Generation of bispecific antibodies by structure-guided redesign of IgG constant regions

Abstract

Bispecific antibodies (BsAbs) form an exciting class of bio-therapeutics owing to their multispecificity. Although numerous formats have been developed, generation of hetero-tetrameric IgG1-like BsAbs having acceptable safety and pharmacokinetics profiles from a single cell culture system remains challenging due to the heterogeneous pairing between the four chains. Herein, we employed a structure-guided approach to engineer mutations in the constant domain interfaces (C_H1-C_L and C_H3-C_H3) of heavy and κ light chains to prevent heavy-light mispairing in the antigen binding fragment (Fab) region and heavy-heavy homodimerization in the Fc region. Transient co-transfection of mammalian cells with heavy and light chains of pre-existing antibodies carrying the engineered constant domains generates BsAbs with percentage purity ranging from 78% to 85%. The engineered BsAbs demonstrate simultaneous binding of both antigens, while retaining the thermal stability, Fc-mediated effector properties and FcRn binding properties of the parental antibodies. Importantly, since the variable domains were not modified, the mutations may enable BsAb formation from antibodies belonging to different germline origins and isotypes. The rationally designed mutations reported in this work could serve as a starting point for generating optimized solutions required for large scale production.

Keywords: EGFR – epidermal growth factor; Fc engineering; Fc receptors (FcR); HER2; bispecific antibody (BsAb); heterodimer; rational design; structure-guided.

Figure 1

Structure-guided redesign of C_H1-C_L interface for BsAb engineering. Expression levels of cognate paired and mispaired monospecific species formed by heavy and light chains of pertuzumab (PTAB) and DL11 (A) without mutations and (B) with mutations. Rendered in (C-F) are the 3D derived models of the C_H1 and C_L domain interface observed in PDB: 3BKY, where the view is perpendicular to the pseudo-2-fold axis. The C_H1 and C_L domains are colored in green and cyan, respectively. The side chains of the modified C_H1-C_L interface residues and WT residues that aid or perturb the C_H1-C_L interface are represented as sticks and labeled. Attractive and repulsive interactions are circled using black and red ovals, respectively. (G–I) Network and two-fold axis relationship of mutants to enhance heterodimerization of C_H3 domains in BsAbs. The view is looking down on the 2-fold axis of symmetry. The C_H3 domains of the two antibodies are colored in green and splitpea, respectively. Potential repulsive interactions that emerge in C_H3 homodimers, such as Glu357- Lys370Glu (G) and Glu357Lys - Lys370, Lys409Arg - Lys370 (H), are highlighted by red oval, whereas the interactions that enhance the heterodimer formation in the BsAb are highlighted by black oval (I). Note that the instance of the interactions that is away from the reader is not shown, for clarity.

Figure 2

Identification of lead C_H3 mutations for Fc heterodimer formation using non-reducing SDS-PAGE analysis. Non-reducing SDS-PAGE of representative Fc engineered antibodies are shown in (A). Protein samples were harvested. Gel was stained with Coomassie blue. Mutations that induce the formation of half-antibody fragments are circled. These include the published knobs-into-holes mutations (lanes 4 & 5) and the computationally engineered variant (lane 6).(B) Identification of compensatory Fc mutations using a three-chain system (2 heavy and 1 light). The Fc mutations shown in column 7 (circled in red) show maximum reduction in the formation of half-antibody fragments, similar to the traditional knob-into-holes mutations (column 3). Lane 1 in A and B contains SeeBlue protein standards.

Figure 3

SDS-PAGE analysis of the two BsAbs compared to parental mAbs. Purified pertuzumab, anti-EGFR/HER2 BsAb, rituximab and anti-CD20/CD20 BsAb were subjected to electrophoresis in 4-12% SDS PAGE under (A) nonreducing condition and (B) reducing condition then stained with Coomassie Blue. Precision Plus Protein Kaleidoscope Prestained Protein Standards (Bio-Rad) was used as molecular weight marker.

Figure 4

Separation of antibodies by size exclusion chromatography. The elution profiles showed a major peak of (A) anti-EGFR/HER2 BsAb, (B) pertuzumab, (C) anti-CD20/CD20 BsAb, and (D) rituximab with a retention time that is consistent with their expected molecular weight of around 144-145 kDa, respectively. All SEC experiments were done in triplicate. Chromatograms shown here are representative of the results.

Figure 5

Intact mass analyses. (A) The deconvoluted MW of the anti-EGFR/HER2 BsAb. (B) The deconvoluted MW of the anti-CD20/CD20 BsAb. MWs in red indicate the theoretical mass with C-terminal lysine clipping and MWs in blue indicate the observed mass. Anti-CD20 monospecific Ab (1) and anti-CD20 monospecific Ab (2) contain Fab corresponding to rituximab and obinutuzumab monoclonal antibodies, respectively.

Figure 6

Far-UV CD spectrum of antibodies. (A) The spectrum of anti-EGFR/HER2 BsAb and its parental mAb (pertuzumab) show a negative band at around 218 nm. (B) The anti-CD20/CD20 BsAb and its parental mAb (rituximab) also show a similar spectrum of negative band as anti-EGFR/HER2 BsAb at approximately 218 nm. All proteins were buffer exchanged in milli-Q water prior analysis and done in triplicate.

Figure 7

Thermal stability analysis of antibodies by differential scanning calorimetry (DSC). (A) The anti-EGFR/HER2 BsAb together with its parental mAb (pertuzumab) show two peaks of similar T_m values corresponding to the C_H2/Fab and C_H3 domains, with an extra peak at T_m 67.52°C. (B) The anti-CD20/CD20 BsAb exhibits similar T_m values for the C_H2/Fab and C_H3 domains when compared to its parental mAb (rituximab).

Figure 8

Fab binding activity of anti-EGFR/HER2 BsAb. (A) Schema of dual-binding ELISA format that was used in this study. (B) The anti-EGFR/HER2 BsAb shows dose-dependent binding consistent with its proposed quaternary structure. (C) Schema of direct HER2 binding ELISA. (D) The binding affinity of BsAb was compared to pertuzumab.The error bars indicate standard deviation from three independent experiments. Graphs were generated using GraphPad Prism version 8.4.3 for Windows.

Figure 9

Fc binding activity of the two BsAbs to their target proteins. (A) SPR sensograms of soluble anti-EGFR/HER2 BsAb to chip-capture FcɣRIIIa (V158), FcɣRIIIa (F158), FcRn and the binding of this BsAb coated on protein-L chip to a soluble C1q protein. (B) SPR sensograms of soluble anti-CD20 BsAb to chip-capture FcɣRIIIa (V158), FcɣRIIIa (F158), FcRn and C1q protein. All Fc receptor SPR bindings were done in triplicate.