CN106890313A

CN106890313A - Medicine for treating pathological myopia

Info

Publication number: CN106890313A
Application number: CN201510958213.XA
Authority: CN
Inventors: 张敏
Original assignee: Innovent Biologics Suzhou Co Ltd
Current assignee: Innovent Biologics Suzhou Co Ltd
Priority date: 2015-12-18
Filing date: 2015-12-18
Publication date: 2017-06-27

Abstract

The present invention relates to biomedicine field, the purposes of a kind of Formulas I a or Ib fusion proteins is specifically provided, in formula, A is the antibody or receptor element for VEGF；B is companion's element；C is the antibody or receptor element for complement；"-" represents the peptide bond or peptide linker of connection said elements, wherein described fusion protein is used to prepare a medicine or preparation, the medicine or preparation are used for (a) and treat and/or prevention pathological myopia；B () is used to improve the diopter of pathological myopia.It is of the invention to be verified by animal pathological myopia disease model, it was demonstrated that the fusion protein can significantly treat pathological myopia.A-B-C (Ia), or C-B-A (Ib).

Description

Medicine for treating pathological myopia

Technical field

The present invention relates to biomedicine field, more particularly to the medicine for treating pathological myopia.This hair Bright medicine contains the active component with treatment pathological myopia effect.

Background technology

Pathological myopia (Pathological myopia, PM) refer to diopter more than -6.00-8.00D, Or axis oculi progressive grows beyond the Eye disease of 26.5mm, often with ball wall tissue is thinning and retinopathy Become (paint crackle, atrophia choroideae et retinae etc.), be to cause one of major reason of low visual acuity and blinding.Generation The illness rate of PM is 1%-4% in the range of boundary, and Asian countries's incidence of disease is higher, wherein more than 40 years old people of China PM illness rates are 2%-10% in group, are the second serious diseases of more than 40 years old crowd's inpairment of vision of China or even blinding Cause.

Pathological myopia develops into certain phase, in fact it could happen that retina choroid new vessels (Choroidal Neovascularization, CNV) bleeding, seepage, retinal detachment, Proliferative vetreoretinopathy Become symptoms such as (Proliferative vitreoretinopathy, PVR), these symptoms can be aggravated further The lesion process of pathological myopia, causes the serious harms such as irreversibility central light loss, is near pathologic Depending on the main cause of patient's blinding.

Rear portion sclera extends, it is thinning be pathological myopia basis.On the basis of sclera excessively extends, arteries and veins There are a series of pathological changes in network film and retina, finally result in choroid and the extensive atrophy denaturation of retina, There is the bleeding of CNV seepage, retinal detachment and proliferative vitreoretinopathy etc. many Plant illness.

Pathological myopia axis oculi extends, and rear portion sclera is constantly extended backward, the thinning formation Portugal of Posterior pole sclera Grape are swollen, so as to cause retinal vessel, ciliary vascular system blood supply insufficiency, induce Posterior pole chronic ischemia and Dysbolism, causes CC that atrophy occurs and hardens, and forms the lacquer cracks (Bruch of concealment Film rupture or defect), subsequent CC endothelial cell, pericyte, fibrocyte and inflammation are thin Born of the same parents etc. can enter subretinal space by these tissue spaces, in layer of retina,pigment epithelium and retina god Through forming neovascular membranes between sensory layer, and then there is CNV seepages and bleeding.Wherein inflammatory factor is also joined With the shaping and development of CNV, the condition such as Posterior pole retinal tissue hypoxic-ischemic, Brunch film ruptures or defect Pigment epithelial cell can be induced to discharge interleukin (such as IL-6, IL-8) and other cell factors (including grow The factor, chemotactic factor (CF) etc.), these cell factors have to inflammatory cells such as macrophage, neutrophil leucocytes Strong chemotaxis, tumor necrosis factor-alpha and IL-1 of macrophage generation etc. then promote iuntercellular to stick The expression of attached molecule -1 and the infiltration of other inflammatory cells.

The extension of pathological myopia axis oculi, vitreum are shunk the coupled layer of retina,limiting,internal of traction and are prolonged together Stretch, extend retina thinning；When retina extension cannot again adapt to the sclera of extension to a certain extent, together When retinal vessel elongation can not adapt to the retina and pore membrane of expansion, will cause between layer of retina point From causing detachment of retina.Axis oculi extension causes rear portion sclera to form staphylomatous position can also be occurred regarding Nethike embrane internal layer is damaged thinning phenomenon, and liquefied vitreum is entered by small internal layer and is accumulated in layer of retina Between, cause retina interlaminar separation, while very thin netted impaired internal layer has to liquefied vitreum necessarily oozing Permeability, can cause to separate and aggravate.Additionally, retina, the choroid of atrophy denaturation also to weaken retina each Contact between layer, causes retina interlaminar separation.During detachment of retina pathological development, on pigment Chrotoplast flow of calcium ions feature substantially, when free intracellular calcium excessive concentration, can be activated various Enzyme, secretes cytokine profiles, exhausts ATP, produces free radical, causes cell Multiple components to destroy, and lures Hair retinal cell apoptosis.Retina departs from do not reset for a long time, vitreous chamber and retina surfaces externally and internally pigment There is migration, conversion, propagation and shrink in epithelial cell and Deiter's cells, will result in PVR.Grind Study carefully and show, it is any to cause ocular inflammatory response or immune response, and discharge into retinal pigment epithelium The pathological factor of vitreous chamber can all promote PVR to develop, and have proven to detect each para-immunity in PVR Globulin, complement component (such as C3), cytokine profiles (such as growth factor, interleukin, cell adhesion Molecule etc.) and T lymphocytes and macrophage etc..Cell factor is by endocrine, autocrine, side point The mode secreted acts on pigment epithelial cell and retinal glial cells, pigment epithelial cell is broken up, The structure of aggregation, chemotactic, transfer and extracellular matrix all plays an important role, and detachment of retina/ischemic Also stimulating cytokine is bred for the generation of anoxic；Vicious circle is so formed between retinal detachment and PVR, The development of PVR is further aggravated, causes more serious inpairment of vision.

However, so far, although this area develops the medicine of some treatment pathological myopias, but by The factor being related in pathological myopia is numerous, and process is complicated, therefore still lacks gratifying effectively curative Thing.Therefore, this area can be used in effectively treating the medicine of pathological myopia in the urgent need to exploitation.

The content of the invention

It is an object of the invention to provide it is a kind of for effectively treatment pathological myopia medicine and its preparation method and Purposes.

In the first aspect of the present invention, there is provided a kind of purposes of fusion protein, the fusion protein has formula Ia or structure described in Formulas I b：

A-B-C (Ia), or

C-B-A (Ib)

Wherein,

A is the antibody or receptor element for VEGF；

B is companion's element；

C is the antibody or receptor element for complement；

Each "-" represents the peptide bond or peptide linker of connection said elements,

Also, described fusion protein is used to prepare a medicine or preparation, and the medicine or preparation are used for (a) Treatment and/or prevention pathological myopia；B () is used to improve the diopter of pathological myopia.

In another preference, the element A is the receptor fragments of VEGF.

In another preference, the sequence such as SEQ ID NO. of the element A:438-629 institute in 1 Show.

In another preference, the described acceptor behaviour complement receptor 1 for complement.

In another preference, described element C is the fragment of complement receptor 1.

In another preference, the sequence of described element C is as in shown in 1-204.

In another preference, described companion's element is the Fc areas of antibody.

In another preference, described antibody Fc district is human IgG1, the Fc of IgG2, IgG3 or IgG4 Area

In another preference, the sequence such as SEQ ID NO. of described element C:205-431 in 1 Shown in companion's element.

In another preference, described fusion protein is Formulas I b structures.

In another preference, the "-" between B and A is flexible joint.

In another preference, the length of above-mentioned peptide linker is 1-30 amino acid, preferably 2-15 Amino acid.

In another preference, described flexible joint such as SEQ ID NO.:In 1 shown in 432-437.

In another preference, the sequence such as SEQ ID NO. of the fusion protein:Shown in 1.

In another preference, described medicine or preparation is additionally operable to treat one or more disease being selected from the group Disease：Pathological myopia merges CNV, pathological myopia and merges punctum luteum hemorrhage, pathological myopia Merge retinal detachment, pathological myopia and merge proliferative vitreoretinopathy.

In another preference, described pathological myopia has following characteristics：Diopter -6.00~ More than -8.00D.

In another preference, described pathological myopia has following characteristics：Axis oculi progressive grows beyond 26.5mm。

In another preference, described pathological myopia has the feature being selected from the group：

I () diopter is in more than -6.00~-8.00D；

(ii) axis oculi progressive grows beyond the Eye disease of 26.5mm；

(iii) with ball wall tissue is thinning and eyeground pathological changes.

In another preference, described eyeground pathological changes are selected from the group：Paint crackle, atrophia choroideae et retinae, Or its combination.

In another preference, described element A is the antibody or the acceptor for VEGF of anti-human VEGF.

In another preference, described fusion protein includes monomer or dimer.

In the second aspect of the present invention, there is provided a kind of medicine box for treating pathological myopia, the medicine box Including：

A () first container, and the fusion protein in first container, the fusion protein have Structure described in Formulas I a or Formulas I b as defined above；

(b) optional utensil for intraocular injection administration；With

C () operation instructions, the specification indicates the fusion protein for treating pathological myopia.

In another preference, the specification also indicates the fusion protein to be used for：(i) reverse axis oculi and Vitreous chamber elongation, atrophy, (ii) reduces punctum luteum hemorrhage, and (iii) reduces new vessels area, and/or (iv) Improve diopter.

In another preference, the specification is indicated：Described administration is intravitreal administration.

In another preference, the specification is indicated：The amount of application of fusion protein is 0.001-10mg/ Ball, preferably 0.01-5mg/ eyeballs.

In the third aspect of the present invention, there is provided a kind of method for treating pathological myopia, including step：Give The object intraocular of needs applies a fusion protein, and the fusion protein has Formulas I a or Formulas I b as defined above Described structure.

In another preference, described administration is intravitreal administration.

In another preference, described amount of application is 0.001-10mg/ eyeballs, preferably 0.01-5mg/ Eyeball.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (such as implementation Example) in specifically describe each technical characteristic between can be combined with each other, so as to constitute new or preferred skill Art scheme.As space is limited, no longer tire out one by one herein and state.

Brief description of the drawings

Fig. 1 shows cavy mask method form deprivation myopia model.

Fig. 2 shows that fusion protein XD-DF can be effectively improved refractive status.

Fig. 3 shows that fusion protein XD-DF can effectively extend axiallength.

Fig. 4 shows that fusion protein XD-DF can be effectively improved VCD.

Fig. 5 show fusion protein XD-DF be effectively reduced CNV areas (μm²)。

Fig. 6 shows that fusion protein XD-DF is effectively reduced the AOD values of vegf expression.

Fig. 7 shows that fusion protein XD-DF can be effectively improved proliferative vitreoretinopathy.

Specific embodiment

The present inventor's process extensively and is in depth studied, by grinding for specific pathological myopia animal model Study carefully, first it was unexpectedly observed that specific " antibody-complement " bifunctional fusion proteins can effectively treat disease Rationality myopia, and with good security.The present invention is completed on this basis.

Specifically, the present inventor merges retina choroid new vessels using specific cavy pathological myopia Model and retinal detachment-proliferative vitreoretinopathy model, have detected fusion protein of the present invention To the effect of pathological myopia, as a result show, fusion protein of the present invention has to pathological myopia significantly to be controlled Treatment is acted on.

Term

As used herein, term " albumen of the present invention ", " fusion protein of the present invention ", " present invention activity Composition ", " fusion protein XD-DF " " XD-DF " are used interchangeably, and refer to a kind of bifunctional fusion of restructuring Albumen, the fusion protein include the antibody of human vessel endothelium growth factor resisting acceptor being merged and People's complement receptor 1.

AOD：Average optical density, average optical density value

AMD：Age-related macular degeneration, AMD

CNV：Choroidal neovascularization, CNV

PVR：Proliferative vitreoretinopathy, proliferative vitreoretinopathy

VEGF：Vascular endothelial growth factor, VEGF

Active component

Active component of the invention is a kind of fusion protein, and the fusion protein has described in Formulas I a or Formulas I b Structure：

A-B-C (Ia), or

C-B-A (Ib)

Wherein,

A is the antibody or receptor element for VEGF；

B is companion's element；

C is the antibody or receptor element for complement；

"-" represents the peptide bond or peptide linker of connection said elements.

A kind of representational fusion protein of the present invention is " vascular endothelial growth factor receptor fragment-anti-for structure Body Fc fragments-people's complement receptor 1 fragment " fusion protein.A kind of typical amino acid sequence such as SEQ ID NO.: Shown in 1.

The structure of the fusion protein is as follows：

1-204 is Complement inhibition domain (fragment from complement receptor 1)

205-431 is companion's element (the Fc fragments from antibody)；

432-437 is flexible joint；

438-629 is VEGF inhibitions domain (fragment from VEGFR)

The present inventor is based on pathological myopia and merges CNV and Proliferative vetreoretinopathy The models such as change, have investigated the therapeutic effect of the pathological myopia that XD-DF is induced model, as a result show, this Invention fusion protein withers to axis oculi and vitreous chamber elongation (vitreum atrophy), punctum luteum hemorrhage, retina cell Die and diopter has good improvement.

The preparation of fusion protein

As used herein, " separation " refers to that material is separated (if natural from its primal environment Material, primal environment is natural surroundings).Such as polynucleotides and polypeptide under the native state in active somatic cell Do not isolate and purify, but same polynucleotides or polypeptide same other things for existing such as from native state Separated in matter, then isolated and purified.

As used herein, " fusion protein of separation " to refer to that fusion protein is substantially free of natural associated therewith Other albumen, lipid, carbohydrate or other materials.Those skilled in the art can be pure with the protein of standard Change technology purified fusion protein.Substantially pure albumen can produce single in non-reducing polyacrylamide gel Master tape.

Present invention also offers the polynucleotides for encoding said fusion protein.Polynucleotides can be DNA Form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can be with It is single-stranded or double-strand.DNA can be coding strand or noncoding strand.

The invention further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid sequence with the present invention The protein fragments of row, analogs and derivatives.The variant of this polynucleotides can be it is natural occur etc. The variant that position variant or non-natural occur.These nucleotide variants include that substitution variants, missing become Allosome and insert variation.As known in the art, allelic variant is an alternative forms for polynucleotides, It is probably substitution, missing or the insertion of one or more nucleotides, but will not be encoded from its is substantially changed The function of polypeptide.

As used herein, term " primer " refers to being matched with template, in the presence of archaeal dna polymerase Can be that starting point carries out the synthesis general name sour with the oligonucleotide of the DNA of template complementation with it.Primer can be Natural RNA, DNA, or any type of natural nucleotide.Primer can even is that non-natural Nucleotides such as LNA or ZNA etc..On primer " generally " (or " substantially ") and template on a chain One special sequence is complementary.Primer must could start to extend with template an abundant complementation of chain, but The sequence of primer need not be with the sequence complete complementary of template.Such as, in a 3' end and the complementary primer of template 5' ends add the preceding paragraph and the not complementary sequence of template, such primer is still generally complementary with template.As long as Having sufficiently long primer can sufficiently be combined with template, and non-fully complementary primer can also be formed with template and drawn Thing-template composite, so as to be expanded.

Each element of fusion protein of the present invention is (such as antibody or receptor fragments, FC fragments and complement for VEGF The fragment of acceptor 1) nucleotides full length sequence or its fragment can generally use PCR TRAPs, recombination method or people The method of work synthesis is obtained.For PCR TRAPs, can be according to published relevant nucleotide sequence, especially Being open reading frame sequence designs primer, and with commercially available cDNA storehouses or by well known by persons skilled in the art CDNA storehouses prepared by conventional method obtain relevant sequence as template, amplification.When sequence is more long, usually Needs are carried out twice or multiple PCR is expanded, and the fragment for then again amplifying each time is spliced by proper order and existed Together.

Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This leads to It is often to be cloned into carrier, then is transferred to cell, then by conventional method from the host cell after propagation Isolated relevant sequence.

Additionally, can also synthesize relevant sequence with artificial synthesized method, when especially fragment length is shorter. Generally, by first synthesizing multiple small fragments, being then attached again can obtain sequence fragment very long.

It is optimized for obtaining gene of the invention using the method for round pcr DNA amplification/RNA.For PCR Primer can be properly selected according to the sequence information of invention disclosed herein, and available conventional method is closed Into.Can be with conventional method as separated by gel electrophoresis and purifying the DNA/RNA fragments for expanding.

The present invention also relates to the carrier comprising polynucleotides of the invention, and with carrier of the invention or fusion The host cell that albumen coded sequence is produced through genetic engineering, and produce egg of the present invention through recombinant technique The method of white matter.

By conventional recombinant DNA technology, can be used to express or raw using polynucleotide sequence of the invention Produce recombinant protein.In general there are following steps：

(1) the polynucleotides (or variant) of coding of the invention albumen of the present invention, or with containing the multinuclear The recombinant expression carrier conversion of thuja acid or suitable host cell of transduceing；

(2) host cell that is cultivated in suitable culture medium；

(3) separated from culture medium or cell, protein purification.

Method well-known to those having ordinary skill in the art can be used for build containing albumen of the present invention DNA sequences encoding with The expression vector of suitable transcription/translation control signal.These methods include recombinant DNA technology in vi, DNA Synthetic technology, In vivo recombination technology etc..Described DNA sequence dna can be effectively connected to appropriate in expression vector In promoter, to instruct mRNA to synthesize.Ribosome bind site of the expression vector also including translation initiation and Transcription terminator.

Additionally, expression vector preferably includes one or more selected markers, to provide for selecting The dihyrofolate reductase of the phenotypic character of the host cell of conversion, such as eukaryotic culture, neomycin resist Property and green fluorescent protein (GFP), or for the tetracycline or amicillin resistance of Escherichia coli.

Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for The appropriate host cell of conversion, allows it to marking protein.

Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells； Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, streptomyces Bacterial cell；Fungal cell's such as yeast；Plant cell；The insect cell of fruit bat S2 or Sf9；CHO、COS、 Or 293 cell zooblast etc..

Converting host cell with recombinant DNA can be carried out with routine techniques well known to those skilled in the art.Work as place When master is for prokaryotes such as Escherichia coli, the competent cell that can absorb DNA can be harvested after exponential phase of growth, Use CaCl₂Method treatment, step used is generally well-known in the art.Another method is to use MgCl₂.Such as Fruit is needed, and conversion can also be carried out with the method for electroporation.When host is eucaryote, following DNA is can select Transfection method：Calcium phosphate precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging Deng.

The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.Root According to host cell used, culture medium used may be selected from various conventional mediums in culture.It is being suitable to host Cultivated under conditions of cell growth.After host cell growth is to appropriate cell density, with suitable Method (such as temperature transition or chemical induction) induces the promoter of selection, and cell is further cultured for into a period of time.

Cell can be expressed or be secreted into protein in the above methods in the cell or on cell membrane Outward.If desired, can utilize its physics, chemistry and other characteristics be separated by various separation methods and Purifying protein.These methods are well-known to those skilled in the art.The example of these methods include but not It is limited to：Conventional renaturation process, (salting-out method), centrifugation, the broken bacterium of infiltration, super is processed with protein precipitant Treatment, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, efficient liquid phase The combination of chromatography (HPLC) and other various LC technologies and these methods.

Antibody

In the present invention, " antibody ", " part " are used interchangeably, refer to specific polyclonal antibody and Monoclonal antibody, especially monoclonal antibody.Here, " specific VEGF antibody " refers to anti-vegf Antibody can be incorporated into VEGF or its fragment.It is preferred that referring to that those can be combined but not with vegf protein or fragment Recognize and be incorporated into the antibody of other non related antigen molecules.Antibody of the invention can be by skill in the art Various technologies are prepared known to art personnel.

The present invention not only includes complete monoclonal or polyclonal antibody, but also including with immunocompetent Antibody fragment, such as Fab' or (Fab)₂Fragment；Heavy chain of antibody；Antibody light chain；Genetically engineered is single-stranded Fv molecules；Or chimeric antibody.

Peptide linker

The invention provides a kind of difunctional fusion protein, it optionally contains peptide linker.Peptide linker is big Small and complexity may influence the activity of albumen.Generally, peptide linker should have enough length and flexible Property, spatially there are enough frees degree to play its function with two albumen for ensureing connection.Avoid simultaneously The influence to the stability of fusion protein such as α spirals or β-pleated sheet is formed in peptide linker.

Too short peptide linker can cause steric hindrance inside fusion protein, influence the correct folding of albumen, mistake Peptide linker long may increase the immunogenicity of fusion protein, it is also possible to influence the activity and function of fusion protein.

The length for connecting peptide is generally 4-44 amino acid, preferably 6-27 amino acid, more preferably 2-15 Individual amino acid.

Pharmaceutical composition and application process

Present invention also offers a kind of composition, it contains the fusion protein of the invention of effective dose, and medicine Acceptable carrier on.Generally, fusion protein of the invention can be formulated in nontoxic, inert and medicine On in acceptable aqueous carrier medium, wherein pH ordinarily be about 5-8, it is preferred that pH is about 6-8.

As used herein, term " effective dose " or " effective dose " refer to that people and/or animal can be produced Function or activity and the amount that can be received by people and/or animal, such as 0.000001-90wt%；Preferably 0.1-50wt%；More preferably, 5-40wt%.

As used herein, the composition of " pharmaceutically acceptable " applies to people and/or mammal and nothing Excessively bad side reaction (such as toxicity, stimulate and allergy), i.e., with rational benefit/risk than Material.Term " pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various figurations Agent and diluent.

Fusion protein of the invention and pharmaceutically may be used that pharmaceutical composition of the invention contains safe and effective amount The carrier of receiving.This kind of carrier includes (but being not limited to)：Salt solution, buffer solution, glucose, water, glycerine, Ethanol, and combinations thereof.Usual pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the invention can To be made into injection form, for example the aqueous solution with physiological saline or containing glucose and other assistant agents is by normal Rule method is prepared.Described pharmaceutical composition is preferably aseptically manufactured.The dosage of active component It is therapeutically effective amount.

The effective dose of fusion protein of the present invention can be with the serious of the pattern and disease to be treated being administered Degree etc. and change.The selection of preferred effective dose can be by those of ordinary skill in the art according to various factors To determine (such as by clinical test).Described factor is included but is not limited to：The medicine of described fusion protein For kinetic parameter such as bioavailability, metabolism, half-life period etc.；The disease to be treated of patient it is serious Degree, the body weight of patient, the immune state of patient, approach of administration etc..Generally, when fusion of the invention Albumen every time with about 0.001mg-10mg/ eyes (preferably 0.01mg-8mg/ eyes, more preferably 0.1-5mg/ eyes) dosage give one or many, so as to obtain gratifying effect.

Main advantages of the present invention include：

A () fusion protein of the present invention can effectively treat pathological myopia, it is near to be especially effectively improved pathologic Depending on diopter.

B () fusion protein of the present invention can not only improve general pathological myopia, or even to proceeding to retina Come off-the pathological myopia in proliferative vitreoretinopathy stage, also there is significant curative effect.

C () fusion protein of the present invention synergistically improves pathologic by suppressing complement system and VEGF, complementation Various different symptoms of myopia, regard including suppression punctum luteum hemorrhage, suppression retinal cell apoptosis, improvement Nethike embrane departs from, suppresses vitreum lesion, suppress axis oculi extension, suppress new vessels generation, improve diopter Deng so as to effectively achieve the target of improvement eyesight.

D () color cavy of animal pattern three of the invention has the feature on congenital myopia eye leopard line shape eyeground, its The development of refractive status is consistent with primate, therefore therapeutic effect more has guidance to anticipate exploitation human drugs Justice.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for The bright present invention rather than limitation the scope of the present invention.The experiment side of unreceipted actual conditions in the following example Method, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to manufactory Condition proposed by business.Unless otherwise indicated, otherwise percentage and number are percentage by weight and parts by weight.

Reagent and instrument

All reagents and instrument are commercially available prod or are prepared with conventional method.

Testing drug (XD-DF)：With the fusion protein of conventional DNA recombination methods, the biological system in commission Xinda Medicine (Suzhou) Co., Ltd produces, sequence such as SEQ ID NO.:Shown in 1, purity >=99%.It is stored in 4 DEG C refrigerator

Negative control (IgG1 Isotype)：Prepared by Innovent Biologics (Suzhou) Inc., be stored in 4 DEG C refrigerator.

Compound tropicamide eye drops 5mg/ml.

Oxybuprocaine hydrochloride eye drops 5ml/20mg.

Ketamine Hydrochloride Inj. 2ml:0.1g.

Hydrochloric acid plug draws piperazine injection eye 1.5ml:30mg.

Ofloxacin eye ointment (Tarivid eye ointment).

Indocyanine green for injection 25mg.

Fluorescein isothiocynate/FITC (Sigma companies).

2% lidocaine injection.

10% antiseptic eye drops.

Tarivid eye ointment.

Chloraldurate.

Rabbit anti-human polyclonal antibody VEGF (Beijing Bo Aosen biotech firms).

SP immunologic combined detection reagent kits/rabbit (Beijing Bo Aosen biotech firms).

DAB colour reagents box (Foochow steps Newbiotics, Inc.).

The goat anti-mouse igg (Beijing Bo Aosen biotech firms) of the marks of Alexa fluor 555.

DAPI dyeing liquors (Beijing Bo Aosen biotech firms).

AEC substrate colour reagents box (health is ShiJi Co., Ltd).

Aqueous mountant (health is ShiJi Co., Ltd).

TUNEL fluorescent dyeing reagents box (Biotium companies).

Cavy VEGF ELISA kit (Biogo companies)

Laboratory apparatus

Retinoscopy mirror (the vision scientific ＆ technical corporation of Suzhou six or six).

Multiwavelength laser machine (Lumenis novas variaXL companies).

Indocyanine green angiography video camera (Topcon 501X).

Fluorescence inverted microscope (Olympus IX81).

Biomicroscope B204LED (Chongqing company of Ao Te optical instruments company).

Surgical operation microscope (TOPCON OMS-610).

Freezing microtome (Leica CM1850).

A-mode diagnostic ultrasonic apparatus (French BIOVISION companies).

Micro syringe (30-gauge, Shanghai glass metrology device factory).

Ophthalmology ultrasound diagnosis instrument (French light too CINESCAN).

Embodiment 1

Pathological myopia experiment merges the foundation of retina choroid new vessels animal model

The color cavy of undercoat three is planted by the big male Britain of three week old, without congenital eye illness, body weight 140-170g, as Animal before screening, is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center.Cavy is under the conditions of ordinary laboratory Raise, drinking water do not limit, add vitamin C in water, 20-22 DEG C of temperature, humidity 60%, 12h/12h is round the clock Circulation.Inspect periodically cavy eye health status.Experimentation strictly observes ARVO principles (The statement of Animals Research in Vision and Ophthalmic)。

Induced by mask method form deprivation and laser photocoagulation, set up pathological myopia-retina choroid new Angiogenic animal models.Method is as follows：

A () mask method form deprivation is induced

Worn after latex balloon is pruned on sobering animal head, covering right eye (experimental eye) carries out shape and feels induction, Left eye is opened wide as control eye.Mask is fixed with stapler, and as growth of animal is timely in experimentation Change mask size, 1 mask position of daily check is ensuring to be completely covered by experimental eye and eyeball not caused Compressing.Fig. 1 shows a guinea pig model for mask method form deprivation myopia.

(b) laser photocoagulation

At the weekend of form deprivation the 6th, all right laser photocoagulations of animal.Lower limb muscles inject ketalar Piperazine mixed liquor (7 is drawn with hydrochloric acid plug:1) anesthetized animal, right eye drop Tropicamide and Phenylephrine ocular fluid mydriasis, at the moment 120D preset lenses are placed, after finding optic disk, under a visual field, is existed with the krypton laser that wavelength is 532nm Optic disk top solidifying two rows of light, often arrange 5 points, totally 10 points.Laser power is 120mW, 50 μm of diameter, Time for exposure is 0.1s.

Embodiment 2

The packet of pathological myopia animal model, treatment and therapeutic evaluation

The present embodiment is used to evaluate therapeutic actions of the XD-DF in cavy pathological myopia model.

2.1 packets and therapeutic scheme

(a) animal packet

Diopter is filtered out by the form deprivation stage>The cavy of -8.00D, using random digits table by globefish Mouse is divided into 4 groups, respectively model group (negative control isotype 0.625mg/ eyeballs), low dosage administration group (XD-DF 0.125mg/ eyeballs), middle dosage administration group (XD-DF 0.625mg/ eyeballs), high dose administration group (XD-DF 1.25mg/ eyeballs), every group of 30 animals.

B () intravitreal is administered

The 2nd day after laser photocoagulation, it is placed under surgical operation microscope under Animal Anesthesia state, uses not damaged microforceps Son separately goes up palpebra inferior, is anaesthetized with Oxybuprocaine hydrochloride eye drops part eyeball surface, then with compound support pyrrole The abundant mydriasis of card amine eye drops.With micro syringe syringe needle, 0.5mm does a puncture orifice from after temporal limbus corneae, Micro syringe perpendicular to eyeball surface from puncture orifice inserting needle, from pupil see needle point after (about 1.5mm), delay Slow injection enters the XD-DF or positive control agent of various dose, and after gently pulling out pin, micro- tweezers gently press from both sides puncture orifice For a moment, it is to avoid medicine outflows, closed with sharp puncture orifice, Tarivid eye ointment is smeared after injection to prevent infection And the cornea equivalent damage caused by eyeball exposure.Whole injection process is careful not to cause vitreous hemorrhage or crystalline substance Bulk damage.

2.2 evaluation methods

For each group experimental animal, gathered data, carries out therapeutic evaluation using the following method.

(a) retinoscopy

Every group of animal is before experiment, shape feels weekend experimental stage 2,4,6, the 7th, 14,28 days after laser Eyes receive retinoscopy.Every group of animal right eye uses Tropicamide and Phenylephrine ocular fluid, every 5 points before inspection shadow Clock drips 1 time, totally 4 times, inspection shadow is carried out by the optist of unknown packet situation under dark room conditions after 1 hour. Inspection shadow operating distance 50cm, correcting lens keeps boresight direction inspection shadow, record knot apart from animal eyes about 5mm Really.

The influence of (b) axiallength and VCD

Using before the super probe measurement guinea-pig studies of special A, shape feel experimental stage the 2nd, 4,6 weekends and laser The right eye axiallength and VCD of the 7th, 14,28 days afterwards.Probe diameter 5mm, ultrasound is differentiated Rate is 0.04mm, and the spread speed for setting ultrasound different medium in guinea pig eye is crystalline lens 1645m/s, its He is position 1540m/s.The 30s intracapsular Oxybuprocaine hydrochloride eye drops surface anesthesias of animal conjunctiva before measurement, survey Fixed animal on the other hand during amount, another hand by alignment probe pupil center, perpendicular to corneal plane.Select ideal Waveform recording, every measures 10 times, and standard deviation is less than 0.04, takes average.

(c) indocyanine green angiography (ICGA)

Before light is solidifying, light it is solidifying after the 7th, 14,28 days, per 5 cavys of group selection, by indocyanine green for injection (6mg/kg) is injected from auricular vein, and row ICGA is checked.

D () CG irrigates choroid tile

5 cavys of each group selection (after ICGA terminates) 7th, 14,28 days after light is solidifying, from auricular vein injection 5mg/ml FITC, are put to death after 15 minutes and take eyeball, and 4% paraformaldehyde is fixed, and 10% is moved to after 24 hours Formaldehyde is fixed, and cornea, crystalline lens, vitreum are removed under disecting microscope, careful to peel off neuroretina layer, By RPE- choroids-sclera complex centered on regarding nipple 5~6 radial incisions of row symmetric row, be placed in On the slide of a small amount of glycerin gelatine of oil dripping, mounting observes CNV, and pass through under laser confocal microscope Image-proplus6.0 specialized images analysis software carries out graphical analysis measurement CNV areas.

E () SABC detects vegf expression

7th, 14,28 days administration groups respectively put to death 5 cavys after light is solidifying, right eye intact eye are won, more than 4% Polyformaldehyde is fixed, and angle of release film is scratched after 1 hour, and crystal is removed after 2 hours, goes to leaching in 20% glucose 24 hours, finally embedded with sucrose-OCT liquid, sample is freezed with liquid nitrogen, -20 DEG C of freezings after freezing completely It is standby.In freezing microtome operate, perpendicular to boresight direction section, to laser facula at start to take up, cut 5 μm of piece thickness.

4 retinas of every group of selection and train of thought membrane structure are continuous and have the section of laser facula, in 56 DEG C of incubators Baking 30 minutes, calf serum room temperature is closed 30 minutes, is separately added into primary antibody (rabbit anti-human VEGF antibody 1:200) overnight, secondary antibody (Alexa Fluor 555 mark goat anti-rabbit igg 1:200), horseradish enzyme mark Streptomysin avidin working solution, AEC colour developings, aqueous mountant mounting.With Image-proplus6.0 specialties Image analysis software carries out graphical analysis, calculates the average optical density value (AOD) of vegf expression.

2.3 experimental results

A () is successfully established model

All results show that after shape feels induction 4 weeks, all zoopery refraction of eyes are both greater than -6.00D；6 Zhou Hou, all zoopery refraction of eyes are both greater than -8.00D, and the control refraction of eye degree without covering is only slight Rise, show that experimental eye has formed pathological myopia.

After laser photocoagulation, model group experiment refraction of eye degree is further deepened, and ICGA is checked and found that light is real after solidifying Test eye occur fluorescence fill, it was demonstrated that CNV is formed, and as time went on, lacquer cracks hypofluorescence occurs, Show there is blutpunkte, illustrate to be successfully established pathological myopia-retina choroid neovascularization model.

The effect of (b) XD-DF Partial Inverse rotexion light states

It can be seen from model group data, the 7th, 14,28 after form deprivation the 2nd, 4,6 weeks and laser photocoagulation Its experimental eye refractive status constantly deteriorates.After XD-DF administrations, compared with before administration, three dosage group dosage Rely on ground Partial Inverse rotexion light state, the 14th day and the 28th day, the dioptric of middle high dose group cavy after light is solidifying Condition improvement has statistical significance, and the 28th day low dose group improves also has statistical significance.At three Between put in high dose group refractive status improve and be slightly better than low dose group, but in the absence of the significant difference (He of table 1 Fig. 2).All data are expressed as means standard deviation.

The refractive status of the administration group of table 1 and model group different time points

^*Using duplicate measurements variance analysis and one-way analysis of variance, and before contrast administration (before laser photocoagulation/shape feel Deprived for the 6th weekend),^*P ＜ 0.05,^**P ＜ 0.01,^***P ＜ 0.001.

The result of table 1 and Fig. 2 shows that comparison model group, fusion protein of the present invention can very significantly change The refractive status of kind pathological myopia animal pattern, solidifying latter 28th day to light, diopter can be aobvious from -12.80 Writing improves to about -5.0 (low dosage) peace treaties -4.0 (middle dosage and high dose).

C () XD-DF shortens the effect of axiallength and VCD

It can be seen from model group data, relative to control eye, form deprivation the 2nd, 4,6 weeks and laser photocoagulation The 7th, 14,28 days experimental eye axis oculi and vitreous chamber all persistently extend afterwards.After XD-DF administrations, with administration Before compare, three dosage can dose-dependant ground Partial Inverse axle and vitreous chamber elongation (vitreum atrophy) in an instant Effect, 14th day and 28 days after light is solidifying, the shortening of middle high dose group cavy axis oculi and Length of the vitreous With significant difference；28th day, low dose group cavy was reversed the effect of vitreum atrophy also to have and counts Learn meaning.The improvement of high dose group guinea-pig studies eye axis oculi and glass body length is superior to low in three time points Dosage group, wherein the difference of the axiallength of the 14th day have statistical significance (table 2 and table 3, Fig. 3 and Fig. 4).All data are expressed as means standard deviation.

The administration group of table 2 and model group different time points axiallength (mm)

^*Using duplicate measurements variance analysis and one-way analysis of variance, and before contrast administration (before laser photocoagulation/shape feel Deprived for the 6th weekend),^*P ＜ 0.05,^**P ＜ 0.01,^***P ＜ 0.001；^#Contrast low dose group,^#P ＜ 0.05,^##P ＜ 0.01,^###P ＜ 0.001.

The administration group of table 3 and model group different time points VCD (mm)

The result of table 2 and Fig. 3, table 3 and Fig. 4 shows that fusion protein of the present invention can very significantly contract The axis oculi and Length of the vitreous of short pathological myopia animal pattern, improve the pathological state of animal.It is solidifying to light The 28th day afterwards, axis oculi and Length of the vitreous approached level before modeling.

D () choroid tile checks that XD-DF reduces the effect of new vessels area

There is fluorescence to manifest around model group experimental eye laser spot after visible ray is solidifying under fluorescence inverted microscope, and mould Type group control eye is then unchanged, shows have new vessels to generate in experimental eye.After XD-DF administrations, with model Control group is compared, and three time points, three dosage can significantly reduce CNV areas, suppresses new vessels generation. Solidifying the 7th day afterwards of light, model group and administration group CNV areas (μm²) it is respectively 237946.9 ± 105264.562,145632.3±132642.896；14th day, laser spot and surrounding had tufted new vessels Generation, area substantially increases, model group and administration group CNV areas (μm²) it is respectively 325684.6 ± 215426.452,215468.0±265875.785；28th day, high fluorescent near laser spot, model group With administration group CNV areas (μm²) be respectively 336528.2 ± 269845.865,225698.8 ± 275692.692, administration group eye CNV areas are significantly greater than model group eye, and difference has significance,statistical (P ＜ 0.05).Significant difference (P ＜ are there is also between high dose and low dosage in three time points after light is solidifying 0.05), then without difference (Fig. 5) between middle high dose.All data are expressed as means standard deviation.

D () XD-DF reduces the effect of vegf expression

After XD-DF administrations, Showed by immune group result, three time points, three administration group experiment eyes retinas The average optical density value of VEGF is substantially reduced, compared with model group, the 7th, 14,28 days models after light is solidifying Between group experimental eye and administration group experimental eye there is significant difference (P ＜ 0.05) in vegf expression.Three after light is solidifying There is significant difference (P ＜ 0.05) in individual time point between high dose and low dosage, and between middle high dose There is no difference (Fig. 6).All data are expressed as means standard deviation.

2.4 conclusions

In the deprivation induced pathological myopia of cavy merges CNV model, three dosage XD-DF all show certain treatment potentiality, including Partial Inverse in an instant axle and vitreous chamber elongation (vitreum withers Contracting), reduce punctum luteum hemorrhage, reduction new vessels area and reduction vegf expression.This shows, of the invention Fusion protein XD-DF can effectively treat pathological myopia merge CNV lesion.

Embodiment 3

Pathological myopia experiment merges the foundation of proliferative vitreoretinopathy animal model

Induced by mask method form deprivation and laser photocoagulation, set up pathological myopia animal model (retina Come off-proliferative vitreopathy model).Method is as follows：

A () mask method form deprivation is induced

With embodiment 1, worn after latex balloon is pruned on sobering animal head, covering right eye (experimental eye) is entered Row shape feels induction, and left eye is opened wide as control eye.Fix mask with stapler, and in experimentation with Growth of animal changes mask size in time, 1 mask position of daily check with ensure to be completely covered by experimental eye and Compressing is not caused to eyeball.

The culture of (b) retinal pigment epithelium

Eyes eyeball is extractd in healthy guinea pig, 2% lidocaine retrobulbar anaesthesia, sterile working.In DEME liquid It is middle to be cut off along annular at 0.5mm after corneoscleral junction, discard anterior ocular segment and vitreum.With 0.25% pancreatin room temperature Lower digestion removes layer of retina,neuroepithelial after 3~5 minutes, after being digested with pancreatin again from cell suspension Retinal pigment epithelium is collected in centrifugation, in cell culture incubator (5%CO₂, 90% humidity, 37 DEG C) in carry out Cellar culture and passage, choose the 4th good generation retinal pigment epithelium of growth conditions, use physiology salt Water is made 1 × 10⁶/ 0.1ml cells are standby, used in 20 minutes, otherwise discard and prepare again.

The retinal pigment epithelium modeling of (c) intravitreal injection

It is placed under surgical operation microscope under Animal Anesthesia state, the upper and lower eye of right eye is separated with the micro- tweezers of not damaged Eyelid, is anaesthetized with Oxybuprocaine hydrochloride eye drops part eyeball surface, is then filled with compound tropicamide eye drops Dispersion pupil, 10% antiseptic eye drops rinses conjunctival sac.With micro syringe syringe needle from temporal limbus corneae 0.5mm does a puncture orifice afterwards, and micro syringe injects cell suspension perpendicular to vitreum from puncture orifice inserting needle 0.1ml.Whole injection process is careful not to cause vitreous hemorrhage or lens lesion that (such as damaging occurs in injection process Wound, is supplied using standby cavy).

Embodiment 4

The present embodiment is regarded for evaluating XD-DF in cavy pathological myopia retinal detachment-proliferative vitreum Therapeutic action in retinopathy.

4.1 animal packets and administration

(a) animal packet

Feel that induction period selects diopter by shape>The cavy (retinoscopy) of -8.00D, using random number Cavy is divided into 4 groups by word table method, respectively model group (negative control 0.625mg unrelated proteins/eyeball), Low dosage administration group (XD-DF 0.125mg/ eyeballs), middle dosage administration group (XD-DF 0.625mg/ eyeballs), High dose administration group (XD-DF 1.25mg/ eyeballs), every group of 12 animals.

B () intravitreal is administered

After vitreous chamber injects cell suspension (retinal pigment epithelium), according to animal packet situation, press Take a picture same injecting method, slowly inject XD-DF the or Isotype 0.01ml of various dose, after gently pulling out pin, Micro- tweezers gently press from both sides puncture orifice for a moment, it is to avoid medicine outflows, and are closed with sharp puncture orifice, and thyrite is smeared after injection Must appropriate eye ointment with prevent infection and eyeball exposure caused by cornea equivalent damage.Whole injection process is careful not to Cause vitreous hemorrhage or lens lesion (such as damaging occurs in injection process, and is supplied using standby cavy).

4.2 evaluation methods

(a) funduscopy

Shape is felt weekend experimental stage 6 (the 0th day), (is located within postoperative 3rd day, 7 days, 14 days, 21 days and 28 days Before dead) ophthalmology ultrasound diagnosis instrument checks for vitreous opacity, whether there is detachment of retina and disengaging degree and Scope, and eyeground accretion zone (every group 12).Lesion is carried out according to propagation degree and retinas Classification, as shown in table 4.

The proliferative vitreoretinopathy of table 4 is classified

(b) fluoroscopic examination retinal cell apoptosis

Postoperative 28th day, animal is put to death, take out eyeball (every group 6), 4% poly is placed in after perforation of cornea 2 hours in formalin, wiped out under microscope and organized outside sclera, be put into 4 DEG C of 15% sucrose solution, sunk 30% sucrose solution, 4 DEG C of refrigerator overnights (12 hours) are placed into behind bottom (about needing 1 hour).2nd day, from Eyeball is taken out in sucrose solution, dry surface sucrose solution is printed with filter paper, embedding liquid with OCT embeds, and waits to organize Cut into slices with liquid nitrogen flash freezer, 5 μm of freezing microtome thickness after submerging and draining bubble.

PBS cleaning frozen section 2 times, is incubated 5 minutes with 100 μ l TUNEL dye solutions, Add 50 μ l TUNEL to react complex systems, 37 DEG C are incubated 2 hours, with X-100 containing Triton and The PBS cleaning of BSA, in fluorescence microscopy Microscopic observation result.3 visuals field are chosen in every section, All cell number amount and apoptotic cell quantity under the visual field are recorded, is averaged.Apoptotic nucleus are dyed to bright Green fluorescence.

The area measurement of (c) intensifier deep

Postoperative 28th day, animal is put to death, extracts eyeball (every group 6), 4% paraformaldehyde fixes 1 week, Take out eyeball to be cut along cornea, optic nerve horizontal plane, flushing, step by step dehydration of alcohol, dimethylbenzene is transparent, leaching Wax, FFPE, the continuous 2 μm of sections of the parallel optic nerve of sagittal plane, routine hematoxylin-eosin stains, 100 Times light Microscopic observation, hyperplasia membrane area is measured with Image-proplus6.0 specialized images analysis software. 5 visuals field are chosen in every section, average.

Vegf expression level in (d) vitreous humor

Postoperative 28th day, animal is put to death, vitreous humor (every group 12) is extracted from vitreous chamber before extracing eyeball, Every group is extracted 1~1.2ml altogether, is detected with kit using double sandwich-ELISA method.

4.3 experimental results

A () is successfully established model

After vitreous chamber retinal pigment epithelium injects 3 days, model group animal starts to occur retina and takes off From, to the 7th day all depart from, scored according to table 4, average lesion classification more than 3, show into Work(sets up model.

B () XD-DF mitigates the effect of vitreoretinopathy

It can be seen from model group data, to administration after the 7th day, it is complete that retina has occurred in model group animal Break-off.After XD-DF administrations, compared with model group, three dosage group dose-dependant ground mitigate vitreum PVR degree, scores according to table 4, counts all administration group experimental eye lesion ranks, and be averaged Value.From the 7-28 days, three dosage mitigated lesion degree and are respectively provided with statistical significance (P ＜ 0.05), middle height Dosage group is slightly better than not existing significant difference (Fig. 7) between low dose group, but three.

C () XD-DF suppresses the effect of retina apoptosis

After XD-DF administrations, by the 28th day, compared with model group, three dosage group dose-dependant ground suppressed to regard The generation of retinulae apoptosis, and with statistical significance (P ＜ 0.05) (table 5).

Experimental eye electron microscopic observation result is shown, nucleus layer is found that abnormal regarding inside and outside experiment eyes retina Retinulae apoptosis feature, including view membranous disc oedema comes off, and pigment epithelial cell vacuole sample changes, cell Film irregularly contraction etc..All data are expressed as means standard deviation.

The XD-DF of table 5 suppresses retinal cell apoptosis

D () XD-DF reduces the effect of hyperplasia membrane area

The streak collagenous fibres of visible red dye in model group experiment vitreum, with retina table in vitreum Face has large stretch of intensifier deep to cover, and hyperplastic tissue is contained within a large amount of new vesselses.

After XD-DF administrations, by the 28th day, compared with model group, in administration group vitreum and retinal surface Large stretch of hyperplastic tissue is showed no, hyperplasia bar rope is had focused largely in vitreous chamber.Three dosages can show Reduction hyperplasia membrane area (P ＜ 0.05) is write, middle high dose group hyperplasia and degree of tissue damage relatively low-dose group are slightly Gently (table 6, without accompanying drawing).All data are expressed as means standard deviation.

The XD-DF of table 6 reduces hyperplasia membrane area

E () XD-DF reduces the effect of vitreous humor VEGF levels

After XD-DF administrations, ELILSA results show, by the 28th day, compared with model group, and three administrations VEGF levels are substantially reduced (P ＜ 0.05) in group experiment vitreous humor, and and dose proportional, show XD-DF can significantly reduce the expression and secretion (table 7, without accompanying drawing) of VEGF.All data are expressed as mean value ± Standard deviation.

Table 7

4.4 experiment conclusions

In the deprivation induced pathological myopia of cavy merges proliferative vitreoretinopathy model, three agent The XD-DF of amount shows certain therapeutic effect, and in dose-effect relationship, including mitigate lesion classification, Suppress retinal cell apoptosis, reduce hyperplasia membrane area, and reduce vitreum vegf expression.These knots Fruit shows, XD-DF can be used for treating that to develop into the pathologic in proliferative vitreoretinopathy stage near Depending on.

Discuss

Research shows, during the pathology occurrence and development of pathological myopia CNV and retinal detachment-PVR VEGF and inflammatory reaction all occupy very important position.

VEGF is the confirmation target spot for treating new vessels, and different from other CNV, pathological myopia choroid is new The blood vessel diameter of angiogenic is smaller with respect to other CNV, and bleeding scope is also more limited to, and late period is then increased with pigment Grow with atrophia choroideae et retinae based on；Additionally, Recent study finds proliferative vitreoretinopathy Vitreum propagation film and preretinal membrane in vegf expression raise, illustrate that VEGF develops with PVR Also there is close relationship.

Inflammatory reaction all played an important role in the morbidity of CNV and PVR.Complement system abnormal activation Complement component (C3, C5 etc.) up-regulated, has mediated various inflammatory reactions afterwards.Three activation of complement activation Approach crosses at C3, and then forms C5 convertase, common to enter end approach eventually.Complement receptor 1 (CR1) It is most important Molecular regulator in Complement Regulatory Protein, the decay of C3 converting Enzymes and C5 convertase can be accelerated And the function of C3b and C4b is inactivated, and the release of inflammatory effector fragment is reduced, further suppress proinflammatory cytokine The expression of the factor (interleukin, chemotactic factor (CF), TNF-α etc.) and the infiltration of macrophage and neutrophil leucocyte, Mitigate body inflammatory reaction, so as to play therapeutic action.

Therefore, at the same for VEGF paths and complement pathway will likely effectively treat pathological myopia CNV and Retinal detachment-PVR, also better than current unipath therapeutic scheme, treatment potentiality should not be underestimated.

XD-DF is Recombinant human vascular endothelial growth factor receptor-antibody-people's complement receptor 1 fusion protein, is A kind of pair of target spot specific fusion protein, can simultaneously target VEGF paths and complement pathway, suppress intraocular VEGF Overexpression and inflammatory reaction, so that complementary, collaboration and more effectively treatment develop into CNV and PVR ranks The pathological myopia of section.

The all documents referred in the present invention are all incorporated as reference in this application, just as each document It is individually recited as with reference to such.In addition, it is to be understood that after above-mentioned instruction content of the invention has been read, Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen Please appended claims limited range.

Claims

1. a kind of purposes of fusion protein, it is characterised in that the fusion protein has Formulas I a or Formulas I b The structure：

A-B-C (Ia), or

C-B-A (Ib)

Wherein,

A is the antibody or receptor element for VEGF；

B is companion's element；

C is the antibody or receptor element for complement；

2. purposes as claimed in claim 1, it is characterised in that the sequence of the fusion protein such as SEQ ID NO.:Shown in 1.

3. purposes as claimed in claim 1, it is characterised in that described medicine or preparation is additionally operable to control One or more disease that treatment is selected from the group：Pathological myopia merges CNV, pathological myopia Merge punctum luteum hemorrhage, pathological myopia merging retinal detachment, pathological myopia to merge proliferative vitreum and regard Retinopathy.

4. purposes as claimed in claim 1, it is characterised in that described pathological myopia has following characteristics： Diopter is in more than -6.00~-8.00D.

5. purposes as claimed in claim 1, it is characterised in that described pathological myopia has following characteristics： Axis oculi progressive grows beyond 26.5mm.

6. purposes as claimed in claim 1, it is characterised in that described pathological myopia has and is selected from the group Feature：

I () diopter is in more than -6.00~-8.00D；

(ii) axis oculi progressive grows beyond the Eye disease of 26.5mm；

(iii) with ball wall tissue is thinning and eyeground pathological changes.

7. purposes as claimed in claim 1, it is characterised in that described eyeground pathological changes are selected from the group： Paint crackle, atrophia choroideae et retinae or its combination.

8. purposes as claimed in claim 1, it is characterised in that described fusion protein include monomer, Or dimer.

9. a kind of medicine box for treating pathological myopia, it is characterised in that the medicine box includes：

A () first container, and the fusion protein in first container, the fusion protein have Structure described in Formulas I a or Formulas I b as defined in claim 1；

(b) optional utensil for intraocular injection administration；With

10. medicine box as claimed in claim 9, it is characterised in that the specification also indicates the fusion Albumen is used for：I () reverses axis oculi and vitreous chamber elongation, atrophy, (ii) reduces punctum luteum hemorrhage, (iii) contracting Subtract new vessels area, and/or (iv) improves diopter.