CN102321173A - Humanized macrophage inhibitory factor 1 monoclonal antibody and application thereof - Google Patents
Humanized macrophage inhibitory factor 1 monoclonal antibody and application thereof Download PDFInfo
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- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 230000005909 tumor killing Effects 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
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Abstract
The invention discloses a humanized macrophage inhibitory factor 1 monoclonal antibody and an application thereof. The antibody comprises: a heavy chain variable region, wherein CDR1, CDR2 and CDR3 amino acid sequences of the heavy chain variable region are amino acid sequences shown in SEQ ID NO 1, 2 and 3; and a light chain variable region, wherein CDR1, CDR2 and CDR3 amino acid sequences of the light chain variable region are amino acid sequences shown in SEQ ID NO 4, 5 and 6. The invention also relates to polynucleotide coding the antibody, a pharmaceutical composition containing the antibody, and an application of the antibody in tumor treatment. The monoclonal antibody provided by the invention has low immunogenicity, high affinity, and strong specificity, and provides new possibility for tumor treatment.
Description
Technical field
The invention belongs to bio-pharmaceutical engineer technology domain, relate to grand antibody of the anti-macrophage inhibition factor 1 of a kind of humanization (MIC-1) monoclonal antibody and application thereof.
Background technology
Macrophage inhibition factor 1 (Macrophage inhibitory cytokine-1; MIC-1) be people's transforming growth factor-beta (Transforming growth factor-β; TGF-β) an important branch member in the superfamily, also be called as growth and differentiation factor-15 (growth differentiation factor-15, GDF-15) etc.; Because of can significantly suppressing activated macrophage, it produces TNF-α gain the name [Bootcov, 1997].
The MIC-1 assignment of genes gene mapping comprises 2 exons (309bp and 891 bp) and an intron (1820 bp) [Fairlie et al, 1999] in karyomit(e) 19p13.1 in the human genome.MIC-1 genes encoding relative molecular mass 25 * 10
3MIC-1 albumen, form by 308 amino acid polypeptides, comprise 29 amino acid signal peptides, 167 amino acid propetides and 112 amino acid maturation zones.Article two, polypeptied chain is sheared by furin appearance proteolytic enzyme after disulfide linkage connects into precursor protein, the final homodimer maturation protein that is connected by disulfide linkage, the molecular weight 25KD [Fairlie LN et al, 1999] of forming.Ripe MIC-1 PE gets into the internal secretion effect [Bauskin et al, 2005] that cytokine is brought into play in blood circulation after going out cell.MIC-1 mainly is present in the cell with precursor forms, under different physiology and pathological conditions, is discharged in the blood after the hydrolysis.
MIC-1 acts on cell surface TGF beta receptor as a kind of cytokine; The signal transmission of wide participation apoptosis, differentiation, propagation and body inflammatory reaction; And increase, promote apoptosis, keep placental function and aspects such as fetal development, adjusting and protection nervus centralis have vital role [Lee DH et al, 2003] suppressing tumour cell.Under the normal physiological state, the MIC-1 high expression level is expressed and in tissues such as prostate gland, colon, kidney, brain, liver, pancreas, be low in placenta tissue.Pathological state such as tumour, acute injury and inflammation, MIC-1 expresses can significantly improve [Bauskin et al, 2006].
MIC-1 mainly is the effect that suppresses tumour through p53, PI3K/AKT/GSK3 signal path, death receptor and the performance of PLAU/uPAR system.In breast cancer cell, MIC-1 causes tumour cell apoptosis and cell to stop at the G1 phase [Bauskin ARet al, 2006] when crossing expression as the target gene of p53; When research colon carcinoma cell line HCT-116, find the target of MIC-1, can promote the apoptosis [Yamaguchi Ket al, 2004] of tumour cell as PI3K/AKT/GSK3 β path; People such as Jang research confirms that then thereby MIC-1 can obviously induce the expression of DR-4 and DE-5 to impel apoptosis of tumor cells to increase and cell viability descends, but the acceptor of MIC-1 effect and conduction path also are not very clear [Jang TJ et al, 2004]; In colon cancer cell, MIC-1 passes through to suppress the activity of PLAU/uPAR system, thereby impels apoptosis of tumor cells [Yang H et al, 2009].
MIC-1 also has propagation, invasion and attack and the transferance that promotes tumour cell simultaneously; People such as Sung discover through the V600EB-Raf signal path and can promote MIC-1 to express and secretion; Thereby promote tumor vascular generation to make tumor proliferation [Sung Jin Huh et al, 2010]; Thereby the trans activation that MIC-1 also can be through promoting the ErBb2 acceptor also follows tyrosine phosphorylations such as EGFR, ErBb2 to activate ERK1/2 and Akt, promotes invasion by tumor cells ability (Kim et al 2008); People such as Lee find that MIC-1 over-expresses in the cancer of the stomach can significantly increase the activity of UPA uPA and uPAR; Raise the uPAu/PAR active system through extracellular signal transduction kinases 1/2 relational approach; The aggressive of inducing tumor cell also promotes the grade malignancy [Lee DH, 2003] of stomach cancer cell.
Also there is certain relation between apositia that MIC-1 is relevant with tumour and the weight loss; MIC-1 can interact with hypothalamic TGF-beta receptor II; Promote ERK-1/2 and proplomela nocortin and suppress the function of neuropeptide tyrosine; Thereby reach control action kou, make the patient that apositia and weight loss and the final patient's of reduction survival time [Johnen et al, 2007] take place appetite.F.E.Wiklund etc. are to having carried out the regression epidemiological study that the meta observation time is respectively 5.3 years and 9.1 years between 876 male sex and the 324 pairs of twinborn serum MIC-1 levels and the mortality ratio; The result shows that MIC-1 can indicate the death (p=0.0001) that the multiple reason that comprises tumour causes; After the interference of getting rid of age, BMI, smoking history; The high serum level group of MIC-1 has been compared tumors of higher mortality risk (OR=2.74,95%CI 1.70-4.22) with the normal level group.
Above-mentioned result of study shows that MIC-1 participates in multiple physiology of body and pathologic process, and brings into play various effects therein.If our imagination is passed through the action pathway of antibody blocking MIC-1, might in tumor treatment, play a role.In carcinoma of the pancreas is example; Use the mouse-anti people MIC-1 antibody abdominal injection treatment carcinoma of the pancreas tumor bearing nude mice (PANC-1 and SW1990) of independent research; Compare the experimental group tumour with contrast and significantly dwindle, the section immunohistochemical analysis shows that CD31 and MMP2 level reduce, and microvessel quantity obviously reduces.Prompting MIC-1 antibody can be through blocking-up MIC-1 and acceptor combine to have brought into play its antitumor action.The MIC-1 monoclonal antibody is expected to provide new treatment to select to tumour patient through the function that suppresses MIC-1.
The mouse monoclonal antibody induces the endogenous anti-antibody can be divided into two types in human body, anti-unique antibody and anti-kind of type antibody.During passing through, the former eliminates the monoclonal antibody binding ability with antigen-binding site; The latter may cause anaphylactic shock in addition except the removing of quickening monoclonal antibody.It is reported that the most mouse source HAMA incidence of property monoclonal antibody medicine in patients such as intestinal cancer is up to 100%, HAMA plays neutralizing effect to the monoclonal antibody medicine that injects, thereby offsets its curative effect.Address these problems, improve from a plurality of links such as antibody quality, antibody physico-chemical property, routes of administration, the main path of avoiding or reduce HAMA to react at present is to make mouse source property monoclonal antibody humanization or development full-length human antibody.But full-length human antibody the time often runs in development that affinity of antibody descends, vigor is not high, poor stability or bottleneck problem such as yield poorly.
In sum; MIC-1 is a very important cytokine; This area is necessary further to research and develop that anti-MIC-1 antibody drug, particularly immunogenicity are low, avidity is high, the Humanized monoclonal antibodies of high specificity, for tumor treatment provides new approach.
Summary of the invention
For addressing the above problem, the invention provides a kind of new MIC-1 monoclonal antibody.The present invention also provides the humanized antibody of said new MIC-1 monoclonal antibody.
The present invention utilizes genetic engineering technique that the mouse source antibody of independent development is transformed, and is keeping on the basis of its antigen-specific, makes its essential amino acid composition from human antibodies, thereby reduces its immunogenicity, becomes the ideal clinical medicine.
Therefore, one aspect of the present invention provides a kind of MIC-1 monoclonal antibody, and it comprises:
Variable region of heavy chain, the CDR1 of this variable region of heavy chain, CDR2 and CDR3 aminoacid sequence are respectively SEQ ID NO:1, the aminoacid sequence shown in 2 and 3; With
Variable region of light chain, the CDR1 of this variable region of light chain, CDR2 and CDR3 aminoacid sequence are respectively SEQ ID NO:4, the aminoacid sequence shown in 5 and 6.
In one embodiment of the invention, the present invention provides the humanization form of above-mentioned MIC-1 monoclonal antibody, and wherein, the framework region aminoacid sequence of variable region of heavy chain is identical with the framework region of people's germline gene site VH4-59 coding; The framework region aminoacid sequence of variable region of light chain is identical with the framework region of people's antibody germline gene site VL 08 coding.
Another aspect of the invention provides a kind of pharmaceutical composition, and it comprises MIC-1 monoclonal antibody of the present invention and pharmaceutical carrier.
The present invention also provides a kind of polynucleotide, its MIC-1 monoclonal antibody of the present invention of encoding on the one hand.
The present invention also provides a kind of expression vector on the one hand, and it comprises polynucleotide of the present invention.
The present invention also provides a kind of host cell on the one hand, and it comprises expression vector of the present invention, and said host cell can be eukaryotic host cell or prokaryotic host cell.
In one embodiment, the present invention also provides the application of MIC-1 monoclonal antibody of the present invention in the medicine of preparation treatment tumour.
According to the knowledge and the experimental data provided by the invention of this area, but the medicable tumour of MIC-1 monoclonal antibody of those skilled in the art's knowledge capital invention comprises carcinoma of the pancreas, the esophageal carcinoma, cancer of the stomach, intestinal cancer, lung cancer, mammary cancer, ovarian cancer and liver cancer.Preferably, said tumour is a carcinoma of the pancreas, the esophageal carcinoma, cancer of the stomach, intestinal cancer and liver cancer.
In one embodiment, MIC-1 monoclonal antibody provided by the invention is an IgG2 type antibody, and with the avidity of MIC-1 be 1 * 10
-9M-1 * 10
-8M.
The present invention is through carrying out humanization modified to the anti-MIC-1 antibody in the mouse source of independent development; Develop have the avidity height, the anti-MIC-1 monoclonal antibody of novel human-derivedization that immunogenicity is low; Antibody of the present invention can be blocked combining of MIC-1 and cell surface receptor; Thereby the transfer and the vasculogenesis that suppress tumour are for the treatment tumour provides a kind of possibility.
Other aspects of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.Sequence in this specification sheets and the sequence table such as inconsistent is as the criterion with the sequence of putting down in writing in the specification sheets.
Description of drawings
From the detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 mouse-anti MIC-1 monoclonal antibody is transplanted tumor-inhibiting action in the people PANC-1 body to nude mice.
Fig. 2 mouse-anti MIC-1 monoclonal antibody is transplanted tumor-inhibiting action in the people SW1990 body to nude mice.
Gross tumor volume variation behind Fig. 3 .PANC-1 tumor-bearing mice MIC-1 antibody administration (my god).
Gross tumor volume variation behind Fig. 4 .SW1990 tumor-bearing mice MIC-1 antibody administration (my god).
Embodiment
The present invention (through the MIC-1 coding region is cloned in the pPIC9K plasmid, expresses through GS115 with the people MIC-1 albumen of Pichia anomala expression; Fusion rotein is through cleer and peaceful SOURCE-30Q anion chromatography purifying on the ammonium sulfate precipitation.PPIC9K plasmid and GS115 bacterial strain are available from Invitrogen company).The female mouse of ordinary method immune health BALB/C (body weight 14～16g).First immunisation adopts MIC-1 and the injection of equivalent Freund's complete adjuvant emulsification pneumoretroperitoneum, and dosage is 100 μ g/.Antigen and Freund's incomplete adjuvant emulsification abdominal injection booster immunization after 14 days are after this whenever at a distance from 2 all booster immunizations 1 time.The blood sampling of immunity beginning in back 10 days mouse tail vein adopts indirect ELISA method to detect serum titer, reaches 1: 10 until serum titer
6More than.Merge the mouse that serum titer was the highest in preceding 3 days and do not add adjuvant tail vein injection 100 μ g antigen booster immunizations.Gather the mouse blood of reinforced immunological after 3 days, separation of serum is as positive control.Aseptic this immune mouse spleen cell of getting; In 10: 1 ratios with immunity after splenocyte and SP2/0 cell in 50%PEG4000 (available from Sigma company) effect fusion down; Divide kind again in the 96 porocyte culture plates of completing nurse cell with the suspension of HAT nutrient solution, put into 37 ℃, 5%CO2 incubator.Adopt indirect elisa method to detect the screening specific antibody, choose the high positive hole of tiring and carry out the limiting dilution assay clone, enlarged culturing and to build strain frozen.Adopt NST-757 to prevent method that hybridoma karyomit(e) is analyzed.
The monoclonal antibody biological characteristics identifies that 1. Dot ELISA identifies the monoclonal antibody specificity: cultivate methanol yeast GS115, and centrifugal collection thalline, deposition suspends with PBS, centrifuging and taking supernatant behind the ultrasonic treatment.A certain size nitrocellulose filter (NC) of clip soaks into deionized water, dries, and gets bacterium liquid 5 μ L points on the NC film, 37 ℃ of dryings 30 minutes, and with the PBST sealing that contains the 100mL/L calf serum, 4 ℃ are spent the night; PBST washing 3 times, 5 minutes/inferior; Immerse again in the mAb solution of working concentration, hatch 2h for 37 ℃, PBST washing 3 times; Change in sheep anti mouse HRP-Ig (G+M) the enzyme labelled antibody solution of working concentration, hatch 1h for 37 ℃, PBST washing 3 times; With the colour developing of DAB colour developing liquid.Establish positive antiserum(antisera) simultaneously as contrast.2. Western blot analyzes: select for use 12% separation gel that MIC-1 and methanol yeast total protein are carried out the SDS-PAGE analysis simultaneously, albumen is transferred on the NC film from sds page again, with the PBS sealing that contains the 50g/L skimmed milk, the room temperature jog spends the night; Behind the thorough washing, add the ascites mAb effect 1h after diluting; Behind the thorough washing, add the sheep anti-mouse igg antibody of the HR mark of working concentration, effect 1h; Behind the thorough washing, with the colour developing of DAB colour developing liquid, zero(ppm) water termination reaction.
Identify the monoclonal antibody subclass with monoclonal antibody subgroup identification test kit.The indirect ELISA that carries out the antibody doubling dilution with different antigen concentrations detects, and draws response curve, calculates the affinity costant of monoclonal antibody with the AC of maximum A450 value one half on the curve, confirms antibody and antigenic binding ability (2 * 10
-9M).Antibody affinity costant (Ka) is calculated [6] by following formula.Ka＝(n-1)/2×(n[Ab]-[Ab]t)。[Ab]: the corresponding AC of A=1/2Amax when the expression antigen concentration is [Ag]; [Ab] t: the corresponding AC of A=1/2Amax when the expression antigen concentration is [Ag] t; [Ag]/[Ag] t: expression antigen concentration; N: be the extension rate between antigen [Ag] and [Ag] t.
The preparation of ascites monoclonal antibody and analyze purifying with the female BALB/C mice in 8 ages in week of aseptic Freund's incomplete adjuvant abdominal injection sensitization of 0.5mL/ dosage, 1 week back injection 2 * 10
6Individual hybridoma.Look the belly situation of expanding of mouse and after 10～14 days, collect ascites, indirect ELISA method is measured the ascites monoclonal antibody and is tired, and BCA test kit method is measured protein concentration.Protein g affinity chromatography purifying behind the purifying employing ammonium sulfate precipitation of ascites carries out the SDS-PAGE swimming to the ascites of aforesaid method purifying, and thin layer scanning is analyzed gel, accounts for the long-pending per-cent of whole districts zone face according to IgG district band, the purity and the recovery of calculating monoclonal antibody.
The preparation of embodiment 2 anti-MIC-1 humanized antibodies
Murine antibody can produce immunological rejection in human body, therefore can only be used for the treatment of acute disease, in case produce the anti-antibody to murine antibody in the human body, promptly loses efficacy as the murine antibody of medicine.In order to overcome this shortcoming, the present invention has carried out humanization to murine antibody, has prepared people-mouse chimeric antibody, and the variable region of this antibody is from murine antibody hybridoma cell strain excretory monoclonal antibody, and the constant region sequence is from people's IgG2.This antibody has kept the antigen-binding specificity of mouse monoclonal antibody, has reduced inductive immunological rejection in human body simultaneously.The step of preparation chimeric antibody is following:
Separation and purification mRNA from above-mentioned hybridoma cell strain uses oligo-dT to obtain cDNA, through PCR method increase respectively light chain of antibody and variable region of heavy chain,
The light chain primer does
5 '-GAYATTG TG MTSACMCARWCTMCA-3 ' (SEQ ID NO:7) and
5‘CTCCAGATGTTAACTGCTCAC3’(SEQ?ID?NO：8)；
The heavy chain primer does
5 '-ATGSARGTNMAGCTGSAGSAGTC-3 ' (SEQ ID NO:9) and
5’-GGTCAAGG?TCACTGGCTCAGG-3’(SEQ?ID?NO：10)。
Wherein R=G or A, Y=T or C, M=A or C, S=G or C, W=T or A, N=G or A or T or C.The PCR product is cloned into respectively in the T carrier checks order; The variable region of heavy chain CDR of mouse source antibody is transplanted on the 4-59 framework, produces humanized variable region of heavy chain.The aminoacid sequence of the CDR1 of variable region of heavy chain, CDR2 and CDR3 is respectively shown in SEQ ID NO:1, SEQ IDNO:2 and SEQ ID NO:3; The weight chain variable region amino acid sequence is shown in SEQ ID NO:11.
SEQ?ID?NO：1：Asp?Gly?Met?Tyr?Thr?Leu?Ala?Lys?Ser?Tyr?Lys?Gly
SEQ?ID?NO：2：Tyr?Ser?His?Ser?Gln?Pro?Trp?Lys?Tyr?Asn?Gly?Leu?Asp?Gly
SEQ?ID?NO：3：Met?Trp?Gly?Asp?Ile?Trp?Asn?Lys?Gly?Gln?Ser?Asp?Ala?Tyr
SEQ?ID?NO：11：
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu?Thr?LeuSer?Leu?Thr?Cys?Thr?Val?Ser?
Asp?Gly?Met?Tyr?Thr?Leu?Ala?Lys?Ser?Tyr?Lys Gly?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?
Tyr?Ser?His?Ser Gln?Pro?Trp?Lys?Tyr?Asn?Gly?Len?Asp?Gly?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?SerLys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?TyrTyr?Cys?Ala?Arg?
Met?Trp?Gly?Asp?Ile?Trp?Asn?Lys?Gly?Gln?Ser?Asp?Ala?Tyr?TrpGly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser。
The variable region of light chain CDR of mouse source antibody is transplanted on the VL08 framework, produces humanized variable region of light chain.The aminoacid sequence of the CDR1 of variable region of light chain, CDR2 and CDR3 is respectively shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.The light chain variable region amino acid sequence is shown in SEQ ID NO:12.
SEQ?ID?NO：4：Ser?Gln?Tyr?Thr?Ser?Ala?Lys?Lys?Gly
SEQ?ID?NO：5：Gly?His?Ser?Trp?Lys?Asn?Gly?Leu?Asp?Tyr
SEQ?ID?NO：6：Tyr?Ile?Gly?Pro?Trp?Gly?Gln?Gln?Leu?Ala?The
SEQ?ID?NO：12：
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?ValThr?Ile?Thr?Cys?
Ser?Gln?Tyr?Thr?Ser?Ala?Lys?Lys?Gly?Trp?Tyr?Gln?Gln?Lys?ProGly?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?
Gly?His?Ser?Trp?Lys?Asn?Gly?Leu Asp?Tyr?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?ThrPhe?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Tyr?Cys?
Tyr?Ile?Gly Pro?Trp?Gly?Gln?Gln?Leu?Ala?The?Phe?Gly?Gly?Gly?Thr?Lys?Val?Asp?Ile?Lys
With ordinary method the humanized antibody variable region of heavy chain is connected to human IgG2's constant region (its encoding sequence is shown in SEQ ID NO:13) and forms full-length gene, the humanization variable region of light chain is connected to people K chain constant region (its encoding sequence is shown in SEQ ID NO:14) and forms full-length gene.Make up the fusion gene of codified chimeric antibody; The encoding sox of this chimeric antibody light chain is inserted into the expression vector PCI-GPT in selective mark (GPT) and gene expression regulation district, and (with the pCI of Promega company plasmid is template; The encoding sox of guanine phosphoribosyl transferase is inserted the MCS acquisition PCI-GPT expression plasmid of PCI) in, this chimeric antibody light chain expression vector pCI-gpt-L obtained; Just the encoding sox of this chimeric antibody heavy chain is inserted into the expression vector PCI-DHFR in selective mark (DHFR) and gene expression regulation district (with the pCI of Promega company plasmid is template; The encoding sox of Tetrahydrofolate dehydrogenase is inserted the MCS acquisition PCI-DHFR expression plasmid of PCI) in, this chimeric antibody heavy chain expression carrier pCI-gpt-H obtained; Method with electrotransfection together imports above-mentioned two carriers among the mammalian cell NS/0.In containing xanthic substratum, screen transfectional cell with mycophenlate mofetil, obtain the cell strain of stable transfection.Carry out the evaluation of secretory antibody according to the method among the embodiment 1, the result shows that chimeric antibody has kept the specificity and the avidity of hybridoma cell strain secrete monoclonal antibody.
SEQ?ID?NO：13
GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCC?CCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ?ID?NO：14
ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
Embodiment 3, the application of anti-MIC-1 antibody in the carcinoma of the pancreas mouse model
The cell in vitro of learning from else's experience is cultivated in nude mouse subcutaneous well-grown human pancreas cancer PANC-1 and the SW1990 of going down to posterity (available from ATCC; Numbering is respectively CRL-1469 and CRL-2172) tumor nodule; Oncocyte liquid is processed in aseptic technique in the super clean bench, and it is subcutaneous to be inoculated in the nude mice armpit, 7x10
6Cell/mouse.Animal is divided into 4 groups respectively at random, 6 every group, totally 8 groups of the labels of weighing, PANC-1 and SW1990.Experiment is respectively model control group, the positive drug key is selected (Gem 50mg/kg) group, mouse-anti hMIC-1 monoclonal antibody high dosage (10mg/kg) group, mouse-anti hMIC-1 monoclonal antibody low dosage (2mg/kg) group.Experimental group gross tumor volume behind inoculated tumour about 80～100mm that grows
3Tail vein injection mouse-anti hMIC-1 monoclonal anti body fluid was each 1 time in the 10th, 13, and tail vein injection mouse-anti hMIC-1 of identical weekly time monoclonal anti body fluid is each 1 time later on, and experiment whole process is injected mouse-anti hMIC-1 monoclonal anti body fluid 8 times altogether.The positive drug key is selected (Gem 50mg/kg) group abdominal injection on the 10th 1 time behind inoculated tumour, identical weekly later on time abdominal injection 1 time, and experiment whole process is injected 4 times altogether.The identical time abdominal injection 0.9% aseptic Nacl injection liquid 10ml/kg of model control group, experiment whole process is injected 8 times altogether.Each organizes administration time beginning in the 10th day behind the inoculated tumour.Daily observation mice with tumor showed with subcutaneous tumor volumes of slide calliper rule survey in per 4 days after experiment begins, after the last administration finishes, and execution animal on the 37th behind tumor inoculation.
Experimental result shows that mouse-anti hMIC-1 monoclonal antibody has obvious antitumor activity (10mg/kg, Biw to human pancreas cancer PANC-1; Iv * 8); Tumor control rate 67.65%, but having dose-effect relationship between knurl effect and dosage, tumor killing effect is superior to key and selects intraperitoneal administration group (Gem 50mg/kg; Qw, ip * 4) (P＜0.01) (table 1, Fig. 1, Fig. 3).Test and finish the back the 6th week to human pancreas cancer PANC-1 experiment, mouse-anti people hMIC-1 monoclonal antibody group (10mg/kg, Biw, iv * 8) animal tumor volume averaging is 559mm
3, and lotus knurl model control group animal tumor volume averaging is 1685mm
3, it is 765mm that key is selected intraperitoneal administration group (Gem 50mg/kg, qw, ip * 4) animal tumor volume averaging
3, gross tumor volume and the remarkable P of the poor heteropole of lotus knurl model control group＜0.01 when the experiment of mouse-anti hMIC-1 monoclonal antibody high dose group finishes.To human pancreas cancer SW1990 experiment, mouse-anti hMIC-1 monoclonal antibody group (10mg/kg, Biw, iv * 8) animal tumor volume averaging is 1045mm
3, and lotus knurl model control group animal tumor volume averaging is 1737mm
3, it is 881mm that key is selected intraperitoneal administration group (Gem 50mg/kg, qw, ip * 4) animal tumor volume averaging
3, gross tumor volume and the remarkable P of lotus knurl model control group comparing difference＜0.05 (Fig. 2, Fig. 4) when the experiment of mouse-anti hMIC-1 monoclonal antibody high dose group finishes.
Table 1: mouse-anti hMIC-1 monoclonal antibody is transplanted the human pancreas cancer tumor-inhibiting action to nude mice and is observed
Δ N.S.: saline water
*Gem or MIC-1 vs contrast
Carry out the transplanted tumor histopathological examination, the down visible lotus knurl model control group cancerous tissue of light microscopic, the tumour cell structure is clear; Cell is complete, and cancer cells is circular or oval, and nuclear is big; Kernel is obvious; Cell differs in size, and cancer cells is separated into the cancer nests that differs in size by fibrous tissue, wherein visible glandular tube, lumen of gland spline structure.The visible tumour cell of mouse-anti hMIC-1 monoclonal antibody group is destroyed, it is thus clear that a large amount of lymphocytic infiltrations are arranged, tumour cell is obviously downright bad, cytolysis down for light microscopic.Show that mouse-anti hMIC-1 monoclonal antibody has obvious restraining effect to tumour cell.
Finishing back paraffin section CD34 immunohistochemical staining result from experiment shows; Mouse-anti hMIC-1 monoclonal antibody group (10mg/kg; Biw, iv * 8) the interior visible form of transplanted tumor tumor tissues is irregular and do not have the new vessel that obvious tube chamber forms, and vascularity is seen with tumor tissues edge more.Blood vessel obviously is less than lotus knurl model control group, and both have significant difference, and microvessel density is significantly higher than mouse-anti hMIC-1 monoclonal antibody group (P＜0.01) in the negative control group transplanted tumor.
Claims (8)
1. MIC-1 monoclonal antibody, it comprises:
Variable region of heavy chain, the CDR1 of this variable region of heavy chain, CDR2 and CDR3 aminoacid sequence are respectively SEQ ID NO:1, the aminoacid sequence shown in 2 and 3; With
Variable region of light chain, the CDR1 of this variable region of light chain, CDR2 and CDR3 aminoacid sequence are respectively SEQ ID NO:4, the aminoacid sequence shown in 5 and 6.
2. MIC-1 monoclonal antibody according to claim 1, wherein, the framework region aminoacid sequence of variable region of heavy chain is identical with the framework region of people's germline gene site VH4-59 coding; The framework region aminoacid sequence of variable region of light chain is identical with the framework region of people's antibody germline gene site VL 08 coding.
3. pharmaceutical composition, it comprises claim 1 or 2 described MIC-1 monoclonal antibody and pharmaceutical carriers.
4. polynucleotide, its MIC-1 monoclonal antibody according to claim 1 and 2 of encoding.
5. expression vector, it comprises the described polynucleotide of claim 4.
6. host cell, it comprises the described expression vector of claim 5.
7. the application of MIC-1 monoclonal antibody according to claim 1 and 2 in the medicine of preparation treatment tumour.
8. application according to claim 7, wherein said tumour comprises carcinoma of the pancreas, the esophageal carcinoma, cancer of the stomach, intestinal cancer, lung cancer, mammary cancer, ovarian cancer and liver cancer, preferred, said tumour is a carcinoma of the pancreas, the esophageal carcinoma, cancer of the stomach, intestinal cancer and liver cancer.
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| PCT/CN2012/079969 WO2013023557A1 (en) | 2011-08-12 | 2012-08-10 | Humanized monoclone antibody of macrophage inhibition factor 1 and use thereof |
| ZA2014/01760A ZA201401760B (en) | 2011-08-12 | 2014-03-11 | Humanized monoclone antibody of macrophage inhibition factor 1 and use thereof |
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| WO2013023557A1 (en) * | 2011-08-12 | 2013-02-21 | 中国医学科学院肿瘤研究所 | Humanized monoclone antibody of macrophage inhibition factor 1 and use thereof |
| CN103616519A (en) * | 2013-11-28 | 2014-03-05 | 张伟 | Application of macrophage inhibition factor 1 in hepatopathy |
| CN103645319A (en) * | 2013-12-20 | 2014-03-19 | 张伟 | Application of macrophage inhibiting factor-1 in colorectal cancer diagnosis |
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| JP2015532271A (en) * | 2012-09-26 | 2015-11-09 | ユリウス・マクシミリアンス−ウニヴェルジテート・ヴュルツブルクＪｕｌｉｕｓ Ｍａｘｉｍｉｌｉａｎｓ−Ｕｎｉｖｅｒｓｉｔａｅｔ Ｗｕｅｒｚｂｕｒｇ | Monoclonal antibody against growth differentiation factor 15 (GDF-15) |
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| WO2013023557A1 (en) | 2013-02-21 |
| CN102321173B (en) | 2013-04-03 |
| ZA201401760B (en) | 2015-12-23 |
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