CN110628717B

CN110628717B - Method for culturing infiltrating T cells

Info

Publication number: CN110628717B
Application number: CN201911029541.6A
Authority: CN
Inventors: 柴勋; 李晓飞
Original assignee: SHANGHAI BIOMED-UNION CO LTD
Current assignee: Shanghai Biomed-union Biotechnology Co.,Ltd.
Priority date: 2019-10-28
Filing date: 2019-10-28
Publication date: 2020-10-30
Anticipated expiration: 2039-10-28
Also published as: CN110628717A

Abstract

A method for culturing infiltrating T cells, comprising the following steps: 1) preparing to form effector T cells, 2) dedifferentiating the effector T cells into TIL-TCM using a dedifferentiation medium, 3) expanding the TCM using an expansion medium. The TIL-TCM cultured by the invention has stronger tumor body tropism and can home to the tumor position; can specifically identify tumor cells and kill tumors; can adapt to the tumor microenvironment; can survive in vivo for a long time, and has long-term anti-tumor effect; a small amount of cells can play a strong anti-tumor role, the culture time is short, the probability of side reaction is low, the operation is simple, and the cell proliferation speed is high.

Description

Method for culturing infiltrating T cells

Technical Field

The invention relates to a method for culturing infiltrating T cells, in particular to a method for culturing tumor infiltrating central memory T cells.

Background

The report of "2012 annual report of tumor registration in China": the incidence of cancer in China is 285.91/10 ten thousand, and the incidence of cancer in cities and rural areas is 303.91/10 ten thousand and 249.98/10 ten thousand respectively. It is estimated that by 2049 years, the incidence of cancer in our country may reach a level of 400/10 ten thousand. The most prominent cause of increased incidence of cancer is aging of the human mouth. The proportion of the aged population in China is 13.6 percent, and the aged population can reach 16 percent after 20 to 30 years. In response, we can understand that the incidence of cancer is always on the rising trend in recent years, and despite the development of current cancer treatment measures, the condition of patients with advanced malignant tumors is still poor, and the 5-year survival rate is less than 15%.

The traditional three tumor treatment methods of operation, chemotherapy and radiotherapy have poor targeting property and large side effect, are easy to generate stronger drug resistance phenomenon, also have the risk of metastasis and relapse, and are difficult to eradicate tumor cells. With the development of tumor biology, molecular biology and immunology, a great deal of research shows that immunotherapy has incomparable superiority to other treatment modes for patients with advanced malignant tumors, so that immunotherapy becomes the fourth tumor treatment method after traditional tumor treatment methods, such as surgery, chemotherapy and radiotherapy, and has wide clinical application prospects.

Tumor immunotherapy is usually referred to as adoptive cellular immunotherapy, which refers to a therapeutic method for treating tumors by infusing autologous or allogeneic specific or non-specific "anti-tumor immune effector cells" to directly kill tumor cells or improve the immune function of the body. The invention mainly researches tumor infiltrating central memory T cells.

Tumor-Infiltrating central memory T cells (TILs), which refer to lymphocytes Infiltrating into tumors, are used to isolate and identify T cells specific for different Tumor-associated antigens. The property of TIL to recognize tumor associated antigens is used for adoptive immune cell therapy of tumors. TIL is separated from melanoma by Rosenberg laboratory, and is applied to patients with advanced metastatic melanoma after being treated by methods such as screening and amplification in vitro, wherein objective treatment response is obtained for nearly 50% of patients. In addition, the number, distribution and type of TILs in tumors vary considerably among different tumor types, and this difference is closely related to the reactivity of the body's immune system to tumors. In recent years, the characterization of TIL in tumors has been used as a criterion for patient prognosis. For example, in colon cancer, the tumor infiltrates higher cell densities of CD3+, CD3+ CD8+ better after recovery, and the criteria are more accurate than the conventional TNM classification. In tumors with higher degrees of malignancy, the expression levels of granzyme and gamma interferon genes are significantly lower than those of the tumors with lower degrees of malignancy, and both components are related to immune cell anti-tumor.

Tumor-specific Central Memory T Cells (TCM), which are the upstream cells in the immune response, are thought to have a longer life span and tumor body tropism in vivo and to play a role in the ligament. After the immune system recognizes the tumor antigen, the TCM is responsible for long-term Memory of the tumor antigen, and continuously generates a large number of Effector Memory T cells (TEMs) for the tumor under the stimulation of the tumor antigen, and further differentiates into a large number of Effector T cells.

365 cancer patients treated by TCM cells in 2013 to 2016 by the chemotherapy department of Mangan swallow, a second subsidiary hospital of North China university prove that TCM has certain effects on prolonging the life cycle of the patients, improving the life quality of the cancer patients, relieving the side effects of chemotherapy and the like. The subsidiary major department of hepatobiliary surgery in the tumor hospital of the academy of Chinese medical science, Zhao hong, indicates that hepatectomy is the most important treatment mode for treating hepatocellular carcinoma, but more than 50% of liver cancer patients relapse within two years after surgery, and the current situation can be improved by TCM treatment. TCM, the latest generation of anti-tumor immune cells, has been introduced into the National Institutes of Health (NIH) cancer center for certification, and is evaluated as the best anti-tumor cell by the us NIH.

At present, the traditional immune cell therapy is a single therapy using TCM or TIL, but no good clinical effect and market application are obtained, and the reasons are as follows:

1. TCM in peripheral blood "remembers" different antigens, and carries out immune response to different diseases and infections, and only a few TCM can identify tumors, so that TCM separated from peripheral blood has limited tumor killing capability, and basically expands some irrelevant cells;

2. with the development of science and technology, people find that only a few parts of the TIL have tumor killing capacity, so how to amplify the tumor specific TIL cells is always the research focus of the cells, and the existing methods such as neoantigen, monoclonal selection and the like are very time-consuming and labor-consuming, have very huge cost and are difficult to popularize;

3. according to the National Institutes of Health (NIH), the reason why TIL needs 1000 billion cells to reach effective concentration and needs to be matched with the infusion of high-dose IL2 to maintain the activity of TIL in endosome is that the traditional TIL is effector T cells which are rapidly exhausted in vivo and cannot be expanded for a long time, so that long-term anti-tumor effect cannot be realized, and the light culture medium with the cell quantity of 1000 billion cells needs hundreds of liters to be expanded, and the cost is conceivably high;

4. large doses of IL2 may have significant side effects on patients;

5. traditional TCM with peripheral blood expansion still cannot infiltrate into tumors even if tumor antigens are memorized due to tumor microenvironment and other reasons.

In summary, the advantages and disadvantages of TIL and TCM are complementary, and if there is a cell that can combine the advantages of 2 and overcome each other, the cell has good therapeutic effect and huge market potential, but because the content of TCM in TIL is very low, there is little, and TCM can not be directly separated from TIL, there is no complete solution at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing a TIL-TCM culture method.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for culturing infiltrating T cells, comprising the following steps: 1) preparing to form effector T cells, 2) dedifferentiating the effector T cells into TIL-TCM using a dedifferentiation medium, 3) expanding the TCM using an expansion medium.

In order to obtain effector T cells, the effector T cells in the step 1) are activated lymphocytes in peripheral blood by using an activation medium.

To activate the activity of effector T cells, the suppression status of effector T cells is relieved, said activation medium comprising a basal medium of 1640 and AIM-V1:1 mix, 55-100ng/ml CD28, 20-60ug/ml PD1 antibody, 70-300ng/ml CD137L, 10-40ng/ml IL2, 2-20% autologous serum.

Preferably, the activation medium comprises a basal medium of 1640 and AIM-V1:1 mix, 80ng/ml CD28, 40ug/ml PD1 antibody, 200ng/ml CD137L, 20ng/ml IL2, 10% autologous serum.

In order to dedifferentiate effector T cells, the dedifferentiation medium in step 2) comprises AIM-V, 3-21mg/ml trimethylolethane, 0.4-2mg/ml BaCL2, 30-80ug/ml everolimus, 1-7mg/ml KCL.

Preferably, the dedifferentiation medium comprises AIM-V, 10mg/ml trimethylolethane, 1mg/ml BaCL₂50ug/ml everolimus, 3mg/ml KCL.

In order to reduce the cost and improve the culture effect, the engineered cells inactivated by irradiation are added in the step 2) and used as trophoblast cells.

Preferably, the proportion of engineered cells that are inactivated by irradiation is 1/100.

In order to improve the amplification efficiency, the amplification medium in step 3) comprises AIM-V, IL2 with the concentration of 9-32ng/ml, and 2-9% autologous serum.

Preferably, the amplification medium comprises AIM-V, 20ng/ml IL2, 5% autologous serum.

Compared with the prior art, the invention has the advantages that:

1. the cultured TIL-TCM has stronger tumor body tropism and can home to the tumor position; can specifically identify tumor cells and kill tumors; can adapt to the tumor microenvironment; can survive in vivo for a long time, and has long-term anti-tumor effect; a small amount of cells can play a strong anti-tumor role, the culture time is short, and the probability of side reaction is low.

2. Simple operation and high cell proliferation speed.

Drawings

FIG. 1 is a diagram of flow cytometry analysis of the experimental group TIL-TCM of example 1.

FIG. 2 is a diagram of flow cytometric analysis of TIL-TCM as a control in example 1.

FIG. 3 is a graph of the in vitro killing rate of breast cancer by TIL-TCM cells and TCM cells of example 2.

FIG. 4 is a venous blood test of nude mice in example 3 after 2 hours of injection.

FIG. 5 is a venous blood test of the nude mice of example 36 days after injection.

FIG. 6 is a graph showing the change in tumor-bearing volume of breast cancer in each mouse in example 4.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

The invention relates to a method for culturing infiltrating T cells, which comprises the following steps: firstly, activating effector cells in the TIL and dedifferentiating the effector cells into the TCM; the purified TIL-TCM is then expanded in large quantities by engineering the cells and allowed to maintain the TCM phenotype in vitro.

The TIL in the tumor tissue is directly separated and amplified, and is differentiated into the TCM through induction activation, so that the TIL remembers the tumor antigen information of a patient and is trained in the tumor microenvironment for a long time. Therefore, the TIL-TCM has extremely strong tumor specificity and tropism, can adapt to the tumor microenvironment specificity, efficiently kills the tumor, and can infiltrate into the tumor.

According to research, other cells in the TIL, such as Treg, gamma T and other immunosuppressive lymphocytes, do not dedifferentiate into the TCM, so that the TCM obtained by dedifferentiating effector T cells in the TIL has an anti-tumor effect through in vitro induction, and the TIL-TCM can be expanded in vitro for a long time, can survive for a long time without the assistance of IL2 in vivo, maintains the function of the TCM and has good activity.

TIL can be activated by tumor cells in vivo, so that it is proliferated and differentiated into effector T cells in vivo in a large amount, and a long-term antitumor effect can be achieved without requiring a huge amount of cells.

Example 1

Tumor tissue samples were obtained from a 34 year old breast cancer patient and approximately 3cm of tumor tissue was surgically removed³Placing into tissue preservation solution; engineering K562 cells stably expressing membrane proteins such as IL7, IL21, IL15, CD137L and the like are constructed.

1. Tumor tissue was removed with forceps and placed in a 10cm dish, quickly sterilized with 75% ethanol for 30s, washed 2 times with PBS, and transferred to another 10cm dish.

2. Cutting tumor tissue into tissue blocks with appropriate size with surgical scissors, fixing the tissue blocks with ophthalmologic forceps, and cutting the tissue blocks into 1mm in the same direction with surgical blade³Size.

3. The cut tissue blocks were uniformly dispersed in a 10cm petri dish and dried for 1 hour.

4. And slowly adding an activating culture medium along the inner wall of the culture dish, and supplementing liquid every other day for relieving the suppression state of the effector T cells.

The activation medium comprises a basic medium mixed by 1640 and AIM-V1:1, 55-100ng/ml CD28, 20-60ug/ml PD1 antibody, 70-300ng/ml CD137L, 10-40ng/ml IL2, and 2-20% autologous serum. AIM-V enables tumors to release antigens, specifically activating effector T cells via the TCR-MHC pathway; CD28 and CD137L can be used as co-stimulators to better activate effector T cells; the PD1 antibody blocks immune checkpoints and thereby relieves tumors of immunosuppression of effector T cells; IL2 is an additive necessary for immune cell expansion; the serum provides nutrition. Preferably, the activation medium comprises a basal medium of 1640 and AIM-V1:1 mix, 80ng/ml CD28, 40ug/ml PD1 antibody, 200ng/ml CD137L, 20ng/ml IL2, 10% autologous serum.

5. On the fifth day, nonadherent cells and tumor tissue blocks are collected, filtered by a 45um filter screen, and the filtered cell suspension and lymphocyte separation liquid are centrifuged at 1:1 and 2200rpm for 15 min.

6. Collecting the middle tunica albuginea layer in a 15ml centrifuge tube, adding physiological saline to 12ml, centrifuging at 1500rpm for 5 min.

7. The supernatant was discarded, the cell pellet was scattered, 12ml of physiological saline was added, and centrifugation was carried out at 1500rpm for 5 min.

8. The supernatant was discarded and resuspended in amplification medium for counting. Cells were adjusted to 1X10^6cells/100ml and inoculated into T175 flasks. The flasks were equally divided into two groups, experimental and control. The experiment group is added with a dedifferentiation culture medium for 24 hours and then is changed into an amplification culture medium. The control group was always in the amplification medium.

The dedifferentiation medium comprises AIM-V, 3-21mg/ml trimethylolethane, 0.4-2mg/ml BaCL₂30-80ug/ml everolimus, 1-7mg/ml KCL, wherein AIM-V is basal medium, trimethylolethane is used as inducing factor for dedifferentiation of effector cells to central memory T cells, BaCl₂The inducing factor for the de-differentiation of effector cells to central memory T cells, the everolimus for maintaining central memory T cells and preventing the re-differentiation into other cells, and the KCL for the inducing factor for the de-differentiation of effector cells to central memory T cells. Preferably, the dedifferentiation medium bagIncluding AIM-V, 10mg/ml trimethylolethane, 1mg/ml BaCL₂50ug/ml everolimus, 3mg/ml KCL.

Amplification medium includes AIM-V, IL2 at 9-32ng/ml, 2-9% autologous serum. Wherein AIM-V is a basic culture medium, IL2 is used for expanding lymphocytes, autologous serum is used for providing essential nutrients for cell culture, and CD28 is used for better activating T lymphocytes. Preferably, the amplification medium comprises AIM-V, 20ng/ml IL2, 5% autologous serum.

9. In the experimental group, 1/100 engineered cells inactivated by irradiation were added to each flask, and the T175 flask was placed at 37 ℃ in a 5% CO2 incubator and replenished every other day.

1/100 parts of PBMC of different healthy persons inactivated by irradiation were added to each flask of the control group, and the T175 flask was placed at 37 ℃ in a 5% CO2 incubator and replenished every other day;

10. on day 21, cells were harvested from experimental and control groups. The harvested cells were analyzed by flow cytometry to show triple positivity for TCM markers CD45RO, CD62L, CCR7 to obtain TIL-TCM purity, the results are shown in fig. 1 and fig. 2.

Analysis of results

As shown in fig. 1, the purity of the chemokine receptor CCR7 expressing cells in the experimental group was as high as 89.8% as shown in fig. 1A, and the purity of the cells double positive (second quadrant) for CD45RO +, CD62L + was as high as 86.4% as shown in fig. 1B. It can be seen that the cells in the experimental group are high-purity TIL-TCM cells, and the cell homing ability and tumor body tropism are strong.

As shown in fig. 2, the control group was shown to have 29.9% purity of CCR7 expressing chemokine receptor in fig. 2A, 45.1% of effector T cells and 40.8% of nondominant cells in the majority of cells obtained in the control group, CD45RO- (first and fourth quadrants), and only 14% of cells with double positive CD45RO +, CD62L + (second quadrant) in fig. 2B. Therefore, the content of TIL-TCM in the control group is very low, and the cell homing ability and tumor body tropism are lost.

Example 2

The tumor tissue sample was obtained from a 57 year old breast cancer patient, and the tumor tissue was surgically removed to about 12cm3 and placed in a tissue preservation solution

1. Preparing target cells:

1.1A portion of the tumor tissue was removed with forceps and placed in a 10cm petri dish, quickly sterilized with 75% ethanol for 30s, and washed 2 times with PBS. Transfer to another 10cm petri dish.

1.2 after removing fat, connective tissue and necrotic tissue from the tumor tissue, the tumor tissue was cut into pieces, transferred to a 50ml centrifuge tube, and digested for 12 hours with 0.2% collagenase, NB 4G.

1.3 the undigested tissue was filtered off with a 200 mesh sieve and the filtrate was inoculated to a cell size of 1X10^6 into a T75 flask to replenish the medium completely.

1.4 when the cells grow to 80%, the cells are digested and transferred to a 24-well plate for culture of target cells.

1.5 taking target cells with good growth state, centrifuging at 1000rpm/min for 5min, resuspending with complete culture medium, counting with Taiwan phenol blue to detect cell viability, and adjusting cell number to 2x10^5/ml and 2ml (50 ul/hole, total 21 holes).

2. Another part of the tumor tissue was cultured according to the method of the experimental group in example 1 to obtain TIL-TCM cells.

3. Preparing effector cells:

taking TIL-TCM cells, resuspending, centrifuging at 1500rpm/min for 5min × 2 times, counting the activity rate of the phloroglucinol blue, resuspending in a complete culture medium, and adjusting the number of the cells to be 4 × 106/ml (according to the maximum effective target ratio of 20:1,50 ul/hole and 6 holes), so as to obtain effector cells for later use. Target cell 10⁴Treatment of effector cells at well site, target-effective ratio E: T ═ 20:1, 10:1, 5:1, 2:1, 1:1, 0.5: 1:

20: 1: adjusting the number of TIL-TCM cells to 4x10⁶1ml, mixed well, 50 ul/well, 6 wells.

10: 1: 500ul of the TIL-TCMT cells at a ratio of 20:1 are taken, 500ul of complete culture medium is added and mixed evenly, 50 ul/hole and 6 holes are formed.

5:1 ratio: 500ul of TIL-TCM cells at a ratio of 10:1 was added to 500ul of complete medium and mixed well at 50 ul/well in 6 wells.

2:1 ratio: 400ul of TIL-TCM cells at a ratio of 5:1 are taken, 600ul of complete culture medium is added, and the mixture is uniformly mixed at 50 ul/hole and 6 holes.

1:1 ratio: 500ul of TIL-TCM cells (2: 1) was added to 500ul of complete medium and mixed well at 50 ul/well in 6 wells.

0.5: 1: 500ul of TIL-TCMT cells in the ratio of 1:1 are taken, 500ul of complete culture medium is added and mixed evenly, 50 ul/hole and 6 holes are formed.

4. TIL-TCM cytotoxicity assay:

and adding the treated TIL-TCM and target cells into a 96-well plate according to different effective target ratios of E to T of 0.5:1, 1:1, 2:1, 5:1, 10:1 and 20:1, and adding complete culture medium into a well with the volume less than 100ul to make up 100 ul. And (3) taking target cells with corresponding cell numbers and TIL-TCM as a control, taking the culture medium with the same volume as a blank control, and adding PBS with the same volume to the peripheral holes of the experimental holes for overnight culture (16-24 h).

5. The following day, 10% CCK-8 was added to each well, incubated at 37 ℃ in a 5% CO2 incubator for 2-4 h, and then measured for OD at 450nm while setting 630nm as a reference wavelength, and the killing rate was calculated and a standard curve was drawn, and the results are shown in FIG. 3.

The killing rate (%) - (control target cell OD value + corresponding control CTL OD value-experimental OD value-blank medium OD value)/(control target cell OD value-blank medium OD value) x 100%

Control group

1. Taking 50ml of peripheral blood of a healthy person, and preparing TCM according to a traditional method:

2.1 gently transferring the collected blood sample into 450 ml centrifuge tubes filled with 20ml of lymphocyte separation liquid, and centrifuging at 2200rpm for 20 min;

2.2 sucking the middle leucocyte layer into a new 50ml centrifuge tube by a Pasteur pipette, and centrifuging for 5min by using 1800rpm of normal saline to wash for 2 times;

2.3 enriching T cells by CD3 magnetic beads, removing other natural immune cells, and washing for 2 times by centrifuging for 5min at 1800rpm of physiological saline;

2.4 using AIM-V culture medium to resuspend the cell sediment and count, and inoculating the cell sediment in a T75 culture bottle according to the density of 2-3 multiplied by 10^ 6/ml; adding 30mL AIM-V, 1000ng/mL interleukin-2, 5ng/mL interleukin-7, 5ng/mL interleukin-15, 3 mug/mL Anti-CD3 antibody and 5% autoplasma, and culturing in a 5% carbon dioxide incubator at 37 deg.C;

2.5 on the seventh day TCM cells were harvested and the killing experiments were performed as described above and standard curves were drawn with the results shown in FIG. 3.

Analysis of results

As shown in fig. 3, the TCM curve is the tumor cell killing rate of the control group cells, since TCM in the peripheral blood of healthy people does not memorize the corresponding tumor antigen, there is no killing effect on the target cells, and the expanded TCM in the peripheral blood has no killing effect on the tumor regardless of the effective target ratio. The TIL-TCM curve in fig. 3 is the tumor cell killing rate of the experimental group cells, and shows that the killing efficiency is 18% when the target ratio of TIL-TCM to breast cancer is 0.5:1, 57% when 1:1, 97.73% when 5:1, close to 100%, and the killing ability is strong.

Example 3

TIL-TCM and TIL prepared in example 1 were injected into two identical weight nude mice with severe immunodeficiency of the same lineage, and the survival of different cells in vivo was observed. The tail venous blood of the nude mice was collected at 2 hours and 6 days, and the content of CD45 (human leukocyte) was measured by flow assay, and the results are shown in fig. 4 and 5.

Analysis of results

FIG. 4 shows venous blood measurements of nude mice 2 hours after injection. As shown in FIG. 4, both TIL-TCM and TIL cells had been circulating in peripheral blood of nude mice 2 hours after infusion of nude mice, wherein the content of TIL cells was 45.1% and the content of TIL-TCM cells was 47.4%, and the ratio of both in peripheral blood cells of mice was not significantly different.

FIG. 5 shows venous blood measurements taken 6 days after injection in nude mice. As shown in FIG. 5, the content of TIL-TCM cells in nude mice was 51.0%, and the cell ratio was slightly increased compared to the data obtained by 2 hours of injection, indicating that TIL-TCM was mostly alive and slightly proliferated in mice. The content of the TIL cells is 9.4%, the cell ratio is obviously reduced, which shows that the TIL cells are obviously metabolized after 6 days in a nude mouse, and only a small part of the cells survive.

Example 4

1. 8 female mice with the severe immunodeficiency (severe immunodeficiency phenotype, no mature T cells, B cells, functional NK cells, deficiency of cytokine signal transmission capability and the like, which are very suitable for transplanting and growing human hematopoietic stem cells and peripheral blood mononuclear cells) of 5-week old and similar body weight are averagely divided into A, B, C, D groups of 4 groups;

inoculating 8 mice with primary breast cancer cells at 1x10E 6/mouse subcutaneously, measuring the tumor size at 7 days, 14 days and 21 times respectively, wherein the cell viability is 93%;

2. on day 21, mice were injected intravenously with immune cells from the tail as follows:

group A is blank group, and normal saline is injected;

group B injected high concentration TIL cells, injected 1X10⁹The TIL cell of (1);

group C injections of low concentration of TIL cells, 1X10⁷The TIL cell of (1);

group D was injected with low concentration TCM-TIL cells, 1X10⁷The TIL-TCM cell of (a);

3. post-infusion reactions were observed: 2 mice in the group B all have reactions such as low temperature and inactivity, wherein 1 mouse dies after 20 hours, and B1 knockout experiment is carried out; another B recovered after 248 hours, and 6 other mice did not respond significantly;

5. measuring the tumor size every 7 days, and drawing a tumor curve, wherein the result is shown in FIG. 6;

6. after 42 days, all survival of 7 mice was observed, in which the tumor bodies of group B and group D disappeared, and the tumor bodies of group C1 mice first shrunk and then grown, and the tumor bodies of the other mice were not significantly different from those of the control group.

Analysis of results

As shown in FIG. 6, the tumors of A1 and A2 mice were in a growing state, and after 35 days, the tumor mass volume of 2 mice exceeded 800mm³(for ethical reasons, 2 mice were sacrificed at this time). B1 shows strong side reaction after injecting TIL cells with high concentration, dies after 20 hours, and eliminates the experiment; b2 tumor mass disappeared after 7 days of injection with high concentration of TIL cells and no recurrence of tumor mass occurred within 42 days of observation period in this experiment. C1 shows no obvious change in tumor after injection of low concentration TIL cells, and still becomes fasterThe tumor volume of the strain is close to 600mm by day 42³(ii) a C2 tumor size was 164mm 7 days after injection of low concentration TIL cells³Reduced to 80mm³Tumor body growth to 110mm at day 35³By day 42, the tumor mass rapidly increased to 320mm³. After D1 injects low-concentration TIL-TCM, the tumor body is continuously reduced and finally disappears; d2 tumor mass disappeared on day 7 after injection of low concentration TIL-TCM cells and no recurrence of tumor mass occurred within 42 days of observation in this experiment.

In summary, the TIL cell requires a large number of cells for treating tumor cells, and has a large side effect; TIL-TCM requires a lower number of cells to be continuously antitumor.

Claims

1. A method for culturing infiltrating T cells, comprising: the method comprises the following steps: 1) preparing and forming effector T cells by utilizing tumor tissues, 2) adding a dedifferentiation culture medium and engineered cells inactivated by irradiation to dedifferentiate the effector T cells into TIL-TCM, wherein the proportion of the engineered cells inactivated by irradiation is 1/100, and 3) amplifying the TCM by using an amplification culture medium;

the dedifferentiation culture medium is AIM-V, trimethylolethane 3-21mg/ml, BaCL 0.4-2mg/ml₂30-80 mu g/ml everolimus and 1-7mg/ml KCl;

the amplification culture medium is AIM-V, IL2 of 9-32ng/ml, and 2-9% autologous serum.

2. The method for culturing infiltrating T-cells according to claim 1, wherein: the effector T cells in the step 1) are activated lymphocytes in peripheral blood by using an activation medium.

3. The method for culturing infiltrating T-cells according to claim 2, wherein: the activation culture medium is a basal culture medium mixed with 1640 and AIM-V1:1, 55-100ng/ml CD28, 20-60 mu g/ml PD1 antibody, 70-300ng/ml CD137L, 10-40ng/ml IL2 and 2-20% autologous serum.

4. The method for culturing infiltrating T-cells according to claim 3, wherein: the activation medium is a basal medium mixed by 1640 and AIM-V1:1, 80ng/ml CD28, 40 mug/ml PD1 antibody, 200ng/ml CD137L, 20ng/ml IL2 and 10% autologous serum.

5. The method for culturing infiltrating T-cells according to claim 1, wherein: the dedifferentiation culture medium is AIM-V, 10mg/ml trimethylolethane and 1mg/ml BaCl₂50ug/ml everolimus, 3mg/ml KCl.

6. The method for culturing infiltrating T-cells according to claim 1, wherein: the amplification medium is AIM-V, IL2 with the concentration of 20ng/ml and 5% of autologous serum.