INTEGRATED
INTERVIEW AT ABL BIO
June 13, 2019
After conducting a series of three labs of gene cloning, expression, and purification, our team wanted to get checked by experts on whether we were on the right track. Thus, on June 13th, we visited Dr. Jinwon Jung and Dr. Sungwon An, head researchers of ABL Bio, to seek help on the current limitations of our study and gain experimental advice.
Dr. Sungwon An told us that the current limitations of our study are threefold: lack of cell specificity, low stability, and impurity. She suggested that fusion specificity can decrease when cell-penetrating peptides and single chain variable fragments are bound together. Also, she said that when scFv non-specifically binds to CPP, it can be potentially dangerous to mammalian cells. She advised us to consider antibody drug conjugation modality and recommended us to assess different cell receptors, affinity, and systemic delivery in order to solve this problem. Furthermore, Dr. An pointed out that scFv is very small, so it may not be able to survive in the cell culture for a sustained period of time. Therefore, she warned us that the CPP-scFv complex could be reduced or the two substances may be separated inside cell culture media or blood due to the different co-localization times. However, due to our time constraints, we were unable to move onto an in vivo trial, but we are taking into consideration Dr. An's suggestions into our project in the future.
When we discussed our future overall procedure, Dr. Jinwon Jung pointed out that immunostaining can indicate the presence of CPP-scFv complex inside the cell, but it cannot show that it is targeting a specific location. He mentioned that after the antibody enters the cell, it needs to show that it can perform a biological phenomenon, proving that it is functional. Hence, he suggested us to demonstrate that our scFv(P5) actually targets the lysozyme using binding assay. Based on his experimental advice, we decided to conduct a size exclusion column (fast protein liquid chromatography) to check whether our antibody is able to bind to the lysozyme, its target. Also, we purified the expressed protein (antibody) through Ni-NTA affinity chromatography, but he said that Ni-NTA can have impurities as the resin has a positive charge, so the impurity and our protein can compete. Thus, our team decided to perform Western blot using the His6-tag on the antibody to further purify the protein.
Dr. Jinwon Jung
Principal Researcher of Antibody-Drug Conjugation & Engineering at ABL Bio
Dr. Sungwon An
Senior Researcher of External development
& Neuro at ABL Bio
INTERVIEW AT CELLTRION
July 16, 2019
On July 16th, Team Korea_HS was able to interview Dr. Oh, the manager of the Innovative Product and Clinical Development team of Celltrion, one of the largest biopharmaceutical companies in South Korea. We asked Dr. Oh what kinds of intracellular target would be appropriate for our project. (Our experiment targeted lysozyme, the only target that has empirical evidence, but it would be too challenging for the antibody to successfully break two cell membranes: it has to break the membrane of lysozymes and the target cell.) He noted that our project should cover the parts that cannot be done by other methods of devices. For instance, the target should be only reached by intracellular targeting devices unlike the ones that can be reached on the cell surface as well. Also, as in terms of the family, he advised, we should avoid using kinase family targets because it can be targeted by the small molecule.
We also asked him about the possible side effects of intracellular antibodies, such as cytotoxic effects, and the solution for them. Dr. Oh first pointed out the fact that empirical evidence lacks in the field of CPP to prove the side effects. However, theoretically, he claimed that CPPs can be dangerous if the antibody attacks the normal cells or fails to target the right molecule. As a solution, he told us to make the CPP specific to the tumor cell environment, which is acidic, in order to make sure the antibody only target tumor cells. Because our CPP-attached hyperstable antibody is functional in a reducing environment, which has its optimal in an acidic condition, we learned that we could apply our project to cancer therapy.
Dr. Choong Seob Oh
Team Leader of the Clinical Development Division at Celltrion
INTERVIEW OF FORMER ONCOLOGIST
September 17, 2019
After completing the wet lab, Team Korea_HS wanted to find a way we could apply our project to cancer therapy. Therefore, on September 17th, Dyanne received an opportunity to meet an oncologist, Dr. Sunghee Ahn, to receive advice on how we could use our CPP-attached hyperstable antibody upon therapeutic usage. Dyanne asked Dr. Sunghee Ahn how our project could be utilized for cancer therapeutics, and she answered that our antibody could target a Ras protein, which stimulates uncontrolled cell growth. By targeting Ras, our antibody would inhibit intracellular signaling of cell growth. She suggested before executing this new idea in a wet lab, our team should attempt a molecular dynamics simulation to predict its feasibility. Based on Dr. Ahn's suggestion, our team was inspired to develop a model of a Ras-targeting hyperstable antibody. Dyanne also asked Dr. Ahn some of the consequences of using Ras-targeting antibodies. She stated that there may be low efficiency of the therapy due to inaccurate delivery of the antibody, and she also mentioned problems dosage effect due to the antibody because too much may potentially kill other cells and too little may pose no difference at all. Although side-effects cannot be indicated through modeling, our team will thoroughly consider these issues in the future when we perform this experiment in an in vivo experiment.
Dr. Sunghee Ahn
Former Oncologist at the University of Texas Southwestern University
