Metabolic Engineering of Glycofusion Bispecific Antibodies for α-Dystroglycanopathies

Figures

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  Figure 1. Producing a glycofusion bispecific antibody for muscle dystrophy. (A) Schematic drawings of the dystrophin-associated glycoprotein complex and dystroglycan (DG) protein [4,5,18]. (B) Schematic drawings of the glycofusion bispecific antibody and the construct designs. The red rectangles in the left schema represent the N-terminal variable domain of an anti-β-DG light chain (VL) or the N-terminal variable domain of the heavy chain (VH). The mucin-like domains are the orange rectangles with the glycan drawings. A linker of (GGGGS)×2 is inserted between the mucin-like domain and the VL. The oval shape with “N” is the N-terminal prodomain of the DAG1 protein. CH1 is antibody heavy chain constant domain 1, and CL is antibody light chain constant domain (CL). Fc contains antibody heavy chain full hinge region and the constant domain 2 and 3. For protein production, the resulting construct DNAs were transfected along with the accessory DNAs encoding human LARGE1 and soluble PACE enzymes into Expi293 cells.

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  Figure 2. The glyco-optimized procedure produced the GBi antibody with a dramatically enhanced matriglycan modification and a better aSEC profile in HEK293 cells. (A) Analytical Diol-200 SEC profiles of GBi antibody derived from the standard production procedure (“standard”) and from the glyco-optimized procedure (Glyco-optimized). (B) Immunoblottings of monoclonal antibody IIH6C4 that recognizes the matriglycan epitopes of α-DG and anti-human IgG1 Fc. Equal volumes of the ProA eluates purified from the conditioned media generated by both production procedures (Lane 1, 2, 5, & 6 from the “Standard” procedure; lane 3, 4, 7, & 8 from the “Glyco-Optimized” procedure) were analyzed in the 4–12% SDS-PAGE under the conditions of reduced (R) and non-reduced (NR). The immunoblotting procedure was performed as described in Section 2.

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  Figure 3. SDS-PAGE and aSEC of GBi antibody derived from either the standard production procedure (“Standard”) or the glyco-optimized production procedure (Glyco-optimized). (A) Coomassie-stained SDS-PAGE of purified GBi antibody. Lane 1 & 2 were from the “Standard” procedure and Lane 3 & 4 were from the “Glyco-optimized” procedure. (B) aSEC on Diol300 column.

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  Figure 4. The glyco-optimized procedure-derived GBi antibody displayed dramatically improved β-DG-laminin bridging ELISA and IIH6C4 ELISA activities. The ELISA assays were performed as described in Section 2. Protein samples were immobilized on the plates at equal amounts during ELISA. (A) β-DG/Antibody (Ab)/human Lamin-211 (hLM211) bridging ELISA (n = 2 ± S.D.). (B) β-DG/Ab/IIH6C4 ELISA (n = 2 ± S.D.).

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  Figure 5. Further characterization of the GBi antibody with a large-scale production under the glyco-optimized procedure. (A) Analytical SEC on Diol 300 with ProA eluates, Fraction pool 1 (P#1), Fraction pool 2 (P#2), Fraction pool 3 (P#3), Fraction pool 4 (P#4), and Fraction pool 5 (P#5). (B) Coomassie-stained SDS-PAGE of ProA eluates (Lane 1 & 2), Fraction pool 1–5 (Lane 3–7). (C) Immunoblotting of IIH6C4 on ProA eluates (Lane 1 & 2) and P#1–5 (Lane 3–9).

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  Figure 6. Different pools of GBi antibody displayed a different activity in the β-DG-laminin bridging ELISA and IIH6C4 ELISA. (A) β-DG/Ab/hLM211 bridging ELISA (n = 2 ± S.D.). (B) β-DG/Ab/IIH6C4 ELISA (n = 2 ± S.D.). Protein samples were immobilized on the plates at equal amounts during ELISA.

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  Figure 7. It is the addition of sugar feeds that increases matriglycan modification of GBi antibody. Co-transfecting either GT1 alone, ST6 alone, GT1 and ST6 together, or in a single vector pJazz, along with the GBi antibody construct into Expi293, were performed. Sugar feeds were added post-transfectionally as indicated. Immunoblotting with IIH6C4 was carried out.

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  Figure 8. Mn2+ played a critical role in producing functionally active GBi antibodies. The GBi antibody constructs were transfected into Expi293 cells. The components of the sugar feeds [uridine (U), manganese ion (M), galactose (G), ManNAc (Man)] were added separately and combined as indicated (A,B). Immunoblotting with IIH6C4 was performed.