CN103319598B - Insulin-like growth factor-1 receptor (IGF-1R) resistant antibody and coding genes and applications thereof - Google Patents

Insulin-like growth factor-1 receptor (IGF-1R) resistant antibody and coding genes and applications thereof Download PDF

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CN103319598B
CN103319598B CN201310176662.XA CN201310176662A CN103319598B CN 103319598 B CN103319598 B CN 103319598B CN 201310176662 A CN201310176662 A CN 201310176662A CN 103319598 B CN103319598 B CN 103319598B
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antibody
variable region
igf
growth factor
seq
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CN103319598A (en
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华绍炳
王炜
余乃绚
王丽江
胡杰锋
侯伟
竺旭峰
李德强
魏栓林
应建军
寿莹佳
吴敏
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Usa California Aihualang Biological Science & Technology Co Ltd
HANGZHOU DETONG BIOLOGICAL TECHNOLOGY Co Ltd
Zhejiang University ZJU
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Usa California Aihualang Biological Science & Technology Co Ltd
HANGZHOU DETONG BIOLOGICAL TECHNOLOGY Co Ltd
Zhejiang University ZJU
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Abstract

The invention discloses an insulin-like growth factor-1 receptor (IGF-1R) resistant antibody and coding genes and applications thereof. Amino acid sequences of three hypervariable regions CDRH1, CDRH2 and CDRH3 of a heavy-chain variable region of the IGF-1R resistant antibody are respectively: GFSFSSFEMN, YISKSGFTIYYADSVKG and SSWAQDFDWLLPFDW, and the amino acid sequences of three hypervariable regions CDRL1, CDRL2 and CDRL3 of light-chain variable region are respectively: RASQGIRNYLA, AASTLQS and QKYNSVPF. Nucleotide sequences of coding genes of the heavy-chain variable region and the light-chain variable region are shown as SEQ ID No.1 and 3. The applications are applications of the IGF-1R resistant antibody in preparation of antitumor drugs. Compared with the prior art, the IGF-1R resistant antibody can effectively inhibit growth of tumor cells with high-expression of the IGF-1R.

Description

Synalbumin like growth factor-1 receptor antibody and encoding gene thereof and application
Technical field
The invention belongs to gene engineering technology field, relate in particular to synalbumin like growth factor-1 receptor antibody and encoding gene thereof and application.
Background technology
IGF-1R (Insulin-like Growth Factor-1Receptor, IGF-1R) is a kind of transmembrane receptor glycoprotein with tyrosine kinase activity.It is made up of four subunits, i.e. alpha subunit outside two born of the same parents, and two β subunits with part in cross-film and born of the same parents.The alpha subunit of IGF-1R and ligand binding and activate the tyrosine kinase activity of β subunit.
IGF-1R structurally has very high similarity with insulin receptor (Insulin Receptor, IR).In cell transduction process, insulin-like growth factor-i (IGF-1) causes the gathering of signal transmission medium with its acceptor (IGF-1R) combination, thereby activates special signal transduction path, comprises ras-raf-MAPK, P13-K/Akt-1 approach etc.Ras-raf-MAPK approach can irritation cell hyperplasia, P13-K/Akt-1 approach is some other functions of active cell, comprise transcribe, metabolism, growth and translation etc.
Research shows, IGF-1R has abnormal high expression in kinds of tumor cells, for example mammary cancer, lung cancer, colorectal carcinoma, carcinoma of the pancreas, bladder cancer, courageous and upright sarcoma (sarcoma), prostate cancer, liver cancer, ovarian cancer, cancer of the stomach etc. (R.Baserga, F.Peruzzi, K.Reiss. (2003). " The IGF-1Receptor in Cancer Biology. " Int.J.Cancer.107:873-877.; M.Chitnis, J.Yuen, A.Protheroe, et al. (2008). " The Type1Insulin-Like Growth Factor Receptor Pathway. " Clinical Cancer Res.14 (20): 6364-6370.; M.Fidler, D.Shersher, J.Borgia, P.Bonomi. (2012). " Targeting the Insulin-Like Growth Factor Receptor Pathway in Lung Cancer:Problems and Pitfalls. " Therap.Adv in Med.Oncol.4 (2): 51-60.; The .2008. such as the Fang Weili " research of IGF-1R and cancer of the stomach generation development relation." " China's digestion magazine " .28 (8): 522-526.; The .2007. such as the Wang Xiaopeng " impact of blocking-up insulin-like growth factor I receptor on liver cancer cell growth." " Chinese of Journal General Surgery " .22 (3): 211-214.).
Some anti-IGF-1 R antibodies (comprising the clinical trial with other chemotherapy means combination therapys) in the clinical trial for the treatment of tumour have shown curative effect (A.Gombos, O.Metzger-Filho, L.Lago, A.Awada-Hussein. (2012). " Clinical Development of Insulin-like Growth Factor Receptor-1 (IGF-1R) Inhibitors:at the Crossroad. " Invest New Drugs.30:2433-2422.; M.Fidler, D.Shersher, J.Borgia, P.Bonomi. (2012). " Targeting the Insulin-Like Growth Factor Receptor Pathway in Lung Cancer:Problems and Pitfalls. " Therap.Adv in Med.Oncol.4 (2): 51-60.; J.Rodon, V.DeSantos, R.Ferry Jr., R.Kurzrock. (2008). " Early Drug Development of Inhibitors of the Insulin-like Growth Factor-I Receptor Pathway:Lessons from the First Clinical Trials. " Mol.Cancer Ther.7:2575-2588.).Therefore the total man source anti-IGF-1 R antibodies that, acquisition can suppress tumor growth seems very meaningful.
Summary of the invention
The invention provides a kind of synalbumin like growth factor-1 acceptor (IGF-1R) antibody, this antibody is total man source anti-IGF-1 R antibodies, has high-affinity with IGF-1R, can be used for the treatment of human body diseases.
Synalbumin like growth factor-1 receptor antibody, is antibody I, and three hypervariable region CDRH1, CDRH2 of its variable region of heavy chain, the aminoacid sequence of CDRH3 are respectively: gFSFSSFEMN, yISKSGFTIYYADSVKG, sSWAQDFDWLLPFDW(SEQ ID No.3～5);
CDRH1 is positioned at 26th～35 of variable region of heavy chain, and CDRH2 is positioned at 50th～66 of variable region of heavy chain, and CDRH3 is positioned at 99th～113 of variable region of heavy chain;
Three hypervariable region CDRL1, CDRL2 of its variable region of light chain, the aminoacid sequence of CDRL3 are respectively rASQGIRNYLA, aASTLQS, qKYNSVPF(SEQ ID No.8～10);
CDRL1 is positioned at 24th～34 of variable region of light chain; CDRL2 is positioned at 50th～56 of variable region of light chain, and CDRL3 is positioned at 89th～96 of variable region of light chain.
On antibody molecule variable region, there is very large variability in the aminoacid sequence of three hypervariable regions, and the region aminoacid sequence between hypervariable region changes less.These three hypervariable regions can form accurate complementation with antigenic determinant on space structure, and the complementary determining region that is therefore otherwise known as (CDR), the CDR of different heavy chains light chain has determined the specificity of antibody to antigen.
Preferably, the aminoacid sequence of the variable region of heavy chain of antibody I is as shown in SEQ ID No.2, and the aminoacid sequence of variable region of light chain is as shown in SEQ ID No.7.
Antibody I can be the antigen-binding portion thereof of whole antibody or whole antibody.Described whole antibody is preferably IgG1 type; Described antigen-binding portion thereof is preferably Fab fragment, Fab ' fragment, F (ab ') 2fragment or single-chain antibody, more preferably single-chain antibody.
Antigen-binding portion thereof had both retained the region that can be combined with antigen-specific, had avoided again the antigenicity of Fc fragment and the side effect that causes; Wherein single-chain antibody has in easy infiltration tumor tissues increases drug level, and immunogenicity is little, in vivo the transformation period of circulation short, be easy to remove, thereby be easy to be connected with toxin or enzyme gene the advantages such as direct adaptive immune toxin or enzyme labelled antibody.
Antigen-binding portion thereof can be prepared by DNA recombinant technology or by enzymatic/chemical cracking whole antibody.The preparation method of the anti-IGF-1R single-chain antibody of the present invention is: adopt the disclosed method of Chinese patent literature that publication number is CN1444651A to build people's single-chain antibody library, and screen anti-IGF-1 R antibodies in this people's single-chain antibody library.
The present invention also provides the encoding gene of described anti-IGF-1 R antibodies, and the nucleotide sequence of antibody I variable region of heavy chain encoding gene is as shown in SEQ ID No.1, and the nucleotide sequence of variable region of light chain encoding gene is as shown in SEQ ID No.6.
The present invention also provides the recombinant vectors or the expression system that contain described encoding gene.The initial carrier of described recombinant vectors is pACT2 or pET27b.
The present invention also provides described anti-IGF-1 R antibodies in the application of preparing in antitumor drug.Anti-IGF-1 R antibodies of the present invention can, specifically in conjunction with people IGF-1R, effectively suppress the growth of the tumour cell of IGF-1R high expression level.Wherein, the nude mouse that colorectal carcinoma is suffered from the people source anti-IGF-1 R antibodies processing that utilizes total length to recombinate is after 20 days, and tumor size is only 50% of control group.
Compared with prior art, beneficial effect of the present invention is:
Anti-IGF-1 R antibodies of the present invention is the total man source anti-IGF-1 R antibodies that can be used for the treatment of human body diseases, and avidity is high, can specific combination people IGF-1R, the effectively growth of inhibition tumor cell.
Brief description of the drawings
Fig. 1 is the structural representation of single-chain antibody of the present invention;
Fig. 2 is the ELISA detected result of the anti-IGF-1R single-chain antibody #IGF1R of the present invention α-2;
Fig. 3 is that anti-IGF-1 R antibodies DB#2 of the present invention suppresses colorectal carcinoma tumor growth result;
Fig. 4 anti-IGF-1 R antibodies DB#2 of the present invention and 5-FU coupling suppress colorectal carcinoma tumor growth result.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
The screening of the anti-IGF-1R single-chain antibody of embodiment 1
Adopt the disclosed method of Chinese patent literature that publication number is CN1444651A to build people's single-chain antibody library, and screen anti-IGF-1R single-chain antibody in this people's single-chain antibody library, specific implementation process is as follows:
1 amplification obtains human antibody heavy chain and variable region of light chain DNA
Taking the poly A+RNA(from people's marrow, people's tire liver, people's spleen and human peripheral leucocytes purchased from Clontech) as template, utilize oligo(dT) and random primer (random primers), use reverse transcriptase test kit (purchased from Clontech), the Methods Instruction providing according to Clontech test kit, by the reverse poly A+RNA cDNA that is transcribed into.
Taking above-mentioned cDNA as template, utilize the primers of a series of identification human antibody heavy chain variable regions (VH) and variable region of light chain (VL) gene, carry out pcr amplification and obtain the DNA sequence dna of variable region of heavy chain and variable region of light chain all in people's antibody.The primer sequence of a series of identification human antibody heavy chains and chain variable region gene is as follows:
First group of 5 '-end primer (SEQ ID No.11～17) for amplification human antibody heavy chain variable region (VH) gene, comprising:
VH1b：5’- CCATACGATGTTCCAGATTACCAGGTGCAGCTGCAGGAGTC(C/G)G-3’；
VH2b：5’- CCATACGATGTTCCAGATTACCAGGTACAGCTGCAGCAGTCA-3’；
VH3b：5’- CCATACGATGTTCCAGATTACCAGGTGCAGCTACAGCAGTGG?G-3’；
VH4b：5’- CCATACGATGTTCCAGATTACGAGGTGCAGCTG(G/T)TGGAG(A/T)C(C/T)-3’；
VH5b：5’- CCATACGATGTTCCAGATTACCAGGTCCAGCT(G/T)GT(A/G)CAGTCTGG-3’；
VH6b：5’- CCATACGATGTTCCAGATTACCAG(A/G)TCACCTTGAAGGAGTCTG-3’；
VH7b：5’- CCATACGATGTTCCAGATTACCAGGTGCAGCTGGTG(C/G)A(A/G)TCTGG-3’；
Second group of 3 '-end primer (SEQ ID No.18～23) for amplification human antibody heavy chain variable region (VH) gene, comprising:
VH1f：5’- GCCGCCTGATCCACCACCGCCTGAGGAGAC(A/G)GTGACCAGGGTG-3’；
VH2f：5’- GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACCAGGGTT-3’；
VH3f：5’- GCCGCCTGATCCACCACCGCCTGAAGAGACGGTGACCATTGT-3’；
VH4f：5’- GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACCGTGGTCC-3’；
VH5f：5’- GCCGCCTGATCCACCACCGCCGGTTGGGGCGGATGCACTCC-3’；
VH6f：5’- GCCGCCTGATCCACCACCGCC(C/G)GATGGGCCCTTGGTGGA(A/G)GC-3’；
The 3rd group of 5 '-end primer (SEQ ID No.24～32) for amplification people's antibody λ-variable region of light chain (V λ) gene, comprising:
VL1b：5’- GGCAGCGGTGGTGGAGGCAGTCAGTCTGT(C/G)(C/G/T)TGACGCAGCCGCC-3’；
VL2b：5’- GGCAGCGGTGGTGGAGGCAGTTCCTATG(A/T)GCTGAC(A/T)CAGCCAC-3’；
VL3b：5’- GGCAGCGGTGGTGGAGGCAGTTCCTATGAGCTGA(C/T)(A/G)CAGC(C/T)ACC-3’；
VL4b：5’- GGCAGCGGTGGTGGAGGCAGTCAGCCTGTGCTGACTCA(A/G)(C/T)C-3’；
VL5b：5’- GGCAGCGGTGGTGGAGGCAGTCAG(A/G/T)CTGTGGTGAC(C/T)CAGGAGCC-3’；
VL6b：5’- GGCAGCGGTGGTGGAGGCAGTCAGCC(A/T)G(G/T)GCTGACTCAGCC(A/C)CC-3’；
VL7b：5’- GGCAGCGGTGGTGGAGGCAGTTCCTCTGAGCTGA(C/G)TCAGGA(C/G)CC-3’；
VL8b：5’- GGCAGCGGTGGTGGAGGCAGTCAGTCTG(C/T)(C/T)CTGA(C/T)TCAGCCT-3’；
VL9b：5’- GGCAGCGGTGGTGGAGGCAGTAATTTTATGCTGACTCAGCCCC-3’；
The 4th group of 3 '-end primer (SEQ ID No.33～34) for amplification people's antibody λ-variable region of light chain (V λ) gene, comprising:
VL1f：5’- GGGGTTTTTCAGTATCTACGATAGGACGGT(C/G)A(C/G)CTTGGTCC-3’；
VL2f：5’- GGGGTTTTTCAGTATCTACGAGAGGACGGTCAGCTGGGTGC-3’；
The 5th group of 5 '-end primer (SEQ ID No.35～38) for amplification people antibody k-variable region of light chain (Vk) gene, comprising:
VK1b：5’- GGCAGCGGTGGTGGAGGCAGTGACATCC(A/G)G(A/G/T)TGACCCAGTCTCC-3’；
VK2b：5’- GGCAGCGGTGGTGGAGGCAGTGAAATTGT(A/G)(A/T)TGAC(A/G)CAGTCTCC-3’；
VK3b：5’- GGCAGCGGTGGTGGAGGCAGTGATATTGTG(A/C)TGAC(C/G/T)CAG(A/T)CTCC-3’；
VK4b：5’- GGCAGCGGTGGTGGAGGCAGTGAAACGACACTCACGCAGTCTC-3’；
The 6th group of 3 '-end primer (SEQ ID No.39～42) for amplification people antibody k-variable region of light chain (Vk) gene, comprising:
VK1f：5’- GGGGTTTTTCAGTATCTACGATTTGATTTCCACCTTGGTCC-3’；
VK2f：5’- GGGGTTTTTCAGTATCTACGATTTGATCTCCA(C/G)CTTGGTCC-3’；
VK3f：5’- GGGGTTTTTCAGTATCTACGATTTGATATCCACTTTGGTCC-3’；
VK4f：5’- GGGGTTTTTCAGTATCTACGATTTAATCTCCAGTCGTGTCC-3’。
, utilize the combination of first group of primer and second group of primer when in amplification people antibody the variable region of heavy chain (VH), have 42 PCR to react; When λ-variable region of light chain (V λ) in amplification people antibody, utilize the combination of the 3rd group of primer and the 4th group of primer, have 18 PCR to react; , utilize the combination of the 5th group of primer and the 6th group of primer when in amplification people antibody the k variable region of light chain (Vk), have 16 PCR to react.
In first group of primer, include yeast two-hybrid carrier pACT2(Hua SB, Luo Y, Qiu M, Chan E, Zhou H, Zhu L. (1998) Gene.215:143-152.) (Hua SB, Qiu M, Chan E, Zhu L, Luo Y. (1997) Plasmid.38:91-96.) the upstream homologous sequence (underscore part) of multiple clone site; In the 4th group and the 6th group of primer, include the downstream homologous sequence (underscore part) of yeast two-hybrid carrier pACT2 multiple clone site; And including connection peptides sequence (underscore part) in second group, the 3rd group and the 5th group of primer, connection peptides is for connecting variable region of heavy chain and the variable region of light chain of antibody.
When amplification, PCR reaction system is all identical with reaction conditions, and PCR reaction system is:
Above-mentioned each component is mixed to be placed in PCR instrument and react.Reaction conditions is: 94 DEG C are unwind 1 minute, anneals 1 minute for 50 DEG C, and 72 DEG C are extended 2.5 minutes, circulate 30 times.
The homologous sequence of 2 connection carrier multiple clone site and connection peptides sequence
(1), taking the human antibody heavy chain variable region DNA of PCR gained in step 1 as template, taking primer 7 and primer 8 as upstream primer and downstream primer, sequence is respectively:
Upstream primer (underscore part is the upstream homologous sequence of carrier pACT2 multiple clone site for primer 7, SEQ ID No.43):
5’-ACCCCACCAAACCCAAAAAAAGAGATCTGTATGGCT TACCC ATACGATGTTCCAGATTAC-3’；
Downstream primer (underscore part is connection peptides anti-chain sequence for primer 8, SEQ ID No.44):
5’-ACTGCCTCCACCACCGCTGCCACCTCCGCCAGATCCTCC GCC GCCTGATCCACCACCGCC-3’；
Carry out pcr amplification, reaction conditions and system are with step 1.React rear acquisition containing the upstream homologous sequence of 5 '-carrier pACT2 multiple clone site and the human antibody heavy chain variable region DNA sequence dna of 3 '-connection peptides anti-chain sequence.
(2) taking the human antibody light chain variable region DNA of PCR gained in step 1 as template, be respectively downstream primer and upstream primer with primer 9 and primer 10, sequence is as follows:
Upstream primer (underscore part is that connection peptides is along chain-ordering for primer 10, SEQ ID No.45):
5’-GGCGGTGGTGGATCAGGCGGCGGAGGATCTGGCGGAGGT GG CAGCGGTGGTGGAGGCAGT-3’；
Downstream primer (underscore part is the downstream homologous sequence of carrier pACT2 multiple clone site for primer 9, SEQ ID No.46):
5’-GAGATGGTGCACGATGCACAGTTGAAGTGAACTTGC GGGGT TTTTCAGTATCTACGA-3’；
Carry out pcr amplification, reaction conditions and system are with step 1.Reacted rear acquisition containing the downstream homologous sequence of 3 '-carrier pACT2 multiple clone site and 5 '-connection peptides the human antibody light chain variable region DNA sequence dna along chain-ordering.
3 connect single-chain antibody
The contain homologous sequence of carrier pACT2 multiple clone site and the human antibody heavy chain variable region DNA of connection peptides sequence and variable region of light chain DNA that pcr amplification in step 2 is obtained mix, taking this hybrid dna as template, respectively taking primer 7 and primer 9 as upstream primer and downstream primer, carry out pcr amplification, reaction conditions and system are with step 1.
As shown in Figure 1, obtain comprising single-chain antibody (scFv) DNA of human antibody heavy chain variable region DNA sequence dna, variable region of light chain DNA sequence dna, carrier pACT2 multiple clone site homologous sequence and connection peptides DNA sequence dna after having reacted.
4 build people's single-chain antibody gene library
Single-chain antibody (scFv) DNA that step 3 is obtained proceeds to yeast strain Y187(MAT α, ura3-52 jointly with the method (Yeast Protocol Handbook, PT3024-1) providing according to former Clontech company through restriction enzyme (Bam HI and Eco RI) yeast two-hybrid carrier pACT2 after treatment, his3-200, ade2-101, lys2-801, trp1-901, leu2-3,112, gal4 Δ, gal80 Δ, met-, URA3::GAL1 uAS-GAL1 tATA-lac Z, MEL1) in, after homologous recombination in cell, single-chain antibody DNA is incorporated on pACT2 carrier, thereby obtains yeast two-hybrid single-chain antibody library, Gal4 active region (Activation Domain, AD) on single-chain antibody DNA fragmentation and pACT2 carrier merges.
For checking the quality in this single-chain antibody gene library, therefrom choose at random 21 clones, Insert Fragment is carried out to sequencing analysis.Analytical results shows, all clones comprise the single-chain antibody DNA fragmentation merging with Gal4, and all single-chain antibody DNA sequence dnas are all unique.
Antibody dna copy number nearly 1 × 10 in the yeast two-hybrid single-chain antibody gene library obtaining through homologous recombination 8individual, can be applicable to yeast-two hybrid technique and screen specific antibody.
5 screening antibodies
(1) antibody screening
End user's IGF-1R (IGF-1R), as antigen, is carried out antibody screening to yeast two-hybrid single-chain antibody gene library.
The DNA of encoding human IGF-1R alpha subunit is recombinated in carrier pGBKT7, build pGBK-IGF1R α.PGBK-IGF1R α coding Gal4DNA calmodulin binding domain CaM (Binding Domain, BD), and merged people IGF-1R alpha subunit at its C end.
After the DNA sequence dna of encoding human IGF-1R alpha subunit is verified, pGBK-IGF1Ra plasmid DNA is transformed into yeast strain AH109(MATa, trp1-901, leu2-3,112, ura3-52, his3-200, gal4 Δ, gal80 Δ, LYS2::GAL1 uAS-GAL1 tATA-HIS3, GAL2 uAS-GAL2 tATA-ADE2, URA3::MEL1 uAS-MEL1 tATA-lac Z); AH109 yeast with pGBK-IGF1R α plasmid can be in the not upper growth of synthetic medium (SD/-W) containing tryptophane.
The MATa type yeast cell (AH109 bacterial strain) containing pGBK-IGF1R α of equivalent and the MAT α type yeast cell (Y187 bacterial strain) that contains single-chain antibody gene library are carried out to co-cultivation, make this two types cell mating combination.Because the pACT2 carrier that is loaded with single-chain antibody gene library contains Leu2 gene, and pGBK-IGF1R α comprises Trp1 gene, and therefore, the yeast cell that comprises two kinds of plasmids can be grown in the not yeast synthetic medium (SD/-LW) containing leucine and Serine.
Exist the interactional cell of scFv/IGF1R α can activate reporter gene ADE2 and the HIS3 being incorporated in strain gene group, thereby make yeast cell can be grown in shortage VITAMIN B4, Histidine, the substratum (SD/-AHLW) of leucine and tryptophane is upper, and forms bacterium colony on this plate culture medium.
(2) specificity antibody screening
1) beta galactosidase enzyme analysis
Due to another reporter gene lac Z that exists the interactional cell of scFv/IGF1R α also can activate to be incorporated in strain gene group, therefore detect beta galactosidase enzyme in yeast cell and whether express the existence that can judge scFv/IGF1R α in yeast cell.
In screening culture medium (SD/-AHLW), altogether pick out 35 bacterium colonies, use beta galactosidase enzyme detection method to detect lac Z and express.Detection method is as follows:
1. will exist the interactional yeast-inoculated of scFv/IGF1Ra to grow to screening culture medium (SD/-AHLW) flat board;
2. yeast colony is transferred on No. five filter paper of Whatman;
3. the filter paper with yeast cell bacterium colony is immersed among liquid nitrogen, make cell rupture;
4. filter paper is taken out from liquid nitrogen, and be placed in the baking oven of 30 DEG C; Repeating step (3) and (4) twice;
5. filter paper is laid in appropriate X-gal solution, and is placed in the incubator of 37 DEG C approximately 15 minutes; Blue if bacterium colony place shows, be indicated as the beta galactosidase enzyme positive, i.e. the lac Z gene expression that is activated.
Wherein, X-gal solution formula is as follows:
16.1g/L?Na 2HPO 4·7H 2O；
5.50g/L?NaH 2PO 4·H 2O；
0.75g/L?KCl；
0.246g/L?MgSO 4·7H 2O；
35mg/L?X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)；
5mM beta-mercaptoethanol; PH7.0.
Detected result: in above-mentioned 35 bacterium colonies picking out, have 22 to be the beta galactosidase enzyme positive, show that reporter gene lacZ has been activated in these bacterium colonies.
2) specific combination analysis
ScFv to the positive bacterium colony of above-mentioned 22 beta galactosidase enzymes carries out specificity analyses, to verify whether single-chain antibody scFv is combined with the α of IGF-1R subunit part specifically.Because the structure of IGF-1R is very similar to insulin receptor (IR), should reject the antibody combining with IR.
The DNA clone of encoding human insulin receptor (IR) alpha subunit is built to pGBKT-IR α to carrier pGBKT7, and pGBKT-IR α coding Gal4DNA calmodulin binding domain CaM (BD) is also held the alpha subunit that has merged people IR at its C.
From the yeast of above-mentioned 22 beta galactosidase enzyme positives, extract the pACT2 plasmid DNA that contains scFv, be more jointly transformed in AH109 yeast cell with pGBKT7 empty carrier DNA, pGBKT-IR α plasmid DNA, pGBKT-Lam plasmid DNA (coding Gal4DNA calmodulin binding domain CaM also merges and has human nuclear fabric layer albumen C at its C end) respectively; Yeast cell after conversion is first laid on SD/-LW plate culture medium grows, and then transfers on SD/-AHLW plate culture medium; The bacterium colony growing is done to beta galactosidase enzyme analysis, be verified as positive bacterium colony and be considered as containing nonspecific scFv, by its rejecting.
After above-mentioned specificity analyses, obtain one people IGF-1R is had to specific single-chain antibody, be numbered #IGF1R α-2, use ABI automatic sequencer to carry out sequencing analysis to this single-chain antibody, result shows:
The DNA sequence dna of variable region of heavy chain, #IGF1R α-2 is as shown in SEQ ID No.1, and aminoacid sequence (SEQ ID No.2) is:
EVQLLETGGGLVQPGGSLRLSCSAS GFSFSSFEMNWVRQAPGKGLEWLS YISKSGFTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SSWAQDFDWLLPFDWWGQGTLVTVSS；
Underscore part is followed successively by three hypervariable region CDRH1, CDRH2, CDRH3(SEQ ID No.3～5);
The DNA sequence dna of the variable region of light chain of #IGF1R α-2 is as shown in SEQ ID No.6, and aminoacid sequence (SEQ ID No.7) is:
ETTLTQSPSSVSAYVGDRVTITC RASQGIRNYLAWYQQKPGKVPNLLIY AASTLQSGVPSRFSGSGSETDFTLTISSLQPEDVATYYC QKYNSVPFTFGPGTKVDIK；
Underscore part is followed successively by three hypervariable region CDRL1, CDRL2, CDRL3(SEQ ID No.8～10).
Embodiment 2 specific detection
1 antibody expression and purifying
The encoding gene of single-chain antibody #IGF1R α-2 is cloned in expression vector pET27b (+), builds and obtain pET27b-IGF1R α;
PET27b-IGF1R α is transformed into and expresses bacterium E.coli BL21 (DE3), and the method IPTG(0.5mM providing according to Novagen company) abduction delivering; In the target protein giving expression to, the N end of scFv is pelB sequence, and pelB sequence can be secreted into the scFv after expressing in the pericentral siphon chamber (periplasmic space) of BL21 (DE3); The C end of scFv contains a HSV marker and 6 × His marker, facilitates the purifying of target protein;
The method that application Qiagen company provides obtains the single-chain antibody of anti-human IGF-1R easily with Ni-NTA column separating purification.
The specificity of 2ELISA test single-chain antibody
The protein of rhIGF-1 R is purchased from Sino Biological Inc..ELISA testing method is as follows:
(1) with the coated 96-orifice plate of IGF-1R, spend the night at 2-8 DEG C;
(2) the 96-orifice plate after coated is made sealing treatment with SuperBlock again;
(3) single-chain antibody #IGF1R α-2 are done in 0.02%BSA after serial dilution, add in the 96-hole that is coated with IGF-1R, be combined with IGF-1R;
(4) after 96-orifice plate is cleaned, add the antibody of the mouse-anti HSV marker of 5000 times of dilutions, be used for detecting the single-chain antibody combining;
(5), after 96-orifice plate is cleaned, add goat dynamics-horseradish peroxidase thing of 10000 times of dilutions;
(6), by after final 96-orifice plate cleaning, application horseradish peroxidase substrate TMB reagent is made color development treatment;
(7) use the sulfuric acid termination reaction of 0.5M, and detect the absorption spectrum of 450nm.
Detected result as shown in Figure 2, shows that single-chain antibody #IGF1R α-2 can be effectively in conjunction with IGF-1 acceptor.
Embodiment 3 anti-IGF-1 R antibodies suppress the growth of tumour
Entrust Sino Biological Inc. to prepare total length recombination human source anti-IGF-1 R antibodies DB#2.DB#2 is IgG1 type, and its variable region sequences is identical with the variable region sequences of single-chain antibody #IGF1R α-2.
According to the method for recording in document (1999. " Inhibition of Epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358:Dynamics of receptor inhibition in situ and antitumor effects in athymic mice. " J.Pharmacol.Exp.Ther.291:739-748.), induced tumor in nude mouse (nu/nu) body.
By adenocarcinoma of colon Colo205 cell (ATCC CCL222) (about 5x10 6individual cell) be subcutaneously injected into induced tumor in nude mouse (nu/nu) body in 3-4 week with Matrigel preparation; Be formed into about 200mm in tumour 3when size, above-mentioned antibody is injected in nude mouse in single dose single (500 μ g/ time, intraperitoneal i.p. injection) mode; Determine the size of tumour with two diameters of vernier caliper measurement tumour, and the volume of the method calculating tumour of recording according to document (" Protocols for screening chemical agents and natural products against animal tumors and other biological systems. " Cancer Chemother.Rep.3:1-104.), gross tumor volume=(length x[width] 2)/2.
Shown in Fig. 3 is the relation with tumor size and time change after antibody DB#2 single dose single treatment, control group physiological saline processing.Detected result shows, compared with control group, the adenocarcinoma of colon Colo205 cell that antibody DB#2 is right has stronger tumor suppression effect.Antibody DB#2 processed after 20 days, and tumor size is only 50% of control group.
Embodiment 4 compositions suppress tumor growth
By adenocarcinoma of colon Colo205 cell (ATCC CCL222) (about 5x10 6individual cell) be subcutaneously injected into induced tumor in nude mouse (nu/nu) body in 3-4 week with Matrigel preparation; Be formed into about 200mm in tumour 3when size, in the time of the 0th, 7,14,21 days, use antibody single dose (500 μ g/ time of embodiment 3, intraperitoneal i.p. injection), 5-FU single dose (100mg/kg, vein i.v. injection), the two combination (antibody+5-FU, vein i.v. injection) process respectively nude mice.
Tumor size changes as shown in Figure 4.Result shows, compared with control group, an antibody DB#2 shot in every 7 days can more effectively suppress the growth of tumour.In addition, with control group, compare by antibody treatment group or independent 5-FU treatment group separately, during by antibody DB#2 and 5-FU coupling, greatly strengthened tumor suppression effect.

Claims (8)

1. synalbumin like growth factor-1 receptor antibody, is characterized in that, is antibody I, and the aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID No.2, and the aminoacid sequence of variable region of light chain is as shown in SEQ ID No.7;
Three hypervariable region CDRH1, CDRH2 of variable region of heavy chain, the aminoacid sequence of CDRH3 are respectively: gFSFSSFEMN, yISKSGFTIYYADSVKG, sSWAQDFDWLLPFDW; Three hypervariable region CDRL1, CDRL2 of its variable region of light chain, the aminoacid sequence of CDRL3 are respectively rASQGIRNYLA, aASTLQS, qKYNSVPF.
2. synalbumin as claimed in claim 1 like growth factor-1 receptor antibody, is characterized in that, described antibody I is the antigen-binding portion thereof of whole antibody or whole antibody.
3. synalbumin as claimed in claim 2 like growth factor-1 receptor antibody, is characterized in that, described whole antibody is IgG1 type.
4. synalbumin as claimed in claim 2 like growth factor-1 receptor antibody, is characterized in that, described antigen-binding portion thereof is Fab fragment, Fab ' fragment, F (ab ') 2fragment or single-chain antibody.
5. the encoding gene of synalbumin as claimed in claim 1 like growth factor-1 receptor antibody, it is characterized in that, the nucleotide sequence of the variable region of heavy chain encoding gene of antibody I is as shown in SEQ ID No.1, and the nucleotide sequence of variable region of light chain encoding gene is as shown in SEQ ID No.6.
6. contain the recombinant vectors of encoding gene as claimed in claim 5.
7. contain the expression system of encoding gene as claimed in claim 5.
8. synalbumin like growth factor-1 receptor antibody as described in as arbitrary in claim 1～4 is in the application of preparing in antitumor drug; It is characterized in that, described antitumor drug is inhibitor against colon carcinoma cells medicine.
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