Monoclonal antibody against papillomavirus type 18 E protein

Abstract

The present invention discloses monoclonal antibodies against papillomavirus type 18 E protein. The present invention provides a heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO:1, a heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO:2, and a heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO:3; a light chain CDR1 with the amino acid sequence shown in SEQ ID NO:9, a light chain CDR2 with the amino acid sequence shown as YTS, and a light chain CDR3 with the amino acid sequence shown in SEQ ID NO:10. The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 together form the complementarity-determining regions of the monoclonal antibodies of the present invention. The present invention also provides corresponding compositions, polynucleotides, vectors, host cells, and corresponding methods and applications.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody against papillomavirus type 18 E protein. Background Art

[0002] Papillomavirus (English: Humanpapillomavirus, abbreviated: HPV) is a non-enveloped DNA virus belonging to the genus Papillomavirus of the family Papillomaviridae. This type of virus infects the epidermis and mucous membranes of the human body. Sometimes HPV can cause warts or even cancer after invading the human body, but most of the time there are no clinical symptoms. Human papillomavirus can be divided into 5 genera based on the differences in their L1 gene DNA sequences. As of September 2023, 231 types of HPV have been identified.

[0003] There are about 30 to 40 types of HPV that can infect the genitals and surrounding skin through sexual intercourse, and some of them can cause genital warts (commonly known as cauliflower). If you are repeatedly infected with certain high-risk HPV types without symptoms such as warts, you may develop precancerous lesions or even invasive cancer. According to research, 99.7% of cervical cancer is caused by infection with HPV. According to data from the International Agency for Research on Cancer, 70% of cervical cancer cases are caused by two high-risk types, HPV-16 and HPV-18.

[0004] HPV infection of the reproductive tract is a long-term process. It can remain latent in cells for several years. Once the opportunity is ripe (such as when the host's immunity is reduced), the latent virus can resume activity. The HPV infection process is usually divided into the latent infection period, subclinical infection period, clinical symptom period and HPV-related tumor period. Cervical cancer also has a series of precursor lesions, namely atypical hyperplasia of the cervical epithelium, which is pathologically called cervical intraepithelial neoplasia (CIN). It is usually divided into three levels according to the severity: mild cervical intraepithelial neoplasia (CIN I), moderate cervical intraepithelial neoplasia (CIN II) and high cervical intraepithelial neoplasia (CIN III). These precancerous lesions may develop into invasive cervical cancer. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0006] The present invention provides, in a first aspect, a monoclonal antibody that specifically binds to the E protein of human papillomavirus type 18. The monoclonal antibody comprises a heavy chain variable region, a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:9, a light chain CDR2 comprising the amino acid sequence shown as YTS, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:10.

[0007] Furthermore, the heavy chain variable region comprises a heavy chain variable region framework region, the heavy chain variable region framework region comprising a heavy chain FR1 of the amino acid sequence shown in SEQ ID NO:4 or its homologous sequence, a heavy chain FR2 of the amino acid sequence shown in SEQ ID NO:5 or its homologous sequence, a heavy chain FR3 of the amino acid sequence shown in SEQ ID NO:6 or its homologous sequence, and a heavy chain FR4 of the amino acid sequence shown in SEQ ID NO:7 or its homologous sequence; the light chain variable region comprises a light chain variable region framework region, the light chain variable region framework region comprising a light chain FR1 of the amino acid sequence shown in SEQ ID NO:11 or its homologous sequence, a light chain FR2 of the amino acid sequence shown in SEQ ID NO:12 or its homologous sequence, a light chain FR3 of the amino acid sequence shown in SEQ ID NO:13 or its homologous sequence, and a light chain FR4 of the amino acid sequence shown in SEQ ID NO:14 or its homologous sequence.

[0008] Furthermore, the monoclonal antibody comprises a heavy chain variable region amino acid sequence of the sequence shown in SEQ ID NO:8 or its homologous sequence and a light chain variable region amino acid sequence of the sequence shown in SEQ ID NO:15 or its homologous sequence.

[0009] Furthermore, the homologous sequence has a homology of more than 60% with the original sequence.

[0010] Furthermore, the homologous sequence has a homology of more than 90% with the original sequence.

[0011] Exemplarily, the homology of the homologous sequence with the original sequence is more than 60%, for example, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, etc.

[0012] Exemplarily, the heavy chain is directly connected to the light chain or connected through a linker peptide.

[0013] In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof targeting the E protein of human papillomavirus type 18 comprises a heavy chain and a light chain.

[0014] For some uses, it is desirable to obtain anti-E protein monoclonal antibodies with high affinity for the E protein. For certain uses, such as therapeutic uses, an affinity of at least about 100 nM is desirable, although antibodies with even higher affinities, such as at least about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM or even higher, may be desired. The specific exemplary anti-E protein monoclonal antibodies disclosed herein exhibit affinities in the range from 10 -6 to 10 -12 M. Anti-E protein monoclonal antibodies with affinities particularly suitable for the specific applications desired can be readily selected therefrom, or generated or designed using a variety of immunogens, complementarity-determining region (CDR) sequences, variable heavy chain VH and variable light chain VL sequences, and the methods described herein. The affinity of any specific anti-E protein monoclonal antibody can be determined using techniques well known in the art or described herein, such as ELISA, isothermal titration calorimetry (ITC), BIAcore, or fluorescence polarization assays.

[0015] Anti-E protein monoclonal antibodies can be of human or non-human origin. Examples of non-human origin anti-E protein monoclonal antibodies include, but are not limited to, those of mammalian origin (such as monkeys, rodents, goats, and rabbits). Humanized anti-E protein monoclonal antibodies are preferred for therapeutic use in humans.

[0016] The "E protein" used in the present invention is the same as the "E protein of human papillomavirus type 18", which refers to the E protein possessed by HPV-18 in HPV. It not only exists in HPV-18 but also in papillomaviruses (including HPV-1, HPV-5, HPV-8, HPV-14, HPV-20, HPV-21, HPV-25, HPV-47, HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-41, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-70, HPV-72). Therefore, in a broad sense, the "E protein" used in the present invention generally refers to the E protein in HPV viruses.

[0017] The second aspect of the present invention provides a composition comprising the monoclonal antibody described in the first aspect of the present invention and a buffer, a stabilizer, a preservative, a non-ionic detergent, and / or an antioxidant.

[0018] Buffers help maintain the pH within a range close to physiological conditions. They can be present at a concentration in the range of about 2 mM to about 50 mM. Buffers suitable for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., sodium citrate - disodium citrate mixture, citric acid - trisodium citrate mixture, citric acid - sodium citrate mixture, etc.), succinate buffers (e.g., succinic acid - sodium succinate mixture, succinic acid - sodium hydroxide mixture, succinic acid - disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid - sodium tartrate mixture, tartaric acid - potassium tartrate mixture, tartaric acid - sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid - sodium fumarate mixture, fumaric acid - disodium fumarate mixture, sodium fumarate - disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid - sodium gluconate mixture, gluconic acid - sodium hydroxide mixture, gluconic acid - potassium gluconate mixture, etc.), oxalate buffers (e.g., oxalic acid - sodium oxalate mixture, oxalic acid - sodium hydroxide mixture, oxalic acid - potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid - sodium lactate mixture, lactic acid - sodium hydroxide mixture, lactic acid - potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid - sodium acetate mixture, acetic acid - sodium hydroxide mixture, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts (such as Tris) can be used.

[0019] Preservatives can be added to retard microbial growth and can be added in an amount in the range of 0.2% - 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, octadecyl dimethyl benzyl ammonium chloride, benzalkonium halides (such as chlorides, bromides, and iodides), chlorhexidine digluconate, and alkyl p-hydroxybenzoates (such as methyl or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0020] Stabilizers (sometimes referred to as "isotonic agents") can be added to ensure the isotonicity of the liquid compositions of the present disclosure, and include polyhydric alcohols, such as trihydric alcohols or higher polyhydric alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a large class of excipients, the functions of which can range from fillers to stabilizing therapeutic agents or additives that help prevent denaturation or adhesion to the walls of the container. Typical stabilizers can be polyhydric alcohols (listed above); amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclic polyols, such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-thioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or fewer residues); proteins, such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides, such as lactose, maltose, sucrose; trisaccharides, such as melezitose; polysaccharides, such as dextran. The stabilizer can be present in an amount of 0.1 to 10,000 parts by weight per part by weight of the active protein.

[0021] Nonionic surfactants or detergents (also referred to as "wetting agents") can be added to aid in dissolving the therapeutic agent, as well as to protect the therapeutic protein from agitation-induced aggregation, and which also allows the formulation to be exposed to a shear stress surface without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), polyols, and polyoxyethylene sorbitan monoethers. The nonionic surfactant can be present in a range of about 0.05 mg / ml to about 1.0 mg / ml, such as about 0.07 mg / ml to about 0.2 mg / ml.

[0022] The third aspect of the present invention provides a polynucleotide encoding the light chain variable region CDR of the monoclonal antibody described in the first aspect of the present invention.

[0023] Furthermore, the polynucleotide of the third aspect of the present invention comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:9, YTS, SEQ ID NO:10 or its degenerate sequence.

[0024] Furthermore, the polynucleotide of the third aspect of the present invention encodes the light chain variable region framework region of the monoclonal antibody described in the first aspect of the present invention.

[0025] Furthermore, the polynucleotide of the third aspect of the present invention comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or its degenerate sequence.

[0026] Furthermore, the polynucleotide of the third aspect of the present invention encodes the light chain variable region of the aforementioned monoclonal antibody.

[0027] Furthermore, the polynucleotide of the third aspect of the present invention encodes the nucleotide sequence of the amino acid sequence shown in SEQ ID NO:15 or its degenerate sequence.

[0028] Furthermore, the degenerate sequence has a homology of more than 60% with the original sequence.

[0029] Furthermore, the degenerate sequence has a homology of more than 90% with the original sequence.

[0030] Exemplarily, the degenerate sequence has a homology of more than 60% with the original sequence, for example, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, etc.

[0031] The fourth aspect of the present invention provides a polynucleotide encoding the heavy chain variable region CDR of the monoclonal antibody described in the first aspect of the present invention.

[0032] Furthermore, the polynucleotide of the fourth aspect of the present invention comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or a degenerate sequence thereof.

[0033] Furthermore, the polynucleotide of the fourth aspect of the present invention encodes the heavy chain variable region framework region of the monoclonal antibody described in the first aspect of the present invention.

[0034] Furthermore, the polynucleotide of the fourth aspect of the present invention comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or a degenerate sequence thereof.

[0035] Furthermore, the polynucleotide of the fourth aspect of the present invention encodes the heavy chain variable region of the monoclonal antibody described in the first aspect of the present invention.

[0036] Furthermore, the polynucleotide of the fourth aspect of the present invention encodes the nucleotide sequence of the amino acid sequence shown in SEQ ID NO:8 or a degenerate sequence thereof.

[0037] Furthermore, the degenerate sequence has a homology of more than 60% with the original sequence.

[0038] Furthermore, the degenerate sequence has a homology of more than 90% with the original sequence.

[0039] The fifth aspect of the present invention provides a vector comprising the polynucleotide described in the third aspect of the present invention or the polynucleotide described in the fourth aspect of the present invention.

[0040] In some embodiments, an expression vector and expression control sequences compatible with the expression host cell used are selected. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or usually both genes are inserted into the same expression vector.

[0041] In some embodiments, the antibody gene is inserted into an expression vector by standard methods (e.g., ligation of the antibody gene fragment and complementary restriction sites on the vector, or blunt-end ligation when restriction sites are absent). Before inserting the light or heavy chain sequence related to the anti-E protein monoclonal antibody, the expression vector may already carry the antibody constant region sequence. For example, one way to convert the VH and VL sequences related to the anti-E protein monoclonal antibody into a full-length antibody gene is to insert them separately into an expression vector that already encodes the heavy chain constant region and the light chain constant region, such that the VH segment is effectively linked to one or more CH segments in the vector, and the VL segment is effectively linked to the CL segment in the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is in-frame with the amino acids of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0042] The sixth aspect of the present invention provides a host cell transformed with a polynucleotide, polynucleotide pair, or vector suitable for expressing the monoclonal antibody described in the first aspect of the present invention.

[0043] In some embodiments, for recombinant expression of an antibody, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cell and optionally secreted into the culture medium in which the host cell is grown, and the antibody can be recovered from the medium. Standard recombinant DNA methods are used to obtain the antibody heavy and light chain genes, incorporate these genes into a recombinant expression vector, and introduce the vector into the host cell, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis, Cold Spring Harbor, N.Y., 1989); Current Protocols in Molecular Biology (edited by Ausubel, F.M. et al., Greene Publishing Associates, 1989); and U.S. Patent No. 4,816,397.

[0044] In some embodiments, the host cell capable of producing a monoclonal antibody against the E protein is a hybridoma. Methods for generating hybridomas are known in the art (see, e.g., Kohler and Milstein, 1975, Nature 256:495), and examples are provided below. Generally, a host animal (such as a mouse) is immunized with an immunogen (such as a peptide of interest) to elicit the development of lymphocytes (such as splenocytes) that produce antibodies capable of specifically binding to the immunogen. Alternatively, isolated lymphocytes (including splenocytes, lymph node cells, or peripheral blood lymphocytes) can be immunized in vitro. The lymphocytes are then fused with an immortalized cell line (such as a myeloma cell line) using a suitable fusogen (such as polyethylene glycol) to form a hybridoma cell line. Suitable immortalized cell lines can be of mammalian origin, such as murine, bovine, or human. The hybridoma cells are then cultured in any suitable medium that contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, when using parental cells lacking hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the fusion can be cultured in a medium containing hypoxanthine, aminopterin, and thymidine ("HAT" medium) that inhibits the growth of parental unfused cells.

[0045] The seventh aspect of the present invention provides a method for preparing the monoclonal antibody described in the first aspect of the present invention, the polynucleotide described in the third aspect of the present invention, or the monoclonal antibody fragment expressed by the polynucleotide described in the fourth aspect of the present invention, comprising: expressing the polynucleotide described in the third aspect of the present invention or the polynucleotide described in the fourth aspect of the present invention in a host cell.

[0046] The eighth aspect of the present invention provides a kit for detecting human papillomavirus type 18 E protein or human papillomavirus, which comprises the monoclonal antibody described in the first aspect of the present invention.

[0047] Furthermore, a detectable label is linked to the monoclonal antibody, and the detectable label does not affect the function of the monoclonal antibody itself.

[0048] Furthermore, the aforementioned monoclonal antibody can be used in the detection of human papillomavirus, for example, to detect the expression of the target in specific cells, tissues, or sera.

[0049] Furthermore, the kit can be used as part of a clinical test method to monitor the development or progression of an immune response, for example, to determine the efficacy of a given treatment regimen.

[0050] In some embodiments, detection can be facilitated by coupling the antibody to a detectable substance or "label". The label can be conjugated directly or indirectly to the anti-E protein monoclonal antibody of the present disclosure. The label can be detectable itself (e.g., radioisotope label, isotope label, or fluorescent label), or in the case of an enzyme label, can catalyze a detectable chemical change in a substrate compound or composition. Examples of detectable substances include a variety of enzymes, cofactors, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron-emitting metals and non-radioactive paramagnetic metal ions using various positron imaging techniques. The detectable substance can be coupled or conjugated to the antibody (or fragment thereof) directly or through an intermediate (e.g., a linker known in the art) using techniques known in the art. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases (such as horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, acetylcholinesterase, glucoamylase, lysozyme, glucose oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Examples of suitable cofactor complexes include streptavidin / biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinyl fluorescein, dansyl chloride, dimethylamine-1-naphthalenesulfonyl chloride, or phycoerythrin, etc.; examples of luminescent substances include luminol; examples of bioluminescent substances include luciferase, fluorescein, and aequorin; examples of suitable isotopic substances include 13 C, 15 N, and deuterium; examples of suitable radioactive substances include 125 I, 131 I, 111 In, or 99 Tc.

[0051] Furthermore, the papillomavirus includes HPV-1, HPV-5, HPV-8, HPV-14, HPV-20, HPV-21, HPV-25, HPV-47, HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-41, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-70, HPV-72.

[0052] Furthermore, the papillomavirus includes HPV-18.

[0053] The ninth aspect of the present invention provides the use of the monoclonal antibody described in the first aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the polynucleotide described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, or the host cell described in the sixth aspect of the present invention in the preparation of a product for detecting papillomavirus, papillomavirus type 18 E protein or a fragment thereof.

[0054] Furthermore, the papillomavirus includes HPV-1, HPV-5, HPV-8, HPV-14, HPV-20, HPV-21, HPV-25, HPV-47, HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-41, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-70, HPV-72.

[0055] Furthermore, the papillomavirus includes HPV-18.

[0056] The tenth aspect of the present invention provides a method for detecting HPV (papillomavirus), which includes the step of contacting a sample to be detected with the monoclonal antibody provided in the first aspect of the present invention.

[0057] The eleventh aspect of the present invention provides a method for treating and preventing HPV (papillomavirus), which includes administering to a subject an effective amount of the monoclonal antibody provided in the first aspect of the present invention for treating the HPV.

[0058] Treatment methods generally include administering to a subject in need of treatment (such as a subject diagnosed with HPV) an effective amount of an anti-E protein monoclonal antibody and / or its pharmaceutical composition that provides a therapeutic benefit. The therapeutic benefits described in more detail below include any improvement in HPV diseases, such as slowing or stopping the progression of HPV, reducing the severity of HPV onset, inhibiting the growth or proliferation of the HPV virus, and reducing the HPV virus serum level in HPV patients. The subject can be human or non-human, including domestic animals (such as cats, dogs, cows, pigs, horses) or non-domestic animals. Preferably, the anti-E protein monoclonal antibody is specific for the HPV of the species being treated. For example, an anti-human E protein antibody is administered to a human patient, and an anti-dog E protein antibody is administered to a canine patient, etc. The subject using anti-human E protein monoclonal antibody therapy can be a patient at any stage of disease progression.

[0059] The twelfth aspect of the present invention provides a method for inhibiting the HPV virus in vitro cells or tissues, which includes exposing the cells or tissues containing the HPV virus to the monoclonal antibody provided in the first aspect of the present invention.

[0060] The term "monoclonal antibody" as used in the present invention can be in the form of a full-length antibody, a multichain or single-chain antibody, a fragment of such an antibody that selectively binds to the E protein (including but not limited to Fab, Fab′, (Fab′)2, Fv, and scFv), a surrobody (including a surrogate light chain construct), a single-domain antibody, a humanized antibody, a camelized antibody, etc. They can also belong to or be derived from any isotype, including for example IgA (such as IgA1 or IgA2), IgD, IgE, IgG (such as IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-E protein antibody is IgG (such as IgG1, IgG2, IgG3, or IgG4).

[0061] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) by any means available or known in the art. A variety of techniques known in the art (including the use of hybridoma technology, recombinant technology, and phage display technology or combinations thereof) can be used to prepare monoclonal antibodies for the present disclosure. In many uses of the present disclosure (including in vivo uses and in vitro detection assays of anti-E protein monoclonal antibodies in humans), chimeric antibodies, primatized antibodies, humanized antibodies, or human antibodies can be suitably used.

[0062] The CDRs used in the present invention are also referred to as hypervariable regions in both the light and heavy chain variable domains. The CDRs are the complementarity-determining regions of a monoclonal antibody. In addition, the more highly conserved portions of the variable domains in a monoclonal antibody are called framework regions (FRs). As is known in the art, depending on the context and the various definitions known in the art, the amino acid positions / boundaries describing the hypervariable regions of an antibody can vary. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered within the hypervariable region under one set of criteria and outside the hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The present disclosure provides antibodies that contain modifications at these hybrid hypervariable positions. The variable domains of the native heavy and light chains each contain four FR regions, which mainly adopt a β-sheet conformation and are connected by three CDRs, which form loop connections and, in some cases, part of a β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs from other chains, contribute to the formation of the antibody target-binding site.

[0063] The present invention also includes monoclonal antibodies for diagnostic and therapeutic applications that are derived, covalently modified, or conjugated to other molecules. For example, but not by way of limitation, derived antibodies include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, protease cleavage, ligation to cell ligands or other proteins, etc. Any of a number of chemical modifications can be carried out by known techniques (including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis with tunicamycin, etc.). In addition, the derivatives can contain one or more non-classical amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 is the electrophoretogram of monoclonal antibody 4F2;

[0065] Figure 2 is the HPLC chromatogram of monoclonal antibody 4F2;

[0066] Figure 3 is the active binding diagram of monoclonal antibody 4F2. DETAILED DESCRIPTION OF THE INVENTION

[0067] The following description of the embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

[0068] Example 1

[0069] 1. Immunogen recombinant expression

[0070] Synthesize the human papillomavirus type 18 E protein sequence, construct it into the pEM5.1 vector; extract the plasmid for transfection; transfect it into HEK293 cells and culture the cells for 7 days; harvest the supernatant, purify it with an Ni column, and concentrate and replace the buffer to obtain the recombinant human papillomavirus type 18 E protein.

[0071] The recombinant human papillomavirus type 18 E protein sequence is from Uniprot,

[0072] The sequence information is as follows:

[0073] MADPEGTDGEGTGCNGWFYVQAIVDKKTGDVISDDEDENATDTGSDMVDFIDTQGTFCEQAELETAQALFHAQEVHNDAQVLHVLKRKFAGGSTENSPLGERLEVDTELSPRLQEISLNSGQKKAKRRLFTISDSGYGCSEVEATQIQVTTNGEHGGNVCSGGSTEAIDNGGTEGNNSSVDGTSDNSNIENVNPQCTIAQLKDLLKVNNKQGAMLAVFKDTYGLSFTDLVRNFKSDKTTCTDWVTAIFGVNPTIAEGFKTLIQPFILYAHIQCLDCKWGVLILALLRYKCGKSRLTVAKGLSTLLHVPETCMLIQPPKLRSSVAALYWYRTGISNISEVMGDTPEWIQRLTIIQHGIDDSNFDLSEMVQWAFDNELTDESDMAFEYALLADSNSNAAAFLKSNCQAKYLKDCATMCKHYRRAQKRQMNMSQWIRFRCSKIDEGGDWRPIVQFLRYQQIEFITFLGALKSFLKGTPKKNCLVFCGPANTGKSYFGMSFIHFIQGAVISFVNSTSHFWLEPLTDTKVAMLDDATTTCWTYFDTYMRNALDGNPISIDRKHKPLIQLKCPPILLTTNIHPAKDNRWPYLESRITVFEFPNAFPFDKNGNPVYEINDKNWKCFFERTWSRLDLHEEEEDADTEGNPFGTFKLRAGQNHRPL(SEQ ID NO:16)

[0074] 2. Immunity

[0075] Immunize mice, perform SP2 / 0 fusion, screen, and subclone. The specific steps are as follows:

[0076] For the first immunization, use Freund's complete adjuvant at a dose of 100 μg per mouse, inject intraperitoneally, with a total dose of 0.5 ml per mouse, and perform the second immunization after a 3-week interval; starting from the second immunization, use Freund's incomplete adjuvant at a dose of 50 μg / 0.5 ml per mouse, and perform the third immunization after a 2-week interval; prepare for cell fusion 10 days after the third injection.

[0077] Take feeder cells, which can be used at 10 5 / well, and plate them one day before fusion at 10 5cells / 100 μl / well; Take the immunized spleen cells of mice and fuse them with the prepared myeloma cells using the fusogen PEG, and plate them into a 96-well cell culture plate that has been added with feeder cells, 100 μl / well.

[0078] Screen positive wells by ELISA detection method, plate recombinant human papillomavirus type 18 E protein overnight; wash the plate, add skim milk powder for blocking, at 37 °C for 1 h; wash the plate, add 100 μl of the supernatant of the 96-well culture medium, incubate at 37 °C for 1 h; wash the plate, add HRP-labeled goat anti-mouse secondary antibody, incubate at 37 °C for 30 min; wash the plate, add the chromogenic solution, develop color for 10 min, add the stop solution, and read the OD450 value; screen cell lines with high expression levels for subcloning.

[0079] 3. Sequence Retrieval

[0080] Collect cells, extract RNA, reverse transcribe, design primers, perform PCR, transform, pick clones, send for sequencing, human papillomavirus type 18 E protein antibody, targeting the E protein. Clone number 4F2, and the specific sequence is shown in Table 1.

[0081] Table 1

[0082]

[0083]

[0084] 4. Antibody Expression

[0085] Expression and purification of monoclonal antibodies:

[0086] (1) Chemically synthesize the screened sequence and clone it into a eukaryotic expression vector.

[0087] (2) Amplify the plasmid and extract the plasmid.

[0088] (3) Transiently transfect the plasmid encoding the antibody into mammalian cells HEK293.

[0089] (4) Collect the supernatant and purify the monoclonal antibody using affinity chromatography.

[0090] Perform transient transfection using HEK293 cells and use PEI for transfection. The expression level of monoclonal antibody 4F2 is 187 mg / L.

[0091] 5. Physicochemical Properties

[0092] 5.1 Gel Electrophoresis Detection

[0093] A. Sample Preparation

[0094] Mix 20 μL of the sample evenly with 5 μL of 5× reducing buffer, heat it at 95 °C for 5 min, and then cool it; mix 20 μL of the sample evenly with 5 μL of 5× non-reducing buffer.

[0095] B. Electrophoresis

[0096] Prepare the gel, add an appropriate amount of electrophoresis buffer, load the sample, and perform electrophoresis.

[0097] C. Staining and Decolorization

[0098] After electrophoresis, put the gel into an appropriate amount of Coomassie Brilliant Blue staining solution and stain it at room temperature for 1 h or longer; pour out the staining solution, add an appropriate amount of Coomassie Brilliant Blue staining decolorization solution, and decolorize it at room temperature for 4 - 24 h. After completion of decolorization, soak it in ddH 2 O, refer to the Marker protein, compare it with the unstained gel, cut out the gel of the required protein component, and collect it. Then separate the protein to be purified from the gel.

[0099] 5.2 HPLC Detection of the Purity of Monoclonal Antibody

[0100] A. Preparation of Mobile Phase

[0101] Add dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride to about 900 mL of purified water, stir to dissolve, make up the volume to 1 L, measure with a pH meter, and ensure that its pH is between 6.2 ± 0.1. Filter with a 0.22 μm filter membrane and store at room temperature.

[0102] B. Sample Preparation

[0103] System suitability sample: Dilute the standard product to 2 mg / mL with the mobile phase;

[0104] Test sample: Dilute the sample to be tested to 2 mg / mL with the mobile phase.

[0105] Perform detection under conventional chromatographic conditions.

[0106] The results are as Figure 1 and Figure 2 shown. The results show that the electrophoresis pattern and the purity detected by liquid phase of the monoclonal antibody 4F2 against human papillomavirus type 18 E protein are both greater than 95%.

[0107] 6. Binding Activity

[0108] Detection of the binding activity of the monoclonal antibody:

[0109] A. Coating: Dilute the E protein of human papillomavirus type 18 to 2 μg / ml with the coating solution, mix well, add it to a 96-well coating plate, 100 μl / well, seal the plate, and incubate overnight at 4 °C.

[0110] B. Wash the plate 3 times. There should be no liquid remaining on the plate after the last wash. Pat the liquid on the surface of the plate dry with absorbent paper.

[0111] C. Blocking: Add 5% milk powder (0.5 g milk powder dissolved in 10 mL DPBS), 300 μL per well, incubate at 37 °C for 1 h, and wash the plate 3 times according to step B.

[0112] D. Gradient dilute the antibody, 100 μL per well, react at 37 °C for 1 h, and wash the plate 3 times according to step B.

[0113] E. Add secondary antibody: Dilute with DPBS at a ratio of 1:2000, add to the 96-well plate, 100 μL per well, react at 37 °C for 1 h, and wash the plate 3 times according to step B.

[0114] F. Color development: Add TMB, 100 μL per well, develop color at room temperature in the dark for 10 min.

[0115] G. Termination: Add 2N H 2 SO 4 , 100 μL per well.

[0116] H. Measure OD450 with an enzyme-linked immunosorbent assay reader within 10 min.

[0117] Coat with VP1 protein, add antibody, add secondary antibody, develop color, read the value, and perform data analysis.

[0118] The results are as Figure 3 shown. The results show that the EC50 of the monoclonal antibody 4F2 against papillomavirus type 18 E protein is 1.5 ng / ml.

Claims

1. A monoclonal antibody that specifically binds to papillomavirus type 18 E protein, the monoclonal antibody comprising a heavy chain variable region, comprising a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO: 3; and a light chain variable region, comprising a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 9, a light chain CDR2 with an amino acid sequence as shown in YTS, and a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO:

10.

2. The monoclonal antibody according to claim 1, wherein the heavy chain variable region comprises a heavy chain variable region framework region, wherein the heavy chain variable region framework region comprises a heavy chain FR1 of the amino acid sequence shown in SEQ ID NO:4 or a homologous sequence thereof, a heavy chain FR2 of the amino acid sequence shown in SEQ ID NO:5 or a homologous sequence thereof, a heavy chain FR3 of the amino acid sequence shown in SEQ ID NO:6 or a homologous sequence thereof, and a heavy chain FR4 of the amino acid sequence shown in SEQ ID NO:7 or a homologous sequence thereof; the light chain variable region comprises a light chain variable region framework region, wherein the light chain variable region framework region comprises a light chain FR1 of the amino acid sequence shown in SEQ ID NO:11 or a homologous sequence thereof, a light chain FR2 of the amino acid sequence shown in SEQ ID NO:12 or a homologous sequence thereof, a light chain FR3 of the amino acid sequence shown in SEQ ID NO:13 or a homologous sequence thereof, and a light chain FR4 of the amino acid sequence shown in SEQ ID NO:14 or a homologous sequence thereof; and the homology between the homologous sequence and the original sequence is greater than 60%.

3. The monoclonal antibody according to claim 1, comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 8 or a homologous sequence thereof and a light chain variable region amino acid sequence of SEQ ID NO: 15 or a homologous sequence thereof, wherein the homology between the homologous sequence and the original sequence is greater than 90%.

4. A composition comprising the monoclonal antibody according to any one of claims 1 to 3 and a buffer, a stabilizer, a preservative, a non-ionic detergent, and / or an antioxidant.

5. A polynucleotide encoding the monoclonal antibody according to any one of claims 1 to 3. A vector comprising the polynucleotide according to claim 5.

7. A host cell transformed with a polynucleotide, a polynucleotide pair or a vector suitable for expressing the monoclonal antibody according to any one of claims 1 to 3.

8. The monoclonal antibody according to any one of claims 1 to 3, or a method for preparing a monoclonal antibody fragment expressed by the polynucleotide according to claim 5, comprising: Express the polynucleotide of claim 5 in a host cell.

9. A kit for detecting papillomavirus type 18 E protein or papillomavirus HPV-18, comprising the monoclonal antibody according to any one of claims 1 to 3.

10. The kit according to claim 9, wherein the monoclonal antibody is linked to a detectable label, and the detectable label does not affect the function of the monoclonal antibody itself.

11. Use of the monoclonal antibody according to any one of claims 1 to 3, the polynucleotide according to claim 5, the vector according to claim 6, or the host cell according to claim 7 in the preparation of a product for detecting HPV-18 and papillomavirus type 18 E protein.

Images