Constant domain-exchanged Fab enables specific light chain pairing in heterodimeric bispecific SEED-antibodies.

Background: Bispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing.

Methods: We have designed a solution in which the C1 and C domain pair in one of the Fab fragments is replaced with a C3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII).

Results: Bispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains.

Conclusion: Incorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties.

General Significance: Our results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with C3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing.