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 available 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 in 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 "disease associated with ocular neovascularization" or "disease associated with ocular fundus neovascularization" refers to the occurrence in the eye or fundus, resulting in the destruction of ocular structure and function by neovascularization, causing 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 the retina at the outer layer of the macula, so that angiogenic factors are generated to stimulate choroidal capillary vessel neogenesis and form subretinal neovascularization. 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 cracks, macular hemorrhage, macular Fuchs plaques, 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 proliferation of retinal capillary bed, often accompanied by fibroplasia and degeneration, and can break through the vitreous membrane inwards and proliferate in the vitreous body, 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 premature infant who has received oxygen for a long period of time, with a low birth weight, within 36 weeks of pregnancy, whose non-vascularized retina undergoes fibrohemangioma proliferation, contraction, and further causes tractional retinal detachment and blindness, a proliferative retinopathy, the formation of new blood vessels playing a dominant role in its pathogenesis.
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. The pharmacologically effective amount results in an improvement in one or more symptoms of the disorder, or prevents the disorder from progressing, or causes the disorder to resolve, 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 illustrate the invention.
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 as prepared with reference to WO96/40210, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO.1 and the light chain amino acid sequence is shown in 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 Limited company, with 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); sbeft (beijing) biotechnology limited,
c57BL/6J mice: scibefu (Beijing) Biotechnology Ltd
Example 1 Effect of cetuximab on laser-induced choroidal angiogenesis in rats
First, experiment method
Laser photocoagulation at the fundus of brown norwegian rats (BN rats), inducing fundus Choroidal Neovascularization (CNV), an animal model similar to human Choroidal neovascularization could be established, and therefore, the animal model was established and relevant experiments were performed under the model.
1. Molding die
The rat is dripped into a beautiful eye (compound tropicamide eye drops) to fully mydriasis, pentobarbital sodium is used for anesthesia, the head of the rat is fixed in front of an ophthalmological laser photocoagulator, CNV modeling is carried out on laser photocoagulation, 6 laser points are irradiated on each eye, and the eye fundus is exposed to bubbles to generate to prompt that the Bruch's membrane is broken and the photocoagulation is successful. 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) JZB09-5mg/ml group, and a positive control group (Aflibercept, Abberizep injection);
animal number: 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
The administration frequency is as follows: 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; 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 and 14d after molding, the total time is 2;
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.
Second, experimental results
The rats are dosed on the same day of CNV modeling, 7d FFA is detected after the model building, as shown in figure 1, obvious laser spots and neovascular leakage can be seen in a PBS group, and the model is prompted to be successfully established; 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 laser spot grading score and the neovascular leakage area of the aflibercept administration group were both significantly reduced, and the difference compared with the PBS group had statistical significance, suggesting that aflibercept has a significant inhibitory effect on laser-induced angiogenesis; 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, the score for laser spot grading and area of neovascular leakage were slightly decreased in JZB09 groups compared to PBS group at 7d after administration; compared with the PBS group, the JZB09 laser spot grading score and the neovascular leakage area are obviously reduced at 14d after the administration, which indicates that 14d and JZB09 after the administration have a relatively obvious inhibition effect on laser-induced angiogenesis.
Example 2 Effect of different concentrations of cetuximab on laser-induced choroidal angiogenesis (CNV) in rats
First, experiment method
1. Molding die
Laser photocoagulation is carried out 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 effect experiment 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) JZB09-5mg/ml group, a JZB09-40mg/ml group, a JZB09-70mg/ml group, and a positive control group (Aflibercept);
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 0d), 7d, 10d and 14d after administration for 4 times per eye;
fluorescent spot grading
Fluorescent spot grading method: level 1: the light spot does not have 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 spot is highly fluorescent, with significant fluorescein leakage beyond the spot edge.
Area of neovascular leakage
Detection time: after FFA detection, namely 7d after molding (administration of 0d), 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.
Second, experimental results
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 7d) 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, as can be seen from fig. 6 and 7, the laser spot grading score and the neovascular leakage area of the test article JZB09 administration group are both obviously reduced, wherein the fluorescence spot grading and the neovascular leakage area difference of the JZB09-40mg/ml dose group and the JZB09-70mg/ml dose group have statistical significance, which indicates that the test article cetuximab-40 mg/ml dose group and the cetuximab-70 mg/ml dose group have obvious regression effect on laser-induced choroidal neovascularization.
Example 3 Effect of cetuximab on mouse retinal neovascularization
First, experiment method
1. Modeling (OIR mouse model)
C57BL/6J mice at postnatal day 7 (D7) were placed in a closed oxygen box along with lactating mothers, with oxygen concentration maintained at 75% + -2%; after 5 days of lactation, day 12 (D12) mice and lactating mothers were returned to room standard (21% oxygen concentration).
2. Group design:
the test group comprises a PBS negative control group, an Abbercept positive control group, an experimental group (cetuximab, JZB09 group, JZB09-5mg/ml group, JZB09-40mg/ml group, JZB09-70mg/ml group) and 5 groups in total, wherein 6 animals are selected 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.
Administration dose: 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.
Second, experimental results
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 avascular area was observed, and no neovascularization was observed, compared to the pbs (ctrl) group, and the difference was statistically significant, indicating that the albuterol inhibits neovascularization and reduces the avascular area.
As shown in fig. 8 and 9, significant reduction in avascular area and no neovascularization was seen in JZB09(IVJ) dose groups compared to pbs (ctrl) group; compared with the aflibercept (IVA) administration group, the avascular area of JZB09(IVJ) dose groups was significantly reduced, and the difference was statistically significant, suggesting that JZB09 may have a stronger effect of narrowing the 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 ((tyrphosin 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 oxidative gene-induced degradation in mice J. Invest optoholm Vis,2013,54(1):864-870) and fig. 3, a-f, administration of the EGFR inhibitor erlotinib can inhibit pathological neovascularization of mouse retina, isonectin B4 stained retinal slides of wild-type mice injected with erlotinib after oil show no difference in P17 blood domain 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.
AG 1478: see Xin J, Yang X, Tao Y, et al, egfr inhibitor, AG1478, inhibitors in fluorescence inhibition and angiogenesis in micro with metabolic recovery [ J ]. Clinical and Experimental Pharmacology and Physiology,2019,46 Experimental results and fig a-D, showing that after administration of AG1478 there is no effect on revascularization compared to untreated pit-associated OIR model, i.e. there is no significant change in the area of the avascular region 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 neovasculature, retinal neovascularization after administration of anti-EGFR antibody (cetuximab) was close to 0, while administration of erlotinib and AG1478, although reduced compared to control neovasculature, was 1.7% ± 0.3, 1.8% respectively, and was significantly more effective in inhibiting neovasculature formation of cetuximab.
SEQUENCE LISTING
<110> Jingze biological medicine (fertilizer combination) Co., Ltd
SHANGHAI JINGZE BIOLOGICAL TECHNOLOGY Co.,Ltd.
<120> use of anti-EGFR antibody
<130> CD3503-22P150621
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Cetuximab heavy chain
<400> 1
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
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Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
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Lys
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Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
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Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn
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Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
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Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
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Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
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Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr
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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210