Anti-HERV-W Env monoclonal antibody 4A6 as well as preparation method and application thereof

Abstract

The invention provides an anti-HERV-W Env monoclonal antibody 4A6. The amino acid sequences of six CDR regions of the antibody are shown as SEQ ID NO.1-6 in sequence; the full length of a heavy chain variable region of the antibody comprises an amino acid sequence as shown in SEQ ID NO.7, and the full length of a light chain variable region of the antibody comprises an amino acid sequence as shown in SEQ ID NO.8. The prepared monoclonal antibody 4A6 is high in purity, high in binding activity and blocking activity and good in affinity, meanwhile, the monoclonal antibody drug has the advantages of being high in targeting performance, small in side effect, capable of being prepared on a large scale and the like, and therefore the monoclonal antibody 4A6 provides a new candidate drug for specific treatment of diseases such as multiple sclerosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a monoclonal antibody 4A6 against HERV-W Env, a preparation method thereof, and an application thereof. Background Art

[0002] The human endogenous retrovirus (HERV) family was discovered in 1982 and accounts for 8% of the human genome. It is the result of exogenous retroviruses invading the human body millions of years ago, infecting human germ cells or embryonic stem cells, integrating their genes into the human genome, and being vertically inherited by offspring. After years of evolution, it has become part of the human genome. This family is divided into three major categories according to its similarity to exogenous retroviruses, and the HERV-W subfamily belongs to Class I. The genome of HERV consists of gag 、 pol 、 env and two long terminal repeats ( LTRs ). gag The LTRs gene encodes the structural parts of matrix, capsid, and nucleocapsid; pol The gag gene encodes the enzymes required for the virus replication process; env The pol gene encodes the envelope protein; LTRs is formed during the reverse transcription process and plays an important regulatory role in virus expression. HERV-W Env is a glycosylated protein encoded by human 7q21.2 chromosome No. 7, with a molecular weight of 73 kDa. Because it can mediate cell-cell fusion, it is also called syncytin-1. It consists of 538 amino acids, including a signal peptide (1-20 aa), an extracellular domain (21-317 aa), and a transmembrane segment (318-538 aa).

[0003] The HERV-W Env protein mainly acts on the human placenta under normal physiological conditions, mediates the occurrence of placental morphology, promotes the fusion of trophoblast cells, and mediates immune tolerance between the fetus and the mother. However, when stimulated by certain factors (such as Epstein-Barr virus, etc.), it will mediate the occurrence of various diseases, such as multiple sclerosis (MS), type I diabetes, mental diseases, and cancers, etc. In multiple sclerosis, the HERV-W Env protein mainly binds to Toll-like receptor 4 (TLR4) on oligodendrocyte precursor cells (OPCs), thereby inhibiting the differentiation of OPC cells and resulting in hindered myelin regeneration. In addition, the HERV-W Env protein can also bind to TLR4 on immune cells, mediate the release of pro-inflammatory factors (such as IL-1β, IL-6, and TNF-α), trigger inflammation, and thus exacerbate the symptoms of MS. In type I diabetes, the HERV-W Env protein binds to TLR4 on the surface of pancreatic islet β cells, causing damage to pancreatic islet β cells, and thus inhibiting insulin secretion. In mental diseases, the HERV-W Env protein can induce the release of nitric oxide (NO) in cells, and nitric oxide plays an important role in the inflammatory state of the brain. In addition, the HERV-W Env protein can also activate the TLR3 and TLR4 pathways in glial cells, inducing inflammation.

[0004] In summary, HERV-W Env mainly causes the occurrence of various diseases by binding to TLR4. Therefore, it is very necessary to develop a monoclonal antibody that can bind to HERV-W Env and block the binding of HERV-W Env to TLR4 for the diagnosis and treatment of diseases. Summary of the Invention

[0005] In view of this, the present invention provides a monoclonal antibody 4A6 that can bind to HERV-W Env and block the binding of HERV-W Env to TLR4.

[0006] One of the purposes of the present invention is to provide: a monoclonal antibody 4A6 against HERV-W Env, and the six CDR regions of the monoclonal antibody 4A6 are specifically as follows: (1) Heavy chain CDR1 contains the amino acid sequence shown in SEQ ID NO.1: GYTFTDYV; (2) Heavy chain CDR2 contains the amino acid sequence shown in SEQ ID NO.2: IFPGTGNI; (3) Heavy chain CDR3 contains the amino acid sequence shown in SEQ ID NO.3: ARWFGTYTWFAY; (4) Light chain CDR1 contains the amino acid sequence shown in SEQ ID NO.4: KSVSTSGYSY; The light chain CDR2 contains the amino acid sequence shown in SEQ ID NO.5: LVS; The light chain CDR3 contains the amino acid sequence shown in SEQ ID NO.6: QHIMSYT.

[0007] Furthermore, the full length of the heavy chain variable region of the monoclonal antibody 4A6 contains the amino acid sequence shown in SEQ ID NO.7, or a sequence with equivalent function formed after substitution, deletion or addition of one or several amino acids to the amino acid sequence shown in SEQ ID NO.7; SEQ ID NO.7 is specifically as follows: PPGAGEAGASVKMSCKASGYTFTDYVITWVKQRTGQGLEWIGEIFPGTGNIYYNEKFKGKATLTADHSSNTAYMQLSSLTSEDSAVYFCARWFGTYTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPE.

[0008] The full length of the light chain variable region of the monoclonal antibody 4A6 contains the amino acid sequence shown in SEQ ID NO.8, or a sequence with equivalent function formed after substitution, deletion or addition of one or several amino acids to the amino acid sequence shown in SEQ ID NO.8; SEQ ID NO.8 is specifically as follows: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIMSYTYDA.

[0009] Furthermore, the monoclonal antibody 4A6 is a murine IgG type antibody.

[0010] Furthermore, the antigen bound by the monoclonal antibody 4A6 is the extracellular domain protein of HERV-W Env, and the amino acid sequence of the extracellular domain protein of HERV-W Env is shown in SEQ ID NO.9; SEQ ID NO. 9 is as follows: APPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLSKGTPTFTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQTGMSDGGGVQDQAREKHVKEVISQLTRVHGTSSPYKGLDLSKLHETLRTHTRLVSLFNTTLTGLHEVSAQNPTNCWICLPLNFRPYVSIPVPEQWNNFSTEINTTSVLVGPLVSNLEITHTSNLTCVKFSNTTYTTNSQCIRWVTPPTQIVCLPSGIFFVCGTSAYRCLNGSSESMCFLSFLVPPMTIYTEQDLYSYVISKPRNKR.

[0011] The second object of the present invention is to provide: A method for preparing the above-mentioned monoclonal antibody 4A6, comprising the following steps: S1. Mix the extracellular domain protein of HERV-W Env with an adjuvant and immunize female BALB / c mice. S2. Take the spleen cells of the immunized mice and fuse them with SP2 / 0 cells, screen and culture to obtain hybridoma cells. S3. Expand the culture of the obtained hybridoma cells, intraperitoneally inject female BALB / c mice, collect ascites, purify and screen after centrifugation to obtain the monoclonal antibody 4A6.

[0012] The third object of the present invention is to provide: A nucleic acid fragment encoding the above-mentioned monoclonal antibody 4A6.

[0013] The fourth object of the present invention is to provide: The application of the above-mentioned monoclonal antibody 4A6 in the preparation of a reagent for detecting HERV-W Env.

[0014] The fifth object of the present invention is to provide: The application of the above-mentioned monoclonal antibody 4A6 in the preparation of a reagent for inhibiting HERV-W Env.

[0015] The sixth object of the present invention is to provide: The application of the above-mentioned monoclonal antibody 4A6 in the preparation of a drug, and the drug is a drug for preventing and / or treating diseases related to HERV-W Env.

[0016] Compared with the prior art, the beneficial effects of the present invention are: Due to the current great difficulty in treating diseases such as multiple sclerosis and type I diabetes, there is an urgent need for more therapeutic drugs to provide more alternative treatment options for the treatment of diseases. Monoclonal antibody drugs have the advantages of strong targeting, small side effects, and large-scale preparation, making monoclonal antibody treatment possible. The monoclonal antibody 4A6 prepared in the present invention has relatively high binding activity (IC 50 is 6.118 ng / mL) and blocking activity (IC 50 is 0.3027 μg / mL), and good affinity (Ka is 5.672E+4 1 / Ms, Kd is 3.856E-5 1 / s, KD is 6.799E-10 M). Therefore, the monoclonal antibody 4A6 obtained in the present invention provides a new candidate drug for the specific treatment of diseases such as multiple sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the SDS-PAGE electrophoresis detection result of monoclonal antibody 4A6; among them, A is non-reducing SDS-PAGE electrophoresis, and B is reducing SDS-PAGE electrophoresis.

[0018] Figure 2 It is the Western-Blot detection result of monoclonal antibody 4A6.

[0019] Figure 3 It is the binding activity detection result of monoclonal antibody 4A6.

[0020] Figure 4 It is the blocking activity detection result of monoclonal antibody 4A6; among them, A is the binding activity of HERV-W Env and TLR4, and B is the monoclonal antibody blocking activity.

[0021] Figure 5 It is the affinity detection result of monoclonal antibody 4A6. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The present invention will be further described in detail below in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well-known to those skilled in the art, and all reagent consumables are commercially available products.

[0023] Example 1 This example provides the preparation of monoclonal antibody 4A6, and the specific steps are as follows: 1. Immunize animals Immunize BALB / c mice with the extracellular domain protein of HERV-W Env expressed by Expi 293F cells (the sequence is shown in SEQ ID NO.9). For the first injection, take 100 μg of the protein, mix it with Freund's complete adjuvant at a volume ratio of 1:1, dilute it to 500 μL, inject 200 μL intraperitoneally, and take the remaining 300 μL and inject it subcutaneously at three points on the back, 100 μL per point. For the second, third, and fourth injections, take 100 μg of the protein, mix it with Freund's incomplete adjuvant at a volume ratio of 1:1, dilute it to 500 μL, and inject it in the same way as the first injection. The interval between each injection is 14 days. Finally, take 100 μg of the protein, dilute it to 100 μL, and perform a tail vein boost. Three days later, select the mouse with the highest serum titer for cell fusion.

[0024] 2. Preparation of hybridoma cells Resuscitate SP2 / 0 cells 7 days before fusion. One day before fusion, take peritoneal macrophages from blank mice to lay feeder cells. On the day of fusion, mix the spleen cells of the mouse with the highest serum titer and SP2 / 0 cells at a cell number ratio of 5:1, centrifuge, add PEG for cell fusion, and incubate the cells at 37°C and 5% CO2 after fusion. On the seventh day after fusion, perform a half-medium change for the hybridoma cells with HT; on the twelfth day after fusion, perform a full-medium change for the hybridoma cells with HT; on the thirteenth day after fusion, screen out the cells with higher binding activity to TLR4 by the indirect ELISA method; on the fourteenth day after fusion, perform the first subcloning by the limited dilution method, and then perform the second and third subclonings every 14 days.

[0025] 3. Preparation of ascites Select hybridoma cells with high binding activity and expand the culture to a T25 flask. Inject 500 μL of Freund's incomplete adjuvant into BALB / c mice 7 days in advance. Adjust the cell density to 2×10 6 cells / mL, and inject 500 μL per mouse. On the tenth day, decapitate the mice to collect ascites. After placing it at 37°C for 2 h, centrifuge at 3000 rpm for 30 min at 4°C, aspirate the middle layer of ascites, and filter and sterilize it with a 0.22 μm filter membrane.

[0026] 4. Purification of antibodies Purify the ascites with a 5 mL protein G pre-packed gravity column. Equilibrate it with 5 column volumes of binding / washing buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.4). Dilute the sample with an equal volume of binding / washing buffer and load it onto the column. Wash it with 10 column volumes of binding / washing buffer. Elute it with 5 column volumes of elution buffer (0.1 M glycine, pH 3.0) in five tubes, and neutralize it with 1 / 10 elution volume of neutralization solution (1 M Tris-HCl, pH 8.5). Ultrafilter and replace the eluate with sterile PBS buffer.

[0027] Example 2 This example provides the functional identification of the monoclonal antibody 4A6 prepared above. The specific steps are as follows: 1. SDS-PAGE detection of monoclonal antibody 4A6 Take 4 μg of the purified monoclonal antibody 4A6 each for non-reducing SDS-PAGE and reducing SDS-PAGE detection. The detection results are as Figure 1 shown. The results show that there are obvious bands at 130 - 180 kDa in non-reducing SDS-PAGE, which is consistent with the molecular weight of the antibody; there are obvious bands at 40 - 55 kDa and 25 - 35 kDa in reducing SDS-PAGE, which is consistent with the molecular weights of the heavy and light chains of the antibody; analyzed by Image J software, the purity is greater than 90%.

[0028] 2. Western-Blot detection of monoclonal antibody 4A6 Take 4 μg of HERV-W Env protein, run it on a gel and transfer the membrane. Block it overnight at 4°C with 5% BSA and then wash the membrane. Add 1 μg / mL of monoclonal antibody 4A6 as the primary antibody, incubate it at 37°C for 1 h and then wash the membrane. Add goat anti-mouse IgG-HRP antibody with a dilution ratio of 1:10000 as the secondary antibody, incubate it at 37°C for 1 h and then wash the membrane and develop the color. The detection results are as Figure 2 shown. The results show that monoclonal antibody 4A6 can specifically bind to the HERV-W Env protein at 55 kDa.

[0029] 3. Indirect ELISA to detect the binding activity of monoclonal antibody 4A6 Using the HERV-W Env protein as the coating antigen, it was diluted to 0.1 μg / mL with bicarbonate buffer, coated on an ELISA plate, incubated overnight at 4°C, then the plate was washed. After blocking with 1% BSA at 37°C for 2 h and washing the plate again, the monoclonal antibody 4A6 was used as the primary antibody with an initial concentration of 40 μg / mL and serially diluted 4-fold for a total of 12 concentration gradients. After incubation at 37°C for 1 h, the plate was washed. Then, goat anti-mouse IgG-HRP antibody diluted to 1:10,000 was added, incubated at 37°C for 1 h, and the plate was washed again. TMB chromogenic solution was added, incubated at 37°C for 15 min, and the stop solution was added. The absorbance A was measured using an ELISA reader. 450nm value. The detection results are as Figure 3 shown. The results showed that the IC 50 of the monoclonal antibody 4A6 against the HERV-W Env protein was 6.118 ng / mL.

[0030] 4. Competitive ELISA for detecting the blocking activity of monoclonal antibody 4A6 Using the HERV-W Env protein as the coating antigen, it was diluted to 0.1 μg / mL with bicarbonate buffer, coated on an ELISA plate, incubated overnight at 4°C, then the plate was washed. After blocking with 1% BSA at 37°C for 2 h and washing the plate again, the TLR4 protein was serially diluted to 8 μg / mL, 6 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and then serially diluted 2-fold for 6 more concentration gradients as the primary antibody and added to the ELISA plate. After incubation at 37°C for 1 h, the plate was washed. Then, anti-TLR4-HRP antibody diluted to 1:10,000 was added, incubated at 37°C for 1 h, and the plate was washed again. TMB chromogenic solution was added, incubated at 37°C for 15 min, and the stop solution was added. The absorbance A was measured using an ELISA reader. 450nm value. The detection results are as Figure 4 shown in Figure A. The results showed that the EC 90 of TLR4 and the HERV-W Env protein was 6 μg / mL.

[0031] Using the HERV-W Env protein as the coating antigen, it was diluted to 0.1 μg / mL with bicarbonate buffer, coated on an ELISA plate, incubated overnight at 4°C, then the plate was washed. After blocking with 1% BSA at 37°C for 2 h and washing the plate again, the TLR4 protein was diluted to 12 μg / mL, and the monoclonal antibody 4A6 was serially diluted with an initial concentration of 4 mg / mL and serially diluted 4-fold for a total of 12 concentration gradients. 50 μL of TLR4 and the monoclonal antibody 4A6 were used as the primary antibody and added to the ELISA plate. After incubation at 37°C for 1 h, the plate was washed. Then, anti-TLR4-HRP antibody diluted to 1:10,000 was added, incubated at 37°C for 1 h, and the plate was washed again. TMB chromogenic solution was added, incubated at 37°C for 15 min, and the stop solution was added. The absorbance A was measured using an ELISA reader. 450nm value. The detection results are as Figure 4As shown in Figure B. The results showed that the IC of the antibody blocking the binding of HERV-W Env to TLR4 50 was 0.3027 μg / mL.

[0032] 5. Affinity detection of monoclonal antibody 4A6 Couple anti-mouse antibody to two channels on the CM5 chip, block the remaining activated sites with 1 M ethanolamine, use HES-EP+ as the experimental buffer, capture 1 μg / mL of the antibody to be tested, dilute HERV-W Env to 0 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM, 60 nM and inject it into the chip. Among them, 20 nM is the repeated concentration. Finally, regenerate the chip with glycine at pH 1.7. The detection results are as Figure 5 shown. The results showed that the binding constant (Ka) of monoclonal antibody 4A6 was 5.672E+4 1 / Ms, the dissociation constant (Kd) was 3.856E-5 1 / s, and the affinity constant (KD) was 6.799E-10 M.

[0033] The above are only the preferred implementation modes of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody 4A6 against HERV-W Env, characterized in that, The six CDR regions of the monoclonal antibody 4A6 are specifically as follows: (1) The heavy-chain CDR1 contains the amino acid sequence shown in SEQ ID NO.1; (2) The heavy-chain CDR2 contains the amino acid sequence shown in SEQ ID NO.2; (3) The heavy-chain CDR3 contains the amino acid sequence shown in SEQ ID NO.3; (4) The light-chain CDR1 contains the amino acid sequence shown in SEQ ID NO.4; (5) The light-chain CDR2 contains the amino acid sequence shown in SEQ ID NO.5; (6) The light-chain CDR3 contains the amino acid sequence shown in SEQ ID NO.

6.

2. The monoclonal antibody 4A6 according to claim 1, wherein The full length of the heavy-chain variable region of the monoclonal antibody 4A6 contains the amino acid sequence shown in SEQ ID NO.7, or a sequence with equivalent function formed by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO.7; The full length of the light-chain variable region of the monoclonal antibody 4A6 contains the amino acid sequence shown in SEQ ID NO.8, or a sequence with equivalent function formed by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO.

8.

3. The monoclonal antibody 4A6 according to claim 1 or 2, characterized in that, The monoclonal antibody 4A6 is a murine IgG type antibody.

4. The monoclonal antibody 4A6 according to claim 1 or 2, characterized in that, The antigen bound by the monoclonal antibody 4A6 is the extracellular domain protein of HERV-W Env.

5. A method for preparing the monoclonal antibody 4A6 according to any one of claims 1-4, comprising the following steps: S1. Mix the extracellular domain protein of HERV-W Env with an adjuvant and immunize female BALB / c mice; S2. Take the spleen cells of the immunized mice and fuse them with SP2 / 0 cells, and screen and culture to obtain hybridoma cells; S3. Expand the culture of the screened hybridoma cells, intraperitoneally inject female BALB / c mice, collect ascites, and purify and screen after centrifugation to obtain the monoclonal antibody 4A6.

6. A nucleic acid fragment encoding the monoclonal antibody 4A6 according to any one of claims 1-4.

7. Use of the monoclonal antibody 4A6 according to any one of claims 1-4 in the preparation of a reagent for detecting HERV-W Env.

8. Use of the monoclonal antibody 4A6 according to any one of claims 1-4 in the preparation of a reagent for inhibiting HERV-W Env.

9. Use of the monoclonal antibody 4A6 according to any one of claims 1-4 in the preparation of a drug, wherein the drug is a drug for preventing and / or treating diseases related to HERV-W Env.

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