Combination of antibody targeting and PTD-mediated intracellular toxin delivery for colorectal cancer therapy

Abstract

The bottlenecks of current chemotherapy in the treatment of colorectal cancer lie in the ineffectiveness of the existing anti-cancer small molecule drugs as well as the dose-limiting toxicity caused by the nonselective action on normal tissues by such drugs. To address these problems, we introduce a novel therapeutic strategy based on tumor targeting using a non-internalizing anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) and intracellular delivery of the extremely potent yet cell-impermeable protein toxin gelonin via the aid of a cell-penetrating peptide (also termed as protein transduction domain; PTD). A chimeric TAT-gelonin fusion protein was genetically engineered, and it displayed remarkably enhanced anti-cancer activity against human colorectal cancer cells, with IC50 values being several orders of magnitude lower than the unmodified gelonin. On the other hand, a chemically synthesized conjugate of heparin and a murine anti-CEA mAb, T84.66 (termed T84.66-Hep) was found able to bind highly specifically to CEA over-expressing LS174T colorectal cancer cells. When mixing together, TAT-gelonin and T84.66-Hep could associate tightly and automatically through an electrostatic interaction between the cationic TAT and anionic heparin. In preliminary in vivo studies using LS174T s.c. xenograft tumor bearing mouse, selective and significantly augmented (58-fold) delivery of TAT-gelonin to the tumor target was observed, when compared with administration of TAT-gelonin alone. More importantly, efficacy studies also revealed that only the TAT-gelonin/T84.66-Hep complex yielded a significant inhibition of tumor growth (46%) without causing gelonin-induced systemic toxicity. Overall, this study suggested a generic strategy to effectively yet safely deliver potent PTD-modified protein toxins to the tumor.

Cell-penetrating peptides: strategies for anticancer treatment

“…This approach was further examined in a LS174T-xenograft model, which resulted in increased intratumoral accumulation and inhibition of tumor growth (46%). This finding demonstrates the potential usefulness of a CPP for enhancing antibody-promoted active targeting [56].…”

“…For example, the TAT-conjugated plant-derived protein toxin geolin binds to a conjugate of heparin (Hep) and a murine anticarcinoembryonic antigen monoclonal antibody (anti-CEA mAb, T84.66) through an electrostatic interaction between TAT and Hep [56]. This complex, when attached to CEA-overexpressing LS174T colorectal cancer cells, slowly released TAT-geolin, which was then internalized by the cancer cells.…”

Molecular tumor targeting of gelonin by fusion with F3 peptide

“…As a fusion partner, TRX is a small 12-kDa redox protein that can be overexpressed to a high level in E coli (accumulation of up to 40% of the cellular proteins) in a soluble form [28] . The utility of TRX as a fusion partner for soluble expression of gelonin fusion proteins has already been reported in our previous publications [24,29] . Herein, we have clearly demonstrated the plausibility of this strategy, as the N-terminal thioredoxin-6×His tagged-gelonin-F3 peptide chimera (F3-Gel) was successfully produced in large quantities as a soluble protein in E coli.…”

PTD-Modified ATTEMPTS for Enhanced Toxin-based Cancer Therapy: An In Vivo Proof-of-Concept Study

“…To this regards, in this research, we explored the feasibility of applying the PTD-modified ATTEMPTS for effective yet safe administration of TAT-Gel, by utilizing a heparin functionalized anti-carcinoembryonic antigen (CEA) mAb, T84.66-Hep that showed feasible tumor targeting of TAT-Gel in our previous studies (24), as the targeting component and protamine as the trigger release agent. After in vitro characterization of the CEA binding specificity and cell internalization of T84.66-Hep, we prepared an antibody/toxin complex by mixing the T84.66-Hep and TAT-Gel.…”