KR102668479B1 - Combined Biomarker Measures for Fibrosis - Google Patents
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- KR102668479B1 KR102668479B1 KR1020187023013A KR20187023013A KR102668479B1 KR 102668479 B1 KR102668479 B1 KR 102668479B1 KR 1020187023013 A KR1020187023013 A KR 1020187023013A KR 20187023013 A KR20187023013 A KR 20187023013A KR 102668479 B1 KR102668479 B1 KR 102668479B1
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Abstract
본 발명은 N-프로테아제에 의한 무손상 타입 III 프로콜라겐의 절단에 의해 형성된 PIIINP의 C-말단 네오-에피토프를 각각 가지며 가닥간 가교에 의해 결합된 PIIINP 가닥 2개 이상을 포함하는, 가교된 PIIINP를 검출하기 위한 샌드위치 면역분석방법을 제공한다. 가교된 PIIINP를 포함하는 생물학적 샘플을, 표면에 결합된 제1 단일클론 항체와 접촉시킨 다음 제2 단일클론 항체와 접촉시키고, 제2 단일클론 항체의 결합을 측정하는 단계를 포함하며, 이들 항체 둘다 PIIINP의 C-말단 서열의 네오-에피토프에 특이적으로 반응한다. 또한, 본 발명은 면역분석을 통해 라이실 산화효소를 타겟팅하는 길항제 약물의 효능을 평가하는 방법 및 제1 단일클론 항체가 결합된 고체 지지체 및 제2 단일클론 항체를 포함하는 키트를 제공한다.The present invention provides a cross-linked PIIINP comprising at least two strands of PIIINP, each having a C-terminal neo-epitope of PIIINP formed by cleavage of intact type III procollagen by N-protease, and joined by interstrand cross-linking. A sandwich immunoassay method for detection is provided. contacting a biological sample comprising cross-linked PIIINP with a first monoclonal antibody bound to a surface and then with a second monoclonal antibody, and measuring binding of the second monoclonal antibody, both antibodies It specifically reacts to the neo-epitope of the C-terminal sequence of PIIINP. Additionally, the present invention provides a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidase through immunoassay and a kit comprising a solid support bound to a first monoclonal antibody and a second monoclonal antibody.
Description
본 발명은 생물학적 샘플에서 가교된 PIIINP를 검출하기 위한 샌드위치 면역분석방법 및 이것을 이용하여 라이실 산화효소 (LOX)를 타겟팅하는 약물의 효능을 평가하기 위한 방법에 관한 것이다. 또한, 본 발명은 샌드위치 면역분석을 수행하기 위한 키트에 관한 것이다.The present invention relates to a sandwich immunoassay method for detecting cross-linked PIIINP in biological samples and a method for evaluating the efficacy of a drug targeting lysyl oxidase (LOX) using the same. Additionally, the present invention relates to a kit for performing a sandwich immunoassay.
섬유증 질환 (표 1에 열거된 질병 포함)은 이환율 및 사망률을 높이는 주 요인으로서, 예를 들어, 간경변의 경우 전세계적으로 해마다 사망자가 800,000명에 달한다 (1).Fibrotic diseases (including those listed in Table 1) are a major cause of morbidity and mortality, with cirrhosis, for example, causing up to 800,000 deaths annually worldwide (1).
표 1. 여러가지 섬유증 질환 (2).Table 1. Various fibrotic diseases (2).
주혈흡충증
지방간염 (알코올성 또는 비-알코올성)viral hepatitis
schistosomiasis
Steatohepatitis (alcoholic or non-alcoholic)
전신성 경화증 (경피증)Idiopathic Pulmonary Fibrosis (IPF)
Systemic sclerosis (scleroderma)
당뇨병
비-치료 고혈압Nephrogenic systemic fibrosis (NSF)
diabetes
Untreated high blood pressure
고혈압
죽상동맥경화증
재협착증heart attack
High blood pressure
atherosclerosis
restenosis
NFSSystemic sclerosis and scleroderma, keloids, hypertrophic scars, burns, hereditary factors
NFS
'섬유증 질환'은 주 증상으로서 또는 부 증상으로서 섬유형성을 유발하는 모든 질환이다. 섬유증은 지속 감염, 자가면역 반응, 알레르기 반응, 화학적 자극 (chemical insult), 방사선 및 조직 손상을 비롯한 다양한 자극들에 의해 유발되는 만성 염증성 반응의 최종 결과이다. 섬유증은 세포외 기질 (ECM)의 축적과 재조직화가 특징적이다. 명백한 병인적 및 임상적 차이가 있음에도 불구하고, 대부분의 만성 섬유증 장애는 공통적으로 성장인자, 단백질분해성 효소, 혈관신생 인자, 및 섬유증식성 사이토카인의 생산을 지속시키는 지속적인 자극원을 가지고 있으며, 이들 자극원은 또한 결합 조직 인자, 특히 콜라겐 및 프로테오글리칸의 침착을 자극하여, 점진적으로 정상적인 조직 구조를 리모델링하고 파괴한다 (3,4). 인간 건강에 대한 섬유증의 상당한 영향력에도 불구하고, 섬유형성 기전을 직접 타겟팅하는 치료제는 현재 승인된 바 없다 (5).'Fibrotic disease' is any disease that causes fibrosis as a main symptom or as a secondary symptom. Fibrosis is the end result of a chronic inflammatory response triggered by a variety of stimuli, including persistent infections, autoimmune reactions, allergic reactions, chemical insults, radiation and tissue damage. Fibrosis is characterized by accumulation and reorganization of extracellular matrix (ECM). Despite apparent etiological and clinical differences, most chronic fibrotic disorders have in common a persistent stimulus that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and profibroproliferative cytokines. Circles also stimulate the deposition of connective tissue factors, particularly collagen and proteoglycans, which gradually remodel and destroy normal tissue architecture (3,4). Despite the significant impact of fibrosis on human health, no therapeutic agents that directly target fibrogenic mechanisms are currently approved (5).
세포외extracellular 기질 (ECM) Matrix (ECM)
ECM은 단백질의 응집체를 형성할 수 있는 능력을 가진 초분자 구조로서, 세포를 3차원 네트워크로 연결하는 다이나믹 스캐폴드를 형성한다. 이 스캐폴드는 프로테아제의 상향 및 하향 조절을 통해 세포-기질 상호작용과 세포 운명을 조절한다 (6). ECM은, 콜라겐, 라미닌, 프로테오글리칸 및 기타 당단백질들의, 다양한 함량 및 조합으로 구성되어, 다양한 생물학적 컴포넌트를 제공하며, 이 컴포넌트들은 개개 조직의 요구를 충족시키는 특이적인 기능을 가진 스캐폴드를 구축하기 위해 프로테아제에 의해 변형될 수 있다 (7).ECM is a supramolecular structure with the ability to form protein aggregates, forming a dynamic scaffold that connects cells into a three-dimensional network. This scaffold regulates cell-matrix interactions and cell fate through up- and down-regulation of proteases (6). The ECM, composed of collagen, laminin, proteoglycans, and other glycoproteins, in varying amounts and combinations, provides a variety of biological components to build scaffolds with specific functions to meet the needs of individual tissues. It can be modified by proteases (7).
콜라겐 타입 I 및 III는 인체의 주요 구조 단백질이다. 콜라겐 타입 III는 심혈관계와 기타 장기에서 콜라겐 타입 I 원섬유 형성에 필수적이다 (8,9). 원섬유의 조립 중에, (동일한 α-체인 3개로 구성되며, 총 분자량이 42 kDa인) 타입 III 프로콜라겐의 N-말단 프로펩타이드는, 성숙 콜라겐이 ECM에 통합되기 전에, 특이적인 N-프로테아제에 의해 절단된다. 절단된 프로펩타이드는 ECM에서 유지되거나 또는 순환계로 방출될 수 있다. 그러나, 프로펩타이드의 절단은 때때로 불완전하여, 프로펩타이드가 분자에 부착된 상태로 남게 된다. 그 결과 비정상적으로 가교된 얇은 원섬유가 만들어지고, 이로 인해 비정상적인 분자는 빠른 대사 전환에 처하게 된다 (10, 11). 따라서, 적절한 샘플에서 타입 III 콜라겐의 N-말단 프로펩타이드의 수준이 콜라겐 타입 III의 형성 및/또는 분해에 대한 마커일 수 있다.Collagen types I and III are the major structural proteins in the human body. Collagen type III is essential for the formation of collagen type I fibrils in the cardiovascular system and other organs (8,9). During the assembly of fibrils, the N-terminal propeptides of type III procollagen (composed of three identical α-chains, with a total molecular weight of 42 kDa) are exposed to specific N-proteases prior to incorporation of mature collagen into the ECM. is cut by Cleaved propeptides may be retained in the ECM or released into circulation. However, cleavage of the propeptide is sometimes incomplete, leaving the propeptide attached to the molecule. The result is abnormally cross-linked thin fibrils that subject the abnormal molecules to rapid metabolic turnover (10, 11). Accordingly, the level of the N-terminal propeptide of type III collagen in an appropriate sample may be a marker for the formation and/or degradation of collagen type III.
ECM의 변형된 구성 성분 및 비-코딩 변형은 조직의 경직성과 무손상 ECM 및 이의 단편의 신호전달 잠재성에 변화를 유발하기 때문에, ECM의 리모델링이 다양한 질병 발생에 중요한 역할을 담당한다. ECM 리모델링은 조직 기능 및 복구에 중요한 선행 조건이며, ECM의 합성 및 분해를 야기하는 효소에 의해 긴밀하게 조절된다.Remodeling of the ECM plays an important role in the development of various diseases because altered components and non-coding modifications of the ECM cause changes in tissue stiffness and signaling potential of the intact ECM and its fragments. ECM remodeling is an important prerequisite for tissue function and repair and is tightly regulated by enzymes that cause the synthesis and degradation of ECM.
섬유증 질환과 같은 질병이 발생하면, ECM의 형성 및 분해 간에 균형이 파괴되어, ECM 조성이 변화된다. 이러한 변화는 조직의 기능을 변화시킨다 (12, 13). PIIINP는 폐 손상 (14), 바이러스성 및 비-바이러스성 간염 (15), 전신 경화증 (16), 혈관 리모델링 (17) 및 신장병 (18)과 같은 수종의 섬유증 질환에 대한 바이오마커로서 사용될 수 있는 것으로 제안되어 있다.When diseases such as fibrotic diseases occur, the balance between formation and degradation of ECM is disrupted, resulting in changes in ECM composition. These changes alter the functioning of the organization (12, 13). PIIINP can be used as a biomarker for several fibrotic diseases, such as lung injury (14), viral and non-viral hepatitis (15), systemic sclerosis (16), vascular remodeling (17), and kidney disease (18). It is proposed that
골격근 조직에서 ECM 리모델링에 대한 제한적인 관심이 존재했다. 랫 모델에서, 콜라겐 유전자 발현 및 생합성 증가가 운동 후 대퇴사두근 및 전경골근에서 확인되었다 (19, 20). 아울러, 혈청내 PIIINP 수준 증가는 운동 후 임상 실험에서 확인되었다 (21). 즉, 골격근 단백질의 리모델링이 순환계 내 PIIINP의 양을 증가시키므로, 조기 근육 동화작용을 검출하기 위한 바이오마커로서 사용될 수 있다. 혈청내 PIIINP 수준은 기존에 테스토스테론 (22), 재조합 인간 성장 호르몬 (23) 또는 이들의 조합 (24, 25)에 대한 근육 조직 반응의 바이오마커로서 제안된 바 있다.There has been limited interest in ECM remodeling in skeletal muscle tissue. In rat models, increased collagen gene expression and biosynthesis have been identified in the quadriceps femoris and tibialis anterior muscles following exercise (19, 20). In addition, increased serum PIIINP levels were confirmed in clinical trials after exercise (21). That is, since remodeling of skeletal muscle proteins increases the amount of PIIINP in the circulation, it can be used as a biomarker to detect early muscle anabolism. Serum PIIINP levels have previously been proposed as a biomarker of muscle tissue response to testosterone (22), recombinant human growth hormone (23), or a combination thereof (24, 25).
간 섬유증에서, 원섬유성 콜라겐 타입 I 및 III는 고도로 상향 조절된다 (26, 27). 타입 III 콜라겐은 섬유증의 초기 단계에 풍부하고, 타입 I 콜라겐의 상향 조절은 섬유증의 후기 단계와 연관되어 있다. 간에서 발생한 섬유증은 콜라겐 침착과 프로펩타이드, 특히 PIIINP의 방출을 초래한다. 결과적으로, PIIINP는 섬유형성에 대해 가장 잘 연구된 마커 중 하나이다 (28,29,30). 수년에 걸쳐, PIIINP를 정량하기 위해, 간경변 검출에 대한 민감도가 최대 94%이고, 특이도가 최대 81%인, 몇가지 방사성면역분석 방법들이 개발되었지만 (31,32); 이전의 분석 방법들은 모두 네오-에피토프 (neo-epitope) 특이성을 가지지 않는다. 또한, PIIINP 정량용으로 현재 상업적으로 이용되는 분석 방법들은 프로콜라겐 또는 프로펩타이드의 내부 서열을 타겟팅하는 다클론 항체 또는 단일클론 항체를 이용하며, 콜라겐 타입 III의 형성 및/또는 분해를 특이적으로 구분하지는 못한다 (31,32).In liver fibrosis, fibrillar collagen types I and III are highly upregulated (26, 27). Type III collagen is abundant in the early stages of fibrosis, and upregulation of type I collagen is associated with the later stages of fibrosis. Fibrosis occurring in the liver results in collagen deposition and release of propeptides, especially PIIINP. As a result, PIIINP is one of the best-studied markers for fibrogenesis (28,29,30). Over the years, several radioimmunoassay methods have been developed to quantify PIIINP, with sensitivity up to 94% and specificity up to 81% for detection of cirrhosis (31,32); None of the previous analysis methods have neo-epitope specificity. In addition, currently commercially available analytical methods for quantifying PIIINP use polyclonal or monoclonal antibodies targeting the internal sequence of procollagen or propeptide, and specifically distinguish the formation and/or degradation of collagen type III. It cannot be done (31,32).
따라서, 콜라겐 타입 III의 형성과 분해를 구분하기 위해, 본 발명자들은 형성 과정시에만 생산되는 네오-에피토프 (즉, 콜라겐 타입 III의 형성시 만들어지지만 콜라겐 타입 III의 분해시에는 만들어지지 않는 단편)를 측정 및 검출하는 것이 필요하다고 생각하게 되었다.Therefore, to distinguish between the formation and degradation of collagen type III, we used a neo-epitope that is produced only during the formation process (i.e., a fragment made during the formation of collagen type III but not during the degradation of collagen type III). It became necessary to measure and detect.
WO 2014/170312에서는 X가 Gly 또는 Pro일 수 있는 C-말단 아미노산 서열 CPTGXQNYSP-COOH (서열번호 4)의 말단 아미노산에 포함된 C-말단 PIIINP 네오-에피토프에 특이적인 단일클론 항체가 개시되었다.In WO 2014/170312, a monoclonal antibody specific for the C-terminal PIIINP neo-epitope contained in the terminal amino acid of the C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4), where X may be Gly or Pro, was disclosed.
Brocks (31)는 변형된 보바인 C-말단 PIIINP 서열 I C* QSCPTGG E NYSP-COOH (서열번호 1) (C* = 아세트아미도 보호된 Cys; Gln이 Glu ( E )으로 치환됨)에 대한 다클론 항체를 개시한 바 있지만, 이 항체는 보바인 PIIINP의 말단 아미노산 C-말단 서열 ICQSCPTGGQNYSP-COOH (서열번호 2)에 비-특이적이며, 아울러 인간 PIIINP를 인지하지 못한다.Brocks (31) described the modified bovine C-terminal PIIINP sequence I C* QSCPTGG E NYSP-COOH (SEQ ID NO: 1) (C* = acetamido protected Cys; Gln replaced with Glu ( E )) Although a polyclonal antibody has been described, this antibody is non-specific for the terminal amino acid C-terminal sequence ICQSCPTGGQNYSP-COOH (SEQ ID NO: 2) of bovine PIIINP and does not recognize human PIIINP.
Bayer (33)는 서열 H2N-GSPGPPGICQSCPTGPQNYSP-COOH (서열번호 3)에 대한 검출제로서 단일클론 항체를 이용하는 샌드위치 ELISA를 개시하였지만, 결합 에피토프가 규정되어 있지 않다.Bayer (33) described a sandwich ELISA using a monoclonal antibody as a detection agent for sequence H 2 N-GSPGPPGICQSCPTGPQNYSP-COOH (SEQ ID NO: 3), but the binding epitope is not defined.
본 출원인은 WO 2014/170312에 개시된 PIIINP의 C-말단 네오-에피토프에 대한 네오-에피토프 특이 항체를 이용한 특이적인 샌드위치 면역분석이 라이실 산화효소 (LOX)를 타겟팅하는 약물, 특히 LOX 길항제 약물의 효능을 평가하는데 유용할 수 있다는 것을 발견하게 되었다. LOX에 의한 효소적 콜라겐 가교 및 프로-콜라겐의 프로세싱은 조직 성숙화 및 안정성에 매우 중요하다. 장기간 섬유증을 앓고 있는 환자의 경우, 콜라겐이 고도로 가교되어, 섬유증이 해소되는 경향이 낮다. LOXL2, 즉 특이적인 LOX는 섬유성 조직에서 병리생리학적 콜라겐 가교에 주요 구동인자이며, 새로운 LOXL2 길항제들이 현재 임상 실험 중에 있다. 따라서, LOX 길항제와 같은 LOX를 타겟팅하는 약물의 효능을 평가하는데 사용할 수 있는 분석은 당연히 약학 산업에 유용한 툴이 될 것이다.The present applicant has demonstrated that a specific sandwich immunoassay using a neo-epitope specific antibody against the C-terminal neo-epitope of PIIINP disclosed in WO 2014/170312 is used to determine the efficacy of drugs targeting lysyl oxidase (LOX), especially LOX antagonist drugs. It was discovered that it could be useful in evaluating . Enzymatic collagen cross-linking and processing of pro-collagen by LOX are very important for tissue maturation and stability. In patients suffering from long-term fibrosis, collagen is highly cross-linked, so the tendency for fibrosis to resolve is low. LOXL2, specifically LOX, is a key driver of pathophysiological collagen cross-linking in fibrous tissues, and novel LOXL2 antagonists are currently in clinical trials. Therefore, an assay that can be used to evaluate the efficacy of drugs targeting LOX, such as LOX antagonists, would naturally be a useful tool for the pharmaceutical industry.
본 발명은 생물학적 샘플에서 가교된 PIIINP를 검출하기 위한 샌드위치 면역분석방법에 관한 것으로서, 가교된 PIIINP는 가닥 간 가교에 의해 결합된 PIIINP 가닥 2개 이상을 포함한다. 본 방법은 가교된 PIIINP를 포함하는 생물학적 샘플을, 표면에 결합된 제1 단일클론 항체와 접촉시키는 단계, 및 제2 단일클론 항체를 첨가하는 단계를 포함하며, 가교된 PIIINP에 포함된 PIIINP의 각 가닥은 N-프로테아제에 의한 무손상 타입 III 프로콜라겐의 절단에 의해 형성된 PIIINP의 C-말단 네오-에피토프를 가진다. 상기한 단일클론 항체 2종은 PIIINP의 C-말단 네오-에피토프에 특이적으로 반응하며, 네오-에피토프는 C-말단 아미노산 서열 CPTGXQNYSP-COOH에 포함되며, 여기서, 여기서 X는 Gly 또는 Pro이다. 본 방법은 제2 단일클론 항체의 결합량을 측정하는 단계를 더 포함한다.The present invention relates to a sandwich immunoassay method for detecting cross-linked PIIINP in a biological sample, wherein the cross-linked PIIINP includes two or more strands of PIIINP joined by inter-strand cross-linking. The method includes contacting a biological sample comprising cross-linked PIIINPs with a first monoclonal antibody bound to a surface, and adding a second monoclonal antibody, wherein each of the PIIINPs included in the cross-linked PIIINPs is added. The strand has the C-terminal neo-epitope of PIIINP formed by cleavage of intact type III procollagen by N-protease. The two monoclonal antibodies described above specifically react to the C-terminal neo-epitope of PIIINP, where the neo-epitope is comprised in the C-terminal amino acid sequence CPTGXQNYSP-COOH, where X is Gly or Pro. The method further includes measuring the binding amount of the second monoclonal antibody.
또한, 본 발명은 라이실 산화효소 (LOX)를 타겟팅하는 길항제 약물의 효능을 평가하는 방법에 관한 것이다. 본 방법은 본원에 기술된 샌드위치 면역분석방법을 이용하여 개체에 길항제 약물을 투여하는 기간 중에 제1 시점 및 이후 하나 이상의 후속 시점에 개체에서 수득한 2종 이상의 생물학적 샘플에서 가교된 PIIINP의 양을 정량하는 단계를 포함한다. 길항제 약물의 투여 기간 중에 제1 시점에서부터 한번 이상의 후속 시점까지 가교된 PIIINP의 양적 감소는 LOX를 타겟팅하는 효과적인 길항제 약물임을 의미한다.The present invention also relates to methods for assessing the efficacy of antagonist drugs targeting lysyl oxidase (LOX). The method uses the sandwich immunoassay method described herein to quantify the amount of cross-linked PIIINP in two or more biological samples obtained from an individual at a first time point and at one or more subsequent time points during the period of administration of the antagonist drug to the individual. It includes steps to: A quantitative decrease in cross-linked PIIINP from a first time point to one or more subsequent time points during administration of the antagonist drug indicates an effective antagonist drug targeting LOX.
본 발명은 본원에 기술된 샌드위치 면역분석방법에 사용하기 위한 키트에 관한 것이다. 키트는 전술한 제1 단일클론 항체와 본원에 기술된 표지된 제2 단일클론 항체가 결합된 고체 지지체를 포함한다.The present invention relates to kits for use in the sandwich immunoassay described herein. The kit includes a solid support to which a first monoclonal antibody described above and a labeled second monoclonal antibody described herein are bound.
도 1: 인간 (서열번호 14) 및 랫 (서열번호 15) 종에서 타겟화된 PIIINP α1 체인 서열의 정렬 (박스로 강조 표시됨). 타입 III 콜라겐의 N-말단 프로펩타이드의 α1 체인 내 대응되는 인간 (-) 및 랫 (~) 서열의 위치. 정렬은 NLP CLUSTALW 소프트웨어를 사용해 수행하였다.
도 2: 단일클론 항체 NB61N62 (레인 1 및 3) 및 NB61N62 + 선택 펩타이드 (레인 2+4)에 의해 인지되는 a) 랫 및 b) 인간 유래의 양수에서 타입 III 콜라겐의 N-말단 프로펩타이드의 구체적인 밴드를 나타낸 웨스턴 블롯. 약 52-60 kDA의 밴드 2개가 랫에서 관찰되는 반면, 인간의 경우 밴드 1개가 관찰되었다. 선택 펩타이드의 추가로 랫 및 인간 모두에서 밴드 세기가 감소하였다.
도 3A-3D: 인간, 설치류 및 마우스 물질에 대한 전형적인 캘리브레이션 곡선 및 네이티브 반응성 (native reactivity)을 나타낸 PRO-C3 ELISA 실시 결과이다. 도 3A: 캘리브레이션 곡선 및 건강한 인간 혈청, 혈장 및 양수 (AF)를 이용한 경쟁적인 PRO-C3 ELISA의 저해. 캘리브레이터 곡선은 76.31 ng/mL에서부터 2배 희석한 반면, 천연 물질은 (--)로 나타낸 바와 같이 1:2에서 1:16까지 희석하여 진행하였다. 도 3B: 캘리브레이션 곡선 및 건강한 랫 혈청, 혈장 및 AF를 이용한 경쟁적인 PRO-C3 ELISA의 저해. 캘리브레이터 곡선은 200 ng/mL에서부터 2배 희석한 반면, 천연 물질은 (--)로 나타낸 바와 같이 비-희석에서 1:8까지 진행하였다. 도 3C: 캘리브레이션 곡선 및 건강한 마우스 혈청 및 혈장을 이용한 경쟁적인 PRO-C3 ELISA의 저해. 캘리브레이터 곡선은 200 ng/mL에서 2배 희석한 반면, 천연 물질은 (--)로 나타낸 바와 같이 비-희석에서 1:4까지 희석하여 진행하였다. 도 3D: 연장된 펩타이드, 즉 C-말단 단부에 아미노산 1개가 추가된 캘리브레이션 펩타이드의 펩타이드 서열을 이용한 PIIINP 네오-에피토프 특이 항체의 네오-에피토프 특이성. 캘리브레이션 곡선, 연장된 펩타이드 및 넌센스 펩타이드를 76.31 ng/mL에서 2배 희석하였다. 신호는 펩타이드 농도의 함수로서 650 nm 백그라운드를 제한 450 nm에서의 광학 밀도로 관찰한다.
도 4: 폐 섬유모세포의 시험관내 모델의 결과 ("scar-in-a-jar").
도 5: 켈로이드의 추출물 및 정상 피부의 추출물에서 Pro-C3X 수준 비교.
도 6A-6B: 간 섬유증 환자에 대한 실험 결과.
도 7: Pro-C3X 분석을 그림으로 도시한 도.
도 8: 알코올성 지방간염 환자에 대한 실험 결과.
도 9: Scar-in-a-Jar 모델에서 10일차에 수집한 상층액의 Pro-C3X 수준. 유의성은 각 조건을 TGF-β 단독을 비교하는 던네트의 다중 비교 검정을 이용한 일원배치분산분석으로 평가하였다. 데이타는 평균 및 SD로 나타낸다. ****p<0.0001. BAPN, β-아미노프로피오니트릴; TGF-β, 형질전환 성장인자 β.Figure 1: Alignment of targeted PIIINP α1 chain sequences in human (SEQ ID NO: 14) and rat (SEQ ID NO: 15) species (highlighted by box). Position of the corresponding human (-) and rat (~) sequences in the α1 chain of the N-terminal propeptide of type III collagen. Alignment was performed using NLP CLUSTALW software.
Figure 2: Specification of the N-terminal propeptide of type III collagen in amniotic fluid of a) rat and b) human origin recognized by monoclonal antibody NB61N62 (lanes 1 and 3) and NB61N62 + selected peptide (lanes 2+4). Western blot showing bands. Two bands of approximately 52-60 kDA were observed in rats, while one band was observed in humans. Addition of the selected peptide decreased the band intensity in both rats and humans.
Figures 3A-3D: PRO-C3 ELISA results showing typical calibration curves and native reactivity for human, rodent and mouse materials. Figure 3A: Calibration curve and inhibition of competitive PRO-C3 ELISA using healthy human serum, plasma and amniotic fluid (AF). The calibrator curve was diluted 2-fold starting from 76.31 ng/mL, while the natural material was diluted from 1:2 to 1:16 as indicated by (--). Figure 3B: Calibration curve and inhibition of competitive PRO-C3 ELISA using healthy rat serum, plasma and AF. The calibrator curve started from 200 ng/mL and was diluted 2-fold, while the native material proceeded from non-diluted to 1:8 as indicated by (--). Figure 3C: Calibration curve and inhibition of competitive PRO-C3 ELISA using healthy mouse serum and plasma. The calibrator curve was diluted 2-fold at 200 ng/mL, while the native material was run from non-diluted to 1:4 diluted, as indicated by (--). Figure 3D: Neo-epitope specificity of the PIIINP neo-epitope specific antibody using the peptide sequence of the extended peptide, i.e. the calibration peptide with one amino acid added to the C-terminal end. Calibration curve, extended peptide and nonsense peptide were diluted 2-fold at 76.31 ng/mL. The signal is observed as an optical density at 450 nm limiting the 650 nm background as a function of peptide concentration.
Figure 4: Results of an in vitro model of lung fibroblasts (“scar-in-a-jar”).
Figure 5: Comparison of Pro-C3X levels in extracts of keloids and extracts of normal skin.
Figures 6A-6B: Results of experiments on patients with liver fibrosis.
Figure 7: A graphical representation of Pro-C3X analysis.
Figure 8: Experimental results for patients with alcoholic steatohepatitis.
Figure 9: Pro-C3X levels in supernatant collected on day 10 in Scar-in-a-Jar model. Significance was assessed by one-way ANOVA using Dunnett's multiple comparison test comparing each condition with TGF-β alone. Data are presented as mean and SD. ****p<0.0001. BAPN, β-aminopropionitrile; TGF-β, transforming growth factor β.
본원에서, 용어 "네오-에피토프"는 폴리펩타이드의 말단에 위치한, 즉, 폴리펩타이드의 N- 또는 C-말단에 위치한 N- 또는 C-말단 펩타이드를 지칭하며, 이의 일반적인 방향을 의미하는 것으로 해석되는 것은 아니다.As used herein, the term “neo-epitope” refers to an N- or C-terminal peptide located at the terminus of a polypeptide, i.e. at the N- or C-terminus of the polypeptide, and is interpreted to mean its general orientation. That is not the case.
본원에서, 용어 "경쟁적인 ELISA"는 경쟁적인 효소-연결된 면역흡착 분석을 지칭하며, 당해 기술 분야의 당업자에게 공지된 기법이다.As used herein, the term “competitive ELISA” refers to competitive enzyme-linked immunosorbent assay, a technique known to those skilled in the art.
본원에서, 용어 "샌드위치 면역분석방법"은 샘플 내 항원을 검출하기 위한 2 이상의 항체의 사용을 의미하며, 당해 기술 분야의 당업자에게 공지된 기법이다.As used herein, the term “sandwich immunoassay” refers to the use of two or more antibodies to detect an antigen in a sample, a technique known to those skilled in the art.
본원에서, 용어 단일클론 항체 NB61N-62는 PIIINP의 C-말단 네오-에피토프에 대한 네오-에피토프 특이 항체를 의미하며, 네오-에피토프는 X가 Gly 또는 Pro인 C-말단 서열 CPTGXQNYSP-COOH (서열번호 4)를 포함한다.As used herein, the term monoclonal antibody NB61N-62 refers to a neo-epitope specific antibody against the C-terminal neo-epitope of PIIINP, wherein the neo-epitope has the C-terminal sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) Includes.
본원에서, 용어 "PRO-C3"는 PIIINP로부터 기원하는 네오-에피토프의 특이적인 결합에 기초하지 않는 당해 기술 분야에 공지된 PIIINP 분석으로부터 본원에 기술된 PIIINP 분석을 구분하기 위해 사용된다.As used herein, the term “PRO-C3” is used to distinguish the PIIINP assay described herein from PIIINP assays known in the art that are not based on specific binding of neo-epitopes originating from PIIINP.
본원에서, 용어 "PRO-C3X" 분석은 가교된 PIIINP를 검출 및 정량하기 위한 본원에 기술된 샌드위치 면역분석방법을 지칭한다.As used herein, the term “PRO-C3X” assay refers to the sandwich immunoassay described herein for detecting and quantifying cross-linked PIIINP.
본 발명의 방법에 사용하기 적합한 단일클론 항체는 WO 2014/170312에 기술되어 있으며, 이는 PIIINP의 C-말단 네오-에피토프와 특이적으로 반응하며, 네오-에피토프는 X가 Gly 또는 Pro인 C-말단 아미노산 서열 CPTGXQNYSP-COOH (서열번호 4)에 포함되며, 단일클론 항체는 C-말단 아미노산 서열의 연장된 버전, 즉 CPTGXQNYSPQZ-COOH (서열번호 5)를 실질적으로 인지 또는 결합하지 않으며, 여기서 Z는 생략되거나 또는 콜라겐 타입 III 서열 중의 하나 이상의 아미노산이다.A monoclonal antibody suitable for use in the method of the present invention is described in WO 2014/170312, which reacts specifically with the C-terminal neo-epitope of PIIINP, wherein the neo-epitope has a C-terminal antibody where X is Gly or Pro. The monoclonal antibody does not substantially recognize or bind the extended version of the C-terminal amino acid sequence, CPTGXQNYSPQZ-COOH (SEQ ID NO: 5), wherein Z is omitted. or is one or more amino acids in the collagen type III sequence.
바람직하게는, 단일클론 항체는, N-프로테아제에 의해 인간 PIIINP의 아미노산 P153-Q154 사이의 Pro-Gln 결합 위치에서 PIIINP의 절단에 의해 무손상 프로콜라겐 타입 III로부터 생성되는, 인간 PIIINP의 네오-에피토프 C-말단 서열 CPTGPQNYSP-COOH (서열번호 6)과 특이적으로 반응한다.Preferably, the monoclonal antibody is a neo-epitope of human PIIINP, produced from intact procollagen type III by cleavage of PIIINP at the Pro-Gln binding site between amino acids P153-Q154 of human PIIINP by N-protease. Reacts specifically with the C-terminal sequence CPTGPQNYSP-COOH (SEQ ID NO: 6).
다른 구현예로, 단일클론 항체는, 설치류 PIIINP의 아미노산 P154-Q155 사이의 Pro-Gln 결합 위치에서 PIIINP의 N-프로테아제 절단에 의해 무손상 프로콜라겐 타입 III로부터 형성되는, 설치류 PIIINP의 네오-에피토프 C-말단 서열 CPTGGQNYSP-COOH (서열번호 7)과 특이적으로 반응할 수 있다.In another embodiment, the monoclonal antibody is directed to the neo-epitope C of rodent PIIINP, formed from intact procollagen type III by N-protease cleavage of PIIINP at the Pro-Gln binding site between amino acids P154-Q155 of rodent PIIINP. -Can react specifically with the terminal sequence CPTGGQNYSP-COOH (SEQ ID NO: 7).
바람직하게는, 연장된 아미노산 서열 CPTGXQNYSPQZ-COOH (서열번호 5)에 대한 단일클론 항체의 친화성에 대한 아미노산 서열 CPTGXQNYSP-COOH (서열번호 4)에 대한 단일클론 항체의 친화성의 비율은 적어도 10 내지 1, 바람직하게는 적어도 100 내지 1, 더 바람직하게는 적어도 1,000 내지 1, 더 바람직하게는 적어도 10,000 내지 1, 더 바람직하게는 적어도 100,000 내지 1, 가장 바람직하게는 적어도 1,000,000 내지 1이다.Preferably, the ratio of the affinity of the monoclonal antibody for the amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of the monoclonal antibody for the extended amino acid sequence CPTGXQNYSPQZ-COOH (SEQ ID NO: 5) is at least 10 to 1, Preferably it is at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1.
바람직하게는, 단일클론 항체는 아미노산 서열 CPTGXQNYS (서열번호 8)을 가진 PIIINP의 C-말단 네오-에피토프의 축소 버전 (shortened version)을 인지 또는 결합하지 않는다.Preferably, the monoclonal antibody does not recognize or bind a shortened version of the C-terminal neo-epitope of PIIINP with amino acid sequence CPTGXQNYS (SEQ ID NO: 8).
바람직하게는, 축소된 아미노산 서열 CPTGXQNYS-COOH (서열번호 8)에 대한 단일클론 항체의 친화성에 대한 아미노산 서열 CPTGXQNYSP-COOH (서열번호 4)에 대한 단일클론 항체의 친화성의 비율은 적어도 10 내지 1, 바람직하게는 적어도 100 내지 1, 더 바람직하게는 적어도 1,000 내지 1, 더 바람직하게는 적어도 10,000 내지 1, 더 바람직하게는 적어도 100,000 내지 1, 가장 바람직하게는 적어도 1,000,000 내지 1이다.Preferably, the ratio of the affinity of the monoclonal antibody for the amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of the monoclonal antibody for the reduced amino acid sequence CPTGXQNYS-COOH (SEQ ID NO: 8) is at least 10 to 1, Preferably it is at least 100 to 1, more preferably at least 1,000 to 1, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1.
본 발명은, 가닥 간 가교에 의해 결합된 PIIINP의 2 이상의 가닥을 포함하는 가교된 PIIINP를 생물학적 샘플에서 검출하기 위한 샌드위치 면역분석방법에 관한 것으로서, 본 방법은,The present invention relates to a sandwich immunoassay method for detecting cross-linked PIIINP comprising two or more strands of PIIINP bound by inter-strand cross-linking in a biological sample, the method comprising:
가교된 PIIINP를 포함하는 생물학적 샘플을 지지체에 결합된 제1 단일클론 항체와 접촉시키는 단계로서, 가교된 PIIINP에 포함된 PIIINP의 각 가닥이 무손상 타입 III 프로콜라겐의 N-프로테아제 절단에 의해 생성된 PIIINP의 C-말단 네오-에피토프를 포함하는, 단계;A step of contacting a biological sample containing cross-linked PIIINP with a first monoclonal antibody bound to a support, wherein each strand of PIIINP included in the cross-linked PIIINP is produced by N-protease cleavage of intact type III procollagen. comprising a C-terminal neo-epitope of PIIINP;
제2 단일클론 항체를 첨가하는 단계; 및adding a second monoclonal antibody; and
제2 단일클론 항체의 결합량을 측정하는 단계를 포함하며,It includes measuring the binding amount of the second monoclonal antibody,
제1 단일클론 항체 및 제2 단일클론 항체 둘다 PIIINP의 C-말단 네오-에피토프에 특이적으로 반응하며, 네오-에피토프는 X가 Gly 또는 Pro인 C-말단 아미노산 서열 CPTGXQNYSP-COOH에 포함된다.Both the first monoclonal antibody and the second monoclonal antibody specifically react with the C-terminal neo-epitope of PIIINP, the neo-epitope being comprised in the C-terminal amino acid sequence CPTGXQNYSP-COOH, where X is Gly or Pro.
바람직하게는, 단일클론 항체는, C-말단 아미노산 서열의 연장된 버전, 즉, Z가 생략되거나 또는 콜라겐 타입 III 서열 중의 하나 이상의 아미노산인 CPTGXQNYSPQZ-COOH에 대해 실질적으로 인지 또는 결합을 나타내지 않는다.Preferably, the monoclonal antibody does not substantially recognize or bind to an extended version of the C-terminal amino acid sequence, i.e., the Z is omitted, or to one or more amino acids in the collagen type III sequence, CPTGXQNYSPQZ-COOH.
본원에 기술된 샌드위치 면역분석방법은 동일한 항체를 포획 항체 및 검출 항체 둘다로 사용하며, 따라서 이중 가닥 펩타이드 (즉, 가교된)를 분석으로 인지할 수 있다.The sandwich immunoassay method described herein uses the same antibody as both the capture and detection antibodies, thus allowing double-stranded peptides (i.e., cross-linked) to be recognized by the assay.
바람직하게는, 샌드위치 면역분석방법을 이용해 생물체액에서 가교된 PIIINP의 양을 정량하며, 생물체액은 혈청, 혈장, 뇨, 양수, 조직 상층액 또는 세포 상층액일 수 있으나, 이들로 한정되는 것은 아니다.Preferably, a sandwich immunoassay method is used to quantify the amount of cross-linked PIIINP in biological fluid, and the biological fluid may be serum, plasma, urine, amniotic fluid, tissue supernatant, or cell supernatant, but is not limited to these.
샌드위치 면역분석방법은 방사성 면역분석, 형광 면역분석 또는 효소-연결된 단일클론 항체일 수 있으나, 이들로 한정되는 것은 아니다.The sandwich immunoassay method may be, but is not limited to, a radioimmunoassay, a fluorescence immunoassay, or an enzyme-linked monoclonal antibody.
바람직한 구현예에서, 제2 단일클론 항체는 제2 단일클론 항체의 결합량을 측정하기 위해 표지될 수 있다.In a preferred embodiment, the second monoclonal antibody can be labeled to measure the amount of binding of the second monoclonal antibody.
바람직하게는, 제2 단일클론 항체는 효소-연결된 항체일 수 있다. 효소는 호스래디시 퍼옥시다제 (HRP)일 수 있으나, 이로 한정되는 것은 아니다.Preferably, the second monoclonal antibody may be an enzyme-linked antibody. The enzyme may be, but is not limited to, horseradish peroxidase (HRP).
바람직하게는, 제2 단일클론 항체는 방사성 표지되거나 또는 형광단이 연결될 수 있다.Preferably, the second monoclonal antibody may be radiolabeled or fluorophore linked.
이들 물질은 본 발명과 함께 사용하기에 바람직한 표지 물질이지만, 비-배타적인 예로, DNA 리포터 또는 전기화학발광 테그 등의 임의의 적합한 표지 시스템이 사용될 수 있는 것으로 생각된다.Although these are preferred labeling materials for use with the present invention, it is contemplated that any suitable labeling system may be used, including, but not limited to, DNA reporters or electrochemiluminescent tags.
다른 구현예에서, 제2 단일클론 항체를 인지하는 추가적인 표지된 항체를 사용해, 제2 단일클론 항체의 결합량을 측정할 수 있다. 추가적인 표지된 항체는 전술한 바와 같은 표지 물질로 표지될 수 있다.In another embodiment, an additional labeled antibody that recognizes the second monoclonal antibody can be used to measure the amount of binding of the second monoclonal antibody. Additional labeled antibodies may be labeled with labeling agents as described above.
본 발명의 바람직한 구현예에서, 샌드위치 면역분석방법은 섬유증 질환의 중증도를 평가하기 위해 본 방법에 의해 측정된 가교된 PIIINP의 양을 질환 중증도가 공지된 섬유성 질환의 표준 샘플과 연관시키는 단계를 더 포함할 수 있다. 이러한 섬유성 질환은 간 질환일 수 있으나, 이로 한정되는 것은 아니다.In a preferred embodiment of the invention, the sandwich immunoassay method further comprises correlating the amount of cross-linked PIIINP measured by the method with a standard sample of fibrotic disease of known disease severity to assess the severity of the fibrotic disease. It can be included. This fibrotic disease may be a liver disease, but is not limited thereto.
다른 측면에서, 본원에 기술된 샌드위치 면역분석방법은 LOX를 타겟팅하는 길항 약물 등의 라이실 산화효소 (LOX)를 타겟팅하는 약물의 효능을 평가하기 위한 방법에 사용될 수 있다.In another aspect, the sandwich immunoassay method described herein can be used in a method for evaluating the efficacy of drugs targeting lysyl oxidase (LOX), such as antagonistic drugs targeting LOX.
이에, 본 발명은 또한 라이실 산화효소 (LOX)를 타겟팅하는 길항제 약물의 효능을 평가하는 방법에 관한 것으로, 본 방법은 적어도 2종 이상의 생물학적 샘플에서 가교된 PIIINP의 양을 정량하기 위해 본원에 기술된 샌드위치 면역분석방법을 이용하는 것을 포함하며, 생물학적 샘플은 개체에 길항제 약물을 투여하는 기간 동안에 제1 시점과 이후 하나 이상의 후속 시점에 개체로부터 수득되며, 길항제 약물을 투여하는 기간 동안 제1 시점에서부터 이후 하나 이상의 후속 시점까지의 가교된 PIIINP의 양적 감소는 상기 길항제 약물이 LOX를 타겟팅하는 효과적인 길항제 약물임을 의미한다.Accordingly, the present invention also relates to a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidase (LOX), the method described herein for quantifying the amount of cross-linked PIIINP in at least two biological samples. A method comprising using a sandwich immunoassay method, wherein the biological sample is obtained from the individual at a first time point and at one or more subsequent time points during the period during which the subject is administered the antagonist drug, wherein the biological sample is obtained from the individual at a first time point and thereafter during the period during which the antagonist drug is administered. Quantitative reduction of cross-linked PIIINP by one or more subsequent time points indicates that the antagonist drug is an effective antagonist drug targeting LOX.
바람직하게는, 본 방법은 길항제 약물의 유효성을 정량한다.Preferably, the method quantifies the effectiveness of an antagonist drug.
바람직하게는, 본 방법은 LOXL2를 타겟팅하는 길항제 약물의 효능을 평가한다.Preferably, the method evaluates the efficacy of an antagonist drug targeting LOXL2.
다른 측면에서, 본 발명은 전술한 제1 단일클론 항체가 결합된 고체 지지체와 전술한 표지된 제2 단일클론 항체를 포함하는, 본원에 기술된 샌드위치 면역분석방법에 사용하기 위한 키트에 관한 것이다.In another aspect, the present invention relates to a kit for use in the sandwich immunoassay method described herein, comprising a solid support to which the above-described first monoclonal antibody is bound and the above-described labeled second monoclonal antibody.
실시예Example
재료 및 일반 고려 사항Materials and General Considerations
실험에 사용되는 모든 시약들은 Merck (Whitehouse Station, NJ, USA) 및 Sigma Aldrich (St. Louis, MO, USA)와 같은 회사에서 구입한 고-품질의 표준 화학제들이다. 단일클론 항체 생산 및 검증에 사용된 합성 펩타이드는 다음과 같다: 1) 면역원성 펩타이드: 오발부민 (OVA)-CGG-CPTGPQNYSP (서열번호 10), 2) 스크리닝 펩타이드: Biotin-CGG-CPTGPQNYSP (서열번호 11), 및 3) 선택 펩타이드: CPTGPQNYSP (서열번호 6). 합성 펩타이드 모두 중국 베이징의 Chinese Peptide Company에서 구입하였다.All reagents used in the experiments were high-quality standard chemicals purchased from companies such as Merck (Whitehouse Station, NJ, USA) and Sigma Aldrich (St. Louis, MO, USA). The synthetic peptides used for monoclonal antibody production and verification are as follows: 1) Immunogenic peptide: Ovalbumin (OVA)-CGG-CPTGPQNYSP (SEQ ID NO: 10), 2) Screening peptide: Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11), and 3) selected peptide: CPTGPQNYSP (SEQ ID NO: 6). All synthetic peptides were purchased from Chinese Peptide Company, Beijing, China.
실시예Example 1 - 단일클론 항체 NB61-N62 1 - Monoclonal antibody NB61-N62
단일클론 항체 제조Monoclonal antibody manufacturing
타입 III 콜라겐의 N-말단 프로펩타이드의 서열을 인간, 랫 및 마우스 종들 간에 정렬시키고, 단백질 블라스팅에 의해 종 간 상동성 및 다른 ECM 단백질들에서 고유성으로 선택하였다. α1 체인 PIIINP에서 아미노산 서열 145'-CPTGPQNYSP-'153 (서열번호 6)은 인간 및 랫 간 상동성이 100%이다 (도 1). 단일클론 항체의 제조는 4-5주령의 Balb/C 마우스에 에멀젼화된 항원 200 ㎕와 PIIINP 네오-에피토프 C-말단 서열 (OVA-CGG-CPTGPQNYSP (서열번호 10)) 50 ㎍을 프레운드의 불완전 보강체를 사용해 피하 주사하여 개시하였다. 안정적인 혈청 역가 수준에 도달할 때까지 2주마다 면역화를 수행하였다. 최고 혈청 역가를 나타낸 마우스를 융합을 위해 선택하였다. 마우스를 1달간 휴식시킨 후, 비장을 단리하기 3일 전에 0.9% 염화나트륨 용액 100 ㎕ 중의 PIIINP 네오-에피토프 C-말단 서열 50 ㎍을 정맥내 부스트 접종하였다. 비장 세포를 SP2/0 골수종 세포와 융합시켜 (34)에 기술된 바와 같이 하이브리도마를 제조하였으며, 세미-매질 방법을 사용해 배양 디쉬에서 클로닝하였다. 클론들은 제한 희석 방법을 적용해 추가적인 증식을 위해 96-웰 마이크로타이터 플레이트에 접종하여, 단일클론을 증식시켰다. 스트렙타비딘-코팅된 플레이트를 사용한 간접 ELISA로 상층액에서 캘리브레이터 펩타이드 및 천연 물질에 대한 반응성을 스크리닝하였다. Biotin-CGG-CPTGPQNYSP (서열번호 11)를 스크리닝 펩타이드로 사용하였으며, 프리 (free) 펩타이드 CPTGPQNYSP (서열번호 6)는 클론에 대한 추가적인 특이성 검정을 위한 캘리브레이터로 사용하였다.The sequence of the N-terminal propeptide of type III collagen was aligned between human, rat and mouse species and selected for cross-species homology and uniqueness among other ECM proteins by protein blasting. The amino acid sequence 145'-CPTGPQNYSP-'153 (SEQ ID NO: 6) in α1 chain PIIINP has 100% homology between human and rat (Figure 1). For the preparation of monoclonal antibodies, 200 μl of emulsified antigen and 50 μg of PIIINP neo-epitope C-terminal sequence (OVA-CGG-CPTGPQNYSP (SEQ ID NO: 10)) were added to 4-5 week old Balb/C mice using Freund's incomplete method. It was initiated by subcutaneous injection using adjuvant. Immunizations were performed every two weeks until stable serum titer levels were reached. The mouse showing the highest serum titer was selected for fusion. After resting the mice for 1 month, they received an intravenous boost dose of 50 μg of PIIINP neo-epitope C-terminal sequence in 100 μl of 0.9% sodium chloride solution 3 days before spleen isolation. Hybridomas were prepared as described (34) by fusing spleen cells with SP2/0 myeloma cells and cloned in culture dishes using the semi-medium method. Clones were inoculated into 96-well microtiter plates for further growth using the limiting dilution method, and monoclones were grown. Supernatants were screened for reactivity to calibrator peptides and natural substances by indirect ELISA using streptavidin-coated plates. Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11) was used as a screening peptide, and the free peptide CPTGPQNYSP (SEQ ID NO: 6) was used as a calibrator for additional specificity testing for the clone.
클론의 특징 규명Characterization of clones
스트렙타비딘-코팅된 마이크로타이터 플레이트에서 2 ng/ml 바이오틴화된 펩타이드 및 증식 중인 단일클론 하이브리도마 세포의 상층액을 이용한 예비 ELISA에서, 펩타이드의 네이티브 반응성 (native reactivity) 및 친화성을 뇨, 혈청 및 양수 (AF)와 같은 여러가지 생물학적 물질을 사용해 인간 및 랫에서 분석하였다. 인간 AF는 2달간 베이징 산부인과 병원의 선택적인 자궁 하부 제왕절개술을 받는 여성 30명으로부터 입수하였다. AF 100-200 ml을 절개 후 직접 수집하고, 양수는 사용할 때까지 -20℃에 보관하였다. 지역 윤리 위원회로부터 실험을 허가받았으며, 모든 여성들에서 샘플 수집 전 서면 동의를 구하였다. 랫 AF는 예정일 2일 전에 임신 중인 Wistar 랫의 자궁에서 배액하였다. 항체 특이성은 선택취소 (deselection) 펩타이드 및 연장된 펩타이드 (즉, 각각 아미노산 10개 치환이 존재하는 캘리브레이터 펩타이드 및 절단부에 아미노산 하나가 부가된 캘리브레이터 펩타이드)를 이용한 예비 분석으로 검사하였다. 단일클론 항체의 이소형은 Clonotyping System-HRP 키트 cat. 5300-05 (Southern Biotech, Birmingham, AL, USA)를 사용해 확인하였다.In a preliminary ELISA using 2 ng/ml biotinylated peptide and supernatants of growing monoclonal hybridoma cells in streptavidin-coated microtiter plates, the native reactivity and affinity of the peptide were determined. , analyzed in humans and rats using various biological materials such as serum and amniotic fluid (AF). Human AF was obtained from 30 women undergoing elective lower uterine cesarean section at Beijing Obstetrics and Gynecology Hospital over a 2-month period. 100-200 ml of AF was collected directly after incision, and amniotic fluid was stored at -20°C until use. Permission for the experiment was obtained from the local ethics committee, and written consent was obtained from all women before sample collection. Rat AF was drained from the uterus of pregnant Wistar rats 2 days before due date. Antibody specificity was tested by preliminary analysis using deselection peptides and extended peptides (i.e., a calibrator peptide with 10 amino acid substitutions each and a calibrator peptide with one amino acid added to the truncation site). The isotype of the monoclonal antibody was determined using the Clonotyping System-HRP kit cat. Confirmation was performed using 5300-05 (Southern Biotech, Birmingham, AL, USA).
항체의 특징 규명Characterization of antibodies
웨스턴 블롯팅 실시 전, 인간 및 랫 AF의 전체 단백질 농도를 비신콘닌산 (BCA, Bicinchoninic acid) 단백질 분석을 제조사의 지침에 따라 사용하여 측정하였다. 간략하게는, BCA를 PBS 중에 2 mg/ml에서부터 2배 희석하여 샘플의 캘리브레이션을 위한 표준 열을 만들었다. 샘플들을 1x 포스페이트-완충화된 염수 (PBS)에 1:4로 희석하고, 샘플 25 ㎕를 마이크로타이터 플레이트에 작동 시약 (시약 A 및 B를 50:1로 혼합) 200 ㎕와 함께 첨가하였다. 내용물을 플레이트 교반기 상에서 30초간 혼합한 다음 37℃에서 30분간 인큐베이션하였다. 인큐베이션 종료 후, 플레이트를 실온으로 냉각시키고, 흡광도를 ELISA 리더에서 562 nm (Molecular Devices, SpectraMax M, CA, USA)에서 측정하였다. 그 후, 랫 또는 인간 AF를 샘플 완충제 (x2) 및 환원제 (x10)와 혼합하고, 10분간 70℃에서 가열한 후 4-20% 트리스-글리신 소듐 도데실 설페이트-폴리아크릴아미드 겔 전기영동 (SDS-page)에 로딩하여 1시간 동안 180V에서 진행하였다. 단백질 밴드를 Invitrogen i-Blot 겔 이동 시스템을 제조사의 지침에 따라 사용해 니트로셀룰로스 막으로 블롯팅하였다. 막을 차단 완충제 (5% 탈지유/Tris-완충화된 염수 + Tween (TBST))에서 밤새 4℃에서 차단 처리하고, 2시간 동안 1 ㎍/ml 호스래디시 퍼옥시다제 (HRP)-접합된 PIIINP 네오-에피토프 특이 단일클론 항체 NB61N-62와 인큐베이션하였다. 과량의 PIIINP 네오-에피토프 캘리브레이터 펩타이드 및 항체를 10:1의 비율로 첨가하여 PIIINP 네오-에피토프 특이 단일클론 항체의 특이성을 조사하고, 1시간 동안 예비 인큐베이션한 후 막에 첨가하여 밤새 인큐베이션하였다. 인큐베이션한 후, 막을 TBST로 4 x 10분 세척하고, 4 ml 화학발광 검출 키트 (ECL)와 인큐베이션한 다음 Amersham Hyperfilm을 사용해 현상하였다.Before Western blotting, the total protein concentration of human and rat AF was measured using the Bicinchoninic acid (BCA) protein assay according to the manufacturer's instructions. Briefly, BCA was diluted 2-fold in PBS from 2 mg/ml to create a standard column for calibration of samples. Samples were diluted 1:4 in 1x phosphate-buffered saline (PBS), and 25 μl of sample was added to a microtiter plate along with 200 μl of running reagent (reagents A and B mixed 50:1). The contents were mixed for 30 seconds on a plate shaker and then incubated at 37°C for 30 minutes. After completion of incubation, the plate was cooled to room temperature, and the absorbance was measured at 562 nm (Molecular Devices, SpectraMax M, CA, USA) in an ELISA reader. Rat or human AF was then mixed with sample buffer (x2) and reducing agent (x10), heated at 70°C for 10 min and then subjected to 4-20% Tris-Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS). -page) and carried out at 180V for 1 hour. Protein bands were blotted to nitrocellulose membranes using the Invitrogen i-Blot gel transfer system according to the manufacturer's instructions. Membranes were blocked overnight at 4°C in blocking buffer (5% skim milk/Tris-buffered saline + Tween (TBST)) and incubated with 1 μg/ml horseradish peroxidase (HRP)-conjugated PIIINP neo- for 2 h. Incubation was performed with the epitope-specific monoclonal antibody NB61N-62. Excess PIIINP neo-epitope calibrator peptide and antibody were added at a ratio of 10:1 to examine the specificity of the PIIINP neo-epitope specific monoclonal antibody, preincubated for 1 hour, and then added to the membrane and incubated overnight. After incubation, the membrane was washed 4 x 10 minutes with TBST, incubated with 4 ml chemiluminescence detection kit (ECL) and developed using Amersham Hyperfilm.
클론 선택 및 특징 규명Clone selection and characterization
서브타입은 IgG1 서브타입으로 확인되었다. 웨스턴 블롯 분석을 통해, PIIINP 네오-에피토프 특이 단일클론 항체 NB61N-62가 랫 양수에서 약 52-60 kDa 분자 크기의 밴드 2개를 인지하는 반면, 인간 양수에서는 약 52 kDa의 밴드 하나만 검출하는 것으로 나타났다. 아울러, 신호는 랫에서 선택 펩타이드에 의해 일부 저해되고, 인간에서 저해될 수 있었다 (도 2). 네이티브 반응성을 ELISA에서 NB61N-62 항체를 사용해 관찰하였다. 인간 혈청, 혈장 및 AF에 대한 네이티브 반응성 뿐만 아니라 설치류 랫, 혈장 및 AF에 대한 네이티브 반응성을 관찰하였다 (도 3A-3C). 신호는 마우스 혈청 및 혈장에서 일부 약간 감소되었다. 경쟁적인 ELISA의 신호는 인간, 설치류 및 마우스 천연 물질에서 각각 1:2에서 1:16, 비-희석에서 1:8 또는 비-희석에서 1:4까지 저해되었다. 천연 물질의 희석 후 3가지 종들 모두에서 대체적으로 킬리브레이터 곡선과 동일한 희석 패턴이 나타났다. 인간 AF는 신호를 최대 100% 저해하였으며; 랫 AF의 경우 80%; 인간 혈청 및 혈장과 랫 혈청의 경우 70%; 랫 혈장의 경우 44%, 마우스 혈청 및 혈장의 경우 35%로 저해하였다. 0 저해는 연장된 펩타이드 (CPTGPQNYSPQ (서열번호 6)) 및 넌센스 펩타이드 (GSPGKDGVRG (서열번호 12))에서 관찰되었다 (도 3D).The subtype was identified as IgG1 subtype. Western blot analysis showed that the PIIINP neo-epitope specific monoclonal antibody NB61N-62 recognized two bands with a molecular size of approximately 52-60 kDa in rat amniotic fluid, while only detecting a single band of approximately 52 kDa in human amniotic fluid. . In addition, the signal was partially inhibited by the selected peptide in rats and could be inhibited in humans (Figure 2). Native reactivity was observed using the NB61N-62 antibody in ELISA. Native reactivity was observed for human serum, plasma, and AF, as well as for rodent rat, plasma, and AF (Figures 3A-3C). The signal was slightly reduced in some cases in mouse serum and plasma. The signal of the competitive ELISA was inhibited 1:2 to 1:16 in human, rodent and mouse native material, 1:8 in undiluted or 1:4 in undiluted, respectively. After dilution of the natural material, a dilution pattern generally identical to the Chillibrator curve appeared in all three species. Human AF inhibited the signal by up to 100%; 80% for rat AF; 70% for human serum and plasma and rat serum; It was inhibited by 44% in rat plasma and 35% in mouse serum and plasma. 0 inhibition was observed with the extended peptide (CPTGPQNYSPQ (SEQ ID NO: 6)) and the nonsense peptide (GSPGKDGVRG (SEQ ID NO: 12)) (Figure 3D).
실시예Example 2 - 2 - NB61NNB61N -62를 이용한 PRO-C3 ELISAPRO-C3 ELISA using -62
항체를 생산하는 하이브리도마로부터 상층액을 수집하고, 단일클론 항체를 HiTrap 친화성 컬럼 (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK)을 사용해 정제한 다음 Lightning-Link™ HRP 접합 키트 (Innova Biosciences, Babraham, Cambridge, UK)를 제조사의 지침에 따라 사용해 HRP로 표지하였다.Supernatants were collected from antibody-producing hybridomas, and monoclonal antibodies were purified using HiTrap affinity columns (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK) followed by Lightning-Link™ HRP conjugation kit (Innova Biosciences). , Babraham, Cambridge, UK) was labeled with HRP using the manufacturer's instructions.
PRO-C3 경쟁적인 ELISA 절차는 다음과 같다: Roche 사의 cat.11940279 96웰 스트렙타비딘-코팅된 ELISA 플레이트를, 코터 완충제 (50mM PBS-BTE + 10% 소르비톨, pH 7.4)에 용해된 바이오틴화 펩타이드 Biotin-CGG-CPTGPQNYSP (서열번호 11)로 코팅하고, 암 조건에서 30분간 20℃에서 인큐베이션한 다음 세척 완충제 (20 mM Tris, 50 mM NaCl, pH 7.2)로 헹구었다. 그 후, 펩타이드 캘리브레이터 또는 샘플 20 ㎕를 적정 웰에 첨가한 다음 인큐베이션 완충제 (50 mM PBS-BTB + 10% LiquidII (Roche), pH 7.4)에 용해된 HRP-접합된 단일클론 항체 NB61N-62 100 ㎕를 첨가하여 4℃에서 20시간 동안 플레이트를 인큐베이션한 다음 세척하였다. 마지막으로, 테트라메틸벤지니딘 (TMB) (Kem-En-Tec cat.: 438OH) 100 ㎕를 첨가하고, 플레이트를 암 조건에서 15분간 20℃에서 인큐베이션하였으며, 반응을 중단시키기 위해 정지 용액 (1% H2SO4) 100 ㎕를 첨가한 다음 플레이트를 ELISA 리더에서 450 nm에서 분석하고, 기준으로서 650 nm에서 분석하였다 (Molecular Devices, SpectraMax M, CA, USA). 캘리브레이션 곡선은 4-모수적 수학 피트 모델 (4-parametric mathematical fit model)을 사용해 작성하였다.The PRO-C3 competitive ELISA procedure is as follows: cat.11940279 96-well streptavidin-coated ELISA plates from Roche were incubated with biotinylated peptides dissolved in Cotter buffer (50mM PBS-BTE + 10% sorbitol, pH 7.4). It was coated with Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11), incubated at 20°C for 30 minutes in dark conditions, and then rinsed with washing buffer (20mM Tris, 50mM NaCl, pH 7.2). Afterwards, 20 μl of peptide calibrator or sample was added to the titration well followed by 100 μl of HRP-conjugated monoclonal antibody NB61N-62 dissolved in incubation buffer (50 mM PBS-BTB + 10% LiquidII (Roche), pH 7.4). was added and the plate was incubated at 4°C for 20 hours and then washed. Finally, 100 μl of tetramethylbenzinidine (TMB) (Kem-En-Tec cat.: 438OH) was added, the plate was incubated at 20°C for 15 minutes in dark conditions, and stop solution (1) was added to stop the reaction. % H 2 SO 4 ) was added and the plate was analyzed at 450 nm in an ELISA reader and at 650 nm as a reference (Molecular Devices, SpectraMax M, CA, USA). The calibration curve was created using a 4-parametric mathematical fit model.
기술적 평가technical evaluation
건강한 혈청 및 혈장 샘플의 2배 희석을 사용해, 직선성 (linearity)을 구하고, 100% 샘플의 회수율로서 계산하였다. 항체 특이성은 100% 캘리브레이터 펩타이드 (CPTGPQNYSP (서열번호 6)), 연장된 펩타이드 (CPTGPQNYSPQ (서열번호 13)) 및 넌센스 펩타이드 (GSPGKDGVRG (서열번호 12))의 회수율로서 계산하였다. 검출 하한 (LLOD)은 표준 K의 21회 측정값들로부터 블랭크의 평균 + 3x표준편차 (SD)로 계산하였다. 검출 상한 (ULOD)은 표준 A의 10회 측정값들의 평균 - 3xSD로서 구하였다. 정량 하한 (LLOQ)은 정확도 30% 미만의 반복적으로 측정되는 최소 농도로서 구하였다. 분석 내 및 분석 간 편차는 QC 샘플 8종의 10회 독립적인 측정을 통해 측정하였으며, 각각의 수행은 샘플의 더블 측정으로 구성된다. 샘플의 정확도는 표준 곡선과 함께 또는 유의한 농도의 인간 양수가 첨가된 건강한 인간 혈청 샘플에서 측정하였으며, 혈청의 이론적인 양의 회수%로서 계산하였다. 헤모글로빈, 혈청 지질 (lipemia) 및 바이오틴이 유의한 농도로 첨가된 건강한 인간 혈청에서 간섭 (interference)을 측정하고, 혈청의 이론적인 양의 회수%로서 계산하였다.Linearity was determined using two-fold dilutions of healthy serum and plasma samples and calculated as recovery of 100% sample. Antibody specificity was calculated as the recovery of 100% calibrator peptide (CPTGPQNYSP (SEQ ID NO: 6)), extended peptide (CPTGPQNYSPQ (SEQ ID NO: 13)) and nonsense peptide (GSPGKDGVRG (SEQ ID NO: 12)). The lower limit of detection (LLOD) was calculated as the mean of the blank + 3x standard deviation (SD) from 21 measurements of standard K. The upper limit of detection (ULOD) was determined as the average of 10 measurements of standard A - 3xSD. The lower limit of quantification (LLOQ) was determined as the minimum concentration that can be measured repeatedly with an accuracy of less than 30%. Intra- and inter-assay variation were measured through 10 independent measurements of eight QC samples, with each run consisting of a double measurement of the sample. Sample accuracy was determined with a standard curve or in healthy human serum samples spiked with significant concentrations of human amniotic fluid and calculated as percent recovery of the theoretical amount of serum. Interference was measured in healthy human serum supplemented with significant concentrations of hemoglobin, serum lipids (lipemia) and biotin and calculated as percent recovery of the theoretical amount of serum.
결과result
인간 PRO-C3 ELISA의 측정 범위는 ULOD 및 LLOQ가 0.867-60.1 ng/ml 범위를 제공하도록 계산함으로써 구하였다. PRO-C3 ELISA의 기술적인 성능은 평균 11.03% 및 4.11%의 허용가능한 분석 간 및 분석 내 편차를 나타내었으며 (표 1), 각각 15% 및 10% 미만의 허용가능한 범위였다.The measurement range of the human PRO-C3 ELISA was determined by calculating the ULOD and LLOQ to give a range of 0.867-60.1 ng/ml. The technical performance of the PRO-C3 ELISA showed acceptable inter- and intra-assay variation of 11.03% and 4.11% on average (Table 1), with acceptable ranges of less than 15% and 10%, respectively.
표 1: 인간 혈청 품질 관리 샘플 # 1-8 (HS1- HS8)을 이용한 PRO-C3 분석에서 분석 간 및 분석 내 편차. 편차는 각 샘플에 대한 10번의 각 측정치들 간의 평균 편차로서 계산하였다.Table 1: Inter- and intra-assay variation in the PRO-C3 assay using human serum quality control samples #1-8 (HS1-HS8). Deviation was calculated as the average deviation between each of the 10 measurements for each sample.
(ng/mL)value
(ng/mL)
희석 회수 (dilution recovery)는 인간, 랫 및 마우스의 건강한 혈청 및 혈장 샘플을 사용해 수행하였다. 희석 회수는 허용가능한 100±20% 회수율 범위내였다 (표 2). 추가적인 희석으로 LLQ 미만의 측정치가 수득되었다.Dilution recovery was performed using healthy serum and plasma samples from humans, rats and mice. Dilution recovery was within the acceptable range of 100 ± 20% recovery (Table 2). Additional dilutions gave measurements below the LLQ.
표 2: 인간, 랫 및 마우스 샘플을 이용한 PRO-C3 분석의 희석 회수율 %. 인간 혈청 (HS), 인간 혈장 (HP), 랫 혈청 (RS), 마우스 혈청 (MS), 마우스 혈장 (MP). Table 2: % dilution recovery for PRO-C3 assay using human, rat and mouse samples. Human serum (HS), human plasma (HP), rat serum (RS), mouse serum (MS), mouse plasma (MP).
ng/mlPIIINP
ng/ml
(n=2)HS
(n=2)
(n=3)HP
(n=3)
(n=10)R.S.
(n=10)
(n=2)M.S.
(n=2)
(n=2)M.P.
(n=2)
혈청 또는 혈장에 캘리브레이터 펩타이드 첨가 결과, 각각의 평균 회수율은 56% 및 55%이었다 (표 3).As a result of adding the calibrator peptide to serum or plasma, the average recovery rates were 56% and 55%, respectively (Table 3).
표 3: 인간 혈청 또는 혈장에서 캘리브레이터 펩타이드, 및 인간 혈청 또는 혈장에서 인간 AF의 첨가 회수율 (spiking recovery). 회수율은 순수한 혈청/혈장 대비 혈청/혈장내 계산된 펩타이드/AF의 회수율%로서 계산하였다. 캘리브레이터 펩타이드의 농도는 38.16 ng/ml (StdB), 19.08 ng/ml (StdC), 9.54 ng/ml (StdD), 4.77 ng/ml (StdE), 2.39 ng/ml (StdF) 및 1.19 ng/ml (StdG)이었다. AF는 1:2에서 시작해 2배 희석으로 첨가하였다.Table 3: Spiking recovery of calibrator peptide in human serum or plasma and human AF in human serum or plasma. The recovery rate was calculated as the percent recovery of peptide/AF calculated in serum/plasma compared to pure serum/plasma. The concentrations of calibrator peptides were 38.16 ng/ml (StdB), 19.08 ng/ml (StdC), 9.54 ng/ml (StdD), 4.77 ng/ml (StdE), 2.39 ng/ml (StdF) and 1.19 ng/ml ( It was StdG). AF was added in two-fold dilutions starting at 1:2.
sRE%Serum (n=3)
sRE%
sRE%Plasma (n=3)
sRE%
sRE%Serum (n=3)
sRE%
sRE%Plasma (n=3)
sRE%
그러나, 건강한 인간 혈청 또는 혈장에 1:2에서 시작한 2배 희석으로 인간 AF 첨가 결과, 각각 평균 회수율은 100% 및 111%였다. 헤모글로빈, 바이오틴 및 혈청지질이 여러가지 농도로 첨가된 혈청에서 간섭은 관찰되지 않았다 (표 4).However, addition of human AF at two-fold dilutions starting at 1:2 to healthy human serum or plasma resulted in average recoveries of 100% and 111%, respectively. No interference was observed in serum containing hemoglobin, biotin, and serum lipids added at various concentrations (Table 4).
표 4: 여러가지 농도로 첨가된 인간 혈청에서 헤모글로빈, 혈청지질 및 바이오틴의 간섭. 모든 데이타는 순수 혈청 대비 회수율%로서 나타낸다.Table 4: Interference of hemoglobin, serum lipids and biotin in human serum spiked at different concentrations. All data are expressed as percent recovery relative to pure serum.
mmol/L RE%hemoglobin
mmol/L RE%
mmol/L RE%serum lipids
mmol/L RE%
ng/L RE%biotin
ng/L RE%
분석물의 안정성은 최대 4번의 냉동/해동 사이클에서 허용가능하였으며, 1회 냉동/해동 사이클과 비교해 회수율은 100±20%이었다 (표 5). The stability of the analyte was acceptable for up to four freeze/thaw cycles, and recovery was 100±20% compared to one freeze/thaw cycle (Table 5).
표 5: 4번의 냉동/해동 사이클에서 인간 혈청 및 혈장 샘플 3종의 안정성. 모든 데이타는 1회 냉동/해동 사이클 대비 평균 회수%로 나타낸다.Table 5: Stability of three human serum and plasma samples over four freeze/thaw cycles. All data are expressed as average recovery percentage compared to one freeze/thaw cycle.
평균 회수율 %serum
Average Recovery %
평균 회수율 %EDTA plasma
Average Recovery %
평균 회수율 %heparin plasma
Average Recovery %
평균 회수율 %citrate plasma
Average Recovery %
실시예Example 3 - 결합 친화성의 비율 결정 3 - Determination of the ratio of binding affinity
타겟 서열에 대한 단일클론 항체의 결합 친화성 : 연장된 또는 축소된 서열에 대한 단일클론 항체의 결합 친화성의 비를 구하기 위해, 각 서열을 합성하였으며, 실시예 2에 기술된 바와 같이 PRO-C3 ELISA에서 캘리브레이터 펩타이드로 사용하였다. 수득한 캘리브레이션 곡선을 사용해 각 서열/항체 조합의 IC50 값을 구한다. IC50[타겟] / IC50[연장된 또는 축소된]의 비는 결합 친화성의 비를 정의한다.Binding affinity of monoclonal antibodies to target sequences: To determine the ratio of binding affinity of monoclonal antibodies to extended or shortened sequences, each sequence was synthesized and subjected to PRO-C3 ELISA as described in Example 2. was used as a calibrator peptide. The IC 50 value of each sequence/antibody combination is determined using the obtained calibration curve. The ratio IC 50 [target] / IC 50 [extended or shortened] defines the ratio of binding affinities.
실시예Example 4 - PRO- 4-PRO- C3XC3X 분석 analyze
전술한 바와 같이, 라이실 산화효소 (LOX)에 의한 효소적 콜라겐 가교 및 프로콜라겐의 프로세싱은 조직 성숙화 및 안정성에 중요하다. 따라서, 효소적 프로세싱하기 전 프로콜라겐 타입 III의 가닥 간 가교 모니터링은 LOX의 생체내 활성을 모니터링하는데 유용함을 입증해 줄 수 있다. 이는, 가교된 PIIINP (즉, 프로콜라겐의 효소적 프로세싱 전, 프로콜라겐 타입 III에서 LOX에 의해 형성된 가닥 간 결합에 의해 결합된 PIIINP 2가닥 이상)를 검출 및 정량함으로써 달성할 수 있다. 순환계에서 검출된 가교된 PIIINP의 높은 수준은 LOX 활성이 더 높다는 것을 의미할 것이다. 즉, LOX를 타겟팅하는 약물, 예를 들어, LOX 길항제 약물에 대한 약물 실험시 가교된 PIIINP의 수준 모니터링은 약물에 유용한 효능 데이타를 제공할 수 있다.As described above, enzymatic collagen cross-linking and processing of procollagen by lysyl oxidase (LOX) is important for tissue maturation and stability. Therefore, monitoring interstrand cross-linking of procollagen type III prior to enzymatic processing may prove useful in monitoring the in vivo activity of LOX. This can be achieved by detecting and quantifying cross-linked PIIINPs (i.e., two or more strands of PIIINPs bound by interstrand bonds formed by LOX in procollagen type III before enzymatic processing of procollagen). The higher levels of cross-linked PIIINP detected in the circulation would indicate higher LOX activity. That is, monitoring the level of cross-linked PIIINP during drug testing for drugs targeting LOX, for example, LOX antagonist drugs, can provide useful efficacy data for the drug.
ELISAELISA
스트렙타비딘-코팅된 플레이트를 1 ㎍/ml 바이오틴화 포획 항체 (바이오틴-연결된 NB61-N62)로 100 ㎕/웰로 코팅하여, 30분간 300 rpm으로 교반하면서 20℃에서 인큐베이션하였다. 플레이트를 세척 완충제 (20 nM TRIS, 50 mM NaCl, pH 7.2)로 5번 헹구었다. 샘플 표준 물질 또는 대조군 (20 ㎕)을 첨가한 다음, 즉시 분석 완충제 100 ㎕를 첨가하고, 20시간 동안 300 rpm으로 교반하면서 4℃에서 인큐베이션하였다. 인큐베이션 후, 플레이트를 세척 완충제로 5번 헹구었다. 1 ㎍/ml HRP-표지된 검출 항체 (HRP-연결된 NB61-N62)를 100 ㎕/웰로 첨가하여, 1시간 동안 300 rpm으로 교반하면서 20℃에서 인큐베이션하였다. 인큐베이션 후, 플레이트를 세척 완충제로 5번 헹구었다. 3,3',5,5'-테트라메틸벤지딘 (TMB) 100 ㎕를 첨가하여 암 조건에서 20℃에서 15분간 인큐베이션하였다. TMB의 효소 반응을 중단시키기 위해, 0.1% 황산 100 ㎕를 첨가하였다. 그런 후, 효소 반응을 이차 곡선 피트 (quadratic curve fit)를 사용해 ELISA 리더에서 판독하였다. 각 ELISA 플레이트에는 키트 대조군 및 사내 품질 관리 샘플을 모두 포함시켜, 분석 간 편차를 모니터링하였다. 샘플들 모두 특이적인 분석의 범위 내에서 측정하였다. 정량 하한 수준 (LLOQ) 미만인 샘플들은 모두 LLOQ 값을 할당하였다.Streptavidin-coated plates were coated at 100 μl/well with 1 μg/ml biotinylated capture antibody (biotin-linked NB61-N62) and incubated at 20°C with agitation at 300 rpm for 30 minutes. Plates were rinsed five times with wash buffer (20 nM TRIS, 50 mM NaCl, pH 7.2). Sample standards or controls (20 μl) were added, followed immediately by 100 μl of assay buffer and incubated at 4°C with agitation at 300 rpm for 20 hours. After incubation, the plate was rinsed five times with wash buffer. 1 μg/ml HRP-labeled detection antibody (HRP-linked NB61-N62) was added at 100 μl/well and incubated at 20°C with agitation at 300 rpm for 1 hour. After incubation, the plate was rinsed five times with wash buffer. 100 ㎕ of 3,3',5,5'-tetramethylbenzidine (TMB) was added and incubated at 20°C for 15 minutes in dark conditions. To stop the enzymatic reaction of TMB, 100 μl of 0.1% sulfuric acid was added. The enzymatic reaction was then read in an ELISA reader using a quadratic curve fit. Each ELISA plate included both kit controls and in-house quality control samples to monitor inter-assay variation. All samples were measured within the scope of the specific analysis. All samples below the lower limit of quantification (LLOQ) were assigned an LLOQ value.
분석의 기술적인 특징은 다음과 같다:Technical features of the analysis include:
혈청 내 혈청: 86%Peptides in serum: 94%
Serum in serum: 86%
결과result
반흔 조직scar tissue
폐 섬유모세포의 시험관내 모델을 이용한 예비 결과 ("scar-in-a-jar"), LOX 패밀리 효소가 PIIINP에서 가교를 담당한다는 것이, 강력하게 시사되었다 (TGF-β는 라이실 옥시다제 (LOX) 효소 활성을 높이는 것으로 당해 기술 분야에 공지되어 있음). 간략하게는, 크라운드 조건 (crowded condition) 및 TGF-β 자극 하에 5일간 폐 섬유모세포를 배양하여 Pro-C3X (즉, 가교된 PIIINP)를 생산하였다. Pro-C3X는 TGF-β와 함께 12일 배양한 후 현저하게 증가하였으며, TGF-β 부재시에는 Pro-C3X가 극소량 관찰되었다 (도 4). 마찬가지로, Pro-C3X는 정상 피부의 추출물과 비교해 켈로이드 추출물에서 증가하였다 (도 5).Preliminary results using an in vitro model of lung fibroblasts (“scar-in-a-jar”) strongly suggest that LOX family enzymes are responsible for cross-linking in PIIINP (TGF-β is lysyl oxidase (LOX ) is known in the art to increase enzyme activity). Briefly, Pro-C3X (i.e., cross-linked PIIINP) was produced by culturing lung fibroblasts for 5 days under crowded conditions and TGF-β stimulation. Pro-C3X significantly increased after culturing with TGF-β for 12 days, and only a small amount of Pro-C3X was observed in the absence of TGF-β (Figure 4). Likewise, Pro-C3X was increased in keloid extracts compared to extracts from normal skin (Figure 5).
간 섬유증liver fibrosis
간 섬유증 환자에 대한 실험은 "Pro-C3X" 분석을 이용해 수행하였으며, 본원에 기술된 "Pro-C3" 경쟁적인 ELISA와 비교하였다. Pro-C3X는 섬유증이 보다 심각한 상황인 질환의 후기에 현저하게 증가하는 것으로 확인되었으며, 질환 초기에는 건강한 대조군과 비슷한 수준이었다 (도 6A). 이와 비교해, Pro-C3 수준은 질환의 모든 단계들에서 상이하였다 (도 6B).Experiments in patients with liver fibrosis were performed using the “Pro-C3X” assay and compared to the “Pro-C3” competitive ELISA described herein. Pro-C3X was found to increase significantly in the later stage of the disease when fibrosis was more severe, and was at a level similar to that of the healthy control group in the early stage of the disease (Figure 6A). In comparison, Pro-C3 levels were different across all stages of the disease (Figure 6B).
Pro-C3X 분석과 Pro-C3 분석 간의 선택성 차이는, Pro-C3X 분석이 가교된 PIIINP만 인지하는 반면, Pro-C3 분석은 가교된 및 비-가교된 PIIINP 둘다를 인지한다는 것에 기초한다. 도 7은 Pro-C3X을 도시한 것으로, 이러한 결론의 근거를 회화적으로 도시한다:The difference in selectivity between the Pro-C3X assay and the Pro-C3 assay is based on the fact that the Pro-C3X assay recognizes only cross-linked PIIINPs, while the Pro-C3 assay recognizes both cross-linked and non-cross-linked PIIINPs. Figure 7 depicts Pro-C3X, pictorially illustrating the basis for this conclusion:
Pro-C3X 분석: 가교된 PIIINP가 존재하는 경우, 제1 항체가 PIIINP의 제1 가닥 상의 유리 에피토프 (free epitope)에 결합하게 될 것이며, 이후 제2 항체가 PIIINP의 제2 가닥의 유리 에피토프에 결합하게 될 것이다. 그러나, 비-가교된 PIIINP가 존재하는 경우, 표면-결합된 항체는 비-가교된 콜라겐 타입 III의 유리 에피토프에 결합하게 되지만, 제2 항체는 결합 에피토프가 이미 점유되어 있어 결합에 실패하게 되며, 그래서 제2 항체 첨가시 신호 발생에는 실패하게 된다. 따라서, Pro-C3X 분석의 신호는 오직 가교된 PIIINP 검출에 의한 것이다.Pro-C3X assay: If cross-linked PIIINP is present, the first antibody will bind to the free epitope on the first strand of PIIINP, and then the second antibody will bind to the free epitope of the second strand of PIIINP. It will be done. However, in the presence of non-crosslinked PIIINP, the surface-bound antibody binds to the free epitope of non-crosslinked collagen type III, but the second antibody fails to bind because the binding epitope is already occupied; Therefore, when the second antibody is added, signal generation fails. Therefore, the signal of the Pro-C3X assay is solely due to detection of cross-linked PIIINP.
반대로, Pro-C3 분석에서 항체는, PIIINP가 가교되거나 또는 가교되지 않든 상관없이, 실질적으로 모두 결합성 유리 에피토프를 포함하는 PIIINP 가닥에 결합할 것이다. 따라서, Pro-C3 분석으로부터 수득한 신호는 가교된 PIIINP 및 비-가교된 PIIINP의 조합 신호 (aggregate signal)이다.Conversely, in the Pro-C3 assay, the antibody will bind to a PIIINP strand containing substantially all binding free epitopes, regardless of whether the PIIINP is cross-linked or not. Therefore, the signal obtained from the Pro-C3 assay is the aggregate signal of cross-linked and non-cross-linked PIIINP.
즉, 이러한 선택성은 LOX를 타겟팅하는 약물의 효능 평가에 있어 Pro-C3X 분석의 사용을 촉구하며; 개체에 약물 투여하는 기간 중에 LOX 활성의 결과인 것으로 보이는 전술한 PIIINP 가교 수준의 모니터링은 약물의 활성을 모니터링하여 약물의 효능을 모니터링하는데 이용할 수 있다.That is, this selectivity urges the use of the Pro-C3X assay in evaluating the efficacy of drugs targeting LOX; Monitoring the level of PIIINP cross-linking described above, which appears to be a result of LOX activity during the period of drug administration to a subject, can be used to monitor the activity of the drug and thus the efficacy of the drug.
알코올성 alcoholic 지방간염Steatohepatitis
알코올성 지방간염 환자에 대한 실험을 "Pro-C3X" 분석을 이용해 수행하고, 본원에 기술된 "Pro-C3" 분석과 비교하였다. 알코올성 지방간염에서, Pro-C3 및 Pro-C3X 둘다 질환의 후기에 증가되었다 (Metavir 2-4). 그러나, 간경변 환자에서 Pro-C3는 MELD (말기 간 질환 모델) 스코어 (P=0.527)와 상관관계가 없었지만, Pro-C3X는 강력한 상관관계가 존재하였다 (상관 계수 3.34, P<0.001). ProC3는 단 알부민과 음의 상관관계가 존재하지만, ProC3X는 알부민, 빌리루빈 및 감마-글루타밀 트랜스펩티다제 (GGT)와 상관관계가 존재하였다 (도 8). 후기 단계에 Pro-C3X 증가는, PIIINP 가교 증가를 시사하며, 이는 간의 반흔 증가와 일치할 것이다 (즉, LOX 활성 증가).Experiments on patients with alcoholic steatohepatitis were performed using the “Pro-C3X” assay and compared to the “Pro-C3” assay described herein. In alcoholic steatohepatitis, both Pro-C3 and Pro-C3X were increased in the later stages of the disease (Metavir 2-4). However, in patients with cirrhosis, Pro-C3 did not correlate with the MELD (Model for End Stage Liver Disease) score (P=0.527), but there was a strong correlation with Pro-C3X (correlation coefficient 3.34, P<0.001). ProC3 only had a negative correlation with albumin, but ProC3X had a correlation with albumin, bilirubin, and gamma-glutamyl transpeptidase (GGT) (Figure 8). Increased Pro-C3X in later stages suggests increased PIIINP cross-linking, which would be consistent with increased liver scarring (i.e. increased LOX activity).
실시예Example 5 5 Scar-in-a-Jar 모델에서 Pro-Pro- in Scar-in-a-Jar model C3XC3X 평가 evaluation
배경 : 섬유증은 발병된 조직 내에 세포외 기질 (ECM)이 축적된 것으로, 이는 장기 부전과 궁극적으로 사멸을 유발할 수 있다. 예를 들어, 형질전환 성장인자 (TGF)-β에 의한 자극 후, 섬유모세포는 ECM 단백질, 특히 콜라겐의 과도한 축적을 야기하는 주 세포 타입이다. 여기서, 본 발명자들은, 콜라겐 형성 및 섬유 형성 중의 가교 형성을 조사하기 위해, Pro-C3X ELISA와 조합하여, ECM 및 가교를 형성하는 것으로 공지된 시험관내 모델 "Scar-in-a-Jar"의 사용을 기술한다. 이 툴은 섬유성 프로세스의 조정을 분석함으로써 새로운 항-섬유성 화합물을 조사하는데 이용할 수 있다. Background : Fibrosis is an accumulation of extracellular matrix (ECM) within affected tissues, which can lead to organ failure and ultimately death. For example, after stimulation with transforming growth factor (TGF)-β, fibroblasts are the predominant cell type that results in excessive accumulation of ECM proteins, especially collagen. Here, we used the in vitro model “Scar-in-a-Jar”, known to form ECM and cross-links, in combination with Pro-C3X ELISA to investigate cross-link formation during collagen formation and fibrogenesis. Describe. This tool can be used to investigate new anti-fibrotic compounds by analyzing the modulation of fibrotic processes.
방법 : 건강한 인간 폐 섬유모세포 (L248)를 세포 30,000 개/웰의 밀도로 접종하여 컨플루언스로 배양하였다. 세포를 0.4% FCS, 225 mg/mL ficoll 70, 150 mg/mL ficoll 400 및 1% 아스코르브산을 포함하는 DMEM에서 18일간 배양하였다. 세포를 1 ng/mL TGF-β로, 단독으로 또는 라이실 옥시다제 (LOX) 저해제 β-아미노프로피오니트릴 (BAPN; 0.02 또는 0.2 mM)을 첨가하여 자극하여, 가교 형성을 저해하였다. ficoll 비-함유 배지에서 배양한 비-자극 세포 또는 세포를 대조군으로 사용하였다. 배지를 3일, 6일, 10일 및 14일에 교체하였다. 섬유모세포 생존성을 AlamarBlue 분석으로 평가하였다. Pro-C3X 수준을 전술한 Pro-C3X 샌드위치 ELISA를 사용해 수집한 상층액에서 분석하였다. Method : Healthy human lung fibroblasts (L248) were seeded at a density of 30,000 cells/well and cultured to confluence. Cells were cultured in DMEM containing 0.4% FCS, 225 mg/mL ficoll 70, 150 mg/mL ficoll 400, and 1% ascorbic acid for 18 days. Cells were stimulated with 1 ng/mL TGF-β, alone or with the addition of the lysyl oxidase (LOX) inhibitor β-aminopropionitrile (BAPN; 0.02 or 0.2 mM) to inhibit cross-link formation. Non-stimulated cells or cells cultured in ficoll-free medium were used as controls. The medium was changed on days 3, 6, 10, and 14. Fibroblast viability was assessed by AlamarBlue assay. Pro-C3X levels were analyzed in collected supernatants using the Pro-C3X sandwich ELISA described above.
결과 : TGF-β 자극은 3일부터 Pro-C3X의 방출을 유도하였으며, Pro-C3X 수준은 10일에 최고 수준에 도달하였으며, 이는 비-자극 세포와 비교해 14배 증가한 수준이었다 (p<0.0001; 도 9). 0.02 mM BAPN 처리는 유의한 효과가 없었으며, 0.2 mM BAPN은 TGF-β 자극 단독에 비해 Pro-C3X 수준의 유의한 감소를 유도하였다 (0.59-배수 변화, p<0.0001; 도 9). Results : TGF-β stimulation induced the release of Pro-C3X starting at day 3, and Pro-C3X levels reached the highest level at day 10, which was a 14-fold increase compared to non-stimulated cells (p<0.0001; Figure 9). Treatment with 0.02mM BAPN had no significant effect, and 0.2mM BAPN induced a significant decrease in Pro-C3X levels compared to TGF-β stimulation alone (0.59-fold change, p<0.0001; Figure 9).
결론: pan LOX 저해제 BAPN은 0.2 mM 농도에서 Pro-C3X 수준을 현저하게 감소시켰으며, 이는 Pro-C3X ELISA가 가교된 에피토프를 분석한다는 것을 의미한다. 따라서, Pro-C3X ELISA를 이용해 섬유모세포 활성을 평가할 수 있으며, 따라서 잠재적인 항-섬유성 화합물을 스크리닝할 수 있다. TGF-β 자극은 가교된 콜라겐 타입 III pro-펩타이드의 마커인 Pro-C3X의 방출을 유도하였다. Conclusions : The pan LOX inhibitor BAPN significantly reduced Pro-C3X levels at 0.2 mM concentration, indicating that the Pro-C3X ELISA analyzes cross-linked epitopes. Therefore, the Pro-C3X ELISA can be used to assess fibroblast activity and thus screen for potential anti-fibrotic compounds. TGF-β stimulation induced the release of Pro-C3X, a marker of cross-linked collagen type III pro-peptide.
결론적으로, Pro-C3X 분석은 콜라겐 타입 III pro-펩타이드의 절단 네오-에피토프와 분자내 가교의 존재를 조합한 2세대 분석이다. 이 분석은 섬유 형성 타임라인에서 다른 프로세스, 즉 반흔에서 콜라겐 분자의 가교를 묘사하므로, 따라서 Pro-C3 측정에 부가적인 정보를 제공해준다. 따라서, LOX 저해제의 사용이 가교된 PIIINP 바이오마커의 존재를 감소시키는 것으로 입증된 바, Pro-C3X 분석을 이용해 LOX를 타겟팅하는 약물, 특히 LOX 길항제/저해제 약물의 효능을 검사할 수 있다.In conclusion, the Pro-C3X assay is a second-generation assay that combines the presence of intramolecular cross-links with a cleaved neo-epitope of collagen type III pro-peptide. This analysis depicts another process in the fibrogenesis timeline, namely the cross-linking of collagen molecules in the scar, and thus provides additional information to Pro-C3 measurements. Therefore, the Pro-C3X assay can be used to test the efficacy of drugs targeting LOX, especially LOX antagonist/inhibitor drugs, as the use of LOX inhibitors has been demonstrated to reduce the presence of cross-linked PIIINP biomarkers.
본 명세서에서, 달리 언급되지 않은 한, '또는'이라는 용어는, '배타적으로 또는'이라는 연산자가 조건들 중 어느 하나만 충족하는 것에 반해, 언급된 조건 중 어느 하나 또는 둘다를 충족시키는 경우를 참값으로 하는 연산자의 의미로 사용된다. 용어 '포함하는'은 '로 이루어진'의 의미 보다는 '비롯한'의 의미로 사용된다. 전술한 모든 종래 기술에 대한 내용들은 원용에 의해 본 명세서에 포함된다. 본원에서 임의의 기존 공개 문헌들에 대한 내용은, 그 교시 내용이 현 시점에 호주 또는 그외 국가들에서 통상적인 일반 지식임을 용인 또는 언급하는 것으로 해석되어서는 안된다.In this specification, unless otherwise stated, the term 'or' refers to the case where one or both of the conditions mentioned are satisfied, whereas the operator 'or' exclusively satisfies only one of the conditions. It is used in the meaning of an operator. The term 'including' is used to mean 'including' rather than 'consisting of'. All contents of the prior art described above are incorporated herein by reference. Reference herein to any existing published documents should not be construed as an admission or statement that the teachings are now common general knowledge in Australia or any other country.
SEQUENCE LISTING <110> Nordic Bioscience A/S <120> PIIINP Neo-epitope Assay <130> P19876WO <150> US15/014,241 <151> 2016-02-03 <160> 15 <170> BiSSAP 1.3.6 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Prior art peptide <220> <221> SITE <222> 2 <223> acetamido protected Cys <400> 1 Ile Cys Gln Ser Cys Pro Thr Gly Gly Glu Asn Tyr Ser Pro 1 5 10 <210> 2 <211> 14 <212> PRT <213> Bovine <220> <223> Bovine PIIINP C-terminal sequence <400> 2 Ile Cys Gln Ser Cys Pro Thr Gly Gly Gln Asn Tyr Ser Pro 1 5 10 <210> 3 <211> 21 <212> PRT <213> Homo sapiens <220> <223> C-terminal PIIINP sequence <400> 3 Gly Ser Pro Gly Pro Pro Gly Ile Cys Gln Ser Cys Pro Thr Gly Pro 1 5 10 15 Gln Asn Tyr Ser Pro 20 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Antibody epitope <220> <221> SITE <222> 5 <223> Xaa can be Pro or Gly <400> 4 Cys Pro Thr Gly Xaa Gln Asn Tyr Ser Pro 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Elongated epitope peptide <220> <221> SITE <222> 5 <223> Xaa can be Pro or Gly <220> <221> SITE <222> 12 <223> Xaa can be absent or can be one or more amino acids of the sequence of collagen type III <400> 5 Cys Pro Thr Gly Xaa Gln Asn Tyr Ser Pro Gln Xaa 1 5 10 <210> 6 <211> 10 <212> PRT <213> Homo sapiens <220> <223> Human PIIINP neo-epitope C-terminal sequence <400> 6 Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 7 <211> 10 <212> PRT <213> Rodentia sp. <220> <223> Rodent PIIINP neo-epitope C-terminal sequence <400> 7 Cys Pro Thr Gly Gly Gln Asn Tyr Ser Pro 1 5 10 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Shortened epitope peptide <220> <221> SITE <222> 5 <223> Xaa can be Pro or Gly <400> 8 Cys Pro Thr Gly Xaa Gln Asn Tyr Ser 1 5 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> 1 <223> Xaa is absent or is biotinylated-Cys Gly Gly <220> <223> Biotinylated peptide <220> <221> SITE <222> 2 <223> Cys is biotinylated if Xaa is absent <400> 9 Xaa Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> 1 <223> Cys has N-terminal bound ovalbumin <220> <223> Ovalbumin bound <400> 10 Cys Gly Gly Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 11 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> 1 <223> Cys is N-terminal biotinylated <220> <223> Biotinylated peptide <400> 11 Cys Gly Gly Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Non-sense peptide <400> 12 Gly Ser Pro Gly Lys Asp Gly Val Arg Gly 1 5 10 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> elongated peptide <400> 13 Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro Gln 1 5 10 <210> 14 <211> 179 <212> PRT <213> Homo sapiens <400> 14 Met Met Ser Phe Val Gln Lys Gly Ser Trp Leu Leu Leu Ala Leu Leu 1 5 10 15 His Pro Thr Ile Ile Leu Ala Gln Gln Glu Ala Val Glu Gly Gly Cys 20 25 30 Ser His Leu Gly Gln Ser Tyr Ala Asp Arg Asp Val Trp Lys Pro Glu 35 40 45 Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Ser Val Leu Cys Asp Asp 50 55 60 Ile Ile Cys Asp Asp Gln Glu Leu Asp Cys Pro Asn Pro Glu Ile Pro 65 70 75 80 Phe Gly Glu Cys Cys Ala Val Cys Pro Gln Pro Pro Thr Ala Pro Thr 85 90 95 Arg Pro Pro Asn Gly Gln Gly Pro Gln Gly Pro Lys Gly Asp Pro Gly 100 105 110 Pro Pro Gly Ile Pro Gly Arg Asn Gly Asp Pro Gly Ile Pro Gly Gln 115 120 125 Pro Gly Ser Pro Gly Ser Pro Gly Pro Pro Gly Ile Cys Glu Ser Cys 130 135 140 Pro Thr Gly Pro Gln Asn Tyr Ser Pro Gln Tyr Asp Ser Tyr Asp Val 145 150 155 160 Lys Ser Gly Val Ala Val Gly Gly Leu Ala Gly Tyr Pro Gly Pro Ala 165 170 175 Gly Pro Pro <210> 15 <211> 178 <212> PRT <213> Rattus <400> 15 Met Met Ser Phe Val Gln Cys Gly Thr Trp Phe Leu Leu Thr Leu Leu 1 5 10 15 His Pro Ser Leu Ile Leu Ala Gln Gln Ser Asn Val Asp Glu Leu Gly 20 25 30 Cys Asn Tyr Leu Gly Gln Ser Tyr Glu Ser Arg Asp Val Trp Lys Pro 35 40 45 Glu Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Ser Val Leu Cys Asp 50 55 60 Asp Ile Met Cys Asp Asp Glu Pro Leu Asp Cys Pro Asn Pro Glu Ile 65 70 75 80 Pro Phe Gly Glu Cys Cys Ala Ile Cys Pro Gln Pro Ser Thr Pro Ala 85 90 95 Pro Val Ile Pro Asp Gly Asn Arg Pro Gln Gly Pro Lys Gly Asp Pro 100 105 110 Gly Pro Pro Gly Ile Pro Gly Arg Asn Gly Asp Pro Gly Leu Pro Gly 115 120 125 Gln Pro Gly Leu Pro Gly Pro Pro Gly Ser Pro Gly Ile Cys Glu Ser 130 135 140 Cys Pro Thr Gly Gly Gln Asn Tyr Ser Pro Gln Phe Asp Ser Tyr Asp 145 150 155 160 Val Lys Ser Gly Val Gly Gly Met Gly Gly Tyr Pro Gly Pro Ala Gly 165 170 175 Pro Pro SEQUENCE LISTING <110> Nordic Bioscience A/S <120> PIIINP Neo-epitope Assay <130>P19876WO <150> US15/014,241 <151> 2016-02-03 <160> 15 <170> BiSSAP 1.3.6 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Prior art peptide <220> <221> SITE <222> 2 <223> acetamido protected Cys <400> 1 Ile Cys Gln Ser Cys Pro Thr Gly Gly Glu Asn Tyr Ser Pro 1 5 10 <210> 2 <211> 14 <212> PRT <213> Bovine <220> <223> Bovine PIIINP C-terminal sequence <400> 2 Ile Cys Gln Ser Cys Pro Thr Gly Gly Gln Asn Tyr Ser Pro 1 5 10 <210> 3 <211> 21 <212> PRT <213> Homo sapiens <220> <223> C-terminal PIIINP sequence <400> 3 Gly Ser Pro Gly Pro Pro Gly Ile Cys Gln Ser Cys Pro Thr Gly Pro 1 5 10 15 Gln Asn Tyr Ser Pro 20 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Antibody epitope <220> <221> SITE <222> 5 <223> Xaa can be Pro or Gly <400> 4 Cys Pro Thr Gly Xaa Gln Asn Tyr Ser Pro 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Elongated epitope peptide <220> <221> SITE <222> 5 <223> Xaa can be Pro or Gly <220> <221> SITE <222> 12 <223> Xaa can be absent or can be one or more amino acids of the sequence of collagen type III <400> 5 Cys Pro Thr Gly Xaa Gln Asn Tyr Ser Pro Gln Xaa 1 5 10 <210> 6 <211> 10 <212> PRT <213> Homo sapiens <220> <223> Human PIIINP neo-epitope C-terminal sequence <400> 6 Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 7 <211> 10 <212> PRT <213> Rodentia sp. <220> <223> Rodent PIIINP neo-epitope C-terminal sequence <400> 7 Cys Pro Thr Gly Gly Gln Asn Tyr Ser Pro 1 5 10 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Shortened epitope peptide <220> <221> SITE <222> 5 <223> Xaa can be Pro or Gly <400> 8 Cys Pro Thr Gly Xaa Gln Asn Tyr Ser 1 5 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> 1 <223> Xaa is absent or is biotinylated-Cys Gly Gly <220> <223> Biotinylated peptide <220> <221> SITE <222> 2 <223> Cys is biotinylated if Xaa is absent <400> 9 Xaa Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> 1 <223> Cys has N-terminal bound ovalbumin <220> <223> Ovalbumin bound <400> 10 Cys Gly Gly Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 11 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> 1 <223> Cys is N-terminal biotinylated <220> <223> Biotinylated peptide <400> 11 Cys Gly Gly Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Non-sense peptide <400> 12 Gly Ser Pro Gly Lys Asp Gly Val Arg Gly 1 5 10 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> elongated peptide <400> 13 Cys Pro Thr Gly Pro Gln Asn Tyr Ser Pro Gln 1 5 10 <210> 14 <211> 179 <212> PRT <213> Homo sapiens <400> 14 Met Met Ser Phe Val Gln Lys Gly Ser Trp Leu Leu Leu Ala Leu Leu 1 5 10 15 His Pro Thr Ile Ile Leu Ala Gln Gln Glu Ala Val Glu Gly Gly Cys 20 25 30 Ser His Leu Gly Gln Ser Tyr Ala Asp Arg Asp Val Trp Lys Pro Glu 35 40 45 Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Ser Val Leu Cys Asp Asp 50 55 60 Ile Ile Cys Asp Asp Gln Glu Leu Asp Cys Pro Asn Pro Glu Ile Pro 65 70 75 80 Phe Gly Glu Cys Cys Ala Val Cys Pro Gln Pro Pro Thr Ala Pro Thr 85 90 95 Arg Pro Pro Asn Gly Gln Gly Pro Gln Gly Pro Lys Gly Asp Pro Gly 100 105 110 Pro Pro Gly Ile Pro Gly Arg Asn Gly Asp Pro Gly Ile Pro Gly Gln 115 120 125 Pro Gly Ser Pro Gly Ser Pro Gly Pro Pro Gly Ile Cys Glu Ser Cys 130 135 140 Pro Thr Gly Pro Gln Asn Tyr Ser Pro Gln Tyr Asp Ser Tyr Asp Val 145 150 155 160 Lys Ser Gly Val Ala Val Gly Gly Leu Ala Gly Tyr Pro Gly Pro Ala 165 170 175 Gly Pro Pro <210> 15 <211> 178 <212> PRT <213> Rattus <400> 15 Met Met Ser Phe Val Gln Cys Gly Thr Trp Phe Leu Leu Thr Leu Leu 1 5 10 15 His Pro Ser Leu Ile Leu Ala Gln Gln Ser Asn Val Asp Glu Leu Gly 20 25 30 Cys Asn Tyr Leu Gly Gln Ser Tyr Glu Ser Arg Asp Val Trp Lys Pro 35 40 45 Glu Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Ser Val Leu Cys Asp 50 55 60 Asp Ile Met Cys Asp Asp Glu Pro Leu Asp Cys Pro Asn Pro Glu Ile 65 70 75 80 Pro Phe Gly Glu Cys Cys Ala Ile Cys Pro Gln Pro Ser Thr Pro Ala 85 90 95 Pro Val Ile Pro Asp Gly Asn Arg Pro Gln Gly Pro Lys Gly Asp Pro 100 105 110 Gly Pro Pro Gly Ile Pro Gly Arg Asn Gly Asp Pro Gly Leu Pro Gly 115 120 125 Gln Pro Gly Leu Pro Gly Pro Pro Gly Ser Pro Gly Ile Cys Glu Ser 130 135 140 Cys Pro Thr Gly Gly Gln Asn Tyr Ser Pro Gln Phe Asp Ser Tyr Asp 145 150 155 160 Val Lys Ser Gly Val Gly Gly Met Gly Gly Tyr Pro Gly Pro Ala Gly 165 170 175 Pro Pro
Claims (16)
상기 가교된 PIIINP가 가닥 간 가교 (inter-strand cross-linking)에 의해 결합된 PIIINP 가닥 2개 이상을 포함하며,
상기 방법이,
가교된 PIIINP를 포함하는 생물학적 샘플을, 표면에 결합된 제1 단일클론 항체와 접촉시키는 단계;
제2 단일클론 항체를 첨가하는 단계; 및
상기 제2 단일클론 항체의 결합량을 측정하는 단계를 포함하며,
상기 가교된 PIIINP에 포함된 PIIINP의 각 가닥이 N-프로테아제에 의한 무손상 타입 III 프로콜라겐 (intact type III procollagen)의 절단에 의해 형성된 PIIINP의 C-말단 네오-에피토프 (C-terminal neo-epitope)를 포함하며,
상기 제1 단일클론 항체와 상기 제2 단일클론 항체가 PIIINP의 C-말단 네오-에피토프에 특이적으로 반응하며,
상기 네오-에피토프는 X가 Gly 또는 Pro인 C-말단 아미노산 서열 CPTGXQNYSP-COOH에 포함되는, 샌드위치 면역분석방법.A sandwich immunoassay method for detecting cross-linked PIIINP (PIIINP) in biological samples,
The cross-linked PIIINP includes two or more PIIINP strands joined by inter-strand cross-linking,
The above method is,
contacting a biological sample comprising cross-linked PIIINP with a first monoclonal antibody bound to the surface;
adding a second monoclonal antibody; and
It includes measuring the binding amount of the second monoclonal antibody,
Each strand of PIIINP included in the cross-linked PIIINP is a C-terminal neo-epitope of PIIINP formed by cleavage of intact type III procollagen by N-protease. Includes,
The first monoclonal antibody and the second monoclonal antibody specifically react with the C-terminal neo-epitope of PIIINP,
The neo-epitope is included in the C-terminal amino acid sequence CPTGXQNYSP-COOH, where X is Gly or Pro.
상기 단일클론 항체가 상기 C-말단 아미노산 서열의 연장된 버전 (elongated version)에 대해 실질적으로 인지 또는 결합하지 않으며,
상기 연장된 버전이, Z가 생략되거나 또는 콜라겐 타입 III 서열 중의 하나 이상의 아미노산인, CPTGXQNYSPQZ-COOH인, 샌드위치 면역분석방법.According to paragraph 1,
the monoclonal antibody does not substantially recognize or bind to an elongated version of the C-terminal amino acid sequence,
The extended version is CPTGXQNYSPQZ-COOH, where Z is omitted or is one or more amino acids of the collagen type III sequence.
상기 샌드위치 면역분석방법이 생물학적 샘플에서 가교된 PIIINP의 양을 정량하는데 사용되는, 샌드위치 면역분석방법.According to claim 1 or 2,
A sandwich immunoassay method, wherein the sandwich immunoassay method is used to quantify the amount of cross-linked PIIINP in a biological sample.
섬유증 질환의 중증도를 평가하기 위해, 상기 방법에 의해 측정된 가교된 PIIINP의 양을 질환 중증도가 공지된 섬유증 질환의 표준 샘플과 연관 (correlating)시키는 단계를 더 포함하는, 샌드위치 면역분석방법.According to paragraph 3,
A sandwich immunoassay method further comprising correlating the amount of cross-linked PIIINP measured by the method with a standard sample of fibrotic disease of known disease severity to assess the severity of fibrotic disease.
상기 섬유증 질환이 간 질환인, 샌드위치 면역분석방법.According to clause 4,
Sandwich immunoassay method, wherein the fibrotic disease is a liver disease.
상기 생물학적 샘플이 생물유체 (biofluid)인, 샌드위치 면역분석방법.According to paragraph 3,
A sandwich immunoassay method, wherein the biological sample is a biofluid.
상기 생물유체가 혈청, 혈장, 뇨, 양수, 조직 상층액 또는 세포 상층액인, 샌드위치 면역분석방법.According to clause 6,
A sandwich immunoassay method, wherein the biological fluid is serum, plasma, urine, amniotic fluid, tissue supernatant, or cell supernatant.
상기 샌드위치 면역분석방법이 방사성 면역분석, 형광 면역분석 또는 효소-연결된 면역흡착 분석인, 샌드위치 면역분석방법.According to paragraph 1,
A sandwich immunoassay method, wherein the sandwich immunoassay method is a radioimmunoassay, a fluorescence immunoassay, or an enzyme-linked immunosorbent assay.
상기 제2 단일클론 항체가 표지된 것인, 샌드위치 면역분석방법.According to paragraph 1,
A sandwich immunoassay method in which the second monoclonal antibody is labeled.
상기 제2 단일클론 항체가 효소-연결된 항체인, 샌드위치 면역분석방법.According to clause 9,
A sandwich immunoassay method, wherein the second monoclonal antibody is an enzyme-linked antibody.
상기 효소가 호스래디시 퍼옥시다제 (horseradish peroxidase, HRP)인, 샌드위치 면역분석방법.According to clause 10,
A sandwich immunoassay method in which the enzyme is horseradish peroxidase (HRP).
상기 제2 단일클론 항체가 방사성 표지되거나 또는 형광단이 연결된 것인, 샌드위치 면역분석방법.According to clause 9,
A sandwich immunoassay method, wherein the second monoclonal antibody is radioactively labeled or fluorophore-linked.
상기 제2 단일클론 항체의 결합량을 측정하기 위해 상기 제2 단일클론 항체를 인지하는 추가적인 표지된 항체를 사용하는, 샌드위치 면역분석방법.According to paragraph 1,
A sandwich immunoassay method using an additional labeled antibody that recognizes the second monoclonal antibody to measure the binding amount of the second monoclonal antibody.
제1항의 샌드위치 면역분석방법을 이용해 2종 이상의 생물학적 샘플에서 가교된 PIIINP의 양을 정량하는 단계를 포함하며,
상기 생물학적 샘플이, 개체에 상기 길항제 약물을 투여하는 기간 동안에, 제1 시점 및 이후의 하나 이상의 시점에서 상기 개체로부터 수득되며,
상기 길항제 약물을 투여하는 기간 동안에 상기 제1 시점부터 상기 이후의 하나 이상의 시점까지 가교된 PIIINP의 양적 감소는, 상기 길항제 약물이 LOX를 타겟팅하는 효과적인 길항제 약물임을 의미하는 것인, 방법.As a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidase (LOX),
It includes the step of quantifying the amount of cross-linked PIIINP in two or more biological samples using the sandwich immunoassay method of paragraph 1,
wherein the biological sample is obtained from the individual at a first time point and at one or more subsequent time points during the period of administration of the antagonist drug to the individual,
A decrease in the quantity of cross-linked PIIINP from the first time point to one or more subsequent time points during the period of administering the antagonist drug indicates that the antagonist drug is an effective antagonist drug targeting LOX.
상기 방법이 LOXL2를 타겟팅하는 길항제 약물의 효능을 평가하는, 방법.According to clause 14,
The method of claim 1, wherein the method assesses the efficacy of an antagonist drug targeting LOXL2.
제1항에 따른 제1 단일클론 항체가 결합된 고체 지지체; 및
제1항에 따른 제2 단일클론 항체를 포함하며,
상기 제2 단일클론 항체가 표지 물질을 포함하는, 키트.As a kit for use in a sandwich analysis method,
A solid support to which the first monoclonal antibody according to claim 1 is bound; and
Comprising a second monoclonal antibody according to claim 1,
A kit wherein the second monoclonal antibody includes a labeling substance.
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