Rice University bioengineer Han Xiao and his team developed the simple pClick technique to attach drugs or other substances to antibodies, the powerful proteins that are central to the bodyâ€™s immune system. Image by Jeff Fitlow
Rice University scientists have found a simple method to attach drugs or other substances to antibodies, the powerful proteins that are central to the bodyâ€™s immune system.
The Rice lab of bioengineer Han Xiao developed a technique called pClick, which uses a cross-linker that snaps to a specific site on antibodies and serves as a bridge to therapeutic molecules or nanomaterials without the need to re-engineer the antibody with harmful chemicals, enzymes or ultraviolet (UV) light.
All of those alternatives are used by companies and researchers to prepare antibodies for conjugation â€“ the process of chemically modifying them.
â€śBut our technology is straightforward, highly efficient and economical,â€ť said Xiao, who joined Rice last year with funding from the Cancer Prevention and Research Institute of Texas (CPRIT). â€śWe use native antibodies with no engineering, no enzyme treatment, no chemical treatment, no UV treatment. People have tried to use all these things before to achieve site-specific conjugation. We donâ€™t need them.â€ť
The research is detailed in the American Chemical Society journal Bioconjugate Chemistry.
Antibodies are dimers, identical proteins that in this case come together in a â€śYâ€ť shape, and the workhorses of the immune system. Their job is to recognize and bind to pathogens, facilitating the invadersâ€™ elimination. Antibodies either roam the blood stream or attach themselves to cells that need protection. Because they are ubiquitous in the body, modifying them is a way to treat disease.
Thatâ€™s not easy, Xiao said. â€śFor the first generation of antibody conjugation, people used targets like lysine or cysteine residues to attach their therapeutic molecules,â€ť Xiao said. â€śBut antibodies are so big, and there are so many lysine and cysteine residues, they can never control their positions. Each batch they make of a drug is different.â€ť
He said second-generation research focused on site-specific conjugation to place therapeutic drugs at a particular location on the antibody. â€śThat let them optimize the position of the drug, but to do so people needed to know the antibodyâ€™s sequence and engineer it,â€ť Xiao said.
â€śOur next-generation work addresses two problems,â€ť he said. â€śFirst, we donâ€™t have to engineer antibodies. We use antibodies from the market for direct, site-specific conjugation. Second, we know exactly where our molecule goes on the antibody.â€ť
The key to proximity-induced conjugation is using non-canonical amino acids (ncAAs), synthetic hooks that can be made to covalently bind to a specific spot on a protein when it gets close enough (the â€śpâ€ť in pClick stands for proximity). Because antibodies are two-protein dimers, ncAAs bind one to a side and provide two spots for enhancement.
Xiao sees pClick as a general strategy for antibody conjugation by industry and academia. He noted tests to link a side chain protein to human and mouse antibodies conjugated with ncAA showed efficiencies of more than 90 percent. Stability tests showed the antibodies did not significantly degrade after two hours of incubation in human serum.
â€śThe regions of the antibody protein we address are conserved across human and mouse, and we demonstrated that pClick works on both,â€ť he said. â€śThis efficiency was a surprise, but itâ€™s important for the antibody-drug conjugate field.â€ť
Xiaoâ€™s Rice lab plans to develop pClick for cancer immunotherapy. â€śWe want to achieve different conjugations, from antibody-drug conjugates all the way to antibody-antibody conjugates, where we would mix two antibodies and they would click together,â€ť he said.
â€śWe think we can have one antibody target the cancer cell, and the other antibody target the immune cell,â€ť Xiao added. â€śThen the antibodies will bring the cells together.â€ť
Rice graduate student Chenfei Yu and postdoctoral researcher Juan Tang are lead authors of the paper. Co-authors are Rice graduate students Axel Loredo and Yuda Chen and undergraduate Aviva Gordon; and Sung Yun Jung, an assistant professor of biochemistry, and Antrix Jain, a research assistant, of Baylor College of Medicine. Xiao is the Norman Hackerman-Welch Young Investigator and an assistant professor of chemistry.
CPRIT and the Robert A. Welch Foundation supported the research.