A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1

Abstract

Edited by Peter CresswellBispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G 1 . We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing saltbridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics.

Opposites attract in bispecific antibody engineering

“…However, applying bsAbs requires maintaining the natural antibody structure and the ability for large-scale production of high-quality material, which has proven challenging with these engineered sequences. A study by De Nardis et al (3) now demonstrates one solution to this problem, facilitated by pairing oppositely charged amino acids across the heterodimer interface.…”

“…These unexpected backbone rearrangements and novel interactions may account for the favorable stability of the molecule, making it a very attractive platform for the development of novel bsAbs. De Nardis et al (3) further demonstrated the utility of the DEKK design to construct a highly stable bsAb targeting HER2 and HER3 and to produce it using known protocols and, under GMP conditions, providing proof-of-concept that the DEKK design can yield bsAbs that can be used in the clinic.…”

“…For bsAbs to be successful, they will need to be amenable to straightforward, high-yield antibody production processes that fit GMP manufacturing conditions but also have physiochemical properties similar to that of naturally occurring antibodies. Design improvements to overcome these critical hurdles, such as the one described by De Nardis et al (3), are important for the success of bsAbs as future therapeutics. …”

“…Bispecific antibodies (bsAbs) 3 represent a fast-growing field of research, with seemingly endless opportunities. Whereas naturally occurring antibodies bind bivalently to one antigen by means of their two Fab arms, bsAbs are engineered to contain the arms of two different antibodies, allowing them to bind simultaneously to two distinct antigens (Fig.…”

“…De Nardis et al (3) first scanned a large series of mutants to identify positively and negatively charged substitutions in the CH3 domain that impaired the formation of homodimers, followed by the introduction of oppositely charged amino acids into the complementary CH3 domain. The L351D/T366K pair in particular resulted in the efficient formation of heterodimers and barely any homodimers.…”