Anti-EGF Nbs single agent and combination with osimertinib potentiates cell viability and colony formation inhibition
To determine the antitumoral activity of anti-EGF Nbs (Nb1 and Nb6), we used human lung adenocarcinoma PC-9 cells, which harbor a Glu746-Ala750 deletion in exon 19, as well as a PC-9 osimertinib-resistant cell line (PC-9-OR4), which was generated by stepwise long-term exposure to the drug [21]. Both cell types were treated for 72 h with the anti-EGF Nbs, in a dose-dependent manner to assess their effect on cellular viability. As shown in Fig. 1, Nb1 and Nb6 cause a small decrease in the viability of PC-9 cells, while their effect on osimertinib-resistant cells is negligible. Next, we assessed whether our anti-EGF Nbs could overcome intrinsic resistance to osimertinib in PC-9 and PC-9-OR4 cells, by combining different doses of osimertinib combined with Nb1 or Nb6. In these assays, the combination treatment resulted in a reduced cellular viability, which was more pronounced for the PC-9 cell line. Among the two antibodies, Nb1 exerted a more potent effect than Nb6, in agreement with their relative binding affinity and especially with their on-target residence time, which is significantly longer for Nb1 [17]. At concentrations below the half-maximal inhibitory concentration (IC50) of each drug (eg. 10 nM of osimertinib plus 10 µM of Nb1), a significant effect on cell viability can be observed. In PC-9-OR4 cells, however, the EGF-withdrawal effect of the Nbs is not able to fully rescue the sensitivity of PC-9 cells to osimertinib.
To study if the effect on cell viability translates into a decreased colony formation capacity, we performed clonogenic assays with PC-9 and PC-9-OR4 cells. As shown in Fig. 2, the Nbs alone did not have a significant effect on colony formation, in agreement with the cell viability results. In contrast, a dose-dependent reduction in colony formation was observed for the combined treatment, showing a clear effect at the dose of 10 µM of Nb1/Nb6 Nbs. Thus, the combination treatment reduces the formation of new colonies to a greater extent than a single treatment strategy.
EGFR signaling pathway is inhibited by the combination of anti-EGF Nbs and osimertinib
To assess the effects of anti-EGF Nbs in expression and activation of proteins related to EGFR-dependent osimertinib resistance [22], Western blotting analyses were conducted. In PC-9 and PC-9-OR4 cells treated with anti-EGF Nbs, a dose-dependent attenuation of EGFR phosphorylation was observed, as well as of downstream molecules Akt and Erk1/2 (Fig. 3). In agreement with the effects observed in the cell viability assay, the combination of osimertinib and Nbs produced a more pronounced effect that suppressed EGFR, Akt and Erk1/2 phosphorylation.
Anti-EGF nanobody treatment modulate the regulation of the expression of proteins involved in osimertinib-resistance
Since the combination of anti-EGF Nbs and osimertinib blocks the phosphorylation of protein kinases downstream of EGFR, which are involved in osimertinib-resistance mechanisms, we aimed at studying the capacity of these Nbs to change the expression of several proteins involved in cell cycle regulation, cancer stem cell (CSC) development and apoptosis, and compared by Western blot the effects of anti-EGF Nbs alone plus osimertinib combination.
To this aim, we selected Nb1, since it shows a more potent and lasting effect on the tumor cells. In PC-9 cells, we observed that Nb1 alone produced a concentration-dependent decrease in the expression of p21, similar to that observed upon osimertinib treatment. A similar reduction was observed for Hes1, a key signature protein that is involved in maintaining quiescent cells and CSCs in a nondividing state. In addition, Nb1 enhanced the expression of the cell-cycle-related protein E2F, which is essential for cell cycle progression. At the higher dose of Nb1, a subtle decrease of AXL was observed, while no effects were shown for the CSC marker Bmi1, nor for the cell cycle protein Rb (Fig. 4). These effects were similar, albeit milder, in PC-9-OR4 cells.
We further assessed that the combination of osimertinib and anti-EGF Nbs combination caused a stronger effect in the expression of several of these proteins. Osimertinib was shown to increase p21 expression, thus confirming its inhibitory effect on cell cycle progression mediated via the inhibition of p53-p21-Cyclin D1 pathway [23]. However, the addition of Nb1 significantly dampened this inhibitory effect. On the other hand, the combination treatment caused a major reduction in the expression levels of Hes1 (Fig. 4), while E2F levels were increased in PC-9 cells treated with the combination. Finally, a clear increment in the cleaved PARP form was observed, which might indicate that the addition of the anti-EGF Nb potentiates the apoptotic effect of osimertinib (Fig. 4).
Anti-EGF Nbs delay the emergence of resistance to osimertinib
Activation of the EGFR pathway is one of the mechanisms associated with intrinsic and acquired resistance to osimertinib. In consequence, we decided to investigate the effects of anti-EGF Nbs on the emergence of resistance to osimertinib in vitro. To this end, low-confluence PC-9 cells growing in 96-well plates were treated with either osimertinib, anti-EGF Nbs, or with the combination treatment. Wells were inspected twice per week and those reaching 50% confluence were scored as positive. We found that the presence of the anti-EGF Nbs significantly delayed the emergence of resistant colonies to osimertinib. In particular, > 60% of the wells with cells treated only with osimertinib reached 50% of confluence at week 16, while only < 40% of wells in the combination treatment group reached the 50% confluence threshold. As in the previous assays, there were no significant differences between Nb1 and Nb6 in the delayed emergence of TKI resistance.