CN115724951B - Antibodies or antigen binding fragments thereof that bind to HPV type 11 and uses thereof - Google Patents

Abstract

The invention provides an antibody combined with 11-type HPV or an antigen-binding fragment thereof 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 having amino acid sequences shown as SEQ ID NO. 1, 2 and 3, respectively, or as shown as SEQ ID NO. 11, 12 and 13, respectively; the heavy chain variable region has heavy chain CDRs consisting of the amino acid sequences shown in SEQ ID NOS 4, 5 and 6, respectively, or CDR-H1, CDR-H2 and CDR-H3 shown in SEQ ID NOS 14, 15 and 16, respectively. The antibody or the antigen binding fragment thereof only specifically reacts with HPV11 type antigen, and has good specificity and neutralization activity.

Description

Antibodies or antigen binding fragments thereof that bind to HPV type 11 and uses 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 papillomaviruses (Human Papillomavirus, HPV) are small non-enveloped DNA viruses, about 200 types exist, more than 40 types of HPV mainly infect skin and mucosal tissues, and more than 40 types of HPV infection can cause human diseases, and HPV can be classified into high-risk types and low-risk types according to the relation between HPV infection and occurrence of cancers, wherein HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and the like are high-risk types, and persistent infection of HPV of high-risk types can cause malignant tumors such as cervical cancer, anal cancer, vaginal cancer and the like. Worldwide, cervical cancer ranks third among high-grade malignant tumors in women, about 60 tens of thousands of women worldwide have new cervical cancer every year, and more than 30 tens of thousands of women die from cervical cancer. Other HPV subtypes belong to low-risk types, mainly cause wart hyperplasia of skin mucous membrane, and are represented by benign lesions such as condyloma acuminatum, flat wart and the like, and no good treatment method is clinically available at present. Epidemiological investigation and clinical studies show that the detection rate of HPV6 and HPV11 types in genital condyloma acuminatum tissues is highest, and statistics show that the ratio of genital warts to recurrent respiratory papillomatosis caused by HPV6 and HPV11 infection exceeds 90 percent.

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

The correct neutralizing epitope and stable VLPs structure are maintained in the whole process of HPV vaccine production, and are the preconditions for guaranteeing the quality and effectiveness of the vaccine. The establishment of accurate, sensitive and specific detection methods in each link of vaccine production process for whole-course monitoring of antigen structure and quantification is not only a technical requirement, but also a guiding principle of regulations. The classical antigen-antibody reaction principle is utilized, the neutralizing antibody is adopted to identify neutralizing epitopes of the antigen, which is an effective means for vaccine quality control, and detection methods such as Western-blot, ELISA, IVRP and the like are used for carrying out identification experiments, antigen content detection and in-vitro effectiveness measurement on antigen components in HPV vaccine stock solutions, semi-finished products and finished products, so that a basis can be provided for vaccine detection release. Therefore, monoclonal neutralizing antibodies are important reagents for quality control of vaccine antigens, and monoclonal antibodies with specificity and neutralization activity have irreplaceable roles in the quality control process of vaccines. In addition, vaccine efficacy detection is an important index for the release of vaccine finished products, at present, HPV vaccine efficacy is evaluated by collecting serum after vaccine immunization of animals and detecting the neutralization titer of the serum by adopting a pseudo-virus neutralization experiment, and although the method can better reflect the neutralization antibody level of the vaccine finished products, the method for detecting the vaccine finished products by gradually eliminating animal experiments has the defects of more animal consumption, larger fluctuation of detection values, long detection period and higher cost and also violates the principle of '3R' (reduction, substitution and optimization) of animal experiments. It has been recognized and used in part practice at present that based on the quality control and characterization of neutralizing antibodies against neutralizing epitopes and in vitro activity of vaccine antigens, an efficient, economical and viable alternative approach has been to use neutralizing antibodies in part or in whole, rather than animal-test-release vaccine products, such as HPV vaccines of merck, national hepatitis a and hepatitis b vaccines.

At present, research reports on HPV11 type neutralizing activity monoclonal antibodies are less than those of other high-risk type HPV antibodies, antibodies with reliable quality exist abroad, but due to limitation of autonomous knowledge rights, domestic manufacturers are difficult to obtain and prepare in large quantities, and a large barrier is brought to the development of domestic multivalent HPV vaccines, so that the development of HPV11 type neutralizing activity monoclonal antibodies for HPV vaccine development and production has important significance.

In view of this, the present invention has been made.

Disclosure of Invention

The first object of the present invention is to provide an antibody or antigen-binding fragment thereof binding to HPV type 11, which specifically reacts only with HPV type 11 antigen, has good specificity and neutralization activity, and alleviates the problem of lack of HPV type 11 neutralizing activity antibodies or antigen-binding fragments thereof in the prior art.

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

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

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 which binds to HPV type 11, characterised by 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 in SEQ ID NO. 1, 2 and 3 or are respectively shown in 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, CDR-H2 and CDR-H3 are shown as SEQ ID NO 4, 5 and 6 or as SEQ ID NO 14, 15 and 16 respectively.

According to another aspect of the present invention, there is also provided 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-described 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 present invention, there is also provided a hybridoma cell line, which is hybridoma cell line 1F9 or hybridoma cell line 1B5;

the hybridoma cell strain 1F9 has a preservation number of: 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 type 11 HPV L1 antigen, the reagent or kit comprising the antibody or antigen-binding fragment thereof which binds to type 11 HPV.

According to another aspect of the present invention, there is provided the use of the antibody or antigen binding fragment thereof which binds to HPV type 11, the biological material, the hybridoma cell line or the reagent or kit for detecting HPV type 11 in (x 1) to (x 4) as follows:

(x 1) vaccine quality control;

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

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

(x 4) preparation of a product for epidemic investigation.

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

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

The identification and characterization of neutralizing epitopes of vaccine antigens by using suitable neutralizing antibodies is an important matter of quality control and release in the vaccine industry, which requires good specificity and neutralizing activity of the antibodies. In order to fully demonstrate the specificity of the obtained HPV 11L 1 antibody or antigen-binding fragment thereof, cross-reaction validation of VLP antigens of HPV6, HPV11, HPV 16, HPV 18, HPV31, HPV 33, HPV45, HPV 52, HPV 58 was first performed by ELISA, indicating that antibodies possessing CDR sequences of the present invention specifically react only with HPV type 11 antigen, indicating excellent specificity. To verify the neutralizing activity of this antibody, pseudovirus neutralization experiments were used, and the results showed that the neutralizing activity titer of hybridoma cell supernatants was as high as 5120.

The antibody or antigen binding fragment thereof which is derived from the CDR sequence and combined with the 11 type HPV has good specificity, sensitivity, linearity and accuracy, can specifically and rapidly identify and quantify HPV 11L 1 protein, evaluate the content and activity of effective antigen components of the vaccine, and is widely applied to the production quality control, clinical etiology detection and epidemic investigation of HPV vaccine, and has important value for the production of 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 that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

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

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, 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 which binds to HPV type 11, the 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 region:

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

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:

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 the above-mentioned CDR-L1 (SEQ ID NO: 1), CDR-L2 (SEQ ID NO: 2) and CDR-L3 (SEQ ID NO: 3) is preferably as shown in SEQ ID NO: 7.

Alternatively, the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NOs 11, 12 and 13 respectively:

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 the above-mentioned CDR-L1 (SEQ ID NO: 11), CDR-L2 (SEQ ID NO: 12) and CDR-L3 (SEQ ID NO: 13) 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, CDR-H3.

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:

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 consisting 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.

Alternatively, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NOs 14, 15 and 16 respectively:

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 consisting of the above-mentioned 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 HPV type 11 contains 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 as follows (a) and (B):

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

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

More preferred embodiments are those in which the monoclonal antibody 1F9,1F9 specifically binding to HPV type 11 secreted by hybridoma cell line 1F9 is murine, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 7, while the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 9, is of the IgG3 subtype and the light chain is of the kappa type. The hybridoma cell line 1F9 has the accession number: CGMCC No.45155.

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

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

More preferred embodiments are those in which the monoclonal antibody 1B5,1B5 which specifically binds to HPV type 11 secreted by hybridoma cell line 1B5 is a murine monoclonal antibody, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 17, while the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 19, is of the IgG1 subtype and the light chain is of the kappa type. The hybridoma cell line 1B5 has a deposit number of: CGMCC No.45156.

The invention obtains the murine monoclonal antibody and hybridoma cell aiming at 11-type HPV through hybridoma technology screening, detects the specificity of the monoclonal antibody through a direct ELISA method, and discovers 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 an ELISPOT instrument through a pseudo-virus neutralization experiment method, and the neutralizing activity of the antibody on HPV11 type pseudo-virus is high. Meanwhile, the heavy chain variable region and the light chain variable region genes of the antibody are sequenced, and CDR regions of the antibody are analyzed, so that the antibody is comprehensively characterized.

The epitopes of the antibodies and the antigens are in one-to-one correspondence, one antigen epitope corresponds to one corresponding antibody, the branching epitopes and the space epitopes of the antigen epitope, most of the neutralizing epitopes are space epitopes, and the neutralizing epitopes of the antigen are structural bases for generating neutralizing antibodies and are also used as the precondition that vaccine candidate antigens excite effective immune responses. Because of the heterogeneity and diversity of antibody production, antibodies having different CDR regions are produced even against the same antigen, and the probability of screening to obtain a high-quality antibody having neutralizing activity is low, the antibody has uniqueness.

It is well known in the art that both binding specificity and avidity of antibodies are determined primarily by CDR sequences, and that amino acid sequences of non-CDR regions can be readily altered to obtain variants with similar biological activity according to well-established and well-known techniques. The monoclonal antibody variant having the CDR sequence identical to the CDR sequence has similar biological activity because of having the CDR sequence identical to the antibody binding to type 11 HPV.

The antigen binding fragment is an antibody fragment of the same specificity as the parent antibody, and may be, for example, but not limited to, F (ab') ₂ One or more of Fab', fab, fv, scFv, dsFv, bispecific antibodies, and antibody minimal recognition units. In addition to the functional fragments described above, any fragment whose half-life has been increased is included.

These functional fragments typically have the same binding specificity as the antibody from which they were derived. It is inferred from the description of the present invention by those skilled in the art 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 methods of cleavage of disulfide bonds by chemical reduction.

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

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

When the antibody binding to HPV type 11 is an intact 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 the CDR regions or light and heavy chain variable regions of antibodies known to those skilled in the art, those skilled in the art can employ methods conventional in the art to obtain different types of antibodies, for example, fusing the variable region genes to corresponding heavy chain or heavy chain constant region encoding genes, and expressing the fusion proteins in host cells to obtain different types of antibodies.

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.

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

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

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

And (3) a carrier: including nucleic acid fragments as described above, the vector may also include portions encoding other constituent elements, such as, but not limited to, encoding regulatory sequences or marker genes, and the like. 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-described vector so that cloning or expression of the above-described vector is achieved.

According to another aspect of the present invention, there are provided two hybridoma cells, hybridoma cell line 1F9 and hybridoma cell line 1B5, respectively, the preservation information being as follows:

hybridoma cell line 1F9: the preservation units are as follows: china general microbiological culture Collection center (CGMCC); the preservation number is: CGMCC No.45155; the preservation unit addresses are: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; the preservation date is: 2022, 05, 12.

Hybridoma cell line 1B5: the preservation units are as follows: china general microbiological culture Collection center (CGMCC); the preservation number is: CGMCC No.45156; the preservation unit addresses are: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; the preservation date is: 2022, 05, 12.

According to another aspect of the present invention there is also provided a reagent or kit for detecting type 11 HPV L1 antigen, the product comprising the antibody or antigen-binding fragment thereof that binds to type 11 HPV. Alternative examples are reagents or kits comprising an antibody that binds to HPV type 11, such as monoclonal antibody 1F9 or monoclonal antibody 1B5; the kit may be, for example, but not limited to, a kit of ELISA detection kit, western Blot detection kit, immunohistochemical detection kit or colloidal gold detection test paper, etc. which is a conventional detection method in the art. It will be appreciated that the reagents may also comprise reagents conventional in the art, for example, and without limitation, one or more of lyoprotectants, buffers and solvents; the kit may also contain 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 support members therefor, and the like.

According to another aspect of the present invention, there is provided the use of the antibody or antigen binding fragment thereof, the biological material, the hybridoma cell line or the reagent or kit for detecting HPV type 11L 1 antigen, examples of which may be, for example, but 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 detection;

(x 4) preparation of a product for epidemic investigation.

The antibody or antigen binding fragment thereof combined with the 11 type HPV has high specificity and neutralization activity on HPV 11L 1VLPs and pseudoviruses, shows that the antibody or antigen binding fragment thereof is an antibody aiming at HPV 11L 1 protein neutralization epitopes, can specifically and rapidly identify and quantify HPV 11L 1 protein based on good antigenicity of the antibody or antigen binding fragment thereof combined with the 11 type HPV, and evaluate the content and activity of effective antigen components of the vaccine, thereby being widely applied to production quality control, clinical etiology detection and epidemic investigation of HPV vaccines and having important value on production of the HPV vaccines and prevention and control of cervical cancer.

In some preferred embodiments, a method of detecting HPV 11L 1 antigen by double-antibody sandwich indirect ELISA is established, using HPV 11L 1 polyclonal antibody coating, e.g., rabbit polyclonal antibody, adding the antigen to be detected for incubation, adding the monoclonal antibody 1F9 or 1B5 conjugated to HPV type 11 for incubation, adding an anti-mouse antibody enzyme-labeled complex, e.g., an anti-mouse IgG enzyme-labeled complex, for performing a display detection.

The result shows that the method has good specificity, sensitivity, linearity and accuracy, can rapidly identify and quantify the HPV 11L 1 antigen, and evaluates the content and activity of the effective antigen component of the vaccine. Because the detection antibody is the neutralizing activity monoclonal antibody, the method can perform good quality control and characterization on the neutralizing epitope of the vaccine antigen, and indicates the immunogenicity of a vaccine finished product to a certain extent, so the method has the potential advantages of evaluating the neutralizing activity of the vaccine by replacing animal experiments, avoids the problems of large detection fluctuation, long detection period and high cost caused by the animal experiments, and simultaneously accords with animal welfare principles advocated by the international society.

The method adopts HPV 11L 1 polyclonal antibody to carry out plate wrapping, can capture antigen to be detected to the greatest extent, improves 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 reading. The traditional double-antibody sandwich ELISA method needs to label one monoclonal antibody, especially when the method is applied to multivalent HPV vaccine, a plurality of types of HPV monoclonal antibodies need to be labeled respectively, so that the workload and the cost are increased.

EXAMPLE 1 preparation of monoclonal antibodies

1.1 immunization of mice

HPV11 type stock solution was mixed with adjuvants CFA and AD11.15, BALB/c mice were immunized, mouse tail blood was taken on day 14, and the evaluation of antibody titer in tail blood was performed using an indirect ELISA method. Coating an ELISA plate with HPV11 type stock solution (1:100 dilution), adding 100 mu L of the ELISA plate into each hole, and reacting at 4 ℃ overnight; the plates were washed 3 times with PBS and blocked with 5% milk-PBS for 1hr at room temperature; washing the plate with PBS solution for 1 time, adding gradient diluted mouse tail blood, and reacting at room temperature for 1hr; washing the plate for 3 times by using a PBS solution, beating, adding an HRP-marked goat anti-mouse Fc secondary antibody diluted 1:2000, reacting for 1hr at room temperature, washing the plate for 5 times by using the PBS solution, beating, adding substrate A solution and substrate B solution with equal volumes, and reacting for 20min under the conditions of light shielding and room temperature; then 50 mu L of stop solution is added, and after even mixing, OD is read on an enzyme labeling instrument ₄₅₀ And OD (optical density) ₆₃₀ Value, output od=od ₄₅₀ -OD ₆₃₀ . Mice with tail blood titers exceeding 1/50000 were taken for subsequent experiments.

1.2 cell fusion and monoclonal screening

The splenocytes from the mice were sacrificed and fused with SP2/0 cells, and were cultured in selection medium as monoclonal cell lines and then cloned, and 9 clones were picked up in 96 well plates. The HPV11 stock solution is used for coating an ELISA plate (1:100 dilution) for monoclonal screening, and the method is the same. A total of 846 clones from 9 96-well plates were initially screened to retain 56 positive clones with OD.gtoreq.1.0. And (3) performing amplification culture on the primary screening positive clone from a 96-well plate to a 48-well plate, performing rescreening after 2-3 days of culture, and retaining 13 strong positive clone strains with the rescreening OD being more than or equal to 1.0.

1.3 clone specific screening

Specific detection of the supernatant of 13 Positive clone cells, wherein 10 total strains 1B5, 1D2, 1F9, 2F8, 2G12, 3H5, 4A2, 4A5, 5C3 and 8H5 are positive grams for specifically recognizing HPV11 VLP antigenThe results of the test are shown in Table 1. The method comprises the following steps: diluting 9 subtype VLP antigens of HPV6, HPV11, HPV 16, HPV 18, HPV31, HPV 33, HPV45, HPV 52 and HPV 58 to 1 mug/mL respectively, adding 100 mu L of each well, and reacting at 4 ℃ overnight; the plates were washed 3 times with PBS and blocked with 5% milk-PBS for 1hr at room temperature; then washing the plate with PBS solution for 1 time, adding hybridoma cell supernatants of different clones diluted by 20 times, and reacting for 1hr at room temperature; washing the plate for 3 times by using PBS solution, beating, adding HRP-marked goat anti-mouse Fc secondary antibody diluted by 1:10000, reacting for 1hr at room temperature, washing the plate for 5 times by using PBS solution, beating, adding substrate A solution and substrate B solution with equal volumes, and reacting for 20min under the conditions of light shielding and room temperature; then 50 mu L of stop solution is added, and after even mixing, OD is read on an enzyme labeling instrument ₄₅₀ And OD (optical density) ₆₃₀ Value, output od=od ₄₅₀ -OD ₆₃₀ 。

TABLE 1 specific ELISA detection of antibodies (OD value. Times.)

* All values are average values of complex pores; VLPs are virus-like particles

1.4 neutralization Activity assay

The cell supernatants of 9 clones of 1B5, 1D2, 1F9, 2F8, 2G12, 4A2, 4A5, 5C3 and 8H5 are all high in neutralization activity, and the detection results are shown in Table 2. The method comprises the following steps: 1) 293FT cells were diluted with DMEM complete medium (containing 10% fetal bovine serum, 1% diabody, 1% L-Glu and 1% G418) and added to 96-well cell culture plates for overnight incubation at 37 ℃; 2) Dilution of HPV6-GFP pseudovirus to the desired concentration (about 400 fluorescent spots per well) with DMEM complete medium-fold ratio; 3) Diluting the supernatant of HPV11 type hybridoma cells to be detected by using DEME complete medium in a multiple ratio; 4) HPV type 11 monoclonal antibody (Gungzhou university) was diluted with DEME complete medium-fold ratio as control group; 5) Mixing 60 mu L of diluted cell supernatant and pseudovirus uniformly, and then placing the mixture into a temperature of 4 ℃ for reaction for 1 hour; 6) Slowly adding 100 μl of the mixed solution into a 96-well cell culture plate paved with 293FT cells, and culturing at 37deg.C for 60-96 hr; 6) The cell culture liquid is discarded, and ELSPOT (AID) is used for reading, wherein the value of fluorescent spots is less than or equal to the average value of positive fluorescent spot points/2 results, and the neutralization activity is judged to be positive. The detection result shows that the received HPV pseudoviruses (three-drug organisms) meet the expected requirement, the number of fluorescent spots in each hole meets the requirement (about 400), the number of fluorescent spots is reduced along with the increase of the dilution factor of the pseudoviruses, and the HPV11 type cell supernatant clones 1B5, 1D2, 1F9, 2F8, 2G12, 4A2, 4A5, 5C3 and 8H5 all have higher neutralization activity, and most of the HPV11 type cell supernatant clones still have neutralization activity after 5120 times of dilution.

Table 2: neutralization activity assay

1.5 ascites preparation and antibody purification

Clones 1F9 and 1B5 are selected according to the affinity, specificity and neutralization activity results of cell supernatant to prepare ascites, hybridoma cells are respectively inoculated with 4 BALB/c mice, the ascites is collected on days 10-14, and the ascites is purified by protein G to obtain purified antibodies.

1.6 antibody sensitivity detection

Clone 1F9 and 1B5 were selected for ascites production and antibody purification according to the affinity, specificity and neutralization activity results of the cell supernatant, and the sensitivity of each of the antibodies 1F9 and 1B5 could reach 0.0005. Mu.g/mL, as shown in Table 3.

TABLE 3 antibody sensitivity detection

Note that: NC is negative control 5% milk-PBS; the positive judgment standard is that the OD value is 2.1 times of the NC value; naN means that the maximum detection limit is exceeded.

1.7 antibody identification

Subtype identification was performed on antibodies 1B5 and 1F9, and the detection results are shown in table 4. Antibody 1B5 is of the IgG1 subtype, the light chain is of the kappa type, and 1F9 is of the IgG3 subtype, the light chain is of the kappa type.

Table 4: antibody subtype identification

Note that: naN indicates that the maximum detection limit is exceeded

Example 2 antibody Gene sequencing and analysis

Extracting total RNA of 1F9 and 1B5 hybridoma cell strains, and carrying out PrimeScript ^TM 1st Strand cDNA Synthesis Kit (Takara, cat#6110A), the antibody VH and VL genes were amplified by RT-PCR, cloned into pUC-19T vector, and sequenced by M13 universal primer on the vector, the results were 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 domain-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 the HPV11 (R150) rabbit polyclonal antibody 200-fold, 500-fold, 1000-fold and 2000-fold with coating solution (carbonate buffer), 100 μl/well, coating (carbonate buffer) at 4 ℃ overnight;

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

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

4) Diluting murine monoclonal antibody corresponding to HPV11 (1F 9 or 1B 5) with 2% skim milk 5000-fold at 37deg.C for 1h at 100 μl/well;

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

6) TMB color development liquid is added, 100 mu L/ml is added, and color development is carried out for 10min at 37 ℃;

7) The reaction was terminated by adding 50. Mu.L/Kong Nong sulfuric acid, and the read values at 450nm and reference wavelength of 620nm were measured by an ELISA reader.

The results are shown in table 5, where AB behaved rabbit polyclonal antibody was 200-fold diluted; CD behaving rabbit polyclonal antibody 500-fold dilution; EF behavior rabbit polyclonal antibody 1000-fold dilution; GH behaved rabbit polyclonal antibody 2000-fold dilution.

TABLE 5 OD values of different dilution of HPV11 type rabbit polyclonal and murine monoclonal antibodies to 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, namely, the dilution factors of the HPV11 type rabbit polyclonal antibody and the mouse monoclonal antibody are respectively 200 and 5000 under the condition of the following antibody reaction concentration, the initial dilution concentration of VLP antigen is 120 mug/mL, the specific detection results are shown in FIG. 1, and the curve parameters are shown in the following table:

TABLE 6

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (9)

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

the light chain variable region has a light chain CDR consisting of CDR-L1, CDR-L2, CDR-L3, and the heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2, CDR-H3;

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

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

2. The antibody or antigen-binding fragment thereof of claim 1, 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;

alternatively, 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.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody that binds to HPV type 11 is secreted by hybridoma cell line 1F9 or hybridoma cell line 1B5;

the hybridoma cell strain 1F9 has a preservation number of: CGMCC No.45155;

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

4. Biological material, characterized by comprising a nucleic acid fragment, a vector or a host cell;

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

(a1) A DNA or RNA encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3;

(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.

5. The biomaterial according to claim 4, wherein the DNA comprises a DNA fragment encoding a light chain variable region as shown in SEQ ID NO. 8 or SEQ ID NO. 18; and/or, a DNA fragment comprising a heavy chain variable region as shown in SEQ ID NO. 10 or SEQ ID NO. 20.

6. The hybridoma cell strain is characterized by being hybridoma cell strain 1F9 or hybridoma cell strain 1B5;

the hybridoma cell strain 1F9 has a preservation number of: CGMCC No.45155;

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

7. A reagent or kit for detecting HPV type 11L 1 antigen, comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.

8. Use of the antibody or antigen binding fragment thereof that binds to HPV type 11 according to any one of claims 1-3, the biomaterial according to claim 4 or 5, the hybridoma cell line according to claim 6 or the reagent or kit for detecting HPV type 11L 1 antigen according to claim 7 in (x 1) - (x 4) as follows:

(x 1) HPV vaccine quality control comprising HPV type 11;

(x 2) preparing a product for HPV vaccine quality control comprising HPV type 11;

(x 3) preparing a product for detection of clinical etiology associated with HPV;

(x 4) preparing a product for an epidemic investigation associated with HPV.

9. The use according to claim 8, wherein the vaccine quality control comprises detection of HPV 11L 1 antigen in HPV vaccine by double-antibody sandwich indirect ELISA, comprising inclusion of HPV 11L 1 polyclonal antibody for inclusion in a panel, followed by addition of the antibody of claim 3, and finally addition of anti-murine enzyme-labeled antibody for color development and reading.