KR101795653B1 - Composition for inhibiting angiogenesis comprising chimeric protein of collagen type II peptide-Aflibercept - Google Patents
Composition for inhibiting angiogenesis comprising chimeric protein of collagen type II peptide-Aflibercept Download PDFInfo
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- KR101795653B1 KR101795653B1 KR1020160061433A KR20160061433A KR101795653B1 KR 101795653 B1 KR101795653 B1 KR 101795653B1 KR 1020160061433 A KR1020160061433 A KR 1020160061433A KR 20160061433 A KR20160061433 A KR 20160061433A KR 101795653 B1 KR101795653 B1 KR 101795653B1
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- chimeric protein
- aflibercept
- peptide
- angiogenesis
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Abstract
Description
본 발명은 콜라겐 타입 II 펩타이드-아플리버셉트(collagen type II peptide-Aflibercept; HYP-GQDGLAGPK-Aflibercept)의 키메라 단백질을 유효성분으로 함유하는 혈관신생 억제용 조성물에 관한 것이다.The present invention relates to a composition for inhibiting angiogenesis, which contains a chimeric protein of collagen type II peptide-Aflibercept (HYP-GQDGLAGPK-Aflibercept) as an active ingredient.
혈관신생(angiogenesis)이란 기존의 미세혈관으로부터 새로운 모세혈관이 형성되는 과정이다. 혈관신생이 정상적으로 일어나는 경우는 배아 발생(embryonic development), 조직재생 및 상처치료, 주기적인 여성의 생식기 계통의 변화인 황체가 발달될 때이며 이러한 경우에도 엄격히 조절되어 진행된다.Angiogenesis is the process by which new capillaries are formed from existing microvessels. The normal occurrence of angiogenesis is when embryonic development, tissue regeneration and wound healing, and development of the luteal system, which is a change in the reproductive system of the periodic woman, are strictly controlled.
성인의 경우, 혈관내피세포는 매우 느리게 자라며, 다른 종류의 세포에 비하여 상대적으로 잘 분열하지 않는다. 혈관신생이 일어나는 과정은 일반적으로 혈관신생 촉진인자의 자극에 의하여 프로테아제로 인한 혈관 기저막의 분해, 혈관 내피세포의 이동, 증식 및 혈관 내피세포 분화에 의한 관강의 형성으로 혈관이 재구성되어 새로운 모세혈관이 생성되는 것으로 이루어진다.In adults, vascular endothelial cells grow very slowly and do not divide relatively well compared to other types of cells. The process of angiogenesis is generally regulated by the stimulation of angiogenesis promoting factors to reconstitute blood vessels by decomposition of blood vessel basement membrane due to protease, migration of vascular endothelial cells, proliferation and formation of vascular endothelial cell differentiation, .
그러나 혈관신생이 자율적으로 조절되지 못하고 병적으로 성장함으로써 야기되는 질환들이 있다. 병리학적 상태에서 나타나는 혈관신생에 관련된 질환으로는 혈관종, 혈관섬유종, 혈관기형 및 심혈관 질환인 동맥경화, 혈관유착, 부종성 경화증이 있고, 혈관신생에 의한 안과 질환으로는 각막이식성 혈관신생, 혈관신생성 녹내장, 당뇨병성 망막증, 신생혈관에 의한 각막 질환, 반점의 변성, 익상편, 망막변성, 후수정체 섬유 증식증, 과립성 결막염 등이 있다.However, there are diseases caused by angiogenesis that can not be controlled autonomously and grows morbidly. The diseases associated with angiogenesis appearing in the pathological state include hemangioma, angiofibroma, vascular anomalies and cardiovascular diseases, arteriosclerosis, vascular adhesion, and edema sclerosis. Ocular diseases caused by angiogenesis include corneal graft angiogenesis, angiogenesis Glaucoma, diabetic retinopathy, corneal diseases caused by neovascularization, denaturation of spots, pterygium, retinal degeneration, posterior capsular hyperplasia, and granular conjunctivitis.
특히, 눈은 가장 혈관이 없는 조직이어서 혈관이 성장하게 된다는 것은 실명을 의미한다. 혈관신생에 의한 안과 질환은 적당한 치료제가 없어 현재 스테로이드나 항생제를 사용하다가 더 진행되면 혈관을 소작(cautery)시키거나 광응혈(photocoagulation) 시키지만 효과가 일시적이며 혈관 증식을 막지 못하므로 재발하게 된다. 따라서 혈관신생을 막아야 근본적인 치료를 할 수 있다.In particular, the eyes are the most vein-free tissues, and blood vessel growth means blindness. Ophthalmologic diseases due to angiogenesis are not suitable treatment, and steroids or antibiotics are used. If they progress further, they cause cautery or photocoagulation of blood vessels, but the effect is temporary and does not prevent vascular proliferation. Therefore, prevention of angiogenesis is essential.
비정상적인 혈관신생으로 인한 질병은 상기 여러 가지 질환의 발병이나 진행과정에 깊이 관련되어 있어 이들 질환의 예방 또는 치료를 위하여 혈관생성을 억제하는 물질을 찾아내려는 연구가 활발히 진행되고 있다.Diseases caused by abnormal angiogenesis are deeply involved in the onset and progress of various diseases, and researches for finding substances that inhibit angiogenesis for the prevention or treatment of these diseases have been actively conducted.
본 발명은 콜라겐 타입 II 펩타이드-아플리버셉트(collagen type II peptide-Aflibercept; HYP-GQDGLAGPK-Aflibercept)의 키메라 단백질을 유효성분으로 함유하는 혈관신생 억제용 조성물을 제공하는데 있다.The present invention provides a composition for inhibiting angiogenesis, which comprises chimeric protein of collagen type II peptide-Aflibercept (HYP-GQDGLAGPK-Aflibercept) as an active ingredient.
상기 목적을 달성하기 위해서, 본 발명은 아플리버셉트(Aflibercept)의 N 말단에 콜라겐 타입 II 펩타이드(collagen type II peptide)가 융합된 키메라 단백질을 제공한다.In order to achieve the above object, the present invention provides a chimeric protein in which a collagen type II peptide is fused to the N-terminus of Aflibercept.
또한, 본 발명은 상기 키메라 단백질을 유효성분으로 함유하는 혈관신생 억제용 약학조성물을 제공한다.The present invention also provides a pharmaceutical composition for inhibiting angiogenesis, comprising the chimeric protein as an active ingredient.
또한, 본 발명은 상기 키메라 단백질을 유효성분으로 함유하는 혈관신생 예방 또는 개선용 건강식품 조성물을 제공한다.The present invention also provides a health food composition for preventing or ameliorating angiogenesis containing the chimeric protein as an active ingredient.
본 발명은 콜라겐 타입 II 펩타이드(collagen type II peptide)-아플리버셉트(Aflibercept)의 키메라 단백질을 유효성분으로 함유하는 혈관신생 억제용 조성물에 관한 것으로서, 아플리버셉트의 N 말단에 콜라겐 타입 II 펩타이드가 융합된 키메라 단백질이 양성 대조군과 비교하여 맥락막 신생혈관 억제에 유의적으로 효과를 나타내는 것을 확인할 수 있었다. 따라서, 상기 키메라 단백질은 안구 신생혈관 질환, 특히 황반변성 질환의 치료에 유용하게 활용될 수 있을 것으로 예상된다.The present invention relates to a composition for inhibiting angiogenesis, which comprises chimeric protein of collagen type II peptide (Aflibercept) as an active ingredient, wherein a collagen type II peptide is added at the N terminus of the influenzacept It was confirmed that the fusion chimeric protein had a significant effect on the inhibition of choroidal neovascularization as compared with the positive control. Thus, the chimeric protein is expected to be useful for the treatment of ocular neovascular diseases, particularly macular degeneration diseases.
도 1은 키메라 단백질(HYP-GQDGLAGPK-Aflibercept) 생산 과정을 나타낸다.
도 2는 키메라 단백질(HYP-GQDGLAGPK-Aflibercept)의 서열 분석 결과를 나타낸다.
도 3은 키메라 단백질(HYP-GQDGLAGPK-Aflibercept)의 발현율과 정제 결과를 나타낸다.
도 4는 Tube formation assay를 통한 키메라 단백질(HYP-GQDGLAGPK-Aflibercept; C-A)의 혈관형성 저해 효능을 나타낸다.
도 5는 맥락막 신생혈관 억제에 미치는 키메라 단백질(HYP-GQDGLAGPK-Aflibercept)의 영향을 나타낸다. A+C: 아플리버셉트(Aflibercept; 0.5 μg) 및 콜라겐 타입 II 펩타이드(HYP-GQDGLAGPK; 5 μg) 혼합물, C-A: 키메라 단백질(HYP-GQDGLAGPK-Aflibercept; 0.2 μg).Figure 1 shows the production process of chimeric protein (HYP-GQDGLAGPK-Aflibercept).
Fig. 2 shows the result of sequencing of the chimeric protein (HYP-GQDGLAGPK-Aflibercept).
Fig. 3 shows the expression rate and purification result of chimeric protein (HYP-GQDGLAGPK-Aflibercept).
Figure 4 shows the antiangiogenic effect of chimeric protein (HYP-GQDGLAGPK-Aflibercept; CA) through the Tube formation assay.
FIG. 5 shows the effect of chimeric protein (HYP-GQDGLAGPK-Aflibercept) on the inhibition of choroidal neovascularization. A + C: mixture of Aflibercept (0.5 μg) and collagen type II peptide (HYP-GQDGLAGPK; 5 μg), CA: chimeric protein (HYP-GQDGLAGPK-Aflibercept;
본 발명은 아플리버셉트(Aflibercept)의 N 말단에 콜라겐 타입 II 펩타이드(collagen type II peptide)가 융합된 키메라 단백질을 제공한다.The present invention provides a chimeric protein in which a collagen type II peptide is fused to the N-terminus of Aflibercept.
상세하게는, 상기 콜라겐 타입 II 펩타이드(collagen type II peptide)는 서열번호 1의 아미노산 서열로 표시될 수 있으나, 이에 제한되는 것은 아니다. 상기 서열번호 1의 아미노산 서열은 "HYP-GQDGLAGPK"의 10개의 아미노산로 이루어져 있으며, "HYP"는 하이드록시 프롤린(Hydroxy proline)을 의미한다.In detail, the collagen type II peptide may be represented by the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. The amino acid sequence of SEQ ID NO: 1 is composed of 10 amino acids of "HYP-GQDGLAGPK", and "HYP" means hydroxy proline.
상세하게는, 상기 콜라겐 타입 II 펩타이드의 C-말단에는 링커(linker)가 결합될 수 있으나, 이에 제한되는 것은 아니다.Specifically, a linker may be bound to the C-terminus of the collagen type II peptide, but the present invention is not limited thereto.
본 발명에 있어서, "링커(linker)"란 기본적으로는 두 개의 서로 다른 융합 파트너 (예를 들어, 생물학적 고분자 등)를 수소 결합, 정전기적 상호작용, 반데르 바알스력, 이황화 결합, 염 브릿지, 소수성 상호작용, 공유결합 등을 이용하여 연결할 수 있는 연결체를 의미하는데, 구체적으로는 생리학적 조건 또는 다른 표준 펩타이드 조건(예를 들면, 펩타이드 정제 조건, 펩타이드 저장 조건)하에서 적어도 하나의 이황화 결합에 참여할 수 있는 적어도 하나의 시스테인을 가질 수 있으며, 단순히 각각의 융합 파트너를 연결하는 역할 이외에도, 융합파트너 사이에 일정한 크기의 간격을 부여하는 역할을 수행하거나 또는 융합체에 유연성 또는 강직성을 제공하는 힌지(hinge)의 역할을 수행할 수 있다. 상기 링커는 비펩타이드 링커 또는 펩타이드 링커일 수 있으며, 펩티드 결합, 이황화 결합 등에 의해서 직접 연결되는 것도 모두 포함될 수 있다.In the present invention, the term "linker" basically refers to two different fusion partners (e.g., biological polymers, etc.) with hydrogen bonds, electrostatic interactions, van der Waals forces, disulfide bonds, Hydrophobic interaction, covalent bond, etc. Specifically, it is preferable to use at least one disulfide bond under physiological conditions or other standard peptide conditions (for example, peptide purification conditions, peptide storage conditions) In addition to simply joining the respective fusion partners, it is also possible to have at least one cysteine capable of participating in the fusion of the fusion partner with a hinge that provides a gap of a certain size between the fusion partners or provides flexibility or rigidity to the fusion body. ) Can play the role of. The linker may be a non-peptide linker or a peptide linker, and may be directly linked by a peptide bond, a disulfide bond or the like.
또한, 본 발명은 상기 키메라 단백질을 유효성분으로 함유하는 혈관신생 억제용 약학조성물을 제공한다.The present invention also provides a pharmaceutical composition for inhibiting angiogenesis, comprising the chimeric protein as an active ingredient.
상세하게는, 상기 혈관신생은 안구의 혈관신생일 수 있으나, 이에 제한되는 것은 아니다. Specifically, the angiogenesis may be angiogenesis of the eye, but is not limited thereto.
상세하게는, 상기 약학조성물은 황반변성, 망막정맥 폐쇄증, 당뇨병성 망막증 및 허혈성 망막증으로 이루어진 군에서 선택되는 질환을 예방 또는 치료할 수 있으나, 이에 제한되는 것은 아니다. Specifically, the pharmaceutical composition may prevent or treat a disease selected from the group consisting of macular degeneration, retinal vein occlusion, diabetic retinopathy and ischemic retinopathy, but is not limited thereto.
본 발명의 약학조성물은 약학조성물 총 100 중량부에 대하여, 상기 키메라 단백질(HYP-GQDGLAGPK-Aflibercept)을 0.01 내지 90 중량부로 함유할 수 있으나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may contain 0.01 to 90 parts by weight of the chimeric protein (HYP-GQDGLAGPK-Aflibercept) per 100 parts by weight of the total amount of the pharmaceutical composition, but is not limited thereto.
본 발명의 한 구체예에서, 상기 약학조성물은 통상적인 방법에 따라 점안제, 주사제, 과립제, 정제, 환제, 캡슐제, 겔, 시럽, 현탁제, 유제, 점적제 및 액제로 이루어진 군에서 선택된 어느 하나의 제형을 사용할 수 있다. In one embodiment of the present invention, the pharmaceutical composition may be administered orally in the form of drops, injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions, Can be used.
본 발명의 다른 구체예에서, 상기 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 윤활제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다.In another embodiment of the present invention, the pharmaceutical compositions may be formulated with suitable carriers, excipients, disintegrants, sweeteners, coatings, swelling agents, lubricants, lubricants, flavors, antioxidants, buffers, , A diluent, a dispersant, a surfactant, a binder, and a lubricant.
구체적으로 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.
상기 약학조성물의 바람직한 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다. The preferred dosage of the pharmaceutical composition may vary depending on the condition and body weight of the subject, the type and degree of disease, the drug form, the administration route and the period, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.
본 발명에 있어서, 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나, 이들 예에 한정되는 것은 아니다.In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.
또한, 본 발명은 상기 키메라 단백질을 유효성분으로 함유하는 혈관신생 예방 또는 개선용 건강식품 조성물을 제공한다.The present invention also provides a health food composition for preventing or ameliorating angiogenesis containing the chimeric protein as an active ingredient.
상기 건강식품 조성물은 분말, 과립, 정제, 캡슐, 시럽, 음료 또는 환의 형태로 제공될 수 있으며, 상기 건강식품 조성물은 유효성분인 본 발명에 따른 키메라 단백질 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health food composition may be provided in the form of a powder, a granule, a tablet, a capsule, a syrup, a beverage or a ring. The health food composition is used together with food or food additives other than the chimeric protein according to the present invention, Can be suitably used according to the method of The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.
상기 건강식품조성물에 함유된 키메라 단백질의 유효용량은 상기 약학조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the chimeric protein contained in the health food composition may be used in accordance with the effective dose of the pharmaceutical composition but may be less than the above range for the purpose of health and hygiene or for long- And it is clear that the active ingredient can be used in an amount exceeding the above range since there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다.There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
하기의 실험예는 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다.The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.
<< 실험예Experimental Example > >
1. One. 키메라chimera 단백질 합성 Protein synthesis
(1) 세포준비(1) preparation of cells
1) 293F cell stock 1 vial을 37℃에서 녹인 후, 50 ml 튜브에 40 ml Freesytle 293 배지(Gibco, 12338)에 첨가하였다.1)
2) 50 ml 튜브는 2000 rpm에서 5분간 원심분리기(Hanil, FLIF1922070)로 원심분리하여 배지를 제거한 뒤 125 ml 플라스크(Nalgene, 4115-0125)에 40 ml의 Freestyle 293 배지(Gibco, 12338)를 채우고 세포를 넣어주었다.2) The 50 ml tube was centrifuged at 2000 rpm for 5 minutes with a centrifuge (Hanil, FLIF1922070) to remove the medium and then filled in a 125 ml flask (Nalgene, 4115-0125) with 40 ml Freestyle 293 medium (Gibco, 12338) Cells were added.
3) 3일 배양(배양 조건: CO2 8%, 습도 60%, 85 rpm) 후 LUNA cell counter (Logos biosystem, L10001)를 이용하여 생존능(viability) 및 살아있는 세포를 측정하여 5×105 cells/ml을 150 ml 배양액이 들어 있는 500 ml 플라스크에서 배양하였다.3) 3-day culture (Culture Conditions: CO 2 8%, 60% humidity, then 85 rpm) to measure viability (viability) and living cells by using a LUNA cell counter (Logos biosystem, L10001 ) 5 × 10 5 cells / ml were cultured in a 500 ml flask containing 150 ml culture medium.
(2) 본 생산(2) Production
1) 항원 배양액 대량 생산 시 형질감염(transfection) 시킬 293F 세포의 조건은 각각 1×106 cells/ml의 농도와 95% 이상의 세포 생존능(cell viability)에서 실시하였으며, 예비발현 생산을 위해 40 ml 배양을 하였다. 1) 293F cells transfected at the time of mass production of the antigen were cultured at a concentration of 1 × 10 6 cells / ml and cell viability of 95% or more, respectively. For the production of preliminary expression, 40 ml culture Respectively.
2) LUNA cell counter를 이용하여 생존능(viability) 및 살아있는 세포를 측정하여 death phase에 넘어가지 않도록 확인하였다. 2) viability and living cells were measured using a LUNA cell counter to confirm that they did not fall into the death phase.
3) 형질감염(transfection) 전날 LUNA를 이용하여 세포수를 측정한 후 40 ml 기준 5×105 cells/ml을 50 ml 튜브에 옮겨 담은 후, 2000 rpm 에서 5분간 원심분리 하였다. 상층액은 제거 후 40ml의 fresh freestyle 293 배지가 들어있는 125ml 플라스크에 재현탁(resuspension)하여 24시간 배양 전후 최종 1×106 cells/ml의 세포수가 되도록 하였다. 3) On the day before transfection, the cells were counted using LUNA. Then, 5 × 10 5 cells / ml of 40 ml was transferred into a 50 ml tube and centrifuged at 2000 rpm for 5 minutes. After removing the supernatant, the supernatant was resuspended in a 125 ml flask containing 40 ml of fresh freestyle 293 medium to allow final cell count to be 1 × 10 6 cells / ml after 24 hours of culture.
4) Polyplex를 만들기 위한 반응액은 40 ml 형질감염(transfection) 기준 1.2 ml의 볼륨으로 실시하였다. 먼저 freestyle 293 배지 600 ul을 2 개의 epp. 튜브에 각각 담고 epp. 튜브당 각각 항원의 DNA 25ug과 PEI (Polysciences, 23966) 100μg을 넣은 후 약 10초간 혼합(vortex) 하였다. 이후 두 튜브를 섞어 준 후 5~10초 간 2번 약하게 혼합(vortex) 하였다. 그런 다음 15~20분 동안 상온에서 반응시킨 후 인버팅하고 세포에 넣어 주었다.4) The reaction solution for making the polyplex was prepared in a volume of 1.2 ml based on 40 ml transfection. First, 600 μl of freestyle 293 medium was added to 2 epp. Respectively. Each tube contained 25 ug of antigen and 100 μg of PEI (Polysciences, 23966), followed by vortexing for about 10 seconds. After mixing the two tubes, they were vortexed 2 times for 5-10 seconds. Then, the reaction was carried out at room temperature for 15 to 20 minutes, then inverted and added to the cells.
5) 형질감염(Transfection) 후 배양 (배양 조건: CO2 8%, 습도 60%, 85 rpm) 6일 경과 후 70% 정도의 생존능(viability)을 확인하였을 때 배양을 정지시키고 세포 제거 단계로 들어갔다(도 1).5) Culture after transfection (culture condition: 8% CO 2 , humidity 60%, 85 rpm) After confirming the viability of about 70% after 6 days, the culture was stopped and the cell was removed (Fig. 1).
(3) 항체 정제(3) Antibody purification
1) 6일 동안 키운 배양액 중 소량은 (20 μl) 발현율을 보기 위해 먼저 웨스턴 블롯을 하였다.1) A small amount (20 μl) of the culture medium grown for 6 days was subjected to western blotting first.
2) 웨스턴 블롯에 이어 배양액을 정제하기 위해 배양액을 8,000 rpm에서 10분간 원심분리를 통하여 세포잔해를 제거해 주고, 배양액을 0.22 μm pore size의 제균필터(Millipore, SCGPT05RE)를 이용하여 필터 해 주었다. 정제를 수행하기 위하여 1개의 칼럼에 protein A sepharose resin slurry(GE Healthcare Life Sciences, Cat. No. 17-1279-04)를 넣어 준 후 100 ml의 DPBS (welgene, LB001)로 레진을 packing하고 세척(washing)을 실시하였다. 2) In order to purify the culture solution after Western blotting, the culture solution was centrifuged at 8,000 rpm for 10 minutes to remove cell debris. The culture solution was filtered using a 0.22 μm pore size filter (Millipore, SCGPT05RE). To perform the purification, the resin was packed with 100 ml of DPBS (welgene, LB001), and washed with 1 ml of protein A sepharose resin slurry (GE Healthcare Life Sciences, Cat. No. 17-1279-04) washing.
3) Packing 된 레진에 필터 된 배지를 걸어 주어 1분당 1 ml로 흘려 주었다(Bio-Rad, EP-1 Econo pump, USA).3) Filtered media was applied to the packed resin and flowed to 1 ml per minute (Bio-Rad, EP-1 Econo pump, USA).
4) 150 ml의 DPBS로 세척(washing)을 수행한 후, 20 ml의 0.1 M glycine-HCl (pH 2.5)(Sigma, 27185-0350)으로 추출하였다. 추출액은 1M Tris-HCl (pH 9.0)(Sigma, TB0194)을 10% 첨가하여 pH를 중화시키고, Amicon Ultra-15(Millipore, UFC901096)를 이용하여 PBS로 버퍼를 바꾸어 주었다. 4) After washing with 150 ml of DPBS, the cells were extracted with 20 ml of 0.1 M glycine-HCl (pH 2.5) (Sigma, 27185-0350). The extract was buffered with PBS using Amicon Ultra-15 (Millipore, UFC901096) by adding 10% 1 M Tris-HCl (pH 9.0) (Sigma, TB0194) to neutralize the pH.
5) 최종 단백질의 정량은 Nano-drop(Nanodrop, ND1000)으로 A280nm에서 수행하였다.5) Quantification of the final protein was performed at A280nm with Nano-drop (Nanodrop, ND1000).
2. 혈관내피세포를 이용한 혈관 형성 억제 효능 평가2. Assessment of angiogenesis inhibition efficacy using vascular endothelial cells
키메라 단백질(HYP-GQDGLAGPK-Aflibercept)의 항혈관형성 효능은 사람망막혈관내피세포(HRMEC)를 이용한 Tubing assay를 통하여 확인하였다. HRMEC 세포를 calcein-AM으로 염색한 후 Matrigel이 코팅된 48-well plate에 seeding 하고, 20 ng/ml 농도의 recombinant human VEGF와 함께 키메라 단백질을 100 ng/ml의 농도로 처리하였다. HRMEC 세포를 37℃ 인큐베이터에서 4시간 방치한 후 형광현미경 (Leica)으로 Tube formation을 관찰하였다.The antiangiogenic effect of chimeric protein (HYP-GQDGLAGPK-Aflibercept) was confirmed by tubing assay using human retinal vascular endothelial cells (HRMEC). HRMEC cells were stained with calcein-AM, seeded on a Matrigel-coated 48-well plate, and treated with recombinant human VEGF at a concentration of 20 ng / ml at a concentration of 100 ng / ml. HRMEC cells were left in a 37 ° C incubator for 4 hours and observed for fluorescence microscopy (Leica).
3. 실험동물 및 맥락막 신생혈관 (3. Experimental Animals and Choroidal Neovascularization CNVCNV ) 모델 제작Model building
C57BL/6 생쥐는 오리엔트 바이오에서 구입하였다. 본 동물 실험은 눈과 시력 연구를 위한 동물 사용에 대하여 인제대학교 의과대학 (No,2013-053)과 ARVO에 승인된 지침에 따라 수행되었다. 6주령의 C57BL/6 생쥐에게 diode green laser (532 nm, 240 mW, 0.1sec, 50 μM, photocoagulator)를 이용하여 망막 시신경 부위에 손상을 주었다. Laser 조사 직후 키메라 단백질(HYP-GQDGLAGPK-Aflibercept), 아플리버셉트(Aflibercept), 콜라겐 타입 II 펩타이드(collagen type II peptide) 및 양성대조군 Avastin을 PBS에 용해하여 하루에 0.2 μg씩 3일간 안구 내에 투여하였다. 각 실험군은 각각 5마리씩 생쥐의 양쪽 눈을 이용하여 상기 실험을 진행하였다.C57BL / 6 mice were purchased from Orient Bio. This animal study was conducted in accordance with the guidelines approved by the University of Inje (No, 2013-053) and ARVO for animal use for eye and vision research. At 6 weeks of age, C57BL / 6 mice were exposed to diode green laser (532 nm, 240 mW, 0.1 sec, 50 μM, photocoagulator) to damage the retinal optic nerve. Immediately after laser irradiation, chimeric proteins (HYP-GQDGLAGPK-Aflibercept), Aflibercept, collagen type II peptide and positive control Avastin were dissolved in PBS and injected into the eye for 3 days in 0.2 μg per day . The experiment was carried out using 5 eyes of each mouse in each experimental group.
4. 망막-맥락막 Flat-mount4. Retinal-choroidal Flat-mount
Laser 조사 14일 후 망막-맥락막 Flat-mount를 통하여 키메라 단백질의 CNV 병변 크기 억제 효능을 평가하였다. 마우스를 마취하고 25 mg/ml의 FITC-dextran을 retro-orbital로 100 μl 주사하였다. 30분 후 마우스를 안락사하고, 안구를 적출하여 10% 포르말린으로 고정한 후 각막과 수정체를 제거하고 Cover glass에 Flat mount 하였다. FITC-dextran에 의해 염색된 신생혈관은 형광 현미경 (Leica)을 통해 관찰하고 그 병변 크기를 Image J program을 통하여 측정하였다.After 14 days of laser irradiation, chimeric proteins were evaluated for the inhibition of CNV lesion size by retinal-choroidal flat-mount. Mice were anesthetized and injected with 100 μl of retro-orbital at 25 mg / ml of FITC-dextran. After 30 minutes, the mouse was euthanized, and the eyeballs were removed and fixed with 10% formalin. Then, the cornea and lens were removed and flat mount on the cover glass. The neovasculature stained with FITC-dextran was observed by fluorescence microscopy (Leica) and the lesion size was measured by Image J program.
< 실시예 1> 콜라겐 타입 II 펩타이드 (collagen type II peptide)와 아플리버셉트(Aflibercept)의 합성 <Example 1> Synthesis of collagen type II peptides (collagen type II peptide) and hurt River septeu (Aflibercept)
아플리버셉트(Aflibercept)의 N 말단(terminal)에 콜라겐 타입 II 펩타이드(collagen type II peptide; HYP-GQDGLAGPK)를 융합(fusion) 시키고, 서열(Sequence)을 확인하였다(도 2). 다음으로, 웨스턴 블롯을 통하여 발현율을 비교하였다. 콜라겐 타입 II 펩타이드(collagen type II peptide)를 아플리버셉트(Aflibercept)의 N-말단에 융합시켰을 때는 23 mg/L 였다(도 3). A collagen type II peptide (HYP-GQDGLAGPK) was fused to the N terminus of Aflibercept to confirm the sequence (Fig. 2). Next, expression rates were compared by Western blotting. When the collagen type II peptide was fused to the N-terminus of Aflibercept, it was 23 mg / L (Fig. 3).
<< 실시예Example 2> 2> 키메라chimera 단백질의 Protein in vitroin vitro 혈관형성 억제 효과 Anti-angiogenic effect
VEGF 처리군은 VEGF 비처리군에 비하여 Tube formation을 상승시켰으나, 키메라 단백질(HYP-GQDGLAGPK-Aflibercept)은 혈관형성을 억제하였으며 VEGF 비처리군의 혈관형성 정도와 거의 동일하였다(도 4).The VEGF-treated group increased Tube formation compared to the VEGF-untreated group, but the chimeric protein (HYP-GQDGLAGPK-Aflibercept) inhibited angiogenesis and was almost identical to that of VEGF-untreated group (FIG.
<< 실시예Example 3> 맥락막 신생혈관 억제에 미치는 3> on the inhibition of choroidal neovascularization 키메라chimera 단백질의 영향 Effect of protein
In vitro Tube formation 실험에서 뛰어난 항혈관형성능을 보인 키메라 단백질(HYP-GQDGLAGPK-Aflibercept)과 양성대조군 Avastin과의 효능 비교를 위하여 laser 조사 14일 후 혈관을 FITC-dextran으로 염색하고 Flat-mount를 통하여 CNV 병변 크기를 측정하였다. 0.2 μg씩 3일 연속 안구내 주사를 하였으며, 키메라 단백질의 효능 비교를 위하여 아플리버셉트(Aflibercept)와 콜라겐 타입 II 펩타이드(collagen type II peptide) 혼합물을 대조군으로 함께 처리하였다. 양성대조군으로는 Avastin을 사용하였다. 키메라 단백질(collagen II peptide-Aflibercept)은 Avastin 처리군 및 Aflibercept와 collagen type II peptide 혼합물 처리군에 비하여 CNV 병변크기 감소에 더 뛰어난 효능을 보였다(도 5). In order to compare the efficacy of chimeric protein (HYP-GQDGLAGPK-Aflibercept) which showed excellent anti-angiogenic ability in in vitro Tube formation experiment and avastin positive control group, 14 days after laser irradiation, blood vessels were stained with FITC-dextran, The lesion size was measured. 0.2 μg injected intraocularly for 3 consecutive days. A mixture of Aflibercept and collagen type II peptide was treated together as a control for comparison of chimeric protein efficacy. Avastin was used as a positive control. The chimeric protein (collagen II peptide-Aflibercept) showed greater efficacy in decreasing CNV lesion size than the Avastin treated group and the Aflibercept and collagen type II peptide treated group (FIG. 5).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> INJE UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION
<120> Composition for inhibiting angiogenesis comprising chimeric
protein of collagen type II peptide-Aflibercept
<130> ADP-2016-0193
<160> 1
<170> KopatentIn 2.0
<210> 1
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Hydroxy proline-GQDGLAGPK
<400> 1
Pro Gly Gln Asp Gly Leu Ala Gly Pro Lys
1 5 10
<110> INJE UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION
<120> Composition for inhibiting angiogenesis comprising chimeric
protein of collagen type II peptide-Aflibercept
<130> ADP-2016-0193
<160> 1
<170> Kopatentin 2.0
<210> 1
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Hydroxy proline-GQDGLAGPK
<400> 1
Pro Gly Gln Asp Gly Leu Ala
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| WO2020099925A3 (en) * | 2018-11-14 | 2020-06-25 | Yuyu Pharma, Inc. | Peptides and pharmaceutical compositions for treating eye diseases |
| JP2020520372A (en) * | 2017-05-17 | 2020-07-09 | ユーユー ファーマ，インコーポレーテッド | Novel peptide and pharmaceutical composition comprising the novel peptide as an active ingredient for the treatment of eye diseases |
| WO2021191689A3 (en) * | 2020-03-05 | 2021-12-23 | Yuyu Pharma, Inc. | Treatment of inflammatory diseases with peptides and pharmaceutical compositions |
| WO2022223140A1 (en) * | 2021-04-23 | 2022-10-27 | Navigo Proteins Gmbh | Fusion proteins with specifity for type ii collagen and vegf-a for the treatment of eye diseases |
| US11613558B2 (en) | 2018-11-14 | 2023-03-28 | Yuyu Pharma, Inc. | Peptides and pharmaceutical compositions for treating eye diseases |
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| US20140179621A1 (en) | 2009-05-01 | 2014-06-26 | Ophthotech Corporation | Methods for Treating or Preventing Ophthalmological Diseases |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2020520372A (en) * | 2017-05-17 | 2020-07-09 | ユーユー ファーマ，インコーポレーテッド | Novel peptide and pharmaceutical composition comprising the novel peptide as an active ingredient for the treatment of eye diseases |
| WO2020099925A3 (en) * | 2018-11-14 | 2020-06-25 | Yuyu Pharma, Inc. | Peptides and pharmaceutical compositions for treating eye diseases |
| US11613558B2 (en) | 2018-11-14 | 2023-03-28 | Yuyu Pharma, Inc. | Peptides and pharmaceutical compositions for treating eye diseases |
| WO2021191689A3 (en) * | 2020-03-05 | 2021-12-23 | Yuyu Pharma, Inc. | Treatment of inflammatory diseases with peptides and pharmaceutical compositions |
| WO2022223140A1 (en) * | 2021-04-23 | 2022-10-27 | Navigo Proteins Gmbh | Fusion proteins with specifity for type ii collagen and vegf-a for the treatment of eye diseases |
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