Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy

Abstract

Human tumors, including gastric cancer, frequently express high levels of epidermal growth factor receptors (EGFRs), which are associated with a poor prognosis. Targeted delivery of anticancer drugs to cancerous tissues shows potential in sparing unaffected tissues. However, it has been a major challenge for drug penetration in solid tumor tissues due to the complicated tumor microenvironment. We have constructed a recombinant protein named anti-EGFR-iRGD consisting of an anti-EGFR VHH (the variable domain from the heavy chain of the antibody) fused to iRGD, a tumor-specific binding peptide with high permeability. Anti-EGFR-iRGD, which targets EGFR and αvβ3, spreads extensively throughout both the multicellular spheroids and the tumor mass. The recombinant protein anti-EGFR-iRGD also exhibited antitumor activity in tumor cell lines, multicellular spheroids, and mice. Moreover, anti-EGFR-iRGD could improve anticancer drugs, such as doxorubicin (DOX), bevacizumab, nanoparticle permeability and efficacy in multicellular spheroids. This study draws attention to the importance of iRGD peptide in the therapeutic approach of anti-EGFR-iRGD. As a consequence, anti-EGFR-iRGD could be a drug candidate for cancer treatment and a useful adjunct of other anticancer drugs.

Three‑dimensional cell culture: A powerful tool in tumor research and drug discovery (Review)

“…Thus, 3D culture may be a powerful tool in tumor and relevant drug research. Marked advances have been made in the basic development of 3D tumor models so far, and prominent studies using 3D tumor cell models to simulate the tumor microenvironment or assess drug delivery in the past 5 years are summarized in Table I (6,8,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Considering the advantages of 3D tumor models, the present review provides an overview of the methods and techniques successfully devised for practicing 3D tumor cell culture.…”

A tumor-penetrating recombinant protein anti-EGFR-iRGD enhance efficacy of paclitaxel in 3D multicellular spheroids and gastric cancer in vivo

“…Antibodies were obtained from Cell Signaling Technology (USA) (for human EGFR and GAPDH antibody). Recombinant proteins anti-EGFR and anti-EGFR-iRGD were prepared as reported previously (Sha et al, 2014). By conjugating at amine groups of lysine, protein was labeled with Fluorescein isothiocyanate (FITC).…”

“…In the previous work, BGC-823 cell line was used to culture the 3D tissue model MCS (Sha et al, 2014). In this study, BGC-823 MCS were exposed to culture medium, recombinant protein alone (100 lg/mL), anti-tumor drugs alone (PTX 4 lg/mL, 5-FU 50 mM, CPT-11 20 mM), antitumor drugs delivered together with protein (100 lg/mL) for 24 h, and then MCS were incubated with fresh culture medium (changed everyday) for another 6 days.…”

“…There are several kinds of small-molecule tyrosine kinase inhibitors (TKIs), such as gefitinib (Iressa), erlotinib (Tarceva) and lapatinib (Tykerb). We previously constructed and purified a bispecific high permeable protein anti-EGFR-iRGD (Sha et al, 2014). Recombinant protein anti-EGFR-iRGD could target both EGFR extracellular domain and integrin avb3/b5, and at the same time, it also had high penetration and could improve other drugs (from 0.6 kDa to 100 nm) penetration into deep zone of gastric cancer 3D multicellular spheroids (MCS).…”

Design and Modification of Anticancer Peptides

“…Then it can be proteolytically cleaved to expose the CRGDK/R sequence on the C-terminal, named C-end Rule (CendR) motif (R/KXXR/K), which has the high affinity to the receptor of neuropilin-1 (NRP-1) and triggers tissue penetration [70]. iRGD has been extensively used as a targeting delivery and penetration tools for nanoparticles [71], peptides [72], monoclonal antibody [73], etc., by chemical conjugated or co-administration [70]. iRGD is one of the most widely used THPs to modify many kind of ACPs, like apoptotic peptide D (KLA) 2 , ATAP, CDD, Thymopentin, TP5, etc.…”