Localized delivery is vital for the successful development of novel and effective therapeutics for the treatment of cancer. The targeting and delivery described herein is based on the pH(Low) Insertion Peptide (pHLIP), a unique delivery peptide that can selectively target tumors in mice and translocate and release cargo molecules intra-cellularly based solely on the low extracellular pH intrinsic to cancer cells. In this study, we investigate the efficacy of pHLIP to target and deliver the highly potent and clinically validated microtubule inhibitor monomethyl auristatin E (MMAE) to cancer cells and breast tumors. We show that pHLIP-MMAE conjugates induce a potent cytotoxic effect (> 90% inhibition of cell growth) in a concentration- and pH-dependent manner after only 2-hour incubation without any apparent disruption of the plasma membrane. pHLIP-MMAE conjugates exhibit between an 11 and 144-fold higher anti-proliferative effect at low pH than at physiological pH, and a pronounced pH-dependent cytotoxicity as compared to free drug. Furthermore, we demonstrate that a pHLIP-MMAE drug conjugate effectively targets triple negative breast tumor xenografts in mice. These results indicate pHLIP-based auristatin conjugates may have an enhanced therapeutic window as compared to free drug, providing a targeting mechanism to attenuate systemic toxicity.
Positively charged antimicrobial peptides have become promising agents for the treatment of cancer by inducing apoptosis though their preferential binding and disruption of negatively charged membranes, such as the mitochondrial membrane. (KLAKLAK)2 is such a peptide but due to its polarity, it cannot cross the cellular membrane and therefore relies on the use of a delivery agent. For targeted delivery, previous studies have relied on cell penetrating peptides, nanoparticles or specific biomarkers. Herein, we investigated the first use of pHLIP to selectively target and directly translocate (KLAKLAK)2 into the cytoplasm of breast cancer cells, based on the acidic tumor micro-environment. With the goal of identifying a lead conjugate with optimized selective cytotoxicity towards cancer cells, we analyzed a family of (KLAKLAK)2 analogs with varying size, polarity and charge. We present a highly efficacious pHLIP conjugate that selectively induces concentration- and pH-dependent toxicity in breast cancer cells.
Even though abnormal expression of G protein-coupled receptors (GPCRs)
and of their ligands is observed in many cancer cells of various origins, only a
few anti-cancer compounds directly act on their signaling. One promising
approach to modulate their activity consists of targeting the receptor
cytoplasmic surfaces interacting with the associated G proteins using peptides
mimicking the intracellular loops of the receptor. Thus, to be fully effective,
the peptide mimics must be selectively targeted to the tumor while sparing
healthy tissues, translocated across the cell membrane and stay anchored to the
cytoplasmic leaflet of the plasma membrane. Here, we introduce a novel way to
selectively target and inhibit the activity of a GPCR in cancer cells under
acidic conditions, such as those found in solid tumors. We find that the
conjugation of a peptide fragment derived from the third intracellular loop of
the Protease Activated Receptor 1 (PAR1) to a peptide that can selectively
target tumors solely based on their acidity (pHLIP), produces a construct
capable of effectively down-regulating PAR1 activity in a concentration - and
pH-dependent manner, and of inducing a potent cytotoxic effect in a panel of
cancer cells that is proportional to the relative level of receptor expression
at the cell surface. This strategy not only allows for a more selective
targeting and specific intracellular delivery than current approaches, but also
offers new possibilities for developing novel anti-cancer drugs targeting
GPCRs.
The targeting of therapeutics specifically to diseased tissue is crucial for the development of successful cancer treatments. The approach here is based on the pH(low) insertion peptide (pHLIP) for the delivery of a potent mitotic inhibitor monomethyl auristatin F (MMAF). We investigated six pHLIP variants conjugated to MMAF to compare their efficacy in vitro against cultured cancer cells. While all pHLIP–MMAF conjugates exhibit potent pH- and concentration-dependent killing, their cytotoxicity profiles are remarkably different. We also show that the lead conjugate exhibits significant therapeutic efficacy in mouse models without overt toxicities. This study confirms pHLIP–monomethyl auristatin conjugates as possible new therapeutic options for cancer treatment and supports their further development.
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