Single-domain antibodies are small proteins derived from the heavy-chain VHH regions of conventional antibodies. Their discovery, by researchers at the Free University of Brussels, in the early 1990s, was based on the observation that antibodies in camels and llamas lack light chains.
Researchers then discovered that VHH regions alone bound to antigens as strongly as the intact antibody and were extremely stable. Therapeutic antibodies have molecular weights of about 150 kDa, whereas single-domain molecules weigh in at under 15 kDa.
Single-domain antibodies overcome problems with mAbs related to their large size, chemical lability, expensive manufacturing, and inability to penetrate into cells.
In 2001 the Free University of Brussels spun off Ablynx, which owns the exclusive commercialization rights for the technology for human therapeutics and diagnostics. Ablynx, which calls its molecules Nanobodies, now boasts a robust pipeline of over 25 programs with three drugs in late Phase II clinical development, four in Phase I, and the remainder in preclinical development. About 50% of these programs are partnered with Pfizer, Merck-Serono, Novartis, and Boehringer Ingelheim.
In May 2011, data released from a Pfizer-led Phase II study of a Nanobody against TNF-alpha demonstrated proof of concept in patients with rheumatoid arthritis, Ablynx reports. “This validates the technology,” notes Ablynx senior research fellow Hilde Revets, Ph.D.
So far Nanobodies have lived up to their promise of manufacturability, solubility, and versatility. Since they comprise only the VHH domain harboring the full antigen-binding capacity of the original heavy chain antibody, Nanobodies may be produced in prokaryotic hosts. Glycosylation has not been an issue thus far, but Dr. Revets notes that undesirable “hot spots” for undesirable post-translational modifications may be deleted out of Nanobodies via sequence optimization.
The strict monomeric behavior and small gene size make Nanobodies ideal building blocks for multivalent (two or more identical Nanobodies on one molecule) or multispecific (two or more different modes of activity) therapeutics. This level of tailorability combined with solubility and ease of manufacturing properly folded protein is difficult to achieve with conventional antibodies, which are already quite large, more difficult to format, and sometimes exhibit solubility issues.
The group at the University of Brussels remains active in this research, as are other academic labs. It is unclear, however, whether Ablynx controls the method of production, the chemical entities, or both.
For example, Ablynx develops nanobodies by vaccinating llamas, collecting blood, isolating mRNA, converting it to DNA, and expressing the protein in yeast. Alpha Universe skips the vaccination step. “Everything is done in vitro, which is less expensive,” says Alexey Zdanovsky, Ph.D., president. Not surprisingly, he would not disclose his method for creating the truncated antibodies, only that it involved “molecular mutagenesis selection.”
Bispecific antibodies provide the potential of two modes of action in one molecule. In early October, X-Body entered into a partnership with Tanabe Research Labs to identify therapeutic target epitopes and develop mono- or bispecific antibodies for autoimmune disorders. X-Body says it will screen its diverse human libraries against target cells to identify specific epitopes and antibody therapeutic candidates.
X-Body screens its human antibody library using its Protein Chain Reaction™, which screens against cell surface targets in their native states on live cells or purified target proteins. According to the company, this “deep sequencing hit analysis” identifies and characterizes rare antibodies with specific therapeutic properties.
“Our nucleic acid display is the most compact approach compared with phage, yeast, or mammalian cell display,” says Tod Woolf, Ph.D., vp of technology development, “and it’s extremely diverse.” The other advantage is that these are fully human antibodies displayed in a mammalian system. “When you use phage display, proteins don’t fold the same way or have the same codon bias.”
X-Body’s library was generated from human donors’ antibodies. Variable chains were amplified using PCR, which captured the full diversity. “Our library is unusual in that it is not engineered,” Dr. Woolf says.
In 2001, a group at Genentech that included senior scientist Mark Dennis, Ph.D., reported increased uptake and activity in the brain of a bispecific mAb. One arm of the antibody binds to the transferrin receptor (TfR), which is upregulated on the surfaces of brain endothelial cells; the other arm inhibits b-secretase 1 (BACE1), an enzyme that transforms amyloid precursor protein into b-amyloid. Amyloid plaques are well-known markers for Alzheimer disease.
mAbs have the potential for treating brain diseases, but the blood-brain barrier excludes them: Just 0.1% of the antibody present in blood enters the brain.
The TfR receptor presents an interesting entry point through the blood-brain barrier. At one time researchers hoped that drugs binding to TfR would be internalized if they adhered to the receptor long enough.
“This receptor-mediated transcytosis strategy was proposed 20 years ago,” Dr. Dennis says. But later studies showed that strong binding tends to keep the antibody trapped inside blood vessels. “So the simple-minded idea is to lower affinity to TfR so the antibody can be released into the brain.”
The Genentech group attenuated TfR affinity by inducing alanine mutations in the complementarity-determining regions of the mAb. In a therapeutic setting, the drug concentration is sufficient to overcome the weaker binding affinity and drive antibody uptake by TfR, which does the rest.
Compared with a control antibody specific for b-secretase 1, the bispecific anti-TfR/anti-BACE1 antibody accumulated in the mouse brain and reportedly led to a significantly greater reduction in b-amyloid in the brains of test mice.