Students engineer antibody for platelet research

MC students engineer antibody for platelet research

April 3, 2017

Maryville College sophomores Morgan Gast ’19 and Victoria Deal ’19 genetically engineered an antibody that binds to TLT-1 and were invited to spend part of their holiday break conducting related research at the University of Puerto Rico (UPR)-Río Piedras.

Their research could mark the beginning of many collaborations between the College’s Natural Science Division and the laboratory of Dr. A. Valance Washington, which studies platelets.

“I was so pleased with all of the student work, and I think Dr. Washington was pleased with our students’ efficiency and their dedication to do the work and do it well,” said Dr. Angelia Gibson, associate professor of chemistry and the students’ advisor for the project. “It is a unique opportunity for our students to participate in research at this level.”

Exploring collaborations

Gibson, who has worked with antibodies and written about therapeutic antibodies, met Washington three years ago at a National Science Foundation-sponsored workshop that encouraged research collaborations between institutions.

“Dr. Washington and I tossed around ideas about small projects that we might be able to take on here [at Maryville College] that his lab doesn’t really have time to work on – projects that I might be able to engage some undergraduates in that would be a benefit to him,” Gibson said. “He had this cloning project that he hadn’t gotten any students to work on and suggested that might be something that we work on here.”

Gibson and Washington kept in contact and from their conversations, an idea for a project emerged that Gibson believed Maryville College students could complete, but not without funding from an outside source. Money was needed for reagents and supplies and possible travel to Puerto Rico.

“The Ledford program is something I had had students work through successfully before, so I thought it would be appropriate to have students apply for Ledford funding,” she said.

Coordinated by the Appalachian College Association (ACA), the Ledford Scholarship program annually offers financial assistance for students who are enrolled at ACA member institutions and conducting summer research in the fields of laboratory and field work, interviews, analyzing special collections and participant observation.

Though Gast and Deal hadn’t even finished their freshman year, Gibson approached them about the project and submitting a proposal for Ledford funding. The professor had gotten to know their interests and abilities well through their participation in the Scots Science Scholars program.

“I knew they were bright, dependable and would be able to learn quickly,” Gibson said.

Deal, a biology major from Huntsville, Ala., is considering veterinary school or a graduate program in biology after finishing her bachelor’s degree.

“I’ve always been interested in STEM fields so I knew this experience would benefit me, whatever avenue I choose,” she said.

Gast, a biochemistry major from Georgetown, Ohio, said she was drawn to an opportunity to learn more from Gibson and also get an early start on a possible Senior Study topic. One of the distinctive features of a Maryville education, the Senior Study requirement calls for students to complete a two-semester research and writing project that is guided by a faculty supervisor.

Learning from the ACA last spring that their proposals had been approved for funding, Gast and Deal began preparing for the research with the help of three other students (biochemistry majors Winode Handagama ’16 and Chehronhai Fozil ’17 and Scots Science Scholar and chemistry major Shania Stephens ’19). The Ledford Scholarships funded their continued work in the Sutton Science laboratory for Grant and Deal throughout the summer, as well as necessary supplies, housing on campus and travel.

“We didn’t know if the research would require a trip to Puerto Rico – we kind of hoped that it might – so we applied for money to travel there or to conferences,” Gibson said.

The research

TLT-1 is a protein that is produced by platelets. Researchers originally thought its main role was to help platelets stick together during the formation of blood clots but now believe it affects the activity of other cell types at wound sites and blood clots.

“We need blood clots to form, stick around and leave after a certain amount of time in the body. When any of the proteins or processes involved in blood clotting are not functioning properly or working at the wrong times, then you have the risk of bleeding or the risk of blood clots forming where they shouldn’t be,” Gibson explained. “The Washington lab has found connections between levels of TLT-1 and disease conditions like atherosclerosis, sepsis and cancer. Platelets are involved in a lot more than just blood clotting, and some of their functions are mediated by TLT-1.”

In scientific terms, the goal of Gast’s and Deal’s research was to convert a partial antibody (known as a single-chain fragment of scFv) to a fully human immunoglobulin G by splicing the genes for the variable heavy and light chains from the TLT-1 scFv to the genes encoding the human IgG1 constant heavy and light chains, respectively.

“Our work was trying to piece together an antibody,” Gast explained. “So we started out with small pieces and tried to put them together. That was our whole goal – to put together this antibody. The antibody was hopefully going to bind to the TLT-1 and block it.”

Describing the antibody as “another molecular tool for studying TLT-1,” Gibson explained that by blocking the TLT-1 protein or by putting florescent tags on the attached antibody, researchers can better track the protein and understand its role in clotting processes.

Gast and Deal worked on the project all summer and into the fall 2016 semester.

‘Big breakthrough’

On Oct. 15, the students texted their professor a photo of a gel that showed the last pieces of the antibody assembled – evidence that they likely had the clone. After purification and sequencing processes were completed, Gibson and her students knew for sure that they were successful.

“It was a big breakthrough for us in that we pieced together these pieces of DNA – a goal that seemed daunting and at times, impossible,” Gibson said. “So, in that respect, it was a huge success for us.”

She said Washington was very excited to hear their news of a successful antibody clone and suggested that Gibson and her MC students plan a trip to his lab to conduct some experiments with it.

“The reagents and cells are all there,” Gibson said of the Washington lab. “We don’t do cell culture work here, and they a lot of mammalian cell culture work. Ultimately, we need mammalian cells to produce the antibody from the recombinant DNA we constructed. They have TLT-1 and all other tools needed to study TLT-1. They also have a flow cytometer, which is an instrument that can be used for studying antibodies and looking at their interactions with cells.

“Scientifically, its significance all depends on how well the antibody works,” she continued. “The lab had the antibody in a partial format, and it binds and inhibits TLT-1 exceptionally well in that format. But the partial format doesn’t do all the things that a full-length antibody can do. Washington’s students – UPR senior Omar Rios and UPR junior Javier Menendez – have demonstrated that when you put the DNA that we spliced together into the mammalial cells, the cells produce a protein that binds to TLT-1 and appears to function like a regular antibody.”

Gibson said comparison tests need to be run on the partial antibody and recombinant full antibody before the significance of the cloned antibody is known.

“Does it bind to TLT-1 as well as the single-chain fragment antibody did? Does it inhibit TLT-1 activity as well as the partial-format antibody, and compared to other full human antibodies, does it function as well in terms of alerting the immune system?” she wondered. “We won’t know how much of a scientific breakthrough the antibody is really until we do all of these tests.”

Time in Puerto Rico

Gibson, Deal and Gast flew to San Juan, P.R., on Dec. 16 and stayed seven days, working six days in the lab. Assisted by Washington, Rios, Menendez and another UPR student, Franklin Staback, Deal and Gast learned the basics of flow cytometry and how to perform Western blots (a technique used to detect the presence of a specific protein).

They also were exposed to the workings of a full-time, active research lab.

“It’s a model that’s more amenable for students coming in and learning from each other. Their job is research,” Gibson said. “They have a budget that can accommodate doing more experiments and giving people more time to learn. The objective there is to learn to be research scientists.”

In their free time, the three from Maryville College explored Río Piedras (one of San Juan’s largest neighborhoods), visited the beach and enjoyed Caribbean cuisine.

Lasting effects

For Gast, the project confirmed for her that spending hours every day in a laboratory isn’t for her.

“This experience was eye-opening, and I’m glad I did it,” she said. “It would have been awful to find out in graduate school that [research science] was the wrong career choice for me.”

For Deal, the project confirmed that she would be happy to pursue veterinary science and a doctorate that would put her in the laboratory regularly. She was recently notified that she received a second grant from the Ledford Scholarship program that will enable her to return to Washington’s lab this coming summer. Currently, Rios and Menendez are continuing to optimize expression of the antibody. During the summer, Deal plans to work with them to characterize its binding activity.

Gibson said the cloning project enhances her teaching.

“Some of the experiments they did over summer, I’ll now integrate those into courses. The details of the experiment are worked out,” she said, adding that her Chemistry 416: Biochemistry and Molecular Biology classes are now using new techniques that she and her students optimized during the cloning project.

Gibson said she is most excited by the possibilities of ongoing collaborations and guiding more Senior Studies that align with her area of expertise and are doable in a two-semester time frame.

“Having this project and this possible avenue for collaboration provides a way for me to plug students into something where they can see real-world, medical implications,” she said. “They can see what the Washington lab is doing, see the findings being published and see the results they’re able to generate on much larger scale.”

Written by Karen Beaty Eldridge '94, Executive Director for Marketing & Communications


Maryville College is ideally situated in Maryville, Tenn., between the Great Smoky Mountains National Park and Knoxville, the state's third largest city. Founded in 1819, it is the 12th oldest institution of higher learning in the South and maintains an affiliation with the Presbyterian Church (USA). Known for offering its students a rigorous and highly personal experience that includes an undergraduate research requirement, Maryville College is a nationally ranked institution of higher learning that successfully joins the liberal arts and professional preparation. Total enrollment for the Fall 2017 semester is 1,181.