CN107868127A - A kind of monoclonal antibody for being used to detect Oncoprotein Expression in histopathologic slide - Google Patents
A kind of monoclonal antibody for being used to detect Oncoprotein Expression in histopathologic slide Download PDFInfo
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- CN107868127A CN107868127A CN201610859309.5A CN201610859309A CN107868127A CN 107868127 A CN107868127 A CN 107868127A CN 201610859309 A CN201610859309 A CN 201610859309A CN 107868127 A CN107868127 A CN 107868127A
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Abstract
The invention provides a kind of monoclonal antibody for cervical carcinoma auxiliary diagnosis and its application, specifically, the invention provides one kind identification high-risk-type (HPV16, HPV18, HPV31, HPV33, HPV58 etc.) HPV positive cervical tissue rabbit resource monoclonal antibody and its application, the antibody can detect the biological marker HPVE7 albumen in tissue (including cervical carcinoma and cervical lesionses) with high specificity, become cervical epithelial tissue so as to distinguish the cervical carcinogenesis tissue related to HPV persistent infections and abnormal cervical or non-cancer, cervical carcinoma that can be caused by the high-risk HPV infection of Accurate Diagnosis, pathologist can give more accurate analysis result according to the expression of HPV E7 albumen to the state of an illness of patient.The rate of missed diagnosis of cervical lesionses can be effectively reduced, and improve injury and the wasting of resources that over-treatment is brought to patient.
Description
Technical Field
The invention belongs to the field of biological medicines, particularly relates to a monoclonal antibody for detecting cancer protein of a cancer pathological section and application thereof, and more particularly relates to detection of HPV E7 protein expression in cancer caused by high-risk HPV.
Background
Cervical cancer is a common malignant tumor of female reproductive systems, is the second place of female malignant tumors, has low survival rate of late cancer for 5 years, and has high morbidity and mortality worldwide. In 1976 zur Hansen suggested that Human Papillomavirus (HPV) may be a sexually transmitted carcinogen, and began to study the relationship between HPV and cervical cancer. Currently, many epidemiology have demonstrated that HPV is the culprit responsible for cervical cancer and can cause a variety of other tumors, including reproductive, breast, digestive and respiratory cancers. zur Hansen also therefore received a nobel prize for physiology or medicine in 2008. In recent years, the spread of HPV in people in China is continuously reduced, and the research work of prevention and treatment of cervical cancer is very important.
80% of women will be infected with HPV virus during their lifetime, and most HPV infections will be self-infected within 1-2 yearsThe body's immune system is cleared and persistent HPV infection will progress to high grade Cervical Intraepithelial Neoplasia (CIN) lesions, such as CIN II and CIN III, and even further to cervical cancer. 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. The pathological morphology of most middle and late cervical cancers is obvious and diagnosis is not difficult. However, diagnosis of early cervical cancer and its precancerous lesions has been the focus of research. In the process of normal cervical epithelium → CIN → cervical cancer, except for corresponding changes of histology and cytology, the structure, function and the like of some genes are changed, and the genes can be used as molecular markers in the process so as to discover and diagnose CIN and cervical cancer at early stage. Such as Ki67, p16INK4AAnd hTERT, etc. are expressed in increasing levels with increasing CIN (Valentina F, Renzo B, Serena B, et al, Detection of HPV E7Oncoviral protein in nuclear division by a municipal solid, apple Immunohistochem Mol Morphol,2013,21(4): 341-350). Recent studies have shown that the tumor suppressor gene p16INK4AOver-expression in most cervical carcinomas and precancerous lesions, p16 is thought to beINK4AThe protein is used as a biological marker for early screening of cervical cancer.
Persistent infection by HPV is the major viral causative agent in cervical intraepithelial carcinogenesis. In china, high risk types HPV16, 18, 31, 33, 52 and 58 are the several subtypes with the highest detection rate of cervical squamous cell carcinoma. Some recent studies have shown that p16INK4AThe overexpression of (A) is closely related to HPV persistent infection and cervical cancer (Kalof AN, Cooper K., p16INK4AIMMunoexpression: defective marker of high-risk HPV and high-grade cervical cancer neoplasia. adv Ant Pathol.2006 Jul; 13(4): 190-4.). In the process of cancer, virus DNA is integrated into the genome of human cells, and the virus oncogenic protein E7 is continuously expressed along with the deletion of the expression control of the E7 protein, so that the cells are continuously differentiated and generate precancerous lesions, the regulation and control of the cells are out of control, and the immortalization occurs. The E7 protein can preferentially bind to and lose the retinoblast oncoprotein pRBAnd (6) alive. Due to p16INK4ANegative feedback regulation exists with pRB quality detection, and after the HPV E7 protein is pRB inactivated, pRB is enabled to be p16INK4AThe negative feedback regulation effect of (1) is lost, thereby leading to p16INK4AHas high positive expression rate in cervical squamous carcinoma with positive HPV infection. 7.2012, the american pathologist association (CAP) and american colposcopy and cervical pathology council (ASCCP) guidelines indicate that p16 can be a marker reflecting the effect of HPV E6/E7 on cell proliferation, with sufficient evidence to suggest recommendations for low-grade anogenital squamous epithelial lesions, suggesting p16 with a specific clone number (E6H4)INK4aThe antibodies serve as biomarkers to detect whether HPV infection affects cell cycle regulation. The clone number is p16 which is globally and uniquely authenticated by IVDINK4aAn antibody. Roche diagnosis CINtec histology p16 (containing p16 antibody E6H4) was marketed in china in 5 months of 2014. Therefore, E7 protein, which is continuously expressed in epithelial cells of patients with high cervical dysplasia caused by HPV infection and cervical cancer, is located at p16in the pathogenesis of cervical cancerINK4aAnd can also serve as a tumor marker for the detection of high-grade cervical lesions and cervical cancer.
The current diagnosis method commonly used for cervical tissues is hematoxylin-eosin staining method (H & E), and although the H & E interpretation is the current standard for grading CIN, the H & E interpretation is easily affected by subjective factors of pathologists and changes of cervical epithelization, atrophy and repair, and the like, so that the H & E interpretation has poor repeatability and low diagnosis accuracy. Clinically, more objective and accurate diagnosis standards are urgently needed. There are two main reasons why there is no suitable antibody for clinical HPV E7 detection: 1. the expression level of HPV protein in clinical tissues or cell samples is low, and high-affinity antibodies are required for detection; 2, HPV viruses cannot survive laboratory culture under current standard tissue culture techniques; 3, the E7 protein has immunosuppression, so that the animal immunized by the E7 protein can not obtain good immune response. Under the condition, a monoclonal antibody of the HPV16E7 protein and a method for detecting the over-expression of the HPV E7 are provided. The monoclonal antibody can specifically bind to HPV 16/18E 7 protein endogenous to tumor cells. The diagnosis CIN I, CIN II and CIN III must be judged according to the form of H & E, by means of the method, precancerous lesions can be accurately known by detecting the biomarker E7 protein, the accuracy of CIN grade judgment is improved, and accurate shunting of patients is ensured so as to adopt appropriate follow-up and treatment measures.
Disclosure of Invention
The invention aims to provide a monoclonal antibody for cervical cancer pre-auxiliary diagnosis 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 E7 protein; more preferably, the antibody is an antibody specific against HPV16E7 protein. Preferably, the antibody also has specific activity against HPV18, HPV31, HPV33, 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 wild-type HPV16E7 protein, or may be a derivative protein of wild-type HPV16E7 protein. The HPV 18E7 protein can be wild-type HPV 18E7 protein or derivative protein of wild-type HPV 18E7 protein. The HPV31E7 protein can be wild-type HPV31E7 protein or derivative protein of wild-type HPV31E7 protein. The HPV 33E 7 protein can be wild-type HPV 33E 7 protein or derivative protein of wild-type HPV 33E 7 protein, the HPV 52E 7 protein can be wild-type HPV 52E 7 protein or derivative protein of wild-type HPV 52E 7 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, HPV 18E7 protein, HPV31E7 protein, HPV 33E 7 protein, HPV 52E 7 and HPV58E7 protein.
In another preferred embodiment, the antibody does not bind to or has a low affinity for other HPV subtypes.
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 E7 protein; more preferably, it is specific against HPV16E7 protein. Preferably, the recombinant protein is also specific to high-risk HPV18, HPV31, HPV33, 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 manufacture of a medicament, a reagent, a detection plate or a kit;
the reagent, assay plate or kit is for:
(1) detecting HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 proteins in the sample; and/or
(2) Detecting endogenous HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 proteins in the tumor cells; and/or
(3) Detecting tumor cells expressing HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 proteins;
the medicament is used for treating or preventing tumors expressing HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 proteins.
In another preferred example, the sample contains HPV16 and/or HPV18 and/or HPV31 and/or HPV33 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 HPV18 and/or HPV31 and/or HPV33 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;
or,
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.
Drawings
FIG. 1 is a diagram showing the result of ELISA detection of HPV16E7 single-chain antibody (scFv) binding to protein.
FIG. 2 shows the result of ELISA detection of antigen binding specificity of HPV16E7 rabbit monoclonal antibody. RAB-016, RAB-017 and RAB-16 only bind to His-HPV16E7, but not to His-HPV16E7 and His-unrelated proteins; RAB-139 was able to bind both His-HPV16E7 and His-HPV 18E7, and not His-unrelated proteins.
FIG. 3 shows the result of HPV16E7 rabbit monoclonal antibody titer ELISA detection. As a result, the RAB-139 antibody titer was slightly superior to other antibodies, followed by RAB-016 and RAB-017.
FIG. 4 shows the result of cross-reaction detection of HPV16E7 rabbit monoclonal antibody in different subtype HPV E7 proteins. The HPV subtypes of RAB-016 capable of binding recombinant protein E7 are 16, 31, 33, 35, 52, 58, 6 and 11, and have certain cross with His-unrelated protein; the HPV subtypes that RAB-017 was able to bind to recombinant protein E7 were 16, 31, 33 (weak), 52, 58 (weak), 6, 11; the HPV subtypes with the ability of RAB-139 to bind recombinant protein E7 are 16, 18, 31, 33, 52, 58.
FIG. 5 is a graph showing the immunohistochemical staining results of paraffin sections of normal cervical tissues and cervical cancer tissues with rabbit monoclonal antibodies RAB-016, RAB-017 and RAB-139. FIG. 5A shows the staining results of rabbit monoclonal antibody RAB-016 in normal and cervical cancer tissues; FIG. 5B shows the staining results of rabbit monoclonal antibody RAB-017 in normal tissues and cervical cancer tissues; FIG. 5C shows the staining results of rabbit anti-RAB-139 in normal tissues and cervical cancer tissues.
FIG. 6 is a graph showing the results of immunohistochemical staining of rabbit mAb RAB-139 in paraffin sections of cervical tissue (chronic inflammation, CIN grade and cervical cancer) samples. The rabbit monoclonal antibody RAB-139 can detect not only HPV E7 protein in cervical cancer, but also CIN-grade HPV E7 protein, and can distinguish benign hyperplasia from malignant hyperplasia samples. Wherein FIG. 6A is a staining result of chronic inflammation with substantially no apparent staining. FIG. 6B shows CIN I staining results, with CIN I-grade specific staining on the left and benign hyperplasia on the right; FIG. 6C shows CIN II staining results, with CIN II specific staining on the left and benign hyperplasia on the right; in FIG. 6D, the left panel shows the staining result of CINIII, and the right panel shows the staining result of cervical cancer.
Detailed Description
The inventor finally obtains a rabbit-derived monoclonal antibody RAB-139 resisting high-risk HPVE7 through extensive and intensive research and a large number of screens. Experimental results show that the rabbit-derived monoclonal antibody aiming at the high-risk HPV 7 protein has high specificity and strong affinity, and has stronger affinity with E7 proteins of high-risk HPV16, 18, 31, 33 and 58. Further research shows that the antibody can also be used for detecting clinical paraffin section pathological samples. The invention provides a method for detecting and/or identifying high-risk HPV E7 protein, which has good stability and high detection sensitivity. The invention also provides a kit containing the antibody.
Specifically, the recombinant His-HPV16E7 fusion protein is adopted to immunize white Japanese auricle rabbits, His-HPV16E7 and another His-labeled unrelated protein are used as screening detection antigens, and after rabbit serum reaches a certain titer, B lymphocytes of lymph nodes are obtained and used 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 identified by antigen binding epitope analysis, antigen subtype cross-reaction analysis, affinity binding identification, immunocytochemical staining (Immunocytochemistry ICC) and immunohistochemical staining (Immunohistochemistry IHC). Through the identification tests, the 1 strain antibody clone RAB-139 passes the test requirements, shows the function of specifically combining high-risk HPV E7 oncoprotein at the protein molecular level, the cell level and the tissue level, most importantly contains a plurality of high-risk HPV subtypes, and increases the application value of the high-risk HPV subtype.
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 HPV 18E7 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 form 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 "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (termed kappa and lambda) according to the amino acid sequence of their constant regions of the heavy chains, the immunoglobulins can be classified into different classes.5 classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2.
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 the high-risk HPVE7 protein, a monoclonal antibody with the variable region chain of the monoclonal antibody for resisting the high-risk HPVE7 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 "VH"may be used interchangeably.
The invention adopts a conventional method to sequence the monoclonal antibody RAB-034 to obtain the sequence information, and the sequence information is described as follows.
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 comprises three complementarity determining regions CDRs including:
CDR1 having an amino acid sequence of GFSLSSYT (SEQ ID NO.:4) and a coding nucleotide sequence of ggattctccctcagtagctataca (SEQ ID NO.: 3);
CDR2, the amino acid sequence of which is ISTGDTT (SEQ ID NO.:6), and the coding nucleotide sequence of which is attagtactggtgataccact (SEQ ID NO.: 5);
CDR3, its amino acid sequence is ARGYGKSSGYSGLNL (SEQ ID NO.:8), its coding nucleotide sequence is, gcgagggggtatggtaaaagtagtggttattctggccttaacttg (SEQ ID NO.: 7).
In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:
QSVEESGGDLVKPGASLTLTCKASGFSLSSYTMGWFRQAPGKGLEYIGAISTGDTTDYTNWAKGRFTISKTSSTTVALQMTSLTAADTATYFCARGYGKSSGYSGLNLWGPGTLVTVSS(SEQ ID NO.:10)；
the coding nucleotide sequence is as follows:
cagtcggtggaggagtccgggggagacctggtcaagcctggggcatccctgacactcacctgcaaagcctctggattctccctcagtagctatacaatgggctggttccgccaggctccagggaaggggctggaatacatcggagccattagtactggtgataccactgactacacgaactgggcgaaaggccgattcaccatctccaaaacctcgtcgaccacggtggctctgcaaatgaccagtctgacagccgcggacacggccacctatttctgtgcgagggggtatggtaaaagtagtggttattctggccttaacttgtggggcccaggtaccctggtcacagtgagctct(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"may be used interchangeably.
In a preferred embodiment of the invention, the light chain variable region of the antibody according to the invention has complementarity determining regions CDRs selected from the group consisting of:
CDR1', the amino acid sequence of which is ESVYSNNY (SEQ ID No.:12),
the coding nucleotide sequence is gagagcgtttatagtaacaactac (SEQ ID NO: 11);
CDR2', the amino acid sequence of which is SAS (SEQ ID NO: 14),
the coding nucleotide sequence is tctgcatcc (SEQ ID NO: 13);
CDR3', having an amino acid sequence of LGSYDCSSTDCFG (SEQ ID NO: 16),
the coding nucleotide sequence is ctaggcagttatgattgtagtagtactgattgttttggt (SEQ ID NO.:15)
In another preferred embodiment, the amino acid sequence of the light chain variable region is:
DPVLTQTASPVSAAVGSTVTISCQSSESVYSNNYLSWFQQKPGQPPKQLIYSASSLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSSTDCFGFGGGTEVVVK(SEQ ID NO.:18)，
the coding nucleotide sequence is as follows:
gaccctgtgctgacccagactgcatcgcccgtgtctgcagctgtgggaagcacagtcaccatcagttgccagtccagtgagagcgtttatagtaacaactacttatcctggtttcagcagaaaccagggcagcctcccaagcaactgatctattctgcatccagtctggcatctggggtctcatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatcagcgacgtgcagtgtgacgatgctgccacttactactgtctaggcagttatgattgtagtagtactgattgttttggtttcggcggagggaccgaggtggtcgtcaaa(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 to HPV16E7 protein, such as a protein or polypeptide having a heavy chain variable region (e.g., the amino acid sequence of SEQ ID No.: 10) and/or a light chain variable region (e.g., 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 located in the variable regions of the heavy and light chains, called the variable regions (CDRs), which are separated by 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction.
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 the HPV16E7 protein binding activity, including 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
| Initial residue(s) | Representative substitutions | Preferred substitutions |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
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, 13, 15, 17, 19 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, 13, 15, 17, 19.
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. And, the polynucleotides that hybridize encode polypeptides having the same biological functions and activities as the mature polypeptides of SEQ ID No.10 and/or SEQ ID No. 18.
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: a calcium phosphate coprecipitation method,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. biological toxins (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 (Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research (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, thereby 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, the 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 TCell hybrids, Elsevier, N.Y.,1981), phage display technology or can be prepared using recombinant DNA methods (U.S. Pat. No.4,816,567) can be used.
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 high-risk type HPVE7 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 invention relates to a method for detecting cervical cancer and risk early warning by using a tissue sample. The method comprises the following steps: obtaining a tissue sample; formalin-fixing a sample, and preparing a paraffin section; detecting high risk HPV E7 oncoprotein levels in the paraffin sectioned samples.
The invention can be used for detecting high-risk HPV E7 oncoprotein in high-risk HPV infection-related cancers, wherein the high-risk HPV infection-related cancers comprise tumors of the urogenital system, such as cervical cancer, bladder cancer, endometrial cancer, penile cancer and the like, small cell lung cancer, melanoma, head and neck tumors and the early stages of the cancers.
The specimen (sample) used in the present invention is a tissue specimen, and a formalin-fixed paraffin section.
Reagent kit
The present invention also provides a kit comprising the antibody (or fragment thereof) of the present invention or the assay plate of the present invention, and in a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, 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 designs and develops a kit for diagnosing and evaluating the conditions related to high-risk HPV infection in a sample from a cervical lesion tissue, and the kit can detect high-risk HPV16E7 oncoprotein in the sample, wherein the sample can be a paraffin section or a frozen section. Cervical tissue is sectioned and used to develop detection kits and in-vitro diagnostic devices for immunological detection of high risk HPV infection in a sample based on non-cytological morphological analysis.
One of the purposes of the invention is to provide a method for detecting the expression of high-risk HPVE7 protein, and the method can be used for detecting HPV infection-related cancers, particularly cervical cancer.
The inventor of the invention creates a rabbit-derived monoclonal antibody RAB-139 aiming at high-risk Human Papilloma Virus (HPV) E7 protein, and researches the application of the rabbit-derived monoclonal antibody RAB-139. The present inventors used the prepared anti-high risk HPVE7 rabbit mab RAB-139 to perform immunohistochemical staining on formalin-fixed paraffin sections of cervical tissues. By means of the high affinity and high specificity of the rabbit monoclonal antibody RAB-139 to the target protein, the RAB-139 can specifically recognize the high-risk HPVE7 protein in cervical cancer tissues and can also recognize the high-risk HPVE7 protein in early cervical lesions. Therefore, the rabbit polyclonal RAB-139 can be used for detecting the cervical cancer in the middle and late stages, can also be used for detecting early cervical lesions with possible malignant change, and provides reliable evidence for clinical diagnosis and cervical lesion progress risk assessment. 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 the high risk type HPVE7 in the sample.
In the method of the present invention, the test sample is preferably a patient having a possibility of cervical lesion of cervical epithelial lesion, or a patient having a lesion of the cervix.
The HPV16E7 is preferably an HPV16E7 protein or fragment thereof. In this case, the step of detecting HPV16E7 is preferably an immunohistochemical staining analysis using HPV16E 7. The anti-HPV16E7 antibody used is preferably an anti-HPV16E7 rabbit monoclonal antibody.
The protein expressed by the oncogene from HPV16E7 adopted by the method for immunodetection is taken as a reliable index of HPV 16-related malignant or premalignant cell occurrence. One of the most useful aspects of the invention is the use in the diagnosis of cervical cancer, squamous epithelial cell lesions and adenocarcinomas and any epithelial cell abnormalities associated with oncogenic HPV16 infection; oncogenic HPV16 infection shown includes 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. The anti-HPV16E7 rabbit monoclonal antibody produced by a eukaryotic expression system or the monoclonal antibody with the same binding activity with the anti-HPV16E7 rabbit monoclonal antibody.
The invention provides a method for distinguishing tissues without HPV oncogenic protein by detecting HPV16E7 protein endogenous to the tissues. And when tissues are fixed by neutral formalin which is widely used in clinic, oncogenic virus proteins can be accurately detected after the tissues are made into paraffin sections, so that the accuracy of CIN grade interpretation can be improved, and patients can be accurately shunted to take appropriate follow-up and treatment measures.
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 HPV E7 protein provided by the invention has high specificity and strong affinity, can be prepared in large scale and has low cost.
(2) The antibody aiming at the HPV16E7 protein can be specifically combined with the HPV16E7 protein and can also be combined with the HPV18, 31, 33, 52 and 59 subtype E7 protein, so that the antibody can be used for broad-spectrum detection of the HPV E7 protein.
(3) The antibody provided by the invention is used in the method for detecting the HPV16E7 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 precancerous risk early warning of related cancers and definite diagnosis of middle and late cancers.
The present invention will be described in further detail with reference to the following 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 specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1
1. Preparation of high-risk Human Papilloma Virus (HPV) E7 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 blank control is set (coated antigen is directly added into enzyme-labeled secondary antibody). His-HPV16E 70.1 mu g/ml wrapper plate, His-unrelated protein 5 mu g/ml wrapper 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 polypeptides are HPV16E7-1 (amino acid sequence SEQ ID NO.:19PTLHEYMLDLQPETTDLYCYEQLNDSSEEE), HPV16E7-27 (amino acid sequence SEQ ID NO.:20LNDSSEEEDEIDGPAGQAEPDRAH), HPV16E 7-5 (amino acid sequence SEQ ID NO.:21KCDSTLRLCVQSTHVDIRTLE), His-Vac (amino acid sequence SEQ ID NO.:22DEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIV) and His18E7-1 (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 scFv strains all have certain specificity, and scFv016, scFv017 and scFv139 are selected for carrying out the next step of antibody production.
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 the scFv antibody genes selected above to construct a full-length rabbit monoclonal antibody gene expression vector, and constructing three 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). Three rabbit monoclonal antibodies were obtained, respectively: RAB-016, RAB-017 and RAB-139. 1.3 specificity of Rabbit monoclonal antibodies
During detection, an indirect ELISA method is adopted: the antigen is selected from His-HPV16E7 and His-HPV 18E7 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 and His-HPV 18E7 fusion protein 0.5 mu g/ml package plate, His-unrelated protein 5 mu g/ml package plate, 4 ℃ overnight, PBST washing and drying, adding 5% skimmed milk powder for sealing, acting at 37 ℃ for 2 hours or 4 ℃ overnight, PBST washing and drying, adding to-be-detected antibody 0.1 mu g/ml, reacting at 37 ℃ for 1 hour, PBST washing and drying, adding to goat Anti-rabbit-HRP secondary antibody (Sigma A0545) (1: 20000), Anti-6 XHis (Abcam ab1187) (1: 10000), reacting at 37 ℃ for 1 hour, after PBST washing and drying, TMB developing color and 2M H2SO4And (6) terminating. The results are shown in FIG. 2: RAB-016, RAB-017 and RAB-139 can bind to His-HPV16E7 but not to His-unrelated protein; meanwhile, RAB-139 has weak cross with His-HPV 18E7, while RAB-016 and RAB-017 can only bind with His-HPV16E7 recombinant protein.
2. Identification of monoclonal antibodies
2.1ELISA detection of Rabbit monoclonal antibody titer
Coating fusion protein His-HPV16E70.5 mu g/ml on a plate, standing overnight at 4 ℃, washing and drying PBST, adding 5% skimmed milk powder for sealing, acting for 2 hours at 37 ℃ or standing overnight at 4 ℃, washing and drying PBST, adding anti-HPV16E7 monoclonal antibodies RAB-016, RAB-017 and RAB-139 with the initial concentration of 1 mu g/ml, diluting in a multiple ratio, reacting for 1 hour at 37 ℃, washing and drying PBST, adding goat anti-rabbit-HRP secondary antibody (SigmaA0545) (1: 20000), reacting for 1 hour at 37 ℃, developing TMB and 2M H after washing and drying PBST2SO4End, OD450 nm. The results are shown in FIG. 3: when His-HPV16E7 is used as antigen, the 3 strains of antibodies all have high binding force.
2.2 Cross-subtype analysis of Rabbit monoclonal antibodies
Identifying the cross reaction of the monoclonal antibody in different subtype HPV E7 protein. And (3) identifying by adopting an ELISA method: the antigen is selected from recombinant proteins, and the HPV subtypes of the recombinant proteins are respectively 16, 18, 31, 33, 35, 45, 52, 58, 6 and 11. Antigen is coated by 0.5 mu g/ml, the temperature is 4 ℃ overnight, after PBST washing and drying, 5% skimmed milk powder is added for sealing, the temperature is 37 ℃ for 2 hours or 4 ℃ overnight, after PBST washing and drying, anti-HPV16E7 monoclonal antibodies RAB-016, RAB-017 and RAB-139(1 mu g/ml) are added for reaction for 1 hour, after PBST washing and drying, goat anti-rabbit-HRP secondary antibody (SigmaA0545) (1: 20000) is added for reaction for 1 hour at 37 ℃, after PBST washing and drying, TMB color development and 2M H are carried out2SO4End, OD450 nm. The results are shown in FIG. 4: the HPV subtypes of RAB-016 capable of binding recombinant protein E7 are 16, 31, 33, 35, 52, 58, 6 and 11, and have certain cross with His-unrelated protein; the HPV subtypes that RAB-017 was able to bind to recombinant protein E7 were 16, 31, 33 (weak), 52, 58 (weak), 6, 11; the HPV subtypes with the ability of RAB-139 to bind recombinant protein E7 are 16, 18, 31, 33, 52, 58.
2.3 detection of HPV16E7 protein in cervical cancer tissue by rabbit-derived monoclonal antibody immunohistochemical staining method
Uses rabbit monoclonal antibodies RAB-016, RAB-017 and RAB-139 as primary antibodies to normal cervical tissues and cervical cancer groups respectivelyThe tissues were subjected to immunohistochemical staining. The method comprises the following steps: soaking pathological paraffin sections in dimethylbenzene twice for 10 minutes each time; then sequentially soaking in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 5 minutes respectively, and finally washing with PBST for three times; the tissue sections were placed in a boiling 0.01M sodium citrate buffer (pH6.0) and autoclaved for 2 minutes. Cooling for 30 minutes at room temperature, and washing for 5 minutes by PBST for three times; addition of 3% H for inactivation of endogenous peroxidase2O2Treating the mixture for 10 minutes at room temperature by using PBS buffer solution; PBST was washed 3 times for 5 minutes each; adding PBST containing 10% calf serum and sealing for 20 minutes at room temperature; discarding the blocking solution, adding 3 antibodies, RAB-016(20ug/ml), RAB-017(20ug/ml) and RAB-139(0.1ug/ml), respectively, and incubating at room temperature for 1 hr; after full washing, adding Anti-Mouse/Rabbit IgG-HRP (DakoK5007) dropwise, and incubating for 30 minutes at room temperature; DAB (Dako K5007) is developed for 3-5 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.
The results are shown in FIG. 5: rabbit monoclonal antibodies RAB-016 and RAB-017 have no staining in normal cervical tissues and cervical cancer tissues; rabbit monoclonal antibody RAB-139 has no specific staining in normal cervical tissue, has obvious brown-yellow staining in cervical cancer tissue, and the staining is located in cytoplasm. In conclusion, only the rabbit-derived monoclonal antibody RAB-139 can specifically recognize HPV16E7 protein in cervical cancer tissues so as to enable cervical cancer part tissues to be specifically stained, and the staining is localized in cytoplasm, so that RAB-139 is selected for further research.
Example 2 detection of cervical lesion tissue by immunohistochemical staining of Rabbit-derived monoclonal antibody RAB-139
According to the antibody screening result, the application of the rabbit-derived monoclonal antibody RAB-139 in detection of cervical precancerous lesion samples is further explored by taking the rabbit-derived monoclonal antibody RAB-139 as a primary antibody. Cervical Intraepithelial Neoplasia (CIN) is a precancerous lesion of cervical cancer, including mild (CIN grade I), moderate (CIN grade II), severe dysplasia, and carcinoma in situ (CIN grade III) of the cervix. Selecting pathological paraffin sections of chronic cervicitis, CIN-I, -II, -III and cervical cancer paraffin sections, and performing immunohistochemical staining detection by using RAB-139, wherein the specific operation refers to the sections 1 and 2.3 of the example. As a result: in the cervical chronic inflammation sample, the positive rate is 16.7%; in CIN I samples, the positive rate is 28.6%; in CIN II samples, the positive rate is 78.6%; in CINII and cervical cancer samples, the positive rate is 100% (Table 1, clinical application data of rabbit monoclonal antibody RAB-139 in paraffin section of cervical tissue). The staining conditions of the samples of the respective grades are listed, and are shown in fig. 6, wherein fig. 6A is the staining result of chronic inflammation, and basically no obvious staining is obtained. FIG. 6B shows CIN I staining results, with CIN I-grade specific staining on the left and benign hyperplasia on the right; FIG. 6C shows CIN II staining results, with CIN II specific staining on the left and benign hyperplasia on the right; in FIG. 6D, the left panel shows the staining result of CIN III, and the right panel shows the staining result of cervical cancer.
TABLE 1 Positive staining rates for various grades of samples in cervical tissue
| Type of sample | Total number of samples | Positive staining | Positive staining rate |
| Chronic inflammation of cervix | 30 | 5 | 16.7％ |
| CINI | 7 | 2 | 28.6％ |
| CIN II | 14 | 11 | 78.6％ |
| CIN III | 8 | 8 | 100％ |
| Cervical cancer | 18 | 18 | 100％ |
Discussion:
the invention adopts the method to prepare the HPV16E7 rabbit-derived monoclonal antibody, selects three rabbit single-chain antibody genes to express the eukaryotic system of the rabbit anti-full-length gene, screens the expressed full-length rabbit monoclonal antibody, and finally selects the monoclonal antibody RAB-139 which has high sensitivity and strong specificity, can identify the endogenous HPV16E7 protein of a tissue slice and has the lowest background. The research shows that the RAB-139 can effectively identify the cancerated cells in the cervical tissue slice sample by combining the high-risk HPV E7 protein related to the cervical cancer. Further research shows that RAB-139 can recognize not only the viral protein in cervical cancer tissue but also the viral protein in lesion tissue (such as CIN II-III grade) before 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; while CIN grade III, the uterus is mainly removed by operation at home, and local treatment is advocated abroad. In clinical practice, the determination of CIN II 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, more than 90 percent of cervical cancer samples can detect the high-risk HPV infection, the method provided by the invention can detect the high-risk HPV in the early CIN stage and at least in the cervical moderate lesion sample, and the high-risk HPV can be used as an index for judging whether the cervical lesion is deteriorated or not, so that whether the CIN patient is operated or not can be shunted, an auxiliary basis is provided for clinical doctors to diagnose and treat the patient, the missed diagnosis rate caused by subjective judgment on tissue morphology can be reduced, and the loss caused by over-treatment can be reduced.
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 (16)
1. An antibody 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;
preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 10.
2. An antibody heavy chain having the heavy chain variable region of claim 1, and
a heavy chain constant region.
3. An antibody 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;
preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 18.
4. An antibody light chain having the light chain variable region of claim 3, and
a light chain constant region.
5. An antibody, wherein said antibody has:
(1) the heavy chain variable region of claim 1; and/or
(2) The light chain variable region of claim 3;
preferably, the antibody has: the heavy chain of claim 2; and/or the light chain of claim 4.
6. A recombinant protein, said recombinant protein having:
(i) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
7. A polynucleotide encoding a polypeptide selected from the group consisting of:
(1) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; 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 heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; 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.
11. A pharmaceutical composition comprising:
(i) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, the recombinant protein of claim 6, or the immunoconjugate of claim 10; and
(ii) a pharmaceutically acceptable carrier.
12. Use of the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, the recombinant protein of claim 6, or the immunoconjugate of claim 10, for the preparation of a medicament, a reagent, a detection panel, or a kit;
the reagent, assay plate or kit is for:
(1) detecting HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 proteins in the sample; and/or
(2) Detecting endogenous HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 proteins in the tumor cells; and/or
(3) Detecting tumor cells expressing HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 proteins;
the medicament is used for treating or preventing tumors expressing HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 proteins.
13. A method of detecting HPV E7 protein in a sample, comprising the steps of:
(1) contacting a sample with the antibody of claim 5;
(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.
14. An assay plate comprising a substrate (support plate) and a test strip comprising an antibody according to claim 5 or an immunoconjugate according to claim 10.
15. A kit, comprising:
(1) a first container comprising the antibody of claim 5; and/or
(2) A second container comprising a secondary antibody against the antibody of claim 5; and/or
(3) A third container comprising a cell lysis reagent;
or,
the kit comprising the assay plate of claim 14.
16. 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 5 or the recombinant protein of claim 6.
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| CN110196332A (en) * | 2019-05-22 | 2019-09-03 | 艾托金生物医药（苏州）有限公司 | A kind of method and its application detecting more hypotype HPV E7 albumen |
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| CN108586607B (en) * | 2018-04-13 | 2020-09-11 | 郑州大学 | Preparation method and application of anti-HPV16 L1 protein monoclonal antibody |
| CN116199772B (en) * | 2022-11-16 | 2023-10-17 | 怡道生物科技(苏州)有限公司 | HPV31 type capsid protein L1 monoclonal antibody, preparation method and application |
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| US20080044809A1 (en) * | 2005-11-15 | 2008-02-21 | Shu-Ling Cheng | Detection method for human pappilomavirus (hpv) and its application in cervical cancer |
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| CN110196332A (en) * | 2019-05-22 | 2019-09-03 | 艾托金生物医药（苏州）有限公司 | A kind of method and its application detecting more hypotype HPV E7 albumen |
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| WO2018059117A1 (en) | 2018-04-05 |
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