The epidermal growth factor receptor (EGFR) is aberrantly activated in many human epithelial cancers. This report presents the preparation, purification, and the anti-cancer potency of an anti-EGFR affibody (ZEGFR 1907')-polyethylenimine (PEI)-polyIC complex (PPEA-polyplex). Surface plasmon resonance analysis showed that the ZEGFR 1907' affibody binds tightly to full-length sEGFR with an average equilibrium dissociation constant, KD, value of 6.74 nM. As expected the PPEA-polyplex does not activate the EGFR kinase, but kills tumor cells expressing medium to high levels of EGFR. The PPEA-polyplex stimulates the release of chemotactic cytokines (e.g., GRO-α, IFN-γ-inducible protein-10) and promoted PBMC-mediated bystander killing of non-treated tumor cells. The PPEA-polyplex also inhibited the growth of human epidermoid vulval carcinoma (A431) xenografts growing in immunocompromised nude mice. Both the in vitro and in vivo results indicate that PPEA-polyplexes have the potential to inhibit the growth of tumors which over-express the EGFR, including colon and breast cancer cells.

Aberrant activation and overexpression of the epidermal growth factor receptor (EGFR) occurs in various solid cancers and often correlates with poor outcome. The clinical benefit from EGFR-targeted therapies is usually short-lived, with resistance being driven by tumor heterogeneity and an immunosuppressive tumor microenvironment (TME). To address these limitations, we developed Targeted Apoptotic Immune Modulators (TAIM), a nonviral nanoparticle platform for the targeted delivery of polyinosine:polycytosine (polyIC), to simultaneously induce tumor cell death and activate antitumor immunity. The first TAIM compound, TAR001, was designed as a systemic treatment against metastatic EGFR-positive solid cancers. Here, we present TAR001's multifaceted mode of action. We demonstrate that TAR001 is selective toward EGFR-overexpressing cancers, provoking a pattern recognition response, apoptosis, cytokine secretion, and antitumor immunity. TAR001 modulates the TME, recruiting and activating both innate and adaptive immune cells. Systemic delivery of TAR001 markedly extends survival and inhibits tumor growth in multiple murine tumor models. TAR001 represents an innovative, safe, multimodal treatment approach with the potential to benefit patients with metastatic head and neck, non-small cell lung cancer, colorectal, renal, and triple-negative breast cancers. This unique modality utilizes a broad range of mechanisms to overcome the tumor's ability to escape apoptosis and immune cell activation.

Abstract Aberrant activation of epidermal growth factor receptor (EGFR) signaling pathways is associated with tumor growth and progression. EGFR overexpression occurs in various types of solid cancers and correlates with poor outcome. Unfortunately, the clinical benefit from EGFR-targeted therapies is usually short-lived, due to the development of resistance. TargImmune has developed first-in-class Targeted Apoptotic Immunomodulators (TAIMs), which harness the body’s anti-viral responses to treat solid tumors. Our novel nanoparticle platform technology, Ta:RNA , selectively delivers synthetic dsRNA, polyinosinic:polycytidylic acid (polyIC) to tumor cells that overexpress a target receptor. PolyIC and other pattern recognition receptor (PRR) agonists have been used as adjuvants or for intratumoral administration but have limited efficacy as single agents. TargImmune’s platform allows systemic delivery of dsRNA, tailoring the nanoparticles to various solid cancers by simply changing the targeting moiety. Here we describe EGFR-Ta:RNA, which is targeted to tumors that overexpress EGFR. EGFR-Ta:RNA does not inhibit EGFR per se, and therefore activation of alternative or downstream oncogenes is not expected to cause resistance. The mechanism of action of EGFR-targeted Ta:RNA nanoparticles was extensively studied in vitro and in vivo, using confocal imaging, large-scale RNA sequencing studies, Western blot analysis and ELISA assays. In vitro, EGFR-Ta:RNA potency and selectivity were established by comparing cells with high receptor and low receptor expression. Imaging studies showed that the nanoparticles are internalized selectively into EGFR-overexpressing cancer cells by receptor-mediated endocytosis. RNAseq studies revealed: (i) a common transcriptional signature in EGFR-Ta:RNA-treated tumours and cancer cells; (ii) enrichment in pathways related to anti-viral response, inflammation, type I interferon signaling, and NFΚB signaling; (iii) enrichment in genes coding for cytokines. Western blot analysis and ELISA assays confirmed that the mechanism of action of EGFR-Ta:RNA is mediated via the induction of PRR downstream signaling, which results in cytokine secretion, apoptosis and immune cell activation. The efficacy of EGFR-Ta:RNA was demonstrated in multiple animal models, including both xenograft and immunocompetent models. The pharmacodynamic effect, exemplified by target engagement and activation of PRR signaling in tumors, was verified in vivo. In conclusion, TargImmune’s EGFR-Ta:RNA nanoparticles deliver polyIC in a tumor-targeted manner to evoke PRR signaling, inducing targeted tumor cell apoptosis as well as immune cell recruitment and activation against the heterogenous tumor. EGFR-Ta:RNA represents a novel, multimodal treatment approach for EGFR overexpressing cancers. Citation Format: Derrick Broka, Caroline De Feyter, Shoshana Klein, Meera Saxena, Joerg Schreiber, Alexei Shir, Michal Skowicki, Yaakov Benenson, Alexander Levitzki, Cornelia G. Palivan, Esteban Pombo-Villar, Babette Schade, Maya Zigler. Novel nanoparticle technology targets the delivery of polyIC to EGFR overexpressing cancers and induces a multimodal anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5799.

PDF file, 1740K, Microvascular density in A375SM NT and Gal-3 shRNA tumors

Supplementary Materials and Methods, Figure Legends 1-6, Table Legend from Expression of Id-1 Is Regulated by MCAM/MUC18: A Missing Link in Melanoma Progression

Supplementary Material and methods

Supplementary Figure 1 from Expression of Id-1 Is Regulated by MCAM/MUC18: A Missing Link in Melanoma Progression

Supplementary Figure illustrating the expression of TLR3 in cell line that deferentially express HER2

PDF file, 1706K, Expression status of LPA receptors in SB-2, WM2664, and A375SM melanoma cells.

Supplementary Methods and Figure Legend from Overexpression of Protease-Activated Receptor-1 Contributes to Melanoma Metastasis via Regulation of Connexin 43

PDF file, 1483K, Transcriptional analysis of autotaxin after silencing galectin-3 by nuclear run-on and dual luciferase assays

Supplementary Figure illustrating the difference in cytokine mRNA and protein expression following treatment of BT-474 with pIC/PPHAffibody and pIC/PPCys

PDF file, 1717K, qRT-PCR of autotaxin and nfat1 mRNA after silencing nfat1 in A375SM melanoma cells

Supplementary Methods and Figures from A Subset of Host B Lymphocytes Controls Melanoma Metastasis through a Melanoma Cell Adhesion Molecule/MUC18-Dependent Interaction: Evidence from Mice and Humans

Supplementary Figure 1 from Overexpression of Protease-Activated Receptor-1 Contributes to Melanoma Metastasis via Regulation of Connexin 43

PDF file, 1628K, Invasion, migration, and soft agar assay of melanoma cells after silencing galectin-3

Supplementary Figure demonstrating the effect of pIC/PPHAffibody treatment on RENCA HER2 cell survival in vitro and in vivo by immunohistochemistry staining for c-caspase-3

PDF file, 61K, Oligos used for experiments