Monoclonal antibody for detecting E6 protein in cervical cancer cell nucleus and application thereof

Abstract

The invention provides a monoclonal antibody for detecting E6 protein in cervical cancer cell nucleus and application of the monoclonal antibody. The invention provides the monoclonal antibody which is high in specificity, strong in affinity, stable in expression, good in repeatability and capable of specifically recognizing the HPV 16E6 protein in a cell nucleus, especially the HPV 16E6 protein, and has the potential of being used for preparing medicines for preventing and treating cervical cancer.

Description

Technical Field

[0001] The present invention belongs to the fields of biological diagnosis and medicine. Specifically, the present invention relates to a monoclonal antibody for detecting E6 protein in the nuclei of cervical cancer cells and its application. Background Art

[0002] Early diagnosis of cervical cancer should adopt a "three-step" procedure, namely: cervical cytology examination, colposcopy, and histopathological examination. Cytology examination is the first step of the "three-step" procedure and is a key link in the prevention and screening of cervical cancer. Although cytological screening has achieved great success in reducing the incidence of cervical cancer, a large number of clinical practices have proved that cervical cancer screening faces the problem of relatively low sensitivity (50-70%) of cytological examination, which is affected by various factors such as sampling, slide preparation, staining, and reading levels.

[0003] Based on the expression of cervical cancer-specific protein markers E6 / E7 oncoproteins in cervical cells as a research hotspot in the field of cervical cancer for screening diagnosis and precision treatment, it is also the future direction of precision diagnosis and targeted treatment of cervical cancer. In the application of clinical detection immunochemistry technology, based on the highly specific binding immune reaction of antigen-antibody, through the reaction principle of enzyme-linked secondary antibody (horse-reddish peroxidase conjugated secondary antibody, HRP-secondary antibody), the enzyme reaction substrate is colored to show the expression of cancer marker antigen protein in cancer cells. Therefore, the immunodetection technology based on cancer markers shows advantages in terms of detection mechanism and technology, and can effectively avoid the low sensitivity caused by cytomorphological detection and the false positives and false negatives caused by the subjectivity of pathologists.

[0004] Currently, there are relatively few products on the market for detecting cervical exfoliated cells based on cytological immunohistochemistry technology. One is a detection product using p16, a cancer surrogate marker, as the target. Since it is produced after the carcinogenic effect of E6 / E7 proteins on cells, there is no necessary relationship with the phenomenon of cell carcinogenesis. Moreover, the expression and activity of E6 / E7 proteins not only have a causal relationship with the occurrence and development of cervical cancer, but also their expression levels increase proportionally with the degree of disease deterioration. Therefore, compared with other non-tumor-specific proteins, such as p16, it has more clinical diagnostic significance. In addition, there are already some domestic and foreign products for detecting E7 and E6 proteins in diseased cells, but due to their own cloning defects, they cannot meet the clinical diagnostic criteria.

[0005] Therefore, there is an urgent need in this field to develop a monoclonal antibody and its application that are highly sensitive and specific and can recognize the nuclear E6 protein in cervical cancer cells infected with multiple HPV types. Summary of the Invention

[0006] The present invention provides a monoclonal antibody that is highly sensitive and highly specific and can recognize the nuclear E6 protein in cervical cancer cells infected with multiple HPV types, and its application.

[0007] In the first aspect of the present invention, there is provided a heavy chain variable region of an antibody, and the heavy chain variable region includes the following three complementarity-determining regions CDR:

[0008] HCDR1: NYGVN SEQ ID NO:5;

[0009] HCDR2: WINRGGSASYASWAKG SEQ ID NO:6; and

[0010] HCDR3: YGDGSLSNI SEQ ID NO:7;

[0011] Wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and / or substituted with at least one amino acid and can retain the binding affinity for the HPV16 E6 protein.

[0012] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1.

[0013] In another preferred example, the HPV16 E6 protein is the nuclear HPV16 E6 protein.

[0014] In the second aspect of the present invention, there is provided a heavy chain of an antibody, and the heavy chain has the heavy chain variable region and the heavy chain constant region as described in the first aspect of the present invention.

[0015] In another preferred example, the heavy chain constant region is human, murine, or rabbit-derived.

[0016] In another preferred example, the heavy chain constant region is the human heavy chain IgG1 constant region.

[0017] In the third aspect of the present invention, there is provided a light chain variable region of an antibody, and the light chain variable region includes the following three complementarity-determining regions CDR:

[0018] LCDR1: QASQSLYNQQNLA SEQ ID NO:8;

[0019] LCDR2: DASELAS SEQ ID NO:9; and

[0020] LCDR3: QGEFTCSGGDCIV SEQ ID NO:10;

[0021] Among them, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and / or substituted with at least one amino acid and can retain the binding affinity of the HPV16 E6 protein.

[0022] In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 3.

[0023] In another preferred embodiment, the HPV16 E6 protein is the HPV16 E6 protein in the cell nucleus.

[0024] In the fourth aspect of the present invention, a light chain of an antibody is provided, and the light chain has the light chain variable region and the light chain constant region as described in the third aspect of the present invention.

[0025] In another preferred embodiment, the light chain constant region is human, murine, or rabbit-derived.

[0026] In the fifth aspect of the present invention, an antibody is provided, and the antibody has:

[0027] (1) The heavy chain variable region as described in the first aspect of the present invention; and / or

[0028] (2) The light chain variable region as described in the third aspect of the present invention;

[0029] Alternatively, the antibody has:

[0030] The heavy chain as described in the second aspect of the present invention; and / or the light chain as described in the fourth aspect of the present invention.

[0031] In another preferred embodiment, the antibody is a specifically anti-HPV antibody; preferably, the antibody is a specifically anti-HPV16 antibody; more preferably, the antibody is a specifically anti-HPV16 E6 protein antibody; more preferably, the antibody is a specifically anti-HPV16 E6 protein antibody in the cell nucleus.

[0032] In another preferred embodiment, the antibody includes: single-chain antibody, double-chain antibody, monoclonal antibody, chimeric antibody (such as human-mouse chimeric antibody), murine antibody, or humanized antibody.

[0033] In another preferred embodiment, the antibody is an IgG-type antibody.

[0034] In the sixth aspect of the present invention, a recombinant protein is provided, and the recombinant protein has:

[0035] (i) The heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention; and

[0036] (ii) Optional tag sequences for assisting expression and / or purification.

[0037] In another preferred embodiment, the tag sequence comprises a 6His tag.

[0038] In another preferred embodiment, the recombinant protein is specifically anti-HPV; preferably, specifically anti-HPV16, HPV31, HPV33, HPV35, HPV52, HPV58; more preferably, specifically anti-HPV16, and most preferably, anti-HPV16E6 protein in the cell nucleus.

[0039] In the seventh aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

[0040] (1) The heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention; or

[0041] (2) The recombinant protein as described in the sixth aspect of the present invention.

[0042] In another preferred embodiment, the polynucleotide has the sequence shown in SEQ ID NO: 2, 4, 11, 12, 13, 14, 15 or 16.

[0043] In the eighth aspect of the present invention, there is provided a vector containing the polynucleotide as described in the seventh aspect of the present invention.

[0044] In another preferred embodiment, the vector includes: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0045] In the ninth aspect of the present invention, there is provided a genetically engineered host cell containing the vector as described in the eighth aspect of the present invention or having the polynucleotide as described in the seventh aspect of the present invention integrated into its genome.

[0046] In the tenth aspect of the present invention, there is provided an immunoconjugate containing:

[0047] (a) The heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention; and

[0048] (b) A conjugate moiety selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0049] In another preferred example, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin), or any form of nanoparticles, etc.

[0050] In the eleventh aspect of the present invention, there is provided a pharmaceutical composition, which contains:

[0051] (i) The heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or the immunoconjugate as described in the tenth aspect of the present invention; and

[0052] (ii) A pharmaceutically acceptable carrier.

[0053] In another preferred example, the pharmaceutical composition is in an injectable form.

[0054] In another preferred example, the pharmaceutical composition is used for preparing a drug for treating tumors, and the tumors are selected from the following group: cervical cancer, bladder cancer, endometrial cancer, vaginal cancer, vulvar cancer, penile cancer, prostate cancer, oral cancer, laryngeal cancer, tonsillar cancer, or a combination thereof.

[0055] In the twelfth aspect of the present invention, there is provided the use of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or the immunoconjugate as described in the tenth aspect of the present invention, for preparing a medicament, reagent, test plate or kit;

[0056] The reagent, test plate or kit is used for:

[0057] (1) Detecting HPV E6 protein in a sample; and / or

[0058] (2) Detecting endogenous HPV E6 protein in tumor cells; and / or

[0059] (3) Detecting tumor cells expressing HPV E6 protein; and / or

[0060] (4) Identify the type of HPV E6 protein;

[0061] The medicament is used for treating or preventing tumors expressing HPV E6 protein.

[0062] In another preferred embodiment, the HPV E6 protein is the HPV E6 protein in the cell nucleus.

[0063] In another preferred embodiment, the HPV E6 protein includes proteins selected from the group consisting of: HPV16 E6 protein, HPV31 E6 protein, HPV33 E6 protein, HPV 35 E6 protein, HPV 52 E6 protein, HPV 58 E6 protein, or a combination thereof; more preferably, specifically against the HPV16 E6 protein in the cell nucleus.

[0064] In another preferred embodiment, the sample contains the HPV16 E6 protein, more preferably the HPV16 E6 protein in the cell nucleus.

[0065] In another preferred embodiment, the tumors include: tumors of the urogenital system, anal cancer, oral cancer, head and neck cancer, head and neck tumors, lung cancer, prostate cancer, colorectal cancer, breast cancer, prostate cancer, or adrenal tumors.

[0066] In another preferred embodiment, the "tumors of the urogenital system" include: cervical cancer, bladder cancer, endometrial cancer, vaginal cancer, vulvar cancer, or penile cancer.

[0067] In another preferred embodiment, the reagent includes a chip and immunomicrospheres coated with antibodies.

[0068] In the thirteenth aspect of the present invention, a method for detecting HPV E6 protein in a sample is provided, and the method includes the steps of:

[0069] (1) Contact the sample with the antibody described in the fifth aspect of the present invention;

[0070] (2) Detect whether an antigen-antibody complex is formed, and the formation of the complex indicates the presence of HPV E6 protein in the sample.

[0071] In another preferred embodiment, in step (2), the detection is performed by ELISA.

[0072] In another preferred embodiment, the HPV E6 protein includes HPV16 E6 protein and / or HPV18 E6 protein.

[0073] In another preferred embodiment, the HPV E6 protein is the HPV E6 protein in the cell nucleus.

[0074] In another preferred embodiment, the HPV E6 protein is the HPV16 E6 protein in the cell nucleus.

[0075] In another preferred embodiment, in step (1), the sample is contacted with two antibodies against the HPV E6 protein, and in step (2), detection is performed by ELISA. At least one of the two antibodies against the HPV E6 protein is the antibody described in the fifth aspect of the present invention.

[0076] In another preferred embodiment, the "antigen-antibody complex" is a "first antibody-antigen-second antibody" ternary complex, wherein the first antibody is the antibody described in the fifth aspect of the present invention, and the binding epitope of the second antibody is different from that of the first antibody.

[0077] In another preferred embodiment, the "antigen-antibody complex" is a "first antibody-antigen-second antibody" ternary complex, wherein the first antibody is the antibody described in the fifth aspect of the present invention, and the binding epitope of the second antibody is different from the binding epitope of the fifth aspect of the present invention.

[0078] In another preferred embodiment, in step (1), after the sample is contacted with the antibody described in the fifth aspect of the present invention, a third antibody against the first antibody is further added to the reaction system, and in step (2), the formation of the "antigen-first antibody-third antibody" complex is detected.

[0079] In another preferred embodiment, the first antibody, the second antibody, or the third antibody is labeled with a detectable label.

[0080] 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.

[0081] In another preferred embodiment, the sample includes: a human or animal tissue sample, a tumor resection sample, and a exfoliated cell sample.

[0082] In another preferred embodiment, the sample is a cervical exfoliated cell sample or a biopsy tissue sample.

[0083] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0084] In another preferred embodiment, the method is an in vitro method.

[0085] In another preferred embodiment, the method further includes the step of (3) analyzing the affinity between the antibody and the antigen.

[0086] In a fourteenth aspect of the present invention, a detection plate is provided, which comprises a substrate (support plate) and a test strip, and the test strip contains the antibody described in the fifth aspect of the present invention or the immunoconjugate described in the tenth aspect of the present invention.

[0087] In another preferred embodiment, the test strip further contains an antigen spotting area.

[0088] In another preferred embodiment, the test strip is sequentially composed of a sample filtering paper, a chromatographic material, a nitrocellulose membrane, and a blotting paper in an overlapping manner.

[0089] In a fifteenth aspect of the present invention, a kit is provided, and the kit includes:

[0090] (1) A first container containing the antibody described in the fifth aspect of the present invention; and / or

[0091] (2) A second container containing a secondary antibody against the antibody described in the fifth aspect of the present invention; and / or

[0092] (3) A third container containing a cell lysis reagent;

[0093] Or,

[0094] The kit contains the detection plate described in the fourteenth aspect of the present invention.

[0095] In another preferred embodiment, the antibody in the first container is labeled with a detectable label.

[0096] In another preferred embodiment, the antibody in the second container is labeled with a detectable label.

[0097] In a sixteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, and the method includes:

[0098] (a) Culturing the host cell described in the ninth aspect of the present invention under suitable expression conditions;

[0099] (b) Isolating the recombinant polypeptide from the culture, and the recombinant polypeptide is the antibody described in the fifth aspect of the present invention or the recombinant protein described in the sixth aspect of the present invention.

[0100] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1The protein electrophoresis results are shown. The estimated molecular weight of the GST-HPV35 E6 recombinant protein is approximately around 45 kDa, the estimated molecular weight of the GST-HPV52 E6 recombinant protein is approximately around 50 kDa, the estimated molecular weight of the GST-HPV18 / 59 E6 recombinant protein is approximately around 60 kDa, and the estimated molecular weight of the GST-N / C HPV16 / 18 E6 recombinant protein is approximately around 35 kDa.

[0102] Figure 2A The ELISA detection of the rabbit serum titer of the monoclonal antibody (ELISA coated antigen MBP-HPV16E6) is shown.

[0103] Figure 2B The ELISA detection of the rabbit serum titer of the monoclonal antibody (ELISA coated antigen MBP-HPV18E6) is shown.

[0104] Figure 3A The ELISA detection results 1 of the monoclonal antibody binding to E6 polypeptide and protein are shown.

[0105] Figure 3B The ELISA detection results 2 of the monoclonal antibody binding to E6 polypeptide and protein are shown.

[0106] Figure 4 The ELISA detection results of the monoclonal antibody binding to HPV E6 protein sites are shown. The antigens are respectively: GST-HPV35 E6, GST-HPV52 E6, GST-HPV18 / 59 E6, GST-HPV16 E6 N-terminal, GST-HPV16 E6 C-terminal, MBP-HPV18 E6, MBP-HPV16 E6.

[0107] Figure 5 The ELISA detection results of the monoclonal antibody binding to HPV E6 polypeptide fragments are shown. The antigens are respectively: peptide 5, peptide 6, peptide 7, peptide 2.

[0108] Figure 6 The immunocytochemical staining tests of CaSki cell mixed with ASC-US (atypical squamous epithelial cells of undetermined significance) clinical samples (CaSki / ASC-US) and HeLa cell mixed with NILM (normal) clinical samples (HeLa / NILM) are shown.

[0109] Figure 7 The chemical staining tests of different concentrations of purified antibody clone 55F8 on 5 kinds of 293T transfected cells are shown; the empty vector 293T cells are used as negative controls.

[0110] Figure 8Shows the ELISA affinity assay of recombinant monoclonal antibodies (protein MBP-HPV16 E6, protein MBP-HPV18 E6).

[0111] Figure 9 Shows the affinity calculation result curves of monoclonal antibody 55F8 and affinity-purified E6 polyclonal antibody affi-poly. Detailed implementation methods

[0112] After extensive and in-depth research and a large number of screenings, the present inventors provided a monoclonal antibody that binds to the HPV16 E6 protein in the nucleus of cervical cancer cells with high affinity. The HPV E6 antibody (55F8) of the present invention can specifically recognize the HPV16 E6 protein, especially the HPV16 E6 protein in the nucleus. Experiments of the present invention showed that the cellular localization of the antibody (55F8) of the present invention is in the nucleus of diseased cells, with clear staining and accurate localization. Therefore, the anti-HPV16 E6 antibody of the present invention can be used for the detection of the E6 protein in the nucleus of cervical cancer cells by cytological immunohistochemical staining, ELISA or other detection techniques. Based on this, the present invention was completed.

[0113] Cervical cancer and HPV

[0114] Cervical cancer is the most common malignant tumor in women, with the incidence ranking fourth among female cancers globally and being the fourth leading cause of cancer-related deaths in women. Among all human tumors, cervical cancer is currently the only tumor with a clear cause and is also the only preventable tumor. More than 99% of cervical cancer cases globally are related to high-risk HPV (Human papillomavirus). The probability of adult women being infected with HPV in their lifetime is 80%. Among Chinese women, 20-30% (varying by region) are positive carriers of HPV (Wang R, Guo X, Wu S et al. Nationwide prevalence of human papillomavirus infection and viral genotype distribution in 37 cities in China. 2015). HPV is the main cause of cervical cancer, but not the only factor, and HPV infection itself does not directly cause cancer. Only persistent high-risk HPV infection will cause precancerous lesions.

[0115] HPV is an enveloped virus containing circular double-stranded DNA. More than 200 subtypes have been discovered so far and are divided into high-risk, intermediate-risk, and low-risk types. There are 14 high-risk HPV types, namely HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Among them, in the phylogenetic tree of HPV subtype classification, high-risk HPV16, 31, 33, 35, 52, and 58 are highly homologous and belong to the same side branch, while high-risk HPV18 and HPV45 are highly homologous and belong to the other side branch (Lidqvist M, Nilsson O et al. Detection of human papillomavirus oncoprotein E7 in liquid-based cytology. 2012). The HPV genome contains three gene regions: the long control region (LCR) that does not encode proteins, the early gene region (E1, E2, E4, E5, E6, E7), and the late gene region (L1, L2).

[0116] Studies have shown that HPV invades and infects the basal cells of the cervical epithelium through damaged cervical epithelium, differentiates and multiplies in the basal cells of the cervical epithelium, and gradually infects the superficial layer of the cervical epithelium. In transient HPV infection, the replication of the viral capsid gene does not affect the normal cell cycle of the host, and the cells will not proliferate out of control. In highly diseased patients with persistent HPV infection, HPV integrates its genes into the host genome and the host cells express the carcinogenic gene products E7 / E6 proteins (Hoppe-Seyler K, Bossler F, Braun JA, Herrmann AL, Hoppe-Seyler F. (2018) Trends Microbiol. 26(2):158-168. The HPV E6 / E7 Oncogenes: Key Factors for Viral Carcinogenesis and Therapeutic Targets). The E6 gene is one of the important carcinogenic genes of high-risk human papillomavirus (hrHPV), and its gene product, the E6 protein, has carcinogenic functions, so it is called E6 Oncoprotein. The E6 protein consists of about 150 amino acids, and its protein has a Cys-x-x-Cys sequence for two zinc finger structures, which is closely related to malignant transformation, transcriptional activation, and interaction with cellular proteins (lliot J. Androphyl, Nancy L. Hubbert, John T. Schiller and Douglas R. Lowy. (1987) The EMBO Journal vol.6 no.4 pp.989-992. Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines). The functions of the E6 oncoprotein mediated by high-risk HPV can regulate cell pathways through multiple ways, establish the possibility of long-term infection in the cellular environment, lead to host gene mutations, and ultimately transform into malignant tumors.

[0117] HPV begins to express the carcinogenic proteins E6 / E7 by integrating into the host cell DNA. Studies have shown that the E6 oncoprotein promotes the degradation of the tumor suppressor protein p53 in normal cells, and the cellular localization of the p53 protein is in the nucleus, indicating that the normal expression of the E6 protein should be in the nucleus; at the same time, another important pathway for the carcinogenic transformation of the E6 protein is its interaction with telomerase, which can cause cell immortality by activating the telomerase complex. Although the carcinogenic pathway of the E6 protein in cells has not been fully clarified, in most cases, the E6 protein plays a related role in the nucleus to promote the carcinogenic transformation of cells.

[0118] Antibody

[0119] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons having the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a number of constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; 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. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0120] As used herein, the term "variable" indicates that certain portions of the variable regions in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions of the variable regions are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which generally assume a β-sheet configuration, connected by three CDRs that form loop structures and in some cases form partial β-sheet structures. The CDRs in each chain are brought into close proximity by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody. The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0121] The "light chains" of vertebrate antibodies (immunoglobulins) can be grouped into one of two distinct classes (called κ and λ) based on the amino acid sequence of their constant regions. Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly five 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, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0122] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for possible minor natural occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, in contrast to conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is 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.

[0123] The present invention also includes monoclonal antibodies having the corresponding amino acid sequences of the monoclonal antibodies against the HPV16 E6 protein described above, monoclonal antibodies having the variable region chains of the monoclonal antibodies against the HPV16 E6 protein, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing hypervariable regions (complementary determining regions, CDRs), provided that the hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light chain and heavy chain of the present invention.

[0124] As is known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the monoclonal antibodies against the HPV16 E6 protein or fragments thereof. The present invention also includes cell surface markers or antigens that bind to the monoclonal antibodies against the HPV16 E6 protein or fragments thereof.

[0125] The present invention includes not only intact monoclonal antibodies but also immunologically active antibody fragments, such as Fab or (Fab')2 fragments; antibody heavy chains; and antibody light chains.

[0126] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0127] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0128] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody (55F8) includes the following three complementary determining regions CDRs:

[0129] HCDR1, whose amino acid sequence is: NYGVN (SEQ ID NO:5), and whose encoding nucleotide sequence is: AACTATGGAGTGAAC (SEQ ID NO:11);

[0130] HCDR2, whose amino acid sequence is: WINRGGSASYASWAKG (SEQ ID NO:6), and whose encoding nucleotide sequence is:

[0131] TGGATTAATCGTGGTGGTAGCGCCTCTTACGCGAGCTGGGCAAAAGGC (SEQ ID NO:12);

[0132] HCDR3, whose amino acid sequence is: YGDGSLSNI (SEQ ID NO:7), and whose encoding nucleotide sequence is: TATGGTGATGGTAGTCTTAGTAACATC (SEQ ID NO:13).

[0133] In a preferred embodiment of the present invention, the light chain variable region of the antibody (55F8) of the present invention has complementarity determining regions CDR selected from the group consisting of:

[0134] LCDR1, whose amino acid sequence is: QASQSLYNQQNLA (SEQ ID NO:8), and whose encoding nucleotide sequence is: CAGGCCAGTCAGAGTCTTTATAACCAGCAAAATTTAGCC (SEQ ID NO:14);

[0135] LCDR2, whose amino acid sequence is: DASELAS (SEQ ID NO:9), and whose encoding nucleotide sequence is: GATGCATCCGAACTGGCATCT (SEQ ID NO:15);

[0136] LCDR3, whose amino acid sequence is: QGEFTCSGGDCIV (SEQ ID NO:10),

[0137] and whose encoding nucleotide sequence is:

[0138] CAAGGCGAATTTACTTGTAGTGGTGGTGATTGTATTGTT (SEQ ID NO:16).

[0139] In another preferred example, the amino acid sequence of the heavy chain variable region of the antibody is:

[0140] METGLRWLLLVAVLKGVQCQSLEESGGRLVKPTDTLTLTCTVSGFSLS NYGVNWVRQAPGKGLE WIGW INRGGSASYASWAKG RFTISKTSTTVDLKIISPTDEDTATYFCAS YGDGSLSNI WGPGTLVTVSS(SEQ ID NO:1):

[0141] In another preferred example, the DNA sequence of the variable region of the antibody heavy chain is:

[0142] ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGT AACTATGGAGTGAAC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGA TGGATTAATCGTGGTGGTA GCGCCTCTTACGCGAGCTGGGCAAAAGGC CGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATTATCAGTCCGACAGATGAGGACACGGCCACCTATTTCTGTGCCAGT TATGGTGATGGTAGTCTTAGTAACATC TGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA(SEQ ID NO:2);

[0143] In another preferred example, the amino acid sequence of the variable region of the antibody light chain is:

[0144] MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAAVGGTVTINC QASQSLYNQQNLA WYQQKPGQSPKLLID DASELAS GVSSRFKGSGSGTQFTLTISGVQCADAATYYC QGEFTCSGGDCIV FGGGTDVVVK(SEQ IDNO:3);

[0145] In another preferred example, the nucleotide sequence encoding the variable region of the antibody light chain is:

[0146] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCGTCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGT CAGAGTCTTTATAACCAGCAAAATTTAGCC TGGTATCAGCAGAAACCAGGGCAGTCTCCCAAGCTCCTGATCGAC G ATGCATCCGAACTGGCATCT GGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGCCGATGCTGCCACTTACTACTGT CAAGGCGAATTTACTTGTAGTGGTGGTGATTGTATT GTT TTCGGCGGAGGGACAGACGTGGTGGTCAAA(SEQ ID NO:4);

[0147] In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and light chain constant region, and the light chain constant region can be murine or human.

[0148] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and all refer to an antibody that specifically binds to the HPV16 E6 protein, such as a protein or polypeptide having a heavy chain and / or a light chain. They may or may not contain a starting methionine.

[0149] In another preferred example, the antibody is a murine or human-mouse chimeric monoclonal antibody against the HPV16 E6 protein, and its heavy chain constant region and / or light chain constant region can be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is the heavy chain constant region or light chain constant region of human IgG1, IgG2, etc.

[0150] The present invention also provides other proteins or fusion expression products having the antibody of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain and a light chain containing variable regions, as long as the variable regions are the same as or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy chain and light chain of the antibody of the present invention.

[0151] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy chain and light chain, called complementarity-determining regions (CDRs), which divide this segment into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures and are spatially close to each other through the β-sheets formed by the intervening FRs. The CDRs on the heavy chain and the corresponding CDRs on the 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.

[0152] The variable regions of the heavy and / or light chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in antigen binding. Accordingly, the present invention includes those molecules having monoclonal antibody light and heavy chain variable regions with CDRs, provided that the CDRs have a homology of more than 90% (preferably more than 95%, most preferably more than 98%) with the CDRs identified herein.

[0153] The present invention includes not only intact monoclonal antibodies, but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of the said antibodies.

[0154] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that extends the polypeptide half-life, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0155] The antibodies of the present invention refer to polypeptides having HPV16E6 protein binding activity and including the above-mentioned CDR regions. The term also includes variant forms of polypeptides containing the above-mentioned CDR regions that have the same function as the antibodies of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar properties or functions, the function of the protein is usually not changed. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0156] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes with the DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained with antiserum against the antibody of the present invention.

[0157] The present invention also provides other polypeptides, such as fusion proteins containing human antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Generally, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0158] In the present invention, "conservative variants of the antibody of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids in the amino acid sequence of the antibody of the present invention with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0159] Table A

[0160]

[0161]

[0162] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as or a degenerate variant of the coding region sequences shown in SEQ ID NO: 1, 3, 5, 6, 7, 8, 9. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but is different from the coding region sequences shown in SEQ ID NO: 2, 4, 11, 12, 13, 14, 15.

[0163] The polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; coding sequences of the mature polypeptides and various additional coding sequences; coding sequences of the mature polypeptides (and optional additional coding sequences) and non-coding sequences.

[0164] The term "polynucleotide encoding a polypeptide" can be a polynucleotide including the polynucleotide encoding this polypeptide, or can also be a polynucleotide further including additional coding and / or non-coding sequences.

[0165] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides described in the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at a lower ionic strength and a higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in SEQ ID NO:1 and / or SEQ ID NO:3.

[0166] The full-length nucleotide sequence or a fragment thereof of the antibody of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0167] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination. This is usually to clone them into a vector, then transfer them into cells, and then isolate the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0168] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then, this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0169] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0170] The host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0171] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated by the CaCl2 method, the steps of which 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 can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0172] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cell. After the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0173] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0174] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or any combination of the above substances.

[0175] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0176] Therapeutic agents that can be bound or conjugated to the antibodies of the present invention include, but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles / nanorods; 5. Virus particles; 6. Liposomes; 7. Nanomagnets; 8. Prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0177] Composition

[0178] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5 - 8, preferably about 6 - 8, although the pH value may vary depending on the nature of the substances to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0179] The pharmaceutical composition of the present invention can be directly used to bind to the HPV16 E6 protein molecule, and thus can be used for the prevention and treatment of tumors. In addition, other therapeutic agents can also be used simultaneously.

[0180] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should be matched with the administration route. The pharmaceutical composition of the present invention can be made into an injectable form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectables and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight - about 5 milligrams / kg body weight per day. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0181] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 8 milligrams / kg body weight. Preferably, the dose is about 10 micrograms / kg body weight - about 1 milligram / kg body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are within the scope of the skills of a skilled physician.

[0182] Preparation of Monoclonal Antibody

[0183] According to different antibody preparation methods, there are mainly three forms: polyclonal antibody (pAb), monoclonal antibody (mAb), and recombinant antibody (rAb). The preparation method of polyclonal antibody is simple and the cost is low, which is the antibody form widely used in early immunoassay. Monoclonal antibody has high affinity, strong specificity, and small differences between different batches. Once the hybridoma cell line is established, the supply of antibodies is continuous, which is suitable for use in immunological method detection. Therefore, in recent years, the application of monoclonal antibody in the development of immunotherapy drugs and immunoassay technology has gradually increased. With the progress of technology, the application of recombinant antibody technology has become more and more extensive.

[0184] The monoclonal antibody of the present invention can be prepared through various technical platforms known to those skilled in the art. For example, the antigen of the present invention can be administered to BALB / c mice to induce an immune response and prepare monoclonal antibodies. For the preparation of monoclonal antibodies, the hybridoma technology route can be adopted (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981), phage display technology, or Single B Cell cloning platform.

[0185] Hybridoma fusion technology involves immunizing an animal, such as a mouse, with B cells (spleen, bone marrow, or peripheral blood) and fusing them with myeloma cells to generate hybridoma clones. Representative myeloma cells are those that are efficiently fusible, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to the culture medium (HAT medium substrate), including myeloma cell lines, such as murine myeloma cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, California, USA) and SP-2, NZ0, or X63-Ag8-653 cells (available from American Type Culture Collection, Rockville, 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., Monoclonal Antibodies Production Techniques and Applications, pages 51-63 (Marcel Dekker, Inc., New York, 1987)].

[0186] The culture supernatant of hybridoma cells is analyzed to detect the production of monoclonal antibodies with the desired specificity, e.g., by in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The location of antibody-expressing cells can be detected using FACS (fluorescence-activated cell sorting). Then, the hybridoma clones can be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) pages 59-103). Media suitable for the growth of hybridoma cells include, for example, DMEM (high glucose) or RPMI-1640 medium or serum-free medium. In addition, hybridoma cells can be injected into animals to grow as ascites tumors. Monoclonal antibodies secreted by the subclones are appropriately isolated from the culture medium, ascites, or serum by conventional immunoglobulin purification processes, which include protein A / G-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0187] The principle of screening single-chain antibodies by phage display technology is to use genetic engineering methods to insert the heavy chain (VH) and light chain variable region (VL) gene fragments of the immunoglobulin IgG gene in immune cells (B cells or immune system stem cells) into the signal peptide coding region of phage or phagemid through a linker, so that the inserted fragment is fused with the phage membrane protein and displayed on the surface of the phage capsid protein, establishing a phage display antibody library. Through the biological panning process of "adsorption-elution-enrichment", positive phage-displayed single-chain antibodies can be screened. According to the different sources of the inserted fragments on the phage (phagemid) vector, phage display antibody libraries can be divided into three categories: immune antibody libraries, natural antibody libraries, and synthetic antibody libraries. Immune antibody libraries use the mRNA in the blood of antigen-immunized animals as a template to synthesize antibody variable regions and insert the variable region genes into specific vectors. Since antigen immunization can stimulate the animal body's lymphocytes to secrete specific antibodies, such antibody libraries contain a large number of genes of induced specific antibodies and can screen high-affinity antibodies targeted, with a greater probability of screening out positive antibodies; directly extract the mRNA in the bone marrow stem cells, peripheral lymphocytes, and spleen B cells of unimmunized animals, reverse transcribe it into cDNA, amplify the antibody variable region genes and connect them to the vector to obtain a natural antibody library. The mRNA of unimmunized animal blood determines the diversity of the antibody library, and its application range is wider, not limited to one or several antigens; the specific reaction of antigen-antibody mainly depends on the spatial conformation of the variable region, and at the molecular level, it depends on the DNA sequences of the CDR (Complementarity Determining Region) of the heavy and light chain variable regions of the antibody. By using genetic engineering methods to artificially modify the gene sequences of the CDR region, the diversity of the antibody library can be greatly improved. The antibody library obtained by this method of genetically modifying the antibody sequence is a synthetic antibody library.

[0188] In the preferred experimental technical route of the present invention, rabbits were immunized, combined with single B cell isolation, screening, and molecular cloning techniques to obtain a rabbit-derived monoclonal antibody against HPV16 E6 protein. In a preferred embodiment of the present invention, the monoclonal antibody was prepared by the Single B Cell cloning method. B cells were obtained by collecting rabbit peripheral blood for PBMC (peripheral blood mononuclear cell) isolation, and B cells were isolated, analyzed, and screened at the single cell level to accurately and efficiently screen out B cells secreting target antibody molecules.

[0189] In a preferred embodiment of the present invention, total RNA is extracted from the positive wells of a cell culture plate with an OD value greater than 1.0 in the ELISA test result. Commercial RNA extraction reagents (Vazyme, R701-01 / 02) can be used, and the total RNA is reverse transcribed into cDNA using commercial universal primers (Prime Script TM 1 st Strand cDNA SynthesisKit, Takara). Subsequently, the heavy and light chain V-region fragments of immunoglobulins are amplified using specific primers for the signal peptide and constant region of immunoglobulin IgG heavy or light chains. The obtained PCR fragments are homologously recombined into the pCDNA3.4 vector, and vector-specific primers are used to perform single-cell sequencing on the inserted fragments. This technical route uses functional activity detection as a screening step and combines high-throughput mode to screen B cell clones, which can increase the screening and identification of antibody molecules with different binding characteristics and affinities, and improve the coverage rate and throughput by more than 100 times.

[0190] In a preferred embodiment of the present invention, the expression and production of monoclonal antibodies use recombinant DNA methods to construct a eukaryotic expression system, transiently transfect HEK293 cells to express antibodies or construct a stable CHO-S cell line, and then the antibodies secreted into the culture medium are purified by an affinity chromatography column (Protein A / G-Sephrose), and the purity can reach more than 95%.

[0191] Application

[0192] The present invention relates to a method for detecting precancerous lesions of cervical cancer in cell samples. The steps of this method are generally as follows: obtaining a cervical exfoliated cell sample after liquid-based preservation; detecting the level of endogenous E6 oncoprotein in high-risk HPV-infected lesion cells in the sample.

[0193] The biomarker used in the present invention is E6 oncoprotein, which is derived from a virus. Since the marker characteristics of the virus in tissues do not occur in uninfected human tissues, the detection of samples of HPV-related cancers is specific.

[0194] The samples used in the present invention may include fixed or preserved cell samples. Suitable preservation media may include one or more mixtures selected from alcohols, aldehydes, ketones, acids, metal ions or mercury, ethers, etc. for preserving cell components. Alcohols include methanol, ethanol, (n- or i-) propanol, (n-, i- or t-) butanol, or highly branched or unbranched alcohols. Aldehydes include formaldehyde, acetaldehyde, glutaraldehyde, etc. Ketones such as acetone can also be used. Acids used in standard sample media include organic acids (acetic acid, trichloroacetic acid, salicylic acid and picric acid) or inorganic acids such as chromic acid. Metals such as silver, copper, chromium, mercury, osmium and uranium may be included in standard sample solutions. Salt solutions such as uranyl acetate, dipotassium dichromate, ammonium sulfate, etc. may be components of the preservation media.

[0195] The liquid-based cell thin sections used in the invention include cell samples prepared by two methods: natural sedimentation method and gradient centrifugation method. For example, an artificially cultured tumor cell line is mixed with a buffer solution, naturally sedimented on a glass slide, and then fixed with a fixative (usually 95% ethanol) to form; exfoliated cells obtained clinically are subjected to gradient centrifugation by a centrifuge, the effective cells are enriched, impurities (such as mucus, blood cell debris, etc.) are removed, and then fixed to form.

[0196] The clinical cell samples used in the present invention are cell mixtures made through special pretreatment. For example, clinical cervical exfoliated cells in a preservation solution are preliminarily mixed, and after passing the detection, they are frozen in a -80°C refrigerator for later use. Therefore, the qualified samples frozen well can be rewarmed and mixed for use during clinical examination.

[0197] Kit

[0198] The present invention also provides a kit containing only the antibody (or its fragment) of the present invention or the detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, an instruction manual, a buffer, etc.

[0199] The present invention further designs a detection kit for detecting high-risk HPV E6 oncoprotein. The kit includes an antibody that recognizes high-risk HPV E6 oncoprotein, general reagents and buffers required for detection, such as various buffers, enzyme-labeled secondary antibodies, detection labels, detection substrates, etc. The antibody is preferably an anti-HPV E6 oncoprotein antibody. The detection kit can be an in vitro diagnostic device.

[0200] The present invention further designs and develops a kit for diagnosing and evaluating HPV infection-related conditions from cervical biopsy samples. The kit can detect high-risk HPV E6 oncoprotein present in the samples. The tissue is fixed with formalin and embedded in paraffin, and is used to develop a detection kit and an in vitro diagnostic device for detecting HPV infection-related tumors in samples based on non-cellular morphology analysis.

[0201] One of the objectives of the present invention is to provide a method for detecting the expression of multiple high-risk HPV E6 proteins, and the method can be used for detecting HPV infection-related cancers, especially cervical cancer.

[0202] The inventors prepared a monoclonal antibody (55F8) against the E6 protein of human papillomavirus HPV16 and studied its immunoreactive properties. Immunocytochemical staining was performed on cervical exfoliated cell samples using this anti-HPV 16E6 monoclonal antibody. The results showed that the antibody had a strong staining reaction in tumor cells caused by hrHPV type viruses, while the background staining of normal cervical cells and general inflammatory cells was clean.

[0203] That is to say, the method of the present invention is a method for detecting tumor markers, characterized in that it includes the step of detecting HPV16 E6 in a sample.

[0204] In the method of the present invention, the detection sample is preferably a patient with possible cervical lesions due to cervical epithelial damage, or a patient with already-occurred cervical lesions.

[0205] The HPV16 E6 immunogen is preferably the full-length protein of HPV16 E6 or its protein fragment, or polypeptide fragment. In this case, the preferred step for using the detection reagent for the HPV16 E6 antibody is to perform immunocytochemical staining analysis using the HPV16 E6 antibody. The anti-HPV16 E6 antibody used is preferably an anti-HPV16 E6 monoclonal antibody. Preferably, the antibody of the present invention can also bind to other multiple high-risk HPV16E6 antigen fragments in addition to the HPV16 E6 immunogen, such as the HPV16E6-N protein antigen, preferably the full-length protein of the high-risk HPV16E6 antigen or its protein fragment, or polypeptide fragment.

[0206] The protein expressed from the high-risk HPV E6 oncogene, including HPV16, used in the immunoassay of the method is a reliable indicator for the occurrence of malignant or pre-malignant cells caused by carcinogenic subtype HPV. One of the most useful aspects of the present invention is its application in the diagnosis of cervical cancer, squamous epithelial cell damage, adenocarcinoma, and any epithelial cell abnormalities related to carcinogenic HPV infection. The indicated carcinogenic HPV16 infection includes koilocytosis; hyperkeratosis; pre-cancerous conditions including intraepithelial neoplasia or intraepithelial lesions; severe dysplasia; and invasive or malignant cancers. In addition to cervical cancer, the detection of E6 oncoprotein is also useful for detecting tumors of the urogenital system such as bladder cancer, endometrial cancer, penile cancer, small cell lung cancer, melanoma, and head and neck tumors.

[0207] Another objective of the present invention is to provide a detection kit by the method of the present invention. The kit can be a diagnostic kit or a research kit.

[0208] The kit of the present invention is a kit for detecting tumor markers, characterized by having a monoclonal antibody against HPV16 E6. The kit of the present invention preferably has general reagents and buffers required for detection, such as various buffers, enzyme-labeled secondary antibodies, detection labels, detection substrates, etc. The antibody is preferably an anti-HPV16 E6 antibody, more preferably a monoclonal antibody against multiple high-risk HPV E6, and particularly preferably a recombinant expression vector of a monoclonal antibody gene against HPV E6 for eukaryotic expression systems obtained by recombinant DNA technology. A monoclonal antibody against high-risk HPV E6 rabbit produced by a eukaryotic expression system or a monoclonal antibody having the same binding activity as the monoclonal antibody against HPV E6 rabbit.

[0209] The present invention provides a method for differentiating tumor cells of cervical non-malignant lesions by detecting endogenous HPV E6 protein in cells, and fixedly matching with cell preservation solutions of various brands widely used clinically, so as to be able to make a diagnosis in the early stage of cancer evolution and provide a basis for timely treatment.

[0210] Furthermore, the present invention also provides a detection kit formed by using the detection method.

[0211] The main advantages of the present invention include:

[0212] (1) The antibody against E6 protein provided by the present invention can specifically bind to the HPV16 E6 protein in the cell nucleus, so this antibody can locate the oncoprotein E6 in the cell nucleus of cervical cancer lesions caused by HPV16 infection.

[0213] (2) The monoclonal antibody and detection method provided by the present invention are applicable to the early diagnosis of related cancers and help in the identification, grading, and evaluation of the disease progression of tumor tissues. More than 50% of cervical cancers are caused by HPV16 type. The antibody of the present invention can specifically bind to the HPV16 E6 protein, identify the HPV16 E6 protein or its epitope containing 5 peptide segments in the cell nucleus of cancer cells, thus helping to more accurately diagnose early cervical cancer.

[0214] (3) The antibody against E6 protein in the cell nucleus provided by the present invention can be used to study the relationship between the distribution of oncoproteins in cervical cells, the degree of cell lesions, and the disease progression.

[0215] (4) Provide risk analysis for HPV16 virus infected patients, which can distinguish "transient infection" or actual precancerous lesions have occurred. HPV16 infection is the most common. However, in the case of transient infection and the virus gene not being integrated into the human genome, the HPV16 E6 protein will not be expressed. In the case of persistent HPV16 infection and gene integration, the E6 protein expression is positive, indicating that the patient infected with the HPV16 virus has developed into cancer or precancerous lesions.

[0216] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0217] Example 1 Preparation and Activity Determination of Monoclonal Antibody Against Human Papillomavirus HPV16 E6

[0218] 1.1 Antigen Design

[0219] Protein antigen design:

[0220] By genetic engineering means, the full-length sequences of HPV16 / 18 E6 are inserted into the prokaryotic expression vector pGEX-4T-1 respectively, so that the HPV16 / 18 E6 protein is constructed into a fusion protein with Maltose binding protein MBP or Glutathione S-transferase GST tags. After the plasmid is activated, it is transformed into prokaryotic Escherichia coli for expression. After IPTG induction at low temperature, the bacterial solution is lysed by ultrasonic waves, and the protein is purified and the concentration and purity are identified.

[0221] The protein electrophoresis results are as Figure 1 :

[0222] The predicted molecular weight of the GST-HPV35 E6 recombinant protein is about 45 kDa, the predicted molecular weight of the GST-HPV52 E6 recombinant protein is about 50 kDa, the predicted molecular weight of the GST-HPV18 / 59 E6 recombinant protein is about 60 kDa, and the predicted molecular weight of the GST-N / C HPV16 / 18 E6 recombinant protein is about 35 kDa. The band position is close to the theoretical value of the protein molecular weight, and the expression level of the E6 protein after induction is normal.

[0223] The protein sequence, or its amino acid sequence, is shown in Table 1 below

[0224] Table 1

[0225]

[0226]

[0227] 1.2 Animal Immunization

[0228] Immunization status: Immunization was carried out with the polypeptide conjugated with KLH. For the dosage of antigen polypeptide and the interval time for each injection, see Table 2 for details:

[0229] Table 2 Animal Immunization Schedule (Polypeptide Antigen)

[0230]

[0231] 1.3 ELISA Detection of Rabbit Serum Titer

[0232] The coated antigens were selected as two recombinant proteins, MBP-HPV16 E6 and MBP-HPV18 E6, respectively, and coated at 0.2 μg / ml, overnight at 4°C. At the same time, a blank control (coated antigen, added with blank antibody diluent) was set. The next day, after washing with PBST and patting dry, 5% skim milk powder was added for room temperature blocking for 2 h. After washing with PBST and patting dry, the post-immunization sera of rabbits R14162, R14163, and R14164 (No. 62, No. 63, No. 64) and the pre-immunization serum (pre) were added respectively, and gradient serial dilutions (serial dilutions at 1:1000, 1:4000, 1:16000, 1:64000, 1:256000) were performed. Incubate at 37°C for 1 h. After washing with PBST and patting dry, goat anti-rabbit-HRP secondary antibody (Sigma A0545) (1:2000) was added respectively, incubated at 37°C for 30 min. After washing with PBST and patting dry, TMB solution was added, and the color development was terminated with 2M H2SO4 after reacting at 37°C for 15 min. Read the absorbance at OD450nm on an enzyme-linked immunosorbent assay reader.

[0233] The ELISA detection results are as Figure 2A and 2B shown: The OD values of the serum of rabbit No. 63 were the highest for both proteins, followed by No. 62; when the dilution ratio was 1:4000, the OD values of No. 62 and No. 63 were both relatively high, while the OD value of No. 64 decreased significantly. Therefore, the sera of rabbits No. 62 and No. 63 were finally selected for the next step of B cell screening.

[0234] 1.4 B Cell Isolation and Clonal Screening

[0235] Collect 15 - 30 ml of fresh peripheral blood from 2 rabbits, draw blood using a sterile tube, then coat the plate with the antigen GST - E6 fusion protein, and enrich specific B cells by binding B cells expressing extracellular antibodies to the E6 antigen protein. The enriched cells are seeded into a 96 - well cell culture plate pre - seeded with feeder cells at a certain cell density. Incubate the plate at 37°C in 5% CO₂. After 6 days of incubation, replace the fresh medium, and collect the supernatant cultured overnight on the 7th day to start testing for the presence of antibodies against the E6 protein using the ELISA binding described below.

[0236] 1.5 ELISA detection of B - cell supernatant clones

[0237] For the coated antigen, select a mixture of 4 E6 naked peptides (peptide 5, peptide 6, peptide 7, peptide 2) for coating, and set the coating concentration to: 4 μg / ml; coat a mixture of MBP - HPV16 E6 and MBP - HPV18 E6 proteins, and set the coating concentration of the E6 protein to: 1 μg / ml; at the same time, set a blank control (coated antigen, add blank antibody diluent). Incubate overnight at 4°C, after washing with PBST and patting dry, add 5% skim milk powder for blocking, incubate at room temperature for 2 h, after washing with PBST and patting dry, add 92 B - cell supernatants respectively, react at 37°C for 1 h, after washing with PBST and patting dry, add goat anti - rabbit - HRP secondary antibody (Sigma A0545) (1:2000) respectively, react at 37°C for 30 min, after washing with PBST and patting dry, add TMB solution, stop the color development with 2M H₂SO₄ after reacting at 37°C for 15 min, and read the absorbance at OD450nm on an ELISA reader.

[0238] Partial results of ELISA are as follows Figure 3A and 3B As shown: Combining ICC and IHC data, preferably clone 54A4, 55D3, 55F8, 58B8, 60H8, 54B10 enter the next stage of recombinant purification.

[0239] 1.6 Sequencing of monoclonal antibody variable regions

[0240] Use RNA extraction reagent (Vazyme, R701 - 01 / 02) to extract the RNA of total cells in wells with OD value greater than 1.0, and use universal primers (Prime Script TM 1 stThe Strand cDNA Synthesis Kit (Takara) was used to reverse transcribe it into cDNA. Subsequently, the V-region fragments of the immunoglobulin heavy and light chains were amplified by antibody signal peptide and constant region-specific primers, and the obtained PCR fragments were recombinated into the pCDNA3.4 vector by homologous recombination. The inserted fragments were sequenced using vector-specific primers. Finally, the unique V-region protein amino acid sequences and plasmids of the clones were obtained.

[0241] 1.7 Small-scale transfection and expression

[0242] The variable region (V-region) sequences of the immunoglobulin genes were cloned into mammalian expression vectors of the IgG heavy chain (HC) and light chain frameworks for expression, and then large-scale transient transfection of CHO cells (Expi-Fectamine CHO Transfection Kit, Gibco) was carried out. After culturing the cells for 48 hours, the supernatant culture medium containing secreted immunoglobulin IgG was collected. The antibody was purified by Protein A / G affinity chromatography, the protein peak effluent was collected, dialyzed with phosphate buffer (PBS), and the antibody protein concentration was measured by ultraviolet spectrophotometer OD260 / OD280, and the antibody titer was detected by indirect ELISA.

[0243] Example 2 Antibody identification

[0244] 2.1 Detection of the binding and localization of the purified antibody to the E6 protein by ELISA

[0245] Seven recombinant proteins, namely GST-HPV16 E6 C-terminal, GST-HPV16 E6 N-terminal, GST-HPV18 / 59 E6, GST-HPV35 E6, GST-HPV52 E6, MBP-HPV16 E6, and MBP-HPV18 E6, were selected as antigens and coated separately. At the same time, a blank control (coated antigen, added with blank antibody diluent) was set. Coating the plate at 1 μg / ml overnight at 4°C. After washing and patting dry with PBST, 5% skim milk powder was added for blocking at room temperature for 2 h. After washing and patting dry with PBST, the purified antibody clone supernatants (54A4; 55D3; 55F8; 58B8; 60H8; 54B10) (1 μg / ml) were added respectively and reacted at room temperature for 1 h. After washing and patting dry with PBST, goat anti-rabbit-HRP secondary antibody (Sigma A0545) (1:2000) was added respectively and reacted at 37°C for 30 min. After washing and patting dry with PBST, TMB solution was added and reacted at 37°C for 15 min, and then the color development was terminated with 2M H2SO4. The absorbance was measured at OD450nm on an enzyme-linked immunosorbent assay (ELISA) reader.

[0246] According to Figure 4Results: Clones 54A4, 55D3, 58B8, 60H8, and 54B10 all had relatively high OD values for recognizing the MBP-18E6 protein and showed no recognition of the GST-tagged protein. Clone 55F8 specifically recognized the HPV 16E6 protein with both GST and MBP tags, and it recognized the N-terminus of the E6 protein. The specificity of clone 55F8 for the target protein was more prominent compared to other clones.

[0247] 2.2 ELISA Detection of the Binding and Localization of Purified Antibodies to E6 Peptides

[0248] Four naked peptides, namely peptide 5, peptide 6, peptide 7, and peptide 2, were selected as antigens and coated separately. A blank control (coated antigen with blank antibody diluent) was also set up. Coating was performed at 1 μg / ml and incubated overnight at 4°C. After washing with PBST and patting dry, 5% skim milk powder was added for blocking at room temperature for 2 h. After washing with PBST and patting dry again, the purified antibody clone supernatants 54A4, 55D3, 55F8, 58B8, 60H8, and 54B10 (1 μg / ml) were added respectively and reacted at room temperature for 1 h. After washing with PBST and patting dry, goat anti-rabbit-HRP secondary antibody (Sigma A0545) (1:2000) was added and reacted at 37°C for 30 min. After washing with PBST and patting dry, TMB solution was added and reacted at 37°C for 15 min, and then the color development was terminated with 2 M H2SO4. The absorbance was read at OD450nm on an ELISA reader.

[0249] According to Figure 5 Results: Clones 54A4, 55D3, 58B8, 60H8, and 54B10 all had relatively high OD values for recognizing peptide 7 and showed little recognition of the other peptide segments. Clone 55F8 only had a relatively high binding affinity for peptide 5.

[0250] 2.3 Immunocytochemical Staining to Detect the Specificity and Sensitivity of Monoclonal Antibodies

[0251] The cervical cancer cell line CaSki cells expressing the HPV16 E6 protein were mixed with ASC-US (atypical squamous epithelial cells of undetermined significance) clinical samples and used here as a positive tumor cell model with overexpression of the HPV16 E7 protein in the state of high cervical lesions. Similarly, the cervical cancer cell line HeLa cells expressing the HPV18 E6 protein were mixed with NILM clinical samples and used here as a tumor cell model with overexpression of the HPV18 E6 protein in the state of high cervical lesions. Immunocytochemical staining experiments were performed on these two types of cells using the monoclonal purified antibodies 54A4, 55D3, 55F8, 58B8, 60H8, and 54B10 respectively. The specific experimental method is as follows:

[0252] After the CaSki and HeLa cells were resuscitated and cultured respectively, an appropriate amount of cell preservation solution was added to the centrifuge tube. The cells in the culture dish were brushed with a cervical sampling brush, and the sampling brush was rinsed in the centrifuge tube. After counting with a cell counting plate, they were stored at 4°C for later use. Among them, the CaSki cells were mixed with ASC-US clinical samples, and the HeLa cells were mixed with NILM clinical samples, both of which were prepared by the centrifugation method (more than 5,000 cells per slide); after the preparation was completed, they were fixed with 95% ethanol for 30 minutes, rehydrated with 50% ethanol for 10 minutes, then heat repaired with EDTA (pH 9.0), cooled, and 3% H2O2 was added for treatment at room temperature for 10 min, and then drained; PBST washing solution was added and washed for 3 min, and then drained; the clone supernatants 54A4, 55D3, 55F8, 58B8, 60H8, 54B10 were added respectively and incubated at room temperature for 30 minutes; PBST washing solution was added and washed 3 times, 3 minutes each time, and then drained; immunochromogenic reagent was added and incubated at room temperature for 30 minutes; PBST washing solution was added and washed 3 times, 3 min each time, and then drained; DAB chromogenic solution was added and reacted at room temperature for 6 minutes, and the staining results were observed under a microscope, and the reaction was terminated by washing with distilled water. The results were observed and recorded under a microscope.

[0253] The results are as Figure 6 shown: Only the rabbit monoclonal antibody 55F8 had good staining intensity for the tumor cell line, and the non-specific background staining was clean, and the other clones had no good specificity.

[0254] In addition, unexpectedly, it was found that the cells of this clone were located in the nuclei of the diseased cells (see the red arrow), with clear staining and accurate localization.

[0255] 2.4 Detection of the coverage type of the 55F8 clone on the FLAG-E6 protein by immunocytochemical staining

[0256] FLAG-E6 plasmids of different HPV subtypes (HPV6, HPV16, HPV18, HPV52, HPV58) were transfected into 293T cells transiently to serve as a tumor cell model with overexpression of a single HPV subtype E6 protein artificially modified. The purified monoclonal antibody 55F8 was used to perform immunocytochemical staining on the 5 transfected 293T cells respectively, and the empty vector 293T cells were used as negative controls. The specific implementation method is as follows:

[0257] After transfection and culturing of 293T cells, an appropriate amount of cell preservation solution was added. The cells in the culture dish were brushed with a cervical sampling brush, and the sampling brush was rinsed in a centrifuge tube. After counting with a cell counting plate, they were stored at 4°C for later use. Among them, both 5 types of transfected 293T cells and untransfected 293T cells were prepared by centrifugation (more than 5000 cells per slide); after preparation, they were fixed with 95% ethanol for 30 minutes, rehydrated with 50% ethanol for 10 minutes, then heat repaired with EDTA (pH 9.0), cooled, and treated with 3% H2O2 at room temperature for 10 min and then drained; washed with PBST washing solution for 3 min and drained; purified antibody clone 55F8 was added respectively, and concentration gradients of 1 μg / ml and 5 μg / ml were set, and incubated at room temperature for 30 minutes; washed with PBST washing solution 3 times, 3 minutes each time, and drained; added immunochromogenic reagent and incubated at room temperature for 30 minutes; washed with PBST washing solution 3 times, 3 min each time, and drained; added DAB chromogenic solution, reacted and developed color at room temperature for 6 minutes, observed the staining result under a microscope, and terminated the reaction with distilled water washing. The result was observed and recorded under a microscope.

[0258] The results were as Figure 7 shown: The staining of rabbit monoclonal antibody 55F8 transfected on transfected HEK-293 cells showed good specificity and staining intensity, could specifically recognize transfected cells expressing E6 protein of HPV16 type, and the staining was localized in the nucleus.

[0259] 2.5 Detection of the ELISA affinity of recombinant monoclonal antibody

[0260] Protein MBP-HPV16 E6 and protein MBP-HPV18 E6 were coated on the plate at 1 μg / ml and incubated overnight at 4°C. After washing and patting dry with PBST, 5% skim milk powder was added for blocking and incubated at room temperature for 2 hours. After washing and patting dry with PBST, purified E6 antibody clone 55F8 was added, with an initial concentration set at 7.5 μg / ml and serially diluted 2-fold, a total of 12 concentration gradients. At the same time, the initial concentration of 55F8 compared with affinity-purified polyclonal antibody Affi-poly (anti-E6 polyclonal antibody) was 15 μg / ml and serially diluted 3-fold. Reacted at room temperature for 1 hour, after washing and patting dry with PBST, goat anti-rabbit-HRP secondary antibody (Sigma A0545) (1:20 000) was added, reacted at 37°C for half an hour, after washing and patting dry with PBST, TMB was developed and terminated with 2M H2SO4, and read at OD450nm.

[0261] The results were as Figure 8 shown: Clone 55F8 only had a high recognition of HPV16 type E6 protein, and the antigen-antibody affinity was relatively high, while there was almost no binding to HPV18 type E6 protein.

[0262] According to Figure 9 the affinity constant was calculated:

[0263] K d (55F8): OD 50 =(3.816 - 0.068) / 2 = 1.874

[0264] When y = 1.874 is substituted into the fitting curve formula: y = -0.0459x 4 -0.2235x 3 +0.1312x 2 +1.8024x + 2.0171

[0265] K d (55F8) = 0.83 nM;

[0266] K d (affi - poly): OD 50 =(3.616 - 0.069) / 2 = 1.7735

[0267] When y = 1.7735 is substituted into the fitting curve formula: y = -0.0622x 4 -0.1922x 3 +0.4392x 2 +1.6761x + 1.1

[0268] K d (affi - poly) = 2.34 nM;

[0269] Since K d is inversely proportional to the affinity constant, it proves that the 55F8 monoclonal antibody has a high affinity and is superior to the E6 polyclonal antibody affi - poly.

[0270] Discussion

[0271] The present invention provides a monoclonal antibody that can recognize the E6 protein in the cell nucleus of cervical epithelial cancer cells caused by high - risk human papillomavirus (hrHPV) and its application in immunohistochemical staining.

[0272] Specifically, the present inventors used the above method to prepare a rabbit monoclonal antibody against HPV16 E6. Through the preliminary screening of the B cell clone culture supernatant on the antigen protein E6 and the tag-unrelated protein, and the immunohistochemical method was used to detect and identify the ability of the antibody clone to recognize the endogenous E6 protein in FFPE-treated cervical cancer tissues. The B cell clone containing the positive antibody was used to extract RNA, and amplification, molecular cloning technology and sequencing technology were used to obtain the immunoglobulin gene, the HV variable region and the LV variable region sequences. Finally, the full-length immunoglobulin IgG was produced in the antibody expression engineering cell line (CHO, 293T cells) by bioengineering technology. The purified antibody clone was identified by immunohistochemical application on clinical exfoliated liquid-based cell samples, and multiple antibody clones were screened, and finally a monoclonal antibody 55F8 with high affinity and capable of specifically binding to Flag-HPV16 E6 with high affinity was obtained, and it can recognize the E6 oncoprotein in the nuclei of high-risk HPV16 lesion cells.

[0273] This study shows that the 55F8 clone can specifically bind to the HPV16 E6 oncoprotein in the nuclei of lesion cells. Through the signal amplification of the secondary antibody and the color development of the catalytic DAB chromogenic substrate, the cancer cells in the cervical exfoliated liquid-based cells or tissue sections show specific brown staining of the cell nuclei, making the cancer cells visually and objectively marked.

[0274] All documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A variable region of an antibody heavy chain, characterized in that, The heavy chain variable region described above includes the following three complementarity determining regions (CDRs): HCDR1: NYGVN SEQ ID NO:5; HCDR2: WINRGGSASYASWAKG SEQ ID NO:6; and HCDR3: YGDGSLSNI SEQ ID NO:7; Wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and / or substituted with at least one amino acid and can retain the binding affinity for the HPV16 E6 protein.

2. An antibody heavy chain, characterized in that, The heavy chain described above has the heavy chain variable region and heavy chain constant region as claimed in claim 1.

3. A variable region of an antibody light chain, characterized in that, The light chain variable region described above includes the following three complementarity determining regions (CDRs): LCDR1: QASQSLYNQQNLA SEQ ID NO:8; LCDR2: DASELAS SEQ ID NO:9; and LCDR3: QGEFTCSGGDCIV SEQ ID NO:10; Wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and / or substituted with at least one amino acid and can retain the binding affinity for the HPV16 E6 protein.

4. An antibody light chain, characterized in that, The light chain described above has the light chain variable region and light chain constant region as claimed in claim 3.

5. An antibody, characterized in that, The antibody has: (1) The heavy chain variable region as claimed in claim 1; and / or (2) The light chain variable region as claimed in claim 3; Alternatively, the antibody has: The heavy chain as claimed in claim 2; and / or the light chain as claimed in claim 4.

6. A recombinant protein, characterized in that, The recombinant protein has: (i) The heavy chain variable region as claimed in claim 1, the heavy chain as claimed in claim 2, the light chain variable region as claimed in claim 3, the light chain as claimed in claim 4, or the antibody as claimed in claim 5; and (ii) Optionally, a tag sequence for assisting expression and / or purification.

7. A polynucleotide, characterized in that, It encodes a polypeptide selected from the group consisting of: (1) The heavy chain variable region as claimed in claim 1, the heavy chain as claimed in claim 2, the light chain variable region as claimed in claim 3, the light chain as claimed in claim 4, or the antibody as claimed in claim 5; or (2) The recombinant protein as claimed in claim 6.

8. A vector, characterized in that, The vector contains the polynucleotide as claimed in claim 7.

9. A genetically engineered host cell, characterized in that, The host cell contains the vector as claimed in claim 8 or the polynucleotide as claimed in claim 7 is integrated into the genome.

10. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) The heavy chain variable region as claimed in claim 1, the heavy chain as claimed in claim 2, the light chain variable region as claimed in claim 3, the light chain as claimed in claim 4, or the antibody as claimed in claim 5; and (b) A conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

Images