WO2018157710A1 - Nav1.9的靶点多肽、与其结合的抗体及抗体片段和相关药物组合物 - Google Patents

Nav1.9的靶点多肽、与其结合的抗体及抗体片段和相关药物组合物 Download PDF

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WO2018157710A1
WO2018157710A1 PCT/CN2018/075597 CN2018075597W WO2018157710A1 WO 2018157710 A1 WO2018157710 A1 WO 2018157710A1 CN 2018075597 W CN2018075597 W CN 2018075597W WO 2018157710 A1 WO2018157710 A1 WO 2018157710A1
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antibody
seq
antibody fragment
fragment
variable region
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杨代常
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武汉大学
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Priority to US16/657,471 priority patent/US11174312B2/en

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    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a target (polypeptide) of Nav1.9, an antibody and/or an antibody fragment which specifically recognizes the above target (polypeptide), and a pharmaceutical composition comprising the above antibody and/or antibody fragment for treating pain and itching And coughing.
  • DRG dorsal root ganglia
  • the generation and conduction of action potentials in neurons depends on voltage-gated sodium channels (VGSCs) located on the cell membrane.
  • VGSCs voltage-gated sodium channels
  • the sodium ion channel When the cell membrane is depolarized, the sodium ion channel is activated, the channel is opened, causing sodium ion influx, and the cell membrane is further depolarized, resulting in the generation of an action potential, which causes pain due to an abnormal action point. Therefore, inhibition of abnormal sodium ion channel activity contributes to the treatment and alleviation of pain.
  • Voltage-gated sodium channels are widely found in cell membranes such as neurons and skeletal muscle cells. They are a class of transmembrane glycoprotein complexes composed of an alpha subunit and several beta subunits.
  • the ⁇ subunit is a functional carrier of sodium ion channel, which is composed of 1700-2000 amino acids, forming 4 domains (Domain, I-IV), and each domain contains 6 transmembrane segments (S1 ⁇ S6). (figure 1).
  • the domains are connected by some large intracellular loops, and the fragments are connected by small extracellular or intracellular loops.
  • S4 is rich in basic amino acid residues and is considered to be a voltage-sensitive component of voltage-gated sodium ion channels.
  • the positive charge on S4 can move clockwise along the axis of S4, changing the sodium ion channel conformation and opening the channel.
  • the pore loop (P-loop) between S5 and S6 forms the outside of the micropore, which is related to the selectivity to sodium ions, and the intracellular portion of the micropores is surrounded by S6.
  • the intracellular loop of the linking domains III and IV acts as an inactivation valve that folds into the intracellular opening of the microwell, blocks the micropore, and inactivates the voltage-gated sodium ion channel. Mutations in the L2 intimal region of domain II can result in a phenotype that loses pain (Nature Genetics, 2013, 45(11): 1399-1404).
  • Nav1 Nav1.1 ⁇ Nav1.9
  • PNS peripheral nervous system
  • SCN11A SCN11A
  • the activation voltage of Nav1.9 is close to the resting membrane potential of neurons (-60 ⁇ -70mV), and has the dynamic characteristics of slow activation and slow deactivation, so it can produce a longer-lasting TTX-R current, which indicates Nav1. 9 can amplify and prolong the response of neurons to threshold-down polarization, triggering action potentials.
  • the activation voltage of Nav1.9 is -80mV.
  • the 811 amino acid mutation in Nav1.9 has produced painless symptoms. Further research on the gene indicates that the gene is one of the sodium ion channels mainly responsible for pain.
  • a first object of the present invention is to provide a voltage sensor paddle region of domain III in Nav1.9 as a target, and to obtain a monoclonal antibody by using a target design polypeptide of the region as an antigen, and binding to a target by a specific antibody
  • the VGSCs ion channel is turned off to suppress pain.
  • the polypeptide has the sequence set forth in SEQ ID NO: 9, and may also be a structurally similar derivative having 80%, 80-85%, 85-90%, 90-95% or 95-99% homology to the polypeptide. sequence.
  • a second object of the present invention is to provide an antibody and an antibody fragment thereof which specifically recognize a target, wherein the heavy chain variable region (VH) has the sequence shown in SEQ ID NO: 7, or is SEQ ID NO:
  • the sequence shown has 80%, 80-85%, 85-90%, 90-95% or 95-99% homologous structurally similar derived sequences
  • its light chain variable region (VL) has SEQ ID NO: a sequence shown by 8, or a sequence similar to the structure shown in SEQ ID NO: 8 having 80%, 80-85%, 85-90%, 90-95% or 95-99% homology
  • the three CDR sequences contained in the heavy chain variable region (SEQ ID NO: 7) and/or the three CDR sequences contained in the light chain variable region (SEQ ID NO: 8) can be optimized to obtain CDR inlays.
  • the heavy chain variable region comprises a CDR sequence of CDRH1 as shown in SEQ ID NO. 1, CDRH2 as shown in SEQ ID NO. 2, and CDRH3 as shown in SEQ ID NO. 3;
  • the CDR sequences contained in the variable region are, for example, the CDRL1 shown in SEQ ID NO. 7, the CDRL2 as shown in SEQ ID NO. 5, and the CDRL3 as shown in SEQ ID NO.
  • the antibody and its antibody fragment light chain constant region may be selected from a kappa chain or a lambda chain, and the heavy chain constant region thereof may be selected from IgM, IgD, IgG I-4, IgA, IgE, and the like;
  • the light chain constant region and the heavy chain constant region species source may be selected from the group consisting of: human antibody constant region, bovine antibody constant region, sheep antibody constant region, canine antibody constant region, porcine antibody constant region, cat source Antibody constant region, equine antibody constant region, and sputum antibody constant region.
  • the antibody and its antibody fragment may be in the form of: whole antibody, Fab, F(ab') 2 , dsFv, scFv, double-stranded antibody, minibody, bispecific antibody, multispecific antibody, chimeric antibody, CDR graft antibody .
  • the antibody and antibody fragment thereof are preferably monoclonal antibodies or antibody fragments.
  • the antibody is a humanized monoclonal antibody.
  • a third object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned antibody or an antibody fragment thereof as an active ingredient, which has analgesic effect and an increased pain threshold effect, can treat pain, Itching and coughing.
  • the voltage sensor paddle region of the domain III of the voltage sensor valve of Nav1.9 is used to screen a polypeptide of a suitable target region as an antigen, which is hydrophilic and For antigenic analysis, a polypeptide with high hydrophilicity and high antigenicity was selected, and its amino acid sequence was DVEFSGEDNAQRIT (SEQ ID NO. 9).
  • the first step is to prepare a monoclonal cell line secreting the antibody.
  • the above polypeptide is chemically synthesized, and the synthetic polypeptide is numbered as C2363BB030-1 (SEQ ID NO. 9), which is conjugated to the carrier protein KLH, and then immunized to the BALB/c mouse, and the immune stimulating body is repeatedly inoculated to generate an immune response.
  • Polyclonal antibodies were produced, blood sampling tests, ELISA tests and evaluations.
  • the polyclonal antibody titer produced by the immunized animal was evaluated by ELISA, and the two animals that met the requirements were finally determined according to the antibody titer of the immunized animal and the specificity of the human neural tissue #1942, #1943 for cell fusion.
  • the spleen cells of two animals were subjected to cell electrofusion with mouse myeloma cells (SP2/0), fused and cultured, and the positive cell lines were screened on the screening medium for antibody titer and tissue specificity.
  • the detection was carried out, and the hybridoma cell strain was screened by using the polypeptide C2363BB030-1 as an antigen, and the positive cell strain was selected for subcloning according to the ELISA detection result. After the obtained subclones are subjected to ELISA detection and specific detection again, subclones which are specific and positive for nerve tissues are selected and cryopreserved.
  • the second step is to sequence the variable region of the native antibody, extract the total RNA of the cell strain, synthesize the cDNA, establish a cDNA library, and perform variable region sequencing.
  • Amplification of a polynucleotide sequence encoding a variable region of an antibody which can integrate DNA sequences encoding VH and VL (which can also be manipulated by RNA sequences encoding variable regions) into the same vector, or integrate them into a vector, respectively.
  • the appropriate host cell is transfected with the vector described above; it is then subjected to sequencing analysis. The sequencing result showed that the DNA sequence of VH was as shown in SEQ ID NO: 10, and the DNA sequence of VL was as shown in SEQ ID NO: 11.
  • the third step is to construct a genetically engineered antibody, and according to different needs, the above-mentioned DNA sequences encoding VH and VL (which may also be CDRs encoding VH and encoding CDRs in VL) are introduced into a suitable host for antibody expression, and the antibody effect is verified.
  • VH and VL which may also be CDRs encoding VH and encoding CDRs in VL
  • the third aspect of the present invention is to detect the biological activity of a monoclonal antibody, firstly expressing the human Nav1.9 gene in ND7/23 cells, extracting total protein from the transiently expressed ND7/23 cell line, and analyzing the antibody by Western Blotting method.
  • the specificity of binding as shown in Figure 4, specifically recognizes the Nav1.9 protein.
  • the second step was to use mice to induce acute inflammatory pain with 5% formalin.
  • the appropriate amount of antibody was injected into the tail vein to detect the effect of antibody on pain relief in pain model mice. The results are shown in Figure 5. After injection of 10 mg/kg of antibody, it had a significant analgesic effect compared to the control.
  • Figure 1 Structure of the sodium ion channel Nav1.9
  • FIG. 4 Western blotting immunogenicity analysis of monoclonal antibodies
  • Figure 5 Analgesic effect of 51H10D12 antibody on 5% formalin-induced acute inflammatory pain in wild-type mice
  • the hydrophilicity and antigenicity analysis were carried out, and the DVEFSGEDNAQRIT sequence was screened.
  • the hydrophilicity and antigenicity were in accordance with the requirements of the antigen, and a fully automatic synthesizer was used.
  • the DVEFSGEDNAQRIT (SEQ ID NO. 9) polypeptide was artificially synthesized.
  • the cleavage reagent is: trifluoroacetic acid + ethanedithiol + phenol + thioanisole + water;
  • cysteine may be added to the end of the polypeptide.
  • the coating antigen is the polypeptide C2363BB030-1; the coating solution is 1*PBS (pH 7.4); the washing buffer: 1*PBS (pH 7.4), 0.05% PBS; the anti-serum is used after the primary antibody is 3; Standard secondary antibody: Peroxidase-AffiniPure Goat Anti-Mouse IgG, Fc ⁇ Fragment Specific (min X Hu, Bov, HrsSr Prot); TMB coloring solution; Stop solution: 1 M hydrochloric acid.
  • Coating The antigen was diluted to 1 ⁇ g/ml with a coating solution, and 100 ⁇ l per well was mixed and added to the strip, covered with a cover film, and left at 4 ° C overnight.
  • Termination reaction The stop solution was added to terminate the reaction.
  • NC is a negative control of unimmunized serum, and the initial dilution factor is 1:1,000.
  • the antiserum after three immunizations was tested the antiserum titer of animal number #1940 was 1:128,000; the antiserum titer of animal number #1939 was 1:512,000; the remaining 3 animals (#1941,# 1942, #1943) anti-serum titer at 1:256,000;
  • Example 2.2 According to the ELISA test results of Example 2.2, combined with the results of tissue specificity, two animals #1942, #1943 were selected for the final elimination, and the spleen cells and tumor cells of the two animals were fused three days later, and the mouse myeloma cells ( SP2/0) and spleen cells were subjected to cell fusion by electrofusion in a ratio of 1:3, and the fused cells were plated into 15 feeder cells using HAT medium, and cultured in a CO 2 incubator.
  • the whole plate was replaced with the culture medium, and the test was carried out by ELISA after 4 hours of liquid exchange.
  • the ELISA specific materials and procedures were identical to the 2.2 animal serum ELISA assay.
  • a total of 15 (numbered from plate 41 to plate 55) 96-well plates, with wells 55H12 (wells H12 of plate 55) set as positive controls, plus 1:1000 dilutions of fused animal serum, the OD value was determined to be 2.215; Well 55G12 (well G12 of plate 55) was set as a negative control, blank medium was added, and the OD value was determined to be 0.088; clones with OD > 0.5 were found.
  • the clones with OD>0.5 of the 68 single-well cells screened for the first time were subjected to the second test (the detection method is the same as above), and the results of the experiment are shown in Table 3. From the 10 clones with the OD value greater than 1.0, 41C12, 47A11, 42A3, 42H10, 48D6, 51H10, 52F11, 52H5, 55E9, 55H7 .
  • the positive selection test was carried out from the selected 10 strains of the second screening (the detection method is the same as above), and the experimental results are shown in Table 4.
  • the above 10 cell lines were expanded into 24-well plates, and 2 ml of supernatant was collected per plant for the next step. confirm.
  • the human nerve tissue is taken for dehydration treatment, and the dehydration treatment is performed by Leica ASP300S.
  • the specific process is as follows:
  • anhydrous ethanol was dehydrated for 30 minutes each time; then dehydrated twice with anhydrous ethanol for 60 minutes each time; then treated with clearing agent for 30 minutes, then treated with clearing agent twice, each time After 60 minutes; treatment with paraffin for 3 times, respectively, 60 minutes, 120 minutes and 180 minutes, using a Leica EG1150 embedding machine for embedding operation, making a wax block, and slicing, the slice thickness was 4 ⁇ m.
  • the nerve tissue sections were baked at 85 ° C for 20 min; the dewaxing agent was treated 3 times for 1 minute each time; the dewaxing was performed 3 times with anhydrous alcohol for 1 minute each time; the water was washed 3 times for 1 minute each time; ER2 (PH) was used.
  • Example 2.3.3 42H10, 51H10, 52F11, 55E9 were selected for subcloning. Four cells were subcloned by limiting dilution method, and the four cells were plated into 96-well feeder cells. After 7-10 days of culture, 12 monoclonal clones were selected for ELISA detection (detection method is the same as above).
  • 51H10D9, 51H10D12, 51H10F8 were selected for the second subcloning (subcloning method is the same as above). After 7-10 days of culture, 12 monoclonal wells were selected for ELISA, and the results showed positive, indicating that these were homozygous singles. clone. Five sub-clone clones were randomly selected, and the titer and subclass of supernatant titers and subclasses were identified (kit Southern Biotech, Cat. No. ST17). The experimental results showed that the OD values of the five strains were consistent, the titer was 1:2430, and the subtype was IgG2b, K type. Finally, 51H10D12 was selected for cell cryopreservation.
  • Antibody fragments of VH and VL were amplified according to the standard operating procedure (SOP) method of GenScript's rapid amplification of cDNA ends (RACE). The amplified antibody fragment was cloned into a standard cloning vector, respectively. Colony PCR was performed to screen for clones with inserts of the correct size. At least 5 colonies with the correct size inserts were sequenced. The sequences of the different clones were aligned and the consensus sequences of these clones were determined.
  • the DNA sequence of VH is thus determined as shown in SEQ ID NO: 10; the DNA sequence of VL is determined as shown in SEQ ID NO:11.
  • amino acid sequence is deduced from the DNA sequences of VH and VL, the VH amino acid sequence is shown in SEQ ID NO: 7, and the VL amino acid sequence is shown in SEQ ID NO: 8.
  • RNA sequence encoding VH is presumed to be as shown in SEQ ID NO: 12, and the RNA sequence encoding VL is shown in SEQ ID NO: 13.
  • the human Nav1.9-HA plasmid (7-8 ⁇ g) was transfected into ND7/23 cells (10 cm cell dishes), cultured at 37 ° C for 10 hours, and then collected in a cell dish and cultured at 29 ° C for 20 hours to collect the cells.
  • the cell lysate was centrifuged at 12000 rpm for 10 minutes at 4 ° C, and the cell lysis supernatant was collected.
  • the cells were incubated with HA tag (1:2000) antibody and 51H10D12 (1:300) antibody for 1 hour at room temperature.
  • the 51H10D12 antibody and the HA tag can detect the hybridization signal between 130-250KD, and the corresponding signal is not detected in the negative control, and the hybridization signal is close to the molecular weight of hNav1.9 of 210kDa, indicating that This antibody recognizes the protein of hNav1.9.

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Abstract

提供了一种抗体或其片段，可特异性结合电压门控钠离子通道Nav1.9α亚基第Ⅲ结构域电压传感器浆的S3-4环，能失活电压传感器阀门，使钠离子不能正常进入神经细胞。还提供了与所述抗体或其片段特异性结合的表位多肽、包含所述抗体或其片段的药物组合物以及所述抗体或其片段在制备治疗疼痛相关疾病中的用途。

Description

Nav1.9的靶点多肽、与其结合的抗体及抗体片段和相关药物组合物 技术领域
本发明涉及Nav1.9的靶点(多肽)，特异性识别上述靶点(多肽)的抗体和/或抗体片段，以及包括上述抗体和/或抗体片段的药物组合物，用于治疗疼痛、瘙痒和咳嗽。
背景技术
疼痛起源于周围神经系统的伤害感受器，而作为一种游离的神经末梢，广泛分布于全身的皮肤、肌肉、关节和内脏组织中，它可以将感受到热的、机械的或化学刺激转化为动作电位，通过神经纤维传递到其位于背根神经节(dorsal root ganglia,DRG)的胞体部分，最终传递到高级神经中枢，从而引起痛觉。而神经元中动作电位的产生和传导又依赖于位于细胞膜上的电压门控钠离子通道(voltage-gated sodium channels,VGSCs)。当细胞膜去极化时，钠离子通道激活，通道打开，引起钠离子内流，使细胞膜进一步去极化，导致动作电位的产生，由于异常的动作点位而产生疼痛感。因此，抑制异常的钠离子通道活动有助于疼痛的治疗、缓解。
电压门控钠离子通道广泛存在于如神经元、骨骼肌细胞的细胞膜上，是一类跨膜糖蛋白复合体，由一个α亚基和数个β亚基构成。其中α亚基是钠离子通道的功能载体，由1700～2000个氨基酸组成，形成了4个结构域(Domain，Ⅰ-Ⅳ)，每个结构域又包含6个跨膜片段(S1～S6)(图1)。结构域之间由一些大的胞内环连接起来，而各片段之间则由一些小的胞外或胞内环相连接。其中，S4上含有丰富的碱性氨基酸残基，被认为是电压门控钠离子通道的电压敏感元件。当细胞膜去极化时，S4上的正电荷可以沿着S4的轴线做顺时针旋转往外迁移，改变钠离子通道构象，使通道打开。S5和S6之间的发夹环(pore loop，P-loop)形成了微孔外部，与对钠离子的选择性有关，微孔的胞内部分则由S6围成。连接结构域Ⅲ和Ⅳ的胞内环起着失活阀门的作用，它可以折叠进入微孔的胞内开口，阻塞微孔，使电压门控钠离子通道失活。在结构域II的L2内膜区域进行突变，可以造成失去疼痛的表型(Nature Genetics，2013，45(11)：1399－1404)。
根据不同可以对进行分类，目前，已经在哺乳动物中鉴定了9种电压门控钠离子通道α亚基，由于其氨基酸序列相似度均大于50％。因此，被认为来自同一家族，命名位Nav1(Nav1.1～Nav1.9)。实验表明它们在神经元中大量特异性表达。其中Nav1.9存在于周围神经系统(peripheral nervous system,PNS)。最近研究表明Nav1与疼痛相关的亚型主要是Nav1.3、Nav1.7、Nav1.8和Nav1.9。其中Nav1.9是主要负责疼痛的重要成员。Nav1.9为TTX-R型，编码基因为SCN11A，主要分布在感受伤害的DRG神经元、三叉神经节和肠肌层神经元。Nav1.9的激活电压接近神经元的静息膜电位(-60～-70mV)，具有缓慢激活、缓慢失活的动力学特征，因此可以产生持续较久的TTX-R电流，这表明Nav1.9可以放大和延长神经元对阈下去极化的反应，引发动作电位。在人体内，Nav1.9的激活电压为-80mV。最近在人体无疼痛的病人中发生在Nav1.9的811位氨基酸突变后，产生了不痛的症状，对该基因进一步研究表明，该基因是主要负责疼痛的钠离子通道之一。
但是临床上普遍应用化学小分子作用(如卡马西平、利多卡因、美西律等)为电压门控钠离子通道抑制剂治疗疼痛，但是它们对电压门控钠离子通道亚型缺乏足够的选择性，因而会产生心脏毒性和中枢神经副作用的弱点。
发明内容
本发明的第一个目的是提供Nav1.9中结构域Ⅲ的电压传感器桨区域作为靶点，利用该区域的靶点设计多肽作为抗原获得单克隆抗体，通过特异性抗体与其靶点结合，可以关闭VGSCs离子通道，从而抑制疼痛。所述多肽具有SEQ ID NO:9所示的序列，也可以是与该多肽具有80％，80～85％，85～90％,90～95％或95～99％同源的结构类似的衍生序列。
本发明的第二个目的是提供特异性识别靶点的抗体及其抗体片段，其重链可变区(VH)具有如SEQ ID NO:7所示的序列，或者与SEQ ID NO:7所示的序列具有80％，80～85％，85～90％,90～95％或95～99％同源的结构类似的衍生序列；其轻链可变区(VL)具有如SEQ ID NO:8所示的序列，或者与SEQ ID NO:8所示的序列具有80％，80～85％，85～90％,90～95％或95～99％同源的结构类似的衍生序列；还可以将重链可变区(SEQ ID NO:7)所含有的3个CDR序列和/或轻链可变区(SEQ ID NO:8)所包含的3个CDR序列，进行优化，获得CDR嵌合 抗体及其抗体片段。所述重链可变区包含的CDR序列为，如SEQ ID NO.1所示的CDRH1、如SEQ ID NO.2所示的CDRH2和如SEQ ID NO.3所示的CDRH3；所述轻链可变区包含的CDR序列为，如SEQ ID NO.7所示的CDRL1、如SEQ ID NO.5所示的CDRL2和如SEQ ID NO.6所示的CDRL3。
所述抗体及其抗体片段轻链恒定区可以选自于κ链或λ链，其重链恒定区可以选自于IgM、IgD、IgG I-4、IgA、IgE等等；
所述轻链恒定区和重链恒定区物种来源可以选自于：人源抗体恒定区、牛源抗体恒定区、羊源抗体恒定区、犬源抗体恒定区、猪源抗体恒定区、猫源抗体恒定区、马源抗体恒定区、驴源抗体恒定区。
所述抗体及其抗体片段结构形式可以是：全抗体、Fab、F(ab’) 2、dsFv、scFv、双链抗体、微抗体、双特异抗体、多特异抗体、嵌合抗体、CDR移植抗体。
所述抗体及其抗体片段优选为单克隆抗体或抗体片段。
所述抗体更优选为人源化单克隆抗体。
本发明的第三个目的是提供一种药物组合物，所述药物组合物含有上述的抗体或其抗体片段作为活性成份，所述药物组合物有镇痛和提高疼痛阈值效果，能治疗疼痛、瘙痒和咳嗽。
根据本发明的第一个方面，根据Nav1.9的晶体结构模型，在Nav1.9的电压传感器阀门的结构域III的电压传感器桨区域筛选合适靶点区域的多肽作为抗原，经过亲水性和抗原性的分析，选择一个具有亲水性好、抗原性高的多肽，其氨基酸序列为DVEFSGEDNAQRIT(SEQ ID NO.9)。
根据本发明的第二个方面，第一步是制备分泌抗体的单克隆细胞株。
化学合成上述多肽，将该合成多肽编号为C2363BB030-1(SEQ ID NO.9)，将其偶联到载体蛋白KLH上，然后免疫BALB/c小鼠，多次接种免疫刺激机体产生免疫应答从而产生多克隆抗体，采血测试、ELISA检测和评价。
通过抗原抗体反应，通过ELISA来评价免疫动物产生的多克隆抗体效价，根据免疫的动物抗体效价和人神经组织的特异性，最终确定符合要求的两只动物#1942,#1943进行细胞融合，取两只动物的脾细胞与小鼠骨髓瘤细胞(SP2/0)进行细胞电融合，融合后进行细胞培养，在筛选培养基上筛选阳性细胞株，对分泌的抗体效价和组织特异性进行检测，利用多肽C2363BB030-1作为抗原筛选 杂交瘤细胞株，根据ELISA检测结果，选择阳性细胞株进行亚克隆。所得亚克隆再次进行ELISA检测和特异性检测后，选择对神经组织具有特异性和阳性的亚克隆，进行细胞冻存。
第二步是对天然抗体的可变区测序，提取细胞株总RNA，合成cDNA，建立cDNA文库，进行可变区测序。扩增编码抗体可变区的多核苷酸序列，可将编码VH和VL的DNA序列(也可用编码可变区的RNA序列来操作)整合到同一个载体，也可将它们分别整合到载体上，用上述载体转染合适的宿主细胞；然后对其进行测序分析。测序结果显示，其VH的DNA序列如SEQ ID NO:10所示，其VL的DNA序列如SEQ ID NO:11所示。
第三步是构建基因工程抗体，根据不同需要，将上述编码VH和VL(也可以是编码VH中CDR和编码VL中CDR)的DNA序列导入合适的宿主进行抗体表达，并验证抗体效果。
本发明的第三个方面是检测单克隆抗体的生物活性，首先在ND7/23细胞瞬时表达人Nav1.9基因，从瞬时表达ND7/23细胞系提取总蛋白质，采用Western Blotting方法，分析该抗体结合的特异性，如图4所示，该抗体能特异性识别Nav1.9蛋白。
第二步是采用小鼠用5％福尔马林诱导急性炎症疼痛造模，经尾静脉注射适量抗体，检测抗体对疼痛模型小鼠镇痛的效果。结果如图5所示，注射10mg/kg抗体后，比对照具有显著的镇痛作用。
有益效果：采用抗体这种生物大分子针对Nav1.9电压门控钠离子通道的电压传感器的靶向的特异性结合，来失活电压传感器阀门，导致不能正常钠离子进入神经细胞，从而达到治疗和缓解疼痛的效果，由于而其具有很好的靶向性，可以克服由于化学小分子药物产生的副作用。
附图说明
图1：钠离子通道Nav1.9的结构图
图2：钠离子通道Nav1.9靶点设计图
图3：单克隆抗体的人神经组织特异性分析
A、42H10；B、51H10；C、52F11；D、55E9
图4：单克隆抗体的Western blotting免疫原性分析
图5：51H10D12抗体对野生型小鼠5％福尔马林诱导的急性炎症疼痛的镇痛效果
具体实施方式
下面以下通过对本发明较佳实施方式的详细描述，说明但不限制本发明。
材料来源：
下述使用的材料和试剂如无特别说明均为商业购买。
实施例1【抗原合成】
根据Nav1.9的氨基酸序列(GenBank No.NP_001274152)和晶体结构模型，进行亲水性和抗原性的分析，筛选出DVEFSGEDNAQRIT序列，其亲水性和抗原性符合抗原的要求，采用全自动合成仪人工合成了DVEFSGEDNAQRIT(SEQ ID NO.9)多肽。
具体步骤如下：
(1)将第一个AA的-COOH用DIEA连接到Cl-Resin,然后将树脂上未反应完的功能团用MeOH封闭；
(2)用DMF洗涤；
(3)用Pip去除第一个AA中-NH 2的保护基团Fmoc，使-NH 2裸露；
(4)用DMF洗涤；
(5)将第二个AA的-COOH用DIC+HOBT活化，然后缩合至第一个AA中的-NH 2上，形成酰胺键；
(6)用DMF洗涤；
(7)用Pip去除第二个AA中-NH 2的保护基团Fmoc，使-NH 2裸露；
(8)用DMF洗涤；
(9)…重复5-8直到最后一个AA的-NH 2裸露；
(10)将多肽从树脂上切割，并切除所有氨基酸的侧链保护基团，切割试剂为：三氟乙酸+乙二硫醇+苯酚+茴香硫醚+水；
(11)将切割液加入乙醚中，使多肽沉淀，离心即得到多肽粗品(C2363BB030-1)；
(12)多肽HPLC的C18制备/分析柱进行纯化，编号为C2363BB030-1，获得纯化的多肽用于免疫动物。
注：为方便多肽偶联，可在此多肽末端额外添加半胱氨酸。
实施例2【单克隆细胞株制备】
2.1 动物免疫
准备弗氏完全佐剂Sigma,F5881和弗氏不完全佐剂(Sigma,F5506)。利用多肽C2363BB030-1的末端-SH将多肽偶联到载体蛋白KLH上，作为免疫原。
选取5只8周龄的雌性BALB/c(动物编号：#1939,#1940,#1941,#1942,#1943)进行3次腹腔免疫，刺激机体产生免疫应答从而产生抗体。初免：50μg/只，三周后进行二次免疫，剂量为50μg/只；在第二次免疫后的2周后进行第三次免疫，剂量为50μg/只，在第三次免疫的1周后进行采血进行抗体检测。
2.2 动物血清ELISA检测
2.2.1 仪器与设备：
洗板机：北京楠华ZDMX
酶标仪：Thermo MultiskanAscent
2.2.2 使用的试剂：
包被抗原为多肽C2363BB030-1；包被液为1*PBS(pH7.4)；洗涤缓冲液：1*PBS(pH7.4)，0.05％PBS；采用一抗为3免后抗血清；酶标二抗：Peroxidase-AffiniPure Goat Anti-Mouse IgG,Fcγ Fragment Specific(min X Hu,Bov,HrsSr Prot)；TMB显色液；终止液：1M盐酸。
具体方法如下：
(1)包被：用包被液将抗原稀释成1μg/ml，混匀后每孔100μl加入板条中，盖上盖板膜，放置4℃过夜。
(2)封闭：取出板子弃去包被液，加入封闭液，盖上盖板膜，37℃恒温箱0.5h。
(3)加一抗：3免抗血清首孔1/1000稀释，然后倍比稀释9个梯度，盖上盖板膜，37℃恒温箱1h。
(4)加二抗：取出酶标板，弃去内液，加入稀释后的酶标二抗，浓度为0.033μg/ml，盖上盖板膜，37℃恒温箱半小时。
(5)显色：取出酶标板，弃去内液，加入显色液，25℃显色13min。
(6)终止反应:加入终止液，终止反应。
(7)加入终止液后，即刻在酶标仪上450nm读数，将OD值大于设定的阴性对照OD值的2.1倍的孔对应的最大稀释度，定为该样品的效价，检测结果如表2所示，NC是未免疫血清的阴性对照，起始稀释倍数是1:1,000。经对三次免疫后的抗血清进行检测，动物编号为#1940的抗血清滴度在1:128,000；动物编号为#1939的抗血清滴度在1:512,000；其余3只动物(#1941,#1942,#1943)的抗血清滴度在1:256,000；
表2.第三次免疫后的血清ELISA检测结果:
动物编号 稀释倍数 No.1939 No.1940 No.1941 No.1942 No.1943
阴性对照 1:1,000 0.087 0.090 0.093 0.143 0.101
稀释1 1:1,000 2.798 2.350 2.404 2.546 2.706
稀释2 1:2,000 2.607 1.980 2.036 2.147 2.339
稀释3 1:4,000 2.557 1.750 1.970 1.830 1.986
稀释4 1:8,000 2.264 1.473 1.522 1.381 1.463
稀释5 1:16,000 1.908 0.956 1.105 0.953 1.024
稀释6 1:32,000 1.453 0.754 0.725 0.595 0.659
稀释7 1:64,000 1.071 0.427 0.513 0.366 0.407
稀释8 1:128,000 0.625 0.274 0.278 0.216 0.261
稀释9 1:256,000 0.368 0.138 0.194 0.164 0.175
稀释10 1:512,000 0.229 0.106 0.128 0.109 0.146
稀释11 空白对照 0.077 0.077 0.077 0.077 0.077
滴度 1:512,000 1:128,000 1:256,000 1:256,000 1:256,000
2.3 细胞融合及杂交瘤细胞株筛选
2.3.1 细胞融合：
根据实施例2.2的ELISA检测结果，结合组织特异性的结果，选择两只动物#1942,#1943进行终免，三天后取该两只动物的脾细胞和瘤细胞融合，小鼠骨髓瘤细胞(SP2/0)与脾脏细胞按照1：3比例，利用电融合的方式进行细胞融合，将融合后的细胞用HAT培养基铺入到15块饲养细胞板中，放入CO 2培养箱培养。
2.3.2 杂交瘤细胞株筛选:
融合后的细胞培养7-10d后，进行整板更换培养液，在换液4h后采用ELISA进行检测。
ELISA具体材料和步骤与2.2动物血清ELISA检测相同。
第一次ELISA筛选：
一共有15个(编号从板41到板55)96孔板，以孔55H12(板55的孔H12)设置为阳性对照，加入融合动物血清1：1000稀释液，其OD值检测为2.215；将孔55G12(板55的孔G12)设置为阴性对照，加入空白培养基，其OD值检测为0.088；找出OD>0.5的克隆。
第二次ELISA筛选：
将第一次筛选的68个单孔细胞的OD>0.5的克隆进行第二次检测(检测方法同上)，实验结果结果如表3所示，从中找出OD值大于1.0的10株克隆，即 41C12、47A11、42A3、42H10、48D6、51H10、52F11、52H5、55E9、55H7。
表3.杂交瘤细胞的第二次ELISA筛选
1 2 3 4 5 6 7 8 9 10 11 12
41C12 41F4 42A3 42D8 42H10 43B1 43B7 44D1 46A6 47A11 48B12 48D6
1.942 0.748 0.966 0.660 1.254 0.524 0.638 0.540 0.629 1.102 0.791 1.362
50C6 51D10 51H9 51H10 51H11 52B4 52C5 52F6 52F11 52H5 52H6 53D9
0.564 0.459 0.834 1.962 0.084 0.588 0.713 0.864 1.118 1.777 0.109 0.808
53E2 55E9 55G5 55H2 55H7 NC PC
0.558 1.294 0.757 0.716 1.148 0.084 1.985
第三次ELISA筛选：
从第二次筛选的选择的10株进行阳性确认检测(检测方法同上)，实验结果如表4所示，将上述10个细胞株扩大到24孔板中，每株收集2ml上清进行下一步确认。
表4杂交瘤细胞的第三次ELISA筛选
1 2 3 4 5 6 7 8 9 10 11 12
41C12 47A11 42A3 42H10 48D6 51H10 52F11 52H5 55E9 55H7 PC NC
2.259 1.454 1.379 1.562 1.574 1.833 1.326 1.748 1.385 1.325 2.025 0.072
2.3.3.细胞学特异性确认
为了确认这些细胞株的是否具有神经组织的特异性，对2.3.2杂交瘤细胞株 筛选过程中第三次ELISA筛选的上清进行免疫组织化学检查。具体实验方法如下：
2.3.3.1组织脱水处理：
取人神经组织进行脱水处理，脱水处理采用徕卡ASP300S，具体流程如下：
采用70％、85％、90％，无水乙醇分别脱水各30分钟；再用无水乙醇脱水2次，每次60分钟；然后透明剂处理30分钟，再用透明剂处理2次，每次60分钟；再用石蜡处理3次，分别为60分钟、120分钟和180分钟，采用徕卡EG1150包埋机进行包埋操作，制作成蜡块，进行切片，切片厚度为4μm。
2.3.3.2原位杂交：
取神经组织切片在85℃烤片20min；脱蜡剂处理3次，每次1分钟；用无水酒精脱蜡3次，每次1分钟；水洗3次，每次1分钟；采用ER2(PH＝9缓冲溶液)热修复20分钟，冷却12分钟，再用水洗3次，每次1分钟；然后封闭30分钟；水洗3次，每次1分钟；加入细胞株的上清孵育30分钟，水洗3次，每次1分钟；采用增强剂孵育8分钟，水洗3次，每次2分钟，加入二抗孵育8分钟；水清洗3次，每次2分钟；DAB显色8分钟；水洗3次，每次1分钟，苏木素染色10分钟；水洗3次，每次1分钟，酒精脱水后风干封片。在奥林巴斯光学显微镜下进行观察。
经光学显微镜观察，42H10,51H10,52F11,55E9细胞株分泌抗体在神经组织的特异性和杂交信号均达到要求。
2.3.4 亚克隆
根据实施例2.3.3的细胞学检测结果，选择42H10,51H10,52F11,55E9进行亚克隆。利用有限稀释法对四株细胞进行亚克隆，将这四株细胞铺入96孔饲养细胞板中，培养7-10d后，每株挑选了12个单克隆进行ELISA检测(检测方法同上)，结果显示，42H10E3、42H10E4、42H10F3、42H10G4、51H10D9、51H10D12、51H10E11、51H10F8、52F11B2、52F11H3、52F11H6、52F11H11、55E9A4、55E9C6、55E9D9、55E9E3等16个细胞株为阳性，其余均为阴性。选择这些阳性细胞株进行第二次确认，经确认，其OD值均较高(见表5)。然后将这些阳性单克隆扩大到24孔板中，每株收集2ml上清进行细胞学确认。经组织免疫化学再次确认，51H10细胞株为免疫组化阳性(图3)。
表5.阳性亚克隆的第二次ELISA检测确认
Figure PCTCN2018075597-appb-000001
选择 51H10D9,51H10D12,51H10F8进行第二次亚克隆(亚克隆方法同上)，培养7-10d后，挑选12个单克隆孔进行ELISA检测，结果显示均为为阳性，表明这些已经是纯合的单克隆。随机选择5株子克隆，对上清液效价和亚类进行效价检测与亚类鉴定(试剂盒Southern Biotech，货号ST17)。实验结果显示5株OD值一致，效价在1：2430，亚型为IgG2b，K型。最终选择 51H10D12进行细胞冻存。
实施例3【抗体测序】
为了确定单克隆抗体序列，选取其中一个单克隆 51H10D12进行测序。按照TRIzol试剂的技术手册从杂交瘤细胞中分离总RNA。然后使用同种型特异性反义引物或通用引物将总RNA逆转录成cDNA，遵循PrimeScript TM第一链cDNA合成试剂盒技术手册。根据GenScript的快速扩增cDNA末端(RACE)的标准操作程序(SOP)方法，扩增VH和VL的抗体片段。分别将扩增的抗体片段克隆入标准克隆载体。进行菌落PCR以筛选具有正确大小的插入片段的克隆。至少对5个具有正确大小的插入片段的菌落进行测序。比对不同克隆的序列，确定这些克隆的共有序列。
由此确定VH的DNA序列，其如SEQ ID NO:10所示；确定VL的DNA序列，其如SEQ ID NO:11所示。
引导序列-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-恒定区-终止密码
Figure PCTCN2018075597-appb-000002
引导序列
Figure PCTCN2018075597-appb-000003
CDR1
Figure PCTCN2018075597-appb-000004
CDR2
Figure PCTCN2018075597-appb-000005
CDR3
Figure PCTCN2018075597-appb-000006
恒定区
Figure PCTCN2018075597-appb-000007
Figure PCTCN2018075597-appb-000008
TGA(SEQ ID NO:10)
引导序列-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-恒定区-终止密码
Figure PCTCN2018075597-appb-000009
引导序列
Figure PCTCN2018075597-appb-000010
CDR1
Figure PCTCN2018075597-appb-000011
CDR2
Figure PCTCN2018075597-appb-000012
Figure PCTCN2018075597-appb-000013
CDR3
Figure PCTCN2018075597-appb-000014
AGTCGTGTGCTTCTTGA
恒定区
Figure PCTCN2018075597-appb-000015
根据VH和VL的DNA序列推导出氨基酸序列，VH氨基酸序列如SEQ ID NO:7所示，VL氨基酸序列如SEQ ID NO:8所示。
引导序列-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-恒定区
Figure PCTCN2018075597-appb-000016
引导序列         CDR1
Figure PCTCN2018075597-appb-000017
CDR2               CDR3
Figure PCTCN2018075597-appb-000018
恒定区
Figure PCTCN2018075597-appb-000019
Figure PCTCN2018075597-appb-000020
(SEQ ID NO:7)
引导序列-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-恒定区
Figure PCTCN2018075597-appb-000021
引导序列           CDR1
Figure PCTCN2018075597-appb-000022
CDR2               CDR3
Figure PCTCN2018075597-appb-000023
恒定区
Figure PCTCN2018075597-appb-000024
推测出编码VH的RNA序列如SEQ ID NO:12所示，编码VL的RNA序列如SEQ ID NO:13所示。
实施例4【抗体的特异性检测】
4.1 hNav1.9抗原的制备
将人Nav1.9-HA质粒(7-8μg)转染ND7/23细胞(10cm细胞皿)中，37℃先培养10小时，然后分到细胞皿中在29℃培养20小时后收集细胞。
4.2 Westernblotting分析
1.吸去细胞培养基后加入2ml灭菌PBS轻轻旋转细胞皿，洗涤细胞两次。
2.加入1mlWestern及IP细胞裂解液，用细胞刮刮取收集细胞置于冰上裂解半小时。
3.用超声破碎仪，超声破碎2次，每次3s。
4.细胞裂解液在4℃下经12000rpm离心10分钟，收集细胞裂解上清。
5.取细胞裂解液上清40微升，经8％的聚丙烯酰胺凝胶电泳。
6.经转膜后，分别用HA tag(1:2000)抗体和51H10D12(1:300)抗体在室温下进行孵育1小时。
7.经PBS清洗三次，每次5分钟；
8.加入地高辛标记的抗鼠抗体抗体(1：20000)在室温先孵育1小时；
9.经PBS清洗三次，每次5分钟；
10.加上地高辛底物，显影5－10分钟。
11.带有进行Western blot杂交。
4.3 Western杂交结果
从图4中可以看出，51H10D12抗体与HA标签可以检测到在130-250KD之间杂交信号，在阴性对照中没有检测到对应的信号，其杂交信号与hNav1.9的分子量210kDa左右接近，说明该抗体能识别hNav1.9的蛋白。
实施例5【51H10D12抗体在野生型小鼠体内镇痛效果】
为了检测51H10D12抗体是否具有镇痛效应，我们采用小鼠5％福尔马林的诱导炎症型痛痛，在静脉注射该抗体后，给予刺激，每5分钟内记录小鼠舔爪和缩爪的时间，评价不同处理对福尔马林诱发的自发炎性疼痛的镇痛效果。
5.1 实验步骤
按照Lee等(2014，Cell 157,1393–1404)的方法进行，取12只野生型小鼠，经过2天的适应后进行试验。随机分为2组，一组为对照组，在尾部静脉注射PBS，一组为实验组，在尾静脉注射10mg/kg的51H10D12抗体，半小时后，后爪皮下注射20μL 5％福尔马林引起急性炎症引起的疼痛，通过每5分钟内记录小鼠舔爪和缩爪的时间，总共45分钟，分别统计分析I相(0-10分钟)和II相(10-45分钟)，I相为急性痛，II相为自发的持续性痛，实验后统计分析这两相以及各时间段内野生型小鼠注射药物与对照组之间的差异。
5.2 实验结果
从上图5可知，野生型小鼠尾静脉注射51H10D12抗体后，经5％福尔马林诱导急性炎症疼痛，该抗体可以减少后爪皮下注射5％Formalin后10-15分钟和30-35分钟内舔爪的总时间，即II相小鼠舔爪的总时间，与阴性对照相比达到显著差异。实验结果表明51H10D12抗体可以缓解野生型小鼠5％Formalin诱导的II相炎性疼痛，与文献报道的效果相当。 序列清单：
Figure PCTCN2018075597-appb-000025
Figure PCTCN2018075597-appb-000026
Figure PCTCN2018075597-appb-000027
Figure PCTCN2018075597-appb-000028
Figure PCTCN2018075597-appb-000029
Figure PCTCN2018075597-appb-000030

Claims (11)

  1. 一种特异结合细胞膜电压门控钠离子通道α亚基Nav1.9的抗体或其抗体片段。
  2. 根据权利要求1所述的抗体或其抗体片段，其特征在于与所述抗体或抗体片段特异结合的靶点为电压门控钠离子通道α亚基的第Ⅲ结构域的电压传感器桨的S3-4环。
  3. 根据权利要求1所述的抗体或其抗体片段，其特征在于：所述抗体或其抗体片段的重链可变区包含的CDR序列为，如SEQ ID NO.1所示的CDRH1、如SEQ ID NO.2所示的CDRH2和如SEQ ID NO.3所示的CDRH3；并且/或
    所述抗体或其抗体片段的轻链可变区包含的CDR序列为，如SEQ ID NO.4所示的CDRL1、如SEQ ID NO.5所示的CDRL2和如SEQ ID NO.6所示的CDRL3。
  4. 根据权利要求3所述的抗体或其抗体片段，其特征在于所述抗体或抗体片段含有如SEQ ID NO.7所示的重链可变区和/或如SEQ ID NO.8所示的轻链可变区。
  5. 根据权利要求1所述的抗体或其抗体片段，其特征在于所述抗体进一步包含抗体恒定区。
  6. 根据权利要求1所述的抗体或其抗体片段，其特征在于所述抗体或抗体片段选自如下结构形式：全抗体、Fab、F(ab’) 2、dsFv、scFv、双链抗体、微抗体、双特异抗体、多特异抗体、嵌合抗体和CDR移植抗体。
  7. 根据权利要求1所述的抗体或其抗体片段，其特征在于所述抗体为单克隆抗体。
  8. 根据权利要求7所述的抗体或其抗体片段，其特征在于所述抗体为人源化抗体。
  9. 一种与权利要求1所述的抗体或其抗体片段特异结合的多肽，其特征在于所述多肽具有如SEQ ID NO.9所示的氨基酸序列。
  10. 一种药物组合物，含有权利要求1～8任一项所述的抗体或其抗体片段。
  11. 权利要求1～8任一项所述的抗体或其抗体片段在制备治疗疼痛相关疾病的药物中的用途。
PCT/CN2018/075597 2017-03-03 2018-02-07 Nav1.9的靶点多肽、与其结合的抗体及抗体片段和相关药物组合物 WO2018157710A1 (zh)

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