Anti-HERV-w env monoclonal antibody 4a6, preparation method therefor, and use thereof

Abstract

Provided is an anti-HERV-W Env monoclonal antibody 4A6. The amino acid sequences of six CDRs of the antibody are sequentially set forth in SEQ ID NOs. 1-6. A full-length heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO. 7, and a full-length light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO. 8. The prepared monoclonal antibody 4A6 has high purity, high binding activity and blocking activity, and good affinity. In addition, the monoclonal antibody drug has the advantages of strong targeting, few side effects, being producible in large quantities, etc. Therefore, the provided monoclonal antibody 4A6 provides a new candidate drug for the specific treatment of diseases such as multiple sclerosis.

Description

A monoclonal antibody 4A6 against HERV-W Env, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a monoclonal antibody 4A6 against HERV-W Env, its preparation method, and its application. Background Technology

[0002] The human endogenous retrovirus (HERV) family, discovered in 1982, comprises 8% of the human genome. Millions of years ago, exogenous retroviruses invaded the human body, infecting germ cells or embryonic stem cells, integrating their genes into the human genome, and vertically inheriting them to offspring. Through long-term evolution, they have become an integral part of the human genome. This family is divided into three main classes based on their similarity to exogenous retroviruses, with the HERV-W subfamily belonging to class I. The HERV genome consists of gag, pol, env, and two long transverse ends (LTRs). The gag gene encodes the structural components of the matrix, capsid, and nucleocapsid; the pol gene encodes enzymes required for viral replication; the env gene encodes envelope proteins; and the LTRs, formed during reverse transcription, play a crucial regulatory role in viral expression. HERV-W Env is a glycosylated protein encoded by human chromosome 7q21.2 with a molecular weight of 73 kDa. Because it can mediate cell-cell fusion, it is also known as syncytin-1. It consists of 538 amino acids, including a signal peptide (1-20 aa), an extracellular segment (21-317 aa), and a transmembrane segment (318-538 aa).

[0003] Under normal physiological conditions, HERV-W Env protein primarily functions in the human placenta, mediating placental morphology, promoting trophoblast cell fusion, and mediating immune tolerance between the fetus and mother. However, when stimulated by certain factors (such as Epstein-Barr virus), it can mediate the development of various diseases, including multiple sclerosis (MS), type 1 diabetes, mental illnesses, and cancer. In MS, HERV-W Env protein mainly binds to Toll-like receptor 4 (TLR4) on oligodendrocyte precursor cells (OPCs), thereby inhibiting OPC cell differentiation and leading to impaired myelin regeneration. In addition, HERV-W Env protein can also bind to TLR4 on immune cells, mediating the release of pro-inflammatory factors (such as IL-1β, IL-6, and TNF-α), triggering inflammation and exacerbating MS symptoms. In type 1 diabetes, HERV-W Env protein binds to TLR4 on the surface of pancreatic β cells, damaging these cells and inhibiting insulin secretion. In mental illnesses, the HERV-W Env protein can induce the release of nitric oxide (NO) in cells, which plays an important role in inflammatory states 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 various diseases by binding to TLR4. Therefore, developing a monoclonal antibody that can bind to HERV-W Env and block the binding of HERV-W Env to TLR4 is essential for the diagnosis and treatment of these diseases. Summary of the Invention

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

[0006] One objective of this invention is to provide a monoclonal antibody 4A6 against HERV-W Env, wherein the six CDR regions of the monoclonal antibody 4A6 are as follows:

[0007] (1) The heavy chain CDR1 contains the amino acid sequence shown in SEQ ID NO.1: GYTFTDYV;

[0008] (2) The heavy chain CDR2 contains the amino acid sequence shown in SEQ ID NO.2: IFPGTGNI;

[0009] (3) The heavy chain CDR3 contains the amino acid sequence shown in SEQ ID NO.3: ARWFGTYTWFAY;

[0010] (4) The light chain CDR1 contains the amino acid sequence shown in SEQ ID NO.4: KSVSTSGYSY;

[0011] (5) The light chain CDR2 contains the amino acid sequence shown in SEQ ID NO.5: LVS;

[0012] (6) The light chain CDR3 contains the amino acid sequence shown in SEQ ID NO.6: QHIMSYT.

[0013] 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 by replacing, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.7;

[0014] SEQ ID NO.7 is as follows: PPGAGEAGASVKMSCKASGYTFTDYVITWVKQRTGQGLEWIGEIFPGTGNIYYNEKFKGKATLTADHSSNTAYMQLSSLTSEDSAVYFCARWFGTYTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPE.

[0015] The full length of the light chain variable region of the monoclonal antibody 4A6 includes the amino acid sequence shown in SEQ ID NO.8, or a sequence with equivalent function formed by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.8;

[0016] SEQ ID NO.8 is as follows: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIMSYTYDA.

[0017] Furthermore, the monoclonal antibody 4A6 is a mouse IgG type antibody.

[0018] Furthermore, the antigen bound by the monoclonal antibody 4A6 is the HERV-W Env extracellular segment protein, and the amino acid sequence of the HERV-W Env extracellular segment protein is shown in SEQ ID NO.9;

[0019] SEQ ID NO.9 is as follows: APPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSKGTPTFTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQTGMSDGGGVQDQAREKHVKEVISQLTRVHGTSSPYKGLDLSKLHETLRTHTRL VSLFNTTTGLHEVSAQNPTNCWICLPLNFRPYVSIPVPEQWNNFSTEINTTSVLVGPLVSNLEITHTSNLTCVKFSNTTYTTNSQCIRWVTPPTQIVCLPSGIFFVCGTSAYRCLNGSSESMCFLSLFLVPPMTIYTEQDLYSYVISKPRNKR.

[0020] The second objective of this invention is to provide a method for preparing the above-mentioned monoclonal antibody 4A6, comprising the following steps:

[0021] S1. Mix the HERV-W Env extracellular fragment protein with an adjuvant and immunize female BALB / c mice.

[0022] S2. Spleen cells from immunized mice were fused with SP2 / 0 cells, screened, and cultured to obtain hybridoma cells;

[0023] S3. The hybridoma cells obtained by screening were expanded and cultured, and then injected intraperitoneally into female BALB / c mice. The ascites fluid was collected, centrifuged, purified, and screened to obtain the monoclonal antibody 4A6.

[0024] The third objective of this invention is to provide a nucleic acid fragment encoding the aforementioned monoclonal antibody 4A6.

[0025] The fourth objective of this invention is to provide the application of the above-mentioned monoclonal antibody 4A6 in the preparation of a reagent for detecting HERV-W Env.

[0026] The fifth objective of this invention is to provide the application of the above-mentioned monoclonal antibody 4A6 in the preparation of a reagent to inhibit HERV-W Env.

[0027] The sixth objective of this invention is to provide the application of the above-mentioned monoclonal antibody 4A6 in the preparation of a drug, wherein the drug is a drug for the prevention and / or treatment of diseases related to HERV-W Env.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] Due to the significant challenges in treating diseases such as multiple sclerosis and type 1 diabetes, there is an urgent need for more therapeutic drugs to provide more treatment options. Monoclonal antibody drugs offer advantages such as strong targeting, fewer side effects, and the ability to be mass-produced, making monoclonal antibody therapy possible. The monoclonal antibody prepared in this invention has 4A6 binding activity (IC50). 50 (6.118 ng / mL) and blocking activity (IC50) 50 The concentrations of the monoclonal antibody 4A6 were all relatively high (0.3027 μg / mL), and the affinity was good (Ka = 5.672E+41 / Ms, Kd = 3.856E-51 / s, KD = 6.799E-10M). Therefore, the monoclonal antibody 4A6 obtained in this invention provides a new candidate drug for the specific treatment of diseases such as multiple sclerosis. Attached Figure Description

[0030] Figure 1 shows the SDS-PAGE electrophoresis results of monoclonal antibody 4A6; where A is non-reducing SDS-PAGE electrophoresis and B is reducing SDS-PAGE electrophoresis.

[0031] Figure 2 shows the Western-Blot results of monoclonal antibody 4A6.

[0032] Figure 3 shows the results of the binding activity assay for monoclonal antibody 4A6.

[0033] Figure 4 shows the results of the blocking activity detection of monoclonal antibody 4A6; where A represents the binding activity of HERV-WEnv to TLR4, and B represents the blocking activity of the monoclonal antibody.

[0034] Figure 5 shows the affinity test results for monoclonal antibody 4A6. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. 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 reagents and consumables are commercially available products.

[0036] Example 1

[0037] This embodiment provides the preparation of monoclonal antibody 4A6, and the specific steps are as follows:

[0038] 1. Immunized animals

[0039] BALB / c mice were immunized with the extracellular fragment of HERV-W Env protein (sequence shown in SEQ ID NO. 9) expressed in Expi 293F cells. For the first injection, 100 μg of protein was mixed with Freund's complete adjuvant at a 1:1 volume ratio, diluted to 500 μL, and 200 μL was injected intraperitoneally. The remaining 300 μL was injected subcutaneously at three points on the back, 100 μL per point. For the second, third, and fourth injections, 100 μg of protein was mixed with Freund's incomplete adjuvant at a 1:1 volume ratio, diluted to 500 μL, and injected in the same manner as the first injection, with each injection spaced 14 days apart. Finally, 100 μg of protein was diluted to 100 μL and administered via tail vein pulse. Three days later, the mice with the highest serum titer were used for cell fusion.

[0040] 2. Preparation of hybridoma cells

[0041] Seven days prior to fusion, SP2 / 0 cells were resuscitated. One day prior to fusion, peritoneal macrophages from blank mice were used to seed the feeder layer. On the day of fusion, spleen cells from mice with the highest serum titers were mixed with SP2 / 0 cells at a cell ratio of 5:1, centrifuged, and PEG was added for cell fusion. After fusion, the cells were incubated at 37°C and 5% CO2. Seven days after fusion, hybridoma cells underwent a half-medium replacement with HT; twelve days after fusion, hybridoma cells underwent a full HT replacement; thirteen days after fusion, cells with high TLR4 binding activity were screened using indirect ELISA; fourteen days after fusion, the first subcloning was performed using limiting dilution, followed by a second and third subcloning every fourteen days.

[0042] 3. Preparation of ascites

[0043] Select hybridoma cells with high binding activity and expand their culture to T25 flasks. Seven days prior to the culture, inject BALB / c mice with 500 μL of Freund's incomplete adjuvant. Adjust the cell density to 2 × 10⁶ cells / mL. 6 The concentration of cells / mL was 500 μL per mouse. On the tenth day, the mice were euthanized by cervical dislocation, and the ascites fluid was collected. After being placed at 37°C for 2 hours, the fluid was centrifuged at 3000 rpm for 30 minutes at 4°C. The middle layer of ascites fluid was collected and filtered through a 0.22 μm filter membrane for sterilization.

[0044] 4. Purify the antibody

[0045] Ascites fluid was purified using a 5 mL protein G pre-packed gravity column. The sample was equilibrated with 5 column volumes of binding / wash buffer (0.15 M NaCl, 20 mM Na₂HPO₄, pH 7.4), diluted with an equal volume of binding / wash buffer, and loaded onto the column. The sample was then washed with 10 column volumes of binding / wash buffer, followed by elution with 5 column volumes of elution buffer (0.1 M glycine, pH 3.0) in five fractions. The eluent was neutralized with 1 / 10 of the elution volume of neutralizing buffer (1 M Tris-HCl, pH 8.5). The eluent was then replaced by ultrafiltration with sterile PBS buffer.

[0046] Example 2

[0047] This embodiment provides the functional identification of the monoclonal antibody 4A6 prepared above, and the specific steps are as follows:

[0048] 1. SDS-PAGE detection of monoclonal antibody 4A6

[0049] Four micrograms of purified monoclonal antibody 4A6 were subjected to non-reducing SDS-PAGE and reducing SDS-PAGE assays. The results are shown in Figure 1. The results showed that non-reducing SDS-PAGE exhibited a distinct band at 130–180 kDa, consistent with the antibody molecular weight; reducing SDS-PAGE showed distinct bands at 40–55 kDa and 25–35 kDa, consistent with the molecular weights of the heavy and light chains; ImageJ software analysis confirmed that the purity of both bands was greater than 90%.

[0050] 2. Western-Blot detection of monoclonal antibody 4A6

[0051] 4 μg of HERV-WEnv protein was sputtered onto a gel and transferred to a membrane. The membrane was blocked overnight at 4°C with 5% BSA, washed, and then 1 μg / mL of monoclonal antibody 4A6 was added as the primary antibody. After incubation at 37°C for 1 hour, the membrane was washed again. Goat anti-mouse IgG-HRP antibody diluted 1:10000 was added as the secondary antibody, and the membrane was incubated at 37°C for 1 hour, washed, and developed. The detection results are shown in Figure 2. The results show that monoclonal antibody 4A6 can specifically bind to the HERV-WEnv protein at 55 kDa.

[0052] 3. Indirect ELISA detection of monoclonal antibody 4A6 binding activity

[0053] Using HERV-W Env protein as the coating antigen, the solution was diluted to 0.1 μg / mL with bicarbonate buffer and coated onto an ELISA plate. After incubation at 4°C overnight, the plate was washed and blocked with 1% BSA at 37°C for 2 hours, followed by washing. Monoclonal antibody 4A6 was used as the primary antibody at a starting concentration of 40 μg / mL, and diluted 4-fold for a total of 12 concentration gradients. After incubation at 37°C for 1 hour, the plate was washed. Goat anti-mouse IgG-HRP antibody diluted to 1:10000 was added, and the plate was incubated at 37°C for 1 hour, followed by washing. TMB chromogenic solution was added, and the plate was incubated at 37°C for 15 minutes. Finally, stop solution was added, and the absorbance A was measured using an ELISA reader. 450nm Value. The detection results are shown in Figure 3. The results show that the IC50 value of monoclonal antibody 4A6 against HERV-WEnv protein is [value missing]. 50 The concentration was 6.118 ng / mL.

[0054] 4. Competitive ELISA detection of the blocking activity of monoclonal antibody 4A6

[0055] Using HERV-W Env protein as the coating antigen, it was diluted to 0.1 μg / mL with bicarbonate buffer and coated onto an ELISA plate. After incubation at 4°C overnight, the plate was washed and blocked with 1% BSA at 37°C for 2 hours, followed by washing. TLR4 protein was diluted to 8 μg / mL, 6 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, with the remaining 2-fold dilutions forming six concentration gradients. These were added to the ELISA plate as primary antibodies and incubated at 37°C for 1 hour, followed by washing. Anti-TLR4-HRP antibody diluted to 1:10000 was added, and the plate was incubated at 37°C for 1 hour, followed by washing. TMB chromogenic solution was added, and the plate was incubated at 37°C for 15 minutes. Stop solution was added, and the absorbance A was measured using an ELISA reader. 450nm Value. The detection results are shown in Figure 4A. The results show that TLR4 and HERV-WEnv protein have an EC50 value. 90 It is 6 μg / mL.

[0056] Using HERV-W Env protein as the coating antigen, it was diluted to 0.1 μg / mL with bicarbonate buffer and coated onto an ELISA plate. After incubation at 4°C overnight, the plate was washed and blocked with 1% BSA at 37°C for 2 hours, followed by washing. TLR4 protein was diluted to 12 μg / mL. Monoclonal antibody 4A6 was diluted 4-fold (12 concentration gradients) starting at 4 mg / mL. 50 μL each of TLR4 and monoclonal antibody 4A6 were added to the ELISA plate as primary antibodies. After incubation at 37°C for 1 hour, the plate was washed. Anti-TLR4-HRP antibody diluted to 1:10000 was added, and the plate was incubated at 37°C for 1 hour, followed by washing. TMB chromogenic solution was added, and the plate was incubated at 37°C for 15 minutes. Stop solution was added, and the absorbance A was measured using an ELISA reader. 450nm Value. The detection results are shown in Figure 4B. The results show that the antibody blocks the IC50 of HERV-WEnv binding to TLR4. 50It is 0.3027 μg / mL.

[0057] 5. Affinity detection of monoclonal antibody 4A6

[0058] The anti-mouse antibody was coupled to two channels on the CM5 chip. The remaining activation sites were blocked with 1M ethanolamine. Using HES-EP+ as the experimental buffer, 1 μg / mL of the antibody was captured. HERV-W Env was diluted to 0 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM, and 60 nM and injected into the chip, with 20 nM being the reproducible concentration. Finally, the chip was regenerated with glycine at pH 1.7. The detection results are shown in Figure 5. The results show that the binding constant (Ka) of monoclonal antibody 4A6 is 5.672E+41 / Ms, the dissociation constant (Kd) is 3.856E-51 / s, and the affinity constant (KD) is 6.799E-10M.

[0059] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection 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 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, characterized in that, 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 replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.

7. The full-length 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 replacing, deleting, or adding 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 mouse IgG type antibody.

4. The monoclonal antibody 4A6 according to claim 1 or 2, characterized in that, The antigen bound to the monoclonal antibody 4A6 is the extracellular segment 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 HERV-W Env extracellular fragment protein with an adjuvant and immunize female BALB / c mice. S2. Spleen cells from immunized mice were fused with SP2 / 0 cells, screened, and cultured to obtain hybridoma cells; S3. The hybridoma cells obtained by screening were expanded and cultured, and then injected intraperitoneally into female BALB / c mice. The ascites fluid was collected, centrifuged, purified, and screened 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. The 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. The use of the monoclonal antibody 4A6 according to any one of claims 1-4 in the preparation of a reagent to inhibit HERV-W Env.

9. The use of the monoclonal antibody 4A6 according to any one of claims 1-4 in the preparation of a medicament, wherein the medicament is a medicament for the prevention and / or treatment of diseases related to HERV-W Env.

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