Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG

Abstract

Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies.

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

“…1A-C) was »3X longer than that used by Lee, 29 Schirrmann 30 and Hust, 31 and about 1.5X longer than that used by Koerber,32 while the linker we used to connect the C-terminus of the first heavy chain to the N-terminus of the second light chain (linker L2 in Fig. 1A-C) was »4.5X longer than the linker used by Lee, 24 Schirrmann 25 and Hust, 31 and 2.5X longer than that used by Koerber. 32 The linkers also contain protease cleavage sites (diamond symbols in Fig.…”

“…Other designs of IgG-Bs have been described, but with the exception of IgG-Bs that are restricted to the same heavy chain on both binding arms 24 and the 'two-in-one' IgG, 25 these other IgG-Bs are either variations or optimizations of the methods described thus far. [26][27][28] Although these approaches generate IgG-Bs, the deviation from the IgG chain sequence and structure remains a substantial issue, [6][7][8][9][10][11][12] and novel antibody engineering solutions are thus still needed.…”

“…The iMab design offers a unique engineering solution to overcome some of the limitations of the described previously IgG-Bs, [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] such as the potential formation of half-antibodies and homodimers, which can be difficult to eliminate and require complex multi-step purification methods.…”

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

“…The low levels of mispaired species are typically removed by chromatography purifications. 26 To simplify the purification process and make it more conducive to platform development, the two unique light chains can be engineered to possess sequence homology from different subfamilies (kappa and lambda). Affinity matrices such as LambdaFabSelect (LFS) and KappaSelect can then be used to selectively capture asymmetric bispecific antibodies that contain at least one light chain within the target subfamily.…”

“…Affinity matrices such as LambdaFabSelect (LFS) and KappaSelect can then be used to selectively capture asymmetric bispecific antibodies that contain at least one light chain within the target subfamily. 26,27 …”