CN103724431B - A kind of people source anti-CD 26 antibodies and application thereof - Google Patents
A kind of people source anti-CD 26 antibodies and application thereof Download PDFInfo
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Abstract
The invention provides a kind of newly can with the high-affinity human antibody of CD26 specific binding, preparation method and application thereof, belong to genetic engineering antibody technical field.CD26 is a kind of ubiquitous multifunctional II type transmembrane glycoprotein, has various biological function, can interact, as ADA, CD45, FAP-alpha etc. with multiple protein.A kind of antibody or its fragment deriving from people source provided by the invention, described antibody or its fragments specific are in conjunction with people CD26, preferred specific binding CD26 extracellular region, does does the aminoacid sequence of described antibody or its fragment comprise and being selected from containing SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID the monoclonal antibody in any region of 6 complementary determining regions of NO:8 mono-group of sequence or the conjugate of its fragment or its fragment, or by aminoacid sequence that amino-acid substitution or modification obtain.The anti-CD26 single-chain antibody that the present invention obtains has high specific with CD26 and is combined, simultaneously also can the obviously propagation of inhibition tumor cell and Invasion and Metastasis.
Description
Technical field
The invention belongs to genetic engineering antibody technical field, especially relate to a kind of newly can with the full human single chain variable fragments antibody of the high-affinity of CD26 specific binding, preparation method and application thereof.
Background technology
CD26 is a kind of ubiquitous multifunctional 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, and relative molecular mass is about 110kDa.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 and hold catalyst structure domain (552 ~ 766), CD26 molecule three-dimensional structure and function closely related.The C of CD26 holds catalyst structure domain to play DPP IV (Dipeptidylpeptidase4, DPPIV) active, can be hydrolyzed multiple substrate in body and play biological action, being rich in halfcystine district can interact with different kinds of molecules in body, thus 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 signal transduction process as costimulatory molecules, also relate to multiple T cell function, comprise T cell and ripe and migration occurs, 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.（Ohnumaetal.(2011)AdvClinChem,53,51-84）
CD26 owing to can interact with multiple protein, as ADA, CD45, FAP-alpha etc., can also in conjunction with ECM, cause increase or the reduction of expressing CD26 cellular infiltration activity, visible CD26 plays a significant role at oncobiology.The expression amount of CD26 can raise in multiple newborn oncocyte surface or serum, and such as, CD26 high expression level is in some offensive T cell malignant tumour, malignant mesothe, nephroncus, some colorectal carcinoma (Havreetal. (2008) FrontBiosci, 13,1634-1645).Some CD26+ colon cancer cell subgroup, CD26+ malignant mesothelioma cells has obvious tumor stem cell feature (Ghanietal. (2011) BiochemBiophysResCommun, 404,735-742andPangetal. (2010) CellStemCell, 6,603-615), therefore CD26 can be used as the molecular marker of kinds of tumors.
From 2012 year-to-date, find many cases and the similar novel coronavirus human coronary virus-EMC(HCoV-EMC of SARS virus) the infected, lethality rate is more than 50%, research and development find the specific combination acceptor CD26 molecule just of this poisoning intrusion human body, CD26 albumen structural conservation between the mankind and many animals, this finds that there is the mechanism helping to illustrate viral infecting both domestic animals and human and propagate, and provides a target spot for potential treatment.Experiment shows, Y'sTherapeutics company exploitation anti-CD26 mono-clonal mouse-anti humanized antibody YS110 can with hCoV-EMC competitive binding CD26, thus stop virus to combine, pathogenic impact (Ohnumaetal. (2013) is produced on human body, JVirol, 87,13892-13899).
Existing multiple mouse source antibody report (Havreetal. (2008) FrontBiosci, 13,1634-1645) in conjunction with CD26, in some CD26 high expression level cancer for the treatment of, and T suppression cell moves, vascularization aspect can play a role.The combination of mouse monoclonal antibody 1F7 and the CD26 of the anti-CD26 of bibliographical information is had to cause cell cycle arrest to limit a little at G1/S, and CD26 induces CD26Jurkat transfectant to be stuck in G1 in combination with Enhanced expressing cyclin matter p21, the formation of CD26+ tumour can be suppressed with 1F7 Antybody therapy, and strengthen survival rate in mouse model.Mouse monoclonal antibody E19 and E26 of other anti-CD26, the cell migration that these antibody exhibits go out to be suppressed to fibrocyte and Traumatic cell to formed individual layer restraining effect, to angiopoietic inhibition and to the formation that intrusion and the kapillary of human skin capillary endothelium newly prop up, there is inhibition.In addition, in the experiment of HCoV-EMC cells infected, anti-CD 26 antibodies, by conjunction with CD26 albumen, significantly can suppress HCoV-EMC poisoning intrusion cell, thus provides potential treatment plan for HCoV-EMC patients with viral infections.
As can be seen here, anti-CD26 monoclonal antibody plays a role in treatment various diseases by the activity changing CD26.But mouse monoclonal antibody human anti-murine antibodies can react (HumanAnti-MouseAntibody, HAMA) when directly applying to human body therapy, and people knows from experience the antibody producing anti-mouse antibody, 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 Chimeric antibody, chimeric antibody is mosaic gene by the V district gene of murine antibody and the C district gene splicing of people's antibody, mouse composition is made to reduce about 70%, and humanized antibody, on the basis of chimeric antibody, the skeleton district (FR) of further employment antibody variable region substitutes mouse FR, greatly reduces the mouse derived components of antibody.But the sequence of residual a small amount of murine antibody still likely potential initiation HAMA.
Summary of the invention
Technical problem to be solved by this invention: the present invention aims to provide that a kind of energy is effective, the human antibody of specific binding people CD26, and in preparation treatment with the expression of CD26, particularly overexpression is the disease of feature, or expresses the novelty teabag in the medicine of disease using CD26 as the disease of viral infection of bind receptor and diagnosis CD26 variability.More particularly:
The present invention's first object is to provide a kind of antibody or its fragment that derive from people 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 of 6 complementary determining regions be selected from containing SEQIDNO:2, SEQIDNO:3, SEQIDNO:4, SEQIDNO:6, SEQIDNO:7, SEQIDNO:8 mono-group of sequence or the conjugate of its fragment or its fragment, or by aminoacid sequence that amino-acid substitution or modification obtain.
Antibody preferably in the present invention or the aminoacid sequence of its fragment are containing, for example the complementary determining region of the variable region of heavy chain of sequence shown in SEQIDNO:2, SEQIDNO:3 and SEQIDNO:4.
Antibody preferably in the present invention or the aminoacid sequence of its fragment are containing, for example the complementary determining region of the variable region of light chain of sequence shown in SEQIDNO:6, SEQIDNO:7 and SEQIDNO:8.
Antibody more preferably 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 SEQIDNO:2, CDRH2 as shown in sequence SEQIDNO:3, the CDRH3 as shown in sequence SEQIDNO:4;
And the aminoacid sequence of its variable region of light chain contains following complementary determining region: the CDRL1 as shown in sequence SEQIDNO:6, the CDRL2 as shown in sequence SEQIDNO:7 and the CDRL3 as shown in sequence SEQIDNO:8.
Antibody more preferably in the present invention or its fragment contain variable region of heavy chain sequence and variable region of light chain sequence as shown in SEQIDNO:5 as shown in SEQIDNO:1.
The present invention's second object is to provide a kind of single-chain antibody deriving from people source, and the aminoacid sequence of described single-chain antibody is as shown in SEQIDNO:11.
The present invention's the 3rd object is to provide a kind of nucleotide sequence of above-mentioned single-chain antibody of encoding, and described nucleotide sequence is as shown in SEQIDNO:10.
The present invention's the 4th object is to provide a kind of expression vector containing above-mentioned nucleotide sequence.
The present invention's the 5th object is to provide a kind of recombinant host bacterium containing above-mentioned expression vector.
The present invention's the 6th object is to provide a kind of method of producing above-mentioned single-chain antibody, comprising:
1) cultivate above-mentioned recombinant host bacterium under suitable conditions and express antibody;
2) then from Host Strains purifying, collect antibody.
The present invention's the 7th object is to provide above-mentioned antibody or the novelty teabag 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 be the method that diagnosis CD26 variability expression disease provides based on antibody, relative disease includes but not limited to autoimmune disease and cancer.
Invention further illustrates:
The anti-CD26 single-chain antibody gene in total man source sequence 744 Nucleotide in the present invention, expection has 248 amino acid.There are 122 amino acid whose variable region of heavy chain (SEQIDNO:1) and 111 amino acid whose variable region of light chain (SEQIDNO:5), between variable region of heavy chain and variable region of light chain, connect (SEQIDNO:9) by 15 amino acid whose flexible peptides.
Expression vector containing CD26 single-chain antibody gene of the present invention and Host Strains all belong to protection scope of the present invention.Increase the primer pair of any fragment of single-chain antibody gene of the present invention 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 and combining also and CD26, thus inhibition tumor cell propagation, invasion and attack and the combination of suppression virus are infected.Specifically, the anti-CD26 single-chain antibody that the present invention obtains can be combined with CD26 high specific, the cell adhesion inhibition tumor cell that in vitro tests display CD26 single-chain antibody mediates by CD26, and CD26 single-chain antibody also possesses the ability of stronger extracorporeal suppression tumor cell growth, as can be seen here, the single-chain antibody that obtains of the present invention can the obviously propagation of inhibition tumor cell and Invasion and Metastasis.
Accompanying drawing explanation
Fig. 1 is the structural representation of single-chain antibody ZHB-2dB3.VH represents heavy chain variable domain (SEQIDNO:1), and VL represents light chain variable domain (SEQIDNO:5), and Linker is the flexible peptide (SEQIDNO:9) connecting VH, VL.
Fig. 2 is the qualification 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 WesternBlot qualification 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 rabbit against murine-HRP antibody (purchased from lifetechnology).
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 CD26 extracellular region protein bag is 1 μ g/mL by concentration, and the phage of screening amplification is taken turns in dilution 4, hatched by the enzyme plate of CD26 extracellular region protein with bag, 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 monoclonal phage ELISA measurement result, selects in mono-clonal to 96 orifice plate and expresses phage antibody, is hatched, anti-M13-HRP antibody test result with bag by the enzyme plate of CD26 extracellular region protein, measures OD
450nm-OD
650nmas end value, mono-clonal and the CD26 extracellular region protein of result display more than 90% produce positive keying action.
Fig. 4 is the qualification figure of single-chain antibody ZHB-2dB3.A is the SDS-PAGE figure of the ZHB-2dB3 after ni-sepharose purification, and Lane1 is standard protein; Lane2 is ZHB-2dB3 sample protein, albumen size strip for the purpose of albumen arrow indication; B is the WesternBlot qualification figure of ZHB-2dB3 after purifying, and primary antibodie is anti-myc mouse-anti, and two resist for rabbit against murine-HRP antibody, and Lane1 is ZHB-2dB3 sample, albumen size strip for the purpose of albumen arrow indication, and Lane2 is standard protein.
Fig. 5 shows the WesternBlot qualification figure that single-chain antibody ZHB-2dB3 is combined with CD26 extracellular region protein.Primary antibodie is anti-myc mouse-anti, and two resist for rabbit against murine-HRP antibody.Lane1 is standard protein, and Lane2 is ZHB-2dB3 and CD26 extracellular region protein keying action, and arrow indication shows and CD26 extracellular region protein band of the same size in Fig. 2.Result shows that anti-CD26 single-chain antibody ZHB-2dB3 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-2dB3 and NCI-H2452 cytosis, and B is that negative control antibody is combined with NCI-H2452 cellular immunofluorescence the negative findings detected, 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, investigates the cell after single-chain antibody effect 12h to the adhesion situation of fibronectin.
OD450(Binding group)-OD450(Blank group)=adherent cell value completely;
OD450(antibody test group)-OD450(Blank group) the cell adhesion value of=sample sets;
IgGcontrol is negative control antibody;
Cell adhesion value/adherent cell the value completely of cell adhesion rate (%)=sample sets
Result shows, compared with negative control IgGcontrol, ZHB-2dB3 and positive control scFv-YS110 all has obvious restraining effect to NCI-H2452 cell adhesion, and ZHB-2dB3 action effect is better than positive control scFv-YS110.
Fig. 8 shows the detected result of single-chain antibody to people mesothelioma NCI-H2452 cell inhibitory effect.Cell is with 1 × 10
4the quantity in/hole is incubated in 96 orifice plates, single-chain antibody ZHB-2dB3, scFv-YS110 and negative control antibody IgG(mouse) act on 48h after, adopt CCK-8 reagent react 30min, measure the absorbance of 450nm, the proliferation inhibition rate of cell represents with the decrement % of OD450nm.Experimental result shows that ZHB-2dB3 and positive control scFv-YS110 all has obvious restraining effect to NCI-H2452, and in concentration dependent, ZHB-2dB3 effect is slightly better than positive control scFv-YS110.
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, single-chain antibody ZHB-2dB3, scFv-YS110 and negative control antibody IgG(mouse) act on 48h after, adopt CCK-8 reagent react 30min, measure the absorbance of 450nm, the proliferation inhibition rate of cell represents with the decrement % of OD450nm.Experimental result shows that ZHB-2dB3 and positive control scFv-YS110 all has obvious restraining effect to HCT116, and in concentration dependent, ZHB-2dB3 effect is slightly better than positive control scFv-YS110.
Embodiment
Definition
" antibody " is can by the immunoglobulin molecules of at least one antigen recognition site specific binding target antigen in variable region, and described target antigen is as sugar, polynucleotide, fat, polypeptide etc.This term not only comprises complete polyclone and monoclonal antibody, also comprises its fragment (such as Fab, Fab ', F(ab ')
2, Fv), single-chain antibody (scFv), its mutant, comprise antibody moiety fusion rotein and arbitrary comprise antigen recognition site other change the immunoglobulin molecules of configurations.Antibody comprises the antibody of arbitrary class, as IgG, IgA or IgM(or its subclass), and antibody does not need for arbitrary certain kinds.
As used herein, " single-chain antibody " refers to immunoglobulin heavy chain variable region (V
h) and variable region of light chain (V
l) the single chain fusion protein that is connected to form by 10 ~ 25 amino acid polypeptides, connection peptides (linker), is rich in glycine and Serine usually, is beneficial to stability and the snappiness of single-chain antibody.Mode of connection can by V
ln end be connected to V
hc-terminal, or on the contrary.Although eliminate constant region and introduce linker, single-chain antibody still remains the specificity of immunoglobulin (Ig) to antigen.
" chimeric antibody and humanized antibody ", in general refers to the antibody in the region be combined with from more than species." chimeric antibody " traditionally comprises from the variable region of mouse (or being rat in some cases) and the constant region from people." humanized antibody " generally refers to inhuman (such as mouse) antibody formation, and it is containing the specific gomphosis immunoglobulin derived from non-human immunoglobulin minmal sequence, immunoglobulin chain or its fragment (such as Fv, Fab, Fab ', F(ab ')
2or other antigen zygote sequence of antibody).Generally speaking, in humanized antibody, except CDR, whole antibody is the polynucleotide encoding of being originated by people, or outside CDR, has identity with the antibody of the polynucleotide encoding of being originated by people.Some or all of CDR is the nucleic acid encoding of being originated by human organism, they is transplanted to the lamella framework region of people antibody variable region to produce antibody, and the specificity of this antibody is determined by the CDR implanted.
As used herein, " human antibody " represents the antibody with the aminoacid sequence corresponding with the aminoacid sequence of the antibody that people produces and/or the antibody prepared by the arbitrary technology preparing human antibody known in the art or disclosed herein.Human antibody can produce by multiple technology known in the art.Such as, human antibody is selected from phage library, and wherein said phage library expresses human antibody.Human antibody also can by introducing transgenic animal to prepare by human immunoglobulin gene's seat (loci), and wherein said transgenic animal are such as by endogenous immunoglobulin Gene Partial or the mouse of putting out a fire completely.Alternatively, prepared by the human B lymphocyte (this bone-marrow-derived lymphocyte can obtain from individual or carry out immunity in vitro) that people's antibody can produce the antibody of anti-target antigen by immortalization.In some embodiments, human antibody is " total man source ", and this represents that antibody comprises people's heavy chain and light chain polypeptide.
With the following Examples the present invention is described now.There is provided these embodiments only for illustration of object, the invention is not restricted to these embodiments, but comprise changing of obviously being produced by instruction provided herein.For carrier construction and plasmid, plasmid is imported host cell and gene, and the detailed description of the ordinary method such as the expression identification of gene product can obtain from various publication, such as " the Molecular Cloning: A Laboratory guide third edition ".The per-cent related in embodiment, wherein solid reagent is weight percentage, and liquid reagent is volume percent.
Portion of material source is illustrated in this:
Clone: HLF cell is the undifferentiated liver cancer cell of people, available from JCRBcellbank (JapaneseCollectionofResearchBioresourcesCellBank).NCI-H2452 is people's mesothelioma cell, available from the American Type Culture Collection council of Chinese Academy of Sciences cell bank/Shanghai Inst. of Life Science, CAS cellular resources center.HCT116 is human colon cancer cell, available from the American Type Culture Collection council of Chinese Academy of Sciences cell bank/Shanghai Inst. of Life Science, CAS cellular resources center.
The reagent such as substratum and damping fluid: RPMI-1640 (GIBCO, Cat#31800022 add NaHCO31.5g/L, glucose2.5g/L, SodiumPyruvate0.11g/L), 90%; High-quality foetal calf serum, (GIBCO) 10%.
2YT substratum: 1L includes Trptone (OXID) 16g, YeastExtract (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 containing 5% or 2% skim-milk (OXID).
0.1%PBST: containing 0.1%Tween20(purchased from Beijing Suo Laibao) PBS.
2%BSA: containing 2%BSA(purchased from MPBiomedicals) 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 agents 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 MedicalResearchCouncil (UK))
The preparation of embodiment 1CD26 extracellular region protein
The object of this research is by Transfected Recombinant Plasmid eukaryotic cell, and G418 resistance screening obtains stable cell strain, and secreting, expressing CD26 extracellular region protein, is purified into CD26 extracellular region protein through affinity protein purification.
CD26 Extracellular domain is the synthetic gene being selected from amino acid 29-766 position " Extracellular " region in Uniprot:P27487 sequence, CD26 Extracellular domain is connected into plasmid p3XFLAG-CMV9, restriction enzyme site is HindIII, XbaI, construction recombination plasmid (J. Pehanorm Brooker. Molecular Cloning: A Laboratory guide. the third edition. Science Press .2002.P68).
Adopt LipofectamineLTXReagent(purchased from Invitrogen) and to specifications method proceed to HLF cell by containing the recombinant plasmid of CD26 Extracellular domain.According to stably transfected cell line construction process (CurrentProtocolsinMolecularBiology, P9.5.5) CD26 extracellular region stable expression cell strain is obtained, be designated as HLF-4D9, serum-free mass propgation, secreting, expressing CD26 extracellular region protein, this albumen is with Flag label, adopt ANTI-FLAGM2AffinityGel(purchased from Sigma-Aldrich), purifying is carried out to serum-free culture supernatant and obtains CD26 extracellular region protein sterling, SDS-PAGE(Fig. 2 A) analyze and WesternBlot (agriculture of J.S. Boneface. fine works Cell Biology Experiment guide. Science Press .2007.P177) qualification (Fig. 2 B), primary antibodie is anti-CD26 mouse-anti (purchased from MBL), two resist for rabbit against murine-HRP antibody (purchased from lifetechnology).
Result as shown in Figure 2, Fig. 2 A is the qualification figure of SDS-PAGE to purifying protein, the single slice (Lane2) of electrophoresis pure level target protein size is obtained through a step affinity chromatography, Fig. 2 B carries out WesternBlot qualification figure to the albumen of purifying, band shown in Lane2 and SDS-PAGE qualification result albumen in the same size, show that CD26 Extracellular domain is through cloning restructuring transfecting eukaryotic cells and expressing, and obtains the pure level CD26 extracellular region protein of electrophoresis after purifying.
The separation of the anti-CD26 single-chain antibody in embodiment 2 total man source
This single-chain antibody shows storehouse from people's single chain antibody phage, adopts the solid phase affinity selection of CD26 extracellular region protein to obtain, and this people's single chain antibody phage shows that storehouse is built by Jiangsu Zhonghong Biopharma Institute Inc..The heavy chain of the antibody that should produce containing people's cell and the phage display library of variable region of light chain build from human peripheral lymphocyte, first run amplification is carried out by using the special primer of antibody variable gene, two take turns amplification adopts heavy chain light chain variable region and flexibly connects peptide gene and be connected to form single-chain antibody gene, and ScFv gene cloning is entered in plasmid pHEN2 transform e. coli tg1, obtain 10
8p.f.u. phage single-chain antibody shows storehouse.(Shen is doubly put forth energy. recombinant antibodies. and Science Press .2005.P107)
By this single-chain antibody library Primary spawn to logarithm growth stage, use M13K07 helper phage infection, in 2YT-AK substratum, 30 DEG C of shaking table overnight incubation.Phage 4%PEG/2.5MNaCl precipitates, and to be resuspended in PBS and to measure antibody library titre, and obtaining titre is 10
11the phage antibody library of p.f.u.(Zhen Yongsu. antibody engineering medicine. Chemical Industry Press .2002.P51) dilute CD26 extracellular region protein to 50 μ g/mL with PBS; Wrap by enzyme plate (Maxi-sorp96, Nunc), blank control wells (not containing CD26 extracellular region protein) is set simultaneously, closes subsequently.Phage antibody library is suspended in 2%MPBS, gets 100 μ L and join in the blank well closed, room temperature joins in the hole containing CD26 extracellular region protein after placing 60min, and room temperature places 2h; 0.1%PBST and PBS washs 10 times respectively, adds 100 μ L0.1M hydrochloric acid (being adjusted to pH2.2 with glycine), shaken at room temperature 10min, 15 μ L1MTris(pH9.0) for neutralizing rapidly the phage eluted; The e. coli tg1 of the phage-infect 5mL logarithmic phase after neutralization, get 100uL, do 1 ~ 3 100 times of serial dilution, then by serial dilution thing paving TYE solid medium (containing 100 μ g/mLAmp and 1% glucose), remaining bacterium liquid adds 20mL again containing 2 × 10
10the 2YT-AG of M13K07 helper phage carries out increasing and preparing phage library, for next round screening process, carries out 4 altogether and takes turns screening.
The antibody fragment being illustrated in phage particle surface is called as phage antibody, this experiment first takes turns the enrichment condition of CD26 specific phage antibody after screening by polyclone Phage-ELISA qualification 4, after identify by monoclonal phage ELISA the phage antibody picking out high-affinity further.
Polyclone Phage-ELISA is identified, envelope antigen i.e. 1 μ g/mLCD26 extracellular region protein, to enzyme plate, after closing, is got the phage obtained after 10 μ L often take turns screening, joined in antigen coated enzyme bar after 2%MPBS dilution.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, adds 100 μ LTMB nitrite ions (purchased from AMRESCO) after washing.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, using the enzyme plate of BSA bag quilt as negative control, along with screening wheel number increases, the combination of phage antibody and CD26 extracellular region protein strengthens, and shows to obtain obvious enrichment with the phage of CD26 specific antibody.
Monoclonal phage ELISA identifies, on the titer determination flat board of random choose third round fourth round screening process, picking mono-clonal is in 96 holes microbial culture plate (purchased from Corning), 2YT substratum (containing 100 μ g/mLAmp and 1% glucose) has been added in every hole, 37 DEG C are cultured to logarithmic phase, and every hole adds 10
9p.f.u.M13K07 helper phage 37 DEG C leaves standstill and infects 30min, cultivates 1h for 37 DEG C.The centrifugal 10min of 1800g, abandons supernatant.Bacterial sediment is resuspended in 200 μ L2YT-AK substratum, 30 DEG C of shaking table overnight incubation.The culture supernatant containing phage that the centrifugal 10min of 1800g next day obtains, with the 20%MPBS incubated at room 1h of 1/10 volume, join in the enzyme plate being coated with recombinant C D26 extracellular region protein again, ELISA qualification (method and reagent are with the qualification of polyclone Phage-ELISA).Measure OD
450and OD
650, and with OD
450-OD
650as last detected result.Select the high clone of reading and carry out determined dna sequence.Fig. 3 B shows the detected result of part monoclonal phage ELSIA, and the mono-clonal of more than 90% shows positive keying action, and show further to take turns screening by 4, the phage with CD26 specific antibody obtains obvious enrichment.Therefrom select the mono-clonal order-checking that 50 readings are high, obtain the single-chain antibody of nucleotide coding sequence as shown in SEQIDNO:10 (being designated as ZHB-2dB3).The aminoacid sequence of corresponding single-chain antibody ZHB-2dB3 is SEQIDNO:11.
The solubility expression of the anti-CD26 single-chain antibody of embodiment 3 and separation and purification
PHEN2 is a difunctional phagemid vector, has amber stop codon (Amber) TAG between detection of expression label (c-mytag) and coat protein gene.If phage-infect amber mutation (SupE) suppressive bacterial strain, as TG1, TAG codon is translated into L-glutamic acid, sequence can read over translation, and antibody fragment and coat protein p3 amalgamation and expression are in phage surface; When phage-infect non-amber mutation suppressive bacterial strain, as HB2151, translate and stop at TAG place, the antibody fragment that soluble type is expressed can be obtained.Antibody fragment C end band has 6 × Histag and c-myctag, is beneficial to purifying and Testing and appraisal.ZHB-2dB3 single-chain antibody adopts the solubility expression in colibacillus periplasm space, and purifying adopts Thief zone method to extract periplasmic albumen, and the separation and purification of recycling affinity chromatography one step obtains the higher target protein of purity.
The little extraction reagent kit of employing plasmid extracts the plasmid (being designated as 2dB3-pHEN2) containing encode single chain antibodies ZHB-2dB3 gene from thalline TG1, for transforming the sub-intestinal bacteria HB2151 of non-suppression, transform and adopt Calcium Chloride Method preparation and transform competent E. coli HB2151(J. Pehanorm Brooker. Molecular Cloning: A Laboratory guide. the third edition. Science Press .2002.P96), after gained transforms, bacterial strain is designated as HB2151-2dB3.
HB2151-2dB3 in 2YT-AG substratum, 37 DEG C of (OD when being cultured to logarithmic phase
600=0.8), add the IPTG of final concentration 1mM, 30 DEG C of overnight induction (16 ~ 20h), express soluble single-chain antibody ZHB-2dB3.6000rpm, 4 DEG C of centrifugal 15min, collect thalline, hypertonic solution (50mMTris-HCl, 20% sucrose, 1mMEDTA, pH8.0) resuspended thalline, slow stirring 1h, 4 DEG C, 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, namely level pad (the Tris50mM containing 5mM imidazoles is adopted, NaCl500mM, pH7.5) 1mL nickel post is balanced, loading after 10 column volumes, again adopt the foreign protein of non-specific binding on the equilibration buffer solution nickel post containing 5mM imidazoles, with the non-specific foreign protein of the equilibration buffer solution containing 50mM imidazoles, finally with the level pad wash-out target protein containing 100mM imidazoles.15%SDS-PAGE detects the sample collected, and WesternBlot identifies single-chain antibody further, and primary antibodie is anti-c-myc mouse-anti (brilliant biological purchased from grace), and two resist for rabbit against murine-HRP antibody.Fig. 4 A to show after purifying containing target protein size strip (arrow indication) in sample, WesternBlot(Fig. 4 B) qualification result is consistent with SDS-PAGE, shows to obtain good purifying through nickel post one step affinity chromatography ZHB-2dB3 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 the specific binding effect in order to verify the anti-CD26 single-chain antibody ZHB-2dB3 that screening obtains and CD26 extracellular region protein.Recombinant C D26 extracellular region protein carries out SDS-PAGE electrophoresis, half-dried robin 1h, albumen is transferred to NC film (purchased from Millipore); Transfer printing terminates, and film room temperature in 5%MPBS is closed 1h; Dilute ZHB-2dB3 single-chain antibody to 1 μ g/mL with 5%MPBS, incubated at room 1h, TBS wash 3 times; 3 times are washed with mouse anti-Myc antibody (brilliant biological purchased from grace) incubated at room 1h, TBS; Gel imaging instrument (ImageQuantLAS4000 after 1h is hatched with rabbit against murine-HRP two is anti-, GE) expose, as shown in Figure 5, in CD26 extracellular region, there is obvious band at target protein size place to result, shows that anti-CD26 single-chain antibody ZHB-2dB3 can specificity be combined with CD26 extracellular region protein.
The immunofluorescence of embodiment 5 soluble single-chain antibody ZHB-2dB3 and people's mesothelioma cell NCI-H2452
CD26 is at people's mesothelioma cell NCI-H2452 cell surface high expression level (Inamotoetal. (2007) ClinCancerRes, 13,4191-200), this experiment is tested and appraised the immunofluorescence of ZHB-2dB3 and NCI-H2452 cell in conjunction with situation, further checking screening obtain single-chain antibody ZHB-2dB3 can be combined with the NCI-H2452 cell-specific of high expression level CD26, simultaneously proof ZHB-2dB3 can with the CD26 specific binding of cell surface.
People's mesothelioma cell NCI-H2452 is digested, centrifugal rear resuspended, 1 × 10
5/ hole bag is by 24 orifice plates by cell attachment 12h, and 4% paraformaldehyde is fixed, and PBS washes 3 times, and 5%MPBS room temperature closes 1h.PBS washes 3 times, different hole adds antibody ZHB-2dB3 or negative control antibody IgG(mouse respectively) (purchased from lifetechnology), hatch 2h for 37 DEG C, 3 times are washed with PBS, ZHB-2dB3 antibody hole adds mouse-anti myc-FITC antibody (purchased from Sigma-Aldrich), control antibodies hole adds sheep anti mouse fluorescence two anti-(purchased from lifetechnology), hatch 1h for 37 DEG C, PBS washs 3 times, fluorescent microscope (Olympus) is observed and takes pictures, result as shown in Figure 6 ZHB-2dB3 single-chain antibody and NCI-H2452 has obvious fluorescence developing, and control antibodies is without obvious fluorescence developing, show that screening the single-chain antibody ZHB-2dB3 obtained can be combined with the NCI-H2452 cell-specific of high expression level CD26, further proof ZHB-2dB3 can with the CD26 specific binding of cell surface.
The anti-CD26 single-chain antibody of embodiment 6 is to the Adhesion inhibiyive effect of people's mesothelioma cell NCI-H2452
CD26 is by the combination with extracellular matrix protein (ECM), the adhesive attraction of mediate tumor cell, fibronectin is one of common ECM, CD26 and the effect of fibronectin generation specific binding, thus the adhesion of mediated cell, and this keying action can block by anti-CD 26 antibodies (Inamotoetal. (2007) ClinCancerRes, 13,4191-200), thus block the adhesive attraction of tumour cell.
YS110 is the humanized antibody of the anti-CD26 mono-clonal mouse-anti of Y'sTherapeutics company exploitation, has carried out the clinical experiment of anti-CD26 high expression level tumour at present.According to heavy chain and the chain variable region gene sequence (CN101282994) of YS110 antibody, the single-chain antibody pattern of complete synthesis structure YS110, namely scFv-YS110 is as the positive control of this experiment, the same ZHB-2dB3 of Expression and purification process.
Adhesion experiment adopts 24 orifice plates, five groups of parallel laboratory tests, often organize 4 multiple holes, wherein one group is blank group (Blank), directly close with 2%BSA, all the other four groups are wrapped by 90min by 10 μ g/mL fibronectin (purchased from BDBiosciences) room temperature 20 ~ 25 DEG C.PBS adds 2%BSA37 DEG C of closed 1h after washing 3 times.Simultaneously by 1 × 10
6five groups of equivalent are divided into after individual NCI-H2452 cell PBS washes 3 times, three groups is antibody test group, cell is respectively with the ZHB-2dB3 containing 50 μ g/mL, positive control scFv-YS110, negative control antibody IgG(mouse) RPMI-1640 medium treatment 2h, other two groups be control group (Binding group, Blank group) with test group containing after the RPMI-1640 medium treatment 2h of equivalent PBS, decile add five groups close after 24 orifice plates in, incubator continue cultivate 12h.With PBS hole flushing 3 times, wash the cell do not adhered to off, every hole adds the CCK-8(of 270 μ LRPMI-1640 and 30 μ L purchased from colleague's chemistry institute), 37 DEG C are continued to cultivate 30min, absorbance value (OD value) is measured at microplate reader 450nm wavelength place, the size of OD value is directly proportional to viable cell quantity, calculates the adhesion rate of cell accordingly.Cell adhesion rate (%)=and [OD(antibody test group)-OD(Blank group)]/[OD(Binding group)-OD(Blank group)].Fig. 7 shows, with the cell after scFv-YS110 and ZHB-2dB3 process, the adhesion of fibronectin is declined, negative control antibody IgG(mouse) then do not affect cell adhesion, show that ZHB-2dB3 can suppress CD26 to the combination of ECM, and ZHB-2dB3 action effect is better than positive control scFv-YS110, suppress the tumor cell adhesion effect of CD26 mediation, thus inhibit tumour cell to the Invasion and Metastasis of other organs.
The anti-CD26 single-chain antibody of embodiment 7 is to the inhibited proliferation of people's mesothelioma cell NCI-H2452
By NCI-H2452 cell 1 × 10
496 porocyte culture plates are inoculated in/hole, 100 μ L/ holes, replace former substratum after cultivating 12h with 1% calf serum medium (containing effect antibody).Cultured cells is divided into three groups, the corresponding effect antibody added is respectively: the single-chain antibody ZHB-2dB3 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, often organizes 5 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 DEG C are continued to cultivate 30min, absorbance value (OD value) is measured at microplate reader 450nm wavelength place, the size of OD value is directly proportional to viable cell quantity, calculates the inhibiting rate of single-chain antibody ZHB-2dB3 on cell proliferation accordingly.As shown in Figure 8, ZHB-2dB3 and positive control scFv-YS110 all has obvious restraining effect to NCI-H2452, and in concentration dependent, ZHB-2dB3 action activity is slightly better than positive control, negative control IgG does not have obvious restraining effect to NCI-H2452, show that ZHB-2dB3 can have restraining effect to the mesothelioma cell propagation of high expression level CD26, can be used as the potential medicine of CD26 high expression level tumour.And be human antibody compared to positive control scFv-YS110, ZHB-2dB3, completely eliminate the immune response of human body to traditional mouse-anti.
The anti-CD26 single-chain antibody of embodiment 8 is to the inhibited proliferation of human colon cancer cell HCT116
Except people's mesothelioma cell NCI-H2452, colon carcinoma cell line HCT116(Abeetal. (2011) BMCCancer of high expression level CD26,2011,11:51) be also used to investigate single-chain antibody to the proliferation inhibiting effect of cell.
By HCT116 cell 8 × 10
496 porocyte culture plates are inoculated in/hole, 100 μ L/ holes, replace former substratum after cultivating 12h with 1% calf serum medium (containing effect antibody).Cultured cells is divided into three groups, the corresponding effect antibody added is respectively: the single-chain antibody ZHB-2dB3 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, often organizes 5 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 DEG C are continued to cultivate 30min, absorbance value (OD value) is measured at microplate reader 450nm wavelength place, the size of OD value is directly proportional to viable cell quantity, calculates the inhibiting rate of single-chain antibody ZHB-2dB3 on cell proliferation accordingly.As shown in Figure 9, ZHB-2dB3 and positive control scFv-YS110 all has obvious restraining effect to HCT116, and in concentration dependent, ZHB-2dB3 action activity is slightly better than positive control, negative control IgG does not have obvious restraining effect to HCT116, show that ZHB-2dB3 can have restraining effect to the Colon Cancer Cells of high expression level CD26, again prove that ZHB-2dB3 can be used as the potential medicine of CD26 high expression level tumour, and compared to positive control scFv-YS110, ZHB-2dB3 is human antibody, eliminates the immune response of human body to traditional mouse-anti.
Claims (7)
1. anti-CD 26 antibodies or its fragment, is characterized in that, the aminoacid sequence of the variable region of heavy chain of described antibody or its fragment is as shown in SEQIDNO:1, and the aminoacid sequence of the variable region of light chain of described antibody or its fragment is as shown in SEQIDNO:5.
2. a single-chain antibody of anti-CD26, is characterized in that, the aminoacid sequence of described single-chain antibody is as shown in SEQIDNO:11.
3. a nucleotide sequence for coding single-chain antibody as claimed in claim 2, it is characterized in that, described nucleotide sequence is as shown in SEQIDNO:10.
4. the expression vector containing, for example nucleotide sequence according to claim 3.
5. one kind contains the recombinant host bacterium of expression vector as claimed in claim 4.
6. produce a method for single-chain antibody as claimed in claim 2, comprising: 1) cultivate recombinant host bacterium as claimed in claim 5 under suitable conditions and express antibody; 2) then from Host Strains purifying, collect antibody.
7. antibody as claimed in claim 1 or 2 or its fragment suppress the novelty teabag in the medicine of the tumor cell proliferation of CD26 high expression level in preparation.
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| 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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| 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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| CD26多克隆抗体的制备与鉴定;宋敏等;《中国输血杂志》;20090725;第22卷(第7期);第535-538页 * |
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