KR20090092202A - ANTIBODY SPECIFICALLY BINDING TO A PHOSPHORYLATED PLC-γ PROTEIN - Google Patents

ANTIBODY SPECIFICALLY BINDING TO A PHOSPHORYLATED PLC-γ PROTEIN

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KR20090092202A
KR20090092202A KR1020080037341A KR20080037341A KR20090092202A KR 20090092202 A KR20090092202 A KR 20090092202A KR 1020080037341 A KR1020080037341 A KR 1020080037341A KR 20080037341 A KR20080037341 A KR 20080037341A KR 20090092202 A KR20090092202 A KR 20090092202A
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이서구
이종서
정준호
심현보
고봉국
구미영
이정원
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이화여자대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

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Abstract

A method for suppressing intracellular signaling uaing an antibody which specifically binds with a phosphorylated protein is provided to suppress intracellular signaling and treat and prevent prostate cancer. An antibody which specifically binds with a phosphorylated PLC-gamma (phospholipase C-gamma) comprises a complementarity determining region (CDR) of heavy chain and CDR of light chain. The CDR of the heavy chain comprises amino acids of the sequence numbers 26 (SEQ ID NO:26), 28, and 30. The CDR of light chain has amino acids of the sequence numbers 32, 34, and 36. The phosphorylated site of phosphorylated PLC-gamma protein is tyrosine.

Description

인산화된 ＰＬＣ-γ 단백질에 특이적으로 결합하는 항체{ANTIBODY SPECIFICALLY BINDING TO A PHOSPHORYLATED PLC-γ PROTEIN}ANTIBODY SPECIFICALLY BINDING TO A PHOSPHORYLATED PLC-γ PROTEIN

본 발명은 인산화된 단백질에 의한 세포 내 신호 전달을 억제하는 방법에 관한 것으로, 보다 구체적으로는, 인산화된 단백질에 특이적으로 결합하는 항체를 세포 내에서 발현시킴으로써 상기 인산화된 단백질에 의한 세포 내 신호 전달을 억제하는 방법 및 상기 항체를 세포 내에서 발현시키기 위한 발현 시스템에 관한 것이다.The present invention relates to a method for inhibiting intracellular signal transduction by a phosphorylated protein, more specifically, intracellular signal by the phosphorylated protein by expressing the antibody in the cell specifically binding to the phosphorylated protein A method of inhibiting delivery and an expression system for expressing said antibody in a cell.

단백질은 DNA상의 유전 정보로부터 전사(transcription) 및 번역(translation) 과정을 거쳐서 만들어진다. 만들어진 단백질은 세포의 구성 성분으로써 세포 골격 형성 등의 기능도 수행하지만, 많은 단백질들은 활성을 가지고 있고, 단백질의 활성 조절은 세포 내 기능 조절을 위한 세포 신호 전달에 매우 중요한 역할을 담당한다. Proteins are made from transcriptional and translational processes from genetic information on DNA. Although the produced protein is a component of the cell and performs functions such as cytoskeletal formation, many proteins have activity, and the activity regulation of the protein plays an important role in cellular signal transduction for the regulation of intracellular function.

단백질에 의한 세포 내 신호 전달의 조절은 단백질 활성의 on/off를 통해 이루어질 수 있으며, 단백질의 활성화 또는 비활성화의 유도를 위해서는 인산화(phosphorylation), 글리코실화 (glycosylation), 메틸화 (methylation), 아세틸화 (acetylation), 단백질-단백질 상호작용 (protein-protein interaction) 등 다양한 변형(modification) 방법이 사용되고 있다. 이와 같이, 번역 후 변형 (post-translational modification)은 세포 내 신호 전달에 매우 중요한 역할을 수행하는 데, 이 중에서 단백질 인산화 (protein phosphorylation)는 세포 외부와 내부의 환경으로부터 유도되는 신호(signal)가 핵 내의 유전자 발현에 영향을 미치는 주된 작용 기전이다 (Manning G, 등, the protein kinase complement of the human genome. Science 298: 1912-1934 (2002)).Regulation of intracellular signal transduction by proteins can be achieved through on / off of protein activity, and phosphorylation, glycosylation, methylation, acetylation ( Various modification methods have been used, such as acetylation and protein-protein interactions. As such, post-translational modification plays an important role in intracellular signal transduction, among which protein phosphorylation is a signal derived from the environment outside the cell and the nucleus. It is the main mechanism of action that affects gene expression within (Manning G, et al., The protein kinase complement of the human genome. Science 298: 1912-1934 (2002)).

이러한 단백질 인산화는 단백질의 활성, 구조, 다른 단백질과의 결합력 등에 영향을 미침으로써 세포 내 신호전달을 담당하는 것으로 알려져 있다 (Hunter T, 등, Tyrosine phosphorylation in cell signaling and disease. Keio J. Med, 51:61-71 (2002)). 분자생물학적 지식이 발달하면서 이러한 기전을 바탕으로 한 새로운 약물들이 개발되고 있다. 대표적인 것이 이러한 단백질의 인산화된 부위와 결합하는 단백질의 활성을 억제하는 방식이다.Such protein phosphorylation is known to be responsible for intracellular signaling by affecting protein activity, structure, and binding ability with other proteins (Hunter T, et al., Tyrosine phosphorylation in cell signaling and disease. Keio J. Med , 51 : 61-71 (2002)). As molecular biology knowledge develops, new drugs based on this mechanism are being developed. Representative is a way of inhibiting the activity of proteins that bind to the phosphorylated sites of these proteins.

세포의 증식을 유도하는 신호전달 단백질인 STAT3 (signal transducers and activators of transcription 3)는, 인터루킨(interleukin, IL) 또는 인터페론-감마 (IFN-γ)와 같은 외부 신호(extracellular signal)에 의해 인산화가 유도되면, 짝을 이루어 (동종이합체(homo-dimer) 또는 이종이합체(hetero-dimer) 형성) 핵 안으로 이동하여 특정 유전자의 발현을 유도한다. 특히, STAT3는 암 유발 인자로 알려지면서 많은 주목을 받고 있다. 실제로, 다양한 암 조직에서 STAT3의 비정상적인 활성화가 보고된 바가 있으며, 이에 따라 STAT3의 비정상적인 활성화를 억제할 수 있는 항암제 개발에 많은 연구자들이 참여하고 있다. 하지만, 아직까지 효과적으로 STAT3를 억제시킬 수 있는 항암제의 개발은 미비한 수준이다 (Frank DA. Mol Med. 1999 Jul; 5(7): 432-456; 및 Buettner R. 등, Clin Cancer Res. 2002 Apr; 8(4): 945-954).STAT3 (signal transducers and activators of transcription 3), a signaling protein that induces cell proliferation, induces phosphorylation by extracellular signals such as interleukin (IL) or interferon-gamma (IFN-γ). When paired, they migrate into the nucleus (homo-dimer or hetero-dimer formation) to induce expression of a particular gene. In particular, STAT3 has received a lot of attention as it is known as a cancer causing factor. In fact, abnormal activation of STAT3 has been reported in various cancer tissues, and thus many researchers have been involved in the development of anticancer drugs that can suppress abnormal activation of STAT3. However, the development of anticancer agents that can effectively inhibit STAT3 is still incomplete (Frank DA. Mol Med . 1999 Jul; 5 (7): 432-456; and Buettner R. et al ., Clin Cancer Res . 2002 Apr; 8 (4): 945-954).

포스포리파제 C (phospholipase C, PLC)는 세포막에 소량 존재하는 인지질인 포스파티딜-이노시톨 4,5-비스포스페이트 (phosphatidyl-inositol 4,5-bisphosphate, PIP2)를 가수분해하여 디아실글리세롤 (diacylglycerol, DG)과 이노시톨 1,4,5-트리포스페이트 (inositol 1,4,5-triphosphate, IP3)를 생성한다. DG는 세포 내에서 프로테인 키나제 C(protein kinase C, PKC)의 활성인자로 작용하며, IP3는 소포체 (endoplasmic reticulum, ER)의 IP3 수용체에 결합함으로써 세포 내의 Ca2 +을 증가시켜 신호를 전달한다. 프로테인 타이로신 키나제 (PTK)가 혈소판-유래 성장 인자 (platelet-derived growth factor, PDGF), 내피세포 성장인자(epidermal growth factor, FGF), 신경 성장 인자(nerve growth factor, NGF) 등과 같은 외부 자극에 의해 활성화되면 PLC-γ 동종효소(isozyme)의 타이로신 잔기가 인산화되어 외부 신호를 세포 안으로 전달하는 것으로 밝혀졌다 (Rhee, S.G. 등, Regulation of phosphoinositide-specific phospholipase C isozymes. J. Biol. Chem. 272: 15045-15048 (1997); 및 Kamat, A. 등, phospholipase C-gamma 1: Regulation of enzyme function and role in growth factor-dependent signal transduction. Cytokine Growth Factor Rev. 8: 109-117 (1997)).Phospholipase C (PLC) hydrolyzes phosphatidyl-inositol 4,5-bisphosphate (PIP2), a small amount of phospholipids present in cell membranes, to diacylglycerol (DG). ) And inositol 1,4,5-triphosphate (inositol 1,4,5-triphosphate, IP3). DG acts as activator of protein kinase C (protein kinase C, PKC) in the cell and, IP3 transmits a signal by increasing the Ca 2 + in the cell by binding to IP3 receptor on the ER (endoplasmic reticulum, ER). Protein tyrosine kinase (PTK) is induced by external stimuli such as platelet-derived growth factor (PDGF), endothelial growth factor (FGF) and nerve growth factor (NGF). Upon activation, tyrosine residues in the PLC-γ isozyme have been phosphorylated to deliver external signals into cells (Rhee, SG et al., Regulation of phosphoinositide-specific phospholipase C isozymes. J. Biol. Chem . 272: 15045) . -15048 (1997); and Kamat, A. like, phospholipase C-gamma 1: Regulation of enzyme function and role in growth factor-dependent signal transduction Cytokine Growth Factor Rev. 8: 109-117 (1997).

세포 내에서 PLC-γ의 활성 증가와 발현량의 증가는 정상 세포의 암화(tumorigenesis)를 유발하는 것으로 알려져 있다 (Peng T. 등, Cardiovasc Res. Jan 17 (2008); 및 Liu J. 등, Ai Zheng, 2007 Sep. 26(9): 957-962).Increasing the activity of PLC-γ and increasing the amount of expression in cells are known to cause normal cell tumorigenesis (Peng T. et al., Cardiovasc). Res . Jan 17 (2008); And Liu J. et al., Ai Zheng , 2007 Sep. 26 (9): 957-962).

항체를 세포 내에서 발현시켜서 표적 항원 단백질의 기능을 억제하는 방식이 보고되고 있다 (A.S.-Y. Lo 등, Therapeutic Antibody . Handbook of Experimental Pharmacology 181). 인트라바디(intrabody)로도 불리는 세포 내 발현 항체 (intracellular antibody)는 번역 후 수준(post-translational level)에서 다양한 표적 항원의 기능을 억제하거나 조절하는 생물공학적인 도구(biotechnological tool)로 이용되고 있다. 세포 내 발현 항체는 세포 내에서 발현되어 세포질(cytosol), 핵(nucleus), 소포체 등을 포함한 다양한 세포내부 (subcellular)에 위치하는 특정 표적 항원에 특이적으로 결합하도록 설계된 항체이다. 예를 들어, 수용체 단백질에 대한 항체를 소포체에 표적화(targeting)하여 발현시킬 경우, 수용체는 발현되지만 세포 외부로 표현(display)이 되지 않아서 발현하지 않은 것과 같은 효과를 볼 수 있다. 또한, 전사 인자에 대한 항체를 세포질 또는 핵에 표적화하여 상기 전사 인자의 발현을 억제하는 보고가 있다.A method of inhibiting the function of target antigen proteins by expressing antibodies in cells has been reported (AS-Y. Lo et al., Therapeutic Antibody . Handbook of Experimental Pharmacology 181 ). Intracellular antibodies, also called intrabodies, are used as biotechnological tools to inhibit or regulate the function of various target antigens at the post-translational level. Intracellular Expression Antibodies are antibodies designed to specifically bind to specific target antigens expressed in cells and located in various subcellular, including cytosol, nucleus, endoplasmic reticulum and the like. For example, when targeting antibodies to the receptor protein to the endoplasmic reticulum (targeting), the expression of the receptor is expressed but not displayed outside the cell (display) can be as effective as not expressed. In addition, there are reports of targeting antibodies to transcription factors in the cytoplasm or nucleus to inhibit expression of the transcription factors.

이러한 세포 내 발현 항체는 다양한 항체 형태로 발현될 수 있지만, 중쇄 및 경쇄의 항원-결합 가변 영역을 링커(interchain linker, ICL)와 연결시켜 만들어진 단일 쇄 항체 (single chain antibody, scFv)가 가장 일반적으로 사용된다. 세포 내 발현 항체는 암, HIV, 자가면역질환 등의 다양한 연구에서 사용되고 있다 (Lo AS 등, Handb Exp Pharmacol. 181: 343-373(2008)).These intracellularly expressed antibodies can be expressed in a variety of antibody forms, but single chain antibodies (scFv) made by linking antigen-binding variable regions of heavy and light chains with an interchain linker (ICL) are most commonly used. Used. Intracellular expression antibodies have been used in various studies such as cancer, HIV, autoimmune diseases (Lo AS et al., Handb) Exp Pharmacol . 181: 343-373 (2008).

그러나, 현재까지 인산화된 단백질에 특이적으로 결합하는 항체를 세포 내에서 발현한 보고는 없다.However, there are no reports of intracellular expression of antibodies that specifically bind to phosphorylated proteins.

따라서, 본 발명의 목적은 인산화된 단백질에 의한 세포 내 신호 전달을 억제하는 방법을 제공하는 것이다.It is therefore an object of the present invention to provide a method for inhibiting intracellular signal transduction by phosphorylated proteins.

본 발명의 다른 목적은 인산화된 단백질에 특이적으로 결합하는 항체를 세포 내에서 발현시키기 위한 발현 시스템을 제공하는 것이다.Another object of the present invention is to provide an expression system for expressing in a cell an antibody that specifically binds to phosphorylated protein.

상기 목적에 따라, 본 발명은 인산화된 단백질에 특이적으로 결합하는 항체를 세포 내에서 발현시킴으로써 상기 인산화된 단백질에 의한 세포 내 신호 전달을 억제하는 방법을 제공한다.In accordance with the above object, the present invention provides a method for inhibiting intracellular signal transduction by the phosphorylated protein by expressing in the cell an antibody that specifically binds to the phosphorylated protein.

상기 다른 목적에 따라, 본 발명은 인산화된 단백질에 특이적으로 결합하는 항체를 암호화하는 핵산 분자를 포함하는, 세포 내에서 상기 항체를 발현시키기 위한 발현 시스템을 제공한다.In accordance with this other object, the present invention provides an expression system for expressing said antibody in a cell, comprising a nucleic acid molecule encoding an antibody that specifically binds to a phosphorylated protein.

본 발명의 방법에 따르면, 인산화된 단백질에 특이적으로 결합하는 항체를 세포 내에서 발현시킴으로써 인산화된 단백질에 의한 세포 내 신호 전달을 억제할 수 있다. 또한, 본 발명의 발현 시스템은 인산화된 단백질에 특이적으로 결합하는 항체를 세포 내에서 특이적으로 고 발현시킬 수 있다. 따라서, 전립선암을 비롯한, 인산화된 단백질에 의한 세포 내 신호 전달에 의해 매개되는 질병의 치료 또는 예방에 유용하게 활용될 수 있다.According to the method of the present invention, intracellular signal transduction by the phosphorylated protein can be inhibited by expressing in the cell an antibody that specifically binds to the phosphorylated protein. In addition, the expression system of the present invention can specifically express high levels of antibodies specifically binding to phosphorylated proteins. Therefore, it can be usefully used for the treatment or prevention of diseases mediated by intracellular signal transduction by phosphorylated proteins, including prostate cancer.

도 1은 세포 내에서 발현되어 인산화된 단백질에 특이적으로 결합할 수 있는 항체의 제조 방법을 개략적으로 도시한 흐름도이다.1 is a flow chart schematically illustrating a method for preparing an antibody that can be expressed in a cell and specifically bind to a phosphorylated protein.

도 2는 pSTAT3에 대한 Fab 항체를 이용한 면역블롯(immunoblot) 결과이다.2 is an immunoblot result using Fab antibody against pSTAT3.

도 3은 pSTAT3에 대한 scFv-Fc 항체를 이용한 면역침강분석(immunoprecipitation) 결과이다.Figure 3 shows the results of immunoprecipitation using scFv-Fc antibody against pSTAT3.

도 4a는 pSTAT3에 대한 scFv 항체가 녹색 형광 단백질 (green fluorescent protein, GFP)과 결합된 형태의 모식도이다.4A is a schematic diagram of a form in which an scFv antibody against pSTAT3 is combined with a green fluorescent protein (GFP).

도 4b는 pSTAT3에 대한 scFv 항체의 결합을 보여주기 위해 면역침강분석을 수행한 후 면역블롯에 의해 확인한 결과이다.Figure 4b is the result confirmed by immunoblot after performing the immunoprecipitation assay to show the binding of the scFv antibody to pSTAT3.

도 4c는 pSTAT3에 대한 scFv 항체가 발현되거나 발현되지 않은 세포에서, IL-6 및 IFN-γ의 처리에 따른 pSTAT3의 핵내 이동을 보여준다.4C shows the nuclear migration of pSTAT3 following treatment with IL-6 and IFN-γ in cells with or without expression of the scFv antibody against pSTAT3.

도 5a는 EGFP가 발현되는 세포에서 Ca2 +의 이동/동원(mobilization)의 정도를 보여준다.Figure 5a shows the degree of movement / mobilization of Ca 2 + in the cell that EGFP was expressed (mobilization).

도 5b는 pPLC-γ에 특이적인 scFv 항체가 발현되는 세포에서 Ca2 +의 이동/동원(mobilization)의 정도를 보여준다.Figure 5b shows the extent of the movement / mobilization of Ca 2 + in the cell that is specific scFv antibody was expressed in pPLC-γ (mobilization).

인산화되는 단백질(상류 단백질, upstream protein)의 인산화된 부위와 결합하는 단백질 (하류 단백질, downstream protein)을 표적화하여 세포 내 신호 전달을 억제하는 기전은 익히 알려져 있다.Mechanisms that inhibit intracellular signal transduction by targeting proteins that bind to phosphorylated sites of phosphorylated proteins (upstream proteins) (downstream proteins) are well known.

이와 달리, 본 발명자들은 인산화되는 단백질을 표적화하여 이와 결합하는 항체를 이용하여 세포 내 신호 전달을 억제하는 방법을 찾기 위해 노력하던 도중, 인산화된 STAT3 및 PLC-γ에 특이적으로 결합하는 항체를 세포 내에서 발현시킨 결과, 인산화된 STAT3는 핵 내 이동이 억제되었으며, 인산화된 PLC-γ의 경우에는 Ca2+의 이동/동원(mobilization)이 억제되었음을 발견하고 본 발명을 완성하게 되었다.In contrast, the present inventors have tried to find a method of inhibiting intracellular signal transduction using an antibody that targets and binds to a protein to be phosphorylated, while the cells specifically bind to an antibody that specifically binds to phosphorylated STAT3 and PLC-γ. As a result of expression in phosphorylated STAT3 was found to inhibit the migration in the nucleus, and in the case of phosphorylated PLC-γ was found to inhibit the migration / mobilization of Ca 2+ to complete the present invention.

이에, 본 발명은 인산화된 단백질에 특이적으로 결합하는 항체를 세포 내에서 발현시킴으로써 상기 인산화된 단백질에 의한 세포 내 신호 전달을 억제하는 방법을 제공한다. Accordingly, the present invention provides a method for inhibiting intracellular signal transduction by the phosphorylated protein by expressing in the cell an antibody that specifically binds to the phosphorylated protein.

바람직하게는, 상기 인산화된 단백질의 인산화된 부위는 타이로신(tyrosine)일 수 있다.Preferably, the phosphorylated site of the phosphorylated protein may be tyrosine.

바람직하게는, 상기 인산화된 단백질은 인산화된 STAT3, PLC-γ 등일 수 있다. Preferably, the phosphorylated protein may be phosphorylated STAT3, PLC-γ and the like.

바람직하게는, 상기 항체는 서열 번호: 8, 10, 12, 14, 16 및 18로 기재된 상보성 결정 영역들(complementarity determining regions, CDRs)을 포함하거나 서열 번호: 26, 28, 30, 32, 34 및 36으로 기재된 CDR들을 포함할 수 있다. 상기 항체는 다양한 항체 형태로 발현될 수 있지만, 중쇄 및 경쇄의 항원-결합 가변 영역을 링커 (interchain linker, ICL)로 연결시켜 만들어진 단일 쇄 항체 (single chain antibody, scFv)가 바람직할 수 있다. 보다 바람직하게는, 상기 항체는 서열 번호: 2 또는 20으로 기재된 아미노산 서열을 가지는 scFv일 수 있다.Preferably, the antibody comprises complementarity determining regions (CDRs) set forth in SEQ ID NOs: 8, 10, 12, 14, 16 and 18 or SEQ ID NOs: 26, 28, 30, 32, 34 and CDRs described as 36. The antibodies can be expressed in various antibody forms, but single chain antibodies (scFv) made by linking the antigen-binding variable regions of the heavy and light chains with an interchain linker (ICL) may be preferred. More preferably, the antibody may be an scFv having an amino acid sequence set forth in SEQ ID NO: 2 or 20.

본 발명의 방법은 상기 항체 또는 이의 항원-결합 부위를 암호화하는 폴리뉴클레오타이드를 포함하는 벡터를 세포 내에 도입시키는 단계를 포함할 수 있다.The method of the present invention may comprise introducing into a cell a vector comprising a polynucleotide encoding said antibody or antigen-binding site thereof.

바람직하게는, 상기 폴리뉴클레오타이드는 서열 번호: 7, 9, 11, 13, 15 및 17로 기재된 뉴클레오타이드 서열들을 포함하는 것일 수 있으며, 보다 바람직하게는 서열 번호: 1의 뉴클레오타이드 서열을 가질 수 있다. 다르게는, 상기 폴리뉴클레오타이드는 서열 번호: 25, 27, 29, 31, 33 및 35로 기재된 뉴클레오타이드 서열들을 포함하는 것일 수 있으며, 보다 바람직하게는 서열 번호: 19의 뉴클레오타이드 서열을 가질 수 있다. Preferably, the polynucleotide may comprise nucleotide sequences set forth in SEQ ID NO: 7, 9, 11, 13, 15 and 17, and more preferably may have the nucleotide sequence of SEQ ID NO: 1. Alternatively, the polynucleotide may comprise nucleotide sequences set forth in SEQ ID NOs: 25, 27, 29, 31, 33 and 35, and more preferably may have the nucleotide sequence of SEQ ID NO: 19.

벡터로는 pEGFPC1(Clontech) 등이 사용될 수 있다. 이외에도 포유동물 세포에서 단백질을 발현할 수 있는 프로모터(promoter)를 갖는 벡터가 이용될 수 있다. As the vector, pEGFPC1 (Clontech) may be used. In addition, a vector having a promoter capable of expressing a protein in mammalian cells may be used.

본 발명의 방법에 따르면, 인산화된 단백질에 특이적으로 결합하는 항체는 인산화된 단백질에 의한 세포 내 신호 전달에 의해 매개되는 질병의 치료 또는 예방이 필요한 대상 세포에서 발현이 유도될 수 있다. 구체적으로, 인산화된 단백질에 의한 세포 내 신호 전달에 의해 매개되는 질병은 전립선암을 포함하는 다양한 암과 면역계 질환 등일 수 있다.According to the method of the present invention, an antibody that specifically binds to a phosphorylated protein can be induced in expression in a subject cell in need of treatment or prevention of a disease mediated by intracellular signal transduction by the phosphorylated protein. Specifically, diseases mediated by intracellular signal transduction by phosphorylated proteins may be various cancers, including prostate cancer, immune system diseases, and the like.

본 발명에서 사용될 수 있는 폴리뉴클레오타이드를 포함하는 벡터를 세포 내에 도입시키는 방법으로는 당 분야에서 외래 유전자를 포함하는 벡터의 세포 내 도입을 위해 사용되는 일반적인 기술들을 이용할 수 있다. 예를 들어, 인산화된 STAT3에 특이적으로 결합하는 항체를 암호화하는 뉴클레오타이드 서열을 플라스미드에 삽입시켜 얻어진 플라스미드 벡터를 자궁경부암 세포주(HeLa)에 형질감염시킬 수 있다.As a method for introducing a vector containing a polynucleotide into a cell, which may be used in the present invention, general techniques used for introducing a vector including a foreign gene into a cell may be used. For example, a plasmid vector obtained by inserting a nucleotide sequence encoding an antibody that specifically binds to phosphorylated STAT3 into a plasmid can be transfected into a cervical cancer cell line (HeLa).

본 발명은 또한 인산화된 단백질에 특이적으로 결합하는 항체를 암호화하는 핵산 분자를 포함하는, 세포 내에서 상기 항체를 발현시키기 위한 발현 시스템을 제공한다.The invention also provides an expression system for expressing said antibody in a cell, comprising a nucleic acid molecule encoding an antibody that specifically binds to a phosphorylated protein.

바람직하게는, 상기 핵산 분자는 서열 번호: 7, 9, 11, 13, 15 및 17로 기재된 뉴클레오타이드 서열들을 포함하는 것일 수 있으며, 보다 바람직하게는 서열 번호: 1의 뉴클레오타이드 서열로 기재되는 폴리뉴클레오타이드일 수 있다. 다르게는, 상기 핵산 분자는 서열 번호: 25, 27, 29, 31, 33 및 35로 기재된 뉴클레오타이드 서열들을 포함하는 것일 수 있으며, 보다 바람직하게는 서열 번호: 19의 뉴클레오타이드 서열로 기재되는 폴리뉴클레오타이드일 수 있다.Preferably, the nucleic acid molecule may comprise nucleotide sequences set forth in SEQ ID NOs: 7, 9, 11, 13, 15 and 17, more preferably polynucleotides described as nucleotide sequences in SEQ ID NO: 1 Can be. Alternatively, the nucleic acid molecule may be one comprising the nucleotide sequences set forth in SEQ ID NOs: 25, 27, 29, 31, 33, and 35, more preferably the polynucleotides set forth in the nucleotide sequence of SEQ ID NO: 19 have.

바람직하게는, 발현 시스템은 발현 키트 (expression kit)와 같은 발현 툴(expression tool)이 이용될 수 있다. 예를 들어, 발현 키트는 인산화된 단백질에 의한 세포 내 신호 전달에 의해 매개되는 질병의 치료 또는 예방이 필요한 대상 세포에서, 인산화된 단백질에 특이적으로 결합하는 항체의 고 발현을 위해 특이적으로 고안된 것일 수 있으며, 상기 항체의 세포 내 발현을 위한 벡터, 트랜스펙션 시약 (transfection reagent), 사용자 매뉴얼 등을 포함할 수 있다.Preferably, the expression system may use an expression tool such as an expression kit. For example, expression kits are specifically designed for high expression of antibodies that specifically bind to phosphorylated proteins in subject cells in need of treatment or prevention of diseases mediated by intracellular signal transduction by phosphorylated proteins. It may be, and may include a vector for intracellular expression of the antibody, a transfection reagent, a user manual, and the like.

도 1은 세포 내에서 발현되어 인산화된 단백질에 특이적으로 결합할 수 있는 항체의 제조 과정을 개략적으로 도시하고 있다. 이하, 도 1을 참조하여 하기 실시예에 의해 본 발명을 더욱 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Figure 1 schematically shows the preparation of an antibody that can be expressed in cells and specifically bind to phosphorylated proteins. Hereinafter, the present invention will be described in more detail with reference to FIG. 1. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

실시예 1: 인산화된 펩타이드에 대한 면역화 및 항체 파지 라이브러리( phage library)의 제작 Example 1: Immunization and antibody phage libraries for the phosphorylated peptides (phage library)

인산화된 펩타이드에 대한 면역반응을 위해, 먼저, STAT3 및 PLC-γ에 대한 각각의 인산화된 펩타이드 pSTAT3 (PGSAAP-pY-LKTKGGGSC; 서열번호: 59) 및 pPLC-γ(RNPGF-pY-VEANPGGGSC; 서열번호: 60)를 합성(Thermo Scientific)하였다. For the immune response to phosphorylated peptides, first, each of the phosphorylated peptides pSTAT3 (PGSAAP-pY-LKTKGGGSC; SEQ ID NO: 59) and pPLC-γ (RNPGF-pY-VEANPGGGSC; SEQ ID NO: 1) for STAT3 and PLC-γ : 60) was synthesized (Thermo Scientific).

이렇게 합성된 펩타이드를, 캐리어 단백질로 사용된 KLH (keyhole limpet hemocyanin, Pierce) 및 OVA (ovalbumin, Sigma)와 접합(conjugation)시킨 후 토끼(New Zealand White, 2.5 kg)에서 면역반응을 유도했다. 이때, 캐리어 단백질에 대한 항체가 만들어질 가능성을 낮추기 위하여 KLH와 OVA를 번갈아 사용하여 면역반응을 유도했다. 면역된 토끼의 혈액을 채취하여 pSTAT3 및 pPLC-γ에 대한 항체가 형성되었는 지를 면역원으로 사용된 펩타이드를 이용한 ELISA 방법으로 확인하였다. The peptide thus synthesized was conjugated with KLH (keyhole limpet hemocyanin, Pierce) and OVA (ovalbumin, Sigma) used as a carrier protein, and induced an immune response in rabbit (New Zealand White, 2.5 kg). At this time, in order to lower the possibility of making an antibody against a carrier protein, KLH and OVA were alternately used to induce an immune response. Blood from immunized rabbits was collected and confirmed by the ELISA method using a peptide used as an immunogen to determine whether antibodies against pSTAT3 and pPLC-γ were formed.

항체 파지 라이브러리 제작은 문헌[참조: Barbas et al., Phage Display : A laboratory manual, Cold Spring Harbor Laboratory Press, Section 2, Chapter 8-9 (2001)]에 따라 다음과 같이 실시하였다. 항체의 형성이 확인된 토끼의 골수(bone marrow) 및 비장(spleen)으로부터 총 RNA를 추출한 뒤 Superscript III First-Strand Synthesis System (Invitrogen)을 이용하여 RT-PCR을 수행하여 cDNA를 합성하였다.Antibody phage library construction is described in Barbas et al., Phage Display : A laboratory manual, Cold Spring Harbor Laboratory Press, Section 2, Chapter 8-9 (2001)]. Total RNA was extracted from the bone marrow and spleen of the rabbit whose antibody was confirmed, and then cDNA was synthesized by performing RT-PCR using Superscript III First-Strand Synthesis System (Invitrogen).

cDNA를 주형으로 하고 하기 표 1 및 2에 기재된 프라이머 서열들을 이용하여 PCR을 수행한 결과, pSTAT3 및 pPLC-γ에 대한 항체의 중쇄 및 경쇄 가변영역에 해당하는 유전자를 확보하였다.PCR was carried out using the cDNA as a template and the primer sequences described in Tables 1 and 2 below. As a result, genes corresponding to the heavy and light chain variable regions of the antibodies against pSTAT3 and pPLC-γ were obtained.

pComb3XTT 벡터(Barbas laboratory)를 주형으로 하고 하기 표 3에 기재된 프라이머 서열들을 이용하여 PCR을 수행한 결과, 인간의 중쇄 불변영역(CH1) 및 경쇄 불변영역(Cκ)에 해당하는 유전자를 확보하였다.PCR was performed using the pComb3XTT vector (Barbas laboratory) as a template and the primer sequences shown in Table 3 below to obtain genes corresponding to the human heavy chain constant region (CH1) and the light chain constant region (Cκ).

이렇게 얻어진 pSTAT3 및 pPLC-γ에 대한 항체의 가변영역 및 불변영역으로부터 하기 표 4 및 5에 수록된 프라이머를 이용하여 오버랩 PCR을 수행하여 Fab (antigen-binding fragment) 형태의 중쇄 및 경쇄 유전자를 확보하였다. From the variable and constant regions of the antibodies against pSTAT3 and pPLC-γ thus obtained, overlap PCR was performed using the primers listed in Tables 4 and 5 to obtain heavy and light chain genes in the form of Fab (antigen-binding fragment).

이를 다시 하기 표 6에 수록된 프라이머를 이용하여 오버랩 PCR을 수행하여 얻어진 PCR 산물(경쇄 가변영역 + 인간 Cκ + 중쇄 가변영역 + CH1)을 pComb3XSS 벡터(Barbas laboratory)에 삽입하여 항체 플라스미드 라이브러리를 제작하였다. The PCR product (light chain variable region + human Cκ + heavy chain variable region + CH1) obtained by performing overlap PCR using the primers listed in Table 6 below was inserted into the pComb3XSS vector (Barbas laboratory) to prepare an antibody plasmid library.

상기 항체 플라스미드 라이브러리를 ER2537 박테리아(New England Biolabs)에 형질전환시킨 다음 VCSM13 헬퍼 파지 (helper phage Stratagene)에 감염시켜 항체 파지 라이브러리를 제작하였다.The antibody plasmid library was transformed into ER2537 bacteria (New England Biolabs) and then infected with VCSM13 helper phage Stratagene to prepare antibody phage libraries.

실시예Example 2:  2: FabFab 클론의 선별  Screening of Clones

실시예 1에서 면역원으로 사용한 pSTAT3 및 pPLC-γ 펩타이드를 소혈청알부민(bovine serum albumin, BSA: Sigma)과 결합시킨 다음 자기 비드 (magnetic bead) (Dynabead M-270 Epoxy, Invitrogen)와 결합하여 펩타이드-BSA-비드를 얻었다. 이렇게 얻어진 펩타이드-BSA-비드와 실시예 1에서 얻어진 파지 라이브러리를 TBS-T-BSA 버퍼(50 mM Tris, 150 mM NaCl, 0.05% Triton X-100, 5% BSA, pH 7.4)에서 실온에서 1시간 동안 반응시켰다. 얻어진 결과물을 TBS-T 버퍼(50 mM Tris, 150 mM NaCl, 0.05% Triton X-100, pH 7.4)를 이용하여 10 분간 1회, 10 분간 5회, 10 분간 10회 세척하여 비특이적으로 결합하는 파지를 제거하고 pH 변화를 통해서 특이적으로 결합한 파지를 확보하였다. PSTAT3 and pPLC-γ peptides used as immunogens in Example 1 were combined with bovine serum albumin (BSA: Sigma) and then magnetic beads (Dynabead M-270 Epoxy, Invitrogen) to bind peptide- BSA-beads were obtained. The peptide-BSA-beads and the phage library obtained in Example 1 were obtained in TBS-T-BSA buffer (50 mM Tris, 150 mM NaCl, 0.05% Triton X-100, 5% BSA, pH 7.4) for 1 hour at room temperature. Reacted for a while. The resultant was washed with TBS-T buffer (50 mM Tris, 150 mM NaCl, 0.05% Triton X-100, pH 7.4) once for 10 minutes, 5 times for 10 minutes and 10 minutes for 10 minutes to bind nonspecifically. Was removed and specifically bound phage was obtained by changing the pH.

이렇게 얻어진 pSTAT3에 대한 Fab 항체의 중쇄 가변영역의 뉴클레오타이드 서열 및 아미노산 서열은 각각 서열번호: 3 및 4와 같았고, 경쇄 가변영역의 뉴클레오타이드 서열 및 아미노산 서열은 각각 서열번호: 5 및 6과 같았다. pPLC-γ에 대한 Fab 항체의 중쇄 가변영역의 뉴클레오타이드 서열 및 아미노산 서열은 각각 서열번호: 21 및 22와 같았고, 경쇄 가변영역의 뉴클레오타이드 서열 및 아미노산 서열은 각각 서열 번호: 23 및 24와 같았다. Fab 항체는 COOH 말단의 His tag와 결합하는 Ni-NTA 수지(Qiagen)를 이용하여 항체를 정제한 후 사용하였다.The nucleotide sequence and amino acid sequence of the heavy chain variable region of the Fab antibody to pSTAT3 thus obtained were as shown in SEQ ID NOs: 3 and 4, respectively, and the nucleotide sequence and amino acid sequence of the light chain variable region were as shown in SEQ ID NOs: 5 and 6, respectively. The nucleotide sequence and amino acid sequence of the heavy chain variable region of the Fab antibody to pPLC- [gamma] were as SEQ ID NOs: 21 and 22, respectively, and the nucleotide sequence and amino acid sequence of the light chain variable region were as SEQ ID NOs: 23 and 24, respectively. Fab antibody was used after purifying the antibody using Ni-NTA resin (Qiagen) that binds to the His tag of the COOH terminal.

실시예 3: 면역블롯(immunoblot, IB)을 이용하여 세포 내에서 인산화된 단백질과 Fab 항체의 결합여부 확인 Example 3: Confirmation of binding of phosphorylated protein and Fab antibody in cells using immunoblot (IB)

실험에 앞서, STAT3가 인산화된 세포를 얻기 위해, HeLa 세포(American Type Culture Collection (ATCC))를 FBS가 포함되지 않은 DMEM 배지(High glucose; HyClone)에서 24 시간 동안 배양한 다음 IFN-α (150 ng/ml)를 15분 동안 처리하였고, PLC-γ가 인산화된 세포를 얻기 위해, NIH3T3 세포(ATCC)를 태 송아지 혈청(fetal calf serum, FCS)이 포함되지 않은 DMEM 배지(High glucose; HyClone)에서 4시간 동안 배양한 다음 PDGF (100 ng/ml)를 20분 동안 처리하였다.Prior to the experiment, in order to obtain STAT3 phosphorylated cells, HeLa cells (American Type Culture Collection (ATCC)) were incubated for 24 hours in DMEM medium (High glucose; HyClone) without FBS, and then IFN-α (150 ng / ml) was treated for 15 minutes, and NIH3T3 cells (ATCC) were treated with DMEM medium (High glucose; HyClone) without fetal calf serum (FCS) to obtain PLC-γ phosphorylated cells. After incubation for 4 hours, PDGF (100 ng / ml) was treated for 20 minutes.

상기와 같이 준비한 HeLa 세포주 및 NIH3T3 세포주에서 세포 추출물을 다음과 같이 준비하였다. 먼저, 세포를 PBS(137 mM NaCl, 2.7 mM KCl, 12 mM Na2HPO4, 1.2 mM KH2PO4, pH 7.4)를 이용하여 2회 세척한 다음 IB 버퍼(20 mM HEPES, Ph 7.2; 20 mM phosphoglycerate; 150 mM NaCl; 10% glycerol; 1% NP-40; 1 mM EDTA; 1 mM EGTA; 1 mM PMSF, 1 uM Leupeptin, 0.3 uM Aprotinin, 0.3 mM Pepstatin)를 첨가하여 4℃에서 30분 동안 방치하였다. 이후, 15,000 rpm에서 15분간 원심분리하여 상층액을 얻었다.Cell extracts were prepared in the HeLa cell line and NIH3T3 cell line prepared as described above. First, cells were washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 12 mM Na 2 HPO 4 , 1.2 mM KH 2 PO 4 , pH 7.4), followed by IB buffer (20 mM HEPES, Ph 7.2; 20 mM phosphoglycerate; 150 mM NaCl; 10% glycerol; 1% NP-40; 1 mM EDTA; 1 mM EGTA; 1 mM PMSF, 1 uM Leupeptin, 0.3 uM Aprotinin, 0.3 mM Pepstatin) for 30 minutes at 4 ° C. It was left to stand. Then, the supernatant was obtained by centrifugation for 15 minutes at 15,000 rpm.

얻어진 세포 추출물에 대해, 실시예 2에서 얻어진 pSTAT3 및 pPLC-γ에 대한 Fab 항체를 1차 항체로 사용하고 HRP 접합 항-HA 항체(Roche)를 2차 항체로 사용하여 면역블롯(immunoblot)을 수행하였다. pSTAT3에 대한 Fab 항체를 이용하여 면역블롯을 수행한 결과가 도 2에 도시되어 있다. On the obtained cell extract, immunoblot was performed using Fab antibodies against pSTAT3 and pPLC-γ obtained in Example 2 as primary antibodies and HRP conjugated anti-HA antibody (Roche) as secondary antibodies. It was. The results of performing the immunoblot using Fab antibodies against pSTAT3 are shown in FIG. 2.

도 2에 도시된 바와 같이, 기존에 보고된 것과 같이, IFN-α 미처리 세포와 달리, IFN-α 처리 세포에서 STAT3 단백질 크기에 해당하는 밴드가 강하게 나타나는 것으로 보아 실시예 2에서 얻어진 항체가 pSTAT3에 특이적으로 결합하는 것을 알 수 있다. As shown in FIG. 2, as previously reported, unlike IFN-α-treated cells, bands corresponding to the STAT3 protein size appear strongly in IFN-α-treated cells, and thus the antibody obtained in Example 2 is expressed in pSTAT3. It can be seen that the specific binding.

실시예Example 4:  4: scFvscFv -- FcFc 미니항체( Mini Antibody ( minibodyminibody )의 제작) Production

pSTAT3 및 pPLC-γ에 대한 scFv-Fc 형태의 미니항체(minibody)를 얻기 위해, 실시예 2에서 얻어진 Fab의 중쇄 및 경쇄 가변영역을 18개의 아미노산 링커(GGSSRSSSSGGGGSGGGG 서열 번호: 58)로 연결하여 단일 쇄 항체(single chain antibody, scFv)를 제작하였다. pSTAT3에 대한 scFv의 6개 CDR의 뉴클레오타이드 서열 및 아미노산 서열을 각각 서열 번호: 7, 9, 11, 13, 15 및 17과 서열 번호: 8, 10, 12, 14, 16 및 18로 기재했으며, pPLC-γ에 대한 scFv의 6개 CDR의 뉴클레오타이드 서열 및 아미노산 서열을 각각 서열 번호: 25, 27, 29, 31, 33 및 35와 서열 번호: 26, 28, 30, 32, 34 및 36으로 기재하였다. 이들 scFv를 링커가 연결된 인간 IgG1의 Fc 부위에 결합시켜 pcDNA3.1 벡터 (Invitrogen)에 클로닝한 다음, 293F 세포(Invitrogen)에 일시적으로 형질감염시켜 (transient transfection) scFv-Fc 형태의 미니항체를 제작하였다. 이렇게 얻어진 미니항체를 단백질-G 비드 (Amersham)을 이용하여 정제한 후 사용하였다.In order to obtain a minibody of scFv-Fc form against pSTAT3 and pPLC-γ, the heavy and light chain variable regions of the Fab obtained in Example 2 were joined by an 18 amino acid linker (GGSSRSSSSGGGGSGGGG SEQ ID NO: 58) to a single chain. An antibody (single chain antibody, scFv) was prepared. The nucleotide and amino acid sequences of the six CDRs of scFv for pSTAT3 are set forth in SEQ ID NOs: 7, 9, 11, 13, 15 and 17 and SEQ ID NOs: 8, 10, 12, 14, 16 and 18, respectively, and pPLC The nucleotide and amino acid sequences of the six CDRs of scFv for -γ are set forth in SEQ ID NOs: 25, 27, 29, 31, 33 and 35 and SEQ ID NOs: 26, 28, 30, 32, 34 and 36, respectively. These scFvs were linked to the Fc region of the linker-linked human IgG1 and cloned into the pcDNA3.1 vector (Invitrogen), and then transiently transfected into 293F cells (Invitrogen) to produce a scFv-Fc-type mini antibody. It was. The mini-antibody thus obtained was purified using protein-G beads (Amersham) and then used.

실시예 5: 면역침강분석(immuunoprecipitation, IP)을 이용한 scFv - Fc 미니항체의 특이성 분석 Example 5 Specificity Analysis of scFv - Fc Mini-Antibodies Using Immunoprecipitation (IP)

면역침강분석을 통해 실시예 4에서 제조된 scFv-Fc 항체의 특이성을 분석하였다.Specificity of the scFv-Fc antibody prepared in Example 4 was analyzed by immunoprecipitation assay.

이를 위해, IFN-α를 처리하거나 처리하지 않은 HeLa 세포 또는 PDGF를 처리하거나 처리하지 않은 NIH3T3 세포에 IP 용 버퍼 (50 mM Tris-HCL, pH7.4; 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mMPMSF, 1 uM Leupeptin, 0.3 uM Aprotinin, 0.3 mM Pepstatin)를 처리하여 세포 용해물(lysate)을 얻고, 이를 동량의 실시예 4에서 얻어진 pSTAT3와 pPLC-γ에 대한 scFv-Fc 항체와 면역침강시켰다. To this end, buffers for IP (50 mM Tris-HCL, pH7.4; 150 mM NaCl, 1% NP-40, 1) in HeLa cells with or without IFN-α or NIH3T3 cells with or without PDGF were treated. treated with mM EDTA, 1 mM PMSF, 1 uM Leupeptin, 0.3 uM Aprotinin, 0.3 mM Pepstatin) to obtain cell lysates, which were obtained with the same amount of scFv-Fc antibodies against pSTAT3 and pPLC-γ obtained in Example 4. Immunoprecipitated.

pSTAT3에 대한 scFv-Fc 항체를 이용한 결과가 도 3에 도시되어 있다. 도 3에 도시된 바와 같이, IFN-α를 처리한 세포 용해물에 보다 많은 양의 pSTAT3가 발현되는 것을 통해서 세포 용해물이 정상적으로 준비되었음을 알 수 있다. 그리고 IFN-α를 처리한 세포 용해물을 이용한 면역침강 결과물에서 보다 많은 STAT3 단백질이 존재하는 것으로 보아 상기 항체가 pSTAT3 단백질과 결합함을 보여준다. The results using the scFv-Fc antibody against pSTAT3 are shown in FIG. 3. As shown in Figure 3, it can be seen that the cell lysate was normally prepared by expressing a greater amount of pSTAT3 in the cell lysate treated with IFN-α. And in the result of immunoprecipitation using IFN-α-treated cell lysate, the presence of more STAT3 protein shows that the antibody binds to pSTAT3 protein.

실시예 6: pSTAT3 에 대한 항체의 세포 내 발현을 통한 pSTAT3 의 핵내 이동 (nuclear translocation) 억제 Example 6: Nuclear transfer of pSTAT3 through the expression of the antibody for the cell pSTAT3 (nuclear translocation) inhibition

인산화된 단백질에 의해 매개되는 세포 신호 전달을, 인산화된 단백질에 특이적인 항체의 세포 내 발현을 통하여 억제가 가능한 지를 확인하기 위해, 우선, pSTAT3에 대한 scFv 항체를 녹색 형광 단백질 (green fluorescent protein, GFP)이 결합된 형태로 제조하였다. 구체적으로, 실시예 4에서 얻어진 pSTAT3에 대한 scFv 항체를 pEGFP-C1 벡터(Clontech)의 XhoI 및 HindIII 제한효소 부위에 클로닝하고 염기서열 분석을 통해 원하는 DNA 염기서열의 존재를 확인하였다. pSTAT3에 대한 scFv의 뉴클레오타이드 서열 및 아미노산 서열을 각각 서열 번호: 1 및 2로 나타내었다. 이러한 항체의 개략적인 구조가 도 4a에 도시되어 있다. 이후, 얻어진 pSTAT3에 대한 scFv 함유 벡터를 HeLa 세포에 형질감염시켰다.To determine whether cellular signaling mediated by phosphorylated proteins can be inhibited through intracellular expression of antibodies specific for the phosphorylated protein, first a scFv antibody against pSTAT3 can be used as a green fluorescent protein (GFP). ) Was prepared in a combined form. Specifically, the scFv antibody against pSTAT3 obtained in Example 4 was cloned into the Xho I and Hind III restriction sites of the pEGFP-C1 vector (Clontech), and sequencing confirmed the presence of the desired DNA sequence. The nucleotide sequence and amino acid sequence of scFv for pSTAT3 are shown in SEQ ID NOs: 1 and 2, respectively. The schematic structure of such an antibody is shown in FIG. 4A. Thereafter, the scFv-containing vector for pSTAT3 obtained was transfected into HeLa cells.

상기 항체가 세포 내에서 정상적으로 발현되어 STAT3와 결합하는 지를 알아보기 위해, 항-GFP 항체(AbFrontier)를 이용하여 면역침강분석을 수행하였다. In order to determine whether the antibody is normally expressed in cells and binds to STAT3, immunoprecipitation analysis was performed using an anti-GFP antibody (AbFrontier).

그 결과, 도 4b에 도시된 바와 같이, 항체를 발현한 세포의 추출물을 이용한면역침강 결과에서만 STAT3가 확인되었으며, 이는 발현된 scFv 항체가 세포 내에서 STAT3와 결합함을 알 수 있다.As a result, as shown in Figure 4b, STAT3 was confirmed only in the results of immunoprecipitation using the extract of the cells expressing the antibody, it can be seen that the expressed scFv antibody binds to the STAT3 in the cell.

pSTAT3에 대한 scFv 항체의 발현을 통한 세포 신호전달의 억제를 확인하기 위해, 앞서 제조된 GFP가 결합된 형태의 pSTAT3에 대한 scFv 항체를 암호화하는 DNA를 HepG2 세포(ATCC)에 형질감염시켰다. 24시간 후에, FBS가 없는 DMEM 배지에서 12시간 동안 세포를 배양하고 IL-6 (50 ng/ml) 및 IFN-γ(100 ng/ml)를 40분 동안 처리하였다. 그 다음, 파라포름알데히드를 이용하여 세포를 고정하고 항-Stat3 항체(Cell Signaling)와 Rhodamine 접합 항-생쥐 항체(Jackson ImmunoResearch Laboratories, Inc.)를 이용하여 면역염색(immunostaining)을 수행하였다. 핵의 위치를 확인하기 위해서 DAPI (4'-6-Diamidino-2-phenylindole; Invitrogen) 염색을 수행한 다음 공초점(confocal) 형광 현미경을 통하여 관찰하였다. To confirm inhibition of cell signaling through expression of scFv antibodies against pSTAT3, DNA encoding scFv antibodies against pSTAT3 in the GFP-bound form prepared previously was transfected into HepG2 cells (ATCC). After 24 hours, cells were incubated for 12 hours in DMEM medium without FBS and treated with IL-6 (50 ng / ml) and IFN-γ (100 ng / ml) for 40 minutes. The cells were then fixed using paraformaldehyde and immunostained using anti-Stat3 antibody (Cell Signaling) and Rhodamine conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc.). DAPI (4'-6-Diamidino-2-phenylindole; Invitrogen) staining was performed to confirm the location of the nucleus and then observed by confocal fluorescence microscopy.

결과가 도 4c에 도시되어 있다. 도 4c의 (1), (2), (3) 및 (4)는 모두 동일한 세포를 대상으로 한 사진으로서, (1)은 항체가 발현되는 세포(녹색으로 표시)와 발현되지 않은 세포를 보여주고, (2)는 세포 내에 존재하는 STAT3(붉은색으로 표시)를 보여주며, (3)은 세포 내에 존재하는 핵 부분(파란색으로 표시)을 보여주며, (4)는 (1), (2) 및 (3)을 조합하여 보여주고 있다. 도 4c에 도시된 바와 같이, 항체가 발현되지 않은 세포들(도 4c의 (1)에서 녹색 신호가 나오지 않는 세포들)에서는 IL-6와 IFN-γ에 의해서 STAT3가 핵으로 이동함이 관찰되었지만, 항체가 발현된 세포(도 4c의 (1)에서 녹색 신호가 나오는 세포)에서는 STAT3의 핵으로의 이동이 억제되었음을 알 수 있다. 이를 통해서 pSTAT3에 특이적인 항체의 세포 내 발현을 통해서 STAT3의 기능이 억제됨을 알 수 있다.The results are shown in Figure 4c. (1), (2), (3) and (4) of FIG. 4C are all photographs of the same cell, and (1) shows cells (not shown in green) and cells not expressing the antibody. (2) shows the STAT3 (in red) present in the cell, (3) shows the nucleus part (in blue) present in the cell, and (4) (1), (2 ) And (3) are shown in combination. As shown in FIG. 4C, in the cells without the antibody expression (cells without green signal in (1) of FIG. 4C), STAT3 is observed in the nucleus by IL-6 and IFN-γ. It can be seen that migration of the STAT3 to the nucleus was suppressed in the cells in which the antibody was expressed (cells showing green signals in (1) of FIG. 4C). This suggests that STAT3 function is inhibited through intracellular expression of pSTAT3 specific antibodies.

실시예 7: pPLC -γ에 대한 scFv 항체의 세포 내 발현을 통한 Ca 2 + 이동/동원(mobilization) 억제 Example 7: Ca 2 + go / mobilization (mobilization) inhibition by expressing within cells of the scFv antibodies to pPLC -γ

pPLC-γ에 특이적인 scFv 항체를 통한 Ca2 + 이동/동원(mobilization)을 확인하기 위해, 실시예 6에 기재된 바와 같이, pPLC-γ에 특이적인 scFv 항체를 녹색 형광 단백질 (GFP)이 결합된 형태로 제조하였다. 이렇게 얻어진 pPLC-γ에 특이적인 scFv 항체의 뉴클레오타이드 서열 및 아미노산 서열을 각각 서열 번호: 19 및 20으로 나타내었다. 얻어진 pPLC-γ에 특이적인 scFv 항체를, 유리-바닥 디쉬(glass-bottom dish)에 접종(seeding)된 NIH3T3 세포에 형질감염시킨 다음 24시간 동안 37℃, 5% CO2 조건으로 배양하였다. FCS가 없는 DMEM에서 4시간 동안 추가 배양하고, Fura-2 (5 uM; Molecular Probe)를 1 시간 동안 처리한 다음 Ca2 +이 없는 상태에서 PDGF (500 ng/ml)를 30초간 처리하였다. 형광 현미경을 통하여 Ca2 + 이동/동원(mobilization)을 확인하였다. 결과가 도 5a 및 5b에 도시되어 있다.in pPLC-γ to determine the Ca 2 + go / mobilized (mobilization) with the specific scFv antibody, Example 6 is, green fluorescent protein (GFP) the specific scFv antibodies in pPLC-γ as coupling described in Prepared in the form. The nucleotide sequence and amino acid sequence of the scFv antibody specific for pPLC-γ thus obtained are shown in SEQ ID NOs: 19 and 20, respectively. The scFv antibody specific for the obtained pPLC-γ was transfected into NIH3T3 cells seeded in a glass-bottom dish and incubated at 37 ° C. and 5% CO 2 conditions for 24 hours. Further incubation for 4 hours in DMEM without FCS, Fura-2 (5 uM; Molecular Probe) for 1 hour followed by PDGF (500 ng / ml) for 30 seconds in the absence of Ca 2 + . Via fluorescence microscopy confirmed the Ca 2 + go / mobilized (mobilization). The results are shown in FIGS. 5A and 5B.

도 5a 및 5b에 도시된 바와 같이, EGFP가 발현되는 세포에서도 약하게 Ca2 + 이동/동원이 감소하였지만, 항체를 발현하는 세포에서 Ca2 + 이동/동원이 크게 감소하였음을 알 수 있다. 이러한 결과는 pPLC-γ에 특이적인 scFv 항체의 세포 내 발현을 통하여 PLC-γ에 의한 신호전달이 억제되었음을 보여준다.As illustrated in Figures 5a and 5b, but weakly in cells that express EGFP is Ca 2 + go / mobilization is reduced, it can be seen in the cells expressing the antibody hayeoteum Ca 2 + go / mobilization is greatly reduced. These results show that signaling by PLC-γ was inhibited through intracellular expression of scFv antibodies specific for pPLC-γ.

<110> AbFRONTIER <120> ANTIBODY SPECIFICALLY BINDING TO A PHOSPHORYLATED PLC-γ PROTEIN <130> FPD/200803-0081 <150> 61/031,433 <151> 2008-02-26 <160> 60 <170> KopatentIn 1.71 <210> 1 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> scFv for pStat3 <220> <221> CDS <222> (1)..(735) <400> 1 gag ctc gtg ctg acc cag act cca tcc ccc gtg tct gca gtt gtg gga 48 Glu Leu Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag tcc agt cag agt gtt tgg ggt aac 96 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly Asn 20 25 30 aac cgc tta tcc tgg tat cag cag aaa cca ggg cag cct ccc agg ctc 144 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu 35 40 45 cta atg tat tat gca tcc aat ctg gca tct ggg gtc tca tcg cgg ttc 192 Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 aaa ggc agt gga tct ggg aca caa ttc act ctc acc atc agc gac gtg 240 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 cag tgt gac gat gct gcc act tac tac tgt caa ggc gga ttt gag tgt 288 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu Cys 85 90 95 agt ggt ggt gat tgt gtt ggt ttc ggc gga ggg acc gag ctg gag atc 336 Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu Ile 100 105 110 cta ggt ggt tcc tct aga tct tcc tcc tct ggt ggc ggt ggc tcg ggc 384 Leu Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 ggt ggt ggg cag tcg gtg gag gag tcc ggg ggt cgc ctg gta gcg cct 432 Gly Gly Gly Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala Pro 130 135 140 gga gga tcc ctg aca ctc acc tgc aca gtc tct gga atc gac ctc agt 480 Gly Gly Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 145 150 155 160 agc gat gca atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa 528 Ser Asp Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170 175 tgg atc gga acg att tat ggt agt gct ggc aca tac tac gcg acc tgg 576 Trp Ile Gly Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp 180 185 190 gcg aaa ggc cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg 624 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 195 200 205 aaa atg acc agt ctg aca acc gag gac acg gcc acc tat ttc tgt acc 672 Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Thr 210 215 220 aga gct ttt agc aac act cga ttg gat ctc tgg ggc cag ggc acc ctg 720 Arg Ala Phe Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu 225 230 235 240 gtc acc atc tct tca tagaa gcttcgaatt ctgcag 756 Val Thr Ile Ser Ser 245 <210> 2 <211> 245 <212> PRT <213> Artificial Sequence <400> 2 Glu Leu Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly Asn 20 25 30 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu 35 40 45 Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu Cys 85 90 95 Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu Ile 100 105 110 Leu Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala Pro 130 135 140 Gly Gly Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 145 150 155 160 Ser Asp Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170 175 Trp Ile Gly Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp 180 185 190 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 195 200 205 Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Thr 210 215 220 Arg Ala Phe Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu 225 230 235 240 Val Thr Ile Ser Ser 245 <210> 3 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of Fab for pStat3 <220> <221> CDS <222> (1)..(342) <400> 3 cag tcg gtg gag gag tcc ggg ggt cgc ctg gta gcg cct gga gga tcc 48 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala Pro Gly Gly Ser 1 5 10 15 ctg aca ctc acc tgc aca gtc tct gga atc gac ctc agt agc gat gca 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asp Ala 20 25 30 atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tgg atc gga 144 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 acg att tat ggt agt gct ggc aca tac tac gcg acc tgg gcg aaa ggc 192 Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 50 55 60 cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg aaa atg acc 240 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 agt ctg aca acc gag gac acg gcc acc tat ttc tgt acc aga gct ttt 288 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Ala Phe 85 90 95 agc aac act cga ttg gat ctc tgg ggc cag ggc acc ctg gtc acc atc 336 Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Ile 100 105 110 tct tca tag 345 Ser Ser <210> 4 <211> 114 <212> PRT <213> Artificial Sequence <400> 4 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala Pro Gly Gly Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asp Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Ala Phe 85 90 95 Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Ile 100 105 110 Ser Ser <210> 5 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Light chain variable region of Fab for pStat3 <220> <221> CDS <222> (1)..(339) <400> 5 gag ctc gtg ctg acc cag act cca tcc ccc gtg tct gca gtt gtg gga 48 Glu Leu Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag tcc agt cag agt gtt tgg ggt aac 96 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly Asn 20 25 30 aac cgc tta tcc tgg tat cag cag aaa cca ggg cag cct ccc agg ctc 144 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu 35 40 45 cta atg tat tat gca tcc aat ctg gca tct ggg gtc tca tcg cgg ttc 192 Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 aaa ggc agt gga tct ggg aca caa ttc act ctc acc atc agc gac gtg 240 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 cag tgt gac gat gct gcc act tac tac tgt caa ggc gga ttt gag tgt 288 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu Cys 85 90 95 agt ggt ggt gat tgt gtt ggt ttc ggc gga ggg acc gag ctg gag atc 336 Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu Ile 100 105 110 cta 339 Leu <210> 6 <211> 113 <212> PRT <213> Artificial Sequence <400> 6 Glu Leu Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly Asn 20 25 30 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu 35 40 45 Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu Cys 85 90 95 Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu Ile 100 105 110 Leu <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR1 of scFv for pStat3 <220> <221> CDS <222> (1)..(15) <400> 7 agc gat gca atg agc 15 Ser Asp Ala Met Ser 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <400> 8 Ser Asp Ala Met Ser 1 5 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> HCDR2 of scFv for pStat3 <220> <221> CDS <222> (1)..(45) <400> 9 acg att tat ggt agt gct ggc aca tac tac gcg acc tgg gcg aaa 45 Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <400> 10 Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of scFv for pStat3 <220> <221> CDS <222> (1)..(27) <400> 11 gct ttt agc aac act cga ttg gat ctc 27 Ala Phe Ser Asn Thr Arg Leu Asp Leu 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Ala Phe Ser Asn Thr Arg Leu Asp Leu 1 5 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of scFv for pStat3 <220> <221> CDS <222> (1)..(18) <400> 13 cag tcc agt cag agt gtt 18 Gln Ser Ser Gln Ser Val 1 5 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <400> 14 Gln Ser Ser Gln Ser Val 1 5 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LCDR2 of scFv for pStat3 <220> <221> CDS <222> (1)..(21) <400> 15 tat gca tcc aat ctg gca tct 21 Tyr Ala Ser Asn Leu Ala Ser 1 5 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <400> 16 Tyr Ala Ser Asn Leu Ala Ser 1 5 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> LCDR3 of scFv for pStat3 <220> <221> CDS <222> (1)..(39) <400> 17 caa ggc gga ttt gag tgt agt ggt ggt gat tgt gtt ggt 39 Gln Gly Gly Phe Glu Cys Ser Gly Gly Asp Cys Val Gly 1 5 10 <210> 18 <211> 13 <212> PRT <213> Artificial Sequence <400> 18 Gln Gly Gly Phe Glu Cys Ser Gly Gly Asp Cys Val Gly 1 5 10 <210> 19 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> scFv for pPLCr <220> <221> CDS <222> (1)..(753) <400> 19 gag ctc gat atg acc cag act cca gcc tct gtg gag gta gct gtg gga 48 Glu Leu Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag gcc agt cag agt gtt tat aag gac 96 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Asp 20 25 30 aac aac tta gcc tgg tat cag cag aaa cca ggg cag cct ccc aag ctc 144 Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 ctg atc tac aag gct tcc act ctg gca tct ggg gtc ccg tcg cgg ttc 192 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 aaa ggc agt gga tct ggg aca cag ttc act ctc acc atc agc gac gtg 240 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 cag tgt gac gat gct acc act tac tac tgt gca ggc ggt tgg aat tct 288 Gln Cys Asp Asp Ala Thr Thr Tyr Tyr Cys Ala Gly Gly Trp Asn Ser 85 90 95 aat gat gat acg ttt gct ttc ggc gga ggg acc gcg gtg gtg gtc aga 336 Asn Asp Asp Thr Phe Ala Phe Gly Gly Gly Thr Ala Val Val Val Arg 100 105 110 ggt ggt tcc tct aga tct tcc tcc tct ggt ggc ggt ggc tcg ggc ggt 384 Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 ggt ggg cag tcg ctg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg 432 Gly Gly Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly 130 135 140 aca ccc ctg aca ctc acc tgc aca gtc tct gga ttc tcc ctc agt aac 480 Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn 145 150 155 160 tac tgg atg aac tgg gtc cgc cag gct cca ggg aag gga ctg gaa tgg 528 Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 165 170 175 atc gga gtc att agt agg agt ggt atc aca ggc tac gcg agc tgg gcg 576 Ile Gly Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala 180 185 190 aaa ggc cga ttc acc atc tcc aaa acc tcg acc aca gtg gat ctg aaa 624 Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys 195 200 205 atc acc agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga 672 Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg 210 215 220 caa tat tat tct ggt tat ggt gat gtt gct tat gtt gac ttt aac ttg 720 Gln Tyr Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu 225 230 235 240 tgg ggc caa ggc acc ctg gtc acc atc tct tca tag 756 Trp Gly Gln Gly Thr Leu Val Thr Ile Ser Ser 245 250 <210> 20 <211> 251 <212> PRT <213> Artificial Sequence <400> 20 Glu Leu Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Asp 20 25 30 Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Thr Thr Tyr Tyr Cys Ala Gly Gly Trp Asn Ser 85 90 95 Asn Asp Asp Thr Phe Ala Phe Gly Gly Gly Thr Ala Val Val Val Arg 100 105 110 Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly 130 135 140 Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn 145 150 155 160 Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 165 170 175 Ile Gly Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala 180 185 190 Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys 195 200 205 Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg 210 215 220 Gln Tyr Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu 225 230 235 240 Trp Gly Gln Gly Thr Leu Val Thr Ile Ser Ser 245 250 <210> 21 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of Fab for pPLCr <220> <221> CDS <222> (1)..(363) <400> 21 cag tcg ctg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 ctg aca ctc acc tgc aca gtc tct gga ttc tcc ctc agt aac tac tgg 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr Trp 20 25 30 atg aac tgg gtc cgc cag gct cca ggg aag gga ctg gaa tgg atc gga 144 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 gtc att agt agg agt ggt atc aca ggc tac gcg agc tgg gcg aaa ggc 192 Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala Lys Gly 50 55 60 cga ttc acc atc tcc aaa acc tcg acc aca gtg gat ctg aaa atc acc 240 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga caa tat 288 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gln Tyr 85 90 95 tat tct ggt tat ggt gat gtt gct tat gtt gac ttt aac ttg tgg ggc 336 Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu Trp Gly 100 105 110 caa ggc acc ctg gtc acc atc tct tca tag 366 Gln Gly Thr Leu Val Thr Ile Ser Ser 115 120 <210> 22 <211> 121 <212> PRT <213> Artificial Sequence <400> 22 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr Trp 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gln Tyr 85 90 95 Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Ile Ser Ser 115 120 <210> 23 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Light chain variable region of Fab for pPLCr <220> <221> CDS <222> (1)..(336) <400> 23 gag ctc gat atg acc cag act cca gcc tct gtg gag gta gct gtg gga 48 Glu Leu Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag gcc agt cag agt gtt tat aag gac 96 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Asp 20 25 30 aac aac tta gcc tgg tat cag cag aaa cca ggg cag cct ccc aag ctc 144 Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 ctg atc tac aag gct tcc act ctg gca tct ggg gtc ccg tcg cgg ttc 192 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 aaa ggc agt gga tct ggg aca cag ttc act ctc acc atc agc gac gtg 240 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 cag tgt gac gat gct acc act tac tac tgt gca ggc ggt tgg aat tct 288 Gln Cys Asp Asp Ala Thr Thr Tyr Tyr Cys Ala Gly Gly Trp Asn Ser 85 90 95 aat gat gat acg ttt gct ttc ggc gga ggg acc gcg gtg gtg gtc aga 336 Asn Asp Asp Thr Phe Ala Phe Gly Gly Gly Thr Ala Val Val Val Arg 100 105 110 <210> 24 <211> 112 <212> PRT <213> Artificial Sequence <400> 24 Glu Leu Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Asp 20 25 30 Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Thr Thr Tyr Tyr Cys Ala Gly Gly Trp Asn Ser 85 90 95 Asn Asp Asp Thr Phe Ala Phe Gly Gly Gly Thr Ala Val Val Val Arg 100 105 110 <210> 25 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR1 of scFv for pPLCr <220> <221> CDS <222> (1)..(15) <400> 25 aac tac tgg atg aac 15 Asn Tyr Trp Met Asn 1 5 <210> 26 <211> 5 <212> PRT <213> Artificial Sequence <400> 26 Asn Tyr Trp Met Asn 1 5 <210> 27 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> HCDR2 of scFv for pPLCr <220> <221> CDS <222> (1)..(45) <400> 27 gtc att agt agg agt ggt atc aca ggc tac gcg agc tgg gcg aaa 45 Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 28 <211> 15 <212> PRT <213> Artificial Sequence <400> 28 Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 29 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of scFv for pPLCr <220> <221> CDS <222> (1)..(48) <400> 29 caa tat tat tct ggt tat ggt gat gtt gct tat gtt gac ttt aac ttg 48 Gln Tyr Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu 1 5 10 15 <210> 30 <211> 16 <212> PRT <213> Artificial Sequence <400> 30 Gln Tyr Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu 1 5 10 15 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of scFv for pPLCr <220> <221> CDS <222> (1)..(18) <400> 31 cag gcc agt cag agt gtt 18 Gln Ala Ser Gln Ser Val 1 5 <210> 32 <211> 6 <212> PRT <213> Artificial Sequence <400> 32 Gln Ala Ser Gln Ser Val 1 5 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LCDR2 of scFv for pPLCr <220> <221> CDS <222> (1)..(21) <400> 33 aag gct tcc act ctg gca tct 21 Lys Ala Ser Thr Leu Ala Ser 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <400> 34 Lys Ala Ser Thr Leu Ala Ser 1 5 <210> 35 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> LCDR3 of scFv for pPLCr <220> <221> CDS <222> (1)..(36) <400> 35 gca ggc ggt tgg aat tct aat gat gat acg ttt gct 36 Ala Gly Gly Trp Asn Ser Asn Asp Asp Thr Phe Ala 1 5 10 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <400> 36 Ala Gly Gly Trp Asn Ser Asn Asp Asp Thr Phe Ala 1 5 10 <210> 37 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat3 and pPLCr <400> 37 gctgcccaac cagccatggc ccagtcggtg gaggagtccr gg 42 <210> 38 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat3 and pPLCr <400> 38 gctgcccaac cagccatggc ccagtcggtg aaggagtccg ag 42 <210> 39 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat3 and pPLCr <400> 39 gctgcccaac cagccatggc ccagtcgytg gaggagtccg gg 42 <210> 40 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat3 and pPLCr <400> 40 gctgcccaac cagccatggc ccagsagcag ctgrtggagt ccgg 44 <210> 41 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for heavy chain variable regions of Fab for pStat3 and pPLCr <400> 41 cgatgggccc ttggtggagg ctgargagay ggtgaccagg gtgcc 45 <210> 42 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 42 gggcccaggc ggccgagctc gtgmtgaccc agactcca 38 <210> 43 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 43 gggcccaggc ggccgagctc gatmtgaccc agactcca 38 <210> 44 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 44 gggcccaggc ggccgagctc gtgatgaccc agactgaa 38 <210> 45 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 45 gggcccaggc ggccgagctc gtgctgactc agtcgccctc 40 <210> 46 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 46 agatggtgca gccacagttc gtttgatttc cacattggtg cc 42 <210> 47 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 47 agatggtgca gccacagttc gtaggatctc cagctcggtc cc 42 <210> 48 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 48 agatggtgca gccacagttc gtttgacsac cacctcggtc cc 42 <210> 49 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 49 agatggtgca gccacagttc ggcctgtgac ggtcagctgg gtccc 45 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for human CH1 <400> 50 gcctccacca agggcccatc ggtc 24 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for human CH1, and antisense primer for heavy chain variable regions of Fab for pStat3 and pPLCr + human CH1 <400> 51 agaagcgtag tccggaacgt c 21 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for human Ck <400> 52 cgaactgtgg ctgcaccatc tgtc 24 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for human Ck and antisense primer for light chain variable regions of Fab for pStat3 and pPLCr + human Ck <400> 53 ggccatggct ggttgggcag c 21 <210> 54 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat3 and pPLCr + human CH1 <400> 54 gctgcccaac cagccatggc c 21 <210> 55 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat3 and pPLCr + human Ck and sense primer for light chain variable regions of Fab for pStat3 and pPLCr + human Ck + heavy chain variable regions of Fab for pStat3 and pPLCr + human CH1 <400> 55 gaggaggagg aggaggaggc ggggcccagg cggccgagct c 41 <210> 56 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr + human Ck + heavy chain variable regions of Fab for pStat3 and pPLCr + human CH1 <400> 56 gaggaggagg aggaggagag aagcgtagtc cggaacgtc 39 <210> 57 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Linker for linkage of heavy and light chains of Fab for pStat3 and pPLCr <220> <221> CDS <222> (1)..(54) <400> 57 ggt ggt tcc tct aga tct tcc tcc tct ggt ggc ggt ggc tcg ggc ggt 48 Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 15 ggt ggg 54 Gly Gly <210> 58 <211> 18 <212> PRT <213> Artificial Sequence <400> 58 Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 15 Gly Gly <210> 59 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Phosphorylated STAT3 (pSTAT3) peptide <220> <221> MOD_RES <222> (7) <223> PHOSPHORYLATION <400> 59 Pro Gly Ser Ala Ala Pro Tyr Leu Lys Thr Lys Gly Gly Gly Ser Cys 1 5 10 15 <210> 60 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Phosphorylated PLCr (pPLCr) peptide <220> <221> MOD_RES <222> (6) <223> PHOSPHORYLATION <400> 60 Arg Asn Pro Gly Phe Tyr Val Glu Ala Asn Pro Gly Gly Gly Ser Cys 1 5 10 15<110> AbFRONTIER <120> ANTIBODY SPECIFICALLY BINDING TO A PHOSPHORYLATED PLC-γ PROTEIN <130> FPD / 200803-0081 <150> 61 / 031,433 <151> 2008-02-26 <160> 60 <170> KopatentIn 1.71 <210> 1 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> scFv for pStat3 <220> <221> CDS (222) (1) .. (735) <400> 1 gag ctc gtg ctg acc cag act cca tcc ccc gtg tct gca gtt gtg gga 48 Glu Leu Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag tcc agt cag agt gtt tgg ggt aac 96 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly Asn 20 25 30 aac cgc tta tcc tgg tat cag cag aaa cca ggg cag cct ccc agg ctc 144 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu 35 40 45 cta atg tat tat gca tcc aat ctg gca tct ggg gtc tca tcg cgg ttc 192 Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 aaa ggc agt gga tct ggg aca caa ttc act ctc acc atc agc gac gtg 240 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 cag tgt gac gat gct gcc act tac tac tgt caa ggc gga ttt gag tgt 288 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu Cys 85 90 95 agt ggt ggt gat tgt gtt ggt ttc ggc gga ggg acc gag ctg gag atc 336 Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu Ile 100 105 110 cta ggt ggt tcc tct aga tct tcc tcc tct ggt ggc ggt ggc tcg ggc 384 Leu Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 ggt ggt ggg cag tcg gtg gag gag tcc ggg ggt cgc ctg gta gcg cct 432 Gly Gly Gly Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala Pro 130 135 140 gga gga tcc ctg aca ctc acc tgc aca gtc tct gga atc gac ctc agt 480 Gly Gly Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 145 150 155 160 agc gat gca atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa 528 Ser Asp Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170 175 tgg atc gga acg att tat ggt agt gct ggc aca tac tac gcg acc tgg 576 Trp Ile Gly Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp 180 185 190 gcg aaa ggc cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg 624 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 195 200 205 aaa atg acc agt ctg aca acc gag gac acg gcc acc tat ttc tgt acc 672 Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Thr 210 215 220 aga gct ttt agc aac act cga ttg gat ctc tgg ggc cag ggc acc ctg 720 Arg Ala Phe Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu 225 230 235 240 gtc acc atc tct tca tagaa gcttcgaatt ctgcag 756 Val Thr Ile Ser Ser 245 <210> 2 <211> 245 <212> PRT <213> Artificial Sequence <400> 2 Glu Leu Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly Asn 20 25 30 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu 35 40 45 Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu Cys 85 90 95 Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu Ile 100 105 110 Leu Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala Pro 130 135 140 Gly Gly Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 145 150 155 160 Ser Asp Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170 175 Trp Ile Gly Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp 180 185 190 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 195 200 205 Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Thr 210 215 220 Arg Ala Phe Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu 225 230 235 240 Val Thr Ile Ser Ser 245 <210> 3 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of Fab for pStat 3 <220> <221> CDS (222) (1) .. (342) <400> 3 cag tcg gtg gag gag tcc ggg ggt cgc ctg gta gcg cct gga gga tcc 48 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala Pro Gly Gly Ser 1 5 10 15 ctg aca ctc acc tgc aca gtc tct gga atc gac ctc agt agc gat gca 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asp Ala 20 25 30 atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tgg atc gga 144 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 acg att tat ggt agt gct ggc aca tac tac gcg acc tgg gcg aaa ggc 192 Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 50 55 60 cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg aaa atg acc 240 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 agt ctg aca acc gag gac acg gcc acc tat ttc tgt acc aga gct ttt 288 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Ala Phe 85 90 95 agc aac act cga ttg gat ctc tgg ggc cag ggc acc ctg gtc acc atc 336 Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Ile 100 105 110 tct tca tag 345 Ser Ser <210> 4 <211> 114 <212> PRT <213> Artificial Sequence <400> 4 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala Pro Gly Gly Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asp Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Ala Phe 85 90 95 Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Ile 100 105 110 Ser Ser <210> 5 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Light chain variable region of Fab for pStat 3 <220> <221> CDS (222) (1) .. (339) <400> 5 gag ctc gtg ctg acc cag act cca tcc ccc gtg tct gca gtt gtg gga 48 Glu Leu Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag tcc agt cag agt gtt tgg ggt aac 96 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly Asn 20 25 30 aac cgc tta tcc tgg tat cag cag aaa cca ggg cag cct ccc agg ctc 144 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu 35 40 45 cta atg tat tat gca tcc aat ctg gca tct ggg gtc tca tcg cgg ttc 192 Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 aaa ggc agt gga tct ggg aca caa ttc act ctc acc atc agc gac gtg 240 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 cag tgt gac gat gct gcc act tac tac tgt caa ggc gga ttt gag tgt 288 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu Cys 85 90 95 agt ggt ggt gat tgt gtt ggt ttc ggc gga ggg acc gag ctg gag atc 336 Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu Ile 100 105 110 cta 339 Leu <210> 6 <211> 113 <212> PRT <213> Artificial Sequence <400> 6 Glu Leu Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly Asn 20 25 30 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu 35 40 45 Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu Cys 85 90 95 Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu Ile 100 105 110 Leu <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR1 of scFv for pStat3 <220> <221> CDS (222) (1) .. (15) <400> 7 agc gat gca atg agc 15 Ser Asp Ala Met Ser 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <400> 8 Ser Asp Ala Met Ser 1 5 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> HCDR2 of scFv for pStat3 <220> <221> CDS (222) (1) .. (45) <400> 9 acg att tat ggt agt gct ggc aca tac tac gcg acc tgg gcg aaa 45 Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <400> 10 Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of scFv for pStat3 <220> <221> CDS (222) (1) .. (27) <400> 11 gct ttt agc aac act cga ttg gat ctc 27 Ala Phe Ser Asn Thr Arg Leu Asp Leu 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Ala Phe Ser Asn Thr Arg Leu Asp Leu 1 5 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of scFv for pStat3 <220> <221> CDS (222) (1) .. (18) <400> 13 cag tcc agt cag agt gtt 18 Gln Ser Ser Gln Ser Val 1 5 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <400> 14 Gln Ser Ser Gln Ser Val 1 5 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LCDR2 of scFv for pStat3 <220> <221> CDS (222) (1) .. (21) <400> 15 tat gca tcc aat ctg gca tct 21 Tyr Ala Ser Asn Leu Ala Ser 1 5 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <400> 16 Tyr Ala Ser Asn Leu Ala Ser 1 5 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> LCDR3 of scFv for pStat3 <220> <221> CDS (222) (1) .. (39) <400> 17 caa ggc gga ttt gag tgt agt ggt ggt gat tgt gtt ggt 39 Gln Gly Gly Phe Glu Cys Ser Gly Gly Asp Cys Val Gly 1 5 10 <210> 18 <211> 13 <212> PRT <213> Artificial Sequence <400> 18 Gln Gly Gly Phe Glu Cys Ser Gly Gly Asp Cys Val Gly 1 5 10 <210> 19 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> scFv for pPLCr <220> <221> CDS (222) (1) .. (753) <400> 19 gag ctc gat atg acc cag act cca gcc tct gtg gag gta gct gtg gga 48 Glu Leu Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag gcc agt cag agt gtt tat aag gac 96 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Asp 20 25 30 aac aac tta gcc tgg tat cag cag aaa cca ggg cag cct ccc aag ctc 144 Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 ctg atc tac aag gct tcc act ctg gca tct ggg gtc ccg tcg cgg ttc 192 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 aaa ggc agt gga tct ggg aca cag ttc act ctc acc atc agc gac gtg 240 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 cag tgt gac gat gct acc act tac tac tgt gca ggc ggt tgg aat tct 288 Gln Cys Asp Asp Ala Thr Thr Tyr Tyr Cys Ala Gly Gly Trp Asn Ser 85 90 95 aat gat gat acg ttt gct ttc ggc gga ggg acc gcg gtg gtg gtc aga 336 Asn Asp Asp Thr Phe Ala Phe Gly Gly Gly Thr Ala Val Val Val Arg 100 105 110 ggt ggt tcc tct aga tct tcc tcc tct ggt ggc ggt ggc tcg ggc ggt 384 Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 ggt ggg cag tcg ctg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg 432 Gly Gly Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly 130 135 140 aca ccc ctg aca ctc acc tgc aca gtc tct gga ttc tcc ctc agt aac 480 Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn 145 150 155 160 tac tgg atg aac tgg gtc cgc cag gct cca ggg aag gga ctg gaa tgg 528 Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 165 170 175 atc gga gtc att agt agg agt ggt atc aca ggc tac gcg agc tgg gcg 576 Ile Gly Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala 180 185 190 aaa ggc cga ttc acc atc tcc aaa acc tcg acc aca gtg gat ctg aaa 624 Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys 195 200 205 atc acc agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga 672 Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg 210 215 220 caa tat tat tct ggt tat ggt gat gtt gct tat gtt gac ttt aac ttg 720 Gln Tyr Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu 225 230 235 240 tgg ggc caa ggc acc ctg gtc acc atc tct tca tag 756 Trp Gly Gln Gly Thr Leu Val Thr Ile Ser Ser 245 250 <210> 20 <211> 251 <212> PRT <213> Artificial Sequence <400> 20 Glu Leu Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Asp 20 25 30 Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Thr Thr Tyr Tyr Cys Ala Gly Gly Trp Asn Ser 85 90 95 Asn Asp Asp Thr Phe Ala Phe Gly Gly Gly Thr Ala Val Val Val Arg 100 105 110 Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly 130 135 140 Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn 145 150 155 160 Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 165 170 175 Ile Gly Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala 180 185 190 Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys 195 200 205 Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg 210 215 220 Gln Tyr Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu 225 230 235 240 Trp Gly Gln Gly Thr Leu Val Thr Ile Ser Ser 245 250 <210> 21 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region of Fab for pPLCr <220> <221> CDS (222) (1) .. (363) <400> 21 cag tcg ctg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 ctg aca ctc acc tgc aca gtc tct gga ttc tcc ctc agt aac tac tgg 96 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr Trp 20 25 30 atg aac tgg gtc cgc cag gct cca ggg aag gga ctg gaa tgg atc gga 144 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 gtc att agt agg agt ggt atc aca ggc tac gcg agc tgg gcg aaa ggc 192 Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala Lys Gly 50 55 60 cga ttc acc atc tcc aaa acc tcg acc aca gtg gat ctg aaa atc acc 240 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga caa tat 288 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gln Tyr 85 90 95 tat tct ggt tat ggt gat gtt gct tat gtt gac ttt aac ttg tgg ggc 336 Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu Trp Gly 100 105 110 caa ggc acc ctg gtc acc atc tct tca tag 366 Gln Gly Thr Leu Val Thr Ile Ser Ser 115 120 <210> 22 <211> 121 <212> PRT <213> Artificial Sequence <400> 22 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr Trp 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gln Tyr 85 90 95 Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Ile Ser Ser 115 120 <210> 23 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Light chain variable region of Fab for pPLCr <220> <221> CDS (222) (1) .. (336) <400> 23 gag ctc gat atg acc cag act cca gcc tct gtg gag gta gct gtg gga 48 Glu Leu Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 ggc aca gtc acc atc aat tgc cag gcc agt cag agt gtt tat aag gac 96 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Asp 20 25 30 aac aac tta gcc tgg tat cag cag aaa cca ggg cag cct ccc aag ctc 144 Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 ctg atc tac aag gct tcc act ctg gca tct ggg gtc ccg tcg cgg ttc 192 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 aaa ggc agt gga tct ggg aca cag ttc act ctc acc atc agc gac gtg 240 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 cag tgt gac gat gct acc act tac tac tgt gca ggc ggt tgg aat tct 288 Gln Cys Asp Asp Ala Thr Thr Tyr Tyr Cys Ala Gly Gly Trp Asn Ser 85 90 95 aat gat gat acg ttt gct ttc ggc gga ggg acc gcg gtg gtg gtc aga 336 Asn Asp Asp Thr Phe Ala Phe Gly Gly Gly Thr Ala Val Val Val Arg 100 105 110 <210> 24 <211> 112 <212> PRT <213> Artificial Sequence <400> 24 Glu Leu Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Asp 20 25 30 Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Thr Thr Tyr Tyr Cys Ala Gly Gly Trp Asn Ser 85 90 95 Asn Asp Asp Thr Phe Ala Phe Gly Gly Gly Thr Ala Val Val Val Arg 100 105 110 <210> 25 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HCDR1 of scFv for pPLCr <220> <221> CDS (222) (1) .. (15) <400> 25 aac tac tgg atg aac 15 Asn Tyr Trp Met Asn 1 5 <210> 26 <211> 5 <212> PRT <213> Artificial Sequence <400> 26 Asn Tyr Trp Met Asn 1 5 <210> 27 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> HCDR2 of scFv for pPLCr <220> <221> CDS (222) (1) .. (45) <400> 27 gtc att agt agg agt ggt atc aca ggc tac gcg agc tgg gcg aaa 45 Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 28 <211> 15 <212> PRT <213> Artificial Sequence <400> 28 Val Ile Ser Arg Ser Gly Ile Thr Gly Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 29 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> HCDR3 of scFv for pPLCr <220> <221> CDS (222) (1) .. (48) <400> 29 caa tat tat tct ggt tat ggt gat gtt gct tat gtt gac ttt aac ttg 48 Gln Tyr Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu 1 5 10 15 <210> 30 <211> 16 <212> PRT <213> Artificial Sequence <400> 30 Gln Tyr Tyr Ser Gly Tyr Gly Asp Val Ala Tyr Val Asp Phe Asn Leu 1 5 10 15 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LCDR1 of scFv for pPLCr <220> <221> CDS (222) (1) .. (18) <400> 31 cag gcc agt cag agt gtt 18 Gln Ala Ser Gln Ser Val 1 5 <210> 32 <211> 6 <212> PRT <213> Artificial Sequence <400> 32 Gln Ala Ser Gln Ser Val 1 5 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LCDR2 of scFv for pPLCr <220> <221> CDS (222) (1) .. (21) <400> 33 aag gct tcc act ctg gca tct 21 Lys Ala Ser Thr Leu Ala Ser 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <400> 34 Lys Ala Ser Thr Leu Ala Ser 1 5 <210> 35 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> LCDR3 of scFv for pPLCr <220> <221> CDS (222) (1) .. (36) <400> 35 gca ggc ggt tgg aat tct aat gat gat acg ttt gct 36 Ala Gly Gly Trp Asn Ser Asn Asp Asp Thr Phe Ala 1 5 10 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <400> 36 Ala Gly Gly Trp Asn Ser Asn Asp Asp Thr Phe Ala 1 5 10 <210> 37 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat 3 and pPLCr <400> 37 gctgcccaac cagccatggc ccagtcggtg gaggagtccr gg 42 <210> 38 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat 3 and pPLCr <400> 38 gctgcccaac cagccatggc ccagtcggtg aaggagtccg ag 42 <210> 39 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat 3 and pPLCr <400> 39 gctgcccaac cagccatggc ccagtcgytg gaggagtccg gg 42 <210> 40 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat 3 and pPLCr <400> 40 gctgcccaac cagccatggc ccagsagcag ctgrtggagt ccgg 44 <210> 41 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for heavy chain variable regions of Fab for pStat3 and pPLCr <400> 41 cgatgggccc ttggtggagg ctgargagay ggtgaccagg gtgcc 45 <210> 42 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat 3 and pPLCr <400> 42 gggcccaggc ggccgagctc gtgmtgaccc agactcca 38 <210> 43 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat 3 and pPLCr <400> 43 gggcccaggc ggccgagctc gatmtgaccc agactcca 38 <210> 44 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat 3 and pPLCr <400> 44 gggcccaggc ggccgagctc gtgatgaccc agactgaa 38 <210> 45 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat 3 and pPLCr <400> 45 gggcccaggc ggccgagctc gtgctgactc agtcgccctc 40 <210> 46 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 46 agatggtgca gccacagttc gtttgatttc cacattggtg cc 42 <210> 47 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 47 agatggtgca gccacagttc gtaggatctc cagctcggtc cc 42 <210> 48 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 48 agatggtgca gccacagttc gtttgacsac cacctcggtc cc 42 <210> 49 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr <400> 49 agatggtgca gccacagttc ggcctgtgac ggtcagctgg gtccc 45 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for human CH1 <400> 50 gcctccacca agggcccatc ggtc 24 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for human CH1, and antisense primer for heavy chain variable regions of Fab for pStat3 and pPLCr + human CH1 <400> 51 agaagcgtag tccggaacgt c 21 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for human Ck <400> 52 cgaactgtgg ctgcaccatc tgtc 24 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for human Ck and antisense primer for light chain variable regions of Fab for pStat3 and pPLCr + human Ck <400> 53 ggccatggct ggttgggcag c 21 <210> 54 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for heavy chain variable regions of Fab for pStat 3 and pPLCr + human CH1 <400> 54 gctgcccaac cagccatggc c 21 <210> 55 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for light chain variable regions of Fab for pStat 3 and pPLCr + human Ck and sense primer for light chain variable regions of Fab for pStat3 and pPLCr + human Ck + heavy chain variable regions of Fab for pStat3 and pPLCr + human CH1 <400> 55 gaggaggagg aggaggaggc ggggcccagg cggccgagct c 41 <210> 56 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for light chain variable regions of Fab for pStat3 and pPLCr + human Ck + heavy chain variable regions of Fab for pStat3 and pPLCr + human CH1 <400> 56 gaggaggagg aggaggagag aagcgtagtc cggaacgtc 39 <210> 57 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Linker for linkage of heavy and light chains of Fab for pStat 3 and pPLCr <220> <221> CDS (222) (1) .. (54) <400> 57 ggt ggt tcc tct aga tct tcc tcc tct ggt ggc ggt ggc tcg ggc ggt 48 Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 15 ggt ggg 54 Gly gly <210> 58 <211> 18 <212> PRT <213> Artificial Sequence <400> 58 Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 15 Gly gly <210> 59 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Phosphorylated STAT3 (pSTAT3) peptide <220> <221> MOD_RES <222> (7) <223> PHOSPHORYLATION <400> 59 Pro Gly Ser Ala Ala Pro Tyr Leu Lys Thr Lys Gly Gly Gly Ser Cys 1 5 10 15 <210> 60 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Phosphorylated PLCr (pPLCr) peptide <220> <221> MOD_RES <222> (6) <223> PHOSPHORYLATION <400> 60 Arg Asn Pro Gly Phe Tyr Val Glu Ala Asn Pro Gly Gly Gly Ser Cys 1 5 10 15

Claims (10)

인산화된 PLC-γ(phospholipase C-gamma) 단백질에 특이적으로 결합하고, 서열 번호: 26, 28 및 30으로 기재된 중쇄 상보성 결정 영역(complementarity determining region, CDR) 및 서열 번호: 32, 34 및 36으로 기재된 경쇄 CDR을 포함하는 것을 특징으로 하는 항체.Specifically binds to phosphorylated phospholipase C-gamma (PLC- [gamma]) proteins and to the heavy chain complementarity determining region (CDR) as set forth in SEQ ID NOs: 26, 28 and 30 and SEQ ID NOs: 32, 34 and 36 An antibody comprising the light chain CDRs described. 제 1 항에 있어서, The method of claim 1, 인산화된 PLC-γ 단백질의 인산화된 부위가 타이로신(tyrosine)인 것을 특징으로 하는 항체.An antibody, wherein the phosphorylated site of the phosphorylated PLC-γ protein is tyrosine. 제 1 항에 있어서,The method of claim 1, 상기 항체가 Fab 항체, ScFv 항체 또는 ScFv-Fc 미니항체인 것을 특징으로 하는 항체.The antibody is a Fab antibody, ScFv antibody or ScFv-Fc mini antibody. 제 3 항에 있어서,The method of claim 3, wherein 상기 Fab 항체가 서열 번호: 22로 기재된 중쇄 가변영역 및 서열 번호: 24로 기재된 경쇄 가변영역을 갖는 것을 특징으로 하는 항체.And said Fab antibody has a heavy chain variable region as set out in SEQ ID NO: 22 and a light chain variable region as set out in SEQ ID NO: 24. 제 3 항에 있어서,The method of claim 3, wherein 상기 ScFv 항체가 서열 번호: 20으로 기재된 아미노산 서열을 가지는 것을 특징으로 하는 항체.And said ScFv antibody has the amino acid sequence set forth in SEQ ID NO: 20. 제 3 항에 있어서,The method of claim 3, wherein 상기 ScFv-Fc 미니항체가 서열 번호: 20으로 기재된 아미노산 서열을 가지는 ScFv에 인간 항체의 Fc 부위가 결합된 것임을 특징으로 하는 항체.The ScFv-Fc mini-antibody is an antibody characterized in that the Fc region of the human antibody is bound to ScFv having the amino acid sequence set forth in SEQ ID NO: 20. 제 1 항의 항체를 암호화하는 폴리뉴클레오타이드.A polynucleotide encoding the antibody of claim 1. 제 7 항에 있어서,The method of claim 7, wherein 서열 번호: 25, 27 및 29로 기재된 중쇄 CDR-코딩 뉴클레오타이드 서열 및 서열 번호: 31, 33 및 35로 기재된 경쇄 CDR-코딩 뉴클레오타이드 서열을 포함하는 것을 특징으로 하는 폴리뉴클레오타이드.A polynucleotide comprising a heavy chain CDR-coding nucleotide sequence set forth in SEQ ID NOs: 25, 27, and 29 and a light chain CDR-coding nucleotide sequence set forth in SEQ ID NOs: 31, 33, and 35. 제 7 항에 있어서,The method of claim 7, wherein 서열 번호: 21의 중쇄 가변영역-코딩 뉴클레오타이드 서열 및 서열 번호: 23의 경쇄 가변영역-코딩 뉴클레오타이드 서열을 포함하는 것을 특징으로 하는 폴리뉴클레오타이드.A polynucleotide comprising the heavy chain variable region-coding nucleotide sequence of SEQ ID NO: 21 and the light chain variable region-coding nucleotide sequence of SEQ ID NO: 23. 제 7 항에 있어서,The method of claim 7, wherein 서열 번호: 19의 뉴클레오타이드 서열을 가지는 것을 특징으로 하는 폴리뉴클레오타이드.A polynucleotide having the nucleotide sequence of SEQ ID NO: 19.
KR1020080037341A 2008-02-26 2008-04-22 ANTIBODY SPECIFICALLY BINDING TO A PHOSPHORYLATED PLC-γ PROTEIN KR20090092202A (en)

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