Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Cartoon representation, SDS-PAGE, reduced western blot and SEC-MALS analyses of the iMab-EI. (A) Cartoon representation of the iMab-EI with linkers connecting the antibody domains shown as red dotted lines. (B) Reduced SDS-PAGE (lanes 1 to 4) and reduced western blot analysis (lanes 5 to 16) of iMab-EI, anti-EGFR and anti-IGF1R antibodies. Molecular mass standards are schematically shown. Lκ, Lλ, H1 and H2 denote anti-EGFR kappa light chain, anti-IGF1R lambda light chain, anti-EGFR heavy chain, anti-IGF1R heavy chain, respectively. FL denotes the full-length iMab-EI. Lane 2 is the anti-EGFR antibody, lane 3 is the anti-IGF1R antibody and lane 4 is the full-length iMab-EI. Lanes 6, 7 and 8 is the reduced western blot probed with an anti-human Fc antibody. Lanes 10, 11 and 12 is the reduced western blot probed with an anti-human kappa antibody. Lane 14, 15 and 16 is the reduced western blot probed with an anti-human lambda antibody. (C) SEC-MALS of the iMab-EI after protein A purification. (D) SEC-MALS of monomeric iMab-EI. The molecular weights in KDa were obtained using SEC-MALS.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: SDS Page, Western Blot, Purification

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Reduced SDS-PAGE, reduced western-blot, SEC-MALS, rRP-HPLC and rLCMS of the iMab-EI after thrombin cleavage. (A) Reduced SDS-PAGE (lanes 1 to 4) and western blot analysis (lanes 5 to 16) of iMab-EI, anti-EGFR and anti-IGF1R antibodies. Molecular mass standards are schematically shown. Lκ, Lλ, H1 and H2 denote anti-EGFR kappa light chain, anti-IGF1R lambda light chain, anti-EGFR heavy chain, anti-IGF1R heavy chain, respectively. Lκ*, Lλ*, H1* and H2* denote the iMab-EI anti-EGFR kappa light chain, anti-IGF1R lambda light chain, anti-EGFR heavy chain, and anti-IGF1R heavy chain, respectively. Lane 2 is the anti-EGFR antibody, lane 3 is the anti-IGF1R antibody and lane 4 is the iMab-EI. Lanes 6, 7 and 8 is the reduced western blot probed with an anti-human Fc antibody. Lanes 10, 11 and 12 is the reduced western blot probed with an anti-human kappa antibody. Lanes 14, 15 and 16 is the reduced western blot probed with an anti-human lambda antibody. The chains of the iMab-EI are migrating slower compared to the chains from the 2 parental antibodies due their slightly higher molecular weight. (B) SEC-MALS analysis of the iMab-EI after thrombin cleavage showed that the iMab-EI is 99% monomer and has a molecular weight resembling that of an intact IgG1. (C) rRP-HPLC of the iMab-EI, (D) anti-IGF1R, and (E) anti-EGFR. The identity of each peak is schematically labeled as shown in panel A. (F) rLCMS of iMab-EI. The chains of the iMab-EI are labeled as shown in the panel A and as in panel C. The molecular masses of the 4 chains are shown in Daltons. The rLCMS was performed after Endo S treatment to trim the N-glycan found at the conserved N297 site in iMab-EI CH2 domain.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: SDS Page, Western Blot, Molecular Weight, Labeling

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: iMab-EI has native interchain disulfide bonds at the hinge and at the heavy-light chains as demonstrated using non-reduced SDS-PAGE. Lane 1 is the molecular weight standards; lane 2 is the anti-IGF1R, lane 3 is the anti-EGFR.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: SDS Page, Molecular Weight

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Non-reduced LCMS of the iMab-EI and the 2 parental antibodies intact glycosylated and after treatment with IdeS, which release the Fc fragment from the F(ab)’2. (A) Intact glycosylated iMab-EI; (B) IdeS-treated iMab-EI; (C) Intact glycosylated anti-EGFR; (D) IdeS-treated anti-EGFR; (E) Intact glycosylated anti-IGF1R; (F) IdeS-treated anti-IGF1R; The deconvoluted mass in Dalton is shown on the x-axis and the ions counts are shown on the y-axis. The molecular weight of each peak is schematically shown with the corresponding glycoform. LCMS show the iMab-EI has correctly formed the interchain disulfide bonds at the hinge and at the heavy and light chains similar to IgG1 parental antibodies.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Molecular Weight

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Isoelectric point (pI) of the iMab-EI and the 2 parental antibodies. (A) iMab-EI pI main peak is 8.43; (B) anti-EGFR antibody pI main peak is 7.72; (C) anti-IGF1R pI main peak is 8.24. pI marker peaks 4.22 and 9.77 are schematically shown.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Marker

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Binding of iMab-EI to EGFR and IGF1R using BIAcore. (A) Concurrent binding to antigens using BIAcore. iMab-EI was immobilized on the BIAcore sensor chip and dual binding was determined by first injecting IGF1R, followed by co-injection of IGF1R and EGFR. (B) Binding kinetics to IGF1R of the iMab-EI and the Fab prepared from the anti-IGF1R antibody. (C) Binding kinetics to EGFR of the iMab-EI and the Fab prepared from the anti-EGFR antibody.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Binding Assay, Injection

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Differential scanning calorimetry (DCS) of the iMab-EI (A), the anti-EGFR (B) and anti-IGF1R (C) antibodies. Transition temperatures are shown as Tm in°C. DSC analysis shows that the iMab-EI has transition temperatures similar to the transition temperatures of the anti-EGFR and anti-IGF1R antibodies, indicating the iMab-EI has a structure similar to the anti-EGFR and anti-IGF1R antibodies. (D) Hydrodynamic radios (Rh) determined using SEC-MALS of the iMab-EI at 0.5, 2 and 6 mg/mL. The Rh of the iMab-EI remains similar at the 3 concentrations tested, which is an indication of structural stability of the iMab-EI.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Differential Scanning Calorimetry

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Schematic representation and data of the EGFR ELISA transfer assay. (A) EGFR was immobilized on the ELISA plate, (B) and incubated with iMab-EI or the 2 parental antibodies. The well volume of the iMab-EI or the parental antibodies pre-absorbed on an EGFR-coated ELISA plates (C) were transferred to an ELISA plate with immobilized IGF1R, (D) followed by detection of binding using an anti-human-lambda-HRP labeled antibody.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Labeling

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Schematic representation and data of the IGF1R ELISA transfer assay. (A) IGF1R was immobilized on the ELISA plate, (B) and incubated with iMab-EI or the 2 parental antibodies. The well volume of the iMab-EI or the parental antibodies pre-absorbed on an IGF1R-coated ELISA plates (C) were transferred to an ELISA plate with immobilized EGFR, (D) followed by detection of binding using an anti-human-kappa-HRP labeled antibody.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Labeling

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: In vivo efficacy of iMab-EI using a xenograft mouse model of a patient derived non-small cell lung cancer. iMab-EI (red curve), isotype non-binding antibody (black curve) and combination of the anti-EGFR and anti-IGF1R antibodies (green curve) were dosed at 10 mg/kg 2 times weekly for 2 weeks. Vehicle-treated mice (blue curve) were used as negative control.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: In Vivo, Derivative Assay, Binding Assay, Negative Control