Nanobody-Based EGFR-Targeting Immunotoxins for Colorectal Cancer Treatment

Abstract

Immunotoxins (ITXs) are chimeric molecules that combine the specificity of a targeting domain, usually derived from an antibody, and the cytotoxic potency of a toxin, leading to the selective death of tumor cells. However, several issues must be addressed and optimized in order to use ITXs as therapeutic tools, such as the selection of a suitable tumor-associated antigen (TAA), high tumor penetration and retention, low kidney elimination, or low immunogenicity of foreign proteins. To this end, we produced and characterized several ITX designs, using a nanobody against EGFR (V_HH 7D12) as the targeting domain. First, we generated a nanoITX, combining V_HH 7D12 and the fungal ribotoxin α-sarcin (αS) as the toxic moiety (V_HHEGFRαS). Then, we incorporated a trimerization domain (TIE^XVIII) into the construct, obtaining a trimeric nanoITX (TriV_HHEGFRαS). Finally, we designed and characterized a bispecific ITX, combining the V_HH 7D12 and the scFv against GPA33 as targeting domains, and a deimmunized (DI) variant of α-sarcin (BsITXαSDI). The results confirm the therapeutic potential of α-sarcin-based nanoITXs. The incorporation of nanobodies as target domains improves their therapeutic use due to their lower molecular size and binding features. The enhanced avidity and toxic load in the trimeric nanoITX and the combination of two different target domains in the bispecific nanoITX allow for increased antitumor effectiveness.

Keywords: antibody engineering; antitumor efficacy; colorectal cancer; immunotoxin; nanobody; α-sarcin.

Figure 1

Schematic diagrams showing the genetic and protein domain arrangements of the nanoITXs V_HHEGFRαS (a), TriV_HHEGFRαS (b), and BsITXαSDI (c). The cDNA constructions appear on the left side of the figure. The schematic representation of the protein motifs of the nanoITXs appears on the right side of the figure, with its molecular size underneath. In both cases, structural and functional domains are highlighted with different colors: α-factor secretion signal peptide (α, yellow), V_HHEGFR (corresponds to V_HH 7D12 [45]) (red), scFvA33 (blue), the 21 aa flexible linkers (gray), the trimerization domain derived from the collagen XVIII (TIE^VIII, orange), the wild-type α-sarcin (αSWT, purple), the non-immunogenic α-sarcin (αSDI, green), and histidine-tag (Histag, black).

Figure 2

Coomassie-blue-stained SDS-PAGE analysis after affinity purification of V_HHEGFRαS (a), TriV_HHEGFRαS (b), and BsITXαSDI (c) and Coomassie blue staining and Western blot analysis (WB) of the purified final fraction of V_HHEGFRαS (d), TriV_HHEGFRαS (e), and BsITXαSDI (f). Western blot analysis was carried out using rabbit anti-α-serum. Notes in gels correspond to the following: MW, prestained molecular weight standard (kDa); VNR, not retained fraction; NaP, washed fraction eluted with sodium phosphate buffer; Imidazole 20 mM, washed fraction eluted with sodium phosphate buffer containing imidazole 20 mM; and different 1 mL fractions eluted with 250 mM imidazole. The original full-length gels and uncropped Western Blot images can be found in Figures S1 and S2.

Figure 3

Structural characterization by far-UV circular dichroism (CD) spectra of V_HHEGFRαS (a), TriV_HHEGFRαS (b), and BsITXαSDI (c). θMRW represents the mean residue weight ellipticities as degree × cm² × dmol⁻¹. The thermal denaturation profiles of V_HHEGFRαS (d) and BsITXαSDI (f) by means of the temperature dependence of the ellipticity at 220 nm. All spectra were carried out at a protein concentration of 0.15 mg/mL in 50 mM sodium phosphate, 0.1 M NaCl, and pH 7.4. Analysis of the trimeric nature of TriV_HHEGFRαS by Superdex 200 FPLC chromatography analysis (e). The eluted protein shows a major symmetric elution peak at the expected volume corresponding to its trimeric size (126 kDa), with the indicated molecular weight measured at the center of the chromatography peak (red curve).

Figure 4

In vitro functional characterization. The ribonucleolytic activity of the toxic domain of V_HHEGFRαS and TriV_HHEGFRαS (a), and BsITXαSDI (b). The arrow indicates the release of the α-fragment, produced by the cleavage of the SRL due to the α-sarcin. In both gels, 2, 6, and 12 pmoles of all three nanoITXs were assayed. C+ represents 2 pmoles of fungal wild-type α-sarcin, whereas in C-, the protein sample was replaced by a buffer. Images were acquired and analyzed using the Gel Doc XR Imaging System and the Quantity One software (BioRad). (c) Quantitation of specific ribonucleolytic activity of the three nanoITXs, expressed as a percentage of α-fragment/RNA 18S ratio, considering 100% to be the ratio obtained by 2 pmol of α-sarcin. Band intensities were quantitated with Quantity One software. The original full-length gels can be found in Figure S3.

Figure 5

In vitro functional characterization of binding activity. ELISA assay (a) against immobilized EGFR (0.5 μg/well), using the three nanoITXs and V_HHEGFR as a positive control (1 µM). Flow cytometry binding assays of V_HHEGFRαS (b) and TriV_HHEGFRαS (c) to EGFR-positive cells (A431 cell line). Curves correspond to cells incubated with a secondary antibody anti-His-Alexa488 (black), 10 nM (red), 100 nM (green), or 1 μM (blue) of each immunotoxin. (d) Comparison between 10 nM of V_HHEGFRαS (purple) or TriV_HHEGFRαS (yellow) in the flow cytometry assay. Binding of BsITXαSDI to EGFR-positive cells (A431) (e) or GPA33-positive cells (SW1222) (f). Curves correspond to cells incubated with a secondary antibody anti-His-Alexa488 (black), 10 nM (red), 100 nM (green), or 1 μM (blue).

Figure 6

In vitro cytotoxicity characterization by MTT viability assays. (a) EGFR-positive A431 cells treated with V_HHEGFRαS for 24, 48, and 72 h; (b) EGFR-positive A431 cells treated for 72 h with TriV_HHEGFRαS (black) compared to V_HHEGFRαS (white); (c) EGFR-positive A431 cells treated for 72 h with BsITXαSDI (black) compared to V_HHEGFRαS (white) and (d) GPA33-positive cells SW1222 treated with BsITXαSDI (black) and scFv-IMTXA33αSDI (white). Measurements were analyzed and plotted (mean ± SD) against untreated controls. In all cases, triplicate samples were carried out. IC₅₀ values were obtained as the protein concentration leading to 50% viability.

Figure 7

In vivo antitumoral activity. (a) Time course of tumor volume growth of SW1222-derived xenografts. Mice were non-treated with PBS (black) or treated with two different doses (25 or 50 µg) of V_HHEGFRαS (labeled in the graph as 25, blue or 50, red). The different doses were administered each 48 h. The vertical dashed line indicates the end of the administration of the antitumoral treatment. Values are represented as means ± sem (standard error of the mean). (b) Kaplan–Meier survival curves. The Kaplan–Meier representation expresses the time to the experimental endpoint (once tumor volume reaches 2000 mm³ of the in vivo assay). The labels in the graph are the same as those used in (a). (c) Statistical analysis of V_HHEGFRαS 25- and V_HHEGFRαS 50-treated tumors compared with vehicle-treated tumors at day 17. In all cases, the experimental groups were composed of 5 mice (n = 5).