WO2022021000A1 - Antigen-binding characteristic epitope and application thereof - Google Patents
Antigen-binding characteristic epitope and application thereof Download PDFInfo
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- WO2022021000A1 WO2022021000A1 PCT/CN2020/104876 CN2020104876W WO2022021000A1 WO 2022021000 A1 WO2022021000 A1 WO 2022021000A1 CN 2020104876 W CN2020104876 W CN 2020104876W WO 2022021000 A1 WO2022021000 A1 WO 2022021000A1
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Definitions
- the invention relates to the field of biomedicine, in particular to a characteristic epitope bound by an antibody and its application.
- Cardiovascular disease is a serious threat to civilization and is the leading cause of death in all population groups. According to the "China Cardiovascular Disease Report 2019" released by the National Cardiovascular Disease Center, the number of cardiovascular disease (CVD) patients in China has reached 290 million, and about 3.5 million people die of cardiovascular disease every year.
- the drugs for lowering cholesterol on the market mainly include statins, cholesterol absorption inhibitors and probucol. Despite the excellent performance of statins in the treatment of cardiovascular disease, with their widespread use, the possible drawbacks have gradually been discovered.
- statins have various side effects, such as causing abnormal blood sugar, muscle toxicity, memory and cognitive impairment in patients, etc. , the incidence of side effects is as high as 20%, serious side effects can lead to rhabdomyolysis and acute renal failure, and a considerable number of patients terminate the treatment because they cannot tolerate the muscle pain caused by the side effects.
- PCSK9 Proprotein convertase subtilisin/kexin type 9
- LDL-R low-density lipoprotein receptor
- PCSK9 inhibitors offer an entirely new therapeutic modality to combat LDL-C and are seen as the biggest advancement in lipid-lowering after statins.
- the advent of PCSK9 inhibitors is a boon for patients who experience severe side effects while taking statins, as well as patients who cannot achieve LDL-C target levels with statin therapy, such as patients with inherited hypercholesterolemia.
- PCSK9 inhibitors can also inhibit NF- ⁇ B channels, thereby reducing the risk of acute coronary syndromes such as thrombosis, inflammation, and activation of vascular endothelial cells.
- potential research projects in the field of PCSK9 inhibitors include inhibitory protein antibodies, siRNA, antisense oligonucleotides, and small molecule inhibitors.
- Monoclonal antibody drugs are currently the main field of PCSK9 inhibitor research due to their strong targeting, high specificity, and low toxicity and side effects. Studies at the animal level showed that after adding an antibody that neutralized PCSK9, the expression level of LDL-R in the liver of the mice was significantly increased, and the concentration of LDL-C in the blood decreased by 30%.
- PCSK9 monoclonal antibody In primates, the PCSK9 monoclonal antibody also showed significant effects, and the effect of reducing LDL-C levels was maintained for more than several weeks. So far, anti-PCSK9 protein monoclonal antibodies have not been found to have relatively obvious toxic side effects, and only minor side effects such as local injection reactions, diarrhea and headache have been reported. Sanofi's Praluent (Alirocumab) and Amgen's Repatha (Evolocumab) are the only humanized PCSK9 antibodies currently approved in the global market. According to Thomson Reuters' list of potential sales of approved drugs in 2015, the sales of the former will reach $4.414 billion by 2019, while the sales of the latter will reach $1.862 billion. There are many CVD patients in my country.
- Antibody drugs are the main direction of new drug research and development, and have been widely used in the diagnosis, prevention and treatment of infectious diseases and biological science research. As of 2015, 48 antibody drugs have been successfully launched, and 7 antibody drugs have been successfully approved from April 2014 to March 2015 alone. In 2015, 6 of the top 10 drugs by global sales were antibody drugs. Since Hamers et al. discovered heavy-chain antibodies that naturally lacked light chains in camel blood in 1993, nanobodies (Nanobodies, Nb) have gradually replaced other small antibodies and have gradually become a hot spot in the development of new antibody drugs. Nb is usually only about 15kDa, about one-tenth of the size of traditional antibodies.
- Nb lacks the Fc segment and light chain.
- the properties enable it to recognize cryptic epitopes or small epitopes that cannot be recognized by traditional antibodies, and avoid complement reactions; in addition, nanobodies have high stability, low toxicity, strong solubility, easy target screening and easy detection in prokaryotic microorganisms. Direct expression, good economy and many other advantages.
- Nb is gradually being developed as a monoclonal antibody drug for disease diagnosis and treatment, and is widely used in the development of enzyme inhibitors, tumor, infection and inflammation biological inhibitors.
- enzyme inhibitors enzyme inhibitors
- tumor infection and inflammation biological inhibitors.
- small-molecule proteins are easily eliminated in vivo.
- the present invention provides a characteristic epitope bound by an antibody such as PCSK9 antigenic epitope and its application.
- the epitopes of the currently marketed drugs that bind to PCSK9 are generally located in the leader region (31-152) and catalytic region (153-425) of PCSK9. When a patient develops resistance or does not respond to such drugs, it is necessary to bind other epitopes of PCSK9. bit of drugs.
- a first aspect of the present invention provides a polypeptide fragment, wherein the polypeptide fragment comprises an amino acid sequence such as X 1 X 2 X 3 X 4 X 5 GX 6 from the N-terminus to the C-terminus , wherein X 1 is S, T, E, Y or F, preferably S; X 2 is T, N, G, S or D, preferably T; X 3 is H, E, Y, T, K, R or A, preferably H; X 4 is G, Y, H, R or M, preferably G; X 5 is A, F, Q, H, I or V, preferably A; X 6 is W or F , preferably W.
- X 1 is S, T, E, Y or F, preferably S
- X 2 is T, N, G, S or D, preferably T
- X 3 is H, E, Y, T, K, R or A, preferably H
- X 4 is G, Y, H, R or
- the amino acid sequence of the polypeptide fragment is such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: :7, SEQ ID NO:8 or SEQ ID NO:9.
- VHH4-Fc refers to a chimeric antibody formed by fusion of Nanobody VHH4 with antibody constant region Fc.
- Patent 1 Application No.: 201711298806.3
- Patent 2 Application No.: PCT/CN2020/074624; domain antibodies and their applications.
- the polypeptide fragment specifically binds to a VHH4 antibody or variant thereof whose amino acid sequence is shown in SEQ ID NO: 23 (VHH4) or shown in SEQ ID NO: 24 (VHH4-hFc).
- the mutation is a deletion, addition or substitution of one or more amino acid residues in the amino acid sequence of the antibody.
- the amino acid sequence similarity of 80% or more with the Nanobody VHH4 or VHH4-Fc is regarded as the range of the antibody that binds to the polypeptide fragment of the present invention. That is, the mutation is at least 80%, 85%, 90%, 95%, 98%, 99% identical to the amino acid sequence of the antibody and retains the function of the antibody.
- the Fc region can refer to either the constant region of human antibodies or the Fc constant region of antibodies of any species, and the target antibody contains all Fc subtypes of any species, that is, IgG1 Fc, IgG2 Fc, etc.
- Any molecular modification related to VHH4 includes but is not limited to methylation modification, glycosylation modification, PEG modification, small molecule coupling such as KLH-VHH4 and dimerization of VHH4 and corresponding light chain to form antibody Fab molecules, etc.
- Fc can also be replaced with any protein fused to VHH4 such as human serum albumin forming HSA-VHH4 or VHH4-HSA.
- the antibody to which the polypeptide fragment can bind is a fusion protein fused to human serum albumin.
- X-ray method crystallography after protein crystallization
- alanine scanning mutagenesis method alanine scanning mutagenesis method
- X-ray method has high identification efficiency and high precision, but there are high cost, antigen-antibody complexes
- the disadvantages of poor crystallization and high requirements for the purity of the complex the alanine scanning mutagenesis method requires a large number of antigen molecules to be mutated, and a large amount of protein, and the expression of amino acids in some parts is extremely low or even impossible to express, and the cost is also high.
- the method of phage display on a random polypeptide library is used to identify the antibody-binding antigenic epitope, and then Western is used for epitope verification, which has the advantages of high efficiency and low cost.
- the second aspect of the present invention provides an isolated nucleic acid molecule encoding the above-mentioned polypeptide fragment.
- a third aspect of the present invention provides an expression vector comprising the isolated nucleic acid molecule as described above; preferably, the base vector (ie, backbone vector) of the expression vector is pMECS.
- the fourth aspect of the present invention provides a transformant, which contains the isolated nucleic acid molecule or expression vector as described above.
- the host cells of the transformants are prokaryotic cells or eukaryotic cells.
- the fifth aspect of the present invention provides a PCSK9 antigen combination, which includes amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 4, and SEQ ID NO: 2.
- amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 4, and SEQ ID NO: 2.
- the sixth aspect of the present invention provides a kind of polypeptide vaccine, it is characterized in that, it is prepared by the following method: will include amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID At least one of the polypeptide fragments shown in NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 is used as Antigen-produced polyclonal antibodies that specifically bind to PCSK9.
- amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID At least one of the polypeptide fragments shown in NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 is used as Antigen-produced polyclonal antibodies that specifically bind to PCSK9.
- the seventh aspect of the present invention provides a ligand, which can specifically bind to the above-mentioned polypeptide fragment, but the ligand is not an amino acid sequence such as SEQ ID NO: 23 or The antibody shown in SEQ ID NO:24.
- the eighth aspect of the present invention provides an inhibitor, which can target and inhibit the isolated nucleic acid molecule as described above.
- the inhibitor is an siRNA or mRNA vaccine.
- the ninth aspect of the present invention provides a kit, which includes the above-mentioned polypeptide fragment, and/or, the above-mentioned ligand.
- the tenth aspect of the present invention provides a method for preparing the above-mentioned polypeptide fragment, which comprises the following steps: culturing the above-mentioned transformant, and expressing it to obtain an expression product.
- the preparation method further includes the step of purifying the expression product.
- the tenth aspect of the present invention provides the polypeptide fragment, the isolated nucleic acid molecule, the expression vector, the transformant, the PCSK9 antigen combination or the The application of the kit described in the preparation of an inhibitor that specifically binds to PCSK9 or a preparation for detecting PCSK9 protein or a PCSK9 vaccine.
- the eleventh aspect of the present invention provides the use of the above-mentioned ligand and the inhibitor in the preparation of a reagent for competitively inhibiting anti-PCSK9 antibody.
- the reagents and raw materials used in the present invention are all commercially available.
- the present invention explores the binding epitopes different from the previous antibodies, and enriches the diversity of the binding epitopes of the anti-PCSK9 antibody.
- the interaction mechanism between the previously developed antibody VHH4-Fc and the target antigen PCSK9 was elucidated, and the interaction epitope was resolved.
- the epitope of the target antibody and PCSK9 antigen binding to each other is expounded, which is conducive to improving the market value of VHH4-Fc, and is also conducive to the further transformation and optimization of VHH4-Fc.
- the new antigenic epitope can also be used to make a polypeptide vaccine against PCSK9; and the polypeptide fragment can be combined with anti-PCSK9 antibodies, which can be used for detection, screening or auxiliary screening of new anti-PCSK9 antibodies to alleviate
- the patient has the problem of strong immunogenicity and other adverse reactions to the primary antibody.
- Fig. 1 is the amino acid sequence of human PCSK9
- FIG. 2 is a schematic diagram of the results of the affinity determination between VHH4-Fc and PCSK9;
- Figure 3 is a differential identification experiment of the novel antibody and Repatha binding epitope
- Fig. 4 is the amino acid sequence comparison schematic diagram of three mutants of human PCSK9 antigen
- FIG. 5 shows Western Blot validation of human PCSK9 and its three mutants.
- the protein A chip capture method based on SPR technology was used to determine its affinity with human PCSK9 (hPCSK9).
- the VHH4-Fc antibody was captured with a Protein A chip, and then 5 different concentrations of hPCSK9 antigen were injected (flowed through) for affinity determination.
- the binding time was set to 120s
- the dissociation time was set to 180s
- the regeneration solution was pH 2.0
- the 100 mM glycine solution, the system solution is 1x PBST.
- the final affinity result is based on the fitting curve given by the built-in evaluation software of Biacore T200, and then the koff and kon values are obtained.
- K D (nM) koff(1/s)/kon(1 /Ms) calculated.
- Rmax is the maximum response value, usually between 0-100RU; Chi 2 value less than or equal to 1/10Rmax is regarded as a reliable measurement result.
- the assay results show that the binding constant of VHH4-Fc and hPCSK9 is 1.872 ⁇ 10 6 , the dissociation constant is 1.288 ⁇ 10 -3 , the Rmax (maximum binding value) is 24.71RU, and the Chi 2 value is 0.693.
- the affinity is 0.6879nM.
- the abscissa refers to the time axis of the antigen-antibody reaction, and the unit is (second, s); the ordinate refers to the response value when the antigen-antibody is bound or dissociated, and the unit is Response units (RU).
- Different curves represent injections of different concentrations of antibody. The affinity of this experiment was tested under the reaction condition of 25°C.
- Embodiment 2 Epitope difference detection
- the purpose of the epitope binning test is to detect the similarities and differences between the screened antibodies and the marketed drug Evolocumab (trade name: Repatha, Repatha) in binding the epitope of human PCSK9.
- Evolocumab trade name: Repatha, Repatha
- two schemes are used to detect the epitope difference between the VHH4 antibody of CN201711298806.3 and PCT/CN2020/074624 (the amino acid sequence of which is shown in SEQ ID NO: 23) and the marketed antibody drug binding target antigen PCSK9 .
- the first scheme is the affinity kinetics detection based on SPR technology.
- the his-tagged human PCSK9 referred to as hPCSK9-his
- VHH4-his After three washes with 1 ⁇ PBST, followed by incubation with 5 ⁇ g/mL hPCSK9-Fc antigen as the “primary antibody” for 1 hour at room temperature; followed by five washes with 1 ⁇ PBST, serially diluted VHH4-his was added to the wells , to the epitope on the human hPCSK9-Fc antigen, if Evolocumab and VHH4-his and other novel antibodies bind the same epitope, VHH4-his will not bind to hPCSK9-Fc and be washed away, on the contrary, VHH4-his Will not be washed off.
- HRP horseradish peroxidase
- FIG. 3 A is a differential detection experiment based on surface plasmon resonance (SPR) to detect VHH4 and marketed drugs binding to human PCSK9 antigenic epitopes.
- SPR surface plasmon resonance
- the method using affinity kinetics is shown.
- the results of the assay, the abscissa is the time axis, and the ordinate is the relative response value; it contains multiple stages, such as the baseline stage (baseline), the capture evolocumab stage (capture evolocumab), the PCSK9 injection and binding stable stage (inject hPCSK9) , VHH4 injection stage (inject VHH4) and dissociation stage (dissociation stage).
- VHH4 double antibody "sandwich” ELISA technology
- the abscissa represents VHH4 at different concentrations
- the ordinate represents the absorbance value of OD450.
- the ELISA plate is first coated with an excess of Evolocumab, followed by the addition of hPCSK9-Fc (as the "primary antibody”), which fully saturates the specific binding site on PCSK9 with Evolocumab, followed by the addition of VHH4-his (as the "secondary antibody”) , if VHH4-his and Evolocumab bind to the same site of PCSK9, it will eventually be completely washed away during washing.
- the experimental results in Figure 3B show that VHH4 and PCSK9 still have a binding curve after Evolocumab saturates their binding sites, confirming that VHH4 novel Antibodies and evolocumab bind different epitopes of PCSK9.
- the right side of FIG. 3B is a schematic diagram of the experimental principle of double-antibody sandwich ELISA.
- Phage libraries of random polypeptides were amplified and rescued using helper phages.
- the monoclonal library saved in step (1) was inserted into 100 mL of medium and cultured to the logarithmic growth phase, and the helper phage with MOI of 20 was added, at room temperature, allowed to stand for 30 min, after low-speed centrifugation, the sediment was suspended with medium, and then into 300 mL of medium and cultured overnight.
- VHH4-Fc protein Take 100ng of VHH4-Fc protein to coat the ELISA plate and incubate at 4°C overnight. The next day, add the rescued VHH4-Fc-specific phage, incubate for 2 h at room temperature; wash the wells 10 times with PBST, add 100 ⁇ L of triethylamine, and incubate for 30 min at room temperature, the collected phage is the obtained phage with affinity for VHH4-Fc. Library; take 10 ⁇ L of infected TG cells and spread them on the plate, which is used to determine the number of clones after screening, and the remaining phage after screening is used for amplification. Repeat panning according to the above steps for three to four rounds of polypeptide screening.
- the ELISA plate was coated with 100ng of VHH4-Fc protein and incubated at 4°C overnight; the next day, 2% BSA was added to block at room temperature for 1 h; the experimental group was added with the phage amplified after each round of panning, and the control group was added with the same amount of wild-type phage , at room temperature, incubate for 2h; wash 10 times with PBST to remove unbound phage; add HRP-labeled anti-M13 antibody, incubate at room temperature for 1h; add chromogenic solution, react in the dark for 10-30min, measure the absorbance, and the absorbance increases with the The number of panning increased gradually and stabilized during the third to fourth rounds of panning, indicating that specific antibodies were enriched.
- the ELISA plate was coated with 100 ng of VHH4-Fc and incubated at 4°C overnight; the phage-coated plate obtained from the last round of screening was taken, and 38 single clones were randomly selected in 1 mL of medium, and incubated at 37°C to log phase.
- VHH4-Fc antibody to be protected, which should include but are not limited to amino acid characteristics: (N-terminal-X 1 X 2 X 3 X 4 X 5 GX 6 -C-terminal), where X represents 20 species Any amino acid among amino acids, but generally speaking, X1 at position 1 is often any amino acid among S, T, E and F; X2 at position 2 is often among T, Y, G, S and D Any one of the amino acids in the 3rd position X3 is often any one of the amino acids in H, E, Y, T, K and A; The 4th position X4 is often any one of the amino acids in G, Y, H, and R ; X5 at position 5 is often any amino acid in A, F, Q, H, I and V; X6 at position 7 is often any amino acid in W and F.
- X1 at position 1 is often any amino acid among S, T, E and F
- X2 at position 2 is often among T, Y, G, S and
- PCSK9-mu02 (repeated loading), PCSK9-mu03 and wild-type PCSK9 (PCSK9-WT); lanes 1-5 and 11-15 are the supernatants of cells expressing PCSK9, lanes 6-10 and 16-20 are The cell lysate precipitates expressing PCSK9 mainly examine whether the expressed target protein is intracellular or extracellular; then the electrophoresis gel is transferred to the membrane for Western Blot verification, mainly to examine whether the truncation mutation of PCSK9 can still be expressed normally (Figure 5A) and whether Can bind to VHH4-hFc (Figure 5B), the results are shown in Figure 5, PCSK9-WT (lanes 5, 10, 15 and 20 at 76kDa) were expressed in cell culture supernatants and pellets, while the other three PCSK9 The mutants were expressed intracellularly (lanes 6-9).
- the truncated mutant control PCSK9-mu01 (lane 16) could still bind to VHH4-hFc after truncation, indicating that the truncated polypeptide was not bound to VHH4-hFc and PCSK9.
- mutant PCSK9-mu02 Although Anti-HA antibody detected intracellular expression (bands in lanes 7 and 8), it almost no longer binds to VHH4-hFc (no bands in lanes 17 and 18)
- the situation of mutant PCSK9-mu03 is similar, although there is intracellular expression detected by Anti-HA antibody (a band at 48kDa in lane 9 in Figure A), but VHH4-hFc no longer binds to PCSK9-mu03 (48kDa in lane 19 in Figure B) There is no band at the place), which indicates that "STHGAGW" (located at positions 447-453 of hPCSK9) is indeed the mutual binding epitope of the target antibody VHH4-hFc and human PCSK9 antigen, or at least a part of the spatial epitope of hPCSK9.
- Example 3 The VHH-Fc in Example 3 was replaced with Evolocumab, and other experimental steps were the same as those in Example 3. Thus, a partial polypeptide that binds with high affinity to Evolocumab was obtained. It can be seen from Table 3 below that the polypeptide fragments that VHH4-Fc binds to Evolocumab are completely different, that is, the two antibodies bind completely different antigenic epitopes.
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Abstract
Description
本发明涉及生物医药领域,具体涉及一种抗体结合的特征性表位及其应用。The invention relates to the field of biomedicine, in particular to a characteristic epitope bound by an antibody and its application.
心血管疾病严重威胁着人类,是所有人口族群的首要死因。据国家心血管病中心发布的《中国心血管病报告2019》显示,我国心血管病(cardiovascular disease,CVD)患病人数已经高达2.9亿,每年约有350万人死于心血管疾病。目前市面上用于降低胆固醇的药物主要有他汀类(statins)、胆固醇吸收抑制剂和普罗布考等。尽管他汀类药物心血管疾病治疗上表现出色,但随着其广泛使用,可能产生的弊端也渐渐被发现。首先,强化他汀治疗患者仍有较高心血管事件残余风险,在2年内发生的风险达22.4%;其次,有大量患者无法耐受他汀类药物,尤其是家族性高胆固醇血症患者,即使接受最大剂量的最有效的他汀类药物治疗,仍然不能达到降低LDL-C浓度的目的;最重要的是,他汀类药物存在多种副作用,如引起患者血糖异常、肌肉毒性、记忆和认知障碍等,副作用的发生率高达20%,严重的副作用会导致横纹肌溶解和急性肾功能衰竭,相当一部分患者因不能忍受副作用带来的肌肉疼痛而终止了治疗。Cardiovascular disease is a serious threat to humanity and is the leading cause of death in all population groups. According to the "China Cardiovascular Disease Report 2019" released by the National Cardiovascular Disease Center, the number of cardiovascular disease (CVD) patients in China has reached 290 million, and about 3.5 million people die of cardiovascular disease every year. At present, the drugs for lowering cholesterol on the market mainly include statins, cholesterol absorption inhibitors and probucol. Despite the excellent performance of statins in the treatment of cardiovascular disease, with their widespread use, the possible drawbacks have gradually been discovered. First, patients on intensive statin therapy still have a high residual risk of cardiovascular events, with a risk of 22.4% occurring within 2 years; second, a large number of patients cannot tolerate statins, especially patients with familial hypercholesterolemia, even if they receive The maximum dose of the most effective statin therapy still cannot achieve the purpose of reducing LDL-C concentration; most importantly, statins have various side effects, such as causing abnormal blood sugar, muscle toxicity, memory and cognitive impairment in patients, etc. , the incidence of side effects is as high as 20%, serious side effects can lead to rhabdomyolysis and acute renal failure, and a considerable number of patients terminate the treatment because they cannot tolerate the muscle pain caused by the side effects.
前蛋白转化酶枯草溶菌素9(Proprotein convertase subtilisin/kexin type 9,PCSK9),枯草蛋白酶亚家族的一种新的前蛋白转化酶,是常染色体显性家族性高胆固醇血症的重要影响因子之一。研究发现,PCSK9除了能影响血浆胆固醇水平,调节神经细胞的凋亡,还与炎症反应有一定的相关性。目前对于PCSK9的研究主要集中在对肝脏脂质代谢的调节功能。前期的研究显示,PCSK9可通过促进肝细胞的低密度脂蛋白受体(low-density lipoprotein receptor,LDL-R)的降解,调节肝脏脂质代谢,进而影响血浆中低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)的水平。但PCSK9存在两种突变类型,功能获得型突变和功能缺失型突变。族群试验显示,若干PCSK9“获得功能”的突变常发生于染色体显性高胆固醇血症的个体,而PCSK9“失去功能”的突变则与血浆胆固醇减少有关,PCSK9功能缺失型突变个体患冠心病的风险明显降低。2005年,Hobbs等在Dallas Heart Study上报道了携带PCSK9无义突变基因的个体中LDL-C水平会比一般人低28%;在2006,Hobbs等又发表PCSK9基因突变对冠心病的作用,该结果基于一项动脉粥样硬化风险调查,他们对9523个白人和3363个非洲裔美国人进行了长达15年的跟踪观察,发现缺失1个或2个PCSK9功能基因的人群的冠心病的发病率显著低于普通人群。Copenhagen Heart Study发现PCSK9基因的功能性缺失会使LDL-C水平下降11-15%,冠心病患病率下降6-46%。Zimbabwe等报道了PCSK9的缺失突变可使非洲女性的LDL-C水平下降27%。PCSK9抑制剂提供了一种全新的治疗模式来对抗LDL-C,被视为他汀类之后降脂领域取得的最大进步。PCSK9抑制剂的出现,为那些服用他汀类药物时出现严重副作用的患者,及他汀类药物治疗无法达到LDL-C目标水平的患者,如遗传性高胆固醇血症患者带来了福音。Proprotein convertase subtilisin/kexin type 9 (PCSK9), a new proprotein convertase of the subtilisin subfamily, is one of the important influencing factors of autosomal dominant familial hypercholesterolemia. one. Studies have found that PCSK9 not only affects plasma cholesterol levels and regulates neuronal apoptosis, but also has a certain correlation with inflammatory responses. The current research on PCSK9 mainly focuses on the regulatory function of hepatic lipid metabolism. Previous studies have shown that PCSK9 can regulate liver lipid metabolism by promoting the degradation of low-density lipoprotein receptor (LDL-R) in hepatocytes, thereby affecting low-density lipoprotein cholesterol (low-density lipoprotein cholesterol) in plasma. density lipoprotein cholesterol, LDL-C). However, there are two types of mutations in PCSK9, gain-of-function mutations and loss-of-function mutations. Population studies have shown that several PCSK9 "gain-of-function" mutations frequently occur in individuals with chromosomally dominant hypercholesterolemia, while PCSK9 "loss-of-function" mutations are associated with reduced plasma cholesterol, and PCSK9 loss-of-function mutations are associated with coronary heart disease. Risk is significantly reduced. In 2005, Hobbs et al. reported in Dallas Heart Study that the LDL-C level in individuals carrying PCSK9 nonsense mutation gene was 28% lower than that of the general population; in 2006, Hobbs et al. published the effect of PCSK9 gene mutation on coronary heart disease, the results Based on an atherosclerosis risk survey, they followed 9523 whites and 3363 African Americans for up to 15 years and found the incidence of coronary heart disease in people who lacked one or two PCSK9 functional genes. significantly lower than the general population. Copenhagen Heart Study found that functional deletion of the PCSK9 gene reduces LDL-C levels by 11-15% and coronary heart disease prevalence by 6-46%. Zimbabwe et al reported that deletion mutations in PCSK9 reduced LDL-C levels by 27% in African women. PCSK9 inhibitors offer an entirely new therapeutic modality to combat LDL-C and are seen as the biggest advancement in lipid-lowering after statins. The advent of PCSK9 inhibitors is a boon for patients who experience severe side effects while taking statins, as well as patients who cannot achieve LDL-C target levels with statin therapy, such as patients with inherited hypercholesterolemia.
PCSK9抑制剂除了能阻止LDL-R回收,还可以抑制NF-κB通道,从而减少血栓、炎症、血管内皮细胞激活等急性冠状动脉综合症的风险。目前PCSK9抑制剂这个领域潜在的研究项目有抑制蛋白抗体、siRNA、反义寡核苷酸以及小分子抑制剂等。单克隆抗体药物因具有靶向性强、特异性高和毒副作用低等特点,是目前PCSK9抑制剂研究的主要领域。在动物水平的研究显示,加入中和PCSK9的抗体之后,小鼠肝脏中的LDL-R表达水平显著上升,血液中LDL-C浓度下降30%。对灵长类动物,PCSK9单克隆抗体也表现出显著的效果,LDL-C水平的下降效果可维持数周以上。到目前为止,还没有发现抗PCSK9蛋白单抗类药物有比较明显的毒副作用,只有报道称出现过局部注射反应、腹泻和头疼等较轻微的副作用。赛诺菲的Praluent(Alirocumab)和安进的Repatha(Evolocumab)是目前全球市场唯二获批的人源化的PCSK9抗体。根据汤森路透对2015年获批药物潜力销售排行榜,到2019年前者的销售规模将达到44.14亿美元,而后者的销售规模将会达到18.62亿美元。我国CVD患者众多,在2015年统计的结果显示,CVD患人数已经高达2.9亿,每年约有350万人死于心血管疾病,然而我国在PCSK9抑制剂领域的研究却严重滞后,完全不能满足CVD患者的需求。In addition to blocking LDL-R recycling, PCSK9 inhibitors can also inhibit NF-κB channels, thereby reducing the risk of acute coronary syndromes such as thrombosis, inflammation, and activation of vascular endothelial cells. At present, potential research projects in the field of PCSK9 inhibitors include inhibitory protein antibodies, siRNA, antisense oligonucleotides, and small molecule inhibitors. Monoclonal antibody drugs are currently the main field of PCSK9 inhibitor research due to their strong targeting, high specificity, and low toxicity and side effects. Studies at the animal level showed that after adding an antibody that neutralized PCSK9, the expression level of LDL-R in the liver of the mice was significantly increased, and the concentration of LDL-C in the blood decreased by 30%. In primates, the PCSK9 monoclonal antibody also showed significant effects, and the effect of reducing LDL-C levels was maintained for more than several weeks. So far, anti-PCSK9 protein monoclonal antibodies have not been found to have relatively obvious toxic side effects, and only minor side effects such as local injection reactions, diarrhea and headache have been reported. Sanofi's Praluent (Alirocumab) and Amgen's Repatha (Evolocumab) are the only humanized PCSK9 antibodies currently approved in the global market. According to Thomson Reuters' list of potential sales of approved drugs in 2015, the sales of the former will reach $4.414 billion by 2019, while the sales of the latter will reach $1.862 billion. There are many CVD patients in my country. According to statistics in 2015, the number of CVD patients has reached 290 million, and about 3.5 million people die of cardiovascular disease every year. However, the research in the field of PCSK9 inhibitors in my country is seriously lagging behind, which cannot meet the requirements of CVD. patient needs.
抗体药物是目前新药研发的主要方向,在传染病的诊断、防治和生物科学研究领域都已得到了广泛的应用。到2015年截止,已经有48个抗体药物成功上市,仅2014年4月至2015年3月期间就有7个抗体药物成功获批。2015年全球销售排名前10的药物中有6个都为抗体药物。自1993年Hamers等在骆驼血液中发现了天然缺失轻链的重链抗体后,纳米抗体(Nanobody,Nb)逐渐取代其他的小型抗体,逐渐成为新型抗体药物研发的热点。Nb通常只有15kDa左右,约传统抗体大小的十分之一,其内部存在二硫键,表面有大量亲水残基,对热和pH有较强的抵抗力;Nb缺乏Fc段和轻链的性质使其能够识别传统抗体无法识别的隐蔽表位或小表位,且避免了补体反应;此外,纳米抗体还具有稳定性高、毒性低、可溶性强、易于靶点筛选和易于在原核微生物中直接表达,经济性好等诸多优势。序列同源性分析显示,骆驼Nb的VHH胚系基因序列和人VH3高度同源,但CDR1和CDR3比人的稍长,CDR3在三级结构中向外凸出,因而推测有更高的抗原结合的特异性和亲和力。 鉴于以上优点,Nb正被逐步开发为疾病诊断和治疗方面的单抗药物,广泛的应用与酶的抑制剂,肿瘤、感染和炎症等生物抑制剂的开发上。然而,纳米抗体微小的体积虽为其治疗功能提供了很多的优势,但小分子蛋白在体内极容易被消除。通过基因工程将Nb改造成靶点酶、跨膜蛋白或者双价化能够有效地提高抗体活力和稳定性等,以达到研究目的。在对抑制病毒复制的研究中发现,双价纳米抗体的有效性至少是单价纳米抗体的60倍,并且在动物体内作用时间更长,有效地延迟了动物的死亡时间。抗体药物的前景巨大,但国内抗体药物仍然处于起步阶段。因此,开发国产化PCSK9抗体抑制剂,满足我国国民对于抗体药物的迫切需求,具有深远而积极的意义。Antibody drugs are the main direction of new drug research and development, and have been widely used in the diagnosis, prevention and treatment of infectious diseases and biological science research. As of 2015, 48 antibody drugs have been successfully launched, and 7 antibody drugs have been successfully approved from April 2014 to March 2015 alone. In 2015, 6 of the top 10 drugs by global sales were antibody drugs. Since Hamers et al. discovered heavy-chain antibodies that naturally lacked light chains in camel blood in 1993, nanobodies (Nanobodies, Nb) have gradually replaced other small antibodies and have gradually become a hot spot in the development of new antibody drugs. Nb is usually only about 15kDa, about one-tenth of the size of traditional antibodies. It has disulfide bonds inside and a large number of hydrophilic residues on the surface, which has strong resistance to heat and pH; Nb lacks the Fc segment and light chain. The properties enable it to recognize cryptic epitopes or small epitopes that cannot be recognized by traditional antibodies, and avoid complement reactions; in addition, nanobodies have high stability, low toxicity, strong solubility, easy target screening and easy detection in prokaryotic microorganisms. Direct expression, good economy and many other advantages. Sequence homology analysis showed that the VHH germline gene sequence of camel Nb is highly homologous to human VH3, but CDR1 and CDR3 are slightly longer than those of human, and CDR3 protrudes outward in the tertiary structure, so it is speculated that there is a higher antigen Binding specificity and affinity. In view of the above advantages, Nb is gradually being developed as a monoclonal antibody drug for disease diagnosis and treatment, and is widely used in the development of enzyme inhibitors, tumor, infection and inflammation biological inhibitors. However, although the tiny size of nanobodies provides many advantages for their therapeutic functions, small-molecule proteins are easily eliminated in vivo. Transforming Nb into target enzyme, transmembrane protein or bivalent through genetic engineering can effectively improve antibody activity and stability, etc., to achieve research purposes. In the study of inhibiting virus replication, it was found that bivalent nanobodies were at least 60 times more effective than monovalent nanobodies, and they acted for a longer time in animals, effectively delaying the time of death of animals. Antibody drugs have great prospects, but domestic antibody drugs are still in their infancy. Therefore, it is of far-reaching and positive significance to develop localized PCSK9 antibody inhibitors to meet the urgent needs of Chinese people for antibody drugs.
2015年,国际药企巨头安进和赛诺菲/再生元先后经FDA批准上市两款PCSK9抑制剂Repatha和Praluent,迄今为止,二者销量逐年升高,每年营收高达5-7亿美元。2014年10月17日,安进控告赛诺菲/再生元的Praluent侵犯了其Repatha三项专利权(专利号为No.US8563698,US8829165和US8859741);最后赛诺菲败诉,他们被禁止在美国销售新的PCSK9抑制剂药物Praluent,由此可见表位保护的重要性,如今Praluent在国际上的销量不足Repatha的一半(据报道,赛诺菲是在以下表位的氨基酸上侵权安进的专利:S153,I154,P155,R194,D238,A239,I369,S372,D374,C375,T377,C378,F379,V380,和S381)。In 2015, the international pharmaceutical giants Amgen and Sanofi/Regeneron were approved by the FDA to launch two PCSK9 inhibitors, Repatha and Praluent. So far, the sales of the two have been increasing year by year, with an annual revenue of US$500-700 million. On October 17, 2014, Amgen sued Sanofi/Regeneron's Praluent for infringing three of its Repatha patents (Patent Nos. US8563698, US8829165 and US8859741); Sanofi lost the case and they were banned from the United States Sales of the new PCSK9 inhibitor drug Praluent, which shows the importance of epitope protection, today Praluent's international sales are less than half of Repatha's (reportedly, Sanofi is infringing Amgen's patent on the amino acid of the following epitope) : S153, I154, P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380, and S381).
发明内容SUMMARY OF THE INVENTION
为解决现有技术中缺乏有效的PCSK9抗原表位及靶向该表位的抗体的技术问题,本发明提供了一种抗体结合的特征性表位例如PCSK9抗原表位及其应用。In order to solve the technical problem of lack of effective PCSK9 antigenic epitope and antibodies targeting the epitope in the prior art, the present invention provides a characteristic epitope bound by an antibody such as PCSK9 antigenic epitope and its application.
目前已经上市的药物结合PCSK9的表位一般都处于PCSK9的前导区(31-152)和催化区(153-425),当病人对此类药物产生耐药或不反应时,需要结合PCSK9其它表位的药物。PCSK9的前导区和催化区内很多关键的表位氨基酸已经被赛诺菲或者安进公司申请专利保护,而PCSK9的C端区域很多氨基酸表位尚未被保护;之前的研究显示VHH4-Fc抗体结合PCSK9的亲和力高达0.6nM,在PCSK9转基因大鼠模型上降脂药效可达约40%的水平,说明该抗体具有极好的应用前景,该抗体所结合的表位也可以用来筛选新的抗PCSK9的抗体。The epitopes of the currently marketed drugs that bind to PCSK9 are generally located in the leader region (31-152) and catalytic region (153-425) of PCSK9. When a patient develops resistance or does not respond to such drugs, it is necessary to bind other epitopes of PCSK9. bit of drugs. Many key epitope amino acids in the leader region and catalytic region of PCSK9 have been patented by Sanofi or Amgen, while many amino acid epitopes in the C-terminal region of PCSK9 have not been protected; previous studies have shown that VHH4-Fc antibody binds The affinity of PCSK9 is as high as 0.6nM, and the lipid-lowering efficacy in PCSK9 transgenic rat model can reach a level of about 40%, indicating that the antibody has excellent application prospects, and the epitope bound by the antibody can also be used to screen new Anti-PCSK9 antibody.
为解决上述技术问题,本发明的第一方面提供了一种多肽片段,其中,所述多肽片段包含从N端至C端如X 1X 2X 3X 4X 5GX 6所示的氨基酸序列,其中,X 1为S、T、E、Y或F,优选为S;X 2为T、N、G、S或D,优选为T;X 3为H、E、Y、T、K、R或A,优选为H;X 4为G、Y、H、R或M,优选为G;X 5为A、F、Q、H、I或V,优选为A;X 6为W或F,优选为W。 In order to solve the above-mentioned technical problems, a first aspect of the present invention provides a polypeptide fragment, wherein the polypeptide fragment comprises an amino acid sequence such as X 1 X 2 X 3 X 4 X 5 GX 6 from the N-terminus to the C-terminus , wherein X 1 is S, T, E, Y or F, preferably S; X 2 is T, N, G, S or D, preferably T; X 3 is H, E, Y, T, K, R or A, preferably H; X 4 is G, Y, H, R or M, preferably G; X 5 is A, F, Q, H, I or V, preferably A; X 6 is W or F , preferably W.
优选地,所述多肽片段的氨基酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:9所示。Preferably, the amino acid sequence of the polypeptide fragment is such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: :7, SEQ ID NO:8 or SEQ ID NO:9.
本发明中阐述的VHH4-Fc对于PCSK9的结合表位处于C端区域(第447-453的:STHGAGW[SEQ ID NO:9])为一新的表位,如图1为PCSK9蛋白氨基酸序列和分区结构示意图。其中VHH4-Fc指的是纳米抗体VHH4融合了抗体恒定区Fc形成的嵌合抗体,VHH4的序列见专利1(申请号:201711298806.3)和专利2(申请号:PCT/CN2020/074624;PCSK9的单域抗体及其应用)。The binding epitope of VHH4-Fc to PCSK9 described in the present invention is in the C-terminal region (No. 447-453: STHGAGW [SEQ ID NO: 9]) as a new epitope, as shown in Figure 1 for the amino acid sequence of PCSK9 protein and Schematic diagram of the partition structure. VHH4-Fc refers to a chimeric antibody formed by fusion of Nanobody VHH4 with antibody constant region Fc. The sequence of VHH4 is shown in Patent 1 (Application No.: 201711298806.3) and Patent 2 (Application No.: PCT/CN2020/074624; domain antibodies and their applications).
优选地,所述多肽片段与VHH4抗体或其变体特异性结合,所述抗体的氨基酸序列如SEQ ID NO:23(VHH4)或如SEQ ID NO:24所示(VHH4-hFc)。Preferably, the polypeptide fragment specifically binds to a VHH4 antibody or variant thereof whose amino acid sequence is shown in SEQ ID NO: 23 (VHH4) or shown in SEQ ID NO: 24 (VHH4-hFc).
更优选地,所述突变为在所述抗体的氨基酸序列中具有一个或多个氨基酸残基的缺失、增加或替换。与纳米抗体VHH4或VHH4-Fc的氨基酸序列相似度80%以上视为与本发明所述的多肽片段结合的抗体的范围。即所述突变与所述抗体的氨基酸序列具有至少80%、85%、90%、95%、98%、99%的同一性并保持所述抗体的功能。More preferably, the mutation is a deletion, addition or substitution of one or more amino acid residues in the amino acid sequence of the antibody. The amino acid sequence similarity of 80% or more with the Nanobody VHH4 or VHH4-Fc is regarded as the range of the antibody that binds to the polypeptide fragment of the present invention. That is, the mutation is at least 80%, 85%, 90%, 95%, 98%, 99% identical to the amino acid sequence of the antibody and retains the function of the antibody.
Fc区既可以指人的抗体的恒定区,也可以指任何物种抗体的Fc恒定区,所述目标抗体包含任何物种的全部Fc亚型,即包含如IgG1 Fc,IgG2 Fc等。任何有关VHH4的分子修饰包括但不限于甲基化修饰、糖基化修饰,PEG修饰,小分子偶联如KLH-VHH4以及VHH4与对应轻链的二聚化形成抗体的Fab分子等。Fc亦可以替换为如人血清白蛋白形成HSA-VHH4或VHH4-HSA等任意融合VHH4的蛋白。由此,所述多肽片段可以结合的抗体为融合有人血清白蛋白的融合蛋白。The Fc region can refer to either the constant region of human antibodies or the Fc constant region of antibodies of any species, and the target antibody contains all Fc subtypes of any species, that is, IgG1 Fc, IgG2 Fc, etc. Any molecular modification related to VHH4 includes but is not limited to methylation modification, glycosylation modification, PEG modification, small molecule coupling such as KLH-VHH4 and dimerization of VHH4 and corresponding light chain to form antibody Fab molecules, etc. Fc can also be replaced with any protein fused to VHH4 such as human serum albumin forming HSA-VHH4 or VHH4-HSA. Thus, the antibody to which the polypeptide fragment can bind is a fusion protein fused to human serum albumin.
鉴定表位有很多种方法:包括蛋白结晶后晶体衍射法(X-ray法)和丙氨酸扫描突变法等;X-ray法鉴定效率高,精度高,但是存在成本高、抗原抗体复合物不好结晶、对复合物纯度要求高等缺点;丙氨酸扫描突变法需要突变大量的抗原分子,需要蛋白量大,而且有些部位氨基酸突变后表达量极低甚至不能表达,成本也很高。本发明中采用了对随机多肽库进行噬菌体展示的方法进行抗体结合抗原表位的鉴定,随后再采用Western进行表位验证,具有效率高,成本低的优势。There are many methods for identifying epitopes: including crystallography after protein crystallization (X-ray method) and alanine scanning mutagenesis method; X-ray method has high identification efficiency and high precision, but there are high cost, antigen-antibody complexes The disadvantages of poor crystallization and high requirements for the purity of the complex; the alanine scanning mutagenesis method requires a large number of antigen molecules to be mutated, and a large amount of protein, and the expression of amino acids in some parts is extremely low or even impossible to express, and the cost is also high. In the present invention, the method of phage display on a random polypeptide library is used to identify the antibody-binding antigenic epitope, and then Western is used for epitope verification, which has the advantages of high efficiency and low cost.
为解决上述技术问题,本发明的第二方面提供了一种分离的核酸分子,其编码如上所述的多肽片段。In order to solve the above technical problems, the second aspect of the present invention provides an isolated nucleic acid molecule encoding the above-mentioned polypeptide fragment.
为解决上述技术问题,本发明的第三方面提供了一种表达载体,其包含如上所述的分离的核酸分子;优选地,所述表达载体的基础载体(即骨架载体)为pMECS。In order to solve the above technical problems, a third aspect of the present invention provides an expression vector comprising the isolated nucleic acid molecule as described above; preferably, the base vector (ie, backbone vector) of the expression vector is pMECS.
为解决上述技术问题,本发明的第四方面提供了一种转化体,其含有如上所述的分 离的核酸分子或表达载体。优选地,所述转化体的宿主细胞为原核细胞或真核细胞。In order to solve the above-mentioned technical problem, the fourth aspect of the present invention provides a transformant, which contains the isolated nucleic acid molecule or expression vector as described above. Preferably, the host cells of the transformants are prokaryotic cells or eukaryotic cells.
为解决上述技术问题,本发明的第五方面提供了一种PCSK9抗原组合,其包括氨基酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:9所示的多肽片段中的两种或多种。In order to solve the above-mentioned technical problems, the fifth aspect of the present invention provides a PCSK9 antigen combination, which includes amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 4, and SEQ ID NO: 2. Two or more of the polypeptide fragments set forth in ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.
为解决上述技术问题,本发明的第六方面提供了一种多肽疫苗,其特征在于,其由以下方法制备而成:将包括氨基酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:9所示的多肽片段中的至少一种作为抗原产生的可特异性结合PCSK9的多克隆抗体。In order to solve the above-mentioned technical problem, the sixth aspect of the present invention provides a kind of polypeptide vaccine, it is characterized in that, it is prepared by the following method: will include amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID At least one of the polypeptide fragments shown in NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 is used as Antigen-produced polyclonal antibodies that specifically bind to PCSK9.
为解决上述技术问题,本发明的第七方面提供了一种配体,所述配体可以特异性结合如上所述的多肽片段,但所述配体不为氨基酸序列如SEQ ID NO:23或在SEQ ID NO:24所示的抗体。In order to solve the above-mentioned technical problems, the seventh aspect of the present invention provides a ligand, which can specifically bind to the above-mentioned polypeptide fragment, but the ligand is not an amino acid sequence such as SEQ ID NO: 23 or The antibody shown in SEQ ID NO:24.
为解决上述技术问题,本发明的第八方面提供了一种抑制剂,其可以靶向抑制如上所述的分离的核酸分子。优选地,所述抑制剂为siRNA或mRNA疫苗。In order to solve the above technical problem, the eighth aspect of the present invention provides an inhibitor, which can target and inhibit the isolated nucleic acid molecule as described above. Preferably, the inhibitor is an siRNA or mRNA vaccine.
为解决上述技术问题,本发明的第九方面提供了一种试剂盒,其包括如上所述的多肽片段,和/或,如上所述的配体。In order to solve the above technical problem, the ninth aspect of the present invention provides a kit, which includes the above-mentioned polypeptide fragment, and/or, the above-mentioned ligand.
为解决上述技术问题,本发明的第十方面提供了一种上述的多肽片段的制备方法,其包括如下步骤:培养如上所述的转化体,使其表达获得表达产物。In order to solve the above-mentioned technical problem, the tenth aspect of the present invention provides a method for preparing the above-mentioned polypeptide fragment, which comprises the following steps: culturing the above-mentioned transformant, and expressing it to obtain an expression product.
优选地,所述的制备方法还包括纯化所述的表达产物的步骤。Preferably, the preparation method further includes the step of purifying the expression product.
为解决上述技术问题,本发明的第十方面提供了一种所述的多肽片段、所述的分离的核酸分子、所述的表达载体、所述的转化体、所述的PCSK9抗原组合或所述的试剂盒在制备特异性结合PCSK9的抑制剂或检测PCSK9蛋白的制剂或PCSK9疫苗中的应用。In order to solve the above technical problems, the tenth aspect of the present invention provides the polypeptide fragment, the isolated nucleic acid molecule, the expression vector, the transformant, the PCSK9 antigen combination or the The application of the kit described in the preparation of an inhibitor that specifically binds to PCSK9 or a preparation for detecting PCSK9 protein or a PCSK9 vaccine.
为解决上述技术问题,本发明的第十一方面提供了一种如上所述的配体、所述的抑制剂在制备竞争性抑制抗PCSK9抗体的试剂中的应用。In order to solve the above technical problems, the eleventh aspect of the present invention provides the use of the above-mentioned ligand and the inhibitor in the preparation of a reagent for competitively inhibiting anti-PCSK9 antibody.
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。On the basis of conforming to common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.
本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are all commercially available.
本发明的积极进步效果在于:The positive progressive effect of the present invention is:
本发明探寻到不同以往抗体的结合表位,丰富了抗PCSK9抗体的结合表位的多样性。阐明了先前研发抗体VHH4-Fc与目标抗原PCSK9的相互作用机制,解析了相互作用表位。阐述了目标抗体和PCSK9抗原相互结合的表位,有利于提升VHH4-Fc该药物的市 场价值,也有利于对VHH4-Fc的进一步改造和优化。更重要的是,还可以利用该新的抗原表位来制作针对PCSK9的多肽疫苗;以及该多肽片段可与抗PCSK9抗体结合,可用于检测、筛选或辅助筛选新的抗PCSK9的抗体,以缓解现有技术中患者对原抗体出现免疫原性强等不良反应的问题。The present invention explores the binding epitopes different from the previous antibodies, and enriches the diversity of the binding epitopes of the anti-PCSK9 antibody. The interaction mechanism between the previously developed antibody VHH4-Fc and the target antigen PCSK9 was elucidated, and the interaction epitope was resolved. The epitope of the target antibody and PCSK9 antigen binding to each other is expounded, which is conducive to improving the market value of VHH4-Fc, and is also conducive to the further transformation and optimization of VHH4-Fc. More importantly, the new antigenic epitope can also be used to make a polypeptide vaccine against PCSK9; and the polypeptide fragment can be combined with anti-PCSK9 antibodies, which can be used for detection, screening or auxiliary screening of new anti-PCSK9 antibodies to alleviate In the prior art, the patient has the problem of strong immunogenicity and other adverse reactions to the primary antibody.
图1为人PCSK9的氨基酸序列;Fig. 1 is the amino acid sequence of human PCSK9;
图2为VHH4-Fc与PCSK9亲和力测定结果示意图;Figure 2 is a schematic diagram of the results of the affinity determination between VHH4-Fc and PCSK9;
图3为新型抗体与Repatha结合抗原表位差异鉴定实验;Figure 3 is a differential identification experiment of the novel antibody and Repatha binding epitope;
图4为人PCSK9抗原三种突变体的氨基酸序列对比示意图;Fig. 4 is the amino acid sequence comparison schematic diagram of three mutants of human PCSK9 antigen;
图5为人PCSK9及其三种突变体的Western Blot验证。Figure 5 shows Western Blot validation of human PCSK9 and its three mutants.
实施例1 VHH4-Fc和人PCSK9亲和力检测Example 1 Affinity detection of VHH4-Fc and human PCSK9
对于Fc标签融合蛋白VHH4-Fc,采用基于SPR技术的Protein A芯片捕获法来测定它与人PCSK9(hPCSK9)之间的亲和力。首先用Protein A芯片捕获VHH4-Fc抗体,再进样(流过)5个不同浓度的hPCSK9抗原进行亲和力的测定,结合时间设置为120s,解离时间设置位180s,再生溶液选取的是pH 2.0的100mM的甘氨酸溶液,系统溶液为1×PBST。如图2,最终的亲和力结果,是基于Biacore T200内置的评价软件给出的拟合曲线,再得出koff和kon值,由公式K D(nM)=koff(1/s)/kon(1/Ms)计算得出。参数说明:Rmax为最大响应值,通常是处于0-100RU之间;Chi 2值小于等于1/10Rmax视为测定结果可靠。测定结果显示:VHH4-Fc与hPCSK9的结合常数是1.872×10 6,解离常数是1.288×10 -3,Rmax(最大结合值)是24.71RU,Chi 2值为0.693,最终拟合得出的亲和力为0.6879nM。 For the Fc tag fusion protein VHH4-Fc, the protein A chip capture method based on SPR technology was used to determine its affinity with human PCSK9 (hPCSK9). First, the VHH4-Fc antibody was captured with a Protein A chip, and then 5 different concentrations of hPCSK9 antigen were injected (flowed through) for affinity determination. The binding time was set to 120s, the dissociation time was set to 180s, and the regeneration solution was pH 2.0 The 100 mM glycine solution, the system solution is 1x PBST. As shown in Figure 2, the final affinity result is based on the fitting curve given by the built-in evaluation software of Biacore T200, and then the koff and kon values are obtained. The formula K D (nM)=koff(1/s)/kon(1 /Ms) calculated. Parameter description: Rmax is the maximum response value, usually between 0-100RU; Chi 2 value less than or equal to 1/10Rmax is regarded as a reliable measurement result. The assay results show that the binding constant of VHH4-Fc and hPCSK9 is 1.872×10 6 , the dissociation constant is 1.288×10 -3 , the Rmax (maximum binding value) is 24.71RU, and the Chi 2 value is 0.693. The affinity is 0.6879nM.
图2中,横坐标指的是抗原抗体反应的时间轴,单位是(秒,s);纵坐标指的是抗原抗体的结合或者解离时的响应值,单位是Response units(RU)。不同的曲线代表进样不同浓度的抗体。本实验的亲和力是在25℃的反应条件下测试得到。In Figure 2, the abscissa refers to the time axis of the antigen-antibody reaction, and the unit is (second, s); the ordinate refers to the response value when the antigen-antibody is bound or dissociated, and the unit is Response units (RU). Different curves represent injections of different concentrations of antibody. The affinity of this experiment was tested under the reaction condition of 25℃.
实施例2表位差异检测
表位差异检测(epitope binning test)的目的是为了检测筛选出的抗体与上市药物Evolocumab(商品名:Repatha,瑞百安)结合人PCSK9表位的异同。本节共采用两种方 案来检测CN201711298806.3和PCT/CN2020/074624的VHH4的抗体(其氨基酸序列如SEQ ID NO:23所示)与已经上市的抗体药物结合靶点抗原PCSK9的表位差异。The purpose of the epitope binning test is to detect the similarities and differences between the screened antibodies and the marketed drug Evolocumab (trade name: Repatha, Repatha) in binding the epitope of human PCSK9. In this section, two schemes are used to detect the epitope difference between the VHH4 antibody of CN201711298806.3 and PCT/CN2020/074624 (the amino acid sequence of which is shown in SEQ ID NO: 23) and the marketed antibody drug binding target antigen PCSK9 .
第一种方案是基于SPR技术的亲和动力学检测,设置Biacore T200的反应温度为室温(即25℃),取一张新的CM5芯片,将上市抗体Evolocumab用1×PBS稀释至1μg/mL,然后偶联至通道2上,偶联水平拟定位约270RU,与此同时,保持1通道为空白对照。接下来,将带his标签的人PCSK9(简称hPCSK9-his)抗原用1×PBS稀释至5μg/mL,设置程序将其作为样本1(Sample 1)自动进样,结合时间设置为90s,如果人PCSK9抗原表面Evolocumab的特异性结合位点全部被饱和,结合和解离曲线都将进入平台期。接着,将抗体VHH4-his(浓度5μg/mL,用1×PBS稀释)作为样本2(Sample 2)继续进样60s,如果VHH4与Evolocumab结合hPCSK9的表位不同,曲线将会继续升高;反之,曲线将继续保持平台期。两种样本的解离时间均设置为120s,再生溶液为pH 2.0的100mM的甘氨酸,系统溶液为1×PBST。The first scheme is the affinity kinetics detection based on SPR technology. Set the reaction temperature of Biacore T200 to room temperature (
第二种方案为双抗体“夹心”ELISA法:取一块新的96孔ELISA板,每孔包被100ng的Evolocumab,条件为4℃过夜。次日用1×PBST洗涤三次后,用3%的BSA封闭96孔板孔底没有结合抗体的位点,条件为室温封闭1小时。用1×PBST洗涤三次后,接着用5μg/mL的hPCSK9-Fc抗原作为“一抗”室温孵育1小时;接着用1×PBST洗涤五次后,用系列倍比稀释的VHH4-his加到孔中,去结合人hPCSK9-Fc抗原上的抗原表位,如果Evolocumab和VHH4-his等新型抗体结合表位相同,VHH4-his将不会再结合hPCSK9-Fc而被洗掉,反之,VHH4-his不会被洗掉。接下来加辣根过氧化物酶(HRP)标记的抗his标签的二抗(1:5000稀释),室温反应1h。随后,用1×PBST洗涤三次后,每孔加入100μL的HRP底物TMB,在室温黑暗的环境中反应5~10min,随后加入50μL 1M的浓硫酸终止反应,用酶标仪读取OD450吸光值。The second protocol is the double-antibody "sandwich" ELISA method: take a new 96-well ELISA plate and coat each well with 100 ng of Evolocumab at 4°C overnight. After washing three times with 1×PBST on the next day, the unbound sites on the bottom of the 96-well plate were blocked with 3% BSA for 1 hour at room temperature. After three washes with 1×PBST, followed by incubation with 5 μg/mL hPCSK9-Fc antigen as the “primary antibody” for 1 hour at room temperature; followed by five washes with 1×PBST, serially diluted VHH4-his was added to the wells , to the epitope on the human hPCSK9-Fc antigen, if Evolocumab and VHH4-his and other novel antibodies bind the same epitope, VHH4-his will not bind to hPCSK9-Fc and be washed away, on the contrary, VHH4-his Will not be washed off. Next, horseradish peroxidase (HRP)-labeled anti-His-tag secondary antibody (1:5000 dilution) was added, and the reaction was carried out at room temperature for 1 h. Then, after washing three times with 1×PBST, add 100 μL of HRP substrate TMB to each well, react in a dark environment at room temperature for 5-10 min, then add 50 μL of 1M concentrated sulfuric acid to stop the reaction, and read the OD450 absorbance value with a microplate reader .
表位差异鉴定结果如图3所示,图3的A为基于表面等离子共振技术(SPR)检测VHH4和上市药物结合人PCSK9抗原表位的差异检测实验,所示为利用亲和动力学的方法测定的结果,横坐标为时间轴,纵坐标为相对响应值;共包含多个阶段,如基线阶段(baseline),捕获Evolocumab阶段(capture evolocumab),PCSK9进样并结合稳定的阶段(inject hPCSK9),VHH4进样阶段(inject VHH4)和解离阶段(dissociation阶段)。在第一个阶段,捕获Evolocumab后,反应曲线稳定在1200RU左右;在PCSK9-his进样后,Evolocumab与PCSK9-his开始结合,并最终稳定在约1350RU,证明PCSK9上Evolocumab的特异性结合位点被其自身饱和,随着VHH4的进样,反应曲线进一步上升,表明PCSK9上除了Evolocumab的特异性结合位点,还存在VHH4的特异性结合位点, 证实VHH4抗体和Evolocumab结合PCSK9的表位不尽相同。The epitope differential identification results are shown in Figure 3. Figure 3 A is a differential detection experiment based on surface plasmon resonance (SPR) to detect VHH4 and marketed drugs binding to human PCSK9 antigenic epitopes. The method using affinity kinetics is shown. The results of the assay, the abscissa is the time axis, and the ordinate is the relative response value; it contains multiple stages, such as the baseline stage (baseline), the capture evolocumab stage (capture evolocumab), the PCSK9 injection and binding stable stage (inject hPCSK9) , VHH4 injection stage (inject VHH4) and dissociation stage (dissociation stage). In the first stage, after capturing Evolocumab, the reaction curve stabilized at around 1200RU; after PCSK9-his injection, Evolocumab began to bind to PCSK9-his and finally stabilized at around 1350RU, proving the specific binding site of Evolocumab on PCSK9 Saturated by itself, with the injection of VHH4, the reaction curve further increased, indicating that in addition to the specific binding site of Evolocumab, there is also a specific binding site for VHH4 on PCSK9, confirming that the epitope of VHH4 antibody and Evolocumab does not bind to PCSK9. all the same.
图3的B为基于双抗体“夹心”ELISA技术检测(VHH4)和上市药物结合人PCSK9抗原表位的差异检测实验,横坐标代表不同浓度的VHH4;纵坐标代表OD450的吸光值。首先是在ELISA板中包被过量的Evolocumab,然后加入hPCSK9-Fc(作为“一抗”),Evolocumab将PCSK9上的特异性结合位点完全饱和,接着加入VHH4-his(作为“二抗”),如果VHH4-his与Evolocumab结合PCSK9的位点相同,最终在洗涤时将完全被洗掉,图3B中实验结果显示,VHH4与PCSK9在Evolocumab饱和其结合位点后仍有结合曲线,证实VHH4新型抗体和Evolocumab结合PCSK9的表位不尽相同。图3B右侧为双抗体夹心ELISA的实验原理示意图。B in Figure 3 is the difference detection experiment based on double antibody "sandwich" ELISA technology (VHH4) and marketed drug binding to human PCSK9 epitope, the abscissa represents VHH4 at different concentrations; the ordinate represents the absorbance value of OD450. The ELISA plate is first coated with an excess of Evolocumab, followed by the addition of hPCSK9-Fc (as the "primary antibody"), which fully saturates the specific binding site on PCSK9 with Evolocumab, followed by the addition of VHH4-his (as the "secondary antibody") , if VHH4-his and Evolocumab bind to the same site of PCSK9, it will eventually be completely washed away during washing. The experimental results in Figure 3B show that VHH4 and PCSK9 still have a binding curve after Evolocumab saturates their binding sites, confirming that VHH4 novel Antibodies and evolocumab bind different epitopes of PCSK9. The right side of FIG. 3B is a schematic diagram of the experimental principle of double-antibody sandwich ELISA.
实施例3表位鉴定实验Example 3 Epitope Identification Experiment
(1)噬菌体展示库的构建(1) Construction of phage display library
取1μL随机多肽库和噬菌体质粒pMECS相连接的DNA产物转化TG感受态细胞(相关分析克隆的步骤参见分子克隆实验指南(第2版)_(美)J.萨姆布鲁克(Joseph Sambrook));37℃复苏2h,梯度稀释至10 1、10 2和10 3,分别取300μL涂布平板,37℃,过夜培养,计算克隆数,约10 5个克隆/平板。采用上述相同的转化方法,大量转化,直到文库的克隆数达到10 7以上。将所有克隆用LB洗脱下,5,000g,离心5min,沉淀用2mL LB悬浮,加入等体积的30%甘油,-80℃冻存。 Take 1 μL of the random polypeptide library and the DNA product linked with the phage plasmid pMECS to transform the TG competent cells (for the relevant analysis and cloning steps, see Molecular Cloning Experiment Guide (2nd Edition)_(US) J. Joseph Sambrook); Resuscitate at 37°C for 2 hours, dilute to 10 1 , 10 2 and 10 3 in a gradient manner, take 300 μL of coated plates respectively, culture at 37° C. overnight, and count the number of clones, about 10 5 clones/plate. Using the same transformation method described above, a large number of transformations were carried out until the number of clones in the library reached more than 10 7 . All clones were eluted with LB, centrifuged at 5,000 g for 5 min, the pellet was suspended with 2 mL of LB, an equal volume of 30% glycerol was added, and frozen at -80°C.
(2)噬菌体扩增和拯救(2) Phage amplification and rescue
采用辅助噬菌体对随机多肽的噬菌体文库进行扩增和拯救。将步骤(1)中保存的单克隆文库接入100mL培养基中培养至对数生长期,加入MOI为20的辅助噬菌体,室温,静置30min,低速离心后,沉淀用培养基悬起,接入300mL培养基中,培养过夜。次日,3,000g离心30min,收集上清,加入PEG沉淀噬菌体,冰上静置30min,3,000g离心30min,沉淀为噬菌体随机多肽库,用PBS悬浮沉淀后,测定其滴度为2.9×10 12pfu/mL。 Phage libraries of random polypeptides were amplified and rescued using helper phages. The monoclonal library saved in step (1) was inserted into 100 mL of medium and cultured to the logarithmic growth phase, and the helper phage with MOI of 20 was added, at room temperature, allowed to stand for 30 min, after low-speed centrifugation, the sediment was suspended with medium, and then into 300 mL of medium and cultured overnight. The next day, centrifuge at 3,000g for 30min, collect the supernatant, add PEG to precipitate the phage, stand on ice for 30min, centrifuge at 3,000g for 30min, the precipitate is a phage random polypeptide library, and the titer is determined to be 2.9×10 12 pfu/mL.
(3)亲和VHH4-Fc的噬菌体库淘洗和拯救(3) Phage library panning and rescue of affinity VHH4-Fc
取100ng VHH4-Fc蛋白包被ELISA板,4℃,过夜孵育。次日,加入拯救出的VHH4-Fc特异的噬菌体,室温,孵育2h;PBST洗孔10次,加入100μL三乙胺,室温,孵育30min,收集的噬菌体即为获得的亲和VHH4-Fc的噬菌体库;取10μL感染TG细胞涂布平板,用于测定筛选后的克隆数测定,剩余的筛选后的噬菌体的用于扩增。按上述如此步骤反复淘选进行三至四轮多肽筛选。Take 100ng of VHH4-Fc protein to coat the ELISA plate and incubate at 4°C overnight. The next day, add the rescued VHH4-Fc-specific phage, incubate for 2 h at room temperature; wash the
(4)ELISA评价可以特异性结合VHH4-Fc的多肽的富集程度(4) ELISA to evaluate the degree of enrichment of polypeptides that can specifically bind to VHH4-Fc
ELISA板包被100ng的VHH4-Fc蛋白,4℃孵育过夜;次日加2%的BSA室温封闭1h;实验组分别加入每轮淘洗后扩增的噬菌体,对照组加入等量野生型的噬菌体,室温,孵育2h;PBST洗10次,以去除没有结合的噬菌体;加入HRP标记的抗M13抗体,室温孵育1h;加入显色液,避光反应10-30min,测吸光值,吸光值随着淘洗次数逐渐上升,并在第三轮到第四轮淘洗时趋于稳定,表明特异性的抗体得到了富集。The ELISA plate was coated with 100ng of VHH4-Fc protein and incubated at 4°C overnight; the next day, 2% BSA was added to block at room temperature for 1 h; the experimental group was added with the phage amplified after each round of panning, and the control group was added with the same amount of wild-type phage , at room temperature, incubate for 2h; wash 10 times with PBST to remove unbound phage; add HRP-labeled anti-M13 antibody, incubate at room temperature for 1h; add chromogenic solution, react in the dark for 10-30min, measure the absorbance, and the absorbance increases with the The number of panning increased gradually and stabilized during the third to fourth rounds of panning, indicating that specific antibodies were enriched.
(5)鉴定VHH4-Fc特异性结合的多肽的阳性克隆(5) Identification of positive clones of polypeptides specifically bound to VHH4-Fc
ELISA板包被100ng的VHH4-Fc,4℃孵育过夜;取最后一轮筛选获得的噬菌体涂布的平板,随机挑取38个单克隆于1mL培养基中,37℃,培养至对数期,加入1mM IPTG诱导过夜;次日,离心收集菌沉,破碎后,5,000g离心15min,收集上清;同时取ELISA板,加2%的BSA室温封闭1h;实验组每孔加入单克隆破碎上清,对照组加入空白TG破碎上清,室温,孵育2h;PBST洗10次,加入鼠抗HA标签的抗体,室温1h;PBST洗3-5次,加入AP标记的抗鼠IgG抗体,室温1h;加入底物,反应10-20min,在酶标仪上读取吸光值;当吸光值与对照孔比值大于2.1(Base line)时,判定为阳性克隆;并将阳性克隆对应孔的大肠杆菌送Sanger测序。The ELISA plate was coated with 100 ng of VHH4-Fc and incubated at 4°C overnight; the phage-coated plate obtained from the last round of screening was taken, and 38 single clones were randomly selected in 1 mL of medium, and incubated at 37°C to log phase. Add 1 mM IPTG to induce overnight; the next day, collect the bacterial sediment by centrifugation, after crushing, centrifuge at 5,000g for 15 min to collect the supernatant; at the same time, take the ELISA plate, add 2% BSA to block at room temperature for 1 hour; add monoclonal crushed supernatant to each well of the experimental group , the control group was added blank TG to break the supernatant, incubated at room temperature for 2 h; washed 10 times with PBST, added mouse anti-HA-labeled antibody for 1 h at room temperature; washed 3-5 times with PBST, added AP-labeled anti-mouse IgG antibody for 1 h at room temperature; Add the substrate, react for 10-20min, and read the absorbance value on the microplate reader; when the ratio of the absorbance value to the control well is greater than 2.1 (Base line), it is judged as a positive clone; and the E. coli in the corresponding hole of the positive clone is sent to Sanger Sequencing.
(6)阳性克隆的序列分析(6) Sequence analysis of positive clones
分析Sanger测序结果显示,共有8种独特型多肽与VHH4-Fc存在特异性结合,如下表2中的黑体字母加下划线代表了表位中可能存在的的固定氨基酸序列,比照PCSK9抗原的氨基酸序列,推断出VHH4-Fc与人PCSK9的作用表位(公共基序)可能为第447-453位的“STHGAGW”(序列位置见图1)。Analysis of Sanger sequencing results showed that a total of 8 idiotype polypeptides have specific binding to VHH4-Fc. The bold letters in the following Table 2 and underlines represent the fixed amino acid sequences that may exist in the epitope. Compared with the amino acid sequence of PCSK9 antigen, It is inferred that the interaction epitope (common motif) between VHH4-Fc and human PCSK9 may be "STHGAGW" at positions 447-453 (see Figure 1 for the sequence position).
我们总结出要保护的VHH4-Fc抗体结合的具体表位,应该包括但不限于氨基酸特征:(N端-X
1X
2X
3X
4X
5GX
6-C端),其中X代表20种氨基酸中的任意一种氨基酸,但一般地来讲,第1位X1经常为S、T、E和F中的任意一种氨基酸;第2位X2经常为T、Y、G、S和D中的任意一种氨基酸;第3位X3经常为H、E、Y、T、K和A中的任意一种氨基酸;第4位X4经常为G、Y、H、和R中的任意一种氨基酸;第5位X5经常为A、F、Q、H、I和V中的任意一种氨基酸;第7位X6经常为W和F中的任意一种氨基酸。
We summarize the specific epitopes bound by the VHH4-Fc antibody to be protected, which should include but are not limited to amino acid characteristics: (N-terminal-X 1 X 2 X 3 X 4 X 5 GX 6 -C-terminal), where X represents 20 species Any amino acid among amino acids, but generally speaking, X1 at
文中所述氨基酸密码子的对照表如下:The comparison table of amino acid codons described in the text is as follows:
表1氨基酸密码子对照表Table 1 Amino acid codon comparison table
表2基于随机多肽库和噬菌体展示技术鉴定到的多肽Table 2 Peptides identified based on random peptide library and phage display technology
实施例4表位验证实验Example 4 Epitope Verification Experiment
随后我们设计了两种有关“ STHGAGW”这一表位的PCSK9分子截断突变体,如下图4列出了三种突变体氨基酸的序列对比,其中标黄的部分为截断的部分氨基酸,其中PCSK9-mu01(SEQ ID NO:20)为截断无关表位的对照,PCSK9-mu02(SEQ ID NO:21)和PCSK9-mu03(SEQ ID NO:22)为截断部分推测表位的突变体。 Then we designed two PCSK9 molecular truncation mutants related to the epitope of " STHGAGW ". Figure 4 below lists the amino acid sequence comparison of the three mutants. mu01 (SEQ ID NO:20) is a control for truncating an irrelevant epitope, and PCSK9-mu02 (SEQ ID NO:21) and PCSK9-mu03 (SEQ ID NO:22) are mutants truncating a portion of the putative epitope.
如图5,将表达以上PCSK9蛋白及其突变体的哺乳细胞培养上清和细胞裂解物进行SDS-PAGE电泳,每五个一组,点样顺序从左到右分别为PCSK9-mu01、PCSK9-mu02、PCSK9-mu02(重复上样)、PCSK9-mu03和野生型PCSK9(PCSK9-WT);其中泳道1-5和11-15为表达PCSK9的细胞培养上清,泳道6-10和16-20为表达PCSK9的细胞裂解沉淀物,主要考察表达的目的蛋白处于胞内还是胞外;后将电泳胶转膜做Western Blot验证,主要考察截断突变后的PCSK9是否仍可以正常表达(图5A)以及是否可以和VHH4-hFc结合(图5B),结果如图5所示,PCSK9-WT(泳道5、10、15和20的76kDa处)在细胞培养上清和沉淀中均有表达,而其他三种PCSK9突变体均在胞内表达(泳道6-9),此外,截断突变对照PCSK9-mu01(泳道16)在截断后仍可以与VHH4-hFc发生结合,说明截断的多肽不是VHH4-hFc和PCSK9结合的关键表位;然而对于突变体PCSK9-mu02,虽然Anti-HA抗体检测胞内有表达(泳道7和8有条带),但与VHH4-hFc几乎不再结合(泳道17和18无条带);突变体PCSK9-mu03情况类似,虽然Anti-HA抗体检测胞内有表达(图A泳道9的48kDa处有条带),但VHH4-hFc与PCSK9-mu03不再结合(图B泳道19的48kDa处无条带),这说明“STHGAGW”(位于hPCSK9的第447-453位)确实为目标抗体VHH4-hFc与人PCSK9抗原的相互结合表位,或至少是hPCSK9空间表位的一部分。As shown in Figure 5, the mammalian cell culture supernatant and cell lysate expressing the above PCSK9 protein and its mutants were subjected to SDS-PAGE electrophoresis. Each group of five, the spotting order from left to right was PCSK9-mu01 and PCSK9-mu02, respectively. , PCSK9-mu02 (repeated loading), PCSK9-mu03 and wild-type PCSK9 (PCSK9-WT); lanes 1-5 and 11-15 are the supernatants of cells expressing PCSK9, lanes 6-10 and 16-20 are The cell lysate precipitates expressing PCSK9 mainly examine whether the expressed target protein is intracellular or extracellular; then the electrophoresis gel is transferred to the membrane for Western Blot verification, mainly to examine whether the truncation mutation of PCSK9 can still be expressed normally (Figure 5A) and whether Can bind to VHH4-hFc (Figure 5B), the results are shown in Figure 5, PCSK9-WT (
对比实施例1 Evolocumab表位鉴定实验Comparative Example 1 Evolocumab epitope identification experiment
将实施例3中的VHH-Fc替换为Evolocumab,其它实验步骤同实施例3。由此获得了与Evolocumab高亲和力结合的部分多肽。由下表3可知VHH4-Fc与Evolocumab结合的多肽片段完全不同,即这两个抗体结合完全不同的抗原表位。The VHH-Fc in Example 3 was replaced with Evolocumab, and other experimental steps were the same as those in Example 3. Thus, a partial polypeptide that binds with high affinity to Evolocumab was obtained. It can be seen from Table 3 below that the polypeptide fragments that VHH4-Fc binds to Evolocumab are completely different, that is, the two antibodies bind completely different antigenic epitopes.
表3基于随机多肽库和噬菌体展示技术鉴定到的多肽Table 3 Peptides identified based on random peptide library and phage display technology
虽然以上描述了本发明的具体实施方式,但是本领域的技术人员应当理解,这些仅是举例说明,在不背离本发明的原理和实质的前提下,可以对这些实施方式做出多种变更或修改。因此,本发明的保护范围由所附权利要求书限定。Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes may be made to these embodiments without departing from the principle and essence of the present invention. Revise. Accordingly, the scope of protection of the present invention is defined by the appended claims.
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