NGR (Asn-Gly-Arg)-targeted delivery of coagulase to tumor vasculature arrests cancer cell growth

Abstract

Induction of selective thrombosis and infarction in tumor-feeding vessels represents an attractive strategy to combat cancer. Here we took advantage of the unique coagulation properties of staphylocoagulase and genetically engineered it to generate a new fusion protein with novel anti-cancer properties. This novel bi-functional protein consists of truncated coagulase (tCoa) and an NGR (GNGRAHA) motif that recognizes CD13 and αvβ3 integrin receptors, targeting it to tumor endothelial cells. Herein, we report that tCoa coupled by its C-terminus to an NGR sequence retained its normal binding activity with prothrombin and avβ3 integrins, as confirmed in silico and in vitro. Moreover, in vivo biodistribution studies demonstrated selective accumulation of FITC-labeled tCoa-NGR fusion proteins at the site of subcutaneously implanted PC3 tumor xenografts in nude mice. Notably, systemic administration of tCoa-NGR to mice bearing 4T1 mouse mammary xenografts or PC3 human prostate tumors resulted in a significant reduction in tumor growth. These anti-tumor effects were accompanied by massive thrombotic occlusion of small and large tumor vessels, tumor infarction and tumor cell death. From these findings, we propose tCoa-NGR mediated tumor infarction as a novel and promising anti-cancer strategy targeting both CD13 and integrin αvβ3 positive tumor neovasculature.

In Vivo Tumor Growth Inhibition and Antiangiogenic Effect of Cyclic NGR Peptide-Daunorubicin Conjugates Developed for Targeted Drug Delivery

“…The effect of conjugates on blood vessel density was higher than on proliferation index. Functional antagonists of CD13/APN inhibit capillary tube formation [22] interfering with highly expressed CD13 on intratumoral blood vessels [10,16,25,26], suppressing the nutrient supply necessary for tumor cell viability and tumor proliferation which inhibition occurs in later stages [35,80].…”

“…For several years, many researches have been conducted to identify molecules that interact with receptors expressed in angiogenic vessels [10][11][12][13][14][15] that play a crucial role in tumor progression. Consequently, two important targets have been found on the new tumor vasculature; integrins and Aminopeptidase N (APN or CD13) [16]. CD13 is an ectoenzyme of 150-240 kDa [17][18][19][20] belonging to the family of zinc metallopeptidases with numerous functions: regulation of hormones and cytokines, cell proliferation, migration and invasion [21].…”

Modulating the tumor microenvironment with new therapeutic nanoparticles: A promising paradigm for tumor treatment

“…The tumor‐homing peptide‐directed iron oxide nanoparticles were further developed to achieve a more effective and safe tumor infarction . In a more recent in vivo study using a novel fusion protein consisting of truncated coagulase and an Asn‐Gly‐Arg (Gly‐Asn‐Gly‐Arg‐Gla‐His‐Gla) motif that recognizes tumor endothelial markers CD13 and α v β 3 integrin receptors to induce tumor infarction, a massive thrombotic occlusion of small and large tumor vessels and subsequent tumor reduction was observed . This provides the further possibility for vessel infarction strategy for potential clinical application.…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Also, CFPS systems employing linear DNA as expression templates (LET) eliminate the need for obligatory cloning steps of previous plasmid‐based methods. The linear template degradation which is problematic without substantial intervention, is fairly common in cell‐free systems, as well (Jahanban‐Esfahlan et al, 2017; Seidi et al, 2018). However, LET‐based CFPS is a practical instrument for high‐throughput studies and screenings of engineered proteins (Schinn, Broadbent, Bradley, & Bundy, 2016).…”