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KR0151408B1 - 항원-특이성 인체 모노클로날 항체의 시헙관내에서의 생성방법 - Google Patents

항원-특이성 인체 모노클로날 항체의 시헙관내에서의 생성방법

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KR0151408B1
KR0151408B1 KR1019910006661A KR910006661A KR0151408B1 KR 0151408 B1 KR0151408 B1 KR 0151408B1 KR 1019910006661 A KR1019910006661 A KR 1019910006661A KR 910006661 A KR910006661 A KR 910006661A KR 0151408 B1 KR0151408 B1 KR 0151408B1
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키넨 고로프 다이아나
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에프.쥐.엠.헤르만스
악조 엔.브이.
에이.쥐.제이.베르미렌
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Abstract

본 발명은 시토킨인터루킨-4(IL-4)과 인터루킨-6(IL-6)중 적어도 하나와 8-구아노신으로 구성된 보조계를 사용하여 엡스타인바르 바이러스(EBV)로 형질전환되고 T-세포가 결핍된 인체말초혈액 임파구로부터 IgG, IgA 및 IgM 항원-특이성인체모노클론항체류를 증폭 생성시키는 방법에 관한 것이다. 또한 본 발명은 상기 보조계와 그것을 함유한 키트도 포함한다.

Description

항원-특이성 인체 모노클로날 항체의 시험관내에서의 생성방법
제1도는 각 아이소타입(isotype)의 면역 글로불린에 대한 T-세포의 영향을 나타낸 그래프이다.
제2도, 제3도 및 제4도는 각 아이소타입의 면역글로불린에 대한 시간경과에 따른 8MG, IL6 및 IL4의 영향을 나타낸 그래프이다.
제5도는 각 아이소타입의 면역 글로불린에 대한 각종 시약의 영향을 나타낸 그래프이다.
본 발명은 시토킨(cytokines)인터루킨-4(IL-4) 및 인터루킨-6(IL-6)중 하나 이상과 8-메르캡토구아노신을 함유하는 보조계(adjuvant system)를 사용하여, 엡스타인 바르 바이러스(EBV)로 형질전환되고 T-세포가 결핍된 인체 말초 혈액임파구로부터, IgG, IgA 및 IgM 항원-특이성 인체 모노클론 항체를 증폭생성시키는 방법에 관한 것이다. 또한 본 발명은 상기 보조계와 그것을 함유한 키트도 포함한다.
마우스로부터 유출해낸 모노클론 항체는 생체내(in vivo)치료 및 진단 방법 과 시험관내(in vitro) 진단 시험에 가장 일반적으로 사용되는 시약이다. 상기한 쥐의 모노클론을 사용하여 우수한 성과를 얻어왔지만 그것의 큰 단점은 상기 항체가 인체내에서 생성되는 항체와 동일하지 않다는 것이다. 종의 상이성 때문에, 인체를 생체내에서 진단 또는 치료함에 있어서 쥐의 모노클론 항체를 사용하는 경우 처치된 환자에게서 항-쥐(anti-murine)항체가 생성되는 것으로 알려져 있다. 상기한 항-쥐 항체가 존재하면, 쥐의 모노클론 항체를 사용한 추가치료나 생체내 진단을 저해할 정도로 환자를 감작시킬 수 있다. 상기 감작상태의 환자에 대한 시험관 내에서의 시험결과, 진단 분석시험에 있어서 쥐의 항체와 항-쥐 항체와의 상호작용으로 인한 오차가 발생할 수도 있는 것으로 나타났다. 상기의 문제점들을 극복하려면, 특히 생체내에서 사용하려면 모노클론 항체는 인체로부터 유도해낸 항체를 선택해야 한다.
비장이나 편도 같은 각종 기관의 세포를 사용하여 인체의 항체를 생성하는 것도 이론적으로는 가능하지만, 말초 혈액으로부터 공급받는 것이 가장 용이하다.
인체 말초 혈액 임파구(PBL)로부터 유도해낸 항원 특이성 모노클론 항체를 생성하는 것이 가능하다는 사실은 본 기술분야에 숙련된 자들에게 공지된 사실이다. 유감스럽게도, 정확한 아이소타입(isotype)을 갖는 항원 특이성 인체 항체를 생성하는 속도가 성공적이지 못하며, 특히 시간이 많이 소모되었다.
모노클로날 항제 생성에 관한 대부분의 연구는 쥐를 모델로 실시되어 왔으며, PBL의 사용은 더 새로운 분야이다. 일반적으로 공여체는 혈액제공 이전에 면역되지 않았고 면역될 수 없기 때문에, PBL의 B세포를 적당한 항원에 노출시킴으로써 시험관내에서만 활성화되어야 한다. 항원에 의해 활성화가 되면 B세포를 계속적으로 증식시키는 사이클이 개시되고 상기 세포는 항체 생성 세포로 변형된다. 일단 상기 세포가 항원에 대한 항체를 생성하면, 일반적으로, IgM 아이소타입이 가장 많이 생성된다는 사실이 알려져 있다. 그러나, 여러 연구에서 선택된 아이소타입은 IgG이다.
시험관내에서 항원을 제공하는 것은 기술적으로 매우 어렵다. 항원 특이성 B세포의 활성화, 증식 및 분화(differentiation)는 C8-치환된 구아닌리보뉴클레오사이드로 알려진 화합물부류를 이용하여 향상시킬 수 있는 것으로 알려져 있다(참조:M.G. 굳만, CB-유도된 구아닌 리보뉴클레오사이드의 면역생물학적 특성, Biomedicine Pharmacotherapy, 37, 344-350(1983); M.G. 굳만등., 유도된 구아닌 뉴클레오사이드: 시험관내 항체 반응을 위한 새로운 보조제 유형, J. of Immuno., 130, 2580-2585(1983); L. 다이엘슨 등., T-세포의존성 항원에 대한 인체 말초성 B 임파구의 시험관내 특이적 면역화에 대한 시토킨의 영향; Immunology., 61, 51-55(1987); M.G. 굳만, CB-치환된 구아닌 리보뉴클레오사이드의 면역 촉진 작용에서의 인산화 반응 및 회수의 역할, J. Immuno, 141, 2394-2399(1988); W.J. 헨넨등., 유럽 특허출원 제0 341 065호, 면역 촉진 구아닌 유도체, 조성물 및 방법). 더욱 활성이 큰 화합물로는 8-브로모구아노신, 8-메르캡토구아노신 및 7-메틸-8-옥소구아노신이 있다. 활성화의 특수한 양태는 정확하게 알려져 있지 않지만, C8-유도된 구아닌 리보뉴클레오사이드, 특히, 8-메트캡토구아노신(8-MG)는 B-세포 활성화를 촉진시켜서 항원 특이성 항체 생성 세포를 생성시키는 능력을 갖는다. 따라서, 배양초기에 PBL중에 존재하는 항원특이성 B-세포의 수가 1/106-107이하인 반면, 8-MG를 첨가하면 시험관 내에서 항원에 대해 반응하는 상기 세포들이 현저하게 클론 확장을 하게 된다.
L. 다니엘슨등(상술함)은 인체 B 임파구의 항원 특이적 활성화에 있어서 시토킨의 영향을 조사하였다. T-세포 결핍된 PBL 및 분리되지 않은 PBL 모두를 항원으로서 헤모시아닌과 보조제로서 마우스의 재조합 IL-1 및 인체의 재조합 IL-2를 다른 시토킨류와 함께 동시에 본 연구에 사용했다. 그 결과 시험관내 비분리 PBL에서 면역반응이 나타나지 않는 것을 발견하였다. T-세포 결핍 PBL에 있어서, 항원 특이성 인체 B세포가 발견되었으며, B 세포 분화인자 및 IL-2를 첨가함으로써 분화 정도가 증진되었다. IL-1을 첨가한 경우에만 무시할 만한 효과가 나타났다. 그러나, B 세포 분화인자(이 경우, 포크워드 마이토겐 T- 세포 치환 치환 인자임)의 사용은 어떠한 반응을 위해서라도 절대적으로 필요하다.
1985년에 굳만과 웨이글은 7-메틸-8-옥소구아노신 및 IL-2를 사용하여 T-세포 결핍된 PBL 의 항원 특이적 주(primary) 항원반응을 시험관내에서 연구하였다. 그 결과, T-세포 결핍된 PBL 시스템에 보조제를 사용하는 경우 항원 특이성 항체반응을 발생시키는 것으로 나타났다. 7-메틸-8-옥소구아노신과 8-MG는 단지 보조제로서 시험되었다. 7-메틸-8-옥소구아노신이 지속적으로 항체 반응을 크게 향상시키는 반면, 8-MG 는 훨씬 적은 반응을 유도했다(M.G. 굳만 등., IL-2와의 상승작용에 있어서 C8-치환된 구아닌 리보뉴클레오사이드의 인체 항체반응 향상 효과, J. Immuno., 135, 3284-3288(1985)).
그후에, 각종 시토킨류가 8-MG 와 상승 작용하여 항원에 대한 B 세포 반응성을 증가시킬 수 있는지 여부를 결정하기 위하여 유사한 연구들이 실시되었다. 쥐의 세포주를 사용했다. 인터페론-감마, 정제된 IL-1, IL-2, IL-3, IL-4 또는 IL-5 로는 상승 효과가 나타나지 않지만 인터페론-알파 및 -베타에서는 상승효과가 나타났다(M.G. 굳만., 액소성 면역시 시토킨과 8-메르캡토구아노신과의 상호작용 : 인터페론과의 상승작용, J. Immuno., 139, 142-146(1987)).
PBL 로부터 인체의 모노클론을 유발시키려는 시도가 직면하게 되는 다른 문제점들은 IgG 및 IgA 아이소타입이 요구되는 것이다. B 세포류를 생성하는 항원 특이성 항체를 영속화(immortalize)시키려는 연구에서는 EBV 로 상기 세포들을 형질전환시킨 것과 같은 전통적인 방법을 사용한다. 유감스럽게도, EBV 형질전환 세포들은 주로 IgM 아이소타입 항체들을 생성한다. 현재까지 알려진 항체보다 실질적으로 소량의 IgM 과 다량의 IgG 및 IgA 을 함유하는 인체 폴리클론 항체를 생성하는 방법이 필요하다. 상기의 인체 모노클론 항체를 생성할 수 있는 세포를 배양하고 클론시킨후 세포 배양물질중에서 인체 모노클론 항체들을 생성하는데 사용한다.
본 발명은 8-메르캡토구아노신(8-MG)과 IL-4, 8-MG 와 IL-6, 또는 8-MG, IL-4 와 IL-6를 함께 배양한, T-세포가 결핍되고 EBV 형질전환된 사람의 말초혈액 임파구를 사용하여, 시험관내에서 주로 IgG 와 IgA 아이소타입 뿐만 아니라 항원 특이성 IgM 의 항원 특이성 인체 모노클론 항체의 생성을 증폭시키는 방법에 관한 것이다. 특히 본 발명은 하기 a) 내지 e)의 단계를 포함한다 :
a) 인체말초 혈액 임파구(PBL)을 수득하는 단계,
b) 표준 방법을 사용하여 상기 PBL 로부터 T-세포를 제거하는 단계,
c) 항원의 존재하에 엡스타인 바르 바이러스(EBV)와 보조제 8-MG 와IL-4, 8-MG 와 IL-6 및 8-MG, IL-4 와 IL-6 로 T-세포 결핍 PBL을 형질전환시키는 단계,
d) 항원 특이성 IgG 와 IgA 항체를 생성하는 세포들을 동정하는 단계, 및
e) 항원 특이성 IgG, IgA 및 IgM 모노클론 항체의 제조에 사용할 수 있는 세포주를 생성하도록 상기에서 동정된 세포들을 클론시키는 단계.
배양시킨 T-세포 결핍되고 EBV 형질전환된 PBL에 8-MG 와 항원을 첨가하면 배양시기 초기에 항원-특이성 클론을 확장시킴으로써 주항체를 생성하는 B 세포의 형질전환 기회가 증가하게 된다. IL-4 와 IL-6를 상기 배양물에 첨가하면, B세포분화 및 아이소타입 전환율을 향상시켜 EBV 로 감염된 PBL 단독에 비해 폴리클론 항체류(PCA)의 생성이 20 내지 40배 증가하게 된다.
사람의 전혈을 헤파린을 함유한 VacutainerTM(미합중국, 뉴저지주, 루서포드, 벡톤 딕킨슨 소재)관에 수거한다. 상기 방법이 전혈을 수득하는 바람직한 방법이지만, 주사바늘과 헤파린 코팅 주사기를 사용하는 것과 같은 임의의 다른 방법도 사용가능하다. 말초 혈액 임파구는 Ficoll-HypaqueTM(스웨덴, 압살라, 파마시아 바이오테크놀로지 그룹 제공)와 같은 밀도 그레디언트를 사용하여 분리한다. 전혈의 나머지 성분으로부터 PBL을 분리할 수 있는 다른 방법들도 사용할 수 있다.
T-세포를 함유한 PBL 은 항원 특이성 항체들을 생성하지 않는 것으로 밝혀졌다. 따라서, 다음 단계는 PBL의 T-세포를 제거하는 단계이다. PBL을 생리적 완충 식염수(PBS)중에 재 현탁시키고, 아미노틸 이소티오우라늄 브로마이드 히드로브로마이드(AET)-양의 적혈구 세포와 혼합하고, 얼음상에 놓은후 재현탁시킨다. 으로우젯(rosetted) T-세포를 Ficoll-HypaqueTM밀토 그레디언트 원심분리로 제거하면 T-세포가 결핍된 PBL이 산출된다. 상기 로우젯 기법은 본 발명의 바람직한 방법으로서, 다른 방법이 PBL로부터 T-세포를 제거하는데 성공적이라면 이러한 방법에 의하여 생성된 PBL도 본 발명에 유용하다.
T-세포 결핍 PBL을 형질전환체에 노출시키면 모노클론 항체를 생성하는 세포주를 연속적으로 증식시킬 수 있다. 임의의 유효한 임파추향성 바이러스나 기타 형질전환 매개체를 사용하여 B-세포를 형질전환 시킴으로써 연속성 배양물중에서 증식할 수 있도록 하고 또한 모노클론 항체 생성하도록 사용할 수 있지만 형질전환매개체로서 EBV를 사용하는 것이 바람직하다.
T-세포 결핍 PBL을 EBV 감염 배양물 상청액중에 재현탁시키고 37℃에서 1 내지 2 시간동안 배양했다. 배양이후, EBV 감염 PBL을 미세역가측정판의 웰에 넣고 바람직한 방법으로 Hur IL-4, HurIL-6(미합중국, 메사츄세츠주, 보스톤, 겐자임 코오포레이숀사 제공), 8-MG와 항원을 첨가한다. 상기 인터루킨 중 하나를 단독으로 첨가하거나 8-MG와 병행하여 첨가할 수도 있다. 인체의 재조합물 이외에 IL-4와 IL-6의 제공원(예, 정제된 인체의 인터루킨, 쥐의 IL-6 및 T-세포 주 또는 T-세포 배양물로부터 수득한 상청액중 발견되는 안터루킨)를 사용할 수도 있다. 배양배지는 10% 태아 송아지 혈청과 젠타마이신이 첨가된 둘베코′스 모디파이드 이글배지와 햄′즈 F12 배지(DMEM/F12)(뉴저지주, 그랜드 아일랜드, 깁코사 판매)인 표준 배지를 사용한다. 상기 배양물을 5% CO2습윤화 배양기중에서 37℃로 항원처리 했다. 4일후, 각웰에 배지를 첨가하고 4일마다 상청액을 제거하며 새로운 배양배지를 첨가한다. 상기 방법을 변형한 방법도 이용될 수 있으며 상기 방법은 바람직한 방법의 하나로서 기술한 것이다.
상청액을 항원 특이성 및 폴리클론 항체 생성에 대하여 시험한다. ELISA 분석결과, 상기 상청액중에서 IgG, IgM 및 IgA 면역글로불린 생성이 시간경과에 따라 증가했으며, IgG 면역글로불린이 최대량으로 생성되는 것으로 나타났다.
상기의 EBV 형질전환 임파구는 공급 세포증으로서 쥐의 대식세포를 이용하여 96개의 웰의 미세역가측정판내에서 제한희석법을 사용하여 클론시킬 수 있다. 몇가지 경우에 있어서 방사선 조사된 PBL를 공급 세포로서 사용하여 클론된 임파구의 증식을 향상시킬 수 있다. 또한, 상기 임파구들은 적당한 융합 짝(partner) 과 융합함으로써 안정한, 모노클론 생성 하이브리도마를 생성할 수 있다.
하기 실시예들은 신규한 본 발명의 방법을 기술하고 있다. 상기 실시예는 단지 본 발명을 기술하기 위해서 제시된 것으로서 본 명세서의 나머지 부분의 내용을 제한하지는 않는다.
[실시예 1]
T-세포 결핍 PBL의 제조
a) 인체의 전혈은 공여자로부터 헤파린을 함유한 VacutainerTM혈액 수거관에 회수했다. 상기의 전혈을 PBS와 1 : 2 비율로 희석하고 Ficoll-Hypaque 상에 가했다. Ficoll-Hypaque 와 전혈을 함유한 관을 20분 동안 400g로 원심분리했다. 황갈색의 코팅층을 Ficoll-Hypaque 상단으로부터 제거하고 PBS로 3회 세척했다.
b) 2주 이상되지 않은 신선한 양의 적혈구 세포(SRBC)를 세척하고 3회 원심분리했다. 최종 세척이후, 압축 SRBC를 37℃에서 15분 동안 3배 부피의 0.14M 아미노틸이소티오우라늄 브로마이드 히드로브로마이드(AET)(pH9.0)와 혼합하여 PBS 중의 AET-SRBC 현탁액을 제조했다.
c) 상기 PBL를 PBS 중의 농도 1×107세포수/ml 로 재현탁하고 동부피의 0.5% AET-SRBC 현탁액과 혼합했다. 상기 혼합물을 5분 동안 500 rpm에서 원심분리했으며, 가볍게 압축된 세포들을 15분 동안 얼음상에 놓는다. 그후 상기 세포들을 가볍게 재현탁시키고 T-세포 결핍 PBL을 Ficoll-Hypaque를 사용하여 분리했다.
[실시예 2]
PBL 배양물의 제조
a) 세포 배양물 상청액을 포함한 EBV를 5 내지 6일된 B95 세포(미합중국, 메릴랜드, 록크빌에 소재한 미합중국 ATCC에서 제공)로부터 수거하고, 다시 사용할 때 까지 -70℃로 냉동시켰다.
b) T-세포 결핍 PBL을 1×106세포수/ml 농도로 상기 EBV 감염 배양 상청액중에 재현탁시키고 1 내지 2 시간 동안 37℃에서 배양한 후 최종농도 2×104세포수/웰로 96웰 미세역가측정판에 가한다. HurIL-4를 최종농도 100유니트/ ml 로 상기 웰에 첨가하지만, 약 5×100 유니트/ml 범위로도 이용가능하다. HurIL-6를 최종 농도 50 유니트/ml로 각 웰에 첨가하며 약 10-100 유니트/ml 범위로 이용가능하다. 0.3ml의 0.1N NaOH에 용해시킨 1.0mM/ml의 8-MG(미합중국, 미주리주, 세인트, 루이스, 시그마사 제공)을 각 웰에 첨가했다. 8-MG를 약 0.3-1.0 mM/ml 범위로 첨가함과 동시에, 항원을 각각 준비된 배양웰에 첨가했다. 오브알부민, 배발암성(carcinoembryonic) 항원(CEA) 및 HT-29(방사선 조사된 결장암세포주)가 항원으로 사용되었다. 24개월에 40㎍의 오브알부민을 접종하고 다른 24개 웰에는 400㎍ 의 오브알부민을 접종했다. 40ng/ml의 CEA를 사용하여 54개 웰의 각각에 접종하고 2×104HT-29 세포수/웰을 다른 60개 웰에 접종물로써 사용했다. 배양물은 실험 과정중 5% CO2습윤화 배양기중에서 37℃로 항온처리했다. 배양개시 4일후, 50ml의 배양배지를 각 웰에 첨가했다. 상기 배양배지는 10% 태아 송아지 혈청과 젠타마이신이 첨가된 DMED/F12로 구성되었다.
그후 4일마다, 100ml의 배양 상청액을 제거하고 100ml의 새로운 배양배지를 각 웰에 첨가했다. 배양 상청액을 폴리클론(PCA) 및 항원-특이성 항체에 대해 분석했다.
[실시예 3]
PCA 제조에 대한 ELISA 분석법
실시예2로부터 얻어진 배양상청액중에 IgG, IgM 및 IgA가 존재하는지는 ELISA로 측정했다. 96-웰 임뮤론 II(Immulon II; Dynatech, 메릴랜드, 샨탈리소재) 플레이트를 0.2㎍/ml 의 염소 항-인체 IgG 또는 IgA 또는 0.1㎍/ml의 염소 항-인체 IgM(Kirkegaard and Perry Laboratories(KPL), 메릴랜드, 게티스버그소재)으로 코팅했다. 블로킹(blocking)시킨 후, 상기 상청액 50㎕를 각 웰에 가하고, 표준 물질로서 펜실베니아, 코크란빌에 소재하는 Cappel Laboratories로부터 구입한 정제된 인체 IgG, IgA 및 IgM 을 사용했다. 플레이트를 37℃에서 1 시간 동안 배양했다. 세척한 후, KPL 로부터 구입한 고추냉이 퍼록시다제로 표시된 염소 항-인체 IgG, IgA, IgM, 카파 및 람다 경쇄를 가한 뒤, 37℃에서 1 시간 동안 배양했다. 세척한 후 KPL 에서 공급된 TMB 효소 기질을 가하고, 플레이트를 실온에서 30 분간 배양했다. 2N 황산을 가해반응을 종료시켰다. 미세역가 측정판 판독기를 사용해 450nm에서 흡광도를 기록했다. 결과는 표1에 수록했다.
아이소타입 폴리클론 항체 특이성 반응은 배지만을 함유한 대조용 배양물에 비해 항원 존재하에 8-MG 및 IL-4 및 IL-6을 함유한 월내에서 증가했다. IgG와 IgA 아이소타입의 증가율은 대부분의 경우 IgM의 증가율보다 컸다.
다음과 같은 농도로 플레이트상에 해당 항원을 코팅시킨 뒤 상기한 바와 유사하게 항원-특이성 ELISA를 실시했다 ; OVA, 10㎍/ml 및 CEA 3.0㎍/ml 결과는 표2에 수록했다.
CEA 에 대해 분석됨
배지만을 함유한 웰에서 얻어진 상청액은 일차 분석법으로 검색할 때 OVA또는 CEA에 대한 항원 특이성 반응을 나타내지 않았다. 반면에 항원으로 자극된 웰에서 얻어진 상청액은 대조용액에 비해 상당히 많은 수의 항원 특이성 웰을 나타내었으며 항원 특이성 항체는 약 11 주 동안의 기간에 해당하는 3차계대 배양된 세포에서도 계속 생산되었다.
[실시예 4]
T-세포 결핍 시스템 및 비결핍 시스템의 비교
a) 본 발명의 방법으로 T-세포 결핍 효과를 측정하기 위해 결핍 PBL 및 비결핍 PBL을 사용한 시스템을 사용했다. PBL을 공여자로부터 수거하여 실시예1에서 기술한 대로 분리했다. 분리된 세포중 1/2을 실시예1에 기술한 대로 AET-SRBC 로 처리하고, 나머지 반은 처리하지 않았다. 2×10 및 5×10 의 농도의 이종의 인체 RBC를 유일한 항원으로 사용한 것 외에는 실시예2에 설명한 대로 실험을 실시했다. 제1도는 배지만을 함유한 대조용으로 사용된 T-세포 결핍 PBL 웰들이 시험된 각각의 아이소타입의 면역 글로불린을 약 10-배 이상 생산했음을 나타내고 있다.
b) 제2도, 제3도와 제4도는 상기 a)에서 설명한 배양물내의 8-MG + IL-6과 8MG + IL-4 의 효과를 비교한 자료를 요약한 것이다. IgM, IgG 와 IgA 면역 글로불린의 생산은 T-세포 결핍 배양물로부터 얻어진 상청액내에서 일정 시간 동안 증가하며, IgG는 상당히 다량으로 생산되었다. IL-4와 8-MG의 조합물은 8MG와 IL-4 각각에 비해 IgG의 생산을 향상시켰다.
비결핍 PBL을 포함하고 있는 배양물은 1㎍/ml 이하의 IgA PCA를 생산했으나, IgA와 IgG의 생산량은 증가하지 않거나 약간 증가했다.
표3에는 T-세포 결핍 배양물 시스템과 비-결핍 배양 시스템 모두에서 20일째에 존재하는 IgG, IgA 및 IgM의 양을 ㎍/ml 의 단위로 나타내었다.
상기 표는 T-세포 결핍 PBL 및 보조제로 8-MG와 IL-4 그리고 8-MG와 IL-6을 함유하고 있는 배양물로부터 얻어진 상청액내 생산된 면역 글로불린의 양이 보조제를 사용하지 않은 비-결핍 PBL 배양물에 비해 20-40배 증가되었음을 나타내고 있다.
c) 제5도는 배지에서만 배양시킨 세포에 비해 8-MG, IL-4 및 IL-6 각각이 T-세포 결핍 PBL 의 폴리글론 항체반응을 증가시킨다는 것을 증명하는 자료를 나타낸 것이다. 8-MG 및 IL-4 의 조합물은 각각의 시약을 단독으로 사용했을 때 관찰된 것 보다 IgG 량을 더 증가시켰다.
[실시예 5]
항원-특이성 항체의 생성
이종(heterologous) 인체 RBC를 유일한 항원으로 웰당 4×10 RBC 의 농도로 사용하여 실시예2에 기술된 실험을 실시했다. 결과는 8-MG가 시험관내 항원-특이성 반응을지시하는 역할을 함을 나타내 주었다.
항-RBC 항체의 존재를 측정하기 위해 적혈구 응집 반응 분석법(HA)이 실시되었다. 최종 배양 상청액을 HA 플레이트중의 1% RBC(각 25㎕)와 혼합하고 1시간 동안 실온에서 배양했다. 상기 플레이트를 45℃ 각도로 기울여서 응집반응을 조사했다. 직접 HA 활성을 보이는 웰을 표시해두고, 플레이트는 PBS로 3회 세척했다. 각 웰에 염소의 항-인체 Ig(1:100)을 가하여 혼합하고 실온에서 1시간 동안 방치했다. 결과는 표4에 나타내었다.
*16 웰/그룹
10개 양성 반응 웰중 9개의 웰의 배양 초기에 8MG를 가했다. 시험되는 8MG를 1mM 의 최대 용량으로 가한 7개의 웰이 HA 양성을 나타내었으며, 이는 상기 시약이 실험실내 항원-특이성 항체 생성을 증가시키는 중요한 역할을 하고 있음을 나타내주는 것이다.
IL-4 및/또는 IL-6을 배양물에 첨가함으로써 8-MG 만을 사용했을 때에 비해 PCA 반응이 증가되었으나(제5도 참조), RBC를 항원으로 사용한 경우 8-MG만을 사용했을 때에 비해 항원-특이성 항체의 생성율은 증가하지 않았다.
[실시예 6]
항원으로서 Molt-3 HIV 용해물(lysate)
본 실험에서는 Molt-3 HIV 용해물을 배양물에 가했다. 8MG와 바이러스 용해물을 가한 웰중에서 64개 웰중 5개가 ELISA에서 Molt-3 및 Molt-3 HIV 용해물과 반응하는 항체를 생성시켰다. IL-6은 시험관내에서 동일한 조합물에 첨가했을 때 16개 웰중 8개로부터 얻어진 상청액이 면역화제에 대해 특이성이 있는 항체를 포함하고 있었다. 이 항원 시스템에서, 림포킨을 첨가함으로써 8-MG 의 면역조절 활성이 증가되었다.
[실시예 7]
인체 PBL의 클로닝
실시예2에 설명한 바와 같은 EBV로 형질전환된 항원 특이성 PBL은 본 기술 분야의 숙련자에게 공지된 제한 희석법을 사용해 클로닝했다. J774 쥐대식 세포 세포주(ATCC TIB67, J774 A.1, 미합중국, 메릴랜드, 록크빌 소재)를 공급 세포층으로 사용한다. 몇몇 경우에서는 방사선을 조사하지 않은 PBL을 공급 세포로 사용할 수 있다.

Claims (14)

  1. a) 인체의 말초 혈액 임파구(PBL)를 분리하고, b) PBL로부터 T-세포를 제거한 후, c) 항원 및 보조제(여기에서, 보조제는 8-메르캡토구아노신, 및 시토킨 IL-4와 IL-6중 하나 이상을 함유한다)의 존재하에서, T-세포 결핍 PBL을 형질전환 매개체로 형질전환시키고, d) 항원 특이성 IgG 또는 IgA 항체를 생성하는 세포를 동정한 후, e) 상기 동정된 세포를 클로닝시킴을 특징으로 하여, 항원 특이성 인체 IgG 또는 IgA 모노클론 항체를 생성하는 인체 말초 혈액 임파구를 시험관내에서 증폭시키는 방법.
  2. 제1항에 있어서, 보조제가 8-메르캡토구아노신, IL-4 및 IL-6 인 방법.
  3. 제1항 또는 제2항에 있어서, 형질전환 매개체가 엡스타인 바르 바이러스(EBV)인 방법.
  4. a) 인체 말초 혈액 임파구(PBL)를 분리하고, b) PBL로부터 T-세포를 제거한 후, c) 항원 및 보조제(여기에서, 보조제는 8-메르캡토구아노신, 및 시토킨 IL-4와 IL-6중 하나 이상을 함유한다)의 존재하에서, T-세포 결핍 PBL을 형질전환 매개체로 형질전환시키고, d) 항원 특이성 IgM 항체를 생성하는 세포를 동정한 후, e) 상기 동정된 세포를 클로닝시킴을 특징으로 하여, 항원 특이성 인체 IgM 모노클론 항체를 생성하는 인체 말초 혈액 임파구를 시험관내에서 증폭시키는 방법.
  5. 제4항에 있어서, 보조제가 8-메르캡토구아노신, IL-4 및 IL-6 인 방법.
  6. 제4항 또는 제5항에 있어서, 형질전환 매채체가 엡스타인 바르 바이러스(EBV)인 방법.
  7. a) 인체 말초 혈액 임파구(PBL)를 분리하고, b) PBL 로부터 T-세포를 제거한 후, c) 항원 및 보조제(여기에서, 보조제는 8-메르캡토구아노신, 및 시토킨 IL-4와 IL-6중 하나 이상을 함유한다)의 존재하에서, T-세포 결핍 PBL을 형질전환 매개체로 형질전환시킴을 특징으로 하여, 인체 면역글로불린을 생성하는 인체 말초 혈액 임파구를 시험관내에서 증폭시키는 방법.
  8. 제7항에 있어서, 형질전환 매채체가 엡스타인 바르 바이러스인 방법.
  9. 8-메르캡토구아노신 및 IL-4를 함유하는 T-세포 결핍되고 형질전환된 인체 말초 혈액 임파구를 이용함을 목적으로 하는, 항원 특이성 면역 글로불린을 생성하는 인체 말초 혈액 임파구를 시험관내에서 증폭시키는데 효과적인 보조 시스템.
  10. 제9항에 있어서, IL-6을 추가로 포함하는 보조 시스템.
  11. 제9항에 있어서, IL-4가 IL-6로 대체된 보조 시스템.
  12. 제9항에 있어서, 약 0.3 내지 1.0 mM/m1 의 8-메르캡토구아노신 및 약 5 내지 100 유니트/m1 의 IL-4를 함유하는 보조 시스템.
  13. 제12항에 있어서, 약 10 내지 100 유니트/ml 의 IL-6 를 추가로 함유하는 보조 시스템.
  14. 8-메르캡토구아노신, 및 시토킨 IL-4와 IL-6 중 하나 이상을 함유하는 항원 특이성 인체 면역 글로불린을 생성하는 인체 말초 혈액 임파구를 시험관내에서 증폭시키기 위한 키트.
KR1019910006661A 1990-04-26 1991-04-25 항원-특이성 인체 모노클로날 항체의 시헙관내에서의 생성방법 Expired - Fee Related KR0151408B1 (ko)

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