WO2020248942A1 - NANOCORPS ANTI-β-NGF ET UTILISATION ASSOCIÉE - Google Patents

NANOCORPS ANTI-β-NGF ET UTILISATION ASSOCIÉE Download PDF

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WO2020248942A1
WO2020248942A1 PCT/CN2020/094938 CN2020094938W WO2020248942A1 WO 2020248942 A1 WO2020248942 A1 WO 2020248942A1 CN 2020094938 W CN2020094938 W CN 2020094938W WO 2020248942 A1 WO2020248942 A1 WO 2020248942A1
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nanobody
seq
ngf
antigen
acid sequence
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PCT/CN2020/094938
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Chinese (zh)
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刘红
宋德勇
邢平平
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山东博安生物技术有限公司
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Priority to CN202080000967.3A priority Critical patent/CN113924315B/zh
Publication of WO2020248942A1 publication Critical patent/WO2020248942A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically relates to ⁇ -NGF nanobodies, coding sequences and can be used to treat and/or prevent ⁇ -NGF-related diseases.
  • Nanobody There is an antibody that naturally lacks light chain and only heavy chain in camel's blood, called heavy chain antibody.
  • the variable region of a cloned heavy chain antibody can be obtained from only one heavy chain variable region, with a size of about 12-15KDa, and only one-tenth of the antibody fragments of traditional IgG-type antibodies, which is called Nanobody.
  • Nanobodies have the advantages of strong tissue penetration, stable physical and chemical properties, easy source, high yield, and expansion of culture by microorganisms.
  • Nerve growth factor is a key survival and maintenance factor in the development of peripheral sympathetic neurons and embryonic sensory neurons and the development of basal forebrain cholinergic neurons (Smeyne et al., Nature 368:246-249 (1994) and Crowley et al., Cell 76: 1001-1011 (1994)).
  • NGF up-regulates the expression of neuropeptides in sensory neurons (Lindsay and Harmer, Nature 337: 362-364 (1989)) and its activity passes through two different membrane-bound receptors: TrkA receptor and p75 common neurotrophin receptor ( Sometimes referred to as "high-affinity” and "low-affinity” NGF receptors) mediated respectively.
  • NGF levels In human patients and in some animal models, an increase in NGF levels has been observed to correlate with a variety of inflammatory diseases. These diseases include systemic lupus erythematosus, multiple sclerosis, psoriasis, arthritis, interstitial cystitis and asthma. Likewise, elevated levels of NGF in peripheral tissues are associated with hyperalgesia and inflammation and are observed in many forms of arthritis.
  • the synovium of patients with rheumatoid arthritis expresses high levels of NGF (Aloe, et al., Arch. Rheum. 35:351-355 (1992)).
  • Increased expression of NGF and high-affinity NGF receptor (TrkA) have been observed in human osteoarthritis chondrocytes (Iannone et al., Rheumatology 41:1413-1418 (2002)).
  • Tanezumab (Chinese patent CN102746399B), an antibody against NGF target, is a nerve growth factor IgG2 antibody blocker, which is jointly conducted by Pfizer and Eli Lilly in clinical trials for the treatment of back pain, cancer-related pain and musculoskeletal Pain, etc.
  • OA osteoarthritis
  • NGF-targeting antibodies currently under research are all IgG-type antibodies with a single form, which cannot yet meet the diverse treatment needs of patients for NGF target-related diseases.
  • the invention provides a Nanobody or an antigen-binding fragment thereof.
  • the Nanobody or antigen-binding fragment thereof comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein:
  • amino acid sequence of CDR1 is SEQ ID NO. 3
  • amino acid sequence of CDR2 is SEQ ID NO. 4
  • amino acid sequence of CDR3 is SEQ ID NO. 5; or
  • amino acid sequence of CDR1 is SEQ ID NO. 6
  • amino acid sequence of CDR2 is SEQ ID NO. 7
  • amino acid sequence of CDR3 is SEQ ID NO. 8;
  • amino acid sequence of CDR1 is SEQ ID NO. 9
  • amino acid sequence of CDR2 is SEQ ID NO. 10
  • amino acid sequence of CDR3 is SEQ ID NO. 11.
  • the sequence of the Nanobody is SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 12.
  • the Nanobody is also called a single domain antibody (sdAb), which refers to an antibody containing only a variable heavy chain, and an antigen-binding fragment refers to any antibody fragment capable of binding to a target antigen.
  • sdAb single domain antibody
  • any of the aforementioned Nanobodies or antigen-binding fragments thereof provided by the present invention can bind to ⁇ -NGF antigen, preferably to human ⁇ -NGF antigen.
  • the Nanobody against ⁇ -NGF antigen provided by the present invention includes a framework region (FR) and a complementarity determining region (CDR).
  • FR framework region
  • CDR complementarity determining region
  • the present invention also provides a multivalent Nanobody comprising any of the aforementioned Nanobodies or antigen-binding fragments thereof.
  • the multivalent Nanobody of the present invention refers to containing two or more Nanobodies.
  • the multivalent Nanobody provided by the present invention includes any of the following Nanobodies or antigen-binding fragments thereof:
  • amino acid sequence of CDR1 is SEQ ID NO. 3
  • amino acid sequence of CDR2 is SEQ ID NO. 4
  • amino acid sequence of CDR3 is the Nanobody or antigen-binding fragment thereof of SEQ ID NO. 5; or
  • the amino acid sequence of CDR1 is SEQ ID NO. 6, the amino acid sequence of CDR2 is SEQ ID NO. 7, and the amino acid sequence of CDR3 is SEQ ID NO. 8 Nanobody or its antigen-binding fragment; or
  • amino acid sequence of CDR1 is SEQ ID NO. 9
  • amino acid sequence of CDR2 is SEQ ID NO. 10
  • amino acid sequence of CDR3 is the Nanobody or antigen binding fragment thereof of SEQ ID NO. 11.
  • the multivalent Nanobody provided by the present invention contains one or more Nanobody whose amino acid sequence is SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 12 or an antigen-binding fragment thereof.
  • any one of the aforementioned multivalent Nanobodies also contains an anti-HSA nanobody (anti-HSA sdAb), and preferably the sequence of the anti-human serum albumin nanobody is SEQ ID NO.13.
  • the Nanobodies may be connected through a linker or directly connected, and the direct connection includes but is not limited to covalent connection.
  • the Nanobodies are connected by a linker.
  • the linker in any of the aforementioned multivalent Nanobodies is an amino acid sequence, preferably the amino acid sequence is AAA, GGGGSGGGS or GGGGSGGGGSGGGGS, more preferably GGGGSGGGS.
  • the multivalent Nanobody provided by the present invention may have the following structure: the first anti- ⁇ -NGF nanobody—linker—anti-human serum albumin nanobody—linker—the second anti- ⁇ -NGF nanobody Antibody.
  • the amino acid sequences of the first and second anti- ⁇ -NGF Nanobodies are selected from SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 12, respectively; preferably the first and second anti- ⁇ -NGF antibodies
  • the amino acid sequence of the ⁇ -NGF Nanobody is SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 12; more preferably, the amino acid sequence of the first and second anti- ⁇ -NGF Nanobodies are both SEQ ID NO.1.
  • the multivalent Nanobody provided by the present invention has a structure of SEQ ID NO. 1—GGGGSGGGS—SEQ ID NO. 13—GGGGSGGGS—SEQ ID NO.1.
  • any of the aforementioned multivalent Nanobodies provided by the present invention can bind ⁇ -NGF antigen, preferably human ⁇ -NGF antigen.
  • the present invention also provides a nucleic acid encoding any of the aforementioned Nanobodies or antigen-binding fragments or multivalent Nanobodies, the Nanobodies or antigen-binding fragments or multivalent Nanobodies comprising:
  • amino acid sequence of CDR1 is SEQ ID NO. 3
  • amino acid sequence of CDR2 is SEQ ID NO. 4
  • amino acid sequence of CDR3 is the Nanobody or antigen-binding fragment thereof of SEQ ID NO. 5; or
  • the amino acid sequence of CDR1 is SEQ ID NO. 6, the amino acid sequence of CDR2 is SEQ ID NO. 7, and the amino acid sequence of CDR3 is SEQ ID NO. 8 Nanobody or its antigen-binding fragment; or
  • amino acid sequence of CDR1 is SEQ ID NO.9
  • amino acid sequence of CDR2 is SEQ ID NO.10
  • amino acid sequence of CDR3 is the Nanobody or antigen-binding fragment thereof of SEQ ID NO.11; or
  • Nanobodies or antigen-binding fragments thereof whose amino acid sequence is SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 12.
  • nucleic acid encoded by the term “nucleic acid” may include encoding nucleic acid, or may also include additional encoding and/or non-encoding sequences.
  • the nucleic acid of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes but is not limited to cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the nucleic acid encoding the Nanobody or its antigen-binding fragment of the present invention includes, but is not limited to, a coding sequence that only encodes the structure of a mature Nanobody or an antigen-binding fragment thereof; a coding sequence that encodes a mature Nanobody or an antigen-binding fragment thereof and various additional codes Sequence; coding sequence (and optional additional coding sequence) and non-coding sequence that encode the structure of mature Nanobody or its antigen-binding fragment.
  • the present invention also relates to nucleic acids that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to nucleic acids that can hybridize to the nucleic acid of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%. Moreover, the polypeptide encoded by the hybridizable nucleic acid has the same biological function and activity as the mature polypeptide.
  • the full-length nucleic acid sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method. Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, Nanobodies and antigen-binding fragments thereof, multivalent Nanobodies, etc.) involved in the present invention include biomolecules in an isolated form.
  • DNA sequences encoding Nanobodies, antigen-binding fragments thereof, multivalent Nanobodies, etc. of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the sequences of the Nanobodies, antigen-binding fragments, multivalent Nanobodies and the like of the present invention through chemical synthesis.
  • the present invention also provides a vector containing nucleic acid encoding any one of the above-mentioned Nanobodies or antigen-binding fragments or multivalent Nanobodies.
  • the vectors include, but are not limited to: viral vectors, such as adenovirus vectors, retroviral vectors, and adeno-associated virus vectors; non-viral vectors, such as plasmids and transposon vectors.
  • the vector is an expression vector.
  • the vector is preferably a plasmid vector, and more preferably a pPICZ ⁇ A vector.
  • the present invention also provides cells for expressing any of the aforementioned Nanobodies or antigen-binding fragments or multivalent Nanobodies, which cells contain encoding any of the aforementioned Nanobodies or antigen-binding fragments or multivalent Nanobodies
  • the cell is a host cell containing the above-mentioned expression vector or nucleic acid.
  • the host cell includes but is not limited to mammalian cells, insect cells, plant cells, fungal cells, prokaryotic cells, and the like.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • the host cell provided by the present invention for expressing the antibody or antigen-binding fragment thereof that binds to the NGF antigen is a Pichia cell.
  • Transformation of host cells with DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • Nanobodies and their antigen-binding fragments, multivalent Nanobodies, etc. in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, its physical, chemical and other characteristics can be used to separate and purify Nanobodies and their antigen-binding fragments, multivalent Nanobodies, etc. through various separation methods. These methods are well known to those skilled in the art.
  • Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • a pharmaceutical composition which comprises the Nanobody or antigen-binding fragment thereof, multivalent Nanobody, nucleic acid, carrier or cell according to any one of the preceding aspects, preferably, the drug
  • the composition also includes a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient includes one or more of the following: pharmaceutically acceptable solvents, dispersants, additives, plastics Shape agent and so on.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium.
  • the formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
  • the present invention also relates to a kit comprising any of the above-mentioned Nanobodies or antibody fragments or multivalent Nanobodies or nucleic acids.
  • the kit further includes a detection reagent, a negative control, and a positive control for detecting ⁇ -NGF antibody reaction.
  • the nanobody of the present invention has a wide range of biological application value and clinical application value, and its application involves the diagnosis and treatment of ⁇ -NGF-related diseases, basic medical research, biological research and other fields.
  • a preferred application is for clinical diagnosis and targeted therapy for ⁇ -NGF.
  • the present invention relates to the use of the Nanobody or antigen-binding fragment, multivalent Nanobody, nucleic acid, carrier or cell of any one of the foregoing aspects in the preparation of drugs for preventing or treating diseases.
  • the present invention relates to the use of the Nanobody or its antigen-binding fragment, multivalent Nanobody, and nucleic acid of any one of the foregoing aspects in the preparation of a diagnostic or inspection kit.
  • the present invention provides a method for preventing or treating diseases, comprising combining the Nanobody or antigen-binding fragment thereof, multivalent Nanobody, nucleic acid, carrier, cell or drug of the present invention in any of the foregoing aspects.
  • the composition is administered to a subject in need.
  • the present invention provides a method for diagnosing or detecting diseases, comprising administering the Nanobody or antigen-binding fragment, multivalent Nanobody, nucleic acid or kit of the present invention to a subject in need or sample.
  • the present invention provides the use of the Nanobody or antigen-binding fragment, multivalent Nanobody, nucleic acid, carrier, cell or pharmaceutical composition of any one of the foregoing aspects for the treatment and prevention of diseases.
  • the present invention provides the use of the Nanobody or antigen-binding fragment, multivalent Nanobody, nucleic acid or kit of any one of the foregoing aspects for detecting or diagnosing diseases.
  • the disease is preferably a ⁇ -NGF-related disease; more preferably pain or inflammation; more preferably postoperative pain, rheumatoid arthritis pain, and osteoarthritis pain.
  • the Nanobody or antigen-binding fragment or multivalent Nanobody provided by the present invention has one or more of the following advantages: the effect of blocking the binding of ⁇ -NGF and TrkA is better, the immunogenicity is low, and the body The half-life is long, and the pain relief/inhibition effect is significant.
  • Figure 1A shows the serum titers of 8TTT and 2389 alpaca after four immunizations (2500 times dilution)
  • Figure 1B shows the serum titer after the 4520 alpaca three immunizations (1000 times dilution)
  • Figure 2 shows the activity of the candidate antibody and ⁇ -NGF protein detected by Elisa
  • Figure 3A shows that the candidate antibody blocks the binding of ⁇ -NGF to TrkA (Method 1)
  • Figure 3B shows that the candidate antibody blocks the binding of ⁇ -NGF to TrkA (Method 2)
  • Figure 4 shows that candidate antibodies block ⁇ -NGF to promote TF-1 cell growth
  • Figure 5 shows the detection of the binding of humanized antibody to ⁇ -NGF protein by Elisa
  • Figure 6 shows that candidate antibodies block the binding of ⁇ -NGF to TrkA
  • Figure 7 shows a schematic diagram of the structure of a multivalent Nanobody
  • Figure 8 shows Pichia pastoris expressing multivalent Nanobody electrophoresis
  • Figure 9 shows the average drug-time curve of each group of cynomolgus monkeys given different antibodies
  • Figure 10 shows the weight of the right hind limbs of each group of animals at different times
  • Recombinant ⁇ -NGF protein (Yiqiao Shenzhou, 11050-HNAC) was used to immunize alpaca with adjuvant.
  • the first immunization was complete Freund's adjuvant
  • the second immunization was incomplete Freund's adjuvant. This time I immunize 3 alpacas, 3-5 times.
  • blood was taken to measure the titer, and the alpaca with higher serum titer was selected for neck blood collection, and lymphocytes were separated for subsequent experiments.
  • the blood titer detected by Elisa method is shown in Figure 1A-B.
  • Nanobody immune library refers to the method of Els Pardon et al. (nature protocols, VOL 9 NO. 3, 2014).
  • the phage libraries established by the alpaca numbered 8TTT/4520/2389 are NGF-8TTT, NGF-4520, and NGF-2389, and the storage capacities are 5*10 8 , 1.4*10 9 , 3.2*10 8 respectively .
  • the clones that specifically bind to ⁇ -NGF are screened by the following two methods:
  • Plate screening Use ⁇ -NGF-His protein (Yiqiao Shenzhou, 11050-HNAC) to coat the plate at 0.2 ⁇ g/well, place overnight at 4°C, block the plate with 2% BSA for 1h the next day, and add the phage library ( 2*10 12 ) Incubate for 2 hours, wash for 4-10 times, and elute the phage that specifically binds to ⁇ -NGF with Elution Buffer (pH 2.2).
  • Magnetic bead screening The ⁇ -NGF protein (Yiqiao Shenzhou, 11050-HNAC) is biotinylated according to conventional procedures, and then combined with Thermo magnetic beads (Invitrogen Dynabeads M-280 Streptavidin, 00355871) and incubated with the phage library , Thereby obtaining a clone that specifically binds to ⁇ -NGF.
  • the binding method of ELISA is as follows:
  • the library that was positive by the phage enzyme-linked immunoassay (Elisa) method was coated on a plate, and a single clone was picked to directly induce expression by IPTG, and the periplasm was extracted for periplasmic detection.
  • the method is as follows: Coat the antigen with a concentration of 0.2 ⁇ g/mL with pH 9.6 CBS ⁇ -NGF (Yiqiao Shenzhou, 11050-HNAC), 100 ⁇ l/well at 4°C overnight; block with 3% skimmed milk powder at 37°C for 1h; add 1 ⁇ g/ml candidate antibody diluted with PBST (PBS+0.05% Tween20) to each well 100 ⁇ l each Incubate at 37°C for 1h; then add Goat pAb to Llama HRP (abcam, ab112786), incubate at 37°C for 1h, develop color for 10 minutes, and read OD450 on the microplate reader.
  • ELISA blocking uses two methods. Among them, antibodies from NGF-4520 are screened by method one, and antibodies against NGF-8TTT, NGF-4520, and NGF-2389 are screened by method two.
  • the specific methods are as follows:
  • Method 1 pH 9.6 CBS coated ⁇ -NGF (Yiqiao Shenzhou, 11050-HNAC) with a concentration of 5 ⁇ g/mL, 100 ⁇ l/well at 4°C overnight; block with 3% skimmed milk powder for 1h, add 50 ⁇ l 2.5 ⁇ g/ml to each well
  • Candidate antibodies of different concentrations 200 ⁇ g/mL, 50 ⁇ g/mL, 12.5 ⁇ g/mL, 0 ⁇ g/mL
  • TrkA-Fc abcam, ab83562
  • PBST PBS+0.05% Tween20
  • Method 2 pH 9.6 CBS coated TRKA (Yiqiao Shenzhou, 11073-H03H) with a concentration of 0.5 ⁇ g/mL, 100 ⁇ l/well 4°C overnight; blocked with 3% skimmed milk powder for 1h; at the same time NGF-Fc-biotin (0.2 ⁇ g /mL) and PBST (PBS+0.05% Tween20) diluted with different concentrations (4 ⁇ g/mL, 1 ⁇ g/mL, 0.25 ⁇ g/mL, 0.0625 ⁇ g/mL, 0.015625 ⁇ g/mL, 0.0039063 ⁇ g/mL) candidate antibodies at 37°C total Incubate for 1 hour, then add to the blocked ELISA plate, and incubate at 37°C for 1 hour; then add streptomycin/HRP and incubate at 37°C for 1 hour; after color development for 10 minutes, read OD450 on the microplate reader.
  • Transform the above cloned plasmid into BL21 competent cells (TAKARA, 9126), select a single clone for culture and induce expression by IPTG, use the permeation method to obtain a crude antibody extract, and prepare a purity of more than 90% by nickel column ion affinity chromatography Nanobody, and determine the antibody concentration by UV280 binding extinction coefficient.
  • Control antibody production According to IMGT data and patent CN 102746399B, the amino acid sequences of the light and heavy chains of Pfizer antibody Tanezumab are as follows. After complete gene synthesis, it was constructed to pCDNA-3.4 and expressed in HEK293 cells.
  • Tanezumab heavy chain sequence (A Tanezumab heavy chain sequence:
  • Tanezumab light chain sequence
  • the purified candidate antibody is tested for its binding activity to determine its binding sensitivity to ⁇ -NGF protein.
  • This test is mainly for the candidate antibody NGF-8TTT-BA22 ⁇ BA81 ⁇ BA85 ⁇ BA91 ⁇ BA94 (hereinafter referred to as BA22 ⁇ BA81 ⁇ BA85 ⁇ BA91 ⁇ BA94), NGF-2389-CA27 ⁇ CA56 ⁇ BA15 ⁇ BA20 ⁇ BA42 ⁇ BA176 ⁇ BA177 (hereinafter referred to as CA27 ⁇ CA56 ⁇ BA15 ⁇ BA20 ⁇ BA42 ⁇ BA176 ⁇ BA177), where 8TTT/2389 represents the library established by alpaca numbers 8TTT and 2389 respectively, CA represents plate screening, and BA represents magnetic bead screening.
  • the method for detecting the binding activity of the candidate antibody to ⁇ -NGF protein by Elisa refers to Example 1.3.
  • the results are shown in Figure 2.
  • the 12 antibodies have similar binding activities.
  • the purified candidate antibody was tested for the activity of blocking the binding of ⁇ -NGF and TrkA, and the method was referred to Example 1.3.
  • Method 2 detects the candidate antibody BA22 ⁇ BA81 ⁇ BA85 ⁇ BA91 ⁇ BA94 ⁇ CA27 ⁇ CA56 ⁇ BA15 ⁇ BA20 ⁇ BA42 ⁇ BA176 ⁇ BA177.
  • concentrations we use multiple concentrations in Method 2
  • four-parameter fitting is performed on the concentration of the antibody to be tested and the absorbance value to obtain an inverse "S"-shaped curve.
  • the IC50 value of the curve is used to evaluate the blocking ability of the antibody.
  • the results are shown in Figure 3B and Table 2. The results show that all 12 strains of antibodies can effectively block the binding of ⁇ -NGF and TrkA.
  • Candidate antibodies block ⁇ -NGF to promote TF-1 cell growth
  • the TF-1 cell line is a human premyeloid cell line, which can be stimulated by exogenous growth factors and cytokines to proliferate.
  • TF-1 cells can express human TrkA receptor and proliferate in response to activation by NGF. Therefore, we used the TF-1 cell proliferation test to further test the in vitro function of the candidate antibody.
  • the method is as follows: Dilute the antibody with detection medium (90% RPMI 1640, 10% FBS) to a total of 4-6 concentrations. Dilute NGF (Yiqiao Shenzhou, 11050-HNAC) to 50ng/mL with detection medium. After mixing the diluted antibody and diluted NGF 1:1, let stand at 37°C for 30 min.
  • CA66, CA30, BA22, BA81, BA176, BA177 can significantly block ⁇ -NGF, thereby inhibiting the growth of TF-1 cells, and its blocking ability is similar to the control antibody Tanezumab.
  • the linker sequence is as follows: GGGGSGGGS
  • NGF-BA22.20 The sequences of NGF-BA22.20, NGF-BA176.10.9, and NGF-CA66.20 were selected to construct multivalent Nanobodies, respectively referred to as N22.20 (the structure is BA22.20-linker-anti-HSA sdAb-linker-BA22. 20), N176.10.9 (structure is BA176.10.9-joint-anti-HSA sdAb-joint-BA176.10.9), N66.20 (structure is CA66.20-joint-anti-HSA sdAb-joint-CA66.20) .
  • the whole gene was synthesized and constructed on the pPICZ ⁇ A vector, the plasmid was linearized with SalI restriction endonuclease and electrotransformed into Pichia pastoris cells, cultured in YPD medium for 22 hours, and methanol was added to induce expression for 3 to 5 days.
  • the expression supernatant was passed through Protein A affinity chromatography, and the antibody concentration was determined by UV280 binding extinction coefficient. The purity of the expression supernatant was detected by SDS-PAGE protein electrophoresis.
  • the method is as follows: Pipette 1mL fermentation broth into a 1.5mL centrifuge tube and centrifuge at 12000rpm for 2min; take 20 ⁇ L of the supernatant and add 20 ⁇ L of 10 ⁇ non-reducing Loading buffer, heat at 80°C for 5min, use 8% -16% polyacrylamide gel for electrophoresis of the sample.
  • the results of electrophoresis are shown in Figure 8. The results show that the purity of the N22.20, N176.10.9, and N66.20 antibodies in the yeast supernatant is relatively high, which is convenient for subsequent large-scale purification.
  • Example 6 ⁇ -NGF antibody cross-reacts with proteins of the same family
  • Tanezumab has a half-life of 8.6 days in cynomolgus monkeys, N176.10.9 has a half-life of 8.4 days, N22.20 has a half-life of 6.2 days, and N66.20 has a half-life of 10 days. Therefore, N66.20 has a longer half-life, can prolong the administration period, and has a longer-lasting therapeutic effect.
  • the specific experimental method is as follows: Coating Tanezumab, N22.20, N66.20, N176.10.9 with CBS coating solution (pH 9.6 carbonic acid solution) at 0.125 ⁇ g/ml, 0.25 ⁇ g/ml, 100 ⁇ l/well at 4 degrees overnight ; Blocked with 3% skimmed milk powder at 37°C for 1h; added 100 ⁇ serum, 100 ⁇ l to each well, incubated at 37°C for 1h; then added Tanezumab-biotin, N66.20-biotin, N22.20-biotin, N176.10.9-biotin, respectively 0.125 ⁇ g/ml, 0.25 ⁇ g/ml, 0.25 ⁇ g/ml, incubate at 37°C for 1h.

Abstract

L'invention concerne un nanocorps ou un nanocorps polyvalent lié à un antigène β-NGF qui peut être utilisé pour traiter, prévenir, détecter ou diagnostiquer des états liés au β-NGF, tels que la douleur ou l'inflammation, et qui est en particulier utilisé pour la douleur après une chirurgie, la douleur de la polyarthrite rhumatoïde et la douleur de l'arthrose.
PCT/CN2020/094938 2019-06-10 2020-06-08 NANOCORPS ANTI-β-NGF ET UTILISATION ASSOCIÉE WO2020248942A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112961244A (zh) * 2021-02-28 2021-06-15 杭州熙源生物技术有限公司 抗TrkA抗体或其抗原结合片段、其制备方法和应用
CN116063532A (zh) * 2021-12-29 2023-05-05 华道(上海)生物医药有限公司 结合抗间皮素的抗体及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004058184A2 (fr) * 2002-12-24 2004-07-15 Rinat Neuroscience Corp. Anticorps anti-ngf et procedes d'utilisation de ces anticorps
WO2009023540A1 (fr) * 2007-08-10 2009-02-19 Regeneron Pharmaceuticals, Inc. Anticorps humains anti-facteur de croissance nerveux humain de haute affinité
WO2010128398A1 (fr) * 2009-05-04 2010-11-11 Pangenetics 110 B.V. Anticorps dirigés contre le facteur de croissance nerveux (ngf) dotés d'une meilleure stabilité in vivo
WO2011049758A1 (fr) * 2009-10-09 2011-04-28 Amgen Inc. Anticorps neutralisants anti-ngf humain en tant qu'inhibiteurs sélectifs de la voie du ngf
WO2012075340A2 (fr) * 2010-12-01 2012-06-07 Alderbio Holdings Llc Compositions anti-ngf et leur utilisation
WO2013184871A1 (fr) * 2012-06-06 2013-12-12 Zoetis Llc Anticorps anti-ngf caninisés et procédés associés

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7569364B2 (en) * 2002-12-24 2009-08-04 Pfizer Inc. Anti-NGF antibodies and methods using same
MX2007008722A (es) * 2005-01-24 2008-02-15 Cambridge Antibody Tech Elementos de aglutinacion especifica para el factor de crecimiento nervioso.
US9067988B2 (en) * 2010-12-01 2015-06-30 Alderbio Holdings Llc Methods of preventing or treating pain using anti-NGF antibodies
CN108178798B (zh) * 2016-12-08 2021-03-30 苏州方德门达新药开发有限公司 pH工程化的NGF抗体及其医药用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004058184A2 (fr) * 2002-12-24 2004-07-15 Rinat Neuroscience Corp. Anticorps anti-ngf et procedes d'utilisation de ces anticorps
WO2009023540A1 (fr) * 2007-08-10 2009-02-19 Regeneron Pharmaceuticals, Inc. Anticorps humains anti-facteur de croissance nerveux humain de haute affinité
WO2010128398A1 (fr) * 2009-05-04 2010-11-11 Pangenetics 110 B.V. Anticorps dirigés contre le facteur de croissance nerveux (ngf) dotés d'une meilleure stabilité in vivo
WO2011049758A1 (fr) * 2009-10-09 2011-04-28 Amgen Inc. Anticorps neutralisants anti-ngf humain en tant qu'inhibiteurs sélectifs de la voie du ngf
WO2012075340A2 (fr) * 2010-12-01 2012-06-07 Alderbio Holdings Llc Compositions anti-ngf et leur utilisation
WO2013184871A1 (fr) * 2012-06-06 2013-12-12 Zoetis Llc Anticorps anti-ngf caninisés et procédés associés

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112961244A (zh) * 2021-02-28 2021-06-15 杭州熙源生物技术有限公司 抗TrkA抗体或其抗原结合片段、其制备方法和应用
CN112961244B (zh) * 2021-02-28 2023-08-29 熙源安健医药（上海）有限公司 抗TrkA抗体或其抗原结合片段、其制备方法和应用
CN116063532A (zh) * 2021-12-29 2023-05-05 华道(上海)生物医药有限公司 结合抗间皮素的抗体及其应用
CN116063532B (zh) * 2021-12-29 2023-11-07 华道(上海)生物医药有限公司 结合抗间皮素的抗体及其应用

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