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 PDFInfo
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- antibody
- variable region
- igf
- growth factor
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 37
- 102100039688 Insulin-like growth factor 1 receptor Human genes 0.000 title abstract description 22
- 101710184277 Insulin-like growth factor 1 receptor Proteins 0.000 title abstract description 9
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 4
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 6
- 230000012010 growth Effects 0.000 claims description 16
- 102000005962 receptors Human genes 0.000 claims description 10
- 108020003175 receptors Proteins 0.000 claims description 10
- 239000000427 antigen Substances 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 claims description 7
- 230000014509 gene expression Effects 0.000 claims description 7
- 206010009944 Colon cancer Diseases 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 239000003112 inhibitor Substances 0.000 claims description 3
- 239000013598 vector Substances 0.000 claims description 3
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 11
- 230000004614 tumor growth Effects 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 4
- 210000004881 tumor cell Anatomy 0.000 abstract description 4
- 206010028980 Neoplasm Diseases 0.000 description 22
- 108020004414 DNA Proteins 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 13
- 238000000034 method Methods 0.000 description 12
- 101001034652 Homo sapiens Insulin-like growth factor 1 receptor Proteins 0.000 description 11
- 230000003321 amplification Effects 0.000 description 11
- 102000005936 beta-Galactosidase Human genes 0.000 description 11
- 108010005774 beta-Galactosidase Proteins 0.000 description 11
- 238000003199 nucleic acid amplification method Methods 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- 210000005253 yeast cell Anatomy 0.000 description 9
- 102000003746 Insulin Receptor Human genes 0.000 description 8
- 108010001127 Insulin Receptor Proteins 0.000 description 8
- 150000001413 amino acids Chemical group 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 238000011144 upstream manufacturing Methods 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000001086 yeast two-hybrid system Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 238000011580 nude mouse model Methods 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 4
- 108010001515 Galectin 4 Proteins 0.000 description 4
- 102100039556 Galectin-4 Human genes 0.000 description 4
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 201000010989 colorectal carcinoma Diseases 0.000 description 4
- 238000007912 intraperitoneal administration Methods 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 206010001167 Adenocarcinoma of colon Diseases 0.000 description 3
- 102000000584 Calmodulin Human genes 0.000 description 3
- 108010041952 Calmodulin Proteins 0.000 description 3
- 108010031794 IGF Type 1 Receptor Proteins 0.000 description 3
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 201000010897 colon adenocarcinoma Diseases 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 101100136076 Aspergillus oryzae (strain ATCC 42149 / RIB 40) pel1 gene Proteins 0.000 description 2
- 101100322915 Caenorhabditis elegans akt-1 gene Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101150094690 GAL1 gene Proteins 0.000 description 2
- 102100028501 Galanin peptides Human genes 0.000 description 2
- 102100024594 Histone-lysine N-methyltransferase PRDM16 Human genes 0.000 description 2
- 101100121078 Homo sapiens GAL gene Proteins 0.000 description 2
- 101000686942 Homo sapiens Histone-lysine N-methyltransferase PRDM16 Proteins 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 2
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 108010017622 Somatomedin Receptors Proteins 0.000 description 2
- 102000004584 Somatomedin Receptors Human genes 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 239000006035 Tryptophane Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000009585 enzyme analysis Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 101150040383 pel2 gene Proteins 0.000 description 2
- 101150050446 pelB gene Proteins 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 201000000498 stomach carcinoma Diseases 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 229960004799 tryptophan Drugs 0.000 description 2
- 230000005760 tumorsuppression Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 102100030981 Beta-alanine-activating enzyme Human genes 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 101100268670 Caenorhabditis elegans acc-3 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000197727 Euscorpius alpha Species 0.000 description 1
- 101150037782 GAL2 gene Proteins 0.000 description 1
- 102100021735 Galectin-2 Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108010009202 Growth Factor Receptors Proteins 0.000 description 1
- 102000009465 Growth Factor Receptors Human genes 0.000 description 1
- 101150009006 HIS3 gene Proteins 0.000 description 1
- 101000773364 Homo sapiens Beta-alanine-activating enzyme Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 101000852815 Homo sapiens Insulin receptor Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000038455 IGF Type 1 Receptor Human genes 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 102100029812 Protein S100-A12 Human genes 0.000 description 1
- 101710110949 Protein S100-A12 Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 101100394989 Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009) hisI gene Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 101150006914 TRP1 gene Proteins 0.000 description 1
- 241000906446 Theraps Species 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000002682 general surgery Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 102000047882 human INSR Human genes 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 102000027257 transmembrane receptors Human genes 0.000 description 1
- 108091008578 transmembrane receptors Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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
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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310176662.XA CN103319598B (en) | 2013-05-13 | 2013-05-13 | Insulin-like growth factor-1 receptor (IGF-1R) resistant antibody and coding genes and applications thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310176662.XA CN103319598B (en) | 2013-05-13 | 2013-05-13 | Insulin-like growth factor-1 receptor (IGF-1R) resistant antibody and coding genes and applications thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN103319598A CN103319598A (en) | 2013-09-25 |
CN103319598B true CN103319598B (en) | 2014-10-22 |
Family
ID=49188681
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201310176662.XA Active CN103319598B (en) | 2013-05-13 | 2013-05-13 | Insulin-like growth factor-1 receptor (IGF-1R) resistant antibody and coding genes and applications thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103319598B (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105315372A (en) * | 2014-06-18 | 2016-02-10 | 中国人民解放军军事医学科学院基础医学研究所 | Preparation and application of anti-insulin-like growth factor receptor (IGF-1R) human-sourced functional antibody LMAb1 |
TWI716400B (en) * | 2015-04-27 | 2021-01-21 | 法商皮爾法伯製藥公司 | Novel igf-1r antibody and its use for the diagnosis of cancer |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1798242B1 (en) * | 2002-01-18 | 2013-01-23 | Pierre Fabre Medicament | Anti-IGF-IR antibodies and their applications |
CA2535071A1 (en) * | 2003-08-13 | 2005-02-24 | Pfizer Products Inc. | Modified human igf-1r antibodies |
EP2322215A3 (en) * | 2004-07-16 | 2011-09-28 | Pfizer Products Inc. | Combination treatment for non-hematologic malignancies using an anti-IGF-1R antibody |
-
2013
- 2013-05-13 CN CN201310176662.XA patent/CN103319598B/en active Active
Also Published As
Publication number | Publication date |
---|---|
CN103319598A (en) | 2013-09-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7041516B2 (en) | Bispecific antibodies or antibody mixtures with common light chains | |
CN103476794B (en) | Antibody against the CSF-1R | |
CN111018986A (en) | Anti-glypican-3 antibody and application thereof | |
MX2010009894A (en) | Antibody against the csf-1 r. | |
CN103319598B (en) | Insulin-like growth factor-1 receptor (IGF-1R) resistant antibody and coding genes and applications thereof | |
CN103319597B (en) | Insulin-like growth factor-1 receptor (IGF-1R) resistant antibody and coding genes and applications thereof | |
CN106831987B (en) | Anti-human HER2 antibody and coding gene and application thereof | |
CN104628859A (en) | Anti-human carcino-embryonic antigen antibody as well as coding gene and application thereof | |
CN112625132B (en) | Nano antibody, coding gene, expression vector, host cell and application thereof | |
Chen et al. | A dual-specific IGF-I/II human engineered antibody domain inhibits IGF signaling in breast cancer cells | |
CN114057880B (en) | DLL3 monoclonal antibody | |
CN102199210B (en) | Anti-interleukin-8 antibody | |
CN107987168B (en) | Single-chain double-specific antibody for resisting VEGF and EGFR and application thereof | |
CN106831986B (en) | Anti-human HER2 antibody and coding gene and application thereof | |
CN112538117B (en) | Anti-human carcinoembryonic antigen antibody and coding gene and application thereof | |
CN103665163B (en) | Anti-osteopontin antibody and application thereof | |
CN101348526A (en) | Anti-interleukin-8 antibody | |
WO2014146487A1 (en) | Anti-cell surface ectopic expression monoclonal antibody, preparation method and use thereof | |
CN101343323A (en) | Anti-interleukins-8 antibody | |
CN102199209B (en) | Anti-interleukin-8 antibody | |
CN114437229B (en) | Preparation and application of CAR T immune cells carrying PD-1 chain antibody and targeting EGFR antigen | |
CN113651889B (en) | anti-EphA 2 fully human bivalent recombinant antibody scFv-Fc | |
CN106279414A (en) | Humanized's anti-amylin antibody and preparation method thereof | |
CN102942630A (en) | Anti-osteopontin antibody | |
CN114369163A (en) | Alpaca source nano antibody combined with human platelet derived growth factor receptor beta |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant |