CN114652826B - Use of anti-EGFR antibodies - Google Patents

Abstract

The invention provides an application of an anti-EGFR antibody in preparation of a medicine for preventing and/or treating diseases related to ocular neovascularization.

Description

Use of anti-EGFR antibodies

Technical Field

The invention belongs to the field of medicines, and relates to a new application of an anti-EGFR antibody.

Background

With the increase of human age, the aggravation of myopia or the development of related metabolic diseases, when fundus diseases such as pathological myopia, age-related macular degeneration, diabetic retinopathy and the like occur, unnecessary blood vessels can be newly generated on the fundus. These new blood vessels invade the original normal ocular fundus tissues such as choroid and retinal photoreceptor cell layer, and the visual function is impaired, and blindness is also caused seriously.

Neovascularization is a common pathological change in many ocular diseases, and commonly occurs in cornea, iris, choroid and retina, resulting diseases including corneal neovascularization, choroidal neovascularization, retinal neovascularization, and the like, and common ocular disorders resulting therefrom include, for example: diabetic Retinopathy (DR), retinopathy of prematurity (ROP), age-related macular degeneration (AMD), including wet AMD and dry AMD, where the pathology of wet AMD changes to choroidal neovascularization), neovascular glaucoma, and the like. Neovascularization can occur in almost all tissues in the eye such as cornea, iris, ciliary body, choroid, retina, macula and optic disk, and it can cause a series of pathological changes such as bleeding, exudation and hyperplasia of tissues at these parts, thus causing the destruction of the structure and function of the eyeball and seriously damaging the visual function. The number of patients with eyeground neovascular diseases is more than 4000 ten thousand, and the number of patients is increasing continuously with the aging degree of population. Patients with ocular fundus neovascular diseases need to be re-diagnosed or injected with relevant drugs almost every month, otherwise the impairment of vision can seriously affect life.

Vascular Endothelial Growth Factor (VEGF) plays an important role in the formation of new blood vessels. Studies have shown that VEGF is currently the most specific and effective growth factor for angiogenesis. Since the 1990 s, drugs targeting VEGF to inhibit the angiogenesis of the ocular fundus by blocking the VEGF signaling pathway have been the focus of development. Ranibizumab (trade name Lucentis) specifically binds to VEGF-Sub>A and has been approved by the fdSub>A in the united states for the treatment of wet age-related macular degeneration and diabetic macular edemSub>A. VEGF Trap-Eye (afliberep or eylene) is an anti-VEGF recombinant protein developed by Regeneron pharmaceutical company in the united states, clinical study results show that the therapeutic effect on DME (diabetic macular edema) is satisfactory, and in 2018, eylene has been approved by the national food and drug administration (CFDA) for the treatment of adult Diabetic Macular Edema (DME). KH902 (combisccept, conbercept) is a VEGFR-Fc recombinant protein developed by chengdu Kang Hong for age-related macular degeneration (AMD), approved for marketing in 12 months in 2013 for the treatment of AMD. Although drugs directed against VEGF targets have made great clinical progress, the regulation of angiogenesis is a very complex dynamic equilibrium process, as angiogenesis is regulated by a variety of factors. The traditional single anti-VEGF therapy still has limitations, and therefore the development of new therapeutic approaches has important clinical significance.

Epidermal Growth Factor Receptor (EGFR), a transmembrane glycoprotein with a total relative molecular weight of 170kDa, is widely distributed in mammalian epithelial cells, and is a member of the ErbB (ErbB 1-4) family, also known as ErbB1. Studies have shown that there is high or abnormal expression of EGFR in many solid tumors. EGFR is involved in the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis and apoptosis.

EGFR consists of 3 parts of an extracellular ligand binding region, a hydrophobic transmembrane domain, and an intracellular kinase region. The intracellular structure of the polypeptide comprises 1 tyrosine kinase domain (tyrosine kinase domain) and a carboxyl terminal tail with a plurality of autophosphorylation sites, and belongs to the receptor tyrosine kinase family (RTKs); the extracellular region is composed of ligand binding sites and 2 cysteine-rich regions and can be combined with various ligands with an exciting function, and after extracellular EGF or TGF-alpha is combined with EGFR, a dimer is formed and is combined with 1 ATP molecule to activate the tyrosine kinase activity of the EGFR so as to enable a plurality of tyrosine sites of an intracellular kinase region to generate autophosphorylation. After EGFR dimerization and phosphorylation, a downstream ras/raf/MAPK (mitogen activated protein kinase) cascade system and a phosphoinositide kinase (phosphoinositide kinase) system are activated to become affinity sites for downstream signal transduction, and various mitotic signals are involved in the transmission from the outside of cells to the inside of the cells, so that the response of the cells to external stimuli, cell proliferation, survival, adhesion, migration and differentiation, cell survival and growth conditions and the like can be effectively regulated. Finally, the complex of EGFR and ligand enters the cell through pinocytosis and is degraded or recycled to the cell surface, completing signal transduction. Therefore, once the function of the tyrosine kinase is hindered, various diseases can be caused.

With the continuous and deep research on the relationship between signal transduction and the abnormality thereof and tumors, the abnormal signal transduction is intervened by intervening treatment on the signal transduction, namely, the abnormal signal transduction is intervened aiming at the link with the abnormality of a signal transduction path in the modes of monoclonal antibodies, immunotoxins, tyrosine kinase inhibitors, antisense nucleic acids and the like, so that the aim of inhibiting the growth of the tumors is fulfilled. The most common treatment modalities are currently: 1. monoclonal antibodies that bind to EGFR, compete for and block binding of ligands such as EGF, TGF- α, and the like; 2. tyrosine kinase inhibitors such as gefitinib, erlotinib, and the like. The EGFR monoclonal antibody competes with endogenous ligands for binding to EGFR, and generates an anti-tumor effect by inhibiting the activation of tyrosine kinase and promoting the internalization of EGFR, and the like, and the anti-EGFR monoclonal antibodies approved to be on the market at present comprise: cetuximab (Cetuximab), nimotuzumab (nimotuzumab), panitumumab (Panitumumab), and Cetuximab (Necitumumab). Can be used for treating colorectal cancer, head and neck cancer and non-small cell lung cancer. Among the antibody EGFR-targeting drugs, cetuximab (Cetuximab) is exclusively a chelator.

The earliest approval by the FDA of Cetuximab (Cetuximab, trade name: cetuximab) in 2004 at 2, 12 months

) The indications are as follows: treating advanced and refractory metastatic colorectal cancer in combination with irinotecan; treating locally or locally advanced squamous cell carcinoma of head and neck in combination with chemoradiotherapy. Cetuximab specifically binds to EGF receptors expressed on the surface of normal cells and a variety of cancer cells and competitively blocks the binding of EGF to other ligands, such as alpha transforming growth factor (TGF-alpha). The cetuximab is an IgG1 monoclonal antibody aiming at an EGF receptor, and after the cetuximab is specifically combined with the EGF receptor, the cetuximab blocks an intracellular signal transduction pathway through the inhibition effect on Tyrosine Kinase (TK) combined with the EGF receptor, thereby inhibiting the proliferation of cancer cells, inducing the apoptosis of the cancer cells and reducing the production of matrix metalloproteinase and vascular endothelial growth factor.

Several studies (hoisting N J, weskamp G, vermaat J, et al. Intra viral injection of TIMP3 or the EGFR inhibitor erlotinib of genes protection from oxidative gene-induced metabolism in mice, [ J ]. Invest Ophthalmol Vis,2013,54 (1): 864-870) have shown that the administration of the EGFR tyrosine kinase inhibitor erlotinib can inhibit pathological neovascularization of mouse retina, likely due to inactivation of ADAM17 and EGFR; xin J, yang X, tao Y, et al, EGFR inhibitor, AG1478, inhibitors in fluorescence uptake and angiogenesis in micro with metabolic retinopathy [ J ]. Clinical and Experimental Pharmacology and Physiology,2019,46A mouse model of type 1 diabetic early retinopathy by injection of chain urezosin and an oxygen-induced terminal diabetic retinopathy (OIR) model characterized by hypoxia-induced vascular remodeling were established, and the hypothesis that the classical EGFR inhibitor AG1478 (Tyrphostin AG 1478) could reduce the pathogenesis of DR was verified in the mouse model. The data indicate that treatment with AG1478 prevents retinal dysfunction in diabetic mice, reduces damage to retinal structure and mitochondrial structure in retinal blood vessels.

Disclosure of Invention

The object of the present invention is to provide a novel use of an anti-EGFR antibody.

According to one aspect of the present invention, without being bound by any theory, monoclonal antibodies targeting EGFR have a very good inhibitory effect on ocular neovasculature, particularly ocular fundus neovasculature, such as choroidal neovasculature and retinal neovasculature, and such antibodies can be used to treat diseases associated with ocular or ocular fundus neovasculature, particularly choroidal neovasculature and retinal neovasculature.

Therefore, the invention provides the application of the anti-EGFR antibody in the preparation of medicines for preventing and/or treating diseases related to ocular neovascularization, in particular diseases related to ocular fundus neovascularization.

According to the present invention, wherein the fundus neovascular-related disease is preferably: choroidal neovascularization associated disorders or retinal neovascularization associated disorders.

The diseases related to the neovascular fundus oculi include but are not limited to diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, retinal periphlebitis, age-related macular degeneration, high myopia macular hemorrhage and central exudative retinal choroidopathy.

According to the present invention, wherein the anti-EGFR antibody is a monoclonal antibody targeting EGFR, including various anti-EGFR antibodies currently marketed or currently being applied for marketing, such as Cetuximab (Cetuximab), nimotuzumab (nimotuzumab), panitumumab (Panitumumab) and nimotuzumab (Necitumumab), preferably Cetuximab.

According to another aspect of the invention, the invention also provides the use of an anti-EGFR antibody in the manufacture of a medicament for reducing the area of an avascular zone of the retina, inhibiting ocular neovascularization and/or causing regression of ocular neovascularization.

According to a further aspect of the invention, the invention also provides a method of treating a disease associated with ocular neovascularization, comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of an anti-EGFR antibody.

According to the present invention, the anti-EGFR antibody or the pharmaceutical composition containing the anti-EGFR antibody can be administered to the whole, part or vitreous of the eye or the affected eye at a prophylactically and/or therapeutically effective dose. The formulation may be any topical formulation suitable for ocular administration.

The invention firstly proves that the anti-EGFR antibody, particularly cetuximab, can effectively inhibit the formation of ocular neovascularization, obviously reduce the area of the retinal avascular region and eliminate the neovascularization, and can be used for treating diseases related to the formation of ocular or ocular fundus neovascularization. The effect is superior to that of monoclonal antibody of VEGF target point and also superior to that of small molecule tyrosine kinase inhibitor.

Drawings

FIG. 1 shows the FFA test results of PBS groups 7 days and 14 days after the rat is administrated on the day of CNV molding,

FIG. 2 shows FFA detection results of Abbericept groups administered on the day of model creation, 7 days and 14 days after model creation of rat CNV;

FIG. 3 shows FFA test results of groups administered on the day of model making of rat CNV, 7 days and 14 days JZB-5 mg/ml after model making;

FIG. 4 is a bar graph of 7-day and 14-day fluorescent spot grading for PBS, abbericept and JZB09-5mg/ml groups;

FIG. 5 bar graph of FFA neovascular leakage area for PBS, abbericept, and JZB09-5mg/ml groups at 7 days and 14 days;

FIG. 6 is a bar graph of pre-dose (0 d), post-dose 7d, post-dose 10d, post-dose 14dFFA fluorescent spot grading in PBS, abelicept, JZB09-5mg/ml, JZB09-40mg/ml, JZB-70 mg/ml;

FIG. 7 is a bar graph of FFA neovascular leakage area before (0 d), 7d, 10d, and 14 days after administration for PBS, aboxipu, JZB-5 mg/ml, JZB09-40mg/ml, JZB09-70 mg/ml;

FIG. 8 is a photograph taken with an Axio imager Z2, after 5 days post-administration, i.e., at day 17 (D17) after birth, with the PBS group, the Abbescept group, the JZB-5 mg/ml group, the JZB09-40mg/ml group, and the JZB-70 mg/ml group, from which mice were sacrificed, and with an eye-retinal-mounted plate, IB4 stained, and a fully automatic upright microscope;

FIG. 9 is a histogram of the area of the avascular area of the PBS group, abbericept group, JZB-5 mg/ml group, JZB09-40mg/ml group, JZB09-70mg/ml group of FIG. 8 as a percentage of the total area of the retina.

Detailed Description

In the present invention, the term "anti-EGFR antibody" refers to a monoclonal antibody targeting EGFR, which binds to EGFR, competes with, and blocks the binding of ligands such as EGF, TGF-alpha, and the like.

In the present invention, the term "Cetuximab" (Cetuximab) is used under the trade name Cetuximab

A recombinant human/mouse chimeric monoclonal antibody can specifically bind to human Epidermal Growth Factor Receptor (EGFR) and competitively inhibit the binding of EGF and other ligands (such as transforming growth factor alpha) to the receptor, and the globally approved indications of the recombinant human/mouse chimeric monoclonal antibody are metastatic head and neck cancer, non-metastatic head and neck cancer, metastatic colorectal cancer, non-small cell lung cancer and the like. The amino acid sequences of the heavy chain and the light chain are shown as SEQ ID NO.1 and SEQ ID NO.2, and the preparation method is described in detail in WO 96/40210.

The antibody of the invention comprises the antibody per se, an analogue of the antibody and a pharmaceutical composition containing the antibody.

The term "ocular neovascularization-associated disease" or "ocular fundus neovascularization-associated disease" refers to a condition occurring in the eye or ocular fundus resulting in structural and functional destruction of the eye by neovascularization, resulting in severe visual impairment. Neovascularization is a common pathological change in many important ocular diseases.

In the present invention, "Choroidal Neovascularization (CNV)", also called subretinal neovascularization, is a proliferative blood vessel derived from choroidal capillaries, which propagates through the breach of Bruch's membrane and proliferates between Bruch's membrane and retinal pigment epithelium, between neuroretina and retinal pigment epithelium, or between retinal pigment epithelium and choroid. CNV formation can be caused by a number of diseases involving the RPE-Bruch membrane-choroidal capillary complex. Degeneration, inflammation, trauma and the like may cause ischemia and hypoxia of retina at the outer layer of macula part, so that angiogenic factors are generated to stimulate choroidal capillary vessels to regenerate to form subretinal new blood vessels. The retinal neovascularization may occur in the early stages without subjective symptoms. As it gradually enlarges, leaks and ruptures to bleed, it can cause hypopsia, distortion of the visual objects, and the appearance of central or lateral dark spots. In the case of the counter-relapsing authors, the macula is severely destroyed, which can lead to permanent vision impairment. The diagnosis can be determined by fundus fluorescence angiography and morphology.

In the present invention, the "choroidal neovascularization-related diseases" include age-related macular degeneration, high myopia macular hemorrhage, central exudative retinochoroidal disease, and the like.

Age-related macular degeneration (AMD) is a pathological aging change in the structure of the macular region. It can be classified into dry (non-exudative) or wet (exudative or neovascular) 2 types. Wet AMD is characterized by choroidal neovascularization, which is the most direct factor affecting vision, as the Bruch's membrane ruptures as the pathological changes continue to progress in the middle and late stages of disease development, and choroidal capillaries pass through the ruptured Bruch's membrane under the RPE or the retinal nerve epithelium, forming Choroidal Neovascularization (CNV). Due to the abnormal structure of the wall of the new blood vessel, the leakage and bleeding of blood vessels often occur, and further the bleeding and edema of macula part are caused to cause a series of changes, aggravate the decline of vision and even suddenly decline of vision by a large margin, which is the main reason of blindness caused by AMD.

High myopia macular hemorrhage: high myopia refers to myopia with a refractive power of more than-6.00D, and is a common eye disease, and the ocular fundus of the eye disease is usually pathologically changed, which is also clinically called pathological myopia or degenerative myopia, and the pathological changes of the high myopia mainly include: vitreous degeneration, posterior scleral staphyloma, macular paint cracking, macular hemorrhage, macular Fuchs spot, and the like. Among them, macular hemorrhage is one of the important complications of high myopia, and is clinically more frequent, often resulting in severe damage to central vision.

Central Exudative Chorioretinopathy (CEC), also known as idiopathic choroidal neovascularization, is one of the more common ophthalmic diseases in young and middle-aged people, and is frequently attacked by a single eye, and after the disease occurs, a patient can cause choroidal neovascularization in a macular region (shown as isolated Choroidal Neovascularization (CNV) in the macular region), and the neovascularization region can cause repeated fluid exudation and hemorrhage to cause macular edema, repeated hemorrhage, exudation and scarring, thus the central vision of the patient is seriously affected

In the present invention, "Retinal neo-vascularization (RNV)" is also called Retinal neovascularization. The tissue of retina outer layer without blood vessel between retina deep capillary bed and choroid capillary bed has blood vessel growth, which refers to retina new blood vessel. It is derived from vascular hyperplasia of retinal capillary bed, often accompanied by fibroplasia and degeneration, and can break through the vitreous membrane inwards and proliferate in the vitreous, becoming the most common cause of vitreous hemorrhage.

In the present invention, the "diseases associated with retinal neovascularization" include diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, retinal periphlebitis and the like.

Diabetic Retinopathy (DR), which is one of the most common microvascular complications of diabetes mellitus and is one of the microvascular complications caused by chronic progressive diabetes mellitus, is characterized by the formation of new blood vessels and the destruction of the retinal blood-retinal barrier (BRB), and is a series of fundus diseases caused by the leakage and blockage of retinal microvasculature, such as microangioma, hard exudation, cotton wool spots, new blood vessels, vitreous proliferation, macular edema and even retinal detachment. DR is classified into proliferative diabetic retinopathy and nonproliferative diabetic retinopathy, based on the presence or absence of abnormal neovascularization from the retina as a criterion.

Retinopathy of prematurity (ROP) refers to a low-birth-weight, long-term oxygen-imbibed preterm infant below 36 weeks gestation whose non-vascularized retina develops fibrohemangioma, which proliferates, contracts, and further causes tractional retinal detachment and blindness, a proliferative retinopathy in which the formation of new blood vessels plays a dominant role.

Retinal vein occlusion is a common ocular fundus vascular disease with long course of disease, long-term retinal ischemia, and induction of neovascularization after ischemia.

Periphlebitis of the retina, also known as Eales disease, manifests initially as periphlebitis of the retina, followed by vascular changes and neovascularization due to retinal ischemia, with or without recurrent vitreous hemorrhage as a final common outcome.

In the present invention, the terms "prophylactically and/or therapeutically effective" and "effective amount" refer to an amount of an agent effective to produce a desired pharmacological, therapeutic or prophylactic result. A pharmacologically effective amount results in amelioration of one or more symptoms of the disorder, or prevents progression of the disorder, or causes regression of the disorder, or prevents the disorder.

The therapeutically effective dose in the methods described herein can be determined by the treating physician. For example, a physician may initiate treatment using the manufacturer's recommended dosages of anti-EGFR antibodies and adjust them based on the physician's observations of the effect of the treatment.

Further guidance is provided in the present application and examples. In addition, clinical trials can be conducted to determine dosages effective in producing a statistically significant therapeutic effect when treating a patient population.

Examples

The following detailed description of the preferred embodiments of the invention, while not limiting the invention, will provide further details.

Material sources are as follows:

unless otherwise specified, all materials described in the examples of the present invention were purchased commercially.

Cetuximab: german Merck Ron pharmaceuticals

Trade name: erbitux, specification 100 mg/20 ml/bottle

Or by reference to WO96/40210, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO. 2.

SEQ ID NO.1

>Cetuximab heavy chain

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.2

>Cetuximab light chain

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In the examples described below, concentrations of 10mg/ml, 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml can be obtained by preparation or concentration of the stock solution.

Abbecept injection: purchased from Bayer medicine health promotion Co Ltd, and has a specification of 40 mg/ml/bottle

The compound tropicamide eye drops have the trade name: melidon (Shentian pharmaceutical company)

Brown norway rats (BN rats); step Bei Fu (Beijing) Biotechnology Inc.,

c57BL/6J mice: si Bei Fu (Beijing) Biotechnology Ltd

Example 1 Effect of cetuximab on laser-induced choroidal angiogenesis in rats

1. Experimental methods

Laser photocoagulation at the fundus of brown norwegian rats (BN rats), induces fundus Choroidal Neovascularization (CNV), can create an animal model similar to human Choroidal neovascularization, and therefore, this animal model was created and relevant experiments were performed under this model.

1. Molding die

Rat drops (compound tropicamide eye drops) are fully mydriasis, pentobarbital sodium is used for anesthesia, the head of the rat is fixed in front of an ophthalmological laser photocoagulation instrument, CNV molding is carried out on laser photocoagulation, 6 laser points are irradiated on each eye, and when bubbles are generated on the eyeground, the Bruch's membrane is broken, and the success of photocoagulation is prompted. And molding and detection are carried out on the right eye.

2. Group design

A negative control group (PBS group), an experimental group (cetuximab, JZB group) JZB-5 mg/ml group, and a positive control group (Aflibercept, abbericept injection);

number of animals: groups 4/group, 3 groups 12 in total, all modeling and testing were performed in the right eye of the rat.

3. Administration of drugs

Administration on the day of molding

The administration mode comprises the following steps: vitreous injection

Administration dose: 4 μ l/eye

Dosing frequency: single administration

4. Detection of

Detection time: after FFA detection, namely 7 days (d) and 14 days (d) after molding, 2 times in total;

fluorescent spot grading

Fluorescent spot grading method: level 1: the light spot does not have high fluorescence; stage 2: the light spot is highly fluorescent but has no fluorescein leakage;

and 3, level: the light spot has high fluorescence and slight fluorescein leakage, and the leakage does not exceed the edge of the light spot; 4, level: the spots are highly fluorescent, and fluorescein leakage is significant, and the leakage exceeds the edges of the spots.

Area of neovascular leakage

Detection time: after FFA detection, namely 7d and 14d after molding, 2 times in total;

area of neovascular leak: the leakage area of each new blood vessel is measured, and the total leakage area of each animal is calculated for comparison.

And observing the influence of the test sample on laser-induced rat choroidal neovascularization according to FFA detection, fluorescent spot grading and neovascularization leakage area.

2. Results of the experiment

The rats are administrated on the same day of model building of the CNV, 7d FFA detection is carried out after model building, as shown in figure 1, obvious laser spots and new blood vessel leakage can be seen in a PBS group, and successful establishment of the model is prompted; after 14d FFA detection, as shown in figures 1, 4 and 5, the grading score of the visible laser spots of the PBS group is increased, and the leakage of the new blood vessels is obviously increased, which indicates that the lesion degree caused by the laser is continuously progressed and has an aggravation trend compared with that of the lesion degree caused by the laser at 14d after the model is made and 7 d.

As shown in fig. 2, 4 and 5, at 7d after administration, compared with the PBS group, the grading score of laser spots and the leakage area of new blood vessels of the aflibercept administration group are both obviously reduced, and the difference of aflibercept administration group and PBS group has statistical significance, which indicates that aflibercept has obvious inhibition effect on angiogenesis caused by laser; at 14d after administration, the laser spot grading score of the aflibercept administration group is not statistically different from that of the PBS group, but the leakage area of the new blood vessels is obviously lower than that of the PBS group, the difference has obvious statistical significance, and the aflibercept has obvious inhibition effect on the angiogenesis caused by laser.

As shown in fig. 3, 4 and 5, 7d after administration, the JZB group laser spot grading score and the area of neovascular leakage were slightly decreased compared to the PBS group; at 14d after administration, compared with the PBS group, the JZB laser speckle grading score and the neovascular leakage area are both obviously reduced, which indicates that 14d after administration and JZB09 have obvious inhibition effect on laser-induced angiogenesis.

Example 2 Effect of different concentrations of cetuximab on laser-induced choroidal angiogenesis (CNV) in rats

1. Experimental methods

1. Molding die

Laser photocoagulation on the fundus of a brown Norway rat (BN rat) to induce fundus Choroidal Neovascularization (CNV), an animal model similar to human Choroidal neovascularization can be established, and an experiment of the effect of cetuximab on inhibiting Choroidal neovascularization is carried out under the model.

Making rat dripping pills (compound tropicamide eye drops) fully mydriasis, performing anesthesia by using sodium pentobarbital, and mixing the rat with the solution

Before the head is fixed in front of an ophthalmologic laser photocoagulation instrument, CNV modeling is carried out on the laser photocoagulation, each eye irradiates 6 laser spots, and the success of photocoagulation is prompted by that bubbles are generated on the fundus to prompt that Bruch's membranes are broken. And molding and detection are carried out on the right eye.

2. Group design

A negative control group (PBS group), an experimental group (cetuximab, JZB group) JZB-5 mg/ml group, JZB09-40mg/ml group, JZB09-70mg/ml group, and a positive control group (Aflibercept, abbericept);

number of animals: groups 8/group, 5 groups 40 in total, all modeling and testing were performed in the right eye of the rat.

3. Administration of drugs

7d after molding

The administration mode comprises the following steps: vitreous injection

Administration dose: 4 μ l/eye

The administration frequency is as follows: single administration

4. Detection of

Detection time: after FFA detection, namely 7d after molding (administration of 0 d), 7d, 10d and 14d after administration for 4 times per eye;

fluorescent spot grading

Fluorescent spot grading method: level 1: the light spot has no high fluorescence; and 2, stage: the light spot is highly fluorescent but has no fluorescein leakage;

and 3, level: the light spot has high fluorescence and slight fluorescein leakage, and the leakage does not exceed the edge of the light spot; 4, level: the spots are highly fluorescent, and fluorescein leakage is significant, and the leakage exceeds the edges of the spots.

Area of neovascular leakage

Detection time: after FFA detection, namely 7d after molding (administration of 0 d), 7d, 10d and 14d after administration for 4 times per eye;

area of neovascular leak: the leakage area of each new blood vessel is measured, and the total leakage area of each animal is calculated for comparison.

And observing the influence of the test sample on laser-induced rat choroidal neovascularization according to FFA detection, fluorescent spot grading and neovascularization leakage area.

2. Results of the experiment

Carrying out FFA detection on 7d (after CNV modeling) and 14d (before administration) of rats, wherein after 0d (before administration), 7d (before administration), 10d (before administration) and 14d (after PBS modeling), obvious laser spots and neovascular leakage are observed, and the model is prompted to be successfully established; FFA detection at 14d (administration of 7 d) after modeling, as can be seen from FIGS. 6 and 7, the grading score of the laser spots in the PBS group is increased, and the leakage of the new vessels is increased, which indicates that the regeneration degree of the choroidal vessels caused by the laser is continuously progressed and is in an aggravation trend compared with the 14d and 7d after modeling.

Compared with the PBS group, 7d, 10d and 14d after administration, the laser spot grading score and the neovascular leakage area of the Abebetocept administration group are obviously reduced, and the difference has statistical significance, so that the Abebetocept is prompted to have a more obvious regression effect on laser-induced choroidal neovascularization.

Compared with the PBS group, 7d, 10d and 14d after administration can be seen from FIGS. 6 and 7, the laser spot grading score and the new blood vessel leakage area of the test article JZB administration group are obviously reduced, wherein the fluorescence spot grading and the new blood vessel leakage area difference of the JZB-40 mg/ml dose group and the JZB-70 mg/ml dose group have statistical significance, which indicates that the test article cetuximab-40 mg/ml dose group and cetuximab-70 mg/ml dose group have obvious regression effect on laser-induced choroidal neovascularization.

Example 3 Effect of cetuximab on mouse retinal neovascularization

1. Experimental methods

1. Model (OIR mouse model)

C57BL/6J mice at postnatal day 7 (D7) and lactating mother mice are placed in a closed oxygen box, and the oxygen concentration is maintained at 75% +/-2%; after 5 days of lactation, day 12 (D12) mice and lactating mothers were returned to room standard (oxygen concentration 21%).

2. Group design:

the test group comprises 5PBS negative control groups, abbercept positive control groups and experimental groups (cetuximab, JZB group, JZB09-5mg/ml group, JZB09-40mg/ml group, JZB-70 mg/ml group), wherein 6 mice are arranged in each group.

3. Administration of drugs

The administration time is as follows: the mice were returned to the standard indoor environment (oxygen concentration 21%) and then administered.

The administration mode comprises the following steps: and (4) injecting a vitreous body.

The administration dose is as follows: 1 μ l/eye.

The administration frequency is as follows: single administration.

4. Detection of

At 5 days post-dose, i.e., day 17 after birth, mice were sacrificed and epiocularly retinal slides were removed, stained with IB4, and photographed with a full-automatic upright microscope (Axio imager Z2), as shown in fig. 8.

2. Results of the experiment

As shown in fig. 8, significant avascular and neovascularization were visible after PBS modeling, suggesting successful model establishment.

As shown in fig. 8 and 9, in the case of the albuterol (IVA) administration group, a significant decrease in the avascular area was observed, and no neovascularization was observed, as compared with the PBS (CTRL) group, and the difference was statistically significant, indicating that the albuterol inhibits neovascularization and can reduce the avascular area.

As shown in fig. 8, 9, significant decrease in avascular area and no neovascularization was seen in JZB (IVJ) dose groups compared to PBS (CTRL) group; compared with the aflibercept (IVA) administration group, JZB (IVJ) each dose group had a significant reduction in avascular area, and the difference was statistically significant, suggesting that JZB may have a stronger effect in reducing avascular area than aflibercept.

Comparative example 1 cetuximab was compared with the effects of other tyrosine kinase inhibitors on the avascular zone of the retina and on neovascularization.

Erlotinib is a small molecule inhibitor acting on Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, and inhibits phosphorylation of intracellular tyrosine kinase related to EGFR;

AG1478 ((Tyrphostin AG-1478)) is a selective EGFR tyrosine kinase inhibitor.

Erlotinib, see Hewing N J, weskamp G, vermaat J, et al, intraviral injection of TIMP3 or the EGFR inhibitor erlotinib of the protection from oxygen-induced rettinopathy in mice J. Invest opthalmol Vis,2013,54 (1): 864-870) and figure 3,a-f, administration of the EGFR inhibitor erlotinib inhibited pathological neovascularization in mouse retinas, isolvectin B4 stained retinal slides in wild-type mice injected with erlotinib after oil showed no difference in P17 avascular zone area after oil, erlotinib: the ratio of the retinal area in the avascular zone was 22.0% + -3.6, and the control 19.0% + -1.7, which showed no change before and after administration of the avascular zone area. The new blood vessel cluster is obviously reduced compared with the control group, erlotinib is 1.7% + -0.3, and the control group: 4.6% ± 1.1.

AG1478: see Xin J, yang X, tao Y, et al, egfr inhibitor, AG1478, inhibitors in fluorescence in filtration and angiogenesis in micro with metabolic recovery [ J ]. Clinical and Experimental Pharmacology and Physiology,2019,46 Experimental results and fig a-D, showing no effect on avascular revascularization after administration of AG1478 compared to the untreated pit associated OIR model, i.e. no significant change in avascular area before and after administration; the neovascular clusters were significantly reduced from the control, which occupied about 1.8% of the area of the retina, whereas the control was about 4.4%.

Cetuximab: referring to example 3, the avascular area significantly decreased after cetuximab administration; the ratio of the area of the avascular region of the cetuximab group to the retina is less than 10%, and the area of the avascular region of the PBS group is about 20%; its neovascular cluster is close to 0. Significant new vascular clusters were visible in the PBS group.

In conclusion, administration of anti-EGFR antibody (cetuximab) significantly reduced the area of the retinal avascular region, whereas administration of erlotinib and AG1478 had no effect on the area of the retinal avascular region; for neovascularization, retinal neovascularization after administration of anti-EGFR antibody (cetuximab) was close to 0, whereas erlotinib and AG1478, although administered less than the control group, were 1.7% ± 0.3, 1.8% neovascularization, respectively, and were significantly more effective in inhibiting neovascularization.

SEQUENCE LISTING

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SHANGHAI JINGZE BIOLOGICAL TECHNOLOGY Co.,Ltd.

<120> use of anti-EGFR antibody

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Claims (8)

1. Use of cetuximab in the preparation of a medicament for the prevention and/or treatment of diseases associated with ocular neovascularization.

2. The use according to claim 1, wherein the ocular neovascular disease is ocular fundus neovascular disease.

3. Use according to claim 2, wherein the fundus neovascular-related disease is a choroidal neovascular-related disease and/or a retinal neovascular-related disease.

4. The use according to claim 2, wherein the disease associated with ocular fundus neovascularization is selected from the group consisting of diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, retinal periphlebitis, age-related macular degeneration, high myopia macular hemorrhage and central exudative retinal choroidopathy.

5. Use of cetuximab in the preparation of an ophthalmic medicament for reducing the area of an avascular zone of the retina, inhibiting ocular neovascularization, and/or causing regression of ocular neovascularization.

6. The use according to any one of claims 1-5, wherein the cetuximab is administered to the eye or affected eye in a topical ophthalmic formulation.

7. The use of claim 6, wherein the formulation is administered to the eye or vitreous of the affected eye.

8. Use according to claim 1 or 5, wherein the cetuximab is at a concentration of 5-80mg/ml.

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