CN109913413B - PD-1 antibody loaded T cell in-vitro culture method, cell preparation and application thereof - Google Patents
PD-1 antibody loaded T cell in-vitro culture method, cell preparation and application thereof Download PDFInfo
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Abstract
The embodiment of the invention discloses a PD-1 antibody loaded T cell in-vitro culture method, a cell preparation and application thereof, relating to the technical field of immunotherapy, in particular to the culture method comprising the following steps: it comprises mixing peripheral blood mononuclear cells with an A-antibody culture medium for primary culture; then, mixing the product obtained by the first culture with a B-type culture medium for the second culture; finally, the product obtained after the second culture is mixed with a K-type culture medium. The culture method can induce lymphocytes separated from whole blood to proliferate and differentiate towards CD8+ T cells in the culture process, the separated T cells can achieve more than 40 times of amplification within 18 days, and the proportion of CD8+ T cells is more than 80%.
Description
Technical Field
The invention relates to the technical field of immunotherapy, and particularly relates to a PD-1 antibody loaded T cell in-vitro culture method, a cell preparation and application thereof.
Background
At present, the cancer still cannot overcome the difficult problem of diseases, and the immunotherapy in 2013 is praised as the method most possibly for curing the cancer by 'science' journal. Immune cell therapy is the key part of immunotherapy, starting from the last 70 th century, LAK cells (peripheral blood is extracted from a human body, lymphocytes are separated from the peripheral blood in vitro, interleukin-2 with a large dose is added for in vitro culture and proliferation, then the lymphocytes with multiple times of proliferation are obtained and named as LAK cells, and then a preparation of the cultured LAK cells which are prepared into cell suspension is infused into the human body intravenously) are the earliest immune cell in vitro culture technology applied to cancer treatment after the immune cells are cultured in vitro.
By about 2000 years, the new generation of immune cells, CIK cells, is applied to clinical cancer treatment, compared with the LAK cell in vitro culture technology, the method mainly adopts CD3 monoclonal antibody and a plurality of cytokines (interleukin-2, plus code-interferon and the like) without stimulating cells by large dose of interleukin-2, but CIK cells have low proliferation multiple, low ratio of killer T cells and unobvious curative effect when used for treating cancers, the culture technology is that CD3 monoclonal antibody is added to complete culture medium to activate cell proliferation, then interleukin-2 is added to follow-up culture to maintain cell proliferation, since the culture can only proliferate various cells mixed together, the ratio of the most important CD8+ cells after the culture is not high (about 50%), the CD8+ T cells are not very lethal (cytotoxic) due to the mixed cells, and the therapeutic effect (anti-cancer cell effect) of the infusion on cancer patients is also not strong.
Then the DC cells are discovered and applied to in vitro culture, and the DC-CIK cells formed by co-culturing the DC cells and the CIK cells technically stimulate the CIK cells to have better proliferation multiple and cell functions by leading the DC cells to present antigen. Therefore, clinical efficacy is better than CIK cells, but CD8+ T cells are also not as lethal (cytotoxic).
In 2017, CAR-T cells are approved in the United states, CAR-T cells are obtained by taking peripheral blood of patients and separating lymphocytes in vitro, then culturing and differentiating the lymphocytes into CD8+ T cells to obtain a certain number, then using adenovirus as a carrier to embed a recognition gene of CD19 into the DNA of the cells to obtain T cells embedded with specific antigen receptors, and the CAR-T cells are named as CAR-T cells, can specifically recognize certain types of B cell leukemia, thereby specifically attacking the cancerated cells of the B cell leukemia, destroying the cancerated cells to treat the leukemia, and have successful cure cases.
In 2017, the PD-1 antibody as a cancer treatment drug is approved to be marketed, two national pharmaceutical companies obtain K in the Shishadong and O in the Guibao, the drug is bombed in the market, and the effective rate can reach 40 percent (the effective rate of cancer drugs such as chemotherapy drugs and targeted drugs is high and is more than ten percent) and is known as an anti-cancer drug.
However, the curative effect of the PD-1 antibody can only be achieved for a part of patients, because PD-L1 is generated by tumors, and other tumors are not caused by PD-L1, or the PD-1 antibody drug has no effect, and the PD-1 antibody drug has great toxic and side effects.
Disclosure of Invention
A first object of the present invention is to provide a method for culturing T cells, which can induce proliferation and differentiation of lymphocytes isolated from whole blood into CD8+ T cells during the culture process, thereby preparing killer T cells, and which can achieve 40-fold or more increase in the isolated T cells within 18 days, and the percentage of CD8+ T cells exceeds 80%.
It is a second object of the present invention to provide a culture medium composition for T cells which can efficiently culture a large number of T cells having lethality in a short period of time.
The third purpose of the invention is to provide a T cell (called PD-1-T cell, PD-1 antibody is added in the late culture period, so that PD-1 molecule on the T cell is combined and sealed by the PD-1 antibody, and the T cell is called PD-1-T cell), the T cell has the advantages of high function of secreting cell factors, strong aggressivity to tumor cells, no influence of PD-L1 of the tumor cells, and broad-spectrum anticancer.
The fourth object of the present invention is to provide a cell preparation comprising the above-mentioned T cells having a strong killing property, which has a better anticancer effect than DC-CIK cells.
The fifth purpose of the invention is to provide the application of the killer T cells or the cell preparation in preparing anti-cancer drugs.
The invention is realized by the following steps:
the embodiment of the invention provides a T cell culture method, which comprises the following steps: culturing peripheral blood mononuclear cells, and adding the PD-1 antibody to the culture solution in the later culture period to continue the culture.
It is noted that the PD-1 antibody drug acts as follows: PD-1 is expressed on T cells (on a membrane) in immune cells, and PD-L1 is expressed on (on a membrane) or secreted (exosome) in tumor cells, however PD-L1 is a PD-1 ligand, and the binding of the PD-L1 can enable the T cells to enter apoptosis and lose functions, so scientists find that the tumor cells escape from T cell killing due to PD-L1, then PD-1 molecules on the T cells are blocked by using a PD-1 antibody, PD-L1 on the tumor cells cannot touch PD-1 on the T cells, and the T cells cannot be contacted with PD-L1 in tumor tissues to die and can kill the cancer cells instead of killing the functions of the cancer cells.
According to the action principle of the PD-1 antibody drug, the PD-1 antibody drug does not directly kill cancer cells, only protects T cells in the body and plays a role in killing cancer cells or T cells. However, the generation of PD-L1 by tumors is only one reason, if the reason is that the PD-1 antibody medicament is used, the curative effect is necessarily good, the melanoma is completely cured after the PD-1 antibody medicament is used, and the tumors disappear. However, if the tumor generation is not caused by PD-L1, the use of the PD-1 antibody drug has no effect and the PD-1 antibody drug has great toxic and side effects.
The immune system of human or animal can eliminate the variant cells (cancer cells), senescent cells and necrotic cells, thus keeping the health of the body, and the immune system performs the immune function of various immune cells (white blood cells), wherein the lymphocytes mainly play a role in eliminating the cancer cells, and the T cells in the lymphocytes, particularly the CD8+ T cells, are the most important immune cells for performing the elimination function.
The invention takes the T cell as a therapeutic substance, adds the PD-1 antibody as a PD-1 molecule on a closed T cell membrane, and does not take the PD-1 antibody as an antibody drug, and meanwhile, the culture method provided by the embodiment of the invention enhances the lethality of the T cell, and can effectively avoid the loss of functions of the T cell due to the PD-L1 possibly existing on the tumor cell and the side effect of the PD-L1 on a user.
Preferably, the culturing includes a first culturing including mixing peripheral blood mononuclear cells with an a-antibody medium; then, mixing the product obtained by the first culture with a B-type culture medium for the second culture; and finally, mixing the product obtained after the second culture with a K-type culture medium for the third culture.
The A-antibody culture medium comprises a complete culture medium, a CD3 monoclonal antibody and one or more of the following components: interleukin-2, vitamin B6, interferon-gamma, interleukin-1B, interleukin-4, L-glutamine, sodium pyruvate, and plasma protein;
the K-type culture medium comprises a complete culture medium, a PD-1 antibody and one or more of the following components: interleukin-2, L-glutamine, sodium pyruvate, and plasma proteins;
the B-type culture medium comprises complete culture medium, CD28 antibody and one or more of the following components: interleukin-2, vitamin C, L-glutamine, sodium pyruvate, and plasma protein.
Further, in the A-antibody culture medium, the final concentration of the CD3 monoclonal antibody is 80-120 ng/ml; preferably, in the A-antibody culture medium, the final concentration of interleukin-2 is 800-1200 u/ml, the final concentration of vitamin B6 is 5-15 ng/ml, the final concentration of interferon-gamma is 15-25 ng/ml, the final concentration of interleukin-1B is 5-15 ng/ml, the final concentration of interleukin-4 is 5-15 ng/ml, the final concentration of L-glutamine is 180-220 ng/ml, the final concentration of sodium cuprate is 15-25 ng/ml, and the final concentration of plasma protein is 40-60 ng/ml.
In the A anti-culture medium, the CD3 monoclonal antibody can activate CD3 molecules on an leukocyte membrane to differentiate the leukocyte to T cells, and interleukin-2 can stimulate the differentiation and proliferation of the leukocyte; vitamin B6 can enhance leukocyte amino acid utilization, activate cell protein synthesis, and enhance cell function; interferon-gamma is capable of differentiating leukocytes into CD8+ T cells; the interleukin-1 b can activate a trace amount of DC cells contained in the separated mononuclear cells, so that the DC cells secrete IL-12 to ensure that the subsequent CD8+ T cells are more lethal; interleukin-4 makes CD4+ T cells change from TH2 subtype to TH1 subtype, thus make CD4+ T cells secrete mainly interferon-gamma, tumor necrosis factor-alpha, make subsequent culture strengthen CD4+ T cells and secrete mainly cytokine function and proliferate for a time, therefore when the in vitro culture reaches 14 days, CD4+ T cells are reduced to 20% from 60% at the beginning gradually; l-glutamine provides cellular energy; sodium cuprate helps cells to utilize energy substances (glucose); plasma proteins are capable of maintaining the extracellular environment and protecting the cell membrane.
5-15 ng/ml of CD28 antibody in a B-type culture medium; preferably, in the B-type culture medium, the final concentration of interleukin-2 is 1800-2200 u/ml, the final concentration of vitamin B6 is 5-15 ng/ml, the final concentration of vitamin C is 5-15 ng/ml, the final concentration of L-glutamine is 180-220 ng/ml, the final concentration of sodium cuprate is 15-25 ng/ml, and the final concentration of plasma protein is 15-25 ng/ml.
Specifically, in a B-type culture medium, the CD28 antibody enhances the function of T cells, and accelerates the proliferation of CD8+ T cells, and the subsequent culture of the CD8+ T cells has stronger killing function and also expresses more PD-1; vitamin C can enhance chemotaxis and deformability of leukocytes, and enhance the ability to destroy bacteria and other abnormal cells. The addition of the cell culture medium helps to eliminate residual impurity cells in initial separation, so that the cultured cell line tends to be pure, and CD8+ T cells are taken as main cells when the culture is finished. In addition, interleukin-2, L-glutamine, sodium pyruvate, and plasma proteins were incubated with A antibody.
In a K-type culture medium, the final concentration of the PD-1 antibody is 5-15 ng/ml; preferably, in the K-type culture medium, the final concentration of interleukin-2 is 1800-2200 u/ml, the final concentration of L-glutamine is 40-60 ng/ml, the final concentration of sodium cuprate is 3-8 ng/ml, and the final concentration of plasma protein is 40-60 ng/ml.
Specifically, the second culture was carried out when cell proliferation was initiated and cell clumps were formed and the cell density gradually increased, and the cell density in the flask was observed under an inverted microscope until the cell density reached 2X 107～5ⅹ107The third culture was performed at one/ml.
Preferably, the number of culture days of the first culture is 3 days, 4 days, 5 days or 6 days, and the number of culture days of the second culture is 10 days, 11 days or 12 days. More preferably, the number of days of culture in the first culture is selected from 3 to 6 days, and the number of days of culture in the second culture is selected from 10 to 12 days.
Further, the culturing method comprises adding the C-type medium after the second culturing or after adding the K-type medium. Preferably, the cell density in the flask is observed under an inverted microscope between the second and third culturing until the cell density reaches 2 x 107～5ⅹ107Add type C medium per ml. After the third cultivation, the cell density reached 2 x 107～5ⅹ107Add type C medium per ml.
Specifically, the C-type culture medium comprises complete culture medium, CD28 antibody and one or more of the following components: interleukin-2, vitamin C, vitamin B6, L-glutamine, sodium pyruvate, and plasma protein;
preferably, in the type C culture medium, the final concentration of the CD28 antibody is 3-8 ng/ml, the final concentration of interleukin-2 is 450-550 u/ml, the final concentration of vitamin B6 is 3-8 ng/ml, the final concentration of vitamin C is 3-8 ng/ml, the final concentration of L-glutamine is 40-60 ng/ml, the final concentration of sodium cuprate is 3-8 ng/ml, and the final concentration of plasma protein is 5-15 ng/ml.
In type C media, vitamin C enhances chemotaxis and deformability of leukocytes, and enhances the ability to destroy bacteria and other abnormal cells. The addition of the cell culture medium helps to eliminate residual impurity cells in initial separation, so that the cultured cell line tends to be pure, CD8+ T cells are taken as the main cells when the culture is finished, and the killing function of the cells is enhanced.
The embodiment of the invention also provides a culture medium combination of the T cells, which comprises an A-type anti-culture medium, a K-type culture medium and a B-type culture medium.
The A-antibody culture medium comprises a complete culture medium, a CD3 monoclonal antibody and one or more of the following components: interleukin-2, vitamin B6, interferon-gamma, interleukin-1B, interleukin-4, L-glutamine, sodium pyruvate, and plasma protein.
The K-type culture medium comprises complete culture medium, PD-1 antibody and one or more of the following components: interleukin-2, L-glutamine, sodium pyruvate, and plasma proteins.
The type B culture medium comprises complete culture medium, CD28 antibody and one or more of the following components: interleukin-2, vitamin C, L-glutamine, sodium pyruvate, and plasma protein.
Further, the culture medium combination can also comprise C-type culture medium.
Preferably, the concentrations of the components of the culture medium in the combination of culture media are referred to the concentrations of the components in the above-mentioned culture method, and are not described in detail herein.
The embodiment of the invention also provides a killer T cell, which is prepared by the culture method.
The embodiment of the invention also provides a cell preparation which comprises the cell back infusion fluid and the T cells prepared by the culture method.
The cell back infusion solution comprises the following components: 80-120 ml of normal saline, 2-7 g of human serum albumin, gentamicin (7-9 ten thousand units), interleukin-2 (9-11 ten thousand units), 8-12 mg of L-glutamine and 3-8 mg of sodium propyl cuprate.
Wherein, the physiological saline acts as isotonic solution; the human serum albumin maintains the cell environment in the transportation process, protects cell membranes and keeps the cell activity; gentamicin prevents contamination during injection; interleukin-2 contributes to survival during cell trafficking; l-glutamine facilitates cellular energy supply during cell transport; the sodium cuprate is helpful for cells to utilize energy substances to maintain normal metabolism in the transportation process.
The embodiment of the invention also provides application of the T cell or the cell preparation in preparing an anti-cancer medicament.
Specifically, the anticancer drug refers to anti-skin cancer, lung cancer, prostate cancer, head and neck cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, adrenal cancer, AIDS-related cancer, soft tissue alveolar sarcoma, astrocytoma, bone cancer, brain and spinal cord cancer, metastatic brain tumor, carotid aneurysm, chondrosarcoma, chordoma, renal chromophobe cell cancer, clear cell cancer, benign fibrosarcoma of the skin, desmoplastic small round cell tumor, ependymoma, ewing's tumor, extraosseous mucoid chondrosarcoma, incomplete fibrogenesis of bone, dysplasia of bone fibers, cancer of gallbladder or bile duct, gestational trophoblastic disease, germ cell tumor, hematologic malignancy, hepatocellular carcinoma, islet cell tumor, kaposi's sarcoma, kidney cancer, lipoma/benign lipoma, liposarcoma/malignant lipoma, A medulloblastoma, meningioma, merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disorders, renal metastasis, rhabdoid tumor, rhabdomyosarcoma, sarcoma, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, bursal sarcoma, testicular cancer, thymus tumor, thyroid metastasis, or uterine cancer;
preferably, the anticancer drug is an anti-cervical cancer drug or an anti-leukemia drug.
The invention has the following beneficial effects:
the embodiment of the invention provides a T cell culture method, which can induce lymphocytes separated from whole blood to proliferate and differentiate towards CD8+ T cells in the culture process, the separated T cells can achieve more than 40 times of amplification within 18 days, and the proportion of CD8+ T cells exceeds 80%.
In addition, the embodiment of the invention also provides a T cell, a culture medium combination thereof, application thereof and a cell preparation, wherein the killer T cell has higher function of secreting cell factors, has high proliferation speed, has stronger aggressivity to tumor cells than DC-CIK cells, and has the advantage of broad-spectrum anticancer.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a comparison of cell counts of DC-CIK cells and PD-1-T cells in comparative example 1 according to the present invention;
FIG. 2 is a graph showing the comparison result of the CD8+ ratio in comparative example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Killer T cells were obtained as follows.
Peripheral blood (150 ml) was drawn or cord blood was collected and mononuclear cells (containing predominantly lymphocytes, but also other white blood cells and small amounts of red blood cells) were isolated using lymphocyte separation and gradient centrifugation.
First culturing
Initial culture the isolated mononuclear cells were mixed with A-antibody medium, placed in a cell culture flask, and incubated at 37 deg.C with 5% CO2The culture is carried out in a constant temperature and humidity carbon dioxide incubator.
The composition of the A anti-culture medium is as follows:
complete culture medium, CD3 monoclonal antibody, interleukin-2, vitamin B6, interferon-gamma, interleukin-1B, interleukin-4, L-glutamine, sodium pyruvate and plasma protein.
Wherein the final concentration of the CD3 monoclonal antibody is 100 ng/ml; preferably, in the A anti-culture medium, the final concentration of interleukin-2 is 1000u/ml, the final concentration of vitamin B6 is 10ng/ml, the final concentration of interferon-gamma is 20ng/ml, the final concentration of interleukin-1B is 10ng/ml, the final concentration of interleukin-4 is 10ng/ml, the final concentration of L-glutamine is 200ng/ml, the final concentration of sodium cuprate is 20ng/ml, and the final concentration of plasma protein is 50 ng/ml.
Second culturing
Culturing in constant temperature and humidity carbon dioxide incubator, observing cell growth every day, and allowing cell proliferation to start and form cell mass with cell density of 3 x 107When the culture medium is added per ml (different from 3 to 6 days of culture), the type B culture medium is added. Specific information on type B medium is as follows.
The B-type culture medium comprises complete culture medium, CD28 antibody, interleukin-2, vitamin C, L-glutamine, sodium pyruvate and plasma protein.
Wherein, the CD28 antibody is 10 ng/ml; preferably, in the B-type medium, the final concentration of interleukin-2 is 1000u/ml, the final concentration of vitamin B6 is 10ng/ml, the final concentration of vitamin C is 10ng/ml, the final concentration of L-glutamine is 100ng/ml, the final concentration of sodium cuprate is 10ng/ml, and the final concentration of plasma protein is 20 ng/ml.
It should be noted that the growth of cells was observed every day after addition of type B medium in the culture, depending on the cell density in the culture medium reaching 3 x 107Adding C-type culture medium at each mlCulturing the medium, and performing flask division according to the culture fluid amount, and culturing for 10 days. Specific information for type C media is as follows:
the type C culture medium comprises complete culture medium, CD28 antibody, interleukin-2, vitamin C, vitamin B6, L-glutamine, sodium pyruvate and plasma protein. Wherein the final concentration of the CD28 antibody is 5ng/ml, the final concentration of interleukin-2 is 500u/ml, the final concentration of vitamin B6 is 5ng/ml, the final concentration of vitamin C is 5ng/ml, the final concentration of L-glutamine is 50ng/ml, the final concentration of sodium cuprate is 5ng/ml, and the final concentration of plasma protein is 10 ng/ml.
Third culturing
After the second culture, the cell density reached 3 x 107At one/ml, K-type medium was added to obtain killer T cells (PD-1-T cells). Specific information on the type K medium is as follows.
The K-type culture medium comprises complete culture medium, PD-1 antibody, interleukin-2, L-glutamine, sodium pyruvate and plasma protein. Wherein the final concentration of the PD-1 antibody is 10 ng/ml; the final concentration of interleukin-2 is 1000u/ml, the final concentration of L-glutamine is 50ng/ml, the final concentration of sodium cuprate is 5ng/ml, and the final concentration of plasma protein is 50 ng/ml.
The growth of the cells was observed daily after addition of K-type medium, depending on the appropriate cell density in the culture (when the cell density reached 3 x 10)7One/ml) was added with C-type medium, and flask division was performed according to the amount of the culture medium.
When the preparation needs to be used, a culture product (namely PD-1-T cells) is separated by a gradient centrifugation method, added into the cell back infusion liquid, mixed uniformly to form a cell suspension, filled into an infusion bottle and packaged to prepare the PD-1-T cell preparation.
Wherein, the cell back transfusion liquid comprises the following components: 100ml of physiological saline, 5g of human serum albumin, gentamicin (8 ten thousand units), interleukin-2 (10 ten thousand units), 10mg of L-glutamine and 5mg of sodium cuprate.
Example 2
The cell preparation provided in example 1 is used for treating cervical cancer patients.
The PET-CT examination result of the cervical cancer patient in 2017, 12 months and 29 days is as follows:
1. after cervical cancer surgery, uterine and bilateral adnexa are deficient; no sign of malignant tumor is seen at the vaginal stump;
2. the left supraclavicular fossa lymph node metastasis seen in the last imaging (2017-7-11) disappears, the left supraclavicular fossa soft tissue is disordered, and the metabolism is slightly increased, and is considered to be changed after treatment;
3. after the peritoneum of the upper abdomen, a plurality of slightly enlarged lymph nodes are removed, the metabolism is slightly increased, and the metabolism is reduced compared with the last imaging considering the lymph node inflammation;
4. 1 nodular hypermetabolic focus on the upper section of the left outer lobe of the liver is mostly considered as a liver metastasis;
5. a nodular hypermetabolic focus is seen beside the right common iliac vessel, and is considered as a lymph node metastasis, and the focus invades an adjacent ureter to cause expansion and effusion of the upper section of the right ureter and the right renal pelvis; impaired right renal function;
6. the right lower abdomen mesentery was slightly thickened and metabolism was slightly increased, considered as post-treatment change;
7. chronic inflammation of the right sphenoid and ethmoid sinus, inflammation of the nasopharynx; multiple lymphadenectasis in bilateral neck, bilateral lung portal and mediastinum; thickening the veins of the double lung; the right superior lobe segment of the right lung is near the sub-pleural benign nodules; radiation pneumonitis in the left lung apex; a little chronic inflammation exists in the lower lingual part of the upper lobe of the left lung and the medial side part of the middle lobe of the right lung; cyst of the right lobe of the liver; bilateral adrenal thickening with physiological concentration; reduction in right kidney volume; systemic subcutaneous multiple lesions; degenerative change of cervical, thoracic and lumbar vertebrae.
CT examination results at 8 months and 22 days in 2018:
1. the change after the self-tributary resection, thickening of the vaginal stump-rectal fascia and soft tissue shadow, and recommending follow-up considering the tumor recurrence and the invasion of the bladder back wall and the lower end of the right ureter;
2. the left inner lobe of the liver is a plurality of similar-circle low-density shadows of the right posterior lobe, and cysts are considered, which is basically the same as the former;
3. the density shadow of the original cystic neck type round soft tissue is not shown at this time; chronic cholecystitis;
4. degenerative change of cervical, thoracic and lumbar vertebrae.
The patient received a first course of PD-1-T cell therapy from 9 months to 15 days in 2018 and a second course of PD-1-T cell therapy from 23 days to 29 months in 2018.
The treatment administration mode in one course of treatment: one bottle of PD-1-T cell preparation contains 25-30 hundred million PD-1-T cells, and one bottle of PD-1-T cell preparation is injected every day, and 7 days are taken as a treatment course continuously.
CT examination results of 11 months and 1 day in 2018 are as follows:
1. the change after the self-tributary resection, thickening of the vaginal stump-rectal fascia and soft tissue shadow, considering the tumor recurrence, and suspicious invasion of the rear wall of the bladder and the lower segment of the right ureter, which are slightly changed compared with the front panel;
2. degeneration of lumbar vertebrae.
According to the analysis of the results, after two courses of PD-1-T cell treatment, the suspected metastasis (cyst) of the liver is completely eliminated, the chronic cholecystitis is also eliminated, and the pelvic lymph node is eliminated, in addition, before the PD-1-T cell treatment is not received, a plurality of lymph nodes are arranged near the neck and the clavicle, the lymph nodes are eliminated after the PD-1-T cell treatment is received, the condition of the disease is obviously alleviated, only lumbar hyperosteogeny is consciously caused to cause pain, and other symptoms do not exist. It is responsible for the condition of cervical cancer that the clinician says it is progressing well.
Example 3
The cell preparation provided in example 1 was validated for its therapeutic effect on leukemia patients.
The leukemia patient is discomfortable in 2017, is confirmed to be the monocytic leukemia (M5) by local hospital examination, and then receives 5 times of chemotherapy, so that the disease condition is relieved, but the blood cells are always low, and the complications such as susceptibility, body discomfort and the like are accompanied. By seeking medical advice from multiple parties, the patient receives CIK immune cell therapy in 7 months in 2018, but the effect is very little, the patient is connected with the applicant in 8 months in 2018, and receives PD-1-T cell therapy of a first treatment course (a treatment course administration mode is that one bottle of PD-1-T cell preparation contains 25-30 hundred million PD-1-T cells, one bottle of PD-1-T cell preparation is injected every day, and 7 continuous days are taken as one treatment course) in 8 days in 9 months in 2018 years. The results before and after treatment are as follows.
Examination results before 9/8 days of PD-1-T cell reinfusion: the patient's white blood cells, red blood cells, hemoglobin, hematocrit, platelets, neutrophils, and lymphocytes were all below normal, and the monocyte, eosinophil, and basophil ratios were all higher than normal.
The results of the examination after the fifth needle (13 days at 9 months) of the first course of PD-1-T cells (one needle per day, 7 needles in total) were reinfused: the patient's red blood cells, hemoglobin and lymphocytes are all below the normal range of values and the values for monocyte, eosinophil and basophil ratios are all above normal.
The results of the patient family members are shown in Table 1:
TABLE 1 test results
| Date | Hemoglobin | White blood cell | Blood platelet | Red blood cell | Neutrophils |
| Range of normal value | 115～150 | 3.5～9.5 | 125～350 | 3.8～5.1 | 1.8～6.3 |
| 9 month and 8 days | 101 | 1.72 | 100 | 3.3 | 0.7 |
| 9 months and 13 days | 113 | 3.39 | 163 | 3.6 | 1.99 |
| 9 month and 21 days | 106 | 2.9 | 164 | 3.26 | 1.8154 |
| 9 month and 28 days | 115 | 2.48 | 126 | 3.61 | 1.63 |
(Note that day 9 and 8 are the results of the hospitalization before the cell transplantation; day 13 and day five (the fifth needle) after the examination; and day 21 and day 28 are the results of the examination after the home-visit).
The results of the examination on day 29/9/2018 are shown in table 2:
TABLE 2 test results
The values of the monocyte fraction, eosinophil fraction and basophil fraction all returned to normal values, and from the above results, it was seen that the number of leukocytes decreased over a period of 15 days after completion of the first course of PD-1-T cell therapy, but the M5 condition was completely alleviated.
And 6, receiving the PD-1-T cell treatment of the second course on 11, 3 days in 2018, drawing blood before returning cells on the same day to check blood routine, and taking the check result on 11, 3 days as the check result before returning cells. The 11 th, 8 th day is the result of the examination after 5 needles/day of transfusion (7 needles in a treatment course and one needle every day). The total number of leukocytes had recovered to normal values, and the results of the 11-month assay are shown in Table 3.
Test results in month 311
| Date | Hemoglobin | White blood cell | Blood platelet | Red blood cell | Neutrophils |
| 115～150 | 3.5～5 | 125～350 | 3.8～5.1 | 1.8～6.3 | |
| 11 month and 3 days | 121 | 1.77 | 132 | 3.59 | 0.92 |
| 11 month and 8 days | 119 | 3.61 | 139 | 3.53 | 2.2 |
| 11 month and 17 days | 121 | 3.0 | 137 | 3.8 | 2.0 |
At present (1 month in 2019), the health condition of patients is good, and discomfort does not exist. A third course of PD-1-T cell treatment was performed on day 17 of 2019, 1 month. Applicants will continue to track the patient treatment.
Some comments are made regarding myeloid leukemia m 5: the myeloid leukemia m5 is the refractory type of acute myeloid leukemia, but with chemotherapy, complete clinical remission is achieved in approximately 70% of patients. From the above results, it is known that allogeneic hematopoietic stem cell transplantation can be performed as early as possible by 2 courses of strengthening after remission, and if the allogeneic hematopoietic stem cell transplantation cannot be performed, the allogeneic hematopoietic stem cell transplantation can only live for three or four months, and active chemotherapy and other drugs can live longer in combination with treatment, which is specifically determined by the state of illness of patients.
The myeloid leukemia m5 belongs to a serious malignant tumor, particularly chromosome, fusion gene and immunity typing are needed, risk factors are analyzed, and the treatment is guided later, the treatment principle is chemotherapy induced remission, and the remission rate is 70%; after remission, most patients relapse if chemotherapy is used, and the prognosis is poor, so a treatment method for radical treatment by allogeneic hematopoietic stem cell transplantation is suggested.
Although the myelocytic leukemia m5 is serious, chemotherapy is actively carried out at the early stage, the patients can still be cured by waiting for proper matching type to carry out hematopoietic stem cell transplantation, and whether the patients need to see the doctors more and more to see the psychology of the patients and the specific conditions of the patients can be cured.
Example 4
The detection of the activity of the PD-1-T cell killing cancer cells provided in example 1 is verified.
The method for detecting the cytocidal activity comprises the following steps:
PD-1-T cells are taken as effector cells, tumor cells are taken as target cells, and the activity of killing the target cells (tumor cells) by the PD-1-T cells is detected. The effective killing efficacy of the PD-1-T cell product is identified by taking the effect/target ratio of 5/1 and 10/1 as one of main indexes for identifying the PD-1-T cell product.
Effector cells the PD-1-T cells prepared in example 1 were effector cells. The target cell is a positive target cell of a cell line 786-O (renal cell carcinoma). A set of comparative examples was set up with DC-CIK cells as effector cells.
Take 1X 104Spreading the target cells in 96-well plate, adding RPMI-1640/10% human AB serum culture solution 200 μ l, 37 deg.C, and 5% CO2Under the culture conditions, the culture was performed overnight (12 hours). The next day, the culture medium in the wells was washed away, and 5-fold and 10-fold effect cells, PD-1-T cells and DC-CIK cells, respectively, were added, and RPMI-1640/10% human AB serum culture medium was added to make the total volume 200. mu.l. 37 ℃ and 5% CO2Under the culture conditions, the culture was carried out for 6 hours and 24 hours, respectively. Three holes per set.
Gently aspirate effector cells that are not in contact with the target cells. After each well was gently washed with the culture solution, the number of viable cells in each well was measured by MTT method.
MTT method for detecting toxic efficiency of killer cells
Preparation of a reagent:
MTT [3- (4, 5-dimethylzol-2-yl) -2,5-diphenyltetrazolium bromide ], 5 mg/ml; DMSO (dimethyl sulfoxide); acidifying isopropanol: HCI was added to the isopropanol to achieve a final HCI concentration of 0.04 mol/L.
The method comprises the following operation steps:
mu.l of MTT solution was added to each cell well after washing. After 4 hours incubation at 37 ℃ a crystalline precipitate was formed. The culture medium and MTT solution were removed, 200. mu.l of DMSO was added, and the precipitate was dissolved in a clear solution by gentle shaking. Add acidified isopropanol 100. mu.l and mix. The OD readings of each well were measured on a microplate reader at a wavelength of 570 nm.
Calculation of the kill Rate
The calculation formula for the target cells cultured adherently is as follows:
the calculation formula for cells cultured in suspension as target cells is as follows:
e: an effector cell; e + T: effector cells + target cells.
The results of the cell killing activity are shown in table 4:
TABLE 4 results of cell lethality test
The in vitro cancer cell killing test is to verify the in vitro cancer cell killing function of the PD-1-T cells cultured in vitro, and the test result shows that the PD-1-T cells have stronger cancer cell killing function compared with DC-CIK cells.
Example 5
The effect of the cell preparation of example 1 was verified.
Experimental methods
PD-1-T cells are cultured for 14 to 20 days, cell back transfusion liquid is added according to the concentration of 2000-3000 ten thousand/ml to prepare PD-1-T cell suspension (namely PD-1-T cell preparation), different blood sources are sampled to prepare PD-1-T cell preparation according to the culture method provided by the embodiment 1 to carry out detection on various cytokines, and the detection result is shown in Table 5 by adopting a multi-factor detection Kit (MultiplexImmunoassay Kit) of Ebioscience.
TABLE 5 examination results of cell preparations
Sampling different blood sources, and preparing PD-1-T cell preparations by the culture method provided by the embodiment 1 to detect transmembrane molecules of cells, and detecting after using a special treatment sample, wherein the detected functional macromolecules on the cells need to be pretreated to release the detected macromolecules, and then the high-precision ELISA detection kit or multi-factor detection kit can be used for detection, and the pretreatment method comprises the following steps: repeatedly freezing and thawing the detection sample to break cells, centrifuging the sample by using a high-speed centrifuge to remove large particle precipitates in cells such as organelles, chromosomes and the like, and taking the centrifuged supernatant solution as the detection sample. The results are shown in Table 6.
TABLE 6 results of detection of transmembrane molecules
As can be seen from tables 5 and 6, the PD-1-T cell finished product (PD-1-T cell preparation) has large difference from the above detection results, the reason for the large difference is that the preparation cell suspension is detected, and the value of the detection result is large difference due to operation difference and storage time difference (the content of the cell suspension is necessarily influenced by the storage time of the cell which continuously produces the cell factors), so that the PD-1-T cell preparation mainly analyzes which cell factors exist and which cell components are expressed.
From the above detection results, it is known that the cell preparation contains many kinds of cytokines, which proves that the cell functions well, maintains good functions after the preparation is prepared, can secrete many kinds of cytokines, and the nonexistent cytokines are: IFN-alpha, the cytokine is secreted predominantly by the Th2 subtype of CD4+ T cells, i.e., without the subtype.
Comparative example 1
Killer T cells (PD-1-T cells) prepared by the culture method provided in example 1 were compared with DC-CIK cells.
Comparison of DC-CIK cells with PD-1-T cells.
Experimental methods
1 part of cord blood was collected, mononuclear cells were separated from the whole blood, and half of the cells were cultured as DC-CIK cells (prior art), and the other half was cultured as PD-1-T cells by the culture method provided in example 1.
Sampling for the first time: sampling after culturing for 0 day, wherein the sampling mode is as follows: counting the total number of cells by a cell counting plate, taking 100 ten thousand just separated mononuclear cells, and detecting the cell surface marker type by a flow cytometer.
And (3) sampling for the second time: culturing for 6 days and sampling; sampling for the third time: culturing for 12 days and sampling; and fourth sampling: culturing for 16 days and sampling; fifth sampling: culturing for 20 days and sampling; sixth sampling: culturing for 24 days and sampling; and seventh sampling: the culture was continued for 27 days and sampled. The sampling mode is as follows: taking 1ml of cell culture suspension, firstly detecting the cell concentration of a sample, then separating out supernatant for detecting cell factors, and detecting cells by a flow cytometer.
And (3) streaming detection items: CD3+, CD4 +; CD3+, CD8 +; CD3+, CD56 +; CD3+, CD28 +; CD3-, CD56 +; CD3-, CD 28; 6 marks. And (3) detecting cytokines: detecting IFN-r, GM-CSF, IL-12, IL-3, TPO, IL-11.
Results of the experiment
Cell proliferation
The results of comparing cell counts of the two culture methods of DC-CIK cells and PD-1-T cells are shown in FIG. 1.
As can be seen from the change in the total number of cells on the days of culture in FIG. 1, the proliferation factor of the PD-1-T cell culture method was more than 180 times for 20 days, while the proliferation factor of the DC-CIK cell culture method was about 80 times for 20 days. The proliferation peak period is 8-16 days.
Flow cytometry detection
The results of cell phenotype detection of DC-CIK cells and PD-1-T cells in different culture times are shown in Table 7.
TABLE 7 results of cell phenotype measurements
Fig. 2 is a graph showing the comparison result of CD8 +. As shown in FIG. 2, CD8+ T cells accounted for 80% or more of the cells cultured for 14 days by the PD-1-T cell culture method.
Cytokine detection
The results of the cytokine secretion assay are shown in Table 8.
TABLE 8 detection results of secreted cytokines
Remarking: day zero is plasma of the whole blood, unit: pg/ml.
As can be seen from Table 8, the secretion of cytokines by PD-1-T cells was stronger in the period from 14 days to 20 days of culture.
From the above test results, the phenotype of PD-1-T cells after 14 days of culture: the proportion of CD3+ is more than 98%, namely more than 98% of T cells, and more than 80% of CD8+ cells, namely PD-1-T cells are mainly cytotoxic T lymphocytes (or killer T lymphocytes); the PD-1-T cell has stronger capability of secreting cell factors than the DC-CIK cell obviously, and reflects that the cell function of the PD-1-T cell is stronger than that of the DC-CIK cell; the PD-1-T cells have faster cell proliferation rate, stronger proliferation times and total number of cells, so that the PD-1-T cells can obtain enough number through in vitro culture, and the PD-1-T cells can obtain larger curative effect in clinical use.
In summary, the embodiments of the present invention provide a method for culturing T cells, which can induce lymphocytes isolated from whole blood to proliferate and differentiate into CD8+ T cells during the culture process, wherein the isolated T cells can increase more than 40 times within 18 days, and the percentage of CD8+ T cells is more than 80%.
In addition, the embodiment of the invention also provides a T cell, a culture medium combination thereof, application thereof and a cell preparation, wherein the killer T cell has higher function of secreting cell factors, has high proliferation speed, has stronger aggressivity to tumor cells than DC-CIK cells, and has the advantage of broad-spectrum anticancer.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for culturing T cells, comprising: culturing peripheral blood mononuclear cells, and adding a PD-1 antibody into a culture solution in the later culture period to continue culturing;
the culture method comprises the following steps: it comprises mixing peripheral blood mononuclear cells with an A-antibody culture medium for primary culture; then, mixing the product obtained by the first culture with a B-type culture medium for the second culture; finally, mixing the product obtained after the second culture with a K-type culture medium for the third culture;
the A-resistant culture medium consists of the following components: complete culture medium, CD3 monoclonal antibody, interleukin-2, vitamin B6, interferon-gamma, interleukin-1B, interleukin-4, L-glutamine, sodium pyruvate and plasma protein; in the A-antibody culture medium, the final concentration of the CD3 monoclonal antibody is 80-120 ng/ml; the final concentration of interleukin-2 is 800-1200 u/ml, the final concentration of vitamin B6 is 5-15 ng/ml, the final concentration of interferon-gamma is 15-25 ng/ml, the final concentration of interleukin-1B is 5-15 ng/ml, the final concentration of interleukin-4 is 5-1 ng/ml, the final concentration of L-glutamine is 180-220 ng/ml, the final concentration of sodium cuprate is 15-25 ng/ml, and the final concentration of plasma protein is 40-60 ng/ml;
the K-type culture medium consists of the following components: complete medium, PD-1 antibody, interleukin-2, L-glutamine, sodium pyruvate and plasma protein; in a K-type culture medium, the final concentration of the PD-1 antibody is 5-15 ng/ml; the final concentration of interleukin-2 is 1800-2200 u/ml, the final concentration of L-glutamine is 40-60 ng/ml, the final concentration of sodium cuprate is 3-8 ng/ml, and the final concentration of plasma protein is 40-60 ng/ml;
the B-type culture medium consists of the following components: complete medium, CD28 antibody, interleukin-2, vitamin C, L-glutamine, sodium pyruvate, and plasma protein; 5-15 ng/ml of CD28 antibody in a B-type culture medium; in a B-type culture medium, the final concentration of interleukin-2 is 1800-2200 u/ml, the final concentration of vitamin B6 is 5-15 ng/ml, the final concentration of vitamin C is 5-15 ng/ml, the final concentration of L-glutamine is 80-120 ng/ml, the final concentration of sodium cuprate is 5-15 ng/ml, and the final concentration of plasma protein is 15-25 ng/ml;
between the second culture and the third culture, and/or after the third culture, adding a C-type culture medium for culture; the type C culture medium consists of the following components: complete medium, CD28 antibody, interleukin-2, vitamin C, vitamin B6, L-glutamine, sodium pyruvate, and plasma protein; in a C-type culture medium, the final concentration of a CD28 antibody is 3-8 ng/ml, the final concentration of interleukin-2 is 450-550 u/ml, the final concentration of vitamin B6 is 3-8 ng/ml, the final concentration of vitamin C is 3-8 ng/ml, the final concentration of L-glutamine is 40-60 ng/ml, the final concentration of sodium cuprate is 3-8 ng/ml, and the final concentration of plasma protein is 5-15 ng/ml.
2. The culture method according to claim 1, wherein the cell proliferation is initiated and cell mass is formed and the cell density reaches 2 x 107～5ⅹ107The second culturing is carried out per ml; when the cell density reaches 2 x 107～5ⅹ107The third culture was performed at one/ml.
3. The culture method according to claim 2, wherein the number of days of culture in the first culture is 3 to 6 days, and the number of days of culture in the second culture is 10 to 12 days.
4. The culture method according to claim 2, wherein the cell density reaches 2 x 10 between the second and third culturing7～5ⅹ107At each ml, C-type medium was added and after the third cultivation, the cell density reached 2 x 107～5ⅹ107At one/ml, type C medium was added.
5. A culture medium combination of T cells is characterized in that the culture medium combination comprises an A anti-culture medium, a K type culture medium and a B type culture medium, and/or a C culture medium;
the A-resistant medium consists of: complete culture medium, CD3 monoclonal antibody, interleukin-2, vitamin B6, interferon-gamma, interleukin-1B, interleukin-4, L-glutamine, sodium pyruvate and plasma protein; in the A-antibody culture medium, the final concentration of the CD3 monoclonal antibody is 80-120 ng/ml; the final concentration of interleukin-2 is 800-1200 u/ml, the final concentration of vitamin B6 is 5-15 ng/ml, the final concentration of interferon-gamma is 15-25 ng/ml, the final concentration of interleukin-1B is 5-15 ng/ml, the final concentration of interleukin-4 is 5-1 ng/ml, the final concentration of L-glutamine is 180-220 ng/ml, the final concentration of sodium cuprate is 15-25 ng/ml, and the final concentration of plasma protein is 40-60 ng/ml;
the K-type culture medium consists of the following components: complete medium, PD-1 antibody, interleukin-2, L-glutamine, sodium pyruvate and plasma protein; in a K-type culture medium, the final concentration of the PD-1 antibody is 5-15 ng/ml; the final concentration of interleukin-2 is 1800-2200 u/ml, the final concentration of L-glutamine is 40-60 ng/ml, the final concentration of sodium cuprate is 3-8 ng/ml, and the final concentration of plasma protein is 40-60 ng/ml;
the type B medium consists of the following components: complete medium, CD28 antibody, interleukin-2, vitamin C, L-glutamine, sodium pyruvate, and plasma protein; 5-15 ng/ml of CD28 antibody in a B-type culture medium; in a B-type culture medium, the final concentration of interleukin-2 is 1800-2200 u/ml, the final concentration of vitamin B6 is 5-15 ng/ml, the final concentration of vitamin C is 5-15 ng/ml, the final concentration of L-glutamine is 80-120 ng/ml, the final concentration of sodium cuprate is 5-15 ng/ml, and the final concentration of plasma protein is 15-25 ng/ml;
type C media consists of: complete medium, CD28 antibody, interleukin-2, vitamin C, vitamin B6, L-glutamine, sodium pyruvate, and plasma protein; in a C-type culture medium, the final concentration of a CD28 antibody is 3-8 ng/ml, the final concentration of interleukin-2 is 450-550 u/ml, the final concentration of vitamin B6 is 3-8 ng/ml, the final concentration of vitamin C is 3-8 ng/ml, the final concentration of L-glutamine is 40-60 ng/ml, the final concentration of sodium cuprate is 3-8 ng/ml, and the final concentration of plasma protein is 5-15 ng/ml.
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