CN103694352A - CD26 antibody and preparation method thereof - Google Patents
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Abstract
The invention provides a new full-humanized antibody which can be specifically combined with CD26 and has high affinity, a preparation method and application thereof, belonging to the technical field of antibodies in genetic engineering. CD26 is a ubiquitous multifunctional type-II transmembrane glycoprotein which has multiple biological functions and can interact with multiple proteins such as ADA, CD45 and FAP-alpha. The humanized antibody or a fragment thereof provided by the invention is specifically combined with human CD26 and preferably specifically combined with the extracellular region of CD26; and the amino acid sequence of the antibody or fragment thereof comprises a monoclonal antibody or fragment thereof or a conjugate of the fragment thereof selected from any area of six supplementary determination regions comprising a group of sequences SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, or comprises an amino acid sequence obtained through amino acid sequence or modification. The CD26 single-chain antibody provided by the invention is combined with CD26 with high specificity, and also can obviously inhibit the proliferation and invasion and metastasis of tumor cells.
Description
Technical field
The invention belongs to genetic engineering antibody technical field, especially relate to a kind of new can with high-affinity human antibody, preparation method and the application thereof of CD26 specific binding.
Background technology
CD26 is a kind of ubiquitous multi-functional II type transmembrane glycoprotein, has various biological function, also can be present in blood plasma with solubilized form.CD26 often exists with homodimer form, and monomer whose is containing 766 amino acid, the about 110kDa of relative molecular mass.Amino-acid residue is from inside to outside divided into 5 parts: intracellular region (1～6), cross-film district (7～28), high glycosylation district (29～323), be rich in halfcystine district (324～551) and C end catalyst structure domain (552～766), CD26 molecule three-dimensional structure and function are closely related.C end catalyst structure domain performance DPP IV (the Dipeptidyl peptidase4 of CD26, DPPIV) activity, can be hydrolyzed multiple substrate performance biological action in body, being rich in halfcystine district can interact with different kinds of molecules in body, thereby participates in immunologic function in body.The effect of CD26 in immunomodulatory is widely studied, CD26 is the molecular marker of T cell activation, also in T cell signalling process as costimulatory molecules, also relate to multiple T cell function, comprise that ripe and migration occurs T cell, cytokine secretion, the antibody that T cell relies on produces, B cell immunoglobulin transition etc.CD26 plays a significant role in the generation evolution of autoimmune disorder, has become the molecular marker of clinical disease, and is considered to treatment or the diagnosis target spot of some immunological disease.（Ohnuma?et?al.(2011)Adv?Clin?Chem,53,51-84）
CD26, owing to interacting with multiple protein, as ADA, CD45, FAP-alpha etc., can also cause expressing increase or the reduction of CD26 cellular infiltration activity in conjunction with ECM, and visible CD26 plays a significant role at oncobiology.The expression amount of CD26 can raise in multiple newborn oncocyte surface or serum, and for example, CD26 high expression level is in some offensive T cell malignancies, malignant mesothe, nephroncus, some colorectal carcinoma (Havre et al. (2008) Front Biosci, 13,1634-1645).Some CD26+ colon cancer cell subgroup, CD26+ malignant mesothe cell has obvious tumor stem cell feature (Ghani et al. (2011) Biochem Biophys Res Commun, 404,735-742and Pang et al. (2010) Cell Stem Cell, 6,603-615), thus CD26 can be used as the molecular marker of kinds of tumors.
The mouse source antibody report of existing multiple combination CD26 (Havre et al. (2008) Front Biosci, 13,1634-1645), in some CD26 high expression level cancer for the treatment of, and suppress cell migration, vascularization aspect can play a role.There are the mouse monoclonal antibody 1F7 of the anti-CD26 of bibliographical information and the combination of CD26 to cause cell cycle arrest to limit a little at G1/S, and the combination of CD26 induces CD26Jurkat transfectant to be stuck in G1 by strengthening express cell cycle regulating protein matter p21, with 1F7 Antybody therapy, can suppress the formation of CD26+ tumour, and in mouse model, strengthen survival rate.Mouse monoclonal antibody E19 and the E26 of other anti-CD26, these antibody show restraining effect that the cell migration that is suppressed to fibrocyte and Traumatic cell forms individual layer, to angiopoietic inhibition and to the intrusion of human skin capillary endothelium and kapillary, formation newly has inhibition.As can be seen here, anti-CD26 monoclonal antibody can play a role by changing the activity of CD26 in treatment various diseases.But mouse monoclonal antibody can produce human antimouse antibody reaction (Human Anti-Mouse Antibody, HAMA) when directly applying to human body therapy, people knows from experience the antibody that produces anti-mouse antibodies, not only can weaken curative effect, also can cause acute sensitivity response.In order to overcome the shortcoming of mouse source monoclonal antibody, genetic engineering antibody technical development has gone out people-mouse chimeric antibody, chimeric antibody is mosaic gene by the V district gene of mouse antibody and the C district gene splicing of people's antibody, make mouse composition reduce by 70% left and right, and humanized antibody, on the basis of chimeric antibody, further the skeleton district (FR) of employment antibody variable region substitutes mouse FR, has greatly reduced the mouse derived components of antibody.But the sequence of residual a small amount of murine antibody is potential initiation HAMA likely still.
Summary of the invention
Technical problem to be solved by this invention: the present invention aim to provide a kind of energy effectively, the human antibody of specific binding people CD26, and the expression with CD26 in preparation treatment, the disease that particularly overexpression is feature, the new purposes in the medicine of diagnosis CD26 variability expression disease.More particularly:
First object of the present invention is to provide antibody or its fragment in a kind of people of deriving from source, described antibody or its fragments specific are in conjunction with people CD26, preferred specific binding CD26 extracellular region, the aminoacid sequence of described antibody or its fragment comprises the monoclonal antibody in any region or the conjugate of its fragment or its fragment that is selected from 6 complementary determining regions containing SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, mono-group of sequence of SEQ ID NO:8, or the aminoacid sequence obtaining by amino-acid substitution or modification.
Preferably the aminoacid sequence of the antibody in the present invention or its fragment is containing the complementary determining region of the variable region of heavy chain just like sequence shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
Preferably the aminoacid sequence of the antibody in the present invention or its fragment is containing the complementary determining region of the variable region of light chain just like sequence shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
The more preferably antibody in the present invention or its fragment, the aminoacid sequence of its variable region of heavy chain contains following complementary determining region: the CDRH1 as shown in sequence SEQ ID NO:2, CDRH2 as shown in sequence SEQ ID NO:3, the CDRH3 as shown in sequence SEQ ID NO:4;
With and the aminoacid sequence of variable region of light chain contain following complementary determining region: the CDRL1 as shown in sequence SEQ ID NO:6, the CDRL2 as shown in sequence SEQ ID NO:7 and the CDRL3 as shown in sequence SEQ ID NO:8.
More preferably the antibody in the present invention or its fragment contain variable region of heavy chain sequence and variable region of light chain sequence as shown in SEQ ID NO:5 as shown in SEQ ID NO:1.
Second object of the present invention is to provide the single-chain antibody in a kind of people of deriving from source, and the aminoacid sequence of described single-chain antibody is as shown in SEQ ID NO:11.
The 3rd object of the present invention is to provide a kind of nucleotide sequence of the above-mentioned single-chain antibody of encoding, and described nucleotide sequence is as shown in SEQ ID NO:10.
The 4th object of the present invention is to provide a kind of expression vector that contains above-mentioned nucleotide sequence.
The 5th object of the present invention is to provide a kind of recombinant host bacterium that contains above-mentioned expression vector.
The 6th object of the present invention is to provide a kind of method of producing above-mentioned single-chain antibody, comprising:
1) under suitable condition, cultivate above-mentioned recombinant host bacterium and express antibody;
2) then from Host Strains purifying, collect antibody.
The 7th object of the present invention is to provide above-mentioned antibody or the new purposes of its fragment in the medicine of preparation treatment CD26 high expression level tumour.Can treat the disease that the expression with CD26, particularly overexpression are feature, and for diagnosis CD26 variability expression disease provides the method based on antibody, relative disease includes but not limited to autoimmune disease and cancer.
Invention further illustrates:
717 Nucleotide of the anti-CD26 single-chain antibody gene in total man source sequence total length in the present invention, expection has 239 amino acid.There are 115 amino acid whose variable region of heavy chain (SEQ ID NO:1) and 109 amino acid whose variable region of light chain (SEQ ID NO:5), between variable region of heavy chain and variable region of light chain, by 15 amino acid whose flexible peptides, connect (SEQ ID NO:9).
The expression vector that contains CD26 single-chain antibody gene of the present invention and Host Strains all belong to protection scope of the present invention.The primer pair of any fragment of amplification single-chain antibody gene of the present invention is also within protection scope of the present invention.
Beneficial effect of the present invention has:
Antibody in the present invention or its fragment have multifrequency nature, comprise in combination also and the ability of CD26.Particularly, the anti-CD26 single-chain antibody that the present invention obtains has high specific with CD26 and is combined, the external cell adhesion can inhibition tumor cell mediating by CD26, extracorporeal suppression tumor cell growth experiment result shows, the single-chain antibody that the present invention obtains is propagation and the Invasion and Metastasis of inhibition tumor cell obviously.
Accompanying drawing explanation
Fig. 1 is the structural representation of single-chain antibody ZHB-pA12.VH represents variable region of heavy chain structural domain (SEQ ID NO:1), and VL represents variable region of light chain structural domain (SEQ ID NO:5), and Linker is for connecting VH, the flexible peptide of VL (SEQ ID NO:9).
Fig. 2 is the evaluation figure of CD26 extracellular region protein.A is the SDS-PAGE figure of albumen after purifying, and Lane1 is standard protein, and Lane2 is CD26 extracellular region protein after purifying (arrow indication); B is the Western Blot evaluation figure of CD26 extracellular region protein after purifying, Lane1 is standard protein, Lane2 is CD26 extracellular region protein after purifying (arrow indication), and primary antibodie is anti-CD26 mouse-anti (purchased from MBL), and two resist for the anti-mouse-HRP of rabbit antibody (purchased from life technology).
Fig. 3 A, Fig. 3 B are the ELISA measurement results that phage antibody is combined with CD26 extracellular region protein.Fig. 3 A is polyclone Phage-ELISA, and the coated concentration of CD26 extracellular region protein is 1 μ g/mL, and the phage of screening amplification is taken turns in dilution 4, hatch with the enzyme plate of coated CD26 extracellular region protein, anti-M13-HRP antibody is hatched again, measures 450nm, 650nm light absorption value, and with OD
450nm-OD
650nmas end value.Result shows, shows and has the phage of CD26 specific single-chain antibody to obtain obvious enrichment.Fig. 3 B is mono-clonal Phage-ELISA measurement result, selects in mono-clonal to 96 orifice plate and expresses phage antibody, hatches with the enzyme plate of coated CD26 extracellular region protein, and anti-M13-HRP antibody test result is measured OD
450nm-OD
650nmas end value, result shows that more than 90% mono-clonal and CD26 extracellular region protein produces positive keying action.
Fig. 4 is the evaluation figure of single-chain antibody ZHB-pA12.A is the SDS-PAGE figure of the ZHB-pA12 after ni-sepharose purification, and Lane2 is ZHB-pA12 sample protein, and albumen arrow indication is target protein size strip; Lane1 is standard protein; B is the Western Blot evaluation figure of ZHB-pA12 after purifying, and primary antibodie is anti-myc mouse-anti, and two resist the antibody for the anti-mouse-HRP of rabbit, and Lane1 is ZHB-pA12 sample, and albumen arrow indication is target protein size strip, and Lane2 is standard protein.
Fig. 5 shows the Western Blot evaluation figure that single-chain antibody ZHB-pA12 is combined with CD26 extracellular region protein.Primary antibodie is anti-myc mouse-anti, and two resist the antibody for the anti-mouse-HRP of rabbit.Lane1 is standard protein, and Lane2 is ZHB-pA12 and CD26 extracellular region protein keying action, arrow indication shown with Fig. 2 in CD26 extracellular region protein band of the same size.Result shows that anti-CD26 single-chain antibody ZHB-pA12 can specificity be combined with CD26 extracellular region protein.
Fig. 6 shows the immunofluorescence figure that single-chain antibody is combined with people's mesothelioma cell NCI-H2452.A is the immunofluorescence of ZHB-pA12 and NCI-H2452 cell, and B is that negative control antibody is combined the negative findings detecting with NCI-H2452 cellular immunofluorescence, and antibody concentration is diluted to 10 μ g/mL by 5%MPBS.
Fig. 7 shows that single-chain antibody suppresses people's mesothelioma cell NCI-H2452 in conjunction with the detected result of ECM.Testing ECM used is fibronectin, and experiment is divided into Binding group, Blank group, antibody test group, the adhesion situation of the cell after investigation single-chain antibody effect 12h to fibronectin.
OD450(Binding group)-OD450(Blank group)=complete adherent cell value;
OD450(antibody test group)-OD450(Blank group) the cell adhesion value of=sample sets;
The negative control antibodies of IgG control;
The cell adhesion value of cell adhesion rate (%)=sample sets/complete adherent cell value
Result shows, compares with negative control IgG control, and ZHB-pA12 and positive control scFv-YS110 all have obvious restraining effect to NCI-H2452 cell adhesion.
Fig. 8 shows the detected result of single-chain antibody to people's mesothelioma NCI-H2452 cell inhibitory effect.Cell is with 1 * 10
4the quantity in/hole is incubated in 96 orifice plates, at single-chain antibody ZHB-pA12, scFv-YS110 and negative control antibody IgG(mouse) act on after 48h, adopt CCK-8 reagent react 30min, the absorbance of measuring 450nm, the proliferation inhibition rate of cell represents with the decrement % of OD450nm.Experimental result shows that ZHB-pA12 and positive control scFv-YS110 all have obvious restraining effect to NCI-H2452, and is concentration dependent.
Fig. 9 shows the detected result of single-chain antibody to human colon carcinoma HCT116 cell inhibitory effect.Cell is with 8 * 10
4the quantity in/hole is incubated in 96 orifice plates, at single-chain antibody ZHB-pA12, scFv-YS110 and negative control antibody IgG(mouse) act on after 48h, adopt CCK-8 reagent react 30min, the absorbance of measuring 450nm, the proliferation inhibition rate of cell represents with the decrement % of OD450nm.Experimental result shows that ZHB-pA12 and positive control scFv-YS110 all have obvious restraining effect to NCI-H2452, and is concentration dependent.
Embodiment
The present invention is described now with the following Examples.The only object for illustrating of these embodiment is provided, the invention is not restricted to these embodiment, but comprise the institute obviously being produced by instruction provided herein, change.Be used for carrier construction and plasmid, plasmid is imported to host cell and gene, and the detailed description of the ordinary methods such as expression identification of gene product can obtain from various publications, < < molecular cloning experiment guide third edition > > for example.The per-cent relating in embodiment, wherein solid reagent is weight percentage, and liquid reagent is volume percent.
Part material source is illustrated in this:
Clone: HLF cell is people's undifferentiated liver cancer cell, available from JCRB cell bank (Japanese Collection of Research Bioresources Cell Bank).NCI-H2452, is people's mesothelioma cell, available from cell bank/Shanghai Inst. of Life Science, CAS cell resource center of typical case's culture collection council of the Chinese Academy of Sciences.HCT116, is human colon cancer cell, available from cell bank/Shanghai Inst. of Life Science, CAS cell resource center of typical case's culture collection council of the Chinese Academy of Sciences.
(GIBCO, Cat#31800022 add NaHCO to the reagent such as substratum and damping fluid: RPMI-1640
31.5g/L, glucose2.5g/L, Sodium Pyruvate0.11g/L), 90%; High-quality foetal calf serum, (GIBCO) 10%.
2YT substratum: 1L includes Trptone (OXID) 16g, Yeast Extract (OXID) 10g, NaCl5g.
2YT-AK substratum: 2YT is containing 100 μ g/mL penbritins and 50 μ g/mL kantlex.
2YT-AG substratum: 2YT is containing 100 μ g/mL penbritins and 2% glucose.
10 * PBS:(is purchased from Beijing Suo Laibao, Cat#P1022).
5%MPBS or 2%MPBS: the PBS. that contains 5% or 2% skim-milk (OXID)
0.1%PBST: contain 0.1%Tween20(purchased from Beijing Suo Laibao) PBS.
2%BSA: contain 2%BSA(purchased from MP Biomedicals) PBS
Amp: penbritin (purchased from the raw work in Shanghai).
Kan: kantlex (purchased from the raw work in Shanghai).
IPTG:(is purchased from Amresco).
Other common reagent example hydrochloric acids, NaCl, Tris, glycine etc. are purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
Bacterial strain: TG1, intestinal bacteria (available from Chinese microorganism strain net); HB2151, intestinal bacteria (available from Chinese microorganism strain net)
Plasmid: p3XFLAG-CMV9(is purchased from Sigma-Aldrich); PHEN2 (available from Medical Research Council (UK))
The preparation of embodiment 1CD26 extracellular region protein
The object of this research is by Transfected Recombinant Plasmid eukaryotic cell, G418 resistance screening acquisition stable cell strain, and secreting, expressing CD26 extracellular region protein, goes out CD26 extracellular region protein through affinity chromatography separation and purification.
CD26 extracellular region gene is the synthetic gene that is selected from region, amino acid 29-766 position " Extracellular " in Uniprot:P27487 sequence, CD26 extracellular region gene is connected into plasmid p3XFLAG-CMV9, restriction enzyme site is Hind III, Xba I, construction recombination plasmid (J. Pehanorm Brooker. molecular cloning experiment guide. the third edition. the .2002.P68 of Science Press).
Adopt Lipofectamine LTX Reagent(purchased from Invitrogen) also method proceeds to HLF cell by the recombinant plasmid containing CD26 extracellular region gene to specifications.According to stably transfected cell line construction process (Current Protocols in Molecular Biology, P9.5.5) obtain CD26 extracellular region stable expression cell strain, be designated as HLF-4D9, serum-free is cultivated in a large number, secreting, expressing CD26 extracellular region protein, this albumen is with Flag label, adopt ANTI-FLAG M2Affinity Gel(purchased from Sigma-Aldrich), serum-free culture supernatant is carried out to purifying and obtain CD26 extracellular region protein sterling, SDS-PAGE(Fig. 2 A) analyze and Western Blot (agriculture of J.S. Boneface. fine works Cell Biology Experiment guide. the .2007.P177 of Science Press) identify (Fig. 2 B), primary antibodie is anti-CD26 mouse-anti (purchased from MBL), two resist for the anti-mouse-HRP of rabbit antibody (purchased from life technology).
Result as shown in Figure 2, Fig. 2 A is the evaluation figure of SDS-PAGE to purifying protein, through a step affinity chromatography, obtain the wall scroll band (Lane2) of the pure level of electrophoresis target protein size, Fig. 2 B carries out Western Blot evaluation figure to the albumen of purifying, band shown in Lane2 and SDS-PAGE qualification result albumen are in the same size, show CD26 extracellular region gene through clone's restructuring transfecting eukaryotic cells and express, after purifying, obtaining the pure level of electrophoresis CD26 extracellular region protein.
The separation of the embodiment 2 anti-CD26 single-chain antibodies in total man source
This single-chain antibody, from people's single-chain antibody phage display library, adopts the affine screening of solid phase of CD26 extracellular region protein to obtain, and this people's single-chain antibody phage display library is built by the Jiangsu red biotechnology Chuan Yao of crowd research institute company limited.The heavy chain of antibody and the phage display library of variable region of light chain that this contains the generation of people's cell build from human peripheral lymphocyte, by using the special primer of antibody variable gene to carry out first run amplification, two take turns amplification adopts heavy chain variable region of light chain to flexibly connect peptide gene and be connected to form single-chain antibody gene, and ScFv gene cloning is entered in plasmid pHEN2 to transform e. coli tg1, obtain 10
8p.f.u. phage single-chain antibody is shown storehouse.(doubly put forth energy in Shen. recombinant antibodies. and the .2005.P107 of Science Press)
This single-chain antibody library Primary spawn, to logarithm growth stage, is infected with M13K07 helper phage, in 2YT-AK substratum, 30 ℃ of shaking table overnight incubation.4%PEG/2.5M NaCl precipitation for phage, and be resuspended in PBS and measure antibody library titre, obtaining titre is 10
11the phage antibody library of p.f.u.(Zhen Yongsu. antibody engineering medicine. the .2002.P51 of Chemical Industry Press) with PBS dilution CD26 extracellular region protein to 50 μ g/mL; Be coated with to enzyme plate (Maxi-sorp96, Nunc), blank hole (not containing CD26 extracellular region protein) is set simultaneously, seal subsequently.Phage antibody library is suspended in to 2%MPBS, gets 100 μ L and join in the blank well of sealing, room temperature is placed after 60min, joins in the hole of containing CD26 extracellular region protein, and room temperature is placed 2h; 0.1%PBST and PBS wash respectively 10 times, add 100 μ L0.1M hydrochloric acid (being adjusted to pH2.2 with glycine), room temperature vibration 10min, 15 μ L1M Tris(pH9.0) for neutralizing rapidly the phage eluting; The e. coli tg1 of the phage-infect 5mL logarithmic phase after neutralization, get 100uL, do 100 times of serial dilutions 1～3 time, then, by serial dilution thing paving TYE solid medium (containing 100 μ g/mL Amp and 1% glucose), remaining bacterium liquid adds 20mL to contain 2 * 10 again
10the 2YT-AG of M13K07 helper phage increases and prepares phage library, for next round screening process, carries out altogether 4 and takes turns screening.
The antibody fragment that is illustrated in phage particle surface is called as phage antibody, this experiment first identifies that by polyclone Phage-ELISA 4 take turns the enrichment condition of CD26 specific phage antibody after screening, after by mono-clonal Phage-ELISA, further identify the phage antibody of picking out high-affinity.
The evaluation of polyclone Phage-ELISA, envelope antigen i.e. 1 μ g/mL CD26 extracellular region protein, to enzyme plate, after sealing, is got the every phage of taking turns the rear acquisition of screening of 10 μ L, and 2%MPBS joins in antigen coated enzyme bar after diluting.Incubated at room 90min, adds mouse-anti M13 phage-HRP antibody (purchased from Yi Qiao Divine Land, Beijing biotech company) incubated at room 1h after washing, after washing, add 100 μ L TMB nitrite ions (purchased from AMRESCO).After incubated at room 10min, 1M dilute sulphuric acid termination reaction.Measure OD
450and OD
650, and with OD
450-OD
650as last detected result.As shown in Figure 3A, the coated enzyme plate of the BSA of usining is as negative control, and along with screening wheel number increases, the combination enhancing of phage antibody and CD26 extracellular region protein, shows to have obtained obvious enrichment with the phage of CD26 specific antibody.
Mono-clonal Phage-ELISA is identified, on the titer determination flat board of random choose third round fourth round screening process, picking mono-clonal is in 96 hole microbial culture plates (purchased from Corning), 2YT substratum (containing 100 μ g/mL Amp and 1% glucose) has been added in every hole, 37 ℃ are cultured to logarithmic phase, and every hole adds 10
937 ℃ of standing infection 30min of p.f.u.M13K07 helper phage, cultivate 1h for 37 ℃.The centrifugal 10min of 1800g, abandons supernatant.Bacterial sediment is resuspended in to 200 μ L2YT-AK substratum, 30 ℃ of shaking table overnight incubation.The culture supernatant containing phage that the centrifugal 10min of 1800g next day obtains, 20%MPBS incubated at room 1h with 1/10 volume, join in the enzyme plate that is coated with recombinant C D26 extracellular region protein, ELISA identifies (method and reagent are identified with polyclone Phage-ELISA) again.Measure OD
450and OD
650, and with OD
450-OD
650as last detected result.Select the clone that reading is high and carry out determined dna sequence.Fig. 3 B has shown the detected result of part mono-clonal phage E LSIA, and more than 90% mono-clonal shows positive keying action, further shows to take turns screening by 4, with the phage of CD26 specific antibody, has obtained obvious enrichment.Therefrom select 50 mono-clonal order-checkings that reading is high, obtain the single-chain antibody (be designated as ZHB-pA12) of nucleotide coding sequence as shown in SEQ ID NO:10.The aminoacid sequence of corresponding single-chain antibody ZHB-pA12 is SEQ ID NO:11.
Solubility expression and the separation and purification of the anti-CD26 single-chain antibody of embodiment 3
PHEN2 is a difunctional phagemid vector, has an amber type terminator codon (Amber) TAG between detection of expression label (c-my tag) and coat protein gene.If phage-infect amber mutation (SupE) inhibition type bacterial strain, as TG1, TAG codon is translated into L-glutamic acid, and sequence can be readed over translation, and antibody fragment and coat protein p3 amalgamation and expression are in phage surface; When the non-amber mutation inhibition of phage-infect type bacterial strain, as HB2151, translation stops at TAG place, can obtain the antibody fragment that solvable type is expressed.Antibody fragment C end, with 6 * His tag and c-myc tag, is beneficial to purifying and detects and identify.ZHB-pA12 single-chain antibody adopts the solubility expression in colibacillus periplasm space, and purifying adopts high osmose process to extract cell periplasm protein, and the one step separation and purification of recycling affinity chromatography obtains the higher target protein of purity.
The little extraction reagent kit of employing plasmid extracts the plasmid (being designated as pA12-pHEN2) that contains coding single-chain antibody ZHB-pA12 gene from thalline TG1, be used for transforming the sub-intestinal bacteria HB2151 of non-inhibition, transform and adopt Calcium Chloride Method preparation and transformed competence colibacillus intestinal bacteria HB2151(J. Pehanorm Brooker. molecular cloning experiment guide. the third edition. the .2002.P96 of Science Press), after gained transforms, bacterial strain is designated as HB2151-pA12.
HB2151-pA12 in 2YT-AG substratum, 37 ℃ of (OD while being cultured to logarithmic phase
600=0.8), add the IPTG of final concentration 1mM, 30 ℃ of inductions are spent the night (16～20h), express soluble single-chain antibody ZHB-pA12.6000rpm, 4 ℃ of centrifugal 15min, collect thalline, hypertonic solution (50mM Tris-HCl, 20% sucrose, 1mM EDTA, pH8.0) resuspended thalline, slowly stir 1h, 4 ℃, the centrifugal 10min of 10000g, pour out supernatant and carry out affinity chromatography (purchased from GE), purification step carries out according to the Standard Operating Procedure of GE, adopt the level pad (Tris50mM that contains 5mM imidazoles, NaCl500mM, pH7.5) balance 1mL nickel post, loading after 10 column volumes, again adopt the foreign protein of non-specific binding on the level pad washing nickel post that contains 5mM imidazoles, with the level pad that contains 50mM imidazoles, wash non-specific foreign protein, finally with the level pad wash-out target protein that contains 100mM imidazoles.15%SDS-PAGE detects the sample of collecting, and Western Blot further identifies single-chain antibody, and primary antibodie is anti-c-myc mouse-anti (brilliant biological purchased from grace), and two resist the antibody for the anti-mouse-HRP of rabbit.Fig. 4 A show after purifying in sample, contain target protein size strip (arrow indication), Western Blot(Fig. 4 B) qualification result is consistent with SDS-PAGE, show to have obtained good purifying through nickel post one step affinity chromatography ZHB-pA12 single-chain antibody.
The Immunoblot that the anti-CD26 single-chain antibody of embodiment 4 is combined with CD26 extracellular region protein identifies
This experiment purpose is in order to verify the anti-CD26 single-chain antibody ZHB-pA12 of screening acquisition and the specific binding effect of CD26 extracellular region protein.Recombinant C D26 extracellular region protein carries out SDS-PAGE electrophoresis, and the half-dried method 1h that turns, is transferred to NC film (purchased from Millipore) by albumen; Transfer printing finishes, by film room temperature sealing 1h in 5%MPBS; With 5%MPBS dilution ZHB-pA12 single-chain antibody to 1 μ g/mL, incubated at room 1h, TBS washing 3 times; With mouse anti-Myc antibody (brilliant biological purchased from grace) incubated at room 1h, TBS washing 3 times; With the anti-mouse-HRP of rabbit bis-is anti-, hatch gel imaging instrument (ImageQuant LAS4000 after 1h, GE) exposure, result as shown in Figure 5, has obvious band at CD26 extracellular region target protein size place, shows that anti-CD26 single-chain antibody ZHB-pA12 can specificity be combined with CD26 extracellular region protein.
The immunofluorescence of embodiment 5 soluble single-chain antibody ZHB-pA12 and people's mesothelioma cell NCI-H2452
CD26 is at people's mesothelioma cell NCI-H2452 cell surface high expression level (Inamoto et al. (2007) Clin Cancer Res, 13,4191-200), this experiment is tested and appraised ZHB-pA12 and is combined situation with the immunofluorescence of NCI-H2452 cell, the single-chain antibody ZHB-pA12 that further checking screening obtains can be combined with the NCI-H2452 of high expression level CD26 cell-specific, prove simultaneously ZHB-pA12 can with the CD26 specific binding of cell surface.
By people's mesothelioma cell NCI-H2452 digestion, centrifugal rear resuspended, 1 * 10
5coated 24 orifice plates in/hole are by cell attachment 12h, and 4% paraformaldehyde is fixed, and PBS washes 3 times, 5%MPBS room temperature sealing 1h.PBS washes 3 times, different holes add respectively antibody ZHB-pA12 or negative control antibody IgG(mouse) (purchased from life technology), hatch 2h for 37 ℃, with PBS washing 3 times, ZHB-pA12 antibody hole adds mouse-anti myc-FITC antibody (purchased from Sigma-Aldrich), control antibodies hole adds sheep anti mouse fluorescence two anti-(purchased from life technology), hatch 1h for 37 ℃, PBS washing 3 times, fluorescent microscope (Olympus) is observed and takes pictures, result as shown in Figure 6 ZHB-pA12 single-chain antibody and NCI-H2452 has obvious fluorescence developing, and control antibodies is without obvious fluorescence developing, the single-chain antibody ZHB-pA12 that shows screening acquisition can be combined with the NCI-H2452 of high expression level CD26 cell-specific, further proof ZHB-pA12 can with the CD26 specific binding of cell surface.
The adherence inhibition effect of the anti-CD26 single-chain antibody of embodiment 6 to people's mesothelioma cell NCI-H2452
CD26 is by the combination with extracellular matrix protein (ECM), the adhesive attraction of mediation tumour cell, fibronectin is one of common ECM, CD26 and the effect of fibronectin generation specific binding, thereby the adhesion of mediated cell, and this keying action can be blocked by anti-CD 26 antibodies (Inamoto et al. (2007) Clin Cancer Res, 13,4191-200), thus blocking-up tumour cell adhesive attraction.
The humanized antibody of the anti-CD26 mono-clonal mouse-anti of YS110 WeiY's Therapeutics company exploitation, has carried out the clinical experiment of anti-CD26 high expression level tumour at present.According to the heavy chain of YS110 antibody and chain variable region gene sequence (CN101282994), the single-chain antibody pattern of complete synthesis structure YS110, scFv-YS110 is as the positive control of this experiment, the same ZHB-pA12 of Expression and purification process.
Adhesion experiment adopts 24 orifice plates, five groups of parallel laboratory tests, 4 every group multiple holes, wherein one group is blank group (Blank), directly with 2%BSA sealing, all the other four groups with 20～25 ℃ of coated 90min of 10 μ g/mL fibronectin (purchased from BD Biosciences) room temperatures.PBS adds 2%BSA37 ℃ of sealing 1h after washing 3 times.Simultaneously by 10 * 10
5individual NCI-H2452 cell is washed and after 3 times, is divided into five groups of equivalent with PBS, three groups is antibody test group, cell is respectively with the ZHB-pA12 containing 50 μ g/mL, positive control scFv-YS110, negative control antibody IgG(mouse) RPMI-1640 medium treatment 2h, other two groups is that control group (Binding group, Blank group) is used with test group containing after the RPMI-1640 medium treatment 2h of equivalent PBS, decile adds in five groups of 24 orifice plates after sealing, and incubator continues to cultivate 12h.With PBS hole flushing 3 times, wash the cell not adhering to off, every hole adds the CCK-8(of 270 μ L RPMI-1640 and 30 μ L purchased from colleague's chemistry institute), 37 ℃ are continued to cultivate 30min, at microplate reader 450nm wavelength place, measure absorbance value (OD value), the size of OD value is directly proportional to viable cell quantity, calculates accordingly the adhesion rate of cell.Cell adhesion rate (%)=and [OD(antibody test group)-OD(Blank group)]/[OD(Binding group)-OD(Blank group)].Fig. 7 shows, cell after processing with scFv-YS110 and ZHB-pA12 declines to the adhesion of fibronectin, negative control antibody IgG(mouse) do not affect cell adhesion, show that ZHB-pA12 can suppress the combination of CD26 to ECM, suppress the tumor cell adhesion effect that CD26 mediates, suppressed the Invasion and Metastasis to other organs of tumour cell.
The inhibited proliferation of the anti-CD26 single-chain antibody of embodiment 7 to people's mesothelioma cell NCI-H2452
By NCI-H2452 cell 1 * 10
496 porocyte culture plates are inoculated in/hole, and former substratum is replaced with 1% calf serum substratum (containing effect antibody) after cultivation 12h in 100 μ L/ holes.Cultured cells is divided into three groups, and the corresponding effect antibody adding is respectively: the single-chain antibody ZHB-pA12 of different concns, positive control scFv-YS110 and negative control antibody IgG(mouse).Effect antibody final concentration is grouped into 0,0.1,1.0,10 μ g/mL, 5 the every group multiple holes of experiment.Incubator continues to cultivate 48h, after observation of cell upgrowth situation, every hole adds the CCK-8(of 10 μ L purchased from colleague's chemistry institute), 37 ℃ are continued to cultivate 30min, at microplate reader 450nm wavelength place, measure absorbance value (OD value), the size of OD value is directly proportional to viable cell quantity, calculates accordingly the inhibiting rate of single-chain antibody ZHB-pA12 on cell proliferation.As shown in Figure 8, ZHB-pA12 and positive control scFv-YS110 all have obvious restraining effect to NCI-H2452, and be concentration dependent, the activity of ZHB-pA12 and positive control is suitable, negative control IgG does not have obvious restraining effect to NCI-H2452, show that ZHB-pA12 can have restraining effect to the mesothelioma cell proliferation of high expression level CD26, can be used as the potential medicine of CD26 high expression level tumour, and than positive control scFv-YS110, ZHB-pA12 is full humanized antibody, has eliminated the impact of foreign protein on human immune system.
The inhibited proliferation of the anti-CD26 single-chain antibody of embodiment 8 to human colon cancer cell HCT116
Except people's mesothelioma cell NCI-H2452, colon carcinoma cell line HCT116(Abe et al. (2011) the BMC Cancer of high expression level CD26,2011,11:51) be also used to investigate the proliferation inhibiting effect of single-chain antibody to cell.
By HCT116 cell 8 * 10
496 porocyte culture plates are inoculated in/hole, and former substratum is replaced with 1% calf serum substratum (containing effect antibody) after cultivation 12h in 100 μ L/ holes.Cultured cells is divided into three groups, and the corresponding effect antibody adding is respectively: the single-chain antibody ZHB-pA12 of different concns, positive control scFv-YS110 and negative control antibody IgG(mouse).Effect antibody final concentration is grouped into 0,0.1,1.0,10 μ g/mL, 5 the every group multiple holes of experiment.Incubator continues to cultivate 48h, after observation of cell upgrowth situation, every hole adds the CCK-8(of 10 μ L purchased from colleague's chemistry institute), 37 ℃ are continued to cultivate 30min, at microplate reader 450nm wavelength place, measure absorbance value (OD value), the size of OD value is directly proportional to viable cell quantity, calculates accordingly the inhibiting rate of single-chain antibody ZHB-pA12 on cell proliferation.As shown in Figure 9, ZHB-pA12 and positive control scFv-YS110 all have obvious restraining effect to HCT116, and be concentration dependent, the activity of ZHB-pA12 and positive control is suitable, negative control IgG does not have obvious restraining effect to HCT116, show that ZHB-pA12 can have restraining effect to the Colon Cancer Cells of high expression level CD26, proving again ZHB-pA12 can be used as the potential medicine of CD26 high expression level tumour, and than positive control scFv-YS110, ZHB-pA12 is full humanized antibody, has eliminated the impact of foreign protein on human immune system.
Claims (10)
1. an anti-CD 26 antibodies or its fragment, it is characterized in that, described antibody or its fragments specific are in conjunction with people CD26, the aminoacid sequence of described antibody or its fragment comprises the monoclonal antibody in any region or the conjugate of its fragment or its fragment that is selected from 6 complementary determining regions containing SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, mono-group of sequence of SEQ ID NO:8, or by the aminoacid sequence that its amino-acid substitution or modification are obtained.
2. anti-CD 26 antibodies as claimed in claim 1 or its fragment, it is characterized in that, the aminoacid sequence of the variable region of heavy chain of described antibody or its fragment contains just like the complementary determining region shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and/or the aminoacid sequence of the variable region of light chain of described antibody or its fragment contains just like the complementary determining region shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
3. anti-CD 26 antibodies as claimed in claim 1 or its fragment, it is characterized in that, the aminoacid sequence of the variable region of heavy chain of described antibody or its fragment is as shown in SEQ ID NO:1, and the aminoacid sequence of the variable region of light chain of described antibody or its fragment is as shown in SEQ ID NO:5.
4. a single-chain antibody of anti-CD26, is characterized in that, the aminoacid sequence of described single-chain antibody is as shown in SEQ ID NO:11.
5. a nucleotide sequence for coding single-chain antibody as claimed in claim 4, is characterized in that, described nucleotide sequence is as shown in SEQ ID NO:10.
6. an expression vector that contains nucleotide sequence as claimed in claim 5.
7. one kind contains the recombinant host bacterium of expression vector as claimed in claim 6.
8. produce a method for single-chain antibody as claimed in claim 4, comprising:
1) under suitable condition, cultivate recombinant host bacterium as claimed in claim 7 and express antibody;
2) then from Host Strains purifying, collect antibody.
9. antibody or its fragment suppress the new purposes in the tumor cell proliferation of CD26 high expression level and the medicine of Invasion and Metastasis in preparation as claimed any one in claims 1 to 3.
10. single-chain antibody as claimed in claim 4 suppresses the new purposes in the tumor cell proliferation of CD26 high expression level and the medicine of Invasion and Metastasis in preparation.
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| CN109535255A (en) * | 2018-12-28 | 2019-03-29 | 江苏众红生物工程创药研究院有限公司 | A kind of anti-human CD26 antibody and its application in detection kit |
| CN109709337A (en) * | 2018-12-28 | 2019-05-03 | 江苏众红生物工程创药研究院有限公司 | The immunologic combined detection reagent kit of people CD26 and its clinical application |
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| CN101172158A (en) * | 2001-05-11 | 2008-05-07 | 得克萨斯州立大学董事会 | Anti-CD26 monoclonal antibodies as therapy for diseases associated with cells expressing CD26 |
| CN101282994A (en) * | 2005-07-22 | 2008-10-08 | Y's治疗有限公司 | Anti-CD26 antibodies and methods of use thereof |
| CN102167744A (en) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | Human anti-CD20 monoclonal antibody and preparation method and application thereof |
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| CN101172158A (en) * | 2001-05-11 | 2008-05-07 | 得克萨斯州立大学董事会 | Anti-CD26 monoclonal antibodies as therapy for diseases associated with cells expressing CD26 |
| CN101282994A (en) * | 2005-07-22 | 2008-10-08 | Y's治疗有限公司 | Anti-CD26 antibodies and methods of use thereof |
| CN102167744A (en) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | Human anti-CD20 monoclonal antibody and preparation method and application thereof |
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| CN109535255A (en) * | 2018-12-28 | 2019-03-29 | 江苏众红生物工程创药研究院有限公司 | A kind of anti-human CD26 antibody and its application in detection kit |
| CN109709337A (en) * | 2018-12-28 | 2019-05-03 | 江苏众红生物工程创药研究院有限公司 | The immunologic combined detection reagent kit of people CD26 and its clinical application |
| CN109709337B (en) * | 2018-12-28 | 2021-09-14 | 江苏众红生物工程创药研究院有限公司 | Immunohistochemical detection kit for human CD26 and clinical application thereof |
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