WO2024140625A1 - Method for screening and/or identifying mutable site in ngf, and method for screening and/or identifying ngf mutant - Google Patents

Method for screening and/or identifying mutable site in ngf, and method for screening and/or identifying ngf mutant Download PDF

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WO2024140625A1
WO2024140625A1 PCT/CN2023/141728 CN2023141728W WO2024140625A1 WO 2024140625 A1 WO2024140625 A1 WO 2024140625A1 CN 2023141728 W CN2023141728 W CN 2023141728W WO 2024140625 A1 WO2024140625 A1 WO 2024140625A1
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ngf
mutant
binding
amino acid
fusion protein
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PCT/CN2023/141728
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French (fr)
Chinese (zh)
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李平
晏丽
周志文
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舒泰神（北京）生物制药股份有限公司
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Publication of WO2024140625A1 publication Critical patent/WO2024140625A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/50—Mutagenesis

Definitions

  • NGF binds to the TrkA receptor (tyrosine kinase receptor) and p75NTR (p75 neurotrophin receptor).
  • TrkA receptor tyrosine kinase receptor
  • p75NTR p75 neurotrophin receptor
  • the TrkA receptor is a functional receptor for NGF. The binding of NGF to the TrkA receptor can promote the survival of neurons (Canu, N., et al. Int J Mol Sci. 18 (2017) 1319).
  • p75NTR has been reported to bind to all neurotrophic factors and is associated with cell apoptosis during the generation of neurons, it is not essential for NGF to achieve its biological activity and function, and may be more of an auxiliary receptor (Barker, PA & Shooter, EM Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to TrkA on PC12 cells. Neuron 13, 203-215 (1994)). It is known that the same phenotype is observed in NGF knockout mice and TrkA knockout mice.
  • NGF plays an important role in the transmission of physiological and pathological pain.
  • Local and systemic administration of NGF can induce hyperalgesia and allodynia (Lewin et al., 1994 Eur. J. Neurosci. 6: 1903-1912).
  • intravenous infusion of NGF produces systemic myalgia, while local administration, in addition to systemic effects, can also cause allodynia at the injection site (Apfel., 1998 Neurology 51: 695-702), which also limits its use to a certain extent. Therefore, current research is also developing recombinant NGF that can reduce pain or be painless during application, so as to increase the dosage and the number of subjects (see, for example, patent CN 109153709 B).
  • non-opioid analgesics such as nonsteroidal anti-inflammatory drugs (NASIDs)
  • NASIDs nonsteroidal anti-inflammatory drugs
  • opioid analgesics are mainly used.
  • NASIDs have a "ceiling effect", and opioids can only effectively relieve less than 30% of non-tumor chronic pain, and 20% of cancer pain patients have opioid tolerance.
  • existing analgesics also have mild to severe adverse reactions, which are particularly evident during long-term use.
  • NGF inhibitors such as anti-NGF antibodies
  • the present application relates to a method for screening and/or identifying mutable sites in NGF, wherein the amino acid mutation at the mutable site will affect the binding of the NGF mutant to one or more anti-NGF antibodies, but still enable the NGF mutant to have biological activity.
  • step (i) uses a homology modeling method to construct and obtain the three-dimensional structure of the NGF antibody, preferably using the Modeller homology modeling method.
  • the method for screening NGF mutants comprises the following steps: (i) obtaining one or more NGF mutants; (ii) screening or identifying a series of NGF mutants from step (i) that have biological activity and have reduced binding to or essentially no binding to anti-NGF antibodies.
  • the NGF mutants described in step (i) above are obtained from an NGF mutant library, or obtained by rational design.
  • the NGF mutant library is obtained by any of the methods for constructing an NGF mutant library as described above, or is selected from the NGF mutant library as described above.
  • the NGF mutant is obtained by screening using a method for screening NGF mutants as described above.
  • the present application relates to an NGF mutant, which comprises amino acid mutations at one or more of the I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 positions relative to the human wild-type mature NGF amino acid sequence.
  • the NGF mutants described herein comprise amino acid mutations at positions S19 and S17 relative to the amino acid sequence of wild-type mature NGF.
  • the NGF mutants described herein comprise amino acid mutations at positions R59 and T83 relative to the amino acid sequence of human wild-type mature NGF.
  • the amino acid mutation at position T56 in the NGF mutant described herein is T56K or T56R.
  • the amino acid mutation at position R59 in the NGF mutant described herein is R59K.
  • the amino acid at position T83 in the NGF mutant described herein is mutated to T83K.
  • the NGF mutant in the fusion protein is connected to Fc, preferably, through a peptide linker, more preferably, the peptide linker is composed of amino acid residues G and S; for example, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 87). In some embodiments, the NGF mutant in the fusion protein is located at the N-terminus and/or C-terminus of Fc.
  • the present application also relates to an isolated nucleic acid encoding any of the NGF mutants described above or a fusion protein comprising an NGF mutant, a vector comprising the nucleic acid, a host cell comprising the nucleic acid or the vector (e.g., CHO cells, HEK 293 cells, Hela cells or COS cells), compositions (e.g., pharmaceutical compositions), kits, and products comprising any NGF mutant described herein or fusion proteins comprising NGF mutants, and methods for preparing the same.
  • a vector comprising the nucleic acid
  • a host cell comprising the nucleic acid or the vector
  • compositions e.g., pharmaceutical compositions
  • kits e.g., and products comprising any NGF mutant described herein or fusion proteins comprising NGF mutants, and methods for preparing the same.
  • the present application relates to a method for treating and/or preventing a disease or condition caused by increased NGF expression and/or enhanced sensitivity to NGF in an individual in need thereof, comprising administering to the individual an effective amount of an anti-NGF antibody and an NGF mutant or a fusion protein comprising an NGF mutant, or a pharmaceutical composition comprising an anti-NGF antibody and an NGF mutant as described above, wherein the NGF mutant has weakened binding or essentially no binding to the anti-NGF antibody while still having biological activity.
  • the diseases or conditions caused by increased NGF expression and/or enhanced sensitivity to NGF described in the above methods include inflammatory pain, postoperative incisional pain, neuropathic pain, fracture pain, gouty joint pain, postherpetic neuralgia, pain caused by burns, cancer pain, osteoarthritis or rheumatoid arthritis pain, sciatica, pain associated with sickle cell crisis, or herpetic neuralgia.
  • the weakened binding of the NGF mutant to the anti-NGF antibody means that the binding activity of the NGF mutant to the anti-NGF antibody is reduced compared with human wild-type mature NGF (SEQ ID NO: 1) or NGF F12E (SEQ ID NO: 83).
  • the binding activity of the NGF mutant with the anti-NGF antibody does not exceed 50% of the binding activity of human wild-type mature NGF or NGF F12E with the same anti-NGF antibody, and is preferably not more than 40%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.6%, 0.5%, 0.2%, 0.1%, 0.07%, 0.02%, 0.01%, 0.001% or less.
  • the NGF mutants do not substantially bind to anti-NGF antibodies.
  • the biological activity of the NGF mutant is characterized by its binding activity to the TrkA receptor.
  • FIG8 shows the results of an in vivo pain threshold test experiment in rats using exemplary NGF mutant and Fc fusion proteins 1A2-Fc and 1A10-Fc.
  • control sequence refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • vector refers to a nucleic acid molecule that is capable of amplifying another nucleic acid molecule to which it is attached.
  • the term includes vectors that are self-replicating nucleic acid structures as well as 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 attached. Such vectors are referred to herein as "expression vectors.”
  • nerve growth factor plays an important role in the transmission of physiological and pathological pain.
  • antagonism of NGF function can prevent hyperalgesia and allodynia in neuropathic pain and chronic inflammatory pain models.
  • animal models of neuropathic pain such as nerve trunk or spinal nerve ligation
  • systemic injection of anti-NGF neutralizing antibodies can prevent both allodynia and hyperalgesia (Ramer et al., 1999, Eur. J. Neurosci. 11: 837-846; and Ro et al., 1999, Pain 79: 265-274).
  • the binding activity between the molecules described in the present application 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), Meso Scale Discovery (MSD) electrochemiluminescence, bead-based multiple immunoassay (MIA), radioimmunoassay (RIA), surface plasmon resonance (SPR), ECL, IRMA, EIA, Biacore assay, Octet analysis, peptide scanning, biomembrane interferometry (BLI), equilibrium dialysis, antigen-binding precipitation, etc.
  • ELISA enzyme-linked immunosorbent assay
  • MSD Meso Scale Discovery
  • MIA bead-based multiple immunoassay
  • RIA radioimmunoassay
  • SPR surface plasmon resonance
  • ECL IRMA
  • EIA IRMA
  • Biacore assay Octet analysis, peptide scanning, biomembrane
  • protein microarrays can be used to measure binding activity.
  • Protein microarrays can be used for large-scale analysis of the interaction, function and activity of wild-type NGF or NGF mutants described herein with their receptors.
  • the protein microarray has a support surface that is combined with a series of capture proteins (e.g., TrkA receptors or their subunits). Fluorescently labeled probe molecules (e.g., wild-type NGF or NGF mutants described herein) are then added to the array and interact with the combined capture proteins, releasing fluorescent signals and reading them by a laser scanner.
  • binding activity can be measured using SPR (Biacore T-200).
  • SPR Biacore T-200
  • anti-human IgG antibodies are coupled to the surface of a CM-5 sensor chip using EDC/NHS chemistry.
  • TrkA-mFc fusion protein or anti-NGF antibody is then used as a capture ligand on the surface.
  • a series of dilutions of wild-type NGF and NGF mutants described herein are bound to the captured ligand, and the binding and dissociation of wild-type NGF or NGF mutants or fusion proteins containing them with TrkA or anti-NGF antibodies can be monitored in real time.
  • the dissociation constant (Kd) and the dissociation rate constant can be determined by kinetic analysis using BIA evaluation software.
  • the binding activity can be measured using an ELISA assay.
  • ELISA has become the most commonly used experimental method by medical laboratories, in vitro diagnostic product manufacturers, regulatory agencies, and the like.
  • EIA Enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding activity is characterized by the EC50 value of the ELISA experiment. The smaller the EC50 value, the stronger the affinity between the molecules and the higher the binding activity.
  • the ratio between the EC50 values can be used as a quantitative indicator of binding activity. For example, in an ELISA test, if the EC50 value between molecules A and B is 10 times the EC50 value between molecules C and B, it means that the binding activity between molecules A and B is 10% of the binding activity between molecules C and B.
  • the "biological activity" of NGF can be characterized from one or more of the following aspects, including but not limited to: the ability to bind to NGF receptors (such as TrkA); the ability to promote TrkA receptor dimerization and/or TrkA receptor autophosphorylation; the ability to activate NGF receptor signaling pathways; the ability to promote cell differentiation, proliferation, survival, growth, and other changes in cell physiology, including changes in neuronal morphology, synaptogenesis, synaptic function, neurotransmitter and/or neuropeptide release, and regeneration after injury (for neurons including peripheral and central neurons); the ability to promote the survival of mouse E13.5 trigeminal neurons and the ability to mediate pain, including post-operative pain.
  • NGF receptors such as TrkA
  • TrkA receptor dimerization and/or TrkA receptor autophosphorylation the ability to activate NGF receptor signaling pathways
  • the biological activity can be characterized by the binding activity of wild-type NGF or NGF mutants to TrkA receptors, see Example 5 below.
  • the biological activity of NGF can be evaluated by TF-1 cell proliferation assay, as described in CN103376248A, CN108727486A or CN114829384A, all of which are incorporated herein by reference, and see also Example 6 below.
  • the biological activity can also be determined based on PC12 cell activity detection experiments, see Example 7 below.
  • the biological activity can also be determined based on the ability to promote the growth of chicken embryo dorsal root ganglia, as described in WO2017157326 or CN114829384A, all of which are incorporated herein by reference, and see also Example 8 below or the ability to promote the growth of neonatal rat superior cervical ganglia (SCG) (see Example 9 below).
  • Any suitable assay protocol known in the art is suitable for testing the biological activity of NGF, NGF mutants, or fusion proteins comprising the same as described herein.
  • NGF's biological activity include: (i) supporting neuronal survival; (ii) promoting neurite outgrowth; (iii) enhancing neurochemical differentiation; (iv) promoting pancreatic beta cell proliferation; (v) inducing innate and/or acquired immunity; (vi) repairing damaged nerve cells and/or preventing damage (e.g., neurotrophic keratitis); (vii) promoting proliferation and/or estrogen secretion of ovarian granulosa cells; (viii) promoting wound healing (e.g., in diabetic neuropathy); (ix) improving the condition of patients with neurotrophic keratitis.
  • 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.
  • the biological activity of wild-type NGF or NGF mutants described herein on cells is determined by immunohistochemistry to locate signal proteins.
  • signal proteins e.g., antibodies to signal proteins themselves or in an activated state can be used. These antibodies have recognition epitopes, including phosphates or other activation conformations.
  • the movement of a specific signal protein e.g., nuclear translocation of a signal molecule
  • GFP green fluorescent protein
  • the biological activity of the wild-type NGF or NGF mutant described herein on cells is detected by western blot.
  • all tyrosine phosphorylated proteins or other phosphorylated amino acids, such as serine or threonine
  • anti-phosphotyrosine antibodies or antibodies against other phosphorylated amino acids
  • the expression of NGF in the wild-type NGF or NGF mutants described herein can be detected in Western blot of the cell lysate obtained after sequential stimulation.
  • the biological activity of the wild-type NGF or NGF mutants described herein on cells can be determined by immunoprecipitation.
  • a primary antibody against a specific signaling protein or all tyrosine phosphorylated proteins is cross-linked to the microbeads.
  • Cells incubated with the wild-type NGF or NGF mutants described herein are lysed in a buffer containing protease inhibitors and then incubated with antibody-coated microbeads. Proteins are separated using SDS electrophoresis and then identified by the Western blot step.
  • GST glutathione S-transferase
  • pulse-down analysis can also be used to determine direct protein-protein (e.g., signaling proteins) interactions.
  • PI 3-kinase signals can also be measured using any suitable method known in the art to reflect the biological activity of NGF.
  • PI 3-kinase signaling can be measured using an antibody specific for phosphorylated S6 ribosomal protein (optionally in combination with flow cytometry analysis).
  • TF-1 cell proliferation assay a serial dilution of a sample (e.g., NGF mutant) and a control group (e.g., human wild-type mature NGF or NGF F12E ) is prepared in a 96-well plate, and then TF-1 cells are added to each well and cultured in a humidified incubator at 37°C and 5% CO2 . After several days of culture (e.g., 3 days), an MTS solution is added to each well of the cell suspension and cultured for 3 hours at 37°C and 5% CO2 .
  • a sample e.g., NGF mutant
  • a control group e.g., human wild-type mature NGF or NGF F12E
  • the absorbance at 490nm and 650nm is measured in a spectrophotometer to illustrate the effects of NGF and NGF mutants on the proliferation of TF-1 cells.
  • the data can be standardized based on the control sample. See Example 6 for an example method.
  • a sample e.g., NGF mutant
  • a control group e.g., PBS, human wild-type mature NGF, or NGF F12E
  • the rats were injected with NGF at the neck of the rats. A few days after the injection, the rats were sacrificed for SCG isolation. SCG can be weighed and the morphology recorded to study the biological activity of wild-type NGF or NGF mutants in promoting the growth of SCG in vivo. See Example 9 for an example method.
  • chick embryo dorsal root ganglia e.g., 8-day-old
  • samples containing different concentrations e.g., NGF mutants
  • control groups e.g., PBS, Mouse NGF, human wild-type mature NGF, NGF F12E
  • Monitor the growth of dorsal root ganglia which can reflect the biological activity of wild-type NGF or NGF mutants in promoting the growth of dorsal root ganglia.
  • the specific biological activity of the sample can also be calculated and expressed in AU/mg.
  • AU/mg the specific activity of the control
  • AU/ml the specific activity of the control
  • AU/ml the specific activity of the control at this dilution point
  • Example 5 in WO2017157326 See Example 5 in WO2017157326.
  • the binding activity to the TrkA receptor it is preferred to use to characterize its biological activity.
  • the NGF mutant has biological activity
  • the biological activity of the NGF mutant is characterized by its binding activity to the TrkA receptor.
  • the binding activity of the NGF mutant to the TrkA receptor is 50% of the binding activity of the wild-type NGF to the TrkA receptor
  • it may mean that the biological activity of the NGF mutant is 50% of the biological activity of the wild-type NGF.
  • the present application provides a method for screening and/or identifying a mutable site in NGF, wherein the amino acid mutation at the mutable site affects the binding of the NGF mutant to one or more anti-NGF antibodies, but still enables the NGF mutant to have biological activity.
  • the amino acid mutation at the mutable site affects the binding of the NGF mutant to the anti-NGF antibody, which means that the NGF mutant containing the above-mentioned amino acid mutation has a weakened binding to the anti-NGF antibody or basically does not bind to the anti-NGF antibody.
  • the method for screening and/or identifying mutable sites in NGF comprises the following steps:
  • step (iii) aligning the binding conformation obtained in step (ii) and the binding conformation of mature NGF and TrkA receptor respectively;
  • step (iv) Based on the comparison results in step (iii), obtain the mutable sites in the NGF.
  • a person skilled in the art may apply any method known in the art to obtain the three-dimensional structure of the anti-NGF antibody, including but not limited to the application of experimental methods, such as X-crystal diffraction and nuclear magnetic resonance (NMR), cryo-electron microscopy, etc., or the application of computational methods to predict its tertiary structure based on the protein sequence.
  • methods for predicting the tertiary structure of proteins include but are not limited to: homology modeling, threading, and ab initio, etc.
  • the homology modeling method includes, but is not limited to SWISS-MODEL, Modeller, or Phyre2.
  • the three-dimensional structure of the anti-NGF antibody described in the present application is obtained by homology modeling.
  • the three-dimensional structure of the anti-NGF antibody described in the present application is obtained by the Modeller homology modeling method.
  • the three-dimensional structure of the anti-NGF antibody obtained in step (i) of the above method can be any antibody or antigen-binding fragment that binds to the NGF target, for example, including, but not limited to, one or more of the following antibodies: Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Biopharma), SSS-40 (Sunshine Guojian) and STC001 (Sultaishen).
  • Tanezumab Pfizer
  • Fasinumab Regeneron
  • Fulranumab Johnson & Johnson
  • MEDI-578 AstraZeneca
  • AK-115 Kerangfang Biopharma
  • SSS-40 Unshine Guojian
  • STC001 Sultaishen
  • step (ii) in the above method a person skilled in the art can use any method known in the prior art to dock the mature NGF crystal structure with the above-obtained three-dimensional structure of the anti-NGF antibody to obtain the binding conformation of the anti-NGF antibody and NGF.
  • the crystal structure of mature NGF is shown in PDB ID: 4EDW.
  • the Discovery studio ZDOCK molecular docking technology is used to molecularly dock the mature NGF crystal structure with the three-dimensional structure of the anti-NGF antibody obtained above to obtain the binding conformation of the anti-NGF antibody and NGF.
  • the predicted results of the binding conformation are displayed according to the scoring function value from high to low, and the other parameters are automatically defaulted by the software.
  • the binding conformation with the highest scoring function value in Discovery studio ZDOCK is selected as the binding conformation of the anti-NGF antibody and NGF.
  • the mutable sites in NGF obtained by comparison refer to amino acid sites in NGF that are located at the binding interface with the anti-NGF antibody but not at the binding interface with the TrkA receptor. In some embodiments, if multiple anti-NGF antibodies are involved, the mutable sites are the intersection or union of multiple groups of mutable sites obtained for various anti-NGF antibodies.
  • the present application provides a mutable site in NGF, and amino acid mutations at the mutable site will affect the binding of the NGF mutant to one or more anti-NGF antibodies, but still enable the NGF mutant to have biological activity.
  • the amino acid mutation affects the binding of the NGF mutant to one or more anti-NGF antibodies, which means that the binding of the NGF mutant containing the above amino acid mutation to the anti-NGF antibody is weakened or substantially does not bind to the anti-NGF antibody.
  • the mutable sites in NGF described herein can be obtained by the screening and/or identification methods of the mutable sites in NGF as described above.
  • the mutable sites in the NGF are a group of mutable sites for a specific anti-NGF antibody, that is, amino acid mutations at the mutable sites will weaken or substantially eliminate the binding of the NGF mutant to the specific anti-NGF antibody.
  • a group of mutable sites for each anti-NGF antibody can be obtained by implementing the method of the present application.
  • the mutable sites in the NGF described in the present application can be the intersection of two or more groups of mutable sites obtained for two or more anti-NGF antibodies, or The latter is the union of two or more groups of mutable sites obtained for two or more anti-NGF antibodies respectively.
  • the mutable sites in NGF described in the present application are a group of mutable sites obtained for any one of the following anti-NGF antibodies: Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Biopharma), SSS-40 (Sunshine Guojian) and STC001 (Shortacion).
  • the mutable sites in NGF described in the present application are sites included in each group of mutable sites obtained for Tanezumab (Pfizer), Fasinumab (Regeneron), MEDI-578 (AstraZeneca), AK-115 (Akeso Biopharma), and STC001 (Sultaisin), respectively.
  • the mutable sites in NGF described in the present application are the union of each group of mutable sites obtained for two or more anti-NGF antibodies selected from the following: Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Biopharma), SSS-40 (Sunshine Guojian) and STC001 (Shortacion).
  • the mutable sites in NGF described in the present application are a set consisting of the groups of mutable sites obtained for Tanezumab (Pfizer), Fasinumab (Regeneron), MEDI-578 (AstraZeneca), AK-115 (Akeso Biopharma) and STC001 (Shortasen), respectively, including all sites in each group of mutable sites.
  • the mutable sites in the NGF described herein can be selected from one or more of the following sites: I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 relative to the amino acid sequence of human wild-type mature NGF.
  • the wild-type mature NGF described herein is human wild-type mature NGF.
  • the amino acid sequence of human wild-type mature NGF described herein is shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the method for constructing the NGF mutant library comprises the following steps: using human wild-type mature NGF as a template, performing amino acid mutations at one or more sites of the mutable sites in NGF to generate a series of NGF mutants, thereby obtaining the NGF mutant library.
  • the amino acid sequence of the human wild-type mature NGF is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the mutable sites in the NGF are obtained by screening and/or identification as described above. In some embodiments, the mutable sites in the NGF are selected from one or more of the following sites: I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 sites relative to the amino acid sequence of human wild-type mature NGF.
  • the NGF mutants in the NGF mutant library contain amino acid mutations at one or more of I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 relative to human wild-type mature NGF.
  • Another aspect of the present application provides a method for screening or identifying NGF mutants, wherein the NGF mutants have weakened binding to one or more anti-NGF antibodies or substantially no binding to anti-NGF antibodies while still having biological activity.
  • the present application provides an NGF mutant, which has a weakened binding to one or more anti-NGF antibodies or substantially no binding to the anti-NGF antibodies, while having biological activity.
  • the NGF mutant is obtained by the NGF mutant screening method as described above.
  • the NGF mutant is identified by the NGF mutant identification method as described above.
  • the NGF mutant is obtained by screening from the NGF mutant library as described above.
  • NGF mutants may include mutations at one or more amino acid sites in the NGF molecule (e.g., relative to human wild-type mature NGF). In some embodiments, NGF mutants include amino acid substitutions at one or more amino acid sites in NGF. In some embodiments, NGF mutants include amino acid deletions or insertions at one or more amino acid sites in NGF. In some embodiments, NGF mutants include modifications of one or more amino acids in NGF.
  • Inserting non-natural amino acids, including synthetic non-natural amino acids, substituted amino acids or one or more D-amino acids, into the NGF mutants of the present application may have a variety of benefits.
  • polypeptides containing L-amino acids polypeptides containing D-amino acids and the like exhibit higher stability in vitro and in vivo. Therefore, when better intracellular stability is required, it is particularly useful to construct polypeptides such as by adding D-amino acids.
  • D-peptides and their analogs are resistant to endogenous peptidases and protease activity, thereby increasing the bioavailability of the molecule and extending its lifespan in vivo when needed.
  • D-peptides and their analogs cannot be effectively processed because the process of presentation to helper T cells by the type II major histocompatibility complex (MHC) is limited, and therefore it is not easy to induce humoral immune responses in subjects.
  • MHC major histocompatibility complex
  • the amino acid sequence variants of the NGF mutant may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the protein or by peptide synthesis.
  • the modifications include, for example, deletion and/or insertion and/or substitution of residues in the amino acid sequence of NGF.
  • the NGF mutants described herein comprise amino acid mutations at one or more of the mutagenic sites described above.
  • the NGF mutants described in the present application comprise amino acid mutations at one or more of S17, S19, K32, T56, R59 and T83 relative to the amino acid sequence of human wild-type mature NGF.
  • the NGF mutants described herein comprise amino acid mutations at positions S19 and S17 relative to the amino acid sequence of human wild-type mature NGF.
  • the NGF mutants described herein comprise amino acid mutations at positions R59 and T83 relative to the amino acid sequence of human wild-type mature NGF.
  • the amino acid at position S17 is mutated to S17R, S17K, S17H or S17E.
  • the amino acid at position S19 is mutated to S19R, S19K or S19F.
  • the amino acid at position K32 is mutated to K32F, K32E, K32N, K32Y, K32M or K32L.
  • the NGF mutant described herein, wherein the amino acid at the T56 position is mutated to T56K or T56R.
  • the amino acid at position R59 is mutated to R59K.
  • the amino acid at position T83 is mutated to T83K.
  • the NGF mutant described in the present application comprises amino acid mutations at positions S17 and K32 relative to the wild-type mature NGF amino acid sequence. In some embodiments, the NGF mutant described in the present application comprising amino acid mutations at positions S17 and K32, wherein the amino acid mutation at position S17 is selected from S17R, S17K, S17H or S17E. In some embodiments, the NGF mutant comprising S17 and K32 sites described herein, wherein the amino acid mutation at the K32 site is selected from K32F, K32E, K32N, K32Y, K32M or K32L.
  • the NGF mutants described herein comprise amino acid mutations S17R and K32E. In some embodiments, the NGF mutants described herein comprise amino acid mutations S17R and K32Y. In some embodiments, the NGF mutants described herein comprise amino acid mutations S17R and K32M. In some embodiments, the NGF mutants described herein comprise amino acid mutations S17R and K32L.
  • the NGF mutants described herein comprise amino acid mutations at the S19 and K32 sites relative to the wild-type mature NGF amino acid sequence of humans. In some embodiments, the NGF mutants described herein comprise amino acid mutations at the S19 and K32 sites, wherein the amino acid mutation at the S19 site is selected from S19R, S19K or S19F. In some embodiments, the NGF mutants described herein comprise S19 and K32 sites, wherein the amino acid mutation at the K32 site is selected from K32F, K32E, K32N, K32Y, K32M or K32L.
  • the NGF mutants described herein comprise amino acid mutations S19R and K32E. In some embodiments, the NGF mutants described herein comprise amino acid mutations S19R and K32Y. In some embodiments, the NGF mutants described herein comprise amino acid mutations S19R and K32M. In some embodiments, the NGF mutants described herein comprise amino acid mutations S19R and K32L.
  • the NGF mutants described herein comprise amino acid mutations at the S19 and S17 sites relative to the wild-type mature NGF amino acid sequence of humans. In some embodiments, the NGF mutants described herein comprise amino acid mutations at the S19 and S17 sites, wherein the amino acid mutation at the S19 site is selected from S19R, S19K or S19F. In some embodiments, the NGF mutants described herein comprise amino acid mutations at the S19 and S17 sites, wherein the amino acid mutation at the S17 site is selected from S17R, S17K, S17H or S17E.
  • the NGF mutants described herein comprise amino acid mutations at positions R59 and T83 relative to the wild-type mature NGF amino acid sequence of humans. In some embodiments, the NGF mutants described herein comprise amino acid mutations R59K and T83K.
  • amino acid sequence of human wild-type mature NGF described in the present application is shown in SEQ ID NO:1 or SEQ ID NO:2.
  • the NGF mutant described in the present application comprises an amino acid sequence shown in any one of SEQ ID NOs: 17-28 and 41-50 or a variant thereof, wherein the variant is different from any one of SEQ ID NOs: 17-28 and 41-50.
  • the amino acid sequences shown have at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • the NGF mutants compared with wild-type NGF, increase the pain threshold of at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%) at one or more time points (e.g., all time points) after administration.
  • the pain threshold of an individual is measured to be about 8, and after administration of wild-type NGF, the pain threshold drops to about 6, and after administration of the NGF mutant, the pain threshold remains at about 8, indicating that compared to wild-type NGF, the pain of the NGF mutant is reduced by about 25%.
  • the NGF mutant comprises mutations as described in patents CN107286233A, WO2017157325A1, and WO2017157326A2, all of which are incorporated herein by reference.
  • the NGF mutant described herein further comprises a mutant F12E relative to human wild-type mature NGF, for example, NGF F12E (SEQ ID NO: 83).
  • the NGF mutant described in the present application comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29-40 and 51-60 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 29-40 and 51-60.
  • the NGF mutants described in the present application have reduced binding to or substantially no binding to anti-NGF antibodies, meaning that compared to wild-type NGF (e.g., human wild-type mature NGF) or NGF F12E , the binding activity of the NGF mutants to the same anti-NGF antibody is reduced, or even no binding to the anti-NGF antibody.
  • wild-type NGF e.g., human wild-type mature NGF
  • NGF F12E e.g., human wild-type mature NGF
  • the binding activity of the NGF mutants to the same anti-NGF antibody is reduced, or even no binding to the anti-NGF antibody.
  • the NGF mutant described in the present application has reduced binding to or essentially no binding to the anti-NGF antibody, which means that in the same detection experiment, its binding activity does not exceed 50% of the binding activity of wild-type NGF (e.g., human wild-type mature NGF) or NGF F12E to the same anti-NGF antibody, for example, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.6%, 0.5%, 0.2%, 0.1%, 0.07%, 0.02%, 0.01%, 0.001%, 0.0001% or less.
  • wild-type NGF e.g., human wild-type mature NGF
  • NGF F12E e.g., human wild-type mature NGF
  • the binding activity with anti-NGF antibodies described in the present application can be detected by ELISA experiments, Biacore experiments, or BLI experiments.
  • the binding activity of the NGF mutants described in the present application with anti-NGF antibodies is detected by ELISA experiments. In other embodiments, the binding activity of the NGF mutants described in the present application with anti-NGF antibodies is characterized by the EC50 value in the ELISA experiment.
  • the NGF mutant described in the present application has weakened binding to or essentially no binding to the anti-NGF antibody, which means that in an ELISA binding experiment, the EC50 value of the binding of the NGF mutant to the anti-NGF antibody is at least 2 times, for example, 2 times, 3 times, 4 times, 5 times, 7 times, 9 times, 10 times, 13 times, 14 times, 15 times, 17 times, 20 times, 25 times, 33 times, 50 times, 100 times, 167 times, 200 times, 500 times, 1000 times, 1400 times, 5000 times, 10000 times, 100000 times, 100000 times or more of the EC50 value of the binding of human wild-type mature NGF (SEQ ID NO: 1 or 2) or NGF F12E (SEQ ID NO: 83) to the same anti-NGF antibody.
  • human wild-type mature NGF SEQ ID NO: 1 or 2
  • NGF F12E SEQ ID NO: 83
  • the EC50 value of the binding of the NGF mutant described in the present application to the anti-NGF antibody is at least 2 times the EC50 value of the binding of human wild-type mature NGF (SEQ ID NO: 1 or 2) or NGF F12E (SEQ ID NO: 83) to the same anti-NGF antibody, for example, 2 times, 3 times, 4 times, 5 times, 7 times, 9 times, 10 times, 13 times, 14 times, 15 times, 17 times, 20 times, 25 times, 33 times, 50 times, 100 times, 167 times, 200 times, 500 times, 1000 times, 1400 times, 5000 times, 10000 times, 100000 times, 1000000 times or more.
  • the NGF mutant described in the present application still has the biological activity of NGF, which means that compared with NGF F12E (SEQ ID NO: 83), it has a biological activity that is substantially equivalent to, higher, or slightly lower than that of NGF F12E (SEQ ID NO: 83).
  • the biological activity described in the present application refers to the ability to induce PC12 cell differentiation in the PC12 cell activity detection experiment. In some embodiments, the biological activity described in the present application refers to the ability to promote the differentiation of chicken embryo dorsal root ganglia in the chicken embryo dorsal root ganglion experiment. In some embodiments, the biological activity described in the present application refers to the ability to promote the growth of rat superior cervical ganglia in the rat superior cervical ganglion experiment.
  • the biological activity of the NGF mutant described in the present application is characterized by its binding activity to the TrkA receptor.
  • the NGF mutant described in the present application has biological activity, which means that the mutant is able to bind to the TrkA receptor.
  • the NGF mutant described in the present application is able to bind to the TrkA receptor, which means that compared with NGF F12E (SEQ ID NO: 83), the binding activity of the NGF mutant to the TrkA receptor is substantially equivalent to, higher than, or slightly lower than that of NGF F12E (SEQ ID NO: 83).
  • the NGF mutant described in the present application is capable of binding to the TrkA receptor, which means that compared with NGF F12E (SEQ ID NO: 83), its binding activity to the TrkA receptor is at least 20% of the binding activity of NGF F12E to the TrkA receptor, for example, at least 21%, 30%, 36%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
  • the EC50 value of the binding of the NGF mutant to the TrkA receptor is 10 ⁇ g/ml, 6 ⁇ g/ml, 5 ⁇ g/ml, 1 ⁇ g/ml, 0.5 ⁇ g/ml, 0.1 ⁇ g/ml, 0.01 ⁇ g/ml or lower.
  • the well plate is coated with the NGF mutant, and anti-NGF antibodies or TrkA are added for detection.
  • a 96-well plate is used, and the concentration of the NGF mutant is 0.1 ⁇ g/well.
  • the fusion protein of the NGF mutant and Fc wherein Fc is from IgG4 Fc, such as human IgG4 Fc.
  • Fc is wild-type IgG4 Fc (e.g., human IgG4 Fc) or a natural mutant thereof.
  • the Fc comprises an S228P mutation relative to wild-type human IgG4 Fc, wherein the numbering is the EU numbering system.
  • a fusion protein of an NGF mutant and Fc from N-terminus to C-terminus or from C-terminus to N-terminus: comprising an NGF mutant and an Fc portion.
  • the optional peptide linker comprises the amino acid sequence (GGGGS)n, wherein n is any one of 1, 2, 3, 4, 5 or 6. In some embodiments, the peptide linker comprises the amino acid sequence (GGGGS) 3 (SEQ ID NO: 87).
  • the Fc part comprises the amino acid sequence SEQ ID NO:85 or its variant, and the variant has at least 90% (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO:85.
  • the fusion protein of the NGF mutant and Fc comprises an amino acid sequence shown in any one of SEQ ID NOs:61-82 or a variant thereof, and the variant has at least 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs:61-82.
  • the Fc portion is from any one of IgA, IgD, IgE, IgG and IgM and their subclasses. Among all immunoglobulins, IgG has the highest serum content and the longest half-life. Unlike other immunoglobulins, IgG can be effectively recovered after binding to Fc receptors (FcRs).
  • the Fc portion is from IgG (e.g., IgG1, IgG2, IgG3 or IgG4). In some embodiments, the Fc portion is from human IgG. In some embodiments, the Fc portion comprises CH2 and CH3. In some embodiments, the Fc portion further comprises all or part of the hinge region.
  • any one and more of the following residues may be substituted with cysteine: heavy chain A118 (EU numbering system) and heavy chain Fc domain S400 (EU numbering system).
  • cysteine heavy chain A118 (EU numbering system) and heavy chain Fc domain S400 (EU numbering system).
  • Cysteine engineered molecules can be produced as described in U.S. Patent No. 7,521,541.
  • the wild-type NGF or NGF mutant and Fc are connected by an optional linker (e.g., a peptide linker, a non-peptide linker).
  • the linker is a flexible linker.
  • the linker is a stable linker. In general, an ideal linker will not affect or significantly affect the correct folding and conformation of the fusion protein of the NGF mutant and Fc described herein.
  • the linker confers flexibility to the fusion protein of the NGF mutant and Fc, retains or improves the biological activity of NGF, and/or does not significantly affect the half-life and/or stability of the fusion protein of the NGF mutant and Fc in vivo.
  • the linker is a stable linker (e.g., cannot be cleaved by a protease, especially a MMP).
  • the linker is a peptide linker.
  • the peptide linker can be of any length.
  • the peptide linker is from 1 to 10 amino acids, from 3 to 18 amino acids, from 1 to 20 amino acids, from 10 to 20 amino acids, from 21 to 30 amino acids, from 1 to 30 amino acids, from 1 to 30 amino acids, from 10 to 30 amino acids, from 1 to 50 amino acids, from 5 to 40 amino acids, from 12 to 18 amino acids, from 4 to 25 amino acids in length.
  • 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 length.
  • the circulating half-life of the fusion protein of the NGF mutant and Fc described herein is at least 5 times (e.g., any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 times, or more) the circulating half-life of the corresponding NGF portion (i.e., comprising only the same NGF mutant without fusion with Fc) or wild-type NGF.
  • the stability of proteins depends mainly on the conformation and colloidal stability of protein molecules. It is generally believed that the first step of non-native protein aggregation, i.e., the most common form of aggregation, is a slight perturbation of the molecular structure, such as partial unfolding of the protein, i.e., conformational change. This is determined by the conformational stability of the protein. In the second step, partially unfolded molecules approach each other under the drive of diffusion and random Brownian motion to form aggregates.
  • the second step is mainly determined by the colloidal stability of the molecules (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).
  • the term "stability” generally refers to maintaining the integrity of biologically active substances (such as proteins) or minimizing their degradation, denaturation, aggregation, or unfolding.
  • a “stable" protein e.g., a long-acting NGF polypeptide as described herein, substantially maintains its physical stability and/or chemical stability and/or biological activity during manufacturing and/or storage.
  • stable protein or formulation
  • a protein e.g., a fusion protein of an NGF mutant and Fc described herein, "retains its physical stability" in a formulation if it shows substantially no signs of instability, such as aggregation, precipitation, and/or denaturation, when measured by visual inspection of color and/or clarity or by UV light scattering or size exclusion chromatography.
  • Aggregation is the process by which individual protein molecules or complexes covalently or non-covalently associate to form aggregates. Aggregation can proceed to the extent that a visible precipitate forms.
  • the stability of proteins depends on other characteristics in addition to the composition of the formulation. For example, stability may be affected by temperature, pressure, humidity, pH, and external radiation.
  • the stability of proteins (e.g., the fusion proteins of NGF mutants and Fc described herein) in protein formulations can be determined by a variety of methods. In some embodiments, protein stability is determined by size exclusion chromatography (SEC). SEC separates analytes according to their hydrodynamic size, diffusion coefficient, and surface properties.
  • SEC can separate the fusion proteins of NGF mutants and Fc described herein in their native three-dimensional conformation from proteins in various denatured states and/or degraded proteins.
  • the stationary phase is typically composed of inert particles filled in a dense three-dimensional matrix in a glass or steel column.
  • the mobile phase can be pure water, an aqueous buffer, an organic solvent, a mixture thereof, or other solvents.
  • the stationary phase particles have pores and/or channels that only allow substances smaller than a certain size to enter. Therefore, large particles are excluded from these pores and channels, but smaller particles are displaced from the mobile phase.
  • the amount of time that particles are immobilized in the fixed pores depends in part on how deeply they can penetrate into the pores. Their displacement from the mobile phase flow causes them to take longer to elute from the column, thus separating the particles based on their size differences.
  • SEC is combined with identification techniques to identify or characterize proteins (e.g., NGF mutants described herein) or fragments thereof.
  • Protein identification and characterization can be accomplished by a variety of techniques, including but not limited to chromatographic techniques, such as high performance liquid chromatography (HPLC), capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), immunoassays, electrophoresis, UV/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.
  • HPLC high performance liquid chromatography
  • CE-SDS capillary electrophoresis with sodium dodecyl sulfate
  • Stability such as the physical stability of a composition or formulation
  • optical attenuation measurements are related to the turbidity of the formulation.
  • the turbidity of a formulation is an inherent property of proteins dissolved in solution, typically determined by turbidimetry and measured in nephelometric turbidity units (NTU).
  • Turbidity for example, as a function of the concentration of one or more components in a solution, e.g., protein and/or salt concentration, is also referred to as the "milky" or “milky appearance" of a formulation.
  • Turbidity can be calculated using a standard curve generated using suspensions of known turbidity.
  • Reference standards for determining the turbidity of a pharmaceutical composition can be based on European Pharmacopoeia standards (European Pharmacopoeia, 4th edition, European Commission Directorate for the Quality of Medicines (EDQM), France). According to the European Pharmacopoeia, a clear solution is defined as having a turbidity less than or equal to that of a control suspension.
  • the turbidity of the control suspension is about 3.
  • turbidity measurements can detect Rayleigh scattering, which generally varies linearly with concentration.
  • Other methods for evaluating the physical stability of pharmaceutical proteins are well known in the art, for example, size exclusion chromatography or analytical ultracentrifugation.
  • stability refers to a formulation containing a fusion protein of an NGF mutant and Fc as described herein having a level of particle formation so low that it is undetectable.
  • level of particle formation so low that it is undetectable refers to a sample 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.
  • particles in the fusion protein formulation of an NGF mutant and Fc are not detected by HIAC analysis or visual analysis.
  • the fusion protein of NGF mutant and Fc involved herein can be further modified to include additional non-protein parts known in the art and easily obtained.
  • Non-protein parts suitable for fusion protein derivatives of NGF mutant and Fc include, but are not limited to, water-soluble polymers.
  • the fusion protein of the NGF mutant and Fc described herein further comprises a tag, wherein the tag is selected from a chromophore, a fluorophore (e.g., coumarin, xanthene, cyanine, pyrene, boron polybenzazaindole, oxazine and its derivatives), a fluorescent protein (e.g., GFP, phycobiliprotein and its derivatives), a phosphorescent dye (e.g., dioxetanes, xanthenes or carbocyanine dyes, lanthanide chelates), a tandem dye (e.g., cyanine-phycobiliprotein derivatives and xanthene-phycobiliprotein derivatives), a particle (e.g., gold clusters, colloidal gold, microspheres, quantum dots), a semi-transparent fluorescent protein (e.g., a fluorescent protein such as GFP, phycobiliprotein and its derivatives
  • the fusion protein of NGF mutant and Fc can be further modified to contain one or more biologically active proteins, polypeptides or fragments thereof.
  • biological activity or “biologically active” are used interchangeably and refer to showing biological activity in vivo to perform a specific function. For example, it may mean binding to a specific biological molecule, such as a protein, DNA, etc., and then promoting or inhibiting the activity of the biological molecule.
  • the biologically active protein or fragment thereof that can be included in the fusion protein of the NGF mutant and Fc as described herein is an antigen binding protein (e.g., an antibody).
  • the biologically active protein or fragment thereof that can be included in the fusion protein of the NGF mutant and Fc as described herein is an antibody mimetic, which is a small engineered protein that contains an antigen binding domain that is pronounced of an antibody (GGeering and Fussenegger, Trends Biotechnol., 33 (2): 65-79, 2015). These molecules are derived from existing human scaffold proteins and consist of a single polypeptide.
  • antibody mimics that can be included in the fusion protein of the NGF mutant and Fc as described herein can be, but are not limited to, a designed ankyrin repeat protein (DARPin; containing 3-5 fully synthetic ankyrin repeat sequences, flanked by N-terminal and C-terminal cap domains), an affinity multimer (avimer; a high affinity protein containing multiple A domains, each domain having a low affinity for the target), or an anticoagulant (based on a lipid scaffold with four accessible loops, the sequence of each loop can be random).
  • DARPin designed ankyrin repeat protein
  • avimer avimer
  • an anticoagulant based on a lipid scaffold with four accessible loops, the sequence of each loop can be random.
  • the biologically active protein or fragment thereof that may be included in the fusion protein of the NGF mutant and Fc as described herein is an armadillo repeat protein (e.g., ⁇ -catenin, ⁇ -importin, plakoglobin, adenomatous polyposis coli (APC)), comprising an armadillo repeat unit (characteristic, the length of the repeated amino acid sequence is about 40 residues).
  • armadillo repeat unit consists of a pair of ⁇ -helices that form a hairpin structure. Multiple repeated copies form the ⁇ -solenoid structure.
  • the present application also relates to an isolated nucleic acid encoding any wild-type NGF, NGF mutant or fusion protein thereof described herein, or a vector comprising the encoding nucleic acid. It also relates to an isolated host cell (e.g., CHO cell, HEK 293 cell, Hela cell or COS cell) comprising a nucleic acid or vector encoding any wild-type NGF, NGF mutant or fusion protein thereof described herein.
  • an isolated host cell e.g., CHO cell, HEK 293 cell, Hela cell or COS cell
  • the fusion protein comprises, from N-terminus to C-terminus or from C-terminus to N-terminus: wild-type NGF or NGF mutant, an optional peptide linker and an Fc portion.
  • the wild-type NGF comprises the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2.
  • the NGF mutant comprises the amino acid sequence shown in any one of SEQ ID NOs:17-60.
  • the optional peptide linker portion comprises the amino acid sequence shown in SEQ ID NO:87.
  • the Fc is from IgG1 Fc or IgG4 Fc.
  • the Fc comprises the amino acid sequence SEQ ID NO:85.
  • the isolated nucleic acid encoding the fusion protein of wild-type NGF and Fc comprises the sequence SEQ ID NO:86.
  • the isolated nucleic acid encoding the fusion protein of NGF mutant and Fc is based on the isolated nucleic acid encoding the fusion protein of wild-type NGF and Fc, and any one or more amino acid mutations described herein are introduced on the basis of the isolated nucleic acid encoding the fusion protein of wild-type NGF and Fc.
  • the fusion protein of the NGF mutant and Fc described in the present application comprises the amino acid sequence shown in any one of SEQ ID NOs:61-82 or a variant thereof, and the variant has at least 80% (for example, at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs:61-82.
  • 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 described in detail, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals.
  • Retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo.
  • Many retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • self-inactivating lentiviral vectors are used.
  • self-inactivating lentiviral vectors carrying construct protein coding sequences 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.
  • 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, including, for example, poly (lactic acid-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly (ethyleneimine) (PEI) and dendrimers. In some embodiments, the vector is a cationic lipid-based non-viral vector, such as cationic liposomes, lipid nanoemulsions and solid lipid nanoparticles (SLN).
  • SBN Sleeping Beauty
  • the vector comprises a selectable marker gene or a reporter gene for selecting cells expressing NGF, NGF mutants, or fusion proteins thereof as described herein from a host cell population transfected with a vector (e.g., a lentiviral vector, a pTT5 vector).
  • a vector e.g., a lentiviral vector, a pTT5 vector.
  • Both the selectable marker and the reporter gene may be surrounded by appropriate regulatory sequences to enable them to be expressed in the host cell.
  • the vector may comprise a transcription and translation terminator, a start sequence, and a promoter for regulating the expression of a nucleic acid sequence.
  • any molecular cloning method known in the art can be used, including, for example, using restriction endonuclease sites and one or more selectable markers to clone nucleic acid into a vector.
  • the nucleic acid is operably linked to a promoter.
  • a variety of promoters for gene expression in prokaryotic cells or eukaryotic cells have been explored, and any promoter known in the art can be used in the present application. Promoters can be roughly divided into constitutive promoters or regulated promoters, such as inducible promoters.
  • the nucleic acid encoding the wild-type NGF, NGF mutant or fusion protein comprising the same as described herein is operably linked to an inducible promoter.
  • Inducible promoters belong to the category of regulatable promoters.
  • Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the host cell or the physiological state of the host cell, an inducer (i.e., an inducing agent) or a combination thereof.
  • the inducing conditions do not induce the expression of endogenous genes in the host cell.
  • Each vector contains various components, which depends on the function of the vector (amplification or expression of heterologous polynucleotides, or both) and the compatibility between the vector and the specific host cell where it is located.
  • 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.
  • plasmid vectors contain replicons and control sequences from species compatible with the host cells, and are used with these host cells.
  • the vector usually carries a replication site, as well as a marker sequence that can provide phenotypic selection in the transformed cells.
  • E. coli is usually transformed using pBR322, a plasmid derived from E. coli.
  • pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, and thus provides a simple method for identifying transformed cells.
  • pBR322 its derivatives or other bacterial plasmids or phages may also contain or be modified to contain promoters that can be used by microorganisms to express endogenous proteins. Carter et al., U.S. Pat. No. 5,648,237 details examples of pBR322 derivatives used to express specific antibodies.
  • phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transformation vectors with these host cells.
  • phage such as GEM TM -11 can be used to prepare recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.
  • a promoter is a non-translated regulatory sequence located upstream (5') of a cistron that regulates downstream gene expression.
  • Prokaryotic promoters are generally divided into two categories, inducible and constitutive.
  • An inducible promoter is a promoter that can respond to changes in culture conditions (e.g., the presence or absence of nutrients or changes in temperature) to initiate and increase transcription levels of a cistron.
  • promoters that can be recognized by potential host cells are all known.Promoter is removed from source DNA by restriction enzyme and the promoter sequence of separation is inserted in the carrier of the present application, and selected promoter is operably connected on the cistron DNA of coded polypeptide.Natural promoter sequence and many heterologous promoters can be used for instructing amplification and/or expression of target gene.In certain embodiments, utilize heterologous promoter, because compared with natural target polypeptide promoter, heterologous promoter usually allows larger transcription, and the output of expressing target gene is higher.
  • Promoters suitable for prokaryotic hosts include PhoA promoter, ⁇ -galactosidase and lactose promoter systems, tryptophan (trp) promoter systems and hybrid promoters, such as tac or trc promoters.
  • PhoA promoter ⁇ -galactosidase and lactose promoter systems
  • tryptophan (trp) promoter systems and hybrid promoters, such as tac or trc promoters.
  • other promoters that are functional in bacteria such as other known bacterial or phage promoters
  • Their nucleic acid sequences have been made public, so that technicians can use linkers or adapters to provide any required restriction sites to connect them to the cistron encoding the target light chain and heavy chain (Siebenlist et al., (1980) Cell 20: 269).
  • each cistron in the recombinant vector contains a secretion signal sequence component, which directly guides the expressed polypeptide to move across the membrane.
  • the signal sequence can be a component of the vector, It can also be a part of the target polypeptide DNA inserted into the vector.
  • the signal sequence selected for this application should be a sequence that can be recognized and processed (i.e., cut by a signal peptidase) by the host cell.
  • production of the protein construct of the present application can occur in the cytoplasm of the host cell, so there is no need for a secretion signal sequence to be present in each cistron.
  • the polypeptide components are expressed, folded, and assembled to form a protein construct in the cytoplasm.
  • Certain host strains e.g., E. coli trxB- strains
  • Escherichia coli cells are used as hosts of the present application.
  • E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 (US Pat. No. 5,639,635) having the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc fepE) degP41 kan R.
  • Other strains and their derivatives such as E. coli 294 (ATCC 31446), E. coli B, E.
  • coli 1776 (ATCC 31537) and E. coli RV308 (ATCC 31608) are also suitable. 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 described in detail, 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 suitable bacteria. For example, when using known plasmids such as pBR322, pBR325, pACYC177 or pKN410 to provide the replicon, Escherichia coli, Serratia or Salmonella are suitable for use as hosts.
  • known plasmids such as pBR322, pBR325, pACYC177 or pKN410 to provide the replicon
  • Escherichia coli, Serratia or Salmonella are suitable for use as hosts.
  • the chromatographic column is coated with a reagent, such as glycerol, to prevent nonspecific adhesion of contaminants.
  • a reagent such as glycerol
  • the solid phase is then washed to remove contaminants that are nonspecifically bound to the solid phase.
  • the target protein construct is recovered from the solid phase by elution.
  • the carrier used for eukaryotic hosts can also be an insert, and this insert encoding signal sequence or has other polypeptides of specific cleavage site at the N-terminal of mature protein or polypeptide.
  • the heterologous signal sequence selected is preferably a sequence recognized and processed (that is, cut by signal peptidase) by host cells.In mammalian cell expression, mammalian signal sequence and virus secretion leader can be obtained, for example, herpes simplex gD signal, all are useful.
  • the DNA in this precursor region is connected with the DNA of the protein construct of encoding the application in reading frame.
  • Selection genes may contain a selection gene, also called a selectable marker.
  • Typical selection genes encode proteins that confer (a) resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic proteins, or (c) provide key nutrients that are not available in complex culture media, such as the gene encoding Bacillus D-alanine racemase.
  • An example of a selection scheme is the use of a drug to stop the growth of host cells. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus survive the selection scheme. Examples of this type of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.
  • Expression vectors used in eukaryotic host cells also contain sequences necessary for transcription termination and stabilization of mRNA. These sequences are usually available from the 5' untranslated region of eukaryotic or viral DNA or cDNA, and occasionally the 3' end. These regions contain nucleotide fragments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide.
  • a suitable transcription termination element is the bovine growth hormone polyadenylated region. See WO94/11026 and the expression vectors disclosed therein.
  • Cell debris can be removed by centrifugation.
  • the supernatant of such expression systems is usually first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration device.
  • Protease inhibitors such as PMSF can be included in any of the above steps to inhibit protein hydrolysis, and antibiotics can be included to prevent the growth of foreign contaminants.
  • the mixture comprising the protein construct of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, with the elution buffer having a pH of about 2.5-4.5, preferably at low salt concentration (eg, from 0-0.25 M salt).
  • composition comprising NGF mutant or its fusion protein
  • the NGF mutant in the pharmaceutical composition of NGF mutant or its fusion protein and anti-NGF antibody, the NGF mutant has weakened binding to the anti-NGF antibody or substantially no binding to the anti-NGF antibody, and the NGF mutant still has biological activity.
  • the NGF mutant is selected from any one of the NGF mutants described above.
  • the present application provides a method for preventing and/or treating NGF-related diseases using an NGF mutant or its fusion protein or a pharmaceutical composition comprising an NGF mutant or its fusion protein.
  • the NGF mutant described in the treatment method has weakened binding to or substantially no binding to an anti-NGF antibody, and the NGF mutant still has biological activity.
  • the NGF mutant is selected from any one or more NGF mutants described in the present application.
  • a disease in an individual e.g., a human being
  • an NGF-related disease such as a neurological disease or a non-neurological disease
  • administering comprising administering to the individual an effective dose of an NGF mutant or a fusion protein thereof (e.g., a fusion protein of an NGF mutant and Fc) or a pharmaceutical composition comprising an NGF mutant or a fusion protein thereof, wherein the NGF mutant has reduced binding to an anti-NGF antibody or substantially no binding to the anti-NGF antibody, and the NGF mutant still has biological activity.
  • an NGF mutant or a fusion protein thereof e.g., a fusion protein of an NGF mutant and Fc
  • a pharmaceutical composition comprising an NGF mutant or a fusion protein thereof, wherein the NGF mutant has reduced binding to an anti-NGF antibody or substantially no binding to the anti-NGF antibody, and the NGF mutant still has biological activity.
  • the NGF mutant or its fusion protein or the pharmaceutical composition comprising the NGF mutant or its fusion protein is administered by intravenous injection, intramuscular injection or subcutaneous injection.
  • NGF-related disease refers to any disease or disorder caused by or associated with impaired NGF receptor signaling (e.g., due to insufficient NGF amounts and/or reduced binding affinity), or a disease or disorder that requires the biological activity of NGF for treatment (e.g., treatment of injuries/damage that require neuronal growth, maintenance, proliferation and/or survival).
  • the method described in this article is suitable for treating neurological diseases and non-neurological diseases.
  • Nervous system diseases include nervous system diseases.
  • Nervous system diseases refer to diseases related to neuronal degeneration or damage of the central and/or peripheral nervous systems.
  • 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 cord injury, acute cerebrovascular disease, brain atrophy, peripheral neuropathy and other diseases characterized by neuronal necrosis or loss, whether central neurons, peripheral neurons or motor neurons, except for nerve damage caused by trauma, burns, renal failure, injury or chemicals/drugs, such as acute cerebrovascular central nervous damage caused by chemicals or drugs.
  • ALS amyotrophic lateral sclerosis
  • Nervous system diseases also include peripheral neuropathy associated with certain diseases, such as neuropathy associated with diabetes, AIDS or chemotherapy.
  • the nervous system disease is selected from multi-infarct dementia, vascular dementia, cognitive impairment caused by organic brain diseases caused by alcoholism, Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's disease, Down syndrome, neurological deafness, Meniere's disease, stroke, ALS, Bell's palsy, diseases involving spinal muscular atrophy, diseases involving paralysis, peripheral neuropathy, nerve damage caused by trauma, nerve damage caused by burns, nerve damage caused by renal dysfunction, nerve damage caused by injury, nerve damage caused by chemotherapy toxicity, nerve damage caused by surgery, nerve damage caused by ischemia, nerve damage caused by infection, nerve damage caused by metabolic diseases, and nerve damage caused by nutritional deficiency.
  • 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-related peripheral neuropathy, and peripheral neuropathy affecting motor neurons.
  • the nervous system disease is selected from neonatal hypoxic-ischemic encephalopathy, cerebral palsy, severe myopathy, neurological deafness, recurrent laryngeal nerve injury, traumatic brain injury, dental nerve injury, stroke, Down 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, retinal related diseases, 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 end
  • the NGF mutant in the method of treating a disease has one or more of the following biological activities: (i) supporting neuronal survival; (ii) promoting neurite outgrowth; (iii) enhancing neurochemical differentiation; (iv) promoting pancreatic ⁇ -cell proliferation; (v) inducing innate and/or acquired immunity; (vi) repairing damaged nerve cells (e.g., corneal nerves) and/or preventing damage (e.g., in neurotrophic keratitis); (vii) promoting proliferation of follicular cells and/or estrogen production; hormone secretion; (viii) promote wound healing (e.g., in diabetic neuropathy); (ix) improve spatial cognition, memory and/or
  • the present application provides a method for preventing and/or treating diseases or conditions caused by increased NGF expression and/or enhanced sensitivity to NGF by using an anti-NGF antibody in combination with an NGF mutant or a fusion protein thereof described in the present application (e.g., a fusion protein of an NGF mutant and Fc), or using a pharmaceutical composition comprising an anti-NGF antibody and an NGF mutant or a fusion protein thereof described in the present application, wherein the NGF mutant has weakened binding to the anti-NGF antibody or substantially no binding to the anti-NGF antibody, and the NGF mutant still has the biological activity of NGF.
  • the NGF mutant described in the treatment method is selected from any one or more NGF mutants described in the present application.
  • the NGF mutant or a fusion protein comprising the same (or a pharmaceutical composition thereof) is administered by intravenous injection, intramuscular injection, or subcutaneous injection.
  • the anti-NGF antibody and the NGF mutant or its fusion protein described herein can be administered to a subject by any suitable means.
  • the anti-NGF antibody and the NGF mutant or its fusion protein are formulated for intravenous injection.
  • the anti-NGF antibody and the NGF mutant or its fusion protein are administered simultaneously, for example, in a single formulation or as separate formulations.
  • the anti-NGF antibody and the NGF mutant or its fusion protein are administered sequentially, for example, as separate formulations.
  • the "diseases or conditions caused by increased NGF expression and/or enhanced sensitivity" described in the present application include but are not limited to acute pain, dental pain, traumatic pain, surgical pain, pain caused by amputation or abscess, burning pain, demyelinating disease, trigeminal neuralgia, cancer, chronic alcoholism, stroke, thalamic pain syndrome, diabetes, acquired immune deficiency syndrome (AIDS), toxins and chemotherapy, general headache, migraine, cluster headache, mixed vascular and non-vascular syndrome, tension headache, general inflammation, arthritis, rheumatism, lupus erythematosus, osteoarthritis, inflammatory bowel disease, irritable bowel syndrome, inflammatory eye disease, inflammatory or unstable bladder disease, psoriasis, skin complaints with inflammatory components, sunburn, etc.
  • the "diseases or conditions with increased NGF expression and/or enhanced sensitivity" described in the present application include inflammatory pain, postoperative incisional pain, neuropathic pain, fracture pain, gouty joint pain, postherpetic neuralgia, pain caused by burns, cancer pain, osteoarthritis or rheumatoid arthritis pain, sciatica, pain associated with sickle cell crisis, or herpetic neuralgia.
  • the present application also provides a method for reducing and/or alleviating adverse reactions during anti-NGF antibody treatment, comprising administering the NGF mutant or its fusion protein described in the present application, or a pharmaceutical composition comprising the NGF mutant or its fusion protein, to a subject who has received, is receiving, or is about to receive anti-NGF antibody treatment, wherein the NGF mutant has weakened binding to the anti-NGF antibody or substantially no binding to the anti-NGF antibody, while still having the biological activity of NGF.
  • the NGF mutant is selected from any one or more NGF mutants described in the present application.
  • the adverse reactions produced when using anti-NGF antibodies for treatment described in the present application include, but are not limited to, adverse reactions related to the nervous system and adverse reactions related to joints.
  • the adverse reactions include: sympathetic nerve damage, osteonecrosis, bone loss, bone damage, joint damage, rapidly progressive osteoarthritis (RPOA), peripheral edema, joint pain, limb pain, peripheral nerve paresthesia, dysesthesia, hyperesthesia, paresthesia, burning pain and tactile pain.
  • RPOA rapidly progressive osteoarthritis
  • the administration of the NGF mutant or its fusion protein, the nucleic acid encoding the NGF mutant or its fusion protein, the vector and host cell comprising the nucleic acid, the pharmaceutical composition comprising the NGF mutant or its fusion protein, or the pharmaceutical composition comprising the NGF mutant or its fusion protein and the anti-NGF antibody at the same time, etc. described in the present application can be carried out in any known and convenient manner, including by injection or infusion.
  • the route of administration is in accordance with known and recognized methods, such as by single or multiple push injections or long-term infusion in an appropriate manner.
  • the dosage and required drug concentration of the pharmaceutical composition of the present application may vary depending on the specific use. Determining the appropriate dosage or route of administration is well within the technical scope of ordinary technicians. Animal experiments provide reliable guidance for determining effective doses for human treatment. Interspecies analogies of effective doses can be made based on the principles of Mordenti, J. and 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.
  • different formulations will be effective for different treatments and different diseases, and the mode of administration intended to treat a specific organ or tissue may be different from that for another organ or tissue.
  • the dose may be administered by one or more separate administrations or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, treatment continues until the symptoms of the disease reach the desired level of suppression.
  • other dosage regimens may be useful. The progress of such treatment is easily monitored by conventional techniques and analysis. The optimum dosage and treatment regimen for a particular patient can be determined by one skilled in the medical arts by monitoring the patient for signs of disease and making adjustments accordingly.
  • the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises an amino acid mutation at one or more of the I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 positions relative to the human wild-type mature NGF amino acid sequence.
  • the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises an amino acid mutation at one or more of S17, S19, K32, T56, R59 and T83 relative to the human wild-type mature NGF amino acid sequence.
  • the NGF mutant in the pharmaceutical composition or disease treatment method described herein comprises an amino acid mutation at position S17R, S17K, S17H or S17E relative to the amino acid sequence of wild-type mature NGF.
  • the NGF mutant in the pharmaceutical composition or disease treatment method described herein comprises an amino acid mutation at position S19 to S19R, S19K or S19F relative to the amino acid sequence of wild-type mature NGF.
  • the NGF mutant in the pharmaceutical composition or method of treating a disease described herein comprises an amino acid mutation at position T83 relative to the amino acid sequence of wild-type mature NGF to T83K.
  • the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises amino acid mutations at the S19 and K32 positions relative to the human wild-type mature NGF amino acid sequence, wherein the mutation at the S19 position is S19R, and the mutation at the K32 position is K32E, K32Y, K32M or K32L.
  • the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises amino acid mutations at the S19 and S17 positions relative to the human wild-type mature NGF amino acid sequence, wherein the mutation at the S19 position is S19R, and the mutation at the S17 position is S17E.
  • the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises an amino acid sequence shown in any one of SEQ ID NOs: 17-60 or a variant thereof, wherein the variant has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 17-60.
  • the fusion protein of the NGF mutant in the pharmaceutical composition or the method for treating a disease described in the present application is a fusion protein of the NGF mutant and Fc.
  • the fusion protein of the NGF mutant and Fc comprises from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: the NGF mutant described herein, an optional peptide linker and an Fc portion.
  • the optional peptide linker comprises the amino acid sequence shown in SEQ ID NO: 87.
  • the Fc portion comprises the amino acid sequence shown in SEQ ID NO: 85 or a variant thereof, wherein the variant has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO: 85.
  • the fusion protein of the NGF mutant and Fc comprises the amino acid sequence shown in any one of SEQ ID NOs:61-82 or a variant thereof, and the variant has at least 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs:61-82.
  • the anti-NGF antibody in the pharmaceutical composition or the method for treating a disease described herein is any anti-NGF antibody disclosed in the prior art.
  • the anti-NGF antibodies described herein include, but are not limited to: Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Biopharma), SSS-40 (Sunshine Guojian) and STC001 (Sutacion).
  • the sequences of the above exemplary anti-NGF antibodies are shown in Table 1.
  • the kit of the present application includes one or more containers containing any NGF mutant or its fusion protein described in the present application, or containing both NGF mutant or its fusion protein and anti-NGF antibody, for example, for treating a disease.
  • it contains instructions describing the administration of NGF mutant or its fusion protein to treat a disease (such as a nervous system disease) or instructions for the combined use of anti-NGF antibody and NGF mutant or its fusion protein to reduce the adverse effects of anti-NGF antibody.
  • the kit may further include a description of selecting an individual (e.g., human) suitable for treatment based on identifying whether the individual has a disease and the stage of the disease.
  • kits related to the use of treating NGF-related diseases or reducing adverse reactions of anti-NGF antibodies typically include information about the dose, dosing schedule, and route of administration for the intended treatment.
  • the container can be a unit dose, bulk package (e.g., multi-dose package), or subunit dose.
  • the kit of the present application The instructions provided in the kit are typically written instructions on a label or drug instructions (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions stored on a disk or CD) are also acceptable.
  • the kit of the present application is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc.
  • 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 injection needle). At least one active agent in the composition is an NGF mutant or a fusion protein containing it as described in the present application.
  • the container may further contain a second pharmaceutically active agent.
  • the kit may optionally provide additional components, such as a buffer and explanatory information.
  • the kit comprises a container and a label or drug instructions on or associated with the container.
  • the kit or article of manufacture includes multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies.
  • the recombinant eukaryotic expression vector pTT5 carrying nucleic acids encoding various NGF mutants and Fc fusion proteins was transfected into 293F cells respectively, and cultured at 37°C, 5% CO 2 , and 120 rpm for 5 days, and the cell culture fluid was collected respectively.
  • TrkA receptor and mFc fusion protein expression vector are the same as those described in 3.1 above.
  • the TrkA receptor extracellular region is fused with mFc, wherein the amino acid sequence of the TrkA receptor extracellular region is shown in SEQ ID NO: 88, mFc is mouse IgG2a Fc, and the amino acid sequence of the TrkA receptor and mFc fusion protein is shown in SEQ ID NO: 89.
  • the cell culture fluids expressing the above-mentioned human wild-type mature NGF or NGF mutant and Fc fusion proteins, TrkA receptor and mFc fusion proteins, and anti-NGF antibodies were collected separately, and after centrifugation, the cell culture supernatant was crudely purified using a HiTrap Protein A HP purification column (the specific purification steps were carried out according to the instructions).
  • the Protein A column was first equilibrated with a 50mM PBS solution (pH 7.2) containing 0.15M NaCl at a flow rate of 150cm/h. The pH of the culture supernatant was adjusted to 7.2, and the sample was loaded at room temperature at a flow rate of 150cm/h.
  • the column was completely purified. After equilibration, 50 mM sodium citrate solution (pH 3.4) was used for elution, and the eluate containing the fusion protein of human wild-type mature NGF or NGF mutant and Fc, the fusion protein of TrkA receptor and mFc, or anti-NGF antibody was collected. Then, based on the different hydrophobicity of the protein, HiTrap TM Butyl HP column (GE Healthcare) was used to further separate it from the host protein.
  • HiTrap TM Butyl HP column GE Healthcare
  • a Superdex 200 gel filtration column (GE Life Sciences) was used to remove the residual aggregates, and purified human wild-type mature NGF or NGF mutant and Fc fusion protein, TrkA receptor and mFc fusion protein, or anti-NGF antibody were obtained respectively.
  • the fusion protein of human wild-type mature NGF and Fc is abbreviated as hNGF-Fc, wherein the amino acid sequence of human wild-type mature NGF is shown in SEQ ID NO: 1, and the amino acid sequence of hNGF-Fc is shown in SEQ ID NO: 94.
  • the fusion protein of TrkA receptor and mFc is abbreviated as TrkA-mFc.
  • NGF F12E -Fc The fusion protein of NGF F12E and Fc is abbreviated as NGF F12E -Fc, wherein the amino acid sequence of NGF F12E is shown in SEQ ID NO:83, and the amino acid sequence of NGF F12E -Fc is shown in SEQ ID NO:84.
  • the method for detecting the binding activity of NGF mutants with different anti-NGF antibodies by ELISA is as follows:
  • anti-NGF antibody for example, Tanezumab, Fasinumab or STC001
  • anti-NGF antibody for example, Tanezumab, Fasinumab or STC001
  • the fusion protein of NGF mutant and Fc (e.g., 1A2-Fc) coated in the well plate is taken as an example.
  • the concentration of 1A2-Fc, a fusion protein of NGF mutant and Fc is 0.1 ⁇ g/well to coat the 96-well plate, overnight at 4°C. Wash the 96-well plate with PBST solution, add 150 ⁇ l 1% BSA-PBS to each well, block at 37°C for 1-2 hours, and then wash with PBST solution. Add 50 ⁇ l of gradient diluted anti-NGF antibody (e.g., STC001) to each well, incubate at 37°C for 1 hour, and wash 6 times with PBST solution.
  • 1A2-Fc a fusion protein of NGF mutant and Fc
  • NGF F12E -Fc binding activity of NGF F12E -Fc with each anti-NGF antibody was detected according to the above method to confirm whether the introduction of mutant F12E into human wild-type mature NGF would affect its binding activity with the corresponding anti-NGF antibody.
  • the ELISA binding curves of hNGF-Fc and NGF F12E -Fc with anti-NGF antibody are shown in Figure 4A and Figure 4B, respectively, and the EC50 values are shown in Table 6.
  • NGF F12E and human wild-type mature NGF have basically the same binding curve trends as the anti-NGF antibody shown in Table 6 ( Figures 4A-4B), and the binding EC50 values are basically equivalent (Table 6).
  • Table 6 The above results show that the introduction of mutation F12E into human wild-type mature NGF will not basically affect its binding activity with anti-NGF antibody.
  • each NGF mutant and Fc fusion protein 1A2-Fc to 1A13-Fc with different anti-NGF antibodies was detected, and the binding EC50 values are shown in Table 7.
  • the ELISA binding curves of the exemplary NGF mutant and Fc fusion protein with anti-NGF antibodies are shown in Figures 4C-4J.
  • the hNGF-Fc fusion protein was used as a control in this experiment.
  • Relative binding activity is used to characterize the change in the binding activity of NGF mutants to anti-NGF antibodies relative to the binding activity of human wild-type mature NGF to the same anti-NGF antibody. For example, if the relative binding activity value is 50%, it means that the binding activity of the NGF mutant to the NGF antibody is 50% of the binding activity of human wild-type mature NGF to the same anti-NGF antibody.
  • the calculation formula for relative binding activity is: the ratio of the binding EC50 value of human wild-type mature NGF to the anti-NGF antibody to the binding EC50 value of the NGF mutant to the same anti-NGF antibody, and the results are shown in Table 7.
  • the exemplary NGF mutants in the present application have weakened binding activity with at least one anti-NGF antibody, for example, mutant 1A7-Fc, whose binding activity with Fasinumab antibody is only 2.10% of that of human wild-type mature NGF; or even basically does not bind to anti-NGF antibodies, for example, mutant 1A10-Fc basically does not bind to MEDI-578 antibody.
  • Example 5 ELISA method to detect the binding activity of NGF mutants to TrkA receptor
  • the method for detecting the binding activity of NGF mutants to TrkA receptor by ELISA is as follows:
  • the binding activity of NGF F12E to TrkA receptor was first detected to confirm whether the introduction of mutant F12E into wild-type mature human NGF would affect its binding activity to TrkA receptor.
  • the ELISA binding EC50 values of hNGF-Fc and NGF F12E -Fc to TrkA receptor are shown in Table 8.
  • NGF F12E still has biological activity and potential therapeutic activity.
  • NGF F12E -Fc for example, 1A7-Fc, 1A11-Fc, 1A8-Fc, 1A13-Fc, 1A12-Fc, 1A6-Fc, 1A2-Fc, 1A10-Fc and 1A3-Fc
  • NGF F12E -Fc for example, 1A4-Fc, 1A5-Fc and 1A9-Fc
  • Example 6 Detection of in vitro biological activity of NGF mutants using TF-1 cell proliferation assay
  • TF-1 cells are promyelocytes of human erythroid leukemia patients.
  • NGF can prolong the survival time of TF-1 cells cultured in vitro, and the survival number of TF-1 cells is positively correlated with the biological activity of NGF. Therefore, the TF-1 cell proliferation method can be used to detect the biological activity of NGF.
  • the specific operation method can refer to the content disclosed in the Chinese patent application publication CN114829384A.
  • TF-1 cells were resuspended in basal medium (RPMI 1640 medium containing 10% FBS) to obtain a suspension containing 6.0 ⁇ 10 4 cells/ml for use.
  • basal medium RPMI 1640 medium containing 10% FBS
  • the samples to be tested were prepared: fusion proteins of various NGF mutants and Fc and positive control sample NGF F12E -Fc, and international standard: hNGF.
  • the concentration gradients of the samples to be tested and NGF F12E -Fc were 200U/ml, 66.66U/ml, 22.22U/ml, 7.4U/ml, 2.47U/ml, and 0.82U/ml, respectively, and they were inoculated in 96-well plates, and then 100 ⁇ l of the above TF-1 cell suspension was added to each well, and cultured in a humidified incubator at 37°C and 5% CO 2 for 72 hours.
  • MTS Promega, Cat# G3581
  • 20 ⁇ l of assay solution was added to each well, and cultured for 3 hours at 37°C and 5% CO 2.
  • the absorbance of the well plate was measured using a spectrophotometer, the data was recorded, and the curve was generated using SoftMAX Pro.
  • PC12 cells were resuspended in basal medium (RPMI 1640 medium containing 0.01% BSA), resuspended and counted in a white transparent bottom 96-well plate, 1.0 ⁇ 10 4 cells/well, 50 ⁇ l per well, and cultured at 37°C, 5% CO 2 for 24 hours.
  • basal medium RPMI 1640 medium containing 0.01% BSA
  • samples to be tested were prepared: various NGF mutants and Fc fusion proteins and positive control: NGF F12E -Fc, and serum-free medium was used as a blank control.
  • 50 ⁇ l of the diluted sample was added to the above 96-well plate, and placed in a humid incubator at 37°C, 5% CO 2 for 6 hours before luciferase detection. After Bright-Glo TM Luciferase Assay was equilibrated to room temperature, 40 ⁇ l/well was added to the above 96-well plate.
  • Luminescence (1000 ms) readings were immediately performed using a microplate reader 2 minutes after cell lysis.
  • GraphPad Prism 5.0 was used to fit the curve and calculate EC50.
  • NGF F12E -Fc or hNGF-Fc samples were diluted to a starting concentration of 54 ng/ml and then diluted 3 times.
  • the reference substances used in this experiment are: Mouse NGF, each tube contains 15000AU, the initial concentration is 6ng/ml, and it is diluted 3 times, 2ng/ml, 0.66ng/ml, 0.22ng/ml, 0.07ng/ml, 0.02ng/ml. After adding 8-day-old chicken embryo dorsal root ganglia to each concentration sample, place it in a 37°C, 5% CO2 incubator, and observe the results after 24 hours.
  • NGF mutant still has the biological activity of NGF after mutation, and its biological activity is basically equivalent to that of hNGF-Fc and NGF F12E -Fc.
  • the fusion proteins of other NGF mutants and Fc were also tested using the same method, and their biological activities were basically equivalent to that of NGF F12E -Fc (data not shown).
  • Newborn Sprague-Dawley (SD) rats were subcutaneously injected with the sample to be tested: each NGF mutant and Fc fusion protein sample (taking 1A2-Fc or 1A10-Fc as an example), positive control: NGF F12E -Fc or negative control: PBS solution, and then the rats were killed and SCG was isolated.
  • Each NGF mutant and Fc fusion protein, NGF F12E -Fc or PBS was injected once a day on day 0, day 1, day 2 and day 3, and SCG was harvested on day 4.
  • the results of the SCG experiment in rats are shown in Figure 7.
  • the exemplary NGF mutant and Fc fusion proteins 1A2-Fc and 1A10-Fc can promote SCG growth.
  • the above results show that whether in the low-dose or high-dose administration group, 1A2-Fc and 1A10-Fc have a significant promoting effect on the growth of SCG compared with the PBS negative control group (**p ⁇ 0.01).
  • 1A2-Fc and 1A10-Fc showed good biological activity, and were basically equivalent to the biological activity of NGF F12E -Fc.
  • other NGF mutants and Fc fusion proteins were also tested, and preliminary results showed that other NGF mutants can also promote SCG growth in rats (data not shown).
  • a pain threshold detection experiment was carried out in rats. First, qualified mice with normal pain response were screened, and a certain dose of the test sample was injected: the fusion protein of each NGF mutant and Fc or the blank control substance: PBS solution. The pain threshold of the mouse's paw reaction was measured by mechanical stimulation, and statistical analysis was performed. The higher the pain threshold, the stronger the mouse's ability to withstand pain. The lower the pain threshold, the more pain it causes in the mouse. In this way, it was determined whether the NGF mutants of the present application still have the effect of reducing pain during the application process.
  • mice were ordered. The mice were male, weighing 30-35g, and were kept in an environment free of special pathogens. All animal experiments were conducted in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • the dynamic plantar tactile instrument (Ugo Basile, Italy, model 37450) was used to screen the experimental animals with an average threshold of 7.5-10 for both feet and a P value of >0.05 for the threshold of the left and right feet of the same mouse as qualified mice.
  • the mice were randomly divided into experimental groups: fusion proteins of various NGF mutants and Fc, blank control group: PBS solution, positive control group: hNGF-Fc and pain relief control group: NGF F12E -Fc, 10 mice in each group.
  • the blank control group was injected with 20 ⁇ l of PBS solution, and the dosage of hNGF-Fc, fusion proteins of NGF mutants and Fc (taking 1A2-Fc and 1A10-Fc as examples) and NGF F12E -Fc was 0.25 ⁇ g/mouse/20 ⁇ l.
  • the drug was administered subcutaneously in the soles of the left and right feet of the mice.
  • the mechanical pain threshold of the mice was measured at 0h before administration, 4h, 24h, and 48h after administration, and the values were recorded.
  • GraphPad Prism software was used for graphing and statistical analysis of the results, and the mechanical pain thresholds of the fusion proteins of NGF mutant and Fc, hNGF-Fc and NGF F12E -Fc groups were compared to see if there were any differences, and the pain-causing properties of the fusion proteins of NGF mutant and Fc were analyzed.
  • the binding ELISA curves of 1A20-Fc and exemplary anti-NGF antibodies are shown in FIG9F .
  • the binding activity of 1A20-Fc to the anti-NGF antibodies Tanezumab, Fasinumab, MEDI-578 and STC001 is weakened or basically does not bind to them.
  • the PC12 cell activity detection method was used to determine the biological activity of the NGF mutant in the presence of the corresponding anti-NGF antibody.
  • the specific operation method is as described in Example 7.
  • a control group was set up for the combined use of hNGF-Fc and the same anti-NGF antibody.

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Abstract

The present application relates to a mutable site in an NGF and a method for screening and/or identifying a mutable site, an NGF mutant library and a construction method therefor, an NGF mutant and a screening and/or identification method therefor, a fusion protein comprising an NGF mutant, a nucleic acid molecule encoding an NGF mutant or a fusion protein, a vector and host cell comprising a nucleic acid molecule, a pharmaceutical composition comprising an NGF mutant or a fusion protein thereof, or a pharmaceutical composition comprising an NGF mutant or a fusion protein thereof and an anti-NGF antibody, and a corresponding treatment method. The present application further provides a preparation method for an NGF mutant and a fusion protein thereof, and the like.

Description

一种筛选和/或鉴定NGF中的可突变位点的方法及其筛选和/或鉴定NGF突变体的方法A method for screening and/or identifying a mutable site in NGF and a method for screening and/or identifying an NGF mutant
提交电子序列表Submit electronic sequence listing
以下提交的电子序列表(文件名称：NGF-seqlist1227.xml，记录日期：2023年12月25日，大小：98KB)的内容通过整体引用并入本文中。The contents of the electronic sequence listing submitted below (file name: NGF-seqlist1227.xml, recording date: December 25, 2023, size: 98KB) are incorporated herein by reference in their entirety.
技术领域Technical Field
本申请涉及NGF中的可突变位点及可突变位点的筛选和/或鉴定方法、NGF突变体库及其构建方法、NGF突变体及其筛选和/或鉴定方法、包含NGF突变体的融合蛋白、编码NGF突变体或融合蛋白的核酸分子、包含核酸分子的载体和宿主细胞，以及包含NGF突变体或其融合蛋白的药物组合物或包含NGF突变体或其融合蛋白和抗NGF抗体的药物组合物和治疗方法，还进一步提供了NGF突变体及其融合蛋白的制备方法等。The present application relates to mutable sites in NGF and methods for screening and/or identifying mutable sites, an NGF mutant library and a method for constructing the same, NGF mutants and methods for screening and/or identifying the same, fusion proteins comprising NGF mutants, nucleic acid molecules encoding NGF mutants or fusion proteins, vectors and host cells comprising nucleic acid molecules, as well as pharmaceutical compositions comprising NGF mutants or their fusion proteins or pharmaceutical compositions comprising NGF mutants or their fusion proteins and anti-NGF antibodies and treatment methods. It also further provides methods for preparing NGF mutants and their fusion proteins.
发明背景Background of the Invention
神经生长因子(NGF)是神经营养因子家族中首个被发现的成员，由意大利科学家Levi-Montlcini于1953年在小鼠肉瘤细胞中首次发现。NGF是一种具有神经元营养和促突起生长双重生物学功能的一种神经细胞生长调节因子，对中枢和外周神经的发育、分化、生长、再生和功能特性的表达均具有重要的调控作用。NGF包含α、β和γ三个亚基，活性区是β亚基，由两条单链通过非共价键结合形成的二聚体。Nerve growth factor (NGF) is the first member of the neurotrophic factor family to be discovered. It was first discovered by Italian scientist Levi-Montlcini in mouse sarcoma cells in 1953. NGF is a neuronal growth regulatory factor with dual biological functions of neuronal nutrition and neurite growth promotion. It plays an important regulatory role in the development, differentiation, growth, regeneration and expression of functional characteristics of central and peripheral nerves. NGF contains three subunits: α, β and γ. The active region is the β subunit, which is a dimer formed by two single chains bound by non-covalent bonds.
天然状况下，NGF与TrkA受体(tyrosine kinase receptor)和p75NTR(p75 neurotrophin receptor)结合。TrkA受体是NGF的功能性受体，NGF与TrkA受体结合能够促进神经元的存活(Canu,N.,et al.Int J Mol Sci.18(2017)1319)。p75NTR虽然被报道与全部的神经营养因子结合，在神经元的产生过程中与细胞凋亡相关，但其对NGF实现其生物学活性和功能并不是必需的，可能更多是作为一个辅助受体(Barker,P.A.&Shooter,E.M.Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to TrkA on PC12 cells.Neuron 13,203-215(1994))。已知在NGF基因敲除小鼠和TrkA基因敲除小鼠中均观察到了相同的表型，据此，认为NGF的生物学活性和生理作用主要通过TrkA受体介导(Conover JC,et al.,Neurotrophin  regulation of the developing nervous system:analyses of knockout mice.Rev Neurosci.1997 Jan-Mar；8(1):13-27)。Under natural conditions, NGF binds to the TrkA receptor (tyrosine kinase receptor) and p75NTR (p75 neurotrophin receptor). The TrkA receptor is a functional receptor for NGF. The binding of NGF to the TrkA receptor can promote the survival of neurons (Canu, N., et al. Int J Mol Sci. 18 (2017) 1319). Although p75NTR has been reported to bind to all neurotrophic factors and is associated with cell apoptosis during the generation of neurons, it is not essential for NGF to achieve its biological activity and function, and may be more of an auxiliary receptor (Barker, PA & Shooter, EM Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to TrkA on PC12 cells. Neuron 13, 203-215 (1994)). It is known that the same phenotype is observed in NGF knockout mice and TrkA knockout mice. Based on this, it is believed that the biological activity and physiological effects of NGF are mainly mediated by TrkA receptors (Conover JC, et al., Neurotrophin regulation of the developing nervous system: analyses of knockout mice. Rev Neurosci. 1997 Jan-Mar; 8(1): 13-27).
已经表明NGF在生理和病理疼痛的传递过程中发挥重要的作用，局部和全身给予NGF能够诱发痛觉过敏和异常性疼痛(Lewin et.al.,1994 Eur.J.Neurosci.6:1903-1912)。在患者中，静脉输注NGF会产生全身肌痛，而局部给药除全身效应外，还会引起注射部位的异常性疼痛(Apfel.,1998 Neurology 51:695-702)，这也在一定程度上限制了它的使用。所以目前研究中也在开发能够在应用过程中降低疼痛或者无痛的重组NGF，从而可以增大使用剂量及受试人群等(参见，例如专利CN 109153709 B)。It has been shown that NGF plays an important role in the transmission of physiological and pathological pain. Local and systemic administration of NGF can induce hyperalgesia and allodynia (Lewin et al., 1994 Eur. J. Neurosci. 6: 1903-1912). In patients, intravenous infusion of NGF produces systemic myalgia, while local administration, in addition to systemic effects, can also cause allodynia at the injection site (Apfel., 1998 Neurology 51: 695-702), which also limits its use to a certain extent. Therefore, current research is also developing recombinant NGF that can reduce pain or be painless during application, so as to increase the dosage and the number of subjects (see, for example, patent CN 109153709 B).
慢性疼痛目前是全球的一个重大的负担，影响着约30％的成年人。目前受到疼痛困扰的人群快速增长，但临床上对于轻至中度疼痛的治疗主要使用非阿片类镇痛药，如非甾体类抗炎药(NASIDs)；对于中至重度疼痛，则主要使用阿片类镇痛药。然而，NASIDs存在“封顶效应”，阿片类药物仅能使不到30％的非肿瘤性慢性疼痛得到有效缓解，20％的癌痛患者存在阿片类药物耐受。此外，现有止痛药还存在轻至重度不良反应，长期用药过程中尤为明显。因此，抗NGF抗体也被认为是疼痛领域具有希望的下一代镇痛药物之一(Kalso E，et al.,Opioids in chronic non-cancer pain:systematic review of efficacy and safety[J].Pain,2004,112(3):372-380)。目前已经报道的抗人NGF抗体包括，但不限于Tanezumab(WO2004/058184)、Fasinumab(WO2009/023540)、Fulranumab(WO2005/019266)、MEDI-578(WO2006/077441)等，但同时抗NGF抗体在临床应用过程中也存在一定的不良反应，包括Tanezumab在临床试验中报道有头痛、上呼吸道感染、感觉异常等(Lane NE et al.,Tanezumab for the treatment of pain from osteoarthritis of the knee.N Engl J Med.2010 Oct 14；363(16):1521-31)，Fulranumab在临床试验中报道有感觉异常、头痛、鼻咽炎等以及神经相关的副反应(Neurologic-related AEs)包括腕管综合征和肌张力障碍(Sanga P et al.,Efficacy,safety,and tolerability of fulranumab,an anti-nerve growth factor antibody,in the treatment of patients with moderate to severe osteoarthritis pain.Pain.2013 Oct；154(10):1910-1919)，Fasinumab在临床试验中报道有关节痛、知觉过敏、肌肉痛、末梢水肿及关节肿胀等(Tiseo PJ et al.,Fasinumab(REGN475),an antibody against nerve growth factor for the treatment of pain:results from a double-blind,placebo-controlled exploratory study in osteoarthritis of the knee.Pain.2014 Jul；155(7):1245-1252)。 Chronic pain is currently a major burden worldwide, affecting about 30% of adults. The number of people suffering from pain is growing rapidly, but clinically, non-opioid analgesics, such as nonsteroidal anti-inflammatory drugs (NASIDs), are mainly used to treat mild to moderate pain; for moderate to severe pain, opioid analgesics are mainly used. However, NASIDs have a "ceiling effect", and opioids can only effectively relieve less than 30% of non-tumor chronic pain, and 20% of cancer pain patients have opioid tolerance. In addition, existing analgesics also have mild to severe adverse reactions, which are particularly evident during long-term use. Therefore, anti-NGF antibodies are also considered to be one of the promising next-generation analgesics in the field of pain (Kalso E, et al., Opioids in chronic non-cancer pain: systematic review of efficacy and safety [J]. Pain, 2004, 112 (3): 372-380). Anti-human NGF antibodies reported so far include, but are not limited to, Tanezumab (WO2004/058184), Fasinumab (WO2009/023540), Fulranumab (WO2005/019266), MEDI-578 (WO2006/077441), etc. However, anti-NGF antibodies also have certain adverse reactions in clinical application, including headache, upper respiratory tract infection, paresthesia, etc. reported in clinical trials of Tanezumab (Lane NE et al., Tanezumab for the treatment of pain from osteoarthritis of the knee. N Engl J Med. 2010 Oct 14; 363(16): 1521-31), paresthesia, headache, nasopharyngitis, etc. reported in clinical trials of Fulranumab, as well as neurological-related AEs including carpal tunnel syndrome and dystonia (Sanga P et al., Efficacy, safety, and Tolerability of fulranumab, an anti-nerve growth factor antibody, in the treatment of patients with moderate to severe osteoarthritis pain. Pain. 2013 Oct; 154(10): 1910-1919), Fasinumab was reported to cause arthralgia, hyperesthesia, myalgia, peripheral edema and joint swelling in clinical trials (Tiseo PJ et al., Fasinumab (REGN475), an antibody against nerve growth factor for the treatment of pain: results from a double-blind, placebo-controlled exploratory study in osteoarthritis of the knee. Pain. 2014 Jul; 155(7): 1245-1252).
综合来看，用抗NGF抗体治疗相关疾病时，最常出现的不良反应主要包括神经系统相关的不良反应(Neurologic-related AEs)和关节相关的不良反应(joint-related AEs)。神经系统相关的不良反应主要包括外周神经感觉症状，包括感觉异常、感觉障碍、感觉亢进、灼烧痛和触摸痛等。已报道的严重的关节相关的不良反应包括骨坏死等(Bannwarth B,et al.,Nerve Growth Factor Antagonists:Is the Future of Monoclonal Antibodies Becoming Clearer？Drugs.2017 Sep；77(13):1377-1387)。虽然目前造成这些不良反应发生的机制尚未被完全阐明，但是已有观点认为，在动物实验中已经验证NGF在成年外周神经系统中的生理作用，包括维持背根神经节感觉神经元的形态稳态和生化功能等(Rask CA.Biological actions of nerve growth factor in the peripheral nervous system.Eur Neurol.1999；41(Suppl.1):14-9)。同时，NGF可以促进组织修复，在软骨修复和荷载诱导的骨形成(load-induced boneformation)中发挥作用，而NGF抑制剂，例如抗NGF抗体的应用，可能破坏了软骨下裂隙患者的骨修复，进而引发骨关节炎的快速进展(Hochberg MC,Tive LA,Abramson SB,et al.When is osteonecrosis not osteonecrosis？Adjudication of reported serious adverse joint events in the tanezumab clinical development program.Arthr Rheumatol.2016；68:382-91)。In general, the most common adverse reactions when anti-NGF antibodies are used to treat related diseases include neurological-related adverse reactions (Neurologic-related AEs) and joint-related adverse reactions (joint-related AEs). Neurological-related adverse reactions mainly include peripheral nerve sensory symptoms, including paresthesia, sensory impairment, hyperesthesia, burning pain and touch pain. Reported serious joint-related adverse reactions include osteonecrosis (Bannwarth B, et al., Nerve Growth Factor Antagonists: Is the Future of Monoclonal Antibodies Becoming Clearer? Drugs. 2017 Sep; 77(13): 1377-1387). Although the mechanism of these adverse reactions has not yet been fully elucidated, it is believed that the physiological effects of NGF in the adult peripheral nervous system have been verified in animal experiments, including maintaining the morphological homeostasis and biochemical function of dorsal root ganglion sensory neurons (Rask CA. Biological actions of nerve growth factor in the peripheral nervous system. Eur Neurol. 1999; 41 (Suppl. 1): 14-9). At the same time, NGF can promote tissue repair and play a role in cartilage repair and load-induced bone formation. The use of NGF inhibitors, such as anti-NGF antibodies, may disrupt bone repair in patients with subchondral clefts, thereby triggering the rapid progression of osteoarthritis (Hochberg MC, Tive LA, Abramson SB, et al. When is osteocrosis not osteocrosis? Adjudication of reported serious adverse joint events in the tanezumab clinical development program. Arthr Rheumatol. 2016; 68: 382-91).
本发明的目的在于，在应用抗NGF抗体进行相应疾病治疗时，在患者中补充能够发挥生物学活性，且生物学活性基本不受所应用的抗NGF抗体影响的NGF突变体，即该突变体与应用的抗NGF抗体结合减弱或基本不结合，该突变体仍能够发挥NGF的生物学活性，从而减轻或避免了应用抗NGF抗体治疗时所产生的不良反应。The purpose of the present invention is to supplement the patient with an NGF mutant that can exert biological activity and whose biological activity is basically unaffected by the anti-NGF antibody used when anti-NGF antibodies are used to treat corresponding diseases, that is, the binding of the mutant to the anti-NGF antibody used is weakened or basically not bound, and the mutant can still exert the biological activity of NGF, thereby reducing or avoiding the adverse reactions caused by the anti-NGF antibody treatment.
本文提及的所有出版物、专利、专利申请和已公开的专利申请所披露的内容，全部以引用方式并入本文。The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety.
发明概述SUMMARY OF THE INVENTION
本申请一方面涉及一种筛选和/或鉴定NGF中可突变位点的方法，其中所述可突变位点上的氨基酸突变会影响NGF突变体与一种或多种抗NGF抗体的结合，但仍能使NGF突变体具有生物学活性。On one hand, the present application relates to a method for screening and/or identifying mutable sites in NGF, wherein the amino acid mutation at the mutable site will affect the binding of the NGF mutant to one or more anti-NGF antibodies, but still enable the NGF mutant to have biological activity.
在一些实施例中，本申请所述的筛选和/或鉴定NGF中可突变位点的方法，包括如下步骤：(i)获得一种或多种抗NGF抗体的三维结构；(ii)将成熟NGF的晶体结构与上述获得的抗NGF抗体的三维结构分别进行对接，以获得一种或多种抗NGF抗体与NGF的结合构象，其中，成熟NGF的晶体结构如PDB ID：4EDW所示；(iii)将 步骤(ii)中获得的结合构象和成熟NGF与TrkA受体的结合构象分别进行比对；(iv)基于步骤(iii)中的比对结果，获得所述NGF中的可突变位点。In some embodiments, the method for screening and/or identifying mutable sites in NGF described in the present application comprises the following steps: (i) obtaining a three-dimensional structure of one or more anti-NGF antibodies; (ii) docking the crystal structure of mature NGF with the three-dimensional structure of the anti-NGF antibody obtained above, respectively, to obtain the binding conformation of one or more anti-NGF antibodies and NGF, wherein the crystal structure of mature NGF is shown in PDB ID: 4EDW; (iii) docking The binding conformation obtained in step (ii) and the binding conformation of mature NGF and TrkA receptor are respectively compared; (iv) based on the comparison results in step (iii), the mutable sites in the NGF are obtained.
在一些实施例中，步骤(i)采用同源建模方法来构建并获得NGF抗体的三维结构，优选应用Modeller同源建模方法。In some embodiments, step (i) uses a homology modeling method to construct and obtain the three-dimensional structure of the NGF antibody, preferably using the Modeller homology modeling method.
在一些实施例中，步骤(ii)采用Discovery studio ZDOCK分子对接技术来将成熟NGF的晶体结构与抗NGF抗体的三维结构进行对接。In some embodiments, step (ii) uses Discovery studio ZDOCK molecular docking technology to dock the crystal structure of mature NGF with the three-dimensional structure of anti-NGF antibody.
在一些实施例中，步骤(iii)中成熟NGF与TrkA受体的结合构象如PDB ID：1WWW所示。In some embodiments, the binding conformation of mature NGF and TrkA receptor in step (iii) is shown in PDB ID: 1WWW.
在一些实施例中，NGF中的可突变位点是指NGF中位于与抗NGF抗体结合界面，但不位于与TrkA受体结合界面的氨基酸位点。在一些实施例中，如果涉及多种抗NGF抗体，则所述可突变位点为针对各种抗NGF抗体所获得的多组可突变位点之间的交集或并集。In some embodiments, the mutable site in NGF refers to an amino acid site in NGF that is located at the binding interface with the anti-NGF antibody but not at the binding interface with the TrkA receptor. In some embodiments, if multiple anti-NGF antibodies are involved, the mutable site is the intersection or union of multiple groups of mutable sites obtained for various anti-NGF antibodies.
本申请一方面涉及一种构建NGF突变体库的方法，其特征在于，所述突变体库中包含的NGF突变体中具有一个或多个可突变位点，在该一个或多个可突变位点上进行氨基酸突变会影响NGF与一种或多种抗NGF抗体的结合，但仍能使NGF突变体具有生物学活性。On the one hand, the present application relates to a method for constructing an NGF mutant library, characterized in that the NGF mutants contained in the mutant library have one or more mutable sites, and amino acid mutations at the one or more mutable sites will affect the binding of NGF to one or more anti-NGF antibodies, but still enable the NGF mutants to have biological activity.
在一些实施例中，构建NGF突变体库的方法包括如下步骤：以人野生型成熟NGF为模板，在上述可突变位点中的一个或多个位点上进行氨基酸突变，产生一系列的NGF突变体，从而获得NGF突变体库。In some embodiments, the method for constructing an NGF mutant library comprises the following steps: using human wild-type mature NGF as a template, performing amino acid mutations at one or more of the above-mentioned mutable sites to generate a series of NGF mutants, thereby obtaining an NGF mutant library.
在一些实施例中，NGF突变体库中包含的NGF突变体所具有的一个或多个可突变位点是通过如前所述的一种筛选和/或鉴定NGF中可突变位点的方法获得。In some embodiments, one or more mutable sites of the NGF mutants contained in the NGF mutant library are obtained by a method for screening and/or identifying mutable sites in NGF as described above.
在一些实施例中，上文所述的可突变位点上的氨基酸突变会影响NGF突变体与抗NGF抗体的结合是指该位点上的氨基酸发生突变会导致NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合。In some embodiments, the amino acid mutation at the above-mentioned mutable site will affect the binding of the NGF mutant to the anti-NGF antibody, which means that the amino acid mutation at this site will cause the NGF mutant to weaken the binding of the anti-NGF antibody or basically not bind to the anti-NGF antibody.
本申请一方面涉及一种NGF突变体库，其包含与一种或多种抗NGF抗体结合减弱或基本不与抗NGF抗体结合，同时具有生物学活性的NGF突变体。 One aspect of the present application relates to an NGF mutant library, which comprises NGF mutants that have reduced binding to one or more anti-NGF antibodies or substantially no binding to anti-NGF antibodies while having biological activity.
在一些实施例中，NGF突变体库采用上文所述的一种构建NGF突变体库的方法构建获得。在一些实施例中，所述NGF突变体库中的NGF突变体进一步包含相对于人野生型成熟NGF的突变F12E。In some embodiments, the NGF mutant library is constructed using a method for constructing an NGF mutant library as described above. In some embodiments, the NGF mutants in the NGF mutant library further comprise a mutation F12E relative to human wild-type mature NGF.
本申请一方面涉及一种筛选或鉴定NGF突变体的方法，其中所述NGF突变体与一种或多种抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，同时具有生物学活性。One aspect of the present application relates to a method for screening or identifying NGF mutants, wherein the NGF mutants have reduced binding to one or more anti-NGF antibodies or substantially no binding to anti-NGF antibodies while having biological activity.
在一些实施例中，筛选NGF突变体的方法包括如下步骤：(i)获得一个或多个NGF突变体；(ii)从步骤(i)中筛选或鉴定具有生物学活性，并且与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的一系列NGF突变体。In some embodiments, the method for screening NGF mutants comprises the following steps: (i) obtaining one or more NGF mutants; (ii) screening or identifying a series of NGF mutants from step (i) that have biological activity and have reduced binding to or essentially no binding to anti-NGF antibodies.
在一些实施中，上述步骤(ii)采用如下任一种方法来获得与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，且具有生物学活性的一系列NGF突变体：(1)首先筛选或鉴定具有生物学活性的NGF突变体，再从中筛选或鉴定与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体；或者(2)首先筛选或鉴定与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体，再从中筛选或鉴定具有生物学活性的NGF突变体；或者(3)分别筛选或鉴定具有生物学活性的NGF突变体，以及与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体，取二者的交集。In some implementations, the above step (ii) adopts any of the following methods to obtain a series of NGF mutants with reduced binding to or essentially no binding to anti-NGF antibodies and having biological activity: (1) first screening or identifying NGF mutants with biological activity, and then screening or identifying NGF mutants with reduced binding to or essentially no binding to anti-NGF antibodies; or (2) first screening or identifying NGF mutants with reduced binding to or essentially no binding to anti-NGF antibodies, and then screening or identifying NGF mutants with biological activity; or (3) separately screening or identifying NGF mutants with biological activity and NGF mutants with reduced binding to or essentially no binding to anti-NGF antibodies, and taking the intersection of the two.
在一些实施中，上述步骤(i)中所述的NGF突变体是从NGF突变体库获得，或者通过理性设计获得。在一些实施例中，所述NGF突变体库通过如上所述的任一项构建NGF突变体库的方法获得，或者选自如上所述的NGF突变体库。In some implementations, the NGF mutants described in step (i) above are obtained from an NGF mutant library, or obtained by rational design. In some embodiments, the NGF mutant library is obtained by any of the methods for constructing an NGF mutant library as described above, or is selected from the NGF mutant library as described above.
在一些实施中，上述步骤(i)中所述的NGF突变体通过表面展示技术进行展示，包括原核展示文库技术和真核展示文库技术，进一步地，可以选择通过酵母表面展示技术、噬菌体表面展示技术、哺乳动物细胞表面展示进术、细菌表面展示技术或杆状病毒表面展示技术进行展示。在一些实施中，上述步骤(ii)中通过MACS、FACS和/或生物学活性测定来筛选或鉴定具有生物学活性的NGF突变体。在一些实施中，上述步骤(ii)中通过MACS、FACS和/或ELISA来筛选与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体。In some implementations, the NGF mutants described in the above step (i) are displayed by surface display technology, including prokaryotic display library technology and eukaryotic display library technology. Further, yeast surface display technology, phage surface display technology, mammalian cell surface display technology, bacterial surface display technology or baculovirus surface display technology can be selected for display. In some implementations, in the above step (ii), MACS, FACS and/or biological activity assays are used to screen or identify NGF mutants with biological activity. In some implementations, in the above step (ii), MACS, FACS and/or ELISA are used to screen NGF mutants with weakened binding to or essentially no binding to anti-NGF antibodies.
本申请一方面涉及一种NGF突变体，其与一种或多种抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，同时具有生物学活性。 One aspect of the present application relates to an NGF mutant, which has a weakened binding to one or more anti-NGF antibodies or substantially no binding to anti-NGF antibodies, while having biological activity.
在一些实施例中，所述NGF突变体采用如上所述的一种筛选NGF突变体的方法筛选获得。In some embodiments, the NGF mutant is obtained by screening using a method for screening NGF mutants as described above.
本申请一方面涉及一种NGF突变体，其包含相对于人野生型成熟NGF氨基酸序列的I31、K32、G33、K34、D93、W21、G23、D24、K50、Y52、T83、H84、F86、R100、R103、D16、S17、S19、T56、R59和R69位点中的一个或多个位点上的氨基酸突变。On the one hand, the present application relates to an NGF mutant, which comprises amino acid mutations at one or more of the I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 positions relative to the human wild-type mature NGF amino acid sequence.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S17、S19、K32、T56、R59和T83中的一个或多个位点上的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at one or more of S17, S19, K32, T56, R59 and T83 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S17和K32位点的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at positions S17 and K32 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列S19和K32位点的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at positions S19 and K32 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列S19和S17位点的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at positions S19 and S17 relative to the amino acid sequence of wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列R59和T83位点的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at positions R59 and T83 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体中S17位点的氨基酸突变为S17R、S17K、S17H或S17E。In some embodiments, the amino acid mutation at the S17 position in the NGF mutant described herein is S17R, S17K, S17H or S17E.
在一些实施例中，本申请所述的NGF突变体中S19位点的氨基酸突变为S19R、S19K或S19F。In some embodiments, the amino acid mutation at position S19 in the NGF mutant described herein is S19R, S19K or S19F.
在一些实施例中，本申请所述的NGF突变体中K32位点的氨基酸突变为K32F、K32E、K32N、K32Y、K32M或K32L。In some embodiments, the amino acid mutation at position K32 in the NGF mutant described herein is K32F, K32E, K32N, K32Y, K32M or K32L.
在一些实施例中，本申请所述的NGF突变体中T56位点的氨基酸突变为T56K或T56R。In some embodiments, the amino acid mutation at position T56 in the NGF mutant described herein is T56K or T56R.
在一些实施例中，本申请所述的NGF突变体中R59位点的氨基酸突变为R59K。In some embodiments, the amino acid mutation at position R59 in the NGF mutant described herein is R59K.
在一些实施例中，本申请所述的NGF突变体中T83位点的氨基酸突变为T83K。 In some embodiments, the amino acid at position T83 in the NGF mutant described herein is mutated to T83K.
在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S17R，以及K32E、K32Y、K32M或K32L。In some embodiments, the NGF mutants described herein comprise the amino acid mutations S17R, and K32E, K32Y, K32M or K32L.
在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S19R，以及K32E、K32Y、K32M或K32L。In some embodiments, the NGF mutants described herein comprise the amino acid mutations S19R, and K32E, K32Y, K32M or K32L.
在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S19R和S17E。In some embodiments, the NGF mutants described herein comprise amino acid mutations S19R and S17E.
在一些实施例中，本申请所述的NGF突变体包含氨基酸突变R59K和T83K。In some embodiments, the NGF mutants described herein comprise amino acid mutations R59K and T83K.
在一些实施例中，本申请所述的NGF突变体进一步包含相对于人野生型成熟NGF的突变F12E。In some embodiments, the NGF mutant described herein further comprises a mutation F12E relative to human wild-type mature NGF.
在一些实施例中，本申请所述的人野生型成熟NGF的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。In some embodiments, the amino acid sequence of human wild-type mature NGF described in the present application is shown in SEQ ID NO:1 or SEQ ID NO:2.
在一些实施例中，本申请所述的NGF突变体包含SEQ ID NOs:17-60中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:17-60中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。In some embodiments, the NGF mutant described in the present application comprises an amino acid sequence shown in any one of SEQ ID NOs: 17-60 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 17-60.
本申请一方面涉及包含上文所述的NGF突变体的融合蛋白，在一些实施例中，所述融合蛋白包含Fc。在一些实施例中，所述Fc包含能够改变效应功能的突变，和/或能够延长半衰期的突变。在一些实施例中，所述Fc来源于人IgG1或IgG4。在一些实施例中，所述Fc来源于人IgG1，优选的包含L234A、L235A和P331S突变，其中所述编号为EU编号系统。在一些实施例中，所述融合蛋白中NGF突变体与Fc连接，优选的，通过肽接头连接，更优选的，所述肽接头由氨基酸残基G和S组成；例如，所述肽接头包含氨基酸序列GGGGSGGGGSGGGGS(SEQ ID NO:87)。在一些实施例中，所述融合蛋白中NGF突变体位于Fc的N端和/或C端。On the one hand, the present application relates to a fusion protein comprising the NGF mutant described above, in some embodiments, the fusion protein comprises Fc. In some embodiments, the Fc comprises mutations that can change effector function, and/or mutations that can extend half-life. In some embodiments, the Fc is derived from human IgG1 or IgG4. In some embodiments, the Fc is derived from human IgG1, preferably comprising L234A, L235A and P331S mutations, wherein the numbering is the EU numbering system. In some embodiments, the NGF mutant in the fusion protein is connected to Fc, preferably, through a peptide linker, more preferably, the peptide linker is composed of amino acid residues G and S; for example, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 87). In some embodiments, the NGF mutant in the fusion protein is located at the N-terminus and/or C-terminus of Fc.
在一些实施例中，本申请所述的包含NGF突变体的融合蛋白，其特征在于，所述融合蛋白包含SEQ ID NOs:61-82中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:61-82中任一所示的氨基酸序列具有至少80％(例如至少80％、85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。In some embodiments, the fusion protein comprising an NGF mutant described in the present application is characterized in that the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 61-82 or a variant thereof, and the variant has at least 80% (for example, at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 61-82.
同时，本申请还涉及编码上文所述任何NGF突变体或包含NGF突变体的融合蛋白的分离的核酸、包含所述核酸的载体、包含所述核酸或载体的宿主细胞(例如， CHO细胞、HEK 293细胞、Hela细胞或COS细胞)、组合物(例如，药物组合物)、试剂盒以及包含本文所述任何NGF突变体或包含NGF突变体的融合蛋白的制品及其制备方法。还涉及使用本文所述任何NGF突变体或其融合蛋白或包含其的药物组合物用于在有需要的个体中(例如，人类)治疗NGF相关的疾病或病症的方法(例如，神经系统疾病，包含例如角膜内皮营养不良、糖尿病神经性病变、糖尿病足溃疡、神经源性皮肤溃疡、压疮、神经营养性角膜溃疡、糖尿病性角膜溃疡和黄斑裂孔等，以及非神经系统疾病，包含例如脾萎缩、脾挫伤、卵巢储备功能下降、卵巢早衰、生精障碍(如少精子症、弱精子症、少弱精子症)、缺血性溃疡、应激性溃疡、类风湿性溃疡、肝纤维化、角膜溃疡、烧伤)的方法。At the same time, the present application also relates to an isolated nucleic acid encoding any of the NGF mutants described above or a fusion protein comprising an NGF mutant, a vector comprising the nucleic acid, a host cell comprising the nucleic acid or the vector (e.g., CHO cells, HEK 293 cells, Hela cells or COS cells), compositions (e.g., pharmaceutical compositions), kits, and products comprising any NGF mutant described herein or fusion proteins comprising NGF mutants, and methods for preparing the same. It also relates to methods for using any NGF mutant described herein or its fusion protein or pharmaceutical compositions comprising it for treating NGF-related diseases or conditions in individuals in need (e.g., humans) (e.g., nervous system diseases, including, for example, corneal endothelial dystrophy, diabetic neuropathy, diabetic foot ulcers, neurogenic skin ulcers, pressure sores, neurotrophic corneal ulcers, diabetic corneal ulcers and macular holes, and non-neurological diseases, including, for example, splenic atrophy, splenic contusion, decreased ovarian reserve function, premature ovarian failure, spermatogenesis disorders (such as oligospermia, asthenozoospermia, oligoasthenozoospermia), ischemic ulcers, stress ulcers, rheumatoid ulcers, liver fibrosis, corneal ulcers, burns).
本申请一方面涉及一种药物组合物，其包含抗NGF抗体和NGF突变体或包含NGF突变体的融合蛋白以及药学上可接受的载体和/或辅料，其中所述NGF突变体与抗NGF抗体结合减弱或基本不结合，同时仍具有生物学活性。On the one hand, the present application relates to a pharmaceutical composition, which comprises an anti-NGF antibody and an NGF mutant or a fusion protein comprising an NGF mutant and a pharmaceutically acceptable carrier and/or excipient, wherein the NGF mutant has weakened binding or essentially no binding to the anti-NGF antibody while still having biological activity.
在一些实施例中，所述的药物组合物中包含的NGF突变体选自如前所述的任一种或多种NGF突变体，或采用如前所述的一种筛选NGF突变体的方法获得。In some embodiments, the NGF mutant contained in the pharmaceutical composition is selected from any one or more NGF mutants as described above, or is obtained by using a method for screening NGF mutants as described above.
本申请一方面涉及一种降低和/或减轻应用抗NGF抗体治疗时的不良反应的方法，包括向已经接受、正在接受或即将接受抗NGF抗体治疗的个体施用NGF突变体或包含NGF突变体的融合蛋白，其中所述NGF突变体与该抗NGF抗体结合减弱或基本不结合，同时仍具有生物学活性。On the one hand, the present application relates to a method for reducing and/or alleviating adverse reactions during anti-NGF antibody treatment, comprising administering an NGF mutant or a fusion protein comprising an NGF mutant to an individual who has received, is receiving, or is about to receive anti-NGF antibody treatment, wherein the NGF mutant has weakened binding or essentially no binding to the anti-NGF antibody while still having biological activity.
在一些实施例中，上述方法中所述的不良反应包括神经系统相关的不良反应和关节相关的不良反应。在一些实施例中，所述不良反应包括：交感神经损伤、骨坏死、骨丢失、骨损伤、关节损伤、快速进展性骨关节炎(RPOA)、外周性水肿、关节痛、四肢疼痛、周围神经感觉异常、感觉迟钝、感觉过敏、灼烧痛和触感痛。In some embodiments, the adverse reactions described in the above methods include adverse reactions related to the nervous system and adverse reactions related to joints. In some embodiments, the adverse reactions include: sympathetic nerve damage, osteonecrosis, bone loss, bone damage, joint damage, rapidly progressive osteoarthritis (RPOA), peripheral edema, joint pain, limb pain, peripheral nerve paresthesia, dysesthesia, hyperesthesia, burning pain and tactile pain.
本申请一方面涉及一种在有需要的个体中治疗和/或预防由NGF表达增加和/或对NGF敏感性增强引起的疾病或病症的方法，包括向个体施用有效量的抗NGF抗体和NGF突变体或包含NGF突变体的融合蛋白或者如前所述的包含抗NGF抗体和NGF突变体的药物组合物，其中所述NGF突变体与该抗NGF抗体结合减弱或基本不结合，同时仍具有生物学活性。 On the one hand, the present application relates to a method for treating and/or preventing a disease or condition caused by increased NGF expression and/or enhanced sensitivity to NGF in an individual in need thereof, comprising administering to the individual an effective amount of an anti-NGF antibody and an NGF mutant or a fusion protein comprising an NGF mutant, or a pharmaceutical composition comprising an anti-NGF antibody and an NGF mutant as described above, wherein the NGF mutant has weakened binding or essentially no binding to the anti-NGF antibody while still having biological activity.
在一些实施例中，上述方法中所述的抗NGF抗体与所述NGF突变体或包含NGF突变体的融合蛋白同时施用或者顺序施用。In some embodiments, the anti-NGF antibody described in the above methods is administered simultaneously or sequentially with the NGF mutant or the fusion protein comprising the NGF mutant.
在一些实施例中，上述方法中所述的由NGF表达增加和/或对NGF敏感性增强引起的疾病或病症包括炎症性疼痛、术后切口疼痛、神经性疼痛、骨折疼痛、痛风关节疼痛、带状疱疹后神经痛、烧伤引起的疼痛、癌症疼痛、骨关节炎或类风湿性关节炎疼痛、坐骨神经痛、与镰状细胞危象相关的疼痛，或疱疹性神经痛。In some embodiments, the diseases or conditions caused by increased NGF expression and/or enhanced sensitivity to NGF described in the above methods include inflammatory pain, postoperative incisional pain, neuropathic pain, fracture pain, gouty joint pain, postherpetic neuralgia, pain caused by burns, cancer pain, osteoarthritis or rheumatoid arthritis pain, sciatica, pain associated with sickle cell crisis, or herpetic neuralgia.
在一些实施例中，上述方法中所述的NGF突变体选自如前所述的突变体库、任一种或多种NGF突变体或者采用如前所述的NGF突变体的筛选方法筛选获得。In some embodiments, the NGF mutants described in the above methods are selected from the mutant library as described above, any one or more NGF mutants, or obtained by screening using the NGF mutant screening method as described above.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述的NGF突变体与抗NGF抗体的结合减弱是指与人野生型成熟NGF(SEQ ID NO：1)或NGFF12E(SEQ ID NO：83)相比，NGF突变体与抗NGF抗体的结合活性降低。In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described in the present application, the weakened binding of the NGF mutant to the anti-NGF antibody means that the binding activity of the NGF mutant to the anti-NGF antibody is reduced compared with human wild-type mature NGF (SEQ ID NO: 1) or NGF F12E (SEQ ID NO: 83).
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述NGF突变体与抗NGF抗体的结合活性不超过人野生型成熟NGF或NGFF12E与相同抗NGF抗体结合活性的50％，优选为不超过40％、30％、20％、10％、8％、7％、6％、5％、4％、3％、2％、1％、0.6％、0.5％、0.2％、0.1％、0.07％、0.02％、0.01％、0.001％或更低。In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described in the present application, the binding activity of the NGF mutant with the anti-NGF antibody does not exceed 50% of the binding activity of human wild-type mature NGF or NGF F12E with the same anti-NGF antibody, and is preferably not more than 40%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.6%, 0.5%, 0.2%, 0.1%, 0.07%, 0.02%, 0.01%, 0.001% or less.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述NGF突变体基本不与抗NGF抗体结合。In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described herein, the NGF mutants do not substantially bind to anti-NGF antibodies.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述NGF突变体具有生物学活性是指与NGFF12E(SEQ ID NO：83)相比，其生物学活性为NGFF12E的至少20％，优选为至少21％、30％、36％、40％、80％、100％、1.5倍、2倍、3倍、4倍、5倍、10倍、15倍、20倍、50倍、100倍或更高。In any method, NGF mutant library, NGF mutant or pharmaceutical composition described in the present application, the NGF mutant has biological activity means that compared with NGF F12E (SEQ ID NO: 83), its biological activity is at least 20% of NGF F12E , preferably at least 21%, 30%, 36%, 40%, 80%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述生物学活性通过NGF生物学活性检测实验测定获得。In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described in the present application, the biological activity is obtained by measuring the NGF biological activity detection experiment.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述NGF生物学活性检测实验选自如下实验中的一种或多种：TrkA受体结合实验、TF-1细胞活性检测实验、PC12细胞活性检测实验、鸡胚背根神经节实验、或大鼠颈上神经节实验。 In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described in the present application, the NGF biological activity detection experiment is selected from one or more of the following experiments: TrkA receptor binding experiment, TF-1 cell activity detection experiment, PC12 cell activity detection experiment, chicken embryo dorsal root ganglion experiment, or rat superior cervical ganglion experiment.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述NGF突变体的生物学活性由其与TrkA受体的结合活性表征。In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described in the present application, the biological activity of the NGF mutant is characterized by its binding activity to the TrkA receptor.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述NGF突变体具有生物学活性是指该突变体能够与TrkA受体结合。In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described in the present application, the NGF mutant having biological activity means that the mutant is able to bind to the TrkA receptor.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述NGF突变体能够与TrkA受体结合是指与NGFF12E(SEQ ID NO：83)相比，其与TrkA受体的结合活性是NGFF12E的至少20％，优选为至少21％、30％、36％、40％、80％、100％、1.5倍、2倍、3倍、4倍、5倍、10倍、15倍、20倍、50倍、100倍或更高。In any method, NGF mutant library, NGF mutant or pharmaceutical composition described in the present application, the NGF mutant is capable of binding to the TrkA receptor, which means that compared with NGF F12E (SEQ ID NO: 83), its binding activity to the TrkA receptor is at least 20% of that of NGF F12E , preferably at least 21%, 30%, 36%, 40%, 80%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述结合或结合活性通过ELISA实验、Biacore实验、BLI实验等进行测定。In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described in the present application, the binding or binding activity is determined by ELISA experiments, Biacore experiments, BLI experiments, etc.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，所述结合活性采用在ELISA实验中的EC50值来表征。In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described herein, the binding activity is characterized by the EC50 value in an ELISA experiment.
本申请所述的任一种方法、NGF突变体库、NGF突变体或药物组合物中，在ELISA实验中，所述NGF突变体与抗NGF抗体的结合EC50值是人野生型成熟NGF或NGFF12E与相同抗NGF抗体的结合EC50值的至少2倍、3倍、5倍、10倍、13倍、14倍、17倍、20倍、25倍、33倍、50倍、100倍、167倍、200倍、500倍、1000倍、1400倍、5000倍、10000倍、100000倍或更高，同时所述NGF突变体与TrkA受体的结合EC50值是NGFF12E与TrkA受体的结合EC50值的至多5倍、3倍、2倍、1倍、70％、60％、50％、35％、30％、25％、20％、10％、7％、5％、2％、1％或更低。In any of the methods, NGF mutant libraries, NGF mutants or pharmaceutical compositions described in the present application, in an ELISA experiment, the EC50 value of the binding of the NGF mutant to an anti-NGF antibody is at least 2 times, 3 times, 5 times, 10 times, 13 times, 14 times, 17 times, 20 times, 25 times, 33 times, 50 times, 100 times, 167 times, 200 times, 500 times, 1000 times, 1400 times, 5000 times, 10000 times, 100000 times or more of the EC50 value of the binding of the NGF mutant to the TrkA receptor. The EC50 value of F12E binding to the TrkA receptor is at most 5-fold, 3-fold, 2-fold, 1-fold, 70%, 60%, 50%, 35%, 30%, 25%, 20%, 10%, 7%, 5%, 2%, 1% or less.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1A所示为STC001与人野生型成熟NGF的结合构象示意图，图1B所示为TrkA受体与人野生型成熟NGF的结合构象示意图，图1C所示为将STC001与人野生型成熟NGF的结合构象与TrkA受体与人野生型成熟NGF的结合构象进行叠合后的构象图。Figure 1A shows a schematic diagram of the binding conformation of STC001 and human wild-type mature NGF, Figure 1B shows a schematic diagram of the binding conformation of TrkA receptor and human wild-type mature NGF, and Figure 1C shows a conformational diagram after superimposing the binding conformation of STC001 and human wild-type mature NGF and the binding conformation of TrkA receptor and human wild-type mature NGF.
图2所示为人野生型成熟NGF的晶体结构，以及针对表1中各抗NGF抗体的多组可突变位点之间交集中的可突变体位点及其所分布的4个区域。 FIG2 shows the crystal structure of wild-type mature human NGF, as well as the mutated sites at the intersection of multiple groups of mutated sites for each anti-NGF antibody in Table 1 and the four regions in which they are distributed.
图3所示为preproNGF的结构示意图。FIG3 is a schematic diagram showing the structure of preproNGF.
图4A所示为hNGF-Fc与多种抗NGF抗体的ELISA结合曲线，图4B所示为NGFF12E-Fc与多种抗NGF抗体的结合ELISA曲线。FIG. 4A shows the ELISA binding curves of hNGF-Fc and various anti-NGF antibodies, and FIG. 4B shows the ELISA binding curves of NGF F12E -Fc and various anti-NGF antibodies.
图4C-图4J所示分别为示例性的NGF突变体与Fc的融合蛋白1A2-Fc、1A10-Fc、1A7-Fc、1A8-Fc、1A9-Fc、1A11-Fc、1A12-Fc、1A13-Fc与多种抗NGF抗体的结合ELISA曲线。Figures 4C-4J show the binding ELISA curves of exemplary NGF mutant and Fc fusion proteins 1A2-Fc, 1A10-Fc, 1A7-Fc, 1A8-Fc, 1A9-Fc, 1A11-Fc, 1A12-Fc, and 1A13-Fc with various anti-NGF antibodies.
图5所示为示例性的NGF突变体与Fc的融合蛋白1A2-Fc和1A10-Fc在TF-1细胞增殖实验中的结果。FIG. 5 shows the results of the TF-1 cell proliferation assay using exemplary NGF mutant and Fc fusion proteins 1A2-Fc and 1A10-Fc.
图6所示为示例性的NGF突变体与Fc的融合蛋白1A2-Fc和1A10-Fc在PC12细胞活性检测实验中的结果。FIG. 6 shows the results of the PC12 cell activity detection experiment using exemplary NGF mutant and Fc fusion proteins 1A2-Fc and 1A10-Fc.
图7所示为不同剂量下的示例性NGF突变体与Fc的融合蛋白1A2-Fc和1A10-Fc在大鼠体内颈上神经节生长实验中的结果。FIG. 7 shows the results of an in vivo superior cervical ganglion growth experiment in rats using exemplary NGF mutant and Fc fusion proteins 1A2-Fc and 1A10-Fc at different doses.
图8所示为示例性的NGF突变体与Fc的融合蛋白1A2-Fc和1A10-Fc在大鼠体内疼痛阈值检测实验中的结果。FIG8 shows the results of an in vivo pain threshold test experiment in rats using exemplary NGF mutant and Fc fusion proteins 1A2-Fc and 1A10-Fc.
图9A所示为ELISA实验中示例性的NGF突变体与Fc的融合蛋白1A17-Fc、1A18-Fc、1A21-Fc和1A22-Fc与STC001的结合曲线。图9B所示为ELISA实验中示例性的NGF突变体与Fc的融合蛋白1A15-Fc、1A16-Fc、1A19-Fc和1A20-Fc与STC001的结合曲线。Figure 9A shows the binding curves of exemplary NGF mutant and Fc fusion proteins 1A17-Fc, 1A18-Fc, 1A21-Fc and 1A22-Fc with STC001 in ELISA experiments. Figure 9B shows the binding curves of exemplary NGF mutant and Fc fusion proteins 1A15-Fc, 1A16-Fc, 1A19-Fc and 1A20-Fc with STC001 in ELISA experiments.
图9C-9G所示分别为ELISA实验中示例性的NGF突变体与Fc的融合蛋白1A15-Fc、1A16-Fc、1A19-Fc、1A20-Fc和1A24-Fc与多种抗NGF抗体的结合曲线。Figures 9C-9G show the binding curves of exemplary NGF mutant and Fc fusion proteins 1A15-Fc, 1A16-Fc, 1A19-Fc, 1A20-Fc and 1A24-Fc with various anti-NGF antibodies in ELISA experiments.
图10A所示为ELISA实验中示例性的NGF突变体与Fc的融合蛋白1A17-Fc、1A18-Fc、1A21-Fc和1A22-Fc与TrkA受体的结合曲线。图10B所示为ELISA实验中示例性的NGF突变体与Fc的融合蛋白1A15-Fc、1A16-Fc、1A19-Fc和1A20-Fc与TrkA受体的结合曲线。图10C所示为ELISA实验中示例性的NGF突变体与Fc的融合蛋白1A24-Fc与TrkA受体的结合曲线。Figure 10A shows the binding curves of exemplary NGF mutant and Fc fusion proteins 1A17-Fc, 1A18-Fc, 1A21-Fc and 1A22-Fc with TrkA receptor in ELISA experiments. Figure 10B shows the binding curves of exemplary NGF mutant and Fc fusion proteins 1A15-Fc, 1A16-Fc, 1A19-Fc and 1A20-Fc with TrkA receptor in ELISA experiments. Figure 10C shows the binding curves of exemplary NGF mutant and Fc fusion protein 1A24-Fc with TrkA receptor in ELISA experiments.
图11A所示为示例性的NGF突变体与Fc的融合蛋白1A7-Fc联合应用Fasinumab在PC12细胞活性检测实验中的结果。图11B所示为示例性的NGF突变体与 Fc的融合蛋白1A13-Fc联合应用AK-115在PC12细胞活性检测实验中的结果。图11C所示为示例性的NGF突变体与Fc的融合蛋白1A12-Fc联合应用STC001在PC12细胞活性检测实验中的结果。图11D所示为示例性的NGF突变体与Fc的融合蛋白1A10-Fc联合应用STC001在PC12细胞活性检测实验中的结果。图11E所示为示例性的NGF突变体与Fc的融合蛋白1A4-Fc和1A5-Fc分别联合应用STC001在PC12细胞活性检测实验中的结果。FIG11A shows the results of an exemplary NGF mutant and Fc fusion protein 1A7-Fc combined with Fasinumab in a PC12 cell activity detection experiment. FIG11B shows the results of an exemplary NGF mutant and The results of the PC12 cell activity detection experiment using the Fc fusion protein 1A13-Fc in combination with AK-115. Figure 11C shows the results of the PC12 cell activity detection experiment using the exemplary NGF mutant and Fc fusion protein 1A12-Fc in combination with STC001. Figure 11D shows the results of the PC12 cell activity detection experiment using the exemplary NGF mutant and Fc fusion protein 1A10-Fc in combination with STC001. Figure 11E shows the results of the PC12 cell activity detection experiment using the exemplary NGF mutant and Fc fusion proteins 1A4-Fc and 1A5-Fc in combination with STC001, respectively.
本发明的详细描述Detailed description of the invention
除非上下文另有明确说明，实施本申请将采用本领域技术范围内的病毒学、免疫学、微生物学、分子生物学和重组DNA技术的常规方法，许多所述方法将在下文详述以供说明。所述技术在文献中有充分解释。参见Current Protocols in Molecular Biology or Current Protocols in Immunology,John Wiley&Sons,New York,N.Y.(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)和其它相似参考资料。Unless the context clearly indicates otherwise, the practice of this application will employ conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of the art, many of which are described in detail below for illustration. Such techniques are fully explained in the literature. See Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (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.
定义如本申请采用的术语“神经生长因子”或“NGF”在本文中可互换使用，包括preproNGF前体，proNGF前体或成熟的NGF(也可称为成熟的β-NGF，或β-NGF)，并且包括其天然变体、同种型、同源物、直系同源物或旁系同源物、片段或其衍生物。NGF可以是截短体、翻译后修饰形式、杂交变体、肽模拟物、生物活性片段、缺失变体、替换变体或插入变体，这些变体至少能在一定程度上维持NGF的生物学活性，例如与NGF受体结合，通过NGF受体传递信号等。如文中应用的NGF可以来自任何有机体，包括所有哺乳动物物种，包括但不限于人、犬、猫、马或牛，优选来自于人。Definitions As used herein, the terms "nerve growth factor" or "NGF" are used interchangeably herein and include preproNGF precursor, proNGF precursor or mature NGF (also referred to as mature β-NGF, or β-NGF), and include natural variants, isoforms, homologs, orthologs or paralogs, fragments or derivatives thereof. NGF may be a truncated form, a post-translationally modified form, a hybrid variant, a peptide mimetic, a biologically active fragment, a deletion variant, a substitution variant or an insertion variant, which variants can maintain the biological activity of NGF at least to a certain extent, such as binding to NGF receptors, transmitting signals through NGF receptors, etc. As used herein, NGF may be from any organism, including all mammalian species, including but not limited to humans, dogs, cats, horses or cattle, preferably from humans.
术语“NGF衍生物”是指一种具有NGF氨基酸序列或NGF类似物的分子，但在一个或多个氨基酸基团、α碳原子、氨基末端或羧基末端上还具有额外的化学修饰。化学修饰包括但不限于，添加化学基团、生成新化学键和移除化学基团。氨基酸侧基 团的修饰包括但不限于赖氨酸的ε氨基酰基化，精氨酸、组氨酸或赖氨酸的N-烷基化，谷氨酸或天冬氨酸的羧基烷基化，谷氨酰胺或天冬酰胺的脱氨。氨基末端的修饰包括但不限于脱氨、N-低烷基，N-二低烷基和N-酰基修饰。羧基末端的修饰包括但不限于酰胺、低烷基酰基、二烷基酰胺和低烷基酯修饰。在一些实施例中，低烷基基团为C1-C4烷基基团。此外，一个或多个侧基或末端基团可以由化学领域的技术人员利用已知的保护基团来保护。氨基酸的α碳可以是单甲基化或二甲基化。在一些实施例中，修饰的NGF包括如聚乙二醇化NGF，或共价修饰的NGF，如糖基化NGF。The term "NGF derivative" refers to a molecule having the amino acid sequence of NGF or an NGF analog, but having additional chemical modifications at one or more amino acid groups, the alpha carbon atom, the amino terminus, or the carboxyl terminus. Chemical modifications include, but are not limited to, adding chemical groups, creating new chemical bonds, and removing chemical groups. Modifications of the amino group include, but are not limited to, acylation of the epsilon amino group of lysine, N-alkylation of arginine, histidine or lysine, carboxyl alkylation of glutamic acid or aspartic acid, and deamination of glutamine or asparagine. Modifications of the amino terminal include, but are not limited to, deamination, N-low alkyl, N-di-low alkyl and N-acyl modifications. Modifications of the carboxyl terminal include, but are not limited to, amide, low alkyl acyl, dialkyl amide and low alkyl ester modifications. In some embodiments, the low alkyl group is a C1-C4 alkyl group. In addition, one or more side groups or terminal groups can be protected by a person skilled in the art of chemistry using known protecting groups. The alpha carbon of the amino acid can be monomethylated or dimethylated. In some embodiments, the modified NGF includes, for example, pegylated NGF, or covalently modified NGF, such as glycosylated NGF.
术语“野生型”是指在一个群体中最经常观察到的基因或基因编码产物(例如，多肽)，因此被设定为正常“normal”或野生型“wild-type”。The term "wild-type" refers to a gene or gene-encoded product (eg, polypeptide) that is most frequently observed in a population and is therefore designated as "normal" or "wild-type".
NGF受体是指由NGF结合或活化的多肽。NGF受体包括任何哺乳动物物种包括但不限于人、犬、猫、马、灵长类动物或牛的TrkA受体及p75NTR受体。NGF receptor refers to a polypeptide bound or activated by NGF. NGF receptors include TrkA receptors and p75NTR receptors of any mammalian species including but not limited to humans, dogs, cats, horses, primates or cattle.
本文所用的术语“TrkA受体(tyrosine kinase receptor)”是NGF的功能性受体，选择性结合NGF。在本领域中也称为原肌球蛋白激酶受体A和1型神经营养酪氨酸激酶受体(NTRK1)。人TrkA受体的示例性非限制性序列包括Genbank登记号NP_001012331(同种型1)、NP_002520(同种型2)和NP_001007793(同种型3)的氨基酸序列。The term "TrkA receptor (tyrosine kinase receptor)" as used herein is a functional receptor for NGF that selectively binds NGF. It is also known in the art as tropomyosin kinase receptor A and neurotrophic tyrosine kinase receptor type 1 (NTRK1). Exemplary non-limiting sequences of human TrkA receptors include the amino acid sequences of Genbank accession numbers NP_001012331 (isoform 1), NP_002520 (isoform 2), and NP_001007793 (isoform 3).
本申请所用的术语“p75神经营养因子受体(p75 neurotrophin receptor，p75NTR)”是指分子量为约75kDa并且结合NGF和其它神经营养蛋白的神经营养蛋白受体，关于p75NTR受体的描述参见于例如Bothwell,M.(1996)Science 272：506-507中。The term "p75 neurotrophin receptor (p75NTR)" used in this application refers to a neurotrophin receptor with a molecular weight of approximately 75 kDa and that binds NGF and other neurotrophins. For a description of the p75NTR receptor, see, for example, Bothwell, M. (1996) Science 272: 506-507.
如本申请所述的氨基酸的“突变”，包括取代、缺失、插入和修饰。可以进行取代、缺失、插入和修饰的任意组合来获得具有期望性质(例如，降低与抗NGF抗体的结合亲和力)的构建体所期望的特性。在一些实施例中，氨基酸的缺失或插入包括在多肽序列的任意位置的缺失或插入。在一些实施例中，氨基酸的取代可以为保守性的氨基酸取代。在另一些实施例中，氨基酸的取代可以为非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。氨基酸的取代还包括应用非天然存在的氨基酸或由20种标准氨基酸的天然存在的氨基酸衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟基赖氨酸)进行替换。在一些实施例中，氨基酸的突变包括氨基酸的取代和缺失的组合。可以利用本领域中公知的遗传或化学方法生成氨基酸突变，包括定点突变、PCR、基因合成、化学修饰等方法。 "Mutation" of amino acids as described herein includes substitution, deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed to obtain the desired properties of the construct with the desired properties (e.g., reducing the binding affinity with anti-NGF antibodies). In some embodiments, the deletion or insertion of amino acids includes deletion or insertion at any position of the polypeptide sequence. In some embodiments, the substitution of amino acids can be conservative amino acid substitution. In other embodiments, the substitution of amino acids can be non-conservative amino acid substitution, that is, one amino acid is replaced with another amino acid having different structures and/or chemical properties. The substitution of amino acids also includes the use of non-natural amino acids or replacement by naturally occurring amino acid derivatives of 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). In some embodiments, the mutation of amino acids includes a combination of substitution and deletion of amino acids. Amino acid mutations can be generated using genetic or chemical methods known in the art, including methods such as site-directed mutagenesis, PCR, gene synthesis, and chemical modification.
如本申请所述的“NGF突变体”，其包括在野生型NGF氨基酸序列(例如但不限于，人野生型成熟NGF，其序列如SEQ ID NO.1或2所示)中包含一个或多个取代、缺失、插入、修饰或者包含其任何形式的组合。The "NGF mutant" as described in the present application includes one or more substitutions, deletions, insertions, modifications or any combination thereof in the wild-type NGF amino acid sequence (for example, but not limited to, human wild-type mature NGF, whose sequence is shown in SEQ ID NO.1 or 2).
在本申请所述的NGF突变体中，氨基酸突变的位置是基于野生型成熟NGF(优选人野生型成熟NGF)氨基酸序列中的氨基酸位置来进行确定的，在一些实施例中，人野生型成熟NGF的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示，可以通过与SEQ ID NO:1或SEQ ID NO:2进行氨基酸序列比对(例如，应用BLAST)，来鉴定NGF突变体中氨基酸发生突变所对应的位置。在一些实施例中，当描述例如S17时，代表相对于野生型成熟NGF(优选人野生型成熟NGF)氨基酸序列的第17位上的氨基酸残基为丝氨酸(S)。“S17位点的突变”代表第17位的丝氨酸残基(S)发生突变。在一些实施例中，当描述NGF突变体时，按照如下方式描述。例如“氨基酸取代”表示为“原始氨基酸残基/发生取代的氨基酸位置/取代后的氨基酸残基”，例如“S17R”代表相对于野生型成熟NGF的氨基酸序列(例如，SEQ ID NO.1或2)的第17位上的丝氨酸残基(S)被精氨酸残基(R)所取代。本申请中关于突变位点组合方案中所述的“+”表示在多个特定的位置同时发生突变，例如，“S17R+K32E”代表相对于野生型成熟的NGF氨基酸序列(例如，SEQ ID NO.1或2)的第17位上的丝氨酸残基(S)被精氨酸残基(R)所取代，以及第32位上的赖氨酸残基(K)被谷氨酸残基(E)所取代。In the NGF mutant described in the present application, the position of the amino acid mutation is determined based on the amino acid position in the amino acid sequence of wild-type mature NGF (preferably human wild-type mature NGF). In some embodiments, the amino acid sequence of human wild-type mature NGF is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the position corresponding to the amino acid mutation in the NGF mutant can be identified by comparing the amino acid sequence with SEQ ID NO: 1 or SEQ ID NO: 2 (for example, using BLAST). In some embodiments, when describing, for example, S17, it means that the amino acid residue at the 17th position relative to the amino acid sequence of wild-type mature NGF (preferably human wild-type mature NGF) is serine (S). "Mutation at the S17 site" means that the serine residue (S) at the 17th position is mutated. In some embodiments, when describing the NGF mutant, it is described as follows. For example, "amino acid substitution" is expressed as "original amino acid residue/substituted amino acid position/substituted amino acid residue", for example, "S17R" represents that the serine residue (S) at position 17 relative to the amino acid sequence of wild-type mature NGF (e.g., SEQ ID NO.1 or 2) is replaced by an arginine residue (R). The "+" described in the mutation site combination scheme in the present application indicates that mutations occur simultaneously at multiple specific positions, for example, "S17R+K32E" represents that the serine residue (S) at position 17 relative to the amino acid sequence of wild-type mature NGF (e.g., SEQ ID NO.1 or 2) is replaced by an arginine residue (R), and the lysine residue (K) at position 32 is replaced by a glutamic acid residue (E).
如本文应用的术语“治疗(treatment)”或“治疗(treating)”是一种为获得有益或期望的结果的方法，包括临床结果。鉴于本申请的目的，所述有益或期望的临床结果包括但不限于下列一种或多种：缓解由疾病引起的一种或多种症状、减少疾病范围、稳定疾病(例如，预防或延缓疾病恶化)、预防或延缓疾病传播、预防或延缓疾病复发、延缓或减缓疾病发展、改善疾病状态、缓解疾病(部分或全部)、减少治疗疾病所需的一种或多种其它药物的剂量、延缓疾病发展、提高生存质量和/或延长生存期。同时，“治疗”还包括减少疾病的病理结果。本申请的方法考虑了这些治疗的任何一个或多个方面。例如，如果一个或多个与该疾病相关的症状得到缓解或消除，包括但不限于减少该疾病引起的症状，提高该疾病患者的生活质量，减少治疗疾病所需的其它药物的剂量，和/或延长个体的生存期，则认为该患者被成功“治疗”。 As used herein, the term "treatment" or "treating" is a method for obtaining a beneficial or desired result, including clinical results. In view of the purpose of this application, the beneficial or desired clinical results include, but are not limited to, one or more of the following: relieving one or more symptoms caused by the disease, reducing the scope of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying disease spread, preventing or delaying disease recurrence, delaying or slowing disease development, improving disease state, alleviating the disease (partially or completely), reducing the dosage of one or more other drugs required for treating the disease, delaying disease development, improving the quality of life and/or prolonging survival. At the same time, "treatment" also includes reducing the pathological results of the disease. The method of the present application takes into account any one or more aspects of these treatments. For example, if one or more symptoms associated with the disease are alleviated or eliminated, including but not limited to reducing the symptoms caused by the disease, improving the quality of life of the patient with the disease, reducing the dosage of other drugs required for treating the disease, and/or prolonging the survival of the individual, the patient is considered to be successfully "treated".
术语“预防(prevent)”和类似的词语，如“预防(prevented)”、“预防(preventing)”等表示预防、抑制或降低疾病或病症复发可能性的方法。它还指延缓疾病或病症的复发或延缓疾病或病症的复发。如本文所用，“预防”和类似词语还包括在疾病或病症复发之前降低疾病或病症的强度、影响、症状和/或负担。The term "prevent" and similar words, such as "prevented", "preventing", etc., refer to methods of preventing, inhibiting, or reducing the likelihood of recurrence of a disease or condition. It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of a disease or condition. As used herein, "preventing" and similar words also include reducing the intensity, effects, symptoms, and/or burden of a disease or condition before the disease or condition recurs.
如本文应用的术语“延缓”疾病的发展表示推迟、阻碍、减缓、减慢、稳定和/或推迟疾病的发展。根据疾病史和/或接受治疗的个体不同，延缓的时间可能不同。一种“延缓”疾病发展的方法是指与不使用该方法相比，在给定时间范围内降低疾病发展概率和/或在给定时间范围内降低疾病程度的方法。这种比较通常基于临床研究，使用统计上显著的个体数。As used herein, the term "delaying" the development of a disease means postponing, hindering, slowing down, slowing down, stabilizing and/or postponing the development of a disease. Depending on the history of the disease and/or the individual being treated, the time of delay may be different. A method of "delaying" the development of a disease refers to a method that reduces the probability of disease development and/or reduces the extent of the disease within a given time frame compared to not using the method. This comparison is usually based on clinical studies using statistically significant numbers of individuals.
如本文中应用的“疼痛”是指任何病因学包括急性及慢性疼痛以及任何有炎性组分的疼痛。疼痛的实例包括外科手术后疼痛、手术后疼痛(包括牙痛)、偏头痛、头痛及三叉神经痛及与烧伤、创伤或肾结石相关疼痛、与外伤(包括外伤性颅脑损伤)相关疼痛、神经性疼痛、与肌肉-骨骼疾病如类风湿性关节炎、骨关节炎、强直性脊柱炎、血清阴性(非类风湿)关节病、非关节性风湿症及关节周病症相关的疼痛以及与癌症相关的疼痛(包括“突破疼痛”及与晚期癌症相关的疼痛)、外周神经病及疱疹后神经痛。有炎性组分疼痛的实例(除以上描述的一些外)包括风湿性疼痛、与粘膜炎及痛经相关的疼痛。As used herein, "pain" refers to any etiology including acute and chronic pain and any pain with an inflammatory component. Examples of pain include postoperative pain, postoperative pain (including toothache), migraine, headache and trigeminal neuralgia and pain associated with burns, trauma or kidney stones, pain associated with trauma (including traumatic craniocerebral injury), neuropathic pain, pain associated with musculoskeletal diseases such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatoid) arthropathy, non-articular rheumatism and periarticular disorders, and pain associated with cancer (including "breakthrough pain" and pain associated with advanced cancer), peripheral neuropathy and post-herpetic neuralgia. Examples of pain with an inflammatory component (in addition to some described above) include rheumatic pain, pain associated with mucositis and dysmenorrhea.
如本文应用的术语“有效量”是指足以治疗特定紊乱、病症或疾病的药物剂量或药物组合物剂量，如改善、减轻、减弱和/或延缓一个或多个症状。在一些实施例中，有效量是足以延缓疾病发展的量。在一些实施例中，有效量是足以预防或延缓疾病复发的量。有效量可在一次或多次施用中给药。在一些实施例中，药物或组合物的有效量可能：(i)支持神经元存活；(ii)促进神经突生长；(iii)增强神经化学分化；(iv)促进TF-1细胞增殖；(v)诱导先天性和/或获得性免疫；(vi)预防或延缓疾病的发生和/或复发；和/或(vii)在一定程度上缓解与该疾病相关的一个或多个症状。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, condition, or disease, such as to improve, alleviate, attenuate, and/or delay one or more symptoms. In some embodiments, an effective amount is an amount sufficient to delay the progression of a disease. In some embodiments, an effective amount is an amount sufficient to prevent or delay the recurrence of a disease. An effective amount may be administered in one or more administrations. In some embodiments, an effective amount of a drug or composition may: (i) support neuronal survival; (ii) promote neurite outgrowth; (iii) enhance neurochemical differentiation; (iv) promote TF-1 cell proliferation; (v) induce innate and/or acquired immunity; (vi) prevent or delay the onset and/or recurrence of a disease; and/or (vii) alleviate one or more symptoms associated with the disease to some extent.
如本文应用的术语“个体”或“主体”是指哺乳动物，包括但不限于人类、牛、马、猫、狗、啮齿动物或灵长类动物。在一些实施例中，所述个体是指人类。As used herein, the term "individual" or "subject" refers to a mammal, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the individual refers to a human.
术语“抗体”包括全长抗体及其抗原结合片段。全长抗体包括两条重链和两条轻链。轻链和重链的可变区负责抗原的结合。两条链中的可变区通常包括3个高变的环，被称为互补决定区(CDRs)(轻链(LC)CDRs包括LC-CDR1、LC-CDR2和LC- CDR3，重链(HC)CDRs包括HC-CDR1、HC-CDR2和HC-CDR3)。本文所披露的抗体或抗原结合片段的CDR边界可通过Kabat,Chothia或Al-Lazikani惯例来定义或识别(Al-Lazikani 1997；Chothia 1985；Chothia 1987；Chothia 1989；Kabat 1987；Kabat 1991)。重链或轻链的3个CDR区插入到被称为框架区(FRs)的侧翼区段之间，所述框架区比CDR区具有更高的保守性，并形成支撑高变环的支架。重链和轻链的恒定区并不参与抗原结合，但展示出多种效应功能。抗体是基于它们重链恒定区的氨基酸序列进行分类的。抗体的五种主要类别或同种型是IgA、IgD、IgE、IgG和IgM，其特征在于分别具有α、δ、ε、γ和μ型重链。几种主要的抗体类别被分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链n)或IgA2(α2重链)。The term "antibody" includes full-length antibodies and antigen-binding fragments thereof. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains usually include three hypervariable loops, called complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC- CDR3, heavy chain (HC) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by heavy chains of type α, δ, ε, γ, and μ, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).
如本文所述，术语“抗原结合片段”是指一种抗体片段，包括，例如，双链抗体、Fab、Fab’、F(ab’)2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv’)、二硫键稳定的双链抗体(ds双链抗体)、单链抗体(scFv)、scFv二聚体(二价双链抗体)，由包含一个或多个CDRs的抗体片段组成的多特异性抗体、单域抗体、纳米抗体、域抗体、二价域抗体或者能够与抗原结合但不包含完整抗体结构的任何其他抗体片段。抗原结合片段还包括包含上述抗体片段的融合蛋白。抗原结合片段能够与亲本抗体或亲本抗体片段(如亲本scFv)结合相同的抗原。在一些实施例中，抗原结合片段可能包括来自特定人抗体的一个或多个CDRs，该CDRs被移植到来自一个或多个不同人抗体的框架区。As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody (scFv), an scFv dimer (a bivalent diabody), a multispecific antibody composed of an antibody fragment comprising one or more CDRs, a single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure. Antigen-binding fragments also include fusion proteins comprising the above antibody fragments. An antigen-binding fragment can bind to the same antigen as a parent antibody or a parent antibody fragment (such as a parent scFv). In some embodiments, an antigen-binding fragment may include one or more CDRs from a specific human antibody that are transplanted to a framework region from one or more different human antibodies.
如本文所述，术语“表位”是指抗体或抗体部分结合的抗原上特定的原子或氨基酸组。如果两种抗体或抗体部分表现出与某抗原竞争性结合，则它们可能结合抗原上相同表位。As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they are likely to bind to the same epitope on the antigen.
如本文所述，术语“CDR”或“互补决定区”是指重链和轻链多肽的可变域内发现的非连续抗原结合位点。在文献Kabat et al.,J.Biol.Chem.252:6609-6616(1977)；Kabat et al.,U.S.Dept.of Health and Human Services,“Sequences of proteins of immunological interest”(1991)；Chothia et al.,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.et al.,J.Mol.Biol.,273:927-948(1997)；MacCallum et al.,J.Mol.Biol.262:732-745(1996)；Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.et al.,Dev.Comp.Immunol.,27:55-77(2003)；和Honegger and Plückthun,J.Mol.Biol.,309:657-670(2001)中已经描述这些特殊的区域，其中当彼此之间互相比较时，这些定义包括氨基酸残基的重合或子 集。然而，采用任何一种定义方式来指示抗体或移植抗体或其变体的CDR，均包括在本文所定义和使用的术语范围之内。表1中列了由上述引用的各篇参考文献所定义的CDR所包括的氨基酸残基的位置，以示比较。CDR预测的算法和结合界面在本领域是已知的，包括，例如Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008)；Ehrenmann F.et al.,Nucleic Acids Res.,38:D301-D307(2010)；和Adolf-Bryfogle J.et al.,Nucleic Acids Res.,43:D432-D438(2015)中均有描述。本段中所引用的参考文献的内容以其整体引用并入本文中，以用于本申请和可能包含在本文中的一个或多个权利要求中。As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable domains of heavy and light chain polypeptides. In the literature, Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest"(1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc M ... These specific regions have been described in Hochschuen et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), wherein these definitions include overlaps or sub-overlaps of amino acid residues when compared to each other. Set. However, any definition used to indicate the CDR of an antibody or a transplanted antibody or a variant thereof is included in the scope of the terms defined and used herein. Table 1 lists the positions of the amino acid residues included in the CDRs defined by the above-cited references for comparison. Algorithms and binding interfaces for CDR prediction are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in the present application and in one or more claims that may be included herein.
表1:CDR定义

1氨基酸残基编号参照上述Kabat et al.中的命名方法
2氨基酸残基编号参照上述Chothia et al.中的命名方法
3氨基酸残基编号参照上述MacCallum et al.中的命名方法
4氨基酸残基编号参照上述Lefranc et al.中的命名方法
5氨基酸残基编号参照上述Honegger and Plückthun中的命名方法Table 1: CDR definitions

1 The amino acid residue numbers refer to the nomenclature of Kabat et al.
2 The amino acid residue numbers refer to the nomenclature of Chothia et al.
3 The amino acid residue numbers refer to the nomenclature of MacCallum et al.
4 The amino acid residue numbers refer to the nomenclature of Lefranc et al.
5 The amino acid residue numbering follows the nomenclature of Honegger and Plückthun above.
术语“嵌合抗体”是指重链和/或轻链的一部分与来自特定种属或属于特定抗体种类或亚类的抗体中的相应序列一致或具有同源性，而这个(些)链的剩余部分与来自另一种属或属于其它抗体种类或亚类的抗体中的相应序列一致或具有同源性的抗体，以及此类抗体的片段，只要其具有本申请中的生物学活性(见U.S.Patent No.4,816,567；and Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851-6855(1984))。The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody from a specific species or belonging to a specific antibody class or subclass, while the remaining portion of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another genus or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they have the biological activity described in the present application (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
“Fv”是包含完整抗原识别及结合位点的最小抗体片段。该片段是由一个重链可变域和一个轻链可变域紧密非共价连接形成的二聚体。通过这两个域的折叠衍生出6个高变环(轻链和重链中各3个环)，所述高变环为抗体提供了用于结合抗原的氨基酸残基，并且赋予抗体与抗原结合的特异性。然而，即使单个可变域(或Fv片段的一 半，其仅包含对抗原具有特异性的3个CDRs，)也具有识别和结合抗原的能力，尽管其亲和力低于完整的结合位点。"Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable domain and a light chain variable domain tightly non-covalently linked. Six hypervariable loops (3 loops each in the light chain and heavy chain) are derived from the folding of these two domains. The hypervariable loops provide the antibody with amino acid residues for binding to the antigen and give the antibody specificity for binding to the antigen. However, even a single variable domain (or a fragment of an Fv) can The half (which contains only three CDRs specific for an antigen) also has the ability to recognize and bind antigen, although its affinity is lower than that of the entire binding site.
“单链Fv”，也可简写成“sFv”或“scFv”，是包含被连接成单一多肽链的VH和VL抗体域的抗体片段。在一些实施例中，scFv多肽进一步包括VH和VL域之间的连接多肽，该连接多肽使得scFv形成抗原结合的理想结构。关于scFv的概述，见Pluckthun in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds.,Springer-Verlag,New York,pp.269-315(1994)。"Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a connecting polypeptide between the VH and VL domains, which enables the scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
术语“双链抗体(diabodies)”是指，在VH和VL域之间采用短接头(例如5～10个残基)构建scFv片段(见上段内容)制备而成的一种小抗体片段，这样就使得可变域在链间而不是链内进行配对，产生一个双价片段，即具有两个抗原结合位点的片段。双特异性的双链抗体是两个“交叉”scFv片段的异二聚体，其中两个抗体的VH和VL域位于不同的多肽链上。在EP 404,097；WO 93/11161；Hollinger et al.,Proc.Natl.Acad.Sci.USA,90:6444-6448(1993)中全面描述了双链抗体。The term "diabodies" refers to a small antibody fragment prepared by constructing an scFv fragment (see above) with a short linker (e.g., 5 to 10 residues) between the VH and VL domains, so that the variable domains pair between chains rather than within the chains, resulting in a bivalent fragment, i.e., a fragment with two antigen binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments, in which the VH and VL domains of the two antibodies are located on different polypeptide chains. Diabodies are fully described in EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
非人源(如啮齿类)抗体的“人源化”形式是嵌合抗体，其包括最少的来自非人源抗体的序列。大多数情况下，人源化抗体是人源免疫球蛋白(受体抗体)，其中受体抗体的高变区(HVR)残基被来自非人源种属例如小鼠、大鼠、兔或非人类灵长类动物的且具有理想的抗体特异性，亲和力和性能的高变区残基所取代(供体抗体)。在某些情况下，人源免疫球蛋白框架区(FR)中的残基被相应的非人源残基所取代。另外，人源化抗体可以包括在受体抗体或供体抗体中均不存在的残基。这些修饰能够进一步改善抗体的性能。通常，人源化抗体会包含基本上所有，至少一个，通常两个可变域，其中所有或基本上所有的高变环均与非人免疫球蛋白的高变环相对应，以及所有或基本上所有的框架区均是人免疫球蛋白序列。人源抗体任选地也还包括免疫球蛋白恒定区(Fc)的至少一部分，通常是人免疫球蛋白的恒定区。具体细节可以参考Jones et al.,Nature 321:522-525(1986)；Riechmann et al.,Nature 332:323-329(1988)；和Presta,Curr.Op.Struct.Biol.2:593-596(1992)。The "humanized" form of a non-human (such as rodent) antibody is a chimeric antibody that includes minimal sequences from non-human antibodies. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which the hypervariable region (HVR) residues of the receptor antibody are replaced by hypervariable region residues from non-human species such as mice, rats, rabbits or non-human primates and have ideal antibody specificity, affinity and performance (donor antibody). In some cases, residues in the framework region (FR) of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues that are not present in either the receptor antibody or the donor antibody. These modifications can further improve the performance of the antibody. Typically, a humanized antibody will contain substantially all, at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework regions are human immunoglobulin sequences. Human antibodies optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For specific details, please refer to Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
术语“恒定结构域”是指免疫球蛋白分子的一部分，相对于免疫球蛋白分子中包含抗原结合位点可变结构域的另一部分，恒定结构域具有更保守的氨基酸序列。恒定结构域包含重链的CH1、CH2和CH3结构域(统称为CH)和轻链的恒定(或CL)结构域。根据免疫球蛋白重链(CH)恒定结构域的氨基酸序列，免疫球蛋白可分为不同 类别或亚型。有五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，重链分别为α、δ、ε、γ和μ。根据CH序列和功能的相对较小差异，将γ和α进一步划分为亚类，例如，人类表达以下亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgA2。The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than another portion of the immunoglobulin molecule that contains the variable domain of the antigen binding site. The constant domain contains the CH1 , CH2 , and CH3 domains (collectively referred to as CH ) of the heavy chain and the constant (or CL ) domain of the light chain. Based on the amino acid sequence of the constant domain of the immunoglobulin heavy chain ( CH ), immunoglobulins can be divided into different Class or subtype. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains of α, δ, ε, γ, and μ, respectively. γ and α are further divided into subclasses based on relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.
人IgG Fc区的“CH1结构域”通常从118位氨基酸延伸到215位氨基酸(EU编号系统)。The " CH1 domain" of a human IgG Fc region typically stretches from amino acid 118 to amino acid 215 (EU numbering system).
“铰链区”通常被定义为从人IgG1的216位Glu延伸到230位Pro(Burton,Molec.Immunol.22:161-206(1985))。通过将形成重链间二硫键的第一个和最后一个半胱氨酸残基置于与IgG1相同位置后，可以使得其他IgG同种型的铰链区与IgG1序列比对。The "hinge region" is generally defined as extending from Glu 216 to Pro 230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine residues that form inter-heavy chain disulfide bonds in the same position as IgG1, the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence.
人IgG Fc区的“CH2结构域”通常从231位氨基酸延伸到340位氨基酸。CH2结构域的独特之处在于，它不会与另一个区域紧密配对，而是在完整的天然IgG分子的两个CH2结构域之间插入了两条N端连接的支链糖链。据推测，糖类可能作为域与域间配对的替代，有助于保持CH2结构域稳定。Burton,Molec Immunol.22:161-206(1985)。The " CH2 domain" of the human IgG Fc region generally extends from amino acid 231 to amino acid 340. The CH2 domain is unique in that it does not pair closely with another region, but rather two N-terminally linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that carbohydrates may serve as a substitute for domain-to-domain pairing, helping to maintain CH2 domain stability. Burton, Molec Immunol. 22: 161-206 (1985).
“CH3”结构域包括在Fc区内从C末端残基延伸到CH2结构域(从341位氨基酸到抗体序列的C末端，通常为IgG的第446或447位氨基酸残基)。The " CH3 " domain includes a region extending from the C-terminal residue to the CH2 domain (from amino acid 341 to the C-terminus of the antibody sequence, generally amino acid residue 446 or 447 for IgG) within the Fc region.
如本文应用的术语“Fc区”、“片段结晶区”、“Fc结构域”或“Fc部分”用于定义免疫球蛋白重链的C端区域，包括天然序列Fc区和变体Fc区。尽管免疫球蛋白重链Fc区的边界可能不同，但人类IgG重链Fc区通常定义为从Cys226位置的氨基酸残基或从Pro230开始，延伸至其羧基末端。Fc区的C端赖氨酸(根据EU编号系统为447残基)可能被移除，例如，在蛋白质的生产或纯化过程中，或通过重组工程编码蛋白质的核酸而移除。用于本文所述构建体的合适的天然序列Fc区包括人类IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4。As used herein, the terms "Fc region", "fragment crystallographic 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 boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during the production or purification of the protein, or by recombinant engineering of the nucleic acid encoding the protein. Suitable native sequence Fc regions for the constructs described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
如本文应用的术语IgG“亚型”或“亚类”是指通过恒定结构域的化学和抗原特性定义的免疫球蛋白的任何亚类。免疫球蛋白主要分为五大类：IgA、IgD、IgE、IgG和IgM，并且其中多个可进一步分为亚类(亚型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。与不同免疫球蛋白类别相对应的的重链恒定结构域分别称为α、 γ、ε、γ和μ。不同类别免疫球蛋白的亚单位结构和三维构型已众所周知，并在Abbas等人的《细胞和分子免疫学》第四版(W.B.Saunders，Co.，2000)中进行了详述。As used herein, the term IgG "subtype" or "subclass" refers to any subclass of immunoglobulins defined by the chemical and antigenic properties of the constant domains. Immunoglobulins are divided into five major classes: IgA, IgD, IgE, IgG, and IgM, and many of these can be further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, IgG2, IgG3, IgG4, IgA1, and IgA2, respectively. γ, ε, γ, and μ. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and described in detail in Abbas et al., Cellular and Molecular Immunology, 4th edition (WB Saunders, Co., 2000).
“Fc受体”或“FcR”描述了与包含Fc的结构(例如，抗体)中的Fc区结合的受体。首选的FcR是人类FcR天然序列。此外，首选的FcR是一种结合IgG抗体(一种γ受体)的FcR，包括FcγRI、FcγRII和FcγRIII等受体亚类，以及这些受体的等位基因变体和可变剪接形式。FcγRII受体包括FcγRIIA(一种“激活受体”)和FcγRIIB(一种“抑制受体”)，它们具有相似的氨基酸序列，主要在胞质结构域有所不同。激活受体FcγRIIA在其胞质结构域中包含免疫受体酪氨酸活化基序(ITAM)。抑制受体FcγRIIB在其胞质结构域中包含免疫受体酪氨酸抑制基序(ITIM)(见于M.Annu.Rev.Immunol.15:203-234(1997))。在Ravetch and Kinet,Annu.Rev.Immunol 9:457-92(1991)；Capel et al.,Immunomethods 4:25-34(1994)和de Haas et al.,J.Lab.Clin.Med.126:330-41(1995)中对FcRs进行了综述。本文中术语“FcR”涵盖其它FcRs，包括那些在未来将被鉴定的FcRs。"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an Fc-containing structure (e.g., an antibody). The preferred FcR is a native human FcR sequence. In addition, the preferred FcR is an FcR that binds to an IgG antibody (a gamma receptor), including receptor subclasses such as FcγRI, FcγRII, and FcγRIII, as well as allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences and differ primarily in the cytoplasmic domain. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (described in M. 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). The term "FcR" herein encompasses other FcRs, including those that will be identified in the future.
术语“Fc受体”或“FcR”也包括新生儿受体FcRn，负责将母体IgG转运给胎儿。Guyer et al.,J.Immunol.117:587(1976)和Kim et al.,J.Immunol.24:249(1994)。测定与FcRn结合的方法已众所周知(参见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.))。可以测定人类FcRn高亲和力结合多肽在体内和血清中与FcRn结合的半衰期，例如，在表达人类FcRn的转基因小鼠或转染的人类细胞系中，或在施用具有变体Fc区的多肽的灵长类动物中。WO 2004/42072(Presta)详述了增强或减弱与FcRs结合的抗体变体。参见Shield et al.,J.Biol.Chem.9(2):6591-6604(2001)。The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transport of maternal IgG to the fetus. Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994). Methods for determining 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 binding to FcRn in vivo and in serum can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with variant Fc regions. WO 2004/42072 (Presta) details antibody variants that enhance or attenuate binding to FcRs. See Shield et al., J. Biol. Chem. 9(2):6591-6604 (2001).
“抗体效应功能”是指那些由包含Fc的结构(例如，抗体)中的Fc区(天然序列Fc区或氨基酸序列变体Fc区)引起的生物活性，并随Fc亚型改变。抗体效应功能的示例包括：C1q结合和补体依赖性细胞毒性(CDC)；Fc受体结合；抗体依赖性细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体的下调(例如，B细胞受体)和B细胞活化。“减少或最小化”抗体效应功能表示与野生型或未经修饰的包含Fc的结构(例如，抗体)相比，减少至少50％(或者60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％或99％)。测定抗体效应功能可由本领域普通技术人员 轻而易举地测定和测量。在优选实施例中，补体结合、补体依赖性细胞毒性和抗体依赖性细胞毒性的抗体效应功能会受到影响。在一些实施例中，通过恒定结构域中的突变消除糖基化来消除效应功能，例如，“无效应功能突变”。在一些实施例中，无效应功能突变体是在CH2区的N297A或DANA突变(D265A+N297A)。Shields et al.,J.Biol.Chem.276(9):6591-6604(2001)。另外，导致效应功能降低或消除的其它突变包括：K322A和L234A/L235A(LALA)。另外，可以通过生产技术减少或消除效应功能，如在不进行糖基化的宿主细胞中(例如，大肠杆菌)或导致糖基化模式改变的宿主细胞中表达，所述糖基化模式改变在促进效应功能方面无效或效果较小(例如，Shinkaw et al.,J.Biol.Chem.278(5):3466-3473(2003))。"Antibody effector functions" refer to those biological activities caused by the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) in an Fc-containing structure (e.g., an antibody), and vary with 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 receptor) and B cell activation. "Reducing or minimizing" antibody effector function means a reduction of at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) compared to a wild-type or unmodified Fc-containing structure (e.g., an antibody). Determination of antibody effector function can be performed by one of ordinary skill in the art. Easily assayed and measured. In preferred embodiments, the antibody effector functions of complement fixation, complement dependent cellular toxicity, and antibody dependent cellular toxicity are affected. In some embodiments, effector function is eliminated by eliminating glycosylation through mutations in the constant domains, e.g., "no effector function mutations". In some embodiments, the no effector function mutant is an N297A or DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). In addition, other mutations that result in reduced or eliminated effector function include: K322A and L234A/L235A (LALA). Additionally, effector function can be reduced or eliminated by production techniques, such as expression in a host cell that does not perform glycosylation (e.g., E. coli) or in a host cell that results in an altered glycosylation pattern that is ineffective or less effective in promoting effector function (e.g., Shinkaw et al., J. Biol. Chem. 278(5):3466-3473 (2003)).
“抗体依赖性细胞介导的细胞毒性”或ADCC是指一种细胞毒性形式，其中分泌型Ig(或配体-Fc结构)与存在于某些细胞毒性细胞(例如，自然杀伤细胞(NK)、中性粒细胞和巨噬细胞)上的Fc受体(FcRs)结合，使这些细胞毒性效应细胞能够特异性结合携带抗原(或携带配体受体)的靶细胞，随后用细胞毒素杀死靶细胞。抗体(或包含Fc的结构)“武装”细胞毒性细胞，且是通过这种机制杀死靶细胞所必需的。介导ADCC的主要细胞，即NK细胞只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。在Ravetch和Kinet,Annu.Rev.Immunol 9:457-92(1991)第464页的Table 2中总结了造血细胞Fc的表达。为评估目标分子的ADCC活性，可以进行体外ADCC试验，如U.S.Pat.No.5,500,362或5,821,337中进行了详述。适用于此类试验的效应细胞包括外周血单核细胞(PBMC)和自然杀伤细胞(NK)。或者，或额外地，目标分子的ADCC活性也可以在体内进行评估，例如，在动物模型中如在Clynes et al.,PNAS(USA)95:652-656(1998)中所公开的。“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 (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing the antigen (or bearing the ligand receptor) and subsequently kill the target cells with cytotoxins. Antibodies (or Fc-containing structures) “arm” the cytotoxic cells and are required for target cell killing by this mechanism. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression by hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay may be performed, as described in detail in U.S. Pat. No. 5,500,362 or 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, or additionally, the ADCC activity of a target molecule may also be assessed in vivo, for example, in an animal model as disclosed in Clynes et al., PNAS (USA) 95: 652-656 (1998).
“补体依赖性细胞毒性”或“CDC”是指在补体存在的情况下裂解靶细胞。经典的补体途径的激活是由补体系统第一组分(C1q)与(合适亚类的)包含Fc的结构结合起始的，所述包含Fc的结构通过Fc融合的配体与其同源受体结合。为了评估补体激活，可以进行CDC试验，如Gazzano-Santoro et al.,J.Immunol.Methods 202:163(1996)中所述。U.S.Pat.No.6,194,551B1和WO99/51642中详述了具有改变的Fc区氨基酸序列从而增加或减少C1q结合能力的抗体变体。这些专利出版物的内容通过引用并入本文。参见Idusogie et al.J.Immunol.164:4178-4184(2000)。 "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 binding of the first component of the complement system (Clq) to an Fc-containing structure (of the appropriate subclass) that binds to its cognate receptor via an Fc-fused ligand. To assess complement activation, a CDC assay may be performed, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Antibody variants with altered Fc region amino acid sequences that increase or decrease Clq binding ability are described in detail in US Pat. No. 6,194,551 B1 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).
如本文应用的术语“特异性结合”、“特异性识别”或“特异性用于”是指可测量和可再现的相互作用，如配体和受体之间的结合，当存在包括生物分子在内的异质分子群的情况下可确定配体的存在。例如，与受体特异性结合的配体，与结合其它受体相比时，在结合目标受体时具有更大的亲和性、亲和力、更容易和/或持续时间更长。在一些实施例中，通过例如放射免疫分析(RIA)法进行测定，配体与无关受体的结合程度小于配体与目标受体结合程度的10％。在一些实施例中，特异性结合靶受体的配体的解离常数(Kd)≤10-5M、≤10-6M、≤10-7M、≤10-8M、≤10-9M、≤10-10M、≤10-11M或≤10-12M。在一些实施例中，配体特异性结合在不同物种中保守的受体。在一些实施例中，特异性结合可以包括但不要求排他性结合。可利用本领域已知的方法通过实验确定配体的结合特异性。如包括但不限于Western blots、ELISA-、RIA-、ECL-、IRMA-、EIA-、BIACORETM-测试和肽扫描。As used herein, the terms "specific binding", "specific recognition" or "specific for" refer to a measurable and reproducible interaction, such as binding between a ligand and a receptor, where the presence of a ligand can be determined in the presence of a heterogeneous population of molecules, including biomolecules. For example, a ligand that specifically binds to a receptor has greater affinity, avidity, is more likely to bind to the target receptor and/or lasts longer than when binding to other receptors. In some embodiments, the degree of binding of the ligand to an unrelated receptor is less than 10% of the degree of binding of the ligand to the target receptor, as determined by, for example, radioimmunoassay (RIA). In some embodiments, the dissociation constant (Kd) of a ligand that specifically binds to a target receptor is ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 M. In some embodiments, a ligand specifically binds to a receptor that is conserved in different species. In some embodiments, specific binding may include but does not require exclusive binding. The binding specificity of a ligand may be determined experimentally using methods known in the art. Such as including but not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE ™ -tests and peptide scanning.
“结合亲和力”通常指分子(例如，配体)的单个结合位点和其结合搭档(例如，受体)之间的非共价相互作用总和的强度。除非另有说明，如本文所用，“结合亲和力”指内在结合亲和力，该内在亲和力可以反映结合对成员之间的1:1相互作用。结合亲和力可以采用Kd、Koff、Kon或Ka表示。如本文所用，术语“Koff”是指配体从配体/受体复合物中解离的速率常数，通过动力学选择装置测定，以s-1为单位来表示。如本文所用，术语“Kon”是指配体与受体结合形成配体/受体复合物的结合速率常数，以M-1s-1为单位来表示。如本文所用，术语解离常数“Kd”是指特定配体-受体相互作用时的解离常数，是指在受体溶液中，配体占据所有受体结合位点的一半并且达到平衡时所需的配体浓度，等于Koff/Kon，以M为单位来表示。测定Kd的前提是所有的结合分子均在溶液中。当受体在细胞膜上的情况下，相应的解离常数用EC50表示，是Kd的一个很好的近似值。结合常数Ka，是解离常数Kd的倒数，以M-1为单位来表示。解离常数(Kd)可以作为反映配体与受体亲和力的指标。使用所述方法得到的Kd值，以M(mol/L)为单位来表示。"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 intrinsic binding affinity, which can reflect a 1:1 interaction between members of a binding pair. Binding affinity can be expressed as Kd, Koff, Kon, or Ka. As used herein, the term "Koff" refers to the rate constant at which a ligand dissociates from a ligand/receptor complex, measured by a kinetic selection device, and expressed in units of s -1 . As used herein, the term "Kon" refers to the binding rate constant at which a ligand binds to a receptor to form a ligand/receptor complex, expressed in units of M -1 s -1 . As used herein, the term dissociation constant "Kd" refers to the dissociation constant for a specific ligand-receptor interaction, and refers to the ligand concentration required when the ligand occupies half of all receptor binding sites in a receptor solution and reaches equilibrium, equal to Koff/Kon, expressed in units of M. The premise for determining Kd is that all binding molecules are in solution. When the receptor is on the cell membrane, the corresponding dissociation constant is expressed as EC50, which is a good approximation of Kd. The binding constant Ka is the reciprocal of the dissociation constant Kd and is expressed in M -1 . The dissociation constant (Kd) can be used as an indicator to reflect the affinity of the ligand to the receptor. The Kd value obtained using the method is expressed in M (mol/L).
半抑制浓度(IC50)是对物质(例如，配体)在抑制特定生物或生化功能中有效性的度量。它表示需要多少特定药物或其它物质(抑制剂，例如，配体)能将给定的生物过程抑制一半。数值通常表示为摩尔浓度。IC50与激动剂药物或其它物质(例如，配体)的“EC50”相当。EC50也代表在体内获得最大效应的50％所需的血浆浓度。如本文所用，“IC50”用于表示在体外中和50％的受体生物活性所需配体的有效浓度。 可通过生物测量测定IC50或EC50，如通过FACS分析(竞争结合试验)抑制配体结合、基于细胞的细胞因子释放试验或放大发光的均相酶联免疫试验(AlphaLISA)。The half inhibitory concentration (IC50) is a measure of the effectiveness of a substance (e.g., a ligand) in inhibiting a specific biological or biochemical function. It indicates how much of a specific drug or other substance (inhibitor, e.g., a ligand) is needed to inhibit a given biological process by half. The values are usually expressed as molar concentrations. The IC50 is equivalent to the "EC50" of an agonist drug or other substance (e.g., a ligand). The EC50 also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. As used herein, "IC50" is used to represent the effective concentration of a ligand required to neutralize 50% of the biological activity of a receptor in vitro. The IC50 or EC50 can be determined by bioassays, such as inhibition of ligand binding by FACS analysis (competition binding assay), cell-based cytokine release assays, or amplified luminescence homogeneous enzyme-linked immunosorbent assays (AlphaLISA).
如本文应用的术语“共价键”是指两个原子之间通过共用一个或多个电子形成的稳定键。共价键的示例包括但不限于肽键和二硫键。如本文所用，“肽键”是指氨基酸的羧基与相邻氨基酸的胺基之间形成的共价键。如本文所用，“二硫键”是指两个硫原子之间形成的共价键，如两个Fc片段通过一个或多个二硫键结合。两个片段之间的一个或多个二硫键可能通过连接两个片段中的硫醇基形成。在一些实施例中，两个Fc片段的一个或多个半胱氨酸之间可能形成一个或多个二硫键。氧化两个硫醇基可形成二硫键。在一些实施例中，共价连接是由共价键直接连接而成的。在一些实施例中，共价连接直接由肽键或二硫键连接。As used herein, the term "covalent bond" refers to a stable bond formed between two atoms by sharing one or more electrons. 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 the carboxyl group of an amino acid and the amine group of an adjacent amino acid. As used herein, "disulfide bond" refers to a covalent bond formed between two sulfur atoms, such as two Fc fragments are bound by one or more disulfide bonds. One or more disulfide bonds between two fragments may be formed by connecting 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. Oxidation of two thiol groups can form a disulfide bond. In some embodiments, the covalent bond is formed by direct connection of covalent bonds. In some embodiments, the covalent bond is directly connected by a peptide bond or a disulfide bond.
肽或多肽序列的“氨基酸序列同一性百分比(％)”或“同源性”被定义为候选序列中与特定多肽或多肽序列中相同氨基酸残基所占的百分比，在序列比对并引入间隙(如果必要)后以实现序列同一性百分比的最大化，并且不考虑任何保守替换作为序列同一性的一部分。为了确定氨基酸序列同一性百分比，可以通过本领域技术范围内的多种比对方式，例如，使用如BLAST、BLAST-2、ALIGN、Megalign(DNASTAR)、或MUSCLE软件等可公开获得的计算机软件。本领域技术人员可以确定用于测量比对的合适参数，包括在所比较的序列的全长上实现最大比对所需的任何算法。The "percentage (%) of amino acid sequence identity" or "homology" of a peptide or polypeptide sequence is defined as the percentage of identical amino acid residues in a candidate sequence to a particular polypeptide or polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to maximize the percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, a variety of alignments within the skill of the art can be used, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.
如本文应用的术语多肽的“C端”是指该多肽的最后一个氨基酸残基，该残基赋予其胺基以与其相邻氨基酸残基的羧基形成肽键。如本文所用，多肽的“N末端”是指该多肽的第一个氨基酸，该残基赋予其羧基以与其相邻氨基酸残基的胺基形成肽键。As used herein, the term "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide that provides its amine group to form a peptide bond with the carboxyl group of its adjacent amino acid residue. As used herein, the "N-terminus" of a polypeptide refers to the first amino acid of the polypeptide that provides its carboxyl group to form a peptide bond with the amine group of its adjacent amino acid residue.
“分离的”多肽是指一种已从其生产环境(例如，天然或重组)的组分中鉴定、分离和/或回收的多肽。优选地，所分离的多肽与其生产环境中的所有其它成分没有关联。生产环境的污染成分，如重组转染细胞产生的污染成分，通常会干扰多肽的研究、诊断或治疗，并且可能包括酶、激素和其它蛋白溶质或非蛋白溶质。在一些实施例中，多肽将被纯化至：(1)按重量计算，多肽含量大于95％，如通过Lowry法确定，在一些实施例中，按重量计算，多肽含量大于99％；(2)通过使用旋杯测序仪达到足以获得至少15个N端残基或内部氨基酸序列的程度；或(3)通过SDS-PAGE在非还原性或还原 性条件下使用考马斯蓝或优选为银染色达到同质性。分离的多肽包括在重组细胞内的原位多肽，因为多肽天然环境中的至少一个要素是不存在的。然而，通常情况下，一个分离的多肽至少要经过一个纯化步骤。An "isolated" polypeptide is one that has been identified, separated, and/or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free from all other components of its production environment. 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. In some embodiments, the polypeptide will be purified to: (1) greater than 95% polypeptide by weight, as determined by the Lowry method, and in some embodiments, greater than 99% polypeptide by weight; (2) to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequence by use of a spinning cup sequenator; or (3) by SDS-PAGE in a non-reducing or reducing medium. Homogeneity is achieved using Coomassie blue or, preferably, silver staining under sterile conditions. An isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one element of the polypeptide's natural environment will not be present. Ordinarily, however, an isolated polypeptide will have been subjected to at least one purification step.
术语“多核苷酸”或“核酸”是任何长度的核苷酸多聚体并且包括DNA及RNA。核苷酸可为脱氧核糖核苷酸、核糖核苷酸、经修饰核苷酸或碱基和/或它们的类似物、或可通过DNA或RNA聚合酶掺入到多聚体中的任何底物。The term "polynucleotide" or "nucleic acid" is a nucleotide polymer of any length and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
如本文所述，术语“分离的核酸”，是指基因组、cDNA或合成来源的核酸或其组合。根据其来源，所述“分离的核酸”(1)与自然界中发现的“分离的核酸”中的全部或部分多核苷酸无关，(2)可与自然状态下不与之相连的多核苷酸可操作性地连接，或(3)在自然界中不作为较长序列的一部分而存在。As used herein, the term "isolated nucleic acid" refers to a nucleic acid of genomic, cDNA or synthetic origin or a combination thereof. Depending on its origin, the "isolated nucleic acid" (1) is unrelated to all or part of a polynucleotide found in the "isolated nucleic acid" in nature, (2) can be operably linked to a polynucleotide to which it is not naturally associated, or (3) does not exist as part of a longer sequence in nature.
一种编码一个构建体(如本申请所述的NGF突变体)的“分离的”核酸分子为一种从至少含一种杂质的核酸分子中鉴定和分离出来的核酸分子，所述杂质核酸分子通常与生产它的环境有关。优选地，所分离的核酸与其生产环境中的所有成分没有关联。如本文所述的编码多肽的分离的核酸分子是以一种并非它在自然界中发现时的形式或形态存在。因此，分离的核酸分子不同于自然存在于细胞中编码本文所述多肽的核酸。一种分离的核酸包括含有该核酸分子的细胞中所含的核酸分子，但该核酸分子存在于染色体外或与其自然染色体位置不同的染色体位置上。An "isolated" nucleic acid molecule encoding a construct (such as the NGF mutant described herein) is a nucleic acid molecule that has been identified and separated from at least one impurity nucleic acid molecule, which is generally associated with the environment in which it is produced. Preferably, the isolated nucleic acid is not associated with all components of its production environment. An isolated nucleic acid molecule encoding a polypeptide as described herein is present in a form or morphology that is not as it is found in nature. Therefore, an isolated nucleic acid molecule is different from a nucleic acid naturally present in a cell that encodes a polypeptide described herein. An isolated nucleic acid includes a nucleic acid molecule contained in a cell containing the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
术语“控制序列”是指在特定宿主生物体中表达可操作连接编码序列所必需的DNA序列。例如，适合原核生物的控制序列包括启动子、任选的操作序列和核糖体结合位点。已知真核细胞利用启动子、多聚腺苷酸化信号和增强子。The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
当核酸与另一个核酸序列建立功能性关系时，所述核酸即为“可操作地连接”。例如，如果前序列或分泌先导物的DNA作为参与多肽分泌的前蛋白表达，则所述DNA可操作连接到多肽的DNA；如果启动子或增强子影响序列的转录，则所述启动子或增强子可操作连接到编码序列；或如果核糖体结合位点处在便于翻译的位置，则所述核糖体结合位点可操作连接到编码序列。一般来说，“可操作连接”意味着连接的DNA序列是连续的，并且，对于分泌先导物，其不仅是连续的并且处于读取阶段(reading phase)。然而，增强子不必是连续的。连接是通过在适宜的限制性位点进行连接来完成的。如果不存在此类位点，则按照常规做法使用合成的寡核苷酸适配体或接头。 A nucleic acid is "operably linked" when it establishes a functional relationship with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA of a polypeptide; if a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably linked to a coding sequence; or if a ribosome binding site is in a position that facilitates translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the DNA sequences being linked are continuous, and, for a secretory leader, they are not only continuous but also in the reading phase. However, enhancers do not have to be continuous. Linking is accomplished by ligating at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used as a matter of routine practice.
如本文应用的术语“载体”是指一种核酸分子能够扩增与其连接的另一核酸分子。该术语包括作为自我复制核酸结构的载体以及被引入已知宿主细胞基因组的载体。某些载体能够指导与之连接的核酸表达。在本文中称此类载体为“表达载体”。As used herein, the term "vector" refers to a nucleic acid molecule that is capable of amplifying another nucleic acid molecule to which it is attached. The term includes vectors that are self-replicating nucleic acid structures as well as 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 attached. Such vectors are referred to herein as "expression vectors."
如本文应用的术语“转染”或“转化”或“转导”是指外源核酸转移或引入宿主细胞的过程。“转染的”或“转化的”或“转导的”细胞是用外源核酸转染、转化或转导的细胞。所述细胞包括原代受试细胞及其子代。As used herein, the term "transfection" or "transformation" or "transduction" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养”可互换使用，指已引入外源核酸的细胞，包括此类细胞的子代。宿主细胞包括“转化体”和“转化细胞”，其中包括原代转化细胞和由此产生的子代，而不考虑传代次数。子代在核酸上可能与母细胞不完全相同，可能含有突变。本文包括在原始转化细胞中筛选或选择与其具有相同功能或生物活性的突变子代。The terms "host cell", "host cell line" and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and the progeny produced therefrom, without regard to the number of passages. The progeny may not be completely identical to the parent cell in nucleic acid and may contain mutations. Screening or selecting mutant progeny having the same function or biological activity as the original transformed cell is included herein.
术语“药物制剂”或“药物组合物”是指一种制剂，所述制剂为使活性成分的生物活性有效的形式，并且不包含对施用所述制剂的受试者具有不可接受毒性的额外成分。这种制剂是无菌的。“无菌”制剂是无菌的或不含任何活的微生物及其孢子。The term "pharmaceutical preparation" or "pharmaceutical composition" refers to a preparation that is in a form that renders the biological activity of the active ingredient effective and that contains no additional ingredients that are unacceptably toxic to a subject to which the preparation is administered. Such preparations are sterile. A "sterile" preparation is sterile or does not contain any living microorganisms or their spores.
术语“药学上可接受的”或“药理学上相容的”是指无生物学活性或者其它不期望性质的材料，例如该材料能够加入到给予患者的药物组合物中，而不会引起显著的不良生物反应，或者，不与组合物中包含的任何其它组分以有害的方式相互作用。药学上可接受的载体或赋形剂优选满足毒理学或制造检测的所需标准和/或包含在美国食品和药品管理局编制的非活性成分指南中。The term "pharmaceutically acceptable" or "pharmacologically compatible" refers to a material that is free of biological activity or other undesirable properties, such as a material that can be added to a pharmaceutical composition administered to a patient without causing a significant adverse biological response, or that does not interact in a harmful manner with any other component contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology or manufacturing testing and/or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.
本申请所述的“交集”是指：设A，B是两个集合，由所有属于集合A且同时属于集合B的元素所组成的集合，叫做集合A与集合B的交集。The "intersection" mentioned in this application means: suppose A and B are two sets, the set consisting of all elements that belong to set A and also belong to set B is called the intersection of set A and set B.
本申请所述的“并集”是指：设A，B是两个集合，把集合A所有的元素和集合B所有的元素合并在一起,而没有其它元素的集合，叫做集合A与集合B的并集。The "union" mentioned in this application means: suppose A and B are two sets, and the set that combines all the elements of set A and all the elements of set B without other elements is called the union of set A and set B.
本申请所述的实施例应理解为包括“由……组成”和/或“基本上由……组成”的实施例。The embodiments described herein should be understood to include "consisting of" and/or "consisting essentially of" embodiments.
本申请中提及“约”为一个数值或参数，包含(和描述)针对该数值或参数本身的变体。例如，涉及“约X”的描述包括“X”的描述。 In this application, "about" is mentioned as a value or parameter, including (and describing) variations of the value or parameter itself. For example, the description involving "about X" includes the description of "X".
本申请中提及“不是”一个数值或参数，通常表示并描述“除了”某一数值或参数之外。例如，该方法不能用于治疗X型癌症，意味着该方法通常用于治疗除X型癌症之外的其它类型。In this application, "not" a value or parameter is mentioned, which usually means and describes "except" a certain value or parameter. For example, the method cannot be used to treat type X cancer, which means that the method is usually used to treat other types of cancer except type X cancer.
如本申请应用的术语“约X-Y”与“约X到约Y”意思相同。As used herein, the term "about X-Y" has the same meaning as "about X to about Y."
除非上下文另有明确说明，本申请说明书和所述权利要求中所采用的单数形式“一”，“一个”和“该”包括复数对象。As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
神经生长因子(NGF)Nerve Growth Factor (NGF)
NGF作为神经营养因子的一类，参与调节某些神经元和其它细胞的生长、维持、增殖和存活等(参见Levi-Montalcini,2004,Progress in Brain Research,vol.146,p.525-527)。NGF存在于多种物种中，在雄性小鼠颌下腺、牛精浆、蛇毒、豚鼠前列腺、人胎盘组织等中含量丰富。小鼠NGF与人类NGF的氨基酸序列同源性高达90％。As a type of neurotrophic factor, NGF is involved in regulating the growth, maintenance, proliferation and survival of certain neurons and other cells (see Levi-Montalcini, 2004, Progress in Brain Research, vol. 146, p. 525-527). NGF exists in many species and is abundant in male mouse submandibular gland, bovine seminal plasma, snake venom, guinea pig prostate, human placenta tissue, etc. The amino acid sequence homology between mouse NGF and human NGF is as high as 90%.
来自小鼠颌下腺的NGF是一种7S蛋白复合物，分子量大约为130kDa，包括3种不同类型的亚基(α、β和γ亚基)，NGF的生物活性与β亚基(也称为2.5sNGF)相关。NGF from mouse submandibular gland is a 7S protein complex with a molecular weight of approximately 130 kDa, including three different types of subunits (α, β and γ subunits). The biological activity of NGF is related to the β subunit (also known as 2.5sNGF).
人类NGF(hNGF)基因位于1号染色体短臂上，完整的NGF外显子具有241个氨基酸，通常被称为preproNGF(SEQ ID NO:90)，包括位于C末端的第240位的R和第241位的A氨基酸残基。preproNGF包含一个信号肽序列(SEQ ID NO:91)、一个前导肽(SEQ ID NO:92)和成熟的NGF序列(β-NGF,SEQ ID NO:1或SEQ ID NO:2)。preproNGF的信号肽在内质网中被切除，形成proNGF(223个氨基酸；SEQ ID NO:93)。proNGF前体在内质网中以同源二聚体形式存在，随后转移至高尔基体。在高尔基体中，再经蛋白酶切除去掉前导肽部分，形成成熟β-NGF二聚体，每个单体含有120个氨基酸残基(SEQ ID NO：2)或118个(缺少C末端的最后两个氨基酸残基R和A，SEQ ID NO：1)氨基酸残基(Rattenholl A,et al.,The pro-sequence facilitates folding of human nerve growth factor from Escherichia coli inclusion bodies.Eur J Biochem.2001 Jun；268(11):3296-303；Seidah NG,et al.,Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases.Biochem J.1996Mar 15；314(Pt 3)(Pt 3):951-60)。在本申请的一些实施例中，优选的成熟NGF单体由118个氨基酸残基组成，其序列如SEQ ID NO：1所示。PreproNGF的结构示意图如图3所示。 The human NGF (hNGF) gene is located on the short arm of chromosome 1. The complete NGF exon has 241 amino acids, usually referred to as preproNGF (SEQ ID NO: 90), including the R at position 240 and the A at position 241 located at the C-terminus. preproNGF contains a signal peptide sequence (SEQ ID NO: 91), a leader peptide (SEQ ID NO: 92) and a mature NGF sequence (β-NGF, SEQ ID NO: 1 or SEQ ID NO: 2). The signal peptide of preproNGF is cleaved in the endoplasmic reticulum to form proNGF (223 amino acids; SEQ ID NO: 93). The proNGF precursor exists as a homodimer in the endoplasmic reticulum and is subsequently transferred to the Golgi apparatus. In the Golgi apparatus, the leader peptide portion is removed by protease to form a mature β-NGF dimer, each monomer containing 120 amino acid residues (SEQ ID NO: 2) or 118 (lacking the last two amino acid residues R and A at the C-terminus, SEQ ID NO: 1) amino acid residues (Rattenholl A, et al., The pro-sequence facilitates folding of human nerve growth factor from Escherichia coli inclusion bodies. Eur J Biochem. 2001 Jun; 268 (11): 3296-303; Seidah NG, et al., Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases. Biochem J. 1996 Mar 15; 314 (Pt 3) (Pt 3): 951-60). In some embodiments of the present application, the preferred mature NGF monomer consists of 118 amino acid residues, and its sequence is shown in SEQ ID NO: 1. The structural schematic diagram of PreproNGF is shown in Figure 3.
NGF与TrkA受体结合，促使受体发生同源二聚化，进而引起酪氨酸激酶片段的自磷酸化，激活PI 3-激酶、ras和PLC信号通路。p75NTR受体同样可以与TrkA形成异源二聚体，该异源二聚体对NGF具有更高的亲和力和特异性。NGF binds to the TrkA receptor, causing the receptor to homodimerize, which in turn causes the autophosphorylation of the tyrosine kinase fragment and activates the PI 3-kinase, ras and PLC signaling pathways. The p75NTR receptor can also form a heterodimer with TrkA, which has a higher affinity and specificity for NGF.
如本申请所述，NGF可以是从各种来源分离出的NGF，如从人体组织或从其它来源，或通过重组或合成方法制备。在一些实施例中，NGF为重组NGF，如重组hNGF(rhNGF)。在一些实施例中，NGF为鼠源NGF，如重组表达的小鼠NGF。As described herein, NGF can be NGF isolated from various sources, such as from human tissue or from other sources, or prepared by recombinant or synthetic methods. In some embodiments, NGF is recombinant NGF, such as recombinant hNGF (rhNGF). In some embodiments, NGF is murine NGF, such as recombinantly expressed mouse NGF.
降低疼痛的NGF突变体NGF mutants that reduce pain
NGF是公认的疼痛靶点，因为它能导致动物和人类疼痛。尤其在成人中，NGF能促进中枢和外周神经元亚群的健康和存活(Huang和Reichardt,Ann.Rev.Neurosci.24:677-736(2001))。NGF也有助于调节这些神经元的功能特性，并能够对被称为伤害感受器的感觉疼痛受体的敏感性或兴奋性进行强直性控制(Priestley et al.,Can.J.Physiol.Pharmacol.80:495-505(2002)；Bennett,Neuroscientist 7:13-17(2001))。伤害感受器感知并向中枢神经系统传递各种有害的刺激，从而产生疼痛感(伤害感受)。NGF受体位于伤害感受器上。在受伤和炎症组织中NGF的表达增加，并在人的疼痛状态下上调。NGF is a recognized pain target because it can cause pain in animals and humans. Especially in adults, NGF can promote the health and survival of central and peripheral neuronal subpopulations (Huang and Reichardt, Ann. Rev. Neurosci. 24: 677-736 (2001)). NGF also helps regulate the functional properties of these neurons and can exert tonic control on 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 sense and transmit various harmful stimuli to the central nervous system, thereby producing a sense of pain (nociception). NGF receptors are located on nociceptors. The expression of NGF is increased in injured and inflamed tissues and is upregulated in human pain states.
广义上的“疼痛”是指一种经验现象，对于体验疼痛的个体而言具有高度的主观性，并且受个体心理状态的影响，包括环境和文化背景。“生理上的”疼痛通常与第三方可感知的刺激有关，这种刺激是造成实际或潜在组织损伤的原因。从这个意义上讲，根据国际疼痛研究协会(IASP)的说法，可以认为疼痛是一种“与实际或潜在组织损伤相关的感觉和情感体验，或用此类损伤的术语来描述”。然而，一些疼痛的例子没有可察觉的原因。例如，精神性疼痛，包括在没有任何外界刺激的情况下感觉到的疼痛，患有心理障碍的人因精神因素有时会持续出现可感知的疼痛、或者引起先前存在的生理疼痛的加剧。"Pain" in a broad sense refers to an empirical phenomenon that is highly subjective to the individual experiencing it and is influenced by the individual's psychological state, including environmental and cultural context. "Physiological" pain is usually associated with a stimulus perceptible to a third party that is the cause of actual or potential tissue damage. In this sense, according to the International Association for the Study of Pain (IASP), pain can be considered a "sensory and affective experience associated with actual or potential tissue damage, or described in terms of such damage." However, some examples of pain have no perceptible cause. For example, psychogenic pain, which includes pain felt in the absence of any external stimulus, and people with psychological disorders sometimes experience persistent perceptible pain due to psychological factors, or exacerbation of pre-existing physiological pain.
疼痛包括伤害性疼痛、神经病理性/神经源性疼痛、突发性疼痛、痛觉超敏、痛觉过敏、感觉过敏、触物感痛、感觉异常、痛觉过度、幻肢痛、精神性疼痛、痛觉缺失、神经痛、神经炎。其它分类包括恶性疼痛、心绞痛疼痛和/或原发性疼痛、复杂区域疼痛综合征I、复杂区域疼痛综合征II。疼痛的类型和症状不必相互排斥。这些术语的定义与IASP一致。 Pain includes nociceptive pain, neuropathic/neurogenic pain, breakthrough pain, allodynia, hyperalgesia, hyperesthesia, allodynia, paresthesia, hyperalgesia, phantom limb pain, psychogenic pain, analgesia, neuralgia, neuritis. Other classifications include malignant pain, anginal pain and/or primary pain, complex regional pain syndrome I, complex regional pain syndrome II. The types and symptoms of pain are not necessarily mutually exclusive. The definitions of these terms are consistent with the IASP.
伤害性疼痛是由外周神经元中的特殊伤害感受器对伤害性刺激作出应答，并将伤害性刺激编码为动作电位而引起的。伤害感受器通常位于Aδ纤维和(多模)C纤维上，是游离的神经末梢，终止于皮肤下方、肌腱、关节和身体器官。背根神经节(DRG)神经元为外周和脊髓之间提供沟通的场所。信号经过脊髓处理，到脑干和丘脑部位，最后到达大脑皮层，在那里它通常(但不总是)引起疼痛感。伤害性疼痛可由多种化学、热、生物(例如，炎症)或机械事件引起，这些事件有可能刺激或损伤身体组织，通常高于伤害感受器中的引起伤害感受活动所需的某种最小强度阈值。Nociceptors are free nerve endings that terminate just below the skin, in tendons, joints, and in body organs. Dorsal root ganglia (DRG) neurons provide a site for communication between the periphery and the spinal cord. The signal is processed by the spinal cord, to the brainstem and thalamus, and finally to the cerebral cortex, where it usually (but not always) causes a sensation of pain. Nociceptive pain can be caused by a variety of chemical, thermal, biological (e.g., inflammation), or mechanical events that have the potential to irritate or damage body tissues, usually above some minimum intensity threshold required to elicit nociceptive activity in the nociceptors.
神经病理性疼痛通常是外周或中枢神经系统功能异常导致的，分别引起外周或中枢神经病理性疼痛。IASP将神经性疼痛定义为神经系统原发性病变或功能障碍引发或导致的疼痛。神经病理性疼痛通常涉及神经系统的实际损伤，尤其是在慢性病中。炎性伤害性疼痛通常是组织损伤和由此引起的炎症过程导致的。神经病理性疼痛可在任何可观察到的组织损伤明显愈合后(例如，数月或数年)仍持续存在。Neuropathic pain is usually caused by abnormal function of the peripheral or central nervous system, causing peripheral or central neuropathic pain, respectively. The IASP defines neuropathic pain as pain caused or caused by primary lesions or dysfunctions of the nervous system. Neuropathic pain usually involves actual damage to the nervous system, especially in chronic diseases. Inflammatory nociceptive pain is usually caused by tissue damage and the resulting inflammatory process. Neuropathic pain can persist after any observable tissue damage has obviously healed (for example, months or years).
在神经病理性疼痛情况下，受影响区域的感觉信息处理过程可能会变得异常，并且通常不会引起疼痛的无害刺激(例如，热刺激、触摸/压力刺激)可能会导致疼痛(即，痛觉超敏)，或有害刺激可能会引起对正常疼痛刺激的过度疼痛感知(即，痛觉过敏)。此外，正常刺激可能会引起类似于电刺痛或电击或“发麻”的感觉(即，感觉异常)和/或不愉快的感觉(即，感觉障碍)。突发性疼痛是先前存在的慢性疼痛的加重。痛觉过敏是一种疼痛综合征，由对刺激的异常疼痛反应引起。在大多数情况下，刺激是重复的，伴随着疼痛阈值增加，所述疼痛阈值被认为是患者能够识别为疼痛的最小疼痛体验。In cases of neuropathic pain, the processing of sensory information in the affected area may become 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 cause excessive pain perception for normally painful stimuli (i.e., hyperalgesia). In addition, normal stimuli may cause sensations similar to electric tingling or shock or "pins and needles" (i.e., paresthesias) and/or unpleasant sensations (i.e., dysesthesias). Breakthrough pain is an exacerbation of pre-existing chronic pain. Hyperalgesia is a pain syndrome caused by an abnormal painful response to a stimulus. In most cases, the stimulus is repetitive and is accompanied by an increase in the pain threshold, which is considered to be the minimum pain experience that the patient can recognize as pain.
神经病理性疼痛的例子包括触觉性痛觉超敏(例如，神经损伤后诱发的)、神经痛(例如，疱疹后(或带状疱疹后)神经痛、三叉神经痛)、反射性交感神经营养不良/烧灼痛(神经创伤)、癌症疼痛(例如，癌症本身或炎症等相关条件引起的疼痛，或由于化疗、手术或放疗等治疗)、假肢痛、嵌压性神经痛(例如，腕管综合征)和神经病变，如外周神经病变(例如，糖尿病、艾滋病、长期饮酒、接触其它毒素(包括许多化疗)、维生素缺乏症，以及其它各种疾病)。神经病理性疼痛包括由于各种原因(例如，外科手术、伤口、带状疱疹、糖尿病性神经病变、腿或手臂截肢、癌症等)导致的神经损伤后神经系统的病理性手术所引起的疼痛。与神经病理性疼痛相关的疾病包括创伤性神经损伤、中风、多发性硬化、脊髓空洞症、脊髓损伤和癌症 Examples of neuropathic pain include tactile allodynia (e.g., induced after nerve injury), neuralgia (e.g., post-herpetic (or post-herpetic) neuralgia, trigeminal neuralgia), reflex sympathetic dystrophy/burning pain (nerve trauma), cancer pain (e.g., pain caused by the cancer itself or related conditions such as inflammation, or due to treatments such as chemotherapy, surgery, or radiation therapy), prosthetic pain, entrapment neuralgia (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 treatments), vitamin deficiencies, and various other diseases). Neuropathic pain includes pain caused by pathological procedures of the nervous system following nerve damage due to various causes (e.g., surgery, wounds, herpes zoster, 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
引起疼痛的刺激通常会引起炎症反应，而炎症反应本身也会导致疼痛。在一些情况下，疼痛似乎是由伤害性和神经性因素的复杂混合因素引起的。例如，慢性疼痛通常包括炎性伤害性疼痛或神经性疼痛，或两者的混合。最初的神经系统功能障碍或损伤可能触发炎症介质的神经释放和随后的神经病理性炎症。例如，偏头痛可以表现为神经性疼痛和伤害性疼痛的混合。此外，肌筋膜疼痛可能继发于来自肌肉的痛觉输入，但异常的肌肉活动可能是神经系统疾病导致的。Pain-causing stimuli often provoke an inflammatory response, which itself can cause pain. In some cases, pain appears to be caused by a complex mixture of nociceptive and neuropathic factors. For example, chronic pain often includes inflammatory nociceptive pain or neuropathic pain, or a mixture of both. Initial nervous system dysfunction or injury may trigger the neural release of inflammatory mediators and subsequent neuropathic inflammation. For example, migraine can present as a mixture of neuropathic and nociceptive pain. In addition, myofascial pain may be secondary to pain input from muscles, but abnormal muscle activity may be the result of nervous system disease.
由于NGF在使用过程中会引起较严重的疼痛，部分病人无法耐受，故造成了使用上的部分受限。NGF参与了疼痛的病理生理过程，通过影响炎症介质的释放、离子通道的开放及促进神经纤维的生长导致疼痛，并通过调控离子通道及分子信号从而参与疼痛的发生发展。有学者推测NGF还可能通过促进致痛物质的表达导致疼痛，并在机体损伤后可改变神经的出芽和再生。目前的研究发现：在人类中，不会引起痛觉过敏的最大剂量为0.03μg/kg(Pettyet al.，1994—29)。但是，如此低的剂量限制了NGF的应用，同时也限制了其适应症的扩大，如用于中枢神经系统。Since NGF can cause severe pain during use, some patients cannot tolerate it, which has caused some restrictions on its use. NGF is involved in the pathophysiological process of pain, causing pain by affecting the release of inflammatory mediators, the opening of ion channels and promoting the growth of nerve fibers, and participating in the occurrence and development of pain by regulating ion channels and molecular signals. Some scholars speculate that NGF may also cause pain by promoting the expression of pain-causing substances, and can change the sprouting and regeneration of nerves after damage to the body. Current studies have found that in humans, the maximum dose that does not cause hyperalgesia is 0.03μg/kg (Pettyet al., 1994-29). However, such a low dose limits the application of NGF and also limits the expansion of its indications, such as use in the central nervous system.
可通过本领域已知的任何方法测量致痛活性，如WO2017157325、WO2017157326和CN108727486A中所述，其全部内容通过引用并入本文。在本申请的一些实施例中，通过痛阈测量疼痛，所述“痛阈值”是疼痛刺激引起应激组织反应的最低值，是机体对外力刺激最大的承受能力，痛阈值越高代表承受疼痛的能力越强，反之越弱。痛阈值降低，代表机体对疼痛的敏感性提高，痛阈值升高，代表机体对疼痛的敏感性降低。The pain-inducing activity can be measured by any method known in the art, such as described in WO2017157325, WO2017157326 and CN108727486A, all of which are incorporated herein by reference. In some embodiments of the present application, pain is measured by pain threshold, and the "pain threshold" is the minimum value of the stress tissue response caused by pain stimulation, which is the maximum tolerance of the body to external force stimulation. The higher the pain threshold, the stronger the ability to withstand pain, and vice versa. The lower the pain threshold, the higher the sensitivity of the body to pain, and the higher the pain threshold, the lower the sensitivity of the body to pain.
例如，引起疼痛的活动可以通过要求患者根据一些不同的量表对所经历的疼痛的质量和强度进行评分来测量。口头疼痛量表使用词语来描述无疼痛、轻度疼痛、中度疼痛和重度疼痛的范围，每一级的分数从0到3。或者，可能会要求患者根据数值疼痛量表从0(无疼痛)到10(最严重疼痛)对其疼痛进行评分。在视觉模拟量表(VAS)上，垂直或水平线包含从无痛到可能的最严重疼痛的文字描述，要求患者在代表其当前疼痛水平的点上标记。McGill疼痛指数使患者能够从一系列简短列表中选择最能描述其疼痛的词语来描述疼痛的质量和强度，例如捶击、灼痛、捏痛。使用VAS或数值量表有困难的成年人(例如，FACES面部或非语言患者)可使用其它疼痛量表，例如，行为评定量表。功能性活性评分通过要求患者执行与疼痛区域相关的任务，来反应患 者因疼痛而受到阻碍的程度。使用这些类型的量表以改善疼痛评分，例如，与野生型相比，疼痛评分的改善潜在地表明待测试NGF突变体引起疼痛的不良反应减少。For example, activities that cause pain can be measured by asking the patient to rate the quality and intensity of the pain experienced on a number of different scales. Verbal pain scales use words to describe a range of no pain, mild pain, moderate pain, and severe pain, with scores from 0 to 3 for each level. Alternatively, patients may be asked to rate their pain on a numerical pain scale from 0 (no pain) to 10 (worst possible pain). On a visual analog scale (VAS), vertical or horizontal lines contain written descriptions from no pain to the worst possible pain, and the patient is asked to mark the point that represents their current pain level. The McGill Pain Index enables patients to describe the quality and intensity of pain by selecting the words that best describe their pain from a short list, such as thumping, burning, pinching. Adults who have difficulty using the VAS or numerical scales (e.g., FACES facial or nonverbal patients) can use other pain scales, such as behavioral rating scales. Functional activity scores reflect the patient's ability to respond to pain by asking the patient to perform tasks related to the area of pain. Using these types of scales to improve pain scores, for example, compared to wild-type, an improvement in pain scores potentially indicates that the NGF mutant being tested is less likely to cause adverse pain.
在一些实施例中，可通过热板法(54-55℃)在小鼠上测试野生型NGF、NGF突变体的致痛活性以确定疼痛阈值。简单来说，将符合反应条件的小鼠麻醉，然后采用神经钳夹法建立小鼠坐骨神经损伤模型，而假手术组仅分离坐骨神经，不钳夹坐骨神经。然后将小鼠分为三组：假手术组、损伤对照组(正常生理盐水)和实验组(例如，应用野生型NGF、NGF突变体进行处理)。每只小鼠的疼痛阈值由舔后脚的潜伏期表示，可在手术前和手术后不同时间点测量。疼痛阈值增加％＝(受伤后第10天的疼痛阈值-受伤前的疼痛阈值)×100％/受伤前的疼痛阈值。In some embodiments, the pain threshold can be determined by testing the pain-inducing activity of wild-type NGF and NGF mutants on mice by the hot plate method (54-55°C). Briefly, mice that meet the reaction conditions are anesthetized, and then the mouse sciatic nerve injury model is established by the nerve clamp method, while the sham operation group only separates the sciatic nerve without clamping the sciatic nerve. The mice are then divided into three groups: a sham operation group, an injury control group (normal saline) and an experimental group (for example, treated with wild-type NGF and NGF mutants). The pain threshold of each mouse is represented by the latency of licking the hind feet, which can be measured at different time points before and after surgery. Pain threshold increase % = (pain threshold on the 10th day after injury - pain threshold before injury) × 100% / pain threshold before injury.
本申请所述的野生型NGF、NGF突变体也可通过测量机械刺激下小鼠的曲爪反应，以确定疼痛阈值。它可以在短期致痛条件下或长期致痛条件下进行测试。简单来说，向符合反应条件的小鼠皮下注射本文所述的载体或本文所述的野生型NGF、NGF突变体(可在不同的浓度下)，然后测量注射后在机械刺激下的曲爪反应(例如，在不同时间点)，这反映了治疗后的疼痛阈值。The wild-type NGF and NGF mutants described in this application can also be used to measure the paw curling reaction of mice under mechanical stimulation to determine the pain threshold. It can be tested under short-term pain conditions or long-term pain conditions. Simply put, the vector described herein or the wild-type NGF and NGF mutants described herein (at different concentrations) are subcutaneously injected into mice that meet the reaction conditions, and then the paw curling reaction under mechanical stimulation after injection is measured (for example, at different time points), which reflects the pain threshold after treatment.
本申请所述的野生型NGF、NGF突变体的致痛活性也可通过行为试验进行测试。例如，给大鼠关节施用本文所述的载体或野生型NGF、NGF突变体(可在不同的浓度下)，然后可通过记录给药后(例如，在不同时间点)的抬腿维持时间和抬腿次数来检测样品是否引起疼痛，以计算抬腿总持续时间。较短的抬腿总时间意味着较少的致痛活性。The pain-inducing activity of wild-type NGF and NGF mutants described in the present application can also be tested by behavioral tests. For example, the carrier described herein or wild-type NGF and NGF mutants (can be at different concentrations) are administered to the rat joints, and then the sample can be tested for pain by recording the duration of leg lifting and the number of leg lifting after administration (for example, at different time points) to calculate the total duration of leg lifting. Shorter total leg lifting time means less pain-inducing activity.
根据专利CN109153709B中所披露内容，通过在人野生型成熟NGF的第12位氨基酸位点处引入突变F12E，获得NGF突变体NGFF12E，该突变体相对于野生型NGF，能够降低NGF在使用过程中产生的疼痛不良反应，该文献的内容在此处以其整体并入到本文之中。在一些实施例中，所述的人野生型成熟NGF的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。在一些实施例中，突变体NGFF12E的氨基酸序列如SEQ ID NO:83所示。According to the disclosure in patent CN109153709B, by introducing the mutation F12E at the 12th amino acid site of human wild-type mature NGF, the NGF mutant NGF F12E is obtained. Compared with wild-type NGF, the mutant can reduce the adverse pain reactions produced by NGF during use. The content of this document is incorporated herein in its entirety. In some embodiments, the amino acid sequence of the human wild-type mature NGF is shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the amino acid sequence of the mutant NGF F12E is shown in SEQ ID NO: 83.
在一些实施例中，与野生型NGF相比，本申请所述的NGFF12E或包含突变F12E的NGF突变体，在受试者中具有减少的或甚至没有致痛活性。在一些实施例中，疼痛是急性疼痛、短期疼痛、持续性或慢性伤害性疼痛、或持续性或慢性神经病理性疼痛。在一些实施例中，与野生型NGF相比，本文所述的NGFF12E或包含突变F12E的 NGF突变体引起的疼痛至少减少10％，例如与野生型NGF相比，疼痛至少减少20％、25％、30％、35％、40％、45％、50％、55％、60％、70％、80％、90％、95％或100％。在一些实施例中，本申请所述的NGFF12E或包含突变F12E的NGF突变体在给受试者施用时不会引起疼痛。In some embodiments, compared to wild-type NGF, the NGF F12E described herein or a NGF mutant comprising a mutant F12E has reduced or even no pain-inducing activity in a subject. 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, compared to wild-type NGF, the NGF F12E described herein or a NGF mutant comprising a mutant F12E has reduced or even no pain-inducing activity in a subject. The pain caused by the NGF mutant is reduced by at least 10%, for example, compared with wild-type NGF, the pain is reduced by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 100%. In some embodiments, the NGF F12E described herein or the NGF mutant comprising the mutant F12E does not cause pain when administered to a subject.
抗NGF抗体Anti-NGF Antibody
目前已经表明神经生长因子在生理性疼痛和病理性疼痛的传递中起重要作用，此外已经证实NGF功能的拮抗作用在神经性疼痛和慢性炎症性疼痛模型中能够防止痛觉过敏和异常性疼痛。例如，在神经性疼痛动物模型(例如神经干或脊神经结扎)中，全身性注射抗NGF中和抗体既能防止异常性疼痛又能防止痛觉过敏(Ramer et al.,1999,Eur.J.Neurosci.11:837-846；以及Ro et al.，1999,Pain 79:265-274)。靶向NGF用于治疗慢性疼痛的概念自1990年以来势头大增(McMahon SB,et al.,The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule.Nat Med 1995；1(8):774e80)，抗NGF抗体也被认为是疼痛领域具有希望的下一代镇痛药物之一，目前涉及到抗NGF抗体的临床试验可以通过在网站www.clinicaltrials.gov中进行查看。其中Tanezumab是第一个开展临床试验的抗NGF抗体，由于其引起的骨坏死严重不良反应，其在用于治疗骨关节炎的临床试验于2010年被FDA暂停。也正是由于抗NGF抗体在治疗疾病过程中所产生的严重不良反应，导致了许多抗NGF抗体项目停止开发，或最终无法通过FDA批准，取得上市申请。关于抗NGF抗体的相关进展及临床试验情况可以参考文献Miller CG,et al.,The current status of imaging in anti-NGF clinical trials.Osteoarthritis Cartilage.2015 Jan；23 Suppl 1:S3-7和Wise BL,et al.,The evolution of nerve growth factor inhibition in clinical medicine.Nat Rev Rheumatol.2021 Jan；17(1):34-46等。It has been shown that nerve growth factor plays an important role in the transmission of physiological and pathological pain. In addition, it has been confirmed that antagonism of NGF function can prevent hyperalgesia and allodynia in neuropathic pain and chronic inflammatory pain models. For example, in animal models of neuropathic pain (such as nerve trunk or spinal nerve ligation), systemic injection of anti-NGF neutralizing antibodies can prevent both allodynia and hyperalgesia (Ramer et al., 1999, Eur. J. Neurosci. 11: 837-846; and Ro et al., 1999, Pain 79: 265-274). The concept of targeting NGF for the treatment of chronic pain has gained momentum since 1990 (McMahon SB, et al., The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule. Nat Med 1995; 1(8): 774e80). Anti-NGF antibodies are also considered to be one of the promising next-generation analgesics in the field of pain. Currently, clinical trials involving anti-NGF antibodies can be viewed on the website www.clinicaltrials.gov. Among them, Tanezumab is the first anti-NGF antibody to be clinically tested. Due to the serious adverse reaction of osteonecrosis caused by it, its clinical trial for the treatment of osteoarthritis was suspended by the FDA in 2010. It is precisely because of the serious adverse reactions caused by anti-NGF antibodies in the treatment of diseases that many anti-NGF antibody projects have stopped development or ultimately failed to pass FDA approval and obtain marketing applications. For the relevant progress and clinical trials of anti-NGF antibodies, please refer to Miller CG, et al., The current status of imaging in anti-NGF clinical trials. Osteoarthritis Cartilage. 2015 Jan; 23 Suppl 1: S3-7 and Wise BL, et al., The evolution of nerve growth factor inhibition in clinical medicine. Nat Rev Rheumatol. 2021 Jan; 17(1): 34-46, etc.
本申请主要涉及的抗NGF抗体包括Tanezumab(辉瑞)、Fasinumab(再生元)、Fulranumab(强生)、MEDI-578(阿斯利康)、AK-115(康方生物)、SSS-40(三生国健)和STC001(舒泰神)，其VH和VL的氨基酸序列如表1所示。关于上述抗体Tanezumab(WO2004/058184)、Fasinumab(WO2009/023540)、Fulranumab(WO2005/019266)、MEDI-578(WO2006/077441)、AK-115(WO2019/201133)、SSS-40(WO2021/218574)，分别在相应的专利中对其相关的性质及治疗用途等进行了披露。 The anti-NGF antibodies mainly involved in this application include Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Bio), SSS-40 (Sunshine Guojian) and STC001 (Sutacion), and the amino acid sequences of their VH and VL are shown in Table 1. Regarding the above-mentioned antibodies Tanezumab (WO2004/058184), Fasinumab (WO2009/023540), Fulranumab (WO2005/019266), MEDI-578 (WO2006/077441), AK-115 (WO2019/201133), SSS-40 (WO2021/218574), their relevant properties and therapeutic uses are disclosed in the corresponding patents.
结合活性及其检测方法Binding activity and its detection method
在一些实施例中，本申请中所述的分子(例如，野生型NGF或NGF突变体)与其结合对象(例如，TrkA受体或抗NGF抗体)之间的结合活性可以通过任何本领域已知的合适的配体结合试验或抗体/抗原结合试验来确定，例如，Western blot、酶联免疫吸附试验(ELISA)、Meso Scale Discovery(MSD)电化学发光、基于珠子的多重免疫分析(MIA)、放射免疫法(RIA)、表面等离子共振(SPR)、ECL、IRMA、EIA、Biacore试验、Octet分析、肽扫描、生物膜干涉技术(BLI)、平衡透析法、结合抗原沉淀法等。例如，通过使用各种标记试剂标记的野生型NGF、NGF突变体、抗NGF抗体或TrkA受体，或其亚单位来进行简单分析。同样可以使用BiacoreX(Amersham Biosciences)来测量，这是一种非处方的测量试剂盒或类似试剂盒，可根据试剂盒随附的用户手册和实验操作方法来操作。在另一些实施例中，分子间的结合活性可以采用分子间的结合亲和力来进行表征。In some embodiments, the binding activity between the molecules described in the present application (e.g., wild-type NGF or NGF mutants) and their binding partners (e.g., TrkA receptors or anti-NGF antibodies) 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), Meso Scale Discovery (MSD) electrochemiluminescence, bead-based multiple immunoassay (MIA), radioimmunoassay (RIA), surface plasmon resonance (SPR), ECL, IRMA, EIA, Biacore assay, Octet analysis, peptide scanning, biomembrane interferometry (BLI), equilibrium dialysis, antigen-binding precipitation, etc. For example, a simple analysis can be performed by using wild-type NGF, NGF mutants, anti-NGF antibodies or TrkA receptors, or their subunits labeled with various labeling agents. It can also be measured using BiacoreX (Amersham Biosciences), which is an over-the-counter measurement kit or a similar kit that can be operated according to the user manual and experimental operation method attached to the kit. In other embodiments, the binding activity between molecules can be characterized by the binding affinity between molecules.
在一些实施例中，可以使用蛋白质微阵列来测量结合活性。蛋白质微阵列可用于大规模分析本文所述的野生型NGF或NGF突变体与其受体的相互作用、功能和活性。蛋白质微阵列具有与一系列捕获蛋白(例如，TrkA受体或其亚单位)结合的支撑表面。随后将荧光标记的探针分子(例如，本文所述的野生型NGF或NGF突变体)添加到阵列中，并且与结合的捕获蛋白相互作用，释放荧光信号并通过激光扫描仪读取。In some embodiments, protein microarrays can be used to measure binding activity. Protein microarrays can be used for large-scale analysis of the interaction, function and activity of wild-type NGF or NGF mutants described herein with their receptors. The protein microarray has a support surface that is combined with a series of capture proteins (e.g., TrkA receptors or their subunits). Fluorescently labeled probe molecules (e.g., wild-type NGF or NGF mutants described herein) are then added to the array and interact with the combined capture proteins, releasing fluorescent signals and reading them by a laser scanner.
在一些实施例中，结合活性可以使用SPR(Biacore T-200)进行测量。例如，使用EDC/NHS化学法将抗人类IgG抗体耦合到CM-5传感器芯片表面。然后使用TrkA-mFc融合蛋白或抗NGF抗体作为该表面的捕获配体。将本文所述的野生型NGF、NGF突变体的一系列稀释液与捕获的配体结合，并且可以实时监测野生型NGF或NGF突变体或包含其的融合蛋白与TrkA或抗NGF抗体的结合和解离。解离常数(Kd)和解离速率常数可通过使用BIA评估软件进行动力学分析来确定。在一些实施例中，分子间的结合活性通过解离常数Kd进行表征，等于Koff/Kon，Kd值越小，代表分子之间的亲和力越强，结合活性越高。在本发明的一些实施例中，可应用Kd数值之间的比值来作为结合活性的量化指标。例如，在SPR测定实验中，应用Biacore T-200测量分子A与分子B之间的解离常数Kd值是分子C与分子B之间的解离常数Kd值的10倍，则代表分子A与分子B之间的结合活性是分子C与分子B之间的结合活性的10％。 In some embodiments, binding activity can be measured using SPR (Biacore T-200). For example, anti-human IgG antibodies are coupled to the surface of a CM-5 sensor chip using EDC/NHS chemistry. TrkA-mFc fusion protein or anti-NGF antibody is then used as a capture ligand on the surface. A series of dilutions of wild-type NGF and NGF mutants described herein are bound to the captured ligand, and the binding and dissociation of wild-type NGF or NGF mutants or fusion proteins containing them with TrkA or anti-NGF antibodies can be monitored in real time. The dissociation constant (Kd) and the dissociation rate constant can be determined by kinetic analysis using BIA evaluation software. In some embodiments, the intermolecular binding activity is characterized by the dissociation constant Kd, which is equal to Koff/Kon. The smaller the Kd value, the stronger the affinity between the molecules and the higher the binding activity. In some embodiments of the present invention, the ratio between the Kd values can be used as a quantitative indicator of binding activity. For example, in an SPR assay, if the dissociation constant Kd between molecules A and B measured by Biacore T-200 is 10 times the dissociation constant Kd between molecules C and B, it means that the binding activity between molecules A and B is 10% of the binding activity between molecules C and B.
在一些实施例中，结合活性可以使用ELISA检测方法测量。ELISA已经成为医学实验室、体外诊断产品制造商、监管机构等最常使用的实验方法。关于ELISA检测方法的发现、检测原理及其操作步骤等内容可参考文献Lequin RM.Enzyme immunoassay(EIA)/enzyme-linked immunosorbent assay(ELISA).Clin Chem.2005 Dec；51(12):2415-8，该文献通过引用将其全部并入到本申请中。在本申请的一些实施例中，所述结合活性通过结合ELISA实验的EC50值进行表征，EC50值越小，代表分子之间的亲和力越强，结合活性越高。在本申请的一些实施例中，可应用EC50数值之间的比值来作为结合活性的量化指标。例如，在ELISA检测实验中，分子A与分子B之间的EC50值是分子C与分子B之间的EC50值的10倍，则代表分子A与分子B之间的结合活性是分子C与B之间的结合活性的10％。In some embodiments, the binding activity can be measured using an ELISA assay. ELISA has become the most commonly used experimental method by medical laboratories, in vitro diagnostic product manufacturers, regulatory agencies, and the like. For the discovery of the ELISA assay, the detection principle, and its operating steps, please refer to the document Lequin RM. Enzyme immunoassay (EIA) / enzyme-linked immunosorbent assay (ELISA). Clin Chem. 2005 Dec; 51 (12): 2415-8, which is incorporated herein by reference in its entirety. In some embodiments of the present application, the binding activity is characterized by the EC50 value of the ELISA experiment. The smaller the EC50 value, the stronger the affinity between the molecules and the higher the binding activity. In some embodiments of the present application, the ratio between the EC50 values can be used as a quantitative indicator of binding activity. For example, in an ELISA test, if the EC50 value between molecules A and B is 10 times the EC50 value between molecules C and B, it means that the binding activity between molecules A and B is 10% of the binding activity between molecules C and B.
NGF的生物学活性及其检测方法Biological activity of NGF and its detection method
如本申请应用的术语，NGF的“生物学活性”可以从以下的一个或多个方面进行表征，包括但不限于：结合NGF受体(如TrkA)的能力；促进TrkA受体二聚化和/或TrkA受体自动磷酸化的能力；活化NGF受体信号途径的能力；促进细胞分化、增殖、存活、生长，以及其它在细胞生理学中的变化，包括在神经元形态、突触发生、突触功能、神经递质和/或神经肽释放及损伤后的再生(对于神经元包括外周及中枢神经元)变化的能力；促进小鼠E13.5三叉神经元存活的能力以及介导疼痛包括手术后疼痛的能力。As the term is used in this application, the "biological activity" of NGF can be characterized from one or more of the following aspects, including but not limited to: the ability to bind to NGF receptors (such as TrkA); the ability to promote TrkA receptor dimerization and/or TrkA receptor autophosphorylation; the ability to activate NGF receptor signaling pathways; the ability to promote cell differentiation, proliferation, survival, growth, and other changes in cell physiology, including changes in neuronal morphology, synaptogenesis, synaptic function, neurotransmitter and/or neuropeptide release, and regeneration after injury (for neurons including peripheral and central neurons); the ability to promote the survival of mouse E13.5 trigeminal neurons and the ability to mediate pain, including post-operative pain.
本申请所述的用于确定野生型NGF或NGF突变体生物学活性的各种方法是本领域已知的。在本申请的一些实施例中，可以通过野生型NGF或NGF突变体与TrkA受体的结合活性来表征其生物学活性，参见下文实施例5。在本申请的另一些实施例中，可通过TF-1细胞增殖试验来评估NGF的生物学活性，如CN103376248A、CN108727486A或CN114829384A中所述，其全部内容通过引用并入本文，也可参见下文实施例6。在本申请的另一些实施例中，生物学活性也可基于PC12细胞活性检测实验进行确定，参见下文实施例7。在本申请的另一些实施例中，生物学活性也可基于促进鸡胚背根神经节生长的能力来确定，如WO2017157326或CN114829384A中所述，其全部内容通过引用并入本文，也可参见下文实施例8或促进新生大鼠颈上神经节(SCG)生长的能力(参见下文实施例9)。本领域已知的任何合适的试验方案均适用于测试本文所述的NGF、NGF突变体或包含其的融合蛋白的生物学活性。 The various methods described in this application for determining the biological activity of wild-type NGF or NGF mutants are known in the art. In some embodiments of the present application, the biological activity can be characterized by the binding activity of wild-type NGF or NGF mutants to TrkA receptors, see Example 5 below. In other embodiments of the present application, the biological activity of NGF can be evaluated by TF-1 cell proliferation assay, as described in CN103376248A, CN108727486A or CN114829384A, all of which are incorporated herein by reference, and see also Example 6 below. In other embodiments of the present application, the biological activity can also be determined based on PC12 cell activity detection experiments, see Example 7 below. In other embodiments of the present application, the biological activity can also be determined based on the ability to promote the growth of chicken embryo dorsal root ganglia, as described in WO2017157326 or CN114829384A, all of which are incorporated herein by reference, and see also Example 8 below or the ability to promote the growth of neonatal rat superior cervical ganglia (SCG) (see Example 9 below). Any suitable assay protocol known in the art is suitable for testing the biological activity of NGF, NGF mutants, or fusion proteins comprising the same as described herein.
生物测定的重点是NGF的生物学活性，并将其以数据的形式进行展现。在生物测定中，例如，在敏感细胞系(例如，原代细胞培养物或对测试样品有依赖和/或响应的体外适应细胞系)或动物模型/患有NGF相关疾病的人上测试样品的活性，并将该活性(例如，细胞增殖)的结果与标准NGF制剂或对照组(例如，人野生型成熟NGF、小鼠NGF或NGFF12E)对比。NGF生物学活性的其它方面包括：(i)支持神经元存活；(ii)促进神经突生长；(iii)增强神经化学分化；(iv)促进胰腺β细胞增殖；(v)诱导先天性和/或获得性免疫；(vi)修复受损的神经细胞和/或预防损伤(例如，神经营养性角膜炎)；(vii)促进卵巢颗粒细胞的增殖和/或雌激素分泌；(viii)促进伤口愈合(例如，在糖尿病神经性病变中)；(ix)改善患有神经退行性疾病(例如，阿尔茨海默病)的受试者的空间认知、记忆和/或学习能力；(x)治疗和/或预防神经退行性病变；(xi)治疗睾丸生精小管萎缩、生精小管生精障碍和/或附睾管细胞碎片；(xii)挽救精子数量和/或活力的降低，或增加精子数量和/或活力(例如，在生精障碍中)；和/或(xiii)改善卵泡数量和/或功能的降低，或提高卵泡数量和/或功能(例如，在卵巢早衰中)。可以使用体外和/或体内试验来测量所有的这些活性，如神经元存活试验或神经突生长试验。The focus of bioassays is the biological activity of NGF and presenting it in the form of data. In bioassays, for example, the activity of a sample is tested on a sensitive cell line (e.g., a primary cell culture or an in vitro adapted cell line that is dependent and/or responsive to the test sample) or an animal model/human with an NGF-related disease, and the results of the activity (e.g., cell proliferation) are compared with a standard NGF preparation or a control group (e.g., human wild-type mature NGF, mouse NGF, or NGF F12E ). Other aspects of NGF's biological activity include: (i) supporting neuronal survival; (ii) promoting neurite outgrowth; (iii) enhancing neurochemical differentiation; (iv) promoting pancreatic beta cell proliferation; (v) inducing innate and/or acquired immunity; (vi) repairing damaged nerve cells and/or preventing damage (e.g., neurotrophic keratitis); (vii) promoting proliferation and/or estrogen secretion of ovarian granulosa cells; (viii) promoting wound healing (e.g., in diabetic neuropathy); (ix) improving the condition of patients with neurotrophic keratitis. (x) treating and/or preventing neurodegenerative lesions; (xi) treating testicular seminiferous tubule atrophy, seminiferous tubule spermatogenesis disorder and/or epididymal tubular cell fragments; (xii) rescuing the reduction of sperm count and/or motility, or increasing sperm count and/or motility (e.g., in spermatogenesis disorder); and/or (xiii) improving the reduction of follicle number and/or function, or increasing follicle 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 neuron survival assays or neurite outgrowth assays.
在本申请的一些实施例中，细胞信号试验也可用于测试本文所述的野生型NGF或NGF突变体的生物活性。各种细胞信号试验试剂盒可商购获得，例如，检测参与信号转导的酶促反应期间产生的分析物，例如ADP、AMP、UDP、GDP和生长因子，或磷酸酶试验，用于量化总的信号蛋白和磷酸化形式的信号蛋白。例如，在将细胞与本文所述的野生型NGF或NGF突变体共培养后，为了确定特定激酶是否具有活性，将细胞裂解物在放射性磷酸盐存在的条件下暴露于酶的已知底物中。通过电泳分离产物(经过或未经过免疫沉淀)，然后将凝胶暴露于X射线胶片以确定蛋白质是否包含同位素。在一些实施例中，本文所述的野生型NGF或NGF突变体在细胞上的生物活性通过免疫组织化学测定以定位信号蛋白。例如，可以使用针对信号蛋白本身或处于激活状态的信号蛋白的抗体。这些抗体具有识别表位，包括磷酸盐或其它激活构象。在一些实施例中，可通过将荧光蛋白基因，例如，绿色荧光蛋白(GFP)，整合到编码所研究蛋白质的基因载体中，从而跟踪特定信号蛋白的移动(例如，信号分子的核易位)。在一些实施例中，本文所述野生型NGF或NGF突变体在细胞上的生物学活性通过western blot检测。例如，所有酪氨酸磷酸化蛋白(或其它磷酸化氨基酸，例如丝氨酸或苏氨酸)都可以用抗磷酸化酪氨酸抗体(或抗其它磷酸化氨基酸的抗体)在按时 间顺序刺激后获得的细胞裂解液的Western blot中检测。在一些实施例中，本文所述野生型NGF或NGF突变体在细胞上的生物学活性可通过免疫沉淀测定。例如，针对特定信号蛋白或所有酪氨酸磷酸化蛋白的一抗与微珠交联。与本文所述的野生型NGF或NGF突变体孵育后的细胞在含有蛋白酶抑制剂的缓冲液中裂解，然后与抗体包被的微珠孵育。使用SDS电泳分离蛋白质，然后通过所述的Western blot步骤鉴定蛋白质。在一些实施例中，还可使用谷胱甘肽S-转移酶(GST)结合或“pull-down”分析，以确定蛋白质-蛋白质(例如，信号蛋白)的直接相互作用。In some embodiments of the present application, cell signaling assays can also be used to test the biological activity of wild-type NGF or NGF mutants described herein. Various cell signaling assay kits are commercially available, for example, to detect analytes produced during enzymatic reactions involved in signal transduction, such as ADP, AMP, UDP, GDP and growth factors, or phosphatase assays for quantifying total signal proteins and phosphorylated forms of signal proteins. For example, after cells are co-cultured with wild-type NGF or NGF mutants described herein, in order to determine whether a specific kinase is active, the cell lysate is exposed to a known substrate of the enzyme in the presence of radioactive phosphate. 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 biological activity of wild-type NGF or NGF mutants described herein on cells is determined by immunohistochemistry to locate signal proteins. For example, antibodies to signal proteins themselves or in an activated state can be used. These antibodies have recognition epitopes, including phosphates or other activation conformations. In some embodiments, the movement of a specific signal protein (e.g., nuclear translocation of a signal molecule) can be tracked by integrating a fluorescent protein gene, such as green fluorescent protein (GFP), into a gene vector encoding the protein under study. In some embodiments, the biological activity of the wild-type NGF or NGF mutant described herein on cells is detected by western blot. For example, all tyrosine phosphorylated proteins (or other phosphorylated amino acids, such as serine or threonine) can be detected by anti-phosphotyrosine antibodies (or antibodies against other phosphorylated amino acids) on a regular basis. The expression of NGF in the wild-type NGF or NGF mutants described herein can be detected in Western blot of the cell lysate obtained after sequential stimulation. In some embodiments, the biological activity of the wild-type NGF or NGF mutants described herein on cells can be determined by immunoprecipitation. For example, a primary antibody against a specific signaling protein or all tyrosine phosphorylated proteins is cross-linked to the microbeads. Cells incubated with the wild-type NGF or NGF mutants described herein are lysed in a buffer containing protease inhibitors and then incubated with antibody-coated microbeads. Proteins are separated using SDS electrophoresis and then identified by the Western blot step. In some embodiments, glutathione S-transferase (GST) binding or "pull-down" analysis can also be used to determine direct protein-protein (e.g., signaling proteins) interactions.
在本申请的一些实施例中，可以通过测量RAS/ERK1/2信号来反映NGF在促进细胞生长方面的生物活性，例如，通过使用本领域已知的任何合适的方法使ERK1/2磷酸化。例如，测量ERK1/2磷酸化可使用该分子磷酸化状态的特异性抗体(可选地结合流式细胞术分析)。例如，将鸡胚胎背根神经节(DRGs)或TF-1细胞与本文所述的野生型NGF或NGF突变体在37℃下孵育。孵育后，立即固定细胞以保持磷酸化状态和渗透性。用抗磷酸化ERK1/2的抗体对细胞进行染色，例如，使用Alexa488偶联的抗ERK1/2pT202/pY204(BD Biosciences)，并通过流式细胞术进行分析。PI 3-激酶信号也可以使用本领域已知的任何适当的方法来测量，以反映NGF的生物活性。例如，可以使用对磷酸化S6核糖体蛋白具有特异性的抗体来测量PI 3-激酶信号转导(可选择结合流式细胞术分析)。In some embodiments of the present application, the biological activity of NGF in promoting cell growth can be reflected by measuring RAS/ERK1/2 signals, for example, by phosphorylating ERK1/2 using any suitable method known in the art. For example, measuring ERK1/2 phosphorylation can use specific antibodies to the phosphorylation state of the molecule (optionally combined with flow cytometry analysis). For example, chicken embryo dorsal root ganglia (DRGs) or TF-1 cells are incubated with wild-type NGF or NGF mutants described herein at 37°C. After incubation, the cells are immediately fixed to maintain phosphorylation status and permeability. The cells are stained with an antibody against phosphorylated ERK1/2, for example, using Alexa488-coupled anti-ERK1/2pT202/pY204 (BD Biosciences), and analyzed by flow cytometry. PI 3-kinase signals can also be measured using any suitable method known in the art to reflect the biological activity of NGF. For example, PI 3-kinase signaling can be measured using an antibody specific for phosphorylated S6 ribosomal protein (optionally in combination with flow cytometry analysis).
本文所述的野生型NGF或NGF突变体的生物学活性也可通过体内或体外实验反映，例如，通过测量指示细胞的增殖，通过测量信号转导的诱导或抑制，通过测量组织体积和/或重量等。The biological activity of the wild-type NGF or NGF mutants described herein can also be reflected by in vivo or in vitro experiments, for example, by measuring the proliferation of indicator cells, by measuring the induction or inhibition of signal transduction, by measuring tissue volume and/or weight, etc.
例如，在TF-1细胞增殖试验中，在96孔板中制备样品(例如，NGF突变体)和对照组(例如，人野生型成熟NGF或NGFF12E)的系列稀释液，然后将TF-1细胞添加到每个孔中，并在37℃、5％CO2的潮湿培养箱中培养。培养几天后(例如，3天)，将MTS溶液添加到细胞悬浮液的每个孔中，在37℃、5％CO2的条件下培养3小时。随后，在分光光度计中测量490nm和650nm下的吸光度，以说明NGF、NGF突变体对TF-1细胞增殖情况的影响。可以根据对照样本对数据进行标准化处理。示例方法参见实施例6。For example, in the TF-1 cell proliferation assay, a serial dilution of a sample (e.g., NGF mutant) and a control group (e.g., human wild-type mature NGF or NGF F12E ) is prepared in a 96-well plate, and then TF-1 cells are added to each well and cultured in a humidified incubator at 37°C and 5% CO2 . After several days of culture (e.g., 3 days), an MTS solution is added to each well of the cell suspension and cultured for 3 hours at 37°C and 5% CO2 . Subsequently, the absorbance at 490nm and 650nm is measured in a spectrophotometer to illustrate the effects of NGF and NGF mutants on the proliferation of TF-1 cells. The data can be standardized based on the control sample. See Example 6 for an example method.
例如，在SCG体内生长试验中，样品(例如，NGF突变体)和对照组(例如，PBS、人野生型成熟NGF或NGFF12E)可通过单次注射或多次注射的方式皮下注射到新 生大鼠颈部。注射几天后，处死这些大鼠进行SCG分离。SCG可以称重并记录形态，以研究野生型NGF或NGF突变体在促进SCG体内生长中的生物活性。示例方法参见实施例9。For example, in an SCG in vivo growth assay, a sample (e.g., NGF mutant) and a control group (e.g., PBS, human wild-type mature NGF, or NGF F12E ) can be injected subcutaneously into a neonate by single or multiple injections. The rats were injected with NGF at the neck of the rats. A few days after the injection, the rats were sacrificed for SCG isolation. SCG can be weighed and the morphology recorded to study the biological activity of wild-type NGF or NGF mutants in promoting the growth of SCG in vivo. See Example 9 for an example method.
例如，在鸡胚背根神经节生长试验中，可将鸡胚背根神经节(例如，8日龄)添加到含有不同浓度的样品(例如，NGF突变体)或对照组(例如，PBS、鼠NGF、人野生型成熟NGF、NGFF12E)的培养基中，并在5％CO2和37℃的饱和湿度培养箱中培养，例如，24小时。监测背根神经节的生长情况，其可以反映野生型NGF或NGF突变体在促进背根神经节生长中的生物学活性。如果实验中包括NGF标准品，也可以计算样品的特定生物学活性，以AU/mg表示。待测样品的比活度(AU/mg)＝对照品的比活度(AU/ml)×[样品的预稀释系数×该稀释点下相应对照品的比活度(AU/ml)/对照品的实际比活度(AU/ml)]。示例方法参见WO2017157326中的实施例5。For example, in a chick embryo dorsal root ganglion growth assay, chick embryo dorsal root ganglia (e.g., 8-day-old) can be added to samples containing different concentrations (e.g., NGF mutants) or control groups (e.g., PBS, Mouse NGF, human wild-type mature NGF, NGF F12E ) in a culture medium and cultured in a saturated humidity incubator at 5% CO2 and 37°C, for example, for 24 hours. Monitor the growth of dorsal root ganglia, which can reflect the biological activity of wild-type NGF or NGF mutants in promoting the growth of dorsal root ganglia. If the NGF standard is included in the experiment, the specific biological activity of the sample can also be calculated and expressed in AU/mg. The specific activity of the sample to be tested (AU/mg) = the specific activity of the control (AU/ml) × [pre-dilution factor of the sample × the specific activity of the corresponding control at this dilution point (AU/ml) / the actual specific activity of the control (AU/ml)]. For an example method, see Example 5 in WO2017157326.
在本申请的一些实施例中，优选采用与TrkA受体的结合活性来表征其生物学活性，例如，当表述“NGF突变体具有生物学活性”时，可意为该NGF突变体能够与TrkA受体结合。在一些实施例中，NGF突变体的生物学活性应用其与TrkA受体的结合活性表征，例如，当表述“NGF突变体与TrkA受体的结合活性是野生型NGF与TrkA受体结合活性的50％时”，可意为NGF突变体的生物学活性是野生型NGF生物学活性的50％。In some embodiments of the present application, it is preferred to use the binding activity to the TrkA receptor to characterize its biological activity. For example, when it is stated that "the NGF mutant has biological activity", it may mean that the NGF mutant is able to bind to the TrkA receptor. In some embodiments, the biological activity of the NGF mutant is characterized by its binding activity to the TrkA receptor. For example, when it is stated that "the binding activity of the NGF mutant to the TrkA receptor is 50% of the binding activity of the wild-type NGF to the TrkA receptor", it may mean that the biological activity of the NGF mutant is 50% of the biological activity of the wild-type NGF.
NGF中可突变位点的筛选和/或鉴定方法Method for screening and/or identifying mutable sites in NGF
本申请提供了一种筛选和/或鉴定NGF中可突变位点的方法，所述可突变位点上的氨基酸突变会影响NGF突变体与一种或多种抗NGF抗体的结合，但仍能使该NGF突变体具有生物学活性。在一些实施例中，所述可突变位点上的氨基酸突变会影响NGF突变体与抗NGF抗体的结合，是指包含上述氨基酸突变的NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合。The present application provides a method for screening and/or identifying a mutable site in NGF, wherein the amino acid mutation at the mutable site affects the binding of the NGF mutant to one or more anti-NGF antibodies, but still enables the NGF mutant to have biological activity. In some embodiments, the amino acid mutation at the mutable site affects the binding of the NGF mutant to the anti-NGF antibody, which means that the NGF mutant containing the above-mentioned amino acid mutation has a weakened binding to the anti-NGF antibody or basically does not bind to the anti-NGF antibody.
在一些实施例中，所述的筛选和/或鉴定NGF中可突变位点的方法包括如下步骤：In some embodiments, the method for screening and/or identifying mutable sites in NGF comprises the following steps:
(i)获得一种或多种抗NGF抗体的三维结构； (i) obtaining the three-dimensional structure of one or more anti-NGF antibodies;
(ii)将成熟NGF的晶体结构与上述获得的抗NGF抗体的三维结构分别进行对接，以获得一种或多种抗NGF抗体与NGF的结合构象，其中，成熟的NGF晶体结构如PDB ID：4EDW所示；(ii) docking the crystal structure of mature NGF with the three-dimensional structure of the anti-NGF antibody obtained above, respectively, to obtain one or more binding conformations of the anti-NGF antibody and NGF, wherein the crystal structure of mature NGF is shown in PDB ID: 4EDW;
(iii)将步骤(ii)中获得的结合构象和成熟NGF与TrkA受体的结合构象分别进行比对；(iii) aligning the binding conformation obtained in step (ii) and the binding conformation of mature NGF and TrkA receptor respectively;
(iv)基于步骤(iii)中的比对结果，获得所述NGF中可突变位点。(iv) Based on the comparison results in step (iii), obtain the mutable sites in the NGF.
在一些实施例中，对于上述方法中的步骤(i)，本领域的技术人员可以应用现有技术中已知的任何方法来获得抗NGF抗体的三维结构，包括但不限于应用实验方法，例如X-晶体衍射和核磁共振(NMR)、冷冻电镜技术等，或者应用计算的方法根据蛋白质序列预测其三级结构。在一些实施例中，蛋白质三级结构预测的方法包括但不限于：同源建模法(Homology Modeling)、归范法(Threading)以及从头预测法(Ab initio)等。在一些实施例中，其中所述同源建模的方法包括，但不限于SWISS-MODEL、Modeller或Phyre2。在一些实施例中，本申请所述的抗NGF抗体的三维结构应用同源建模方法获得。在一些优选实施例中，本申请所述的抗NGF抗体的三维结构通过Modeller同源建模方法获得。In some embodiments, for step (i) in the above method, a person skilled in the art may apply any method known in the art to obtain the three-dimensional structure of the anti-NGF antibody, including but not limited to the application of experimental methods, such as X-crystal diffraction and nuclear magnetic resonance (NMR), cryo-electron microscopy, etc., or the application of computational methods to predict its tertiary structure based on the protein sequence. In some embodiments, methods for predicting the tertiary structure of proteins include but are not limited to: homology modeling, threading, and ab initio, etc. In some embodiments, the homology modeling method includes, but is not limited to SWISS-MODEL, Modeller, or Phyre2. In some embodiments, the three-dimensional structure of the anti-NGF antibody described in the present application is obtained by homology modeling. In some preferred embodiments, the three-dimensional structure of the anti-NGF antibody described in the present application is obtained by the Modeller homology modeling method.
在一些实施例中，对于上述方法中的步骤(i)所述的获得抗NGF抗体的三维结构，其可以为任一结合NGF靶点的抗体或抗原结合片段，例如，包括，但不限于如下抗体中的一种或多种：Tanezumab(辉瑞)、Fasinumab(再生元)、Fulranumab(强生)、MEDI-578(阿斯利康)、AK-115(康方生物)、SSS-40(三生国健)以及STC001(舒泰神)。上述抗NGF抗体的VH和VL的氨基酸序列如表1中所示。In some embodiments, the three-dimensional structure of the anti-NGF antibody obtained in step (i) of the above method can be any antibody or antigen-binding fragment that binds to the NGF target, for example, including, but not limited to, one or more of the following antibodies: Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Biopharma), SSS-40 (Sunshine Guojian) and STC001 (Sultaishen). The amino acid sequences of VH and VL of the above anti-NGF antibodies are shown in Table 1.
在一些实施例中，对于上述方法中的步骤(ii)，本领域技术人员可以应用现有技术中已知的任何方法将成熟NGF晶体结构与上述获得的抗NGF抗体三维结构进行对接，以获得抗NGF抗体与NGF的结合构象。在一些优选实施例中，成熟NGF的晶体结构如PDB ID：4EDW所示。In some embodiments, for step (ii) in the above method, a person skilled in the art can use any method known in the prior art to dock the mature NGF crystal structure with the above-obtained three-dimensional structure of the anti-NGF antibody to obtain the binding conformation of the anti-NGF antibody and NGF. In some preferred embodiments, the crystal structure of mature NGF is shown in PDB ID: 4EDW.
在一些优选实施例中，应用Discovery studio ZDOCK分子对接技术将成熟NGF晶体结构与上述获得的抗NGF抗体三维结构进行分子对接，以获得抗NGF抗体与NGF的结合构象。 In some preferred embodiments, the Discovery studio ZDOCK molecular docking technology is used to molecularly dock the mature NGF crystal structure with the three-dimensional structure of the anti-NGF antibody obtained above to obtain the binding conformation of the anti-NGF antibody and NGF.
在进一步的一些优选实施例中，应用Discovery studio ZDOCK进行分子对接时，根据评分函数值由高到低，显示结合构象的预测结果，其它参数软件自动默认。在进行分子对接时，本领域技术人员可以通过实验研究和查阅现有资料等，确定受体、配体的氨基酸结合域。在一些优选实施例中，选择Discovery studio ZDOCK中评分函数值最高的结合构象作为抗NGF抗体与NGF的结合构象。In some further preferred embodiments, when using Discovery studio ZDOCK for molecular docking, the predicted results of the binding conformation are displayed according to the scoring function value from high to low, and the other parameters are automatically defaulted by the software. When performing molecular docking, those skilled in the art can determine the amino acid binding domains of receptors and ligands through experimental research and consulting existing data. In some preferred embodiments, the binding conformation with the highest scoring function value in Discovery studio ZDOCK is selected as the binding conformation of the anti-NGF antibody and NGF.
在一些实施例中，上述方法中的步骤(iii)中所述的NGF与TrkA受体的结合构象如PDB ID：1WWW所示。在一些实施例中，对于上述方法中的步骤(iii)，将获得的NGF与抗NGF抗体的结合构象和成熟NGF与TrkA受体的结合构象通过叠合后进行比对。In some embodiments, the binding conformation of NGF and TrkA receptor described in step (iii) of the above method is shown in PDB ID: 1WWW. In some embodiments, for step (iii) of the above method, the obtained binding conformation of NGF and anti-NGF antibody and the binding conformation of mature NGF and TrkA receptor are compared by superposition.
在一些实施例中，对于上述方法中的步骤(iv)，通过比对获得的NGF中的可突变位点是指NGF中位于与抗NGF抗体结合界面，但不位于与TrkA受体结合界面的氨基酸位点。在一些实施例中，如果涉及多种抗NGF抗体，则所述可突变位点为针对各种抗NGF抗体所获得的多组可突变位点之间的交集或并集。In some embodiments, for step (iv) in the above method, the mutable sites in NGF obtained by comparison refer to amino acid sites in NGF that are located at the binding interface with the anti-NGF antibody but not at the binding interface with the TrkA receptor. In some embodiments, if multiple anti-NGF antibodies are involved, the mutable sites are the intersection or union of multiple groups of mutable sites obtained for various anti-NGF antibodies.
NGF中的可突变位点Mutable sites in NGF
本申请一方面提供了NGF中的可突变位点，在所述可突变位点上进行氨基酸突变会影响NGF突变体与一种或多种抗NGF抗体的结合，但仍能使NGF突变体具有生物学活性。在一些实施例中，所述氨基酸突变会影响NGF突变体与一种或多种抗NGF抗体的结合是指包含上述氨基酸突变的NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合。On the one hand, the present application provides a mutable site in NGF, and amino acid mutations at the mutable site will affect the binding of the NGF mutant to one or more anti-NGF antibodies, but still enable the NGF mutant to have biological activity. In some embodiments, the amino acid mutation affects the binding of the NGF mutant to one or more anti-NGF antibodies, which means that the binding of the NGF mutant containing the above amino acid mutation to the anti-NGF antibody is weakened or substantially does not bind to the anti-NGF antibody.
在一些实施例中，本申请所述的NGF中的可突变位点可以通过如前所述的NGF可突变位点的筛选和/或鉴定方法获得。In some embodiments, the mutable sites in NGF described herein can be obtained by the screening and/or identification methods of the mutable sites in NGF as described above.
在一些实施例中，所述的NGF中可突变位点为针对某个特定的抗NGF抗体的一组可突变位点，即在该可突变位点上进行氨基酸突变会使NGF突变体与该特定抗NGF抗体结合减弱或基本不结合。在一些实施例中，如果涉及两种或两种以上的抗NGF抗体，针对每一个抗NGF抗体，通过实施本申请中的方法均可分别获得针对每一个抗NGF抗体的一组可突变位点，例如，本申请所述的NGF中的可突变位点可以为针对两种或两种以上抗NGF抗体所分别获得的两组或两组以上可突变位点之间的交集或 者为针对两种或两种以上抗NGF抗体分别所获得的两组或两组以上可突变位点之间的并集。In some embodiments, the mutable sites in the NGF are a group of mutable sites for a specific anti-NGF antibody, that is, amino acid mutations at the mutable sites will weaken or substantially eliminate the binding of the NGF mutant to the specific anti-NGF antibody. In some embodiments, if two or more anti-NGF antibodies are involved, for each anti-NGF antibody, a group of mutable sites for each anti-NGF antibody can be obtained by implementing the method of the present application. For example, the mutable sites in the NGF described in the present application can be the intersection of two or more groups of mutable sites obtained for two or more anti-NGF antibodies, or The latter is the union of two or more groups of mutable sites obtained for two or more anti-NGF antibodies respectively.
在一些实施例中，本申请所述的NGF中的可突变位点为分别针对如下任一种抗NGF抗体：Tanezumab(辉瑞)、Fasinumab(再生元)、Fulranumab(强生)、MEDI-578(阿斯利康)、AK-115(康方生物)、SSS-40(三生国健)以及STC001(舒泰神)所获得的一组可突变位点。In some embodiments, the mutable sites in NGF described in the present application are a group of mutable sites obtained for any one of the following anti-NGF antibodies: Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Biopharma), SSS-40 (Sunshine Guojian) and STC001 (Shortacion).
在一些实施例中，本申请所述的NGF中的可突变位点为针对选自如下两种或两种以上的抗NGF抗体：Tanezumab(辉瑞)、Fasinumab(再生元)、Fulranumab(强生)、MEDI-578(阿斯利康)、AK-115(康方生物)、SSS-40(三生国健)以及STC001(舒泰神)所分别获得的各组可突变位点的交集。In some embodiments, the mutable sites in NGF described in the present application are the intersection of the groups of mutable sites obtained for two or more anti-NGF antibodies selected from: Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Biopharma), SSS-40 (Sunshine Guojian) and STC001 (Shortacion).
例如，在一些实施例中，本申请所述的NGF中的可突变位点为针对Tanezumab(辉瑞)、Fasinumab(再生元)、MEDI-578(阿斯利康)、AK-115(康方生物)以及STC001(舒泰神)所分别获得的各组可突变位点中均包含的位点。For example, in some embodiments, the mutable sites in NGF described in the present application are sites included in each group of mutable sites obtained for Tanezumab (Pfizer), Fasinumab (Regeneron), MEDI-578 (AstraZeneca), AK-115 (Akeso Biopharma), and STC001 (Sultaisin), respectively.
在一些实施例中，本申请所述的NGF中的可突变位点为针对选自如下两种或两种以上的抗NGF抗体：Tanezumab(辉瑞)、Fasinumab(再生元)、Fulranumab(强生)、MEDI-578(阿斯利康)、AK-115(康方生物)、SSS-40(三生国健)以及STC001(舒泰神)所分别获得的各组可突变位点的并集。In some embodiments, the mutable sites in NGF described in the present application are the union of each group of mutable sites obtained for two or more anti-NGF antibodies selected from the following: Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Biopharma), SSS-40 (Sunshine Guojian) and STC001 (Shortacion).
例如，在一些实施例中，本申请所述的NGF中的可突变位点为针对Tanezumab(辉瑞)、Fasinumab(再生元)、MEDI-578(阿斯利康)、AK-115(康方生物)以及STC001(舒泰神)所分别获得的各组可突变位点一起组成的集合，包含各组可突变位点中的所有位点。For example, in some embodiments, the mutable sites in NGF described in the present application are a set consisting of the groups of mutable sites obtained for Tanezumab (Pfizer), Fasinumab (Regeneron), MEDI-578 (AstraZeneca), AK-115 (Akeso Biopharma) and STC001 (Shortasen), respectively, including all sites in each group of mutable sites.
在一些实施例中，本申请所述的NGF中的可突变位点可选自如下位点中的一个或多个：相对于人野生型成熟NGF氨基酸序列的I31、K32、G33、K34、D93、W21、G23、D24、K50、Y52、T83、H84、F86、R100、R103、D16、S17、S19、T56、R59和R69位点。In some embodiments, the mutable sites in the NGF described herein can be selected from one or more of the following sites: I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的野生型成熟NGF为人野生型成熟NGF。在一些实施例中，本申请所述的人野生型成熟NGF的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。 In some embodiments, the wild-type mature NGF described herein is human wild-type mature NGF. In some embodiments, the amino acid sequence of human wild-type mature NGF described herein is shown in SEQ ID NO: 1 or SEQ ID NO: 2.
NGF突变体库的构建方法Method for constructing NGF mutant library
本申请提供了一种构建NGF突变体库的方法，所述突变体库中包含的NGF突变体中具有一个或多个可突变位点，在该一个或多个可突变位点上进行氨基酸突变会影响NGF与一种或多种抗NGF抗体的结合，但仍能使NGF突变体具有生物学活性。在一些实施例中，所述在一个或多个可突变位点上进行氨基酸突变会影响NGF突变体与抗NGF抗体的结合，是指在该位点上的氨基酸突变会导致NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合。The present application provides a method for constructing an NGF mutant library, wherein the NGF mutants contained in the mutant library have one or more mutable sites, and amino acid mutations at the one or more mutable sites will affect the binding of NGF to one or more anti-NGF antibodies, but still enable the NGF mutants to have biological activity. In some embodiments, the amino acid mutations at one or more mutable sites will affect the binding of the NGF mutant to the anti-NGF antibody, which means that the amino acid mutations at the sites will cause the NGF mutant to weaken the binding to the anti-NGF antibody or substantially not bind to the anti-NGF antibody.
在一些实施例中，所述构建NGF突变体库的方法包括如下步骤：以人野生型成熟NGF为模板，在NGF中可突变位点的一个或多个位点上进行氨基酸突变，产生一系列的NGF突变体，从而获得NGF突变体库。在一些实施例中，所述人野生型成熟NGF的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。In some embodiments, the method for constructing the NGF mutant library comprises the following steps: using human wild-type mature NGF as a template, performing amino acid mutations at one or more sites of the mutable sites in NGF to generate a series of NGF mutants, thereby obtaining the NGF mutant library. In some embodiments, the amino acid sequence of the human wild-type mature NGF is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
在一些实施例中，所述的NGF中可突变位点通过如前所述方法筛选和/或鉴定获得。在一些实施例中，所述的NGF中可突变位点选自如下位点中的一个或多个：相对于人野生型成熟NGF氨基酸序列的I31、K32、G33、K34、D93、W21、G23、D24、K50、Y52、T83、H84、F86、R100、R103、D16、S17、S19、T56、R59和R69位点。In some embodiments, the mutable sites in the NGF are obtained by screening and/or identification as described above. In some embodiments, the mutable sites in the NGF are selected from one or more of the following sites: I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 sites relative to the amino acid sequence of human wild-type mature NGF.
NGF突变体库NGF mutant library
本申请提供了一种NGF突变体库，其包含与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，同时具有生物学活性的NGF突变体。在一些实施例中，所述NGF突变体库通过如前所述的NGF突变体库的构建方法获得。The present application provides an NGF mutant library, which comprises NGF mutants that have reduced binding to or substantially no binding to anti-NGF antibodies and have biological activity. In some embodiments, the NGF mutant library is obtained by the method for constructing the NGF mutant library as described above.
在一些具体实施例中，所述NGF突变体库中的NGF突变体包含相对于人野生型成熟NGF的I31、K32、G33、K34、D93、W21、G23、D24、K50、Y52、T83、H84、F86、R100、R103、D16、S17、S19、T56、R59和R69中的一个或多个位点上的氨基酸突变。In some specific embodiments, the NGF mutants in the NGF mutant library contain amino acid mutations at one or more of I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 relative to human wild-type mature NGF.
在一些实施例中，所述NGF突变体库中的NGF突变体进一步包含相对于人野生型成熟NGF的突变F12E。In some embodiments, the NGF mutants in the NGF mutant library further comprise a mutation F12E relative to human wild-type mature NGF.
在一些实施例中，所述人野生型成熟NGF的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。 In some embodiments, the amino acid sequence of the human wild-type mature NGF is shown as SEQ ID NO:1 or SEQ ID NO:2.
NGF突变体筛选或鉴定方法Method for screening or identifying NGF mutants
本申请的另一方面还提供了一种NGF突变体的筛选或鉴定方法，其中所述NGF突变体与一种或多种抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，同时仍具有生物学活性。Another aspect of the present application provides a method for screening or identifying NGF mutants, wherein the NGF mutants have weakened binding to one or more anti-NGF antibodies or substantially no binding to anti-NGF antibodies while still having biological activity.
在一些实施例中，本申请所述的NGF突变体的筛选方法，包括如下步骤：In some embodiments, the method for screening NGF mutants described in the present application comprises the following steps:
(i)获得一个或多个NGF突变体；(i) obtaining one or more NGF mutants;
(ii)从步骤(i)中筛选或鉴定具有生物学活性，并且与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的一系列NGF突变体。(ii) Screening or identifying a series of NGF mutants having biological activity and having reduced binding to or substantially no binding to anti-NGF antibodies from step (i).
在一些实施例中，对于上述筛选方法中的步骤(ii)，采用如下方法获得与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，且仍具有生物学活性的一系列NGF突变体：(1)首先筛选或鉴定具有生物学活性的NGF突变体，再从中筛选或鉴定与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体。(2)首先筛选或鉴定与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体，再从中筛选或鉴定具有生物学活性的NGF突变体。(3)分别筛选或鉴定具有生物学活性的NGF突变体，以及与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体，取二者的交集。In some embodiments, for step (ii) in the above screening method, the following method is used to obtain a series of NGF mutants that have reduced binding to or substantially no binding to anti-NGF antibodies and still have biological activity: (1) First screen or identify NGF mutants with biological activity, and then screen or identify NGF mutants with reduced binding to or substantially no binding to anti-NGF antibodies. (2) First screen or identify NGF mutants with reduced binding to or substantially no binding to anti-NGF antibodies, and then screen or identify NGF mutants with biological activity. (3) Separately screen or identify NGF mutants with biological activity and NGF mutants with reduced binding to or substantially no binding to anti-NGF antibodies, and take the intersection of the two.
在一些实施例中，对于上述筛选方法中的步骤(i)中的NGF突变体是从NGF突变体库获得或者通过理性设计获得，例如，所述NGF突变体库可以通过如前所述的NGF突变体库的构建方法获得。在一些实施例中，所述的NGF突变体库选自如前所述的NGF突变体库。In some embodiments, the NGF mutants in step (i) of the above screening method are obtained from an NGF mutant library or by rational design. For example, the NGF mutant library can be obtained by the construction method of the NGF mutant library as described above. In some embodiments, the NGF mutant library is selected from the NGF mutant library as described above.
在一些实施例中，对于上述筛选方法步骤(i)中的NGF突变体通过表面展示技术进行展示，包括原核展示文库技术和真核展示文库技术，进一步地，可以选择通过酵母表面展示技术、噬菌体表面展示技术、哺乳动物细胞表面展示进术、细菌表面展示技术或杆状病毒表面展示技术进行展示。在一些实施例中，上述筛选方法步骤(i)中的NGF突变体库通过酵母表面展示系统进行展示。例如，本领域已知多种方法用于产生酵母展示文库以及筛选这些文库来获得所需结合特性的重组蛋白分子。这些方法例如在Cherf GM,Cochran JR.Applications of Yeast Surface Display for Protein Engineering.Methods Mol Biol.2015；1319:155-75；Shibasaki S,Maeda H,Ueda M.Molecular display  technology using yeast--arming technology.Anal Sci.2009 Jan；25(1):41-9；和Shenoy A,Barb AW.Recent Advances Toward Engineering Glycoproteins Using Modified Yeast Display Platforms.Methods Mol Biol.2022；2370:185-205等中进行了描述。In some embodiments, the NGF mutants in step (i) of the above screening method are displayed by surface display technology, including prokaryotic display library technology and eukaryotic display library technology. Further, yeast surface display technology, phage surface display technology, mammalian cell surface display technology, bacterial surface display technology or baculovirus surface display technology can be selected for display. In some embodiments, the NGF mutant library in step (i) of the above screening method is displayed by a yeast surface display system. For example, a variety of methods are known in the art for generating yeast display libraries and screening these libraries to obtain recombinant protein molecules with desired binding properties. These methods are described, for example, in Cherf GM, Cochran JR. Applications of Yeast Surface Display for Protein Engineering. Methods Mol Biol. 2015; 1319: 155-75; Shibasaki S, Maeda H, Ueda M. Molecular display technology using yeast--arming technology. Anal Sci. 2009 Jan; 25(1): 41-9; and Shenoy A, Barb AW. Recent Advances Toward Engineering Glycoproteins Using Modified Yeast Display Platforms. Methods Mol Biol. 2022; 2370: 185-205, etc.
在一些实施例中，对于上述鉴定方法中的步骤(ii)，可以通过本申请所述的任一种结合活性检测方法来筛选或鉴定NGF与抗NGF抗体的结合活性，确认该突变体与抗NGF抗体的结合减弱或基本不结合。在一些实施例中，上述筛选方法步骤(ii)中应用MACS、FACS和/或ELISA来筛选与抗NGF抗体结合减弱或基本不结合的NGF突变体。In some embodiments, for step (ii) in the above identification method, the binding activity of NGF and anti-NGF antibody can be screened or identified by any binding activity detection method described in the present application to confirm that the binding of the mutant to the anti-NGF antibody is weakened or substantially non-binding. In some embodiments, in step (ii) of the above screening method, MACS, FACS and/or ELISA are used to screen NGF mutants that have weakened or substantially non-binding binding to the anti-NGF antibody.
在一些实施例中，对于上述鉴定方法中的步骤(ii)，可以通过本申请所述的任一种生物学活性检测方法来检测NGF突变体的生物学活性，确认该NGF突变体仍具有生物学活性。在一些实施例中，上述筛选方法中的步骤(ii)中，可应用MACS、FACS和/或生物学活性测定来筛选或鉴定具有生物学活性的NGF突变体。In some embodiments, for step (ii) in the above identification method, the biological activity of the NGF mutant can be detected by any biological activity detection method described in the present application to confirm that the NGF mutant still has biological activity. In some embodiments, in step (ii) in the above screening method, MACS, FACS and/or biological activity assays can be used to screen or identify NGF mutants with biological activity.
NGF突变体NGF mutants
本申请另一方面提供了NGF突变体，所述NGF突变体与一种或多种抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，同时具有生物学活性。在一些实施例中，所述NGF突变体通过如前所述的NGF突变体筛选方法获得。在一些实施例中，所述NGF突变体通过如前所述的NGF突变体鉴定方法进行鉴定。在一些实施例中，所述NGF突变体是从如前所述的NGF突变体库中筛选获得的。On the other hand, the present application provides an NGF mutant, which has a weakened binding to one or more anti-NGF antibodies or substantially no binding to the anti-NGF antibodies, while having biological activity. In some embodiments, the NGF mutant is obtained by the NGF mutant screening method as described above. In some embodiments, the NGF mutant is identified by the NGF mutant identification method as described above. In some embodiments, the NGF mutant is obtained by screening from the NGF mutant library as described above.
在一些实施例中，本申请所述的NGF突变体包含如前所述的一个或多个可突变位点上的氨基酸突变，其中所述氨基酸突变会影响其与抗NGF抗体结合，但仍能使NGF突变体具有生物学活性。在一些实施例中，所述氨基酸突变会影响其与抗NGF抗体结合是指该氨基酸突变导致NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合。In some embodiments, the NGF mutant described in the present application comprises an amino acid mutation at one or more mutable sites as described above, wherein the amino acid mutation affects its binding to an anti-NGF antibody, but still enables the NGF mutant to have biological activity. In some embodiments, the amino acid mutation affects its binding to an anti-NGF antibody, which means that the amino acid mutation causes the NGF mutant to weaken its binding to the anti-NGF antibody or substantially not bind to the anti-NGF antibody.
NGF突变体可包括在NGF分子中(例如，相对于人野生型成熟NGF)的一个或多个氨基酸位点的突变。在一些实施例中，NGF突变体包括在NGF中的一个或多个氨基酸位点的氨基酸取代。在一些实施例中，NGF突变体包括在NGF中的一个或多个氨基酸位点的氨基酸缺失或插入。在一些实施例中，NGF突变体包括在NGF中一个或多个氨基酸的修饰。 NGF mutants may include mutations at one or more amino acid sites in the NGF molecule (e.g., relative to human wild-type mature NGF). In some embodiments, NGF mutants include amino acid substitutions at one or more amino acid sites in NGF. In some embodiments, NGF mutants include amino acid deletions or insertions at one or more amino acid sites in NGF. In some embodiments, NGF mutants include modifications of one or more amino acids in NGF.
在一些实施例中，与野生型NGF(例如人野生型成熟NGF)相比，本申请所述的NGF突变体具有至少一个氨基酸突变，例如氨基酸的取代、缺失、插入、修饰，或者包含上述突变形式的任意组合。In some embodiments, compared with wild-type NGF (eg, human wild-type mature NGF), the NGF mutants described herein have at least one amino acid mutation, such as amino acid substitution, deletion, insertion, modification, or any combination thereof.
在一些实施例中，NGF突变体还可以包含一个或多个保守的氨基酸取代。“保守取代”是指应用另一种氨基酸进行取代，所述另一氨基酸与被取代氨基酸带有相同的净电荷和大致相同的大小和形状。当侧链上的碳原子和杂原子总数相差不超过4个时，带有脂肪族或取代脂肪族氨基酸侧链的氨基酸大小大致相同。当其侧链上的分支数相差不超过1个时，氨基酸的形状大致相同。在侧链上具有苯基或取代苯基的氨基酸，可以认为其大小和形状大致相同。除非另有说明，保守取代优选应用天然氨基酸。In some embodiments, the NGF mutant may also contain one or more conservative amino acid substitutions. "Conservative substitution" refers to the use of another amino acid for substitution, wherein the other amino acid has the same net charge and approximately the same size and shape as the substituted amino acid. When the total number of carbon atoms and heteroatoms on the side chain does not differ by more than 4, the amino acids with aliphatic or substituted aliphatic amino acid side chains are approximately the same size. When the number of branches on their side chains does not differ by more than 1, the shape of the amino acids is approximately the same. Amino acids with phenyl or substituted phenyl on the side chain can be considered to have approximately the same size and shape. Unless otherwise stated, conservative substitutions preferably use natural amino acids.
“氨基酸”在本文中以其最广泛的定义使用，即包括天然存在的氨基酸也包括非天然存在的氨基酸，包括氨基酸的类似物和衍生物。后者包括含有氨基酸部分的分子。根据这一宽泛的定义，本领域的技术人员会发现，本文所述氨基酸包括，例如，形成蛋白质的天然L-氨基酸；D-氨基酸；化学修饰的氨基酸，如氨基酸类似物和衍生物；不形成蛋白质的天然氨基酸，如正亮氨酸、β-丙氨酸、鸟氨酸、GABA等；以及本领域已知的具有氨基酸特征的化学合成的化合物。如本文所用，术语“蛋白质形成”是指可以通过代谢途径合成细胞的肽、多肽或蛋白质的氨基酸。"Amino acid" is used herein in its broadest sense, i.e., includes naturally occurring amino acids as well as non-naturally occurring amino acids, including analogs and derivatives of amino acids. The latter include molecules containing amino acid moieties. According to this broad definition, those skilled in the art will find that the amino acids described herein include, for example, natural L-amino acids that form proteins; D-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; natural amino acids that do not form proteins, such as norleucine, β-alanine, ornithine, GABA, etc.; and chemically synthesized compounds with amino acid characteristics known in the art. As used herein, the term "protein formation" refers to amino acids that can be synthesized into peptides, polypeptides or proteins of cells through metabolic pathways.
将包括合成的非天然氨基酸、取代氨基酸或一种或多种D-氨基酸在内的非天然氨基酸插入到本申请的NGF突变体中可具有多种益处。与含有L-氨基酸的多肽相比，含有D-氨基酸的多肽等在体外和体内表现出更高的稳定性。因此，当需要更好的细胞内稳定性时，如通过加入D-氨基酸进行多肽的构建是特别有用的。特别是D-肽和其类似物对内源性肽酶和蛋白酶活性有抵抗力，从而在需要时提高分子的生物有效性并延长其在体内的寿命。此外，D-肽和其类似物不能被有效加工，这是由于II型主要组织相容性复合体(MHC)呈递给辅助T细胞的过程受限，因此不易在受试者中诱导体液免疫反应。Inserting non-natural amino acids, including synthetic non-natural amino acids, substituted amino acids or one or more D-amino acids, into the NGF mutants of the present application may have a variety of benefits. Compared with polypeptides containing L-amino acids, polypeptides containing D-amino acids and the like exhibit higher stability in vitro and in vivo. Therefore, when better intracellular stability is required, it is particularly useful to construct polypeptides such as by adding D-amino acids. In particular, D-peptides and their analogs are resistant to endogenous peptidases and protease activity, thereby increasing the bioavailability of the molecule and extending its lifespan in vivo when needed. In addition, D-peptides and their analogs cannot be effectively processed because the process of presentation to helper T cells by the type II major histocompatibility complex (MHC) is limited, and therefore it is not easy to induce humoral immune responses in subjects.
表A为保守型取代。在表A“取代示例”标题下提供了更多实质性取代，如下文关于氨基酸侧链类别部分进一步详述。氨基酸可以根据常见的侧链性质分类：(1)疏水性：正亮氨酸、Met、Ala、Val、Leu、Ile；(2)中性亲水性：Cys、Ser、Thr、Asn、Gln；(3)酸性：Asp、Glu；(4)碱性：His、Lys、Arg；(5)影响链方向的残基：Gly、 Pro；(6)芳香族：Trp、Tyr、Phe。非保守型取代需要将这些类别中的一个成员替换为另一类别的成员。可以将氨基酸取代引入到蛋白质构建体中，并筛选符合上文所述所需活性的产品。Table A is a conservative substitution. More substantial substitutions are provided under the heading "Examples of Substitutions" in Table A, as further described in detail below in the section on amino acid side chain categories. Amino acids can be classified according to common side chain properties: (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 affect chain direction: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions require replacing a member of one of these classes with a member of another class. Amino acid substitutions can be introduced into protein constructs and screened for products that meet the desired activity described above.
表A氨基酸取代
Table A Amino Acid Substitutions
如本申请所述，NGF突变体的氨基酸序列变体可能是通过在编码蛋白质的核酸序列中引入适当的修饰或通过肽合成来制备的。所述修饰包括，例如，在NGF的氨基酸序列中进行缺失和/或插入和/或取代残基。可以通过缺失、插入和取代的任何组合来获得最终的构建体，只要最终构建体具有所需的特性，例如，与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，但仍具有NGF的生物学活性、保留或改进的配体-受体结合、保留或增强的生物活性(例如，促进神经元的生长、维持和/或存活)、保留或增强半衰期、保留/降低ADCC/CDC，保留或减少致痛活性等。As described in the present application, the amino acid sequence variants of the NGF mutant may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the protein or by peptide synthesis. The modifications include, for example, deletion and/or insertion and/or substitution of residues in the amino acid sequence of NGF. The final construct can be obtained by any combination of deletion, insertion and substitution, as long as the final construct has the desired properties, for example, the binding to the anti-NGF antibody is weakened or substantially not bound to the anti-NGF antibody, but still has the biological activity of NGF, retains or improves the ligand-receptor binding, retains or enhances the biological activity (for example, promotes the growth, maintenance and/or survival of neurons), retains or enhances the half-life, retains/reduces ADCC/CDC, retains or reduces the pain-inducing activity, etc.
在一些实施例中，本申请所述的NGF突变体包含如前所述的可突变位点中的一个或多个位点上的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at one or more of the mutagenic sites described above.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF(例如，如SEQ ID NO：1或2中所示序列)的I31、K32、G33、K34、D93、W21、G23、D24、K50、Y52、T83、H84、F86、R100、R103、D16、S17、S19、T56、R59和R69位点中的一个或多个位点上的氨基酸突变。 In some embodiments, the NGF mutants described herein comprise amino acid mutations at one or more of the I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 positions relative to human wild-type mature NGF (e.g., the sequence shown in SEQ ID NO: 1 or 2).
在一些优选实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S17、S19、K32、T56、R59和T83中的一个或多个位点上的氨基酸突变。In some preferred embodiments, the NGF mutants described in the present application comprise amino acid mutations at one or more of S17, S19, K32, T56, R59 and T83 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S17和K32位点的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at positions S17 and K32 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S19和K32位点的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at positions S19 and K32 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S19和S17位点的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at positions S19 and S17 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的R59和T83位点的氨基酸突变。In some embodiments, the NGF mutants described herein comprise amino acid mutations at positions R59 and T83 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的NGF突变体，其中S17位点的氨基酸突变为S17R、S17K、S17H或S17E。In some embodiments, in the NGF mutant described herein, the amino acid at position S17 is mutated to S17R, S17K, S17H or S17E.
在一些实施例中，本申请所述的NGF突变体，其中S19位点的氨基酸突变为S19R、S19K或S19F。In some embodiments, in the NGF mutant described herein, the amino acid at position S19 is mutated to S19R, S19K or S19F.
在一些实施例中，本申请所述的NGF突变体，其中K32位点的氨基酸突变为K32F、K32E、K32N、K32Y、K32M或K32L。In some embodiments, in the NGF mutant described herein, the amino acid at position K32 is mutated to K32F, K32E, K32N, K32Y, K32M or K32L.
在一些实施例中，本申请所述的NGF突变体，其中所述T56位点的氨基酸突变为T56K或T56R。In some embodiments, the NGF mutant described herein, wherein the amino acid at the T56 position is mutated to T56K or T56R.
在一些实施例中，本申请所述的NGF突变体，其中R59位点的氨基酸突变为R59K。In some embodiments, in the NGF mutant described herein, the amino acid at position R59 is mutated to R59K.
在一些实施例中，本申请所述的NGF突变体，其中T83位点的氨基酸突变为T83K。In some embodiments, in the NGF mutant described herein, the amino acid at position T83 is mutated to T83K.
在一些实施例中，本申请所述的NGF突变体，其包含相对于人野生型成熟NGF氨基酸序列S17和K32位点的氨基酸突变。在一些实施例中，本申请所述的包含S17和K32位点氨基酸突变的NGF突变体，其中S17位点的氨基酸突变选自S17R、 S17K、S17H或S17E。在一些实施例中，本申请所述的包含S17和K32位点的NGF突变体，其中K32位点的氨基酸突变选自K32F、K32E、K32N、K32Y、K32M或K32L。In some embodiments, the NGF mutant described in the present application comprises amino acid mutations at positions S17 and K32 relative to the wild-type mature NGF amino acid sequence. In some embodiments, the NGF mutant described in the present application comprising amino acid mutations at positions S17 and K32, wherein the amino acid mutation at position S17 is selected from S17R, S17K, S17H or S17E. In some embodiments, the NGF mutant comprising S17 and K32 sites described herein, wherein the amino acid mutation at the K32 site is selected from K32F, K32E, K32N, K32Y, K32M or K32L.
在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S17R和K32E。在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S17R和K32Y。在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S17R和K32M。在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S17R和K32L。In some embodiments, the NGF mutants described herein comprise amino acid mutations S17R and K32E. In some embodiments, the NGF mutants described herein comprise amino acid mutations S17R and K32Y. In some embodiments, the NGF mutants described herein comprise amino acid mutations S17R and K32M. In some embodiments, the NGF mutants described herein comprise amino acid mutations S17R and K32L.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列S19和K32位点的氨基酸突变。在一些实施例中，本申请所述的包含S19和K32位点氨基酸突变的NGF突变体，其中S19位点的氨基酸突变选自S19R、S19K或S19F。在一些实施例中，本申请所述的包含S19和K32位点的NGF突变体，其中K32位点的氨基酸突变选自K32F、K32E、K32N、K32Y、K32M或K32L。In some embodiments, the NGF mutants described herein comprise amino acid mutations at the S19 and K32 sites relative to the wild-type mature NGF amino acid sequence of humans. In some embodiments, the NGF mutants described herein comprise amino acid mutations at the S19 and K32 sites, wherein the amino acid mutation at the S19 site is selected from S19R, S19K or S19F. In some embodiments, the NGF mutants described herein comprise S19 and K32 sites, wherein the amino acid mutation at the K32 site is selected from K32F, K32E, K32N, K32Y, K32M or K32L.
在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S19R和K32E。在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S19R和K32Y。在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S19R和K32M。在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S19R和K32L。In some embodiments, the NGF mutants described herein comprise amino acid mutations S19R and K32E. In some embodiments, the NGF mutants described herein comprise amino acid mutations S19R and K32Y. In some embodiments, the NGF mutants described herein comprise amino acid mutations S19R and K32M. In some embodiments, the NGF mutants described herein comprise amino acid mutations S19R and K32L.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列S19和S17位点的氨基酸突变。在一些实施例中，本申请所述的包含S19和S17位点氨基酸突变的NGF突变体，其中S19位点的氨基酸突变选自S19R、S19K或S19F。在一些实施例中，本申请所述的包含S19和S17位点的NGF突变体，其中S17位点的氨基酸突变选自S17R、S17K、S17H或S17E。In some embodiments, the NGF mutants described herein comprise amino acid mutations at the S19 and S17 sites relative to the wild-type mature NGF amino acid sequence of humans. In some embodiments, the NGF mutants described herein comprise amino acid mutations at the S19 and S17 sites, wherein the amino acid mutation at the S19 site is selected from S19R, S19K or S19F. In some embodiments, the NGF mutants described herein comprise amino acid mutations at the S19 and S17 sites, wherein the amino acid mutation at the S17 site is selected from S17R, S17K, S17H or S17E.
在一些实施例中，本申请所述的NGF突变体包含氨基酸突变S19R和S17E。In some embodiments, the NGF mutants described herein comprise amino acid mutations S19R and S17E.
在一些实施例中，本申请所述的NGF突变体包含相对于人野生型成熟NGF氨基酸序列R59和T83位点的氨基酸突变。在一些实施例中，本申请所述的NGF突变体包含氨基酸突变R59K和T83K。In some embodiments, the NGF mutants described herein comprise amino acid mutations at positions R59 and T83 relative to the wild-type mature NGF amino acid sequence of humans. In some embodiments, the NGF mutants described herein comprise amino acid mutations R59K and T83K.
在一些实施例中，本申请所述的人野生型成熟NGF的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。In some embodiments, the amino acid sequence of human wild-type mature NGF described in the present application is shown in SEQ ID NO:1 or SEQ ID NO:2.
在一些实施例中，本申请所述的NGF突变体包含SEQ ID NOs:17-28和41-50中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:17-28和41-50中任一所 示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。In some embodiments, the NGF mutant described in the present application comprises an amino acid sequence shown in any one of SEQ ID NOs: 17-28 and 41-50 or a variant thereof, wherein the variant is different from any one of SEQ ID NOs: 17-28 and 41-50. The amino acid sequences shown have at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology.
在一些实施例中，本申请所述的NGF变体，与野生型NGF相比，所述NGF变体减少了在使用过程中产生的疼痛，或为无痛的。在一些实施例中，所述NGF突变体，与野生型NGF相比，所述NGF突变体减少了至少5％(例如至少10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或100％)的疼痛，如在给药后的一个或多个时间点(例如，全部时间点)减少了至少5％的疼痛。在一些实施例中，所述NGF突变体，与野生型NGF相比，NGF突变体在给药后的一个或多个时间点(例如，全部时间点)增加了至少5％(例如至少10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或100％)的疼痛阈值。例如，在一些实施例中，测量个体的疼痛阈值约为8，施用野生型NGF后，疼痛阈值降至约6，施用NGF突变体后，疼痛阈值保持在8左右，说明相比于野生型NGF，施用NGF突变体的疼痛减少约25％。在一些实施例中，NGF突变体包含如专利CN107286233A、WO2017157325A1和WO2017157326A2中所述的突变，其全部内容通过引用并入本文。In some embodiments, the NGF variants described in the present application, compared with wild-type NGF, reduce the pain generated during use, or are painless. In some embodiments, the NGF mutants, compared with wild-type NGF, reduce pain by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%), such as reducing pain by at least 5% at one or more time points (e.g., all time points) after administration. In some embodiments, the NGF mutants, compared with wild-type NGF, increase the pain threshold of at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%) at one or more time points (e.g., all time points) after administration. For example, in some embodiments, the pain threshold of an individual is measured to be about 8, and after administration of wild-type NGF, the pain threshold drops to about 6, and after administration of the NGF mutant, the pain threshold remains at about 8, indicating that compared to wild-type NGF, the pain of the NGF mutant is reduced by about 25%. In some embodiments, the NGF mutant comprises mutations as described in patents CN107286233A, WO2017157325A1, and WO2017157326A2, all of which are incorporated herein by reference.
在一些实施例中，本申请所述的NGF突变体进一步包含相对于人野生型成熟NGF的突变F12E，例如，NGFF12E(SEQ ID NO:83)。In some embodiments, the NGF mutant described herein further comprises a mutant F12E relative to human wild-type mature NGF, for example, NGF F12E (SEQ ID NO: 83).
在一些实施例中，本申请所述的NGF突变体包含SEQ ID NOs:29-40和51-60中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:29-40和51-60中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。In some embodiments, the NGF mutant described in the present application comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29-40 and 51-60 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 29-40 and 51-60.
在一些实施例中，本申请中所述的NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，是指与野生型NGF(例如，人野生型成熟NGF)或NGFF12E相比，NGF突变体与相同的抗NGF抗体的结合活性降低，甚至不与抗NGF抗体结合。In some embodiments, the NGF mutants described in the present application have reduced binding to or substantially no binding to anti-NGF antibodies, meaning that compared to wild-type NGF (e.g., human wild-type mature NGF) or NGF F12E , the binding activity of the NGF mutants to the same anti-NGF antibody is reduced, or even no binding to the anti-NGF antibody.
在一些实施例中，本申请中所述的NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合是指在相同的检测实验中，其结合活性不超过野生型NGF(例如，人野生型成熟NGF)或NGFF12E与相同抗NGF抗体的结合活性的50％，例如40％、35％、30％、25％、20％、15％、10％、9％，8％，7％，6％，5％，4％，3％，2％、1％、0.6％、0.5％、0.2％、0.1％、0.07％、0.02％、0.01％、0.001％、0.0001％或更低。 In some embodiments, the NGF mutant described in the present application has reduced binding to or essentially no binding to the anti-NGF antibody, which means that in the same detection experiment, its binding activity does not exceed 50% of the binding activity of wild-type NGF (e.g., human wild-type mature NGF) or NGF F12E to the same anti-NGF antibody, for example, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.6%, 0.5%, 0.2%, 0.1%, 0.07%, 0.02%, 0.01%, 0.001%, 0.0001% or less.
在一些实施例中，本申请所述的NGF突变体与抗NGF抗体的结合活性不超过野生型NGF(例如，人野生型成熟NGF)或NGFF12E与相同抗NGF抗体的结合活性的50％，例如40％、35％、30％、25％、20％、15％、10％、9％，8％，7％，6％，5％，4％，3％，2％、1％、0.6％、0.5％、0.2％、0.1％、0.07％、0.02％、0.01％、0.001％、0.0001％或更低。In some embodiments, the binding activity of the NGF mutants described herein to anti-NGF antibodies is no more than 50% of the binding activity of wild-type NGF (e.g., human wild-type mature NGF) or NGF F12E to the same anti-NGF antibody, for example, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.6%, 0.5%, 0.2%, 0.1%, 0.07%, 0.02%, 0.01%, 0.001%, 0.0001% or less.
在一些实施例中，本申请所述的与抗NGF抗体的结合活性可以选择通过ELISA实验、Biacore实验或BLI实验等进行检测。In some embodiments, the binding activity with anti-NGF antibodies described in the present application can be detected by ELISA experiments, Biacore experiments, or BLI experiments.
在一些实施例中，本申请所述的与抗NGF抗体的结合活性通过ELISA实验进行检测。在另一些实施例中，本申请所述的NGF突变体与抗NGF抗体的结合活性采用ELISA实验中的EC50值来表征。In some embodiments, the binding activity of the NGF mutants described in the present application with anti-NGF antibodies is detected by ELISA experiments. In other embodiments, the binding activity of the NGF mutants described in the present application with anti-NGF antibodies is characterized by the EC50 value in the ELISA experiment.
在一些优选实施例中，本申请所述的NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合是指在ELISA结合实验中，所述NGF突变体与抗NGF抗体的结合EC50值是人野生型成熟NGF(SEQ ID NO：1或2)或NGFF12E(SEQ ID NO：83)与相同抗NGF抗体的结合EC50值的至少2倍，例如2倍、3倍、4倍、5倍、7倍、9倍、10倍、13倍、14倍、15倍、17倍、20倍、25倍、33倍、50倍、100倍、167倍、200倍、500倍、1000倍、1400倍、5000倍、10000倍、100000倍、1000000倍或更高。In some preferred embodiments, the NGF mutant described in the present application has weakened binding to or essentially no binding to the anti-NGF antibody, which means that in an ELISA binding experiment, the EC50 value of the binding of the NGF mutant to the anti-NGF antibody is at least 2 times, for example, 2 times, 3 times, 4 times, 5 times, 7 times, 9 times, 10 times, 13 times, 14 times, 15 times, 17 times, 20 times, 25 times, 33 times, 50 times, 100 times, 167 times, 200 times, 500 times, 1000 times, 1400 times, 5000 times, 10000 times, 100000 times, 100000 times or more of the EC50 value of the binding of human wild-type mature NGF (SEQ ID NO: 1 or 2) or NGF F12E (SEQ ID NO: 83) to the same anti-NGF antibody.
在一些实施例中，本申请所述的NGF突变体与抗NGF抗体的结合EC50值是人野生型成熟NGF(SEQ ID NO：1或2)或NGFF12E(SEQ ID NO：83)与相同抗NGF抗体的结合EC50值的至少2倍，例如2倍、3倍、4倍、5倍、7倍、9倍、10倍、13倍、14倍、15倍、17倍、20倍、25倍、33倍、50倍、100倍、167倍、200倍、500倍、1000倍、1400倍、5000倍、10000倍、100000倍、1000000倍或更高。In some embodiments, the EC50 value of the binding of the NGF mutant described in the present application to the anti-NGF antibody is at least 2 times the EC50 value of the binding of human wild-type mature NGF (SEQ ID NO: 1 or 2) or NGF F12E (SEQ ID NO: 83) to the same anti-NGF antibody, for example, 2 times, 3 times, 4 times, 5 times, 7 times, 9 times, 10 times, 13 times, 14 times, 15 times, 17 times, 20 times, 25 times, 33 times, 50 times, 100 times, 167 times, 200 times, 500 times, 1000 times, 1400 times, 5000 times, 10000 times, 100000 times, 1000000 times or more.
在一些实施例中，本申请的方法、NGF中的可突变位点、NGF突变体库以及NGF突变体中所述的NGF突变体仍具有NGF的生物学活性。在一些实施例中，所述NGF突变体仍具有的NGF的生物学活性包含本申请所述的任一种NGF生物学活性。在一些实施例中，所述NGF突变体的生物学活性由本申请所述的任一种NGF生物学活性检测实验测定获得，例如，包括，但不限于TrkA受体结合实验、TF-1细胞活性检测实验、PC12细胞活性检测实验、鸡胚背根神经节实验、或大鼠颈上神经节实验。在一些优选实施例中，所述NGF突变体的生物学活性由TrkA受体结合实验测定获得。 In some embodiments, the NGF mutants described in the methods of the present application, the mutable sites in NGF, the NGF mutant library, and the NGF mutants still have the biological activity of NGF. In some embodiments, the biological activity of NGF still possessed by the NGF mutants includes any one of the NGF biological activities described in the present application. In some embodiments, the biological activity of the NGF mutants is obtained by any one of the NGF biological activity detection experiments described in the present application, for example, including, but not limited to, TrkA receptor binding experiments, TF-1 cell activity detection experiments, PC12 cell activity detection experiments, chicken embryo dorsal root ganglion experiments, or rat superior cervical ganglion experiments. In some preferred embodiments, the biological activity of the NGF mutants is obtained by TrkA receptor binding experiments.
在一些实施例中，本申请所述的NGF突变体仍具有NGF的生物学活性，是指与NGFF12E(SEQ ID NO：83)相比，其具有与之基本相当，更高，或稍低的生物学活性。In some embodiments, the NGF mutant described in the present application still has the biological activity of NGF, which means that compared with NGF F12E (SEQ ID NO: 83), it has a biological activity that is substantially equivalent to, higher, or slightly lower than that of NGF F12E (SEQ ID NO: 83).
在一些实施例中，本申请所述的NGF突变体仍具有NGF的生物学活性是指在相同的检测实验中，其生物学活性是NGFF12E生物学活性的至少20％，优选为至少20％，例如至少21％、30％、36％、40％、50％、60％、70％、80％、90％、100％、1.5倍、2倍、3倍、4倍、5倍、7倍、10倍、15倍、20倍、50倍、100倍或更高。In some embodiments, the NGF mutant described in the present application still has the biological activity of NGF, which means that in the same detection experiment, its biological activity is at least 20% of the biological activity of NGF F12E , preferably at least 20%, for example at least 21%, 30%, 36%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
在一些实施例中，本申请所述的NGF突变体的生物学活性为NGFF12E生物学活性的至少20％，优选为至少20％，例如至少21％、30％、36％、40％、50％、60％、70％、80％、90％、100％、1.5倍、2倍、3倍、4倍、5倍、7倍、10倍、15倍、20倍、50倍、100倍或更高。在一些实施例中，本申请所述的NGF突变体的生物学活性可以用体外实验进行测定。在一些实施例中，本申请所述的具有生物学活性，是指在TF-1细胞增殖实验中能够促进TF-1细胞增殖的活性。在一些实施例中，本申请所述的具有生物学活性，是指在PC12细胞活性检测实验中能够诱导PC12细胞分化的能力。在一些实施例中，本申请所述的具有生物学活性，是指在鸡胚背根神经节实验中能够促进鸡胚背根神经节分化。在一些实施例中，本申请所述的具有生物学活性，是指在大鼠颈上神经节实验中能够促进大鼠颈上神经节生长。In some embodiments, the biological activity of the NGF mutant described in the present application is at least 20% of the biological activity of NGF F12E , preferably at least 20%, for example at least 21%, 30%, 36%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 15 times, 20 times, 50 times, 100 times or higher. In some embodiments, the biological activity of the NGF mutant described in the present application can be determined by in vitro experiments. In some embodiments, the biological activity described in the present application refers to the activity that can promote the proliferation of TF-1 cells in the TF-1 cell proliferation experiment. In some embodiments, the biological activity described in the present application refers to the ability to induce PC12 cell differentiation in the PC12 cell activity detection experiment. In some embodiments, the biological activity described in the present application refers to the ability to promote the differentiation of chicken embryo dorsal root ganglia in the chicken embryo dorsal root ganglion experiment. In some embodiments, the biological activity described in the present application refers to the ability to promote the growth of rat superior cervical ganglia in the rat superior cervical ganglion experiment.
在一些实施例中，本申请所述的NGF突变体的生物学活性由其与TrkA受体的结合活性表征。在一些实施例中，本申请所述的NGF突变体具有生物学活性，是指该突变体能够与TrkA受体结合。在一些实施例中，本申请所述的NGF突变体能够与TrkA受体结合是指与NGFF12E(SEQ ID NO：83)相比，所述NGF突变体与TrkA受体的结合活性与之基本相当或更高或稍低。In some embodiments, the biological activity of the NGF mutant described in the present application is characterized by its binding activity to the TrkA receptor. In some embodiments, the NGF mutant described in the present application has biological activity, which means that the mutant is able to bind to the TrkA receptor. In some embodiments, the NGF mutant described in the present application is able to bind to the TrkA receptor, which means that compared with NGF F12E (SEQ ID NO: 83), the binding activity of the NGF mutant to the TrkA receptor is substantially equivalent to, higher than, or slightly lower than that of NGF F12E (SEQ ID NO: 83).
在一些实施例中，本申请所述的NGF突变体能够与TrkA受体结合是指与NGFF12E(SEQ ID NO：83)相比，其与TrkA受体的结合活性是NGFF12E与TrkA受体结合活性的至少20％，例如至少21％、30％、36％、40％、50％、60％、70％、80％、90％、100％、1.5倍、2倍、3倍、4倍、5倍、7倍、10倍、15倍、20倍、50倍、100倍或更高。In some embodiments, the NGF mutant described in the present application is capable of binding to the TrkA receptor, which means that compared with NGF F12E (SEQ ID NO: 83), its binding activity to the TrkA receptor is at least 20% of the binding activity of NGF F12E to the TrkA receptor, for example, at least 21%, 30%, 36%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
在一些实施例中，本申请所述的NGF突变体仍具有NGF的生物学活性是指其与TrkA受体的结合活性是NGFF12E(SEQ ID NO：83)与TrkA受体结合活性的至少 20％，例如至少21％、30％、36％、40％、50％、60％、70％、80％、90％、100％、1.5倍、2倍、3倍、4倍、5倍、7倍、10倍、15倍、20倍、50倍、100倍或更高。In some embodiments, the NGF mutant described in the present application still has the biological activity of NGF, which means that its binding activity to the TrkA receptor is at least 1:1 of the binding activity of NGF F12E (SEQ ID NO: 83) to the TrkA receptor. 20%, for example at least 21%, 30%, 36%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
在一些实施例中，本申请所述的NGF突变体与TrkA受体的结合活性是NGFF12E(SEQ ID NO：83)与TrkA受体结合活性的至少20％，例如至少21％、30％、36％、40％、50％、60％、70％、80％、90％、100％、1.5倍、2倍、3倍、4倍、5倍、7倍、10倍、15倍、20倍、50倍、100倍或更高。In some embodiments, the binding activity of the NGF mutant described in the present application to the TrkA receptor is at least 20% of the binding activity of NGF F12E (SEQ ID NO: 83) to the TrkA receptor, for example, at least 21%, 30%, 36%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
在一些实施例中，本申请所述的NGF突变体与TrkA受体的结合活性通过ELISA实验进行检测。在另一些实施例中，本申请所述的NGF突变体与TrkA受体的结合活性采用ELISA实验中的EC50值来表征。In some embodiments, the binding activity of the NGF mutants described in the present application to the TrkA receptor is detected by ELISA experiments. In other embodiments, the binding activity of the NGF mutants described in the present application to the TrkA receptor is characterized by the EC50 value in the ELISA experiment.
在一些实施例中，本申请所述的NGF突变体仍具有NGF的生物学活性是指在ELISA结合实验中，其与TrkA受体的结合EC50值是NGFF12E(SEQ ID NO：83)与TrkA受体的结合EC50值的至多5倍，例如5倍、4倍、3倍、2倍、1倍、80％、70％、60％、50％、40％、35％、30％、25％、20％、10％、7％、5％、2％、1％或更低。In some embodiments, the NGF mutant described in the present application still has the biological activity of NGF, which means that in an ELISA binding experiment, its EC50 value for binding to the TrkA receptor is at most 5 times the EC50 value for binding to the TrkA receptor of NGF F12E (SEQ ID NO: 83), for example, 5 times, 4 times, 3 times, 2 times, 1 times, 80%, 70%, 60%, 50%, 40%, 35%, 30%, 25%, 20%, 10%, 7%, 5%, 2%, 1% or less.
在一些实施例中，本申请所述的NGF突变体与TrkA受体的结合EC50值是NGFF12E(SEQ ID NO：83)与TrkA受体的结合EC50值的至多5倍，例如5倍、4倍、3倍、2倍、1倍、80％、70％、60％、50％、40％、35％、30％、25％、20％、10％、7％、5％、2％、1％或更低。In some embodiments, the EC50 value of the binding of the NGF mutant described in the present application to the TrkA receptor is at most 5 times the EC50 value of the binding of NGF F12E (SEQ ID NO: 83) to the TrkA receptor, for example, 5 times, 4 times, 3 times, 2 times, 1 times, 80%, 70%, 60%, 50%, 40%, 35%, 30%, 25%, 20%, 10%, 7%, 5%, 2%, 1% or less.
在一些实施例中，本申请中所述的NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，但其仍具有NGF的生物学活性，是指所述NGF突变体与抗NGF抗体的结合活性是人野生型成熟NGF或NGFF12E与相同抗NGF抗体的结合活性的至多50％，例如40％、35％、30％、25％、20％、15％、10％、9％，8％，7％，6％，5％，4％，3％，2％、1％、0.6％、0.5％、0.2％、0.1％、0.07％、0.02％、0.01％、0.001％、0.0001％或更低，且所述NGF突变体与TrkA受体结合活性是NGFF12E与TrkA受体结合活性的至少20％，例如至少21％、30％、36％、40％、50％、60％、70％、80％、90％、100％、1.5倍、2倍、3倍、4倍、5倍、7倍、10倍、15倍、20倍、50倍、100倍或更高。In some embodiments, the NGF mutant described in the present application has weakened binding to or essentially no binding to anti-NGF antibody, but still has the biological activity of NGF, which means that the binding activity of the NGF mutant to anti-NGF antibody is at most 50% of the binding activity of human wild-type mature NGF or NGF F12E to the same anti-NGF antibody, for example, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.6%, 0.5%, 0.2%, 0.1%, 0.07%, 0.02%, 0.01%, 0.001%, 0.0001% or lower, and the binding activity of the NGF mutant to TrkA receptor is NGF F12E has at least 20% of the binding activity to the TrkA receptor, such as at least 21%, 30%, 36%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
在一些实施例中，本申请所述的NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，但其仍具有NGF的生物学活性，是指在ELISA检测实验中， 所述NGF突变体与抗NGF抗体的结合EC50值是人野生型成熟NGF或NGFF12E与相同抗NGF抗体的结合EC50值的至少2倍，例如2倍、3倍、4倍、5倍、7倍、9倍、10倍、13倍、14倍、15倍、17倍、20倍、25倍、33倍、50倍、100倍、167倍、200倍、500倍、1000倍、1400倍、5000倍、10000倍、100000倍、1000000倍或更高，同时所述NGF突变体与TrkA受体的结合EC50值是NGFF12E与TrkA受体的结合EC50值的至多5倍，例如5倍、4倍、3倍、2倍、1倍、80％、70％、60％、50％、40％、35％、30％、25％、20％、10％、7％、5％、2％、1％或更低。In some embodiments, the NGF mutant described in the present application has a weakened binding to the anti-NGF antibody or substantially no binding to the anti-NGF antibody, but still has the biological activity of NGF, which means that in the ELISA test, The EC50 value of the binding of the NGF mutant to the anti-NGF antibody is at least 2 times, for example, 2 times, 3 times, 4 times, 5 times, 7 times, 9 times, 10 times, 13 times, 14 times, 15 times, 17 times, 20 times, 25 times, 33 times, 50 times, 100 times, 167 times, 200 times, 500 times, 1000 times, 1400 times, 5000 times, 10000 times, 100000 times, 1000000 times or more of the EC50 value of the binding of the NGF mutant to the TrkA receptor. The EC50 value of F12E binding to the TrkA receptor is up to 5-fold, e.g., 5-fold, 4-fold, 3-fold, 2-fold, 1-fold, 80%, 70%, 60%, 50%, 40%, 35%, 30%, 25%, 20%, 10%, 7%, 5%, 2%, 1% or less.
在一些实施例中，本申请所述的NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，但其仍具有NGF的生物学活性，是指在ELISA检测实验中，所述NGF突变体与一种或多种抗NGF抗体的结合EC50值为1μg/ml、2μg/ml、3μg/ml、5μg/ml、10μg/ml。100μg/ml、300μg/ml、1000μg/ml或更高，同时所述NGF突变体与TrkA受体的结合EC50值为10μg/ml、6μg/ml、5μg/ml、1μg/ml、0.5μg/ml、0.1μg/ml、0.01μg/ml或更低。在一些实施例中，在所述ELISA检测实验中，以NGF突变体包被孔板，加入抗NGF抗体或TrkA进行检测。在进一步优选的实施例中，在所述ELISA检测实验中，使用96孔板，且所述NGF突变体的浓度为0.1μg/孔。在另一些实施例中，在所述ELISA检测实验中，以抗NGF抗体或TrkA包被孔板，加入NGF突变体进行检测。在进一步优选的实施例中，在所述ELISA检测实验中，使用96孔板，且所述抗NGF抗体或TrkA的浓度为0.1μg/孔。In some embodiments, the NGF mutant described in the present application has a weakened binding to the anti-NGF antibody or basically does not bind to the anti-NGF antibody, but it still has the biological activity of NGF, which means that in the ELISA detection experiment, the EC50 value of the binding of the NGF mutant to one or more anti-NGF antibodies is 1μg/ml, 2μg/ml, 3μg/ml, 5μg/ml, 10μg/ml. 100μg/ml, 300μg/ml, 1000μg/ml or higher, and the EC50 value of the binding of the NGF mutant to the TrkA receptor is 10μg/ml, 6μg/ml, 5μg/ml, 1μg/ml, 0.5μg/ml, 0.1μg/ml, 0.01μg/ml or lower. In some embodiments, in the ELISA detection experiment, the well plate is coated with the NGF mutant, and anti-NGF antibodies or TrkA are added for detection. In a further preferred embodiment, in the ELISA detection experiment, a 96-well plate is used, and the concentration of the NGF mutant is 0.1 μg/well. In other embodiments, in the ELISA detection experiment, the well plate is coated with anti-NGF antibody or TrkA, and the NGF mutant is added for detection. In a further preferred embodiment, in the ELISA detection experiment, a 96-well plate is used, and the concentration of the anti-NGF antibody or TrkA is 0.1 μg/well.
包含NGF突变体的融合蛋白Fusion proteins containing NGF mutants
本申请另一方面还提供包含本文所述NGF突变体的融合蛋白。在一些实施例中，本申请所述的NGF突变体可以与其它蛋白进行融合，以增加NGF突变体在体内的半衰期或提高NGF突变体在体内的稳定性，例如，通过增加分子的大小和/或降低肾清除率来达到上述目的。在一些优选实施例中，本申请所述的NGF突变体与Fc进行融合。在另一些优选实施例中，本申请所述的NGF突变体与人血清白蛋白(HSA)进行融合。在另一些优选实施例中，本申请所述的NGF突变体与一些短肽(例如，XTEN)进行融合。On the other hand, the present application also provides fusion proteins comprising the NGF mutants described herein. In some embodiments, the NGF mutants described in the present application can be fused with other proteins to increase the half-life of the NGF mutants in vivo or improve the stability of the NGF mutants in vivo, for example, by increasing the size of the molecule and/or reducing the renal clearance rate to achieve the above purpose. In some preferred embodiments, the NGF mutants described in the present application are fused with Fc. In other preferred embodiments, the NGF mutants described in the present application are fused with human serum albumin (HSA). In other preferred embodiments, the NGF mutants described in the present application are fused with some short peptides (e.g., XTEN).
NGF突变体与Fc的融合蛋白：Fusion protein of NGF mutant and Fc:
Fc融合蛋白可通过重组DNA技术产生，其中哺乳动物免疫球蛋白(例如，IgG)的Fc结构域的翻译阅读框被融合到另一蛋白以产生新的单个重组多肽。Fc融合 蛋白通常作为二硫键连接的二聚体产生，其由位于Fc结构域中铰链区的二硫键连接在一起。Fc fusion proteins can be produced by recombinant DNA technology, in which the translational reading frame of the Fc domain of a mammalian immunoglobulin (e.g., IgG) is fused to another protein to produce a new single recombinant polypeptide. The protein is typically produced as a disulfide-linked dimer held together by a disulfide bond located in the hinge region of the Fc domain.
可以将NGF突变体融合于Fc的N端和/或C端。优选地，将NGF突变体融合于Fc的N端。在一些实施例中，NGF突变体与Fc之间通过肽键直接融合。在一些实施例中，NGF突变体与Fc之间包含肽接头。The NGF mutant can be fused to the N-terminus and/or C-terminus of Fc. Preferably, the NGF mutant is fused to the N-terminus of Fc. In some embodiments, the NGF mutant is directly fused to Fc via a peptide bond. In some embodiments, a peptide linker is included between the NGF mutant and Fc.
在一些实施例中，所述NGF突变体与Fc的融合蛋白，其中Fc来自IgG1 Fc，如人IgG1 Fc。在一些实施例中，Fc为野生型IgG1 Fc(例如，人IgG1 Fc)或其天然突变体。在一些实施例中，Fc为IgG1自然变体(例如，IGHG1*03，相对于IGHG1*05包含D239E和L241M双突变)。在一些实施例中，所述Fc相对野生型人IgG1 Fc，包含L234A、L235A和P331S突变，其中所述编号为EU编号系统。在一些实施例中，所述Fc包含氨基酸序列SEQ ID NO:85或其变体，所述变体与氨基酸序列SEQ ID NO:85具有至少90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％))序列同源性。In some embodiments, the NGF mutant is a fusion protein with Fc, wherein Fc is from IgG1 Fc, such as human IgG1 Fc. In some embodiments, Fc is a wild-type IgG1 Fc (e.g., human IgG1 Fc) or a natural mutant thereof. In some embodiments, Fc is a natural variant of IgG1 (e.g., IGHG1*03, comprising a double mutation of D239E and L241M relative to IGHG1*05). In some embodiments, the Fc comprises L234A, L235A and P331S mutations relative to wild-type human IgG1 Fc, wherein the numbering is the EU numbering system. In some embodiments, the Fc comprises an amino acid sequence of SEQ ID NO: 85 or a variant thereof, wherein the variant has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence of SEQ ID NO: 85.
在一些实施例中，所述NGF突变体与Fc的融合蛋白，其中Fc来自IgG4 Fc，如人IgG4 Fc。在一些实施例中，Fc为野生型IgG4 Fc(例如，人IgG4 Fc)或其天然突变体。在一些实施例中，所述Fc相对野生型人IgG4 Fc，包含S228P突变，其中所述编号为EU编号系统。In some embodiments, the fusion protein of the NGF mutant and Fc, wherein Fc is from IgG4 Fc, such as human IgG4 Fc. In some embodiments, Fc is wild-type IgG4 Fc (e.g., human IgG4 Fc) or a natural mutant thereof. In some embodiments, the Fc comprises an S228P mutation relative to wild-type human IgG4 Fc, wherein the numbering is the EU numbering system.
在一些实施例中，涉及一种NGF突变体与Fc的融合蛋白，从N端到C端或从C端到N端：包含NGF突变体和Fc部分。In some embodiments, it relates to a fusion protein of an NGF mutant and Fc, from N-terminus to C-terminus or from C-terminus to N-terminus: comprising an NGF mutant and an Fc portion.
在一些实施例中，涉及一种NGF突变体与Fc的融合蛋白，从N端到C端或从C端到N端：包含NGF突变体、可选的肽接头和Fc部分。In some embodiments, it relates to a fusion protein of an NGF mutant and Fc, from N-terminus to C-terminus or from C-terminus to N-terminus: comprising an NGF mutant, an optional peptide linker and an Fc portion.
在一些实施例中，所述的NGF突变体与Fc的融合蛋白中，NGF突变体包含SEQ ID NOs:17-60中的任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:17-60中的任一所示的氨基酸序列具有至少90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。In some embodiments, in the fusion protein of the NGF mutant and Fc, the NGF mutant comprises an amino acid sequence shown in any one of SEQ ID NOs: 17-60 or a variant thereof, and the variant has at least 90% (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 17-60.
在一些实施例中，所述的NGF突变体与Fc的融合蛋白中，可选的肽接头包含氨基酸序列(GGGGS)n，并且其中n为1、2、3、4、5或6中的任何一个。在一些实施例中，肽接头包含氨基酸序列(GGGGS)3(SEQ ID NO:87)。 In some embodiments, in the fusion protein of NGF mutant and Fc, the optional peptide linker comprises the amino acid sequence (GGGGS)n, wherein n is any one of 1, 2, 3, 4, 5 or 6. In some embodiments, the peptide linker comprises the amino acid sequence (GGGGS) 3 (SEQ ID NO: 87).
在一些实施例中，所述的NGF突变体与Fc的融合蛋白中，Fc部分包含氨基酸序列SEQ ID NO:85或其变体，所述变体与SEQ ID NO:85具有至少90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％))序列同源性。In some embodiments, in the fusion protein of the NGF mutant and Fc, the Fc part comprises the amino acid sequence SEQ ID NO:85 or its variant, and the variant has at least 90% (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO:85.
在一些实施例中，所述的NGF突变体与Fc的融合蛋白包含SEQ ID NOs:61-82中的任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:61-82中的任一所示的氨基酸序列具有至少80％(例如至少80％、85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。In some embodiments, the fusion protein of the NGF mutant and Fc comprises an amino acid sequence shown in any one of SEQ ID NOs:61-82 or a variant thereof, and the variant has at least 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs:61-82.
Fc部分Fc part
如本文所述，在NGF突变体与Fc的融合蛋白的N端或C端包含Fc部分。As described herein, the Fc portion is included at the N-terminus or C-terminus of the fusion protein of the NGF mutant and Fc.
在一些实施例中，Fc部分来自IgA、IgD、IgE、IgG和IgM及其亚类中的任何一种。在所有免疫球蛋白中，IgG的血清含量最高，半衰期最长。与其它免疫球蛋白不同，在与Fc受体(FcRs)结合后，IgG可有效回收。在一些实施例中，Fc部分来自IgG(例如，IgG1、IgG2、IgG3或IgG4)。在一些实施例中，Fc部分来自人IgG。在一些实施例中，Fc部分包含CH2和CH3。在一些实施例中，Fc部分进一步包含全部或部分铰链区。在一些实施例中，Fc部分来自人IgG1或人IgG4。在一些实施例中，Fc部分的两个亚基通过一个或多个(例如，1、2、3、4或更多)二硫键二聚化。在一些实施例中，Fc部分的每个亚基都包含全长Fc序列。在一些实施例中，Fc部分的每个亚基都包含N端截短的Fc序列，如截短的Fc部分含有较少的N端半胱氨酸，以减少二聚化过程中二硫键的错配。在一些实施例中，Fc部分在N端被截短，例如，缺失完整免疫球蛋白Fc部分的前1、2、3、4、5、6、7、8、9或10个氨基酸。在一些实施例中，Fc部分包含一个或多个突变，如插入、缺失和/或取代。In some embodiments, the Fc portion is from any one of IgA, IgD, IgE, IgG and IgM and their subclasses. Among all immunoglobulins, IgG has the highest serum content and the longest half-life. Unlike other immunoglobulins, IgG can be effectively recovered after binding to Fc receptors (FcRs). In some embodiments, the Fc portion is from IgG (e.g., IgG1, IgG2, IgG3 or IgG4). In some embodiments, the Fc portion is from human IgG. In some embodiments, the Fc portion comprises CH2 and CH3. In some embodiments, the Fc portion further comprises all or part of the hinge region. In some embodiments, the Fc portion is from human IgG1 or human IgG4. In some embodiments, the two subunits of the Fc portion are dimerized by one or more (e.g., 1, 2, 3, 4 or more) disulfide bonds. In some embodiments, each subunit of the Fc portion comprises a full-length Fc sequence. In some embodiments, each subunit of the Fc portion comprises an N-terminal truncated Fc sequence, such as a truncated Fc portion containing less N-terminal cysteine to reduce the mismatch of disulfide bonds during dimerization. In some embodiments, the Fc portion is truncated at the N-terminus, for example, the first 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the intact immunoglobulin Fc portion are deleted. In some embodiments, the Fc portion comprises one or more mutations, such as insertions, deletions and/or substitutions.
在一些实施例中，Fc部分能够为NGF突变体与Fc的融合蛋白提供如本文所述的高生物活性、长半衰期和低免疫毒性(例如，ADCC和/或CDC)。In some embodiments, the Fc portion can provide the fusion protein of NGF mutant and Fc with high biological activity, long half-life, and low immunotoxicity (eg, ADCC and/or CDC) as described herein.
通过Fc部分，包含Fc的蛋白可以激活补体并与Fc受体(FcRs)相互作用。这种固有的免疫球蛋白特性已经被认为是不利的，因为NGF突变体与Fc的融合蛋白可能靶向表达Fc受体的细胞，而不是优选的表达NGF受体的细胞，而且进一步考虑到Fc融合蛋白的半衰期长，由于全身毒性，使其难以应用于治疗。因此，在一些实施例中，Fc部分被改造(例如，包含一个或多个氨基酸突变)以改变其与FcR的结合，特别是改变与Fcγ受体(负责ADCC)的结合和/或改变效应功能，如改变抗体依赖性细 胞介导的细胞毒性(ADCC)、抗体依赖型细胞吞噬作用(ADCP)和/或补体依赖性细胞毒性(CDC)。优选地，此类氨基酸突变不会减少与FcRn受体(负责半衰期)的结合。Through the Fc portion, proteins containing Fc can activate complement and interact with Fc receptors (FcRs). This inherent immunoglobulin property has been considered disadvantageous because fusion proteins of NGF mutants with Fc may target cells expressing Fc receptors rather than the preferred cells expressing NGF receptors, and further considering the long half-life of Fc fusion proteins, it is difficult to use them in therapy due to systemic toxicity. Therefore, in some embodiments, the Fc portion is modified (e.g., comprising one or more amino acid mutations) to alter its binding to FcR, in particular to alter its binding to Fcγ receptors (responsible for ADCC) and/or to alter effector functions, such as altering antibody-dependent cell proliferation. Preferably, such amino acid mutations do not reduce binding to the FcRn receptor (responsible for half-life).
Fc部分(例如，人类IgG1 Fc)发生突变，以去除一个或多个效应功能，如ADCC、ADCP或CDC，以下称为“无效应”或“几乎无效应”Fc部分。例如，在一些实施例中，Fc部分为一种无效应人IgG1 Fc，所述人IgG1 Fc包含一个或多个以下突变(如在每一个Fc亚基中)：L234A、L235A和P331S。The Fc portion (e.g., human IgG1 Fc) is mutated to remove one or more effector functions, such as ADCC, ADCP or CDC, hereinafter referred to as a "non-effector" or "almost non-effector" Fc portion. For example, in some embodiments, the Fc portion is a non-effector human IgG1 Fc, wherein the human IgG1 Fc comprises one or more of the following mutations (e.g., in each Fc subunit): L234A, L235A and P331S.
IgG1 Fc中K322A、L234A和L235A的组合足以完全消除FcγR和C1q的结合(Hezareh et al.,J Virol 75,12161-12168,2001)。MedImmune发现一组三突变L234F/L235E/P331S具有非常相似的效应(Oganesyan et al.,Acta Crystallographica 64,700-704,2008)。在一些实施例中，Fc部分包含IgG1 Fc结构域N297上的糖基化修饰，已知这是最优FcR相互作用所必需的。Fc部分修饰可以是Wang等人提到的任何合适的IgG Fc工程。(“IgG Fc engineering to modulate antibody effector functions,”Protein Cell.2018 Jan；9(1):63-73)，其内容全部通过引用并入本文。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). MedImmune found that a set of triple mutations L234F/L235E/P331S had very similar effects (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 required for optimal FcR interaction. The Fc portion modification can 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.
在一些实施例中，如本文所述，包含NGF突变体与Fc的融合蛋白不具有ADCC和/或CDC，或不具有可检测的ADCC和/或CDC。在一些实施例中，如本文所述，NGF突变体与Fc的融合蛋白与包含相同NGF部分但为野生型或未修饰Fc部分的NGF与Fc的融合蛋白相比，ADCC和/或CDC减少了至少5％(如至少为10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或100％中的任何一个)In some embodiments, as described herein, the fusion protein comprising a NGF mutant and Fc has no ADCC and/or CDC, or has no detectable ADCC and/or CDC. In some embodiments, as described herein, the fusion protein of a NGF mutant and Fc has at least 5% (e.g., at least any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) reduced ADCC and/or CDC compared to a fusion protein of NGF and Fc comprising the same NGF portion but a wild-type or unmodified Fc portion.
糖基化变体Glycosylation variants
在一些实施例中，通过改变Fc部分或NGF突变体与Fc的融合蛋白来增加或减少构建体的糖基化程度。可以通过改变氨基酸序列在Fc部分中加入或删除糖基化位点，以创建或移除一个或多个糖基化位点。In some embodiments, the glycosylation level of the construct is increased or decreased by altering the Fc portion or the fusion protein of the NGF mutant and Fc. Glycosylation sites can be added or deleted in the Fc portion by altering the amino acid sequence to create or remove one or more glycosylation sites.
哺乳动物细胞产生的天然含Fc的蛋白通常包含一个分支链的双触角寡糖，所述双触角寡糖通常通过N-键连接到Fc的CH2结构域的Asn297上。参见Wright et al.,TIBTECH 15:26-32(1997)。寡糖可以包括各种碳水化合物，例如，甘露糖、N-乙酰葡萄糖胺(GlcNAc)、半乳糖和唾液酸，以及附着在双触角寡糖结构的“茎”上GlcNAc的岩藻糖。在一些实施例中，可对Fc部分中的寡糖进行修饰以产生某些改良的特性。 The natural Fc-containing proteins produced by mammalian cells usually contain a branched chain of biantennary oligosaccharides, which are usually connected to Asn297 of the CH2 domain of Fc by N-bonds. See Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose attached to the GlcNAc on the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the Fc part can be modified to produce certain improved properties.
在一些实施例中，如本文所述的Fc部分或NGF突变体与Fc的融合蛋白具有碳水化合物结构，该结构缺乏附着(直接或间接)在Fc部分的岩藻糖。例如，在这种Fc部分或NGF突变体与Fc的融合蛋白中的岩藻糖含量可能从1％至80％、从1％至65％、从5％至65％或从20％至40％。如WO 2008/077546所述，岩藻糖的含量通过MALDI-TOF质谱测量连接到Asn297上的糖链内岩藻糖平均含量相对于附着在Asn297(例如，复合、杂交和高甘露糖结构)上的所有糖结构的总和来确定的。Asn297是指位于Fc区297位的天冬酰胺残基(Fc区残基EU编号系统)；然而，由于Fc区的微小序列变化，Asn297也可位于297位的上游或下游约±3个氨基酸，即在294和300位之间。这类岩藻糖基化变体可能具有增强的ADCC功能。参见US Patent Publication Nos.US 2003/0157108(Presta,L.)，US 2004/0093621(Kyowa Hakko Kogyo Co.,Ltd)。与“去岩藻糖基化”或“岩藻糖缺乏”的抗体变体相关的出版物示例包括：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)。能够产生去岩藻糖基化含Fc蛋白的细胞系的示例包括缺乏蛋白岩藻糖基化功能的Lec13 CHO细胞(Ripka et al.,Arch.Biochem.Biophys.249:533-545(1986)；US Pat Appl No US 2003/0157108 A1,Presta,L；和WO 2004/056312 A1,Adams et al.,尤其是实施例11)，和基因敲除细胞系，如α-1,6-岩藻糖基转移酶基因、FUT8基因敲除的CHO细胞(参见Yamane-Ohnuki et al.,Biotech.Bioeng.87:614(2004)；Kanda,Y.et al.,Biotechnol.Bioeng.,94(4):680-688(2006)；和WO2003/085107)。In some embodiments, the Fc portion or the fusion protein of NGF mutant and Fc as described herein has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc portion. For example, the fucose content in such Fc portion or the fusion protein of NGF mutant and Fc may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. As described in WO 2008/077546, the fucose content is determined by measuring the average fucose content within the sugar chain attached to Asn297 by MALDI-TOF mass spectrometry relative to the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid and high mannose structures). Asn297 refers to the asparagine residue located at position 297 of the Fc region (EU numbering system for residues in the Fc region); however, due to minor sequence variations in the Fc region, Asn297 may also be located about ±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 "defucosylated" 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 2004/01098 65; 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 defucosylated Fc-containing proteins include Lec13 CHO cells that 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. ., especially Example 11), and gene knockout cell lines, such as CHO cells with α-1,6-fucosyltransferase gene and FUT8 gene knockout (see Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); and WO2003/085107).
效应功能变体Effector function variants
在一些实施例中，本申请提供了一种Fc，其具有一些但非全部Fc的效应功能，使得其成为某种应用的理想候选，例如，在该应用中，NGF突变体与Fc的融合蛋白在体内的半衰期很重要，但某些效应功能(如CDC和ADCC)是非必需的或有害的。可以在体外或体内进行细胞毒性试验，以确定CDC和/或ADCC活性的减少/耗尽。例如，可以进行Fc受体(FcR)结合试验，以确保Fc部分或NGF突变体与Fc的融合蛋白缺乏FcγR结合(因此可能缺乏ADCC活性)，但保留FcRn结合能力。介导ADCC的主 要细胞，自然杀伤细胞(NK)，仅表达FcγRIII，然而单核细胞表达FcγRI、FcγRII和FcγRIII。Ravetch和Kinet,Annu.Rev.Immunol.9:457-492(1991)第464页的表2中汇总了FcR在造血细胞上的表达。U.S.Patent No.5,500,362(参见Hellstrom,I.et al.,Proc.Nat’l Acad.Sci.USA 83:7059-7063(1986))和Hellstrom,I.et al.,Proc.Nat’l Acad.Sci.USA 82:1499-1502(1985)；5,821,337(见于Bruggemann,M.et al.,J.Exp.Med.166:1351-1361(1987))中详述了用于评估目标分子ADCC活性的体外试验的非限制性示例。或者，可采用非放射性检测方法(参见用于流式细胞术的ACTITM非放射性毒性试验(CellTechnology,Inc.Mountain View,CA)和CytoTox非放射性毒性试验(Promega,Madison,WI))。适用于这种检测的效应细胞包括外周血单核细胞(PBMC)和NK细胞。此外，也可以在体内评估目标分子的ADCC活性，例如，在如Clynes et al.,Proc.Nat’l Acad.Sci.USA 95:652-656(1998)所公开的动物模型中。也可进行C1q结合试验以确定长效NGF多肽不能与C1q结合并因此缺失CDC活性。参见WO 2006/029879和WO 2005/100402中C1q和C3c结合酶联免疫吸附试验。可以进行CDC试验以评估补体活性(参见Gazzano-Santoro et al.,J.Immunol.Methods 202:163(1996)；Cragg,M.S.et al.,Blood 101:1045-1052(2003)和Cragg,M.S.和M.J.Glennie,Blood 103:2738-2743(2004))。可以利用本领域已知的方法进行FcRn结合和体内清除/半衰期测定(参见Petkova,S.B.et al..,Int’l.Immunol.18(12):1759-1769(2006))。In some embodiments, the present application provides an Fc that has some but not all of the effector functions of Fc, making it an ideal candidate for a certain application, for example, in which the half-life of the fusion protein of NGF mutant and Fc in vivo is important, but certain effector functions (such as CDC and ADCC) are non-essential or deleterious. Cytotoxicity assays can be performed in vitro or in vivo to determine the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the Fc portion or the fusion protein of NGF mutant and Fc lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The main effector functions that mediate ADCC are The main cell, natural killer (NK) cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). US Patent No. 5,500,362 (see Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)) details non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules. Alternatively, non-radioactive detection methods (see ACTI TM non-radioactive toxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA) and CytoTox Non-radioactive toxicity test (Promega, Madison, WI). Effector cells suitable for such an assay include peripheral blood mononuclear cells (PBMC) and NK cells. In addition, ADCC activity of the target molecule can also be assessed in vivo, for example, in an animal model as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to determine that the long-acting NGF polypeptide cannot bind to C1q and therefore lacks CDC activity. See WO 2006/029879 and WO 2005/100402 for C1q and C3c binding enzyme-linked immunosorbent assays. 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 assays can be performed using methods known in the art (see Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).
效应功能降低的Fc部分包含那些在Fc区第238、265、269、270、297、327和329位中一个或多个残基的取代(U.S.专利No.6,737,056)。这类Fc突变体包括在氨基酸位点265、269、270、297和327中的两个或更多位点上的取代，包括所谓的将265和297残基替换成丙氨酸的“DANA”Fc突变体(US专利No.7,332,581)。详述了增强或减少与FcRs的结合的某些抗体变体(参见U.S.专利No.6,737,056；WO 2004/056312,和Shields et al.,J.Biol.Chem.9(2):6591-6604(2001)。在一些实施例中，改造Fc区以改变(即，增加或减少)C1q结合和/或CDC，例如US专利No.6,194,551、WO 99/51642和Idusogie et al.,J.Immunol.164:4 178-4184(2000)中所述的。The Fc portion with reduced effector function comprises those substitutions of one or more residues in positions 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutants in which residues 265 and 297 are replaced with alanine (U.S. Patent No. 7,332,581). Certain antibody variants that enhance or reduce binding to FcRs are described in detail (see U.S. 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 is engineered to alter (i.e., increase or decrease) C1q binding and/or CDC, such as described in U.S. Patent No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164:4 178-4184 (2000).
在一些实施例中，Fc部分包含一个或多个氨基酸替换，这增加了半衰期和/或增强了与新生儿Fc受体(FcRn)的结合。半衰期增加和与新生儿FcRn结合增强了的抗体负责将母体IgGs转运给胎儿(Guyer et al.,J.Immunol.117:587(1976)和Kim et al.,J.Immunol.24:249(1994))，并在US2005/0014934A1(Hinton等)中进行了详述。那些 包含带有一个或多个替换的Fc部分的抗体因此增加了Fc部分与FcRn的结合。这类Fc变体包括那些带有一个或多个Fc区残基替换的变体，例如，Fc区434残基替换(US专利No.7,371,826)。In some embodiments, the Fc portion comprises one or more amino acid substitutions that increase half-life and/or enhance binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and enhanced binding to neonatal FcRn are responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and are described in detail in US 2005/0014934A1 (Hinton et al.). Antibodies comprising an Fc portion with one or more substitutions thus have increased binding of the Fc portion to FcRn. Such Fc variants include those with one or more Fc region residue substitutions, for example, Fc region 434 residue substitutions (US Patent No. 7,371,826).
参见Duncan和Winter,Nature 322:738-40(1988)；U.S.专利No.5,648,260；U.S.专利No.5,624,821；和WO 94/29351关于Fc结构域变体的其它示例。See Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 for other examples of Fc domain variants.
半胱氨酸工程变体Cysteine engineered variants
在一些实施例中，NGF突变体与Fc的融合蛋白中可能需要创建半胱氨酸工程的Fc部分，其中Fc部分的一个或多个残基被半胱氨酸残基取代。在一些实施例中，取代残基出现在Fc部分或NGF突变体与Fc的融合蛋白上易接近的位点。通过将这些残基替换成半胱氨酸，活性硫醇基因此被定位在Fc部分或NGF突变体与Fc的融合蛋白的易接近位点，并可用于将分子与其它部分共轭，如药物部分或接头-药物部分，以创建NGF突变体与Fc的融合蛋白共轭物。在一些实施例中，下列残基中的任一个和多个可能被半胱氨酸取代：重链A118(EU编号系统)和重链Fc结构域S400(EU编号系统)。半胱氨酸工程分子可以按照U.S.Patent No.7,521,541中所述的那样产生。In some embodiments, it may be desirable to create a cysteine engineered Fc portion in a fusion protein of an NGF mutant and Fc, wherein one or more residues of the Fc portion are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites on the Fc portion or the fusion protein of an NGF mutant and Fc. By replacing these residues with cysteine, the active thiol groups are thereby positioned at accessible sites on the Fc portion or the fusion protein of an NGF mutant and Fc and can be used to conjugate the molecule to other moieties, such as a drug moiety or a linker-drug moiety, to create a fusion protein conjugate of an NGF mutant and Fc. In some embodiments, any one and more of the following residues may be substituted with cysteine: heavy chain A118 (EU numbering system) and heavy chain Fc domain S400 (EU numbering system). Cysteine engineered molecules can be produced as described in U.S. Patent No. 7,521,541.
接头Connectors
在一些实施例中，野生型NGF或NGF突变体和Fc之间通过一个可选的接头(例如，肽接头、非肽接头)连接。在一些实施例中，该接头为一种柔性接头。在一些实施例中，该接头为一种稳定接头。一般来说，理想的接头不会影响或显著影响本文所述的NGF突变体与Fc的融合蛋白的正确折叠和构象。优选地，该接头赋予了NGF突变体与Fc的融合蛋白灵活性，保留或提高了NGF的生物学活性，和/或不显著影响NGF突变体与Fc的融合蛋白在体内的半衰期和/或稳定性。在一些实施例中，接头为稳定接头(例如，不能被蛋白酶切割，特别是MMP切割)。In some embodiments, the wild-type NGF or NGF mutant and Fc are connected 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. In general, an ideal linker will not affect or significantly affect the correct folding and conformation of the fusion protein of the NGF mutant and Fc described herein. Preferably, the linker confers flexibility to the fusion protein of the NGF mutant and Fc, retains or improves the biological activity of NGF, and/or does not significantly affect the half-life and/or stability of the fusion protein of the NGF mutant and Fc in vivo. In some embodiments, the linker is a stable linker (e.g., cannot be cleaved by a protease, especially a MMP).
在一些实施例中，该接头为肽接头。肽接头可以是任意长度。在一些实施例中，肽接头长度为从1个到10个氨基酸，从3个到18个氨基酸，从1个到20个氨基酸，从10个到20个氨基酸，从21个到30个氨基酸，从1个到30个氨基酸，从10个到30个氨基酸，从1个到50个氨基酸，从5个到40个氨基酸，从12个到18个氨基酸，从4个到25个氨基酸。在一些实施例中，该肽接头长度为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个氨基酸中的任何一个。在一些 实施例中，该肽接头长度为21、22、23、24、25、26、27、28、29或30个氨基酸中的任何一个。在一些实施例中，该肽接头长度为31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个氨基酸中的任何一个。优选地，如本申请所述的NGF突变体与Fc的融合蛋白在体内的功能和稳定性通过添加肽接头来优化，以防止潜在的不期望的结构域的相互作用。在一些实施例中，接头长度不超过防止不期望的结构域相互作用和/或优化生物功能和/或稳定性所必需的长度。在一些实施例中，肽接头的长度最多为30个氨基酸，例如最多20个氨基酸，或最多15个氨基酸。在一些实施例中，接头长度为5个到30个氨基酸，或5个到15个氨基酸。In some embodiments, the linker is a peptide linker. The peptide linker can be of any length. In some embodiments, the peptide linker is from 1 to 10 amino acids, from 3 to 18 amino acids, from 1 to 20 amino acids, from 10 to 20 amino acids, from 21 to 30 amino acids, from 1 to 30 amino acids, from 1 to 30 amino acids, from 10 to 30 amino acids, from 1 to 50 amino acids, from 5 to 40 amino acids, from 12 to 18 amino acids, from 4 to 25 amino acids in length. In some embodiments, 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 length. In some In an embodiment, the peptide linker length is any one of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. In some embodiments, the peptide linker length 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. Preferably, the function and stability of the fusion protein of NGF mutant and Fc as described in the present application in vivo are optimized by adding a peptide linker to prevent the interaction of potential undesirable domains. In some embodiments, the linker length does not exceed the length required to prevent undesirable domain interactions and/or optimize biological function and/or stability. In some embodiments, the length of the peptide linker is up to 30 amino acids, for example, 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 15 amino acids.
肽接头可以具有天然存在的序列，也可以具有非天然存在的序列。例如，可以使用来自抗体重链铰链区的序列作为接头。例如，参见WO1996/34103。在一些实施例中，肽接头为人类IgG1、IgG2、IgG3或IgG4铰链。在一些实施例中，肽接头为突变的人IgG1、IgG2、IgG3或IgG4铰链。在一些实施例中，接头是柔性接头。典型的柔性接头富含甘氨酸-丝氨酸聚合物(GGGGS)n，其中n为至少是1的整数，例如n为1、2、3、4、5或6中的一个整数，优选地n为2到6中的整数，更优选地n为整数3或4)、甘氨酸-丙氨酸聚合物、丙氨酸-丝氨酸聚合物和其它本领域已知的柔性接头。甘氨酸和甘氨酸-丝氨酸聚合物为相对非结构化的，并且因此可以作为组分之间的一种中性链。甘氨酸比丙氨酸拥有更多的phi-psi空间，且相比具有更长侧链的残基，受到的限制更少(见于Scheraga,Rev.Computational Chem.11 173-142(1992))。柔性接头的示例包括但不限于GGGGSGGGGSGGGGS(SEQ ID NO:87)等。一般来说，本领域技术人员会意识到所设计的NGF突变体与Fc的融合蛋白可以包括全部或部分柔性接头，使得接头可以包括一个柔性接头部分以及一个或多个提供较少柔性结构的部分，以提供一个理想的NGF突变体与Fc的融合蛋白的结构和功能。Peptide linkers can have naturally occurring sequences or non-naturally occurring sequences. For example, sequences from the hinge region of the antibody heavy chain can be used as linkers. For example, see WO1996/34103. In certain embodiments, peptide linkers are human IgG1, IgG2, IgG3 or IgG4 hinges. In certain embodiments, peptide linkers are mutated human IgG1, IgG2, IgG3 or IgG4 hinges. In certain embodiments, linkers are flexible linkers. Typical flexible linkers are rich in glycine-serine polymers (GGGGS) n, wherein n is an integer of at least 1, such as n is an integer in 1, 2, 3, 4, 5 or 6, preferably n is an integer in 2 to 6, more preferably n is an integer 3 or 4), glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore can be used as a kind of neutral chain between components. Glycine has more phi-psi space than alanine and is less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Examples of flexible linkers include, but are not limited to, GGGGSGGGGSGGGGS (SEQ ID NO: 87), etc. In general, those skilled in the art will recognize that the designed fusion protein of NGF mutant and Fc may include all or part of a flexible linker, so that the linker may include a flexible linker portion and one or more portions that provide a less flexible structure to provide an ideal structure and function of a fusion protein of NGF mutant and Fc.
其它关于接头的考虑因素包括对产生的NGF突变体与Fc的融合蛋白的物理或药代动力学性质的影响，例如溶解性、亲脂性、亲水性、疏水性、稳定性(或多或少的稳定性以及计划内的降解)、刚性、柔韧性、免疫原性、NGF与受体结合、胶体或脂质体的结合能力等。Other considerations regarding linkers include the effect on the physical or pharmacokinetic properties of the resulting NGF mutant and Fc fusion protein, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stability and planned degradation), rigidity, flexibility, immunogenicity, NGF binding to receptors, ability to bind to colloids or liposomes, etc.
药代动力学(PK)Pharmacokinetics (PK)
药代动力学(PK)是指药物(例如，本文所述的NGF突变体或包含NGF突变体的融合蛋白)在向受试者施用后的吸收、分布、代谢和排泄。可用于确定临床效用 的药代动力学参数包括但不限于血清/血浆浓度、随时间变化的血清/血浆浓度、最大血清/血浆浓度(Cmax)、达到最大浓度的时间(Tmax)、半衰期(t1/2)、给药间隔内浓度-时间曲线下的面积(AUCτ)等。Pharmacokinetics (PK) refers to the absorption, distribution, metabolism, and excretion of a drug (e.g., an NGF mutant or a fusion protein comprising an NGF mutant described herein) after administration to a subject. Can be used to determine clinical efficacy The pharmacokinetic parameters include, but are not limited to, serum/plasma concentration, serum/plasma concentration changing with time, maximum serum/plasma concentration (C max ), time to reach maximum concentration (T max ), half-life (t 1/2 ), area under the concentration-time curve within the dosing interval (AUC τ ), etc.
在一些实施例中，在个体的血液、血浆或血清样本中测量如本文所述的NGF、NGF突变体或其融合蛋白的PK曲线。在一些实施例中，使用质谱技术(例如，LC-MS/MS或ELISA)测量个体中如本文所述的NGF突变体或NGF突变体与Fc的融合蛋白多肽的PK曲线。可通过本领域已知的任何方法在PK曲线上进行PK分析，例如，非室间分析，使用PKSolver V2软件(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)。In some embodiments, the PK curve of NGF, NGF mutants or their fusion proteins as described herein is measured in individual blood, plasma or serum samples. In some embodiments, the PK curve of NGF mutants or fusion protein polypeptides of NGF mutants and Fc as described herein is measured in individuals using mass spectrometry (e.g., LC-MS/MS or ELISA). PK analysis can be performed on the PK curve by any method known in the art, for example, non-compartmental analysis, using PKSolver V2 software (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).
“C”表示受试者血浆、血清或任何合适的体液或组织中的药物(例如，NGF突变体与Fc的融合蛋白)浓度，通常表示为每单位体积的质量，例如纳克/毫升。为方便起见，血清或血浆中的药物浓度在本文中称为“血清浓度”或“血浆浓度”。给药后任何时间(例如，NGF突变体与Fc的融合蛋白，例如静脉注射、腹腔注射或皮下注射)的血清/血浆浓度称为Ctime或Ct。给药期间的最大血清/血浆药物浓度被称为Cmax；Cmin是指给药间隔结束时的最小血清/血浆药物浓度；Cave指给药间隔期间的平均浓度。"C" represents the concentration of the drug (e.g., a fusion protein of an NGF mutant and Fc) in the subject's plasma, serum, or any suitable body fluid or tissue, usually expressed as mass per unit volume, such as nanograms per milliliter. For convenience, the drug concentration in serum or plasma is referred to herein as "serum concentration" or "plasma concentration." The serum/plasma concentration at any time after administration (e.g., a fusion protein of an NGF mutant and Fc, such as intravenous, intraperitoneal, or subcutaneous injection) is referred to as C time or C t . The maximum serum/plasma drug concentration during administration is referred to as C max ; C min refers to the minimum serum/plasma drug concentration at the end of the dosing interval; Cave refers to the average concentration during the dosing interval.
术语“生物利用度”是指药物(例如，NGF突变体与Fc的融合蛋白)通过体循环，从而进入作用部位的程度或速率。The term "bioavailability" refers to the extent or rate at which a drug (eg, a fusion protein of an NGF mutant and Fc) passes through systemic circulation and thereby enters the site of action.
“AUC”是血清/血浆浓度-时间曲线下的面积，被认为是对生物利用度最可靠的测量方式，如给药间隔内浓度-时间曲线下的面积(AUCτ)，“总暴露”或“一段时间内的总药物暴露”(AUC0-last或AUC0-inf)，给药后t时间的浓度-时间曲线下的面积(AUC0-t)等。"AUC" is the area under the serum/plasma concentration-time curve and is considered to be the most reliable measure of bioavailability, such as the area under the concentration-time curve during the dosing interval (AUCτ), "total exposure" or "total drug exposure over a period of time" (AUC 0-last or AUC 0-inf ), the area under the concentration-time curve at time t after dosing (AUC 0-t ), etc.
血清/血浆浓度峰值时间(Tmax)是给药(例如，NGF突变体与Fc的融合蛋白)后达到血清/血浆浓度(Cmax)峰值的时间。The peak serum/plasma concentration time (T max ) is the time to reach the peak serum/plasma concentration (C max ) after administration (eg, a fusion protein of an NGF mutant and Fc).
半衰期(t1/2)是指在血浆或血清(或其它生物基质)中测得的药物浓度(例如，NGF突变体与Fc的融合蛋白)降至其在特定时间点的浓度或量的一半所需的时间。例如，静脉给药后，由于药物的分布和消除，血浆或血清中的药物浓度下降。在静脉给药后血浆或血清药物浓度随时间变化的曲线中，第一阶段或快速下降阶段被认为主 要是由于分布导致，而后期的下降通常较慢，主要是由于消除导致，尽管这两个过程在这两个阶段都会发生。分布被认为是在足够的时间后完成的。一般来讲，消除半衰期由血浆/血清浓度-时间曲线的终末期或消除(主要)阶段决定。参见Michael Schrag和Kelly Regal，“临床前药物开发毒理学综合指南”的“第3章-药代动力学和毒代动力学”，2013。The half-life (t 1/2 ) is the time required for the concentration of a drug (e.g., a fusion protein of a NGF mutant and Fc) measured in plasma or serum (or other biological matrix) to drop to half of its concentration or amount at a specific time point. For example, after intravenous administration, the concentration of the drug in plasma or serum decreases due to the distribution and elimination of the drug. In the curve of plasma or serum drug concentration over time after intravenous administration, the first phase or rapid decline phase is considered to be the main The majority of the decline is due to distribution, while the later decline is usually slower and is primarily due to elimination, although both processes occur during both phases. Distribution is considered complete after sufficient time has elapsed. In general, the elimination half-life is determined by the terminal or elimination (primary) phase of the plasma/serum concentration-time curve. See Michael Schrag and Kelly Regal, “Chapter 3 - Pharmacokinetics and Toxicokinetics” of “A Comprehensive Guide to Toxicology in Preclinical Drug Development,” 2013.
在一些实施例中，本文所述NGF突变体与Fc的融合蛋白具有至少5小时的半衰期(例如，静脉注射、皮下注射或肌肉注射，如给人类注射)，如至少为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或300小时中的任一个，或更长。In some embodiments, the fusion protein of the NGF mutant and Fc described herein has a half-life of at least 5 hours (e.g., intravenous, subcutaneous or intramuscular injection, such as injection into humans), such as at least any 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 longer.
在一些实施例中，本文所述的NGF突变体与Fc的融合蛋白的循环半衰期至少为相应的NGF部分(即，只包含相同的NGF突变体，而不与Fc融合)或野生型NGF的循环半衰期的5倍，(例如，6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40、50、60、70、80、90或100倍中的任一个，或以上)。In some embodiments, the circulating half-life of the fusion protein of the NGF mutant and Fc described herein is at least 5 times (e.g., any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 times, or more) the circulating half-life of the corresponding NGF portion (i.e., comprising only the same NGF mutant without fusion with Fc) or wild-type NGF.
稳定性stability
在一些实施例中，本文所述的野生型NGF、NGF突变体或包含其的融合蛋白(可在不同的浓度下)具有优异的稳定性，例如物理稳定性、化学稳定性和/或生物稳定性。在一些实施例中，本文所述的NGF突变体与Fc的融合蛋白具有优异的热稳定性，例如高熔解温度(Tm)和/或高聚集起始温度(Tagg)。在一些实施例中，本文所述NGF突变体与Fc的融合蛋白在加速应力(例如，高温)下具有优异的稳定性，例如较少或没有片段化、聚集体形成和/或聚集体增量。In some embodiments, the wild-type NGF, NGF mutants, or fusion proteins comprising the same (which may be at different concentrations) described herein have excellent stability, such as physical stability, chemical stability, and/or biological stability. In some embodiments, the fusion proteins of the NGF mutants and Fc described herein have excellent thermal stability, such as high melting temperature (Tm) and/or high aggregation initiation temperature (Tagg). In some embodiments, the fusion proteins of the NGF mutants and Fc described herein have excellent stability under accelerated stress (e.g., high temperature), such as less or no fragmentation, aggregate formation, and/or aggregate increment.
蛋白质的稳定性，特别是对聚集的敏感性，主要取决于蛋白质分子的构象和胶体稳定性。一般认为，非天然蛋白质聚集的第一步，即最普遍的聚集形式，是分子结构的轻微扰动，例如，蛋白质的部分去折叠，即构象变化。这是由蛋白质的构象稳定性决定的。在第二步中，部分未折叠的分子在扩散和随机布朗运动的驱动下靠近，形成聚集。第二步主要由分子的胶体稳定性决定(参见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)。如本文所用，术语“稳定性”通常是指维持生物活性物质(如蛋白质)的完整性或尽量减少其降解、变性、聚集或去折叠。如本文所用，“改良的稳定性”通常意味着，在已知会导致降解、变性、 聚集或去折叠的条件下，与对照组蛋白质(例如，其它NGF-Fc融合蛋白)相比，目标蛋白质(例如，本文所述的NGF突变体与Fc的融合蛋白)保持更佳的稳定性。The stability of proteins, especially their sensitivity to aggregation, depends mainly on the conformation and colloidal stability of protein molecules. It is generally believed that the first step of non-native protein aggregation, i.e., the most common form of aggregation, is a slight perturbation of the molecular structure, such as partial unfolding of the protein, i.e., conformational change. This is determined by the conformational stability of the protein. In the second step, partially unfolded molecules approach each other under the drive of diffusion and random Brownian motion to form aggregates. The second step is mainly determined by the colloidal stability of the molecules (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 biologically active substances (such as proteins) or minimizing their degradation, denaturation, aggregation, or unfolding. As used herein, "improved stability" generally means that under conditions known to cause degradation, denaturation, Under conditions of aggregation or unfolding, the target protein (eg, the fusion protein of the NGF mutant described herein and Fc) maintains better stability compared to a control protein (eg, other NGF-Fc fusion proteins).
差示扫描量热法(DSC)和差示扫描荧光法(DSF)是本领域众所周知的技术，用于预测蛋白质制剂的稳定性。具体而言，这些技术可用于确定给定制剂中蛋白质的去折叠温度(Tm)。将给定制剂中蛋白质的高Tm测量值与可用于长期、稳定储存的更可靠和稳定的蛋白质制剂相关联是本领域的标准做法。Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) are well known techniques in the art for predicting the stability of protein formulations. Specifically, these techniques can be used to determine the unfolding temperature (Tm) of a protein in a given formulation. It is standard practice in the art to associate a high Tm measurement for a protein in a given formulation with a more reliable and stable protein formulation that can be used for long-term, stable storage.
一种“稳定的”蛋白质(或制剂)，例如，本文所述的长效NGF多肽，基本上在制造过程中和/或储存时保持其物理稳定性和/或化学稳定性和/或生物活性。本领域有多种用于测量蛋白质稳定性的分析技术，并在Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed.,Marcel Dekker,Inc.,New York,N.Y.,Pubs.(1991)和Jones,A.(1993)Adv.Drug Delivery Rev.10:29-90。10:29-90中进行了综述。例如，在一个实施例中，蛋白质的稳定性根据溶液中单体蛋白质的百分比确定，其中降解(例如，片段化)和/或聚集蛋白质的百分比较低。优选地，蛋白质(或制剂)在室温(约30℃)或40℃下稳定至少1个月和/或在约2-8℃下稳定至少6个月，或至少1年或至少2年。此外，该蛋白质(或制剂)优选在冷冻(例如-70℃)和融解后稳定，以下称为“冻/融循环”。A "stable" protein (or formulation), e.g., a long-acting NGF polypeptide as described herein, substantially maintains its physical stability and/or chemical stability and/or biological activity during manufacturing and/or storage. There are a variety of analytical techniques in the art for measuring protein stability and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90. 10:29-90. For example, in one embodiment, the stability of a protein is determined based on the percentage of monomeric protein in a solution, wherein the percentage of degraded (e.g., fragmented) and/or aggregated protein is lower. Preferably, the protein (or formulation) is stable at room temperature (about 30°C) or 40°C for at least 1 month and/or at about 2-8°C for at least 6 months, or at least 1 year or at least 2 years. In addition, the protein (or formulation) is preferably stable after freezing (e.g. -70°C) and thawing, hereinafter referred to as "freeze/thaw cycle".
一种蛋白质，例如，本文所述的NGF突变体与Fc的融合蛋白，如果在颜色和/或透明度的目视检查或通过紫外光散射或尺寸排阻色谱法测量时，基本上没有不稳定迹象，例如聚集、沉淀和/或变性，则这种蛋白质在制剂中“保持其物理稳定性”。聚集是单个蛋白质分子或复合物共价或非共价结合形成聚集体的过程。聚集可以进行到形成可见沉淀的程度。A protein, e.g., a fusion protein of an NGF mutant and Fc described herein, "retains its physical stability" in a formulation if it shows substantially no signs of instability, such as aggregation, precipitation, and/or denaturation, when measured by visual inspection of color and/or clarity or by UV light scattering or size exclusion chromatography. Aggregation is the process by which individual protein molecules or complexes covalently or non-covalently associate to form aggregates. Aggregation can proceed to the extent that a visible precipitate forms.
一种蛋白质，例如，本文所述的NGF突变体与Fc的融合蛋白，如果在给定时间内的化学稳定性使得该蛋白质仍然保持其生物活性(例如，如上文“生物活性”小节所述)，则该蛋白质在制剂中“保持其化学稳定性”。化学稳定性可以通过例如检测和量化蛋白质的化学变化形式来评估。化学变化可能涉及尺寸改变(例如，剪切)，可使用尺寸排阻色谱法、SDS-PAGE和/或基质辅助激光解吸电离/飞行时间质谱(MALDI/TOF MS)进行评估。其它类型的化学变化包括电荷变化(例如，由于脱酰胺或氧化而发生的变化)，例如，可通过离子交换色谱法进行评估。A protein, e.g., a fusion protein of an NGF mutant and Fc described herein, "retains its chemical stability" in a formulation if the chemical stability over a given period of time is such that the protein still retains its biological activity (e.g., as described above in the "Biological Activity" section). Chemical stability can be assessed, for example, by detecting and quantifying chemically altered forms of the protein. Chemical changes may 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 changes include charge changes (e.g., changes due to deamidation or oxidation), which can be assessed, for example, by ion exchange chromatography.
一种蛋白质，例如，本文所述的NGF突变体与Fc的融合蛋白，如果药物制剂中的蛋白质对于其预期目的具有生物活性，则该蛋白质在制剂中“保留其生物活性”。 例如，如果制剂中蛋白质的生物学活性为在制备制剂时显示的生物活性的30％、20％或10％(在分析误差范围内)内，则该蛋白保留了其生物活性。A protein, for example, a fusion protein of an NGF mutant and Fc described herein, "retains its biological activity" in a pharmaceutical formulation if the protein in the formulation is biologically active for its intended purpose. For example, a protein has retained its biological activity if the biological activity of the protein in the formulation is within 30%, 20%, or 10% (within the analytical error) of the biological activity exhibited when the formulation was prepared.
本领域技术人员已知，蛋白质(例如，本文所述的NGF突变体与Fc的融合蛋白)的稳定性除制剂的组成外还取决于其它特性。例如，稳定性可能受到温度、压力、湿度、pH值和外部辐射的影响。蛋白质制剂中蛋白质(例如，本文所述的NGF突变体与Fc的融合蛋白)的稳定性可通过多种方法确定。在一些实施例中，通过尺寸排阻色谱法(SEC)确定蛋白质稳定性。SEC根据分析物(例如，蛋白质等大分子)的流体力学尺寸、扩散系数和表面性质来分离分析物。因此，例如，SEC可将本文所述的天然三维构象的NGF突变体与Fc的融合蛋白与处于各种变性状态的蛋白质和/或已降解的蛋白质分离。在SEC中，固定相通常由填充在玻璃或钢柱内致密的三维基质中的惰性颗粒组成。流动相可以是纯水、水性缓冲液、有机溶剂、它们的混合物或其它溶剂。固定相颗粒具有小孔和/或通道，仅允许小于一定尺寸的物质进入。因此，大颗粒被排除在这些孔隙和通道之外，但较小颗粒从流动相中转移。颗粒被固定在固定孔隙中的时间在一定程度上取决于它们能渗透到孔隙中的深度。它们从流动相液流中转移会导致其从色谱柱中洗脱所需的时间更长，因此颗粒之间基于其大小差异而进行分离。It is known to those skilled in the art that the stability of proteins (e.g., the fusion proteins of NGF mutants and Fc described herein) depends on other characteristics in addition to the composition of the formulation. For example, stability may be affected by temperature, pressure, humidity, pH, and external radiation. The stability of proteins (e.g., the fusion proteins of NGF mutants and Fc described herein) in protein formulations can be determined by a variety of methods. In some embodiments, protein stability is determined by size exclusion chromatography (SEC). SEC separates analytes according to their hydrodynamic size, diffusion coefficient, and surface properties. Thus, for example, SEC can separate the fusion proteins of NGF mutants and Fc described herein in their native three-dimensional conformation from proteins in various denatured states and/or degraded proteins. In SEC, the stationary phase is typically composed of inert particles filled in a dense three-dimensional matrix in a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, a mixture thereof, or other solvents. The stationary phase particles have pores and/or channels that only allow substances smaller than a certain size to enter. Therefore, large particles are excluded from these pores and channels, but smaller particles are displaced from the mobile phase. The amount of time that particles are immobilized in the fixed pores depends in part on how deeply they can penetrate into the pores. Their displacement from the mobile phase flow causes them to take longer to elute from the column, thus separating the particles based on their size differences.
在一些实施例中，SEC与鉴定技术相结合，以鉴定或表征蛋白质(例如，本文所述的NGF突变体)或其片段。蛋白质鉴定和表征可通过多种技术完成，包括但不限于色谱技术，例如高效液相色谱(HPLC)、十二烷基硫酸钠毛细管电泳(CE-SDS)、免疫分析、电泳、紫外/可见/红外光谱、拉曼光谱、表面增强拉曼光谱、质谱、气相色谱、静态光散射(SLS)、傅里叶变换红外光谱(FTIR)、圆二色谱(CD)、尿素诱导蛋白质去折叠技术、固有色氨酸荧光、差示扫描量热法和/或ANS蛋白结合。In some embodiments, SEC is combined with identification techniques to identify or characterize proteins (e.g., NGF mutants described herein) or fragments thereof. Protein identification and characterization can be accomplished by a variety of techniques, including but not limited to chromatographic techniques, such as high performance liquid chromatography (HPLC), capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), immunoassays, electrophoresis, UV/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.
稳定性，例如组合物或制剂的物理稳定性，可通过本领域众所周知的方法评估，包括测量样品的表观光衰减(吸光度或光密度)。这种光衰减测量与制剂的浑浊度有关。制剂的浑浊度是溶解在溶液中的蛋白质的固有特性，通常通过比浊法测定，并以比浊法浊度单位(NTU)测量。Stability, such as the physical stability of a composition or formulation, can be assessed by methods well known in the art, including measuring the apparent optical attenuation (absorbance or optical density) of a sample. Such optical attenuation measurements are related to the turbidity of the formulation. The turbidity of a formulation is an inherent property of proteins dissolved in solution, typically determined by turbidimetry and measured in nephelometric turbidity units (NTU).
浊度，例如，作为溶液中一种或多种成分浓度的函数，例如，蛋白质和/或盐浓度，也被称为制剂的“乳白色”或“乳白色外观”。浊度可以通过使用已知浊度的悬浮液生成的标准曲线来计算。确定药物组合物浑浊度的参考标准可基于欧洲药典标准(欧洲药典，第四版，欧洲委员会药品质量理事会(EDQM)，斯特拉斯堡，法 国)。根据欧洲药典标准，澄清溶液定义为浊度小于或等于对照悬浮液。根据欧洲药典标准，该对照悬浮液的浊度约为3。在没有关联或非理想效应的情况下，浊度测量可以检测到瑞利散射，瑞利散射通常随浓度线性变化。用于评估药物蛋白质的物理稳定性的其它方法在本领域是公知的，例如，尺寸排阻色谱法或分析性超速离心法。Turbidity, for example, as a function of the concentration of one or more components in a solution, e.g., protein and/or salt concentration, is also referred to as the "milky" or "milky appearance" of a formulation. Turbidity can be calculated using a standard curve generated using suspensions of known turbidity. Reference standards for determining the turbidity of a pharmaceutical composition can be based on European Pharmacopoeia standards (European Pharmacopoeia, 4th edition, European Commission Directorate for the Quality of Medicines (EDQM), Strasbourg, France). According to the European Pharmacopoeia, a clear solution is defined as having a turbidity less than or equal to that of a control suspension. According to the European Pharmacopoeia, the turbidity of the control suspension is about 3. In the absence of correlation or non-ideal effects, turbidity measurements can detect Rayleigh scattering, which generally varies linearly with concentration. Other methods for evaluating the physical stability of pharmaceutical proteins are well known in the art, for example, size exclusion chromatography or analytical ultracentrifugation.
在一些实施例中，稳定性指含有本文所述的NGF突变体与Fc的融合蛋白的制剂具有低至检测不到的微粒形成水平。本文所用的短语“低至检测不到的微粒形成水平”是指通过HIAC分析或目视分析确定，含有小于30个微粒/ml、小于20个微粒/ml、小于15个微粒/ml、小于10个微粒/ml、小于5个微粒/ml、小于2粒/ml或小于1粒/ml的样品。在一些实施例中，通过HIAC分析或目视分析未检测到NGF突变体与Fc的融合蛋白制剂中的微粒。In some embodiments, stability refers to a formulation containing a fusion protein of an NGF mutant and Fc as described herein having a level of particle formation so low that it is undetectable. The phrase "level of particle formation so low that it is undetectable" as used herein refers to a sample 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, particles in the fusion protein formulation of an NGF mutant and Fc are not detected by HIAC analysis or visual analysis.
NGF突变体与Fc的融合蛋白衍生物Fusion protein derivatives of NGF mutants and Fc
在一些实施例中，本文所涉及的NGF突变体与Fc的融合蛋白可被进一步修饰，以包含本领域已知且容易获得的额外的非蛋白部分。适于NGF突变体与Fc的融合蛋白衍生物的非蛋白部分包括但不限于水溶性聚合物。水溶性聚合物的非限制性示例包括但不限于聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二氧环戊烷、聚-1,3,6-三氧戊烷、乙烯/马来酸酐共聚物，聚酰胺酸(均聚物或无规共聚物)、葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、环氧丙烷/环氧乙烷共聚物、聚氧乙烯基多元醇(例如甘油)、聚乙烯醇及其混合物。由于聚乙二醇丙醛在水中的稳定性，其在制造中可能具有优势。聚合物可以是任何分子量的，并且可以是支链或非支链的。连接到NGF突变体与Fc的融合蛋白的聚合物的数量可能不同，如果连接了多个聚合物，则它们可以是相同或不同的分子。一般来说，用于衍生化的聚合物的数量和/或类型可基于以下考虑来确定，包括但不限于待改进的NGF突变体与Fc的融合蛋白的特定性质或功能，NGF突变体与Fc的融合蛋白衍生物是否会在特定条件下用于治疗等。In some embodiments, the fusion protein of NGF mutant and Fc involved herein can be further modified to include additional non-protein parts known in the art and easily obtained. Non-protein parts suitable for fusion protein derivatives of NGF mutant and Fc 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxolane, ethylene/maleic anhydride copolymers, polyamic acid (homopolymer or random copolymer), dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylene polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Due to the stability of polyethylene glycol propionaldehyde in water, it may have advantages in manufacturing. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the fusion protein of NGF mutant and Fc may vary, and if multiple polymers are attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions of the fusion protein of NGF mutant and Fc to be improved, whether the fusion protein derivative of NGF mutant and Fc will be used for treatment under specific conditions, etc.
在一些实施例中，本文所述的NGF突变体与Fc的融合蛋白进一步包含标签，所述标签选自发色团、荧光团(例如，香豆素、氧杂蒽、菁、芘、硼聚苯并氮杂吲哚、恶嗪及其衍生物)、荧光蛋白(例如GFP、藻胆蛋白及其衍生物)，磷光染料(例如，二氧杂环丁烷、氧杂蒽或碳菁染料、镧系螯合物)、串联染料(例如，菁-藻胆蛋白衍生物和氧杂蒽-藻胆蛋白衍生物)、粒子(例如，金簇、胶体金、微球、量子点)、半 抗原、酶(例如，过氧化物酶、磷酸酶、糖苷酶、荧光素酶)和放射性同位素(例如，125I、3H、14C、32P)。In some embodiments, the fusion protein of the NGF mutant and Fc described herein further comprises a tag, wherein the tag is selected from a chromophore, a fluorophore (e.g., coumarin, xanthene, cyanine, pyrene, boron polybenzazaindole, oxazine and its derivatives), a fluorescent protein (e.g., GFP, phycobiliprotein and its derivatives), a phosphorescent dye (e.g., dioxetanes, xanthenes or carbocyanine dyes, lanthanide chelates), a tandem dye (e.g., cyanine-phycobiliprotein derivatives and xanthene-phycobiliprotein derivatives), a particle (e.g., gold clusters, colloidal gold, microspheres, quantum dots), a semi-transparent fluorescent protein (e.g., a fluorescent protein such as GFP, phycobiliprotein and its derivatives), a fluorescent protein such as GFP, a phycobiliprotein and its derivatives, ... Antigens, enzymes (eg, peroxidase, phosphatase, glycosidase, luciferase), and radioactive isotopes (eg, 125 I, 3 H, 14 C, 32 P).
在一些实施例中，NGF突变体与Fc的融合蛋白可被进一步修饰以包含一种或多种生物活性蛋白质、多肽或其片段。如本文所用，“生物活性”或“生物学上的活性”可互换使用，是指在体内显示生物活性以执行特定功能。例如，它可能意味着与特定生物分子结合，如蛋白质、DNA等，然后促进或抑制该生物分子的活性。在一些实施例中，生物活性蛋白质或其片段包括为预防或治疗疾病或症状作为活性药物施用给患者的蛋白质和多肽，以及用于诊断目的的蛋白质和多肽，例如用于诊断试验或体外检测的酶，以及给患者施用以预防疾病的蛋白质和多肽，例如疫苗。在一些实施例中，生物活性蛋白质或其片段具有免疫刺激/免疫调节、膜转运或酶活性。在一些实施例中，生物活性蛋白、多肽或其片段为酶、激素、生长因子、细胞因子或其混合物。在一些实施例中，生物活性蛋白质、多肽或片段可特异性识别目标肽(例如抗原或其它蛋白质)。In some embodiments, the fusion protein of NGF mutant and Fc can be further modified to contain one or more biologically active proteins, polypeptides or fragments thereof. As used herein, "biological activity" or "biologically active" are used interchangeably and refer to showing biological activity in vivo to perform a specific function. For example, it may mean binding to a specific biological molecule, such as a protein, DNA, etc., and then promoting or inhibiting the activity of the biological molecule. In some embodiments, biologically active proteins or fragments thereof include proteins and polypeptides administered to patients as active drugs to prevent or treat diseases or symptoms, as well as proteins and polypeptides for diagnostic purposes, such as enzymes for diagnostic tests or in vitro detection, and proteins and polypeptides administered to patients to prevent diseases, such as vaccines. In some embodiments, biologically active proteins or fragments thereof have immunostimulatory/immunomodulatory, membrane transport or enzymatic activity. In some embodiments, biologically active proteins, polypeptides or fragments thereof are enzymes, hormones, growth factors, cytokines or mixtures thereof. In some embodiments, biologically active proteins, polypeptides or fragments can specifically recognize target peptides (e.g., antigens or other proteins).
在一些实施例中，可包含在本文所述的NGF突变体与Fc的融合蛋白中的生物活性蛋白质或其片段是抗原结合蛋白质(例如，抗体)。在一些实施例中，可包含在如本文所述的NGF突变体与Fc的融合蛋白中的生物活性蛋白质或其片段是抗体模拟物，其是一种使人联想到抗体的包含抗原结合域的小型工程蛋白质，(GGeering和Fussenegger,Trends Biotechnol.,33(2):65-79,2015)。这些分子来自现有的人类支架蛋白，由单一多肽组成。可包含在如本文所述的NGF突变体与Fc的融合蛋白中的抗体模拟物示例可以是但不限于设计的锚蛋白重复蛋白(DARPin；包含3-5个完全合成的锚蛋白重复序列，两侧为N端和C端帽结构域)，一种亲和力多聚体(avimer；一种高亲和力蛋白质，包含多个A结构域，每个结构域对靶点的亲和力较低)，或一种抗凝素(基于脂质支架，具有四个可接近的环，每个环的序列可以随机)。在一些实施例中，可包含在如本文所述的NGF突变体与Fc的融合蛋白中的生物活性蛋白质或其片段是犰狳重复蛋白(例如，β-连环蛋白、α-导入蛋白、斑珠蛋白、大肠腺瘤性息肉病(APC))，包含犰狳重复单元(特性，重复氨基酸序列的长度约为40个残基)。每个犰狳重复单元由一对形成发夹结构的α螺旋组成。多个重复拷贝形成了所述的α螺线管结构。犰狳重复蛋白能够结合不同类型的肽，依赖于肽主链的恒定结合方式，而不需要特定的保守侧链或与肽的游离N-或C-末端相互作用。通过残基识别肽残基的 可能性，再加上重复蛋白的内在模块性，使得犰狳重复蛋白有望成为肽结合通用支架的候选。In some embodiments, the biologically active protein or fragment thereof that can be included in the fusion protein of the NGF mutant and Fc as described herein is an antigen binding protein (e.g., an antibody). In some embodiments, the biologically active protein or fragment thereof that can be included in the fusion protein of the NGF mutant and Fc as described herein is an antibody mimetic, which is a small engineered protein that contains an antigen binding domain that is reminiscent of an antibody (GGeering and Fussenegger, Trends Biotechnol., 33 (2): 65-79, 2015). These molecules are derived from existing human scaffold proteins and consist of a single polypeptide. Examples of antibody mimics that can be included in the fusion protein of the NGF mutant and Fc as described herein can be, but are not limited to, a designed ankyrin repeat protein (DARPin; containing 3-5 fully synthetic ankyrin repeat sequences, flanked by N-terminal and C-terminal cap domains), an affinity multimer (avimer; a high affinity protein containing multiple A domains, each domain having a low affinity for the target), or an anticoagulant (based on a lipid scaffold with four accessible loops, the sequence of each loop can be random). In some embodiments, the biologically active protein or fragment thereof that may be included in the fusion protein of the NGF mutant and Fc as described herein is an armadillo repeat protein (e.g., β-catenin, α-importin, plakoglobin, adenomatous polyposis coli (APC)), comprising an armadillo repeat unit (characteristic, the length of the repeated amino acid sequence is about 40 residues). Each armadillo repeat unit consists of a pair of α-helices that form a hairpin structure. Multiple repeated copies form the α-solenoid structure. The armadillo repeat protein is able to bind to different types of peptides, relying on a constant 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. Recognition of peptide residues by residue This possibility, coupled with the intrinsic modularity of repeat proteins, makes armadillo repeat proteins promising candidates for universal scaffolds for peptide binding.
编码NGF突变体或其融合蛋白的分离的核酸及其载体Isolated nucleic acid encoding NGF mutant or its fusion protein and vector thereof
本申请还涉及编码本文所述任何野生型NGF、NGF突变体或其融合蛋白的分离的核酸，或包含该编码核酸的载体。还涉及包含编码本文所述任何野生型NGF、NGF突变体或其融合蛋白的核酸或载体的分离的宿主细胞(例如，CHO细胞、HEK 293细胞、Hela细胞或COS细胞)。The present application also relates to an isolated nucleic acid encoding any wild-type NGF, NGF mutant or fusion protein thereof described herein, or a vector comprising the encoding nucleic acid. It also relates to an isolated host cell (e.g., CHO cell, HEK 293 cell, Hela cell or COS cell) comprising a nucleic acid or vector encoding any wild-type NGF, NGF mutant or fusion protein thereof described herein.
在一些实施例中，涉及一种编码野生型NGF与Fc的融合蛋白或NGF突变体与Fc的融合蛋白的分离的核酸，所述融合蛋白从N端到C端或从C端到N端：包含野生型NGF或NGF突变体、可选的肽接头和Fc部分。在一些实施例中，野生型NGF包含氨基酸序列SEQ ID NO:1或SEQ ID NO:2。在一些实施例中，NGF突变体包含SEQ ID NOs:17-60中的任一所示的氨基酸序列。在一些实施例中，可选的肽接头部分包含SEQ ID NO:87所示的氨基酸序列。在一些实施例中，所述Fc来自IgG1 Fc或IgG4 Fc。在一些实施例中，所述Fc包含氨基酸序列SEQ ID NO:85。In some embodiments, it relates to an isolated nucleic acid encoding a fusion protein of wild-type NGF and Fc or a fusion protein of NGF mutant and Fc, wherein the fusion protein comprises, from N-terminus to C-terminus or from C-terminus to N-terminus: wild-type NGF or NGF mutant, an optional peptide linker and an Fc portion. In some embodiments, the wild-type NGF comprises the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the NGF mutant comprises the amino acid sequence shown in any one of SEQ ID NOs:17-60. In some embodiments, the optional peptide linker portion comprises the amino acid sequence shown in SEQ ID NO:87. In some embodiments, the Fc is from IgG1 Fc or IgG4 Fc. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO:85.
在一些实施例中，涉及一种编码野生型NGF与Fc的融合蛋白或NGF突变体与Fc的融合蛋白的分离的核酸。在一些实施例中，编码野生型NGF与Fc的融合蛋白的分离的核酸包含序列SEQ ID NO:86。在一些实施例中，编码NGF突变体与Fc的融合蛋白的分离核酸以编野生型NGF与Fc的融合蛋白的分离的核酸为母本，在其基础上引入本文所述的任一种或多种氨基酸突变。在一些实施例中，所述的NGF突变体的氨基酸序列包含SEQ ID NOs:17-60中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:17-60中任一所示的氨基酸序列具有至少90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。在一些实施例中，本申请所述的NGF突变体与Fc的融合蛋白包含SEQ ID NOs:61-82中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:61-82中任一所示的氨基酸序列具有至少80％(例如至少80％、85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。In some embodiments, it relates to an isolated nucleic acid encoding a fusion protein of wild-type NGF and Fc or a fusion protein of NGF mutant and Fc. In some embodiments, the isolated nucleic acid encoding the fusion protein of wild-type NGF and Fc comprises the sequence SEQ ID NO:86. In some embodiments, the isolated nucleic acid encoding the fusion protein of NGF mutant and Fc is based on the isolated nucleic acid encoding the fusion protein of wild-type NGF and Fc, and any one or more amino acid mutations described herein are introduced on the basis of the isolated nucleic acid encoding the fusion protein of wild-type NGF and Fc. In some embodiments, the amino acid sequence of the NGF mutant comprises the amino acid sequence shown in any one of SEQ ID NOs:17-60 or a variant thereof, and the variant has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs:17-60. In some embodiments, the fusion protein of the NGF mutant and Fc described in the present application comprises the amino acid sequence shown in any one of SEQ ID NOs:61-82 or a variant thereof, and the variant has at least 80% (for example, at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs:61-82.
在一些实施例中，包含编码本文所述任何NGF、NGF突变体或包含其的融合蛋白的核酸的载体适于在真核细胞中复制和整合，如哺乳动物细胞(例如，CHO细胞、 HEK 293细胞、Hela细胞、COS细胞)。在一些实施例中，载体是一种病毒载体。在一些实施例中，载体是非病毒载体，如pTT5。In some embodiments, a vector comprising a nucleic acid encoding any of the NGF, NGF mutants, or fusion proteins thereof 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.
已经开发出许多基于病毒的系统用于将基因转移到哺乳动物细胞中。病毒载体的示例包括但不限于腺病毒载体、腺相关病毒载体、慢病毒载体、逆转录病毒载体、单纯疱疹病毒载体及其衍生物。病毒载体技术是本领域公知的，例如，在Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)中，及其它病毒学和分子生物学手册中均对其进行了详述。逆转录病毒为基因传递系统提供了一个方便的平台。可使用本领域已知的技术将异源核酸插入载体并包装在逆转录病毒微粒中。然后，可分离重组病毒并在体外或离体条件下将其输送至工程哺乳动物细胞。许多逆转录病毒系统是本领域已知的。在一些实施例中，使用腺病毒载体。许多腺病毒载体是本领域已知的。在一些实施例中，使用慢病毒载体。在一些实施例中，使用自失活慢病毒载体。例如，携带构建体蛋白编码序列的自失活慢病毒载体可以用本领域已知的实验方法进行包装。所得的慢病毒载体可用于使用本领域已知的方法转导至哺乳动物细胞。来自逆转录病毒(如慢病毒)的载体是实现长期基因转导的合适工具，因为它们允许转基因长期、稳定地整合并在子代细胞中的繁殖。慢病毒载体也具有低免疫原性，并且可以转导非增殖细胞。Many virus-based systems have been developed for transferring 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 described in detail, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to 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, self-inactivating lentiviral vectors carrying construct protein coding sequences 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 from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transduction because they allow long-term, stable integration of transgenes and propagation in daughter cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.
在一些实施例中，载体是非病毒载体。在一些实施例中，载体是pTT5载体。在一些实施例中，载体是转座子，例如睡美人(SB)转座子系统或PiggyBac转座子系统。在一些实施例中，载体是基于聚合物的非病毒载体，包括，例如，聚(乳酸-羟基乙酸共聚物)(PLGA)和聚乳酸(PLA)、聚(乙烯亚胺)(PEI)和树枝状大分子。在一些实施例中，载体是基于阳离子脂质的非病毒载体，如阳离子脂质体、脂质纳米乳和固体脂质纳米粒(SLN)。在一些实施例中，载体是基于肽的非病毒基因载体，例如聚-L-赖氨酸。适用于基因组编辑的任何已知非病毒载体都可用于将编码NGF、NGF突变体或包含其的融合蛋白的核酸引入宿主细胞。参见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和Zhao S.et al.,“PiggyBac transposon vectors:the tools of the human gene editing.”Transl.Lung Cancer Res.(2016)5(1):120-125，通过引用并入本文。在一些实施例中，通过物理方法将编码本文所述NGF、NGF突变体或包含其的融合蛋白的任何一个或多个核酸或载体引入宿 主细胞(例如，CHO、HEK 293、Hela或COS)，包括但不限于电穿孔、声穿孔、光穿孔、磁转染、水穿孔。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, including, for example, poly (lactic acid-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly (ethyleneimine) (PEI) and dendrimers. 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 nucleic acids encoding NGF, NGF mutants or fusion proteins comprising them into host cells. See 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, incorporated herein by reference. In some embodiments, any one or more nucleic acids or vectors encoding NGF, NGF mutants, or fusion proteins thereof described herein are introduced into the host by physical methods. Host cells (e.g., CHO, HEK 293, Hela, or COS), including but not limited to electroporation, sonoporation, photoporation, magnetoporation, hydroporation.
在一些实施例中，载体包含可选择的标记基因或报告基因，用于从载体(例如，慢病毒载体、pTT5载体)转染的宿主细胞群中选择出表达本文所述的NGF、NGF突变体或包含其的融合蛋白的细胞。可选择的标记和报告基因都可能被适当的调控序列包围，以使其能够在宿主细胞中表达。例如，载体可包含转录和翻译终止子、起始序列和用于调节核酸序列表达的启动子。In some embodiments, the vector comprises a selectable marker gene or a reporter gene for selecting cells expressing NGF, NGF mutants, or fusion proteins thereof as described herein from a host cell population transfected with a vector (e.g., a lentiviral vector, a pTT5 vector). Both the selectable marker and the reporter gene may be surrounded by appropriate regulatory sequences to enable them to be expressed in the host cell. For example, the vector may comprise a transcription and translation terminator, a start sequence, and a promoter for regulating the expression of a nucleic acid sequence.
可使用本领域已知的任何分子克隆方法，包括，例如，使用限制性内切酶位点和一个或多个可选择的标记将核酸克隆到载体中。在一些实施例中，核酸可操作地连接到启动子上。，已经探索出多种用于原核细胞或真核细胞(例如，哺乳动物细胞)中进行基因表达的启动子，并且本领域已知的任何启动子都可用于本申请。启动子大致可分为组成型启动子或调控型启动子，如诱导型启动子。Any molecular cloning method known in the art can be used, including, for example, using restriction endonuclease sites and one or more selectable markers to clone nucleic acid into a vector. In certain embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters for gene expression in prokaryotic cells or eukaryotic cells (e.g., mammalian cells) have been explored, and any promoter known in the art can be used in the present application. Promoters can be roughly divided into constitutive promoters or regulated promoters, such as inducible promoters.
在一些实施例中，编码本文所述的野生型NGF、NGF突变体或包含其的融合蛋白的核酸可操作地连接到组成型启动子上。组成型启动子允许异源基因(也称为转基因)在宿主细胞中组成型表达。本文所考虑的启动子示例包括但不限于CMV启动子(CMV)、人类延伸因子-1α(hEF1α)、泛素C启动子(UbiC)、磷酸甘油激酶启动子(PGK)、猿猴病毒40早期启动子(SV40)、鸡β-肌动蛋白启动子与CMV早期增强子(CAGG)偶联，罗氏肉瘤病毒(RSV)启动子、多瘤病毒增强子/单纯疱疹胸苷激酶(MC1)启动子、β肌动蛋白(β-ACT)启动子、“骨髓增生性肉瘤病毒增强子、阴性对照区缺失、d1587rev引物结合位点取代(MND)”启动子。大量研究中已广泛比较了这些组成型启动子在驱动转基因表达方面的效率。在一些实施例中，编码本文所述的野生型NGF、NGF突变体或包含其的融合蛋白的核酸可操作地连接到CMV启动子上。In some embodiments, the nucleic acid encoding the wild-type NGF, NGF mutant or fusion protein comprising it described herein is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Examples of promoters contemplated herein include, but are not limited to, CMV promoter (CMV), human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), simian virus 40 early promoter (SV40), chicken β-actin promoter coupled to CMV early enhancer (CAGG), Roche sarcoma virus (RSV) promoter, polyoma virus enhancer/herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, "myeloproliferative sarcoma virus enhancer, negative control region deletion, d1587rev primer binding site substitution (MND)" promoter. The efficiency of these constitutive promoters in driving transgenic expression has been extensively compared in a large number of studies. In some embodiments, the nucleic acid encoding the wild-type NGF, NGF mutant, or fusion protein comprising the same described herein is operably linked to a CMV promoter.
在一些实施例中，编码本文所述的野生型NGF、NGF突变体或包含其的融合蛋白的核酸可操作地连接到诱导型启动子。诱导型启动子属于调控型启动子的范畴。诱导型启动子可被一个或多个条件诱导，如物理条件、宿主细胞的微环境或宿主细胞的生理状态、诱导剂(即诱导药剂)或其组合物。在一些实施例中，诱导条件不诱导宿主细胞中内源性基因的表达。在一些实施例中，诱导条件选自：诱导剂、辐射(如 电离辐射、光)、温度(如热)、氧化还原状态和宿主细胞的激活状态。在一些实施例中，诱导型启动子可以是NFAT启动子、启动子或NFκB启动子。In some embodiments, the nucleic acid encoding the wild-type NGF, NGF mutant or fusion protein comprising the same as described herein is operably linked to an inducible promoter. Inducible promoters belong to the category of regulatable promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the host cell or the physiological state of the host cell, an inducer (i.e., an inducing agent) or a combination thereof. In some embodiments, the inducing conditions do not induce the expression of endogenous genes in the host cell. In some embodiments, the inducing conditions are selected from: an inducer, radiation (e.g., ionizing radiation, light), temperature (such as heat), redox state, and activation state of the host cell. In some embodiments, the inducible promoter can be a NFAT promoter, Promoter or NFκB promoter.
制备方法Preparation
本申请还涉及制备本文所述任何野生型NGF、NGF突变体或包含其的融合蛋白的方法。因此，在一些实施例中，涉及一种制备野生型NGF、NGF突变体或包含其的融合蛋白的方法，包括：(a)在可有效表达所编码的野生型NGF、NGF突变体或包含其的融合蛋白的条件下，培养包含编码本文所述任何野生型NGF、NGF突变体或包含其的融合蛋白的核酸或载体的宿主细胞(例如，CHO细胞、HEK 293细胞、Hela细胞或COS细胞)；和(b)从所述宿主细胞获得表达的野生型NGF、NGF突变体或包含其的融合蛋白。在一些实施例中，步骤(a)的方法进一步包含产生宿主细胞，所述宿主细胞包含编码本文所述的野生型NGF、NGF突变体或包含其的融合蛋白的核酸或载体。本文所述的野生型NGF、NGF突变体或包含其的融合蛋白可使用本领域已知或本文所述的任何方法制备。The present application also relates to a method for preparing any wild-type NGF, NGF mutant, or fusion protein comprising the same as described herein. Therefore, in some embodiments, it relates to a method for preparing wild-type NGF, NGF mutant, or fusion protein comprising the same, comprising: (a) culturing a host cell (e.g., CHO cell, HEK 293 cell, Hela cell, or COS cell) containing a nucleic acid or vector encoding any wild-type NGF, NGF mutant, or fusion protein comprising the same as described herein under conditions that can effectively express the encoded wild-type NGF, NGF mutant, or fusion protein comprising the same; and (b) obtaining the expressed wild-type NGF, NGF mutant, or fusion protein comprising the same from the host cell. In some embodiments, the method of step (a) further comprises producing a host cell, wherein the host cell contains a nucleic acid or vector encoding the wild-type NGF, NGF mutant, or fusion protein comprising the same as described herein. The wild-type NGF, NGF mutant, or fusion protein comprising the same as described herein can be prepared using any method known in the art or described herein.
在一些实施例中，本文所述的野生型NGF、NGF突变体或包含其的融合蛋白用真核细胞表达，如哺乳动物细胞。在一些实施例中，本文所述的野生型NGF、NGF突变体或包含其的融合蛋白用原核细胞表达。当本文所述的野生型NGF、NGF突变体或包含其的融合蛋白用原核细胞表达时，例如，所产生的proNGF-(可选肽接头)-Fc部分不能被加工成前导肽序列。因此，当用于原核细胞表达时，可将编码长效NGF多肽的核酸设计为不含编码前导肽序列的核酸序列。In some embodiments, the wild-type NGF, NGF mutants, or fusion proteins comprising the same as described herein are expressed in eukaryotic cells, such as mammalian cells. In some embodiments, the wild-type NGF, NGF mutants, or fusion proteins comprising the same as described herein are expressed in prokaryotic cells. When the wild-type NGF, NGF mutants, or fusion proteins comprising the same as described herein are expressed in prokaryotic cells, for example, the proNGF-(optional peptide linker)-Fc portion produced cannot be processed into a leader peptide sequence. Therefore, when used for prokaryotic cell expression, the nucleic acid encoding the long-acting NGF polypeptide can be designed to not contain a nucleic acid sequence encoding a leader peptide sequence.
1.原核细胞的重组产物1. Recombinant products of prokaryotic cells
a)载体构建a) Vector construction
可以使用标准重组技术获得编码本申请所述的蛋白质构建体的多核酸序列。可以使用核苷酸合成仪或PCR技术合成多核苷酸。一旦获得编码多肽的序列，将其插入能够在原核宿主中复制和表达异源多核苷酸的重组载体中。本领域中已知且可使用的许多载体都可用于本申请。选择合适的载体主要取决于要插入载体的核酸大小以及载体所转化的特定宿主细胞。每个载体包含各种成分，这取决于该载体的功能(异源多核苷酸的扩增或表达，或两者兼有)及该载体与其所在的特定宿主细胞之间的兼容性。 载体组件通常包括但不限于：复制起始位点、选择标记基因、启动子、核糖体结合位点(RBS)、信号序列、异源核酸插入和转录终止序列。The polynucleotide sequences encoding the protein constructs described herein can be obtained using standard recombinant techniques. Polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once the sequence encoding the polypeptide is obtained, it is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors known and available in the art can be used for the present application. The selection of a suitable vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell transformed by the vector. Each vector contains various components, which depends on the function of the vector (amplification or expression of heterologous polynucleotides, or both) and the compatibility between the vector and the specific host cell where it is located. 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.
一般来说，质粒载体含有来自于与宿主细胞相容物种的复制子和控制序列，，其与这些宿主细胞一起使用。载体通常携带一个复制位点，以及能够在转化细胞中提供表型选择的标记序列。例如，大肠杆菌通常使用pBR322转化，pBR322是一种来源于大肠杆菌的质粒。pBR322包含编码氨苄青霉素(Amp)和四环素(Tet)抗性的基因，并因此提供了识别转化细胞的简单方法。pBR322、其衍生物或其它细菌质粒或噬菌体也可含有或经修饰后含有可被微生物用于表达内源性蛋白质的启动子。Carter et al.,U.S.Pat.No.5,648,237详述了用于表达特定抗体的pBR322衍生物的示例。In general, plasmid vectors contain replicons and control sequences from species compatible with the host cells, and are used with these host cells. The vector usually carries a replication site, as well as a marker sequence that can provide phenotypic selection in the transformed cells. For example, E. coli is usually transformed using pBR322, a plasmid derived from E. coli. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, and thus provides a simple method for identifying transformed cells. pBR322, its derivatives or other bacterial plasmids or phages may also contain or be modified to contain promoters that can be used by microorganisms to express endogenous proteins. Carter et al., U.S. Pat. No. 5,648,237 details examples of pBR322 derivatives used to express specific antibodies.
此外，包含与宿主微生物相容的复制子和控制序列的噬菌体载体可作为转化载体与这些宿主细胞一起使用。例如，噬菌体如GEMTM-11可用于制备重组载体，所述重组载体可用于转化易感宿主细胞，如大肠杆菌LE392。In addition, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transformation vectors with these host cells. For example, phage such as GEM ™ -11 can be used to prepare recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.
启动子是一段位于顺反子上游(5′)的非翻译调控序列，可调节下游基因表达。原核启动子通常分为两类，诱导型和组成型。诱导型启动子是一种可以响应培养条件改变(例如，营养素的存在或缺乏或温度的变化)，从而启动并提高顺反子转录水平的启动子。A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that regulates downstream gene expression. Prokaryotic promoters are generally divided into two categories, inducible and constitutive. An inducible promoter is a promoter that can respond to changes in culture conditions (e.g., the presence or absence of nutrients or changes in temperature) to initiate and increase transcription levels of a cistron.
可被潜在宿主细胞识别的许多启动子都是公知的。通过限制性内切酶将启动子从源DNA中移出并将分离的启动子序列插入本申请的载体中，所选启动子可操作地连接到编码多肽的顺反子DNA上。天然启动子序列和许多异源启动子都可用于指导靶基因的扩增和/或表达。在一些实施例中，利用异源启动子，因为与天然靶多肽启动子相比，异源启动子通常允许更大的转录，并且表达靶基因的产量更高。Many promoters that can be recognized by potential host cells are all known.Promoter is removed from source DNA by restriction enzyme and the promoter sequence of separation is inserted in the carrier of the present application, and selected promoter is operably connected on the cistron DNA of coded polypeptide.Natural promoter sequence and many heterologous promoters can be used for instructing amplification and/or expression of target gene.In certain embodiments, utilize heterologous promoter, because compared with natural target polypeptide promoter, heterologous promoter usually allows larger transcription, and the output of expressing target gene is higher.
适用于原核宿主的启动子包括PhoA启动子、半乳糖苷酶和乳糖启动子系统、色氨酸(trp)启动子系统和杂交启动子，如tac或trc启动子。然而，在细菌中具有功能的其它启动子(如其它已知的细菌或噬菌体启动子)也适用。它们的核酸序列已经公开，从而使技术人员能够使用接头或适配子来提供任何所需的限制位点将它们连接到编码目标轻链和重链的顺反子上(Siebenlist et al.,(1980)Cell 20:269)。Promoters suitable for prokaryotic hosts include PhoA promoter, β-galactosidase and lactose promoter systems, tryptophan (trp) promoter systems and hybrid promoters, such as tac or trc promoters. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleic acid sequences have been made public, so that technicians can use linkers or adapters to provide any required restriction sites to connect them to the cistron encoding the target light chain and heavy chain (Siebenlist et al., (1980) Cell 20: 269).
在一些实施例中，重组载体内的每个顺反子都包含一个分泌信号序列组分，该组分直接引导所表达的多肽跨膜转移。一般来说，信号序列可以是载体的组成部分， 也可以是插入载体的靶多肽DNA的一部分。为本申请而选择的信号序列应为能被宿主细胞识别和加工(即，被信号肽酶切割)的序列。对于不能识别和加工异源多肽固有信号序列的原核宿主细胞，信号序列被原核信号序列所取代，该原核信号序列选自，例如，碱性磷酸酶、青霉素酶、Ipp或热稳定性肠毒素II(STII)先导物、LamP、PhoE、，PelB、OmpA和MBP。In some embodiments, each cistron in the recombinant vector contains a secretion signal sequence component, which directly guides the expressed polypeptide to move across the membrane. Generally speaking, the signal sequence can be a component of the vector, It can also be a part of the target polypeptide DNA inserted into the vector. The signal sequence selected for this application should be a sequence that can be recognized and processed (i.e., cut by a signal peptidase) by the host cell. For prokaryotic host cells that cannot recognize and process the intrinsic signal sequence of the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) leaders, LamP, PhoE, PelB, OmpA and MBP.
在一些实施例中，生产本申请的蛋白质构建体可发生在宿主细胞的细胞质中，因此不需要在每个顺反子内都存在分泌信号序列。在一些实施例中，多肽组分被表达、折叠和组装以在细胞质内形成蛋白质构建体。某些宿主菌株(例如，大肠杆菌trxB-菌株)提供有利于二硫键形成的细胞质条件，从而允许表达的蛋白质亚基适当折叠和组装。参见Proba和Pullthun,Gene,159:203(1995)。In some embodiments, production of the protein construct of the present application can occur in the cytoplasm of the host cell, so there is no need for a secretion signal sequence to be present in each cistron. In some embodiments, the polypeptide components are expressed, folded, and assembled to form a protein construct in the cytoplasm. Certain host strains (e.g., E. coli trxB- strains) provide cytoplasmic conditions that are conducive to disulfide bond formation, thereby allowing the expressed protein subunits to fold and assemble appropriately. See Proba and Pullthun, Gene, 159: 203 (1995).
b)原核宿主细胞b) Prokaryotic host cells
适于表达本申请的蛋白质的原核宿主细胞包括古细菌和真细菌，如革兰氏阴性菌或革兰氏阳性菌。可用细菌的示例包括大肠杆菌(例如，大肠杆菌)、杆菌(例如，枯草杆菌)、肠杆菌、假单胞菌(例如，铜绿假单胞菌)、鼠伤寒沙门氏菌、粘质沙雷氏菌、克雷伯菌、变形杆菌、志贺菌、根瘤菌、透明颤菌或副球菌。在一些实施例中，使用革兰氏阴性细胞。在一些实施例中，大肠杆菌细胞作为本申请的宿主。大肠杆菌菌株示例包括菌株W3110(Bachmann,Cellular and Molecular Biology,vol.2(Washington,D.C.:American Society for Microbiology,1987),pp.1190-1219；ATCC Deposit No.27,325)及其衍生物，包括具有基因型W3110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompT A(nmpc fepE)degP41 kanR的菌株33D3(U.S.Pat.No.5,639,635)。其它菌株及其衍生物，如E.coli 294(ATCC 31446)、E.coli B、E.coli 1776(ATCC 31537)和E.coli RV308(ATCC 31608)也同样适用。这些例子是说明性的，而不是限制性。构建任何上述提及的已知基因型的细菌衍生物的方法在本领域已知，并在例如Bass et al.,Proteins,8:309-314(1990)中进行了详述。考虑到复制子在细菌细胞中的可复制性，通常需要选择合适的细菌。例如，当使用公知的质粒如pBR322、pBR325、pACYC177或pKN410来提供复制子时，大肠杆菌、沙雷氏菌或沙门氏菌适于用作宿主。Prokaryotic host cells suitable for expressing the protein of the present application include archaea and true bacteria, such as gram-negative bacteria or gram-positive bacteria. Examples of available bacteria include Escherichia coli (e.g., Escherichia coli), bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreoscilla or Paracoccus. In certain embodiments, gram-negative cells are used. In certain embodiments, Escherichia coli cells are used as hosts of the present application. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 (US Pat. No. 5,639,635) having the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc fepE) degP41 kan R. Other strains and their derivatives, such as E. coli 294 (ATCC 31446), E. coli B, E. coli 1776 (ATCC 31537) and E. coli RV308 (ATCC 31608) are also suitable. 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 described in detail, 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 suitable bacteria. For example, when using known plasmids such as pBR322, pBR325, pACYC177 or pKN410 to provide the replicon, Escherichia coli, Serratia or Salmonella are suitable for use as hosts.
通常，宿主细胞应分泌最少量的蛋白水解酶，并需要在细胞培养物中适当加入额外的蛋白酶抑制剂。 Generally, the host cells should secrete minimal amounts of proteolytic enzymes and additional protease inhibitors should be appropriately included in the cell culture.
c)蛋白生产c) Protein production
用上述表达载体转化宿主细胞，并在经适当改良的传统营养培养基中培养，以诱导启动子、选择转化子或扩增编码所需序列的基因。转化是指将DNA导入原核宿主，使DNA可以作为染色体外的元件或通过染色体整合进行复制。根据所用的宿主细胞，使用适合此类细胞的标准技术进行转化。采用氯化钙的钙处理通常用于含有大量细胞壁屏障的细菌细胞。另一种转化方法采用聚乙二醇/二甲基亚砜。另一种技术是电穿孔。Host cells are transformed with the above expression vectors and cultured in conventional nutrient media modified appropriately to induce promoters, select transformants, or amplify genes encoding the desired sequences. Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA can replicate as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. Calcium treatment using calcium chloride is commonly used for bacterial cells that contain a large cell wall barrier. Another transformation method uses polyethylene glycol/dimethyl sulfoxide. Another technique is electroporation.
用于生产本申请的蛋白质构建体的原核细胞在本领域已知的且适于培养所选宿主细胞的培养基中生长。合适的培养基包括luria broth(LB)和必要的营养补充剂。在一些实施例中，培养基还包含基于表达载体的结构所选择的选择剂，以选择性地允许包含表达载体的原核细胞生长。例如，将氨苄青霉素添加到培养基中，用于表达氨苄青霉素抗性基因的细胞生长。Prokaryotic cells used to produce the protein constructs of the present application are grown in a culture medium known in the art and suitable for culturing the selected host cells. Suitable culture media include luria broth (LB) and necessary nutritional supplements. In some embodiments, the culture medium also contains a selection agent selected based on the structure of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium for the growth of cells expressing an ampicillin resistance gene.
除碳源、氮源和无机磷酸盐源外，任何必要的补充剂也可单独或作为与其它补充剂或介质(如复合氮源)的混合物以适当的浓度加入。可选地，培养基可包含一种或多种选自谷胱甘肽、半胱氨酸、胱胺、硫代甘氨酸、二硫代赤藓糖醇和二硫苏糖醇的还原剂。原核宿主细胞在合适的温度下培养。例如，对于大肠杆菌的生长，优选的温度范围为20℃至39℃，更优选的为25℃至37℃，甚至更优选的为30℃。培养基的pH值可以是5到9之间的任何pH值，主要取决于宿主生物。对于大肠杆菌来说，pH值优选为6.8至7.4，且更优选为7.0。In addition to carbon sources, nitrogen sources and inorganic phosphate sources, any necessary supplements may also be added at appropriate concentrations, either alone or as a mixture with other supplements or media (such as a complex nitrogen source). Optionally, the culture medium may contain one or more reducing agents selected from glutathione, cysteine, cystamine, thioglycine, dithioerythritol and dithiothreitol. Prokaryotic host cells are cultured at a suitable temperature. For example, for the growth of Escherichia coli, the preferred temperature range is 20°C to 39°C, more preferably 25°C to 37°C, and even more preferably 30°C. The pH value of the culture medium can be any pH value between 5 and 9, depending primarily on the host organism. For Escherichia coli, the pH value is preferably 6.8 to 7.4, and more preferably 7.0.
如果在本申请的表达载体中使用诱导型启动子，则在适合启动子激活的条件下诱导蛋白质表达。在本申请的一方面，PhoA启动子用于控制多肽的转录。因此，转化的宿主细胞在磷酸盐限制培养基中培养以进行诱导。优选地，磷酸盐限制培养基为C.R.A.P培养基(参见Simmons et al.,J.Immunol.Methods(2002),263:133-147)。根据所采用的载体结构，可使用本领域已知的多种其它诱导剂，这在本领域是已知的。If an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation. In one aspect of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is C.R.A.P medium (see Simmons et al., J. Immunol. Methods (2002), 263: 133-147). Depending on the vector structure used, a variety of other inducers known in the art can be used, which is known in the art.
本申请所表达的蛋白质构建体分泌到宿主细胞的周质中并从中回收。蛋白质回收通常涉及破坏微生物，通常通过渗透压休克、超声处理或裂解等方式。一旦细胞被破坏，可通过离心或过滤去除细胞碎片或整个细胞。例如，可通过亲和树脂色谱法进一步纯化蛋白质。或者，蛋白质可以被运输到培养基中并在其中被分离出来。可从培养基中移除细胞，过滤和浓缩培养基上清液以进一步纯化所产生的蛋白质。所表达的 多肽可通过聚丙烯酰胺凝胶电泳(PAGE)和Western blot试验等常用方法进一步分离和鉴定。The protein constructs expressed in the present application are secreted into the periplasm of the host cells and recovered therefrom. Protein recovery generally involves disrupting the microorganism, usually by osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. For example, the protein can be further purified by affinity resin chromatography. Alternatively, the protein can be transported to a culture medium and isolated therein. The cells can be removed from the culture medium, and the culture supernatant can be filtered and concentrated to further purify the produced protein. The expressed The polypeptides can be further separated and identified by common methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.
或者，通过发酵过程大规模生产蛋白质。各种大规模补料分批发酵程序可用于生产重组蛋白。大规模发酵的容量至少为1000升，最好为1000至100000升。这些发酵罐使用搅拌器叶轮来分配氧气和营养物质，特别是葡萄糖(首选碳/能源)。小规模发酵通常指发酵罐中的发酵，其容量体积不超过100升，范围从1升到100升。Alternatively, proteins are produced on a large scale by fermentation processes. Various large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. Large-scale fermentations have a capacity of at least 1,000 liters, preferably 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source). Small-scale fermentation generally refers to fermentation in fermenters with a capacity volume of no more than 100 liters, ranging from 1 liter to 100 liters.
在发酵过程中，通常是细胞在合适的条件下生长至所需密度后开始诱导蛋白质表达，例如，在OD550约为180-220时，此时细胞处于早期静止期。根据所采用的载体结构，可使用本领域已知和上文所述的多种诱导剂。细胞在诱导前可以生长较短的时间。细胞通常诱导约12-50小时，尽管可以使用可能更长或更短的诱导时间。In the fermentation process, the induction of protein expression is usually initiated after the cells have grown to a desired density under appropriate conditions, for example, at an OD 550 of about 180-220, when the cells are in the early stationary phase. Depending on the vector structure used, a variety of inducing agents known in the art and described above can be used. The cells can be grown for a shorter period of time before induction. The cells are typically induced for about 12-50 hours, although a longer or shorter induction time may be used.
为了提高本申请的蛋白质构建体的产量和质量，可以改良各种发酵条件。例如，为了提高分泌多肽的正确组装和折叠，可以使用过表达伴侣蛋白的附加载体来共转化宿主原核细胞，如Dsb蛋白(DsbA、DsbB、DsbC、DsbD或DsbG)或FkpA(具有伴侣活性的肽脯氨酰顺反异构酶)。已证明伴侣蛋白有助于促进细菌宿主细胞中所产生的异源蛋白正确折叠和溶解。Chen et al.,(1999)J Bio Chem 274:19601-19605；Georgiou et al.,U.S.Pat.No.6,083,715；Georgiou et al.,U.S.Pat.No.6,027,888；Bothmann和Pluckthun(2000)J.Biol.Chem.275:17100-17105；Ramm和Pluckthun(2000)J.Biol.Chem.275:17106-17113；Arie et al.,(2001)Mol.Microbiol.39:199-210。In order to improve the yield and quality of the protein constructs of the present application, various fermentation conditions can be improved. For example, in order to improve the correct assembly and folding of secreted polypeptides, additional vectors that overexpress chaperone proteins can be used to co-transform host prokaryotes, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD or DsbG) or FkpA (a peptide prolyl cis-trans isomerase with chaperone activity). Chaperone proteins have been shown to help promote the correct folding and dissolution of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem 274: 19601-19605; Georgiou et al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. 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.
为了最大限度地减少表达的异源蛋白质(尤其是蛋白水解敏感的蛋白质)的水解，某些缺乏蛋白水解酶的宿主菌株可用于本申请。例如，宿主细胞菌株可经修饰使得编码已知细菌蛋白酶的基因发生基因突变，如蛋白酶III、OmpT、DegP、Tsp、蛋白酶I、蛋白酶Mi、蛋白酶V、蛋白酶VI及其组合物。可以使用一些大肠杆菌蛋白酶缺失菌株，在Joly et al.,(1998),supra；Georgiou et al.,U.S.Pat.No.5,264,365；Georgiou et al.,U.S.Pat.No.5,508,192；Hara et al.,Microbial Drug Resistance,2:63-72(1996)中进行了详述。In order to minimize the hydrolysis of expressed heterologous proteins (especially proteolytically sensitive proteins), certain host strains lacking proteolytic enzymes can be used in the present application. For example, the host cell strain can be modified to cause 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. Some E. coli protease deletion strains can be used, which are described in detail in Joly et al., (1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996).
缺乏蛋白水解酶且用过表达一种或多种伴侣蛋白的质粒转化的大肠杆菌菌株，可在编码本申请所述的蛋白质构建体的表达系统中作为宿主细胞。 E. coli strains lacking proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins can be used as host cells in expression systems encoding the protein constructs described in this application.
d)蛋白纯化d) Protein purification
进一步纯化本文所生产的蛋白质构建体，以获得用于进一步分析和使用的基本上均匀的制剂。可采用本领域已知的标准蛋白质纯化方法。以下程序为适用的纯化程序示例：免疫亲和柱或离子交换柱上的分馏、乙醇沉淀、反相液相色谱HPLC、二氧化硅或阳离子交换树脂(如DEAE)色谱、色谱聚焦、SDS-PAGE、硫酸铵沉淀和凝胶过滤，例如，葡聚糖凝胶G-75。The protein constructs produced herein are further purified to obtain substantially uniform preparations for further analysis and use. Standard protein purification methods known in the art can be used. The following procedures are examples of applicable purification procedures: fractionation on an immunoaffinity column or ion exchange column, ethanol precipitation, reversed phase liquid chromatography HPLC, silica or cation exchange resin (such as DEAE) chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and gel filtration, for example, Sephadex G-75.
在一些实施例中，固定在固相上的蛋白质A被用于蛋白质构建体的免疫亲和纯化，所述蛋白质构建体包含本申请所述的Fc区域。蛋白A为来自金黄色葡萄球菌的42kDa表面蛋白，其与含Fc的结构具有很高的结合亲和力，例如，本文所述的NGF突变体与Fc的融合蛋白。Lindmark et al.,(1983)J.Immunol.Meth.62:1-1。固定蛋白质A的固相优选包含玻璃或二氧化硅表面的柱子，更优选为可控孔径玻璃柱或硅酸柱。在某些应用中，色谱柱涂有试剂，如甘油，以防止污染物的非特异性粘附。然后清洗固相以去除非特异性结合到固相的污染物。最后，通过洗脱从固相中回收目标蛋白构建体。In some embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of a protein construct comprising an Fc region as described herein. Protein A is a 42 kDa surface protein from Staphylococcus aureus that has a high binding affinity to Fc-containing structures, for example, the fusion protein of the NGF mutant and Fc described herein. Lindmark et al., (1983) J. Immunol. Meth. 62: 1-1. The solid phase on which protein A is fixed preferably comprises a column having a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In certain applications, the chromatographic column is coated with a reagent, such as glycerol, to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants that are nonspecifically bound to the solid phase. Finally, the target protein construct is recovered from the solid phase by elution.
2.真核细胞的重组产物2. Recombinant products of eukaryotic cells
对于真核表达，载体组分通常包括但不限于下列一个或多个：信号序列、复制起始点、一个或多个标记基因、增强子元件、启动子和转录终止序列。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)信号序列元件a) Signal sequence elements
用于真核宿主的载体还可以是一个插入物，该插入物编码信号序列或在成熟蛋白或多肽的N端具有特定裂解位点的其它多肽。选择的异源信号序列优选为由宿主细胞识别和加工(即，被信号肽酶切割)的序列。在哺乳动物细胞表达中，可以获得哺乳动物信号序列以及病毒分泌先导物，例如，单纯疱疹gD信号，都是有用的。该前体区域的DNA在阅读框中与编码本申请的蛋白质构建体的DNA连接。The carrier used for eukaryotic hosts can also be an insert, and this insert encoding signal sequence or has other polypeptides of specific cleavage site at the N-terminal of mature protein or polypeptide.The heterologous signal sequence selected is preferably a sequence recognized and processed (that is, cut by signal peptidase) by host cells.In mammalian cell expression, mammalian signal sequence and virus secretion leader can be obtained, for example, herpes simplex gD signal, all are useful.The DNA in this precursor region is connected with the DNA of the protein construct of encoding the application in reading frame.
b)复制起始点b) Origin of replication
一般来说，哺乳动物表达载体不需要复制起始点元件(SV40起点通常仅因其包含早期启动子而使用)。 Generally, mammalian expression vectors do not require an origin of replication element (the SV40 origin is often used only because it contains the early promoter).
c)选择基因元件c) Selecting genetic elements
表达和克隆载体可包含选择基因，也称为选择标记。典型的选择基因编码以下蛋白质：(a)对抗生素或其它毒素具有抗药性的蛋白质，例如，氨苄青霉素、新霉素、甲氨蝶呤或四环素、(b)补体营养缺陷蛋白质或(c)提供复杂培养基无法提供的关键营养素的蛋白质，如编码杆菌D-丙氨酸消旋酶的基因。Expression and cloning vectors may contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that confer (a) resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic proteins, or (c) provide key nutrients that are not available in complex culture media, such as the gene encoding Bacillus D-alanine racemase.
选择方案的一个示例是利用药物来阻止宿主细胞的生长。那些异源基因成功转化的细胞产生一种具有抗药性的蛋白质，从而在选择方案中存活下来。这种优势选择的示例使用药物新霉素、霉酚酸和潮霉素。An example of a selection scheme is the use of a drug to stop the growth of host cells. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus survive the selection scheme. Examples of this type of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.
适用于哺乳动物细胞的可选择的标记的另一个示例是，那些能够识别有能力携带编码本申请所述蛋白质构建体核酸的细胞的可选择的标记，如DHFR、胸苷激酶、金属硫蛋白-I和-II，优选为灵长类金属硫蛋白基因、腺苷脱氨酶、鸟氨酸脱羧酶等。Another example of a selectable marker suitable for mammalian cells is one that can identify cells that are competent to carry the nucleic acid encoding the protein construct described in the present application, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
例如，用DHFR选择基因转化的细胞，首先将所有转化子培养在含有甲氨蝶呤(Mtx)的培养基中进行鉴定，甲氨蝶呤是DHFR的竞争性拮抗剂。当使用野生型DHFR时，适用的宿主细胞为缺乏DHFR活性的中国仓鼠卵巢(CHO)细胞系(例如，ATCC CRL-9096)。For example, for cells transformed with a DHFR selection gene, all transformants are first identified by culturing in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity (e.g., ATCC CRL-9096).
或者，用编码多肽的DNA序列、野生型DHFR蛋白和另一选择标记，如氨基糖苷3′-磷酸转移酶(APH)转化或共转化的宿主细胞(尤其是含有内源性DHFR的野生型宿主)，可通过在含有选择标记物的培养基中的细胞生长来选择，所述选择标记物例如氨基糖苷类抗生素，例如，卡那霉素、新霉素或G418。参见U.S.Pat.No.4,965,199。Alternatively, host cells transformed or co-transformed with a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and another selection marker, such as aminoglycoside 3'-phosphotransferase (APH), especially wild-type hosts containing endogenous DHFR, can be selected by growing the cells in a medium containing a selection marker, such as an aminoglycoside antibiotic, for example, kanamycin, neomycin or G418. See U.S. Pat. No. 4,965,199.
d)启动子元件d) Promoter element
表达和克隆载体通常包含一个启动子，所述启动子被宿主识别，并可操作地连接到编码所需多肽序列的核酸上。几乎所有真核基因都有一个富含AT的区域，位于转录起始点上游约25到30个碱基。在许多基因转录起始点上游70到80个碱基处发现的另一个序列为CNCAAT区域，其中N可以是任何核苷酸。在大多数真核生物的3′端有一个AATAAA序列，该序列可能是编码序列3′端增加多聚A尾的信号。所有这些序列都可以插入到真核表达载体。 Expression and cloning vectors usually include a promoter, which is recognized by the host and operably connected to the nucleic acid encoding the desired polypeptide sequence. Almost all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream of the transcription start point. Another sequence found at 70 to 80 bases upstream of many gene transcription start points is the CNCAAT region, in which N can be any nucleotide. At the 3' end of most eukaryotic organisms, there is an AATAAA sequence, which may be a signal for increasing the poly A tail at the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.
哺乳动物宿主细胞载体中的多肽转录受启动子控制，例如，通过从病毒基因组获得的启动子，如多瘤病毒、鸡痘病毒、腺病毒(如腺病毒2)、牛乳头状瘤病毒、禽肉瘤病毒、巨细胞病毒、逆转录病毒、乙型肝炎病毒和最优选的猿猴病毒40(SV40)，来自异源哺乳动物的启动子，例如，肌动蛋白启动子或免疫球蛋白启动子，来自热休克启动子，前提是此类启动子与宿主细胞系统兼容。Transcription of the polypeptide in a mammalian host cell vector is controlled by a promoter, for example, by promoters obtained from viral genomes, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, from heat shock promoters, provided that such promoters are compatible with the host cell systems.
SV40病毒早期和晚期启动子作为SV40限制性片段便于获得，该限制性片段也包含SV40病毒复制起始点。人类巨细胞病毒的即刻早期启动子作为HindIII E限制性片段便于获得。U.S.Pat.No.4,419,446中公开了一种使用牛乳头瘤病毒作为载体在哺乳动物宿主中表达DNA的系统。U.S.Pat.No.4,601,978中详述了对该系统的改良。参见Reyes et al.,Nature 297:598-601(1982)，关于在单纯疱疹病毒胸苷激酶启动子控制下人类干扰素cDNA在小鼠细胞中的表达。或者，可以使用劳斯肉瘤病毒长末端重复序列作为启动子。The SV40 virus early and late promoters are readily available as SV40 restriction fragments that also contain the SV40 virus replication start site. The immediate early promoter of the human cytomegalovirus is readily available as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. Improvements to this system are detailed in U.S. Pat. No. 4,601,978. See Reyes et al., Nature 297:598-601 (1982), for expression of human interferon cDNA in 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)增强子元件e) Enhancer elements
高等真核生物对编码本申请蛋白质构建体DNA的转录通常通过在载体中插入增强子序列而增加。在哺乳动物基因中已经发现了许多增强子序列(珠蛋白、弹性蛋白酶、白蛋白、α-甲胎蛋白和胰岛素)。然而，通常使用的是真核细胞病毒的增强子。示例包括复制起始点末端(100-270bp)的SV40增强子、巨细胞病毒早期启动子增强子、复制起始点末端的多瘤病毒增强子和腺病毒增强子。参见Yaniv,Nature 297:17-18(1982)，关于激活真核生物启动子的增强元件。增强子可在多肽编码序列的5′或3′处拼接到载体上，但优选位于启动子的5′处。Transcription of the 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 have been found in mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). However, enhancers of eukaryotic cell viruses are commonly used. Examples include the SV40 enhancer at the end of the replication origin (100-270bp), the cytomegalovirus early promoter enhancer, the polyoma enhancer at the end of the replication origin, and the adenovirus enhancer. See Yaniv, Nature 297:17-18 (1982) for enhancing elements that activate eukaryotic promoters. The enhancer can be spliced into the vector at 5′ or 3′ of the polypeptide coding sequence, but is preferably located at 5′ of the promoter.
f)转录终止元件f) Transcription termination element
真核宿主细胞(酵母、真菌、昆虫、植物、动物、人类或其它多细胞生物的有核细胞)中使用的表达载体也包含转录终止和稳定mRNA所必需的序列。这些序列通常可从真核或病毒DNA或cDNA的5′端非翻译区获得，偶尔为3′端。这些区域包含核苷酸片段，所述核苷酸片段在编码多肽的mRNA的未翻译部分作为多聚腺苷酸片段被转录。一个合适的转录终止元件为牛生长激素多聚腺苷酸区域。参见WO94/11026和其中所公开的表达载体。 Expression vectors used in eukaryotic host cells (nucleated cells of yeast, fungi, insects, plants, animals, humans or other multicellular organisms) also contain sequences necessary for transcription termination and stabilization of mRNA. These sequences are usually available from the 5' untranslated region of eukaryotic or viral DNA or cDNA, and occasionally the 3' end. These regions contain nucleotide fragments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. A suitable transcription termination element is the bovine growth hormone polyadenylated region. See WO94/11026 and the expression vectors disclosed therein.
g)宿主细胞的选择和转化g) Host cell selection and transformation
用于克隆或表达本文所述载体中的DNA的合适宿主细胞包括本文所述的高等真核细胞，包括脊椎动物宿主细胞。脊椎动物细胞的培养繁殖(组织培养)已成为常规程序。有用的哺乳动物宿主细胞系示例为SV40转化的猴肾CV1系(COS-7，ATCC CRL 1651)；来源于猴肾组织的COS成纤维细胞样细胞系；人类胚胎肾系(293或293细胞亚克隆，用于悬浮培养生长，Graham et al.,J.Gen Virol.36:59(1977))；乳仓鼠肾细胞(BHK，ATCC CCL 10)；中国仓鼠卵巢细胞/-DHFR(CHO，Urlaub et al.,Proc.Natl.Acad.Sci.USA 77:4216(1980))；小鼠塞尔托力氏细胞(TM4，Mather,Biol.Reprod.23:243-251(1980))；猴肾细胞(CV1 ATCC CCL 70)；非洲绿猴肾细胞(VERO-76，ATCC CRL-1587)；人宫颈癌细胞(HELA，ATCC-ccl2)；犬肾细胞(MDCK，ATCC-ccl34)；水牛-大鼠肝细胞(BRL 3A，ATCC CRL 1442)；人类肺细胞(W138，ATCC CCL 75)；人类肝细胞(Hep G2，HB 8065)；小鼠乳腺肿瘤(MMT 060562，ATCC CCL51)；TR1细胞(Mather et al.,Annals N.Y.Acad.Sci.383:44-68(1982))；MRC5细胞；FS4细胞和人类肝癌细胞系(Hep G2)。Suitable host cells for cloning or expressing the DNA in the vectors described herein include higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) is routine procedure. Examples of useful mammalian host cell lines are SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); COS fibroblast-like cell line derived from monkey kidney tissue; human embryonic kidney line (293 or 293 cell subclones for suspension culture growth, 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 ... cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC-ccl2); canine kidney cells (MDCK, ATCC-ccl34); buffalo-rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells and human hepatoma cell line (Hep G2).
用上述表达载体或克隆载体转化宿主细胞以产生蛋白质结构，并在经适当改良的常规营养培养基中培养，以诱导启动子、选择转化子或扩增编码所需序列的基因。Host cells are transformed with the above-described expression vectors or cloning vectors to produce the protein construct and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify genes encoding the desired sequences.
h)培养宿主细胞h) Cultivating host cells
用于生产本申请的蛋白质构建体的宿主细胞可在多种培养基中培养。商用培养基，如Ham's F10(Sigma)、最小基本培养基((MEM)，Sigma)、RPMI-1640(Sigma)和Dulbecco's修饰的Eagle's Medium((DMEM)，Sigma)适合培养宿主细胞。此外，Ham et al.,Meth.Enz.58:44(1979)、Barnes et al.,Anal.Biochem.102:255(1980)、U.S.Pat.No.4,767,704；4,657,866；4,927,762；4,560,655或5,122,469、WO 90/03430、WO 87/00195或U.S.Pat.Re.30,985所述的任何培养基都可用作宿主细胞的培养基。这些培养基中的任何一种都可以根据需要补充激素和/或其它生长因子(如胰岛素、转铁蛋白或表皮生长因子)、盐(如氯化钠、钙、镁和磷酸盐)、缓冲液(如HEPE)、核苷酸(如腺苷和胸苷)、抗生素(如庆大霉素TM药物)、微量元素(定义为通常最终浓度在微摩尔范围内存在的无机化合物)和葡萄糖或等效能源。任何其它必要的补充剂也可在本领域技术人员已知的适当浓度下加入。培养条件，如温度、 pH等，是那些先前宿主细胞表达所使用过的条件，并且对于普通技术人员来说显而易见。Host cells used to produce the protein constructs of the present application can be cultured in a variety of culture media. Commercial culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any culture 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 as a culture medium for host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPE), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug gentamicin ™ ), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be added at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, The pH, etc., are those conditions previously used for expression in the host cell and will be apparent to the ordinarily skilled artisan.
i)蛋白纯化i) Protein purification
当使用重组技术时，本申请的蛋白质构建体可在细胞内、周质中产生，或直接分泌到培养基中。如果蛋白质构建体在细胞内产生，第一步是通过离心或超滤去除微粒碎片(即宿主细胞或裂解片段)。Carter et al.,Bio/Technology 10:163-167(1992)详述了分离分泌到大肠杆菌周质中的抗体的程序。简而言之，细胞体在醋酸钠(pH 3.5)、EDTA和苯甲基磺酰氟(PMSF)存在的条件下融解约30分钟。细胞碎片可通过离心去除。当蛋白质构建体分泌到培养基中，此类表达系统的上清液通常首先使用市面上的蛋白质浓缩过滤器进行浓缩，例如，Amicon或Millipore Pellicon超滤装置。蛋白酶抑制剂如PMSF可包含在上述任何步骤中，以抑制蛋白质水解，且可包含抗生素以防止外来污染物的生长。When recombinant techniques are used, the protein constructs of the present application can be produced intracellularly, in the periplasm, or directly secreted into the culture medium. If the protein construct is produced intracellularly, the first step is to remove particulate debris (i.e., host cells or cleavage fragments) by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) details the procedure for isolating antibodies secreted into the periplasm of E. coli. Briefly, the cell bodies are lysed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. When the protein construct is secreted into the culture medium, the supernatant of such expression systems is usually first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration device. Protease inhibitors such as PMSF can be included in any of the above steps to inhibit protein hydrolysis, and antibiotics can be included to prevent the growth of foreign contaminants.
可以使用例如羟基磷灰石色谱、凝胶电泳、透析和亲和色谱法来纯化从细胞中制备的蛋白质组合物，其中亲和色谱法是优选的纯化技术。蛋白质A作为亲和配体的适用性取决于在含Fc蛋白构建体中存在的任何免疫球蛋白Fc结构域的种类和亚型。蛋白质A可用于纯化基于含1、2或4重链的人类免疫球蛋白的含Fc蛋白质(Lindmark et al.,J.Immunol.Meth.62:1-13(1983))。推荐蛋白质G用于所有小鼠亚型和人类3型(Guss et al.,EMBO J.5:15671575(1986))。连接亲和配体的基质通常是琼脂糖，但也有其它基质。与琼脂糖相比，机械稳定的基质(如可控孔径玻璃或聚(苯乙烯-二乙烯基)苯)可以实现更快的流速和更短的处理时间。Bakerbond ABXTMresin可用于纯化包含CH3结构域的蛋白质构建体(J.T.Baker,Phillipsburg,N.J.)。其它蛋白质纯化技术也同样适用，如离子交换柱分馏、乙醇沉淀、反相液相色谱HPLC、硅胶层析、肝素SEPHAROSETM色谱、阴离子或阳离子交换树脂(如聚天冬氨酸柱)、色谱聚焦、SDS-PAGE和硫酸铵沉淀，取决于要回收的蛋白质构建体。Protein compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and subtype of any immunoglobulin Fc domain present in the Fc-containing protein construct. Protein A can be used to purify Fc-containing proteins based on human immunoglobulins containing 1, 2, or 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse subtypes and human type 3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is usually agarose, but there are other matrices. Compared with agarose, mechanically stable matrices (such as controlled pore glass or poly (styrene-divinyl) benzene) can achieve faster flow rates and shorter processing times. Bakerbond ABX™ resin can be used to purify protein constructs containing CH3 domains (JT Baker, Phillipsburg, NJ). Other protein purification techniques are also suitable, such as ion exchange column fractionation, ethanol precipitation, reverse phase liquid chromatography HPLC, silica gel chromatography, heparin SEPHAROSE ™ chromatography, anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation, depending on the protein construct to be recovered.
在任何初步纯化步骤之后，可以对包含目标蛋白质构建体和污染物的混合物进行低pH疏水相互作用色谱，洗脱缓冲液pH值为约2.5-4.5，优选地，在低盐浓度下进行(例如，从0-0.25M盐)。 Following any preliminary purification steps, the mixture comprising the protein construct of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, with the elution buffer having a pH of about 2.5-4.5, preferably at low salt concentration (eg, from 0-0.25 M salt).
药物组合物Pharmaceutical composition
本申请一方面涉及包含本申请所述的任何NGF突变体或其融合蛋白(例如，NGF突变体与Fc的融合蛋白)的药物组合物。One aspect of the present application relates to a pharmaceutical composition comprising any NGF mutant or a fusion protein thereof (eg, a fusion protein of an NGF mutant and Fc) described in the present application.
本申请一方面涉及包含本申请所述的任何NGF突变体或其融合蛋白(例如NGF突变体与Fc的融合蛋白)与抗NGF抗体的药物组合物。One aspect of the present application relates to a pharmaceutical composition comprising any NGF mutant or a fusion protein thereof (eg, a fusion protein of an NGF mutant and Fc) described in the present application and an anti-NGF antibody.
在一些实施例中，本申请所述这些药物组合物可以任选的进一步包含药学上可接受的载体和/或辅料。在一些实施例中，所述可接受的载体和/或辅料包括赋形剂和/或稳定剂。In some embodiments, the pharmaceutical compositions described herein may optionally further comprise a pharmaceutically acceptable carrier and/or excipient. In some embodiments, the acceptable carrier and/or excipient include an excipient and/or a stabilizer.
本申请所述的药物组合物优选为稳定的，其中所含蛋白质在储存时基本上保持其物理和化学稳定性和完整性。本领域有各种用于测量蛋白质稳定性的分析技术，并在Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed.,Marcel Dekker,Inc.,New York,N.Y.,Pubs.(1991)和Jones,A.Adv.Drug Delivery Rev.10:29-90(1993)中进行了综述。稳定性可以在选定的温度和选定的时间段内进行测量。对于加速筛选，可将制剂在40℃下保存2周至1个月，在此期间测量稳定性。如果制剂在2-8℃下储存，通常制剂应在30℃或40℃下稳定至少1个月，和/或在2-8℃下稳定至少2年。如果制剂在30℃下储存，通常制剂应在30℃下稳定至少2年，和/或在40℃下稳定至少6个月。例如，储存期间的聚集程度可作为蛋白质稳定性的指标。在一些实施例中，本文所述的NGF突变体或包含其的融合蛋白(例如，NGF突变体与Fc的融合蛋白)的稳定制剂中可包含少于10％(优选少于5％)的本文所述的聚集体。The pharmaceutical compositions described herein are preferably stable, wherein the proteins contained therein substantially maintain their physical and chemical stability and integrity during storage. There are various analytical techniques in the art for measuring protein stability and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature and a selected time period. For accelerated screening, the formulation can be stored at 40°C for 2 weeks to 1 month, during which stability is measured. 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. If the formulation is stored at 30°C, the formulation should generally be stable at 30°C for at least 2 years, and/or at 40°C for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability. In some embodiments, a stable formulation of an NGF mutant described herein or a fusion protein comprising the same (e.g., a fusion protein of an NGF mutant with Fc) may contain less than 10% (preferably less than 5%) of the aggregates described herein.
在一些实施例中，药物组合物具有至少15天的保质期，如至少20天、1个月、2个月、3个月、6个月、1年、2年、3年或更长的保质期，例如，在2-25℃(如2-8℃)下。如本文所用，“保质期”是指当药物制剂在特定储存条件下储存时，例如2-8℃，药物制剂中的活性成分如治疗性蛋白质(例如，本申请所述的NGF突变体与Fc的融合蛋白)发生最小降解(例如，不超过5％降解，如不超过4％、3％，或2％降解)的储存期。用于评估蛋白质或制剂稳定性的示例性技术包括尺寸排阻色谱(SEC)-HPLC以检测，例如，聚集、反相液相色谱(RP)-HPLC以检测，例如，蛋白质片段、离子交换HPLC以检测，例如，蛋白质电荷变化、质谱、荧光光谱、圆二色(CD)光谱、傅里叶变换红外光谱(FT-IR)和拉曼光谱以检测蛋白质构象变化。所有这些技术均可单独或组合使用，以评估药物制剂中蛋白质的降解情况，并确定该制剂的保质期。 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 longer, for example, at 2-25°C (such as 2-8°C). As used herein, "shelf life" refers to the storage period during which the active ingredient in the pharmaceutical formulation, such as a therapeutic protein (e.g., a fusion protein of a NGF mutant and Fc described herein), undergoes minimal degradation (e.g., no more than 5% degradation, such as no more than 4%, 3%, or 2% degradation) when the pharmaceutical formulation is stored under specific storage conditions, such as 2-8°C. Exemplary techniques for assessing protein or formulation stability include size exclusion chromatography (SEC)-HPLC to detect, for example, aggregation, reverse phase liquid chromatography (RP)-HPLC to detect, for example, protein fragments, ion exchange HPLC to detect, for example, protein charge changes, mass spectrometry, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy to detect protein conformational changes. All of these techniques can be used alone or in combination to assess the degradation of proteins in a pharmaceutical formulation and determine the shelf life of the formulation.
为了使药物组合物可用于体内给药，它们必须是无菌的。可通过无菌滤膜过滤使药物组合物无菌。所述药物组合物通常放置在具有无菌接入口的容器中，例如，具有可被皮下注射针刺穿的塞子的静脉注射溶液袋或小瓶。In order for pharmaceutical compositions to be useful for in vivo administration, they must be sterile. Pharmaceutical compositions can be sterilized by filtration through a sterile filter membrane. The pharmaceutical compositions are typically placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.
根据所治疗的特定适应症的需要，本文所述的药物组合物还可以包含不止一种活性化合物，优选为具有互补活性，不会对彼此产生不利影响的化合物。此类分子以适当的数量组合在一起以达到预期目的。Depending on the needs of the specific indication to be treated, the pharmaceutical compositions described herein may also contain more than one active compound, preferably compounds with complementary activities that do not adversely affect each other. Such molecules are combined together in appropriate amounts to achieve the intended purpose.
在一些实施例中，药物组合物装在一次性使用的小瓶中，如一次性密封小瓶。在一些实施例中，药物组合物装在可多次使用的小瓶中。在一些实施例中，药物组合物散装在容器中。在一些实施例中，药物组合物是冷冻保存的。In some embodiments, the pharmaceutical composition is contained in a disposable vial, such as a disposable sealed vial. In some embodiments, the pharmaceutical composition is contained in a vial that can be used multiple times. In some embodiments, the pharmaceutical composition is bulked in a container. In some embodiments, the pharmaceutical composition is stored frozen.
药物组合物可以通过将本文所述的具有所需纯度的野生型NGF、NGF突变体或包含其的融合蛋白(例如，NGF突变体与Fc的融合蛋白)与可选的药学上可接受的载体、赋形剂或稳定剂(Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980))混合制备，成冻干制剂或水溶液的形式。The pharmaceutical composition can be prepared by mixing the wild-type NGF, NGF mutant or fusion protein containing the same (for example, a fusion protein of NGF mutant and Fc) having the desired purity described herein with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized preparation or an aqueous solution.
可通过将冻干的NGF突变体或包含其的融合蛋白(例如，NGF突变体与Fc的融合蛋白)溶解在稀释剂中，使蛋白质均匀分散，从而制备重组制剂。适于在本申请中使用的医药上可接受的(对人体施用安全且无毒)稀释剂示例包括但不限于无菌水、注射用抑菌水(BWFI)、pH缓冲溶液(例如，磷酸盐缓冲盐水)、无菌盐水，林格溶液或葡萄糖溶液，或盐和/或缓冲剂的水溶液。The recombinant formulation can be prepared by dissolving the lyophilized NGF mutant or a fusion protein comprising the same (e.g., a fusion protein of the NGF mutant and Fc) in a diluent to uniformly disperse the protein. Examples of pharmaceutically acceptable (safe and non-toxic to human administration) diluents suitable for use in the present application 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 glucose solution, or aqueous solutions of salts and/or buffers.
在一些实施例中，本申请所述的药物组合物基本上由(例如，由......组成)本文所述的NGF突变体或包含其的融合蛋白(例如，NGF突变体与Fc的融合蛋白)和可选的药学上可接受的载体组成。在一些实施例中，本申请所述的药物组合物基本上由(例如，由......组成)本文所述的NGF突变体或包含其的融合蛋白(例如，NGF突变体与Fc的融合蛋白)、抗NGF抗体和可选的药学上可接受的载体组成。在一些实施例中，本申请所述的药物组合物不包含任何宿主细胞(例如CHO)蛋白质。In some embodiments, the pharmaceutical composition described herein consists essentially of (e.g., consists of) the NGF mutant described herein or a fusion protein comprising it (e.g., a fusion protein of an NGF mutant and Fc) and an optional pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition described herein consists essentially of (e.g., consists of) the NGF mutant described herein or a fusion protein comprising it (e.g., a fusion protein of an NGF mutant and Fc), an anti-NGF antibody and an optional pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition described herein does not contain any host cell (e.g., CHO) protein.
包含NGF突变体或其融合蛋白的药物组合物Pharmaceutical composition comprising NGF mutant or its fusion protein
本申请一方面涉及包含所述NGF突变体或其融合蛋白(例如，NGF突变体与Fc的融合蛋白)的药物组合物，这些药物组合物可以任选的进一步包含药学上可接受的载体和/或辅料。 On one hand, the present application relates to pharmaceutical compositions comprising the NGF mutant or its fusion protein (eg, a fusion protein of the NGF mutant and Fc), and these pharmaceutical compositions may optionally further comprise a pharmaceutically acceptable carrier and/or excipient.
在一些实施例中，在NGF突变体或其融合蛋白的药物组合物中，其中所述的NGF突变体与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，同时该NGF突变体仍具有生物学活性。在另一些实施例中，其中所述的NGF突变体选自如前所述的任一种NGF突变体。In some embodiments, in the pharmaceutical composition of NGF mutant or its fusion protein, the NGF mutant has weakened binding to anti-NGF antibody or substantially no binding to anti-NGF antibody, and the NGF mutant still has biological activity. In other embodiments, the NGF mutant is selected from any one of the NGF mutants described above.
包含NGF突变体或其融合蛋白与抗NGF抗体的药物组合物Pharmaceutical composition comprising NGF mutant or its fusion protein and anti-NGF antibody
本申请一方面涉及包含NGF突变体或其融合蛋白(例如，NGF突变体与Fc的融合蛋白)和抗NGF抗体的药物组合物。In one aspect, the present application relates to a pharmaceutical composition comprising an NGF mutant or a fusion protein thereof (eg, a fusion protein of an NGF mutant and Fc) and an anti-NGF antibody.
在一些实施例中，在NGF突变体或其融合蛋白与抗NGF抗体的药物组合物中，其中所述的NGF突变体与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，同时该NGF突变体仍具有生物学活性。在另一些实施例中，所述NGF突变体选自如前所述的任一种NGF突变体。In some embodiments, in the pharmaceutical composition of NGF mutant or its fusion protein and anti-NGF antibody, the NGF mutant has weakened binding to the anti-NGF antibody or substantially no binding to the anti-NGF antibody, and the NGF mutant still has biological activity. In other embodiments, the NGF mutant is selected from any one of the NGF mutants described above.
在一些实施例中，在NGF突变体或其融合蛋白与抗NGF抗体的药物组合物中，其中所述的抗NGF抗体选自如表1中所示的抗NGF抗体中的一种或多种。In some embodiments, in the pharmaceutical composition of NGF mutant or its fusion protein and anti-NGF antibody, the anti-NGF antibody is selected from one or more of the anti-NGF antibodies shown in Table 1.
疾病的治疗方法Treatment of the disease
NGF相关疾病的治疗方法Treatment of NGF-related diseases
本申请一方面提供了一种应用NGF突变体或其融合蛋白或者包含NGF突变体或其融合蛋白的药物组合物预防和/或治疗NGF相关疾病的方法。在一些实施例中，该治疗方法中所述的NGF突变体与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，同时该NGF突变体仍具有生物学活性。在另一些实施例中，所述NGF突变体选自本申请中所述的任一种或多种NGF突变体。On the one hand, the present application provides a method for preventing and/or treating NGF-related diseases using an NGF mutant or its fusion protein or a pharmaceutical composition comprising an NGF mutant or its fusion protein. In some embodiments, the NGF mutant described in the treatment method has weakened binding to or substantially no binding to an anti-NGF antibody, and the NGF mutant still has biological activity. In other embodiments, the NGF mutant is selected from any one or more NGF mutants described in the present application.
在一些实施例中，涉及一种治疗个体(例如，人类)疾病(例如，NGF相关疾病，如神经系统疾病或非神经系统疾病的方法，包含向个体施用有效剂量的NGF突变体或其融合蛋白(例如，NGF突变体与Fc的融合蛋白)或者包含NGF突变体或其融合蛋白的药物组合物，所述NGF突变体与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，同时该NGF突变体仍具有生物学活性。In some embodiments, it relates to a method for treating a disease in an individual (e.g., a human being) (e.g., an NGF-related disease, such as a neurological disease or a non-neurological disease), comprising administering to the individual an effective dose of an NGF mutant or a fusion protein thereof (e.g., a fusion protein of an NGF mutant and Fc) or a pharmaceutical composition comprising an NGF mutant or a fusion protein thereof, wherein the NGF mutant has reduced binding to an anti-NGF antibody or substantially no binding to the anti-NGF antibody, and the NGF mutant still has biological activity.
在一些实施例中，NGF突变体或其融合蛋白或者包含NGF突变体或其融合蛋白的药物组合物通过静脉注射、肌肉注射或皮下注射给药。 In some embodiments, the NGF mutant or its fusion protein or the pharmaceutical composition comprising the NGF mutant or its fusion protein is administered by intravenous injection, intramuscular injection or subcutaneous injection.
本文所用的术语“NGF相关疾病”是指因NGF受体信号传导受损引起或与之相关的任何疾病或紊乱(如由于NGF数量不足和/或结合亲和力降低)，或需要NGF生物活性进行治疗的疾病或紊乱(如治疗时需要神经元生长、维持、增殖和/或存活的伤害/损伤)。The term "NGF-related disease" as used herein refers to any disease or disorder caused by or associated with impaired NGF receptor signaling (e.g., due to insufficient NGF amounts and/or reduced binding affinity), or a disease or disorder that requires the biological activity of NGF for treatment (e.g., treatment of injuries/damage that require neuronal growth, maintenance, proliferation and/or survival).
本文所述方法适用于治疗神经系统疾病(neurological disease)和非神经系统疾病(non-neurological disease)。The method described in this article is suitable for treating neurological diseases and non-neurological diseases.
神经系统疾病包括神经系统疾病(nervous system disease)。神经系统疾病是指与中枢和/或外周神经系统的神经元变性或损伤相关的疾病。神经系统疾病的具体示例包括但不限于阿尔茨海默病、帕金森病、亨廷顿病、中风、肌萎缩侧索硬化(ALS)、面神经炎、颅脑或脊髓损伤、急性脑血管病、脑萎缩、外周神经病变和其它以神经元坏死或缺失为特征的疾病，无论是中枢神经元、外周神经元还是运动神经元，除了由外伤、烧伤、肾功能衰竭、损伤或化学品/药物引起的神经损伤，如由化学品或药物引起的急性脑血管中枢神经损伤。神经系统疾病还包括与某些疾病相关的外周神经病变，如与糖尿病、艾滋病或化疗相关的神经病变。在一些实施例中，神经系统疾病选自多发性梗死性痴呆、血管性痴呆、由酒精中毒引起的器质性脑疾病引起的认知障碍、阿尔茨海默病、帕金森病、癫痫、多发性硬化症、亨廷顿舞蹈病、唐氏综合征、神经性耳聋、梅尼埃病、中风、ALS、贝尔氏麻痹、涉及脊髓肌萎缩的疾病、涉及瘫痪的疾病、外周神经病变、创伤所致神经损伤、烧伤所致神经损伤、肾功能障碍所致神经损伤、损伤所致神经损伤、化疗毒副作用所致神经损伤、手术导致的神经损伤、缺血导致的神经损伤、感染导致的神经损伤、代谢疾病导致的神经损伤以及营养缺乏导致的神经损伤。在一些实施例中，神经系统疾病是从糖尿病外周神经病变、毒素诱导外周神经病变、化疗诱导外周神经病变、HIV相关外周神经病变和影响运动神经元的外周神经病变组成的组中选择的外周神经病变。在一些实施例中，神经系统疾病选自新生儿缺氧缺血性脑病、脑瘫、重症肌病、神经性耳聋、喉返神经损伤、创伤性脑损伤、牙神经损伤、脑卒中、唐氏综合征、ALS，多发性硬化、脊髓肌萎缩、弥漫性脑损伤、胸腺发育不良、视神经挫伤、滤泡发育不良、脊髓损伤、青光眼、神经营养性角膜炎、视神经损伤、视神经脊髓炎、视网膜相关疾病、尿失禁、阿尔茨海默病、帕金森病、亨廷顿病、痴呆、，高血压脑出血神经功能障碍、脑小血管疾病、 急性缺血性中风、角膜内皮营养不良、糖尿病足溃疡、神经源性皮肤溃疡、压疮、神经营养性角膜溃疡、糖尿病角膜溃疡和黄斑裂孔。Nervous system diseases include nervous system diseases. Nervous system diseases refer to diseases related to neuronal degeneration or damage of the central and/or peripheral nervous systems. 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 cord injury, acute cerebrovascular disease, brain atrophy, peripheral neuropathy and other diseases characterized by neuronal necrosis or loss, whether central neurons, peripheral neurons or motor neurons, except for nerve damage caused by trauma, burns, renal failure, injury or chemicals/drugs, such as acute cerebrovascular central nervous damage caused by chemicals or drugs. Nervous system diseases also include peripheral neuropathy associated with certain diseases, such as neuropathy associated with diabetes, AIDS or chemotherapy. In some embodiments, the nervous system disease is selected from multi-infarct dementia, vascular dementia, cognitive impairment caused by organic brain diseases caused by alcoholism, Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's disease, Down syndrome, neurological deafness, Meniere's disease, stroke, ALS, Bell's palsy, diseases involving spinal muscular atrophy, diseases involving paralysis, peripheral neuropathy, nerve damage caused by trauma, nerve damage caused by burns, nerve damage caused by renal dysfunction, nerve damage caused by injury, nerve damage caused by chemotherapy toxicity, nerve damage caused by surgery, nerve damage caused by ischemia, nerve damage caused by infection, nerve damage caused by metabolic diseases, and nerve damage caused by 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-related peripheral neuropathy, and peripheral neuropathy affecting motor neurons. In some embodiments, the nervous system disease is selected from neonatal hypoxic-ischemic encephalopathy, cerebral palsy, severe myopathy, neurological deafness, recurrent laryngeal nerve injury, traumatic brain injury, dental nerve injury, stroke, Down 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, retinal related diseases, 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.
非神经系统疾病包括脾脏萎缩、脾挫伤、卵巢储备减少、卵巢早衰(POF)、卵巢过度刺激综合征、卵巢残余综合征、卵巢卵泡发育不良、生精障碍(例如，少精子症(oligozoospermia或oligospermia)、弱精子症、少弱精子症、无精子症、畸形精子症、少弱畸精子症(oligoasthenoteratozoospermia，OAT综合征))、缺血性溃疡、应激性溃疡、类风湿性溃疡、肝纤维化、角膜溃疡、烧伤、口腔溃疡和腿部静脉溃疡。Non-neurological diseases include splenic atrophy, splenic contusion, diminished ovarian reserve, premature ovarian failure (POF), ovarian hyperstimulation syndrome, ovarian remnant syndrome, ovarian follicular hypoplasia, spermatogenic disorders (e.g., oligozoospermia (oligospermia or oligospermia), asthenozoospermia, oligoasthenozoospermia, azoospermia, teratozoospermia, oligoasthenoteratozoospermia (OAT syndrome)), ischemic ulcers, stress ulcers, rheumatoid ulcers, liver fibrosis, corneal ulcers, burns, oral ulcers, and venous leg ulcers.
在一些实施例中，治疗疾病(例如，NGF相关疾病，如神经系统疾病(例如，糖尿病神经性病变、阿尔茨海默症或神经营养性角膜炎)或非神经系统疾病(例如，卵巢早衰或生精障碍)的方法中的NGF突变体具有以下一种或多种生物活性：(i)支持神经元存活；(ii)促进神经突生长；(iii)增强神经化学分化；(iv)促进胰腺β细胞增殖；(v)诱导先天性和/或获得性免疫；(vi)修复受损的神经细胞(例如，角膜神经)和/或防止损伤(例如，在神经营养性角膜炎中)；(vii)促进卵泡细胞的增殖和/或雌激素分泌；(viii)促进伤口愈合(例如，在糖尿病神经性病变中)；(ix)改善患有神经退行性疾病(例如，阿尔茨海默病)的受试者的空间认知、记忆和/或学习能力；(x)治疗和/或预防神经退行性病变；(xi)治疗睾丸生精小管萎缩、生精小管生精障碍和/或附睾管细胞碎片；(xii)挽救精子数量和/或活力的降低，或增加精子数量和/或活力(例如，在生精障碍中)；(xiii)预防/逆转卵泡数量和/或功能的降低，或提高卵泡数量和/或功能(例如，在卵巢早衰中)；和/或(xiv)延长患者生存期。In some embodiments, the NGF mutant in the method of 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 spermatogenesis disorder) has one or more of the following biological activities: (i) supporting neuronal survival; (ii) promoting neurite outgrowth; (iii) enhancing neurochemical differentiation; (iv) promoting pancreatic β-cell proliferation; (v) inducing innate and/or acquired immunity; (vi) repairing damaged nerve cells (e.g., corneal nerves) and/or preventing damage (e.g., in neurotrophic keratitis); (vii) promoting proliferation of follicular cells and/or estrogen production; hormone secretion; (viii) promote wound healing (e.g., in diabetic neuropathy); (ix) improve spatial cognition, memory and/or learning ability in subjects with neurodegenerative diseases (e.g., Alzheimer's disease); (x) treat and/or prevent neurodegenerative diseases; (xi) treat testicular seminiferous tubule atrophy, seminiferous tubule spermatogenesis disorder and/or epididymal tubular cell fragmentation; (xii) rescue the decrease in sperm count and/or motility, or increase sperm count and/or motility (e.g., in spermatogenesis disorder); (xiii) prevent/reverse the decrease in follicle number and/or function, or increase follicle number and/or function (e.g., in premature ovarian failure); and/or (xiv) prolong patient survival.
由NGF表达增加和/或对NGF敏感性增强引起的疾病或病症的治疗方法Methods for treating diseases or conditions caused by increased NGF expression and/or enhanced sensitivity to NGF
本申请一方面提供了一种应用抗NGF抗体联合本申请所述的NGF突变体或其融合蛋白(例如，NGF突变体与Fc的融合蛋白)，或者应用包含抗NGF抗体和本申请所述的NGF突变体或其融合蛋白的药物组合物来预防和/或治疗由NGF表达增加和/或对NGF敏感性增强所引起的疾病或病症的方法，其中所述的NGF突变体与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，同时该NGF突变体仍具有NGF的生物学活性。在一些实施例中，该治疗方法中所述的NGF突变体选自本申请中所述的任一种或多种NGF突变体。在一些实施例中，NGF突变体或包含其的融合蛋白(或其药物组合物)通过静脉注射、肌肉注射或皮下注射给药。 On the one hand, the present application provides a method for preventing and/or treating diseases or conditions caused by increased NGF expression and/or enhanced sensitivity to NGF by using an anti-NGF antibody in combination with an NGF mutant or a fusion protein thereof described in the present application (e.g., a fusion protein of an NGF mutant and Fc), or using a pharmaceutical composition comprising an anti-NGF antibody and an NGF mutant or a fusion protein thereof described in the present application, wherein the NGF mutant has weakened binding to the anti-NGF antibody or substantially no binding to the anti-NGF antibody, and the NGF mutant still has the biological activity of NGF. In some embodiments, the NGF mutant described in the treatment method is selected from any one or more NGF mutants described in the present application. In some embodiments, the NGF mutant or a fusion protein comprising the same (or a pharmaceutical composition thereof) is administered by intravenous injection, intramuscular injection, or subcutaneous injection.
可以通过任何合适的方式向受试者施用所述的抗NGF抗体和本文所述的NGF突变体或其融合蛋白(例如，NGF突变体与Fc的融合蛋白)。在一些实施例中，所述抗NGF抗体和NGF突变体或其融合蛋白被配制成静脉内注射施用。在一些实施例中，所述抗NGF抗体与NGF突变体或其融合蛋白同时施用，例如在单一制剂中或者作为单独的制剂同时施用。在一些实施例中，所述抗NGF抗体和NGF突变体或其融合蛋白顺序地施用，例如，作为单独的制剂。The anti-NGF antibody and the NGF mutant or its fusion protein described herein (e.g., a fusion protein of an NGF mutant and Fc) can be administered to a subject by any suitable means. In some embodiments, the anti-NGF antibody and the NGF mutant or its fusion protein are formulated for intravenous injection. In some embodiments, the anti-NGF antibody and the NGF mutant or its fusion protein are administered simultaneously, for example, in a single formulation or as separate formulations. In some embodiments, the anti-NGF antibody and the NGF mutant or its fusion protein are administered sequentially, for example, as separate formulations.
本申请所述的“由NGF表达增加和/或敏感性增强引起的疾病或病症”包括但并不限于急性疼痛、牙齿疼痛、外伤疼痛、手术疼痛、截肢或脓肿引起的疼痛、灼痛、脱髓鞘疾病、三叉神经痛、癌症、慢性酒精中毒、中风、丘脑疼痛综合症、糖尿病、获得性免疫缺陷综合症(AIDS)、毒素和化疗、一般头痛、偏头痛、丛集性头痛、混合血管和非血管综合征、紧张性头痛、一般炎症、关节炎、风湿病、红斑狼疮、骨关节炎、炎症性肠病、肠易激综合征、炎症性眼病、炎症性或不稳定的膀胱病、银屑病、有炎症成分的皮肤不适、晒伤、心肌炎、皮炎、肌炎、神经炎、胶原血管疾病、慢性炎症疾病、炎症性疼痛及相关的痛觉过敏和痛觉超敏、神经性疼痛及相关的痛觉过敏和痛觉超敏、糖尿病神经病变疼痛、灼痛、交感神经维持性疼痛、传入神经阻滞综合征、哮喘、上皮组织损伤或功能障碍、单纯性疱疹，呼吸系统、泌尿生殖系统、胃肠道或血管区域的内脏运动紊乱，伤口、烧伤、过敏性皮肤反应、瘙痒症、白癜风、一般胃肠道疾病、结肠炎、胃溃疡、十二指肠溃疡、血管舒缩性或过敏性鼻炎、或支气管疾病、痛经、消化不良、胃食管反流、胰腺炎和内脏痛。The "diseases or conditions caused by increased NGF expression and/or enhanced sensitivity" described in the present application include but are not limited to acute pain, dental pain, traumatic pain, surgical pain, pain caused by amputation or abscess, burning pain, demyelinating disease, trigeminal neuralgia, cancer, chronic alcoholism, stroke, thalamic pain syndrome, diabetes, acquired immune deficiency syndrome (AIDS), toxins and chemotherapy, general headache, migraine, cluster headache, mixed vascular and non-vascular syndrome, tension headache, general inflammation, arthritis, rheumatism, lupus erythematosus, osteoarthritis, inflammatory bowel disease, irritable bowel syndrome, inflammatory eye disease, inflammatory or unstable bladder disease, psoriasis, skin complaints with inflammatory components, sunburn, etc. Injury, myocarditis, dermatitis, myositis, neuritis, collagen vascular disease, chronic inflammatory disease, inflammatory pain and related hyperalgesia and allodynia, neuropathic pain and related hyperalgesia and allodynia, diabetic neuropathy pain, causalgia, sympathetically maintained pain, deafferentation syndrome, asthma, epithelial tissue damage or dysfunction, herpes simplex, visceral motor disorders of the respiratory, genitourinary, gastrointestinal or vascular regions, wounds, burns, allergic skin reactions, pruritus, vitiligo, general gastrointestinal diseases, colitis, gastric ulcer, duodenal ulcer, vasomotor or allergic rhinitis, or bronchial diseases, dysmenorrhea, dyspepsia, gastroesophageal reflux, pancreatitis and visceral pain.
在一些实施例中，本申请所述的“NGF表达增加和/或敏感性增强的疾病或病症”包括炎症性疼痛、术后切口疼痛、神经性疼痛、骨折疼痛、痛风关节疼痛、带状疱疹后神经痛、烧伤引起的疼痛、癌症疼痛、骨关节炎或类风湿性关节炎疼痛、坐骨神经痛、与镰状细胞危象相关的疼痛，或疱疹性神经痛。In some embodiments, the "diseases or conditions with increased NGF expression and/or enhanced sensitivity" described in the present application include inflammatory pain, postoperative incisional pain, neuropathic pain, fracture pain, gouty joint pain, postherpetic neuralgia, pain caused by burns, cancer pain, osteoarthritis or rheumatoid arthritis pain, sciatica, pain associated with sickle cell crisis, or herpetic neuralgia.
降低和/或减轻应用抗NGF抗体治疗时的不良反应的方法Method for reducing and/or alleviating adverse reactions during treatment with anti-NGF antibodies
本申请还提供一种降低和/或减轻应用抗NGF抗体治疗时不良反应的方法，包括向已经接受、正在接受或即将接受抗NGF抗体治疗的受试者施用本申请所述的NGF突变体或其融合蛋白，或包含NGF突变体或其融合蛋白的药物组合物，其中所述NGF突变体与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，同时仍具有NGF的生物 学活性。在一些实施例中，所述NGF突变体选自本申请所述的任一种或多种NGF突变体。The present application also provides a method for reducing and/or alleviating adverse reactions during anti-NGF antibody treatment, comprising administering the NGF mutant or its fusion protein described in the present application, or a pharmaceutical composition comprising the NGF mutant or its fusion protein, to a subject who has received, is receiving, or is about to receive anti-NGF antibody treatment, wherein the NGF mutant has weakened binding to the anti-NGF antibody or substantially no binding to the anti-NGF antibody, while still having the biological activity of NGF. In some embodiments, the NGF mutant is selected from any one or more NGF mutants described in the present application.
本申请所述的应用抗NGF抗体治疗时产生的不良反应包括但不限于神经系统相关的不良反应和关节相关的不良反应。在一些实施例中，所述不良反应包括：交感神经损伤、骨坏死、骨丢失、骨损伤、关节损伤、快速进展的骨关节炎(RPOA)、外周性水肿、关节痛、四肢疼痛、周围神经感觉异常、感觉迟钝、感觉过敏、感觉异常、灼烧痛和触感痛。The adverse reactions produced when using anti-NGF antibodies for treatment described in the present application include, but are not limited to, adverse reactions related to the nervous system and adverse reactions related to joints. In some embodiments, the adverse reactions include: sympathetic nerve damage, osteonecrosis, bone loss, bone damage, joint damage, rapidly progressive osteoarthritis (RPOA), peripheral edema, joint pain, limb pain, peripheral nerve paresthesia, dysesthesia, hyperesthesia, paresthesia, burning pain and tactile pain.
本申请所述的NGF突变体或其融合蛋白、编码NGF突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、包含NGF突变体或其融合蛋白的药物组合物、或同时包含NGF突变体或其融合蛋白与抗NGF抗体的药物组合物等的给药方式可以以任何已知和方便的方式进行，包括通过注射或输液。给药途径依照已知和公认的方法，如通过单次或多次推注或以适当方式长时间输液。The administration of the NGF mutant or its fusion protein, the nucleic acid encoding the NGF mutant or its fusion protein, the vector and host cell comprising the nucleic acid, the pharmaceutical composition comprising the NGF mutant or its fusion protein, or the pharmaceutical composition comprising the NGF mutant or its fusion protein and the anti-NGF antibody at the same time, etc. described in the present application can be carried out in any known and convenient manner, including by injection or infusion. The route of administration is in accordance with known and recognized methods, such as by single or multiple push injections or long-term infusion in an appropriate manner.
本申请的药物组合物的剂量和所需药物浓度可根据特定用途而变化。确定合适的给药剂量或给药途径完全属于普通技术人员的技术范围。动物实验为确定人类治疗的有效剂量提供了可靠的指导。可以依据Mordenti,J.和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中的原则进行有效剂量的种间类推。The dosage and required drug concentration of the pharmaceutical composition of the present application may vary depending on the specific use. Determining the appropriate dosage or route of administration is well within the technical scope of ordinary technicians. Animal experiments provide reliable guidance for determining effective doses for human treatment. Interspecies analogies of effective doses can be made based on the principles of Mordenti, J. and 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.
当使用NGF突变体、包含NGF突变体的融合蛋白、编码NGF突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、包含NGF突变体或其融合蛋白的药物组合物、或同时包含NGF突变体或其融合蛋白与抗NGF抗体的药物组合物在体内给药时，治疗有效量可取决于例如治疗情境和目标。本领域普通技术人员将理解，用于治疗的适当剂量水平将取决于被递送的分子、治疗的适应症、施用途径和患者的体型(体重，体表面或器官的大小)和/或状况(年龄和一般健康状况)而变化。在本申请的范围内，不同的制剂将对不同的治疗和不同的疾病有效，并且旨在治疗特定器官或组织的给药方式可能与针对另一器官或组织的方式不同。此外，剂量可通过一次或多次单独给药或持续输注给药。对于几天或更长时间的重复给药，根据病情，治疗持续到疾病症状达到预期的抑制程度为止。或者，其它剂量方案可能有用。这种治疗的进展很容易通过常规技术和分析进行监测。特定患者的最佳剂量和治疗方案可以由医学领域的技术人员通过监测患者的疾病体征并相应地进行调整以确定。 When NGF mutants, fusion proteins comprising NGF mutants, nucleic acids encoding NGF mutants or their fusion proteins, vectors and host cells comprising such nucleic acids, pharmaceutical compositions comprising NGF mutants or their fusion proteins, or pharmaceutical compositions comprising both NGF mutants or their fusion proteins and anti-NGF antibodies are administered in vivo, the therapeutically effective amount may depend on, for example, the therapeutic context and goals. One of ordinary skill in the art will appreciate that appropriate dosage levels for treatment will vary depending on the molecule being delivered, the indication for treatment, the route of administration, and the patient's size (weight, size of body surface or organs) and/or condition (age and general health). Within the scope of the present application, different formulations will be effective for different treatments and different diseases, and the mode of administration intended to treat a specific organ or tissue may be different from that for another organ or tissue. In addition, the dose may be administered by one or more separate administrations or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, treatment continues until the symptoms of the disease reach the desired level of suppression. Alternatively, other dosage regimens may be useful. The progress of such treatment is easily monitored by conventional techniques and analysis. The optimum dosage and treatment regimen for a particular patient can be determined by one skilled in the medical arts by monitoring the patient for signs of disease and making adjustments accordingly.
在一些实施例中，本申请所述的疾病的治疗方法中的NGF突变体包含本申请所述的任一种或多种NGF突变体。In some embodiments, the NGF mutant used in the treatment methods of the diseases described herein comprises any one or more NGF mutants described herein.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的I31、K32、G33、K34、D93、W21、G23、D24、K50、Y52、T83、H84、F86、R100、R103、D16、S17、S19、T56、R59和R69位点中一个或多个位点上的氨基酸突变。In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises an amino acid mutation at one or more of the I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 positions relative to the human wild-type mature NGF amino acid sequence.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S17、S19、K32、T56、R59和T83中的一个或多个位点上的氨基酸突变。In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises an amino acid mutation at one or more of S17, S19, K32, T56, R59 and T83 relative to the human wild-type mature NGF amino acid sequence.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S17和K32位点的氨基酸突变。In some embodiments, the NGF mutant in the pharmaceutical composition or method of treating a disease described herein comprises amino acid mutations at positions S17 and K32 relative to the amino acid sequence of wild-type mature human NGF.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S19和K32位点的氨基酸突变。In some embodiments, the NGF mutant in the pharmaceutical composition or method of treating a disease described herein comprises amino acid mutations at positions S19 and K32 relative to the amino acid sequence of wild-type mature NGF.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S19和S17位点的氨基酸突变。In some embodiments, the NGF mutant in the pharmaceutical composition or method of treating a disease described herein comprises amino acid mutations at positions S19 and S17 relative to the amino acid sequence of wild-type mature NGF.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的R59和T83位点的氨基酸突变。In some embodiments, the NGF mutant in the pharmaceutical composition or method for treating a disease described herein comprises amino acid mutations at positions R59 and T83 relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S17位点的氨基酸突变为S17R、S17K、S17H或S17E。In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described herein comprises an amino acid mutation at position S17R, S17K, S17H or S17E relative to the amino acid sequence of wild-type mature NGF.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S19位点的氨基酸突变为S19R、S19K或S19F。In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described herein comprises an amino acid mutation at position S19 to S19R, S19K or S19F relative to the amino acid sequence of wild-type mature NGF.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的K32位点的氨基酸突变为K32F、K32E、K32N、K32Y、K32M或K32L。 In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises an amino acid mutation at the K32 position relative to the human wild-type mature NGF amino acid sequence to K32F, K32E, K32N, K32Y, K32M or K32L.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的T56位点的氨基酸突变为T56R或T56K。In some embodiments, the NGF mutant in the pharmaceutical composition or method of treating a disease described herein comprises an amino acid mutation at position T56 relative to the amino acid sequence of wild-type mature NGF to T56R or T56K.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的R59位点的氨基酸突变为R59K。In some embodiments, the NGF mutant in the pharmaceutical composition or method of treating a disease described herein comprises an amino acid mutation at position R59 relative to the amino acid sequence of wild-type mature NGF to R59K.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的T83位点的氨基酸突变为T83K。In some embodiments, the NGF mutant in the pharmaceutical composition or method of treating a disease described herein comprises an amino acid mutation at position T83 relative to the amino acid sequence of wild-type mature NGF to T83K.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S17和K32位点的氨基酸突变，其中S17位点的突变为S17R，和K32位点的突变为K32E、K32Y、K32M或K32L。In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises amino acid mutations at the S17 and K32 positions relative to the human wild-type mature NGF amino acid sequence, wherein the mutation at the S17 position is S17R, and the mutation at the K32 position is K32E, K32Y, K32M or K32L.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S19和K32位点的氨基酸突变，其中S19位点的突变为S19R，和K32位点的突变为K32E、K32Y、K32M或K32L。In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises amino acid mutations at the S19 and K32 positions relative to the human wild-type mature NGF amino acid sequence, wherein the mutation at the S19 position is S19R, and the mutation at the K32 position is K32E, K32Y, K32M or K32L.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的S19和S17位点的氨基酸突变，其中S19位点的突变为S19R，和S17位点的突变为S17E。In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises amino acid mutations at the S19 and S17 positions relative to the human wild-type mature NGF amino acid sequence, wherein the mutation at the S19 position is S19R, and the mutation at the S17 position is S17E.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含相对于人野生型成熟NGF氨基酸序列的R59和T83位点的氨基酸突变，其中R59位点的突变为R59K，和T83位点的突变为T83K。In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises amino acid mutations at the R59 and T83 positions relative to the human wild-type mature NGF amino acid sequence, wherein the mutation at the R59 position is R59K, and the mutation at the T83 position is T83K.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体进一步包含相对于人野生型成熟NGF氨基酸序列的F12E突变。In some embodiments, the NGF mutant in the pharmaceutical composition or method of treating a disease described herein further comprises a F12E mutation relative to the amino acid sequence of human wild-type mature NGF.
在一些实施例中，所述人野生型成熟NGF的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。In some embodiments, the amino acid sequence of the human wild-type mature NGF is shown as SEQ ID NO:1 or SEQ ID NO:2.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体包含SEQ ID NOs:17-60中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:17-60中任一所示的氨基酸序列具有至少90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。 In some embodiments, the NGF mutant in the pharmaceutical composition or disease treatment method described in the present application comprises an amino acid sequence shown in any one of SEQ ID NOs: 17-60 or a variant thereof, wherein the variant has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 17-60.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的NGF突变体的融合蛋白为NGF突变体与Fc的融合蛋白。在一些实施例中，所述NGF突变体与Fc的融合蛋白从N端到C端或从C端到N端包含：本文所述的NGF突变体、可选的肽接头和Fc部分。在一些实施例中，可选的肽接头包含SEQ ID NO:87所示的氨基酸序列。在一些实施例中，Fc部分包含SEQ ID NO:85所示的氨基酸序列或其变体，所述变体具有与SEQ ID NO:85至少90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。在一些实施例中，所述NGF突变体与Fc的融合蛋白包含SEQ ID NOs:61-82中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:61-82中任一所示的氨基酸序列具有至少80％(例如至少80％、85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。In some embodiments, the fusion protein of the NGF mutant in the pharmaceutical composition or the method for treating a disease described in the present application is a fusion protein of the NGF mutant and Fc. In some embodiments, the fusion protein of the NGF mutant and Fc comprises from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: the NGF mutant described herein, an optional peptide linker and an Fc portion. In some embodiments, the optional peptide linker comprises the amino acid sequence shown in SEQ ID NO: 87. In some embodiments, the Fc portion comprises the amino acid sequence shown in SEQ ID NO: 85 or a variant thereof, wherein the variant has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO: 85. In some embodiments, the fusion protein of the NGF mutant and Fc comprises the amino acid sequence shown in any one of SEQ ID NOs:61-82 or a variant thereof, and the variant has at least 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with the amino acid sequence shown in any one of SEQ ID NOs:61-82.
在一些实施例中，本申请所述的药物组合物或疾病的治疗方法中的抗NGF抗体为现有技术中披露的任一抗NGF抗体。在一些实施例中，本申请所述的抗NGF抗体包括，但不限于：Tanezumab(辉瑞)、Fasinumab(再生元)、Fulranumab(强生)、MEDI-578(阿斯利康)、AK-115(康方生物)、SSS-40(三生国健)以及STC001(舒泰神)。在一些实施例中，上述示例性抗NGF抗体的序列如表1中所示。In some embodiments, the anti-NGF antibody in the pharmaceutical composition or the method for treating a disease described herein is any anti-NGF antibody disclosed in the prior art. In some embodiments, the anti-NGF antibodies described herein include, but are not limited to: Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Kangfang Biopharma), SSS-40 (Sunshine Guojian) and STC001 (Sutacion). In some embodiments, the sequences of the above exemplary anti-NGF antibodies are shown in Table 1.
制品及试剂盒Products and kits
进一步涉及包含本申请所述的任何NGF突变体或其融合蛋白的试剂盒、单位剂量和制品，或者同时包含本申请所述的任何NGF突变体或其融合蛋白和抗NGF抗体的试剂盒、单位剂量和制品。在一些实施例中，涉及包含本文所述的任一种药物组合物的试剂盒，并且优选地提供其使用说明，如用于治疗本文所述的疾病(例如，神经系统疾病)。Further relates to a kit, unit dose and product comprising any NGF mutant or its fusion protein described in the present application, or a kit, unit dose and product comprising any NGF mutant or its fusion protein described in the present application and an anti-NGF antibody at the same time. In some embodiments, it relates to a kit comprising any of the pharmaceutical compositions described herein, and preferably provides instructions for its use, such as for the treatment of diseases described herein (e.g., neurological diseases).
本申请的试剂盒包括一个或多个包含本申请所述的任何NGF突变体或其融合蛋白或者同时包含NGF突变体或其融合蛋白和抗NGF抗体的容器，例如，用于治疗疾病。例如，包含描述施用NGF突变体或其融合蛋白以治疗疾病(如神经系统疾病)的说明书或者联合应用抗NGF抗体和NGF突变体或其融合蛋白用于降低抗NGF抗体不良反应的说明书。试剂盒可能进一步包含基于识别个体是否患有疾病和疾病阶段来选择适合治疗的个体(例如，人类)的描述。与治疗NGF相关的疾病或降低抗NGF抗体不良反应的使用相关的说明通常包括关于预期治疗的剂量、给药计划和给药途径的信息。容器可以是单位剂量、散装包装(例如，多剂量包装)或亚单位剂量。本申请的 试剂盒中提供的说明通常是标签或药品说明书上的书面说明(例如，试剂盒中包括的纸张)，但机器可读说明(例如，存储在磁盘或光盘上的说明)也是可以接受的。本申请的试剂盒采用合适的包装。合适的包装包括但不限于小瓶、瓶子、罐子、软包装(例如，密封的聚酯薄膜或塑料袋)等。还考虑与特定装置结合使用的包装，如输液装置如微型泵。试剂盒可具有无菌接入端口(例如，容器可为静脉注射溶液袋或具有可被皮下注射针刺穿的塞子的小瓶)。该组合物中的至少一种活性剂是如本申请所述的NGF突变体或包含其的融合蛋白。容器可进一步包含第二种医药活性剂。试剂盒可选择性地提供附加组份，如缓冲液和解释信息。一般来说，试剂盒包含一个容器和容器上或与容器相关的标签或药品说明书。The kit of the present application includes one or more containers containing any NGF mutant or its fusion protein described in the present application, or containing both NGF mutant or its fusion protein and anti-NGF antibody, for example, for treating a disease. For example, it contains instructions describing the administration of NGF mutant or its fusion protein to treat a disease (such as a nervous system disease) or instructions for the combined use of anti-NGF antibody and NGF mutant or its fusion protein to reduce the adverse effects of anti-NGF antibody. The kit may further include a description of selecting an individual (e.g., human) suitable for treatment based on identifying whether the individual has a disease and the stage of the disease. Instructions related to the use of treating NGF-related diseases or reducing adverse reactions of anti-NGF antibodies typically include information about the dose, dosing schedule, and route of administration for the intended treatment. The container can be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The kit of the present application The instructions provided in the kit are typically written instructions on a label or drug instructions (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions stored on a disk or CD) are also acceptable. The kit of the present application is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. Packaging used in conjunction with specific devices, such as infusion devices such as micropumps, is also considered. 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 injection needle). At least one active agent in the composition is an NGF mutant or a fusion protein containing it as described in the present application. The container may further contain a second pharmaceutically active agent. The kit may optionally provide additional components, such as a buffer and explanatory information. Generally, the kit comprises a container and a label or drug instructions on or associated with the container.
因此，本申请还涉及制品，包括小瓶(如密封小瓶)、瓶、罐、软包装等。该制品包含容器和容器上或与容器相关的标签或药品说明书。合适的容器包括，例如，瓶子、小瓶、注射器等。容器可以由多种材料制成，如玻璃或塑料。一般来说，容器容纳的组合物可有效治疗本文所述疾病或紊乱(如神经系统疾病)，并且可具有无菌接入端口(例如，容器可为静脉注射溶液袋或具有可被皮下注射针刺穿的塞子的小瓶)。标签或药品说明书表明该组合物用于治疗个体的特定病症。标签或药品说明书进一步包含向个体施用组合物的说明。标签可能会注明重构和/或使用的说明。容纳药物组合物的容器可以是多次使用的小瓶，允许重构制剂重复施用(例如，2-6次施用)。药品说明书是指通常包含在治疗产品商业包装中的说明书，其中包含有关使用此类治疗产品的适应症、用法、剂量、给药、禁忌症和/或警告信息。此外，制品可能进一步包含第二容器，包含药学可接受的缓冲液，如注射用抑菌水(BWFI)、磷酸盐缓冲盐水、林格溶液和葡萄糖溶液。从商业和用户角度来看，可能进一步包括其它需要的材料，包括其它缓冲液、稀释剂、过滤器、针头和注射器。Therefore, the present application also relates to articles, including vials (such as sealed vials), bottles, cans, flexible packages, etc. The article comprises a container and a label or drug instructions on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be made of a variety of materials, such as glass or plastic. In general, the composition contained in the container can effectively treat the diseases or disorders described herein (such as nervous system diseases), and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The label or drug instructions indicate that the composition is used to treat a specific condition of an individual. The label or drug instructions further include instructions for administering the composition to an individual. The label may indicate instructions for reconstitution and/or use. The container for holding the pharmaceutical composition can be a vial for multiple uses, allowing the reconstituted preparation to be repeatedly administered (for example, 2-6 administrations). Drug instructions refer to instructions usually contained in the commercial packaging of therapeutic products, which contain indications, usage, dosage, administration, contraindications and/or warning information about the use of such therapeutic products. In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. From a commercial and user perspective, other required materials may be further included, including other buffers, diluents, filters, needles and syringes.
试剂盒或制品包括多个单位剂量的药物组合物和使用说明书，包装数量足以在药房中储存和使用，例如，医院药房和复方药房。The kit or article of manufacture includes multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies.
实施例Example
以下实施例旨在纯粹作为本申请的示例，因此不应被视为以任何方式限制本申请。以下实施例和详述是以说明的方式提供的，而非限制的方式。 The following examples are intended to be purely exemplary of the present application and therefore should not be considered to limit the present application in any way.The following examples and detailed description are provided by way of illustration and not by way of limitation.
实施例1：筛选并获得NGF中的可突变位点Example 1: Screening and obtaining mutagenic sites in NGF
通过如下方法筛选NGF中的可突变位点，所述可突变位点上的氨基酸突变会使NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，但仍具有NGF的生物学活性。The mutable sites in NGF are screened by the following method. The amino acid mutation at the mutable site will weaken the binding of the NGF mutant to the anti-NGF antibody or substantially prevent the NGF mutant from binding to the anti-NGF antibody, but the mutant still has the biological activity of NGF.
选取目前国内外主要的几个抗NGF抗体，即Tanezumab(辉瑞)、Fasinumab(再生元)、Fulranumab(强生)、MEDI-578(阿斯利康)、AK-115(康方生物)、SSS-40(三生国健)以及STC001(舒泰神)作为示例，以完成本发明。上述抗NGF抗体的VH及VL的氨基酸序列如表1所示。Several major anti-NGF antibodies at home and abroad, namely Tanezumab (Pfizer), Fasinumab (Regeneron), Fulranumab (Johnson & Johnson), MEDI-578 (AstraZeneca), AK-115 (Akeso Bio), SSS-40 (Sunshine Guojian) and STC001 (Sultaishen) are selected as examples to complete the present invention. The amino acid sequences of VH and VL of the above anti-NGF antibodies are shown in Table 1.
表1

Table 1

以STC001抗体为示例，进行以下步骤：Taking STC001 antibody as an example, perform the following steps:
(i)应用Modeller同源建模方法对STC001抗体进行同源建模，构建STC001抗体的三维结构。(i) The Modeller homology modeling method was used to perform homology modeling on the STC001 antibody and construct the three-dimensional structure of the STC001 antibody.
(ii)应用Discovery studio ZDOCK软件将人野生型成熟NGF的晶体结构与STC001抗体的三维结构进行对接，通过软件评分函数对所得到的结合构象进行排序， 获得STC001与人野生型成熟NGF的结合构象。其中人野生型成熟NGF的晶体结构如PDB ID：4EDW所示。(ii) The crystal structure of human wild-type mature NGF was docked with the three-dimensional structure of the STC001 antibody using the Discovery studio ZDOCK software, and the resulting binding conformations were ranked using the software scoring function. The binding conformation of STC001 and human wild-type mature NGF was obtained. The crystal structure of human wild-type mature NGF is shown in PDB ID: 4EDW.
(iii)对比分析STC001与人野生型成熟NGF的结合构象以及TrkA受体与人野生型成熟NGF的结合构象，例如，可以将STC001与人野生型成熟NGF的结合构象与TrkA受体与人野生型成熟NGF的结合构象进行叠合后进行对比分析，挑选出NGF中位于与STC001结合界面，但不位于与TrkA受体结合界面的一系列氨基酸位点，该一系列氨基酸位点即为NGF中针对STC001的可突变位点。其中TrkA受体与人野生型成熟NGF的结合构象如PDB ID：1WWW所示。(iii) Comparative analysis of the binding conformation of STC001 to human wild-type mature NGF and the binding conformation of TrkA receptor to human wild-type mature NGF. For example, the binding conformation of STC001 to human wild-type mature NGF and the binding conformation of TrkA receptor to human wild-type mature NGF can be superimposed and then compared and analyzed to select a series of amino acid sites in NGF that are located at the binding interface with STC001 but not at the binding interface with TrkA receptor. This series of amino acid sites is the mutable sites in NGF for STC001. The binding conformation of TrkA receptor to human wild-type mature NGF is shown in PDB ID: 1WWW.
STC001与人野生型成熟NGF的结合构象示意图如图1A所示；TrkA受体与人野生型成熟NGF的结合构象示意图如图1B所示；将STC001与人野生型成熟NGF的结合构象与TrkA受体与人野生型成熟NGF的结合构象进行叠合后的构象图如图1C所示。其中，本申请所述的NGF中的可突变位点的位置是相对于人野生型成熟NGF的氨基酸序列确定的。示例性的人野生型成熟NGF的氨基酸序列如表2所示。The schematic diagram of the binding conformation of STC001 and human wild-type mature NGF is shown in Figure 1A; the schematic diagram of the binding conformation of TrkA receptor and human wild-type mature NGF is shown in Figure 1B; the conformation diagram after superimposing the binding conformation of STC001 and human wild-type mature NGF with the binding conformation of TrkA receptor and human wild-type mature NGF is shown in Figure 1C. Among them, the position of the mutable site in NGF described in this application is determined relative to the amino acid sequence of human wild-type mature NGF. The amino acid sequence of an exemplary human wild-type mature NGF is shown in Table 2.
表2
Table 2
对于上述表1中其他几个抗NGF抗体，也分别按照上述步骤进行相应的分析(相关分析图谱未显示)。针对各个抗体，均分别获得了一系列可突变位点。从上述分别获得的一系列可突变位点中，挑选出针对各个抗NGF抗体所共有的可突变位点，即将所获得的针对各个抗NGF抗体的可突变位点取交集。根据人野生型成熟NGF的晶体结构(如PDB ID：4EDW所示)，上述共有的可突变位点分别位于如下四个区域：区域1包含I31、K32、G33、K34和D93位点；区域2包含W21、G23、D24、K50和 Y52位点；区域3包含T83、H84、F86、R100和R103位点；区域4包含D16、S17、S19、T56、R59和R69位点(如图2所示)。For the other anti-NGF antibodies in Table 1 above, the corresponding analysis was performed according to the above steps (the relevant analysis graphs are not shown). For each antibody, a series of mutable sites were obtained. From the series of mutable sites obtained above, the common mutable sites for each anti-NGF antibody were selected, that is, the obtained mutable sites for each anti-NGF antibody were intersected. According to the crystal structure of human wild-type mature NGF (as shown in PDB ID: 4EDW), the common mutable sites are located in the following four regions: Region 1 includes I31, K32, G33, K34 and D93 sites; Region 2 includes W21, G23, D24, K50 and Y52 site; region 3 includes T83, H84, F86, R100 and R103 sites; region 4 includes D16, S17, S19, T56, R59 and R69 sites (as shown in Figure 2).
实施例2：NGF突变体库的构建及筛选Example 2: Construction and screening of NGF mutant library
(1)构建NGF突变体酵母展示文库，并从中筛选和获得具有生物学活性，但与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体(1) Constructing a yeast display library of NGF mutants, and screening and obtaining NGF mutants having biological activity but with reduced binding to or substantially no binding to anti-NGF antibodies
以人野生型成熟NGF(SEQ ID NO:1)为模板，分别对实施例1中所获得的NGF中一个或多个可突变位点上的氨基酸残基进行饱和突变，构建若干NGF突变体，并将这些突变体展示到酵母细胞表面，构建NGF突变体酵母展示文库。Using human wild-type mature NGF (SEQ ID NO: 1) as a template, saturation mutations were performed on the amino acid residues at one or more mutagenic sites in the NGF obtained in Example 1 to construct several NGF mutants. These mutants were displayed on the surface of yeast cells to construct an NGF mutant yeast display library.
分别通过MACS和FACS从该酵母展示文库中分选出与TrkA受体结合的酵母细胞，然后将上述获得的酵母细胞再通过FACS分选出分别与各个抗NGF抗体结合减弱或基本不与其结合的酵母细胞。Yeast cells binding to TrkA receptor were sorted out from the yeast display library by MACS and FACS, respectively. Then, yeast cells obtained as above were sorted out by FACS to obtain yeast cells whose binding to each anti-NGF antibody was weakened or basically not bound thereto.
最后通过FACS单克隆鉴定出既能与TrkA受体结合，同时与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的单克隆，对挑选的单克隆进行测序。针对不同的抗NGF抗体，最终筛选获得若干NGF突变体，选择其中的12个突变体进行后续实验。上述NGF突变体及其对应的抗NGF抗体如表3所示，即所述的NGF突变体与其对应的抗NGF抗体结合减弱或基本不与其结合。NGF突变体的具体氨基酸序列如表4所示。Finally, monoclonal clones that can bind to TrkA receptors and have weakened binding to or basically no binding to anti-NGF antibodies were identified through FACS monoclonal analysis, and the selected monoclonal clones were sequenced. For different anti-NGF antibodies, several NGF mutants were finally screened and obtained, and 12 of them were selected for subsequent experiments. The above-mentioned NGF mutants and their corresponding anti-NGF antibodies are shown in Table 3, that is, the NGF mutants have weakened binding to or basically no binding to their corresponding anti-NGF antibodies. The specific amino acid sequences of the NGF mutants are shown in Table 4.
表3

table 3

表4

Table 4

(2)在上述NGF突变体中进一步引入突变F12E：(2) Further introducing mutation F12E into the above NGF mutant:
为了减轻NGF突变体在治疗过程中所引起的疼痛副作用，在表4所示的NGF突变体中进一步引入突变F12E。该突变是本领域中已知的能够减轻NGF疼痛副作用的突变，且在人野生型成熟NGF中引入突变F12E后(下称NGFF12E)，该突变体仍具有生物学活性和治疗活性，具体描述参见专利CN109153709 B。同时经实验验证，在人野生型成熟NGF中引入突变F12E后，并未对其与抗NGF抗体的结合活性产生影响(具体参见实施例4的结果)。本实施例后续实验中所采用的NGF突变体均是包含突变F12E的突变体，分别命名为1A2-1A13，其氨基酸序列如表5所示。In order to reduce the pain side effects caused by NGF mutants during treatment, mutation F12E was further introduced into the NGF mutants shown in Table 4. This mutation is a mutation known in the art that can reduce the pain side effects of NGF, and after the introduction of mutation F12E into human wild-type mature NGF (hereinafter referred to as NGF F12E ), the mutant still has biological activity and therapeutic activity. For specific descriptions, see patent CN109153709 B. At the same time, it has been experimentally verified that after the introduction of mutation F12E into human wild-type mature NGF, its binding activity with anti-NGF antibodies was not affected (see the results of Example 4 for details). The NGF mutants used in the subsequent experiments of this example are all mutants containing mutation F12E, named 1A2-1A13, and their amino acid sequences are shown in Table 5.
表5


table 5


实施例3：蛋白的表达及纯化Example 3: Protein expression and purification
3.1 NGF突变体与Fc融合蛋白的表达3.1 Expression of NGF mutant and Fc fusion protein
表达载体的构建：以构建人野生型成熟NGF与Fc融合蛋白的表达载体为示例，委托上海杰瑞生物工程有限公司北京分公司合成编码preproNGF-Fc融合蛋白的核酸序 列(SEQ ID NO:86)，其中preproNGF的结构示意图如图3所示，并将其克隆到pSC-T载体中，其中Fc为人IgG1 Fc，包含突变L234A、L235A和P331S(根据EU编号系统编号)，其氨基酸序列如SEQ ID NO:85所示。采用分别携带有HindIII和XhoI限制性酶切位点的PCR引物扩增编码preproNGF-Fc的核酸序列，然后将PCR产物亚克隆到真核表达载体pTT5中，即构建成功。在编码preproNGF-Fc的核酸序列中分别引入表5中所示的突变，通过定点突变PCR，分别构建各个NGF突变体与Fc融合蛋白的重组真核表达载体。Construction of expression vector: Taking the construction of expression vector of human wild-type mature NGF and Fc fusion protein as an example, Shanghai Jerry Biotechnology Co., Ltd. Beijing Branch was commissioned to synthesize the nucleic acid sequence encoding preproNGF-Fc fusion protein. Column (SEQ ID NO:86), wherein the schematic diagram of the structure of preproNGF is shown in Figure 3, and it is cloned into the pSC-T vector, wherein Fc is human IgG1 Fc, comprising mutations L234A, L235A and P331S (numbered according to the EU numbering system), and its amino acid sequence is shown in SEQ ID NO:85. The nucleic acid sequence encoding preproNGF-Fc was amplified using PCR primers carrying HindIII and XhoI restriction enzyme sites, and then the PCR product was subcloned into the eukaryotic expression vector pTT5, and the construction was successful. The mutations shown in Table 5 were introduced into the nucleic acid sequence encoding preproNGF-Fc, and the recombinant eukaryotic expression vectors of each NGF mutant and Fc fusion protein were constructed by site-directed mutagenesis PCR.
将携带编码各个不同NGF突变体与Fc融合蛋白核酸的重组真核表达载体pTT5分别转染293F细胞，37℃、5％CO2、120rpm培养5天，分别收集细胞培养液。The recombinant eukaryotic expression vector pTT5 carrying nucleic acids encoding various NGF mutants and Fc fusion proteins was transfected into 293F cells respectively, and cultured at 37°C, 5% CO 2 , and 120 rpm for 5 days, and the cell culture fluid was collected respectively.
3.2 TrkA受体与mFc融合蛋白的表达3.2 Expression of TrkA receptor and mFc fusion protein
构建TrkA受体与mFc融合蛋白表达载体的操作步骤如同上文3.1中所述。简言之，将TrkA受体胞外区与mFc进行融合，其中TrkA受体胞外区的氨基酸序列如SEQ ID NO:88所示，mFc为小鼠IgG2a Fc，TrkA受体与mFc融合蛋白的氨基酸序列如SEQ ID NO:89所示。The steps for constructing the TrkA receptor and mFc fusion protein expression vector are the same as those described in 3.1 above. In brief, the TrkA receptor extracellular region is fused with mFc, wherein the amino acid sequence of the TrkA receptor extracellular region is shown in SEQ ID NO: 88, mFc is mouse IgG2a Fc, and the amino acid sequence of the TrkA receptor and mFc fusion protein is shown in SEQ ID NO: 89.
3.3抗NGF抗体的表达3.3 Expression of anti-NGF antibodies
根据相关文献或专利中的记载，将表1中所示的抗NGF抗体分别构建成具有人IgG1或IgG4的重链恒定区和人kappa或lambda轻链恒定区的抗体分子。委托上海杰瑞生物工程有限公司北京分公司分别合成编码表1中所示的抗NGF抗体VL及VH的核酸序列，分别构建于真核表达载体pTT5-L(包含kappa恒定区或lambda恒定区)和pTT5-H1(包含IgG1重链恒定区)或pTT5-H4(包含IgG4重链恒定区)中。分别提取表达相应抗体轻重链的质粒，共转染293F细胞，37℃、5％CO2、120rpm培养5天，收集培养液。According to the records in relevant literature or patents, the anti-NGF antibodies shown in Table 1 were constructed into antibody molecules with heavy chain constant regions of human IgG1 or IgG4 and human kappa or lambda light chain constant regions. The Beijing branch of Shanghai Jerry Biotechnology Co., Ltd. was commissioned to synthesize the nucleic acid sequences encoding the anti-NGF antibodies V L and V H shown in Table 1, respectively, and constructed in eukaryotic expression vectors pTT5-L (containing kappa constant region or lambda constant region) and pTT5-H1 (containing IgG1 heavy chain constant region) or pTT5-H4 (containing IgG4 heavy chain constant region). The plasmids expressing the light and heavy chains of the corresponding antibodies were extracted respectively, co-transfected into 293F cells, cultured at 37°C, 5% CO 2 , 120rpm for 5 days, and the culture fluid was collected.
3.4蛋白纯化：3.4 Protein purification:
分别收集上述表达的人野生型成熟NGF或NGF突变体与Fc的融合蛋白、TrkA受体与mFc的融合蛋白以及抗NGF抗体的细胞培养液，离心后，采用HiTrap Protein A HP纯化柱将细胞培养液上清进行粗纯化(具体纯化步骤按照说明书进行)。简言之：Protein A柱首先用含有0.15M NaCl的50mM的PBS溶液(pH 7.2)进行平衡，流速150cm/h。将培养液上清的pH调整至7.2，室温上样，流速150cm/h。随后，柱子完全 平衡后，应用50mM的柠檬酸钠(sodium citrate)溶液(pH 3.4)进行洗脱，收集含有人野生型成熟NGF或NGF突变体与Fc的融合蛋白、TrkA受体与mFc的融合蛋白或抗NGF抗体的洗脱液。然后基于蛋白质不同的疏水性，使用HiTrapTM Butyl HP柱(GE Healthcare)进一步将其与宿主蛋白分离。然后使用Superdex 200凝胶过滤柱(GE Life Sciences)去除残余聚集物，分别获得纯化的人野生型成熟NGF或NGF突变体与Fc的融合蛋白、TrkA受体与mFc的融合蛋白或抗NGF抗体。在如下实验中，人野生型成熟NGF与Fc的融合蛋白简写成hNGF-Fc，其中人野生型成熟NGF的氨基酸序列如SEQ ID NO:1所示，hNGF-Fc的氨基酸序列如SEQ ID NO:94所示。TrkA受体与mFc的融合蛋白简写成TrkA-mFc。NGFF12E与Fc的融合蛋白简写成NGFF12E-Fc，其中NGFF12E的氨基酸序列如SEQ ID NO:83所示，NGFF12E-Fc的氨基酸序列如SEQ ID NO:84所示。The cell culture fluids expressing the above-mentioned human wild-type mature NGF or NGF mutant and Fc fusion proteins, TrkA receptor and mFc fusion proteins, and anti-NGF antibodies were collected separately, and after centrifugation, the cell culture supernatant was crudely purified using a HiTrap Protein A HP purification column (the specific purification steps were carried out according to the instructions). In brief: The Protein A column was first equilibrated with a 50mM PBS solution (pH 7.2) containing 0.15M NaCl at a flow rate of 150cm/h. The pH of the culture supernatant was adjusted to 7.2, and the sample was loaded at room temperature at a flow rate of 150cm/h. Subsequently, the column was completely purified. After equilibration, 50 mM sodium citrate solution (pH 3.4) was used for elution, and the eluate containing the fusion protein of human wild-type mature NGF or NGF mutant and Fc, the fusion protein of TrkA receptor and mFc, or anti-NGF antibody was collected. Then, based on the different hydrophobicity of the protein, HiTrap TM Butyl HP column (GE Healthcare) was used to further separate it from the host protein. Then, a Superdex 200 gel filtration column (GE Life Sciences) was used to remove the residual aggregates, and purified human wild-type mature NGF or NGF mutant and Fc fusion protein, TrkA receptor and mFc fusion protein, or anti-NGF antibody were obtained respectively. In the following experiment, the fusion protein of human wild-type mature NGF and Fc is abbreviated as hNGF-Fc, wherein the amino acid sequence of human wild-type mature NGF is shown in SEQ ID NO: 1, and the amino acid sequence of hNGF-Fc is shown in SEQ ID NO: 94. The fusion protein of TrkA receptor and mFc is abbreviated as TrkA-mFc. The fusion protein of NGF F12E and Fc is abbreviated as NGF F12E -Fc, wherein the amino acid sequence of NGF F12E is shown in SEQ ID NO:83, and the amino acid sequence of NGF F12E -Fc is shown in SEQ ID NO:84.
实施例4：ELISA法检测NGF突变体与抗NGF抗体的结合活性Example 4: ELISA method to detect the binding activity of NGF mutants and anti-NGF antibodies
ELISA法检测NGF突变体与不同抗NGF抗体结合活性的方法如下所述：The method for detecting the binding activity of NGF mutants with different anti-NGF antibodies by ELISA is as follows:
根据实验需要可以选择在孔板中包被NGF突变体与Fc的融合蛋白(例如1A2-Fc至1A13-Fc)，加入抗NGF抗体(例如，Tanezumab、Fasinumab或STC001)进行检测；或者在孔板中包被抗NGF抗体(例如，Tanezumab、Fasinumab或STC001)，加入NGF突变体与Fc的融合蛋白(例如1A2-Fc至1A13-Fc)进行检测。According to experimental needs, you can choose to coat the fusion protein of NGF mutant and Fc (for example, 1A2-Fc to 1A13-Fc) in the well plate, add anti-NGF antibody (for example, Tanezumab, Fasinumab or STC001) for detection; or coat the anti-NGF antibody (for example, Tanezumab, Fasinumab or STC001) in the well plate, add the fusion protein of NGF mutant and Fc (for example, 1A2-Fc to 1A13-Fc) for detection.
本实施例中以孔板中包被NGF突变体与Fc的融合蛋白(例如，1A2-Fc)为例。简言之，按照NGF突变体与Fc的融合蛋白1A2-Fc的浓度为0.1μg/孔来包被96孔板，4℃过夜。用PBST溶液洗涤96孔板，每孔加入150μl 1％BSA-PBS，37℃封闭1-2小时，再用PBST溶液洗涤。每孔加入50μl梯度稀释的抗NGF抗体(例如，STC001)，37℃孵育1小时后，用PBST溶液洗涤6次。每孔加入100μL按照1:4000稀释的anti-human kappa-AP(Southernbiotech,2064-04)，37℃孵育1小时，用PBST溶液洗涤6次。每孔加入65μL pNPP(Southernbiotech，0421-01)，37℃孵育30分钟，分析ELISA结果(OD405)，并通过PRISM生成结合曲线。In this embodiment, the fusion protein of NGF mutant and Fc (e.g., 1A2-Fc) coated in the well plate is taken as an example. In short, the concentration of 1A2-Fc, a fusion protein of NGF mutant and Fc, is 0.1 μg/well to coat the 96-well plate, overnight at 4°C. Wash the 96-well plate with PBST solution, add 150 μl 1% BSA-PBS to each well, block at 37°C for 1-2 hours, and then wash with PBST solution. Add 50 μl of gradient diluted anti-NGF antibody (e.g., STC001) to each well, incubate at 37°C for 1 hour, and wash 6 times with PBST solution. Add 100 μL of anti-human kappa-AP (Southernbiotech, 2064-04) diluted 1:4000 to each well, incubate at 37°C for 1 hour, and wash 6 times with PBST solution. Add 65 μL pNPP (Southernbiotech, 0421-01) to each well and incubate at 37°C for 30 min. Analyze ELISA results (OD405) and generate binding curves using PRISM.
4.1 ELISA法检测NGFF12E与抗NGF抗体的结合活性4.1 ELISA to detect the binding activity of NGF F12E and anti-NGF antibody
首先按照上述的方法检测NGFF12E-Fc分别与各个抗NGF抗体的结合活性，以确认在人野生型成熟NGF中引入突变F12E是否会对其与相应的抗NGF抗体的结合活 性产生影响。hNGF-Fc以及NGFF12E-Fc与抗NGF抗体的ELISA结合曲线分别如图4A和图4B所示，EC50值如表6所示。First, the binding activity of NGF F12E -Fc with each anti-NGF antibody was detected according to the above method to confirm whether the introduction of mutant F12E into human wild-type mature NGF would affect its binding activity with the corresponding anti-NGF antibody. The ELISA binding curves of hNGF-Fc and NGF F12E -Fc with anti-NGF antibody are shown in Figure 4A and Figure 4B, respectively, and the EC50 values are shown in Table 6.
表6
Table 6
如上结果显示，NGFF12E和人野生型成熟NGF，二者分别与表6中所示的抗NGF抗体的结合曲线趋势基本一致(图4A-4B)，结合EC50值基本相当(表6)。以上结果表明，在人野生型成熟NGF中引入突变F12E，基本不会影响其与抗NGF抗体的结合活性。As shown in the above results, NGF F12E and human wild-type mature NGF have basically the same binding curve trends as the anti-NGF antibody shown in Table 6 (Figures 4A-4B), and the binding EC50 values are basically equivalent (Table 6). The above results show that the introduction of mutation F12E into human wild-type mature NGF will not basically affect its binding activity with anti-NGF antibody.
4.2 ELISA法检测NGF突变体与抗NGF抗体的结合活性4.2 ELISA to detect the binding activity of NGF mutants and anti-NGF antibodies
按照上述的方法，检测各个NGF突变体与Fc的融合蛋白：1A2-Fc至1A13-Fc与不同抗NGF抗体的结合活性，其结合EC50值如表7所示，示例性的NGF突变体与Fc的融合蛋白与抗NGF抗体的ELISA结合曲线如图4C-4J所示，hNGF-Fc融合蛋白作为本实验的对照。According to the above method, the binding activity of each NGF mutant and Fc fusion protein: 1A2-Fc to 1A13-Fc with different anti-NGF antibodies was detected, and the binding EC50 values are shown in Table 7. The ELISA binding curves of the exemplary NGF mutant and Fc fusion protein with anti-NGF antibodies are shown in Figures 4C-4J. The hNGF-Fc fusion protein was used as a control in this experiment.
应用相对结合活性表征NGF突变体与抗NGF抗体的结合活性相对于人野生型成熟NGF与相同抗NGF抗体的结合活性的变化。例如，若相对结合活性数值为50％，则意味着该NGF突变体与NGF抗体的结合活性是人野生型成熟NGF与相同抗NGF抗体结合活性的50％。相对结合活性的计算公式为：人野生型成熟NGF与抗NGF抗体的结合EC50值与NGF突变体与相同的抗NGF抗体的结合EC50值的比值，结果如表7所示。Relative binding activity is used to characterize the change in the binding activity of NGF mutants to anti-NGF antibodies relative to the binding activity of human wild-type mature NGF to the same anti-NGF antibody. For example, if the relative binding activity value is 50%, it means that the binding activity of the NGF mutant to the NGF antibody is 50% of the binding activity of human wild-type mature NGF to the same anti-NGF antibody. The calculation formula for relative binding activity is: the ratio of the binding EC50 value of human wild-type mature NGF to the anti-NGF antibody to the binding EC50 value of the NGF mutant to the same anti-NGF antibody, and the results are shown in Table 7.
表7



“/”代表未进行检测，“基本不结合”表示实际结果显示NGF突变体与相应的抗NGF
抗体基本不结合，而软件未准确拟合出EC50实际数值Table 7



“/” indicates that no detection was performed, and “substantially no binding” indicates that the actual results show that NGF mutants bind to the corresponding anti-NGF
The antibody barely binds, and the software does not accurately fit the actual EC50 value
上述结果表明，与人野生型成熟NGF或NGFF12E相比，本申请中的示例性的NGF突变体与至少一种抗NGF抗体的结合活性减弱，例如突变体1A7-Fc，其与Fasinumab抗体的结合活性仅为人野生型成熟NGF的2.10％；甚至基本不与抗NGF抗体结合，例如突变体1A10-Fc基本不与MEDI-578抗体结合。The above results indicate that compared with human wild-type mature NGF or NGF F12E , the exemplary NGF mutants in the present application have weakened binding activity with at least one anti-NGF antibody, for example, mutant 1A7-Fc, whose binding activity with Fasinumab antibody is only 2.10% of that of human wild-type mature NGF; or even basically does not bind to anti-NGF antibodies, for example, mutant 1A10-Fc basically does not bind to MEDI-578 antibody.
实施例5：ELISA法检测NGF突变体与TrkA受体的结合活性Example 5: ELISA method to detect the binding activity of NGF mutants to TrkA receptor
ELISA法检测NGF突变体与TrkA受体结合活性的方法如下所述：The method for detecting the binding activity of NGF mutants to TrkA receptor by ELISA is as follows:
按照TrkA-mFc融合蛋白的浓度为0.1μg/孔来包被96孔板，4℃过夜。用PBST溶液洗涤96孔板，每孔加入150μl 1％BSA-PBS，37℃封闭1-2小时，再用PBST溶液洗涤。每孔加入50μl梯度稀释的待检测NGF突变体与Fc的融合蛋白(例如1A2-Fc至1A13-Fc)，37℃孵育1小时后，用PBST溶液洗涤6次。每孔加入100μL按照1:4000稀释羊抗人Fc-AP(Southernbiote，2048-04)，37℃孵育1小时，用PBST溶液洗涤6次。每孔加入65μL pNPP(Southernbioteh，0421-01)，37℃孵育30分钟，分析ELISA结果(OD405)，并通过PRISM生成结合曲线。Coat 96-well plates with TrkA-mFc fusion protein at a concentration of 0.1 μg/well at 4°C overnight. Wash the 96-well plates with PBST solution, add 150 μl 1% BSA-PBS to each well, block at 37°C for 1-2 hours, and then wash with PBST solution. Add 50 μl of gradient dilutions of the fusion protein of the NGF mutant to be tested and Fc (e.g. 1A2-Fc to 1A13-Fc) to each well, incubate at 37°C for 1 hour, and wash 6 times with PBST solution. Add 100 μL of goat anti-human Fc-AP (Southernbiote, 2048-04) diluted 1:4000 to each well, incubate at 37°C for 1 hour, and wash 6 times with PBST solution. Add 65 μL pNPP (Southernbioteh, 0421-01) to each well, incubate at 37°C for 30 minutes, analyze ELISA results (OD405), and generate binding curves using PRISM.
5.1 ELISA法检测NGFF12E与TrkA受体的结合活性5.1 ELISA to detect the binding activity of NGF F12E to TrkA receptor
按照上述的方法，首先检测NGFF12E与TrkA受体的结合活性，以确认在人野生型成熟NGF中引入突变F12E是否会对其与TrkA受体的结合活性产生影响。hNGF-Fc以及NGFF12E-Fc与TrkA受体的ELISA结合EC50值如表8所示。According to the above method, the binding activity of NGF F12E to TrkA receptor was first detected to confirm whether the introduction of mutant F12E into wild-type mature human NGF would affect its binding activity to TrkA receptor. The ELISA binding EC50 values of hNGF-Fc and NGF F12E -Fc to TrkA receptor are shown in Table 8.
表8
Table 8
上述实验结果显示，与人野生型成熟NGF相比，NGFF12E与TrkA受体的结合EC50值是人野生型成熟NGF与TrkA受体结合EC50值的约4倍(即其与TrkA受体的结合活性为人野生型NGF的约25％)。上述结果表明，引入突变F12E会降低人野生型成熟NGF与TrkA受体的结合活性。尽管如此，根据现有技术(例如，参见CN109153709B)，以及本申请如下生物学活性检测实验中的结果(例如，参见实施例6及图5)，NGFF12E仍具有生物学活性以及潜在的治疗活性。The above experimental results show that compared with human wild-type mature NGF, the EC50 value of NGF F12E binding to the TrkA receptor is about 4 times the EC50 value of human wild-type mature NGF binding to the TrkA receptor (i.e., its binding activity to the TrkA receptor is about 25% of that of human wild-type NGF). The above results indicate that the introduction of the mutant F12E will reduce the binding activity of human wild-type mature NGF to the TrkA receptor. Nevertheless, according to the prior art (for example, see CN109153709B), and the results of the following biological activity detection experiments of the present application (for example, see Example 6 and Figure 5), NGF F12E still has biological activity and potential therapeutic activity.
5.2 ELISA法检测不同NGF突变体与TrkA受体的结合活性5.2 ELISA to detect the binding activity of different NGF mutants to TrkA receptor
按照上述的方法，检测NGF突变体与Fc的融合蛋白：1A2-Fc至1A13-Fc与TrkA受体的结合活性，其在ELISA结合实验中的EC50值如表9所示，NGFF12E-Fc融合蛋白作为本实验的对照。According to the above method, the binding activity of NGF mutant and Fc fusion proteins: 1A2-Fc to 1A13-Fc with TrkA receptor was detected. The EC50 values in the ELISA binding experiment are shown in Table 9. NGF F12E -Fc fusion protein was used as the control of this experiment.
应用相对结合活性表征NGF突变体与TrkA受体的结合活性相对于NGFF12E与TrkA受体的结合活性的变化。相对结合活性的计算公式为：NGFF12E与TrkA受体的结合EC50值与NGF突变体与TrkA受体的结合EC50值的比值，结果如表9中所示。例如1A7-Fc的相对结合活性为4.28，则表示1A7与TrkA受体的结合活性约为NGFF12E与TrkA受体的结合活性的4.28倍、1A9-Fc的相对结合活性为36％，则表示1A9与TrkA受体的结合活性约为NGFF12E与TrkA受体的结合活性的36％。Relative binding activity was used to characterize the change in the binding activity of NGF mutants to TrkA receptor relative to the binding activity of NGF F12E to TrkA receptor. The calculation formula of relative binding activity is: the ratio of the binding EC50 value of NGF F12E to TrkA receptor to the binding EC50 value of NGF mutants to TrkA receptor, and the results are shown in Table 9. For example, the relative binding activity of 1A7-Fc is 4.28, which means that the binding activity of 1A7 to TrkA receptor is about 4.28 times that of NGF F12E to TrkA receptor, and the relative binding activity of 1A9-Fc is 36%, which means that the binding activity of 1A9 to TrkA receptor is about 36% of the binding activity of NGF F12E to TrkA receptor.
表9

Table 9

表9中的数据表明，通过ELISA结合实验测定，与NGFF12E-Fc相比，部分NGF突变体与Fc的融合蛋白与TrkA受体的结合活性不低于NGFF12E-Fc(例如，1A7-Fc、1A11-Fc、1A8-Fc、1A13-Fc、1A12-Fc、1A6-Fc、1A2-Fc、1A10-Fc和1A3-Fc、)或至少达到NGFF12E-Fc的21％(例如1A4-Fc、1A5-Fc和1A9-Fc)。The data in Table 9 show that, as determined by ELISA binding experiments, the binding activity of some NGF mutants and Fc fusion proteins to TrkA receptors is no less than that of NGF F12E -Fc (for example, 1A7-Fc, 1A11-Fc, 1A8-Fc, 1A13-Fc, 1A12-Fc, 1A6-Fc, 1A2-Fc, 1A10-Fc and 1A3-Fc) or at least reaches 21% of NGF F12E -Fc (for example, 1A4-Fc, 1A5-Fc and 1A9-Fc).
上述NGF突变体进一步通过如下所述的活性检测实验(例如，实施例6-9)来验证其生物学活性，其中对于与TrkA受体的结合活性低于NGFF12E的突变体(例如，1A4-Fc和1A5-Fc)进一步通过实验验证其在与抗NGF抗体联合应用时的生物学活性(例如，实施例12)。The biological activity of the above-mentioned NGF mutants was further verified by the activity detection experiments described below (for example, Examples 6-9), among which the mutants whose binding activity to the TrkA receptor was lower than that of NGF F12E (for example, 1A4-Fc and 1A5-Fc) were further verified by experiments when used in combination with anti-NGF antibodies (for example, Example 12).
实施例6：TF-1细胞增殖法检测NGF突变体的体外生物学活性Example 6: Detection of in vitro biological activity of NGF mutants using TF-1 cell proliferation assay
为了验证NGF突变体的生物学活性，应用TF-1细胞增殖法进行检测，TF-1细胞是人红系白血病患者的前髓细胞，在无血清和GM-CSF存在的条件下，NGF能延长离体培养的TF-1细胞的存活时间，并且TF-1细胞的存活数量与NGF生物学活性具有正相关性，因此可应用TF-1细胞增殖法进行NGF生物学活性的检测。具体操作方法可参考中国专利申请公开文本CN114829384A中所披露内容。In order to verify the biological activity of NGF mutants, the TF-1 cell proliferation method was used for detection. TF-1 cells are promyelocytes of human erythroid leukemia patients. In the absence of serum and GM-CSF, NGF can prolong the survival time of TF-1 cells cultured in vitro, and the survival number of TF-1 cells is positively correlated with the biological activity of NGF. Therefore, the TF-1 cell proliferation method can be used to detect the biological activity of NGF. The specific operation method can refer to the content disclosed in the Chinese patent application publication CN114829384A.
简言之，将TF-1细胞重悬于基础培养基中(RPMI 1640培养基含10％FBS)，以获得含有6.0×104个细胞/ml的悬浮液，备用。制备待测样品：各个NGF突变体与Fc的融合蛋白以及阳性对照样品NGFF12E-Fc，国际标准品：hNGF。待测样品以及NGFF12E-Fc的浓度梯度依次为200U/ml、66.66U/ml、22.22U/ml、7.4U/ml、2.47U/ml、0.82U/ml，分别接种于96孔板中，然后，每孔加入100μl上述的TF-1细胞悬浮液，在37℃、5％CO2的潮湿培养箱中培养72小时。应用MTS(Promega,Cat#G3581)进行检测，每孔加入20μl分析溶液，在37℃、5％CO2下培养3小时。使用分光光度计测量孔板的吸光度，记录数据，并通过SoftMAX Pro生成曲线。Briefly, TF-1 cells were resuspended in basal medium (RPMI 1640 medium containing 10% FBS) to obtain a suspension containing 6.0×10 4 cells/ml for use. The samples to be tested were prepared: fusion proteins of various NGF mutants and Fc and positive control sample NGF F12E -Fc, and international standard: hNGF. The concentration gradients of the samples to be tested and NGF F12E -Fc were 200U/ml, 66.66U/ml, 22.22U/ml, 7.4U/ml, 2.47U/ml, and 0.82U/ml, respectively, and they were inoculated in 96-well plates, and then 100μl of the above TF-1 cell suspension was added to each well, and cultured in a humidified incubator at 37°C and 5% CO 2 for 72 hours. MTS (Promega, Cat# G3581) was used for detection, 20 μl of assay solution was added to each well, and cultured for 3 hours at 37°C and 5% CO 2. The absorbance of the well plate was measured using a spectrophotometer, the data was recorded, and the curve was generated using SoftMAX Pro.
TF-1细胞增殖实验的结果如图5所示，示例性的NGF突变体1A2-Fc和1A10-Fc能够促进TF-1细胞的增殖，具有良好的生物学活性，并且其生物学活性甚至比阳性对照NGFF12E-Fc更高。其他NGF突变体也采用相同的方法进行检测，也都能够促进TF-1细胞的增殖，具有良好的生物学活性(数据未显示)。 The results of the TF-1 cell proliferation experiment are shown in Figure 5. The exemplary NGF mutants 1A2-Fc and 1A10-Fc can promote the proliferation of TF-1 cells and have good biological activity, and their biological activity is even higher than that of the positive control NGF F12E -Fc. Other NGF mutants were also tested using the same method and were also able to promote the proliferation of TF-1 cells and had good biological activity (data not shown).
实施例7：PC12细胞活性实验检测NGF突变体的体外生物学活性Example 7: PC12 cell activity assay to detect in vitro biological activity of NGF mutants
NGF突变体的生物学活性也可以通过PC12细胞活性实验来检测，PC12细胞系是来源于成年大白鼠肾上腺髓质嗜铬细胞瘤的细胞系，NGF具有刺激轴突生成和维持神经元细胞存活的作用。PC12细胞表面存在NGF高亲和力受体TrkA，与NGF结合可使PC12细胞停止分裂，长出突起，转化成神经元样细胞。且在一定的NGF浓度范围内，突起的长度以及长突起的细胞数与NGF浓度呈正相关，因此，PC12细胞可以用于NGF生物学活性检测。The biological activity of NGF mutants can also be detected by PC12 cell activity experiments. The PC12 cell line is a cell line derived from pheochromocytoma in the adrenal medulla of adult rats. NGF has the function of stimulating axonal generation and maintaining the survival of neuronal cells. There is a high-affinity receptor TrkA for NGF on the surface of PC12 cells. Binding to NGF can stop PC12 cells from dividing, grow protrusions, and transform into neuron-like cells. And within a certain range of NGF concentrations, the length of the protrusions and the number of cells with long protrusions are positively correlated with the NGF concentration. Therefore, PC12 cells can be used for NGF biological activity detection.
简言之，第一天将PC12细胞重悬于基础培养基中(RPMI 1640培养基含0.01％BSA)，重悬计数铺白色透底96孔板，1.0×104个/孔，每孔50μl，37℃、5％CO2培养24小时。第二天制备待测样品：各种NGF突变体与Fc的融合蛋白以及阳性对照品：NGFF12E-Fc，无血清培养基作为空白对照。待测样品(以1A2-Fc和1A10-Fc为例)以及NGFF12E-Fc的最高浓度为12.5μg/ml，5倍梯队稀释到最低浓度160pg/ml，取50μl稀释样品加入到上述96孔板中，放入37℃、5％CO2的潮湿培养箱中培养6小时后进行荧光素酶检测。Bright-GloTM Luciferase Assay平衡至室温后，40μl/孔加至上述96孔板中，细胞裂解2分钟后立即用酶标仪进行Luminescence(1000ms)读数，使用GraphPad Prism 5.0进行曲线的拟合并计算EC50。Briefly, on the first day, PC12 cells were resuspended in basal medium (RPMI 1640 medium containing 0.01% BSA), resuspended and counted in a white transparent bottom 96-well plate, 1.0×10 4 cells/well, 50 μl per well, and cultured at 37°C, 5% CO 2 for 24 hours. On the second day, the samples to be tested were prepared: various NGF mutants and Fc fusion proteins and positive control: NGF F12E -Fc, and serum-free medium was used as a blank control. The highest concentration of the samples to be tested (taking 1A2-Fc and 1A10-Fc as examples) and NGF F12E -Fc was 12.5 μg/ml, and the lowest concentration was 160 pg/ml after 5-fold dilution. 50 μl of the diluted sample was added to the above 96-well plate, and placed in a humid incubator at 37°C, 5% CO 2 for 6 hours before luciferase detection. After Bright-Glo ™ Luciferase Assay was equilibrated to room temperature, 40 μl/well was added to the above 96-well plate. Luminescence (1000 ms) readings were immediately performed using a microplate reader 2 minutes after cell lysis. GraphPad Prism 5.0 was used to fit the curve and calculate EC50.
PC12细胞活性检测实验的结果如图6所示，示例性的NGF突变体1A2-Fc和1A10-Fc能够诱导PC12细胞分化，具有良好的生物学活性，并且其生物学活性与阳性对照NGFF12E-Fc相当或略高。其他NGF突变体也采用相同的方法进行检测，也都能够诱导PC12细胞分化，具有良好的生物学活性(数据未显示)。The results of the PC12 cell activity detection experiment are shown in Figure 6. The exemplary NGF mutants 1A2-Fc and 1A10-Fc can induce PC12 cell differentiation and have good biological activity, and their biological activity is equivalent to or slightly higher than that of the positive control NGF F12E -Fc. Other NGF mutants were also detected using the same method and were also able to induce PC12 cell differentiation and had good biological activity (data not shown).
实施例8：鸡胚背根神经节培养法检测NGF突变体的体外生物学活性Example 8: Detection of in vitro biological activity of NGF mutants by chicken embryo dorsal root ganglion culture
NGF突变体的生物学活性也可以通过鸡胚背根神经节培养方法进行检测，NGF能够促进交感神经元和发育中的感觉神经元成熟与分化，其中对促进神经细胞突起伸长作用最为明显。鸡胚背根神经节培养法是NGF生物学活性测定的经典方法，该法最早由Levi-Montalcini于1954年创立。具体操作方法可参考国际专利申请公开文本WO2017157326A中所披露内容。The biological activity of NGF mutants can also be detected by chicken embryo dorsal root ganglion culture method. NGF can promote the maturation and differentiation of sympathetic neurons and developing sensory neurons, among which the effect on promoting the elongation of nerve cell processes is the most obvious. Chicken embryo dorsal root ganglion culture method is a classic method for determining the biological activity of NGF, which was first created by Levi-Montalcini in 1954. For specific operating methods, please refer to the contents disclosed in the international patent application publication WO2017157326A.
简言之，将NGF突变体与Fc的融合蛋白(以1A2-Fc、1A3-Fc或1A10-Fc为例)、NGFF12E-Fc或hNGF-Fc样品进行稀释，起始浓度为54ng/ml，进行3倍倍比稀 释，依次为18ng/ml、6ng/ml、2ng/ml、0.66ng/ml、0.22ng/ml。本实验中所用参考品为鼠NGF，每支装量为15000AU，起始浓度为6ng/ml，进行3倍倍比稀释，依次为2ng/ml、0.66ng/ml、0.22ng/ml、0.07ng/ml、0.02ng/ml。分别在每个浓度的样品中加入8日龄的鸡胚背根神经节后，置于37℃、5％CO2培养箱中，24小时后观察结果。Briefly, the fusion protein of NGF mutant and Fc (taking 1A2-Fc, 1A3-Fc or 1A10-Fc as an example), NGF F12E -Fc or hNGF-Fc samples were diluted to a starting concentration of 54 ng/ml and then diluted 3 times. The reference substances used in this experiment are: Mouse NGF, each tube contains 15000AU, the initial concentration is 6ng/ml, and it is diluted 3 times, 2ng/ml, 0.66ng/ml, 0.22ng/ml, 0.07ng/ml, 0.02ng/ml. After adding 8-day-old chicken embryo dorsal root ganglia to each concentration sample, place it in a 37℃, 5% CO2 incubator, and observe the results after 24 hours.
以生长最好时每毫升待测样品中NGF的含量作为1个活性单位(AU)。从出现阴性对照结果的稀释度开始往回数第3和第4的两个稀释度中取生长最好的作为判定终点计算效价。The NGF content in each milliliter of the sample under test when the growth is the best is taken as 1 activity unit (AU). The best growth is taken as the endpoint for calculating the titer from the third and fourth dilutions starting from the dilution with the negative control result.
NGF比活的计算公式为：待测样品的比活(AU/mg)＝参考品活性(AU/ml)×[样品预稀释倍数×对应参考品稀释点处的活性(AU/ml)/参考品实测活性(AU/ml)]The calculation formula of NGF specific activity is: specific activity of the sample to be tested (AU/mg) = activity of the reference product (AU/ml) × [pre-dilution multiple of the sample × activity at the corresponding reference product dilution point (AU/ml) / measured activity of the reference product (AU/ml)]
鸡胚背根神经节培养实结果如表10所示，示例性的NGF突变体1A2-Fc、1A3-Fc和1A10-Fc均能够促进鸡胚背根神经节生长，具有良好的生物学活性。The results of the chicken embryo dorsal root ganglion culture are shown in Table 10. The exemplary NGF mutants 1A2-Fc, 1A3-Fc and 1A10-Fc can all promote the growth of chicken embryo dorsal root ganglia and have good biological activity.
该实验结果说明NGF突变体在经过突变后仍然具有NGF的生物学活性，并且与hNGF-Fc以及NGFF12E-Fc的生物学活性基本相当。其它NGF突变体与Fc的融合蛋白也采用相同的方法进行检测，其生物学活性与NGFF12E-Fc基本相当(数据未显示)。The experimental results show that the NGF mutant still has the biological activity of NGF after mutation, and its biological activity is basically equivalent to that of hNGF-Fc and NGF F12E -Fc. The fusion proteins of other NGF mutants and Fc were also tested using the same method, and their biological activities were basically equivalent to that of NGF F12E -Fc (data not shown).
表10
Table 10
实施例9：NGF突变体在大鼠体内的生物学活性检测Example 9: Detection of biological activity of NGF mutants in rats
NGF突变体的生物学活性可以通过大鼠体内颈上神经节生长实验进行检测。颈上神经节(SCG)是由大约30000个神经元组成的组织，是对NGF最敏感的组织之 一，尤其是在产前和产后发育期间。将不同的NGF突变体与Fc的融合蛋白样品分别注射到大鼠SCG中，并在注射后不同时间点测量SCG大小，以评估NGF突变体与Fc的融合蛋白在体内促进SCG生长的活性。The biological activity of NGF mutants can be detected by the superior cervical ganglion growth assay in rats. The superior cervical ganglion (SCG) is composed of approximately 30,000 neurons and is one of the most sensitive tissues to NGF. First, especially during prenatal and postnatal development. Different NGF mutant-Fc fusion protein samples were injected into rat SCG, and the SCG size was measured at different time points after injection to evaluate the activity of NGF mutant-Fc fusion protein in promoting SCG growth in vivo.
新生Sprague-Dawley(SD)大鼠颈部皮下注射待测样品：各个NGF突变体与Fc的融合蛋白样品(以1A2-Fc或1A10-Fc为例)、阳性对照品：NGFF12E-Fc或阴性对照品：PBS溶液，然后处死大鼠，分离SCG。分别在第0天、第1天、第2天和第3天每天注射一次各个NGF突变体与Fc的融合蛋白、NGFF12E-Fc或PBS，在第4天收获SCG。NGFF12E-Fc的注射剂量为405μg/kg，NGF突变体与Fc的融合蛋白注射剂量分为高剂量组405μg/kg，以及低剂量组162μg/kg。Newborn Sprague-Dawley (SD) rats were subcutaneously injected with the sample to be tested: each NGF mutant and Fc fusion protein sample (taking 1A2-Fc or 1A10-Fc as an example), positive control: NGF F12E -Fc or negative control: PBS solution, and then the rats were killed and SCG was isolated. Each NGF mutant and Fc fusion protein, NGF F12E -Fc or PBS was injected once a day on day 0, day 1, day 2 and day 3, and SCG was harvested on day 4. The injection dose of NGF F12E -Fc was 405 μg/kg, and the injection dose of NGF mutant and Fc fusion protein was divided into a high-dose group of 405 μg/kg and a low-dose group of 162 μg/kg.
简言之，断头后，将大鼠头部固定在手术台上，用棉球吸干血液，首先定位气管和枕骨大孔，然后定位气管斜后侧的颈动脉鞘组织，用微型镊子将其取出，放入含有PBS的培养皿中，然后在解剖显微镜下分离SCG。用纸巾去除分离的SCG表面的多余液体，然后将SCG置于干净的表面皿上测量重量。记录的数据采用Student t检验进行分析。**表示与PBS处理组相比具有显著差异；n.s.表示与PBS处理组相比“无显著差异”。Briefly, after decapitation, the rat head was fixed on the operating table, blood was blotted with cotton balls, and the trachea and foramen magnum were first located, and then the carotid sheath tissue obliquely behind the trachea was located, which was removed with micro forceps and placed in a culture dish containing PBS, and then the SCG was isolated under a dissecting microscope. Excess liquid on the surface of the isolated SCG was removed with a paper towel, and then the SCG was placed on a clean surface dish to measure the weight. The recorded data were analyzed using the Student t test. ** indicates a significant difference compared with the PBS-treated group; n.s. indicates "no significant difference" compared with the PBS-treated group.
大鼠体内SCG实验结果如图7所示，示例性的NGF突变体与Fc的融合蛋白1A2-Fc和1A10-Fc均能够促进SCG生长。上述结果表明，无论在低剂量或高剂量给药组中，与PBS阴性对照组相比，1A2-Fc和1A10-Fc均对SCG的生长产生了显著的促进作用(**p<0.01)。在162μg/kg的低剂量时，1A2-Fc和1A10-Fc即表现出良好的生物学活性，并且与NGFF12E-Fc的生物学活性基本相当。同时对其它NGF突变体与Fc的融合蛋白也进行了检测，初步的结果显示其他NGF突变体在大鼠体内也能够促进SCG生长(数据未显示)。The results of the SCG experiment in rats are shown in Figure 7. The exemplary NGF mutant and Fc fusion proteins 1A2-Fc and 1A10-Fc can promote SCG growth. The above results show that whether in the low-dose or high-dose administration group, 1A2-Fc and 1A10-Fc have a significant promoting effect on the growth of SCG compared with the PBS negative control group (**p<0.01). At a low dose of 162μg/kg, 1A2-Fc and 1A10-Fc showed good biological activity, and were basically equivalent to the biological activity of NGF F12E -Fc. At the same time, other NGF mutants and Fc fusion proteins were also tested, and preliminary results showed that other NGF mutants can also promote SCG growth in rats (data not shown).
实施例10：NGF突变体在大鼠体内的疼痛阈值检测实验Example 10: Pain threshold detection experiment of NGF mutants in rats
为了检测上述NGF突变体在应用的过程中是否仍具有减低疼痛的效果，在大鼠体内进行疼痛阈值检测实验。首先筛选对痛觉反应正常的合格小鼠，注射一定剂量的待测样品：各个NGF突变体与Fc的融合蛋白或空白对照品：PBS溶液，通过机械刺激测定小鼠曲爪反应的疼痛阈值，进行统计分析，疼痛阈值越高代表小鼠承受疼痛的能力越强，疼痛阈值降低表明在小鼠中导致了疼痛，以此确定本申请的NGF突变体在应用的过程中是否仍具有减低疼痛的效果。 In order to detect whether the above-mentioned NGF mutants still have the effect of reducing pain during the application process, a pain threshold detection experiment was carried out in rats. First, qualified mice with normal pain response were screened, and a certain dose of the test sample was injected: the fusion protein of each NGF mutant and Fc or the blank control substance: PBS solution. The pain threshold of the mouse's paw reaction was measured by mechanical stimulation, and statistical analysis was performed. The higher the pain threshold, the stronger the mouse's ability to withstand pain. The lower the pain threshold, the more pain it causes in the mouse. In this way, it was determined whether the NGF mutants of the present application still have the effect of reducing pain during the application process.
具体操作如下：订购SPF级别CD-1小鼠，小鼠规格为雄性，体重30-35g，并饲养在无特殊病原体的环境中。所有动物实验都是依照研究机构动物护理和使用委员会(IACUC)方案和指导进行的。使用动态足底触觉仪(意大利/Ugo Basile，型号37450)筛选两足平均阈值在7.5-10之间且同一只小鼠左右脚阈值P值>0.05的实验动物为合格小鼠。将小鼠随机分组，分为实验组：各个NGF突变体与Fc的融合蛋白、空白对照组：PBS溶液、阳性对照组：hNGF-Fc和减痛对照组：NGFF12E-Fc，每组10只。空白对照组注射PBS溶液20μl，hNGF-Fc、NGF突变体与Fc的融合蛋白(以1A2-Fc和1A10-Fc为例)和NGFF12E-Fc的给药剂量为0.25μg/只/20μl。给药方式为在小鼠左右脚足底皮下给药。分别在给药前0h，给药后4h、24h、以及48h对小鼠进行机械痛阈值测定，并且记录数值。采用GraphPad Prism软件进行图表绘制及结果统计分析，分别比较NGF突变体与Fc的融合蛋白、hNGF-Fc与NGFF12E-Fc各组之间机械痛阈值是否有差异，分析NGF突变体与Fc的融合蛋白的致痛性。The specific operation is as follows: SPF CD-1 mice were ordered. The mice were male, weighing 30-35g, and were kept in an environment free of special pathogens. All animal experiments were conducted in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC). The dynamic plantar tactile instrument (Ugo Basile, Italy, model 37450) was used to screen the experimental animals with an average threshold of 7.5-10 for both feet and a P value of >0.05 for the threshold of the left and right feet of the same mouse as qualified mice. The mice were randomly divided into experimental groups: fusion proteins of various NGF mutants and Fc, blank control group: PBS solution, positive control group: hNGF-Fc and pain relief control group: NGF F12E -Fc, 10 mice in each group. The blank control group was injected with 20μl of PBS solution, and the dosage of hNGF-Fc, fusion proteins of NGF mutants and Fc (taking 1A2-Fc and 1A10-Fc as examples) and NGF F12E -Fc was 0.25μg/mouse/20μl. The drug was administered subcutaneously in the soles of the left and right feet of the mice. The mechanical pain threshold of the mice was measured at 0h before administration, 4h, 24h, and 48h after administration, and the values were recorded. GraphPad Prism software was used for graphing and statistical analysis of the results, and the mechanical pain thresholds of the fusion proteins of NGF mutant and Fc, hNGF-Fc and NGF F12E -Fc groups were compared to see if there were any differences, and the pain-causing properties of the fusion proteins of NGF mutant and Fc were analyzed.
大鼠体内疼痛阈值检测实验结果如图8所示，在给药剂量为0.25μg/只时，注射4h后，1A2-Fc的痛阈值与hNGF-Fc相当，而1A10-Fc和NGFF12E-Fc痛阈值相当，均高于hNGF-Fc；注射24h后，1A2-Fc、1A10-Fc和NGFF12E-Fc痛阈值均高于hNGF-Fc。上述实验结果表明，相比于hNGF-Fc，本申请中的NGF突变体与Fc的融合蛋白以及NGFF12E-Fc所引起的疼痛副作用明显减轻。此外，1A2-Fc、1A10-Fc与NGFF12E-Fc相比，其所引起的疼痛程度基本相同，即并未引起额外的疼痛。The results of the pain threshold detection experiment in rats are shown in Figure 8. When the dosage was 0.25 μg/rat, 4 hours after injection, the pain threshold of 1A2-Fc was equivalent to that of hNGF-Fc, while the pain thresholds of 1A10-Fc and NGF F12E -Fc were equivalent, both higher than hNGF-Fc; 24 hours after injection, the pain thresholds of 1A2-Fc, 1A10-Fc and NGF F12E -Fc were all higher than hNGF-Fc. The above experimental results show that compared with hNGF-Fc, the pain side effects caused by the fusion protein of the NGF mutant and Fc and NGF F12E -Fc in this application are significantly reduced. In addition, compared with NGF F12E -Fc, the degree of pain caused by 1A2-Fc and 1A10-Fc is basically the same, that is, no additional pain is caused.
同时也对其它NGF突变体与Fc的融合蛋白的痛阈值进行了检测，相比于人野生型成熟NGF，各个NGF突变体所带来的疼痛副作用均显著减轻(具体实验数据未显示)。At the same time, the pain thresholds of other NGF mutants and Fc fusion proteins were also tested. Compared with wild-type mature human NGF, the pain side effects caused by each NGF mutant were significantly reduced (specific experimental data not shown).
实施例11：NGF的组合突变体Example 11: Combination mutants of NGF
筛选获得的示例性NGF组合突变体及其对应的抗NGF抗体如表11所示，即所述的NGF组合突变体与其对应的抗NGF抗体之间的结合减弱或基本不与其结合。其氨基酸序列如表12所示。The exemplary NGF combination mutants obtained by screening and their corresponding anti-NGF antibodies are shown in Table 11, that is, the binding between the NGF combination mutants and their corresponding anti-NGF antibodies is weakened or substantially non-binding. The amino acid sequences thereof are shown in Table 12.
表11

Table 11

表12

Table 12

同样，在上述突变体中也进一步引入突变F12E，获得如下NGF突变体1A15-1A24，其氨基酸序列如表13所示，后续均基于突变体1A15-1A24进行实验。进一步应用ELSIA法验证了部分NGF组合突变体分别与TrkA受体和抗NGF抗体的结合活性。Similarly, mutation F12E was further introduced into the above mutants to obtain the following NGF mutants 1A15-1A24, whose amino acid sequences are shown in Table 13. Subsequent experiments were all based on mutants 1A15-1A24. The ELSIA method was further used to verify the binding activity of some NGF combination mutants to TrkA receptor and anti-NGF antibody.
表13

Table 13

11.1 NGF组合突变体与抗NGF抗体的结合活性11.1 Binding activity of NGF combination mutants and anti-NGF antibodies
采用ELISA法来检测上述各个组合突变体与Fc的融合蛋白与抗NGF抗体的结合活性，具体的操作步骤参见实施例4。The ELISA method was used to detect the binding activity of the fusion proteins of the above-mentioned combined mutants and Fc with anti-NGF antibodies. For specific operating steps, please refer to Example 4.
1A17-Fc、1A18-Fc、1A21-Fc和1A22-Fc与STC001的结合ELISA曲线如图9A所示，1A15-Fc、1A16-Fc、1A19-Fc和1A20-Fc与STC001的ELISA结合曲线如图9B所示。由图可知，上述NGF组合突变体均与STC001抗体的结合活性减弱或基本不与之结合。The ELISA binding curves of 1A17-Fc, 1A18-Fc, 1A21-Fc and 1A22-Fc to STC001 are shown in Figure 9A, and the ELISA binding curves of 1A15-Fc, 1A16-Fc, 1A19-Fc and 1A20-Fc to STC001 are shown in Figure 9B. As can be seen from the figure, the binding activity of the above-mentioned NGF combination mutants to the STC001 antibody is weakened or basically does not bind to it.
1A15-Fc与示例性的抗NGF抗体的结合ELISA曲线如图9C所示，由图可知，1A15-Fc与抗NGF抗体Fasinumab、MEDI-578和STC001的结合活性减弱或基本不与之结合。 The binding ELISA curves of 1A15-Fc and exemplary anti-NGF antibodies are shown in FIG9C . As can be seen from the figure, the binding activity of 1A15-Fc to the anti-NGF antibodies Fasinumab, MEDI-578 and STC001 is weakened or it basically does not bind to them.
1A16-Fc与示例性的抗NGF抗体的结合ELISA曲线如图9D所示，由图可知，1A16-Fc与抗NGF抗体Fasinumab、MEDI-578和STC001的结合活性减弱或基本不与之结合。The binding ELISA curves of 1A16-Fc and exemplary anti-NGF antibodies are shown in FIG9D . As can be seen from the figure, the binding activity of 1A16-Fc to the anti-NGF antibodies Fasinumab, MEDI-578 and STC001 is weakened or it basically does not bind to them.
1A19-Fc与示例性的抗NGF抗体的结合ELISA曲线如图9E所示，由图可知，1A19-Fc与抗NGF抗体Tanezumab、MEDI-578和STC001的结合活性减弱或基本不与之结合。The binding ELISA curves of 1A19-Fc and exemplary anti-NGF antibodies are shown in FIG9E . As can be seen from the figure, the binding activity of 1A19-Fc to the anti-NGF antibodies Tanezumab, MEDI-578 and STC001 is weakened or it basically does not bind to them.
1A20-Fc与示例性的抗NGF抗体的结合ELISA曲线如图9F所示，由图可知，1A20-Fc与抗NGF抗体Tanezumab、Fasinumab、MEDI-578和STC001的结合活性减弱或基本不与之结合。The binding ELISA curves of 1A20-Fc and exemplary anti-NGF antibodies are shown in FIG9F . As can be seen from the figure, the binding activity of 1A20-Fc to the anti-NGF antibodies Tanezumab, Fasinumab, MEDI-578 and STC001 is weakened or basically does not bind to them.
1A24-Fc与示例性的抗NGF抗体的结合ELISA曲线如图9G所示，由图可知，1A24-Fc与抗NGF抗体Fulranumab和MEDI-578的结合活性减弱或基本不与之结合。The binding ELISA curves of 1A24-Fc and exemplary anti-NGF antibodies are shown in FIG9G . As can be seen from the figure, the binding activity of 1A24-Fc to the anti-NGF antibodies Fulranumab and MEDI-578 is weakened or 1A24-Fc does not bind to them at all.
11.2 NGF组合突变体与TrkA受体的结合活性11.2 Binding activity of NGF combination mutants to TrkA receptor
采用ELISA法检测上述各个组合突变体与Fc的融合蛋白与TrkA受体的结合活性，具体的操作步骤参见实施例5。The binding activity of the fusion proteins of the above-mentioned combined mutants and Fc to the TrkA receptor was detected by ELISA method. For specific operation steps, see Example 5.
1A17-Fc、1A18-Fc、1A21-Fc和1A22-Fc与TrkA受体的结合ELISA曲线如图10A所示，1A15-Fc、1A16-Fc、1A19-Fc和1A20-Fc与TrkA受体的结合ELISA曲线如图10B所示，1A24-Fc与TrkA受体的结合ELISA曲线如图10C所示。由图可知，上述NGF组合突变体均能够与TrkA受体结合，并且其与TrkA受体的结合活性与NGFF12E相当或更高。The binding ELISA curves of 1A17-Fc, 1A18-Fc, 1A21-Fc and 1A22-Fc to TrkA receptor are shown in Figure 10A, the binding ELISA curves of 1A15-Fc, 1A16-Fc, 1A19-Fc and 1A20-Fc to TrkA receptor are shown in Figure 10B, and the binding ELISA curve of 1A24-Fc to TrkA receptor is shown in Figure 10C. As can be seen from the figure, the above-mentioned NGF combination mutants can bind to TrkA receptor, and their binding activity to TrkA receptor is equivalent to or higher than that of NGF F12E .
11.3 NGF组合突变体的生物学活性11.3 Biological activities of NGF combination mutants
以1A15-Fc、1A16-Fc和1A23-Fc为示例，应用鸡胚法检测各个NGF组合突变体的生物学活性，具体操作步骤如实施例8中所述，检测结果示于表14中。Taking 1A15-Fc, 1A16-Fc and 1A23-Fc as examples, the chicken embryo method was used to detect the biological activity of each NGF combination mutant. The specific operation steps are as described in Example 8, and the test results are shown in Table 14.
表14中的结果表明，1A15-Fc、1A16-Fc以及1A23-Fc的生物学活性与hNGF-Fc和NGFF12E-Fc相当。 The results in Table 14 indicate that the biological activities of 1A15-Fc, 1A16-Fc and 1A23-Fc are comparable to those of hNGF-Fc and NGF F12E -Fc.
表14
Table 14
也采用相同的方法对其它的NGF组合突变体进行了生物学活性检测，初步的结果表明其他的NGF组合突变体也具有相应的生物学活性，能够促进鸡胚背根神经节生长(数据未展示)。The same method was also used to detect the biological activities of other NGF combination mutants. Preliminary results showed that other NGF combination mutants also had corresponding biological activities and could promote the growth of chicken embryo dorsal root ganglia (data not shown).
实施例12：抗NGF抗体与NGF突变体的联合应用Example 12: Combined use of anti-NGF antibodies and NGF mutants
为了验证本申请的NGF突变体的生物学活性是否会被与之对应的抗NGF抗体所拮抗，设计如下实验：共同施用抗NGF抗体和NGF突变体与Fc的融合蛋白后，采用如实施例7或8中所述的PC12细胞活性检测实验或鸡胚法，检测NGF突变体的生物学活性。In order to verify whether the biological activity of the NGF mutant of the present application would be antagonized by the corresponding anti-NGF antibody, the following experiment was designed: after co-administering the anti-NGF antibody and the fusion protein of the NGF mutant and Fc, the biological activity of the NGF mutant was detected by the PC12 cell activity detection experiment or the chicken embryo method as described in Example 7 or 8.
12.1同时在抗NGF抗体存在下检测NGF突变体的生物学活性(PC12细胞活性检测)：12.1 Simultaneously detecting the biological activity of NGF mutants in the presence of anti-NGF antibodies (PC12 cell activity detection):
采用PC12细胞活性检测方法测定在对应的抗NGF抗体存在的情况下NGF突变体的生物学活性，具体操作方法如实施例7中所述。在实验中设立联合应用hNGF-Fc和相同的抗NGF抗体的对照组。在本实验中以6组联合应用：1A7-Fc联合应用Fasinumab、1A13-Fc联合应用AK-115、1A12-Fc联合应用STC001、1A10-Fc联合应用STC001、1A4-Fc联合应用STC001和1A5-Fc联合应用STC001为示例，其中抗NGF抗体的浓度为1.6μg/ml，NGF突变体与Fc的融合蛋白以及hNGF-Fc的最高浓度为6.25μg/ml，5倍梯度稀释到最低浓度80pg/ml。96孔板各孔加好样品，放入37℃，CO2 培养箱进行培养6小时后进行荧光素酶检测，并使用GraphPad Prism 5.0进行曲线的拟合并计算EC50。The PC12 cell activity detection method was used to determine the biological activity of the NGF mutant in the presence of the corresponding anti-NGF antibody. The specific operation method is as described in Example 7. In the experiment, a control group was set up for the combined use of hNGF-Fc and the same anti-NGF antibody. In this experiment, 6 groups of combined applications were used: 1A7-Fc combined with Fasinumab, 1A13-Fc combined with AK-115, 1A12-Fc combined with STC001, 1A10-Fc combined with STC001, 1A4-Fc combined with STC001 and 1A5-Fc combined with STC001 as examples, where the concentration of anti-NGF antibody was 1.6μg/ml, the highest concentration of NGF mutant and Fc fusion protein and hNGF-Fc was 6.25μg/ml, and it was diluted 5 times to the lowest concentration of 80pg/ml. Add samples to each well of the 96-well plate and place it at 37°C, CO 2 Luciferase assay was performed after 6 h of incubation in the incubator, and GraphPad Prism 5.0 was used to fit the curve and calculate the EC50.
其中，1A7-Fc联合应用Fasinumab的结果如图11A所示，1A13-Fc联合应用AK-115的结果如图11B所示，1A12-Fc联合应用STC001的结果如图11C所示，1A10-Fc联合应用STC001的结果如图11D所示，1A4和1A5分别联合应用STC001的结果如图11E所示。具体的EC50值如表15所示。Among them, the results of 1A7-Fc combined with Fasinumab are shown in Figure 11A, the results of 1A13-Fc combined with AK-115 are shown in Figure 11B, the results of 1A12-Fc combined with STC001 are shown in Figure 11C, the results of 1A10-Fc combined with STC001 are shown in Figure 11D, and the results of 1A4 and 1A5 combined with STC001 are shown in Figure 11E. The specific EC50 values are shown in Table 15.
由上述实验结果可以看出，与hNGF-Fc相比，当联合应用抗NGF抗体时，示例性的NGF突变体与Fc的融合蛋白在PC12细胞活性检测实验中仍具有良好的生物学活性，其生物学活性未被抗NGF抗体所拮抗。It can be seen from the above experimental results that compared with hNGF-Fc, when used in combination with anti-NGF antibodies, the fusion protein of the exemplary NGF mutant and Fc still has good biological activity in the PC12 cell activity detection experiment, and its biological activity is not antagonized by anti-NGF antibodies.
表15
Table 15
12.2在抗NGF抗体存在下检测NGF突变体的生物学活性(鸡胚法检测)：12.2 Detection of biological activity of NGF mutants in the presence of anti-NGF antibodies (chicken embryo method):
采用鸡胚法测定在对应的抗NGF抗体存在的情况下，NGF突变体的生物学活性(以1A2-Fc、1A3-Fc、1A10-Fc和1A13-Fc为例)，其中鸡胚法检测的具体操作步骤参见实施例8，同时加入不同浓度的抗NGF抗体。在实验中设立联合应用NGFF12E-Fc和相同抗NGF抗体对照组，联合应用hNGF-Fc和相同抗NGF抗体对照组，以及联合应用参考品鼠NGF和相同抗NGF抗体对照组。 The chicken embryo method was used to determine the biological activity of NGF mutants (taking 1A2-Fc, 1A3-Fc, 1A10-Fc and 1A13-Fc as examples) in the presence of the corresponding anti-NGF antibodies. The specific operation steps of the chicken embryo method are shown in Example 8, and different concentrations of anti-NGF antibodies were added at the same time. In the experiment, a control group of combined application of NGF F12E -Fc and the same anti-NGF antibody, a control group of combined application of hNGF-Fc and the same anti-NGF antibody, and a control group of combined application of reference Mouse NGF and the same anti-NGF antibody control group.
以STC001联合应用1A2-Fc、1A3-Fc或1A10-Fc为示例，分别检测了在STC001的终浓度分别为0μg/ml、0.1μg/ml、0.5μg/ml、1μg/ml、10μg/ml、或50μg ml时，NGF突变体的生物学活性。Taking the combined use of STC001 with 1A2-Fc, 1A3-Fc or 1A10-Fc as an example, the biological activity of NGF mutants was detected when the final concentration of STC001 was 0 μg/ml, 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, 10 μg/ml, or 50 μg ml.
其结果如表16-1所示。The results are shown in Table 16-1.
表16-1
Table 16-1
由表16-1数据可以看出，当不应用抗NGF抗体时，NGF突变体与Fc的融合蛋白和hNGF-Fc或NGFF12E-Fc均能发挥生物学活性，且它们的生物学活性基本相当。然而，当抗体的浓度逐渐增加，直到用量达到50μg/ml时，在不同的抗体浓度下，示例性的NGF突变体1A2-Fc、1A10-Fc和1A3-Fc仍具有生物学活性，能够促进鸡胚背根神经节生长，且即便抗体浓度的不断增加，其仍能保持生物学活性，相比之下，野生型hNGF-Fc或NGFF12E-Fc的生物学活性则被显著抑制。It can be seen from the data in Table 16-1 that when anti-NGF antibodies are not used, the fusion protein of NGF mutant and Fc and hNGF-Fc or NGF F12E -Fc can exert biological activity, and their biological activities are basically equivalent. However, when the concentration of the antibody is gradually increased until the dosage reaches 50 μg/ml, at different antibody concentrations, the exemplary NGF mutants 1A2-Fc, 1A10-Fc and 1A3-Fc still have biological activity and can promote the growth of chicken embryo dorsal root ganglia, and even if the antibody concentration continues to increase, it can still maintain biological activity. In contrast, the biological activity of wild-type hNGF-Fc or NGF F12E -Fc is significantly inhibited.
表16-2为其它NGF突变体与对应的抗NGF抗体联合应用时，通过鸡胚法测定的NGF突变体的生物学活性。其中hNGF-Fc联合应用相同的抗NGF抗体作为对照，由表16-2数据要以看出，当同时应用相应的抗NGF抗体时，hNGF-Fc的生物学活性完全被拮抗，而本申请NGF突变体仍能够发挥NGF的生物学活性，能够促进鸡胚背根神经节生长。Table 16-2 shows the biological activity of NGF mutants measured by the chicken embryo method when other NGF mutants were used in combination with the corresponding anti-NGF antibodies. hNGF-Fc was used in combination with the same anti-NGF antibody as a control. It can be seen from the data in Table 16-2 that when the corresponding anti-NGF antibody was used at the same time, the biological activity of hNGF-Fc was completely antagonized, while the NGF mutant of the present application was still able to exert the biological activity of NGF and promote the growth of dorsal root ganglia in chicken embryos.
表16-2


“/”代表未检测Table 16-2


“/” means not detected
上述PC12细胞活性检测实验以及鸡胚背根神经节实验的结果说明，本申请的NGF突变体与相应的抗NGF抗体联合应用时，NGF突变体仍能发挥生物学活性，即本申请的NGF突变体与相应的抗NGF抗体联合应用时，能够减轻抗NGF抗体治疗相关疾病时对体内NGF的过度拮抗所带来的不良反应。 The results of the above-mentioned PC12 cell activity detection experiment and chicken embryo dorsal root ganglion experiment indicate that when the NGF mutant of the present application is used in combination with the corresponding anti-NGF antibody, the NGF mutant can still exert biological activity, that is, when the NGF mutant of the present application is used in combination with the corresponding anti-NGF antibody, the adverse reactions caused by excessive antagonism of NGF in the body when the anti-NGF antibody is used to treat related diseases can be reduced.

Claims (87)

  1. 一种筛选和/或鉴定NGF中可突变位点的方法，其中所述可突变位点上的氨基酸突变会影响NGF突变体与一种或多种抗NGF抗体的结合，但仍能使NGF突变体具有生物学活性。A method for screening and/or identifying mutable sites in NGF, wherein the amino acid mutation at the mutable site will affect the binding of the NGF mutant to one or more anti-NGF antibodies, but still enable the NGF mutant to have biological activity.
  2. 根据权利要求1中所述的方法，其包括如下步骤：The method according to claim 1, comprising the steps of:
    (i)获得一种或多种抗NGF抗体的三维结构；(i) obtaining the three-dimensional structure of one or more anti-NGF antibodies;
    (ii)将成熟NGF的晶体结构与上述获得的抗NGF抗体的三维结构分别进行对接，以获得一种或多种抗NGF抗体与NGF的结合构象，其中，成熟NGF的晶体结构如PDB ID：4EDW所示；(ii) docking the crystal structure of mature NGF with the three-dimensional structure of the anti-NGF antibody obtained above, respectively, to obtain the binding conformation of one or more anti-NGF antibodies and NGF, wherein the crystal structure of mature NGF is shown in PDB ID: 4EDW;
    (iii)将步骤(ii)中获得的结合构象和成熟NGF与TrkA受体的结合构象分别进行比对；(iii) aligning the binding conformation obtained in step (ii) and the binding conformation of mature NGF and TrkA receptor respectively;
    (iv)基于步骤(iii)中的比对结果，获得所述NGF中的可突变位点。(iv) Based on the comparison results in step (iii), obtain the mutable sites in the NGF.
  3. 根据权利要求2中所述的方法，其中所述步骤(i)中采用同源建模方法来构建并获得抗NGF抗体的三维结构；优选地，所述同源建模方法为Modeller同源建模方法。According to the method described in claim 2, wherein in step (i), a homology modeling method is used to construct and obtain the three-dimensional structure of the anti-NGF antibody; preferably, the homology modeling method is the Modeller homology modeling method.
  4. 根据权利要求2或3中所述的方法，其中所述步骤(ii)中采用Discovery studio ZDOCK分子对接技术来将成熟NGF的晶体结构与抗NGF抗体的三维结构进行对接。According to the method according to claim 2 or 3, in the step (ii), the Discovery studio ZDOCK molecular docking technology is used to dock the crystal structure of mature NGF with the three-dimensional structure of the anti-NGF antibody.
  5. 根据权利要求2-4中任一项所述的方法，其中所述成熟NGF与TrkA受体的结合构象如PDB ID：1WWW所示。The method according to any one of claims 2-4, wherein the binding conformation of the mature NGF and TrkA receptor is as shown in PDB ID: 1WWW.
  6. 根据权利要求2-5中任一项所述的方法，其特征在于，所述可突变位点是指NGF中位于与抗NGF抗体结合界面，但不位于与TrkA受体结合界面的氨基酸位点；进一步地，如果涉及多种抗NGF抗体，则所述可突变位点为针对各种抗NGF抗体所获得的多组可突变位点之间的交集或并集。The method according to any one of claims 2-5 is characterized in that the mutable site refers to an amino acid site in NGF that is located at the binding interface with the anti-NGF antibody but not at the binding interface with the TrkA receptor; further, if multiple anti-NGF antibodies are involved, the mutable site is the intersection or union of multiple groups of mutable sites obtained for various anti-NGF antibodies.
  7. 一种构建NGF突变体库的方法，其特征在于，所述突变体库中包含的NGF突变体中具有一个或多个可突变位点，在该一个或多个可突变位点上进行氨基酸突变会影响NGF与一种或多种抗NGF抗体的结合，但仍能使NGF突变体具有生物学活性。A method for constructing an NGF mutant library, characterized in that the NGF mutants contained in the mutant library have one or more mutable sites, and amino acid mutations at the one or more mutable sites will affect the binding of NGF to one or more anti-NGF antibodies, but still enable the NGF mutants to have biological activity.
  8. 根据权利要求7中所述的方法，其包括如下步骤：以人野生型成熟NGF为模板，在上述可突变位点中的一个或多个位点上进行氨基酸突变，产生一系列的NGF突变体，从而获得NGF突变体库。According to the method described in claim 7, it comprises the following steps: using human wild-type mature NGF as a template, performing amino acid mutations at one or more of the above-mentioned mutable sites to produce a series of NGF mutants, thereby obtaining an NGF mutant library.
  9. 根据权利要求7或8中所述的方法，其中所述可突变位点采用如权利要求1-6中任一项所述的方法获得。 The method according to claim 7 or 8, wherein the mutable site is obtained by the method according to any one of claims 1 to 6.
  10. 根据权利要求1-9中任一项所述的方法，其特征在于，所述可突变位点上的氨基酸突变会影响NGF突变体与抗NGF抗体的结合是指该位点上的氨基酸发生突变会导致NGF突变体与抗NGF抗体的结合减弱或基本不与抗NGF抗体结合。The method according to any one of claims 1-9 is characterized in that the amino acid mutation at the mutable site affects the binding of the NGF mutant to the anti-NGF antibody, which means that the amino acid mutation at the site will cause the binding of the NGF mutant to the anti-NGF antibody to be weakened or basically not bind to the anti-NGF antibody.
  11. 一种NGF突变体库，其包含与一种或多种抗NGF抗体结合减弱或基本不与抗NGF抗体结合，同时具有生物学活性的NGF突变体。A NGF mutant library comprises NGF mutants that have reduced binding to one or more anti-NGF antibodies or substantially no binding to anti-NGF antibodies while having biological activity.
  12. 根据权利要求11中所述的NGF突变体库，其由权利要求7-10中任一项所述的方法构建获得。The NGF mutant library according to claim 11 is constructed by the method according to any one of claims 7-10.
  13. 根据权利要求11或12中所述的NGF突变体库，其所包含的NGF突变体进一步包含相对于人野生型成熟NGF的突变F12E。According to the NGF mutant library according to claim 11 or 12, the NGF mutants contained therein further comprise the mutation F12E relative to human wild-type mature NGF.
  14. 一种筛选或鉴定NGF突变体的方法，其中所述NGF突变体与一种或多种抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，同时具有生物学活性。A method for screening or identifying NGF mutants, wherein the NGF mutants have reduced binding to one or more anti-NGF antibodies or substantially no binding to anti-NGF antibodies while having biological activity.
  15. 根据权利要求14中所述的方法，其包括如下步骤：The method according to claim 14, comprising the steps of:
    (i)获得一个或多个NGF突变体；(i) obtaining one or more NGF mutants;
    (ii)从步骤(i)中的NGF突变体中筛选或鉴定具有生物学活性，并且与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体。(ii) Screening or identifying NGF mutants from the NGF mutants in step (i) that have biological activity and have reduced binding to or substantially no binding to anti-NGF antibodies.
  16. 根据权利要求15中所述的方法，其中所述步骤(ii)采用如下方法完成：The method according to claim 15, wherein step (ii) is performed by:
    (1)首先筛选或鉴定具有生物学活性的NGF突变体，再从中筛选或鉴定与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体；或者，(1) first screening or identifying NGF mutants with biological activity, and then screening or identifying NGF mutants with reduced binding to or substantially no binding to anti-NGF antibodies; or,
    (2)首先筛选或鉴定与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体，再从中筛选或鉴定具有生物学活性的NGF突变体；或者，(2) first screening or identifying NGF mutants that have reduced binding to or substantially no binding to anti-NGF antibodies, and then screening or identifying NGF mutants with biological activity; or,
    (3)分别筛选或鉴定具有生物学活性的NGF突变体，以及与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体，取二者的交集；(3) respectively screening or identifying NGF mutants with biological activity and NGF mutants with reduced binding to or substantially no binding to anti-NGF antibodies, and taking the intersection of the two;
    最终筛选或鉴定出与抗NGF抗体结合减弱或基本不与抗NGF抗体结合，且具有生物学活性的一系列NGF突变体。Finally, a series of NGF mutants with reduced or essentially no binding to anti-NGF antibodies and biological activity are screened or identified.
  17. 根据权利要求14-16中任一项所述的方法，其中所述步骤(i)中的NGF突变体是从NGF突变体库获得或者通过理性设计获得。The method according to any one of claims 14-16, wherein the NGF mutant in step (i) is obtained from an NGF mutant library or through rational design.
  18. 根据权利要求17中所述的方法，其中所述NGF突变体库由权利要求7-10中任一项所述的方法获得，或者选自权利要求11-13中任一项所述的NGF突变体库。The method according to claim 17, wherein the NGF mutant library is obtained by the method of any one of claims 7-10, or is selected from the NGF mutant library of any one of claims 11-13.
  19. 根据权利要求15中所述的方法，其中所述步骤(i)中的NGF突变体通过表面展示技术进行展示，包括原核展示文库技术和真核展示文库技术；进一步优选地，通过酵 母表面展示技术、噬菌体表面展示技术、哺乳动物细胞表面展示进术、细菌表面展示技术或杆状病毒表面展示技术进行展示。The method according to claim 15, wherein the NGF mutant in step (i) is displayed by surface display technology, including prokaryotic display library technology and eukaryotic display library technology; further preferably, by enzyme The display can be carried out by using mother surface display technology, phage surface display technology, mammalian cell surface display technology, bacterial surface display technology or baculovirus surface display technology.
  20. 根据权利要求15中所述的方法，其中所述步骤(ii)中通过MACS、FACS和/或生物学活性测定来筛选或鉴定具有生物学活性的NGF突变体。The method according to claim 15, wherein in step (ii), NGF mutants with biological activity are screened or identified by MACS, FACS and/or biological activity assays.
  21. 根据权利要求15中所述的方法，其中所述步骤(ii)中通过MACS、FACS和/或ELISA来筛选与抗NGF抗体结合减弱或基本不与抗NGF抗体结合的NGF突变体。The method according to claim 15, wherein in step (ii), MACS, FACS and/or ELISA are used to screen NGF mutants that have reduced binding to or essentially no binding to anti-NGF antibodies.
  22. 一种NGF突变体，其与一种或多种抗NGF抗体的结合减弱或基本不与抗NGF抗体结合，同时具有生物学活性。An NGF mutant has reduced binding to one or more anti-NGF antibodies or substantially no binding to anti-NGF antibodies, while having biological activity.
  23. 根据权利要求22中所述的NGF突变体，其由权利要求14-21中任一项所述的方法筛选获得。The NGF mutant according to claim 22 is obtained by screening according to the method described in any one of claims 14-21.
  24. 根据权利要求22或23中所述的NGF突变体，其进一步包含相对于人野生型成熟NGF的突变F12E。The NGF mutant according to claim 22 or 23, further comprising a mutation F12E relative to human wild-type mature NGF.
  25. 一种融合蛋白，其包括权利要求22-24中任一项所述的NGF突变体。A fusion protein comprising the NGF mutant according to any one of claims 22-24.
  26. 一种药物组合物，其包含抗NGF抗体和NGF突变体或包含NGF突变体的融合蛋白以及药学上可接受的载体和/或辅料，其中所述NGF突变体与该抗NGF抗体结合减弱或基本不与该抗NGF抗体结合，同时仍具有生物学活性。A pharmaceutical composition comprising an anti-NGF antibody and an NGF mutant or a fusion protein comprising an NGF mutant and a pharmaceutically acceptable carrier and/or excipient, wherein the NGF mutant has a weakened binding to the anti-NGF antibody or substantially no binding to the anti-NGF antibody while still having biological activity.
  27. 根据权利要求26中所述的药物组合物，其中所述NGF突变体采用如权利要求14-21中任一项所述的方法筛选获得或选自权利要求22-24中任一项所述的NGF突变体。The pharmaceutical composition according to claim 26, wherein the NGF mutant is obtained by screening using the method described in any one of claims 14-21 or is selected from the NGF mutant described in any one of claims 22-24.
  28. 一种降低和/或减轻应用抗NGF抗体治疗时的不良反应的方法，包括向已经接受、正在接受或即将接受抗NGF抗体治疗的个体施用NGF突变体或包含NGF突变体的融合蛋白，其中所述NGF突变体与该抗NGF抗体结合减弱或基本不与该抗NGF抗体结合，同时仍具有生物学活性。A method for reducing and/or alleviating adverse reactions during anti-NGF antibody treatment, comprising administering an NGF mutant or a fusion protein comprising an NGF mutant to an individual who has received, is receiving, or is about to receive anti-NGF antibody treatment, wherein the NGF mutant has weakened binding to the anti-NGF antibody or essentially no binding to the anti-NGF antibody while still having biological activity.
  29. 根据权利要求28中所述的方法，其中所述不良反应包括神经系统相关的不良反应和关节相关的不良反应。The method according to claim 28, wherein the adverse reactions include nervous system-related adverse reactions and joint-related adverse reactions.
  30. 根据权利要求29中所述的方法，其中所述不良反应包括：交感神经损伤、骨坏死、骨丢失、骨损伤、关节损伤、快速进展性骨关节炎(RPOA)、外周性水肿、关节痛、四肢疼痛、周围神经感觉异常、感觉迟钝、感觉过敏、灼烧痛和触感痛。The method of claim 29, wherein the adverse reactions include: sympathetic nerve damage, osteonecrosis, bone loss, bone damage, joint damage, rapidly progressive osteoarthritis (RPOA), peripheral edema, joint pain, limb pain, peripheral nerve paresthesia, dysesthesia, hyperesthesia, burning pain and tactile pain.
  31. 一种在有需要的个体中治疗和/或预防由NGF表达增加和/或对NGF敏感性增强引起的疾病或病症的方法，包括向个体施用有效量的抗NGF抗体和NGF突变体或包 含NGF突变体的融合蛋白，其中所述NGF突变体与该抗NGF抗体结合减弱或基本不与该抗NGF抗体结合，同时仍具有生物学活性；或者施用如权利要求26或27中所述的药物组合物。A method for treating and/or preventing a disease or condition caused by increased NGF expression and/or enhanced sensitivity to NGF in an individual in need thereof, comprising administering to the individual an effective amount of an anti-NGF antibody and an NGF mutant or a combination thereof A fusion protein containing an NGF mutant, wherein the NGF mutant has weakened binding to the anti-NGF antibody or essentially no binding to the anti-NGF antibody while still having biological activity; or administering the pharmaceutical composition as described in claim 26 or 27.
  32. 根据权利要求31中所述的方法，其中所述抗NGF抗体与所述NGF突变体或包含NGF突变体的融合蛋白同时施用或者顺序施用。The method according to claim 31, wherein the anti-NGF antibody and the NGF mutant or the fusion protein comprising the NGF mutant are administered simultaneously or sequentially.
  33. 根据权利要求31或32中所述的方法，其中所述疾病或病症包括炎症性疼痛、术后切口疼痛、神经性疼痛、骨折疼痛、痛风关节疼痛、带状疱疹后神经痛、烧伤引起的疼痛、癌症疼痛、骨关节炎或类风湿性关节炎疼痛、坐骨神经痛、与镰状细胞危象相关的疼痛，或疱疹性神经痛。The method of claim 31 or 32, wherein the disease or condition comprises inflammatory pain, postoperative incisional pain, neuropathic pain, fracture pain, gouty joint pain, postherpetic neuralgia, pain caused by burns, cancer pain, osteoarthritis or rheumatoid arthritis pain, sciatica, pain associated with sickle cell crisis, or herpetic neuralgia.
  34. 根据权利要求28-33中任一项所述的方法，其中所述NGF突变体采用如权利要求14-21中任一项所述的方法筛选获得或选自权利要求22-24中任一项所述的NGF突变体。The method according to any one of claims 28-33, wherein the NGF mutant is obtained by screening using the method according to any one of claims 14-21 or is selected from the NGF mutant according to any one of claims 22-24.
  35. 根据权利要求10，14-21，28-34中任一项所述的方法，权利要求11-13中任一项所述的NGF突变体库，权利要求22-24中任一项所述的NGF突变体，权利要求25中所述的融合蛋白，或权利要求26或27中所述的药物组合物，其中所述NGF突变体与抗NGF抗体的结合减弱是指与人野生型成熟NGF(SEQ ID NO：1)或NGFF12E(SEQ ID NO：83)相比，NGF突变体与抗NGF抗体的结合活性降低。According to the method described in any one of claims 10, 14-21, 28-34, the NGF mutant library described in any one of claims 11-13, the NGF mutant described in any one of claims 22-24, the fusion protein described in claim 25, or the pharmaceutical composition described in claim 26 or 27, wherein the reduced binding of the NGF mutant to the anti-NGF antibody means that the binding activity of the NGF mutant to the anti-NGF antibody is reduced compared with human wild-type mature NGF (SEQ ID NO: 1) or NGF F12E (SEQ ID NO: 83).
  36. 根据权利要求35中所述的方法、NGF突变体库、NGF突变体、包含NGF突变体的融合蛋白或药物组合物，其中所述NGF突变体与抗NGF抗体的结合活性不超过人野生型成熟NGF或NGFF12E与相同抗NGF抗体结合活性的50％，例如不超过40％、30％、20％、10％、8％、7％、6％、5％、4％、3％、2％、1％、0.6％、0.5％、0.2％、0.1％、0.07％、0.02％、0.01％、0.001％或更低。According to the method, NGF mutant library, NGF mutant, fusion protein or pharmaceutical composition described in claim 35, wherein the binding activity of the NGF mutant with the anti-NGF antibody does not exceed 50% of the binding activity of human wild-type mature NGF or NGF F12E with the same anti-NGF antibody, for example, not more than 40%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.6%, 0.5%, 0.2%, 0.1%, 0.07%, 0.02%, 0.01%, 0.001% or less.
  37. 根据权利要求1-10，14-21，28-34中任一所述的方法，权利要求11-13中任一项所述的NGF突变体库，权利要求22-24中任一项所述的NGF突变体，权利要求25中所述的融合蛋白，或权利要求26或27中所述的药物组合物，其中所述NGF突变体基本不与抗NGF抗体结合。The method according to any one of claims 1-10, 14-21, 28-34, the NGF mutant library according to any one of claims 11-13, the NGF mutant according to any one of claims 22-24, the fusion protein described in claim 25, or the pharmaceutical composition described in claim 26 or 27, wherein the NGF mutant does not substantially bind to anti-NGF antibodies.
  38. 根据权利要求1-10，14-21，28-34中任一所述的方法，权利要求11-13中任一项所述的NGF突变体库，权利要求22-24中任一项所述的NGF突变体，权利要求25中所述的融合蛋白，或权利要求26或27中所述的药物组合物，其中所述NGF突变体具有生物学活性是指与NGFF12E(SEQ ID NO：83)相比，其生物学活性为NGFF12E的 至少20％，例如至少21％、30％、36％、40％、80％、100％、1.5倍、2倍、3倍、4倍、5倍、10倍、15倍、20倍、50倍、100倍或更高。The method according to any one of claims 1-10, 14-21, 28-34, the NGF mutant library according to any one of claims 11-13, the NGF mutant according to any one of claims 22-24, the fusion protein according to claim 25, or the pharmaceutical composition according to claim 26 or 27, wherein the NGF mutant has biological activity means that its biological activity is compared with NGF F12E ( SEQ ID NO: 83), and its biological activity is At least 20%, such as at least 21%, 30%, 36%, 40%, 80%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
  39. 根据权利要求1-10，14-21，28-34中任一所述的方法，权利要求11-13中任一项所述的NGF突变体库，权利要求22-24中任一项所述的NGF突变体，权利要求25中所述的融合蛋白，或权利要求26或27中所述的药物组合物，其中所述生物学活性通过NGF生物学活性检测实验测定获得。According to the method described in any one of claims 1-10, 14-21, 28-34, the NGF mutant library described in any one of claims 11-13, the NGF mutant described in any one of claims 22-24, the fusion protein described in claim 25, or the pharmaceutical composition described in claim 26 or 27, wherein the biological activity is obtained by measuring the NGF biological activity detection experiment.
  40. 根据权利要求1-10，14-21，28-34中任一所述的方法，权利要求11-13中任一项所述的NGF突变体库，权利要求22-24中任一项所述的NGF突变体，权利要求25中所述的融合蛋白，或权利要求26或27中所述的药物组合物，其中所述NGF生物学活性检测实验选自如下实验中的一种或多种：TrkA受体结合实验、TF-1细胞活性检测实验、PC12细胞活性检测实验、鸡胚背根神经节实验、或大鼠颈上神经节实验。According to any method described in claims 1-10, 14-21, 28-34, the NGF mutant library described in any one of claims 11-13, the NGF mutant described in any one of claims 22-24, the fusion protein described in claim 25, or the pharmaceutical composition described in claim 26 or 27, wherein the NGF biological activity detection experiment is selected from one or more of the following experiments: TrkA receptor binding experiment, TF-1 cell activity detection experiment, PC12 cell activity detection experiment, chicken embryo dorsal root ganglion experiment, or rat superior cervical ganglion experiment.
  41. 根据权利要求1-10，14-21，28-34中任一所述的方法，权利要求11-13中任一项所述的NGF突变体库，权利要求22-24中任一项所述的NGF突变体，权利要求25中所述的融合蛋白，或权利要求26或27中所述的药物组合物，其中所述NGF突变体的生物学活性由其与TrkA受体的结合活性表征。The method according to any one of claims 1-10, 14-21, 28-34, the NGF mutant library according to any one of claims 11-13, the NGF mutant according to any one of claims 22-24, the fusion protein described in claim 25, or the pharmaceutical composition described in claim 26 or 27, wherein the biological activity of the NGF mutant is characterized by its binding activity to the TrkA receptor.
  42. 根据权利要求1-10，14-21，28-34中任一所述的方法，权利要求11-13中任一项所述的NGF突变体库，权利要求22-24中任一项所述的NGF突变体，权利要求25中所述的融合蛋白，或权利要求26或27中所述的药物组合物，其中所述NGF突变体具有生物学活性是指该突变体能够与TrkA受体结合。According to the method described in any one of claims 1-10, 14-21, 28-34, the NGF mutant library described in any one of claims 11-13, the NGF mutant described in any one of claims 22-24, the fusion protein described in claim 25, or the pharmaceutical composition described in claim 26 or 27, wherein the NGF mutant has biological activity means that the mutant is able to bind to the TrkA receptor.
  43. 根据权利要求42中所述的方法、NGF突变体库、NGF突变体、包含NGF突变体的融合蛋白或药物组合物，其中所述NGF突变体能够与TrkA受体结合是指与NGFF12E(SEQ ID NO：83)相比，其与TrkA受体的结合活性是NGFF12E的至少20％，例如至少21％、30％、36％、40％、80％、100％、1.5倍、2倍、3倍、4倍、5倍、10倍、15倍、20倍、50倍、100倍或更高。According to the method, NGF mutant library, NGF mutant, fusion protein or pharmaceutical composition described in claim 42, wherein the NGF mutant is capable of binding to the TrkA receptor means that compared with NGF F12E (SEQ ID NO: 83), its binding activity to the TrkA receptor is at least 20% of NGF F12E , for example, at least 21%, 30%, 36%, 40%, 80%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 50 times, 100 times or more.
  44. 根据权利要求35-37，40-43中任一项所述的方法、NGF突变体库、NGF突变体、包含NGF突变体的融合蛋白或药物组合物，其中所述结合或结合活性通过ELISA实验、Biacore实验、BLI实验等进行测定。According to any one of claims 35-37, 40-43, the method, NGF mutant library, NGF mutant, fusion protein comprising NGF mutant or pharmaceutical composition, wherein the binding or binding activity is determined by ELISA experiment, Biacore experiment, BLI experiment, etc.
  45. 根据权利要求44中所述的方法、NGF突变体库、NGF突变体、包含NGF突变体的融合蛋白或药物组合物，其中所述结合活性采用在ELISA实验中的EC50值来表征。According to the method, NGF mutant library, NGF mutant, fusion protein comprising NGF mutant or pharmaceutical composition described in claim 44, wherein the binding activity is characterized by the EC50 value in an ELISA experiment.
  46. 根据权利要求45中所述的方法、NGF突变体库、NGF突变体、包含NGF突变体的 融合蛋白或药物组合物，其中，在ELISA实验中，所述NGF突变体与抗NGF抗体的结合EC50值是人野生型成熟NGF或NGFF12E与相同抗NGF抗体的结合EC50值的至少2倍、3倍、5倍、10倍、13倍、14倍、17倍、20倍、25倍、33倍、50倍、100倍、167倍、200倍、500倍、1000倍、1400倍、5000倍、10000倍、100000倍或更高，同时所述NGF突变体与TrkA受体的结合EC50值是NGFF12E与TrkA受体的结合EC50值的至多5倍、3倍、2倍、1倍、70％、60％、50％、35％、30％、25％、20％、10％、7％、5％、2％、1％或更低。The method according to claim 45, the NGF mutant library, the NGF mutant, the NGF mutant containing the NGF mutant A fusion protein or pharmaceutical composition, wherein in an ELISA experiment, the EC50 value of the binding of the NGF mutant to the anti-NGF antibody is at least 2 times, 3 times, 5 times, 10 times, 13 times, 14 times, 17 times, 20 times, 25 times, 33 times, 50 times, 100 times, 167 times, 200 times, 500 times, 1000 times, 1400 times, 5000 times, 10000 times, 100000 times or more of the EC50 value of the binding of the NGF mutant to the TrkA receptor. The EC50 value of F12E binding to the TrkA receptor is at most 5-fold, 3-fold, 2-fold, 1-fold, 70%, 60%, 50%, 35%, 30%, 25%, 20%, 10%, 7%, 5%, 2%, 1% or less.
  47. 一种NGF突变体，其包含相对于人野生型成熟NGF氨基酸序列的I31、K32、G33、K34、D93、W21、G23、D24、K50、Y52、T83、H84、F86、R100、R103、D16、S17、S19、T56、R59和R69位点中的一个或多个位点上的氨基酸突变。A NGF mutant comprising amino acid mutations at one or more of the I31, K32, G33, K34, D93, W21, G23, D24, K50, Y52, T83, H84, F86, R100, R103, D16, S17, S19, T56, R59 and R69 positions relative to the human wild-type mature NGF amino acid sequence.
  48. 根据权利要求47中所述的NGF突变体，其包含相对于人野生型成熟NGF氨基酸序列的S17、S19、K32、T56、R59和T83中的一个或多个位点上的氨基酸突变。According to the NGF mutant described in claim 47, it comprises amino acid mutations at one or more of S17, S19, K32, T56, R59 and T83 relative to the human wild-type mature NGF amino acid sequence.
  49. 根据权利要求47或48中所述的NGF突变体，其包含相对于人野生型成熟NGF氨基酸序列的S17和K32位点的氨基酸突变。The NGF mutant according to claim 47 or 48, comprising amino acid mutations at positions S17 and K32 relative to the amino acid sequence of human wild-type mature NGF.
  50. 根据权利要求47或48中所述的NGF突变体，其包含相对于人野生型成熟NGF氨基酸序列S19和K32位点的氨基酸突变。The NGF mutant according to claim 47 or 48, comprising amino acid mutations at positions S19 and K32 relative to the amino acid sequence of human wild-type mature NGF.
  51. 根据权利要求47或48中所述的NGF突变体，其包含相对于人野生型成熟NGF氨基酸序列S19和S17位点的氨基酸突变。The NGF mutant according to claim 47 or 48, comprising amino acid mutations at positions S19 and S17 relative to the amino acid sequence of human wild-type mature NGF.
  52. 根据权利要求47或48中所述的NGF突变体，其包含相对于人野生型成熟NGF氨基酸序列R59和T83位点的氨基酸突变。The NGF mutant according to claim 47 or 48 comprises amino acid mutations at positions R59 and T83 relative to the amino acid sequence of human wild-type mature NGF.
  53. 根据权利要求47-49、51中任一项所述的NGF突变体，其中所述S17位点的氨基酸突变为S17R、S17K、S17H或S17E。The NGF mutant according to any one of claims 47-49 and 51, wherein the amino acid mutation at the S17 site is S17R, S17K, S17H or S17E.
  54. 根据权利要求47、48、50或52中所述的NGF突变体，其中所述S19位点的氨基酸突变为S19R、S19K或S19F。The NGF mutant according to claim 47, 48, 50 or 52, wherein the amino acid mutation at the S19 position is S19R, S19K or S19F.
  55. 根据权利要求47-50中任一项所述的NGF突变体，其中所述K32位点的氨基酸突变为K32F、K32E、K32N、K32Y、K32M或K32L。The NGF mutant according to any one of claims 47-50, wherein the amino acid mutation at the K32 position is K32F, K32E, K32N, K32Y, K32M or K32L.
  56. 根据权利要求47或48中所述的NGF突变体，其中所述T56位点的氨基酸突变为T56K或T56R。The NGF mutant according to claim 47 or 48, wherein the amino acid at the T56 position is mutated to T56K or T56R.
  57. 根据权利要求47、48或52中所述的NGF突变体，其中所述R59位点的氨基酸突变为R59K。 The NGF mutant according to claim 47, 48 or 52, wherein the amino acid at the R59 position is mutated to R59K.
  58. 根据权利要求47、48或52中所述的NGF突变体，其中所述T83位点的的氨基酸突变为T83K。The NGF mutant according to claim 47, 48 or 52, wherein the amino acid at the T83 position is mutated to T83K.
  59. 根据权利要求49中所述的NGF突变体，其包含氨基酸突变S17R，以及K32E、K32Y、K32M或K32L。The NGF mutant according to claim 49, comprising the amino acid mutation S17R, and K32E, K32Y, K32M or K32L.
  60. 根据权利要求50中所述的NGF突变体，其包含氨基酸突变S19R，以及K32E、K32Y、K32M或K32L。The NGF mutant according to claim 50, comprising the amino acid mutation S19R, and K32E, K32Y, K32M or K32L.
  61. 根据权利要求51中所述的NGF突变体，其包含氨基酸突变S19R和S17E。The NGF mutant according to claim 51, comprising amino acid mutations S19R and S17E.
  62. 根据权利要求52中所述的NGF突变体，其包含氨基酸突变R59K和T83K。The NGF mutant according to claim 52, comprising amino acid mutations R59K and T83K.
  63. 根据权利要求47-62中任一项所述的NGF突变体，其特征在于，所述NGF突变体进一步包含相对于人野生型成熟NGF氨基酸序列的突变F12E。The NGF mutant according to any one of claims 47-62 is characterized in that the NGF mutant further comprises a mutation F12E relative to the human wild-type mature NGF amino acid sequence.
  64. 根据权利要求47-63中任一项所述的NGF突变体，其中所述人野生型成熟NGF的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。The NGF mutant according to any one of claims 47-63, wherein the amino acid sequence of the human wild-type mature NGF is as shown in SEQ ID NO:1 or SEQ ID NO:2.
  65. 根据权利要求47-64中任一项所述的NGF突变体，其特征在于，所述NGF突变体包含SEQ ID NOs:17-60中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:17-60中任一所示的氨基酸序列具有至少约90％序列同源性。The NGF mutant according to any one of claims 47-64 is characterized in that the NGF mutant comprises an amino acid sequence shown in any one of SEQ ID NOs: 17-60 or a variant thereof, and the variant has at least about 90% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 17-60.
  66. 一种包含权利要求47-65中任一项所述的NGF突变体的融合蛋白。A fusion protein comprising the NGF mutant described in any one of claims 47-65.
  67. 根据权利要求66中所述的融合蛋白，其包含Fc；优选地，其中Fc包含能够改变效应功能的突变，和/或能够延长半衰期的突变；更优选地，所述Fc来源于人IgG1或人IgG4。The fusion protein according to claim 66, comprising Fc; preferably, wherein Fc comprises a mutation that can alter effector function and/or a mutation that can extend half-life; more preferably, the Fc is derived from human IgG1 or human IgG4.
  68. 根据权利要求67中所述的融合蛋白，其中所述Fc来源于人IgG1；优选地包含L234A、L235A和P331S突变，其中所述编号为EU编号系统。The fusion protein according to claim 67, wherein the Fc is derived from human IgG1; preferably comprises L234A, L235A and P331S mutations, wherein the numbering is the EU numbering system.
  69. 根据权利要求66-68中任一项所述的融合蛋白，其中所述NGF突变体与Fc连接；优选的，所述NGF突变体与Fc通过肽接头连接；更优选的，所述肽接头由氨基酸残基G和S组成；例如，所述肽接头包含氨基酸序列GGGGSGGGGSGGGGS(SEQ ID NO:87)。The fusion protein according to any one of claims 66-68, wherein the NGF mutant is connected to Fc; preferably, the NGF mutant is connected to Fc via a peptide linker; more preferably, the peptide linker consists of amino acid residues G and S; for example, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:87).
  70. 根据权利要求67-70中任一项所述的融合蛋白，其中所述NGF突变体位于Fc的N端和/或C端。The fusion protein according to any one of claims 67-70, wherein the NGF mutant is located at the N-terminus and/or C-terminus of Fc.
  71. 一种包含NGF突变体的融合蛋白，其特征在于，所述融合蛋白包含SEQ ID NOs:61-82中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:61-82中任一所示的氨基酸序列具有至少80％序列同源性。 A fusion protein comprising an NGF mutant, characterized in that the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs:61-82 or a variant thereof, and the variant has at least 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs:61-82.
  72. 一种编码权利要求47-65中任一项所述的NGF突变体或权利要求66-71中任一项所述的融合蛋白的核酸。A nucleic acid encoding the NGF mutant of any one of claims 47-65 or the fusion protein of any one of claims 66-71.
  73. 一种包含权利要求72中所述核酸的载体。A vector comprising the nucleic acid of claim 72.
  74. 一种包含权利要求73中所述载体的宿主细胞。A host cell comprising the vector of claim 73.
  75. 一种制备NGF突变体或包含NGF突变体的融合蛋白的方法，包括：A method for preparing an NGF mutant or a fusion protein containing an NGF mutant, comprising:
    (a)在能有效表达NGF突变体或包含NGF突变体的融合蛋白的条件下培养权利要求71中所述的宿主细胞；并且(a) culturing the host cell of claim 71 under conditions that effectively express the NGF mutant or a fusion protein comprising the NGF mutant; and
    (b)从宿主细胞中获得所表达的NGF突变体或包含NGF突变体的融合蛋白。(b) Obtaining the expressed NGF mutant or the fusion protein comprising the NGF mutant from the host cell.
  76. 一种药物组合物，其包含权利要求47-65中任一项所述的NGF突变体，权利要求66-71中任一项所述的融合蛋白，权利要求72中所述的核酸，权利要求73中所述的载体，权利要求74中所述的宿主细胞，或应用权利要求75中所述方法制备的NGF突变体或包含NGF突变体的融合蛋白，以及药学上可接受的载体和/或辅料。A pharmaceutical composition comprising the NGF mutant described in any one of claims 47-65, the fusion protein described in any one of claims 66-71, the nucleic acid described in claim 72, the vector described in claim 73, the host cell described in claim 74, or the NGF mutant or the fusion protein comprising the NGF mutant prepared by the method described in claim 75, and a pharmaceutically acceptable carrier and/or excipient.
  77. 一种在有需要的个体中治疗和/或预防与NGF相关的疾病或病症的方法，包括向个体施用权利要求47-65中任一项所述的NGF突变体，权利要求66-71中任一项所述的融合蛋白，权利要求72中所述的核酸、权利要求73中所述的载体、权利要求74中所述的宿主细胞、或应用权利要求75中所述的方法制备的NGF突变体或其融合蛋白或权利要求76中所述的药物组合物。A method for treating and/or preventing a disease or condition associated with NGF in an individual in need thereof, comprising administering to the individual an NGF mutant as described in any one of claims 47-65, a fusion protein as described in any one of claims 66-71, a nucleic acid as described in claim 72, a vector as described in claim 73, a host cell as described in claim 74, or an NGF mutant or its fusion protein prepared by the method as described in claim 75, or a pharmaceutical composition as described in claim 76.
  78. 根据权利要求77中所述的方法，其中所述疾病或病症包括神经系统疾病；优选地，所述神经系统疾病包含新生儿缺氧缺血性脑病、脑瘫、危重症肌病、神经性耳聋、喉返神经损伤、创伤性脑损伤、牙神经损伤、脑卒中、唐氏综合征、肌萎缩性脊髓侧索硬化症，多发性硬化、脊髓性肌萎缩、弥漫性脑损伤、胸腺发育不良、视神经挫伤、滤泡发育不良、脊髓损伤、青光眼、神经营养性角膜炎、视神经损伤、视神经脊髓炎、视网膜相关疾病、尿失禁、阿尔茨海默症、帕金森病、亨廷顿病、痴呆、高血压脑出血神经功能障碍、脑小血管疾病、急性缺血性中风、角膜内皮营养不良、糖尿病神经性病变、糖尿病足溃疡、神经源性皮肤溃疡、压疮、神经营养性角膜溃疡、糖尿病性角膜溃疡和黄斑裂孔。The method according to claim 77, wherein the disease or condition comprises a nervous system disease; preferably, the nervous system disease comprises neonatal hypoxic-ischemic encephalopathy, cerebral palsy, critical illness myopathy, neurological deafness, recurrent laryngeal nerve injury, traumatic brain injury, dental nerve injury, stroke, Down syndrome, amyotrophic 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 diseases, 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 sore, neurotrophic corneal ulcer, diabetic corneal ulcer and macular hole.
  79. 根据权利要求78中所述的方法，其中所述疾病或病症包括非神经系统疾病；优选地，所述非神经系统疾病包含脾萎缩、脾挫伤、卵巢储备功能下降、卵巢早衰、卵巢过度刺激综合征、卵巢残余综合征、卵巢卵泡发育不良、生精障碍(如少精子 症、弱精子症、少弱精子症)、缺血性溃疡、应激性溃疡、类风湿性溃疡、肝纤维化、角膜溃疡、烧伤、口腔溃疡和下肢静脉溃疡。The method according to claim 78, wherein the disease or condition comprises a non-neurological disease; preferably, the non-neurological disease comprises spleen atrophy, spleen contusion, decreased ovarian reserve function, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian remnant syndrome, ovarian follicle dysplasia, spermatogenesis disorder (such as oligospermia), disease, asthenozoospermia, oligoasthenozoospermia), ischemic ulcers, stress ulcers, rheumatoid ulcers, liver fibrosis, corneal ulcers, burns, oral ulcers and venous ulcers of the lower limbs.
  80. 一种药物组合物，其包含抗NGF抗体和NGF突变体或包含NGF突变体的融合蛋白以及药学上可接受的载体和/或辅料，其中所述NGF突变体选自如权利要求47-65中任一项所述的NGF突变体，所述包含NGF突变体的融合蛋白选自如权利要求66-71中任一项所述的融合蛋白。A pharmaceutical composition comprising an anti-NGF antibody and an NGF mutant or a fusion protein comprising an NGF mutant and a pharmaceutically acceptable carrier and/or excipient, wherein the NGF mutant is selected from the NGF mutant described in any one of claims 47 to 65, and the fusion protein comprising an NGF mutant is selected from the fusion protein described in any one of claims 66 to 71.
  81. 一种降低和/或减轻应用抗NGF抗体治疗时的不良反应的方法，包括向已经接受、正在接受或即将接受抗NGF抗体治疗的受试者施用NGF突变体或包含NGF突变体的融合蛋白，其中所述NGF突变体选自如权利要求47-65中任一项所述的NGF突变体，所述包含NGF突变体的融合蛋白选自如权利要求66-71中任一项所述的融合蛋白。A method for reducing and/or alleviating adverse reactions during anti-NGF antibody treatment, comprising administering an NGF mutant or a fusion protein comprising an NGF mutant to a subject who has received, is receiving, or is about to receive anti-NGF antibody treatment, wherein the NGF mutant is selected from the NGF mutant described in any one of claims 47-65, and the fusion protein comprising the NGF mutant is selected from the fusion protein described in any one of claims 66-71.
  82. 根据权利要求81中所述的方法，其中所述不良反应包括神经系统相关的不良反应和关节相关的不良反应。The method according to claim 81, wherein the adverse reactions include nervous system-related adverse reactions and joint-related adverse reactions.
  83. 根据权利要求82中所述的方法，其中所述不良反应包括：交感神经损伤、骨坏死、骨丢失、骨损伤、关节损伤、快速进展性骨关节炎(RPOA)、外周性水肿、关节痛、四肢疼痛、周围神经感觉异常、感觉迟钝、感觉过敏、灼烧痛和触感痛。The method of claim 82, wherein the adverse reactions include: sympathetic nerve damage, osteonecrosis, bone loss, bone damage, joint damage, rapidly progressive osteoarthritis (RPOA), peripheral edema, joint pain, pain in the limbs, peripheral nerve paresthesia, dysesthesia, hyperesthesia, burning pain and tactile pain.
  84. 一种在有需要的个体中治疗和/或预防由NGF表达增加和/或对NGF敏感性增强引起的疾病或病症的方法，包括向个体施用有效量的抗NGF抗体和NGF突变体或包含NGF突变体的融合蛋白，其中所述NGF突变体选自如权利要求47-65中任一项所述的NGF突变体，所述包含NGF突变体的融合蛋白选自如权利要求66-71中任一项所述的融合蛋白，或者施用如权利要求80中所述的药物组合物。A method for treating and/or preventing a disease or condition caused by increased NGF expression and/or enhanced sensitivity to NGF in an individual in need thereof, comprising administering to the individual an effective amount of an anti-NGF antibody and an NGF mutant or a fusion protein comprising an NGF mutant, wherein the NGF mutant is selected from the NGF mutant described in any one of claims 47-65, the fusion protein comprising the NGF mutant is selected from the fusion protein described in any one of claims 66-71, or administering the pharmaceutical composition described in claim 80.
  85. 根据权利要求84中所述的方法，其中抗NGF抗体与NGF突变体或包含NGF突变体的融合蛋白同时施用；或者顺序施用。The method according to claim 84, wherein the anti-NGF antibody and the NGF mutant or the fusion protein comprising the NGF mutant are administered simultaneously; or administered sequentially.
  86. 根据权利要求84或85中所述的方法，其中所述疾病或病症包括炎症性疼痛、术后切口疼痛、神经性疼痛、骨折疼痛、痛风关节疼痛、带状疱疹后神经痛、烧伤引起的疼痛、癌症疼痛、骨关节炎或类风湿性关节炎疼痛、坐骨神经痛、与镰状细胞危象相关的疼痛，或疱疹性神经痛。The method of claim 84 or 85, wherein the disease or condition comprises inflammatory pain, postoperative incisional pain, neuropathic pain, fracture pain, gouty joint pain, postherpetic neuralgia, pain caused by burns, cancer pain, osteoarthritis or rheumatoid arthritis pain, sciatica, pain associated with sickle cell crisis, or herpetic neuralgia.
  87. 根据权利要求80中所述的药物组合物或权利要求81-86中任一项所述的方法，其中所述抗NGF抗体选自说明书表1中所示的任一抗NGF抗体。 According to the pharmaceutical composition described in claim 80 or the method of any one of claims 81-86, the anti-NGF antibody is selected from any anti-NGF antibody shown in Table 1 of the specification.
PCT/CN2023/141728 2022-12-28 2023-12-26 Method for screening and/or identifying mutable site in ngf, and method for screening and/or identifying ngf mutant WO2024140625A1 (en)

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