CN111073852A - NK cell culture solution and culture method - Google Patents
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Abstract
The invention relates to the technical field of cell culture, in particular to an NK cell culture solution and a culture method thereof, and discloses the NK cell culture solution, wherein IFN-gamma and IL-15 are compounded in the culture solution, IL-2, IL-1 α and CD3 monoclonal antibodies are compounded, the combined action is favorable for stimulating the efficient conversion of peripheral blood mononuclear cells to NK cells, the multiplying power of the NK cells is increased, more NK cells are obtained, the contents of CD3-CD56+ and CD3+ CD56+ are high, and the killing activity is high, so that the NK cell culture solution provided by the invention can obviously improve the cytotoxic activity while increasing the amplification quantity of the NK cells.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to an NK cell culture solution and a culture method.
Background
NK cells are important immune cells which participate in the body to resist tumor and virus infection and immunoregulation, have no MHC restriction in the process of killing target cells, do not depend on antibodies, and effectively kill various tumor cells through direct and indirect ways. However, the number of NK cells in the peripheral blood of normal human body is small, and only accounts for 10% -20% of the total number of mononuclear cells in the peripheral blood. In recent years, cell immunotherapy has achieved breakthrough progress, and NK cell therapy has achieved certain clinical efficacy. At present, how to obtain a large amount of high-quality and high-purity NK cells is one of the most critical problems in clinical application.
In addition to cytokine stimulation, a culture solution used in the currently common in-vitro NK cell amplification method is also a method for amplifying NK cells by adopting trophoblast cells, and inactivated K562 tumor cell strains and peripheral blood mononuclear cells are required to be added for mixed culture so as to induce and amplify the NK cells. However, because the tumor cells are added into the culture solution, the quality control cannot be carried out in the later period, potential safety hazards are introduced, the application to clinical treatment is difficult, and the ethical barrier which is difficult to overcome exists.
Disclosure of Invention
The invention provides an NK cell culture solution and a culture method, and solves the problems that in the existing process of amplifying NK cells by adopting trophoblasts, inactivated K562 tumor cell strains and peripheral blood mononuclear cells need to be added for mixed culture to induce and amplify the NK cells, so that quality control cannot be performed in the later period, and potential safety hazards are introduced.
The invention provides an NK cell culture solution, which comprises: culture solution A and culture solution B;
the culture solution A comprises: IFN-gamma, IL-15 and basal medium;
the culture solution B comprises IL-2, IL-1 α, a CD3 monoclonal antibody and a basal culture medium.
In the invention, IFN-gamma is interferon gamma, IL-15 is interleukin 15, IL-2 is interleukin 2, and IL-1 α is interleukin 1 α.
IL-15 is not only similar in spatial structure to IL-2, but IL-15R and IL-2R share β c and gammac subunits, and can promote the differentiation of hematopoietic progenitor cells into CD56+ NK cells IL-15 can enhance the release of IFN-gamma from NK cells IL-15 can up-regulate the expression of NKG2D activation receptor, enhance the expression of cytotoxic molecules such as TRAIL and perforin, and thereby enhance the lytic activity of NK cells.
IL-2 is mainly secreted by T cells, and promotes the proliferation and differentiation and effector functions of T cells, NK cells, B cells and peripheral blood mononuclear cells in an autocrine and paracrine mode. IL-2 can also induce KIR receptor expression of NK cell subset of KIR-, C lectin receptor NKG2D and natural killer toxicity receptor NKp44 expression, and enhance release of IFN-gamma by NK cells.
IFN-gamma has the effect of up-regulating the expression of IL-2R on the surface of peripheral blood lymphocytes, thus enhancing the sensitivity and intensity of T cells to IL-2 proliferative responses.
IL-1 α may also mediate the upregulation of IL-2R expression on the surface of peripheral blood lymphocytes.
The invention compounds IFN-gamma and IL-15, compounds IL-2, IL-1 α and CD3 monoclonal antibody, and the combined action is favorable for stimulating the transformation of peripheral blood mononuclear cells to NK cells.
In the present invention, the NK cell culture solution further comprises: a culture solution C; the culture solution C comprises: IL-2, IL-15 and basal medium.
The NK cell culture solution of the present invention further comprises: PD-1.
The monoclonal antibody of the invention is a monoclonal antibody which is bound to programmed death receptor-1 (PD-1), and blocks PD-1 pathway mediated immune suppression reaction including anti-tumor immune reaction by blocking the interaction between PD-1 and ligands PD-L1 and PD-L2 thereof. The present invention was added on day 13 of NK cell culture,
in the present invention, the final concentration of PD-1 is 0.5 to 1.25. mu.g/ml, preferably 1 to 1.25. mu.g/ml, and more preferably 1.25. mu.g/ml. According to the invention, the culture solution IL-2 and IL-15 are compounded to further stimulate the peripheral blood mononuclear cells to induce the NK cell transformation.
In the invention, the final concentration of IFN-gamma in the culture solution A is 2500-5000 IU/ml, more preferably 4000-5000 IU/ml, and further preferably 5000 IU/ml;
the final concentration of the IL-15 in the culture solution A is 10-100 mu g/ml, more preferably 20-50 mu g/ml, and further preferably 50 mu g/ml;
the final concentration of the IL-2 in the culture solution B is IL-2: 500-1000 IU/ml, more preferably 900-1000 IU/ml, and further preferably 1000 IU/ml;
the final concentration of the IL-1 α in the culture solution B is 2.5-5 ng/ml, more preferably 4-5 ng/ml, and further preferably 5 ng/ml;
the final concentration of the CD3 monoclonal antibody in the culture solution B is 250-500 ng/ml, more preferably 400-500 ng/ml, and even more preferably 500 ng/ml.
In the present invention, the NK cell culture solution further comprises: the culture solution C or D is preferably culture solution D.
In the present invention, the culture solution C includes: IL-2, IL-15 and basal medium. The final concentration of the IL-2 in the culture solution C is 500-1000 IU/ml, more preferably 1000 IU/ml.
The final concentration of the IL-15 in the culture solution C is 10-100 mu g/ml, more preferably 20-50 mu g/ml, and even more preferably 50 mu g/ml.
The culture solution D comprises: IL-2 and basal medium. The final concentration of the IL-2 in the culture solution D is 500-1000 IU/ml, and preferably 1000 IU/ml.
In the process of culturing the NK cells, the culture solution A, the culture solution B and the culture solution C or the culture solution D are sequentially adopted to stimulate the conversion of the peripheral blood mononuclear cells to the NK cells, so that higher NK cell number and cytotoxic activity can be obtained.
In the invention, basic culture media in the culture solution A, the culture solution B, the culture solution C and the culture solution D are lymphocyte serum-free culture media;
the plasma is autologous plasma. The autologous plasma is used as a nutrient substance, so that the use of fetal calf serum is avoided, exogenous pyrogen and sensitization pollution are reduced, and the culture cost is reduced.
The invention also provides an NK cell culture kit, which comprises the culture solution A, the culture solution B, PD-1 and the culture solution C or the culture solution D;
the culture solution B comprises IL-2, IL-1 α, CD3 monoclonal antibody and a basal culture medium;
the culture solution C comprises: IL-2, IL-15 and basal medium;
the culture solution D comprises: IL-2 and basal medium.
The final concentrations of each component in the culture solution A, the culture solution B, the culture solution C and the culture solution D in the NK cell culture kit of the invention have been specifically explained in the above technical scheme, and are not described herein again.
The invention also provides a culture method of the NK cells, which comprises the following steps:
step 1: on day0, the peripheral blood single cells were inoculated into the culture solution a for culture;
step 2: culturing to day1, adding culture solution B, and continuing culturing;
and step 3: culturing for 2, 4, 8 and 11 days, respectively adding culture solution C or D, and continuing culturing;
and 4, step 4: adding PD-1 to continue culturing until day 13 or day 14, and culturing until day15 to obtain NK cells;
the culture solution B comprises IL-2, IL-1 α, CD3 monoclonal antibody and a basal culture medium;
the culture solution C comprises: IL-2, IL-15 and basal medium;
the culture solution D comprises: IL-2 and basal medium.
In step 1 of the present invention, the amount of single cells in peripheral blood inoculated is 2X 106～1×107One/ml, preferably 2X 106～4×106One per ml.
In step 3 of the present invention, it is preferable to add the culture solution D to the cells cultured up to 2, 4, 8 and 11 days to continue the culture.
Step 4 of the present invention preferably includes the addition of PD-1 on day 13, which induces NK cell antibody-specific cytotoxicity.
In the invention, the cells obtained by adopting the NK cell culture solution and the PD-1 culture are PD-NK cells.
In the present invention, before step 1, the method further comprises: separation of peripheral blood mononuclear cells.
In the present invention, the isolation of peripheral blood mononuclear cells comprises the following steps:
step a: centrifuging human peripheral blood, and diluting the centrifuged red blood cells to obtain red blood cell diluent;
step b: and adding lymphocyte separation liquid into the erythrocyte diluent, taking a peripheral blood mononuclear cell layer, and then sequentially washing and centrifuging to obtain the peripheral blood mononuclear cell.
In the invention, the rotation speed of the centrifugation in the step a is 2000-3000 rpm, the time is 10-15 min, preferably 2500rpm, 10 min; and after the peripheral blood is centrifuged, the obtained upper plasma can be inactivated in water bath and stored at 4 ℃ for later use, wherein the water bath inactivation temperature is 55.75-6.3 ℃, and the time is 25-30 min, preferably 56.0 ℃ and 30 min.
In step b of the present invention, the volume ratio of the red blood cell diluent to the lymphocyte separation medium is preferably 1: 1, and the total volume of the erythrocyte diluent and the lymphocyte separation solution is not more than two thirds of the volume of the required centrifugal tube; the rotation speed of the centrifugation is 1800 rpm-2000 rpm, the time is 5 min-10 min, preferably 1800rpm, 10 min.
The washing in the step b of the invention is specifically as follows: and (3) resuspending the sediment obtained after centrifugation by using a cleaning solution, and then centrifuging at 1500-2000 rpm for 5-10 min, preferably 1600rpm for 5 min.
In the method for culturing NK cells of the present invention, plasma is added after the culture medium is added on days 1, 4 and 8 of the culture.
According to the technical scheme, the invention has the following advantages:
the invention provides an NK cell culture solution, IFN-gamma and IL-15 are compounded in the culture solution, IL-2, IL-1 α and CD3 monoclonal antibodies are compounded, the combined action is favorable for stimulating the efficient transformation of peripheral blood mononuclear cells to NK cells, the multiplying power of the NK cells is increased, more NK cells are obtained, the content of CD3-CD56+ and CD3+ CD56+ is high, and the killing activity is high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.
FIG. 1 is a graph showing the results of flow assay on day0 of NK cell culture according to example 1 of the present invention;
FIG. 2 is a graph showing the results of flow assay on day 14 of NK cell culture according to example 1 of the present invention;
FIG. 3 is a graph showing the results of flow assay on day 14 of NK cell culture according to example 2 of the present invention;
FIG. 4 is a graph showing the results of flow assay on day 14 of NK cell culture provided in example 3 of the present invention;
FIG. 5 is a graph showing the results of flow assay on day 14 of NK cell culture according to example 4 of the present invention.
Detailed Description
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The cleaning liquids used in the embodiment of the invention are all as follows: 0.9% sodium chloride injection.
In the examples of the present invention, IFN-. gamma.was purchased from Shanghai Biotechnology Ltd, IL-15 was purchased from Setarian Okinawa (Beijing) bioscience technology Ltd, IL-2 was purchased from Shandong spring harbor pharmaceutical industry Ltd, IL-1 α was purchased from Guangzhou DongRui technology Ltd, CD3 monoclonal antibody was purchased from Baori doctor Biotechnology (Beijing) Ltd, lymphocyte serum-free medium was purchased from Guangzhou Haichin Biotechnology Ltd, and A549 tumor cells were preserved from Guangzhou Yanghua medicine technology Ltd.
Example 1
This example is the culture of NK cells
(1) Isolation of peripheral blood mononuclear cells:
(1.1) Whole blood separation and plasma treatment
Collecting 5ml of human peripheral venous blood, transferring to a centrifuge tube, centrifuging at 2500rpm for 10min, transferring upper layer plasma to a new centrifuge tube, performing heat inactivation in a 56.0 deg.C water bath for 30min, and storing in a 4 deg.C refrigerator;
(1.2) isolation of peripheral blood mononuclear cells: and (3) adding an equal volume of cleaning solution into the separated lower cell layer in the step (1.1) for dilution, and uniformly blowing. Adding 5ml of Ficoll into a 15ml centrifuge tube, inclining the centrifuge tube by 45 degrees, slowly adding the diluted cells to the upper layer of the Ficoll along the tube wall at a constant speed, and centrifuging at 2500rpm for 20 min; most of the washing liquid is sucked out, and the residual plasma mixed layer is discarded. Absorbing and transferring the residual plasma mixed layer, the peripheral blood mononuclear cell layer and the Ficoll layer of the residual cleaning solution into a new 15ml centrifuge tube, and adding the cleaning solution to the maximum value of the scale of the centrifuge tube;
(1.3) peripheral blood mononuclear cell washing: the centrifugal tube obtained after the completion of (1.2) was centrifuged at 1800rpm for 10 min. Discarding the supernatant, resuspending the cells with a washing solution, and adding the washing solution to the maximum scale of the centrifuge tube;
(1.4) obtaining peripheral blood mononuclear cells: centrifuge at 1600rpm for 5min and discard the supernatant. The precipitate is the peripheral blood mononuclear cells, and counting;
(2) directionally expanding NK cells:
(2.1) centrifugation of post heat inactivation plasma: and (3) taking out the blood plasma subjected to heat inactivation in the step (1.1) from the refrigerator, centrifuging for 5min at 4000rpm, transferring the supernatant into a new centrifugal tube, and placing the centrifugal tube in the refrigerator at 4 ℃ for standby.
(2.2)Day0
And adding IFN-gamma and IL-15 into the lymphocyte serum-free culture medium, and uniformly mixing to obtain a culture solution A, wherein the final concentrations of the IFN-gamma and the IL-15 in the culture solution A are 5000IU/ml and 50 mu g/ml respectively.
Resuspending (1.4) the step of peripheral blood mononuclear cells (1X 10 inoculum size of peripheral blood mononuclear cells) with 5ml of culture A7Pieces/ml), the cell suspension was transferred to a T25 flask and placed at 37 ℃ in 5% CO2The culture box is used for culturing for 24 hours.
(2.3)Day1
And adding IL-2, IL-1 α and CD3 monoclonal antibodies into the lymphocyte serum-free culture medium, and uniformly mixing to obtain a culture solution B, wherein the final concentrations of the IL-2, IL-1 α and CD3 monoclonal antibodies in the culture solution B are 1000IU/ml, 5ng/ml and 500ng/ml respectively.
2.5ml of culture solution B was added to the flask of step (2.2). Adding 0.5ml (2.1) of heat-inactivated plasma into the culture flask, and standing at 37 deg.C and 5% CO2The cultivation was continued for 24 h.
(2.4) periodically rotating bottles and replenishing liquid:
and adding IL-2 and IL-15 into the lymphocyte serum-free culture medium, and uniformly mixing to obtain a culture solution C, wherein the final concentrations of the IL-2 and the IL-15 in the culture solution C are 1000IU/ml and 50 mu g/ml respectively.
Day 2: 5ml of culture medium C was supplemented;
day 4: transfer all cells from the T25 flask to a new T75 flask and replenish culture medium C to a total volume of 40ml, and 1ml (1.1) of centrifuged heat-inactivated plasma;
day 8: transfer all cells from the T75 flask to a new T175 flask and replenish medium C to a total volume of 100ml, and 0.5ml (1.1) of centrifuged heat-inactivated plasma;
day 11: supplementing 50ml of culture solution C;
(2.5)Day13
adding 0.2ml PD-1 reagent with concentration of 5mg/ml into the culture flask of step (2.4), placing at 37 deg.C and 5% CO2Continuously culturing in the incubator;
(3) day 15: collecting cells
Example 2
This example is the culture of NK cells
This example differs from example 1 in that: (2.4) regular bottle rotating and liquid supplementing steps:
and adding IL-2 and IL-15 into the lymphocyte serum-free culture medium, and uniformly mixing to obtain a culture solution C, wherein the final concentrations of the IL-2 and the IL-15 in the culture solution C are 1000IU/ml and 50 mu g/ml respectively.
And adding IL-2 into the lymphocyte serum-free culture medium, and uniformly mixing to obtain a culture solution D, wherein the final concentration of the IL-2 in the culture solution D is 1000 IU/ml.
Day 2: 5ml of culture medium C was supplemented;
day 4: the whole cells from the T25 flask were transferred to a 15ml centrifuge tube, centrifuged at 1000rpm for 5min, the supernatant discarded, and washed 2 times with washing solution. Resuspend cells with medium D, transfer all to a new T75 flask, and replenish medium D to a total volume of 40ml, and heat inactivated plasma after centrifugation in 1ml (1.1);
day 8: transfer all cells from the T75 flask to a new T175 flask and supplement with medium D to a total volume of 100ml, and 0.5ml (1.1) of heat-inactivated plasma after centrifugation;
day 11: 50ml of culture D was supplemented.
Example 3
This example is the culture of NK cells
This example differs from example 1 in that: (2.4) periodically rotating bottles and replenishing liquid:
and adding IL-2 into the lymphocyte serum-free culture medium, and uniformly mixing to obtain a culture solution D, wherein the final concentration of the IL-2 in the culture solution D is 1000 IU/ml.
Day 2: 5ml of culture solution D was supplemented;
day 4: transfer all cells from the T25 flask to a new T75 flask and supplement medium D to a total volume of 40ml, and 1ml (1.1) of centrifuged heat-inactivated plasma;
day 8: transfer all cells from the T75 flask to a new T175 flask and supplement medium D to a total volume of 100ml, and 0.5ml (1.1) of centrifuged heat-inactivated plasma;
day 11: 50ml of culture D was supplemented.
Example 4
This example is the culture of NK cells
This example differs from example 1 in that: (2.2) Day0 and (2.4) regular bottle transfer and fluid infusion
(2.2)Day0：
And adding IFN-gamma into the lymphocyte serum-free culture medium, and uniformly mixing to obtain a culture solution E, wherein the final concentration of the IFN-gamma in the culture solution E is 5000 IU/ml.
Resuspending (1.4) the step of peripheral blood mononuclear cells (1X 10 inoculation amount of peripheral blood mononuclear cells) with 5ml of culture medium E7Pieces/ml), the cell suspension was transferred to a T25 flask and placed at 37 ℃ in 5% CO2Culturing for 24 hours in an incubator;
(2.4) periodically rotating bottles and replenishing liquid:
and adding IL-2 into the lymphocyte serum-free culture medium, and uniformly mixing to obtain a culture solution D, wherein the final concentration of the IL-2 in the culture solution D is 1000 IU/ml.
Day 2: 5ml of culture solution D was supplemented;
day 4: transfer all cells from the T25 flask to a new T75 flask and supplement medium D to a total volume of 40ml, and 1ml (1.1) of centrifuged heat-inactivated plasma;
day 8: transfer all cells from the T75 flask to a new T175 flask and supplement medium D to a total volume of 100ml, and 0.5ml (1.1) of centrifuged heat-inactivated plasma;
day 11: 50ml of culture D was supplemented.
Example 5
Transferring the cell suspension collected in the examples 1-4 to 3 50ml centrifuge tubes, centrifuging for 5min at 2000rmp, and removing the supernatant; the cells were washed 3 times by adding wash solution and combining the cell suspension into one tube. Sampling 0.1ml for detecting endotoxin, and sampling 0.5ml for next cell counting, activity rate detection, flow detection and tumor killing activity test (selecting A549 lung cancer cells as target cells).
The results of counting NK cells at day0 and day 14 are shown in Table 1, and the fold expansion of cells was higher in examples 1 and 2 than in examples 3 and 4, but the fold expansion was smaller in examples 1 and 2. NK cell flow results are shown in FIGS. 1-5 and Table 2, and examples 1, 2 and 3 are higher in CD3-CD56+ and CD3+ CD56+ than example 2.
The result of the NK cell killing activity detection is shown in Table 3, the effective target ratio of NK cells is 20: the cell killing rate in 1 time reaches 92.86 percent, which is obviously higher than that in examples 1-4.
In conclusion, the NK cell culture solution provided by the invention can increase the amplification quantity of NK cells and simultaneously remarkably improve the cytotoxic activity.
TABLE 1 fold expansion of cells
TABLE 2 NK cell flow assay results
| CD3-CD56+ | CD3+CD56+ | |
| DAY-0 | 5.44 | 6.58 |
| Example 1 | 10.82 | 19.92 |
| Example 2 | 9.33 | 13.21 |
| Example 3 | 21.22 | 12.08 |
| Example 4 | 10.25 | 16.68 |
TABLE 3 NK cell killing Activity assay
| Effective target ratio | 20：1 | 10：1 |
| Example 1 | 92.86％ | 64.47％ |
| Example 2 | 63.19％ | 60.62％ |
| Example 3 | 77.54％ | 71.67％ |
| Example 4 | 47.40％ | 61.17％ |
The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. An NK cell culture solution, comprising: culture solution A and culture solution B;
the culture solution A comprises: IFN-gamma, IL-15 and basal medium;
the culture solution B comprises IL-2, IL-1 α, a CD3 monoclonal antibody and a basal culture medium.
2. The NK cell culture solution according to claim 1, wherein the final concentration of IFN- γ in the culture solution A is 2500-5000 IU/ml;
the final concentration of the IL-15 in the culture solution A is 10-100 mu g/ml;
the final concentration of the IL-2 in the culture solution B is 500-1000 IU/ml;
the final concentration of the IL-1 α in the culture solution B is 2.5-5 ng/ml;
the final concentration of the CD3 monoclonal antibody in the culture solution B is 250-500 ng/ml.
3. The NK cell culture solution according to claim 1, further comprising: culture solution C or culture solution D;
the culture solution C comprises: IL-2, IL-15 and basal medium;
the culture solution D comprises: IL-2 and basal medium.
4. The NK cell culture solution according to claim 3, wherein the final concentration of the IL-2 in the culture solution C is 500-1000 IU/ml;
the final concentration of the IL-15 in the culture solution C is 10-100 mu g/ml;
the final concentration of the IL-2 in the culture solution D is 500-1000 IU/ml.
5. The NK cell culture solution according to any one of claims 1 to 4, further comprising: autologous plasma;
the basic culture medium is a lymphocyte serum-free culture medium.
6. The NK cell culture solution according to any one of claims 1 to 4, further comprising: PD-1.
7. An NK cell culture kit comprising the culture solution A, the culture solution B and PD-1 according to claim 1, and a culture solution C or a culture solution D;
the culture solution B comprises IL-2, IL-1 α, CD3 monoclonal antibody and a basal culture medium;
the culture solution C comprises: IL-2, IL-15 and basal medium;
the culture solution D comprises: IL-2 and basal medium.
8. A method for culturing NK cells, comprising the steps of:
step 1: on day0, the peripheral blood single cells were inoculated into the culture solution a for culture;
step 2: culturing to day1, adding culture solution B, and continuing culturing;
and step 3: culturing for 2, 4, 8 and 11 days, respectively adding culture solution C or D, and continuing culturing;
and 4, step 4: adding PD-1 to continue culturing until day 13 or day 14, and culturing until day15 to obtain NK cells;
the culture solution B comprises IL-2, IL-1 α, CD3 monoclonal antibody and a basal culture medium;
the culture solution C comprises: IL-2, IL-15 and basal medium;
the culture solution D comprises: IL-2 and basal medium.
9. The culture method according to claim 8, wherein the amount of the single cells in peripheral blood inoculated is 2X 106～1×107One per ml.
10. The culture method according to claim 8, wherein the final concentration of PD-1 is 0.5 to 1.25. mu.g/ml.
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