Symmetry breaking: bispecific antibodies, the beginnings, and 50 years on.

The archetypes The era of modern antibody therapy begins with Rodney Porter’s Nobel Prize-winning report on the basic structure of the immunoglobulin molecule. Using papain for digestion of a 7s rabbit antibody preparation, he obtained two almost identical antigen-binding fragments (Fab) that blocked antigen precipitation by the parent antibody while a third, easily crystallizable fragment (Fc) was found inactive (1). From a historian’s viewpoint, it is striking that, almost simultaneously with these first insights into the antibody’s structure and function, the idea of an artificially constructed bispecific antibody was born. Without a clear image of the now heraldic Y and its underlying tetrameric symmetry, Alfred Nisonoff had the vision of combining two different antigen binding sites in one molecule. He had used pepsin instead of Porter’s papain to generate univalent Fab fragments that specifically inhibited antigen precipitation. Discussing his findings, he speculated about the future experiments, “to attempt to prepare antibody of mixed specificity” (2). It took him just another year to realize the idea of a F(ab’)2 molecule with dual specificity: this he obtained under mild reoxidation from a mixture of univalent fragments of anti-BGG (bovine gamma globulin) and anti-OVA (ovalbumin) antibodies (3). Several publications later and in collaboration with Hugh Fudenberg, Nisonoff elegantly proved the bispecificity of the reassociated F(ab’)2 by direct visual evidence (4). Coupling BGG and OA to human and chicken red cells, respectively, the authors demonstrated under the microscope how the two easily distinguishable red cells got agglutinated by the bispecific fragment. Besides Nisonoff ’s early bispecific fragment antibody, one may cite a much older bispecific antibody, not man-made, whose biological effects had already been described in allergic patients in the 1940s because of its peculiar blocking activity on other antibodies; its true nature, however, had remained completely unknown. Part of the mystery was solved when Rob C. Aalberse and co-workers found elevated IgG4 antibodies in sera of beekeepers who were chronically exposed to phospholipase of bee venom (PLA2). These full-size IgG4 antibodies were “functionally monovalent,” i.e., they blocked other PLA2 antibodies (5). The antibodies had exchanged Fab arms and consisted of two heavy and light chain pairs, each one derived from a different IgG antibody, as was only later discovered. The stochastic nature of the posttranslational formation of bispecific IgG4 molecules could later be demonstrated in vivo with recombinant IgG4 antibodies against defined allergens; excess irrelevant IgG4 prevented the formation of hybrid antibodies almost completely (6). The anti-inflammatory activity of the functionally monovalent IgG4 was shown in a rhesus monkey model with experimental myasthenia gravis. Whether the recently described broad spectrum of IgG4-related diseases in man is causally related to the Fab arm exchange remains to be demonstrated (7). The mechanism of IgG4 Fab arm exchange has been extensively studied by Aalberse and colleagues with a sensitive real-time FRET assay suggesting that the exchange occurs in vivo under specific local redox conditions (8). This story—retold here as an aside without any conceit of hindsight—and the newly recognized systemic condition of IgG4-related disease may hold some clues for a better understanding of therapy with bispecific antibodies. This extension of the bispecific story into much earlier eras was deeply hidden when Nisonoff and Fudenberg were proving the bispecificity of the F(ab’)2 by agglutination experiments. Their vision anticipated in a way the action of today’s recombinant bispecific antibodies designed to retarget effector cells at cancer cells. However, the Nisonoff approach would have remained without any traceable consequences had Lloyd Old not given it a serious try. Together with Ulrich Hämmerling, Old used the original F(ab’)2 procedure to develop a bispecific antibody addressing mouse immunoglobulin and ferritin, thus generating a universal reagent to detect immunoglobulin on the surface of mouse lymphocytes by electron microscopy (9). The low yield of the original Nisonoff-Rivers method apparently prevented its broader application.