Mini-antibodies discovered in sharks and camels could lead to drugs for cancer and other diseases

Helen Dooley admits that she often gets puzzled responses when she describes her work. "People say, ‘You bleed sharks for a living?’"

That's an overstatement, but every couple of weeks she and a helper drop by several large fiberglass tanks at the Institute of Marine and Environmental Technology on the Inner Harbor in Baltimore, Maryland. They net a cat shark or nurse shark and wrestle it into a small pool of water that contains a mild sedative. The drug calms the shark so they can lift it from the water and puncture a vein in its tail. Drawing a few milliliters of blood "doesn't take more than a few seconds," Dooley says, after which they return the animal to its aquarium to recover. "They're usually swimming about perfectly normally, and looking for food, after only a minute or so."

Dooley, an immunologist at the University of Maryland (UMD) School of Medicine in Baltimore, has been tapping shark blood for 2 decades for the same reason that other researchers have been draining the veins of llamas, camels, and their relatives. All those animals pump out unusual, diminutive antibodies that are only about half the size of the conventional versions.

Researchers have known about those tiny proteins since the late 1980s, when scientists at the Free University of Brussels (VUB) stumbled across them. But, "Since 2012, the field has really taken off," says biochemist Hidde Ploegh of Boston Children's Hospital. Compared with conventional antibodies, the molecules and even tinier fragments of them, often called nanobodies, are easier for researchers to make, more durable, and more soluble. Small antibodies can work inside cells, and their size allows them to wend deep into tissues, which regular antibodies have a hard time penetrating.

Those qualities have established the molecules as valuable research tools. "As a biochemist, I find them exceedingly useful," Ploegh says. He and his colleagues have deployed them for tasks as diverse as tracking a key immune protein in the body, neutralizing plant viruses, and labeling cancer cells. But the antibodies' most illuminating research role may be binding to and stabilizing wobbly proteins so that researchers can probe their architecture. "What they've been fantastic for is determining crystal structures," says evolutionary immunologist Martin Flajnik, also of UMD's School of Medicine. Indeed, a llama nanobody was key to structure work that won a recent Nobel Prize.

Miniature antibodies are also starting to prove their worth in patients. Later this year, the first nanobody treatment—derived from a llama small antibody—is expected to receive approval in some countries for use in people with a rare clotting disease. More than 40 similar therapies are in the works for diseases as varied as lupus, lung infections, and cancer. Conventional antibodies are mainstays of diagnosis and staple therapies for a host of diseases, but molecular biologist Nick Devoogdt, also of VUB, predicts that their petite cousins will take over in uses "where conventional antibodies are less optimal."

Small antibodies first came to light by accident, when students at VUB objected to analyzing human blood for a lab exam because they worried about the possibility of contracting a disease. They also refused to kill a mouse to obtain its blood, recalls Serge Muyldermans, a molecular biologist at the university. Some rummaging around, Muyldermans says, turned up an alternative: a stash of frozen dromedary camel serum collected to study the animals' parasites.

But another apparent problem cropped up when the students finished analyzing the blood. Along with normal antibodies, they had sifted out what appeared to be an undersize version of the molecules. "We thought that they had done something wrong," Muyldermans says. So he and other scientists at the university investigated further. An analysis of blood from zoo animals in the same evolutionary family, including a Bactrian camel and a llama, revealed that all had the same diminutive antibodies.

The researchers unveiled their discovery in a 1993 Nature paper and pointed out a key difference between the small antibodies and their full-size counterparts. A conventional antibody consists of four protein strands—two heavy chains that form the backbone of the Y-shaped molecule and two light chains that cling to the outside of the prongs. The mini-antibodies retain roughly the same shape but are missing the light chains. How the unusual molecules might benefit the animals wasn't clear, but when Dooley learned about them a few years later, she was fascinated. For her Ph.D. research at the University of Aberdeen in the United Kingdom, she set out to investigate those immune oddities.

Downsizing antibodies Human blood teems with conventional antibodies—bulky, Y-shaped proteins that home in on bacteria and viruses. The small antibodies produced by sharks and the camel family differ from those immune molecules not only in size, but also in their structure and binding ability. No light chains ~15 kDa Heavy chains Target molecule Molecular weight: ~150 kilodaltons (kDa) Typical antibody Small antibody Nanobody binding Nanobody A labmade fragment of a camel or llama small antibody that shares its binding ability. Two light and two heavy chains intertwine to make a protein that can identify and affix to bits of pathogens or other molecules. This slimmed-down variety lacks light chains but can still bind to its targets. Because of its binding style, a nanobody can fit into crevices on molecules. ~90 kDa Light chains Deep binding pocket Small antibodies and nanobodies Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit. Typical antibody Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam. Sed ut perspiciatis unde omnis. Small antibody Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusan - tium doloremque. Nanobody Sed ut perspiciatis unde omnis iste nataus error sit voluptatem accusantium doloremque accusantium natus error. Nanobody binding Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque accusantium doloremque.

She asked VUB researchers for help obtaining samples, and they arranged for colleagues in Morocco to immunize a few camels and send her some blood. After the first shipment arrived, however, the animals mysteriously disappeared, possibly because they were stolen, she says. "I was a year into my Ph.D., and I had no Ph.D. project." But she soon found an alternative to camels. In 1995, Flajnik and colleagues fished an unconventional antibody out of blood from a nurse shark. Like the camel versions, the shark antibody was smaller than the regular variety and lacked light chains. Dooley contacted Flajnik and finished her Ph.D. research on the shark molecules. Then she continued the work as a postdoc in his lab. "That's when we started to dig into the nitty-gritty of shark antibodies," she says.

Humans and mice occasionally churn out antibodies that contain only heavy chains, but researchers think they are duds produced by malfunctioning B cells, the immune factories for such proteins. In contrast, the undersize antibodies of sharks and the camel family are not half-finished rejects. Even though they lack the light chains that help regular antibodies recognize and grab antigens, they can bind tightly to their targets with great specificity, and they appear to be a key part of the animals' response to pathogens. When Dooley and Flajnik injected immune-stimulating antigens into nurse sharks, they discovered that within a few months—the shark immune system is slower to react than ours—the animals were churning out a variety of small antibodies that targeted the foreign molecules. "It's the major antibody used in the nurse shark," Flajnik says.

He and other researchers speculate that the minute antibodies enable immune systems to counteract a broader range of pathogens. Conventional antibodies excel at sticking to flat surfaces on viral and bacterial molecules. Heavy-chain–only antibodies "are skinny and might be able to penetrate canyons and crevices that regular antibodies couldn't get into," says structural immunologist K. Christopher Garcia of the Stanford University School of Medicine in Palo Alto, California.

Small antibodies must have an important role, Muyldermans notes, because they emerged independently in as many as three different lineages: sharks, camels, and probably another group of sharklike fishes. But just what advantage they confer on those animals is a mystery. Scientists know little about shark pathogens, for example, and aren't even sure how to tell when sharks are sick, Dooley says. "They don't just curl up in the corner." Even in camels and their kin, whose diseases are better understood, researchers haven't identified which pathogens the small antibodies combat. At the moment, Muyldermans says, "We have no clue" why the molecules evolved.

That hasn't stopped scientists from putting them to work. For most medical and research applications, they prune the small antibodies, leaving only the antigen-binding tip. Nanobodies, as they're called if they come from camels and their relatives, have several handy attributes, Ploegh says. For one, the well-studied bacterium Escherichia coli can make them. "You can express them in E. coli in exceptionally high yield, and they are easy to purify," he says. In contrast, producing working full-size antibodies in E. coli has proved to be difficult, so researchers typically generate them from more expensive cultures of mammalian cells.

Nanobodies also remain functional within cells, whereas conventional antibodies typically fall apart in the cytoplasm. "Suddenly we can do smart things [inside cells] with antibodies," says developmental biologist Markus Affolter of the University of Basel in Switzerland. One clever use, he says, is "a completely new way to manipulate proteins."

He and his colleagues, for example, harnessed nanobodies to eliminate specific proteins from cells. The team started by engineering the cells to produce a nanobody that, on its nonbinding end, carried molecules that direct proteins to the cell's garbage disposal. The other end of the nanobody recognized and attached to a specific protein. Stimulating a cell to manufacture the nanobody caused the protein target to disappear in as little as 3 hours, the researchers reported in 2012.

Depleting a protein by inducing mutations in its gene, a standard approach in basic research, banishes it for good. In contrast, the nanobody technique allows scientists to delete and then restore the molecule, probing its role in more detail. In 2015, for example, a team led by researchers at Baylor College of Medicine in Houston, Texas, engineered fruit flies to generate a nanobody that grabs the protein Dunce—which is essential for insects' ability to learn and remember—and promotes its destruction. The researchers then trained the flies to avoid a certain odor. When the team spurred the flies to start making the nanobody against Dunce, levels of the protein plummeted and the insects became dumber—they had a harder time learning to avoid the odor. When the team shut off production of the antibody, Dunce's abundance bounced back, and the flies became smart again.

Because nanobodies reach deep into the nooks and crannies of molecules they bind to, the fragments have also become favorites for researchers trying to stabilize floppy molecules so they can be crystallized, a first step in determining their structure. In perhaps their most impressive performance, nanobodies enabled Brian Kobilka of Stanford's School of Medicine and his colleagues to capture the first structure of an activated G protein–coupled receptor (GPCR). Those cell membrane proteins relay a multitude of signals into the cell: The adrenaline that makes your heart race, the morphine that eases a patient's pain after surgery, the bitter flavor in a cup of coffee—all act through GPCRs, which also are the targets of about one-third of drugs. Researchers had tried for about 20 years to get crystal structures of typical, switched-on GPCRs, but the molecules were too shifty.

Then Kobilka met structural biologist Jan Steyaert of VUB at a conference, and the two decided to try nanobodies. The researchers and their colleagues injected a llama with the human β2-adrenergic receptor, which responds to adrenaline, and from the animal's blood prepared a set of nanobodies that clamped onto the receptor. In a 2011 Nature paper, the scientists revealed that one nanobody locked the GPCR into an active shape, allowing them to determine its crystal structure. "That was a devastatingly effective use" of the molecules, Garcia says, as evidenced by the fact that in 2012, Kobilka shared the Nobel Prize in Chemistry.

Like Kobilka and Steyaert, researchers who need a supply of small antibodies or nanobodies often start with llamas, camels, or sharks that have been immunized with particular antigens. In response, the animals' immune cells begin to make small antibodies that recognize and fasten onto the antigen. Blood cells isolated from the animals allow scientists to obtain the genes for those antibodies, which they can then insert into bacteria or other microorganisms to synthesize large quantities of the immune molecules. But the animals can take a month or more to respond to the antigen and spawn new small antibodies, and llamas and their relatives won't manufacture versions that target some human proteins that are very similar to their own, notes structural biologist Andrew Kruse of Harvard Medical School in Boston.

Earlier this year, he and his colleagues unveiled a synthetic nanobody library that they say overcomes those limitations. Instead of relying on animals as the source of antibody genes, the researchers synthesized DNA sequences to craft their own. Using the published structures and corresponding amino acid sequences of known nanobodies as a guide, they created more than 100 million custom nanobody genes. They slipped the genes into yeast cells, which served as factories for the molecules. Testing showed that "the fully synthetic library is performing at least as well as animal immunization," Kruse says. He and his colleagues will send it to any academic researchers who pay the shipping costs. Kruse says they are dispatching about 10 to 15 packages a week.

Muyldermans and Steyaert, however, contend that animal-derived nanobodies bind to their targets better than entirely labmade alternatives, so they're sticking with the old-fashioned method. "You never change a winning team, and these llamas are on my team," Steyaert says.

Llamas—along with sharks and camels—may soon be aiding patients. The Ghent, Belgium–based company Ablynx, a spinoff from the original group that discovered the unorthodox antibodies, has already completed a phase III effectiveness trial of one such protein, caplacizumab, for the rare disease acquired thrombotic thrombocytopenic purpura, in which many blood clots can trigger strokes, organ failure, or death. The llama-derived molecule works by latching onto and inactivating a blood protein named von Willebrand factor that promotes clotting—and it's much more clingy than traditional antibodies, notes Edwin Moses, Ablynx's CEO. The company, which presented the positive results of the trial in December 2017 at the American Society of Hematology conference in Atlanta, has applied for approval to sell the drug in Europe and plans to do the same in the United States later this year.

At least seven other small antibody–derived treatments have reached clinical trials—targeting diseases such as rheumatoid arthritis, psoriasis, and lupus—and more than 30 other treatments are under development. Most of those molecules are derived from the antibodies of camels and their relatives, but the first shark-based drug, produced by the Melbourne, Australia–based firm AdAlta, should enter clinical trials later this year, says Mick Foley, the company's chief scientific officer. The drug is meant to alleviate lung fibrosis, a stiffening of the organs caused by the buildup of scar tissue.

Researchers also hope the unique properties of small antibodies will enable them to pry open the blood-brain barrier, an obstacle to treating many brain diseases because it rebuffs most large molecules, including standard antibodies and many other drugs. Ossianix, a Philadelphia, Pennsylvania–based biotech, has crafted a shard of a shark small antibody that binds to a receptor controlling access across the barrier. By stimulating the receptor, the fragment might open the way for drugs such as rituximab, a cancer-killing conventional antibody, to cross into the brain, says Ossianix CEO Frank Walsh.

Other applications beckon. Researchers can equip small antibodies with therapeutic cargoes, such as other drugs or cancer-killing radioactive compounds, without making them so massive that they can't infiltrate tissues or tumors. In addition, the kidneys rapidly filter the molecules out of the blood for excretion—a benefit when the cargo is radioactive. Diminutive antibodies can also be fused to radioactive or fluorescent tracers to illuminate tumors or even guide surgery. To visualize a tumor, for example, "you need small molecules that bind tightly" to the cancer, Devoogdt says. Rapid clearance of a tracer is a plus, as well, because it reduces background noise that can leave the tumor harder to discern. He and his colleagues, along with several other groups, have performed safety trials on nanobodies as tumor-visualizing agents, and they hope to start testing the molecules' therapeutic usefulness in patients next year.

Small antibodies do have some drawbacks for medicine, researchers caution. The rapid excretion of the molecules can be a downside if they leave the body before patients receive their full benefit. Another shortcoming, notes biochemist Jan Gettemans of Ghent University, is that unlike many important drugs, including statins and the anti-HIV drug azidothymidine, the antibodies can't enter cells on their own. Researchers can get them into the cytoplasm by genetically altering cells to produce them, but that's not feasible for most treatments. And although brief studies suggest small antibodies are safe for patients, "there is no clear experience in humans … for a long period of time," Gettemans says. But he and other researchers think the small antibodies' advantages outweigh their shortcomings and are confident that they "will be part of the armamentarium," as Walsh puts it.

Meanwhile, the mysteries they pose continue to entrance Dooley. She did a stint in drug development—4 years working on a pharmaceutical company's small-antibody project—but these days when she takes a tube of shark blood back to her lab, she's usually trying to answer basic questions such as how the animals switch on their antibody-manufacturing cells. She has become fond of the sharks, too. "They are beautiful animals to work with." If someone did steal the camels she was originally planning to study, she says, "they really did me a favor."

*Correction, 11 May, 2:10 p.m.: An earlier version of this story misstated Hidde Ploegh's affiliation.