WO2019238120A1 - 一种治疗hpv持续感染引起疾病的药物及制备方法与应用 - Google Patents

一种治疗hpv持续感染引起疾病的药物及制备方法与应用 Download PDF

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WO2019238120A1
WO2019238120A1 PCT/CN2019/091260 CN2019091260W WO2019238120A1 WO 2019238120 A1 WO2019238120 A1 WO 2019238120A1 CN 2019091260 W CN2019091260 W CN 2019091260W WO 2019238120 A1 WO2019238120 A1 WO 2019238120A1
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cell expansion
cells
hpv infection
external
hpv
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English (en)
French (fr)
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刘军
王苏鸣
王驹
李祥宇
卢玲琳
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上海鸣大生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells

Definitions

  • the invention belongs to the field of biotechnology and its clinical application technology, and particularly relates to a medicine for treating diseases caused by continuous HPV infection, and a preparation method and application thereof.
  • Human papillomavirus is a genus of papilloma vacuole virus A belonging to the Papillomaviridae family. It is a spherical DNA virus that can stiffen squamous epithelium of human skin and mucous membranes. It shows symptoms such as common warts and condyloma acuminata. As the incidence of genital warts in venereal diseases increases and cervical cancer, anal cancer, and esophageal cancer increase, human papillomavirus (HPV) infection has attracted increasing attention. Skin-type HPV infection rates are very common, such as the common warts, toe warts, and flat warts mentioned above. High-risk HPV infections and low-risk HPV infections of the external genitals cause cervical cancer and genital warts. Genital warts caused by HPV infection account for 15-20% of global STDs.
  • HPV infection is the underlying cause of cervical cancer. HPV usually disappears on its own after infection. Only persistent high-risk human papillomavirus infection is the root cause of cervical cancer. As a common sexually transmitted disease, cervical infection with high-risk HPV is very common, HPV infections are mostly transient, and 10% -15% of patients are persistently infected. Many studies have shown that persistent high-risk HPV infection is a necessary condition for cervical lesions. HPV is ubiquitous in the population. A survey in the United States showed that 1/3 of female college students who have sex can detect HPV-DNA, and 70% of women have a chance to contract high-risk HPV once in their lifetime. The prevalence of HPV is estimated to be 25% -39%, depending on the age group, area of residence, and testing methods.
  • the detection rate of HPV-DNA in precancerous lesions is 70% -90%.
  • the grade of intraepithelial neoplasia is 20% -50%. According to scientific research, the correlation between the occurrence of esophageal cancer and HPV infection reaches 60% -80%.
  • the object of the present invention is to provide a medicine for treating HPV infection or a disease caused by persistent HPV infection, and a preparation method and application thereof.
  • the medicine can be directly applied by external application, which can better treat HPV infection or diseases caused by continuous HPV infection and reduce patient suffering.
  • an immune cell expansion suspension for preparing a medicament for treating HPV infection or a disease caused by HPV infection.
  • the immune cell expansion suspension is a main medicinal component.
  • the immune cell expansion suspension refers to a supernatant obtained after the immune cells are expanded and cultured and separated. Therefore, the immune cell expansion suspension may also be referred to as a suspension obtained by centrifugation of the immune cell expansion.
  • the immune cell expansion suspension does not include immune cells.
  • X-VIVO15 can be used as the culture medium during the expansion culture.
  • the density of immune cells reaches 2.0 ⁇ 10 6 cells / mL or more.
  • the density of immune cells reaches 3.0 ⁇ 10 6 cells / mL or more.
  • the density of immune cells reaches 4.0 ⁇ 10 6 cells / mL or more.
  • the method used in the expansion is selected from a cytokine stimulation method, an antibody stimulation method, or a trophoblast cell expansion method.
  • the cytokine stimulation method refers to: using cytokines such as IL-2, IL-12, IL-18, 4-1BB, IL-21, and IL-15 to stimulate and induce amplification.
  • the main cells obtained were a mixture of NKT cells and T cells.
  • the antibody stimulation method refers to: using antibodies such as CD16 monoclonal antibody, IL-2, IL-12, IL-21 / IL-15 to induce stimulation and amplification.
  • the main cells obtained were still mixed cells of NKT cells and T cells.
  • the trophoblast expansion method refers to: using tumor cells as the trophoblast cell mother, transfecting 4-1BB, IL-21 or IL-15, CD86, etc. to the surface of the cell membrane, and then irradiating them to destroy Treatment in live mode, added to the culture system at a certain ratio, co-stimulated with IL-12, IL-18, and CD16 monoclonal antibodies to induce expansion, and the purity of the NK cells obtained is generally greater than 70%. The proportion of mixed cells of NKT and T cells decreased.
  • the immune cell expansion suspension is a NK cell expansion suspension.
  • the NK cell expansion suspension refers to a supernatant obtained after NK cells are expanded and cultured and separated. Therefore, the NK cell expansion suspension can also be referred to as a suspension obtained by centrifugation after NK cell expansion.
  • the NK cell expansion suspension does not contain NK cells.
  • the NK cells can be obtained from monocyte culture.
  • the monocytes are derived from peripheral blood, umbilical cord blood, or placental blood.
  • the HPV infection means that the HPV test is positive.
  • the disease caused by HPV infection refers to a disease caused by persistent HPV infection.
  • the disease caused by persistent HPV infection is cervical neoplasia (CIN), genital warts, or cervical cancer.
  • the medicament is an external preparation.
  • the medicament is an external application preparation.
  • the medicine is a gel for external use.
  • the main medicinal component of the external gel preparation is: an immune cell expansion suspension.
  • the external gel preparation contains a pharmaceutically acceptable carrier excipient in addition to the main medicinal ingredient.
  • carrier excipients may be conventional excipients for external gel preparations, such as carbomer.
  • the amount of auxiliary materials can be determined by those skilled in the art according to actual needs. For example, it may account for 3-5% (mass percentage) of the entire external gel preparation.
  • the method for preparing a gel preparation for external use includes the steps of adding a certain amount of carbomer to the immune cell expansion suspension for dissolution, and adjusting the pH value to 6-6.5 by using a 0.1M sodium hydroxide solution. During the process, stir slowly and evenly. Fill into pre-made PE injection packaging, seal, pack, and store at 4-10 ° C.
  • the drug is a topical infiltration preparation.
  • the main medicinal component of the external infiltration preparation is: an immune cell expansion suspension.
  • a sterile medical cotton ball is infiltrated into the immune cell expansion suspension to form an external infiltration preparation.
  • the sterile medical cotton ball impregnated with the immune cell expansion suspension that is, the external infiltration preparation can be directly applied or covered on the patient's cervix.
  • a method for treating HPV infection or a disease caused by HPV infection comprising the steps of: covering or smearing a medicament on a patient's cervix with a drug having an immune cell expansion suspension as a main medicinal component.
  • the medicine using the immune cell expansion suspension as the main medicinal ingredient may be an external infiltration preparation.
  • a sterile medical cotton ball is infiltrated into the immune cell expansion suspension to form an external infiltration preparation.
  • the immune cell expansion suspension (that is, the suspension obtained by centrifugation after the expansion of the immune cells) can be directly applied or covered on the cervix of the patient by means of a medical cotton ball infiltration.
  • the drug using the immune cell expansion suspension as the main medicinal ingredient may be an external gel preparation.
  • an externally applied gel preparation i.e., a suspension obtained by centrifuging the immune cell after expansion
  • a main medicinal ingredient is directly applied to the cervix of a patient.
  • each treatment course includes several days, and multiple treatment courses are continuous.
  • the specific method of applying medicine can be selected according to the severity of the disease. For example, HPV-positive infections can be applied every other day for 15 consecutive days for 3 consecutive sessions. For diseases caused by continuous HPV infection: CIN or genital warts, etc., you can apply it once a day for 15 days for 3 consecutive sessions.
  • the treatment mechanism of the method is: the immune cell expansion suspension contains a large amount of immune cells secreted during the expansion culture process, such as interferon, cell necrosis factor, etc., and the disposable wash cell expansion suspension acts on the patient's cervix Site, passive immunity can be used to treat HPV infection and further control and treat other diseases caused by infection.
  • a medicament for treating HPV infection or a disease caused by HPV infection is provided, and the main medicinal effect component is an immune cell expansion suspension.
  • the HPV infection means that the HPV test is positive.
  • the disease caused by HPV infection refers to a disease caused by persistent HPV infection.
  • the disease caused by persistent HPV infection is cervical neoplasia (CIN), genital warts, or cervical cancer.
  • the medicament is an external preparation.
  • the medicament is an external application preparation.
  • the medicine is a gel for external use.
  • the main medicinal component of the external gel preparation is: an immune cell expansion suspension.
  • the external gel preparation contains a pharmaceutically acceptable excipient in addition to the main medicinal ingredient.
  • excipients may be conventional excipients for external gel preparations, such as carbomer.
  • the amount of auxiliary materials can be determined by those skilled in the art according to actual needs. For example, it may account for 3-5% (mass percentage) of the entire external gel preparation.
  • the method for preparing a gel preparation for external use includes the steps of adding a certain amount of carbomer to the immune cell expansion suspension for dissolution, and adjusting the pH value to 6-6.5 by using a 0.1M sodium hydroxide solution. During the process, stir slowly and evenly. Fill into pre-made PE injection packaging, seal, pack, and store at 4-10 ° C.
  • the drug is a topical infiltration preparation.
  • the main medicinal component of the external infiltration preparation is: an immune cell expansion suspension.
  • a sterile medical cotton ball is infiltrated into the immune cell expansion suspension to form an external infiltration preparation.
  • the sterile medical cotton ball impregnated with the immune cell expansion suspension that is, the external infiltration preparation can be directly applied or covered on the patient's cervix.
  • the present invention has the following beneficial effects:
  • the present invention finds for the first time that using the immune cell expansion suspension as the main medicinal ingredient smeared on the cervix and mainly taking isolation and isolation for further infection contact opportunities can treat HPV infection or diseases caused by continuous HPV infection, making HPV positive patients Overcast.
  • Positive patients with HPV infection treated by the method can achieve a negative rate of over 50% after 7-8 treatments, and a negative rate of over 90% after 15-20 treatments, and can be effectively controlled and treated HPV-induced cervical neoplasia (CIN) and the recurrence or metastasis of cervical cancer can prevent and treat cervical cancer.
  • CIN HPV-induced cervical neoplasia
  • the treatment mechanism of the present invention is: secreting a large amount of interferon, cytonecrosis factor, etc. through the immune cell culture process, at the same time, the immune cells actively kill after contacting the infected cells, while secreting cytokines to kill the infected cells, and further Antibodies are mediated through antigen stimulation, and the goals of treating HPV infection and further controlling and treating other diseases caused by infection are achieved through active and passive immunity.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention use conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the technical field. Conventional technology. These technologies have been fully explained in the existing literature. For details, refer to MOLECULAR CLONING: Sambrook et al. MANUAL, Second Edition, Cold Spring Harbor Laboratories Press, 1989 and Third Employment Edition, 2001; Ausubel et al.
  • Interleukin 2 (Beijing Shuanglu Pharmaceutical, 1 million IU / bottle), lymphocyte serum-free medium X-VIVO15 (Lonza), CD16 monoclonal antibody (BD Company of the United States), IL-21-K562 trophoblast cells (Nanjing Suning Kang); T75 cell culture flask (NUCK), T175 cell culture flask (NUCK), cell culture bag GT-T610A (Takara) and pipette (NUCK).
  • Biological safety cabinet Thermo
  • Thermo Scientific Thermo Scientific
  • STorvall ST16 / 16R desktop high-performance centrifuge carbon dioxide incubator
  • carbon dioxide incubator Thermo, 3111
  • inverted biological microscope Motic, AE-31
  • automatic cell counter Invitrogen, Countess
  • mobile Fluid Thermo
  • GE WAVE wave bioreactor peristaltic pump and ultrafiltration membrane package (10kd, 40kd) and clamp (PALL).
  • NK cells in immune cells are selected for expansion and culture, and the supernatant is an NK cell expansion suspension (also referred to as a suspension obtained by NK cell expansion after centrifugation), and the lower cells are after expansion.
  • NK cells also referred to as a suspension obtained by NK cell expansion after centrifugation
  • the NK cell expansion suspension can be used as a main medicinal component.
  • the expanded NK cells can also be used as the main medicinal component.
  • the NK cell expansion suspension and the method for preparing the expanded NK cells include the following steps:
  • Mononuclear cells can be obtained by apheresis, peripheral blood, placental blood, umbilical cord blood collection or lymphocyte separation tube separation.
  • Peripheral blood mononuclear cells are used in this embodiment.
  • peripheral blood mononuclear cells were taken for cell culture, and a cytokine combined with a trophoblast was used to stimulate and induce the culture.
  • the cells were inoculated into a T75 square bottle with a density of 1.0-1.5 ⁇ 10 6 cells / mL.
  • trophoblast cells 1.0 ⁇ 10 5 cells / mL, IL-2 100U / ml, IL12 20ng / ml, gradually expand the culture according to the expansion of the culture, and the fluid replacement is transferred to the T175 culture until the 2L culture bag.
  • NK cells reached a 2L culture system 13-14 days after expansion, and the cell density reached more than 2.0 ⁇ 10 6 cells / mL, and the purity of NK cells was greater than 90%. After centrifugation at 400G for 15 minutes, the supernatant and lower cells were collected, respectively. The obtained supernatant is a suspension of NK cells after expansion.
  • the obtained NK cell expansion suspension is a main medicinal component and can be prepared into an infiltration preparation. It is directly applied or covered on the patient's cervix by means of medical cotton ball infiltration.
  • the preparation method of the infiltration preparation using the NK cell expansion suspension as the main medicinal ingredient specifically includes the following steps:
  • the NK cell expansion suspension obtained above was filtered through 40kd to obtain an intermediate product, which was stored together with a sterile medical cotton ball in a 50ml medical sealed container and stored at 4-10 ° C as an infiltration preparation.
  • Treatment subjects Volunteers, a total of 21, all females, aged between 20 and 50 years old, are those who have clinically sustained HPV infection.
  • Treatment method Apply cotton wool infiltration of NK cell expansion suspension directly or daily to volunteers' cervical area;
  • NK cells in immune cells are selected for expansion and culture, and the supernatant is an NK cell expansion suspension (also referred to as a suspension obtained by NK cell expansion after centrifugation), and the lower cells are after expansion.
  • NK cells also referred to as a suspension obtained by NK cell expansion after centrifugation
  • the NK cell expansion suspension can be used as a main medicinal component.
  • the expanded NK cells can also be used as the main medicinal component.
  • the NK cell expansion suspension and the method for preparing the expanded NK cells include the following steps:
  • Mononuclear cells can be obtained by apheresis, peripheral blood, placental blood, umbilical cord blood collection or lymphocyte separation tube separation.
  • Peripheral blood mononuclear cells are used in this embodiment.
  • peripheral blood mononuclear cells were taken for cell culture, and a cytokine combined with a trophoblast was used to stimulate and induce the culture.
  • the cells were inoculated into a T75 square bottle with a density of 1.0-1.5 ⁇ 10 6 cells / mL.
  • trophoblast cells 1.0 ⁇ 10 5 cells / mL, IL-2 100U / ml, IL12 20ng / ml, gradually expand the culture according to the expansion of the culture, and the fluid replacement is transferred to the T175 culture until the 2L culture bag.
  • NK cells reached a 2L culture system 13-14 days after expansion, and the cell density reached more than 2.0 ⁇ 10 6 cells / mL, and the purity of NK cells was greater than 90%. After centrifugation at 400G for 15 minutes, the supernatant and lower cells were collected, respectively. The obtained supernatant is a suspension of NK cells after expansion.
  • the obtained NK cell expansion suspension is a main medicinal component, and can be prepared into a gel preparation for external use for cervical application.
  • the preparation method of the external gel preparation using the NK cell expansion suspension as the main medicinal ingredient specifically includes the following steps:
  • the NK cell expansion suspension obtained above was filtered through 40kd to obtain an intermediate product, and then a certain amount of carbomer was added to the immune cell expansion suspension to dissolve, and the pH value was adjusted to 6 with 0.1M sodium hydroxide solution. 6.5, stir slowly and evenly during the process. Fill into pre-made PE injection packaging, seal, pack, and store at 4-10 ° C.
  • the external gel preparation of immune cell expansion suspension is clinically applied to the treatment of persistent HPV infection:
  • Treatment subjects Volunteers, a total of 15 women, aged 20 to 50 years, were clinically infected with HPV.
  • Treatment method Apply NK cell expansion suspension as the main medicinal ingredient to the volunteer's cervix every day or every other day
  • the negative rate is the percentage of negative patients to the number of participants.
  • the NK cell expansion suspension is a topical gel preparation with the main medicinal effect component; the index observation: the normal leucorrhea flora is normal; the result of one month review: HPV negative (2017.10.20); the result of three month review: HPV negative (2017-11.17).

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Abstract

本发明公开了使用免疫细胞扩增获得的上清悬液为主要药效成分涂抹于宫颈部位，治疗HPV感染或由于HPV持续感染所引起的疾病。通过所述方法治疗的HPV感染的阳性患者，经7-8次治疗后，转阴率达到30％以上，经过15到20次治疗后转阴率可达到50％以上，并且可有效控制和治疗HPV引起的宫颈瘤样变(CIN)以及宫颈癌复发或转移，达到预防和治疗宫颈癌的效果。

Description

一种治疗HPV持续感染引起疾病的药物及制备方法与应用 技术领域
本发明属于生物技术及其在临床上应用技术领域，具体涉及一种治疗HPV持续感染引起疾病的药物及制备方法与应用。
背景技术
人乳头瘤病毒(human papillomavirus，HPV)是一种属于乳多空病毒科的乳头瘤空泡病毒A属，是球形DNA病毒，能硬气人体皮肤黏膜的鳞状上皮增生。表现为寻常疣、尖锐湿疣等症状，随着性病中尖锐湿疣的发病率上升和宫颈癌、肛门癌、食道癌等的增多，人乳头瘤病毒(HPV)感染越来越引起关注。皮肤型的HPV人群感染率非常普遍，如上述常见的寻常疣、趾疣、扁平疣等，引起注意的高危型HPV感染和外生殖器的低危型HPV感染造成的宫颈癌和生殖器疣，据统计在全球性病中，HPV感染引起的生殖器疣占15-20％。
HPV感染是宫颈癌的根本致病因素。HPV通常在感染后能自行消失，只有持续的高危型人乳头瘤病毒感染才是导致宫颈癌的根本原因。作为一种常见的性传播疾病，宫颈感染高危型HPV是非常普遍的，HPV感染多为短暂性的，有10％-15％的患者呈持续性感染状态。许多研究显示持续性高危型HPV感染是发生宫颈病变的必要条件。HPV在人群中普遍存在，美国一调查显示1/3有性行为的女大学生可检测到HPV-DNA，70％的妇女一生中有1次机会感染高危型HPV。HPV的现患病率估计为25％-39％，主要取决于年龄组、居住地区及检测手段。生殖道HPV感染的高峰年龄是l8-28岁，35岁后5％-10％为高危HPV持续感染状态，持续感染HPV高危型病毒的妇女发生宫颈上皮内瘤样病变(cervical intraepithelial neoplasia，CIN)的风险增加100-300倍。持续感染HPV高危型病毒是引致并维持宫颈上皮重度不典型增生(cervical intraepithelial neoplasia 3，CIN II)病变的必要条件。世界不同地区所开展的病例对照研究显示：HPV感染在宫颈癌发病中的相对危险度50-100，而HPV16、18感染的相对危险度更是高达100-500。在几乎全部宫颈癌标本中均能检测到高危组HPV的存在，在癌前病变(即Ⅱ、Ⅲ级上皮内瘤样病变)中HPV-DNA的检出率为70％-90％，在I级上皮内瘤变中为20％-50％。另据科学研究表明食道癌的发生与HPV感染相关度达到60％-80％。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种治疗HPV感染或治疗HPV持续感染引起疾病的药物及其制备方法与应用。所述药物可直接通过外用涂抹的方式，即可更好地治疗HPV感染或由于HPV持续感染引起的疾病，减轻病患痛苦。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供免疫细胞扩增悬液用于制备治疗HPV感染或HPV感染引起的疾病的药物的用途。
一种实施方式中，所述免疫细胞扩增悬液为主要药效成分。
一种实施方式中，所述免疫细胞扩增悬液是指免疫细胞经扩增培养、分离后获得的上清。因此，所述免疫细胞扩增悬液亦可称为：免疫细胞扩增后离心获得的悬液。
需要说明的是，所述免疫细胞扩增悬液中不含免疫细胞。
一种实施方式中，扩增培养时，培养基可采用X-VIVO15。
一种实施方式中，扩增培养后，免疫细胞密度达到2.0×10 6个细胞/mL以上。
一种实施方式中，扩增培养后，免疫细胞密度达到3.0×10 6个细胞/mL以上。
一种实施方式中，扩增培养后，免疫细胞密度达到4.0×10 6个细胞/mL以上。
所述扩增时可采用现有技术中已有的扩增方法。
一种实施方式中，所述扩增时所采用的方法选自：细胞因子刺激法、抗体刺激方法或滋养层细胞扩增法。
一种实施方式中，所述细胞因子刺激法是指：采用IL-2、IL-12、IL-18，4-1BB，IL-21、IL-15等细胞因子进行刺激诱导扩增。主要获得的细胞为NKT细胞和T细胞的混合细胞。
一种实施方式中，所述抗体刺激方法是指：采用CD16单抗、IL-2、IL-12、IL-21/IL-15等抗体进行诱导刺激扩增。主要获得的细胞仍为NKT细胞和T细胞的混合细胞。
一种实施方式中，滋养细胞扩增法是指：使用肿瘤细胞作为滋养层细胞母体，将4-1BB，IL-21或IL-15、CD86等转染到细胞膜表面，再经过辐照等灭活方式处理，按一定比例加入培养体系中，与IL-12、IL-18、CD16单抗共同刺激诱导扩增，获得的NK细胞纯度一般可大于70％。NKT和T细胞的混合细胞占比减小。
一种实施方式中，所述免疫细胞扩增悬液为NK细胞扩增悬液。所述NK细胞扩增悬液是指NK细胞经扩增培养、分离后获得的上清。因此，所述NK细胞扩增悬液亦可称之为NK细胞扩增后离心获得的悬液。
需要说明的是，所述NK细胞扩增悬液中不含有NK细胞。
一种实施方式中，NK细胞可以是由单核细胞培养获得。
一种实施方式中，所述单核细胞来源于外周血、脐带血或胎盘血等。
一种实施方式中，所述HPV感染是指HPV检测为阳性。
一种实施方式中，所述HPV感染引起的疾病是指HPV持续感染引起的疾病。
一种实施方式中，HPV持续感染引起的疾病为宫颈瘤样变(CIN)、尖锐湿疣或宫颈癌。
一种实施方式中，所述药物为外用制剂。
一种实施方式中，所述药物为外用涂抹制剂。
一种实施方式中，所述药物为外用凝胶剂。
一种实施方式中，所述外用凝胶制剂的主要药效成分为：免疫细胞扩增悬液。
一种实施方式中，所述外用凝胶制剂中除了主要药效成分外，还含有药学上可接受的载体辅料。这些载体辅料可以是用于外用凝胶制剂的常规辅料，例如卡波姆。辅料的用量，本领域技术人员可根据实际需求来确定。例如，可占整个外用凝胶制剂的3-5％(质量百分比)。
一种实施方式中，所述外用凝胶制剂的制备方法，包括步骤：将一定量的卡波姆加入免疫细胞扩增悬液溶解，采用0.1M的氢氧化钠溶液调节PH值至6-6.5，过程中缓慢均匀搅拌。灌装入预制PE注射包装内，封口，包装，储存于4-10℃。
一种实施方式中，所述药物为外用浸润制剂。
一种实施方式中，外用浸润制剂的主要药效成分为：免疫细胞扩增悬液。
一种实施方式中，无菌医用棉球浸润于所述免疫细胞扩增悬液，形成外用浸润制剂。
使用时，将浸润了所述免疫细胞扩增悬液的无菌医用棉球，也就是外用浸润制剂直接涂抹或覆盖于患者宫颈部位即可。
本发明的第二方面，提供一种治疗HPV感染或HPV感染引起疾病的方法，包括步骤：将以免疫细胞扩增悬液为主要药效成分的药物覆盖或涂抹于患者的宫颈部位。
一种实施方式中，以免疫细胞扩增悬液为主要药效成分的药物可以是外用浸润制剂。
一种实施方式中，无菌医用棉球浸润于所述免疫细胞扩增悬液，形成外用浸润制剂。
一种实施方式中，免疫细胞扩增悬液(亦即免疫细胞扩增后离心获得的悬液)可通过医用棉球浸润的方式，直接涂抹或覆盖于患者宫颈部位。
一种实施方式中，以免疫细胞扩增悬液为主要药效成分的药物可以是外用凝胶制剂。
一种实施方式中，免疫细胞扩增悬液(亦即免疫细胞扩增后离心获得的悬液)为主要药效成分制备成的外用凝胶制剂直接涂抹于患者宫颈部位。
一种实施方式中，可隔一定时间涂抹一次，每疗程包括数日，连续多个疗程。具体可根据疾病轻重来选择合适的上药方法。例如，对于HPV阳性感染，可隔日涂抹一次，每疗程15日，连续3个疗程。对于由于HPV持续感染引起的疾病：CIN或尖锐湿疣等，可每日涂抹一次，每疗程15日，连续3个疗程。
所述方法的治疗机制为：免疫细胞扩增悬液中含有大量的免疫细胞在扩增培养过程中所分泌大量的如干扰素、细胞坏死因子等，免洗细胞扩增悬液作用于患者宫颈部位，可通过被动免疫达到治疗HPV感染及进一步控制和治疗由于感染引起的其它疾病的目的。
本发明的第三方面，提供一种治疗HPV感染或HPV感染引起疾病的药物，主要药效成分为免疫细胞扩增悬液。
一种实施方式中，所述HPV感染是指HPV检测为阳性。
一种实施方式中，所述HPV感染引起的疾病是指HPV持续感染引起的疾病。
一种实施方式中，HPV持续感染引起的疾病为宫颈瘤样变(CIN)、尖锐湿疣或宫颈癌。
一种实施方式中，所述药物为外用制剂。
一种实施方式中，所述药物为外用涂抹制剂。
一种实施方式中，所述药物为外用凝胶剂。
一种实施方式中，所述外用凝胶制剂的主要药效成分为：免疫细胞扩增悬液。
一种实施方式中，所述外用凝胶制剂中除了主要药效成分外，还含有药学上可接受的辅料。这些辅料可以是用于外用凝胶制剂的常规辅料，例如卡波姆。辅料的用量，本领域技术人员可根据实际需求来确定。例如，可占整个外用凝胶制剂的3-5％(质量百分比)。
一种实施方式中，所述外用凝胶制剂的制备方法，包括步骤：将一定量的卡波姆加入免疫细胞扩增悬液溶解，采用0.1M的氢氧化钠溶液调节PH值至6-6.5，过程中缓慢均匀搅拌。灌装入预制PE注射包装内，封口，包装，储存于4-10℃。
一种实施方式中，所述药物为外用浸润制剂。
一种实施方式中，外用浸润制剂的主要药效成分为：免疫细胞扩增悬液。
一种实施方式中，无菌医用棉球浸润于所述免疫细胞扩增悬液，形成外用浸润制剂。
使用时，将浸润了所述免疫细胞扩增悬液的无菌医用棉球，也就是外用浸润制剂直接涂抹或覆盖于患者宫颈部位即可。
与现有技术相比，本发明具有如下有益效果：
本发明首次发现，使用免疫细胞扩增悬液为主要药效成分涂抹于宫颈部位、主要采取隔离和隔绝进一步感染接触机会，可治疗HPV感染或由于HPV持续感染所引起的疾病，使HPV阳性患者转阴。通过所述方法治疗的HPV感染的阳性患者，经7-8次治疗后，转阴率达到50％以上，经过15到20次治疗后转阴率可达到90％以上，并且可有效控制和治疗HPV引起的宫颈瘤样变(CIN)以及宫颈癌复发或转移，达到预防和治疗宫颈癌的效果。至今，通过免疫细胞相关外用涂抹使HPV持续感染快速转阴以及预防和治疗宫颈癌的方法国内外均无报道，此方法为国内外首创。
具体实施方式
本发明的治疗机制为：通过免疫细胞培养过程分泌大量的如干扰素、细胞坏死因子等，同时免疫细胞在接触被感染的细胞后主动杀伤，同时分泌细胞因子杀伤受侵染的细胞，并进一步通过抗原刺激介导出抗体，通过主动和被动免疫共同达到治疗HPV感染及进一步控制和治疗由于感染引起的其它疾病的目的。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN  MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987 and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
本发明的实验材料及其来源：
1、实验环境：GMP实验室里的生物安全柜中操作；10000级洁净区域，生物安全柜100级。
2、试剂及耗材：
白介素2(北京双鹭药业，100万IU/瓶)、淋巴细胞无血清培养基X-VIVO15(Lonza)、CD16单克隆抗体(美国BD公司)、IL-21-K562滋养层细胞(南京苏宁康)；T75细胞培养瓶(NUCK)、T175细胞培养瓶(NUCK)、、细胞培养袋GT-T610A(Takara)和移液管(NUCK)。
3、本发明的实施例进行试验时所采用的设备包括：
生物安全柜(Thermo)、Thermo Scientific Sorvall ST16/16R台式高性能离心机、二氧化碳培养箱(Thermo，3111)、倒置生物显微镜(Motic，AE-31)、自动细胞计数仪(Invitrogen，Countess)、移液器(Thermo)和GE WAVE波浪生物反应器，蠕动泵及超滤膜包(10kd、40kd)及夹具(PALL)。
实施例1 免疫细胞扩增悬液的浸润制剂及其制备和应用
本实施例选用免疫细胞中的NK细胞进行扩增培养，分离，上清为NK细胞扩增悬液(亦可称之为NK细胞扩增后离心获得的悬液)，下层细胞为扩增后的NK细胞。所述NK细胞扩增悬液可作为主要药效成分。所述扩增后的NK细胞也可作为主要药效成分。
具体地，NK细胞扩增悬液、扩增后的NK细胞的制备方法包括如下步骤：
(1)单核细胞采集与获得：
可采用单采、外周血、胎盘血、脐带血采集后淋巴细胞分离液分离或淋巴细胞分离管分离获得单核细胞。
本实施例中所使用的是外周血单核细胞；
(2)单核细胞诱导培养与NK细胞扩增：
取外周血单核细胞4.0×10 7个进行细胞培养，采用细胞因子结合滋养层刺激诱导培养的方法，接种于T75方瓶，密度为1.0-1.5×10 6个细胞/mL，培养基以X-VIVO15为基础，其中滋养层细胞1.0×10 5个细胞/mL，IL-2 100U/ml,IL12 20ng/ml根据培养扩增情况，逐步扩大培养，补液转移到T175培养，直至2L培养袋。
NK细胞扩增后13-14天达到2L培养体系，且细胞密度达到2.0×10 6个细胞/mL以上，NK细胞纯度大于90％，经400G离心15分钟，分别收集上清液和下层细胞，所得上清液即为NK细胞扩增后悬液。
获得的NK细胞扩增悬液为主要药效成分，可制备成浸润制剂。通过医用棉球浸润的方式，直接涂抹或覆盖于患者宫颈部位。
以上述NK细胞扩增悬液为主要药效成分的浸润制剂的制备方法具体包括如下步骤：
将上述获得的NK细胞扩增悬液，经40kd过滤后获得中间品，与无菌医用棉球共同放入50ml医用密封容器中4-10℃储存，作为浸润制剂。
取出后可直接使用。
NK细胞扩增悬液为主要药效成分的外用浸润制剂临床应用于HPV持续感染的治疗：
治疗对象：志愿者，一共21名，均为女性，年龄为20～50岁之间，为临床上已经患HPV持续感染的人群。
治疗方式：每日或隔日采用棉球浸润NK细胞扩增悬液直接涂抹于志愿者的宫颈部位；
治疗效果判断：PCR技术检测HPV-DNA阴性。
治疗结果统计：一共21名患者中，采用本实施例中的治疗方法治疗后，其中2人未复查，5人仍为阳性。采用本实施例中的治疗方法治疗的HPV感染的阳性患者，经7-8次治疗后，转阴率达到31.8％以上。经过15到20次治疗后转阴率可达到52.6％以上。通过治疗随访获知，可有效控制和治疗HPV引起的宫颈瘤样变(CIN)以及宫颈癌复发或转移，达到预防和治疗宫颈癌的效果。
典型案例：
1、志愿者1-1，年龄44岁，病史：2016年10月宫颈癌筛查hpv 52型阳性，2017年1月宫颈活检：正常，2017年7月21日复查hpv52型阳性；治疗方式：2017.9.15-9.27隔日 一次宫颈涂抹本实施例中的NK细胞扩增悬液的外用浸润制剂；指标观察：白带正常菌群正常；一个月复查结果：HPV阳性(2017.10.13)；三个月复查结果：HPV阴性(2017.11.17)。
2、志愿者1-2，年龄32岁，病史：2016年10宫颈癌筛查hpv阳性；2017年09月8日宫颈TCT：正常，HPV82阳性；治疗方式：2017.9.15-9.27隔日一次宫颈涂抹本实施例中的NK细胞扩增悬液的外用浸润制剂；指标观察：白带白色分泌物菌群正常；一个月复查：HPV阳性(2017.10.12)；三个月复查(2017.11.15)。
3、志愿者1-3，年龄46岁，病史：2016年10参加中国宫颈癌筛查hpv阳性；2017年1月宫颈活检：正常；2017.09.26复查hpv23分型：低危44阳性；治疗方法：2017.9.19-9.25每日一次宫颈涂抹本实施例NK细胞扩增悬液的外用浸润制剂；指标观察：白带正常菌群正常；一个月复查：HPV阴性(2017.10.12)；三个月复查：HPV阴性(2017.11.15)。
4、志愿者1-4，年龄42岁，病史：2016年10参加中国宫颈癌筛查hpv阳性；未行阴道镜+活检，未行任何处理。2017年08月25宫颈TCT：正常；活检：正常；复查hpv23分型：高危18、66、68阳性，低危6、11阳性；治疗方法：2017.9.15-9.27隔日一次宫颈涂抹本实施例NK细胞扩增悬液的外用浸润制剂；指标观察：白带正常菌群正常；一个月复查：HPV阴性(2017.10.13)；三个月复查：HPV阴性(2017.11.13)。
5、志愿者1-5，年龄35岁，病史：2017.4.13宫颈TCT：非典型细胞；2017.9.26复查hpv23分型：低危43阳性；治疗方法：2017.9.19-9.25每日一次宫颈涂抹本实施例NK细胞扩增悬液的外用浸润制剂；指标观察：白带正常菌群正常；一个月复查：HPV阳性(2017.10.12)；三个月复查：HPV阴性(2017.11.13)。
实施例2 免疫细胞扩增悬液的浸润制剂及其制备和应用
本实施例选用免疫细胞中的NK细胞进行扩增培养，分离，上清为NK细胞扩增悬液(亦可称之为NK细胞扩增后离心获得的悬液)，下层细胞为扩增后的NK细胞。所述NK细胞扩增悬液可作为主要药效成分。所述扩增后的NK细胞也可作为主要药效成分。
具体地，NK细胞扩增悬液、扩增后的NK细胞的制备方法包括如下步骤：
(1)单核细胞采集与获得：
可采用单采、外周血、胎盘血、脐带血采集后淋巴细胞分离液分离或淋巴细胞分离管分离获得单核细胞。
本实施例中所使用的是外周血单核细胞；
(2)单核细胞诱导培养与NK细胞扩增：
取外周血单核细胞4.0×10 7个进行细胞培养，采用细胞因子结合滋养层刺激诱导培养的方法，接种于T75方瓶，密度为1.0-1.5×10 6个细胞/mL，培养基以X-VIVO15为基础，其中滋养层细胞1.0×10 5个细胞/mL，IL-2 100U/ml,IL12 20ng/ml根据培养扩增情况，逐步扩大培养，补液转移到T175培养，直至2L培养袋。
NK细胞扩增后13-14天达到2L培养体系，且细胞密度达到2.0×10 6个细胞/mL以上，NK细胞纯度大于90％，经400G离心15分钟，分别收集上清液和下层细胞，所得上清液即为NK细胞扩增后悬液。
获得的NK细胞扩增悬液为主要药效成分，可制备成外用凝胶制剂进行宫颈涂抹外用。
以上述NK细胞扩增悬液为主要药效成分的外用凝胶制剂的制备方法具体包括如下步骤：
将上述获得的NK细胞扩增悬液，经40kd过滤后获得中间品，然后将一定量的卡波姆加入免疫细胞扩增悬液溶解，采用0.1M的氢氧化钠溶液调节PH值至6-6.5，过程中缓慢均匀搅拌。灌装入预制PE注射包装内，封口，包装，储存于4-10℃。
免疫细胞扩增悬液的外用凝胶制剂临床应用于HPV持续感染的治疗：
治疗对象：志愿者，一共15名，均为女性，年龄为20～50岁，为临床上已经患HPV持续感染的人群。
治疗方法：每日或隔日采用NK细胞扩增悬液为主要药效成分的外用凝胶制剂涂抹于志愿者的宫颈部位
治疗效果判断：转阴是指PCR技术是检测HPV-DNA阴性。
转阴率是指转阴患者数量与参加试验者数量的百分比。
治疗结果统计：一共15名患者中，采用本实施例中的治疗方法治疗后，其中1人未复查，6人两次检查为阳性，HPV感染的阳性患者，经7-8次治疗后，转阴率达到33.3％以上。经过15到20次治疗后转阴率可达到53.3％以上。通过治疗随访获知，可有效控制和治疗HPV引起的宫颈瘤样变(CIN)以及宫颈癌复发或转移，达到预防和治疗宫颈癌的效果。
典型案例：
1、志愿者2-1，年龄39岁，病史：2016年11月检查hpv18型阳性；2017年7月28 日宫颈TCT：正常，HPV39阳性；治疗方式：2017.9.20-9.27每日一次宫颈涂抹本实施例NK细胞扩增悬液为主要药效成分的外用凝胶制剂；指标观察：白带正常菌群正常；一个月复查结果：HPV阴性(2017.10.20)；三个月复查结果：HPV阴性(2017-11.17)。
2、志愿者2-2，年龄29岁，病史：2017年9月20日查hpv高危52、68型阳性、低危81阳性、疑似高危73阳性；治疗方式：2017.9.15-9.27隔日一次宫颈涂抹本实施例NK细胞扩增悬液为主要药效成分的外用凝胶制剂；指标观察：白带正常菌群正常；一个月复查结果：未复查；三个月复查结果：HPV阴性(2017-11.15)。
3、志愿者2-3，年龄：42岁；病史：2016年08宫颈癌筛查hpv16型阳性；2017年01月宫颈TCT：正常；治疗方法：2017.9.20-9.26每日一次宫颈涂抹本实施例NK细胞扩增悬液为主要药效成分的外用凝胶制剂；指标观察：白带正常菌群正常；一个月复查结果：HPV阳性(2017.10.12)；三个月复查结果：HPV阴性(2017.11.15)。
4、志愿者2-4，年龄34岁，病史：2017.09.14hpv高危51、58型阳性；治疗方法：2017.9.15-10.13隔日一次宫颈涂抹本实施例NK细胞扩增悬液为主要药效成分的外用凝胶制剂；指标观察：白带正常菌群正常；一个月复查：HPV阴性(2017.10.13)；三个月复查：HPV阴性(2017.11.13)。
5、志愿者2-5，年龄45岁，病史：2016.10参加中国宫颈癌筛查hpv阳性；2017.01正常；阴道药物，具体用药不详；2017.09.5复查hpv 23分型：高危52、58阳性；低危81阳性；治疗方法：2017.9.20-9.26每日一次本实施例NK细胞扩增悬液为主要药效成分的外用凝胶制剂；指标观察：白带正常菌群正常；一个月复查：HPV阳性(2017.10.12)；三个月复查：HPV阴性(2017.11.13)。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (18)

  1. 免疫细胞扩增悬液用于制备治疗HPV感染或HPV感染引起的疾病的药物的用途。
  2. 根据权利要求1所述的用途，其特征在于，所述免疫细胞扩增悬液为主要药效成分。
  3. 根据权利要求1所述的用途，其特征在于，所述免疫细胞扩增悬液是指免疫细胞经扩增培养、分离后获得的上清。
  4. 根据权利要求1所述的用途，其特征在于，扩增培养后，免疫细胞密度达到2.0×10 6个细胞/mL以上；优选地，扩增培养后，免疫细胞密度达到3.0×10 6个细胞/mL以上；再优选地，扩增培养后，免疫细胞密度达到4.0×10 6个细胞/mL以上。
  5. 根据权利要求1所述的用途，其特征在于，所述扩增时所采用的方法选自：细胞因子刺激法、抗体刺激方法或滋养层细胞扩增法。
  6. 根据权利要求1所述的用途，其特征在于，所述免疫细胞扩增悬液为NK细胞扩增悬液，所述NK细胞扩增悬液是指NK细胞经扩增培养、分离后获得的上清。
  7. 根据权利要求1所述的用途，其特征在于，NK细胞由单核细胞培养获得；进一步地，所述单核细胞来源于外周血、脐带血或胎盘血。
  8. 根据权利要求1所述的用途，其特征在于，所述HPV感染是指HPV检测为阳性。
  9. 根据权利要求1所述的用途，其特征在于，所述HPV感染引起的疾病是指HPV持续感染引起的疾病。
  10. 根据权利要求9所述的用途，其特征在于，HPV持续感染引起的疾病为宫颈瘤样变、尖锐湿疣或宫颈癌。
  11. 根据权利要求1所述的用途，其特征在于，所述药物为外用制剂，进一步地，所述药物为外用涂抹制剂。
  12. 根据权利要求11所述的用途，其特征在于，所述药物为外用凝胶剂或外用浸润制剂。
  13. 根据权利要求11所述的用途，其特征在于，所述外用凝胶剂含有免疫细胞扩增悬液和药学上可接受的载体辅料；所述外用浸润制剂包括免疫细胞扩增悬液和浸润了免疫细胞扩增悬液的无菌医用棉球。
  14. 一种治疗HPV感染或HPV感染引起疾病的方法，包括步骤：将以免疫细胞扩增悬液为主要药效成分的药物覆盖或涂抹于患者的宫颈部位。
  15. 一种治疗HPV感染或HPV感染引起疾病的药物，主要药效成分为免疫细胞扩增悬液。
  16. 根据权利要求15所述的药物，其特征在于，所述药物为外用制剂，进一步地，所述药物为外用涂抹制剂。
  17. 根据权利要求15所述的药物，其特征在于，所述药物为外用凝胶剂或外用浸润制剂。
  18. 根据权利要求15所述的药物，其特征在于，所述外用凝胶剂含有免疫细胞扩增悬液和药学上可接受的载体辅料；所述外用浸润制剂包括免疫细胞扩增悬液和浸润了免疫细胞扩增悬液的无菌医用棉球。
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