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Scripps Research article describes new way to discover therapeutic antibodies
LA JOLLA, Calif.óWith an eye toward staking a claim in the fast-growing therapeutic antibody market, scientists at the Scripps Research Institute have found a new technique that enables researchers to search large antibody libraries and select those with desired biological effects.
The technique, described in an article published Aug. 20 in an early edition of the journal Proceedings of the National Academy of Sciences (PNAS), also provides for the creation of unusual, asymmetric antibodies whose capabilities extend beyond those of natural antibodies. The Scripps effort was led by Richard A. Lerner, a Scripps immunochemistry professor and a member of the institute's Department of Molecular Biology who has been working for 20 years to develop techniques for generating very large libraries of combinatorial antibodies and quickly isolating those that can bind to a desired target.
Two decades ago, Lerner and his laboratory at Scripps, in parallel with the group of Sir Gregory Winter at the Laboratory of Molecular Biology in England, developed the first techniques for generating those large libraries and quickly isolating the desired combinatorial antibodies.
"Antibodies are currently an important therapeutic option for treatment of a wide variety of diseases," writes Lerner.
Indeed, lab-grown antibodies and library generation techniques have been used to find antibodies to treat cancer, arthritis, transplant rejection and other conditions.
"The field of immunochemistry has now turned its attention to more challenging goals, such as the generation of broadly neutralizing antiviral antibodies where a useful molecule may be very rare," Lerner continues.
This frequency problem, writes Lerner, has been largely solved by the advent of combinatorial antibody libraries, a repertoire from which one can select more than 1,000 different members. This approach has been used in the study of flu viruses where the selection of rare antibodies has led to the discovery of new modes of virus neutralization, with the hopes of eventually generating a universal vaccine.
However, even with a solution to the frequency problem, isolation of an antibody whose function goes beyond simple binding is still a less-than-perfect, two-step process where one first screens for binding and then function, Lerner notes.
"To generate agonist antibodies, the difficulty is compounded by the fact that the secondary screen for function can only be carried out in eukaryotic cellular systems where antigen presentation may be constrained by the milieu or by the concentration of the target protein, compared with screening in vitro against highly purified molecules," he writes. "Thus, for agonists, the power of a process that begins as a selection from a vast diversity of antibodies expressed in either yeast or phage, for example, is dampened by the bottleneck of the secondary screen where one essentially studies each antibody individually. Paradoxically, the complexity of the secondary screen is proportional to the success of the first screen, which in phage systems can yield thousands of candidate antibodies."
The technique created by the Scripps team allows researchers to select directly for functional antibodies in eukaryotic cells. The scientists constructed a combinatorial antibody library in lentiviruses where, after infection, antibodies are efficiently expressed inside cells and also secreted so that both intracellular and extracellular targets can be accessed.
"The initial goal of our method was to express as large an antibody library as possible inside eukaryotic cells where the antibodies can be either contained in the cytoplasm or secreted," Lerner writes. "To accomplish this in a way that gives the greatest degree of freedom, we used both M-13 phage and lentivirus vectors that were constructed so that the antibody genes could be easily interchanged."
The Scripps scientists demonstrated the power of the technique by using it to find an asymmetric antibody that almost perfectly mimics the activity of erythropoietin (EPO), a medically valuable hormone.
Although other research teams have studied the intracellular expression of single antibodies to a known target, the Scripps team's approach is very different in that it can study the consequences of the expression of massive numbers of proteins, each with different binding specificities, inside a population of cells, Lerner notes.
"The real power of this technique is its ability to help us discover the unknown," he says, emphasizing that the Scripps technique can be used not just against known targets such as the EPO receptor, but also against cellular functions involving targets that have not yet been found.
"The method may be important to identify new therapeutic targets, even when they are not addressable by antibodies," Lerner adds. "Thus, when molecules that are exclusively intracellular are identified, they may be novel targets for small-molecule therapeutics, which could be especially important in cancer, where targets may be exclusive to certain types of cancer or even tumors isolated from individual patients."
Lerner's colleagues, postdoctoral researcher Hongkai Zhang and Ian A. Wilson, a structural biology professor at Scripps and an expert on the structure of the EPO receptor, contributed to the PNAS article.