CN105524166A - Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof - Google Patents

Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof Download PDF

Info

Publication number
CN105524166A
CN105524166A CN201410508253.XA CN201410508253A CN105524166A CN 105524166 A CN105524166 A CN 105524166A CN 201410508253 A CN201410508253 A CN 201410508253A CN 105524166 A CN105524166 A CN 105524166A
Authority
CN
China
Prior art keywords
antibody
protein
variable region
heavy chain
chain variable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201410508253.XA
Other languages
Chinese (zh)
Other versions
CN105524166B (en
Inventor
常小迦
施丽君
时成龙
韩凤丽
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Etoki Bio Pharmaceutical (suzhou) Co Ltd
Original Assignee
Etoki Bio Pharmaceutical (suzhou) Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Etoki Bio Pharmaceutical (suzhou) Co Ltd filed Critical Etoki Bio Pharmaceutical (suzhou) Co Ltd
Priority to CN201410508253.XA priority Critical patent/CN105524166B/en
Publication of CN105524166A publication Critical patent/CN105524166A/en
Application granted granted Critical
Publication of CN105524166B publication Critical patent/CN105524166B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Peptides Or Proteins (AREA)

Abstract

The present invention provides a monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof. According to the present invention, with the monoclonal antibody, the cervical cancer biomarker HPV18E7 protein in the tumor cells can be detected in a highly specific manner so as to distinguish between the cancerous cervical epithelial cells and the abnormal cervical epithelial cells or non-cancerous cervical epithelial cells and provide basis for the accurate diagnose of the cancer caused by the HPV infection, such that the treatment can be timely taken, the cancer occurrence or cancer spreading can be prevented, and the suffering of patients can be alleviated.

Description

Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof
Technical Field
The invention belongs to the field of biological diagnosis and medicines, and particularly relates to a monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof.
Background
Cervical cancer is a common malignancy in the female reproductive system, second-place in female malignancies. In 1949, Human Papillomavirus (HPV) particles were first observed by stuuss in verrucous body leachate under an electron microscope. After HaraldZurHausen proposed that HPV could be a sexually transmitted carcinogen in 1976, the study of the relationship between HPV infection and cervical cancer became a popular topic of research in the etiology of tumor viruses. A large number of researches find that the human papilloma virus HPV is an extremely powerful cause of cervical cancer and can also cause various other tumors including cancers of the reproductive tract, mammary gland, digestive tract and respiratory tract. 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.
Most women infect genital HPV throughout their lives, with genital HPV infection reaching as high as 10.4% worldwide. Most HPV infections are cleared by the autoimmune system within 1-2 years, and persistent HPV infections will progress to high grade Cervical Intraepithelial Neoplasia (CIN) lesions, such as CIN2 and CIN3, 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. Therefore, early diagnosis and prevention of HPV infection are important breakthrough ports for reducing mortality of diseases related to cervical cancer and the like and reducing the cost of cervical cancer treatment. At present, the cervical cancer screening mainly adopts pap smear test to detect the cytological morphology of cervical exfoliated cells. The cytomorphological detection has certain subjectivity, difficult flaking, poor repeatability in batch and between batches and higher false positive and false negative rates, so the sensitivity for detecting early cancers is low, the missed diagnosis rate is high, and the positive detection rate is only 30-50%; in some areas, pap smear tests have been replaced by Liquid Based Cytology (LBC), a new technique for semi-or fully automated specimen processing that automatically analyzes test specimens and provides the remaining cell samples for analysis of other HPV infections. Another clinically used molecular assay, the HPV dna assay (e.g. HC2), can aid cytological detection of the presence of high risk HPV viruses. Although HPV dna detection is more sensitive than cytomorphological detection, it is not possible to distinguish between transient infection or persistent infection of HPV, and both sensitivity and specificity of the detection method are essential in the course of malignant evolution of tumors. It cannot be used to detect cancer because it does not accurately reflect the occurrence of cervical cancer (the cervical cancer screening guidelines updated separately in the us and at the end of 2012 of our country consistently point out "screening by HPV detection alone in people of any age group is not recommended").
HPV is a species-specific epitheliotropic virus, a small DNA virus with double-stranded closed loops, containing about 8000 base pairs. These included 8 early open reading frames (E1-E8), 2 late reading frames and 1 non-coding long control region. In the early open reading frame, the E6 and E7 genes were most important for cell growth stimulation. The research of Ziegent (2003) and the like has proved that the HPVE6 and E7 genes have cell transformation function and are potential oncogenes, the HPVE6 and E7 proteins which are encoded into oncoproteins can transform mouse epithelial cells in vitro and can also enable human epithelial cells to be immortalized, and the continuous expression of the HPVE6 and E7 proteins is necessary for maintaining the immortalization of in vitro cultured cells. Therefore, the early expression proteins E6 and E7 of high-risk HPV play an important role in the development of cervical cancer. During carcinogenesis, virus DNA is integrated into human cell genome, and E6 and E7 proteins are continuously expressed in epithelial cells of patients with high cervical dysplasia and cervical cancer along with the loss of expression control of E6 and E7 proteins. E7 is a tumor-origin protein, has strong antigenicity, can inactivate tumor cancer suppressor pRb, and finally causes uncontrolled cell growth, resulting in cell immortalization and canceration. This makes E7 a tumor marker for the detection of high-grade cervical lesions and carcinoma.
Currently, clinical immunohistochemical detection of HPV infection mainly uses HPVL1 and other auxiliary biomarkers, such as p16INK4A, Ki67, hTERT, etc. (Valentinaf, Renzob, SerenaB, etc., DetectionnofHPVE7 OncoviralproteininecainerVille, Byanewantside, 2013,21(4): 341-350). There are three main reasons for the lack of suitable antibodies for clinical HPV detection: 1, the HPV protein has low expression level in clinical tissues or cell samples, and needs high-affinity antibodies for detection; 2, HPV viruses cannot survive laboratory culture under current standard tissue culture techniques; 3, E7 protein has immunosuppression, so that animals immunized by the E7 protein cannot obtain good immune response, and the prepared antibody has no specificity to the E7 protein due to cross reaction with other HPV proteins.
Therefore, there is a need to provide a simple and objective method for detecting cancers (particularly cervical cancer) infected with high-risk HPV types.
Disclosure of Invention
The invention aims to provide a monoclonal antibody capable of identifying HPV18 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 or murine 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. 14,
CDR2' shown in SEQ ID NO. 16, and
CDR3' as shown in SEQ ID NO. 18.
In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 20.
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 or murine 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 18; more preferably, the antibody is an antibody specific against HPV18E7 protein.
In another preferred embodiment, the antibody comprises: a single chain antibody, a diabody, a monoclonal antibody, a chimeric antibody (e.g., a human-murine chimeric antibody), a murine antibody, or a humanized antibody.
In another preferred embodiment, the antibody has the following properties:
(1) the affinity with HPV18E7 protein is less than or equal to 2.67 nM; and
(2) does not bind to HPV16E7 protein.
The HPV18E7 protein can be wild type HPV18E7 protein or derivative protein of wild type HPV18E7 protein. The HPV16E7 protein can be wild type HPV16E7 protein or derivative protein of wild type HPV16E7 protein.
In another preferred embodiment, the antibody is a monoclonal antibody capable of specifically distinguishing HPV18E7 protein from HPV16E7 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) can specifically bind to protein containing HPV18E7 amino acids 44-58.
In another preferred embodiment, the antibody is an IgG 1-type antibody.
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 18; more preferably, it is specific against HPV18E7 protein.
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, 13, 15, 17, or 19.
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, prostatic cancer, colorectal cancer, breast cancer, large intestine cancer, prostatic cancer, cervical 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 HPV18E7 protein in the sample; and/or
(2) Detecting endogenous HPV18E7 protein in the tumor cells; and/or
(3) Detecting tumor cells expressing HPV18E7 protein; and/or
(4) Identifying the type of HPV;
the medicament is used for treating or preventing tumors expressing HPV18E7 protein.
In another preferred embodiment, the sample contains HPV18E7 protein.
In another preferred embodiment, the tumor comprises: a tumor of the urogenital system, a small cell lung cancer, a melanoma or a head and neck tumor, a gastric cancer, a liver cancer, a leukemia, a kidney tumor, a lung cancer, a small intestine cancer, a bone cancer, a prostate cancer, a colorectal cancer, a breast cancer, a large intestine cancer, a prostate cancer, or an adrenal tumor.
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 invention, there is provided a method of detecting HPVE7 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 HPVE7 protein in the sample.
In another preferred example, the detection is performed by ELISA method in step (2).
In another preferred embodiment, the HPV E7 protein comprises an HPV18E7 protein.
In another preferred example, in step (1), the sample is contacted with two antibodies against the HPVE7 protein, at least one of which is an antibody according to the fifth aspect of the present invention, and the detection is performed 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, the second antibody is a monoclonal antibody produced by a hybridoma cell line CGMCC NO. 5714.
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.
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 shows the result of ELISA detection of the antigen binding specificity of HPV18E7 monoclonal antibody MAB-MAB-F2, and the details of the experiment are shown in the sections of examples 1 and 2.2: MAB-F2 was able to specifically bind HPV18E7 recombinant protein but not HPV16E7 recombinant protein.
FIG. 2 shows the result of ELISA detection of the epitope-binding amino acid sequence region of HPV18E7 monoclonal antibody, and the details of the experiment are shown in the sections of examples 1 and 2.3: FIG. 2A is a schematic diagram of HPV18E7 amino acid sequence, and FIG. 2B is a diagram of ELISA detection result. MAB-F2 was able to bind to His-HPV18E7 recombinant protein as well as the polypeptides HPV18E7-1 and HPV18E7-2, but not to the polypeptide His-HPV16E7 recombinant protein.
FIG. 3 is a diagram showing the result of detection of HPV18E7 monoclonal antibody protein binding affinity assay ELISA, and the details of the experiment are shown in the sections of examples 1 and 2.4: the coating concentrations of His-HPV18E7 are respectively 8 mug/ml, 2 mug/ml, 0.5 mug/ml and 0.125 mug/ml, the incubation concentration of MAB-F2 is diluted by a factor from 8 mug/ml, and the total is sevenGradient, substitution into Kaff＝(n-1)/2(n[Ab′]-[Ab]) Six K values were calculated and averaged to give MAB-F2 with 2.67nM protein level affinity.
FIG. 4 is a diagram showing the result of IP-WB detection of the HPV18E7 monoclonal antibody MAB-F2 on the endogenous HPV18E7 of Hela cells, and the details of the experiment are shown in the sections of example 1 and 2.5: mIgG as a negative control failed to bind HPV18E7 in the tumor cell lysate. The mAbMAB-F2 was able to bind HPV18E7 in tumor cell lysates, and therefore a band was evident at 15 kDa. Wherein the bands around 50kDa and 25kDa are the heavy and light chains of the antibodies used in the IP process.
FIG. 5 is a diagram showing the result of HPV18E7 monoclonal antibody immunocytochemical staining detection, and details of the experiment are shown in example 1, section 2.6: a is the result of C-33A cell staining, B is the result of Hela cell staining, C is the result of CaSki cell staining, monoclonal antibody MAB-F2 has strong staining reaction with Hela cells, but has no staining with C-33A cells and CaSki cells.
FIG. 6 is a graph showing the results of immunocytochemical staining of cervical cancer cell lines C-33A and Hela cells fixed in different fixative solutions by using HPV18E 7-specific monoclonal antibody MAB-F2; see example 2 for specific experimental details; C-33A cells do not have immune reaction with HPV18E7 monoclonal antibody MAB-F2; after mixing the two cells, HPV18E7 monoclonal antibody MAB-F2 enables Hela color (red arrow) accounting for 10% of the total number of cells and negative cells C-33A have no staining; 100% of Hela cells fixed in suspension have strong immune response with HPV18E7 monoclonal antibody MAB-F2. 4% paraformaldehyde and LBC fixative had no effect on the specificity of HPV18E7 monoclonal antibody MAB-F2 in ICC assay.
Detailed Description
The inventor unexpectedly obtains an anti-HPVE 7 monoclonal antibody MAB-F2 through extensive and intensive research and a large number of screens, and experimental results show that the monoclonal antibody aiming at the HPVE7 protein has high specificity and strong affinity, and can obviously distinguish the HPV18E7 protein in cells. The monoclonal antibody can be specifically combined with HPV18E7 protein, but has no combination activity with HPV16E7 protein, so that the antibody not only can be used for detecting HPV18E7 protein, but also can be used for identifying different HPV types. The invention also provides a method for detecting and/or identifying the HPV18E7 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 invention adopts recombinant GST-HPV18E7 fusion protein to immunize mice, and uses another fusion protein His-HPV18E7 as a screening detection antigen. The positive hybridoma clones are screened out, can secrete antibody IgG, can be combined with recombinant protein His-HPV18E7, and are specifically combined with HPVE7 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, affinitybinding characterization, WesternBlot, immunoprecipitation, immunocytochemistry staining (immunocytochemistry icc), tissue section immunostaining (immunocytochemistry ihc) methods. Through the identification tests, the 1 strain antibody clone MAB-F2 passes the test requirements, and shows the function of specifically binding high-risk HPVE7 oncoprotein at the protein molecular level, the cell level and the tissue level.
In order to screen out an antibody reagent with clinical examination value, the monoclonal antibody is further used for a detection test of cervical cancer cell strains. Finally, a monoclonal antibody capable of specifically recognizing HPV18E7 positive cervical epithelial cells was obtained, and based on the specificity of the antibody, methods capable of distinguishing cervical epithelial cancer cells from normal cells were developed, which methods were sufficient in terms of sensitivity to identify those patients with early-stage cervical cancer, and in terms of specificity to distinguish between normal and malignant tumor cells.
In a preferred embodiment of the present invention, the amino acid sequence of the HPV16E7 protein is as follows:
HGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP(SEQIDNO.:1)。
in a preferred embodiment of the present invention, the amino acid sequence of the HPV18E7 protein is as follows:
MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ(SEQIDNO.: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, NIHPubl. 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 designated α, 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, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated with 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 HPV18E7 protein, a monoclonal antibody with the variable region chain of the monoclonal antibody for resisting HPV18E7 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-HPV18E7 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.
As used herein, the term "variable region" is used interchangeably with "Complementarity 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, the amino acid sequence of which is GFTFNRYD (SEQ ID NO.:4), and the coding nucleotide sequence of which is ggattcactttcaatagatatgac (SEQ ID NO.: 3);
CDR2, the amino acid sequence of which is IRNDGSKT (SEQ ID NO.:6), and the coding nucleotide sequence of which is atcagaaatgatggtagtaagact (SEQ ID NO.: 5);
CDR3, the amino acid sequence of which is TRDSYGTVFAY (SEQ ID NO.:8), and the coding nucleotide sequence of which is acaagggactcctatggtaccgtgtttgcttac (SEQ ID NO.: 7).
In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:
LVQSGGGSVKPGGSLKLSCAASGFTFNRYDMSWFRQSPEKRLEWVAEIRNDGSKTHYSDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCTRDSYGTVFAYWGQGTLVTVSA(SEQIDNO.:10)；
the coding nucleotide sequence is as follows:
Ctggtgcagtcagggggaggctcagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcaatagatatgacatgtcttggtttcgccagtctccagagaagaggctggagtgggtcgcagagatcagaaatgatggtagtaagactcactattcagacactgtgacgggccgattcaccatctccagagacaatgccaagaacaccctgtacctggaaatgagcagtctgaggtctgaggacacggccatgtattactgtacaagggactcctatggtaccgtgtttgcttactggggccaagggactctggtcactgtctctgcag(SEQIDNO.: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 of murine or human origin.
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 has complementarity determining regions CDRs selected from the group consisting of:
CDR1', the amino acid sequence of which is QSLLYSNGKTY (SEQ ID NO.:14), and the coding nucleotide sequence of which is cagagcctcttatatagtaatggaaaaacctat (SEQ ID NO.: 13);
CDR2', the amino acid sequence of which is LVS (SEQ ID NO.:16), the coding nucleotide sequence of which is ctggtgtct (SEQ ID NO.:15)
CDR3', the amino acid sequence of which is VQGTHFPQT (SEQ ID NO.:18), and the coding nucleotide sequence of which is gtgcaaggtacacattttcctcagacg (SEQ ID NO.:17)
In another preferred embodiment, the amino acid sequence of the light chain variable region is:
VMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPQTFGGGTKLEIK(SEQIDNO.:20)，
the coding nucleotide sequence is as follows:
gtcatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagtcaagtcagagcctcttatatagtaatggaaaaacctatttgaattggttattacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacactgaaaatcagcagagtggaggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctcagacgttcggtggaggcaccaagctggaaatcaaac(SEQIDNO.:19)。
in a preferred embodiment of the invention, the light chain of the antibody comprises the light chain variable region and a light chain constant region, which may be murine or human.
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 HPV18E7 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.: 20). They may or may not contain the initial methionine.
In another preferred embodiment, the antibody is a murine or human murine chimeric monoclonal antibody directed against the HPV18E7 protein, and its heavy chain constant region and/or light chain constant region may be humanized heavy chain constant region or light chain constant region. 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 HPV18E7 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 coding region sequence encoding the mature polypeptide may be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO. 3, 5, 7, 9, 13, 15, 17, 19. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences that encode a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but which differ from the coding region sequence 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 polypeptide coded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide 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 host cells with recombinant DNA is well known to those skilled in the artAccording to conventional techniques. 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 (cancermetastassisreviews) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, cancer immunology and immunotherapy (cancer immunology) 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 immunologyand immunotherapy)53, 345; Halin et al, 2003, cancer research (cancer research)63, 3202); 4. gold nanoparticles/nanorods (Lapotko et al, 2005, cancer communications (cancerlettes) 239, 36; Huang et al, 2006, journal of the american chemical society 128, 2115); 5. viral particles (Peng et al, 2004, Gene therapy (Genotherapy) 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.
Hybridoma cell strain
The invention also provides a hybridoma cell strain capable of producing the monoclonal antibody against the HPVE7 protein; preferably, the invention provides a hybridoma cell strain with high titer aiming at the HPV18E7 protein monoclonal antibody.
After obtaining the hybridoma producing the monoclonal antibody against the HPVE7 protein of the present invention, those skilled in the art can conveniently prepare the antibody using the hybridoma cell line. In addition, the structure of the antibody of the present invention (e.g., the heavy chain variable region and the light chain variable region of the antibody) can be easily known by those skilled in the art, and then the monoclonal antibody of the present invention can be prepared by recombinant methods.
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, Inmonclonal antibodies and cell hybrids, Elsevier, N.Y.,1981) 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 institutional cell distribution center, san diego, california, usa), and SP-2, NZ0, or X63-Ag8-653 cells (available from american typeculture, 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 (PrinciplsasandPractice), 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 invention provides a monoclonal antibody aiming at an HPVE7 protein, in particular to a monoclonal antibody aiming at an HPV16E7 protein. In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. Taking supernatant fluid of hybridoma cell culture, carrying out saturated ammonium sulfate precipitation to obtain IgG, and purifying the antibody obtained by crude extraction through an affinity chromatography column (protein G-Sepharose).
In a preferred embodiment of the invention, the monoclonal antibody is prepared by a method for producing the monoclonal antibody by Balb/C mouse ascites. The hybridoma cells were inoculated into the abdominal cavity of the sensitized mice, and significant abdominal distension was observed within 2-4 weeks. Ascites is extracted, and after crude extraction by a saturated ammonium sulfate precipitation method, the crude antibody is purified by an affinity chromatography column (protein G-Sepharose).
Labeled immunoglobulins
In a preferred embodiment of the invention, the immunoglobulin is provided with a detectable label. More preferably, the marker is selected from the group consisting of: a colloidal gold label, a colored label, or a fluorescent label.
The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the monoclonal antibody of HPV18E7 protein is labeled with colloidal gold to obtain a colloidal gold-labeled monoclonal antibody.
The HPV18E7 protein monoclonal antibody has good specificity and high titer.
Test plate and material therefor
The detection plate can be made of detection plate materials commonly used in the field by adopting a conventional detection plate preparation method.
The immunoassay plate for detecting HPV18E7 protein of the invention comprises a test strip and a support plate for supporting the test strip, for example, a PVC polyester rubber plate and the like can be adopted; the test strip is formed by sequentially overlapping sample filtering paper, chromatographic materials, a nitrocellulose membrane and absorbent paper, and the overlapped part can be fixedly connected by adopting a conventional method, such as an adhesive tape and the like; wherein: the chromatographic material is pre-coated with a monoclonal antibody or polyclonal antibody of HPVE7 protein marked by colloidal gold or colored marks, preferably the monoclonal antibody of HPVE7 protein marked by colloidal gold, and an adsorption detection line and a quality control line are arranged on a nitrocellulose membrane;
in a preferred embodiment: the HPVE7 protein monoclonal antibody pre-coated with the colloidal gold label on the chromatography material is pre-coated by adopting a HPVE7 protein monoclonal antibody solution with the colloidal gold label concentration of 0.5-1.5mg/ml, and the coating amount is 50 mul/cm2(ii) a The preferred concentration is 0.5 or 1.5mg/ml, 50. mu.l/cm2；
Detection method and result judgment
And (3) flatly placing the detection plate, dropping the sample on the filter sample paper, and observing the chromatographic result within 3-5 min after the sample is about 120 mu l. And judging the result according to the position of the appearing stripe.
Negative: the quality control area and the detection area both have obvious colored bands and are shown as negative;
positive: a clear color band appears only in the quality control area, and no color band appears in the detection area, which is shown as positive;
and (4) invalidation: the quality control area and the detection area have no color band or no color band appears in the quality control area and a color band appears in the detection area, which indicates that the detection method is wrong or the detection plate is deteriorated or invalid, and the detection plate is required to be replaced for detection.
Method and sample
The present invention relates to methods for detecting cervical cancer in cytolytic samples of cells and/or tissue. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of HPV oncoprotein in the lysed 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. Without support based on cellular information, diagnostic methods for cancer are limited to cases only at the protein molecule level, where the marker or the level of the marker should be specific for the situation to be detected. The biomarkers employed in the present invention are HPV oncoproteins, which are derived from viruses, and the detection of HPV infection can be accomplished in a sample solution because the nature of the marker 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 Tripathimaging "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.
In addition, samples that are subjected to cell lysis immediately after they are obtained can be used in the methods disclosed herein. The samples are lysed immediately after they are obtained, during which morphological information is lost and the protein molecular information of the samples is preserved. The sample may be transferred directly from the body of the individual into a solution containing suitable detergents and preservatives. Using appropriate reagents in the lysis medium, the molecular components of the starting material can be preserved and do not degrade. E.g., by using enzyme inhibitors but enzyme activity degradation is minimized. Thus, a solution of the test sample in the lysis medium may, upon dissolution, exhibit the protein molecular properties of the test sample.
According to the present invention, the sample may be dissolved in any suitable lysis medium. The lysis medium may be, for example, urea, formamide, aqueous solutions of detergents, for example anionic detergents (such as SDS, N-dodecanol creatine sodium, deoxycholate sodium, alkylaryl sulphonates, long-chain (aliphatic) alcohol sulphates, olefin sulphates and sulphonates, alpha-olefin sulphates and sulphonates, sulphate glycerol-acid esters, sulphate ethers, sulphosuccinates, alkane sulphonates, phosphate esters, alkyl isothionates, sucrose esters), cationic detergents (such as cetyltrimethylammonium chloride), non-ionic detergents (such as Tween 20, NonidetP-40, TritonX-100, NP-40, lgepalCA-630, N-octyl-glycoside) or amphoteric detergents (such as CHAPS, 3-dodecyl-dimethylamine-propane-1-sulphonic acid, dodecanol dimethylamine oxide) and/or amphoteric detergents (such as CHAPS, 3-dodecyl-dimethylamine-propane-1-sulphonic acid, dodecanol dimethylamine oxide), such as sodium hydroxide or potassium hydroxide. Generally any suitable liquid may be used as a solvent for the lysis medium of the present invention. The liquid may be organic or inorganic and may be a pure liquid, a liquid mixture or a liquid containing a solution of the substance and may contain other substances to enhance the properties of the solvent. In an embodiment of the invention, the formulation of the lysis medium is 50mM Tris-HCl,150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS.
The lysis medium for dissolving a sample according to the present invention may further contain one or more agents that prevent degradation of components in the starting material. The component includes, for example, an enzyme inhibitor, such as a protease inhibitor. In an embodiment of the invention, the sample is lysed directly. Protease inhibitors may include, for example, serine protease inhibitors, cysteine protease inhibitors, aspartic protease inhibitors, metalloprotease inhibitors, acid protease inhibitors, alkaline protease inhibitors, or neutral protease inhibitors. In the embodiment of the present invention, Pepstatin, Leupeptin, (aprotinin) and 100. mu.g/ml PMSF (phenylmethylsulfonyl fluoride) are used as protease inhibitors.
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 present invention further designs a detection kit for detecting the level of HPV oncoprotein, which comprises an antibody recognizing HPV oncoprotein, a lysis medium for solubilizing a sample, general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The antibody is preferably an anti-HPV 7 antibody, more preferably an anti-HPV16E7 antibody. The test kit may be an in vitro diagnostic device.
The present invention further contemplates the development of a kit for diagnostic assessment of conditions associated with HPV infection from a sample solution, which can detect HPV oncoproteins present in the sample solution, wherein the cell preservation fluid used to preserve the sample can be, for example, a cell preservation fluid used in a liquid-based cytology procedure. The cells are lysed in a suitable lysis medium and used to develop detection kits and in-vitro diagnostic devices for the detection of HPV infection-associated tumors from lysed samples on a non-cellular assay basis.
One of the purposes of the invention is to provide a method for detecting the expression of HPV18E7 protein, and the method can be used for detecting HPV infection-related cancers, particularly cervical cancer.
The inventor of the invention prepares a monoclonal antibody MAB-F2 aiming at human papilloma virus HPV18E7 protein and researches the reactivity of the monoclonal antibody, wherein the antigen binding epitope peptide of the monoclonal antibody is between amino acid 44 and 58, and the antigen binding epitope peptide comprises an amino acid sequence VNHQHLPARRAEPQR. When the monoclonal antibody MAB-F2 against HPV18E7 was used to perform immunocytochemical staining on a cervical cancer cell line C-33A which does not express HPV, a human cervical cancer cell line CaSki which expresses HPV16E7, and a human cervical cancer cell line Hela which expresses HPV18E7, the results were found to be: MAB-F2 showed strong staining reaction with HPV18E7 positive cell Hela, and reaction with HPV16E7 positive cell CaSki and C-33A cell not expressing HPV protein.
Furthermore, the present inventors used the prepared anti-HPV18E7 monoclonal antibody MAB-F2 to perform immunocytochemical staining of a cervical cancer cell line fixed with 4% paraformaldehyde or LBC fixative for several days. The expression of HPV18E7 was not detected in human cervical cancer cell line C-33A without HPVDNA, and when the proportion of positive tumor cells was as low as 10% after Hela cells and C-33A cells were mixed in a ratio (1:9), the presence of positive tumor cells was also specifically detected by detecting HPV18E7 inside tumor cells, MAB-F2, and both of the fixatives had no effect on the determination of positive and negative results. 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 HPV18E7 in 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 cell is preferably a cervical exfoliated cell.
In the method of the present invention, the sample is preferably a patient who may suffer from cervical lesions, or a patient whose cervix has already suffered from lesions.
Preferably, the HPV18E7 is an HPV18E7 protein or fragment thereof. In this case, the step of detecting HPV18E7 is preferably an immunocytochemical staining assay using HPV18E 7. The anti-HPV18E7 antibody used is preferably an anti-HPV18E7 monoclonal antibody.
The protein expressed by the oncogene from HPV18E7 used in the immunodetection of the method serves as a reliable indicator of HPV 18-associated malignancy or premalignancy. One of the most useful aspects of the invention is its use in the diagnosis of cervical cancer, squamous epithelial cells and adenocarcinomas and any epithelial abnormalities associated with oncogenic HPV18 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 HPV18E7 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-HPV18E7 monoclonal antibody. The kit of the present invention preferably further comprises general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, and the like. The antibody is preferably an anti-HPV18E7 antibody, more preferably an anti-HPV18E7 monoclonal antibody, and particularly preferably a mouse anti-HPV18E7 monoclonal antibody produced by hybridoma MAB-F2 or a monoclonal antibody having binding activity equivalent to that of the mouse anti-HPV18E7 monoclonal antibody.
The invention provides a method for distinguishing HPV18E7 infected tumor cells from tumor cells without HPV DNA (deoxyribonucleic acid) by detecting HPV18E7 protein endogenous to the cells. The method is based on that when HPV18E7 positive tumor cells account for 10% of the total number of cells, the positive cells can be still accurately detected, and when the cells are fixed in LBC fixing solution which is widely used clinically, the positive cells can be still accurately detected, 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 HPV18E7 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 HPV18E7 protein can be specifically combined with the HPV18E7 protein, but not combined with the HPV16E7 protein, so that the antibody not only can be used for detecting the HPV E7 protein, but also can be used for identifying different HPV types.
(3) The antibody provided by the invention is used in the method for detecting the HPVE7 protein, the stability is good, the detection sensitivity is extremely high, and the binding affinity with the HPV18E7 protein reaches 2.67 nM.
(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: ColdSpringHarbor laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1
1. Preparation of human papilloma virus HPV18E7 monoclonal antibody
1.1 animal immunization
Female BALB/C mice at 6-8 weeks were selected and the immunogen was GST-HPV18E7 protein, and the immunization program is shown in Table 1. The tail breaking blood collection of the mice is carried out before each immunization, and Hi is adoptedsHPV18E7 as detection antigen for detecting mouse serum titer by indirect ELISA method. And taking splenocytes to fuse when the serum titer of the immunized mice reaches the maximum value and does not rise any more.
Table 1 mouse immunization procedure
1.2 cell fusion and culture
Spleen cells of immunized mice were collected by a conventional method and fused with myeloma cells SP2/0 at a ratio of: SP2/0 is 5:1, the fused cells are subpackaged in 96-well culture plates and selectively cultured in a constant temperature incubator at 37 ℃ and 5% CO 2. After fusion, the liquid is completely changed by HAT culture medium for 3 times; ELISA detection was performed when the hybridoma cells grew over a microscope field.
1.3 screening of Positive hybridomas
During detection, indirect ELISA screening is adopted: the antigen is His-HPV18E7 fusion protein, 2 mu g/ml plate wrapping is adopted, the temperature is kept overnight at 4 ℃, PBST is washed and dried, 5% of skimmed milk powder is added for sealing, the temperature is kept at 37 ℃ for 2h or 4 ℃ for overnight, after PBST is washed and dried, hybridoma supernatant is added, positive (P), negative (N) and blank control (enzyme-labeled secondary antibody is directly added in blank), the reaction is carried out for 1h at 37 ℃, after PBST is washed and dried, goat anti-mouse-HRP secondary antibody (SigmaA2554) (1:10000) is added for reaction for 45-60 min at 37 ℃, after PBST is washed and dried, TMB color development and 2MH are carried out2SO4And (6) terminating.
And (4) taking the positive hole with the OD value larger than 0.6 as the ELISA screening result, and rechecking. After two times of continuous detection, the positive hole cells are amplified, and are frozen and subcloned in time.
1.4 establishment of hybridoma cell lines
For the wells which were positive in all of the 2 screenings, subcloning was performed by limiting dilution, a 96-well cell culture plate was taken, and 150. mu.l HAT medium was added to each well; the positive wells to be subcloned were gently pipetted into suspension, and 100. mu.l of cell suspension was pipetted into a 96-well cell plate and diluted in multiple ratios from the first well. Counting cells, selecting wells with about 100 cells, adding the wells into a sample adding groove containing 6ml HAT culture solution, adding the wells into a cell plate with a feeder layer according to 100 mu l/well, and adding the first three columns; then 3ml HAT culture medium is added, 100 mul/hole is added into a cell plate paved with a feeder layer, and three rows are added; then 5ml HAT medium was added, 100. mu.l/well was added to the feeder cells, followed by six columns. When the positive rate of each plate reaches 100%, a stable cell strain can be obtained. Transfer to cell culture plates, expand culture and bulk cryopreservation.
And screening to obtain a positive hybridoma cell strain MAB-F2.
1.5 Mass production of monoclonal antibodies (ascites production)
Culturing hybridoma cells (MAB-F2), immunizing BALB/C female mouse with liquid paraffin in advance at 8-10 weeks, and performing intraperitoneal injection with 1X106And (4) after about 10 days of hybridoma cells per mouse and the abdomen of the mouse obviously expands to a certain degree, extracting ascites by using a 9-gauge needle. The harvested ascites fluid was coated with His-HPV18E7 and ascites titer reached 1: more than 10,000. The ascites fluid is purified by protein affinity column chromatography after being processed by fibrin removal and salting out. Collecting protein peak effluent, dialyzing with Phosphate Buffer Solution (PBS), measuring antibody protein concentration with ultraviolet spectrophotometer OD260,280 to be 0.7-1.5mg/ml, indirect ELISA detection result shows: the titer of the purified monoclonal antibody is more than 1: 10000.
2. Identification of monoclonal antibodies
2.1 identification of monoclonal antibody Ig subclasses:
the purified monoclonal antibody is diluted by PBS1:10000 and is operated according to the specification of a subtype identification kit of Sigma company, and the monoclonal antibody MAB-F2 is IgG 1.
2.2ELISA detection of anti-HPV18E7 monoclonal antibody specificity and cross-reactivity:
antigen respectively selects His-HPV18E7(HPV18E7 amino acid sequence SEQ ID NO.:2MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ) and His-HPV16E7(HPV16E7 amino acid sequence SEQ ID NO.:1HGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP) fusion protein, 2 mu g/ml wrapping plate, 4 ℃ overnight, PBST is washed and dried, 5% skimmed milk powder is added for sealing, 37 ℃ is acted for 2h or 4 ℃ overnight, PBST is washed and dried, anti-HPV18E7 monoclonal antibody MAB-F2(1 mu g/ml) is added, 37 ℃ is acted for 1h, PBST is washed and dried, goat anti-mouse-HRP secondary antibody (SigmaA2554) (1:10000) is added for reaction at 37 ℃ for 45-60 min, after PBST is washed and dried, TMB is developed and MH 2MH is used2SO4End, OD450 nm. The results are shown in FIG. 1: MAB-F2 was only able to specifically bind HPV18E7 recombinant protein and not HPV16E 7.
2.3 antigen binding epitope analysis of Anti-HPV18E7 monoclonal antibody
Identifying the epitope amino acid sequence region of the monoclonal antibody in the HPV18E7 antigen protein. And (3) identifying by adopting an ELISA method: the antigen is selected from polypeptide or recombinant protein, wherein the polypeptide is HPV18E7-1 (amino acid sequence SEQ ID NO.:11SDSEEENDEIDGVNHQHLPARRAEPQRH), HPV18E7-2 (amino acid sequence SEQ ID NO.:12IDGVNHQHLPARRAEPQR), the recombinant protein is His-HPV18E7 and His-HPV16E7, the protein antigen is coated on a plate with the concentration of 4 mu g/ml, the polypeptide antigen is coated on a plate with the concentration of 2 mu g/ml, the temperature is 4 ℃ overnight, after PBST is washed and patted dry, 5% skimmed milk powder is added for sealing, the temperature is 37 ℃ for acting for 2h or 4 ℃ overnight, after PBST is washed and patted dry, anti-HPV18E7 monoclonal antibody MAB-F2(1 mu g/ml) is added, the temperature is 37 ℃ for reacting for 1h, after PBST is washed and patted dry, a goat anti-mouse-HRP secondary antibody (SigmaA2554) (1:10000) is added for reacting at 37 ℃ for 45-60 MH min, after PBST is2SO4End, OD450 nm. The results are shown in FIG. 2: since MAB-F2 can bind to the polypeptides HPV18E7-1 and HPV18E7-2, the binding epitope of MAB-F2 and HPV18E7 is preliminarily presumed to be amino acids 41-55, while the binding amino acid sequence of MAB-F2 and HPV18E7 is located between VNHQHLPARRAEPQR, namely amino acids 44-58 of HPV18E7, since MAB-F2 can only specifically bind to HPV18E7 recombinant protein and not to HPV16E7 recombinant protein, and the amino acids 41-43 are present in both recombinant proteins.
2.4Anti-HPV18E7 monoclonal antibody protein binding affinity assay
Monoclonal antibody protein binding affinity constant assays were performed according to the method established by Beatty et al (j. davidbeatty, barbarbarabag. beattyandwilliamg. vlahos 1987). His-HPV18E7 protein is coated with the concentrations of 8. mu.g/ml, 2. mu.g/ml, 0.5. mu.g/ml and 0.125. mu.g/ml, the concentration ratio of the four is 1:4:16:64, primary antibody MAB-F2 antibody is added, the concentration is diluted from 8. mu.g/ml in a double ratio, and seven gradients are used for carrying out ELISA detection. Substituting the obtained value into formula Kaff＝(n-1)/2(n[Ab′]-[Ab]) Calculation of affinity constant, [ Ab ] in the formula]And [ Ab']The antibody concentrations that give half the absorbance when the antigen concentrations were Ag and Ag', respectively, are plotted at half the maximum OD (i.e., OD 50%) of the curve. Wherein n is Ag/Ag'. Then, two-by-two comparison is carried out, when n is 4, 3K values can be obtained, when n is 16, 2K values can be obtained, when n is 64, 1K value can be obtained, and the average of 6K values is obtained as the final result. The results are shown in FIG. 3: the binding affinity of MAB-F2 to His-HPV18E7 recombinant protein was calculated to be 2.67 nM.
2.5IP-WB detection of monoclonal antibody binding to tumor cell endogenous HPV18E 7:
take 5x1050.5ml of cell lysate containing 1% Triton X-100,1mM EDTA, 50mM NaCl, 0.1(m/v) SDS, 1% Sodiumdeoxyholate, 1mM PMSF, 2. mu.g/ml aprotinin, 1. mu.g/ml Leupitptin and 1. mu.g/ml pepstatin was added to the HPV18E7 positive cervical cancer cell line Hela cells, wherein all protease inhibitors were added just before use. After the cells are placed on ice for cracking for 30min, the cells are centrifuged at 12500rpm and 4 ℃ for 10min, and the supernatant is collected, namely the target protein extract.
Mu.g of monoclonal antibody MAB-F2 or mIgG was added to the cell lysis supernatant and incubated at 4 ℃ for two hours, followed by 15. mu.l of proteina A/Gboads 4 ℃ overnight. On the next day, after washing the beads twice with the cell lysate containing no protease inhibitor, 1xSDS loading buffer was added for immunoblotting detection, and the membrane was transferred for 35min with NC membrane at 100V. After 5% skim milk powder is blocked for 2h, MAB-F2 or mIgG protein diluted by 5% skim milk powder is added, the antibody is used at the concentration of 2 mu g/ml, the mixture is incubated overnight at 4 ℃, after being washed for 4 times by TBS containing 0.1% Tween, Anti-MouseIgG (Fcspecfic) -peroxiaseentaody (SigmaA2554) (1:1000) is added, the mixture is incubated for 1h at room temperature, DAB (Wuhan Boussd SA2024) is added for color development, and the result is shown in figure 4, and at the molecular weight indicated by an arrow, the MAB-F2 antibody specifically recognizes HPV16E7 protein endogenous to Hela cells.
2.6 immunocytochemical staining to detect expression of HPV18E7 in cervical cancer cell lines:
the immunocytochemical staining test of the cervical cancer cell line Hela cell expressing HPV18E7 protein, the cervical cancer cell line CaSki cell expressing HPV16E7 protein and the cervical cancer cell line C-33A cell not containing HPVDNA was carried out with monoclonal antibody MAB-F2. The specific experimental method is as follows:
CaSki, Hela, C-33A cells were plated on poly-L-Lysine treated coverslips at 37 ℃ with 5% CO2Culturing for 24h, taking out the cover glass, and fixing for 30min at room temperature by using 4% paraformaldehyde fixing solution; adding TBS lotion for washing for 5min, and spin-drying; adding TBS buffer solution containing 0.3% TritonX-100, standing at room temperature for 15min to allow cell membrane permeation; adding TBS lotion for washing for 5min, and spin-drying; addition of 1% H for inactivation of endogenous peroxidase2O2The TBS buffer solution is treated for 5min at room temperature and is dried; adding TBS lotion for washing for 5min, and spin-drying; adding 10% FBS/TBS blocking solution, sealing overnight, and spin-drying; adding mAbanti-HPV18E7MAB-F2(5 mu g/ml) and incubating at 37 ℃ for 2 h; adding TBS/0.1% Tween washing solution, washing for 5 times, each time for 5min, and spin-drying; adding Anti-MouseIgG (Fcspecific) -peroxiaseentaody (SigmaA2554) (1:1000) secondary antibody, and incubating at 37 ℃ for 1 h; adding TBS/0.1% Tween washing solution, washing for 5 times, each time for 5min, and spin-drying; adding DAB color development solution (ZLI-9017, China fir Jinqiao, Beijing), reacting at room temperature for 10min, and washing with distilled water to terminate the reaction. The results were observed under a microscope and recorded.
The results are shown in FIG. 5, and microscopic examination shows: the HPV18E7 monoclonal antibody MAB-F2 has strong immunochemical staining reaction only with a cervical cancer cell strain Hela expressing HPV18E7 protein, but has no staining reaction with CaSki cells expressing HPV16E7 and a cervical cancer cell strain C-33A not expressing HPV protein. Consistent with the previous ELISA detection result, MAB-F2 has strong binding ability with the HPV18E7 endogenous to the cell, namely can specifically recognize the HPV18E7 protein endogenous to the cell.
Example 2
Immunocytochemical staining detects the specificity of HPV18E7 monoclonal antibody MAB-F2 in suspension-immobilized cervical cancer cell lines:
the cervical cancer cell strain Hela cells expressing HPV18E7 protein and the cervical cancer cell strain C-33A cells without HPVDNA are fixed by different cell fixing solutions and then are subjected to immunocytochemical staining test by using a monoclonal antibody MAB-F2. The specific experimental method is as follows:
collecting C-33A cells and Hela cells, fixing with 4% paraformaldehyde or LBC fixing solution for several days, and collecting 50000 cells, wherein the cervical cancer Hela cells and negative control C-33A cells are mixed at a certain ratio, and the ratio of Hela cells to C-33A cells is 1:9,1:19,1:49,1:99,1:499 and 1: 999. Mixing, placing on a poly-L-Lysine treated cover glass, and air-drying at room temperature to fix cells on the cover glass; adding TBS lotion for washing for 5min, and spin-drying; adding TBS buffer solution containing 0.3% TritonX-100, standing at room temperature for 15min to allow cell membrane permeation; adding TBS lotion for washing for 5min, and spin-drying; addition of 1% H for inactivation of endogenous peroxidase2O2The TBS buffer solution is treated for 5min at room temperature and is dried; adding TBS lotion for washing for 5min, and spin-drying; adding 10% FBS/TBS blocking solution, sealing overnight, and spin-drying; adding mAbanti-HPV16E7MAb-F2(2 mu g/ml) and incubating at 37 ℃ for 2 h; adding TBS/0.1% Tween washing solution, washing for 5 times, each time for 5min, and spin-drying; adding Anti-MouseIgG (Fcspecific) -peroxiaseentaody (SigmaA2554) (1:1000) secondary antibody, and incubating at 37 ℃ for 1 h; adding TBS/0.1% Tween 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 for 10min, and washing with distilled water to terminate the reaction; adding hematoxylin staining solution (Biyuntian C0107) for staining for 2min, soaking in tap water to wash off excessive staining solution for about 10min, washing with distilled water for another time (several seconds), observing and recording under microscope.
Microscopic examination shows that: the HPV18E7 specific monoclonal antibody MAB-F2 has strong immunochemical staining reaction only with the cervical cancer cell strain Hela expressing HPV18E7 protein, and has no immunological staining reaction with the cervical cancer cell strain C-33A without HPVDNA. When HeLa cells are mixed with C-33A cells at the ratio of 1:9, MAB-F2 can specifically bind to HeLa cells but not to C-33A cells, namely MAB-F2 can still accurately distinguish negative cells from positive cells when positive tumor cells account for 10% of the total cells. In addition, the cell immobilization by using 4% paraformaldehyde or LBC stationary liquid has no influence on the judgment of negative and positive staining in the ICC detection.
Therefore, the result shows that after the tumor cells are fixed in advance by using 4% paraformaldehyde or LBC fixing solution for several days, the HPV18E7 monoclonal antibody MAB-F2 can still specifically detect the HPV18E7 protein in the tumor cells, namely, the chemical staining result of the immunocytes is not affected by the fixing solution for several days, such as the staining reduction or the background enhancement. In addition, when the proportion of HPV18E7 cells is as low as 10%, the MAB-F2 antibody can still accurately detect target cells, and the detection method is proved to have good specificity and sensitivity. Because the wide LBC detection technology is currently used clinically, the residual cell sample can be provided for other HPV infection analysis, and in the embodiment, the HPV18E 7-related cervical cancer cells can be accurately detected after the LBC is fixed for several days, so the method can be further developed and used for detecting the related malignant tumor caused by clinical HPV18E7 persistent infection.
Discussion:
the inventor adopts the method to prepare the HPV18E7 monoclonal antibody, screens more than 1000 strains of cells, screens 8 strains of cells with better specificity and no cross reaction with HPV16E7 protein, turns 5 strains to negative after subcloning, and still turns 3 strains to positive. Finally, only the monoclonal antibody MAB-F2 can be used for IP-WB detection and immunocytochemistry detection, and has great value in clinical detection application.
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 and the heavy chain constant region of claim 1.
3. An antibody light chain variable region having Complementarity Determining Regions (CDRs) selected from the group consisting of:
CDR1' shown in SEQ ID NO. 14,
CDR2' shown in SEQ ID NO. 16, and
18, CDR3' shown in SEQ ID NO;
preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 20.
4. An antibody light chain comprising the light chain variable region and the light chain constant region of claim 3.
5. An antibody, wherein said antibody has: the heavy chain variable region of claim 1; and/or the light chain variable region of claim 3;
alternatively, 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 HPV18E7 protein in the sample; and/or
(2) Detecting endogenous HPV18E7 protein in the tumor cells; and/or
(3) Detecting tumor cells expressing HPV18E7 protein; and/or
(4) Identifying the type of HPV;
the medicament is used for treating or preventing tumors expressing HPV18E7 protein.
13. A method of detecting HPVE7 protein in a sample, the method 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 HPVE7 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.
CN201410508253.XA 2014-09-28 2014-09-28 Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof Active CN105524166B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410508253.XA CN105524166B (en) 2014-09-28 2014-09-28 Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410508253.XA CN105524166B (en) 2014-09-28 2014-09-28 Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof

Publications (2)

Publication Number Publication Date
CN105524166A true CN105524166A (en) 2016-04-27
CN105524166B CN105524166B (en) 2020-07-10

Family

ID=55766721

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410508253.XA Active CN105524166B (en) 2014-09-28 2014-09-28 Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof

Country Status (1)

Country Link
CN (1) CN105524166B (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107556379A (en) * 2016-07-01 2018-01-09 艾托金生物医药（苏州）有限公司 Identify monoclonal antibody and its application of high-risk HPV E7 albumen
CN107868127A (en) * 2016-09-28 2018-04-03 艾托金生物医药（苏州）有限公司 A kind of monoclonal antibody for being used to detect Oncoprotein Expression in histopathologic slide
CN107987167A (en) * 2017-12-15 2018-05-04 艾比玛特医药科技（上海）有限公司 A kind of monoclonal antibody of rna plymerase ii transcription subunit 37 e mediators and its application
CN108218983A (en) * 2018-03-01 2018-06-29 珠海美华医疗科技有限公司 Monoclonal antibody, Test paper and the detection kit of anti-HPV18 types E7 albumen
CN108276491A (en) * 2018-02-05 2018-07-13 源道隆（苏州）医学科技有限公司 monoclonal antibody capable of specifically recognizing HPV 18L 1 protein and application thereof
CN109021097A (en) * 2017-06-08 2018-12-18 艾托金生物医药（苏州）有限公司 A kind of monoclonal antibody and its application identifying HPV18 and/or HPV45
CN109575130A (en) * 2018-12-03 2019-04-05 艾托金生物医药（苏州）有限公司 A kind of monoclonal antibody and its preparation and application detecting HPV18 E7 albumen
CN114605552A (en) * 2022-03-09 2022-06-10 上海交通大学医学院 PRL2 antibody, and preparation method and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103254307A (en) * 2012-02-16 2013-08-21 艾托金生物医药（苏州）有限公司 HPV18E7 monoclonal antibody, hybridoma cell strain and application
CN103740741A (en) * 2014-01-22 2014-04-23 北京工业大学 HPV18 E and E7 fusion gene mutant as well as related biological material and coding protein thereof
CN103864936A (en) * 2012-12-11 2014-06-18 中国疾病预防控制中心病毒病预防控制所 HPV18 type L2NE7E6 fusion protein gene, and expression vector, preparation method, strain and use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103254307A (en) * 2012-02-16 2013-08-21 艾托金生物医药（苏州）有限公司 HPV18E7 monoclonal antibody, hybridoma cell strain and application
CN103864936A (en) * 2012-12-11 2014-06-18 中国疾病预防控制中心病毒病预防控制所 HPV18 type L2NE7E6 fusion protein gene, and expression vector, preparation method, strain and use thereof
CN103740741A (en) * 2014-01-22 2014-04-23 北京工业大学 HPV18 E and E7 fusion gene mutant as well as related biological material and coding protein thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
唐艳: "HPV18型E7抗原B细胞表位的预测与鉴定", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107556379A (en) * 2016-07-01 2018-01-09 艾托金生物医药（苏州）有限公司 Identify monoclonal antibody and its application of high-risk HPV E7 albumen
CN107556379B (en) * 2016-07-01 2021-05-28 艾托金生物医药（苏州）有限公司 Monoclonal antibody for identifying high-risk HPV E7 protein and application thereof
CN107868127A (en) * 2016-09-28 2018-04-03 艾托金生物医药（苏州）有限公司 A kind of monoclonal antibody for being used to detect Oncoprotein Expression in histopathologic slide
CN109021097A (en) * 2017-06-08 2018-12-18 艾托金生物医药（苏州）有限公司 A kind of monoclonal antibody and its application identifying HPV18 and/or HPV45
CN107987167A (en) * 2017-12-15 2018-05-04 艾比玛特医药科技（上海）有限公司 A kind of monoclonal antibody of rna plymerase ii transcription subunit 37 e mediators and its application
CN107987167B (en) * 2017-12-15 2021-07-06 艾比玛特医药科技（上海）有限公司 Monoclonal antibody of RNA polymerase II transcription subunit 37e mediator and application thereof
CN108276491A (en) * 2018-02-05 2018-07-13 源道隆（苏州）医学科技有限公司 monoclonal antibody capable of specifically recognizing HPV 18L 1 protein and application thereof
CN108276491B (en) * 2018-02-05 2021-01-12 南京薇熙生物医药科技有限公司 Monoclonal antibody capable of specifically recognizing HPV18L1 protein and application thereof
CN108218983A (en) * 2018-03-01 2018-06-29 珠海美华医疗科技有限公司 Monoclonal antibody, Test paper and the detection kit of anti-HPV18 types E7 albumen
CN109575130A (en) * 2018-12-03 2019-04-05 艾托金生物医药（苏州）有限公司 A kind of monoclonal antibody and its preparation and application detecting HPV18 E7 albumen
CN114605552A (en) * 2022-03-09 2022-06-10 上海交通大学医学院 PRL2 antibody, and preparation method and application thereof
CN114605552B (en) * 2022-03-09 2023-12-01 上海交通大学医学院 Antibody for diagnosing inflammatory bone diseases and preparation method thereof

Also Published As

Publication number Publication date
CN105524166B (en) 2020-07-10

Similar Documents

Publication Publication Date Title
CN105504049B (en) The relevant HPV E7 protein monoclonal antibody of cervical carcinoma and its application
CN105524166B (en) Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof
CN105131113B (en) For cervical carcinoma detection and the grand antibody of list being classified and its application
CN105837689B (en) anti-CD19 monoclonal antibody and preparation method thereof
KR20130108069A (en) Monoclonal antibodies against her2 antigens, and uses therefor
CN109081868B (en) Monoclonal antibody targeting Zika virus envelope protein conserved epitope and application thereof
WO2016173558A1 (en) Preparation and use of anti-norovirus gii.4 type murine monoclonal antibody
CN106366186B (en) Monoclonal antibody for identifying HPV16 positive tumor cells and application thereof
WO2018059117A1 (en) Monoclonal antibody for detecting oncoprotein expression in pathologic tissue section
CN107556379B (en) Monoclonal antibody for identifying high-risk HPV E7 protein and application thereof
KR20080077279A (en) Mcm6 and mcm7 monoclonal antibodies and methods for their use in the detection of cervical disease
CN108727488B (en) Preparation and application of anti-norovirus GII.17 monoclonal antibody
CN105866420A (en) Method and apparatus for detecting immunogen
CN109575130B (en) Monoclonal antibody for detecting HPV18E7 protein and preparation and application thereof
CN109593131B (en) Monoclonal antibody for resisting 14-3-3 eta protein and application thereof
EP4293044A1 (en) Anti-trpv6 monoclonal antibody and application thereof
CN109021097A (en) A kind of monoclonal antibody and its application identifying HPV18 and/or HPV45
CN109957014B (en) Preparation and application of anti-norovirus GII.3 murine monoclonal antibody
CN109897109B (en) Monoclonal antibody of antitumor marker protein and application thereof
CN114685670A (en) CLDN18.2 antibodies and uses thereof
CN116410305A (en) Monoclonal antibody for detecting HPV 16E 6 protein in cervical cancer pathological tissue
CN116410321A (en) Monoclonal antibody for detecting endogenous protein HPV 16E 6 in cervical cancer cells, and preparation and application thereof
CN116410306A (en) Monoclonal antibody for detecting cervical cancer protein HPV E6 and application thereof
CN113321730B (en) CLDN18.2 antibodies and uses thereof
CN111333720B (en) anti-HPV 16E7 protein monoclonal antibody 79A11, hybridoma cell strain, and preparation method and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
PE01 Entry into force of the registration of the contract for pledge of patent right
PE01 Entry into force of the registration of the contract for pledge of patent right

Denomination of invention: Monoclonal antibody for recognizing HPV18 positive cervical epithelial cancer cells and its application

Effective date of registration: 20211020

Granted publication date: 20200710

Pledgee: Jiangsu Suzhou Rural Commercial Bank Co.,Ltd. science and Technology Financial Industrial Park sub branch

Pledgor: ATTOGEN BIOMEDICAL (SUZHOU) Inc.,Ltd.

Registration number: Y2021320010426

PC01 Cancellation of the registration of the contract for pledge of patent right
PC01 Cancellation of the registration of the contract for pledge of patent right

Granted publication date: 20200710

Pledgee: Jiangsu Suzhou Rural Commercial Bank Co.,Ltd. science and Technology Financial Industrial Park sub branch

Pledgor: ATTOGEN BIOMEDICAL (SUZHOU) Inc.,Ltd.

Registration number: Y2021320010426

PE01 Entry into force of the registration of the contract for pledge of patent right
PE01 Entry into force of the registration of the contract for pledge of patent right

Denomination of invention: Monoclonal antibodies for identifying HPV18 positive cervical epithelial cancer cells and their application

Granted publication date: 20200710

Pledgee: Suzhou Beiming Intelligent Manufacturing Co.,Ltd.

Pledgor: ATTOGEN BIOMEDICAL (SUZHOU) Inc.,Ltd.

Registration number: Y2024990000122