How to Make Bispecific Antibodies

Abstract

This protocol describes the production of bispecific F(ab’)₂ antibody derivatives (BsAbs) by the linking of two Fab/tc fragments via their hinge region SH groups using the bifunctional crosslinker o-phenylenedimaleimide (o-PDM) as described by Glennie et al. (1,2). The procedure is illustrated in Fig. 1. The first step is to obtain F(ab’)₂ from the two parent IgG antibodies. Methods for digestion of IgG to F(ab’)₂ are described in Chapter 151. Fab’ fragments are then prepared from the two F(ab’)₂ species by reduction with thiol, thus exposing free SH groups at the hinge region (three SH-groups for mouse IgG1 and IgG2a antibodies) (see Note 1). One of the Faba species (Fab’-A) is selected for alkylation with o-PDM. Because o-PDM has a strong tendency to crosslink adjacent intramolecular SH-groups, two of the three hinge SH-groups will probably be linked together, leaving a single reactive maleimide group available for conjugation Fig. 1; see Note 2). Excess o-PDM is then removed by column chromatography, and the Fab’-A(mal) is mixed with the second reduced Fab’ (Fab’-B) under conditions favoring the crosslinking of the maleimide and SH groups. When equal amounts of the two parent Fab’ species are used, the major product is bispecific F(ab’)₂, resulting from the reaction of one Fab’-A(mal) with one of the SH groups at the hinge of Fab’-B. Increasing the proportion of Fab’-A(mal) in the reaction mixture results in a significant amount of F(ab’)₃ product by the reaction of two molecules of Fab’-A(mal) with two free SH-groups at the hinge of a single Fab’-B molecule (see Note 3). The remaining free SH groups on Fab’-B are alkylated, and the F(ab’)₂ bispecific antibody product (Fab’-A×Fab’-B) is separated by gel filtration chromatography. Each stage of the procedure is checked by HPLC.