LA JOLLA, Calif.—With an eye toward staking a claim in thefast-growing therapeutic antibody market, scientists at the Scripps ResearchInstitute have found a new technique that enables researchers to search largeantibody libraries and select those with desired biological effects.
The technique, described in an article published Aug. 20 inan early edition of the journal Proceedingsof the National Academy of Sciences (PNAS), also provides for the creationof unusual, asymmetric antibodies whose capabilities extend beyond those ofnatural antibodies. The Scripps effort was led by Richard A. Lerner, a Scrippsimmunochemistry professor and a member of the institute's Department ofMolecular Biology who has been working for 20 years to develop techniques forgenerating very large libraries of combinatorial antibodies and quicklyisolating those that can bind to a desired target.
Two decades ago, Lerner and his laboratory at Scripps, inparallel with the group of Sir Gregory Winter at the Laboratory of MolecularBiology in England, developed the first techniques for generating those largelibraries and quickly isolating the desired combinatorial antibodies.
"Antibodies are currently an important therapeutic optionfor treatment of a wide variety of diseases," writes Lerner.
Indeed, lab-grown antibodies and library generationtechniques have been used to find antibodies to treat cancer, arthritis,transplant rejection and other conditions.
"The field of immunochemistry has now turned its attentionto more challenging goals, such as the generation of broadly neutralizingantiviral antibodies where a useful molecule may be very rare," Lernercontinues.
This frequency problem, writes Lerner, has been largelysolved by the advent of combinatorial antibody libraries, a repertoire from whichone can select more than 1,000 different members. This approach has been usedin the study of flu viruses where the selection of rare antibodies has led tothe discovery of new modes of virus neutralization, with the hopes ofeventually 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 aless-than-perfect, two-step process where one first screens for binding andthen function, Lerner notes.
"To generate agonist antibodies, the difficulty iscompounded by the fact that the secondary screen for function can only becarried out in eukaryotic cellular systems where antigen presentation may beconstrained by the milieu or by the concentration of the target protein,compared with screening in vitroagainst highly purified molecules," he writes. "Thus, for agonists, the powerof a process that begins as a selection from a vast diversity of antibodiesexpressed in either yeast or phage, for example, is dampened by the bottleneckof the secondary screen where one essentially studies each antibodyindividually. Paradoxically, the complexity of the secondary screen isproportional to the success of the first screen, which in phage systems canyield thousands of candidate antibodies."
The technique created by the Scripps team allows researchersto select directly for functional antibodies in eukaryotic cells. Thescientists constructed a combinatorial antibody library in lentiviruses where,after infection, antibodies are efficiently expressed inside cells and alsosecreted so that both intracellular and extracellular targets can be accessed.
"The initial goal of our method was to express as large anantibody library as possible inside eukaryotic cells where the antibodies canbe either contained in the cytoplasm or secreted," Lerner writes. "Toaccomplish this in a way that gives the greatest degree of freedom, we usedboth M-13 phage and lentivirus vectors that were constructed so that theantibody genes could be easily interchanged."
The Scripps scientists demonstrated the power of thetechnique by using it to find an asymmetric antibody that almost perfectlymimics the activity of erythropoietin (EPO), a medically valuable hormone.
Although other research teams have studied the intracellularexpression of single antibodies to a known target, the Scripps team's approachis very different in that it can study the consequences of the expression ofmassive numbers of proteins, each with different binding specificities, insidea population of cells, Lerner notes.
"The real power of this technique is its ability to help usdiscover the unknown," he says, emphasizing that the Scripps technique can beused not just against known targets such as the EPO receptor, but also againstcellular functions involving targets that have not yet been found.
"The method may be important to identify new therapeutictargets, even when they are not addressable by antibodies," Lerner adds. "Thus,when molecules that are exclusively intracellular are identified, they may benovel targets for small-molecule therapeutics, which could be especiallyimportant in cancer, where targets may be exclusive to certain types of canceror even tumors isolated from individual patients."
Lerner's colleagues, postdoctoral researcher Hongkai Zhangand Ian A. Wilson, a structural biology professor at Scripps and an expert onthe structure of the EPO receptor, contributed to the PNAS article.
