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. 2021 Jan-Dec;13(1):1914883.
doi: 10.1080/19420862.2021.1914883.

Anti-tumoral activity of the Pan-HER (Sym013) antibody mixture in gemcitabine-resistant pancreatic cancer models

Emilia Rabia  1 , Véronique Garambois  1 , Julie Hubert  1 , Marine Bruciamacchie  1 , Nelly Pirot  1   2 , Hélène Delpech  1 , Morgane Broyon  1   2 , Charles Theillet  1 , Pierre-Emmanuel Colombo  3 , Nadia Vie  1 , Diego Tosi  1   3 , Celine Gongora  1 , Lakhdar Khellaf  3 , Marta Jarlier  3 , Nina Radosevic-Robin  4 , Thierry Chardès  1 , André Pèlegrin  1 , Christel Larbouret  1
Affiliations

Anti-tumoral activity of the Pan-HER (Sym013) antibody mixture in gemcitabine-resistant pancreatic cancer models

Emilia Rabia et al. MAbs. 2021 Jan-Dec.

Abstract

Chemoresistance, particularly to gemcitabine, is a major challenge in pancreatic cancer. The epidermal growth factor receptor (EGFR) and human epidermal growth factor receptors 2 and 3 (HER2, HER3) are expressed in many tumors, and they are relevant therapeutic targets due to their synergistic interaction to promote tumor aggressiveness and therapeutic resistance. Cocktails of antibodies directed against different targets are a promising strategy to overcome these processes. Here, we found by immunohistochemistry that these three receptors were co-expressed in 11% of patients with pancreatic adenocarcinoma. We then developed gemcitabine-resistant pancreatic cancer cell models (SW-1990-GR and BxPC3-GR) and one patient-derived xenograft (PDX2846-GR) by successive exposure to increasing doses of gemcitabine. We showed that expression of EGFR, HER2 and HER3 was increased in these gemcitabine-resistant pancreatic cancer models, and that an antibody mixture against all three receptors inhibited tumor growth in mice and downregulated HER receptors. Finally, we demonstrated that the Pan-HER and gemcitabine combination has an additive effect in vitro and in mice xenografted with the gemcitabine-sensitive or resistant pancreatic models. The mixture of anti-EGFR, HER2 and HER3 antibodies is a good candidate therapeutic approach for gemcitabine-sensitive and -resistant pancreatic cancer.

Keywords: EGFR; HER2; HER3; Pan-Her; chemoresistance; gemcitabine; pancreatic cancer.

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Figures

Figure 1.
Figure 1.
Co-expression of EGFR, HER2 and HER3 in pancreatic cancer models. a. Western blot analysis of EGFR, HER2 and HER3 expression in the BxPC-3, SW1990, CFPAC and HPAC pancreatic cancer cell lines, two cell lines derived from the PDX P7054, P4604 (C-PDX), and the PDX P2846, using the relevant antibodies. GAPDH served as loading control. Quantification of each protein band was normalized to GAPDH. b. EGFR, HER2 and HER3 expression was assessed by IHC in 45 FFPE tumor samples from patients with pancreatic cancer. The number and the percentage of positive (Pos) tumor samples are shown. c. Examples of EGFR, HER2 and HER3 positive and negative cases
Figure 2.
Figure 2.
Development of gemcitabine-resistant pancreatic cancer models. a. Gemcitabine sensitivity of the gemcitabine-resistant (GR; gray) and parental (sensitive; wt; black) pancreatic cancer cell lines HPAC, CFPAC, BxPC3 and SW1990 was assessed in vitro using the MTS assay. Cells were plated in 96-well plates in complete medium and incubated with increasing doses of gemcitabine for 5 days (n = 3). The IC50 of each cell line is indicated in the graphs. b. Gemcitabine sensitivity of BxPC3-GR and SW1990-GR cells were tested in vivo in xenografted mice. Mice harboring 200 mm3 BxPC3-GR and SW1990-GR cell xenografts were treated with 100 mg/kg gemcitabine, or vehicle, twice per week for 3 weeks. Mice were euthanized when tumors reached 1500 mm3 and Kaplan-Meier curves were computed. The treatment period is highlighted in blue. c. In vivo generation of the P2846-GR. Two mice (8363 and 8364) were xenografted with the PDX P2846 and treated with two cycles of 50 mg/kg gemcitabine followed by two cycles of 100 mg/kg gemcitabine until tumors continued to grow during treatment with 100 mg/kg gemcitabine. Tumor volume was checked throughout the experiment. d. Gemcitabine sensitivity of PDX P2846-GR and parental PDX 2846 was tested in vivo in xenografted mice. Mice were treated with 100 mg/kg of gemcitabine, or vehicle, twice per week for 3 weeks. Mice were euthanized when tumors reached 1500 mm3 and Kaplan-Meier curves were computed. The treatment period is highlighted in blue
Figure 3.
Figure 3.
Expression of EGFR, HER2, HER3 and their ligands in gemcitabine-resistant pancreatic cancer models. a. Western blot analysis of EGFR, HER2 and HER3 expression in the gemcitabine-resistant BxPC3-GR and SW1990-GR and parental (wt) cancer cell lines, using the relevant antibodies. b. Western blot analysis of EGFR, HER2 and HER3 expression in the gemcitabine-resistant P2846-GR and parental P2846 (wt) PDX, using the relevant antibodies. GAPDH served as the loading control. Quantification of each protein band was normalized to GAPDH or β tubulin and then represented as fold relative to WT cells band. c. Immuno fluorescence analysis of EGFR, HER2 and HER3 expression in PDX P2846 WT and GR. The pictures are representative image (X40; X80). Quantification of fluorescence (for HER receptors) was done by ImageJ (4 randomly images/tumors) on four different tumors of each condition and normalized with the fluorescence of DAPI (nucleus). FI (fluorescence intensity) represents a mean average of fluorescence of 4 randomly images per tumors. d. Q-PCR analysis of EGFR, HER2, HER3 and EGF and NRG1 expression in SW1990 wt and SW1990-GR cells (upper panel), BxPC3 wt and BxPC3-GR cells (lower panel) and PDX P2846 WT and GR (upper panel). Concentrations were then normalized to the concentration of the housekeeping gene 18S rRNA, and expressed relatively to untreated samples
Figure 4.
Figure 4.
In vitro effect of Pan-HER on cell proliferation of gemcitabine-resistant pancreatic cancer cell lines. a. Gemcitabine-resistant SW1990-GR and parental SW1990 cells, b. Gemcitabine-resistant BxPC3-GR and parental BxPC3 cells, and c the C-PDX P4604 and P7054 were plated in 96-well plates in complete medium and incubated with increasing doses of Pan-HER (0.1 to 100 μg/ml) (n = 3) for 5 days. Cell proliferation was analyzed using the MTS assay. Results are indicated as the percentage of cell relative to untreated cells
Figure 5.
Figure 5.
In vivo effect of Pan-HER on tumor growth of gemcitabine-resistant pancreatic cancer cell xenografts and PDX. Mice were xenografted with gemcitabine-resistant SW1990-GR (a), BxPC3-GR (b) and PDX 2846-GR (c). d The parental gemcitabine-sensitive PDX P2846 was used as a control. When tumors reached 150 mm3, xenografted mice (n = 10/group) were treated with 50 mg/kg Pan-HER (gray curve) or vehicle (black curve) twice per week for 3 weeks. Tumor size was measured throughout the experiment (upper panel) and Kaplan Meyer curves were computed (lower panels). The treatment period is highlighted in blue
Figure 6.
Figure 6.
Effect of Pan-HER on EGFR, HER2 and HER3 expression in PDX P2846 WT and GR. a. HER receptors expression was analyzed by immunofluorescence on tumors treated with Pan-HER or the vehicle. The photos are representative image (X40; X80). b. Expression quantification was done by ImageJ on four tumors for each condition of treatments
Figure 7.
Figure 7.
Effect of the Pan-HER plus gemcitabine combination on pancreatic cancer models. a In vitro interaction of gemcitabine and Pan-HER. BxPC3, C-PDX P4604 and SW1990 cells were exposed to the Pan-HER + gemcitabine combination at the indicated concentrations for five days. Cell proliferation was measured using the SRB colorimetric assay (n = 3). Results are presented as the percentage of proliferating cells relative to untreated cells (proliferation matrices; left panel). The interaction (synergy vs additivity) between the tested drugs was investigated using dose-response matrices (right panel). In the blue matrices, values indicate the percentage of surviving cells. In the synergism matrice, the green color indicates antagonist combinations, the red the synergistic combination and the black cells show additive combinations. b In vivo effect of the gemcitabine and Pan-HER combination on tumor growth in mice xenografted with C-PDX P4604 (left panels) and SW1990 cells. Mice (n = 10/group) were treated with Pan-HER (25 mg/kg) or/and gemcitabine (50 mg/kg) twice per week for 3 weeks. Tumor growth (upper panels) was monitored and Kaplan Meyer curves (middle panels) were computed at the end of experiment. Survival statistics are in the table (lower panel)
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This work was supported by the Programme Investissement d’Avenir (grant agreement: Labex MabImprove, ANR-10-LABX-53-01) and ANR DUAL PancHER and the “Agence Nationale de la Recherche” (grant agreement: ANR-16-CE17-002).