / or the individual receiving the treatment. A method of "delaying" disease progression refers to a method that reduces the probability of disease progression within a certain time range and / or reduces the extent of disease within a certain time range compared to the absence of the method. Such comparisons are usually based on clinical studies using a statistically significant number of individuals.

[0028] As used herein, the term "effective amount" refers to a dose of a drug or a dose of a pharmaceutical composition sufficient to treat a particular disorder, disease, or condition, e.g., ameliorate, alleviate, attenuate, and / or delay one or more symptoms. In some embodiments, an effective amount is an amount sufficient to delay progression of the disease. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence of the disease. An effective amount may be an amount administered in one or more doses. In some embodiments, an effective amount of a drug or composition is sufficient to (i) support neuronal survival, (ii) promote neurite outgrowth, (iii) enhance neurochemical differentiation, (iv) promote proliferation of pancreatic beta cells, (v) induce innate and / or adaptive immunity, (vi) prevent or delay onset and / or recurrence of the disease, and / or (vii) alleviate to some extent one or more symptoms associated with the disease.

[0029] As used herein, an "individual" or "subject" refers to a mammal, including but not limited to a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual refers to a human.

[0030] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to another portion of the immunoglobulin molecule, including the variable domain of the antigen-binding site. The constant domain includes the CH1, CH2 and CH3 domains (commonly referred to as CH) of the heavy chain and the CHL (or CL) domain of the light chain. Depending on the amino acid sequence of the immunoglobulin heavy chain (CH) constant domain, immunoglobulins can be classified into different classes or subtypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with the heavy chains being α, δ, ε, γ and μ, respectively. Based on relatively minor differences in CH sequence and function, γ and α are further classified into subclasses, e.g., in humans, the following subclasses are expressed: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

[0031] As used herein, the terms "Fc region", "fragment crystalline region", "Fc domain" or "Fc portion" are used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the section of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as beginning at the amino acid residue at position Cys226 or Pro230 and extending to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the protein or by recombinant engineering of the nucleic acid encoding the protein. Suitable native sequence Fc regions for use in the constructs described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0032] As used herein, the term IgG "subtype" or "subclass" refers to any subclass of immunoglobulin defined by the chemical and antigenic properties of the constant domains. Immunoglobulins are classified into five main classes: IgA, IgD, IgE, IgG, and IgM, some of which may be further classified into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, E, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and are described in detail in Abbas et al., Cellular and Molecular Immunology, 4th Edition (WB Saunders, Co., 2000).

[0033] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region in an Fc-containing structure (e.g., an antibody). A preferred FcR is a human FcR native sequence. Additionally, preferred FcRs are those that bind IgG antibodies (gamma receptors), including receptor subclasses such as FcγRI, FcγRII, and FcγRIII, as well as allelic variants of these receptors and alternatively spliced ​​forms thereof. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994) and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). As used herein, the term "FcR" encompasses other FcRs, including those that may be identified in the future.

[0034] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus. See Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994). Methods for measuring binding to FcRn are well known (see Ghetie and Ward, Immunol. Today 18:(12):592-8 (1997); Ghetie et al., Nature Biotechnology 15(7):637-40 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6 (2004); WO 2004 / 92219 (Hinton et al.)). The half-life of human FcRn high affinity binding polypeptides that bind to FcRn in vivo and in serum can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with variant Fc regions. WO2004 / 42072 (Presta) details antibody modifications that enhance or attenuate binding to FcRs. See Shield et al., J.Biol.Chem.9(2):6591-6604(2001).

[0035] "Antibody effector function" refers to a biological activity exerted by an Fc region (either a native sequence Fc region or an amino acid sequence variant Fc region) in an Fc-containing structure (e.g., an antibody) and varies according to the Fc subtype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors) and B cell activation. "Reducing or minimizing" an antibody effector function means reducing by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) compared to that of a wild-type or unmodified Fc-containing structure (e.g., an antibody). Antibody effector functions can be readily determined and measured by one of skill in the art. In preferred embodiments, the antibody effector functions of complement binding, complement dependent cytotoxicity and antibody-dependent cellular cytotoxicity can be affected. In some embodiments, effector function is eliminated by a mutation in the constant domain that eliminates glycosylation, e.g., an "effector function null mutation." In some embodiments, the effector function null mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region. See Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). Other mutations that result in reduced or eliminated effector function include K322A and L234A / L235A (LALA). Effector function can also be reduced or eliminated by production techniques such as expression in a host cell that does not glycosylate (e.g., E. coli) or that results in altered glycosylation patterns that are ineffective or less effective in promoting effector function (see, e.g., Shinkaw et al., J. Biol. Chem. 278(5):3466-3473 (2003)).

[0036] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig (or ligand-Fc structures) bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer cells (NK), neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing (or ligand-receptor-bearing) target cells and subsequently kill the target cells with cytotoxins. Antibodies (or Fc-containing structures) are required to "arm" the cytotoxic cells to kill the target cells by this mechanism. The primary cells mediating ADCC are NK cells, which express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Hematopoietic cell Fc expression is summarized in Table 2 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991) p. 464. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, as detailed in US Pat. No. 5,500,362 or 5,821,337. Suitable effector cells for this type of assay include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model as disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0037] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an Fc-containing structure (of the appropriate subclass) that is bound to a cognate receptor via a ligand fused to Fc. To assess complement activation, a CDC assay can be performed as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Antibody variants in which the amino acid sequence of the Fc region has been altered to increase or decrease C1q binding capacity are described in detail in US Pat. No. 6,194,551B1 and WO 99 / 51642. The contents of these patent publications are incorporated herein by reference. See Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0038] As used herein, the terms "specifically bind," "specifically recognize," or "specifically used" refer to a measurable and reproducible interaction, e.g., binding between a ligand and a receptor, where the presence of the ligand can be determined when a heterogeneous population of molecules, including biomolecules, is present. For example, a ligand that specifically binds to a receptor binds to the receptor of interest with higher affinity, higher avidity, easier, and / or longer duration than it binds to other receptors. In some embodiments, the extent of ligand binding to unrelated receptors is less than 10% of the extent of ligand binding to the receptor of interest, as measured, e.g., by radioimmunoassay (RIA) methods. In some embodiments, the equilibrium dissociation constant of a ligand that specifically binds to a target receptor is (K d )≦10 -5 M, ≦10 -6 M, ≦10 -7 M, ≦10 -8 M, ≦10 -9 M, ≦10 -10 M, ≦10 -11 M or ≦10 -12In some embodiments, the ligand specifically binds to a conserved receptor in different species. In some embodiments, specific binding includes, but does not require, exclusive binding. The binding specificity of the ligand can be determined experimentally using methods known in the art. Examples include Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE. TM -tests and peptide scans.

[0039] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a ligand) and its binding partner (e.g., a receptor). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that can reflect a 1:1 interaction between members of a binding pair. Binding affinity is expressed as the K d , K off , K on Or K a As used herein, the term "K off " refers to the rate constant for dissociation of the ligand from the ligand / receptor complex, as determined by a kinetic selection apparatus, s -1 As used herein, the term "K on " refers to the binding rate constant at which a ligand binds to a receptor to form a ligand / receptor complex, and M -1 s -1 As used herein, the equilibrium dissociation constant "K d The term "dissociation constant" refers to the dissociation constant for a particular ligand-receptor interaction, the concentration of ligand required to reach equilibrium when the ligand occupies half of the total receptor binding sites in the receptor solution, K off / K on and is expressed in units of M. d Measurement of EC assumes that all bound molecules are in solution. If the receptor is on the cell membrane, the corresponding dissociation rate constant is EC 50 It is expressed as K d is a good approximation of the affinity constant Ka is the dissociation constant K d is the reciprocal of M -1 The dissociation constant (K d ) is used as an index reflecting the affinity between the ligand and the receptor. The K d Values ​​are expressed in units of M (mol / L).

[0040] Half inhibitory concentration (IC 50 IC ) is a measure of the effectiveness of a substance (e.g., a ligand) in inhibiting a particular biological or biochemical function; it represents the amount of a particular drug or other substance (inhibitor, e.g., ligand) required to inhibit a particular biological process by half, and is usually expressed as a molar concentration. 50 is the "EC value" of an agonist drug or other substance (e.g., a ligand). 50 " EC 50 As used herein, "IC" may also refer to the plasma concentration required to obtain 50% of the maximal effect in vivo. 50 " is used to indicate the effective concentration of a ligand required to neutralize 50% of a receptor's biological activity in vitro. 50 or EC 50 can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competitive binding assay), cell-based cytokine release assays or amplified luminescence proximity homogeneous assay binding immunosorbent assay (AlphaLISA).

[0041] As used herein, "covalent bond" refers to a stable bond formed by sharing one or more electrons between two atoms. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, "peptide bond" refers to a covalent bond formed between a carboxy group of an amino acid and an amine group of an adjacent amino acid. As used herein, "disulfide bond" refers to a covalent bond formed between two sulfur atoms, for example, two Fc fragments are linked by one or more disulfide bonds. One or more disulfide bonds between two fragments may be formed by linking thiol groups in the two fragments. In some embodiments, one or more disulfide bonds may be formed between one or more cysteines of two Fc fragments. The disulfide bond may be formed by oxidation of two thiol groups. In some embodiments, the covalent linkage is a direct covalent linkage. In some embodiments, the covalent linkage is a direct peptide bond or disulfide bond.

[0042] The "percentage (%) of amino acid sequence identity" or "homology" of a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular polypeptide or polypeptide sequence, maximizing the percentage of sequence identity after the sequences are aligned and gaps are introduced (if necessary), and not taking into account any conservative substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, various alignment aspects within the skill of the art can be performed using publicly available computer software, such as, for example, BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Appropriate parameters to be used for alignment measurements, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared, can be determined by one of ordinary skill in the art.

[0043] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide that provides an amine group for forming a peptide bond with the carboxy group of the adjacent amino acid residue. As used herein, the "N-terminus" of a polypeptide refers to the first amino acid of the polypeptide that provides a carboxy group for forming a peptide bond with the amine group of the adjacent amino acid residue.

[0044] An "isolated" polypeptide refers to a polypeptide that has been identified, isolated, and / or recovered from a component of the environment (e.g., natural or recombinant) for which it was produced. An isolated polypeptide is preferably free from association with all other components in the environment for which it was produced. Contaminating components of the production environment, such as those produced by recombinant transfected cells, often interfere with the study, diagnosis, or treatment of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. (1) In some embodiments, the polypeptide is purified to a polypeptide content of greater than 95% by weight, e.g., in some embodiments, the polypeptide content is greater than 99% by weight as determined by the Lowry method. (2) In some embodiments, the polypeptide is purified sufficiently to obtain at least 15 N-terminal residues or internal amino acid sequence using a spinning cup sequencer. (3) In some embodiments, the polypeptide is purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions with Coomassie Blue or preferred silver stain. An isolated polypeptide includes the polypeptide in situ within a recombinant cell, since at least one component of the polypeptide's natural environment is absent. Ordinarily, however, an isolated polypeptide will be subjected to at least one purification step.

[0045] An "isolated" nucleic acid molecule encoding a construct (such as a NGF polypeptide as described herein) is one that has been identified and isolated from a nucleic acid molecule that contains at least one impurity that is typically associated with the environment in which it is produced. An isolated nucleic acid is preferably not associated with all components of the environment in which it is produced. For example, an isolated nucleic acid molecule encoding a polypeptide as described herein is present in a form or configuration that is not found in nature. Thus, an isolated nucleic acid molecule is different from a nucleic acid encoding a polypeptide as described herein that is naturally present in a cell. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0046] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are also known to utilize promoters, polyadenylation signals, and enhancers.

[0047] "Operably linked" means that a nucleic acid establishes a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA would be operably linked to a polypeptide DNA if it is expressed as a preprotein involved in the secretion of the polypeptide. A promoter or enhancer would be operably linked to a coding sequence if it affects the transcription of the sequence. Alternatively, a ribosome binding site would be operably linked to a coding sequence if it is in a position that facilitates translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous as well as in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at suitable restriction sites. If no such sites exist, conventionally synthesized oligonucleotide aptamers or linkers are used.

[0048] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid molecule to which it is linked. This term includes vectors that are self-replicating nucleic acid structures and vectors that are introduced into the genome of a known host cell. Certain vectors are capable of directing the expression of nucleic acids to which they are linked. Such vectors are referred to herein as "expression vectors."

[0049] As used herein, the terms "transfect" or "transformation" or "transduction" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. Such cells include the primary test cell and its progeny.

[0050] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny regardless of the number of passages. The progeny may contain mutations and not be identical in nucleic acid to the parent cell. Included herein are mutant progeny that have the same function or biological activity as screened or selected from the originally transformed cell.

[0051] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that effectively utilizes the biological activity of the active ingredient and that does not contain additional ingredients that are unacceptably toxic to the subject to which it is administered. Such a preparation is sterile. A "sterile" preparation is one that is sterile or free of any living microorganisms or their spores.

[0052] It should be understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0053] As used herein, "about" refers to a value or parameter and includes (and describes) variants relative to the value or parameter itself. For example, a statement about "about X" includes the statement of "X."

[0054] As used herein, a reference to a value or parameter "is not" is generally intended to mean "excluding" the particular value or parameter. For example, a reference to a method that cannot be used to treat cancer type X means that the method is typically used to treat cancers other than cancer type X.

[0055] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0056] As used in this specification and the appended claims, the singular forms "a," "one," "the," and "the" may include plural referents unless the context clearly dictates otherwise. II. Long-acting NGF Polypeptides

[0057] One aspect of the present invention relates to a sustained-release NGF polypeptide comprising, from the N-terminus to the C-terminus, an NGF portion and an Fc portion. In some embodiments, the sustained-release NGF polypeptide comprises, from the N-terminus to the C-terminus, an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3), and an Fc portion derived from IgG1 Fc or IgG4 Fc. In some embodiments, the NGF portion comprises (or consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1). In some embodiments, the long-acting NGF polypeptide has a half-life of at least 10 hours (e.g., at least any of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, and 100 hours) when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection. In some embodiments, the long-acting NGF polypeptide causes less pain (e.g., reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to an NGF portion comprising the amino acid sequence of SEQ ID NO:4.

[0058] In some embodiments, the NGF portion is fused to the Fc portion via a peptide linker. In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, a peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc or IgG4 Fc. In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence (GGGGS)n (SEQ ID NO: 70, where n is 1, 2, 3, 4, 5, or 6). In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 68 or 69. In some embodiments, the NGF portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO:1. In some embodiments, when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection, the long-acting NGF polypeptide has a half-life of at least about 10 hours (e.g., at least about any of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 hours). In some embodiments, the long-acting NGF polypeptide causes less pain (e.g., reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to an NGF portion comprising the amino acid sequence of SEQ ID NO:4.

[0059] In some embodiments, the Fc portion is derived from IgG1 Fc, such as human IgG1 Fc. In some embodiments, the Fc portion is wild-type IgG1 Fc (e.g., human IgG1 Fc) or a naturally occurring variant thereof. In some embodiments, the Fc portion is derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8. In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 7 or 8. In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the Fc portion lacks the first 5 amino acids of SEQ ID NO: 7 or 8.In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) mutations at one or more positions selected from E233, L234, L235, G236, G237, N297, A327, A330, and P331 relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising one or more mutations selected from E233P, L234V, L234A, L235A, L235E, G236del, G237A, N297A, A327G, A330S, and P331S relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the Fc portion further lacks the first five amino acids of SEQ ID NO: 7 or 8. In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence (GGGGS)n (SEQ ID NO:70, where n is 1, 2, 3, 4, 5, or 6). In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO:68 or 69.In some embodiments, the NGF portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 1. In some embodiments, the long-acting NGF polypeptide has a half-life of at least 10 hours (e.g., a half-life of at least one of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, and 100 hours) when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection. In some embodiments, the long-acting NGF polypeptide causes less pain (e.g., reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to an NGF portion comprising the amino acid sequence of SEQ ID NO: 4.

[0060] In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) mutations at positions L234, L235, and P331 relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the Fc portion further lacks the first 5 amino acids of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) the L234A, L235A and P331S mutations relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8).In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) L234A, L235A and P331S mutations relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8), and further lacking the first 5 amino acids of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 11 or 12. In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence (GGGGS)n (SEQ ID NO: 70, where n is 1, 2, 3, 4, 5, or 6). In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 68 or 69. In some embodiments, the NGF portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 1. In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising (or consists essentially of, or consists of) the amino acid sequence set forth in any one of SEQ ID NOs: 62-64.In some embodiments, the long-acting NGF polypeptide has a half-life of at least 10 hours (e.g., a half-life of at least one of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, and 100 hours) when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection. In some embodiments, the long-acting NGF polypeptide causes less pain (e.g., reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to an NGF portion comprising the amino acid sequence of SEQ ID NO:4.

[0061] In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) mutations at positions E233, L234, L235, G236, A327, A330, and P331 relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the Fc portion further lacks the first 5 amino acids of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) the E233P, L234V, L235A, G236del, A327G, A330S, and P331S mutations relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8).In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) E233P, L234V, L235A, G236del, A327G, A330S and P331S mutations relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8), and further missing the first 5 amino acids of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 15 or 16. In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence (GGGGS)n (SEQ ID NO: 70, where n is 1, 2, 3, 4, 5, or 6). In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 68 or 69. In some embodiments, the NGF portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 1. In some embodiments, the invention relates to a sustained-release NGF polypeptide comprising (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO:66.In some embodiments, the long-acting NGF polypeptide has a half-life of at least 10 hours (e.g., a half-life of at least one of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, and 100 hours) when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection. In some embodiments, the long-acting NGF polypeptide causes less pain (e.g., reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to an NGF portion comprising the amino acid sequence of SEQ ID NO:4.

[0062] In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) mutations at positions L234, L235, G237, A330, and P331 relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the Fc portion further lacks the first 5 amino acids of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) the L234A, L235E, G237A, A330S, and P331S mutations relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8).In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) L234A, L235E, G237A, A330S and P331S mutations relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8), and further lacking the first 5 amino acids of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence (GGGGS)n (SEQ ID NO: 70, where n is 1, 2, 3, 4, 5, or 6). In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 68 or 69. In some embodiments, the NGF portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 1. Thus, in some embodiments, the sustained-release NGF polypeptide comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 65.In some embodiments, the long-acting NGF polypeptide has a half-life of at least 10 hours (e.g., a half-life of at least one of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, and 100 hours) when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection. In some embodiments, the long-acting NGF polypeptide causes less pain (e.g., reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to an NGF portion comprising the amino acid sequence of SEQ ID NO:4.

[0063] In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1-4 (e.g., any one of SEQ ID NOs: 1-3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68-99 or any one of SEQ ID NOs: 68-72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) one mutation at position N297 relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the Fc portion further lacks the first 5 amino acids of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8) and comprising (or consisting essentially of, or consisting of) one N297A mutation relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8).In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG1 Fc comprising the amino acid sequence of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8), comprising (or consisting essentially of, or consisting of) one N297A mutation relative to SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8), and further lacking the first 5 amino acids of SEQ ID NO: 7 or 8 (e.g., SEQ ID NO: 8). Thus, in some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence (GGGGS)n (SEQ ID NO: 70, where n is 1, 2, 3, 4, 5, or 6). In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 68 or 69. In some embodiments, the NGF portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 1. In some embodiments, the invention relates to a sustained-release NGF polypeptide comprising (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 61.In some embodiments, the long-acting NGF polypeptide has a half-life of at least 10 hours (e.g., a half-life of at least one of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, and 100 hours) when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection. In some embodiments, the long-acting NGF polypeptide causes less pain (e.g., reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to an NGF portion comprising the amino acid sequence of SEQ ID NO:4.

[0064] In some embodiments, the Fc portion is derived from an IgG4 Fc, such as human IgG4 Fc. In some embodiments, the Fc portion is a wild-type IgG4 Fc (e.g., human IgG4 Fc) or a naturally occurring variant thereof. In some embodiments, the Fc portion is derived from an IgG4 Fc comprising the amino acid sequence of SEQ ID NO: 17. Thus, in some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 17. In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG4 Fc comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the Fc portion lacks the first 5 amino acids of SEQ ID NO: 17. In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG4 Fc comprising the amino acid sequence of SEQ ID NO: 17 and comprising (or consisting essentially of, or consisting of) mutations at one or more positions selected from S228, F234, and L235 relative to SEQ ID NO: 17.In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG4 Fc comprising the amino acid sequence of SEQ ID NO: 17 and comprising (or consisting essentially of, or consisting of) mutations at positions S228, F234, and L235 relative to SEQ ID NO: 17. In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG4 Fc comprising the amino acid sequence of SEQ ID NO: 17 and comprising (or consisting essentially of, or consisting of) one or more mutations selected from S228P, F234A and L235A relative to SEQ ID NO: 17. In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion derived from IgG4 Fc comprising the amino acid sequence of SEQ ID NO: 17 and comprising (or consisting essentially of, or consisting of) S228P, F234A, and L235A mutations relative to SEQ ID NO: 17. In some embodiments, the Fc portion further lacks the first 5 amino acids of SEQ ID NO: 17.Thus, in some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, an optional peptide linker (e.g., any one of SEQ ID NOs: 68 to 99, or any one of SEQ ID NOs: 68 to 72), and an Fc portion comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 18. In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence (GGGGS)n (SEQ ID NO: 70, where n is 1, 2, 3, 4, 5, or 6). In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 68 or 69. In some embodiments, the NGF portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 1. In some embodiments, the present invention relates to a long-acting NGF polypeptide comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 67. In some embodiments, the long-acting NGF polypeptide has a half-life of at least 10 hours (e.g., a half-life of at least one of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, and 100 hours) when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection. In some embodiments, the long-acting NGF polypeptide causes less pain (e.g., reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to an NGF portion comprising the amino acid sequence of SEQ ID NO: 4.

[0065] In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising (or consisting essentially of, or consisting of) the amino acid sequence set forth in any one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, and 46. In some embodiments, the present invention relates to a sustained-release NGF polypeptide comprising (or consisting essentially of, or consisting of) the amino acid sequence set forth in any one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, and 46, and not including the signal peptide sequence of SEQ ID NO:6. In some embodiments, the long-acting NGF polypeptide has a half-life of at least 10 hours (e.g., a half-life of at least one of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, and 100 hours) when administered to an individual (e.g., a human) by intravenous, intramuscular, or subcutaneous injection. In some embodiments, the long-acting NGF polypeptide causes less pain (e.g., reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to an NGF portion comprising the amino acid sequence of SEQ ID NO:4. NGF part

[0066] The initial NGF expressed is a 7S, 130 kDa complex consisting of three proteins, α-NGF, β-NGF and γ-NGF (ratio 2:1:2). The γ subunit of the complex cleaves the N-terminus of the β subunit as a serine protease, thereby activating the protein to functional NGF. The human NGF gene is located on the short arm of chromosome 1, and the complete NGF exon encodes 241 amino acids, commonly referred to as the preproNGF precursor (SEQ ID NO:50). The preproNGF precursor contains a signal peptide sequence (SEQ ID NO:6), a propeptide (SEQ ID NO:5) and the mature NGF sequence (β-NGF, SEQ ID NO:4). The signal peptide of the preproNGF precursor is cleaved in the endoplasmic reticulum to form the proNGF precursor (223 amino acids, SEQ ID NO:54). The proNGF precursor exists as a homodimer in the endoplasmic reticulum and then translocates to the Golgi apparatus. In the Golgi apparatus, the dimer of the proNGF precursor is cleaved by furin at three furin motifs in the propeptide. Mature β-NGF dimers are formed by cleavage of the furin motifs located at the -1 and -2 positions of the mature NGF sequence by furin, and each monomer contains 118 or 120 amino acids. The mature β-NGF dimer is then transported outside the cell. Some of the uncleaved proNGF precursor is also secreted outside the cell. The structure of NGF is shown in Figure 2.

[0067] NGF is abundantly present in various species, including the submandibular gland of male mice, bovine seminal plasma, snake venom, guinea pig prostate, and human placental tissue. Mouse NGF and human NGF share a high amino acid sequence identity of 90%.

[0068] Binding of NGF to either tropomyosin receptor kinase A (TrkA) or the low affinity NGF receptor (LNGFR / p75NTR) is associated with neurodegenerative diseases. Binding of NGF to the TrkA receptor promotes receptor homodimerization and autophosphorylation of the tyrosine kinase fragment, activating the PI3-kinase, ras and PLC signaling pathways. The p75NTR receptor can form heterodimers similar to TrkA that have higher affinity and specificity for NGF. Meanwhile, proNGF binds to p75NTR and sortilin with high affinity to generate a signaling complex. The signaling complex recruits NRAGE and Rac to activate the JNK signaling cascade, which mainly promotes apoptosis. Binding of proNGF to p75NTR can also promote activation of NFκB, which can induce neuronal survival.

[0069] As used herein, the term "NGF moiety" refers to an NGF molecule, its species variants, fragments, mutants, or derivatives thereof. The NGF moiety may be a truncated version, a post-translationally modified version, a hybrid variant, a peptidomimetic, a biologically active fragment, a deletion variant, a substitution variant, or an insertion variant. These variants are capable of retaining, at least to some extent, the parent NGF's activity of binding to the NGF receptor to induce signaling by the NGF receptor. As used herein, "parent NGF" or "parent NGF" refers to an NGF reference sequence from which an NGF moiety is designed, modified, or derivatized.

[0070] In some embodiments, the NGF portion is wild-type NGF. In some embodiments, the NGF portion is an NGF natural variant. In some embodiments, the NGF portion is an NGF analog, for example, an NGF containing mutations of 6 or fewer amino acids (e.g., 6, 5, 4, 3, 2, and 1 amino acid). In some embodiments, the NGF portion is an NGF derivative. As used herein, the term "NGF derivative" refers to a molecule having an NGF amino acid sequence or an NGF analog, but may further have chemical modifications added at one or more amino acid groups, alpha carbon atoms, amino termini, or carboxyl termini. Chemical modifications include, but are not limited to, the addition of chemical groups, the creation of new chemical bonds, and the removal of chemical groups. Modifications of amino acid side groups include, but are not limited to, epsilon aminoacylation of lysine; N-alkylation of arginine, histidine, or lysine; alkylation of the carboxy group of glutamic acid or aspartic acid; deamination of glutamine or asparagine. Amino-terminal modifications include, but are not limited to, deamination, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Carboxyl-terminal modifications include, but are not limited to, amide, lower alkyl acyl, dialkyl amide, and lower alkyl ester modifications. In some embodiments, the lower alkyl group is a C1-C4 alkyl group. Additionally, one or more side groups or terminal groups may be protected with a protecting group known to those skilled in the chemical arts. The alpha carbon of the amino acid may be monomethylated or dimethylated. In some embodiments, the NGF moiety is a modified NGF, such as pegylated NGF, or a covalently modified NGF, such as glycosylated NGF.

[0071] The NGF portion can be derived from any organism, for example, from mammals, including, but not limited to, livestock (e.g., cows, sheep, goats, cats, dogs, donkeys, horses, etc.), primates (e.g., humans and non-human primates such as monkeys and chimpanzees), rabbits, and rodents (e.g., mice, rats, gerbils, hamsters, etc.).

[0072] In some embodiments, the NGF moiety is human NGF (hNGF). In some embodiments, the NGF moiety is wild-type (wt) hNGF. In some embodiments, the NGF moiety is an hNGF natural variant. In some embodiments, the NGF moiety is an hNGF analog, such as hNGF containing six or fewer amino acid mutations (e.g., six, five, four, three, two, or one amino acid mutations). In some embodiments, the NGF moiety is an hNGF derivative. The NGF moiety applied in this application may be any one of many active fragments, analogs, and derivatives of hNGF known in the art.

[0073] As described herein, the NGF portion may be NGF isolated from various sources, such as human tissue or other sources, or may be NGF prepared by recombinant or synthetic methods. In some embodiments, the NGF portion is recombinant NGF, such as recombinant hNGF (rhNGF). In some embodiments, the NGF portion is mouse NGF, such as recombinant mouse NGF.

[0074] In some embodiments, the NGF portion is full-length NGF. In some embodiments, the NGF portion is a functional fragment of NGF. The functional fragment of NGF is capable of producing most or all of the biological activity of a full-length NGF molecule, for example, most or all of the biological activity of full-length β-NGF. In some embodiments, the NGF portion is preproNGF (e.g., human preproNGF) or an active fragment thereof, i.e., includes the signal peptide, the propeptide, and full-length or a fragment of β-NGF. In some embodiments, the NGF portion comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 47-50. In some embodiments, the NGF portion comprises proNGF (e.g., human proNGF) or an active fragment thereof, i.e., includes the propeptide and full-length or a fragment of β-NGF. In some embodiments, the NGF portion comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 51-54. In some embodiments, the NGF portion comprises mature NGF or an active fragment thereof, i.e., full-length or an active fragment of β-NGF (e.g., human β-NGF). In some embodiments, the NGF portion comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-4. In some embodiments, the NGF portion is wild-type human β-NGF (SEQ ID NO: 4). In some embodiments, the NGF portion is wild-type human β-NGF (SEQ ID NO: 3) with the last two amino acids truncated. In some embodiments, the NGF portion comprises a signal peptide at the N-terminus of β-NGF, derived from a different or the same NGF molecule. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the NGF portion comprises a propeptide at the N-terminus of β-NGF. In some embodiments, the propeptide comprises the amino acid sequence of SEQ ID NO: 5.

[0075] In some embodiments, the NGF portion is a mutant or variant of NGF, e.g., an NGF variant or mutant capable of producing most or all of the biological activity of wild-type NGF. The mutant NGF portion may include a mutation at one or more amino acid sites in an NGF molecule (e.g., mature β-NGF). In some embodiments, the mutant NGF portion may include an amino acid substitution at one or more amino acid sites in NGF. In some embodiments, the mutant NGF portion includes an amino acid deletion or insertion at one or more amino acid sites in NGF. In some embodiments, the mutant NGF portion includes a modification of one or more amino acids in NGF.

[0076] In some embodiments, the NGF portion has one or more conservative amino acid sequence substitutions. A "conservative substitution" refers to a substitution of another amino acid with the same net charge and approximately the same size and shape as the amino acid being replaced. Amino acids having aliphatic or substituted aliphatic amino acid side chains are considered to be approximately the same size if the total number of carbon and heteroatoms in the side chains differs by no more than four. Amino acids are considered to be approximately the same shape if the number of branches in their side chains differs by no more than one. Amino acids having phenyl or substituted phenyl groups in their side chains are considered to be approximately the same size and shape. Unless otherwise specified, it is preferred to use natural amino acids for conservative substitutions. See the "Amino Acid Substitutions" section below.

[0077] "Amino acid" is used herein in its broadest definition, i.e., it includes both naturally occurring and non-naturally occurring amino acid sequences, as well as amino acid analogs and derivatives. The latter includes molecules that contain an amino acid moiety. The amino acids described herein include, for example, naturally occurring L-amino acids involved in protein formation; D-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; naturally occurring amino acids that do not participate in protein formation, such as norleucine, β-alanine, ornithine, and GABA; as well as chemically synthesized compounds that have amino acid properties known in the art. This is where the skilled artisan diverges according to the broad definition. As used herein, the term "protein formation" refers to an amino acid that can synthesize a cellular peptide, polypeptide, or protein through a metabolic pathway.

[0078] The insertion of synthetic non-natural amino acids, substituted amino acids, or non-natural amino acids, including one or more D-amino acids, into the sustained-release NGF polypeptides (or NGF moieties) of the present invention provides various advantages. Polypeptides containing D-amino acids have shown higher stability in vitro and in vivo compared to polypeptides containing L-amino acids. Therefore, constructing a polypeptide by adding D-amino acids is particularly useful for achieving good intracellular stability. In particular, D-peptides and their analogs are resistant to endogenous peptidase and protease activity, and are therefore used to improve the bioavailability of the molecule and extend its life span in vivo, as required. However, D-peptides and their analogs are difficult to elicit a humoral immune response in subjects because they cannot be efficiently processed due to limited presentation to helper T cells by type II major histocompatibility complex (MHC).

[0079] In some embodiments, the NGF portion is a mutant or NGF variant and reduces or eliminates side effects (e.g., pain) to a greater extent than wild-type NGF. In some embodiments, the mutant or NGF variant NGF portion reduces pain by at least 5% (e.g., at least any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%) compared to wild-type NGF, e.g., reduces pain by at least 5% at one or more time points (e.g., all time points) after administration. In some embodiments, the mutant or NGF variant NGF portion increases the pain threshold by at least 5% (e.g., at least any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%) compared to wild-type NGF. For example, in some embodiments, an individual's pain threshold is about 8, and after administration of wild-type NGF, the pain threshold is reduced to about 6, but after administration of a mutant or NGF variant (or a long-acting NGF polypeptide comprising the same as described herein), the pain threshold is maintained at about 8, e.g., the pain is reduced by about 25% or the pain threshold is increased by about 25%. In some embodiments, the NGF portion is a mutant or NGF variant described in patents CN107286233A, WO2017157325 and WO2017157326, the entire contents of which are incorporated herein by reference. In some embodiments, the NGF portion comprises a F12E mutation relative to the human wild-type β-NGF sequence (SEQ ID NO: 4). In some embodiments, the NGF portion comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the NGF portion comprises a F12E mutation relative to the human wild-type β-NGF sequence (SEQ ID NO: 4) and the last two amino acids are truncated. In some embodiments, the NGF portion comprises the amino acid sequence set forth in SEQ ID NO:1 (hereinafter also referred to as "mNGF118").

[0080] As described herein, amino acid sequence variants of the NGF portion or sustained-release NGF polypeptide may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the protein or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues in the amino acid sequence of the NGF portion or sustained-release NGF polypeptide. The final construct may be obtained by deletion, insertion, substitution, or any combination thereof, so long as it has the desired properties, such as retained or improved ligand-receptor binding, retained or enhanced biological activity (e.g., promoting neuronal growth, maintenance, proliferation and / or survival), retained or enhanced half-life, retained or reduced ADCC / CDC, retained or reduced pain-inducing activity.

[0081] Conservative substitutions are shown in Table A. Many more substantial substitutions are provided in Table 1 under the heading "Examples of Substitutions," as further detailed in the following section on amino acid side chain classes. Amino acids are classified according to their general side chain properties as follows: (1) hydrophobic Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic Cys, Ser, Thr, Asn, Gln; (3) acidic Asp, Glu; (4) basic His, Lys, Arg; (5) residues that influence chain orientation Gly, Pro; and (6) aromatic Trp, Tyr, Phe. Non-conservative substitutions require the replacement of a member of one of these categories with a member of another category. Amino acid substitutions can be introduced into protein constructs and screened to obtain products that meet the desired activity as described above.

[0082] [Table 1]

[0083] As described herein, the sustained-release NGF polypeptide comprises an Fc portion at the C-terminus. In some embodiments, the Fc portion is derived from any one of IgA, IgD, IgE, IgG, and IgM, and subclasses thereof. Among all immunoglobulins, IgG has the highest serum content and the longest half-life. Unlike other immunoglobulins, IgG may be effectively recycled after binding to Fc receptors (FcRs). In some embodiments, the Fc portion is derived from IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc portion is derived from human IgG. In some embodiments, the Fc portion comprises CH2 and CH3. In some embodiments, the Fc portion further comprises all or a portion of a hinge region. In some embodiments, the Fc portion is derived from human IgG1 or human IgG4. In some embodiments, the two subunits of the Fc portion are dimerized through one or more (e.g., 1, 2, 3, 4, or more) disulfide bonds. In some embodiments, the Fc portion comprises a full-length Fc sequence in each subunit. In some embodiments, the Fc portion comprises an N-terminal truncated Fc sequence in each subunit, e.g., each subunit comprises a truncated Fc domain with fewer N-terminal cysteines, such that disulfide bond mispairing during dimerization is reduced. In some embodiments, the Fc portion is truncated at the N-terminus, e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of an intact immunoglobulin Fc domain are deleted. In some embodiments, the Fc portion comprises one or more mutations, such as insertions, deletions, and / or substitutions.

[0084] The present invention anticipates that screening will provide Fc fragments capable of providing high biological activity, long half-life and low immunotoxicity (e.g., ADCC and / or CDC) to sustained-release NGF polypeptides as described herein.

[0085] Due to the Fc domain, Fc-containing proteins may activate complement and interact with Fc receptors (FcRs). Such inherent immunoglobulin properties are considered to be disadvantageous, as they may cause NGF-Fc fusion proteins to target cells expressing Fc receptors instead of targeting preferred cells expressing NGF receptors. Furthermore, Fc fusion proteins have a long half-life and systemic toxicity, making them difficult to apply therapeutically. Thus, in some embodiments, the Fc portion is engineered (e.g., includes one or more amino acid mutations) to modify binding to FcR, in particular to modify binding to Fcγ receptors (involved in ADCC) and / or effector functions, e.g., to modify antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Preferably, such amino acid mutations do not reduce binding to FcRn receptors (involved in half-life).

[0086] An Fc portion (e.g., human IgG1 Fc) mutated to remove one or more effector functions such as ADCC, ADCP, or CDC is hereinafter referred to as an "effector-free" or "nearly effector-free" Fc portion. In some embodiments, the Fc portion is an effector-free human IgG1 Fc that contains one or more selected mutations, for example, L234A, L235E, G237A, A330S, and P331S (e.g., in each Fc subunit). The combination of K322A, L234A, and L235A in IgG1 Fc is sufficient to completely abolish FcγR and C1q binding (Hezareh et al., J Virol 75, 12161-12168, 2001). It was found by MedImmune that the triple mutation L234F / L235E / P331S has a very similar effect (Oganesyan et al., Acta Crystallographica 64,700-704,2008). In some embodiments, the Fc portion comprises a glycosylation modification on N297 of the IgG1 Fc domain, which is known to be necessary for optimal FcR interaction. The Fc portion modification may be any suitable IgG Fc engineering mentioned by Wang et al., “IgG Fc Engineering to Modulate Antibody Effector Functions,” Protein Cell. 2018 Jan;9(1):63-73, the contents of which are incorporated herein by reference in their entirety.

[0087] In some embodiments, the long-acting NGF polypeptide has no or no detectable ADCC and / or CDC as described herein. In some embodiments, the long-acting NGF polypeptide has at least 5% (at least one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%) reduced ADCC and / or CDC compared to an NGF-Fc construct comprising the same NGF portion but being a wild-type or unmodified Fc fragment as described herein. Glycosylation variants

[0088] In some embodiments, the Fc portion or the sustained-release NGF polypeptide is modified to increase or decrease the degree of glycosylation of the construct. The amino acid sequence can be modified to add or remove glycosylation sites in the Fc portion, creating or removing one or more glycosylation sites.

[0089] Natural Fc-containing proteins produced by mammalian cells typically contain biantennary oligosaccharides that are typically linked to Asn297 of the CH2 domain of the Fc region via an N-linkage. See Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc on the "stem" of the biantennary oligosaccharide. In some embodiments, the oligosaccharides in the Fc portion can be modified to produce specific improved properties.

[0090] In some embodiments, the Fc portion or sustained-release NGF polypeptide described herein has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc portion. For example, such an Fc portion or sustained-release NGF polypeptide may have a fucose content therein ranging from 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. As described in WO2008 / 077546, the fucose content is determined by measuring the average content of fucose in the glycan linked to Asn297 relative to the sum of all glycan structures linked to Asn297 (e.g., complex, hybrid, and high mannose structures) using MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue at position 297 of the Fc region (using the EU numbering system for Fc region residues). However, due to minor sequence variations in the Fc region, Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have enhanced ADCC function. See US Patent Publication Nos. US 2003 / 0157108 (Presta, L.), US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "afucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing afucosylated Fc-containing proteins include Lec13 CHO cells, which lack protein fucosylation function (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., see especially Example 11), knockout cell lines such as alpha-1,6-fucosyltransferase gene and FUT8 knockout CHO cells (see Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107). Effector function mutants

[0091] In some embodiments, the present invention uses Fc portions or long-acting NGF polypeptides that have some but not all of the Fc effector functions, making them ideal candidates for certain applications in which the in vivo half-life of the long-acting NGF polypeptide is important, but certain effector functions (such as CDC and ADCC) are not essential or are deleterious. To determine the reduction / depletion of CDC and / or ADCC activity, cytotoxicity assays can be performed in vitro or in vivo. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the Fc portion or long-acting NGF polypeptide lacks FcR binding (and thus may lack ADCC activity) but retains FcRn binding ability. The primary cells mediating ADCC are natural killer cells (NK) which express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 2 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991) at page 464. Non-limiting examples of in vitro assays that can be used to assess ADCC activity of a molecule of interest include those described in US Patent No. 5,500,362 (Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, 2002). 83:7059-7063 (1986) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods (ACTI for flow cytometry) can be used. TM Non-radioactive toxicity assay (CellTechnology, Inc. Mountain View, CA) and CytoTox 96 RNon-radiotoxicity assays (see Promega, Madison, WI) may be used. Suitable effector cells for this detection include peripheral blood mononuclear cells (PBMC) and NK cells. In addition, the ADCC activity of the molecule of interest may be evaluated in vivo, for example in the animal model described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to determine that a sustained-release NGF polypeptide lacks CDC activity because it cannot bind C1q. See, for example, WO2006 / 029879 and WO2005 / 100402 for enzyme-linked immunosorbent assays combining C1q and C3c. CDC assays can be performed to assess complement activity (see Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003) and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0092] Fc portions with reduced effector function include substitutions at one or more of the residues at positions 238, 265, 269, 270, 297, 327, and 329 in the Fc region (US Patent No. 6,737,056). Such Fc variants include substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, and also include the so-called "DANA" Fc variants in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581). Certain antibody variants with enhanced or diminished binding to FcRs are described in detail in US Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001). In some embodiments, the Fc region has been modified to alter (i.e., increase or decrease) C1q binding and / or CDC, as described in US Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4 178-4184 (2000).

[0093] In some embodiments, the Fc portion contains one or more amino acid substitutions resulting in improved half-life and / or binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and binding to neonatal FcRn are involved in the transfer of maternal IgGs to the fetus (see Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and are also described in detail in US2005 / 0014934A1 (Hinton et al.). Those antibodies that contain an Fc domain with one or more substitutions improve the binding of the Fc region to FcRn. Such Fc variants include those with one or more residue substitutions in the Fc region, for example, substitution of residue 434 in the Fc region (US Patent No. 7,371,826).

[0094] For further examples of Fc domain variants, see Duncan and Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94 / 29351. Cysteine ​​engineered mutants

[0095] In some embodiments, it may be desirable to create a cysteine ​​engineered Fc portion or long-acting NGF polypeptide in which one or more residues of the Fc domain are replaced with cysteine ​​residues. In some embodiments, the replaced residues appear at accessible sites on the Fc portion or long-acting NGF polypeptide. By replacing these residues with cysteine, active thiol groups are placed at accessible sites on the Fc portion or long-acting NGF polypeptide and can be used to attach the molecule to other moieties, such as drug moieties or linker-drug moieties, to create long-acting NGF polypeptide conjugates. In some embodiments, any one or more residues of the heavy chain A118 (EU numbering system) and the heavy chain Fc domain S400 (EU numbering system) may be replaced with cysteine. Cysteine ​​engineered molecules can be generated as described in US Patent No. 7,521,541.

[0096] In some embodiments, the Fc portion is derived from IgG1 Fc. In some embodiments, the Fc portion is derived from human IgG1 Fc. In some embodiments, the Fc portion is derived from wild-type IgG1 Fc (IGHG1*05). In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO:7. In some embodiments, the Fc portion is an IgG1 natural variant (e.g., IGHG1*03, which comprises a double mutation of D239E and L241M relative to IGHG1*05). In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO:8. In some embodiments, the Fc portion does not include the hinge region of gG1 Fc. In some embodiments, the Fc portion comprises up to 5 amino acids truncated from the N-terminus of IgG1 Fc, for example, the first, the first 2, the first 3, the first 4, or the first 5 amino acids truncated from the N-terminus of IgG1 Fc. In some embodiments, the Fc portion comprises one or more effector-free and / or deglycosylation mutations. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) a mutation at one or more positions selected from E233, L234, L235, G236, G237, N297, A327, A330, and P331 relative to SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises one or more mutations selected from E233P, L234V, L234A, L235A, L235E, G236del, G237A, N297A, A327G, A330S, and P331S relative to SEQ ID NO: 7 or 8. In some embodiments, the Fc portion further deletes the first (N-terminal) 5 amino acids of SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) a mutation at position N297 relative to SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) a N297A mutation relative to SEQ ID NO: 7 or 8.In some embodiments, the Fc portion comprises (or consists essentially of or consists of) the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the Fc portion comprises (or consists essentially of or consists of) mutations at positions L234, L235 and P331 relative to SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises (or consists essentially of or consists of) the L234A, L235A and P331S mutations relative to SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises (or consists essentially of or consists of) the amino acid sequence of SEQ ID NO: 11 or 12. In some embodiments, the Fc portion comprises (or consists essentially of or consists of) mutations at positions L234, L235, G237, A330 and P331 relative to SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises (or alternatively consists essentially of, or consists of) the L234A, L235E, G237A, A330S, and P331S mutations relative to SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises (or alternatively consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the Fc portion comprises (or alternatively consists essentially of, or consists of) mutations at positions E233, L234, L235, G236, A327, A330, and P331 relative to SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises (or alternatively consists essentially of, or consists of) the E233P, L234V, L235A, G236del, A327G, A330S, and P331S mutations relative to SEQ ID NO: 7 or 8. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO:15 or 16.

[0097] In some embodiments, the Fc portion is derived from IgG4 Fc. In some embodiments, the Fc portion is derived from human IgG4 Fc. In some embodiments, the Fc portion is wild-type IgG4 Fc. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 17. In some embodiments, the Fc portion is an IgG4 natural variant. In some embodiments, the Fc portion does not comprise the hinge region of IgG4. In some embodiments, the Fc portion comprises a truncation of up to 5 amino acids from the N-terminus of IgG4, such as a truncation of the first, the first 2, the first 3, the first 4, or the first 5 amino acids from the N-terminus of IgG4. In some embodiments, the Fc portion comprises one or more effector-free and / or deglycosylation mutations. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) a mutation at one or more positions selected from S228, F234, and L235 relative to SEQ ID NO: 17. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) mutations at positions S228, F234, and L235 relative to SEQ ID NO: 17. In some embodiments, the Fc portion comprises one or more mutations selected from S228P, F234A, and L235A relative to SEQ ID NO: 17. In some embodiments, the Fc portion comprises mutations selected from S228P, F234A, and L235A relative to SEQ ID NO: 17. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 18. In some embodiments, the Fc portion further deletes the first (N-terminal) 5 amino acids of SEQ ID NO: 17. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 19 or 20. Linker

[0098] The NGF portion and the Fc portion are linked by an optional linker (e.g., a peptide linker, a non-peptide linker). In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a stable linker. An ideal linker generally does not affect or does not significantly affect the correct folding and conformation of the long-acting NGF polypeptide described herein. Preferably, the linker provides flexibility to the long-acting NGF polypeptide, retains or improves the biological function of NGF, and / or does not significantly affect the half-life and / or stability of the long-acting NGF polypeptide in vivo. In some embodiments, the linker is a stable linker (e.g., one that cannot be cleaved by proteases, particularly MMPs).

[0099] In some embodiments, the linker is a peptide linker. The peptide linker may be of any length. In some embodiments, the length of the peptide linker is 1 to 10 amino acids, 3 to 18 amino acids, 1 to 20 amino acids, 10 to 20 amino acids, 21 to 30 amino acids, 1 to 30 amino acids, 10 to 30 amino acids, 1 to 50 amino acids, 5 to 40 amino acids, 12 to 18 amino acids, or 4 to 25 amino acids. In some embodiments, the length of the peptide linker is any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the length of the peptide linker is any one of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the peptide linker is any one of 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Preferably, the sustained-release NGF polypeptides described herein are optimized for in vivo function and stability by adding a peptide linker to prevent potential undesired domain interactions. In some embodiments, the linker length is no longer than necessary to prevent undesired domain interactions and / or optimize biological function and / or stability. In some embodiments, the peptide linker length is up to 30 amino acids, e.g., up to 20 amino acids, or up to 15 amino acids. In some embodiments, the linker length is 5 to 30 amino acids, or 5 to 18 amino acids.

[0100] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from an antibody heavy chain hinge region can be used as a linker. For an example, see WO1996 / 34103. In some embodiments, the peptide linker is a human IgG1, IgG2, IgG3 or IgG4 hinge. In some embodiments, the peptide linker is a mutated human IgG1, IgG2, IgG3 or IgG4 hinge. In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G)n (SEQ ID NO:73), glycine-serine polymers (e.g., (GS)n (SEQ ID NO:74), (GGS)n (SEQ ID NO:75), (GGGS)n (SEQ ID NO:76), (GGS)n(GGGS)n (SEQ ID NO:77), (GSGGS)n (SEQ ID NO:78), (GGSGS)n (SEQ ID NO:79) or (GGGGS)n (SEQ ID NO:70), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore function as neutral chains between components. Glycine has more phi-psi space than alanine and has residues with longer side chains, which are less restrictive (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)).Examples of flexible linkers include GG (SEQ ID NO: 86), GGSG (SEQ ID NO: 87), GGSGG (SEQ ID NO: 88), GSGSG (SEQ ID NO: 89), GSGGG (SEQ ID NO: 90), GGGSG (SEQ ID NO: 91), GSSSG (SEQ ID NO: 92), GGSGGS (SEQ ID NO: 93), SGGGGS (SEQ ID NO: 94), GGGGS (SEQ ID NO: 95), (GA)n (SEQ ID NO: 96, where n is an integer of at least 1), GRAGGGGAGGGG (SEQ ID NO: 97), GRAGGG (SEQ ID NO: 98), GSGGGSGGGGSGGGGS (SEQ ID NO: 80). , GGGSGGGGSGGGGS (SEQ ID NO: 81), GGGSGGSGGS (SEQ ID NO: 82), GGSGGSGGSGGSGGG (SEQ ID NO: 83), GGSGGSGGGGSGGGGS (SEQ ID NO: 84), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 85), GGGGSGGGGSGGGGGS (SEQ ID NO: 68), GGGGGGSGGGGSGGGGSA (SEQ ID NO: 69), GSGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 71), KTGGGSGGGS (SEQ ID NO: 72), and the like. In some embodiments, the linker comprises the sequence ASTKGP (SEQ ID NO: 99). Those skilled in the art will generally recognize that designed long-acting NGF polypeptides can contain all or a portion of flexible linkers to provide the ideal long-acting NGF polypeptide structure and function, allowing the linker to include one flexible linker portion and one or more portions that provide a less flexible structure. In some embodiments, the peptide linker is serine-glycine rich. In some embodiments, the peptide linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 68 to 72. In some embodiments, the peptide linker comprises an amino acid sequence (GGGGS)n (SEQ ID NO: 70, wherein n is any one of 1, 2, 3, 4, 5, and 6, preferably n is an integer from 2 to 6, and more preferably n is an integer of 3 or 4). In some embodiments, the peptide linker comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 68 or 69.

[0101] Other considerations for the linker include its effect on the physical or pharmacokinetic properties of the resulting sustained-release NGF polypeptide, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable and deliberate degradation), rigidity, flexibility, immunogenicity, binding to the NGF moiety / NGF receptor, colloid or liposome binding capacity, etc. binding affinity

[0102] The binding affinity of a molecule (e.g., an NGF moiety or an NGF polypeptide containing an NGF moiety) to its binding partner (e.g., an NGF receptor such as TrkA) can be determined by any suitable ligand-binding assay or antibody / antigen-binding assay known in the art, such as Western blot, enzyme-linked immunosorbent assay (ELISA), mesoscale discovery (MSD) electrochemiluminescence, bead-based multiplex immunoassay (MIA), RIA, surface plasmon resonance (SPR), ECL, IRMA, EIA, Biacore assay, octet analysis, peptide scan, etc. For example, the analysis can be easily performed using the NGF moiety, the NGF polypeptide containing an NGF moiety or its receptor (e.g., TrkA), or its subunits labeled with various labeling reagents, or BiacoreX (Amersham Biosciences). Note that BiacoreX is a commercially available measurement kit or a similar kit, and can be operated according to the instruction manual and experimental operation method provided with the kit.

[0103] In some embodiments, the interaction, function and activity of the NGF moieties or long-acting NGF polypeptides described herein with their receptors are analyzed on a large scale using protein microarrays, which have a support surface that binds a series of capture proteins (e.g., NGF receptors or subunits thereof). Fluorescently labeled probe molecules (e.g., NGF moieties or long-acting NGF polypeptides described herein) are then added to the array, allowed to interact with the bound capture proteins, and emit a fluorescent signal that is read by a laser scanner.

[0104] Binding affinity can also be measured using SPR (Biacore T-200). For example, anti-human IgG antibodies are bound to the surface of a CM-5 sensor chip using EDC / NHS chemistry. Human TrkA-Fc fusion protein is then used as the capture ligand for this surface. A dilution series of NGF moieties or sustained-release NGF polypeptides described herein are allowed to bind to the captured ligand, and the association and dissociation of NGF with TrkA can be monitored in real time. The dissociation constant (K d ) and dissociation rate constants can be determined by kinetic analysis using BIA evaluation software.

[0105] In some embodiments, the NGF portion or sustained-release NGF polypeptide described herein has a binding Kd to its receptor (e.g., TrkA) or a subunit thereof of ≦10 -5 M, ≦10 -6 M, ≦10 -7 M, ≦10 -8 M, ≦10 -9 M, ≦10 -10 M, ≦10 -11 M and ≦10 -12 In some embodiments, wild-type NGF has a binding K d is a binding K between an NGF portion or sustained-acting NGF polypeptide described herein and the receptor (e.g., TrkA) or a subunit thereof. d In some embodiments, the NGF moiety or sustained-release NGF polypeptide described herein has a binding K of about 1.5 times (e.g., at least one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, and 1000 times) to its receptor (e.g., P75) or a subunit thereof. d The binding K between wild-type NGF and its receptor (e.g., p75) or its subunits is dor is at least about two times (e.g., at least about any one of 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, and 1000 times).

[0106] In some embodiments, the NGF moiety comprises a mutation or modification (e.g., a post-translational modification) that reduces the K of binding between the mutated / modified NGF moiety (or long-acting NGF polypeptide) and its receptor (e.g., TrkA) or a subunit thereof. d is the K of binding between wild-type NGF and its receptor (e.g., TrkA). d In some embodiments, the NGF portion contains a mutation or modification (e.g., post-translational modification) that reduces the K of binding between wild-type NGF and its receptor (e.g., P75) or a subunit thereof. d is the K of binding between the mutated / modified NGF moiety (or long-acting NGF polypeptide) and its receptor (e.g., p75). d or is at least about two times (e.g., at least about any one of 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, and 1000 times). biological activity

[0107] The various methods described herein for determining the biological activity of NGF, NGF portion or sustained-release NGF polypeptide are known in the art. For example, biological activity can be evaluated by TF-1 cell proliferation test as described in CN103376248A and CN108727486A (the entire contents of which are incorporated herein by reference). See Example 4 below. Biological activity can also be determined based on the ability to promote the growth of superior cervical ganglion (SCG) in neonatal rats (see Example 5 below) or the ability to promote the growth of chick embryo dorsal root ganglion (see WO2017157326, the entire contents of which are incorporated herein by reference). Biological activity may also be determined based on whether there is: i) improved wound healing in animal models / patients with diabetic neuropathy (see, e.g., Example 7); ii) improved spatial awareness, memory and / or learning ability in animal models / patients with Alzheimer's disease (see, e.g., Example 8); iii) improved proliferation and / or estrogen secretion in ovarian granulosa tumor cell lines and / or improved follicle reduction in premature ovarian failure animal models / patients (see, e.g., Example 9); iv) rescue of sperm count and / or reduction in motility, and / or therapeutic effect on testicular seminiferous tubule atrophy, impaired seminiferous tubule spermatogenesis and / or epididymal duct cell fragmentation in animal models / patients with impaired spermatogenesis (see, e.g., Example 10); or v) restoration of damaged corneal integrity (e.g., by sodium fluorescein staining test) and / or rescue of damaged corneal nerves (e.g., by measuring corneal nerve length) in animal models / patients with neurotrophic keratitis (see, e.g., Example 11). It should be noted that any suitable assay protocol known in the art can be applied to detect the biological activity of the NGF portions or sustained-acting NGF polypeptides described herein.

[0108] Bioassays focus on the biological activity of NGF and use it as a readout. In bioassays, the activity of the samples is tested in sensitive cell lines (e.g., primary cell cultures or in vitro adapted cell lines that depend and / or respond to the test sample) or animal models / humans with NGF-related diseases, and the results of the activity (e.g., cell proliferation) are compared to standard NGF preparations or controls (e.g., mouse NGF, mNGF118, or known long-acting NGF polypeptides). Other aspects of NGF's biological activity include (i) supporting neuronal survival; (ii) promoting neurite outgrowth; (iii) enhancing neurochemical differentiation; (iv) promoting proliferation of pancreatic β-cells; (v) inducing innate and / or adaptive immunity; (vi) repairing damaged neuronal cells and / or preventing damage (e.g., neurotrophic keratitis); (vii) promoting proliferation and / or estrogen secretion of ovarian granulosa tumor cell lines; (viii) promoting wound healing (e.g., in diabetic neuropathy); (ix) treating neurodegenerative diseases (e.g., in atherosclerosis, glaucoma, and glaucoma). (x) improving spatial awareness, memory and / or learning ability in subjects with rheumatoid arthritis (such as rheumatoid arthritis); (x) treating and / or preventing neurodegenerative diseases; (xi) treating testicular seminiferous tubule atrophy, impaired spermatogenesis in seminiferous tubules and / or epididymal duct cell fragments; (xii) relieving reduced sperm count and / or motility or increasing sperm count and / or motility (e.g., in impaired spermatogenesis); and / or (xiii) improving reduced follicular number and / or function or increasing follicular number and / or function (e.g., in premature ovarian failure). All of these activities can be measured using in vitro and / or in vivo assays such as neuronal survival assays or neurite outgrowth assays.

[0109] For example, in a TF-1 cell proliferation test, a sample (e.g., a sustained-release NGF polypeptide) and a control (e.g., a vector or SuTaiSheng R Prepare serial dilutions of mouse NGF (NGF) and then add TF-1 cells to each well and incubate at 37 °C, 5% CO. 2 After culturing for several days (e.g., 3 days), add the MTS solution to each well of the cell suspension and incubate at 37 °C, 5% CO 2The cells were incubated for 3 hours under the conditions of 490 nm and 650 nm. The absorbance was then measured by a spectrophotometer to show how the NGF portion or the long-acting NGF polypeptide promotes TF-1 cell proliferation. The data can be normalized to the control sample. See Example 4 for an exemplary method.

[0110] Cell signaling assays can also be used to measure the biological activity of the NGF moieties or sustained-release NGF polypeptides described herein. Various commercially available cell signaling assay kits can be used to detect analytes such as ADP, AMP, UDP, GDP, growth factors, etc., generated during enzymatic reactions involved in signal transduction, or to perform phosphatase assays to quantify total and phosphorylated forms of signaling proteins. For example, after co-incubation of cells with the NGF moieties or sustained-release NGF polypeptides described herein, the cell lysates are exposed to a known matrix of enzymes in the presence of radioactive phosphate to determine whether a particular kinase is active. The products are separated by electrophoresis (with or without immunoprecipitation), and the gel is then exposed to X-ray film to determine whether the protein contains an isotope. In some embodiments, the location of the signaling protein is determined by measuring the biological activity of the NGF moieties or sustained-release NGF polypeptides described herein in cells by immunohistochemistry. For example, antibodies against the signaling protein itself or against the signaling protein in its activated state can be used. These antibodies have a recognition epitope that includes a phosphate or other activated conformation. In some embodiments, a fluorescent protein gene, e.g., green fluorescent protein (GFP), can be incorporated into a gene vector encoding a protein of interest to track the movement of a particular signaling protein (e.g., nuclear translocation of a signaling molecule). In some embodiments, the cellular biological activity of the NGF portion or long-acting NGF polypeptide described herein is detected by Western blot. For example, all tyrosine phosphorylated proteins (or other phosphorylated amino acids, such as serine and threonine) can be detected by Western blot of cell lysates obtained after time-dependent stimulation with anti-phosphotyrosine antibodies (or antibodies against other phosphorylated amino acids). In some embodiments, the cellular biological activity of the NGF portion or long-acting NGF polypeptide described herein can be determined by immunoprecipitation.For example, a primary antibody against a specific signaling protein or all tyrosine phosphorylated proteins is crosslinked to the beads. Cells incubated with the NGF moieties or sustained NGF polypeptides described herein are then lysed in a buffer containing protease inhibitors and then incubated with the antibody-coated beads. Proteins are then isolated using SDS-electrophoresis and identified by the Western blot step described above. In some embodiments, direct protein-protein (e.g., signaling protein) interactions may be determined by using glutathione S-transferase (GST) binding or "pull-down" assays.

[0111] For example, to reflect the biological activity of NGF in promoting cell growth, RAS / ERK1 / 2 signaling can be measured, for example, ERK1 / 2 phosphorylation can be performed using any suitable method known in the art. For example, to measure ERK1 / 2 phosphorylation, specific antibodies can be used that are phosphorylated for this molecule (optionally combined with flow cytometry analysis). For example, chick embryo dorsal root ganglia (DRGs) or TF-1 cells are incubated with the NGF moiety or long-acting NGF polypeptide described herein at 37°C. Immediately after incubation, the cells are fixed to maintain the phosphorylation state and permeability. The cells are stained with an antibody against phosphorylated ERK1 / 2, for example, Alexa488-conjugated anti-ERK1 / 2 pT202 / pY204 (BD Biosciences), and analyzed by flow cytometry. To reflect the biological activity of NGF, PI 3-kinase signaling can also be measured using any suitable method known in the art. For example, PI 3-kinase signaling can be measured using antibodies specific for phosphorylated S6 ribosomal protein, optionally coupled with flow cytometry analysis.

[0112] The biological activity of the NGF portions or sustained-release NGF polypeptides described herein can also be reflected by in vivo or in vitro experiments, such as measuring, for example, the proliferation of indicator cells, induction or inhibition of signal transduction, or tissue volume and / or weight.

[0113] For example, to study the biological activity of the NGF moiety or the sustained release NGF polypeptide in promoting SCG in vivo growth, a sample (e.g., sustained release NGF polypeptide) and a control (e.g., PBS or SuTaiSheng) are injected into the neck of neonatal rats in a single or multiple injections in an SCG in vivo growth assay. R Mouse NGF) can be subcutaneously injected into the rats and several days after injection, the rats can be sacrificed to isolate the SCG, which can then be weighed and the morphology recorded (see Example 5 for an exemplary method).

[0114] In addition, to reflect the biological activity of the NGF portion or the sustained-release NGF polypeptide in promoting dorsal root ganglion growth, various concentrations of the sample (e.g., sustained-release NGF polypeptide) or the control group (e.g., PBS or SuTaiSheng R Supplement chick embryo dorsal root ganglia (e.g., 8 days old) in a medium containing mouse NGF and incubate at 37°C for 24 hours at 5% CO. 2 and 37°C saturated humidity incubator for 24 hours, and monitor the growth status of dorsal root ganglia. If an NGF standard is used in the experiment, the specific biological activity of the sample may be calculated as AU / mg. Specific activity of test sample (AU / mg) = specific activity of reference substance (AU / ml) x [pre-dilution factor of sample x specific activity of corresponding reference substance at this dilution point (AU / ml) / actual specific activity of reference substance (AU / ml)]. For an exemplary method, see Example 5 of WO2017157326.

[0115] In some embodiments, the NGF portion (or sustained-release NGF polypeptide) described herein comprises a mutation or modification (e.g., a post-translational modification) such that its biological activity is maintained / enhanced / reduced as compared to wild-type NGF (or a polypeptide comprising wild-type NGF). In some embodiments, the mutated or modified NGF portion or sustained-release NGF polypeptide described herein has similar (e.g., equal) or at least twice as much (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 5000, or 10,000 times or more) biological activity (e.g., promotion of cell growth) as compared to wild-type NGF (or a polypeptide comprising wild-type NGF). In some embodiments, the long-acting NGF polypeptides described herein have similar (e.g., equal) or at least 1.1-fold (e.g., at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold or more) biological activity compared to the NGF portion (e.g., the corresponding NGF portion of the long-acting NGF polypeptide). Pharmacokinetics (PK)

[0116] Pharmacokinetics (PK) refers to the absorption, distribution, metabolism, and excretion of a drug (e.g., an NGF moiety or sustained-release NGF polypeptide described herein) administered to a subject. Pharmacokinetic parameters that can be used to determine clinical utility include serum / plasma concentration, serum / plasma concentration over time, peak serum / plasma concentration (C max ), time to reach maximum concentration (T max ), elimination half-life (t 1 / 2 ), area under the concentration-time curve within the dosing interval (AUCτ), and the like.

[0117] A drug, such as an NGF portion or a sustained-release NGF polypeptide described herein or a control drug (e.g., SuTaiSheng RTechniques for obtaining PK curves of mouse NGF) are known in the art. See Heller et al., Annu Rev Anal Chem, 11, 2018; Ghandforoush Sattari et al., J Amino Acids, Article ID 346237, Volume 2010. In some embodiments, the PK curve of the NGF portion or long-acting NGF polypeptide described herein is measured in an individual's blood, plasma or serum sample. In some embodiments, the PK curve of the NGF portion or long-acting NGF polypeptide described herein in an individual is measured by mass spectrometry techniques (e.g., LC-MS / MS or ELISA). Also, PK analysis, such as non-compartmental analysis, can be performed on the PK curves using PKSolver V2 software by any method known in the art (Zhang Y. et al., "PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel," Comput Methods Programs Biomed. 2010; 99(3): 306-1). For an exemplary method, see Example 6.

[0118] "C" represents the concentration of drug (e.g., NGF moiety or sustained-release NGF polypeptide) in the subject's plasma, serum, or any suitable bodily fluid or tissue, and is usually expressed as mass per unit volume, such as nanograms per milliliter. For convenience, the concentration of drug in serum or plasma is referred to herein as the "serum concentration" or "plasma concentration." The serum / plasma concentration at any time point after administration (e.g., NGF moiety or sustained-release NGF polypeptide, e.g., intravenous, peritoneal, or subcutaneous injection) is expressed as the C time Or C t The maximum serum / plasma drug concentration during the administration period is called C max It is called C min C refers to the minimum serum / plasma drug concentration at the end of the dosing interval. ave refers to the average concentration during the dosing interval.

[0119] The term "bioavailability" refers to the extent or rate at which a drug (eg, an NGF moiety or a sustained-release NGF polypeptide) passes through the systemic circulation and reaches a site of action.

[0120] "AUC" refers to the area under the serum / plasma concentration-time curve, specifically the area under the concentration-time curve within the dosing interval (AUCτ), "total exposure" or "total drug exposure over time" (AUC 0-last or AUC 0-inf ), the area under the concentration-time curve up to t hours after administration (AUC 0-t ), which is considered to be the most reliable scale of bioavailability.

[0121] Time to reach maximum serum / plasma concentration (T max ) is the maximum serum / plasma concentration (C) after administration (e.g., of an NGF moiety or a sustained-release NGF polypeptide). max ) is the time it takes to reach

[0122] Elimination half-life (t 1 / 2 ) refers to the time required for the concentration of a drug (e.g., an NGF moiety or a sustained-release NGF polypeptide) measured in plasma or serum (or other biological matrix) to fall to half of its concentration or amount at a particular time point. For example, as the drug distributes and disappears after intravenous administration, the concentration of the drug in plasma or serum falls. In the time-dependent curve of the plasma or serum drug concentration after intravenous administration, both distribution and disappearance can occur in two phases, although the initial or rapid decline phase is mainly due to distribution and the subsequent decline phase is usually slower and mainly due to elimination. Distribution is assumed to be complete after a sufficient time has elapsed. The elimination half-life is generally determined by the terminal or elimination (main) phase of the plasma / serum concentration-time curve. See Michael Schrag and Kelly Regal, Comprehensive Guide to Toxicology in Preclinical Drug Development, Chapter 3 - Pharmacokinetics and Toxicokinetics, 2013.

[0123] In some embodiments, the long-acting NGF polypeptides described herein have a half-life (e.g., intravenous, subcutaneous, or intramuscular injection, such as injection into a human) of at least 5 hours, e.g., at least any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 hours, or more. In some embodiments, the long-acting NGF polypeptides described herein have a half-life (e.g., intravenous, such as injection into a human) of 5 hours to 300 hours, e.g., any one of 8 hours to 100 hours, 10 hours to 60 hours, 15 hours to 60 hours, and 20 hours to 58 hours. In some embodiments, the long-acting NGF polypeptides described herein are administered as a single dose, such as a single intravenous injection or infusion, a single intramuscular injection, or a single subcutaneous injection. In some embodiments, the long-acting NGF polypeptides described herein have a circulating half-life of about 55 hours.

[0124] In some embodiments, the NGF portion has a half-life of 1 hour to 2.5 hours, e.g., a half-life of 1.5 hours to 2.4 hours. In some embodiments, the circulating half-life of the long-acting NGF polypeptide described herein is 5-fold greater than the circulating half-life of the corresponding NGF portion (i.e., the NGF portion of the long-acting NGF polypeptide that is not fused to Fc) or wild-type NGF, e.g., any one or more of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, and 100-fold greater than the circulating half-life of the corresponding NGF portion or wild-type NGF. Pain-inducing activity

[0125] NGF is known as a pain target because it can induce pain in animals and humans. NGF may promote the health and survival of central and peripheral nerve subsets, especially in adults (Huang and Reichardt, Ann. Rev. Neurosci. 24:677-736 (2001)). NGF also contributes to regulating the function and properties of these neurons, and can exert a tonic control over the sensitivity or excitability of sensory pain receptors called nociceptors (Priestley et al., Can. J. Physiol. Pharmacol. 80:495-505 (2002); Bennett, Neuroscientist 7:13-17 (2001)). Nociceptors perceive various noxious stimuli and transmit them to the central nervous system, resulting in pain (nociception). Nociceptors have NGF receptors. NGF expression is increased in injured and inflamed tissues and is upregulated in human pain conditions. NGF-induced nociception and pain are mediated by the high-affinity NGF receptor, trkA (tyrosine receptor kinase A) (Sah et al., Nat. Rev. Drug Disc. 2:460-72 (2003)).

[0126] "Pain" is broadly defined as an experiential phenomenon, highly subjective to the individual experiencing it, and influenced by the individual's psychological state, including the environment and cultural background. "Physiological" pain is often associated with a stimulus perceived by a third party that causes actual or potential tissue damage. Pain, in this sense, is "a sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage," according to the International Association for the Study of Pain (IASP). However, there are some examples of pain without an identifiable cause. One example is psychogenic pain, which involves the aggravation of existing physical pain due to psychogenic factors or a perceptible pain syndrome that sometimes persists in people with psychological disorders, in the absence of any so-called evidence that causes perceptible pain.

[0127] Pain includes nociceptive pain, neuropathic / neurogenic pain, breakthrough pain, allodynia, hyperalgesia, hyperesthesia, dysesthesias, dysesthesia, hyperalgesia, phantom limb pain, psychogenic pain, anesthesia, neuralgia, and neuritis. Other classifications include malignant pain, angina pain and / or primary pain, complex regional pain syndrome I, and complex regional pain syndrome II. Pain types and symptoms are not necessarily mutually exclusive. The definitions of these terms are consistent with the IASP.

[0128] Nociceptive pain is caused by specialized nociceptors in peripheral nerve neurons that respond to noxious stimuli and encode them as action potentials. Nociceptors are free nerve endings, typically A-delta and (polymodal) C-fibers, that terminate beneath the skin, in tendons, joints, and in organs of the body. Dorsal root ganglion (DRG) neurons provide a site of communication between the periphery and the spinal cord. Signals that are processed in the spinal cord, reach brainstem and thalamic sites, and finally reach the cerebral cortex, where they often, but not always, cause pain. Nociceptive pain can be caused by a variety of chemical, thermal, biological (e.g., inflammation), and mechanical events that can stimulate or damage body tissues, and generally exceed a certain minimum threshold of intensity required to cause nociceptive activity in nociceptors.

[0129] Neuropathic pain usually results from abnormal functioning of the peripheral or central nervous system, causing peripheral neuropathic pain or central neuropathic pain, respectively, and is defined by IASP as pain caused or caused by a primary lesion or dysfunction of the nervous system. Neuropathic pain is often accompanied by actual damage to the nervous system, especially in chronic disease. Inflammatory nociceptive pain usually results from tissue damage and the inflammatory process it causes. Neuropathic pain may persist (e.g., months or years) after the visible damage to tissue has apparently healed.

[0130] When neuropathic pain is present, sensory information processing by the affected area becomes abnormal and innocuous stimuli that do not normally cause pain (e.g., heat, touch / pressure) may cause pain (i.e., allodynia) or noxious stimuli may induce an exaggerated pain perception (i.e., hyperalgesia) in response to normal painful stimuli. Furthermore, normal stimuli may cause sensations similar to electrical tingling or shocks or "numbness" (i.e., paresthesia) and / or unpleasant sensations (i.e., dysesthesias). Breakthrough pain is an exacerbation of existing chronic pain. Hyperalgesia is a pain syndrome caused by an abnormal pain response to a stimulus that is often repetitive and associated with an increase in the pain threshold, which is considered the minimum pain that the patient can identify as a painful experience.

[0131] Examples of neuropathic pain include tactile allodynia (e.g., induced after nerve injury), neuralgia (e.g., postherpetic (or postherpetic) neuralgia, trigeminal neuralgia), reflex sympathetic dystrophy / causalgia (nerve trauma), cancer pain (e.g., pain due to the cancer itself or associated conditions such as inflammation, or pain due to treatments such as chemotherapy, surgery, or radiation therapy), phantom limb pain, entrapment neuropathy (e.g., carpal tunnel syndrome), and neuropathies such as peripheral neuropathy (e.g., diabetes, AIDS, chronic alcohol use, exposure to other toxins (including many chemotherapy drugs), vitamin deficiencies, and various other diseases). Neuropathic pain includes pain caused by pathological operations of the nervous system following nerve injury from various causes (e.g., surgery, wounds, shingles, diabetic neuropathy, leg or arm amputation, cancer, etc.). Diseases associated with neuropathic pain include traumatic nerve injury, stroke, multiple sclerosis, syringomyelia, spinal cord injury and cancer.

[0132] Pain-inducing stimuli often induce an inflammatory response, which may itself cause pain. Pain may also be caused by a complex mixture of nociceptive and neuropathic factors. For example, chronic pain usually includes a mixture of inflammatory nociceptive pain or neuropathic pain, or both. An initial nervous system dysfunction or injury may cause neural release of inflammatory mediators and subsequent neuropathic inflammation. For example, migraine headaches may manifest as a mixture of neuropathic and nociceptive pain. Additionally, myofascial pain may be secondary to nociceptive input from muscle, while abnormal muscle activity may be the result of nervous system disease.

[0133] In some embodiments, the long-acting NGF polypeptides described herein have reduced or no activity in inducing pain in a subject compared to the corresponding NGF portion without the Fc fusion, wild-type NGF, or other NGF-Fc fusion proteins not described herein (hereinafter referred to as "control NGF constructs"). In some embodiments, the pain is acute pain, short-term pain, persistent or chronic nociceptive pain, or persistent or chronic neuropathic pain. In some embodiments, the long-acting NGF polypeptides described herein cause at least 10% less pain compared to a control NGF construct (e.g., wild-type β-NGF), and cause at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 100% less pain compared to a control NGF construct. In some embodiments, the long-acting NGF polypeptides described herein do not cause pain when administered to a subject.

[0134] Pain-inducing activity can be measured by any method known in the art, such as those described in WO2017157325, WO2017157326 and CN108727486A, the entire contents of which are incorporated herein by reference. In some embodiments, pain is measured by pain threshold. The higher the pain threshold, the less pain.

[0135] For example, pain-inducing activities can be measured by asking patients to rate the quality and intensity of pain experienced on various rating scales. A verbal pain rating scale describes the degree of pain as no pain, mild pain, moderate pain, and severe pain verbally, as scores of 0 to 3, respectively. Alternatively, patients may be asked to rate their pain according to a numerical pain rating scale ranging from 0 (no pain) to 10 (worst pain). A visual analog scale (VAS), with a vertical or level line on which there are words representing pain from no pain to the pain considered to be the worst, asks patients to place a mark on a point that represents the level of pain at that time. According to the McGill Pain Index, patients can describe both the quality and intensity of pain by selecting the word that best describes their pain from a series of short lists, such as punching, burning, pinching, etc. In adults who have difficulty using the VAS or numerical scales (e.g., FACES facial or non-verbal patients), other pain rating scales, such as behavioral rating scales, can be used. Functional activity scores reflect the degree to which pain interferes with a patient by asking the patient to perform tasks related to the painful area. These types of rating scales are used to improve pain scores. For example, improved pain scores potentially indicate that the test NGF construct (e.g., the sustained-release NGF polypeptide described herein) causes less pain side effects compared to the control NGF construct.

[0136] In some embodiments, the pain-inducing activity of the sustained-release NGF polypeptides described herein can be measured in mice by the hot plate method (54-55°C) to determine the pain threshold. Briefly, after anesthetizing mice that meet the reaction conditions, a mouse sciatic nerve injury model is established by the nerve clamp method, whereas the sham-operated group is isolated without clamping the sciatic nerve. Mice are then divided into three groups: a sham-operated group, an injury control group (normal saline), and an experimental group (treated with the sustained-release NGF polypeptides described herein and / or treated with a control NGF such as mouse β-NGF). The pain threshold of each mouse is represented by the latency to lick its hind paw, which can be measured at various time points before and after surgery. % increase in pain threshold = (pain threshold 10 days after injury - pain threshold before injury) x 100% / pain threshold before injury.

[0137] The pain-inducing activity of the long-acting NGF polypeptides described herein can also be tested in mice by measuring the paw flexion response of the mice to mechanical stimulation to determine the pain threshold, and can be measured under short-term and long-term pain-inducing conditions. Briefly, mice that meet the responsive conditions are subcutaneously injected with the vectors described herein or the long-acting NGF polypeptides described herein (or control NGF, such as mouse β-NGF; performed at various concentrations), and then the paw flexion response to mechanical stimulation after injection (e.g., at different time points) is measured to reflect the pain threshold after treatment.

[0138] The pain-inducing activity of the long-acting NGF polypeptide described herein can also be tested by behavioral assay.For example, administer the vector described herein or the long-acting NGF polypeptide (or control NGF such as mouse β-NGF; carried out at various concentrations) to the joints of rats, then detect whether the test drug causes pain by recording the duration of leg lifting and the number of leg lifting after administration (e.g., at different time points), and calculate the total duration of leg lifting.A shorter total time of leg lifting means less pain-inducing activity. stability

[0139] In some embodiments, the sustained NGF polypeptides described herein have good stability, such as physical stability, chemical stability and / or biological stability. In some embodiments, the sustained NGF polypeptides described herein have good thermal stability, such as high melting temperature (Tm) and / or high aggregation onset temperature (Tagg). In some embodiments, the sustained NGF polypeptides described herein have good stability under accelerated stress (e.g., high temperature), such as low or no fragmentation, aggregate formation and / or increase in aggregates.

[0140] Protein stability, particularly susceptibility to aggregation, depends primarily on the conformational and colloidal stability of the protein molecule. The first step in the aggregation of non-native proteins, the most common form of aggregation, is believed to be a slight perturbation of the molecular structure, e.g., partial unfolding, i.e., conformational change, of the protein, and is determined by the conformational stability of the protein. In the second step, the partially unfolded molecules come into close proximity to form aggregates, driven by diffusion and random Brownian motion. The second step is determined primarily by the colloidal stability of the molecule (see Chi et al., Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony stimulating factor. Protein Science, 2003 May;12(5):903-913). As used herein, the term "stability" generally refers to maintaining the integrity of a bioactive substance (e.g., a protein) or minimizing degradation, denaturation, aggregation, or unfolding. As used herein, "improved stability" means that a protein of interest (e.g., a sustained-release NGF polypeptide described herein) is more stable than a control protein (e.g., another NGF-Fc fusion protein) under conditions that may cause degradation, denaturation, aggregation, or unfolding.

[0141] Differential scanning calorimetry (DSC) and differential scanning fluorescence (DSF) are well known in the art as tools for predicting the stability of protein formulations, and specifically can be used to determine the unfolding temperature (Tm) of a protein in a given formulation. It is standard practice in the art to correlate high quality Tm measurements of a protein in a given formulation with reliable and stable protein formulations available for long-term storage stability.

[0142] A "stable" protein (or formulation), such as a sustained release NGF polypeptide as described herein, substantially retains its physical and / or chemical stability and / or biological activity during manufacture and / or storage. There are various analytical techniques in the art for measuring protein stability, as reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90. For example, in one embodiment, protein stability is determined based on the percentage of monomeric protein in solution, and has a relatively low percentage of degraded (e.g., fragmented) and / or aggregated protein. Preferably, the protein (or formulation) is stable at room temperature (about 30° C.) or 40° C. for at least one month, and / or stable for at least six months, or at least one year, or at least two years at about 2-8° C. Furthermore, the protein (or formulation) is preferably stable after freezing (e.g., at −70° C.) and thawing (hereinafter referred to as a “freeze-thaw cycle”).

[0143] A protein, such as a sustained release NGF polypeptide as described herein, is said to "retain its physical stability" in a formulation if it is substantially free of signs of instability, such as aggregation, precipitation, and / or denaturation, as measured by visual inspection of color and / or clarity, or by ultraviolet light scattering or size exclusion chromatography. Aggregation is the process by which individual protein molecules or complexes form aggregates by covalent or non-covalent association, which may proceed to the extent that a visible precipitate is formed.

[0144] A protein, such as a sustained release NGF polypeptide as described herein, "retains its chemical stability" in a formulation if the chemical stability over a given period of time still maintains its biological activity (e.g., as described in the "Biological Activity" section above). Chemical stability can be assessed, for example, by detecting and quantifying chemically altered forms of the protein. Chemical alterations can also involve size changes (e.g., shearing) and can be assessed using size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical alterations include, for example, charge changes (e.g., deamidation or oxidation), and can be assessed, for example, by ion exchange chromatography.

[0145] A protein, such as a sustained release NGF polypeptide as described herein, can be said to "retain its biological activity" in a pharmaceutical formulation if it exhibits biological activity for a desired purpose. For example, a protein retains its biological activity in the formulation if the biological activity is 30%, 20%, or 10% (within analytical error) of the biological activity when formulated into the formulation.

[0146] It is known to those skilled in the art that the stability of a protein (e.g., a sustained-release NGF polypeptide described herein) depends on other properties than the composition of the formulation. For example, the stability may be affected by temperature, pressure, humidity, pH value, and external radiation. The stability of a protein (e.g., a sustained-release NGF polypeptide described herein) in a protein formulation can be determined in various ways. In some embodiments, the stability of a protein is determined by size exclusion chromatography (SEC). SEC separates analytes (e.g., macromolecules such as proteins) based on their hydrodynamic size, diffusion coefficient, and surface properties. Thus, for example, SEC can separate the sustained-release NGF polypeptide described herein in its native three-dimensional conformation from various denatured and / or degraded states of the protein. In SEC, the stationary phase is typically composed of inert particles packed into a dense three-dimensional matrix in a glass or steel column, and the mobile phase is pure water, aqueous buffer, organic solvent, mixtures thereof, or other solvents. The particles of the stationary phase have small wells and / or channels that only materials smaller than a certain size can enter. Thus, larger particles are removed from these pores and channels, while smaller particles are transferred from the mobile phase. The time that a particle remains immobilized in the stationary pores depends on how much of the particle can enter the pores. By being transferred from the mobile phase stream, the time required to elute from the chromatographic column increases, so that the particles become separated based on size differences. See the exemplary method provided in Example 3.

[0147] In some embodiments, a protein (e.g., a sustained-release NGF polypeptide described herein) or a fragment thereof is identified or characterized by combining SEC with an identification technique. Protein identification and characterization means are varied and include, but are not limited to, chromatographic techniques such as high performance liquid chromatography (HPLC), sodium dodecyl sulfate capillary electrophoresis (CE-SDS), immunoassays, electrophoresis, ultraviolet / visible / infrared spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy, mass spectrometry, gas chromatography, static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS protein binding.

[0148] In some embodiments, the sample formulation (e.g., comprising a sustained release NGF polypeptide described herein) and the control formulation (e.g., comprising another NGF-Fc fusion protein or standard) are optionally measured prior to the processing step to determine the content of monomer, aggregated and / or fragmented protein (and / or percentage increase in fragments, percentage increase in aggregates, etc.), as described in Example 3 below. Each protein formulation then undergoes a processing step. For example, each protein formulation is stored at a particular temperature (e.g., 40° C., 25° C., or 5° C.) for an extended period of time (e.g., 3 months, 6 months, 12 months, or more). In some embodiments, the protein formulation undergoes a physical stress test, such as an agitation stress test. In some embodiments, the protein formulation undergoes an accelerated stability test, for example, processed under accelerated stresses including high temperature (e.g., 40° C.), high humidity, and / or low pH values. In some embodiments, the protein formulation undergoes freeze-thaw cycles. In some embodiments, samples of the same protein preparation are subjected to different treatments, e.g., stored at different temperatures for a period of time. After the treatment steps, the protein preparation is measured to determine the content of monomers, aggregates, and / or fragments of the protein (and / or the percentage increase in fragments, the percentage increase in aggregates, etc.). In some embodiments, the protein preparation is treated under continuous heating, e.g., increasing the temperature from about 20° C. to about 95° C. (e.g., at a heating rate of about 0.3° C. / min), to measure the melting temperature (Tm) and / or the onset temperature of aggregation (Tagg). Tm and Tagg can be measured by changes in fluorescence absorbance and light scattering at 266 nm / 473 nm, respectively, using a fluorescent protein analyzer. The higher the Tm, the higher the thermal stability. The higher the Tagg, the less likely it is to aggregate. In some embodiments, the sustained release NGF polypeptides described herein have a Tm of 50°C or greater, e.g., a Tm of any one of 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 66°C, 67°C, 68°C, 69°C, 70°C, or 75°C.In some embodiments, the sustained release NGF polypeptides described herein have a Tagg temperature of 50°C or higher, e.g., any one of 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, or 85°C.

[0149] Stability, e.g., the physical stability of a composition or formulation, can be assessed by methods well known in the art, such as measuring the apparent light attenuation (absorbance or optical density) of a sample. This light attenuation measurement is related to the turbidity of the formulation, which is an intrinsic property of proteins dissolved in solution and is measured by nephelometry in Nephelometric Turbidity Units (NTU).

[0150] Turbidity, also referred to as the "milky whiteness" or "milky appearance" of a formulation, as a function of the concentration of one or more components (e.g., protein and / or salt) in the solution, can be calculated using a standard curve made from suspensions of known turbidity. To measure the turbidity of a pharmaceutical composition, the European Pharmacopoeia standard (European Pharmacopoeia, 4th edition, European Commission Directorate for the Quality of Medical Products (EDQM), Strasbourg, France) can be used as a reference standard. According to the European Pharmacopoeia standard, a clear solution is defined as having a turbidity equal to or less than that of a control suspension, which has a turbidity of about 3. In the absence of correlation or non-ideal effects, turbidity measurements usually involve the possibility of detecting Rayleigh scattering, which varies linearly with concentration. Other methods for evaluating the physical stability of pharmaceutical proteins are well known in the art, such as size exclusion chromatography or analytical ultracentrifugation.

[0151] In some embodiments, stability refers to formulations comprising the sustained release NGF polypeptides described herein having low levels of particle formation that are undetectable.As used herein, the phrase "low levels of particle formation that are undetectable" refers to samples containing less than 30 particles / ml, less than 20 particles / ml, less than 15 particles / ml, less than 10 particles / ml, less than 5 particles / ml, less than 2 particles / ml, or less than 1 particle / ml as determined by HIAC analysis or visual analysis.In some embodiments, no particles are detected in the sustained release NGF polypeptide formulations by HIAC analysis or visual analysis.

[0152] "Substantial protein aggregation" refers to a level of protein aggregation in a protein preparation that is significantly higher than the level of protein aggregation in a control protein preparation, which may be the same protein preparation before storage or processing (e.g., before being subjected to unstable conditions such as high temperature, humidity, pH, and / or long-term storage) or a different protein preparation (e.g., another NGF-Fc fusion protein, or an NGF portion not fused to Fc) measured under the same conditions.

[0153] "Substantially free of protein aggregation" means that the protein (or formulation) of the present invention does not have a significantly higher level or percentage of protein aggregation than a control formulation, for example, the protein (or formulation) has an aggregation level of less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1%. Protein aggregation levels can be determined using standard techniques known in the art, such as those described in Example 3 herein. In some embodiments, the sustained release NGF polypeptides described herein are substantially free of protein aggregation (e.g., in accelerated stability tests). In some embodiments, the sustained release NGF polypeptide has up to 15% protein aggregation, e.g., up to 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% protein aggregation (e.g., under accelerated stability test conditions, e.g., heating). In some embodiments, the sustained release NGF polypeptide described herein is free of protein aggregation (e.g., under accelerated stability test conditions, e.g., heating). In some embodiments, the sustained release NGF polypeptide has no more than 15% increase in aggregates, e.g., no more than 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% increase in aggregates (e.g., under accelerated stability test conditions, e.g., accelerated heating). In some embodiments, the stability is measured by SEC. In some embodiments, the stability is measured by CE-SDS.

[0154] In some embodiments, stability refers to reduced fragmentation of the sustained release NGF polypeptides described herein. As used herein, the term "undetectably low fragmentation levels" refers to a sample that contains 80%, 85%, 90%, 95%, 98% or 99% or more of the total protein, e.g., a single peak as determined by HPSEC, or multiple peaks (e.g., a peak with the same number of subunits) as determined by reduced capillary gel electrophoresis (rCGE) that represent undegraded protein or undegraded fragments thereof, and does not contain other single peaks that account for more than 5%, 4%, 3%, 2%, 1%, or 0.5% of the total protein. As used herein, the term "reduced capillary gel electrophoresis" refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in Fc-containing proteins, such as the sustained release NGF polypeptides described herein. In some embodiments, the sustained release NGF polypeptide has 0%-15% fragments, e.g., 0%-12% fragments (e.g., under accelerated stability test conditions, e.g., accelerated heating). In some embodiments, the sustained release NGF polypeptide has no more than 30% fragments, e.g., no more than 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% fragments (e.g., under accelerated stability test conditions, e.g., accelerated heating). In some embodiments, the sustained release NGF polypeptide has no fragments (e.g., under accelerated stability test conditions, e.g., accelerated heating). In some embodiments, the sustained release NGF polypeptide does not exhibit an increase in fragments of more than 30%, e.g., an increase in fragments of more than 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (e.g., under accelerated stability testing conditions such as accelerated heating).In some embodiments, the sustained release NGF polypeptide has at least 75% of the main peak, for example, at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the main peak (e.g., under accelerated stability test conditions such as accelerated heating). In some embodiments, the stability is measured by SEC. In some embodiments, the stability is measured by CE-SDS. Long-acting NGF polypeptide derivative

[0155] In some embodiments, the sustained release NGF polypeptides referred to herein can be further modified to include additional non-protein moieties that are known in the art and readily available. Suitable non-protein moieties for sustained release NGF polypeptide derivatives include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxolane, ethylene / maleic anhydride copolymers, polyamic acid (homopolymer or random copolymer), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylene polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers linked to the sustained-release NGF polypeptide may vary, and when multiple polymers are linked, the polymers may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular property or function of the sustained-release NGF polypeptide to be improved, whether the sustained-release NGF polypeptide derivative is to be used for treatment under a particular condition, etc.

[0156] In some embodiments, the sustained release NGF polypeptides described herein may be coupled to any of a wide variety of chromophores, fluorophores (e.g., coumarins, xanthenes, cyanines, pyrenes, borapolyazaindacenes, oxazines, and derivatives thereof), fluorescent proteins (e.g., GFP, phycobiliproteins, and derivatives thereof), phosphorescent dyes (e.g., dioxetane, xanthene, or carbocyanine dyes, lanthanide chelates), tandem dyes (e.g., cyanine-phycobiliprotein derivatives, and xanthene-phycobiliprotein derivatives), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), haptens, enzymes (e.g., peroxidases, phosphatases, glycosidases, luciferases), and radioisotopes (e.g., 125 I, 3 H, 14 C. 32 P).

[0157] In some embodiments, the sustained-release NGF polypeptide can be further modified to include one or more biologically active proteins, polypeptides, or fragments thereof. As used herein, "biological activity" or "biological activity" are used interchangeably and refer to exhibiting biological activity in vivo to perform a specific function. For example, it means binding to a specific biomolecule, such as a protein, DNA, and promoting or inhibiting the activity of the biomolecule. In some embodiments, biologically active proteins or fragments thereof include proteins and polypeptides administered to a patient as active pharmaceutical agents for the prevention or treatment of a disease or condition, proteins and polypeptides used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro detection, and proteins and polypeptides administered to a patient to prevent a disease, such as a vaccine. In some embodiments, the biologically active protein or fragment thereof has immune stimulating / immunomodulating, membrane transport, or enzymatic activity. In some embodiments, the biologically active protein, polypeptide, or fragment thereof is an enzyme, hormone, growth factor, cytokine, or mixture thereof. In some embodiments, the biologically active protein, polypeptide, or fragment is capable of specifically recognizing a peptide of interest (eg, an antigen or other protein).

[0158] In some embodiments, the biologically active protein or fragment thereof that may be included in the sustained release NGF polypeptides described herein is an antigen-binding protein (e.g., an antibody). In some embodiments, the biologically active protein or fragment thereof that may be included in the sustained release NGF polypeptides described herein is an antibody mimetic, a small artificial protein that contains an antigen-binding domain reminiscent of an antibody (G Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and are composed of a single polypeptide. Antibody mimetics that may be included in the sustained-release NGF polypeptides described herein include, but are not limited to, designed ankyrin repeat proteins (DARPins; containing 3-5 fully synthetic ankyrin repeat sequences and flanked by N- and C-terminal cap domains), affinity multimers (avimers; high-affinity proteins containing multiple A domains with low affinity for the target), or anticoagulants (based on a lipid scaffold with four accessible and randomly sequenced possible loops). In some embodiments, biologically active proteins or fragments thereof that may be included in the sustained-release NGF polypeptides described herein are armadillo repeat proteins (e.g., β-catenin, α-importin, plakoglobin, adenomatous polyposis (APC)) that contain armadillo repeat units (characteristic, the repeat amino acid sequence is about 40 residues in length). Each armadillo repeat unit is composed of a pair of α-helices that form a hairpin structure. The alpha solenoid structure is formed by multiple copies of the repeat. Armadillo repeat proteins can bind to a wide variety of peptides, relying on the fixed binding mode of the peptide backbone, without the need for specific conserved side chains or interactions with the free N- or C-termini of the peptide. The possibility of recognizing peptide residue by residue, and the inherent modularity of repeat proteins, make it anticipated that armadillo repeat proteins can be used as a versatile scaffold for peptide binding. III. Vectors encoding sustained-release NGF polypeptides

[0159] The present invention also relates to an isolated nucleic acid encoding any of the long-acting NGF polypeptides described herein, a vector comprising a nucleic acid encoding any of the long-acting NGF polypeptides described herein. The present invention also relates to an isolated host cell (e.g., CHO cell, HEK 293 cell, Hela cell or COS cell) comprising a nucleic acid encoding any of the long-acting NGF polypeptides described herein, or a vector comprising a nucleic acid encoding any of the long-acting NGF polypeptides described herein. In some embodiments, the isolated nucleic acid further encodes a signal peptide sequence (e.g., SEQ ID NO: 6) at the N-terminus of the long-acting NGF polypeptide. In some embodiments, the isolated nucleic acid further encodes a propeptide sequence (e.g., SEQ ID NO: 5) at the N-terminus of the long-acting NGF polypeptide. In some embodiments, the isolated nucleic acid further encodes a signal peptide sequence (e.g., SEQ ID NO: 6) followed by a propeptide sequence (e.g., SEQ ID NO: 5) at the N-terminus of the long-acting NGF polypeptide.

[0160] Thus, in some embodiments, the present invention relates to an isolated nucleic acid encoding a sustained-release NGF polypeptide comprising an NGF portion comprising (or consisting essentially of, or consisting of) an amino acid sequence shown in any one of SEQ ID NOs: 1 to 4 (e.g., any one of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, and an Fc portion derived from IgG1 Fc or IgG4 Fc. In some embodiments, the present invention relates to an isolated nucleic acid encoding a sustained-release NGF polypeptide comprising (or consisting essentially of, or consisting of) an amino acid sequence shown in any one of SEQ ID NOs: 61 to 67. In some embodiments, the isolated nucleic acid further comprises a nucleic acid sequence encoding a signal peptide sequence of SEQ ID NO: 6 at the 5'-terminus. In some embodiments, the isolated nucleic acid further comprises a nucleic acid sequence encoding a propeptide sequence of SEQ ID NO: 5 at the 5'-terminus. In some embodiments, the isolated nucleic acid (from 5' to 3') comprises a nucleic acid sequence encoding a signal peptide sequence of SEQ ID NO: 6 followed by a nucleic acid sequence encoding a propeptide sequence of SEQ ID NO: 5 located at the 5'-terminus. The amino acid sequence is further included.

[0161] In some embodiments, the present invention relates to an isolated nucleic acid encoding a sustained-release NGF polypeptide comprising (or consisting essentially of, or consisting of) the amino acid sequence set forth in any one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, and 46. In some embodiments, the present invention relates to an isolated nucleic acid encoding a sustained-release NGF polypeptide comprising (or consisting essentially of, or consisting of) the amino acid sequence set forth in any one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, and 46, without the signal peptide sequence of SEQ ID NO: 6. In some embodiments, the present invention relates to an isolated nucleic acid comprising (or consisting essentially of, or consisting of) the nucleic acid sequence set forth in any one of SEQ ID NOs: 33, 35, 37, 39, 41, 43, and 45.

[0162] In some embodiments, the vector comprising the nucleic acid encoding any of the sustained-release NGF polypeptides described herein is suitable for replication and integration in eukaryotic cells, such as mammalian cells (e.g., CHO cells, HEK 293 cells, Hela cells, COS cells). In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector, such as pTT5.

[0163] A number of virus-based systems have been developed to introduce genes into mammalian cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, herpes simplex viral vectors, and derivatives thereof. Viral vector technology is well known in the art and is detailed, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other handbooks of virology and molecular biology. Retroviruses provide a convenient platform for gene delivery systems. Using techniques known in the art, heterologous nucleic acid can be inserted into vectors and packaged into retroviral particles. Recombinant viruses can then be isolated and delivered to genetically engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, a self-inactivating lentiviral vector carrying the protein coding sequence of the construct can be packaged using experimental methods known in the art. The resulting lentiviral vector can be used to transduce mammalian cells using methods known in the art. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transduction because they allow long-term and stable integration of the transgene and propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0164] In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a pTT5 vector. In some embodiments, the vector is a transposon, such as the Sleeping Beauty (SB) transposon system or the PiggyBac transposon system. In some embodiments, the vector is a polymer-based non-viral vector, such as poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly(ethyleneimine) (PEI), or a dendrimer. In some embodiments, the vector is a cationic lipid-based non-viral vector, such as cationic liposomes, lipid nanoemulsions, and solid lipid nanoparticles (SLN). In some embodiments, the vector is a peptide-based non-viral gene vector, such as poly-L-lysine. Any known non-viral vector suitable for genome editing can be used to introduce the nucleic acid encoding the long-acting NGF polypeptide into the host cell. See, for example, Yin H. et al., Nature Rev. Genetics (2014) 15:521-555; Aronovich EL et al., "The Sleeping Beauty transposon system: a non-viral vector for gene therapy." Hum. Mol. Genet. (2011) R1: R14-20 and Zhao S. et al., "PiggyBac transposon vectors: the tools of the human gene editing." Transl. Lung Cancer Res. (2016) 5(1):120-125, which are incorporated herein by reference. In some embodiments, any one or more nucleic acids or vectors encoding the sustained-release NGF polypeptides described herein are introduced into a host cell (e.g., CHO, HEK 293, Hela, or COS) by physical means, including, but not limited to, electroporation, sonoporation, photoporation, magnetofection, and hydroporation.

[0165] In some embodiments, the vector comprises a selectable marker gene or a reporter gene for selecting cells expressing the long-acting NGF polypeptide described herein from a population of host cells transfected with the vector (e.g., lentiviral vector, pTT5 vector). Both the selectable marker and the reporter gene can be surrounded by appropriate regulatory sequences so that they can be expressed in the host cell. For example, the vector may comprise transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the nucleic acid sequence.

[0166] The nucleic acid can be cloned into the vector by any molecular cloning method known in the art, for example, using a restriction endonuclease site and one or more optional markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been developed to drive gene expression in prokaryotic or eukaryotic cells (e.g., mammalian cells). Any promoter known in the art can be used in the present invention. Promoters are broadly classified into constitutive promoters and regulatable promoters, such as inducible promoters.

[0167] In some embodiments, the nucleic acid encoding the sustained-acting NGF polypeptide described herein is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also known as a transgene) to be constitutively expressed in a host cell. Examples of promoters herein include, but are not limited to, CMV promoter (CMV), human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), chicken β-actin promoter associated with CMV early enhancer (CAGG), Rous sarcoma virus (RSV) promoter, polyoma enhancer / herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, and myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer binding site substitution (MND) promoter. The efficiency with which these constitutive promoters drive the expression of transgenes has been widely compared in many studies. In some embodiments, a nucleic acid encoding a long-acting NGF polypeptide described herein is operably linked to a CMV promoter.

[0168] In some embodiments, the nucleic acid encoding the sustained-acting NGF polypeptide described herein is operably linked to an inducible promoter. Inducible promoters belong to the category of regulated promoters. Inducible promoters can be induced by one or more conditions, such as a physical condition, a host cell microenvironment or a host cell physiological state, an inducing agent (i.e., an inducing drug), or a combination thereof. In some embodiments, the inducing condition does not induce the expression of an endogenous gene in the host cell. In some embodiments, the inducing condition is selected from an inducing agent, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, and an activation state of the host cell. In some embodiments, the inducible promoter is an NFAT promoter, TETON promoter, or a combination thereof. R The promoter may be a NFκB promoter. IV. Manufacturing method

[0169] The present invention also relates to a method for making any of the long-acting NGF polypeptides described herein.Thus, in some embodiments, the method for preparing a long-acting NGF polypeptide comprises the steps of: (a) culturing a host cell (e.g., CHO cell, HEK 293 cell, Hela cell or COS cell) comprising a nucleic acid or vector encoding any of the long-acting NGF polypeptides described herein under conditions that allow effective expression of the encoded long-acting NGF polypeptide; and (b) obtaining the expressed long-acting NGF polypeptide from said host cell.In some embodiments, step (a) further comprises producing a host cell comprising a nucleic acid or vector encoding the long-acting NGF polypeptide described herein.The long-acting NGF polypeptides described herein can be made using any method known in the art or described herein.For an exemplary method, see Example 1.

[0170] In some embodiments, the long-acting NGF polypeptide described herein is expressed in eukaryotic cells, such as mammalian cells. In some embodiments, the long-acting NGF polypeptide described herein is expressed in prokaryotic cells. When the long-acting NGF polypeptide described herein is expressed in prokaryotic cells, the produced proNGF-(optional linker)-Fc part cannot be processed into a propeptide sequence. Therefore, when used for prokaryotic cell expression, the nucleic acid encoding the long-acting NGF polypeptide can be designed without the nucleic acid sequence encoding the propeptide sequence (e.g., SEQ ID NO: 5). 1. Recombinant products of prokaryotic cells a) Vector construction

[0171] Polynucleic acid sequences encoding the protein constructs of the present invention can be obtained using standard recombinant techniques. Polynucleotides can be synthesized using nucleotide synthesizers or PCR techniques. Once a sequence encoding a polypeptide is obtained, it is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors known in the art can be used in the present invention. The vector is appropriately selected primarily based on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transfected with the vector. Each vector contains various components depending on the function of the vector (amplification or expression of heterologous polynucleotides, or both) and the compatibility of the vector with the particular host cell in which it resides. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[0172] Generally, plasmid vectors contain replicon and control sequences derived from species compatible with the host cell and are used with these host cells. Vectors usually have a replication site and marker sequences capable of providing phenotypic selection in transformed cells. For example, E. coli is commonly transformed with pBR322, a plasmid derived from E. coli. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, providing an easy way to identify transformed cells. pBR322, its derivatives, or other bacterial plasmids or phages may contain or be modified to contain promoters used by the microorganism to express endogenous proteins. Examples of pBR322 derivatives used to express specific antibodies are detailed in Carter et al., US Pat. No. 5,648,237.

[0173] In addition, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transformation vectors with these host cells. TMPhages such as -11 may be used to generate recombinant vectors that are used to transform susceptible host cells such as E. coli LE392.

[0174] A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that controls downstream gene expression. Prokaryotic promoters are generally divided into two classes: inducible and constitutive. Inducible promoters are promoters that initiate and increase transcription levels of a cistron in response to changes in culture conditions (e.g., the presence or absence of nutrients or a change in temperature).

[0175] Many promoters recognized by potential host cells are well known. The promoter sequence is mobilized and isolated from the source DNA by restriction endonucleases and inserted into the vector of the present application so that it is operably linked to the cistron DNA encoding the promoter polypeptide. Either the native promoter sequence or many heterologous promoters can be used to direct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they allow more transcription and higher yields of expressed target gene compared to the native target polypeptide promoter.

[0176] Suitable promoters for prokaryotic hosts include the PhoA promoter, -galactosidase and lactose promoter systems, tryptophan (trp) promoter systems, and hybrid promoters such as the tac and trc promoters. However, other promoters functional in bacteria (e.g., other known bacterial or phage promoters) may also be applied. The nucleic acid sequences are disclosed, so that one skilled in the art can provide any desired restriction sites using linkers or aptamers and ligate them to the cistrons encoding the light and heavy chains of interest (Siebenlist et al., (1980) Cell 20:269).

[0177] In some embodiments, each cistron in a recombinant vector contains a secretory signal sequence component that directs transmembrane translocation of an expressed polypeptide. In general, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the present invention is one that can be recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In prokaryotic host cells that are unable to recognize and process the native signal sequence of a heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) leader, LamP, PhoE, PelB, OmpA and MBP.

[0178] In some embodiments, production of the protein constructs of the present application can occur in the cytoplasm of the host cell, and therefore the presence of a secretion signal sequence in either cistron is not required. In some embodiments, the polypeptide components are expressed, folded, and assembled to form the protein construct in the cytoplasm. Some host strains (e.g., E. coli trxB-strains) provide cytoplasmic conditions that favor disulfide bond formation, thereby allowing the expressed protein subunits to be properly folded and assembled. See Proba and Pullthun, Gene, 159:203 (1995). b) Prokaryotic host cells

[0179] Suitable prokaryotic host cells for expressing the proteins of the present application include archaebacteria and eubacteria, such as gram-negative or gram-positive bacteria. Examples of bacteria that can be used include Escherichia coli, Bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobia, Vitreosilla, and Paracoccus. In some embodiments, gram-negative cells are used. In some embodiments, Escherichia coli cells are used as hosts in the present invention. Examples of E. coli strains include the W3110 strain (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27, 325) and its derivatives, which have the following genotypes: W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc fepE) degP41 kan R Examples of suitable bacterial strains include the 33D3 strain (US Pat. No. 5,639,635) having the nucleotide sequence 1141-1141. Other strains and their derivatives may also be applied, such as E. coli 294 (ATCC 31446), E. coli B, E. coli 1776 (ATCC 31537) and E. coli RV308 (ATCC 31608). These examples are illustrative and not limiting. Methods for constructing bacterial derivatives of any of the above-mentioned known genotypes are known in the art and are detailed, for example, in Bass et al., Proteins, 8:309-314 (1990). Considering the replicability of the replicon in bacterial cells, it is usually necessary to select an appropriate bacterium. For example, when known plasmids such as pBR322, pBR325, pACYC177 or pKN410 are used to provide the replicon, E. coli, Serratia or Salmonella are used as hosts.

[0180] In general, host cells secrete minimal amounts of proteolytic enzymes, requiring appropriate addition of additional protease inhibitors to the cell culture. c) Protein Production

[0181] Host cells are transfected with the expression vectors described above and cultured in conventional nutrient media modified appropriately to induce promoters, select transformants, or amplify genes encoding the desired sequences. Transfection means introducing DNA into a prokaryotic host, allowing the DNA to replicate as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transfection is performed using standard techniques applicable to such cells. Typically, calcium treatment with calcium chloride is used for bacterial cells with extensive cell wall barriers. Alternative transfection methods include the use of polyethylene glycol / dimethyl sulfoxide, electroporation.

[0182] Prokaryotic cells used to produce the protein constructs of the invention are grown in media known in the art and suitable for the culture of the selected host cells. Suitable media include Luria Broth (LB) and necessary nutritional supplements. In some embodiments, the media further includes a selection agent selected based on the construction of the expression vector to selectively allow growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the media for the growth of cells expressing an ampicillin resistance gene.

[0183] In addition to the carbon source, nitrogen source and inorganic phosphate source, any necessary supplements may also be added to the medium in appropriate concentrations, either alone or in mixture with other supplements or media (e.g., complex nitrogen sources). Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol. The prokaryotic host cells are cultured at a suitable temperature. For example, for the growth of E. coli, the temperature range is preferably 20°C to 39°C, more preferably 25°C to 37°C, and even more preferably 30°C. The pH value of the medium is mainly determined by the host organism, but may be any pH value between 5 and 9. For E. coli, the pH value is preferably 6.8 to 7.4, more preferably 7.0.

[0184] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter. In one embodiment of the present invention, the PhoA promoter is used to control the transcription of the polypeptide. Thus, the transformed host cell is induced by culturing in a phosphate-limiting medium. The phosphate-limiting medium is preferably CRAP medium (see Simmons et al., J. Immunol. Methods (2002), 263:133-147). However, as known in the art, various other inducers known in the art may be used depending on the vector structure used.

[0185] The protein constructs expressed in the present invention are secreted into the periplasm of the host cells and recovered therefrom. Recovery of the protein usually involves disrupting the microorganisms by means such as osmotic shock, sonication, lysis, etc. Once the cells are disrupted, cell fragments or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported to the medium and isolated there. The cells can be removed from the medium and the medium supernatant can be filtered and concentrated to further purify the produced protein. The expressed polypeptides can be further isolated and identified by common methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot tests.

[0186] Alternatively, proteins are produced on a large scale through fermentation processes. Various large-scale fed-batch fermentation procedures are available for recombinant protein production. Large-scale fermentation has a volume of at least 1000 liters, preferably 1000-100000 liters, and uses fermenters with impellers to distribute oxygen and nutrients, especially glucose (carbon / energy preferred). Small-scale fermentation generally refers to fermentation in fermenters with a volume of 100 liters, ranging from 1 liter to 100 liters.

[0187] During fermentation, induction of protein expression usually begins after cells have grown to a desired density under appropriate conditions, e.g., at OD 550 The cells are in early stationary phase when the ΔΨ is about 180-220. Depending on the vector construct used, various inducers known in the art and described above can be used. Prior to induction, the cells can be grown for a short period of time. Typically, the cells are induced for about 12-50 hours, although longer or shorter induction times may be used.

[0188] Various fermentation conditions can be modified to improve the yield and quality of the protein constructs of the present invention. For example, to improve the correct assembly and folding of secreted polypeptides, the host prokaryotic cells can be co-transformed with additional vectors to overexpress chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD or DsbG) and FkpA (peptidyl prolyl cis-trans isomerase with chaperone activity). Chaperone proteins have been shown to be useful in promoting the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al.,(1999)J Bio Chem 274:19601-19605;Georgiou et al.,USPat.No.6,083,715;Georgiou et al. al.,USPat.No.6,027,888;Bothmann and Pluckthun(2000)J.Biol.Chem.275:17100-17105;Ramm and Pluckthun(2000)J.Biol.Chem.275:17106-17113;Arie et al.,(2001)Mol.Microbiol.39:199-210.

[0189] To minimize hydrolysis of expressed heterologous proteins, especially proteolytically sensitive proteins, certain host strains lacking proteolytic enzymes can be used in the present invention. For example, the host cell strain can be engineered to allow for genetic mutations in genes encoding known bacterial proteases, such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof. Also, several E. coli protease-deficient strains can be used, as detailed in Joly et al., (1998), supra; Georgiou et al., US Pat. No. 5,264,365; Georgiou et al., US Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0190] As host cells in expression systems encoding the protein constructs described in this invention, E. coli strains that are deficient in proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins can be used. d) Protein purification

[0191] The protein constructs produced by the present invention can be further purified using standard protein purification techniques known in the art to obtain substantially homogeneous preparations for further analysis and use. Examples of suitable purification procedures include fractionation through immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase liquid chromatography (HPLC), silica or cation exchange resin (e.g., DEAE) chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and filtration through gels such as dextran gel G-75.

[0192] In some embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of protein constructs containing an Fc region as described herein. Protein A is a 42 kDa surface protein from Staphylococcus aureus that has high binding affinity for Fc-containing structures, such as the sustained release NGF polypeptides described herein. Lindmark et al., (1983) J. Immunol. Meth. 62:1-1. The solid phase for immobilizing Protein A is preferably a column containing a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the chromatographic column is coated with a reagent such as glycerol to prevent nonspecific attachment of contaminants. The solid phase is then washed to remove contaminants nonspecifically bound to the solid phase. Finally, the protein construct of interest is recovered from the solid phase by elution. 2. Eukaryotic recombinant products

[0193] For eukaryotic expression, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. a) Signal sequence element

[0194] Vectors for eukaryotic hosts may contain an insert encoding a signal sequence or other polypeptide that has a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For mammalian cell expression, mammalian signal sequences and viral secretory leaders, such as the herpes simplex gD signal, are available. The DNA of this precursor region is ligated in reading frame to the DNA encoding the protein construct of the present application. b) Origin of replication

[0195] Generally, the origin of replication element is not needed for mammalian expression vectors (although the SV40 origin is often used only because it contains the early promoter). c) Selection Gene Element

[0196] Expression and cloning vectors can contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement an auxotrophic deficiency, or (c) provide a vital nutrient that cannot be provided in complex media, such as the gene encoding Bacillus D-alanine racemase.

[0197] An example of a selection scheme is the use of drugs to inhibit the growth of host cells. Those cells that are successfully transfected with a heterologous gene survive the selection scheme because they produce a protein that confers drug resistance. Examples of such dominant selection include the drugs neomycin, mycophenolic acid, and hygromycin.

[0198] Further examples of suitable selectable markers for mammalian cells are selectable markers that allow the identification of cells competent to take up nucleic acid encoding the protein constructs described in this application, such as DHFR, thymidine kinase, metallothionein-I and -II, and preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0199] For example, for cells transfected with the DHFR selection gene, all transformants are identified by culturing in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a Chinese hamster ovary (CHO) cell line lacking DHFR activity (e.g., ATCC CRL-9096) can be used as the host cell.

[0200] Alternatively, the DNA sequence encoding the polypeptide, wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), and co-transfected host cells (particularly endogenous DHFR-containing wild-type hosts) may be used to select by growing the cells in medium containing a selectable marker, e.g., an aminoglycoside antibiotic such as kanamycin, neomycin, or G418. See US Pat. No. 4,965,199. d) Promoter Element

[0201] Expression and cloning vectors generally contain a promoter that is recognized by the host and is operably linked to a nucleic acid encoding the desired polypeptide sequence. Most eukaryotic genes have an AT-rich region about 25-30 bases upstream from the transcription origin. Another sequence, a CNCAAT region (where N can be any nucleotide), is often found 70-80 bases upstream from the transcription origin of the gene. In addition, most eukaryotes have an AATAAA sequence at the 3' end that may be a signal for adding a poly A tail to the 3' end of the coding sequence. These sequences can be inserted into eukaryotic expression vectors. See section "III. Vectors Encoding Long-Acting NGF Polypeptides" above.

[0202] Transcription of the polypeptide in the mammalian host cell vector is controlled by a promoter, which may be, for example, a promoter derived from a viral genome such as polyoma, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and optimally Simian Virus 40 (SV40), a promoter derived from a heterologous mammalian species such as the actin promoter or an immunoglobulin promoter, or a heat shock promoter, provided that it is compatible with the host cell system.

[0203] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is disclosed in US Pat. No. 4,419,446. Improvements on this system are further detailed in US Pat. No. 4,601,978. See Reyes et al., Nature 297:598-601 (1982), in which interferon cDNA is expressed in human mouse cells under the control of the herpes simplex virus thymidine kinase promoter. Alternatively, the Rous sarcoma virus long terminal repeat sequence can be used as a promoter. e) Enhancer elements

[0204] Transcription of DNA encoding the protein construct of the present application by higher eukaryotes is usually increased by inserting an enhancer sequence into the vector. Many enhancer sequences (globin, elastase, albumin, α-fetoprotein, and insulin) have been found in mammalian genes. Usually, enhancers from eukaryotic cells viruses are used. Examples include the SV40 enhancer at the end of the replication origin (100-270 bp), the cytomegalovirus early promoter enhancer, the polyoma enhancer at the end of the replication origin, and adenovirus enhancers. See Yaniv, Nature 297:17-18 (1982) for enhancing elements that activate eukaryotic promoters. The enhancer can be spliced ​​into the vector 5' or 3' to the polypeptide coding sequence, but is preferably located 5' from the promoter. f) Transcription termination element

[0205] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells of other multicellular organisms) further contain sequences necessary for the transcription termination and stabilization of mRNA. These sequences are usually available from the 5' and occasionally the 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide fragments which are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding a polypeptide. A suitable transcription termination element is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein. g) Host cell selection and transfection

[0206] Suitable host cells for cloning or expressing the DNA in the vectors described herein include the higher eukaryotic cells described herein, such as vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); COS fibroblast-like cell lines derived from monkey kidney tissue; human embryonic kidney lines (293 or 293 cell subclones for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC-ccl2); canine kidney cells (MDCK, ATCC-ccl34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells, and a human hepatoma cell line (Hep G2).

[0207] Host cells are transfected with the above-described expression or cloning vectors to produce the protein constructs and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the desired sequences. h) Cultivation of host cells

[0208] The host cells used to produce the protein constructs of the present invention can be cultured in a variety of media. Commercially available media include Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma), etc., which are suitable for culturing the host cells. In addition, as the medium for the host cells, any medium described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655 or 5,122,469, WO 90 / 03430, WO 87 / 00195 or US Pat. Re. 30,985 can be used. These media may contain, as necessary, hormones and / or other growth factors (e.g., insulin, transferrin, epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, phosphate), buffers (e.g., HEPE), nucleotides (e.g., adenosine, thymidine), antibiotics (e.g., GENTAMYCIN, TM The culture medium may be supplemented with nutrients, such as nutrient solutions, drugs, trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source, and any other necessary supplements may be added at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, will be those used for expression in the host cells, as will be apparent to those skilled in the art. i) Protein purification

[0209] When using recombinant techniques, the protein constructs of the invention may be produced intracellularly in the periplasm or may be secreted directly into the medium. If the protein constructs are produced intracellularly, isolation is first achieved by removing particulate fragments (i.e., host cells or lysed fragments) by centrifugation or ultrafiltration. A procedure for isolating antibodies secreted into the periplasm of E. coli is detailed in Carter et al., Bio / Technology 10:163-167 (1992). Briefly, cell bodies are lysed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes, and cell fragments are removed by centrifugation. If the protein constructs are secreted into the medium, the supernatants of such expression systems are usually first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration devices. Any of the above procedures may include the addition of protease inhibitors such as PMSF to inhibit proteolysis and / or antibiotics to prevent the growth of adventitious contaminants.

[0210] Protein compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, preferably affinity chromatography purification techniques. Depending on the species and subtype of immunoglobulin Fc domain present in the Fc-containing protein construct, protein A may be applied as an affinity ligand. Protein A can be used to purify Fc-containing proteins based on human immunoglobulins containing one, two or four heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for mouse subtypes and human type 3 (Guss et al., EMBO J. 5:15671575 (1986)). Agarose is usually used as a matrix for binding affinity ligands, but other matrices may also be used. Mechanically stable matrices (e.g., controlled pore glass and poly(styrene-divinyl)benzene) can be used to purify protein constructs containing CH3 domains, allowing faster flow rates and shorter processing times than agarose. Bakerbond ABXTM resin can be used to purify protein constructs containing CH3 domains (JT Baker, Phillipsburg, NJ). Depending on the protein constructs to be recovered, ion exchange column fractionation, ethanol precipitation, reversed phase liquid chromatography (HPLC), silica gel chromatography, heparin SEPHAROSE, etc. may also be used. TM Other protein purification techniques, such as chromatography, anion or cation exchange resins (eg, polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, may also be used.

[0211] After any pre-purification steps, the mixture containing the protein construct of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, such as using an elution buffer with a pH value of about 2.5 to 4.5, preferably performed at a low salt concentration (e.g., 0 to 0.25 M salt). V. Pharmaceutical Compositions

[0212] Furthermore, the present invention relates to a pharmaceutical composition comprising any of the sustained-release NGF polypeptides described herein, and optionally a pharma- ceutical acceptable carrier and / or excipient. Thus, in some embodiments, the present invention relates to a pharmaceutical composition comprising a sustained-release NGF polypeptide comprising an NGF portion comprising an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 from the N-terminus to the C-terminus, and an Fc portion derived from IgG1 Fc or IgG4 Fc, and optionally a pharma- ceutical acceptable carrier. The pharmaceutical composition can be prepared by mixing the sustained-release NGF polypeptides described herein having the desired purity in the form of a lyophilized formulation or an aqueous solution, and optionally a pharma- ceutical acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0213] The recombinant formulation can be prepared by dissolving the lyophilized long-acting NGF polypeptide in a diluent to uniformly disperse the protein. Examples of pharma- ceutically acceptable (safe and non-toxic when administered to humans) diluents for use in the present invention include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution, or aqueous solutions of salts and / or buffers.

[0214] In some embodiments, the pharmaceutical composition comprises a homogeneous population of the long-acting NGF polypeptides described herein. By homogeneous population, it is meant that the long-acting NGF polypeptides are identical to each other, for example, the same long-acting NGF polypeptide structure, the same NGF portion, the same linker (if any), and the same Fc portion. In some embodiments, the long-acting NGF polypeptides in the pharmaceutical composition are at least 70% homogeneous (for example, at least any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%).

[0215] In some embodiments, the pharmaceutical composition consists essentially of (e.g., consists of) a sustained-release NGF polypeptide described herein and optionally a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition does not include a proNGF-Fc or preproNGF-Fc fusion protein. In some embodiments, the pharmaceutical composition includes up to 5% (e.g., up to any of 4%, 3%, 2%, and 1%) of a proNGF-Fc or preproNGF-Fc fusion protein. In some embodiments, the pharmaceutical composition does not include host cell (e.g., CHO) proteins.

[0216] The pharmaceutical composition is preferably stable, and the protein contained therein essentially retains its physical stability, chemical stability and integrity during storage. To measure protein stability, various analytical techniques are available in the art, as reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991); Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability may be measured at a selected temperature and for a selected time. For accelerated screening, stability may be measured during storage of the formulation at 40°C for 2 weeks to 1 month. If the formulation is stored at 2-8°C, the formulation should generally be stable at 30°C or 40°C for at least 1 month and / or stable at 2-8°C for at least 2 years. When the formulation is stored at 30° C., the formulation should generally be stable at 30° C. for at least 2 years and / or at least 6 months at 40° C. An indicator of protein stability can be, for example, the degree of aggregation during storage. In some embodiments, a stable formulation of the sustained release NGF polypeptide described herein may contain less than 10% (preferably less than 5%) of the sustained release NGF polypeptide described herein.

[0217] In some embodiments, the pharmaceutical composition has a shelf life of at least 15 days, such as at least 20 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or more, at, for example, 2-25° C. (e.g., 2-8° C.). As used herein, "shelf life" refers to a pharmaceutical formulation that has minimal degradation (e.g., no more than 5% degradation, specifically no more than 4%, 3%, or 2% degradation) of an active ingredient, such as a therapeutic protein (e.g., a sustained-release NGF polypeptide described herein), during the storage period when the pharmaceutical formulation is stored under a particular storage condition, e.g., at 2-8° C. Exemplary techniques for assessing the stability of a protein or formulation include size exclusion chromatography (SEC)-HPLC to detect aggregation, reversed-phase liquid chromatography (RP)-HPLC to detect protein fragments, ion exchange HPLC to detect changes in protein charge, mass spectrometry, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy to detect changes in protein conformation. These techniques can be used alone or in combination to assess protein degradation in pharmaceutical formulations and determine the shelf life of the formulations. In some embodiments, the pharmaceutical formulations of the invention exhibit no more than 5% (e.g., no more than 4%, 3%, 2%, or 1%) degradation (e.g., fragmentation, aggregation, or unfolding) within at least 15 days (e.g., at least 20 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, or more) when stored at 2-8°C.

[0218] Acceptable vectors, excipients, or stabilizers are those that are non-toxic to subjects at the dosages and concentrations employed and include, for example, buffers; antioxidants such as ascorbic acid, methionine, vitamin E, sodium metabisulfite, etc.; preservatives, isotonicity agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., zinc-protein complexes); chelating agents such as EDTA, and / or non-ionic surfactants.

[0219] Examples of physiologically acceptable vectors include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than 10 residues); proteins such as serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, and dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or TWEEN. TM Polyethylene glycol (PEG), PLURONICS TM and other nonionic surfactants.

[0220] Buffers are used to control the pH value within a range that optimizes the therapeutic effect, especially when stability is dependent on the pH value. The buffer is preferably present in a concentration range of about 50 mM to about 250 mM. Suitable buffers for the present application include organic and inorganic acids and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, or acetate. Additionally, the buffer may be a histidine or trimethylamine salt, such as Tris.

[0221] Preservatives are added to prevent microbial growth and are usually present in the range of 0.2% to 1.0% (w / v). For example, the addition of a preservative can facilitate the production of a formulation for multiple uses (multiple doses). Preservatives suitable for the present application include, for example, octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzalkonium chloride (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butanol, or benzyl alcohol; alkyl parabens such as methyl and propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0222] Tonicity agents, also called "stabilizers", are used to adjust and maintain the tonicity of the liquid in the composition. When used with large charged biomolecules (e.g., proteins), tonicity agents are often called "stabilizers" because they interact with the charged groups on the amino acid side chains, reducing the possibility of inter- and intra-molecular interactions. Tonicity agents may be present in any amount between 0.1% and 25% (by weight), preferably 1% and 5%, taking into account the relative amounts of other ingredients. Tonicity agents are preferably polyhydric sugar alcohols, more preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0223] Other excipients include one or more of the following: (1) bulking agents, (2) dissolution enhancers, (3) stabilizers, and (4) agents to prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (as described above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myo-inositose, galactose, galactitol, glycerin, cyclohexanol (e.g., inositol), and polyethylene glycol. or sugar alcohols; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, and other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin and dextran.

[0224] The presence of a non-ionic surfactant or detergent (also called a "wetting agent") contributes to the solubilization of the protein and protects it from aggregation due to agitation. This allows the formulation to be exposed to shear surfaces without denaturing the active protein. The non-ionic surfactant is present in the range of 0.05 mg / ml to 1.0 mg / ml, preferably 0.07 mg / ml to 0.2 mg / ml.

[0225] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC R Polyol, TRITON R , polyoxyethylene sorbitan monoether (TWEEN R -20, TWEEN R-80, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents include sodium dodecyl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0226] The pharmaceutical composition must be sterile in order to be used for in vivo administration. The pharmaceutical composition can be made sterile by filtration through a sterile filter. The pharmaceutical composition is usually placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0227] It can be prepared as a sustained release formulation. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (US Pat. No. 3,773,919), copolymers of L-glutamic acid and L-ethyl glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT TM (injectable microspheres composed of poly(lactic-co-glycolic acid) and leuprolide acetate), and poly-D-(-)-3-hydroxybutyrate.

[0228] Depending on the needs of the particular indication being treated, the pharmaceutical compositions described herein may contain more than one active compound, preferably compounds with complementary activities that do not adversely affect each other. Such molecules are combined in appropriate amounts to achieve the desired purpose.

[0229] The active ingredient may also be encapsulated in, for example, hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules in microcapsules prepared by gel techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences.

[0230] In some embodiments, the pharmaceutical composition is packaged in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is packaged in a reusable vial. In some embodiments, the pharmaceutical composition is packaged in bulk in a container. In some embodiments, the pharmaceutical composition is stored frozen. VI. Methods of Treating Diseases

[0231] The long-acting NGF polypeptides and compositions thereof (e.g., pharmaceutical compositions) described herein are useful in a variety of applications, including diagnosis, molecular detection, and therapy. In some embodiments, the present invention relates to a method for treating a disease (e.g., an NGF-related disease, such as a nervous system disease) in an individual (e.g., a human), comprising administering to the individual an effective amount of any long-acting NGF polypeptide or pharmaceutical composition thereof described herein. As used herein, the term "NGF-related disease" refers to any disease or disorder caused by or associated with impaired NGF receptor signaling (e.g., due to insufficient amounts of NGF and / or reduced binding affinity), or a disease or disorder that requires the biological activity of NGF for treatment (e.g., injury / injury that requires neuronal growth, maintenance, proliferation, and / or survival when treated). In some embodiments, the long-acting NGF polypeptide (or pharmaceutical composition thereof) is administered by intravenous, intramuscular, or subcutaneous injection.

[0232] Thus, in some embodiments, the present invention relates to a method for treating a disease (e.g., an NGF-related disease such as a nervous system disease (e.g., diabetic neuropathy, Alzheimer's disease, or neurotrophic keratitis) or a non-nervous system disease (e.g., premature ovarian failure or spermatogenic disorder)) in an individual (e.g., human), comprising administering to the individual an effective amount of a sustained-release NGF polypeptide (or a pharmaceutical composition thereof) comprising an NGF portion comprising (or consisting essentially of, or consisting of) an amino acid sequence shown in any one of SEQ ID NOs: 1 to 4 (e.g., any of SEQ ID NOs: 1 to 3) from the N-terminus to the C-terminus, and an Fc portion derived from IgG1 Fc or IgG4 Fc. In some embodiments, the present invention relates to a method for treating a disease (e.g., an NGF-related disease such as a nervous system disease (e.g., diabetic neuropathy, Alzheimer's disease, or neurotrophic keratitis) or a non-nervous system disease (e.g., premature ovarian failure or spermatogenic disorder)) in an individual (e.g., human), comprising administering to the individual an effective amount of a sustained-release NGF polypeptide (or a pharmaceutical composition thereof) comprising (or consisting essentially of, or consisting of) an amino acid sequence shown in any one of SEQ ID NOs: 61 to 67. In some embodiments, the sustained-release NGF polypeptide (or a pharmaceutical composition thereof) is administered by intravenous injection, intramuscular injection, or subcutaneous injection.

[0233] The methods described herein are applicable to the treatment of neurological and non-neurological diseases.

[0234] Nervous system disease refers to diseases associated with neuronal degeneration or damage in the central and / or peripheral nervous system. Specific examples of nervous system diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, amyotrophic lateral sclerosis (ALS), facial neuritis, craniocerebral or spinal trauma, acute cerebrovascular disease, brain atrophy, peripheral neuropathy, and other diseases characterized by neuronal necrosis or loss, as well as central neuron, peripheral neuron, or motor neuron damage (excluding trauma, burns, renal failure, injury, or chemical / drug-induced nerve damage, e.g., acute cerebrovascular central nerve damage caused by chemical / drug). Nervous system diseases further include peripheral neuropathy associated with certain diseases, such as diabetes, AIDS, or chemotherapy-related neuropathy. In some embodiments, the nervous system disease is selected from the group consisting of multi-infarct dementia, vascular dementia, cognitive impairment due to organic brain disease caused by alcoholism, Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's chorea, Down's syndrome, nerve deafness, Meniere's disease, stroke, ALS, Bell's palsy, diseases involving spinal muscular atrophy, diseases involving paralysis, peripheral neuropathy, nerve damage due to trauma, nerve damage due to burns, nerve damage due to renal dysfunction, nerve damage due to injury, nerve damage due to toxic side effects of chemotherapy, nerve damage due to surgery, nerve damage due to ischemia, nerve damage due to infection, nerve damage due to metabolic disease, and nerve damage due to nutritional deficiency. In some embodiments, the nervous system disease is a peripheral neuropathy selected from the group consisting of diabetic peripheral neuropathy, toxin-induced peripheral neuropathy, chemotherapy-induced peripheral neuropathy, HIV-associated peripheral neuropathy, and peripheral neuropathy affecting motor neurons.In some embodiments, the nervous system disease is selected from the group consisting of neonatal hypoxic-ischemic encephalopathy, cerebral palsy, critical illness myopathy, neurogenic deafness, recurrent laryngeal nerve injury, traumatic brain injury, dental nerve injury, stroke, Down's syndrome, ALS, multiple sclerosis, spinal muscular atrophy, diffuse brain injury, thymic dysplasia, optic nerve contusion, follicular dysplasia, spinal cord injury, glaucoma, neurotrophic keratitis, optic nerve injury, neuromyelitis optica, retina-related disease, urinary incontinence, Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, hypertensive cerebral hemorrhage neurological dysfunction, cerebral small vessel disease, acute ischemic stroke, corneal endothelial dystrophy, diabetic foot ulcer, neurogenic skin ulcer, pressure ulcer, neurotrophic corneal ulcer, diabetic corneal ulcer, and macular hole.

[0235] Non-nervous system diseases include splenic atrophy, splenic contusion, diminished ovarian reserve, premature ovarian failure (POF), ovarian hyperstimulation syndrome, ovarian remnant syndrome, follicular dysplasia, spermatogenic disorders (e.g., oligozoospermia or oligospermia, asthenozoospermia, oligoasthenozoospermia, azoospermia, teratozoospermia, oligoasthenoteratozoospermia (OAT syndrome)), ischemic ulcers, stress ulcers, rheumatic ulcers, liver fibrosis, corneal ulcers, burns, oral ulcers, and venous leg ulcers.

[0236] In some embodiments, the method for treating a disease (e.g., an NGF-related disease, such as a neurological disease (e.g., diabetic neuropathy, Alzheimer's disease, or neurotrophic keratitis) or a non-neurological disease (e.g., premature ovarian failure or spermatogenic disorders)) has one or more of the following biological activities: (i) supporting neuronal survival; (ii) promoting neurite outgrowth; (iii) enhancing neurochemical differentiation; (iv) promoting pancreatic beta cell proliferation; (v) inducing innate and / or adaptive immunity; (vi) repairing damaged nerve cells (e.g., corneal nerves) and / or preventing damage (e.g., during neurotrophic keratitis); (vii) promoting follicular cell proliferation and / or estrogen proliferation (e.g., during neurotrophic keratitis); (vii) promoting ovarian fibrosis (e.g., fibroblast growth factor (EF) and / or fibroblast growth factor (EF)). (viii) promoting secretion (e.g., during diabetic neuropathy); (ix) improving spatial awareness, memory and / or learning ability in subjects with neurodegenerative diseases (e.g., Alzheimer's disease); (x) treating and / or preventing neurodegenerative diseases; (xi) treating testicular seminiferous tubule atrophy, impaired seminiferous tubule spermatogenesis and / or epididymal duct cell fragments; (xii) relieving reduced sperm count and / or motility or increasing sperm count and / or motility (e.g., during impaired spermatogenesis); (xiii) preventing / reversing reduced follicular number and / or function or increasing follicular number and / or function (e.g., during premature ovarian failure); and / or (xiv) prolonging patient survival. In some embodiments, the methods of supporting neuronal survival mediated by sustained-release NGF polypeptides or pharmaceutical compositions described herein can achieve neuronal survival rates of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, the methods of promoting neurite outgrowth mediated by a sustained release NGF polypeptide or pharmaceutical composition described herein can promote neurite outgrowth by at least two-fold (including, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50-fold or more). In some embodiments, the methods of enhancing neurochemical differentiation mediated by a sustained release NGF polypeptide or pharmaceutical composition described herein can promote neurochemical differentiation by at least two-fold (including, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50-fold or more).In some embodiments, the methods of promoting pancreatic β-cell proliferation mediated by a sustained-release NGF polypeptide or pharmaceutical composition described herein can promote pancreatic β-cell proliferation by at least two-fold (including, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50-fold or more). In some embodiments, the methods of inducing ovulation mediated by a sustained-release NGF polypeptide or pharmaceutical composition described herein can enhance ovulation by at least two-fold (including, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50-fold or more). In some embodiments, the methods of inducing innate and / or adaptive immunity mediated by the long-acting NGF polypeptides or pharmaceutical compositions described herein can induce at least 1.1-fold (e.g., at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50-fold or more) innate and / or adaptive immunity. In some embodiments, methods of repairing and / or preventing neuronal damage mediated by a sustained-release NGF polypeptide or pharmaceutical composition described herein can repair and / or prevent at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) neuronal damage, or have at least 1.1-fold (e.g., at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50-fold or more) repair and / or prevention effect. In some embodiments, the methods of promoting ovarian granulosa cell proliferation and / or estrogen secretion mediated by the sustained-release NGF polypeptides or pharmaceutical compositions described herein can promote ovarian granulosa cell proliferation and / or estrogen secretion by at least 1.1 fold (e.g., at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 fold or more).In some embodiments, the method of promoting wound healing mediated by the sustained release NGF polypeptide or pharmaceutical composition described herein can promote wound healing by at least 1.1-fold (e.g., at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50-fold or more). In some embodiments, the method of improving spatial cognition, memory, and / or learning ability of a patient with a neurodegenerative disease (e.g., Alzheimer's disease) mediated by the sustained release NGF polypeptide or pharmaceutical composition described herein can improve spatial cognition, memory, and / or learning ability by at least 1.1-fold (e.g., including at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50-fold or more). In some embodiments, the method of treating and / or preventing neurodegenerative diseases mediated by the sustained release NGF polypeptide or pharmaceutical composition described herein can treat and / or prevent at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) of neurodegenerative diseases. In some embodiments, the method of treating testicular seminiferous tubule atrophy, seminiferous tubule spermatogenesis disorder and / or epididymal duct cell fragments mediated by the sustained release NGF polypeptide or pharmaceutical composition described herein can treat at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) of testicular seminiferous tubule atrophy, seminiferous tubule spermatogenesis disorder and / or epididymal duct cell fragments. In some embodiments, the methods of relieving reduced sperm count and / or motility mediated by the sustained-release NGF polypeptides or pharmaceutical compositions described herein can rescue at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) of the reduction in sperm count and / or motility.In some embodiments, the method of increasing sperm count and / or motility mediated by the sustained release NGF polypeptide or pharmaceutical composition described herein can increase sperm count and / or motility by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more). In some embodiments, the method of relieving a decrease in follicle number and / or function mediated by the sustained release NGF polypeptide or pharmaceutical composition described herein can relieving a decrease in follicle number and / or function by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, the methods of increasing follicle number and / or function mediated by the sustained release NGF polypeptides or pharmaceutical compositions described herein can increase follicle number and / or function by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more). In some embodiments, the methods of extending the survival time of an individual (e.g., a human) mediated by the sustained release NGF polypeptides or pharmaceutical compositions described herein can extend the survival time of an individual by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months or 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more.

[0237] Administration of the sustained release NGF polypeptide or pharmaceutical composition thereof described herein can be by any convenient method, including injection or infusion. The route of administration is according to known and recognized methods, such as single or multiple bolus injections or chronic infusion in an appropriate manner. The sustained release NGF polypeptide or pharmaceutical composition thereof can be administered orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, intravaginally, intrarectally, intraocularly, topically, intraarterially, intradermally, intranodal, intracavitary, intramedullary, intrathecal, intraventricularly, intracerebral, intraspinal, intrathecal, intralesional, or intraocularly. In some embodiments, the sustained release NGF polypeptide or pharmaceutical composition thereof is administered systemically. In some embodiments, the sustained release NGF polypeptide or pharmaceutical composition thereof is administered to an individual by injection (e.g., intravenous infusion). Injection techniques used in immunotherapy are known in the art (see Rosenberg et al., New Eng. J. of Med. 319:1676 (1988)). In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered to an individual by intradermal or subcutaneous (i.e., under the skin) injection. For subcutaneous injection, a syringe can be used to inject the long-acting NGF polypeptide or pharmaceutical composition thereof. Other devices for administering the long-acting NGF polypeptide or pharmaceutical composition thereof include, for example, injection devices; injection pens; auto-injector devices, needle-free devices; and subcutaneous patch delivery systems. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered by intravenous injection. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is injected directly into the brain or spine. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered locally to the site of injury or damage, for example, directly into wounded tissue. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered by sustained release or sustained release technology.

[0238] The dosage and desired drug concentration of the pharmaceutical composition of the present invention may vary depending on the specific application. The dosage or route of administration can be appropriately determined by those skilled in the art within their capabilities. The effective dosage for human treatment can be determined by reliable information provided by animal experiments. Interspecies analogy of the effective dosage can be made according to the principles of Mordenti, J. Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.

[0239] When in vivo administration of sustained-release NGF polypeptide or pharmaceutical composition thereof is used, typical dosages may vary from 0.01 μg to 10 mg per kg of mammalian body weight, depending on the route of administration and the type of mammal. It is contemplated within the scope of this application that different formulations may be effective for different treatments and different diseases, and that the route of administration used to treat a particular organ or tissue may differ from the route of administration to another organ or tissue. Furthermore, the dosage may be administered by one or more individual administrations, or by continuous infusion. In the case of repeated administration over several days or more, the treatment may be sustained to the extent that the disease symptoms are suppressed as desired, depending on the disease state. However, other dosage schemes may also be useful. The progress of this treatment may be easily monitored by conventional techniques and assays. In some embodiments, the sustained-release NGF polypeptide or pharmaceutical composition thereof is administered at a dose of 0.01 μg / kg to 10 mg / kg, for example, at any dose in the range of 0.01 μg / kg to 1 μg / kg, 1 μg / kg to 100 μg / kg, 100 μg / kg to 500 μg / kg, 500 μg / kg to 1 mg / kg, 1 mg / kg to 10 mg / kg, or 0.01 μg / kg to 1 mg / kg. In some embodiments, the sustained-release NGF polypeptide or pharmaceutical composition thereof is administered at a dose of 0.01 μg to 1000 μg per individual (e.g., human), for example, at any dose in the range of 0.01 μg to 1 μg, 1 μg to 500 μg, 500 μg to 1000 μg, 1 μg to 300 μg, or 100 μg to 1000 μg per individual.

[0240] In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered once (e.g., bolus injection). In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered multiple times (e.g., 2, 3, 4, 5, 6 or more). When administered multiple times, they may be administered by the same or different routes, and may be administered at the same site or other sites. The long-acting NGF polypeptide or pharmaceutical composition thereof may be administered as frequently as once a day to once a year. The interval between administrations may be any time within the range of 24 hours to one year, and may be irregular (e.g., with tumor progression). In some embodiments, there is no break in the administration schedule. The optimal dosage and treatment schedule for a particular patient can be determined by those skilled in the medical arts by monitoring the patient's disease symptoms and adjusting accordingly. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof described herein is administered once a day (daily administration), once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, or once a year. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered once every 3 days. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered once a week. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered once a month. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered by infusion once a day. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered by infusion three times a day. In some embodiments, the long-acting NGF polypeptide or pharmaceutical composition thereof is administered by infusion five times a day.

[0241] In some embodiments, the sustained-release NGF polypeptide or pharmaceutical composition thereof is administered in divided doses, such as 2, 3, 4, 5 or more doses. In some embodiments, the divided doses are administered over a period of 1 week, 1 month, 2 months, 3 months or more. In some embodiments, the dose is evenly divided. In some embodiments, the divided doses are 20%, 30% and 50% of the total dose. In some embodiments, the interval between successive divided doses is 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months or more. When administered repeatedly over several days or more, the treatment is sustained to the extent that the disease symptoms are suppressed as desired, depending on the condition. However, other dosing schemes may be useful. The progress of such treatment is easily monitored by conventional techniques and assays. VII. Products and Kits

[0242] The invention also relates to kits, unit doses, and articles of manufacture comprising any of the sustained-release NGF polypeptides described herein. In some embodiments, the invention relates to kits comprising any one of the pharmaceutical compositions described herein, and preferably the kits are provided with instructions for use thereof, such as for use in treating disorders (e.g., neurological disorders) described herein.

[0243] The kits of the present invention include one or more containers containing a sustained-release NGF polypeptide as described herein, for example for use in treating a disease. For example, instructions are included that explain administering the sustained-release NGF polypeptide to treat a disease (e.g., a neurological disease). The kits may further include instructions for selecting an individual (e.g., a human) suitable for treatment based on identifying whether the individual suffers from a disease and the stage of the disease. Instructions for use of the sustained-release NGF polypeptide generally include information regarding the expected dose, dosing schedule, and route of administration of the treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. Instructions provided in the kits of the present invention are typically written instructions on a label or insert (e.g., a paper included in the kit), but may also be machine-readable instructions (e.g., instructions stored on a magnetic or optical disk). The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The packaging may be a package for use in combination with a particular device, such as an infusion device, such as a minipump. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle), with the proviso that at least one active agent in the composition is a sustained-release NGF polypeptide as described herein. The container may further include a second pharmacoactive agent. The kit may provide additional components, such as buffers and interpretive information, as needed. In general, the kit includes a container and a label or package insert on or associated with the container.

[0244] Thus, the present invention also relates to articles of manufacture, including vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like. The articles of manufacture include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be made of a variety of materials, including glass and plastic. In general, the container contains a composition effective for treating a disease or disorder described herein (e.g., a neurological disease) and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a particular condition in an individual. The label or package insert further includes instructions for administering the composition to an individual. The label may include reconstitution and / or use instructions. The container containing the pharmaceutical composition may be a multi-use vial that can be used repeatedly (e.g., 2-6 administrations) for formulation reconstitution. Package insert refers to instructions typically included in commercial packaging for a therapeutic product, which contain information regarding the indications, usage, dosage, administration, contraindications, and / or warnings for the use of such therapeutic product. Additionally, the product may further include a second container comprising a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, dextrose solution, or the like, and may further include other necessary materials from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, etc.

[0245] The kit or article of manufacture includes a plurality of unit doses of the pharmaceutical composition and instructions for use, packaged in an amount sufficient for storage and use in a pharmacy, such as a hospital pharmacy or compounding pharmacy. EXAMPLES

[0246] The following examples are intended to serve as illustrations of the present invention and therefore should not be construed as limiting the present invention. It is to be noted that the following examples and detailed description are provided by way of illustration and not by way of limitation. Example 1: Preparation of NGF Polypeptides Plasmid construction

[0247] The construction of the plasmid is described by taking the preproNGF-Fc fusion protein 2-118-L3Fc10-M1-5 (SEQ ID NO: 34) as an example. Plasmids were constructed using a similar method for other preproNGF-Fc fusion proteins and the control prepro-mNGF118 (SEQ ID NO: 47). However, the control prepro-mNGF118 was a preproNGF with an F12E mutation, truncating two amino acids (Arg-Ala) at the C-terminus of the β-NGF moiety. The FD-G4Fc (see CN105273087A) and WM-G24Fc (see CN106008722A) constructs served as controls. NGF-1-15M7 (rhNGF-Fc1), NGF-L3Fc10M7-5 (rhNGF-Li-Fc1), 2-1-15M7 (rhNGF-(F12E)-Fc1) and NGF-4-12PAA (rhNGF-Fc4) were constructed as described in WO2017157325. The structures of the different NGF polypeptides are shown in Table 2 and the sequence alignment of the Fc portion is shown in Figures 1A-1F.

[0248] [Table 2]

[0249] Nucleic acids encoding various preproNGF-Fc fusion proteins (e.g., "2-118-L3Fc10-M1-5") or prepro-mNGF118 controls were synthesized and cloned into pSC-T vectors (e.g., "pSC-2-118-L3Fc10-M1-5") (synthesized and cloned by Shanghai Jierui Bio-Engineering Co., Ltd. Beijing Branch). Nucleic acids encoding various preproNGF-Fc fusion proteins or prepro-mNGF118 controls were amplified using PCR primers with HindIII and XhoI restriction sites, respectively, and then the PCR products were subcloned into endogenous eukaryotic expression vector pTT5 (e.g., "pTT5-2-118-L3Fc10-M1-5"). Recombinant protein expression

[0250] 293F cells were transfected with the eukaryotic expression vector pTT5 carrying nucleic acid encoding various preproNGF-Fc fusion proteins (e.g., pTT5-2-118-L3Fc10-M1-5) or prepro-mNGF118 control and incubated at 37°C, 8% CO 2 The cells were cultured at 120 rpm for 5 days, and the supernatant containing the expressed protein was collected. Recombinant protein purification The expressed NGF-Fc fusion protein was first pre-purified by Protein A affinity purification and then purified by HiTrap TM The mature mNGF118 control was purified using a HiTrap column (GE Healthcare) to remove residual aggregates and obtain the mature NGF-Fc fusion protein. TM After purification on a Butyl HP column (GE Healthcare), the proteins were purified on a Superdex 200 gel filtration column (GE Life Sciences). The proteins were >90% pure as determined by SDS-PAGE. Example 2: Thermal stability study of mature NGF-Fc fusion protein

[0251] The changes in fluorescence absorbance and light scattering of the samples during heating were measured at 266 nm / 473 nm using a fluorescent protein analyzer UNcle (Uncheed Labs), and the melting temperature (Tm) and aggregation onset temperature (Tagg) of the samples were calculated, respectively. The initial temperature was set to 20°C, the final temperature to 95°C, the heating rate was set to 0.3°C / min, and measurements were repeated three times for each sample. The results are summarized in Table 3.

[0252] [Table 3]

[0253] Melting temperature (Tm) As shown in Table 3, among all mature NGF-Fc fusion proteins containing an IgG4-derived Fc fragment (FD-G4Fc, WM-G24Fc, NGF-4-12PAA, and 2-118-L3G4-BM), and thus among all mature NGF-Fc fusion proteins detected, 2-118-L3G4-BM had the highest melting temperature (Tm), i.e., the best thermal stability. Among all mature NGF-Fc fusion proteins containing an IgG1-derived Fc fragment, 2-118-L3Fc10-M1-5 had a lower Tm (ie, worse thermal stability) than the other proteins that showed similar Tms. Agglomeration start temperature (Tagg)

[0254] As shown in Table 3, the mature NGF-Fc fusion proteins containing the IgG1-derived Fc fragment (except for 2-118-L3Fc10-M1-5) had higher Tagg than the mature NGF-Fc fusion proteins containing the IgG4-derived Fc fragment, indicating that the mature NGF-Fc fusion proteins containing the IgG1-derived Fc fragment are less likely to aggregate during heating.

[0255] 2-118-L3G4-BM had the highest Tagg among all mature NGF-Fc fusion proteins containing IgG4-derived Fc fragments, indicating that 2-118-L3G4-BM has superior anti-aggregation performance during heating compared to other IgG4-derived Fc fusion constructs.

[0256] 2-118-L3Fc10-M1-5 had the lowest Tagging (worst anti-aggregation performance during heating) among all mature NGF-Fc fusion proteins containing IgG1-derived Fc fragments, whereas NGF-1-15M7 and 2-1-15M7 showed relatively low Tagging, and the remaining IgG1-derived Fc fusion constructs showed similar high Tagging. Example 3: Accelerated stability study of mature NGF-Fc fusion protein

[0257] Mature NGF-Fc fusion protein was diluted in PBS to a final concentration of 2 mg / ml and incubated at 40° C. Samples were taken at days 0 (indicated as "0 h" in the figure), 3, 7, 9, and 14 of the incubation period and stored at -80° C. Degradation and aggregation of samples were detected by size exclusion chromatography (SEC) and sodium dodecyl sulfate capillary electrophoresis (CE-SDS). Size Exclusion Chromatography (SEC) Detection

[0258] SEC separates molecules according to their size as they pass through a resin packed in a column. Protein samples were centrifuged at 10,000 g for 5 min at 4°C, and the precipitate was resuspended in PBS. Waters R ACQUITY UPLC R Using an H-Class Bio Tunable UV (TUV) detector, 80-100 μl of samples transferred to a 384-well plate were detected under the following conditions: injection volume 20 μl, wavelength 280 nm, flow rate 0.25 ml / min, total run time 17 min. The mobile phase buffer was 100 mM PB (80 mM Na 2 HPO 4 , 20 mM NaH 2 PO4 ), 300 mM NaCl, 10% acetonitrile, pH 7.2.

[0259] As shown in Table 3 and Figures 3A-3D, among all mature NGF-Fc fusion proteins containing IgG4-derived Fc fragments, 2-118-L3G4-BM and FD-G4Fc were more stable than WM-G24Fc and NGF-4-12PAA in terms of increased aggregates and fragment generation. During accelerated stress, 2-118-L3G4-BM and FD-G4Fc had fragment percentages less than 2.1%, while NGF-4-12PAA was obviously fragmented with a fragment percentage reaching 49.59% (Figure 3A). WM-G24Fc showed a significant increase in aggregates (aggregate percentage reaching 49.15%) and also showed obvious fragmentation during accelerated stress (Figure 3C). The monomer percentages of 2-118-L3G4-BM and FD-G4Fc were over 85%, while the monomer percentages of WM-G24Fc and NGF-4-12PAA decreased significantly over time. FD-G4Fc formed more aggregates and was more fragmented during accelerated stress than 2-118-L3G4-BM, indicating that 2-118-L3G4-BM had better anti-fragmentation and anti-aggregation activity than FD-G4Fc.

[0260] As shown in Table 4 and Figures 3E to 3M, among all mature NGF-Fc fusion proteins containing IgG1-derived Fc fragments, 2-118-L3Fc10-M3-5, 2-118-L3Fc10-M5-5, 2-118-L3Fc10-M7-5 and NGF-118-L3Fc10-M3-5 were more stable than other IgG1-derived Fc fusion proteins during accelerated stress, with undetectable (0%) fragment formation and less increase in aggregates. 2-118-L3Fc10-M1-5 was more stable than or comparable to IgG1-derived Fc fusion proteins such as NGF-1-15M7, NGF-L3Fc10M7-5 and 2-1-15M7.

[0261] [Table 4]

[0262] Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) detection method In capillary electrophoresis, samples are separated in a capillary according to their electrophoretic mobility, which varies with the charge and size of the molecules. First, 40 μl of 1× sample buffer and 10 μl of protein sample were mixed in a centrifuge tube to obtain a 50 μl mixture with a final protein concentration of 0.4 μg / μl. To the mixture, 1 μl of reconstituted 25× Internal Standard was added, followed by 2.5 μl of 250 mM iodoacetamide. The entire mixture was vortexed and incubated at 70°C for 10 min, cooled, mixed well and centrifuged. 50 μl of treated sample supernatant was transferred to a 96-well plate. The 96-well plate was centrifuged at 1000 g for 10 min and then placed in the Maurice system (ProteinSimple) for CE-SDS detection according to standard laboratory methods.

[0263] As shown in Table 5 and Figures 4A-4D, among all mature NGF-Fc fusion proteins containing IgG4-derived Fc fragments, NGF-4-12PAA and WM-G24Fc were very obviously fragmented during accelerated stress, while 2-118-L3G4-BM was least fragmented. This is consistent with the SEC results. FD-G4Fc (Figure 4B) showed an aggregate peak to the right of the main peak, whereas 2-118-L3G4-BM (Figure 4D) showed no aggregate peak. Thus, consistent with the SEC results, 2-118-L3G4-BM had better stability during accelerated stress than other IgG4-derived Fc fusion proteins.

[0264] As shown in Table 5 and Figures 4E-4M, among all mature NGF-Fc fusion proteins containing Fc fragments derived from IgG1, 2-118-L3Fc10M7-5, NGF-118-L3Fc10-M3-5 and 2-118-L3Fc10-M3-5 showed the best stability during accelerated stress, with no fragment peaks (see Figures 4I, 4J and 4M) and 0% increase in fragments in the samples on day 14. 2-118-L3Fc10-M5-5 showed a low increase in fragments (5.8% increase in fragments on day 14) and good stability. In contrast, other NGF-Fc fusion proteins containing Fc fragments derived from IgG1 (e.g., NGF-L3Fc10M7-5) were prone to fragment formation.

[0265] [Table 5]

[0266] The results of SEC and CE-SDS were summarized as follows: 1) Among all mature NGF-Fc fusion proteins containing IgG4-derived Fc fragments, 2-118-L3G4-BM had better accelerated stability than FD-G4Fc, and also had significantly better accelerated stability than WM-G24Fc and NGF-4-12PAA; 2) Among all mature NGF-Fc fusion proteins containing IgG1-derived Fc fragments, 2-118-L3Fc10M5-5 had good accelerated stability. qualitatively different from the above, but more importantly, 2-118-L3Fc10-M3-5, NGF-118-L3Fc10-M3-5 and 2-118-L3Fc10M7-5 have the best accelerated stability compared to all other constructs; 3) mature NGF-Fc fusion proteins containing an IgG1-derived Fc fragment have better anti-aggregation properties under accelerated stress conditions compared to mature NGF-Fc fusion proteins containing an IgG4-derived Fc fragment. Example 4: TF-1 cell proliferation assay to evaluate the biological activity of NGF-Fc fusion proteins

[0267] A TF-1 cell proliferation assay was used to detect the biological activity of various NGF-Fc fusion proteins. TF-1 cells are a factor-dependent human erythroleukemia cell line. TF-1 cells were resuspended in basal medium (RPMI 1640 medium + 10% FBS) and diluted at 5.0 × 10 4 The result was a suspension of cells / ml. R A mouse NGF (standard control) standard solution was prepared, and test solutions of various NGF-Fc fusion proteins and control solutions of mNGF118 (mutant β-NGF 118aa) and rhNGF (recombinant human wild-type β-NGF 120 amino acids, SEQ ID NO: 4, prepared and purified as described in Example 1) were prepared so that the final protein was 200U / ml x 100μl / well in a pre-labeled 96-well plate. The standard control (Su Tai Sheng R 5.0 × 10 cells were added to each well of a 96-well plate containing mouse NGF, test NGF-Fc fusion protein, or control solution. 4 Add 100 μl of TF-1 cell suspension at 100 cells / ml and incubate at 37 °C, 5% CO 2 The cells were cultured for 72 hours in a humidified incubator at 37 °C. R Add 20 μl of the assay solution in AQueous One Solution Cell Proliferation Assay (Promega, Cat# G3581) to each well of the cell suspension and incubate at 37°C, 5% CO 2 The wells were incubated at 490 nm and 650 nm for 3 h. The absorbance of the wells was measured at 490 nm and 650 nm using a spectrophotometer. The recorded data were compared with the standard NGF control (Su Tai Sheng R Normalized to mouse NGF.

[0268] As shown in FIG. 5A, in all mature NGF-Fc fusion proteins containing Fc fragments derived from IgG1 (rhNGF, mNGF118 and SuTaiSheng R2-118-L3Fc10-M3-5 was the most biologically active of all NGF constructs, including mouse NGF. In accelerated stability studies (see Example 3), 2-118-L3Fc10-M3-5 was the most biologically active of the three most stable constructs (2-118-L3Fc10-M3-5, NGF-118-L3Fc10-M3-5, and 2-118-L3Fc10M7-5). As shown in FIG. 5B, all mature NGF-Fc fusion proteins containing IgG4-derived Fc fragments have biological activity in promoting the proliferation of TF-1 cells. 2-118-L3G4-BM also showed a higher biological activity than the standard control SuTaiSheng R It showed biological activity comparable to that of mouse NGF. Example 5: Biological activity testing of various NGF-Fc fusion proteins in rats

[0269] The superior cervical ganglion (SCG) is a tissue composed of approximately 30,000 neurons and is one of the most sensitive tissues to NGF, especially during prenatal and postnatal development. In a TF-1 cell proliferation assay (see Example 4), certain NGF-Fc fusion proteins containing IgG1- or IgG4-derived Fc fragments showed very high biological activity. Various NGF-Fc fusion proteins were injected into rat SCG and SCG size was measured at various time points after injection to evaluate the activity of NGF-Fc fusion proteins in promoting SCG proliferation in vivo.

[0270] Neonatal Sprague-Dawley (SD) rats were injected with various NGF-Fc fusion proteins or NGF control (Su Tai Sheng R Mouse NGF or mutant NGF118) was subcutaneously injected into the rats, and then the rats were sacrificed to isolate SCGs. PBS infusion served as a negative control. As shown in Figure 6A, NGF control protein (SuTaiSheng RMouse NGF or mutant NGF118) or PBS was injected once a day on days 0, 1, 2 and 3, then SCGs were obtained on day 4. 2-118-L3Fc10-M3-5 or 2-118-L3G4-BM was injected once at the same dose on day 0, then SCGs were obtained on day 4. Briefly, after decapitation, the rat's head was fixed on the operating table, blood was sucked up with a cotton ball, the trachea and foramen magnum were first found, then the carotid sheath tissue on the oblique posterior side of the trachea was found, the SCGs were taken out with microtweezers, and the SCGs were isolated under a dissecting microscope after being placed in a Petri dish containing PBS. The excess liquid on the surface of the isolated SCGs was removed with tissue paper, and then the SCGs were placed on a clean surface dish and weighed. The morphology of the SCGs is shown in Figure 6B. The recorded data was analyzed by Student's t-test. ** indicates significant difference compared with the PBS-treated group, and ns indicates "no significant difference" compared with the PBS-treated group. As shown in Figure 6C, at a dose of 2 nM, mutant β-NGF 118aa (mNGF118), Su Tai Sheng R Mouse NGF and 2-118-L3Fc10-M3-5 had no significant promoting effect on SCG proliferation compared to the PBS negative control group, and the difference was not statistically significant. Meanwhile, 2-118-L3G4-BM significantly promoted SCG proliferation compared to the PBS control group (**p<0.01). At a dose of 5 nM, the NGF control group (SuTaiSheng R Both mouse NGF or mNGF118) and NGF-Fc fusion proteins (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) significantly promoted SCG proliferation in vivo compared to the PBS-treated group (**p<0.01), and no significant differences were found in the activity of promoting SCG proliferation among the four tested NGF proteins (ns indicates p>0.05) (Figure 6D). Thus, even at a dose of 2 nM, 2-118-L3G4-BM was significantly suppressed by the SuTaiSheng RCompared with mouse NGF or mutant β-NGF 118aa (mNGF118), a single subcutaneous injection showed superior activity in promoting SCG proliferation in vivo. At a high dose (5 nM), a single treatment with either 2-118-L3G4-BM or 2-118-L3Fc10-M3-5 showed similar activity in promoting SCG proliferation. Example 6: Pharmacokinetic (PK) studies of various NGF polypeptides in rats

[0271] Various NGF constructs were injected into adult rats to detect the PK profile.

[0272] Twenty-four male SD rats (6-8 weeks old, approximately 250g-300g / rat) were randomly divided into three groups (8 rats per group) and intramuscularly injected with 2-118-L3Fc10-M3-5, 2-118-L3G4-BM, and mNGF118 (mutant β-NGF 118 amino acids without Fc fusion) at 235μg / kg, respectively. 150μl of retroorbital venous blood was collected before (0 hours) or 1, 4, 8, 24, 48, 72, 120, 168, 216, and 288 hours after injection, respectively. After blood collection, plasma was separated, and then NGF content was detected with Human NGF Matched ELISA Antibody Pair Set (Sino Biological, SEK11050). Plot the average ODs values ​​of the standards from 450 nm to 630 nm on the Y-axis and the concentration of the standards on the X-axis. 2 A linear equation of the standard curve was generated with a requirement of >0.98.The plasma concentration of each sample was then calculated according to the linear equation of the standard curve.GraphPad Prism 5.0 was used to generate semi-logarithmic plots of sample concentration versus time, Phoenix WinNonlin 6.2 was used for PK analysis, and GraphPad Prism 5.0 was used to plot the half-life scatter plots.

[0273] As shown in Figure 7A, after a single intramuscular injection, the NGF-Fc fusion protein group had higher plasma concentrations over time compared to the mNGF118 control group not fused to Fc. After a single intramuscular injection, 2-118-L3G4-BM showed similar plasma concentrations over time as 2-118-L3Fc10-M3-5. As shown in Figure 7B, 2-118-L3G4-BM (55 hours) and 2-118-L3Fc10-M3-5 (55 hours) had similar half-lives, but were significantly longer (about 31-fold) than the mNGF118 control group not fused to Fc (half-life 1.75 hours). These results suggest that a single dose of 2-118-L3G4-BM or 2-118-L3Fc10-M3-5 enhanced SCG proliferation in vivo, whereas continuous injections of the NGF control group not fused to Fc (Su Tai Sheng R The reason why it showed activity similar to that of mouse NGF or mNGF118) became clearer (see FIG. 6D).

[0274] The half-lives of wtNGF120 (i.e., "rhNGF", human wild-type β-NGF 120 amino acids, SEQ ID NO: 4), NGF-1-15M7 (rhNGF-Fc1), NGF-L3Fc10M7-5 (rhNGF-Li-Fc1), 2-1-15M7 (rhNGF-(F12E)-Fc1) and NGF-4-12PAA (rhNGF-Fc4) are detailed in Table 2 of WO2017157325 and summarized in Table 6. The half-life of mNGF118 detected in this experiment (1.75 hours) was similar to the half-life of wtNGF120 detected in WO2017157325 (1.8 hours). As shown in Table 6, the NGF-1-15M7 (rhNGF-Fc1), NGF-L3Fc10M7-5 (rhNGF-Li-Fc1), and 2-1-15M7 (rhNGF-(F12E)-Fc1) constructs had in vivo half-lives that were more than 17-fold longer than the wtNGF120 or mNGF118 controls without Fc fusion, and 1.4-fold longer than NGF-4-12PAA (rhNGF-Fc4). The half-life of 2-118-L3G4-BM (55 h) detected here was nearly identical to that of 2-118-L3Fc10-M3-5 (55 h), both of which were approximately 31-fold longer than the Fc-fusion-free wtNGF120 or mNGF118 controls, and was also much longer than all previously measured mature NGF-Fc fusion proteins, including IgG1- or IgG4-derived Fc fragments.

[0275] [Table 6]

[0276] Example 7: Promotion of wound healing in diabetic neuropathy by NGF-Fc fusion protein Diabetic neuropathy is one of the common chronic complications associated with diabetes, and patients suffer from delayed wound healing, various degrees of local infection, ulcers, anthrax, and the risk of amputation of the toes and lower limbs. This example describes a study on the therapeutic effect of NGF-Fc fusion protein on an animal model of diabetic neuropathy (e.g., assessed based on wound healing). CD-1 mice were obtained from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Standard methods were adopted to establish the diabetic animal model (see, for example, Graiani G.et al., Nerve growth factor promotes reparative angiogenesis and inhibits endothelial apoptosis in cutaneous wounds of Type 1 diabetic mice. Diabetologia. 2004, 47(6):1047-54). Four weeks after diabetes induction, the mice were anesthetized and a disposable skin punch was used to obtain one full-thickness skin wound with a diameter of 4 mm on each side between the shoulder blades. 50 μg / ml SuTaiSheng R Mouse NGF, mNGF118, or NGF-Fc fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM) was administered at a dose of 20 μl / infusion to the right wound. An equal volume of PBS infusion was administered to the left wound (as a negative control). PBS, SuTaiSheng R Mouse NGF or mNGF118 was administered once a day on day 0 (after puncturing the mouse's back), day 1, day 2, and day 3. 2-118-L3Fc10-M3-5 or 2-118-L3G4-BM was administered once at the same dose on day 0. The wound area immediately after puncturing was measured and recorded as the wound area on day 0, and the wound area was measured on days 4 and 7 to calculate the wound healing rate. The recorded data was analyzed by Student's t-test and plotted as a vertical bar graph by GraphPad Prism 8.0.1.

[0277] As shown in Figure 8, wound healing was improved in all groups on day 7 compared with day 4. R Mouse NGF, mNGF118 and NGF-Fc fusion proteins (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) all significantly promoted diabetic wound healing compared with the PBS negative control (p<0.01). Specifically, on day 4, the mean wound area in the PBS-treated group was significantly larger than that in the SuTaiSheng RThe results were approximately 1.3 times higher than those of the mouse NGF, mNGF118 or NGF-Fc fusion protein treatment groups. R It was shown that mouse NGF, mNGF118 or NGF-Fc fusion protein could effectively improve the delayed wound healing defect of diabetic mice. Furthermore, on the 7th day, the wound healing rate of diabetic mice administered NGF-Fc fusion protein was significantly improved by 1.0%. R The results showed that the NGF-Fc fusion protein was able to inhibit the inflammatory response of NGF (e.g., SuTaiSheng R It was shown to have a superior in vivo therapeutic effect than mouse NGF or mNGF118). Example 8: Therapeutic effect of NGF-Fc fusion protein on Alzheimer's disease

[0278] Alzheimer's disease is a degenerative disease of the central nervous system with progressive memory loss as the main clinical symptom, which often occurs in elderly people and has a complex etiology. This example describes the study of the therapeutic effect of NGF-Fc fusion protein against AD in vivo (e.g., as assessed by the behavioral changes of animals).

[0279] Wistar rats were employed to establish an AD animal model using standard methods (see, for example, "Wenk GL, Harrington CA, Tucker DA, et al. Basal forebrain neurons and memory: a biochemical, histological and behavioural study of differential vulnerability to ibotenate and quisqualate. Behav Neurosci, 1992, 106: 909-923."). Briefly, Wistar rats were stereotaxically injected with ibotenic acid (IBO). Two days after IBO injection, the AD model rats were anesthetized and placed in a supine position. 150 μg / ml NGF (Su Tai Sheng RMouse NGF or mNGF118 (experimental group) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) was administered intranasally at a total dose of 100 μl / dose. AD model rats administered with the same volume of PBS served as negative controls. NGF or PBS was administered once a day for 7 consecutive days. NGF-Fc fusion protein was administered once only on the first day. On the 7th day, behavioral changes of rats were evaluated by Morris water maze test. Briefly, using a Morris water maze apparatus, rats were trained to climb onto the platform before the experiment, and on the day of the experiment, the time it took the rat to find the platform (latency time; the time from entering the water to climbing onto the platform) and the number of times the rat crossed the original platform position within 120 s after the platform was removed were recorded. The recorded data were analyzed by Student's t-test.

[0280] As shown in Table 7, Su Tai Sheng R AD model rats treated with mouse NGF, mNGF118 or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) showed a significantly shorter time to find the platform (*p<0.05) and a significantly increased number of times crossing the platform compared to the negative control group (*p<0.05). Therefore, either NGF or NGF-Fc fusion protein described herein can effectively improve the spatial cognition, memory and learning ability of AD model rats. Furthermore, AD model rats treated with 2-118-L3G4-BM or 2-118-L3Fc10-M3-5 showed a significantly improved spatial cognition, memory and learning ability compared to the negative control group. R The time it took to find the platform was shorter and the frequency of crossing the platform was higher than that of rats treated with mouse NGF or mNGF.118 These data indicate that the NGF-Fc fusion protein has a superior in vivo therapeutic effect against AD.

[0281] [Table 7]

[0282] Example 9: Therapeutic effect of NGF-Fc fusion protein on premature ovarian failure Premature ovarian failure (POF) is often accompanied by low estrogen levels, high follicle-producing hormone levels, and high gonadotropin levels, and refers to spontaneous amenorrhea in women under 40 years of age due to ovarian insufficiency, with complex etiology and mechanisms. This example describes an in vitro human ovarian granulosa tumor cell line (KGN) proliferation assay and KGN estrogen secretion assay, as well as a study on the effect of NGF-Fc fusion protein on the treatment of POF using a POF model rat.

[0283] In the KGN proliferation test, 100 ul of KGN suspension (1 × 10 4 The serum-free DMEM medium was replaced before the experiment. After medium replacement, the cells were added to each well of the 96-well plate at a final concentration of 10 μg / mL. R Mouse NGF, mNGF118 or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) was added to the wells of the experimental groups, respectively, and no treatment was performed on the negative control group after changing the medium. Four duplicate wells were set up for each group, and after 48 hours, 10uL of CCK-8 (DOJINDO, #CK04) was added to each well to measure the viable cells. After 1 hour of incubation, the absorbance value at 450nm was measured, and the recorded data was analyzed by Student's t-test, and the vertical bar graphs were plotted using GraphPad Prism 8.0.1.

[0284] As shown in Figure 9A, NGF (Su Tai Sheng REither mouse NGF or mNGF118 or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) significantly promoted the proliferation of KGN compared to the PBS negative control group (p<0.05). In contrast, KGN treated with NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) had a slightly higher proliferation rate compared to KGN treated with NGF.

[0285] For the KGN estrogen secretion assay, 1 × 10 5 Cells were seeded at 1000 x g / well (cell confluence was approximately 80%) and then replaced with serum-free medium. After medium replacement, the final concentration in the medium was adjusted to 10 μg / ml by adding SuTaiSheng R Mouse NGF, mNGF118, or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) was added to the wells of the experimental group, respectively. The negative control group was not treated after changing the medium. Four duplicate wells were set up in each group, incubated for 18 hours, washed twice, and then 2.2 × 10 -8 After treatment with M testosterone (Beijing Solarbio Life Science & Technology Co.,Ltd, #IT0110) and 0.01IU / ml ovine follicle-stimulating hormone (National Health Physics Program, Ovine FSH) for 24 hours, the supernatant was collected and diluted 1.6-fold, and the absorbance value at 450 nm was measured using an estrogen measurement kit (KGE014) manufactured by R&D Systems, and the secreted estrogen concentration was calculated. The recorded data were analyzed by Student's t-test, and a column graph was plotted using GraphPad Prism 8.0.1.

[0286] As shown in Figure 9B, NGF (Su Tai Sheng REither mouse NGF or mNGF118 or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) significantly promoted KGN estrogen secretion compared to the negative control group (p<0.05). In contrast, KGN treated with NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) showed slightly higher estrogen secretion compared to KGN treated with NGF.

[0287] 4-vinylcyclohexene diepoxide (VCD) can selectively destroy primordial and primary follicles in the ovaries of female mice, but not affect secondary and antral follicles, resulting in POF in female mice. To further study the in vivo effect of NGF-Fc fusion protein in treating POF, VCD was intraperitoneally injected into SD rats for two consecutive weeks to establish a POF rat model (see, for example, Muhammad FS et al., Effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague-Dawley rats: ovarian, clinical, and pathologic outcomes[J]. Comp Med, 2009, 59(1):46-59.). NGF administration was performed at the beginning of model establishment and recorded as day 1. Subcutaneous injection was adopted, and NGF (Su Tai Sheng R The injection dose of mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) in the experimental group was 10 μg / kg bw, and the same amount of sterile saline was used as the negative control. RMouse NGF, mNGF118 or sterile saline was administered every other day, and NGF-Fc fusion proteins (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) were administered once a week. After 42 days, all rats were euthanized, and ovarian tissues were fixed and then routinely embedded in paraffin, sectioned and stained with H&E (hematoxylin and eosin), and follicles were counted at all levels. Recorded data were analyzed by Student t-test, and column graphs were plotted using GraphPad Prism 8.0.1.

[0288] As shown in Figure 9C, Su Tai Sheng R Mouse NGF, mNGF118 and NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) all significantly increased the number of primary follicles compared to the negative control group (p<0.05), demonstrating their excellent effect in reversing the reduction in primary follicle number caused by POF. R POF rat models treated with mouse NGF, mNGF118, or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) had higher numbers of primordial and secondary follicles compared to the negative control group. Example 10: Therapeutic effect of NGF-Fc fusion protein on asthenozoospermia

[0289] Asthenozoospermia is mainly manifested as a decrease in sperm count and / or a decrease in sperm motility. Sperm are formed in the seminiferous tubules of the testis by a series of divisions and differentiations of germ cells with the ability to proliferate, and heat stress may affect the division, differentiation and spermatogenesis of proliferating cells. This Example describes a study of the therapeutic effect of NGF-Fc fusion protein on asthenozoospermia (oligozoospermia and asthenozoospermia) in a mouse spermatogenesis disorder model. In this experiment, C57BL / 6JSHjh mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were used. The experimental group was administered 20 μg / kg bw / injection of NGF (SuTaiSheng R The mice were injected with 60 μg / kg bw / dose of mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5), respectively. The normal control group or spermatogenesis disorder model control group was injected with an equal amount of 0.9% sodium chloride infusion. The day of the first administration (NGF, NGF-Fc fusion protein, or sodium chloride) was designated as day 1. To construct an animal model of spermatogenesis disorder caused by heat stress in mouse testes, the mice were anesthetized 4 hours after the first administration, and after the mouse testes descended into the scrotum, the lower abdomen (hind legs, tail, and scrotum) of the mice in the spermatogenesis disorder model control group, NGF experimental group, and NGF-Fc fusion protein experimental group were immersed in a constant temperature water bath at 42°C for 30 minutes, and the lower abdomen (hind legs, tail, and scrotum) of the mice in the normal control group was immersed in a constant temperature water bath at 25°C for 30 minutes. Su Tai Sheng R Mouse NGF or mNGF118 was administered once every other day, and 2-118-L3G4-BM or 2-118-L3Fc10-M3-5 was administered twice a week. The normal control group or model control group was injected with an equal amount of 0.9% sodium chloride infusion every other day. The administration was carried out for a total of 5 weeks. On the 37th day after administration, the mice were euthanized, the left tail of the epididymis was collected and weighed, and placed in M199 culture medium preheated to 37°C, cut into pieces and incubated in an incubator at 37°C for 5 minutes, the sperm suspension was aspirated, diluted 1:6 with M199 culture medium, and the diluted solution was taken after uniform mixing, and the sperm count and sperm motility were detected using a TOX IVOS sperm analyzer. The recorded data were analyzed by Student's t-test.

[0290] As shown in Table 8, the sperm count and sperm motility of the spermatogenic disorder model control group were significantly lower than those of the normal control group, indicating that the animal model was successfully established. RThe sperm count and sperm motility of mice in the experimental groups treated with mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3Fc10-M3-5 and 2-118-L3G4-BM) were significantly increased compared to the spermatogenic disorder model control group. These results demonstrated that subcutaneous injection of NGF or NGF-Fc fusion protein could effectively rescue the reduced sperm count and sperm motility in spermatogenic disorders (e.g. asthenozoospermia, oligozoospermia, and oligozoospermia).

[0291] [Table 8]

[0292] To further study the therapeutic effects of NGF and NGF-Fc, the right testis and epididymis of the euthanized mice were collected and weighed, then fixed in 10% neutral formalin, embedded, sectioned and stained with H&E to evaluate histopathological lesions. As shown in Table 8, the effect of NGF (SuTaiSheng R Mouse NGF or mNGF118) and NGF-Fc fusion proteins (2-118-L3Fc10-M3-5 and 2-118-L3G4-BM) showed significant therapeutic effects on testicular seminiferous tubule atrophy, impaired spermatogenesis in the seminiferous tubules, and epididymal duct cell fragmentation symptoms caused by heat stress.

[0293] Furthermore, the NGF-Fc fusion protein showed therapeutic effects (sperm count, sperm motility, histopathology) equal to or greater than those of NGF.

[0294] [Table 9]

[0295] Example 11: Therapeutic effect of NGF-Fc fusion protein on neurotrophic keratitis Neurotrophic keratitis is a degenerative disease caused by impaired healing of the corneal epithelium and is primarily characterized by decreased corneal sensitivity. This example describes a study of the therapeutic effect of NGF-Fc fusion protein on neurotrophic keratitis in a rat model of neurotrophic keratitis (e.g., by corneal sodium fluorescein staining test and corneal nerve length measurement).

[0296] To establish an animal model of neurotrophic keratitis, 3-day-old SD rats were subcutaneously injected with 8 mg / ml capsaicin solution (Shanghai McLean Biochemical Technology Co., Ltd., #C10831884) at a dose of 50 μl / rat. Two weeks after capsaicin injection, the rats were subcutaneously injected with 60 μg / ml NGF (SuTaiSheng R Mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM) was administered in the form of eye drops to both eyes at approximately 2-hour intervals, 6 times a day, 20 μl / eye / time. As a negative control, the same amount of 0.9% sodium chloride solution was administered at the same frequency. The first day of administration was recorded as D1. The administration was continued for 2 weeks, and each group was administered once on D15.

[0297] Next, corneal sodium fluorescein staining was performed to evaluate the therapeutic effect of NGF-Fc fusion protein. The corneal sodium fluorescein staining score can directly indicate the integrity and the degree of damage of the cornea. Intact corneas are not stained, only damaged corneas are stained, and the higher the score, the higher the degree of corneal damage. Briefly, sodium fluorescein solution (3 μL, 0.5%) was dropped into the animal's eyes to stain for 1.5 min. Then, the animal's conjunctival sac was washed three times consecutively with 1.25 mL of sterile saline approximately every 10 s. After each wash, the residual saline around the animal's eyes was blotted with tissue paper. After 5 min of staining, the ocular surface was observed with a slit lamp (+ cobalt blue filter), photographed, and scored. The improved NEI fluorescent staining grading method was used as the scoring standard. Specifically, each cornea was divided into five regions (1-central region, 2-superior, 3-temporal, 4-nasal, 5-inferior), and each region was scored with a maximum of 8 points. Here, 0 points indicated no staining, 1 point indicated that the area of ​​punctate staining was 1%-25% of the area of ​​the corresponding region, 2 points indicated that the area of ​​punctate staining was 26%-50% of the area of ​​the corresponding region, 3 points indicated that the area of ​​punctate staining was 51%-75% of the area of ​​the corresponding region, and 4 points indicated that the area of ​​punctate staining was 76%-100% of the area of ​​the corresponding region. If the stained region was dense and / or had obvious fusion in the region, an additional score of 1, 2, 3, or 4 was further awarded depending on the area of ​​the corresponding region that occupied the stained area. That is, 1 point was given for a stained area of ​​1% to 25%, 2 points for a stained area of ​​26% to 50%, 3 points for a stained area of ​​51% to 75%, and 4 points for a stained area of ​​76% to 100%. The maximum total score for each eye was 40 points. A total of four measurements were made on days 0 (before the first treatment), 4, 8, and 14. A total score of corneal sodium fluorescein staining was calculated. The recorded data were processed with SPSS 13.0, and column graphs were plotted using GraphPad Prism 8.0.1.

[0298] As shown in FIG. 10A, NGF (Su Tai Sheng RThe experimental groups treated with mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) had significantly lower corneal sodium fluorescein staining scores in the neurotrophic keratitis rat model than the negative control group (p<0.01 on days 4 and 8; p<0.001 on day 14), indicating that NGF (SuTaiSheng R It was shown that mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) could significantly restore the integrity of the injured cornea. In contrast, corneas treated with NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) had slightly lower corneal sodium fluorescein staining scores compared to corneas treated with NGF.

[0299] Corneal nerve counting test was further carried out to study its therapeutic effect. On the 15th day, 1 hour after administration, the rats were euthanized, the right eyeball was harvested, and the cornea was enucleated along the limbus, washed, flattened, stained and fixed on a slide glass. The morphology of the corneal nerve fibers was observed under an optical microscope (×200 magnification), the cornea was radially divided into four lobes from the center, and the clear field with the most corneal nerves was selected and photographed from each of the four lobes, the length of the corneal nerve in each field was measured, and the average length of the corneal nerve in the four fields was taken as the final result. The data was processed using SPSS13.0 and plotted as a column graph by GraphPad Prism 8.0.1.

[0300] As shown in FIG. 10B, NGF (Su Tai Sheng R Neurotrophic keratitis rat models treated with mouse NGF or mNGF118) or NGF-Fc fusion proteins (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) had significantly longer mean corneal nerve length than the negative control group (p<0.05). RThe average corneal nerve length treated with mouse NGF, mNGF118, 2-118-L3G4-BM and 2-118-L3Fc10-M3-5 was approximately 1.14, 1.14, 1.21 and 1.18 times that of the negative control group, respectively. R We demonstrated that mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) could effectively ameliorate the corneal nerve damage caused by neurotrophic keratitis.

[0301] Sequence Listing SEQ ID NO:1 (mutated human β-NGF, 118aa) SSSHPIFHRGEESVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR SEQ ID NO:2 (mutated human β-NGF, 120aa) SSSHPIFHRGEESVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA SEQ ID NO:3 (wild type human β-NGF, 118aa) SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR SEQ ID NO:4 (wild type human β-NGF, 120aa) SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA SEQ ID NO:5 (NGF polypeptide, 103aa) EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKR SEQ ID NO:6 (NGF signal peptide, 18aa) MSMLFYTLITAFLIGIQA SEQ ID NO:7 (human wild type IgG1 Fc IGHG1*05) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:8 (human wild type IgG1 Fc IGHG1*03, naturally occurring variant) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD...

Claims

1. A sustained-release nerve growth factor (NGF) polypeptide comprising, from the N-terminus to the C-terminus, an NGF portion and an Fc portion, wherein the NGF portion consists of the amino acid sequence shown in SEQ ID NO: 1, and the Fc portion is derived from IgG1 Fc or IgG4 Fc.

2. the NGF portion is fused to an Fc portion via a polypeptide linker, The polypeptide linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 68-72, and / or 2. The sustained-release NGF polypeptide of claim 1, wherein the polypeptide linker comprises an amino acid sequence (GGGGS)n (SEQ ID NO: 70, where n is any one of 1, 2, 3, 4, 5 and 6).

3. the Fc portion is derived from an IgG1 Fc; the Fc portion comprises the amino acid sequence of SEQ ID NO: 7 or 8; and / or the Fc portion comprises a mutation at one or more positions selected from E233, L234, L235, G236, G237, N297, A327, A330, and P331 relative to SEQ ID NO:8; and / or the Fc portion comprises one or more mutations selected from E233P, L234V, L234A, L235A, L235E, G236del, G237A, N297A, A327G, A330S, and P331S relative to SEQ ID NO:8; and / or 3. The sustained-release NGF polypeptide according to claim 1, wherein the Fc portion further lacks the first five amino acids of the amino acid sequence of SEQ ID NO: 7 or 8.

4. the Fc portion comprises the L234A, L235A, and P331S mutations relative to SEQ ID NO:8, and / or 4. The sustained-release NGF polypeptide of claim 3, wherein the Fc portion comprises the amino acid sequence of SEQ ID NO: 11 or 12.

5. 5. The sustained-release NGF polypeptide of claim 4, wherein the sustained-release NGF polypeptide comprises the amino acid sequence shown in SEQ ID NO:

62.

6. the Fc portion comprises E233P, L234V, L235A, G236del, A327G, A330S, and P331S mutations relative to SEQ ID NO:8; and / or 4. The sustained-release NGF polypeptide of claim 3, wherein the Fc portion comprises the amino acid sequence of SEQ ID NO: 15 or 16.

7. 7. The sustained-release NGF polypeptide of claim 6, wherein the sustained-release NGF polypeptide comprises the amino acid sequence of SEQ ID NO:

66.

8. the Fc portion comprises the following mutations relative to SEQ ID NO:8: L234A, L235E, G237A, A330S, and P331S; and / or 4. The sustained-release NGF polypeptide of claim 3, wherein the Fc portion comprises the amino acid sequence of SEQ ID NO: 13 or 14.

9. 9. The sustained-release NGF polypeptide of claim 8, wherein the sustained-release NGF polypeptide comprises the amino acid sequence of SEQ ID NO:

65.

10. and / or the Fc portion comprises a N297A mutation relative to SEQ ID NO:8; and / or 4. The sustained-release NGF polypeptide of claim 3, wherein the Fc portion comprises the amino acid sequence of SEQ ID NO: 9 or 10.

11. 11. The sustained-release NGF polypeptide of claim 10, wherein the sustained-release NGF polypeptide comprises the amino acid sequence of SEQ ID NO:

61.

12. the Fc portion is derived from an IgG4 Fc; and / or the Fc portion comprises the amino acid sequence of SEQ ID NO: 17; and / or the Fc portion comprises a mutation at one or more positions selected from S228, F234 and L235 relative to SEQ ID NO: 17; and / or the Fc portion comprises one or more mutations selected from S228P, F234A and L235A relative to SEQ ID NO: 17; and / or 3. The sustained-release NGF polypeptide of claim 1 or 2, wherein the Fc portion comprises the amino acid sequence of SEQ ID NO:

18.

13. 13. The sustained-release NGF polypeptide of claim 12, wherein the sustained-release NGF polypeptide comprises the amino acid sequence of SEQ ID NO:

67.

14. has a half-life of at least 10 hours when administered to a human individual by intravenous, intramuscular, intraocular or subcutaneous injection; and / or 3. The sustained-release NGF polypeptide of claim 1 or 2, which causes less pain compared to an NGF polypeptide comprising an NGF portion having the amino acid sequence of SEQ ID NO: 3 or 4.

15. An isolated nucleic acid encoding the sustained-release NGF polypeptide according to any one of claims 1 to 14.

16. A vector comprising the isolated nucleic acid of claim 15.

17. A host cell comprising the vector of claim 16.

18. A pharmaceutical composition comprising a sustained-release NGF polypeptide according to any one of claims 1 to 14, or an isolated nucleic acid according to claim 15, or a vector according to claim 16, or a host cell according to claim 17, and a pharma- ceutically acceptable carrier and / or excipient.

19. for use in treating an NGF-related disorder in an individual, The NGF-related disorder is a neurological disorder or a non-neurological disorder; The nervous system disease is selected from the group consisting of neonatal hypoxic-ischemic encephalopathy, cerebral palsy, critical illness myopathy, neurogenic deafness, recurrent laryngeal nerve injury, traumatic brain injury, dental nerve injury, stroke, Down's syndrome, amyotrophic spinal lateral sclerosis, multiple sclerosis, spinal muscular atrophy, diffuse brain injury, thymic dysplasia, optic nerve contusion, follicular dysplasia, spinal cord injury, glaucoma, neurotrophic keratitis, optic nerve injury, neuromyelitis optica, retina-related disease, urinary incontinence, Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, hypertensive cerebral hemorrhage neurological dysfunction, cerebral small vessel disease, acute ischemic stroke, corneal endothelial dystrophy, diabetic neuropathy, diabetic foot ulcer, neurogenic skin ulcer, pressure ulcer, neurotrophic corneal ulcer, diabetic corneal ulcer, and macular hole; and / or 19. The pharmaceutical composition of claim 18, wherein the non-neurological disease is selected from the group consisting of splenic atrophy, splenic contusion, diminished ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian remnant syndrome, follicular dysplasia, spermatogenic disorders, rheumatic ulcers, and burns.

20. The pharmaceutical composition described in claim 18, wherein the spermatogenesis disorder is selected from the group consisting of oligozoospermia, asthenozoospermia and oligozoospermia.

21. The pharmaceutical composition is administered at a dose of 0.01 μg to 1000 μg per individual, and / or the pharmaceutical composition is administered at a dosing frequency of once per week or once per month; and / or 20. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is administered orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, intravaginally, intrarectally, or intraocularly, or is administered topically.

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