A NEW NATURAL KILLER CD16+ CELL AND NATURAL KILLER CD16+ CELL CULTIVATION METHOD - US20230036481A1 (2023)

FIELD OF THE INVENTION

[0001]The present invention relates to a CD16⁺natural killer cell and a CD16 culture method⁺nature's killer cell; specifically refers to a CD16⁺Killer cell line that does not contain a genetically engineered polynucleotide encoding the CD16 receptor and a culture method capable of mass proliferation of CD16⁺natural killer cells and maintenance of CD16 expression.

BACKGROUND OF THE INVENTION

[0002]Natural killer (NK) cells are lymphocytes that are an important part of the innate immune system and are best studied in the innate defense against viral infections and in the surveillance of tumor cells. In humans, NK cells are classically characterized by the absence of the T cell receptor complex (CD3⁻) and the presence of a neural cell adhesion molecule (CD56⁺). There are two major subsets of NK cells in human peripheral blood, with the majority (>90%) of peripheral blood NK cells being CD3⁻CD56^darkenCD16⁺NK cells and the minority (10%) of peripheral blood NK cells are CD3⁻CD56^hellCD16⁻NK cells (Orange JS, 2013).

[0003]The CD16 receptor (FcγRIII; it is a receptor for the Fc region of IgG and can bind to the Fc portion of IgG antibodies) is required for antibody-dependent cellular cytotoxicity (ADCC) processes mediated by human NK cells are carried out. In humans, the polynucleotide encoding CD16 receptor is located on the q arm of chromosome 1 at position 1q23.3 Human NK cells expressing CD16 receptor can kill many types of target cells such as cancer cells, tumor cells and infected cells by HIV through ADCC processes (Rezvani K and Rouce RH, 2015; Littwitz-Salomon et al, 2016; Eileen Scully and Galit Alter, 2016). Taking tumor cells as an example, tumor cells expressing tumor-associated antigens (e.g. human epidermal growth factor receptor 2, termed HER2) can bind to endogenous IgG antibodies or clinically approved therapeutic IgG antibodies directed against tumor-associated antigens (such as trastuzumab, rituximab or cetuximab). Once CD 16 receptors (IgG Fc receptor FcγRIII) expressed by an NK cell bind to the Fc region of endogenous IgG antibodies or clinically approved therapeutic IgG antibodies, an NK cell-mediated ADCC becomes triggered and the NK cell releases cytotoxic factors that cause tumor cell death (Rezvani K and Rouce RH, 2015).

[0004]There are two main methods of treating cancer with CD16⁺NK cells. The first method includes the following steps: (a) obtaining autologous or allogeneic blood; (b) isolate autologous or allogeneic primary CD16⁺Natural killer cells (primary CD16⁺NK cells) from autologous or allogeneic blood; (c) autologous or allogeneic primary CD16 proliferate⁺NK cells in vitro; and (d) inject proliferated autologous or allogeneic primary CD16⁺NK cells back into a cancer patient's veins so that there is enough CD16⁺NK cells in cancer patient to release cytotoxic factors that cause cancer cell death through ADCC process. Due to the fact that primary CD16⁺The NK cell ages and dies even after several weeks of short-term culture, it is necessary to continuously maintain the primary CD16⁺Autologous or allogeneic blood NK cell for long-term treatment. In addition, studies have shown that in all cultured cells obtained from high-purity CD16 culture⁺NK cell population (i.e. the amount of CD16 cells⁺If the number of NK cells is equal to or greater than 99%, only 10% of the cells express CD16 for 4 days using the conventional method. In other words, the current CD16 culture method⁺NK cells in vitro cannot induce NK cells to stably express CD16 after proliferation. Therefore, not only does this method have difficulties in maintaining the primary CD16 source⁺NK cells, but also lack of method for stable proliferation of CD16⁺NK cells in vitro. These problems often make it difficult for cancer patients to get adequate CD16 counts.⁺NK cells and it is difficult to smoothly treat the cancer every time. In addition, the above-mentioned method also has to face the problem of difficulty in controlling the potency caused by individual cell differences.

[0005]The second method involves an NK-92 cell line (ATCC Accession Number CRL-2407). The NK-92 cell line is a CD16⁻Natural killer cell line isolated from the blood of a 50-year-old Caucasian male suffering from malignant non-Hodgkin's lymphoma. The NK-92 cell line can be continuously subcultured without senescence and death problems, and this NK-92 cell line is not tumorigenic to immunocompromised mice. Once irradiated with γ-rays, it is also non-carcinogenic to allogeneic humans, so it has a certain degree of applicability. However, since the NK-92 cell line does not express the CD16 receptor, it is unable to destroy cancer cells through the ADCC process. Therefore, the second method mentioned above requires the genetic transfer of the CD16 receptor gene to the NK-92 cell line via transgenic technology in order to obtain a CD16-NK-92 transgenic cell line capable of expressing the CD16 receptor and to exercise ADCC. Then the CD16-transfected NK-92 cell line is injected into the vein of the cancer patient; therefore there are enough CD16⁺natural killer cells in cancer patient to release cytotoxic factors that cause cancer cell death through ADCC process. Unfortunately, the medical community and the general public are concerned about the long-term safety of transgenic immune cells in the human circulatory system. Therefore, the development of the above-mentioned method is limited to a considerable extent.

[0006]Consequently, there is still an urgent need for a non-transgenic, non-tumorigenic cell line that can be continuously subcultured and a culture method capable of massively expanding CD16.⁺NK maintains CD16 expression.

[0007]Furthermore, the inventors of the present invention believe that the NK cell line has potential to be further applied in cell therapy specifically targeting abnormal cells. Therefore, there is a need to develop conjugation of an antigen-binding moiety with an NK cell line based on chemical conjugation technology or to develop a genetically modified NK cell to express an antigen-binding complex for cell therapy that specifically targets abnormal cells .

SUMMARY OF THE INVENTION

[0008]The present invention provides a natural killer cell line that can be continuously subcultured without the problem of senescence or death.

[0009]The second object of the present invention is to provide a natural killer cell which can still stably express CD16.⁺Recipients after at least 3 months of proliferation.

[0010]Another object of the present invention is to provide a CD16⁺natural killer cell line that does not contain a genetically modified polynucleotide encoding the CD16 receptor.

[0011]Another object of the present invention is to provide a CD16⁺natural killer cell line that is non-tumorigenic to immunocompromised mice.

[0012]Another object of the present invention is to provide a CD16⁺natural killer cell line that is not carcinogenic to an allogeneic human after γ-ray exposure.

[0013]Another object of the present invention is to provide a culture method for mass propagation of CD16⁺natural killer cells.

[0014]Another object of the present invention is to provide a culture method that enables CD16⁺natural killer to stably express CD16 receptor after proliferation.

[0015]Another object of the present invention is to provide a composition substantially enriched in human CD16⁺natural killer cells in which the human CD16 count⁺natural killer cells in the composition is at least 5×10⁵and human CD16⁺natural killer cells are in an amount equal to or greater than 5% in number based on the total number of cells in the composition as 100%; the human CD16⁺natural killer cell with the following properties: retention of proliferative ability after subculture for at least 3 months.

[0016]Another object of the present invention is to provide a natural killer cell line conjugated with an antigen-binding moiety or an antibody based on chemical conjugation technology.

[0017]Another object of the present invention is to provide an NK cell genetically modified to express an antigen-binding complex.

[0018]Another object of the present invention is to provide an antigen-specific NK cell line for cell therapy that specifically targets abnormal cells.

[0019]Another purpose of the present invention is to provide a method for obtaining an antigen-specific NK cell line that does not contain a genetically modified polynucleotide encoding the CD16 receptor.

[0020]Another object of the present invention is to provide a method of treating cancer, tumor, autoimmune disease, neuronal disease, human immunodeficiency virus (HIV) infection, hematopoietic cell related diseases, metabolic syndrome, pathogenic disease, viral infection or bacterial infection.

[0021]The present invention provides a human natural killer cell having the following properties: (i) expressing a CD16 receptor; (ii) retain its ability to reproduce after subcultivation for at least 3 months; and (iii) comprising an expressed polynucleotide sequence encoding the CD16 receptor, wherein the expressed polynucleotide sequence encoding the CD16 receptor is not synthetically, genetically modified and/or intentionally introduced into cells.

[0022]Preferably, the human natural killer cell is capable of proliferating for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months after subculture months, 10 months or 11 months.

[0023]Preferably, the human natural killer cell is capable of proliferation for at least 1 year, 2 years or 3 years after subculture.

[0024]Preferably, the human natural killer cell is nontumorogenic in an immunocompromised mouse.

[0025]Preferably, the immunocompromised mouse is a SCID/Beige, a NOD/SCID, an NSG, or a nude mouse.

[0026]The present invention further provides a composition substantially enriched in human CD16⁺natural killer cells in which the human CD16 count⁺natural killer cells in the composition is at least 5×10⁵and human CD16⁺natural killer cells are in an amount equal to or greater than 5% in number based on the total number of cells in the composition as 100%; the human CD16⁺natural killer cell having all of the following properties: (i) expressing a CD16 receptor, (ii) retaining its ability to proliferate after subculture for at least 3 months, and (iii) comprising an expressed polynucleotide sequence encoding the CD16 receptor wherein the sequence polynucleotide encoding the CD16 receptor is not synthetic, is not genetically modified and/or is not intentionally introduced into cells.

[0027]Preferably the human CD16 number⁺The composition of natural killer cells is 5 × 10⁵-5×10⁹.

[0028]Preferably the human CD16 number⁺The composition of natural killer cells is 1 × 10⁶, 1,1 × 10⁶, 5×10⁶, 5,1 × 10⁶, 1×10⁷, 1,1 × 10⁷, 5×10⁷, 5,1 × 10⁷, 1×10⁸, 1,1 × 10⁸, 5×10⁸, 5,1 × 10⁸, 1×10⁹, 1,1 × 10⁹, or 5×10⁹.

[0029]Preferably the total amount is human CD16⁺natural killer cells is 5%-100% based on the total number of cells in the composition as 100%.

[0030]Preferably human CD16⁺Natural killer cells are at or above 5%, 7%, 9%, 10%, 12%, 15%, 19%, 20%, 22%, 25%, 29%, 30%, 32%, 35%, 39% , 40%, 42%, 45%, 49%, 50%, 52%, 55%, 59%, 60%, 62%, 65%, 69%, 70%, 72%, 75%, 79%, 80 %, 82%, 85%, 89% or 95% by number based on the total number of cells in the composition as 100%.

[0031]Preferably, the CD16 receptor is a CD16a receptor or a CD16b receptor.

[0032]Preferably, the expressed polynucleotide sequence encoding the CD16a receptor or the CD16b receptor is not synthetic, is not genetically modified, and/or is not intentionally introduced into cells.

[0033]Method of obtaining a composition substantially enriched in human CD16⁺natural killer cells; the method comprising: (a) obtaining a human peripheral blood natural killer cell population derived from the cell population having ATCC accession number CRL-2047; (b) contacting the human peripheral blood natural killer cell population with an antibody specific for a CD16 receptor; and (c) separating the cells specifically bound by the antibody, thereby obtaining the composition substantially enriched in human CD16⁺natural killer cells; where human CD16⁺natural killer cell comprises an expressed polynucleotide encoding a CD16 receptor, and the expressed polynucleotide sequence encoding the CD16 receptor is not synthetic, not genetically modified, and/or is not intentionally introduced into cells.

[0034]Preferably human CD16⁺Natural killer cells are able to retain their ability to proliferate for at least 3 months after subculture.

[0035]Preferably human CD16⁺Natural killer cells can retain their proliferative ability after subculture for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months Months.

[0036]Preferably human CD16⁺Natural killer cells are capable of maintaining their proliferative ability for at least 1 year, 2 years, or 3 years after subculture.

[0037]Preferably, the expressed polynucleotide sequence encoding the CD16 receptor is not synthetic, is not genetically modified, and/or is not intentionally introduced into cells.

[0038]Preferably in the composition is human CD16⁺Natural killer cells are at or above 80% in number based on the total number of cells in the composition as 100%.

[0039]Preferably human CD16⁺natural killer cells are present in a number of 5% or more based on the total number of cells in the composition than 100%.

[0040]Preferably human CD16⁺Natural killer cells are at or above 50% in number based on the total number of cells in the composition as 100%.

[0041]The concentration of glucose dissolved in the culture medium is preferably greater than 1500 mg/l.

[0042]Preferably, the culture medium is fully aerated, the dissolved oxygen concentration in the culture medium is maintained in a stable range, or the dissolved glucose concentration in the culture medium is 1500-5000 mg/l.

[0043]The glucose concentration dissolved in the culture medium is preferably 2500, 2501, 3500, 3501, 4000 or 4500 mg/l.

[0044]Preferably the human CD16 number⁺natural killer cells in the composition is at least 5×10⁵and human CD16⁺natural killer cells are present in a number of 5% or more based on the total number of cells in the composition than 100%.

[0045]Preferably the human CD16 number⁺The composition of natural killer cells is 5 × 10⁵-5×10⁹.

[0046]Preferably the human CD16 number⁺The composition of natural killer cells is 1 × 10⁶, 1,1 × 10⁶, 5×10⁶, 1×10⁷, 1,1 × 10⁷, 5×10⁷, 5,1 × 10⁷, 1×10⁸, 1,1 × 10⁸, 5×10⁸, 5,1 × 10⁸, 1×10⁹, 1,1 × 10⁹, 5×10⁹, 1×10¹⁰, 1,1 × 10¹⁰, 5×10¹⁰, 1×10¹¹, 1,1 × 10¹¹, 5×10¹¹, 5,1 × 10¹¹, 1×10¹², 1,1 × 10¹², 5×10¹², 5,1 × 10¹², 1×10¹³, 1,1 × 10¹⁴, 5×10¹⁴, 1×10¹⁵, 1,1 × 10¹⁵, 5×10¹⁵, 1×10¹⁶, 1,1 × 10¹⁶, 5×10¹⁶, 5,1 × 10¹⁶, 1×10¹⁷, 1,1 × 10¹⁷, 5×10¹⁷, 5,1 × 10¹⁷, 1×10¹⁸, 1,1 × 10¹⁸, 5×10¹⁸, 1×10¹⁹, 1,1 × 10¹⁹, 5×10¹⁹, 1×10²⁰, 1,1 × 10²⁰, 5×10²⁰, 5,1 × 10²⁰, 1×10²¹, 1,1 × 10²¹, 5×10²¹, 5,1 × 10²¹, 1×10²², 1,1 × 10²², 5×10²², 1×10²³, 1,1 × 10²³, 5×10²³, 1×10²⁴, 1,1 × 10²⁴, 5×10²⁴, 5,1 × 10²⁴, 1×10²⁵, 1,1 × 10²⁵, 5×10²⁵, 5,1 × 10²⁵, 1×10²⁶, 1,1 × 10²⁶, 5×10²⁶, 1×10²⁷, 1,1 × 10²⁷, 5×10²⁷, 1×10²⁸, 1,1 × 10²⁸, 5×10²⁸, 5,1 × 10²⁸, 1×10²⁹, 1,1 × 10²⁹, 5×10²⁹, 5,1 × 10²⁹, 1×10³⁰, 1,1 × 10³⁰, 5×10³⁰, 1×10³¹, 1,1 × 10³¹, 5×10³¹, 1×10³², 1,1 × 10³², 5×10³², 5,1 × 10³², 1×10³³, 1,1 × 10³³, 5×10³³, 5,1 × 10³³, 1×10³⁴, 1,1 × 10³⁴, 5×10³⁴, 1×10³⁵, 1,1 × 10³⁵, 5×10³⁵, 1×10³⁶, 1,1 × 10³⁶, 5×10³⁶, 5,1 × 10³⁶, 1×10³⁷, 1,1 × 10³⁷, 5×10³⁷, 5,1 × 10³⁷, 1×10³⁸, 1,1 × 10³⁸, 5×10³⁸, 1×10³⁹, 1,1 × 10³⁹, 5×10³⁹, 5,1 × 10³⁹, 1×10⁴⁰, 1,1 × 10⁴⁰, 5×10⁴⁰. Preferably the human CD16 number⁺The composition of natural killer cells is 1 × 10⁶-1×10⁴¹.

[0047]Preferably the total number of human CD16 cells⁺natural killer cells is 5%-100% based on the total number of cells in the composition as 100%.

[0048]Preferably human CD16⁺Natural killer cells are at or above 5%, 7%, 9%, 10%, 12%, 15%, 19%, 20%, 22%, 25%, 29%, 30%, 32%, 35%, 39% , 40%, 42%, 45%, 49%, 50%, 52%, 55%, 59%, 60%, 62%, 65%, 69%, 70%, 72%, 75%, 79%, 80 %, 82%, 85%, 89% or 95% by number based on the total number of cells in the composition as 100%.

[0049]The present invention further provides a method for culturing and expanding human CD16⁺natural killer cells; the method comprising: (x) contacting human CD16 in a container⁺natural killer cells with a culture medium comprising human platelet lysate and IL-2; and (y) culturing the cells for several days; where human CD16⁺natural killer cell comprises an expressed polynucleotide encoding a CD16 receptor, and the expressed polynucleotide sequence encoding the CD16 receptor is not synthetic, not genetically modified, and/or is not intentionally introduced into cells.

[0050]The concentration of glucose dissolved in the culture medium is preferably greater than 1500 mg/l.

[0051]Preferably, step (y) comprises sub-steps: (y1) culturing the cells for at least one day; and (y2) subculturing the cells for at least 3 months.

[0052]Preferably, step (y2) consists in subculturing the cells for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months or 11 months.

[0053]Preferably step (y2) consists in subculturing the cells for at least 1 year, 2 years or 3 years.

[0054]Preferably human CD16⁺Natural killer cells are able to retain their ability to proliferate for at least 3 months after subculture.

[0055]Preferably human CD16⁺Natural killer cells can retain their proliferative ability after subculture for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months Months.

[0056]Preferably human CD16⁺Natural killer cells are capable of maintaining their proliferative ability for at least 1 year, 2 years, or 3 years after subculture.

[0057]Preferably, the expressed polynucleotide sequence encoding the CD16 receptor is not synthetic, is not genetically modified, and/or is not intentionally introduced into cells.

[0058]Preferably human CD16⁺Natural killer cells express the CD2 molecule (CD2⁺).

[0059]Preferably human CD16⁺the natural killer cell expresses NKp44, NKp46, NKG2D or CD107a.

[0060]The present invention provides a human natural killer cell having the following properties: (i) expressing a CD16 receptor; (ii) retain its ability to reproduce after subcultivation for at least 3 months; and (iii) comprising an expressed CD16A gene encoding the CD16 receptor, wherein the expressed CD16A gene is located on the q-arm of chromosome 1.

[0061]Preferably, the expressed CD16A gene is located on the q arm of chromosome 1 at position 1q23.3.

[0062]The present invention provides a human natural killer cell having the following properties: (i) expressing a CD16 receptor; (ii) retain its ability to reproduce after subcultivation for at least 3 months; and (iii) comprising an expressed CD16A gene encoding the CD16 receptor, wherein the polynucleotide sequence of the expressed CD16A gene is not synthetic, is not genetically modified and/or is not intentionally introduced into cells.

[0063]Preferably, the expressed CD16A gene polynucleotide sequence is not synthetic, is not genetically modified, and/or is not intentionally introduced into cells.

[0064]The present invention provides a human natural killer cell which is (A) deposited with NPMD under NITE accession number BP-03017; or (B) having the following characteristics:

[0065]i) expresses a CD16 receptor;

[0066]ii) retain its ability to proliferate after subculture for at least 3 months; It is

[0067]iii) x) excluding synthetic, genetically modified and/or intentionally introduced polynucleotide encoding the CD16 receptor, or y) using the ddPCR system to analyze the genomic DNA of the cell, the ratio of the probe-detectable DNA molecule CD16 F176F to the CD16 F176V probe -detectable DNA molecule is greater than 1, wherein the CD16 F176F probe sequence is SEQ ID NO:11 and the CD16 F176V probe sequence is SEQ ID NO:12.

[0068]Preferably, the CD16 receptor is a CD16a receptor or a CD16b receptor.

[0069]Preferably, an expressed polynucleotide encoding the CD16 receptor is located on the q-arm of chromosome 1 at position 1q23.3.

[0070]Preferably, the cell is non-tumorigenic in an immunocompromised mouse.

[0071]Preferably, the cell is non-tumorigenic in an allogeneic individual after being irradiated with γ-rays.

[0072]Preferably, a polynucleotide encoding the CD16 receptor comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 19.

[0073]Preferably, the CD16 receptor comprises an amino acid sequence of SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 20.

[0074]Preferably, the cell further comprises an inactive tumor suppressor gene or a mutated and highly expressed oncogene.

[0075]Preferably, the cell is capable of mediating an antibody dependent cellular cytotoxicity (ADCC) response and the cell is a male cell.

[0076]Preferably, the cell further comprises at least one exogenous targeting moiety complexed to the cell, the exogenous targeting moiety comprising a targeting moiety characterized by: (a) exhibiting specific binding to a biological marker in a target cell; (b) is not a nucleic acid; and (c) it is not produced by the cell.

[0077]Preferably, the exogenous targeting moiety is complexed to the cell via an interaction between a first ligand conjugated to the targeting moiety and a second ligand conjugated to the cell.

[0078]Preferably the first linker is a first polynucleotide or the second linker is a second polynucleotide.

[0079]Preferably the targeting moiety comprises an antigen binding moiety.

[0080]Preferably, the first polynucleotide comprises a single-stranded region.

[0081]Preferably, the first linker is a first polynucleotide and the second linker is a second polynucleotide.

[0082]Preferably, the first linker and the second linker are selected from the group consisting of: a DNA binding domain and a DNA target; a leucine zipper and target DNA; biotin and avidin; biotin and streptavidin; calmodulin and calmodulin-binding protein; a hormone and a hormone receptor; lectin and a carbohydrate; a cell membrane receptor and a receptor ligand; an enzyme and a substrate; an antigen and an antibody; an agonist and an antagonist; polynucleotide hybridization sequences; an aptamer and a target; and a zinc finger and target DNA.

[0083]Preferably, the first linker comprises a first reactive group and the second linker comprises a second reactive group, and wherein the cell is linked to the targeting moiety via a covalent bond formed by a reaction between the second reactive group and the first reactive group is complexed.

[0084]Preferably the targeting moiety comprises an antigen binding moiety.

[0085]Preferably the second linker comprises a PEG region.

[0086]Preferably, the targeting moiety and the cell are separated by a length of 1 nm to 400 nm, or the targeting moiety and the cell are by a length of 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 separated , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 130, 170, 200, 230, 270, 300, 330 or 370mm.

[0087]Preferably, the exogenous targeting moiety comprises an antigen-binding moiety, and the antigen-binding moiety binds to a cancer antigen, glycolipid, glycoprotein, differentiation antigen cluster present on cells of a hematopoietic lineage, gamma-glutamyl transpeptidase, adhesion protein, hormone, growth factor, cytokine , ligand receptor, ion channel, membrane-bound form of a µ-immunoglobulin. -chain, alpha-fetoprotein, C-reactive protein, chromogranin A, epithelial mucin antigen, human epithelial-specific antigen, Lewis(a) antigen, multidrug resistance-related protein, neu oncogene protein, neuron-specific enolase, glycoprotein P, Multidrug Resistance Related Antigen, p170, Multidrug Resistance Related Antigen, Prostate Specific Antigen, NCAM, Ganglioside Molecule, MART-1, Heat Shock Protein, SialylTn, Tyrosinase, MUC-1, HER-2 /neu, KSA, PSMA, p53, RAS, EGF -R, VEGF, MAGE or other target antigen (marker) expressed by a target cell.

[0088]Preferably, the antigen-binding moiety is an antibody against a cancer antigen selected from HER2/neu (ERBB2), HER3 (ERBB3), EGFR, VEGF, VEGFR2, GD2, CTLA4, CD19, CD20, CD22, CD30, CD33 (Siglec-3 ), CD52 (CAMPATH-1 antigen), CD326 (EpCAM), CA-125 (MUC16), MMP9, DLL3, CD274 (PD-L1), CEA, MSLN (Mesothelin), CA19-9, CD73, CD205 (DEC205) , CD51, c-MET, TRAIL-R2, IGF-1R, CD3, MIF, folate receptor alpha (FOLR1), CSF1, OX-40, CD137, TfR, MUC1, CD25 (IL-2R), CD115 (CSF1R), IL1B, CD105 (Endoglin), KIR, CD47, CEA, IL-17A, DLL4, CD51, Angiopoietin 2, Neuropilin-1, CD37, CD223 (LAG-3), CD40, LIV-1 (SLC39A6), CD27 (TNFRSF7) , CD276 (B7-H3), Trop2, Claudin1 (CLDN1), PSMA, TIM-1 (HAVcr-1), CEACAM5, CD70, LY6E, BCMA, CD135 (FLT3), APRIL, TF(F3) , Nectin-4, FAP, GPC3, FGFR3, killer cell immunoglobulin-like receptors (KIRs), a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, and combinations thereof.

[0089]Preferably, the targeting moiety is conjugated to the first polynucleotide using a linking group, wherein the linking group is an NHS ester, another activated ester, an alkyl or acyl halide, a bifunctional crosslinker, or a maleimide group.

[0090]Preferably, the first polynucleotide or second polynucleotide comprises a sequence selected from 20-mer poly-CA, 20-mer poly-GGTT, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 8; 9, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 , SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 7 and SEQ ID NO: 10.

[0091]Preferably, the binding affinity of the targeting moiety for the biomarker is less than 250 nM or the binding affinity of the targeting moiety for the biomarker is 5 nM, 10 nM, 40 nM, 90 nM, 130 nM or 170 nM.

[0092]Preferably, the length of the first polynucleotide or the length of the second polynucleotide is 4 nt to 500 nt. Preferably, the length of the first polynucleotide or the length of the second polynucleotide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100, 120, 160, 220, 300, 400 or 480 NT.

[0093]Preferably the binding affinity between the first ligand and the second ligand is less than 250 nM. Preferably the binding affinity between the first ligand and the second ligand is 5 nM, 10 nM, 40 nM, 90 nM, 130 nM or 170 nM.

[0094]Preferably, the first linker or the second linker is conjugated to a functional group native to the targeting moiety or a cell surface, where the native functional group is an amino acid, a sugar or an amine.

[0095]Preferably the targeting moiety is a peptide, protein or aptamer.

[0096]The present invention provides a composition substantially enriched in human CD16⁺natural killer cells in which the human CD16 count⁺natural killer cells in the composition is at least 5×10⁵and human CD16⁺natural killer cells are in an amount equal to or greater than 5% in number based on the total number of cells in the composition as 100%; the human CD16⁺natural killer cell is (A) deposited with NPMD with NITE accession number BP-03017; or (B) having the following characteristics:

[0097]i) expresses a CD16 receptor,

[0098]ii) retain its ability to proliferate for at least 3 months after subculture, and

[0099]iii) x) excluding synthetic, genetically modified and/or intentionally introduced polynucleotide encoding the CD16 receptor, or y) using the ddPCR system to analyze the genomic DNA of the cell, the ratio of the probe-detectable DNA molecule CD16 F176F to the CD16 F176V probe -detectable DNA molecule is equal to or greater than 1, wherein the CD16 F176F probe sequence is SEQ ID NO:11 and the CD16 F176V probe sequence is SEQ ID NO:12.

[0100]Preferably, the CD16 receptor is a CD16a receptor or a CD16b receptor.

[0101]Preferably, a polynucleotide encoding the CD16 receptor is located on the q arm of chromosome 1 at position 1q23.3.

[0102]Preferably human CD16⁺Natural killer cells are not tumorigenic in an immunocompromised mouse.

[0103]Preferably human CD16 after being irradiated with γ-rays⁺Natural killer cells are not tumorigenic in an allogeneic individual.

[0104]Preferably, a polynucleotide encoding the CD16 receptor comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 19.

[0105]Preferably, the CD16 receptor comprises an amino acid sequence of SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 20.

[0106]Preferably human CD16⁺the natural killer cell further comprises an inactive tumor suppressor gene or a mutated and highly expressed oncogene.

[0107]Preferably human CD16⁺the natural killer cell is capable of mediating an antibody dependent cellular cytotoxicity (ADCC) response and the cell is a male cell.

[0108]Preferably human CD16⁺Natural killer cell further comprises at least one exogenous targeting moiety complexed to human CD16⁺A natural killer cell, wherein the exogenous targeting moiety comprises a targeting moiety characterized by (a) having specific binding to a biological marker in a target cell; (b) is not a nucleic acid; and (c) is not produced by human CD16⁺natural killer cell.

[0109]Preferably, the exogenous targeting moiety is complexed to human CD16⁺natural killer cell through an interaction between a first ligand conjugated to the targeting moiety and a second ligand conjugated to human CD16⁺natural killer cell.

[0110]Preferably the first linker is a first polynucleotide or the second linker is a second polynucleotide.

[0111]Preferably the targeting moiety comprises an antigen binding moiety.

[0112]Preferably, the first polynucleotide comprises a single-stranded region.

[0113]Preferably, the first linker is a first polynucleotide and the second linker is a second polynucleotide.

[0114]Preferably, the first linker and the second linker are selected from the group consisting of: a DNA binding domain and a DNA target; a leucine zipper and target DNA; biotin and avidin; biotin and streptavidin; calmodulin and calmodulin-binding protein; a hormone and a hormone receptor; lectin and a carbohydrate; a cell membrane receptor and a receptor ligand; an enzyme and a substrate; an antigen and an antibody; an agonist and an antagonist; polynucleotide hybridization sequences; an aptamer and a target; and a zinc finger and target DNA.

[0115]Preferably, the first linker comprises a first reactive group and the second linker comprises a second reactive group, and wherein the human CD16⁺the natural killer cell is complexed to the targeting moiety via a covalent bond formed by a reaction between the second reactive group and the first reactive group.

[0116]Preferably the targeting moiety comprises an antigen binding moiety.

[0117]Preferably the second linker comprises a PEG region.

[0118]Preferably the targeting fraction and human CD16⁺Natural killer cells are separated by a length of 1 nm to 400 nm.

[0119]Preferably, the exogenous targeting moiety comprises an antigen-binding moiety, and the antigen-binding moiety binds to a cancer antigen, glycolipid, glycoprotein, differentiation antigen cluster present on cells of a hematopoietic lineage, gamma-glutamyl transpeptidase, adhesion protein, hormone, growth factor, cytokine , ligand receptor, ion channel, membrane-bound form of a µ-immunoglobulin. -chain, alpha-fetoprotein, C-reactive protein, chromogranin A, epithelial mucin antigen, human epithelial-specific antigen, Lewis(a) antigen, multidrug resistance-related protein, neu oncogene protein, neuron-specific enolase, glycoprotein P, Multidrug Resistance Related Antigen, p170, Multidrug Resistance Related Antigen, Prostate Specific Antigen, NCAM, Ganglioside Molecule, MART-1, Heat Shock Protein, SialylTn, Tyrosinase, MUC-1, HER-2 /neu, KSA, PSMA, p53, RAS, EGF -R, VEGF, MAGE or other target antigen (marker) expressed by a target cell.

[0120]Preferably, the antigen-binding moiety is an antibody against a cancer antigen selected from HER2/neu (ERBB2), HER3 (ERBB3), EGFR, VEGF, VEGFR2, GD2, CTLA4, CD19, CD20, CD22, CD30, CD33 (Siglec-3 ), CD52 (CAMPATH-1 antigen), CD326 (EpCAM), CA-125 (MUC16), MMP9, DLL3, CD274 (PD-L1), CEA, MSLN (Mesothelin), CA19-9, CD73, CD205 (DEC205) , CD51, c-MET, TRAIL-R2, IGF-1R, CD3, MIF, folate receptor alpha (FOLR1), CSF1, OX-40, CD137, TfR, MUC1, CD25 (IL-2R), CD115 (CSF1R), IL1B, CD105 (Endoglin), KIR, CD47, CEA, IL-17A, DLL4, CD51, Angiopoietin 2, Neuropilin-1, CD37, CD223 (LAG-3), CD40, LIV-1 (SLC39A6), CD27 (TNFRSF7) , CD276 (B7-H3), Trop2, Claudin1 (CLDN1), PSMA, TIM-1 (HAVcr-1), CEACAM5, CD70, LY6E, BCMA, CD135 (FLT3), APRIL, TF(F3) , Nectin-4, FAP, GPC3, FGFR3, ICAM-1 (CD54), ROBO1, NKG2D Ligands, CD123, CS1/SLAMF7/CD319/CRACC, CD7, CD142 (Platelet Tissue Factor, Factor III, Tissue Factor), CD38, CD138, EGFR VIII, EGFR, EGFR806, EGFR family member, PD-1, ROR1, CSPG4, CLL-1 (CLEC12A), CD147, PSCA, EPHA2, GPRC5D, CD133, B7H6, DSC2, AE1 (SLC4A1), GUCY2C, CDH17, HPSE, CD24, MUC4, AFP- L3, SP17, DCLK1, CAIX (CA9), IL13RA2, IL13Ra, CD56, CD44v6, TCR beta chain, Chlorotoxin Ligands, Claudin-6, Claudin-18.2, EIIIB (Fibronectin), Glypican-1 (GPC1), PLAP (placental alkaline phosphatase), uPAR, HCMV glycoprotein B (gB), HLA-DR (Lym1 antibody target), tumor-associated integrin αvβ6, LunX, integrin αvβ3, folate receptor beta (FRβ), LILRB4, MISIIR (Mullerian substance type 2 receptor Inhibitor), 5T4, CD83 Ligand, HBsAg, CD171 (L1-CAM), TAG72 (TAG72 (Tumor Associated Glycoprotein 72)), B7-H4, CD166 (ALCAM), AC133 (PROM1), LeY, CD13 (TIM1) , CD117, TEM8 (ANTXR1), CD26, IL13Ra2, IGF1R, Muc3a, IL1RAP, TSLPR (CRLF2), LMP1, Siglec7, Siglec9, Epstein-Barr Virus gp350, CD1a, CLEC14A, MAGE-A1, MAGE-A4, Neurofilament M ( NEFM), HERV-K env protein, HLA-A*0201/NY-ESO-1(157-165) peptide, 2B4, TACI (TNFRSF13B), CD32A(131R), AXL, Lewis Y, CD80, CD86, ROR2, Killer cell immunoglobulin-like receptors (KIRs), a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, and combinations thereof.

[0121]Preferably, the targeting moiety is conjugated to the first polynucleotide using a linking group, wherein the linking group is an NHS ester, another activated ester, an alkyl or acyl halide, a bifunctional crosslinker, or a maleimide group.

[0122]Preferably, the first polynucleotide or second polynucleotide comprises a sequence selected from 20-mer poly-CA, 20-mer poly-GGTT, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 8; 9, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 , SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 7 and SEQ ID NO: 10.

[0123]Preferably, the binding affinity of the targeting moiety for the biomarker is less than 250 nM.

[0124]Preferably, the length of the first polynucleotide or the length of the second polynucleotide is 4 nt to 500 nt.

[0125]Preferably the binding affinity between the first ligand and the second ligand is less than 250 nM.

[0126]Preferably, the first linker or the second linker is conjugated to a functional group native to the targeting moiety or a surface of human CD16⁺natural killer cell where the native functional group is an amino acid, a sugar or an amine.

[0127]Preferably the targeting moiety is a peptide, protein or aptamer.

[0128]The present invention provides a method of obtaining a composition substantially enriched in human CD16⁺natural killer cells; the method comprising: (a) obtaining a human peripheral blood natural killer cell population derived from the cell population having ATCC accession number CRL-2407; (b) contacting the human peripheral blood natural killer cell population with an antibody specific for a CD16 receptor; and (c) separating the cells specifically bound by the antibody, thereby obtaining the composition substantially enriched in human CD16⁺natural killer cells; where human CD16⁺the natural killer cell is: (A) deposited with the NPMD under NITE accession number BP-03017; or (B) having the following characteristics:

[0129]i) expresses a CD16 receptor, and

[0130]ii) x) excluding synthetic, genetically modified and/or intentionally introduced polynucleotides encoding the CD16 receptor, or y) using the ddPCR system to analyze the genomic DNA of the cell, the ratio of the probeable DNA molecule CD16 F176F to CD16 F176V probe-detectable DNA molecule is equal to or greater than 1, wherein the CD16 F176F probe sequence is SEQ ID NO: 11 and the CD16 F176V probe sequence is SEQ ID NO: 12.

[0131]Preferably, the antibody is specific for at least one of a CD16a receptor and a CD16b receptor.

[0132]Preferably human CD16⁺Natural killer cells are able to retain their ability to proliferate for at least 1 week after subculture.

[0133]Preferably, an expressed polynucleotide encoding the CD16 receptor is located on the q-arm of chromosome 1 at position 1q23.3.

[0134]Preferably human CD16⁺Natural killer cells are not tumorigenic in an immunocompromised mouse.

[0135]Preferably human CD16 after being irradiated with γ-rays⁺Natural killer cells are not tumorigenic in an allogeneic individual.

[0136]Preferably, a polynucleotide encoding the CD16 receptor comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 19.

[0137]Preferably, the CD16 receptor comprises an amino acid sequence of SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 20.

[0138]Preferably human CD16⁺the natural killer cell further comprises an inactive tumor suppressor gene or a mutated and highly expressed oncogene.

[0139]Preferably in the composition is human CD16⁺natural killer cells are present in a number of 5% or more based on the total number of cells in the composition than 100%.

[0140]Preferably human CD16⁺Natural killer cells are capable of mediating an antibody-dependent cellular cytotoxicity (ADCC) response, and human CD16⁺Natural killer cells are male cells.

[0141]Preferably, step (c) comprises sub-steps: (c1) separating cells that are specifically bound by the antibody; (c2) contacting the cells specifically bound by the antibody in a vessel with a culture medium comprising human platelet lysate and IL-2; and (c3) culturing the cells for several days, thereby obtaining the composition substantially enriched in human CD16⁺natural killer cells.

[0142]Preferably the container is G-Rex culture devices.

[0143]Preferably, the container includes a bottom for seeding cells, and the bottom is air permeable and water impermeable.

[0144]The concentration of glucose dissolved in the culture medium is preferably 1500-5000 mg/l.

[0145]Preferably the human CD16 number⁺natural killer cells in the composition is at least 5×10⁵and human CD16⁺natural killer cells are present in a number of 5% or more based on the total number of cells in the composition than 100%.

[0146]The present invention provides a method for culturing and expanding human CD16⁺natural killer cells; wherein method (x) comprises contacting human CD16 in a container⁺natural killer cells with a culture medium comprising 0.5-10% v/v human platelet lysate and 100-3000 IU/mL LIL-2; and (y) culturing the cells for several days. Preferably, the culture medium comprises 1% by volume, 2% by volume, 3% by volume, 4% by volume, 5% by volume, 6% by volume, 7% by volume, 8% by volume %, 9%, 10%, 11%, 12%, 13%, 14% or 15% by volume human platelet lysate. Preferably, the culture medium comprises 0.5-20% by volume human platelet lysate. Preferably, the culture medium comprises 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2100, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 IU/mL IL-2.

[0147]Preferably the container is G-Rex culture devices.

[0148]Preferably, the container includes a bottom for seeding cells, and the bottom is air permeable and water impermeable.

[0149]The concentration of glucose dissolved in the culture medium is preferably 1500-5000 mg/l.

[0150]Preferably, step (y) comprises sub-steps: (y1) culturing the cells for at least one day; and (y2) subculturing the cells for at least 1 month.

[0151]Preferably human CD16⁺Natural killer cells are able to retain their ability to proliferate for at least 3 months after subculture.

[0152]Preferably human CD16⁺the natural killer cell is: (A) deposited with the NPMD under NITE accession number BP-03017; or (B) having the following characteristics:

[0153]i) expresses a CD16 receptor, and

[0154]ii) x) excluding synthetic, genetically modified and/or intentionally introduced polynucleotide encoding the CD16 receptor, or y) using the ddPCR system to analyze the genomic DNA of the cell, the ratio of the probe-detectable DNA molecule CD16 F176F to the CD16 F176V probe -detectable DNA molecule is equal to or greater than 1, wherein the CD16 F176F probe sequence is SEQ ID NO:11 and the CD16 F176V probe sequence is SEQ ID NO:12

[0155]Preferably human CD16⁺Natural killer cells are not tumorigenic in an immunocompromised mouse.

[0156]Preferably human CD16 after being irradiated with γ-rays⁺Natural killer cells are not tumorigenic in an allogeneic individual.

[0157]Preferably, a polynucleotide encoding the CD16 receptor comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 19.

[0158]Preferably, the CD16 receptor comprises an amino acid sequence of SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 20.

[0159]Preferably human CD16⁺the natural killer cell further comprises an inactive tumor suppressor gene or a mutated and highly expressed oncogene.

[0160]The present invention provides a method of treating cancer, autoimmune disease, neuronal disease, human immunodeficiency virus (HIV) infection, hematopoietic cell-related disease, metabolic syndrome, pathogenic disease, viral infection or bacterial infection comprising administering a composition comprising an effective amount of a human natural killer cell for an individual in need thereof; the human natural killer cell is (A) deposited with NPMD with NITE accession number BP-03017; or (B) having the following characteristics:

[0161]i) expresses a CD16 receptor,

[0162]ii) maintaining its ability to proliferate after subculture for at least 3 months and iii) x) no synthetic, genetically modified and/or intentionally introduced polynucleotide encoding the CD16 receptor, or y) using the ddPCR system to analyze genomic DNA of human natural killer cell, the ratio of the DNA molecule detectable by the CD16 F176F probe to the DNA molecule detectable by the CD16 F176V probe is greater than 1, wherein the sequence of the CD16 F176F probe is SEQ ID NO: 11 and the sequence of the CD16 F176V probe is SEQ ID NO: 12.

[0163]Preferably, the human natural killer cell further comprises at least one exogenous targeting moiety complexed to the human natural killer cell, the exogenous targeting moiety comprising a targeting moiety characterized by: (a) exhibiting specific binding to a biological marker in a target cell; (b) is not a nucleic acid; and (c) it is not produced by the human natural killer cell.

[0164]Preferably, the exogenous targeting moiety is complexed with the human natural killer cell via an interaction between a first ligand conjugated with the targeting moiety and a second ligand conjugated with the human natural killer cell.

[0165]Preferably the first linker is a first polynucleotide or the second linker is a second polynucleotide.

[0166]Preferably the targeting moiety comprises an antigen binding moiety.

[0167]Preferably, the first polynucleotide comprises a single-stranded region.

[0168]Preferably, the first linker is a first polynucleotide and the second linker is a second polynucleotide.

[0169]Preferably, the first linker and the second linker are selected from the group consisting of: a DNA binding domain and a DNA target; a leucine zipper and target DNA; biotin and avidin; biotin and streptavidin; calmodulin and calmodulin-binding protein; a hormone and a hormone receptor; lectin and a carbohydrate; a cell membrane receptor and a receptor ligand; an enzyme and a substrate; an antigen and an antibody; an agonist and an antagonist; polynucleotide hybridization sequences; an aptamer and a target; and a zinc finger and target DNA.

[0170]Preferably, the first linker comprises a first reactive group and the second linker comprises a second reactive group, and wherein the human natural killer cell is targeted to the targeting via a covalent bond formed by a reaction between the second reactive group and the first reactive group -Unit is complexed group.

[0171]Preferably the targeting moiety comprises an antigen binding moiety.

[0172]Preferably the second linker comprises a PEG region.

[0173]Preferably, the targeting moiety and the human natural killer cell are separated by a length of 1 nm to 400 nm.

[0174]Preferably, the exogenous targeting moiety comprises an antigen-binding moiety, and the antigen-binding moiety binds to a cancer antigen, glycolipid, glycoprotein, differentiation antigen cluster present on cells of a hematopoietic lineage, gamma-glutamyl transpeptidase, adhesion protein, hormone, growth factor, cytokine , ligand receptor, ion channel, membrane-bound form of a µ-immunoglobulin. -chain, alpha-fetoprotein, C-reactive protein, chromogranin A, epithelial mucin antigen, human epithelial-specific antigen, Lewis(a) antigen, multidrug resistance-related protein, neu oncogene protein, neuron-specific enolase, glycoprotein P, Multidrug Resistance Related Antigen, p170, Multidrug Resistance Related Antigen, Prostate Specific Antigen, NCAM, Ganglioside Molecule, MART-1, Heat Shock Protein, SialylTn, Tyrosinase, MUC-1, HER-2 /neu, KSA, PSMA, p53, RAS, EGF -R, VEGF, MAGE or other target antigen (marker) expressed by a target cell.

[0175]Preferably, the antigen-binding moiety is an antibody against a cancer antigen selected from HER2/neu (ERBB2), HER3 (ERBB3), EGFR, VEGF, VEGFR2, GD2, CTLA4, CD19, CD20, CD22, CD30, CD33 (Siglec-3 ), CD52 (CAMPATH-1 antigen), CD326 (EpCAM), CA-125 (MUC16), MMP9, DLL3, CD274 (PD-L1), CEA, MSLN (Mesothelin), CA19-9, CD73, CD205 (DEC205) , CD51, c-MET, TRAIL-R2, IGF-1R, CD3, MIF, folate receptor alpha (FOLR1), CSF1, OX-40, CD137, TfR, MUC1, CD25 (IL-2R), CD115 (CSF1R), IL1B, CD105 (Endoglin), KIR, CD47, CEA, IL-17A, DLL4, CD51, Angiopoietin 2, Neuropilin-1, CD37, CD223 (LAG-3), CD40, LIV-1 (SLC39A6), CD27 (TNFRSF7) , CD276 (B7-H3), Trop2, Claudin1 (CLDN1), PSMA, TIM-1 (HAVcr-1), CEACAM5, CD70, LY6E, BCMA, CD135 (FLT3), APRIL, TF(F3) , Nectin-4, FAP, GPC3, FGFR3, ICAM-1 (CD54), ROBO1, NKG2D Ligands, CD123, CS1/SLAMF7/CD319/CRACC, CD7, CD142 (Platelet Tissue Factor, Factor III, Tissue Factor), CD38, CD138, EGFR VIII, EGFR, EGFR806, EGFR family member, PD-1, ROR1, CSPG4, CLL-1 (CLEC12A), CD147, PSCA, EPHA2, GPRC5D, CD133, B7H6, DSC2, AE1 (SLC4A1), GUCY2C, CDH17, HPSE, CD24, MUC4, AFP- L3, SP17, DCLK1, CAIX (CA9), IL13RA2, IL13Ra, CD56, CD44v6, TCR beta chain, Chlorotoxin Ligands, Claudin-6, Claudin-18.2, EIIIB (Fibronectin), Glypican-1 (GPC1), PLAP (placental alkaline phosphatase), uPAR, HCMV glycoprotein B (gB), HLA-DR (Lym1 antibody target), tumor-associated integrin αvβ6, LunX, integrin αvβ3, folate receptor beta (FRβ), LILRB4, MISIIR (Mullerian substance type 2 receptor Inhibitor), 5T4, CD83 Ligand, HBsAg, CD171 (L1-CAM), TAG72 (TAG72 (Tumor Associated Glycoprotein 72)), B7-H4, CD166 (ALCAM), AC133 (PROM1), LeY, CD13 (TIM1) , CD117, TEM8 (ANTXR1), CD26, IL13Ra2, IGF1R, Muc3a, IL1RAP, TSLPR (CRLF2), LMP1, Siglec7, Siglec9, Epstein-Barr Virus gp350, CD1a, CLEC14A, MAGE-A1, MAGE-A4, Neurofilament M ( NEFM), HERV-K env protein, HLA-A*0201/NY-ESO-1(157-165) peptide, 2B4, TACI (TNFRSF13B), CD32A(131R), AXL, Lewis Y, CD80, CD86, ROR2, Killer cell immunoglobulin-like receptors (KIRs), a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, and combinations thereof.

[0176]The HER2/neu cancer antigen (ERBB2) is an antigen encoded by the HER2/neu (ERBB2) gene. The HER2/neu (ERBB2) gene, located on but not limited to chromosome 17 q arm 12, encodes a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. This protein does not have its own ligand-binding domain and therefore cannot bind growth factors. The gene ID on the NCBI is 2064, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/2064.

[0177]The HER3 cancer antigen (ERBB3) is an antigen encoded by the HER3 gene (ERBB3). The HER3 gene (ERBB3), located on, but not limited to, chromosome 12 q arm 13.2, encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. This membrane-bound protein has a neuregulin-binding domain but no active kinase domain. Therefore, it can bind to this ligand but cannot transmit the signal to the cell through protein phosphorylation. This protein does not have its own ligand-binding domain and therefore cannot bind growth factors. The gene ID on the NCBI is 2065, but not limited to. See https://www.ncbi.nlm.nih.gov/gene/2065.

[0178]The EGFR cancer antigen is an antigen encoded by the EGFR gene. The EGFR gene is located on, but not limited to, the 11.2 arm of chromosome 7p. The protein encoded by this gene is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor, thereby inducing receptor dimerization and tyrosine autophosphorylation, leading to cell proliferation. The gene ID on the NCBI is 1956, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/1956.

[0179]The VEGF cancer antigen is an antigen encoded by the VEGF gene. The VEGF gene is located on, but not limited to, chromosome 6. Arm 21.1 is a member of the PDGF/VEGF growth factor family. It encodes a heparin-binding protein present as a disulfide-linked homodimer. This growth factor induces the proliferation and migration of vascular endothelial cells and is essential for physiological and pathological angiogenesis. Disruption of this gene in mice led to abnormal formation of embryonic blood vessels. This gene is upregulated in many known tumors and its expression correlates with tumor stage and progression. Elevated levels of this protein are found in patients with POEMS syndrome, also known as Crow's fukase syndrome. The Gene ID on the NCBI is 7422, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/7422.

[0180]The cancer antigen VEGFR2 is an antigen encoded by the VEGFR2 gene. The VEGFR2 gene is located on, but not limited to, chromosome 4 q arm 12 and encodes one of the two VEGF receptors. This receptor, known as the kinase insertion domain receptor, is a type III tyrosine kinase receptor. It functions as the major mediator of VEGF-induced endothelial proliferation, survival, migration, tubular morphogenesis, and budding. The gene ID on the NCBI is 3791, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/3791.

[0181]The carcinogenic antigen GD2 is a disialoganglioside expressed in tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with severely restricted expression in normal tissues, mainly in the cerebellum and peripheral nerves in humans. The gene ID on the NCBI is 6644, but not limited to that. See https://www.immunol.org/content/181/9/6644.

[0182]The CTLA4 cancer antigen is an antigen encoded by the CTLA4 gene. The CTLA4 gene, located on but not limited to chromosome 2 q arm 33.2, is a member of the immunoglobulin superfamily and encodes a protein that transmits an inhibitory signal to T cells. The protein contains a V domain , a transmembrane domain and a cytoplasmic tail. Alternative transcriptional splicing variants encoding different isoforms have been characterized. The membrane-bound isoform functions as a homodimer linked by a disulfide bond, while the soluble isoform functions as a monomer. The Gene ID on the NCBI is 1493, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/1493.

[0183]The CD19 cancer antigen is an antigen encoded by the CD19 gene. The CD19 gene, located on but not limited to the 11.2 arm of chromosome 16p, encodes a member of the immunoglobulin gene superfamily. Expression of this cell surface protein is restricted to B cell lymphocytes. This protein is a reliable marker for pre-B cells, but its expression decreases during differentiation of terminal B cells into antibody-secreting plasma cells. The protein has two extracellular N-terminal Ig-like domains separated by a non-Ig-like domain, a hydrophobic transmembrane domain, and a large C-terminal cytoplasmic domain. The gene ID on the NCBI is 930, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/930.

[0184]The CD20 cancer antigen is an antigen encoded by the CD20 gene. The CD20 gene, located on but not limited to chromosome 11 q arm 12.2, encodes a membrane-spanning member of the 4A gene family. Members of this nascent family of proteins are characterized by shared structural features and similar intron/exon junction boundaries, and display unique expression patterns between hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that is involved in the development and differentiation of B cells into plasma cells. The Gene ID on the NCBI is 931, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/931.

[0185]The CD22 cancer antigen is an antigen encoded by the CD22 gene. The CD22 gene, located on but not limited to chromosome 19 q arm 13.12, is a molecule belonging to the SIGLEC family of lectins that specifically binds to sialic acid with an immunoglobulin ( Ig) domain is located. The presence of Ig domains makes CD22 a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling. The Gene ID on the NCBI is 933, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/933.

[0186]The CD30 cancer antigen is an antigen encoded by the CD30 gene. The CD30 gene, located on but not limited to the p36.22 arm of chromosome 1, encodes a member of the TNF receptor superfamily. This receptor is expressed by activated T and B cells but not by resting B cells. TRAF2 and TRAF5 can interact with this receptor and mediate the signal transduction that leads to activation of NF-kappaB. This receptor is a positive regulator of apoptosis and has also been shown to limit the proliferative potential of self-reactive CD8 effector T cells and protect the body from autoimmunity. The gene ID on the NCBI is 943, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/943.

[0187]The cancer antigen CD33 (Siglec-3) is an antigen encoded by the CD33 (Siglec-3) gene. The CD33 gene (Siglec-3), located inter alia on chromosome 19 q arm 13.41, encodes a transmembrane receptor that is expressed in cells of the myeloid lineage. It binds to sialic acids, making it a member of the SIGLEC family of lectins. The Gene ID on the NCBI is 945, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/945.

[0188]The CD52 cancer antigen (CAMPATH-1 antigen) is an antigen encoded by the CD52 gene (CAMPATH-1 antigen). The CD52 gene (CAMPATH-1 antigen), located but not limited to the p36.11 arm of chromosome 1, encodes a glycoprotein present on the surface of mature lymphocytes but not on the stem cells from which these lymphocytes originate. CD52 binds to the ITIM-bearing sialic acid-binding lectin (immune receptor tyrosine-based motif inhibitor) SIGLEC10. The gene ID is 1043, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/1043.

[0189]The CD326 cancer antigen (EpCAM) is an antigen encoded by the CD326 gene (EpCAM). The CD326 (EpCAM) gene, located in but not limited to arm 21 of chromosome 2p, encodes a carcinoma-associated antigen and is a member of a family that includes at least two type I membrane proteins, normal epithelial cells and gastrointestinal carcinomas and as a homotype functions calcium-independent cell adhesion molecule. The antigen is used as a target for immunotherapeutic treatment of human carcinoma. The gene ID on the NCBI is 4072, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4072.

[0190]Cancer antigen CA-125 (MUC16) is an antigen encoded by the CA-125 (MUC16) gene. The CA-125 gene (MUC16), located on but not limited to arm 13.2 of chromosome 19p, encodes a protein that is a member of the mucin family. Mucins are high molecular weight O-glycosylated proteins that play an important role in the formation of a protective mucosal barrier and are found on the apical surfaces of epithelia. The encoded protein is a membrane-bound mucin containing an extracellular domain at its amino terminus, a large tandem repeat domain, and a transmembrane domain with a short cytoplasmic domain. The Gene ID on the NCBI is 94025, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/94025.

[0191]The MMP9 cancer antigen is an antigen encoded by the MMP9 gene. The MMP9 gene, located on but not limited to chromosome 20 q arm 13.12, encodes a 92 kDa type IV collagenase, 92 kDa gelatinase, or gelatinase B (YELLOW), is a matrixin, a class of enzymes that belong to the zinc-zinc family. Metalloproteinases involved in the breakdown of the extracellular matrix. The Gene ID on the NCBI is 4318, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4318.

[0192]The DLL3 cancer antigen is an antigen encoded by the DLL3 gene. The DLL3 gene, located on but not limited to chromosome 19 q arm 13.2, encodes a member of the Delta protein-ligand family. This family functions as Notch ligands characterized by a DSL domain, EGF repeats, and a transmembrane domain. The gene ID on the NCBI is 10683, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/10683.

[0193]The cancer antigen CD274 (PD-L1) is an antigen encoded by the CD274 (PD-L1) gene. The CD274 (PD-L1) gene, located on but not limited to the 24.1 arm of chromosome 9p, encodes an immunosuppressive receptor ligand expressed by hematopoietic and non-hematopoietic cells such as T cells and B cells and different tumor types is expressed cells . The encoded protein is a type I transmembrane protein that has V-type and C-type immunoglobulin domains. The interaction of this ligand with its receptor inhibits T cell activation and cytokine production. The gene ID on the NCBI is 29126, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/29126.

[0194]The CEA cancer antigen is an antigen encoded by the CEA gene. The CEA gene, located on but not limited to chromosome 19 q arm 13.2, encodes a cell surface glycoprotein that is the founding member of the carcinoembryonic antigen (CEA) family of proteins. The encoded protein is used as a clinical biomarker for gastrointestinal cancer and may promote tumor development through its role as a cell adhesion molecule. In addition, the encoded protein can regulate cell differentiation, apoptosis and polarity. The gene ID on the NCBI is 1048, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/1048.

[0195]The MSLN (mesothelin) cancer antigen is an antigen encoded by the MSLN (mesothelin) gene. The MSLN (mesothelin) gene, located on but not limited to chromosome 16 p arm 13.3, encodes a pre-proprotein that is proteolytically processed to produce two protein products, megakaryocyte potentiating factor and mesothelin. Megakaryocyte-elevating factor acts as a cytokine capable of stimulating the formation of megakaryocyte colonies in the bone marrow. Mesothelin is a glycosylphosphatidylinositol-anchored cell surface protein that can function as a cell adhesion protein. This protein is overexpressed in epithelial mesothelioma, ovarian cancer and in certain squamous cell carcinomas. The gene ID on the NCBI is 10232, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/10232.

[0196]The CD73 cancer antigen is an antigen encoded by the CD73 gene. The CD73 gene, located on but not limited to chromosome 6 q arm 14.3, encodes a plasma membrane protein that catalyzes the conversion of extracellular nucleotides into membrane-permeable nucleosides. The encoded protein is used as a determinant of lymphocyte differentiation. Defects in this gene can lead to calcification of joints and arteries. The Gene ID on the NCBI is 4907, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4907.

[0197]The CD205 cancer antigen (DEC205) is an antigen encoded by the CD205 gene (DEC205). The CD205 (DEC205) LY75 gene, located on but not limited to chromosome 2 q-arm 24.2, encodes a CD205 or DEC-205 protein. The gene ID on the NCBI is 4065, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4065.

[0198]The CD51 cancer antigen is an antigen encoded by the CD51 gene. The CD51 gene, located on but not limited to chromosome 2 q arm 32.1, encodes the integrin alpha chain family. Integrins are heterodimeric integral membrane proteins composed of an alpha subunit and a beta subunit that function in adhesion and cell surface signaling. The encoded preproprotein undergoes proteolytic processing to generate light and heavy chains that make up the V-alpha subunit. This subunit associates with the beta-1, beta-3, beta-5, beta-6, and beta -8 subunits.Alpha V and Beta 3 subunits is also known as the vitronectin receptor. This integrin can regulate angiogenesis and cancer progression. The gene ID on the NCBI is 3685, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3685)

[0199]The c-MET cancer antigen is an antigen encoded by the c-MET gene. The c-MET gene, located on but not limited to chromosome 7 q arm 31.2, encodes a member of the tyrosine kinase receptor protein family and the MET proto-oncogene product. The encoded preproprotein is proteolytically processed to generate alpha and beta subunits, which are linked by disulfide bonds to form the mature receptor. Further processing of the beta subunit leads to the formation of the M10 peptide, which has been shown to reduce pulmonary fibrosis. Binding of its ligand, hepatocyte growth factor, induces dimerization and activation of the receptor, which plays a role in cell survival, embryogenesis, migration, and cell invasion. Mutations in this gene are associated with papillary renal cell carcinoma, hepatocellular carcinoma, and several types of head and neck cancer. Amplification and overexpression of this gene is also linked to several human cancers. The Gene ID on the NCBI is 4233, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4233)

[0200]TRAIL-R2 cancer antigen is an antigen encoded by the TRAIL-R2 gene. The TRAIL-R2 gene, located but not limited to arm 21.3 of chromosome 8p, encodes a member of the TNF receptor superfamily and contains an intracellular death domain. This receptor can be activated by tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL/APO-2L) and transduces an apoptosis signal. Studies in FADD-deficient mice have suggested that FADD, a death domain containing an adapter protein, is required for FADD-mediated apoptosis. The Gene ID on the NCBI is 8795, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/8795.

[0201]The IGF-1R cancer antigen is an antigen encoded by the IGF-1R gene. The IGF-1R gene, located on but not limited to chromosome 15 q arm 26.3, encodes a high affinity receptor-binding insulin-like growth factor. It has tyrosine kinase activity. Insulin-like growth factor receptor I plays a crucial role in transformation events. Precursor fission produces alpha and beta subunits. It is highly overexpressed in most malignant tissues, where it acts as an antiapoptotic agent and improves cell survival. The gene ID on the NCBI is 3480, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3480.

[0202]The MIF cancer antigen is an antigen encoded by the MIF gene. The MIF gene, located on but not limited to chromosome 22 q arm 11.23, encodes a lymphokine involved in cell-mediated immunity, immune regulation and inflammation. It plays a role in regulating macrophage function in host defenses by suppressing the anti-inflammatory effects of glucocorticoids. This lymphokine and the JAB1 protein form a complex in the cytosol near the peripheral plasma membrane, which may indicate an additional role in integrin signaling. The Gene ID on the NCBI is 4282, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4282)

[0203]The cancer antigen folate receptor alpha (FOLR1) is an antigen encoded by the folate receptor alpha gene (FOLR1). The folate receptor alpha gene (FOLR1), located on but not limited to chromosome 11q, arm 13.4, encodes a member of the folate receptor family. This gene product is a secreted protein anchored to membranes via a glycosyl phosphatidylinositol linkage or in a soluble form. Mutations in this gene have been linked to neurodegeneration due to impaired cerebral folate transport. The gene ID on NCBI is 2348, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/2348.

[0204]The CSF1 cancer antigen is an antigen encoded by the CSF1 gene. The CSF1 gene, located but not limited to the p13.3 arm of chromosome 1, encodes a cytokine that controls macrophage production, differentiation, and function. The active form of the protein is found extracellularly as a disulfide-linked homodimer and is thought to be produced by proteolytic cleavage of membrane-bound precursors. The encoded protein may be involved in the development of the placenta. The Gene ID on the NCBI is 1435, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/1435.

[0205]The CSF1 cancer antigen is an antigen encoded by the CSF1 gene. The OX-40 gene, located on but not limited to the p36.33 arm of chromosome 1, encodes a member of the TNF receptor superfamily. This receptor has been shown to activate NF-kappaB through its interaction with the adapter proteins TRAF2 and TRAF5. Knockout studies in mice indicated that this receptor promotes expression of the apoptosis inhibitors BCL2 and BCL21L1/BCL2-XL, thereby suppressing apoptosis. Knockout studies have also suggested a role for this receptor in the CD4+ T cell response as well as in T cell dependent B cell proliferation and differentiation. The gene ID on the NCBI is 7293, but not limited thereto . See https://www.ncbi.nlm.nih.gov/gene/7293.

[0206]The CD137 cancer antigen is an antigen encoded by the CD137 gene. The CD137 gene, located on but not limited to the p36.23 arm of chromosome 1, encodes a member of the TNF receptor superfamily. This receptor contributes to clonal expansion, survival and development of T cells. It can also induce proliferation in peripheral monocytes, enhance T cell apoptosis induced by TCR/CD3-triggered activation and regulate CD28 costimulation, to promote Th1 cell responses. Expression of this receptor is induced by activation of lymphocytes. TRAF adapter proteins have been shown to bind to this receptor and relay the signals leading to NF-kappaB activation. The Gene ID on the NCBI is 3604, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/3604.

[0207]The TfR cancer antigen is an antigen encoded by the TfR gene. The TfR gene, located on but not limited to chromosome 3q arm 29, encodes a cell surface receptor required for cellular iron uptake through the process of receptor-mediated endocytosis. This receptor is necessary for erythropoiesis and neurodevelopment. The Gene ID on the NCBI is 7037, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/7037.

[0208]The MUC1 cancer antigen is an antigen encoded by the MUC1 gene. The MUC1 gene, located on but not limited to chromosome 1 q arm 22, encodes a membrane-bound protein that is a member of the mucin family. Mucins are O-glycosylated proteins that play an essential role in forming protective mucosal barriers on epithelial surfaces. These proteins also play a role in intracellular signaling. This protein is expressed on the apical surface of epithelial cells lining the mucosal surfaces of many different tissues, including the lung, breast, stomach, and pancreas. This protein is proteolytically cleaved into alpha and beta subunits that form a heterodimeric complex. The N-terminal alpha subunit functions in cell adhesion and the C-terminal beta subunit is involved in cell signaling. Overexpression, aberrant intracellular localization, and changes in glycosylation of this protein have been associated with carcinoma. The gene ID on the NCBI is 4582, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/4582.

[0209]The CD25 cancer antigen (IL-2R) is an antigen encoded by the CD25 gene (IL-2R). The CD25 (IL-2R) gene, located inter alia on the 15.1 arm of chromosome 10p, encodes the IL-2 alpha receptor (IL2RA) and together with the common gamma beta chain (IL2RB) forms (IL2RG) the IL2 receptor high affinity. The alpha homodimeric chains (IL2RA) give rise to low affinity receptors, while the beta homodimeric chains (IL2RB) produce an intermediate affinity receptor. Normally an integral membrane protein, soluble IL2RA, was isolated and characterized as the result of extracellular proteolysis. The gene ID on NCBI is 3559, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3559.

[0210]The CD115 cancer antigen (CSF1R) is an antigen encoded by the CD115 gene (CSF1R). The CD115 gene (CSF1R), located on but not limited to chromosome 5 q arm 32, encodes the receptor for colony stimulating factor 1, a cytokine that controls macrophage production, differentiation and function. This receptor mediates most if not all of the biological effects of this cytokine. Ligand binding activates the receptor kinase through a process of oligomerization and transphosphorylation. The encoded protein is a transmembrane receptor tyrosine kinase and a member of the CSF1/PDGF receptor family of protein tyrosine kinases. The Gene ID on the NCBI is 1436, but not limited to. See https://www.ncbi.nlm.nih.gov/gene/1436.

[0211]The IL1B cancer antigen is an antigen encoded by the IL1B gene. The IL1B gene, located on but not limited to chromosome 2 q arm 14.1, encodes a member of the cytokine family interleukin 1. This cytokine is produced by activated macrophages as a proprotein that is proteolytically processed by caspase 1 to its active form ( CASP1/ICE). This cytokine is an important mediator of the inflammatory response and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. Induction of cyclooxygenase-2 (PTGS2/COX2) by this cytokine in the central nervous system (CNS) contributes to hypersensitivity to inflammatory pain. Likewise, IL-1B has been implicated in the pathogenesis of human osteoarthritis. The gene ID on the NCBI is 3553, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3553.

[0212]The CD105 (endoglin) cancer antigen is an antigen encoded by the CD105 (endoglin) gene. The CD105 (endoglin) gene, located on but not limited to chromosome 9 q arm 34.11, encodes a homodimeric transmembrane protein that is a major glycoprotein of the vascular endothelium. This protein is part of the transforming growth factor beta receptor complex and binds beta1 and beta3 peptides with high affinity. Mutations in this gene cause hereditary hemorrhagic telangiectasia, also known as Osler-Rendu-Weber syndrome1, an autosomal dominant multisystemic vascular dysplasia. This gene may also be involved in preeclampsia and various types of cancer. The Gene ID on NCBI is 2022, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/2022.

[0213]The CD47 cancer antigen is an antigen encoded by the CD47 gene. The CD47 gene, located but not limited to chromosome 3 q arm 13.12, encodes a membrane protein involved in the increase in intracellular calcium concentration that occurs after cell adhesion to the extracellular matrix. The encoded protein is also a receptor for the C-terminal cell-binding domain of thrombospondin and may play a role in membrane transport and signal transduction. This gene has a wide tissue distribution and its expression is reduced in Rh erythrocytes. The Gene ID on the NCBI is 961, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/961.

[0214]The CEA cancer antigen is an antigen encoded by the CEA gene. The CEA gene, located on but not limited to chromosome 19q, arm 13.2, encodes a member of the family of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which are used by various bacterial pathogens to attach to host cells and penetrate into them. The encoded transmembrane protein directs the phagocytosis of several bacterial species in dependence on the small GTPase Rac. It is believed to play an important role in the innate immune system's control of specific human pathogens. The gene ID on the NCBI is 1084, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/1084.

[0215]The IL-17A cancer antigen is an antigen encoded by the IL-17A gene. The IL-17A gene, located on but not limited to the 12.2 arm of chromosome 6p, encodes a member of the IL-17 receptor family that includes five members (IL-17RA-E), and that encoded protein is a pro-inflammatory cytokine produced by activating T cells. IL-17A-mediated downstream pathways induce the production of inflammatory molecules, chemokines, antimicrobial peptides, and remodeling proteins. The encoded protein has critical implications for host defenses, cellular trafficking, immune modulation and tissue repair, playing a key role in inducing innate immunity. This cytokine stimulates non-hematopoietic cells and promotes chemokine production, thereby attracting myeloid cells to sites of inflammation. This cytokine also regulates the activities of NF-kappaB and mitogen-activated protein kinases and can stimulate expression of IL6 and cyclooxygenase-2 (PTGS2/COX-2), in addition to increasing nitric oxide (NO) production. IL-17A plays a key role in several infectious, inflammatory, autoimmune and cancer diseases. High levels of this cytokine have been linked to several chronic inflammatory diseases, including rheumatoid arthritis, psoriasis, and multiple sclerosis. The Gene ID on the NCBI is 3605, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/3605.

[0216]The DLL4 cancer antigen is an antigen encoded by the DLL4 gene. The DLL4 gene, located on but not limited to chromosome 15 q arm 15.1, encodes a homologue ofDrosophilaDelta Gen. The Delta gene family encodes Notch ligands characterized by a DSL domain, EGF repeats, and a transmembrane domain. The Gene ID on NCBI is, but not limited to, 54567. See https://www.ncbi.nlm.nih.gov/gene/54567.

[0217]The CD51 cancer antigen is an antigen encoded by the CD51 gene. The CD51 gene, located on but not limited to chromosome 2 q-arm 32.1, encodes the product belonging to the integrin alpha chain family. Integrins are heterodimeric integral membrane proteins composed of an alpha subunit and a beta subunit that function in adhesion and cell surface signaling. The encoded preproprotein undergoes proteolytic processing to generate light and heavy chains that make up the V-alpha subunit. This subunit associates with the beta-1, beta-3, beta-5, beta-6, and beta -8 subunits.Alpha V and Beta 3 subunits is also known as the vitronectin receptor. This integrin can regulate angiogenesis and cancer progression. The gene ID on the NCBI is 3685, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3685.

[0218]The angiopoietin-2 cancer antigen is an antigen encoded by the angiopoietin-2 gene. The angiopoietin-2 gene, located on, but not limited to, the 23.1 arm of chromosome 8p, encodes the product that leads to angiopoietin -Family of growth factors. The protein encoded by this gene is an angiopoietin 1 antagonist, and both angiopoietin 1 and angiopoietin 2 are ligands for the endothelial receptor TEK tyrosine kinase. . The encoded protein affects angiogenesis during embryogenesis and tumorigenesis, disrupts the vascular remodeling capacity of angiopoietin 1, and can induce endothelial cell apoptosis. The gene ID on the NCBI is 285, but not limited to this. See https://www.ncbi.nlm.nih.gov/gene/285.

[0219]The Neuropilin-1 cancer antigen is an antigen encoded by the Neuropilin-1 gene. The Neuropilin 1 gene, located in arm 11.22 of chromosome 10p among others, encodes one of two neuropilins that contain specific protein domains that allow them to participate in several different types of signaling pathways that control cell migration. Neuropilins contain a large N-terminal extracellular domain composed of complement-fixing domains, coagulation factor V/VIII and meprin. These proteins also contain a short membrane-spanning domain and a small cytoplasmic domain. Neuropilins bind many ligands and several types of co-receptors; they affect cell survival, migration and attraction. Some of the ligands and co-receptors bound by neuropilins are vascular endothelial growth factor (VEGF) and members of the semaphorin family. The Gene ID on the NCBI is 8829, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/8829.

[0220]The CD37 cancer antigen is an antigen encoded by the CD37 gene. The CD37 gene, located on but not limited to chromosome 19 q arm 13.33, encodes a transmembrane member4Superfamily, also known as the tetraspanin family. Most of these members are cell surface proteins characterized by the presence of four hydrophobic domains. Proteins mediate signal transduction events that play a role in the regulation of cell development, activation, growth, and motility. This encoded protein is a cell surface glycoprotein that is known to form complexes with integrins and other transmembrane proteins.4Superfamily proteins. May play a role in T cell-B cell interactions. The Gene ID on the NCBI is 951, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/951.

[0221]Cancer antigen CD223 (LAG-3) is an antigen encoded by the CD223 (LAG-3) gene. The CD223 (LAG-3) gene, located but not limited to arm 13.31 of chromosome 12p, encodes lymphocyte activating protein 3, which belongs to the Ig superfamily and contains 4 Ig-like extracellular domains. The Gene ID on the NCBI is 3902, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/3902.

[0222]The CD40 cancer antigen is an antigen encoded by the CD40 gene. The CD40 gene, located on but not limited to the 13.12 arm of chromosome 20, encodes a receptor on antigen-presenting cells of the immune system and is responsible for mediating a variety of immune and inflammatory responses, including the cell-dependent immunoglobulin class , essential switching T, development of memory B cells and germinal center formation. The Gene ID on the NCBI is 958, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/958.

[0223]Cancer antigen LIV-1 (SLC39A6) is an antigen encoded by the LIV-1 gene (SLC39A6). The LIV-1 gene (SLC39A6), located inter alia on chromosome 18 q arm 12.2, encodes a protein belonging to a subfamily of proteins that exhibit structural properties of zinc transporters. The gene ID on NCBI is 25800, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/25800.

[0224]The CD27 cancer antigen (TNFRSF7) is an antigen encoded by the CD27 (TNFRSF7) gene. The CD27 gene (TNFRSF7), located in but not limited to arm 13.31 of chromosome 12p, encodes a member of the TNF receptor superfamily. This receptor is necessary for the generation and long-term maintenance of T-cell immunity, binds to CD70 ligand, and plays a key role in regulating B-cell activation and immunoglobulin synthesis. This receptor transmits signals that lead to the activation of NF-kappaB and MAPK8/INK. The adapter proteins TRAF2 and TRAF5 have been shown to mediate the signaling process of this receptor. The gene ID on the NCBI is 939, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/939.

[0225]The cancer antigen CD276 (B7-H3) is an antigen encoded by the CD276 (B7-H3) gene. The CD276 gene (B7-H3), located on but not limited to arm 24.1 of chromosome 15q, encodes a protein belonging to the immunoglobulin superfamily and is believed to be involved in the regulation of the by T-cell mediated immune response is ubiquitously expressed in normal tissues and solid tumors, the protein is preferentially expressed only in tumor tissues. The gene ID on the NCBI is 80381, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/80381.

[0226]The Trop2 cancer antigen is an antigen encoded by the Trop2 gene. The Trop2 gene, located on but not limited to the p 32.1 arm of chromosome 1, encodes a carcinoma-associated antigen. This antigen is a cell surface receptor that transmits calcium signals. The gene ID on the NCBI is 4070, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4070.

[0227]The cancer antigen Claudin1 (CLDN1) is an antigen encoded by the Claudin1 (CLDN1) gene. The Claudin1 (CLDN1) gene, located on but not limited to chromosome 17 p arm 13.1, encodes a member of the Claudin family. Claudins are integral membrane proteins and components of tight junction chains. Tight junction filaments serve as a physical barrier to prevent solutes and water from freely passing through the paracellular space between epithelial or endothelial cell layers, and also play a critical role in maintaining cell polarity and signaling. Differential expression of this gene has been observed in different types of malignancies including but not limited to breast cancer, ovarian cancer, hepatocellular carcinoma, urinary tumor, prostate cancer, lung cancer, head and neck cancer, thyroid carcinoma, etc. The gene ID on the NCBI is 1366. See https://www.ncbi.nlm.nih.gov/gene/1366.

[0228]The PSMA cancer antigen is an antigen encoded by the PSMA gene. The PSMA gene, located on but not limited to chromosome 11, p-arm 11.12, encodes a type II transmembrane glycoprotein belonging to the M28 peptidase family. The protein acts as a glutamate carboxypeptidase on various alternative substrates, including the nutrient folate and the neuropeptide N-acetyl-1-aspartyl-1-glutamate, and is expressed in various tissues such as the prostate, central and peripheral nervous system, and kidney. A mutation in this gene may be associated with impaired intestinal absorption of dietary folates, leading to low blood folate levels and consequent hyperhomocysteinemia. Expression of this protein in the brain may be involved in several pathological conditions associated with glutamate excitotoxicity. In the prostate, the protein is upregulated in cancer cells and used as an effective diagnostic and prognostic indicator of prostate cancer. The gene ID on the NCBI is 2346, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/2346.

[0229]The cancer antigen TIM-1 (HAVcr-1) is an antigen encoded by the TIM-1 gene (HAVcr-1). The TIM-1 gene (HAVcr-1), located inter alia on chromosome 5 q arm 33.3, encodes a membrane receptor for the human hepatitis A virus (HHAV) and TIMD4. The encoded protein may be involved in alleviating asthma and allergic diseases. The Gene ID on NCBI is 26762, but is not limited to this. See https://www.ncbi.nlm.nih.gov/gene/26762.

[0230]The CEACAM5 cancer antigen is an antigen encoded by the CEACAM5 gene. The CEACAM5 gene, located on but not limited to chromosome 19 q arm 13.2, encodes a cell surface glycoprotein that is the founding member of the carcinoembryonic antigen (CEA) family of proteins. The encoded protein is used as a clinical biomarker for gastrointestinal cancer and may promote tumor development through its role as a cell adhesion molecule. In addition, the encoded protein can regulate cell differentiation, apoptosis and polarity. The gene ID on the NCBI is 1048, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/1048.

[0231]The CD70 cancer antigen is an antigen encoded by the CD70 gene. Located on, but not limited to, the p13.3 arm of chromosome 19, the CD70 gene encodes a cytokine belonging to the tumor necrosis factor (TNF) family of ligands. This cytokine is a ligand for TNFRSF27/CD27. It is a surface antigen on activated but not quiescent T and B lymphocytes. It induces co-stimulated T cell proliferation, increases cytolytic T cell formation and contributes to T cell activation. This cytokine also plays a role in the regulation of B cell activation, natural killer cell cytotoxic function and of immunoglobulin synthesis. The gene ID on the NCBI is 970, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/970.

[0232]LY6E cancer antigen is an antigen encoded by the LY6E gene. The LY6E gene, located on but not limited to chromosome 8 q arm 24.3, encodes a protein whose increased expression is associated with poor survival outcomes in various malignancies, as determined by a search of more than 130 published clinical studies on gene expression studies was determined in specimens from cancerous tissue and adjacent normal tissue. The gene ID on the NCBI is 4061, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4061.

[0233]BCMA cancer antigen is an antigen encoded by the BCMA gene. The BCMA gene, located in but not limited to the p13.13 arm of chromosome 16, encodes a member of the TNF receptor superfamily. This receptor is preferentially expressed on mature B lymphocytes and may be important in B cell development and the autoimmune response. This receptor has been shown to specifically bind to the tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B/TALL-1/BAFF) and leads to activation of NF-kappaB and MAPK8/INK. This receptor also binds to several members of the TRAF family and can therefore transmit signals for cell survival and proliferation. The Gene ID on the NCBI is 608, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/608.

[0234]CD135 cancer antigen (FLT3) is an antigen encoded by the CD135 (FLT3) gene. The CD135 (FLT3) gene, located on but not limited to chromosome 13 q arm 12.2, encodes a class III tyrosine kinase receptor that regulates hematopoiesis. This receptor is activated by binding of the fms-related tyrosine kinase-3 ligand to the extracellular domain, which induces the formation of homodimers in the plasma membrane, leading to autophosphorylation of the receptor. Activated receptor kinase subsequently phosphorylates and activates several cytoplasmic effector molecules in pathways involved in apoptosis, proliferation, and differentiation of hematopoietic cells in the bone marrow. Mutations that result in constitutive activation of this receptor result in acute myeloid leukemia and acute lymphoblastic leukemia. The Gene ID on the NCBI is 2322, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/2322.

[0235]The cancer antigen APRIL is an antigen encoded by the APRIL gene. The APRIL gene, located but not limited to arm 22.33 of chromosome 9q, encodes a member of the tumor necrosis factor superfamily whose alternative name, A Proliferation Inducing Ligand, shares the same acronym as ANP32B. The Gene ID on the NCBI is 10541, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/10541.

[0236]The cancer antigen Nectin-4 is an antigen encoded by the Nectin-4 gene. The nectin-4 gene, located on but not limited to chromosome 1 q arm 23.3, encodes a member of the nectin family. The encoded protein contains two immunoglobulin-like C2-like (Ig-like) domains and one Ig-like V-like domain. It is involved in cell adhesion through transhomophilic and -heterophilic interactions. It is a single pass type I membrane protein. The soluble form is produced by proteolytic cleavage on the cell surface by the metalloproteinase ADAM17/TACE. The secreted form is found in both breast tumor cell lines and in breast tumor patients. Mutations in this gene are the cause of ectodermal dysplasia syndactyly syndrome type 1, an autosomal recessive disorder. The Gene ID on the NCBI is 81607, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/81607.

[0237]The FAP cancer antigen is an antigen encoded by the FAP gene. The FAP gene, located on but not limited to chromosome 2 q arm 24.2, encodes a homodimeric integral membrane gelatinase belonging to the serine protease family. It is selectively expressed in reactive stromal fibroblasts of epithelial cancer, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas. This protein is thought to be involved in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis. The Gene ID on the NCBI is 2191, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/2191.

[0238]The GPC3 cancer antigen is an antigen encoded by the GPC3 gene. The GPC3 gene, located on but not limited to q-arm 26.2 of the X chromosome, encodes a membrane-associated protein core replaced with a variable number of heparan sulfate chains. via a glycosylphosphatidylinositol linkage. These proteins may play a role in controlling cell division and regulating growth. The protein encoded by this gene can bind to and inhibit CD26 dipeptidyl peptidase activity and induce apoptosis in certain cell types. Deletion mutations in this gene are associated with Simpson-Golabi-Behmel syndrome, also known as Simpson dysmorphic syndrome. The gene ID on the NCBI is 2719, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/2719.

[0239]The FGFR3 cancer antigen is an antigen encoded by the FGFR3 gene. The FGFR3 gene, located but not limited to arm 16.3 of chromosome 4p, encodes a member of the fibroblast growth factor receptor (FGFR) family, with its amino acid sequence being highly conserved between members and between different species. Members of the FGFR family differ from each other in their ligand affinity and tissue distribution. A representative full-length protein would consist of an extracellular region composed of three immunoglobulin-like domains, a single transmembrane hydrophobic segment, and a cytoplasmic tyrosine kinase domain. The extracellular portion of the protein interacts with fibroblast growth factors and initiates a cascade of downstream signals that affect mitogenesis and differentiation. This particular family member binds acidic and basic fibroblastic growth hormone and plays a role in bone development and maintenance. Mutations in this gene lead to craniosynostosis and various types of skeletal dysplasia. The Gene ID on the NCBI is 2261, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/2261.

[0240]The ROBO1 cancer antigen is an antigen encoded by the ROBO1 gene. The ROBO1 gene, located but not limited to arm 12.3 of chromosome 3p, encodes an integral membrane protein that functions in axon guidance and neuronal progenitor cell migration. This receptor is activated by SLIT family proteins, resulting in a repelling effect on the targeting of glioma cells in the developing brain. The product of this gene is a member of the immunoglobulin gene superfamily. The Gene ID on the NCBI is 6091, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/6091.

[0241]NKG2D ligands comprise a diverse family of ligands that include class I MHC chain-related proteins A and B and UL-16-binding proteins, where ligand-receptor interactions are used to activate NK and T-cells. Ligand is important for the immune system's recognition of stressed cells and therefore this protein and its ligands are therapeutic targets for the treatment of immune diseases and cancer. The NKG2D receptor is encoded by the KLRK1 gene, located on, but not limited to, the 13.2 arm of chromosome 12p. The gene ID of KLRK1 on the NCBI is, but not limited to, 22914. See https://www.ncbi.nlm.nih.gov/gene/22914.

[0242]The CD123 cancer antigen is an antigen encoded by the CD123 gene. The CD123 gene, located on but not limited to p-arm 22.33 of the X chromosome and p-arm 11.2 of the Y chromosome, encodes a specific interleukin-3 subunit of a heterodimeric cytokine receptor. The receptor consists of a ligand-specific alpha subunit and a signal transducer beta subunit that share receptors for interleukin 3 (IL3), colony stimulating factor 2 (CSF2/GM-CSF), and interleukin 5 (IL5). The binding of this protein to IL3 is dependent on the beta subunit. The beta subunit is activated by ligand binding and is required for IL3 biological activities. This gene and the gene encoding colony stimulating factor 2 receptor alpha chain (CSF2RA) form a cluster of cytokine receptor genes in a pseudoautosomal X-Y region on chromosomes X or Y. The NCBI gene ID is 3563, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3563.

[0243]The cancer antigen SLAMF7 (CS1/CS319) is an antigen encoded by the SLAMF7 gene (CS1/CS319). The SLAMF7 gene (CS1/CS319), located on but not limited to chromosome 1 q arm 23.3, encodes a robust marker for normal plasma cells and malignant plasma cells in multiple myeloma. Unlike CD138 (the traditional plasma cell marker), CD319/SLAMF7 is much more stable and allows for robust isolation of malignant plasma cells from delayed or even cryopreserved samples. The Gene ID on the NCBI is, but not limited to, 57823. See https://www.ncbi.nlm.nih.gov/gene/57823.

[0244]The CD7 cancer antigen is an antigen encoded by the CD7 gene. The CD7 gene, located on but not limited to chromosome 17 q arm 25.3, encodes a transmembrane protein that is a member of the immunoglobulin superfamily. This protein is found in thymocytes and mature T cells. It plays an essential role in T cell interactions and also in T/B cell interaction during early lymphoid development. The gene ID on the NCBI is 924, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/924.

[0245]The cancer antigen CD142 (coagulation factor F3 III) is an antigen encoded by the gene CD142 (coagulation factor F3 III). The CD142 gene (F3 coagulation factor III), located on but not limited to chromosome 1 p-arm 21.3, encodes coagulation factor III, which is a cell surface glycoprotein. This factor enables cells to initiate blood coagulation cascades and acts as a high affinity receptor for coagulation factor VII. The resulting complex provides a catalytic event responsible for the initiation of specific limited proteolysis-coagulation protease cascades. Unlike the other cofactors of these protease cascades, which circulate as non-functional precursors, this factor is a fully functional potent initiator when expressed on cell surfaces such as monocytes. There are 3 distinct domains of this factor: extracellular, transmembrane and cytoplasmic. Platelets and monocytes have been shown to express this coagulation factor under procoagulant and proinflammatory stimuli, and an important role in HIV-associated coagulopathy has been described. The gene ID on the NCBI is 2152, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/2152.

[0246]The CD38 cancer antigen is an antigen encoded by the CD38 gene. The CD38 gene, located on, but not limited to, the p15.32 arm of chromosome 4, encodes a nonlineage-restricted type II transmembrane glycoprotein, cyclic adenosine-5'-diphosphate-ribose , an intracellular messenger that mobilizes, synthesizes, and hydrolyzes calcium ions. The release of soluble protein and the ability of the membrane-bound protein to be internalized indicate extracellular and intracellular functions for the protein. This protein has an N-terminal cytoplasmic tail, a single transmembrane domain and a C-terminal extracellular region with four N-glycosylation sites. Analysis of the crystal structure shows that the functional molecule is a dimer, with the central part containing the catalytic site. It is used as a prognostic marker for patients with chronic lymphocytic leukemia. The Gene ID on the NCBI is 952, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/952.

[0247]The cancer antigen CD138 (SDC1, syndecan) is an antigen encoded by the CD138 (SDC1, syndecan) gene. The CD138 gene (SDC1, syndecan), located in but not limited to arm 24.1 of chromosome 2p, encodes a transmembrane heparan sulfate proteoglycan (type I) and is a member of the syndecan proteoglycan family. Syndecans mediate cell attachment, cell signaling, and cytoskeletal organization, and syndecan receptors are required for internalization of HIV-1's tat protein. Syndecan-1 protein functions as an integral membrane protein and is involved in cell proliferation, cell migration, and cell-matrix interactions through its receptor for extracellular matrix proteins. Altered syndecan-1 expression has been demonstrated in several different tumor types. Although several transcriptional variants may exist for this gene, only two have been fully described to date. The gene ID on the NCBI is 6382, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/6382.

[0248]The EGFR cancer antigen is an antigen encoded by the EGFR gene. The EGFR gene, located on but not limited to the 11.2 arm of chromosome 7p, encodes a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor, thereby inducing receptor dimerization and tyrosine autophosphorylation, leading to cell proliferation. Mutations in this gene are associated with lung cancer. The gene ID on the NCBI is 1956, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/1956.

[0249]Cancer antigen PD-1 (CD279, PDCD1, hPD-1) is an antigen encoded by the PD-1 gene (CD279, PDCD1, hPD-1). The PD-1 gene (CD279, PDCD1, hPD-1), located on but not limited to chromosome 2 q arm 37.3, encodes an immunoinhibitory receptor that is expressed on activated T cells; is involved in the regulation of T cell functions, including effector CD8+ T cells. In addition, this protein can also promote the differentiation of CD4+ T cells into regulatory T cells. PDCD1 is expressed in many types of tumors, including melanoma, and has been shown to play a role in antitumor immunity. In addition, this protein has been shown to be involved in protection against autoimmunity, but it may also contribute to the inhibition of potent antitumor and antimicrobial immunity. The Gene ID on the NCBI is 5133, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/5133.

[0250]The ROR1 cancer antigen is an antigen encoded by the ROR1 gene. The ROR1 gene, located but not limited to arm 31.3 of chromosome 1p, encodes an orphan tyrosine kinase-like receptor that modulates neurite outgrowth in the central nervous system. The encoded protein is a glycosylated type I membrane protein belonging to the ROR subfamily of cell surface receptors. It is a pseudokinase that lacks catalytic activity and can interact with non-canonical Wnt signaling. This gene is highly expressed during early embryonic development but is expressed at very low levels in adult tissues. Increased expression of this gene is associated with B-cell chronic lymphocytic leukemia. The gene ID on the NCBI is, but not limited to, 4919. See https://www.ncbi.nlm.nih.gov/gene/4919.

[0251]The CSPG4 cancer antigen is an antigen encoded by the CSPG4 gene. The CSPG4 gene, located on but not limited to the 24.2 arm of chromosome 15q, encodes a human melanoma-associated chondroitin sulfate proteoglycan that has a role in stabilizing cell-substrate interactions during early events melanoma cell shedding via the endothelial bases of the membranes. CSPG4 is a full membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The gene ID on the NCBI is 1464, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/1464.

[0252]The cancer antigen CLL-1 (CLEC12A) is an antigen encoded by the CLL-1 gene (CLEC12A). The CLL-1 (CLEC12A) gene, located on but not limited to the p13.31 arm of chromosome 12, encodes a member of the C-type lectin/C-type lectin-like domain ( CTL/CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-to-cell signaling, glycoprotein turnover, and roles in inflammation and the immune response. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced ​​transcriptional variants of this gene have been described, but the full nature of some of these variants has not been determined. This gene is closely linked to other members of the CTL/CTLD superfamily in the natural killer gene complex region of chromosome 12p13. The gene ID on the NCBI is 160364, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/160364.

[0253]CD147 cancer antigen (BSG-basigin) is an antigen encoded by the CD147(BSG-basigin) gene. The CD147 gene (BSG-basigin), located in but not limited to arm 13.3 of chromosome 19p, encodes a plasma membrane protein important in spermatogenesis, embryo implantation, neural network formation and tumor progression. Basigin is also a member of the immunoglobulin superfamily that is ubiquitously expressed in various tissues. The gene ID on the NCBI is 682, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/682.

[0254]The PSCA cancer antigen is an antigen encoded by the PSCA gene. The PSCA gene, located but not limited to chromosome 8 q-arm 24.3, encodes a cell membrane glycoprotein anchored to glycosylphosphatidylinositol. Not only is it highly expressed in the prostate, but also in the bladder, placenta, colon, kidney and stomach. This gene is upregulated in a large proportion of prostate cancers and is also found in bladder and pancreatic cancers. This gene contains a polymorphism that results in an upstream initiation codon in some individuals; This polymorphism is believed to be linked to a risk of certain types of gastric and bladder cancer. The gene ID on NCBI is 8000, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/8000.

[0255]The EPHA2 cancer antigen is an antigen encoded by the EPHA2 gene. The EPHA2 gene, located on but not limited to the p36.13 arm of chromosome 1, encodes a protein that binds to ephrin A ligands. This gene belongs to the ephrin receptor subfamily of the protein tyrosine kinase family. EPH and EPH-related receptors have been implicated in mediating developmental events, particularly in the nervous system. Receptors in the EPH subfamily typically have a single kinase domain and an extracellular region containing a Cys-rich domain and 2 repeats of fibronectin type III. Ephrin receptors are divided into 2 groups based on the similarity of their extracellular domain sequences and their affinities for binding ephrin A and ephrin B ligands. Mutations in this gene are the cause of certain genetic cataract diseases. The Gene ID on the NCBI is 1969 but not limited to. See https://www.ncbi.nlm.nih.gov/gene/1969.

[0256]The GPRC5D cancer antigen is an antigen encoded by the GPRC5D gene. The GPRC5D gene, located on but not limited to the 13.1 arm of chromosome 12p, encodes a member of the G protein-coupled receptor family. The gene ID in NCBI is, but is not limited to, 55507. See https://www.ncbi.nlm.nih.gov/gene/55507.

[0257]The cancer antigen CD133 (PROM1, AC133) is an antigen encoded by the CD133 (PROM1, AC133) gene. The CD133 gene (PROM1, AC133), located on but not limited to the p15.32 arm of chromosome 4, encodes a pentaspane transmembrane glycoprotein. The protein is located on membrane ridges and is commonly expressed in adult stem cells, which are thought to maintain stem cell properties by suppressing differentiation. Mutations in this gene have been shown to lead to retinitis pigmentosa and Stargardt's disease. The expression of this gene is also linked to several types of cancer. This gene is expressed from at least five alternative promoters that are expressed in a tissue-dependent manner. The Gene ID on the NCBI is 8842, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/8842.

[0258]The B7H6 cancer antigen (NCR3LG1) is an antigen encoded by the B7H6 (NCR3LG1) gene. The B7H6 gene (NCR3LG1), located on but not limited to the 15.1 arm of chromosome 11, encodes a natural killer cell cytotoxicity receptor 3. B7H6 belongs to the B7 family (see MIM 605402) and is selectively expressed in tumor cells expressed. The interaction of B7H6 with NKp30 (NCR3; MIM 611550) leads to activation and cytotoxicity of natural killer (NK) cells. The gene ID in the NCBI is 374383, but is not limited to this. See https://www.ncbi.nlm.nih.gov/gene/374383.

[0259]The cancer antigen DSC2 (DG2, DSC3) is an antigen encoded by the DSC2 gene (DG2, DSC3). The DSC2 gene (DG2, DSC3), located on but not limited to chromosome 18 q arm 12.1, encodes a member of the desmocholine protein subfamily. Desmocholines, along with desmogleins, are cadherin-like transmembrane glycoproteins that are major components of the desmosome. Desmosomes are cell-to-cell junctions that help resist shear forces and are found in high concentrations in cells subjected to mechanical stress. This gene is clustered with other members of the desmocholine family on chromosome 18. Mutations in this gene are associated with arrhythmogenic right ventricular dysplasia-11, and decreased protein expression has been described in several cancers. The Gene ID on the NCBI is 1824, but not limited to. See https://www.ncbi.nlm.nih.gov/gene/1824.

[0260]The cancer antigen AE1 (SLC4A1) is an antigen encoded by the AE1 gene (SLC4A1). The AE1 gene (SLC4A1), located inter alia on chromosome 17 q arm 21.31, encodes part of the anion exchanger (AE) family and is expressed in the plasma membrane of erythrocytes, where it functions as a chloride/bicarbonate exchanger, the Carbon dioxide is involved in the transport of carbon from the tissues to the lungs. The protein comprises two domains that differ structurally and functionally. The 40 kDa N-terminal domain is located in the cytoplasm and acts as a binding site for the red blood cell skeleton by binding to ankyrin. The glycosylated C-terminal membrane-associated domain contains 12–14 transmembrane segments and performs stilbene disulfonate-sensitive anion exchange transport. The cytoplasmic tail at the C-terminal end of the membrane domain binds carbonic anhydrase II. The encoded protein associates with the glycophorin A protein of the red blood cell membrane, and this association promotes correct folding and translocation of the exchanger. This protein is predominantly dimeric, but forms tetramers in the presence of ankyrin. Many mutations in this gene are known in humans, and these mutations can lead to two types of diseases: destabilization of the red blood cell membrane, leading to hereditary spherocytosis, and defective renal acid secretion, leading to distal renal tubular acidosis. Other mutations that do not cause disease result in new blood group antigens that form the Diego blood group system. Southeast Asian ovalocytosis (SAO, Melanesian ovalocytosis) results from the heterozygous presence of a deletion in the encoded protein and is present in areas wherePlasmodium falciparumMalaria is endemic. A null mutation in this gene is known to result in very severe anemia and nephrocalcinosis. The Gene ID on the NCBI is 6521, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/6521.

[0261]The cancer antigen GUCY2C (GC-C, MUCIL) is an antigen encoded by the gene GUCY2C (GC-C, MUCIL). The GUCY2C (GC-C, MUCIL) gene, located on but not limited to the 12.3 arm of chromosome 12p, encodes a transmembrane protein that acts as a receptor for the endogenous peptides guanylin and uroguanylin, and the term stableE colienterotoxin. The encoded protein activates the cystic fibrosis transmembrane conductance regulator. Mutations in this gene are associated with familial diarrhea (autosomal dominant) and meconium ileus (autosomal recessive). The gene ID on NCBI is 2984, but not limited to this. See https://www.ncbi.nlm.nih.gov/gene/2984.

[0262]The cancer antigen CDH17 (HPT1) is an antigen encoded by the CDH17 (HPT1) gene. The CDH17 (HPT1) gene, located on but not limited to chromosome 8 q arm 22.1, encodes a member of the cadherin superfamily, genes that encode calcium-dependent membrane-associated glycoproteins. The encoded protein resembles cadherin, consisting of an extracellular region containing 7 cadherin domains and a transmembrane region, but lacking the conserved cytoplasmic domain. The protein is a component of the gastrointestinal and pancreatic ducts and functions as an intestinal proton-dependent peptide transporter in the first step of the oral absorption of many medically important peptide-based drugs. Protein may also play a role in the morphological organization of the liver and intestines. The Gene ID on the NCBI is 1015, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/1015.

[0263]The HPSE cancer antigen is an antigen encoded by the HPSE gene. The HPSE gene, located in but not limited to arm 21.23 of chromosome 4q, encodes an enzyme that cleaves heparan sulfate proteoglycans to allow cell movement through extracellular matrix remodeling. In addition, this cleavage can release bioactive molecules from the extracellular matrix. Several transcriptional variants encoding different isoforms were found for this gene. The gene ID on the NCBI is 10855, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/10855.

[0264]The CD24 cancer antigen is an antigen encoded by the CD24 gene. The CD24 gene, located on but not limited to chromosome 6 q arm 21, encodes a sialoglycoprotein that is expressed in mature granulocytes and B cells and modulates growth and differentiation signals for these cells. The precursor protein is cleaved into a short 32 amino acid mature peptide that is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) linkage. This gene was absent from previous genome assemblies but is correctly located on chromosome 6. Untranscribed pseudogenes have been assigned to chromosomes 1, 15, 20 and Y. The gene ID in the NCBI is 100133941, but is not limited to this. See https://www.ncbi.nlm.nih.gov/gene/100133941.

[0265]The MUC4(ASGP) cancer antigen is an antigen encoded by the MUC4(ASGP) gene. The MUC4 gene (ASGP), located on but not limited to chromosome 3 q arm 29, encodes the major components of mucus, mucin, covering epithelial surfaces such as the trachea, colon and cervix, expressed in mature granulocytes and B -cells and growth modulates differentiation signals to these cells. The precursor protein is cleaved into a short 32 amino acid mature peptide that is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) linkage. This gene was absent from previous genome assemblies but is correctly located on chromosome 6. Untranscribed pseudogenes have been assigned to chromosomes 1, 15, 20 and Y. The gene ID on the NCBI is 4585, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/4585.

[0266]The AFP-L3 cancer antigen is an antigen encoded by the AFP-L3 gene. The AFP-L3 gene isculinary lensesAgglutinin-binding alpha-fetoprotein (AFP) isoform (LCA), a substance commonly used in triple testing during pregnancy and to screen patients with chronic liver disease for hepatocellular carcinoma (HCC). AFP can be fractionated by affinity electrophoresis into 3 glycoforms: L1, L2 and L3 based on lectin reactivityculinary lensesagglutinin (LCA). AFP-L3 binds tightly to LCA via an additional 1-6 fucose residue attached to the reducing end of N-acetylglucosamine; this is in contrast to the L1 isoform.

[0267]Cancer antigen SP17 (SPA17) is an antigen encoded by the SP17 (SPA17) gene. The SP17 gene (SPA17), located on but not limited to chromosome 11 q arm 24.2, encodes a protein present on the cell surface. The N-terminus shares sequence similarity with the human cAMP-dependent protein kinase A (PKA) type II alpha regulatory subunit (RIIa), while the C-terminus has a calmodulin IQ binding motif. The central part of the protein has carbohydrate-binding motifs and is probably involved in cell-cell adhesion. The protein was originally characterized by its involvement in sperm binding to the zona pellucida of the oocyte. Recent studies indicate that it is also involved in additional cell-cell adhesion functions such as B. Immune cell migration and metastasis. A retrotransposed pseudogene is present on chromosome 10q22. The Gene ID on NCBI is 53340, but is not limited to this. See https://www.ncbi.nlm.nih.gov/gene/53340.

[0268]The DCLK1 cancer antigen is an antigen encoded by the DCLK1 gene. The DCLK1 gene, located on but not limited to chromosome 13 q arm 13.3, encodes a member of the protein kinase superfamily and the doublecortin family. The protein encoded by this gene contains two N-terminal doublecortin domains that bind to microtubules and regulate microtubule polymerization, a C-terminal serine/threonine protein kinase domain that shows substantial homology to the Ca2+-dependent protein kinase/calmodulin domain, and a serine/proline-rich domain between the doublecortin and protein kinase domains that mediate multiple protein-protein interactions. The microtubule polymerization activity of the encoded protein is independent of its protein kinase activity. The encoded protein is involved in several different cellular processes including neuronal migration, retrograde transport, neuronal apoptosis and neurogenesis. This gene is upregulated by a brain-derived neurotrophic factor and is associated with memory and general cognitive ability. Several transcriptional variants generated using two alternative promoters and alternative splicing have been reported, but the full nature and biological validity of some variants have not been defined. These variants encode different isoforms that are expressed differently and have different kinase activities. The Gene ID on the NCBI is 9201, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/9201.

[0269]The CAIX cancer antigen (CA9) is an antigen encoded by the CAIX gene (CA9). The CAIX gene (CA9), located in but not limited to arm 13.3 of chromosome 9p, encodes a transmembrane protein and is one of two known isoenzymes of tumor-associated carbonic anhydrase. It is expressed in all clear cell renal carcinomas but is not detected in normal kidneys or most other normal tissues. May be involved in cell proliferation and transformation. The gene ID on the NCBI is 768, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/768.

[0270]The IL13RA2 cancer antigen gene (IL13Ra2), located on, but not limited to, X chromosome q-arm 23, encodes a subunit of the interleukin-13 receptor complex. This protein binds IL13 with high affinity, but it lacks a cytoplasmic domain and does not appear to function as a signaling mediator. It is reported to play a role in the internalization of IL13. The gene ID on NCBI is 3598, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3598.

[0271]The CD56 cancer antigen (NCAM1) is an antigen encoded by the CD56 (NCAM1) gene. The CD56 (NCAM1) gene, located on but not limited to chromosome 11 q arm 23.2, encodes a cell adhesion protein that is a member of the immunoglobulin superfamily. The encoded protein is involved in cell-cell and cell-matrix interactions during development and differentiation. The encoded protein plays a role in nervous system development by regulating neurogenesis, neurite outgrowth, and cell migration. This protein is also involved in the expansion of T lymphocytes, B lymphocytes and natural killer (NK) cells, which play an important role in immune surveillance. This protein plays a role in signal transduction, interacting with fibroblast growth factor receptors, N-cadherin, and other components of the extracellular matrix, and triggering signaling cascades involving FYN focal adhesion kinase (FAK), a mitogen-activated protein kinase (MAPK), and phosphatidylinositol are involved in 3-kinase (PI3K). A prominent isoform of this gene, the cell surface molecule CD56, plays a role in several myeloproliferative disorders such as acute myeloid leukemia, and differential expression of this gene is associated with differential disease progression. The gene ID on NCBI is 4684, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/4684.

[0272]The CD44v6 cancer antigen is an antigen encoded by the CD44v6 gene. The CD44v6 gene, located on but not limited to chromosome 11p, arm 13, encodes a cell surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. It is a receptor for hyaluronic acid (HA) and can also interact with other ligands such as osteopontin, collagens and matrix metalloproteinases (MMPs). This protein is involved in a variety of cellular functions, including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis. Transcripts for this gene undergo complex alternative splicing resulting in many functionally distinct isoforms, however, the nature of some of these full-length variants has not been determined. Alternative splicing underlies the structural and functional diversity of this protein, which may be implicated in tumor metastasis. The gene ID on the NCBI is 960, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/960.

[0273]The cancer antigen claudin-6 is an antigen encoded by the claudin-6 gene. The Claudin 6 gene, located on but not limited to chromosome 16p, arm 13.3, encodes a component of tight junction chains that is a member of the Claudin family. The protein is an integral membrane protein and one of the entry cofactors for the hepatitis C virus. Gene methylation may be involved in esophageal tumorigenesis. This gene is adjacent to another member of the CLDN9 family on chromosome 16. Tight junctions represent a mode of cell-to-cell adhesion in epithelial or endothelial cell layers that form continuous seals around cells and serve as a physical barrier to prevent the passage of solutes and water freely through the paracellular space. These connections consist of sets of continuous network filaments in the outward cytoplasmic cusp with complementary grooves in the inward extracytoplasmic cusp. The Gene ID on the NCBI is 9074, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/9074.

[0274]Glypican-1 (GPC1) cancer antigen is an antigen encoded by the Glypican-1 (GPC1) gene. The Glypican-1 (GPC1) gene, located on but not limited to chromosome 2 q arm 37.3, encodes a component of cell-surface heparan sulfate proteoglycans, which consist of a membrane-associated protein core surrounded by a variable number of heparan sulfate family related integral membrane proteoglycans (GRIPS) contain a core protein anchored to the cytoplasmic membrane via a glycosylphosphatidylinositol bond. These proteins may play a role in controlling cell division and regulating growth. The Gene ID on the NCBI is 2817, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/2817.

[0275]PLAP (ALPP) cancer antigen is an antigen encoded by the PLAP (ALPP) gene. The PLAP (ALPP) gene, located on but not limited to chromosome 2 q arm 37.1, encodes an alkaline phosphatase, a metalloenzyme that catalyzes the hydrolysis of phosphoric acid monoesters. It belongs to a multigene family consisting of four alkaline phosphatase isoenzymes. The enzyme acts as a homodimer and has a catalytic site containing one magnesium ion and two zinc ions necessary for its enzymatic function. One of the main sources of this enzyme is the liver and as such it is one of several indicators of liver damage in various clinical conditions. In pregnant women, this protein is mainly expressed in placental and endometrial tissues, however, strong ectopic expression has been detected in ovarian adenocarcinoma, serous cystadenocarcinoma and other ovarian cancer cells. The gene ID on the NCBI is 250, but not limited to this. See https://www.ncbi.nlm.nih.gov/gene/250.

[0276]The uPAR cancer antigen (PLAUR) is an antigen encoded by the uPAR (PLAUR) gene. The uPAR gene (PLAUR), located on but not limited to chromosome 19 q arm 13.31, encodes the receptor for urokinase plasminogen activator and is likely to affect many related normals due to its role in localizing and promoting plasmin formation and pathological processes, activation of cell surface plasminogen and localization of extracellular matrix degradation. It binds to the proprotein and mature forms of urokinase plasminogen activator and allows activation of the receptor-bound proenzyme by plasmin. The protein lacks transmembrane or cytoplasmic domains and can be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) moiety near its carboxy terminus after cleavage of the nascent polypeptide. However, in some cell types, a soluble protein is also produced. Alternative splicing results in multiple transcriptional variants encoding different isoforms. The gene ID on the NCBI is 5329, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/5329.

[0277]Cancer LunX Antigen (BPIFAI) is an antigen encoded by the LunX gene (BPIFAI). The LunX gene (BPIFAI), located in, but not limited to, arm 11.21 of chromosome 20q, encodes the human homologue of murine Plunc and, like the mouse gene, is specifically expressed in the upper airway and nasopharyngeal regions. The encoded antimicrobial protein exhibits antibacterial activity against gram-negative bacteria. It is thought to be involved in inflammatory responses to irritants in the upper airways and may also serve as a potential molecular marker to detect micrometastasis in non-small cell lung cancer. The gene ID on the NCBI is 51297, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/51297.

[0278]The folate receptor beta (FRβ, FOLR2) cancer antigen is an antigen encoded by the folate receptor beta (FRβ, FOLR2) gene. The folate receptor beta (FRβ, FOLR2) gene, located on but not limited to chromosome 11 q arm 13.4, encodes a member of the folate receptor (FOLR) family and these genes exist in a cluster on chromosome 11. The members of this gene family have high affinity for folic acid and several reduced folic acid derivatives and mediate the release of 5-methyltetrahydrofolate in cells. This protein shares 68% and 79% sequence homology with the FOLR1 and FOLR3 proteins, respectively. Although this protein was originally thought to be placenta specific, it may also be present in other tissues and play a role in methotrexate transport in synovial macrophages in patients with rheumatoid arthritis. The gene ID on the NCBI is 2350, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/2350.

[0279]The LILRB4 (ILT3, CD85K) cancer antigen is an antigen encoded by the LILRB4 (ILT3, CD85K) gene. The LILRB4 (ILT3, CD85K) gene, located on but not limited to chromosome 19q, arm 13.42, encodes a member of the leukocyte immunoglobulin-like receptor (LIR) family, located in a gene cluster in the chromosomal region 19q13.4 can be found. The encoded protein belongs to the class B LIR receptor subfamily, which contains two or four extracellular immunoglobulin domains, one transmembrane domain, and two to four cytoplasmic tyrosine-based immunoreceptor inhibitor motifs (ITIMs). The receptor is expressed on immune cells, where it binds to class I MHC molecules on antigen-presenting cells and transmits a negative signal that inhibits the stimulation of an immune response. The receptor can also function in capturing and presenting antigens. It is thought to control inflammatory responses and cytotoxicity to focus the immune response and limit self-reactivity. The gene ID on the NCBI is 11006, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/11006.

[0280]The cancer antigen MISIIR (Müllerian Inhibitory Substance Type 2 Receptor, AMHR2) is an antigen encoded by the MISIIR (Müllerian Inhibitory Substance Type 2, AMHR2) gene. The MISIIR gene (Müller inhibitor receptor type 2, AMHR2), which is located on chromosome 12 q arm 13.13, encodes the receptor for the anti-Müllerian hormone (AMH), which, in addition to testosterone, leads to male gender differentiation AMH and testosterone are produced by different cells in the testicles and have different effects. Testosterone promotes the development of the male genitalia, while binding of AMH to the encoded receptor prevents the development of the mullerian ducts in the uterus and fallopian tubes. Mutations in this gene are associated with persistent Mueller-Giant syndrome type II. The gene ID on the NCBI is 269, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/269.

[0281]Cancer antigen 5T4 (TPBG) is an antigen encoded by the 5T4 gene (TPBG). The 5T4 gene (TPBG), located on but not limited to chromosome 6 q arm 14.1, encodes a leucine-rich transmembrane glycoprotein that may be involved in cell adhesion. The encoded protein is an oncofetal antigen specific to trophoblast cells. In adults, this protein is highly expressed in many tumor cells and is associated with poor clinical outcomes in several cancer types. The Gene ID on the NCBI is 7162, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/7162.

[0282]CD83 Ligand is encoded by the CD83 Ligand gene. The CD83 ligand gene, located in but not limited to arm 23 of chromosome 6p, encodes a single-pass type I membrane protein and a member of the immunoglobulin receptor superfamily. The encoded protein may be involved in the regulation of antigen presentation. A soluble form of this protein can bind to dendritic cells and inhibit their maturation. The Gene ID on the NCBI is 9308, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/9308.

[0283]The CD171 (L1-CAM) cancer antigen is an antigen encoded by the CD171 (L1-CAM) gene. The CD171 gene (L1-CAM), located on, but not limited to, arm 28 of the X chromosome q, encodes an axonal glycoprotein that belongs to the immunoglobulin supergene family. The ectodomain, which consists of multiple immunoglobulin-like domains and fibronectin-like repeats (type III), is linked to a conserved cytoplasmic domain by a single transmembrane sequence. This cell adhesion molecule plays an important role in nervous system development, including neuronal migration and differentiation. Mutations in the gene cause X-linked neurological syndromes known as CRASH (Corpus Callosum Hypoplasia, Retardation, Aphasia, Spastic Paraplegia, and Hydrocephalus). The Gene ID on NCBI is 3897, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/3897.

[0284]The B7-H4 cancer antigen (VTCN1) is an antigen encoded by the B7-H4 (VTCN1) gene. The B7-H4 gene (VTCN1), located on but not limited to the 13.1 p12 arm of chromosome 1p, encodes a protein belonging to the B7 family of costimulatory proteins. Proteins from this family are present on the surface of antigen-presenting cells and interact with ligands attached to receptors on the surface of T cells. Studies have shown that high levels of the encoded protein correlate with tumor progression. A pseudogene of this gene is located on chromosome 20. The gene ID on the NCBI is, but not limited to, 79679. See https://www.ncbi.nlm.nih.gov/gene/79679.

[0285]The CD166 cancer antigen (ALCAM) is an antigen encoded by the CD166 (ALCAM) gene. The CD166 (ALCAM) gene, located on but not limited to chromosome 3 q arm 13.11, encodes the activated leukocyte cell adhesion molecule (ALCAM), also known as CD166 (differentiation cluster 166), which is a member of a subfamily of receptors for immunoglobulins with five immunoglobulin-like domains (VVC2C2C2) is in the extracellular domain. This protein binds to the CD6 T cell differentiation antigen and is involved in cell adhesion and migration processes. The Gene ID on the NCBI is 214, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/214.

[0286]CD13 cancer antigen (ANPEP) is an antigen encoded by the CD13 gene (ANPEP). The CD13 (ANPEP) gene, located on but not limited to chromosome 15 q arm 26.1, encodes a well-known enzyme called aminopeptidase N. Aminopeptidase N is located in the microvillar membrane of the small intestine and kidney and also in other plasma membranes . In the small intestine, aminopeptidase N plays a role in the final digestion of peptides generated by protein hydrolysis by gastric and pancreatic proteases. Its function in proximal tubular epithelial cells and other cell types is less clear. The large carboxy-terminal extracellular domain contains a consensus pentapeptide sequence characteristic of members of the zinc-binding metalloproteinase superfamily. The enzyme is thought to be involved in the metabolism of regulatory peptides by a variety of cell types, including renal and small intestinal tubular epithelial cells, macrophages, granulocytes, and CNS synaptic membranes. The Gene ID on the NCBI is 290, but not limited to this. See https://www.ncbi.nlm.nih.gov/gene/290.

[0287]The CD117 cancer antigen is an antigen encoded by the CD117 gene. The CD117 gene, located on but not limited to chromosome 4 q arm 12, encodes a tyrosine kinase receptor. This gene was originally identified as a homologue of the v-Kit feline sarcoma virus oncogene and is often referred to as the c-Kit proto-oncogene. The canonical form of this glycosylated transmembrane protein has an N-terminal extracellular region with five immunoglobulin-like domains, a transmembrane region and an intracellular tyrosine kinase domain at the C-terminus. ), this protein phosphorylates several intracellular proteins that play a role in the proliferation, differentiation, migration, and apoptosis of many cell types and therefore play important roles in hematopoiesis, stem cell maintenance, gametogenesis, melanogenesis, as well as in development, migration, and mast cell function. This protein can be membrane-bound or soluble. Mutations in this gene are associated with gastrointestinal stromal tumors, mast cell disease, acute myeloid leukemia, and piebaldism. The gene ID on the NCBI is 3815, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3815.

[0288]The cancer antigen TEM8 (ANTXR1) is an antigen encoded by the TEM8 (ANTXR1) gene. The TEM8 gene (ANTXR1), located inter alia in arm 13.3 of chromosome 2p, encodes a type I transmembrane protein and is a tumor-specific endothelial marker associated with colorectal cancer. It has also been shown that the encoded protein is a docking protein or receptor forBacillus anthracisToxin, the causative agent of the disease, anthrax. Binding of the protective antigen (PA) component of the tripartite anthrax toxin to this receptor protein mediates delivery of the toxin components to the cytosol of cells. Once inside the cell, the other two components of the anthrax toxin, edema factor (EF) and lethal factor (LF), disrupt normal cellular processes. The gene ID on the NCBI is 84168, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/84168.

[0289]The CD26 (DPP4) cancer antigen is an antigen encoded by the CD26 (DPP4) gene. The CD26 (DPP4) gene, located on but not limited to chromosome 2 q-arm 24.2, encodes dipeptidyl peptidase 4, which is identical to the adenosine deaminase 2 complex protein and the T cell activation antigen CD26. It is an intrinsic type II transmembrane glycoprotein and a serine exopeptidase that cleaves proline X dipeptides from the N-terminus of polypeptides. Dipeptidyl peptidase 4 is heavily involved in glucose and insulin metabolism and immune regulation. The Gene ID on the NCBI is 1803, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/1803.

[0290]The IGF1R cancer antigen is an antigen encoded by the IGF1R gene. The IGF1R gene, located on but not limited to chromosome 15 q arm 26.3, encodes a high affinity receptor-binding insulin-like growth factor. It has tyrosine kinase activity. Insulin-like growth factor receptor I plays a crucial role in transformation events. Precursor fission produces alpha and beta subunits. It is highly overexpressed in most malignant tissues, where it acts as an antiapoptotic agent and improves cell survival. The gene ID on the NCBI is 3480, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3480.

[0291]The Muc3a cancer antigen (MUC3A) is an antigen encoded by the Muc3a (MUC3A) gene. The Muc3a gene (MUC3A), located on but not limited to chromosome 7 q arm 22.1, encodes epithelial glycoproteins, some of which are secreted and others membrane-bound. Each of the genes contains at least one large tandem repeat sequence domain that encodes the serine and/or threonine residue-rich peptide sequence that carries most of the O-linked glycosylation. The gene ID in the NCBI is 4584, but is not limited thereto. See https://www.ncbi.nlm.nih.gov/gene/4584.

[0292]The cancer antigen IL1RAP (IL1R3) is an antigen encoded by the IL1RAP gene (IL1R3). The IL1RAP gene (IL1R3), located on but not limited to chromosome 3 q-arm 28, encodes a component of the interleukin-1 receptor complex that initiates signaling events leading to the activation of interleukin-1 responsive genes , in membrane-bound and soluble isoforms that differ in their C-terminus. The ratio of soluble to membrane-bound forms increases during the induction or stress phase of the acute phase. The gene ID on the NCBI is 3556, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/3556.

[0293]The TSLPR cancer antigen (CRLF2) is an antigen encoded by the TSLPR gene (CRLF2). The TSLPR (CRLF2) gene, located on X chromosome p-arm 22.33 and Y chromosome p-arm 11.2, encodes, but is not limited to, a member of the type I cytokine receptor family. The encoded protein is a receptor for thymic stromal lymphopoietin (TSLP). Along with the interleukin-7 receptor (IL7R), the encoded protein and TSLP activate the STAT3, STATS, and JAK2 signaling pathways that control processes such as cell proliferation and the development of the hematopoietic system. Rearrangement of this gene with the immunoglobulin heavy chain gene (IGH) on chromosome 14 or with the purinoceptor gene P2Y 8 (P2RY8) on the same X or Y chromosomes is associated with acute lymphoblastic leukemia of B parents (ALL) and Down -ALL associated syndrome. The Gene ID on the NCBI is 64109, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/64109.

[0294]The cancer antigen LMP1 (LMP3, PDLIM7) is an antigen encoded by the LMP1 gene (LMP3, PDLIM7). The LMP1 gene (LMP3, PDLIM7), located on but not limited to chromosome 5 q-arm 35.3, encodes a member of a protein family consisting of conserved PDZ and LIM domains. transcription and development and in the cytoskeleton interaction. The LIM domains of this protein bind to protein kinases, while the PDZ domain binds to actin filaments. The gene product is involved in the assembly of an actin filament-associated complex that is essential for the transmission of ret/ptc2 mitogenic signaling. The biological function is likely that of an adapter, with the PDZ domain localizing LIM-binding proteins to actin filaments of skeletal muscle and non-muscle tissues. The gene ID on the NCBI is 9260, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/9260.

[0295]The Siglec7 (SIGLEC7, CD328) cancer antigen is an antigen encoded by the Siglec7 gene (SIGLEC7, CD328). The Siglec7 gene (SIGLEC7, CD328), located on but not limited to chromosome 19 q arm 13.41, encodes a protein encoded by the SIGLEC7 gene in humans. SIGLEC7 has also been referred to as CD328 (differentiation cluster 328). The gene ID on the NCBI is 27036, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/27036.

[0296]The Siglec9 cancer antigen (CD329) is an antigen encoded by the Siglec9 (CD329) gene. The Siglec9 gene (CD329), which is located inter alia on chromosome 19 q arm 13.41, encodes a protein which is encoded by the SIGLEC9 gene in humans. The gene ID on NCBI is 27180, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/27180.

[0297]The CD1a (CD1, HTA1) cancer antigen is an antigen encoded by the CD1a (CD1, HTA1) gene. The CD1a gene (CD1, HTA1), located on, but not limited to, chromosome 1 q arm 23.1, encodes a member of the CD1 family of transmembrane glycoproteins that are structurally related to major histocompatibility complex (MHC) proteins and form heterodimers with beta-2 microglobulin. CD1 proteins primarily mediate the presentation of lipid and glycolipid antigens of intrinsic or microbial origin to T cells. The human genome contains five genes of the CD1 family, arranged in a cluster on chromosome 1. Localization and specificity for particular lipid ligands. The protein encoded by this gene is found in the plasma membrane and in the recycling vesicles of the initial endocytic system. The Gene ID on the NCBI is 909, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/909.

[0298]The cancer antigen CLEC14A (C14orf27, CEG1) is an antigen encoded by the gene CLEC14A (C14orf27, CEG1). The CLEC14A gene (C14orf27, CEG1), located on but not limited to chromosome 14 q arm 21.1, encodes a member of the C-type lectin/C-type lectin domain (CTL/CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-to-cell signaling, glycoprotein turnover, and roles in inflammation and the immune response. This family member plays a role in cell-cell adhesion and angiogenesis. Functions in filopodia formation, cell migration and tube formation. Because it is present in higher concentrations in tumor endothelium than in normal tissue endothelium, it is considered a candidate for vascular tumor targeting. The gene ID on the NCBI is 161198, but not limited to. See https://www.ncbi.nlm.nih.gov/gene/161198.

[0299]The cancer antigen MAGE-A1 (MAGEA1) is an antigen encoded by the gene MAGE-A1 (MAGEA1). The MAGE-A1 gene (MAGEA1), located in, but not limited to, arm 28 of the X chromosome q, encodes a member of the MAGEA gene family. Members of this family encode proteins with 50 to 80% sequence identity to each other. The promoters and first exons of the MAGEA genes show considerable variability, suggesting that the existence of this gene family allows the same function to be expressed under different transcriptional controls. The MAGEA genes are clustered at chromosomal position Xq28. They have been linked to some hereditary diseases such as congenital dyskeratosis. The gene ID on the NCBI is 4100, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4100.

[0300]The cancer antigen MAGE-A4 (MAGEA4) is an antigen encoded by the MAGE-A4 (MAGEA4) gene. The MAGE-A4 gene (MAGEA4), located in, but not limited to, arm 28 of the X chromosome q, encodes a member of the MAGEA gene family. Members of this family encode proteins with 50 to 80% sequence identity to each other. The promoters and first exons of the MAGEA genes show considerable variability, suggesting that the existence of this gene family allows the same function to be expressed under different transcriptional controls. The MAGEA genes are clustered at chromosomal position Xq28. They have been linked to some hereditary diseases such as congenital dyskeratosis. Several variants encoding the same protein have been found for this gene. The Gene ID on the NCBI is 4103, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4103.

[0301]Neurofilament M cancer antigen (NEFM) is an antigen encoded by the neurofilament M gene (NEFM). The neurofilament M gene (NEFM), located in but not limited to arm 21.2 of chromosome 8p, encodes the middle neurofilament protein. This protein is commonly used as a biomarker for neuronal damage. Neurofilaments are heteropolymers of Type IV intermediate filaments composed of light, medium, and heavy chains. Neurofilaments comprise the axoskeleton and functionally maintain neuronal caliber. They can also play a role in intracellular transport to axons and dendrites. The Gene ID on the NCBI is 4741, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4741.

[0302]Cancer antigen 2B4 (CD244) is an antigen encoded by the 2B4 (CD244) gene. The 2B4 (CD244) gene, located on but not limited to chromosome 1 q arm 23.3, encodes a cell surface receptor that is expressed on natural killer (NK) cells (and some T cells) and restricted death of the non -main histocompatibility complex mediated (MIC). The interaction between NK cells and target cells through this receptor is thought to modulate the cytolytic activity of NK cells. The gene ID on the NCBI is 51744, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/51744.

[0303]TACI cancer antigen (TNFRSF13B, CD267) is an antigen encoded by the TACI gene (TNFRSF13B, CD267). The TACI gene (TNFRSF13B, CD267), located in, but not limited to, arm 11.2 of chromosome 17p, encodes a lymphocyte-specific member of the tumor necrosis factor (TNF) receptor superfamily. It interacts with calcium modulator and cyclophilin ligand (CAML). The protein induces activation of the transcription factors NFAT, AP1 and NF-kappa-B and plays a crucial role in humoral immunity by interacting with a TNF ligand. This gene is located in the Smith-Magenis syndrome region on chromosome 17. The gene ID on the NCBI is 23495 but is not limited to this. See https://www.ncbi.nlm.nih.gov/gene/23495.

[0304]The CD32A (FCGR2A, IGFR2, CD32) cancer antigen is an antigen encoded by the CD32A (FCGR2A, IGFR2, CD32) gene. The CD32A gene (FCGR2A, IGFR2, CD32), located on but not limited to chromosome 1 q arm 23.3, encodes a member of a family of Fc immunoglobulin receptor genes found on the surface of many immune response cells . The protein encoded by this gene is a cell surface receptor found on phagocytic cells such as macrophages and neutrophils and is involved in the process of phagocytosis and elimination of immune complexes. The gene ID on NCBI is 2212, but is not limited to this. See https://www.ncbi.nlm.nih.gov/gene/2212.

[0305]The cancer antigen AXL (ARK, UFO) is an antigen encoded by the AXL gene (ARK, UFO). The AXL gene (ARK, UFO), located on but not limited to chromosome 19 q arm 13.2, encodes a member of the Tyro3-Axl-mer receptor tyrosine kinase (TAM) subfamily. The encoded protein has an extracellular domain consisting of two immunoglobulin-like motifs at the N-terminus followed by two fibronectin type III motifs. It transmits signals from the extracellular matrix to the cytoplasm by binding to the growth inhibition-specific vitamin K-dependent protein 6 (Gas6). This gene can be involved in multiple cellular functions including growth, migration, aggregation and anti-inflammatory in different cell types. The gene ID on the NCBI is 558, but not limited to that. See https://www.ncbi.nlm.nih.gov/gene/558.

[0306]The CD80 (B7, BB1, CD28LG) cancer antigen is an antigen encoded by the CD80 (B7, BB1, CD28LG) gene. The CD80 gene (B7, BB1, CD28LG), located on but not limited to chromosome 3 q arm 13.33, encodes a membrane receptor that is activated by binding of CD28 or CTLA-4. The activated protein induces the proliferation of T cells and the production of cytokines. This protein may act as a receptor for adenovirus subgroup B and play a role in lupus neuropathy. The Gene ID on the NCBI is 941, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/941.

[0307]The CD86 cancer antigen (B70, B&-2, CD28LG2) is an antigen encoded by the CD86 gene (B70, B&-2, CD28LG2). The CD86 gene (B70, B&-2, CD28LG2), located on but not limited to chromosome 3 q arm 13.33, encodes a type I membrane protein that is a member of the immunoglobulin superfamily. This protein is expressed by antigen-presenting cells and is the ligand for two proteins on the cell surface of T cells, the CD28 antigen and the cytotoxic T lymphocyte-associated protein 4. Binding of this protein to the CD28 antigen ceases costimulatory signal for T-cell activation. Binding of this protein to the cytotoxic T-lymphocyte-associated protein 4 down-regulates T-cell activation and decreases the immune response. The Gene ID on the NCBI is 942, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/942.

[0308]The cancer antigen ROR2 (BDB, NTRKR2) is an antigen encoded by the ROR2 gene (BDB, NTRKR2). The ROR2 gene (BDB, NTRKR2), located on but not limited to chromosome 9 q arm 22.31, encodes a tyrosine kinase receptor protein and a type I transmembrane protein belonging to the ROR subfamily of cell surface receptors. The protein may be involved in the initial formation of chondrocytes and may be required for cartilage and growth plate development. Mutations in this gene can cause type B brachydactyly, a skeletal disorder characterized by hypoplasia/aplasia of the distal phalanges and nails. In addition, mutations in this gene can cause the autosomal recessive form of Robinow syndrome, which is characterized by skeletal dysplasia with generalized shortening of the limb bones, segmental spinal defects, brachydactyly, and dysmorphic facial features. The Gene ID on the NCBI is 4920, but not limited to it. See https://www.ncbi.nlm.nih.gov/gene/4920.

[0309]Preferably, the targeting moiety is conjugated to the first polynucleotide using a linking group, wherein the linking group is an NHS ester, another activated ester, an alkyl or acyl halide, a bifunctional crosslinker, or a maleimide group.

[0310]Preferably, the first polynucleotide or second polynucleotide comprises a sequence selected from 20-mer poly-CA, 20-mer poly-GGTT, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 8; 9, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 , SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 7 and SEQ ID NO: 10.

[0311]Preferably, the binding affinity of the targeting moiety for the biomarker is less than 250 nM. Preferably, the binding affinity of the targeting moiety for the biomarker is 5 nM, 10 nM, 40 nM, 90 nM, 130 nM or 170 nM.

[0312]Preferably, the length of the first polynucleotide or the length of the second polynucleotide is 4 nt to 500 nt. Preferably, the length of the first polynucleotide or the length of the second polynucleotide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100, 120, 160, 220, 300, 400 or 480 NT.

[0313]Preferably the binding affinity between the first ligand and the second ligand is less than 250 nM. Preferably, the binding affinity between the first linker and the second linker is 2, 10, 25, 50, 62, 70, 85, 100, 102, 110, 125, 150, 162, 170, 185, 200, 202, 210, 225 or 250nM.

[0314]Preferably, the first linker or the second linker is conjugated to a functional group native to the targeting moiety or a cell surface, where the native functional group is an amino acid, a sugar or an amine.

[0315]Preferably the targeting moiety is a peptide, protein or aptamer.

[0316]Preferably, the CD16 receptor is a CD16a receptor or a CD16b receptor.

[0317]Preferably, an expressed polynucleotide encoding the CD16 receptor is located on the q-arm of chromosome 1 at position 1q23.3.

[0318]Preferably, the cell is non-tumorigenic in an immunocompromised mouse.

[0319]Preferably, the cell is non-tumorigenic in an allogeneic individual after being irradiated with γ-rays.

[0320]Preferably, a polynucleotide encoding the CD16 receptor comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 19.

[0321]Preferably, the CD16 receptor comprises an amino acid sequence of SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 20.

[0322]Preferably, the human natural killer cell further comprises an inactive tumor suppressor gene or a highly expressed and mutated oncogene.

[0323]Preferably, the human natural killer cell is capable of mediating an antibody-dependent cellular cytotoxicity (ADCC) response and the human natural killer cell is a male cell.

[0324]Preferably, the number of human natural killer cells in the composition is at least 5×10⁵and human natural killer cells in an amount equal to or greater than 5% in number based on the total number of cells in the composition as 100%.

[0325]Preferably the subject is a human.

[0326]Preferably, the method is for the selective treatment of cancer acanthoma, acinar cell carcinoma, acoustic neuroma, acral tiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid leukemia dendritic cell leukemia, acute myeloid leukemia Leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adrenal cortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, lymphoma AIDS-related, alveolar soft tissue sarcoma, ameloblastic fibroma, Anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendectomy cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma , bile duct cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchoalveolar carcinoma, brown tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile carcinoma, carcinoma of unknown primary site , carcinosarcoma, Castleman's disease, embryonic central nervous system tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma , chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic Myeloproliferative disease, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colon cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonic carcinoma , endodermal sinus tumor, endometrial carcinoma, endometrial cancer, endometrioid tumor, T cell-associated lymphoma enteropathy, ependymoblastoma , ependymoma, epitheloid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing family tumor, Ewing family sarcoma, Ewing disease sarcoma , extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal Carcinoid tumor, Gastrointestinal stromal tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational trophoblastic tumor, Giant cell bone tumor, Glioblastoma multiforme, Glioma, Cerebral glioma, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy cell leukemia, Hairy cell leukemia, Head and neck cancer, Head and cervical cancer, breast cancer heart, hemangioblastoma, hemangiopericytoma, hemangiosarcoma, hematological malignancy, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, Islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, larynx cancer, larynx cancer, lentigo malignant melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphepithelioma, Lymphocytic Leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant Fibrous Histiocytoma, Malignant Bone Fibrous Histiocytoma, Malignant Glioma, Malignant Mesothelioma, Malignant Peripheral Nerve Sheath Tumor, Malignant Rhabdoid Tumor, Malignant Triton Cell Tumor, MALT Lymphoma Lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloblastoma, medulloepithelioma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell carcinoma with occult primary tumor, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer , mucinous tumor, multiple endocrine neoplasia syndrome, myeloma, myelodysplastic disease, myelodysplastic syndromes, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neoplasia, neurinoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma, Non-Melanoma. Skin Cancer, Non-Small Cell Lung Cancer, Eye Oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic Nerve Sheath Meningioma, Oral Cancer, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Ovarian Cancer, Ovarian Epithelial Cancer, Germ Cell Tumor Of Ovarian, Ovarian Tumor With Low Malignant Potential, Paget's Disease Of Breast, Pancoast Tumor, Bauchspeicheldrüsenkrebs, Bauchspeicheldrüsenkrebs, papillärer Schilddrüsenkrebs, Papillomatose, Paragangliom, Nasennebenhöhlenkrebs, Nebenschilddrüsenkrebs, Peniskrebs, perivaskulärer Epitheloidzelltumor, Rachenkrebs, Phäochromozytom , Zirbeldrüsenparenchymtumor intermediärer Differenzierung, Pineoblastom, Pituyzytom, Hypophysenadenom, Hypophysentumor, Plasmazellneoplasma, pleuropulmonales Blastom, Polyembryom, Vorläufer-T-lymphoblastisches Lymphom, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular cancer, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, peritoneal pseudomyxoma, rectal cancer, renal cell carcinoma, airway carcinoma involving the NUT gene in chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter -Transformation, Sacrococcygeal teratoma, Salivary gland cancer , Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Spinal cord stromal tumor, Sexuality, Sezary syndrome, Signet ring cell carcinoma, Skin cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small Cell Lymphoma, Small Bowel Cancer, Partial Sarcoma, Somatostatinoma, Fuliginous Wart, Spinal Cord Tumor, Spinal Tumor, Splenic Marginal Zone Lymphoma, Squamous Cell Carcinoma, Gastric Cancer, Superficially Spreading Melanoma, Supratentorial Primitive Neuroectodermal Tumor, Epithelial-stroma Surface Tumor, Synovial Sarcoma, Leukemia Acute T -cell lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, terminal lymphocytic cancer, testicular cancer, thecoma, pharyngeal cancer, thymic carcinoma, thymoma, Thyroid Cancer, Transitional Cell Carcinoma of Renal Pelvis and Ureter, Transitional Cell Carcinoma, Urachal Carcinoma, Urethral Cancer, Urogenital Neoplasia, Uterine Sarcoma, Choroidal Melanoma, Vaginal Cancer, Verner-Morrison Syndrome, Verrucous Carcinoma B. Optic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Warthin's Tumor, Wilms' Tumor, other cancers and combinations thereof.

[0327]Preferably, the biological marker is selected from carbohydrates, glycolipids, glycoproteins, CD antigens (differentiation group) present in cells of a hematopoietic lineage such as CD2, CD4, CD8, CD21, etc.), γ-glutamyl transpeptidase; an adhesion protein (e.g., ICAM-1, ICAM-2, ELAM-1, VCAM-1); hormone, growth factor, cytokine and other receptor ligands; ion channels; and the membrane-bound form of a μ-immunoglobulin. Chain.

[0328]Preferably, the CD (cluster of differentiation) antigens present on cells of a hematopoietic lineage are CD2, CD4, CD8, CD21 or other CD (cluster of differentiation) antigens.

[0329]Preferably the adhesion protein is ICAM-1, ICAM-2, ELAM-1, VCAM-1 or another adhesion protein.

[0330]Preferably, the cell further comprises a synthetic, genetically modified and/or intentionally delivered polynucleotide encoding the chimeric antigen receptor (CAR) comprising a target-binding single-chain variable fragment (scFv) against a selected CD2 antigen (example clone: ​​AICD2.M1 and AICD2 .M2), CD3 delta, CD3 epsilon (clone example: OKT3), CD3 gamma, CD4 (clone example: DP81), CD7 (clone example: T3-3A1), CD8a, CD8 (clone example: SK1), CD11a (ITGAL, clone example: S6F1 ), CD11b (ITGAM, clone example: 17aba), CD11c (ITGAX, clone example: Bu15), CD11d (ITGAD), CD 18 (ITGB2, clone example: 23F2G), CD 19 ( B4, clone example: FMC63, SJ25C1), CD27 (TNFRSF7 , clone example: M-T271), CD28 (pattern clone: ​​KOL-2), CD29 (ITGB1, example clone: ​​AJ2), CD30 (TNFRSF8, example clone: ​​5F11), CD40 (TNFRSF5, example clone: ​​G28-5), CD48 (SLAMF2, Clone example: MEM-102), CD49a (ITGA1, clone example: mAQC2), CD49d (ITGA4, clone example: P3E3 ), CD49f (ITGA6, clone example: FW14-14), CD66a (CEACAM1), CD66b (CEACAM8, clone example BW 250/183 ), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5, clone example: CE25) , CD69 (CLEC2, example clone: ​​FN50), CD79A (alpha chain associated with B cell antigen receptor complex, example clone: ​​HM47), CD79B (Beta chain associated with B cell antigen receptor complex, clone example: CB3-1), CD84 (SLAMF5, clone example: CD84.1.21), CD96 (Tactile, clone example: TH -111), CD100 (SEMA4D, clone example: A8), CD103 (ITGAE, clone example: 2E7), CD134 (OX40 , clone example: Ber-ACT35), CD137 (4-1BB, clone example: 4B4-1-1), CD150 ( SLAMF1, example clone: ​​A12), CD158A (KIR2DL1, example clone: ​​HP-DM1) , CD158B1 (KIR2DL2, example clone: ​​DX27), CD158B2 (KIR2DL3, example clone: ​​DX27), CD158C (KIR3DP1), CD158D (KIRDL4, example clone: ​​mAb 33), CD158F1 (KIR2DL5A, clone example: UP-R1), CD158F2 (KIR2DL5B, clone example: UP-R1), CD158K (KIR3DL2), CD160 (clone example: BY55), CD162 (SELPLG, clone example: KPL-1), CD226 ( DNAM1, clone example: 11A8) , CD229 (SLAMF3, clone example: HLy-9.1.25), CD244 (SLAMF4, clone example: 2-69), CD247 (CD3-zeta, clone example: 6B10.2), CD258 (LIGHT, clone example: T5-39), CD268 (BAFFR, example clone: ​​11C1), CD270 (TNFSF14, example clone: ​​122), CD272 (BTLA, example clone: ​​M1H26), CD276 (B7-H3, example clone: ​​DCN.70), CD279 (PD-1 , example clone: ​​4B9), CD314 (NKG2D, example clone: ​​1D11), CD319 (SLAMF7, example clone: ​​162.1), CD335 (NK-p46, example clone: ​​9E2), CD336 (NK -p44, example clone: ​​P44-8), CD337 ( NK-p30, clone example: P30-15), CD352 (SLAMF6, clone example: NT-7), CD353 (SLAMF8, clone example: Cr24.1), CD355 (CRTAM), CD357 (TNFRSF18, clone example: 108-17), inducible T cell costimulator (ICOS), LFA-1 (CD11a/CD18), NKG2C, DAP-10, ICAM-1 (clone example: 1A6), NKp80 (KLRF1, clone example: 5D12), IL-2R beta (clone example: TU27 ), IL-2R gamma (clone example: AE.C9), IL-7R alpha (clone example: A019D5), LFA-1, SLAMF9 , LAT (clone example: W15102A), GADS Claudin-6 (GrpL), SLP-76 (LCP2 , clone example: H76), PAG1/CBP, a CD83 ligand, Fc gamma receptor, MHC molecule class 1, class 2 MHC molecule , TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptors, Toll-like Receptor, HER2 (example clone: ​​4D5), BCMA (example clone: ​​C11D5.3), PD-L1 (example clone: ​​PD-L1.1), VEGFR2 (example clone: ​​2C6), TCR b chain, and combinations thereof.

[0331]Preferably, an antigen-targeted scFv can be derived from a monoclonal antibody produced in spleen cells of immunized mice, a Fab phage display system, and applications are not limited to acquisition of scFv clones.

[0332]Preferably, proteins and DNA sequences of an antigen target-binding scFv can be obtained from public databases including GeneDatabase (https://www.ncbi.nlm.nih.gov/nuccore/), EMBL-EBI (https://www.ncbi.nlm.nih.gov/nuccore/), EMBL-EBI (https://www.ncbi.nlm.nih.gov/nuccore/). /www.ebi .ac.uk/),

[0333]ScFv against B cell maturation antigen (BCMA) derived from monoclonal antibodies (clone example C11D5.3) is generated in female RBF mice immunized against BCMA-Fc/KLH conjugate protein. Clone C11D5.3 is an immunoglobulin G (IgG) subclass 1 that specifically binds to naïve B cells, plasma cells, and/or memory B cells. The DNA sequence of C11D5.3 scFv is SEQ ID NO: 48 and the protein sequence of C11D5.3 scFv is SEQ ID NO: 49.

[0334]ScFv against human epidermal growth factor receptor 2 (HER2)-derived monoclonal antibody (clone example 4D5) is produced in BALB/c mice immunized against A431 cells of human squamous cell carcinoma cell line administered intraperitoneally. The mice with the highest serum titer are then boosted with purified A431 membrane extracts administered intravenously. Splenocytes derived from the immunized mice are fused with the mouse myeloma line X63.Ag8.653 and the 4D5 clone is selected against HER2. Monoclonal antibody binds to the extracellular domain of p185^HER2no cross-reactivity with other epidermal growth factor receptors. The 4D5 scFv DNA sequence is SEQ ID NO: 50 and the 4D5 scFv protein sequence is SEQ ID NO: 51.

[0335]Anti-intercellular adhesion molecule 1 (ICAM-1) ScFv derived from monoclonal antibody (clone example 1A6) is constructed by a structure-guided complementarity determining region (CDR) transplantation method. By sequence alignment of the CDR region of the crystal structure of the PDB antibody fragment id. 1A3R and 1A14, the final structure of the antibodies is determined by the energy minimization process provided by the Swiss PDB software. The clone-encoded plasmid is transformed into TOP10 cells and the lysed cell pellets are passed over a Ni+ chelating column for further purification according to the manufacturer's instructions. The 1A6 scFv DNA sequence is SEQ ID NO: 52 and the 1A6 scFv protein sequence is SEQ ID NO: 53.

[0336]Anti-programmed cell death protein 1 (PD-1) ScFv derived from a monoclonal antibody (clone example 4B9) is generated in BALB/c mice immunized against mitomycin-treated L929-PD-1 cells. Splenocytes from immunized mice are fused with cells from the murine myeloma cell line SP2/0 and the PD-1 clone 4B9 is selected for its strong reactivity against L929 PD-1 cells, not L929/mock. The DNA sequence of C11D5.3 scFv is SEQ ID NO: 54 and the protein sequence of C11D5.3 scFv is SEQ ID NO: 55.

[0337]The ScFv against vascular endothelial growth factor receptor 2 (VEGFR2)-derived monoclonal antibody (clone example 2C6) is obtained from a large human Fab phage display library (containing 3.7 × 10¹⁰clones) through multiple rounds of selection against the receptor protein containing the insertion domain of the immobilized recombinant kinase (KDR). The 2C6 scFv DNA sequence is SEQ ID NO: 56 and the 2C6 scFv protein sequence is SEQ ID NO: 57.

[0338]Preferably, sequences of scFv clones can be synthesized and constructed according to public databases such as NCBI Nucleotide (https://www.ncbi.nlm.nih.gov/nuccore).

[0339]Preferably, the cell is capable of mediating an antibody dependent cellular cytotoxicity (ADCC) response.

[0340]The present invention provides a method of treating cancer, autoimmune disease, neuronal disease, human immunodeficiency virus (HIV) infection, hematopoietic cell-related disease, metabolic syndrome, pathogenic disease, viral infection or bacterial infection comprising administering a composition comprising an effective amount of a human natural killer cell for an individual in need thereof; the human natural killer cell comprises a synthetic, genetically modified and/or intentionally delivered polynucleotide encoding the chimeric antigen receptor (CAR) comprising a target-binding single-chain variable fragment (scFv) against a selected antigen from CD2, CD3 delta, CD3 epsilon, CD3 gamma , CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1 ) , CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3 ) , CD66e (CEACAM5), CD69 (CLEC2), CD79A (alpha chain-associated B cell antigen receptor complex), CD79B (beta chain associated B cell antigen receptor complex), CD84 (SLAMF5), CD96 (tactile), CD100 (SEMA4D) , CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3) , CD158C (KIR3DP1), CD158D (KIRDL4) , CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 ( LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), T-cell inducible costimulator (ICOS), LFA-1 (CD11a/CD18 ), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R Beta, IL-2R Gamma, IL-7R Alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2 ), PAG1/ CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK Cell receptors, Toll-like receptor, HER2, BCMA B. PD-L1 and combinations thereof, and the human natural killer cell is (A) deposited at NPMD with NITE accession number BP-03017; or (B) having the following characteristics:

[0341]i) expresses a CD16 receptor,

[0342]ii) retain its ability to proliferate for at least 3 months after subculture, and

[0343]iii) x) excluding synthetic, genetically modified and/or intentionally introduced polynucleotides encoding the CD16 receptor, or y) using the ddPCR system to analyze the genomic DNA of human natural killer cells, the ratio of the DNA molecule encoded by the CD16 F176F probe is detectable The DNA molecule detectable by the CD16 F176V probe is equal to or greater than 1, wherein the sequence of the CD16 F176F probe is SEQ ID NO: 11 and the sequence of the CD16 F176V probe is SEQ ID NO: 12.

[0344]Preferably, the number of human natural killer cells in the composition is at least 5×10⁵and human natural killer cells in an amount equal to or greater than 5% in number based on the total number of cells in the composition as 100%.

[0345]Preferably the subject is a human.

[0346]Preferably, the method is for the selective treatment of cancer acanthoma, acinar cell carcinoma, acoustic neuroma, acral tiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid leukemia dendritic cell leukemia, acute myeloid leukemia Leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adrenal cortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, lymphoma AIDS-related, alveolar soft tissue sarcoma, ameloblastic fibroma, Anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendectomy cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma , bile duct cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchoalveolar carcinoma, brown tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile carcinoma, carcinoma of unknown primary site , carcinosarcoma, Castleman's disease, embryonic central nervous system tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma , chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic Myeloproliferative disease, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colon cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonic carcinoma , endodermal sinus tumor, endometrial carcinoma, endometrial cancer, endometrioid tumor, T cell-associated lymphoma enteropathy, ependymoblastoma , ependymoma, epitheloid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing family tumor, Ewing family sarcoma, Ewing disease sarcoma , extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal Carcinoid tumor, Gastrointestinal stromal tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational trophoblastic tumor, Giant cell bone tumor, Glioblastoma multiforme, Glioma, Cerebral glioma, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy cell leukemia, Hairy cell leukemia, Head and neck cancer, Head and cervical cancer, breast cancer heart, hemangioblastoma, hemangiopericytoma, hemangiosarcoma, hematological malignancy, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, Islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, larynx cancer, larynx cancer, lentigo maligna melanoma, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoblastic Leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant Fibrous Histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant Peripheral Nerve Sheath Tumor, Malignant Rhabdoid Tumor, Malignant Triton Tumor, MALT Lymphoma, Mantle Cell Lymphoma -Leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell carcinoma with occult primary tumor, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndromes, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neoplasia, neurinoma, neuroblastoma , neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-melanoma. Skin cancer, non-small cell lung cancer, ocular oncology, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, low malignant ovarian tumor, Paget's disease of the breast, Pancoast Tumor, Bauchspeicheldrüsenkrebs, Bauchspeicheldrüsenkrebs, papillärer Schilddrüsenkrebs, Papillomatose, Paragangliom, Nasennebenhöhlenkrebs, Nebenschilddrüsenkrebs, Peniskrebs, perivaskulärer Epithelioidzelltumor, Rachenkrebs, Phäochromozytom , Zirbeldrüsenparenchymtumor intermediärer Differenzierung, Pineoblastom, Pituizytom, Hypophysenadenom, Hypophysentumor, Plasmazellneoplasie, Pleuropulmonales Blastom, Polyembryom, Vorläufer-T-lymphoblastisches Lymphoma, Central lymphoma of the primary nervous system, Primary effusion lymphoma, Primary hepatocellular cancer, Primary liver cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Peritoneal pseudomyxoma, Rectal cancer, Renal cell carcinoma, Airway carcinoma involving the NUT gene on chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma , Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma , schwannomatosis, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord stromal tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma , small cell lung cancer, small cell lymphoma, small bowel cancer, soft tissue sarcoma, somatostatinoma, sooty wart, spinal cord tumor, spine tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, epithelial surface stromal tumor, synovial sarcoma, acute lymphoblastic T-cell Leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphocytic cancer, testicular cancer, thecoma, pharyngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional cell carcinoma, urethral cancer, urethral cancer, urogenital neoplasia, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, macroglobulinemia, Waldenstrom's tumor, Warthin's tumor, Wilms' tumor, other cancers and combinations thereof.

[0347]The present invention provides a human cell with cytotoxic capacity, which has the following properties:

[0348]i) carriers of a CD3 phenotype⁻CD56⁺and expresses a CD16 receptor; It is

[0349]ii) comprising at least one antigen-binding complex on the cell membrane, the antigen-binding complex being an agent for inducing a cytotoxic activity of the cell by specific binding by an antigen selected from cancer antigen, glycolipid, glycoprotein, differentiation antigen cluster present in cells of a hematopoietic lineage, Antigenic peptide bound to major histocompatibility complex, gamma-glutamyl transpeptidase, adhesion protein, hormone, growth factor, cytokine, receptor ligand, ion channel, form bound to membrane of a μ-immunoglobulin. -chain, alpha-fetoprotein, C-reactive protein, chromogranin A, epithelial mucin antigen, human epithelial-specific antigen, Lewis(a) antigen, multidrug resistance-related protein, neu oncogene protein, neuron-specific enolase, glycoprotein P, Multidrug Resistance Related Antigen, p170, Multidrug Resistance Related Antigen, Prostate Specific Antigen, NCAM, Ganglioside Molecule, MART-1, Heat Shock Protein, SialylTn, Tyrosinase, MUC-1, HER-2 /neu, KSA, PSMA, p53, RAS, EGF -R, VEGF, MAGE or other target antigen (marker) expressed by a target cell; wherein the cell is not genetically modified from the natural killer cell having ATCC accession number CRL-2407.

[0350]Preferably, the CD16 receptor is a CD16a receptor or a CD16b receptor.

[0351]Preferably, an expressed polynucleotide encoding the CD16 receptor is located on the q-arm of chromosome 1 at position 1q23.3.

[0352]Preferably, the cell is non-tumorigenic in an immunocompromised mouse.

[0353]Preferably, the cell is non-tumorigenic in an allogeneic individual after being irradiated with γ-rays.

[0354]Preferably, an expressed polynucleotide encoding the CD16 receptor comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 19.

[0355]Preferably, the CD16 receptor comprises an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 20.

[0356]Preferably, the cell further comprises an inactive tumor suppressor gene or a mutated and highly expressed oncogene.

[0357]Preferably, the cell is capable of mediating an antibody dependent cellular cytotoxicity (ADCC) response and the cell is a male cell.

[0358]Preferably, the cell is a natural killer cell genetically engineered to express the antigen-binding complex.

[0359]Preferably the antigen is a cancer antigen selected from HER2/neu (ERBB2), HER3 (ERBB3), EGFR, VEGF, VEGFR2, GD2, CTLA4, CD19, CD20, CD22, CD30, CD33 (Siglec-3), CD52 (CAMPATH - 1 antigen), CD326 (EpCAM), CA-125 (MUC16), MMP9, DLL3, CD274 (PD-L1), CEA, MSLN (Mesothelin), CA19-9, CD73, CD205 (DEC205), CD51, c- MET, TRAIL-R2, IGF-1R, CD3, MIF, folate receptor alpha (FOLR1), CSF1, OX-40, CD137, TfR, MUC1, CD25 (IL-2R), CD115 (CSF1R), IL1B, CD105 (Endoglin ) , KIR, CD47, CEA, IL-17A, DLL4, CD51, Angiopoietin 2, Neuropilin-1, CD37, CD223 (LAG-3), CD40, LIV-1 (SLC39A6), CD27 (TNFRSF7), CD276 (B7-H3 ), Trop2, Claudin1 (CLDN1), PSMA, TIM-1 (HAVcr-1), CEACAM5, CD70, LY6E, BCMA, CD135 (FLT3), APRIL, TF(F3), Nectin-4, FAP, GPC3, FGFR3 , ICAM-1 (CD54), ROBO1, NKG2D Ligand, CD123, CS1/SLAMF7/CD319/CRACC, CD7, CD142 (Platelet Tissue Factor, Factor III, Tissue Factor), CD38, CD138, EGFR VIII, EGFR, EGFR806, EGFR family member, PD -1, ROR1, CSPG4, CLL-1 (CLEC12A), CD147, PSCA, EPHA2, GPRC5D, CD133, B7H6, DSC2, AE1 (SLC4A1), GUCY2C, CDH17, HPSE, CD24, MUC4, AFP-L3 , SP17, DCLK1 , CAIX (CA9), IL13RA2, IL13Ra, CD56, CD44v6, TCR beta chain, chlorotoxin ligands, claudin-6, claudin-18.2, EIIIB (fibronectin), glypican-1 (GPC1)), PLAP (placental alkaline phosphatase ), uPAR, HCMV glycoprotein B (gB), HLA-DR (Lym1 antibody target), tumor-associated αvβ6 integrin, LunX, αvβ3 integrin, folate receptor beta (FRβ), LILRB4, MISIIR (Müller type 2 receptor Inhibitor Substance), 5T4, CD83 Ligand, HBsAg, CD171 (L1-CAM), TAG72 (TAG72 (tumor-associated glycoprotein 72)), B7-H4, CD166 (ALCAM), AC133 (PROM1), LeY, CD13 (TIM1 ), CD117, TEM8 (ANTXR1), CD26, IL13Ra2, IGF1R, Muc3a, IL1RAP, TSLPR (CRLF2), LMP1, Siglec7, Siglec9, Epstein-Barr virus gp350, CD1a, CLEC14A, MAGE-A1, MAGE-A4, neurofilament M (NEFM), HERV- K env protein, HLA-A*0201/NY-ESO-1(157-165) peptide, 2B4, TACI (TNFRSF13B), CD32A(131R), AXL, Lewis Y, CD80, CD86, ROR2, a killer immunoglobulin-like receptors (KIRs), a T cell receptor, a major histocompatibility complex protein, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, and combinations thereof, or the antigen binding complex is a Chimeric Antigen Receptor (CAR).

[0360]Preferably, the NK3.3 natural killer cell and the cell line are from different individuals.

[0361]Preferably, the cell is from an individual with cancer.

[0362]Preferably the cell is from a Caucasian male.

[0363]Preferably, the cell and the natural killer cell having ATCC Accession Number CRL-2407 are from the same individual.

[0364]Preferably, the cell retains its ability to proliferate after subculture for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year. 2 years, 3 years or 4 years.

[0365]Preferably, the antigen-binding complex is produced by the cell.

[0366]Preferably, the cell further has the ability to secrete IL-15, the ability to secrete IL-18, the ability to secrete IL-21, the ability to secrete IL-2, or the ability to secrete another proliferation-inducing cytokine or a combination of these.

[0367]Preferably, the cell still carries a CD2 phenotype⁺.

[0368]Preferably, the cell still carries a CD45 phenotype⁺.

[0369]Preferably, the cell still carries a selected CD4 phenotype⁺, CD25⁺, NKp30⁺, NKG2D⁺, NKp44⁺, NKp46⁺, CD27⁺, OX40⁺, CD107a⁺, NKG2A⁺, PD-1⁺, TIGIT⁺, SIRPα⁺, CD158⁺and their combination.

[0370]Preferably, the antigen binding complex comprises the CD3 zeta subunit (CD3).

[0371]Preferably, the antigen binding complex further comprises the CD28 subunit, ICOS subunit (CD278), 4-1BB subunit (CD137), OX40 subunit (CD134), CD27 subunit, CD40 subunit, CD40L subunit, TLRs subunit or a other costimulatory molecule expressed by at least one of the effector cells or a combination thereof.

[0372]Preferably, the cell further comprises a synthetic, genetically engineered and/or intentionally delivered polynucleotide encoding a single-chain target-binding variable fragment (scFv) against the antigen, and the single-chain target-binding variable fragment is at least one subunit of the antigen-binding complex .

[0373]Preferably, a DNA sequence from the cell's chromosome is at least 90% or 95% similar to the DNA sequence from the corresponding natural killer cell chromosome deposited with NPMD under NITE accession number BP-03017. Preferably, a DNA sequence from the cell's chromosome is at least 99%, 99.99% or 99.995% similar to the DNA sequence from the corresponding natural killer cell chromosome deposited with NPMD under NITE accession number BP-03017.

[0374]Preferably, the chromosome DNA sequence is a chromosome 17 DNA, a chromosome 19 DNA sequence, a chromosome 22 DNA sequence, a chromosome 4 DNA sequence, a chromosome 18 DNA sequence , a Y chromosome DNA or an X chromosome DNA sequence Chromosome DNA sequence is a chromosome 1 DNA sequence, a chromosome 2 DNA sequence, a chromosome 5 DNA sequence, a DNA of chromosome 6, a DNA sequence of chromosome 7, a DNA sequence of chromosome 8, a DNA sequence of chromosome 9, a DNA sequence of chromosome 10, DNA sequence of chromosome 11, DNA sequence of chromosome 12, DNA Sequence of Chromosome 13 DNA, Chromosome 14 DNA Sequence, Chromosome 15 DNA Sequence, Chromosome 16 DNA Sequence, Chromosome 20 DNA Sequence, Chromosome 21 DNA Sequence, Chromosome 3 DNA Sequence.

[0375]Preferably, the entire genome of the cell is at least 99.995% similar to the entire genome of the natural killer cell deposited with the NPMD under NITE accession number BP-03017.

[0376]Preferably, the cell does not contain synthetic, genetically modified, and/or intentionally introduced polynucleotides encoding the CD16 receptor.

[0377]Preferably, using the ddPCR system to analyze the genomic DNA of the cell, the ratio of the DNA molecule detectable by the CD16 F176F probe to the DNA molecule detectable by the CD16 F176V probe is equal to or greater than 1, the sequence of the CD16 probe is F176F is SEQ ID NO: 11 and CD16 F176V probe sequence is SEQ ID NO: 12.

[0378]The present invention provides a composition substantially enriched in human cells having cytotoxic capacity, wherein the number of human cells in the composition is at least 5x10⁵and human cells are in an amount equal to or greater than 5% by number based on the total number of cells in the composition as 100%; The human cell has the following characteristics:

[0379]i) carriers of a CD3CD56 phenotype⁺and expresses a CD16 receptor, and

[0380]ii) comprising at least one antigen-binding complex on the cell membrane, the antigen-binding complex being an agent for inducing a cytotoxic activity of the cell by specific binding by an antigen selected from cancer antigen, glycolipid, glycoprotein, differentiation antigen cluster present in cells of a hematopoietic lineage, Antigenic peptide bound to major histocompatibility complex, gamma-glutamyl transpeptidase, adhesion protein, hormone, growth factor, cytokine, receptor ligand, ion channel, form bound to membrane of a μ-immunoglobulin. -chain, alpha-fetoprotein, C-reactive protein, chromogranin A, mucin epithelial antigen, human epithelial-specific antigen, Lewis(a) antigen, multidrug resistance-related protein, neu oncogene protein, neuron-specific enolase, Glycoprotein P, Multidrug Resistance Related Antigen, p170, Multidrug Resistance Related Antigen, Prostate Specific Antigen, NCAM, Ganglioside Molecule, MART-1, Heat Shock Protein, SialylTn, Tyrosinase, MUC-1, HER-2 /neu, KSA, PSMA, p53, RAS , EGF -R, VEGF, MAGE or other target antigen (marker) expressed by a target cell, which cell is not genetically modified from the natural killer cell having accession number ATCC CRL-2407.

[0381]Preferably, the CD16 receptor is a CD16a receptor or a CD16b receptor.

[0382]Preferably, an expressed polynucleotide encoding the CD16 receptor is located on the q-arm of chromosome 1 at position 1q23.3.

[0383]Preferably, the human cells are nontumorogenic in an immunocompromised mouse.

[0384]Preferably, human cells are non-tumorigenic in an allogeneic individual after being irradiated with γ-rays.

[0385]Preferably, an expressed polynucleotide encoding the CD16 receptor comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 19.

[0386]Preferably, the CD16 receptor comprises an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 20.

[0387]Preferably, the human cell further comprises an inactive tumor suppressor gene or a mutated and highly expressed oncogene.

[0388]Preferably, the human cell is capable of mediating an antibody dependent cellular cytotoxicity (ADCC) response and the cell is a male cell.

[0389]Preferably, the cell is a natural killer cell genetically engineered to express the antigen-binding complex.

[0390]Preferably the antigen is a cancer antigen selected from HER2/neu (ERBB2), HER3 (ERBB3), EGFR, VEGF, VEGFR2, GD2, CTLA4, CD19, CD20, CD22, CD30, CD33 (Siglec-3), CD52 (CAMPATH - 1 antigen), CD326 (EpCAM), CA-125 (MUC16), MMP9, DLL3, CD274 (PD-L1), CEA, MSLN (Mesothelin), CA19-9, CD73, CD205 (DEC205), CD51, c- MET, TRAIL-R2, IGF-1R, CD3, MIF, folate receptor alpha (FOLR1), CSF1, OX-40, CD137, TfR, MUC1, CD25 (IL-2R), CD115 (CSF1R), IL1B, CD105 (Endoglin ) , KIR, CD47, CEA, IL-17A, DLL4, CD51, Angiopoietin 2, Neuropilin-1, CD37, CD223 (LAG-3), CD40, LIV-1 (SLC39A6), CD27 (TNFRSF7), CD276 (B7-H3 ), Trop2, Claudin1 (CLDN1), PSMA, TIM-1 (HAVcr-1), CEACAM5, CD70, LY6E, BCMA, CD135 (FLT3), APRIL, TF(F3), Nectin-4, FAP, GPC3, FGFR3 , ICAM-1 (CD54), ROBO1, NKG2D Ligand, CD123, CS1/SLAMF7/CD319/CRACC, CD7, CD142 (Platelet Tissue Factor, Factor III, Tissue Factor), CD38, CD138, EGFR VIII, EGFR, EGFR806, EGFR family member, PD -1, ROR1, CSPG4, CLL-1 (CLEC12A), CD147, PSCA, EPHA2, GPRC5D, CD133, B7H6, DSC2, AE1 (SLC4A1), GUCY2C, CDH17, HPSE, CD24, MUC4, AFP-L3 , SP17, DCLK1 , CAIX (CA9), IL13RA2, IL13Ra, CD56, CD44v6, TCR beta chain, chlorotoxin ligands, claudin-6, claudin-18.2, EIIIB (fibronectin), glypican-1 (GPC1)), PLAP (placental alkaline phosphatase ), uPAR, HCMV glycoprotein B (gB), HLA-DR (Lym1 antibody target), tumor-associated αvβ6 integrin, LunX, αvβ3 integrin, folate receptor beta (FRβ), LILRB4, MISIIR (Müller type 2 receptor -Inhibitor substance), 5T4, CD83 Ligand, HBsAg, CD171 (L1-CAM), TAG72 (TAG72 (Tumor Associated Glycoprotein 72)), B7-H4, CD166 (ALCAM), AC133 (PROM1), LeY, CD13 (TIM1) , CD117, TEM8 (ANTXR1), CD26, IL13Ra2, IGF1R, Muc3a, IL1RAP, TSLPR (CRLF2), LMP1, Siglec7, Siglec9, Epstein-Barr Virus gp350, CD1a, CLEC14A, MAGE-A1, MAGE -A4, Neurofilament M (NEFM), HERV-K env protein, HLA-A*0201/NY-ESO-1(157-165) peptide, 2B4, TACI (TNFRSF13B), CD32A(131R), AXL, Lewis Y, CD80, CD86 , ROR2, a killer cellular immunoglobulin-like receptors (KIRs), a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, and combinations thereof, or the complex of antigen binding is a chimeric antigen receptor (CAR).

[0391]Preferably, the human cell and the NK3.3 natural killer cell line are from different individuals.

[0392]Preferably, the human cell is from an individual with cancer.

[0393]Preferably, the human cell is from a Caucasian male.

[0394]Preferably, the human cell and the natural killer cell having ATCC Accession Number CRL-2407 are from the same individual.

[0395]Preferably, the human cell retains its ability to proliferate after subculture for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years or 4 years.

[0396]Preferably, the number of human cells in the composition is 5x10⁵-5×10⁹.

[0397]Preferably, the number of human cells in the composition is 1×10⁶, 1,1 × 10⁶, 5×10⁶, 1×10⁷, 1,1 × 10⁷, 5×10⁷, 5,1 × 10⁷, 1×10⁸, 1,1 × 10⁸, 5×10⁸, 5,1 × 10⁸, 1×10⁹, 1,1 × 10⁹, 5×10⁹, 1×10¹⁰, 1,1 × 10¹⁰, 5×10¹⁰, 1×10¹¹, 1,1 × 10¹¹, 5×10¹¹, 5,1 × 10¹¹, 1×10¹², 1,1 × 10¹², 5×10¹², 5,1 × 10¹², 1×10¹³, 1,1 × 10¹⁴, 5×10¹⁴, 1×10¹⁵, 1,1 × 10¹⁵, 5×10¹⁵, 1×10¹⁶, 1,1 × 10¹⁶, 5×10¹⁶, 5,1 × 10¹⁶, 1×10¹⁷, 1,1 × 10¹⁷, 5×10¹⁷, 5,1 × 10¹⁷, 1×10¹⁸, 1,1 × 10¹⁸, 5×10¹⁸, 1×10¹⁹, 1,1 × 10¹⁹, 5×10¹⁹, 1×10²⁰, 1,1 × 10²⁰, 5×10²⁰, 5,1 × 10²⁰, 1×10²¹, 1,1 × 10²¹, 5×10²¹, 5,1 × 10²¹, 1×10²², 1,1 × 10²², 5×10²², 1×10²³, 1,1 × 10²³, 5×10²³, 1×10²⁴, 1,1 × 10²⁴, 5×10²⁴, 5,1 × 10²⁴, 1×10²⁵, 1,1 × 10²⁵, 5×10²⁵, 5,1 × 10²⁵, 1×10²⁶, 1,1 × 10²⁶, 5×10²⁶, 1×10²⁷, 1,1 × 10²⁷, 5×10²⁷, 1×10²⁸, 1,1 × 10²⁸, 5×10²⁸, 5,1 × 10²⁸, 1×10²⁹, 1,1 × 10²⁹, 5×10²⁹, 5,1 × 10²⁹, 1×10³⁰, 1,1 × 10³⁰, 5×10³⁰, 1×10³¹, 1,1 × 10³¹, 5×10³¹, 1×10³², 1,1 × 10³², 5×10³², 5,1 × 10³², 1×10³³, 1,1 × 10³³, 5×10³³, 5,1 × 10³³, 1×10³⁴, 1,1 × 10³⁴, 5×10³⁴, 1×10³⁵, 1,1 × 10³⁵, 5×10³⁵, 1×10³⁶, 1,1 × 10³⁶, 5×10³⁶, 5,1 × 10³⁶, 1×10³⁷, 1,1 × 10³⁷, 5×10³⁷, 5,1 × 10³⁷, 1×10³⁸, 1,1 × 10³⁸, 5×10³⁸, 1×10³⁹, 1,1 × 10³⁹, 5×10³⁹, 5,1 × 10³⁹, 1×10⁴⁰, 1,1 × 10⁴⁰, 5×10⁴⁰. Preferably, the number of human cells in the composition is 1×10⁶-1×10⁴¹.

[0398]Preferably the total number of human cells is 5%-100% based on the total number of cells in the composition as 100%. Preferably, the human cells are in an amount equal to or greater than 5%, 7%, 9%, 10%, 12%, 15%, 19%, 20%, 22%, 25%, 29%, 30%, 32% , 35%, 39%, 40%, 42%, 45%, 49%, 50%, 52%, 55%, 59%, 60%, 62%, 65%, 69%, 70%, 72% , 75 %, 79%, 80%, 82%, 85%, 89% or 95% by number based on the total number of cells in the composition as 100%.

[0399]Preferably, the antigen-binding complex is produced by the human cell.

[0400]Preferably, the human cell further has IL-15 secreting ability, IL-18 secreting ability, IL-21 secreting ability, IL-2 secreting ability, or other proliferation-inducing cytokine secreting ability or one combination of them.

[0401]Preferably, the human cell still carries a CD2 phenotype⁺.

[0402]Preferably, the human cell still carries a CD45 phenotype⁺.

[0403]Preferably, the human cell still carries a selected CD4 phenotype⁺, CD25⁺, NKp30⁺, NKG2D⁺, NKp44⁺, NKp46⁺, CD27⁺, OX40⁺, CD107a⁺, NKG2A⁺, PD-1⁺, TIGIT⁺, SIRPα⁺, CD 158⁺and their combination.

[0404]Preferably, the antigen binding complex comprises the CD3 zeta subunit (CD3).

[0405]Preferably, the antigen binding complex further comprises the CD28 subunit, ICOS subunit (CD278), 4-1BB subunit (CD137), OX40 subunit (CD134), CD27 subunit, CD40 subunit, CD40L subunit, TLRs subunit or a other costimulatory molecule expressed by at least one of the effector cells or a combination thereof.

[0406]Preferably, the cell further comprises a synthetic, genetically engineered and/or intentionally delivered polynucleotide encoding a single-chain target-binding variable fragment (scFv) against the antigen, and the single-chain target-binding variable fragment is at least one subunit of the antigen-binding complex .

[0407]Preferably, a human cell chromosomal DNA sequence is at least 90% or 95% similar to the corresponding natural killer cell chromosomal DNA sequence deposited with NPMD under NITE accession number BP-03017.

[0408]Preferably, a DNA sequence from the cell's chromosome is at least 99%, 99.99% or 99.995% similar to the DNA sequence from the corresponding natural killer cell chromosome deposited with NPMD under NITE accession number BP-03017.

[0409]Preferably, the chromosome DNA sequence is a chromosome 17 DNA, a chromosome 19 DNA sequence, a chromosome 22 DNA sequence, a chromosome 4 DNA sequence, a chromosome 18 DNA sequence , a Y chromosome DNA or an X chromosome DNA sequence Chromosome DNA sequence is a chromosome 1 DNA sequence, a chromosome 2 DNA sequence, a chromosome 5 DNA sequence, a DNA of chromosome 6, a DNA sequence of chromosome 7, a DNA sequence of chromosome 8, a DNA sequence of chromosome 9, a DNA sequence of chromosome 10, DNA sequence of chromosome 11, DNA sequence of chromosome 12, DNA Sequence of Chromosome 13 DNA, Chromosome 14 DNA Sequence, Chromosome 15 DNA Sequence, Chromosome 16 DNA Sequence, Chromosome 20 DNA Sequence, Chromosome 21 DNA Sequence, Chromosome 3 DNA Sequence.

[0410]Preferably, the entire genome of the cell is at least 99.995% similar to the entire genome of the natural killer cell deposited with the NPMD under NITE accession number BP-03017.

[0411]Preferably, the cell does not contain synthetic, genetically modified, and/or intentionally introduced polynucleotides encoding the CD16 receptor.

[0412]Preferably, using the ddPCR system to analyze the genomic DNA of the cell, the ratio of the DNA molecule detectable by the CD16 F176F probe to the DNA molecule detectable by the CD16 F176V probe is equal to or greater than 1, the sequence of the CD16 probe is F176F is SEQ ID NO: 11 and CD16 F176V probe sequence is SEQ ID NO: 12.

[0413]The present invention provides a method of obtaining a composition substantially enriched in human cells; the procedure includes:

[0414](a) Obtaining a population of human CD16⁺natural killer cells; It is

[0415](b) delivery of a polynucleotide encoding the antigen-binding complex comprising a single-chain target-binding variable fragment (scFv) to the antigen on human CD16⁺natural killer cells, thereby obtaining the composition substantially enriched in human cells;

[0416]where human CD16⁺The natural killer cell has the following properties:

[0417]i) a human CD16 chromosomal DNA sequence⁺the natural killer cells are at least 90% or 95% similar to the DNA sequence of the corresponding chromosome of the natural killer cell deposited with the NPMD under NITE accession number BP-03017, and

[0418]ii) not genetically modified from natural killer cells having ATCC accession number CRL-2407.

[0419]Preferably, the antigen binding complex comprises a CD3 zeta peptide (CD3).

[0420]Preferably, the antigen binding complex further comprises CD28 peptide, ICOS peptide (CD278), 4-1BB peptide (CD137), OX40 peptide (CD134), CD27 peptide, CD40 peptide, CD40L peptide, TLRs peptide or another molecular peptide costimulator expressed by at least one of the effector cells or a combination thereof.

[0421]Preferably, the number of human cells in the composition is at least 5x10⁵, and human cells in an amount equal to or greater than 5% in number based on the total number of cells in the composition as 100%.

[0422]Preferably, the number of human cells in the composition is 5x10⁵-5×10⁹.

[0423]Preferably, the number of human cells in the composition is 1×10⁶, 1,1 × 10⁶, 5×10⁶, 1×10⁷, 1,1 × 10⁷, 5×10⁷, 5,1 × 10⁷, 1×10⁸, 1,1 × 10⁸, 5×10⁸, 5,1 × 10⁸, 1×10⁹, 1,1 × 10⁹, 5×10⁹, 1×10¹⁰, 1,1 × 10¹⁰, 5×10¹⁰, 1×10¹¹, 1,1 × 10¹¹, 5×10¹¹, 5,1 × 10¹¹, 1×10¹², 1,1 × 10¹², 5×10¹², 5,1 × 10¹², 1×10¹³, 1,1 × 10¹⁴, 5×10¹⁴, 1×10¹⁵, 1,1 × 10¹⁵, 5×10¹⁵, 1×10¹⁶, 1,1 × 10¹⁶, 5×10¹⁶, 5,1 × 10¹⁶, 1×10¹⁷, 1,1 × 10¹⁷, 5×10¹⁷, 5,1 × 10¹⁷, 1×10¹⁸, 1,1 × 10¹⁸, 5×10¹⁸, 1×10¹⁹, 1,1 × 10¹⁹, 5×10¹⁹, 1×10²⁰, 1,1 × 10²⁰, 5×10²⁰, 5,1 × 10²⁰, 1×10²¹, 1,1 × 10²¹, 5×10²¹, 5,1 × 10²¹, 1×10²², 1,1 × 10²², 5×10²², 1×10²³, 1,1 × 10²³, 5×10²³, 1×10²⁴, 1,1 × 10²⁴, 5×10²⁴, 5,1 × 10²⁴, 1×10²⁵, 1,1 × 10²⁵, 5×10²⁵, 5,1 × 10²⁵, 1×10²⁶, 1,1 × 10²⁶, 5×10²⁶, 1×10²⁷, 1,1 × 10²⁷, 5×10²⁷, 1×10²⁸, 1,1 × 10²⁸, 5×10²⁸, 5,1 × 10²⁸, 1×10²⁹, 1,1 × 10²⁹, 5×10²⁹, 5,1 × 10²⁹, 1×10³⁰, 1,1 × 10³⁰, 5×10³⁰, 1×10³¹, 1,1 × 10³¹, 5×10³¹, 1×10³², 1,1 × 10³², 5×10³², 5,1 × 10³², 1×10³³, 1,1 × 10³³, 5×10³³, 5,1 × 10³³, 1×10³⁴, 1,1 × 10³⁴, 5×10³⁴, 1×10³⁵, 1,1 × 10³⁵, 5×10³⁵, 1×10³⁶, 1,1 × 10³⁶, 5×10³⁶, 5,1 × 10³⁶, 1×10³⁷, 1,1 × 10³⁷, 5×10³⁷, 5,1 × 10³⁷, 1×10³⁸, 1,1 × 10³⁸, 5×10³⁸, 1×10³⁹, 1,1 × 10³⁹, 5×10³⁹, 5,1 × 10³⁹, 1×10⁴⁰, 1,1 × 10⁴⁰, 5×10⁴⁰. Preferably, the number of human cells in the composition is 1×10⁶-1×10⁴¹.

[0424]Preferably the total number of human cells is 5%-100% based on the total number of cells in the composition as 100%. Preferably, the human cells are in an amount equal to or greater than 5%, 7%, 9%, 10%, 12%, 15%, 19%, 20%, 22%, 25%, 29%, 30%, 32% , 35%, 39%, 40%, 42%, 45%, 49%, 50%, 52%, 55%, 59%, 60%, 62%, 65%, 69%, 70%, 72% , 75 %, 79%, 80%, 82%, 85%, 89% or 95% by number based on the total number of cells in the composition as 100%.

[0425]Preferably, a DNA sequence from the cell's chromosome is at least 99%, 99.99% or 99.995% similar to the DNA sequence from the corresponding natural killer cell chromosome deposited with NPMD under NITE accession number BP-03017.

[0426]Preferably, the chromosome DNA sequence is a chromosome 17 DNA, a chromosome 19 DNA sequence, a chromosome 22 DNA sequence, a chromosome 4 DNA sequence, a chromosome 18 DNA sequence , a Y chromosome DNA or an X chromosome DNA sequence Chromosome DNA sequence is a chromosome 1 DNA sequence, a chromosome 2 DNA sequence, a chromosome 5 DNA sequence, a DNA of chromosome 6, a DNA sequence of chromosome 7, a DNA sequence of chromosome 8, a DNA sequence of chromosome 9, a DNA sequence of chromosome 10, DNA sequence of chromosome 11, DNA sequence of chromosome 12, DNA Sequence of Chromosome 13 DNA, Chromosome 14 DNA Sequence, Chromosome 15 DNA Sequence, Chromosome 16 DNA Sequence, Chromosome 20 DNA Sequence, Chromosome 21 DNA Sequence, Chromosome 3 DNA Sequence.

[0427]Preferably, the entire genome of the cell is at least 99.995% similar to the entire genome of the natural killer cell deposited with the NPMD under NITE accession number BP-03017.

[0428]The present invention provides a method for culturing and expanding human cells; the method comprising

[0429](x) contacting the human cells in a container with a culture medium comprising 0.5-10% by volume human platelet lysate and 100-3000 IU/ml LIL-2; It is

[0430](y) culturing the cells for several days;

[0431]Preferably, the container includes a bottom for seeding cells, and the bottom is air permeable and water impermeable.

[0432]Step (y) preferably comprises sub-steps:

[0433](y1) culturing the cells for at least one day; It is

[0434](y2) Subculturing the cells for at least 1 month.

[0435]The present invention provides a method of treating cancer, tumor, autoimmune disease, neuronal disease, human immunodeficiency virus (HIV) infection, hematopoietic cell-associated diseases, metabolic syndrome, pathogenic disease, viral infection or bacterial infection, comprising administering a A composition comprising an effective amount of the cell for a patient in need thereof.

[0436]Preferably the antigen is a cancer antigen.

[0437]Preferably the method is for the treatment of cancer or tumor.

[0438]Preferably, the method is for treating acanthoma, acinar cell carcinoma, acoustic neuroma, acral tiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, myeloid leukemia, acute dendritic leukemia cell leukemia, acute myeloid leukemia Leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenal cortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, soft partial alveolar sarcoma, ameloblastic fibroma , anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendectomy cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, baso-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini's Duct carcinoma, bile duct cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, tumor Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt's disease lymphoma, cancer of unknown primary location, carcinoid tumor, carcinoma, carcinoma in situ, penile carcinoma, carcinoma of unknown primary location, carcinosarcoma, Castleman's disease, embryonic central nervous system tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, chronic neutrophilic leukemia, clear cell cancer, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B cell lymphoma , dysembryoplastic neuroepithelial tumor, embryonic carcinoma, cancer of the endodermal cavity, endometrial carcinoma, endometrial cancer, endometrioid tumor, T-cell lymphoma associated with enteropathy, ependymoblastoma, ependymoma, epitheloid sarcoma, erythroleukemia, esophageal cancer, esthetic euroblastoma, tumor of the Ewing family, sarcoma of Ewing -Family, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal -Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal Stromal Tumor, Germ Cell Tumor, Germinoma, Gestational Choriocarcinoma, Gestational Trophoblastic Tumor, Bone Cell Tumor, Giant Tumor, Glioblastoma Multiforme, Glioma, Gliomatosis Cerebri, Glomus- tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, hemangiosarcoma, hematological malignancy, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and Ovarian Cancer Syndrome, Hodgkin Lymphoma, Hodgkin Lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory Breast Cancer, Intraocular Melanoma, Islet Cell Tumor Carcinoma, Islet Cell Tumor, Juvenile Myelomonocytic Leukemia, Kaposi's Sarcoma, Kaposi's Sarcoma, Kidney Cancer, Klatskin's Tumor, Krukenberg's Tumor, Laryngeal Cancer , Laryngeal Cancer, Lentigo-Melanoma Malignant, Leukemia, Leukemia, Lip And Oral Cavity Cancer, Liposarcoma, Lung Cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphocytic Leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant Fibrous Histiocytoma, Malignant Fibrous Histiocytoma Of Bone, Malignant glioma, malignant mesothelioma, malignant tumor of peripheral nerve sheath of bone, malignant rhabdoid tumor, malignant Triton's tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloblastoma, medulloepithelioma , melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cervical cancer with occult primary tumor, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndromes, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cavity cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neoplasia, neurinoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-Hodgkin's Lymphoma, non-melanoma. Skin Cancer, Non-Small Cell Lung Cancer, Eye Oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic Nerve Sheath Meningioma, Oral Cancer, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Ovarian Cancer, Ovarian Epithelial Cancer, Germ Cell Tumor Of Ovarian, Ovarian Tumor With Low Malignant Potential, Paget's Disease Of Breast, Pancoast Tumor, Bauchspeicheldrüsenkrebs, Bauchspeicheldrüsenkrebs, papillärer Schilddrüsenkrebs, Papillomatose, Paragangliom, Nasennebenhöhlenkrebs, Nebenschilddrüsenkrebs, Peniskrebs, perivaskulärer Epitheloidzelltumor, Rachenkrebs, Phäochromozytom , Zirbeldrüsenparenchymtumor intermediärer Differenzierung, Pineoblastom, Pituyzytom, Hypophysenadenom, Hypophysentumor, Plasmazellneoplasma, pleuropulmonales Blastom, Polyembryom, Vorläufer-T-lymphoblastisches Lymphom, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular cancer, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, peritoneal pseudomyxoma, rectal cancer, renal cell carcinoma, airway carcinoma involving the NUT gene in chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter -Transformation, Sacrococcygeal teratoma, Salivary gland cancer , Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Spinal cord stromal tumor, Sexuality, Sezary syndrome, Signet ring cell carcinoma, Skin cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small Cell Lymphoma, Small Bowel Cancer, Partial Sarcoma, Somatostatinoma, Fuliginous Wart, Spinal Cord Tumor, Spinal Tumor, Splenic Marginal Zone Lymphoma, Squamous Cell Carcinoma, Gastric Cancer, Superficially Spreading Melanoma, Supratentorial Primitive Neuroectodermal Tumor, Epithelial-stroma Surface Tumor, Synovial Sarcoma, Leukemia Acute T -cell lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, terminal lymphocytic cancer, testicular cancer, thecoma, pharyngeal cancer, thymic carcinoma, thymoma, Thyroid cancer, Transitional cell carcinoma of renal pelvis and ureter, Transitional cell carcinoma, Urachal carcinoma, Urethral cancer, Urogenital neoplasia, Uterine sarcoma, Choroidal melanoma, Vaginal cancer, Verner-Morrison syndrome, Verrucous carcinoma, Optic pathway glioma, Vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor .

[0439]Preferably, the method is for treating a solid tumor.

[0440]Preferably, the method is for treating a liquid tumor.

[0441]The term "oNK" refers to (a) isolated non-transgenic human CD16⁺a natural killer cell line derived from the natural killer cell population of human peripheral blood having ATCC accession number CRL-2407; (b) non-transgenic human CD16⁺natural killer cell line obtained by culturing the cell of (a) for several days with the culture method disclosed in Embodiments 2.1; (c) the cell deposited with the NPMID with accession number NITE BP-03017; or (d) a human natural killer cell having the following characteristics:

[0442]i) expresses a CD16 receptor;

[0443]ii) retain its ability to proliferate after subculture for at least 3 months; It is

[0444]iii) x) excluding synthetic, genetically modified and/or intentionally introduced polynucleotide encoding the CD16 receptor, or y) using the ddPCR system to analyze the genomic DNA of the cell, the ratio of the probe-detectable DNA molecule CD16 F176F to the CD16 F176V probe -detectable DNA molecule is equal to or greater than 1, wherein the CD16 F176F probe sequence is SEQ ID NO:11 and the CD16 F176V probe sequence is SEQ ID NO:12

DETAILED DESCRIPTION OF THE INVENTION

[0488]What follows is a detailed description using the embodiments of the present invention and the techniques and features of the present invention, however, these embodiments are not intended to limit the invention, any changes and modifications to be made without departing from the spirit and scope of the invention anyone familiar with this technology is intended to be included within the scope of the invention.

Modality 1: Obtaining CD16⁺Natural killer cell line that does not contain a genetically modified polynucleotide encoding the CD16 receptor

[0489]Please askFIGO.1.FIGO.1Figure 12 is the flow chart for obtaining a CD16⁺natural killer cell line that does not contain a genetically modified polynucleotide encoding the CD16 receptor. The method for obtaining a non-transgenic human CD16⁺natural killer cell line in the present invention comprises at least the following steps:

[0490]steps11: obtaining a human peripheral blood natural killer cell population derived from a cell population having ATCC accession number CRL-2407; steps12: contacting the human peripheral blood natural killer cell population with an antibody specific for a CD16 receptor; steps13: Separation of cells that are specifically bound by the antibody, yielding CD16⁺natural killer cell line that does not contain a genetically modified polynucleotide encoding the CD16 receptor.

[0491]Best in step S12the CD16 receptor is a CD16a receptor.

[0492]Preferably, flow cytometry, pellets, or any antibody-modified surface material is used to separate cells that are specifically bound by the antibody in step S13.

[0493]Preferably, the term “CD16⁺natural killer cell line not containing a genetically engineered polynucleotide encoding the CD16 receptor" refers to non-genetically engineered human CD16⁺natural killer cell line and/or human CD16⁺natural killer cell line lacking a synthetic or exogenous polynucleotide sequence encoding the CD16 receptor.

[0494]The detailed description of the preferred embodiment is elaborated below.

Modality 1.1 CD16 label and classification⁺Natural killer cell line that does not contain a genetically modified polynucleotide encoding the CD16 receptor

[0495]This modality consists of an experimental group and a control group. The human peripheral blood natural killer cell population ATCC accession number CRL-2407 was centrifuged at a speed of 100-1000 xg for 3-5 minutes. The supernatant was removed and the human peripheral blood natural killer cell population was resuspended in buffer. The human peripheral blood natural killer cell population was evenly distributed in the cell culture plates of the control group and the experimental group. The human peripheral blood natural killer cell population of the experimental group was cultured in the cell culture plates containing cell culture medium (DMEM culture medium, Eagle's minimal essential medium alpha modification or XVIVO 10 culture medium) 0.5%-30% (volume percent, vol. , v/v) human platelet lysate and 100-3000 IU/mL interleukin 2 (IL-2) and then mixed with fluorescently labeled antibody CD16 (CD16 -PE-Cy7, an antibody against the CD16a receptor and CD16b receptor) to to target cells expressing the CD16 receptor in the natural killer cell population of human peripheral blood; while the human peripheral blood natural killer cell population of the control group was mixed with an equal volume of the buffer. Cells from the experimental and control groups were centrifuged separately, the supernatant was removed, and a sorting buffer was added to adjust the cell concentration to 1×10⁷cells per ml. Finally, the cell population of the experimental and control groups was analyzed using a cell classifier.

[0496]If the buffer was presorted buffer, sample preparation buffer for flow cytometry, or Dulbecco's phosphate-buffered saline (DPBS). The sorting buffer was pre-sort buffer, sample preparation buffer for flow cytometry, or Dulbecco's phosphate-saline buffer (DPBS) supplemented with fetal bovine serum (FBS). The cell sorter was, for example, a Becton Dickinson-FACSAria lllu model flow cytometer.

[0497]Preferably, the screening buffer comprises 0.1-10% (volume percent, volume percent, v/v) fetal bovine serum (fetal bovine serum, FBS).

[0498]Preferably, the sorting time is 1 hour and the sorting speed is 50 to 70,000 events/second.

[0499]After using the Forward Scatter (FSC) and Side Scatter (SSC) cell sorter to analyze 10,000 particles in the control group and the experimental group, respectively, out of the 10,000 particles in the control group, 6,771 particles were cells (which is the amount of cells 67, 7% when the total number of particles is 100%), and 6944 particles of the 10,000 particles of the experimental group were cells (when the total number of particles is 100%, the amount of cells is 69.4%).

[0500]The results of fluorescence analysis of the control group cells are shown in FIGFIGO.2A,FIGO.2AFigure 12 is the two-dimensional scatter plot showing the human peripheral blood natural killer cell population without CD16 fluorescently labeled antibody labeling, the human peripheral blood natural killer cell population being derived from cell population numbered ATCC CRL-2407 deposit; The result of fluorescence analysis of cells from the experimental group is shown in FIGFIGO.2B,FIGO.2BFigure 12 is the two-dimensional dot plot showing the human peripheral blood natural killer cell population with the fluorescently labeled antibody label CD16, the human peripheral blood natural killer cell population being derived from the ATCC CRL- number cell population. 2407 bail.

[0501]EmFIGO.2AeFIGO.2B, the abscissa is the relative value of PE-Cy7 fluorescence intensity (fluorescence-labeled CD16 antibody used in this experiment emits PE-Cy7 fluorescence), and the ordinate is the relative value of forward scatter intensity (FSC).

[0502]The results aFIGO.2Ashow that all 6771 cells analyzed in the control group did not emit PE-Cy7 fluorescence (0 cells in the rectangular region). Thus, in the absence of the CD16-PE-cy7 fluorescent antibody label, there was no other light irradiated with similar wavelengths as the PE-Cy7 fluorescent dye, interfering with the experimental outcome of control group cells.

[0503]The results aFIGO.2Bshow that most of the 6944 analyzed cells in the experimental group showed no PE-Cy7 fluorescence and only few cells showed PE-Cy7 fluorescence (there are only 174 cells in the rectangular area). Thus, it is known that 6944 of the 10,000 particles in the experimental group are cells, of which 174 cells have the CD16 receptor, which means that only 1.7% of the particles are cells expressing the CD16 receptor (174÷10000 = 1.7% ) and only 2.5–2.6% of the cells are CD16 receptor-expressing cells (174÷6944≈2.6%). In the experimental group, the cell concentration based on the condition is 1 × 10⁷Cells per ml, each ml of cell solution in the experimental group contained approximately 2.6 × 10⁵Cells expressing the CD16 receptor.

[0504]Cells expressing the CD16 receptor were separated from cells in the experimental group to obtain highly pure CD16⁺(hereinafter referred to as "cleaned CD16⁺cell population”, “isolated oNK” or “isolated non-transgenic human CD16⁺natural killer cell line".)

[0505]Please askFIGO.2C,FIGO.2CFigure 12 is a two-dimensional dot plot depicting the cell expressing the CD16 receptor isolated from the natural killer cell population of human peripheral blood by staining with fluorescently labeled CD16 antibody. The results aFIGO.2Cshow that the majority of cells on purified CD16⁺the cell population emits PE-Cy7 fluorescence and the purity of the cells expressing the CD16 receptor reaches 99%.

[0506]The aforementioned cells expressing the CD16 receptor on purified CD16⁺cell population are non-transgenic cells; all cells mentioned above express the CD16 receptor on purified CD16⁺Cell population has the property of CD3⁻CD56⁺they can be continuously subcultured after analysis and are non-tumorigenic; therefore, the aforementioned cell expresses the CD16 receptor on purified CD16⁺Cell population is a novel non-transgenic human CD16⁺natural killer cell line.

Modality 2: culture of human CD16⁺natural killer cells

[0507]Please askFIGO.3.FIGO.3Figure 12 is the flow chart of human CD16 culture⁺natural killer cells. The method of culturing human CD16⁺Natural killer cells involves at least the following steps:

[0508]steps21: Obtained from human CD16⁺natural killer cells;

[0509]steps22: In container, in contact with human CD16⁺natural killer cells with a culture medium comprising human platelet lysate and IL-2; It is

[0510]steps23: Growing human CD16⁺natural killer cells for several days to proliferate human CD16⁺natural killer cells.

[0511]The following describes a specific modality for culturing a non-transgenic human CD16⁺natural killer cell line by the present invention, but the application of the invention is not limited thereto, which means that the invention can also be used to cultivate other human CD16⁺natural killer cells. For example primary CD16⁺natural killer cell isolated from autologous or allogeneic blood, transgenic NK-92 cell line CD16 or other human CD16⁺natural killer cells.

Modality 2.1 Non-transgenic human CD16 culture⁺Natural killer cell line

[0512]steps21': The cleaned CD16⁺the cell population (the proportion of cells expressing the CD16 receptor was as high as 99%) sorted by modality 1 was centrifuged and the supernatant was removed.

[0513]steps22': After resuspending the cells with 1 ml of cell culture medium, the cell suspension was placed in a first container so that the first container contains 6 54×10⁵Non-transgenic human CD16⁺natural killer cell line in 40 ml cell culture medium; Cell culture medium comprises: 0.5%-30% (volume percent, volume percent, v/v) human platelet lysate; 100-3000 IU/mL Interleukin 2 (IL-2); and DMEM (Dulbecco's Modified Eagle's Medium) culture medium, alpha modification of Eagle's Minimal Essential Medium or XVIVO 10 culture medium.

[0514]steps23': After several days of cultivation, a composition substantially enriched in human CD16⁺Natural killer cells were obtained and substantially enriched in composition with human CD16⁺natural killer cells, the number of non-transgenic human CD16 cells⁺the lineage of natural killer cells is at least 5×10⁵; several days are, for example, 1 day to 3 years.

[0515]Preferably, the multiple days are 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years or 3 years.

[0516]Preferably, the cell culture medium comprises 0.5%, 1%, 1.5%, 1.6%, 2%, 2.5%, 2.6%, 3%, 3.5%, 3.6%, 4% , 4.5%, 4.6%, 5.0% , 5.1%, 5.5%, 5.6%, 6%, 6.1%, 6.5%, 6.6%, 7th %, 7.1%, 7.5%, 7.6%, 8%, 8.1%, 8.5%, 8.6%, 9%, 9.1%, 9, 5%, 9, 6% or 10% (volume percent, vol%, v/v) human platelet lysate.

[0517]Preferably, the cell culture medium comprises 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2100, 21000, 2500, 2600, 2700, 2800, 2900 or 3000 IU/mL IL-2.

[0518]Preferably step S23' also includes substeps:

[0519]steps231': After several days of culture, the cell number in the cell culture medium reached the first cell number, and the first cell number was 1.25 × 10⁶~5×10⁶;

[0520]steps232': The cell suspension was placed in a second container to make the number of cells in the second container equal to the first number of cells; after several days of culture, the cell number reached the second cell number, and the second cell number was 5 × 10⁷~1 × 10⁹; e

[0521]steps233': The cell suspension was placed in a third container to bring the cell count in the third container to the second cell count; after several days of culture, the cell count reached the third cell count to obtain a composition substantially enriched in human CD16⁺natural killer cells; for example, the number in the third cell was 5×10⁹, or 1×10⁴⁰.

[0522]Where the first container was, for example, a T25 cell culture flask (T25 flask) or a 6-well G-Rex cell culture plate. The second and third vessels comprise a gas-permeable but water-impermeable membrane, or the second and third vessels can completely aerate the dissolved oxygen concentration or maintain the dissolved glucose concentration in the culture medium at 1500-5000 mg/EU. Preferably, the second container was, for example, G-Rex 100M bottle (product number 81100, WILSON WOLF, USA), the third container was, for example, G-Rex 500M (product number 85500S, WILSON WOLF, USA). For instructions on using the G-Rex 6M 6-well cell culture plate, G-Rex 100M flask and G-Rex 500M, see the product manual for these flasks.

[0523]in the S levels23'e S231'S233’, 0.5%-30% (volume percent, vol%, v/v) human platelet lysate and 100-3000 IU/ml interleukin 2 (IL-2) were added to a medium to culture the cells. And the medium is, for example, DMEM culture medium (Dulbecco's modified Eagle's medium), alpha-modification of Eagle's minimal essential medium, XVIVO 10 culture medium or X-VIVO medium 10 for serum-free hematopoietic cells.

[0524]in the S levels23'e S231'S233' The cells were incubated at 37°C and 5% carbon dioxide.

Modality 2.2 Demonstration of cell viability of cultured cells obtained from Modality 2.1

[0525]Each sample of the cell suspensions obtained by culturing on different days with the culture method disclosed in Embodiment 2.1 was mixed with an equal volume of trypan blue, and the cell number and cell survival rate were observed.

[0526]The experimental results showed that after culturing for 7, 16, 21, 28, 37, 42, 49, 65, 92, 97, 103, 134, 166, 184 and 202 days, the number of cells was 1.61 × 10, respectively reached⁶, 1,01×10⁹, 2,53×10⁹, 5,06×10⁹, 1,01×10¹⁰, 1,62×10¹⁰, 3,24×10¹⁰, 1,13×10¹¹, 1,81×10′⁵, 3,25×10¹⁶, 6,50 × 10¹⁷, 1,35 × 10²², 3,24×10²⁷, 1,30 × 10³³, e 1,04 × 10³⁹. Please askFIGO.4.FIGO.4shows that cell viability at 7, 16, 21, 28, 37, 42, 49, 65, 92, 97, 103, 134, 166, 184 and 202 days of culturing CD16 non-transgenic humans is 84-97% was held⁺natural killer cell line. This is how non-transgenic human CD16 is cultured⁺the natural killer cell line with the culture method of the present invention can expand the number of cells by at least 1.59×10 6³³folds [(1.04×10³⁹)÷(6,54×10⁵)≈1,59×10³³], effectively maintaining cell viability rate after proliferation.

Modality 3: cell state detection and cell surface markers

Modality 3.1 Long-term culture of non-transgenic human CD16⁺Natural killer cell line by the culture method of the present invention

[0527]There are two experimental tests in this modality. The first batch of purified CD16⁺cell population and the second batch of purified CD16⁺Cell population (the proportions of cells expressing the CD16 receptor in both batches reached 99%) were sorted by the method of modality 1.1, then the first batch of purified CD16⁺cell population and the second batch of purified CD16⁺Populations of cells were each cultured by the culture method of embodiment 2.1 to obtain the cell suspensions of the first experimental run and the cell suspensions of the second experimental run. The first batch of purified CD16⁺cell population was cultured for a total of 35 days, while the second batch of purified CD16⁺Cell population was cultured for at least a longer period until day 202.

Modality 3.2 Determination of the status of cultured cells

[0528]Each sample of cell suspensions obtained at different time points in modality 3.1 was centrifuged; the supernatant was removed, the cells were resuspended in the buffer and then mixed with 1 µl propidium iodide (PI). Cell sorters or flow cytometers were used to determine if cells were stained with propidium iodide to determine the percentage of cells undergoing apoptosis or dead.

Modality 3.3 Detection of CD56, CD3 and CD2 surface markers from cultured cells

[0529]Each sample of cell suspensions obtained at different time points in modality 3.1 was centrifuged; the supernatant was removed, the cells were resuspended in the buffer and then treated with 1 µL of fluorescently labeled antibody CD56 (cat. no. 318304, Biolegend, USA), 14, fluorescently labeled antibody CD3 (cat. no. 300410, Biolegend, USA) and 1 µl ;l fluorescently labeled CD2 antibody (cat. no. 300222, Biolegend, USA) to simultaneously label cells expressing the CD56 molecule, the CD3 molecule and/or the CD2 molecule. Finally, the cell sorter or flow cytometer was used to analyze whether the cells showed CD56 molecules, CD3 molecules and/or CD2 molecules, and the proportion of cells with different cell surface manufacturers was calculated.

Modality 3.4 Detection of CD16 surface markers from cultured cells

[0530]Each sample of cell suspensions obtained at different time points in modality 3.1 was centrifuged; the supernatant was removed, the cells were resuspended in the buffer and then mixed with 1 µl of fluorescently labeled CD16 antibody (cat. no. 302016, Biolegend, USA) to label cells expressing the CD16 receptor. Finally, the cell sorter was used to analyze whether cells showed CD16 receptor and the percentage of cells with CD16 receptor was calculated.

Modality 3.5 Detection of the cytotoxic function of cultured cells

[0531]The xCELLigence Real Time Cellular Analysis System (xCELLigence RTCA system, ACEA Biosciences Inc., USA) was used in this modality to demonstrate the cytotoxic capacity of the cultured cell relative to the target cells. This modality included a 96-well xCELLigence E-Plate to perform the cytotoxicity assay, and the wells in the xCELLigence E-Plate were divided into control wells, experimental wells, and control wells for maximal target cell lysis. The effector cells used in this modality were the cell suspensions obtained by culturing at different times in Modality 3.1, and the target cells were the cell line SK-OV-3 (HTB-77, purchased from ATCC), which is a cancer of an adherent type ovarian cell line. SK-OV-3 cells were seeded into the control well, experimental well and control well with maximal target cell lysis such that each well contained 20,000 SK-OV-3 cells and then allowed to settle for 30 minutes.

[0532]A sample of the cell suspension obtained in modality 3.1 was added to the experimental well and the ratio of the number of effector cells to the number of SK-OV-3 cells (target cells) was 2, 5 and 10; an equal tenth volume of lysis buffer added to the cell suspension sample in the control well for maximum target cell lysis; no sample or lysis buffer was added to the control well. The xCELLigence E-Plate was placed on the xCELLigence Real Time Cell Analysis System to detect real-time changes in cell index (CI) under the condition of 37°C and 5% carbon dioxide.

[0533]The following applies: The greater the number of target cells attached to the underside of the xCELLigence E-Plate, the greater the index of cells recognized by the xCELLigence Real Time Cell Analysis System. Therefore, the cell index can be used to convert the percentage of target cells that are lysed in the experimental well. The formula for converting the cell count to the percentage of target cells lysed in the experimental well is:

[0000]Percent Target Cell Lysed (%) = 1 - [(Experimental Well Cell Index - Control Well Cell Index with Maximum Target Cell Lysis) - (Control Well Cell Index - Control Well Cell Index Target Cell Maximum Lysis Control)] × 100%

[0534]See Table 1 and Table 2. Table 1 shows the results of the cell suspensions obtained in the first run and Table 2 shows the results of the cell suspensions obtained in the second run.

[0535]In Table 1, the first column "Day" indicates the number of cultivation days; the second column "PI" indicates the percentage of cells that have undergone apoptosis or died based on the total number of cells in the cell suspension as 100%; because natural killer cells, CD4⁺T cells and CD8⁺All T cells show CD56+ (Pernick, N, 2018), hence the third column is “CD56⁺' indicates the percentage of the total number of natural killer cells, CD4⁺T cells and CD8⁺T cells based on the total number of cells in the cell suspension as 100%; since all T cells display CD3+ (Pernick, N, 2018), the fourth column is “CD3⁻” indicates the percentage of cells other than T-cells based on the total number of cells in the cell suspension as 100%; since natural killer cells, peripheral blood T cells and most thymocytes have CD2⁺(Pernick, N, 2018) and the cells to be overcome in modality 3 are from peripheral blood, so the fifth column “CD2⁺” indicates the percentage of the total number of natural killer cells and T cells based on the total number of cells in the cell suspension as 100%; the sixth column “CD56⁺CD3⁻” indicates the percentage of natural killer cells based on the total number of cells in the cell suspension as 100%; the seventh column “CD56⁺CD2⁺” indicates the percentage of the total number of natural killer cells and T cells based on the total number of cells in the cell suspension as 100%; since natural killer cells and macrophages present CD16⁺(Pernick, N, 2018) and CD16 is involved in antibody-dependent cellular cytotoxicity (ADCC), the eighth column “CD16⁺” indicates the percentage of the total number of natural killer cells and macrophages with ADCC function based on the total number of cells in the cell suspension as 100%; the ninth column “CD56⁺CD16⁺' indicates the percentage of natural killer cells with ADCC function relative to the total number of natural killer cells (i.e. CD56⁺CD3⁻cells) than 100%.

[0536]The indication of the first to eighth columns in Table 2 is the same as in Table 1; if the ninth column "killing test" marks the symbol "✓", this indicates that the cytotoxic function of the cells in the cell suspension at the given time was tested simultaneously and confirmed that the cells have a cytotoxic function.

[0537]Table 1 shows that (1) in the cell suspension obtained after the first batch of purified CD16⁺Cell population (where the proportion of human CD16⁺Natural killer cell line is up to 99%) has been cultured for 7 to 35 days, the percentage of cells undergoing apoptosis or dead is 5.65% to 7.34%, so the percentage of cell survival during culture is 92.66 % is up to 94.35%; (2) in the cell suspension obtained after the first batch of purified CD16⁺Cell population was cultured for 7~35 days, the percentage of the total number of natural killer cells, CD4⁺T cells and CD8⁺T cell is 99.08~99.56% based on the total number of cells in the cell suspension as 100%; (3) in the cell suspension obtained after the first batch of purified CD16⁺the cell population was cultured for 7~35 days, the percentage of cells other than T cells is 99.88~100% based on the total number of cells in the cell suspension as 100%; (4) in the cell suspension obtained after the first batch of purified CD16⁺the cell population was cultured for 7 to 35 days, the percentage of the total number of natural killer cells and T cells is 98.08 to 99.22% based on the total number of cells in the cell suspension as 100%; (5) in the cell suspension obtained after the first batch of purified CD16⁺the cell population was cultured for 7~35 days, the percentage of natural killer cells is 98.21~98.76% based on the total number of cells in the cell suspension as 100%; (6) in the cell suspension obtained after the first batch of purified CD16⁺the cell population was cultured for 7~35 days, the percentage of the total number of natural killer cells and T cells is 98.78~99.33% based on the total number of cells in the cell suspension as 100%; (7) in the cell suspension obtained after the first batch of purified CD16⁺the cell population was cultured for 7~35 days, the percentage of the total number of natural killer cells and macrophages with ADCC function is 90.17~92.36% based on the total number of cells in the cell suspension as 100%; (8) in the cell suspension obtained after the first batch of purified CD16⁺cell population was cultured for 7~35 days, the percentage of natural killer cells with ADCC function is 88.79~92.11% based on the total number of natural killer cells (i.e. CD56⁺CD3⁻cell) than 100%.

[0538]Table 2 shows that (1) in the cell suspension obtained after the second batch of purified CD16⁺Cell population (where the proportion of human CD16⁺Natural killer cell line is as high as 99%) has been cultured for 7-202 days, the percentage of cells undergoing apoptosis or dead is 2.7%-10.5%, so the percentage of cell survival during culture is 89 is .5%-97.3%; (2) in the cell suspension obtained after the second batch of purified CD16⁺Cell population was cultured for 7-202 days, the percentage of the total number of natural killer cells, CD4⁺T cells and CD8⁺T cell is 98.85%-99.65% based on the total number of cells in the cell suspension as 100%; (3) in the cell suspension obtained after the second batch of purified CD16⁺the cell population was cultured for 7-202 days, the percentage of cells other than T cells is 99.82%-100% based on the total number of cells in the cell suspension as 100%; (4) in the cell suspension obtained after the second batch of purified CD16⁺the cell population was cultured for 7-202 days, the percentage of the total number of natural killer cells and T cells is 94.5%-99.68% based on the total number of cells in the cell suspension as 100%; (5) in the cell suspension obtained after the second batch of purified CD16⁺the cell population was cultured for 7-202 days, the percentage of natural killer cells is 97.65%-99.05% based on the total cell number in the cell suspension as 100%; (6) in the cell suspension obtained after the second batch of purified CD16⁺the cell population was cultured for 7-202 days, the percentage of the total number of natural killer cells and T cells is 97.83%-99.61% based on the total number of cells in the cell suspension as 100%; (7) in the cell suspension obtained after the second batch of purified CD16⁺the cell population was cultured for 7-202 days, the percentage of the total number of natural killer cells and macrophages with ADCC function is 83.88%-94.04% based on the total number of cells in the cell suspension as 100%; (8) The cell in the cell suspension obtained after the second batch of purified CD16⁺Cell population cultured for 7-202 days was confirmed to have a cytotoxic function.

[0539]The cell suspension obtained by culturing for 28 days using the culture method disclosed in Modality 2.1 has been deposited with the NPMD under accession number NITE BP-03017. The results disclosed in this invention show that the oNK cell line can retain its ability to proliferate after subculture for at least 3 months and may therefore comprise dysregulated genes responsible for cell growth control (e.g. the oNK cell line can inactive tumor suppressor gene, or a mutated and highly expressed oncogene).

Modality 3.6 Detection of activation markers, inhibitory markers and other NK cell markers from cultured cells

[0540]In this embodiment, cell suspensions obtained by culturing for 93 days by the culture method disclosed in Embodiment 2.1 (referred to as oNK suspension cultured for 93 days) were used. The cells in the cell suspensions were evenly divided into 19 groups. Cells from the first group were centrifuged; the supernatant was removed, the cells were resuspended in buffer, then treated with 1 µl of fluorescently labeled antibody CD56 (cat. no. 318304, Biolegend, USA), 1 µl of fluorescently labeled antibody CD3 (cat. no. 300410, Biolegend, USA) and 1 µl ;l fluorescently labeled CD2 antibody (cat. no. 300222, Biolegend, USA) to simultaneously label cells expressing the CD56 molecule, the CD3 molecule and/or the CD2 molecule. Finally, the cell sorter or flow cytometer was used to analyze whether the cells showed CD56 molecules, CD3 molecules and/or CD2 molecules, and the proportion of cells with different cell surface manufacturers was calculated.

[0541]Cells from the other 18 groups were centrifuged; the supernatant was removed, the cells were resuspended in buffer, then with 1 µL each of CD16 fluorescently labeled antibody (Cat. #302016, Biolegend, USA), CD45 fluorescently labeled antibody (Cat. #368512, Biolegend, USA), CD4 fluorescently labeled antibody (Cat. #300514, Biolegend, USA), CD8 fluorescently labeled antibody (Cat. #344706, Biolegend, USA), CD19 fluorescently labeled antibody (Cat. #302210, Biolegend, USA), CD25 fluorescently labeled antibody (Cat. # 302614, Biolegend, USA), Fluorescently labeled antibody NKp30 (Cat. No. 325214, Biolegend, USA), Fluorescently labeled antibody NKG2D (Cat. No. 320812, Biolegend, USA), NKp44 fluorescently labeled antibody (Cat. No. 325116, Biolegend, USA ), NKp46 fluorescently labeled antibody (Cat. #331916, Biolegend, USA), CD27 fluorescently labeled antibody (Cat. #47-0279-42, Invitrogen, USA), OX40 fluorescently labeled antibody (Cat. #350004, Biolegend, USA), fluorescently labeled antibody CD107a (cat. no. 350004, Biolegend, USA). In the. 328630, Biolegend, USA), NKG2A Fluorescence-labeled Antibody (Cat# FAB1059P, R&D Systems, USA), PD-1 Fluorescence-labeled Antibody (Cat# 367406, Biolegend, USA), TIGIT- Fluorescently labeled antibody (Cat. No. 372704, Biolegend, USA), Fluorescently labeled antibody SIRPα (Cat. No. 372104, Biolegend, USA) and Fluorescently labeled antibody CD158 (Cat. No. FAB1848P, R&D Systems, USA).

[0542]Finally, the cell sorter was used to analyze whether cells have CD16 receptor, CD45 marker, CD4 marker, CD8 marker, CD19 marker, CD25 marker, NKp30 marker, NKG2D marker, NKp44 marker, NKp46 Markers CD27 markers, OX40 markers, CD107a markers showed , NKG2A markers, PD-1 markers, TIGIT markers, SIRPα markers and CD158 markers.

[0543]Among these markers are CD16, CD25, NKp30, NKG2D, NKp44, NKp46 and CD107a activation markers, while NKG2A, PD-1, TIGIT, SIRPα. CD27, OX40 and CD158 are inhibitory markers. Based on the knowledge of those skilled in the art, expression of activating markers potentiates antitumor activity of NK cells, while expression of inhibitory markers potentiates inhibition of NK cell function.

[0544]See Table 3. Table 3 shows the test results of activation markers, inhibition markers and other NK cell markers of cell suspensions obtained in modalities 2.1.

[0545]Table 3 shows that purified CD16⁺Populations express CD56 (98.0 ± 0.2%), CD2 (99.5 ± 0.2%), CD45 (99.7 ± 0.1%), CD4 (0.8 ± 0.3%), CD3 (0.0 ± 0.0%), CD8 (0.0 ± 0.0%), CD19 (0.0 ± 0.0%), CD16 (85.7 ± 7.0%), CD25 ( 42.3 ± 13.1%), NKp30 (93.6 ± 4.3%), NKG2D (46.1 ± 17.4%), NKp44 (75.1 ± 13.3%), NKp46 (46, 4 ± 16.9%), CD27 (0.62 ± 0.08%), OX40 (0.11 ± 0.03%), CD107a (96.1 ± 4.3%), NKG2A (0.14 ± 0.15%), PD-1 (27.0 ± 19.4%), TIGIT (4.3 ± 6.5%), SIRPα (3.2 ± 3.0%) and CD158 (0.4 ± 0.3%). All of the above cells expressing the CD16 receptor on purified CD16⁺Cell population has the property of CD3⁻CD56⁺after analysis. Preferably, the aforesaid cells express the CD16 receptor on purified CD16⁺Cell population is positive for CD2, CD45 and CD4 and negative for CD8 and CD19. CD4 positivity is an unexpected result.

Modality 4: Non-tumorigenicity of non-transgenic human CD16⁺Natural killer cell line

[0546]In this modality, six to eight week old female BALB/c nude mice (purchased from The Jackson Laboratory or BioLasco, Taiwan) were used. 30 mice were randomly assigned to six groups, which were SK-OV-3 group, Raji group, Daudi group, oNK group, γ-irradiated ACE-oNK group and DPBS group.

[0547]A human ovarian cancer cell line "SK-OV-3" (acquired from ATCC; accession number is ATCC HTB-77), human B-lymphoblastoid cell lines "Raji" (acquired from ATCC; accession number is ATCC CCL-86) and "Daudi" (acquired from ATCC; accession number is ATCC CCL-213), a cell suspension obtained by culturing for 88 days with the culture method disclosed in Embodiments 2.1 (88-day cultured oNK suspension of the present invention, referred to as oNK suspension cultured for 88 days) and a suspension of γ-irradiated ACE-oNK cells were used in this embodiment The method for preparing the γ-irradiated ACE-oNK cell suspension will be described below.

[0548]γ-irradiated ACE-oNK cell suspension: the cell suspension obtained by culturing for 84 days with the culture method disclosed in Embodiment 2.1 (84-day-cultured oNK-suspension of the present invention, referred to as 84-day-cultured oNK-day-suspension ) were gamma-irradiated at a dose of 10 Gy. After binding of trastuzumab to cells in the 84-day γ-irradiated cultured ONK suspension using a cell ligand and a trastuzumab ligand that are complementary, a suspension of γ-irradiated ACE-ONK cells was obtained.

[0549]The procedure for binding trastuzumab to cells (e.g. natural killer cells, cells cultured in OSC suspension for 60 days, cells cultured in OSC suspension for 60 days with γ-rays) was as follows: (A) The preparation step the cell ligand and binding of the cell ligand to the cell to produce a cell-ssDNA conjugate; (B) The step of preparing the trastuzumab linker and binding the trastuzumab linker to trastuzumab to prepare the trastuzumab-ssDNA conjugate; (C) Mix the cell-ssDNA conjugate and the trastuzumab-ssDNA conjugate to combine the cell-ssDNA conjugate and the trastuzumab-ssDNA conjugate through the cell linker and its complementary sequence in the trastuzumab linker to make trastuzumab- prepare conjugated cells.

[0550]Wherein step (A) of preparing the cell ligand and binding the cell ligand to the cell comprises the following steps (a1)~(a4):

[0551]Step (a1) A first single-stranded DNA was obtained, wherein the sequence of the first single-stranded DNA was SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0552]Step (a2) The 5' end of the first single-stranded DNA was modified with thiol-modified first single-stranded DNA at the 5' end to obtain a cell ligand stock. Cell Ligand Strain is also commercially available from Integrated DNA Technologies. The actual modification methods are known or will be apparent to those skilled in the art (Zimmermann, J, 2010).

[0553]Step (a3) ​​10-500 µl cell ligand stock and 0.1-10 µl NHS-maleimide (commercially available from Fisher Scientific) were mixed and incubated for 1-60 min(s).

[0554]Step (a4) The mixture obtained in step (a3) ​​was mixed at 1 × 10⁶-1×10⁸cells and incubated for 1-60 minutes to obtain a cell-ssDNA conjugate.

[0555]Step (B) of preparing the trastuzumab linker and binding the trastuzumab linker to trastuzumab comprises the following steps (b1) to (b4):

[0556]Step (b1) A second single-stranded DNA was obtained, wherein the sequence of the second single-stranded DNA was SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 and the sequence of the second single-stranded DNA single-stranded DNA is the complementary one strand to the first single-stranded DNA.

[0557]Step (b2) The 5' end of the second single-stranded DNA was modified with thiol-modified second single-stranded DNA at the 5' end to obtain a trastuzumab linker stock. Trastuzumab Linker Stock is also commercially available from Integrated DNA Technologies. The actual modification methods are known or will be apparent to those skilled in the art (Zimmermann, J, 2010).

[0558]Step (b3) 10-500 µl trastuzumab linker stock and 0.1-10 µl NHS-maleimide (commercially available from Fisher Scientific) were mixed and incubated for 1-60 min(s).

[0559]Step (b4) The mixture obtained in step (b3) was mixed with 10-100 µl of trastuzumab stock solution (commercially available from Roche) and incubated for 10 minutes to 3 hours to obtain trastuzumab-ssDNA conjugate.

[0560]The cell-ssDNA conjugate and the trastuzumab-ssDNA conjugate were mixed to obtain trastuzumab-conjugated cells, e.g. B. Cells in the γ-irradiated ACE-oNK cell suspension.

[0561]1×10⁷SK-OV-3-Zellen, 1 × 10⁷Raji-Zellen, 1x10⁷Daudi cells, 1x10⁷Cells in the oNK suspension cultured for 60 days and 1 × 10⁷Cells in suspension of γ-irradiated ACE-oNK cells were each suspended in 100 µl of Dulbecco's phosphate buffered saline (DPBS) to obtain various cell suspensions. Cell suspensions and 100 μl of DPBS were injected subcutaneously into BALB/c female nude mice in the SK-OV-3 group, the Raji group, the Daudi group, the oNK group, the γ-irradiated ACE-oNK group and the DPBS group implanted on day 0. Tumor growth in each mouse was observed on day 14, day 21, day 24, day 42 and day 59, and the mice were sacrificed on day 59.

[0562]See Table 4. Table 4 shows the results of tumor formation in nude mice xenografted with various cell lines.

[0563]Table 4 shows that there was no tumor formation in mice from the DPBS groups (negative control group) throughout the study period (0/5, 0%), while all five mice from the SK-OV-3 group (positive control group ) developed tumors (5/5, 100%). In mice xenografted with the Daudi lymphoma cell line, 4 out of 5 mice in the Daudi group developed tumors (4/5, 80%) that persisted until the end of the study (Day 59). In mice xenografted with the Raji lymphoma cell line, 1 in 5 (1/5) mice had a detectable tumor before day 42 but then reverted to an unmeasurable size at the end of the study.

[0564]In mice xenografted with oNK cells or γ-irradiated ACE-oNK cells of the present invention, there was no tumor formation in mice in the oNK group and γ-irradiated ACE-oNK group (0/ 5.0%). . These study results demonstrate that non-irradiated oNK cells and irradiated ACE-oNK cells conjugated with trastuzumab are nontumorigenic and safe for future clinical use and disease treatment.

Modality 5: Cytotoxicity of non-transgenic cultured human CD16⁺Natural killer cell line against various cancer cells

[0565]The experimental procedure of this modality is almost the same as that of Modality 3.5, except that (1) the effector cell used in this modality is the cell suspension obtained by culturing for 104 days with the culture method disclosed in Modality 2.1 (Cultivate oNK suspension for 104 days of the present invention, referred to as a suspension of oNK cultured for 104 days, in which the proportion of human CD16⁺the natural killer cell line is 82.51%), (2) the target cells used in this modality are SK-OV-3 (a human ovarian cancer cell line), SK-BR-3 (a human ovarian cancer cell line, human breast cancer ), OVCAR-3 (a human ovarian cancer cell line), MCF-7 (a human breast cancer cell line), A549 (a human lung cancer cell line), and T24 (a human breast cancer cell line). ; and (3) the ratio of the number of effector cells to the number of target cells is 1:1, 2:1 and 5:1 (ET1, ET2 and ET5).

[0566]Please askFIGO.5.FIGO.5Figure 12 is the bar graph showing the cytotoxicity of cultured non-transgenic human CD16⁺Natural killer cell line against various cancer cells.

[0567]The results of this study demonstrate that cultured non-transgenic CD16⁺natural killer cell line kills 4.33 ± 3.43% at 92.98 ± 1.06% SK-OV-3, 12.23 ± 0.09% at 87.88 ± 0.01% SK-BR-3, 47 .78 ± 0.09% to 81.30 ± 0.52% of OVCAR-3, 27.02 ±5.05% to 85.15 ± 0.01% of MCF-7, 31, 68 ± 3.00% to 90.74 ± 0.22% of A549 and 27.77 ± 1.57% to 37.09 ± 2.21% of T24 in the ratio of the number of effector cells to the number of cells target is 1:1 to 5:1 (ET1 to ET5). Thus, the cultured non-transgenic CD16+ natural killer cell line exhibits potent cytotoxicity against several types of cancer cells.

Modality 6: Comparison of cytotoxic activity between cultured non-transgenic human CD16⁺Natural killer cell line and NK-92 cell line

[0568]The experimental procedure of this modality is almost the same as that of modality 3.5, except that (1) the effector cell used in this modality is the cell suspension obtained by culturing for 33 days with the culture method disclosed in Embodiments 2.1 (33 days cultured oNK suspension of the present invention) refers to the 33-day cultured oNK suspension in which the proportion of human CD16⁺Natural killer cell line is 91.74%), or {circle around (2)} the human peripheral blood natural killer cell population with accession number ATCC CRL-2407 (refers to NK-92 suspension since the level of NK-92 cells in the line is at least 98% as shown in FIGFIGO.2Band the NK-92 cell line is a CD16⁻natural killer cell line); and (2) the ratio of the number of effector cells (the total number of cells in the oNK suspension cultured for 33 days or the total number of cells in the NK-92 suspension) to the number of SK-OV-3 cells (target cells) is 2:1 (ET2).

[0569]Please askFIGO.6.FIGO.6Figure 13 is the bar graph showing the comparison of cytotoxic function between cultured non-transgenic human CD16⁺natural killer cell line and NK-92 cell line for killing cancer cells.FIGO.6shows that the cell line NK-92 (a CD16⁻natural killer cell line and therefore unable to destroy cancer cells through the ADCC process) killed only 2.40 ± 5.52% of the cancer cells, while oNK cells (non-transgenic human CD16⁺natural killer cells not associated or co-cultured with IgG antibodies directed against tumor-associated antigens and therefore not activated to induce the ADCC response) killed 49.68 ± 1.19% of the cancer cells.

[0570]Thus, the result shows that: compared to NK-92 cells (NK-92 is a CD16⁻natural killer cell line and therefore unable to destroy cancer cells through the ADCC process), oNK cells that have not been activated to induce the ADCC response can cause an approximately 21-fold increase in cytotoxicity (49,68 ÷2.4 = 21). This is an unexpected result.

[0571]Furthermore, based on this result, applicant believes that after isolating human CD16⁺Natural killer cell line from oNK suspension cultured for 33 days (cultured oNK) and CD16 isolation⁻natural killer cell line (NK-92) from the NK-92 suspension, similar unexpected results can be observed.

Modality 7: Comparison of cytotoxic activity between different levels of non-transgenic human CD16⁺Natural killer cell line

[0572]The experimental procedure of this embodiment is almost the same as for Modality 3.5, except that (1) the effector cell used in this embodiment is the cell suspension obtained by culturing for X days with the culture method disclosed in Embodiments 2.1 (oNK suspension, cultured on day X of the present invention in which the level of human CD16⁺the natural killer cell line is 8.91%, denoted as a suspension with a small number of oNK cells), {circle around (2)} the cell suspension obtained by culturing for Y days with the culture method disclosed in 2.1 (modalities ( cultured Y-oNK per day of the present invention in which the proportion of human CD16⁺the natural killer cell line is 64.15%, referred to as the suspension with an average number of oNK cells), {circle around (3)} the cell suspension obtained by culturing for Z days with the culture method disclosed in 2.1 (cultured Z - oNK per day of the present invention, in which the proportion of human CD16⁺the natural killer cell lineage is 91.74%, designated as a suspension with a high number of oNK cells), {circle around (4)} the natural killer cell population of human peripheral blood having ATCC accession number CRL-2407 (designated NK-92 Suspension in which the proportion of the NK-92 cell line is at least 98% as shown in FIGFIGO.2Band the NK-92 cell line is a CD16⁻natural killer cell line), {circle around (5)} suspension with low number of ACE-oNK-HER2 cells, {circle around (6)} suspension with medium number of ACE-oNK-HER2 cells or {circle around (7) } Suspension with large numbers of ACE-oNK-HER2 cells; and (2) the ratio of the number of effector cells (the total cells in the low-oNK cell-number suspension, in the medium-oNK cell-number suspension, in the high-oNK cell-number suspension, in the NK- 92, in the suspension with a small number of ACE-oNK-HER2 cells, in the suspension with an intermediate number of ACE-oNK-HER2 cells, or in the suspension with a large number of ACE-oNK-HER2 cells) for the number of SKOV-3 cells (target cells) is 2:1 (ET2).

[0573]The method for preparing the ACE-oNK-HER2 cell count low suspension, the ACE-oNK-HER2 cell count medium suspension and the ACE-oNK-HER2 cell count high suspension was described below.

[0574]The ACE-oNK-HER2 Low-Cell Suspension: The whole cells in the “oNK-Low-Cell-Suspension” were linked to trastuzumab using a cell linker and a trastuzumab-linker, which are complementary and hence the low-cell suspension of ACE-oNK-HER2 cells was obtained, in which the proportion of ACE-oNK-HER2 cells is about 8.91%.

[0575]The ACE-oNK HER2 medium number suspension: The whole cells in the “oNK medium cell suspension” were ligated to trastuzumab using a cell linker and a trastuzumab linker that are complementary and therefore the medium number suspension of ACE-oNK HER2 cells were obtained, in which the proportion of ACE-oNK-HER2 cells is about 64.15%.

[0576]The ACE-oNK-HER2 cell high suspension: The whole cells in the "oNK cell high-suspension" were linked to trastuzumab using a cell linker and trastuzumab linker, which are complementary, and therefore the high cell suspension of ACE-oNK-HER2 cells was obtained in which the proportion of ACE-oNK-HER2 cells is approximately 91.74%.

[0577]The procedure for binding of trastuzumab to cells (cells in suspension with low oNK cell counts, medium oNK cell counts, or suspension with high oNK cell counts) is the same as in modality 4.

[0578]Please askFIGO.7AeFIGO.7B.FIGO.7AFigure 12 is the bar graph showing the comparison of cytotoxic activity between different numbers of non-transgenic human CD16⁺Natural killer cell line for killing cancer cells.FIGO.7BFigure 12 is the bar graph showing the comparison of cytotoxic activity between different numbers of non-transgenic human CD16 conjugated with anti-HER2 antibody⁺natural killer cell line to kill cancer cells through the ADCC process.

[0579]FIGO.7Ashows that the cell line NK-92 (a CD16⁻natural killer cell line and therefore unable to destroy cancer cells through the ADCC process) killed only 2.40 ± 5.52% of the cancer cells; small number of oNK cells (non-transgenic human CD16⁺natural killer cells not associated or co-cultured with IgG antibodies directed against tumor-associated antigens and therefore not activated to induce the ADCC response) killed 25.00 ± 3.60% of the cancer cells; average number of oNK cells (non-transgenic human CD16⁺natural killer cells not associated or co-cultured with IgG antibodies directed against tumor-associated antigens and therefore not activated to induce the ADCC response) killed 47.60 ± 6.80% of the cancer cells; large number of oNK cells (non-transgenic human CD16⁺natural killer cells not associated or co-cultured with IgG antibodies directed against tumor-associated antigens and therefore not activated to induce the ADCC response) killed 49.68 ± 1.19% of the cancer cells.

[0580]Thus, the result shows that: compared to NK-92 cells (NK-92 is a CD16⁻natural killer cell line and therefore unable to destroy cancer cells through the ADCC process), the suspension with low numbers of oNK cells (in which the proportion of human CD16⁺natural killer cell line 8.91%) is sufficient to cause an approximately 10-fold increase in cytotoxicity (25 ± 2.4 = 10). This is an unexpected result. Therefore, it indicated that human CD16⁺the natural killer cell line is sufficient in number to kill cancer cells at 5% or more based on the total number of cells in the composition than 100%. Based on this result, the Applicant believes that a similar unexpected result may be observed in clinical trials.

[0581]The result also shows that the suspension contains a medium or large number of oNK cells (in which the proportion of human CD16⁺natural killer cell line is 64.15% or 91.74%) can cause an approximately 20- to 21-fold increase in cytotoxicity (47.60 ± 2.4 = 20; 49.68 ± 2.4 = 21). Therefore, it suggests that the more human CD16⁺natural killer cell line, the more cancer cells are killed and then plateau like human CD16⁺natural killer cell line in an amount equal to about 60%-65% by number based on the total number of cells in the composition as 100%. Based on this result, the Applicant believes that a similar result could be observed in clinical trials.

[0582]FIGO.7Bshows that a small number of oNK cells killed 25.00 ± 3.60% of the cancer cells; mean number of oNK cells killed 47.60 ± 6.80% of cancer cells; large number of oNK cells killed 49.68 ± 1.19% of cancer cells; a small number of ACE-oNK-HER2 cells killed 63.70 ± 5.00% of the cancer cells; mean number of ACE-oNK-HER2 cells killed 62.00 ± 4.00% of cancer cells; Large numbers of ACE-oNK-HER2 cells killed 73.9 ± 11.80% of the cancer cells.

[0583]Thus, the result shows the following: if non-transgenic human CD16⁺The natural killer cell line obtained by the culture of the present invention was linked to antibodies directed against tumor-associated antigens (such as trastuzumab) using a cell linker and an antibody linker (such as trastuzumab linker) that are complementary and therefore activated can induce the ADCC response, the cytotoxic effect increased significantly by 14.4%-38.7% (62.00%-47.60%=14.4%; 63.70%-25.00%=38 .7%).

[0584]The result also shows that oNK cells complexed with an exogenous targeting moiety (e.g. oNK cells conjugated with anti-HER2 antibodies) in an amount of at least 5% are sufficient to target cancer cells to kill by the ADCC process; also shows that the more the exogenous targeting moiety is complexed with the oNK cell, the more cancer cells are killed by the ADCC process and reach a first plateau when the exogenous targeting moiety is equal to the oNK cell in an amount about 5%-10% by number is complexed, based on the total number of cells in the composition as 100%. Based on this result, the Applicant believes that a similar result could be observed in clinical trials.

Modality 8: Comparison of cytotoxic activity between non-transgenic human CD16 conjugated with anti-HER2 antibody⁺Natural killer cell line and anti-HER2 antibody co-cultured non-transgenic human CD16⁺Natural killer cell line

[0585]The experimental procedure of this embodiment is almost the same as for modality 3.5, except that (1) the effector cell used in this embodiment are cell suspensions obtained by culturing for 55 days with the culture method disclosed in modalities 2.1 (refers to 55-day cultured oNK suspension) or {circle around (2)} cell suspension containing ACE-oNK-HER2 cells (the total number of cells in “55 days cultured oNK suspension” was linked to trastuzumab using a cell linker and a trastuzumab linker that are complementary are as described in embodiment 4); (2) the ratio of the number of effector cells (the total number of cells in the cultured 55-day oNK suspension or the total number of cells in the cell suspension containing ACE-oNK-HER2 cells) to the number of SK-OV -3 cells ( -target) is 1:1 (ET1), 2:1 (ET2) or 5:1 (ET5); and (3) in the experimental wells for the 55-day cultured oNK suspension, an equivalent amount of trastuzumab as the total amount of trastuzumab bound to cells in the cell suspension with ACE-oNK HER2 cells at an E:T Ratio of 1 (0.55 ng), 2 (1.10 ng), and 5 (2.75 ng). The detailed procedure has been described below.

[0586]The wells in the xCELLigence E-Plate were divided into control wells, ACE-oNK-HER2 ET1 experimental well, ACE-oNK-HER2 ET2 experimental well, ACE-oNK-HER2 ET5 experimental well, oNK and Herceptin ET1 experimental well, oNK and Herceptin ET2 experimental well, oNK and Herceptin ET5 experimental well and target cell maximal lysis control well. SK-OV-3 cells were seeded into the control well, ACE-oNK-HER2 ET1 well, ACE-oNK-HER2 ET2 well, ACE-oNK-HER2 ET5 well, oNK and Herceptin ET1 well, oNK and Herceptin ET2 experimental well, oNK and Herceptin ET5 experimental well and target cell maximal lysis control well such that each well contains 20,000 SK-OV-3 cells and then allowed to stand for 30 minutes.

[0587]20,000, 40,000 or 100,000 cells in the cell suspension containing ACE-oNK-HER2 cells were seeded into ACE-oNK-HER2 ET1 test well, ACE-oNK-HER2 ET2 test well and ACE-oNK-HER2 ET5 test well, respectively; therefore, the ratio of the number of effector cells (the total number of cells in the cell suspension containing ACE-oNK-HER2 cells) to the number of SKOV-3 cells (target cells) was 1, 2, and 5.

[0588]Either 20,000, 40,000 or 100,000 cells in the oNK suspension cultured for 55 days and 0.55, 1.10 or 2.75 ng of trastuzumab (an antibody against the HER2 protein with the product name Herceptin was purchased from Roche Swiss) were added to the oNK and Herceptin ET1 Trial Well, oNK and Herceptin ET2 Trial Well, and oNK and Herceptin ET5 Trial Well, respectively. Therefore, the ratio of the number of effector cells (the total number of cells in the oNK suspension cultured for 55 days) to the number of SK-OV-3 cells (target cells) was 1, 2, and 5; the amount of trastuzumab in the oNK and Herceptin ET1 Trial Well, oNK and Herceptin ET2 Trial Well, and oNK and Herceptin ET5 Trial Well, respectively, corresponded to the total amount of cell-bound trastuzumab in the ACE -oNK- HER2 ET1 well experimental wells , ACE-oNK-HER2 ET2 experimental well and ACE-oNK-HER2 ET5 experimental well.

[0589]Please askFIGO.8.FIGO.8Figure 12 is the bar graph showing the comparison of cytotoxic function between non-transgenic human CD16 conjugated with anti-HER2 antibody⁺natural killer cell line and non-transgenic human CD16 co-cultured with anti-HER2 antibody⁺natural killer cell line to kill cancer cells through the ADCC process.FIGO.8shows that oNK cells co-cultured with IgG antibodies directed against tumor-associated antigens (and therefore activated to induce the ADCC response) only 0.00±2.10%, 7.30± 1.40% or 71.8 ± 2.10% of cancer cells in an E:T ratio of 1, 2, and 5, respectively, while ACE-oNK-HER2 cells associated (conjugated) with IgG antibodies were that were directed against tumor-associated antigens (and therefore activated to elicit the ADCC response) killed 31.40 ± 1.10%, 65.60 ± 1.00%, or 99.10 ± 1.30% of Cancer cells at an E:T ratio of 1.2 and 5, respectively.

[0590]Thus, the result shows that: compared to oNK cells co-cultured with IgG antibodies directed against tumor-associated antigens, ACE-oNK-HER2 cells were linked (conjugated) with IgG antibodies directed against tumor-associated antigens , a 9-fold increase in cytotoxicity at lower doses (ET1 with 0.55 ng trastuzumab or ET2 with 1.10 ng trastuzumab; 65.60÷7.30=9; 31.40÷0.00=∞; ∞ is a symbol representing an infinitely large number). That is, “CD16 binding⁺natural killer cells with antitumor antigen antibody” (e.g. binding of oNK cultured with trastuzumab) leads to an unexpected result and binds CD16⁺Anti-Tumor Antigen Antibody Natural Killer Cells make therapy more effective and safer based on lower dose treatment.

[0591]Furthermore, based on this result, applicant believes that after isolating human CD16⁺oNK suspension of natural killer cell line cultured for 55 days (cultured oNK) and isolation of trastuzumab-bound CD16⁺natural killer cells (ACE-oNK-HER2 cells) from the cell suspension with ACE-oNK-HER2 cells, a similar unexpected result can be observed.

Modality 9: Genomic DNA detection of non-transgenic human CD16⁺Natural killer cell line

Modality 9.1 Detection of the DNA sequence coding for the CD16 receptor by Digital Droplet PCR (ddPCR)

[0592]Digital bead PCR (ddPCR) was used in this modality to detect the DNA sequence encoding the CD16 receptor from cultured non-transgenic human CD16⁺natural killer cell line in the present invention (oNK) or CD16 transgenic NK-92 cell line (yNK).

[0593]Cell suspensions obtained by culturing for M days with the culture method disclosed in Modalities 2.1 (referred to as oNK suspension cultured for M days) and CD16 transgenic NK-92 cell line (acquired from ATCC with accession number ATCC PTA-6967; cf. as yNK ) were used in this modality. yNK genomic DNA and cells in oNK suspension cultured in M ​​days were isolated using Blood & Cell Culture DNA Mini Kit (purchased from Qiagen).

[0594]yNK probe or oNK probe: 50 ng of genomic DNA isolated from yNK or oNK suspension cultured at days M was mixed with 10 µl of ddPCR™ Supermix for Probes (2×) (catalog number 1863026; purchased from Bio-Rad) , 1 µL mixed BstXI restriction enzyme (product name BstXI; catalog number R0113S; form purchased from BioLabs), and 1 µL CD16 F176F hydrolysis probe and CD16 F176V hydrolysis probe mix (Assay ID: C_25815666_10; purchased from ThermoFisher; a context string

[0595]Control sample without template: water, 10 µl ddPCR™ Supermix for Probes (2×), 1 µl BstXI restriction enzyme and 1 µl CD16 F176F hydrolysis probe and CD16 F176V hydrolysis probe mix were mixed and the final volume is 20 µl.

[0596]ddPCR experiments were performed using the QX100/QX200 Droplet Digital PCR (ddPCR) system (purchased from Bio-Rad). First, the samples are placed in a QX100 or QX200 Droplet Generator (a machine on the QX100/QX200 Droplet Digital PCR System) to split each sample into 15,000-20,000 drops (nanoliter sized drops).

[0597]Second, the wells in the 96-well plate (product name: DG8 cartridge; purchased from Bio-Rad) were divided into no-template control well, yNK well and oNK well, and these wells are for no-template control group (NTC group ), yNK group and oNK group, respectively. The nanolyzed no-template control sample, yNK sample, and oNK sample were transferred to the no-template control well, yNK well, and oNK well, respectively.

[0598]Third, for the PCR amplification procedure, the thermocycling conditions were 95°C for 10 min, 45 cycles of 95°C for 15 s, and 60°C for 1 min, followed by 98°C for 10 min and then holding at 4°C The ramp rate for each step was set to 2°C/s.

[0599]The CD16 F176F hydrolysis probe is a probe labeled with the FAM reporter fluorophore and the CD16 F176V hydrolysis probe is a probe labeled with the VIC reporter fluorophore. The main steps in the PCR amplification process are denaturation, annealing and extension. During annealing, the hydrolysis probe (such as CD16 F176F hydrolysis probe or CD16 F176V hydrolysis probe) binds to the target sequence; then, during extension, the labeled reporter at the 5' end of the probe is cleaved and the free reporter fluoresces. The sequence of the F176F CD16 hydrolysis probe is SEQ ID NO: 11 and is therefore expected to be able to detect the DNA sequence encoding the CD16 receptor located on the q arm of chromosome 1 located at position 1q23.3; the sequence of the F176V-CD16 hydrolysis probe is SEQ ID NO: 12 and is expected to be able to detect the synthetic DNA sequence in yNK.

[0600]Note that the DNA sequence encoding the CD16 receptor, located on the q-arm of chromosome 1 at position 1q23.3 in oNK, is transcribed into CD16-F176F mRNA and then into the CD16-F176F Protein would be translated where the DNA sequence encoding the CD16 receptor is the q-arm of chromosome 1 at position 1q23.3 in oNK comprises SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:19 ; the CD16 F176F mRNA sequence comprises SEQ ID NO: 13; the sequence of the CD16 F176F protein comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:14 or SEQ ID NO:20. The synthetic DNA sequence encoding the CD16 receptor on yNK would be transcribed into CD16-F176V mRNA and then translated into CD16-F176V protein and the CD16-F176V mRNA sequence is SEQ ID NO: 15; the sequence of the CD16 F176V protein is SEQ ID NO: 16.

[0601]Next, the drops for droplet reading were read using a QX100/QX200 droplet reader (a QX100/QX200 droplet digital PCR system machine) in which the drops for fluorescence reading were individually spaced and therefore each droplet was analyzed individually using a two-color detection system (set to detect FAM and VIC). Positive droplets that contain at least one copy of the target DNA molecule (e.g., a DNA molecule detected by the CD16 F176F hydrolysis probe or a DNA molecule detected by the CD16 F176V hydrolysis probe) point compared to negative droplets increased fluorescence.

[0602]Please askFIGO.9.FIGO.9Figure 12 is the bar graph showing the genotype comparison between non-transgenic human CD16⁺natural killer cell line and the CD16 transgenic NK-92 cell line.

[0603]In the NTC group, out of a total of 14,568 droplets (events) collected, there was only 1 positive droplet containing DNA molecules detectable by the CD16-F176F hydrolysis probe and 4 positive droplets containing DNA molecules detectable by were detectable by the CD16-F176V hydrolysis probe. In the yNK group, there were 6,737 positive droplets containing DNA molecules detectable by CD16 F176F hydrolysis probe and 8,152 positive droplets containing DNA molecules detectable by CD16 F176V hydrolysis probe in total 14,230 collected droplets (events). In the oNK group, there were 7637 positive droplets containing DNA molecules detectable by CD16 F176F hydrolysis probe and 5333 positive droplets containing DNA molecules detectable by CD16 F176V hydrolysis probe in total 14230 collected droplets (events).

[0604]Thus, the result shows that when using the ddPCR system to analyze the genomic DNA of yNK cells, the ratio of the DNA molecule detectable by the CD16 F176F hydrolysis probe to the DNA molecule detectable by the CD16 F176V hydrolysis probe is 0.83 ( 6737÷8152=0.83), while the ddPCR system was used to analyze genomic DNA from oNK cells, the ratio of DNA molecules detectable by the CD16 F176F hydrolysis probe to DNA molecules detectable by of the CD16 F176V hydrolysis probe were detectable, 1.43 (7637÷5333 = 1.43).

[0605]Namely, using ddPCR system to analyze human CD16 genomic DNA⁺natural killer (oNK) cell line in the present invention, the ratio of DNA molecule detectable by CD16 F176F hydrolysis probe to DNA molecule detectable by CD16 F176V hydrolysis probe is equal to or greater than 1 ( the number of DNA molecules detectable by the CD16 F176F probe ÷ the number of DNA molecules detectable by the CD16 F176V probe ≥1).

[0606]Furthermore, based on this result, applicant believes that after isolating human CD16⁺In the oNK suspension natural killer cell line cultured in dia M (cultured oNK), a similar result can be observed.

[0607]Based on the applicant's experience, other hydrolysis probes having the sequence SEQ ID NO: 17 or SEQ ID NO: 18 can detect the DNA sequence encoding the CD16 receptor in other CD16 transgenic NK cells.

Modality 9.2 Detection of the DNA sequence encoding the CD16 receptor by fluorescence in situ hybridization (FISH)

[0608]Two-color fluorescence in situ hybridization (FISH) has been used in this modality to detect transgenic, synthetic, genetically engineered, or intentionally introduced DNA sequences encoding the CD16a receptor in human natural killer cells.

[0609]Cultured non-transgenic human CD16⁺The natural killer cell line in the present invention (oNK) is used as an example to show the result of the human cell with no transgenic, synthetic, genetically modified or intentionally introduced DNA sequence encoding CD16 receptor, while the CD16 transgenic NK - Cell line 92 (yNK) is used as an example to show the result of a human cell with transgenic, synthetic, genetically modified or intentionally introduced DNA sequence encoding the CD16 receptor.

[0610]In detail, the isolated CD16⁺NK cells (oNK cells) from cell suspensions obtained by culturing for N days with the culture method disclosed in Modalities 2.1 (referred to as oNK suspension cultured for N days) and CD16 transgenic NK-92 cell line (purchased from ATCC with the Depot number is ATCC PTA-6967, referred to as yNK) were used in this embodiment.

[0611]Kallioniemi published the details of the two-color fluorescence in situ hybridization (FISH) method in 1996, and a brief extract is given below.

[0612]First 1×10 cores⁷yNK cells or oNK cells (CD16⁺NK cells isolated on N days from the cultured oNK suspension are prepared according to protocols used in DNA flow cytometry (Kallioniemi et al., 1996; Vindelov et al., 1983). In detail, the cell pellet is incubated in a hypotonic detergent solution and briefly digested with trypsin.

[0613]Second, the nuclei are spotted onto slides, air dried, and fixed in methanol and acetic acid.

[0614]Third, target nuclei are treated with proteinase K or other proteolytic enzymes prior to hybridization to improve probe penetration.

[0615]Fourth, denaturation of the target nuclei is usually performed by immersing the slides in a denaturing solution (70% formamide, 2×SSC) for 2 to 4 minutes at 70°C, followed by ethanol fixation and dehydration. Denaturation time and temperature must be optimized according to the characteristics of the target cells.

[0616]Fifth, prior to hybridization, 20-60 ng of FISH fluorescent dye-labeled first FCGR3A probe (a test probe capable of detecting the entire human DNA sequence encoding the CD16a receptor; purchased from Empire Genomics), 20-60 ng of the second fluorescent Labeled chromosome 1 control probe (a reference probe; purchased from Empire Genomics) and blocking DNA (unlabeled Cot-1 or placenta DNA) are added to a formamide-based hybridization buffer. It is necessary to use blocking DNA when the probe contains repetitive sequences that hybridize to multiple sites in the genome. The hybridization mixture is heated at 70°C for 5 min to denature the probe fragments and then applied to the target slide; a coverslip is applied and sealed with rubber cement. Hybridization is performed overnight at 37°C in a humid chamber.

[0617]Sixth, unbound probes are washed away.

[0618]Seventh, target nuclei are counterstained with a DNA stain, typically propidium iodide or DAPI.

[0619]Hybridizations are evaluated with a high quality regular epifluorescence microscope. Almost all newer microscope models from well-known manufacturers (Zeiss, Leitz, Olympus and Nikon) are suitable for gene-specific FISH analysis; the 60× plan apos or other objectives where chromatic aberrations are carefully corrected are preferred. The number of test and reference probe signals is evaluated from at least 100 randomly selected cores across the sled. Only morphologically intact and non-overlapping nuclei are counted. Because the nuclei are three-dimensional, it is necessary to move the focus along the depth of the nuclei to get the correct signal count.

[0620]Various formats are commonly used to report gene-specific FISH results, for example: (1) the number of test probe signals per cell; (2) the number of signals per cell from the test probe divided by that from the reference probe; or (3) the percentage of cells in which the signal number of the test probe is present in greater or lesser number of copies than the reference probe.

[0621]Please askFIGO.10A-10E.FIGO.10A-10EFigure 1 illustrates the principle by which two-color FISH analysis with a single-colored labeled CD16a receptor gene-specific test probe and a different-colored labeled reference probe can be used to detect transgenic, synthetic, genetically engineered, or intentionally introduced DNA sequences encoding can CD16a receptor in human natural killer cells.

[0622]Based on the applicant's experience, the number of signals from the FCGR3A FISH probe (a test probe that contains the entire human DNA sequence encoding the CD16a receptor) per oNK cell would be 2 (the actual number of gene copies per cell) and oNK's default two-color FISH would look likeFIGO.10A(normal pattern indicating the outcome of a human cell with no transgenic, synthetic, genetically engineered, or intentionally introduced DNA sequence encoding the CD16a receptor). The number of FCGR3A FISH probe signals (a test probe capable of detecting the entire human DNA sequence encoding the CD16a receptor) per yNK cell can be greater than 2, and the two-color FISH pattern of yNK would look like thisFIGO.10B-10E(CD16 transgenic pattern indicating the outcome of a human cell with transgenic, synthetic, genetically modified or intentionally introduced DNA sequence encoding the CD16a receptor).

Modality 10: Effect of freezing and thawing on survival of non-transgenic human CD16⁺Natural killer cell line

[0623]The cleaned CD16⁺Cell population (the proportion of cells expressing the CD16 receptor was as high as 99%) was sorted by the method of modality 1.1 and then CD16 purified⁺Cell population was cultured for 21 days by the Modality 2.1 culture method (purified CD16⁺cell population was subcultured 8 times). The cell solution sample was mixed with an equal volume of trypan blue, then subjected to cell counting, and the cell survival rate was found to be 95%. Take a sufficient amount of the cell solution that contained 2 × 10⁷viable cells, perform the following freezing and thawing procedures.

[0624]Freezing Procedure: Centrifuge the cell solution containing 2 × 10⁷viable cells, removed the supernatant and resuspended the cell with 1 ml frozen medium (CryoStor® CS10 Freeze Media, contains 10% v/v DMSO, BioLife Solutions, USA). The cell suspension was placed in a cryovial, and the cryovial was placed in the CoolCell Cell freezer (Corning USA), then the CoolCell Cell freezer was stored in a refrigerator at -80°C overnight (which increased by 1°C per minute fell off). ). The cryovial was transferred and stored in liquid nitrogen for 17 days.

[0625]Thawing procedure: Place the cryotube in a 37°C water bath to quickly thaw the cell suspension and mix 1 mL of cell suspension with 9 mL of cell culture medium in modality 2.1. After mixing a sample of the cell mixture with an equal volume of trypan blue, cell number and cell viability were observed.

[0626]Experimental results showed that 1.95×10⁷the cell survived after thawing and the recovery rate was 97.5% [(1.95 × 10⁷)÷(2×10⁷) × 100% = 97.5%], and the cell survival rate was 96%, with no significant difference in viability (95%) before freezing.

Modality 11: Cytotoxic Activity of Non-Transgenic Human CD16⁺Natural killer cell line

[0627]The experimental method of this modality is almost the same as that of Modality 3.5 except that (1) the effector cells used in this modality are Ctrl oNK cells, Ctrl yNK cells, ACE-oNK cells or ACE-yNK cells; and (2) the ratio of the number of effector cells to the number of SKOV-3 cells (target cells) is 2:1 (ET2) or 5:1 (ET5).

[0628]Ctrl oNK cell: Ctrl oNK cells are the cell population cultured after purified CD16⁺Cell populations (where the proportion of non-transgenic human CD16⁺Natural killer cell line rate is up to 99%) were cultured for 26 days using Modality 2.1 method.

[0629]Ctrl yNK cell: Ctrl yNK cells are the CD16 transgenic cell line NK-92 (purchased from ATCC; accession number is ATCC PTA-6967);

[0630]ACE-oNK cell: ACE-oNK cells are cells obtained by linking trastuzumab (an antibody against the HER2 protein, product name Herceptin, purchased from Roche Swiss) to Ctrl-oNK cells using a cell linker and a trastuzumab Linkers that are complementary are obtained.

[0631]ACE-yNK cell: ACE-yNK cells are cells formed by binding trastuzumab (an antibody against the HER2 protein, product name Herceptin, purchased from Roche Swiss) to Ctrl-yNK cells using a cell linker and a trastuzumab Linkers that are complementary are obtained.

[0632]The procedure for binding trastuzumab to natural killer cells (e.g. Ctrl-oNK cells or Ctrl-yNK cells) is as follows: (A) The step of producing the cell ligand and binding the cell ligand to the natural killer cell to produce a NK-conjugated ssDNA; (B) The step of preparing the trastuzumab linker and binding the trastuzumab linker to trastuzumab to prepare the trastuzumab-ssDNA conjugate; (C) Mix the NK-ssDNA conjugate and the trastuzumab-ssDNA conjugate to combine the NK-ssDNA conjugate and the trastuzumab-ssDNA conjugate via the cell linker and its complementary sequence in the trastuzumab linker to obtain trastuzumab- conjugated natural killer cells (eg, ACE-oNK or ACE-yNK cells).

[0633]Wherein step (A) of preparing the cell ligand and binding the cell ligand to the natural killer cell comprises the following steps (a1)~(a4):

[0634]Step (a1) A first single-stranded DNA was obtained, wherein the sequence of the first single-stranded DNA was SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0635]Step (a2) The 5' end of the first single-stranded DNA was modified with thiol-modified first single-stranded DNA at the 5' end to obtain a cell ligand stock. Cell Ligand Strain is also commercially available from Integrated DNA Technologies. The actual modification methods are known or will be apparent to those skilled in the art (Zimmermann, J, 2010).

[0636]Step (a3) ​​10-500 µl of cell ligand stock and 0.1-10 µl of NHS-maleimide (commercially available from Fisher Scientific) were mixed and incubated for 1-60 min(s).

[0637]Step (a4) The mixture obtained in step (a3) ​​was mixed at 1 × 10⁶-1×10⁸natural killer cells and incubated for 1-60 minutes to obtain the NK-ssDNA conjugate.

[0638]Step (B) of preparing the trastuzumab linker and binding the trastuzumab linker to trastuzumab comprises the following steps (b1) to (b4):

[0639]Step (b1) A second single-stranded DNA was obtained, wherein the sequence of the second single-stranded DNA was SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 and the sequence of the second single-stranded DNA single-stranded DNA is the complementary one strand to the first single-stranded DNA.

[0640]Step (b2) The 5' end of the second single-stranded DNA was modified with the thiol-modified second single-stranded DNA at the 5' end to obtain the trastuzumab linker stem. Trastuzumab Linker Stock is also commercially available from Integrated DNA Technologies. The actual modification methods are known or will be apparent to those skilled in the art (Zimmermann, J, 2010).

[0641]Step (b3) 10-500 µl trastuzumab linker stock and 0.1-10 µl NHS-maleimide (commercially available from Fisher Scientific) were mixed and incubated for 1-60 min(s).

[0642]Step (b4) The mixture obtained in step (b3) was mixed with 10-100 µl of trastuzumab stock solution (commercially available from Roche) and incubated for 10 minutes to 3 hours to obtain trastuzumab-ssDNA conjugate.

[0643]Please askFIGO.11.FIGO.11Figure 12 is the bar graph showing the cytotoxic function of non-transgenic human CD16⁺natural killer cell line to kill cancer cells through the ADCC process.FIGO.11shows that regardless of the ratio of the number of effector cells to the number of SKOV-3 cells (target cells) is 2:1 (ET2) or 5:1 (ET5), non-transgenic human CD16⁺Natural killer cell lines (Ctrl-oNK cells) not activated by trastuzumab killed 60% to 65% of cancer cells, while non-transgenic human CD16 was activated by trastuzumab⁺The natural killer cell line (ACE-oNK cells) killed 95% to 100% of the cancer cells. Hence non-transgenic human CD16⁺the natural killer cell line obtained by the culture of the present invention actually has the cytotoxic function of killing cancer cells and if it is non-transgenic human CD16⁺the natural killer cell line obtained by the culture of the present invention was activated to induce the ADCC response, the cytotoxic effect was significantly increased by at least 30% (95% - 65% = 30%; p<0.05).

[0644]Please askFIG.12to z12B.FIGO.12AFigure 12 is the bar graph showing the comparison of cytotoxic function between non-transgenic human CD16⁺natural killer cell line and CD16 transgenic cell line NK-92 for killing cancer cells at different effector (E) to target (T) ratios; It isFIGO.12BFigure 12 is the bar graph showing the comparison of cytotoxic function between non-transgenic human CD16⁺natural killer cell line and CD16 transgenic NK-92 cell line to kill cancer cells through the ADCC process at different effector (E) to target (T) ratios.

[0645]The results ofFIGO.12Ashow that when the ratio of the number of effector cells to the number of SK-OV-3 cells (target cells) is 5:1 (ET5) and is not activated by trastuzumab, non-transgenic human CD16⁺natural killer cell lines (Ctrl-oNK cell) kill 70% of cancer cells, while the CD16 transgenic NK-92 cell line (Ctrl yNK) kills 72% of cancer cells, there was no significant difference between the two groups (p > 0.05 ). Thus, when the ratio of the number of effector cells to the number of SK-OV-3 cells (target cells) is 5:1 (ET5), the cytotoxic function of non-transgenic human CD16 is⁺the natural killer cell line obtained by the culture of the present invention was non-inferior to the CD16 transgenic cell line NK-92. In other words, compared to the CD16-NK-92 transgenic cell line, the non-transgenic human CD16 cell line⁺the natural killer cell line obtained by the method of the present invention is not only safe but also has the same cytotoxic effect.

[0646]The results aFIGO.12Bshow that trastuzumab-activated non-transgenic human CD16 is 2:1 (ET2) or 5:1 (ET5) regardless of the ratio of the number of effector cells to the number of SK-OV-3 cells (target cells).⁺the natural killer cell line (ACE-oNK cells) killed 95% of the cancer cells, while the CD16-activated CD16 transgenic cell line (ACE-yNK) also killed 95% of the cancer cells, and there was no significant difference between the two groups (p > 0.05). Thus, the cytotoxic function is mediated by the ADCC process of non-transgenic human CD16⁺the natural killer cell line obtained by the culture method of the present invention was non-inferior to the CD16 transgenic cell line NK-92. In other words, compared to the CD16-NK-92 transgenic cell line, the non-transgenic human CD16 cell line⁺the natural killer cell line obtained by the method of the present invention is not only safe but also has the same cytotoxic effect on killing cancer cells by the ADCC method.

Modality 12: cultivation of non-transgenic human CD16⁺Natural killer cell line with different concentrations of human platelet lysate

[0647]The experimental method of this modality is almost the same as that of Modality 2.1 except that (1) in step S22' All cells in the cell suspensions obtained by culturing for 9 days with the culture method disclosed in Embodiment 2.1 (referred to as 9-day cultured oNK suspension) were cultured in this embodiment, and the number of cells in the first vessel in step S22Don't 5×10⁶; and (2) the cell culture medium comprises 500 IU/mL IL-2 and {circle around (1)} 2.5% human platelet lysate, {circle around (2)} 5.0% human platelet lysate, {circle around (3)} } 10.0% human platelet lysate or {circle around (4)} 5.0% human serum (does not include human platelet lysate).

[0648]The experimental procedure for detecting cell number, cell viability and CD16 surface marker of cells cultured in this embodiment is the same as in Modality 2.2 and 3.4.

[0649]Please askFIGO.13A-13C.FIGO.13A-13CFigure 12 are line graphs showing the effect of human platelet lysate on total cell number, cell viability and maintenance of CD16 expression after different days of human CD16 culture, respectively⁺natural killer cell line.

[0650]FIGO.13Ashowed that after 14 days of cultivation, the number of non-transgenic human CD16⁺Natural killer cells grown in cell culture medium without human platelet lysate (but containing 5.0% human serum), 2.5% human platelet lysate, 5.0% human platelet lysate and 10.0% human platelet lysate were 4.7 × 10⁸, 6,49×10⁸, 1,01×10⁹, e 1,74 × 10⁹respectively. Thus, the result shows that: compared to cell culture medium without human platelet lysate (but comprising 5.0% human serum), human platelet lysate can cause a 3.7-fold increase (17.4 ÷ 4.7 = 3.7 ). That is, human platelet lysate produces an unexpected result and human platelet lysate produces non-transgenic human CD16⁺Natural killer cells expand greatly. In addition, these results suggested that Formula 3 (comprising 10.0% human platelet lysate) was better than the other formulas for human CD16⁺Expansion of natural killer cells.

[0651]FIGO.13Bshowed that after 7 days of cultivation, the cell viability of non-transgenic human CD16⁺Natural killer cells grown in cell culture medium lacking human platelet lysate (but containing 5.0% human serum), 2.5% human platelet lysate, 5.0% human platelet lysate, and 10.0% human platelet lysate were found in 92%, 88% , 92% retained. or 92%. Cell viability of non-transgenic human CD16 after 14 days of cultivation⁺Natural killer cells grown in cell culture medium lacking human platelet lysate (but containing 5.0% human serum), 2.5% human platelet lysate, 5.0% human platelet lysate, and 10.0% human platelet lysate were found in 94%, 90% , 92% retained. or 93%. So the result shows: human CD16⁺Natural killer cells that have not been treated with human platelet lysate have viability similar to human CD16⁺Natural killer cells treated with 2.5%-10.0% human platelet lysate.

[0652]FIGO.13Cshowed that after culturing in cell culture medium without human platelet lysate (but comprising 5.0% human serum), 2.5% human platelet lysate, 5.0% human platelet lysate, or 10.0% human platelet lysate for 7 days, the percentage of CD16⁺Cells were maintained at 83.55%, 84.15%, 82.81%, and 83.95%, respectively. After culturing in cell culture medium without human platelet lysate (but containing 5.0% human serum), 2.5% human platelet lysate, 5.0% human platelet lysate, or 10.0% human platelet lysate for 14 days, the percentage of CD16⁺Cells were maintained at 80.72%, 80.74%, 78.07% and 80.76%, respectively. Thus, the result shows that: 2.5%-10% human platelet lysate keeps CD16 similar⁺Population as no human platelet lysate (consisting of 5.0% human serum).

[0653]Furthermore, based on this result, applicant believes that after isolating human CD16⁺Natural killer cell line of oNK suspension cultured for 9 days (cultured oNK), a similar result can be observed.

Modality 13: cultivation of non-transgenic human CD16⁺Natural killer cell line with different concentrations of IL-2

[0654]The experimental method of this modality is almost the same as that of Modality 2.1 except that (1) in step S22' All cells in the cell suspensions obtained by culturing for 9 days with the culture method disclosed in Embodiment 2.1 (referred to as 9-day cultured oNK suspension) were cultured in this embodiment, and the number of cells in the first vessel in step S22Don't 5×10⁶; and (2) the cell culture medium comprises 5.0% human platelet lysate and {circle around (1)} 100 IU/mL IL-2, {circle around (2)} 200 IU/mL IL-2, {circle around (3) } 500 IU/mL IL-2, {circle around (4)} 750 IU/mL IL-2 or {circle around (5)} 1000 IU/mL IL-2.

[0655]Note that both IL-2 and human platelet lysate were required for human CD16 expansion⁺natural killer cells. In this mode 1.8 × 10⁷IU/ml IL-2 believe igual has 1.1 mg/ml IL-2. Portanto, 100 IU/ml IL-2 believe igual a 0.0612 μg/ml IL-2; 200 IU/mL IL-2 believe igual to 0.1224 μg/mL IL-2; 500 IU/mL IL-2 believe igual to 0.306 μg/mL IL-2; 750 IU/mL IL-2 believe igual to 0.459 μg/mL IL-2; e 1000 IU/ml IL-2 believe igual contains 0.612 μg/ml IL-2.

[0656]The experimental procedure for detecting cell number, cell viability and CD16 surface marker of cells cultured in this embodiment is the same as in Modality 2.2 and 3.4.

[0657]Please askFIGO.14A-14F.FIGO.14A-14Fare line graphs showing the effect of IL-2 on total cell number, cell viability, and maintenance of CD16 expression, respectively, after different days of culturing human CD16⁺natural killer cell line.

[0658]FIGO.14A-14Bshowed that IL-2 levels did not affect non-transgenic human CD16⁺Expansion of natural killer cells. Note that the cells were reseeded on day 7 and continued to expand until day 11; the expansion process was repeated every 11 days.

[0659]FIGO.14C-14Dshowed that IL-2 level did not affect cell viability of non-transgenic human CD16⁺natural killer cells.

[0660]FIGO.14E-14Fshowed that after 40 days of cultivation in cell culture medium containing 100-200 IU/ml IL-2, the percentage of CD16 decreased⁺Cells fell to less than 20%. On the other hand, after 40 days of cultivation in cell culture medium containing 500-1000 IU/ml IL-2, the percentage of CD16⁺Cells increased to 80%. That is, 500-1000 IU/ml IL-2 produces an unexpected result and 500-1000 IU/ml IL-2 produces CD16⁺Population holds a lot.

[0661]Furthermore, based on this result, applicant believes that after isolating human CD16⁺similar results can be observed with natural killer cell lines from the oNK suspension (cultured oNK) cultured for 9 days.

Modality 14: cultivation of non-transgenic human CD16⁺Natural killer cell line in different containers

[0662]The experimental method of this modality is almost the same as that of Modality 2.1 except that (1) in step S22' All cells in the cell suspensions obtained by culturing for 9 days with the culture method disclosed in Embodiment 2.1 (referred to as 9-day cultured oNK suspension) were cultured in this embodiment, and the number of cells in the first vessel in step S22Don't 5×10⁶; (2) the cell culture medium comprises 500 IU/ml IL-2 and 5.0% human platelet lysate; and (3) the containers used in this embodiment are air-permeable containers such as a G-Rex 6-well culture plate or air-tight containers such as T25 cell culture flasks.

[0663]The experimental procedure for detecting cell number, cell viability and CD16 surface marker of cells cultured in this embodiment is the same as in Modality 2.2 and 3.4.

[0664]Please askFIGO.15A-15C.FIGO.15A-15Care the line graphs showing the effect of the air permeable container on total cell number, cell viability and maintenance of CD16 expression after different days of human CD16 culture, respectively⁺natural killer cell line.

[0665]FIGO.15Ashowed that after 14 days of cultivation, the number of non-transgenic human CD16⁺Natural killer cells grown in non-air-permeable and air-permeable containers were 3.1 × 10⁸e 1,01×10⁹and Thus, the result shows the following: Compared to cells grown in a non-air permeable container, an air permeable container can produce a 3.26-fold increase (10.1 ÷ 3.1 = 3.26). That is, the air permeable container produces an unexpected result and the air permeable container produces non-transgenic human CD16⁺Natural killer cells expand greatly.

[0666]FIGO.15Bshowed that after 7 days of cultivation, the cell viability of non-transgenic human CD16⁺Natural killer cells cultured in non-air permeable containers and air permeable containers were maintained at 87% and 92%, respectively. Cell viability of non-transgenic human CD16 after 14 days of cultivation⁺Natural killer cells cultured in non-air permeable containers and air permeable containers were maintained at 88% and 92%, respectively. So the result shows: human CD16⁺Natural killer cells grown in air-permeable containers have better viability than human CD16⁺natural killer cells cultivated in a non-air permeable container.

[0667]FIGO.15Cshowed that after culturing in a non-air-permeable container and an air-permeable container for 7 days, the percentage of CD16⁺Cells were maintained at 82.63% and 82.81%, respectively. After 14 days of cultivation in a non-air permeable container and an air permeable container, the percentage of CD16⁺Cells were maintained at 83.79% and 88.07%, respectively. Thus, the result shows the following: The air-permeable container holds CD16 similarly⁺Population as an impermeable container.

[0668]Furthermore, based on this result, applicant believes that after isolating human CD16⁺Natural killer cell line of oNK suspension cultured for 9 days (cultured oNK), a similar result can be observed.

Modality 15: Preparation of exogenous targeting moiety-oNK-complexed cells

[0669]In this embodiment applicant produces an oNK cell complexed with an exogenous targeting moiety having at least one exogenous targeting moiety complexed thereto. The exogenous targeting moiety includes a targeting moiety exhibiting specific binding to a biological marker on a target cell, and the targeting moiety capable of binding to a biological marker selected from cancer antigen, glycolipid, glycoprotein, differentiation antigen cluster expressed in cells of a hematopoietic lineage are present antigen, gamma-glutamyl transpeptidase, adhesion protein, hormone, growth factor, cytokine, ligand receptor, ion channel, membrane-bound form of a μ-immunoglobulin. -chain, alpha-fetoprotein, C-reactive protein, chromogranin A, epithelial mucin antigen, human epithelial-specific antigen, Lewis(a) antigen, multidrug resistance-related protein, neu oncogene protein, neuron-specific enolase, glycoprotein P, Multidrug Resistance Related Antigen, p170, Multidrug Resistance Related Antigen, Prostate Specific Antigen, NCAM, Ganglioside Molecule, MART-1, Heat Shock Protein, SialylTn, Tyrosinase, MUC-1, HER-2 /neu, KSA, PSMA, p53, RAS, EGF -R, VEGF, MAGE or other target antigen (marker) expressed by a target cell. The targeting moiety is not a nucleic acid and is not produced by the oNK cell complexed with the exogenous targeting moiety.

[0670]The procedure for binding a targeting moiety (such as trastuzumab directed against the HER2 protein) to oNK cells is as follows: (A) The step of producing the cell ligand and binding the cell ligand to the natural killer cell to produce an NK - conjugated ssDNA; (B) The step of preparing the targeting entity linker (such as trastuzumab linker) and joining the targeting entity linker to the targeting entity to form the targeting entity-ssDNA conjugate; (C) Mix the NK-ssDNA conjugate and the targeting moiety-ssDNA conjugate to form the NK-ssDNA conjugate and the targeting moiety-ssDNA conjugate via the cell linker and its complementary sequence on the targeting moiety- combine linkers to form the complex. conjugated exogenous targeting moiety of natural killer cells (e.g. ACE-oNK cells or ACE-yNK cells).

[0671]Wherein step (A) of preparing the cell ligand and binding the cell ligand to the natural killer cell comprises the following steps (a1)~(a4):

[0672]Step (a1) A first single-stranded DNA was obtained, wherein the sequence of the first single-stranded DNA was SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0673]Step (a2) The 5' end of the first single-stranded DNA was modified with thiol-modified first single-stranded DNA at the 5' end to obtain a cell ligand stock. Cell Ligand Strain is also commercially available from Integrated DNA Technologies. The actual modification methods are known or will be apparent to those skilled in the art (Zimmermann, J, 2010).

[0674]Step (a3) ​​10-500 µl cell ligand stock and 0.1-10 µl NHS-maleimide (commercially available from Fisher Scientific) were mixed and incubated for 1-60 min(s).

[0675]Step (a4) The mixture obtained in step (a3) ​​was mixed at 1 × 10⁶-1×10⁸natural killer cells and incubated for 1-60 minutes to obtain the NK-ssDNA conjugate.

[0676]Step (B) of preparing the linker of the targeting entity and binding the ligand of the targeting entity to the targeting entity comprises the following steps (b1)-(b4):

[0677]Step (b1) A second single-stranded DNA was obtained, wherein the sequence of the second single-stranded DNA was SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 and the sequence of the second single-stranded DNA single-stranded DNA is the complementary one strand to the first single-stranded DNA.

[0678]Step (b2) The 5' end of the second single-stranded DNA was modified as a thiol-modified second single-stranded DNA at the 5' end to obtain the targeting residue linker stem. Linker stock for targeting units is also commercially available from Integrated DNA Technologies. The actual modification methods are known or will be apparent to those skilled in the art (Zimmermann, J, 2010).

[0679]Step (b3) 10-500 µL of target fraction ligand strain and 0.1-10 µL of NHS-maleimide (commercially available from Fisher Scientific) were mixed and incubated for 1-60 minute(s). Step (b4) The mixture obtained in step (b3) was mixed with 10-100 µl of targeting fraction stock (commercially available from Roche) and incubated for 10 minutes to 3 hours to obtain the targeting fraction-ssDNA conjugate .

[0680]The targeting moiety can be a peptide, protein or aptamer, where the protein can be an antibody against a cancer antigen selected from HER2/neu (ERBB2), HER3 (ERBB3), EGFR, VEGF, VEGFR2, GD2, CTLA4, CD19, CD20 , CD22, CD30, CD33 (Siglec-3), CD52 (CAMPATH-1 antigen), CD326 (EpCAM), CA-125 (MUC16), MMP9, DLL3, CD274 (PD-L1), CEA, MSLN (Mesothelin ), CA19-9, CD73, CD205 (DEC205), CD51, c-MET, TRAIL-R2, IGF-1R, CD3, MIF, folate receptor alpha (FOLR1), CSF1, OX-40, CD137, TfR, MUC1, CD25 (IL-2R), CD115 (CSF1R), IL1B, CD105 (Endoglin), KIR, CD47, CEA, IL-17A, DLL4, CD51, Angiopoietin 2, Neuropilin-1, CD37, CD223 (LAG-3), CD40, LIV-1 (SLC39A6), CD27 (TNFRSF7), CD276 (B7-H3), Trop2, Claudin1 (CLDN1), PSMA, TIM-1 (HAVcr-1), CEACAM5, CD70, LY6E, BCMA, CD135 (FLT3), APRIL B. TF(F3), Nectin-4, FAP, GPC3, FGFR3, killer cell immunoglobulin-like receptors (KIRs), a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, and combinations thereof.

[0681]Preferably, the targeting moiety is an antigen-binding moiety or an antibody such as trastuzumab (an antibody against the HER2 protein, product name Herceptin, was obtained from Roche, Switzerland).

[0682]According to the disclosures shown in Embodiments 7, 8, 11 and 15, andFIG.7B,8,11, z12B, one skilled in the art would understand the chemical process to produce the NK cell lineage antigen-binding moiety conjugation (conjugation between the antigen-binding moiety and the NK cell line) and would also understand the NK cell lineage application of the antigen-binding moiety Conjugation in cell therapy specifically targeting abnormal cells.

Modality 15.1 Cytotoxicity of complexed oNK cells of an exogenous targeting moiety against solid tumor

[0683]Human ovarian cancer cell line expressing SKOV3 luciferase (SKOV3-Luc, a Her2⁺cell lines; (catalog number AKR-232, obtained from CELL BIOLABS Inc) were injected intraperitoneally into each of 15 female NOG mice (Jackson Laboratory) on day 0. Five mice from each group were treated with oNK (cells in cell suspensions obtained by culturing with the culture Method disclosed in embodiment 2.1), the ACE-oNK-HER2 cells disclosed in embodiment 7 (oNK cells complexed with exogenous targeting unit) or vehicle (cell medium only, such as fresh growth medium described in embodiment 16.1) on days 0, 3, 5, 11 and 18. Luminescence was detected by AMI HTX (Spectral Imaging) at the end of the experiment.

[0684]The inventors of the present invention expect that oNK cells complexed with exogenous targeting moieties will exert superior efficacy against solid ovarian tumors, and compared with oNK cells, oNK cells complexed with exogenous targeting moieties can , cause greater cytotoxicity and prolong life.

Modality 16: Production of oNK cells expressing the “antigen-binding complex”.

[0685]The method of producing oNK cells comprising a synthetic, genetically engineered and/or intentionally delivered polynucleotide encoding an antigen-binding complex comprising a target-binding single-chain variable fragment (scFv) to the target antigen disclosed in this embodiment in that the target antigen is selected from CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD) , CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f ( ITGA6), CD66a (CEACAM1) , CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (alpha chain associated with B cell antigen receptor complex) , CD79B (alpha chain associated with the beta chain B cell antigen receptor complex), CD84 (SLAMF5), CD96 (tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 ( 4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA ), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6 ), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18) , Inducible T cell costimulator (ICOS), LFA-1 (CD11a/CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1) , IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, a CD83 ligand, Fc gamma receptor , MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, NK cell receptor activation, a toll-like receptor, HER2, BCMA, PD-L1, VEGFR2, TCR b chain and combinations thereof.

[0686]Preferably, the antigen binding complex is a chimeric antigen receptor (CAR) that is designed similar to a chimeric antigen receptor on a chimeric antigen receptor T cell (CAR T cell).

[0687]On the membrane of naturally occurring antigen-specific T cells there are both (1) T cell receptors responsible for the specific recognition of fragments of antigens presented by HLA and (2) costimulatory molecules. Both the antigen-binding signal and sufficient costimulatory signals are required for full activation of antigen-specific T cells; where costimulatory signals are induced by binding the costimulatory molecules through their ligands, which are normally displayed on the target cell membrane (Weinkove et al., 2019).

[0688]Examples of costimulatory molecules expressed by T cells include CD28 subunit, ICOS subunit (CD278), 4-1BB subunit (CD137), OX40 subunit (CD134), CD27 subunit, CD40 subunit, CD40L subunit, TLRs subunit or other costimulatory molecules (Weinkove et al. others, 2019).

[0689]The synthetic chimeric antigen receptor (CAR) combines variable regions of an antibody with intracellular signaling components derived from the T cell receptor complex and therefore allows for the rerouting of T cell cytotoxicity against an antigen on any HLA background (the HLA Antigen processing and presentation is not required) (Weinkove et al., 2019).

[0690]First-generation CARs contain only intracellular CD3 (as CD3 zeta, also known as CD3c). Second generation CARs also contain an intracellular signaling domain of a costimulatory molecule (like CD28), and third generation CARs contain more than one intracellular signaling domain of a costimulatory molecule (like the combination of CD28 and 4-1BB) (Weinkove et al., 2019) .

[0691]Take CD19 as an example to explain the method of producing oNK comprising a synthetic, genetically modified and/or intentionally delivered polynucleotide encoding a chimeric antigen receptor (CAR) that targets a single chain variable fragment (scFv) against CD19 as included below.

[0692]FIGO.16A-16Gdemonstrate the CD19 CAR constructs. The construct comprises a synthetic, genetically modified and/or intentionally delivered polynucleotide encoding a chimeric antigen receptor (CAR) comprising a target-binding single chain variable fragment (scFv) against CD19.

[0693]FIGO.17Figure 11 illustrates the method of producing oNK comprising a synthetic, genetically modified and/or intentionally introduced polynucleotide encoding a chimeric antigen receptor (CAR) as shown in Figure 12FIGO.16A-16G. The method for establishing CD16 expressing human CAR⁺Natural killer cells involves at least the following steps:

[0694]steps31: Generate the CAR constructs based on transfection, electroporation, or lentivirus;

[0695]steps32: transfection, electroporation or infection of oNK cells with CAR construct;

[0696]steps33: Enrichment of oNK cells expressing CAR with an antibody specific for the marker or antigen.

Modality 16.1: Prepare oNK cells expressing the chimeric antigen receptor (CAR).

[0697]The following describes a specific modality for establishing a human CD16 expressing CAR⁺anti-CD19 natural killer cell line that does not contain a genetically modified polynucleotide encoding the CD16 receptor (such as oNK cells expressing anti-CD19 CAR) by the present invention, but the application of the invention is not limited to what the Invention also means can be used to establish human CD16 expressing CAR⁺natural killer cell lines against other CAR target antigens that do not contain genetically engineered polynucleotides encoding the CD16 receptor. For example, the invention can also be used to establish human CD16 expressing CAR⁺natural killer cell line against CD70, GPC3 or PD-L1 that does not contain a genetically modified polynucleotide encoding the CD16 receptor. In the present invention, the inventors expect that the larger the binding capacity of human CD16 expressing CAR⁺natural killer cell line against a CAR target antigen (such as anti-CD 19 oNK cells expressing CAR against the recombinant protein CD19), the greater the cytotoxicity of human CD16 expressing CAR⁺natural killer cell line against target cells expressing the target CAR antigen.

[0698]No Step S31B. Anti-CD19 CAR nucleotide sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47 is chemically synthesized by Synbio Technologies. A polymerase chain reaction (PCR) kit (M05305, New England Biolabs) is used to amplify the synthesized anti-CD19 CAR nucleotide to a sufficiently large amount. Restriction enzyme (such as HinDIII, EcoRI or BamHI) is used to cut the synthesized anti-CD19 CAR nucleotide and a vector such as pBudCE linearized vector (ThermoFisher Scientific), pMAXCloning vector (Lonza), pCD810A-1 or pCD510B- 1 (system of life sciences). A ligase (such as Taq DNA ligase) is mixed with the synthesized, restricted, enzyme-digested anti-CD19 CAR nucleotide and the restricted, enzyme-digested vector to perform ligation reactions and an "anti-CD19 CAR plasmid comprising the Myc gene (or anti-CAR plasmid to promote -CD19-spanning tag)” formation Actual methods for making CAR constructs based on transfection, electroporation or lentivirus (such as anti-CD19 CAR plasmid or anti-CD19 CAR plasmid comprising the Myc gene or anti-CD19 CAR plasmid comprising Tag) are known or obvious to those skilled in the art (US Patent No. 7,446,179; WO 2015157252; US Patent No. 7,446,179).

[0699]No Step S32Anti-CD19 CAR plasmids comprising the Myc gene (or tag-comprising anti-CD19 CAR plasmid) are transfected, electroporated or transduced into oNK to obtain oNK expressing anti-CD19 CAR.

[0700]For example, cell suspensions obtained by culturing with the culture method disclosed in Embodiment 2.1 (referred to as cultured oNK suspension) were harvested (cfFIGO.18A) and transfected (as Lipofectamine 2000, Lipofectamine 3000, ThermoFisher Scientific), electroporated (as P3 Primary Cell Nucleofector Kit, SF Cell line Nucleofector Kit, Lonza) or transduced (as Lentivirus, Retrovirus) with CAR construct. The CAR-expressing oNK were expanded with fresh growth medium (such as DMEM culture medium, Eagles minimal essential medium alpha modification, or XVIVO 10 culture medium containing 0.5%-30% (vol%, vol%, v /v) Human platelets comprised lysed and 100-3000 IU/ml Interleukin 2 (IL-2)) and cultured at 37°C in a G-Rex plate.

Modality 16.2: Enrichment of oNK cells expressing the chimeric antigen receptor (CAR).

[0701]No Step S33, to enrich for the CAR-expressing oNK cells, the cells obtained in step S32were labeled with recombinant CD19 protein (cat. no. 11880-H08H from Sino Biological or FITC-conjugated CD19 protein with cat. no. CD9-HF2H2 from ACROBiosystem or APC-conjugated recombinant CD19 protein with cat. no . CD19-3309HA from Creative stained BioMart) and fluorescence-conjugated anti-Myc antibody (Novus Biologicals) (cfFIGO.18B). The CAR-expressing oNK cells were further enriched by cell sorter (BD Bioscience) or antifluorescence MicroBeads (Miltenyl Biotec) to obtain the anti-CD19 CAR-expressing oNK cells (CAR19-oNK) (see Figure 18C) and to fresh oNK expanded growth medium (such as DMEM culture medium, Eagle's minimal essential medium alpha modification, or XVIVO 10 culture medium containing 0.5%-30% (volume percent, %vol, v/v) human platelet lysate and 100-3000 IU/mL Interleukin 2 includes (IL-2)) and grown at 37°C on a G-Rex plate.

[0702]The results for the fluorescence analysis of the cultured oNK cell suspension without the transudate CAR anti-CD19 construct are shown in FIGFIGO.18A;FIGO.18Ais the two-dimensional scatter plot representing Myc⁺Population of cells with CD19 binding activity in the cultured oNK cell suspension lacking the transudate anti-CD19 CAR construct. The results for fluorescence analysis of the suspension of oNK cells cultured with the transudate CAR-anti-CD19 construct are shown in FIGFIGO.18B;FIGO.18BFigure 12 is the two-dimensional dot plot depicting the population of Myc+ cells with CD19-binding activity in the suspension of oNK cells cultured with the transudate CAR-anti-CD19 construct.

[0703]The results aFIGO.18Ashow that oNK cell suspension cultured without the transduced anti-CD19 CAR construct shows no fluorescence signal from expressed Myc marker and bound human recombinant CD19 protein. There is 0.44% double positive background signal in the cultured oNK cell suspension with no anti-CD19 CAR construct transduced.

[0704]The results aFIGO.18Bshow that the oNK cell suspension cultured with the transudated anti-CD19 CAR construct exhibits a 9.06% double positive fluorescence signal of the expressed Myc marker and the bound human recombinant CD19 protein.

[0705]Please askFIGO.18C.FIGO.18Cis the two-dimensional scatter plot representing isolated Myc⁺Cells with CD19 binding activity isolated from the cell suspension as shown in FIGFIGO.18Bby labeling labeled recombinant CD19 protein and fluorescently conjugated anti-Myc antibody. The results aFIGO.18Cshow that the suspension of cultured oNK cells isolated with the CAR-anti-CD19 construct were transudatedFIGO.18Bshow 82.27% double positive fluorescence signal from expressed Myc tag and bound human recombinant CD19 protein.

[0706]Preferably, the anti-CD19 CAR nucleotide sequence comprises a sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or other anti-CD19 CAR nucleotide sequence.

[0707]The method of making CAR plasmids and lentiviral particles comprising CAR plasmids is known or will be known to those skilled in the art.

[0708]In addition, CAR plasmids and lentiviral particles comprising CAR plasmids are commercially available. For example, lentiviral particles comprising the anti-CD19 CAR plasmid can be purchased from Creative Biolabs (this anti-CD19 CAR construct comprises the CD19 scFv domain, the CD28 domain and the CD3 zeta domain; Cat No. VP-CAR-LC61); lentiviral particles comprising the anti-BCMA-CAR plasmid can be purchased from Creative Biolabs (Cat. # VP-CAR-LC534); lentiviral particles comprising the anti-HER2-CAR plasmid can be purchased from Creative Biolabs (Cat. # VP-CAR-LC834); lentiviral particles comprising the anti-PD-L1 CAR plasmid can be purchased from Creative Biolabs (Cat# CAR-ZP1471); lentiviral particles comprising the anti-VEGFR2-CAR plasmid can be purchased from Creative Biolabs (Cat# VP-CAR-LC616); lentiviral particles comprising the anti-TCR b-chain CAR plasmid can be purchased from Creative Biolabs (Cat # VP-TCR-YC160); lentiviral particles comprising the anti-ICAM-1 CAR plasmid can be purchased from Creative Biolabs (Cat.# CAR-ZP7800); lentiviral particles comprising the anti-PD-1 CAR plasmid can be purchased from Creative Biolabs (Cat# VP-CAR-LC412).

[0709]Cell suspensions obtained by culturing with the culture method disclosed in Embodiment 2.1 (referred to as cultured oNK suspension) can be transfected (as Lipofectamine 2000, Lipofectamine 3000, ThermoFisher Scientific), electroporated (as P3 Primary Cell Nucleofector Kit, SF Kit Nucleofector- Cell line) , Lonza) or transduced (such as lentiviruses, retroviruses) with any of these CAR plasmids or lentiviral particles comprising CAR plasmids to obtain oNK cells expressing CAR against CD2, CD3-Delta, CD3-Epsilon , CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (alpha chain associated B cell antigen receptor complex), CD79B (beta chain associated B cell antigen receptor complex), CD84 (SLAMF5), CD96 (tactile) , CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40) , CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D ( KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46) , CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), T-cell inducible costimulator (ICOS), LFA-1 (CD11a /CD18), NKG2C, DAP-10, ICAM-1, NKp80 ( KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, a Toll-like receptor, HER2, BCMA, PD-L1, VEGFR2, TCR b chain, or combinations thereof.

Modality 17: Non-tumorigenicity of oNK cells expressing CAR

[0710]In this modality, six to eight week old female BALB/c nude mice (purchased from The Jackson Laboratory or BioLasco, Taiwan) were used. 25 mice were randomized into five groups, which were SK-OV-3 group, Daudi group, oNK group, CAR19-oNK group and DPBS group.

[0711]A human ovarian cancer cell line "SK-OV-3" (purchased from ATCC; accession number is ATCC HTB-77), human B-lymphoblastoid cell lines "Daudi" (purchased from ATCC; accession number is ATCC CCL-213), a cell suspension, obtained by culturing for 101 days with the culture method disclosed in Embodiment 2.1 (oNK suspension cultured for 101 days of the present invention, referred to as oNK suspension cultured for 101 days) and an anti-CD19 CAR-expressing oNK cell suspension (a cell suspension obtained by culturing oNK cells for 25 days with the culture method disclosed in Embodiments 2.1 and then performing the transduction method and the CAR-expressing cell isolation method as disclosed in Embodiments 16.1 and 16.2 with reference to up to a suspension of CAR19-oNK cells cultured for 25 days) were used in this embodiment.

[0712]1×10⁷SK-OV-3-Zellen, 1 × 10⁷Daudi cells, 1x10⁷Cells in the 101 days cultured oNK suspension and 1 × 10⁷Cells in suspension of CAR19-oNK cells cultured for 25 days were each suspended in 100 L of Dulbecco's phosphate buffered saline (DPBS) to obtain various cell suspensions. Cell suspensions and 100 µl of DPBS were implanted subcutaneously into female BALB/crude mice in SK-OV-3 group, Daudi group, oNK group, CAR19-oNK group, and DPBS group on day 0, respectively. Tumor growth in each mouse was observed on day 21, day 24, day 42 and day 59, and the mice were sacrificed on day 59.

[0713]See Table 5. Table 5 shows the results of tumor formation in nude mice xenografted with various cell lines.

[0714]Table 5 shows that there was no tumor formation in mice from the DPBS groups (negative control group) throughout the study period (0/5.0%), while all five mice from the SK-OV-3 group (positive control group ) developed tumors (5/5, 100%). In mice xenografted with the Daudi lymphoma cell line, 4 out of 5 mice in the Daudi group developed tumors (4/5, 80%) that persisted until the end of the study (Day 59).

[0715]In mice xenografted with oNK cells or oNK cells expressing anti-CD19-CAR of the present invention, there was no tumor formation in mice in the oNK group and in the CAR19-oNK group throughout the study period (0/5, 0%). . These study results demonstrate that non-irradiated oNK cells and CAR-expressed oNK cells are non-tumorigenic and safe for future clinical applications and disease treatment.

Modality 18: Analyze the cytotoxicity of oNK cells expressing CAR against target cells in vitro

Modality 18.1 CD19 binding activity of CAR19-oNK cells

[0716]Effector oNK (cell suspensions obtained by culturing for 70 days with the culture method disclosed in embodiment 2.1; also referred to as oNK suspension, cultured for 70 days) and CAR19-oNK (CAR19-oNK cell suspension, cultured for 70 days, obtained by culturing of oNK cells for 70 days using the culture method disclosed in Embodiment 2.1 and then performing the transduction process and the process of isolating CAR expressing cells as disclosed in Embodiments 16.1 and 16.2, wherein the transduction process is based on pseudo-CD19-expressing CAR lentivirals Particle-based made from pCD810A-1 and lentivirus-based CAR-CD19 constructs as shown in FIGFIGO.16A) were each co-incubated with fluorescence-conjugated recombinant CD19 protein (cat. no. CD9-HF2H2-25 µg from Acro Biosystems).

[0717]The results for the CD19 binding activity of oNK and CAR19-oNK are shown in FIGFIGO.19A;FIGO.19AFigure 12 is the histogram showing CD19 binding activity of oNK and CAR19-oNK.

[0718]The results aFIGO.19Ashow that oNK cells expressing anti-CD19-CAR (CAR19-oNK) elicit increased CD19-binding activity compared to oNK cells and can therefore elicit increased cytotoxicity against CD19⁺B cell lymphoma.

Modality 18.2 cytotoxicity of CAR19-oNK cells against CD19⁺B cell lymphoma

[0719]Effector oNK (cell suspensions obtained by culturing for 70 days with the culture method disclosed in embodiment 2.1; also referred to as oNK suspension, cultured for 70 days) and CAR19-oNK (CAR19-oNK cell suspension, cultured for 70 days, obtained by culturing of oNK cells for 70 days using the culture method disclosed in Embodiment 2.1 and then performing the transduction process and the process of isolating CAR expressing cells as disclosed in Embodiments 16.1 and 16.2, wherein the transduction process is based on pseudo-CD19-expressing CAR lentivirals Particle-based made from pCD810A-1 and lentivirus-based CAR-CD19 constructs as shown in FIGFIGO.16A) were each probed with target cells expressing Raji-luciferase (Raji-Luc, a CD19⁺B cell lymphoma cell line expressing the luciferase gene) at E:T ratios of 0.2:1, 0.5:1, 1:1, 2:1 and 5:1 for 1 hour. D-luciferin (consumable luciferase substrate purchased from GoldBio) was added to the cells and incubated at 37°C for 10 minutes. The luminescence of each well was detected using HTX Multi-Mode Reader (BioTek) and calculated as percentage of cytotoxicity relative to the luminescence obtained from untreated Raji-Luc cells; the luminescence being the catalytic product of luciferase. Statistics were analyzed using Student's t-test. **, p<0.01; ***, p<0.001; ****, p<0.0001. The actual methods of this experiment are known or will be apparent to those skilled in the art (Rigo V, 2017).

[0720]See 19B.FIGO.19BFigure 13 is the bar graph showing the comparison of cytotoxic function between oNK and CAR19-oNK for killing CD19⁺B-cell lymphoma in different effector (E) to target (T) ratios.FIGO.19Bshows that oNK 0.0 ± 0.0%, 8.5 ± 3.8%, 27.3 ± 2.48%, 38.4 ± 3.6% and 62.7 ± 3.2% of CD19 kills⁺B-cell lymphoma in the ratio of the number of effector cells to the number of target cells of 0.2:1, 0.5:1, 1:1, 2:1 and 5:1 (ET0.2 to ET5), respectively; CAR-19 oNK kills 13.9 ± 1.2%, 45.9 ± 2.5%, 64.1 ± 2.5%, 80.0 ± 7.4% and 91.4 ± 1.9% of CD19⁺B-cell lymphoma in the ratio of the number of effector cells to the number of target cells of 0.2:1, 0.5:1, 1:1, 2:1 and 5:1 (ET0.2 to ET5), respectively.FIGO.19Bshows that oNK cells expressed by anti-CD19-CAR (CAR19-oNK) cause increased cytotoxicity against CD19 compared to oNK cells⁺B-cell lymphoma in different E:T ratios.

[0721]In addition, the result shows that: CAR19 oNK cells can cause an approximately ∞-fold increase in cytotoxicity compared to oNK cells (13.1÷0=∞). This is an unexpected result.

Modality 18.3 CAR19-oNK cell has no off-target cytotoxicity

[0722]Effector oNK (cell suspensions obtained by culturing for 69 days with the culture method disclosed in embodiment 2.1; also referred to as oNK suspension, cultured for 69 days) and CAR19-oNK (CAR19-oNK cell suspension, cultured for 69 days, obtained by culturing of oNK cells for 69 days using the culture method disclosed in Embodiment 2.1 and then performing the transduction process and the process of isolating cells expressing CAR as disclosed in Embodiments 16.1 and 16.2, wherein the transduction process was based on pseudolentiviral particles derived from pCD810A -1 and lentivirus-based CAR-CD19 constructs as shown in FIGFIGO.16A) were each co-incubated with the calcein-labeled K562 target cell (CCL-243 purchased from ATCC; K562 is a CD19).⁻cancer cell line) at E:T ratios of 0.2:1, 0.5:1, 1:1, 2:1 and 5:1 for 2.5 hours; where calcein is a fluorescent dye. Calcein-labeled K562 lysed with Triton X-100 was defined as 100% lysed control. Fluorescence signals from dead cells in the supernatant of each sample and 100% lysed control were detected by 490 nm excitation and 520 nm emission with the HTX Multi-Mode Reader.

[0723]Please askFIGO.20.FIGO.20Figure 12 is the bar graph showing the comparison of cytotoxic function between oNK and CAR19-oNK for killing CD19 cancer cells at different effector (E) to target (T) ratios.

[0724]FIGO.20shows that CAR19-oNK exerts no potent increased cytotoxicity against CD19⁻Cancer cell line compared to parental oNK cells. Therefore, CAR19-oNK has no off-target cytotoxicity.

Modality 19: Analyze the cytotoxicity of oNK cells expressing CAR against target cells in vivo

Modality 19.1 Cytotoxicity of oNK cells expressing CAR against liquid tumor

[0725]1×10⁵Target cell expressing Raji luciferase (Raji-Luc, a CD19⁺B-cell lymphoma cell lines; CCL-86, ATCC) were each injected intravenously into 15 female immunocompromised NSG mice (Jackson Laboratory) on day 0. Five mice from each group were treated with 5x10⁶oNK (cells in cell suspensions obtained by culturing for 117 days with the culture method disclosed in embodiment 2.1; also referred to as oNK suspension cultured for 117 days), CAR19-oNK (CAR19-oNK cell suspension cultured for 117 days obtained by Cultivating oNK cells for 117 days using the culture method disclosed in Embodiments 2.1 and then performing the transduction process and the CAR-expressing cell isolation process as disclosed in Embodiments 16.1 and 16.2, wherein the transduction process was based on anti-CD19 CAR-expressing pseudolentiviral particles made from pCD810A-1 and lentivirus-based CAR-CD19 constructs as shown in FIGFIGO.16A) or vehicle (cell medium only, such as fresh growth medium described in modality 16.1) on days 0, 3, 7 and 10. Luminescence was monitored by AMI HTX (Spectral Imaging) on ​​days 0, 4, 7, 11 detected, 14 and 18.

[0726]FIGO.21Aare the fluorescence images of tumor cells in mice on days 4, 7, 11, 14 and 18.FIGO.21Ashows that CAR19-oNK exerts superior efficacy against lymphoma cells.

[0727]FIGO.21Bis the luminescence statistical analysis shown in FIGFIGO.21Anach dem Mixed-Effects-Modell. *, p < 0,05; ****, p<0,0001.FIGO.21Bshows: Compared to oNK cells, CAR19-oNK cells can produce an approximately 11.6-fold increase in cytotoxicity (13.8 × 10⁸1,2 × 10⁸=11.6). This is an unexpected result.

[0728]FIGO.21Cthe survival rate of rats is shown in FIGFIGO.21A.FIGO.21Cshows a significantly prolonged mouse survival rate in mice treated with CAR19-oNK.FIGO.21Cshows that: Compared to oNK cell treatment, CAR19 oNK treatment can prolong life by 20 to 80 times. This is an unexpected result.

[0729]FIG.21to z21Bshow that CAR19-oNK cells elicit significantly increased potency against CD19 compared to vehicle⁺B cell lymphoma.

Modality 19.2 Cytotoxicity of oNK cells expressing CAR against solid tumors

[0730]The human ovarian cancer cell line expressing SKOV3 luciferase (SKOV3-Luc, a HER2⁺cell lines; Catalog number AKR-232, obtained from CELL BIOLABS Inc) were injected intraperitoneally on day 0 into 15 female NSG mice (Jackson Laboratory). Five mice from each group were treated with oNK (cells in cell suspensions obtained by culturing with the culture method disclosed in embodiment 2.1), CARHER2-oNK (CARHER2-oNK cell suspension obtained by culturing oNK cells with the culture methods disclosed in embodiment 2.1 and then performing the transduction process with the pseudolentiviral particles expressing anti-HER2-CAR) or vehicle (cell B. fresh growth medium described in modality 16.1) on days 0, 4, 7, 10, 14 and 17. Luminescence was detected by AMI HTX (Spectral Imaging) weekly and at the end of the experiment.

[0731]The inventors of the present invention expect that CARHER2-oNK exerts superior potency against solid ovarian tumors, and compared to oNK cells, CARHER2-oNK cells could cause greater cytotoxicity and prolong life.

Modality 20: Monitoring long-term cell viability, cell proliferation, CD19 binding activity, and cell surface markers of CAR19-oNK

Modality 20.1 Cell Viability and Cell Proliferation of CAR19-oNK

[0732]CAR19-oNK (Cell suspensions obtained by 4- to 83-day CAR-oNK suspension culture generated by transduction of cultured oNK with pseudolentiviral particles expressing anti-CD19-CAR, prepared from pCD810A-1 and CAR-based CD19 constructs in lentiviruses, as shown in FIGFIGO.16A) was used in this modality. The culture method for growing CAR-oNK is almost the same as Modality 2 but IL-2 in step S22may not be required. Therefore, the culture procedure for culturing CAR-oNK includes at least the following step:

[0733]steps41: receipt of CAR19-oNK;

[0734]steps42: contacting the CAR19-oNK with a culture medium comprising human platelet lysate on day 0 in the recipient; It is

[0735]steps43: Growing CAR19-oNK for several days to propagate CAR19-oNK.

[0736]Detailed conditions can be found in modality 2.1, modality 11 and modality 13 and can still be found in modality 12.

[0737]Each sample of cell suspensions obtained by culturing CAR19-oNK for different days was mixed with an equal volume of trypan blue and viability and cell number were observed. Viability was determined by dividing the number of viable cells by the total number of cells.

Modality 20.2 Detection of CD56, CD3 and CD2 surface markers from cultured CAR19-oNK

[0738]Each sample of cell suspensions obtained at different time points in modality 20.1 was centrifuged; the supernatant was removed, the cells were resuspended in buffer and then treated with 1 µL of fluorescently labeled antibody CD56 (cat. no. 318304, Biolegend, USA), 1 µL of fluorescently labeled antibody CD3 (cat. no. 300410, Biolegend, USA) and 1 µl ;l fluorescently labeled CD2 antibody (cat. no. 300222, Biolegend, USA) to simultaneously label cells expressing the CD56 molecule, the CD3 molecule and/or the CD2 molecule. Finally, the cell sorter or flow cytometer was used to analyze whether the cells showed CD56 molecules, CD3 molecules and/or CD2 molecules, and the proportion of cells with different cell surface manufacturers was calculated.

Modality 20.3 Detection of CD16 expression from cultured CAR19-oNK

[0739]Each sample of cell suspensions obtained at different time points in modality 20.1 was centrifuged; the supernatant was removed, the cells were resuspended in the buffer and then mixed with 1 µl of fluorescently labeled CD16 antibody (Cat. No. 302016, Biolegend, USA) to label cells expressing CD16. Finally, the cell sorter or flow cytometer was used to analyze whether the cells showed CD16 and the percentage of cells with CD16 was calculated.

Modality 20.4 Demonstration of the binding capacity of cultured CAR19-oNK to recombinant CD19 protein

[0740]Each sample of cell suspensions obtained at different time points in modality 20.1 was centrifuged; the supernatant was removed, the cells were resuspended in buffer and then treated with labeled recombinant CD19 protein (cat. no. 11880-H08H from Sino Biological, or FITC-conjugated recombinant CD19 protein with cat. no. CD9-HF2H2 from ACROBiosystem, or Protein Recombinant APC-conjugated CD19 cat # CD19-3309HA from Creative BioMart). Finally, the cell sorter or flow cytometer was used to analyze whether cells bound recombinant CD19 protein and the percentage of cells binding recombinant CD19 protein was calculated.

[0741]FIGO.22AFigure 12 is the line graph showing cell viability, CD19 binding activity and cell surface markers of CAR19-oNK within 83 days of culture.FIGO.22Ashows that cell viability was maintained at 88-95% after 4, 7, 11, 14, 21, 27, 34, 41, 48, 55, 62, 69, 76 and 83 days of CAR19-oNK culture; CD2 and CD56 were maintained at >99% after 4, 21, 41, 48, 69, 76 and 83 days of CAR19-oNK culture; CD3 was maintained at <2% after 4, 41, 48, 69, 76 and 83 days of CAR19-oNK culture. Our data show that CD16 gradually decreased by 84% after 4, 7, 11, 14, 21, 27, 41, 48, 55, 62, 69, 76 and 83 days of CAR19-oNK culture.

[0742]FIGO.22BFigure 12 is the line graph showing the proliferation of CAR19-oNK in 83 days of culture.FIGO.22Bshows that culturing CAR19-oNK with the culturing method of the present invention can maintain stable expansion within 83 days. Note that the cells were reseeded on day 7 and continued to expand until day 11; the expansion process was repeated every 11 days.

[0743]Cells carrying a CD3 phenotype⁻CD56⁺CD16⁺having recombinant CD19 protein binding activity are CAR19-oNK of the present invention. These CAR19-oNK cells can be isolated by a cell sorter (BD Bioscience).

Modality 21 Detection of CAR19-oNK cytokine secretion

[0744]CAR19-oNK cell suspensions (Cell suspensions obtained by 50-day CAR-oNK suspension culture generated by transduction of cultured oNK with pseudolentiviral particles expressing anti-CD19-CAR, prepared from pCD810A-1 and CD19-CAR-based constructs lentivirus , comprising IL-15 expression domain as shown inFIGO.16A) and Ctrl-oNK cell suspensions (obtained by culturing for 50 days with the culture method disclosed in Embodiment 2.1; also referred to as oNK culture suspension for 50 days) were used in this embodiment. The supernatant of CAR19 oNK cell suspensions and Ctrl oNK cell suspensions (parental oNK cell suspensions) was detected by an enzyme-linked immunosorbent assay (ELISA) (D1500, R&D Systems) and the IL-15 concentration in each sample was calculated using the standard curve with the intrapolation method. The actual procedures of this experiment are known or will be apparent to those skilled in the art (D1500 Handbook, R&D Systems).

[0745]FIGO.23Figure 13 is the bar graph showing IL-15 secretion by CAR19-oNK.FIGO.23shows that CAR19-oNK cells secreted IL-15, while no IL-15 was detectable in parental oNK cells. Therefore, it demonstrated the successful production and processing of IL-15 by CAR19-oNK.

[0746]For oNK cells transduced with the CD19-CAR construct comprising an IL-18 expression domain, an IL-21 expression domain, an IL-2 expression domain, or other proliferation-inducing cytokine expression domain, such transduced oNK are -Cells capable of secreting IL-18, IL-21, IL-2 or another proliferation-inducing cytokine.

Modality 22: Analyze the independence of supplemented cytokines in CAR19-oNK culture

[0747]One million CAR19-oNK (cultured cell suspensions for 56 days CAR-oNK suspension generated by transduction of cultured oNK with pseudolentiviral particles expressing anti-CD19-CAR produced from pCD810A-1 and CAR-based CD19 constructs in lentiviruses, as shown inFIGO.16A) and Ctrl-oNK (cell suspensions obtained by culturing for 56 days with the culture method disclosed in embodiment 2.1; also referred to as 56-day cultured oNK suspension) were seeded and cultured in the presence of 500, 100, 10 and 0 IU/ml IL -2. The detailed conditions are given in modality 2.1 and modality 12. The cells were subcultured by replacing the culture with fresh oNK growth medium containing the appropriate concentration of IL-2 every 4-7 days, depending on the cell concentration. Fresh ONK growth medium is a cell culture medium comprising: (1) 0.5%-30% (volume percent, vol%, v/v) human platelet lysate; (2) appropriate concentration of IL-2; and (3) DMEM culture medium (Dulbecco's Modified Eagle Medium), Eagle alpha modification of Minimal Essential Medium, or XVIVO 10 culture medium.

[0748]FIGO.24Figure 12 is the line graph showing the effect of IL-2 on the fold increase in total cell number after different days of CAR19-oNK culturing.FIGO.24shows that CAR19-oNK can be cultured and maintained in the presence of IL-15 secreted by CAR19-oNK. The results showed that CAR19-oNK can grow in medium lacking IL-2, while Ctrl-oNK can only grow in medium containing IL-2.

Embodiment 23: Detection of the ability of CAR-expressing oNK cells to bind to their specific recombinant protein

[0749]oNK cells expressing CAR comprising a single-chain target-binding variable fragment (scFv) against a target antigen (such as BCMA) were centrifuged; the supernatant was removed, the cells were resuspended in the buffer and then mixed with the labeled recombinant target antigen protein (such as labeled recombinant BCMA protein). Finally, the cell sorter or flow cytometer was used to analyze whether the cells bound the target recombinant antigen protein (such as BCMA) and the percentage of cells binding the target recombinant antigen protein (such as BCMA) was calculated.

[0750]If the cells have the recombinant protein-binding activity of the target antigen (such as BCMA), oNK cells expressing CAR against the target antigen will be successfully grown.

[0751]Based on the results shown in Embodiments 18 and 19, the inventors of the present invention believe that oNK cells transduced with the CAR construct against CD19 or against any of the target antigens disclosed in the invention have increased cytotoxicity against cancer cells, tumor fluid and solid tumor expressing the target antigen without off-target cytotoxicity.

[0752]Preferably, the target antigen is CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2). CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1 ), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (alpha chain associated B cell antigen receptor complex), CD79B (antigen receptor -complex-associated beta chain), CD84 (SLAMF5), CD96 (tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1) , CD158B1 (KIR2DL2), CD158B2 (KIR2DL3). ), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4 ), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 ( SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), T-cell inducible costimulator (ICOS), LFA-1 (CD11a/CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT , GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein Immunoglobulin protein, a cytokine receptor, an integrin, NK cell receptor activation, a toll-like receptor, HER2, BCMA, PD-L1, VEGFR2, TCR b chain, and combinations thereof.

[0753]Preferably, oNK cells expressing CAR comprise at least one antigen-binding complex on the cell membrane, the antigen-binding complex comprising a means of inducing a cytotoxic activity of the cell through specific binding by a selected target antigen to cancer antigen, glycolipid, glycoprotein, differentiation antigen moiety present in cells of a hematopoietic lineage, antigenic peptide associated with the major histocompatibility complex, gamma-glutamyltranspeptidase, adhesion protein, hormone, growth factor, cytokine, receptor ligand, ion channel, membrane-bound form of a µ-immunoglobulin. -chain, alpha-fetoprotein, C-reactive protein, chromogranin A, epithelial mucin antigen, human epithelial-specific antigen, Lewis(a) antigen, multidrug resistance-related protein, neu oncogene protein, neuron-specific enolase, glycoprotein P, Multidrug Resistance Related Antigen, p170, Multidrug Resistance Related Antigen, Prostate Specific Antigen, NCAM, Ganglioside Molecule, MART-1, Heat Shock Protein, SialylTn, Tyrosinase, MUC-1, HER-2 /neu, KSA, PSMA, p53, RAS, EGF -R, VEGF, MAGE or other target antigen (marker) expressed by a target cell.

[0754]Preferably, the antigen-binding complex comprises a single-chain target-binding variable fragment (scFv) against the target antigen.

[0755]Preferably, the target antigen is a cancer antigen selected from HER2/neu (ERBB2), HER3 (ERBB3), EGFR, VEGF, VEGFR2, GD2, CTLA4, CD19, CD20, CD22, CD30, CD33 (Siglec-3), CD52 (CAMPATH- 1 antigen), CD326 (EpCAM), CA-125 (MUC16), MMP9, DLL3, CD274 (PD-L1), CEA, MSLN (Mesothelin), CA19-9, CD73, CD205 (DEC205), CD51, c - MET, TRAIL-R2, IGF-1R, CD3, MIF, folate receptor alpha (FOLR1), CSF1, OX-40, CD137, TfR, MUC1, CD25 (IL-2R), CD115 (CSF1R), IL1B, CD105 ( Endoglin) , KIR, CD47, CEA, IL-17A, DLL4, CD51, Angiopoietin 2, Neuropilin-1, CD37, CD223 (LAG-3), CD40, LIV-1 (SLC39A6), CD27 (TNFRSF7), CD276 (B7-H3 ), Trop2, Claudin1 (CLDN1), PSMA, TIM-1 (HAVcr-1), CEACAM5, CD70, LY6E, BCMA, CD135 (FLT3), APRIL, TF(F3), Nectin-4, FAP, GPC3, FGFR3, ICAM-1 (CD54), ROBO1, NKG2D Ligand, CD123, CS1/SLAMF7/CD319/CRACC, CD7, CD142 (Platelet Tissue Factor, Factor III, Tissue Factor), CD38, CD138, EGFR VIII, EGFR, EGFR806, EGFR Family Member, PD-1, ROR1, CSPG4, CLL-1 (CLEC12A), CD147, PSCA, EPHA2, GPRC5D, CD133, B7H6, DSC2, AE1 (SLC4A1), GUCY2C, CDH17, HPSE, CD24, MUC4, AFP-L3, SP17, DCLK1, CAIX (CA9), IL13RA2, IL13Ra, CD56, CD44v6, TCR beta chain, chlorotoxin ligands, Claudin-6, Claudin-18.2, EIIIB (fibronectin), Glypican-1 (GPC1), PLAP (placental alkaline phosphatase ), uPAR, HCMV glycoprotein B (gB), HLA-DR (Lym1 antibody target), tumor-associated integrin αvβ6, LunX, integrin αvβ3, folate receptor beta (FRβ), LILRB4, MISIIR (Mullerian type 2 receptor inhibitor substance ) , 5T4, CD83 Ligand, HBsAg, CD171 (L1-CAM), TAG72 (TAG72 (tumor-associated glycoprotein 72)), B7-H4, CD166 (ALCAM), AC133 (PROM1), LeY, CD13 (TIM1), CD117, TEM8 (ANTXR1), CD26, IL13Ra2, IGF1R, Muc3a, IL1RAP, TSLPR (CRLF2), LMP1, Siglec7, Siglec9, Epstein-Barr Virus gp350, CD1a, CLEC14A, MAGE-A1, MAGE-A4, Neurofilament M ( NEFM), HERV -K env protein, HLA-A*0201/NY-ESO-1(157-165) peptide, 2B4, TACI (TNFRSF13B), CD32A(131R), AXL, Lewis Y, CD80, CD86, ROR2, a killer cell immunoglobulin-like receptors (KIRs), a T cell receptor, a major histocompatibility complex protein, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin activating NK cell receptor, and combinations thereof.

[0756]Preferably, the antigen binding complex is a chimeric antigen receptor (CAR).

[0757]Preferably, a DNA sequence from the cell's chromosome is at least 90% or 95% similar to the DNA sequence from the corresponding natural killer cell chromosome deposited with NPMD under NITE accession number BP-03017.

[0758]Preferably, a DNA sequence from the cell's chromosome is at least 99%, 99.99% or 99.995% similar to the DNA sequence from the corresponding natural killer cell chromosome deposited with NPMD under NITE accession number BP-03017.

[0759]Preferably, the chromosome DNA sequence is a chromosome 17 DNA, a chromosome 19 DNA sequence, a chromosome 22 DNA sequence, a chromosome 4 DNA sequence, a chromosome 18 DNA sequence , a Y chromosome DNA or an X chromosome DNA sequence Chromosome DNA sequence is a chromosome 1 DNA sequence, a chromosome 2 DNA sequence, a chromosome 5 DNA sequence, a DNA of chromosome 6, a DNA sequence of chromosome 7, a DNA sequence of chromosome 8, a DNA sequence of chromosome 9, a DNA sequence of chromosome 10, DNA sequence of chromosome 11, DNA sequence of chromosome 12, DNA Sequence of Chromosome 13 DNA, Chromosome 14 DNA Sequence, Chromosome 15 DNA Sequence, Chromosome 16 DNA Sequence, Chromosome 20 DNA Sequence, Chromosome 21 DNA Sequence, Chromosome 3 DNA Sequence.

[0760]Preferably, the entire genome of the cell is at least 99.995% similar to the entire genome of the natural killer cell deposited with the NPMD under NITE accession number BP-03017.

[0767]http://www.pathologyoutlines.com/topic/cdmarkerscd56.html

[0768]http://www.pathologyoutlines.com/topic/cdmarkerscd3.html

[0769]http://www.pathologyoutlines.com/topic/cdmarkerscd2.html

[0777]The above descriptions are only the preferred embodiments of the present invention and are not intended to limit the scope of the patent application of the present invention. Therefore, any change or modification that does not depart from the spirit disclosed herein must be embraced within the scope of patent application for the present invention.