CN109913413A - A kind of T cell extracorporeal culturing method, its cell preparation and its application loading PD-1 antibody - Google Patents

Abstract

The embodiment of the invention discloses a kind of T cell extracorporeal culturing method, its cell preparation and its applications for loading PD-1 antibody, it is related to immunotherapy techniques field, specifically, the cultural method is the following steps are included: it includes mixing peripheral blood mononuclear cells with the anti-culture medium of A to carry out first time culture；Then, obtained product will be cultivated for the first time, and second of culture of progress is mixed with Type B culture medium；Finally, the product obtained after second is cultivated is mixed with K-type culture medium.The lymphocyte that the cultural method can induce whole blood to isolate breaks up to CD8+T cell proliferation during the cultivation process, and the T cell isolated can reach 40 times or more of amplification within 18 days, and the accounting of CD8+T cell is more than 80%.

Description

It is a kind of load the T cell extracorporeal culturing method of PD-1 antibody, its cell preparation and its Using

Technical field

The present invention relates to immunotherapy techniques fields, train in vitro in particular to a kind of T cell for loading PD-1 antibody The method of supporting, its cell preparation and its application.

Background technique

Current cancer is also the failure to capture disease problem, and immunotherapy in 2013 is described as by " science " magazine and is most possibly controlled More the method for cancer.Immune cell therapy is the intermediate portions in immunotherapy, and immune cell therapy is from the last century 70's Start, earliest is that LAK cell (extracts peripheral blood from human body, isolates lymphocyte from peripheral blood in vitro, large dosage is added Proleulzin carries out in vitro culture proliferation and then obtains the lymphocytes of proliferation several times being named as LAK cell, then having cultivated At LAK cell be made into the preparation venoclysis human body of cell suspension), after LAK cell is earliest immunocyte in vitro culture Immunocyte Vitro Culture Techniques applied to treatment of cancer.

By 2000 or so, immunocyte-CIK cell of new generation was applied to clinical cancer therapy, compared with LAK cell body Outer culture technique does not mainly stimulate cell with large dosage of proleulzin, using CD3 monoclonal antibody and cytokine profiles (interleukin- 2, raise the price-interferon etc.), but CIK cell increment multiple is low, and killer T cell ratio is low, then curative effect is not when using for cancer It obviously, is that CD3 monoclonal antibody active cell proliferation is added on complete medium in culture technique, then follow-up cultivation adds white Jie Element -2 maintains cell Proliferation, and culture in this way can only make to mix various cells together and all be proliferated, therefore cultivate the main CD8+ completed Cell accounting is not high (50% or so), there are various cells secretion different cytokines together and interacts due to mixing cell and leads Cause lethal (cytotoxicity) of CD8+T cell not it is strong also just interior the risen therapeutic effect of infusion cancer patient body (to inhibiting tumor cell Effect) it is also not strong.

DC cell finds and is applied in vitro culture, and DC- made of DC cell and CIK cell co-cultivation later CIK cell is technically to make DC cell offer antigenic action stimulation CIK cell to make the more preferable proliferation times of CIK cell and cell Function.Therefore, clinical efficacy is better than CIK cell, but lethal (cytotoxicity) of CD8+T cell be not equally strong.

2017, for CAR-T cell in the granted listing in the U.S., CAR-T cell was to extract patient's peripheral blood in-vitro separation to go out to drench Bar cell, then cultivates and makes it to CD8+T cell differentiation and obtain certain amount, then make carrier CD19 using adenovirus The identification genetic insertion cell DNA and obtain insertion specific antigenic receptors T cell be named as CAR-T cell, the CAR-T is thin Born of the same parents' energy certain type of B cell leukemia of specific recognition, thus the cancerous tumor cell of this B cell leukemia of specific attack, Cancerous tumor cell destroys and treats this leukaemia, and has and successfully cure case.

2017, PD-1 antibody was obtained as the granted listing for the treatment of cancer drug, You Liangjia national corporation of medicine first is that silent The K in husky east, another is the O medicine of Shi Guibao, and drug listing is created much of a stir, effective percentage up to 40% (chemotherapeutic, targeting medicine etc. More than ten) the very high ability percent of effective percentage of cancer drug is known as anticancer god's medicine.

But PD-1 antibody energy curative effect can only be directed to a part of patient, be since tumour produces PD-L1, for other Caused by tumour is not due to due to PD-L1 or PD-1 antibody drug is no any effect, and PD-1 antibody Drug has very big toxic side effect.

Summary of the invention

The first object of the present invention is to provide a kind of cultural method of T cell, which can induce whole blood point The lymphocyte separated out breaks up to CD8+T cell proliferation during the cultivation process, thus the killer T cell being prepared, and point The T cell separated out can reach 40 times or more of amplification within 18 days, and the accounting of CD8+T cell is more than 80%.

The second object of the present invention is that the culture medium for providing a kind of T cell combines, and culture medium combination can effectively exist It is turned out in a short time with a large amount of T cells with lethality.

The third object of the present invention is to provide a kind of T cell, and (referred to as PD-1-T cell, it is anti-that PD-1 is added in the phase after incubation Body, make PD-1 molecule in T cell by PD-1 antibody in conjunction with and close, referred to as PD-1-T cell), the T cell is with higher The function of secrete cytokines, it is stronger for tumor cell invasiveness, and do not influenced by the PD-L1 of tumour cell, having can The advantages of broad spectrum anticancer.

The fourth object of the present invention is to provide a kind of cell preparation comprising has above-mentioned lethal stronger T cell, has There is anticancer therapeutic more better than DC-CIK cell.

The fifth object of the present invention is to provide above-mentioned killer T cell or cell preparation in cell preparation in preparation anticancer Application in drug.

The present invention is implemented as follows:

The embodiment of the invention provides a kind of cultural methods of T cell comprising following steps: the single core of human peripheral blood is thin Born of the same parents cultivate, and the phase adds PD-1 antibody into culture solution and continues to cultivate after incubation.

It should be noted that PD-1 antibody drug works in this way: being expressed (on film) in the T cell in immunocyte PD-1, and express (on film) on tumour cell or secret out of (excretion body) PD-L1, however PD-L1 is PD-1 ligand, they combine This T cell can be made just to enter programmed cell death and lose function, the T so scientist's discovery tumour cell is escaped because there is PD-L1 Cell chase, then PD-1 molecule in T cell is closed using PD-1 antibody, it is thin that the PD-L1 on tumour cell cannot touch T PD-1 on born of the same parents, such T cell will not be contacted and dead anti-executable removing cancer cell function into tumor tissues by PD-L1 Kill cancer cell.

By PD-1 antibody drug action principle as it can be seen that PD-1 antibody drug is without directly killing cancer cell effect, it It is only the T cell protected inside body, plays the role of the still T cell for killing cancer cell.But tumour generates PD-L1 One of reason uses PD-1 antibody drug certainty good curative effect if it is this reason, and former US President obtained melanin Tumor is fully recovered after using PD-1 antibody drug, tumor disappearance.But if tumour generation is not caused by PD-L1 reason, then making It is no any effect with PD-1 antibody drug, and PD-1 antibody drug has very big toxic side effect.

The immune system of human body or animal, can remove cell (cancer cell), the aging, non-viable non-apoptotic cell of variation, thus make machine Body is kept fit, and it is exactly various immunocytes (leucocyte) that immune system, which executes immune function, wherein lymphocyte is Cancerous tumor cell effect of removing is primarily served, T cell, especially CD8+T cell are that function is removed in most important execution in lymphocyte Immunocyte.

The present invention regard T cell itself as therapeutic substance, and PD-1 antibody is added as PD-1 points on closing T cell film Son, rather than using PD-1 antibody as antibody drug itself, meanwhile, cultural method provided in an embodiment of the present invention strengthens T cell The lethality of itself, T cell can be effectively prevented because on tumour cell PD-L1 that may be present and loss of function and Side effect of the PD-L1 to user.

Preferably, which includes mixing peripheral blood mononuclear cells with the anti-culture medium of A to carry out first time culture； Then, obtained product will be cultivated for the first time, and second of culture of progress is mixed with Type B culture medium；Finally, after cultivating for second Obtained product is mixed with K-type culture medium carries out third time culture.

The anti-culture medium of A includes complete medium, CD3 monoclonal antibody and one or more of component: proleulzin, Vitamin B6, interferon-γ, interleukin-1 b, interleukin-4, L-Glutamine, the third bronze medal acid sodium and plasma protein；

The K-type culture medium includes complete medium, PD-1 antibody and one or more of component: interleukin- 2, L-Glutamine, the third bronze medal acid sodium and plasma protein；

It include complete medium, CD28 antibody and one or more of component: Bai Jie in the Type B culture medium Element -2, vitamin C, L-Glutamine, the third bronze medal acid sodium and plasma protein.

Further, in the anti-culture medium of A, final concentration of 80~120ng/ml of CD3 monoclonal antibody；Preferably, in the anti-culture of A In base, final concentration of 800~1200u/ml of proleulzin, final concentration of 5~15ng/ml of vitamin B6, interferon-γ Final concentration of 5~15ng/ml of final concentration of 15~25ng/ml, interleukin-1 b, final concentration of 5~15ng/ of interleukin-4 Ml, final concentration of 180~220ng/ml of L-Glutamine, final concentration of 15~25ng/ml of the third bronze medal acid sodium, plasma protein Final concentration of 40~60ng/ml.

It should be noted that in the anti-culture medium of A, CD3 monoclonal antibody can CD3 molecule on activated leukocyte film, make leucocyte Break up to T cell, proleulzin being capable of exciting leukocyte differentiation proliferation；Vitamin B6 can strengthen the utilization of leucocyte amino acid, The synthesis of active cell protein is to strengthen cell function；Interferon-γ can make leucocyte to CD8+T cell differentiation；Bai Jie Element -1b can be activated after separation in mononuclearcell containing micro DC cell, and DC cell secretion IL-12 is allowed to make subsequent CD8+ T cell is more lethal；Interleukin-4 changes CD4+T cell from TH2 hypotype to TH1 hypotype, so that CD4+T cell be made to secrete Based on interferon-γ, tumor necrosis factor-alpha, make follow-up cultivation strengthen CD4+T cell based on secrete cytokines function and Proliferation is secondary, therefore when in vitro culture was to 14 days, 60% is gradually reduced to 20% when CD4+T cell is by originating；L-Glutamine Cellular energy is provided；Third bronze medal acid sodium has synergidae to utilize energy matter (glucose)；Plasma protein is able to maintain that extracellular environment And protect cell membrane.

In Type B culture medium, 5~15ng/ml of CD28 antibody；Preferably, in Type B culture medium, the end of proleulzin is dense Degree be 1800~2200u/ml, final concentration of 5~15ng/ml of vitamin B6, ascorbic final concentration of 5~15ng/ml, Final concentration of 180~220ng/ml of L-Glutamine, final concentration of 15~25ng/ml of the third bronze medal acid sodium, the end of plasma protein Concentration is 15~25ng/ml.

Specifically, in Type B culture medium, the antibody strengthened T cell function of CD28, and accelerate CD8+T cell Proliferation, after The continuous culture stronger killing ability of CD8+T cell also expresses more PD-1；Vitamin C can enhance the chemotaxis and deformation energy of leucocyte Power improves and destroys bacterium cell ability abnormal with other.Addition, which helps, removes incipient separation residual heteroproteose cell, makes to cultivate cell System tends to be pure, makes culture when completing based on CD8+T cell.In addition, proleulzin, L-Glutamine, the third bronze medal acid sodium and Plasma protein is the same as the anti-culture medium of A.

In K-type culture medium, final concentration of 5~15ng/ml of PD-1 antibody；Preferably, in K-type culture medium, Bai Jie The final concentration of the bronze medal acid sodium of final concentration of 1800~2200u/ml, final concentration of 40~60ng/ml of L-Glutamine, third of element -2 For 3~8ng/ml, final concentration of 40~60ng/ml of plasma protein.

Specifically, start and formed cell mass to cell proliferation, and carry out when cell density is gradually increased cultivating for second, The cell density in culture bottle is observed under inverted microscope, reaches 2 X 10 to cell density⁷~5 X 10⁷Third is carried out when a/ml Secondary culture.

Preferably, the cultivated days of the first time culture are 3 days, 4 days, 5 days or 6 days, the culture day of second of culture Number is 10,11 days or 12 days.Further preferably, it is selected in cultivated days 3~6 days of the first time culture, second The cultivated days of culture are to select in 10~12 days.

Further, in second of culture or after K-type culture medium is added, the cultural method includes described in addition C-type culture medium.Preferably, it between second of culture and third time culture, is observed in culture bottle under inverted microscope Cell density reaches 2 X 10 to cell density⁷~5 X 10⁷C-type culture medium is added in a/ml.After third time is cultivated, to thin Born of the same parents' density reaches 2 X 10⁷~5 X 10⁷C-type culture medium is added in a/ml.

Specifically, c-type culture medium includes complete medium, CD28 antibody and one or more of component: Bai Jie Element -2, vitamin C, vitamin B6, L-Glutamine, the third bronze medal acid sodium and plasma protein；

Preferably, in c-type culture medium, final concentration of 3~8ng/ml of CD28 antibody, proleulzin it is final concentration of 450~550u/ml, final concentration of 3~8ng/ml of vitamin B6, ascorbic final concentration of 3~8ng/ml, L- glutamy Final concentration of 40~60ng/ml of amine, final concentration of 3~8ng/ml of the third bronze medal acid sodium, plasma protein final concentration of 5~ 15ng/ml。

In c-type culture medium, vitamin C can enhance the chemotaxis and deformability of leucocyte, improve and destroy bacterium and its His abnormal cell ability.Addition, which helps, removes incipient separation residual heteroproteose cell, so that cultured cells system is tended to be pure, so that culture Based on CD8+T cell and its killing ability is enable to strengthen when completion.

The embodiment of the present invention also provides a kind of culture medium combination of T cell comprising have the anti-culture medium of A, K-type culture medium with And Type B culture medium.

The anti-culture medium of A includes complete medium, CD3 monoclonal antibody and one or more of component: proleulzin, dimension life Plain B6, interferon-γ, interleukin-1 b, interleukin-4, L-Glutamine, the third bronze medal acid sodium and plasma protein.

K-type culture medium includes complete medium, PD-1 antibody and one or more of component: proleulzin, L- Glutamine, the third bronze medal acid sodium and plasma protein.

Include complete medium, CD28 antibody and one or more of component in Type B culture medium: proleulzin, Vitamin C, L-Glutamine, the third bronze medal acid sodium and plasma protein.

Further, culture medium combination can also include c-type culture medium.

Preferably, the concentration of component of the culture medium in culture medium combination is referring to the concentration of component in above-mentioned cultural method, This is repeated no more.

The embodiment of the invention also provides a kind of killer T cells, are made by above-mentioned cultural method.

The embodiment of the invention also provides a kind of cell preparations comprising has cell to feed back liquid and above-mentioned cultural method system The T cell obtained.

It includes following components that cell, which feeds back liquid: 80~120ml physiological saline, 2~7g human serum albumin, gentamicin (7 ~9 ten thousand units), proleulzin (9~110,000 unit), 8~12mg L-Glutamine and 3~8mg the third bronze medal acid sodium.

Wherein, physiological saline isotonic solution acts on；Human serum albumin maintains cellular environment in transportational process, protects cell Film keeps cell activity；It is polluted when the anti-injection of gentamicin；Proleulzin, which helps in cellular transport processes, survives；L- glutamy Amine is convenient for providing cellular energy in cellular transport processes；Third bronze medal acid sodium has synergidae to maintain using energy matter during transportation Eubolism.

The embodiment of the present invention also provides above-mentioned T cell or above-mentioned cell preparation and is preparing the application in anticancer drug.

Specifically, anticancer drug refers to anti-cutaneum carcinoma, lung cancer, prostate cancer, head and neck cancer, cervical carcinoma, thyroid cancer, plastic Cell plastid tumor, glioma, leukaemia, lymthoma, adrenal, AIDS associated cancer, alveolar soft part sarcoma, star Cytoma, osteocarcinoma, brain and spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, kidney chromophobe cell tumor, Clear cell carcinoma, skin Benign Fibrous Histiocytoma, desmoplastic small round cell tumor, ependymoma, You Yin The outer myxoid chondrosarcoma of tumor, bone, the generation of bone imperfection fiber, Fibrous dysplasia of bone, gall-bladder or cholangiocarcinoma, gestation are nourished Cell disease, germinoma, hematologic malignancies, hepatocellular carcinoma, islet-cell tumour, Kaposi sarcoma, kidney, fat Tumor/benign lipoma, embryonal-cell lipoma/pernicious lipoma, medulloblastoma, meningioma, merkel's cells cancer, it is multiple in Secrete tumor formation, Huppert's disease, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, mamillary Thyroid cancer, parathyroid adenoma, paediatric cancer, Peripheral Nerve Sheath Tumors, pheochromocytoma, hypophysoma, prostate cancer, rear uvea Melanoma, rare blood disease, kidney metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, soft tissue sarcoma, squamous cell Cancer, gastric cancer, synovial bursa sarcoma, carcinoma of testis, thymic carcinoma, thymoma, Thyroid metastasis cancer or uterine cancer drug；

Preferably, the anticancer drug is medicament for resisting cervical cancer or anti-leukemia medicine.

The invention has the following advantages:

The embodiment of the invention provides a kind of cultural method of T cell, which can induce whole blood to isolate Lymphocyte during the cultivation process to CD8+T cell proliferation break up, the T cell isolated can reach within 18 days 40 times with On amplification, and the accounting of CD8+T cell is more than 80%.

In addition, the embodiment of the invention also provides a kind of T cell, the combination of its culture medium, its application and a kind of cell systems Agent, the function of killer T cell secrete cytokines with higher, value-added speed is fast, for tumor cell invasiveness compared with DC-CIK cell is strong, and having the advantages that being capable of broad spectrum anticancer.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is the ratio of the cell count of DC-CIK cell and two kinds of cultural methods of PD-1-T cell in comparative example 1 of the present invention Relatively result；

Fig. 2 is CD8+ accounting comparison result figure in comparative example 1 of the present invention.

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

Feature and performance of the invention are described in further detail with reference to embodiments.

Embodiment 1

The preparation method of killer T cell is as follows.

150ml peripheral blood is extracted, or collects bleeding of the umbilicus, isolates list using lymphocyte separation medium and gradient centrifugation procedure A nucleus (mainly containing lymphocyte, there are also other leucocytes and a small amount of red blood cells).

It cultivates for the first time

Starting culture will separate mononuclearcell and mix with the anti-culture medium of A, be fitted into cell culture square vase, be placed on 37 DEG C, 5%CO₂Constant temperature and humidity carbon dioxide incubator in cultivate.

The component of the anti-culture medium of A is as follows:

Complete medium, CD3 monoclonal antibody, proleulzin, vitamin B6, interferon-γ, interleukin-1 b, interleukin-4, L- Glutamine, the third bronze medal acid sodium and plasma protein.

Wherein, the final concentration of 100ng/ml of CD3 monoclonal antibody；Preferably, in the anti-culture medium of A, the final concentration of proleulzin For 1000u/ml, the final concentration of 10ng/ml of vitamin B6, the final concentration of 20ng/ml of interferon-γ, the end of interleukin-1 b Concentration is the bronze medal acid sodium of 10ng/ml, the final concentration of 10ng/ml of interleukin-4, the final concentration of 200ng/ml of L-Glutamine, third Final concentration of 20ng/ml, the final concentration of 50ng/ml of plasma protein.

Second of culture

It is cultivated in constant temperature and humidity carbon dioxide incubator, observes cell growth status daily, simultaneously to cell Proliferation starting It forms cell mass and cell density reaches 3 X 10⁷(differing within cultivated days 3-6 days) adds Type B culture medium when a/ml.Type B training The specifying information for supporting base is as follows.

It include complete medium, CD28 antibody, proleulzin, vitamin C, L-Glutamine, the third bronze medal in Type B culture medium Sour sodium and plasma protein.

Wherein, CD28 antibody 10ng/ml；Preferably, in Type B culture medium, the final concentration of 1000u/ml of proleulzin, The final concentration of 10ng/ml of vitamin B6, ascorbic final concentration of 10ng/ml, the final concentration of 100ng/ of L-Glutamine Ml, the final concentration of 10ng/ml of the third bronze medal acid sodium, the final concentration of 20ng/ml of plasma protein.

It should be noted that culture observes cell growth status after Type B culture medium is added daily, visual cell is in culture solution Density reaches 3 X 10⁷When a/ml, then c-type culture medium is added, and sub-bottle is carried out according to Culture liquid measure, cultivated to 10 days.C-type The specifying information of culture medium is as follows:

C-type culture medium includes complete medium, CD28 antibody, proleulzin, vitamin C, vitamin B6, L- glutamy Amine, the third bronze medal acid sodium and plasma protein.Wherein, the final concentration of 5ng/ml of CD28 antibody, the final concentration of 500u/ of proleulzin Ml, the final concentration of 5ng/ml of vitamin B6, ascorbic final concentration of 5ng/ml, the final concentration of 50ng/ of L-Glutamine Ml, the final concentration of 5ng/ml of the third bronze medal acid sodium, the final concentration of 10ng/ml of plasma protein.

Third time is cultivated

After having cultivated for the second time, when cell density reaches 3 X 10⁷When a/ml, it is thin that addition K-type culture medium obtains lethal T Born of the same parents' (PD-1-T cell).The specifying information of K-type culture medium is as follows.

K-type culture medium includes complete medium, PD-1 antibody, proleulzin, L-Glutamine, the third bronze medal acid sodium and blood Starch albumen.Wherein, the final concentration of 10ng/ml of PD-1 antibody；The final concentration of 1000u/ml of proleulzin, L-Glutamine Final concentration of 50ng/ml, the final concentration of 5ng/ml of the third bronze medal acid sodium, the final concentration of 50ng/ml of plasma protein.

The cell growth status of observation observation daily is added after K-type culture medium, and visual cell's density in culture solution is appropriate (when thin Born of the same parents' density reaches 3 X 10⁷When a/ml) addition c-type culture medium, and sub-bottle is carried out according to Culture liquid measure.

To needs in use, isolating cultured products (i.e.) PD-1-T cell by gradient centrifugation procedure, cell is added and feeds back It is mixed into cell suspension in liquid and is packed into infusion bottle encapsulation, PD-1-T cell preparation is made.

Wherein, it includes following components that cell, which feeds back liquid: 100ml physiological saline, 5g human serum albumin, gentamicin (80,000 Unit), proleulzin (100,000 unit), 10mg L-Glutamine and 5mg the third bronze medal acid sodium.

Embodiment 2

Curative effect of the cell preparation that embodiment 1 is provided for cervical cancer patient.

Cervical cancer patient PET-CT inspection result on December 29th, 2017 are as follows:

1. after operation for cervical carcinoma, uterus and bilateral attachment are lacked such as；Vaginal Stump has no that malignant tumour levies picture；

2. last time images nest Metastatic Lymph Nodes on left collarbone seen in (2017-7-11), this time imaging disappears, left side Supraclavicular fossa soft tissue disorder, metabolism are slightly increased, and are changed after being thought of as treatment；

3. multiple slightly increase lymph nodes are removed in after upper abdomen peritonaeum, metabolism is slightly increased, and lymph node inflammation is thought of as, with last time Imaging is reduced compared to metabolism；

4. 1 nodositas hypermetabolism lesion of left lateral lobe of liver upper section, is thought of as hepatic metastases stove more；

5. seeing nodositas hypermetabolism lesion by the total blood vessel of right side ilium, Metastatic Lymph Nodes are thought of as, which invades adjacent Ureter leads to upper section and right renal pelvis expansion, hydrops in the ureter of right side；Right impaired renal function；

6. right lower abdomen mesentery slightly thickens, metabolism is slightly increased, and is changed after being thought of as treatment；

7. right side sphenoid sinus and sieve chronic infection of sinus, nasopharynx inflam mation；Multiple lymph in bilateral neck, bilateral hilus pulumonis and mediastinum Tie inflammatory hyperplasia；Double lung increased bronchovascular shadows；Benign lesser tubercle under the nearly pleura of superior lobe of right lung apical segment；Left apex pulmonis radiation pneumonitis；It is left Leaf segmentum lingulare inferius and a little chronic inflammation of medial segment of right middle lobe of lung on lung；Right lobe of liver tumour；Bilateral adrenal glands thickening is with physiology Property it is dense poly-；Right kidney volume-diminished；The subcutaneous Multiple of whole body；Neck Thoracolumbar disk regression deformation.

The CT examination result on the 22nd of August in 2018:

1. Zi Gong cuts off Postoperative changes, Vaginal Stump-Mesorectal fascia thickens and soft tissue shadow, considers tumor recurrence, And posterior wall of urinary bladder and right side ureter lower end can be invaded, it is proposed that follow-up；

2. the left inside leaf of liver considers tumour, substantially ditto with the number of sheets similar round low-density shadow behind the right side；

3. former gallbladder neck similar round soft-tissue density shadow, this has no display；Chronic cholecystitis；

4. neck Thoracolumbar disk regression deformation.

The patient received the PD-1-T cell therapy of first course for the treatment of, on October 23rd, 2018-on September-15 days 9,2018 Receive within 29th the PD-1-T cell therapy of second course for the treatment of.

One course of therapy administration mode: one bottle of PD-1-T cell preparation contains hundred million PD-1-T cells of 25-30, injects daily One bottle of PD-1-T cell preparation, continuous 7 days as a course for the treatment of.

On November 1st, 2018, CT examination result was as follows:

1. Zi Gong cuts off Postoperative changes, Vaginal Stump-Mesorectal fascia thickens and soft tissue shadow, considers tumor recurrence, Less compared with anter variation, posterior wall of urinary bladder and right side Distal Ureteral is suspicious is invaded；

2. lumbar retrogression.

It is analyzed by the above results, doubtful transfer (tumour) disappears completely at liver after the PD-1-T cell therapy of two courses for the treatment of It removes, chronic cholecystitis is also eliminated, and pelvic lymph node is eliminated, in addition, neck, clavicle are attached before not receiving PD-1-T cell therapy Closely there is many places lymph node, eliminated after receiving PD-1-T cell therapy, and the state of an illness obviously mitigates, consciously only lumbar vertebrae bone increases Life enables it have pain, other are simultaneously asymptomatic.It is responsible for clinician and claims the disease progression of its cervical carcinoma good.

Embodiment 3

Curative effect of the cell preparation for leukaemic of the offer of embodiment 1 is provided.

The leukaemic is diagnosed as monocytic leukemia (M5) to local hospital inspection in the end of the year 2017 discomfort, it It being followed by by 5 chemotherapy, the state of an illness is eased, but haemocyte is low always, with easy infection, the complication such as uncomfortable.Through excessive Side seeks medical advice, and in July, 2018 receives CIK immune cell therapy, but produces little effect, and in August, 2018 relates to applicant, and in 2018 (a course of therapy administration mode: one bottle of PD-1-T cell preparation contains hundred million PD-1-T of 25-30 on September 8, first courses for the treatment of of receiving Cell injects one bottle of PD-1-T cell preparation daily, and continuous 7 days as a course for the treatment of) PD-1-T cell therapy.Knot before and after treatment Fruit is as follows.

Inspection result before September 8 days feedback PD-1-T cells: leucocyte, red blood cell, hemoglobin, the red blood cell ratio of patient Appearance, blood platelet, neutrophil leucocyte and lymphocyte are below normal value, monocyte ratio, eosinophil ratio with And the numerical value of basophilic granulocyte ratio is above normal value.

Feed back (September 13 days) inspection result after the 5th needle of first course for the treatment of PD-1-T cell (a daily needle, totally 7 needle): Red blood cell, hemoglobin and the lymphocyte of patient is below range of normal value, monocyte ratio, eosinophil ratio The numerical value of rate and basophilic granulocyte ratio is above normal value.

The inspection result that families of patients arranges is as shown in table 1:

1 testing result of table

Date	Hemoglobin	Leucocyte	Blood platelet	Red blood cell	Neutrophil leucocyte
						Range of normal value	115~150	3.5~9.5	125~350	3.8~5.1	1.8~6.3
September 8 days	101	1.72	100	3.3	0.7
						September 13 days	113	3.39	163	3.6	1.99
September 21 days	106	2.9	164	3.26	1.8154
						September 28 days	115	2.48	126	3.61	1.63

(explanation: September 8th are results hospitalized to have a thorough examination before beating cell；5th day on the 13rd (the 5th needle) inspection result afterwards；21 days, 28 are inspection results after going home).

The inspection result on the 29th of September in 2018 is as shown in table 2:

2 testing result of table

The numerical value of monocyte ratio, eosinophil ratio and basophilic granulocyte ratio restores normal value, By the above results it can be seen that have after the PD-1-T cell therapy for completing first course for the treatment of by 15 day time leucocyte numerical value Declined, but M5 state of an illness complete incidence graph.

Receive the PD-1-T cell therapy of second course for the treatment of on November 3rd, 2018, blood of taking a blood sample before same day feedback cell is normal Rule, following 3 daily inspection result in November are to feed back inspection result before cell.November 8 be fed back 5 needles/day (course for the treatment of totally 7 needle, A daily needle) inspection result afterwards.Recovered normal value, the testing result in November are as shown in table 3 for total white blood cells.

The testing result in 3 November of table

Date	Hemoglobin	Leucocyte	Blood platelet	Red blood cell	Neutrophil leucocyte
							115~150	3.5~5	125~350	3.8~5.1	1.8~6.3
November 3	121	1.77	132	3.59	0.92
						November 8	119	3.61	139	3.53	2.2
November 17	121	3.0	137	3.8	2.0

At present (in January, 2019) patient health in order, no discomfort.The PD- of progress on January 17th, the 2019 third course for the treatment of 1-T cell therapy.Applicant will continue to track patient's treatment condition.

Make some explanations about myelocytic leukemia m5: myelocytic leukemia m5 belong in acute myeloid leukemia compared with Refractory type, but by chemotherapy, about 70% patient can achieve complete incidence graph clinically.By the above results It is found that again by the reinforcings of 2 courses for the treatment of after alleviating, row Allogeneic Hematopoietic Stem Cell Transplantation as early as possible, if not can be carried out transplanting, that It may can only live three or four months, actively carrying out the drugs partner treatments such as chemotherapy may survive relatively long, with specific reference to patient Depending on the state of an illness.

Myelocytic leukemia m5 belongs to more serious malignant tumour, specifically also need to do chromosome, fusion and Immunophenotyping analyzes risk factor, and treatment after instructing, principle of reatment is: chemotherapy inducer remission, remission rate is 70%；Alleviate After treat, if with chemotherapy, most patients recurrence, prognosis is bad, it is therefore proposed that Allogeneic Hematopoietic Stem Cell Transplantation radical cure is controlled Treatment method.

Although myelocytic leukemia m5, than more serious, discovery actively carries out chemotherapy early, suitable distribution type is waited Carry out hematopoietic stem cell transplantation, still can fully recover, can cure not merely to see the doctor more to see patient phychology and The specific state of an illness of patient.

Embodiment 4

Verify the PD-1-T cell killing cancer cell viability examination that embodiment 1 provides.

The detection method of Execution vigor:

It is effector cell with PD-1-T cell, tumour cell is target cell, detects PD-1-T cell killing target cell (tumour Cell) vigor.It is identification PD-1- with 5/1,10/1 effect/target than effective killing efficiency to identify PD-1-T cell products One of T cell product main indicator.

Effector cell is that the PD-1-T cell that embodiment 1 is prepared is effector cell.Target cell is with cell line 786-O (the saturating cell cancer of kidney) is positive target cell.Control one group of comparative example of setting, using DC-CIK cell as effector cell.

Take 1 × 10⁴A target cell is laid in 96 orifice-plate microporosities, adds 200 μ l of RPMI-1640/10% people's AB serum free culture system liquid, 37 DEG C, 5%CO₂Under condition of culture, overnight incubation (12 hours).Next day washes away the training liquid in micropore, respectively plus 5 times and 10 times Effector cell-PD-1-T cell, DC-CIK cell, and add RPMI-1640/10% people AB serum free culture system liquid, make total volume 200 μl.37 DEG C, 5%CO₂Under condition of culture, cultivate 6 hours and 24 hours respectively.Every group of three holes.

Gently suck the effector cell not being in contact with target cell.Each hole is rinsed with training liquid, it is thin to detect each hole survival later Born of the same parents' number, is detected with mtt assay.

Mtt assay detection killing cell toxicant efficiency

The preparation of reagent:

MTT [3- (4,5-dimethyiazol-2-yl) -2,5-diphenyltetrazoliumbromide], 5mg/ml； DMSO (dimethyl sulfoxide)；Acidification isopropanol: HCI is added in isopropanol, HCI final concentration is made to reach 0.04mol/L.

Operating procedure:

20 μ l MTT liquid are added in each cell micropore after washing.After 37 DEG C incubate 4 hours, there is the generation of crystalline deposit object.It goes Liquid and MTT liquid are trained, 200 μ l of DMSO is added, jog makes sediment be dissolved into transparency liquid.HCl dropwise 100 μ l of isopropanol, makes to mix It closes.The OD reading in each hole is surveyed under 570nm wavelength in microplate reader.

The calculating of killing rate

To as follows as the calculating fraction of target cell using the cell of adhere-wall culture:

To as follows as the calculation formula of target cell using the cell for the culture that suspends:

E: effector cell；E+T: effector cell+target cell.

The testing result of Execution vigor is as shown in table 4:

The testing result of 4 Execution vigor of table

The test of Cytotoxicity in vitro cancer cell is the Cytotoxicity in vitro cancer cell function of verifying the PD-1-T cell of in vitro culture, examination It tests the result shows that the killing cancer cell of PD-1-T versus's DC-CIK cell is with better function.

Embodiment 5

Verify the effect of cell preparation in embodiment 1.

Experimental method

PD-1-T cell culture is added cell according to the concentration of ten thousand/ml of 2000-3000 and feeds back between -20 days 14 days PD-1-T cell suspension (as PD-1-T cell preparation) is made in liquid, samples the culture side that different blood sources are provided according to embodiment 1 The PD-1-T cell preparation of method production does cytokine profiles detection, using (the Multiplex of Ebioscience company, the U.S. Immunoassay Kit) multiple-factor detection kit detected, and testing result is as shown in table 5.

The testing result of 5 cell preparation of table

The PD-1-T cell preparation for sampling the cultural method production that different blood sources are provided according to embodiment 1 does the cross-film of cell The detection of molecule, using the detection after specially treated sample, by function macromolecular on be detected as cell, it is necessary to be located in advance Reason allows the macromolecular separate out of detection, can just be detected with high-precision ELISA detection kit or multiple-factor detection kit, Preprocess method: it will test sample and carry out multigelation, so that clasmatosis makes cell using supercentrifuge Centrifuge A sample The intracellular bulky grain precipitating such as device and chromosome is removed, and supernatant solution after centrifugation is as test sample.Testing result such as table 6 It is shown.

The testing result of 6 transmembrane molecule of table

By table 5 and table 6 it is found that PD-1-T cells finished product (PD-1-T cell preparation) is from above-mentioned testing result comparison in difference Greatly, difference big reason detection is preparation cell suspension, because difference in operation and resting period difference is not (since cell stops to produce cell Factor resting period length necessarily affects content in cell suspension) cause the value difference of testing result different big, therefore this detection PD-1- T cell preparation Main Analysis is there are which cell factor and expresses which cell component.

By above-mentioned testing result it is found that cell factor proves that cell function is good containing there are many kinds of in cell preparation, making Agent also keeps good function after carrying out, and can secrete cytokine profiles, and the cell factor being not present has: IFN-alpha, the cell The factor is mainly the Th2 hypotype secretion of CD4+T cell, namely is free of the hypotype.

Comparative example 1

The killer T cell (PD-1-T cell) for the cultural method preparation that embodiment 1 is provided is mutual with DC-CIK cell Comparison.

DC-CIK cell is compared with PD-1-T cell.

Experimental method

Collect 1 cord blood takes a semicell to make DC-CIK cell culture (existing skill after whole blood separates monocyte Art), the other half does PD-1-T cell culture using the cultural method that embodiment 1 provides.

First sub-sampling: it samples within culture 0 day, sampling mode: cell counting board counts total number of cells, then takes 1,000,000 Mononuclearcell just is isolated, makees flow cytomery cell surface marker type.

Second sub-sampling: culture was sampled to 6 days；Third sub-sampling: culture was sampled to 12 days；4th sub-sampling: culture is extremely It samples within 16 days；5th sub-sampling: culture was sampled to 20 days；6th sub-sampling: culture was sampled to 24 days；7th sub-sampling: culture It was sampled to 27 days.Sampling mode is equal are as follows: takes suspension culture 1ml, supernatant is then demultiplex out in the cell concentration of first test sample Make cytokines measurement, cell makees flow cytomery.

Flow cytometer detection project: CD3+, CD4+；CD3+,CD8+；CD3+,CD56+；CD3+,CD28+；CD3-,CD56+； CD3-,CD28；6 marks.Cytokines measurement: detection IFN-r, GM-CSF, IL-12, IL-3, TPO, IL-11.

Experimental result

Cell Proliferation

The comparison result of the cell count of two kinds of cultural methods of DC-CIK cell and PD-1-T cell is as shown in Figure 1.

By the total number of cells variation of Fig. 1 cultivated days it can be seen that Lai the cell culture 20 of PD-1-T cell culture method It proliferation times are greater than 180 times, and about 80 times of the proliferation times of the cell culture 20 days of the training approach of DC-CIK cell. The peak of proliferation phase is -16 days 8 days.

Flow cytomery

DC-CIK cell and two kinds of cultural method difference incubation time cell phenotype testing results of PD-1-T cell are as shown in table 7.

7 cell phenotype testing result of table

CD8+ accounting comparison result figure please refers to attached drawing 2.By attached drawing 2 it is found that the cell of PD-1-T cell culture method is trained It supports to after 14 days, 80% or more CD8+T cell accounting.

Cytokines measurement

The testing result of secrete cytokines is as shown in table 8.

The testing result of 8 secrete cytokines of table

Remarks: the zero day blood plasma for the whole blood, unit: pg/ml.

As shown in Table 8, stronger in the period PD-1-T cell secretion of cytokines of culture to 14 days to 20 days.

From the point of view of above-mentioned testing result, PD-1-T cell culture was to 14 days later phenotypes: CD3+ accounting is 98% or more, Namely 98% or more T cell, and 80% or more the cell namely PD-1-T cell of CD8+ be with cytotoxic T lymphocyte (or Claim: cytotoxic T lymphocyte) based on；Its secrete cytokines ability of PD-1-T cell is obviously strong compared with DC-CIK cell, reflection The cell function of PD-1-T cell is strong compared with DC-CIK cell；The cell proliferation rate of PD-1-T cell is very fast, proliferation times and thin Born of the same parents' sum is stronger, therefore can obtain sufficient amount by vitro culture PD-1-T cell, will will obtain in clinical use bigger Curative effect.

In conclusion the cultural method can induce whole blood the embodiment of the invention provides a kind of cultural method of T cell The lymphocyte isolated breaks up to CD8+T cell proliferation during the cultivation process, and the T cell isolated can reach within 18 days To 40 times or more of amplification, and the accounting of CD8+T cell is more than 80%.

In addition, the embodiment of the invention also provides a kind of T cell, the combination of its culture medium, its application and a kind of cell systems Agent, the function of killer T cell secrete cytokines with higher, value-added speed is fast, for tumor cell invasiveness compared with DC-CIK cell is strong, and having the advantages that being capable of broad spectrum anticancer.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Claims (10)

1. a kind of cultural method of T cell characterized by comprising Cytokines in Peripheral Blood Mononuclear is cultivated, and is being cultivated Later period adds PD-1 antibody into culture solution and continues to cultivate；

Its preferably, the cultural method is the following steps are included: it includes mixing peripheral blood mononuclear cells with the anti-culture medium of A Carry out first time culture；Then, obtained product will be cultivated for the first time, and second of culture of progress is mixed with Type B culture medium；Finally, The product obtained after second is cultivated is mixed with K-type culture medium carries out third time culture；

The anti-culture medium of A includes complete medium, CD3 monoclonal antibody and one or more of component: proleulzin, dimension life Plain B6, interferon-γ, interleukin-1 b, interleukin-4, L-Glutamine, the third bronze medal acid sodium and plasma protein；

The K-type culture medium includes complete medium, PD-1 antibody and one or more of component: proleulzin, L- Glutamine, the third bronze medal acid sodium and plasma protein；

Include complete medium, CD28 antibody and one or more of component in the Type B culture medium: proleulzin, Vitamin C, L-Glutamine, the third bronze medal acid sodium and plasma protein.

2. cultural method according to claim 1, which is characterized in that in the anti-culture medium of A, CD3 monoclonal antibody it is final concentration of 80~120ng/ml；Preferably, in the anti-culture medium of A, final concentration of 800~1200u/ml of proleulzin, vitamin B6 Final concentration of 5~15ng/ml, final concentration of 5~15ng/ of final concentration of 15~25ng/ml of interferon-γ, interleukin-1 b Ml, final concentration of 5~1ng/ml of interleukin-4, final concentration of 180~220ng/ml of L-Glutamine, the end of the third bronze medal acid sodium Concentration is 15~25ng/ml, final concentration of 40~60ng/ml of plasma protein；

In Type B culture medium, 5~15ng/ml of CD28 antibody；Preferably, in Type B culture medium, proleulzin it is final concentration of 1800~2200u/ml, final concentration of 5~15ng/ml of vitamin B6, ascorbic final concentration of 5~15ng/ml, L- paddy Final concentration of 80~120ng/ml of glutamine, final concentration of 5~15ng/ml of the third bronze medal acid sodium, plasma protein it is final concentration of 15~25ng/ml；

In K-type culture medium, final concentration of 5~15ng/ml of PD-1 antibody；Preferably, in K-type culture medium, proleulzin The bronze medal acid sodium of final concentration of 1800~2200u/ml, final concentration of 40~60ng/ml of L-Glutamine, third final concentration of 3 ~8ng/ml, final concentration of 40~60ng/ml of plasma protein.

3. cultural method according to claim 2, which is characterized in that start and formed cell mass and thin to cell proliferation Born of the same parents' density reaches 2 X 10⁷~5 X 10⁷A/ml carries out described second and cultivates；Reach 2 X 10 to cell density⁷~5 X 10⁷ Third time culture is carried out when a/ml；

Preferably, the cultivated days of the first time culture are 3~6 days, and the cultivated days of second of culture are 10~12 days.

4. cultural method according to claim 3, which is characterized in that cultivate it with third time in second of culture Between, and/or, it further include that c-type culture medium is added to be cultivated after third time is cultivated；

Preferably, between second of culture and third time culture, when cell density reaches 2 X 10⁷~5 X 10⁷A/ml When, c-type culture medium is added, after third time is cultivated, when cell density reaches 2 X 10⁷~5 X 10⁷When a/ml, c-type is added Culture medium；

C-type culture medium includes complete medium, CD28 antibody and one or more of component: proleulzin, vitamin C, vitamin B6, L-Glutamine, the third bronze medal acid sodium and plasma protein.

5. cultural method according to claim 4, which is characterized in that in c-type culture medium, CD28 antibody it is final concentration of 3~8ng/ml, final concentration of 450~550u/ml of proleulzin, final concentration of 3~8ng/ml of vitamin B6, vitamin C The bronze medal acid sodium of final concentration of 3~8ng/ml, final concentration of 40~60ng/ml of L-Glutamine, third final concentration of 3~8ng/ Ml, final concentration of 5~15ng/ml of plasma protein.

6. a kind of culture medium of T cell combines, which is characterized in that it includes the anti-culture medium of A, K-type culture medium and Type B culture Base；

The anti-culture medium of A includes complete medium, CD3 monoclonal antibody and one or more of component: proleulzin, dimension life Plain B6, interferon-γ, interleukin-1 b, interleukin-4, L-Glutamine, the third bronze medal acid sodium and plasma protein；

The K-type culture medium includes complete medium, PD-1 antibody and one or more of component: proleulzin, L- Glutamine, the third bronze medal acid sodium and plasma protein；

Include complete medium, CD28 antibody and one or more of component in the Type B culture medium: proleulzin, Vitamin C, L-Glutamine, the third bronze medal acid sodium and plasma protein；

Preferably, culture medium combination further includes c-type culture medium, c-type culture medium include complete medium, CD28 antibody with And one or more of component: proleulzin, vitamin C, vitamin B6, L-Glutamine, the third bronze medal acid sodium and blood plasma egg It is white.

7. a kind of T cell, which is characterized in that it is made by the described in any item cultural methods of Claims 1 to 5, or using power Benefit require 6 described in culture medium combination culture obtain.

8. a kind of cell preparation, which is characterized in that it includes that cell feeds back liquid and T cell as claimed in claim 7.

9. T cell as claimed in claim 7 or cell preparation as claimed in claim 8 are preparing answering in anticancer drug With.

10. application according to claim 9, which is characterized in that the anticancer drug is anti-cutaneum carcinoma, lung cancer, prostate Cancer, head and neck cancer, cervical carcinoma, thyroid cancer, spongioblastoma, glioma, leukaemia, lymthoma, adrenal, AIDS Associated cancer, alveolar soft part sarcoma, astrocytoma, osteocarcinoma, brain and spinal cord cancer, metastatic brain tumor, carotid body tumor, Chondrosarcoma, kidney chromophobe cell tumor, clear cell carcinoma, skin Benign Fibrous Histiocytoma, promotees connective tissue proliferation at chordoma Property small round cell neoplasm, ependymoma, ewing's tumor, the outer myxoid chondrosarcoma of bone, bone imperfection fiber occur, bone fibres hair Educate exception, gall-bladder or cholangiocarcinoma, gestational trophoblastic disease, germinoma, hematologic malignancies, hepatocellular carcinoma, pancreas islet Cytoma, Kaposi sarcoma, kidney, lipoma/benign lipoma, embryonal-cell lipoma/pernicious lipoma, medulloblastoma, Meningioma, merkel's cells cancer, the formation of Multiple Endocrine tumor, Huppert's disease, myelodysplastic syndrome, at nerve Cytoma, neuroendocrine tumor, papillary thyroid carcinoma, parathyroid adenoma, paediatric cancer, Peripheral Nerve Sheath Tumors, thermophilic chromium are thin Born of the same parents' tumor, hypophysoma, prostate cancer, rear uveal melanoma, rare blood disease, kidney metastatic carcinoma, rhabdoid tumor, band muscle Tumor, sarcoma, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial bursa sarcoma, carcinoma of testis, thymic carcinoma, thymoma, Thyroid metastasis cancer Or the drug of uterine cancer；

Preferably, the anticancer drug is medicament for resisting cervical cancer or anti-leukemia medicine.