CN101104640A - Preparation for anti human PD-L1 monoclonal antibody and application thereof - Google Patents
Preparation for anti human PD-L1 monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention relates to anti-human PD-L1 monoclonal antibodies 2H11 and 10E10, a preparation method of the monoclonal antibodies and the applications of the monoclonal antibodies in inhibiting apoptosis of a specific CTL which is related to tumor and is induced by PD-L1, and in detecting PD-L1 molecules in gastric cancer tissues which is used as a prognostic judgment biological index.
Description
FIELD OF THE INVENTION
The present invention relates to anti human PD-L 1 monoclonal antibody 2H11 and 10E10, its preparation method and these monoclonal antibodies be at the apoptosis that suppresses the relevant PD-L1 inductive specific CTL of tumour, and at PD-L1 molecular level that production is used for detecting stomach organization with the application in the test kit of the judgement of carrying out the cancer of the stomach prognosis.
The background of invention
Known have many receptor-ligand binding all to participate in inducing, setting up and regulate antigen specific immune reaction.For activated T cell reaction effectively, need two signals usually at least.Wherein the MHC-antigenic compound on T cell antigen receptor (TCR) identification antigen presenting cell (APC) is the antigen-specific signal so that first signal to be provided, also necessary non-antigen-specific, the restrictive second signal of non-MHC that obtains the costimulatory molecules interaction back generation of T cell and APC expression.Second signal is costimulatory signal or costimulatory signal.Lack costimulatory signal if the antigen-specific signal is only arranged, the T cell will show as reactionless or immune tolerance state, even cause apoptosis.As seen, costimulatory signal is that the amplification of T cell clone, differentiation and performance biological effect institute are requisite.Therefore can think, first signal deciding specificity of T cell activation, can second signal then determine the T cell-mediated immune responses effectively carry out (Noelle RJ, et al., Proc.Natl.Acad.Sci.USA.89:6550,1992; Allen RC et al., Science.259:990,1993).
In recent years, Protocols in Molecular Biology is widely used in immunology research, and costimulatory molecules is constantly found.According to its structure, these costimulatory moleculeses can be divided into two classes: a class is tumour necrosis factor/Tumor Necrosis Factor Receptors (TNF/TNFR) superfamily, comprises CD40/CD154, CD27/CD27L, CD30/CD30L, 4-1BB/4-1BBL, OX40/PD-L1, RANK/RANKL and Fas/FasL etc.Another kind of is immunoglobulin superfamily, as (Korthauer U et al., Nature, 361:539,1993) such as B7-CD28/CTLA4, LAF1-ICAM-1/ICAM-2/ICAM-3, ICOS-GL50, CD2/LFA-3.These costimulatory moleculeses are with the mode conducted signal of acceptor and ligand interaction.Acceptor generally is expressed in different cell surfaces with part, and common one is the persistence expression, and one is the inducible expression.In the different steps of immunne response, these molecules participate in the signal conduction in mode unique and that be associated separately, regulate and control the immunne response of body jointly.
The I type transmembrane glycoprotein that human PD-L 1 is clone's in 1999 the about 30-35KD of molecular weight, be made up of 290 amino acid is one of member of tumour necrosis factor (TNF) superfamily.The human PD-L 1 molecule mainly is expressed on static and activated T cell, B cell, scavenger cell and the dendritic cell (DCs), and non-Lymphoid tissue cell surfaces such as the heart, placenta, blood vessel epithelium, liver, lung, kidney and skeletal muscle.In addition, PD-L1 all has expression on cancerous tissue cells such as lung cancer, liver cancer, mammary cancer, squamous cell carcinoma and ovarian cancer, and many cancerous tissues also can be made the up-regulated of PD-L1 after inducing.But in many cases, healthy tissues must just be expressed PD-L1 after inducing.
In the propagation reactivation process of T cell, when TCR and CD3 are in the optimal activation state, start the phosphorylation of PD-1 cytoplasmic domain ITIM tyrosine residues by PD-1-PD-L1 approach (as subsidiary signal), impel combining of SHP-2 Phosphoric acid esterase and SHP-2, cause the activation of SHP-2, thereby the propagation of suppressor T cell (particularly some previous activated T cells), and the effect of antagonism CD28-B7-2 approach under certain condition.Research shows further that also the PD-1-PD-L1 approach is to CD8
+T cell inhibiting effect comparison CD4
+T cell more obvious, and this effect is the IL-2 dose-dependently.The PD-1-PD-L1 approach also influences the secretion of a lot of Th1 and Th2 cytokines when suppressing the T cells with antigenic specificity activation and proliferation, for example this approach can be reduced IL-2 and IFN-γ secretion.
Studies show that in recent years, the PD-1-PD-L1 signal path can play a significant role in the process of mediation anti-tumor immune response and autoimmune disorder.But many antigenic non-Lymphoid tissues of carrying autoantibody identification are high expression level PD-L (especially time PD-L1) all, the up-regulated that PD-L again can the surperficial PD-1 of inductive effect cell (mainly being T, B cell) simultaneously, thereby at the negativity regulating and controlling effect of the inflammation part performance PD-1/PD-L of various autoimmune disease approach.Behind mouse PD-1 gene knockout, cause the disappearance of non-Lymphoid tissues such as heart, kidney and the intercellular PD-1/PD-L approach of T, B, make autoreactivity CD8
+The T cell is easier to be activated and to move to target tissues such as the heart and kidney, thereby causes autoantibody to produce and local tissue damage.Therefore, the PD-1/PD-L approach is as a kind of negativity control methods, plays a significant role in the inducing of peripheral tolerance and autoimmune disease.PD-L1 is high expression level on the cancer in many endotheliums source and tumour cell, and the PD-1/PD-L1 approach is to CD8
+The activation and proliferation of T cell has the obvious suppression effect again, therefore points out many cancers and tumor tissues to escape CD8 by this approach
+The lethal effect of T cell (CTL) weakens antitumor immunity of organism and replys.Nearest animal vivo test finds, the PD-L1 that tumour is relevant be expressed as tumour immunity identification and immune clearance provides the intrinsic approach, block the efficient that this path can improve the CTL killing tumor cell effectively.Therefore, anti-PD-L1 monoclonal antibody might become the ideal diagnosis and the therapeutical agent of clinical intervention autoimmune disorder, graft-rejection and tumour.
Although relevant PD-L1 receptor-ligand all is that part is known in conjunction with preparation and their other immunologic functions of right T cytositimulation function, PD-L1 antibody, do not appear in the newspapers as new anti human PD-L 1 monoclonal antibody of the present invention and relatively simple preparation method thereof.And anti human PD-L 1 monoclonal antibody of the present invention has some different biological function.
Goal of the invention
An object of the present invention is to provide anti human PD-L 1 monoclonal antibody 2H11 and 10E10.
According to the preferred embodiments of the invention, respectively the hybridoma cell line excretory of CGMCC No.1637 and CGMCC No.1636 when said anti human PD-L 1 monoclonal antibody 2H11 and 10E10.
Another object of the present invention provides the method for preparing the anti human PD-L 1 monoclonal antibody that is defined as above, and this method comprises:
(1) the transgenosis cell strain L929/PD-L1 of the high expression level human PD-L 1 molecule of usefulness pre-preparation or human breast cancer cell strain MDA-MB-231 are as the immunogen immune animal;
(2) separate the splenic lymphocyte of immunized animal and merge with the myeloma cell being suitable for producing under the condition of hybridoma;
(3) screen and cultivate the hybridoma that as above obtains;
(4) from the ascites fluid of animal of cell culture fluid or inoculation hybridoma, separate and the required monoclonal antibody of purifying.
According to the preferred embodiments of the invention, the deposit number that wherein produces the hybridoma cell strain of anti human PD-L 1 monoclonal antibody 2H11 and 10E10 is respectively CGMCC No.1637 and CGMCCNo.1636.
A further object of the present invention provides the hybridoma cell line that deposit number is respectively CGMCC No.1636 and CGMCCNo.1637.
A further object of the present invention provides anti human PD-L 1 monoclonal antibody 2H11 and the application of 10E10 in external evoked cytotoxic T lymphocyte activation and proliferation that is defined as above.
A further object of the present invention provides anti human PD-L 1 monoclonal antibody 2H11 and the application of 10E10 in the efficient that strengthens the specific CTL killing tumor cell that is defined as above.
A further object of the present invention provides the anti human PD-L 1 monoclonal antibody 2H11 that is defined as above and 10E10 as the Biological Detection index, the application in cancer of the stomach prognosis judgement and individualized treatment.
Brief Description Of Drawings
Fig. 1 shows the structure synoptic diagram of L929/PD-L1 transgenic cell.
Fig. 2 shows the identification to PD-L1 molecule on the transgenic cell with flow cytometry anti human PD-L 1 monoclonal antibody 2H11 and 10E10.The negative contrast in wherein transparent peak, one anti-is mouse IgG, two anti-ly are the sheep anti-mouse igg of fluorescein PE mark.The black peak shows the result of transgenic cell and the reaction of anti human PD-L 1 monoclonal antibody, wherein first antibody is respectively commercialization anti human PD-L 1 monoclonal antibody MIHI (positive control) and anti human PD-L 1 monoclonal antibody 2H11 of the present invention and 10E10, and second antibody is the sheep anti-mouse igg of fluorescein PE mark.
Fig. 3 shows 2H11 and the chromosomal karyotyping of 10E10 hybridoma cell strain (* 1000 times).
Fig. 4 shows with the identification of flow cytometry 10E10 to the PD-L1 molecule on activated T, B cell and the sophisticated DC.
A: show the identification of monoclonal antibody of the present invention to PD-L1 molecule on the activating T cell.The negative contrast in transparent peak: cell at first reacts with mouse IgG, adds the sheep anti-mouse igg of two anti-fluorescein FITC marks then, and the mouse-anti people CD25 that adds fluorescein PE mark at last directly marks monoclonal antibody; The black peak is the sheep anti-mouse igg effect that adds fluorescein FITC mark after the B cell reacts with 2 strain anti human PD-L 1 monoclonal antibody 2H11 and 10E10 respectively again.
B: show the identification of monoclonal antibody of the present invention to LPS activatory B cell.The negative contrast in wherein transparent peak: cell at first reacts with mouse IgG, adds the sheep anti-mouse igg of two anti-fluorescein FITC marks then, and the mouse-anti people CD69 that adds fluorescein PE mark at last directly marks monoclonal antibody; The black peak is the sheep anti-mouse igg effect that adds fluorescein FITC mark after the B cell reacts with 2 strain anti human PD-L 1 monoclonal antibody 2H11 and 10E10 respectively again.
C: show the identification of monoclonal antibody of the present invention to mature dendritic cell (DC).The negative contrast in wherein transparent peak: cell at first with mouse IgG reaction, two anti-ly are the streptavidin (Streptavidin-PE) of PE mark; The black peak is that DC adds the Streptavidin-PE effect respectively with after biotin labeled two strain anti human PD-L 1 monoclonal antibody 2H11 of the present invention and the 10E10 reaction again.
Fig. 5 shows the antigen site with flow cytometry monoclonal antibody 2H11 and 10E10 identification.
Last figure shows 2H11 identification and 10E10 and commercialization antibody MIH1 site all inequality.Figure below shows 10E10 identification and 2H11 site inequality, but the site almost completely identical with MIH1.
Fig. 6 shows Western blot immunoblotting assay.
The 1st road is small component albumen Marker, and the 2nd, 5,8 roads are L929/PD-L1 membranin extract; 3rd, 6,9 roads are L929/mock membranin extract; 4th, 7,10 roads are MDA-MB-231 membranin extract.Mouse IgG is as negative control.Antibody 10E10 and 2H11 all can discern L929/PD-L1 and MDA-MB-231 membrane surface molecule amount is the protein molecular of 32KD.
Fig. 7 shows the external evoked T cell proliferation with mtt assay analysis monoclonal antibody 10E10.Wherein ordinate zou is the OD value, and X-coordinate is the differential responses group.A: be the independent stimulating group of monoclonal antibody CD3.B: be 1.25 μ g/ml antibody stimulating group.C: be 2.5 μ g/ml antibody stimulating group.D: be 5 μ g/ml antibody stimulating group.E: be 10 μ g/ml antibody stimulating group.Wherein series one is mouse IgG control group, and series two is 10E10 effect group.
Fig. 8 shows with the apoptosis detection method and analyzes 10E10 and the 2H11 promoter action to the CTL killing tumor cell.Wherein use MDA-MB-231 and MDA-MBB-435/PD-L1 as target cell.
A figure shows the apoptosis-induced effect of the PD-L1 molecule of MDA-MB-231 and MDA-MBB-435/PD-L1 expression to specific CTL; B figure and C scheme to show respectively antibody 10E10 and the 2H11 blocking effect to above-mentioned apoptosis phenomenon.Mouse IgG is as negative control, and the activity of antibody is 0-10 μ g/ml.
Fig. 9 shows the expression of using PD-L1 molecule in monoclonal antibody 2H11 and the 10E10 detection stomach organization with immunohistochemical method, the dependency of PD-L1 and patients with gastric cancer pathological change and survival rate.
Summary of the invention
The present invention relates to tumor necrosis factor superfamily member's conjugated protein or polypeptide, more particularly, the present invention relates to anti human PD-L 1 monoclonal antibody 2H11 and 10E10, its preparation method and these monoclonal antibodies in external evoked T cell activation propagation, strengthen specific CTL and kill and wound in the tumour cell of high expressed PD-L1 molecule, and a kind of important application of biological indicator in cancer of the stomach prognosis judgement and individualized treatment. The invention further relates to and use anti-PD-L1 monoclonal antibody to produce the pharmaceutical composition that is used for improving people or mammiferous immune response level.
Said in this specification " PD-1 " refers to the protein of expressing on the T cell surface of antigen activation; The PD-1 part refers to the protein that can be combined with the PD-1 receptor-specific of expressing on some mammalian cell.
Term " anti-PD-L1 antibody " can be PD-L1 specific monoclonal or polyclonal antibody or their immunologic competence part. Among the present invention, preferably monoclonal antibody, particularly mouse anti human PD-L1 antibody.
Can prepare anti human PD-L 1 monoclonal antibody 2H11 of the present invention and 10E10 (referring to Kohler and Milrtein, Nature 256:495-96,1975 according to conventional method known in the art; Harlow and Lane, Aatibodies, A Laboratory, Cold Spring Harbor Laboritory, 1988). According to the preferred embodiments of the invention, preferably use the expression vector be carried the human PD-L 1 gene to transform, and can under suitable condition of culture, efficiently express the recombinant cell of human PD-L 1 polypeptide as the immunogene of preparation monoclonal antibody of the present invention.
Therefore, in order to prepare anti human PD-L 1 monoclonal antibody of the present invention, at first make up the transgenic cell (L929/PD-L1) of high expressed human PD-L 1. The transgenic cell L929/PD-L1 of high expressed human PD-L 1 molecule has stronger immunogenicity, and the steric configuration of expressed antigen molecule can be exposed to surface of cell membrane with nature, thus the more effectively immune response of excitating organism. In addition, because the L929 cell is the fibroblast of mouse, other molecules on its surface have relatively weak antigenicity, therefore will reduce the generation of non-specific antibody with this cell as immunogene, make the screening of monoclonal antibody more convenient and greatly improve positive rate.
In order to prepare the L929/PD-L1 cell, can be according to DNA operating technology well known to those skilled in the art (for example referring to Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbour, 1989) carry out gene separation, nucleotide fragments cutting be connected, identification, transformation and the cultivation of the structure of clone and expression vector and amplification, nucleotide sequence. For example, at first, use the RT-PCR technology from the T cell of activation, to amplify the human PD-L 1 gene. With the PD-L1 gene Retroviral Vector pEGZ-Term that packs into, obtain recombinant retroviral vector pEGZ-Term/PD-L1. Then, use recombinant retroviral vector pEGZ-Term/PD-L1 with helper virus pHIT456 and pHIT60 cotransfection 293T cell. After 48 hours, collection contains the 293T cell culture supernatant of virion and infects L929 cell (ATCC Cat.No.CCL-1), consecutive infection 3 days with it. At last, with the selection Screening of Media that contains Zeocin, it is enough large to treat that the resistance monoclonal grows to, and picking monoclonal colony enlarges cultivation. The PD-L1 expression rate of the transgenic cell that obtains as stated above is up to more than 97%.
Can be according to conventional method known in the art (Kohler and Milstein, Nature 265:495-497,1975) preparation anti human PD-L 1 monoclonal antibody 2H11 of the present invention and 10E10. Briefly, with transgenic cell L929/PD-L1 or breast carcinoma cell strain MDA-MB-231 immune balb/c mice. When the antibody level of serum of immunized animal reached peak value, the splenocyte of separating animal's also prepared single cell suspension. In case of necessity, can use immune adsorption method screening splenocyte. For example, splenocyte suspension can be added in PD-L1 the antigen protein coated flat board or aperture, the B cell of expressing PD-L1 polypeptid specificity immunoglobulin (Ig) namely is attached on the flat board, and can not be washed off by remaining suspension. Then can collect resulting B cell or splenocyte that all dissociate, and for example merge to form hybridoma with myeloma under the inducing of polyethylene glycol at suitable fusion agent, and for example cultivate the hybridoma that merges with screening in the HAT culture medium at selective medium. Then, identify required positive resisting cell strain with methods such as flow cytometry, Western blotting, immuno-precipitations. (in the mouse ascites) cultivates the hybridoma of selected secretion anti human PD-L 1 antibody in external (for example in tissue culture flasks or porous fibre reactor) or body, and collects and the required antibody of purifying from cell culture fluid or mouse ascites liquid.
The flow cytometry analysis result shows that anti human PD-L 1 monoclonal antibody 2H11 provided by the invention and 10E10 all can identify the PD-L1 molecule of expressing on activation T, B cell and the ripe DC surface to some extent. Competition inhibition test result shows that further anti human PD-L 1 monoclonal antibody 2H11 of the present invention and 10E10 can identify incomplete same antigen site. Therefore, might utilize 2H11 and 10E10 for the preparation of the kit that detects soluble human PD-L1.
In order to identify the biological function of anti human PD-L 1 monoclonal antibody of the present invention, the present invention at first carries out Activated in Vitro T test cell line. Experimental result shows that anti human PD-L 1 monoclonal antibody 2H11 of the present invention and 10E10 all can hinder the PD-1/PD-L1 combination, relies on the propagation of mode inducer T lymphocyte with dosage, and can act synergistically with anti-human CD3 excitated type monoclonal antibody performance.
As everyone knows, tumour is to monitor the result that malignant proliferation occurs because of the cell escape from immune. Immune evasion mechanism is because the specific for tumour antigen T cell of body is in immune tolerance state. The Partial tumors cell surface is crossed the PD-L1 molecule of expression and thereby the acceptor interaction on the tumour-specific CTL is induced the CTL apoptosis, kills and wounds so that tumour cell has been escaped. In our test, use monoclonal antibody 2H11 and 10E10 can effectively block this effect, greatly improve the lethal effect of CTL, and have dose dependent. Therefore, might utilize exciting type anti-human PD-L1 monoclonal antibody 2H11 of the present invention and 10E10 to change near the state of CTL tumour cell, and the reaction of excitating organism specificity antineoplastic immunity, to remove and the control tumour.
Although hemopoietic system and non-hematopoietic system cancer cell are all expressed PD-L1 (Dong.H., Zhu.G.et al. Nature Med.1999), but the expression intensity of tumour cell is apparently higher than normal cell (Yoshiko Iwai, Masayoshi Ishida et al.PNAS 2002). We use the tissue specimen that monoclonal antibody 2H11 of the present invention and 10E10 have detected the patients with gastric cancer of 100 examples, the result shows, the expression of PD-L1 and patient age, sex, tumor locus, types of organization are irrelevant, and closely related with tumor size, tumor invasive depth, lymphatic metastasis, patients with gastric cancer prognosis. Our experimental data shows, the positive expression rate (58.8%) of the patients with gastric cancer PD-L1 of life cycle＜2 year is significantly higher than the patients with gastric cancer (25.5%) of life cycle＞5 year. Include in the correlative factor variable analysis such as the expression of tumor size, invasive depth, lymphatic metastasis, PD-L1, the expression of prompting PD-L1 is to affect the cancer of the stomach independent factor of life cycle. Therefore, utilize the present invention can effectively detect the expression of the PD-L1 molecule in the stomach organization, in order to as the judgement of cancer of the stomach prognosis and the biological indicator of selecting as individualized treatment. Simultaneously, infer that PD-L1 antibody might be used to clinical as a kind of curing gastric cancer agent.
Below describe the present invention for example in detail by non-limiting example.Those skilled in the art can not deviate under essence spirit of the present invention and the principle prerequisite, change or change some technology contents or the sport technique segment of describing in this specification sheets.But one will understand that change that these are parallel or change all will be included within the claim scope that awaits the reply of the present invention.
Embodiment 1: the preparation of anti human PD-L 1 monoclonal antibody
Present embodiment is described the preparation method of anti human PD-L 1 monoclonal antibody 2H11 and 10E10.
(1) foundation of transgenic cell L929/PD-L1 and L929/mock
(a) clone of human PD-L 1 gene: use TRIZOL reagent (Gibco, BRL) total RNA of extracting activating T cell.Method according to reverse transcription test kit (MBI, the U.S.) working instructions are recommended becomes cDNA with the mRNA reverse transcription.Getting 5 μ l cDNA is template, uses positive strand primer to carry out pcr amplification (72 ℃ were extended 1 minute for 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, and 30 circulations were extended 5 minutes for back 72 ℃).Behind the purifying, at T
4The PCR product is connected with the pMD18-T carrier to spend the night.Connect product transformed competence colibacillus bacterium Top10 with gained, and the positive colony of screening is carried out PCR and enzyme cut and identify and dna sequencing.The result shows that the human PD-L 1 cDNA sequence of registering among cDNA sequence that is obtained and the GenBank is in full accord.
(b) construction of recombinant defective retroviral vector: respectively with endonuclease PstI and correct pMD18-T/PD-L1 plasmid and the retroviral vector pEGZ-Term (37 ℃, 4 hours) of EcoR I digestion order-checking.Recovery contains the fragment of goal gene, and with connecting product transformed competence colibacillus bacterium.After identify confirming with aforesaid method, the recombinant retroviral vector that is obtained named be pEGZ-Term/PD-L1.
(c) structure of the L929 transgenic cell of stably express human PD-L 1: according to flow process shown in Figure 1, at first with test kit (Invitrogen, the U.S.) liposome method of operational manual recommendation, with recombinant retroviral vector pEGZ-Term/PD-L1 and helper virus pHIT456 and pHIT60 (modern immunology, 2004,24 (2): 99-103) 60-70% confluent growth 293T cell in blocks in (volume ratio 2: 1: 1) cotransfection 6 orifice plates.Collect the 293T culture supernatant that contains virion after 48 hours, infect the L929 cell with it.Add polybrene to final concentration be 8ng/ml, continues 37 ℃ of infection 6 hours.Add 1640 substratum (liquid is changed in the 2ml/ hole every other day) that contain 10% foetal calf serum (FCS) again.Collecting cell after the superinfection 3 times places 2 weeks of screening and culturing in the selection substratum that contains Zeocin (Invitrogen, 500 μ g/ml) with the cell culture of dilution in 1: 6.Treat the resistant cell clonal growth to enough big, picking mono-clonal bacterium colony carries out enlarged culturing.Preparation is as the transgenic cell L929/mock of negative control simultaneously.Flow cytometer detects and shows that the proteic expression rate of human PD-L 1 is about 99% on the L929/PD-L1 cytolemma.
(2) preparation of anti human PD-L 1 monoclonal antibody
The transgenic cell (L929/PD-L1) that uses the high expression level PD-L1 molecule as above obtain is as immunogen, three immunization Balb/c mouse (10
7/ 500 μ l/ are only) (3 weeks at interval).After the last immunity the 4th day, get mouse spleen cell and SP2/0 myeloma cell strain and carry out cytogamy (totally 20 96 orifice plates).With the positive contrast of L929/PD-L1 of high expression level PD-L1 molecule and the negative contrast of L929/mock of expression PD-L1 molecule, hybridoma culture supernatant is carried out preliminary screening, Identification of Fusion Protein and Ig subgroup identification (referring to Fig. 2) with indirect immunofluorescence.Positive colony proves that clone's positive rate reaches about 95% behind 3 limiting dilutions.Behind multiple sieve and subclone, obtain stably to secrete the hybridoma cell strain of specific murine anti human PD-L 1, and be named as 2H11 respectively.
The breast carcinoma cell strain MDA-MB-231 that uses natural high expression level PD-L1 molecule has obtained another strain cell strain 10E10 as immunogen with same method.
These hybridomas after external continue to go down to posterity (more than 40 generations), secreting specificity antibody stably still.Chromosome analysis to hybridoma cell strain 2H11 and 10E10 shows that the chromosome number of this three strain of hybridoma is 80-110 (referring to Fig. 3).
(3) production of anti human PD-L 1 monoclonal antibody and CHARACTERISTICS IDENTIFICATION
(a) induce method manufacture order clonal antibody in the ascites body that adopts this chamber to set up.Get the 6-8 female Balb/c mouse in age in week, intraperitoneal injects Pristane (0.5ml/ only).Inoculation hybridoma (1 * 10 in one all pneumoretroperitoneums
7/ only), the equal-volume mixture (0.2ml/) of intraperitoneal injection Pristane and freund 's incomplete adjuvant once more simultaneously.Gather in the crops ascites after 5-10 days, and the centrifuging and taking supernatant is in-80 ℃ of preservations.
(b) purifying of ascitic type monoclonal antibody and quantitative.Ascites fluid is after the removal scleroproein and the processing of saltouing, with Protein G affinity column chromatography method purifying.Collect the protein peak effluent liquid, it is 0.8～10mg/ml that antibody protein concentration is measured with 751 ultraviolet spectrophotometers in phosphate buffered saline buffer (PBS) dialysis back.Indirect immunofluorescence is analyzed, and tiring of monoclonal antibody is more than 1: 1000 behind the purifying.
(c) Ig subgroup identification.Adopt test paper rapid determination (Argen company) method, identify the Ig subclass, the result shows that 2H11 and 10E10 are mouse IgG1 type.
(d) competitive inhibition of antibody recognition antigen site test.At L929/PD-L1 cell (5 * 10
5/ pipe) adds monoclonal antibody (every pipe 2 μ g) respectively in the suspension, hatched 45 minutes for 4 ℃.After washing cell, add biotin labeled monoclonal antibody and Streptavidine-PE successively, and respectively 4 ℃ hatched 30 minutes.Flow cytometry analysis is used in the washing back once more, establishes the positive and negative control simultaneously.As shown in Figure 5, monoclonal antibody 2H11 partly blocks combining of PD-L1 molecule on monoclonal antibody 10E10 and the L929/PD-L1 cell.Show 2H11 and the different PD-L1 antigen site of 10E10 identification thus.
(f) the Western trace of monoclonal antibody of the present invention is identified.Extracting L929/PD-L1 epicyte protein spends the night with 4 ℃ of sealings of 5% skim-milk.Added the antibody purified incubated at room next day 1 hour.With the unconjugated antibody of TBST solution flush away, add two of AP mark and resist 30 minutes.Hatch the back and add the substrate colour developing.As shown in Figure 6, the L929/PD-L1 cell all can combine with the PD-L1 protein-specific, forms positive band.Show that this two strains monoclonal antibody all has the good specificity of identification PD-L1 molecule.
Embodiment 2: external evoked T cell activation of anti human PD-L 1 monoclonal antibody 2H11 and 10E10 and propagation
Present embodiment describe with
3H-TdR mixes method and detects monoclonal antibody 2H11 and external evoked T cell activation of 10E10 and proliferation function.
(1) T cell preparation: get normal volunteer's peripheral blood 100ml (deriving from Red Cross blood station, Suzhou), separate (Ficoll) and obtain mononuclearcell.After washing cell, be diluted to 3 * 10 with the RPMI1640 substratum that contains 10% calf serum
6/ ml.Then cell is added and cultivate 2 hours (5%CO in 6 well culture plates (2ml/ hole)
2, 37 ℃).The sucking-off suspension cell promptly obtains PBMC gently.Separate according to conventional sheep red blood cell (SRBC) combined techniques then and obtain T cell (purity＞90%).
(2) external evoked T cell proliferation: wrap by 96 orifice plates (4 ℃ are spent the night) with excitated type CD 3-resisting monoclonal antibody (0.5 μ g/ml).Next day, in aperture, add the T cell of purifying and be divided into 5 groups: (A) be the independent stimulating group of monoclonal antibody CD3 (0.5 μ g/ml); (B) be 1.25 μ g/ml monoclonal antibody 2H11 or 10E10 stimulating group; (C) be 2.5 μ g/ml monoclonal antibody 2H11 or 10E10 stimulating group.D: be 5 μ g/ml monoclonal antibody 2H11 or 10E10 stimulating group.E: be 10 μ g/ml monoclonal antibody 2H11 or 10E10 stimulating group.Every group of mouse IgG homotype control group of establishing corresponding dosage.Stimulate after 3 days with
3The H-TdR method of mixing detects T cell proliferation situation.By data shown in Figure 7 as can be seen, two strain monoclonal antibodies of the present invention all can rely on obvious inducing T cell activation of mode and propagation with dosage.
Embodiment 3: anti human PD-L 1 monoclonal antibody 2H11 and 10E10 promote the external breast cancer cell that kills and wounds of specific CTL
Present embodiment is described respectively the enhancement to the external killing tumor cells target cell of CTL (breast cancer cell) with Flow cytometry monoclonal antibody 2H11 and 10E10.
(1) preparation of specific CTL: get normal volunteer's peripheral blood 200ml (deriving from Red Cross blood station, Suzhou), separate obtaining mononuclearcell.After washing cell, with containing the RPMI1640 substratum of 10% calf serum (FCS) with cell dilution to 3 * 10
6/ ml, and place 6 well culture plates (2ml/ hole) to cultivate 2 hours (5%CO
2, 37 ℃).Sucking-off suspension cell gently then, adding the RPMI1640 substratum that contains GM-CSF (100ng/ml), IL-4 (50ng/ml) and 10%FCS in culture plate continues to cultivate, changed liquid once in 3 days, MDA-MB-231 cell and MDA-MB-435 cell that adding in the 6th day is handled through mitomycin.Two days later, take out the antigenic DC of load and separate co-culture of cells, the specific CTL that results are activated after 3 days from the T of same peripheral blood.
(2) CTL killing tumor cell: get the CTL of as above preparation and MDA-MB-231 cell and the MDA-MB-435 cell of handling through mitomycin, added in the 96 orifice plate apertures by 50: 1 and cultivate altogether.In aperture, add monoclonal antibody 2H11 and the 10E10 that concentration is respectively 1.25 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml simultaneously.The mouse IgG homotype that each group is established respective concentration in contrast.Cultivate after 3 days, detect apoptotic cell through AnexinV and PI dyeing.As shown in Figure 8, MDA-MB-231 effect group after adding different concns antibody, has all reduced the apoptosis rate of CTL to some extent, and the MDA-MB-435 group does not then have considerable change.The result shows that antibody 2H11 and 10E10 can strengthen CTL killing and wounding tumour cell.
Embodiment 4: the biological indicator of judging observation of curative effect with anti human PD-L 1 monoclonal antibody 2H11 and 10E10 as the cancer of the stomach prognosis
Present embodiment is described with monoclonal antibody 2H11 and 10E10 level as biological indicator, carries out cancer of the stomach prognosis judgement and curative effect monitoring with the immunohistochemical methods method.Simultaneously, the test kit that is used for cancer of the stomach prognosis judgement and curative effect monitoring that uses Monoclonal Antibody of the present invention is described.
(1) sample source: 102 routine samples are taken from the cancer of the stomach patient with operation of hospitalizing in the period of the 1998-1999.All do not accept chemotherapy or radiotherapy before all patient's arts.
(2) immunostaining and assessment: use known ElivisionTM plus immunohistochemical staining: behind the paraffin section de-waxing and aquation with sample to be checked, with PBS (pH 7.4) flushing three times, each 3 minutes.Get a certain amount of pH 6.0 citrate buffers and in beaker, heat dewaxing, be cooled to room temperature after, respectively wash twice with distilled water and PBS successively, each 3 minutes.Every section adds 1 or 50 μ l 3%H
2O
2, hatched under the room temperature 10 minutes, with the blocking-up endogenous peroxydase.Section drips first antibodies with three (each 3 minutes) backs of PBS flushing, hatches under the room temperature to spend the night in 60 minutes or 4 ℃.With after the PBS flushing three times, every section adds 50 μ l polymkeric substance tougheners (reagent A) again, hatched under the room temperature 20 minutes and PBS flushing 3 times after, drip the sheep anti-mouse igg antibody (reagent B) of horseradish peroxidase-labeled again, hatched under the room temperature 30 minutes.After the PBS flushing and adding the freshly prepared DBA colour developing of 100 μ l liquid, observed 3～10 minutes in microscopically.Positive in showing the particle of pale brown color.Behind the distilled water flushing, redye with Hematorylin.Section is transparent with ethanol dehydration drying and dimethylbenzene, uses the neutral gum mounting then.
The result judges: obviously be colored as the positive with tumour cell matter.The obvious painted positive cell of PD-L1 is less than 20% for expressing feminine gender in the stomach organization, is that expression is positive greater than 20%.
(3) carry out data analysis with the SPSS10.0 statistical software: on the statistics, with X
2Method of inspection is expressed between the positive and negative group of organizing PD-L1 and is compared.Adopt multivariate Logistic regression method to analyze the relation of prognosis and factors.The result is as shown in table 1.
Table 1: the relation of prognosis of gastric cancer situation and patients with gastric cancer cancer cells PD-L1 expression level
| Project | Total routine number | Positive routine number (positive rate %) | X 2 | P |
| Sex | ||||
| The men and women | 75 27 | 32(42.7) 11(40.7) | 0.030 | 0.862 |
| Age | ||||
| ＞60 years old＜60 years old | 64 38 | 28(43.8) 15(39.5) | 0.179 | 0.672 |
| The cancer of the stomach position | ||||
| In 1/3 stomach of Weishang under 1/3 |
31 30 41 | 13(41.9) 11(36.7) 19(46.3) | 0.666 | 0.717 |
| Types of organization | ||||
| Differentiated hangs down differentiated | 72 30 | 33(45.8) 10(33.3) | 1.357 | 0.244 |
| Invasive depth | ||||
| Do not invade and dark flesh layer is invaded and |
30 72 | 6(20.0) 37(51.4) | 8.556 | 0.003 |
| Nodus lymphoideus transferring rate | ||||
| Negative positive | 54 48 | 15(27.8) 28(58.3) | 9.730 | 0.002 |
| Lifetime | ||||
| ＜2 years＞5 years | 51 51 | 30(58.8) 13(25.5) | 11.619 | 0.001 |
| The tumour size | ||||
| ＜5cm ＞5cm | 55 47 | 18(32.7) 25(53.2) | 4.352 | 0.037 |
By the data shown in the table 1 as can be seen, the cytolemma of stomach organization and cytoplasm all show the positive expression of PD-L1 molecule, and positive rate reaches 42.2%.And, data analysis shows, lifetime greater than 5 years with lifetime, there were significant differences less than the expression of PD-L1 molecule in the patients with gastric cancer cancerous tissue cell in 2 years: be significantly higher than lifetime greater than the patients with gastric cancer (25.5%) in 5 year less than the The positive expression rate (58.8%) of patient PD-L1 molecule in stomach organization in 2 years lifetime.Statistical analysis prompting PD-L1 molecule is closely related in the prognosis of the expression of stomach cancer cell and patients with gastric cancer.Therefore, can use monoclonal antibody 10E10 box 2H11 to prepare the immunohistochemistry test kit, be applied to the detection of clinical stomach organization.
The hybridoma sample preservation
Particularly produce the accidental opportunity of the hybridoma screening of monoclonal antibody of the present invention with high yield in view of positive hybridoma, also be as replenishing to this specification sheets text description part, the applicant in Chinese common micro-organisms preservation administrative center (CGMCC) preservation produce two hybridoma cell strains of monoclonal antibody 2H11 of the present invention and 10E1 respectively, their deposit number is respectively CGMCC No.1637 and CGMCC No.1636
Claims (6)
1. anti human PD-L 1 monoclonal antibody 2H11 and 10E10 are characterised in that said anti human PD-L 1 monoclonal antibody 2H11 and 10E10 are is respectively the hybridoma cell strain excretory of CGMCC No.1637 and CGMCCNo.1636 by deposit number.
2. according to the anti human PD-L 1 monoclonal antibody of claim 1, be characterised in that these antibody are monoclonal antibodies of anti-soluble human PD-L1.
3. according to the anti human PD-L 1 monoclonal antibody of claim 1, the deposit number that wherein produces the hybridoma cell strain of anti human B 7-H 3 monoclonal antibody 2H11 and 10E10 is respectively CGMCC No.1637 and CGMCC No.1636.
4. the application in external evoked T cell activation propagation according to the anti human PD-L 1 monoclonal antibody 2H11 of claim 1 and 10E10.
5. according to the anti human PD-L 1 monoclonal antibody 2H11 of claim 1 and the 10E10 application in strengthening the tumour cell that specific CTL kills and wounds high expression level PD-L1 molecule.
According to the anti human PD-L 1 monoclonal antibody 2H11 of claim 1 and 10E10 as the Biological Detection index, the application in cancer of the stomach prognosis judgement and individualized treatment.
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