Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

Abstract

The promise of bispecific antibodies (bsAbs) to yield more effective therapeutics is well recognized; however, the generation of bsAbs in a practical and cost-effective manner has been a formidable challenge. Here we present a technology for the efficient generation of bsAbs with normal IgG structures that is amenable to both antibody drug discovery and development. The process involves separate expression of two parental antibodies, each containing single matched point mutations in the CH3 domains. The parental antibodies are mixed and subjected to controlled reducing conditions in vitro that separate the antibodies into HL half-molecules and allow reassembly and reoxidation to form highly pure bsAbs. The technology is compatible with standard large-scale antibody manufacturing and ensures bsAbs with Fc-mediated effector functions and in vivo stability typical of IgG1 antibodies. Proof-of-concept studies with HER2×CD3 (T-cell recruitment) and HER2×HER2 (dual epitope targeting) bsAbs demonstrate superior in vivo activity compared with parental antibody pairs.

Fig. 1.

Controlled Fab-arm exchange (cFAE). (A) Schematic representation of in vitro IgG4 Fab-arm exchange in dynamic equilibrium. IgG4 molecules containing permissive hinges (S228) and CH3 domains (R409) are incubated in the presence of reduced glutathione (GSH). (B) Schematic representation of in vitro generation of bsAbs by cFAE. IgG1 molecules containing stable WT hinges, and matched CH3 domain mutations are incubated in the presence of 2-MEA. (C) In silico model for the CH3_K409R(blue)–CH3_WT(orange) heterodimer interface. Modeling was done using PDB entry 1L6× for IgG1 Fc (28) and described previously (21). Only relevant side chains are shown in stick representation. The K409R substitution is depicted in green. Structural images were generated with PyMOL software (DeLano Scientific). (D) Matched CH3 mutations were identified by site-directed mutagenesis of residues Y407, L368, F405, K370, and D399. Bispecific antibodies obtained by cFAE (25 mM 2-MEA, 90 min at 37 °C) were assessed by dual-binding ELISA. Signals were normalized to that of the combination of IgG1-K409R-CD20 × IgG1-F405L-EGFR (orange bar). Residues present in WT IgG1 are indicated by arrows. Mixtures of IgG4-CD20 × IgG4-EGFR were included as reference (gray dashed line). Data represents the mean ± SEM of at least two separate experiments at a total antibody concentration of 1 μg/mL.

Fig. 2.

Defining the conditions for cFAE. (A) Generation of bsAbs by cFAE (25 mM 2-MEA) at different temperatures as a function of time was assessed by dual-binding ELISA. One representative experiment is shown. Data represent dual-binding at total antibody concentrations of 1 μg/mL. (B) Generation of bsAbs by cFAE (90 min at 37 °C) in response to increasing 2-MEA concentrations was assessed by dual-binding ELISA (black symbols, left y axis) and ESI-MS (blue symbols, right y axis). Mixtures of IgG4 molecules (circles) were included as controls. Data represent mean ± SEM of at least two separate experiments at a total antibody concentration of 1 μg/mL. Shaded areas represent peak overlap that precludes accurate species identification by ESI-MS. (C and D) Representative deconvoluted ESI-MS spectra (C) and CIEX profiles (D) of parental mAbs and the bsAb product bsIgG1-EGFRxCD20 obtained by cFAE (25 mM 2-MEA, 90 min at 37 °C). (E) Matched CH3 mutations were introduced into several other human mAbs (see Tables S1 and S2 for more details), and the generation of bsAbs by cFAE (25 mM 2-MEA, 90 min at 37 °C) was monitored by CIEX.

Fig. 3.

Large-scale generation of bsIgG1 by cFAE. (A) CIEX profiles of equimolar mixtures of IgG1-EGFR-F405L and IgG1-CD20-K409R before (Upper) and after (Lower) addition of 50 mM 2-MEA and subsequent incubation at 15 °C, 25 °C, or 37 °C for 5 or 24 h. (B) Nonreduced SDS/PAGE of bsIgG1 batches described in A: 5 h at 15 °C (lane 1), 24 h at 15 °C (lane 2), 5 h at 25 °C (lane 3), 24 h at 25 °C (lane 4), 5 h at 37 °C (lane 5), and 24 h at 37 °C (lane 6). Product-related impurities resulting from loss of light (L) and heavy (H) chains are labeled on the right. (C) CIEX profiles of starting materials (Upper) and end products (Lower) obtained by cFAE (5 h at ambient temperature) at indicated scales. (D) Reduced (Left) and nonreduced (Right) SDS/PAGE of bsIgG1 batches described in C: IgG1-b12 assay control (lane 1), IgG1-F405L-EGFR (lane 2), IgG1-K409R-CD20 (lane 3), 25-L test run at 1 g/L (lane 4), 25-L run at 20 g/L (lane 5). (E) HP-SEC profiles of starting materials (Upper) and end product (Lower) of 25-L test run at 1 g/L (dashed gray line) and 25-L run at 20 g/L (solid black line). Colors correspond with C. (F–H) Six-month stability at 15 g/L of the heterodimer bsIgG1 (25-L run at 20 g/L) in PBS, pH 7.4, buffer at 5 °C (lane 2; gray dashed lines), 25 °C (lane 3; green dashed lines), and 40 °C (lane 4; magenta dashed lines) as assessed by reduced (Left) and nonreduced (Right) SDS/PAGE (F), CIEX (G), and HP-SEC (H). Reference sample (t = 0) is included (lane 1; solid black line).

Fig. 4.

Bispecific IgG1 generated by cFAE retains WT IgG1-like functionality. (A and B) CDC of Daudi cells incubated for 45 min at 37 °C with serial diluted antibodies in the presence of 20% pooled human serum. (C and D) ADCC of Daudi target cells incubated for 4 h at 37 °C with serial diluted antibodies in the presence of PBMC effector cells (E:T ratio of 100:1). Symbols correspond to A and B. (E and F) Pharmacokinetics of bsIgG1-EGFRxCD20 and WT IgG1 antibody in SCID mice. BsIgG1-EGFR×CD20 plasma concentrations were determined by dual-binding ELISA (E). Data represent mean concentrations ± SEM. Plasma clearance (F) was calculated from these data and compared with that of WT IgG1-CD20 (gray triangles) and IgG1-EGFR (gray diamonds), for which plasma concentrations were determined with antibody-specific ELISAs. Data points represent individual mice.

Fig. 5.

Bispecific Abs show improved in vivo efficacy in selected animal models. (A) T cell–mediated cytotoxicity of AU565 target cells cocultured for 2 d at 37 °C with PBMC effector cells (E:T ratio 5:1) in the presence of serial diluted bsIgG1-N297Q-CD3×HER2_169. Data represent mean ± SEM of three experiments. Percentage T cells in PBMCs were 18.9%, 38.5%, and 59.8%. (B and C) Prophylactic treatment of NCI-N87 tumors in an s.c. xenograft model in NOD-SCID mice reconstituted with human PBMCs. Mice were inoculated with 5 × 10⁶ NCI-N87 cells mixed with 5 × 10⁶ PBMCs, randomized (n = 3–4 per group) and treated i.p. (B) or i.v. (C) 1 h thereafter. Mice receiving PBMCs from donors showing nonspecific killing of tumor cells in the absence of antibody (alloreaction) were excluded from analysis. Mice were treated with 4 mg/kg bsIgG1-N297Q-CD3×b12 or indicated doses of bsIgG1-N297Q-CD3×HER2₁₆₉, supplemented with IgG1-b12 to a total antibody dose of 4 mg/kg (B) or with 0.5 mg/kg bsIgG1-N297Q-CD3×b12 or indicated doses of bsIgG1-N297Q-CD3xHER2₁₆₉ (C). Data indicate mean tumor volumes ± SEM (red arrowheads indicate treatment days). (D and E) Treatment of established NCI-N87 tumors in an s.c. xenograft model in SCID mice. Mice were treated with a 40-mg/kg i.p. dose of total antibody on day 7, followed by a 20-mg/kg i.p. dose on day 14 (red arrowheads indicate treatment days). Data represent mean tumor volumes ± SEM (D) and time to progression (as defined by mice with tumor volumes ≤ 400 mm³) (E). The experiment was repeated twice with similar results. Statistical significance was determined by one-way ANOVA (Tukeys multiple comparison) in B–D and by log-rank Mantel-Cox analysis in E (*P < 0.05; **P < 0.01; ***P < 0.001).