Tumor Activated Cell Penetrating Peptides to Selectively Deliver Immune Modulatory Drugs

Abstract

Recent advances in immunotherapy have revolutionized cancer therapy. Immunotherapies can engage the adaptive and innate arms of the immune system. Therapeutics targeting immune checkpoint inhibitors (i.e., CTLA-4; PD-1, and PD-L1) have shown efficacy for subsets of cancer patients by unleashing an adaptive antitumor immune response. Alternatively, small molecule immune modulators of the innate immune system such as toll-like receptor (TLR) agonists are being developed for cancer therapy. TLRs function as pattern recognition receptors to microbial products and are also involved in carcinogenesis. Reisquimod is a TLR 7/8 agonist that has antitumor efficacy. However, systemic delivery free resiquimod has proven to be challenging due to toxicity of nonspecific TLR 7/8 activation. Therefore, we developed a targeted peptide-drug conjugate strategy for systemic delivery of resiquimod. We designed an activatable cell penetrating peptide to deliver resiquimod specifically to the tumor tissue while avoiding normal tissues. The activatable cell penetrating peptide (ACPP) scaffold undergoes enzymatic cleavage by matrix metalloproteinases 2/9 in the extracellular matrix followed by intracellular lysosomal cathepsin B mediated release of the free resiquimod. Importantly, when conjugated to ACPP; the tumor tissue concentration of resiquimod was more than 1000-fold greater than that of surrounding non-cancerous tissue. Moreover, systemic ACPP-resiquimod delivery produced comparable therapeutic efficacy to localized free resiquimod in syngeneic murine tumors. These results highlight a precision peptide-drug conjugate delivery.

Keywords: cell penetrating peptides; matrix metalloproteinases; targeted drug delivery; toll-like receptor ligand.

Figure 1

Tumor targeted activatable cell penetrating peptides. Model for activatable cell penetrating peptide tumor localization. Polycationic cell penetrating peptide (+) and autoinhibitory polyanionic peptide (−) are connected by a metalloproteinase (MMP) 2/9 sensitive peptide linker (PLGC(Me)AG).

Figure 2

Activatable cell penetrating peptide (ACPP) scaffold blocks cell penetrating peptide (CPP) uptake by murine tumor cells. (A) Structural representation of Cy5 labeled CPP and ACPP. (B) Murine cancer cells exposed to Cy5 labeled CPP and ACPP probes. Cells imaged for Cy5 fluorescence (Magenta). Nuclei stained with DAPI (Blue).

Figure 3

ACPPs are cleaved in situ within murine tumors. (A) Structural representation of ratiometric activatable cell penetrating peptide probe with intervening MMP-2/9 protease sensitive amino acid sequence. (B) Mice with subcutaneous syngeneic LL2 tumors tail vein injected with 10 nanomoles ratiometric ACPP. In situ mouse imaging for Cy5:Cy7 emission ratio shown. Cy5:Cy7 emission ratio pseudocolor scale bar shown on far right. (C) Confocal imaging of tissue from LL2 tumor bearing mice injected with ratiometric ACPP. Tissue section containing tumor and adjacent muscle imaged for Cy5 (Red). Nuclei DAPI stained (Blue).

Figure 4

Conjugating resiquimod to ACPP. Chemical synthesis attaches resiquimod to a MC-VC-PABC linker.

Figure 5

ACPP selectively delivers resiquimod to tumor tissue. (A) Structural representation of resiquimod conjugated to ACPP. (B) Resiquimod drug concentrations within tissue. Mice with B16 tumors intravenously (i.v.) injected with ACPP-resiquimod. Tumor and muscle drug concentrations quantitated by LC-MS/MS. Individual tissue concentrations plotted from muscle and tumor. **** p < 0.0001.

Figure 6

Antitumor efficacy of ACPP conjugated resiquimod. (A) Mice with B16 tumors intra-tumorally (IT) injected with free resiquimod. Mouse survival plotted and statistical significances calculated using Log-rank (Mantel–Cox) test. (B) Mice with B16 tumors treated IT with free resiquimod or ACPP conjugated resiquimod given IT or intravenously (IV). Tumors measured twice a week and plotted as mean tumor volume ± SEM. Statistical significances calculated using two-way ANOVA. * p < 0.05, ** p < 0.01.