Expression and function of immunotherapeutic recombinant proteins through transient and transgenic plant expression systems

다국어 초록 (Multilingual Abstract)

The factors to consider in the development of vaccines and antibodies (immunetherapeutic proteins) for cancer treatment are their bio-reactivity, stability and most importantly, a reasonable cost for their mass-production. In general, recombinant immunotherapeutic proteins such as vaccines and antibodies have been produced through host cells such as mammalian cells, fungi, and bacteria. The therapeutics produced in this way have high reactivity and stability, however, there are some limitations in that expensive production costs occur in the process of culturing and purification, and advanced technology is required. However, plants are attractive as a new host that can overcome the limitations of existing therapeutic protein expression hosts. Since they have several advantages such as an cost-effectiveness and easy production systems, ease of storage through seeds, and reduced risk of human pathogenic contamination, unlike conventional hosts. The technology for expression and production in plants based on genetic recombination technology is called ‘plant molecular farming (PMF)’. PMF has been developed to produce highly valuable proteins including recombinant vaccines, antibodies, and enzymes.
The first chapter of this study is a review of plant expression systems and their ability to produce recombinant vaccines and antibodies. In this chapter, the detailed information on plant transformation methods, fragment crystallizable (Fc) fusion proteins, and the advantages and limitations of plant-derived immunotherapeutic proteins is included.
The second chapter demonstrates the expression of plant-derived colorectal cancer vaccine candidates GA733-Fc and GA733-FcK using transient transformation and validation of the vaccine candidate through various in vitro and in vivo functional analyses. To produce GA733-Fc and GA733-FcK using the transient expression method, I cloned a fusion gene of GA733 and human immunoglobulin G Fc fragment (GA733-Fc), and a KDEL endoplasmic reticulum (ER) retention motif sequence was attached to the C-terminus of Fc to increase protein expression and avoid the plant-specific glycosylation (pEAQ-HT GA733-Fc and pEAQ-HT GA733-FcK). These cloned plasmids were transformed into Agrobacterium tumefaciens (LBA4404, Ag/GA733-Fc and Ag/GA733-FcK) for infiltration to leaves of Nicotiana benthamiana. In order to temporally and spatially optimize the harvest conditions of infiltrated leaves, I harvested by dividing various conditions [1-10 days post-infiltration (dpi) and leaf positions (top, middle, and base)]. Then, I performed western blot and polymerase chain reaction (PCR) using each dpi harvested leaf and its isolated genomic DNA (gDNA) for optimization target protein expression level. And according to the established conditions, mass produced GA733-Fc and GA733-FcK were successfully purified with high purity using protein A resin. Furthermore, I performed surface plasmon resonance (SPR), and Enzyme-linked immunosorbent assay (ELISA) for in vitro function analysis as vaccine candidates. In addition, high-performance liquid chromatography (HPLC) analysis was performed to confirm a high-mannose type N-glycan structure of GA733-FcK. In this chapter, in vivo generation of GA733-specific antibodies were analyzed in BALB/c mice immunized by GA733-Fc and GA733-FcK. ELISA with sera from the vaccinated mice confirmed that the plant-derived GA733-Fc and GA733-FcK produced both GA733-specific antibodies. These results suggest that GA733-FcK has a higher effect as a vaccine candidate than GA733-Fc in vitro and in vivo as well as increase the expression level in plants.
The third chapter demonstrates the expression and function of anti-HER2 VHH-FcK, the recombinant anti-HER2-positive breast cancer llama antibody fused to FcK in the plant expression system compared to the commercial Trastuzumab (Herceptin), anti-HER2-positive breast cancer antibody. Trastuzumab-resistant HER2-positive breast cancer is considered the biggest drawback of Trastuzumab. Thus, I performed various in vitro analyses to compare the function of anti-HER2 VHH-FcK and Trastuzumab (Herceptin) as antibodies against HER2-positive breast cancer cells BT-474 and Trastuzumab resistant mutant cell line BT-474 (BT-474-TR). So SPR was performed to analyze the binding affinity of each antibody to the HER2 protein in protein level. In the result of binding affinity, it was confirmed that Trastuzumab had a significantly higher binding activity to HER2 protein than that of anti-HER2 VHH-FcK. I also performed various biological antibody functional analyses to compare the biological property of anti-HER2 VHH-FcK with Trastuzumab. In MTT assay, it was confirmed that the cell viability of both BT-474 and BT-474-TR cells treated with anti-HER2 VHH-FcK was lower than that of Trastuzumab suggesting that anti-HER2 VHH-FcK has a greater effect as an antibody on the inhibition of cell proliferation, cytotoxicity and growth of BT-474 and BT-474-TR cells than Trastuzumab. Transwell migration assay confirmed that the inhibition of metastasis in vitro was higher than that of trastuzumab when treated with anti-HER2 VHH-FcK. In addition, when anti-HER2 VHH-FcK treated both BT-474 and BT-474-TR cells, the degradation of HER2 protein on the cell surface was confirmed by western blot using cell lysis treated with anti-HER2 VHH-FcK and Trastuzumab. In cell ELISA, it was confirmed that both cell lines treated with anti-HER2 VHH-FcK was bound stronger than that of Trastuzumab. Atomic Force Microscopy (AFM) physically confirmed that the binding probability (%) was higher than that of Trastuzumab. In various in vitro experimental results, it was confirmed that when Trastuzumab was treated to BT-474-TR cell, its function as an antibody was significantly reduced compared to that of BT-474 cell, whereas the anti-cancer activity of anti-HER2 VHH-FcK was maintained at a similar level between BT-474 and BT-474-TR. Therefore, it suggests that plant-derived anti-HER2 VHH-FcK has potential as an alternative treatment that could overcome the limitations of the existing commercial anti-HER2-positive breast cancer antibody, Trastuzumab.
Overall, this thesis suggests that colorectal cancer vaccine candidates GA733-Fc, GA733-FcK and anti-HER2 positive breast cancer llama antibody, anti-HER2 VHH-FcK, can be successfully produced through plant expression systems. And the various in vitro and in vivo function results suggest that plant-derived immunotherapeutic proteins can be an alternative solution to overcome the limitations of existing biopharmaceuticals.

국문 초록 (Abstract)

일반적으로 백신과 항체와 같은 재조합 치료용 단백질은 동물 세포, 곰팡이, 박테리아 등의 숙주를 통해 생산되고 있다. 이렇게 생산된 단백질은 높은 반응성과 안정성을 갖지만 배양 및 정제 과정에서 고가의 생산비용이 발생하며 고도화된 기술이 필요하다는 한계점이 존재한다. 그러나 최근, 기존 시스템의 한계점에 대응하여 치료용 재조합 단백질 발현을 위한 시스템으로 식물이 많은 가능성을 보이며 각광을 받고 있다. 식물은 기존 생산 플랫폼과 달리, 동물세포 배양에 비해 빠르고 인수공통병원체에 대한 감염 우려가 없어 안전하며 생산 비용이 저렴하며 보관이 용이하다는 다양한 이점을 갖는다.
암세포를 제거하는 결정적인 역할을 하는 것이 T 세포인데, 이 T 세포가 활성 하기 위해서는 종양 관련 항원(TAA)를 인식하는 과정이 필수적이기에 TAA의 선택이 암 백신 개발의 가장 중요하다고 볼 수 있다. 본 연구의 2장에서는 대장암의 백신 치료를 위해 면역 반응을 유도할 수 있는 종양 관련 항원(TAA)으로 대장암 세포 표면에 과발현 되어있는 당 단백질 GA733을 식물에서 발현하고자 하였다. 대장암에 과발현 되어있는 TAA인 GA733을 이에 본 연구에서는 GA733을 면역글로불린G의 Fc영역과의 결합을 통한 정제의 용이성, 면역반응 증대, Fc 수용체와의 상호작용 등의 장점을 통해 많은 각광을 받고 있는 Fc융합 기술과 접목하였다(GA733-Fc). 뿐만 아니라, 식물에서 생산되는 단백질의 발현 수준을 극대화하고, 투여 시 알러지 반응을 일으킬 수 있는 식물 특이적 당의 생성을 막기 위해 KDEL서열을 부착한 형태인 GA733-FcK를 transient 형질전환 방법을 이용하여 담배식물에서 생산하였다. Transient 형질전환을 위해 사전 연구를 통해 식물에서의 이종 단백질 발현 수준 증진의 우수성이 증명된 콩과 작물 바이러스 기반의 pEAQ-HT를 발현 백터로 사용하였다. 클로닝된 GA733-Fc와 GA733-FcK 유전자 플라스미드는 아그로박테리움(LBA4404)에 형질전환을 통해 매개체로 삼아 담배식물(Nicotiana benthamiana) 잎에 주사기를 이용하여 침윤 시켰다. 침윤된 잎의 목표 단백질 대량생산을 위한 수확 조건을 시간적·공간적으로 최적화하기 위해 다양한 수확 날짜 (1~10일, dpi)와 식물체 잎의 위치(상·중·하위) 별로 수확했다. 목표 단백질 발현 수준을 최적화하기 위해, 각각의 dpi별로 수확한 잎에서 추출한 genomic DNA를 사용한 중합효소 연쇄 반응(PCR)과 웨스턴블랏 결과, GA733-Fc와 GA733-FcK는 모두 상위에 위치한 잎에서 침윤된 지 5일차(5 dpi)에 가장 높은 발현 수준을 나타냈다. 이러한 결과는 KDEL과 상부의 잎에서 이종단백질의 발현 수준을 증대시키는 데 관여한다는 것을 시사한다. 이러한 조건을 바탕으로 정제를 수행 하였으며, SPR을 통해 GA733-FcK의 Fc 감마 수용체 I (CD64)간의 결합력이 GA733-Fc보다 높다는 것을 확인하였으며, 효소면역정량법(ELISA)을 통해 GA733-FcK가 동물세포에서 생산된 상업용 GA733-FcM보다 높은 GA733 항체에 결합 반응을 나타내는 것을 확인하였다. 그리고 당 구조를 파악하기 위해 HPLC 분석을 수행하여 GA733-FcK에는 KDEL에 의해 알러지 반응을 유도할 수 있는 식물 특이적 당 구조가 생성되지 않은 high-mannose 형태를 갖는다는 것을 확인하였다. 아울러, in vivo에서의 GA733-Fc와 GA733-FcK는 BALB/c에 정제된 단백질을 접종하여 얻은 혈청을 이용하여 효소면역정량법(ELISA)를 수행 함으로서 식물 유래 GA733-Fc와 GA733-FcK는 GA733 특이적 항체를 모두 생산 할 수 있다는 것을 확인하였다. 그리고 이러한 결과는 GA733-FcK는 생산량의 증대 뿐만 아니라 Fc 융합 단백질과 백신 후보 물질로서 GA733-Fc보다 높은 효과가 있다는 것을 시사한다.
본 연구의 3장은 서구화된 식습관 등으로 인한 여성 발병률 1위인 유방암 중, 20-25%정도에 해당하는 Human Epidermal growth factor Receptor 2 (HER2) 양성 유방암의 항체를 치료효과 증진을 위한 구조 변경 후 식물에서 생산하여 기존 상업용 치료제인 Trastuzumab (Herceptin)에 대체 가능성에 대하여 다루고자 하였다. HER2 양성 유방암의 치료는 종양의 생존, 전이, 증식 등을 촉진하는 역할을 하는 불량한 예후 인자이며, 이에 대한 항체가 주된 치료방법으로 사용되어왔으며, 가장 많이 이용되는 표적치료제가 Trastuzumab이다. 그러나 환자 중 약 50%가 Trastuzumab에 대한 내성을 나타내어 새로운 치료제가 필요한 상황이다. 본 장에서는 식물에서 라마 항체 기반의 새로운 HER2 양성 유방암 항체 'anti-HER2 VHH-FcK'를 발현 및 생산하였으며, Trastuzumab 내성을 갖는 BT-474-TR 세포 에서의 항체 활성 평가를 분석 하였다. SPR을 통해 HER2 단백질과 결합력 비교를 통해 단백질-단백질 상호 작용 단계에서 Trastuzumab이 anti-HER2 VHH-FcK보다 높은 결합력을 보유하고 있다는 것을 확인하였다. 그러나 HER2 양성 유방암 세포 BT-474와 BT-474-TR을 이용한 다양한 항체 활성 평가 (MTT, transwell cell migration, Cell ELISA, cell western blot)을 통해 BT-474에 대한 항체 활성은 anti-HER2 VHH-FcK과 Trastuzumab이 비슷한 수준을 보인 반면, BT-474-TR 세포에 대한 Trastuzumab의 암세포 생존, 전이, 유방암 세포 표면에 과발현 되어있는 HER2 단백질과의 결합 능력 및 이동성 저해 등에 관한 항체 활성이 크게 감소하였으며, anti-HER2 VHH-FcK는 BT-474와 비슷한 활성 수준을 유지하는 것을 확인하였다. 뿐만 아니라, 원자 힘 현미경(AFM)을 이용하여 물리학적인 결합력의 힘을 분석한 결과, anti-HER2 VHH-FcK는 Trastuzumab과 달리 BT-474와 BT-474-TR 모두에 높은 binding probability를 유지한다는 것을 확인하였다. 이러한 결과들은 anti-HER2 VHH-FcK는 Trastuzumab 내성 HER2 양성 유방암 세포에서 항체 활성이 유지된다는 것을 의미한다.
종합적으로, 본 논문에서는 식물 발현 시스템을 통해 생산된 대장암 백신 후보물질 GA733-Fc와 GA733-FcK, 그리고 라마 항체 모티브의 HER2 양성 유방암 항체 anti-HER2 VHH-FcK는 성공적으로 생산될 수 있으며, in vitro 환경에서 다양한 항체 활성 실험을 통해 각각의 백신, 항체로서 기능이 확인되어, 기존에 사용 되던 바이오 의약품들의 한계점을 극복할 수 있는 대안이 될 수 있음을 시사한다.

목차 (Table of Contents)

• Chapter I. Production system of plant-derived recombinant therapeutic proteins 1
• 1. Abstract 2
• 2. Introduction 3
• 3.1 Plant expression system as bioreactor for production of recombinant therapeutic protein 5
• 3.2 Strategies for production of recombinant therapeutic protein in plant 9
• 3.3 Glycosylation in plant 18
• 3.4 Structural characteristics of antibody and various recombinant antibodies 24
• 3.5 Engineering of Fc fusion protein in plant expression system 30
• 4. Conclusion 33
• Chapter II. Transient expression of GA733-Fc and GA733-FcK, a recombinant colorectal cancer vaccine candidate in vitro and in vivo functional analysis in plants 34
• 1. Abstract 35
• 2. Introduction 38
• 3. Materials and Methods 48
• 3.1 Construction of recombinant GA733-Fc and GA733-FcK in pEAQ-HT for plant transient expression 48
• 3.2 Plant for agroinfiltaration 53
• 3.3 Agroinfiltartion using the syringe method 53
• 3.4 Harvesting of agroinfiltrated leaves by days post-infiltration (dpi) 55
• 3.5 Genomic DNA (gDNA) isolation and Polymerase Chain Reaction (PCR) 55
• 3.6 Protein extraction and immunoblot analysis 57
• 3.7 Purification of GA733-IgG Fc and GA733-IgG FcK proteins from agroinfiltrated leaves 59
• 3.8 Dialysis and SDS-PAGE for quantitative analysis 61
• 3.9 Surface Plasmon Resonance (SPR) analysis to confirm the binding activity of Fc gamma receptor (Fcγ receptor) with plant derived GA733-Fc and GA733-FcK 62
• 3.10 N-glycan analysis 63
• 3.11 Enzyme-linked immunosorbent assay (ELISA) analysis 66
• 3.12 Animals 67
• 3.13 Immunization of mice 68
• 3.14 ELISA analysis to evaluate humoral response 72
• 4. Results 74
• 4.1 Morphological state changes of each tissue position leaves after agroinfiltration 74
• 4.2 Confirmation of GA733-Fc and GA733-FcK genes insertion in agroinfiltrated leaves of Nicotiana benthamiana 78
• 4.3 Confirmation of GA733-Fc and GA733-FcK expression level according to each leaf tissue position 82
• 4.4 Purification of GA733-Fc and GA733-FcK from agroinfiltrated N. benthamiana plant leaves 86
• 4.5 Binding affinities of GA733-Fc and GA733-FcK proteins to FcγRI (CD64) 90
• 4.6 N-glycan analysis of GA733-Fc and GA733-FcK using HPLC 94
• 4.7 Comparison of antibody binding affinity of GA733-Fc and GA733-FcK 97
• 4.8 GA733 antigen specific IgG response in mice immunized with GA733-Fc and GA733-FcK 101
• 5. Discussion 107
• Chapter III. Anti-breast cancer activity of plant-derived anti-HER2 VHH-FcK antibody against HER2-positive breast cancer cell lines BT-474 and Trastuzumab resistant BT-474-TR 124
• 1. Abstract 125
• 2. Introduction 128
• 3. Materials and Methods 137
• 3.1 Plant vector establishment and plant transformation to express anti-HER2 VHH-FcK protein 137
• 3.2 Genomic DNA isolation and Polymerase Chain Reaction (PCR) 143
• 3.3 Western blot for quantitative analysis 144
• 3.4 Plant growth for mass production and purification of anti-HER2 VHH-FcK 145
• 3.5 Dialysis and SDS-PAGE for quantitative analysis 147
• 3.6 Surface Plasmon Resonance (SPR) 148
• 3.7 Transwell migration assay 149
• 3.8 ELISA assay to confirm binding activity of anti-HER2 VHH-FcK to Fc gamma receptor (Fcγ receptor, CD16a) 150
• 3.9 HER2-positive breast cancer cell line BT-474 and BT-474-trastuzumab resistant (BT-474-TR) cell culture 151
• 3.10 Conjugation of anti-HER2 VHH-FcK and Trastuzumab to atomic force microscopy (AFM) cantilever tip 152
• 3.11 Atomic Force Microscopy (AFM) analysis and Single-cell force spectroscopy (SCFS) 154
• 3.12 Western blot using cellular protein 155
• 3.13 Cell viability assay 157
• 3.14 Cell ELISA 159
• 4. Results 161
• 4.1 PCR analysis for confirmation of anti-HER2 VHH-FcK gene insertion in Nicotiana tabacum plant 161
• 4.2 Western blot analysis to confirm purification of anti-HER2 VHH-FcK protein expression level in purified fractions from transgenic tobacco plant 162
• 4.3 Quantitative analysis of purified anti-HER2 VHH-FcK 166
• 4.4 Binding affinities of anti-HER2 VHH-FcK to the HER2 protein 169
• 4.5 Transwell assay to compare the cell migration inhibition activity between anti-HER2 VHH-FcK and Trastuzumab against BT-474 and BT-474-TR 173
• 4.6 Binding activity of anti-HER2 VHH-FcK to Fc gamma receptor (Fcγ receptor, CD16a) 179
• 4.7 Analysis of HER2 protein degradation of HER2-positive breast cancer cells 183
• 4.8 Inhibition effect of anti-HER2 VHH-FcK on proliferation of HER2-posiive breast cancer cells 187
• 4.9 ELISA to confirm the binding capacity and affinity of anti-HER2 VHH-FcK to HER2-positive breast cancer cells (BT-474 and BT-474-TR) 191
• 4.10 Binding probability analysis for anti-HER2 VHH-FcK and Trastuzumab to BT-474 and BT-474-TR through single-cell force spectroscopy (SCFS) 195
• 5. Discussion 199
• References 211
• 국문초록 246

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