CN1299833A - Human vessel endothelium growth factor resisting single stranded antibody and its preparation - Google Patents
Human vessel endothelium growth factor resisting single stranded antibody and its preparation Download PDFInfo
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Abstract
The present invention relates to single chain antibody (ScFv) of antihuman vascular endothelial growth factor (VEGF) for curing tumor of human body. Its aesign and preparation. It prepares VEGF synthetic peptide to establish antihuman VEGF monoclonal antibody (MAb) hybridoma cell strain. SAid MAb possesses stable bioactivity and obvious tumor-inhibiting action. Said invention make RT-PCR amplificatino of light and heavy chain genes of MAb variable region, and uses DNA fragment to implement connection to constitute antihuman VEGF ScFv gene and finally obtain the high-effective expres of ScFv. The tests show that said antihuman VEGF ScFv is small in molecular weight, possesses strong power for penetrating tumor ittue, its immunogenicity is low, so that it possesses the tumor-resisting function.
Description
The present invention relates to be used for the treatment of the antibody of human tumor, especially for single-chain antibody of human vessel endothelium growth factor resisting and preparation method thereof.
Tumor growth relies on vasculogenesis, suppresses tumor-blood-vessel growth and can contain tumor growth effectively.Vascular endothelial growth factor (Vascular Endothelial Growth Factor VEGF) plays a part most critical in the induced tumor angiogenesis, therefore, the blocking VEGF activity can significantly suppress tumor growth.In " Inhibition of vascular endothelial growth factor-induced angiogenesissuppresses tumor growth in vivo. " (Nature, 1993; 362 (6423): 841-844)) Kim etc. discloses anti-VEGF monoclonal antibody first and can suppress the mice with tumor tumor growth in.But mouse source monoclonal antibody easily brings out human antimouse antibody (HAMA) immune response, is difficult to use in clinical.At " Humanization of an anti-vascular endothelial factor monoclonal antibody for the therapy of solid tumorsand other disorders. " (Cancer Res.1997; Leonard etc. clones antibody variable gene 57:4593-4599) from the hybridoma cell strain of secreting above-mentioned monoclonal antibody.Made up people-mouse chimeric antibody Fab, build by computer mould again and the rite-directed mutagenesis method, with the Fab humanization. obtain low immunogenicity, the humanization Fab that keeps simultaneously former parental antibody biologic activity again, but Fab is a heterodimer is not suitable for forgiving great expression, is not easy to make up antibody fusion protein.That research is maximum at present is single-chain antibody (ScFv), and its superiority is to pass through the inclusion body great expression, is easy to the genetically engineered operation, especially is easy to make up antibody fusion protein.Recently, U.S.'s recombinant DNA board of consultants (RAC) approved 2 clinical trials for the treatment of disease with single-chain antibody.But do not see the report that anti-people VEGF single-chain antibody is arranged so far as yet.
Purpose of the present invention provides a kind of molecular weight little, strong to the tumor tissues penetration power in order to overcome above-mentioned weak point of the prior art just, and low human vessel endothelium growth factor resisting (VEGF) single-chain antibody (ScFv) of autoimmunization source property and preparation method thereof, this human vessel endothelium growth factor resisting single stranded antibody reaches the purpose of the neoplasm growth that suppresses tumor-blood-vessel growth by its high expression level that checks body tumor tissue's vascular endothelial growth factor.
The gene of the human vessel endothelium growth factor resisting single stranded antibody that the present invention is designed and aminoacid sequence are:
60GAG?GTG?CAG?CTT?CTG?GAG?TCT?GGG?GCA?GAG?CTT?GTG?AAG?CCA?GGG?GCC?TCA?GTC?AAG?TTGGlu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Ala?Glu?Leu?Val?Lys?Pro?Gly?Ala?Ser?Val?Lys?Leu
120TCC?TGC?ACA?GCT?TCT?GGC?TTC?AAC?ATT?AAA?GAC?ACC?TAT?ATG?CAC?TGG?GTG?AAG?CAG?AGGSer?Cys?Thr?Ala?Ser?Gly?Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Met?His?Trp?Val?Lys?Gln?Arg
CDR1
180CCT?GAA?CAG?GGC?CTG?GAG?TGG?ATT?GGA?AGG?ATT?GAT?CCT?GCG?AAT?GGT?AAT?ACT?AAA?TATPro?Glu?Gln?Gly?Leu?Glu?Tro?Ile?Gly?Arg?Ile?Asp?Pro?Ala?Asn?Gly?Asn?Thr?Lys?Tyr
52a????????????CDR2
240GAC?CCG?AAG?TTC?CAG?GGC?AAG?GCC?ACT?ATA?ACA?GCA?GAC?ACA?TCC?TCC?AAC?ACA?GCC?TACAsn?Pro?Lys?Phe?Gln?Gly?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr
300CTG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?ACT?GCC?GTC?TAT?TAC?TGT?GCT?AGG?CCA?TCTGln?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Pro?Ser
82a?82b?82c
360ATT?TAC?TAC?GGT?AGT?AAC?CAC?TGG?TAC?TTC?GAT?GTC?TGG?GGC?GCA?GGA?ACC?TCA?GTC?ACCIle?Tyr?Tyr?Gly?Ser?Asn?His?Trp?Tyr?Phe?Asp?Val?Trp?Gly?Ala?Gly?Thr?Ser?Val?Thr???CDR3???????????100a100b100c100d100e100fGTC?TCC?TCA?GGT?GGA?GGC?GGT?TCA?GGC?GGA?GGT?GGC?TCT?GGC?GGT?GGC?GGA?TCGVal?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
Linker
60GAC?ATT?GTG?CTG?ACA?CAG?TTT?CCT?GCT?TCC?CTT?AGC?GTA?TTT?TTG?GGG?CAG?AGG?GCC?ACCAsp?Ile?Val?Leu?Thr?Gln?Pro?Pro?Ala?Ser?Leu?Ser?Val?Pro?Leu?Gly?Gln?Arg?Ala?Thr
120ATT?TCA?TGC?AGG?GCC?AGC?CAA?AGT?GTC?AGT?ACA?TAT?GGC?TAT?AGT?TAT?ATG?CAC?TGG?AACIle?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Thr?Tyr?Gly?Tyr?Ser?Tyr?Met?His?Trp?Asn
27a?27b?27e?27d?CDR1
180CAA?CAG?AAA?CCA?GGA?CAG?CCA?CCC?AGA?CTC?CTC?ATT?TAT?CTT?GTA?TCC?AAC?CTA?GAA?TTTGln?Gln?Lys?Pro?Gly?Gln?Pro?Pro?Arg?Leu?Leu?Ile?Tyr?Leu?Val?Ser?Asn?Leu?Glu?Phe
CDR2
240GGG?GTC?CCT?GCC?AGG?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACA?GAC?TTC?ACC?CTC?AAC?ATC?CATGly?Val?Pro?Ala?Arg?Pro?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Ser?Thr?Leu?Asn?Ile?His
300CCT?GTG?GAG?GAG?GAG?GAT?GCT?GCA?ACC?TAT?TAC?TGT?CAG?CAC?ATT?AGG?GAG?CTT?CCG?TACPro?Val?Glu?Glu?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?His?Ile?Arg?Glu?Leu?Pro?Tyr
CDR3
ACG?TTC?GGA?GGG?GGG?ACC?AAG?CTG?GAA?ATC?AAA
Thr?Pro?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys
Above-mentioned human vessel endothelium growth factor resisting single stranded antibody contains 249 amino acid, and molecular weight is 26648.1 dalton, and iso-electric point is 5.90, is made up of three parts, i.e. light, heavy chain of antibody variable region and binding peptide.This three part is finished identification and conjugated antigen (VEGF) jointly, the bioactive effect of performance blocking VEGF.Its tomograph is seen Fig. 1.
The method for preparing above-mentioned human vessel endothelium growth factor resisting single stranded antibody is: prior art protein secondary structure prediction scheme, hydrophilic scheme and antigenic index scheme are combined, designed with 26 one section polypeptide that amino-acid residue is a template of people VEGF189N end, and in ABI431 solid phase automatic DNA synthesizer DNA (Applied-Biosystems PE company, the U.S.) upward synthetic, purity is 97.4%.Set up synthetic peptide monoclonal antibody (MAb) hybridoma cell strain (name and be E11) of anti-people VEGF according to a conventional method.The E11 hybridoma cell strain is cultivated, gone down to posterity, be inoculated in mouse peritoneal after resuspended, draw ascites after 12 days.Then adopt the anti-people VEGF MAb in the quick batch of purification via adsorption-based process mouse ascites fluid, and adopt ultra-filtration membrane dialysis mode to concentrate.After measured, anti-people VEGF MAb content is 2.4mg/ml, belongs to IgG2b (γ, κ 2b).This MAb possesses good antigen-specific and antigens with higher avidity, and can suppress vasculogenesis and the tumor growth of people's cheek cancer BCaCD885 and rat meat knurl S180 effectively.On this basis, to aforesaid well-grown, can continue, the hybridoma cell strain E11 (vitro culture half a year) of the anti-people VEGF of stably excreting MAb extracts total RNA, respectively at VL and two and five primers of VH gene design, then carry out pcr amplification, cloned the variable region gene fragment of MAb.Dna fragmentation ((GGGGS) with coding wetting ability peptide linker
3) the E11 monoclonal antibody is light, heavy chain variable region gene connection, PET-15YV (Novagen company) carrier is gone in transduction, then imports in the e. coli bl21 (DE3) and expresses.And then at carrying out the production technique improvement with the anti-people VEGF ScFv expression system [PET-15YV/BL21 (DE3)] of inclusion body (IBS) formal representation, the preparation links such as separation, sex change and renaturation of inclusion body.The inclusion body that has obtained to account for bacterial protein expression amount 52.9% efficiently expresses the high yield with 48mg/ml.Anti-people VEGF ScFv adopts Westemblot, and indirect elisa method and competitive ELISA method confirm and can synthesize peptide, VEGF with VEGF respectively
165The specificity combination, the reaction titre reaches 8 * 10 respectively
-3With 5 * 10
-5And its avidity to VEGF is close with former parent's monoclonal antibody.Simultaneously, also determine anti-people VEGF ScFv one of important attribute-iso-electric point is 5.90.Also resist people VEGF single-chain antibody the variation of transplanting cheek cancerous tissue penetration power and be detained in lotus people cheek cancer nude mice serum is carried out corresponding comparative studies with anti-people VEGF monoclonal antibody (MAb) respectively.Found that: anti-people VEGF ScFv cheek cancerous tissue penetration power (speed) is much higher than anti-people VEGF MAb (48: 1); Serum clearance rate in its 1 hour is about 10 times of anti-people VEGF MAb.These presentation of results: anti-people VEGF ScFv is as a kind of successful small molecules genetic engineering antibody, have to the tumor tissues penetration power strong and in serum short characteristic of residence time, be the antibody materials of a kind of ideal immunotherapy of tumors and radioimmunotherapy and the ideal carrier of radioautograph imaging, have purposes widely.
The present invention compared with prior art has following advantage:
1. molecular weight is little, and is strong to the solid tumor penetration power;
2. be easy to make up antibody fusion protein;
3. be suitable for the inclusion body great expression;
4. preparation procedure is simple, easy handling, efficient height;
5. easier being eliminated of immune complex that forms with antigen is fit to the effect of performance antibody blocking VEGF;
6. be used for intracellular immunity, can be used as a kind of scheme of gene therapy;
7. labelled nuclide can be used for immune imaging diagnosis and treatment;
8. immunogenicity is little, reduces the possibility of HAMA reaction, and further humanization.
Below be the drawing explanation of Figure of description:
Fig. 1 is that the three-dimensional mould of the anti-people VEGF of the present invention single-chain antibody (ScFv) is built structure iron.Yellow is an antigen recognition site among the figure, and green is the skeleton district, and pink colour is a connection peptides, Lignt Chain: light chain; Heavy Chain: heavy chain; Linker: connection peptides.
Fig. 2 is the tumor-inhibiting action of the anti-people VEGF of the present invention monoclonal antibody (MAb) to mouse BCaCD885 transplanted tumor.1. physiological saline groups among the figure; 2.MAb intraperitoneal injection group (100 μ g/d); 3.MAb drug administration by injection group under the knurl perithelium (200 μ g/d).
Fig. 3 is the tumor-inhibiting action of the anti-people VEGF of the present invention monoclonal antibody (MAb) to mouse S180 transplanted sarcoma.1. physiological saline groups among the figure; 2.MAb intraperitoneal injection group (100 μ g/d); 3.MAb intraperitoneal injection group (200 μ g/d); 4.MAb drug administration by injection group under the knurl perithelium (200 μ g/d); 5.5-Fu intraperitoneal injection group.
Fig. 4 is VH VL pcr amplification product of the present invention gel electrophoresis.A:VH among the figure; B:Marker is followed successively by 657 from the bottom to top, (458,434), 328,289,267bp C:VL.
Fig. 5 is that recombinant plasmid PET15-YV enzyme of the present invention is cut evaluation.A:Nco I among the figure+Not I+BamH I is downcut VL and VH+Linker; B:Marker.
Fig. 6 is the structure of secreting, expressing plasmid PET15-YV of the present invention.
Fig. 7 is the structure of the anti-people VEGF of the present invention single-chain antibody gene sequencing vector.
Fig. 8 is expression and the purifying 15%SDS-PAGE result of PET15-YV of the present invention.A among the figure: low molecular weight protein (LMWP) standard; The full bacterium of B:PET15-YV; The C:PET15-YV supernatant; The D:PET15-YV inclusion body.
Fig. 9 is that the low differentiation of the present invention cheek cancerous tissue VEGF immunohistochemical staining (ScFv) cancer nests is dark-brown.
The present invention is described in further detail below with reference to embodiment:
The design and the preparation of the synthetic peptide of embodiment 1. people VEGF189: prior art protein secondary structure prediction scheme, hydrophilic scheme and antigenic index scheme are combined, designed with 26 one section polypeptide that amino-acid residue is a template of people VEGF189N end, comprehensively judge according to first three items, be summarized as table one, find 6 continuity epi-positions altogether, be designated as: P1 (10-22), P2 (27-36), P3 (39-49), P4 (66-75), P5 (78-95), P6 (103-112).People VEGF189 the 75th amino acids is a glutamine, is glycosylation site, and polysaccharide may be covered this site, so P4 can not consider.Be to increase success ratio, the P1 of first-selected N-end, because of end more pliable and tougher, more hydrophilic than the centre.In conjunction with the VEGF secondary structure, 21-25 has a β-corner, be everlasting because of corner and make recognition site in known protein matter and the polypeptide, so binding peptide should be designed to P10-25, peptide segment length and its antigenicity are proportional, final synthetic peptide is defined as N-end 1-26 residue (26 is halfcystine, for the covalent attachment point of carrier proteins).Below be the aminoacid sequence of people VEGF189:
APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFK
PSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSF
LQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVPCG
PCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR
Table 1
The synthetic peptide of anti-people VEGF is gone up synthetic at ABI431 solid phase automatic DNA synthesizer DNA (Applied-Biosystems PE company, the U.S.), purity is 97.4%.
| Scheme | The VEGF epi-position of prediction | |||||
| Antigenic index | 10-19 | 27-36 | 40-49 | 66-75 | 82-91 | 103-112 |
| Hydrophilic | 15-21 | 31-37 | 42-48 | 76-82 | 91-97 | |
| Accessible degree | 7-13 | 18-24 | 36-42 | 70-76 | 84-90 | 107-113 |
| Comprehensive judgement | 10-22 | 27-36 | 39-49 | 66-75 | 78-95 | 103-112 |
The preparation of the foundation of synthetic peptide monoclonal antibody (MAb) hybridoma cell strain of embodiment 2. anti-people VEGF and the synthetic peptide MAb of anti-people VEGF: adopt the prepared synthetic peptide of embodiment 1 as antigen, set up synthetic peptide monoclonal antibody (MAb) hybridoma cell strain (name and be E11) of anti-people VEGF according to a conventional method.The E11 hybridoma cell strain is cultivated, gone down to posterity, be inoculated in mouse peritoneal after resuspended, draw ascites after 12 days.Then adopt the anti-people VEGF MAb in the quick batch of purification via adsorption-based process mouse ascites fluid, and adopt ultra-filtration membrane dialysis mode to concentrate.After measured, anti-people VEGF MAb content is 2.4mg/ml, belongs to IgG2b (γ, κ 2b).This MAb possesses good antigen-specific and antigens with higher avidity, and can suppress vasculogenesis and the tumor growth (seeing Fig. 2, Fig. 3) of people's cheek cancer BCaCD885 and rat meat knurl S180 effectively.
Structure, expression and the activity identification of embodiment 3. anti-people VEGF single-chain antibodies (ScFv): on the basis of embodiment 1 and embodiment 2, to aforesaid well-grown, can extract total RNA by hybridoma cell strain E11 (vitro culture half a year) lasting, the anti-people VEGFMAb of stably excreting, respectively at VL and two and five primers of VH gene design, then carry out pcr amplification, cloned the variable region gene fragment of MAb.Anti-people VEGF monoclonal antibody light chain and variable region of heavy chain Nucleotide and deduced amino acid sequence thereof are as follows:
60
GAG?GTG?CAG?CTT?CTG?GAG?TCT?GGG?GCA?GAG?CTT?GTG?AAG?CCA?GGG?GCC?TCA?GTC?AAG?TTG
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Ala?Glu?Leu?Val?Lys?Pro?Gly?Ala?Ser?Val?Lys?Leu
120
TCC?TGC?ACA?GCT?TCT?GGC?TTC?AAC?ATT?AAA?GAC?ACC?TAT?ATG?CAC?TGG?GTG?AAG?CAG?AGG
Ser?Cys?Thr?Ala?Ser?Gly?Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Met?His?Trp?Val?Lys?Gln?Arg
CDR1
180
CCT?GAA?CAG?GGC?CTG?GAG?TGG?ATT?GGA?AGG?ATT?GAT?CCT?GCG?AAT?GGT?AAT?ACT?AAA?TAT
Pro?Glu?Gln?Gly?Leu?Glu?Trp?lle?Gly?Arg?Ile?Asp?Pro?Ala?Asn?Gly?Asn?Thr?Lys?Tyr
52a?????????????CDR2
240
GAC?CCG?AAG?TTC?CAG?GGC?AAG?GCC?ACT?ATA?ACA?GCA?GAC?ACA?TCC?TCC?AAC?ACA?GCC?TAC
Asn?Pro?Lys?Phe?Gln?Gly?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr
300
CTG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?ACT?GCC?GTC?TAT?TAC?TGT?GCT?AGG?CCA?TCT
Gln?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Pro?Ser
82a?82b?82c
360
ATT?TAC?TAC?GGT?AGT?AAC?CAC?TGG?TAC?TTC?GAT?GTC?TGG?GGC?GCA?GGA?ACC?TCA?GTC?ACC
Ile?Tyr?Tyr?Gly?Ser?Asn?His?Trp?Tyr?Phe?Asp?Val?Trp?Gly?Ala?Gly?Thr?Ser?Val?Thr
CDR3???????????100a100b100c100d100e100f
GTC?TCC?TCA?GGT?GGA?GGC?GGT?TCA?GGC?GGA?GGT?GGC?TCT?GGC?GGT?GGC?GGA?TCG
Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
Linker
60
GAC?ATT?GTG?CTG?ACA?CAG?TTT?CCT?GCT?TCC?CTT?AGC?GTA?TTT?TTG?GGG?CAG?AGG?GCC?ACC
Asp?Ile?Val?Leu?Thr?Gln?Pro?Pro?Ala?Ser?Leu?Ser?Val?Pro?Leu?Gly?Gln?Arg?Ala?Thr
120
ATT?TCA?TGC?AGG?GCC?AGC?CAA?AGT?GTC?AGT?ACA?TAT?GGC?TAT?AGT?TAT?ATG?CAC?TGG?AAC
Ile?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Thr?Tyr?Gly?Tyr?Ser?Tyr?Met?His?Trp?Asn
27a?27b?27c?27d?CDR1
180
CAA?CAG?AAA?CCA?GGA?CAG?CCA?CCC?AGA?CTC?CTC?ATT?TAT?CTT?GTA?TCC?AAC?CTA?GAA?TTT
Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro?Arg?Leu?Leu?Ile?Tyr?Leu?Val?Ser?Asn?Leu?Glu?Phe
CDR2
240
GGG?GTC?CCT?GCC?AGG?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACA?GAC?TTC?ACC?CTC?AAC?ATC?CAT
Gly?Val?Pro?Ala?Arg?Pro?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Ser?Thr?Leu?Asn?Ile?His
300
CCT?GTG?GAG?GAG?GAG?GAT?GCT?GCA?ACC?TAT?TAC?TGT?CAG?CAC?ATT?AGG?GAG?CTT?CCG?TAC
Pro?Val?Glu?Glu?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?His?Ile?Arg?Glu?Leu?Pro?Tyr
CDR3
ACG?TTC?GGA?GGG?GGG?ACC?AAG?CTG?GAA?ATC?AAA
Thr?Pro?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys
Table 2
Dna fragmentation ((GGGGS) with coding wetting ability peptide linker
3) the E11 monoclonal antibody is light, heavy chain variable region gene connection, PET-15YV (Novagen company) carrier is gone in transduction, then imports in the e. coli bl21 (DE3) and expresses.
(1) anti-people VEGF single-chain antibody (ScFv) is gene constructed:
According to light, the restriction enzyme mapping of variable region of heavy chain and the restriction enzyme site of carrier construction PET-15S (containing E-tag), P5-P8 in the design PCR primer (table 2).VH is a primer with P5, P6, and P6 contains the connection peptides sequence.VL is primer with P7-P8, is that template is carried out pcr amplification with the VH and the VL that have cloned respectively.Amplified production reclaims through the electrophoresis purifying.The VH of purifying and VL enzyme respectively cut, connect after the repurity, with P5, the amplification of P8 performing PCR, promptly obtain the ScFv complete genome sequence at last.
(2) genetic expression of anti-people VEGF single-chain antibody (ScFv)
ScFv and carrier PET-15S used respectively carry out enzyme with a pair of enzyme (Nco I, Not I) and cut, connect the back and transform DH5 α, bacterium liquid PCR selects positive colony, the capable restriction analysis of extracting plasmid.To PET15-YV be transformed BL21 (DE3) through the plasmid called after PET15-YV of screening.Select positive colony.The inducing culture of expression strain, SDS-PAGE, carrying out ultrasonic bacteria breaking, get supernatant and precipitation respectively, determine the form of expression product.Dilution method renaturation single-chain antibody.1. the acquisition 5000 * g of inclusion body collected 100ml thalline (1mM IPTG induces, and cultivates 3.0 hours for 37 ℃) in centrifugal 15 minutes.With 5ml GTE (0.3mmol/L sucrose; 25mmol/L Tris.HCL, pH8.0; 25mmol/L EDTA) resuspended, recentrifuge is received bacterium, and is resuspended with 3mlGTE.Add N,O-Diacetylmuramidase (10mg/ml) 400 μ l, put upside down mixing 30 minutes; Add 100 μ l Sodium desoxycholates (40mg/ml), 30 μ l MgCl
2(1mol/L), 10 μ l DNase I (1mg/ml), mixing to thalline by thick thinning.Centrifugal 10 minutes of 12000rpm abandons supernatant, and precipitation is with STET (0.1mol/L NaCl-10mmol/L ThisHCl pH8.0), and 1mmol/L EDTA, 0.5%Triton-X 100) washing 3 times, use the TE solution washing 2 times of 3.5mol/L urea again.5000 * g abandoned precipitation in centrifugal 5 minutes; 12000rpm abandoned supernatant in centrifugal 10 minutes.Be precipitated as inclusion body, be used for sex change, renaturation.2. sex change, renaturation: with 1ml sex change liquid (0.1mol/L TrisHCl, pH8.0; 6mol/L) Guanidinium hydrochloride; 2mmol/L EDTA; 0.5% beta-hydroxy ethanol) dissolving inclusion body, room temperature was placed 2.0 hours, and centrifugal 15 minutes of 30000 * g gets supernatant, and the protein content of working sample is adjusted into 10mg/ml with sex change liquid with its concentration.Get supernatant 1ml, join renaturation solution (0.1mol/L TrisHCl, the pH8.0 of 100ml fast; 0.5mol/L the L-arginine, 50 μ lmol/L CuSO
42mmol/L EDTA), 10 ℃ are incubated 24 hours at least.3. dialyse, concentrate: sample is packed in the dialysis tubing, with 2000ml dialyzate (0.1mol/L urea; 20mmol/L TrisHCl, pH8.0; 25mmol/L NaCl) stirs the companion's in 4 ℃ of dialysis 12 hours, and with bar magnet.Use PBST (PBS+1%Triton-X100) dialysis 24 hours again.20%PEG20000 (PBS preparation) concentrates.
(3) sequencing of anti-people VEGF single-chain antibody (ScFy)
The bacterial classification of correct expression band will be obtained, extracting plasmid PET15-YV, ScFv sequence is wherein cut the EcoR I with a pair of enzyme, the Xhol I) under, PUC19 cuts with EcoR I and Sal I enzyme, the sticky end complementation of cutting because of Xhol I and Sal I enzyme, therefore, ScFy can be cloned among the PUC19, carry out the full-automatic bidirectional order-checking.
(4) activity identification and the immunohistochemical methods of anti-people VEGF single-chain antibody (ScFv)
Get cheek cancerous tissue sample, had better differentiation (high, middle differentiation) 16 examples, differentiation degree poor (low differentiation) 4 examples, totally 20 examples.The paraffin section of the about 5 μ m in dyeing back, the dewaxing back is earlier through 0.3%H
2O
2Methanol solution soaks 30 ', drips normal sheep serum then, 37 ℃ down 10 ', one anti-be ScFv renaturation stoste and mouse Anti-E tag antibody successively, two anti-be the anti-mouse-HRP of rabbit, 1 hour after scouring of room temperature reaction is to contain DAB and H
2O
2Substrate solution colour developing, microscopically brown dye positively, dark-brown is a strong positive.
Through above experiment gained result:
1. E11 variable region gene (VH, clone VL) and sequencing
Through RT-PCR, VL and VH all increase, 3 ' the end primer of amplification VH is GH4, the band position, district that agarose gel electrophoresis shows both and estimates that size conforms to respectively about 430 and 370.The sequencing result, VL full length gene 333bp, 111 amino acid of encoding, ownership mouse chain variable region gene III subgroup.VH full length gene 369bp, 123 amino acid of encoding, ownership murine heavy chain variable region gene II (A) subgroup.(see figure 4)
2. the structure of human vessel endothelium growth factor resisting single stranded antibody gene
According to variable region gene sequence design primer P5-P8 light, heavy chain, respectively VL and VH are increased, wherein the edge joint peptide (GGGGS) of variable region of heavy chain
3Gene links to each other.All introduced BamH I site because of the 3 ' end of VH and the 5 ' end of VL, connected through the T4 ligase enzyme, use P5, the P8 performing PCR increases, and promptly obtains complete ScFv complete genome sequence.Electrophoresis result is 760 band.In ScFv gene cloning and expression carrier PET-15 (S), choose positive colony after transforming, the extracting plasmid enzyme restriction is identified (Fig. 5).Qualification result shows, VH-linker-VL connected and directed cloning in PET-15S, called after PET15-YV.The structure of PET15-YV such as (see figure 6).
The sequencing of 3. anti-people VEGF single-chain antibody gene
The structure of single-chain antibody gene sequencing carrier is seen Fig. 7.The DNA automatic sequencing proves that ScFv gene order reading frame is correct among the PET15-YV that obtains to express.
The expression of 4. anti-people VEGF single-chain antibody gene
The PET15-YV of BL21 (DE3) competence through containing single-chain antibody gene transforms, and under the inducing of 0.2mmol/L IPTG final concentration, mainly the form with inclusion body obtains great expression.Inclusion body accounts for 40% of total protein.SDS-PAGE is presented near molecular weight 30KD place a strongly expressed band (see figure 8).Inclusion body is dialysed, is concentrated after the sex change renaturation.
The activity identification of 5. anti-people VEGF single-chain antibody
Use immunohistochemical method, detect the specific reaction of renaturing inclusion bodies product to the paraffin section of 20 routine cheek cancers and healthy tissues, detected result shows that ScFv is identical with former parental antibody to the specific reaction of cheek cancerous tissue.Do not see the positive reaction that except that the high differentiation of 4 examples sample 16 examples of surplusing are positive reaction, wherein the low differentiation of 4 examples sample is strong positive.(see figure 9)
Embodiment 4: gene and aminoacid sequence according to the resulting human vessel endothelium growth factor resisting single stranded antibody of embodiment 1-3 are:
60
GAG?GTG?CAG?CTT?CTG?GAG?TCT?GGG?GCA?GAG?CTT?GTG?AAG?CCA?GGG?GCC?TCA?GTC?AAG?TTG
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Ala?Glu?Leu?Val?Lys?Pro?Gly?Ala?Ser?Val?Lys?Leu
120
TCC?TGC?ACA?GCT?TCT?GGC?TTC?AAC?ATT?AAA?GAC?ACC?TAT?ATG?CAC?TGG?GTG?AAG?CAG?AGG
Ser?Cys?Thr?Ala?Ser?Gly?Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Met?His?Trp?Val?Lys?Gln?Arg
CDR1
180
CCT?GAA?CAG?GGC?CTG?GAG?TGG?ATT?GGA?AGG?ATT?GAT?CCT?GCG?AAT?GGT?AAT?ACT?AAA?TAT
Pro?Glu?Gln?Gly?Leu?Glu?Trp?Ile?Gly?Arg?Ile?Asp?Pro?Ala?Asn?Gly?Asn?Thr?Lys?Tyr
52a?????????????CDR2
240
GAC?CCG?AAG?TTC?CAG?GGC?AAG?GCC?ACT?ATA?ACA?GCA?GAC?ACA?TCC?TCC?AAC?ACA?GCC?TAC
Asn?Pro?Lys?Phe?Gln?Gly?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr
300
CTG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?ACT?GCC?GTC?TAT?TAC?TGT?GCT?AGG?CCA?TCT
Gln?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Pro?Ser
82a?82b?82c
360
ATT?TAC?TAC?GGT?AGT?AAC?CAC?TGG?TAC?TTC?GAT?GTC?TGG?GGC?GCA?GGA?ACC?TCA?GTC?ACC
Ile?Tyr?Tyr?Gly?Ser?Asn?His?Trp?Tyr?Phe?Asp?val?Trp?Gly?Ala?Gly?Thr?Ser?Val?Thr
CDR3???????????100a100b100c100d100e100f
GTC?TCC?TCA?GGT?GGA?GGC?GGT?TCA?GGC?GGA?GGT?GGC?TCT?GGC?GGT?GGC?GGA?TCG
Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
Linker
60
GAC?ATT?GTG?CTG?ACA?CAG?TTT?CCT?GCT?TCC?CTT?ACC?GTA?TTT?TTG?GGG?CAG?AGG?GCC?ACC
Asp?Ile?Val?Leu?Thr?Gln?Pro?Pro?Ala?Ser?Leu?Ser?Val?Pro?Leu?Gly?Gln?Arg?Ala?Thr
120
ATT?TCA?TGC?AGG?GCC?AGC?CAA?AGT?GTC?AGT?ACA?TAT?GGC?TAT?AGT?TAT?ATG?CAC?TGG?AAC
Ile?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Thr?Tyr?Gly?Tyr?Ser?Tyr?Met?His?Trp?Asn
27a?27b?27c?27d?CDR1
180
CAA?CAG?AAA?CCA?GGA?CAG?CCA?CCC?AGA?CTC?CTC?ATT?TAT?CTT?GTA?TCC?AAC?CTA?GAA?TTT
Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro?Arg?Leu?Leu?Ile?Tyr?Leu?Val?Ser?Asn?Leu?Glu?Phe
CDR2
240
GGG?GTC?CCT?GCC?AGG?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACA?GAC?TTC?ACC?CTC?AAC?ATC?CAT
Gly?Val?Pro?Ala?Arg?Pro?5er?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Ser?Thr?Leu?Asn?Ile?His
300
CCT?GTG?GAG?GAG?GAG?GAT?GCT?GCA?ACC?TAT?TAC?TGT?CAG?CAC?ATT?AGG?GAG?CTT?CCG?TAC
Pro?Val?Glu?Glu?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?His?Ile?Arg?Glu?Leu?Pro?Tyr
CDR3
ACG?TTC?GGA?GGG?GGG?ACC?AAG?CTG?GAA?ATC?AAA
Thr?Pro?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys
Embodiment 5: the three-dimensional structure mould of anti-people VEGF single-chain antibody is built: according to embodiment 4 resulting human vessel endothelium growth factor resisting single stranded antibodies, its total length is 249 amino-acid residues.The N-end is VH structural domain (123 residues), and the centre is joining region (15 residue), and the C-end is VL structural domain (111 residues).The primary structure sequence is (the line sequence is the joining region):
EVQLLESGAELVKPGASVKFSCTASGFNIKDTYMHWVKQRPEQGLEWIGR
IDPRNGNTKYLGLNQGKATITATDSSNTAYQQLSSLTSEDTAVYYCARPSI
YYGSSHSYFDVWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQFPASLSVF
LGQRATISCRASQSVSTYGSYMHWNQQKPGQPPRLLIYLVSNLEFGVPARF
SGSGSGTDSTLNIHPVEEEDAATYYCQHIRELPYTFGGGTKLEIK
Antibody is light, the mould of heavy chain three-dimensional structure is built the homologous protein mould that mainly utilizes Biosym and built system (homology) and carry out on SGI computer graphical workstation.Its key step is as follows: (Brookhaven protein data bank PDB) searches for homologous protein in the storehouse at protein data; With the reverse (LOOP district) of homologous protein structure stack with definite structure conserved regions (SCR district) and body structure surface; Mould builds albumen and the homologous protein sequence alignment is built proteic SCR district and LOOP district to determine mould; Side chain is installed; Mould is built structure carry out molecular mechanics and Dynamics Optimization; Mould is built structure carry out soundness verification.Find embodiment 4 resulting human vessel endothelium growth factor resisting single stranded antibodies by three parts, form promptly that antibody variable region is light, heavy chain and binding peptide.This three part is finished identification and conjugated antigen (VEGF) jointly, the bioactive effect of performance blocking VEGF.Its tomograph is seen Fig. 1.
Embodiment 6. human vessel endothelium growth factor resisting single stranded antibodies can be as template, reconstruct (comprising the humanization scheme) according to its nucleotide sequence and amino acid primary structure and three-dimensional space configuration, become the antibody materials of immunotherapy of tumors and radioimmunotherapy.
Claims (4)
1. human vessel endothelium growth factor resisting single stranded antibody, it is characterized in that: the gene and the aminoacid sequence of 1.1 human vessel endothelium growth factor resistings (VEGF) single-chain antibodies (ScFv) are:
GAG?GTG?CAG?CTT?CTG?GAG?TCT?GGG?GCA?GAG?CTT?GTG?AAG?CCA?GGG?GCC?TCA?GTC?AAG?TTG
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Ala?Glu?Leu?Val?Lys?Pro?Gly?Ala?Ser?Val?Lys?Leu
120
TCC?TGC?ACA?GCT?TCT?GGC?TTC?AAC?ATT?AAA?GAC?ACC?TAT?ATG?CAC?TGG?GTG?AAG?CAG?AGG
Ser?Cys?Thr?Ala?Ser?Gly?Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Met?His?Trp?Val?Lys?Gln?Arg
CDR1
180
CCT?GAA?CAG?GGC?CTG?GAG?TGG?ATT?GGA?AGG?ATT?GAT?CCT?GCG?AAT?GGT?AAT?ACT?AAA?TAT
Pro?Glu?Gln?Gly?Leu?Glu?Trp?Ile?Gly?Arg?Ile?Asp?Pro?Ala?Asn?Gly?Asn?Thr?Lys?Tyr
52a?????????????CDR2
240
GAC?CCG?AAG?TTC?CAG?GGC?AAG?GCC?ACT?ATA?ACA?GCA?GAC?ACA?TCC?TCC?AAC?ACA?GCC?TAC
Asn?Pro?Lys?Phe?Gln?Gly?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr
300
CTG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?ACT?GCC?GTC?TAT?TAC?TGT?GCT?AGG?CCA?TCT
Gln?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Pro?Ser
82a?82b?82c
360
ATT?TAC?TAC?GGT?AGT?AAC?CAC?TGG?TAC?TTC?GAT?GTC?TGG?GGC?GCA?GGA?ACC?TCA?GTC?ACC
Ile?Tyr?Tyr?Gly?Ser?Asn?His?Trp?Tyr?Phe?Asp?Val?Trp?Gly?Ala?Gly?Thr?Ser?Val?Thr
CDR3???????????100a100b100c100d100e100f
GTC?TCC?TCA?GGT?GGA?GGC?GGT?TCA?GGC?GGA?GGT?GGC?TCT?GGC?GGT?GGC?GGA?TCG
Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
Linker
60
GAC?ATT?GTG?CTG?ACA?CAG?TTT?CCT?GCT?TCC?CTT?AGC?GTA?TTT?TTG?GGG?CAG?AGG?GCC?ACC
Asp?Ile?Val?Leu?Thr?Gln?Pro?Pro?Ala?Ser?Leu?Ser?Val?Pro?Leu?Gly?Gln?Arg?Ala?Thr
120
ATT?TCA?TGC?AGG?GCC?AGC?CAA?AGT?GTC?AGT?ACA?TAT?GGC?TAT?AGT?TAT?ATG?CAC?TGG?AAC
Ile?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Thr?Tyr?Gly?Tyr?Ser?Tyr?Mer?His?Trp?Asn
27a?27b?27c?27d?CDR1
180
CAA?CAG?AAA?CCA?GGA?CAG?CCA?CCC?AGA?CTC?CTC?ATT?TAT?CTT?GTA?TCC?AAC?CTA?GAA?TTT
Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro?Arg?Leu?Leu?Ile?Tyr?Leu?Val?Ser?Asn?Leu?Glu?Phe
CDR2
240
GGG?GTC?CCT?GCC?AGG?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACA?GAC?TTC?ACC?CTC?AAC?ATC?CAT
Gly?Val?Pro?Ala?Arg?Pro?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Ser?Thr?Leu?Asn?Ile?His
300
CCT?GTG?GAG?GAG?GAG?GAT?GCT?GCA?ACC?TAT?TAC?TGT?CAG?CAC?ATT?AGG?GAG?CTT?CCG?TAC
Pro?Val?Glu?Glu?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?His?Ile?Arg?Glu?Leu?Pro?Tyr
CDR3
ACG?TTC?GGA?GGG?GGG?ACC?AAG?CTG?GAA?ATC?AAA
Thr Pro Gly Gly Gly Thr Lys Leu Glu Ile Lys1.2 human vessel endothelium growth factor resisting single stranded antibody contains 249 amino acid, and molecular weight is 26648.1 roads
Er Dun, iso-electric point is that 5.90,1.3 above-mentioned human vessel endothelium growth factor resisting single stranded antibodies are made up of three parts, promptly antibody variable region is light, heavy
Chain and binding peptide, this three part is finished identification and conjugated antigen (VEGF) jointly, the performance blocking-up
The bioactive effect of VEGF, its tomograph is seen Fig. 1,
2. preparation method who obtains the described human vessel endothelium growth factor resisting single stranded antibody of claim 1: it is characterized in that:
2.1 the design and the preparation of the synthetic peptide of people VEGF189: with prior art protein secondary structure prediction side
Case, hydrophilic scheme and antigenic index scheme combine, and have designed with 26 ammonia of people VEGF189N end
The sour residue of base is one section polypeptide of template, comprehensively judges according to first three items, and adopts solid state chemistry to close
Established law is closed on ABI431 solid phase automatic DNA synthesizer DNA (Applied-Biosystems PE company, the U.S.)
Become, purity is 97.4%, and people VEGF189 aminoacid sequence is:
APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFK
PSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSF
LQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVPCG
The foundation and the anti-people of synthetic peptide monoclonal antibody (MAb) hybridoma cell strain of the anti-people VEGF of PCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR2.2
The preparation of the synthetic peptide MAb of VEGF: adopt 2.1 prepared synthetic peptides as antigen, routinely
Method set up synthetic peptide monoclonal antibody (MAb) hybridoma cell strain of anti-people VEGF (name into
E11)。The E11 hybridoma cell strain is cultivated, gone down to posterity, be inoculated in mouse peritoneal, 12 after resuspended
Ascites is drawn in it back, then adopts the anti-people VEGF in the quick batch of purification via adsorption-based process mouse ascites fluid
MAb, and adopt ultra-filtration membrane dialysis mode to concentrate, after measured, anti-people VEGF MAb content
Be 2.4mg/ml, belong to IgG2b (γ, κ 2b), this MAb possesses good antigen-specific and higher
Antigen avidity, and can suppress the blood of people's cheek cancer BCaCD885 and rat meat knurl S180 effectively
Pipe generates and tumor growth, structure, expression and the activity identification of 2.3 anti-people VEGF single-chain antibodies (ScFv): 2.1 and 2.2
On the basis,, can continue aforesaid well-grown, the hybridization of the anti-people VEGF of stably excreting MAb
Tumor cell strain E11 (vitro culture half a year) extracts total RNA, establishes at VL and VH gene respectively
Count two and five primers, then carry out pcr amplification, cloned the variable region gene sheet of MAb
Section is with the dna fragmentation ((GGGGS) of coding wetting ability peptide linker
3) with the E11 monoclonal antibody
Gently, heavy chain variable region gene connects, PET-15YV (Novagen company) carrier is gone in transduction, then
Import in the e. coli bl21 (DE3) and express, and then at inclusion body (IBS) form table
The anti-people VEGF ScFv expression system [PET-15YV/BL21 (DE3)] that reaches, inclusion body
Preparation links such as separation, sex change and renaturation are carried out the production technique improvement, have obtained to account for the bacterial protein table
The inclusion body of the amount of reaching 52.9% efficiently expresses the high yield with 48mg/ml,
Anti-people VEGF monoclonal antibody variable region of heavy chain Nucleotide and deduced amino acid sequence thereof as
Down:
60
GAG?GTG?CAG?CTT?CTG?GAG?TCT?GGG?GCA?GAG?CTT?GTG?AAG?CCA?GGG?GCC?TCA?GTC?AAG?TTG
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Ala?Glu?Leu?Val?Lys?Pro?Gly?Ala?Ser?Val?Lys?Leu
120
TCC?TGC?ACA?GCT?TCT?GGC?TTC?AAC?ATT?AAA?GAC?ACC?TAT?ATG?CAC?TGG?GTG?AAG?CAG?AGG
Ser?Cys?Thr?Ala?Ser?Gly?Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Met?His?Trp?Val?Lys?Gln?Arg
CDR1
180
CCT?GAA?CAG?GGC?CTG?GAG?TGG?ATT?GGA?AGG?ATT?GAT?CCT?GCG?AAT?GGT?AAT?ACT?AAA?TAT
Pro?Glu?Gln?Gly?Leu?Glu?Trp?Ile?Gly?Arg?Ile?Asp?Pro?Ala?Asn?Gly?Asn?Thr?Lys?Tyr
52a?????????????CDR2
240
GAC?CCG?AAG?TTC?CAG?GGC?AAG?GCC?ACT?ATA?ACA?GCA?GAC?ACA?TCC?TCC?AAC?ACA?GCC?TAC
Asn?Pro?Lys?Phe?Gln?Gly?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr
300
CTG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?ACT?GCC?GTC?TAT?TAC?TGT?GCT?AGG?CCA?TCT
Gln?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Pro?Ser
82a?82b?82c
360
ATT?TAC?TAC?GGT?AGT?AAC?CAC?TGG?TAC?TTC?GAT?GTC?TGG?GGC?GCA?GGA?ACC?TCA?GTC?ACC
Ile?Tyr?Tyr?Gly?Ser?Asn?His?Trp?Tyr?Phe?Asp?Val?Trp?Gly?Ala?Gly?Thr?Ser?Val?Thr
CDR3???????????100a100b100c100d100e100f
GTC?TCC?TCA
The anti-people VEGF of Val Ser Ser monoclonal antibody variable region of light chain Nucleotide and deduced amino acid sequence thereof are as follows:
60
GAC?ATT?GTG?CTG?ACA?CAG?TTT?CCT?GCT?TCC?CTT?AGC?GTA?TTT?TTG?GGG?CAG?AGG?GCC?ACC
Asp?Ile?Val?Leu?Thr?Gln?Pro?Pro?Ala?Ser?Leu?Ser?Val?Pro?Leu?Gly?Gln?Arg?Ala?Thr
120
ATT?TCA?TGC?AGG?GCC?AGC?CAA?AGT?GTC?AGT?ACA?TAT?GGC?TAT?AGT?TAT?ATG?CAC?TGG?AAC
Ile?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Thr?Tyr?Gly?Tyr?Ser?Tyr?Met?His?Trp?Asn
27a?27b?27c?27d?CDR1
180
CAA?CAG?AAA?CCA?GGA?CAG?CCA?CCC?AGA?CTC?CTC?ATT?TAT?CTT?GTA?TCC?AAC?CTA?GAA?TTT
Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro?Arg?Leu?Leu?Ile?Tyr?Leu?Val?Ser?Asn?Leu?Glu?Phe
CDR2
240
GGG?GTC?CCT?GCC?AGG?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACA?GAC?TTC?ACC?CTC?AAC?ATC?CAT
Gly?Val?Pro?Ala?Arg?Pro?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Ser?Thr?Leu?Asn?Ile?His
300
CCT?GTG?GAG?GAG?GAG?GAT?GCT?GCA?ACC?TAT?TAC?TGT?CAG?CAC?ATT?AGG?GAG?CTT?CCG?TAC
Pro?Val?Glu?Glu?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?His?Ile?Arg?Glu?Leu?Pro?Tyr
CDR3
ACG?TTC?GGA?GGG?GGG?ACC?AAG?CTG?GAA?ATC?AAA
Thr?Pro?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys
3. according to the described a kind of human vessel endothelium growth factor resisting single stranded antibody of claim 1, it is characterized in that: this single-chain antibody can be used as the carrier of radioimmunotherapy and radioautograph imaging.
4. according to the described a kind of human vessel endothelium growth factor resisting single stranded antibody of claim 1, it is characterized in that: this single-chain antibody can be as template, reconstruct (comprising the humanization scheme) according to its nucleotide sequence and amino acid primary structure and three-dimensional space configuration, become the antibody materials of immunotherapy of tumors and radioimmunotherapy.
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|---|---|---|---|
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1648939A2 (en) * | 2003-08-01 | 2006-04-26 | Genentech, Inc. | Anti-vegf antibodies |
| CN100392080C (en) * | 2003-07-18 | 2008-06-04 | 中国医学科学院血液学研究所 | Variable region of light chain and heavy chain genes of anti-KDR monoclonal antibody and its use |
| CN102212135A (en) * | 2011-01-14 | 2011-10-12 | 中国科学院北京基因组研究所 | Anti-vascular endothelial growth factor monoclonal antibody and application thereof |
| CN102485753A (en) * | 2010-12-03 | 2012-06-06 | 上海杰隆生物工程股份有限公司 | Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity |
| CN103992405A (en) * | 2008-03-26 | 2014-08-20 | 宜康公司 | Anti-vegf antibody |
| US10072075B2 (en) | 2015-09-23 | 2018-09-11 | Genentech, Inc. | Optimized variants of anti-VEGF antibodies and methods of treatment thereof by reducing or inhibiting angiogenesis |
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| CN109021103A (en) * | 2017-06-12 | 2018-12-18 | 上海睿智化学研究有限公司 | Antibody of human vessel endothelium growth factor resisting and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100392080C (en) * | 2003-07-18 | 2008-06-04 | 中国医学科学院血液学研究所 | Variable region of light chain and heavy chain genes of anti-KDR monoclonal antibody and its use |
| EP1648939A2 (en) * | 2003-08-01 | 2006-04-26 | Genentech, Inc. | Anti-vegf antibodies |
| CN103992405A (en) * | 2008-03-26 | 2014-08-20 | 宜康公司 | Anti-vegf antibody |
| CN103992405B (en) * | 2008-03-26 | 2016-08-17 | 宜康公司 | Anti-VEGF antibodies |
| CN102485753A (en) * | 2010-12-03 | 2012-06-06 | 上海杰隆生物工程股份有限公司 | Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity |
| CN102212135B (en) * | 2011-01-14 | 2013-01-09 | 中国科学院北京基因组研究所 | Anti-vascular endothelial growth factor monoclonal antibody and application thereof |
| CN102212135A (en) * | 2011-01-14 | 2011-10-12 | 中国科学院北京基因组研究所 | Anti-vascular endothelial growth factor monoclonal antibody and application thereof |
| US10072075B2 (en) | 2015-09-23 | 2018-09-11 | Genentech, Inc. | Optimized variants of anti-VEGF antibodies and methods of treatment thereof by reducing or inhibiting angiogenesis |
| US10899828B2 (en) | 2015-09-23 | 2021-01-26 | Genentech, Inc. | Optimized variants of anti-vegf antibodies and methods of use thereof in treatment |
| US10906968B2 (en) | 2015-09-23 | 2021-02-02 | Genentech, Inc. | Polynucleotides encoding optimized variants of anti-VEGF antibodies |
| CN109021103A (en) * | 2017-06-12 | 2018-12-18 | 上海睿智化学研究有限公司 | Antibody of human vessel endothelium growth factor resisting and its preparation method and application |
| CN110320364A (en) * | 2018-03-30 | 2019-10-11 | 洛阳普莱柯万泰生物技术有限公司 | A kind of double antibodies sandwich gold-immunochromatographyreagent reagent for assay box, and the preparation method and application thereof |
| CN108776181A (en) * | 2018-04-16 | 2018-11-09 | 上海老盛昌配送有限公司 | A kind of detection method of clenbuterol hydrochloride |
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