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. 2022 Dec 13;23(24):15820.
doi: 10.3390/ijms232415820.

Expanding the Therapeutic Window of EGFR-Targeted PE24 Immunotoxin for EGFR-Overexpressing Cancers by Tailoring the EGFR Binding Affinity

Sei-Yong Jun  1 , Dae-Seong Kim  1 , Yong-Sung Kim  1   2
Affiliations

Expanding the Therapeutic Window of EGFR-Targeted PE24 Immunotoxin for EGFR-Overexpressing Cancers by Tailoring the EGFR Binding Affinity

Sei-Yong Jun et al. Int J Mol Sci. .

Abstract

Immunotoxins (ITs), which are toxin-fused tumor antigen-specific antibody chimeric proteins, have been developed to selectively kill targeted cancer cells. The epidermal growth factor receptor (EGFR) is an attractive target for the development of anti-EGFR ITs against solid tumors due to its overexpression on the cell surface of various solid tumors. However, the low basal level expression of EGFR in normal tissue cells can cause undesirable on-target/off-tumor toxicity and reduce the therapeutic window of anti-EGFR ITs. Here, based on an anti-EGFR monobody with cross-reactivity to both human and murine EGFR, we developed a strategy to tailor the anti-EGFR affinity of the monobody-based ITs carrying a 24-kDa fragment of Pseudomonas exotoxin A (PE24), termed ER-PE24, to distinguish tumors that overexpress EGFR from normal tissues. Five variants of ER-PE24 were generated with different EGFR affinities (KD ≈ 0.24 nM to 104 nM), showing comparable binding activity for both human and murine EGFR. ER/0.2-PE24 with the highest affinity (KD ≈ 0.24 nM) exhibited a narrow therapeutic window of 19 pM to 93 pM, whereas ER/21-PE24 with an intermediate affinity (KD ≈ 21 nM) showed a much broader therapeutic window of 73 pM to 1.5 nM in in vitro cytotoxic assays using tumor model cell lines. In EGFR-overexpressing tumor xenograft mouse models, the maximum tolerated dose (MTD) of intravenous injection of ER/21-PE24 was found to be 0.4 mg/kg, which was fourfold higher than the MTD (0.1 mg/kg) of ER/0.2-PE24. Our study provides a strategy for the development of IT targeting tumor overexpressed antigens with basal expression in broad normal tissues by tailoring tumor antigen affinities.

Keywords: PE24 toxin; affinity variants; anti-EGFR monobody; immunotoxin; on-target/off-tumor toxicity; therapeutic index; therapeutic window.

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Conflict of interest statement

The authors declare that they have no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Generation and characterization of ER-PE24 IT variants with different affinities for EGFR. (A) Schematic view of the ER-PE24 IT construction (lower panel), and predicted structure via AlphaFold (AlphaFold v.2.0) (upper panel). The anti-EGFR monobody (Green) was fused to the N-terminus of the B-cell epitope-removed PE24 toxin, LO10 PE24 (Cyan), via a GGS linker. (B) SDS-PAGE analyses of the purified ER-PE24 ITs (each 5 μg loading) under non-reducing conditions. The gel was stained with Coomassie Blue R-250. (C) Representative binding isotherms of the immobilized EGFR-ECD-Fc in relation to soluble ER-PE24 IT, as measured by bio-layer interferometry. The concentrations of ER-PE24 IT analyzed are indicated using different colors. The binding kinetics parameters are listed in Table 1. (D) Concentration-dependent binding activities of ER-PE24 ITs to human or murine EGFR-ECD, as determined by ELISA. Error bars represent the mean ± SEM (n = 3).
Figure 2
Figure 2
Cell surface binding activities of ER-PE24 ITs based on the affinity for EGFR and EGFR expression levels on cells. (A) Representative histograms showing EGFR expression levels on the surface of indicated cell lines determined using flow cytometry. Negative control was used only with the secondary antibody (2nd Ab only) without the anti-EGFR primary antibody. The relative mean fluorescence intensity (MFI) was calculated by comparison with the MFI of negative control and is presented in each histogram. (B–D) Concentration-dependent cell surface binding activities of ER-PE24 ITs to three different cell lines, EGFR high-expressing A431 cells (B), EGFR low-expressing HT29 cells (C), and EGFR negative SW620 cells (D), determined by flow cytometry. The relative MFI was calculated by comparison with the MFI measured with or without ER-PE24 (indicated by the dashed black line). In (B–D), each value represents the mean ± SEM (n = 3).
Figure 3
Figure 3
ER-PE24 ITs have different therapeutic windows in vitro depending on the anti-EGFR affinity. (A) In vitro cell cytotoxicity assay of ER-PE24 variants in EGFR high-expressing A431 cells, EGFR low-expressing HT29 cells, and EGFR negative SW620 cells. Each cell line was cultured with the indicated ER-PE24 ITs at various concentrations for 72 h, before the determination of cell viability using the CellTiter-Glo assay. Error bars represent the mean ± SD (n = 3). (B) The therapeutic index of each ER-PE24 was calculated by dividing [IC50 against HT29 cells] by [IC50 against A431 cells]. (C) The therapeutic window of each ER-PE24 was estimated by the range between the IC20 values against A431 and HT29, i.e., [IC20 against A431 cells]–[IC20 against HT29 cells]. The values of the therapeutic index and therapeutic window of each ER-PE24 variant are listed in Table 2.
Figure 4
Figure 4
ER/21-PE24 with an intermediate EGFR affinity has a much broader therapeutic window than ER/0.2-PE24 with the highest EGFR affinity. (A,B) Tumor growth (left), mouse body weight (middle), and survival curves (right), as measured during treatment of A431 tumor-bearing mice (TBM) initiated at the tumor volume of ~100 to 120 mm3 with either i.v. injection of ER/0.2-PE24 (A) or ER/21-PE24 (B) at a dose of 0.1, 0.2, 0.4, and 0.6 mg/kg every other day (indicated by red arrows). The vehicle indicates PBS buffer as control. If TBM death occurs in more than 2 TBMs from each regimen, treatment was stopped, as indicated by the arrow in the panel. Symbols and error bars represent the mean ± SEM. Data were pooled from two independent experiments with four mice per group. (C) Serum levels of liver enzymes (ALT and AST) were measured in each group of TBM at the endpoint of treatment. Each symbol represents the value obtained from individual mice, and the bar graphs present the mean ± SD (n = 5 per group). In (A–C), **, p < 0.01; ****, p < 0.0001 versus the vehicle-treated TBM. ns, not significant. (D) Hematoxylin and eosin (H&E) staining of representative tissue sections from the liver and kidney of each group of TBM was performed at the end of treatment. Abnormal histological features suggestive of hepatic toxicity or renal toxicity are indicated using arrows of different colors [Inflammatory cell infiltration (red), tubular necrosis (green), interstitial hemorrhage (blue)]. Magnification, ×200; scale bar, 100 μm.
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