Method for detecting effectiveness of immunity and medical use of lymphocytes or natural killer cells

Abstract

Some embodiments of the present disclosure provide a method for detecting immunity efficacy.SOLUTION: The method for detecting the effectiveness of immunity comprises: calculating the percentage of the number of each T cell subset in lymphocytes and the percentage of the number of each NK cell subset in lymphocytes to obtain a plurality of actual percentages of T cell subsets and a plurality of actual percentages of NK cell subsets; and determining whether the effectiveness of immunity is normal according to the actual percentages of T cell subsets and the actual percentages of NK cell subsets, so as to provide a more comprehensive test for the functions of various aspects of the immune system. Some embodiments of the present disclosure further provide use of a plurality of lymphocytes in the preparation of a pharmaceutical composition for treating a cancer in an individual, wherein the actual total number of T cells or the actual total number of natural killer cells is controlled within a theoretical range, thereby improving the efficacy of treating the cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a method and system for detecting the effectiveness of immunity, and the use of lymphocytes or natural killer cells for preparing a pharmaceutical composition for treating cancer in an individual. [Background technology]

[0002] Human blood accounts for approximately one-thirteenth of body weight (i.e., a 65 kg body has approximately 5 liters of blood, with lymphocytes accounting for approximately 3.5 to 10 billion per liter). Different lymphocytes have different activities and functions and work together to resist foreign pathogens or internal lesions (e.g., cancer) and maintain physiological balance within the body. However, traditional analytical indicators for evaluating immune effectiveness are limited and cannot comprehensively indicate the status of the immune system.

[0003] Currently, clinically, the choice of cell number in immune cell therapy is generally determined by the physician's experience and the type of cell. However, due to the large differences in immune efficacy between different human bodies, the efficacy of immune cell therapy varies greatly, which also poses a bottleneck in the application of existing immune cell therapies in clinical treatment. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, it is necessary to provide a more complete method for detecting the effectiveness of immunity and to control the immunity of different human individuals to the same degree before immunotherapy. [Means for solving the problem]

[0005] In some embodiments of the present disclosure, the method includes the steps of providing an in vitro sample from an individual comprising a plurality of lymphocyte cells, including a plurality of T cells and a plurality of natural killer cells; classifying the T cells into a plurality of T cell subsets according to the presence or absence of expression of a plurality of first antigens, including CD28; and classifying the natural killer cells into a plurality of natural killer cell subsets according to the presence or absence of expression of a plurality of second antigens, including CD16; calculating the percentage of the number of each of the T cell subsets among the lymphocyte cells and the percentage of the number of each of the natural killer cell subsets among the lymphocyte cells, to determine the actual percentage of the plurality of T cell subsets and the plurality of natural killer cell subsets. and determining whether the actual percentages of each T cell subset meet the reference range for the T cell subset, and whether the actual percentages of each natural killer cell subset meet the reference range for the natural killer cell subset; and determining that the individual's immune efficacy is normal if the actual percentages of all of the T cell subsets meet the reference range for the T cell subset and the actual percentages of all of the natural killer cell subsets meet the reference range for the natural killer cell subset.

[0006] In some embodiments, the T cell subsets include early helper T cells, senescent helper T cells, regulatory helper T cells, early killer T cells, senescent killer T cells, or a combination thereof, wherein early helper T cells are CD3+, CD4+, CD45RA+, CD62L+, and CD28+, senescent helper T cells are CD3+, CD4+, CD45RO+, and CD62L+, regulatory helper T cells are CD3+, CD4+, CD25+, FoxP3+, and CD39+, early killer T cells are CD8+, CD27+, CD45RA+, CD62L+, and CD127+, and senescent killer T cells are CD8+, CD27+, CD45RO+, CD62L+, and CD57+.

[0007] In some embodiments, the reference range for T cell subsets includes a reference range for early helper T cells, and the reference range for early helper T cells is between 20% and 100%.

[0008] In some embodiments, the reference range for a T cell subset includes a reference range for regulatory helper T cells, and the reference range for regulatory helper T cells is between 1% and 15%.

[0009] In some embodiments, the reference range for the T cell subset includes a reference range for early killer T cells, and the reference range for early killer T cells is between 15% and 100%.

[0010] In some embodiments, the reference range for a T cell subset includes a reference range for senescent killer T cells, and the reference range for senescent killer T cells is 0% to 50%.

[0011] In some embodiments, the reference ranges for T cell subsets are derived from physiological values ​​of a group of healthy individuals, which are human individuals aged 5 to 85 years who are not affected by immunodeficiency syndromes or hyperimmune syndromes.

[0012] In some embodiments, the method further comprises calculating the percentage of the number of CD28 positive cells in the initial helper T cells, the senescent helper T cells, and the regulatory helper T cells, where the sum of the percentages of the number of initial helper T cells, the senescent helper T cells, and the regulatory helper T cells is 100%, to obtain an actual percentage of CD28 positive cells in the helper T cells; and determining whether the actual percentage of CD28 positive cells in the helper T cells meets the reference range for CD28 positive cells in the helper T cells.

[0013] In some embodiments, the reference range for CD28 positivity in helper T cells is 80% to 100%.

[0014] In some embodiments, the reference range for CD28 positivity in helper T cells is derived from physiological values ​​in a group of healthy individuals, which are human individuals aged 5 to 85 years who are not affected by immunodeficiency syndrome or hyperimmune syndrome.

[0015] In some embodiments, the natural killer cell subsets include killer natural killer cells and regulatory natural killer cells, wherein the killer natural killer cells are CD16+, CD34+, CD56+, CD94+, and CD117+, and the regulatory natural killer cells are CD34+, CD56+, CD94+, and CD117+.

[0016] In some embodiments, the reference range for natural killer cell subsets includes a reference range for regulatory natural killer cells, wherein the reference range for regulatory natural killer cells is 0% to 20%.

[0017] In some embodiments, the reference range for regulatory natural killer cells is derived from physiological values ​​in a group of healthy individuals, the healthy individuals being human individuals aged 5 to 85 years who are not affected by immunodeficiency syndromes or hyperimmune syndromes.

[0018] In some embodiments, the method further includes measuring the actual value of the natural killer cell killing ability of the natural killer cells by co-culturing the natural killer cells with a plurality of cancer cells at a specific ratio and calculating the percentage of the dead number of the cancer cells to obtain the actual value of the natural killer cell killing ability of the natural killer cells; and determining whether the actual value of the natural killer cell killing ability meets the reference range of the natural killer cell killing ability, where when the number ratio is 6.25, the reference range of the natural killer cell killing ability is 0% to 58.8%, when the number ratio is 12.5, the reference range of the natural killer cell killing ability is 1.7% to 88.7%, when the number ratio is 25, the reference range of the natural killer cell killing ability is 17.4% to 100%, and when the number ratio is 50, the reference range of the natural killer cell killing ability is 35.3% to 100%.

[0019] In some embodiments, the method further comprises the steps of cryopreserving each T cell subset if the actual percentage of each T cell subset meets the reference range for the T cell subset, and cryopreserving each natural killer cell subset if the actual percentage of each natural killer cell subset meets the reference range for the natural killer cell subset.

[0020] In some embodiments, the step of cryopreserving each T cell subset or each natural killer cell subset comprises a step of sorting the T cell subset or natural killer cell subset by immunobead cell sorting, and a step of cryopreserving the T cell subset or natural killer cell subset.

[0021] In some embodiments according to the present disclosure, there is provided a system for detecting the effectiveness of immunity, the system including a processor and a memory, wherein the memory stores a plurality of computer program instructions, which, when executed by the processor, cause the processor to perform the following steps: accessing in vitro sample data including antigen data on the surface of a plurality of sample cells from the individual; using a cell subset database including classification information of T cell subsets and classification information of natural killer cell subsets to generate count data of a plurality of T cell subsets and count data of a plurality of natural killer cell subsets based on the in vitro sample data, wherein the classification information of the T cell subsets includes a classification index according to the presence or absence of expression of a plurality of first antigens including CD28, and the classification information of the natural killer cell subsets includes a classification index according to the presence or absence of expression of a plurality of second antigens including CD16; and using an immunity effectiveness evaluation database including reference range data of a plurality of T cell subsets and reference range data of a plurality of natural killer cell subsets to generate immune effectiveness data based on the count data of the T cell subsets and the count data of the natural killer cell subsets.

[0022] In some embodiments, the first antigen further comprises CD3, CD4, CD8, CD25, CD27, CD39, CD45RO, CD45RA, CD57, CD62L, CD127, FoxP3, or a combination thereof, and the second antigen further comprises CD34, CD56, CD94, CD117, or a combination thereof.

[0023] In some embodiments, the system further includes an output module coupled to the processor for receiving the immunity efficacy data and outputting it as an immunity efficacy report.

[0024] In some embodiments, the immunity efficacy report includes an immunity efficacy field and a plurality of immunity analysis indicator fields.

[0025] Some embodiments of the present disclosure relate to the use of a plurality of lymphocytes in the preparation of a pharmaceutical composition for treating cancer in an individual, the lymphocytes comprising a plurality of T cells, a plurality of natural killer cells, or a combination thereof, and the treatment includes the steps of: (a) generating a deviation value of the total number of T cells between the actual total number of T cells and a theoretical range of the total number of T cells, and generating a deviation value of the total number of natural killer cells between the actual total number of natural killer cells and the theoretical range of the total number of natural killer cells; and (b) administering the pharmaceutical composition comprising the T cells to the individual so that the actual total number of T cells of the individual satisfies the theoretical range of the total number of T cells based on the deviation value of the total number of T cells, thereby increasing the total number of natural killer cells. and administering to the individual a pharmaceutical composition comprising said natural killer cells so that the actual total number of natural killer cells in the individual satisfies the theoretical range of the total number of natural killer cells based on the number deviation value.

[0026] In some embodiments, the theoretical ranges of the total number of T cells and the theoretical ranges of the total number of natural killer cells are derived from physiological values ​​of a group of healthy individuals, which are human individuals aged 5 to 85 years who are not affected by immunodeficiency syndrome or immunohypersensitivity syndrome.

[0027] In some embodiments, the theoretical range for the total number of T cells is 700 cells / microliter to 2500 cells / microliter, and the theoretical range for the total number of natural killer cells is 100 cells / microliter to 300 cells / microliter.

[0028] In some embodiments, the treatment includes the steps of: (a) classifying the lymphocyte cells into a plurality of T cell subsets according to the presence or absence of expression of a plurality of first antigens; and (b) classifying the lymphocyte cells into a plurality of natural killer cell subsets according to the presence or absence of expression of a plurality of second antigens; (c) generating deviation values ​​for T cell subsets between actual percentages of T cell subsets and theoretical ranges of T cell subsets; and (d) generating deviation values ​​for natural killer cell subsets between actual percentages of natural killer cell subsets and theoretical ranges of natural killer cell subsets; wherein the actual percentages of T cell subsets are a function of the number of each T cell subset in the lymphocyte cells. and (c) administering to the individual a pharmaceutical composition comprising a T cell subset such that the actual percentages of the individual's T cell subsets satisfy the theoretical range of T cell subsets based on the deviation values ​​of the T cell subsets, and administering to the individual a pharmaceutical composition comprising a natural killer cell subset such that the actual percentages of the individual's natural killer cell subsets satisfy the theoretical range of natural killer cell subsets based on the deviation values ​​of the natural killer cell subsets.

[0029] In some embodiments, the theoretical ranges of T cell subsets and the theoretical ranges of natural killer cell subsets are derived from physiological values ​​of a group of healthy individuals, which are human individuals aged 5 to 85 years who are not affected by immunodeficiency syndrome or immunohyperactivity syndrome.

[0030] In some embodiments, the T cell subsets include early helper T cells, regulatory helper T cells, early killer T cells, or a combination thereof, wherein early helper T cells are CD3+, CD4+, CD45RA+, CD62L+, and CD28+, regulatory helper T cells are CD3+, CD4+, CD25+, FoxP3+, and CD39+, and early killer T cells are CD8+, CD27+, CD45RA+, CD62L+, and CD127+.

[0031] In some embodiments, the theoretical range of T cell subsets includes a theoretical range of early helper T cells, a theoretical range of regulatory helper T cells, a theoretical range of early killer T cells, or a combination thereof, wherein the theoretical range of early helper T cells is 35% to 100%, the theoretical range of regulatory helper T cells is 3% to 10%, and the theoretical range of early killer T cells is 20% to 100%.

[0032] In some embodiments, the natural killer cell subsets include regulatory natural killer cells, which are CD34+, CD56+, CD94+, and CD117+.

[0033] In some embodiments, the theoretical range of natural killer cell subsets includes a theoretical range of regulatory natural killer cells that is 0% to 10%.

[0034] In some embodiments, the treatment includes the steps of: (a) classifying the lymphocyte cells into a plurality of T cell subsets according to the presence or absence of expression of a plurality of first antigens, the T cell subsets comprising a first T cell subset and a second T cell subset; (b) generating a deviation value of the T cell subset proportion between the actual value of the proportion of the two T cell subsets and a theoretical range of the proportion of the two T cell subsets, the actual value of the proportion of the two T cell subsets being the ratio of the numbers between the first T cell subset and the second T cell subset; and (c) administering to the individual the pharmaceutical composition comprising the first T cell subset or the second T cell subset based on the deviation value of the T cell subset proportions so that the actual value of the individual's two T cell subset proportions falls within the theoretical range of the proportion of the two T cell subsets.

[0035] In some embodiments, the theoretical ranges of the proportions of the two T cell subsets are derived from physiological values ​​of a group of healthy individuals, which are human individuals aged 5 to 85 years who are not affected by immunodeficiency syndrome or immunohypersensitivity syndrome.

[0036] In some embodiments, when the first T cell subset is CD4-positive T cells and the second T cell subset is CD8-positive T cells, the theoretical ratio of the two T cell subsets is 1:1 to 5:1.

[0037] In some embodiments, the treatment further comprises administering immune cell therapy to the individual after administering to the individual a pharmaceutical composition comprising the T cells or a pharmaceutical composition comprising the natural killer cells.

[0038] In some embodiments, the immune cell therapy comprises natural killer cell therapy, cytokine-induced killer cell immunotherapy, γδ T cell therapy, dendritic cell therapy, tumor-infiltrating lymphocyte therapy, chimeric antigen receptor T cell therapy, or a combination thereof.

[0039] In some embodiments, the lymphocytes are derived from an individual and are obtained by in vitro culture.

[0040] In some embodiments, the cancer comprises colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer, hepatocellular carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, basal cell carcinoma, cutaneous squamous cell carcinoma, bile duct carcinoma, Merkel cell carcinoma, or a combination thereof.

[0041] Some embodiments of the present disclosure provide a use of a plurality of natural killer cells for preparing a pharmaceutical composition for treating cancer in an individual, the treatment comprising: (a) measuring the actual value of the killing ability of the natural killer cells, obtained by co-culturing the natural killer cells with a plurality of cancer cells at a certain ratio and generating a percentage of the number of dead cancer cells to obtain the actual value of the killing ability of the natural killer cells; (b) generating a deviation value of the killing ability of the natural killer cells between the actual value of the killing ability of the natural killer cells and the theoretical range of the killing ability of the natural killer cells, based on the actual value of the killing ability of the natural killer cells and the theoretical range of the killing ability of the natural killer cells; and (c) administering a pharmaceutical composition comprising the natural killer cells to the individual so that the actual value of the killing ability of the individual satisfies the theoretical range of the killing ability of the natural killer cells, based on the deviation value of the killing ability of the natural killer cells.

[0042] In some embodiments, when the number ratio is 6.25, the theoretical range of natural killer cell killing capacity is 0% to 58.8%; when the number ratio is 12.5, the theoretical range of natural killer cell killing capacity is 1.7% to 88.7%; when the number ratio is 25, the theoretical range of natural killer cell killing capacity is 17.4% to 100%; and when the number ratio is 50, the theoretical range of natural killer cell killing capacity is 35.3% to 100%.

[0043] In some embodiments, in step (a), when the number ratio is 50 and leukocyte interleukin-2 is simultaneously added at an effective concentration of 100 international units / milliliter and co-cultured with the natural killer cells and the cancer cells, the theoretical range of the killing ability of the natural killer cells is 57.4% to 100%.

[0044] In some embodiments, the treatment includes the steps of generating a deviation value of the total number of natural killer cells between the actual total number of natural killer cells and the theoretical range of the total number of natural killer cells, and administering a pharmaceutical composition containing the natural killer cells to the individual based on the deviation value of the killing ability of natural killer cells and the deviation value of the total number of natural killer cells so that the actual total number of the individual's natural killer cells satisfies the theoretical range of the total number of natural killer cells, thereby causing the actual value of the killing ability of the individual's natural killer cells to satisfy the theoretical range of the killing ability of natural killer cells.

[0045] In some embodiments, the treatment further includes the steps of: classifying the natural killer cells into a plurality of natural killer cell subsets according to the presence or absence of expression of a plurality of specific antigens, the natural killer cell subsets including regulatory natural killer cells, the regulatory natural killer cells being CD34+, CD56+, CD94+, and CD117+; generating a deviation value of regulatory natural killer cells between the actual percentage of regulatory natural killer cells and the theoretical range of regulatory natural killer cells, the actual percentage of regulatory natural killer cells being the percentage of the number of regulatory natural killer cells among lymphocytes; and administering a pharmaceutical composition containing the regulatory natural killer cells to the individual based on the deviation value of the killing ability of natural killer cells and the deviation value of regulatory natural killer cells, so that the actual percentage of the individual's regulatory natural killer cells satisfies the theoretical range of killing ability of regulatory natural killer cells, thereby causing the actual value of the killing ability of the individual's natural killer cells to satisfy the theoretical range of killing ability of natural killer cells. [Brief explanation of the drawings]

[0046] The following description of the drawings will make the above and other objects, features, advantages and embodiments of the present invention more comprehensible. [Figure 1] 1 is a flowchart of a method for detecting the effectiveness of immunity in some embodiments of the present disclosure. [Figure 2] 1 is a flowchart of steps performed by a processor of an immune efficacy detection system in some embodiments of the present disclosure. [Figure 3] FIG. 1 is a schematic diagram of an immune efficacy detection system according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0047] In order to provide a complete and detailed description of the present invention, the following detailed description of the embodiments and specific examples of the present invention will be provided. However, these are not the only ways to implement or apply the specific examples of the present invention. The disclosed examples can be combined or substituted where beneficial, and one example can be added to another without further description or explanation. In the following description, numerous specific details are provided to enable the reader to fully understand the following examples. However, the embodiments of the present invention may be practiced without these specific details.

[0048] In this context, unless otherwise limited, "a," "an," and "the above" generally refer to one or more. As used herein, the terms "comprise," "include," "have," and similar words should be understood to refer to stated features, regions, integers, steps, operations, elements, and / or components, but not to the exclusion of other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0049] In this text, deviation refers to the difference obtained by subtracting two numbers. For example, the deviation of the total number of T cells between the actual total number of T cells and the theoretical range of the total number of T cells is the difference between the actual total number of T cells and the theoretical range of the total number of T cells.

[0050] Although the methods disclosed herein are described below using a series of operations or steps, the order in which the operations or steps are presented should not be construed as limiting the present invention. For example, certain operations or steps may be performed in a different order and / or simultaneously with other steps. It should be noted that not all operations, steps, and / or features need be performed to achieve embodiments of the present invention. Furthermore, each operation or step described herein may include multiple sub-steps or actions.

[0051] See Figure 1. A method 100 for detecting the effectiveness of immunity according to some embodiments of the present disclosure includes steps S110 to S140.

[0052] First, step S110 provides an in vitro sample of an individual, the in vitro sample including a plurality of lymphocyte cells, the lymphocyte cells including T cells and natural killer cells.

[0053] In some embodiments, the in vitro sample is human blood.

[0054] Next, in step S120, lymphocyte cells are classified into a plurality of T cell subsets according to the presence or absence of expression of a plurality of first antigens including CD28, and into a plurality of natural killer cell subsets according to the presence or absence of expression of a plurality of second antigens including CD16.

[0055] In some embodiments, the first antigen further comprises CD3, CD4, CD8, CD25, CD27, CD39, CD45RO, CD45RA, CD57, CD62L, CD127, FoxP3, or a combination thereof. In some embodiments, the second antigen further comprises CD34, CD56, CD94, CD117, or a combination thereof.

[0056] Furthermore, since CD28 is a surface antigen of early helper cells in T cell subsets and CD16 is a surface antigen of killer natural killer cells, step S120 makes it possible to classify at least T cell subsets into early helper cells and non-early helper cells, and natural killer cell subsets into killer natural killer cells and non-killer natural killer cells.

[0057] In some embodiments, sorting the T cells into multiple T cell subsets comprises sorting the T cells into early helper T cells (CD3+CD4+CD45RA+CD62L+CD28+), senescent helper T cells (CD3+CD4+CD45RO+CD62L+), regulatory helper T cells (CD3+CD4+CD25+FoxP3+CD39+), early killer T cells (CD8+CD27+CD45RA+CD62L+CD127+), senescent killer T cells (CD8+CD27+CD45RO+CD62L+CD57+), or a combination thereof.

[0058] In some embodiments, natural killer cell subsets include killer natural killer cells (CD16+CD34+CD56+CD94+CD117+) and regulatory natural killer cells (CD34+CD56+CD94+CD117+).

[0059] Next, step S130 calculates the percentage of the number of each T cell subset among lymphocytes and the percentage of the number of each natural killer cell subset among lymphocytes to obtain actual percentages of the plurality of T cell subsets and actual percentages of the plurality of natural killer cell subsets. In some embodiments, the actual percentages of the plurality of T cell subsets include the actual percentage of early helper T cells, the actual percentage of senescent helper T cells, the actual percentage of regulatory helper T cells, the actual percentage of early killer T cells, the actual percentage of senescent killer T cells, or a combination thereof. In some embodiments, the actual percentages of the plurality of natural killer cell subsets include the actual percentage of killer natural killer cells, the actual percentage of regulatory natural killer cells, or a combination thereof.

[0060] Next, step S140 determines whether the actual percentage of each T cell subset meets the reference range for each T cell subset, and determines whether the actual percentage of each natural killer cell subset meets the reference range for each natural killer cell subset.

[0061] In some embodiments, the reference ranges for T cell subsets and natural killer cell subsets are derived from physiological values ​​of a healthy population, where the healthy population is human individuals aged 5 to 85 years who are free from immunodeficiency syndromes (e.g., acquired immunodeficiency syndromes) and immune hyperactivity syndromes (e.g., autoimmune diseases).

[0062] In some embodiments, the reference range for each T cell subset or the reference range for each natural killer cell subset includes the corresponding acceptable range and theoretical range, i.e., the reference range for a T cell subset includes the acceptable range for the T cell subset and the theoretical range for the T cell subset, and the reference range for a natural killer cell subset includes the acceptable range for the natural killer cell subset and the theoretical range for the natural killer subset.

[0063] If the actual percentages of all T cell subsets all meet the reference ranges for the corresponding T cell subsets and the actual percentages of all natural killer cell subsets all meet the reference ranges for the corresponding natural killer cell subsets (e.g., the actual percentages of all T cell subsets and the actual percentages of all natural killer cell subsets all meet their respective accepted or theoretical ranges), the immunological efficacy is determined to be normal. In some embodiments, the immunological efficacy is further determined to be good if the actual percentages of all T cell subsets all meet the theoretical ranges for the corresponding T cell subsets and the actual percentages of all natural killer cell subsets all meet the theoretical ranges for the corresponding natural killer cell subsets.

[0064] If the actual percentage of any T cell subset does not meet the reference range for the corresponding T cell subset, or if the actual percentage of any natural killer cell subset does not meet the reference range for the natural killer cell subset, the effectiveness of the immune system is determined to be low.

[0065] In some embodiments, the reference ranges for T cell subsets include a reference range for early helper T cells, a reference range for regulatory helper T cells, a reference range for early killer T cells, or a reference range for senescent killer T cells. The reference range for early helper T cells is 20% to 100%, with 20% < acceptable range < 35%, and a theoretical range of 35% to 100%. The reference range for regulatory helper T cells is 1% to 15%, with 1% < acceptable range < 3% and 10% < acceptable range ≤ 15%, and a theoretical range of 3% to 10%. The reference range for early killer T cells is 15% to 100%, with 15% < acceptable range < 20%, and a theoretical range of 20% to 100%. The reference range for senescent killer T cells is 0% to 50%, with 30% < acceptable range ≤ 50%, and a theoretical range of 0% to 30%.

[0066] In some embodiments, the reference ranges for natural killer cell subsets include a reference range for regulatory natural killer cells, which is 0% to 20%, with 10% < acceptable range ≦ 20%, and a theoretical range of 0% to 10%.

[0067] Furthermore, depending on whether the actual percentages of each T cell subset and each natural killer cell subset meet the reference ranges of the corresponding T cell subsets and natural killer cell subsets, the immune system's defense against each disease and the risk of contracting a particular disease are further analyzed.

[0068] For example, if the actual percentages of early helper T cells and early killer T cells are within their respective reference ranges (the reference range for early helper T cells or the reference range for early killer T cells), it indicates good protection against new pathogens. If the actual percentage of early killer T cells is within the reference range for early killer T cells, it indicates good inhibitory ability against cancer. If the actual percentage of regulatory helper T cells is higher than the reference range for regulatory helper T cells, it will excessively inhibit other immune cells, resulting in a high mortality rate in cancer patients. If the actual percentage of senescent killer T cells is higher than the reference range for senescent killer T cells, it indicates aging or dysfunction of the immune system. If the actual percentage of regulatory natural killer cells is higher than the reference range for regulatory natural killer cells, it will excessively inhibit the activity of killer natural killer cells, resulting in a high probability of the individual being infected with chronic viruses.

[0069] In some embodiments, method 100 includes calculating the percentage of the number of CD28 positive cells in the initial helper T cells, the senescent helper T cells, and the regulatory helper T cells (the proportion of CD28 positive (CD28+) cells in the helper T cells, i.e., the percentage of the number of initial helper T cells among the helper T cells), assuming that the sum of the percentages of the number of senescent helper T cells and regulatory helper T cells is 100%, to obtain an actual percentage of CD28 positive cells in the helper T cells; and determining whether the actual percentage of CD28 positive cells in the helper T cells meets the reference range for CD28 positive cells in the helper T cells, thereby determining the risk of an individual suffering from an autoimmune disease.

[0070] In some embodiments, the reference range for CD28 positivity in helper T cells is derived from physiological values ​​in a group of healthy individuals, which are human individuals aged 5 to 85 years who are not affected by immunodeficiency syndrome or hyperimmune syndrome.

[0071] In some embodiments, the reference range for CD28 positivity (CD28+) in helper T cells is greater than 80% to 100%, with 80%≦acceptable range<90%, and the theoretical range is 90% to 100%. Therefore, if the actual percentage of CD28 positivity (CD28+) in helper T cells is less than 80%, there is a high probability of suffering from an autoimmune disease.

[0072] In some embodiments, method 100 includes steps of measuring the actual value of the natural killer cell killing ability of natural killer cells (the actual value of the natural killer cell killing ability is calculated by co-culturing the calculated natural killer cells with cancer cells at a specific ratio and calculating the percentage of cancer cell deaths to obtain the actual value of the natural killer cell killing ability), and determining whether the actual value of the natural killer cell killing ability meets the reference range of the natural killer cell killing ability, whereby if the actual value of the natural killer cell killing ability falls within the reference range of the natural killer cell killing ability, it indicates that the natural killer cell has excellent inhibitory ability against cancer. For example, when the number ratio is 6.25, the reference range for the killing ability of natural killer cells is 0% to 58.8%, when the number ratio is 12.5, the reference range for the killing ability of natural killer cells is 1.7% to 88.7%, when the number ratio is 25, the reference range for the killing ability of natural killer cells is 17.4% to 100%, and when the number ratio is 50, the reference range for the killing ability of natural killer cells is 35.3% to 100%. In some embodiments, the reference range for the killing ability of natural killer cells is derived from physiological values ​​of a healthy group, and the healthy group is human individuals aged 5 to 85 who are not suffering from immunodeficiency syndrome or hyperimmune syndrome.

[0073] In some embodiments, natural killer cells and human immortalized myeloid leukemia cells (K-562 cells) are co-cultured at different ratios, and the rate at which K-562 cells die is measured to evaluate the killing ability of natural killer cells. The ratio of IL-2 to K-562 cells may be 6.25, 12.5, 25, or 50.

[0074] Furthermore, by adding leukocyte interleukin 2 (IL-2), the activity of regulatory natural killer cells (which inhibit the activity of killer natural killer cells) is completely inhibited, and the activity of killer natural killer cells is acquired, allowing further analysis of the maximum activity of natural killer cells.

[0075] In some embodiments, the method 100 further comprises calculating the T cell and natural killer cell content of the in vitro sample (e.g., how many T cells and natural killer cells are present per milliliter of blood) to analyze the overall status of the immune system. For example, a total natural killer cell count (the number of natural killer cells in a whole blood sample per microliter) of less than 50 (less than 50) indicates a high risk of developing cancer.

[0076] In some embodiments, method 100 further comprises the steps of cryopreserving each T cell subset if the actual percentage of each T cell subset meets the reference range for each T cell subset, and cryopreserving each natural killer cell subset if the actual percentage of each natural killer cell subset meets the reference range for each natural killer cell subset.

[0077] In some embodiments, if the actual percentages of the natural killer cell subsets and the actual values ​​of the killing capacity of the natural killer cells are both within the corresponding reference ranges, the natural killer cell subsets (e.g., killer natural killer cells and regulatory natural killer cells) are cryopreserved.

[0078] In some embodiments, T cell subsets or natural killer cell subsets (e.g., T cell subsets such as early helper T cells, senescent helper T cells, regulatory helper T cells, early killer T cells, or senescent killer T cells, or natural killer cell subsets such as killer natural killer cells or regulatory natural killer cells) can be selected using immunobead cell sorting, and the T cell subsets or natural killer cell subsets are cryopreserved. Conventional methods such as immunoprecipitation and centrifugation are prone to damaging cells, making subculture and cryopreservation unsustainable. In contrast, cells isolated by immunobead cell sorting can retain activity even after multiple sorting cycles, making them advantageous for expansion and storage.

[0079] In some embodiments, T cell subsets or natural killer cell subsets can be selected using a positive selection system, a negative selection system, or a combination thereof. In some embodiments, CD3, CD4, CD8, CD25, CD27, CD28, CD39, CD45, CD45RO, CD45RA, CD57, CD62L, CD127, FoxP3, or a combination thereof can be used to select early helper T cells, senescent helper T cells, regulatory helper T cells, early killer T cells, and senescent killer T cells. In some embodiments, CD16, CD34, CD56, CD94, CD117, or a combination thereof can be used to select killer natural killer cells and regulatory natural killer cells.

[0080] Some embodiments of the present disclosure further provide a system for detecting the effectiveness of immunity, the system including a processor and a memory, wherein a plurality of computer program instructions are stored in the memory, and when the computer program instructions are executed by the processor, the processor performs flow 200 including steps S210 to S230, see FIG. 2.

[0081] First, step S210 accesses in vitro sample data that includes antigen data on the surface of a plurality of sample cells from an individual. In some embodiments, the sample cell surface antigen data is analyzed by a flow cytometer.

[0082] Next, see step S220. Using a cell subset database, data on the number of multiple T cell subsets and data on the number of multiple natural killer cell subsets are generated based on the in vitro sample data. The cell subset database includes classification information for T cell subsets and natural killer cell subsets, where the classification information for T cell subsets includes classification indexes based on the presence or absence of expression of multiple first antigens including CD28, and the classification information for natural killer cell subsets includes classification indexes based on the presence or absence of expression of multiple second antigens including CD16.

[0083] In some embodiments, the first antigen further comprises CD3, CD4, CD8, CD25, CD27, CD39, CD45RO, CD45RA, CD57, CD62L, CD127, FoxP3, or a combination thereof, and the second antigen further comprises CD34, CD56, CD94, CD117, or a combination thereof.

[0084] In some embodiments, the T cell subset classification information includes antigen expression information for each T cell subset, for example, primary helper T cells are CD3+CD4+CD45RA+CD62L+CD28+, and natural killer cell subset classification information includes antigen expression information for each natural killer cell subset, for example, regulatory natural killer cells are CD34+CD56+CD94+CD117+.

[0085] In some embodiments, the T cell subset count data and the natural killer cell subset count data include the percentage of each T cell subset or each natural killer cell subset count among lymphocytes, e.g., the percentage of early helper T cells among lymphocytes.

[0086] Next, see step S230. Using an immunity effectiveness assessment database, immunity effectiveness data is generated based on the data on the number of T cell subsets and the data on the number of natural killer cell subsets. The immunity effectiveness assessment database includes reference range data for multiple T cell subsets (e.g., including the reference ranges for T cell subsets set forth in method 100 above) and reference range data for multiple natural killer cell subsets (e.g., the reference ranges for natural killer cell subsets set forth in method 100 above).

[0087] In some embodiments, the system further includes an output module connected to the processor that receives the immune efficacy data and outputs it as an immune efficacy report (e.g., displayed on a display screen) to evaluate the immune efficacy of the individual and determine whether to cryopreserve the cells, and the immune efficacy report includes an immune efficacy field (describes the degree of immune efficacy) and multiple immune analysis index fields (including the proportions of each cell subset).

[0088] In some embodiments, the reference range data for each T cell subset or the reference range data for each natural killer cell subset includes corresponding acceptable range data and theoretical range data, i.e., the reference range data for the T cell subset includes acceptable range data for the T cell subset and theoretical range data for the T cell subset, and the reference range data for the natural killer cell subset includes acceptable range data for the natural killer cell subset and theoretical range data for the natural killer subset.

[0089] If the number data for all T cell subsets meet the reference range data for the corresponding T cell subsets and the number data for all natural killer cell subsets meet the reference range data for the corresponding natural killer cell subsets (e.g., the number data for all T cell subsets and the number data for all natural killer cell subsets meet their respective acceptable range data or theoretical range data), the immunological efficacy field will state that the immunological efficacy is normal. In some embodiments, if the number data for all T cell subsets meet the theoretical range data for the corresponding T cell subsets and the number data for all natural killer cell subsets meet the theoretical range data for the corresponding natural killer cell subsets, the immunological efficacy is further determined to be good.

[0090] If the data for the number of any T cell subset does not meet the reference range data for the corresponding T cell subset, or if the data for the number of natural killer cell subsets does not meet the reference range data for the natural killer cell subset, the effectiveness of immunity is judged to be low.

[0091] In some other embodiments, the reference range data for each T cell subset or the reference range data for each natural killer cell subset is theoretical range data.

[0092] Some embodiments of the present disclosure provide use of a plurality of lymphocytes in preparing a pharmaceutical composition for treating cancer in an individual, the lymphocytes comprising a plurality of T cells, a plurality of natural killer cells, or a combination thereof, and the treatment comprises: (a) generating a total number deviation value of T cells between the actual total number of T cells and the theoretical range of the total number of T cells, and a total number deviation value of natural killer cells between the actual total number of natural killer cells and the theoretical range of the total number of natural killer cells; and (b) administering the pharmaceutical composition comprising the T cells to the individual so that the actual total number of T cells of the individual satisfies the theoretical range of the total number of T cells based on the total number deviation value of T cells, and administering the pharmaceutical composition comprising natural killer cells to the individual so that the actual total number of natural killer cells of the individual satisfies the theoretical range of the total number of natural killer cells based on the total number deviation value of natural killer cells.

[0093] It has been found that individuals suffering from cancer often have low immune efficacy due to a deficiency in the actual total number of T cells, the actual total number of natural killer cells, or both. The present disclosure adjusts the efficacy of an individual's immune system to a healthy state by controlling the actual total number of T cells and the actual total number of natural killer cells within a theoretical range, thereby improving the individual's immune system's resistance to cancer and enhancing the efficacy of cancer treatment. Unlike conventional radiation therapy or chemotherapy, the use of the present disclosure does not damage autologous cells. Furthermore, the use of the present disclosure can be used in conjunction with other immune cell therapies. By accurately controlling the actual total number of T cells and the actual total number of natural killer cells, the efficacy of immune cell therapy can be enhanced and side effects such as an overreaction (e.g., cytokine storm) due to estimation errors in the actual total number of T cells or the actual total number of natural killer cells can be avoided.

[0094] In some embodiments, the theoretical ranges of the total number of T cells and the theoretical ranges of the total number of natural killer cells are derived from physiological values ​​of a group of healthy individuals, which are human individuals aged 5 to 85 years who are not affected by immunodeficiency syndrome or immunohypersensitivity syndrome.

[0095] In some embodiments, the theoretical range for the total number of T cells is 700 cells / microliter to 2500 cells / microliter (e.g., 700 cells / microliter, 800 cells / microliter, 900 cells / microliter, 1000 cells / microliter, 1100 cells / microliter, 1200 cells / microliter, 1300 cells / microliter, 1400 cells / microliter, 1500 cells / microliter, 1600 cells / microliter, 1700 cells / microliter, 1800 cells / microliter). The average number of T cells is typically 1000 cells / microliter, 1900 cells / microliter, 2000 cells / microliter, 2100 cells / microliter, 2200 cells / microliter, 2300 cells / microliter, 2400 cells / microliter, 2500 cells / microliter, or any value between the above ranges, and the theoretical range for the total number of natural killer cells is 100 cells / microliter to 300 cells / microliter (e.g., 100 cells / microliter, 200 cells / microliter, 300 cells / microliter, or any value between the above ranges). If the actual total number of T cells is lower than the theoretical range for the total number of T cells, the actual total number of natural killer cells is lower than the theoretical range for the total number of natural killer cells, or both conditions exist simultaneously, the individual's immune efficacy may be reduced. Conversely, if the actual total number of T cells is higher than the theoretical range for the total number of T cells, the actual total number of natural killer cells is higher than the theoretical range for the total number of natural killer cells, or both conditions exist simultaneously, autoimmunity may become excessive.

[0096] In some embodiments, the T cell count deviation or natural killer cell count deviation is combined with the individual's blood content (e.g., a 65 kg individual has a blood volume of approximately 5 liters) to generate the appropriate dose of T cells or natural killer cells to be administered.

[0097] In some embodiments, the treatment includes the steps of: (a) classifying the lymphocyte cells into a plurality of T cell subsets according to the presence or absence of expression of a plurality of first antigens; and classifying the lymphocyte cells into a plurality of natural killer cell subsets according to the presence or absence of expression of a plurality of second antigens; (b) generating deviation values ​​of T cell subsets between actual percentages of T cell subsets and theoretical ranges of T cell subsets, and generating deviation values ​​of natural killer cell subsets between actual percentages of natural killer cell subsets and theoretical ranges of natural killer cell subsets, wherein the actual percentages of T cell subsets are determined by the number of each T cell subset in the lymphocyte cells. and the actual percentages of the natural killer cell subsets are the percentages of the number of each natural killer cell subset in the lymphocytes; and (c) administering to the individual a pharmaceutical composition comprising the T cell subsets such that the actual percentages of the T cell subsets in the individual fulfill the theoretical range of T cell subsets based on the deviation values ​​of the T cell subsets, and administering to the individual a pharmaceutical composition comprising the natural killer cell subsets such that the actual percentages of the natural killer cell subsets in the individual fulfill the theoretical range of natural killer cell subsets based on the deviation values ​​of the natural killer cell subsets.

[0098] Furthermore, by administering a pharmaceutical composition containing the T cell subset or natural killer cell subset to an individual based on the deviation value of the T cell subset or the deviation value of the natural killer cell subset, the actual total number of T cells or the actual total number of natural killer cells can be adjusted, while the T cell subsets and natural killer cell subsets that control anti-cancer and killing functions in the immune system can be further adjusted, thereby achieving more precise control over immune efficacy.

[0099] In some embodiments, the theoretical ranges of T cell subsets and the theoretical ranges of natural killer cell subsets are derived from physiological values ​​of a group of healthy individuals, which are human individuals aged 5 to 85 years who are not affected by immunodeficiency syndrome or immunohyperactivity syndrome.

[0100] In some embodiments, depending on the individual's blood content (e.g., a 65 kg individual has a blood volume of approximately 5 liters), the deviation values ​​of the T cell subsets and the actual total number of T cells or the deviation values ​​of the natural killer cell subsets and the actual total number of natural killer cells are combined to generate the appropriate dosage to administer for each T cell subset or natural killer subset.

[0101] In some embodiments, the first antigen comprises CD3, CD4, CD8, CD25, CD27, CD28, CD39, CD45RO, CD45RA, CD57, CD62L, CD127, FoxP3, or a combination thereof. In some embodiments, the second antigen comprises CD16, CD34, CD56, CD94, CD117, or a combination thereof. In some embodiments, sorting the T cells into multiple T cell subsets comprises sorting the T cells into early helper T cells (CD3+CD4+CD45RA+CD62L+CD28+), senescent helper T cells (CD3+CD4+CD45RO+CD62L+), regulatory helper T cells (CD3+CD4+CD25+FoxP3+CD39+), early killer T cells (CD8+CD27+CD45RA+CD62L+CD127+), senescent killer T cells (CD8+CD27+CD45RO+CD62L+CD57+), or a combination thereof.

[0102] In some embodiments, natural killer cell subsets include killer natural killer cells (CD16+CD34+CD56+CD94+CD117+) and regulatory natural killer cells (CD34+CD56+CD94+CD117+).

[0103] In some embodiments, the actual percentages of T cell subsets include the actual percentage of early helper T cells, the actual percentage of senescent helper T cells, the actual percentage of regulatory helper T cells, the actual percentage of early killer T cells, the actual percentage of senescent killer T cells, or a combination thereof. In some embodiments, the actual percentages of natural killer cell subsets include the actual percentage of killer natural killer cells, the actual percentage of regulatory natural killer cells, or a combination thereof.

[0104] In some embodiments, the theoretical range of T cell subsets includes a theoretical range of early helper T cells, a theoretical range of regulatory helper T cells, a theoretical range of early killer T cells, or a theoretical range of senescent killer T cells. The theoretical range of early helper T cells is 35% to 100%, the theoretical range of regulatory helper T cells is 3% to 10%, the theoretical range of early killer T cells is 20% to 100%, and the theoretical range of senescent killer T cells is 0% to 30%.

[0105] In some embodiments, the theoretical range of natural killer cell subsets includes a theoretical range of regulatory natural killer cells that is 0% to 10%.

[0106] In some embodiments, because early helper T cells and early killer T cells can provide the immune system with protection against cancer cells, if the actual percentage of early helper T cells or the actual percentage of early killer T cells is lower than the respective theoretical ranges, the individual's immune system will be less effective at killing cancer cells. Administering the deficient early helper T cells or early killer T cells to an individual can restore the individual's immune efficacy to a healthy state and enhance the immune system's anti-cancer effectiveness.

[0107] In some embodiments, regulatory helper T cells are used to inhibit other lymphocytes (e.g., other T cell subsets) and balance the efficacy of the immune system. Therefore, if the actual percentage of regulatory helper T cells is higher than the theoretical range of regulatory helper T cells, other lymphocytes will be excessively inhibited, resulting in reduced immune efficacy and increased mortality when an individual suffers from cancer. Removing excess regulatory helper T cells in an individual can restore the individual's immune efficacy to a healthy state and enhance the immune system's anti-cancer efficacy.

[0108] In some embodiments, senescent killer T cells represent the senescent state of an individual's immune system, and therefore, a higher actual percentage of senescent killer T cells than the theoretical range of senescent killer T cells indicates senescence or dysfunction of the immune system. Removal of excess senescent killer T cells in an individual can restore the individual's immune efficacy to a healthy state and enhance the immune system's anti-cancer efficacy.

[0109] In some embodiments, regulatory natural killer cells are used to inhibit other lymphocytes (e.g., killer natural killer cells) and balance the efficacy of the immune system. Therefore, if the actual percentage of regulatory natural killer cells is higher than the theoretical range of regulatory natural killer cells, other lymphocytes will be excessively inhibited, immune efficacy will be reduced, and the mortality rate when an individual is infected with cancer will increase. By removing excess regulatory natural killer cells in an individual, the individual's immune efficacy can be restored to a healthy state and the immune system's anti-cancer efficacy can be improved.

[0110] In some embodiments, the treatment includes the steps of: (a) classifying the lymphocyte cells into a plurality of T cell subsets according to the presence or absence of expression of a plurality of first antigens, the T cell subsets including a first T cell subset and a second T cell subset; (b) generating a deviation value of the T cell subset proportion between the actual value of the two T cell subset proportions and a theoretical range of the two T cell subset proportions, the actual value of the two T cell subset proportions being the ratio of numbers between the first T cell subset and the second T cell subset; and (c) administering to the individual a pharmaceutical composition including the first T cell subset or the second T cell subset based on the deviation value of the T cell subset proportions so that the actual value of the individual's two T cell subset proportions falls within the theoretical range of the two T cell subset proportions.

[0111] In some embodiments, the theoretical ranges of the proportions of the two T cell subsets are derived from physiological values ​​of a group of healthy individuals, which are human individuals aged 5 to 85 years who are not affected by immunodeficiency syndrome or immunohypersensitivity syndrome.

[0112] In some embodiments, when the first T cell subset is CD4-positive T cells (CD4+ T cells) and the second T cell subset is CD8-positive T cells (CD8+ T cells), the theoretical range of the ratio of the two T cell subsets (theoretical range of the CD4 / CD8 ratio) is 1:1 to 5:1. Because CD4-positive T cells can activate early killer T cells, it is known that when the actual CD4 / CD8 ratio is lower than 1:1, the individual will exhibit a weakened immune system. A pharmaceutical composition containing CD4-positive T cells is administered to an individual, and an appropriate dosage of the CD4-positive T cells is determined based on the individual's blood content, the actual number of CD4-positive T cells (the content of CD4-positive T cells in the blood), and the corresponding theoretical range.

[0113] In some embodiments, the treatment further comprises administering immune cell therapy to the individual after administering to the individual a pharmaceutical composition comprising the T cells or a pharmaceutical composition comprising the natural killer cells. In some embodiments, the immune cell therapy includes natural killer cell therapy, cytokine-induced killer cell therapy, γδ T cell therapy, dendritic cell therapy, tumor infiltrating lymphocytes (TIL) therapy, chimeric antigen receptor T cell therapy, or the like. T cell) therapy or a combination thereof.

[0114] Furthermore, none of the conventional immune cell therapies involves a step of comparing the deviation of actual values ​​of immune analysis indicators, such as the total number of T cells, the total number of natural killer cells, the percentage of T cell subsets, the percentage of natural killer cell subsets, or the CD4 / CD8 ratio, from their corresponding theoretical ranges in advance, nor does it involve a step of adjusting the actual values ​​of immune analysis indicators to their theoretical ranges based on the deviation of the immune analysis indicators by culturing lymphocytes, such as T cells, natural killer cells, specific T cell subsets, or specific natural killer cell subsets, in vitro and then returning them to an individual, or by removing lymphocytes, such as T cells, natural killer cells, specific T cell subsets, or specific natural killer cell subsets, from the individual.As a result, the efficacy of existing immune cell therapies varies greatly depending on the efficacy of an individual's immune system (e.g., due to a weakened immune system or an autoimmune disease).

[0115] In contrast, the use of the present disclosure can ensure that immune cell therapy is performed after an individual's immune efficacy has returned to a good state, thereby increasing the success rate of immune cell therapy.

[0116] For example, before performing T cell-related γδ T cell therapy, dendritic cell therapy, tumor-infiltrating lymphocyte therapy, or chimeric antigen receptor T cell therapy, the actual total number of T cells can be adjusted to within the theoretical range of the total number of T cells, and the actual proportion of T cell subsets can be adjusted to within the theoretical range of the proportion of T cell subsets based on the deviation value of the total number of T cells and the deviation value of the T cell subsets, thereby restoring the immune efficacy of T cells to a healthy state and improving the therapeutic effect of immune cells.

[0117] In another embodiment, before performing natural killer cell-related natural killer cell therapy or cytokine-induced killer cell immunotherapy, the actual total number of natural killer cells can be adjusted to the theoretical range of the total number of natural killer cells and the actual value of the proportion of natural killer cell subsets can be adjusted to the theoretical range of the proportion of natural killer cell subsets based on the deviation value of the total number of natural killer cells and the deviation value of the natural killer cell subsets, so that the natural killer cells can restore immune efficacy to a healthy state and enhance the therapeutic effect of immune cells.

[0118] In some embodiments, the lymphocytes are derived from an individual in need of treatment and obtained by in vitro culture. For example, before an individual receives treatment, they are screened from the individual's own blood and specific lymphocytes are cryopreserved. In the future, when cancer is treated, they can be directly thawed, screened, expanded, cultured, and returned to the individual. By pre-cryopreserving specific lymphocytes, not only can the flow of lymphocytes required for treatment be reduced, but also immune rejection between different individuals can be avoided.

[0119] In some embodiments, the cancer comprises colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer, hepatocellular carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, basal cell carcinoma, cutaneous squamous cell carcinoma, bile duct carcinoma, Merkel cell carcinoma, or a combination thereof.

[0120] The present disclosure further provides a use of a plurality of natural killer cells for preparing a pharmaceutical composition for treating cancer in an individual, the treatment comprising: (a) measuring the actual value of the natural killer cell killing ability of the natural killer cells, wherein the actual value of the natural killer cell killing ability is generated by co-culturing the natural killer cells with a plurality of cancer cells at a specific ratio and calculating the percentage of the cancer cell deaths to obtain the actual value of the natural killer cell killing ability; (b) generating a deviation value of the natural killer cell killing ability between the actual value of the natural killer cell killing ability and the theoretical range of the natural killer cell killing ability based on the deviation value of the natural killer cell killing ability; and (c) administering a pharmaceutical composition containing the natural killer cells to the individual so that the actual value of the individual's natural killer cell killing ability satisfies the theoretical range of the natural killer cell killing ability based on the deviation value of the natural killer cell killing ability.

[0121] It has been found that when an individual is a cancer patient, the killing ability of natural killer cells can be reduced in efficacy or lead to excessive autoimmunity due to an insufficient or excessive number of natural killer cells or insufficient activity of natural killer cells. The present disclosure regulates the actual value of the killing ability of natural killer cells within a theoretical range (e.g., by controlling the actual total number of natural killer cells within a theoretical range or by activating natural killer cells within an individual), thereby adjusting the efficacy of the individual's immune system to a healthy state and enhancing the ability of the individual's autoimmune system to resist cancer, thereby improving the efficacy of cancer treatment. Compared to conventional radiation therapy or chemotherapy, the use of the present disclosure does not damage autologous cells. Furthermore, the use of the present disclosure may be used in combination with other immune cell therapies, and precisely controlling the killing ability of natural killer cells can improve the efficacy of immune cell therapies.

[0122] In some embodiments, the theoretical range of natural killer cell killing capacity is derived from physiological values ​​in a healthy population, where the healthy population is human individuals aged 5 to 85 years who are not affected by immunodeficiency syndrome or immunohyperactivity syndrome.

[0123] In some embodiments, when the number ratio is 6.25, the theoretical range of natural killer cell killing capacity is 0% to 58.8%; when the number ratio is 12.5, the theoretical range of natural killer cell killing capacity is 1.7% to 88.7%; when the number ratio is 25, the theoretical range of natural killer cell killing capacity is 17.4% to 100%; and when the number ratio is 50, the theoretical range of natural killer cell killing capacity is 35.3% to 100%.

[0124] In some embodiments, in step (a), when natural killer cells and cancer cells are co-cultured with leukocyte interleukin 2 (IL-2) at a number ratio of 50 and an effective concentration of 100 International Units (IU / ml) (referred to as the maximum activity group of natural killer cells or the IL-2 control group), the theoretical range of natural killer cell killing capacity is 57.4% to 100%. Note that at this effective concentration, IL-2 completely inhibits the activity of regulatory natural killer cells (which inhibit the activity of killer natural killer cells), and therefore the actual value of the natural killer cell killing capacity obtained under these conditions reflects the activity of an individual's killer natural killer cells, i.e., the maximum activity of natural killer cells.

[0125] In some embodiments, the treatment includes the steps of generating a deviation value of the total number of natural killer cells between the actual total number of natural killer cells and the theoretical range of the total number of natural killer cells, and administering a pharmaceutical composition containing the natural killer cells to the individual based on the deviation value of the killing ability of natural killer cells and the deviation value of the total number of natural killer cells so that the actual total number of the individual's natural killer cells satisfies the theoretical range of the total number of natural killer cells, thereby causing the actual value of the killing ability of the individual's natural killer cells to satisfy the theoretical range of the killing ability of natural killer cells.

[0126] In some embodiments, the treatment further includes the steps of: classifying the natural killer cells into a plurality of natural killer cell subsets according to the presence or absence of expression of a plurality of specific antigens, the natural killer cell subsets including regulatory natural killer cells, the regulatory natural killer cells being CD34+, CD56+, CD94+, and CD117+; generating a deviation value of regulatory natural killer cells between the actual percentage of regulatory natural killer cells and the theoretical range of regulatory natural killer cells, the actual percentage of regulatory natural killer cells being the percentage of the number of regulatory natural killer cells among lymphocytes; and administering a pharmaceutical composition containing the regulatory natural killer cells to the individual based on the deviation value of the killing ability of natural killer cells and the deviation value of regulatory natural killer cells, so that the actual percentage of the individual's regulatory natural killer cells satisfies the theoretical range of killing ability of regulatory natural killer cells, thereby causing the actual value of the killing ability of the individual's natural killer cells to satisfy the theoretical range of killing ability of natural killer cells.

[0127] Furthermore, by analyzing the trends in natural killer cell killing capacity at different numerical ratios and combining them when the numerical ratio is 50, it is possible to combine this with the natural killer cell killing capacity measured when IL-2 is added (maximum activity of natural killer cells) to provide an overview of the immune efficacy of natural killer cells.

[0128] For example, if the proportion of natural killer cells co-cultured with cancer cells increases, but the actual killing power of the natural killer cells in each group is lower than the theoretical range, and the actual killing power of the natural killer cells in the most active natural killer cell group (IL-2 control group) is also lower than the theoretical range, there will be a deviation in the killing power of the natural killer cells between the actual killing power of the natural killer cells and the theoretical range of the killing power of the natural killer cells.

[0129] Next, the actual total number of natural killer cells can be further analyzed. If the actual total number of natural killer cells is lower than the corresponding theoretical range, natural killer cells can be administered to the individual so that the actual total number of natural killer cells meets the theoretical range of the total number of natural killer cells, and the actual killing ability of the individual's natural killer cells meets the theoretical range of killing ability of natural killer cells. On the other hand, the actual percentage of regulatory natural killer cells can also be analyzed at the same time. If the actual percentage of regulatory natural killer cells is higher than the corresponding theoretical range, it indicates that the efficacy of natural killer cells is excessively inhibited. By removing regulatory natural killer cells with an excessively high proportion so that the actual percentage of regulatory natural killer cells meets the corresponding theoretical range, the actual killing ability of natural killer cells can be returned to the theoretical range of killing ability of natural killer cells.

[0130] In another embodiment, if the proportion of natural killer cells is high and the actual killing capacity of natural killer cells in each group is lower than the theoretical range, but the actual killing capacity of natural killer cells in the most active natural killer cell group (IL-2 control group) is within the theoretical range, this indicates insufficient natural killer cell activity. Natural killer cells can be first selected from an individual, and then subjected to IL-2 activation culture. After confirming that the actual killing capacity of activated natural killer cells has returned to the corresponding theoretical range, they can be returned to the individual to improve immune efficacy.

[0131] In some embodiments, the treatment further comprises administering immune cell therapy to the individual after administering the pharmaceutical composition comprising the natural killer cells to the individual, in some embodiments, the immune cell therapy comprises natural killer cell therapy, cytokine-induced killer cell immunotherapy, γδ T cell therapy, dendritic cell therapy, tumor-infiltrating lymphocyte therapy, chimeric antigen receptor T cell therapy, or a combination thereof.

[0132] Furthermore, while the efficacy of existing immune cell therapies varies greatly depending on the individual's immune system (e.g., due to a weakened immune system or an autoimmune disease), the use of the present disclosure can ensure that immune cell therapy is administered only after the individual's natural killer cell killing ability has returned to a good state, thereby increasing the success rate of immune cell therapy.

[0133] For example, before performing natural killer cell therapy, the actual total number of natural killer cells can be adjusted to the theoretical range of the total number of natural killer cells based on the actual value of the killing ability of natural killer cells and based on the deviation value of the total number of natural killer cells, the deviation value of regulatory natural killer cells, or both, and the actual value of the proportion of natural killer cell subsets can be adjusted to the theoretical range of the proportion of natural killer cell subsets, or both can be performed simultaneously, so that the actual value of the killing ability of natural killer cells falls within the theoretical range of the killing ability of natural killer cells.

[0134] In some embodiments, natural killer cells are derived from an individual in need of treatment and obtained by in vitro culture. For example, before an individual receives treatment, specific lymphocytes are screened from the individual's own blood and cryopreserved. In the future, when cancer is treated, they can be directly thawed, screened, expanded, cultured, and returned to the individual. This not only reduces the flow of natural killer cells required for treatment, but also avoids immune rejection between different individuals.

[0135] In some embodiments, the cancer comprises colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer, hepatocellular carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, basal cell carcinoma, cutaneous squamous cell carcinoma, bile duct carcinoma, Merkel cell carcinoma, or a combination thereof.

[0136] To further illustrate the method and system for detecting the effectiveness of immunity and the use of lymphocytes to prepare a pharmaceutical composition for treating cancer in an individual (a method for treating cancer by controlling lymphocyte content) according to various embodiments of the present disclosure, the following examples are provided for illustrative purposes only and are not intended to limit the present invention.

[0137] 1. Method and system for detecting the effectiveness of immunity

[0138] 1. Detection of immune cell types and antigen expression status (detection of actual values ​​of immune analysis indicators of T cells and natural killer cells)

[0139] First, 100 microliters of whole blood sample from a human is placed in a test tube containing an appropriate concentration of anticoagulant, which can be heparin or ethylenediaminetetraacetic acid (EDTA), and when EDTA is added, the weight percentage of EDTA action is 0.1%.

[0140] Based on the antigens (classification markers) shown in Table 1, specific antibodies with fluorescent groups (e.g., fluorescein isothiocyanate (FITC)) are selected, and depending on the antigen distribution location (e.g., cell surface antigens or intracellular antigens), immunostaining is performed in combination with an appropriate method to label specific cells and classify them into multiple cell subsets.

[0141] [Table 1]

[0142] If the selected antigen is a cell surface antigen (e.g., CD3, CD4, CD8, CD25, CD27, CD34, CD39, CD45RO, CD45RA, CD56, CD57, CD62L, CD94, CD117, and CD127), the following surface antigen staining step is carried out.

[0143] Add 100 microliters of fluorescently labeled antibody (e.g., Invitrogen Flow Cytometry Antibodies) to a test tube containing 100 microliters of whole blood sample, resulting in a 1:1 volume ratio of whole blood sample to antibody and a 50-100x antibody dilution ratio. Then, incubate at 2-8°C for 30 minutes to 1 hour with rotation, away from light. Next, wash the cells in the whole blood sample with a flow cytometer staining buffer (Invitrogen eBioscience Flow Cytometry Staining Buffer, product number 00-4222-26) to obtain the first cell staining solution.

[0144] If the selected antigens include surface antigens and intracellular antigens (e.g., FoxP3), the above surface antigen staining step is performed, followed by the following steps.

[0145] Add 2 milliliters of fixative / lysis solution (Invitrogen eBioscience 1-Step Fix / Lyse Solution (10X), product number 00-5333-54) at room temperature to 100 microliters of the first cell staining solution, and mix by inversion to lyse red blood cells so that they do not interfere with subsequent flow cytometry analysis. Next, incubate the cells at room temperature for 15 to 60 minutes in the dark to obtain the treatment solution.

[0146] The treated solution was centrifuged at 500×g for 5 minutes at room temperature to pellet the cells, and the supernatant was removed.

[0147] In a dark environment, suspend the cells in 2 ml of permeabilization solution (prepared by diluting 10 times with Invitrogen eBioscience Permeabilization Buffer (10X) (product number 00-8333-56)). Next, centrifuge at 500 x g for 5 minutes at room temperature, remove the supernatant, and repeat the process of adding permeabilization solution and removing the supernatant by centrifugation.

[0148] Suspend the cells in 100 microliters of flow cytometry solution and add 100 microliters of a fluorescently labeled antibody (e.g., Invitrogen Flow Cytometry Antibodies) at a dilution ratio of 50-100. Incubate at room temperature, protected from light, for 20-60 minutes. Wash the cells with another 2 milliliters of flow cytometry solution, remove the supernatant by centrifugation, and then suspend the cells in 500 microliters of flow cytometry solution to obtain a second cell stain.

[0149] Using a flow cytometer, antigen data on the sample cell surface is acquired according to the presence or absence of expression of cell classification markers (see Table 1), and T cell subsets and natural killer cell subsets are classified to generate the content of T cells and natural killer cells in the whole blood sample (the actual total number of T cells and the actual total number of natural killer cells), the proportion of T cell subsets and each natural killer cell subset in lymphocytes (the actual percentage of T cell subsets and the actual percentage of natural killer cell subsets), and the proportion of CD4+ T cells / CD8+ T cells. Additionally, if necessary, the proportion of T cells bearing a specific marker antigen (e.g., CD28) among all T cells is analyzed.

[0150] Furthermore, the classification markers used in this application are multi-checked using dedicated antigens for each T cell subset and each natural killer cell subset, further improving the accuracy of classification.

[0151] in particular,

[0152] Regarding early helper T cells, we used dual markers, CD45RA (a marker for early T cells) and CD28 (a marker for early helper T cells), to verify the efficacy of these cells. In addition, we also used CD62L (a marker for non-regulatory helper T cells in T cells) to eliminate regulatory helper T cells and reduce the probability of regulatory helper T cells being mixed in.

[0153] Regarding aging helper T cells, we verified this by using CD45RO (a marker for aging T cells) on helper T cells, and also reduced the probability of regulatory helper T cells being mixed in via CD62L.

[0154] Regarding regulatory helper T cells, the accuracy of regulatory helper T cell subpopulation can be improved by using the triple antigens CD25, FoxP3, and CD39 specific to regulatory helper T cells for verification.

[0155] Regarding early killer T cells, the early cell marker CD45RA was used to classify early T cells from killer T cells (CD8), and the dedicated marker CD127 for early killer T cells was also used for double verification, which can improve the accuracy of early killer T cell classification.

[0156] Regarding senescent killer T cells, the senescent cell marker CD45RO was used to classify senescent T cells from killer T cells (CD8). In addition, the dedicated marker for senescent killer T cells, CD57, was used for double verification to improve the accuracy of classification of senescent killer T cells.

[0157] For killer natural killer cells and regulatory natural killer cells, both are separated by CD16 (a marker for killer natural killer cells), and both are multiplexed via the natural killer cell-specific markers CD34, CD56, CD94, and CD117.

[0158] 2. Detection of the actual killing capacity of natural killer cells

[0159] Because natural killer cells are the first line of defense against viruses and cancer, detecting the killing ability of natural killer cells can be used to evaluate the innate immune ability to resist viruses and cancer, and the detection of the killing ability of natural killer cells includes the following steps:

[0160] First, natural killer cells (e.g., obtained by isolating natural killer cells from a whole blood sample using an appropriate antibody selected according to Table 1 above) and K-562 cells are co-cultured at different ratios (effector cell / target cell = ET ratio, e.g., natural killer cells / K-562 = 6.25, 12.5, 25, 50), and the rate at which K-562 cells die is analyzed using a flow cytometer to evaluate the killing ability of the natural killer cells and determine their health and anti-cancer ability.

[0161] In addition, because regulatory natural killer cells inhibit the activity of killer natural killer cells, IL-2 (effective concentration: 100 International Units / milliliter = 100 IU / milliliter) is further added under the condition that the E:T ratio is 50 to inhibit the activity of regulatory natural killer cells. By comparing with a group to which IL-2 was not added, the activity of killer natural killer cells is evaluated, the maximum activity of natural killer cells is analyzed, and it is possible to determine whether natural killer cells are in a state of immune inhibition.

[0162] Specifically, when the ET ratio increases from 6.25 to 50 in a linear fashion, the percentage of K-562 cell death (or the number of K-562 cell deaths) increases in an arithmetic progression, indicating that natural killer cells are healthy and vigorous, and have the ability to eliminate and protect cancer cells. Conversely, if the ET ratio continues to increase but the percentage of K-562 cell death (or the number of K-562 cell deaths) is lower than the reference range and does not show an arithmetic progression upward trend, it indicates that natural killer cells (especially killer natural killer cells) are not healthy and lack the ability to kill and protect against cancer.

[0163] To further clarify the status of natural killer cells, the percentage of K-562 cell death in the IL-2 control group and the number of regulatory natural killer cells can be simultaneously referenced.

[0164] In the IL-2 control group, the percentage of K-562 cell death was lower than the reference range, and a high proportion of regulatory natural killer cells indicates an autoimmune state (common causes include cancer and chronic viral infection). Conversely, a normal proportion of regulatory natural killer cells indicates an immunocompromised state (common causes include aging (especially over 65 years of age), drug effects (including, for example, immunosuppressants or chemotherapy), radiation therapy, or excessive radiation damage).

[0165] Furthermore, when the percentage of K-562 cell deaths in the IL-2 control group fell within the reference range, it indicated that natural killer cells were stimulated and activated to produce anti-cancer toxic activity, meaning that although the primordial activity of natural killer cells was lower than that of normal people, it did not affect the body's health.

[0166] Therefore, by testing the killing ability of natural killer cells, the anti-cancer killing ability and health of natural killer cells can be effectively and quantitatively evaluated, providing a more accurate analysis for clinical disease diagnosis.

[0167] 3.Analysis Immunity Effectiveness

[0168] The types of immune cells (T cell subsets and natural killer cell subsets) and antigen expression status in the whole blood sample are obtained according to step 1 above, and the actual percentages of each cell subset (T cell subsets and natural killer cell subsets) in lymphocytes are calculated. After obtaining the actual values ​​of the killing ability of natural killer cells according to steps 1 and 2 above, the effectiveness of the immune system is judged according to Tables 2 to 4 below. If all the actual values ​​fall within the reference range (e.g., the theoretical range or the acceptable range), the effectiveness of the immune system is judged to be normal. Furthermore, if the actual values ​​(e.g., the actual percentages) of each cell subset fall within the theoretical range, the effectiveness of the immune system is judged to be good. However, if any of the actual values ​​(e.g., the actual percentages) do not fall within the reference range (i.e., the theoretical range and the acceptable range), the effectiveness of the immune system is judged to be low.

[0169] [Table 2]

[0170] [Table 3]

[0171] [Table 4]

[0172] Furthermore, the method for analyzing the effectiveness of immunity can also be implemented in a specific system 300. See Figure 3. The system 300 includes a memory 310, a processor 320, and an output module 330.

[0173] First, using an electronic device such as a personal computer, smartphone, or server, the antigen data on the surface of the sample cells (in the case of antigen expression on cells in a whole blood sample) obtained by flow cytometer analysis in "Detection of immune cell type and antigen expression state" above is transmitted as a file, and the natural killer cell killing ability data obtained by "Detection of natural killer cell killing ability" above 2 is transmitted to and stored in memory 310 (e.g., read-only memory, flash memory, disk, or cloud database).

[0174] The system 300 further includes a processor 320 connected to the memory 310, which may include a central processor, an image processor, a microprocessor, etc., including a processing unit including, for example, a multi-core or a combination of multiple processing units, and the processor 320 accesses the sample cell surface antigen data and natural killer cell killing activity data in the memory 310 to perform the following analyses:

[0175] First, the processor 320 analyzes the antigen data on the surface of the sample cells based on the cell subset database 312 (including the classification information of T cell subsets and natural killer cell subsets in Table 1), classifies the cells in the whole blood sample into multiple cell subsets (including multiple T cell subsets and multiple natural killer cell subsets), calculates the percentage of the number of each cell subset among lymphocytes, and obtains data on the number of T cell subsets and data on the number of natural killer cell subsets.

[0176] Next, the processor 320 analyzes the data on the number of T cell subsets, the data on the number of natural killer cell subsets, and the natural killer cell killing capacity data based on the immune effectiveness evaluation database 314 (including the reference range data for T cell subsets, the reference range data for natural killer cell subsets, and the reference range data for natural killer cell killing capacity in Tables 2 to 4), and generates immune effectiveness data.

[0177] The system 300 further includes an output module 330 coupled to the processor 320, which outputs the immunopotency efficacy data as an immunopotency efficacy report for display on a display screen (not shown) or transmission to another electronic device for evaluating whether to cryopreserve a particular T cell subset or a particular natural killer cell subset.

[0178] 2. Isolation and cryopreservation of target cells

[0179] Based on the results of the analyses 1 and 3 above, it is determined whether a specific T cell subset or a specific natural killer cell subset is to be cryopreserved. For example, if the actual percentage of early helper T cells meets the reference range for early helper T cells, the early helper T cells are cryopreserved.

[0180] 1. Target Cell Isolation

[0181] Target cells (specific T cell subsets or specific natural killer cell subsets) are isolated by immunobead cell sorting, which includes positive and negative sorting systems.

[0182] A positive selection system (Invitrogen's Dynal (R) positive i isolation kits and FlowComp TM The selection flow uses antibodies with specially modified biotin (FlowComp TM The process involves marking target cells with the Antibody Mix in the kit series products, and then using streptavidin-containing positive selection magnetic beads (FlowComp TM FlowComp in kit series products TM Dynabeads TM ) to bind the streptavidin and biotin on the positive selection magnetic beads; and selecting cells that bind to the positive selection magnetic beads using magnetic force, removing the positive selection magnetic beads that bind to the cells, and obtaining the target cells.

[0183] Negative isolation (Untouched) TM The selection flow uses a mixture of multiple antibodies with biotin (Negative isolation (Untouched)). TM The process involves marking the cells to be removed with the antibody mix in the kits, and negative isolation (Untouched) magnetic beads containing streptavidin. TM ) kits Depletion MyOne TM SA Dynabeads (R) ) and negative selection magnetic beads The streptavidin and biotin on the beads are bound to each other, and the cells to be removed are sieved using magnetic force to select target cells that are not bound to the negative selection magnetic beads.

[0184] Below, examples of methods for selecting each T cell subset and natural killer cell subset are given.

[0185] (1) Regulatory helper T cells (CD3+CD4+CD25+FoxP3+CD39+)

[0186] CD3+CD4+ is used to screen for helper T cells (including early helper T cells, senescent helper T cells and regulatory helper T cells), and CD25+CD45- is used to screen for regulatory helper T cells.

[0187] The selection process for CD3+ cells is specifically as follows (taking the positive selection system as an example):

[0188] First, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples and then cultured at a cell density of 5x10 using a separation solution. 6 The PBMC separation solution was prepared at a concentration of cells / milliliter. The separation solution was a calcium- and magnesium-ion-free phosphate buffer solution (purchased from Gibco, product number 14190) supplemented with 0.1% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA).

[0189] Next, Invitrogen Dynabeads (R) FlowComp TM The Hum an CD3 kit (product number 113-65D) was used to separate cells expressing CD3, which specifically includes the following steps:

[0190] Mix 25 microliters of a specially modified biotin-containing CD3 antibody (Antibody Mix in the kit) with 500 microliters of PBMC separation solution and incubate at 2-8°C for 10 minutes to allow the CD3 antibody to bind to CD3-expressing cells. Then wash the PBMCs with 2 milliliters of separation solution, centrifuge at 350xg for 8 minutes, and remove the supernatant.

[0191] Next, PBMCs were suspended in 1 milliliter of isolation solution and then added to 75 microliters of positive selection magnetic beads (FlowComp® in the kit). TM Dynabeads TM ) and incubate with rotation at room temperature (approximately 25°C) for 15 minutes to obtain a positive mixture.

[0192] Next, place the positive mixture on a magnet for at least one minute and remove the supernatant, leaving behind the magnetic bead-labeled cells. After separating the magnetic bead-labeled cells from the magnet, add 1 milliliter of separation solution to suspend the magnetic bead-labeled cells. Place the magnetic bead-labeled cells on a magnet for at least one minute and remove the supernatant.

[0193] Next, add 1 milliliter of magnetic bead removal solution (FlowComp TM Add the magnetic bead release buffer and rotate for 10 minutes at room temperature. Place the magnetic bead-labeled cells in magnetic bead removal solution on a magnet for 1 minute, then transfer the supernatant (CD3-expressing cells) to a new centrifuge tube and centrifuge at 350 x g for 8 minutes to pellet the cells. Remove the supernatant and collect the CD3-expressing cells.

[0194] Next, the corresponding set is selected, and by a process similar to that described above, CD4+ cells (CD3+CD4+ cells) are further selected from CD3+ cells, followed by further selection of CD25+ cells (CD3+CD4+CD25+ cells) from the CD3+CD4+ cells, and then CD45+ cells are selected and removed from the CD3+CD4+CD25+ cells to obtain CD45- cells (CD3+CD4+CD25+CD45- cells), thereby obtaining regulatory helper T cells.

[0195] (2) Early helper T cells (CD3+CD4+CD45RA+CD62L+CD28+) or senescent helper T cells (CD3+CD4+CD45RO+CD62L+CD28-)

[0196] Referring to the specific selection process of CD3+ cells described above, helper T cells (including early helper T cells, senescent helper T cells, and regulatory helper T cells) are selected using CD3+CD4+, early helper T cells and senescent helper T cells are selected using CD25-CD45+ (here, CD45+ refers to at least cells expressing CD45RA or CD45RO, where CD45RA and CD45RO are different isomers of CD45), and finally early helper T cells (CD28+) or senescent helper T cells (CD28-) are selected using CD28+ or CD28-.

[0197] (3) Early killer T cells or senescent killer T cells

[0198] Referring to the specific selection process of CD3+ cells described above, killer T cells (including early killer T cells and senescent killer T cells) are selected using CD3+CD8+, and early killer T cells (CD57-) or senescent killer T cells (CD57+) are selected using CD57- or CD57+.

[0199] (4) Killer natural killer cells or regulatory natural killer cells

[0200] Referring to the specific selection process for CD3+ cells described above, natural killer cell subsets (including killer natural killer cells and regulatory natural killer cells) are screened using CD34+CD56+, and killer natural killer cells (CD16+) or regulatory natural killer cells (CD16-) are screened using CD16+ or CD16-.

[0201] (5) Simultaneous isolation of T cell subsets and natural killer cell subsets from a single blood sample

[0202] The classification markers selected in this application enable sequential separation of each T cell subset and natural killer cell subset from the same blood sample, eliminating the need to separate multiple blood samples to screen each cell subset, thereby saving screening time and consumables, improving screening efficiency, and reducing quantification errors. Furthermore, the selection of the markers in this invention allows blood samples to be screened for two antigens at a time, and the number of separation steps can be controlled within the cell's tolerance range (2-3 sorting steps) to avoid damaging the cells.

[0203] Therefore, the markers selected in this application can improve the applicability and accuracy of clinical detection, see the following flow chart for details.

[0204] For T cells

[0205] (1) Helper T cells:

[0206] First, CD3 and CD4 were used to separate helper T cells (CD3 is a T cell marker, and CD4 is a helper T cell marker). Then, CD45RA (an early T cell marker) and CD62L (a marker for non-regulatory helper T cells in T cells), CD45RO (a marker for senescent T cells) and CD62L, and CD25 and FoxP3 (both are markers for regulatory helper T cells) were used to initially separate early helper T cells, senescent helper T cells, and regulatory helper T cells, respectively. Finally, CD28 (an early helper T cell marker) and CD39 (a regulatory helper T cell marker) were used to further test the cells that expressed CD45RA and CD62L or CD25 and FoxP3, respectively, to obtain early helper T cells and regulatory helper T cells.

[0207] (2) Killer T cells:

[0208] After screening the helper T cells, the remaining cells are then screened using CD8 and CD27 (both killer T cell markers) to separate killer T cells, and CD45RA (early T cell marker) and CD62L (a marker for non-regulatory helper T cells in T cells), CD45RO (senescent T cell marker) and CD62L are used to initially separate early killer T cells and senescent killer T cells, respectively. Finally, CD127 (early killer T cell marker) and CD57 (senescent killer T cell marker) are used to test the early killer T cells and senescent killer T cells, respectively, to obtain early killer T cells and senescent killer T cells.

[0209] Regarding natural killer cells,

[0210] After isolating T cell subsets, the unseparated cells are further screened for CD16 (a killer natural killer cell marker) and CD34 (a natural killer cell marker) to initially isolate killer natural killer cells. Killer T cells are then screened for multiple times using CD56, CD94, and CD117 (all natural killer cell markers) to obtain killer natural killer cells. The remaining cells are then screened for regulatory T cells using CD34, CD56, CD94, and CD117, respectively.

[0211] 2. Cryopreservation of Target Cells

[0212] The target cells selected in the above 4.1 "Isolation of target cells" are subcultured, and the culture medium containing the target cells (select the appropriate culture medium depending on the type of cells) is centrifuged to precipitate the cells. After the supernatant is removed, the target cells are further suspended in a culture medium containing 5% by weight of dimethyl sulfoxide (DMSO) to adjust the target cell density to 1 x 10 6 / ml ~ 5x10 6After diluting to 1 / ml, the solution is divided into cryopreservation tubes and stored at 4°C for 10 to 30 minutes, -20°C for 30 minutes, and -80°C for 16 to 18 hours, and finally transferred to liquid nitrogen for long-term frozen storage, or placed in a programmed temperature-reducing chamber (e.g., 1 to 3°C per minute) until the temperature drops to -80°C, after which the solution is placed in liquid nitrogen for long-term frozen storage.

[0213] If the cells are stored at -20°C for more than an hour, the ice crystals will be too large, reducing cell viability. Alternatively, the previous steps of 4°C and -20°C can be skipped and the cells can be stored directly at -80°C, however, this will also reduce cell viability.

[0214] Since each T cell subset and natural killer cell subset has a different function and plays a different role in the immune system (for example, early helper T cells are useful in resisting new pathogens, while killer natural killer cells can inhibit cancer), the method and system for detecting immune effectiveness disclosed herein classifies T cell subsets (for example, classifying early helper T cells expressing CD28) with a first antigen including CD28, classifies natural killer cell subsets (for example, classifying killer natural killer cells expressing CD16) with a second antigen including CD16, calculates the actual percentage in lymphocytes, and analyzes the effectiveness of immunity, providing a more comprehensive test of the function of each aspect of the immune system.

[0215] 3. Methods for controlling lymphocyte content and treating cancer

[0216] 1.Detecting the actual values ​​of each immunoassay indicator

[0217] The actual values ​​of the immunoassay indicators of T cells and natural killer cells are detected by the method 1 above, and the actual values ​​of the killing ability of natural killer cells are detected by the methods 1 and 2 above.

[0218] 2. Adjust the actual values ​​of individual immunoassay indicators to the theoretical range

[0219] 2.1. Adjusting the actual values ​​of T cell immune assay indicators to the theoretical range

[0220] According to Table 2 above, generate deviation values ​​between the actual values ​​of each immune analysis index obtained by 3 and 1 above and the corresponding theoretical range, and then generate appropriate dosages of T cells or specific T cell subsets to be replenished or removed from the individual based on the actual values ​​of the individual's blood volume or other related immune analysis indexes.

[0221] Specifically, the following example will be taken as an example of a cancer patient weighing 65 kg and having a total blood volume of 5 liters.

[0222] Actual total number of T cells is 500 / microliter (theoretical range of total number of T cells is 700 When the T cell dose range is determined to be between 1x10 and 2500 cells / microliter, the appropriate dose range for T cell replenishment is 1x10. 9 ~1x10 10 pieces [(700 - 500) pieces / microliter) x 5 liters = 1 x 10 9 , (2500-500) pieces / microliter) x 5 liters = 1 x 10 10 By adjusting the actual total number of T cells to within the theoretical range, it is possible to improve the individual's immune strength, and further increase the safety and efficacy of the subsequent combined immune cell therapy.

[0223] When the number of CD4+ T cells is detected to be 200 cells / microliter and the number of CD8+ T cells is detected to be 250 cells / microliter, the actual CD4 / CD8 ratio is 0.8:1 (the theoretical range of the CD4 / CD8 ratio is 1:1 to 5:1). Because CD4+ T cells can activate early killer T cells, if the CD4+ T cell ratio is too low, the individual will exhibit a weakened immune system, and if directly treated with immune cell therapy (e.g., dendritic cell therapy or chimeric antigen receptor T cell therapy), the anti-cancer effect will be low. To improve immune efficacy, first, the CD4+ T cells in the individual are positively screened, and then further expanded and cultured to obtain 2.5 x 10 CD4+ T cells in the individual.8 Pieces [(250 - 200) pieces / microliter x 5 liters = 2.5 x 10 8 )] and bring the actual CD4 / CD8 ratio back to at least 1:1. Adjusting the actual CD4 / CD8 ratio to the theoretical range will increase the number of CD4-positive T cells, activate early killer T cells, and improve the individual's anti-cancer killing ability.

[0224] If the actual percentage of early helper T cells is detected to be 25% (theoretical range of early helper T cells is ≥ 35% and the theoretical range of total T cells is 700 cells / microliter to 2500 cells / microliter), the individual will exhibit a state of immunocompromise. To improve the efficacy of the immune system, 3.5 x 10 early helper T cells isolated and obtained from the individual will be used. 8 pieces~1.25x10 9 Pieces [(35-25)% x 5 liters x 700 pieces / microliter = 3.5 x 10 8 , (35-25)% x 5 liters x 2500 particles / microliter = 1.25 x 10 9 ] and then reintroduced into the individual to restore the actual percentage of initial helper T cells to at least 35% or more. Adjusting the actual percentage of initial helper T cells to within the theoretical range can reboot the individual's immune anti-cancer mechanism, improve the individual's anti-cancer killing ability, and further increase the success rate when combined with other immune cell therapies.

[0225] Detecting an actual percentage of regulatory helper T cells of 15% (theoretical range of regulatory helper T cells is 3%-10%) and an actual total number of T cells of 2000 / microliter indicates an excess of regulatory helper T cells to inhibit other lymphocytes, inhibiting the individual's immune anti-cancer efficacy. 8 Regulatory helper T cells [(15-10)% x 5 liters x 2000 cells / microliter = 5 x 10 8It is necessary to remove [number of cells], which will improve the condition of excessively inhibited immune function. Specifically, peripheral blood mononuclear cells (PBMCs) are first extracted from an individual through hemapheresis, and then at least 3.33 x 10 PBMCs are extracted through lymphopheresis. 9 pieces (5x10 8 CD4-positive T cells (15%) are positively selected, and then CD25-positive regulatory helper T cells are removed by negative selection, after which the remaining PBMCs are returned to the individual.

[0226] Conversely, if the actual percentage of regulatory helper T cells is 2% (theoretical range of regulatory helper T cells is 3%-10%) and the actual total number of T cells is found to be 2000 / microliter, this indicates that there are too few regulatory helper T cells to inhibit other lymphocytes, potentially leading to a state of autoimmunity. 8 pieces~8x10 8 Regulatory helper T cells are expressed in an individual [(3-2)% x 5 liters x 2000 cells / microliter = 1 x 10 8 , (10-2)% x 5 liters x 2000 particles / microliter = 8 x 10 8 ], which can improve autoimmune conditions and inhibit immune function. Specifically, PBMCs are first extracted from individuals using a blood separator, and then positively selected regulatory helper T cells are selected using lymphocyte separation. Then, 1x10 regulatory helper T cells are selected. 8 pieces~8x10 8 After amplifying and culturing the cells to individual cells, they are returned to the individual.

[0227] If the actual percentage of early killer T cells is 15% (theoretical range for early killer T cells is >20%) and the actual total number of T cells is 2000 / microliter, indicating a lack of efficacy of the immune system in killing cancer cells, early killer T cells should be isolated from the individual and at least 5x10 8Number of early killer T cells [(20-15)% x 5 liters x 2000 cells / microliter = 5 x 10 8 These cells need to be amplified and cultured, and then returned to the individual, which can restart the individual's immune anti-cancer mechanism, improve the individual's anti-cancer ability, and further increase the success rate when combined with other immune cell therapies.

[0228] 2.2. Adjusting the actual values ​​of natural killer cell immunoassay indicators to the theoretical range

[0229] According to Table 3 above, the deviation value between the actual value of each immune analysis indicator obtained by 3 and 1 above and the corresponding theoretical range is generated, and then the appropriate dosage of natural killer cells or natural killer cell subsets to be replenished or removed is generated according to the individual's blood volume.

[0230] First, the actual values ​​of the killing capacity of natural killer cells obtained by the method 3.1 above are compared with the theoretical range in Table 4 above to determine the proportion of each number and the tendency of the actual values ​​of the killing capacity of natural killer cells when IL-2 is added, and the deviation from the theoretical range. If the actual values ​​of the killing capacity of natural killer cells are not within the theoretical range, the deviation between the actual values ​​of each immune analysis indicator related to natural killer cells obtained by the method 3.1 above and the theoretical range corresponding to Table 3 above is calculated. Next, an appropriate dosage of natural killer cells or a specific natural killer cell subset to be replenished or removed is calculated based on the individual's blood volume, thereby optimizing the killing capacity of natural killer cells and further increasing the success rate when combined with other immune cell therapies.

[0231] Theoretically, when the E:T ratio increases from, say, 6.25 to 50, the number of cancer cells killed by natural killer cells increases arithmetically, indicating that an individual's natural killer cells can normally kill cancer cells. Furthermore, natural killer cells can be stimulated with IL-2 to generate maximum activity and compared with a control group with an E:T ratio of 50, which can be used to determine whether an individual is in a state of immunosuppression.

[0232] Specifically, taking the example of a cancer patient weighing 65 kg and estimating that the total blood volume is 5 liters, the following three possible situations are provided for illustration:

[0233] 1. Although the E:T ratio is elevated, the actual values ​​of the killing capacity of natural killer cells in the E:T ratios of group 4 are lower than the theoretical range, and do not show a trend of linear increase. At the same time, the actual values ​​of the killing capacity of natural killer cells in the maximum activity group of natural killer cells (IL-2 control group) are lower than the theoretical range, and furthermore, by comparing the actual percentage of regulatory natural killer cells obtained by 3.1 above with the theoretical range corresponding to Table 3, if the actual percentage of regulatory natural killer cells is high,

[0234] It has been shown that an excess of regulatory natural killer cells usually inhibits the ability of killer natural killer cells to kill cancer cells. In this case, regulatory natural killer cells with an excessively high percentage are removed according to the actual percentage of regulatory natural killer cells and the corresponding theoretical range in Table 3.

[0235] For example, if the actual percentage of regulatory natural killers is 15% (theoretical range for regulatory natural killers is ≦10%) and the actual total number of natural killer cells is 5×10 8 Individually In some cases, regulatory natural killer cells are over-expressed. To improve the immune system's ability to kill cancer cells, 2.5x10 7 pieces (5x10 8 It is necessary to remove regulatory natural killer cells (NKCs), which account for 5% of the total number of cells. Specifically, PBMCs are first extracted from the individual using a blood separator, and then NKCs with CD56 positivity and CD3 negativity are positively selected using lymphocyte separation. The number of NKCs is 1.67 x 10 8 pieces (2.5x10 7 / 0.15=1.67x10 8 ) and then further negative selection to obtain CD16-negative After removal of the regulatory natural killer cells, the remaining natural killer cells (which are primarily killer natural killer cells) are returned to the individual.

[0236] 2. As mentioned above, the E:T ratio is similarly increased, but if the actual values ​​of the killing ability of natural killer cells in the E:T ratios of group 4 are lower than the theoretical range, there is no tendency for the ratio to increase linearly. At the same time, if the actual value of the killing ability of natural killer cells in the maximum activity group of natural killer cells (IL-2 control group) is lower than the theoretical range, and furthermore, by comparing the actual percentage of regulatory natural killer cells obtained in 3.1 above with the theoretical range corresponding to Table 3, if the actual percentage of regulatory natural killer cells is within the theoretical range,

[0237] This usually indicates that the individual is in a severely immunocompromised state, and common causes include (1) aging (especially in humans over 65 years of age), (2) drug effects (e.g., immunosuppressants or chemotherapy), and (3) radiation therapy or excessive radiation damage. Typically, such individuals have a low actual total number of natural killer cells. Natural killer cells can be expanded and cultured and then reintroduced into the individual to increase the actual total number of natural killer cells and improve the individual's immune function. In some other embodiments, when comparing the actual percentage of regulatory natural killer cells obtained by 3.1 above with the corresponding theoretical range in Table 3, the actual total number of natural killer cells can also be compared with the corresponding theoretical range. Before performing the natural killer cell reintroduction process, it is confirmed whether the actual total number of natural killer cells falls within the theoretical range.

[0238] For example, if the actual total number of natural killer cells after detection is 80 cells / microliter (the theoretical range of the total number of natural killer cells is 100 cells / microliter), it indicates that the killing ability of natural killer cells is insufficient. In order to improve the individual's killing ability against cancer cells, the actual total number of natural killer cells should be increased to at least 1x10 8 Pieces [(100 - 80) pieces / microliter x 5 liters = 1 x 10 8The cells need to be amplified and cultured in a separate container, and then returned to the individual.

[0239] 3. Similarly, if the E:T ratio increases, but the actual values ​​of the killing ability of natural killer cells in the E:T ratios of Group 4 are lower than the theoretical range and do not show a linear increase trend, and at the same time, the actual value of the killing ability of natural killer cells in the maximum activity group of natural killer cells (IL-2 control group) falls within the theoretical range,

[0240] Although the activity of natural killer cells in an individual is lower than that of a normal person, they can be stimulated and activated to improve their ability to kill cancer cells. To achieve this, natural killer cells are first selected from an individual, and then cultured in IL-2 to activate them. After confirming that the actual killing activity of the natural killer cells has returned to the theoretical range, they can be returned to the individual.

[0241] Therefore, by analyzing the actual value of the killing ability of natural killer cells, the actual total number of natural killer cells, and the actual percentage of regulatory natural killer cells and the corresponding theoretical range, a more comprehensive analysis of the state of the anti-cancer killing ability of natural killer cells can be provided. Furthermore, based on the analysis results, the content of natural killer cells or regulatory natural killer cells in an individual can be correspondingly adjusted, or natural killer cells activated by IL-2 can be added, so that the killing ability of natural killer cells can be restored to the theoretical range, thereby improving the success rate when subsequently combined with other immune cell therapies (e.g., natural killer cell therapies).

[0242] Although the embodiments have been disclosed above, they are not intended to limit the scope of the present disclosure, and anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure is determined by the scope of the following claims. [Explanation of symbols]

[0243] 100 ways 200 Flow S110, S120, S130, S140, S210, S220, S230 process 300 System 310 memory 312 Cell Subset Database 314 Immunity Effectiveness Evaluation Database 320 processor 330 Output Module

Claims

1. 1. A pharmaceutical composition for treating cancer in an individual, comprising: Contains multiple lymphocytes, The lymphocytes may include a plurality of T cells, a plurality of natural killer cells, or a combination thereof. fruit, The treatment of cancer comprises: (a) generating a deviation value of the total number of T cells between the actual total number of T cells and the theoretical range of the total number of T cells; The actual total number of natural killer cells and the theoretical range of the total number of natural killer cells were compared. generating a standard deviation score for the total number of neural killer cells; (b) determining whether the actual total number of T cells in the individual satisfies the theoretical range of total number of T cells; and administering the pharmaceutical composition containing the T cells to the individual based on the deviation value of the total number of the T cells. Administer Based on the deviation value of the total number of natural killer cells, the natural killer cell count of the individual is determined. The individual cells are then cultured so that the actual total number of cells meets the theoretical range of the total number of natural killer cells. administering the pharmaceutical composition comprising the natural killer cells to a body; A pharmaceutical composition comprising:

2. The theoretical range of the total number of T cells is 700 cells / microliter to 2500 cells / microliter. The theoretical range of the total number of natural killer cells is 100 cells / microliter. The pharmaceutical composition according to claim 1, characterized in that the number of cells is in the range of 300 to 300 cells / microliter.

3. The treatment of cancer comprises: (a) dividing the lymphocytes into a plurality of T cell subsets depending on the presence or absence of expression of a plurality of first antigens; and classifying the lymphocytes into a plurality of natural groups depending on the presence or absence of expression of a plurality of second antigens. classifying the cells into a primary killer cell subset; (b) Comparison of the actual percentages of T cell subsets with the theoretical ranges of T cell subsets. Generate a deviation score for each subset to compare the actual percentage of natural killer cell subsets with the natural killer cell subsets. The theoretical range of natural killer cell subsets was calculated using the deviation values ​​of natural killer cell subsets. The actual percentage of each T cell subset in the lymphocytes is determined by the percentage of each T cell subset. The actual percentage of the natural killer cell subset is the percentage of the number of natural killer cell subsets. a step of determining the percentage of the number of each of the natural killer cell subsets in the total lymphocytes; , and (c) determining whether the actual percentage of said T cell subset in said individual is greater than or equal to the theoretical percentage of said T cell subset; and administering the T cell subsets to the individual based on the deviation values ​​of the T cell subsets so as to meet a target range. administering said pharmaceutical composition comprising a cell subset; Based on the deviation value of the natural killer cell subset, the natural killer cell subset of the individual is determined. The actual percentage of killer cell subsets is within the theoretical range of the natural killer cell subsets. the pharmaceutical composition comprising the natural killer cell subset in the individual so as to satisfy the range of Administering a substance The pharmaceutical composition of claim 1 further comprising:

4. The T cell subsets include early helper T cells, regulatory helper T cells, early killer T cells, and cells or combinations thereof, The early helper T cells are CD3+, CD4+, CD45RA+, CD62L+, and are CD28+, The regulatory helper T cells are CD3+, CD4+, CD25+, FoxP3+, and C D39+, The primary killer T cells are CD8+, CD27+, CD45RA+, CD62L+, and The pharmaceutical composition according to claim 3, characterized in that it is CD127+.

5. The theoretical range of the T cell subsets includes the theoretical range of early helper T cells, regulatory helper T cells, and a theoretical range of primary T cells, a theoretical range of primary killer T cells, or a combination thereof; The theoretical range of the primary helper T cells is 35% to 100%, and the regulatory helper T The theoretical range for the primary killer T cells is 3% to 10%, and the theoretical range for the primary killer T cells is 20% to 5. The pharmaceutical composition according to claim 4, characterized in that it is 100%.

6. The natural killer cell subset includes regulatory natural killer cells, Human natural killer cells are CD34+, CD56+, CD94+, and CD117+. The pharmaceutical composition according to claim 3,

7. The theoretical range of the natural killer cell subset is 0% to 10%.

7. The pharmaceutical composition according to claim 6, characterized in that it contains a theoretical range of neural killer cells.

8. The treatment of cancer comprises: (a) dividing the lymphocytes into a plurality of T cell subsets depending on the presence or absence of expression of a plurality of first antigens; the T cell subsets are classified into a first T cell subset and a second T cell subset. a process including (b) Actual values ​​of two T cell subset proportions and theoretical ranges of two T cell subset proportions The deviation value of the T cell subset ratio from the surrounding range is generated, and the actual ratio of the two T cell subsets is calculated. The value is the ratio of the number between the first T cell subset and the second T cell subset. process, and (c) determining the two types of T cell subsets of the individual based on the deviation values ​​of the T cell subset proportions; so that the actual value of the subset ratio satisfies the theoretical range of the ratio of the two T cell subsets. the medicament comprising the first T cell subset or the second T cell subset in the individual; Administering the composition 2. The pharmaceutical composition of claim 1, further comprising:

9. the first T cell subset is CD4-positive T cells, and the second T cell subset is are CD8-positive T cells, the theoretical range of the ratio of the two T cell subsets is 1:1 The pharmaceutical composition according to claim 8, characterized in that the ratio is 5:1 or less.

10. The pharmaceutical composition of claim 1, wherein the cancer treatment further comprises administering to the individual the pharmaceutical composition comprising the T cells or the pharmaceutical composition comprising the natural killer cells, followed by administering to the individual immune cell therapy.

11. The immune cell therapy includes natural killer cell therapy and cytokine-induced killer cell immunotherapy. therapy, γδT cell therapy, dendritic cell therapy, tumor infiltrating lymphocyte therapy, chimeric antigen receptor T cell therapy 11. The pharmaceutical composition according to claim 10, characterized in that it comprises a method or a combination thereof.

12. The lymphocytes are derived from the individual and are obtained by in vitro culture. The pharmaceutical composition of claim 1.

13. The cancers include colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, and uterine cancer. Cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer , esophageal cancer, head and neck cancer, salivary gland cancer, hepatocellular carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, basal cell cancer, cutaneous squamous cell carcinoma, bile duct carcinoma, Merkel cell carcinoma, or a combination thereof. The pharmaceutical composition according to claim 1 .

14. 1. A pharmaceutical composition for treating cancer in an individual, comprising: Contains multiple natural killer cells The treatment of cancer comprises: (a) co-culturing the natural killer cells and a plurality of cancer cells at a certain ratio; The percentage of cancer cell deaths is generated to obtain the actual killing capacity of the natural killer cells. By obtaining the actual value of the killing ability of the natural killer cells, measuring the (b) the actual value of the killing ability of the natural killer cells and the killing ability of the natural killer cells Based on the theoretical range, the actual value of the killing ability of the natural killer cells and the natural To generate deviations of natural killer cell killing capacity within the theoretical range of killer cell killing capacity, and (c) determining the natural killer cell killing ability of the individual based on the deviation value of the natural killer cell killing ability; The actual killing capacity of natural killer cells does not exceed the theoretical range of the killing capacity of natural killer cells. administering to said individual said pharmaceutical composition comprising said natural killer cells so as to A pharmaceutical composition comprising:

15. When the ratio of the numbers is 6.25, the theoretical range of the killing ability of the natural killer cells is is 0% to 58.8%, and when the ratio of the numbers is 12.5, the natural killer The theoretical range of cell killing capacity is 1.7% to 88.7%, and when the ratio of these numbers is 25, In this case, the theoretical range of the killing ability of the natural killer cells is 17.4% to 100%. If the ratio of these numbers is 50, the theoretical range of killing capacity of the natural killer cells is 35. The pharmaceutical composition according to claim 14, characterized in that it is between 3% and 100%.

16. In step (a), the ratio of the numbers is 50 and the working concentration is 100 international units / milliliter. The natural killer cells and the cancer cells were simultaneously added with leukocyte interleukin 2. When co-cultured with cells, the theoretical range of killing capacity of the natural killer cells is 57.4% to 1.

16. The pharmaceutical composition according to claim 15, characterized in that it is 00%.

17. The treatment of cancer comprises: Comparison of the actual total number of natural killer cells with the theoretical range of the total number of natural killer cells generating a standard deviation score for the total number of neural killer cells; The deviation value of the killing ability of the natural killer cells and the deviation value of the total number of the natural killer cells Based on the value, the actual total number of natural killer cells in the individual is determined to be - the total number of cells before including the natural killer cells in the individual so as to satisfy the theoretical range. By administering the pharmaceutical composition, the actual value of the killing ability of the natural killer cells of the individual is increased. a step of satisfying the theoretical range of the killing ability of the natural killer cells; 16. The pharmaceutical composition of claim 15, further comprising:

18. The treatment of cancer comprises: The natural killer cells are differentiated into a plurality of natural killer cells depending on the presence or absence of expression of a plurality of antigens. The natural killer cell subset is classified into regulatory natural killer cell subsets. - cells, and the regulatory natural killer cells are CD34+, CD56+, CD94+ and CD117+; Actual percentage of regulatory natural killer cells and theoretical category of regulatory natural killer cells generating a deviation value of regulatory natural killer cells relative to the surrounding area, and The actual percentage is the percentage of the number of regulatory natural killer cells in lymphocytes. A process, and The deviation value of the killing ability of the natural killer cells and the deviation value of the regulatory natural killer cells Based on the difference value, the actual percentage of the regulatory natural killer cells in the individual is determined to be the regulatory natural killer cells. and administering to the individual the regulatory natural killer cells so as to meet the theoretical range of regulatory natural killer cells. By administering the pharmaceutical composition containing killer cells, the natural killer cells of the individual are increased. the actual value of the killing ability of the natural killer cells falls within the theoretical range of the killing ability of the natural killer cells.

16. The pharmaceutical composition of claim 15, further comprising:

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