Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG

Abstract

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.

FIGURE 1.

a, ribbon representation of an antibody Fc region crystal structure (PDB code 1L6X) (21). Only the CH3 domain is involved in protein-protein interaction. b, the figure shows the backbone trace of the CH3 domain interface structure with residues involved in the domain-domain interaction. The interface residues were identified using a distance cutoff (4.5 Å) method. Structurally conserved and buried (solvent accessible surface area ≤10%) residues are shown in the ball-and-stick model. Solvent-exposed or structurally not conserved residues are shown in stick representation. The analysis is based on the IgG1 crystal structure that determined at high resolution (1.65 Å; 1L6X). c, crystal structure of the CH3 domain homodimer with one domain shown in ribbon representation and the other domain shown in wire model. The Lys⁴⁰⁹ (K409′ in the second domain) and Asp³⁹⁹ (D399′ in the second) residues are shown in ball-and-stick model to illustrate each pairwise interaction that is represented twice in the structure. This is due to the 2-fold symmetry present in the CH3-CH3 domain interaction. Schematics showing electrostatic interactions in the wild type (d) and charge-pair mutant (e) were designed as an example to enhance heterodimer formation and hinder homodimer formation. In the case of wild type, the charge-charge interaction favors both heterodimer and homodimer formation giving them equal probability. In the case of charge-pair mutant, the charge-charge interaction favors heterodimer and disfavors homodimer formation.

FIGURE 2.

a, schematic drawing of the two constructs used in the co-transfection experiments in 293 cells. The first construct encodes a scFv-Fc fusion. The second construct encodes a dummy Fc, which does not have any domain attached to it. The products of the co-transfection experiments with different Fc variants were analyzed by SDS-PAGE under non-reduced conditions and Western blot as shown in b–d. b, effects of single charge-pair mutations of the Asp³⁹⁹–Lys⁴⁰⁹′ interaction pair on Fc heterodimerization. Products of either single transfection or cotransfections of Fc variants are shown. All four single charge-pair variants (D399R/K409′E, D399K/K409′E, D399R/K409′D, and D399K/K409′D) led to higher yield for the scFv-Fc/Fc heterodimer (49 to 60%) compared with the homodimers (7 to 23%). The construct for the wild type Fc contained an extra tag of 33 amino acids, so its products appeared larger on SDS-PAGE. c, effects of charge-pair mutation combinations on Fc heterodimerization. The K409D:K392D/D399′K:E356′K mutation combination and the WT/WT are highlighted. The T366W (knob) and T366′S:L368′A:Y407′V (hole) mutation combination was included as a control. d, the effects of charge-pair mutation combinations on Fc homodimer and heterodimer formations. The products of single transfection (homodimer formation) and cotransfection (homodimers and heterodimer) for each Fc variants were shown. The single transfection of the triple mutant constructs (K409D:K392D:K370D and D399′K:E356′K:E357′K) did not yield any product of homodimer. However, cotransfection of the two triple mutant constructs led a heterodimer formation. Note the Fc sequence used in the constructs is derived from human IgG1 non- (a) allotype, which has a Glu at position 356. The crystal structure (1L6X) used for the computational analysis is from a different IgG1 allotype, which has Asp at position 356.

FIGURE 3.

a, schematic drawing of bispecific scFv-Fc generated by using the Fc heterodimer with charge-pair mutations (left panel) and SDS-PAGE analysis (right panel) of the purified bispecific scFv-Fc heterodimer in non-reduced (lane 1) and reduced (lane 2) conditions. Anti-CD3 scFv OKT3 (VH1/VL1 in red) was fused to the human Fc backbone with D399′K:E356′K mutations. Anti-TARTK scFv (VH2/VL2 in blue) was fused to human Fc with K409D:K392D mutations. b, the total ion chromatogram of deglycosylated bispecific scFv-Fc heterodimer using peptide:N-glycosidase F (PNGase F). Both heterodimer and peptide:N-glycosidase F peaks were seen in the 55-min gradient using a diphenyl. The deconvoluted mass spectrum result of deglycosylated bispecific scFv-Fc heterodimer is shown in the lower panel. The heterodimer with average mass of 107468.74 is seen, and no homodimers were detected in the mass analysis. The calculated heterodimer average mass is 107466.21. c, the binding of bispecific TARTKxCD3 scFv-Fc heterodimer to U87-TARTK cells and T cells (J45.01) by fluorescence-activated cell sorter. OKT3 huIgG1 and TARTK huIgG1 were used as controls. d, specific cytotoxic activity of bispecific TARTKxCD3 scFv-Fc on TARTK expressing cells. U87-TARTK cells (left panel) or U87 cells (right panel) were incubated with peripheral blood mononuclear cells in the presence of serial dilutions of bispecific TARTKxCD3 scFv-Fc heterodimer (red line), a control bispecific scFv-Fc heterodimer that binds to CD3 and an unrelated antigen (green line), and anti-TARTK huIgG1 (blue line) for 48 h. Percent of cell lysis was calculated and plotted. e, dose-dependent effect of bispecific TARTKxCD3 scFv-Fc heterodimer on the outgrowth of U87-TARTK glioma in NOD/SCID mice. U87-TARTK tumor cells were mixed 1:1 with purified T cells and innoculated subcutaneously. The indicated dose of bispecific TARTKxCD3 scFv-Fc heterodimer or Dulbecco's phosphate-buffered saline (DPBS) control were administrated via tail vein injections on days 0 and 4. Tumor growth curve derived from 10 animals is shown.

FIGURE 4.

a, schematic drawing of the monovalent IgG (left panel) and SDS-PAGE analysis of monovalent IgG preparations using wild type Fc (middle panel) and heterodimer Fc with charge-pair mutations (right panel). The content of culture supernatant (S) and eluted fractions (EL) after the protein A column of each preparation were examined under reduced conditions. The bands corresponding to LC-Fc and HC-Fc are indicated by arrows, respectively. b, anti-mouse TNFR1 14D2 monovalent IgG, either as human Fc (blue) or mouse Fc heterodimer (red), exhibited good potency in blocking TNF-mediated cytotoxicity in L929 assays. 14D2 mouse IgG1 was used as a control. IC₅₀ values were estimated from the percentage cell lysis curves and listed. c, 14D2 monovalent IgGs were free of agonistic activity. The cellular viability of L929 cells was assessed by fluorescence readout in the presence of 14D2 monovalent IgG, as human Fc (blue) or mouse Fc heterodimer (red), and 14D2 mouse IgG1 and control IgG (anti-FLAG) without TNF. d, 14D2 monovalent IgGs had a similar pharmacokinetics profile as full IgG in mouse serum.