WO2022021000A1 - Épitope caractéristique de liaison à l'antigène et son utilisation - Google Patents
Épitope caractéristique de liaison à l'antigène et son utilisation 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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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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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
L'invention concerne un fragment polypeptidique et son procédé de préparation. Le fragment polypeptidique comprend une séquence d'acides aminés telle que représentée dans X1X2X3X4X5GX6 de l'extrémité N-terminale à l'extrémité C-terminale, dans laquelle X1 représente S, T, E, Y ou F; X2 représente T, N, G, S ou D; X3 représente H, E, Y, T, K, R ou A; X4 représente G, Y, H, R ou M; X5 représente A, F, Q, H, I ou V; X6 représente W ou F. L'invention concerne également une molécule d'acide nucléique isolée codant pour le polypeptide, et la préparation d'un vaccin polypeptidique pour PCSK9 utilisant le polypeptide. Le fragment polypeptidique peut être lié à un anticorps anti-PCSK9, et peut être utilisé pour la détection, le criblage ou le criblage auxiliaire pour un nouvel anticorps anti-PCSK9, de sorte que le problème d'une réaction indésirable, par exemple, une forte immunogénicité d'un patient sur un anticorps d'origine, est atténué.
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