WO2018153262A1 - 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 PDFInfo
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Definitions
- the present invention relates to a target polypeptide of Nav1.9, an antibody and/or antibody fragment which binds to the above polypeptide, and a pharmaceutical composition comprising the above antibody and/or antibody fragment for the treatment of pain, itching and cough.
- 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 II in Nav1.9 as a target, obtain a monoclonal antibody by using the polypeptide as an antigen, and close the VGSCs ion channel by binding the specific antibody to its target. Thereby inhibiting pain.
- the polypeptide has the sequence set forth in SEQ ID NO: 1, and may also be a structurally similar derivative sequence having 80%, 85%, 90% or 95% homology to the polypeptide.
- a second object of the present invention is to provide an antibody and an antibody fragment thereof which specifically recognize a target having a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 2 or having 80% of the polypeptide. 85%, 90% or 95% homologous structurally similar derived sequence; its light chain variable region (VL) has the sequence set forth in SEQ ID NO: 3, or 80%, 85%, 90 with the polypeptide % or 95% homologous structurally similar derived sequence; three CDR sequences and/or light chain variable regions (SEQ ID NO: 3) contained in the heavy chain variable region (SEQ ID NO: 2) can also be removed The three CDR sequences contained were transplanted to obtain CDR-grafted antibodies and antibody fragments thereof.
- VH heavy chain variable region
- VL light chain variable region
- the heavy chain variable region comprises a CDR sequence of CDRH1 as set forth in SEQ ID NO. 4, CDRH2 as set forth in SEQ ID NO. 5, and CDRH3 as set forth in SEQ ID NO.
- 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. 8, 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, 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 antibodies or antibody fragments in monoclonal form.
- 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.
- a suitable polypeptide is screened as an antigen in the voltage sensor paddle region of domain II of the voltage sensor valve of Nav1.9, via hydrophilicity and antigenicity.
- a polypeptide having high hydrophilicity and high antigenicity was selected, and its amino acid sequence was LNSFSNEERNGNLE (SEQ ID NO. 1).
- the first step is to prepare a monoclonal cell line secreting the antibody.
- the polyclonal antibody titer of the immunized animal was evaluated by ELISA, and according to the antibody titer of the immunized animal and the specificity of the nerve tissue, two animals #1955, #1958 were finally determined to perform cell fusion, and two were taken.
- the spleen cells of the animal were electrofused with the mouse myeloma cells (SP2/0), and the cells were cultured after fusion, and the positive cell lines were screened on the screening medium to detect the titer and tissue specificity of the secreted antibody.
- the hybridoma cell line was screened by using the polypeptide C8079BB030-1 as an antigen, and according to the ELISA test results, 5A2, 9D2, and 15F5 were subcloned by the limiting dilution method.
- the second step is to sequence the variable region of the native antibody, extract the total RNA of the 5A2F9-4 cell line, 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 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 present invention is directed to the clinical application of chemical small molecule effects (such as carbamazepine, lidocaine, mexiletine, etc.) for the treatment of pain by voltage-gated sodium ion channel inhibitors, but their lack of voltage-gated sodium ion channel subtypes Sufficient selectivity, resulting in weakness in cardiotoxicity and central nervous side effects.
- chemical small molecule effects such as carbamazepine, lidocaine, mexiletine, etc.
- Figure 1 Structure of the sodium ion channel Nav1.9
- FIG. 3 Antibodies secreted by 5A2F8 and 5A2F9 cell lines are specific for neural tissue
- Figure 4 Western blotting to verify antibody specificity
- HA-hNav1.9 is ND7/23 cells transfected with HA-hNav1.9 plasmid
- COS-7 HA-M was a negative control for COS-7 cells transfected with HA-label
- ND7/23 is an untransfected cell negative control
- Figure 5 Analgesic effect of 5A2F9-4 antibody on acute inflammatory pain induced by 5% formalin in wild type mice.
- the cleavage reagent is: trifluoroacetic acid + ethanedithiol + phenol + thioanisole + water;
- the coating antigen was polypeptide C8079BB030-1; the coating solution was 1*PBS (pH 7.4); the washing buffer: 1*PBS (pH 7.4), 0.05% PBS; the anti-serum was used after the primary antibody was 3; Standard secondary antibody: Peroxidase-AffiniPure Goat Anti-Mouse IgG, Fc ⁇ Fragment Specific (min X Hu, Bov, Hrs Sr 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 added to the slats after mixing, and the cover film was placed 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.
- Example 2.2 According to the ELISA test results of Example 2.2, combined with the tissue-specific results, two animals #1955, #1958 were selected for terminal elimination, and the spleen cells and tumor cells of the two animals were fused three days later, and mouse myeloma cells (SP2) were used. /0) Cell fusion was performed by electrofusion in a 1:3 ratio with spleen cells, and the fused cells were plated into 15 feeder cells using HAT medium, and cultured in a CO 2 incubator.
- SP2 mouse myeloma cells
- 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 96-well plates were set as positive control with well 15H12 (15th whole plated well H12), and 1:1 fusion of fused animal serum was added.
- the OD value was 2.398; the well was 15G12 (15th)
- the whole plate of the well G12) was set as a negative control, and the blank medium was added, and the OD value detection result was 0.085; the results showed that 68 of the 14 96-well 1440 cell species had an OD value greater than 1.0.
- the clones of OD>1.0 of 68 single-well cells screened for the first time were subjected to a second test (the detection method is the same as above).
- the results of the experiment showed that the OD values of 20 cell lines were close to the positive control, and the OD value was above 2.1. . They are 2D11,3E2,3F3, 3F11, 5A2,5B4, 5E4, 7C8,9B10, 9D2, 9G9,10A7,10C1,11A2,11B6,12D12, 13B4, 13H7, 15F5, 15H3.
- the human nerve tissue was taken for dehydration treatment, and the dehydration treatment was carried out using Leica ASP300S.
- the specific procedure was as follows: 70%, 85%, 90%, anhydrous ethanol were respectively dehydrated for 30 minutes; then dehydrated with absolute ethanol for 2 times, each time for 60 minutes. Then, the transparent agent was treated for 30 minutes, and then treated with a transparent agent twice for 60 minutes each time; and then treated with paraffin three times for 60 minutes, 120 minutes, and 180 minutes, respectively, and the Leica EG1150 embedding machine was used for embedding operation.
- a wax block was prepared and sliced to a thickness of 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.
- 5A2, 9D2, and 15F5 were selected for subcloning.
- Three cells were subcloned by limiting dilution method, and the three 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). It was shown that 14 cell lines such as 5A2E9, 5A2F8, 5A2F9, 9D2A10, 9D2C9, 9D2F7, 9D2F8, 15F5B9, 15F5B11, 15F5D10, and 15F5G6 had OD values higher than 2.2 and the others were negative.
- 5A2F8 and 5A2F9 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. Both are positive, indicating that these are already homozygous monoclonal. 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 1:2400, and the subtypes were IgGa and K. Finally, three cells, 5A2F8-5, 5A2F9-1, and 5A2F9-4, were 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.
- the leader sequence is underlined by a dashed line, and the CDR sequence is underlined by a solid line.
- the amino acid sequence is deduced from the DNA sequences of VH and VL, the VH amino acid sequence is shown in SEQ ID NO: 2, and the VL amino acid sequence is shown in SEQ ID NO: 3.
- the leader sequence is underlined by a dashed line, and the CDR sequence is underlined by a solid line.
- RNA sequence encoding VH is SEQ ID NO: 12
- RNA sequence encoding VL is 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 cultured in a cell dish at 29 ° C for 20 hours to collect cells ND7 / 23: HA-h Nav1.9.
- the HA tag plasmid was transfected into COS-7 cells in the same manner to obtain COS-7:HA-M 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 5A2F9-4 (1:300) antibody for 1 hour at room temperature.
- the 5A2F9-4 antibody and the HA-tagged antibody can detect a hybridization signal between 130-250 KD, and no corresponding signal is detected in the negative control, and the hybridization signal and the molecular weight of hNav1.9 are about 210 kDa. Close, indicating that the antibody recognizes the protein of hNav1.9.
- mice 12 wild-type mice were taken and tested after 2 days of adaptation. They were randomly divided into 2 groups, one group was the control group, PBS was injected intravenously in the tail, and one group was the experimental group. 10 mg/kg of 5A2F9-4 antibody was injected into the tail vein. After half an hour, the hind paw was injected subcutaneously with 20 ⁇ L of 5% ⁇ . Marlin caused pain caused by acute inflammation, and the time of the mouse paw and paw withdrawal was recorded every 5 minutes for a total of 45 minutes, and the phase I (0-10 minutes) and phase II (10-45 minutes) were statistically analyzed. Phase I was acute pain, and phase II was spontaneous persistent pain. After the experiment, the differences between the two phases and the injection of the wild type mice in the wild type mice and the control group were statistically analyzed.
- Fig. 5 wild type mice were injected with 5A2F9-4 antibody in the tail vein, and acute inflammatory pain was induced by 5% formalin.
- the antibody can reduce the hind paws by subcutaneous injection of 5% Formalin for 10-15 minutes and 30-40.
- the results showed that 5A2F9-4 antibody can alleviate phase II inflammatory pain induced by 5% of Formalin in wild-type mice, which is equivalent to the reported results.
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Abstract
本发明提供了一种靶向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,其特异结合的靶点为电压门控钠离子通道α亚基的第II结构域的电压传感器桨的S3-4环。所述抗体或其抗体片段能失活电压传感器阀门,使得钠离子不能正常进入神经细胞,从而达到治疗和缓解疼痛的效果。
Description
本发明涉及Nav1.9的靶点多肽,结合上述多肽的抗体和/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。
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。
发明内容
本发明的第一个目的是提供Nav1.9中结构域II的电压传感器桨区域作为靶点,利用该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:1所示的序列,也可以是与该多肽具有80%,85%,90%或95%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:2所示的序列,或者与该多肽具有80%,85%,90%或95%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:3所示的序列,或者与该多肽具有80%,85%,90%或95%同源的结构类似的衍生序列;还可以取出重链可变区(SEQ ID NO:2)所含有的3个CDR序列和/或轻链可变区(SEQ ID NO:3)所包含的3个CDR序列,进行移植,获得CDR移植抗体及其抗体片段。所述重链可变区包含的CDR序列为,如SEQ ID NO.4所示的CDRH1、如SEQ ID NO.5所示的CDRH2和如SEQ ID NO.6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.8所示的CDRL2和如SEQ ID NO.9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、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形式的抗体或抗体片段。
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II的电压传感器桨区域筛选合适的多肽作为抗原,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LNSFSNEERNGNLE(SEQ ID NO.1)。
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(在其C端额外添加一个半胱氨酸),将该合成多肽编号为C8079BB030-1,将其偶联到载体蛋白KLH上,然后免疫BALB/c小鼠,多次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来评价免疫动物的多克隆抗体效价,根据免疫的动物抗体效价和神经组织的特异性,最终确定两只动物#1955,#1958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,对分泌的抗体的效价和组织特异性进行检测,利用多肽C8079BB030-1作为抗原筛选杂交瘤细胞株,根据ELISA检测结果,选择5A2,9D2,15F5采用有限稀释法进行亚克隆。
再次进行ELISA检测和特异性检测后,只有5A2细胞对神经组织具有特异性和阳性,最终选择细胞5A2F9-1和5A2F9-4进行细胞冻存。
第二步是对天然抗体的可变区测序,提取5A2F9-4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,编码其VH的DNA序列如SEQ ID NO:10所示,编码其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。
本发明是针对临床上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,因而产生心脏毒性和中枢神经副作用的弱点。采用生物大分子针对Nav1.9电压门控钠离子通道的电压传感器的靶向抗体,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。
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3:5A2F8和5A2F9细胞株分泌的抗体对神经组织特异性
图4:western blotting验证抗体特异性
ND7/23:HA-hNav1.9为转染了HA-hNav1.9质粒的ND7/23细胞;
COS-7:HA-M为转染了HA标签的COS-7细胞作阴性对照;
ND7/23为未转染的细胞阴性对照;
图5:5A2F9-4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。
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,在疼痛位点811附近,进行亲水性和抗原性的分析,筛选出LNSFSNEERNGNLE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LNSFSNEERNGNLEC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(C8079BB030-1);
(12)多肽HPLC的C18制备/分析柱进行纯化,编号为C8079BB030-1,获得纯化的多肽用于免疫动物。
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C8079BB030-1的C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:#1954,#1955,#1956,#1957,#1958)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 周天后进行二次免疫,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,在三免疫的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 Multiskan Ascent
2.2.2使用的试剂:
包被抗原为多肽C8079BB030-1;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,FcγFragment Specific(min X Hu,Bov,Hrs Sr 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#1955,#1956的抗血清滴度在1:128,000;动物编号为#1954的抗血清滴度在1:64,000;其余2只动物(#1957,#1958)的抗血清滴度在1:16,000;
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.1954 | No.1955 | No.1956 | No.1957 | No.1958 |
阴性对照 | 1:1,000 | 0.072 | 0.066 | 0.086 | 0.088 | 0.104 |
稀释1 | 1:1,000 | 3.219 | 2.88 | 3.067 | 2.915 | 2.74 |
稀释2 | 1:2,000 | 3.125 | 2.733 | 2.847 | 2.579 | 2.322 |
稀释3 | 1:4,000 | 3.087 | 2.714 | 2.57 | 2.22 | 2.031 |
稀释4 | 1:8,000 | 2.47 | 2.541 | 2.456 | 1.677 | 1.565 |
稀释5 | 1:16,000 | 2.135 | 2.314 | 2.352 | 1.105 | 1.068 |
稀释6 | 1:32,000 | 1.73 | 1.942 | 1.988 | 0.675 | 0.647 |
稀释7 | 1:64,000 | 1.242 | 1.578 | 1.553 | 0.384 | 0.417 |
稀释8 | 1:128,000 | 0.843 | 1.077 | 1.052 | 0.207 | 0.233 |
稀释9 | 1:256,000 | 0.471 | 0.654 | 0.649 | 0.162 | 0.155 |
稀释10 | 1:512,000 | 0.296 | 0.373 | 0.387 | 0.119 | 0.118 |
稀释11 | 空白对照 | 0.072 | 0.072 | 0.072 | 0.072 | 0.072 |
滴度 | 1:512,000 | 1:512,000 | 1:512,000 | 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,结合组织特异性结果,选择两只动物#1955,#1958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96孔板,以孔15H12(第15个整板的孔H12)设置为阳性对照,加入融合动物血清1:1000稀释液,其OD值检测结果为2.386;将孔15G12(第15个整板的孔G12)设置为阴性对照,加入空白培养基,其OD值检测结果0.085;结果显示,15块96孔的1440个细胞种有68个的OD值大于1.0。
第二次ELISA筛选:
将第一次筛选的68个单孔细胞的OD>1.0的克隆进行第二次检测(检测方 法同上),实验结果结果显示,20个细胞株的OD值接近阳性对照,其OD值在2.1以上。它们分别是
2D11、3E2、3F3、3F11、
5A2、5B4、5E4、
7C8、9B10、
9D2、9G9、10A7、10C1、11A2、11B6、12D12、
13B4、13H7、
15F5、15H3。
第三次ELISA筛选:
从第二次筛选的选择OD值较高的20株进行阳性确认检测(检测方法同上):,实验结果显示,只有10个细胞株的OD值大于2.0,它们是
2D11、3E2、
3F3、5A2、5B4、7C8、9B10、9D2、13B4、15F5。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。
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
2D11、3E2、3F3、
5A2、5B4、7C8、9B10、9D2、13B4、15F5一共10个细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,有三个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,结果见下表。
克隆号 | 特异性 | 杂交信号 |
5A2 | ** | ** |
9D2 | ** | * |
15F5 | ** | * |
2.3.4亚克隆
根据实施例2.3.3的细胞学检测结果,选择5A2、9D2、15F5进行亚克隆。利用有限稀释法对三株细胞进行亚克隆,将这三株细胞铺入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,5A2E9、5A2F8、5A2F9、9D2A10、9D2C9、9D2F7、9D2F8、15F5B9、15F5B11、15F5D10、15F5G6等14个细胞株的OD值高于2.2以上,其余均为阴性。选择这些进行第二次确认,经确认,其OD值均大于2.2。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。经组织免疫化学确认,只有5A2F8和5A2F9细胞株为免疫组化阳性(见图3)。
由于免疫组化阳性的三个克隆来自于同一克隆,因此,选择5A2F8,5A2F9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1:2400,亚型为IgGa,K型。最终选择三株细胞5A2F8-5,5A2F9-1,5A2F9-4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5A2F9-4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。其中作虚线下划线的为先导序列,作实线下划线的为CDR序列。
先导序列-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
先导序列-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
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:2所示,VL氨基酸序列如SEQ ID NO:3所示。其中作虚线下划线的为先导序列,作实线下划线的为CDR序列。
先导序列-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
先导序列-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
推测出编码VH的RNA序列即SEQ ID NO:12,编码VL的RNA序列即SEQ ID NO:13。
实施例4【抗体的特异性检测】
4.1hNav1.9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ND7/23:HA-h Nav1.9。
同样方法将HA标签质粒转染COS-7细胞,得到COS-7:HA-M细胞。
4.2Westernblotting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5A2F9-4(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. 3Western杂交结果
从图4中可以看出,5A2F9-4抗体与HA标签抗体可以检测到在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【5A2F9-4抗体在野生型小鼠体内镇痛效果】
为了检测5A2F9-4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5A2F9-4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
从图5可知,野生型小鼠尾静脉注射5A2F9-4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30-40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5A2F9-4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:
Claims (10)
- 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。
- 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II结构域的电压传感器桨的S3-4环。
- 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.4所示的CDRH1、如SEQ ID NO.5所示的CDRH2和如SEQ ID NO.6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.7所示的CDRL1、如SEQ ID NO.8所示的CDRL2和如SEQ ID NO.9所示的CDRL3。
- 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.2所示的重链可变区和/或如SEQ ID NO.3所示的轻链可变区。
- 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。
- 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。
- 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。
- 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.1所示的氨基酸序列。
- 一种药物组合物,含有权利要求1所述的抗体或其抗体片段。
- 权利要求1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。
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EP18758448.7A EP3594234A4 (en) | 2017-02-21 | 2018-02-07 | NAV1.9 TARGET POLYPEPTIDE, COMBINED ANTIBODIES AND ANTIBODY FRAGMENTS AND RELATED PHARMACEUTICAL COMPOSITION |
CN201880024589.5A CN110662763B (zh) | 2017-02-21 | 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 |
CA3062486A CA3062486A1 (en) | 2017-02-21 | 2018-02-07 | Nav1.9 target polypeptide, antibody and antibody fragment combined with same, and related pharmaceutical composition |
US16/657,515 US11136382B2 (en) | 2017-02-21 | 2019-10-18 | Antibodies and antibody fragments for binding to NAV1.9 polypeptides and methods for using same to treat pain-related disease |
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CN114805497B (zh) * | 2021-01-29 | 2023-09-22 | 李威 | 一种靶向钠离子通道蛋白Nav1.4的新型多肽NaV-B及其应用 |
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WO2014207402A1 (fr) * | 2013-06-28 | 2014-12-31 | Galderma Research & Development | Anticorps dirigés contre le canal sodique nav1.9 humain et leurs utilisations en diagnostic |
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US9139648B1 (en) * | 2014-07-15 | 2015-09-22 | Kymab Limited | Precision medicine by targeting human NAV1.9 variants for treatment of pain |
Non-Patent Citations (3)
Title |
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LEE ET AL., CELL, vol. 157, 2014, pages 1393 - 1404 |
NATURE GENETICS, vol. 45, no. 11, 2013, pages 1399 - 1404 |
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