CN106065033A - The preparation of a kind of cytokine fusion antibody and application thereof - Google Patents
The preparation of a kind of cytokine fusion antibody and application thereof Download PDFInfo
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- CN106065033A CN106065033A CN201610159826.1A CN201610159826A CN106065033A CN 106065033 A CN106065033 A CN 106065033A CN 201610159826 A CN201610159826 A CN 201610159826A CN 106065033 A CN106065033 A CN 106065033A
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- antibody
- vegfr2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
Abstract
The invention belongs to genetic engineering antibody technical field, specifically disclose antibody JZB00 and the fusion antibody JZA01 of interferon-ALPHA (IFN α), the Preparation Method And The Use of a kind of targeted human VEGF (VEGFR2, also known as KDR).The invention also discloses the heavy chain of JZA01 and the aminoacid sequence of light chain immunoglobulin molecule.Present invention also offers the construction method of JZA01 heavy chain and light chain gene, transfection CHO cell, limiting dilution assay picking monoclonal, obtain fusion antibody by eukaryotic cell secreting, expressing, affinitive layer purification afterwards.The fusion antibody of the present invention can specifically bind to VEGFR2, tumor neovasculature generation can be suppressed, the interferon part of coupling can also play direct tumor-killing effect and immunoregulation effect, does not produce toxic and side effects simultaneously, thus preferably suppresses the growth of tumor.This fusion antibody can suppress people's venous endothelial cell (HUVEC) and the propagation of Partial tumors cell in vitro.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of can be with the full people of high-affinity of human VEGFR-3 2 specific bond
Source fusion antibody, this antibody, based on the full length antibody and IFN α of anti-vegf R2 of Quan Renyuan, is built into by gene recombinaton
Fusion gene, utilizes flexible peptide to be coupled together by two sections of albumen.On the one hand this antibody can suppress human vascular endothelial growth factor to be subject to
The activation of body 2, thus suppress tumor neovasculature generation, on the other hand targeting makes IFN be enriched in tumor and tumor is new
Angiogenic position, plays direct tumor-killing effect and immunoregulation effect, and does not produce toxic and side effects, and it is anti-to be that one has
Angiogenic activity and the genetic engineering antibody of anti-tumor activity.
Background technology
The development that occurs of tumor cell be unable to do without the nutrition supply of new vessels, and the growth of new vessels be unable to do without various
Cytokine and the interaction of receptor thereof.Now there are some researches show, VEGF/vascular endothelial growth factor receptor
(VEGF/VEGFR) signal path has critical regulation effect for the generation of new vessels.VEGFR2 is as the master of VEGF
Want receptor, the propagation of regulation vascular endothelial cell, survive, migrate and the change of permeability.
Human vascular endothelial growth factor receptor 2 (vascular endothelial growth factor receptor2,
Also referred to as VEGFR2 or KDR, Flk-1, referred to herein as " VEGFR2 ") it is the transmembrane glycoprotein of 230kDa, belongs to receptor tyrosine
Kinases superfamily, is the major function receptor of angiogenesis factor (VEGF), is mainly distributed in vascular endothelial cell and lymph
In chrotoplast, VEGF stimulating endothelial cell propagation, increase the effect of vascular permeability and new vascular generation mainly by combining and
Activating VEGFR2 to realize, VEGFR2 also has expression at some tumor cell surfaces simultaneously.VEGFR2 is the cross-film of a kind of type III
Protein kinase, the VEGFR2 gene of the mankind is positioned at chromosome 4q11-q12,1356 aminoacid of encoding full leng receptor, VEGFR2
It is translated into when cell interior and there are about 150kDa, and there is no obvious glycosylated protein, be then passed through a series of glycosyl
Change processes, and there are about 230kDa time ripe, expresses on after birth.VEGFR2 is formed owing to relating to tumor, has become as antitumor
The specific target target for the treatment of.
Targeting VEGFR2 drug main to have two classes: a class is to directly act on recipient cell inner section in case stop signal conducts
The tyrosine kinase inhibitor (TKI) of synthesis, mainly include Sunitinib, Ceritinib etc.；Another kind of is to block part
It is attached to the monoclonal antibody (mAb) of receptor ectodomain, including Lei Molu monoclonal antibody (Ramucirumab) etc..
Lei Molu monoclonal antibody (Ramucirumab), trade name Cyramza) it is the humanization IgG1 monoclonal of human VEGFR-3 resistant 2
Antibody, it was used for treating late gastric cancer or stomach esophageal junction adenocarcinoma by FDA's approval in 2014.
Interferon (interferon, IFN) is one group has the reactive protein (mainly glycoprotein) of several functions, is
A kind of cytokine produced by mononuclear cell and lymphocyte.Interferon has the antiviral of wide spectrum, anti-tumor activity, simultaneously
Also can strengthen natural killer cell (NK cell), macrophage and the vigor of T lymphocyte, thus play immunoregulation effect,
It it is one of anti-tumor biological product currently mainly.But interferon is because of the shortcomings such as half-life toxic and side effects short, systemic is strong limit
Make its application clinically.Some long-acting interferons are the most had listed, though internal the half of interferon can be improved
Decline the phase, but can not well reduce its systematicness toxic and side effects.By gene recombination technology by interferon and targeting VEGFR2
Full length antibody coupling, both can extend interferon Half-life in vivo, simultaneously can be by the targeting of antibody by interferon
It is enriched in tumor and tumor neogenetic blood vessels position, reduces its system toxic and side effects, make it more effectively play antitumor and immunity
Regulation effect, produces a kind of novel fusion antibody with anti-angiogenesis activity and anti-tumor activity.
Summary of the invention
Goal of the invention
The present invention provides the fusion antibody of a kind of Human anti-human VEGFR2 with potential medical science and pharmacy value.This
The feature of bright albumen is specific binding human VEGFR-3 2 extracellular region, can suppress Human umbilical vein endothelial cells HUVEC in vitro
Propagation and the increment of Partial tumors cell.
Technical scheme
A kind of fusion antibody of anti-vegf R2 in full people source, it is characterised in that: with the full length antibody JZB00 of anti-vegf R2 and
Based on IFN α albumen, both are connected with flexible peptide by gene recombination technology.
Wherein chain is connected with flexible peptide by the heavy chain of the full length antibody of anti-vegf R2 and IFN α albumen and forms, its amino
Acid sequence is SEQ NO.1；Another is the light chain of full length antibody of anti-vegf R2, and its aminoacid sequence is SEQ NO.2.
The nucleic acid of a kind of separation, it is characterised in that the fusion antibody that this nucleic acid coding is above-mentioned.
A kind of expression vector, containing above-mentioned nucleic acid.
A kind of recombinant host cell, containing above-mentioned expression vector.
The antibody of any of the above-described or the application of antibody fragment, be optionally combined with VEGFR2 or suppress VEGFR2 with
The combination of VEGFR2 part or neutralization VEGFR2.
The antibody fragment of any of the above-described, selected from single-chain antibody, Fab, scFv, double antibody and three antibody.
The antibody of any of the above-described or the conjugate of antibody fragment.
The antibody of any of the above-described or the mutant of antibody fragment.
The application in terms of oncotherapy of the targeting VEGFR2 fusion antibody.
Invention further illustrates:
In the present invention full people source anti-vegf R2 fusion antibody heavy chain by the heavy chain of the full length antibody of anti-vegf R2 and IFN α with
Flexible peptide (GGGGS) connects, and light chain is the light chain of the full length antibody of anti-vegf R2.
Expression vector and host cell containing VEGF R2 fusion antibody gene of the present invention belong to this
The protection domain of invention.Expand fusion antibody gene of the present invention any fragment primer to also protection scope of the present invention it
In.
It is a further object to provide one and can express vascular endothelial growth factor receptor 2 above-mentioned with purification
The method of antibody.
Western Blot identifies the isolated and purified JZA01 obtained；Flow cytometer detection JZA01 and high expressed VEGFR2 cell strain
The combination of HUVEC；Detection fusion antibody adds the growth inhibited effect to people venous endothelial cell HUVEC and Partial tumors cell.
Vascular endothelial growth factor receptor 2 and the people's venous endothelial cell HUVEC vascular surface endothelium that the present invention obtains
Growth factor acceptor 2 has high specific and combines, and vitro inhibition HUVEC and Partial tumors cell growth assay result show, this
The fusion antibody that invention obtains can substantially suppress HUVEC and the growth of Partial tumors cell.The present invention for treatment and diagnosis with
The disease that the expression of VEGF R2 antigen, particularly overexpression are characterized provides treatment based on antibody
Method, relevant disease includes but not limited to autoimmune disease and cancer.
Accompanying drawing explanation
Fig. 1 is JZA01 gene recombinaton analysis chart, and recombination includes that heavy chain gene size is about 1932bp, light chain gene size
It is about 762bp.
Fig. 2 is SDS-PAGE protein electrophorese figure, and the JZA01 describing fermentation expression is carried out point by ProteinA affinity column
Result from purification.A is the non-reduced electrophoresis of JZA01, and B is JZA01 reduction electrophoresis.
Fig. 3 is JZA01Western Blot qualification figure after purification, and A is Non, and detection antibody is anti-human (H&
L)-HRP；B is reduced form, and one resists for anti-humanIFN α, and detection antibody is the sheep anti-mouse antibody of HRP labelling；C is reduction
Type, detection antibody is anti-human (H&L)-HRP.
Fig. 4 is that JZA01 with HUVEC cell streaming is combined figure, describes the combination situation of JZA01 and surface endothelial cell antigens, knot
Conjunction rate is about 39%, suitable with parent monoclonal antibody JZB00.
Fig. 5 is a curve chart, describes the JZA01 growth inhibited effect to HUVEC cell.
Fig. 6 is a curve chart, describes the JZA01 growth to tetra-kinds of tumor cells of MDA-MB-231, HepG2, U937 and NCI-N87
Inhibitory action.
The present invention provides the fusion antibody of a kind of Human anti-human VEGFR2 with potential medical science and pharmacy value.This
The feature of bright albumen is specific binding human VEGFR-3 2 extracellular region, can suppress Human umbilical vein endothelial cells HUVEC in vitro
Propagation and the increment of Partial tumors cell.
Technical scheme
A kind of fusion antibody of anti-vegf R2 in full people source, it is characterised in that: with the full length antibody JZB00 of anti-vegf R2 and
Based on IFN α albumen, both are connected with flexible peptide by gene recombination technology.
Wherein chain is connected with flexible peptide by the heavy chain of the full length antibody of anti-vegf R2 and IFN α albumen and forms, its amino
Acid sequence is SEQ NO.1；Another is the light chain of full length antibody of anti-vegf R2, and its aminoacid sequence is SEQ NO.2.
The nucleic acid of a kind of separation, it is characterised in that the fusion antibody that this nucleic acid coding is above-mentioned.
A kind of expression vector, containing above-mentioned nucleic acid.
A kind of recombinant host cell, containing above-mentioned expression vector.
The antibody of any of the above-described or the application of antibody fragment, be optionally combined with VEGFR2 or suppress VEGFR2 with
The combination of VEGFR2 part or neutralization VEGFR2.
The antibody fragment of any of the above-described, selected from single-chain antibody, Fab, scFv, double antibody and three antibody.
The antibody of any of the above-described or the conjugate of antibody fragment.
The antibody of any of the above-described or the mutant of antibody fragment.
The application in terms of oncotherapy of the targeting VEGFR2 fusion antibody.
Invention further illustrates:
In the present invention full people source anti-vegf R2 fusion antibody heavy chain by the heavy chain of the full length antibody of anti-vegf R2 and IFN α with
Flexible peptide (GGGGS) connects, and light chain is the light chain of the full length antibody of anti-vegf R2.
Expression vector and host cell containing VEGF R2 fusion antibody gene of the present invention belong to this
The protection domain of invention.Expand fusion antibody gene of the present invention any fragment primer to also protection scope of the present invention it
In.
It is a further object to provide one and can express vascular endothelial growth factor receptor 2 above-mentioned with purification
The method of antibody.
Western Blot identifies the isolated and purified JZA01 obtained；Flow cytometer detection JZA01 and high expressed VEGFR2 cell strain
The combination of HUVEC；Detection fusion antibody adds the growth inhibited effect to people venous endothelial cell HUVEC and Partial tumors cell.
Vascular endothelial growth factor receptor 2 and the people's venous endothelial cell HUVEC vascular surface endothelium that the present invention obtains
Growth factor acceptor 2 has high specific and combines, and vitro inhibition HUVEC and Partial tumors cell growth assay result show, this
The fusion antibody that invention obtains can substantially suppress HUVEC and the growth of Partial tumors cell.The present invention for treatment and diagnosis with
The disease that the expression of VEGF R2 antigen, particularly overexpression are characterized provides treatment based on antibody
Method, relevant disease includes but not limited to autoimmune disease and cancer.
Accompanying drawing explanation
Embodiment 4 anti-vegf R2 fusion antibody is combined with the streaming of HUVEC cell
Digestion HUVEC cell, is divided into 1 × 106Often group, 1500rpm, 4 DEG C of centrifugal 5min, PBS+2% washes three times, respectively with
100nM JZA01,100nM JZB00 and skimmed milk solution 4 DEG C effect 1h.1500rpm, 4 DEG C of centrifugal 5min, PBS+2%FBS
Wash three times, act on 1h with Donkey Anti-Human IgG (H+L)-FITC antibody (Life Sciences) 4 DEG C.1500rpm、
4 DEG C of centrifugal 5min, PBS+2% wash three times, use PBS re-suspended cell, flow cytomery after being centrifuged for the last time.
The growth inhibited effect to HUVEC and Partial tumors cell of the embodiment 5 anti-vegf R2 fusion antibody
Cell is prepared as 3 × 104Cells/ml cell suspension, inoculates 96 porocyte culture plates, and 100 μ l/ holes, at 37 DEG C
5%CO2Incubator is cultivated 24h.With 2% hyclone culture medium compounding pharmaceutical, have JZA01, JZB00 and IFN α three groups,
Often group arranges 2.5nM, 5nM, 10nM, 20nM, 40nM, 80nM, 160nM, 320nM totally 8 concentration (0.01nM, 0.1nM, 1nM,
10nM, 100nM, 200nM totally 6 concentration).The cell supernatant discarded that will cultivate, adds medicine according to packet, and every hole 100 μ L is every
The group each concentration of medicine arranges three parallel holes, continues to cultivate 72h, and every hole adds the MTT of 11 μ l, and 37 DEG C are continued to cultivate 4h, little
The heart inclines supernatant, and every hole adds 150 μ l DMSO, measures absorbance value (OD value), count at microplate reader 570nm/630nm wavelength
Calculate the suppression ratio of medicine cell proliferation.The suppression ratio evaluation that the biological activity of JZA01 is grown by cell.Suppression ratio=(1-is real
Test group OD value/matched group OD value) × 100%.
Claims (10)
1. the fusion antibody of the human vessel endothelium growth factor resisting receptor 2 in a full people source, it is characterised in that:
Based on the full length antibody JZB00 and interferon (IFN α) of anti-vegf R2, will with flexible peptide by gene recombination technology
Both albumen connect.
The fusion antibody of the human vessel endothelium growth factor resisting receptor 2 in a kind of full people source the most according to claim 1, it is special
Levy and be: wherein chain is connected with flexible peptide by the heavy chain of the full length antibody of anti-vegf R2 and IFN α albumen and forms, its amino
Acid sequence is SEQ NO.1；Another chain is the light chain of the full length antibody of anti-vegf R2, and its aminoacid sequence is SEQ NO.2.
3. the nucleic acid separated, it is characterised in that the anti-human blood vessel endothelium in a kind of full people source of this nucleic acid coding claim 1
The fusion antibody of growth factor acceptor 2.
4. an expression vector, containing the nucleic acid described in claim 5.
5. a host cell, containing the expression vector described in claim 6.
6. according to antibody or the application of antibody fragment of any one of claim 1 or 2, it is characterised in that optionally with VEGFR2
In conjunction with or suppression VEGFR2 Yu VEGFR2 part combination or neutralization VEGFR2.
7. the antibody fragment of any one of claim 1 or 2, selected from single-chain antibody, Fab, scFv, double antibody and three antibody.
8. the antibody of any one of claim 1 or 2 or the conjugate of antibody fragment.
9. the antibody of any one of claim 1 or 2 or the mutant of antibody fragment.
10. the application in terms of oncotherapy of the fusion antibody described in claim 1.
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| CN201610159826.1A CN106065033A (en) | 2016-03-17 | 2016-03-17 | The preparation of a kind of cytokine fusion antibody and application thereof |
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| CN201610159826.1A CN106065033A (en) | 2016-03-17 | 2016-03-17 | The preparation of a kind of cytokine fusion antibody and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106854248A (en) * | 2016-12-09 | 2017-06-16 | 中国药科大学 | A kind of preparation and its application of cell factor mutant fusion antibody |
| CN108997489A (en) * | 2018-09-04 | 2018-12-14 | 中国药科大学 | Interferon mutant and interferon mutant fusion antibody and preparation method thereof, application |
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| EP1292334A4 (en) * | 2000-06-22 | 2003-11-19 | Idec Pharma Corp | Bispecific fusion protein and method of use for enhancing effector cell killing of target cells |
| US7456257B2 (en) * | 2003-02-18 | 2008-11-25 | Merck Patent Gmbh | Fusion proteins of interferon alpha muteins with improved properties |
| WO2014139468A1 (en) * | 2013-03-15 | 2014-09-18 | Admark Healthcare, Llc | Fusion protein molecules and method of use |
| CN104203982A (en) * | 2011-10-28 | 2014-12-10 | 特瓦制药澳大利亚私人有限公司 | Polypeptide constructs and uses thereof |
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2016
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1292334A4 (en) * | 2000-06-22 | 2003-11-19 | Idec Pharma Corp | Bispecific fusion protein and method of use for enhancing effector cell killing of target cells |
| US7456257B2 (en) * | 2003-02-18 | 2008-11-25 | Merck Patent Gmbh | Fusion proteins of interferon alpha muteins with improved properties |
| CN104203982A (en) * | 2011-10-28 | 2014-12-10 | 特瓦制药澳大利亚私人有限公司 | Polypeptide constructs and uses thereof |
| WO2014139468A1 (en) * | 2013-03-15 | 2014-09-18 | Admark Healthcare, Llc | Fusion protein molecules and method of use |
Non-Patent Citations (1)
| Title |
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| XUAN C等: "Targeted delivery of interferon-alpha via fusion to anti-CD20 results in potent antitumor activity against B-cell lymphoma", 《BLOOD》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106854248A (en) * | 2016-12-09 | 2017-06-16 | 中国药科大学 | A kind of preparation and its application of cell factor mutant fusion antibody |
| CN108997489A (en) * | 2018-09-04 | 2018-12-14 | 中国药科大学 | Interferon mutant and interferon mutant fusion antibody and preparation method thereof, application |
| CN108997489B (en) * | 2018-09-04 | 2021-08-27 | 中国药科大学 | Interferon mutant and interferon mutant fusion antibody, and preparation method and application thereof |
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Application publication date: 20161102 |