CN111228472A - Method for improving gastric cancer by TIL immune cells - Google Patents

Abstract

The invention discloses a method for improving gastric cancer by TIL immune cells, which comprises the steps of firstly removing tumors from a patient body, transporting tumor tissues to a GMP laboratory under the sterile and cold chain conditions, and separating to obtain initial tumor infiltrating lymphocytes; culturing the initial tumor infiltrating lymphocytes under proper conditions, amplifying by ten thousand times, further purifying, and freezing; recovering and expanding tumor infiltrating lymphocyte to 8 × 10⁷Knocking down PD-1 by using a lentivirus vector RNAi technology; further expansion of tumor infiltrating lymphocytes to 10¹⁰More than one; finally, the tumor infiltrating lymphocytes are back-transfused to the patients, and the average cell amount of the tumor infiltrating lymphocytes injected each time is 7.3 multiplied by 10⁸And (4) respectively. The invention has high efficiencyThe ability to kill, can discern multiple antigen targets, use autoimmune cell, the side effect is not had, and TIL immunotherapy can be used to the relapse or metastatic late stage gastric cancer patient.

Description

Method for improving gastric cancer by TIL immune cells

Technical Field

The invention belongs to the technical field of immune cell therapy, and particularly relates to a method for improving gastric cancer through TIL immune cells.

Background

Gastric cancer (gastric cancer) is a malignant tumor originated from gastric mucosal epithelium, the incidence rate of the gastric cancer is the first in various malignant tumors in China, the incidence rate of the gastric cancer is obviously different regionally, and the incidence rate of the gastric cancer is obviously higher in northwest and east coastal areas of China than in south areas. The good hair age is more than 50 years old, and the ratio of the incidence rates of men and women is 2: 1. gastric cancer tends to be younger due to changes in dietary structure, increased working pressure, infection with helicobacter pylori, and the like. Gastric cancer can occur in any part of the stomach, more than half of which occur in antrum, greater curvature, lesser curvature, and anterior and posterior walls. Most of gastric cancers belong to adenocarcinoma, have no obvious symptoms in the early stage, or have nonspecific symptoms such as epigastric discomfort, eructation and the like, are often similar to the symptoms of chronic stomach diseases such as gastritis, gastric ulcer and the like, and are easy to ignore, so the early diagnosis rate of the gastric cancers in China is still low at present. The prognosis of gastric cancer is related to the pathological stage, location, tissue type, biological behavior, and therapeutic measures of gastric cancer.

The causes of gastric cancer are: 1. regional environment and dietary life factors: the incidence of gastric cancer is obviously different regionally, and the incidence rate of gastric cancer is obviously higher in northwest and east coastal areas of China than in south areas. The incidence rate of far-end gastric cancer in people who eat smoked and salted foods for a long time is high, and is related to the high content of carcinogens or precancers such as nitrite, mycotoxin and polycyclic aromatic hydrocarbon compounds in the foods; smokers have a 50% higher risk of stomach cancer than non-smokers. 2. Helicobacter pylori (Hp) infection: the Hp infection rate of adults in high-incidence areas of gastric cancer in China is more than 60%. Helicobacter pylori can promote the conversion of nitrate into nitrite and nitrosamine to cause cancer; chronic inflammation of gastric mucosa caused by Hp infection and environmental pathogenic factors accelerate excessive proliferation of mucosal epithelial cells, so that distortion and carcinogenesis are caused; toxic products CagA and VacA of helicobacter pylori may have cancer promotion effect, and the detection rate of anti-CagA antibodies in gastric cancer patients is obviously higher than that of general people. 3. Precancerous lesions: gastric diseases include gastric polyps, chronic atrophic gastritis and gastric remnants after gastric resection, all of which may be associated with various degrees of chronic inflammatory processes, metaplasia or atypical hyperplasia of the gastric mucosa and intestinal epithelium, and may turn into cancer. Precancerous lesion refers to pathological and histological change of gastric mucosa which is easy to become cancerous, and is a boundary pathological change in the process of transforming benign epithelial tissue into cancer. The dysplasia of gastric mucosa epithelium belongs to precancerous lesions, which can be divided into light, medium and heavy according to the heterotypic degree of cells, and the severe dysplasia is sometimes difficult to distinguish from early gastric cancer with good differentiation. 4. Genetic and genetic: genetic and molecular biological researches show that the incidence of gastric cancer of relatives with blood relationship of gastric cancer patients is 4 times higher than that of the relatives in a control group. The canceration of gastric cancer is a process of multi-factor, multi-step, and multi-stage development, involving changes in oncogenes, cancer suppressor genes, apoptosis-related genes, metastasis-related genes, and the like, and the forms of gene changes are also diverse.

The preventive measures for gastric cancer include: 1. the food changes the dietary structure, and is eaten by vegetables, fruits, beans, milk, fresh fish, meat and eggs, and advocates to eat garlic and green tea. 2. Bad eating habits are changed, the overeating caused by heavy drinking is avoided, and three meals are untimely; fast, hot and hard eating is not suitable; the smoked food is eaten less, and the high-salt diet is avoided. 3. Less strong liquor, no smoking. 4. The mildew prevention and removal work of the grains is well done, and the sanitation of the edible water is protected. 5. Can be used for treating gastric ulcer, chronic gastritis, and helicobacter pylori infection in stomach. 6. The general investigation of the gastric cancer is carried out on high-risk people and high-incidence areas.

Gastric cancer is classified by gross morphology: 1. early gastric cancer: it refers to a gastric cancer in which cancer tissues are localized in the gastric mucosa and submucosa, and can be classified into a bulge type, a flat type and a recess type according to the morphology of naked eyes. 2. Advanced gastric cancer: it refers to the gastric cancer whose cancer tissue infiltration depth exceeds the submucosal layer, and can be classified into polyp type, localized ulcer type, infiltration ulcer type and diffuse infiltration type. Gastric cancer is classified by histopathology: can be divided into adenocarcinoma, adenosquamous carcinoma, squamous carcinoma, carcinoid, etc., and the majority is gastric adenocarcinoma. Adenocarcinoma can be classified into papillary carcinoma, tubular adenocarcinoma, poorly differentiated adenocarcinoma, mucinous adenocarcinoma, and signet ring cell carcinoma according to the tissue structure. According to the differentiation degree of cells, the cells can be divided into 3 types of high differentiation, medium differentiation and low differentiation. They can be classified into intestinal type and gastric type according to their tissue origin. Stomach cancer is classified by the diseased site: it can be classified into gastric fundus and cardia cancer, gastric antrum cancer, etc., and the gastric cancer at different sites determines the difference of the operation types.

In the early stage of gastric cancer, most patients have no obvious symptoms, and few patients have nausea, vomiting or upper gastrointestinal symptoms similar to ulcer diseases, so that sufficient attention is hardly paid. As the tumor grows, the stomach function is affected, but the symptoms are obvious, but the specificity is lacked. Pain and weight loss are the most common clinical symptoms of advanced gastric cancer. Patients often have more definite upper gastrointestinal symptoms, such as upper abdominal discomfort, fullness after eating, aggravation of upper abdominal pain along with the progress of the disease, appetite reduction and hypodynamia. It is also characterized by different tumor sites. Carcinoma of the gastric fundus of the cardia may have poststernal pain and progressive dysphagia; gastric cancer near the pylorus shows pyloric obstruction. When the tumor destroys blood vessels, there may be symptoms of digestive tract hemorrhage such as hematemesis and dark stool; if the tumor invades the pancreatic capsule, persistent pain radiating to the back of the waist can appear; perforation of tumor ulcers can cause severe pain and even signs of peritoneal irritation; jaundice can occur when hepatic lymph node metastasis occurs in the tumor or when the common bile duct is pressed; when distant lymph nodes metastasize, the swollen lymph node can be reached on the left clavicle. Patients with advanced gastric cancer often have the symptoms of anemia, emaciation, malnutrition, even cachexia and the like.

The following pathways exist for the spread and metastasis of gastric cancer: 1. direct infiltration: carcinoma of the gastric fundus of the cardia and stomach, which is apt to invade the lower part of the esophagus, may infiltrate into the duodenum. After the gastric cancer with poor infiltrative growth of differentiation breaks through serosa, the gastric cancer is easy to diffuse to adjacent organs such as omentum, colon, liver, pancreas and the like. 2. Blood circulation transfer: in the advanced stage, cancer cells enter the portal vein or systemic circulation and spread to other parts of the body, forming metastases. The common organs with metastasis include liver, lung, pancreas, and bones, and liver metastasis is usually considered. 3. And (3) peritoneal planting and transferring: when the stomach cancer tissue infiltrates outside the serosa, tumor cells fall off and are planted on the peritoneum and the visceral serosa to form metastatic nodules. Peritoneal seeding most easily occurs in the upper abdomen, mesentery. The implantation in the rectum and bladder is the sign of late gastric cancer. Metastatic carcinoma of the anterior fovea rectum can be found by digital rectal examination; ovarian metastatic tumors can develop in female patients with gastric cancer. 4. Lymphatic metastasis: lymph metastasis is the main metastasis way of gastric cancer, the lymph metastasis rate of the gastric cancer in the advanced stage is as high as about 70%, and lymph metastasis can also occur in early stage gastric cancer. The lymph node metastasis rate of gastric cancer is positively correlated with the depth of invasion of the cancer foci. Lymph node metastasis from gastric carcinoma is usually gradual, but also abrupt lymph metastasis can occur, i.e., no metastasis at the first station and metastasis at the second station. Terminal stage gastric cancer may metastasize to the supraclavicular lymph nodes via the chest catheter, or to the umbilicus via the ligamentum teres hepaticum.

The medical history, physical examination and laboratory examination are in accordance with the characteristics of the gastric cancer, and the gastric cancer can be clinically diagnosed by finding space-occupying lesion by X-ray barium double radiography or endoscopy, but the final diagnosis of the gastric cancer is also determined according to biopsy or cytological examination results. The following cases are required to be examined in time: 1. patients with gastric ulcer still have no improvement after strict internal medicine treatment; 2. persons with epigastric discomfort or pain, no apparent rhythmicity, and with apparent anorexia and emaciation after age 40; 3. the patients over 40 years old with chronic atrophic gastritis or atypical hyperplasia and more recent symptoms; 4. the former has history of chronic gastric disease, and the stool occult blood examination shows that the stool occult blood is positive for more than 2 weeks; 5. stomach polyp greater than 2 cm. After the qualitative diagnosis of gastric cancer is obtained by gastroscopy and biopsy, a series of imaging examinations are also needed to perform staged diagnosis of gastric cancer. Accurate staging is important for making a reasonable treatment plan, judging prognosis and evaluating curative effect. The higher the stage, the later the disease condition and the shorter the survival period.

The prior art methods for treating gastric cancer include the following: 1. and (3) surgical treatment: radical operation, which consists in excising part or all of the stomach including the cancer focus and possibly infiltrated stomach wall in one piece, removing lymph nodes around the stomach in one piece according to the clinical staging standard, and reconstructing the digestive tract. Palliative surgery, where the primary focus cannot be removed, is performed to relieve symptoms caused by complications such as obstruction, perforation, bleeding, etc., such as gastrojejunostomy, jejunostomy, puncture repair, etc. 2. Chemotherapy: can be used before, during and after radical operation to prolong life. The advanced gastric cancer patient adopts a proper amount of chemotherapy, can slow down the development speed of the tumor and improve symptoms, and has certain recent effect. In principle, auxiliary chemotherapy is not needed after the early gastric cancer radical operation, and the following cases are in need of auxiliary chemotherapy: the malignancy degree of the pathological type is high; the area of the cancer focus is more than 5 cm; multiple cancer foci; the age is below 40 years. Chemotherapy is needed for patients with recurrence after advanced gastric cancer radical surgery, palliative surgery and radical surgery. The common administration routes of gastric cancer chemotherapy comprise oral administration, intravenous administration, peritoneal cavity administration, arterial intubation region perfusion administration and the like. Common oral chemotherapeutics include tegafur, efonidine, fluocinolone, and the like. The commonly used intravenous chemotherapy drugs include fluorouracil, mitomycin, cisplatin, adriamycin, etoposide, calcium formyltetrahydrofolate, and the like. In recent years, new chemotherapeutic drugs such as paclitaxel, platinum oxalate, topoisomerase inhibitor, and Hirodada are used for treating gastric cancer. 3. Targeted therapy: targeted therapy can be aimed at damaging cancer cells and reducing normal cell damage. At present, the types and the effects of the gastric cancer targeted therapeutic drugs are limited. The targeted therapeutic medicine mainly comprises an epidermal growth factor receptor inhibitor, an angiogenesis inhibitor, a cell cycle inhibitor, an apoptosis promoter, a matrix metalloproteinase inhibitor and the like. 4. Other treatments: immunotherapy for gastric cancer includes non-specific biological response modifiers such as BCG, lentinan, etc.; cytokines such as interleukins, interferons, tumor necrosis factors, etc.; and adoptive immunotherapy such as killing cells after Lymphocyte Activation (LAK) and the like. Anti-angiogenic genes are the most studied gene therapy methods and may play a role in the treatment of gastric cancer. 5. Supporting treatment: aims to relieve the pain of patients, improve the life quality and prolong the life cycle. Including analgesia, anemia correction, appetite improvement, nutritional status improvement, obstruction relief, ascites control, psychotherapy, and the like. The prognosis of gastric cancer is related to the pathological stage, location, tissue type, biological behavior, and therapeutic measures of gastric cancer. The prognosis of early gastric cancer after treatment is better. Gastric cardia cancer and proximal gastric cancer at the suprastomach 1/3 have a poorer prognosis than gastric body and distal gastric cancer. Women have a better prognosis than men. The postoperative effect of the gastric cancer patients over 60 years old is better, and the prognosis is poor below 30 years old.

Tumor immunotherapy is the most popular, even the hottest, cancer treatment for these years. In the case of tumor immunotherapy, it is likely that many patients with advanced cancer would benefit from the PD-1/PD-L1 antibody, K drug, O drug, and domestic PD-1 antibody. However, in addition to PD-1/PD-L1, tumor immunotherapy also includes immunocytotherapy, such as the well-known CAR-T technique, which achieves tumor control by reinfusing specific CAR-T cells into cancer patients. Gastric cancer is generally used to control the progression of the disease, prevent the disease from further worsening, and prolong the life of the patient as much as possible, based on the refractory nature of gastric cancer. In addition to CAR-T, immune cell therapy also includes TCRT therapy/TIL cell therapy, and it would be a good news to gastric cancer patients if a TIL cell technology in immune cell therapy could be invented to improve the quality of life of gastric cancer patients.

Disclosure of Invention

The invention provides a method for improving gastric cancer through TIL immune cells in order to overcome the defects in the prior art.

The invention is realized by the following technical scheme: the invention discloses a method for improving gastric cancer through TIL immune cells, which comprises the following specific operation processes:

(1) first, a tumor is excised from a patient, the tumor tissue is transported to a GMP laboratory under sterile, cold chain conditions, and isolated to yield 10⁴～10⁶An initial tumor infiltrating lymphocyte;

(2) culturing the initial tumor infiltrating lymphocytes under proper conditions, amplifying by ten thousand times and further purifying to 10⁸Performing primary seed cell culture, and performing cryopreservation under conditions of 10 cells per group and 10 cells per group⁷Storing each cell/branch at the temperature of liquid nitrogen-196 ℃;

(3) recovering and expanding tumor infiltrating lymphocyte to 8 × 10⁷Knocking down PD-1 by using a lentivirus vector RNAi technology;

(4) further expanding the tumor-infiltrating lymphocytes to one thousand to ten thousand times, so that the tumor-infiltrating lymphocytes are further expanded to 10¹⁰More than one;

(5) finally, the tumor infiltrating lymphocytes are back-transfused to the patients, and the average cell amount of the tumor infiltrating lymphocytes injected each time is 7.3 multiplied by 10⁸And (4) respectively.

T cells are named after fierce because of book formation and belong to important immune cells, and the main functions of the T cells are mediated cellular immunity and the immune response of an organism is regulated. T cells are derived from hematopoietic stem cells, develop and differentiate in thymus, migrate to thymus-dependent regions of peripheral immune organs after maturation, and colonize, and account for 65-70% of the total number of lymphocytes in peripheral blood. The thymus-dependent lymphocyte T cell is formed by differentiating a bone marrow-derived lymphocyte stem cell in thymus, and the T cell with mature thymus development is transferred to peripheral lymphoid organs or lymphoid tissues and is kept in a relatively resting state before being stimulated by no contact with specific antigen molecules, so that the T cell is called an initial T cell. Once stimulated by the corresponding antigens, the antigens are converted into large lymphocytes which are active in metabolism and have the diameter of 15-20 mu m, and the large lymphocytes are proliferated and differentiated. The majority differentiate into effector T cells, acquire the functions of migration, cytokine production and other effects, and the minority form memory T cells. The effector T cells have short life span and have the function of killing target cells. Memory T cells can last for years, even for life, and when the body is again stimulated by the same antigen, they can rapidly transform and proliferate, forming a large number of effector T cells, initiating a more intense immune response, and maintaining the body's immunity to the antigen for a longer period of time. Since effector T cells can directly kill target cells, the immunity in which T cells participate is called cellular immunity.

Doctor of the national cancer institute surgical department, maine roleb (Steve Rosenberg), pioneered the use of tumor infiltrating lymphocytes. TIL tumor-infiltrating lymphocytes are cells derived from the infiltration of tumor tissue, and are known immune cells that are capable of specifically recognizing and attacking cancer. The application of the tumor infiltrating lymphocytes in the invention in improving the gastric cancer is to separate and amplify the lymphocytes in breast tumor tissues and then return the lymphocytes to patients, thus achieving the purpose of improving the quality of life of the patients with the gastric cancer.

The tumor infiltrating lymphocyte is a T cell which is infiltrated in a tumor tissue and can effectively identify a tumor cell neoantigen, and the T cell which can identify the tumor cell neoantigen has the capability of efficiently killing tumor cells, including tumor passage cells of other parts transferred from the focus. The advantages of tumor infiltrating lymphocytes are: compared with other immunotherapy methods, the tumor infiltrating lymphocytes have the advantages of more tumor antigen recognition targets, less side effects, more solid tumors which can be treated, stronger curative effect and the like.

The invention has the beneficial effects that: ASCO published data in 2019 month 5: the objective remission rate of the TIL immune cells for treating solid tumors reaches 44 percent, and the disease control rate reaches 89 percent. Advantages of the TIL immunotherapy of the invention over other treatment methods: 1. surgical resection method: the advantages are that: is capable of directly excising the focus; disadvantages are that: has the risk of bleeding or infection, has long healing time, and can not be operated if the tumor spreads or metastasizes, thereby injuring the primordial qi of the patient. 2. Radiotherapy: the advantages are that: accurate, high-efficiency, small side effect and the like; disadvantages are that: only aiming at specific parts, some parts can not be used, and the traditional Chinese medicine composition has harm to normal tissue cells and low side effect. 3. Chemotherapy: the advantages are that: the tumor cells can be effectively killed and reduced when the tumor spreads or metastasizes; disadvantages are that: the side effect is large, the immune system loss is serious, the infection risk is increased, and the drug resistance is easy to generate. 4. Targeted drug therapy: the advantages are that: the method is accurate and efficient; disadvantages are that: only aims at a single antigen target, and is easy to generate drug resistance and side effects. The TIL immunotherapy disclosed by the invention has high killing capacity, can identify a plurality of antigen targets, uses autoimmune cells, has no side effect, and can be used for patients with recurrent or metastatic late gastric cancer.

Table 1: TIL immune cell therapy has advantages over other immunotherapies.

Detailed Description

The present invention will be described in detail with reference to specific embodiments.

The invention discloses a method for improving gastric cancer through TIL immune cells, which comprises the following specific operation processes: (1) first, a tumor is excised from a patient, the tumor tissue is transported to a GMP laboratory under sterile, cold chain conditions, and isolated to yield 10⁴～10⁶An initial tumor infiltrating lymphocyte; (2) culturing the initial tumor infiltrating lymphocytes under proper conditions, amplifying by ten thousand times and further purifying to 10⁸Freezing the primary seed cells, and storingWith the condition that 10 of the above-mentioned groups are contained in the total amount of 10⁷Storing each cell/branch at the temperature of liquid nitrogen-196 ℃; (3) recovering and expanding tumor infiltrating lymphocyte to 8 × 10⁷Knocking down PD-1 by using a lentivirus vector RNAi technology; (4) further expanding the tumor-infiltrating lymphocytes to one thousand to ten thousand times, so that the tumor-infiltrating lymphocytes are further expanded to 10¹⁰More than one; (5) finally, the tumor infiltrating lymphocytes are back-transfused to the patients, and the average cell amount of the tumor infiltrating lymphocytes injected each time is 7.3 multiplied by 10⁸And (4) respectively.

The improvement case is as follows: 1 patient with 52-year-old brain metastasis and gastric cancer who had undergone resection and chemotherapy for gastric cancer and had an average injection of TIL cells of 7.3X 10⁸After 17 months, the tumor disappeared from the medical image, and the patient was clinically free.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Claims (1)

1. A method of ameliorating gastric cancer by TIL immune cells, comprising: the specific operation flow of the application of the tumor infiltrating lymphocytes in improving the gastric cancer is as follows:

(1) first, a tumor is excised from a patient, the tumor tissue is transported to a GMP laboratory under sterile, cold chain conditions, and isolated to yield 10⁴～10⁶An initial tumor infiltrating lymphocyte;

(2) culturing the initial tumor infiltrating lymphocytes under proper conditions, amplifying by ten thousand times and further purifying to 10⁸Performing primary seed cell culture, and performing cryopreservation under conditions of 10 cells per group and 10 cells per group⁷Storing each cell/branch at the temperature of liquid nitrogen-196 ℃;

(3) recovering and expanding tumor infiltrating lymphocyte to 8 × 10⁷Knocking down PD-1 by using a lentivirus vector RNAi technology;

(4) tumor infiltrating lymphocytesFurther expanding to one thousand to ten thousand times, further expanding the tumor infiltrating lymphocytes to 10¹⁰More than one;

(5) finally, the tumor infiltrating lymphocytes are back-transfused to the patients, and the average cell amount of the tumor infiltrating lymphocytes injected each time is 7.3 multiplied by 10⁸And (4) respectively.