WO2019047520A1 - 抗pl2l60蛋白抗体在制备抗肿瘤药物中的应用及治疗肿瘤的方法 - Google Patents

抗pl2l60蛋白抗体在制备抗肿瘤药物中的应用及治疗肿瘤的方法 Download PDF

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WO2019047520A1
WO2019047520A1 PCT/CN2018/082380 CN2018082380W WO2019047520A1 WO 2019047520 A1 WO2019047520 A1 WO 2019047520A1 CN 2018082380 W CN2018082380 W CN 2018082380W WO 2019047520 A1 WO2019047520 A1 WO 2019047520A1
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cancer
kao3
antibody
tumor
pl2l60
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French (fr)
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高建新
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上海易范科生物科技有限公司
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Priority to US16/346,631 priority patent/US20200055950A1/en
Priority to JP2019517939A priority patent/JP2020500153A/ja
Publication of WO2019047520A1 publication Critical patent/WO2019047520A1/zh

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    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present disclosure belongs to the field of biomedicine, relates to an agent for inhibiting ectopic expression of PIWIL2 gene, an anti-PIWIL2 gene ectopically expressed protein antibody for preparing an antitumor drug, and particularly relates to an application of an anti-PL2L60 protein antibody for preparing an antitumor drug, And a pharmaceutical composition comprising the above reagent or antibody, and a method of treating a tumor.
  • Cancer immunotherapy applies immunological principles and methods to improve the immunogenicity of tumor cells and sensitivity to effector cell killing, stimulate and enhance the body's anti-tumor immune response, and use immune cells and effector molecules to infuse the host, synergy
  • the body's immune system kills tumors, inhibits tumor growth, and has good therapeutic effects in various types of cancer, and is therefore receiving more and more attention.
  • the targets of cancer immunotherapy are largely individualized, with most targets being distributed only in a few types of cancer, not all types of cancer. Moreover, these molecular targets are also not cancer specific and are essential for the function of normal cells. Therefore, the current bottleneck in immunotherapy is the lack of specific and broad-spectrum targets or antigens that are widely expressed in hematopoietic and solid cancers.
  • the objects of the present disclosure include the use of an agent for inhibiting ectopic expression of the PIWIL2 gene in the preparation of an antitumor drug, the use of an anti-PIWIL2 gene ectopically expressed protein antibody for the preparation of an antitumor drug, and a novel antitumor drug combination. And a method of treating tumors.
  • the present disclosure may employ the following technical solutions:
  • a first aspect of the disclosure includes the use of an agent that inhibits ectopic expression of a PIWIL2 gene for the preparation of an anti-tumor drug.
  • a second aspect of the disclosure includes the use of an ectopically expressed protein antibody that provides an anti-PIWIL2 gene for the preparation of an anti-tumor drug.
  • the above antitumor drugs include drugs against lymphoma, melanoma, breast cancer, lung cancer, liver cancer, bladder cancer, cervical cancer, prostate cancer, gastric cancer, leukemia, colorectal cancer, colon cancer, ovarian cancer or testicular germ cell tumors.
  • the ectopically expressed protein antibody against the PIWIL2 gene provided by the present disclosure is an anti-PL2L protein antibody, and may be, for example, an anti-PL2L80, PL2L80A, PL2L60, PL2L60A, PL2L50 or PL2L40 protein antibody, preferably an anti-PL2L60 protein antibody.
  • PL2L60 is mainly expressed in precancerous stem cells (pCSCs) as well as various types of human and murine tumor cell lines, and its expression level is much higher than that of full length PIWIL2.
  • PL2L60 can promote the survival and proliferation of tumor cells by up-regulating STAT3 and BCL2 genes in vitro, and can also promote tumorigenesis in coordination with NF- ⁇ B, which may represent a common pathway for tumor development in various tissues.
  • peptides derived from PL2L60 can serve as a strong source of immunity against a variety of cancer types.
  • PL2L protein especially anti-PL2L60 monoclonal antibody, exhibits a unique ability to directly induce cancer cell apoptosis and inhibit cell proliferation and arrest cell cycle.
  • PL2L60 protein is pCSC-induced naturally occurring tumor immunity (NOTI).
  • NOTI tumor immunity
  • One of the targets may be a common target for cancer immunotherapy.
  • the anti-PL2L60 protein antibody provided by the present disclosure is preferably a KAO3 monoclonal antibody, and the KAO3 monoclonal antibody sequence is shown in SEQ ID NO: 1.
  • the anti-PL2L60 protein antibody is an antibody that inhibits STAT3 or BCL2 gene activation.
  • the KAO3 antibody has the potential to prepare PL2L protein-specific CRA-T cells.
  • a third aspect of the present disclosure includes providing an antitumor pharmaceutical composition which can be composed of an agent which inhibits ectopic expression of the PIWIL2 gene, and a pharmaceutically acceptable excipient, and can also be ectopically expressed by an anti-PIWIL2 gene Protein antibody and pharmaceutically acceptable excipient composition.
  • a fourth aspect of the present disclosure includes a method of treating a tumor comprising: inhibiting ectopic expression of a PIWIL2 gene.
  • the tumor may include any one of lymphoma, melanoma, breast cancer, lung cancer, liver cancer, bladder cancer, cervical cancer, prostate cancer, gastric cancer, leukemia, colorectal cancer, colon cancer, ovarian cancer and testicular germ cell tumor.
  • lymphoma lymphoma
  • melanoma breast cancer
  • lung cancer liver cancer
  • bladder cancer cervical cancer
  • prostate cancer gastric cancer
  • leukemia colorectal cancer
  • colon cancer colon cancer
  • ovarian cancer testicular germ cell tumor.
  • the present disclosure demonstrates in a large number of experiments that the widespread expression of PL2L protein in various cancer types makes it an ideal broad-spectrum target for solid and hematopoietic stem cell immunotherapy, and is capable of exhibiting direct induction of cancer from various types of PL2L proteins.
  • the PL2L60 protein which has apoptosis and a unique ability to inhibit cell proliferation and cell cycle, has also developed an anti-PL2L60 mAb (KAO3) against this protein.
  • KAO3 anti-PL2L60 mAb
  • the present disclosure provides a new basis for the preparation of anti-tumor drugs using anti-PL2L60 mAb.
  • Figure 1 shows the expression of PL2L60 protein in cancer cells.
  • (A) is the flow cytometry to determine the binding activity of anti-PL2L60 monoclonal antibody: flow cytometry analysis of plate cultured cell lines (2C4, 326t-4, MDA-MB-231, A549 and HeLa) surface PL2L60 Protein expression.
  • Mouse B16 cell lines include LLC, E14, 326t-4 and 2C4; human cell lines include HCT116, HeLa, HepG2, A549 and MDA-MB-231; (FG): expression of mouse and human tumor cell line PL2L60 protein Quantitative analysis.
  • Figure 2 shows that anti-PL2L60 mAb KAO3 inhibits proliferation of cancer cells and induces apoptosis in vitro.
  • A observed the morphology of cancer cells after 48 hours of treatment with mAb KAO3 under phase contrast microscope, the bar represents 25 ⁇ m;
  • B flow cytometry against cancer cells treated with PL2L60 monoclonal antibody for 48 hours; Inhibition of cancer cells by anti-PL2L60 mAb at the end of 48 hours of culture;
  • D Summary of the dose-dependent effect of mAb KAO3 supernatant ( ⁇ m/ml) on induction of apoptosis in cancer cells.
  • Figure 3 is a graph of cell cycle distribution.
  • A is the cell cycle distribution of 2C4, 326T-4, MDA-MB-231, A549 and HeLa cell lines treated with KAO3;
  • B the ratio of cells in the G0/G1 phase between the control group and the treatment group.
  • C proportion of cells in the S phase;
  • D proportion of cells in the G2/M phase.
  • FIG. 4 shows the effect of KAO3 pretreated cancer cells on tumor incidence.
  • (A)-(E) were the incidence of tumors after KAO3 pretreatment and IgG control pretreatment of 2C4, 326T-4, MDA-MB-231, A549 and HeLa cell lines, respectively, * represents p ⁇ 0.05, *** Representative p ⁇ 0.001.
  • Figure 5 shows the effect of KAO3 on the treatment of tumor-bearing mice at different stages.
  • A, D, G, J, and M tumor size of mice inoculated with KAO3 culture supernatant pretreatment or medium (R10F) control pretreated cancer cells, followed by control treatment, wherein A: 2C4; D : 326T-4; G: MDA-MB-231; J: A549; M: HeLa;
  • B, E, H, K, and N mouse tumor of control medium (R10F) control pretreated cancer cells Size, followed by KAO3 or control treatment.
  • FIG. 6 shows the results of an experimental analysis of complement dependent cytotoxicity (CDC).
  • the CDC experiment was performed in the five cells mentioned in the present disclosure, wherein (A) is a histogram of PI positive cells.
  • (B) is a statistical analysis of PI positive cells. The percentage of dead cells was positively correlated with the anti-tumor efficacy of the anti-PL2L60 mAb.
  • Mouse pCSCs 2C4 and human breast cancer cell line MDA-MB-231 showed the strongest oncolytic effect in CDC experiments, followed by mouse lymphoma cell line 326T-4 and human lung cancer cell line A549.
  • the human cervical cancer cell line HeLa has the weakest CDC effect.
  • the present disclosure relates generally to the use of an agent that inhibits the ectopic expression of the PIWIL2 gene in the preparation of an antitumor drug and the use of an ectopically expressed protein antibody against the PIWIL2 gene in the preparation of an antitumor drug.
  • the above PIWIL2 is usually expressed in the testis, but can also be activated in somatic cells after DNA damage, and promotes DNA repair by remodeling chromatin, and therefore plays a key role in stem cell self-renewal and maintenance.
  • Ectopic expression of the PIWIL2 gene is usually expressed in the testis, but can also be activated in somatic cells after DNA damage, and promotes DNA repair by remodeling chromatin, and therefore plays a key role in stem cell self-renewal and maintenance.
  • PIWIL2 can promote tumorigenesis by regulating several signal transduction pathways, and inhibits apoptosis death of tumor cells by activation of the Stat3/Bcl-XL pathway.
  • most of the PIWIL2-specific antibodies obtained commercially are unable to resolve full-length PIWIL2 from their variants. It is worth noting that in primary breast cancer and cervical cancer, the full-length PIWIL2 protein is mainly detected in apoptotic tumor cells, but not in living tumor cells.
  • variant PL2L proteins of PIWIL2 (such as PL2L60) are abundantly detected in various types of tumor tissues and tumor cell lines, indicating that the tumorigenic function of PIWIL2 is mainly mediated by PIWIL2 variants.
  • PIWIL2 There are many variants of PIWIL2, including PL2L80, PL2L80A, PL2L60, PL2L60A, PL2L50 and PL2L40. Some variants appear to be transcribed by an intragenic promoter rather than a canonical promoter. Although full-length PIWIL2 can mediate DNA repair, as a barrier gene for tumorigenesis initiation and promote apoptotic cell death in tumor tissues, variants such as PL2L60 and PL2L60A promote tumorigenesis. In the above variants, PL2L60 is expressed primarily in precancerous stem cells (pCSCs) as well as in various types of human and murine tumor cell lines, at levels much higher than full length PIWIL2.
  • pCSCs precancerous stem cells
  • PL2L60 promotes tumor cell survival and proliferation by up-regulating STAT3 and BCL2 genes. It can also be coordinated with NF- ⁇ B to promote tumorigenesis and may represent a common pathway for tumor development in various types of tissues. Importantly, peptides derived from PL2L60 can serve as strong immunogens for targeting various types of cancer. In addition, PL2L60 was also detected in testicular cells of mice, indicating its role in gametogenesis or development.
  • PIWIL2 (PL2L60) protein, a product of the anabolically activated PIWIL2 gene, is widely expressed in various hematopoietic and solid tumors and mediates tumorigenesis by promoting tumor cell proliferation and inhibiting apoptosis.
  • tumors can be treated by inhibiting ectopic expression of the PIWIL2 gene.
  • An agent that inhibits ectopic expression of the PIWIL2 gene or an ectopically expressed antibody against the PIWIL2 gene can also be used to prepare an antitumor drug.
  • the ectopically expressed protein antibody against the PIWIL2 gene of the present disclosure is a PL2L protein antibody, and may include, for example, at least one of anti-PL2L80, PL2L80A, PL2L60, PL2L60A, PL2L50, and PL2L40 protein antibodies.
  • the ectopically expressed protein antibody against the PIWIL2 gene of the present disclosure is an anti-PL2L60 protein antibody.
  • the inventors of the present disclosure mainly developed three monoclonal antibodies (mAbs) against PIWIL2: mAb KAO1, mAb KAO2, and mAb KAO3.
  • mAb KAO2 and mAb KAO3 have a stronger affinity for PL2L60 than mAbKAO1, but since mAbKAO3 specifically targets PL2L60, it is considered a potential therapeutic candidate for the treatment of cancer. Therefore, the anti-PL2L60 protein antibody used in the present disclosure is preferably a KAO3 monoclonal antibody, and the KAO3 monoclonal antibody sequence is shown in SEQ ID NO: 1.
  • anti-PL2L60 protein antibody of the present disclosure may also be an antibody that inhibits STAT3 or BCL2 gene activation.
  • an antitumor pharmaceutical composition which may be composed, for example, of the above-mentioned agent for inhibiting ectopic expression of the PIWIL2 gene, and a pharmaceutically acceptable adjuvant, or may be composed of the above-mentioned anti-PIWIL2 gene.
  • a ectopically expressed protein antibody and a pharmaceutically acceptable excipient composition may be composed, for example, of the above-mentioned agent for inhibiting ectopic expression of the PIWIL2 gene, and a pharmaceutically acceptable adjuvant, or may be composed of the above-mentioned anti-PIWIL2 gene.
  • pharmaceutically acceptable excipients refer to excipients and additives used in the production of pharmaceuticals and formulation. It is a substance contained in a pharmaceutical preparation other than the active ingredient. In addition to shaping, acting as a carrier and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, and controlled release.
  • the pharmaceutical excipient may include, for example, at least one of a filler, a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a film coating material, a dropping base, and a condensate.
  • a filler for example, at least one of a filler, a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a film coating material, a dropping base, and a condensate.
  • a filler for example, at least one of a filler, a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a film coating material, a dropping base, and a condensate.
  • starch dextrin, lactose, microcrystalline cellulose, sugar alcohol, ethanol, cement, polyethylene glycol, methyl cellulose, sodium carboxymethyl starch, magnesium
  • the antibody in the pharmaceutical composition can be referred to the above anti-PL2L protein antibody, preferably a KAO3 monoclonal antibody.
  • the anti-PL2L protein antibody may also be a therapeutic antibody that targets PL2L60 and/or inhibits STAT3 and BCL2 activation.
  • amino acid sequence of the antibody in the pharmaceutical composition provided by the present disclosure may be a sequence including SEQ ID NO: 1 and/or a substitution and/or deletion and/or addition via multiple amino acid residues. Derived sequence obtained and having the same biological activity as SEQ ID NO: 1.
  • the present disclosure also provides a method of treating a tumor comprising: inhibiting ectopic expression of the PIWIL2 gene.
  • the ectopic expression of the PIWIL2 gene is inhibited by the above KAO3 monoclonal antibody.
  • the tumors involved herein include any of lymphoma, melanoma, breast cancer, lung cancer, liver cancer, bladder cancer, cervical cancer, prostate cancer, gastric cancer, leukemia, colorectal cancer, colon cancer, ovarian cancer, and testicular germ cell tumors.
  • lymphoma, melanoma breast cancer, lung cancer, liver cancer, bladder cancer, cervical cancer, prostate cancer, gastric cancer, leukemia, colorectal cancer, colon cancer, ovarian cancer, and testicular germ cell tumors.
  • SCID mice 8 to 12 weeks of mice were used and these mice were housed in animal detoxification facilities.
  • Human cell lines human breast cancer cell line MDA-MB-231, human lung cancer cell line A549 and human cervical cancer cell line HeLa were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 0.1 mg/ml penicillin-streptomycin (Gibco).
  • Mouse lymphoma cell lines 2C4 and 326T-4 were prepared in the laboratory: the cells were placed in a humidified incubator containing 5% CO 2 (v/v) and maintained at R10F (RPMI 1640 plus 10 mmol) at 37 °C. /L fetal bovine serum supplemented with 5 mmol/L glutamine, 50 mmol/L 2-methylacetophenone, 100 U/mL penicillin and 100 mg/ml streptomycin).
  • the anti-PL2L60 monoclonal antibody (KAO3 mAb, isoform IgM) was prepared in the laboratory according to the preparation procedure of the monoclonal antibody, and the sequence is shown in SEQ ID NO: 1.
  • the preparation process of the monoclonal antibody in the present disclosure may include, for example:
  • Immunization of animals is the process of immunizing mice with the antigen of interest to produce sensitized B lymphocytes in mice.
  • Female Balb/c mice, 6-8 weeks old, are generally selected for immunization according to a pre-established immunization protocol.
  • the antigen enters the peripheral immune organs through the blood circulation or lymph circulation, stimulates the corresponding B lymphocyte clones, activates, proliferates, and differentiates into sensitized B lymphocytes.
  • mice were sacrificed by carbon dioxide gas, the spleen was aseptically removed, and spleen cell suspension was prepared by extrusion grinding in a dish.
  • the prepared syngeneic myeloma cells are mixed with mouse spleen cells in a certain ratio, and a fusogenic agent polyethylene glycol is added. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells.
  • HAT selective medium can be employed.
  • HAT medium unfused myeloma cells are unable to synthesize DNA by a salvage pathway and die due to the lack of hypoxanthine-guanine-phosphoribosyltransferase.
  • unfused lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they cannot survive long-term survival in vitro and gradually die.
  • hybridoma cells grown in HAT medium Only a small number of hybridoma cells grown in HAT medium are cells secreting a predetermined specific monoclonal antibody, and therefore, screening and cloning must be performed.
  • clonal culture of hybridoma cells can be performed, for example, by limiting dilution.
  • positive hybridoma cells that produce the desired monoclonal antibody are screened and cloned and amplified. After comprehensively identifying the immunoglobulin type, subclass, specificity, affinity, epitope of the recognized antigen and its molecular weight of the monoclonal antibody secreted by the monoclonal antibody, it is frozen in time.
  • a large number of monoclonal antibodies can be prepared by in vivo in vivo induction and in vitro culture.
  • the in vivo induction method includes: taking Balb/c mice, first pre-treatment by intraperitoneal injection of 0.5 ml of liquid paraffin or pristane. After 1-2 weeks, the hybridoma cells were inoculated intraperitoneally. Hybridoma cells proliferate in the peritoneal cavity of mice and produce and secrete monoclonal antibodies. About 1-2 weeks, the mouse's abdomen is enlarged. A large amount of monoclonal antibody can be obtained by taking ascites with a syringe.
  • the in vitro culture method comprises: placing the hybridoma cells in a culture flask for culture. During the culture process, the hybridoma cells produce and secrete monoclonal antibodies, collect the culture supernatant, and centrifuge to remove the cells and fragments thereof to obtain the desired monoclonal antibodies.
  • the inventors of the present disclosure have developed three monoclonal antibodies (mAbs) against PIWIL2: mAb KAO1, mAb KAO2, and mAb KAO3. Since mAbKAO3 is specifically directed to PL2L60, the KAO3 monoclonal antibody is preferably used in the following assays.
  • tumor cells were collected, and tumor cells were resuspended in PBS at a cell concentration of 5 ⁇ 10 6 /ml, and added to a 96-well plate at 0.2 mL of a cell suspension (1 ⁇ 10 6 cells/well). Rotate the cells (1000 rpm, 5 minutes) and discard the supernatant.
  • Anti-PL2L60 monoclonal antibody KAO3 was added to 100 ⁇ L of PBS and spun for 5 seconds. The samples were incubated for 30 minutes at 4 °C. The cells were washed twice with PBS. The supernatant was discarded, and 100 ⁇ l of PBS containing 1% paraformaldehyde was added to each well to fix the cells.
  • tumor cells cultured on coverslips were fixed in 2% paraformaldehyde for 20 minutes, then washed, followed by blocking with PBS containing 1% bovine serum albumin for 30 minutes.
  • Cells were incubated with anti-PL2L60 mAb (KAO3, 1:100 dilution) in 1% bovine serum albumin at room temperature. After 1 hour of incubation, the cells were washed and co-incubated with FITC goat anti-mouse antibody (IgM, Bioligend). The nuclei were stained with a stain such as 4',6-diamidino-2-phenylindole (DAPI, 1:500).
  • DAPI 4',6-diamidino-2-phenylindole
  • the cell samples Prior to harvesting with trypsin, the cell samples were washed twice in cold PBS and then lysed with protein extraction reagent. The total protein concentration of the whole cell lysate was determined using a BCA protein assay kit (Beyotime, Shanghai, China), and then the protein was separated using a 12% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane.
  • the cells were incubated with specific primary antibody at 4 ° C overnight, and the membrane was washed with TBS-T for 5 minutes, and the washing was repeated three times. Thereafter, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse IgM secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour at room temperature, followed by washing with TBST for 5 minutes, three times. The membrane was then analyzed using an ECL chemiluminescence detection system (Bio-Rad).
  • BSA bovine serum albumin
  • anti-PIWIL2 polyclonal antibody prepared by our group
  • images were obtained from Kodak Imaging Station 2000R (Eastman Kodak, USA).
  • CCK-8 assay The effect of anti-PL2L60 mAb (KAO3) on cell viability was assessed using CCK-8 assay (Dojindo, Japan).
  • CCK-8 assay cells were mixed with concentration gradients (1, 2, 4, and 8 ⁇ g/ml) of KAO3 in a final volume of 200 ⁇ L of 10% FBS medium using the same concentration of IgG as a control.
  • Serially diluted PL2L60 antibody or control IgG was then seeded at a density of 2 x 10 3 cells/well in flat-bottom 96-well plates.
  • the cells were fixed in frozen 75% ethanol, and subjected to cell cycle analysis staining in PBS with a propidium iodide (PI) solution containing 100 ⁇ g/ml ribonuclease (Tiangen Biotech) and 50 ⁇ g/mL PI (Biolegend). The percentage of cells at each stage of the cell cycle was measured by a C6 flow cytometer (BD).
  • PI propidium iodide
  • mice Five x 10 6 cells per 200 ⁇ L PBS were injected into the groin of SCID mice. After the tumor occurred, the mice carrying the tumor were randomly divided into four groups: group 1: control ⁇ control group; group 2: control ⁇ KAO3; group 3: KAO3 ⁇ control group; group 4: KAO3 ⁇ KAO3. KAO3 or isotype IgG was injected in situ for two weeks. The length and width of the tumor were measured with a caliper every 2 days, and when the tumor reached a diameter of 2 cm, the mouse was euthanized.
  • CDC Complement dependent cytotoxicity
  • the plate was incubated at 37 ° C for 3 hours and then the cell viability reagent was added. Triton 1X-100 was added to the wells of control cells to establish maximum lysis control. After incubating for 1 hour at 37 ° C, fluorescence was measured by a fluorescence microscope (Olymplus, Japan).
  • analysis parameters, the analysis conditions, the reagents used, and the like involved in the above analysis methods can be appropriately adjusted according to the specific conditions and conditions of the experiment.
  • PIWIL2 protein By immunohistochemistry, the full-length PIWIL2 protein was mainly detected in apoptotic tumor cells in primary breast cancer and cervical cancer, but almost no detectable in living tumor cells.
  • PIWIL2 variant PL2L proteins (such as PL2L60) are abundantly detected in various types of tumor tissues and tumor cell lines, suggesting that the tumorigenic function of PIWIL2 may be mediated primarily by PIWIL2 variants.
  • PL2L proteins such as PL2L60
  • PL2L protein on the cell surface of various types of tumor cell lines using a monoclonal antibody (mAb KAO3) against human and mouse homologous PIWIL2 peptides.
  • mAb KAO3 monoclonal antibody
  • PL2L proteins were detected on the surface of tumor cell lines by flow cytometry and fluorescence microscopy (Fig. 1A-B), including mouse pre-hematopoietic cancer stem cells (pCSCs) 2C4 and cancer stem cells (CSC).
  • pCSCs mouse pre-hematopoietic cancer stem cells
  • CSC cancer stem cells
  • Fig. 1C Intracellular immunofluorescence analysis
  • Fig. 1C Intracellular immunofluorescence analysis
  • Fig. 1D-G Western blotting data
  • PL2L60 total protein was highly expressed in various types of human and murine tumor cell lines, and the expression level was highest in the cytoplasm of pCSCs 2C4 (Fig. 1D-G).
  • PL2L60 can promote tumor cell survival and proliferation by up-regulating STAT3 and BCL2 genes, and it can also promote tumorigenesis in coordination with NF- ⁇ B protein. Therefore, we have developed antibodies that inhibit the proliferation of cancer by producing monoclonal antibodies against PL2L60. There is currently no public report on anti-PL2L60 antibodies that can directly inhibit proliferation and induce apoptosis. In the current study, we examined whether the newly produced anti-PL2L60 mAb has this ability.
  • KAO3 mAb can induce apoptosis in cancer cells, such as MDA-MB-231, A549, HeLa, 2C4 and 326T-4 (Fig. 2).
  • cancer cells such as MDA-MB-231, A549, HeLa, 2C4 and 326T-4 (Fig. 2).
  • the morphology of cancer cells in the control group and the experimental group were basically the same. The control cells grew well and the shape was polygonal. After 48 hours of culture, the size of the control cells was uniform. After 48 hours of treatment with anti-PL2L60mAb KAO3, cell chromatin loss was observed under a high power microscope, the number of cells was significantly reduced, the cell morphology was irregular, and some cells became round. Visible nuclear chromosomes and reduced cytoplasmic vacuoles were observed under a high power microscope (Fig. 2A).
  • Fig. 2B and Fig. 2C The number of apoptotic cells in the semi-independent cells in the suspension increased dramatically (Fig. 2B and Fig. 2C). Specifically, cell viability was reduced after anti-PL2L60 antibody treatment at different doses for 48 hours (1, 2, 4, and 8 ⁇ l/well of mAb KAO3 supernatant) (Fig. 2D).
  • KAO3 mAb induces cell cycle arrest in cancer cells
  • Figure 3A shows the cell cycle distribution of 2C4, 326T-4, MDA-MB-231, A549 and HeLa after treatment with anti-PL2L60 mAb (KAO3).
  • the ratio of G0/G1 phase of cancer cells decreased slightly after treatment with anti-PL2L60 mAb (KAO3) for 48 h, and the percentage of cells in S phase showed little change.
  • the number of cells in the G2/M phase is increased.
  • A549 cells have the largest number of G2/M phase cells.
  • the proportion of G0/G1 and G2/M phases increased sharply, and the percentage of cells in S phase decreased sharply in MDA-MB-231 and HeLa cells.
  • the maximal changes were observed in G0/G1, S and G2/M phase 2C4 cells.
  • There was no significant change in 326T-4 cells (Fig. 3B - Fig. 3D).
  • PI/FACS analysis indicated that the anti-PL2L60 mAb (KAO3) caused significant G2/M arrest in five cancer cell
  • KAO3 anti-PL2L60 monoclonal antibody
  • human and mouse cancer cells were suspended in the culture supernatant of KAO3 hybridoma or culture medium, and then inoculated into SCID mice, which were treated with isotype IgG as a control.
  • SCID mice which were treated with isotype IgG as a control.
  • tumor-bearing mice in different treatment groups were divided into two groups. One group of tumor-bearing mice were injected with KAO3 in situ every two days, and the other group was injected with isotype IgG as a control, and tumor growth was observed.
  • mice inoculated with KAO3-pretreated MDA-MB-231 cells were 50%, compared with 83% in the control group.
  • the tumor incidence rate of the other three cell lines was 100%, and there was no difference between the KAO3 pretreatment group and the control group (Fig. 4A - Fig. 4E).
  • CSCs mouse hematopoietic stem cells
  • 326T-4 326T-4
  • Fig. 5D-Fig. 5F The same treatment of mouse hematopoietic stem cells (CSCs; clone 326T-4) and 326T-4 also resulted in varying degrees of tumorigenicity or tumor growth inhibition in SCID mice (Fig. 5D-Fig. 5F).
  • mAb KAO3 also recognizes human PL2L60 protein, human breast cancer cell line MDA-MB-231, human lung cancer cell line A549 and human cervical cancer cell line were treated with the culture supernatant of hybridoma KAO3 by the same method as 2C4 pCSC. HeLa, and similar results were observed, that is, KAO3 mAb was also effective in inhibiting tumorigenesis of human cancer cells and growth of established tumors in SCID mice (Fig. 5G-Fig. 5P). The results indicate that mAb KAO3 can effectively kill or inhibit cancer cells in humans and mice.
  • mAb KAO3 does not appear to be limited to two tumors, as the same treatment of murine hematopoietic stem cells (CSCs; clone 326T-4), human lung cancer cell line A549 and human cervical cancer cell line HeLa also results in different degrees of tumorigenicity. Or tumor growth inhibition of SCID mice. These results confirm our findings that KAO3 has a higher therapeutic efficacy than the control in reducing tumor growth. KAO3 has been shown to inhibit the growth of tumor growth in humans and mice in vivo.
  • the anti-tumor therapeutic effect is positively correlated with the expression level of PL2L60 protein on the surface of tumor cells on complement-dependent cytotoxicity, because in the presence of complement in vitro, mAb can kill tumor cells. Therefore, the PL2L60 protein is also a suitable target for passive immunotherapy of cancer.
  • mice pCSC line 2C4 and the human breast cancer cell line MDA-MB-231 showed the strongest oncolytic effect in the CDC experiment, followed by the mouse lymphoma cell line 326T-4 and the human lung cancer cell line A549.
  • the human cervical cancer cell line HeLa has the weakest CDC effect (Fig. 6A-B).
  • mAb KAO3 we used our laboratory-developed mAb KAO3 to test whether PL2L protein is a common target for cancer immunotherapy.
  • Differential expression of mAb KAO3 in inhibiting tumorigenesis and tumor growth appears to be associated with expression of surface KAO3-specific antigen (ie, PL2L protein) between cancer cell lines (Fig. 1A - Fig. 1B), but not with intracellular expression (Fig. 1C- Figure 1G), because the ability of KAO3 mAb to inhibit tumorigenesis correlates with the percentage of surface KAO3+ cells.
  • the HeLa and 326T-4 cell lines contained less KAO3+ cells than other lines and were less sensitive to mAb KAO3 treatment ( Figure 5). Therefore, the therapeutic efficacy of mAb KAO3 is determined by the amount of surface PL2L protein expression.
  • anti-PL2L60 antibodies inhibit tumors in vivo remains unclear. Unpublished studies have demonstrated that anti-PL2L60 antibodies directly induce apoptosis in cancer cells in a clinical setting, so it is unclear whether this antibody can directly inhibit tumor cell proliferation. In the present study, KAO3 mAb treatment inhibited proliferation of human and mouse cancer cells and significantly increased the percentage of cells in the G2/M phase of the cell cycle in a dose-dependent manner, particularly the percentage of 2S4 cells in the S phase was significantly reduced.
  • KAO3 has a strong inhibitory effect on the cell growth of cancer cells.
  • KAO3 induces cell cycle arrest in a dose-dependent manner in the G2/M phase, followed by progression to apoptosis (Fig. 2–3), which may be associated with microtubule depolymerization or inhibition of nuclear translocation of NF- ⁇ B and STAT3, Further, the transcription activity is inhibited.
  • KAO3 effectively delays tumor growth ( Figure 4-5).
  • Our results provide direct evidence that our anti-PL2L60 antibody inhibits tumor cell proliferation and induces apoptosis.
  • PL2L protein is an effective target for cancer immunotherapy.
  • mAb KAO3 recognizes surface PL2L proteins expressed on various types of tumor cell lines, including, for example, lymphoma, melanoma, breast cancer, lung cancer, cervical cancer, liver cancer, bladder cancer, prostate cancer, and gastric cancer. , leukemia, colorectal cancer, colon cancer, ovarian cancer or testicular germ cell tumors.
  • mAb KAO3 can effectively inhibit tumorigenesis in humans and mice by inducing cell cycle arrest in G2/M phase and activation of complement, and inhibit tumor cell proliferation in vitro. Its inhibition is closely related to the number of surface KAO3+ cells in tumor cell lines.
  • the low expression of PL2L60 on the cell surface may have a significant response to the treatment of KAO3 in vitro and in vivo, which is related to the dry/progenitor cell properties of these cancer cells.
  • High expression of pCCs with PL2L60 is more sensitive to the tumorigenic inhibition of mAb KAO3, as KAO3 mAb inhibits tumor stem/progenitor cells more effectively than non-stem cell cancer cells.
  • cancer cells with low PL2L60 in cell lines are associated with lower tumor growth rates. This suggests that cancer cells carrying PL2L60 may represent stem/progenitor cancer cells at various developmental stages, or the only targets representing the KAO3 mAb.
  • PL2L60 may be a promising target antigen for cancer treatment.
  • all data indicate that the antitumor activity of the KAO3 monoclonal antibody is significant.
  • PL2L60 proteins particularly anti-PL2L60 mAbs (KAO3), which are capable of exhibiting unique ability to directly induce apoptosis of cancer cells and inhibit cell proliferation and cell cycle, are obtained from various types of PL2L proteins.
  • KAO3 anti-PL2L60 mAbs
  • Treatment of human or mouse tumor cells with KAO3 at the time of inoculation is effective in inhibiting tumorigenesis in mice.
  • injection of KAO3 into established tissues such as lymphoma, breast cancer, lung cancer and cervical cancer can significantly inhibit tumor growth and prolong the survival of tumor-bearing mice.
  • KAO3 The inhibitory effect of KAO3 is related to the KAO3-specific antigen expressed on the surface of tumor cells. KAO3 induces tumor cell apoptosis by arresting the cell cycle, inhibiting DNA synthesis, and activating complement in the G2/M phase. Therefore, the anti-PL2L60 mAb (KAO3) is a potential therapeutic candidate for the treatment of cancer.

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Abstract

本发明提供了抑制PIWIL2基因异位表达的试剂以及抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用。本公开还提供了抗肿瘤的药物组合物以及治疗肿瘤的方法。通过实验证明，在接种时用KAO3处理人或小鼠肿瘤细胞能够抑制小鼠肿瘤的发生。此外，将KAO3注射入已建立的淋巴瘤、乳腺癌、肺癌和子宫颈癌中，能够抑制肿瘤生长，并延长荷瘤小鼠的存活。KAO3的抑制效应与肿瘤细胞表面表达的KAO3特异性抗原相关。KAO3通过在G2/M期阻滞细胞周期、抑制DNA合成和活化补体来诱导肿瘤细胞凋亡。

Description

抗PL2L60蛋白抗体在制备抗肿瘤药物中的应用及治疗肿瘤的方法
相关申请的交叉引用
本申请要求于2017年09月07日提交中国专利局的申请号为201710800022.X、名称为“抗PL2L60蛋白抗体在制备抗肿瘤药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开属于生物医药领域，涉及抑制PIWIL2基因异位表达的试剂、抗PIWIL2基因的异位表达蛋白抗体制备抗肿瘤药物中的应用，具体涉及包括抗PL2L60蛋白抗体在制备抗肿瘤药物中的应用，以及含有上述试剂或抗体的药物组合物，以及治疗肿瘤的方法。
背景技术
“癌症免疫治疗”应用免疫学原理和方法，提高肿瘤细胞的免疫原性和对效应细胞杀伤的敏感性，激发和增强机体抗肿瘤免疫应答，并应用免疫细胞和效应分子输注宿主体内，协同机体免疫系统杀伤肿瘤、抑制肿瘤生长，在多种类型癌症中的具有良好治疗功效，因此正受到越来越多的关注。
然而，癌症免疫治疗的靶标在很大程度上是个体化的，大多数靶标仅分布在少数几种类型的癌症中，而非所有类型得癌症中。此外，这些分子靶标也不具癌症特异性，其对正常细胞的功能也是必需的。因此，目前的免疫治疗的瓶颈是缺乏在造血和实体癌症中广泛表达的，特异也广谱的靶标或抗原。
发明内容
本公开的目的包括提供抑制PIWIL2基因异位表达的试剂在制备抗肿瘤药物中的应用，抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用，以及一种新的抗肿瘤药物组合物，以及一种治疗肿瘤的方法。
为实现本公开的上述目的中的至少一个目的，本公开可采用以下技术方案：
本公开的第一方面包括提供抑制PIWIL2基因异位表达的试剂在制备抗肿瘤药物中的应用。
本公开的第二方面包括提供抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用。
上述抗肿瘤药物包括抗淋巴瘤、黑素瘤、乳腺癌、肺癌、肝癌、膀胱癌、宫颈癌、前列腺癌、胃癌、白血病、结肠直肠癌、结肠癌、卵巢癌或睾丸生殖细胞肿瘤的药物。
进一步地，本公开提供的抗PIWIL2基因的异位表达蛋白抗体为抗PL2L蛋白抗体，例如可以为抗PL2L80，PL2L80A，PL2L60，PL2L60A，PL2L50或PL2L40蛋白抗体，优选抗PL2L60蛋白抗体。
在上述变体中，PL2L60主要在癌前干细胞(pCSCs)以及各种类型的人和鼠肿瘤细胞系中表达，其表达水平远高于全长PIWIL2。PL2L60可以通过上调STAT3和BCL2基因体外促进肿瘤细胞的存活和增殖，也可以与NF-κB协调促进肿瘤的发生，这可能代表了各种组织中肿瘤发展的共同途径。重要的是，衍生自PL2L60的肽可以作为针对各种癌症类型的强免疫源。研究结果表明，PL2L蛋白，特别是抗PL2L60单抗表现出直接诱导癌细胞凋亡和抑制细胞增殖、阻滞细胞周期的独特能力，此外，PL2L60蛋白是pCSC诱导的天然发生肿瘤免疫(NOTI)的靶标之一，可能是癌症免疫治疗的常见靶标。
进一步地，本公开提供的抗PL2L60蛋白抗体优选为KAO3单克隆抗体，KAO3单克隆抗体序列如SEQ ID NO:1所示。
进一步地，由于PL2L60蛋白可以通过上调STAT3和BCL2基因体外促进肿瘤细胞的存活和增殖，所以抗PL2L60蛋白抗体为抑制STAT3或BCL2基因激活的抗体。
进一步地，KAO3抗体具有制备PL2L蛋白特异的CRA-T细胞的潜力。
本公开的第三方面包括提供一种抗肿瘤的药物组合物，该药物组合物可以由抑制PIWIL2基因异位表达的试剂以及药学上可接受的辅料组成，也可以由抗PIWIL2基因的异位表达蛋白抗体以及药学上可接受的辅料组成。
本公开的第四方面包括提供一种治疗肿瘤的方法，包括：抑制PIWIL2基因异位表达。
在上述治疗肿瘤的方法中，优选以KAO3单克隆抗体抑制PIWIL2基因异位表达，所述KAO3单克隆抗体序列如SEQ ID NO:1所示。
其中，肿瘤可以包括淋巴瘤、黑素瘤、乳腺癌、肺癌、肝癌、膀胱癌、宫颈癌、前列腺癌、胃癌、白血病、结肠直肠癌、结肠癌、卵巢癌和睾丸生殖细胞肿瘤中的任意一种。
本公开的作用与效果包括：
本公开通过大量实验证实了PL2L蛋白在各种癌症类型中的广泛表达使其成为实体和造血干细胞免疫治疗的理想广谱靶标，并从各种类型的PL2L蛋白中获得了能够表现出直接诱导癌细胞凋亡和抑制细胞增殖、细胞周期的独特能力的PL2L60蛋白，同时针对该蛋白研究出了抗PL2L60 mAb(KAO3)。
通过实验证明，在接种时用KAO3处理人或小鼠肿瘤细胞能够有效抑制小鼠肿瘤的发生。此外，将KAO3注射入已建立的如淋巴瘤，乳腺癌，肺癌和子宫颈癌等中，能够显著抑制肿瘤生长，并延长荷瘤小鼠的存活。KAO3的抑制效应与肿瘤细胞表面表达的KAO3特异性抗原相关。KAO3通过在G2/M期阻滞细胞周期、抑制DNA合成和活化补体来诱导肿瘤细胞凋亡。因此，抗PL2L60 mAb(KAO3)是治疗癌症的潜在治疗候选药物。
因此，本公开为用抗PL2L60 mAb制备抗肿瘤药物提供了新的依据。
附图说明
图1为PL2L60蛋白在癌细胞中的表达结果。其中，(A)是流式细胞仪测定抗PL2L60单克隆抗体的结合活性结果：流式细胞仪分析平板培养的细胞系(2C4，326t-4，MDA-MB-231，A549和HeLa)表面PL2L60蛋白的表达。采用cells tripper收获细胞，在4℃条件下用PL2L60mAb染色1小时，随后用APC缀合的山羊抗小鼠IgM染色，使用BDC6软件进行分析；(B)为免疫荧光染色检测的PL2L60在肿瘤细胞表面的表达；(C)为免疫荧光染色检测的PL2L60在癌细胞系中的细胞内表达；(D)和(E)为免疫印迹法结果：Western blotting分析平板中的癌细胞PL2L60蛋白的表达。小鼠B16细胞系包括LLC，E14，326t-4和2C4；人类细胞系包括HCT116、HeLa、HepG2、A549和MDA-MB-231；(F-G)：对小鼠和人类肿瘤细胞株PL2L60蛋白表达的定量分析。
图2为抗PL2L60 mAb KAO3在体外抑制癌细胞的增殖和诱导细胞凋亡。其中，(A)相差显微镜下观察到mAb KAO3处理48小时后癌细胞的形态学，棒代表25μm；(B)流式细胞术对抗PL2L60单克隆抗体处理48小时后的癌细胞进行分析；(C)抗PL2L60 mAb在48小时培养结束时对癌细胞的抑制作用；(D)mAb KAO3上清液(μm/ml)对诱导癌细胞凋亡的剂量依赖性作用的总结。
图3为细胞周期分布图。其中，(A)为2C4,326T-4,MDA-MB-231,A549 and HeLa细胞系经KAO3处理后的细胞周期分布图；(B)对照组和治疗组处于G0/G1期细胞比例对比。(C)处于S期的细胞比例；(D)处于G2/M期的细胞比例。
图4为KAO3预处理癌细胞对肿瘤发生率的影响。其中，(A)-(E)分别为KAO3预处理和IgG对照预处理2C4,326T-4,MDA-MB-231,A549 and HeLa细胞系后肿瘤发生率，*代表p<0.05，***代表p<0.001。
图5为KAO3对不同阶段的荷瘤小鼠处理的效果。其中，(A，D，G，J和M)：接种KAO3培养上清预处理或培养基(R10F)对照预处理的癌细胞的小鼠肿瘤大小，随后接受对照治疗，其中A:2C4；D:326T-4；G:MDA-MB-231；J:A549；M:HeLa；(B，E，H，K,and N)：接种培养基(R10F)对照预处理的癌细胞的小鼠肿瘤大小，随后接受KAO3或对照治疗。B:2C4；E:326T-4；H:MDA-MB-231；K:A549；N:HeLa；(C，F，I，L，O)接种KAO3预处理的癌细胞的小鼠肿瘤大小，随后接受KAO3或对照治疗，C:2C4；F:326T-4；I:MDA-MB-231；L:A549；O:HeLa；(P)不同处理后的肿瘤细胞的大小。2C4:35天；326T-4:30天；MDA-MB-231:100天；A549:100天；HeLa:100天。
图6为补体依赖细胞毒性(CDC)实验分析结果。CDC实验在本公开提到的5种细胞中进行，其中(A)为PI阳性细胞直方图。(B)为PI阳性细胞的统计学分析。死亡细胞的百分比与抗PL2L60 mAb的抗肿瘤功效呈正相关。小鼠pCSCs 2C4和人乳腺癌细胞系MDA-MB-231在CDC实验中显示出最强的溶瘤作用，其次是小鼠淋巴瘤细胞系326T-4和人肺癌细胞系A549。人宫颈癌细胞系HeLa的CDC效应最弱。
具体实施方式
为使公开明实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面结合实施例和附图对本公开进行详细描述。但下列实施例不应看作对本公开范围的限制。
本公开主要涉及抑制PIWIL2基因异位表达的试剂在制备抗肿瘤药物中的应用以及抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用。
上述PIWIL2通常在睾丸中表达，但在DNA损伤后也可以在体细胞中被激活，并通过重塑染色质促进DNA修复，因此，其在干细胞的自我更新和维持中起关键作用。在各种原发性肿瘤和肿瘤细胞系，包括乳腺癌，肺癌、肝癌、膀胱癌、宫颈癌，前列腺癌、胃癌，白血病，结肠直肠癌，结肠癌，卵巢癌和睾丸生殖细胞肿瘤中已观察到PIWIL2基因的异位表达。
PIWIL2可以通过调节几种信号转导途径来促进肿瘤发生，并通过Stat3/Bcl-XL途径的激活抑制肿瘤细胞的凋亡死亡。然而，通过商业途径获取的大多数PIWIL2特异性抗体无法从其变体分辨全长PIWIL2。值得说明的是，在原发性乳腺癌和子宫颈癌中，全长PIWIL2蛋白主要在凋亡性肿瘤细胞中检测到，但在活体肿瘤细胞中检测不到。相比之下，PIWIL2的变体PL2L蛋白(如PL2L60)在各种类型的肿瘤组织和肿瘤细胞系中被大量检测到，表明PIWIL2的致瘤功能主要由PIWIL2变体介导。
PIWIL2有多种变体，包括PL2L80，PL2L80A，PL2L60，PL2L60A，PL2L50和PL2L40等蛋白。一些变体似乎被基因内启动子而不是规范启动子转录。虽然全长PIWIL2可以介导DNA修复，作为肿瘤发生起始的屏障基因，并促进肿瘤组织中的凋亡性细胞死亡，但其变体如PL2L60和PL2L60A却可促进肿瘤发生。在上述变体中，PL2L60主要在癌前期干细胞(pCSCs)以及各种类型的人和鼠肿瘤细胞系中表达，其水平远高于全长PIWIL2。在体外实验中，PL2L60通过上调STAT3和BCL2基因，来促进肿瘤细胞的存活和增殖。它也可以与NF-κB协调以促进肿瘤发生，可能代表了各种类型组织中肿瘤发展的共同途径。重要的是，衍生自PL2L60的肽可以作为靶向各种类型癌症的强免疫原。此外，PL2L60在小鼠的睾丸细胞中也被检测到，表明其在配子发生或发育中的作用。
发明人发现，异化激活的PIWIL2基因的产物——类PIWIL2(PL2L60)蛋白广泛地在各种造血和实体瘤中表达，并通过促进肿瘤细胞增殖和抑制细胞凋亡来介导肿瘤的发生。
因此，可通过抑制PIWIL2基因异位表达来治疗肿瘤。也即可将抑制抑制PIWIL2基因异位表达的试剂或者抗PIWIL2基因的异位表达蛋白抗体应用于制备抗肿瘤药物。
可选地，本公开涉及的抗PIWIL2基因的异位表达蛋白抗体为PL2L蛋白抗体，例如可以包括抗PL2L80，PL2L80A，PL2L60，PL2L60A，PL2L50和PL2L40蛋白抗体中的至少一种。
优选地，本公开涉及的抗PIWIL2基因的异位表达蛋白抗体为抗PL2L60蛋白抗体。
本公开的发明人主要研发了三种针对PIWIL2的单克隆抗体(mAbs)：mAb KAO1、mAb KAO2、和mAb KAO3。在免疫组织化学染色测定和蛋白质印迹分析中，mAb KAO2和mAb KAO3对PL2L60具有比mAbKAO1更强的亲和力，但由于mAbKAO3专门针对PL2L60，所以被视为治疗癌症的潜在治疗候选药物。故，本公开所用的抗PL2L60蛋白抗体优选为KAO3单克隆抗体，该KAO3单克隆抗体序列如SEQ ID NO:1所示。
此外，本公开所涉及的抗PL2L60蛋白抗体还可以为抑制STAT3或BCL2基因激活的抗体。
进一步地，本公开还提供了一种抗肿瘤的药物组合物，该药物组合物例如可以由上述抑制PIWIL2基因异位表达的试剂以及药学上可接受的辅料组成，也可以由上述抗PIWIL2基因的异位表达蛋白抗体以及药学上可接受的辅料组成。
其中，药学上可接受的辅料指生产药品和调配处方时使用的赋形剂和附加剂。是除活性成分以外，包含在药物制剂中的物质。药用辅料除了赋形、充当载体、提高稳定性外，还具有增溶、助溶、缓控释等重要功能。
作为可选地，药用辅料例如可以包括填充剂、稀释剂、湿润剂、粘合剂、崩解剂、润滑剂、薄膜衣材料、滴丸基质和冷凝液中的至少一种。具体地，可参照淀粉、糊精、乳糖、微晶纤维素、糖醇、乙醇、胶浆、聚乙二醇、甲基纤维素、羧甲基淀粉钠、硬脂酸镁、微粉硅胶、滑石粉和柠檬酸三乙酯等。
药物组合物中的抗体可参照上述抗PL2L蛋白抗体，优选为KAO3单克隆抗体。并且，抗PL2L蛋白抗体还可以为靶向PL2L60和/或抑制STAT3和BCL2激活的治疗性抗体。
值得说明的是，本公开所提供的药物组合物中抗体的氨基酸序列可以是包括由SEQ ID NO:1所示的序列和/或经过多个氨基酸残基的取代和/或缺失和/或添加得到的且与SEQ ID NO:1具有相同生物活性的衍生序列。
此外，本公开还提供了一种治疗肿瘤的方法，其包括：抑制PIWIL2基因异位表达。优选地，以上述KAO3单克隆抗体抑制PIWIL2基因异位表达。此处所涉及的肿瘤包括淋巴瘤、黑素瘤、乳腺癌、肺癌、肝癌、膀胱癌、宫颈癌、前列腺癌、胃癌、白血病、结肠直肠癌、结肠癌、卵巢癌和睾丸生殖细胞肿瘤中的任意一种。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，物质的百分比和份数均按体积计算。
本公开中所用的材料如下：
严重联合免疫缺陷(SCID)小鼠：使用8～12周的小鼠，这些小鼠被饲养在动物无害化设施中。
人类细胞系：人乳腺癌细胞系MDA-MB-231，人肺癌细胞系A549和人宫颈癌细胞系HeLa均获自美国典型培养物保藏中心(ATCC,Manassas,VA,USA)。将细胞保持在补充有10％胎牛血清(Gibco)和0.1mg/ml青霉素-链霉素(Gibco)的DMEM(Gibco)中。
小鼠淋巴瘤细胞系2C4和326T-4在实验室中自行制备：将细胞放置在含有5％CO 2(v/v)的加湿培养箱中，在37℃下保持在R10F(RPMI 1640加10mmol/L胎牛血清，补充有5mmol/L谷氨酰胺，50mmol/L 2-甲基苯乙酮，100U/mL青霉素和100mg/ml链霉素)内。
抗PL2L60单克隆抗体(KAO3mAb，同种型IgM))按照单克隆抗体的制备流程在实验室中自行制备，序列如SEQ ID NO:1所示。
可参照地，本公开中单克隆抗体的制备流程例如可以包括：
(1)免疫动物
免疫动物是用目的抗原免疫小鼠，使小鼠产生致敏B淋巴细胞的过程。一般选用6-8周龄雌性Balb/c小鼠，按照预先制定的免疫方案进行免疫注射。抗原通过血液循环或淋巴循环进入外周免疫器官，刺激相应B淋巴细胞克隆，使其活化、增殖，并分化成为致敏B淋巴细胞。
(2)细胞融合
采用二氧化碳气体处死小鼠，无菌操作取出脾脏，在平皿内挤压研磨，制备脾细胞悬液。将准备好的同系骨髓瘤细胞与小鼠脾细胞按一定比例混合，并加入促融合剂聚乙二醇。在聚乙二醇作用下，各种淋巴细胞可与骨髓瘤细胞发生融合，形成杂交瘤细胞。
(3)选择性培养
选择性培养的目的是筛选融合的杂交瘤细胞，可选地，例如可采用HAT选择性培养基。在HAT培养基中，未融合的骨髓瘤细胞因缺乏次黄嘌呤-鸟嘌呤-磷酸核糖转移酶，不能利用补救途径合成DNA而死亡。未融合的淋巴细胞虽具有次黄嘌呤-鸟嘌呤-磷酸核糖转移酶，但其本身不能在体外长期存活也逐渐死亡。只有融合的杂交瘤细胞由于从脾细胞获得了次黄嘌呤鸟嘌呤磷酸核糖转移酶，并具有骨髓瘤细胞能无限增殖的特性，因此能在HAT培养基中存活和增殖。
(4)杂交瘤阳性克隆的筛选与克隆化
在HAT培养基中生长的杂交瘤细胞，只有少数是分泌预定特异性单克隆抗体的细胞，因此，必须进行筛选和克隆化。可选地，例如可采用有限稀释法进行杂交瘤细胞的克隆化培养。采用灵敏、快速、特异的免疫学方法，筛选出能产生所需单克隆抗体的阳性杂交瘤细胞，并进行克隆扩增。经过全面鉴定其所分泌单克隆抗体的免疫球蛋白类型、亚类、特异性、亲和力、识别抗原的表位及其分子量后，及时进行冻存。
(5)单克隆抗体的大量制备
单克隆抗体的大量制备可采用动物体内诱生法和体外培养法。其中，体内诱生法包括：取Balb/c小鼠，首先腹腔注射0.5ml液体石蜡或降植烷进行预处理。1-2周后，腹腔内接种杂交瘤细胞。杂交瘤细胞在小鼠腹腔内增殖，并产生和分泌单克隆抗体。约1-2周，可见小鼠腹部膨大。用注射器抽取腹水，即可获得大量单克隆抗体。此外，体外培养法包括：将杂交瘤细胞置于培养瓶中进行培养。在培养过程中，杂交瘤细胞产生并分泌单克隆抗体，收集培养上清液，离心去除细胞及其碎片，即可获得所需要的单克隆抗体。
本公开的发明人共研发了三种针对PIWIL2的单克隆抗体(mAbs)：mAb KAO1、mAb KAO2、和mAb KAO3。由于mAbKAO3专门针对PL2L60，故以下分析方法均优选采用KAO3单克隆抗体。
以下对本公开所用到的分析方法以及实验结果进行详细说明。
一、分析方法
流式细胞仪分析
可参照地，例如可用0.25％胰蛋白酶-EDTA(1mM；Invitrogen)将癌细胞解离1-3分钟，用细胞分选缓冲液(含有1％胎牛血清的PBS)洗涤细胞，然后在4℃下用抗PL2L60mAbs孵育1小时。将细胞与藻红蛋白缀合的山羊抗小鼠IgM(1:250稀释；Bioligend)在4℃孵育30分钟。最终洗涤后，将细胞用含1％FBS的PBS重悬浮，并通过流式细胞术(BD，San Jose，CA，USA)进行分析。细胞表面和细胞内免疫荧光染色
对于表面染色，收集肿瘤细胞，在PBS中重悬肿瘤细胞，细胞浓度为5×10 6/ml，以0.2mL细胞悬液(1×10 6个细胞/孔)加入96孔板中。旋转细胞(1000rpm，5分钟)并丢弃上清液。在100μLPBS中加入抗PL2L60单克隆抗体KAO3，并旋转5秒。将样品在4℃下孵育30分钟。用PBS洗涤细胞两次。弃去上清液，加入含有1％多聚甲醛的100μlPBS至每孔，以固定细胞。
对于细胞内染色，将在盖玻片上培养的肿瘤细胞在2％多聚甲醛中固定20分钟，然后洗涤，随后用含1％牛血清白蛋白的PBS封闭30分钟。在室温下，细胞与抗-PL2L60mAb(KAO3,1:100稀释)在1％牛血清白蛋白中孵育。孵育1小时后，洗涤细胞并与FITC山羊抗小鼠抗体(IgM，Bioligend)共孵育。用染色剂，例如4',6-二脒基-2-苯基吲哚(DAPI，1:500)对细胞核染色。
Western blot
在用胰蛋白酶收获之前，将细胞样品在冷PBS中洗涤两次，然后用蛋白质提取试剂裂解细胞。使用BCA蛋白测定试剂盒(Beyotime，Shanghai，China)测定全细胞裂解物的总蛋白浓度，然后使用12％聚丙烯酰胺凝胶分离蛋白质，并转移到聚二氟乙烯膜上。
在TBS/吐温20(TBST)中用5％牛血清白蛋白(BSA)封闭后，在4℃下用特异性一抗孵育过夜，用TBS-T洗涤膜5分钟，洗涤重复三次。之后，将膜与辣根过氧化物酶缀合的抗小鼠IgM二抗(Santa Cruz Biotechnology，Santa Cruz，CA，USA)在室温下孵育1小时，随后用TBST洗涤5分钟，重复三次。然后用ECL化学发光检测系统(Bio-Rad)对膜进行分析。
在本研究中使用以下抗体：抗PIWIL2多克隆抗体(由我们组制备)，并且图像由Kodak Imaging Station 2000R(Eastman Kodak，USA)获得。
CCK-8分析
使用CCK-8分析(Dojindo，Japan)评估抗PL2L60mAb(KAO3)对细胞活力的影响。为了进行CCK-8测定，将细胞与成浓度梯度(1,2,4和8μg/ml)的KAO3在终体积为200μL的含10％FBS培养基中混合，使用相同浓度的IgG作为对照。然后将连续稀释的PL2L60抗体或对照IgG以2×10 3个细胞/孔的密度接种在平底96孔板上。孵育48小时后，将10μL CCK-8试剂移液到96孔测定板(孔中含有100μL新鲜无酚红培养基)的每个孔中，将板在37℃，5％CO 2的湿润气氛中孵育1-4小时。然后使用
Figure PCTCN2018082380-appb-000001
Max M5系列(Molecular Devices)记录490nm处的吸光度(A)。
细胞活力计算为(A 样品-A 空白)/(A 对照-A 空白)×100％。所有实验重复至少三次，每次实验一式三份。
细胞凋亡测定
在用KAO3处理24小时后，收获细胞并用预冷却的PBS洗涤两次。将IgG2a同种型对照(Biolegend)稀释，然后与含有膜联蛋白V(Biolegend)和碘化丙啶(Sigma)的混合物在结合缓冲液中黑暗中孵育15分钟。使用膜联蛋白V-APC和PI检测凋亡细胞，并使用流式细胞仪(BD，C6)进行分析。早期凋亡细胞用Annexin V-APC细胞凋亡检测试剂盒(Biolegend)进行测定。用碘化丙啶(PI，Sigma)染色检测凋亡细胞核。共进行三次独立实验。
细胞周期分析
将细胞固定在冷冻的75％乙醇中，在PBS中，用含有100μg/ml核糖核酸酶(Tiangen Biotech)和50μg/mL PI(Biolegend)的碘化丙啶(PI)溶液进行细胞周期分析染色，细胞周期各阶段的细胞百分数通过C6流式细胞仪(BD)测量。
抗肿瘤试验
将细胞(每200μLPBS中的细胞浓度为5×10 6)注射到SCID小鼠的腹股沟处。待肿瘤发生后，将携带肿瘤的小鼠随机分为四组：组1：对照→对照组；组2：对照→KAO3；组3：KAO3→对照组；组4：KAO3→KAO3。KAO3或同种型IgG原位注射两周。每2天用卡尺测量肿瘤的长度和宽度，当肿瘤直径达到2cm时，小鼠被安乐死。
补体依赖性细胞毒性(CDC)分析
补体依赖性细胞毒性(CDC)靶细胞以5×10 4个细胞/孔的浓度接种于平板内。测试抗体以指定的最终浓度加入到活化或热灭活(60℃，30分钟)人血清(25％最终血清浓度；Pathway Diagnostics，Dorking，UK)中。
将平板在37℃下孵育3小时，然后加入细胞活力试剂。将Triton1X-100加入到对照细胞的孔中以建立最大裂解控制。在37℃孵育1小时后，通过荧光显微镜(Olymplus，Japan)测量荧光。
所有的实验数据均来自至少三次独立实验。实验数值以平均值±标准差(SD)的形式表示，实验组与相应的对照通过student t检验(Student’s t-test)进行比较。三组及以上的组通过单因素方差分析(ANOVA)进行比较。当p值小于0.05时，差异被认为是显着的。*表示p值<0.05；**表示p值<0.01；***表示p值<0.001。使用Kaplan-Meier生存曲线进行存活分析，并使用对数秩检验来测试组之间的显着差异，通过Spearman分析测定相关系数。
值得说明的是，上述分析方法中所涉及的分析参数、分析条件和所使用的试剂等均可根据实验具体条件和情况进行适当调整。
二、实验结果
蛋白在癌细胞中的表达
通过免疫组织化学检测，在原发性乳腺癌和子宫颈癌中，全长PIWIL2蛋白主要在凋亡性肿瘤细胞中检出，但活体肿瘤细胞中几乎无检出。相比之下，PIWIL2变体PL2L蛋白(如PL2L60)在各种类型的肿瘤组织和肿瘤细胞系中大量检测到，表明PIWIL2的致瘤功能可能主要由PIWIL2变体介导。因此，PL2L蛋白在各种癌症类型中的广泛表达使其成为实体和造血干细胞免疫治疗的理想广谱靶标。
为了验证这一假设，我们首先使用单克隆抗体(mAb KAO3)对人类和小鼠同源的PIWIL2肽来研究PL2L蛋白在各种类型肿瘤细胞系的细胞表面上的表达。除了细胞质外，还通过流式细胞术和荧光显微镜(图1A-B)在肿瘤细胞系的表面上检测到PL2L蛋白，包括小鼠造血前癌干细胞(pCSCs)系2C4和癌症干细胞(CSC)系326T-4和人乳腺癌细胞系MDA-MB-231，肺癌细胞系A549和宫颈癌细胞系HeLa。
细胞内免疫荧光分析(图1C)显示，Pl2L60蛋白主要在各种类型的人和鼠肿瘤细胞的细胞质中表达，不同癌细胞无显著性差异。蛋白质印迹(Western blotting)数据显示，PL2L60总蛋白在各种类型的人和鼠肿瘤细胞系中高度表达，在pCSCs 2C4的细胞质中的表达量最高(图1D-G)。
KAO3 mAb的细胞毒性
在体外实验中，PL2L60可以通过上调STAT3和BCL2基因来促进肿瘤细胞的存活和增殖，它还可以与NF-κB蛋白协调促进肿瘤发生。因此，我们已经开发出通过产生针对PL2L60的单克隆抗体来抑制癌症增殖的抗体。目前还没有关于可以直接抑制增殖并诱导凋亡的抗PL2L60抗体的公开报道。在目前的研究中，我们检查了新产生的抗PL2L60 mAb是否具有这种能力。
KAO3mAb能诱导癌细胞凋亡，如MDA-MB-231，A549，HeLa，2C4和326T-4(图2)。KAO3治疗前，对照组和实验组癌细胞形态基本一致，对照细胞生长良好，形状多边形，培养48h后，对照细胞的大小均匀。在用抗PL2L60mAb KAO3处理细胞48小时后，在高功率显微镜下观察到细胞染色质丢失，细胞数明显减少，细胞形态不规则，有些细胞变圆。在大功率显微镜下观察到可见的核染色体和减少的细胞质空泡(图2A)。悬浮液中的半独立细胞，凋亡细胞数量急剧增加(图2B和图2C)。具体地说，在抗PL2L60抗体以不同剂量处理48小时(1,2,4和8μl/孔的mAb KAO3上清液)后，细胞活力降低(图2D)。
这些结果表明，抗PL2L60 mAb KAO3可显着抑制五种癌细胞株的细胞增殖(图2D)。总体而言，这些结果表明抗PL2L60 mAb(KAO3)可能通过诱导凋亡途径诱导癌细胞的细胞毒活性。
KAO3 mAb诱导癌细胞中的细胞周期停滞
图3A显示了抗PL2L60 mAb(KAO3)处理后的2C4，326T-4，MDA-MB-231，A549和HeLa的细胞周期分布。与对照细胞相比，用抗PL2L60 mAb(KAO3)处理48h后，癌细胞G0/G1期的比例略有降低，S期细胞百分比几乎没有变化。G2/M期细胞数量增加。A549细胞的G2/M期细胞数量最多。G0/G1和G2/M期的比例急剧增加，S期细胞百分比在MDA-MB-231和HeLa细胞中急剧下降。G0/G1，S和G2/M期2C4细胞发生最大变化。 326T-4细胞没有显着变化(图3B-图3D)。简而言之，PI/FACS分析表明，抗PL2L60 mAb(KAO3)在五种癌细胞系中引起显著的G2/M期停滞。
用抗PL2L60 mAb KAO3治疗异种移植瘤
由于抗PL2L60单克隆抗体(KAO3)治疗破坏了癌细胞生长并在体外诱导了癌细胞凋亡，因此研究了KAO3在体内是否可以用于直接抑制肿瘤生长。为了观察KAO3对肿瘤细胞致瘤性的影响，肿瘤治疗方案分为两个阶段：一个是肿瘤发生的初期阶段，另一个是肿瘤治疗阶段。
首先，将人和小鼠癌细胞悬浮在KAO3杂交瘤或培养基的培养上清液中，然后接种到SCID小鼠中，细胞用同种型IgG处理作为对照。肿瘤形成后，不同治疗组的荷瘤小鼠分为两组，每两天将一组荷瘤小鼠原位注射KAO3，另一组注射同种型IgG作为对照，观察到肿瘤生长。
在肿瘤发生的最初阶段，当平均肿瘤直径接近0.5cm时，对不同组的肿瘤发生率进行计数。我们发现SCID小鼠中pCSCs的肿瘤发生几乎完全被KAO3mAb抑制。在观察的150天内，只有33％的小鼠(3/9)在接种后第10,67和118天发生肿瘤。，相比之下，对照组的所有小鼠(100％)在接种后3周内均发生肿瘤。用KAO3预处理的MDA-MB-231细胞接种的小鼠的肿瘤发生率为50％，而对照组为83％。其他三种细胞系的肿瘤发生率为100％，KAO3预处理组与对照组无差异(图4A-图4E)。
肿瘤生长动力学的进一步分析显示，来自KAO3预处理的pCSCs的一种肿瘤生长速度比来自对照(中等处理)的pCSCs的肿瘤生长相对较慢(图5A)。对照组肿瘤被注射50μlKAO3mAb上清液后，被mAb显著抑制(图5B)。来自被KAO3mAb预处理的pCSCs，并且在第67和118天生长出的肿瘤几乎完全被mAb抑制(图5C)。结果表明，mAb KAO3可有效预防pCSCs的肿瘤发生，抑制已建立肿瘤的生长。小鼠造血干细胞(CSCs；克隆326T-4)和326T-4的相同治疗也导致SCID小鼠中不同程度的致瘤性或肿瘤生长抑制(图5D-图5F)。
由于mAb KAO3也识别人PL2L60蛋白，用处理2C4 pCSC系相同的方法，用杂交瘤KAO3的培养上清液处理人乳腺癌细胞系MDA-MB-231，人肺癌细胞系A549和人宫颈癌细胞系HeLa，并观察到了类似的结果，即、KAO3mAb也可以有效抑制人类癌细胞的肿瘤发生和SCID小鼠中已建立的肿瘤的生长(图5G-图5P)。结果表明，mAb KAO3可以有效地杀死或抑制人和小鼠的癌细胞。
mAb KAO3的作用似乎并不局限于两种肿瘤，因为鼠类造血干细胞(CSCs；克隆326T-4)，人肺癌细胞系A549和人宫颈癌细胞系HeLa的相同治疗也使不同程度的致瘤性或SCID小鼠的肿瘤生长抑制作用。这些结果证实了我们的发现，KAO3在降低肿瘤生长方面具有比对照更高的治疗功效。KAO3显示能够抑制体内人和小鼠肿瘤生长的生长。
mAb KAO3的补体依赖性细胞毒性(CDC)
抗肿瘤治疗效果与补体依赖性细胞毒性的肿瘤细胞表面PL2L60蛋白的表达水平正相关，因为在体外补体存在下，mAb可以杀死肿瘤细胞。因此，PL2L60蛋白也是癌症被动免疫治疗的合适靶标。
结果表明，小鼠pCSC系2C4和人乳腺癌细胞系MDA-MB-231在CDC实验中显示出最强的溶瘤作用，其次是小鼠淋巴瘤细胞系326T-4和人肺癌细胞系A549。人宫颈癌细胞系HeLa的CDC效应最弱(图6A-B)。这些结果与PL2L60蛋白在癌细胞表面的表达水平一致。进一步表明抗PL2L60的mAb KAO3是肿瘤细胞表面的PL2L60蛋白的靶标，KAO3可通过CDC依赖机制发挥其抗肿瘤作用。
综上，靶向PL2L60和/或抑制STAT3和BCL2激活的治疗性抗体的发展具有消除肿瘤的巨大潜力。
在本研究中，我们使用自己实验室开发的mAb KAO3来检验PL2L蛋白是否是癌症免疫治疗的常见靶标。mAb KAO3抑制肿瘤发生和肿瘤生长的差异表达似乎与癌细胞系(图1A-图1B)之间的表面KAO3特异性抗原(即PL2L蛋白)的表达相关，但与细胞内表达无关(图1C-图1G)，因为KAO3mAb抑制肿瘤发生的能力与表面KAO3+细胞的百分比相关。HeLa和326T-4细胞系比其他品系含有较少的KAO3+细胞，对mAb KAO3处理的敏感性较低(图5)。因此，mAb KAO3的治疗功效由表面PL2L蛋白质表达量决定。
抗PL2L60抗体在体内抑制肿瘤的机制仍不清楚。没有公布的研究已经证明了在临床环境中抗PL2L60抗体直接诱导癌细胞凋亡，因此目前尚不清楚这种抗体是否可以直接抑制肿瘤细胞增殖。本研究中，KAO3 mAb处理抑制人和小鼠癌细胞的增殖，并以剂量依赖的方式显着提高细胞周期G2/M期细胞的百分比，特别是处于S期的2S4细胞的百分比显著降低。
我们研究了KAO3的抗肿瘤作用，其对癌细胞的细胞生长具有很强的抑制作用。我们证明KAO3剂量依赖性地在G2/M期诱导细胞周期停滞，随后进展为细胞凋亡(图2-图3)，其可能与微管解聚或抑制NF-κB和STAT3核易位相关，进而抑制转录活性。在肿瘤异种移植模型中，KAO3有效地延缓肿瘤生长(图4-图5)。我们的研究结果提供了我们的抗PL2L60抗体能够抑制肿瘤细胞增殖并诱导凋亡的直接证据。
结果表明，PL2L蛋白是癌症免疫治疗的有效靶标。我们首次发现mAb KAO3能够识别在各种类型的肿瘤细胞系上表达的表面PL2L蛋白质，例如可包括淋巴瘤，黑素瘤，乳腺癌，肺癌，子宫颈癌，肝癌，膀胱癌，前列腺癌，胃癌，白血病，结肠直肠癌，结肠癌，卵巢癌或睾丸生殖细胞肿瘤。mAb KAO3通过诱导G2/M期的细胞周期停滞以及补体的激活，在体内能够有效抑制人和小鼠肿瘤发生，在体外肿能够抑制瘤细胞增殖。其抑制作用与肿瘤细胞系中表面KAO3+细胞的数量密切相关。
PL2L60在细胞表面的低表达有可能在体外和体内对KAO3的治疗产生显著反应，这与这些癌细胞的干/祖细胞性质有关。pCCs高水平表达PL2L60对mAb KAO3的致瘤性抑制更敏感，因为KAO3mAb对肿瘤干细胞/祖细胞的抑制比对非干细胞癌细胞的作用更有效。值得注意的是，细胞系中荷PL2L60低的癌细胞与较低的肿瘤生长率有关。这表明携带PL2L60的癌细胞可能代表各种发育阶段的干细胞/祖细胞癌细胞，或代表KAO3 mAb的唯一靶标。这些结果验证了PL2L蛋白在肿瘤发生中起关键作用，因此是癌症免疫治疗的有效常用靶标。我们的发现为癌症免疫治疗癌症的治疗提供了一个新的场所。
总之，PL2L60可能是一个有希望的癌症治疗的目标抗原。作为PL2L60特定的抗原表位，所有的数据表明KAO3单克隆抗体的抗肿瘤活性是显著的。
以上显示和描述了本公开的基本原理、主要特征和本公开的优点。本行业的技术人员应该了解，本公开不受上述实施例的限制，上述实施例和说明书中描述的只是说明本公开的原理，在不脱离本公开精神和范围的前提下本公开还会有各种变化和改进，这些变化和改进都落入要求保护的本公开范围内。本公开要求保护范围由所附的权利要求书及其等同物界定。
工业实用性：
本公开提供的PL2L蛋白在各种癌症类型中的广泛表达使其成为实体和造血干细胞免疫治疗的理想广谱靶标。从各种类型的PL2L蛋白中获得了能够表现出直接诱导癌细胞凋亡和抑制细胞增殖、细胞周期的独特能力的PL2L60蛋白，特别是抗PL2L60 mAb(KAO3)。在接种时用KAO3处理人或小鼠肿瘤细胞能够有效抑制小鼠肿瘤的发生。此外，将KAO3注射入已建立的如淋巴瘤，乳腺癌，肺癌和子宫颈癌等中，能够显著抑制肿瘤生长，并延长荷瘤小鼠的存活。KAO3的抑制效应与肿瘤细胞表面表达的KAO3特异性抗原相关。KAO3通过在G2/M期阻滞细胞周期、抑制DNA合成和活化补体来诱导肿瘤细胞凋亡。因此，抗PL2L60 mAb(KAO3)是治疗癌症的潜在治疗候选药物。

Claims (18)

  1. 抑制PIWIL2基因异位表达的试剂在制备抗肿瘤药物中的应用。
  2. 抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用。
  3. 根据权利要求2所述的抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用，其特征在于：
    其中，所述的抗PIWIL2基因的异位表达蛋白抗体为抗PL2L蛋白抗体。
  4. 根据权利要求3所述的抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用，其特征在于：
    其中，所述抗PL2L蛋白抗体包括抗PL2L80，PL2L80A，PL2L60，PL2L60A，PL2L50和PL2L40蛋白抗体中的至少一种。
  5. 根据权利要求4所述的抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用，其特征在于：
    其中，所述抗PL2L蛋白抗体为抗PL2L60蛋白抗体。
  6. 根据权利要求5所述的抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用，其特征在于：
    其中，所述抗PL2L60蛋白抗体为KAO3单克隆抗体，所述KAO3单克隆抗体序列如SEQ ID NO:1所示。
  7. 根据权利要求5所述的抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用，其特征在于：
    其中，所述抗PL2L60蛋白抗体为抑制STAT3或BCL2基因激活的抗体。
  8. 根据权利要求2-7任一项所述的抗PIWIL2基因的异位表达蛋白抗体在制备抗肿瘤药物中的应用，其特征在于：
    其中，所述肿瘤包括乳腺癌、肺癌、肝癌、膀胱癌、宫颈癌、前列腺癌、胃癌、白血病、结肠直肠癌、结肠癌、卵巢癌和睾丸生殖细胞肿瘤中的任意一种。
  9. 一种抗肿瘤的药物组合物，其特征在于：
    由权利要求1所述的抑制PIWIL2基因异位表达的试剂以及药学上可接受的辅料组成。
  10. 一种抗肿瘤的药物组合物，其特征在于：
    由权利要求2所述的抗PIWIL2基因的异位表达蛋白抗体以及药学上可接受的辅料组成。
  11. 根据权利要求9或10所述的药物组合物，其特征在于，所述辅料包括填充剂、稀 释剂、湿润剂、粘合剂、崩解剂、润滑剂、薄膜衣材料、滴丸基质和冷凝液中的至少一种；
    可选地，所述辅料包括淀粉、糊精、乳糖、微晶纤维素、糖醇、乙醇、胶浆、聚乙二醇、甲基纤维素、羧甲基淀粉钠、硬脂酸镁、微粉硅胶、滑石粉和柠檬酸三乙酯中的至少一种。
  12. 根据权利要求10所述的药物组合物，其特征在于，所述抗体为抗PL2L蛋白抗体。
  13. 根据权利要求10-12任一项所述的药物组合物，其特征在于，所述抗体包括抗PL2L80，PL2L80A，PL2L60，PL2L60A，PL2L50和PL2L40蛋白抗体中的至少一种。
  14. 根据权利要求10-13任一项所述的抗体，其特征在于，所述抗体为抗PL2L60蛋白抗体。
  15. 根据权利要求14所述的抗体，其特征在于，所述抗PL2L60蛋白抗体为KAO3单克隆抗体，所述KAO3单克隆抗体序列如SEQ ID NO:1所示。
  16. 根据权利要求10-12任一项所述的抗体，其特征在于，所述抗体为靶向PL2L60和/或抑制STAT3和BCL2激活的治疗性抗体。
  17. 根据权利要求10-14任一项所述的抗体，其特征在于，所述抗体的氨基酸序列包括由SEQ ID NO:1所示的序列和/或经过多个氨基酸残基的取代和/或缺失和/或添加得到的且与SEQ ID NO:1具有相同生物活性的衍生序列。
  18. 一种治疗肿瘤的方法，其特征在于，包括：抑制PIWIL2基因异位表达；优选地，以KAO3单克隆抗体抑制PIWIL2基因异位表达，所述KAO3单克隆抗体序列如SEQ ID NO:1所示；
    所述肿瘤包括乳腺癌、肺癌、肝癌、膀胱癌、宫颈癌、前列腺癌、胃癌、白血病、结肠直肠癌、结肠癌、卵巢癌和睾丸生殖细胞肿瘤中的任意一种。
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