Polypeptidic Taxol-Tropins: Targeting paclitaxel to the tumor microenvironment
Authors: M. Gordon Erlinda , Liu Seiya , P. Chawla Sant , L. Hall Frederick 
Published: November 2021

Background and Rationale: Although PTX is widely used as a single chemotherapeutic agent and in various combination regimens, its clinical utility is hindered by acquired drug resistance and serious dose-limiting side effects that result from the ungoverned biodistribution of the taxane. Hypothesis: Conceptually, the precision, validity, and efficiency of paclitaxel delivery to tumor compartments might be substantially improved by “actively targeting” the exposed collagenous (XC-) proteins presented within the tumor microenvironment (TME)—XC-proteins physically exposed by the pathologic biochemical processes of tumor invasion, reactive stroma formation, and neo-angiogenesis. Objective: An adaptive bioengineering approach aims to apply pathotropic tumor-targeting functionality to paclitaxel (PTX), a powerful cytotoxic taxane which exhibits anti-tubulin / anti-mitotic / anti-cancer activities against a broad range of solid tumors. Materials and Methods: Synthetic peptide XC-targeting probes (< 40 aa) and polypeptide aptamers (40 to 53 aa), 85 - 99% purity, were prepared by 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis, purified by high performance liquid chromatography (HPLC), and verified by mass spectrometry and amino acid analysis, and the XC-targeting probes were FITC-labeled. Analysis of fluorescence in XC-binding assays was visualized with an Ultra Bright Blue Light trans-illuminator equipped with an amber filter; photo-documentation was provided by a Leica V-Lux 1 digital camera; and comparative fluorescence was quantified using a Quantus benchtop fluorimeter (Promega). The tumor-targeting properties of Taxol-Tropins were tested in vitro by Taxol-aptamer binding assays and collagen-agarose binding assays and the bioactivities of PTX bound non-covalently toTaxol-Tropin aptamers were tested on XC-agarose beads. Further, the tumor targeting property of the Taxol-Tropin aptamers was tested in vivo in a murine model of metastatic cancer. Results: Here we report on the first actively targeted delivery of paclitaxel utilizing bifunctional polypeptide targeting onco-aptamers, called Taxol-Tropins, which: (i) bind PTX upon simple mixing with suitably high affinities and; (ii) bind exposed XC-proteins, thereby promoting enhanced partitioning and drug delivery into the TME. The bifunctional peptide sequence-optimized Taxol-Tropins bound tightly non-covalently to PTX and also exhibited high affinity and selectivity for XC-agarose beads in vitro. Importantly, the cytotoxic bioactivity of the Taxol-Tropin-bound-PTX molecule was well preserved in cellulo, as was demonstrated by cytocidal activity observed in MDA-MB-231 breast cancer cell cultures. Tumor-targeted PTX delivery by Taxol-Tropin onco-aptamers in vivo was modeled by subcutaneous xenografts of human pancreatic cancer in nude mice: where intense fluorescence of the PTX probe was observed in tumors of mice injected with the Taxol-Tropin-bound-PTX within minutes after intravenous injection, but not in untreated mice or mice treated with non-targeted PTX probe. Conclusions: These results demonstrate the feasibility of pro-actively targeting PTX, a clinically important small molecule, using Taxol-Tropins: synthetic polypeptide onco-aptamers, revealing optimized drug binding sequences and structural modifications pertinent to further clinical development of the tumor-targeting platform which may indeed shift the Therapeutic Index of PTX to one of greater clinical efficacy at lower drug doses.