Our Team
Our team Korea_HS is made up of 14 passionate high school students, each living in different places like the United States, South Korea, and Hong Kong. We came together as a team inspired by synthetic biology and the profound effects it can have on the world. Despite being from different high schools, our team was able to successfully come together and work towards our common goal in the iGEM competition. From this opportunity we were able to make so many worldwide connections and learn from people with professional expertise. Moreover, with our knowledge, we were able to contribute back to our community and spread information. For our team Korea_HS, this project was truly an unforgettable and most importantly, enjoyable experience.
Abstract: Design and characterization of hyper stable cell-penetrating scFv targeting RAS
In the modern-day world, antibodies are often being developed as therapeutic agents. However, the targets of currently used antibodies are restricted to cell surface proteins due to their inability to go into the cell and maintain their function in the reducing environment of the cell. Thus we have chosen a hyperstable single-chain variable fragment(scFv(P5)) as a scaffold and engineered it to recognize human RAS protein by changing residues in the antigen-binding site(scFv(RAS)). We attached a cell-penetrating peptide to the N-terminus as well to ensure that the antibody could go into the cell(CPP-scFv(RAS)). We cloned, expressed, purified, and tested if CPP-scFv(RAS) works as designed. The development of antibodies that can work inside the cell will dramatically broaden the range of target molecules and the diseases that can be treated with therapeutic antibodies.
Team:Korea HS/Poster
Poster: Korea_HS
Poster Template
This poster template will let you create an interactive poster! The poster is divided in two parts: the visual overview on the left and the documentation on the right. The visual overview is broken down into sections that the user can click on. When a user clicks on a poster section in the visual overview, the documentation on the right will display the text and graphics associated with this section of the poster. You can find the documentation on how to use this template, as well as an example here: https://2020.igem.org/Competition/Deliverables/Poster
Cell-Penetrating Hyperstable Antibody scFv(Ras)
Problem Statement/Inspiration
Cancer is still an unbeaten obstacle in human biology. About 1 to 6 deaths are caused by cancer as it is the second leading cause of death responsible for the loss of 9.6 million people in 2018, according to the World Health Organization (WHO).
Unfortunately, the current cancer therapies developed in response to combat cancer have failed to tackle the root problem of cancer, which lies in the mutation of intracellular cell proliferation pathways such as RAS.
Limitation to current antibody therapeutics are
While these methods do not effectively cure cancer, and side effects of cytotoxicity weaken the patient's immune system making one prone to other diseases. We questioned: Is there a way to develop a cancer therapy that directly interferes with the mutated intracellular antigen's protein-protein interaction and has minimal effect on normal cells?
Unfortunately, the current cancer therapies developed in response to combat cancer have failed to tackle the root problem of cancer, which lies in the mutation of intracellular cell proliferation pathways such as RAS.
Limitation to current antibody therapeutics are
- Due to the lack of target specificity, current Monoclonal antibodies (mAbs) are not effective in stopping the uncontrolled cell proliferation while potentially causing infections and cardiotoxicity.
- CPP tagged scFv antibodies can overcome the limitation of monoclonal antibodies.
- The antibody engages in target-specific binding; it avoids binding to any other cellular components that can lead to side effects.
While these methods do not effectively cure cancer, and side effects of cytotoxicity weaken the patient's immune system making one prone to other diseases. We questioned: Is there a way to develop a cancer therapy that directly interferes with the mutated intracellular antigen's protein-protein interaction and has minimal effect on normal cells?
Goals
- Engineer the Hyperstable Cpp tagged scFv(F8) model from last year to graft RAS, an intracellular cell proliferation pathway.
- Utilize the cell penetrating model created last year to use the mechanism to treat cancer.
- Overcome the limits of current cancer therapeutics and design a cancer therapy that tackles the root cause of cancer.
Experiment
1. Cloning
2. Expression
3. Purification
4. Characterization
2. Expression
3. Purification
4. Characterization
Results
- Results show that CPP-scFv(Ras) successfully binds to HRas(G12V).
- The CPP-scFv(Ras) successfully cuts the HRas(G12V) protein interaction, which effectively stops cell proliferation of the mutant cell.
- CPP-scFv(Ras) and intracellular antibodies have promising possibilities for the development of cancer therapeutics based on its characteristic antigen specificity and increase in range of target molecules.
Protein-Protein Docking
Through protein-protein docking modeling, we were able to observe the binding characteristics of scFv(Ras) and hRas(G12V) by comparing the binding model with that of 2vH5 (the initial binding structure mimicked by antibody when engineering). The specific modeling opens possibilities for future study and development of the hyperstable CPP-tagged scFv(Ras) antibody model for clinical/commercial use.
Parts
The function of our Part Collection is to create a cell-penetrating peptide(CPP) tagged single-chain variable fragment (scFv(Ras)) that successfully binds to Human Ras G12V (HRas G12V).
Single chain variable fragment (scFV) recognizing Ras - Part:BBa_K3573000
ScFv(RAS) is a single chain variable fragment that is designed to recognize human RAS protein. This ScFv(RAS) was engineered from scFv(F8) that is hyperstable and is functional in the reducing environment inside the cell.
Single chain variable fragment with CPP recognizing Ras- Part:BBa_K3573001
Last year, the Korea_HS team modelled the structure of CPP-scFv(RAS) using the program Modeller. Modeller searched the database and used similar structures (PDB ID: 5B3N, 6NJL, 5GS3, 6G8R and 3UYP) to build a homology model. Based on the results of the homology modelling, we have decided to synthesize the gene for CPP-scFv(RAS) for protein production and binding assays. CPP (BBa_K3090000) was attached to scFv(RAS)(BBa_3573000) for entry into the cell.
Human Ras G12V mutant - Part:BBa_K3573002
Ras signaling is involved in numerous cellular functions, including cell proliferation, apoptosis, migration, fate specification, and differentiation. A key Ras effector pathway is the mitogen-activated protein kinase (MAPK), Raf-MEK- ERK pathway. The oncogenic RAS protein is involved in a major signaling pathway mediated through the guanosine triphosphate (GTP) bound active and guanosine diphosphate (GDP) inactive states. When RAS or the downstream effectors such as PI3K and RALGDS is mutated, the RAS protein is left in a constant active state leading to abnormal cell division.
Single chain variable fragment (scFV) recognizing Ras - Part:BBa_K3573000
ScFv(RAS) is a single chain variable fragment that is designed to recognize human RAS protein. This ScFv(RAS) was engineered from scFv(F8) that is hyperstable and is functional in the reducing environment inside the cell.
Single chain variable fragment with CPP recognizing Ras- Part:BBa_K3573001
Last year, the Korea_HS team modelled the structure of CPP-scFv(RAS) using the program Modeller. Modeller searched the database and used similar structures (PDB ID: 5B3N, 6NJL, 5GS3, 6G8R and 3UYP) to build a homology model. Based on the results of the homology modelling, we have decided to synthesize the gene for CPP-scFv(RAS) for protein production and binding assays. CPP (BBa_K3090000) was attached to scFv(RAS)(BBa_3573000) for entry into the cell.
Human Ras G12V mutant - Part:BBa_K3573002
Ras signaling is involved in numerous cellular functions, including cell proliferation, apoptosis, migration, fate specification, and differentiation. A key Ras effector pathway is the mitogen-activated protein kinase (MAPK), Raf-MEK- ERK pathway. The oncogenic RAS protein is involved in a major signaling pathway mediated through the guanosine triphosphate (GTP) bound active and guanosine diphosphate (GDP) inactive states. When RAS or the downstream effectors such as PI3K and RALGDS is mutated, the RAS protein is left in a constant active state leading to abnormal cell division.
Future
- Mutation of intracellular proteins like Ras can lead to cancerous tumors. We made scFv(Ras), an engineered form of scFv(F8) made to target the Ras protein by grafting antigen-binding sites of the anti-Ras antibody.
- This approach to cancer therapy aims to disable specific mutant antigens in the cell's intracellular environment. Unlike conventional cancer therapy that aims to kill the cancerous cells in numbers, the scFv(Ras) compromises individual protein interaction within the cell. The method is comparatively effective and less lethal on surrounding normal cells caused by cytotoxicity. The promising target specificity and binding affinity of scFv(F8) justify scFv(Ras) therapeutic possibilities while the technology widens the specific target mutant antigens for effective cancer treatment.
- More research on the novel antibody cancer therapy before being applied to the real world by investigating factors such as binding characteristics through modeling/wet lab
- Mathematical modeling of the optimal dose and concentration during local administration of the antibody needs to be calculated. This process is necessary to mitigate any possible side effects.
- Optimizing therapeutic and doing cost-effective analysis for effective commercialization.
Human Practices
1. Interview
We interviewed three pharmaceutical experts and discussed our project for integrated human practices. They provided us with guidance on our project's future direction and its potential impacts in the pharmaceutical industry. It made us aware of the potential challenges we must overcome, such as CPP safety and potential side effects.
2. Pamplet
We created and distributed the pamphlet to about 30 people. The pamphlet contained information about our team’s project, synthetic biology, and iGEM.
3. Survey
We created a survey in Korean and English and delivered it in ten different countries for public engagement. The survey was created to see how familiar synthetic biology is to the public. It was completed by people in different age groups, countries, and levels of education with total 173 participants.
4. Presentation
Our members held informative sessions with high school students about therapeutic antibodies as well as our project. We also had a Q&A session with students.
5. KSCY
Jina attended the Korea Scholar’s Conference for Youth (KSCY) and shared our team’s project, including a presentation of our objective, experiment, results, model, and future to raise awareness to scholars and proffesors.
We interviewed three pharmaceutical experts and discussed our project for integrated human practices. They provided us with guidance on our project's future direction and its potential impacts in the pharmaceutical industry. It made us aware of the potential challenges we must overcome, such as CPP safety and potential side effects.
2. Pamplet
We created and distributed the pamphlet to about 30 people. The pamphlet contained information about our team’s project, synthetic biology, and iGEM.
3. Survey
We created a survey in Korean and English and delivered it in ten different countries for public engagement. The survey was created to see how familiar synthetic biology is to the public. It was completed by people in different age groups, countries, and levels of education with total 173 participants.
4. Presentation
Our members held informative sessions with high school students about therapeutic antibodies as well as our project. We also had a Q&A session with students.
5. KSCY
Jina attended the Korea Scholar’s Conference for Youth (KSCY) and shared our team’s project, including a presentation of our objective, experiment, results, model, and future to raise awareness to scholars and proffesors.
Collaboration
- UNILausanne : As our first collaboration team, they gave us an idea for a public board, in which team members could be updated on what progress each sub-team had made.
- DTU Denmark : Our team asked questions about what they were doing for Human Practice and general tips in the competition because of their prior experience.
- Imperial College London : Imperial College London first suggested the idea of Mathematical Modelling for our team because it would be a chance to display information outside of the experiment.
- NJU China : NJU China’s objectives were very similar to ours in the way that they were also developing a therapy for cancer.
- Queen’s University : Besides being a collaboration, Queen’s University was very eager to mentor us in how our experiment could be carried out and possible solutions if it didn’t work.
Events
- Mentorship Meeting with Saniya
- Korea Meetup (Hosted by Korea_HS)
We held collaboration sessions from early July to late September. We met with five different university teams and Saniya as part of the iGEM mentorship program. Overall, the sessions were helpful because we were able to receive tips from more experienced biologists.
Our team was able to host a great event was the Korea Meetup, by gathering teams in Korea, including Korea-SIS and KSA_KOREA. Here, we were able to meet other team members in person and learn about what other teams were doing from the same country.
Safety
Regarding Covid-19
Project Safety
Specific materials including potential risks
Followed the 10 Lab Safety Rules
- Temperature check required
- Mask worn all the time and disposed after single use
- Every member’s temperature and visit date recorded
Project Safety
- E. coli was used to overexpress scFv and Ras proteins, but our project remained at an experimental stage without any human experimentation.
- The disposal of every chemical followed government guidelines.
Specific materials including potential risks
- E. coli K12 : Bacteria that were purchased, and it has very low risks to humans and the environment (completely nonpathogenic chassis).
- HEK 293T : Human cell line obtained from lab and has very low risks to humans and environment.
Followed the 10 Lab Safety Rules
- Wear protective gear including safety goggles and gloves
- Never eat or drink in the laboratory
- Never taste or breathe in chemicals directly
- Properly dispose lab waste
- Be fully aware of the building’s evacuation procedures
- Know where the lab’s safety equipment is located, including fire extinguishers, safety showers, and first aid kits
- Work in properly ventilated areas
- Be approved or trained by the supervisor of all equipment before first use
- Never leave an ongoing experiment unattended
- Never put unused chemicals back into the original container
Attribution
- Professor Jungwoo Choe
- Sunho Hong
- Joseph Kim
- Dyanne Ahn
- iGEM Mentor Saniya
- Mr. Ho-juhn Song
- Mr. Taegwon Oh
- Mr. Kim Min Soo
- Team DTU-Denmark
- Team Imperial College London
- Team NJU China
- Team Queen’s University
- Team UNILausanne
- The University of Seoul Natural Science Research Institute
Reference
Desiderio, Angiola, et al. “A Semi-Synthetic Repertoire of Intrinsically Stable Antibody Fragments Derived from a Single- Framework Scaffold.” Academic Press, 2001.
Donini, Marcello, et al. “Engineering Stable Cytoplasmic Intrabodies within Designed Specificity.” Elsevier Science Ltd, 2003.
Tanaka, Tomoyuki, and Terence H. Rabbitts. “Functional Intracellular Antibody Fragments Do Not Require Invariant Intra-Domain Disulfide Bonds.” Elsevier Ltd., 2007.
Tanaka, Tomoyuki, et al.“ Tumour Prevention by a Single Antibody Domain Targeting the Interaction of Signal Transduction Proteins with RAS.” MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK, The Embo Journal, 2007.
Tanaka, Tomoyuki, et al. “Single Domain Intracellular Antibodies: A Minimal Fragment For Direct In Vivo Selection of Antigen-Specific Intrabodies.” Elsevier Ltd, 2003
Tavladoraki, Paraskevi, et al. “A Single-Chain Antibody Fragment Is Functionally Expressed in the Cytoplasm of Both Escherichia Coli and Transgenic Plants.” FEBS, 1999.
Yu, Wanting. “Highly Efficient Cellular Uptake of a Cell-Penetrating Peptide ( CPP) Derived from the Capsid Protein of Porcine Circovirus Type 2.” Research Center of Reverse Vaccinology (RCRV), College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China, ¶State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China, and **Tsinghua-Peking Joint Center for Life Science, Tsinghua University, ASBMB, 2018.