CN108129566A - Target high-affinity C- type single domain antibodies of mesothelin and preparation method and application - Google Patents
Target high-affinity C- type single domain antibodies of mesothelin and preparation method and application Download PDFInfo
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- CN108129566A CN108129566A CN201711496467.XA CN201711496467A CN108129566A CN 108129566 A CN108129566 A CN 108129566A CN 201711496467 A CN201711496467 A CN 201711496467A CN 108129566 A CN108129566 A CN 108129566A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/524—CH2 domain
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The invention discloses a kind of high-affinity C type single domain antibodies for targeting mesothelin and preparation method and application.The present invention is the mutant m01s using human antibody IgG constant region CH2 structural domains as skeleton, build polypeptide libraries, it is obtained again by antigen selection of mesothelin, such antibody molecule amount is smaller, relative to overall length monoclonal antibody, with better tissue permeability and combine there are steric effect epitope ability, for treat and diagnose some mesothelin height expression tumour (such as adenocarcinoma ovaries) in addition oral medicine can be developed into；Secondly, its plasma half-life is likely to be breached 10 hours；Finally, it can be expressed in prokaryotic expression system, production cost is low, the period is short.And there is good inhibiting effect for the ovary adenocarcinoma cells OVCAR 3 of mesothelin height expression by the F3 that the present invention is filtered out from such antibody, application prospect is good.
Description
Technical field
The present invention relates to biotechnology, in particular to a kind of high-affinity C- types single domain antibody for targeting mesothelin and
Preparation method and application.
Background technology
Mesothelin (mesothelin, MSLN) is a kind of Tumor Differentiation antigen newfound in recent years.Its precursor is sugared phosphorus
Fatty acyl group alcohol (GPI) anchoring, glycosylated cells surface protein can be divided by the N- ends that furin protease hydrolytics are 31kD
Type albumen and the C- terminal proteins of 40kD are secreted, the latter mainly exists in the form of the GPI anchorings that film combines, limited to be distributed in normally
Mesothelial cell and superficial epithelial cells, therefore it is named as mesothelin.Mesothelin limited expression in the normal tissue, it is high in certain tumours
Expression：Such as cancer of pancreas (100% case), adenocarcinoma ovaries (70% case), adenocarcinoma of lung (50% case) and malignant mesothelioma
(100% case) etc..Mesothelin induces tumorigenic mechanism that may include following three aspects：First, mesothelin can be with
Promote peritoneal seeding and the transfer of tumour cell by interacting with Mucin1 6 (i.e. CA125)；Second, mesothelin
It can promote survival and the proliferation of tumour cell by NF- κ B signals access；Finally, the expression of mesothelin can enhance tumour cell pair
Certain specific drugs are (such as：TNF-α, taxol, platinum-cyclophosphamide compound) tolerance (Tang Z, et al.,
Anticancer Agents Med Chem.,2013)。
It can be seen that mesothelin is the ideal targets for being used for oncotherapy.At present, have several for anti-mesothelin
Drug candidate is carrying out clinical test, including Anti-mesothelin antibodies coupling immunotoxin, high-affinity mouse/people's inosculating antibody mesothelin
Antibody and Anti-mesothelin antibodies coupling drug and tumor vaccine, T cell adoptive immunotherapy for mesothelin etc..
In the research process of antibody, it has been found that the molecular weight of full length antibody is larger (~150kD), makes their tissue
Permeability is poor, it is also difficult to spatially have the critical epitopes of steric effect with reference to some, so as to influence activity.The strategy of solution
First, full length antibody is minimized, a series of antibody fragments with binding function are thus developed：Such as Fab (50-
60kD), the miniaturizations such as single-chain antibody (scFv, 20-30kD), variable fragments of heavy chain (VH, 12-15kD) (single domain antibody) resist
Body.These antibody fragments have better tissue permeability and combine the antigen table there are steric effect relative to overall length monoclonal antibody
The ability of position.
However, either Fab, scFv or VH, since they all lack Fc sections, so as to can not with Fc receptors (such as
Neonatal Fc receptor, FcRn) etc. be combined.Viewpoint thinks IgG in vivo with very long half-lift, (one week arrived at present
Several weeks) the reason of be that IgG can be combined by Fc sections in acid pH (pH 6.0) and FcRn, be released in neutral pH (pH 7.4)
(this, which is combined, is known as the combination that pH is relied on), so as to complete the process of its circular regeneration.Due to lacking this combination, Fab, scFv and
The half-life period of VH in vivo usually only has a few minutes to dozens of minutes, and which limits their further applications.Therefore it needs
Continue to research and develop the structure and the screening for specific antigen that molecular weight is small, and the skeleton that plasma half-life is grown is used for antibody library.
Recently, a kind of new skeleton based on antibody Fc section CH2 structural domains, which is considered being hopeful researching and developing novel single domain, resists
Body, referred to as C- types single domain antibody.Structure (the PDB of CH2 (~12kDa)：1HZH)(Prabakaran P,et al.,Acta
Crystallogr D Biol Crystallogr., 2008) it is similar to VH, mainly it is made of seven beta sheets (A to G), by
Three loop and two α-helix are connected, and contain a pair of natural disulfide bond.Three complementations of three loop areas and VH are determined
Determine that area (complementarity determining region, CDR) is similar, mutation can be introduced and carry out the structure of antibody library
It builds.Due to being a part of Fc, Fc and the part of Fc receptors (such as neonatal Fc receptor, FcRn) etc. that CH2 contains
Binding site.By improved CH2 skeletons, it is possible to and FcRn be combined with each other.The C- screened based on this CH2 skeletons
Type single domain antibody can have economic benefits and social benefits valency：Antigen can either be combined, and such as FcRn equimoleculars (extending plasma half-life) can be combined,
So as to fulfill the function of (or part is realized) full length antibody.This is also that CH2 skeletons compare the similar skeleton of other molecular weight (such as
VH main advantage).
The stability of CH2 is weaker, needs to optimize.The mutant of a CH2 is obtained by introducing a pair of of disulfide bond
M01, with respect to CH2, Tm values improve nearly 20 DEG C (Gong R, et al., J Biol Chem., 2009)；By the N- for clipping CH2
Seven of end are in the amino acid of random state, a truncated mutant CH2s are obtained, with respect to CH2, the resistant to aggregation of CH2s
Ability is significantly improved (Gong R, et al., Mol Pharm., 2013).Obtained after the two is combined one it is new
Mutant m01s (Gong R, et al., J Biol Chem., 2011) (Fig. 1), its Tm values are 30 DEG C higher than the Tm values of CH2, tool
There are better soluble-expression and protease resistant.What is more important, relative to CH2, m01s has preferably dependent on pH's
With the binding ability of people FcRn, the plasma half-life in different animals model up to 10 hours or so, considerably longer than with its point
Son measures similar other structures domain (plasma half-life of such as VH only has a few minutes) (Gehlsen K, et al., MAbs, 2012).
Therefore, m01s can be used for the construction and screening of antibody library as an ideal skeleton.
Invention content
The purpose of the present invention is to provide a kind of high-affinity C- type single domain antibodies for targeting mesothelin and preparation method thereof
With application, which verifies through ELISA, flow cytometry, can be with antigen mesothelin used in screening and cell
The mesothelin on surface is combined；There is direct repression to the cancer cell multiplication of mesothelin height expression.
To achieve the above object, the present invention provides a kind of high-affinity C- type single domain antibodies for targeting mesothelin, it is
Using the mutant m01s of human antibody IgG constant region CH2 structural domains as skeleton, polypeptide and protein library are built, then with mesothelin
It is obtained for antigen selection.
In said program, it can realize that the polypeptide of antibody screening and protein library may be used to the present invention, such as yeast
Display libraries etc., in the prior art, it is preferred to use phage display library.
Preferably, the C- types single domain antibody is F3, it possesses tri- loop areas of BC, DE, FG, the ammonia of the Loop BC
Base acid sequence is that the amino acid sequence of Seq ID No.1, the Loop DE are seq ID No.3, the amino of the Loop FG
Acid sequence is Seq ID No.5.
Optionally, the gene order of the coding Loop BC of the F3 is seq ID No.2, encodes the gene sequence of Loop DE
Seq ID No.4 are classified as, the gene order of coding Loop FG is Seq ID No.6.
Preferably, the amino acid full length sequence of the F3 is Seq ID No.7.
Optionally, the full length gene sequence of the F3 is Seq ID No.8.
Optionally, the mutation of base and amino acid is carried out to regions of the F3 in addition to loop, obtained with F3 function classes
It should belong to scope of the invention like antibody.Relative to the Core Feature loop areas of F3, other regions are connected and roll over as skeleton
Folded effect, simple mutation and to the function of F3 without substantial effect, should be regarded as identical with the present invention.
The present invention also provides the preparation method of the high-affinity C- type single domain antibodies of above-mentioned targeting mesothelin, including as follows
Step：
(1) carrier for expression of eukaryon containing mesothelin gene sequence is built, transfection mammalian cell expresses mesothelin,
It isolates and purifies to obtain mesothelin；
(2) it is that antigen is screened with the mesothelin of purifying from the phage display library using m01s as skeleton, number was taken turns
Afterwards, the clone of the specific binding of a high-affinity is obtained；
(3) expression and purification clone obtains the high-affinity C- type single domain antibodies of targeting mesothelin.
Present invention further teaches the high-affinity C- type single domain antibodies of above-mentioned targeting mesothelin, are preparing high expression mesothelin
Application in the inhibitor of tumour cell；Or the application in high expression mesothelin tumour cell detection reagent is prepared, such as prepare
For detection probe.
Include the C- types single domain antibody being prepared into the various albumen containing the antibody and based on this in above application
Derivative (for example being coupled other molecules) of antibody etc., is prepared into fusion antibody or coupled antibody for another example；Fusion antibody is this hair
The peptide fragment of bright antibody and several amino acid includes but are not limited to itself, after the fusions of the Fc sections of antibody to the fusion of protein
It obtains；Coupled antibody include but are not limited to antibody of the present invention and radioactive isotope, it is toxin conjugated after obtain.
Beneficial effects of the present invention：By from m01s be skeleton phage display library in, with mammalian cell express between
Pi Su is antigen, and screening has obtained a kind of high-affinity C- type single domain antibodies for targeting mesothelin；First, relative to overall length monoclonal antibody
(molecular weight~150kD), such antibody molecule amount is smaller, has better tissue permeability and combines resisting there are steric effect
The ability of former epitope, for treating and diagnosing the tumour (such as adenocarcinoma ovaries) of some mesothelin height expression or even can develop into
Oral medicine；Secondly, because its m01s skeleton being based on has the binding ability with FcRn, its plasma half-life may also reach
10 hours, this is just longer than based on VHEtc. (usually a few minutes to more than ten plasma half-life of the single domain antibody of other skeletons
Minute)；Third, can be expressed in prokaryotic expression system, production cost is low, the period is short.And by the present invention from such antibody
The F3 filtered out has good inhibiting effect for the ovary adenocarcinoma cells OVCAR-3 of mesothelin height expression, and application prospect is good.
Description of the drawings
The structure diagram of the mutant m01s of the CH2 structural domains of Fig. 1 behaviour IgG antibodies.
Fig. 2 is the SDS electrophoretograms of the mesothelin of purifying.
Fig. 3 is the SDS electrophoretograms of the C- type single domain antibodies F3 of purifying.
Fig. 4 is the dose-effect curve that the F3 that ELISA is measured and mesothelin combine.
Fig. 5 is the dose-effect curve that F3, F3-1, F3-2 that ELISA is measured and mesothelin combine.
Fig. 6 is that flow cytometry detects the streaming figure that F3 is combined with the mesothelin that OVCAR-3 cell surfaces are expressed.
Fig. 7 is that CCK8 methods detect activity suppression effect contrast figures of the F3 to OVCAR-3 cell Proliferations.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.Following embodiment is with this
Implemented under premised on inventive technique scheme, give detailed embodiment and specific operating process, but the present invention
Protection domain is not limited to following embodiments.
Embodiment 1：The gene of monoclonal mesothelin
About 2 × 10 are inoculated in 6 orifice plates of culture cell6A Proliferation of Human Ovarian Cell OVCAR-3 cultivates 12~16h, cell
Grow up to individual layer.Culture medium is removed, cell total rna, obtained cell are extracted with Trizol Reagent (Invitrogen companies)
Total serum IgE is dissolved in 50 water of the μ L without RNA enzyme.Then M-MLV Reverse Transcriptase kits (Promega companies) are used, using random
Primer, reverse transcription obtain cDNA.
According to the gene order (GenBank No.AY743922) of mesothelin, its amino acid sequence is analyzed, designs mesothelin
Extracellular domain (amino acid 296-600) (Feng Y, the et al., Mol of the forward and reverse primer amplification mesothelin polypeptide of gene
Cancer Ther.,2009)：Forward primer GGAATTC(horizontal line is labeled as EcoR I digestions position to TG GAAGTGGAGAAGACAGC
Point)；Reverse primer CGAGCCGTCCCCGAGAGG (1), CCGCTCGAG(horizontal line is labeled as Xho I digestions position to CCGTCCCC (2)
Point).PCR is first carried out by template of cDNA with forward primer and reverse primer 1, obtains mesothelin gene segment, then drawn with forward direction
Object carries out PCR with reverse primer 2 by template of mesothelin gene segment, so as to obtain containing EcoR I and Xho I restriction enzyme sites
Mesothelin gene segment.
Agarose gel electrophoresis recycles mesothelin gene segment, with EcoR I and Xho I double digestions mesothelin gene and load
Mesothelin gene after recycling with carrier pSecTag2A connect, converts by body pSecTag2A, carrier construction pSecTag2A-
MSLN, upgrading grain is used for subsequent experimental from sequencing identification sequence correctly clone.
Embodiment 2：It expresses and purifies mesothelin
By 293F cells, (control cell density is 5 × 10 within 1 day before transfection5A/ml) inoculated and cultured 40mL cell liquid,
37 DEG C, 8%CO2, overnight incubation in the constant incubator of 135r/min.40 μ g plasmids is taken to be placed in the Falcon pipes of 14mL.Add
Enter 4mLD-PBS, gently mixing 3 times, every amalgamation in 6 seconds time.60 μ L (2mg/mL) PEI are added in, as stated above mixing.It will be mixed
It closes object and is stored at room temperature 20-30min, drop by drop add in cell, mixture is made to be come into full contact with as possible with cell.Matter will be transfected
The cell of grain, is placed in 37 DEG C, 8%CO2, cultivate in the constant incubator of 135r/min.
Cell sample is observed, and collects transfection for 24 hours, 48h, 72h, the cell sample of 96h, 120h, with mouse source anti-His
Pass through protein immunoblot as primary antibody (used carrier provides 6 × His Tag labels in the C-terminal of expressed albumen and is used to detect)
(Western Blot) detects the expression of mesothelin.
It detects after 293F cell transfectings pSecTag2A-MSLN after expression mesothelin, expands cell culture and advised with transfection
Mould, great expression mesothelin protein.Culture medium supernatant is collected, mesothelin is purified with Ni-NTA fillers (GE companies), then uses and cut
It stays the ultra-filtration centrifuge tube (Merck Millipore companies) that molecular weight is 10kD that mesothelin is concentrated by ultrafiltration, is verified through SDS-PAGE
Its purity, the results are shown in Figure 2, and swimming lane 1 is mesothelin.
Embodiment 3：The structure of phage display library is to screen
M01s is the mutant of the CH2 structural domains of human antibody IgG, relative to CH2, the seven amino acid quilt of its N- ends
It truncates to enhance resistant to aggregation ability, and the combination of the pH dependences of FcRn enhances；In addition to the disulfide bond (Native of itself
Disulfide bond) outside, also contain a pair of artificial disulfide bond (Engineered disulfide bond) to enhance stabilization
Property.Three loop areas contained by m01s are Loop BC, Loop DE and Loop FG respectively, they can be used for introduce mutation from
And build antibody library.
Using m01s as skeleton (its gene order is Seq ID No.10, amino acid sequence is Seq ID No.9), according to
There is the method structure phage library of document (Gong R, et al., PLoS ONE, 2012), between mammalian cell expression
Pi Su is screened for antigen, is screened by 4 wheels, is obtained the clone of an enrichment, be named as F3.
By sequencing, the amino acid full length sequence of F3 is：Seq ID No.7, coding DNA full length sequence are：Seq ID
No.8；
The amino acid sequence of F3 and m01s is in 3 loop areas difference, three loop areas of F3, the ammonia of Loop BC
Base acid sequence is Seq ID No.1, and gene order is Seq ID No.2；The amino acid sequence for encoding Loop DE is Seq ID
No.3, gene order are Seq ID No.4；The amino acid sequence for encoding Loop FG is Seq ID No.5, gene order Seq
ID No.6。
Embodiment 4：The expression and purification of F3
F3 is expressed and purified according to existing document (Gong R, et al., Methods Mol Biol., 2012).
Protokaryon F3 expression vectors are built, are transformed into E.coli HB2151.Strain is inoculated in the SB culture mediums containing 100 μ g/ml ammonia benzyls
(tryptone containing 30g, 20g yeast extracts and 10g MOPS, 7.0) pH value is adjusted to NaOH in, treat OD in 1L culture mediums600
IPTG to final concentration of 200 μ g/ml is added in when reaching 0.7~1.0, induced expression 14 is carried out under conditions of 37 DEG C, 220rpm
~16h.4 DEG C, thalline were collected by centrifugation by 6000rpm, 15min, abandons culture medium, precipitation is resuspended in Buffer A (50mM Tris-
HCL, 450mM NaCL, pH 8.0) in, then supernatant is collected by centrifugation after polymyxin B (polymyxin B) is handled 1 hour.With
Ni-NTA resins (QIAGEN companies) purify F3, verify its purity through SDS-PAGE, as shown in Figure 3.Then use molecular cut off
F3 is concentrated by ultrafiltration in ultra-filtration centrifuge tube (Merck Millipore companies) for 3kD.It is marked containing 6 × His the C- ends of obtained F3
Label and FLAG labels.
Embodiment 5：ELISA measures the combination of F3 and mesothelin
Mesothelin (2 μ g/mL) is coated on elisa plate, with PBS+3%milk in 37 DEG C of closings after 4 DEG C of overnight incubations
1h.Add in the F3 that is serially diluted, 37 DEG C be incubated 2 hours after washed four times with PBST (PBS+0.05%Tween 20), then plus horseradish
The anti-FLAG monoclonal antibodies of mouse of peroxidase (HRP) label are washed four times with PBST, added after 37 DEG C are incubated 1 hour
ABTS is detected.Skeleton m01s is as negative control.As shown in figure 4, the EC that F3 and mesothelin combine50For 26nM, m01s is not
It can be combined with mesothelin.
With existing Chinese invention granted patent " the CN201310667427.2 a kind of nano antibody and its coding of anti-mesothelin
The purposes of gene and the nano antibody " is compared, in the prior art the EC of disclosed mesothelin antibody50For 273nM, antibody of the present invention
Combination activity it is much higher.
Embodiment 6：ELISA measures the combination of F3, F3-1, F3-2 and mesothelin
The process of experimental example 5 is repeated, F3 is only replaced with into F3-1 and F3-2.
F3-1 is the difference for employing antibody and F3 that m01 (introducing the CH2 mutant that a pair of of disulfide bond obtains) is skeleton
It is not clip the seven amino acid of N- ends；Its amino acid and DNA sequence dna are respectively：Seq ID No.11 and 12.
F3-2 is to employ antibody that CH2 is skeleton and F3 difference lies in the seven amino acid for not clipping N- ends,
Also without introducing artificial disulfide bond；Its amino acid and DNA sequence dna are respectively：Seq ID No.13 and 14.
F3-1 and F3-2 may be used DNA it is artificial synthesized after again expression way obtain, preparation method is the prior art, is not done
It repeats.
Experimental result is as shown in figure 5, the combination activity of F3-1 and F3-2 can not show a candle to F3, and under study for action, inventor has found F3-
1 resistance to aggregation will be substantially weaker than F3, and the skeleton of F3-2 is unstable.
Embodiment 7：The combination of the mesothelin of flow cytometry detection F3 and OVCAR-3 cell surface expression
Take OVCAR-3 cells (about 1 × 106It is a), it is incubated 2 hours at 37 DEG C with F3, PBSA, PBS add after being respectively washed 3 times
Enter the anti-FLAG monoclonal antibodies of mouse, 37 DEG C are incubated 1 hour.Fluorescence secondary antibody (Texas is added in after being respectively washed 3 times with PBSA, PBS
The sheep anti-mouse igg of Red couplings), after 37 DEG C are incubated 1h, then it is respectively washed with PBSA, PBS and washes 3 times after in flow cytometer
On analyzed.Using m01s (skeleton) as negative control.The results are shown in Figure 6, it was demonstrated that F3 can combine high expression mesothelin
The surface of cell (people ovary adenocarcinoma cells OVCAR-3).
Embodiment 8：CCK8 methods detect inhibitory activity of the F3 to OVCAR-3 cell Proliferations
By the OVCAR-3 (1 × 10 of 100 μ L4A/hole) 96 orifice plate of cell inoculation, place incubator (37 DEG C, 5%CO2) in
Cultivate 16-18h.The F3 protein solutions of various concentration are added in as experimental group, with the cell normally cultivated (containing culture medium, nothing
Albumen) as a control group (PBS), for skeleton m01s as negative control, every group sets 3 parallel controls.Culture for 24 hours, 48H and 72h
Afterwards, the culture solution in reject culture hole, every hole add in 100 μ L cell culture fluids and 10 μ L CCK-8 reagents, are placed in culture respectively
1h is incubated in case, the absorbance at 450nm, i.e. A450 are measured with microplate reader.According to the A450 of measure, calculated according to formula thin
Born of the same parents' survival rate, so as to which reflection candidate clone is for the inhibitory activity of tumor cell proliferation indirectly, the results are shown in Figure 7, between F3 pairs
Skin element high-expression cell line (such as OVCAR-3) has direct repression.
Sequence table
<110>Wuhan Virology Institute,Chinan academy of Sciences
<120>Target high-affinity C- type single domain antibodies of mesothelin and preparation method and application
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His Asn Ala Lys Thr Lys Pro Arg Asp Asp Tyr Asp Tyr Asn Ser Tyr
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tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga tgattacgat 180
tacaactcct atcgtgtggt cagcaaactc accgtcctgc accaggactg gctgaatggc 240
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tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 180
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 240
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<213> F3-1
<400> 12
gcacctgaac tcctgggggg accgtcagtc ttgtgcttcc ccccaaaacc caaggacacc 60
ctcatgatct cccggacccc tgaggtcaca tgcgtggtga aagattatta ctaccacgct 120
agtcctattg ctaagttcaa atggtacgtg gacggcgtgg aggtgcataa tgccaagaca 180
aagccgcggg atgattacga ttacaactcc tatcgtgtgg tcagcaaact caccgtcctg 240
caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaccat tcatagcaat 300
acccatatca gtattcccat cgagtgcacc atctccaaag ccaaa 345
<210> 13
<211> 115
<212> PRT
<213> F3-2
<400> 13
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Leu Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Lys Asp Tyr Tyr Tyr His Ala Ser Pro Ile Ala Lys Phe Lys Trp
35 40 45
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Asp
50 55 60
Asp Tyr Asp Tyr Asn Ser Tyr Arg Val Val Ser Lys Leu Thr Val Leu
65 70 75 80
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Thr
85 90 95
Ile His Ser Asn Thr His Ile Ser Ile Pro Ile Glu Lys Thr Ile Ser
100 105 110
Lys Ala Lys
115
<210> 14
<211> 345
<212> DNA
<213> F3-2
<400> 14
gcacctgaac tcctgggggg accgtcagtc ttgctcttcc ccccaaaacc caaggacacc 60
ctcatgatct cccggacccc tgaggtcaca tgcgtggtga aagattatta ctaccacgct 120
agtcctattg ctaagttcaa atggtacgtg gacggcgtgg aggtgcataa tgccaagaca 180
aagccgcggg atgattacga ttacaactcc tatcgtgtgg tcagcaaact caccgtcctg 240
caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaccat tcatagcaat 300
acccatatca gtattcccat cgagaaaacc atctccaaag ccaaa 345
Claims (10)
1. a kind of high-affinity C- type single domain antibodies for targeting mesothelin, it is characterised in that：It is with human antibody IgG constant regions
The mutant m01s of CH2 structural domains is skeleton, builds polypeptide and protein library, then obtain by antigen selection of mesothelin.
2. the high-affinity C- type single domain antibodies of mesothelin are targeted according to claim 1, it is characterised in that：The polypeptide and
Protein library is phage display library.
3. the high-affinity C- type single domain antibodies of mesothelin are targeted according to claim 1, it is characterised in that：The C- types list
Domain antibodies are F3, it possesses tri- loop areas of BC, DE, FG, and the amino acid sequence of the Loop BC is Seq ID No.1, described
The amino acid sequence of Loop DE is that the amino acid sequence of seq ID No.3, the Loop FG are Seq ID No.5.
4. the high-affinity C- type single domain antibodies of mesothelin are targeted according to claim 3, it is characterised in that：The volume of the F3
The gene order of code Loop BC is seq ID No.2, and the gene order of coding Loop DE is Seq ID No.4, encodes Loop
The gene order of FG is Seq ID No.6.
5. the high-affinity C- type single domain antibodies of mesothelin are targeted according to claim 3, it is characterised in that：The ammonia of the F3
Base acid full length sequence is Seq ID No.7.
6. the high-affinity C- type single domain antibodies of mesothelin are targeted according to claim 3, it is characterised in that：The base of the F3
Because full length sequence is Seq ID No.8.
7. the high-affinity C- type single domain antibodies of mesothelin are targeted according to claim 3, it is characterised in that：Loop is removed to F3
Region in addition carries out the mutation of base and amino acid, and obtains and the functionally similar antibody of F3.
8. targeting the preparation method of the high-affinity C- type single domain antibodies of mesothelin described in claim 1, include the following steps：
(1) carrier for expression of eukaryon containing mesothelin gene sequence is built, transfection mammalian cell expresses mesothelin, separation
Purifying obtains mesothelin；
(2) it is that antigen is screened with the mesothelin of purifying from the phage display library using m01s as skeleton, after number wheel,
Obtain the clone of the specific binding of a high-affinity；
(3) expression and purification clone obtains the high-affinity C- type single domain antibodies of targeting mesothelin.
9. targeting the high-affinity C- type single domain antibodies of mesothelin described in claim 1, preparing, high expression mesothelin tumour is thin
Application in the inhibitor of born of the same parents or the application in high expression mesothelin tumour cell detection reagent is prepared.
10. the application of the high-affinity C- type single domain antibodies of targeting mesothelin according to claim 6, including by the C- types
Single domain antibody is prepared into the albumen containing the antibody and the derivative based on the antibody.
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| CN114685666A (en) * | 2020-12-28 | 2022-07-01 | 浙江纳米抗体技术中心有限公司 | Anti-mesothelin nano antibody and application thereof |
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