Anti-EGFR Recombinant Antibody (Imgatuzumab) (CAT#: TAB-753)

Figure 1 In vitro ADCC analysis of imgatuzumab, cetuximab or the combination.

PBMCs as effector cells were added to the untreated H441 (A), H292 (B) or A549 (C) target cells at a ratio of 25:1. ADCC responses were normalized to the ADCC response of the highest imgatuzumab concentration due to variations in responses between PBMCs of different donors. Absolute ADCC responses ranged between 12 and 53%. (*P < 0.05, **P < 0.01, ***P < 0.001 cetuximab vs imgatuzumab). (D) A549 cells were pretreated with imgatuzumab alone (20 µg/mL) or combined with cetuximab (20 µg/mL total) in culture medium for 72 hours. Next, cells with (open triangle and open square) or without (solid triangle and solid square) antibody pretreatment for 72 hours were washed and the ADCC assay was performed using PBMCs as effector cells and A549 cells as target cells at a ratio of 25:1. Data points are mean ± SD (n = 3).

Kol, A., van Scheltinga, A. T., Pool, M., Gerdes, C., de Vries, E., & de Jong, S. (2017). ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells. Oncotarget, 8(28), 45432.

Figure 2 In vitro growth-inhibitory effect of anti-EGFR monoclonal antibody treatment.

Cells were seeded in 12-wells plates and allowed to adhere overnight. Next, H292, H322 and A549 cells were treated with the anti-EGFR monoclonal antibodies (20 µg/ mL total) for the indicated days under normal growth conditions without additional EGF (medium with 10% FCS) or in medium containing 10% FCS with 1 or 10 ng/mL EGF. Data points are mean + SD. All experiments were performed in triplicate.

Kol, A., van Scheltinga, A. T., Pool, M., Gerdes, C., de Vries, E., & de Jong, S. (2017). ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells. Oncotarget, 8(28), 45432.

Figure 3 Cellular localization and endocytic trafficking of anti-EGFR monoclonal antibodies in SW-1573.

Kol, A., van Scheltinga, A. T., Pool, M., Gerdes, C., de Vries, E., & de Jong, S. (2017). ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells. Oncotarget, 8(28), 45432.

Figure 4 Effects of TKI treatment on monoclonal antibody-induced internalization and degradation.

(A) Western blot analysis of the effect of 24 hours anti-EGFR monoclonal antibody treatment (20 µg/mL total) with or without erlotinib (10 µM) on EGFR total protein levels in SW-1573, H292 and A549. Data points are mean + SD. (*P < 0.05, **P < 0.01, ***P < 0.001 compared to untreated control; $ P < 0.05, $ P < 0.01, $$ P < 0.001 compared to erlotinib treated control). (B) SW-1573 cells were pretreated for 24 hours with the EGFR TKI inhibitor erlotinib (10 µM). Cells were subsequently surface labeled with the anti-EGFR monoclonal antibodies (20 µg/mL total) on ice and incubated at 37 °C for 4 hours, and then analyzed for the non-internalized EGFR-antibody complexes (NI) and cell surface expression (SE). The lower the amount of non-internalized EGFR-antibody complexes, the higher the amount of internalized antibodyEGFR complexes. Surface expression of the control and erlotinib treated cells were both set at 100%. 24 hours erlotinib treatment resulted in a slight upregulation (10 – 15 % increase). Data points are mean ± SD (n = 3). (*P < 0.05).

Kol, A., van Scheltinga, A. T., Pool, M., Gerdes, C., de Vries, E., & de Jong, S. (2017). ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells. Oncotarget, 8(28), 45432.

Figure 5 Influence of monoclonal antibody binding on EGFR internalization and surface expression.

SW-1573 (A) and H292 cells (B) were surface labeled with the anti-EGFR monoclonal antibodies on ice and incubated at 37 °C for the indicated times, and then analyzed for the non-internalized EGFR-antibody complexes (left) and cell surface expression (right). The lower the amount of noninternalized EGFR-antibody complexes, the higher the amount of internalized antibody-EGFR complexes. The surface expression at t=0 was set at 100%. The non-internalized and surface expression were determined as described in Materials and Methods (n=3). Data points are mean ± SD. (**P < 0.01, ***P < 0.001 combination vs imgatuzumab; $ P < 0.01, $$ P < 0.001 combination vs cetuximab; two-way ANOVA followed by Bonferroni post-test).

Kol, A., van Scheltinga, A. T., Pool, M., Gerdes, C., de Vries, E., & de Jong, S. (2017). ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells. Oncotarget, 8(28), 45432.

Figure 6 Western blot analysis of the effect of 24 hours anti-EGFR monoclonal antibody treatment on EGFR total protein levels and downstream signaling under normal growth conditions or 15 min EGF stimulation (10 ng/mL).

The immunoreactive spots were quantified by densitometric analysis and normalized using actin (see also Supplementary Figure 2). Values are expressed as fold increase versus control + SD. All experiments were performed in triplicate.

Kol, A., van Scheltinga, A. T., Pool, M., Gerdes, C., de Vries, E., & de Jong, S. (2017). ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells. Oncotarget, 8(28), 45432.

Figure 7 Effect of anti-EGFR monoclonal antibody treatment on EGFR surface expression levels.

(A) Flow cytometric analysis of imgatuzumab and cetuximab binding alone or in combination in H322, SW-1573, H441, H292 and A549 cells. (B) H322, SW-1573, H292, H441 and A549 cells were treated with the anti-EGFR monoclonal antibodies (20 µg/mL total) for 72 hours. Surface expression levels were determined using flow cytometry. The surface expression in untreated control cells was set at 100% both for the single antibodies and the combination. (*P < 0.05, **P < 0.01 combination vs imgatuzumab; $ P < 0.05, $ P < 0.01, $$ P < 0.001 combination vs cetuximab; unpaired t-test). Data points are mean + SD (n = 3).

Kol, A., van Scheltinga, A. T., Pool, M., Gerdes, C., de Vries, E., & de Jong, S. (2017). ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells. Oncotarget, 8(28), 45432.

Figure 8 EGFR expression analysis and immunohistochemistry.

(A) Ex vivo tissue analysis. Mean vessel density (MVD), EGFR and hematoxylin and eosin immunohistochemical staining of A431, A549 and H441 tumors. (B) In vitro flow cytometric analysis of EGFR membrane expression in A431, A549 and H441 cells. (C) Ex vivo human EGFR extracellular domain levels in A431, A549 and H441 tumor tissue lysates. (D) EGFR histo-score (H-score) for A431, A549 and H441 tumors. (E) Mean vessel density score for A431, A549 and H441 tumors.

Pool, M., Kol, A., Lub-de Hooge, M. N., Gerdes, C. A., de Jong, S., de Vries, E. G., & van Scheltinga, A. G. T. (2016). Extracellular domain shedding influences specific tumor uptake and organ distribution of the EGFR PET tracer 89Zr-imgatuzumab. Oncotarget, 7(42), 68111.

Figure 9 EGFR analysis of plasma, liver and cell lysates.

(A) Human EGFR extracellular domain concentration in plasma of A431, A549 and H441 xenograft bearing and non-tumor bearing control mice. (B) Human EGFR extracellular domain level in liver tissue lysates of A431, A549 and H441 xenograft bearing and non-tumor bearing control mice. (C) Human EGFR extracellular domain concentration in serum-free supernates of confluent cell cultures of A431, A549 and H441 cells. (D) Human EGFR extracellular domain concentration in serum-containing supernates of confluent cell cultures of A431, A549 and H441 cells, only statistical significances against A431 are shown for clarity.

Pool, M., Kol, A., Lub-de Hooge, M. N., Gerdes, C. A., de Jong, S., de Vries, E. G., & van Scheltinga, A. G. T. (2016). Extracellular domain shedding influences specific tumor uptake and organ distribution of the EGFR PET tracer 89Zr-imgatuzumab. Oncotarget, 7(42), 68111.

Figure 1 Anti-EGFR Recombinant Antibody (TAB-753) in SDS-PAGE

SDS-PAGE analysis of TAB-753 in reduced (Lane 2) and non-reduced (Lane 1) conditions.

Figure 2 Anti-EGFR Recombinant Antibody (TAB-753) in HPLC

The purity of TAB-753 was greater than 95% as determined by SEC-HPLC.