Next generation Fc scaffold for multispecific antibodies

Abstract

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.

Keywords: Biochemistry; Bioengineering; Biomolecular engineering; Structural biology.

Graphical abstract

Figure 1

Structural analysis of SEFL2.2 Fc (A) Schematic representation of WT Fc (PDB: 1OQO) and SEFL2.2 Fc (PDB: 7LUR). (B) Superimposition of WT Fc (gray) with SEFL2.2 Fc (green), shown in cartoon representation with N297 glycan shown in sticks and C292-C302 in yellow spheres. Distances between the CH2 N-termini were measured using the Cα of G237 of each chain. Distances between the CH3 C-termini were measured using Cα of L443 of each chain. (C) Superimposition of CH3/CH3 dimers. (D) Superimposition of CH2/CH2 dimers and glycan interface. (E) Conformational shift of glycan binding residues (in sticks) in the CH2. (F) Non-canonical disulfide bond representation. (G) 2Fo-Fc electron density map for non-canonical disulfide bond countered at 1.0ϭ. (H) Rosetta energy score of WT Fc and 49 variants with different candidate disulfides.

Figure 2

The mechanism for loss of Fcγ receptor binding by SEFL2.2 Fc (A) Complex structures of WT IgG1 Fc binding to Fcγ receptors modeled onto the surface of transmembrane domain (TMD). (B) Model of SEFL2.2 Fc interface with FcγRI using the WT Fc (not depicted) as a reference. (C) The surface areas (Ų) buried on the FcγRI, FcγRII, and FcγRIII by WT Fc and SEFL2.2 Fc.

Figure 3

Structural analysis of CPMv1 Fc (A) Schematic representation of SEFL2.2 Fc (PDB: 7LUR) and CPMv1 Fc (PDB: 7LUS). (B) Superimposition of CPMv1 Fc (orange and wheat) with SEFL2.2 Fc (green), shown in cartoon representation. Distances between the CH2 N-termini were measured using the Cα of G237 of each chain. Distances between the CH3 C-termini were measured using Cα of L443 of each chain. (C) Superimposition of CH3/CH3′ dimers. (D–F) Surface representation of CH3/CH3′ dimer interface with key residues shown in sticks. Dotted lines represent the distances between side-chains.

Figure 4

Screening of CPM variants in the CH3/CH3′ interface (A) Diagram representation of the three positions (reference Edelman Unit) available for the design of CPMs. (B) Schematic representation of format used for CPMs screening. (C) Expression and analytical assessment of the 43 CPM variants and SEFL2.2 control. (D) Trends of expression and analytical assessment per number of charge pairs.

Figure 5

Purification profiles for selected CPM variants (A) Schematic representation of format used for CPM screening with the CPMs represented in red and blue and a DEVD site inserted into the upper hinge of one HC. (B) Expression levels of CPM variants after harvest. (C) CEX profiles for CPM variants assessed with buffers at pH 5.0 and pH 5.6. Arrows highlight main peaks.

Figure 6

Stability assessment of selected CPM variants. (A and B) Determination of protein species by analytical SEC after stress tests. (C) DSC thermogram for CPM variants. On top, schematic representation of T_m1 and T_m2 as result of SEFL2.2 and CPM engineering.

Figure 7

Characterization of CPMv1 and v103 by HDX-MS (A) Schematic representation of SEFL2.2 Fc with CPMv1 and CPMv103. (B) Structure of CPMv1 CH3/CH3′ interface with selected residues shown in spheres. (C) HDX plots showing the level of deuterium incorporation in both CPMv1 and CPMv103 variants.

Figure 8

Rational deployment of CPMv103 in Hetero-IgG molecules (A) Protein A yields of CPMv103 and CPMv103BCD each in both versions, swapping and not-swapping, applied to 4 Hetero-IgG molecules. (B) Characterization of impurity levels (½-Ab species) measured by SEC and LC-MS. The ½-Ab species with over 3% detected (dash line) were characterized by LC-MS and highlighted with schematics.