CN112213488A - Application of PL2L60 protein in preparation of kit for early screening or diagnosis of tumor - Google Patents

Abstract

The invention relates to the technical field of medicines, provides application of PL2L60 protein as a tumor diagnosis marker, and particularly relates to application of PL2L60 protein in preparation of a kit for early screening or diagnosis of tumors, wherein a tumor screening or diagnosis reagent refers to a reagent for screening or diagnosis of tumor stem cells or tumor progenitor cells, and the tumors refer to leukemia, lymphoma, breast cancer, cervical cancer, lung cancer, pancreatic cancer, colon cancer or liver cancer. Research results show that the PL2L60 protein has tumor specificity and tumor broad spectrum, particularly has specific expression on the surface of tumor stem/progenitor cells, takes PL2L60 as a target, not only can research and develop high-efficiency antibody drugs, cell therapy technology and tumor vaccines, but also can research and develop early tumor diagnosis reagents, contributes to the progress of tumor diagnosis and treatment, is beneficial to promoting clinical tumor immunotherapy, and has wide clinical application prospect.

Description

Application of PL2L60 protein in preparation of kit for early screening or diagnosis of tumor

Technical Field

The invention belongs to the field of biomedical detection, relates to application of PL2L60 protein as a tumor marker, and particularly relates to application of PL2L60 protein antibody in preparation of a kit for early screening or diagnosis of tumors.

Background

Currently, immunotherapy is a hot spot for exploring new methods for tumor treatment. There is increasing evidence that the cure for tumors requires the cooperation of immunotherapy. Immunotherapy mainly includes antibody-mediated passive immunotherapy and active immunotherapy characterized by the activation of tumor-specific immune cells. Active immunotherapy may include: (1) specific tumor vaccines, (2) immune biological modulators (Immunomodulators) and (3) immune cell therapy. Currently, the antibody drugs targeting PD-1 are used for clinical immunotherapy. Other therapies such as immune cell therapy are not satisfactory. Even CART therapy is only a transitional approach to tumor immunotherapy.

However, immunotherapy targeting PD-1 has significant limitations. It was initially thought that anti-PD-1 antibodies block immune checkpoints in anti-tumor immune responses and can activate the anti-tumor immune system. This assumption has proved to be false in practice. The fact is that the anti-PD-1 antibody kills tumor cells directly by recognizing PD-1 expressed on the surface of tumor cells and activating ADCC pathway, rather than by blocking the PD-1/PD-L1 immunosuppressive point. Since most tumors do not express PD-1, the use of anti-PD-1 antibody drugs is greatly limited. Furthermore, the only one tumor vaccine (actually immune cells) product, PROVENGE (sipuleucELT), approved by the FDA for treatment of advanced prostate cancer at 29/4/2010, is subject to failure due to its poor clinical efficacy. In summary, both passive and active immunotherapy suffer from a bottleneck that is currently difficult to overcome due to the lack of tumor-specific broad-spectrum tumor antigens.

To overcome the bottleneck of current immunotherapy, Tumor-specific and Tumor-broad-spectrum Tumor biomarkers (TSA) need to be found. At present, tumor targets which are researched and developed by domestic and foreign medicine enterprises generally do not have tumor specificity and tumor broad spectrum, so that milestone breakthrough is difficult to be realized in the aspect of tumor immunotherapy. Currently, the breakthrough of tumor immunotherapy needs to overcome the following several bottlenecks:

(1) there is a need for tumor-specific and broad-spectrum Targets (TSAs), particularly those that can be expressed on tumor stem/progenitor cells;

(2) preparing corresponding high-efficiency cytotoxic antibody (passive immunotherapy);

(3) having a polypeptide sequence that is not MHC-restricted and can be efficiently presented by an Antigen Presenting Cell (APC);

(4) techniques for culturing, expanding, and culturing immune cells that are immune-normal (immunocampent) and do not develop immune tolerance (immunolerant) include dendritic cells (Dendriticell; DC) and Killer cells (Killer).

The era of tumor immunotherapy is really coming if these bottlenecks are overcome. However, no document has been available in the prior art on genes or proteins that can be specifically expressed in various tumors.

Disclosure of Invention

The inventor of the invention discovers and identifies a tumor stem/progenitor cell specific protein PL2L60 through a plurality of years of intensive research, and the protein is a novel tumor stem cell marker. In addition, a high-efficiency anti-PL 2L60 antibody is prepared, a polypeptide with stronger immunogenicity is screened from PL2L60 protein, and PL2L60 is proved to be an important target for tumor immunotherapy in animal experiments. The tumor immunotherapy scheme based on PL2L60 can break through the bottleneck of the current immunotherapy, and effectively solve the problems.

PL2L60 is the product of the differentiation activation of the germ stem cell gene PIWIL2 in tumors PIWIL2 is generally expressed in germ stem cells and is involved in nuclear reprogramming or chromatin remodeling (chromatinremModification) through the piwi-piRNA pathway during spermatogenesis; the activity of retrotransposons can also be regulated by DNA methylation. However, it is not normally expressed in somatic cells. When somatic cells are exposed to an oncogenic environment and DNA is damaged, the PIWIL2 gene is activated and participates in the repair of DNA damage, but the over-expression of PIWIL2 induces the apoptosis of tumor cells. Thus, PIWIL2 functions as a Tumor Barrier Gene (TBG).

However, in an oncogenic environment, when the promoter in PIWIL2 gene is activated, a catabolically activated protein such as PL2L60 is produced. PL2L60 is not expressed in normal cells, is mainly expressed in transformed cells and tumor stem/progenitor cells, and promotes the generation and development of tumors. The tumor stem cell is a seed cell for generating and developing tumors, and the aim of inhibiting and even curing the tumors can be achieved by inhibiting the tumor stem cell.

Tumor stem cells can be transformed from normal cells into tumor initiating stem cells (TICs), via precancerous stem cells (pCSC) and Cancer Stem Cells (CSC) into Cancer Progenitor Cells (CPC), the latter becoming the main seed cell for tumor development. PL2L60 is expressed in precancerous stem cells, cancer progenitor cells and tumor cell lines, and the expression level is positively correlated with the proliferation capacity of tumor cells. More importantly, PL2L60 is expressed in various hematologic and solid tumors with tumor specificity and broad spectrum. An immunotherapy scheme designed by taking PL2L60 as a target can effectively prevent and treat various blood cancers and solid tumors.

PL2L60 is expressed at high levels in precancerous stem cells and is a potent target for natural anti-tumor immunity (NATI). anti-PL 2L60 mAb recognized both human and mouse PL2L60, and treatment of pre-cancerous stem cells with mAb anti-PL 2L60 (KAO2 and KAO3 clones) effectively blocked their tumorigenic capacity in immunodeficient mice, so the results obtained from mice were easily reproducible in humans. Recently, we found that PL2L60 can also be expressed on the surface of a subset of tumor cell lines in addition to the cytosol, and these cells were identified to express PL2L60 on the surface of tumor cells with the properties of tumor stem/progenitor cells, suggesting that the tumor-inhibiting effects of mabs KAO2 and KAO3 may act by targeting tumor stem/progenitor cells.

The precancerous stem cells can induce natural antitumor immunity and inhibit the tumorigenic effect of the precancerous stem cells. This function is that the immune system functions by targeting PL2L60 expressed by pre-cancerous stem cells, since priming dendritic cells (DC-PL2L tumor vaccine) with PL2L60 polypeptide can activate anti-tumor immunity, achieving the same effect as that induced by pre-cancerous stem cells.

Therefore, the PL2L60 protein has the potential to be used as a tumor marker and a tumor target.

The invention aims to provide application of PL2L60 protein as a molecular marker of tumor, in particular application of PL2L60 protein in preparing a reagent or a kit for early screening or diagnosing tumor.

The invention provides an application of PL2L60 protein in preparing a reagent or a kit for early screening or diagnosing tumor.

The screening or diagnosis tumor reagent refers to a reagent for screening or diagnosis of tumor stem cells or tumor progenitor cells.

The kit comprises a reagent for screening or diagnosing the tumor stem cells or the tumor progenitor cells.

The tumor refers to leukemia, lymph cancer, breast cancer, cervical cancer, lung cancer, pancreatic cancer, colon cancer or liver cancer.

Further, in the application of the PL2L60 protein in preparing a reagent or a kit for early screening or diagnosis of Brachyury positive tumors, the reagent comprises a PL2L60 protein antibody and a reagent for quantitative or semi-quantitative detection of the expression level of PL2L60 protein, and the kit comprises a PL2L60 protein antibody and a reagent for quantitative or semi-quantitative detection of the expression level of PL2L60 protein.

Preferably, the anti-PL 2L60 protein antibody is KAO3 monoclonal antibody, and the sequence of the KAO3 monoclonal antibody is shown as SEQ ID NO: 1.

Action and Effect of the invention

According to the invention, a large number of experiments prove that the PL2L60 protein is not expressed in normal cells, is mainly expressed in transformed cells and tumor stem/progenitor cells to promote the generation and development of tumors, while the tumor stem cells or the tumor progenitor cells are seed cells for the generation and development of the tumors, the diagnosis of early tumors can be realized by detecting the expression level of the PL2L60 protein, and the purpose of inhibiting and even curing the tumors can be achieved by inhibiting the tumor stem cells through the PL2L60 protein antibody.

In addition, research results show that the PL2L60 protein has tumor specificity and tumor broad spectrum, particularly has specific expression on the surface of tumor stem/progenitor cells, and by taking PL2L60 as a target, not only can high-efficiency antibody drugs, cell therapy technologies and tumor vaccines be developed, but also tumor early diagnosis reagents can be developed, thereby contributing to the progress of tumor diagnosis and treatment, being beneficial to promoting clinical tumor immunotherapy and having wide clinical application prospects.

Drawings

FIG. 1 is a diagram showing the activation of the promoter in the PIWIL2 gene and its products, (A) is the expression of PIWIL2 gene; (B) is the expression of mRNA; (C) is the expression of a protein;

FIG. 2 shows a comparison of the difference in expression between PL2L60 protein and PIWIL2 gene;

FIG. 3 shows the results of high expression of PL2L60 protein in various human and mouse leukemia and tumor cell lines;

FIG. 4 shows the expression result of PL2L60 protein on the surface of tumor cell membrane;

FIG. 5 shows the results of PL2L60 expression on the surface of KAO3 +;

FIG. 6 shows the results of comparing tumorigenic capacity of KAO3+ cells to KAO 3-cells;

FIG. 7 is a graph of PL2L60 protein overexpression versus the number of KAO3+ tumor cells;

FIG. 8 shows the results of the validation that PL2L60 protein can improve the proliferation and migration ability of tumor cells;

FIG. 9 is a graph showing that PL2L60 protein can improve tumorigenic ability of tumor cells;

FIG. 10 shows the result of natural tumor immunity (NOTI) induced by the pre-cancerous stem cells;

FIG. 11 is a graph of the results of NOTI induction as a function specific to precancerous stem cells;

FIG. 12 is a graph of the results of pre-cancerous stem cell induced NOTI as non-tissue specific;

FIG. 13 is a graph showing the results of DC-PL2L vaccine inducing high-efficiency anti-tumor immunity and inhibiting lung metastasis;

FIG. 14 is a graph showing the results of PL2L60 monoclonal antibody (KAO2) effectively inhibiting tumor cell tumorigenesis and metastasis;

FIG. 15 is a graph showing the effect of PL2L60 monoclonal antibody (KAO3) on the inhibition of tumor growth in human and mouse;

FIG. 16 is a graph of the results of the induction of apoptosis in tumor cells by KAO3 antibody;

FIG. 17 is a graph showing the results of KAO3 monoclonal antibody activating human complement to kill human and mouse tumor cells.

Detailed Description

The present invention will be described in detail below with reference to examples and the accompanying drawings. The following examples should not be construed as limiting the scope of the invention.

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Percentages and parts of material are by volume unless otherwise indicated.

The materials used in the present invention are as follows:

severe Combined Immunodeficiency (SCID) mice: mice were used for 8-12 weeks and were housed in an animal decontamination facility.

Human cell lines: the human breast cancer cell line MDA-MB-231, the human lung cancer cell line A549 and the 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 0.1mg/ml penicillin-streptomycin (Gibco).

Mouse lymphoma cell lines 2C4 and 326T-4 were self-prepared in the laboratory: the cells were placed in a humidified incubator containing 5% CO2(v/v) and maintained at 37 ℃ in R10F (RPMI1640 plus 10mmol/L fetal bovine serum supplemented with 5mmol/L glutamine, 50 mmol/L2-methylacetophenone, 100U/mL penicillin and 100mg/mL streptomycin).

anti-PL 2L60 monoclonal antibody (KAO3mAb, isotype IgM)) was prepared in the laboratory by itself according to the conventional protocol for the preparation of monoclonal antibodies, the sequence shown in SEQ ID NO: 1.

The analytical methods and experimental results used in the present invention are described in detail below.

The expression of PL2L60 protein has tumor specificity and tumor broad spectrum

As shown in FIG. 1, there are multiple promoters within the PIWIL2 gene, which are normally silenced in somatic cells and activated in tumor cells. Wherein the PL2L60 protein is the main product of PIWIL2 gene catabolism activation in tumor cells.

Expression of PL2L60 protein with tumor specificity as shown in fig. 2, (a) shows that a PL2L60 specific monoclonal antibody can recognize PIWIL2 and PL2L proteins in testis (mouse) and tumor cells (human): KAO1 primarily recognizes PIWIL2 in the testes; KAO2 and KAO3 recognize PIWIL2 and PL2L40 in testis and PL2L60 and PL2L50 in human tumor cells (SW 480); PIWIL2 was expressed at very low levels in tumor cells.

(B) High levels of PL2L protein (2,4) were shown to be expressed in primary breast (1-2) and cervical (3-4) carcinoma tissues, with PIWIL2 being expressed only in a few apoptotic cells (1, 3; indicated by arrows).

(C) It is shown that PIWIL2 is not expressed (1, indicated by arrows) or is expressed only in a small number of apoptotic cells (3, indicated by arrows) in metastatic cancers of breast tissue (1-2) and lymph nodes (3-4), but PL2L60 is expressed in almost all metastatic cancer cells (2, 4).

(D) PL2L60+ cells were shown to simultaneously express NF-kB: (1) single staining with PL2L 60; (2) NF-kB single dyeing; (3) PL2L60 and NF-kB double staining; (4) after enlarging the cells in the square of FIG. 3, the arrows indicate PL2L60 and NF-kB double positive cells.

The tumor spectrum of PL2L60 protein expression is shown in FIG. 3, and FIGS. 3(A) to 3(D) show the expression results of PL2L60 protein in various human and mouse leukemia and tumor cell lines, including leukemia cells, lymphoma, breast cancer, cervical cancer, lung cancer, pancreatic cancer, colon cancer, liver cancer, etc. HLMVEC-SvanHLVEC-Pri was an early transformed cell used as a negative control. The level of PIWIL2 was very low in all cell lines, suggesting expression was only in a few apoptotic cells, consistent with the results in figure 2. The results confirmed that PL2L60 expression has tumor specificity and tumor broad spectrum.

(II) PL2L60 promotes the generation and development of tumor stem cells

The tumor cells with cell membrane expressing PL2L60 have the characteristics of stem cells. The KAO3+ cell has strong tumorigenic capacity, and the tumor cell over-expressing PL2L60 and KAO3+ cell are increased, the proliferation capacity is improved, and the invasion, migration and spheroid generating capacity are enhanced, and the characteristics are related to the tumor stem cell. The results show that PL2L60+ CD44+ tumor cells have the characteristics of stem cells, and can promote the generation and development of tumor stem cells. The results of the specific experimental verification are shown in fig. 4 to 9:

FIG. 4 shows the expression result of PL2L60 protein on the surface of tumor cell membrane. Generally, PL2L60 is thought to be expressed in the cytoplasm or nucleus, but the inventors have recently found that part of the tumor cell membrane surface also expresses PL2L60 and verified using flow cytometry, immunofluorescence microscopy, and immunoblotting (WB) techniques, respectively: (A) flow cytometry techniques showed that the surface of human (MBA-231, HeLa and A549) and mouse (2C4 and 326T-4) tumor cells also expressed PL2L60(KAO3 +); (B-C) immunofluorescence microscopy confirmed that PL2L60 was expressed on the Surface of tumor cells (Surface). Note that: intracellular KAO3+ cells (C) more than cell membrane KAO3+ cells (B); (D-E) immunoblotting technique (WB) demonstrated that various tumor cells of mouse (D) and human (E) expressed PL2L 60; (F-G) is the results of semi-quantitative analysis of panels D & E.

After that, KAO3+ and KAO 3-breast cancer cells (MBA-MD231) were separated by flow cytometry, and immunoblotting analysis was performed on the separated cell membranes, and as a result, as shown in FIG. 5, it was confirmed that PL2L60 protein was contained in KAO3+ cell membranes, and it was confirmed that KAO3+ cell surface expressed PL2L 60.

FIG. 6 shows the results of a comparison of tumorigenic capacity of KAO3+ cells and KAO 3-cells. Cells of KAO3+ and KAO 3-on the cell surface were isolated from mouse (A) tumor cells (B16: melanoma; EL-4: lymphoma; LLC: lung cancer) and human (B) tumor cells (MDA-MB-231: breast cancer; A549: lung cancer; HeLa: cervical cancer), and inoculated into mouse tumor cells to immunocompromised B6 mice and human tumor cells to immunocompromised nude mice. Tumor sizes were observed and examined regularly. The results show that: KAO3+ tumor cells have a higher tumorigenicity than KAO 3-. In contrast to in vivo experiments, KAO3+ cells were not readily viable when cultured in vitro, suggesting that KAO3+ tumor cells, like stem cells, require a suitable niche (niche) for viability.

FIG. 7 is a graph of the relationship between PL2L60 protein overexpression and KAO3+ tumor cell number, showing that PL2L60 overexpression increases KAO3+ tumor cells, promoting cell proliferation: (A) PL2L60 gene (GTX-920) was stably transferred into human breast cancer tumor cells (MDA-MB-231 and MCF-7), and the control group was transferred into GFP gene. The results showed that the cells into which PL2L60 gene was transferred doubled the KAO3+ cells characteristic of tumor stem cells. (B) The MDA-MB-231-GFP and MDA-MB-231-GTX920 cells are transfected by siRNA-E7 and siRNA-E21 to knock out PIWIL2(E7) mRNA and PL2L60(E21) mRNA respectively, and the result shows that the PIWIL2mRNA knock-out has no inhibition effect on cell proliferation and can even promote the tumor cell proliferation; however, knock-out of PL2L60mRNA significantly inhibited cell proliferation. It was confirmed again that PIWIL2 expression had an inhibitory effect on tumors, while PIWIL2 catabolic activation could promote tumorigenesis and tumor development by inducing tumor stem cells.

FIG. 8 shows that PL2L60 increased the proliferation and migration ability of tumor cells. The real-time imaging system detects that: overexpression of PL2L60(GTX-920) by breast cancer cells MDA-MB-231 and MCF-7 led to accelerated cell proliferation (A, B) and enhanced invasion and migration capabilities (C, D).

Figure 9 shows that PL2L60 increases the tumorigenic capacity of tumor cells: PL2L60(GTX-920) promoted spheroid formation and growth of breast cancer cells (MDA-MB-231 and MCF-7) as shown in Panel A and Panel B; as shown by panels C and D, PL2L60 promoted tumorigenesis.

(III) the precancerous stem cells induce a broad spectrum of natural tumor immunity (NOTI)

PL2L60 is a tumor-specific broad-spectrum marker, immunogenic or not, and is of great significance in tumor immunotherapy. The precancerous stem cells express high levels of PL2L60 protein, which is an early stage of tumor stem cell development and has the potential for benign and malignant differentiation. The results of the study on the relationship between the precancerous stem cells and the natural tumor immunity in this section are shown in FIGS. 10 to 12:

FIG. 10 shows that precancerous stem cells can induce natural tumor immunity (NOTI). In panels a and B, pre-cancerous stem cells (2C4, 3B5C, and 3B6C) and lymphoma cell line EL-4 were inoculated into C57BL/6 mice (n ═ 6), respectively, and the mice inoculated with the pre-cancerous stem cells remained tumor-free after 200 days, but the mice inoculated with EL-4 grew tumor on day seven after inoculation;

in panels C and D, mice pre-seeded with pre-cancerous stem cells (3B5C, 3B6C) were re-seeded with EL-4 cells at 210 days, and the tumorigenic capacity of EL-4 was inhibited in these mice. Of these, 50% or more of mice did not develop tumors (C), and even tumor-developing mice, tumors were very small (D). The results indicate that precancerous stem cells can induce NOTI, preventing tumor progression.

Figure 11 shows that induction of NOTI is a unique function of precancerous stem cells: (A) b6 mice were inoculated 8 weeks later with live cells of pre-cancerous stem cells and cell lysates (5x106), respectively, followed by B16F10 melanoma cells (7x 105). The results show that both viable cells and cell lysates are effective in inducing NOTI. (B) B6C mice were immunized with lysates of pre-cancerous stem cells and lymphoma EL-4 cells at various concentrations and inoculated 8 weeks later with B16F10 cells. The results show that only the precancerous stem cell lysate induced NOTI, and that EL-4 cells did not have this ability. The cell number of lysates was correlated with the intensity of NOTI induced by the lysates.

Figure 12 shows that pre-cancerous stem cell induced NOTI is non-tissue specific: (A) mice were inoculated 8 weeks later with lysates of pre-cancerous stem cells (pCSC, lymphoma of origin), EL-4 and B16F10 tumor cells, and with B16F10 tumor cells. The result shows that only the cancer stem cell lysate induces the strongest NOTI, and the growth of the melanoma can be completely inhibited in more than 60 percent of mice; (B) panel A tumor size of experiment. The results show that NOTI after vaccination with pre-cancerous stem cell lysates is broad-spectrum and similarly inhibitory to melanoma and lymphoma (fig. 11).

(IV) PL2L60 protein is a major component of the induction of NOTI in precancerous stem cells

DC-PL2L vaccine is used to induce high-efficiency antitumor immunity and inhibit lung metastasis cancer. Bone marrow Dendritic Cells (DCs) were sensitized with PL2L polypeptide Pa or Pb, C57BL/6 mice were inoculated subcutaneously, B16F10 was injected intravenously after 4 weeks, the mice were sacrificed two weeks later to collect lung tissue, paraffin sections, and metastatic tumors or foci of inflammation (Counts/section) were counted under a microscope. As shown by the results in fig. 13: pb polypeptides induce a greater incidence of noti (a) tumors than Pa polypeptides; (B) metastases and foci of inflammation; (C) micrographs at different magnifications after staining of H & E lung tissue. And (3) upper row: a focus of inflammation; and (3) lower row: metastasis of tumors.

(V) PL2L60 targeting monoclonal antibodies KAO2 and KAO3 can kill mouse and human tumor cells, and effectively inhibit the tumor forming ability of human and mouse tumor cells

FIG. 14 shows that PL2L60 monoclonal antibody (KAO2) effectively inhibited tumor cell tumorigenesis and metastasis. The pre-cancer stem cells (2C4G2) were treated with mab KAO2 culture supernatant (100 μ l) or culture medium and inoculated subcutaneously into the groin of SCID mice (n-3/group). The results show that: cells treated with KAO2 did not grow into tumors (A & B), but all cells treated with medium grew into tumors (B & C). To evaluate the effect of KAO2 mab on metastases, B16F10 melanoma cells were treated with KAO2 culture supernatant and injected intravenously into B6 mice. Two weeks later mice were sacrificed and lung tissue was collected for examination of metastases. The results showed that the metastatic potential of KAO2 treated B16F10 was significantly inhibited (D), and the metastases were significantly lower microscopically in lung tissue of mice injected with KAO2 treated B16F10 cells than in the untreated group.

FIG. 15 shows that treatment of human and mouse tumors with KAO3 monoclonal antibody was effective in inhibiting the growth of various tumors. The mouse precancerous stem cell 2C4, the cancer stem cell 326T-4 and the human tumor cells (breast cancer: MDA-MB-231; lung cancer: A549; cervical cancer: HeLa) are respectively treated by KAO3 monoclonal antibody culture supernatant or culture medium and then inoculated with the tumor formation rate and the tumor size of the immunodeficient mouse (SCID) in the observation period. Meanwhile, growing tumors were treated with KAO3 antibody at 1.0CM diameter size and control group with culture supernatant (Ctrl). As shown in panels a-D, the KAO3 antibody was effective in inhibiting tumor production in humans and mice, and also in inhibiting tumor growth when treated (panels E and F).

Figure 16 shows that antibodies to KAO3 can induce apoptosis in tumor cells. Human and mouse tumor cells were examined for their effect on cell viability 24 hours after treatment with varying concentrations of KAO3 monoclonal antibody supernatant (0,1,2,4,8 μ l/well). (A) Flow cytometry detects apoptotic cell examples. (B) Statistics of the effect of KAO3 treatment on tumor cell viability; (C) statistics of the effect of different concentrations on apoptosis and (D) micrographs of cells in culture air. The results show that: both human and mouse tumor cells were treated with the KAO3 antibody to induce apoptosis to varying degrees. These apoptotic cells may be tumor stem cells.

FIG. 17 shows that KAO3 monoclonal antibody can activate human complement to kill human and mouse tumor cells. (A) Tumor cells were examined for apoptotic cells using flow cytometry after one hour incubation with KAO3 and human complement. (B) After 3 hours of incubation of tumor cells with KAO3 and human complement, cell activity was measured and the percentage of apoptotic cells was determined. The results show that: KAO3 is effective in inducing apoptosis in tumor cells.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (2)

1. The application of PL2L60 protein antibody in preparing reagent or kit for early screening or diagnosing tumor is KAO3 monoclonal antibody, the sequence of the KAO3 monoclonal antibody is shown in SEQ ID NO. 1, and the screening or diagnosing tumor reagent refers to the reagent for screening or diagnosing tumor stem cells or tumor progenitor cells.
2. 2. The use of claim 1, wherein the tumor is leukemia, lymphoma, breast cancer, cervical cancer, lung cancer, pancreatic cancer, colon cancer or liver cancer.

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