Next generation Fc scaffold for multispecific antibodies

Abstract

Summary Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3′ interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies.

Decreasing hydrophobicity or shielding hydrophobic areas of CH2 attenuates low pH-induced IgG4 aggregation

“…The CH2 domain is an important and unique component of human IgG. It has unique properties, 1), CH2 connects with the lower hinge region, and they are relatively hydrophobic and structurally flexible, which is necessary for interaction with Fcγ receptors ( Sibéril et al, 2006 ; Kiyoshi et al, 2015 ); 2), the CH2-CH2 inter-chain interaction is mediated by N-glycans, not by direct protein interaction ( Kayser et al, 2011 ; Estes et al, 2021 ); 3), CH2 and CH3 interact with FcRn, which is mediated by electrostatic forces and hydrophobic interaction; 4), CH2 is glycosylated at N297, which partially but not completely covers hydrophobic patches on CH2 ( Kayser et al, 2011 ; Kiyoshi et al, 2017 ). N-glycosylation contributes to antibody stability and the tendency to aggregation.…”

Design and engineering of bispecific antibodies: insights and practical considerations

“…Many asymmetric bsAbs rely on heterodimerization of two different HC, thus spiking interest in HC steering platforms for promoting HC heterodimerization. Numerous HC steering platforms have been developed ( Liu et al, 2017 ), of which the majority is industry-originated, and functions by creating complementary interfaces in the CH3 domains ( Ridgway et al, 1996 ; Atwell et al, 1997 ; Merchant et al, 1998 ; Davis et al, 2010 ; Gunasekaran et al, 2010 ; Moore et al, 2011a ; Choi et al, 2013 ; Labrijn et al, 2013 ; Von Kreudenstein et al, 2013 ; Choi et al, 2015a ; Choi et al, 2015b ; Leaver-Fay et al, 2016 ; De Nardis et al, 2017 ; Skegro et al, 2017 ; Estes et al, 2021 ), which are key in governing the antibody structural integrity ( Feige et al, 2010 ; Bertz et al, 2013 ). The mutations in the structurally important CH3 domain are therefore also known to often affect the bsAb thermal stability ( Pomarici et al, 2022 ).…”

Protocol for high-throughput cloning, expression, purification, and evaluation of bispecific antibodies

“…Golden Gate Assembly provides a seamless and orderly strategy to clone multiple DNA fragments into a mammalian expression vector ( Figure 1 ) ( Engler et al., 2008 , 2009 ; Estes et al., 2021 ; Gong et al., 2021 ). The pTT5 vector is a suitable vector for both bacterial cloning as well as protein expression in mammalian hosts.…”

“…This protocol enables the rapid screening of large panels of bispecific molecules to identify top candidates for further development. For complete details on the use and execution of this protocol, please refer to Estes et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Estes et al. (2021) .…”