CN1724655A - Preparation method of lymphocyte and immune therapeutic agent - Google Patents
Preparation method of lymphocyte and immune therapeutic agent Download PDFInfo
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- CN1724655A CN1724655A CNA2004100707377A CN200410070737A CN1724655A CN 1724655 A CN1724655 A CN 1724655A CN A2004100707377 A CNA2004100707377 A CN A2004100707377A CN 200410070737 A CN200410070737 A CN 200410070737A CN 1724655 A CN1724655 A CN 1724655A
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Abstract
The invention relates to an activation proliferation method for lymphocyte and the cell immunity therapeutic agent. It uses IL-2 and IL-12 to activate lymphocyte, especial NK cell and T cell to enhance the response ability of non-specificity NK cell and specificity T cell and the killing activity. The technology supplies a manufacture method for proliferation and immunity therapeutic.
Description
Affiliated technical field
The present invention relates to a kind of lymphocytic activationa and proliferation method and method and the cellular immunotherapeutic agents waited until thus.
Background technology
Treatment for cancer has many immunotherapies now.All respects in anticarcinogenic effect cell, cytokine, anticancrin and knurl seedling and gene therapy research all have obvious progress at present, and have obtained certain technological breakthrough and clinical efficacy.Such as for improving cancer patients's self immunizing power, drop into the immuno-chemical substance of numerous species, as cytokine classes such as Interferon, rabbit, interleukins, or the immunity effect agent.Wherein the Activiation method of the inducing of cytokine, anticarcinogenic effect cell is the focus of research.The network of the inducing of the effective antitumor factor, cytokine and formation thereof, not only at immune cell activated, induce cytokine and regulate in the reaction of whole antitumor immune and play an important role, the propagation transferance that also has direct inhibition tumour cell is the important preparation of tumor biotherapy.At present cytokines such as IL-2, G-CSF, GM-CSF, IFN-α, IFN-γ and IFN-β have been applied to clinically, but really have the cytokine of produce effects killing ability few in number, satisfactory for result also seldom, even have also have very strong side effect.So need to seek effectively treatment method for cancer.
Now, have clinically multiple immunocyte is taken out external, after the activationa and proliferation again with defeated intravital methods of treatment.The method of representative has the LAK cell, CAT cell, TCL cell, til cell etc.
The LAK cell is with the blood cell of blood cell separator with a few hours separation patient, can obtain 5 * 10
5～5 * 10
10Individual monocyte is by the stimulation acquisition LAK cell of interleukin-22.At last, the LAK cell that obtains and a large amount of interleukin-22s are fed back in the body.Consequently the cancer to a part of cancer patients, part kind has result of treatment.But the side effect of severes such as Most patients can be with heating, aversion to cold, feel sick, vomiting, expiratory dyspnea even skin erythema.Owing of this method a little is to need a large amount of lymphocytes, and fails back the caused severe side effect of intravital cytokine simultaneously with lymphocyte.
The CAT cell therapy is with a spot of tip lymphocyte, by solidified anti-cd 3 antibodies activated lymphocyte, makes it propagation with the interleukin-22 irritation cell then.This therapeutics has the cancerous swelling of making to dwindle or the report that disappears.But a part of cell activity is not wherein fully played and is utilized.
The til cell therapy is with in the cancerous swelling or the lymphocyte around the cancerous swelling, with and the same method of LAK cell, lymphocyte and interleukin-22 cultivated together drop into afterwards human body.Compare with the CAT cell therapy, the til cell therapy is stronger to the cognitive ability of cancer cells.
The CTL cell therapy is similar with the CAT cell therapy, also is with a spot of tip lymphocyte, by solidified anti-cd 3 antibodies activated lymphocyte, makes it propagation with the interleukin-22 irritation cell then.But add some tumour specific antigens simultaneously, stimulate lymphocyte to make it that tumour cell is had specific cognitive ability repeatedly.Kill capability to cancer cells is greater than LAK cell therapy and CAT cell therapy.
More than these therapies all be to be conceived to how to improve the T cell activity, and ignored the utility value of the NK cell that occupies significant proportion in the lymphocyte.
The NK cell accounts for the 5-7% of total number of peripheral blood.In human body, the antitumor action of the antitumor action of T cell and NK cell has complementary action.The T cell depends on the antigenic prompting of class I to the injury effect of tumour cell, and the NK cell can act on the tumour cell that might can not be identified because class I antigen reduces.The NK cell has the initial stage of important effect, particularly metastatic carcinoma particularly important to the transfer of cancer cells.
IL-12 is a kind of glycoprotein, and molecular weight is 75kd.By B cell and single ball is that cell produces, and it is characterized in that the NK cell is had tangible activation, and the propagation and the lethal effect of promotion NK cell that can be powerful are a kind of NK cell stimulating factor.Simultaneously, IL-12 can also promote the propagation and the lethal effect of T cell, significantly strengthens the killing activity of LAK cell, promotes the responsibility of specific CTL.IL-12 has inducing T cell and a large amount of secretion of gamma-IFN of NK cell, increase the ability of various cytokine receptors of NK cell expressing and adhesion molecule, can start the Th0 cell again to the Th1 cells whose development, synthetic, the collaborative SCF that suppresses IL-4 mediation IgE induces the generation of medullary cell, IL-12 all plays a significant role in congenital and acquired antineoplastic immune, has good anti-cancer applications and is worth.
IL-12 directly drops in the body, can produce severe side effect.Can't play the effect of killing and wounding cancer cells.Activate the purpose that the NK cell then can reach the treatment cancer with IL-12.
Summary of the invention
The problem that solves
Improve the quantity of T cell and NK cell simultaneously and to the killing activity of cancer cells.When cancerous swelling is played therapeutic action, also can effectively prevent cancer cells diffusion and transfer in vivo.
Present technique provides a kind of lymphocytic cultural method, relates in optimized technical scheme the cultural method of lymphocyte under the condition that anti-cd 3 antibodies exists.
Present technique provides a kind of lymphocytic cultural method, relates in optimized technical scheme the cultural method of lymphocyte under the condition of anti-cd 3 antibodies and IL-12 existence.
Present technique provides a kind of lymphocytic cultural method, relates in optimized technical scheme the cultural method of lymphocyte under the condition of anti-cd 3 antibodies and IL-2 and IL-12 existence.
Present technique provides a kind of lymphocytic cultural method, relates in optimized technical scheme the cultural method of lymphocyte under the condition of IL-12 and IL-2 existence.
Present technique provides a kind of cellular immunization preparation, in optimized technical scheme, relate to above-mentioned lymphocyte or based on above-mentioned lymphocyte, or as the part of treatment means, be used for cancer or infect the immunity therapeutic preparation of treatment usefulness such as disease.
Experimental technique
The lymphocyte that present technique is used, be not limited only to peripheral blood lymphocyte, also can be used for the lymphocyte that epithelium lymphocyte, the interior lymphocyte infiltration of tumour, malignant ascite endolymph cell, pernicious hydrothorax endolymph cell or the like are present in all kinds in the human body.Not special restriction, but peripheral blood lymphocyte is owing to obtaining easily and convenient separation, so more commonly used.The number of cell is with 1 * 10
6～5 * 10
7Individual being advisable.
With above-mentioned cell, under the environment of the existence of anti-cd 3 antibodies, cultivate.CD3 is present in the surface of T cell, by the mediation of CD3 molecule, will stimulate information to be sent to cell interior, and activating cells makes cell begin increment.CD3 is the important cell surface molecule of antigen understanding.And the numerator mediated cytositimulation of CD3 is then realized by anti-cd 3 antibodies.
Anti-cd 3 antibodies can also can be solidified it by the form of free state.The latter's better effects if.The curing of anti-cd 3 antibodies can be dissolved with the physiological saline or the phosphoric acid buffer of sterilization, and concentration is 0.1～100 μ g/ml.For well, common culturing bottle gets final product solidified carrier with materials such as plastics, glass.Curing is that above-mentioned anti-cd 3 antibodies solution is added in the assimilation container, and 4～37 ℃ left standstill 2～24 hours.Container after the curing places 4-30 ℃ of preservation.Clean with physiological saline at normal temperature during use.
Present technique uses IL-12 as stimulating factor.IL-12 physiological saline, phosphoric acid buffer, or nutrient solution dissolving, concentration is 1～2000 international unit/ml.
Present technique uses IL-2 as common stimulating factor.IL-2 physiological saline, phosphoric acid buffer, or nutrient solution dissolving, concentration is 1～2000 international unit/ml.
Lymphocytic nutrient solution does not have special requirement, all can so long as be applicable to the nutrient solution of lymphocyte growth.Such as RPMI-164O, AIM-V, DMEM, IMDM etc.Can add fetal bovine serum as required, bovine serum albumin or people's serum.Lymphocyte is suspended in the nutrient solution that contains IL-2 and IL-12, is added in the container that solidified with anti-cd 3 antibodies.
Cultural method carries out according to common cell culture processes.General requirement is at CO
2Carry out CO in the incubator
2Concentration is 1-10%.Temperature is 30-40 ℃, incubation time 2-20 days.Answer the growing state of observation of cell between incubation period, and the quantity of counting cells.Generally after cultivating a couple of days, nutrient solution can yellowing.Should add a certain amount of new nutrient solution this moment, replenishes a certain amount of IL-2 and IL-12 solution simultaneously.
Under the condition that anti-cd 3 antibodies exists, long-term cultivation is unfavorable for lymphocytic a large amount of propagation, therefore rises in value to a certain degree the time at lymphocyte, cell should be moved on to the environment that does not have anti-cd 3 antibodies to exist and cultivate down.3-7 days after cultivation normally move into culturing cell in the gas permeability culture bag and to cultivate.
In this stage, the propagation of cell need constantly be added fresh nutrient solution, and fresh IL-2 and IL-12 solution.Cell concn is 1 * 10
5～1 * 10
8/ ml.
Present technique mainly is to induce the increment of NK cell, improves the lytic activity of the killer cell of NK cell, and induces the IFN-γ of NK cell and the secretion capacity of relevant cell element.The latter is the immune response of activating cells mediation further.Can improve non-specific lymphocytic response capacity, improve non-specific kill capability, both can kill and wound cancer cells, the stronger ability that prevents cancer metastasis is arranged again cancer cells.
Present technique can also be rised in value by inducer T lymphocyte, improves specificity lymphocyte reaction and high degree of specificity ctl response.
Present technique can also be induced CD4
+T cell, CD8
+The propagation of T cell.
Present technique can also improve the kill capability to tumour by increasing the ability of various cytokine receptors of NK cell expressing and adhesion molecule.
As a kind of therapeutical agent, the quantity of cell is 1 * 10
5～1 * 10
12Individual.For the validity of proof technical solution of the present invention, can test according to following method
The optimum implementation of present technique
Present technique is the lymphocyte that should choose.Test used lymphocyte, can be peripheral blood lymphocyte, also available be the lymphocyte that lymphocyte infiltration, malignant ascite endolymph cell, pernicious hydrothorax endolymph cell or the like are present in all kinds in the human body in epithelium lymphocyte, the tumour.Because peripheral blood lymphocyte is obtained and convenient separation easily, so peripheral blood lymphocyte commonly used.The number of cell is with 1 * 10
6～50 * 10
6Individual being advisable.
The curing of anti-cd 3 antibodies can be dissolved with the physiological saline or the phosphoric acid buffer of sterilization, and concentration is 0.1～100 μ g/ml, is advisable with 1～10 μ g/ml.Solidified carrier is advisable with materials such as plastics, glass, and common culturing bottle gets final product.Curing is that above-mentioned anti-cd 3 antibodies solution is added in the curing vessel, and 4～37 ℃ left standstill 2～24 hours.Container after the curing places 4 ℃ of refrigerators to preserve.Clean with physiological saline at normal temperature during use.
Present technique uses IL-12 as stimulating factor.IL-12 physiological saline, phosphoric acid buffer, or nutrient solution dissolving, concentration is advisable with 1～2000 international unit/ml.
Present technique uses IL-2 as common stimulating factor.IL-2 physiological saline, phosphoric acid buffer, or nutrient solution dissolving, concentration is advisable with 10～2000 international unit/ml with 1～2000 international unit/ml.
Lymphocytic nutrient solution will be applicable to the nutrient solution of lymphocyte growth.Such as RPMI-1640, AIM-V, DMEM, IMDM etc.Can add fetal bovine serum as required, bovine serum albumin or people's serum.Lymphocyte is suspended in the nutrient solution that contains IL-2 and IL-12, is added to in the anti-cd 3 antibodies solidified container.
Cultural method carries out according to common cell culture processes.General requirement is at CO
2Carry out CO in the incubator
2Concentration is 1-10%, is advisable with 5%.Temperature is 30-40 ℃, is advisable with 37 ℃.Incubation time 2-21 days, be advisable with 7-14 days.Answer the growing state of observation of cell between incubation period, and the quantity of counting cells.Generally at incubation time, be generally 3 days after, nutrient solution can yellowing.Should add a certain amount of new nutrient solution this moment, replenishes a certain amount of IL-2 and IL-12 solution simultaneously, is advisable with isoconcentration.
Under the condition that CD3 antibody exists, long-term cultivation is unfavorable for lymphocytic a large amount of propagation, therefore rises in value to a certain degree the time at lymphocyte, cell should be moved on to the environment that does not have anti-cd 3 antibodies to exist and cultivate down.3-7 days after cultivation normally move into culturing cell in the gas permeability culture bag and to cultivate, and are advisable with 5-7 days.
In this stage, the propagation of cell need constantly be added fresh nutrient solution, and fresh IL-2 and IL-12 solution.Cell concn is 1 * 10
5～100 * 10
5Individual/ml, with 5 * 10
5～10 * 10
5Individual/ml is advisable.
After cultivating end, cell centrifugation is reclaimed, cell is cleaned the back and suspended with physiological saline, and is standby.
As a kind of therapeutical agent, the quantity of cell is 1 * 10
5～1 * 10
12, with 1 * 10
7～1 * 10
11Individual being advisable.
Beneficial effect
Present technique can provide a lymphocyte that cancer cell is had greater activity, larger amt. Jointly to do by T cell and NK cell With and the experiment. Both can be used for the treatment of cancer, more can prevent the diffusion of cancer cell.
Claims (5)
1. a lymphocytic cultural method relates to the cultural method of lymphocyte under the condition that anti-cd 3 antibodies exists.
2. a lymphocytic cultural method relates to the cultural method of lymphocyte under the condition of anti-cd 3 antibodies and IL-12 existence.
3. a lymphocytic cultural method relates to the cultural method of lymphocyte under the condition of anti-cd 3 antibodies and IL-2 and IL-12 existence.
4. a lymphocytic cultural method relates to the cultural method of lymphocyte under the condition of IL-12 and IL-2 existence.
5. cellular immunization preparation, by claim 1, claim 2, claim 3, claim 4 or, the prepared cell of the described lymphocyte cultural method of claim 5 is main body or is the therapeutic preparation of the part of treatment means.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100707377A CN1724655A (en) | 2004-07-23 | 2004-07-23 | Preparation method of lymphocyte and immune therapeutic agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100707377A CN1724655A (en) | 2004-07-23 | 2004-07-23 | Preparation method of lymphocyte and immune therapeutic agent |
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|---|---|
| CN1724655A true CN1724655A (en) | 2006-01-25 |
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|---|---|---|---|
| CNA2004100707377A Pending CN1724655A (en) | 2004-07-23 | 2004-07-23 | Preparation method of lymphocyte and immune therapeutic agent |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969731A (en) * | 2016-07-29 | 2016-09-28 | 解西河 | Method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid |
| CN114667152A (en) * | 2019-11-01 | 2022-06-24 | 首尔大学校产学协力团 | Composition for improving immunological activity and method thereof |
-
2004
- 2004-07-23 CN CNA2004100707377A patent/CN1724655A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969731A (en) * | 2016-07-29 | 2016-09-28 | 解西河 | Method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid |
| CN105969731B (en) * | 2016-07-29 | 2019-10-22 | 青岛市中心医院 | A method of High Fragmentation activity til cell is largely prepared using pernicious Pleural effusions |
| CN114667152A (en) * | 2019-11-01 | 2022-06-24 | 首尔大学校产学协力团 | Composition for improving immunological activity and method thereof |
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