CN107266572A - Anti- PD L1 protein monoclonal antibodies and application thereof - Google Patents

Abstract

The present invention relates to biological technical field, a kind of hybridoma cell strain (deposit number is CGMCC No.13826), and the monoclonal antibody OR 5E3 that thus hybridoma cell strain is produced are disclosed.The application in being used to detect the immune detection instrument of PD L1 albumen is being prepared the invention further relates to monoclonal antibody OR 5E3, the 5E3 of OR containing monoclonal antibody immunologic combined detection reagent kit, and applications of the monoclonal antibody OR 5E3 in the kit for marked tumor is prepared.Monoclonal antibody of the present invention can be combined with PD L1 protein-specifics, and with other intracellular albumen no cross reactions, significantly improve PD L1 protein immunizations detection specificity and reliability.

Description

Anti- PD-L1 protein monoclonal antibodies and application thereof

Technical field

The present invention relates to biological technical field, and in particular to can specific bond PD-L1 albumen monoclonal antibody OR-5E3, Produce the cell line of the monoclonal antibody and apply the diagnostic method of the antibody and purposes.

Background technology

The ligand 1 of programmed cell death factors 1 (processed death-1ligand 1, PD-L1) be also known as B7-H1, CD274, is an inhibition costimulatory molecules by CD274 gene codes, is that Dong is equal to 1999 from human gene bank Search what is found and confirm in placenta cdna library when B7.1 and B7.2 immune globulin variable regions and constant region homolgous molecule. PD-L1 belongs to I type transmembrane glycoproteins, there is 290 amino acid, including extracellular region, hydrophobic transmembrane and afterbody cytoplasmic domain.PD-L1 eggs White then wide expression such as activates T and B cell, macrophage, BMDC, thymic epithelial cell cell in lymphocyte And the position such as heart and placenta, while also high expression in the immunocyte in kinds of tumor cells and tumor microenvironment, such as Lung cancer, breast cancer, the cancer of the esophagus, lymthoma etc..

PD-L1 plays more crucial role in immunological regulation.The PD-L1 of tumor cells expression is with expression in activation T Negativity costimulatory molecules PD-1 on cell is combined, and can suppress T cell propagation, and promote the apoptosis of activating T cell.In recent years, Substantial amounts of research shows that PD-L1 can be examined as a kind of targeted therapy of important molecular marker in various tumours and prognosis Played an important role in disconnected.

At present clinically generally with immunohistochemistry (Immunohistochemistry, IHC) experiment detection tumor group The expression situation of PD-L1 in inner tumour cell is knitted, the core of its experimental method is specific binding PD-L1 monoclonal antibody, The quality of its performance directly decides the sensitivity and specificity entirely detected.Therefore, a kind of spy in higher sensitivity is developed The opposite sex has great importance for the monoclonal antibody of PD-L1 albumen.

The content of the invention

In view of this, it is anti-it is an object of the invention to provide a kind of monoclonal of the higher PD-L1 albumen of binding specificity Body, and its preparing the application in being used to detect the immune detection instrument of PD-L1 albumen.

The invention provides a kind of hybridoma cell strain, China Committee for Culture Collection of Microorganisms is preserved in commonly micro- Bio-Centers (referred to as CGMCC), preservation date is on April 7th, 2017, and deposit number is CGMCC No.13826.

Present invention also offers a kind of monoclonal antibody OR-5E3 of specific binding PD-L1 albumen, by above-mentioned hybridoma Cell line is produced.

The preparation method of monoclonal antibody of the present invention is as follows：

(1) screening and preparation of monoclonal antibody：Using chemical synthesis PD-L1 intracellular region polypeptide fragment 260-290aa (Hangzhoupro State Chinese Peptide Co., Ltd.) immune new zealand white rabbit (being purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.), take Rabbit spleen cell is merged with the strain of 240E rabbit bone marrow knurls like cell, and limiting dilution assay obtains monoclonal, and ELISA method screening is positive Hybridoma, acquisition can secrete the hybridoma cell strain of anti-PD-L1 specific antibodies, be named as OR-5E3, hypotype is accredited as IgG；Antibody is prepared by serum free medium, PD-L1 monoclonal antibodies are obtained by affinity column purifying.Pass through immune group Change the sensitivity and specificity of the experimental verification monoclonal antibody.

Further the specificity of said monoclonal antibody is verified using OriGene high-density proteins chip：

Lysate is overexpressed on OriGene high-density protein chips comprising 10,000 HEK293T cell proteins (not including PD-L1 albumen), every kind of protein lysate has the repetition of two copies on chip.Protein lysate is fine in nitric acid by trace On the plain film of dimension.The positioning of each clock protein lysate can be accurately positioned by Excel file.Albumen point on protein chip For 40 sub- matrixes, each there are some references on the matrix of Asia, by referring to, the content of albumen on each chip point can be quantified, The repeatability of each immune response data is monitored, and positions the direction of positive signal.

The monoclonal antibody OR-5E3 and said chip are hybridized and determine positive signal site by the present invention, are as a result shown Show：Monoclonal antibody OR-5E3 of the present invention specifically binds PD-L1 albumen, and with other albumen no cross reactions.

Prepared present invention also offers monoclonal antibody OR-5E3 for detecting in the immune detection instrument of PD-L1 albumen Application.

Specifically, the immune detection instrument is kit, chip or test paper.

In the particular embodiment, the invention provides a kind of immunologic combined detection reagent kit, including above-mentioned monoclonal is anti- Body OR-5E3, can detect the expression situation of PD-L1 in immunocyte and tumour cell.

Prepared present invention also offers said monoclonal antibody for detecting PD-L1 in immunocyte and tumour cell Application in the kit of protein level.

Wherein described tumour be specifically including but not limited to lymthoma, lung cancer, liver cancer, colorectal cancer, melanoma, kidney and Breast cancer etc..

Compared with prior art, the invention provides a kind of hybridoma cell strain, (deposit number is CGMCC No.13826 the monoclonal antibody OR-5E3 that), and thus hybridoma cell strain is produced.Present invention also offers monoclonal antibody OR-5E3 is preparing the application in being used to detect the immune detection instrument of PD-L1 albumen, and the OR-5E3's containing monoclonal antibody is immune Groupization detection kit, and monoclonal antibody OR-5E3 PD-L1 in preparing for labelled immune cell and tumour cell Application in the kit of protein level.Monoclonal antibody of the present invention is anti-without intersecting with 10000 kinds of other albumen on chip Should, significantly improve PD-L1 protein immunizations detection specificity and reliability, be widely used in the immunocyte of various samples with And in tumour cell PD-L1 detection.

Preservation information

Classification And Nomenclature for the hybridoma cell strain OR-5E3 of preservation is：Ligand 1 (the PD- of rabbit-anti people's programmed cell death factor 1 L1) monoclonal hybridoma strain；

Depositary institution's full name：China Committee for Culture Collection of Microorganisms's common micro-organisms center；

Depositary institution is referred to as：CGMCC；

Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；

Preservation date：On April 7th, 2017；

Deposit number：CGMCC No.13826.

Brief description of the drawings

In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing There is the accompanying drawing used required in technology description to be briefly described.

Fig. 1 shows IHC testing result figures of the embodiment 2PD-L1 monoclonal antibodies OR-5E3 on PD-L1 positive cells, with The expression of PD-L1 albumen in the HEK293T cells of OR-5E3 detection transient transfection PD-L1 plasmids, left figure is transfection empty carrier The IHC testing results of HEK293T cells, right figure is the IHC testing results of the HEK293T cells of transfection pCMV6-PD-L1 plasmids (primary antibody is PD-L1 monoclonal antibodies OR-5E3,1:100)；

Fig. 2 shows that (primary antibody is PD-L1 monoclonal antibodies OR-5E3,1 to the placenta tissue SABC testing result figure of embodiment 2: 100)；

Fig. 3 shows that (primary antibody is PD-L1 monoclonal antibodies OR-5E3,1 to the cancerous lung tissue SABC testing result figure of embodiment 2: 100)；

Fig. 4 shows that (primary antibody is PD-L1 monoclonal antibodies OR- to the melanoma tissue SABC testing result figure of embodiment 2 5E3,1:100)；

Fig. 5 shows that (primary antibody is PD-L1 monoclonal antibodies OR-5E3,1 to embodiment 3OriGene protein chip qualification results figure:100；Two Resist the donkey anti-rabbit IgG marked for Alexa 647-, 1:500).

Embodiment

Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, Obviously, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based in the present invention Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, all Belong to the scope of protection of the invention.

The preparation and screening of embodiment 1, PD-L1 monoclonal antibodies

According to the standard method chemical synthesis PD-L1 intracellular region polypeptide fragments bought in Zhongtai Bio-Chem. Co., Ltd., Hangzhou 260-290aa (hereinafter referred to as PD-L1 antigens) is to new zealand white rabbit (Beijing Vital River Experimental Animals Technology Co., Ltd.) It is immunized.Specific method is as follows：

1st, animal immune：PD-L1 antigens are emulsified with complete Freund's adjuvant, and it is new that 6-8 week old is immunized using subcutaneous injection method Western orchid White Rabbit, for 500 μ g/ only, interval carries out being immunized for second immunizing dose after two weeks, is emulsified with incomplete Freund's adjuvant, Immunizing dose is 250 μ g/.It is immune to take blood to determine serum titer with ELISA method gradient dilution afterwards twice；It is according to result determination No booster immunization, chooses antibody titer highest rabbit and carries out cell fusion.

2nd, cell fusion：The 240E rabbit bone marrow knurls like cell strain that myeloma cell is originated using new zealand white rabbit, fusion When be in exponential phase；Take and rabbit spleen is immunized, lymphocyte single cell suspension is made；Rabbit splenic lymphocytes and myeloma are thin Born of the same parents are with 1:10 mixing, 50%PEG (PH 8.0) 1mL of 37 DEG C of dropwise addition adds incomplete culture medium and remaining terminate liquid, and centrifugation is abandoned Add the suspension of HAT culture mediums after supernatant to mix, MC constant volumes are dispensed into 3.5cm culture dishes, are put in wet box, be placed in 50mL 37 DEG C, 5%CO₂Cultivated in constant incubator.

3rd, screen and clone：Fusion selects cell clone in 7-10 days, and ELISA tests are carried out using PD-L1 antigens.Mark Cell line number.Positive hole cell is carried out to determine ELISA values, picking OD within 5-6 days after limiting dilution, each limiting dilution₂₈₀It is positive The higher monoclonal hole of value carries out limiting dilution, until it is the positive that ELISA, which determines the complete hardened fruit of 96 orifice plates,.Picking positive value is high Monoclonal singling.Its correspondence fusion plate cell line is OR-5E3.

4th, the preparation and purification of cell conditioned medium monoclonal antibody：By DMEM medium cultures of the cell line OR-5E3 containing 15% serum Cultivate, spread cultivation to about 4 × 10 in 10cm culture dishes⁷When individual, 800rpm centrifugation 5min abandon supernatant and cell are transferred into 2L turns In bottle, serum free medium is added, it is about 3 × 10 to make cell density⁵Individual/ml.Continue after cultivating 1~2 week, work as cell mortality (now cell density is about 1-2 × 10 when reaching 60%-70%⁶Individual/ml), collect cell suspension 6000rpm high speed centrifugations 20min, takes supernatant, and affinity chromatography carries out supernatant purifying, and corresponding post material is selected according to antibody die mould, and monoclonal antibody OR-5E3 hypotypes are IgG1, is purified using protein G.Monoclonal antibody concentration mensuration after purification, packing (100uL/ is managed, and concentration is 1mg/ml), It is stored in 4-8 DEG C.

Embodiment 2, PD-L1 monoclonal antibodies OR-5E3 detect for the SABC of primary antibody

(1), experimental method：

1st, the 293T cells, placenta, lung cancer and the melanoma tissue block that are overexpressed empty carrier and PD-L1 plasmids is taken to enter respectively Row FFPE, is cut into slices using SAKURA histotomes, and tissue thickness is 4 μm.

2nd, dewaxing and aquation：Analyze pure 3 × 10min of dimethylbenzene, absolute ethyl alcohol 3 × 10min, 95% ethanol 5min, 85% Ethanol 5min, 75% ethanol 5min, deionized water immersion 3min × 3 time

3rd, antigen retrieval buffers (1mM EDTA, pH9.0 10mM Tris-HCL buffer solutions) pressure cooker hot high pressure is added to repair 2.5min, when high pressure pot temperature is down to about 90 DEG C, opens pressure cooker, takes out sample, then naturally cool to room temperature.Deionization Water soaks 3min × 3 time.

4th, using 3% hydrogen peroxide deactivation tissue endogenous peroxydase, it is stored at room temperature 10min.Deionized water is soaked 5min × 3 time.

5th, plus confining liquid (Normal Goat Serum of PBS+5% skimmed milk powers+5%), 37 DEG C of incubation 60min.

6th, confining liquid is removed, is not rinsed, PD-L1 monoclonal antibodies (OR-5E3), thinner ratio is added：1:100, carried out using confining liquid Dilution.It is placed in wet box, 37 DEG C of incubation 60min.PBST (0.1%Tween-20) is washed 2 times, and 5min is washed every time.PBST (0.02%Tween-20) is washed 1 time, and 5min is washed every time.

7th, reagent PV8000 in polymer HRP staining kits is added dropwise and (is purchased from Beijing company of Zhong Shan Golden Bridge, Catlog No.PV-8000), 37 DEG C are incubated 15 minutes.Washed 3 times using PBS, each 5min.

8th, developed the color using DAB solution (Zhong Shan Golden Bridge ZLI-9019), develop the color 3~10min.Distill water washing.

9th, haematoxylin redyeing nucleus 2min, distilled water rinsing, the differentiation of 1% hydrochloric acid.Distilled water is rinsed 3 times, is stored at room temperature 1min。

10th, it is dehydrated and transparent：75% ethanol 5min, 100% ethanol 5min x 3 times, 85% ethanol 5min, 95% ethanol 5min, 100% 3 × 5min of ethanol；3 × 5min of dimethylbenzene, neutral gum mounting.

11st, microscopy, is shown in Fig. 1, Fig. 2, Fig. 3 and Fig. 4.

(2), experimental result：

From Fig. 1 results, PD-L1 albumen in the 293T cells for being overexpressed PD-L1 plasmids in specific cell film and Cytoplasmic expression, and without specificity coloring in the 293T cells for being overexpressed empty carrier.

From Fig. 2 results, PD-L1 albumen trophocyte in placenta tissue is in specific cell film and cytoplasm table Reach.

From Fig. 3 results, tumour cell of the PD-L1 albumen in cancerous lung tissue is in specific cell film and cytoplasm table Reach.

From Fig. 4 results, tumour cell of the PD-L1 albumen in melanoma tissue is in specific cell film and cell Matter is expressed.

As a result show that monoclonal antibody OR-5E3 of the present invention being capable of specific recognition placenta, lung cancer and melanoma PD-L1 albumen in tissue.

The specific proteins chip detection of embodiment 3, monoclonal antibody OR-5E3

Lysate is overexpressed on OriGene high-density protein chips comprising 10,000 HEK293T cell proteins (not containing PD-L1 albumen), every kind of protein lysate has the repetition of two copies on chip.Protein lysate is fine in nitric acid by trace On the plain film of dimension.The positioning of each clock protein lysate can be accurately positioned by Excel file.Albumen point on protein chip For 40 sub- matrixes, each there are some references on the matrix of Asia, by referring to, the content of albumen on each chip point can be quantified, The repeatability of each immune response data is monitored, and positions the direction of positive signal.It is to use OriGene albumen below (OriGene Cat PA100001) chip carries out the experimental method of OR-5E3 Identification of the antibodies experiments：

1st, a protein chip is placed in 50mL centrifuge tubes, infiltrates chip using 40mL deionized waters, be placed on shaking table, Mixed at room temperature 30 minutes.Deionized water is discarded, chip is balanced using 10mLPBST.Room temperature treatment 10 minutes.

2nd, add 40mL5% skim milks (being diluted with PBST) into 50mL centrifuge tubes to be placed on shaking table, room temperature envelope Close 30 minutes.

3rd, primary antibody OR-5E3 is diluted using confining liquid (5% skim milk), thinner ratio is classified as 1：100.

4th, clean sealed membrane is pasted on experimental bench, 250-300 μ L primary antibodies is added dropwise on sealed membrane.

5th, protein chip is extracted out from confining liquid, by the one of protein chip NC films down, contacted from one side of chip Antibody, is slowly slided, by surface tension of liquid, and antibody will slowly infiltrate chip NC films, until whole NC films infiltration is in primary antibody In solution.Whole operation process avoids producing bubble.Chip is moved on under 4 DEG C of environment, stood, primary antibody is incubated overnight.In chip Upper capping culture dish lid, sticks a hygenic towelette thereon, causes antibody to evaporate to prevent from being incubated for a long time.

6th, chip was moved in 50mL centrifuge tubes in second day, chip is rinsed twice using PBST, remove unnecessary antibody.Make Chip is washed with 40mL PBST (0.1%Tween-20), is placed on shaking table and is well mixed, wash three times, 5min is washed every time.

7th, the donkey anti-rabbit IgG of secondary antibody Alexa 647- marks is diluted using confining liquid (5% skim milk), dilution ratio is 1：500.

8th, according to above-mentioned steps 4, step 5 carries out secondary antibody and is incubated operation.Incubation at room temperature 1 hour.Aluminium foil is used above chip Paper is covered, to prevent signal bleaching.

9th, according to above-mentioned steps 6, chip is washed using PBST.

10th, using deionized water rinsing chip, to remove remnants salinity and denaturant.

11st, drying at room temperature chip, it is ensured that chip is completely dried.

12nd, fluorescence signal is read using chip scanner.

13rd, chip direction and the site of positive signal are determined according to BSA-Cy3 and BSA-Cy5.

14th, correspondence protein lysate ID is found out according to positive signal site, according to lysate database information, found pair Answer protein name, NCBI typings number (accession number), protein I D, the information such as albumen size.As a result Fig. 5 is seen.

Fig. 5 results show that all albumen are not combined with antibody OR-5E3 on protein chip, show of the present invention anti- Body OR-5E3 has certain specificity.

Claims (7)

1. a kind of monoclonal antibody OR-5E3 of specific binding PD-L1 albumen, is CGMCC No.13826's by deposit number Hybridoma cell strain is produced.

2. a kind of hybridoma cell strain, its deposit number is CGMCC No.13826.

3. monoclonal antibody as claimed in claim 1 is being prepared for detecting answering in the immune detection instrument of PD-L1 albumen With.

4. application according to claim 3, the immune detection instrument is kit, chip or test paper.

5. a kind of immunologic combined detection reagent kit, including the monoclonal antibody described in claim 1.

6. application of the monoclonal antibody as claimed in claim 1 in the kit for marked tumor cell is prepared.

7. application according to claim 6, described tumour is to include melanoma and lung cancer etc..