CN-121991891-A - Method for improving TIL production quality by regulating and controlling immune metabolism through deprivation of aspartic acid

CN121991891ACN 121991891 ACN121991891 ACN 121991891ACN-121991891-A

Abstract

The invention provides a method for improving the production quality of TIL by controlling immune metabolism through deprivation of aspartic acid, which comprises an initial amplification stage and a rapid amplification stage, wherein cells are changed into a deprivation of aspartic acid culture medium for culture in the late culture stage of the initial amplification stage and/or the rapid amplification stage until the cells in each stage are harvested. The method can enhance the proliferation capacity of the TIL on the basis of the existing TIL amplification flow, reduce the risk of insufficient proliferation power and limited amplification in the amplification process, and obtain the TIL amplification product meeting the feedback requirement. The method can also improve immune effect of amplification final product, thereby improving anti-tumor immune effect of the feedback TIL preparation, improving TIL metabolism state, improving mitochondrial function and reducing oxidative stress level, and being beneficial to maintaining effective and continuous anti-tumor reaction in vivo after feedback.

Inventors

LI CHAO
ZHANG YAN
Shu xiaogang
ZHANG YINING
CHEN JINHUANG
CHENG XUKAI
JIANG MI
SONG JIA
FANG FEIFEI
CHANG KE
ZHU XINYUE

Assignees

武汉麦迪克生物科技有限公司
华中科技大学同济医学院附属协和医院

Dates

Publication Date: 20260508
Application Date: 20260209

Claims (10)

1. A method for improving TIL production quality by controlling immune metabolism through deprivation of aspartic acid is characterized by comprising an initial amplification stage and a rapid amplification stage, wherein cells are changed into a deprived aspartic acid culture medium for culture at the later culture stage of the initial amplification stage and/or the rapid amplification stage until the cells at each stage are harvested.
2. The method for improving the production quality of TIL by controlling immune metabolism by deprived aspartic acid according to claim 1, wherein the initial amplification stage comprises the following steps when cells are changed to a deprived aspartic acid culture medium for culture in a late culture stage of the initial amplification stage: a1, taking isolated tumor tissue, cutting the tumor tissue into small blocks, and inoculating the small blocks into a complete culture medium for culture; a2, culturing until 3 days before the expected harvest day, and continuously culturing the cells by changing the liquid into an aspartic acid deprivation culture medium; A3, culturing for 3 days by adopting an aspartic acid deprivation culture medium, and then harvesting TIL cells; the rapid amplification stage comprises the following steps: B1, adding TIL cells and feeder cells obtained in the initial expansion stage into a complete culture medium, and adding a stimulator for culturing, wherein the day is counted as day 0; B2, culturing until the 6 th day, and carrying out amplification culture by adopting a complete culture medium until TIL cells are harvested.
3. The method for improving the production quality of TIL by controlling immune metabolism by deprivation of aspartic acid according to claim 1, wherein the initial amplification stage comprises the following steps when cells are changed to a culture medium in which deprivation of aspartic acid is carried out in a late stage of culture in the rapid amplification stage: a11, taking isolated tumor tissues, cutting the tumor tissues into small blocks, inoculating the small blocks into a complete culture medium, culturing the small blocks until the harvest date, and harvesting TIL cells; the rapid amplification stage comprises the following steps: b11, adding TIL cells and feeder cells obtained in the initial expansion stage into a complete culture medium, and adding a stimulator for culturing, wherein the day is counted as day 0; B12, culturing until the 6 th day, performing amplification culture by adopting a complete culture medium, and replacing the cell with a culture medium containing deprived aspartic acid for continuous amplification culture 3 days before the expected harvest day; b13, obtaining TIL cells after amplifying culture for 3 days by adopting an aspartic acid deprived culture medium.
4. The method for improving the production quality of TIL by controlling immune metabolism by depriving aspartic acid according to claim 1, wherein the method is characterized in that the initial amplification stage comprises the following steps when cells are changed into a deprived aspartic acid culture medium for culture in the initial amplification stage and the late culture stage of the rapid amplification stage: a1, taking isolated tumor tissue, cutting the tumor tissue into small blocks, and inoculating the small blocks into a complete culture medium for culture; a2, culturing until 3 days before the expected harvest day, and continuously culturing the cells by changing the liquid into an aspartic acid deprivation culture medium; A3, culturing for 3 days by adopting an aspartic acid deprivation culture medium, and then harvesting TIL cells; the rapid amplification stage comprises the following steps: b11, adding TIL cells and feeder cells obtained in the initial expansion stage into a complete culture medium, and adding a stimulator for culturing, wherein the day is counted as day 0; B12, culturing until the 6 th day, performing amplification culture by adopting a complete culture medium, and replacing the cell with a culture medium containing deprived aspartic acid for continuous amplification culture 3 days before the expected harvest day; b13, obtaining TIL cells after amplifying culture for 3 days by adopting an aspartic acid deprived culture medium.
5. The method for improving the production quality of TIL by controlling immune metabolism through deprivation of aspartic acid according to any one of claims 1 to 4, wherein the total culture time of cells in the initial amplification stage is 2 to 4 weeks, and the total culture time of cells in the rapid amplification stage is 13 to 14 days.
6. The method for improving the production quality of TIL by controlling immune metabolism by depriving aspartic acid according to any one of claims 2 to 4, wherein the complete medium in each step comprises basal medium, 6000 IU/ml IL-2, 10% human AB serum and 1% gentamicin; The deprived aspartic acid culture medium comprises a basic culture medium with the L-aspartic acid content of 0-2 mg/L, 6000 IU/ml IL-2, 10% human AB serum and 1% gentamicin.
7. The method for improving the production quality of TIL by controlling immune metabolism by depriving aspartic acid according to claim 6, wherein the basal medium is RPMI-1640 medium.
8. The method for improving the production quality of TIL by controlling immune metabolism by depriving aspartic acid according to any one of claims 2 to 4, wherein in step B1 or B11, the feeder cells are irradiated peripheral blood mononuclear cells; the ratio of TIL cells to feeder cells is 2-3×10 8 feeder cells matched with every 200 ten thousand TIL cells. The stimulus is an anti-CD 3 antibody, and the final concentration of the stimulus is 30 ng/ml.
9. The method of enhancing the quality of TIL production by controlling immune metabolism by depriving aspartic acid according to any one of claims 2 to 4, wherein in step A2 or B12, the estimated harvest date is determined based on the time required for TIL to be expected to grow up to the well of an orifice plate; in step B12, the estimated harvest date is determined based on the time the number of TILs meets the patient reinfusion dose requirement.
10. A TIL obtained according to the method of any of claims 1-9, wherein the TIL CD8 + T cells account for 80% or more, CD4 + T cells account for 14% or more, the proportion of expressed gzmb+ is 32% or more, the proportion of expressed tnfα+ is 610% or more, the proportion of expressed ifnγ+ is 50% or more, and the proportion of expressed PD-1+ to TCF-1+ is 310% or more.

Description

Method for improving TIL production quality by regulating and controlling immune metabolism through deprivation of aspartic acid Technical Field The invention relates to the technical field of biological medicines, in particular to a method for improving the production quality of TIL by regulating and controlling immune metabolism through deprivation of aspartic acid. Background TIL (tumor-infiltrating lymphocytes) therapy is a novel cellular immunotherapy that has been remarkably progressed in the field of tumor treatment in recent years, and this technique is based on the patient's own immune system, attacks tumor cells by extracting, expanding and reinfusion tumor-infiltrating lymphocytes (TILs), exhibiting high specificity and safety. 1. The technical principle is that TILs which are infiltrated and show anti-tumor activity are extracted from tumor tissues of a patient by TIL therapy, and returned after laboratory culture and amplification so as to activate and enhance immune response to target tumor cells. 2. Development history the study of this therapy began in the 80 s of the last century, and Steven a. Rosenberg teaches for the first time that TILs were able to inhibit metastasis of malignant melanoma cells, with technological advances, moving towards clinical use and achieving remarkable results. 3. The technical characteristics include high specificity (capable of identifying and attacking tumor cells), high safety (low immune rejection risk) and remarkable curative effect (good performance in melanoma, lung cancer, breast cancer and other solid tumors). 4. Clinical application the TIL therapy has been developed in a number of clinical trials worldwide and has been approved by the FDA for use in unresectable or metastatic melanoma, showing good application prospects. 5. With the continuous progress of technology, TIL therapy is expected to break through in the treatment of more tumor types, and becomes an important means for tumor immunotherapy, and researchers continue to optimize the preparation process and the treatment method, so that the curative effect and the safety are improved, the cost is reduced, and more patients benefit. In conclusion, the TIL therapy has important application value and wide development prospect in the field of tumor treatment. Aiming at the amplification method of TIL, a novel culture and in-vitro amplification method of primary liver cancer tumor infiltrating lymphocytes is disclosed in the patent document with the application number of CN 202110292616. The method comprises the following steps of 1, cleaning the surface of a liver cancer tissue specimen and removing adipose tissues, 2, cutting the tissues into small blocks, inoculating the small blocks into an orifice plate, culturing by adopting a primary culture medium, 3, after tumor-infiltrating lymphocytes climb out of the orifice plate for a certain amount, mixing the tumor-infiltrating lymphocytes with feeder cells, inoculating the feeder cells and adding the feeder cells into a complete culture medium for continuous culture, and 4, collecting the cells and freezing the cells after the feeder cells at least partially die after the culture to obtain the tumor-infiltrating lymphocytes. The method is quick, reliable and high in cost performance, can obtain enough high-activity lymphocytes in a short time, is suitable for cell treatment (such as ACT), and provides a good foundation for treatment methods such as CART, TCRT and the like. The patent application CN202380015383 discloses that TIL is obtained by first subjecting TIL derived from tumor tissue to at least one stage of in vitro amplification, and in each in vitro amplification stage, co-culturing the in vitro amplified and/or non-in vitro amplified TIL with T cell activator and/or T cell growth factor for a certain period of time, and then with feeder cells. The method can effectively improve the quantity and activity of TIL, provides a new medicine foundation for tumor treatment, and has important clinical application value. However, the existing TIL culture methods still have the following disadvantages: 1) The amplification process has strong dependence on starting materials, insufficient proliferation activity is easy to cause limited amplification, the existing TIL preparation flow usually comprises two stages of initial amplification (pre-REP, about 2-4 weeks) and rapid amplification (REP, about 14 days), the whole culture period is long, and the amplification effect is greatly dependent on the tumor sample quality, the tumor tissue quantity and the quantity and functional state of the initial TIL. For samples with limited tumor tissue quantity or low TIL infiltration degree, the conditions of insufficient proliferation power, slow amplification speed, limited amplification and the like often occur in the amplification process, and cell products meeting the feedback requirement are difficult to stably obtain. 2) The amplification end product is eas