Preclinical evaluation of a COVID-19 vaccine candidate based on a recombinant RBD fusion heterodimer of SARS-CoV-2.

Current COVID-19 vaccines have been associated with a decline in infection rates, prevention of severe disease, and a decrease in mortality rates. However, SARS-CoV-2 variants are continuously evolving, and development of new accessible COVID-19 vaccines is essential to mitigate the pandemic. Here, we present data on preclinical studies in mice of a receptor-binding domain (RBD)-based recombinant protein vaccine (PHH-1V) consisting of an RBD fusion heterodimer comprising the B.1.351 and B.1.1.7 SARS-CoV-2 variants formulated in SQBA adjuvant, an oil-in-water emulsion. A prime-boost immunisation with PHH-1V in BALB/c and K18-hACE2 mice induced a CD4+ and CD8+ T cell response and RBD-binding antibodies with neutralizing activity against several variants, and also showed a good tolerability profile. Significantly, RBD fusion heterodimer vaccination conferred 100% efficacy, preventing mortality in SARS-CoV-2 infected K18-hACE2 mice, but also reducing Beta, Delta and Omicron infection in lower respiratory airways. These findings demonstrate the feasibility of this recombinant vaccine strategy.

A comparison of non-biologically active truncated EGF (EGFt) and full-length hEGF for delivery of Auger electron-emitting 111In to EGFR-positive breast cancer cells and tumor xenografts in athymic mice.

INTRODUCTION: EGFt is a truncated form of human epidermal growth factor (hEGF) that is non-biologically active but retains binding and internalization into EGFR-positive cells. Our aim was to compare EGFt and hEGF for delivery of (111)In to human breast cancer (BC) cells and tumors and evaluate its cytotoxicity against EGFR-positive BC cells, mediated by the Auger electron emissions of (111)In. METHODS: The binding, internalization and nuclear localization of EGFt and hEGF in MDA-MB-468 human BC cells were first assessed by confocal fluorescence microscopy. Subcellular fractionation was then used to quantify the cellular and nuclear uptake of (111)In-EGFt and (111)In-hEGF in MDA-MB-468 cells. The effect of exposure in vitro to (111)In-EGFt or (111)In-hEGF on the clonogenic survival of MDA-MB-468 (10(6) EGFR/cell) or MCF-7 cells (10(4) EGFR/cell) was determined. The pharmacokinetics and tumor and normal tissue biodistribution of (111)In-EGFt was compared to (111)In-hEGF in CD-1 athymic mice with s.c. MDA-MB-468 and MCF-7 tumors. Nuclear importation in MDA-MB-468 tumors was determined ex vivo by subcellular fractionation. RESULTS: Fluorescently-labeled EGFt and hEGF were bound, internalized and localized in the nucleus of MDA-MB-468 cells. Binding of (111)In-EGFt to MDA-MB-468 cells was 8-fold lower than (111)In-hEGF, but nuclear importation as a proportion of cell-bound (111)In was 3.6-fold greater than (111)In-hEGF. Nuclear uptake of (111)In-EGFt was lower than (111)In-hEGF when differences in cell binding were taken into account. The cytotoxicity of (111)In-EGFt (1.0MBq/mL; 10 nmols/L) against MDA-MB-468 cells was 9-fold lower than (111)In-hEGF but only 2-fold lower at a higher concentration (1.85 MBq/mL; 40 nmols/L). (111)In-EGFt and (111)In-hEGF exhibited greater cytotoxicity against MDA-MB-468 cells than MCF-7 cells. (111)In-EGFt was eliminated more slowly from the blood of tumor-bearing mice and exhibited lower liver uptake but higher kidney accumulation. Uptake of (111)In-EGFt in MDA-MB-468 tumors was 2.2-fold lower than (111)In-hEGF, and was blocked by anti-EGFR monoclonal antibody, nimotuzumab. Nuclear uptake into MDA-MB-468 tumor cells was higher for (111)In-EGFt than (111)In-hEGF, but when the lower tumor uptake of (111)In-EGFt was considered, there were no overall differences. CONCLUSION: We conclude that the absence of biological activity of EGFt makes it attractive for delivery of Auger electron-emitting (111)In to EGFR-overexpressing BC, but its lower cellular and tumor uptake would limit its effectiveness compared to (111)In-hEGF. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: (111)In-EGFt may reduce the adverse effects previously observed in patients administered (111)In-hEGF since it is not biologically active, but its lower uptake by BC cells and tumors would limit its effectiveness for treatment of breast cancer.

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

Androgen-independent prostate cancer cells circumvent EGFR inhibition by overexpression of alternative HER receptors and ligands.

The deregulation of the epidermal growth factor receptor (EGFR) pathway plays a major role in the pathogenesis of prostate cancer (PCa). However, therapies targeting EGFR have demonstrated limited effectiveness in PCa. A potential mechanism to overcome EGFR blockade in cancer cells is the autocrine activation of alternative receptors of the human EGFR (HER) family through the overexpression of the HER receptors and ligands. In the present study, we were interested in analyzing if this intrinsic resistance mechanism might contribute to the inefficacy of EGFR inhibitors in PCa. To this end, we selected two androgen-independent human prostate carcinoma cell lines (DU145 and PC3) and established DU145 erlotinib-resistant cells (DUErR). Cells were treated with three EGFR inhibitors (cetuximab, gefinitib and erlotinib) and the sensitivity to each treatment was assessed. The gene expression of the four EGFR/HER receptors and seven ligands of the HER family was analyzed by real-time PCR prior to and after each treatment. The receptors expression and activation were further characterized by flow cytometry and western blot analysis. EGFR inhibition rapidly induced enhanced gene expression of the EGF, betacellulin and neuregulin-1 ligands along with HER2, HER3 and HER4 receptors in the DU145 cells. In contrast, slight changes were observed in the PC3 cells, which are defective in the phosphatase and tensin homolog (PTEN) tumor suppressor gene. In the erlotinib-resistant DUErR cells, the expression of HER2 and HER3 was increased at mRNA and protein levels together with neuregulin-1, leading to enhanced HER3 phosphorylation and the activation of the downstream PI3K/Akt survival pathway. HER3 blockage by a monoclonal antibody restored the cytostatic activity of erlotinib in DUErR cells. Our results confirm that the overexpression and autocrine activation of HER3 play a key role in mediating the resistance to EGFR inhibitors in androgen-independent PCa cells.