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

Journal: The Journal of Biological Chemistry

Article Title: Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer *

doi: 10.1074/jbc.M116.754960

Figure Lengend Snippet: Bioinformatic identification and experimental validation of RTKs mediating trastuzumab resistance. A, forest plot of the concordance index estimates of survival risk for each RTK in HER2 patients. Data are ordered and presented as the mean ± S.E. Higher concordance index numbers predict worse outcome. The most significant p values are also shown; *, p < 0.05; **, p < 0.01; ***, p < 0.001. B–D, SKBR3, BT474, or AU565 breast cancer cells engineered to stably express the indicated RTKs were cultured for 2 days in increasing amounts of lapatinib, and then cell viability was measured and plotted as a function of drug concentration. Error bars, S.E. Results are summarized in E. NR, no rescue; P, partial rescue; R, rescue. F–H, Kaplan-Meier curves of IGF1R, TYRO3, and PDGFRb in each of the HER2 molecular subtype. Gene expression is split on the median into low and high classifications, and data are presented as the probability of relapse-free survival or distant metastasis-free survival versus time in months.

Article Snippet: Phospho-Akt (Ser-473, 4060), Akt (4691), ERK (4695), phospho-IGF1R (Tyr-980, 4568), IGF1R (9750), phospho-PDGFRb (Tyr-740, 3168), TYRO3 (5585), p21 (2947), and p27 (2552) were from Cell Signaling.

Techniques: Stable Transfection, Cell Culture, Concentration Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer *

doi: 10.1074/jbc.M116.754960

Figure Lengend Snippet: RTKs mediating resistance are up-regulated in drug resistant tumors and cell lines as a result of HER2 inhibition. A and B, IGF1R, PDGFRb, and TYRO3 expression levels increase as a result of trastuzumab (TRA) treatment in drug-resistant breast cancer cells. rBT474 (A) or MDA-MB-361 (B) cells were cultured for 72 h in the presence of 1, 3, or 10 μm trastuzumab, after which cells were harvested and protein lysates were immunoblotted using the indicated antibodies. C–E, representative images and quantification of IGF1R, PDGFRb, and TYRO3 immunohistochemistry in resistant and sensitive tumors before and after neoadjuvant trastuzumab therapy. Samples were obtained from HER2-positive breast cancers by mammotome biopsy before therapy and from surgery after treatment. n = 19 sensitive tumors; 13 resistant tumors. Error bars, S.D.; ***, p < 0.01. See supplemental Table S3 for exact p values. F, frequency of RTK overexpression after HER2 inhibition based on immunohistochemistry analysis of unresponsive patient tumors. A green bar represents up-regulation of the indicated RTK in an individual tumor.

Article Snippet: Phospho-Akt (Ser-473, 4060), Akt (4691), ERK (4695), phospho-IGF1R (Tyr-980, 4568), IGF1R (9750), phospho-PDGFRb (Tyr-740, 3168), TYRO3 (5585), p21 (2947), and p27 (2552) were from Cell Signaling.

Techniques: Inhibition, Expressing, Cell Culture, Immunohistochemistry, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer *

doi: 10.1074/jbc.M116.754960

Figure Lengend Snippet: IGF1R, TYRO3, and PDGFRb activity is necessary to sustain signal transduction and tumor cell proliferation in response to HER2 inhibition. A, SKBR3 cells expressing TYRO3, IGF1R, or PDGFRb were treated with lapatinib (Lap) for 24 h, and then proteins were resolved by SDS-PAGE and immunoblotted using antibodies specific for the indicated signaling proteins. B, synergistic inhibition of trastuzumab-resistant cell growth by HER2 and IGF1R chemical inhibitors. rBT474 cells were cultured for 72 h in the presence of increasing concentrations of lapatinib, AEW541, or lapatinib plus AEW541 and then metabolically labeled with 2 μCi of [3H]thymidine for 16 h, after which DNA was precipitated and thymidine incorporation was measured by scintillation counting. C, rBT474 cells were cultured as in B but treated with the PDGFRb inhibitor sunitinib instead of AEW541. Error bars, S.E.; ***, p < 0.05. D and E, combined HER2 and IGF1R inhibition delays mammary tumor formation in mice. Immunodeficient BALB/c nude mice were orthotopically injected with rBT474 breast cancer cells, and, after allowing 1 week for tumor formation, were treated daily with vehicle, lapatinib, or lapatinib plus AEW541. Tumor sizes were measured twice weekly using vernier calipers until day 25, at which point mice were sacrificed, and dissected tumors were photographed (E). Mean tumor volumes ± S.E. are shown in D; ***, p < 0.05 (n = 4 mice/group).

Article Snippet: Phospho-Akt (Ser-473, 4060), Akt (4691), ERK (4695), phospho-IGF1R (Tyr-980, 4568), IGF1R (9750), phospho-PDGFRb (Tyr-740, 3168), TYRO3 (5585), p21 (2947), and p27 (2552) were from Cell Signaling.

Techniques: Activity Assay, Transduction, Inhibition, Expressing, SDS Page, Cell Culture, Metabolic Labelling, Labeling, Injection

Journal: The Journal of Biological Chemistry

Article Title: Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer *

doi: 10.1074/jbc.M116.754960

Figure Lengend Snippet: Effective anti-HER2 therapy results in tumor cell senescence. A–C, representative images and quantification of Ki67 (A), p16 (B), and p21 (C) immunohistochemistry in resistant and sensitive tumors before and after neoadjuvant trastuzumab therapy. Samples were obtained from HER2-positive breast cancers by mammotome biopsy before therapy and from surgery after treatment. Error bars, S.D.; ***, p < 0.01. Exact p values can be found in supplemental Table S3. D–F, combined pharmacological inhibition of HER2 and IGF1R leads to cellular senescence in drug-resistant breast cancer cells. MDA-MB-361 cells were treated for 72 h with trastuzumab (TRA) and AEW541 as indicated, and cellular senescence was assayed by SA-βgal staining (D) and immunoblotting cell lysates with antibodies specific for the senescence-associated proteins p16 and p21 (E). F, rBT474 breast cancer cells were grown as tumor xenografts in BALB/c nude mice (n = 4/group). Following 1 week of tumor establishment, mice were treated with either vehicle, lapatinib (50 mg/kg/day), or lapatinib combined with AEW541 (both at 50 mg/kg/day) for 18 days. Frozen tumor sections were stained with hematoxylin and eosin to examine tumor histology or for SA-βgal activity to detect cellular senescence.

Article Snippet: Phospho-Akt (Ser-473, 4060), Akt (4691), ERK (4695), phospho-IGF1R (Tyr-980, 4568), IGF1R (9750), phospho-PDGFRb (Tyr-740, 3168), TYRO3 (5585), p21 (2947), and p27 (2552) were from Cell Signaling.

Techniques: Immunohistochemistry, Inhibition, Staining, Western Blot, Activity Assay