CN105820247A

CN105820247A - Method for screening PD-1 antibody

Info

Publication number: CN105820247A
Application number: CN201410853531.5A
Authority: CN
Inventors: 赵杰; 张成海; 朱玲巧
Original assignee: Three Country Kin Pharmaceutical (shanghai) Ltd By Share Ltd
Current assignee: Three Country Kin Pharmaceutical (shanghai) Ltd By Share Ltd; Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date: 2014-12-31
Filing date: 2014-12-31
Publication date: 2016-08-03

Abstract

The invention relates to a method for screening PD-1 antibody. The method comprises the following steps: respectively constructing full-length genes of human PD-1 and PD-L1 into a lentiviral expression vector; respectively transfecting to T and B lymphoblast cell lines; screening stably expressed lymphocyte; adding PD-1 monoclonal antibody and T cell superantigen SEE to carry out co-incubation for 48 h; and finally screening required PD-1 monoclonal antibody according to content of IL-2 in a supernatant. In comparison with an experiment of SEB stimulation of whole blood for IL-2 secretion, the method of the invention has more excellent repeatability and accuracy, experimental cost is remarkably reduced, and various potential risks brought by operation of human blood are decreased.

Description

A kind of method screening PD-1 antibody

Technical field

A kind of method that the present invention relates to screening antibodies, is specifically related to a kind of method screening PD-1 antibody.

Background technology

The activation startup of T cell and effect at least need two kinds of signals to exist during playing, the first letter Number be antigenic specificity, by major histocompatibility complex (Major histocompatibility complex, And antigenic peptides in connection is identified by φt cell receptor (T cell receptor, TCR) and ties MHC) After conjunction, TCR complex in T cell, conduct activation signal；The second activation signal is antigen non-specific Property, it is by multiple auxiliary in antigen presenting cell (antigen presenting cell, APC) and T cell Pairing and interaction between molecule and provide, using the teaching of the invention it is possible to provide a class T cell of the second activation signal Memebrane protein is otherwise known as costimulatory molecules, and wherein CD28 is that a kind of expression stimulates altogether the important of T cell surface Molecule, plays a significant role in T cell activation.After TCR with MHC-antigenic peptide complexes is combined, If costimulatory molecules lacks, by causing T cell to be incompetent state to special antigen expression, not only can not It is activated, and activation signals specific to antigen is not replied；If costimulatory molecules such as CD28 with After B7 molecule combines and activates, can the activation of mediate T cell in multiple levels.The T cell of activation is permissible The form of autocrine and paracrine produces interleukin II (interleukin 2；IL-2), cell table is acted on The receptor IL2R in face, stimulates the propagation of T cell further, thus ready for playing effector function.

The immunoglobulin superfamily I type transmembrane glycopeptide that PD-1 (CD279) is made up of 288 aminoacid In vain, it is initially to clone from the mouse Tcell hybridoma of apoptosis, is considered relevant to apoptosis and names For programmed death-1 (programmed death-1, PD-1).PD-1 is in the B cell activated, T cell Inhibition member (Okazaki etc. (2002) Curr Opin with the CD28 family expressed on myeloid cell Immunol 14:391779-82；Bennett etc. (2003) J Immunol 170:711-8), this family also includes CD28, CTLA-4, ICOS and BTLA etc..PD-1 is at B cell, T cell and the myeloid cell of activation Upper expression (Okazaki etc. (2002) Curr.Opin.Tmmuno1.14:391779-82；Bennett etc. (2003) J Immunol 170:711-8).PD-1 contain close to film immunity receptor Tyrosine Inhibitory Motifs (ITIM) and Conversion motif (ITSM) based on tyrosine (Thomas, M.L. (1995) J Exp Medl away from film 81:1953-6；Vivier, E and Daeron, M (1997) Immunol Todayl8:286-91).Although with CTLA-4 structure is similar to, but PD-1 albumen for want of mediates CD28/CTLA-4 with B7-1/B7-2 and combines MYPPPY sequence, and the mediation FDPPPF sequence that combines of ICOS with ICOS-L, thus tying Notable difference is there is with CD28, CTLA-4 and ICOS on structure.Therefore, the combination of PD-1 and part is very Special, the B7 family molecule with other does not produces cross coupled.

Identify two kinds of parts of PD-1, be PD-L1 (B7-H1) and PD-L2 (B7-DC) respectively (Latchman etc. (2001) Nat Immuno 12:261-8；Carter etc. (2002) Eur J Immuno132:634-43).PD-L1 belongs to B7 family, has IgV and IgC sample district, cross-film district and born of the same parents Slurry district afterbody, PD-L1 interacts with the receptor PD-1 in its T cell, and the negativity in immunne response regulates and controls Aspect plays an important role；This molecule has the distribution expression pattern of broadness, and wide expression is in antigen presenting cell (APCs), activation T/B cell, macrophage, placental trophoblast, cardiac muscle endothelium and thymic cortex epithelium are thin Born of the same parents.PD-L1 all can detect that the expression of PD-L1 albumen, and many cancer groups in many mankind tumor tissues Knit the PD-L1 expression in compared with normal tissue and substantially raise (Dong et al. (2002) Nat.Med 8:787-9). Application immunohistochemical method, successively breast carcinoma, pulmonary carcinoma, gastric cancer, intestinal cancer, the esophageal carcinoma, ovarian cancer, The mankind tumor tissues such as cervical cancer, renal carcinoma, bladder cancer, cancer of pancreas, glioma, melanoma detect The expression of PD-L1 albumen, and the expression of PD-L1 and the clinic of patient and prognosis be closely related.Many Research all shows that PD-L1 is relevant to the Immune escaping mechanism of tumor.Research shows, tumor cell and tumor The PD-L1 that APC in microenvironment expresses all can be special through PD-1/PD-L1 signal path suppression tumor antigen Property T cell activation, lower T cell mediation tumor immune response.Experimental results demonstrate, block PD-1/PD-L1 signal can promote the increment of specific for tumour antigen T cell, plays killing tumor cell Effect, effectively suppresses tumor growth.Therefore, intervene PD-1/PD-L1 signal to be expected to become immunotherapy of tumors New Policy.

At present, the model that PD-1 monoclonal antibody carries out in-vitro evaluation generally requires the people using fresh anticoagulant Blood or the leukocyte separated from human blood, human blood source is relatively difficult, and the most relatively costly but also existence is dived Biohazardous.Therefore, set up a kind of reliable and stable, can Long Term Passages is cultivated in vitro cell The problem that the function of PD-1 antibody, always those skilled in the art are anxious to be resolved evaluated by model.

Summary of the invention

It is an object of the invention to, it is provided that a kind of method utilizing passage cell screening PD-1 monoclonal antibody, The method can effectively filter out specific binding PD-1 and promote lymphocyte to divide the monoclonal anti that must go out IL-2 Body, and the inventive method is without using blood.

For achieving the above object, the present invention is by the following technical solutions:

A kind of method screening PD-1 monoclonal antibody, comprises the following steps:

1, people PD-1 and PD-L1 full-length gene are building up in Lentiviral；

2, the PD-1 expression vector built is stably transfected into T lymph by the method for slow-virus transfection thin Born of the same parents' strain；The PD-L1 expression vector built is stably transfected into B lymph by the method for slow-virus transfection thin Born of the same parents' strain；

3, filter out the T lymphocyte of stable expression PD-1 with antibiotic G418 and stably express PD-L1 Bone-marrow-derived lymphocyte；

4, the T lymphocyte stably expressing PD-1 and the bone-marrow-derived lymphocyte stably expressing PD-L1 are according to properly Ratio mixing after, add the PD-1 monoclonal antibody of gradient dilution and T cell superantigen SEE, then altogether With hatching 48 hours；

5, with the IL-2 content in ELISA method detection cells and supernatant, sieve according to the height of IL-2 content PD-1 monoclonal antibody needed for choosing.

It is also preferred that the left described carrier is pLV-IRES-Neo carrier.

It is also preferred that the left in step 4, described T lymphocyte and bone-marrow-derived lymphocyte are mixed according to the number of cells ratio of 1:1 Close.

It is also preferred that the left described T lymphocyte strain is selected from the condition of culture in vitro deriving from mice, rat or people The T lymphocyte strain that can stably pass on down；Described bone-marrow-derived lymphocyte select good strains in the field for seed from derive from mice, rat or People cultivates the bone-marrow-derived lymphocyte strain that can stably pass in vitro.Preferably described T lymphocyte strain behaviour T Lymphocyte strain Jurkat；Described bone-marrow-derived lymphocyte strain is human B lymphocyte strain Raji.

Experiment shows, PD-1 monoclonal antibody Nivolumab can be obviously enhanced Raji cell and SEE stimulates The IL-2 of Jurkat cell secretes, and this effect has dose dependent；It addition, this method and SEB The experiment of stimulation of whole secretion IL-2 is compared, more superior in terms of repeatability and degree of accuracy, and significantly drops Low experimental cost, decreases the various potential risks that operator's blood brings, has reached the purpose of the present invention.

Accompanying drawing explanation

Fig. 1 is the relative expression of Jurkat cell surface PD-1 after transfection；

Fig. 2 is the relative expression of Raji cell surface PD-L1 after transfection；

Fig. 3 is the SEB stimulation of whole experiment of anti-PD-1 antibody；

Fig. 4 is the cell mixing reaction that SEE stimulates Jurkat-PD-1 and Raji-PD-L1.

Detailed description of the invention

Following example, experimental example are to further illustrate the present invention, should not be construed as limitation of the present invention. Embodiment does not include the detailed description to conventional method, as those are for carrier construction and the method for plasmid, will compile The gene of code albumen is inserted into such carrier and the method for plasmid or the method that plasmid introduces host cell.This The method of sample is it is well known that to person having ordinary skill in the art and in many publications all It is described, such as: Sambrook, J., Fritsch, E.F.and Maniais, T. (1989) Molecular Cloning:A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

The structure of embodiment 1. people source PD-1, PD-L1 and PD-L2 expression vector and the expression of antibody

The full-length gene of people source PD-1 and PD-L1 is bought from Sino Biological Inc., its Sequence with in Gene Bank announce sequence identical (ID be respectively as follows: NM_005018.2 and NM_014143.2).PCR all uses high-fidelity DNA polymerase (such as: Takara companyHS DNA Polymerase).Amplification PD-1 and PD-L1 full-length gene reaction condition according to Archaeal dna polymerase manufacturer's description is rationally arranged: 95 DEG C 20 seconds；55 DEG C 10 seconds, 72 DEG C of 1 point/kb；30 Individual circulation.Amplification PD-1 and PD-L1 full-length gene the primer sequence is as follows: PD-1 forward primer: TATGAATTCGCCACCATGCAGATCC CACAGGCGCCCTG, PD-1 reverse primer: ATAGCGGCCGCTCAGAGGGGCCAAGAGCAGTGTCC；PD-L1 forward primer: TATGAATTCGCCACCATGAGGATATTTGCTGTCTTTAT, PD-L1 reverse primer: ATAGCGGCCGCTTACGTCTCCTCCAAATGTGTAT.PCR primer is through agarose gel electricity Swimming purification reclaims and is cloned in pLV-IRES-Neo carrier (purchased from Beijing English luxuriant industry limited public affairs of biotechnology Department), confirm after sequence verification to obtain correct clone.Correct clone in this example is denoted as respectively PLV-IRES-Neo-PD-1 and pLV-IRES-Neo-PD-L1.

PD-1 monoclonal antibody Nivolumab (Bristol-Myers Squibb company as positive control Product) and the aminoacid sequence announced according to WHO of the sequence of Pembrolizumab (http://www.who.int/medicines/publications/druginformation/en/) is raw by Suzhou gold only intelligence The full genome synthesis of thing Technology Service Co., Ltd.The coded sequence of light chain of antibody and heavy chain is building up to pCHO1.0 In carrier, expression vector transfected CHO-S cells (Life technologies Products) and recombiant protein The description that provides according to cell supplier of expression and purification method implement.

The packaging of embodiment 2. slow virus carrier and transfected target cells

Packaging plasmid pH1, pH2 of slow virus is purchased from Inovogen Tech Co., Ltd., its coding Slow-virus transfection and the necessary albumen of integration, HEK293 cell is as the host cell of packaging virus granule. With Lipofectamine2000 (purchased from Life Technologies) by pLV-IRES-Neo-PD-1 and PLV-IRES-Neo-PD-L1 respectively with the mixture (quality of pH1 Yu pH2 of packaging plasmid pH1 and pH2 Ratio is 1:1) it is transfected in HEK293 cell, expression vector, the mass ratio of packaging plasmid are 1:1.Turn Dye concretely comprises the following steps: transfects first 24 hours and is inoculated in 6 orifice plates by HEK293 cell, every hole 2ml cell Suspension totally 10⁶Individual cell；By Lentiviral that 1.5 μ l concentration are 1 μ g/ μ l and 1.5 μ l during transfection Concentration is that the slow virus packaging plasmid of 1 μ g/ μ l is diluted to 150 μ lOptiPRO^TMSFM is (purchased from Life Technologies), in, mix gently；10 μ l transfection reagent Lipofectamine2000 are diluted to 150μlOptiPRO^TMIn SFM, mix gently；Immediately by slow for the Lipofectamine2000 solution after dilution Slowly join in the DNA solution after dilution, add fashionable rifle head and be dipped under liquid level and slowly drain；Vertical I.e. turning upside down and make solution mix for several times, incubated at room temperature about 5 minutes is so that DNA-liposome complex shape Become；300 μ l DNA-Lipofectamine2000 complexes drop-wise are joined 6 orifice plates of above-mentioned cultivation cell In, limit edged jog culture plate；Cell culture after transfection is placed in 37 DEG C, 5%CO₂Saturated humidity Cell culture incubator is cultivated 48 hours.

Target cell transfection process is as follows: after 48 hours, with 0.45 μm Millex-HV filter, (Millipore produces Product) filter the HEK293 cell culture supernatant after transfecting, the supernatant filtered is added separately to Jurkat With in Raji cell culture, the cell density of Jurkat and Raji is 5 × 10⁵Individual/ml；It is subsequently adding and turns Dye reinforcing agent Polybrene (Sigma Products) so that it is final concentration of 5 μ g/ml；Continue to be placed in 37 DEG C, 5%CO₂Saturated humidity cell culture incubator cultivate 48 hours；Metainfective Jurkat and Raji of centrifugal collection Cell, respectively with the RPMI1640 complete culture solution containing 1mg/mlG418 (the raw chemical product in Shanghai) (RPMI1640 minimal medium+10% hyclone；Both of which be purchased from Life Technologies) resuspended carefully Born of the same parents, continue to cultivate 7 days；After 7 days, will be killed by G418 for proceeding to the cell of exogenous gene, and turn The cell of dye exogenous gene because simultaneously expression alien gene and neomycin resistance gene (its product can be by G418 inactivates) and survive and constantly breed.The Jurkat cell of stable transfection PD-1 and turning the most at last The Raji cell having contaminated PD-L1 is respectively designated as Jurkat-PD1 and Raji-PD-L1.

The expression of embodiment 3. flow cytometry detection cell surface destination protein

Jurkat-PD1 cell is expressed the situation flow cytometer of PD-1 and is determined, only transfects the Jurkat of empty carrier As compared with control cells, specifically comprise the following steps that the various cells of centrifugal collection, with containing 5% (mass fraction) cattle Sero-abluminous phosphate buffer (hereinafter referred to as PBSB:135mM NaCl, 2.7mM KCl, 1.5mM KH₂PO₄, 8mM K₂HPO₄, 5% bovine serum albumin；PH 7.2) cell density is adjusted to 5 × 10⁵ Individual/ml；Being divided into two parts by cell suspension, every part of 1ml, a copy of it adds final concentration of 1 μ g/ml's Nivolumab (PD-1 monoclonal antibody, prepared by this laboratory internal), another part is not added with；Incubated at room 1 After hour, centrifugal collecting cell abandons supernatant, is washed three times by cell with PBS, and last every part of sample is used 1mlPBSB is resuspended；Adding the goat anti-human igg two of FITC labelling, anti-(Fc is special；Sigma Products), Incubated at room 30 minutes；Centrifugal collecting cell abandons supernatant, is washed three times by cell with PBS, finally uses PBS Re-suspended cell；Flow cytomery fluorescence intensity.Experimental result is as it is shown in figure 1, the PD-1 of Jurkat-PD1 Expression is apparently higher than the Jurkat cell only transfecting empty carrier.

Raji-PD-L1 cell is expressed the situation flow cytometer of PD-L1 and is determined, only transfects the Raji of empty carrier Cell, respectively as compared with control cells, specifically comprises the following steps that the various cells of centrifugal collection, with containing 5% Ox blood serum Cell density is adjusted to 5 × 10 by albuminous PBS⁵Individual/ml；Just cell suspension is divided into two parts, every part 1ml, a copy of it adds PD-1-hFc (PD-1 extracellular fragment and the Fc of human IgG of final concentration of 3 μ g/ml The fusion protein of section, R&D Products), another part is not added with；After incubated at room 1 hour, centrifugal collection Cell abandons supernatant, is washed three times by cell with PBS, and last every part of sample 1mlPBSB is resuspended；Add FITC The goat anti-human igg two of labelling is anti-, and (Fc is special；Sigma Products), incubated at room 30 minutes；Centrifugal Collect cell and abandon supernatant, with PBS, cell is washed three times, finally use PBS re-suspended cell；Flow cytometer Fluorescence intensity.Experimental result is as in figure 2 it is shown, the PD-L1 expression of Raji-PD-L1 is apparently higher than only The Raji cell of transfection empty carrier.

Embodiment 4. staphylococcal enterotoxin B stimulation of whole is tested

Staphylococcal enterotoxin B (Staphylococcal Enterotoxin B, SEB) is that a kind of T cell is super anti- Former, directly can be combined with the TCR-V district of MHC-II quasi-molecule and some hypotype, T cell sum can be stimulated 5～20%, there is strong activation T cell (CD4⁺T cell) effect.The purpose of this embodiment It is intended to detect PD-1 monoclonal antibody and can further enhance the T cell of SEB activation to interleukin The secretion of (interleukin 2, IL-2).It is embodied as step as follows: by serum-free RPMI1640 culture medium The fresh anticoagulation picking up from healthy volunteer is diluted by 1:5, then the blood diluted is inoculated into 96 holes Round bottom culture plate (Coring Products), every hole 100 μ l；50 μ l gradient concentrations are added in respective aperture PD-1 monoclonal antibody Nivolumab or Pembrolizumab, be placed in cell culture incubator and hatch 30-60 and divide Clock；Every hole adds 50 μ l SEB so that it is final concentration of 100ng/ml；It is placed in cell culture incubator continuation training Support 2-4 days；Take cell culture supernatant enzyme linked immunosorbent assay (enzyme-linked immunosorbent Assay, ELISA) measure the content of IL-2 in supernatant.

ELISA method measure IL-2 content in supernatant to be embodied as step as follows: the IL-2 capture of rat anti-human Antibody (BD Biosciences Products) is diluted to 2 μ g/ml with 0.05M carbonate buffer solution (pH 9.6), The antibody-solutions after the 100 above-mentioned dilutions of μ l is added in the every hole of 96 hole ELISA Plate (Corning Products), is placed in 37 DEG C of calorstats hatch 2 hours；By the lavation buffer solution (phosphate-buffered containing 0.05%Tween-20 Liquid) wash 3 times, each 3 minutes；It is subsequently adding confining liquid (the 0.05M carbonate containing 5% defatted milk powder Buffer；PH 9.6), in 4 DEG C of fridge overnight；Next day, discard solution in hole, wash 3 with lavation buffer solution Secondary, each 3 minutes；100 μ l supernatants of dilution 4 times are added in the most coated above-mentioned reacting hole, put 37 DEG C Hatch 1 hour；After washing with lavation buffer solution, add the IL-2 that the biotinylated rat of concentration 1 μ g/ml is anti-human Detection antibody (BD Biosciences Products) in each reacting hole, every hole 100 μ l, hatch 1 for 37 DEG C Hour；After lavation buffer solution washing, add the Streptavidin of the horseradish peroxidase-labeled of concentration 1 μ g/ml (Streptavidin-HRP, BD Biosciences Products) in each reacting hole, every hole 100 μ l, 37 DEG C Hatch 1 hour；After washing with lavation buffer solution, the tmb substrate adding Extemporaneous in each reacting hole is molten Liquid 100 μ l develops the color, and places 10～30 minutes for 37 DEG C；In each reacting hole, add 2M sulphuric acid 50 μ l terminate anti- Should；Put SpectraMax i3 microplate reader (U.S.'s Molecular Devices Products) and measure 450nm ripple Strong point light absorption value.

Experimental result (show the meansigma methods independently repeating experiment three times) as shown in Figure 3, two positive controls Antibody Nivolumab and Pembrolizumab test at this in all can the secretion of effective stimulus IL-2, it SEB stimulation of whole test in EC₅₀It is respectively 52.25 and 55.13 (units ng/ml), their 95% Confidence interval is respectively 27.41-99.61 and 25.87-117.5, additionally their R²(quality of reflection degree of fitting, Numerical value is the best closer to 1 explanation degree of fitting) it is respectively 0.9374 and 0.9020.

Embodiment 5. aureus enterotoxin E stimulates the experiment of Jurkat-PD-1 and Raji-PD-L1 cell mixing

Aureus enterotoxin E (Staphylococcal Enterotoxin E, SEE) is also that a kind of T cell surpasses Antigen, directly can be combined with the TCR-V district of MHC-II quasi-molecule and some hypotype, have strong activation T Cell (CD4⁺T cell) effect.Jurkat cell derives from the T _ Lymphoid Leukemic Cells of people, and Raji Deriving from the B _ Lymphoid Leukemic Cells of people, they remain the character of T and bone-marrow-derived lymphocyte the most respectively, I.e. Jurkat expresses TCR and Raji expresses MHC-II quasi-molecule, and SEE can be in combination with on two kinds of cells TCR and MHC-II quasi-molecule, thus activate Jurkat cell and make it secrete IL-2.The mesh of this embodiment Be intended to detect PD-1 monoclonal antibody can further enhance SEE and Raji activate Jurkat cell dialogue The secretion of interleukin (interleukin 2, IL-2).It is embodied as step as follows:

Centrifugal collection Jurkat-PD-1 and Raji-PD-L1 cell respectively, with complete containing 800 μ g/ml The density of two kinds of cells is all adjusted to 2 × 10 by RPMI1640 culture medium⁵Individual/ml, then by two kinds of cells etc. Volume mixture, adds SEE and makes its final concentration of 100ng/ml, be inoculated into 96 holes after mixing in mixed culture In round bottom culture plate (Coring Products), every hole 150 μ l；50 μ l gradient concentrations are added in respective aperture PD-1 monoclonal antibody Nivolumab or Pembrolizumab, be placed in cell culture incubator continuation cultivation 2 My god；Take cell culture supernatant ELISA method and measure the content of IL-2 in supernatant.ELISA method detection IL-2 The method of concentration is identical with described in embodiment 4.

Experimental result (show the meansigma methods independently repeating experiment three times) as shown in Figure 4, and two positive controls resist Jurkat-PD-1 and the Raji-PD-L1 cell mixing that body Nivolumab and Pembrolizumab stimulates at SEE is anti- Ying Zhongjun can the secretion of effective stimulus IL-2, their EC₅₀It is respectively 47.94 and 44.22 (units ng/ml), Their 95% confidence interval is respectively 39.55-58.11 and 35.11-55.70, additionally their R²It is respectively 0.9957 and 0.9929.It addition, our experimental result also shows, only transfect empty carrier Jurkat and The cell mixing reaction of Raji-PD-L1, and the mixing of Jurkat-PD-1 and the Raji that only transfects empty carrier is thin Born of the same parents react and all can not secrete by the further of effective stimulus IL-2, and this explanation transfects in two kinds of cells the most simultaneously PD-1 and PD-L1 is the essential condition setting up this model, and accordingly result is not illustrated.

The result of Fig. 3 and 4 illustrates that the cell mixing reaction model that we set up is wanted in terms of collimation and degree of accuracy It is better than the Whole blood experiments that SEB stimulates.

Claims

1. the method screening PD-1 monoclonal antibody, comprises the following steps:

1), people PD-1 and PD-L1 full-length gene are building up in Lentiviral respectively；

2), the PD-1 expression vector built is stably transfected into T lymphocyte strain by the method for slow-virus transfection； The PD-L1 expression vector built is stably transfected into bone-marrow-derived lymphocyte strain by the method for slow-virus transfection；

3), filter out the T lymphocyte of stable expression PD-1 with antibiotic G418 and stably express the B of PD-L1 Lymphocyte；

4), the T lymphocyte of the stable expression PD-1 after screening and stably express the bone-marrow-derived lymphocyte of PD-L1 and press After mixing according to suitable ratio, add PD-1 monoclonal antibody and the T cell superantigen SEE of gradient dilution, The most jointly hatch 48 hours；

5) the IL-2 content, in detection cells and supernatant, the PD-1 needed for screening according to the height of IL-2 content Monoclonal antibody.

Method the most according to claim 1, wherein, described carrier is pLV-IRES-Neo carrier.

Method the most according to claim 1 and 2, wherein, T lymphocyte described in step 4 and B lymph Cell is according to the number of cells ratio mixing of 1:1.

Method the most according to claim 1 and 2, wherein, described T lymphocyte strain behaviour T lymph is thin Born of the same parents strain Jurkat.

Method the most according to claim 1 and 2, wherein, described bone-marrow-derived lymphocyte strain behaviour B lymph is thin Born of the same parents strain Raji.