阅读说明：本技术 Hpv58l1的单克隆中和抗体及其应用 (Monoclonal neutralizing antibody of HPV58L1 and application thereof ) 是由 魏颖颖 邹国宝 刘欣欣 郭光华 蔺皓 宋高尚 沈江卫 于 2019-12-30 设计创作，主要内容包括：本发明涉及抗体药物技术领域,尤其涉及HPV58L1的单克隆中和抗体及其应用。本发明提供了HPV58的特异性的单克隆中和抗体以及产生该抗体的杂交瘤细胞系,利用其中的一株中和活性效价最高的单克隆中和抗体,采用间接ELISA法、双抗夹心ELISA法、免疫杂交法等,可广泛应用于临床样本中HPV58病毒的感染检测；利用其中的一株中和活性效价最高的单克隆中和抗体,制备成阴道凝胶、阴道清洗液、外用洗液、阴道栓剂、冻干粉、阴道泡腾片等,用于HPV58感染患者的治疗,能有效提高HPV58患者的临床转阴率。本发明对女性的健康发展和公共卫生防控均具有重要的意义。(The invention relates to the technical field of antibody medicines, in particular to a monoclonal neutralizing antibody of HPV58L1 and application thereof. The invention provides specific monoclonal neutralizing antibodies of HPV58 and a hybridoma cell line for producing the antibodies, wherein one monoclonal neutralizing antibody with the highest neutralizing activity titer is utilized, and an indirect ELISA method, a double-antibody sandwich ELISA method, an immune hybridization method and the like are adopted, so that the monoclonal neutralizing antibodies can be widely applied to infection detection of HPV58 virus in clinical samples; the monoclonal neutralizing antibody with the highest neutralizing activity titer is used for preparing vaginal gel, vaginal cleaning solution, external washing solution, vaginal suppository, freeze-dried powder, vaginal effervescent tablets and the like, is used for treating patients infected with HPV58, and can effectively improve the clinical negative conversion rate of patients infected with HPV 58. The invention has important significance for the health development and public health prevention and control of women.)

1. A monoclonal antibody characterized by having a sequence selected from the group consisting of,

the amino acid sequence of at least one of the CDR regions of the heavy chain has the amino acid sequence shown as SEQ ID NO.1, 2 or 3 or a sequence with at least 80% sequence identity with the amino acid sequence;

wherein at least one of the CDR regions of the light chain has an amino acid sequence as set forth in SEQ ID NO 4, 5 or 6 or a sequence having at least 80% sequence identity thereto.

2. The monoclonal antibody according to claim 1,

the heavy chain comprises three CDR regions, and the amino acid sequences of the CDR regions respectively have the amino acid sequences shown as SEQ ID NO 1, 2 and 3;

the light chain comprises three CDR regions, and the amino acid sequences of the CDR regions respectively have the amino acid sequences shown in SEQ ID NO. 4, 5 and 6.

3. The monoclonal antibody according to claim 1 or 2,

the amino acid sequences of the 4 FR regions of the heavy chain have the amino acid sequences shown as SEQ ID NO 7, 8, 9 and 10 respectively;

the amino acid sequences of the 4 FR regions of the light chain have the amino acid sequences shown as SEQ ID NOS: 11, 12, 13 and 14, respectively.

4. The monoclonal antibody according to any one of claims 1 to 3,

the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NO 15;

the light chain variable region has an amino acid sequence shown in any one of SEQ ID NO 16.

5. The monoclonal antibody of any one of claims 1-4, wherein the heavy chain constant region is of the IgG1 type and the light chain constant region is of the kappa type.

6. The monoclonal antibody according to any one of claims 1 to 5, which is produced by a hybridoma cell line having a accession number of CGMCCNo.19187.

7. A polynucleotide encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1 to 6.

8. A nucleic acid vector comprising a polynucleotide encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1 to 6.

9. A recombinant host comprising the nucleic acid vector of claim 8.

10. A method for producing the monoclonal antibody according to any one of claims 1 to 6, comprising:

culturing the recombinant host of claim 9 to induce expression of the monoclonal antibody;

or culturing hybridoma cell strain with preservation number of CGMCC No.19187 to obtain the monoclonal antibody.

11. A chemically or biologically labeled monoclonal antibody according to any one of claims 1 to 6.

12. A conjugate prepared by conjugating the monoclonal antibody of any one of claims 1 to 6 or the monoclonal antibody of claim 11 to a solid or semi-solid medium.

13. Use of the monoclonal antibody of any one of claims 1 to 6, the monoclonal antibody of claim 11 and/or the conjugate of claim 12 for the preparation of a product for detecting HPV58 type.

14. A kit comprising the monoclonal antibody of any one of claims 1 to 6, the monoclonal antibody of claim 11 and/or the conjugate of claim 12.

15. Use of the monoclonal antibody of any one of claims 1 to 6, the monoclonal antibody of claim 11 and/or the conjugate of claim 12 for the preparation of a formulation against infection by HPV58 type virus.

16. Use of the monoclonal antibody according to any one of claims 1 to 6, the monoclonal antibody according to claim 11 and/or the conjugate according to claim 12 for the preparation of a formulation for the prevention and treatment of tumors and/or warts.

17. Use according to claim 16,

the tumor cell is cervical cancer, vulvar cancer, vaginal cancer, anal cancer, rectal cancer, oral cancer, tonsil cancer, penis cancer, prostatic cancer or bladder cancer;

the warts are genital warts, flat warts or common warts.

18. A medicament or vaccine comprising a monoclonal antibody according to any one of claims 1 to 6, a monoclonal antibody according to claim 11 and/or a conjugate according to claim 12.

Technical Field

The invention relates to the technical field of antibody medicines, in particular to a monoclonal neutralizing antibody of HPV58L1 and application thereof.

Background

Human Papilloma Virus (HPV) is a spherical double-stranded DNA virus, the diameter of virus particles is 55-60 nm, the nucleocapsid is in 20-face symmetry and consists of pentamers of 72 major capsid proteins L1 and a minor capsid protein L2. Mainly invade epithelial tissues and cells of human bodies, can cause squamous epithelial proliferation of skin mucous membranes of human bodies, and further induce various benign and malignant hyperplasia lesions. High risk HPV infections are associated with the development of various malignancies, and low risk HPV infections cause anal and genital warts.

High-risk types of HPV include 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82 and the like, which can cause Cervical Intraepithelial Neoplasia (CIN) and cervical cancer, wherein HPV16 and HPV18 are the most common oncogenic types, and monoclonal antibodies thereof are more researched, as in the Chinese patent: a monoclonal antibody against HPV16L1 protein, a preparation method and application thereof (publication No. CN105367652B), a monoclonal antibody capable of specifically recognizing HPV18L1 protein and application thereof (publication No. CN 108276491A).

Statistical data of 13.79 ten thousand people in tumor hospital of Chinese medical academy of sciences show that 7 provinces and cities are provided in Beijing, Shanghai, Jiangsu, Zhejiang, Hunan, Shanxi and Sichuan, and the detection rates of different high-risk HPV are in the following sequence: HPV16, 52, 58, 53, 39, 56, 51, 18, 59, 33, 66, 35, 31, 82, 68, 45, 26; zhejiang Taizhou hospital, based on the detection data of 3.80 ten thousand people, the detection rate sequence of high-risk HPV is as follows: HPV52, 16, 58, 39, 18, 56, 51, 33, 59, 68, 66, 31, 35, 45; the detection data of 40311 in southeast of China are HPV16 (29.9%), HPV52 (18.6%), HPV58 (17.1%), HPV18 (10.1%) and HPV33 (7.53%), respectively.

Qingdao central hospital and Qingdao tumor hospital, 1664 patients including CIN, SCC, adenocarcinoma, HPV16, 52, 31, 33, 58, 51 occurred more in CIN1-3, SCC; among adenocarcinomas, HPV18 occurs most frequently; in 1336 cases of invasive cervical cancer in Hunan, the detection rates of various high-risk types are respectively as follows: HPV16 (50.6%), HPV58 (12.4%), HPV52 (10.9%), HPV18 (7.3%); among northern cervical disease patients in Xinjiang, the detection rate of each high-risk type is as follows: HPV16 (44.0%), HPV53 (28.9%), 52 (25.3%), 58 (22.3%).

As can be seen from the above data, HPV58 is second only to HPV16 and HPV52 in the cases of HPV infection and cervical cancer patients, but the research on monoclonal antibodies thereof is very few at present, which limits the detection of HPV58 virus and the treatment of HPV58 virus by using monoclonal antibodies.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a monoclonal neutralizing antibody against HPV58L1 and its application, wherein the monoclonal neutralizing antibody can be widely applied to infection detection of HPV58 virus in clinical samples, and can be used for treatment of patients infected with HPV 58.

The monoclonal antibody provided by the invention has the advantages of high specificity,

the amino acid sequence of at least one of the CDR regions of the heavy chain has the amino acid sequence shown as SEQ ID NO.1, 2 or 3 or a sequence with at least 80% sequence identity with the amino acid sequence;

wherein at least one of the CDR regions of the light chain has an amino acid sequence as set forth in SEQ ID NO 4, 5 or 6 or a sequence having at least 80% sequence identity thereto.

In the invention, the heavy chain comprises three CDR regions, and the amino acid sequences of the CDR regions respectively have the amino acid sequences shown as SEQ ID Nos. 1, 2 and 3;

the light chain comprises three CDR regions, and the amino acid sequences of the CDR regions respectively have the amino acid sequences shown in SEQ ID NO. 4, 5 and 6.

Wherein the sequence shown in SEQ ID NO.1 is GFTFSIANMS;

the sequence shown as SEQ ID NO.2 is YINPGTGDTFVHDKF;

the sequence shown in SEQ ID NO. 3 is FGSNFFDY;

the sequence shown in SEQ ID NO. 4 is KASQDIYYIGA;

the sequence shown in SEQ ID NO. 5 is GAYALET;

the sequence shown in SEQ ID NO. 6 is QQYSTTPWT.

In the present invention, in the monoclonal antibody,

the heavy chain comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown as SEQ ID NO. 7, 8, 9 or 10, or a sequence with at least 80 percent of sequence homology with the FR region;

the light chain comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown as SEQ ID NO. 11, 12, 13 or 14, or a sequence with at least 80% of sequence homology with the FR region.

In the present invention, in the monoclonal antibody,

the amino acid sequences of the 4 FR regions of the heavy chain have the amino acid sequences shown as SEQ ID NO 7, 8, 9 and 10 respectively;

the amino acid sequences of the 4 FR regions of the light chain have the amino acid sequences shown as SEQ ID NOS: 11, 12, 13 and 14, respectively.

In the present invention, the sequence having at least 80% sequence homology is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids from the original sequence, wherein the plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.

In the present example, the heavy chain variable region of the monoclonal antibody comprises the CDR regions shown in SEQ ID NO.1, 2 or 3 and the FR regions shown in SEQ ID NO. 7, 8, 9 and 10.

In the present example, the light chain variable region of the monoclonal antibody comprises the CDR regions shown in SEQ ID NO. 4, 5 or 6 and the FR regions shown in SEQ ID NO. 11, 12, 13 and 14.

In some embodiments, the heavy chain variable region of the monoclonal antibody comprises SEQ ID NO 7, SEQ ID NO 1, SEQ ID NO 8, SEQ ID NO 2, SEQ ID NO 9, SEQ ID NO 3, SEQ ID NO 10;

in some embodiments, the light chain variable region of the monoclonal antibody comprises SEQ ID NO 11, SEQ ID NO 4, SEQ ID NO 12, SEQ ID NO 5, SEQ ID NO 13, SEQ ID NO 6, SEQ ID NO 14, in that order.

In some embodiments, the heavy chain variable region of the monoclonal antibody has the amino acid sequence set forth in any one of SEQ ID NOs 15; the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO 16.

In the present invention, the heavy chain constant region of the monoclonal antibody is of the IgG1 type, and the light chain constant region is of the K type.

Drawings

FIG. 1 shows the antibody purification effect (M: molecular weight Marker; 1: denatured antibody; 2: antibody).

Biological preservation Instructions

58-4A3, classified name, hybridoma cell, which is preserved in China general microbiological culture Collection center of China general microbiological culture Collection management Committee in 2019, 12.5.12.4.A microbial research institute, China academy of sciences, No. 3, West Lu 1, North Chen, south China, Beijing, and the preservation number is CGMCC No. 19187.

In a specific embodiment, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC No. 19187.

The monoclonal antibody provided by the invention takes a pseudovirus particle (VLP) coated by HPV58L1 protein as immunogen, immunizes a mouse, obtains a hybridoma cell strain capable of continuously and stably secreting anti-HPV 58 by cell fusion and screening by adopting a hybridoma technology, and prepares the monoclonal antibody. The monoclonal antibody obtained by the invention has good sensitivity and specificity, and experiments show that the monoclonal antibody and IC of HPV58VLP₉₀As low as 2ng/mL, there was no cross-reaction with the other 10 types tested (HPV6, 11, 16, 18, 31, 33, 45, 52, 59, 68). Therefore, the antibody can be applied to clinical diagnosis of HPV58 type and treatment or prevention of HPV58 type virus infection.

The invention also provides polynucleotides encoding the monoclonal antibodies or functional fragments thereof.

The functional fragment includes at least one of the CDR regions of the heavy chain or at least one of the CDR regions of the light chain.

Specifically, the polynucleotide sequence encoding the CDR region shown in SEQ ID NO.1 is shown in SEQ ID NO. 17;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO.2 is shown in SEQ ID NO. 18;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO. 3 is shown in SEQ ID NO. 19;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO. 4 is shown in SEQ ID NO. 20;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO. 5 is shown in SEQ ID NO. 21;

the polynucleotide sequence for coding the CDR region shown in SEQ ID NO. 6 is shown in SEQ ID NO. 22;

in some embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody provided by the invention is as set forth in SEQ ID NO: shown at 23. The polynucleotide sequence for coding the variable region of the monoclonal antibody light chain is shown in SEQ ID NO. 24.

In some embodiments of the invention, the nucleotide sequence shown in any one of SEQ ID NOs 17 to 24 is a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotide sequences.

The invention also provides a nucleic acid vector comprising nucleotides encoding the monoclonal antibody or functional fragment thereof.

The invention also provides a recombinant host containing the nucleic acid vector.

The nucleic acid vectors of the present invention are capable of producing the antibodies expressed in vivo in a host, referred to herein as a recombinant host. The host cell is selected from escherichia coli, yeast, insect cells or mammalian cells.

The invention provides two preparation methods of the monoclonal antibody, one method is as follows: culturing the recombinant host of the invention, and inducing the expression of the monoclonal antibody;

the second is as follows: culturing the hybridoma cell strain with the preservation number of CGMCC No.19187 to obtain the monoclonal antibody.

The invention also provides said monoclonal antibody chemically or biologically labeled.

The chemical label is an isotope, an immunotoxin and/or a chemical drug;

the biomarker is a biotin, avidin, or enzyme label.

The enzyme label is preferably horseradish peroxidase or alkaline phosphatase.

The immunotoxin is preferably aflatoxin, diphtheria toxin, pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe agglutinin, modeccin, PAP, nystatin, gelonin or luffa toxin.

The monoclonal antibody is a conjugate prepared by coupling with a solid medium or a semisolid medium.

The solid phase medium or semi-solid medium refers to any support to which the monoclonal antibody or labeled monoclonal antibody of the present invention can be attached, including but not limited to nitrocellulose membrane, polyvinylidene fluoride (PVDF) membrane, iPDMS chip, microwell plate, polystyrene plate, microparticle, microcarrier, gel, etc.

The monoclonal antibody, the labeled monoclonal antibody and/or the conjugate are applied to preparation of products for detecting HPV58 types.

The HPV58 type detection product can be used as an auxiliary diagnostic tool for HPV58 virus infection, and can be a reagent combination or a kit, for example.

The research of the invention shows that the HPV58L 1/VLP/virus can be detected by using the monoclonal neutralizing antibody of HPV58 provided by the invention and adopting an indirect ELISA method, a double-antibody sandwich ELISA method or an immunoblotting method and the like.

The invention also provides a kit for detecting HPV58, which comprises the monoclonal antibody, the labeled monoclonal antibody and/or the conjugate.

The invention provides a diagnosis method of HPV58 infection, which is used for detection by the kit provided by the invention.

In some embodiments, kits for indirect ELISA detection of HPV58 are provided, comprising: the monoclonal antibody of the invention. The kit also comprises an enzyme label plate, a coating solution, a confining solution, an enzyme-labeled secondary antibody, a washing solution, a luminescent substrate and a stop solution. Wherein the coating solution is 0.01M carbonate buffer solution with pH of 9.6; the blocking solution was 0.01M PBS buffer pH 7.2 containing 2% BSA; the enzyme-labeled secondary antibody is goat anti-mouse IgM labeled by horseradish peroxidase; the washing solution was 0.01M PBS buffer pH7.4 containing 0.05% Tween 20; luminescent substrate TMB, stop solution is 2M H₂SO₄。

In this example, the method of diagnosis of HPV58 infection was indirect. The method specifically comprises the following steps: diluting a sample with a coating solution, coating the sample on an enzyme label plate, washing, sealing, adding the monoclonal antibody, incubating, reacting with an enzyme-labeled secondary antibody and a luminescent substrate in sequence, and determining OD (optical density) after terminating the reaction_450nmThe value is obtained.

In some embodiments, a kit for detecting HPV58 by a double antibody sandwich ELISA method is provided, comprising: polyclonal antibodies against HPV58 VLPs and monoclonal antibodies described herein. Further comprising: an ELISA plate, a coating solution, a confining solution, a washing solution, a luminescent substrate and a stop solution.

In this example, the diagnostic method for HPV58 infection was a double antibody sandwich ELISA.

In some embodiments, a kit for detecting HPV58 by immunoblotting is provided, wherein the kit comprises the monoclonal antibody of the invention, and further comprises a PVDF membrane, a blocking solution, TBST, an enzyme-labeled secondary antibody and ECL. Specifically, the enzyme-labeled secondary antibody is goat anti-mouse IgM labeled by horseradish peroxidase.

In this example, the diagnostic method for HPV58 infection was immunoblotting.

The monoclonal antibody, the labeled monoclonal antibody and/or the conjugate disclosed by the invention are applied to preparation of an anti-HPV 58 type virus infection preparation.

The monoclonal antibody, the marked monoclonal antibody and/or the conjugate are applied to preparation of a preparation for preventing and treating tumors and/or warts.

The tumor cell is cervical cancer, vulvar cancer, vaginal cancer, anal cancer, rectal cancer, oral cancer, tonsil cancer, penis cancer, prostatic cancer or bladder cancer;

the warts are genital warts, flat warts or common warts.

The invention also provides a medicament or vaccine comprising the monoclonal antibody, the labeled monoclonal antibody and/or the conjugate.

The preparation form of the medicine is vaginal gel, vaginal cleaning solution, external washing solution, vaginal suppository, freeze-dried powder or vaginal effervescent tablets.

In some embodiments, the pharmaceutical dosage form is a vaginal gel comprising a monoclonal antibody of the invention and an excipient comprising: disodium edetate, carbomer 974P, polycarbophil AA-1, glycerol, benzyl alcohol or triethanolamine.

In some embodiments, the pharmaceutical dosage form is vaginal irrigation solution comprising the monoclonal antibody of the invention and an excipient comprising: glycerol and physiological saline.

In some embodiments, the pharmaceutical dosage form is a topical lotion comprising the monoclonal antibody of the invention and an adjuvant comprising: glycerol, purified water, ethanol and a foaming agent. And may also include a fragrance.

In some embodiments, the pharmaceutical dosage form is a vaginal suppository comprising the monoclonal antibody of the invention and an adjuvant comprising: purified water, gelatin, glycerol and mannitol.

In some embodiments, the pharmaceutical dosage form is a lyophilized powder comprising the monoclonal antibody of the invention and an adjuvant comprising: bovine serum albumin and trehalose.

In some embodiments, the pharmaceutical dosage form is a vaginal effervescent tablet comprising the monoclonal antibody of the invention and an excipient comprising: sodium bicarbonate, ethanol solution containing 20% PEG6000, citric acid, vitamin C, lactose, microcrystalline cellulose, hydroxypropyl methylcellulose, silica gel micropowder, and magnesium stearate.

The invention also provides a method for preventing and treating HPV58 infection, and the medicine or the vaccine is administered.

The invention provides specific monoclonal neutralizing antibodies of HPV58 and a hybridoma cell line for producing the antibodies, wherein one monoclonal neutralizing antibody with the highest neutralizing activity titer is utilized, and an indirect ELISA method, a double-antibody sandwich ELISA method, an immune hybridization method and the like are adopted, so that the monoclonal neutralizing antibodies can be widely applied to infection detection of HPV58 virus in clinical samples; the monoclonal neutralizing antibody with the highest neutralizing activity titer is used for preparing vaginal gel, vaginal cleaning solution, external washing solution, vaginal suppository, freeze-dried powder, vaginal effervescent tablets and the like, is used for treating patients infected with HPV58, and can effectively improve the clinical negative conversion rate of patients infected with HPV 58. The invention has important significance for the health development and public health prevention and control of women.