Method for diagnosing cancer and diagnostic kit using measurement of NK cell activity

Abstract

To provide a method usable for diagnosis and evaluation of various types of cancer in view of the unceasing need for new methods for this purpose and useful kits and reagents for practicing such methods.SOLUTION: A method and a diagnostic kit for cancer diagnosis together with useful compositions for measuring NK cell activity serve to facilitate monitoring how in vivo immunity systems are changing by way of measuring NK cell activity in blood to determine cancer affection. Therefore, cancer affection can be easily predicted by using blood specimens taken from the suspected patient.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of Korean Patent Application No. 2011-0012983, filed Feb. 14, 2011, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] 1. Technical field of the invention The present invention relates to a method for diagnosing cancer and a diagnostic kit that utilizes the measurement of NK cell activity.

[0003] 2. Consideration of Related Technologies Natural killer (NK) cells are involved in innate immunity to eliminate pathogens and cancer cells, and are known to secrete interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-1β (MIP-1β), and other molecules that regulate adaptive immunity. When NK cells encounter other cells, their major histocompatibility complexes (MHCs) have a mechanism to send signals into the NK cells to attack these abnormal cells through their molecular actions when MHC class 1 is absent, such as in cancer cells, or when the shape of MHC class is abnormal, such as in virus-infected cells. However, NK cells have been reported to have impaired function and differentiation capacity in various types of cancer, and therefore NK cell activity is closely related to the survival of cancer cells. Therefore, extensive research has been conducted to increase the number or activity of NK cells for cancer immunotherapy.

[0004] Meanwhile, methods for diagnosing cancer mainly involve detecting the presence of cancer from graphic images obtained using computed tomography (CT), magnetic resonance imaging (MRI) or X-rays. However, these tests are usually performed only when there is a strong need for the patient to undergo the test due to pain or inconvenience, and only in certain tissues, so the presence of cancer may be overlooked. Methods for determining the risk of cancer using blood tests have been developed, but their use as a method for diagnosing cancer is limited. This is because the methods are performed using blood tumor markers, for example for prostate, colon, ovarian, pancreatic or liver cancer, and therefore the patient may appear to be positive for cancer when the etiological factor is in the corresponding organ rather than the cancer. There are also attempts to diagnose cancer using antibodies, but such attempts are limited to certain types of cancer.

[0005] Thus, there is a continuing need for new methods for diagnosing various types of cancer. Summary of the Invention

[0006] It is therefore an object of the present invention to provide methods that can be used to diagnose and assess cancer, as well as kits and reagents useful in such methods.

[0007] As one aspect of the present invention, there is provided a method for measuring NK cell activity, the method comprising stimulating NK cells in a blood sample, thereby artificially activating the NK cells to produce NK cell-secreted cytokines, and measuring the amount of NK cell-secreted cytokines in the blood sample.

[0008] In certain non-limiting embodiments, the blood sample may be a whole blood sample, peripheral blood mononuclear cells (PBMCs) or NK cells.

[0009] In further embodiments, stimulation of NK cells may be performed by incubating the blood sample with at least one stimulatory cytokine, including interleukin 2, interleukin 12, interleukin 15, interleukin 18, or a combination thereof, or by incubating the blood sample with lipopolysaccharides (LPSs) or polyinosinic:polycytidylic acid (Poly I:C).

[0010] In certain embodiments, the NK cell secreted cytokines may include interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), or macrophage inflammatory protein-1β (MIP-1β).

[0011] In a further non-limiting embodiment of this method, macrophage inflammatory protein-1β (MIP-1β) can be used as a control group to compare NK cell activation with that of normal subjects.

[0012] Also, in certain embodiments, the method may be carried out using at least one stimulatory cytokine fused to a stabilizing peptide. For example, and without wishing to be limiting, the stabilizing peptide may be a C-terminal acidic tail domain peptide of the synuclein family. In such embodiments, the stabilizing peptide may comprise amino acid residues 103-115 (SEQ ID NO:22), amino acid residues 114-126 (SEQ ID NO:23), amino acid residues 119-140 (SEQ ID NO:24) or amino acid residues 130-140 (SEQ ID NO:25) of the C-terminal acidic tail domain of α-synuclein, amino acid residues 85-134 of the C-terminal acidic tail domain of β-synuclein (SEQ ID NO:27), amino acid residues 96-127 of the C-terminal acidic tail domain of γ-synuclein (SEQ ID NO:29), or amino acid residues 96-127 of the C-terminal acidic tail domain of synoretin (SEQ ID NO:29).

[0013] In a further embodiment, the step of stimulating the NK cells in the blood sample, thereby artificially activating the NK cells to produce NK cell-secreted cytokines, is performed in a medium containing a carrier protein, e.g., serum albumin protein.

[0014] The method is particularly useful for detecting the onset or recurrence of cancer. In such embodiments, a decrease in the amount of NK cell secreted cytokines in a subject compared to levels in normal individuals is indicative of the onset or recurrence of cancer.

[0015] As a further aspect of the present invention, a kit for measuring NK cell activity is provided, which includes an agent for stimulating NK cells in a blood sample, thereby artificially activating the NK cells to produce NK cell-secreted cytokines, and which may be useful for carrying out the methods described above, including for detecting the onset or recurrence of cancer.

[0016] In further non-limiting embodiments of the kit, the NK cell secreted cytokine can be interferon-gamma (IFN-γ) or tumor necrosis factor-alpha (TNF-α).

[0017] In further embodiments, the agent for stimulating NK cells in a blood sample and artificially activating the NK cells to produce NK cell-secreted cytokines may comprise at least one stimulatory cytokine, LPS or poly I:C, wherein said at least one stimulatory cytokine comprises one or more of interleukin 2, interleukin 12, interleukin 15, interleukin 18.

[0018] The described kits may, in certain embodiments, also include one or more of the following: an anti-INF-γ antibody, an anti-TNF-α antibody, and an anti-MIP-1β antibody. Without wishing to be limiting in any way, the kit may further include instructions for comparing the amount of NK cell secreted cytokines in a subject with levels in normal individuals, where a reduction in the level of NK cell secreted cytokines in the subject is indicative of cancer development or recurrence.

[0019] As a further aspect of the invention, there is provided a fusion protein comprising a cytokine linked to a C-terminal acidic tail domain peptide of the synuclein family, the cytokine being any of interleukin 2, interleukin 12, interleukin 15 or interleukin 18.

[0020] In certain non-limiting embodiments of the described fusion proteins, the synuclein family C-terminal acidic tail domain peptide may comprise amino acid residues 103-115 (SEQ ID NO:22), amino acid residues 114-126 (SEQ ID NO:23), amino acid residues 119-140 (SEQ ID NO:24) or amino acid residues 130-140 (SEQ ID NO:25) of the C-terminal acidic tail domain of α-synuclein, amino acid residues 85-134 of the C-terminal acidic tail domain of β-synuclein (SEQ ID NO:27), amino acid residues 96-127 of the C-terminal acidic tail domain of γ-synuclein (SEQ ID NO:29), or amino acid residues 96-127 of the C-terminal acidic tail domain of synuclein (SEQ ID NO:29).

[0021] Compositions comprising the fusion proteins are also provided.

[0022] Also provided herein is a cancer diagnostic kit comprising any of the above fusion proteins or compositions.

[0023] The cancer diagnostic kit may, in certain non-limiting embodiments, also comprise at least one of the following antibodies: an anti-INF-γ antibody, an anti-TNF-α antibody, and an anti-MIP-1β antibody.

[0024] Also provided herein is a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. Without wishing to be limiting, the polypeptide may have a higher percent identity to the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10, including 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0025] Oligonucleotides encoding the above fusion proteins and polypeptides are also provided. For example, oligonucleotides are provided that comprise a nucleic acid sequence having at least 80% identity with the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9, or their complementary strands. Such oligonucleotides may have a higher percent identity, including, without limitation, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9, or their complementary strands.

[0026] Vectors containing the above-described oligonucleotides are also provided, as are host cells containing such vectors or oligonucleotides. [Brief description of the drawings]

[0027] The above and other objects, features and advantages of the present invention will become more apparent to those skilled in the art from the detailed description of illustrative embodiments thereof taken in conjunction with the drawings.

[0028] [Figure 1]FIG. 1 is a schematic diagram showing the fusion products of SP peptides fused to either the N-terminus or C-terminus of cytokines including hIL2, hIL12, hIL15 and hIL18.

[0029] [Diagram 2] FIG. 2 is a photograph showing the results of electrophoresis of purified SP fusion proteins.

[0030] [Diagram 3] FIG. 3 shows the activity of artificially activated NK cells in normal subjects by analysis of the amount of interferon-γ produced when NK cells were stimulated with a single cytokine (FIG. 3A) or a combination of cytokines (FIGS. 3B-3D).

[0031] [Figure 4] FIG. 4 is a graph showing cytokines secreted from activated NK cells by sandwich ELISA.

[0032] [Diagram 5] FIG. 5 shows a comparison of protein activity (A) and stability (B) between SP IL-2 and IL-2.

[0033] [Figure 6] FIG. 6 shows NK cell activity in normal subjects and cancer patients treated separately with SP IL-2 (10 ng / ml) (condition A) and SP IL-2 (5 ng / ml) + IL-12 (5 ng / ml) (condition B).

[0034] [Figure 7] FIG. 7 is a graph showing the ability of NK cells to secrete interferon-γ following stimulation with IL2 in T cells, NK cells, whole blood and PBMCs.

[0035] [Figure 8]FIG. 8 is a graph showing the change in the amount of interferon-γ secreted from NK cells of normal subjects stimulated with LPS.

[0036] [Figure 9] FIG. 9 is a graph showing the change in the ability of NK cells to secrete interferon-γ due to different concentrations of IL12 and IL15 treated and different medium compositions.

[0037] [Figure 10] FIG. 10 is a graph showing the change in the amount of secreted interferon-γ according to the stage of cancer progression.

[0038] [Figure 11] FIG. 11 shows the results of analysis, using an ELISA plate, of interferon-γ produced from NK cells of normal subjects stimulated with cytokines.

[0039] [Figure 12] FIG. 12 shows the results of flow cytometry of whole blood donated from normal subjects stimulated with cytokines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description of the Invention The present invention relates to a method, a kit and a reagent for diagnosing cancer by utilizing the interaction between cancer and NK cells.

[0041] For this purpose, a method for measuring NK cell activity is provided, which comprises stimulating NK cells in a blood sample, thereby artificially activating the NK cells to produce NK cell-secreted cytokines, and measuring the amount of NK cell-secreted cytokines in the blood sample.

[0042] Based on the observation that NK cell activity is decreased in cancer patients, the present inventors have found that the onset of cancer can be screened primarily by measuring NK cell activity. The method described herein makes it possible to determine whether NK cells function normally by artificially stimulating NK cells, and to measure the activation level of NK cells by detecting changes in the amount of NK cell-secreted cytokines present in a blood sample, which is different from other methods that simply measure the number of NK cells or the amount of cytokines originally present in a blood sample. For example, in conventional methods for measuring the activation level of NK cells, 51 Cr release assay is used as a method for measuring target-specific cytotoxicity. However, this method of measuring NK cell activity requires the use of radioisotopes, which makes measurement and analysis difficult, complicated, and expensive. Therefore, the assay is not suitable for use in a primary cancer screening / testing method that can easily diagnose cancer. On the other hand, according to the present invention, NK cell activity can be measured by stimulating NK cells to produce NK cell-secreted cytokines and quantifying the produced NK cell-secreted cytokines, so that subjects with reduced NK cell activity can be advantageously screened as subjects suffering from or at risk of suffering from cancer.

[0043] According to the present invention, blood samples may include, but are not limited to, whole blood, peripheral blood mononuclear cells (PBMCs), and NK cells drawn from a subject. Although PBMCs or NK cells may be used intact instead of whole blood, in certain embodiments, the use of whole blood may be advantageous due to simpler procedures and lower costs.

[0044] In the present invention, the term "subject" refers to a mammal suspected of having cancer or having a recurrence of cancer, or in which it is desired to determine the incidence or recurrence of cancer.

[0045] NK cells present in a blood sample are generally in an inactive state. According to the present invention, at least one cytokine, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C) can be used as an agent (also referred to herein as an agonist or activator) that stimulates the NK cells in the blood sample and artificially activates the NK cells to produce NK cell-secreted cytokines. Here, the cytokines used to activate the NK cells can be interleukin 2, interleukin 12, interleukin 15 and interleukin 18, or a combination thereof. It is widely known in the art that interleukin 2, interleukin 12, interleukin 15, interleukin 18, LPS or poly I:C stimulates the NK cells to produce NK cell-secreted cytokines. Thus, according to one exemplary embodiment of the present invention, stimulation of NK cells may be performed by incubating the blood sample with at least one cytokine, including interleukin 2, interleukin 12, interleukin 15 and / or interleukin 18, or by incubating the blood sample with LPS or poly I:C.

[0046] In one non-limiting embodiment, NK cell stimulation may be performed by incubating blood sample with interleukin 2. Interleukin 2 is one of the cytokines secreted by T cells, and is known to be related to the activation of NK cells by T cells in the adaptive immune response in vivo. Interleukin 2 is also a cytokine that is commonly and widely used to activate NK cells in vitro. Therefore, NK cell stimulation may be performed by incubating blood sample with interleukin 2.

[0047] In other non-limiting embodiments, NK cell stimulation may be performed by incubating blood samples with interleukin 2 and interleukin 12. In early cancer patients, NK cell activity may be low, but T cell activity may be high. In contrast, in late cancer patients, not only NK cell activity but also T cell activity may be low. Interleukin 12 is involved in the activation of not only NK cells but also T cells. Therefore, if treated with interleukin 12 together with interleukin 2, the cytokines secreted by NK cells are added to the cytokines secreted by T cell stimulation. Therefore, it is possible to evaluate not only the anti-cancer immunity of NK cells but also the overall level of immunity, and use this level as a marker representing the degree of cancer progression or the prognosis of cancer treatment. Interleukin 15 and interleukin 18 are cytokines secreted by activated dendritic cells and macrophages, which induce the activation and proliferation of NK cells during in vitro natural immune responses. In particular, when interleukin-12 is combined with interleukin-15 or interleukin-18, a relatively small amount of interleukin-12 can be used to stimulate the secretion of NK cell-secreted cytokines in NK cells. Therefore, stimulation of NK cells can be effectively achieved by incubating a blood sample with interleukin-12 and interleukin-15, or with interleukin-12 and interleukin-18.

[0048] According to the present invention, the value of NK cell secreted cytokines is used as a measure to evaluate NK cell activity. In the present invention, "NK cell secreted cytokines" refers to cytokines secreted from NK cells, particularly cytokines from activated NK cells by artificial stimulation. In one embodiment, the NK cell secreted cytokine is at least one cytokine selected from the group of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein-1β (MIP-1β). Interferon-γ is secreted from NK cells, dendritic cells, Tc cells, Th1 cells, etc., and is known as a cytokine that plays an important role in natural and adaptive immunity for controlling cancer. In addition, tumor necrosis factor-alpha (TNF-α) kills cancer cells, and is also involved in killing foreign invaders such as bacteria, inducing T cell activation, and serves as a supplementary factor for antibody production from B cells. Therefore, for example, if the value of interferon-gamma or tumor necrosis factor-alpha is smaller than that of interferon-gamma or tumor necrosis factor-alpha from a normal person, this indicates that there is a problem with NK cell activity for controlling cancer. Therefore, it is possible to determine NK cell activity by comparing the amount of interferon-gamma or tumor necrosis factor-alpha secreted from artificially activated NK cells with the amount of interferon-gamma or tumor necrosis factor-alpha from a normal person.

[0049] On the other hand, macrophage inflammatory protein-1β (MIP-1β) can be used as a control group to compare the activation of NK cells. As shown in the following example, the levels of macrophage inflammatory protein-1β (MIP-1β) are similarly high in both normal subjects and cancer patients. Therefore, macrophage inflammatory protein-1β (MIP-1β) can be used to analyze NK cell activity in normal subjects and cancer patients, or can be used as a control group for analysis using a cancer diagnostic kit.

[0050] Quantification of NK cell secreted cytokines may be performed by any method known in the art, but the present invention is not limited thereto. For example, quantification of interferon-γ may be performed using interferon-γ enzyme-linked immunosorbent assay (interferon-γ ELISA).

[0051] Also, at least one cytokine, including interleukin 2, interleukin 12, interleukin 15 or interleukin 18, used as an agent that stimulates NK cells in a blood sample and serves to artificially activate the NK cells to produce NK cell-secreted cytokines, may be in the form of a fusion protein with a stabilizing peptide.

[0052] Interleukin-2, interleukin-12, interleukin-15 or interleukin-18 in the form of a fusion protein with a stabilizing peptide can provide similar biological activity and increased storage stability compared to the biological activity and storage stability of wild-type interleukin-2, interleukin-12, interleukin-15 or interleukin-18. For example, when a cytokine is bound to the stabilizing peptide, the cytokine retains its native activity while remaining stable despite changes in the environment, such as lyophilization.

[0053] The stabilizing peptide may be linked to the N- or C-terminus of interleukin-2, interleukin-12, interleukin-15 or interleukin-18, and the fusion protein may be prepared using known methods for preparing fusion proteins.

[0054] According to one exemplary embodiment, the C-terminal acidic tail (acidic tail amino acid sequence of alpha-synuclein, ATS) domain peptide of the synuclein family can be used as a stabilizing peptide capable of binding to interleukin 2, interleukin 12, interleukin 15 or interleukin 18, but the present invention is not limited thereto. Korean Patent No. 10-0506766 discloses that ATS peptides confer resistance to environmental stresses to fusion partner proteins.

[0055] According to one exemplary embodiment, the stabilizing peptides that can be used herein include stabilizing peptides selected from amino acid residues 103-115, 114-126, 119-140, and 130-140 of the C-terminal acidic tail domain of α-synuclein, amino acid residues 85-134 of the C-terminal acidic tail domain of β-synuclein, amino acid residues 96-127 of the C-terminal acidic tail domain of γ-synuclein, and amino acid residues 96-127 of the C-terminal acidic tail domain of synoretin. In the present invention, the amino acid sequence of the ATS peptide, the ATS peptide, and the method for preparing the fusion protein containing the same peptide can be performed using the method disclosed in Korean Patent No. 10-0506766. With reference to the following examples, it is shown that interleukin-2, interleukin-12, interleukin-15, or interleukin-18 fused with the ATS peptide are highly stable and exhibit activity similar to that of the wild type when the cytokine is activated by T lymphocytes.

[0056] In one embodiment, the step of stimulating NK cells in a blood sample, thereby artificially activating NK cells to produce NK cell secreted cytokines, can be carried out in a medium containing a carrier protein. The carrier protein serves to stabilize cytokines such as interleukin 2, interleukin 12, interleukin 15 or interleukin 18, which are used as agents to stimulate NK cells in a blood sample, thereby artificially activating NK cells to produce NK cell secreted cytokines, thereby inducing NK cells to produce more NK cell secreted cytokines. In a particular embodiment, the carrier protein can be, but is not limited to, bovine serum albumin or human serum albumin.

[0057] In addition, the method for measuring NK cell activity can be used to screen for the onset or recurrence of cancer.

[0058] NK cell activity can be measured by comparing the amount of NK cell secreted cytokines secreted from artificially activated NK cells with the amount of NK cell secreted cytokines from normal subjects. In this case, if the amount of NK cell secreted cytokines is less than that of NK cell secreted cytokines from normal subjects, NK cell activity is considered to be decreased. Therefore, it is possible to determine the risk of cancer or recurrence of cancer. If NK cell activity is decreased compared to normal subjects, the subject can be primarily classified as a patient suspected of having cancer or a patient with cancer recurrence. In addition, the onset or recurrence of cancer can be diagnosed through further diagnostic methods such as CT, MRI, or positron emission tomography (PET), which are commonly used for cancer diagnosis, or through final tissue examination. Although the method according to the present invention is not a method for finally diagnosing cancer, the method has the excellent advantage of being able to primarily screen for the onset or recurrence of cancer using blood.

[0059] The present invention also provides a kit for measuring NK cell activity, which comprises an agent such as an agonist or activator that stimulates NK cells in a blood sample and artificially activates the NK cells to produce NK cell-secreted cytokines. The kit for measuring NK cell activity can be used to easily carry out the method for measuring NK cell activity.

[0060] In the kit for measuring NK cell activity, the agent that stimulates NK cells and artificially activates NK cells to produce NK cell-secreted cytokines may be at least one cytokine, LPS or poly I:C, and the cytokine may be selected from the group consisting of interleukin 2, interleukin 12, interleukin 15 and interleukin 18. In addition to the agent that stimulates NK cells and artificially activates NK cells to produce NK cell-secreted cytokines such as interferon-γ, the cancer diagnostic kit may contain additional components for measuring NK cell activity, such as antibodies and substrates for quantifying NK cell-secreted cytokines. In one embodiment, the kit of the present invention further comprises at least one antibody selected from the group of anti-INF-γ antibody, anti-TNF-α antibody, and anti-MIP-1β antibody.

[0061] The antibody in the kit according to the present invention may be immobilized on a solid substrate. The antibody can be immobilized using various methods described in the literature (Antibodies: A Laboratory Manual, Harlow & Lane; Cold Spring Harbor, 1988). Suitable solid substrates may include cell culture plates, ELISA plates, tubes and polymeric films. In addition, solid substrates include rods, synthetic glass, agarose beads, cups, flat packs, or other films or coatings supported by or attached to a solid support.

[0062] The kit according to the present invention may also include reagents used for immunological analysis using an antibody that selectively recognizes a NK cell secreted cytokine, such as interferon-γ. Immunological analysis may include any method that can measure the binding of an antigen to an antibody according to the present invention. Such methods are known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassay, ELISA, immunoblot, Farr assay, precipitin reaction, turbidimetry, immunodiffusion, countercurrent electrophoresis, single radial immunodiffusion, and immunofluorescence.

[0063] Reagents used for immunological analysis include a suitable carrier, a label capable of producing a detectable signal, a lysing agent, a detergent, and, if the labeling material is an enzyme, the reagents may also include a substrate that allows the enzyme activity to be measured, and a reaction quenching agent. Suitable carriers may include, but are not limited to, soluble carriers, such as one of the physiologically available buffers known in the art (e.g., PBS), or insoluble carriers, such as polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesins, cross-linked dextran, polysaccharides and magnetic particles obtained by coating the surface of latex with metal, as well as other paper, glass, metal, agarose and combinations thereof.

[0064] As a label capable of generating a detectable signal, an enzyme, a fluorescent substance, a luminescent substance, and a radioactive substance can be used. As an enzyme, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, malate dehydrogenase, glucose 6-phosphate dehydrogenase, invertase, etc. can be used, fluorescein isothiocyanate or phycobiliprotein can be used as a fluorescent substance, isoluminol or lucigenin can be used as a luminescent substance, and I 131 , C 14 Alternatively, H3 can be used as a radioactive material. However, in addition to the exemplary materials, any material that can be used for immunological analysis can be used here.

[0065] The present invention further provides a fusion protein comprising a cytokine linked to a C-terminal acidic tail domain peptide of the synuclein family, where the cytokine may be interleukin-2, interleukin-12, interleukin-15 or interleukin-18. As described above, the fusion protein can be used as an agent that serves to stimulate NK cells and artificially activate NK cells to produce NK cell-secreted cytokines, and provides increased stability compared to wild-type interleukin-2, interleukin-12, interleukin-15 or interleukin-18 despite environmental changes such as lyophilization or long-term storage.

[0066] According to one exemplary embodiment, the fusion protein may be a fusion protein in which interleukin 2 is linked to a C-terminal acidic tail domain peptide of the synuclein family.

[0067] According to another exemplary embodiment, the fusion protein may be a fusion protein in which interleukin-12 is linked to a C-terminal acidic tail domain peptide of the synuclein family.

[0068] According to yet another exemplary embodiment, the fusion protein may be a fusion protein in which interleukin 15 is linked to a C-terminal acidic tail domain peptide of the synuclein family.

[0069] According to yet another exemplary embodiment, the fusion protein may be a fusion protein in which interleukin 18 is linked to a C-terminal acidic tail domain peptide of the synuclein family.

[0070] In the fusion protein, the synuclein family C-terminal acidic tail domain peptide may also be selected from amino acid residues 103-115, amino acid residues 114-126, amino acid residues 119-140 and amino acid residues 130-140 of the C-terminal acidic tail domain of α-synuclein, amino acid residues 85-134 of the C-terminal acidic tail domain of β-synuclein, amino acid residues 96-127 of the C-terminal acidic tail domain of γ-synuclein and amino acid residues 96-127 of the C-terminal acidic tail domain of synuclein.

[0071] The present invention also provides a use of the fusion protein for activating NK cells. As described above, the fusion protein can be used to activate NK cells in blood and promote the secretion of NK cell-secreted cytokines.

[0072] Thus, the present invention provides a composition for activating NK cells, wherein the composition comprises at least one fusion protein selected from the group consisting of interleukin 2 bound to a C-terminal acidic tail domain peptide of the synuclein family, interleukin 12 bound to a C-terminal acidic tail domain peptide of the synuclein family, interleukin 15 bound to a C-terminal acidic tail domain peptide of the synuclein family, and interleukin 18 bound to a C-terminal acidic tail domain peptide of the synuclein family.

[0073] According to one exemplary embodiment, the synuclein family C-terminal acidic tail domain peptide may be selected from amino acid residues 103-115, amino acid residues 114-126, amino acid residues 119-140 and amino acid residues 130-140 of the C-terminal acidic tail domain of α-synuclein, amino acid residues 85-134 of the C-terminal acidic tail domain of β-synuclein, amino acid residues 96-127 of the C-terminal acidic tail domain of γ-synuclein and amino acid residues 96-127 of the C-terminal acidic tail domain of synuclein.

[0074] The composition for activating NK cells may also contain, in addition to the cytokine fused to the stabilizing peptide, a buffer capable of maintaining and storing the fusion protein.

[0075] Furthermore, the present invention provides a cancer diagnostic kit comprising at least one fusion protein selected from the group consisting of interleukin 2 bound to a C-terminal acidic tail domain peptide of the synuclein family, interleukin 12 bound to a C-terminal acidic tail domain peptide of the synuclein family, interleukin 15 bound to a C-terminal acidic tail domain peptide of the synuclein family, and interleukin 18 bound to a C-terminal acidic tail domain peptide of the synuclein family. As described above, when a blood sample taken from a subject is incubated with the fusion protein, NK cells in the blood sample are activated. Therefore, NK cell activity in a subject can be measured by quantifying interferon-γ produced by the activation of NK cells, and thus cancer can be primarily diagnosed by classifying a subject having a lower NK cell activity than that of a normal person as a patient suffering from cancer or at risk of having cancer recurrence.

[0076] According to one exemplary embodiment, the synuclein family C-terminal acidic tail domain peptide may be selected from amino acid residues 103-115, amino acid residues 114-126, amino acid residues 119-140 and amino acid residues 130-140 of the C-terminal acidic tail domain of α-synuclein, amino acid residues 85-134 of the C-terminal acidic tail domain of β-synuclein, amino acid residues 96-127 of the C-terminal acidic tail domain of γ-synuclein and amino acid residues 96-127 of the C-terminal acidic tail domain of synuclein.

[0077] In addition to the fusion protein, the cancer diagnostic kit may contain further components used to perform the diagnostic method according to the present invention, such as antibodies and substrates for quantifying NK cell secreted cytokines. These components are described above in connection with the kit for measuring NK cell activity. Instructions for using these components in the above-mentioned method may also be included in the kit.

[0078] It will be apparent that these and other features, aspects and advantages of preferred embodiments of the present invention will be more fully described in the following examples. It should also be understood that these examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art will appreciate that other equivalents and modifications can be made without departing from the scope of the invention as set forth in the claims. EXAMPLES

[0079] example Preparation Example 1 Construction of expression vector carrying stabilized peptide-IL fusion protein To prepare IL-2, IL-12, IL-15 or IL-18 fused with a stabilizing peptide, an expression vector was constructed. A peptide containing amino acid residues 119-140 of α-synuclein (SEQ ID NO: 23, hereinafter referred to as "SP") was used as the stabilizing peptide. cDNAs of IL2, IL12p35, IL12p40, IL15 and IL-18 were obtained by isolating total RNA from human lymphocytes using a total RNA extraction kit (Invitron Biotechnology) and reverse transcribing the total RNA using reverse transcriptase (Invitrogen). The obtained cDNAs were used as templates and amplified by PCR using the following primers specific to each cDNA gene.

number

[0080] FIG. 1 is a schematic diagram showing the constructs of the fusion products of the above cytokines with SP, including IL2, IL12p35, IL12p40, IL15, and IL-18. As illustrated in the figure, the SP-hIL2 fusion product was constructed by sequentially subcloning the PCR-amplified genes encoding hIL2 and amino acid residues 119-140 of α-synuclein into the pRSETA expression vector. The SP-hIL12p40 fusion product was constructed by sequentially subcloning the PCR-amplified genes encoding hIL12p40 and amino acid residues 119-140 of α-synuclein into the pVL1393 expression vector. The SP-hIL12p35 fusion product was constructed by sequentially subcloning the PCR-amplified genes encoding SP-hIL12p35 and amino acid residues 119-140 of α-synuclein into the pVL1393 expression vector. The hIL15-SP fusion product was constructed by sequentially subcloning PCR-amplified genes encoding hIL15 and amino acid residues 119-140 of α-synuclein into the pRSETA expression vector. The SP-hIL18 fusion product was constructed by sequentially subcloning PCR-amplified genes encoding hIL18 and amino acid residues 119-140 of α-synuclein into the pRSETA expression vector. The sequences of all constructs were confirmed by DNA sequencing.

[0081] The nucleic acid and amino acid sequences of the SP-hIL2 fusion product are set forth in SEQ ID NOs: 1 and 2, respectively. The nucleic acid and amino acid sequences of the SP-hIL12p40 fusion product are set forth in SEQ ID NOs: 3 and 4, respectively. The nucleic acid and amino acid sequences of the SP-hIL12p35 fusion product are set forth in SEQ ID NOs: 5 and 6, respectively. As shown in FIG. 1, a 6×His-tag sequence is included in each vector for the purpose of isolation and purification of the virally expressed SP-hIL12p40 and SP-hIL12p35 fusion products. The nucleic acid and amino acid sequences of the hIL15-SP fusion product are set forth in SEQ ID NOs: 7 and 8, respectively. And the nucleic acid and amino acid sequences of the SP-hIL18 fusion product are set forth in SEQ ID NOs: 9 and 10, respectively.

[0082] Preparation Example 2 Expression and purification of recombinant SP fusion proteins The expression vector constructed to express the recombinant SP-hIL2 protein was transformed into Escherichia coli BL21(DE3)RIPL (Invitrogen) and then incubated. The culture was centrifuged at 10,000 rpm for 10 minutes to obtain a cell pellet. The cell pellet was resuspended in phosphate-buffered saline (PBS, pH 7.4) and then homogenized by sonication. The SP fusion protein expressed in an insoluble form in E. coli was subjected to a refolding procedure and then purified using an ion-exchange resin.

[0083] The two expression vectors constructed to express the recombinant SP-hIL12 protein were transfected into sf21 cells, an insect cell line, respectively, to produce viral cultures, which were then simultaneously transfected into an insect sf21 cell line to produce the heterodimeric IL12p70 protein, in which IL12p40 is bound to IL12p35, and then purified.

[0084] The expression vector constructed to express recombinant hIL15-SP protein was transformed into E. coli BL21(DE3)RIPL (Invitrogen) and then incubated. The culture was centrifuged at 10,000 rpm for 10 minutes to obtain a cell pellet. The cell pellet was resuspended in PBS (pH 7.4) and then homogenized by sonication. The SP fusion protein expressed in soluble form in E. coli was purified using ion-exchange resin.

[0085] The expression vector constructed to express the recombinant SP-hIL18 protein was transformed into E. coli BL21(DE3)RIPL (Invitrogen) and then incubated. The culture was centrifuged at 10,000 rpm for 10 minutes to obtain a cell pellet. The cell pellet was resuspended in PBS (pH 7.4) and then homogenized by sonication. The SP fusion protein expressed in soluble form in E. coli was purified using ion-exchange resin.

[0086] The purified SP fusion proteins (3 μg) were electrophoresed using 15% SDS-PAGE to confirm the final purified proteins (Figure 2: (a) SP-hIL2 protein (ATGen, Cat# ATGK04), (b) IL15-SP protein (ATGen, Cat# ATGK06) and (c) SP-IL18 protein (ATGen, Cat# ATGK07)).

[0087] Experimental Example 1 : Identification of cytokines capable of activating NK cells in whole blood 1 ml of whole blood from normal subjects and 1 ml of RPMI1640 medium were placed in a 24-well cell culture plate, mixed with 10 ng / ml of each recombinant human interleukin IL-2, IL-12, IL-15 and IL-18, and then cultured for 24 hours. After 24 hours of culture, the supernatant was collected and the amount of interferon-γ in the supernatant was measured using a sandwich ELISA method (Figure 3A). As a result, the cytokines secreted by NK cells in the blood samples of normal subjects were not detected because they were in trace amounts, but when the blood samples were treated with at least one of IL-2, IL-12, IL-15 and IL-18, the levels of cytokines secreted by NK cells in the blood samples increased. It was found that when the blood samples were treated with the NK cell stimulator alone, the levels of interferon-γ in the blood samples increased, especially in the IL-2-treated group and the IL-12-treated group (Figure 3A).

[0088] Also, 1 ml of whole blood from normal subjects and 1 ml of RPMI1640 medium were placed in a 24-well cell culture plate, treated with various combinations of recombinant human interleukins as shown in Figure 3B (10 ng / ml each), and cultured for 24 hours. After 24 hours of culture, the supernatants were collected and the levels of interferon-γ were measured in the same manner as above. It was found that when whole blood was treated with various combinations of NK cell stimulators, the levels of interferon-γ increased, especially in the presence of IL-2+IL-12 (Figure 3B).

[0089] Furthermore, to measure the levels of interferon-γ after treatment with a combination of IL-12 and IL-15, whole blood was treated with NK cell stimulators at the concentrations shown in Figure 3C and cultured for 24 hours. After 24 hours of culture, the supernatants were harvested and the levels of interferon-γ were measured in the same manner as above.

[0090] To measure the levels of interferon-γ after combined IL-12 and IL-18 treatment, whole blood was also treated with NK cell stimulators at the concentrations shown in Figure 3D and cultured for 24 hours. After 24 hours of culture, the supernatants were harvested and the levels of interferon-γ were measured in the same manner as above.

[0091] Experimental Example 2 : Identification of the types of cytokines secreted by NK cells artificially activated with IL-2 Whole blood samples were obtained from 61 normal subjects and 50 cancer patients. 1 ml of whole blood and 1 ml of RPMI1640 medium were placed in a 24-well cell culture plate, treated with 10 ng / ml recombinant human interleukin SP IL-2, and then cultured for 24 hours. After 24 hours of culture, the supernatant was collected, and then the levels of interferon-γ, TNF-α, and MIP-1β were measured using a sandwich ELISA method. As a result, as shown in Figure 4, it was confirmed that interferon-γ and TNF-α were secreted in smaller amounts from whole blood samples from normal subjects than from cancer patients, while MIP-1β was secreted from whole blood samples from normal subjects and cancer patients.

[0092] For in vitro diagnostic reagents used for disease testing, various validation techniques are used. Here, normal range and cut-off analysis are generally used. Normal range is a reference range used to measure the mean and standard deviation of each sample group, and cut-off analysis is a method to measure clinical sensitivity and clinical specificity by calculating the estimated value of in vitro diagnostic reagent. Clinical sensitivity means the probability that the result of the diagnostic test will be demonstrated to be positive if the patient has the disease, and clinical specificity means the probability that the result of the diagnostic test will be demonstrated to be negative if the patient does not have the disease.

[0093] It is assumed that the cutoff values ​​are set to positive and negative values ​​when the cutoff values ​​are greater than 10% and less than 10%, respectively. In that case, the clinical sensitivity and clinical specificity were measured using the cutoff analysis. The results are shown in Table 1. [Table 1]

[0094] In a group of cancer patients and normal subjects, IFN-γ was measured to have a sensitivity of 98.4% and a specificity of 98%. TNF-α was measured to have a sensitivity of 90.9% and a specificity of 69%, which was lower than that of IFN-γ, but the latest developed cancer diagnostic kits have a specificity of at most 20-30%. Therefore, it is expected that TNF-α, which has a specificity of about 70% or more, can also be used as a marker for cancer diagnostic kits to measure NK cell activity.

[0095] Experimental Example 3 Comparison of stability of SP IL-2 and IL-2 To compare the stability of SP IL-2 and IL-2, whole blood samples were collected from two individuals. 1 ml of each of the obtained whole blood samples and 1 ml of RPMI1640 medium were placed in a 24-well cell culture plate, and then SP IL-2 and IL-2 were added, mixed thoroughly, and then cultured for 24 hours. After 24 hours of culture, the supernatant was collected, and the level of interferon-γ in the supernatant was measured using a sandwich ELISA method. The results of the activity analysis of IL-2 and SP IL-2 showed that there was no difference in the activity of the two proteins (Figure 5A). However, when whole blood was treated with SP IL-2 but not with IL-2 under each whole blood culture condition, it was confirmed that NK cells were activated by SP IL-2, thereby increasing the level of interferon-γ (Figure 5B). This indicates that there is no difference in the activity of the two proteins, but the application of SP increases the stability of IL-2.

[0096] Experimental Example 4 Comparison of NK cell activity from normal subjects and cancer patients depending on conditions for stimulating NK cells 1 ml of whole blood samples from 20 normal subjects and 48 terminal cancer patients (stages 3-4) were placed in 24-well cell culture plates with 1 ml of RPMI1640 medium, and each sample was divided into two subgroups, which were treated with SP IL-2 (10 ng / ml) (condition A) and SP IL-2 (5 ng / ml) + IL-12 (5 ng / ml) (condition B), respectively, and then cultured for 24 hours. After culture, the supernatants were collected and the levels of interferon-γ were measured using a sandwich ELISA method.

[0097] As a result, in the case of condition A, as shown in FIG. 6, it was found that about 90% of normal subjects had high interferon-γ levels, but the majority of cancer patients had low interferon-γ levels. In the case of condition B, it was also found that normal subjects had high interferon-γ levels, but the majority of cancer patients had low interferon-γ levels. However, compared with the case of condition A, the high interferon-γ levels in the case of condition B were higher in cancer patients. When whole blood samples are treated with only SP IL-2, only NK cells are specifically activated (see Experimental Example 5 and FIG. 7 below), but when whole blood samples are treated with a combination of SP IL-2 and IL-12, NK cells appear to be activated together with T cells. Therefore, it seems that the level of interferon-γ is increased by the activation of T cells. Therefore, it is considered that high interferon-γ levels may be observed in some cancer patients who still have T cell activity. If cancer patients have low interferon-γ levels even when treated with condition B, it can be inferred that the anti-cancer immunity of NK cells and general systemic immunity are decreased in cancer patients, which could be used as an important marker for determining the progression and prognosis of cancer.

[0098] Experimental Example 5 : Comparison of NK cell activity between normal subjects and cancer patients according to the type of blood sample To determine the difference in the interferon-γ secretion ability by IL2 depending on the type of blood sample from normal subjects, the following experiment was performed. (a) The interferon-γ secretion ability of NK cells to IL2 1 ng / ml from T cells, (b) the interferon-γ secretion ability of NK cells to IL2 1 ng / ml from NK cells, (c) the interferon-γ secretion ability of NK cells to IL2 1 ng / ml from whole blood, and (d) the interferon-γ secretion ability of NK cells depending on the IL2 concentration from PBMCs were measured. The results are shown in Figure 7. Interferon-γ was measured in the same manner as above. As a result, the amount of interferon-γ secreted by IL2 activation in T cells changed, but was not significantly different from that of the untreated group, so T cells were not suitable for use as a blood sample. There was a significant difference in the amount of interferon-γ in whole blood, PBMCs, and NK cells compared to the amount of interferon-γ in the untreated group. Therefore, whole blood, PBMCs and NK cells were evaluated as suitable blood samples for application in the method and kit according to the present invention.

[0099] Experimental Example 6 : Comparison of NK cell activity from healthy subjects induced by LPS As another example of an agent that serves to stimulate NK cells in a blood sample and artificially activate NK cells to produce interferon-γ, LPS was used to measure the amount of interferon-γ from human whole blood. As shown in Figure 8, it was revealed that the secretion of interferon-γ was induced by 50 ng / ml LPS, indicating that NK cells can be artificially activated to produce interferon-γ even when they are stimulated with a non-specific agonist such as LPS.

[0100] Experimental Example 7 Stimulation of NK cells by hIL12 and hIL15 fused with stabilized peptides As a tube for incubating NK cells, a tube containing sodium heparin, an anticoagulant (BD), was purchased and used to prevent blood clotting. 5 ml of whole blood was collected and placed in a tube containing an anticoagulant (sodium heparin). 1 ml of the obtained whole blood was mixed with RPMI1640 medium, and SP-hIL2 / hIL12, an NK cell activator, was added to it. The obtained mixture was incubated at 37°C for 16 to 24 hours. The stimulation of NK cells in whole blood by SPhIL2 and hIL12 fused with stabilized peptides was determined by measuring the amount of interferon-γ in the blood incubated according to the method described in the above experimental example.

[0101] We also measured the amount of interferon-γ secreted depending on the culture conditions of whole blood. As shown in Figure 9, when NK cells were incubated in PBS containing a carrier protein such as bovine serum albumin, the ability of NK cells to secrete interferon-γ was increased compared to when NK cells were incubated in PBS.

[0102] Experimental Example 8 : Differences in interferon-γ secretion depending on the stage of cancer progression To determine the amount of secreted interferon-γ according to the stage of cancer progression, whole blood from cancer patient 1 (a patient who had fully recovered from breast cancer), cancer patient 2 (a patient suspected of having a brain tumor), and a normal subject was incubated in RPMI1640 medium supplemented with IL12 100ng / ml and IL15 1000ng / ml for 24 hours, and the amount of secreted interferon-γ was measured as described above. Whole blood was also subjected to flow cytometry.

[0103] As a result, as shown in Figure 10, the ability to secrete interferon-γ was confirmed in the order of normal subjects, cancer patient 1, and cancer patient 2. Therefore, it was confirmed that the amount of secreted interferon-γ differs depending on the stage of cancer progression. From these facts, it was found that the method according to the present invention can be used to measure the amount of interferon-γ secreted by NK cells in a blood sample, and thereby predict the onset and stage of cancer, or predict the recurrence of cancer.

[0104] Experimental Example 9 Quantification of interferon-γ produced by NK cell stimulation As a tube for incubating NK cells, a tube containing sodium heparin, an anticoagulant, was purchased (BD) and used to prevent blood clotting. 5 ml of whole blood was collected from eight normal subjects and placed in a tube containing an anticoagulant (sodium heparin). 1 ml of the obtained whole blood was mixed with RPMI1640 medium, and SP-hIL12 / hIL15-SP bound to a stabilizing peptide was added to it. The obtained mixture was incubated at 37°C for 16 to 24 hours.

[0105] Whole blood from eight normal subjects incubated at 37°C was centrifuged at 1500-2000g to obtain serum as the supernatant. Then, 150-200μl of serum was collected and subjected to interferon-γ ELISA. 0.05% Tween primary antibody (anti-human interferon-γ monoclonal antibody, ATGen, Cat# ATGK02) was diluted in a coating buffer (0.1 sodium carbonate, pH 9.5) at a ratio of 1:1000. The diluted primary antibody was divided into 96-well microtiter ELISA plates (Nunc Maxisorp, NUNC, Naperville, IL) in a volume of 100μl / well and maintained at 4°C for 16-18 hours. After that, the solution in the plate was removed and the plate was washed with washing solution (PBS containing 0.05% Tween20) in a volume of 400μl / well. In this case, washing was performed three times. Then, PBS containing 10% fetal bovine serum (FBS) was divided into portions at 300 μl / well and kept at room temperature for 1 hour. After that, the solution in the plate was removed, and the plate was washed with PBST (PBS solution containing 0.05% Tween 20) at 400 μl / well. In this case, washing was performed three times. The 96-well microtiter ELISA plate coated with the primary antibody was sealed and stored at 4° C. for use.

[0106] Interferon-γ standard solution (PBS containing 200ng of recombinant human interferon-γ (ATGen, Cat# IFG4001) and 0.05% Proclin300) was diluted and dispensed into a 96-well microtiter ELISA plate coated with primary antibody in a volume of 100μl / well, and the patient serum prepared in the experimental phase was dispensed in a volume of 100μl / well, and then kept at room temperature for 2 hours. [Table 2]

[0107] After 2 hours, the solution in the 96-well microtiter ELISA plate was removed, and the plate was washed with 400 μl / well of washing solution. In this case, washing was performed three times. Then, the secondary antibody (biotinylated anti-human interferon-γ monoclonal antibody (ATGen Cat# ATGK03)) was diluted in the dilution solution at a ratio of 1:500, divided in a volume of 100 μl / well, and then kept at room temperature for 1 hour. Then, the solution in the plate was removed, and the plate was washed three times with 400 μl / well of washing solution. The HRP-conjugated streptavidin solution (Thermo Scientific, Cat# 21130) was diluted in the dilution solution at a ratio of 1:3000, divided in a volume of 100 μl / well, and then kept at room temperature for 30 minutes. Then, the diluted HRP-conjugated streptavidin solution was divided into the ELISA plate and incubated for 1 hour. After 1 hour of incubation, the solution in the 96-well microtiter ELISA plate was removed and the plate was washed three times with a washing solution in an amount of 400 μl / well.

[0108] 1 mg of tetramethylbenzidine (TMB) was dissolved in 1 ml of dimethyl sulfoxide (DMSO), and the resulting mixture was diluted with 9 ml of 0.05 M citrate phosphate buffer to prepare a substrate solution. The substrate solution was then divided into plates at a volume of 100 μl / well and kept at room temperature for 30 minutes.

[0109] The reaction was stopped by adding a reaction stop solution (2N dilute sulfuric acid solution) in an amount of 100 μl / well, and the resulting reaction solution was measured at 450 nm using an ELISA reader.

[0110] The interferon-γ secretion ability of NK cells measured using whole blood from eight normal subjects is shown in Figure 11. These results indicate that when whole blood is stimulated with cytokines, the immune cells present in the blood are effectively activated to induce the secretion of interferon-γ.

[0111] In addition, whole blood from eight normal subjects was stimulated with cytokines and then subjected to flow cytometry. The results are shown in Figure 12. These results revealed that NK cells exhibited cytotoxicity when activated by whole blood stimulation. CD56 is a marker for NK cells, and CD107a is a marker indicating that NK cells secrete cytotoxic granules. The interferon-γ secretion results in Figure 11 significantly correlated with the cytotoxicity results by NK cells in Figure 12, indicating that the interferon-γ secretion ability of NK cells stimulated with whole blood indirectly represents the cytotoxicity of NK cells.

[0112] According to the present invention, the onset or recurrence of cancer can be diagnosed, for example, by monitoring changes in the in vivo immune system in a subject suffering from or suspected of having cancer and measuring NK cell activity in blood. Therefore, the present invention can be useful for predicting the onset or recurrence of cancer using a blood sample from a subject.

[0113] Although exemplary embodiments have been disclosed herein, it should be understood that other variations are possible. Such variations should not be regarded as a departure from the scope of the exemplary embodiments of the invention, and all such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

[0114] All documents cited herein are hereby incorporated by reference.

Claims

1. A method for measuring natural killer (NK) cell activity to aid in the diagnosis of cancer onset or recurrence, comprising: stimulating NK cells in the whole blood sample by incubating the whole blood sample with an agent including interleukin 12 and interleukin 15, thereby artificially activating the NK cells to produce and secrete NK cell-secreted cytokines; and measuring the amount of NK cell secreted cytokines secreted into a whole blood sample and using said amount as a measure for assessing NK cell activity; Including, NK cell-secreted cytokines include interferon gamma (IFN-γ); The method.

2. The method of claim 1, wherein the measurement of the amount of NK cell secreted cytokines is performed by enzyme-linked immunosorbent assay (ELISA) or other immunological analysis.

3. Interleukin-12 and / or interleukin-15 are in the form of a fusion protein with a stabilizing peptide, the stabilizing peptide is a C-terminal acidic tail domain peptide of the synuclein family; the stabilizing peptide comprises amino acid residues 103-115 (SEQ ID NO:22), amino acid residues 114-126 (SEQ ID NO:23), amino acid residues 119-140 (SEQ ID NO:24) or amino acid residues 130-140 (SEQ ID NO:25) of the C-terminal acidic tail domain of alpha-synuclein, amino acid residues 85-134 of the C-terminal acidic tail domain of beta-synuclein (SEQ ID NO:27), amino acid residues 1-127 of gamma-synuclein (SEQ ID NO:28), or amino acid residues 96-127 of the C-terminal acidic tail domain of gamma-synuclein (SEQ ID NO:29); The method of claim 1.

4. 2. The method of claim 1, wherein the step of stimulating NK cells in a whole blood sample, thereby artificially activating the NK cells to produce and secrete NK cell-secreted cytokines, is performed in a solution containing a carrier protein.

5. A decrease in the amount of NK cell secreted cytokines in a subject compared to levels in a normal individual is an indication of cancer onset or recurrence. The method of claim 1.

6. The interleukin-15 is in the form of a fusion protein with a stabilizing peptide, The fusion protein comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:8; The method of claim 1.

7. 2. The method according to claim 1, wherein the stimulation of NK cells is carried out by incubating the whole blood sample with interleukin 12 or interleukin 12 in the form of a fusion protein with a stabilizing peptide, and interleukin 15 or interleukin 15 in the form of a fusion protein with a stabilizing peptide.

8. 10. The method of claim 1, wherein NK cell activity is monitored for an increase or decrease in NK cell activity over time.

9. 2. The method of claim 1, wherein measuring NK cell activity comprises comparing a measured amount of NK cell secreted cytokines secreted in a whole blood sample from the subject with a measured amount of NK cell secreted cytokines secreted in a whole blood sample from a normal individual.

10. The method of claim 1 , wherein the whole blood sample is a human whole blood sample.

11. The method of claim 1, wherein the NK cells in the whole blood sample are in an inactivated state prior to the stimulation step.

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