Korean J Urol Oncol 2012;10(2):41-49 식물및곤충세포발현시스템을이용한암진단및치료용항체생산 중앙대학교의과대학의학부, 1 국립농업과학원농식품자원부, 2 원광대학교자연과학대학생명과학부 이정환ㆍ김득수ㆍ이재혁ㆍ명순철ㆍ황경아 1 ㆍ추영국 2 ㆍ고기성 Production of Cancer Diagnostic and Therapeutic mab Using Plant and Insect Expression Systems Jeong-Hwan Lee, Deuk-Su Kim, Jaehyouk Lee, Soon-Chul Myung, Kyung-A Hwang 1, Young-Kug Choo 2, Kisung Ko Department of Medicine, Medical Research Institute, College of Medicine, Chung-Ang University, Seoul, 1 Department of Agrofood Resources, National Academy of Agricultural Science, Rural Development Administration, Suwon, 2 Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan, Korea Therapeutic proteins can prevent or treat wide ranges of diseases from cancer and viral or bacterial infections. Production of the therapeutic proteins has been well established mainly in mammalian and bacterial cells including transgenic organisms by recombinant DNA techniques. Recent plant and insect biotechnology and advanced molecular immunology have established molecular biofarming system as an alternative way to produce recombinant pharmaceutical proteins such as immunotherapeutic monoclonal antibodies (mab). The plants and insects as an expression system have several advantages, which include the lack of animal pathogenic contaminants, low cost of production, and ease of agricultural scale-up compared to other currently available systems. Thus, the paradigm of plant is being shifted as a food source to so-called plant bioreactor for production of therapeutic proteins. Currently, we have successfully developed a plant and insect cell expression system for production of anti-cancer and anti-virus monoclonal antibodies. The effective heterologous production system for recombinant therapeutics requires the appropriate expression machinery with optimal combination of transgene expression regulatory conditions such as control of transcriptional and post transcriptional events. In this paper, the possibility of targeting proteins to the ER and possibly storing them to the protein storage vacuoles with glycosylation modification are discussed in plant and insect cells. (Korean J Urol Oncol 2012;10:41-49) Key Words: Plant, Insect cell, Therapeutic proteins 서 론 논문접수일 :2012 년 7 월 31 일, 수정일 :2012 년 8 월 4 일, 채택일 :2012 년 8 월 7 일교신저자 : 고기성, 중앙대학교의과대학의학부서울시동작구흑석로 84, 156-756 Tel: 02-820-5666, Fax: 02-813-5387 E-mail: ksko@cau.ac.kr 본논문은농산수산식품기술기획평가원 (Code#111096-03-1-SB010), 한국연구재단 (NRF-2011-002 6969), 농촌진흥청 (Code#PJ007492), 차세대바이오그린 21 (grant#pj00906) 의지원에의해서수행됨. 이형발현시스템 (heterologous expression system) 은 recombinant DNA 기술을응용하여특정목적을가진단백질을생산해낼수있다는중요한이점을가지고있다. 특히, 식물세포와곤충세포를이용한이형발현시스템은비뇨기계관련암및여러질병의예방과치료에도움을줄수있는치료용단백질을생산할수있다는이점이있다. 본논문에서는현재항체및의료용단백질을생산하는시스템으 41
42 대한비뇨기종양학술지 : 제 10 권제 2 호 2012 로는박테리아, 효모, 곤충세포, 동물세포배양시스템등이이용되고있다. 1 이러한발현시스템들은다른종간에발생할수있는병원균이나바이러스등 2차감염의위험성이있으며, 생산된단백질을분리하고정제하는데에매우어려우며, 그비용또한매우높다. 이러한발현시스템들의문제점으로인해전세계적으로요구되는의료용단백질의수요를충족시켜주지못하고있는실정이다. 2,3 현재전세계적으로전립선암이크게증가하고있는추세이며, 전립선특이항원이전립선관련진단에사용됨에따라전립선암진단율은더욱더증가하고있다. 4 전립선암은임상적기준에따라매우다양한데이러한전립선암의치료는그양상에따라매우다양하다. 5 따라서전립선암환자의암진단적양상을구분하는일은치료방법을선택하는데매우중요하다. 이러한전립선암의양상을정확히파악하여야고위험군환자의수명을연장시킬뿐아니라저위험군환자에서는불필요한치료로인한비용과합병증을낮출수있다. 6,7 이렇게다양한특징을가진전립선암에대응하기위해새로운암진단및치료방법으로재조합의료용단백질생산시스템의개발이요구되고있으며, 기존의의료용단백질생산시스템을보완하기위한방법으로최근식물생명공학기술과첨단분자생물학지식을이용하여식물또는곤충세포에서의료용단백질을대량생산하기위한연구가진행되고있다. 3,6-9 식물과곤충세포는인간과유사한 post-translational modification system을가지고있어단백질및당구조가흡사한고가의의료용단백질을생산할수있으며, 식물같은경우인간및동물바이러스등과같은병원체에의한 2차감염의우려가없고, 곤충세포도포유류세포에는감염성이없는곤충세포특이바이러스 (baculovirus) 를이용하기때문에안전하다고할수있다. 본리뷰에서는식물과곤충세포를이용한이형발현시스템이비뇨기관련암질병의진단및치료기술에도움을줄수있는가능성을설명하고자한다. 식물시스템을이용한항체의생산진핵세포인동물세포와곰팡이, 그리고원핵세포인박테리아등을일반적으로이용하여연구, 진단및치료의료목적으로고부가가치를갖는단백질인단일클론항체 (therapeutic monoclonal antibody) 를생산정제하여얻는다. 1-3 하지만이러한항체단백질생산방법은발현조건의영향으로단백질의발현, 정제, 및분리가상황에따라어려운점이있으며, 고가의 upstream processing 과정으로인해생산비용이높을수있다는단점들을가지고있다. 때문에최근발전하는식물생명공학기술과재조합단백질공학기술을 바탕으로진단및치료용항체생산을할수있는식물단백질발현시스템에대한연구가활발히진행되고있다 (Table 1). 10,11 지금까지의식물세포배양기술은대부분이 2차대사산물의생산이었으며, 이는자연적으로식물체가생산하는물질의생산능력제고에지나지않았지만, 식물을이용한형질전환식물체가보고된이후로, 4,5 식물발현시스템을이용하여진단및치료용단백질들을발현하려하는연구를수행하여이를이용한고부가가치의다양한의료용단백질들을안전하고적은비용으로쉽게생산규모를확대하여생산할수있는식물단백질발현시스템개발이진행되고있다. 12,13 식물을이용한치료용단클론항체생산은사람에서발견되는병원균에의한발병위험요소의감소, 식물의다양하고빠른바이오매스증가율과높은수확률, 원핵미생물을이용한시스템에서는볼수없는 glycosylation과진핵미생물보다동물세포에가까운 post-translational modification 과정이포함된다는장점을가지고있어인간이나동물에게유용한당단백질생산및생산된단백질의높은생물학적활성과안전성을확보할수있다. 그리고식물은종자나다른저장기관에서의생산을통한보존의편리성과같은경제적측면등에있어서도많은장점을가진다. 8 반면, 동물세포배양및실험동물을이용한항체및유용단백질의생산은동물유래단백질의구조우수성에도불구하고 host cell 이갖고있는인수공통병원성의감염우려가있다는단점이있어서쉽게사용하기에용이하지않다. 때문에이러한단점들을극복할수있는식물을유용단백질발현시스템으로의개발을위해있는많은연구대상이되고있다. Table 1. Monoclonal antibodies recently produced in transgenic plants and insect cells Plant Soybean, rice sf-9 sf-9 sf-9 Insect Monoclonal antibody type (target) References IgG (Streptococus mutans) Ma et al. (1998) IgG (herpes simplex virus) Zeitlin et al. (1998) IgG (hepatitis B virus) Valdes et al. (2003) IgG (rabies) Ko et al. (2003) IgG (colorectal cancer) Ko et al. (2005) IgG (breast cancer) Brodzik et al. (2006) Monoclonal antibody type (target) IgG (African cassava mosaic virus) IgG (colorectal cancer) IgG (colorectal cancer) References Reavy et al. (2000) Park et al. (2005) Song et al. (2005)
이정환외 : 식물및곤충세포발현시스템을이용한암진단및치료용항체생산 43 Fig. 1. N-glycosylation processing of glycoproteins in plant, human, insect and yeast. 고등식물들은진핵세포로서동물세포가가지고있는세포기관인 Endoplasmic reticulum ( 소포체 ) 및 Golgi complex ( 골지체 ) 를가지고있어당단백질의활성 (bioactivity) 에있어서중요한단백질조합 (assembly), 단백질접힘 (folding) 및당화 (glycosylation) 과정을할수있다. 비록, 식물과동물에따른코돈선호도 (cordon usage) 나 post translation 과정중 glycosylation process의차이가있기는하나, 미생물과비교해볼때식물은동물세포와매우유사한점을더많이가지고있다 (Fig. 1). 이러한다양한생명체유래재조합항체공학분야는초기의카이메릭항체 (chimeric antibody) 나인간화항체 (humanized antibody) 의개발단계를거쳐최근에는항체의탈면역화 (deimmunization) 기술이나형질전환생쥐및 phage display 기술을이용한인간항체 (fully human antibody) 개발단계까지발전하고있다. 7 이러한기술을바탕으로차세대항체발현시스템이라고할수있는식물을이용하여항암 (anti-cancer) 혹은항바이러스 (anti-virus) 효능을가진단일클론항체 (monoclonal antibody) 를대량생산하기위한연구가활발하게진행중이다. 14 하지만, 식물단백질발현시스템이가지고있는위에서언급한여러장점들에도불구하고형질전환기술에의해식물세포로이동한재조합단백질유전자의낮은발현과식물조직배양후식물바이오매스를높이기위한종자를얻기까지는걸리는시간이다소오래걸리는점등의단점을가 지고있다. 이와같이단일클론항체의생산을위해서식물단백질발현시스템을상업화한다는것은많은문제점을해결이우선시되지않는다면많은어려움이발생될수있다. 이러한문제점을해결하기위하여유전자재조합단계에서발현하고자하는특정단백질생산을궁극적으로높일수있는강력한식물벡터프로모터의개발및이용, 바이오매스를빠른시일내에높일수있는식물단백질발현시스템의확립, 뿐만아니라단백질발현후발생하는변성억제및식물세포내의저장효율성증가등을위한연구가필요하다. 15-17 식물유래치료용항체생산을위한기술적고려현재생명공학산업에서는인간및동물에서발병하는질병에대한진단및치료에널리사용되는면역단백질인항체를생산하기위하여주로미생물, 곤충및동물세포배양시스템을이용하고있다. 12 미생물단백질발현시스템은박테리아를이용하여빠른시일내에다량의단백질생산이용이하다는것이장점이지만, 번역후당화가이루어져야활성을갖게되는대부분의의료용당단백질의특성상, 당화시킬수있는능력을없는미생물의경우최종적으로얻는항체가생리활성을지니지못한단백질을발현하게된다. 9 또한, 동물세포경우는번역후변형이필요한당단백질및항체등의생산에주된방법으로이루어지고있으나
44 대한비뇨기종양학술지 : 제 10 권제 2 호 2012 높은생산비용이필요하다는단점을갖고있고동물세포내의인수공통병원체 2차감염위험이발생할수있어새로운대체단백질발현시스템이필요하다. 18 이에따라기존생산시스템에대한대체방법으로식물단백질발현시스템을활용한단백질생산기술에대한기대와관심연구가활발히진행되고있다. 위에서언급한바와같이식물시스템은동물세포배양과유사한번역후당화에의하여미생물에서생산이불가능한고부가가치의의료용당단백질생산이가능하며, 식물의양적생물자원의증감을자유자재로할수있고, 항체얻기까지의요구되는생산비용을획기적으로절감할수있기때문에기존에사용된동물, 미생물의단백질생산시스템의단점을보완하고식물의특성을이용한안전한단백질발현기술로발전시킬수있을것으로예상하고있다. 19,20 하지만식물단백질발현시스템은식물고유의당화과정을거치기때문에인간유래당단백질인항체의당구조를식물의특이적인당구조로변형시킬수있는문제점이있다. 이는당구조를인간화하지못한다면식물유래항체가인체와포유류유래당구조의차이로인해식물을이용한치료용단일클론항체의상업화기술개발은어려울수있다. 21,22 때문에식물의특이적인당화과정을인간화하기위한연구가활발히진행되고있다. 23,24 인체나포유동물유래의항체에는 fucose가 α(1,6) 결합으로 N-acetyl glucosamine에연결되어있는반면식물유래항체에는 α(1,3) 결합으로연결되어있어있다. 이러한식물특이적 fucose는 allergenic response를유발할수있는 residue로보고있어제거해야한다. 25 또한, 식물은 allergenic response를일으킬수있는 β(1,3) xylose가 mannose에연결되어있어이또한인간화당구조를갖는항체를발현하기위해서는제거해야할 glycan residue 중에하나이다. 특히, 암치료를목적으로하는단일클론항체는무엇보다도항암활성을갖는것이중요하다. 식물과동물들을이용한발현시스템이라고하더라도인간에서유래한항체가가져야할능력을갖는단백질을발현해야한다. 때문에단순히발현률만높이는연구뿐만아니라항암활성을높을수있도록단백질및당구조변경에대한연구가최근활발히이루어지고있다. 26,27 항체의항암활성에주로영향을주는것은암세포를특이적으로인지하는항체-항원반응과면역세포를유도하여암세포를괴멸시키는 ADCC (antibody-dependent cell cytotoxicity) 이다. ADCC는항체에붙어있는당구조에의해영향을받을수있다. 항체의 Fc와면역세포의 Fc receptor간의상호작용은이러한 ADCC에중요한역할을하는데당구조에 α(1,6)-fucose가존재하지않을경우 Fc와 Fc receptor간의상호작용을증가시켜 ADCC를높일수있다. 따라서, 식물에서의경우 α(1,3)-fucose를제거하는대신당 구조의인간화를위해 N-glcan 구조에 α(1,6)-fucose를넣을필요가없다. 식물 N-glcyan에는 sialic acid가존재하지않는 residue이다. 하지만항체의안정성에중요한 sialic acid나 galactose의존재는항암활성에는직접적으로영향을주지는않지만필요하다. 최근식물에도 sialylation을위한당화과정의경로가존재한다는보고가있어 sialic acid를 terminal N-glycan에부착하기위한연구를지속적으로진행해오고있다. 하지만식물세포내의 sialylation을위한당화과정자체뿐만아니라 sialic acid에대한전구물질또한중요한요소이기에이에대한연구또한필요하다. 28 식물을이용한단백질발현시스템으로의료용항체를생산할수있는기술에대한연구가많이이루어지고있으나, 아직까지는기존의동물세포를이용한항체생산시스템에비해단백질의발현율이비교적낮아상업화에어려움이있다. 일반적으로식물에서의료용단백질유전자를발현할때 cauliflower mosaic virus (CaMV 35S) promoter를많이이용하게되는데, 이경우의료용단백질은전체 total soluble protein (TSP) 의 0.0001-0.1% 정도의낮은발현율을보인다. 때문에적절한 inducible promoter의개발이나조직특이적으로발현하게하는고발현식물벡터 promoter의적용을통해높은발현율을유도하여야한다. 29 또한, 대부분의의료용유전자는동물혹은인간유래이므로식물에서발현할경우식물에적합한유전자의코돈 (codon) 최적화를통해동물유래재조합단백질의발현율을높여야한다. 30 식물세포내에서도세포질에서의발현뿐만아니라세포밖으로단백질을분비할수있도록 default secretion과정에필요한 endoplasmic reticulum signal peptide를재조합단백질에붙여발현할수있는 amino acid sequence를디자인하기도한다. 31 또한, 세포내 endoplasmic reticulum이나엽록체등의세포소기관으로의단백질축적등의기술을이용하여의료용단백질의발현율및항체생산수율을증폭시킬수가있다. 32,33 의료용항체의식물세포내에서세포외부로의분비는단백질분비과정에관련된신호펩타이드 (signal peptide) 에의해서소포체내로들어가시작되며, 소포체에서 folding과 assembly 및당화과정이이루어진후, 단백질이소포체보유신호 KDEL signal peptide (ER retention signal) 나다른세포소기관으로이동시키는신호가없으면골지체로이동되어외부로분비된다 (Fig. 2). 34 식물세포의소포체내에단백질을보유하게 KDEL signal peptide를항체중쇄의 C-terminal에부착하여소포체에축적시키는경우항체의생산량이 10-100배정도증가하게된다. 35 앞에서언급한바와같이식물단백질의당단백질이 N-당화부위에부착되는 xylose와 fucose 당은인간에투여시 allergy 반응을유발하
이정환외 : 식물및곤충세포발현시스템을이용한암진단및치료용항체생산 45 Fig. 2. Antibody structure and its components affecting biofunctional properties. 는주요항원으로서, 식물유래의의료용단백질이임상에사용되기위해서는면역학적으로과민반응을일으키지않게하는전략이필요하다. 이러한 allergy 유발원인인 xylose와 fucose를동시에제거할수있는방법은의료용당단백질인항체를소포체에머물게하여더이상복잡한당이부착되지않게하거나골지체내 xylosyltransferase나 fucosyltransferase와같은당화관련효소의발현을억제하는방법등이있다. 36 부가적으로식물특이적 glycan에 β(1,4)- galactose를추가하게되면 xylose나 fucose가붙게되는것을억제할수있다. 37 최근에는항원에특이적으로작용하는부분인항체의중쇄와경쇄의가변부위 (variable region) 만을폴리펩타이드 (polypeptide) linker로연결한 scfv (single chain variable fragment) 와 Fc 지역만을제외한나머지부분을포함하는 Fab (antigen binding fragment) 를제작하여식물유래의료용단백질의당구조에의한면역부작용을제거하기위해항체단백질의구조중필요한부분만을발현하기도한다. BACULOVIRUS EXPRESSION VECTOR SYSTEM (BEVS) 인간과동물의병을고치거나예방하는데쓰이는의료용단백질은다양한발현시스템으로만들어져왔다. 38-40 의료를위한단백질을만드는데사용되는발현시스템은포유류, 식물, 곤충, 균류, 박테리아그리고생물체에유전자도 입을위한재조합 DNA 기술을사용함으로써생산되었다. 곤충세포에서는베큘로바이러스를유전자전달체로이용하여의료용단백질을생산하는데사용되었다. 지금까지 500종이상의다양한베큘로바이러스가발견되었고, 감염이가능한숙주범위로는곤충과같은무척추동물들로제한된다. 41 그러므로베큘로바이러스는포유류세포또는다른척추동물의세포에감염될수없다. Monoclonal antibody (mab) 를포함한의료용단백질은종양, 염증등에매우밀접한관련이있는당단백질이다. BEVS는재조합단백질발현을위한가장다재다능하고강력한진핵세포벡터체제중하나가되었다. BEVS는곤충세포에특이적으로감염되는베큘로바이러스의특성을이용한재조합단백질발현시스템이다. 조밤나방 (armyworm; 학명 : Spodoptera frugiperda) 에서유래한 cell line을주로이용하며, Autographa californica 핵다면체바이러스 (AcMNPV) 로불리는바이러스를이용한다. 41 특정곤충종에만제한적으로감염되는 virus의특성상 mammalian cell에서증식하지않고, 박테리아, 효모등다른세포발현시스템과비교하여매우높은효율로재조합단백질을발현시킬수있다. 이러한이점때문에 BEVS은항체 (mab) 와같은치료용재조합단백질 (therapeutic recombinant protein) 연구뿐만아니라다른여러기능성단백질생산에필수적인시스템으로여겨진다. 원핵세포발현시스템의대표적인 E. coli 발현시스템의경우단백질의기능에필수적인당화과정 (glycosylation), 인산화반응 (phosphorylation) 등진핵세포에서만일어나는 posttranslation modification이진행되지않아대부분의단백질이불활성화된다. 또한비용적인측면에서 CHO cell과같은동물세포단백질발현시스템보다저렴하다는이점도가지고있다. 한편, 포유류와 BEVS 모두 GPCRs와같은 transmembrane protein의발현을위해일상적으로이용되고있으나, 단지발현적인측면만보면 BEVS가포유류보다는낮다. 42 Bac-to-Bac R 베큘로바이러스발현시스템을이용한항암항체생산 Bac-to-Bac R 베큘로바이러스발현시스템은 Invitrogen회사에서개발된시스템으로서베큘로바이러스셔틀벡터 (shuttle vector) 에유전자발현카셋트 (gene expression cassette) 의위치-특이성전위 (site-specific transposition) 를기반으로하여대장균을증식시켜바이러스벡터를얻는원리이다 (Fig. 3). 43 이시스템은 Monsanto R 의연구원에의해처음개발되었으며, Bac-to-Bac R 은단시간에효과적으로재조합베큘로바이러스를얻을수있다는장점이있다. 44 곤충세포
46 대한비뇨기종양학술지 : 제 10 권제 2 호 2012 Fig. 3. Schematic diagram of production of anti-cancer monoclonal antibody (mab) using Baculovirus expression vector system (BEVS) (modified from Ahn et al., 2008). 주는기본적으로 Sf-9 cell line (Spodoptera frugiperda) 을이용하며, pfastbac 계열의다양한 modified vector를이용하여곤충세포에서재조합단백질을생산을위한바이러스벡터 (Bacmid) 를생산할수있다. 바이러스벡터인 Bacmid를제작하기위해서는형질전환 E.coli의한종류인 DH10Bac을사용한다. pfastbac vector에목적유전자를삽입하여 DH10Bac 대장균에형질전환을실시하면 DH10Bac 내에서베큘로바이러스벡터인 bacmid가형성된다. 형성된 bacmid 를분리해내어 Bac-to-Bac R 시스템에따라곤충세포주 (Sf-9) 에형질도입 (transfecrion) 을실시하면약 3일후곤충세포내에서베큘로바이러스가형성된다 (Fig. 3). 이시스템을이용하여실제로항암항체를생산하였다. 대장암등몇몇암세포의세포표면에특이적으로과발현되는 Epidermal cell adhesion molecule (EpCAM) 의한종류인 GA733-2를인지하는단일항체 (mab) 를타겟으로하여항대장암단일항체 (mab CO17-1A) 를생산하였다. 45 곤충세포특성상 post-translation modification이가능하기때문에단일항체와같은당단백질을생산할수있고, 이러한특성을이용하여 ER retention signal인 KDEL을삽입하여생산된단일항체를높은수준으로얻을수있다. 45,46 또한 KDEL이삽입된단일항체의당구조 (glycan structure) 는 high-mannose 형태를가지기때문에곤충특이적인당구조를갖지않아이종간면역거부반응 (immune rejection response) 을어느정도억제할수있을것으로기대된다. 45,46 비뇨기관련암질병진단및치료목적으로서 BEVS의효용가치앞서언급된이형발현시스템중곤충세포를이용한 BEVS의의료용단백질생산시스템은비뇨기관련암질병의진단및치료에적용될수있다. 특히, 전립선암의성장을억제하고, 세포사멸에이르게할수있는단일항체의개발이야말로전립선암을치료할수있는최적의방법이라고사료된다. 전립선암의치료방법을개발하기위해단일항체를이용한전립선암억제연구가활발히진행되고있다. 47-49 앞에서말한바와같이비뇨기암에효능이검증된단일항체가밝혀지고유전자가확보가되면단일항체의유전자를기존연구를통해구축된 BEV 항체발현시스템에발현벡터 cloning으로쉽게적용하여곤충세포에서생산할수있을것으로본다. 물론, 여기에서몇가지전제조건이있다. 첫째, 발현하고자하는단일항체의항암효능이검증되어야한다, 둘째, 동물이나인간유전자에서유래한것이므로곤충세포에서효율적인발현을위해서는 condon optimization이필요하다. 셋째, 곤충세포에서생산된항체라하더라도임상치료를위해서는안전성이필요하다. 특히, 곤충특이적당구조를피하고인간특이적당구조를갖는항체를얻는것이중요한관건이라할수있다. 넷째, 높은생산성을위해서는곤충세포의효율적인바이오매스증식이중
이정환외 : 식물및곤충세포발현시스템을이용한암진단및치료용항체생산 47 요하지만정제과정에서의높은비용은어는단일항체발현과정에서제일중요한부분이다. 때문에, 경제적인정제시스템을개발구축하여야한다. 이러한이유로본연구자들은이에대한곤충발현시스템개발, 당구조변형, 발현및정제과정최적화등과관련하여다양한연구결과들을토대로암치료항체및진단용항체발현연구를하고있다. 11,45 이러한시스템이확보될경우최근전이성신장암에쓰일수있는 Bevacizumab의경쇄와중쇄유전자를곤충발현벡터에있는 Pph와 P10 promoter에각각발현할수있도록 cloning 하여우선적으로발현여부와활성검증을통해 BEV 시스템에적용가능여부를확인할수있을것으로본다. 47 임상암진단및치료는환자본인에게수술이나화학약물투여로인한신체적, 정신적스트레스를최소한으로할수있다는장점이있다. 그러나이러한치료용단일항체는 CHO cell 발현시스템과같은동물세포발현시스템을통해생산할경우그생산비용과급격한수요증가로인한수요량충족을만족시킬수가없다. 13 그렇기때문에 BEVS와같은이형발현시스템을적극활용한다면경제적이고안전한환경에서대량의의료용단백질을생산할수있을것으로기대된다. 그러나안정적인단일항체의정제방법, 곤충특이적인당단백질의당구조변형등몇가지문제점의해결이요구된다. 결 식물및곤충시스템을이용한의료용항체의생산은경제적으로이득이며, 기타미생물이나포유동물발현시스템과비교시인간에게감염되는병원균이결여되어있어안전하다는장점을가지고있다. 때문에식물및곤충세포를이용하여다양한의료용단백질뿐만아니라인간질병에대한면역치료용단일클론항체를생산할수있는식물단백질발현시스템이상업적으로경쟁력을가질수있다. 특히비뇨기계관련암및질병의예방과치료에많은도움을줄수있는진단및치료용항체를생산하는이형발현시스템개발을위한연구를계속진행해나간다면저렴한생산비용으로안전한의료용항체를대량생산할수있을것으로본다. 론 REFERENCES 1. Ma JKC, Vine ND. Plant expression systems for the production of vaccines. Curr Top Microbiol Immunol 1999;236:275-92 2. Ma JK, Drake PM, Christou P. The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 2003;4:794-805 3. Ko K, Steplewski Z, Glogowska M, Koprowski H. Inhibition of tumor growth by plant-derived mab. Proc Natl Acad Sci USA 2005;102:7026-30 4. Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP, et al. Expression of bacterial genes in plant cells. Proc Natl Acad Sci USA 1983;80:4803-7 5. Horsch RB, Fraley RT, Rogers SG, Sanders PR, Lloyd A, Hoffman N. Inheritance of functional foreign genes in plants. Science 1984;223:496-8 6. Gomord W, Chamberlain P, Jefferis R, Faye L. Biopharmaceutical production in plants: problems, solutions and opportunities. Trends in Biotechno 2005;23:559-65 7. Little M, Kipriyanov SM, Le Gall F, Moldenhauer G. Of mice and men: hybridoma and recombinant antibodies. Immunol 2000;21:364-70 8. Breedveld FC. Therapeutic monoclonal antibodies. Lancet 2000;355:735-40 9. Miele L. Plants as bioreactors for biopharmaceuticals: regulatory considerations. Trends Biotechnol 1997;15:45-50 10. Ko K, Steplewski Z, Glogowska M, Koprowski H. Inhibition of tumor growth by plant-derived mab. Proc Natl Acad Sci USA 2005;102:7026-30 11. Park DY, Lee JH, So YK, Kim YK, Ko K, Park SW, et al. Optimization of expression conditions for production of Anti-colorectal cancer monoclonal antibody CO17-1A in baculovirus-insect cell system. Hybridoma 2011;30:419-26 12. Kusnadi AR, Evangelista RL, Hood EE, Howard J, Nikolov ZL. Production and purification of two recombinant proteins from transgenic corn. Biotechnol Progress 1998;14:149-55 13. Ko K, Koprowski H. Plant biopharming of monoclonal antibodies. Virus Res 2005;111:93-100 14. Brodzik R, Glogowska M, Bandurska K, Okulicz M, Deka D, Ko K, et al. Plant-derived anti-lewis Y mab exhibits biological activities for efficient immunotherapy against human cancer cells. Proc Natl Acad Sci USA 2006;103:8804-9 15. Tekoah Y, Ko K, Koprowski H, Harvey DJ, Wormald MR, Dwek RA, et al. Controlled glycosylation of therapeutic antibodies in plants. Arch Biochem Biophys 2004;426:266-78 16. Bevaart L, Jansen MJH, van Vugt MJ, Verbeek JS, van de Winkel JG, Leusen JH. The high-affinity IgG receptor, FcgRI, plays a central role in antibody therapy of experimental melanoma. Cancer Res 2006;66:1261-4 17. Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants. Nature 1989;342:76-8 18. Bakker H, Bardor M, Molthoff JW, Gomord V, Elbers I, Stevens LH, et al. Galactose-extended glycans of antibodies produced by transgenic plants. Proc Natl Acad Sci USA 2001;98:2899-904 19. Doran P. Foreign protein production in plant tissue cultures. Current Opinion in Biotechnol 2000;11:199-204
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