WO2019028980A1 - 抗人乳头瘤病毒和抗cd人源化的双靶向抗体及其组合物、制备方法和应用 - Google Patents

抗人乳头瘤病毒和抗cd人源化的双靶向抗体及其组合物、制备方法和应用 Download PDF

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WO2019028980A1
WO2019028980A1 PCT/CN2017/103683 CN2017103683W WO2019028980A1 WO 2019028980 A1 WO2019028980 A1 WO 2019028980A1 CN 2017103683 W CN2017103683 W CN 2017103683W WO 2019028980 A1 WO2019028980 A1 WO 2019028980A1
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human
antibody
murine
heavy chain
light chain
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French (fr)
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包晟
杨荣鉴
谢亦武
曹天福
龚芹
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深圳市雅臣智能生物工程有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody

Definitions

  • the present invention relates to the technical field of genetic engineering, and more particularly to a dual-targeted antibody against human papillomavirus and anti-CD humanization, and a composition, preparation method and application thereof.
  • HPV infection is most common among young people between the ages of 18 and 28. It can be said that more than 50% of all people who are active in sexual life will develop genital HPV infection at some point in their lives. The vast majority of women with a ratio of up to 80% have had HPV infection before the age of 50. According to statistics from the World Health Organization, the total number of infected people worldwide is about 630 million, accounting for 10% of the total population, of which women are the overwhelming majority; this is a shockingly large number.
  • HPV infections have no signs and symptoms; however, the virus can be transmitted through sexual life, which increases the risk of widespread transmission of the virus.
  • Teen who has sex before the age of 16 and who has multiple sexual partners with his or her spouse will become a high-risk group of HPV.
  • people with low immunity for whatever reason are also susceptible to HPV, such as women with multiple pregnancies, HIV (HIV), organ transplants or chemotherapy, and smoking. , drug and nutrition Good women wait.
  • HPV infection and induced cervical cancer constitute a great threat to women's lives. Therefore, research and development of a new drug that can prevent and treat human papillomavirus (HPV) infection, which is both preventable and treatable, is a major event affecting the health and life of hundreds of millions of women around the world. It is also a front of the international medical science interface. important topic.
  • the technical solution adopted by the present invention to solve the technical problem is: a dual-targeting antibody against human papillomavirus and anti-CD humanization, including a full-length gene sequence of a mouse-human chimeric antibody light chain cloned in a vector and a mouse a human chimeric antibody heavy chain full-length gene sequence obtained by sequencing the antibody light chain obtained by immunization with a related antigen, the murine-human chimeric antibody heavy chain
  • the long gene sequence is obtained by sequencing the antibody heavy chain obtained after immunization with the relevant antigen.
  • the vector is a gene vector or an expression vector.
  • the invention also provides a composition comprising the above-described anti-human papillomavirus and anti-CD humanized dual targeting antibody and a pharmaceutically acceptable adjuvant.
  • the method before the step S1, the method further comprises S0, preparing a two-hybrid Tumor cells:
  • Purification of anti-human papillomavirus (HPV) and anti-CD humanization by purification of purified virus-like particles (VLPs or cVLPs) and recombinant human CD3 protein or other types of recombinant human CD protein by mouse immunization and cell fusion Hybridoma cells with dual targeting antibodies.
  • VLPs or cVLPs virus-like particles
  • recombinant human CD3 protein or other types of recombinant human CD protein by mouse immunization and cell fusion Hybridoma cells with dual targeting antibodies.
  • step S1 the method specifically includes:
  • the total RNA of the two-hybridoma cells was extracted, cDNA was reverse transcribed, and the sequence of the murine antibody light chain variable region and the murine antibody heavy chain variable region were amplified respectively.
  • the total RNA was extracted by lymphocytes and prepared by reverse transcription. cDNA, amplifying a human antibody light chain constant region and a human antibody heavy chain constant region sequence, respectively;
  • step S2 specifically:
  • the murine-human chimeric antibody light chain full-length gene sequence and the murine-human chimeric antibody heavy chain full-length gene sequence are cloned by a viral gene vector or a non-viral gene vector, and are large by insect cells or other cells.
  • Scale-scale fermentation production obtained after purification to obtain genotype anti-human papillomavirus and anti-CD humanized double targeting antibody;
  • the viral gene vector comprises baculovirus;
  • the non-viral gene vector comprises plasmid DNA, micro Circular DNA, naked DNA, liposome and liposome complex, cationic high polymer, ligand-mediated targeting vector, free carrier;
  • the present invention also provides the above-mentioned anti-human papillomavirus and anti-CD humanized dual targeting antibody in the preparation of a medicament for eliminating human papillomavirus (HPV) infection, and/or for preventing and treating cervical cancer, Applications in disinfection products, health products and medical devices.
  • HPV human papillomavirus
  • anti-human papillomavirus and anti-CD humanized dual targeting antibody of the present invention are further described below with reference to the examples:
  • the present invention provides an anti-human papillomavirus and an anti-CD humanized dual targeting antibody comprising a murine-human chimeric antibody light chain full-length gene sequence cloned in a vector and a murine-human chimeric An antibody heavy chain full-length gene sequence obtained by sequencing the antibody light chain obtained by immunization with a related antigen, wherein the murine-human chimeric antibody heavy chain full-length gene sequence is passed The antibody obtained after immunization with the relevant antigen was obtained by heavy chain sequencing.
  • the vector may be a gene vector or an expression vector, but is not limited thereto.
  • the present invention humanizes the above-mentioned dual targeting antibody, and then produces the antibody protein by large-scale fermentation by CHO cells, or expresses in an insect cell expression system and mass-produces high-purity containing human papillomavirus and anti-CD humanization.
  • a dual-targeted antibody which uses a high-purity anti-human papillomavirus or an anti-CD humanized dual-targeted antibody in the form of a protein or gene to suppress human papillomavirus (HPV), thereby eliminating human papillomavirus ( HPV), the purpose of prevention and adjuvant treatment of cervical cancer.
  • the preparation method of the above-mentioned anti-human papillomavirus and anti-CD humanized double targeting antibody is as follows.
  • Methods for preparing dual targeting antibodies include hybridoma technology, chemical cross-linking methods, and genetic engineering techniques.
  • the present invention is described by taking hybridoma technology and genetic engineering technology as an example, but is not limited to the following description.
  • L1, L2 and E6, E7 protein expressed in eukaryotic cells or prokaryotic cells L1, L2 and E6, E7 can also be chimeric and expressed in true Nuclear cells or prokaryotic cells, self-assembled into virus-like particles (VLPs) and purified to obtain purified virus-like particles (VLPs or cVLPs);
  • HPV virus is at least one of the following: HPV 6, 11, 16, 18, 31, 33, 45, 52, 58.
  • Other types of HPV viruses can also be selected according to different HPV viruses that are popular in the local area;
  • Recombinant human CD3 protein or other recombinant human CD protein is purified or purchased by using E. coli to express human CD3 recombinant protein or other human CD recombinant protein;
  • VLPs or cVLPs purified virus-like particles
  • recombinant human CD protein human CD3 recombinant protein or other kinds of human CD recombinant protein
  • HPV neoplastic virus
  • variable region sequence and constant region sequence extracting total RNA of the two-hybridoma cells, preparing cDNA by reverse transcription, and respectively amplifying the sequences of light chain and heavy chain of the variable region of the mouse antibody; taking lymph The cells extract total RNA, reverse-transcribe cDNA, and respectively amplify the light chain and heavy chain sequences of the constant region of the human antibody;
  • Cloning of the full-length gene of the light chain and the heavy chain overlapping and extending the murine antibody light chain variable region and the human antibody light chain constant region to obtain a murine-human chimeric antibody light chain full-length gene sequence;
  • the murine antibody heavy chain variable region and the human antibody heavy chain constant region are obtained, and a murine-human chimeric antibody heavy chain full-length gene sequence is obtained;
  • the preparation process of the genotype anti-human papillomavirus and the anti-CD humanized dual targeting antibody comprises: the murine-human chimeric antibody light chain full-length gene sequence and the murine-human chimeric antibody are heavy
  • the full-length gene sequence of the chain is cloned by a viral gene vector or a non-viral gene vector; then, it is mass-produced by insect cell fermentation, and purified to obtain genotype anti-human papillomavirus and anti-CD humanized double target.
  • the preparation process of the protein type anti-human papillomavirus and the anti-CD humanized double targeting antibody comprises: the mouse-human chimeric antibody light chain full-length gene sequence and the mouse-human chimeric antibody
  • the full-length gene sequence of the heavy chain is cloned under the two promoters of the expression vector pFast-Bac-Dual to obtain a recombinant plasmid; the recombinant plasmid is transfected into CHO cells or expressed in an insect cell expression system to obtain an anti-protein type antibody.
  • Human papillomavirus (HPV) And anti-CD humanized dual targeting antibodies are examples of the human papillomavirus (HPV) And anti-CD humanized dual targeting antibodies.
  • Expanded culture select highly expressed stable transfected cell lines, ferment by stepwise amplification of cell reactor, filter and collect supernatant and remove cells and debris;
  • Antibody purification The supernatant was captured by cation exchange chromatography to capture genotypes of anti-human papillomavirus (HPV) and anti-CD humanized dual targeting antibodies, which were further purified and purified by anion exchange chromatography. The buffer was replaced by ultrafiltration to prepare a protein of high purity microcirculation DNA against human papillomavirus (HPV) and an anti-CD humanized dual targeting antibody.
  • HPV human papillomavirus
  • anti-CD humanized dual targeting antibody The buffer was replaced by ultrafiltration to prepare a protein of high purity microcirculation DNA against human papillomavirus (HPV) and an anti-CD humanized dual targeting antibody.
  • the excipient comprises one or more of excipients, fillers, solvents, solubilizers, surfactants, and capsule excipients.
  • the formulation is a vaginal gel, a suppository, a lotion, and the like.
  • the preparation is an injection, a tablet, a spray, a powder, a chewing gum, an oral solution or a capsule, a dressing, and the like.
  • the aforementioned genotype or protein type of anti-human papillomavirus (HPV) and anti-CD humanized dual targeting antibody (or nanoliposome) are at least one of water for injection and powder injection. It can be used for the following purposes: those who have been infected with human papillomavirus (HPV) "yang to yin"; prevent cervical cancer patients from recurrence after surgery or after chemotherapy and chemotherapy; prevention and adjuvant treatment of cervical cancer.
  • the anti-human papillomavirus (HPV) and anti-CD humanized dual targeting antibodies of the present invention incorporate four cutting-edge technologies for global antibody engineering to carry out four innovations.
  • HPV vaccine has only preventive effect and has no therapeutic effect. It only has certain preventive effect on people who have not been infected with HPV virus. The effect of injecting this vaccine on people who have been infected with the HPV virus will be greatly reduced, and the vast majority of women have been infected with the HPV virus before the age of 50. Obviously, the vaccine has a limited preventive effect. It is also unrealistic to require every woman, especially a young and healthy adolescent, to be forced to inject the vaccine before it is infected; this vaccine does not give the injector a lifelong immunization.
  • HPV vaccine is ineffective, and HPV is recurring after surgical resection of cervical cancer.
  • the vaccine is also powerless. While the present invention alters the conventional manner in which an injection vaccine stimulates the production of antibodies in the human body, a passive immunization method in which a highly active antibody is directly introduced into the human body is employed. Since the anti-human papillomavirus (HPV) and the anti-CD humanized dual targeting antibody of the present invention belong to the class of monoclonal antibodies, the titer of the monoclonal antibody is generally higher than the antibody produced by the vaccine in vivo.
  • HPV anti-human papillomavirus
  • the anti-CD humanized dual targeting antibody of the present invention belong to the class of monoclonal antibodies, the titer of the monoclonal antibody is generally higher than the antibody produced by the vaccine in vivo.
  • the anti-human papillomavirus and the anti-CD humanized double-targeted antibody provided by the invention can completely eliminate the HPV virus in the body and effectively block the HPV virus infection, and can quickly realize the "yang to yin". It can also prevent recurrence after resection; it can also produce corresponding antibodies according to different models of human papillomavirus (HPV) popular in various countries, so as to achieve targeted killing of "check-in” and achieve better results.
  • HPV human papillomavirus
  • the anti-human papillomavirus and anti-CD humanized dual targeting antibodies provided by the present invention are a better alternative to vaccines and are an innovation in traditional prevention methods.
  • Double-targeted antibody increases a specific antigen-binding site compared with the common antibody, and exhibits the following advantages in treatment: the two antigen-binding sites respectively bind to human papillomavirus (HPV) ) and the immune cells to produce a bridging effect, which can directly interact with human papillomavirus (HPV), activate the functions of immune cells such as T cells and NK cells, increase the concentration of local NK cells or T cells, and enhance the killing ability of effect molecules.
  • HPV human papillomavirus
  • murine monoclonal antibodies have a strong antibody effect, if a mouse monoclonal antibody is injected directly into the human body, it will induce an anti-mouse antibody reaction (meaning that the murine monoclonal antibody has a heterologous protein to other animals).
  • the immunogenicity is quickly eliminated in the circulatory system of the human body, resulting in a short half-life in the body.
  • the invention modifies the prepared anti-human papillomavirus and the anti-CD humanized double targeting antibody, and retains the high affinity of the antigen and the antibody, and removes most of the mouse gene sequence to humanize the gene.
  • Microcircle DNA vector When the anti-human papillomavirus and the anti-CD humanized double-targeted antibody are cloned using a non-viral gene vector, microcircle DNA expression can be specifically selected.
  • the microcircle DNA is a small supercoiled plasmid, and the prokaryotic skeleton DNA which produces a "dumb (sleeping effect)" is removed from the ordinary plasmid, thereby greatly increasing the expression level and sustainable expression.
  • the use of the microcircle DNA vector requires only the L-arabinose concentration of 1/100 to 1/1000 in the original system, which can greatly reduce the cost; at the same time, the microcircle DNA itself is not infectious, and does not have hidden dangers to biosafety. The sequence, such as the antibiotic gene, is therefore very safe.
  • microcircle DNA vector Another feature of the microcircle DNA vector is that the antibody is directly transfected into the human body, and can be continuously replicated and expressed by the protein synthesis mechanism in the human body, and the microcircular DNA anti-human papillomavirus and anti-CD humanized are continuously produced.
  • the dual-targeted antibody achieves a long-term passive immune effect, and solves the problem that the antibody protein needs to be repeatedly injected due to a short half-life. In this way, human papillomavirus (HPV) in the human body can be completely eliminated, and the goal of eradicating cervical cancer can be achieved.
  • HPV human papillomavirus
  • HPV viruses such as 6, 11, 16, 18, 31, 33, 45, 52 and 58 can be divided into three groups, namely 16, 18, 31 as a group (high risk group), 45 (high risk), 6 (common), 11 (common) are a group (mixed group), and 33, 52, 58 are a group (middle) Risk group).
  • HPV6, 11, 16, 18, 31, 33, 45, 52, 58 and other L1 proteins were used as detection antigens, and the anti-human papillomavirus and anti-antibody were detected by "ELISA” method (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • the concentration of the anti-human papillomavirus and anti-CD humanized dual targeting antibody solution in the test sample was 1 mg/mL.
  • Antibody binding titer detection of CD against human papillomavirus (HPV) and anti-CD humanized dual targeting antibodies Take the CD3 antigen as an example.
  • the concentration of anti-human papillomavirus (HPV) and anti-CD humanized dual targeting antibody solution in the test sample was 1 mg/mL.
  • the prepared anti-human papillomavirus (HPV) and anti-CD humanized double targeting antibody have high antibody binding titers to the CD3 antigen.
  • HPV human papillomavirus
  • HPV human papillomavirus
  • Lipofectamine 2000 Transfected mammalian cells 293FT cells were pre-plated to a 24-well cell culture plate (1.5 ⁇ 10 5 /well) 5 h in advance. 1 ⁇ g of pN312EGFP was transfected into pre-plated cells, and the liposome Lipofectamine 2000 transfection method was found in the Invitrogen operating manual. After 12 hours, it was replaced with complete medium, and observation and detection were performed after 48 hours.
  • Identification epitopes of monoclonal antibodies that are reactive with VLP and have poor reactivity with samples after VLP boiling should be The specific spatial conformation is related; the recognition epitope of the good monoclonal antibody with the VLP boiled sample should be unaffected by the spatial structure and belong to the linear epitope.
  • Example 4 Neutralization experiment of anti-human papillomavirus (HPV) and anti-CD humanized dual targeting antibody
  • Example 5 The anti-human papillomavirus (HPV) and the anti-CD humanized double-targeted antibody injection prepared in the following Example 7 were used to test positive for HPV antibody in women.
  • HPV anti-human papillomavirus
  • Example 7 The anti-CD humanized double-targeted antibody injection prepared in the following Example 7 were used to test positive for HPV antibody in women.
  • Control group normal saline was injected, one injection per week, and three consecutive injections.
  • follow-up observations were performed as required, and the patients were followed up for 30 days to observe the high-risk HPV infection.
  • the treatment group reviewed 96 cases of HPV negative, 0 cases were ineffective, and the conversion rate was 100%. In the control group, 7 cases of HPV were negative, and 89 cases were ineffective. The conversion rate was 7.3%.
  • Test subjects age 21 to 45 years old, no major organ disease, no history of drug allergy and no allergic constitution), women with sexual life history, confirmed by HPV antibody test and liquid-based cytology A patient who is positive. A total of 256 cases. They were randomly divided into treatment group and control group, 128 cases each.
  • control group no drug treatment, according to the regulations, follow-up observation throughout the follow-up observation, on the 30th day to review, observe the high-risk HPV infection turned negative. Review HPV high-risk DNA detection and typing.
  • the treatment group reviewed 105 cases of HPV, and 23 cases were ineffective. The conversion rate was 82.0%. In the control group, 9 cases of HPV were negative, and 119 cases were ineffective. The conversion rate was 7.0%.
  • Example 7 Injection of anti-human papillomavirus (HPV) and anti-CD humanized dual targeting antibody
  • the antibody solution B was added to the mixed solution A in a fine stream while stirring, and sufficiently dissolved to obtain a solution C.
  • Solution C was sterilized by filtration using a sterilized 0.45 ⁇ m tandem 0.22 ⁇ m microporous filter. It is potted in a sterile container in a sterile potting machine; light inspection, leak detection, printing, and packaging are shipped.
  • Example 8 Production of a gel against human papillomavirus (HPV) and anti-CD humanized dual targeting antibody
  • distilled water is heated to 90 ° C, then add S-40 and K-30 dispersant and lactose respectively, stir for more than 30 minutes, dissolve evenly; then cool to 60 ° C, slowly add Tween (drop ) and peppermint oil and glycerin (drop), mix at low speed for 60 minutes to fully dissolve; keep the temperature at 60 ° C, forming solution A;
  • seaweed extract pharmaceutical grade glycerin, peppermint oil, flavor and 403, 503 foaming agent are sterilized by ultraviolet light irradiation for 24 hours, and sealed aseptically;
  • Example 10 Production of an effervescent tablet against human papillomavirus (HPV) and anti-CD3 humanized dual targeting antibody
  • the blister pack is sealed, and the outer casing of the aluminum-plastic composite film is sealed, and the box is packed and packed into the warehouse.
  • Example 11 Production of a tablet against human papillomavirus (HPV) and anti-CD3 humanized dual targeting antibody
  • carboxymethyl cellulose is dispersed in 30% ethanol to make a 1% ethanol solution
  • Example 12 Production of anti-human papillomavirus (HPV) and anti-CD3 humanized dual targeting Hard capsules of antibodies.
  • Example 13 Production of soft capsules against human papillomavirus (HPV) and anti-CD3 humanized dual targeting antibodies
  • anti-human papillomavirus (HPV) and anti-CD3 humanized double-targeted antibody with high-purity protein type are first dissolved in distilled water cooled to room temperature after high-temperature sterilization, fully Stir well to prepare antibody aqueous solution B;
  • the prepared nanocapsule solution C is added to a soft capsule machine to form a rolling die, 300 mg per tablet;

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Abstract

本发明提供了一种抗人乳头瘤病毒和抗CD人源化的双靶向抗体及其组合物、制备方法和应用，所述抗体包括克隆于载体的鼠-人嵌合抗体轻链全长基因序列和鼠-人嵌合抗体重链全长基因序列，所述载体包括病毒类基因载体和非病毒基因载体，所述鼠-人嵌合抗体轻链全长基因序列和重链全长基因分别通过相关抗原免疫后得到的抗体轻链和重链测序得到。

Description

抗人乳头瘤病毒和抗CD人源化的双靶向抗体及其组合物、制备方法和应用 技术领域
本发明涉及基因工程的技术领域，更具体地说，涉及一种抗人乳头瘤病毒和抗CD人源化的双靶向抗体及其组合物、制备方法和应用。
背景技术
宫颈癌是严重危害妇女健康和生命的疾病，全球每年约有50万新发病例，25万人因宫颈癌死亡，而且这数字在逐年增长，是引起妇女死亡的第二大原因。现代医学在大量试验研究的基础上，己证实子宫颈癌源自一种人乳头瘤病毒(HPV)，这是目前人类所有癌症中唯一病因明确的癌症。
大量检测资料显示，HPV感染在18～28岁之间的青年人中最常见。可以说，所有在性生活上活跃的人，50％以上会在其一生中的某个时刻发生生殖器HPV感染。而比例高达80％的绝大多数妇女，在50岁之前有过HPV感染。世界卫生组织的统计数字表明，全球被感染总数约达到6.3亿人之多，占人口总数10％，其中女性占绝大多数；这是一个令人触目惊心的大数字。
比较麻烦的是，多数HPV感染没有体征和症状；但是，病毒却可以通过性生活传染，这就增加了这种病毒广泛传播的危险性。凡16岁前过早就有性生活的人，自己或配偶有多个性伴侣的人，就成为HPV的高危人群。此外，无论何种原因导致免疫力处于低下状况的人，也都属于HPV病毒的易感者，如多孕早产的妇女、HIV病毒(艾滋病毒)感染者、器官移植者或化疗患者，以及吸烟、吸毒和营养不 良的妇女等。
可是，迄今为止，医药界还没有开发出用于治疗HPV感染的有效的抗病毒药物，抗生素和其它杀菌药、消毒药对HPV病毒完全没有作用。目前，除了欧美国家推出的HPV疫苗作为预防用外，还没有可用于治疗HPV感染的药物；对已经发生的HPV感染更没有任何根除方法，现有的治标方法仅为去除外生的疣体；而且，各种暂时的处置手段均有复发的可能。一旦受到高危类型HPV病毒感染，最终往往会发展成为癌症。而HPV疫苗只有预防作用，并没有治疗作用，它仅仅对没有感染过HPV病毒的人有一定的预防效果，对于已受到HPV病毒感染的人注射这种疫苗是无能为力的。而绝大多数妇女，在50岁之前都曾受到HPV病毒感染，因此这种疫苗的预防作用也很有限。而要求每个女性特别是年青健康的青少年在未受到感染前都要强制注射这种疫苗也不太现实；何况，这种疫苗并不能使注射者获得终身免疫。更困扰的是，疫苗接种者仍有可能感染所接种的疫苗未包含的其它种类人乳头瘤病毒。
中国宫颈癌病例占全球的28％以上。我国对3381例女性生殖道人乳头瘤病毒检测结果分析的一份研究报告显示，3381例女性中HPV阳性率高达18.75％。根据一项调查，在全球范围内，平均每分钟即检查出一例新发病例，每两分钟就有一名女性死于宫颈癌。
近年来，年轻妇女宫颈癌的发病率明显增加，且预后差，这一趋势已经引起众多外学者的高度重视。在年轻妇女中，HPV感染率如此之高，是导致宫颈癌年轻化的一个非常重要的因素之一，特别是深圳等沿海城市每三个女性就有一人女性感染HPV病毒。
对于全球6.3亿受人乳头瘤病毒(HPV)感染的人群，目前既无药可治，也没有任何根除方法，现有疫苗又存在种种缺陷。最令人惊悚的是，其中20％受高危型人乳头瘤病毒(HPV)感染者如不提前进行干预，往往会发展成为癌症。显而易见，这是笼罩在亿万女性头上 的魔咒！
由于人乳头瘤病毒(HPV)感染以及诱发的宫颈癌已构成对女性生命安全的极大威胁。因此，研究开发一种可对抗人乳头瘤病毒(HPV)感染，既可预防又可治疗的新药，是关系到全球亿万女性健康和生命的大事，也是摆在国际医学科学界面前的一项重要课题。
发明内容
本发明的目的在于提供一种抗人乳头瘤病毒和抗CD人源化的双靶向抗体及其组合物、制备方法和应用，直击宫颈癌防治的痛点，化解人乳头瘤病毒感染者“阳转阴”以及手术后复发的难题。
本发明解决技术问题所采用的技术方案是：一种抗人乳头瘤病毒和抗CD人源化的双靶向抗体，包括克隆于载体的鼠-人嵌合抗体轻链全长基因序列和鼠-人嵌合抗体重链全长基因序列，所述鼠-人嵌合抗体轻链全长基因序列通过相关抗原免疫后得到的抗体轻链测序获取，所述鼠-人嵌合抗体重链全长基因序列通过相关抗原免疫后得到的抗体重链测序获取。
在本发明的双靶向抗体中，所述载体为基因载体或表达载体。
本发明还提供一种组合物，包含上述的抗人乳头瘤病毒和抗CD人源化的双靶向抗体和药学上可接受的辅料。
本发明还提供了一种抗人乳头瘤病毒和抗CD人源化的双靶向抗体的制备方法，包括：
S1、制备获得鼠-人嵌合抗体轻链全长基因序列和鼠-人嵌合抗体重链全长基因序列；
S2、将所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列克隆于载体，获得抗人乳头瘤病毒和抗CD人源化的双靶向抗体。
在本发明的制备方法中，在步骤S1之前还包括S0、制备双杂交 瘤细胞：
利用HPV病毒亚单位病毒衣壳蛋白或者HPV病毒其它蛋白表达于真核细胞或原核细胞中，或者将该衣壳蛋白和其它蛋白互相嵌合表达于真核细胞或原核细胞中,自行装配成病毒样颗粒(VLPs)并加以纯化，得到纯化的病毒样颗粒(VLPs或cVLPs)；
利用大肠杆菌表达人CD3重组蛋白或其它种类人CD重组蛋白并纯化重组人CD3蛋白和其它种类重组人CD蛋白；
将纯化的病毒样颗粒(VLPs或cVLPs)和重组人CD3蛋白或其它种类重组人CD蛋白经过小鼠免疫和细胞融合，制得可分泌抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的杂交瘤细胞。
在本发明的制备方法中，在步骤S1中，具体包括：
提取双杂交瘤细胞的总RNA，反转录制备cDNA，分别扩增鼠源抗体轻链可变区和鼠源抗体重链可变区的序列；取淋巴细胞提取其总RNA，反转录制备cDNA，分别扩增人源抗体轻链恒定区和人源抗体重链恒定区序列；
重叠延伸所述鼠源抗体轻链可变区和所述人源抗体轻链恒定区，获得鼠-人嵌合抗体轻链全长基因序列；重叠延伸所述鼠源抗体重链可变区和所述人源抗体重链恒定区，获得鼠-人嵌合抗体重链全长基因序列。
在本发明的制备方法中，在步骤S1中还具体包括：
根据鼠源抗体轻链可变区和鼠源抗体重链可变区序列及人源抗体轻链恒定区和人源抗体重链恒定区序列，设计引物：
利用引物LV-F/LV-R与HV-F/HV-R分别扩增鼠源抗体轻链可变区和鼠源抗体重链可变区的序列；利用引物HC-F/HC-R与LC-F/LC-R分别扩增人源抗体轻链恒定区和人源抗体重链恒定区的序列；
利用引物LV-F/LC-R重叠延伸扩增所述鼠源抗体轻链可变区和所述人源抗体轻链恒定区；利用引物HV-F/HC-R重叠延伸扩增所述 鼠源抗体重链可变区和所述人源抗体重链恒定区。
在本发明的制备方法中，在步骤S2中，具体包括：
所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列，以病毒类基因载体或非病毒基因载体进行克隆，通过昆虫细胞或其它细胞大规模发酵生产，经纯化后获得基因型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体；所述病毒类基因载体包括杆状病毒；所述非病毒基因载体包括质粒DNA、微环DNA、裸DNA、脂质体和脂质体复合物、阳离子高聚物、配体介导的靶向载体、游离载体；
或者，将所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列分别克隆于表达载体的两个启动子之下获得重组质粒；将所述重组质粒转染CHO细胞或在昆虫细胞表达系统中表达，获得蛋白型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体；
或者，将所述鼠-人嵌合抗体轻链全长基因使用限制酶双酶切克隆于表达载体的一个启动子之下；将所述鼠-人嵌合抗体重链全长基因使用限制酶双酶切的另一个启动子之下，获得蛋白型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体。
本发明还提供了上述的抗人乳头瘤病毒和抗CD人源化的双靶向抗体在制备用于清除人乳头瘤病毒(HPV)感染、和/或用于预防和治疗宫颈癌的药物、消毒产品、保健品和医疗器械中的应用。
本发明还提供了上述的组合物在制备用于清除人乳头瘤病毒(HPV)感染、和/或用于预防和治疗宫颈癌的药物、消毒产品、保健品和医疗器械中的应用。
实施本发明的抗人乳头瘤病毒和抗CD人源化的双靶向抗体及其组合物、制备方法和应用，具有以下有益效果：本发明采用抗人乳头瘤病毒和抗CD人源化的双靶向抗体可以预防和辅助治疗宫颈癌，改变了目前接种疫苗预防宫颈癌的传统模式，与传统抗体相比，在抗体 活性、组织渗透率、杀灭人乳头瘤病毒(HPV)效率、脱靶率和临床适应症等指标方面都具有明显优势，还可显著降低治疗成本，同时避免了人体对异种蛋白抗体的排斥。
具体实施方式
下面结合实施例，对本发明的抗人乳头瘤病毒和抗CD人源化的双靶向抗体及其组合物、制备方法和应用作进一步说明：
本发明提供一种抗人乳头瘤病毒和抗CD人源化的双靶向抗体，该双靶向抗体包括克隆于载体的鼠-人嵌合抗体轻链全长基因序列和鼠-人嵌合抗体重链全长基因序列，所述鼠-人嵌合抗体轻链全长基因序列通过相关抗原免疫后得到的抗体轻链测序获取，所述鼠-人嵌合抗体重链全长基因序列通过相关抗原免疫后得到的抗体重链测序获取。其中，载体可以为基因载体或表达载体，但不限于此。
本发明把上述双靶向抗体人源化，然后通过CHO细胞进行规模化发酵生产抗体蛋白，或在昆虫细胞表达系统中表达并大量生产高纯度含有抗人乳头瘤病毒和抗CD人源化的双靶向抗体的载体，再采用蛋白或基因形式的高纯度抗人乳头瘤病毒和抗CD人源化的双靶向抗体抑灭人乳头瘤病毒(HPV)，从而达到消除人乳头瘤病毒(HPV)，预防和辅助治疗宫颈癌的目的。
上述抗人乳头瘤病毒和抗CD人源化的双靶向抗体的制备方法如下所述。
由于人乳头瘤病毒(HPV)具有明显的种系特异性，不能在动物体内感染，也不能作体外细胞培养，因而给免疫抗原的制备带来很大难度。但是，本发明所采用方法可解决这个难题。
制备双靶向抗体的方法有杂交瘤技术、化学交联法和基因工程技术等。本发明以杂交瘤技术和基因工程技术为例说明，但不限于以下说明。
S0、制备双杂交瘤细胞：
利用HPV病毒晚期编码基因L1与L2亚单位病毒衣壳蛋白或者HPV病毒早期基因E6、E7蛋白表达于真核细胞或原核细胞中，也可将L1、L2和E6、E7互相嵌合表达于真核细胞或原核细胞中,自行装配成病毒样颗粒(VLPs)并加以纯化，得到纯化的病毒样颗粒(VLPs或cVLPs)；
上述的HPV病毒为如下中的至少一种：HPV6，11，16，18，31，33，45，52，58。还可根据当地流行的不同HPV病毒，选择其它型号的HPV病毒；
利用大肠杆菌表达人CD3重组蛋白或其它种类人CD重组蛋白并纯化或者外购重组人CD3蛋白和其它种类重组人CD蛋白；
制备双杂交瘤细胞：将纯化的病毒样颗粒(VLPs或cVLPs)和重组人CD蛋白(人CD3重组蛋白或其它种类人CD重组蛋白)经过小鼠免疫和细胞融合，制得可分泌抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的杂交瘤细胞。
S2、可变区序列与恒定区序列的克隆：提取所述双杂交瘤细胞的总RNA，反转录制备cDNA，分别扩增鼠源抗体可变区的轻链、重链的序列；取淋巴细胞提取其总RNA，反转录制备cDNA，分别扩增人源抗体恒定区的轻链、重链序列；
轻链与重链全长基因的克隆：重叠延伸所述鼠源抗体轻链可变区和所述人源抗体轻链恒定区，获得鼠-人嵌合抗体轻链全长基因序列；重叠延伸所述鼠源抗体重链可变区和所述人源抗体重链恒定区，获得鼠-人嵌合抗体重链全长基因序列；
其中，根据鼠源抗体可变区的轻链和重链序列及人源抗体恒定区的轻链和重链序列，设计特异性引物；
利用引物LV-F/LV-R与HV-F/HV-R分别扩增鼠源抗体可变区的轻链、重链的序列；利用引物HC-F/HC-R与LC-F/LC-R分别扩增人 源抗体恒定区的轻链、重链序列；
利用引物LV-F/LC-R重叠延伸扩增所述鼠源抗体轻链可变区和所述人源抗体轻链恒定区；利用引物HV-F/HC-R重叠延伸扩增所述鼠源抗体重链可变区和所述人源抗体重链恒定区。
S2、制备抗人乳头瘤病毒和抗CD人源化的双靶向抗体：将所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列克隆于基因载体，扩增后纯化获得基因型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体；或者，将所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列克隆于表达载体并在表达系统中表达，获得蛋白型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体。
采用常规的分子生物学的基因克隆方法设计并构建、表达可同时靶向人乳头瘤病毒(HPV)和CD(人CD3重组蛋白或其它种类人CD重组蛋白)的人源化双靶向抗体。
其中，基因型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体的制备过程包括：所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列，以病毒类基因载体或非病毒基因载体进行克隆；然后，通过昆虫细胞发酵大规模生产，经纯化后获得基因型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体；
所述病毒类基因载体包括杆状病毒；所述非病毒基因载体包括质粒DNA、微环DNA、裸DNA、脂质体和脂质体复合物、阳离子高聚物、配体介导的靶向载体、游离载体。
其中，蛋白型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体的制备过程包括：将所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列分别克隆于表达载体pFast-Bac-Dual的两个启动子之下获得重组质粒；将所述重组质粒转染CHO细胞或在昆虫细胞表达系统中表达，获得蛋白型的抗人乳头瘤病毒(HPV) 和抗CD人源化的双靶向抗体。
也可以将所述鼠-人嵌合抗体轻链全长基因使用BamH Ⅰ/EcoR Ⅰ双酶切克隆于表达载体pFast-Bac-Dual的一个启动子之下；将所述鼠-人嵌合抗体重链全长基因使用Xho Ⅰ/Nhe Ⅰ双酶切的另一个启动子之下。
在本发明提供的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体制备方法中，还包括：
扩大培养：挑选高表达的稳定转染细胞株，通过逐级放大的细胞反应器发酵，过滤收集上清液并去除细胞和碎片；
抗体纯化：将所述上清液通过阳离子交换层析法捕获基因型的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体，通过阴离子交换层析法进一步纯化和精制，再通过超滤方法进行缓冲液置换，制成高纯度微环DNA的蛋白型的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体。
在本发明提供的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的制备方法中，采用如下操作处理制得的基因型或蛋白型的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体：加入保护剂制备成单抗或抗体蛋白液体制剂，或者脂质体化处理制备成纳米脂质体单抗或抗体蛋白，或者干燥成抗体或抗体蛋白干粉。
另外，本发明还提供一种组合物，该组合物包含前述的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体以及药学上可接受的辅料。需要说明的是，该组合物可以是由前述的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体和辅料制备成的各种药物制剂，也可以是制备的医疗器械，如试剂盒等；也可以是制备的保健用品和消毒产品等。
前述的基因型或蛋白型的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体(或纳米脂质体化)可以制成各种稳定制剂，包括但 不限于下述制剂：
优选地，辅料包括赋形剂、填充剂、溶剂、助溶剂、表面活性剂和胶囊辅料中一种或多种。
优选地，该制剂为阴道凝胶、栓剂、洗液等。
优选地，该制剂为注射剂、片剂、喷雾剂、粉剂、、口香糖、口服液剂或胶囊、敷料等。
前述的基因型或蛋白型的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体(或纳米脂质体化)制成注射用水针剂和粉针剂中的至少一种。其可用于以下用途：已受到人乳头瘤病毒(HPV)感染者“阳转阴”；防止宫颈癌患者手术后或放化疗后复发；预防和辅助治疗宫颈癌。
或者，前述的基因型或蛋白型的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体(或纳米脂质体化)添加辅料或基料制成凝胶、栓剂、洗液、片剂、泡腾片、胶囊剂、软胶囊、医疗器械中的至少一种，可制备用于消除人乳头瘤病毒(HPV)感染以及预防和辅助治疗宫颈癌的生物药。
本发明的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体融合全球抗体工程的尖端技术进行四项创新。
(1)改变疫苗预防的传统方式：目前世界各国都是采用HPV疫苗预防宫颈癌，但是，HPV疫苗只有预防作用，并没有治疗作用，它仅仅对没有感染过HPV病毒的人有一定的预防效果，对于已受到HPV病毒感染的人注射这种疫苗的效果将大打折扣，而绝大多数妇女，在50岁之前都曾受到HPV病毒感染，显然，这种疫苗的预防作用很有限。而要求每个女性特别是年青健康的青少年在未受到感染前都要强制注射这种疫苗也不太现实；何况，这种疫苗并不能使注射者获得终身免疫。另外，对于在“两癌筛查”中呈“阳性”的受HPV病毒感染者，HPV疫苗是无效的，对于宫颈癌手术切除后复发的问题HPV 疫苗也是无能为力的。而本发明改变了注射疫苗刺激人体产生抗体的传统方式，而采用直接向人体内输入高活性抗体的被动免疫方式。由于本发明的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体属于单克隆抗体一类，单克隆抗体的效价比注射疫苗在体内产生的抗体的效价普遍高200倍以上(注：注射疫苗在体内产生抗体的效价﹥1:40即合格，双靶向抗体效价1:1万-1:100万)。更重要的是，本发明提供的抗人乳头瘤病毒和抗CD人源化的双靶向抗体可彻底清除体内的HPV病毒并有效阻断HPV病毒感染，既可迅速实现“阳转阴”，又可杜绝切除手术后复发；还能根据各国流行的人乳头瘤病毒(HPV)不同型号生产相对应的抗体，实现“对号入座”靶向杀灭，达到更好效果。因此，本发明提供的抗人乳头瘤病毒和抗CD人源化的双靶向抗体是超越疫苗的更佳选择，是对传统预防方式的创新。
(2)双靶向抗体：双靶向抗体与普通抗体相比增加了一个特异性抗原结合位点，在治疗方面表现出了以下优势：两个抗原结合位点分别结合人乳头瘤病毒(HPV)和免疫细胞而产生桥接作用，既可直接与人乳头瘤病毒(HPV)作用，又能激活T细胞和NK细胞等免疫细胞的功能，提高局部NK细胞或T细胞浓度，增强效应分子杀伤能力，更有效杀死人乳头瘤病毒及感染细胞；发挥免疫细胞和抗原递呈细胞的协调刺激作用，释放细胞因子，增强抑灭人乳头瘤病毒作用；两个抗原结合域增强抗体的特异性，降低脱靶毒性等副作用。
双靶向抗体与传统抗体相比在组织渗透率、杀灭人乳头瘤病毒(HPV)效率、脱靶率和临床适应症等指标方面都具有明显优势。鉴于其治疗效果是普通抗体的100-1,000倍，使用剂量可降为原来的1/2,000，因此还可显著降低治疗成本。
(3)抗体人源化：将双靶向抗体人源化，保留鼠源抗体活性区基因序列使结合抗原能力不变，用人源恒定区基因序列代替鼠源恒定区基因序列，避免了人体对异种蛋白抗体的排斥。
鼠源性的单克隆抗体虽然具有很强的抗体效应，但是如果将鼠源性的单克隆抗体直接注射到人体内会诱发抗鼠抗体反应(指鼠源性单抗对其他动物体具有异种蛋白的免疫原性)，在人体内的循环系统中会很快被清除掉，造成在体内半衰期较短。本发明将所制备的抗人乳头瘤病毒和抗CD人源化的双靶向抗体进行改造，保留抗原与抗体结合的高亲和力的基础上，去除大部分鼠源基因序列，以人源化基因序列代替，这样就减少了鼠单抗的异物性和免疫原性，降低了鼠源性抗体在人体内的抗抗体反应并且加强了抗体在人体内的ADCC作用(抗体依赖细胞介导的细胞毒性作用)，增加了其半衰期的长度。使得本发明的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的作用时间与作用效果都得到了增强。
(4)微环DNA载体：抗人乳头瘤病毒和抗CD人源化的双靶向抗体采用非病毒基因载体进行克隆时，可特别选用微环DNA表达。
微环DNA是较小的超螺旋质粒，从普通质粒中移掉了产生“哑巴(睡觉)效应”的原核骨架DNA，从而大大提高表达水平并且可持续表达。另外，采用微环DNA载体只需要原来系统中1/100至1/1000的L-arabinose浓度，可大幅度降低成本；同时，微环DNA本身不具有感染性，不含对生物安全性有隐患的序列，如抗生素基因，因此十分安全。采用微环DNA载体还有一个特点，即抗体进入人体直接转染，可利用人体内的蛋白合成机制不断复制和表达，源源不断地生产微环DNA抗人乳头瘤病毒和抗CD人源化的双靶向抗体，达到长期的被动免疫效果，解决了抗体蛋白因半衰期短而需反复注射的问题。这样，就可彻底清除人体内的人乳头瘤病毒(HPV)，实现消灭宫颈癌的目标。
可将6、11、16、18、31、33、45、52和58等九种型HPV病毒分为三组，分别是16、18、31为一组(高危组)，45(高危)、6(常见)、11(常见)为一组(混合组)，33、52、58为一组(中 危组)。
可应用微环DNA作为载体分成三个优化组合进行表达，每个优化组合分别表达三种抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体。在实际应用中这些组合的数目以及每个组合所表达的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的种类、数量可任意变化。
下面通过具体实施例进行详细说明。
实施例1：
抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体对有代表性人乳头瘤病毒(HPV)的抗体结合效价检测。
分别采用HPV6，11，16，18，31，33，45，52，58等型L1蛋白作为检测抗原，用“ELISA”方法(酶联免疫法)检测所制得的抗人乳头瘤病毒和抗CD人源化的双靶向抗体的抗体效价，结果如下表所示：
Figure PCTCN2017103683-appb-000001
Figure PCTCN2017103683-appb-000002
注：测试样本中的抗人乳头瘤病毒和抗CD人源化的双靶向抗体溶液浓度为1mg/mL。
从以上检测结果可看出，所制备的抗人乳头瘤病毒(HPV)和抗CD人源化双靶向抗体对有代表性人乳头瘤病毒(HPV)L1蛋白抗原都有很高的抗体结合效价。
实施例2：
抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体对CD的抗体结合效价检测。以CD3抗原为例。
采用CD3作为检测抗原，用“ELISA”方法(酶联免疫法)检测所制得的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的抗体效价，结果如下表所示：
Figure PCTCN2017103683-appb-000003
注：测试样本中的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体溶液浓度为1mg/mL。
从以上检测结果可看出，所制备的抗人乳头瘤病毒(HPV)和抗CD人源化双靶向抗体对CD3抗原有很高的抗体结合效价。
实施例3：
人乳头瘤病毒(HPV)假病毒的制备：
以人乳头瘤病毒(HPV)16型假病毒的制备为例说明，但不限于以下说明。其它型号的人乳头瘤病毒(HPV)的假病毒制备可采用以下方法或其它方法，本文就不赘述。
(1)外购质粒、细胞及单克隆抗体质粒p16L1h和p16L2h，分别含有经密码子优化后的HPV16L1和L2表达元件。构建报告 质粒pN312EGFP。以上三种质粒按照常规方法进行CsCl密度梯度超速离心纯化。外购细胞株293FT，培养于含10％FBS的DMEM完全培养基中。外购HPV16单克隆抗体V5、U4、E70和D9。
(2)磷酸钙共转染构建HPV16假病毒将293FT细胞预先铺于10cm细胞培养皿(7×106/dish)，5h后将质粒p16L1h、p16L2h和pN312EGFP用磷酸钙方法进行共转染，用量均为40μg。磷酸钙转染方法：提前5h将293FT细胞预铺至24孔细胞培养板(1.5×105/well)或10cm细胞培养皿(7×106/dish),将0.5mol/L的CaCl2溶液与等体积的ddH2O混合，加入适量质粒，再缓慢加入2倍体积的2×HeBS(pH＝6.97)溶液中并吹泡混合均匀，静置5min后将混合液加至预铺的293FT细胞孔中，12h后更换为完全培养基。同时对同样数量的细胞转染40μg的pN312EGFP作为对照。48h后进行观察并收集和裂解细胞，将收集的细胞裂解上清置-80℃保存。
(3)脂质体Lipofectamine2000转染哺乳动物细胞提前5h将293FT细胞预铺至24孔细胞培养板(1.5×105/well)。取1μgpN312EGFP转染到预铺的细胞中，脂质体Lipofectamine2000转染方法见Invitrogen公司操作手册。12h后更换为完全培养基，48h后进行观察和检测。
(4)HPV16L1的蛋白印迹实验(Western blotting)取少量细胞裂解上清样品进行10％SDS2PAGE电泳后转移到硝酸纤维膜，按常规方法进行蛋白印迹实验。使用的一抗为HPV16L1单克隆抗体D9，二抗为辣根过氧化物酶(HRP)标记的羊抗鼠IgG。
(5)Dot blotting鉴定单抗识别表位的构象依赖性将原核表达的HPV16L1VLP样品和经煮沸变性解聚后的VLP样品(0.3μg/μL)，分别进行10倍梯度稀释后点样到硝酸纤维膜上，用5％脱脂奶封闭后与不同的单抗分别进行反应，显色方法参照蛋白印迹实验。与VLP反应性好而与VLP煮沸后的样品反应性差的单抗的识别表位应与 特定的空间构象有关；与VLP煮沸后的样品好的单抗的识别表位应不受空间结构的影响，属于线性表位。
(6)假病毒颗粒的纯化和透射电镜观察
在NVT65离心管(Beckman)中加9mL的30％OptiPrep和1.0mL收集的细胞裂解液，剩余的离心管空隙用离心介质补充，16℃下62000r/min离心5h(NVT65转头，Beckman超速离心机)。取出，用针头穿刺离心管底部收集离心组分，每份约250μL，以蛋白印迹实验方法检测各组分中的L1蛋白活性。取活性最高的组分上样到铜网，用2％的磷钨酸染色15min，用JEM2100透射电镜(日本电子公司)观察颗粒。
(7)感染滴度测定
收集获得的细胞裂解液用10％DMEM进行10倍梯度稀释(10-2～10-6)，分别取100μL稀释液感染铺于24孔细胞培养板中的293FT细胞(1.5×105/well)，对照细胞裂解液也做同样的稀释感染。培养48h后用流式细胞仪(Beckman Coulter EPICS XL)检测孔中表达EGFP的细胞数量，测定假病毒的滴度(TU/mL)。
实施例4：抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体中和实验
将293FT细胞铺于96孔细胞培养板中(1.5×104/well)。5h后进行中和实验，将三种优化组合的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体样品分别用10％DMEM从10倍起进行连续倍比稀释，然后取50μL分别与50μL稀释于10％DMEM的其中一种型号的HPV假病毒液(moi＝0.1)混合。4℃孵育1h后分别加入预铺有293FT细胞的96孔细胞培养板中，37℃培养48h后用流式细胞仪分别检测各孔细胞的感染率。感染率为细胞样品在阳性区中的细胞数量百分率减去未感染的对照细胞样品在阳性区的数量百分率。感染抑制率＝(1-阻断孔的感染率/未阻断孔的感染率)×100％。20倍稀释后能达到 50％以上感染抑制率的稀释倍比衡量所制备的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的中和病毒效价。结果如下：
Figure PCTCN2017103683-appb-000004
实施例5：采用下述实施例7制备的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体注射剂使HPV抗体检测为阳性的妇女“阳转阴”试验
(1)试验对象：年龄21～45岁，无重大脏器疾病者，无药物过敏史和无过敏体质者)，有性生活史的妇女，经HPV抗体检测和液基细胞学检查，确诊为为阳性的患者。共192例。随机分为治疗组和对照组各96例。
(2)试验方法
治疗组：采用抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体注射剂进行治疗。每周注射一针，连续注射三针。按要求接受全程追踪随访观察，患者入组30天后复诊，观察高危型HPV感染转阴情况。复查HPV高危型DNA检测及分型。
对照组：注射生理盐水，每周注射一针，连续注射三针。按要求接受全程追踪随访观察，患者入组30天后复诊，观察高危型HPV感染转阴情况。复查HPV高危型DNA检测及分型。
(3)追踪随访
患者按要求入组后，接受口头知情并详细填写各项临床观察表，第30天作相应检查，并将结果详细填入观察表中。如果在治疗过程中出现药物不良反应要详细填入表中，出现严重反应，立即上报相应部门并及时处理。
(4)治疗疗效判定标准
治愈为高危型HPV检测完全阴性。无效为HPV检测仍为为阳性。
(5)结果
试验30天后复诊，治疗组复查HPV转阴96例，无效0例，转阴率为100％。对照组复查HPV转阴7例，无效89例，转阴率为7.3％。
实施例6：采用下述实施例8制备的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体阴道凝胶使HPV抗体检测为阳性的妇女“阳转阴”试验
(1)试验对象：年龄21～45岁，无重大脏器疾病者，无药物过敏史和无过敏体质者)，有性生活史的妇女，经HPV抗体检测和液基细胞学检查，确诊为为阳性的患者。共256例。随机分为治疗组和对照组各128例。
(2)试验方法
治疗组：患者月经干净后开始使用抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体阴道凝胶，2.5g/支，每晚使用一支，放置阴道深处，第30天复诊，观察高危型HPV感染转阴情况。复查HPV高危型DNA检测及分型。
对照组：不用药物治疗，均按规定，按要求接受全程追踪随访观察，第30天复诊，观察高危型HPV感染转阴情况。复查HPV高危型DNA检测及分型。
(3)追踪随访
患者按要求入组后，接受口头知情并详细填写各项临床观察表，第30天作相应检查，并将结果详细填入观察表中。如果在治疗过程 中出现药物不良反应要详细填入表中，出现严重反应，立即上报相应部门并及时处理。
(4)治疗疗效判定标准
治愈为高危型HPV检测完全阴性。无效为HPV检测仍为为阳性。
(5)结果
试验30天后复诊，治疗组复查HPV转阴105例，无效23例，转阴率为82.0％。对照组复查HPV转阴9例，无效119例，转阴率为7.0％。
实施例7：生产抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的注射剂
将所制得的抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体制成一种新型注射剂，供“两癌筛查”检出为阳性的受人乳头瘤病毒(HPV)感染者注射，或者用于宫颈癌患者辅助治疗以及手术后阻止复发。具体配方和生产工艺如下：
配方：
Figure PCTCN2017103683-appb-000005
工艺：
(1)将配方量注射用水加0.1％针用活性炭，60℃搅拌15min；脱炭过滤；滤液密闭加热100℃30min，冷却至室温备用；
(2)一边搅拌一边分别将配方量磷酸二氢钠一水合物和磷酸氢二钠七水合物以及氯化钠加入占配方量90％的经活性炭处理过的灭菌注射用水中，加热溶解；另取聚山梨醇酯-80，加入PEG400混匀；在搅拌下加入上述混合液中；再加0.1％针用活性炭，60℃搅拌15min,脱炭过滤；滤液密闭加热100℃30min，冷却至室温备用，得混合液A；
(3)一边搅拌一边将抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体纯化液加入占配方量10％的经活性炭处理过的灭菌注射用水，充分溶解，得微环DNA抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体溶液B。
(4)一边搅拌一边将抗体溶液B以细流加于混合液A中，充分溶解，得溶液C。
(5)用0.1mol的NaOH溶液调节溶液C的pH值至6.0～7.5。
(6)采用已灭菌的0.45μm串联0.22μm微孔滤膜将溶液C过滤除菌。于无菌灌封机中灌封于无菌容器中；灯检、检漏、印字、包装出厂。
实施例8：生产抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的凝胶
配方：
Figure PCTCN2017103683-appb-000006
Figure PCTCN2017103683-appb-000007
工艺：
(1)将配方量乳糖、羟乙基纤维素(HEC)、薄荷油、香精用紫外光照射灭菌消毒24小时，无菌密封备用；
(2)按配方量蒸馏水加热至至90℃，再分别加入S-40和K-30分散剂以及乳糖，搅拌30分钟以上，溶解均匀；再冷却至60℃，分别缓慢加入吐温(滴加)和薄荷油以及甘油(滴加)，低速揽拌60分钟至充分溶解；保持温度在60℃，形成溶液A；
(3)将溶液A加热至60℃，保持温度在60℃，一边搅拌一边将医药级尿囊素加入溶液A中，低速搅拌60min，形成溶液B；
(4)将配方量表皮生长因子用适量95％的酒精溶解，一边搅拌一边加热至60℃，直至完全溶解；保持温度在60℃，得表皮生长因子酒精溶液C；
(5)一边搅拌一边将溶液C滴加入溶液B中，继续高速搅拌60min，温度始终控制在50-60℃，直至完全溶解，然后冷却至室温，得溶液D；
(6)一边搅拌一边将蛋白型的抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体加入溶液D中，低速搅拌60min，直至完全混合均匀，得溶液E；
(7)一边搅拌一边将香精(玫瑰花香精)加入溶液E中，低速搅拌60min，直至完全溶解，得溶液F；
(8)一边搅拌一边将配方量羟乙基纤维素(HEC)加入溶液F中，低速搅拌60min，直至完全溶解，形成均质稳定的溶液，测试溶液粘稠度，适合后，得溶液G；
(9)用pH计测量溶液G的pH值，用柠檬酸调节pH值至4.5±0.1；
(10)静置一夜，直至上部泡沫全部消溶后，再将溶液G分装在清洗消毒过的塑料桶中，贴上标签出厂。
实施例9：生产抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体的洗液
配方：
Figure PCTCN2017103683-appb-000008
工艺：
(1)将配方量海藻精华、医药级甘油、薄荷油、香精以及403、503发泡剂用紫外光照射灭菌消毒24小时，无菌密封备用；
(2)将配方量蒸馏水加热至90℃，停留15分钟；再冷却至60℃，一边搅拌一边先后加入海藻精华和医药级甘油，低速揽拌30分钟，直至完全溶解，冷却至室温，得溶液A；
(3)一边搅拌一边将香精(玫瑰花香精)加入溶液A中，低速搅拌60min，直至完全溶解，得溶液B；
(4)将403加热至80℃，然后一边搅拌一边将503徐徐滴加入到403中，低速搅拌30min，得到溶液C；
(5)保持溶液C温度在80℃，一边搅拌一边将薄荷油徐徐滴加入到溶液C中，低速搅拌60min，得到溶液D；
(6)将溶液D加热至90℃高温灭菌5min，然后冷却至室温；
(7)一边搅拌一边将溶液B缓慢地加到溶液D中，低速搅拌60min；如未形成均质稳定的乳状溶液，则需延长搅拌时间，制得溶 液E；
(8)一边搅拌一边将抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体缓慢地加到溶液E中，低速搅拌60min；如未形成均质稳定的乳状溶液，则需延长搅拌时间，制得溶液F；
(9)用pH计测量溶液F的pH值，采用柠檬酸或pH4.5磷酸氢二钠–柠檬酸缓冲液调节pH值至4.5±0.1；
(10)静置一夜，直至上部泡沫全部消溶后，再将溶液F液分装在清洗消毒过的塑料容器中，贴上标签出厂。
实施例10：生产抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体的泡腾片
配方：
Figure PCTCN2017103683-appb-000009
工艺：
(1)将NaHCO360℃干燥2h，加20％PEG乙醇液适量制软材，14目尼龙筛制粒60℃干燥备用；
(2)枸椽酸、乳糖、MCC混匀80目粉碎，加于2％HPMC醇 液中搅匀；
(3)加入高纯度蛋白型抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体、氢氧化铝混匀过一次14目筛，再加硬脂酸镁混匀压片即得；
(4)检验合格后水泡眼包装，外套铝塑复合膜塑料袋密封，装盒，装箱入库。
实施例11：生产抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体的片剂
配方：
Figure PCTCN2017103683-appb-000010
工艺：
(1)山梨糖醇过60目筛两次备用；
(2)羧甲基纤维素分散于30％乙醇中制成1％乙醇溶液；
(3)将(1)项用适量(2)项制软材，14目筛网制粒，60℃通风干燥，18目筛整粒；用40目筛筛出适量细粉与高纯度蛋白型的抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体充分混匀；
(4)再拌入硬脂酸镁，一起与整批颗粒混合均匀，密闭4小时以上；
(5)压片机压片，每片600mg；
(6)检验合格后，包装，全验出厂。
实施例12：生产抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向 抗体的硬胶囊。
配方：
Figure PCTCN2017103683-appb-000011
工艺：
(1)将高纯度抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体和药用葡萄糖按重量比1:1比例混合，充分搅拌均匀；
(2)用胶囊充填机将抗体的蛋白粉和药用葡萄糖混合物定量分装入药用胶囊，每粒装300mg；
(3)将胶囊装瓶，检验出厂。
实施例13：生产抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体的软胶囊
配方：
Figure PCTCN2017103683-appb-000012
工艺：
(1)将吐温加热至50℃，一边搅拌一边将大豆油徐徐滴加入到吐温中，高速(3000r/min)搅拌15min，冷却到室温，得到溶液A；
(2)将配方量蒸馏水采用高压蒸气灭菌，加热到121-126℃，压力升至102.9kPa(1.05kg/cm2)，停留60min，然后冷却到室温；
(3)将配方量高纯度蛋白型的抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体先用高温灭菌后冷却到室温的蒸馏水溶解，充分 搅拌均匀，制成抗体水溶液B；
(4)一边搅拌一边缓慢地将抗人乳头瘤病毒(HPV)和抗CD3人源化的双靶向抗体水溶液B加入到溶液A中，高速(3000r/min)搅拌60min以上，得到溶液C；经检测，溶液C中80％粒径在2～50μm范围，平均收率可达91.4％；
(5)将所制成的纳米微囊溶液C加入软胶囊机滚压模切断成型，每粒300mg；
(6)将压模成型的软胶囊干燥定型；
(7)采用铝塑包装机包装；
(8)纸盒包装，检验合格出厂。
应当理解的是，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进或变换都应属于本发明所附权利要求的保护范围之内。

Claims (10)

  1. 一种抗人乳头瘤病毒和抗CD人源化的双靶向抗体，其特征在于，包括克隆于载体的鼠-人嵌合抗体轻链全长基因序列和鼠-人嵌合抗体重链全长基因序列，所述鼠-人嵌合抗体轻链全长基因序列通过相关抗原免疫后得到的抗体轻链测序获取，所述鼠-人嵌合抗体重链全长基因序列通过相关抗原免疫后得到的抗体重链测序获取。
  2. 根据权利要求1所述的双靶向抗体，其特征在于，所述载体为基因载体或表达载体。
  3. 一种组合物，其特征在于，包含权利要求1或2所述的抗人乳头瘤病毒和抗CD人源化的双靶向抗体和药学上可接受的辅料。
  4. 一种抗人乳头瘤病毒和抗CD人源化的双靶向抗体的制备方法，其特征在于，包括：
    S1、制备获得鼠-人嵌合抗体轻链全长基因序列和鼠-人嵌合抗体重链全长基因序列；
    S2、将所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列克隆于载体，获得抗人乳头瘤病毒和抗CD人源化的双靶向抗体。
  5. 根据权利要求4所述的制备方法，其特征在于，在步骤S1之前还包括S0、制备双杂交瘤细胞：
    利用HPV病毒亚单位病毒衣壳蛋白或者HPV病毒其它蛋白表达于真核细胞或原核细胞中，或者将该衣壳蛋白和其它蛋白互相嵌合表达于真核细胞或原核细胞中,自行装配成病毒样颗粒(VLPs)并加以纯化，得到纯化的病毒样颗粒(VLPs或cVLPs)；
    利用大肠杆菌表达人CD3重组蛋白或其它种类人CD重组蛋白并纯化重组人CD3蛋白和其它种类重组人CD蛋白；
    将纯化的病毒样颗粒(VLPs或cVLPs)和重组人CD3蛋白或其它种类重组人CD蛋白经过小鼠免疫和细胞融合，制得可分泌抗人乳头瘤病毒(HPV)和抗CD人源化的双靶向抗体的杂交瘤细胞。
  6. 根据权利要求5所述的制备方法，其特征在于，在步骤S1中，具体包括：
    提取双杂交瘤细胞的总RNA，反转录制备cDNA，分别扩增鼠源抗体轻链可变区和鼠源抗体重链可变区的序列；取淋巴细胞提取其总RNA，反转录制备cDNA，分别扩增人源抗体轻链恒定区和人源抗体重链恒定区序列；
    重叠延伸所述鼠源抗体轻链可变区和所述人源抗体轻链恒定区，获得鼠-人嵌合抗体轻链全长基因序列；重叠延伸所述鼠源抗体重链可变区和所述人源抗体重链恒定区，获得鼠-人嵌合抗体重链全长基因序列。
  7. 根据权利要求6所述的制备方法，其特征在于，在步骤S1中还具体包括：
    根据鼠源抗体轻链可变区和鼠源抗体重链可变区序列及人源抗体轻链恒定区和人源抗体重链恒定区序列，设计引物：
    利用引物LV-F/LV-R与HV-F/HV-R分别扩增鼠源抗体轻链可变区和鼠源抗体重链可变区的序列；利用引物HC-F/HC-R与LC-F/LC-R分别扩增人源抗体轻链恒定区和人源抗体重链恒定区的序列；
    利用引物LV-F/LC-R重叠延伸扩增所述鼠源抗体轻链可变区和所述人源抗体轻链恒定区；利用引物HV-F/HC-R重叠延伸扩增所述鼠源抗体重链可变区和所述人源抗体重链恒定区。
  8. 根据权利要求4所述的制备方法，其特征在于，在步骤S2中，具体包括：
    所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列，以病毒类基因载体或非病毒基因载体进行克隆，通 过昆虫细胞或其它细胞大规模发酵生产，经纯化后获得基因型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体；所述病毒类基因载体包括杆状病毒；所述非病毒基因载体包括质粒DNA、微环DNA、裸DNA、脂质体和脂质体复合物、阳离子高聚物、配体介导的靶向载体、游离载体；
    或者，将所述鼠-人嵌合抗体轻链全长基因序列和所述鼠-人嵌合抗体重链全长基因序列分别克隆于表达载体的两个启动子之下获得重组质粒；将所述重组质粒转染CHO细胞或在昆虫细胞表达系统中表达，获得蛋白型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体；
    或者，将所述鼠-人嵌合抗体轻链全长基因使用限制酶双酶切克隆于表达载体的一个启动子之下；将所述鼠-人嵌合抗体重链全长基因使用限制酶双酶切的另一个启动子之下，获得蛋白型的抗人乳头瘤病毒和抗CD人源化的双靶向抗体。
  9. 权利要求1或2所述的抗人乳头瘤病毒和抗CD人源化的双靶向抗体在制备用于清除人乳头瘤病毒(HPV)感染、和/或用于预防和治疗宫颈癌的药物、消毒产品、保健品和医疗器械中的应用。
  10. 权利要求3所述的组合物在制备用于清除人乳头瘤病毒(HPV)感染、和/或用于预防和治疗宫颈癌的药物、消毒产品、保健品和医疗器械中的应用。
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