보고서 정보
주관연구기관 |
국립농업과학원 National Institute of Agricultural Sciences |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2015-02 |
과제시작연도 |
2013 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201500010641 |
과제고유번호 |
1395030688 |
사업명 |
농축산물부가가치향상 |
DB 구축일자 |
2015-07-11
|
DOI |
https://doi.org/10.23000/TRKO201500010641 |
초록
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Ⅳ. 연구개발결과
1. 식물복합항체의 유효성 평가 연구
식물복합항체의 반응특이성을 확인한 결과, 식물복합항체는 대장암과 유방암 세포에만 특이적으로 반응하는 것을 확인하였고, 암세포 특이마커에도 잘 반응하는 것을 확인하였다. 이러한 식물복합항체의 항체-항원 결합반응은 기존 동물항체와 유사한 활성을 나타냄을 확인하였다. 또한, 식물복합항체의 암세포 억제활성을 확인결과, 식물복합항체는 caspase 활성을 촉진하여 apoptosis 유도를 증가시키는 것을 확인하였다. 개발된 식물복합항체에 적합한 간접효소면역분석법(indirec
Ⅳ. 연구개발결과
1. 식물복합항체의 유효성 평가 연구
식물복합항체의 반응특이성을 확인한 결과, 식물복합항체는 대장암과 유방암 세포에만 특이적으로 반응하는 것을 확인하였고, 암세포 특이마커에도 잘 반응하는 것을 확인하였다. 이러한 식물복합항체의 항체-항원 결합반응은 기존 동물항체와 유사한 활성을 나타냄을 확인하였다. 또한, 식물복합항체의 암세포 억제활성을 확인결과, 식물복합항체는 caspase 활성을 촉진하여 apoptosis 유도를 증가시키는 것을 확인하였다. 개발된 식물복합항체에 적합한 간접효소면역분석법(indirect ELISA)을 개발하여 다양한 시료(혈액, 암조직, 암세포 등)에 대한 적용가능성을 확인하였다.
2. 고효율 식물복합항체 생산법 구축
한 식물에서 대장암을 인지하는 항체와 유방을 인지하는 항체를 동시에 발현하여 복합항체의 형태를 통해 두 가지의 항원을 표적으로 하는 복합항체를 발현했다. 타가수분으로 얻은 F1씨앗에서 각각의 항체의 유전자를 확인하였으며, 하나의 식물에서 두 가지의 항체가 발현하는 것을 확인하였다. 당구로 분석을 통해 ER type의 당구조로 주를 이루는 것을 확인했으며, 항원에 대한 인지능도 높은 상호작용을 보이는 것으로 확인하였다.
3. 동물모델에서 복합항체의 표적지향성 평가연구
본 연구에서는 식물소재 복합항체 (multiple mAbPCO17-1A×BR55)의 대장암에 대한 항암효과가 시판되고 있는 대장암 동물항체(anti-EpCAM) 보다 높게 나타남을 확인하였으며, xenograft(이종이식)동물모델에서의 대장암 및 유방암에 대한 항암효과를 확인한 결과 식물소재복합항체 (multiple mAbPCO17-1A×BR55)의 유의성 있는 종양성장 억제 (항암효과)를 확인하였다. Azoxymethan(AOM)에 의한 원발성 대장암 유발 동물모델에서 식물소재 복합항체 (multiple mAbPCO17-1A×BR55)는 유의성 있는 항암효과를 가지고 있음을 확인하였다.
4. 식물복합항체 기반 암 진단키트 개발
Photolithography 방법을 이용하여 디자인된 암표지 단백질 검출 면역 진단칩 은 크게 반응 용액이 들어가는 마이크로 채널 부위, 암표지 단백질 항체가 결합된 glass bead를 저장하고 최종 신호를 관찰하는 마이크로 챔버 부위, 반응 용액이 나오는 마이크로 채널 부위로 구분된다. 암표지 단백질 검출 면역 진단칩에 암표지 단백질 재조합 단백질과 대장암세포주의 세포 용해물을 각각 적용하여 성능 검증 실험을 두 가지 측면에서 수행하였다. 첫 번째는 암표지 재조합 단백질에 반응여부이고 두 번째는 대장암 세포주의 단백질 추출물에 대한 반응여부를 확인하는 것으로 재조합 단백질 70 ㎍에 대하여 강한 반응을 나타냈으며, 암표지 단백질의 농도가 상대적으로 낮은 대장암세포주의 단백질 추출물에서도 양성 반응이 관찰되는것을 확인하였다.
Abstract
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Targeted therapeutics such as monoclonal antibodies(mAbs) have proven successful as cancer drugs. mAbs are powerful new treatment options for cancers, and immune-modulatory antibodies have also recently achieved remarkable clinical success. Currently, transgenic plants have proven to be an efficient
Targeted therapeutics such as monoclonal antibodies(mAbs) have proven successful as cancer drugs. mAbs are powerful new treatment options for cancers, and immune-modulatory antibodies have also recently achieved remarkable clinical success. Currently, transgenic plants have proven to be an efficient production system for the expression of functional therapeutic proteins. Plant-derived mAbs have the same advantages, namely, the lack of animal pathogenic contaminants, low cost of production, and ease of agricultural scale-up compared with the conventional fermentation methods. In this study, we have established plant production system to express two different targeting mAbs in a single transgenic plant through crossing fertilization between two different transgenic plants expressing anti-colorectal cancer mAbCO17-1A and anti-breast cancer mAbBR55, respectively. And we analyzed the biological properties of the plant-derived multiple mAb in transgenic tobacco plants compared to the animal-derived mAb. In addition, we demonstrated the feasibility of mAbs from Solanaceae as a detection reagent for point-of-care diagnostics.
First, we have established plant production system to express two different targeting mAbs. The F1 seedlings were obtained cross fertilization between the two transgenic parental plants. The presence of heavy chain (HC) and light chain (LC) of both mAbs in the F1 seedling plant were probed by immunoblot analyses, respectively. The decision of multiple mAbs in a single plant were confirmed to have relatively stronger detection of HC and LC protein bands size compared to the transgenic plant expressing individual mAb. N-glycan structure analysis was confirmed the similar glycan structures between individual mAb and multiple mAb. Kinetic analysis with antigen interaction using surface plasmon resonance (SPR) was showed that multiple mAb was halved binding affinity to the antigen in comparison with the individual mAb. Migration assay indicated that mammalian-derived mAb CO17-1A, plant-derived mAb CO17-1A and plant-derived mAb CO×BR was similar the inhibition of metastasis. These results indicate that the F1 seedling transgenic plant produced both of mAbPCO and mAbPBR of having a different binding site, however, HC and LC of each anti-colorectal cancer and anti-breast cancer mAbs were assembled in single plant, resulting in chimerism in HC and LC assembly. Thus, the seedlings with presence and transcription of HC and LC genes of both mAbs were selected, and the selected seedlings were confirmed to have relatively stronger density of HC and LC protein bands compared to the transgenic plant expressing only each mAb. These results indicate that the F1 seedling plant with carrying both mAb genes was established.
Second, we demonstrated that the biological properties of plant-derived mAbs compared to the animal-derived mAbs. The biological activities of plant-derived mAbs were assessed in cell proliferation, caspase activation, binding and neutralizing activities. Plant-derived mAbs and animal-derived mAbs exhibited similar binding and neutralizing activities for GA733 and SW620. And also, blocking GA733 by mAbP suppressed cell proliferation, and increased caspase activation in coloectal cancer cells. Our results show that plant-derived mAbs acts as a inhibitor of cell proliferation and cell survival in cancer cells like animal-derived mAbs. The results suggest that plant-derived mAbs may have potential as therapeutic agents for treating the cancer. And also, we developed an indirect ELISA for the rapid detection of cancer.
Third, we studied for the anticancer effects of plant-derived multiple mAbs in animal models. We confirmed the higher anticancer activity of plant-derived multiple mAbs than EpCAM in cancer cell lines and xenograft colorectal cancer animal model. And also, we confirmed the significant anticancer activity of plant-derived muliple mAbs than EpCAM in AOM-induced colorectal cancer animal model.
Fourth, we demonstrated the feasibility of monoclonal antibodies from Solanaceae as a detection reagent for point-of-care diagnostics. To accomplish it, we designed a novel glass-bead packed microfluidic device platform for detection of antigen present on the surface of the intestinal and breast cancer. Compared to commercially available animal-originated antibodies, the plant-originated ones have a similar binding ability to the target antigen in crude extract of intestinal cancer cells as well as mouse tumor tissues.
Taken together, plant crossing fertilization can be applied to generate an efficient production system expressing multiple monoclonal antibodies for immunotherapy in a single plant. And the plant-derived mAbs utilized in the field of cancer treatment technology. And the latter ones can be use for diagnostic purpose in the future. With further acquirement of intellectual properties related to the plant-derived antibodies, the antibodies could be successfully launches as a product in the market of antibodies.
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