CN111116740B

CN111116740B - Monoclonal neutralizing antibody of HPV33L1 and application thereof

Info

Publication number: CN111116740B
Application number: CN201911398464.1A
Authority: CN
Inventors: 邹国宝; 魏颖颖; 郭光华; 蔺皓; 刘欣欣; 宋高尚; 沈江卫
Original assignee: Zhongsheng Fangzheng Bio Tech Co ltd
Current assignee: Zhongsheng Fangzheng Bio Tech Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2022-05-31
Anticipated expiration: 2039-12-30
Also published as: CN111116740A

Abstract

The invention relates to the technical field of antibody medicines, in particular to a monoclonal neutralizing antibody of HPV33L1 and application thereof. The invention provides specific monoclonal neutralizing antibodies of HPV33 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 HPV33 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 HPV33, and can effectively improve the clinical negative conversion rate of patients infected with HPV 33. The invention has important significance for the health development and public health prevention and control of women.

Description

Monoclonal neutralizing antibody of HPV33L1 and application thereof

Technical Field

The invention relates to the technical field of antibody medicines, in particular to a monoclonal neutralizing antibody of HPV33L1 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 a variety of 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).

Among 105 cervical cancer patient samples in the Kyoho, the HPV16 detection rate was the highest (58.3%), the HPV33 detection rate followed immediately thereafter (25.0%), and the HPV18 ranked in the third place (8.4%); in 700 cervical neoplastic lesion samples in mexico, HPV detection was ranked as HPV33 (33.1%), HPV16 (16.6%), HPV18 and HPV51 (6.7% each); in 185 Malaysia cervical cancer patient samples, HPV16 (35.7%), HPV18 (26.0%), HPV58 (9.1%), HPV33 (7.1%); in 12816 cervical swab samples in southeast region of China, 2856 cases (22.3%) are positive for HPV, and the top five cases are HPV18 (18.2%), HPV52 (14.1%), HPV16 (11.9%), HPV58 (10.6%) and HPV33 (5.5%); among 2309 cervical cancer patients in the eastern region of China, 90.86% are positive for HPV, wherein the most common types are HPV16, HPV18, HPV58, HPV53 and HPV 33; 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; 1336 cases of invasive cervical cancer patients in Hunan province in China have the most common HPV types: HPV16 (50.6%), HPV58 (12.4%), HPV52 (10.9%), HPV18 (7.3%), HPV33 (5.5%), HPV59 (4.2%), HPV39 (4.0%), HPV 61 (3.4%), HPV31 (3.3%), HPV56 (3.2%).

The data show that the HPV33 is a common HPV type in cervical cancer patients both abroad and domestically, but the research on the monoclonal antibody is very few at present, which limits the detection of HPV33 virus and HPV33 virus treatment by using the monoclonal antibody.

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 HPV33L1 and its application, wherein the monoclonal neutralizing antibody can be widely applied to infection detection of HPV33 virus in clinical samples, and can be used for treatment of patients infected with HPV 33.

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 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.

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

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

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

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

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

the sequence shown as SEQ ID NO. 6 is QQYWSSPWT;

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 in SEQ ID NO 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 an amino acid sequence as set forth in any one of SEQ ID NO 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 IgG2b type, and the light chain constant region is of the lambda type.

In a specific embodiment, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC No. 19190.

The monoclonal antibody provided by the invention takes a pseudovirus particle (VLP) coated by HPV33L1 protein as immunogen, immunizes a mouse, obtains a hybridoma cell strain capable of continuously and stably secreting anti-HPV 33 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 has IC90 as low as 2ng/mL with HPV33VLP and has no cross reaction with other 10 types (HPV6, 11, 16, 18, 31, 45, 52, 58, 59 and 68) tested. Therefore, the antibody can be applied to clinical diagnosis of HPV33 type and treatment or prevention of HPV33 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.19190 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 HPV33 types.

The HPV33 type detection product can be used as an auxiliary diagnostic tool for HPV33 virus infection, and can be a reagent combination or a kit, for example.

The research of the invention shows that the HPV33L 1/VLP/virus can be detected by using the monoclonal neutralizing antibody of HPV33 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 HPV33, which comprises the monoclonal antibody, the labeled monoclonal antibody and/or the conjugate.

The invention provides a diagnosis method of HPV33 infection, which is used for detection by the kit provided by the invention.

In some embodiments, kits for indirect ELISA detection of HPV33 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 HPV33 infection was indirect. Utensil for cleaning buttockThe body includes: 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 HPV33 by a double antibody sandwich ELISA method is provided, comprising: polyclonal antibodies against HPV33 VLPs and monoclonal antibodies of the invention. 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 HPV33 infection was a double antibody sandwich ELISA.

In some embodiments, a kit for detecting HPV33 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 HPV33 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 33 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 HPV33 infection, and the medicine or the vaccine is administered.

The invention provides specific monoclonal neutralizing antibodies of HPV33 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 HPV33 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 HPV33, and can effectively improve the clinical negative conversion rate of patients infected with HPV 33. The invention has important significance for the health development and public health prevention and control of women.

Drawings

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

Biological preservation Instructions

The biological material 33-2B6 is classified and named as hybridoma cell, which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 12.5.2019, with the address of microorganism institute of China academy of sciences No. 3, West Lu No.1 Hospital, North Cheng, Chaoyang, Beijing and the preservation number of CGMCC No. 19190.

Detailed Description

The invention provides a monoclonal neutralizing antibody of HPV33L1 and application thereof, and a person skilled in the art can realize the monoclonal neutralizing antibody by appropriately modifying process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to definitions and terminology in this field, the expert may refer in particular to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

An "antibody" refers to a protein composed of one or more polypeptides that specifically bind to an antigen. One form of antibody constitutes the basic building block of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains are joined together and are responsible for binding to antigen, while the constant regions are responsible for the effector functions of the antibody.

The "variable region" of an antibody heavy or light chain is the N-terminal mature region of the chain, and the amino acid composition and arrangement of the variable region varies with the specificity of the antibody. Amino acid residues in certain regions of the variable region, known as the hypervariable region, which is the site of specific binding of Ig to an antigenic determinant and is also known as the complementarity determining region, the CDR region, exhibit greater variability, while other regions of the variable region, known as the framework region, are structurally relatively stable.

The types of antibodies currently known include kappa and lambda light chains, as well as alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or other type equivalents thereof. Full-length immunoglobulin "light chains" (about 25kDa or about 214 amino acids) comprise a single NH domain₂A variable region of approximately 110 amino acids at the end, and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin "heavy chains" (about 50kDa or about 450-550 amino acids) also contain a variable region (about 116 amino acids), and one of the heavy chain constant regions, e.g., gamma (about 330 amino acids).

"antibody" includes any isotype of antibody or immunoglobulin, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like. The antibodies can also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.

In the present invention, the "polynucleotide" refers to a biological macromolecular compound formed by polymerizing a plurality of nucleotides, which can be ribonucleic acid or deoxyribonucleic acid and their modifications, including double-stranded or single-stranded DNA, cDNA, RNA, mRNA, etc., and can be circular or linear, or can be a part of a circular vector or a fragment in a genome.

In the present invention, the "nucleic acid vector" refers to a recombinant DNA molecule comprising a desired coding sequence and suitable nucleic acid sequences necessary for the expression of the operably linked coding gene in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotic cells include promoters, optionally including operator sequences, ribosome binding sites and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers and termination and polyadenylation signals. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, the plasmid integrates into the genome. In the present specification, "plasmid" and "vector" may sometimes be used interchangeably, since plasmids are the most commonly used form of vector at present. However, the present invention is intended to include such other forms of expression vectors which serve equivalent functions, which are or will become known in the art, including, but not limited to: plasmids, phage particles, viral vectors and/or simply potential genomic inserts.

In the present invention, a "host cell" is generally a prokaryotic or eukaryotic host containing a nucleic acid vector and/or a gene of interest. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are competent to replicate the vector encoding the protein or to express the desired protein.

The term "monoclonal antibody" refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

In the invention, the method for detecting HPV33 type virus is carried out by immunology. The immunological detection is a technology for qualitatively, quantitatively or locally researching intracellular antigens (polypeptides and proteins) or antibodies by applying the principle of immunological basic principle-antigen-antibody reaction, namely the principle of specific combination of antigen and antibody and developing color development agents (fluorescein, enzyme, metal ions and isotopes) for labeling the antibodies through chemical reaction, and the technology comprises but is not limited to enzyme-linked immunoassay (indirect, direct or double antibody sandwich method), immunofluorescence, radioimmunoassay, immunoblot, immunohistochemistry, co-immunoprecipitation, chromatin co-precipitation and the like.

The medicament of the invention contains at least one functional component and also comprises a pharmaceutically acceptable carrier. The functional component comprises the antibody provided by the invention. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffered solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifying agents or buffer substances as additives.

The test materials and instruments adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 establishment of hybridoma cells

1. Animal immunization

Preparing HPV33 type L1 protein virus packaging particles by using an escherichia coli expression system, using the virus packaging particles as antigens, mixing the antigens with a Freund's complete adjuvant in equal volume for the first immunization, fully emulsifying, injecting subcutaneously by points, wherein the injection amount of each Balb/c mouse is 100 mu g each time, and immunizing 3 mice; and on the 7 th day, 14 th day and 21 day of the first immunization, adopting emulsion of antigen and Freund's incomplete adjuvant to carry out boosting immunization, collecting blood through tail veins of mice on the 14 th day, separating serum, detecting antibody titer by using an indirect ELISA method, wherein the antibody titer of the serum of the mice is respectively more than 1:32000, 1:8000 and 1:16000, and selecting the No.1 mouse with the highest titer for fusion.

The indirect ELISA method was performed as follows: plate-coated with 200 ng/well of HPV33VLP, and 100. mu.L of PBS solution containing 5% skim milk powder was added to the Negative Control (NC) well, overnight at 4 ℃; discarding the liquid in the hole, washing the plate with PBS for 3 times, adding PBS solution containing 5% skimmed milk powder, sealing at 200 μ L/hole for 1 hr at room temperature; discarding the liquid in the wells, washing the plate with PBS 1 time, adding primary antibody (tail blood is diluted 2 times from 1: 500 to 1:32000 in gradient, hybridoma cell culture supernatant is diluted 1:1, mouse ascites is diluted 10 times from 1:1000 to 1:100 in gradient, purified antibody is diluted 2 times from 1 μ g/mL to 0.0005 μ g/mL), and incubating at room temperature for 1 hour; discarding the liquid in the hole, washing the plate with PBS 3 times, adding HRP-conjugated goat anti-mouse IgG Fc (diluted 1: 2000 times), and incubating at room temperature for 1 hr; washing the plate with PBS for 5 times, adding substrate A and B solution, 50 μ L/hole, reacting for 30min at room temperature in dark place; adding 1mol/L sulfuric acid to terminate the reaction, and measuring the A450 value by using a microplate reader. If the A450 value is more than 2 times larger than the negative control, the positive can be judged initially.

2. Preparation and screening of hybridoma cells

The mouse No.1 with the highest antibody titer was taken for fusion. 3 days prior to fusion, the immunogen was boosted with Freund's incomplete adjuvant. Spleen cells from mice were harvested conventionally and fused with SP2/0 cells at a 5:1 ratio with 500g/L PEG 4000. Selectively culturing with HAT culture solution, taking supernatant after 10-15 days of fusion, and screening positive hybridoma cell strains by adopting an indirect ELISA method. 135 positive cell strains are screened from 384 cell strains, 96 cell strains with higher activity are selected, after 10 generations of culture, 5 cell strains capable of stably secreting HPV33L1-VLP antibody are selected, and indirect ELISA results are shown in Table 1.

TABLE 1 Indirect ELISA test results for cell line supernatants stably secreting HPV33L1-VLP antibody

Clone number	1C9	2B6	2H3	3G8	3H12	Negative of
							A450	1.752	2.014	1.312	1.687	1.498	0.035

3. Antibody specificity detection in hybridoma cell secretory supernatants

Using indirect ELISA, the envelope antigens were HPV6, 11, 16, 18, 31, 33, 45, 52, 58, 59, 68L 1VLP proteins, 100 ng/well, respectively. The detection results of the antibodies secreted by the 5 hybridoma cells in the cell supernatant show that the two hybridoma cells of 2B6 and 3G8 only react positively to HPV33L1-VLP protein, and the detection results of the HPV of other types are negative, and the 2 hybridoma cells are determined to be type-specific antibodies. Combining the detection results in the table 1 and the table 2, the specific antibody 2B6 with the highest titer is selected for subsequent development.

Table 2: detection result of antibody specificity in hybridoma cell secretion supernatant

Clone number	1C9	2B6	2H3	3G8	3H12
						6L1VLP	-	-	-	-	-
11L1VLP	-	-	-	-	-
						16L1VLP	-	-	-	-	+
18L1VLP	-	-	-	-	-
						31L1VLP	+	-	-	-	-
33L1VLP	+	+	+	+	+
						45L1VLP	-	-	-	-	-
52L1VLP	-	-	+	-	+
						58L1VLP	-	-	-	-	-
59L1VLP	-	-	-	-	-
						68L1VLP	-	-	-	-	-

Example 2: preparation and characterization of monoclonal antibodies

1. Preparation of monoclonal antibody against HPV33 from ascites of mouse

Adult BALB/c mice were selected, and the peritoneal cavity inoculated with hybridoma cells 33-2B6, 1X 10 per mouse⁶-2×10⁶When the abdomen is obviously enlarged and the hand touches the abdomen, the skin is tense, and the 16-gauge needle can be used for collecting ascites. The ascites is centrifuged (13000r/min30 min), cell components and other precipitates are removed, the supernatant is collected, the antibody is purified by a ProteinG affinity column, the ascites and the titer of the purified antibody are detected by an indirect ELISA method, and the result shows that the ascites is positive when diluted by 10 ten thousand times and the purified antibody is still positive when diluted to 1 ng/mL.

2. Determination of antibody purity

The purified antibody was subjected to 12% SDS-PAGE, which showed a purity of 95% or more, and the result is shown in FIG. 1, wherein 1. the denatured antibody has two bands of light chain and heavy chain, and 2. the antibody has a single band.

3. Antibody subtype identification

The subtype of antibody produced by hybridoma cells 33-2B6 was identified using various antibodies against mouse immunoglobulin subtypes (IgG1, IgG2a, IgG2B, IgG3, IgA, IgM) by indirect ELISA and was shown to be IgG 2B.

4. Determination of antibody light chain and heavy chain variable region sequences

Extracting mRNA of 33-2B6 hybridoma cells, performing reverse transcription to form cDNA, performing high-fidelity PCR amplification by using variable region universal primers, inserting PCR product fragments into a T vector for DNA sequence determination, and determining the gene sequence of the variable region of 33-2B 6: the heavy chain is shown as SEQ ID NO.23, and the light chain is shown as SEQ ID NO. 24. The obtained nucleotide sequence is translated into an amino acid sequence. 33-2B 6: the heavy chain variable region is shown as SEQ ID NO.15, and the light chain variable region is shown as SEQ ID NO. 16.

Using the sequences identified above, various genetically engineered antibodies, such as recombinant antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, diabodies, and the like, can be prepared by known antibody engineering techniques, while retaining the biological properties of the monoclonal antibody from which it is derived.

Example 3: antibody neutralization activity assay

The neutralizing activity of the antibody was tested by a pseudovirus-cell neutralization model.

Antibodies were first diluted to 1000ng/mL with PBS, then the antibodies were diluted to 0.06ng/mL with a 2-fold gradient, 50 μ L of each concentration of antibody was incubated with 50 μ L of HPV6, 11, 16, 18, 31, 33, 45, 52, 58, 59, 68 pseudovirus at the appropriate concentration in 96-well plates for one hour at 4 ℃, and negative serum controls, positive serum controls, cell controls, and pseudovirus controls were set. The mixtures were then added to 96-well plates previously plated with 293FT cells and incubated in a cell incubator for 72 hours. And then observing the fluorescence, collecting cells, detecting the fluorescence by using flow cytometry, calculating the fluorescence inhibition rate according to the fluorescence inhibition rate (1-experimental group/control group) multiplied by 100 percent if the fluorescence inhibition rate has the inhibition effect, and respectively taking the fluorescence inhibition rates of more than 50 percent and 90 percent as the neutralization titer of the monoclonal antibody to the pseudovirus. The result shows that the antibody only has an inhibiting effect on HPV33 pseudovirus, the 50% inhibition rate concentration is 0.5ng/mL, and the 90% inhibition rate concentration is 2 ng/mL; has no inhibitory effect on HPV6, 11, 16, 18, 31, 45, 52, 58, 59 and 68 pseudoviruses. The antibody 33-2B6 is shown to be an HPV33 virus neutralizing antibody with high titer and good specificity.

Example 4: application of monoclonal antibody 33-2B6 in HPV33 virus diagnosis

Using the monoclonal antibody 33-2B6 obtained in example 2, indirect ELISA, double antibody sandwich ELISA, immunoblotting, etc. can be used to detect HPV33L 1/VLP/virus content in a sample.

1. Determination of HPV33L 1/VLP/Virus content in samples by Indirect ELISA

(1) Properly diluting a detection sample in 0.01M carbonate buffer solution with pH9.6, setting multiple wells at 100 mu L/well, setting a sample diluent as a negative control well and standard substances with different concentrations as a positive control well, covering a sealing plate membrane, and incubating at room temperature for 2h or coating overnight at 4 ℃.

(2) The liquid was emptied and the residual liquid was patted dry and the plates were washed three times with 0.01M pH7.4PBS-Tween 20 wash.

(3) Blocking with 2% BSA-0.01M PBS pH 7.2 for 1 hour.

(4) The plate was washed 3 times as above.

(5) The monoclonal antibody 33-2B6 was added thereto at a 1: 100-fold dilution, and the mixture was reacted at room temperature for 2 hours in a 0.1 mL/well.

(6) After washing the plate 3 times, horseradish peroxidase-labeled goat anti-mouse IgM was added at 0.1 mL/well and reacted at room temperature for 1 hour.

(7) After soaking the plate in the washing solution for 5min, adding a substrate (3,3',5,5' -tetramethylbenzidine, TMB) after washing the plate for 3 times, and reacting for 5-10 minutes at room temperature in a dark place.

(9)2M H₂SO₄The reaction was terminated and the OD value was measured at 450 nm.

(10) Based on the assay data, the HPV33L 1/VLP/virus content in the sample was determined against a standard curve.

2. Double antibody sandwich ELISA for determination of HPV33 VLP/virus content in samples

The monoclonal antibody 33-2B6 is used as a capture antibody to prepare a detection antibody capable of specifically recognizing HPV33 VLP/virus, other related detection reagents known in the field are prepared, and the content of HPV33 VLP/virus in a sample is determined by using a double-antibody sandwich ELISA.

The preparation method of the detection antibody capable of specifically recognizing HPV33 VLP/virus comprises the following steps: polyclonal antibodies against HPV33 VLPs were prepared by immunizing new zealand white rabbits with HPV33 VLPs. The specific method comprises the following steps: at week 0, the primary immunization was performed by thoroughly emulsifying 500. mu.L of 0.2mg/mL HPV33VLP solution with 500. mu.L Freund's complete adjuvant to form a water-in-oil emulsion, and performing multiple dorsal subcutaneous injections to immunize 2 New Zealand white rabbits. The booster immunization was performed 4 times at 2 weeks, 5 weeks, 8 weeks, and 11 weeks, respectively, each timeImmunizing each time with 500 μ L of 0.2mg/mL HPV33VLP solution and 500 μ L of emulsion of Freund's incomplete adjuvant; blood sampling detection is carried out at 6 weeks, 9 weeks and 12 weeks. Indirect ELISA results showed that both new zealand white rabbits immunized with HPV33VLP 5 induced up to 2.0x10⁷And (3) high-titer antiserum with dilution times, wherein the polyclonal serum is purified and is named pAb-33 as a detection antibody in the development of a detection reagent.

By combining with other related detection reagents known in the field and by condition exploration and optimization, the kit for ELISA is constructed by using the monoclonal antibody 33-2B6 as a capture antibody and pAb-33 as a detection antibody. Through detection of patient samples, the double-antibody sandwich ELISA detection kit is not found to identify patient samples of other HPV high-risk subtypes (such as HPV16, HPV18, HPV26, HPV31, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82), and only specifically identifies HPV33 patient samples. Therefore, the ELISA can be used for clinical rapid detection of HPV33 infection.

3. Detection of HPV33L1 protein in sample by immunoblotting

The monoclonal antibody 33-2B6 is used as a primary antibody, Western Blotting is utilized to detect HPV33L1 protein in a sample, and the specific steps are as follows:

(1) SDS Polyacrylamide gel electrophoresis methods are described in Oseber et al, eds (molecular biology laboratory Manual) (science Press 1998). The electrophoresis is terminated by adopting 10 percent separation gel and 5 percent concentrated gel under the electrophoresis condition of 150V voltage and the distance between the bromophenol blue dye band and the bottom edge of the gel is about 1.5 cm.

(2) Electrotransfer: see e.g.Oseber et al, eds molecular biology laboratory Manual (science Press 1998). It was transferred to a PVDF (0.45 μm) membrane by electrotransfer.

(3) Immunoblotting: after the electric membrane transfer is finished, the membrane is sealed in 5% skimmed milk powder sealing solution for 1 hour at room temperature, the monoclonal antibody (1 mug/mL) prepared by the invention is used as a primary antibody, the membrane is incubated for 2 hours at room temperature or reacted overnight at 4 ℃, and the membrane is washed for 3 times (10 min each time) by TBST (TBS added with 0.5% Tween-20).

(4) And (3) using horseradish peroxidase-labeled goat anti-mouse IgM as a secondary antibody, reacting for 2h at room temperature, washing by the method, acting for 1min by ECL, and exposing and imaging in a full-automatic chemiluminescence imager (Bio-Rad Versa doc 5000 MP).

Example 5 formulations for prevention or treatment of HPV33 Virus infection

The monoclonal neutralizing antibody 33-2B6 obtained in example 2 was used to prepare vaginal gel, vaginal washing solution, external washing solution, vaginal suppository, lyophilized powder, vaginal effervescent tablet, etc., for prevention and treatment of HPV infection.

1. Preparation of monoclonal neutralizing antibody 33-2B6 vaginal gel

HPV vaginal gel is prepared according to the following proportion and process: adding 0.2-0.8 wt% of edetate disodium, 0.5-1.5 wt% of carbomer 974P, 0.1-0.3 wt% of polycarbophil AA-1 and 4-8 wt% of glycerol into purified water, heating, stirring uniformly and fully swelling, adding 1-7 wt% of benzyl alcohol and 5ng/mL of HPV33 monoclonal antibody 33-2B6 when the mixture is cooled to room temperature, fully stirring uniformly, finally adding 0.6-1.0 wt% of triethanolamine, and stirring while adding to form homogeneous gel. 3mL of HPV vaginal gel prepared above was loaded into a disposable vaginal syringe.

The product can be used for female vagina/genital tract with positive HPV33 virus. The monoclonal neutralizing antibody 33-2B6 in the gel is specifically combined with HPV33 virus, so that further infection of HPV33 virus is effectively blocked, and the HPV33 virus is discharged out of a body under the wrapping of carbomer gel, so that the purpose of turning negative is achieved.

2. Preparation of vaginal wash solution containing monoclonal neutralizing antibody 33-2B6

Preparing the vaginal cleaning solution according to the following proportion and process: fully dissolving glycerol (4 wt% -8 wt%), HPV33 monoclonal antibody 33-2B6 (the final concentration is 10ng/mL) in physiological saline, filtering and sterilizing, and filling 3 mL/piece into a disposable vagina cleaning device.

The product can be used for cleaning vagina/genital tract of female with positive HPV33 virus. The monoclonal neutralizing antibody 33-2B6 in the washing liquid is specifically combined with the HPV33 virus, so that the further infection of the HPV33 virus is effectively blocked, and the HPV33 virus is discharged out of the body, thereby achieving the purpose of negative conversion.

3. Preparation of monoclonal neutralizing antibody 33-2B6 external lotion

The external lotion is prepared according to the following proportion and process: fully melting glycerol (4 wt% -8 wt%) and HPV33 monoclonal antibody 33-2B6 (the final concentration is 20ng/mL) into purified water, and uniformly mixing; adding 0.1-1 wt% of rose essence into 0.5-1 wt% of ethanol for dissolving, adding 1-3 wt% of natural plant foaming agent for uniformly mixing, adding a mixed solution of glycerol/HPV 33 monoclonal antibody 33-2B6 under stirring, uniformly stirring, filtering and sterilizing. The external lotion was poured into 100mL bottles of plastic with a pump head.

The product is used for cleaning male and female genital parts in daily life, and can be applied to private parts by applying on private parts, kneading gently, and washing for a moment, so as to effectively prevent HPV33 virus infection.

4. Preparation of vaginal suppository containing monoclonal neutralizing antibody 33-2B6

The pessary is prepared according to the following proportion and process: taking 300g of purified water, heating to 90 ℃, adding 200g of gelatin for full swelling, cooling and maintaining the temperature at about 60 ℃, adding 500g of glycerol, stirring uniformly while adding, adding 600g of mannitol while stirring to fully dissolve the glycerol gelatin when the water in the glycerol gelatin matrix is evaporated to 1000g, cooling to below 40 ℃, adding HPV33 monoclonal antibody 33-2B6 (the final concentration is 5ng/g), stirring uniformly at low speed, pouring into a suppository mold, standing at low temperature conventionally, taking out, and carrying out plastic packaging.

The product is used for female vagina/genital tract positive by HPV33 virus, and monoclonal neutralizing antibody 33-2B6 in the suppository is specifically combined with HPV33 virus, so that further infection of HPV33 virus is effectively blocked, and HPV33 virus is discharged out of body, thereby achieving the purpose of turning negative.

5. Preparation of monoclonal neutralizing antibody 33-2B6 freeze-dried powder

The freeze-dried powder is prepared according to the following proportion and process: fully dissolving HPV33 monoclonal antibody 33-2B6 (the final concentration is 5ng/mL), bovine serum albumin (0.1 mg/mL-1 mg/mL) and trehalose (3 wt% -10 wt%) in purified water, filling 3 mL/support in a penicillin bottle, freeze-drying for 3-4 hours under the pressure of 5.0mBar, sealing and storing.

The product is used for cleaning female vagina in daily families or medical institutions, when in use, 3mL of physiological saline is added for full dissolution, the mixture is injected into vagina for cleaning through a hose, wherein a monoclonal neutralizing antibody 33-2B6 is specifically combined with HPV33 virus, further infection of HPV33 virus is effectively blocked, and the HPV33 virus is discharged out of a body, so that the purpose of turning negative is achieved.

6. Preparation of monoclonal neutralizing antibody 33-2B6 vaginal effervescent tablet

Preparing the vaginal effervescent tablets according to the following proportion and process: (1) drying sodium bicarbonate (35 wt%) at 60 deg.C for 2 hr, adding 20 wt% PEG6000 ethanol solution to obtain soft material, granulating with 14 mesh nylon sieve, and drying at 60 deg.C; (2) 11 wt% of citric acid, 25 wt% of vitamin C, 12 wt% of lactose and 15 wt% of microcrystalline cellulose, adding into 2% hydroxypropyl methyl cellulose alcohol solution, stirring uniformly to prepare soft materials, granulating by a 14-mesh nylon sieve, and drying at 60 ℃ for later use; (3) adding 1g of monoclonal neutralizing antibody 33-2B6 lyophilized powder, 8g of aerosil and 8g of magnesium stearate, mixing uniformly, pressing into 1000 tablets, and packaging by plastic-aluminum blister.

The product is used for vaginal/genital tract packing of women with positive HPV33 virus, is convenient to use, and the monoclonal neutralizing antibody 33-2B6 in the effervescent tablet is specifically combined with the HPV33 virus, so that further infection of the HPV33 virus is effectively blocked, and the purpose of turning negative is achieved.

Safety testing of HPV33 Virus infection treatment/prevention formulations

According to the 'disinfection technical specification' 2002 edition, the vagina mucosa irritation test is carried out on the vagina gel, vagina cleaning solution, external washing solution, vaginal suppository, freeze-dried powder and vaginal effervescent tablets prepared by the method, the experimental object is a rabbit, the irritation reaction is negative, and the safety meets the requirements.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

<110> Zhongsheng Fangzheng Biotechnology Ltd

<120> HPV33L1 monoclonal neutralizing antibody and application thereof

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Claims

1. Monoclonal antibody targeting the HPV33L1 protein,

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

the amino acid sequences of CDR regions 1-3 of the light chain are respectively shown in SEQ ID NO. 4, 5 and 6.

2. The monoclonal antibody according to claim 1,

the amino acid sequences of the FR regions 1-4 of the heavy chain are respectively shown as SEQ ID NO 7, 8, 9 and 10;

the amino acid sequences of the FR regions 1-4 of the light chain are respectively shown as SEQ ID NO 11, 12, 13 and 14.

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

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 15;

the amino acid sequence of the light chain variable region is shown in SEQ ID NO 16.

4. The monoclonal antibody of claim 3, wherein the heavy chain constant region is of the IgG2b type and the light chain constant region is of the lambda type.

5. The monoclonal antibody of claim 4, produced by a hybridoma cell line having a accession number of CGMCC No. 19190.

6. A polynucleotide encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1 to 5.

7. A nucleic acid vector comprising a polynucleotide encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1 to 5.

8. A recombinant host cell comprising the nucleic acid vector of claim 7.

9. A method for producing the monoclonal antibody according to any one of claims 1 to 5, comprising:

culturing the recombinant host cell of claim 8 to induce expression of the monoclonal antibody;

or culturing hybridoma cell strain with preservation number of CGMCC No.19190 to obtain the monoclonal antibody.

10. A chemically or biologically labeled monoclonal antibody according to any one of claims 1-5.

11. A conjugate prepared by conjugating the monoclonal antibody of any one of claims 1 to 5 or the monoclonal antibody of claim 10 to a solid medium or a semi-solid medium.

12. Use of the monoclonal antibody of any one of claims 1 to 5, the monoclonal antibody of claim 10 and/or the conjugate of claim 11 for the preparation of a product for detecting HPV33 type virus.

13. A kit for detecting HPV33, comprising the monoclonal antibody of any one of claims 1-5, the monoclonal antibody of claim 10 and/or the conjugate of claim 11.