CN105983093A - Applications of angiogenic factor fusion protein in preparing medicines for treating eye diseases relevant to angiogenesis - Google Patents
Applications of angiogenic factor fusion protein in preparing medicines for treating eye diseases relevant to angiogenesis Download PDFInfo
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Abstract
The invention relates to various applications of angiogenic factor fusion protein in preparing medicines for treating eye diseases relevant to angiogenesis, such as diabetic retinopathy and age-related macular degeneration. More specifically, the invention relates to applications of fusion protein adopting the VEGF receptor and the FGF receptor in preparing the medicines for treating eye diseases relevant to angiogenesis.
Description
Technical field
The present invention relates to multiple angiogenic factor fusion protein in preparation for treatment and angiogenesis
The relevant application in ocular disease medicine, such as diabetic retinopathy, age related macular degeneration
Deng.More particularly it relates to vegf receptor and the fusion protein of FGF receptor and/or FGF
Receptor fusion protein application in preparation is used for the ocular disease medicine that treatment is relevant to angiogenesis.
Background technology
Optical fundus new vessels is the severe complication of multiple eye, and new vessels can be at cornea, iris
The almost ophthalmic such as corpus ciliare, choroid, retina, macula lutea and optic disc institute is middle in a organized way to be occurred, its energy
The tissue enough causing these positions is hemorrhage, ooze out and a series of pathological changes such as hypertrophy, thus results in eye
Spherical structure and the destruction of function, seriously damage visual function.This series of retinopathy includes diabetes
Retinopathy (diabetic retinopathy, DR), retinopathy of prematurity (retinopathy
Ofprematurity, ROP), age-related macular degeneration (age-related macular
Degeneration, AMD) etc., have a strong impact on vision, even blinding.
Diabetic retinopathy (DR, Diabetic Retinopathy) be diabetes (DM,
Diabetes Mellitus) common microvascular complication, it is one of the severe complication of diabetes,
Pathogenesis is complicated, and blind rate is high, is also one of the major reason of the blinding oculopathy day after tomorrow.Clinically,
Typically DR is divided into 3 classes: the first kind is non-proliferative type DR (NPDR, Non-Proliferative
Diabetic Retinopathy)；Equations of The Second Kind is proliferous type DR (PDR, Proliferative Diabetic
Retinopathy), this type DR is more serious, and blind rate is high, and 50%PDR patient can be in 5 years
Interior blinding；3rd class is diabetic macular edema (DME, Diabetic Macular Edema), if
Treatment has blind danger not in time.In the DR of these three type, cause DR patient's vision
Mainly PDR and DME gone down and lose.The pathological characters of DR is retinal neovascularization shape
Become and BRB (blood-retinal barrier) destroys.The basic lesion of DR is that capillary endothelial cell breaks
Bad, the selectivity minimizing of pericyte even disappears, cell migration, hypertrophy, and new vessels is formed, goes out
Blood, ooze out, film propagation, tractional detachment of retina be until blind.
Age-related macular degeneration is that with age increase, sickness rate rises one and causes center to regard
The disease that power declines, is also called age-related macular degeneration, is clinically in old people's retinopathy
Commonly encountered diseases, is also the oculopathy of a kind of serious infringement central vision, yellow with the retina that vision is extremely sensitive
Degenerative change and the new vessels of macular area are generated as feature.Clinically according to choroid with or without new hemopoietic
Pipe stretches under retinal pigment epithelium, and AMD is divided into atrophic type and exudative type (atrophic type
Be referred to as dryness, exudative be also called moist).
Research shows, VEGF is current known somatomedin the most special to angiogenesis, maximally effective
[1,2].Since generation nineteen ninety, with VEGF as target, suppressed by blocking VEGF signal path
The medicine of optical fundus blood vessel new life becomes the focus of exploitation.Ranibizumab (Ranibizumab, trade name
Lucentis) may specifically bind VEGF A, obtain U.S. FDA approval moist for treating
Age-related macular degeneration and diabetic macular edema [3].VEGF trap-eye (VEGF Trap-Eye)
(VEGF Trap or Eylea) is the anti-vegf restructuring egg of Regeneron drugmaker of U.S. exploitation
In vain, clinical study results shows that the curative effect to DME is satisfactory, has submitted treatment DME and has supplemented
Application.KH902 (Compaq is western general, Conbercept) be Chengdu Kang Hong company exploitation for old age
The VEGFR-Fc recombiant protein of degeneration of macula (AMD), in December, 2013 approval listing is used for
Treatment AMD [4].Lucentis with VEGF as target etc. be used successfully to DR clinical research and
Clinical practice showing, VEGF is main effectively one of target of DR.
Although the medicine for VEGF target makes great progress clinically, due to blood vessel
New life is regulated by the multiple factor, and the regulation and control of angiogenesis are a sufficiently complex dynamic equilibrium
Journey.Existing medicine there is also significant limitation in clinical treatment, improves the most further
The clinical therapeutic efficacy of anti-angiogenic rebirth is the problem that researcher needs to solve, and is also to research and develop next
Emphasis for anti-angiogenic drugs.
Fibroblast growth factor (FGF) is the growth factor family of heparin-binding, moves in suckling
In thing, it has 22 family members (FGF 1-14,16-23).FGF is in various biological functionally
There is important function, such as cell proliferation, break up, migrate, angiogenesis and tumor occur.It leads to
Cross combination and activated cell surface FGF receptor (FGFR) plays its various biological function [5].
Fibroblast growth factor acceptor (fibroblast growth factor receptor, FGFR) is
The receptor combined with FGF family member.A part of involved in diseases process in FGFR.
Summary of the invention
Present inventors have surprisingly discovered that the antagonizing vessel new life factor (particularly antagonism FGF and/
Or dual antagonism VEGF and FGF) high molecular weight protein medicine can preferably suppress the new of blood vessel
Raw, obtain more preferable therapeutic effect clinically.The present inventor by build multiple comprise different
VEGFR fragment, FGFR fragment and the fusion protein of Ig G1Fc, it is thus achieved that to VEGF and
FGF (especially FGF-2) has the fusion protein and to FGF (especially of high affinity
FGF-2) there is the fusion protein of high affinity.And, inventors demonstrated that the fusion of the present invention
Albumen therapeutic effect when for treating angiogenesis associated ocular disease is significantly better than prior art
In for the mono-target of VEGF medicine (such as the U.S. Regeneron company research and development
VEGF-Trap).Especially, the fusion protein of the present invention is at diabetic retinopathy (such as STZ
The DR rat model of induction), age relevant degeneration of macula (such as laser-induced Rhesus Macacus choroid
New vessels inhibition), retinopathy of prematurity (such as hyperoxia induction retinopathy OIR
Mouse model) in show the therapeutical effect being unexpectedly significantly better than the mono-target agent of VEGF.
In an aspect, the invention provides the fusion protein of VEGFR and FGFR
(VEGFR-FGFR fusion protein) is used for, in preparation, the ocular disease that treatment is relevant to angiogenesis
Medicine in application.
Especially, the VEGFR-FGFR fusion protein of the present invention comprises: the second of VEGFR1
Ig spline structure territory, the 3rd Ig spline structure territory of VEGFR2, it is derived from the middle of FGFR Ig spline structure territory
The part in functional sequence district, FGFR the 2nd Ig spline structure territory and FGFR the 3rd Ig spline structure territory.
In some embodiments, contained in the fusion protein of the present invention FGFR Ig sample that is derived from is tied
The aminoacid sequence of the part of intermediate functional sequence area, structure territory is corresponding to starting point in SEQ ID NO:1
It is the aminoacid sequence of the 162nd amino acids to terminal for the aminoacid selected from the 119th to 151,
It is preferably SEQ ID NO:1 the 134th to 162,145 to 162 or 151 to 162 institutes
The aminoacid sequence shown, the 145th to 162 shown aminoacid of more preferably SEQ ID NO:1
Sequence.
In other embodiments, the described fusion protein of the present invention also comprises immunoglobulin Fc
District, preferably human IgG1 Fc district, the most described human IgG1 Fc district has and SEQ ID NO:4 phase
Corresponding aminoacid sequence.
In other embodiments, described VEGFR1 the 2nd Ig spline structure territory has: with SEQ
The 151st to 214 corresponding aminoacid sequence of ID NO:2, described VEGFR2 the 3rd Ig sample
Domain has: with the 224th to 320 corresponding aminoacid sequence of SEQ ID NO:3, institute
State FGFR1 the 2nd Ig spline structure territory to have: relative with SEQ ID NO:1 the 163rd to 247
The aminoacid sequence answered, and described FGFR1 the 3rd Ig spline structure territory has: with SEQ ID NO:1
270th to 359 corresponding aminoacid sequence.
In other embodiments, the VEGFR-FGFR fusion protein of the present invention has SEQ
The aminoacid sequence of ID NO:5.
In other embodiment, the described ocular disease relevant to angiogenesis is selected from: age phase
Closing property degeneration of macula such as atrophic type AMD and Exudative AMD, diabetic retinopathy is such as
Non-proliferative type DR, proliferous type DR and DME, diabetic macular edema, premature retinal
Disease and retinal vessel occlusion.
In an aspect, the invention provides FGFR fusion protein in preparation for treatment and blood
Application in the medicine of the ocular disease that pipe new life is correlated with.Preferably, present invention also offers
VEGFR-FGFR fusion protein and FGFR fusion protein are used for treatment and angiogenesis in preparation
Application in the medicine of relevant ocular disease.Especially, the FGFR fusion protein bag of the present invention
Containing being derived from the part of intermediate functional sequence area, FGFR Ig spline structure territory, FGFR the 2nd Ig sample knot
Structure territory and FGFR the 3rd Ig spline structure territory.
In yet another aspect, the invention provides VEGFR-FGFR fusion protein and/or FGFR
Fusion protein is preparation answering in the medicine improving short-term retina injury in disease subject
With.In some embodiments, described short-term retina injury is withered in minimizing retinal blood pipe network
Die cell quantity, to reduce the seepage of blood-retina barrier, suppression retinal gliocytes reactive
Hypertrophy and improve the ultrastructure of neural retina and retinal vessel.
In yet another aspect, the invention provides VEGFR-FGFR fusion protein and/or FGFR
Fusion protein is preparation answering in the medicine improving long-term retina injury in disease subject
With.In some embodiments, described long-term retina injury selected from improve retinal barrier seepage with
And suppress thickening of capillary basement membrane.
In yet another aspect, the invention provides VEGFR-FGFR fusion protein and/or FGFR
Fusion protein is used for improving retina in retinopathy of prematurity object without blood vessel perfusion area in preparation
Or reduce the application in the medicine of retinal neovascularization cell check figure.
In yet another aspect, the invention provides VEGFR-FGFR fusion protein and/or FGFR
Fusion protein preparation in object reduce retinal endothelial cell propagation, divide a word with a hyphen at the end of a line and/or
Application in the medicine of segment dislocation.
In yet another aspect, the invention provides VEGFR-FGFR fusion protein and/or FGFR
Fusion protein, it is for treating the ocular disease relevant to angiogenesis.
In yet another aspect, the invention provides VEGFR-FGFR fusion protein and/or FGFR
Fusion protein, it is for improving short-term retina injury in disease subject.In some embodiments
In, described short-term retina injury is selected from reducing the quantity of apoptotic cell, reduction in retinal blood pipe network
The seepage of blood-retina barrier, suppression retinal gliocytes reactive hyperplasia and improvement nerve
Retina and the ultrastructure of retinal vessel.
In yet another aspect, the invention provides VEGFR-FGFR fusion protein and/or FGFR
Fusion protein, it is for improving long-term retina injury in disease subject.In some embodiments
In, described long-term retina injury is selected from improving retinal barrier seepage and suppression Capillary Basement
Thickening of film.
In yet another aspect, the invention provides VEGFR-FGFR fusion protein and/or FGFR
Fusion protein, its for improve in retinopathy of prematurity object retina without blood vessel perfusion area or
Reduce retinal neovascularization cell check figure.
In yet another aspect, the invention provides VEGFR-FGFR fusion protein and/or FGFR
Fusion protein, it is for reducing retinal endothelial cell propagation, dividing a word with a hyphen at the end of a line and/or manage in object
Chamber is formed.
In yet another aspect, the invention provides the eye disease that treatment is relevant to angiogenesis in object
Sick method, it include to described subject according to the present invention's
VEGFR-FGFR fusion protein and/or FGFR fusion protein.
In yet another aspect, the invention provides in disease subject, improve short-term retina injury
Method, it includes the VEGFR-FGFR according to the present invention to described subject
Fusion protein and/or FGFR fusion protein.In some embodiments, described short-term retina damages
Wound selected from reduce the quantity of apoptotic cell in retinal blood pipe network, reduce blood-retina barrier seepage,
Suppression retinal gliocytes reactive hyperplasia and improve the super of neural retina and retinal vessel
Micro structure.
In yet another aspect, the invention provides in disease subject, improve long-term retina injury
Method, it includes the VEGFR-FGFR according to the present invention to described subject
Fusion protein and/or FGFR fusion protein.In some embodiments, described long-term retina damages
Wound is selected from improving retinal barrier seepage and suppressing thickening of capillary basement membrane.
In yet another aspect, the invention provides improve in retinopathy of prematurity object retina without
Vascular perfusion district or the method reducing retinal neovascularization cell check figure, it includes to described object
The VEGFR-FGFR fusion protein according to the present invention of administering therapeutic effective dose and/or FGFR melt
Hop protein.
In yet another aspect, the invention provides in object, reduce retinal endothelial cell increasing
Growing, divide a word with a hyphen at the end of a line and/or the method for segment dislocation, it includes the root to described subject
VEGFR-FGFR fusion protein and/or FGFR fusion protein according to the present invention.
It is an object of the present invention to provide VEGFR and FGFR dual-target fusion protein in system
The application of the medicine in the ocular disease that standby treatment is relevant with new vessels.Especially, with new vessels
Relevant ocular disease includes but not limited to: age-related macular degeneration such as atrophic type AMD and
Exudative AMD, diabetic retinopathy such as non-proliferative type DR, proliferous type DR and DME,
Diabetic macular edema, retinopathy of prematurity, retinal vessel occlusion etc..
VEGFR and the FGFR dual-target fusion protein of the present invention can have following structure: bag
Containing at least one derived from the part of VEGFR extracellular region and at least one derived from FGFR born of the same parents outside
The part in district, the wherein said part derived from VEGFR extracellular region comprises: the of VEGFR1
Two Ig spline structure territories and the 3rd spline structure territory of VEGFR2, described derived from FGFR extracellular region
Part consist of: be derived from intermediate functional sequence area, FGFR Ig spline structure territory part,
FGFR the 2nd Ig spline structure territory and FGFR the 3rd Ig spline structure territory, wherein said FGFR Ig
Intermediate functional sequence area, spline structure territory is an Ig spline structure territory and the 2nd Ig sample in FGFR albumen
Sequence between domain, and described in be derived from intermediate functional sequence area, FGFR Ig spline structure territory
The starting point in SEQ ID NO:1 that corresponds partly to be to end selected from the aminoacid of the 119th to 151
Point is the aminoacid sequence of the 162nd amino acids, is directly connected between said structure territory and/or part
And/or connected by joint.
Preferably, the dual-target fusion protein of the present invention has the amino as described in SEQ ID NO:5
Acid sequence.
In one embodiment, at the short-term retina injury model of STZ induced diabetic rats
In, VEGFR and the FGFR dual-target fusion protein of the present invention can substantially reduce diabetes and regard
The quantity of apoptotic cell in retinal vasculature net, significantly reduces oozing of diabetes blood-retina barrier in early days
Leakage, hence it is evident that suppression retinal gliocytes reactive hyperplasia.Improve neural retina and retinal vessel
Ultrastructure.
In another embodiment, at the long-term retina injury mould of STZ induced diabetic rats
In type, VEGFR and the FGFR dual-target fusion protein of the present invention can improve diabetes rat
Retinal barrier Seepage, thickening of suppression DR capillary basement membrane.
In another embodiment, VEGFR and the FGFR dual-target fusion protein energy of the present invention
Enough be effectively improved hyperoxia induction OIR Mouse Retina without blood vessel perfusion area, significantly reduce retina
New vessels cell check figure.
In further embodiment, VEGFR and the FGFR dual-target fusion protein of the present invention
Significantly reduce the retinal endothelial cell propagation of high sugar and VEGF+FGF induction, divide a word with a hyphen at the end of a line and manage
Chamber is formed.
In yet, in Rhesus Macacus choroidal neovascularization model, the present invention's
VEGFR and FGFR dual-target fusion protein can improve fluorescence leakage phenomenon.
Accompanying drawing illustrates:
Fig. 1 .VF28 apoptotic inhibitory action to trypsinization retinal vessels in rats net
*p<0.05,**p<0.01,***p<0.001。
Fig. 2. ivens blue laws detection blood-retina barrier seepage: before injection azovan blue dyestuff, rat
Form is normal (A).After injection dyestuff, the eye of rat, auricle, limb end, and tail
All present blueness (B).She in blood (C) and retina (D) is detected with standard curve method detection
Train of thought indigo plant content, then respectively organizes the azovan blue content in retina according to computing formula quantitative analysis
(E)。*p<0.05,***p<0.001。
Fig. 3 .GFAP dyeing each group of neural retina Glial Activation of detection.
Fig. 4. Electronic Speculum detection neural retina and the early changes of retinal vessel: Electronic Speculum detects and contrasts respectively
Process group choroidal artery, photoreceptor cell，photosensory cell acromere, outer nuclear layer, and the ultrastructure of retinal vessel.
Fig. 5 VF28 is to diabetic retinal tissue in rat VEGF (Fig. 5 A) and FGF (Fig. 5 B) albumen water
Flat impact, * p < 0.05, * * p < 0.01.
The impact (* p < 0.05) of Fig. 6 VF28 gene mRNA expression each on diabetic retinal tissue in rat.
A:ICAM-1mRNA；B:TNF-α mRNA.
Fig. 7 .FITC-dextran retrobulbar injection shows the labelling to each group of retinal vessels in rats net.
Fig. 8. the PAS coloration result of trypsinization retinal capillary net: normal retina blood capillary
Net has no or seldom sees acellular capillary structure.Under the diabetic retina vasoganglion visual field visible many
Individual acellular capillary structure (shown in black arrow).
Fig. 9. ivens blue laws detection blood-retina barrier seepage: (A) and (B) respectively detects blood
With the standard curve of azovan blue content in retina；(C) it is ivens in each group of rat retina
The quantitative analysis of blue content.*p<0.05,**p<0.01.
Figure 10. transmission electron microscope detection retinal capillary base film thickness: (A) is each group of rat view
The representative picture of film blood capillary transmission electron microscope, wherein capillary basement membrane is with white arrow mark
Go out.(B) it is the quantitative analysis of each group of capillary basement membrane thickness；*p<0.05,**p<0.01,
***p<0.001。
Figure 11. the change of Western blotting (Western Blot) detection VEGF Yu FGF protein level:
(A) (B) represents each group of VEGF and FGF protein quantification respectively.*p<0.05,**p<
0.01,***p<0.001
Figure 12. the change of Western blotting detection FLK protein level: bar diagram represents FLK protein quantification
Analyze.*p<0.05,**p<0.01,***p<0.001
Figure 13. fluorescence fundus angiography shows each group of Mouse Retina vascular condition: each group mice is after ball
After injection high molecular FITC-dextran, the representative picture of inner nuclear layer retina is as shown in (A).
The occupied area proportional quantities fractional analysis of retinal nonperfusion district is as shown in (B).***p<0.001.
Figure 14 HE dyeing each group of Mouse Retina new vessels cell check figure of detection: (A) is each group of view
The representative picture of film paraffin section HE dyeing.(B) it is that averagely every retina of each group of mice is cut
The quantitative analysis of sheet new vessels cell check figure.***p<0.001.
Figure 15. Western blotting result detection PEDF, the change of vegf protein level.
Figure 16. high sugar stimulates the number change of lower retinal endothelial cell.24h (A) after high sugar stimulation,
48h (B), 72h (C), the number change of each process group RF/6A cell is as shown in the figure.*p<0.05,
**p<0.01,***p<0.001。
Figure 17. under high sugar stimulates, the migration assays of retinal endothelial cell.(A) it is each process group
Representative picture after cell migration, after violet staining.(B) it is determining of each group of transitional cell
Component analysis.***p<0.001.
Figure 18. under high sugar stimulates, the retinal blood endothelial tube after scratch test 6h (A) and 12h (B)
The quantitative analysis of cell.
Figure 19. under high sugar stimulates, RF/6A cell segment dislocation is tested.(A) it is the representative of each process group
Property picture.(B) it is the quantitative analysis of each composition tube chamber.**p<0.01,***p<0.001.
Figure 20 .VF28 stimulates the impact of lower RF/6A cell proliferation, retinal vessel to VEGF+FGF
The number change of endotheliocyte.*p<0.05,***p<0.001.
Under Figure 21 .VEGF and the FGF factor stimulate, dividing a word with a hyphen at the end of a line of retinal endothelial cell.(A) it is
After violet staining, each representative picture organizing cell.(B) it is determining of each group of transitional cell quantity
Component analysis result.*p<0.05,***p<0.001.
Figure 22. under VEGF and the FGF factor stimulates, the segment dislocation of RF/6A cell.(A) it is
The representative picture of each process group cell.(B) it is the quantitative analysis of each composition tube chamber quantity.***p
<0.001。
Figure 23. under high sugar stimulates, VEGF and FGF gene and albumen table in retinal endothelial cell
The dynamic change reached.Under the stimulation of high sugar (33mM) culture medium, VEGF in RF/6A cell
As shown in (A) in transcriptional level trend over time with FGF gene.VEGF and FGF
As shown in (B) and (C) respectively in the expression of protein level.
Figure 24. in Rhesus Macacus laser-induced choroidal neovascularization model of hyperplasia, each administration group is to eye fluorescence leakage
Improvement situation.
Detailed description of the invention:
Definition:
Unless otherwise defined, all scientific and technical terminologies used herein have those of ordinary skill in the art institute
The identical meanings understood.About definition and the term of this area, professional specifically refers to Current
Protocols in Molecular Biology(Ausubel).The abbreviation of amino acid residue is this area
The standard 3 referring to one of 20 conventional l-amino acids letter used by and/or 1 letter code.
Although the digital scope shown in the broad scope of the present invention and parameter approximation, but it is embodied as
Numerical value shown in example is recorded the most accurately.But, any numerical value the most necessarily contains
Certain error, it is present in the measurement by each of which caused by standard deviation.It addition, herein
Disclosed all scopes are interpreted as containing any and all subrange wherein comprised.Such as record
The scope of " 1 to 10 " is considered as comprising appointing of (comprising end points) between minima 1 and maximum 10
What and all subranges；It is to say, it is all with minima 1 or subrange more initial, such as
1 to 6.1, and with maximum 10 or the subrange of less termination, such as 5.5 to 10.It addition,
Any list of references being referred to as " being expressly incorporated herein " is interpreted as being integrally incorporated with it.
It is further noted that as used in this description, singulative includes answering of its referent
Number form formula, unless clear and clear and definite is limited to a referent.Term "or" can with term " and/
Or " exchange use, the most clearly indicate.
Terms used herein " VEGFR-FGFR fusion protein " can with " VEGFR and
The fusion protein of FGFR " exchange use, represent vascular endothelial growth factor receptor or its fragment and become
The fusion protein that bfgf receptor or its segment composition obtain.In some particular implementation sides
In case, the VEGFR-FGFR fusion protein of the present invention is VEGFR-FGFR-Fc fusion protein,
The i.e. fusion protein of VEGFR, FGFR and Fc.In some embodiments, the present invention
VEGFR-FGFR fusion protein is to separate.Preferably, the VEGFR-FGFR of the present invention melts
Hop protein is soluble fusion protein.Preferably, in the VEGFR-FGFR fusion protein of the present invention
Each domain and/or part are joined directly together or are connected by joint.
Terms used herein " FGFR fusion protein " be expressed as bfgf receptor or
Its fragment merges, with other protein fragments, the fusion protein obtained.In some particular, this
The FGFR fusion protein of invention is the fusion egg of FGFR-Fc fusion protein, i.e. FGFR and Fc
In vain.In some embodiments, the FGFR fusion protein of the present invention is to separate.Preferably,
The FGFR fusion protein of the present invention is soluble fusion protein.Preferably, the FGFR of the present invention
In fusion protein, each domain and/or part are joined directly together or are connected by joint.It is highly preferred that
The FGFR fusion protein of the present invention is FGF trap.
Terms used herein " Fc ", " Fc district ", " Fc fragment " or " immunoglobulin Fc
District " etc. represent immunoglobulin FC, in the present invention, the described preferred human IgG1 in Fc district
Fc district.
Term " Fc fusion protein " refers to the binding specificity of heterologous protein permanent with immunoglobulin
The antibody sample molecule that the effector function of constant domain combines.From a structural point, Fc merges egg
Comprise aminoacid sequence and the immunoglobulin constant domains sequence with required binding specificity in vain
Row.Fc fusion protein molecule generally comprises the binding site of receptor or part.Described immunoglobulin
Constant domain sequence can be derived from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or
IgG-4 hypotype, IgA (includes IgA-1 and IgA-2), IgE, IgD or IgM.
Terms used herein " solubility " albumen refer to biology relevant temperature, pH level
With the albumen of water soluble solution under osmotic pressure." soluble fusion protein " used herein represents should
Fusion protein does not comprise cross-film district and intracellular region.
As used herein, term " separation " refers to following material and/or entity, (1) and a primiparity
(in natural surroundings and/or in test is arranged) and its at least some component phase being associated time raw
Separate and/or (2) are by manually producing, prepare and/or manufacturing.Separate material and/or entity can with extremely
Few about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about
80%, at the beginning of its of about 90%, about 95%, about 98%, about 99%, basic 100% or 100%
Other components beginning to be associated are separated.
Term " part " and " fragment " is interchangeable refers to polypeptide, nucleic acid or the one of other molecule construction thing
Part.
Terms used herein " VEGFR " refers to vascular endothelial growth factor receptor, and it can be
VEGFR1, VEGFR2 and/or VEGFR3.Preferably, the VEGFR in the present invention is
The VEGFR of VEGFR1 and/or VEGFR2, preferably people.
Terms used herein " FGFR " refers to fibroblast growth factor acceptor, and it can be
FGFR1, FGFR2, FGFR3 and/or FGFR4.Preferably, the FGFR in the present invention is
FGFR1, more preferably people FGFR1.
Terms used herein " Ig spline structure territory " represents immunoglobulin like domain, and it is visible
In multiple proteins family, participate in various biological function, including cell-ECM identification, cell table
Face receptor, immunologic function etc..
Terms used herein " VEGFR the oneth Ig spline structure territory " represents in VEGFR albumen
First Ig spline structure territory from N end, preferably VEGFR1 albumen first Ig sample from N end
Domain (herein referred as VEGFR1 the oneth Ig spline structure territory) or VEGFR2 albumen are from N end
Play first Ig spline structure territory (herein referred as VEGFR2 the oneth Ig spline structure territory), there is example
Aminoacid such as SEQ ID NO:2 the 32nd to 123 or SEQ ID NO:3 the 46th to 110
Sequence.Similarly, VEGFR the 2nd Ig spline structure territory has such as SEQ ID NO:2 the 151st
To the aminoacid sequence of 214 or SEQ ID NO:3 the 141st to 207, VEGFR the 3rd
Ig spline structure territory has the 230th to 327 or SEQ ID NO:3 of such as SEQ ID NO:2
The aminoacid sequence of 224 to 320, VEGFR the 4th Ig spline structure territory has such as SEQ ID
The aminoacid sequence of the 335th to 421 or SEQ ID NO:3 of NO:2 the 328th to 414,
VEGFR the 5th Ig spline structure territory such as has SEQ ID NO:2 the 428th to 553 or SEQ
The aminoacid sequence that ID NO:3 is the 421st to 548, VEGFR the 6th Ig spline structure territory has
The 556th to 654 or SEQ ID NO:3's of such as SEQ ID NO:2 the 551st to 660
Aminoacid sequence, VEGFR the 7th Ig spline structure territory have such as SEQ ID NO:2 the 661st to
The aminoacid sequence of 747 or SEQ ID NO:3 the 667th to 753.Preferably, described
VEGFR can be VEGFR1 or VEGFR2.
Terms used herein " FGFR the oneth Ig spline structure territory " or " FGFR1 the oneth Ig sample
Domain " represent in FGFR or FGFR1 albumen first Ig spline structure territory from N end, its
There is the aminoacid sequence of such as SEQ ID NO:1 the 40th to 118.Similarly, term " FGFR
2nd Ig spline structure territory " or " the 2nd Ig spline structure territory " represent in FGFR albumen from N end
Second Ig spline structure territory, it has the aminoacid of such as SEQ ID NO:1 the 163rd to 247
Sequence；Term " FGFR the 3rd Ig spline structure territory " or " the 3rd Ig spline structure territory " represent FGFR
In albumen from N end first Ig spline structure territory, it has such as SEQ ID NO:1 the 270th
To the aminoacid sequence of 359.Preferably, described FGFR is FGFR1, FGFR the oneth Ig
Spline structure territory is FGFR1 the oneth Ig spline structure territory, and FGFR the 2nd Ig spline structure territory is FGFR1
2nd Ig spline structure territory, FGFR the 3rd Ig spline structure territory is FGFR1 the 3rd Ig spline structure territory.
Terms used herein " intermediate functional sequence area, FGFR Ig spline structure territory " or " middle
Functional sequence district " represent in FGFR albumen an Ig spline structure territory and the 2nd Ig spline structure territory it
Between sequence, it is preferable that IFS sequence has corresponding to SEQ ID NO:1 the 118th to 162
Aminoacid sequence.Inventors have surprisingly discovered that, described intermediate functional sequence area for
The function in Ig spline structure territory has significant impact.Described FGFR albumen preferred FGFR1 (SEQ
ID NO:1), particularly people FGFR1 albumen.The aminoacid sequence of people's FGFR1 albumen sees
SEQ ID NO:1。
The partial sequence of hFGFR1 is given below, and dash area represents each Ig spline structure territory successively,
Can be found in http://www.ncbi.nlm.nih.gov/protein/AAH15035.1
MWSWKCLLFWAVLVTATLCTARPSPTLPEQAQPWGAPVE SDALPSSEDDDDDDDS
SSEEKETDNTKPNPVAPYWTSPEKMEKK RSPHRPILQAGLPANKTVALGS LE
ER
The aminoacid sequence of FGFR1 sees SEQ ID NO:1.
The partial sequence of hVEGFR1 is given below, and dash area represents each Ig spline structure successively
Territory, is natural catenation sequence between each domain, can be found in
Http:// www.uniprot.org/uniprot/P17948:
SKLKD IYIFISDTGRPFVEMYSEIPEIIHMTE LYKTNYLTHRQTNTI IYDKAFI LIVNVK DV IRDQEA VQGTSDKSNLE
The aminoacid sequence of VEGFR1 sees SEQ ID NO:2.
The partial sequence of hVEGFR2 is given below, and dash area represents each Ig spline structure successively
Territory, is natural catenation sequence between each domain, can be found in
http://www.uniprot.org/uniprot/P35968
ASVGLPSVSLDLPRLSIQKDILTIKA ASVIYVYVQDYRSPFIASVSDQHGVVYITE SYQSIMYIVVVVGYRI FVRVHEK LVVYVP RG VLE
RVA IEGA
QEKTNLE
The aminoacid sequence of VEGFR2 sees SEQ ID NO:3.
Term as used herein " object " refers to mammal, such as the mankind but it also may be that other moves
Thing, as dynamic in domestic animal (such as Canis familiaris L., cat etc.), domestic animal (such as cattle, sheep, pig, horse etc.) or experiment
Thing (such as monkey, rat, mice, rabbit, Cavia porcellus etc.).
Terms used herein " joint ", " peptide linker ", " catenation sequence " or " joint sequence "
Represent the short aminoacid that each domain comprised by fusion protein of the present invention and/or region are connected
Sequence, it is generally 0-20 amino acid long, preferably 2-10 aminoacid.
Terms used herein fusion protein or part or domain " corresponding with SEQ ID NO:N
Aminoacid sequence " represent, described fusion protein or part or domain have substantially such as SEQ ID
Aminoacid sequence shown in NO:N, wherein contains less than 1,2,3,4,5,10
Or 20 aminoacid replace, add or lack, the most described fusion protein or partly or structure
Territory have with aminoacid sequence at least 80% shown in SEQ ID NO:N, 90%, 93%, 95%,
97%, the homogeneity of 98% or 99%, it is highly preferred that described fusion protein or part or domain
There is aminoacid sequence shown in SEQ ID NO:N.
The VEGFR-FGFR-Fc fusion protein of the present invention also can comprise post translational modification.Such
Modification includes but not limited to acetylation, carboxylated, glycosylation, phosphorylation, esterified and be acylated.Knot
Really, modified VEGFR-FGFR-Fc fusion protein can comprise non-amino acid component, the most poly-
Ethylene glycol, lipid, polysaccharide or monosaccharide and phosphoric acid.Such non-amino acid component pair
The effect of VEGFR-FGFR-Fc fusion protein function can be as herein for other
Test as described in VEGFR-FGFR-Fc fusion protein variant.When
When VEGFR-FGFR-Fc fusion protein produces in cell, post translational processing folds for correct
And/or protein function may also be important.Different cell (such as CHO, HeLa,
MDCK, 293, WI38, NIH-3T3 or HEK293) have for these translate after activity
Specific cells machine and peculiar mechanism, and different cells can be selected to guarantee
The correct of VEGFR-FGFR-Fc fusion protein is modified and processing.
Fusion protein as herein described can be produced by any methods known in the art.Such as chemistry closes
Become or produce from expression of nucleic acid.Can be according to perfect normal fluid known in the art or preferred solid phase
The peptide that peptide symthesis method is easily prepared for the present invention (see, e.g. J.M.Stewart and J.
D.Young,Solid Phase Peptide Synthesis,2nd edition,Pierce Chemical
Company, Rockford, Illinois (1984), in M.Bodanzsky and A.Bodanzsky,
The Practice of Peptide Synthesis,Springer Verlag,New York(1984)).Can
To use techniques known in the art to produce described fusion protein to be included in described albumen being positioned at expection
In peptide sequence in cysteine residues between form one or more intramolecular crosslinking and (see example
Such as United States Patent (USP) No.5478925).It addition, protein described herein can be by described albumen
The C end of matter or N end add cysteine or biotin carries out conventional modification.
Terms used herein " therapeutically effective amount " or " effective dose " refer to be enough to show that it is for being executed
Dosage by object benefit.The actual amount used, and speed and the time course used can depend on
The own situation of institute's therapist and the order of severity.The prescription (the such as decision etc. to dosage) for the treatment of is
It is general practitioner and the responsibility of other doctor eventually and relies on it and make a decision, generally consider the disease treated
Disease, the situation of individual patients, site of delivery, application process and for doctor known other
Factor.
Terms used herein " VF28 " represents specific VEGFR-FGFR fusion protein, its
Comprise the 2nd Ig spline structure territory of VEGFR1, the 3rd Ig spline structure territory of VEGFR2, be derived from
The part of intermediate functional sequence area, FGFR Ig spline structure territory, FGFR the 2nd Ig spline structure territory,
FGFR the 3rd Ig spline structure territory and Fc fragment.VF28 is that 28#VEGFR-FGFR merges egg
White abbreviation, its building process and other information can be found in CN102219859A.Specifically, VF28
Aminoacid sequence as shown in SEQ ID NO:5.
Terms used herein " FGF trap " refers to FGFR-Fc fusion protein, and it can be used as
The trap of FGF, thus antagonism FGF.Specifically, FGF trap used in the embodiment of the present invention
Being 26#FGFR fusion protein, its building process and other information can be found in CN102219860A.
Specifically, the aminoacid sequence such as SEQ ID NO:6 of FGF trap used in the embodiment of the present invention
Shown in.
Terms used herein " VEGF trap " refers to VEGFR-Fc fusion protein, and it can use
Make the trap of VEGF, thus antagonism VEGF.Specifically, VEGF used in the embodiment of the present invention
Trap is the anti-vegf recombiant protein of Regeneron drugmaker of U.S. exploitation, and it can be from market
On buy.
The VEGFR-FGFR fusion protein of the present invention is open according to Chinese patent application
Method disclosed in CN102219860A and CN102219859A builds, wherein said patent
Application is open to be integrally incorporated herein by quoting.
Embodiment is that the present invention will be further elaborated and explains, and is not construed as this
Bright restriction.
The protective effect to the early stage retina injury of STZ induced diabetic rats of embodiment 1VF28
Method: 8-10 week old SD rat, body weight 200-220g is (purchased from army of the Chinese People's Liberation Army
Thing Academy of Medical Sciences Experimental Animal Center), 2%STZ (streptozotocin, Streptozotocin,；
Amresco Chemical Co., USA) inject induction generation diabetes mould with the dosage of 45mg/kg
Type；Separately set Normal group, inject isopyknic sodium citrate buffer by body weight.Little in 72
Time after monitor blood sugar level, with animal blood glucose higher than 20mmol/L for modeling Success criteria, include in
Diabetes (DM) experimental group；Modeling successful DM rat is randomly divided into model control group, VF28
Dosage (3 μ g/ eye) in low dosage (1 μ g/ eye), VF28, VF28 high dose (9 μ g/ eye),
FGF trap (FGF trap) (3 μ g/ eye) and VEGF trap (VEGF trap) (are purchased from
Regeneron/Bayer) (3 μ g/ eye), often group 15.
VF28 drug level is 10mg/mL, and specification 1mL/ is propped up；VEGF trap concentration is
40mg/mL, specification 1mL/ is propped up；FGF trap concentration is that 5mg/mL, 1mL/ prop up.VF28、
VEGF trap and FGF trap the most all uses physiological saline solution to be diluted to required concentration
Use, place at ice bath after dilution and used in 24 hours.At 2-8 before all medicines are undiluted
DEG C preserve.
After modeling 1 week and 4 weeks, each rats underwent intravitreal of each group is administered, every injection
Volume is 3 μ L.Different pharmaceutical group rat, respective medicine, model are injected in slow glass chamber respectively
Matched group and rats in normal control group give equal-volume normal saline.After diabetes 6 weeks, detect view
Film apoptosis of vascular endothelial cell；The expression of glial cell marker gene GFAP in detection retina；
Detection blood-retina barrier seepage situation；Detection retinal vessel and neural Ultrastructural early stage
Change；Use FGF and the VEGF factor in ELISA method detection retinal tissue homogenate
Protein content (detection kit is purchased from R&D company) and employing RT-PCR method detection
The mrna expression of VEGF, FGF, ICAM-1 and TNF α is to studying VF28 to glycosuria
The molecular mechanism of sick vascular endothelial injury protective effect in early days.
Result: in the course of disease of 6 weeks, DM rat is basic, normal, high through intravitreal through twice
3 dosage VF28 (1,3 and 9 μ g/ eye) or medicine VEGF-trap (3 μ g/ eye) or FGF are fallen into
Trap (3 μ g/ eye), result is as follows:
1) quantity of apoptotic cell during VF28 can substantially reduce diabetic retina vasoganglion, and identical
The VF28 of dosage (3 μ g/ eye) is significantly better than VEGF trap (see figure 1)；
2) high dose VF28 significantly reduces the seepage of diabetes blood-retina barrier in early days (see Fig. 2
Shown in)；
3) each dosage of VF28 all can substantially suppress retinal gliocytes reactive hyperplasia (see Fig. 3
Shown in)；
4) VF28 each dosage group all can improve neural retina and retinal vessel ultrastructure (see
Shown in Fig. 4)；
5) for the vegf protein level raised notable in diabetes in early days retina, VF28 is low,
Middle and high dosage all significantly inhibits effect (as shown in Figure 5)；
6) for the FGF protein level (non-limiting) increased in diabetes in early days retina,
In VF28, low dosage significantly lower FGF protein level, and VF28 high dose and FGF trap
Can be down to close with the FGF level of Normal group (as shown in Figure 5)；
7) ICAM-1 on the expression of mRNA, in diabetes in early days rat retina
Significantly raise with the mRNA of TNF α；VF28 and FGF trap significantly lower ICAM-1 and
TNF α mRNA level in-site (as shown in Figure 6).
The protective effect to the long-term retina injury of STZ induced diabetic rats of embodiment 2VF28
Method: 8-10 week old SD rat, body weight 200-220g is (purchased from army of the Chinese People's Liberation Army
Thing Academy of Medical Sciences Experimental Animal Center) raise in conventional environment, as in embodiment 1, method is carried out
Glycemia Decline；Modeling successful DM rat is randomly divided into model control group, VF28 low dosage (2
μ g/ eye), dosage (6 μ g/ eye), VF28 high dose (18 μ g/ eye), FGF in VF28
Trap (12 μ g/ eye) and VEGF trap (12 μ g/ eye), and set Normal group simultaneously.?
After modeling the 5th week and the 9th week, each organize each rat intravitreal to respective medicine, be administered
Method is with embodiment 1.After diabetes 12 weeks, observe the leakage scenarios of retinal blood pipe network, blood-regard
Retinal barrier seepage situation, detects the presence of the thin blood vessel structure of cell membrane；Retinal capillary basement membrane
The change thickened；And detect FGF, VEGF and FLK factor protein content in retina homogenate
Change.
Result: in the course of disease of 12 weeks, DM rat through low through intravitreal twice, in,
High 3 dosage VF28 (2,6 and 18 μ g/ eyes, difference suitable 13.5,40.6 and 122pmoL/
Eye) or VEGF trap (12 μ g/ eyes, 124pmoL/ eye) or FGF trap (12 μ g/ eyes,
117pmoL/ eye), result is as follows:
1) retrobulbar injection high molecular FITC-dextran labelling diabetic retinal tissue in rat blood only
The retinal blood pipe network of pipe network, Normal group and other each treatment group all can not labelling.Result shows
VF28 can significantly improve blood-retina barrier seepage (shown in Fig. 7).
2) PAS coloration result display VF28 (the 2-18 μ g/ of trypsinization retinal capillary net
Eye) and FGF trap (12 μ g/ eye) all can significantly improve diabetic retina acellular capillary
Structure (as shown in Figure 8).
3) in azovan blue is tested, VF28 and FGF trap all can significantly improve diabetes rat
The phenomenon of retinal barrier seepage, and the effect of the close VF28 of molar dose is significantly better than VEGF
Trap (P < 0.05) (as shown in Figure 9).
4) VF28 and FGF trap all can significantly inhibit thickening of DR capillary basement membrane, and
The effect of the VF28 that molar dose is close is significantly better than VEGF trap and FGF trap (P < 0.05)
(as shown in Figure 10).
5) Western blotting result shows, VEGF and FGF in diabetic groups retina homogenate
Content dramatically increases, and VF28 each dosage group can significantly reduce the expressing quantity of VEGF, VF28
High dose group can significantly reduce the protein content (as shown in Figure 11) of FGF.
6) on the other hand, Western blotting result shows, the master of mediation VEGF signal transduction pathway
Wanting receptor FLK (having another name called VEGFR2), in diabetic retina, protein level significantly reduces,
And in each intervention group of VF28 in dose-dependently increasing, return to the most right in middle high dose group
Similar according to group level.In VEGF trap group, the protein level of FLK is significantly higher than Normal group
Level, FGF trap group, FLK level is similar to Normal group.(as shown in Figure 12).
7) VF28 that molar dose is close is to long-term diabetic rat retinal capillary basement membrane
The inhibitory action thickened is significantly better than VEGF trap and FGF trap；Even to diabetic groups retina
VEGF and the FGF content dramatically increased in serosity all has significant inhibitory action, and VEGF falls into
Trap and FGF trap all can only significantly inhibit the exception of respective target and raise, to non-target without effect.
These researchs show the biological targeting medicine of the identical molar dose of confined space injection within the eye, act on double
The VF28 of target (VEGF+FGF) is better than single target agent VEGF trap (target VEGF)
With FGF trap (target FGF).
Embodiment 3 observes the VF28 intervention to retinal neovascularization in the OIR mice that hyperoxia is induced
Effect
Method: the C57 used by experiment is female, neonatal rat is purchased from military medicine section of the Chinese People's Liberation Army
Institute's Experimental Animal Center, neonatal rat age in days 3-5d.OIR mice is placed in when being born 7 days (P7)
Raising 5 days in the airtight oxygen cabin of high oxygen treatment, Normal group is raised under normal oxygen.Deliver from godown at P12
After, OIR mice every immediately through intravitreal low (0.5 μ g), high (1 μ g) dosage VF28,
And VEGF trap (0.5 μ g) and FGF trap (0.5 μ g), eyes inject, every injection
Volume is 1 μ L, and according to injection medicine different grouping；Normal group injecting normal saline, institute
Mice is had to raise under normal oxygen condition 5 days after injection again.By retrobulbar injection FITC-dextran
Row fluorescence fundus angiography, observes retinal vessel change, measures the relative of retinal nonperfusion district
Area；The detection nuclear number of new vessels；PEDF and VEGF egg in detection eye homogenate
The change of Bai Hanliang.
Result: OIR mice 2 dosage VF28 (0.5 low through intravitreal through single, high
With 1 μ g/ eye) or medicine VEGF trap (0.5 μ g/ eye) or FGF trap (0.5 μ g/ eye),
Result is as follows:
1) fluorescence fundus angiography display OIR Mouse Retina occurs without perfusion area and a large amount of arrangement
Disorderly new vessels, and the retinal vein of tortuous expansion；Each pharmaceutical intervention group without perfusion area
Relative area be substantially less than OIR mice, VF28 to retina without the improvement result of blood vessel perfusion area
It is significantly better than FGF trap group (as shown in Figure 13).
2) after detecting each group of Mouse Retina new vessels cell check figure result of the test display high oxygen treatment,
Mouse Retina new vessels check figure more normal oxygen group dramatically increases.VF28, VEGF trap and FGF
Trap all can significantly reduce OIR Mouse Retina new vessels check figure, with the VF28 group of dosage
It is significantly better than VEGF trap group and FGF trap group (as shown in Figure 14).
3) Western blotting result shows, PEDF and VEGF in OIR little rathole homogenate in
Contrary change, shows as that VEGF level significantly raises and PEDF significantly reduces；VF28
(0.5-1 μ g/ eye) can significantly reduce VEGF level and make PEDF dramatically increase reactively
(P < 0.001vs model group)；FGF trap (0.5 μ g/ eye) can dramatically increase PEDF level
(P < 0.05vs model group) but the rise on VEGF does not affect (as shown in Figure 15).
Therefore, VF28, in OIR mouse model, can be effectively improved retina without blood vessel perfusion area,
Significantly reduce retinal neovascularization cell check figure.
Embodiment 4VF28 is external stimulates lower retinal endothelial cell to high sugar and VEGF+FGF
Pharmacodynamic study and molecular mechanism thereof
Method: utilize in vitro under the retinal endothelial cell VF28 effect that has been scale-model investigation
Cell behavior changes.Monkey retinal endothelial cell RF/6A (is purchased from Chinese Academy of Sciences typical case
Culture collection committee cell bank) it is placed in 33mmol/L high glucose medium, respectively with in real time
PCR and ELISA detection 12,24,36, VEGF and FGF gene and the table of albumen after 48h
Reach situation；Stimulate 24 at high sugar, 48, after 72h, VEGF (80ng/mL)+FGF (70ng/mL)
After the factor stimulates 48h hour, the VF28 (0.025,0.08 and 0.24 μ g/ μ L) of detection various dose
Impact on RF/6A cell proliferation；After filtering out optimal VF28 dosage from cell proliferation test,
It is added separately in culture medium make final concentration be by VF28, VEGF trap, FGF trap medicine
0.08 μ g/ μ L, is respectively adopted Transwell, Matrigel and scratch experiment detects these medicines to height
Dividing a word with a hyphen at the end of a line and the effect of segment dislocation of RF/6A cell under sugar and the stimulation of the VEGF+FGF factor.
Result: (see Figure 16-23)
1), in testing in vitro, VF28 and FGF trap all can significantly reduce high sugar and VEGF+FGF
The retinal endothelial cell of induction is bred, is divided a word with a hyphen at the end of a line and segment dislocation (see Figure 16-23).
2) completely unexpectedly, the effect of FGF trap suppression vascular endothelial cell proliferation is better than respectively
Plant VF28 and the VEGF trap of dosage.(see Figure 16,20)
3) under under the VEGF+FGF factor stimulates, Transwell migration assays and high sugar stimulate
In scratch test, FGF trap and VF28 can significantly inhibit the shifting of retinal endothelial cell
OK, both is significantly stronger than VEGF trap.(see Figure 18,21)
4) mRNA of VEGF and FGF gene in retinal endothelial cell under high sugar stimulates
All raise with protein level. (see Figure 23).
5) VF28 is allowed to retinal endothelial cell by acting on VEGF and FGF target
Segment dislocation inhibitory action is close with VEGF trap, to the propagation of retinal endothelial cell with divide a word with a hyphen at the end of a line
Inhibitory action is close with FGF trap, and VF28 combines both of VEGF trap and FGF trap
Action advantage.
The fusion protein of embodiment 5 present invention is to laser-induced Rhesus Macacus choroidal neovascularization inhibitory action
Method: laboratory animal Rhesus Monkeys in Anesthesia, mydriasis occiput are fixed on before ophthalmology laser light coagulates instrument,
Macular area is coagulated through full skiascope light.Light is solidifying around central fovea of macula, and every irradiates 6～8 points.
Laser parameter: spot diameter 50 μm, energy 0.6～0.7W, time of exposure 0.05s.Laser photocoagulation
Within latter 2～3 weeks, do 1 fluoresecein angiography and optical coherence tomography (OCT) checks, as
Modeling success or not basis for estimation.
VF28 gives 0.5 through eyes vitreous body single injection, 0.25,0.05mg/ eye, give simultaneously
FGF trap, VEGF trap, 0.5mg/ eye, model control group gives infusion liquid BSS Plus (Alcon
Company), intravitreal injection, single-dose.Before light is solidifying, light solidifying after the same day, be administered before,
Fluorescein angiography (FFA) inspection in 7,14 days after administration.Fluorescent spot is carried out 1-4 level classification
Mark and calculate Fluorescein Leakage area improvement rate.
Experimental result: make by the improvement of fluorescent spot area and fluorescence leakage area being evaluated medicine
By effect, result as shown in figure 24: compared with model group (not being administered group), the fusion of the present invention
Albumen (VF28 and FGF trap) significantly improves fluorescence leakage, though and model group is administered 14 days
Have no progress but after 28 days fluorescence leakage substantially increase.
VF28 drug effect presents certain dosage correlation, i.e. 0.5mg/ eye is better than 0.25mg/ eye and is better than
0.05mg/ eye.
List of references
[1]Ferrara N,Gerber HP,LeCouter J.The biology of VEGF and its
receptors.Nat Med.2003,9:669-76.
[2]Ferrara N.Vascular endothelial growth factor as a target for
anticancer therapy.Oncologist.2004,1:2-10.
[3]Rodrigues EB,Farah ME,Maia M,Penha FM,Regatieri C,Melo
GB et al.Therapeutic monoclonal antibodies in ophthalmology.Prog
Retin Eye Res 2009；28(2):117-44.
[4]Li X,Xu G,Wang Y,Xu X,Liu X,Tang S et al.Safety and Efficacy
of Conbercept in Neovascular Age-Related Macular Degeneration:Results
from a 12-Month Randomized Phase 2 Study:AURORA Study.
Ophthalmology 2014.
[5]Eswarakumar,Lax I,Schlessinger J.Cellular signaling by fibroblast
growth factor receptors.Cytokine Growth Factor Rev.2005,16:139-149.
Claims (12)
1.VEGFR-FGFR fusion protein is used for, in preparation, the eye that treatment is relevant to angiogenesis
Application in the medicine of disease, described fusion protein comprises: the 2nd Ig spline structure territory of VEGFR1,
The 3rd Ig spline structure territory of VEGFR2, it is derived from intermediate functional sequence area, FGFR Ig spline structure territory
Part, FGFR the 2nd Ig spline structure territory and FGFR the 3rd Ig spline structure territory.
Application the most according to claim 1, wherein said fusion protein also comprises immunoglobulin Fc
District, preferably human IgG1 Fc district, the most described human IgG1 Fc district has and SEQ ID NO:4 phase
Corresponding aminoacid sequence.
Application the most according to claim 1, wherein:
Described VEGFR1 the 2nd Ig spline structure territory has: with SEQ ID NO:2 the 151st to 214
The aminoacid sequence that position is corresponding,
Described VEGFR2 the 3rd Ig spline structure territory has: with SEQ ID NO:3 the 224th to 320
The aminoacid sequence that position is corresponding,
Described FGFR1 the 2nd Ig spline structure territory has: with SEQ ID NO:1 the 163rd to 247
The aminoacid sequence that position is corresponding, and
Described FGFR1 the 3rd Ig spline structure territory has: with SEQ ID NO:1 the 270th to 359
The aminoacid sequence that position is corresponding.
Application the most according to claim 1, wherein said VEGFR-FGFR fusion protein has
The aminoacid sequence of SEQ ID NO:5.
Application the most according to claim 1, the wherein said ocular disease choosing relevant to angiogenesis
From: age-related macular degeneration such as atrophic type AMD and Exudative AMD, diabetes view
Film pathological changes such as non-proliferative type DR, proliferous type DR and DME, diabetic macular edema, early
Newborn baby's retinopathy and retinal vessel occlusion.
6.VEGFR-FGFR fusion protein regards for improving short-term in disease subject in preparation
Application in the medicine of nethike embrane damage, described fusion protein comprises: the 2nd Ig sample knot of VEGFR1
Structure territory, the 3rd Ig spline structure territory of VEGFR2, it is derived from FGFR Ig spline structure territory intermediate functional
The part of sequence area, FGFR the 2nd Ig spline structure territory and FGFR the 3rd Ig spline structure territory.
Application the most according to claim 6, wherein said short-term retina injury is selected from reducing view
The quantity of apoptotic cell, the seepage of reduction blood-retina barrier, suppression retina glue in film vasoganglion
Cell plastid reactive hyperplasia and improve the ultrastructure of neural retina and retinal vessel.
8.VEGFR-FGFR fusion protein regards for improving in disease subject for a long time in preparation
Application in the medicine of nethike embrane damage, described fusion protein comprises: the 2nd Ig sample knot of VEGFR1
Structure territory, the 3rd Ig spline structure territory of VEGFR2, it is derived from FGFR Ig spline structure territory intermediate functional
The part of sequence area, FGFR the 2nd Ig spline structure territory and FGFR the 3rd Ig spline structure territory.
Application the most according to claim 8, wherein said long-term retina injury is selected from improving view
Envelope barrier seepage and suppression the thickening of capillary basement membrane.
Application the most according to claim 1, wherein said is derived from merit in the middle of FGFR Ig spline structure territory
Can the aminoacid sequence of part of property sequence area be selected from the corresponding to starting point in SEQ ID NO:1
The aminoacid of 119 to 151 is the aminoacid sequence of the 162nd amino acids to terminal, is preferably
SEQ ID NO:1 the 134th to 162,145 to 162 or 151 to 162 shown amino
Acid sequence, the 145th to 162 shown aminoacid sequence of more preferably SEQ ID NO:1.
11.VEGFR-FGFR fusion protein is used for changing in retinopathy of prematurity object in preparation
Kind retina is without answering in the medicine of blood vessel perfusion area or reduction retinal neovascularization cell check figure
With, described fusion protein comprises: the 2nd Ig spline structure territory of VEGFR1, the 3rd of VEGFR2 the
Ig spline structure territory, it is derived from the part of intermediate functional sequence area, FGFR Ig spline structure territory, FGFR
2nd Ig spline structure territory and FGFR the 3rd Ig spline structure territory.
12.VEGFR-FGFR fusion protein is used for reducing in retinal vessel in object in preparation
Epithelial cell proliferation, dividing a word with a hyphen at the end of a line and/or application in the medicine of segment dislocation, described fusion protein comprises:
The 2nd Ig spline structure territory of VEGFR1, the 3rd Ig spline structure territory of VEGFR2, it is derived from FGFR
The part of intermediate functional sequence area, Ig spline structure territory, FGFR the 2nd Ig spline structure territory and FGFR
3rd Ig spline structure territory.
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| CN107099589A (en) * | 2017-04-28 | 2017-08-29 | 赵晨 | LncRNA ZNF503 AS1 prepare the application in the kit of diagnosis retinal degenerative disease |
| WO2021004549A1 (en) * | 2019-07-11 | 2021-01-14 | 滨州医学院 | Use for dual target vascular inhibitor in preparing drugs for preventing or treating fibrosis |
| CN116715749A (en) * | 2023-03-20 | 2023-09-08 | 吉林大学 | VEGF activity inhibition protein with specific binding capacity with collagen and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107099589A (en) * | 2017-04-28 | 2017-08-29 | 赵晨 | LncRNA ZNF503 AS1 prepare the application in the kit of diagnosis retinal degenerative disease |
| CN107099589B (en) * | 2017-04-28 | 2020-03-10 | 赵晨 | Application of LncRNA ZNF503-AS1 in preparation of kit for diagnosing retinal degenerative disease |
| WO2021004549A1 (en) * | 2019-07-11 | 2021-01-14 | 滨州医学院 | Use for dual target vascular inhibitor in preparing drugs for preventing or treating fibrosis |
| CN116715749A (en) * | 2023-03-20 | 2023-09-08 | 吉林大学 | VEGF activity inhibition protein with specific binding capacity with collagen and preparation method and application thereof |
| CN116715749B (en) * | 2023-03-20 | 2024-04-09 | 吉林大学 | VEGF activity inhibition protein with specific binding capacity with collagen and preparation method and application thereof |
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