Novel method for preparing natural killer cells and method for treating liver cancer by using same

Abstract

The present invention relates to a novel method for preparing natural killer cells and a method for treating liver cancer by using the natural killer cells obtained therefrom. Specifically, the present invention provides the novel preparation method enabling the mass-culturing of natural killer cells in a short period of time, wherein the natural killer cells prepared using the method have high anticancer efficacy, and thus can be used as an anticancer therapy.

Description

A novel method for producing natural killer cells and a method for treating liver cancer using the same.

[0001] The present invention relates to a novel method for producing natural killer cells and a method for treating liver cancer using the natural killer cells obtained thereby. Specifically, the present invention provides a novel method for producing natural killer cells capable of culturing large quantities in a short period of time, and the natural killer cells produced by the method can be used as an anticancer therapy due to their high anticancer efficacy.

[0002]

[0003] Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide. Treatment for HCC is generally determined by tumor stage. In Korea, hepatic arterial infusion chemotherapy (HAIC) is particularly effective for the treatment of locally advanced HCC.

[0004] Because intra-arterial chemotherapy (IAC) delivers drugs through the hepatic artery, it allows direct drug delivery to tumor cells and can result in higher efficacy and lower toxicity compared to systemic therapy. 5-Fluorouracil (5-FU) and cisplatin are the most commonly used chemotherapeutic agents in IAC, and they have been reported to have immunomodulatory effects as potent inducers of natural killer cell activity.

[0005] Natural killer cells (NK cells) are essential components of the liver's innate immune system, accounting for 30-50% of intrahepatic lymphocytes. Significant reductions in the number and function of NK cells have been previously reported in patients with hepatocellular carcinoma (HCC). Specifically, a decrease in tumor-infiltrating NK cells has been associated with poor survival in advanced HCC. Thus, intrahepatic NK cells play a crucial role in immune surveillance of HCC. Therefore, overcoming NK cell dysfunction and restoring NK cell activity are crucial for the successful treatment of HCC.

[0006] NK cells play an important role in the immune system, but in cancer patients, the proportion of NK cells among peripheral blood lymphocytes is significantly reduced, so there are limitations in overcoming NK cell dysfunction and inducing recovery of activity without a separate culture and activation process.

[0007] Accordingly, the present inventors established a novel method for producing NK cells by culturing NK cells in large quantities and finding a combination of cytokines that can restore their activity, and completed the present invention by finding a novel treatment method that can effectively treat hepatocellular carcinoma by inducing NK cell activity through intrahepatic artery chemotherapy and then injecting NK cells produced by the novel production method.

[0008]

[0009] The purpose of the present invention is to provide a method for producing natural killer cells that can rapidly proliferate in vitro and are active against tumors.

[0010] The purpose of the present invention is to provide a composition containing the above natural killer cells.

[0011] The present invention aims to provide a method for treating a cancer subject, comprising a step of administering the above natural killer cells.

[0012]

[0013] The present invention comprises the steps of (a) isolating peripheral blood mononuclear cells from a liver cancer patient;

[0014] (b) culturing the isolated peripheral blood mononuclear cells in the presence of IL-2, IL-21, and 4-1BBL and K562 cell lines; and

[0015] (c) Provided is a method for producing isolated natural killer cells, comprising a step of culturing isolated peripheral blood mononuclear cells in the presence of IL-2 and IL-15.

[0016] In one embodiment, the K562 cell line may be irradiated with 100 Gy gamma rays.

[0017] In one embodiment, the IL-2 concentration of step (b) may be from 10 U / mL to 30 U / mL.

[0018] In one embodiment, the IL-2 concentration in step (b) may be 10 U / mL.

[0019] In one embodiment, the IL-21 concentration in step (b) may be 5 ng / mL.

[0020] In one embodiment, the 4-1BBL concentration of step (b) may be 10 ng / mL.

[0021] In one embodiment, the incubation period of step (b) may be up to 7 days.

[0022] In one embodiment, the IL-2 concentration of step (c) can be from 100 U / mL to 1000 U / mL.

[0023] In one embodiment, the IL-2 concentration in step (c) may be 100 U / mL.

[0024] In one embodiment, the IL-15 concentration in step (c) may be 10 U / mL.

[0025] In one embodiment, the incubation period of step (c) may be up to 12 days.

[0026] The present invention provides a composition for treating liver cancer, which comprises a composition comprising natural killer cells manufactured by the above manufacturing method, administered once a day for 5 consecutive days.

[0027] In one embodiment, the second administration may be administered once a day for 5 consecutive days 4 weeks after the start of the administration.

[0028] In one embodiment, the composition may 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).

[0029] In one embodiment, the composition comprises 2.5 Х 10 8 Inside 12 Х 10 8 It may contain natural killer cells of dogs, preferably 8 Х 10 8 Inside 12 Х 10 8 It may contain natural killer cells of dogs, more preferably 10 Х 10 8 It may contain natural killer cells of the dog.

[0030] In one embodiment, the composition may be administered to a patient undergoing intraarterial chemotherapy.

[0031] In one embodiment, the intraarterial chemotherapy may be cisplatin, 5-FU, or a combination thereof.

[0032] In one embodiment, the liver cancer may be hepatocellular carcinoma.

[0033] The present invention provides a method for treating a liver cancer patient by administering a composition containing natural killer cells manufactured by the above manufacturing method to the liver cancer patient once a day for 5 consecutive days.

[0034] In one embodiment, the second administration may be administered once a day for 5 consecutive days 4 weeks after the start of the administration.

[0035] In one embodiment, the treatment may 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).

[0036] In one embodiment, the composition comprises 2.5 Х 10 8 Inside 12 Х 10 8 It may contain natural killer cells of dogs, preferably 8 Х 10 8 Inside 12 Х 10 8 It may contain natural killer cells of dogs, more preferably 10 Х 10 8 It may contain natural killer cells of the dog.

[0037] In one embodiment, the patient may be a patient who has received intraarterial chemotherapy.

[0038] In one embodiment, the liver cancer may be hepatocellular carcinoma.

[0039]

[0040] The novel method for producing natural killer cells of the present invention enables the mass proliferation and culture of natural killer cells from peripheral blood mononuclear cells in a relatively short period of time. Furthermore, natural killer cells obtained through this method exhibit a high response rate in patients with hepatocellular carcinoma, making them an effective liver cancer treatment.

[0041]

[0042] Figure 1 is a schematic diagram of the manufacturing process of the natural killer cell composition (Vax-NK / HCC) of the present invention.

[0043] Figure 2 is a schematic diagram of the treatment schedule for phase 1 clinical trial Vax-NK / HCC.

[0044] 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 1 clinical trials.

[0045] Figure 4 is a graph showing the changes in (A) AFP and (B) PIVKA-II during or after Vax-NK / HCC treatment.

[0046] Figure 5 is a schematic diagram of the treatment schedule for phase 2 clinical trial Vax-NK / HCC.

[0047] Figure 6 is a Kaplan Meier Plot graph of A) Independent Review Committee judgment and B) Investigator judgment for duration of response (DOR) after HAIC, Vax-NK / HCC treatment.

[0048] Figure 7 is a Kaplan-Meier Plot graph of A) Independent Review Committee judgment and B) Investigator judgment for time to tumor progression (TTP) after HAIC, Vax-NK / HCC treatment.

[0049] Figure 8 is a Kaplan Meier Plot graph for overall survival (OS) after HAIC and Vax-NK / HCC treatment.

[0050] Figure 9 is an image of a patient who received Vax-NK / HCC treatment.

[0051]

[0052] Hereinafter, the present invention and embodiments will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various forms and is not limited to the embodiments and embodiments described herein.

[0053] The term "peripheral blood mononuclear cells" as used herein, also referred to as "PBMCs" or "peripheral blood monocytes", typically refers to mononuclear cells isolated from peripheral blood. Peripheral blood mononuclear cells can be obtained using a known method appropriately selected by a person skilled in the art, such as the Ficoll-Hypaque density gradient method or the Lymphoprep density gradient method.

[0054] The term “proliferation” as used herein means an increase in the number of cells.

[0055] The term "natural killer cell" as used herein, also referred to as NK cell, refers to a cell that morphologically has granules in its cytoplasm and can eliminate tumor cells or virus-infected cells.

[0056] The term "cytokine" as used herein refers to an immune activating cytokine that can be used to induce differentiation of peripheral blood mononuclear cells into natural killer cells, and may preferably be IL-2, IL-15, IL-21, 4-1BBL or a combination thereof.

[0057] The term "trophoblast" as used herein refers to a cell that cannot proliferate but is capable of metabolism and produces metabolic substances to help other cells proliferate, and may preferably be K562.

[0058] The term "K562" as used herein refers to cells derived from lymphoblast cells of the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia, and is available from ATCC (ATCC CCL-243).

[0059] The term "objective response rate (ORR)" as used herein refers to the proportion of patients who exhibit tumor reduction of a predefined amount or greater.

[0060] The term "disease control rate (DCR)" as used herein refers to the proportion of patients who achieved a complete response, a partial response, or no progression of disease.

[0061] The term "duration of response (DOR)" as used herein refers to the duration of response during which the size of the tumor is maintained.

[0062] The term "time to tumor progression (TTP)" as used herein means the period of time it takes for a tumor to increase in size or for death to occur.

[0063] The term “overall survival (OS)” as used herein means the period from the start of treatment to death.

[0064] The term “quality of life (EORTC QLQ-C30)” as used herein means the quantified quality of life assessed by the EORTC QLQ-C30.

[0065] Natural killer cells according to the present invention can be administered via any conventional route to reach the target tissue. For example, they can be administered intra-hepatic, but are not limited thereto.

[0066] The term “intra-arterial administration” as used herein refers to a method of administering a drug directly into the hepatic artery.

[0067] The term "transhepatic arterial infusion chemotherapy" or "intrahepatic arterial chemotherapy" used herein means a therapy that administers an anticancer drug through intrahepatic artery administration, and the anticancer drug may preferably be 5-fluorouracil (5-FU) and cisplatin.

[0068] The term “combination administration” as used herein means administering two or more types of active ingredients simultaneously or sequentially.

[0069] The natural killer cells of the present invention may be administered in combination with other anticancer agents appropriately selected by those skilled in the art. For example, they may be administered in combination with immune checkpoint inhibitors, but are not limited thereto.

[0070] The term "prevention" as used herein means any action that inhibits or delays the onset of a disease by administering a composition, and "treatment" means any action that improves or beneficially changes the symptoms of a subject suspected of or suffering from a disease by administering a composition.

[0071]

[0072] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0073]

[0074] [Manufacturing Example 1]

[0075] Ex vivo natural killer cell (Vax-NK / HCC) production

[0076]

[0077] In vitro natural killer cells were prepared as shown in Fig. 1, and specifically, as follows.

[0078] The patient's blood was dispensed into 50 mL tubes (15 mL each) and diluted with 30 mL of 1X PBS. Peripheral blood mononuclear cells were isolated using density gradient centrifugation (20°C, 1200 g, 25 min) using Lymphoprep (Serumwerk Bernburg AG, Bernburg, Germany). Specifically, after removing the supernatant, the cell layer was collected in 50 mL tubes (less than 15 mL), suspended in 1X PBS (less than 45 mL in total), and centrifuged (20°C, 500 g, 5 min). The supernatant was removed, suspended in medium (RPMI1640, 10% FBS, 4 mM L-Glutamine), and centrifuged (20°C, 500 g, 5 min). Afterwards, the cell number was measured using a hematocytometer, and peripheral blood mononuclear cells were collected. For subsequent production, some peripheral blood mononuclear cells were centrifuged (20℃ 500g, 5 min) and suspended in cryopreservation solution (50% FBS, 40% RPMI1640, 10% DMSO) to produce 2x10 8 Cells / mL were dispensed into vials for cryopreservation. Afterwards, frozen cells for the second method were dispensed into plastic vials for cryopreservation (Cryogenic vials), stored in a cell freezing container, and frozen by storing in a freezer below -70℃ for more than 2 hours. The vials after freezing were transferred to an LN2 tank and stored at below -150℃.

[0079] K562 (ATCC, USA), a feeder cell line for NK cell culture, was transferred to a 50-mL tube and centrifuged (20°C, 500 g, 5 min). The harvested cell line was irradiated with 100 Gy of gamma rays using an irradiator. After irradiation, the cells were washed twice with medium (RPMI1640, 10% FBS, 4 mM L-Glutamine).

[0080] Afterwards, the isolated peripheral blood mononuclear cells were mixed with 100 Gy irradiated trophoblast cell lines at a ratio of 6:1 and cultured in 20 mL of complete medium (RPMI1640, 10% FBS, 4 mM L-Glutamine). IL-2 (10 U / mL), IL-21 (5 ng / mL), and 4-1BBL (10 ng / mL) were added to promote the proliferation of natural killer cells, and the medium was dispensed into T75 flasks or half of the medium was replaced and cultured for 7 days in a 5% CO2 incubator. The medium (RPMI1640, 10% FBS, 4 mM L-Glutamine) was dispensed and replaced half of the medium at approximately 2-day intervals and cultured from day 0 to day 7.

[0081] On the 7th day of culture, the cultured natural killer cells were divided into two T75 flasks and transferred, and new medium (RPMI1640, 10% FBS, 4 mM L-Glutamine) was added to make 50 mL per flask. After adding 100 U / mL of IL-2 and 10 U / mL of IL-15, the cells were cultured in a 5% CO2 incubator. The medium (RPMI1640, 10% FBS, 4 mM L-Glutamine) was added in portions and replaced by half every two days, and the cells were cultured for 11 days.

[0082] On the 11th to 13th day of culture, a certain amount of medium was harvested from each T75 flask and transferred to a 50 mL tube, and cytokine residual test, DMSO content test, mycoplasma negativity test, sterility test, and adventitious virus negativity test were performed.

[0083] On the 14th to 18th day of culture, natural killer cells were collected from the designated culture vessel, transferred to a 50 mL tube, and centrifuged (20℃, 500g, 5 min). After removing the supernatant, the cells were suspended with Hartmann's solution, washed once, and centrifuged (20℃, 500g, 5 min). After removing the supernatant, the cells were washed once more with Hartmann's solution, the supernatant was removed, Hartmann's solution was added, the cell number was counted, and centrifuged (20℃, 500g, 5 min). 10 mL of human serum albumin (albumin 20% 100 mL / vial) and 490 mL of Hartmann's solution were suspended and added to Hartmann's solution at a ratio of 1X10 9 After dispensing cells ± 20% / 500mL, they were stored in the refrigerator.

[0084]

[0085] [Example 1]

[0086] Safety evaluation of natural killer cells in patients receiving intra-arterial chemotherapy

[0087]

[0088] The safety of natural killer cells in patients receiving intra-arterial chemotherapy was evaluated as follows.

[0089] The natural killer cell composition (Vax-NK / HCC) manufactured by the method of Manufacturing Example 1 above was administered to patients selected based on the following criteria.

[0090] 1) Patients aged 18 years or older diagnosed with hepatocellular carcinoma

[0091] 2) Patients with Child-Pugh class A (Points 5-6) or upper B (up to Point 7 is included as upper B)

[0092] 3) Patients who achieved SD, PR, or CR in the mRECIST evaluation after completing two cycles of intra-arterial chemotherapy administered at the advanced stage of liver cancer

[0093] 4) Patients for whom surgical resection is not possible

[0094] 5) Patients who do not respond to transarterial chemoembolization

[0095] 6) Patients with multiple intrahepatic tumors or vascular invasion who are expected not to respond to sorafenib

[0096] 7) Patients whose organ function is maintained as neutrophil count (ANC) ≥ 1,500 / μl, platelets (PLTs) ≥ 75,000 / μl, hemoglobin (Hb) ≥ 9 g / dL, serum AST and ALT ≤ 5 x ULN, serum creatinine ≤ 1.5 x ULN, INR < 1.7 or PT < 4 seconds, serum albumin > 3.5 g / dL, and serum bilirubin < 2 mg / dL.

[0097] 8) Those who are able to perform daily activities (patients with ECOG physical activity level of 0 or 1)

[0098] 9) A patient or a patient representative who has given written consent to participate in this clinical trial.

[0099] Additionally, those who met the following criteria were excluded from the evaluation.

[0100] 1) Those who received chemotherapy, radiotherapy, immunotherapy, hormone therapy, local tumor treatment, or targeted therapy within 4 weeks of the screening period.

[0101] 2) Patients with Child-Pugh class B or C

[0102] 3) Patients who can be cured with local treatment

[0103] 4) Patients who have undergone liver transplantation

[0104] 5) Patients with varices or non-healing ulcers at risk of bleeding on endoscopy, or patients with bleeding of NCI CTCAE grade ≥ 3 observed within 4 weeks of screening.

[0105] 6) Patients with difficulty performing daily activities (ECOG physical activity level ≥ 2)

[0106] 7) Patients who received immunotherapy 6 months prior to recruitment

[0107] 8) Patients with other malignant tumors diagnosed or treated within the past 5 years

[0108] 9) Patients with hypersensitivity to 5-FU and Cisplatin

[0109] 10) Patients with severe or uncontrolled cardiovascular disease (ejection fraction < 0.5 or severe arrhythmia)

[0110] 11) Patients with active infections such as sepsis

[0111] 12) Pregnant or lactating women or women of childbearing potential who are not using appropriate contraception

[0112] 13) Patients with autoimmune diseases

[0113] 14) Patients deemed unsuitable as clinical trial subjects by the researcher

[0114] Among the patients selected through the above criteria, those who had a response level of SD or higher by mRECIST evaluation after completing two cycles of hepatic arterial infusion chemotherapy using 5-FU and cisplatin before initiation of Vax-NK / HCC administration were selected as candidates for Vax-NK / HCC administration. After completing the third administration, the selected patients underwent leukapheresis, and natural killer cells (Vax-NK / HCC) were cultured for 14 days and injected at daily intervals the day after the end of four cycles of hepatic arterial infusion chemotherapy, for a total of five days. Hepatic arterial infusion chemotherapy consists of four consecutive days of injection of 5-FU and cisplatin followed by a three-week rest period, and these drugs are slowly infused over two hours through a port connected to a catheter inserted into the hepatic artery. The phase 1 Vax-NK / HCC treatment schedule was set up as shown in Figure 2.

[0115] A phase 1 clinical trial was conducted on patients with advanced liver cancer who were not suitable for or failed surgical resection, transarterial chemoembolization, or sorafenib treatment, and on the combination of hepatic arterial infusion chemotherapy and Vax-NK / HCC. After four cycles of hepatic arterial infusion chemotherapy (5-fluorouracil, cisplatin), patients received 2.5 х 10 cycles of Vax-NK / HCC. 8 , 5 Х 10 8 , 10 Х 10 8 The cell dose was administered once daily for five consecutive days for a total of five doses to three, three, and five subjects, respectively. The results were published in the June 2022 issue of Frontiers in Immunology. The median age of the subjects enrolled in this clinical trial was 56.6 years (range, 43-71 years), and two subjects were aged 65 years or older. The characteristics of the clinical trial subjects are shown in Table 1 below.

[0116]

[0117]

[0118] The majority of enrolled subjects were male (90.9%) and classified as Child-Pugh A (81.8%). The proportions of BCLC stages B and C were similar (54.5% and 45.5%). Eight subjects (72.7%) had multinodular HCC, and three subjects (27.3%) had invasive HCC. In addition, seven subjects (63.7%) had large tumors measuring 5 cm or more in diameter. The most common prior treatments received by the subjects were surgical resection and chemoembolization. Hepatitis B was diagnosed in 54.5% of the subjects, and hepatitis C was diagnosed in 27.3%.

[0119] The purity, activating receptor, and cytotoxic function of Vax-NK / HCC isolated from 11 enrolled subjects are shown in Table 2 below. Specifically, NK (CD3) in the manufactured Vax-NK / HCC - CD56 +) cells reached about 80-90%, and T (CD3 + CD56 - ) cells, NKT (CD3 + CD56 + ) cells, and other (CD3 - CD56 - ) cells were approximately 7%, 5%, and 3%, respectively. NK cells also expressed high levels of activating receptors such as CD16, CD69, CD94, and NKG2D. Their function was evaluated by cytotoxicity against K562, and they exhibited at least 70% cytotoxicity. Therefore, it was confirmed that the manufactured Vax-NK / HCC were highly pure and activated NK cells.

[0120]

[0121]

[0122] Hematological and non-hematological toxicities are shown in Tables 3 and 4 below. Specifically, the most common hematological toxicity after transarterial chemotherapy and Vax-NK / HCC administration was anemia (45.5%, 5 patients), of which one subject had grade 3 or higher. Neutropenia occurred in two subjects (18.2%), corresponding to grades 3 and 4, respectively, but recovered after appropriate treatment with granulocyte colony-stimulating factor (G-CSF). In addition, one subject developed grade 2 thrombocytopenia, and some subjects also experienced hematological adverse events (hyperkalemia, hypomagnesemia, and increased creatinine). On the other hand, non-hematological toxicities observed during the clinical study included nausea (63.6%), fatigue (27.3%), and rhinorrhea (18.2%). However, all drug-related adverse events were related to intraarterial chemotherapy, and none of them were considered Vax-NK / HCC-related.

[0123]

[0124]

[0125] In a clinical trial involving 11 enrolled patients, the objective response rate (ORR) was 63.6% (7 patients), as shown in Table 5 below. Of these, complete response (CR) occurred in 36.4% (4 patients) and partial response (PR) occurred in 27.3% (3 patients). In addition, stable disease (SD) occurred in 18.2% of patients, resulting in a disease control rate (DCR) of 81.8% (9 patients).

[0126]

[0127]

[0128] Also, as shown in Figure 3, the median progression-free survival was 10.3 months (95% confidence interval: 6.8–13.7), and the median 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.

[0129] As shown in Fig. 4, before and after Vax-NK / HCC administration, the changes in blood concentrations of liver cancer markers AFP (α-fetoprotein) and PIVKA-II (vitamin K absence or antagonist-II) were examined. The median values ​​of AFP and PIVKA-II at the initial screening were 305.8 IU / ml and 182 mAU / ml, respectively. After hepatic arterial infusion chemotherapy and Vax-NK / HCC administration, the two liver cancer markers decreased to 15.4 IU / ml and 49.0 mAU / ml, respectively.

[0130]

[0131] [Example 2]

[0132] Evaluation of the efficacy and safety of natural killer cells in patients receiving intra-arterial chemotherapy.

[0133]

[0134] To evaluate the efficacy and safety of natural killer cells (Vax-NK / HCC) in patients receiving intra-arterial chemotherapy, the objective response rate (ORR) as judged by the independent review committee was evaluated for the combination of hepatic arterial chemotherapy and Vax-NK / HCC using mRECIST tumor assessment in patients with advanced hepatocellular carcinoma. In addition, 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) as judged by the investigators were evaluated for the combination of hepatic arterial chemotherapy and Vax-NK / HCC in patients with hepatocellular carcinoma.

[0135] When selecting patients for the above evaluation, the selection criteria are as follows.

[0136] 1. Patients aged 19 years or older diagnosed with hepatocellular carcinoma

[0137] 2. Child-Pugh class A (Points 5-6) or upper B (Points 7 and up are included as upper B)

[0138] 3. Patients with advanced liver cancer that is not amenable to surgical resection or refractory to local liver treatment who received 2 cycles of intra-arterial hepatic infusion chemotherapy (HAIC) and achieved stable disease (SD), partial response (PR), or complete response (CR) in the mRECIST evaluation [however, the minimum total dose per cycle of HAIC administration is 5-fluorouracil (5-FU) 2000 mg / m 2 and cisplatin 60 mg / m 2 Although recommended above, the dose of HAIC administered according to the off-label anticancer therapy (5-FU 850 mg / m) may be adjusted at the investigator's discretion. 2 with cisplatin 35 mg / m 2) can be reduced up to .]

[0139] 4. Patients who meet the following criteria:

[0140] 1) Absolute neutrophil count (ANC) ≥ 1,000 / μL, platelets (PLTs) ≥ 50,000 / μL, hemoglobin (Hb) ≥ 9 g / dL,

[0141] 2) Serum aspartate transaminase (AST) and alanine transaminase (ALT) ≤ 5 Х upper limit of normal (ULN), serum creatinine ≤ 1.5 Х ULN

[0142] 3) International normalized ratio (INR) < 1.7

[0143] 4) Those who are able to perform daily activities (patients with a physical activity level of 0 or 1 according to the Eastern Cooperative Oncology Group [ECOG])

[0144] 5) A patient or a patient representative who has given written consent to participate in this clinical trial.

[0145] Based on the above criteria, indications for performing transarterial chemotherapy include patients who have not responded to two transarterial chemoembolizations (TACE) within 6 months, patients who have failed or cannot use first-line and second-line systemic treatments such as sorafenib, lenvatinib, regorafenib, nivolumab, cabozantinib, ramucirumab, atezolizumab, and bevacizumab, or patients with portal vein invasion.

[0146] Additionally, those who met the following criteria were excluded from the evaluation.

[0147] 1. Those who received chemotherapy, radiotherapy, immunotherapy (monoclonal antibody, cytokine therapy, etc.), hormone therapy, local tumor treatment, or targeted therapy within 4 weeks prior to screening.

[0148] 2. Patients with extrahepatic metastases

[0149] 3. Patients with Child-Pugh class B or C

[0150] 4. Patients who can be cured with local treatment

[0151] 5. Patients who have undergone liver transplantation

[0152] 6. Patients with difficulty performing daily activities (ECOG physical activity level ≥ 2)

[0153] 7. Patients who received immunotherapy within 6 months prior to screening

[0154] 8. Patients with other malignant tumors diagnosed or treated within the past 5 years.

[0155] 9. Patients with hypersensitivity to 5-fluorouracil (5-FU) and cisplatin

[0156] 10. Patients with varices or non-healing ulcers at risk of bleeding on endoscopy, or patients with bleeding of grade ≥3 or higher according to the National Cancer Institute (NCI)-common terminology criteria for adverse events (CTCAE ver5.0) within 4 weeks prior to screening.

[0157] 11. Patients with severe or uncontrolled cardiovascular disease

[0158] 12. Patients with active infections such as sepsis

[0159] 13. Pregnant or lactating women or women of childbearing potential who are not using appropriate contraception

[0160] 14. Patients with autoimmune diseases

[0161] 15. Patients deemed unsuitable as clinical trial subjects by the investigator.

[0162] After two cycles of 5 FU, cisplatin hepatic artery infusion chemotherapy, subjects for Vax-NK / HCC administration were selected through mRECIST tumor assessment in the screening stage (complete response, partial response, stable lesion). The selected subjects received two additional cycles of hepatic artery infusion chemotherapy (HAIC) and leukapheresis, and Vax-NK / HCC was manufactured as in Manufacturing Example 1.

[0163] As shown in Figure 5, subjects who completed a total of 4 cycles of intraarterial chemotherapy began Vax-NK / HCC administration 3 days later. Vax-NK / HCC administration was performed for a total of 2 cycles, each cycle consisting of 4 weeks. Vax-NK / HCC was administered once daily for 5 consecutive days from the first day of each cycle. However, Vax-NK / HCC administration for the second cycle was performed only for subjects whose mRECIST tumor evaluation results at the time of completion of the first cycle (visit 8) were stable lesions or higher.

[0164] After completion of the dosing period, efficacy and safety follow-up was conducted at 8-week intervals until disease progression (PD), the start of subsequent chemotherapy, or the end of the entire trial (6 months after the first dose of the investigational drug in the last subject). Survival follow-up was conducted until the subject's death or the end of the entire trial.

[0165] A phase 2 clinical trial was conducted on patients who failed or could not use first-line or second-line systemic treatments such as surgical resection, transarterial chemoembolization, or sorafenib, and on the combination of hepatic arterial infusion chemotherapy and Vax-NK / HCC. After receiving four cycles of hepatic arterial infusion chemotherapy, 10 Х 10 8 A total of 17 patients were administered Vax-NK / HCC at a dose of 100 mg. The results were presented at the 2023 AASLD and KSMO conferences, respectively. Of the 17 enrolled subjects, 16 were included in the efficacy analysis group. Patient characteristics are presented in Table 6.

[0166]

[0167]

[0168] The median age of the subjects enrolled in this clinical trial was 58.5 years (range, 42-75), and all but one subject was male (93.7%). ECOG performance status was 0 (18.7%) or 1 (81.3%), and Child-Pugh A and B were 93.7% and 6.3%, respectively. BCLC stage B comprised 11 subjects (68.7%), which was very similar to the characteristics of the subjects who participated in the phase 1 clinical trial. As in the phase 1 clinical trial, most of the prior treatments included surgical resection and chemoembolization, and three subjects received systemic therapy such as sorafenib, lenvatinib, and bevacizumab + atezolizumab.

[0169] The purity, activating receptor, and cytotoxic function of Vax-NK / HCC isolated from 16 enrolled subjects are shown in Table 7 below. NK (CD3) in the manufactured Vax-NK / HCC - CD56 + ) cells accounted for approximately 90%, and T (CD3 + CD56 - ) cells, NKT (CD3 + CD56 + ) cells, and other (CD3 - CD56 - ) cells were less than about 7%, 5%, and 3%, respectively. In addition, they showed high cytotoxicity against K562.

[0170]

[0171]

[0172] All 17 enrolled subjects were included in the safety analysis group. As shown in Tables 8 and 9 below, 134 adverse events occurred in 15 of the 17 subjects after obtaining informed consent, of which 37 adverse events of grade 3 or higher occurred in 8 subjects. The most frequent adverse events related to hematological toxicity were thrombocytopenia (41.18%) and neutropenia (35.29%), while non-hematological toxicity-related adverse events included nausea (47.06%) and constipation (29.41%).

[0173] .

[0174]

[0175]

[0176]

[0177] As shown in Table 10 below, all serious adverse events that occurred after obtaining informed consent were grade 3 or higher, occurring in 7 cases in 5 of 17 subjects. Hepatic encephalopathy (11.76%), and dizziness, neutropenia, leukopenia, portal hypertension, and traffic accidents all occurred in 5.88% of subjects. However, none of the serious adverse events reported in the study were related to Vax-NK / HCC.

[0178]

[0179]

[0180] For the 16 subjects included in the efficacy analysis, the objective response rates determined by the independent review committee and the investigators were 68.7% and 62.5%, respectively, as shown in Table 11 below, and the disease control rate was 100% in both cases. This is almost identical to the objective response rate of 63.6% in the Phase 1 clinical trial involving 11 subjects. In particular, in the Phase 2 clinical trial, the objective response rate determined by the independent review committee was used as the primary endpoint for objective imaging evaluation, and this result was almost identical to the objective response rate determined by the investigators.

[0181]

[0182]

[0183] To determine the duration of response (DOR) for all subjects who showed a treatment response, an analysis was conducted, and the results are shown in Figure 6. The median DOR (mDOR) determined by the independent review committee and the investigators was 12.65 months (95% confidence interval: 4.60-22.21) and 10.48 months (95% confidence interval: 4.85-NR), respectively. In addition, as shown in Figure 7, the median time to tumor (mTTP) as judged by the independent review committee and investigators was NR (95% confidence interval: 6.80-NR) and 16.82 months (95% confidence interval: 6.70-18.46), respectively. This is an improved figure compared to the mPFS of 10.3 months in phase 1 liver cancer, and it is thought that it may be related to the therapeutic effect of administering one additional cycle of Vax-NK / HCC in phase 2 liver cancer compared to phase 1 liver cancer.

[0184] Additionally, the median overall survival (mOS) for the 16 subjects included in the efficacy analysis group was 24.05 months (95% confidence interval: 17.15-NR), as shown in Figure 8 . Additionally, one subject who achieved a complete response (CR) continued to demonstrate a CR from the imaging assessment (baseline) in March 2020 to June 2023, as shown in Figure 9 .

Claims

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

2. A method for producing isolated natural killer cells, wherein the K562 cell line is irradiated with 100 Gy of gamma rays in the first paragraph.

3. A method for producing isolated natural killer cells, characterized in that the IL-2 concentration in step (b) in paragraph 1 is 10 U / mL to 30 U / mL.

4. A method for producing isolated natural killer cells, characterized in that the IL-2 concentration in step (b) is 10 U / mL in the first paragraph.

5. A method for producing isolated natural killer cells, characterized in that the IL-21 concentration in step (b) is 5 ng / mL in the first paragraph.

6. A method for producing isolated natural killer cells, characterized in that the concentration of 4-1BBL in step (b) is 10 ng / mL in the first paragraph.

7. A method for producing isolated natural killer cells, characterized in that in step (b) in paragraph 1, the cells are cultured for up to 7 days.

8. A method for producing isolated natural killer cells, characterized in that the IL-2 concentration in step (c) in paragraph 1 is 100 U / mL to 1000 U / mL.

9. A method for producing isolated natural killer cells, characterized in that the IL-2 concentration in step (c) in paragraph 1 is 100 U / mL.

10. A method for producing isolated natural killer cells, characterized in that the IL-15 concentration in step (c) is 10 U / mL in the first paragraph.

11. A method for producing isolated natural killer cells, characterized in that in step (c) in paragraph 1, the cells are cultured for up to 12 days.

12. A composition for treating liver cancer, comprising a composition containing natural killer cells manufactured by the method of paragraph 1, administered once a day for 5 consecutive days.

13. A composition for treating liver cancer, wherein the second administration is administered once a day for 5 consecutive days, 4 weeks after the start of the administration in paragraph 12.

14. A composition for treating liver cancer, wherein the composition in claim 12 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. In the 12th paragraph, the composition is 2.5 Х 10 8 12 Х 10 8 A composition for treating liver cancer, comprising natural killer cells of a dog.

16. In the 12th paragraph, the composition is 8 Х 10 8 12 Х 10 8 A composition for treating liver cancer, comprising natural killer cells of a dog.

17. In the 12th paragraph, the composition is 10 Х 10 8 A composition for treating liver cancer, comprising natural killer cells of a dog.

18. A composition for treating liver cancer, wherein the composition is administered to a patient who has received intravenous hepatic artery infusion chemotherapy in paragraph 12.

19. A composition for treating liver cancer, wherein the intra-arterial chemotherapy in claim 18 is cisplatin, 5-FU, or a combination thereof.

20. A composition for treating liver cancer, wherein the liver cancer in claim 12 is hepatocellular carcinoma.

21. A method for treating a liver cancer patient by administering a composition containing natural killer cells prepared by the method of paragraph 1 to the patient once a day for 5 consecutive days.

22. A method for treating a patient with liver cancer by administering the second dose once a day for 5 consecutive days 4 weeks after the start of the administration in paragraph 21.

23. A method for treating a patient with liver cancer, wherein the method 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) in claim 21.

24. In the 21st paragraph, the composition is 2.5 Х 10 8 12 Х 10 8 A method for treating a patient with liver cancer, comprising natural killer cells of a dog.

25. In the 21st paragraph, the composition comprises 8 Х 10 8 12 Х 10 8 A method for treating a patient with liver cancer, comprising natural killer cells of a dog.

26. In the 21st paragraph, the composition is 10 Х 10 8 A method for treating a patient with liver cancer, comprising natural killer cells of a dog.

27. A method for treating a liver cancer patient in claim 21, wherein the patient is a patient who has received intravenous hepatic artery infusion chemotherapy.

28. A method for treating a liver cancer patient, wherein the liver cancer is hepatocellular carcinoma in claim 21.

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