CN102321173B - 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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- 230000019491 signal transduction Effects 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 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
- 230000005760 tumorsuppression 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 the 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 transforming growth factor-beta (Transforming growth factor-β, TGF-β) an important branch member in the superfamily, be also referred to as GDF-15 (growth differentiation factor-15, GDF-15) etc., because 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, formed by 308 amino acid polypeptides, comprise 29 amino acid signal peptides, 167 amino acid propetides and 112 amino acid maturation zones.Article two, polypeptide chain is sheared by furin sample proteolytic enzyme after disulfide linkage connects into precursor protein, the final homodimer maturation protein that is connected by disulfide linkage, molecular weight 25KD[Fairlie LN et al, 1999 of forming].Ripe MIC-1 protein excretion enters 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, is discharged in the blood after the hydrolysis under different physiology and pathological conditions.
MIC-1 as a kind of Cytokine in cell surface TGF beta receptor; the signal transmission of wide participation apoptosis, differentiation, propagation and body inflammatory reaction; and increase at inhibition tumor cell, promote apoptosis, keep placental function and the aspects such as fetal development, adjusting and protection nervus centralis have vital role [Lee DH et al, 2003].Under the normal physiological state, the MIC-1 high expression level is expressed and be low in the tissues such as prostate gland, colon, kidney, brain, liver, pancreas 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 by p53, PI3K/AKT/GSK3 signal β 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 that MIC-1 as the target of PI3K/AKT/GSK3 β path, can promote the apoptosis [Yamaguchi Ket al, 2004] of tumour cell; The 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, the people such as Sung study and find can promote MIC-1 to express and secretion by the V600EB-Raf signal path, 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 by promoting the ErBb2 acceptor also follows the tyrosine phosphorylations such as EGFR, ErBb2 to activate ERK1/2 and Akt, promotes the invasive ability (Kim et al 2008) of tumour cell; The people such as Lee find that MIC-1 overexpression in the cancer of the stomach can significantly increase the activity of UPA uPA and uPAR, raise the uPAu/PAR active system by 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 the apositia of MIC-1 and Tumor-assaciated 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 the control action kou to appetite, make the patient that apositia and weight loss and the final patient's of reduction survival time [Johnen et al, 2007] occur.F.E.Wiklund etc. are respectively the regression epidemiological study of 5.3 years and 9.1 years to having carried out the meta observation time 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 many reasons that comprises tumour causes, after the interference of getting rid of age, BMI, smoking history, the high serum level group of MIC-1 is compared with the normal level group higher tumor mortality risk (OR=2.74,95%CI 1.70-4.22).
Above-mentioned result of study shows, the MIC-1 participation multiple physiology of body and pathologic process, and bring into play therein various effects.If our imagination is passed through the action pathway of antibody blocking MIC-1, might in the treatment of tumour, play a role.Be example in carcinoma of the pancreas, 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, the experimental group tumour is significantly dwindled compared with the control, and the section immunohistochemical analysis shows that CD31 and MMP2 level reduce, and microvessel quantity obviously reduces.Prompting MIC-1 antibody can have been brought into play its antitumor action by the combination of blocking-up MIC-1 and acceptor.The MIC-1 monoclonal antibody is expected to provide new treatment to select to tumour patient by the function that suppresses MIC-1.
The mouse monoclonal antibody induces the endogenous anti-antibody can be divided into two classes 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 accelerating monoclonal antibody.It is reported, the HAMA incidence of most mouse monoclonal antibody medicines in the patients such as intestinal cancer is up to 100%, and 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 at present or reduce HAMA to react is to make mouse monoclonal antibody humanization or development full-length human antibody.But full-length human antibody often runs in when development that affinity of antibody descends, vigor is not high, poor stability or the 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 the treatment of tumour 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 described 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 desirable 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 the aminoacid sequence shown in the SEQ ID NO:1,2 and 3; With
Variable region of light chain, the CDR1 of this variable region of light chain, CDR2 and CDR3 aminoacid sequence are respectively the aminoacid sequence shown in the SEQ ID NO:4,5 and 6.
In one embodiment of the invention, the invention provides the humanization form of above-mentioned MIC-1 monoclonal antibody, 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 described 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 knowledge and the experimental data provided by the invention of this area, those skilled in the art as can be known medicable tumour of MIC-1 monoclonal antibody of the present invention comprise carcinoma of the pancreas, the esophageal carcinoma, cancer of the stomach, intestinal cancer, lung cancer, mammary cancer, ovarian cancer and liver cancer.Preferably, described tumour is 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 IgG2 type antibody, and with the avidity of MIC-1 be 1 * 10
-9M-1 * 10
-8M.
The present invention is by carrying out humanization modified to the anti-MIC-1 antibody in the mouse source of independent development, develop and have the anti-MIC-1 monoclonal antibody of novel human-derivedization that avidity is high, immunogenicity is low, antibody of the present invention can be blocked the combination 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 apparent 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 to nude mice Transplanted Human PANC-1 in vivo antitumor effect.
Fig. 2 mouse-anti MIC-1 monoclonal antibody is to nude mice Transplanted Human SW1990 in vivo antitumor effect.
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 preparation of the monoclonal antibody of embodiment 1 hybridoma cell strain secretion
The present invention (by 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 by 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 every 2 all booster immunizations 1 time.The blood sampling of immunity beginning in rear 10 days mouse tail vein adopts indirect ELISA method to detect serum titer, until serum titer reaches 1: 10
6Above.Merge the mouse that serum titer was the highest in front 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 the lower fusion of 50%PEG4000 (available from Sigma company) effect, divide again kind 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 higher positive hole of tiring and carry out the limiting dilution assay clone, enlarged culturing and to build strain frozen.Adopt colchicine to prevent method that hybridoma karyomit(e) is analyzed.
Monoclonal antibody Identification of Biological Characteristics 1. Dot ELISA is identified the monoclonal antibody specificity: cultivate methanol yeast GS115, and centrifugal collection thalline, precipitation 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/time; 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; Develop the color with the DAB nitrite ion.Establish simultaneously positive antiserum(antisera) in contrast.2. Western blot analyzes: select 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; Fully after the washing, add the ascites mAb effect 1h after diluting; Fully after the washing, add the sheep anti-mouse igg antibody of the HR mark of working concentration, effect 1h; Fully after the washing, with the colour developing of DAB nitrite ion, distilled 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 antibody concentration of maximum A450 value one half on the curve, determines the binding ability (2 * 10 of antibody and antigen
-9M).Antibody affinity costant (Ka) is calculated as follows [6].Ka＝(n-1)/2×(n[Ab]-[Ab]t)。[Ab]: antibody concentration corresponding to A=1/2Amax when the expression antigen concentration is [Ag]; [Ab] t: antibody concentration corresponding to 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, injection 2 * 10 after 1 week
6Individual hybridoma.The belly situation of expanding depending on mouse was collected ascites after 10～14 days, indirect ELISA method is measured the ascites monoclonal antibody and 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 whole districts with the per-cent of area according to IgG district band, calculates purity and the rate of recovery of 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 for murine antibody in the human body, namely 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 the monoclonal antibody of murine antibody hybridoma cell strain secretion, and the constant region sequence is from people's IgG2.This antibody has kept the antigen-binding specificity of mouse monoclonal antibody, has reduced simultaneously the immunological rejection of inducing in human body.The step of preparation chimeric antibody is as follows:
Separation and purification mRNA from above-mentioned hybridoma cell strain uses oligo-dT to obtain cDNA, by PCR method increase respectively light chain of antibody and variable region of heavy chain,
The light chain primer is
5 '-GAYATTG TG MTSACMCARWCTMCA-3 ' (SEQ ID NO:7) and
5‘CTCCAGATGTTAACTGCTCAC3’(SEQ?ID?NO：8)；
The heavy chain primer is
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 gene of this chimeric antibody light chain is inserted into the expression vector PCI-GPT in selective mark (GPT) and gene expression regulation district (take the pCI of Promega company plasmid as template, the encoding gene of guanine phosphoribosyl transferase is inserted the multiple clone site acquisition PCI-GPT expression plasmid of PCI) in, this chimeric antibody light chain expression vector pCI-gpt-L obtained; Just the encoding gene of this chimeric antibody heavy chain is inserted into the expression vector PCI-DHFR in selective mark (DHFR) and gene expression regulation district (take the pCI of Promega company plasmid as template, the encoding gene of Tetrahydrofolate dehydrogenase is inserted the multiple clone site 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 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 made in aseptic technique in the super clean bench, is inoculated in the nude mice armpit subcutaneous, 7x10
6Cell/mouse.Animal is divided into respectively 4 groups 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 approximately 80～100mm that grows
3The 10th, 13 tail vein injection mouse-anti hMIC-1 monoclonal anti body fluid each 1 time, later same time tail vein injection mouse-anti hMIC-1 monoclonal anti body fluid each 1 time weekly, 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, later same time abdominal injection 1 time weekly, and experiment whole process is injected 4 times altogether.Model control group same time abdominal injection 0.9% aseptic Nacl injection liquid 10ml/kg, experiment whole process is injected 8 times altogether.Each organizes administration time from behind the inoculated tumour the 10th day.Surveyed a subcutaneous tumor volumes with slide calliper rule in per 4 days after experiment beginning, daily observation mice with tumor performance after the last administration finishes, was put to death animal on the 37th behind tumor inoculation.
Experimental result shows, mouse-anti hMIC-1 monoclonal antibody has obvious antitumor activity (10mg/kg to human pancreas cancer PANC-1, Biw, iv * 8), tumor control rate 67.65% has dose-effect relationship between tumor suppression effect and dosage, and tumor killing effect is better than key and selects intraperitoneal administration group (Gem 50mg/kg, qw, ip * 4) (P＜0.01) (table 1, Fig. 1, Fig. 3).After testing the end of the 6th week human pancreas cancer PANC-1 is tested, 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 observed nude mice Transplanted Human carcinoma of the pancreas tumor-inhibiting action
Δ N.S.: physiological saline
*Gem or MIC-1 vs contrast
Carry out the transplanted tumor histopathological examination, visible lotus knurl model control group cancerous tissue under the light microscopic, the tumour cell structure is clear, cell is complete, and cancer cells is circular or oval, and nuclear is large, kernel is obvious, cell size does not wait, 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, and as seen a large amount of lymphocytic infiltrations are arranged under the light microscopic, and tumour cell is obviously downright bad, cytolysis.Show that mouse-anti hMIC-1 monoclonal antibody has obvious restraining effect to tumour cell.
Finishing rear paraffin section CD34 immunohistochemical staining result from experiment shows, mouse-anti hMIC-1 monoclonal antibody group (10mg/kg, Biw, iv * 8) new vessel that visible form is irregular and the obvious tube chamber of nothing forms in the transplanted tumor tumor tissues, vascularity is common with tumor tissues edge.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 the aminoacid sequence shown in the SEQ ID NO:1,2 and 3; With
Variable region of light chain, the CDR1 of this variable region of light chain, CDR2 and CDR3 aminoacid sequence are respectively the aminoacid sequence shown in the SEQ ID NO:4,5 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 polynucleotide claimed in claim 4.
6. host cell, it comprises expression vector claimed in 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 is selected from carcinoma of the pancreas, the esophageal carcinoma, cancer of the stomach, intestinal cancer and liver cancer.
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| DK2900263T3 (en) * | 2012-09-26 | 2019-07-29 | Univ Wuerzburg J Maximilians | MONOCLONAL ANTIBODIES AGAINST GROWTH AND DIFFERENTIAL FACTOR 15 (GDF-15) |
| ES2778201T3 (en) | 2013-11-21 | 2020-08-10 | Brigham & Womens Hospital Inc | Compositions and methods for treating pulmonary hypertension |
| CN103616519A (en) * | 2013-11-28 | 2014-03-05 | 张伟 | Application of macrophage inhibition factor 1 in hepatopathy |
| CN103645319B (en) * | 2013-12-20 | 2016-05-18 | 张伟 | The application of GDF-15 in diagnosis of colorectal carcinoma |
| GB2524553C (en) * | 2014-03-26 | 2017-07-19 | Julius-Maximilians-Universitãt Würzburg | Monoclonal antibodies to growth and differentiation factor 15 (GDF-15), and uses thereof for treating cancer cachexia |
| CA2943694A1 (en) | 2014-03-26 | 2015-10-01 | Julius-Maximilians-Universitat Wurzburg | Monoclonal antibodies to growth and differentiation factor 15 (gdf-15), and uses thereof for treating cancer cachexia and cancer |
| GB2524552B (en) * | 2014-03-26 | 2017-07-12 | Julius-Maximilians-Universitãt Wurzburg | Monoclonal antibodies to growth and differentiation factor 15 (GDF-15), and use thereof for treating cancer |
| AU2015296037B2 (en) * | 2014-08-01 | 2021-04-29 | The Brigham And Women's Hospital, Inc. | Methods and compositions relating to treatment of pulmonary arterial hypertension |
| CN104974988B (en) * | 2015-07-31 | 2016-08-24 | 南京麦科林生物医药科技有限公司 | Anti-pancreatic cancer monoclonal antibody and application thereof |
| EP3423094A4 (en) * | 2016-02-29 | 2019-11-06 | Eli Lilly and Company | Gfral receptor therapies |
| CA3215737A1 (en) | 2021-03-31 | 2022-10-06 | Cambridge Enterprise Limited | Therapeutic inhibitors of gdf15 signalling |
| CN116444667B (en) * | 2023-06-13 | 2023-09-01 | 上海驯鹿生物技术有限公司 | GDF 15-targeted fully-humanized antibody and application thereof |
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| CN101852804A (en) * | 2010-03-29 | 2010-10-06 | 中国医学科学院病原生物学研究所 | New applications of antibody of GDF15 (Growth differentiation factor 15) protein |
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| CN102321173A (en) | 2012-01-18 |
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