CN107556379B - Monoclonal antibody for identifying high-risk HPV E7 protein and application thereof - Google Patents

Abstract

The invention provides a monoclonal antibody for identifying high-risk HPV E7 positive cervical epithelial cancer cells and application thereof, wherein the antibody can detect a cervical cancer biomarker HPV E7 protein in tumor cells with high specificity, and covers high-risk subtypes such as high-risk HPV16, HPV31, HPV35, HPV52 and HPV58, so that cancerous cervical epithelial cells and cervical abnormal or non-cancerous cervical epithelial cells can be distinguished, basis is provided for doctors to accurately diagnose cancer caused by HPV infection, missed diagnosis rate of high-level cervical lesions can be effectively reduced, sufficient time and basis are provided for clinics to diagnose and treat patients, and early-stage cervical disease detection and early intervention are improved. At the same time, unnecessary colposcopy can be reduced and avoided.

Description

Monoclonal antibody for identifying high-risk HPV E7 protein and application thereof

Technical Field

The invention belongs to the field of biological diagnosis and medicine, and particularly relates to a monoclonal antibody for identifying Human Papilloma Virus (HPV)16 subtype positive tumor cells including human cervical epithelial cancer cells and application thereof.

Background

Cervical cancer is the second most common female malignancy, with approximately 50 million women globally diagnosed with cervical cancer each year, with more than half of them dying as a result. Early symptoms of cervical cancer are not evident and there is a long, reversible, pre-cancerous period in the progression. Statistically, about 20% of low-grade cervical lesions will turn into high-grade lesions, and 30% of them will further turn into malignant tumors if not treated in time. It takes about 10 years to develop from a general cervical cancer precursor to cervical cancer, and if the cancer precursor can be diagnosed early, the occurrence of cancer can be prevented.

After Harald Zur Hausen, a German scientist, suggested that HPV may be a sexually transmitted carcinogen in 1976, the study of the relationship between HPV infection and cervical cancer became a popular topic of research on the etiology of tumor viruses. Harald zur Hausen received the Nobel biomedical prize in 2008 because of the demonstration that HPV virus is the causative agent of cervical cancer. There are over 100 subtypes of HPV viruses known at present, and they are classified into two broad categories according to the severity of carcinogenicity of viruses: low risk HPV (non-cancer associated type): the most common subtypes include HPV6, 11, 42, 43 and 44; high risk HPV (cancer-associated type): the most common subtypes include HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. Wherein the high-risk HPV16, 31, 33, 35, 52, 58 is highly homologous to the same side branch in the HPV subtype classification evolutionary tree, and the high-risk HPV18 and HPV45 are highly homologous to the other side branch (Lidqvist M, nilsso O et al detection of human papillomavir oncoprotein E7in liquid-based cytology.2012). More than 99% of cervical cancer cases worldwide are associated with high-risk HPV. Adult women have a probability of infecting HPV in their lifetime of 80%, and 20-30% (region-by-region) of the population of women in China are HPV positive carriers (Wang R, Guo X, Wu S et al. national wide prediction of human papillomavir infection and viral genetic distribution in 37cities in China.2015). However, HPV infection is not equal to cervical cancer, more than 80% of positive carriers of transient infection can be self-healed within 1-2 years, and only a small part of positive carriers can progress to cervical lesion or cancer. The American Cancer Society (ACS), american colposcopy and cervical pathology society (ASCCP) and american clinical pathology society (ASCP) 2012 suggested that the best strategy for cervical cancer screening would be to both identify precancerous lesions that may progress to invasive cancer, and avoid probing and unnecessary treatment of transient HPV infections and their corresponding benign lesions that do not necessarily have malignant progression.

For many years, the diagnosis of cervical intraepithelial neoplasia and cervical cancer, which is common internationally, has mainly followed the "three-step" diagnostic procedure, i.e. cervical cytology, colposcopy and histopathological examination. Cervical cytology examination is a commonly used screening method for cervical intraepithelial neoplasia and cervical cancer, and early lesions can be found. However, the sensitivity of the cytological method for detecting CIN-II and higher-level lesions is low, the cytology has no very good specificity to precancerous lesions, the result difference among laboratories is large, and the screening intervals are frequent. Cytological interpretation requires that morphological grounds have irrevocable subjectivity and causes extremely heavy workload on pathologists. These factors greatly restrict the efficiency and coverage of early diagnosis and individualized treatment of cervical cancer in China.

In recent years, new detection means based on biomarkers have been widely regarded for clinical application. Cell proliferation associated protein p16^INK4aThe application of the cervical cancer detection alternative molecular target is widely regarded by clinicians in the field of cervical cancer diagnosis at home and abroad, and the clinical application of the molecular target is beneficial to improving the interpretation accuracy of high-grade precancerous lesions (CIN-II and CIN-III) and the consistency of diagnosis results among pathologists. Research shows that p16 is generated during cell transformation caused by high-risk HPV persistent infection^INK4aThe relation between high expression and cervical cancer is realized through a signal path activated by high-risk HPV E7 protein. Middleton Kate et al, in studies on HPV diagnostic marker selection during tumor progression, showed that HPV E7 protein is expressed in large amounts in high-grade lesions and is positively correlated with the degree of carcinogenesis (Middleton K., et al, Organization of human papillomavir productive progression products a basic for selection of diagnostic markers. 2003). Theoretical and extensive research data indicate that the E7 protein can be used as a specific tumor molecular marker for detecting high-grade cervical injury and cervical cancer.

Therefore, in order to achieve an early and accurate diagnosis of cervical lesions, it is desirable to provide a method that does not rely on conventional morphological examinations and molecular detection for high-risk HPV infections. The method should be able to specifically identify high-grade cervical lesions present in all patient populations (improving the coverage of high-risk HPV subtype detection as much as possible), and in particular to be able to shunt cases in which the morphological detection is classified as a low grade lesion, but in fact is a high grade lesion, or has a significant probability of developing a high grade lesion. There is therefore a need in the art for specific, reliable diagnostic methods that are capable of detecting high-grade cervical disease and shunting those who are positive for HPV detection.

Disclosure of Invention

The invention aims to provide a monoclonal antibody capable of identifying high-risk HPV E7 protein positive cervical epithelial cancer cells and application thereof.

In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO.4,

CDR2 shown in SEQ ID NO.6, and

CDR3 shown in SEQ ID NO. 8.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 10.

In a second aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region as described in the first aspect of the invention, an

A heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.

In a third aspect of the present invention, there is provided a light chain variable region of an antibody, said light chain variable region having complementarity determining regions CDRs selected from the group consisting of:

CDR1' shown in SEQ ID NO.12,

CDR2' of SEQ ID NO.14, and

CDR3' shown in SEQ ID NO. 16.

In another preferred embodiment, the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 18.

In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having the light chain variable region as described in the third aspect of the invention, and

a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.

In a fifth aspect of the invention, there is provided an antibody having:

(1) a heavy chain variable region according to the first aspect of the invention; and/or

(2) A light chain variable region according to the third aspect of the invention.

In another preferred embodiment, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.

In another preferred embodiment, the antibody is an antibody specific to HPV; preferably, the antibody is an antibody specific against HPV 16; more preferably, the antibody is an antibody specific against HPV16E7 protein. The antibody also has the function of specifically resisting HPV31, HPV35, HPV52 and HPV58E7 proteins.

In another preferred embodiment, the antibody comprises: single chain antibodies, diabodies, monoclonal antibodies, chimeric antibodies (e.g., human rabbit chimeric antibodies), murine antibodies, rabbit antibodies, or humanized antibodies.

In another preferred example, the "HPV 16E7 protein" may be a wild-type HPV16E7 protein, or a derivative protein of the wild-type HPV16E7 protein. The HPV31E7 protein can be wild-type HPV31E7 protein or derivative protein of wild-type HPV31E7 protein, the HPV35E7 protein can be wild-type HPV35E7 protein or derivative protein of wild-type HPV35E7 protein, the HPV52E7 protein can be wild-type HPV52E7 protein or derivative protein of wild-type HPV52E7 protein, and the HPV58E7 protein can be wild-type HPV58E7 protein or derivative protein of wild-type HPV58E7 protein.

In another preferred example, the antibody is a monoclonal antibody capable of specifically binding to HPV16E7 protein, HPV31E7 protein, HPV35E7 protein, HPV52E7 protein and HPV58E7 protein.

In another preferred embodiment, the antibody does not bind to or has a low affinity for other HPV subtypes.

In another preferred embodiment, the antibody further has the following properties:

(3) the binding epitope capable of binding HPV16E7 specifically binds HPV16E7 is amino acids 5-34.

In a sixth aspect of the present invention, there is provided a recombinant protein having:

(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein is specific against HPV; preferably, specific anti-HPV 16; more preferably, it is specific against HPV16E7 protein. Preferably, the recombinant protein is also specifically resistant to high-risk HPV31, HPV35, HPV52 and HPV58 proteins.

In a seventh aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or

(2) A recombinant protein according to the sixth aspect of the invention.

In another preferred embodiment, the polynucleotide has the sequence shown in SEQ ID No.3, 5, 7, 9, 11, 13, 15, or 17.

In an eighth aspect of the invention, there is provided a vector comprising a polynucleotide according to the seventh aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

According to a ninth aspect of the invention, there is provided a genetically engineered host cell comprising a vector or genome according to the eighth aspect of the invention into which has been integrated a polynucleotide according to the seventh aspect of the invention.

In a tenth aspect of the invention, there is provided an immunoconjugate comprising:

(a) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and

(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.

In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or an immunoconjugate according to the tenth aspect of the invention; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in the form of injection.

In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating tumors selected from the group consisting of: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, endometrial cancer, penis cancer, adrenal gland tumor, or bladder tumor.

In a twelfth aspect of the invention, there is provided a use of the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, or the immunoconjugate according to the tenth aspect of the invention for the preparation of a medicament, a reagent, a detection plate or a kit;

the reagent, assay plate or kit is for:

(1) detecting HPV16 and/or HPV31 and/or HPV35 and/or HPV52 and/or HPV58E7 proteins in the sample; and/or

(2) Detecting endogenous HPV16 and/or HPV31 and/or HPV35 and/or HPV52 and/or HPV58E7 proteins in the tumor cells; and/or

(3) Detecting tumor cells expressing HPV16 and/or HPV31 and/or HPV35 and/or HPV52 and/or HPV58E7 proteins;

the medicament is used for treating or preventing tumors expressing HPV16 and/or HPV31 and/or HPV35 and/or HPV52 and/or HPV58E7 proteins.

In another preferred example, the sample contains HPV16 and/or HPV31 and/or HPV35 and/or HPV52 and/or HPV58E7 proteins.

In another preferred embodiment, the tumor comprises: tumors of the urogenital system, tumors of the respiratory system, tumors of the digestive system, comprising: cervical cancer, endometrial cancer, penile cancer, small cell lung cancer, melanoma or head and neck tumors, gastric cancer, liver cancer, leukemia, kidney tumors, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, or adrenal tumors.

In another preferred embodiment, the "tumor of urogenital system" includes: cervical cancer, bladder cancer, endometrial cancer, or penile cancer.

In another preferred embodiment, the reagent comprises a chip and immune microparticles coated with antibodies.

In a thirteenth aspect of the present invention, there is provided a method for detecting HPV E7 protein in a sample, the method comprising the steps of:

(1) contacting the sample with an antibody according to the fifth aspect of the invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex indicates the presence of HPV E7 protein in the sample.

In another preferred example, the detection is performed by ELISA method in step (2).

In another preferred example, the HPV E7 protein comprises HPV16 and/or HPV31 and/or HPV35 and/or HPV52 and/or HPV58E7 protein.

In another preferred example, in step (1), the sample is contacted with two antibodies against HPV E7 protein, at least one of which is an antibody according to the fifth aspect of the present invention, and detected by ELISA in step (2).

In another preferred embodiment, the "antigen-antibody complex" is a "first antibody-antigen-second antibody" ternary complex, wherein the first antibody is an antibody according to the fifth aspect of the invention and the binding epitope of the second antibody is different from the binding epitope of the first antibody.

In another preferred embodiment, after the sample is contacted with the antibody according to the fifth aspect of the present invention in step (1), a third antibody against the first antibody is further added to the reaction system, and the formation of an "antigen-first antibody-third antibody" complex is detected in step (2).

In another preferred embodiment, the first antibody, the second antibody or the third antibody carries a detectable label thereon.

In another preferred embodiment, the detectable label is a biotin label, a colloidal gold label, a horseradish peroxidase label, a radionuclide label, or a fluorescein label.

In another preferred example, the sample comprises: human or animal tissue samples, tumor resection samples, exfoliated cell samples.

In another preferred embodiment, the method is used for non-diagnostic purposes.

In a fourteenth aspect of the present invention, there is provided a test plate comprising a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the present invention or an immunoconjugate according to the sixth aspect of the present invention.

In another preferred embodiment, the test strip further comprises an antigen-sampling region.

In another preferred embodiment, the test strip is formed by overlapping the filter paper, the chromatographic material, the nitrocellulose membrane and the absorbent paper in sequence.

In a fifteenth aspect of the present invention, there is provided a kit comprising:

(1) a first container comprising an antibody according to the fifth aspect of the invention; and/or

(2) A second container comprising a secondary antibody against the antibody of the fifth aspect of the invention; and/or

(3) A third container comprising a cell lysis reagent;

alternatively, the first and second electrodes may be,

the kit comprises the detection plate according to the fourteenth aspect of the present invention.

In another preferred embodiment, the antibody in the first container is detectably labeled.

In another preferred embodiment, the antibody in the second container is detectably labeled.

In a sixteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) culturing a host cell according to the ninth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a diagram showing the result of ELISA detection of HPV16E7 single-chain antibody (scFv) binding to protein and/or polypeptide.

FIG. 2 shows the result of ELISA detection of HPV16E7 rabbit monoclonal antibody activity. RAB-01, RAB-016, RAB-023 and RAB-017 can be specifically combined with His-HPV16E7 recombinant protein with high affinity and can not be combined with irrelevant protein with His label.

FIG. 3 shows the result of HPV16E7 rabbit monoclonal antibody titer ELISA detection. As a result, the antibody titer of RAB-01 was optimal, and that of RAB-023 and RAB-016 was low, and that of RAB-017 was low.

FIG. 4 shows the result of ELISA detection of HPV16E7 rabbit monoclonal antibody antigen binding specificity. The RAB-016, RAB-023 and RAB-017 can be specifically combined with His-HPV16E7 recombinant protein.

FIG. 5 shows the result of ELISA detection of the HPV16E7 monoclonal antibody binding epitope amino acid sequence region. FIG. 5A is a schematic diagram of HPV16E7 amino acid sequence, and FIG. 5B is a diagram of ELISA detection result.

FIG. 6 is a graph showing the result of immunochemical staining of HPV16E7 monoclonal antibody on reptile cells. FIG. 6A shows the results of RAB-01 staining CaSki and C-33A slide cells; FIG. 6B shows the result of RAB-023 staining CaSki and C-33A slide cells; FIG. 6C shows the results of RAB-016 staining CaSki and C-33A slide cells; FIG. 6D shows the result of RAB-017 staining CaSki and C-33A cells. Monoclonal antibodies RAB-023 and RAB-017 both stained specifically with CaSki cells, but not with C-33A cells. Monoclonal antibodies RAB-01 and RAB-016 stained both CaSki cells, but also strongly non-specifically stained C-33A cells.

FIG. 7 shows the result of detection of liquid-based cervical cancer cell lines. The results of immunocytochemical staining of CaSki and C-33A cells treated with the novel Boehringer's cell fixative using HPV16E 7-specific monoclonal antibodies RAB-023 and RAB-017 are shown in FIG. 7. FIG. 7A shows the result of RAB-023 staining CaSki and C-33A cells; FIG. 7B shows the result of RAB-017 staining CaSki and C-33A cells. Monoclonal antibody RAB-023 has specific staining reaction with CaSki cells treated by liquid-based stationary liquid, but has no staining with C-33A cells. The monoclonal antibody RAB-017 did not stain specifically with CaSki and C-33A cells treated with a liquid-based fixative.

FIG. 8 shows the result of HPV16E7 rabbit monoclonal antibody antigen subtype analysis ELISA detection. The RAB-023 can specifically bind to His-HPV16E7, His-HPV31E7, His-HPV35E7, His-HPV52E7 and His-HPV58E7 recombinant proteins.

FIG. 9 is a graph showing the result of immunohistochemical staining of HPV16E7 rabbit monoclonal antibody RAB-023 in CIN grade and paraffin sections of cervical cancer samples. Rabbit monoclonal antibody RAB-023 can detect HPV E7 protein in cervical carcinoma and also detect CIN-level HPV E7 protein; FIG. 9A shows weak staining in CIN I, FIG. 9B shows strong staining in CIN II-III, and FIG. 9C shows the detection result of cervical cancer samples. By using RAB-023 antibody, the staining degree of HPV E7 protein positive cell tissue is detected to be positively correlated with the disease degree of cancer development and the cancer stage (i.e. disease stage). Figure 9D shows a clear dark brown staining in cervical cancer tissue.

Detailed Description

The inventor obtains an anti-HPV E7 monoclonal antibody RAB-023 through extensive and intensive research and a large number of screens, and experimental results show that the monoclonal antibody aiming at the HPV E7 protein has strong affinity, not only can be specifically combined with the HPV16E7 protein in cells, but also can be used for detecting high-risk HPV31, HPV35, HPV52 and HPV58E7 proteins. Further research shows that the antibody can also be used for detecting clinical paraffin section pathological samples. The invention also provides a method for detecting and/or identifying the HPV E7 protein, which has good stability and extremely high detection sensitivity. The invention also provides a kit containing the antibody and a detection plate.

Specifically, the recombinant His-HPV16E7 fusion protein is adopted to immunize white Japanese auricle rabbits, and His-HPV16E7 and another His-labeled unrelated protein are used as screening detection antigens to obtain B lymphocytes for constructing a phage total antibody library. Techniques for producing specific antibodies by phage display are well known in the art. And (3) introducing the positive antibody strain Fv gene obtained by screening into a eukaryotic expression system, expressing the rabbit full-length antibody, and specifically binding the HPV E7 fusion protein in ELISA detection.

In addition to the identification of ELISA binding to recombinant protein antigens, positive monoclonal antibodies were further subjected to antigen subtype cross-reaction analysis, affinity binding identification, Immunocytochemistry staining (Immunocytochemistry ICC). Through the identification tests, the antibody clone RAB-023 passes through the test requirements and shows the function of specifically binding high-risk HPV E7 oncoprotein at the protein molecular level and the cell level.

In order to screen out an antibody reagent with clinical examination value, the monoclonal antibody is further subjected to a liquid-based cervical cancer cell line and cervical cancer clinical sample immunohistochemical detection test, and finally a monoclonal antibody RAB-023 capable of specifically recognizing HPV E7 positive cervical epithelial cells is obtained. Based on the specificity of the antibody, methods capable of identifying human cervical epithelial canceration or precancerous cells are developed, and the methods can be used for auxiliary cell morphology or HPV molecular detection and differential diagnosis.

In a preferred embodiment of the present invention, the amino acid sequence of the HPV16E7 protein is as follows:

HGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP(SEQ ID NO.1)。

in a preferred embodiment of the present invention, the amino acid sequence of the HPV18E7 protein is as follows:

MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ(SEQ ID NO.2)。

as used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

As used herein, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the rabbit monoclonal antibodies herein are obtained by constructing full-length rabbit monoclonal antibody gene expression vectors by molecular biology methods after screening phage libraries, transferring the vectors into eukaryotic expression systems, and harvesting cell supernatants after culture, and are free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The invention also comprises a monoclonal antibody with the corresponding amino acid sequence of the monoclonal antibody for resisting HPV16E7 protein, a monoclonal antibody with the variable region chain of the monoclonal antibody for resisting HPV16E7 protein, and other proteins or protein conjugates and fusion expression products with the chains. Specifically, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having light and heavy chains with hypervariable regions (complementarity determining regions, CDRs) so long as the hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the monoclonal antibody to HPV16E7 protein or a fragment thereof. The invention also comprises a cell surface marker or antigen combined with the anti-HPV16E7 protein monoclonal antibody or the fragment thereof.

The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab')₂A fragment; an antibody heavy chain; the light chain of the antibody.

As used herein, the terms "heavy chain variable region" and "V_H"may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody (RAB-023) comprises three complementarity determining region CDRs:

CDR1, the amino acid sequence of which is GFSLSSYT (SEQ ID NO.4), the coding nucleotide sequence of which is ggattctccctcagtagctataca (SEQ ID NO. 3);

CDR2, the amino acid sequence of which is ISTGGTT (SEQ ID NO.6), the coding nucleotide sequence of which is attagtactggtggtaccact (SEQ ID NO. 5);

CDR3, the amino acid sequence of which is ARGYGKGSGYSGLNL (SEQ ID NO.8), and the coding nucleotide sequence of which is gcgagggggtatggtaaaggtagtggttattctggccttaacttg (SEQ ID NO. 7).

In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:

QSVEESGGDLVKPGASLTLTCKASGFSLSSYTMGWFRQAPGKGLEYIGAISTGGTTDYANWAKGRFTISKTSSTTVALQMTSLTAADTATYFCARGYGKGSGYSGLNLWGPGTLVTVSS(SEQ ID NO.10)；

the coding nucleotide sequence is as follows:

cagtcggtggaggagtccgggggagacctggtcaagcctggggcatccctgacactcacctgcaaagcctctggattctccctcagtagctatacaatgggctggttccgccaggctccagggaaggggctggaatacatcggagccattagtactggtggtaccactgactacgcgaactgggcgaaaggccgattcaccatctccaaaacctcgtcgaccacggtggctctgcaaatgaccagtctgacagccgcggacacggccacctatttctgtgcgagggggtatggtaaaggtagtggttattctggccttaacttgtggggcccaggtaccctggtcacagtgagctct(SEQ ID NO.9)。

in a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and a heavy chain constant region, which may be murine, human or rabbit.

As used herein, the terms "light chain variable region" and "VL" are used interchangeably.

In a preferred embodiment of the invention, the light chain variable region of the antibody according to the invention (RAB-023) has complementarity determining regions CDRs selected from the group consisting of:

CDR1', the amino acid sequence of which is ESVYNNY (SEQ ID NO.12), and the coding nucleotide sequence of which is gagagcgtttataataacaactac (SEQ ID NO. 11);

CDR2', having an amino acid sequence of RAS (SEQ ID NO.14) and a coding nucleotide sequence of agggcatcc (SEQ ID NO.13)

CDR3', having an amino acid sequence of LGSYDCSSAGCFA (SEQ ID NO.16) and a coding nucleotide sequence of ctaggcagttatgattgtagtagtgctggttgttttgct (SEQ ID NO.15)

In another preferred embodiment, the amino acid sequence of the light chain variable region is:

DPMLTQTASPVSAAVGSTVTISCQSSESVYNNNYLSWFQQKPGQPPKQLIYRASSLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSSAGCFAFGGGTELEIL(SEQ ID NO.18)，

the coding nucleotide sequence is as follows:

gaccctatgctgacccagactgcatcgcccgtgtctgcagctgtgggaagcacagtcaccatcagttgccagtccagtgagagcgtttataataacaactacttatcctggtttcagcagaaaccagggcagcctcccaagcaactgatctacagggcatccagtctggcatctggggtctcatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatcagcgacgtgcagtgtgacgatgctgccacttactactgtctaggcagttatgattgtagtagtgctggttgttttgctttcggcggagggaccgagctggagatccta(SEQ ID NO.17)。

in a preferred embodiment of the present invention, the light chain of the antibody comprises the light chain variable region and the light chain constant region, and the light chain constant region may be of murine, human or rabbit origin.

In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and refer to an antibody that specifically binds HPV16E7 protein, such as a protein or polypeptide having a heavy chain variable region (such as the amino acid sequence of SEQ ID No. 10) and/or a light chain variable region (such as the amino acid sequence of SEQ ID No. 18). They may or may not contain the initial methionine.

In another preferred embodiment, the antibody is a rabbit or human rabbit chimeric monoclonal antibody against HPV16E7 protein, wherein the heavy chain constant region and/or the light chain constant region may be humanized. More preferably, the humanized heavy or light chain constant region is that of human IgG1, IgG2, or the like.

The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains with variable regions, provided that the variable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy and light chains of the antibody of the invention.

In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

The invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having HPV16E7 protein binding activity, comprising the CDR regions described above. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region as shown in SEQ ID NO.3, 5, 7, 9, 11, 13, 15, 17 or may be a degenerate variant. As used herein, "degenerate variant" means in the present invention a nucleic acid sequence which encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differs from the sequence of the coding region as set forth in SEQ ID No.3, 5, 7, 9, 11, 13, 15, 17.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in SEQ ID NO.10 and/or SEQ ID NO. 20.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 5. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 6. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-described antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the invention can be directly used for binding HPV16E7 protein molecules, thus being used for preventing and treating tumors. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Preparation of monoclonal antibodies

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the antigens of the invention can be administered to an animal to induce the production of monoclonal antibodies. For Monoclonal Antibodies, they can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol.6:511,1976; Kohler et al, Eur. J. Immunol.6:292,1976; Hammerling et al, In Monoclonal Antibodies and T Cell hybrids, Elsevier, N.Y.,1981), phage display technology or can be prepared using recombinant DNA methods (U.S. Pat. No.4,816,567).

Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to medium (HAT medium matrix), including myeloma Cell lines, such as murine myeloma Cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, san diego, california, usa), and SP-2, NZ0, or X63-Ag8-653 cells (available from American Type Culture Collection, rockwell, maryland, usa). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [ Kozbor, j.immunol., 133: 3001 (1984); brodeur et al, Techniques for the Production and use of Monoclonal Antibodies (Monoclonal Antibodies Production Techniques and Applications), pp 51-63 (Marcel Dekker, Inc., New York, 1987).

The medium in which the hybridoma cells are grown is assayed to detect the production of monoclonal antibodies of the desired specificity, e.g., by in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA). The location of the antibody-expressing cells can be detected by FACS. The hybridoma clones can then be subcloned by limiting dilution procedures (subcloned) and grown by standard methods (Goding, Monoclonal Antibodies): Principles and Practice (Principles and Practice), Academic Press (1986) pp 59-103). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow in animals as ascites tumors.

The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The phage display technology is a screening technology, exogenous polypeptide or protein and capsid protein of phage are fused and expressed, the fusion protein is displayed on the surface of virus particle, and DNA coding the fusion is located in virus particle, so that direct connection is established between a large number of polypeptides and DNA coding sequences thereof, and polypeptide ligands of various target molecules (antibody, enzyme, cell surface receptor, etc.) can be rapidly identified by panning.

The invention provides a monoclonal antibody aiming at HPV E7 protein, in particular a monoclonal antibody aiming at HPV16E7 protein. In a preferred embodiment of the present invention, the monoclonal antibody is screened by phage display technology, a eukaryotic expression system is constructed by recombinant DNA method to express the antibody, and the antibody secreted in the culture medium is purified by affinity chromatography column (Protein A-Sepharose).

Method and sample

The present invention relates to methods for detecting cervical cancer in cell and/or tissue samples. The method comprises the following steps: obtaining a cell and/or tissue sample; detecting the level of HPV oncoprotein in the sample. The sample used in the method of the present invention may be any sample comprising cells present in a cell preservation solution, as used in liquid-based cytology methods.

The invention can be used for detecting HPV oncoprotein in HPV infection-related cancers, wherein the cancers related to HPV infection comprise tumors of genitourinary systems such as cervical cancer, bladder cancer, endometrial cancer, penile cancer and the like, small cell lung cancer, melanoma, head and neck tumors and the prophase stages of the cancers.

According to the present invention, the use of molecular markers for HPV oncoproteins may support or even replace cytological and/or histological detection methods. In special cases, protein molecular markers can be used as diagnostic tools without the support of further cell-based morphological tests. The biomarkers employed in the present invention are HPV oncoproteins, which are derived from viruses, and are specific for the detection of HPV-infected samples, since the nature of the markers present in the tissue by the virus does not occur in uninfected human tissue.

Samples (specimens) used in the present invention include cells, tissue samples, and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus biopsies as used in the present invention may comprise e.g. resection samples of tumours, tissue samples prepared by endoscopic methods or needle biopsy of organs.

Samples for use in the present invention may include fixed or preserved cell or tissue samples. The cell or tissue sample may be, for example, preserved in a standard sample collection, storage or transport medium, such as those commercially available preservation media known to those skilled in the art (formalin, Cytyc "PreservCyt" or Tripath Imaging "Cytorich", etc.). Suitable cell preservation media may include one or more mixtures selected from alcohols, aldehydes, ketones, acids, metal ions or mercury, ethers, and the like, for preserving cellular components. The alcohol includes methanol, ethanol, (n-or iso-) propanol, (n-, iso-or tert-) butanol or highly branched or unbranched alcohols. Aldehydes include formaldehyde, acetaldehyde, glutaraldehyde, and the like. Ketones such as acetone may also be used. Acids used in standard sample media include organic acids (acetic, trichloroacetic, salicylic and picric) or inorganic acids such as chromic acid. Standard sample solutions may include metals such as silver, copper, chromium, mercury, osmium, and uranium. Salt solutions such as uranyl acetate, potassium dichromate, ammonium sulfate, and the like may be components of the preservation medium.

Reagent kit

The present invention also provides a kit comprising an antibody (or fragment thereof) of the present invention or an assay plate of the present invention, and in a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, and the like.

The invention further designs a detection kit for detecting the high-risk HPV E7 oncoprotein, which comprises an antibody for identifying the high-risk HPV E7 oncoprotein, general reagents and buffers required for detection, such as various buffers, enzyme-linked labeled secondary antibodies, detection labels, detection substrates and the like. The antibody is preferably an anti-HPV E7 antibody, more preferably an anti-HPV16E7 monoclonal antibody. The test kit may be an in vitro diagnostic device.

The invention further relates to the development of a kit for diagnostic assessment of conditions associated with HPV infection from a cervical exfoliated cell sample, which can detect the high risk HPV E7 oncoprotein present in the sample, wherein the cell preservation solution used to preserve the sample can be, for example, a cell preservation solution in a liquid-based cytology method. Cells are fixed in an appropriate cell preservation solution and used for development of a detection kit and an in vitro diagnostic apparatus for detecting HPV infection-associated tumor of a specimen based on non-cell morphological analysis.

The invention aims to provide a method for detecting the expression of high-risk HPV E7 protein, and the method can be used for detecting HPV infection-related cancers, particularly cervical cancer.

The present inventors have prepared a monoclonal antibody RAB-023 directed against the HPV16E7 protein of human papillomavirus and studied its reactivity. When the anti-HPV16E7 monoclonal antibody RAB-023 is used for immunocytochemical staining on a cervical cancer cell line C-33A not expressing HPV and a human cervical cancer cell line CaSki expressing HPV16E7, the results show that: RAB-023 has no response to C-33A cells not expressing HPV protein, and has stronger staining response to CaSki which expresses HPV16E 7.

The present inventors used the prepared anti-HPV16E7 monoclonal antibody RAB-023 to perform immunocytochemical staining on cervical cancer cell lines fixed by aldehydes (paraformaldehyde fixing solution). CaSki and C-33A slide cells were fixed separately, and by detecting HPV16E7 inside tumor cells, it was found that: RAB-023 has no response to C-33A reprinting cells which do not express HPV protein, and has stronger staining response to positive reprinting cells CaSki which express HPV16E 7.

Furthermore, the present inventors performed immunohistochemical staining of formalin-fixed paraffin tissue sections of cervical cancer using the prepared anti-HPV16E7 monoclonal antibody RAB-023. The results show that RAB-023 can specifically bind to cervical cancer cells and stain the cervical cancer cells obviously, but not bind to normal cervical tissue cells. Based on the finding, the inventors of the present invention have completed the present invention.

That is, the method of the present invention is a method for detecting a tumor marker, characterized in that: comprising the step of detecting HPV16E 7in the sample.

In the method of the present invention, the test sample is preferably exfoliated cells collected from a specimen, or a culture of the tissue or a section of the tissue, or may be suspended cells prepared from a tissue collected from a specimen or a culture of the tissue. In addition, the cells are preferably sections of cervical tissue.

In the method of the present invention, the sample is preferably a patient having a possibility of cervical lesion due to cervical epithelial damage, or a patient having a lesion in the cervix.

Preferably, the HPV16E7 is an HPV16E7 protein or fragment thereof. In this case, the step of detecting HPV16E7 is preferably an immunocytochemical staining assay using HPV16E 7. The anti-HPV16E7 antibody used is preferably an anti-HPV16E7 monoclonal antibody.

The protein expressed by the oncogene from HPV16E7 used in the immunodetection of the method serves as a reliable indicator of HPV 16-associated malignancy or premalignancy. One of the most useful aspects of the invention is its use in the diagnosis of cervical cancer, squamous epithelial cell lesions and adenocarcinomas and any epithelial abnormalities associated with oncogenic HPV16 infection, including hollow cell disease; hyperkeratosis; precancerous conditions including intraepithelial neoplasia or intraepithelial lesions; high dysplasia; and invasive or malignant cancers. In addition to cervical cancer, detection of HPV16E7 is useful for detection of genitourinary tumors such as bladder cancer, endometrial cancer, penile cancer, small cell lung cancer, melanoma, and head and neck tumors.

Another object of the present invention is to provide a detection kit by the method of the present invention. The kit may be a diagnostic kit or a research kit.

The kit of the present invention is a kit for detecting a tumor marker, and is characterized by having an anti-HPV16E7 monoclonal antibody. The kit of the invention preferably also comprises general reagents and buffers required for detection, such as various buffers, enzyme-linked labeled secondary antibodies, detection labels, detection substrates and the like. The antibody is preferably an anti-HPV16E7 antibody, more preferably an anti-HPV16E7 monoclonal antibody, and particularly preferably an anti-HPV16E7 monoclonal antibody gene recombinant expression vector for a eukaryotic expression system is obtained by phage display and recombinant DNA technology. An anti-HPV16E7 rabbit monoclonal antibody produced by a eukaryotic expression system or a monoclonal antibody having binding activity equivalent to that of the anti-HPV16E7 rabbit monoclonal antibody.

The invention provides a method for distinguishing tumor cells without HPVDNA by detecting HPV16E7 protein endogenous to the cells. And positive cells can be accurately detected after the cells are fixed in neutral formalin fixing solution which is widely adopted clinically, so that diagnosis can be made at the early stage of cancer evolution, and a basis is provided for timely treatment.

Furthermore, the invention also provides a detection kit formed by adopting the detection method.

The main advantages of the invention are:

(1) the antibody aiming at the HPV16E7 protein provided by the invention has high specificity and strong affinity, and can be prepared in large scale, and the quality of the monoclonal antibody is easy to control.

(2) The antibody aiming at the HPV16E7 protein can be specifically combined with the HPV16E7 protein and can also be combined with high-risk HPV31, 35, 52 and 58E7 proteins, so that the antibody can be used for simultaneously detecting HPV16 and/or HPV31, 35, 52 and 58E7 proteins.

(3) The antibody provided by the invention is used in the method for detecting the HPV E7 protein, and has good stability and extremely high detection sensitivity.

(4) The monoclonal antibody and the detection method provided by the invention are suitable for early diagnosis of related cancers and large-scale patient screening, and can be used for monitoring relapsing patients.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1

1. Preparation of human papilloma virus HPV16E7 rabbit monoclonal antibody

1.1 screening of Single chain antibodies (scFv)

The rabbit was immunized with His-HPV16E7 recombinant protein and titer detection was performed with His-HPV16E7 recombinant protein and His-unrelated protein. The rabbit B lymphocytes were isolated to obtain immunoglobulin genes. Cloning out the complete set of variable region genes of the B cell to assemble a phage antibody library. The constructed phage antibody library was panned using recombinant protein His-HPV16E 7. Enrichment through three rounds of elutriation; determining the titer of the phage; plaque amplification; DNA sequencing; the screened target molecule binding peptides were detected by ELISA. The ELISA detection screening selects recombinant protein His-HPV16E7, and uses His-unrelated protein to set negative control (N), Anti-6 XHis antibody to set coated His antigen positive control (P), and simultaneously sets blank control (coated antigen is directly added into enzyme-labeled secondary antibody). His-HPV16E70.1 mu g/ml coated plate, His-unrelated protein 5 mu g/ml coated plate, 4 ℃ overnight, PBST washing and drying, adding 5% skimmed milk powder for sealing, acting at room temperature for 2h or 4 ℃ overnight, PBST washing and drying, and adding the antibody to be detected 5 mu g/ml. After the reaction is carried out for 1h at 37 ℃, after PBST is washed and dried, an Anti-Flag-HRP secondary antibody (Sigma A8592) (1: 10000) and Anti-6 XHis (Abcam ab1187) (1: 10000) are respectively added, the reaction is carried out for 60min at 37 ℃, and after PBST is washed and dried, TMB coloration and 2M H2SO4 are stopped. And (3) performing an ELISA screening result by taking the positive result that the OD value of the antibody to be detected in the reaction of the recombinant protein His-HPV16E7 is more than 2, and performing a recheck. Screening to obtain 8 single-chain antibodies: scFv001, scFv016, scFv017, scFv020, scFv023, scFv034, scFv133, scFv 139.

The epitope amino acid sequence region bound by this 8 scFv was preliminarily identified. And (3) identifying by adopting an ELISA method: the antigen selects recombinant protein and polypeptide, wherein the recombinant protein is His-HPV16E 7; the polypeptide is HPV16E7-1 (amino acid sequence SEQ ID NO.19PTLHEYMLDLQPETTDLYCYEQLNDSSEEE), HPV16E7-2 (amino acid sequence SEQ ID NO.20LNDSSEEEDEIDGPAGQAEPDRAH), HPV16E7-3 (amino acid sequence SEQ ID NO.21KCDSTLRLCVQSTHVDIRTLE), His-Vac (amino acid sequence SEQ ID NO.22DEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIV) and HPV18E7-1 (amino acid sequence SEQ ID NO.23SDSEEENDEIDGVNHQHLPARRAEPQRH), respectively. And grouping the 8 scFvs according to the different binding capacity of the scFvs and the amino acid sequence. The results are shown in FIG. 1: the 8 scFvs were divided into three groups, where scFv001 was a single group, scFv016, scFv023, scFv034 and scFv139 were a group, and scFv017, scFv020 and scFc133 were a group. Finally, 4 scFv strains were selected for full-length rabbit mab expression: scFv001, scFv023, scFv016 and scFv017 were selected respectively.

1.2 production of Rabbit monoclonal antibodies

Eukaryotic in vitro expression techniques for producing rabbit monoclonal antibodies are well known in the art. Firstly, according to the Fc sequence of the rabbit antibody gene library, combining with the Fv antibody gene selected above, constructing a full-length rabbit monoclonal antibody gene expression vector, and constructing four eukaryotic expression vectors. The expression vector was transiently transferred to HEK293F cells through liposome, and culture supernatant was collected 72 hours later and purified by affinity chromatography (Protein A-Sepharose). Obtaining four strains of rabbit monoclonal antibodies, which are respectively: RAB-01, RAB-016, RAB-023 and RAB-017. The obtained monoclonal antibodies were sequenced using methods conventional in the art.

1.3 detection of Positive Rabbit monoclonal antibodies

During detection, indirect ELISA screening is adopted: the antigen is His-HPV16E7 fusion protein. And a His-unrelated protein is used for setting a negative control (N) of the antibody to be detected, an Anti-6 XHis antibody is used for setting a coated His antigen positive control (P), and a blank control (the coated antigen is directly added into an enzyme-labeled secondary antibody) is also set. His-HPV16E7 fusion protein 0.1,1, 5. mu.g/ml plate, His-unrelated protein 5. mu.g/ml plate, 4 ℃ overnight, PBST washingAfter patting dry, adding 5% skimmed milk powder for sealing, acting at 37 ℃ for 2h or overnight at 4 ℃, washing and patting dry PBST, adding 0.1 mu g/ml of antibody to be detected, reacting at 37 ℃ for 1h, washing and patting dry PBST, then respectively adding goat Anti-rabbit-HRP secondary antibody (Sigma A0545) (1: 20000), Anti-6 XHis (Abcam ab1187) (1: 10000), reacting at 37 ℃ for 60min, developing TMB and developing 2M H after washing and patting dry PBST₂SO₄And (6) terminating. And (3) performing retest on the ELISA screening result by taking the positive result that the OD value of the reaction of the antibody to be detected on the coating antigen of 0.1 mu g/ml is more than 1 and the OD value of the cross reaction of the antibody to be detected on the His-unrelated protein is less than 0.1. The results are shown in FIG. 2: RAB-01, RAB-016, RAB-023 and RAB-017 can be combined with His-HPV16E7 but not with His-unrelated protein, namely all positive antibody strains.

2. Identification of monoclonal antibodies

2.1ELISA detection of Rabbit monoclonal antibody titer

Coating fusion protein His-HPV16E70.5 μ g/ml, standing overnight at 4 deg.C, washing and drying PBST, adding 5% skimmed milk powder for sealing, acting at 37 deg.C for 2h or 4 deg.C for overnight, washing and drying PBST, adding anti-HPV16E7 monoclonal antibodies RAB-01, RAB-016, RAB-023, RAB-017 with initial concentration of 1 μ g/ml, diluting in multiple proportion, reacting at 37 deg.C for 1h, washing and drying PBST, adding goat anti-rabbit-HRP secondary antibody (Sigma A0545) (1: 20000), reacting at 37 deg.C for 60min, washing and drying PBST, developing TMB and 2M H₂SO₄End, OD450 nm. The results are shown in FIG. 3: when His-HPV16E7 is used as antigen, the titer of the monoclonal antibody RAB-01 is better than that of other monoclonal antibodies, RAB-023 and RAB-016 are lower than that of RAB-017.

2.2ELISA detection of anti-HPV16E7 rabbit monoclonal antibody specificity and Cross-reactivity:

the antigen is His-HPV18E7 and His-HPV16E7 fusion proteins respectively, and Anti-6 XHis antibody is selected to coat His antigen positive control (P), and blank control is set at the same time (the coating antigen is directly added into enzyme-labeled secondary antibody). 0.5 mu g/ml plate-wrapping, 4 ℃ overnight, PBST washing and drying, adding 5% skimmed milk powder for sealing, acting at 37 ℃ for 2h or 4 ℃ overnight, PBST washing and drying, respectively adding anti-HPV16E7 rabbit monoclonal antibody RAB-01, RAB-016, RAB-023, RAB-017(1 mu g/ml), reacting at 37 ℃ for 1h, PBST washing and dryingAdding goat Anti-rabbit-HRP secondary antibody (Sigma A0545) (1: 20000), Anti-6 XHis (Abcam ab1187) (1: 10000), reacting at 37 deg.C for 60min, washing with PBST, drying, developing TMB, and 2M H₂SO₄End, OD450 nm. The results are shown in FIG. 4: the RAB-01, RAB-016, RAB-023 and RAB-017 can be combined with HPV16E7 recombinant protein, and the RAB-01 can be combined with HPV18E7 recombinant protein.

2.3 antigen binding epitope analysis of Anti-HPV16E7 Rabbit monoclonal antibody

Identifying the epitope amino acid sequence region of the monoclonal antibody in the HPV16E7 antigen protein. And (3) identifying by adopting an ELISA method: the antigen is selected from polypeptide or recombinant protein, wherein the polypeptide is His-Vac, HPV16E7-1, HPV16E7-2 and HPV16E7-3 respectively; the recombinant protein is His-HPV16E7 protein antigen. Recombinant protein antigen 0.5 mug/ml coated plate, polypeptide antigen 2 mug/ml coated plate, 4 ℃ overnight, PBST washing and drying, adding 5% skimmed milk powder for sealing, acting at 37 ℃ for 2h or 4 ℃ overnight, PBST washing and drying, adding anti-HPV16E7 monoclonal antibody RAB-01, RAB-016, RAB-023, RAB-017(1 mug/ml), reacting at 37 ℃ for 1h, PBST washing and drying, adding goat anti-rabbit-HRP secondary antibody (Sigma A0545) (1: 20000), reacting at 37 ℃ for 60min, after PBST washing and drying, TMB color development 2M H₂SO₄End, OD450 nm.

The results are shown in FIG. 5: RAB-01 is only able to bind to the recombinant protein His-HPV16E7, so it was preliminarily speculated that RAB-01 recognizes the spatial conformation of the recombinant protein HPV16E 7. The binding epitopes of RAB-016, RAB-023 and RAB-017 specifically bind to HPV16E7 are amino acids 5-34.

2.4 rabbit monoclonal antibody immunocytochemical staining method for detection of cervical cancer cell line expressing HPV16E 7:

cervical cancer cell line CaSki cells expressing HPV16E7 protein were used herein as a tumor cell model (positive control) for E7 protein overexpression in highly cervical lesion states. Cervical cancer cell line C-33A cells without HPV DNA were used as negative control. Immunocytochemical staining tests were performed on these two cells using rabbit monoclonal antibodies RAB-01, RAB-016, RAB-023, and RAB-017, respectively. The specific experimental method is as follows:

CaSki, C-33A cells are respectively planted in the 24-hole fineOn cover glass in cell culture plates, 5% CO at 37 ℃₂Culturing for 24h, removing the culture medium, carefully rinsing with PBS twice, and spin-drying; fixing with 4% paraformaldehyde fixing solution at room temperature for 30 min; to prevent non-specific background staining, the coverslip was not allowed to dry during staining. Adding 0.3% Triton X-100(in PBS) to permeate cell membrane for 15 min at room temperature; addition of 3% H for inactivation of endogenous peroxidase₂O₂PBS, treating at room temperature for 5min, and spin-drying; adding PBS lotion, washing for 5min, and spin-drying; adding 10% FBS/PBST sealing liquid, sealing for 1h, and spin-drying; rabbit monoclonal antibodies RAB-01(20,10, 5. mu.g/ml), RAB-016(20,10, 5. mu.g/ml), RAB-023(20,10, 5. mu.g/ml), RAB-17(20,10, 5. mu.g/ml) were added, and incubated overnight at 4 ℃; adding PBST washing solution, washing for 5 times, each time for 5min, and spin-drying; adding goat anti-rabbit-HRP secondary antibody (Sigma A0545) (1:1000) and incubating at 37 ℃ for 1 h; adding PBST washing solution, washing for 5 times, each time for 5min, and spin-drying; adding DAB color development solution (ZLI-9017, China fir Jinqiao in Beijing), reacting at room temperature, observing the dyeing result under a microscope, and washing with distilled water to terminate the reaction. The results were observed under a microscope and recorded.

The results are shown in FIG. 6, and microscopic examination shows: HPV16E7 monoclonal antibodies RAB-023(5 mu g/ml) and RAB-017(5 mu g/ml) have specific immunochemical staining reaction with a cervical cancer cell line CaSki expressing HPV16E7 protein, but have no staining reaction with a cervical cancer cell line C-33A not expressing HPV protein. However, RAB-01 and RAB-016 showed immunochemical staining reactions with the cervical cancer cell line CaSki expressing HPV16E7 protein, and showed immunochemical staining reactions with the same intensity as that with the cervical cancer cell line C-33A not expressing HPV protein. Namely, only two rabbit monoclonal antibodies of RAB-023 and RAB-017 can specifically recognize HPV16E7 protein endogenous to cells.

Example 2 detection of HPV16E7 protein expression in liquid-based fixative-immobilized tumor cells Using immunocytochemistry staining

CaSki and C-33A cells were collected in PBS by centrifugation, and after removing excess PBS, TCT fixative was added for 30 min. And respectively smearing the fixed CaSki cells and the C-33A cells on a glass slide, immediately soaking in 95% ethanol for 30min, and air-drying overnight.

Drying the air-dried tumor cellsAfter the cover glass is placed in 50% ethanol for 10min, the cover glass is transferred to deionized water for at least 30 s. To prevent non-specific background staining, the coverslip was not allowed to dry during staining. Placing the cell slide glass treated by deionized water into a Tris-EDTA (pH 9.0) repairing solution, performing thermal repairing at 95-99 ℃ for 10min, and then performing room temperature rewarming for 20min together with the repairing solution; spin-drying, and washing with PBST lotion for 5 min; spin-drying, adding 3% H for inactivating endogenous peroxidase₂O₂PBS for 10min at room temperature; spin-drying, and washing with PBST lotion for 5 min; spin-drying, adding 10% FBS/PBST) closed liquid, and sealing for 1h at room temperature; spin-drying, respectively adding rabbit monoclonal antibodies RAB-023(10 mu g/ml) and RAB-017(10 mu g/ml), and incubating for 1h at room temperature; adding PBST washing solution, and washing for 5 times, each for 5 min; spin-drying, adding goat anti-rabbit-HRP secondary antibody (Sigma A0545) (1:1000) and incubating at 37 deg.C for 1 h; adding PBST washing solution, and washing for 5 times, each for 5 min; spin-drying, adding DAB color development solution (ZLI-9017, China fir Jinqiao, Beijing), reacting at room temperature, observing the dyeing result under microscope, and washing with distilled water to terminate the reaction. The results were observed under a microscope and recorded.

The results are shown in FIG. 7, and microscopic examination shows: when tumor cells are fixed by TCT stationary liquid, only the monoclonal antibody RAB-023 can specifically detect the cervical cancer cells expressing HPV16E7 protein, but RAB-017 cannot be combined with HPV16E7 protein in the tumor cells after the liquid-based stationary liquid is fixed.

Example 3ELISA detection of Rabbit monoclonal antibody RAB-023 binding to HPV high-risk subtype analysis

The antigen is respectively selected from His-HPV18E7, His-HPV16E7, His-HPV31E7, His-HPV35E7, His-HPV45E7, His-HPV52E7 and His-HPV58E7 fusion proteins, and Anti-6 XHis antibody is selected to coat a His antigen positive control (P), and meanwhile, a blank control is set (the coating antigen is directly added into an enzyme-labeled secondary antibody). 0.5 mu g/ml plate package, 4 ℃ overnight, PBST washing and drying, adding 5% skimmed milk powder for sealing, acting at 37 ℃ for 2h or 4 ℃ overnight, PBST washing and drying, respectively adding Anti-HPV E7 rabbit monoclonal antibody RAB-023, RAB-034(1 mu g/ml), reacting at 37 ℃ for 1h, PBST washing and drying, respectively adding goat Anti-rabbit-HRP secondary antibody (Sigma A0545) (1: 20000), Anti-6 XHis (Abcam ab1187) (1: 10000), reacting at 37 ℃ for 60min, and PBST washing and drying, TMB developing colorAnd 2M H₂SO₄The reading at OD450nm was terminated.

The results are shown in FIG. 8: the RAB-023 can specifically bind to His-HPV16E7, His-HPV31E7, His-HPV35E7, His-HPV52E7 and His-HPV58E7 recombinant proteins, namely the RAB-023 can bind to HPV31, 35, 52 and 58 subtypes which have high homology with HPV16 on an evolutionary tree, but not bind to an HPV18 branch with homology of less than 40 percent.

Example 4 detection of cervical lesion tissue by immunohistochemical staining of Rabbit-derived monoclonal antibody RAB-023

Further explores the application of rabbit monoclonal antibody RAB-023 in the aspect of cervical tissue immunohistochemical detection. Uses rabbit monoclonal antibody RAB-023 as a primary antibody to carry out immunohistochemical staining on normal cervical tissues and cervical lesion tissues of different grades. The method comprises the following steps: soaking pathological paraffin sections in dimethylbenzene twice for 10 minutes each time; then soaking in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 5min, and washing twice with TBST; the tissue sections were placed in a boiling 0.01M sodium citrate buffer (pH6.0) and autoclaved for 5 minutes. Cooling for 30 minutes at room temperature, washing for three times with TBST, 5 minutes each time; addition of 3% H for inactivation of endogenous peroxidase₂O₂The TBST buffer of (5) is treated for 10 minutes at room temperature; TBST wash 3 times for 5 minutes each; adding TBST containing 10% calf serum, and sealing at room temperature for 15 min; the blocking solution was discarded, and antibody RAB-023(8ug/ml) was added and incubated for 30 minutes at room temperature; after full washing, adding Anti-Mouse/Rabbit IgG-HRP (Dako K5007) dropwise, and incubating for 30 minutes at room temperature; DAB (Beijing China fir Jinqiao ZLI-9017) is developed for 5 minutes (observed under a microscope) after being fully washed, and is washed for 5 minutes by running water; counter staining with hematoxylin for 1 min, washing with tap water, and soaking in tap water for 10 min; dehydrating step by step, and sequentially soaking in 70% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol for 5 min; finally, soaking the mixture twice in dimethylbenzene for 5 minutes each time; then sealing the neutral gum into a piece; and finally, observing and photographing through a microscope.

Microscopic examination is shown in fig. 9: rabbit monoclonal antibody RAB-023 has no specific staining in normal cervical tissue (9A), the staining of monoclonal antibody in tissue is gradually deepened along with the increase of cervical lesion grade, and the staining is obviously dark brown in cervical cancer tissue (9D), and the staining is located in cytoplasm. The combination of HPV E7 protein reported in the literature and cervical lesion degree shows a positive correlation, and the rabbit-derived monoclonal antibody RAB-023 can specifically recognize HPV E7 protein in cervical syndromic tissues so as to enable the cervical lesion tissues to show specific staining, and the staining is localized in cytoplasm.

Discussion:

the invention adopts the method to prepare the HPV16E7 rabbit-derived monoclonal antibody, four rabbit single-chain antibody genes are selected to express a eukaryotic system of a rabbit anti-full-length gene, the expressed full-length rabbit monoclonal antibody is screened, and finally, the monoclonal antibody RAB-023 which has strong specificity, can identify clinically-commonly used aldehydes, tumor cells fixed by liquid-based stationary liquid and tissue slices and endogenous HPV E7 protein and has the lowest background is selected. The research shows that the RAB-023 can effectively identify the cancerated cells in the cervical tissue slice sample by combining with the HPV E7 protein related to the high-risk type cervical cancer. Further research shows that RAB-023 not only can identify the viral protein in the cervical cancer tissue, but also can identify the viral protein in lesion tissues (such as CIN II-III grade) before the cervical cancer. CIN encompasses all precancerous lesions and carcinoma in situ, reflecting a series of pathological changes that progress continuously in the development of cervical cancer, namely, cervical dysplasia (light → medium → heavy) → carcinoma in situ → early invasive carcinoma. CIN is a continuously progressive lesion, but there is also a possibility of reversal, either cervical dysplasia or carcinoma in situ, with only minimal reversal of carcinoma in situ. In clinic, the follow-up observation can be carried out on the CIN I grade patient with young, fertility requirement and small lesion range, and the local treatment such as frozen laser is adopted for the CIN II grade patient; for the above CIN II grade pathological changes, the uterus is mainly removed by operation at home, and local treatment is advocated at foreign countries. In clinical practice, the decision on CINII level is often more controversial due to the subjectivity of the pathologist. Because the high-risk HPV is closely related to the occurrence of cervical cancer, the high-risk HPV infection can be detected in more than 99 percent of cervical cancer samples, and the method provided by the invention can detect the high-risk HPV E7 protein in the early CIN stage and at least in the cervical moderate lesion samples, so as to indicate whether the cervical lesion is deteriorated or not, provide an auxiliary basis for clinical doctors to diagnose and treat patients, and reduce the missed diagnosis rate caused by subjective judgment on tissue morphology and the loss caused by over-treatment.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (15)

1. An antibody that specifically binds HPV E7 protein, wherein said antibody has a heavy chain variable region and a light chain variable region, and wherein said heavy chain variable region comprises the following three complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO.4,

CDR2 shown in SEQ ID NO.6, and

CDR3 shown in SEQ ID NO. 8;

the light chain variable region has Complementarity Determining Regions (CDRs) selected from the group consisting of:

CDR1' shown in SEQ ID NO.12,

CDR2' of SEQ ID NO.14, and

CDR3' shown in SEQ ID NO. 16.

2. The antibody of claim 1, wherein said heavy chain variable region has the amino acid sequence set forth in SEQ ID No. 10.

3. The antibody of claim 1, wherein the light chain variable region has the amino acid sequence of SEQ ID No. 18.

4. The antibody of claim 1, wherein said HPV E7 protein is selected from the group consisting of: an HPV16E7 protein, an HPV31E7 protein, an HPV35E7 protein, an HPV52E7 protein, an HPV58E7 protein, or a combination thereof.

5. The antibody of claim 1, wherein said antibody is a rabbit derived antibody, a human rabbit chimeric antibody, or a humanized antibody.

6. A recombinant protein, said recombinant protein having:

(i) the antibody of claim 1; and

(ii) a tag sequence to facilitate expression and/or purification.

7. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) the antibody of claim 1; or

(2) The recombinant protein of claim 6.

8. A vector comprising the polynucleotide of claim 7.

9. A genetically engineered host cell comprising the vector or genome of claim 8 having the polynucleotide of claim 7 integrated therein.

10. An immunoconjugate, comprising:

(a) the antibody of claim 1; and

(b) a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.

11. A pharmaceutical composition comprising:

(i) the antibody of claim 1, the recombinant protein of claim 6, or the immunoconjugate of claim 10; and

(ii) a pharmaceutically acceptable carrier.

12. Use of the antibody of claim 1, the recombinant protein of claim 6, or the immunoconjugate of claim 10 for the preparation of a medicament, a reagent, a test plate, or a kit;

the reagent, assay plate or kit is for:

(1) detecting HPV16, HPV31, HPV35, HPV52, and/or HPV58E7 protein in the sample;

(2) detecting endogenous HPV16, HPV31, HPV35, HPV52, HPV58, and/or E7 proteins in the tumor cell;

(3) detecting tumor cells expressing HPV16, HPV31, HPV35, HPV52, and/or HPV58E7 proteins;

the medicament is used for treating or preventing tumors expressing HPV16, HPV31, HPV35, HPV52 and/or HPV58E7 proteins.

13. An assay plate comprising a substrate and a test strip comprising the antibody of claim 1 or the immunoconjugate of claim 10.

14. A kit, comprising:

(1) a first container comprising the antibody of claim 1;

(2) a second container comprising a secondary antibody against the antibody of claim 1; and

(3) a third container comprising a cell lysis reagent;

alternatively, the first and second electrodes may be,

the kit comprising the assay plate of claim 13.

15. A method of making a recombinant polypeptide, the method comprising:

(a) culturing the host cell of claim 9 under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being the antibody of claim 1 or the recombinant protein of claim 6.

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