CN106957823A - Anti- PD L2 protein monoclonal antibodies and application thereof - Google Patents
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- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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Abstract
The present invention relates to biological technical field, anti-PD L2 protein monoclonal antibodies hybridoma and its anti-PD L2 monoclonal antibodies of generation and application are disclosed.Anti- PD L2 protein monoclonal antibody hybridomas deposit number of the present invention is CGMCC NO：13285.The hybridoma that the present invention is provided can stably excreting produce anti-PD L2 protein monoclonal antibodies, and combined with PD L2 protein-specifics, and with nearly 10000 kinds other albumen no cross reactions on protein chip, specificity, accuracy and the reliability of PD L2 protein immunizations detection are significantly improved, the detection of PD L2 in various tumor tissues and its microenvironment is widely used in.
Description
Technical field
The present invention relates to biological technical field, and in particular to anti-PD-L2 protein monoclonal antibodies hybridoma and its production
Raw anti-PD-L2 monoclonal antibodies and application.
Background technology
The part 2 (processed death-1ligand 2, PD-L2) of programmed cell death factors 1 is also known as B7-DC,
By one of the PDCD1LG2 gene codes I type transmembrane glycoprotein constituted containing 274 amino acid residues, including extracellular region, dredge
Water transmembrane region and afterbody cytoplasmic domain, its amino acid identity with PD-L1 is up to 40%, but both also have certain difference.
The distribution of PD-L2 albumen has limitation, only in the film of macrophage, BMDC and some B cell subclass
Surface expression, but there are some researches show under the stimulation of specific microenvironment, PD-L2 is in other panimmunity cells and non-exempts from recent years
High expression in expression, such as lung cancer, breast cancer, thymoma tumour cell can be induced in epidemic disease cell.
PD-L2 plays more crucial role in immunological regulation.The PD-L2 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-L2 can be as a kind of targeted therapy of important molecular marker in various tumours and prognosis
Played an important role in diagnosis.
At present clinically generally with immunohistochemistry (Immunohistochemistry, IHC) experiment detection tumor group
The expression situation of PD-L2 in inner tumour cell is knitted, the core of its experimental method is specific binding PD-L2 monoclonal antibody,
The quality of its performance directly decides the sensitivity and specificity entirely detected.Therefore, a kind of binding specificity is developed higher
The monoclonal antibody for PD-L2 albumen have great importance.
The content of the invention
In view of this, it is an object of the invention to provide anti-PD-L2 protein monoclonal antibodies hybridoma and its generation
Anti- PD-L2 monoclonal antibodies and application, the combination for improving monoclonal antibody and PD-L2 albumen that the hybridoma is produced is special
Property is simultaneously applied in the preparation of Related product.
To achieve the above object, the present invention provides following technical scheme：
Anti- PD-L2 protein monoclonal antibodies hybridoma, deposit number is CGMCC NO：13285.
Anti- PD-L2 protein monoclonal antibodies hybridoma of the present invention is prepared by following methods：
(1) screening and preparation of monoclonal antibody：By buying OriGene companies PD-L2 recombinant protein (article No.s
TP324141) as immunogene, BALB/c mouse is immunized, takes Mouse spleen cells to be merged with SP2/0 cells, it is limited dilute
Interpretation of the law obtains monoclonal, and ELISA method screening positive hybridoma cell, acquisition can secrete the hybridoma of anti-PD-L2 specific antibodies
Cell line；Antibody is prepared by serum free medium, PD-L2 monoclonal antibodies are obtained by affinity column purifying.By exempting from
The sensitivity and specificity of the epidemic disease group experimental verification monoclonal antibody.
After prepared by the above method, the hybridoma for being capable of the anti-PD-L2 monoclonal antibodies of stably excreting is filtered out, is ordered
Entitled UMAB223, hypotype is accredited as IgG1, and is preserved on November 22nd, 2016 Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica,
Deposit number is CGMCC NO：13285.
Meanwhile, the present invention also provides a kind of anti-PD-L2 protein monoclonal antibodies, is CGMCC NO by deposit number：13285
Hybridoma secretion produce.The art conventional method can be used by preparing anti-PD-L2 protein monoclonal antibodies, such as logical
Cross serum free medium and prepare antibody, PD-L2 monoclonal antibodies are obtained by affinity column purifying.
Deposit number of the present invention is CGMCC NO：13285 hybridoma chromosome stabilityX, it secretes what is produced
Anti- PD-L2 protein monoclonal antibodies are IgG1 types, and potency is higher.The SABC testing result of the monoclonal antibody shows,
It can be in specific recognition cancerous lung tissue, thymus gland tumor tissue and hyperplastic prostate tissue PD-L2 albumen.
Meanwhile, the specific test for detecting the monoclonal antibody using OriGene high-density proteins chip shows, of the present invention
Monoclonal antibody energy specific recognition PD-L2 albumen, and with nearly 10000 kinds other albumen no cross reactions.
Therefore, it is CGMCC NO present invention also offers deposit number：13285 hybridoma is preparing anti-PD-L2
Application and secreted anti-PD-L2 protein monoclonal antibodies in protein monoclonal antibody are preparing detection PD-L2 albumen
Application in immune detection product.
Preferably, the immune detection product is kit, test paper or chip.
The present invention also provides a kind of kit of SABC detection, including deposit number is CGMCC NO：13285 it is miscellaneous
The anti-PD-L2 protein monoclonal antibodies for handing over oncocyte secretion to produce.The immunity detection reagent can detect tumor tissues and its
PD-L2 expression situation in microenvironment.
In addition, it is CGMCC NO that the present invention, which also provides deposit number,：The anti-PD- that 13285 hybridoma secretion is produced
Application of the L2 protein monoclonal antibodies in the kit for preparing marked tumor tissue and its microenvironment medium size lymphocyte.
Preferably, the tumour includes lung cancer and thymoma.
The hybridoma that the present invention is provided can stably excreting produce anti-PD-L2 protein monoclonal antibodies, and with PD-L2 eggs
White specific binding, and with nearly 10000 kinds other albumen no cross reactions on protein chip, significantly improve PD-L2 albumen and exempt from
Specificity, accuracy and the reliability of epidemic disease detection, are widely used in the detection of PD-L2 in various tumor tissues and its microenvironment.
Biological deposits information explanation
Classification And Nomenclature for the hybridoma cell strain UMAB223 of preservation is：The anti-human part 2 of the programmed cell death factor 1 of mouse
(PD-L2) monoclonal hybridoma strain；
Depositary institution's title：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 November 22nd, 2016；
Deposit number：CGMCC No.13285.
Brief description of the drawings
Fig. 1 show the PD-L2 protein SDS-PAGE result figures bought in embodiment 1；
(primary antibody is that UMAB223 secretes the PD-L2 produced to the cancerous lung tissue SABC testing result figure of embodiment illustrated in fig. 22
Monoclonal antibody, 1:500)；
(primary antibody is that UMAB223 secretes the PD- produced to the thymoma histogenic immunity group testing result figure of embodiment illustrated in fig. 32
L2 monoclonal antibodies, 1:500)；
(primary antibody is that UMAB223 secretes generation to the hyperplastic prostate tissue SABC testing result figure of embodiment illustrated in fig. 42
PD-L2 monoclonal antibodies, 1:500)；
Embodiment illustrated in fig. 53 is overexpressed the Flow cytometry result figure (primary antibody of the 293T living cells of PD-L2 plasmids
The PD-L2 monoclonal antibodies produced, 1 are secreted for UMAB223:50)；
(primary antibody is that UMAB223 secretes the PD-L2 produced to embodiment illustrated in fig. 6 4OriGene protein chip qualification results figure
Monoclonal antibody, 1:100；Secondary antibody is the donkey anti-mouse IgG that Alexa 647- are marked, 1:500).
Embodiment：
The invention discloses anti-PD-L2 protein monoclonal antibodies hybridoma and its anti-PD-L2 monoclonals of generation are anti-
Body and application, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular
It is that all similar replacements and change are apparent to those skilled in the art, they are considered as being included in this
Invention.The product of the present invention and application are described by preferred embodiment, and related personnel can substantially not depart from this
Method described herein and application are modified in the content of the invention, spirit and scope or suitably change are with combining, come realize with
Using the technology of the present invention.
The anti-PD-L2 protein monoclonal antibodies hybridoma and its anti-PD-L2 of generation provided below with regard to the present invention is mono-
Clonal antibody and application are described further.
Embodiment 1:The preparation screening of anti-PD-L2 monoclonal antibody hybridoma cells and its monoclonal antibody
Buy in OriGene companies total length PD-L2 recombinant proteins (article No. TP324141, hereinafter referred to as PD-L2 resist
Original, as shown in Figure 1) be used to B6/C57 mouse (Beijing Vital River Experimental Animals Technology Co., Ltd.) are immunized.Specifically
Method is as follows：
1st, animal immune：Purified PD-L2 antigens are emulsified with complete Freund's adjuvant, using subcutaneous or abdominal cavity injection side
6-8 week old BALB/c mouses are immunized in method, and for 50 μ g/ only, interval carries out being immunized for second immunizing dose after two weeks, with incomplete
Freund's adjuvant is emulsified, and immunizing dose is 50 μ g/.It is immune to take tail blood to determine serum effect with ELISA method gradient dilution afterwards twice
Valency；Booster immunization is determined whether according to result, antibody titer highest mouse is chosen and carries out cell fusion.
2nd, cell fusion：Myeloma cell uses the sp2/0 that BALB/c originates, and exponential phase is in during fusion；Take
Immune mouse spleen, is made lymphocyte single cell suspension；Mouse spleen lymphocyte is with myeloma cell with 1:5-1:10 mix
Close, 37 DEG C 50%PEG (PH 8.0) 1mL is added dropwise, add incomplete culture medium and remaining terminate liquid, centrifugation, which is abandoned after supernatant, to be added
Enter HAT culture mediums to suspend mixing, MC constant volumes are dispensed into 3.5cm culture dishes, are put in wet box to 50mL, be placed in 37 DEG C, 5%
CO2Cultivated in constant incubator.
3rd, screen and clone：Fusion selects cell clone in 7-10 days, is carried out using the PD-L2 recombinant proteins of purifying
ELISA is tested.Mark cell line number.Positive hole cell is carried out to determine ELISA within 5-6 days after limiting dilution, each limiting dilution
Value, the higher monoclonal hole of picking OD280 positive values carries out limiting dilution, until it is sun that ELISA, which determines the complete hardened fruit of 96 orifice plates,
Property.The high monoclonal singling of picking positive value.Its correspondence fusion plate cell line is UMAB223.
4th, the preparation and purification of cell conditioned medium monoclonal antibody：The DMEM culture mediums containing 15% serum are used to train cell line UMAB223
Support and cultivated in 10cm culture dishes, spread cultivation to about 4 × 107When individual, 800rpm centrifugation 5min abandon supernatant and are transferred to cell
In 2L rolling bottles, serum free medium is added, it is about 3 × 10 to make cell density5Individual/ml.Continue after cultivating 1~2 week, work as cell
(now cell density is about 1-2 × 10 when the death rate reaches 60%-70%6Individual/ml), collect cell suspension 6000rpm high
Speed centrifugation 20min, takes supernatant, and affinity chromatography carries out supernatant purifying, and corresponding post material, cell line are selected according to antibody subtype
The monoclonal antibody hypotype that UMAB223 is produced is 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：The monoclonal antibody that UMAB223 secretions are produced detects for the SABC of primary antibody
(1), experimental method：
1st, take lung cancer, thymoma and hyperplastic prostate tissue block to carry out FFPE respectively, use SAKURA histotomies
Machine is cut into slices, 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, pH8 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.Go from
Sub- 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-L2 monoclonal antibodies (UMAB223 secretions are produced), thinner ratio is added：1:50, use envelope
Liquid is closed to be diluted.It is placed in wet box, 37 DEG C of incubation 60min.PBST (0.1%Tween-20) is washed 2 times, every time washing
5min.PBST (0.02%Tween-20) is washed 1 time, and 5min is washed every time.
7th, reagent PV8000 (Zhong Shan Golden Bridge, article No. PV-8000) in polymer HRP staining kits, 37 DEG C of incubations is added dropwise
15 minutes.Washed 3 times using PBS, each 5min.
8th, developed the color using DAB solution (Zhong Shan Golden Bridge, article No. 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. 2, Fig. 3 and Fig. 4.
(2), experimental result：
From Fig. 2 results, PD-L2 albumen is in specific cell film and cytoplasm table in the tumour cell of cancerous lung tissue
Reach, as indicated in the figures by an arrow tumour cell.
From Fig. 3 results, PD-L2 albumen is in specific cell film and cell on the tumour cell in thymus gland tumor tissue
Matter is expressed, as indicated in the figures by an arrow tumour cell.
From Fig. 4 results, PD-L2 albumen is in specific cell film expression in hyperplastic prostate tissue glandular epithelium,
Glandular epithelium as indicated in the figures by an arrow.
As a result show monoclonal antibody that UMAB223 of the present invention produces can specific recognition lung cancer, thymus gland tumor tissue and
PD-L2 albumen in hyperplastic prostate tissue.
Embodiment 3：The Flow cytometry for the monoclonal antibody that UMAB223 secretions are produced
(1), experimental method：
1st, cell is prepared：Prepare people HEK293 cell lines (buying from ATCC) to be incubated at containing 5%CO237 DEG C of cell culture
In case.
2nd, transfectional cell：By PD-L2 eukaryon expression plasmid RC224141 (article No. RC224141) transfections purchased from OriGene
After 293T cells, it is incubated at containing 5%CO237 DEG C of cell culture incubators in.
3rd, harvesting：Horizontal centrifuge 800-1000rpm/5min is centrifuged after cell dissociation, and PBS washes cell 2 times,
800rpm is centrifuged；Living cells precipitation is managed with PBS constant volumes to 400ul-EP, prepares cell suspension.
4th, it is incubated antibody：By PD-L2 mouse monoclonal antibodies UMAB223 (working concentration is 10ug/ml) and negative control antibody point
Other above two cell suspension is incubated 1 hour altogether in ice bath.
5th, it is incubated secondary antibody：Above-mentioned cell-antibody mixed liquor is washed into 3 times, 800rpm/5min with PBS；Then donkey is added
Anti- mouse Alexa488 marks secondary antibody (Jackson, Catlog No.715-545-151) 200ul/ holes, ice bath 1 hour.
6th, flow cytometry analysis after washing：Washed with PBS 3 times, 800rpm/5min, it is slow afterwards plus containing PI (1000X)
Fliud flushing 250ul/ holes are suspended, and cell suspension is passed through into flow cytometry analysis.
7th, according to feminine gender, positive, blank control carries out interpretation of result and judgement, sees Fig. 5.
(2), experimental result：
From Fig. 5 results, PD-L2 mouse monoclonal antibodies UMAB223 can be overexpressed PD-L2 293T by Flow cytometry
Blue line represents negative control antibody signal in the PD-L2 albumen (red line) of liver cell surface, figure.
Embodiment 4：The specific proteins chip detection for the monoclonal antibody that UMAB223 secretions are produced
Lysate is overexpressed comprising 10000 HEK293T cell proteins on OriGene high-density protein chips, per hatching egg
White lysate has the repetition of two copies on chip.Protein lysate is by trace on nitrocellulose filter.Each clock egg
The positioning of white lysate can be accurately positioned by Excel file.Albumen is divided into 40 sub- matrixes on protein chip, often
There are some references on individual sub- matrix, by referring to the content of albumen on each chip point can be quantified, monitoring is immune anti-every time
The repeatability of data is answered, and positions the direction of positive signal.It is using OriGene albumen (OriGene Cat below
PA100001) the method that chip carries out the Identification of Monoclonal Antibodies experiment that UMAB223 secretions are produced：
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 UMAB223 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 anti-mouse IgG of donkey 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. 6 is seen.
Fig. 6 results show that UMAB223 can be specifically bound with the PD-L2 protein sites on protein chip, and in protein chip
On other all albumen and UMAB223 secrete the monoclonal antibody produced without binding signal, show monoclonal antibody of the present invention with
Other nearly 10000 kinds of albumen no cross reactions.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (8)
1. anti-PD-L2 protein monoclonal antibodies hybridoma, it is characterised in that deposit number is CGMCC NO：13285.
2. deposit number is CGMCC NO：13285 hybridoma answering in anti-PD-L2 protein monoclonal antibodies are prepared
With.
3. anti-PD-L2 protein monoclonal antibodies, it is characterised in that by deposit number be CGMCC NO：13285 hybridoma
Secretion is produced.
4. deposit number is CGMCC NO：The anti-PD-L2 protein monoclonal antibodies that 13285 hybridoma secretion is produced are in system
Application in the immune detection product of standby detection PD-L2 albumen.
5. apply according to claim 4, it is characterised in that the immune detection product is kit, test paper or chip.
6. a kind of kit of SABC detection, it is characterised in that including deposit number be CGMCC NO：13285 hybridization
The anti-PD-L2 protein monoclonal antibodies that oncocyte secretion is produced.
7. deposit number is CGMCC NO：The anti-PD-L2 protein monoclonal antibodies that 13285 hybridoma secretion is produced are in system
Application in the kit of standby marked tumor tissue and its microenvironment medium size lymphocyte.
8. apply according to claim 7, it is characterised in that the tumour includes lung cancer and thymoma.
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| CN107286240A (en) * | 2017-08-02 | 2017-10-24 | 北京大学 | Anti- CTRP4 monoclonal antibodies and its application |
| CN111902156A (en) * | 2018-03-23 | 2020-11-06 | 得克萨斯州大学系统董事会 | Human PD-L2 antibodies and methods of use thereof |
| CN114075288A (en) * | 2020-08-18 | 2022-02-22 | 正大天晴药业集团股份有限公司 | anti-PD-L2 antibody |
| US12325747B2 (en) | 2017-10-11 | 2025-06-10 | Board Of Regents, The University Of Texas System | Human PD-L1 antibodies and methods of use therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US12325747B2 (en) | 2017-10-11 | 2025-06-10 | Board Of Regents, The University Of Texas System | Human PD-L1 antibodies and methods of use therefor |
| CN111902156A (en) * | 2018-03-23 | 2020-11-06 | 得克萨斯州大学系统董事会 | Human PD-L2 antibodies and methods of use thereof |
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| CN114075288A (en) * | 2020-08-18 | 2022-02-22 | 正大天晴药业集团股份有限公司 | anti-PD-L2 antibody |
| CN114075288B (en) * | 2020-08-18 | 2025-03-28 | 正大天晴药业集团股份有限公司 | Anti-PD-L2 antibodies |
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Application publication date: 20170718 |