CN103804497A - Anti-human Delta like4 monoclonal antibody - Google Patents
Anti-human Delta like4 monoclonal antibody Download PDFInfo
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Abstract
本发明涉及一种抗人Delta like4(hD114)单克隆抗体。本发明提供的单克隆抗体或其片段的特征在于：可以与hD114特异性结合；可以阻断D114对人脐静脉内皮细胞(HUVEC)增殖的抑制；同时，可以在鸡胚尿囊膜模型中促进毛细血管的增殖。本发明具体地公开了抗人Delta like4单克隆抗体的筛选、其制备方法。本发明同时还公开抗人Delta like4单克隆抗体的重链可变区和轻链可变区的核苷酸和氨基酸序列，包括对应于互补决定区CDR1、CDR2和CDR3的核苷酸和氨基酸序列。The invention relates to an anti-human Delta like4 (hD114) monoclonal antibody. The monoclonal antibody or its fragment provided by the present invention is characterized in that: it can specifically bind to hD114; it can block the inhibition of D114 on the proliferation of human umbilical vein endothelial cells (HUVEC); Proliferation of capillaries. The invention specifically discloses the screening and preparation method of anti-human Delta like4 monoclonal antibody. The present invention also discloses the nucleotide and amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-human Delta like4 monoclonal antibody, including the nucleotide and amino acid sequences corresponding to the complementarity determining regions CDR1, CDR2 and CDR3 .
Description
Technical field
The present invention is specifically related to biological technical field, specifically, the present invention relates to a kind of new anti-human Delta like4 monoclonal antibody.
Background technology
From Folkman
[1]since " the tumor-blood-vessel growth dependence theory " of proposition in 1971, anti-tumor neovascularization becomes one of Critical policies of oncotherapy.At present, reach by vegf blocker (vascular endothelial growth factor, VEGF) and principal recipient VEGFR2 thereof the object that suppresses tumor angiogenesis
[2].But clinical study is found: kinds of tumors has antagonistic action to VEGF and VEGFR2 inhibitor, can not effectively suppress the growth of tumour
[3].
Delta like ligand4 (D114) is extensively present in without in vertebra and vertebrates body, by interacting with its acceptor Notch, activate Notch signal and conduct in cell, and regulate the expression of downstream target gene, thereby participate in the vital process such as growth, growth of organism.Research is found, D114/Notch is bringing into play important regulating effect in tumor-blood-vessel growth, suppress D114/Notch signal conduction in tumor tissues, can cause a large amount of non-functional new vesseles of generation in tumor tissues, can not, effectively for tumor tissues provides oxygen and nutritive substance, finally can lead oncogenic growth and be suppressed.And the signal conduction of D114/Notch, can suppress VEGF inhibitor to be controlled the growth of the tumor tissues of antagonism equally in blocking-up tumor tissues
[4,5], therefore, the signal conduction that suppresses D114/Notch is the important channel that suppresses tumor growth.
The present invention utilizes hybridoma technology, take recombinant human Delta like4 (rhD114) as antigen, and immune BALB/c mouse, anti-human Delta like4 (hD114) monoclonal antibody of acquisition high-affinity and biologic activity.
Reference:
1.Folkman?J.Tumor?angiogenesis：therapeutic?implication.J?Invest?Dermato1.1972，59：40-43.
2.Hicklin?DJ.Ellis?LM.Role?ofthe?vascular?endothelial?growth?factor?pathway?in?tumor?growth?and?angiogenesis.J?clin?Onco1.2005.23：1011-1027.
3.Jain?RK，Duda?DG，Clark?JW，et?a1.Lessons?from?phase?III?clinical?trials?on?anti-VEGF?therapy?for?cancer.Nat?Clin?Pract?Onco1.2006，3：24-40.
4.Ridgway?J，Zhang?G，Wu?Y，et?a1.Inhibition?of?D114signalling?inhibits?tumour?growth?by?deregulating?angiogenesis.Nature.2006，444：1083-1087.
5.Noguera-Troise?I，Daly?C，Papadopoulos?NJ，et?aI.Blockade?ofD114inhibits?tumor?growth?by?promoting?non-productive?angiogenesis.Nature.2006，444：1032-1037.
Summary of the invention
The invention provides a kind of anti-hD114 monoclonal antibody with potential medical science and pharmacy value, and related protein and nucleotide sequence.To achieve these goals, the invention provides following technical scheme.
The invention provides a kind of anti-hD114 monoclonal antibody, it comprises variable region of heavy chain and variable region of light chain, it is characterized in that, variable region of heavy chain has SEQ ID NO:2,18,34,50, aminoacid sequence shown in 66 and 82, variable region of light chain has SEQ ID NO:10,26,42, aminoacid sequence shown in 58,74 and 90.
The present invention provides a kind of DNA molecular simultaneously, this DNA molecular contains SEQ IDNO:1,17,33,49, the nucleotide sequence of the described monoclonal antibody of the coding shown in 65 and 81 variable region of heavy chain, and SEQ IDNO:9,25,41, the nucleotide sequence of the described monoclonal antibody of the coding shown in 57,73 and 89 variable region of light chain.
The invention provides heavy chain complementary determining region CDR1, the CDR2 of anti-hD114 monoclonal antibody and amino acid and the nucleotide sequence of CDR3 and variable region of light chain CDR1, CDR2 and CDR3.
The invention provides the preparation method of preparation said monoclonal antibody.
Anti-hD114 monoclonal antibody provided by the present invention all can be brought into play its biological function in vivo and in vitro effectively.
Accompanying drawing explanation
Fig. 1. anti-hD114 monoclonal antibody gene nucleic acid electrophoresis figure, A: heavy chain gene; B: light chain gene;
Fig. 2. anti-hD114 monoclonal antibody titer of ascites, P/N is that rhD114 group light absorption value/Control organizes light absorption value, in the time of P/N > 2, ELISA result is positive;
Fig. 3. the purified rear SDS-PAGE figure of anti-hD114 monoclonal antibody;
Fig. 4 .SPR measures anti-hD114 monoclonal antibody avidity matched curve figure;
Fig. 5 .Western Bloting identifies the specific binding of anti-hD114 monoclonal antibody and rhD114;
Fig. 6. the inhibition of anti-hD114 monoclonal antibody blocking-up Delta like4 Human umbilical vein endothelial cells (HUVEC) propagation;
Fig. 7. anti-hD114 monoclonal antibody promotes chick chorioallantoic membrane blood vessel hyper-proliferative.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.But should be appreciated that enumerating these embodiment is in order to play an illustration, and be not for limiting the present invention.
The preparation of embodiment 1, anti-hD114 monoclonal antibody:
(1) immune mouse
Take recombinant human Delta like4 (rhD114) as immunogen, mix with quick antibody adjuvant equal-volume, concussion mixes, intramuscular injection immune mouse, every injected in mice 100 μ l.Within the 21st day, press the same manner booster immunization one pin.Within the 35th day, carry out according to a conventional method antigen and impact immunity.After 3 days, put to death mouse, take out spleen.
(2) cytogamy
To the spleen taking out, prepare spleen single cell suspension by ordinary method, and by trypan blue method detection cytoactive, determine cytoactive > 90%.Spleen cell is mixed to centrifugal co-precipitation with SP2/0 murine myeloma cell 5:1, and under 37 ℃ of C water bath condition, PEG (1450) carries out cytogamy.Time of fusion adds fresh serum free medium to stop merging after 3 minutes.After centrifugation, cell is joined and contain in 20%FCS1 × HATDMEM substratum 96 orifice plates.Within every 3 days, change fresh culture 1 time, to growing clone.
(3) colony screening
Within the 14th day, take out supernatant liquor, by indirect elisa method mensuration antibody expression situation.Select positive hole subclone, under normal condition, go down to posterity, and subclone is carried out in positive hole, until institute's selected clone is for all positive.
The amplification of embodiment 2, anti-hD114 variable region of mab gene
According to the conservative property of mouse antibody genes, adopt following primer to clone the variable region sequences of anti-hD114 monoclonal antibody:
Amplification variable region of heavy chain 5 ' end primer:
1.ctt?ccg?gaattc?SAR?GTN?MAG?CTG?SAG?SAG?TC
2.ctt?ccg?gaattc?SAR?GTN?MAG?CTG?SAG?SAG?TCW?GG
Amplification variable region of heavy chain 3 ' end primer:
gga?agatct?CTT?GAC?CAG?GCA?TCC?TAGAGT?CA
Amplification Kappa variable region of light chain 5 ' end primer:
gg?gag?ctc?GAYATT?GTG?MTS?ACM?CAR?WCT?MCA
Amplification Kappa variable region of light chain 3 ' end primer:
ggtgcatgc?GGATACAGTTGG?TGCAGCATC
In above-mentioned primer: R=A, G; Y=C, T; M=A, C; K=G, T; S=C, G; W=A, T; V=A, C, G; N=A, C, G.
Adopt Trizol total RNA extraction agent box, the total R N of extracting and purifying A from the hybridoma cell strain obtaining, whole operation steps is undertaken by producer's book that furnishes an explanation.
In 1% agarose electrophoresis, the RNA of above-mentioned purifying is carried out after quality evalution, carry out reverse transcription according to reverse transcription test kit specification sheets and obtain cDNA.
Enter respectively performing PCR amplification with above-mentioned primer and c D N A and obtain V L and V H fragment.PCR reaction conditions is as follows:
Reaction system:
Reaction conditions: 94 ℃ of denaturation 5min, loop parameter is 94 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, complete after 35 circulations 72 ℃ of 10min.
Complete after PCR reaction, in 1% agarose electrophoresis, increased product is identified, find that VH and VL reaction system only have a band within the scope of molecular weight approximately 300 to 500bp, see accompanying drawing 1.Use glue to reclaim test kit and reclaim object band, and insert in pMD19-T carrier, and be converted into bacillus coli DH 5 alpha, be then applied to contain on the antibiotic agar plate of ammonia benzyl and screen.Select 10 positive colonies and carry out PCR evaluation, and deliver to the order-checking of order-checking company.Finally use IMGT database to analyze the antibody sequence obtaining.
The preparation of embodiment 3, anti-hD114 monoclonal antibody
500 μ l paraffin oil sensitization BALB/c mouse in 8 week age are after 7 days, abdominal injection 10
6individual logarithmic phase hybridoma is to mouse peritoneal.After one week, extract mouse ascites.And measure titer of ascites with indirect elisa method, it is tired and reaches 3,000,000 left and right, sees accompanying drawing 2.
The separation and purification of embodiment 4, anti-hD114 monoclonal antibody
4 ℃ of centrifugal 20min of 5000g, remove ascites inner cell and other sedimentable matter, then use 0.22 μ m membrane filtration.Carry out purifying with reference to Hi-Trap ProteinA post specification sheets.After purifying, antibody concentration reaches 1.98mg/ml, and uses sex change electrophoresis to identify, sees accompanying drawing 3.
The avidity of embodiment 5, anti-hD114 monoclonal antibody is measured
With reference to specification sheets, rhD114 is caught to NTA sensing chip, concrete grammar is as follows: adopt the PBS/0.05%o P20 buffer salt solution that filters also degasification as moving phase solution, by NTA sensor chip Module-embedding BIAcore system; In flow chamber, inject Ni2
+activation chip, then contains His label protein toward interior injection of flow chamber, makes it to be caught on chip, then inject ligandin, the cohesive process between monitoring objective albumen and part in flow cell.Finally use EDTA wash-out regeneration chip.Detailed process is as following table 1:
Finally measured result is carried out to matching, the avidity of extrapolating anti-hD114 monoclonal antibody is 3pM, the results are shown in shown in accompanying drawing 4.
The biological activity determination of embodiment 6, anti-hD114 monoclonal antibody
(1) immunoblot experiment
Immunoblot experiment shows, above-mentioned anti-hD114 monoclonal antibody can with hD114 specific binding, the results are shown in accompanying drawing 5.
(2) inhibition of anti-hDIi4 monoclonal antibody blocking-up rhD114 to HUVEC cell proliferation
In 96 orifice plates, add 100 μ l to be diluted to the rhD114 of 1 μ g/mi with carbonate buffer solution, 4 ℃ of C are coated with and spend the night.Next day, PBS washes twice, in every hole, adds 4000 Human umbilical vein endothelial cells, and the antibody of different concns, 3 multiple holes of each concentration; Prepare not 96 orifice plates of coated rhD114 simultaneously, in every hole, add 4000 Human umbilical vein endothelial cells, and the antibody of different concns, 3 multiple holes of each concentration.With 37 ℃, 5%CO
2middle cultivation 72h, mtt assay detects the propagation of cell, the results are shown in accompanying drawing 6.
(3) anti-hDI14 monoclonal antibody promotes chick chorioallantoic membrane capillary vessel hyper-proliferative
Fertilized eggs is placed in 37 ℃, and humidity is about in 55% incubator cultivates 6 days.Then use medical curved nickel to window, the area of window is about 1cm
2left and right, overnight incubation in incubator.Within second day, add different medicine carrying filter paper: D114 (1 μ g/ml, 100 μ l), anti-human Deltalike4 antibody (100ng/ml, 100 μ l), the position of tablet take dereliction blood vessel as good, physiological saline compares; Each drug level all does 10 eggs.Chicken embryo after dosing is hatched 48 hours in 37 ℃.Then, add the about 1.5ml of stationary liquid (methyl alcohol: acetone=1:1), and static 15min.Finally, cut chorioallantoic membrane with medical curved scissors, after cleaning with physiological saline, be laid on slide glass and take pictures.The results are shown in accompanying drawing 7.
Claims (18)
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| CN105384819A (en) * | 2015-12-17 | 2016-03-09 | 中国药科大学 | Anti-human Delta-like4 humanized antibody and preparation and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101490084A (en) * | 2006-06-06 | 2009-07-22 | 健泰科生物技术公司 | anti-DLL 4 antibodies and methods of use thereof |
| CN101611055A (en) * | 2006-12-14 | 2009-12-23 | 里珍纳龙药品有限公司 | People's antibody of anti-people DELTA sample part 4 |
| CN102264763A (en) * | 2008-09-19 | 2011-11-30 | 米迪缪尼有限公司 | Antibodies directed to dll4 and uses thereof |
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| CN101490084A (en) * | 2006-06-06 | 2009-07-22 | 健泰科生物技术公司 | anti-DLL 4 antibodies and methods of use thereof |
| CN101611055A (en) * | 2006-12-14 | 2009-12-23 | 里珍纳龙药品有限公司 | People's antibody of anti-people DELTA sample part 4 |
| CN102264763A (en) * | 2008-09-19 | 2011-11-30 | 米迪缪尼有限公司 | Antibodies directed to dll4 and uses thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105384819A (en) * | 2015-12-17 | 2016-03-09 | 中国药科大学 | Anti-human Delta-like4 humanized antibody and preparation and application thereof |
| CN105384819B (en) * | 2015-12-17 | 2018-09-21 | 中国药科大学 | A kind of 4 humanized antibodies of anti-human Delta-like and its preparation and application |
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