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David Ruth
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Mike Williams
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Antibodies get easy upgrade with pClick

Rice U. scientists develop method to quickly enhance immune-system proteins for treatment, research

HOUSTON – (Nov. 5, 2018) – Rice University scientists have found a simple method to attach drugs or other substances to antibodies, the powerful proteins that are central to the body’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.

A proximity-induced antibody conjugation enables a covalent bond to form between functional molecules and a defined lysine residue of native antibodies without performing antibody engineering, according to Rice University scientists. This conjugation method, named pClick, allows a rapid and efficient site-specific functionalization of antibodies from different species and subclasses. Courtesy of the Xiao Lab

All of those alternatives are used by companies and researchers to prepare antibodies for conjugation – the process of chemically modifying them.

“But our technology is straightforward, highly efficient and economical,” said Xiao, who joined Rice last year with funding from the Cancer Prevention and Research Institute of Texas (CPRIT). “We 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 don’t need them.”

The research is detailed in the American Chemical Society journal Bioconjugate Chemistry.

From left, Rice University bioengineer Han Xiao, graduate student Chenfei Yu and postdoctoral researcher Juan Tang monitor a sample in Xiao’s lab. The researchers have developed pClick, a new technique to attach drugs or other substances to antibodies. Photo by Jeff Fitlow

Antibodies are dimers, identical proteins that in this case come together in a “Y” shape, and the workhorses of the immune system. Their job is to recognize and bind to pathogens, facilitating the invaders’ 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.

That’s not easy, Xiao said. “For the first generation of antibody conjugation, people used targets like lysine or cysteine residues to attach their therapeutic molecules,” Xiao said. “But 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.”

He said second-generation research focused on site-specific conjugation to place therapeutic drugs at a particular location on the antibody. “That let them optimize the position of the drug, but to do so people needed to know the antibody’s sequence and engineer it,” Xiao said.

“Our next-generation work addresses two problems,” he said. “First, we don’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.”

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 “p” in pClick stands for proximity). Because antibodies are two-protein dimers, ncAAs bind one to a side and provide two spots for enhancement.

A technique developed at Rice University to simplify the process of antibody conjugation called pClick does not require antibody engineering or ultraviolet or chemical treatment, thus enabling the attachment of functional molecules, including small molecules, polymers, nanomaterials and proteins to most antibodies used for research and therapy. Courtesy of the Xiao Lab

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.

“The regions of the antibody protein we address are conserved across human and mouse, and we demonstrated that pClick works on both,” he said. “This efficiency was a surprise, but it’s important for the antibody-drug conjugate field.”

Xiao’s Rice lab plans to develop pClick for cancer immunotherapy. “We 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,” he said.

“We think we can have one antibody target the cancer cell, and the other antibody target the immune cell,” Xiao added. “Then the antibodies will bring the cells together.”

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.

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Read the abstract at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.8b00680.

Follow Rice News and Media Relations via Twitter @RiceUNews.

Related materials:

Xiao Lab: http://xiao.rice.edu/People/hanxiao/hanxiao.html

Department of Chemistry: https://chemistry.rice.edu

Wiess School of Natural Sciences: https://naturalsciences.rice.edu

Images for download:

http://news.rice.edu/files/2018/11/1105_ANTIBODY-1-WEB-27ez0u1.jpg

A proximity-induced antibody conjugation enables a covalent bond to form between functional molecules and a defined lysine residue of native antibodies without performing antibody engineering, according to Rice University scientists. This conjugation method, named pClick, allows a rapid and efficient site-specific functionalization of antibodies from different species and subclasses. (Credit: Xiao Lab/Rice University)

http://news.rice.edu/files/2018/11/1105_ANTIBODY-2-WEB-2ncw5vd.jpg

These images show the results of testing of an antibody-fluorophore conjugate prepared using pClick, developed at Rice University. The conjugate was demonstrated by visualizing antigens on breast cancer cell surfaces. A set of varied cells were incubated with a green fluorophore conjugate and stained with a red fluorescent membrane-staining dye and a blue fluorescent nuclear stain. The fluorescent imaging shows that cell surface-associated green fluorescence was exhibited only by breast cancer cells with the specific antigen. (Credit: Xiao Lab/Rice University)

http://news.rice.edu/files/2018/11/1105_ANTIBODY-3-WEB-2j5jekb.jpg

Figure 3: pClick, a technique developed at Rice University to simplify the process of antibody conjugation, does not require antibody engineering or ultraviolet or chemical treatment, thus enabling the attachment of functional molecules, including small molecules, polymers, nanomaterials and proteins to most antibodies used for research and therapy. (Credit: Xiao Lab/Rice University)

http://news.rice.edu/files/2018/11/1105_ANTIBODY-4-web-1pbcwf0.jpg

From left, Rice University bioengineer Han Xiao, graduate student Chenfei Yu and postdoctoral researcher Juan Tang monitor a sample in Xiao’s lab. The researchers have developed pClick, a new technique to attach drugs or other substances to antibodies. (Credit: Jeff Fitlow/Rice University)

http://news.rice.edu/files/2018/11/1105_ANTIBODY-5-web-1v7t87z.jpg

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 body’s immune system. (Credit: Jeff Fitlow/Rice University)

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