A novel manufacturing method of natural killer cells and liver cancer treatment using thereof

Abstract

This invention relates to a novel method for preparing natural killer cells and a treatment method for liver cancer using natural killer cells obtained by this method. Specifically, this invention provides a novel method for preparing natural killer cells that can culture large quantities in a short time. The natural killer cells prepared by the above method can be used as an anti-cancer therapy due to their high anti-cancer effect.

Description

[Technical Field]

[0001] This invention relates to a novel method for preparing natural killer cells and a liver cancer treatment method using natural killer cells obtained by this method. Specifically, this invention provides a novel method for preparing natural killer cells that can culture large quantities in a short time. The natural killer cells prepared by the above method can be used as an anti-cancer therapy due to their high anti-cancer effect. [Previous Technology]

[0002] Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer-related deaths. Treatment for HCC is usually determined by tumor staging. In South Korea, hepatic arterial infusion chemotherapy (HAIC) is used, especially in the treatment of locally advanced HCC.

[0003] Intrahepatic artery infusion therapy (IAI) delivers drugs directly to tumor cells via the hepatic artery, exhibiting higher efficacy and lower toxicity compared to systemic therapy. 5-Fluorouracil (5-fluorouracil; 5-FU) and cisplatin are the most commonly used chemotherapeutic agents in IAI, and they have been reported to have immunomodulatory effects as potent inducers of natural killer cell activity.

[0004] Natural killer cells (NK cells) are essential structural elements of the liver's innate immune system, accounting for 30% to 50% of intrahepatic lymphocytes. Reports indicate a significant reduction in the number and function of NK cells in patients with hepatocellular carcinoma (HCC), particularly a decrease in tumor-infiltrating NK cells, which is associated with reduced survival in advanced HCC. Therefore, intrahepatic NK cells play a crucial role in immune surveillance against HCC. Thus, overcoming NK cell dysfunction and restoring NK cell activity are essential for successful treatment of HCC and for effective immune defense against the disease.

[0005] Although natural killer cells play such an important role in the immune system, the proportion of natural killer cells in peripheral blood lymphocytes of cancer patients is significantly reduced. Without additional culture or initiation processes, the induction of natural killer cell dysfunction and the restoration of its activity will be limited.

[0006] Therefore, the inventors established a novel method for preparing natural killer cells by finding a combination of cytokines that can culture natural killer cells in large quantities and restore their activity. Furthermore, they found a novel treatment method that can effectively treat hepatocellular carcinoma by perfusing natural killer cells prepared by the novel method after inducing their activity with intrahepatic artery anticancer perfusion therapy. Thus, the present invention was completed. [Summary of the Invention]

[0007] The problem the invention aims to solve

[0008] The purpose of this invention is to provide a method for preparing natural killer cells that can rapidly proliferate in vitro and are active against tumors.

[0009] The object of the present invention is to provide a composition comprising the above-mentioned natural killer cells.

[0010] The object of the present invention is to provide a method for treating a cancer subject, comprising the step of administering the aforementioned natural killer cells. Technical means to solve the problem.

[0011] The present invention provides a method for preparing isolated natural killer cells, comprising: step (a) isolating peripheral blood mononuclear cells from a liver cancer patient; step (b) culturing the isolated peripheral blood mononuclear cells in the presence of IL-2, IL-21, 4-1BBL and K562 cell lines; and step (c) culturing the isolated peripheral blood mononuclear cells in the presence of IL-2 and IL-15.

[0012] In one embodiment, the K562 cell line described above may be a cell line irradiated with 100 Gy of γ rays.

[0013] In one embodiment, the IL-2 concentration in step (b) can be from 10 U / mL to 30 U / mL.

[0014] In one embodiment, the IL-2 concentration in step (b) can be 10 U / mL.

[0015] In one embodiment, the concentration of IL-21 in step (b) can be 5 ng / mL.

[0016] In one embodiment, the concentration of 4-1BBL in step (b) can be 10 ng / mL.

[0017] In one embodiment, the culture time in step (b) can be up to 7 days.

[0018] In one embodiment, the IL-2 concentration in step (c) can be from 100 U / mL to 1000 U / mL.

[0019] In one embodiment, the IL-2 concentration in step (c) can be 100 U / mL.

[0020] In one embodiment, the IL-15 concentration in step (c) can be 10 U / mL.

[0021] In one embodiment, the culture time in step (c) can be up to 12 days.

[0022] The present invention provides a composition for treating liver cancer, wherein the composition comprising natural killer cells prepared by the above preparation method is administered once daily for 5 consecutive days.

[0023] In one embodiment, a second administration may be given 4 weeks after the start of the above administration, once a day for 5 consecutive days.

[0024] In one embodiment, the above composition can improve objective response rate (ORR), disease control rate (DCR), duration of response (DOR), time to tumor progression (TTP), overall survival (OS), or quality of life (EORTC QLQ-C30).

[0025] In one embodiment, the above composition may contain 2.5 × 10⁸ to 12 × 10⁸ natural killer cells, preferably 8 × 10⁸ to 12 × 10⁸ natural killer cells, and more preferably 10 × 10⁸ natural killer cells.

[0026] In one embodiment, the above composition can be administered to a patient who has received hepatic artery infusion chemotherapy.

[0027] In one embodiment, the above-mentioned hepatic artery infusion chemotherapy may be cisplatin, 5-fluorouracil, or a combination thereof.

[0028] In one embodiment, the liver cancer described above may be hepatocellular carcinoma.

[0029] The present invention provides a method for treating liver cancer patients by administering a first dose of the following to the liver cancer patients: a composition comprising natural killer cells prepared by the above preparation method is administered once daily for 5 consecutive days.

[0030] In one embodiment, a second administration may be given 4 weeks after the start of the above administration, once a day for 5 consecutive days.

[0031] In one embodiment, the above-described treatment method can improve objective response rate (ORR), disease control rate (DCR), duration of response (DOR), time to tumor progression (TTP), overall survival (OS), or quality of life (EORTC QLQ-C30).

[0032] In one embodiment, the above composition may contain 2.5 × 10⁸ to 12 × 10⁸ natural killer cells, preferably 8 × 10⁸ to 12 × 10⁸ natural killer cells, and more preferably 10 × 10⁸ natural killer cells.

[0033] In one embodiment, the above-mentioned patient may be a patient who has received hepatic artery infusion chemotherapy.

[0034] In one embodiment, the aforementioned liver cancer may be hepatocellular carcinoma. (Effectiveness compared to prior art)

[0035] The novel natural killer cell preparation method of the present invention can proliferate and culture natural killer cells in large quantities from peripheral blood mononuclear cells in a short period of time. Furthermore, the natural killer cells obtained by the above method show a high response rate in patients with hepatocellular carcinoma, and can therefore be effectively used as a therapeutic agent for liver cancer.

Implementation Method

[0045] Hereinafter, the present invention and its embodiments will be described in detail with reference to the accompanying drawings, so as to enable those skilled in the art to implement the present invention. However, the present invention can be implemented in many forms and is not limited to the embodiments and examples described herein.

[0046] The term "peripheral blood mononuclear cell" as used in this specification, also known as "PBMC" or "peripheral blood mononuclear leukocyte," generally refers to mononuclear cells isolated from peripheral blood. Peripheral blood mononuclear cells can be obtained using well-known methods appropriately selected by those skilled in the art, such as the Ficoll-Hypaque density gradient method or the Lymphoprep density gradient method.

[0047] The term “proliferation” as used in this specification refers to an increase in the number of cells.

[0048] The term “natural killer cell” used in this specification is also called NK cell. Morphologically, it refers to a cell with granules in its cytoplasm, which is a cell that can eliminate tumor cells or cells infected by viruses.

[0049] The term “cytokine” as used in this specification refers to an immune-initiating cytokine that can be used in the process of differentiating peripheral blood mononuclear cells into natural killer cells, preferably IL-2, IL-15, IL-21, 4-1BBL or a combination thereof.

[0050] The term “nutritive cell” as used in this specification refers to a cell that, although it cannot proliferate, can metabolize and produce metabolic substances to help the proliferation of other cells. Preferably, it can be K562.

[0051] The term “K562” used in this specification refers to lymphoblast cells derived from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia, which can be obtained from ATCC (ATCC CCL-243).

[0052] The term “objective response rate (ORR)” as used in this specification refers to the proportion of patients who show a reduction in tumor size above a predefined amount.

[0053] The term “Disease Control Rate (DCR)” as used in this specification refers to the proportion of patients who show a complete response, a partial response, or no disease progression.

[0054] The term “duration of response (DOR)” as used in this specification refers to the period of response during which the tumor size is maintained.

[0055] The term “time to tumor progression (TTP)” as used in this specification refers to the time elapsed until the tumor size increases or until death.

[0056] The term “overall survival (OS)” as used in this specification refers to the time elapsed from the start of treatment to death.

[0057] The term “quality of life (EORTC QLQ-C30)” used in this specification refers to the numerical quality of life assessed by EORTC QLQ-C30.

[0058] The natural killer cells of the present invention can be administered using all conventional routes to reach the target tissue. For example, they can be administered into the hepatic artery, but are not limited thereto.

[0059] The term “intra-arterial administration” as used in this specification refers to a method of administration in which the drug is administered directly into the hepatic artery.

[0060] The terms “hepatic artery infusion chemotherapy” or “intrahepatic artery anticancer infusion therapy” used in this specification refer to a treatment method that administers anticancer agents via intrahepatic artery administration. Preferably, the anticancer agents may be 5-fluorouracil (5-FU) and cisplatin.

[0061] The term “combined administration” as used in this specification refers to the administration of two or more active ingredients simultaneously or sequentially.

[0062] The natural killer cells of the present invention can be administered in combination with other anticancer agents suitably selected by those skilled in the art to which this invention pertains. For example, they can be administered in combination with immune checkpoint inhibitors, but are not limited thereto.

[0063] The term “prevention” as used in this specification means all actions that suppress or delay the onset of a disease by administering the composition, and “treatment” means all actions that improve or change the symptoms of an individual suspected of having a disease or having a disease to a state of cure by administering the composition.

[0065] Hereinafter, the present invention will be described in more detail by way of examples, but the following examples are for illustrative purposes only and are not intended to limit the present invention.

[0067] Preparation Example 1

[0068] Preparation of in vitro natural killer cells (Vax-NK / HCC)

[0070] The in vitro natural killer cells were prepared as shown in Figure 1, as detailed below.

[0071] Peripheral blood mononuclear cells were isolated by aliquoting 15 mL of the patient's blood into a 50 mL test tube and diluted with 30 mL of 1× phosphate-buffered saline (PBS) using density gradient centrifugation (20°C, 1200 g, 25 min) on a Lymphoprep (Serumwerk Bernburg AG, Bernburg, Germany). Specifically, after removing the supernatant, less than 15 mL of the cell layer was collected into a 50 mL test tube and resuspended in 1× phosphate-buffered saline to a total volume of less than 45 mL before centrifugation (20°C, 500 g, 5 min). After removing the supernatant, the cells were resuspended in culture medium (RPMI 1640, 10% fetal bovine serum (FBS), 4 mM L-glutamine) and centrifuged again (20°C, 500 g, 5 min). The cell count was then measured using a hematocytometer, and peripheral blood mononuclear cells were collected. For the next stage of production, a portion of peripheral blood mononuclear cells were centrifuged (20°C, 500g, 5 minutes), resuspended in cryopreservation medium (50% fetal bovine serum, 40% RPMI 1640, 10% dimethyl sulfoxide (DMSO)), and aliquoted at 2 × 10⁸ cells / mL into cryogenic vials. The frozen cells for Method 2 were then aliquoted into cryogenic vials and stored in cell freezing containers at -70°C or below for at least 2 hours to freeze. The cryogenic vials were then transferred to an LN2 tank and stored at -150°C or below.

[0072] K562 (ATCC, USA) used as a feeder cell line for culturing natural killer cells was transferred to a 50 mL test tube and centrifuged (20°C, 500 g, 5 min). The obtained cell line was irradiated with 100 Gy of gamma rays using an irradiation apparatus. After irradiation, the cells were washed twice with culture medium (RPMI 1640, 10% fetal bovine serum, 4 mM L-glutamine).

[0073] Then, the isolated peripheral blood mononuclear cells were mixed with nutrient cell lines irradiated with 100 Gy at a ratio of 6:1 and cultured in 20 mL of complete culture medium (RPMI 1640, 10% fetal bovine serum, 4 mM L-glutamine). Natural killer cells were expanded by adding IL-2 (10 U / mL), IL-21 (5 ng / mL), and 4-1BBL (10 ng / mL), and then aliquoted into T75 flasks (Flask) with half the medium added or replaced, and cultured in a 5% CO2 incubator for 7 days. The culture medium (RPMI 1640, 10% fetal bovine serum, 4 mM L-glutamine) was aliquoted at approximately 2-day intervals, with half the medium replaced, from day 0 to day 7.

[0074] Natural killer cells cultured to day 7 were divided into two aliquots and transferred to two T75 flasks. New culture medium (RPMI 1640, 10% fetal bovine serum, 4 mM L-glutamine) was added to each flask to make a volume of 50 mL. After adding 100 U / mL IL-2 and 10 U / mL IL-15, the cells were cultured in a 5% CO2 incubator. The culture medium (RPMI 1640, 10% fetal bovine serum, 4 mM L-glutamine) was added at approximately 2-day intervals, with half of the medium replaced. The cells were cultured for 11 days.

[0075] On days 11 to 13 of culture, the prescribed culture medium was obtained from each T75 flask and transferred to 50 mL test tubes for cytokine residue test, dimethyl sulfoxide content test, mycoplasma negative test, sterility test and foreign virus negative test.

[0076] From day 14 to day 18 of culture, natural killer cells were collected in the prescribed culture container and transferred to 50 mL test tubes for centrifugation (20°C, 500 g, 5 min). After removing the supernatant, the cells were resuspended in Hartmann's solution and washed once, then centrifuged (20°C, 500 g, 5 min). After removing the supernatant, the cells were washed once more with Hartmann's solution, and after removing the supernatant and adding Hartmann's solution to count the cells, the cells were centrifuged (20°C, 500 g, 5 min). The cells were resuspended in 10 mL of human serum albumin (20% albumin, 100 mL / vial) and 490 mL of Hartmann's solution and aliquoted into Hartmann's infusion solution at 1×10⁹ cells ± 20% / 500 mL, then refrigerated.

[0078] Example 1

[0079] Evaluating the safety of natural killer cells in patients who have received intrahepatic artery infusion therapy for cancer.

[0081] The safety of natural killer cells in patients who have received intrahepatic artery anticancer infusion therapy was evaluated in the following manner.

[0082] The natural killer cell composition (Vax-NK / HCC) prepared by the method of Preparation Example 1 described above was administered to patients selected according to the following criteria.

[0083] 1) Patients aged 18 and above diagnosed with hepatocellular carcinoma

[0084] 2) Patients with Child-Pugh classification A (5-6 points) or upper B (including B up to 7 points)

[0085] 3) Patients who, after completing two cycles of intrahepatic artery infusion therapy for advanced hepatocellular carcinoma, are assessed by mRECIST as having SD, PR, or CR.

[0086] 4) Patients whose surgery is not possible

[0087] 5) Patients who do not respond to transarterial chemoembolization

[0088] 6) Patients with multiple intrahepatic tumors or those expected to be unresponsive to sorafenib due to vascular invasion.

[0089] 7) Organ function: neutrophil count (ANC) ≥1500 / µl, platelet count (PLTs) ≥75000 / µl, hemoglobin (Hb) ≥9g / dL, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤5×ULN, serum creatinine ≤1.5×ULN, INR <1.7 or PT <4 seconds, serum albumin >3.5g / dL, serum bilirubin <2mg / dL.

[0090] 8) Patients who can take care of themselves in daily living (patients with an ECOG physical activity level of 0 or 1)

[0091] 9) Patients who have given written consent to participate in this clinical trial.

[0092] Furthermore, personnel meeting the following criteria are removed from the assessment subjects.

[0093] 1) Patients who received chemotherapy, radiotherapy, immunotherapy, hormone therapy, local tumor treatment or targeted therapy within 4 weeks during the screening period.

[0094] 2) Patients with Child-Pugh classification of lower B or C

[0095] 3) Patients who can be cured through local treatment

[0096] 4) Patients who have undergone liver transplantation

[0097] 5) Patients with varicose veins or non-therapeutic ulcers that pose a risk of bleeding during endoscopy, or patients who have been observed to have bleeding of NCI CTCAE grade ≥3 within 4 weeks of screening.

[0098] 6) Patients who have difficulty taking care of themselves in daily living (ECOG physical activity level ≥2)

[0099] 7) Patients who received immunotherapy 6 months prior to recruitment

[0100] 8) Patients who have been diagnosed with other malignant tumors or have received treatment for them within the past 5 years.

[0101] 9) Patients with allergies to 5-fluorouracil and cisplatin

[0102] 10) Patients with severe or unmanageable cardiovascular disease (ejection fraction <0.5 or severe arrhythmia)

[0103] 11) Patients with active infections such as sepsis

[0104] 12) Pregnant women, breastfeeding women, or women of childbearing age who are not using appropriate contraception.

[0105] 13) Patients with autoimmune diseases

[0106] 14) Patients deemed unsuitable by researchers to be subjects in clinical trials

[0107] Among patients selected according to the above criteria, those who had completed two cycles of hepatic artery infusion chemotherapy with 5-FU and cisplatin and whose efficacy was assessed as SD or better by mRECIST before the start of Vax-NK / HCC administration were selected as Vax-NK / HCC recipients. Selected patients, after completing the third dose, received natural killer cells (Vax-NK / HCC) prepared by leukapheresis and cultured for 14 days, starting the day after the end of four cycles of hepatic artery infusion chemotherapy, at one-day intervals for a total of 5 days. One cycle of hepatic artery infusion chemotherapy consisted of four consecutive days of injections of 5-fluorouracil and cisplatin followed by a three-week rest period. These drugs were administered via a slow, 2-hour infusion through an interface connected to a catheter inserted into the hepatic artery. The clinical phase I Vax-NK / HCC treatment regimen was set as shown in Figure 2.

[0108] A phase I clinical trial was conducted on patients with advanced hepatocellular carcinoma who were unsuitable for surgical resection, transarterial chemoembolization, or sorafenib treatment, or who had failed these treatments, to investigate the combination of hepatic artery infusion chemotherapy and Vax-NK / HCC therapy. After receiving four cycles of hepatic artery infusion chemotherapy (5-fluorouracil, cisplatin), Vax-NK / HCC was administered to 3, 3, and 5 subjects, respectively, at cell doses of 2.5 × 10⁸, 5 × 10⁸, and 10 × 10⁸, once daily for 5 consecutive days for a total of 5 days. The results were published in the June 2022 issue of *Frontiers in Immunology*. The median age of the enrolled participants in this clinical trial was 56.6 years (range 43–71 years), including two patients aged 65 years and older. The characteristics of the clinical trial participants are shown in Table 1 below.

[0110] Table 1 characteristic Number of patients (%) Median age range 56.6 (43-71) gender male 10 (90.9) female 1 (9.1) ECOG physical activity level 0 2 (18.2) 1 9 (81.8) Child-Pugh classification A 9 (81.8) B 2 (18.2) BCLC Installment B 6 (54.5) C 5 (45.5) Tumor size <5cm 4 (36.4) 5–10cm 4 (36.4) ≥10cm 3 (27.3) Large vessel infiltration yes 5 (45.5) no 6 (54.5) Etiology Hepatitis B virus / Hepatitis C virus / Unknown 6 (54.5) / 3 (27.3) / 2 (18.2) (HBV / HCV / unknown) Extrahepatic metastasis whether 0 (0.0) / 11 (100) Pre-treatment Surgical removal + chemoembolization 4 (36.4) Surgical removal + radiotherapy 1 (9.1) Chemoembolization + sorafenib 1 (9.1) Chemoembolization 3 (27.3) none 2 (18.2) Liver cancer morphology Multinodular 8 (72.7) wettability 3 (27.3) Alpha-fetoprotein (AFP) ≥400 μg / L ≥400μg / L 6 (54.5) <400μg / L 5 (45.5)

[0111] The majority of registered subjects were male (90.9%), with Child-Pugh classification A (81.8%). The proportions of BCLC stages B and C were similar: 54.5% and 45.5%, respectively. Eight subjects (72.7%) had multinodular hepatocellular carcinoma, and three (27.3%) had invasive hepatocellular carcinoma. Furthermore, seven subjects (63.7%) had tumors larger than 5 cm in diameter. The subjects' initial treatments were primarily surgical resection and chemoembolization; 54.5% had hepatitis B, and 27.3% were diagnosed with hepatitis C.

[0112] The purity, initiation receptors, and cytotoxicity of Vax-NK / HCCs isolated and prepared from 11 registered subjects are shown in Table 2 below. Specifically, the proportion of natural killer (CD3-CD56+) cells in the prepared Vax-NK / HCCs was approximately 80-90%, while T (CD3+CD56-) cells, natural killer T (NKT) (CD3+CD56+) cells, and other (CD3-CD56-) cells accounted for approximately 7%, 5%, and 3%, respectively. Furthermore, the natural killer cells expressed active receptors such as CD16, CD69, CD94, and NKG2D at high levels. Their function was evaluated by cytotoxicity against K562 cells, demonstrating cytotoxicity exceeding 70%. Therefore, the prepared Vax-NK / HCCs are high-purity and well-initiated natural killer cells.

[0114] Table 2 Patient ID No. Purity (%) Cell surface markers (%) Cytotoxicity (%) CD3 - CD56 + (NK cells) CD3 + CD56 - (T cells) CD3 + CD56 + (NKT cells) CD3 - CD56 - (others) CD16 + CD69 + CD94 + NKG2D + 1 90.4 6.4 2.4 0.6 99.2 79.2 98.3 94.2 74 2 90.0 6.6 1.4 1.7 96.5 81.4 98.1 99.1 79 3 89.8 6.0 2.1 1.9 92.7 83.3 98.8 97.2 70 4 89.5 5.5 2.1 2.6 98.9 94.3 96.4 100 74 5 89.3 6.3 2.4 1.8 99.9 97.3 98.8 90.8 80 6 90.2 5.7 2.1 1.8 98.2 92.3 98.3 99.2 78 7 92.6 4.1 3.0 0.2 99.6 95.0 99.6 100 90 8 91.7 5.4 2.5 0.3 99.6 97.1 99.6 100 84 9 96.9 1.4 1.3 0.1 99.9 82.2 93.3 100 85 10 90.7 6.6 1.7 0.8 98.4 94.3 99.5 99.4 82 11 91.1 5.4 1.7 1.7 99.3 79.9 98.5 99.3 94

[0115] Hematologic and non-hematologic toxicities are shown in Tables 3 and 4 below. Specifically, the most common hematologic toxicity following hepatic artery infusion chemotherapy and Vax-NK / HCC administration was anemia (45.5%, 5 patients), one of whom had grade 3 or higher. Two patients experienced neutropenia (18.2%), grade 3 and 4, respectively, but recovered after appropriate treatment with granulocyte colony-stimulating factor (G-CSF). One patient also experienced grade 2 thrombocytopenia, and several patients experienced hematologic abnormalities (hyperkalemia, hypomagnesemia, elevated creatinine). Conversely, non-hematologic toxicities observed during the clinical study were nausea (63.6%), fatigue (27.3%), and rhinorrhea (18.2%). However, all drug-related abnormalities were associated with hepatic artery infusion chemotherapy, and none were considered abnormalities related to Vax-NK / HCC.

[0117] Table 3 Blood abnormality cases All levels Level 3 anomaly (Hematologic adverse events) quantity(%) quantity(%) Anemia 5 (45.5) 1 (9.1) hyperkalemia (Hyperkalemia) 4 (36.4) 1 (9.1) Neutropenia (Neutropenia) 2 (18.2) 2 (18.2) Thrombocytopenia (Thrombocytopenia) 1 (9.1) 0 (0) Elevated creatinine (Increased creatinine) 1 (9.1) 0 (0) hypomagnesemia (Hypomagnesemia) 1 (9.1) 0 (0)

[0118] Table 4 Non-hematological abnormalities (Non-hematologic adverse events) All levels Level 3 anomaly quantity(%) quantity(%) Nausea 7 (63.6) 0 (0) Fatigue 3 (27.3) 2 (18.2) Rhinorrhea 2 (18.2) 0 (0) Ascites 1 (9.1) 0 (0) Pain 1 (9.1) 0 (0) Headache 1 (9.1) 0 (0) Myalgia (muscle pain) 1 (9.1) 0 (0) Vomiting 1 (9.1) 0 (0) Dizziness 1 (9.1) 0 (0)

[0119] In a clinical trial involving 11 enrolled patients, as shown in Table 5 below, the objective response rate (ORR) was 63.6% (7 patients), of which the complete response (CR) was 36.4% (4 patients) and the partial response (PR) was 27.3% (3 patients). Furthermore, stable disease (SD) occurred in 18.2% of the patients, and the disease control rate (DCR) was 81.8% (9 patients).

[0121] Table 5 Treatment response rate Number of patients (%) Complete reaction (CR) 4 (36.4) Partial response (PR) 3 (27.3) Stable disease (SD) 2 (18.2) Disease progression (PD) 2 (18.2) Objective Response Rate (ORR) 7 (63.6) Disease Control Rate (DCR) 9 (81.8)

[0122] Furthermore, as shown in Figure 3, the intermediate progression-free survival was 10.3 months (confidence interval: 6.8–13.7), and the intermediate overall survival was 41.6 months (95% confidence interval: 16.0–67.2). The progression-free survival rates at 12 and 24 months were 36.4% and 9.1%, respectively, and the overall survival rates at 12 and 24 months were 72.7% and 54.5%, respectively.

[0123] Figure 4 shows the changes in serum concentrations of alpha-fetoprotein (AFP) and PIVKA-II (vitamin K absence or antagonist-II), which are markers of liver cancer, before and after Vax-NK / HCC administration. The initial median values ​​for AFP and PIVKA-II were 305.8 IU / ml and 182 mAU / ml, respectively. After hepatic artery infusion chemotherapy and Vax-NK / HCC administration, the liver cancer markers decreased to 15.4 IU / ml and 49.0 mAU / ml, respectively.

[0125] Example 2

[0126] To evaluate the efficacy and safety of natural killer cells in patients who have received intrahepatic artery infusion therapy for cancer.

[0128] To evaluate the efficacy and safety of natural killer cells (Vax-NK / HCC) in patients who have received intra-arterial chemotherapy for cancer, the objective response rate (ORR) of patients with advanced hepatocellular carcinoma was assessed by mRECIST tumor assessment using independent review committees. Additionally, the objective response rate (ORR), disease control rate (DCR), duration of response (DOR), time to tumor progression (TTP), overall survival (OS), and quality of life (EORTC QLQ-C30) of patients with hepatocellular carcinoma were assessed by trial personnel.

[0129] When selecting patients for the above assessment, the selection criteria are as follows.

[0130] 1. Patients aged 19 years or older diagnosed with hepatocellular carcinoma

[0131] 2. Patients with Child-Pugh classification A (5-6 points) or upper B (including upper B up to 7 points)

[0132] 3. Patients with advanced hepatocellular carcinoma who are not surgically resectable or do not respond to local liver therapy and who have undergone two cycles of hepatic artery infusion chemotherapy (HAIC) and whose mRECIST assessment is equivalent to stable disease (SD), partial response (PR), or complete response (CR) (it is recommended that the overall minimum dose of HAIC for one cycle be ≥2000 mg / m² of 5-fluorouracil and ≥60 mg / m² of cisplatin, but may be reduced to the HAIC dose exceeding the permissible anticancer therapy (850 mg / m² of 5-fluorouracil and 35 mg / m² of cisplatin) at the discretion of the trial personnel).

[0133] 4. Patients who meet the following criteria

[0134] 1) Absolute neutrophil count (ANC) ≥ 1000 / μL, platelet count (PLTs) ≥ 50000 / μL, hemoglobin (Hb) ≥ 9g / dL

[0135] 2) Serum aspartate transaminase (AST) and alanine transaminase (ALT) ≤ 5 × upper limit of normal (ULN), serum creatinine ≤ 1.5 × ULN

[0136] 3) International normalized ratio (INR) < 1.7

[0137] 4) Patients who can take care of themselves in daily life (patients with a physical activity level of 0 or 1 according to the Eastern Cooperative Oncology Group (ECOG))

[0138] 5) Patients who have given written consent to participate in this clinical trial.

[0139] In the above criteria, the indications for hepatic artery infusion chemotherapy are patients who have not responded to two transarterial chemoembolization (TACE) treatments within 6 months, patients who have failed or are not eligible for one or two systemic therapies such as sorafenib, lenvatinib, regorafenib, nivolumab, cabozantinib, ramucirumab, atezolizumab, bevacizumab, etc., or patients with portal vein invasion.

[0140] Furthermore, patients meeting the following criteria were excluded during the assessment.

[0141] 1. Patients who received chemotherapy, radiotherapy, immunotherapy (monoclonal antibodies, cytokine therapy, etc.), hormone therapy, local tumor treatment, or targeted therapy within 4 weeks during the screening period.

[0142] 2. Patients with extrahepatic metastases

[0143] 3. Patients with a Child-Pugh classification of lower B or C.

[0144] 4. Patients who can be cured through local treatment

[0145] 5. Patients who have undergone liver transplantation

[0146] 6. Patients who have difficulty taking care of themselves in daily living (ECOG physical activity level ≥2)

[0147] 7. Patients who received immunotherapy within 6 months prior to screening.

[0148] 8. Patients diagnosed with or treated for other malignant tumors within the past 5 years.

[0149] 9. Patients with hypersensitivity to 5-fluorouracil and cisplatin

[0150] 10. Patients with varicose veins or non-therapeutic ulcers that pose a risk of bleeding during endoscopy, or patients who have observed bleeding ≥ 3 according to the National Cancer Institute (NCI) common terminology criteria for adverse events (CTCAE ver5.0) within 4 weeks prior to screening.

[0151] 11. Patients with severe or unmanageable cardiovascular disease

[0152] 12. Patients with active infections such as sepsis.

[0153] 13. Pregnant women, breastfeeding women, or women of childbearing age who are not using appropriate contraception.

[0154] 14. Patients with autoimmune diseases

[0155] 15. Patients deemed unsuitable by researchers to be subjects in clinical trials.

[0156] After two cycles of 5-fluorouracil and cisplatin-based hepatic artery infusion chemotherapy, Vax-NK / HCC candidates (complete response, partial response, stable lesions) were selected during the screening phase by mRECIST tumor assessment. The selected candidates received an additional two cycles of hepatic artery infusion chemotherapy (HAIC), and Vax-NK / HCC was prepared using the same method as in Preparation Example 1 after leukapheresis.

[0157] As shown in Figure 5, for subjects who completed a total of 4 cycles of hepatic artery infusion chemotherapy, Vax-NK / HCC administration was initiated three days later. Vax-NK / HCC administration was performed for a total of 2 cycles, with each cycle consisting of 4 weeks. Each cycle consisted of Vax-NK / HCC administered once daily for 5 consecutive days starting from day 1. However, the 2 cycles of Vax-NK / HCC administration were limited to subjects whose mRECIST tumor assessment results were stable or better at the end of one cycle (day 8 of the follow-up visit).

[0158] After the dosing period, disease progression (PD), efficacy, and safety will be monitored at 8-week intervals until the start of subsequent anticancer therapy or the completion of the entire clinical trial (6 months after the first dose of the last clinical trial drug for the trial subject). Survival will be tracked until the death of the trial subject or the completion of the entire clinical trial.

[0159] A phase II clinical trial of hepatic artery infusion chemotherapy combined with Vax-NK / HCC was conducted in patients who had failed or were ineligible for one or two prior systemic therapies, such as surgical resection, transarterial chemoembolization, or sorafenib. A total of 17 patients received 10 × 10⁸ doses of Vax-NK / HCC after four cycles of hepatic artery infusion chemotherapy. Results were presented at the AASLD and KSMO conferences in 2023. Of the 17 enrolled patients, 16 were included in the efficacy analysis group. Patient characteristics are shown in Table 6 below.

[0161] Table 6 characteristic Number of patients (%) Median age range 58.5 (42-75) gender male 15 (93.7) female 1 (6.3) ECOG physical activity level 0 3 (18.7) 1 13 (81.3) Child-Pugh classification A 15 (93.7) B 1 (6.3) BCLC Installment B 11 (68.7) C 5 (31.3) Pre-treatment Surgical removal + chemoembolization 3 (18.7) Surgical excision + high-frequency excision 1 (6.2) Surgical excision 1 (6.2) Surgical removal + bevacizumab + atezolizumab + high-frequency excision + mistletoe 1 (6.2) Chemoembolization + radiation therapy 1 (6.2) Chemoembolization + high-frequency ablation 1 (6.2) Chemoembolization + sorafenib 1 (6.2) Chemoembolization + Lenvatinib + Radiation Therapy 1 (6.2) Chemoembolization 4 (25) none 2 (12.5) Alpha-fetoprotein (AFP) ≥400 μg / L ≥400μg / L 3 (18.7) <400μg / L 13 (81.3)

[0162] The median age of the subjects enrolled in this clinical trial was 58.5 years (range 42-75), and all but one were male (93.7%). ECOG activity was 0 (18.7%) or 1 (81.3%), and Child-Pugh classification was A and B, respectively, at 93.7% and 6.3%. Eleven subjects (68.7%) had BCLC stage B, which is very similar to the characteristics of the subjects participating in Phase I clinical trials. Initial treatment was the same as in Phase I clinical trials, mostly surgical resection and chemoembolization, including three subjects who received systemic therapy such as sorafenib, lenvatinib, or bevacizumab + atezolizumab.

[0163] The purity, initiation receptor, and cytotoxicity of Vax-NK / HCC isolated and prepared from 16 registered subjects are shown in Table 7 below. The proportion of natural killer (CD3-CD56+) cells in the prepared Vax-NK / HCC was approximately 90%, while T (CD3+CD56-) cells, natural killer T (CD3+CD56+) cells, and other (CD3-CD56-) cells were less than approximately 7%, 5%, and 3%, respectively. Furthermore, it exhibited high cytotoxicity against K562.

[0165] Table 7 Patient number purity(%) Cell surface markers (%) Cytotoxicity CD3 - CD56 + (Natural killer cells (NK cells)) CD3 + CD56 - (T cells) CD3 + CD56 + (Natural killer T cells (NKT cells)) CD3 - CD56 - (Others) CD16 + CD69 + CD94 + NKG2D + 1 89.5 7.0 3.0 0.6 96.2 97.9 99.8 99.9 72.1 2 88.6 6.3 4.8 0.2 99.2 100.0 100.0 100.0 87.7 3 91.5 5.5 2.1 0.9 98.5 87.2 98.4 99.2 83.1 4 96.2 2.2 0.9 0.7 99.4 94.9 99.3 99.8 73.8 5 95.2 3.4 1.0 0.4 98.5 71.4 97.7 98.1 82.8 6 86.1 6.7 4.2 3.0 94.7 80.0 72.6 83.0 70.0 7 89.2 7.0 3.0 0.9 98.8 82.4 98.9 99.6 92.5 8 89.4 6.6 3.2 0.8 99.4 85.4 98.6 99.5 87.4 9 92.4 4.9 1.3 1.4 99.5 92.9 98.7 99.8 93.0 10 88.1 6.5 4.8 0.5 98.1 92.2 99.8 99.8 95.2 11 89.4 7.0 3.4 0.2 96.9 79.8 95.4 99.3 77.7 12 93.9 3.9 2.0 0.2 99.3 98.4 99.4 99.9 71.0 13 89.1 6.4 4.1 0.5 98.6 88.4 98.9 99.9 72.6 14 87.7 6.9 5.0 0.4 99.7 97.2 98.9 99.2 78.7 15 90.1 6.6 1.1 2.3 98.3 85.9 98.6 99.9 77.2 16 96.9 1.2 1.7 0.1 98.9 94.6 100.0 100.0 83.3

[0166] All 17 registered subjects were included in the safety analysis group. As shown in Tables 8 and 9 below, 134 abnormal events occurred in 15 of the 17 subjects after signing the consent form, of which 37 were grade 3 or higher abnormal events in 8 subjects. The most common hematologic toxicity-related abnormalities were thrombocytopenia (41.18%) and neutropenia (35.29%), while non-hematologic toxicity-related abnormalities included nausea (47.06%) and constipation (29.41%).

[0168] Table 8 Cases of blood abnormalities (Hematologic adverse events) All levels Level 3 or above quantity(%) quantity(%) Thrombocytopenia (Platelet count decreased) 7 (41.18) 5 (29.41) Neutrophil count decreased 6 (35.29) 6 (35.29) Leukopenia (decreased white blood cell count) 4 (25.53) 3 (17.65) The international normalized ratio increased. 2 (11.76) 0 0 Elevated blood bilirubin levels 2 (11.76) 1 (5.88) hypomagnesemia (Hypomagnesaemia) 2 (11.76) 1 (5.88) Elevated aspartate aminotransferase (AST) 1 (5.88) 0 0 Decreased red blood cell count 1 (5.88) 0 0 hypoalbuminemia (Hypoalbuminaemia) 1 (5.88) 0 0 Hyperglycemia 1 (5.88) 1 (5.88) Hyperkalemia 1 (5.88) 0 0 hypocalcemia (Hypocalcaemia) 1 (5.88) 0 0 Hypoglycemia 1 (5.88) 0 0 hyponatremia (Hyponatraemia) 1 (5.88) 0 0 hypophosphatemia (Hypophosphataemia) 1 (5.88) 1 (5.88)

[0169] Table 9 Non-hematological abnormalities (Non-hematologic adverse events) All levels Level 3 and above quantity(%) quantity(%) nausea (Nausea) 8 (47.06) 0 0 Loss of appetite (Decreased appetite) 6 (35.29) 1 (5.88) Constipation 5 (29.41) 0 0 stomach ache (Abdominal pain) 4 (23.53) 0 0 indigestion (Dyspepsia) 4 (23.53) 0 0 Headache 4 (23.53) 0 0 Chills 3 (17.65) 0 0 Fever (Pyrexia) 3 (17.65) 0 0 Anemia 3 (17.65) 3 (17.65) Vomiting 2 (11.76) 0 0 Dizziness 2 (11.76) 1 (5.88) Hepatic encephalopathy (Hepatic encephalopathy) 2 (11.76) 2 (11.76) Pain in the limbs (Pain in extremity) 2 (11.76) 0 0 Pruritus 2 (11.76) 0 0 Coronavirus disease (COVID-19) 2 (11.76) 0 0 upper abdominal pain (Abdominal pain in the upper body) 1 (5.88) 0 0 bloating (Abdominal distension) 1 (5.88) 0 0 ascites 1 (5.88) 0 0 Diarrhea (Diarrhoea) 1 (5.88) 0 0 upper abdominal discomfort (Epigastric discomfort) 1 (5.88) 0 0 Periodontal disease (Periodontal disease) 1 (5.88) 0 0 Portal hypertensive gastropathy 1 (5.88) 1 (5.88) Weight gain (Weight increased) 1 (5.88) 0 0 Catheter site hemorrhage 1 (5.88) 0 0 Pain 1 (5.88) 0 0 Back pain 1 (5.88) 0 0 Flank pain 1 (5.88) 0 0 Muscle weakness (Muscular weakness) 1 (5.88) 0 0 Iron deficiency anemia (Iron deficiency anaemia) 1 (5.88) 1 (5.88) Rash 1 (5.88) 0 0 Bloodshot eyes (Ocular hyperaemia) 1 (5.88) 0 0 Blurred vision (Vision blurred) 1 (5.88) 0 0 atrial fibrillation (Atrial fibrillation) 1 (5.88) 0 0 Jaundice 1 (5.88) 0 0 Traffic accidents (Road traffic accident) 1 (5.88) 1 (5.88) Insomnia 1 (5.88) 0 0 Male breast development (Gynaecomastia) 1 (5.88) 0 0 Difficulty breathing (Dyspnoea) 1 (5.88) 0 0 Hyperaemia 1 (5.88) 0 0

[0170] As shown in Table 10 below, all major abnormal events occurring after the consent form were grade 3 or higher, occurring in 7 out of 5 of the 17 subjects. These were hepatic encephalopathy (11.76%), dizziness, neutropenia, leukopenia, portal hypertensive gastropathy, and traffic accidents, each occurring in 5.88% of the cases. However, none of the major abnormal events reported in the relevant studies were associated with Vax-NK / HCC.

[0172] Table 10 Significant anomalies Level 3 and above (Serious adverse events) quantity(%) Hepatic encephalopathy 2 (11.76) Dizziness 1 (5.88) Neutrophil count decreased 1 (5.88) Leukopenia (decreased white blood cell count) 1 (5.88) Portal hypertensive gastropathy 1 (5.88) Traffic accident 1 (5.88)

[0173] As shown in Table 11 below, the objective response rates judged by the independent review committee and the trial personnel for the 16 subjects included in the efficacy analysis were 68.7% and 62.5%, respectively, with a disease control rate of 100% for both. This is almost consistent with the objective response rate of 63.6% in the Phase I clinical trial with 11 subjects. In particular, in the Phase II clinical trial, the objective response rate judged by the independent review committee was used as a primary assessment indicator for objective imaging evaluation, and the result was almost consistent with the objective response rate judged by the trial personnel.

[0174] Table 11 Treatment response rate Number of patients (%) Independent review committee judgment Test personnel judge Best Overall Response (BOR) Complete reaction (CR) 3 (18.7) 6 (37.5) Partial response (PR) 8 (50) 4 (25) Stable disease (SD) 5 (31.2) 6 (37.5) Disease progression (PD) 0 0 Objective Response Rate (ORR) 11 (68.7) 10 (62.5) Disease Control Rate (DCR) 16 (100) 16 (100)

[0175] An analysis was conducted to confirm the duration of response (DOR) for all subjects who showed a treatment response, and the results are shown in Figure 6. The intermediate response duration (mDOR) judged by the independent review committee and the trial personnel was 12.65 months (95% confidence interval: 4.60–22.21) and 10.48 months (95% confidence interval: 4.85–NR), respectively. Furthermore, as shown in Figure 7, the time to intermediate tumor (mTTP) judged by the independent review committee and the trial personnel was NR (95% confidence interval: 6.80–NR) and 16.82 months (95% confidence interval: 6.70–18.46), respectively. This is an improvement compared to the mPFS of 10.3 months for stage I hepatocellular carcinoma, suggesting that an additional cycle of Vax-NK / HCC administration may be associated with treatment efficacy in stage II hepatocellular carcinoma compared to stage I.

[0176] Furthermore, as shown in Figure 8, the median overall survival (mOS) of the 16 subjects included in the efficacy analysis group was 24.05 months (95% confidence interval: 17.15-NR). Additionally, as shown in Figure 9, one of the subjects with complete response (CR) was shown to have shown a complete response from the imaging assessment in March 2020 (baseline) until June 2023. [Simplified Explanation of the Diagram]

[0036] Figure 1 is a schematic diagram of the preparation process of the natural killer cell composition (Vax-NK / HCC) of the present invention.

[0037] Figure 2 is a schematic diagram of the treatment regimen for Phase I clinical Vax-NK / HCC.

[0038] Figure 3 is a graph showing the results of progression-free survival (PFS) and overall survival (OS) analysis using the Kaplan-Meier method in Phase I clinical trials.

[0039] Figure 4 is a graph showing the changes in AFP (part A) and PIVKA-II (part B) during or after Vax-NK / HCC treatment.

[0040] Figure 5 is a schematic diagram of the treatment regimen for Vax-NK / HCC in clinical stage II.

[0041] Figure 6 is a Kaplan-Meier plot of the independent review committee’s judgment (Part A) and the trial personnel’s judgment (Part B) on the duration of response (DOR) after HAIC and Vax-NK / HCC treatment.

[0042] Figure 7 shows the Kaplan-Mel curves of the independent review committee's judgment (Part A) and the trial personnel's judgment (Part B) on time to tumor progression (TTP) after HAIC and Vax-NK / HCC treatment.

[0043] Figure 8 is a Kaplan-Mel curve of overall survival (OS) after HAIC and Vax-NK / HCC treatment.

[0044] Figure 9 shows images of patients who have received Vax-NK / HCC treatment.

Claims

1. A method for preparing isolated natural killer cells, comprising: Step (a): Isolate peripheral blood mononuclear cells from liver cancer patients; Step (b) involves mixing the isolated peripheral blood mononuclear cells with the K562 cell line and culturing the mixture in a medium supplemented with IL-2, IL-21 and 4-1BBL; and step (c) involves culturing the peripheral blood mononuclear cells cultured in step (b) in the presence of IL-2 and IL-15.

2. The method for preparing isolated natural killer cells as described in claim 1, wherein, The K562 cell line mentioned above was irradiated with 100 Gy of γ rays.

3. The method for preparing isolated natural killer cells as described in claim 1, wherein, The IL-2 concentration in step (b) is 10 U / mL to 30 U / mL.

4. The method for preparing isolated natural killer cells as described in claim 1, wherein, The IL-2 concentration in step (b) is 10 U / mL.

5. The method for preparing isolated natural killer cells as claimed in claim 1, wherein, The concentration of IL-21 in step (b) is 5 ng / mL.

6. The method for preparing isolated natural killer cells as claimed in claim 1, wherein, The concentration of 4-1BBL in step (b) is 10 ng / mL.

7. The method for preparing isolated natural killer cells as claimed in claim 1, wherein, In step (b), culture for 7 days.

8. The method for preparing isolated natural killer cells as claimed in claim 1, wherein, The IL-2 concentration in step (c) is 100 U / mL to 1000 U / mL.

9. The method for preparing isolated natural killer cells as claimed in claim 1, wherein, The IL-2 concentration in step (c) is 100 U / mL.

10. The method for preparing isolated natural killer cells as claimed in claim 1, wherein, The IL-15 concentration in step (c) is 10 U / mL.

11. The method for preparing isolated natural killer cells as claimed in claim 1, wherein, Culture for 12 days in step (c).

12. A composition for treating liver cancer, wherein, The composition comprises natural killer cells prepared by the method described in claim 1, and the composition is administered once daily for 5 consecutive days.

13. The composition for treating liver cancer as claimed in claim 12, wherein, Four weeks after the start of the above-mentioned administration, a second dose was administered once daily for five consecutive days.

14. The composition for treating liver cancer as claimed in claim 12, wherein, The above composition improves objective response rate (ORR), disease control rate (DCR), duration of response (DOR), time to tumor progression (TTP), overall survival (OS), or quality of life (EORTC QLQ-C30).

15. The composition for treating liver cancer as claimed in claim 12, wherein, The above composition contains 2.5 × 10⁸ to 12 × 10⁸ natural killer cells.

16. The composition for treating liver cancer as claimed in claim 12, wherein, The above composition contains 8 × 10⁸ to 12 × 10⁸ natural killer cells.

17. The composition for treating liver cancer as claimed in claim 12, wherein, The above composition contains 10 × 10⁸ natural killer cells.

18. The composition for treating liver cancer as claimed in claim 12, wherein, The above composition was administered to patients who had received hepatic artery infusion chemotherapy.

19. The composition for treating liver cancer as claimed in claim 18, wherein, The aforementioned hepatic artery infusion chemotherapy consists of cisplatin, 5-fluorouracil, or a combination thereof.

20. The composition for treating liver cancer as claimed in claim 12, wherein, The liver cancer mentioned above is hepatocellular carcinoma.

21. A composition for treating liver cancer, wherein, The composition comprises natural killer cells prepared by the method described in claim 1, and the composition has a concentration of 1×10⁹ cells ± 20% per 500 mL.

22. The composition of claim 21, wherein, The composition was prepared by suspending the natural killer cells in 10 mL of human serum albumin and 490 mL of Hartmann's solution.

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