Solving the light chain mismatch of IgG-like bispecific antibody by utilizing 2A peptide based FaBody platform

Abstract

Bispecific antibodies (BsAbs), engineered to target multiple antigens or epitopes simultaneously, promise enhanced therapeutic efficacy over traditional monoclonal antibodies (mAbs). However, the complex BsAbs structure presents significant production challenges, particularly chain mismatch issues. This study presents a novel approach utilizing 2A peptides within a FaBody platform to address light chain mismatches in IgG-like BsAbs. By leveraging self-cleaving 2A peptides, stable expression in mammalian cells significantly improves the accuracy of antibody chain assembly. This strategy markedly enhances the production of correctly assembled IgG-like BsAbs, providing a promising solution to critical challenges in BsAbs drug development.

Keywords: 2A peptides; Bispecific antibodies; Fabody platform; Light chain mismatch.

Fig. 1

Expression of bispecific antibodies in vitro, facilitated by the 2A peptide.

Fig. 2

(a) Different designs of anti-HER2/cMet bispecific antibody; (b) Predictive models of anti-HER2/cMet bispecific antibodies of different designs (generated by ESMFold); (c) Enlarged view of the anti-cMet side’s Fab region.

Fig. 3

Characterization and functional data of purified r-GS, d-F2A, l-F2A and r-F2A. (a) Summary of protein yield; (b) Protein SDS-PAGE electropherograms (non-reduced gel on the left and reduced gel on the right. Full-length gels are presented in Figure S1); (c) HPLC chromatogram with relative purity; (d) Protein Tm value (Raw data are presented in Figure S2); (e) ELISA binding results (hHER2-his antigen test left and hcMet-his antigen test right. Raw data are presented in Table S1).

Fig. 4

The affinity results of the HER2/cMet bispecific antibody produced by 2A peptide-mediated production and the corresponding parent molecule and control. (a) The affinity results of d-F2A; (b) The affinity results of l-F2A; (c) The affinity results of r-F2A; (d) The affinity results of d-GS; (e) The affinity results of N-linker; (f) The affinity results of Anti-HER2 Dimer; (g) The affinity results of Anti-cMet Dimer; (h) The affinity results of Anti-HER2 Monomer; (i) The affinity results of Anti-cMet Monomer.

Fig. 5

Characterization and functional data of d-F2A protein at different amounts of furin enzyme. (a) Summary of molecular yields under different transfection ratios; (b) Protein SDS-PAGE electropherograms (non-reduced gel on the left and reduced gel on the right. Full-gels are presented in Figure S3); (c) HPLC chromatogram with relative purity; (d) Protein Tm value (Raw data are presented in Figure S4); (e) Bridging-ELISA binding results (Raw data are presented in Table S2).

Fig. 6

Mass Spectrometric analysis of d-F2A protein. (a) Total Ion Current Chart Total Ion Chromatogram (TIC); (b) Non-denatured Reduction Mass Spectrometry was used to detect the residue of amino acids at the ends of light and heavy chains; (c) Disulfide bond determination.

Fig. 7

Characterization and functional data of purified double-ScFab proteins with different 2A linkers. (a) Summary of molecular yields with different 2A peptides; (b) Protein SDS-PAGE electropherograms (non-reduced gel on the left and reduced gel on the right. Full-length gels are presented in Figure S5); (c) HPLC chromatogram with relative purity; (d) Protein Tm value (Raw data are presented in Figure S6); (e) Bridging-ELISA binding results of purified double-ScFab proteins with different furin recognization sequence and 2A linkers (Raw data are presented in Table S3); (f) Western blot results; (g) Proliferation inhibition experiment result of HER2-positive cells (Raw data are presented in Table S4).

Fig. 8

Non-denatured Reduction Mass Spectrum (MS) of purified double-ScFab proteins with different 2A linkers. (a) Non-denatured Reduced Mass Spectrum of ${\mathrm{LC}}_{\mathrm{HER2}}$; (b) Non-denatured Reduction Mass Spectrum of ${\mathrm{LC}}_{\mathrm{cMet}}$; (c) Non-denatured Reduction Mass Spectra of ${\mathrm{HC}}_{\mathrm{HER2}}$ and ${\mathrm{HC}}_{\mathrm{cMet}}$.

Fig. 9

Composition of d-T2A RRKR 1:1:2 analyzed by Nonreduced Intact Mass analysis.

Fig. 10

The potential of the Fabody platform in constructing multispecific antibodies. (a) Different designs of bispecific antibody produced by Fabody platform; (b) Protein SDS-PAGE electropherograms (non-reduced gel on the left and reduced gel on the right. Full-length gels are presented in Figure S7–Figure S8); (c) HPLC chromatogram with relative purity; (d) Protein Tm value (Raw data are presented in Figure S9–Figure S11).

Fig. 11

The potential of the Fabody platform in constructing multispecific antibodies.