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Bispecific antibodies have been generated in many different formats and it is becoming clear that rational design alone cannot create optimal functionalities. Here the authors introduce the high throughput methodology, Format Chain Exchange (FORCE), to enable combinatorial generation of bispecific antibodies.

In the vein of Daniel Defoe’s Robinson Crusoe (1719), the German writer Johann Gottfried Schnabel (1692–1748) wrote a four-volume Robinsonade novel, Die Insel Felsenburg [The Island Felsenburg], which was published between 1731 and 1743. Schnabel’s novel became extremely popular in Germany, as it tells the story of a group of shipwrecked settlers who, in the spirit of protestant piety, establish an ideal state on the beautiful island on which they are stranded. One day, they discover a hidden cave, where they find a well-preserved mummified man, sitting in a stone chair at a table. On a tin board, this man, Don Cyrillo de Valaro, had engraved important information for posterity: namely that he was born on 9 August 1475, came to the island on 14 November 1514, and recorded his recollection on 27 June 1606. His writing ends as follows: ‘I am still alive, however close to death, June 28. 29. and 30. and still July 1., 2. 3., 4. By recording every day that he was still alive, Don Cyrillo, the only inhabitant on the island at the time, managed to do what no autobiographer could ever complete: record his death. One could even go so far as to say that his method typifies a life-writing model – documenting the days of one’s life in the face of inevitable death. In the context of Schnabel’s novel, this episode is remarkable in so far as the most prominent entertainment of the island’s inhabitants is to tell one another about their lives. In the evening, when their work is done, they come together – and there is no TV or internet – and tell their stories. Remarkably enough, their stories are full of sex and crime – aspects of life that are banned from the virtuous island. The story of Don Cyrillo de Valaro and the settlers is fiction, of course. However, it triggers the question as to how ‘real’ autobiographers deal with or even describe their own deaths.

Bispecific antibodies (bsAbs) with avidity-enhanced specificity can be used to address target cells with increased specificity, ideally binding efficiently to cells that express two cognate antigens, yet not to cells that express only one of those. Building blocks required to generate such bsAbs are binders that recognize the two antigens with high specificity yet with various (including very low monovalent) affinities. The herein described ‘back-to-germline’ (B2G) procedure defines such derivatives. It converts parent antibodies with high specificity to derivatives that retain specificity but modulate affinity. The approach defines mutations to be introduced into antibody complementarity-determining regions (CDRs) regions without requiring structures of antibody-antigen complexes. Instead, it reverses the B-cell maturation process that increases affinities, with preference on CDR residues with high antigen contact probability. Placing germline residues at those positions generates VH and VL domains and Fv-combinations thereof that retain specificities but are ‘de-matured’ to different degrees. De-maturation influences on-rates and off-rates, and can produce entities with extremely low affinity for which binding can only be detected in bivalent formats. A comparison with alanine replacement in CDRs (so far, the most frequently applied technology) indicates that B2G may be more reliable/predictable without introduction of stickiness or poly-reactivity. The applicability for generating sets of affinity-modulated monospecific variants is exemplarily shown for antibodies that bind CD138, Her2/neu, and EGFR.