CN115724951A - Antibody or antigen binding fragment thereof binding to HPV type 11 and application thereof - Google Patents

Abstract

The invention provides an antibody or an antigen binding fragment thereof combined with 11-type HPV (human papilloma Virus) and application thereof, and relates to the technical field of antibodies. The antibody or antigen-binding fragment thereof that binds to HPV type 11 comprises a light chain variable region and/or a heavy chain variable region; the light chain variable region has a light chain CDR consisting of CDR-L1, CDR-L2 and CDR-L3 with amino acid sequences shown as SEQ ID NO 1, 2 and 3 respectively, or SEQ ID NO 11, 12 and 13 respectively; the heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2, CDR-H3 having amino acid sequences shown in SEQ ID NOS: 4, 5 and 6, respectively, or SEQ ID NOS: 14, 15 and 16, respectively. The antibody or the antigen binding fragment thereof only reacts with HPV11 type antigen specifically, and has good specificity and neutralization activity.

Description

Antibody or antigen binding fragment thereof binding to HPV type 11 and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an antibody combined with 11-type HPV or an antigen-binding fragment thereof and application thereof.

Background

Human Papilloma Virus (HPV) is a small non-enveloped DNA virus, currently, there are more than 200 types, HPV mainly infects skin and mucosal tissues, wherein more than 40 types of HPV infection can cause Human diseases, and HPV can be divided into high-risk type and low-risk type according to the relationship between HPV infection and cancer occurrence, wherein HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 and the like are high-risk type, and the persistent infection of high-risk HPV can cause malignant tumors such as cervical cancer, anal cancer and vaginal cancer. Worldwide, cervical cancer ranks third in high-incidence malignant tumors in women, about 60 million new cervical cancers in women occur every year worldwide, and more than 30 million women die of cervical cancer. Other HPV subtypes belong to low-risk types, mainly cause wart hyperplasia of skin mucosa and are shown as benign lesions such as condyloma acuminatum and flat wart, and no good treatment method exists clinically at present. Epidemiological survey and clinical research show that the detection rate of HPV6 and HPV11 types in genital condyloma acuminatum tissues is the highest, and statistics show that the ratio of genital warts to recurrent respiratory papillomatosis caused by HPV6 and HPV11 infection is more than 90%.

The most effective method for preventing HPV infection is to inoculate HPV vaccine, 5 cervical cancer vaccines which are on the market at home and abroad at present all take HPV main capsid protein L1 as effective antigen, are assembled into Virus-like particles (VLPs) by gene recombination technology under certain conditions, and are prepared into vaccines by being supplemented with different adjuvants. Based on successful marketing and wide application of recombinant HPV L1VLPs vaccines, the L1VLPs are fully shown to highly reduce the structure of natural HPV, retain key neutralizing epitopes of natural viruses, have antigenicity and immunogenicity the same as or similar to those of wild homoviruses, can induce high-titer neutralizing antibodies, and further well prevent HPV persistent infection, cervical cancer and other related diseases. The HPV vaccines currently on the market, the tetravalent vaccine "Gardasil" and the nine-valent vaccine "Gardasil 9" from Merck and the HPV vaccines developed more than bivalent at home and abroad mostly contain HPV type 6 vaccine components.

In the whole process of HPV vaccine production, correct neutralization epitopes and stable VLPs structures are kept, which is the premise of ensuring the quality and effectiveness of the vaccine. Establishing accurate, sensitive and specific detection methods in all links of vaccine production process to carry out whole-course monitoring on antigen structure and quantification is not only a technical requirement, but also a guiding principle of regulation. The identification of antigen neutralizing epitopes by using neutralizing antibodies by using a classical antigen-antibody reaction principle is an effective means for vaccine quality control, and antigen components in HPV vaccine stock solution, semi-finished products and finished products are subjected to identification experiments, antigen content detection and antigen in-vitro efficacy determination by Western-blot, ELISA, IVRP and other detection methods, so that a basis can be provided for vaccine detection. Therefore, the monoclonal neutralizing antibody is an important reaction reagent for the quality control of the vaccine antigen, and the monoclonal antibody with specificity and neutralizing activity has irreplaceable effect in the quality control process of the vaccine. In addition, vaccine efficacy detection is an important index for releasing vaccine finished products, the HPV vaccine efficacy is evaluated by collecting serum after immunizing animals with vaccines and detecting the neutralizing titer of the serum by adopting a pseudovirus neutralization experiment at present, although the method can better reflect the neutralizing antibody level of the vaccine finished products, the method also has the defects of more animal dosage, larger fluctuation of detection values, long detection period and higher cost, and simultaneously violates the '3R' (reduction, substitution and optimization) principle of animal experiments, some enterprises at home and abroad have responded to the advocated international organization and gradually eliminate the method for detecting the vaccine finished product efficacy by the animal experiments. Quality control and characterization based on neutralizing antibodies against neutralizing epitopes and in vitro activity of vaccine antigens, which are currently recognized and used in partial practice, has been an efficient, economical and feasible alternative, e.g., HPV vaccines by merck, usa, and domestic hepatitis a and b vaccines, all of which have been released for vaccine production using neutralizing antibodies, partially or fully, rather than animal experiments.

At present, the research reports on the HPV11 type neutralizing active monoclonal antibody relative to other high-risk type HPV antibodies are few, and the antibodies with reliable quality are available abroad, but due to the limitation of independent knowledge right, domestic manufacturers are difficult to obtain and prepare a large amount of antibodies, which brings a large barrier to the development of domestic multivalent HPV vaccines, so that the development of the HPV11 type neutralizing active monoclonal antibody for HPV vaccine development and production has important significance.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide an antibody or an antigen binding fragment thereof which can be combined with the HPV type 11, wherein the antibody or the antigen binding fragment thereof which can be combined with the HPV type 11 can only be specifically reacted with the HPV type 11 antigen, has good specificity and neutralization activity, and relieves the problem of lacking of HPV type 11 neutralization activity antibodies or antigen binding fragments thereof in the prior art.

The second object of the present invention is to provide a biomaterial or a hybridoma cell line capable of encoding or producing the above-mentioned antibody or antigen-binding fragment thereof that binds to HPV type 11.

The third object of the present invention is to provide use of the above antibody or antigen-binding fragment thereof binding to HPV type 11, the above biomaterial or the above hybridoma cell line.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention, there is provided an antibody or antigen-binding fragment thereof that binds to HPV type 11, comprising a light chain variable region and/or a heavy chain variable region;

the light chain variable region is provided with a light chain CDR consisting of CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 are respectively shown as SEQ ID NO 1, 2 and 3, or respectively shown as SEQ ID NO 11, 12 and 13;

the heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 are respectively shown as SEQ ID NO.4, 5 and 6, or respectively shown as SEQ ID NO. 14, 15 and 16.

According to another aspect of the invention, the invention also provides a biological material comprising a nucleic acid fragment, a vector or a host cell;

the nucleic acid fragment is selected from (a 1) or (a 2):

(a1) DNA or RNA encoding the above antibody or antigen-binding fragment thereof that binds to HPV type 11;

(a2) A nucleic acid fragment complementary to the nucleic acid fragment defined in (a 1);

the vector comprises the nucleic acid fragment;

the host cell is transformed with the vector.

According to another aspect of the invention, the invention also provides a hybridoma cell strain which is a hybridoma cell strain 1F9 or a hybridoma cell strain 1B5;

the preservation number of the hybridoma cell strain 1F9 is as follows: CGMCC No.45155;

the preservation number of the hybridoma cell strain 1B5 is as follows: CGMCC No.45156.

According to another aspect of the present invention, there is also provided a reagent or kit for detecting HPV type 11L1 antigen, comprising the antibody or antigen-binding fragment thereof that binds to HPV type 11.

According to another aspect of the present invention, the present invention further provides the antibody or antigen-binding fragment thereof binding to HPV type 11, the biomaterial, the hybridoma cell strain or the reagent or kit for detecting HPV type 11, and applications of the antibody or antigen-binding fragment thereof binding to HPV type 11 in the following (x 1) to (x 4):

(x 1) vaccine quality control;

(x 2) preparing a product for vaccine quality control;

(x 3) preparing a product for clinical etiology testing;

(x 4) preparing a product for epidemiological investigation.

Compared with the prior art, the invention has the following beneficial effects:

the invention obtains the mouse-derived monoclonal antibody aiming at the HPV type 11 through screening by a hybridoma technology, and the antibody has high specificity and neutralization activity on HPV11L 1VLPs and pseudoviruses, which indicates that the antibody is an antibody aiming at HPV11L1 protein neutralization epitopes. Sequencing the gene of the antibody obtained by screening, and analyzing the CDR region of the antibody to obtain the light chain CDR with amino acid sequences shown as SEQ ID NO. 1, 2 and 3 or respectively shown as SEQ ID NO. 11, 12 and 13; and heavy chain CDRs whose amino acid sequences are shown in SEQ ID NOS 4, 5 and 6, respectively, or in SEQ ID NOS 14, 15 and 16, respectively.

The identification and characterization of the neutralizing epitope of the vaccine antigen by using a suitable neutralizing antibody is an important content for quality control and release in the vaccine industry, and thus the antibody is required to have good specificity and neutralizing activity. In order to fully prove the specificity of the obtained HPV11L1 antibody or antigen-binding fragment thereof, the cross-reaction verification of VLP antigens of HPV6, HPV11, HPV 16, HPV 18, HPV31, HPV 33, HPV45, HPV 52 and HPV 58 is firstly carried out by using an ELISA method, which shows that the antibody with the CDR sequences of the invention is specifically reacted with HPV11 type antigens only, and the excellent specificity is shown. In order to verify the neutralizing activity of the antibody, a pseudovirus neutralizing experiment is adopted, and the result shows that the neutralizing activity titer of hybridoma cell supernatant is as high as 5120.

The antibody or antigen binding fragment thereof which is derived based on the CDR sequence obtained by screening and is combined with the HPV type 11 has good specificity, sensitivity, linearity and accuracy, can specifically and quickly identify and quantify the HPV11L1 protein, evaluates the content and activity of the effective antigen components of the vaccine, is widely applied to the production quality control, clinical etiology detection and epidemic investigation of the HPV vaccine, and has important value on the production of the HPV vaccine and the prevention and control of cervical cancer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a standard curve for detection of VLP antigen after 200 and 5000 fold dilution of HPV type 11 rabbit polyclonal antibody and murine monoclonal antibody, respectively.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

According to one aspect of the present invention, there is provided an antibody or antigen-binding fragment thereof that binds to HPV type 11, said antibody or antigen-binding fragment thereof comprising at least one light chain variable region or one heavy chain variable region, or both light chain variable regions and heavy chain variable regions.

Light chain variable region:

the light chain variable region has a light chain CDR consisting of CDR-L1, CDR-L2 and CDR-L3.

The amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 are respectively shown in SEQ ID NO 1, 2 and 3:

CDR-L1:RASQSIGTSIH(SEQ ID NO:1)

CDR-L2:YASESIS(SEQ ID NO:2)

CDR-L3:QQSSNWPYT(SEQ ID NO:3)

the amino acid sequence of the light chain variable region comprising the light chain CDR consisting of CDR-L1 (SEQ ID NO: 1), CDR-L2 (SEQ ID NO: 2) and CDR-L3 (SEQ ID NO: 3) described above is preferably as shown in SEQ ID NO: 7.

Or the amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 are respectively shown as SEQ ID NO:11, 12 and 13:

CDR-L1:RASQSIGTNIH(SEQ ID NO:11)

CDR-L2:YASESVS(SEQ ID NO:12)

CDR-L3:QQSNNWPYT(SEQ ID NO:13)。

the amino acid sequence of the light chain variable region comprising the light chain CDR consisting of CDR-L1 (SEQ ID NO: 11), CDR-L2 (SEQ ID NO: 12) and CDR-L3 (SEQ ID NO: 13) described above is preferably as shown in SEQ ID NO: 17.

Heavy chain variable region:

the heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2 and CDR-H3.

The amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 are respectively shown in SEQ ID NO.4, 5 and 6:

CDR-H1:NYLIE(SEQ ID NO:4)

CDR-H2:VINPGSGGSNYNENFKG(SEQ ID NO:5)

CDR-H3:PIYYGNPWFAY(SEQ ID NO:6)

the amino acid sequence of the heavy chain variable region comprising the heavy chain CDR composed of the above-mentioned CDR-H1 (SEQ ID NO: 4), CDR-H2 (SEQ ID NO: 5) and CDR-H3 (SEQ ID NO: 6) is preferably as shown in SEQ ID NO: 9.

Or the amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 are respectively shown as SEQ ID NO:14, 15 and 16:

CDR-H1:NYLIE(SEQ ID NO:14)

CDR-H2:VINPGSGGFNYNEKFKG(SEQ ID NO:15)

CDR-H3:PIYYGYPGFAY(SEQ ID NO:16)

the amino acid sequence of the heavy chain variable region comprising the heavy chain CDR composed of the above CDR-H1 (SEQ ID NO: 14), CDR-H2 (SEQ ID NO: 15) and CDR-H3 (SEQ ID NO: 16) is preferably as shown in SEQ ID NO: 19.

In some preferred embodiments, the antibody or antigen-binding fragment thereof that binds to HPV type 11 comprises both a light chain variable region and a heavy chain variable region. When both the light chain variable region and the heavy chain variable region are contained, the light chain CDR and the heavy chain CDR are preferably combined in two ways as follows (A) and (B):

(A) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 which form the light chain CDR of the light chain variable region are respectively shown as SEQ ID NO 1, 2 and 3; meanwhile, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 which form the heavy chain CDR of the heavy chain variable region are respectively shown in SEQ ID NO.4, 5 and 6.

In this combination, the preferred embodiment is that the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 7, while the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 9.

More preferably, the 11 type HPV specific binding monoclonal antibodies 1F9 and 1F9 secreted and produced by the hybridoma cell strain 1F9 are murine monoclonal antibodies, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 7, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9, the light chain variable region is of an IgG3 subtype, and the light chain is of a kappa type. The preservation number of the hybridoma cell strain 1F9 is as follows: CGMCC No.45155.

(B) The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 which form the light chain CDR of the light chain variable region are respectively shown in SEQ ID NO. 11, 12 and 13; meanwhile, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 which form the heavy chain variable region heavy chain CDR are respectively shown in SEQ ID NO. 14, 15 and 16.

In this combination, the preferred embodiment is such that the amino acid sequence of the light chain variable region is shown in SEQ ID NO:17, while the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19.

More preferably, the monoclonal antibodies 1B5 and 1B5 which are secreted and produced by the hybridoma cell strain 1B5 and specifically bind to the HPV type 11 are murine monoclonal antibodies, the amino acid sequence of the light chain variable region is shown as SEQ ID NO:17, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:19, the light chain variable region is of an IgG1 subtype, and the light chain is of a kappa type. The preservation number of the hybridoma cell strain 1B5 is as follows: CGMCC No.45156.

The invention obtains the mouse-derived monoclonal antibody and the hybridoma cell aiming at the 11-type HPV by screening through a hybridoma technology, detects the specificity of the monoclonal antibody through a direct ELISA method, finds that the antibody has no cross reaction with antigens such as HPV6, HPV 16, HPV 18, HPV31, HPV 33, HPV45, HPV 52, HPV 58 and the like, and has good specificity. The neutralizing activity of the antibody is detected by a pseudovirus neutralizing experimental method of an ELISPOT instrument, and the neutralizing activity to HPV11 type pseudovirus is shown to be high. And simultaneously, the heavy chain variable region gene and the light chain variable region gene of the antibody are sequenced, the CDR regions of the heavy chain variable region gene and the light chain variable region gene are analyzed, and the antibody is comprehensively characterized.

The neutralizing epitope of the antigen is a structural basis for generating the neutralizing antibody and is also a premise for stimulating effective immune response as a vaccine candidate antigen. Due to the heterogeneity and diversity of antibody production, antibodies with different CDR regions are produced even against the same antigen, and screening for good quality antibodies with neutralizing activity has a low probability, and the antibodies are unique.

It is well known in the art that both the binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and that variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established, well-known techniques of the art. The monoclonal antibody variants of the present invention having CDR sequences identical to those described above have similar biological activities because they have CDR sequences identical to those of the HPV type 11-binding antibody of the present invention.

An antigen-binding fragment is an antibody fragment of the same specificity as the parent antibody, and can be, for example, but not limited to, F (ab') ₂ One or more of, fab', fab, fv, scFv, dsFv, diabody, and antibody minimal recognition unit. In addition to the above functional fragments, any fragment having an increased half-life is also included.

These functional fragments typically have the same binding specificity as the antibody from which they are derived. Those skilled in the art will conclude from the disclosure of the present invention that the antigen-binding fragments of the present invention can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds.

Antigen-binding fragments can also be obtained by peptide synthesis by recombinant genetic techniques also known to those skilled in the art or by, for example, an automated peptide synthesizer; or by expressing a gene encoding the above functional fragment in a host cell.

The antibody or antigen-binding fragment thereof that binds to HPV type 11 provided by the present invention removes CDR regions, and the remaining sequence source species include, but are not limited to, one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

The CDR regions of the antibody or antigen-binding fragment thereof of the present invention that bind to HPV type 11 are derived from mouse. The whole sequence of the antibody or the antigen binding fragment thereof can be derived from a mouse, and the monoclonal antibodies 1F9 and 1B5 provided by the embodiment of the invention are murine antibodies; alternatively, the framework portion of the antibody or antigen-binding fragment thereof from which the CDR regions are removed, such as the light chain constant region, the heavy chain constant region, or the light chain variable region and the heavy chain variable region from which the CDR regions are removed, may also be selected from amino acid sequences of non-murine origin, such as, but not limited to, a human-murine chimeric antibody or a humanized antibody.

When the antibody binding to HPV type 11 is a whole antibody molecule, the antibody type may be, for example, but not limited to, igG1, igG2a, igG2b, igG2c, igG3, igG4, igA, igM, igE or IgD. Given that the CDR regions or the light and heavy chain variable regions of an antibody are known to those skilled in the art, those skilled in the art can obtain different types of antibodies using methods routine in the art, such as fusion of a variable region gene with a corresponding heavy or heavy chain constant region encoding gene and expression of the fusion protein in a host cell to obtain different types of antibodies.

According to another aspect of the invention, there is also provided a biological material comprising a nucleic acid fragment, a vector or a host cell.

Nucleic acid fragment(s): selected from (a 1) or (a 2)

(a1) An antibody or antigen-binding fragment thereof that encodes binding to HPV type 11; in some preferred embodiments, the nucleic acid fragment is DNA comprising a DNA fragment encoding the variable region of the light chain having the sequence set forth in SEQ ID NO 8 or SEQ ID NO 18; and/or a DNA fragment encoding the heavy chain variable region having the sequence shown in SEQ ID NO 10 or SEQ ID NO 20.

(a2) And a nucleic acid fragment complementary to the nucleic acid fragment defined in (a 1).

Carrier: including nucleic acid fragments as described above, the vector may also include portions encoding other components, such as but not limited to, regulatory sequences or marker genes, etc. The vector may be, for example, but not limited to, a prokaryotic expression vector, a eukaryotic expression vector, or an insect expression vector.

Host cell: the host cell is transformed with the above-mentioned vector so as to clone or express the above-mentioned vector.

According to another aspect of the present invention, the present invention further provides two hybridoma cells, namely hybridoma cell line 1F9 and hybridoma cell line 1B5, with the following preservation information:

hybridoma cell line 1F9: the preservation unit is as follows: china general microbiological culture Collection center (CGMCC); the preservation number is: CGMCC No.45155; the address of the preservation unit is as follows: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 12/05/2022.

Hybridoma cell line 1B5: the preservation unit is as follows: china general microbiological culture Collection center (CGMCC); the preservation number is: CGMCC No.45156; the address of the preservation unit is as follows: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 12/05/2022.

According to another aspect of the present invention, there is also provided a reagent or kit for detecting HPV type 11L1 antigen, said product comprising said antibody or antigen-binding fragment thereof binding to HPV type 11. Alternatively, the reagent or kit comprises an antibody which binds to HPV type 11, such as monoclonal antibody 1F9 or monoclonal antibody 1B5; the kit can be, but is not limited to, a kit which is a conventional detection method in the field, such as an ELISA detection kit, a Western Blot detection kit, an immunohistochemical detection kit or a colloidal gold detection test paper. It is understood that the reagents may also comprise reagents conventional in the art, such as but not limited to including one or more of lyoprotectants, buffering substances and solvents; the kit may further comprise reagents or consumables conventional in the art, such as but not limited to one or more of buffers, blocking solutions, secondary antibodies, chromogenic substances, labels and reaction substrates, magnetic particles, test strips and supporting members thereof.

According to another aspect of the present invention, the present invention further provides applications of the antibody or the antigen binding fragment thereof, the biological material, the hybridoma cell strain or the reagent or the kit for detecting the 11-type HPV L1 antigen, and examples of the applications include, but are not limited to, the following (x 1) to (x 4):

(x 1) vaccine quality control;

(x 2) preparing a product for vaccine quality control;

(x 3) preparing a product for clinical etiology testing;

(x 4) preparing a product for epidemiological investigation.

The antibody or the antigen binding fragment thereof combined with the 11-type HPV, which is provided by the invention, has high specificity and neutralization activity on HPV11L 1VLPs and pseudoviruses, and shows that the antibody or the antigen binding fragment thereof is an antibody aiming at HPV11L1 protein neutralization epitopes, and based on the good antigenicity of the antibody or the antigen binding fragment thereof combined with the 11-type HPV, the HPV11L1 protein can be specifically and quickly identified and quantified, so that the content and the activity of effective antigen components of the vaccine can be evaluated, and the antibody or the antigen binding fragment thereof can be widely applied to the production quality control, clinical etiology detection and epidemic investigation of HPV vaccines, and has important values on the production of the HPV vaccines and the prevention and control of cervical cancer.

In some preferred embodiments, a method for detecting HPV11L1 antigen by double-antibody sandwich indirect ELISA is established, HPV11L1 polyclonal coating, such as rabbit polyclonal antibody, is added to the antigen to be detected for incubation, the monoclonal antibody 1F9 or 1B5 combined with the 11 type HPV is added for incubation, and anti-mouse antibody enzyme-labeled complex, such as anti-mouse IgG enzyme-labeled complex, is added for display detection.

The result shows that the method has good specificity, sensitivity, linearity and accuracy, can quickly identify and quantify the HPV11L1 antigen, and evaluates the content and activity of the effective antigen components of the vaccine. Because the detection antibody is the neutralizing activity monoclonal antibody, the method can well control and characterize the neutralizing epitope of the vaccine antigen, and indicate the immunogenicity of the vaccine finished product to a certain extent, so that the method has the potential advantage of replacing animal experiments to evaluate the neutralizing activity of the vaccine, avoids the problems of large detection fluctuation, long detection period and high cost caused by animal experiments, and also conforms to the animal welfare principle advocated by the international society.

The method adopts HPV11L1 polyclonal antibody to carry out plate wrapping, can capture the antigen to be detected to the utmost extent, improves the sensitivity, adds the antigen to be detected, then adds HPV11 type monoclonal antibody (1F 9 or 1B 5), and finally adds anti-mouse enzyme labeled antibody to carry out color development and value reading. The traditional double-antibody sandwich ELISA method needs to label one monoclonal antibody, especially needs to label a plurality of types of HPV monoclonal antibodies when being applied to multivalent HPV vaccines, increases workload and cost, and adopts an anti-mouse enzyme-labeled antibody as a universal chromogenic antibody when the invention aims at quantitative detection of different multivalent antigens of the multivalent HPV vaccines, thereby greatly improving detection convenience and reducing cost.

EXAMPLE 1 preparation of monoclonal antibodies

1.1 immunization of mice

HPV11 type stock solution is mixed with adjuvants CFA and AD11.15 respectively, BALB/c mice are immunized, tail blood of the mice is taken on day 14, and the antibody titer in the tail blood is evaluated by using an indirect ELISA method. Adopting HPV11 type stock solution to coat an enzyme label plate (1; washing the plate 3 times with PBS solution, blocking with 5% millik-PBS at room temperature for 1hr; washing the plate with PBS solution for 1 time, adding the mouse tail blood diluted in gradient, and reacting at room temperature for 1hr; then washing the plate 3 times by using PBS solution, beating to dry, adding HRP-labeled goat anti-mouse Fc secondary antibody diluted by 1:2000, reacting at room temperature for 1hr, washing the plate 5 times by using PBS solution, beating to dry, adding substrate A solution and B solution with equal volume, reacting at room temperature in a dark place for 20min; then adding 50 mu L of stop solution, uniformly mixing, and reading OD on an enzyme-linked immunosorbent assay (ELISA) instrument ₄₅₀ And OD ₆₃₀ Value, output OD = OD ₄₅₀ -OD ₆₃₀ . And taking mice with tail blood titers exceeding 1/50000 for subsequent experiments.

1.2 cell fusion and monoclonal screening

Splenocytes of the killed mice are respectively fused with SP2/0 cells, cultured in a selective medium to be monoclonal cell strains, then selected for cloning, and 9 clones of 96-well plates are selected in total. Monoclonal screening was performed using HPV type 11 stock-coated elisa plates (1. A total of 56 positive clones with OD ≥ 1.0 were retained by primary screening from 846 clones in 9 96-well plates. And (3) performing amplification culture on the primary screened positive clones from a 96-well plate to a 48-well plate, performing re-screening after culturing for 2-3 days, and reserving the 13 strong positive clones with the re-screening OD being more than or equal to 1.0.

1.3 clone-specific screening

Cell supernatants of 13 positive clones were specifically tested, wherein 10 positive clones in total, 1B5, 1D2, 1F9, 2F8, 2G12, 3H5, 4A2, 4A5, 5C3 and 8H5, were specifically identified HPV11 VLP antigen, and the test results are shown in Table 1. The method comprises the following steps: respectively diluting VLP antigens of 9 subtypes including HPV6, HPV11, HPV 16, HPV 18, HPV31, HPV 33, HPV45, HPV 52 and HPV 58 to 1 mu g/mL, adding 100 mu L of VLP antigens into each hole, and reacting at 4 ℃ overnight; washing the plate 3 times with PBS solution, blocking with 5% milk-PBS at room temperature for 1hr; washing the plate with PBS solution for 1 time, adding 20 times diluted hybridoma cell supernatant of different clones, and reacting at room temperature for 1hr; then washing the plate 3 times with PBS solution, patting dry, adding HRP-labeled goat anti-mouse Fc secondary antibody diluted by 1; then adding 50 mu L of stop solution, uniformly mixing, and reading OD on an enzyme-linked immunosorbent assay (ELISA) instrument ₄₅₀ And OD ₆₃₀ Value, output OD = OD ₄₅₀ -OD ₆₃₀ 。

TABLE 1 specific ELISA detection of antibodies (OD values)

* All values are the average of duplicate wells; VLPs are virus-like particles

1.4 neutralization Activity assay

The detection result is shown in table 2, and the cell supernatants of 9 clones, 1B5, 1D2, 1F9, 2F8, 2G12, 4A2, 4A5, 5C3 and 8H5, have high neutralizing activity through the verification of a pseudovirus neutralizing experiment. The method comprises the following steps: 1) Diluting 293FT cells with DMEM complete medium (containing 10% fetal bovine serum, 1% double antibody, 1% L-Glu and 1% G418) and adding to 96-well cell culture plates, and culturing overnight at 37 ℃; 2) Diluting HPV6-GFP pseudovirus to the required concentration in DMEM complete medium in multiple proportions (approximately 400 fluorescent spots per well); 3) Diluting the supernatant of the HPV11 type hybridoma to be detected by using a DEME complete culture medium in a multiple ratio; 4) HPV type 11 monoclonal antibody (university of Bingzhou) was diluted in multiple ratios with DEME complete medium as a control group; 5) Respectively taking 60 mu L of diluted cell supernatant and pseudovirus, uniformly mixing, and then placing the mixture into a reactor to react for 1 hour at 4 ℃; 6) Slowly adding 100 mu L of mixed solution into a 96-hole cell culture plate paved with 293FT cells, and culturing at 37 ℃ for 60-96 hours; 6) Discarding the cell culture solution, and reading with ELSPOT (AID), wherein the number of fluorescence spots is not more than the average value of the number of positive fluorescence spots/2 result to judge that the neutralization activity is positive. The detection result shows that the received HPV pseudoviruses (three-drug organisms) meet the expected requirements, the number of fluorescent spots in each well meets the requirements (about 400), the number of fluorescent spots is also reduced along with the increase of the dilution multiple of the pseudoviruses, the HPV11 type cell supernatant clone strains 1B5, 1D2, 1F9, 2F8, 2G12, 4A2, 4A5, 5C3 and 8H5 have higher neutralizing activity, and most of the HPV11 type cell supernatant clones still have the neutralizing activity after being diluted by 5120 times.

Table 2: neutralization Activity assay

1.5 ascites preparation and antibody purification

Selecting clones 1F9 and 1B5 according to the affinity, specificity and neutralization activity results of cell supernatants for ascites preparation, inoculating 4 BALB/c mice with hybridoma cells respectively, collecting ascites on 10-14 days, and purifying the ascites by using protein G to obtain a purified antibody.

1.6 antibody sensitivity detection

Clones 1F9 and 1B5 were selected for ascites preparation and antibody purification based on the results of affinity, specificity and neutralizing activity of the cell supernatants, and the sensitivity of both antibodies 1F9 and 1B5 was 0.0005. Mu.g/mL, as shown in Table 3.

TABLE 3 antibody sensitivity detection

Note: NC as negative control 5% mill-PBS; the positive judgment standard is that the OD value is more than 2.1 times of the NC value; naN indicates the maximum detection limit is exceeded.

1.7 antibody type identification

The antibodies 1B5 and 1F9 were subtype-identified and the results are shown in Table 4. Antibody 1B5 is of the IgG1 subtype, the light chain is of the kappa type, while 1F9 is of the IgG3 subtype, and the light chain is of the kappa type.

Table 4: antibody subtype identification

Note: naN indicates exceeding of the maximum detection limit

Example 2 antibody Gene sequencing and analysis

Extracting total RNA of 1F9 and 1B5 hybridoma cell lines, and performing PrimeScript ^TM 1st Strand cDNA Synthesis Kit (Takara, cat # 6110A) reverse transcription, amplification of antibody VH and VL genes by RT-PCR, cloning to pUC-19T vector, sequencing by M13 universal primer on the vector, the results are as follows:

monoclonal antibody 1F9

2.1 VH Region-1F 9 (Leader sequence-Variable Region)

1)DNA Sequence:417bp(SEQ ID NO:10)

ATGGAATGGAGCGGAGTCTTTATCTTTCTCCTGTCAGTAACTGCAGGTGTTCACTCCCAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACCAATTACTTGATAGAGTGGATAAGGCAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTAGTAACTACAATGAGAACTTCAAGGGCAAGGCAACACTGACTACAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGACCAATCTACTATGGTAACCCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

2)Amino Acid Sequence:139aa(SEQ ID NO:9)

MEWSGVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWIRQRPGQGLEWIGVINPGSGGSNYNENFKGKATLTTDKSSSTAYMQLSSLTSDDSAVYFCARPIYYGNPWFAYWGQGTLVTVSA

CDR-H1:NYLIE(SEQ ID NO:4)

CDR-H2:VINPGSGGSNYNENFKG(SEQ ID NO:5)

CDR-H3:PIYYGNPWFAY(SEQ ID NO:6)

2.2 VL Region-1F 9 (Leader sequence-Variable Region)

1)DNA Sequence:381bp(SEQ ID NO:8)

ATGGTATCCACACCTCAGTTCCTTGTATTTTTGCTTTTCTGGATTCCAGCCTCCAGAGGTGACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACACTGGTTTCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGCTTATTACTGTCAACAAAGTAGTAACTGGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

2)Amino Acid Sequence:127aa(SEQ ID NO:7)

MVSTPQFLVFLLFWIPASRGDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWFQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIAAYYCQQSSNWPYTFGGGTKLEIK

CDR-L1:RASQSIGTSIH(SEQ ID NO:1)

CDR-L2:YASESIS(SEQ ID NO:2)

CDR-L3:QQSSNWPYT(SEQ ID NO:3)

Monoclonal antibody 1B5

2.3 VH Region-1B 5 (Leader sequence-Variable Region)

1)DNA Sequence:417bp(SEQ ID NO:20)

ATGGAATGGAGCGGAGTCTTTATCTTTCTCCTGTCAGTAACTGCAGGTGTTCACTCCCAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTACTTGATAGAGTGGGTAAAACAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTTTTAACTACAATGAGAAGTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGACCCATCTACTATGGTTACCCTGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

2)Amino Acid Sequence:139aa(SEQ ID NO:19)

MEWSGVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGFNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARPIYYGYPGFAYWGQGTLVTVSA

CDR-H1:NYLIE(SEQ ID NO:14)

CDR-H2:VINPGSGGFNYNEKFKG(SEQ ID NO:15)

CDR-H3:PIYYGYPGFAY(SEQ ID NO:16)

2.4 VL Region-1B 5 (Leader Sequence-Variable Region) 1) DNA Sequence:381bp (SEQ ID NO: 18)

ATGGTATCCACACCTCAGTTCCTTGTATTTTTGCTTTTCTGGATTCCAGCCTCCAGAGGTGACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTGTCTCTGGGATCCCTTCCAGGTTTCGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAACTGGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

2)Amino Acid Sequence:127aa(SEQ ID NO:17)

MVSTPQFLVFLLFWIPASRGDILLTQSPAILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESVSGIPSRFRGSGSGTDFTLSINSVESEDIADYYCQQSNNWPYTFGGGTKLEIK

CDR-L1:RASQSIGTNIH(SEQ ID NO:11)

CDR-L2:YASESVS(SEQ ID NO:12)

CDR-L3:QQSNNWPYT(SEQ ID NO:13)

EXAMPLE 3 use of monoclonal antibodies

3.1 establishment of double antibody Sandwich Indirect ELISA method

The method comprises the following specific steps:

1) Diluting HPV11 (R150) rabbit polyclonal antibody 200 times, 500 times, 1000 times and 2000 times with coating solution (carbonate buffer), 100 uL/hole, 4 ℃, coating (carbonate buffer), and standing overnight;

2) Adding 5% skimmed milk (PBS), sealing at 37 deg.C for 2 hr at 200 μ L/well;

3) 2% skim milk diluted HPV type 11 VLP stock (R621 (11) 180725P) to 120. Mu.g/mL, then 3 fold diluted to 11 gradients, 100. Mu.L/well, 37 ℃,1h;

4) Diluting corresponding HPV11 (1F 9 or 1B 5) murine monoclonal antibody with 2% skim milk 5000 times, 100 μ L/well, 37 deg.C, 1h;

5) 2% skim milk 5000 times/10000 times diluted goat anti-mouse lgG-HRP secondary antibody (BIO-RAD, 100. Mu.L/well, 37 ℃,1h;

6) Adding TMB developing solution, 100 μ L/ml, developing at 37 deg.C for 10min;

7) Adding 50 mu L/hole concentrated sulfuric acid to terminate the reaction, and measuring the reading value at 450nm and the reference wavelength of 620nm by using an enzyme-labeling instrument.

The results are shown in table 5, where AB behavior is a 200-fold dilution of rabbit polyclonal antibody; diluting the rabbit polyclonal antibody with CD behavior by 500 times; diluting the EF behavior rabbit polyclonal antibody by 1000 times; GH behavior rabbit polyclonal antibody is diluted 2000 times.

TABLE 5 different dilution multiple of HPV type 11 rabbit polyclonal antibody and murine monoclonal antibody for detecting OD value of VLP antigen

	1	2	3	4	5	6	7	8	9	10	11	12
													A	3.267	3.236	3.262	3.128	2.929	2.295	1.355	0.672	0.283	0.129	0.071	0.06
B	3.302	3.171	3.168	3.045	2.668	2.188	1.284	0.685	0.276	0.119	0.071	0.053
													C	3.228	3.191	3.194	3.001	2.636	2.061	1.016	0.591	0.231	0.095	0.053	0.041
D	3.248	3.153	3.182	2.978	2.552	1.995	1.141	0.55	0.232	0.099	0.054	0.039
													E	3.267	3.151	3.155	2.951	2.385	1.839	1.105	0.518	0.194	0.083	0.046	0.035
F	3.247	3.122	3.1	2.924	2.465	1.827	1.036	0.431	0.191	0.083	0.044	0.034
													G	3.202	3.103	3.058	2.718	2.365	1.735	0.868	0.357	0.131	0.059	0.031	0.027
H	3.401	3.249	3.278	3.047	2.476	1.811	0.948	0.374	0.142	0.06	0.033	0.031

The detection results are shown by a 4-parameter curve, and have good linear relation under the following antibody reaction concentration conditions, namely the dilution times of the HPV11 type rabbit polyclonal antibody and the mouse monoclonal antibody are respectively 200 and 5000, the VLP antigen initial dilution concentration is 120 mu g/mL, the specific detection results are shown in figure 1, and the curve parameters are shown in the following table:

TABLE 6

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (11)

1. An antibody or antigen-binding fragment thereof that binds to HPV type 11, comprising a light chain variable region and/or a heavy chain variable region;

the light chain variable region is provided with a light chain CDR consisting of CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences of the CDR-L1, the CDR-L2 and the CDR-L3 are respectively shown as SEQ ID NO 1, 2 and 3, or respectively shown as SEQ ID NO 11, 12 and 13;

the heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 are respectively shown as SEQ ID NO.4, 5 and 6, or respectively shown as SEQ ID NO. 14, 15 and 16.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region has the amino acid sequence shown in SEQ ID No. 7 or SEQ ID No. 17;

and/or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9 or SEQ ID NO. 19.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a light chain variable region and a heavy chain variable region;

the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 which form the light chain CDR of the light chain variable region are respectively shown as SEQ ID NO 1, 2 and 3; and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 which form the heavy chain CDR of the heavy chain variable region are respectively shown in SEQ ID NO.4, 5 and 6;

or the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 which form the light chain CDR of the light chain variable region are respectively shown in SEQ ID NO. 11, 12 and 13; and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 which form the heavy chain variable region heavy chain are respectively shown in SEQ ID NO. 14, 15 and 16.

4. The antibody or antigen-binding fragment thereof of claim 3, comprising a light chain variable region and a heavy chain variable region;

the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9;

or the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19.

5. The antibody or the antigen-binding fragment thereof according to claim 4, wherein the antibody that binds to HPV type 11 is secreted from hybridoma cell line 1F9 or hybridoma cell line 1B5;

the preservation number of the hybridoma cell strain 1F9 is as follows: CGMCC No.45155;

the preservation number of the hybridoma cell strain 1B5 is as follows: CGMCC No.45156.

6. Biological material comprising a nucleic acid fragment, a vector or a host cell;

the nucleic acid fragment is selected from (a 1) or (a 2):

(a1) DNA or RNA encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5;

(a2) A nucleic acid fragment complementary to the nucleic acid fragment defined in (a 1);

the vector comprises the nucleic acid fragment;

the host cell is transformed with the vector.

7. The biomaterial according to claim 6, wherein the DNA comprises a DNA fragment encoding a light chain variable region having the sequence shown in SEQ ID NO 8 or SEQ ID NO 18; and/or, a DNA fragment encoding the heavy chain variable region having the sequence shown in SEQ ID NO 10 or SEQ ID NO 20.

8. A hybridoma cell strain, wherein the hybridoma cell strain is a hybridoma cell strain 1F9 or a hybridoma cell strain 1B5;

the preservation number of the hybridoma cell strain 1F9 is as follows: CGMCC No.45155;

the preservation number of the hybridoma cell strain 1B5 is as follows: CGMCC No.45156.

9. A reagent or kit for detecting HPV L1 type 11 antigen, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

10. Use of the antibody or antigen-binding fragment thereof binding to HPV type 11 according to any one of claims 1 to 5, the biomaterial according to claim 6 or 7, the hybridoma cell line according to claim 8, or a reagent or kit for detecting HPV type 11 in the following (x 1) to (x 4):

(x 1) vaccine quality control;

(x 2) preparing a product for vaccine quality control;

(x 3) preparing a product for clinical etiology testing;

(x 4) preparation of products for epidemiological investigations.

11. The use of claim 10, wherein the vaccine quality control comprises detection of HPV11L1 antigen in HPV vaccine by double antibody sandwich indirect ELISA, which comprises plate-coating with HPV11L1 polyclonal antibody, adding the antibody of claim 5, and adding anti-mouse enzyme labeled antibody for development and reading.

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