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, 12 (1), 1685350

Design and Characterization of Mouse IgG1 and IgG2a Bispecific Antibodies for Use in Syngeneic Models

Affiliations

Design and Characterization of Mouse IgG1 and IgG2a Bispecific Antibodies for Use in Syngeneic Models

Feng Wang et al. MAbs.

Abstract

The development of antibody therapeutics relies on animal models that accurately recapitulate disease biology. Syngeneic mouse models are increasingly used with new molecules to capture the biology of complex cancers and disease states, and to provide insight into the role of the immune system. The establishment of syngeneic mouse models requires the ability to generate surrogate mouse counterparts to antibodies designed for humans. In the field of bispecific antibodies, there remains a dearth of technologies available to generate native IgG-like mouse bispecific antibodies. Thus, we engineered a simple co-expression system for one-step purification of intact mouse IgG1 and IgG2a bispecific antibodies from any antibody pair. We demonstrated proof of concept with CD3/CD20 bispecific antibodies, which highlighted both the quality and efficacy of materials generated by this technology.

Keywords: Mouse bispecific antibodies; T-cell engaging bispecific antibody; co-expression; electrostatic steering; inter-chain disulfide bond shifting; syngeneic mouse model.

Figures

Figure 1.
Figure 1.
Design and engineering of bispecific mIgG1 and mIgG2a antibodies. (a) Location of two salt bridges (E356:K439 and D398:K309) in the CH3 domain of mG1 (PDB: 3HKF). (b) Switching residues to opposite charge still maintains salt bridges between heterodimer (a’- b’), but causes electrostatic clashes between homodimer (a’ – a’ and b’ – b’). (c) Sequence alignment of human (IgG1) and murine (IgG1 and IgG2a) CH3 domain. Salt bridge E357:K370 in human is missing in murine IgGs. Underlined residues were mutated to a human when the third salt bridge was included. (d) Testing bispecific heavy chain design in co-expression. Percentage of heterodimeric heavy chain pair were quantified via mass spectrometry. In mIgG1, K/D and designs with additional engineered salt-bridge produce complete heterodimer. In mIgG2a, K/D design produces 95% heterodimer with 5% HC1:HC1 homodimer. Designs with additional engineered salt bridge enhances bispecific HC formation to completion. (e) Superposition of bispecific mG2aH4 Fc (green) with mIgG2a wild-type Fc (pale red, PDB: 3zo0) and mIgG2a cFAE bispecific (blue, PDB: 5VAA). (f) Two Fc orientations exist in crystal packing, resulting in an AB and BA average structure at bispecific mutations. (g) Sequence alignment of human kappa light chain with DuetMab design and murine kappa light chain. S121 from CL and Y122 from CH1 (PDB: 1IGT) were selected for Cys substitutions to form inter-chain disulfide bonds.
Figure 2.
Figure 2.
Purification and optimization of a panel of mG1 and mG2a bispecific antibodies. Intact mass spectrometry has conducted a panel of (a) mG1KD D265A, (b) mG2aH4, and mG2aH4 D265A bispecific antibodies transfected at equal DNA ratios for all four polypeptide chains. For constructs that exhibited some degree of mispairing DNA transfection ratios were optimized (asterisks) to restore complete bispecific formation.
Figure 3.
Figure 3.
Co-expression and purification of CD3/CD20 bispecific antibodies. Intact mass spectrometry of (a) CD3/CD20 mG1KD D265A and (b) CD3/CD20 mG2aH4 treated with PNGase overnight. CD3/CD20 mG2aH4 was not completely deglycosylated, hence the presence of one or more G0F glycans in addition to the mass of the correct bispecific. (c) CD3/CD20 mG1KD D265A and (d) CD3/CD20 mG2aH4 were analyzed by SDS-PAGE in both non-reducing (lanes C-1 and D-1) and 50 mM dithiothreitol reducing (lanes C-3 and D-3) conditions. Asterisk (*) denotes glycosylated products, which run at a greater molecular weight. CD3/CD20 mG1KD D265A and CD3/CD20 mG2aH4 were also analyzed by SEC-MALS (e and f, respectively). High molecular weight (HMW) and low molecular weight (LMW) species were calculated as a percentage of the total area under the curve. Sedimentation coefficients for CD3/CD20 mG1KD D265A and CD3/CD20 mG2aH4 as measured by analytical ultracentrifugation (g and h, respectively).
Figure 4.
Figure 4.
CD3/CD20 bispecific antibodies engage T-cells and target CD20+ cells in vitro. To assess CD20 binding, CD3/CD20, Neg/CD20, CD3/Neg, or Neg/Neg bispecific antibodies in both (a) mG1KD D265A and (b) mG2aH4 backbones were incubated with CHO-CD20 cells and assessed by flow cytometry (mean ± SD, n = 3). To assess CD3 binding, CD3/CD20 or Neg/CD20 bispecific antibodies in both (c) mG1KD D265A and (d) mG2aH4 backbones were incubated with primary T-cells and assessed by flow cytometry (mean ± SD, n = 3). T-cell-mediated depletion of A20 cells were conducted at a 1:5 T:E ratio for 72 h with CD3/CD20, Neg/CD20, or CD3/Neg in both (e) mG1KD D265A (EC50 = 1.85 nM) and (f) mG2aH4 (EC50 = 0.77 nM) backbones. The depletion of CellTrace Violet (CTV) labeled A20 cells were measured by flow cytometry (mean ± SD, n = 3).
Figure 5.
Figure 5.
CD3/CD20 bispecific antibodies activate T-cells through direct conjugation of T-cells and B-cells. (a) Total splenocytes were incubated with CD3/CD20 and CD3/Neg in both mG1KD D265A and mG2aH4 backbones for 24 h. T-cell activation was assessed by CD25 staining followed by flow cytometry (mean ± SD, n = 3). (b) B/T-cell conjugates were captured by flow cytometry following incubation of total splenocytes with CD3/CD20, Neg/CD20, or CD3/Neg in the mG1KD D265A backbone for 1.5 h. The total percentage of conjugates were calculated from Σ(CD19+CD4+, CD19+CD8+, CD19+CD4+CD8+)/total events (mean ± SD, n = 3). Representative flow cytometry plots at (c) 2.26E-4 ug/ml and (d) 40 ug/ml CD3/CD20 antibody depicts the concentration-dependent increase of conjugated CD4+CD19+ B-cells and T-cells (top right quadrant).
Figure 6.
Figure 6.
CD3/CD20 bispecific antibodies deplete B-cells in vivo. Wild-type C57BL/6 mice were treated with a single 1 mg/kg dose of CD3/CD20 or CD3/Neg in both mG1KD D265A and mG2aH4 D265A. (a) After 7 days, CD19 + B-cells were measured by flow cytometry (mean ± SD, n = 4). (b) In a second study, wild-type C57BL/6 were treated with a single 1 mg/kg dose of CD3/CD20 mG2aH4 D265A, CD3/Neg mG2aH4 D265A, or CD20 mG2a. Circulating B-cells were measured by flow cytometry at 0, 1, 2, 3, 4, and 7 days (mean ± SD, n = 4). (c) Spleens were harvested at day 7 and B-cells measured by flow cytometry (mean ± SD, n = 4). (d) Pharmacokinetic (PK) data were collected from wild-type C57BL/6 treated with 1 mg/kg CD3/CD20 or CD3/Neg in both mG1KD D265A, mG2aH4, and mG2aH4 D265A backbones. Serum concentrations were measured at 0, 1, 7, 14, and 21 days.

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