EGFR-overexpressing Cancer Cells Research Articles

Abstract 5799: Novel nanoparticle technology targets the delivery of polyIC to EGFR overexpressing cancers and induces a multimodal anti-tumor response

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.

Dual-Function Antibody Conjugate-Enabled Photoimmunotherapy Complements Fluorescence and Photoacoustic Imaging of Head and Neck Cancer Spheroids.

Several molecular-targeted imaging and therapeutic agents are in clinical trials for image-guided surgery and photoimmunotherapy (PIT) for head and neck cancers. In this context, we have previously reported the development, characterization, and specificity of a dual-function antibody conjugate (DFAC) for multimodal imaging and photoimmunotherapy (PIT) of EGFR-overexpressing cancer cells. The DFAC reported previously and used in the present study comprises an EGFR-targeted antibody, cetuximab, conjugated to benzoporphyrin derivative (BPD) for fluorescence imaging and PIT and a Si-centered naphthalocyanine dye for photoacoustic imaging. We report here the evaluation and performance of DFAC in detecting microscopic cancer spheroids by fluorescence and photoacoustic imaging along with their treatment by PIT. We demonstrate that while fluorescence imaging can detect spheroids with volumes greater than 0.049 mm3, photoacoustic imaging-based detection was possible even for the smallest spheroids (0.01 mm3) developed in the study. When subjected to PIT, the spheroids showed a dose-dependent response, with smaller spheroids (0.01 and 0.018 mm3) showing a complete response with no recurrence when treated with 100 J/cm2. Together our results demonstrate the complementary imaging and treatment capacity of DFAC. This potentially enables fluorescence imaging to assess the presence of tumor on a macroscopic scale, followed by photoacoustic imaging for delineating tumor margins guiding surgical resection and elimination of any residual microscopic disease by PIT, in a single intraoperative setting.

Anti-EGFR bioengineered bacterial outer membrane vesicles as targeted immunotherapy candidate in triple-negative breast tumor murine model

Cancer immunotherapy employing checkpoint inhibitors holds great promise across diverse cancers; nonetheless, a substantial proportion of patients (ranging from 55 to 87%) remain unresponsive to this treatment. To amplify therapeutic efficiency, we propose a synergistic therapeutic strategy that entails the deployment of targeted nano-sized particles carrying Toll-like receptor (TLR) agonists to the tumor site. This innovative approach seeks to activate intratumoral antigen-presenting cells using bioengineered outer membrane vesicles (OMVs) derived from gram-negative bacteria. These OMVs possess inherent attributes of surface-exposed immune stimulators and TLR-activating components, rendering them intriguing candidates for investigation. These OMVs were meticulously designed to selectively target cancer cells exhibiting an overexpression of epidermal growth factor receptor (EGFR). To gauge the precision of this targeting, the conducted affinity-based assays aimed at determining the equilibrium dissociation constant of the single-chain variable fragment employed for this purpose. In vitro experiments confirmed the OMVs' proficiency in adhering to EGFR-overexpressed cancer cells. Moreover, the evaluation extended to an in vivo context, where the therapeutic effect of nanovesicles was appraised within the tumor microenvironment of the triple-negative breast cancer mouse model. Notably, both intraperitoneal and intratumoral administrations of nanovesicles exhibited the ability to activate natural killer cells and skew M2 macrophage towards an M1 phenotype. The combined scrutiny of in vitro and in vivo findings underscores the potential efficiency of OMVs as a promising strategy for future anti-tumor endeavors.

EGFR-Targeted Cellular Delivery of Therapeutic Nucleic Acids Mediated by Boron Clusters

New boron carriers with high boron content and targeted cancer-cell delivery are considered the first choice for boron neutron capture therapy (BNCT) for cancer treatment. Previously, we have shown that composites of antisense oligonucleotide and boron clusters are functional nanoparticles for the downregulation of expression of epidermal growth factor receptor (EGFR) and can be loaded into EGFR-overexpressing cancer cells without a transfection factor. In this study, we hypothesize that free cellular uptake is mediated by binding and activation of the EGFR by boron clusters. Proteomic analysis of proteins pulled-down from various EGFR-overexpressing cancer cells using short oligonucleotide probes, conjugated to 1,2-dicarba-closo-dodecaborane (1,2-DCDDB, [C2B10H12]) and [(3,3'-Iron-1,2,1',2'-dicarbollide)-] (FESAN, [Fe(C2B9H11)2]-), evidenced that boron cage binds to EGFR subdomains. Moreover, inductively coupled plasma mass spectrometry (ICP MS) and fluorescence microscopy analyses confirmed that FESANs-highly decorated B-ASOs were efficiently delivered and internalized by EGFR-overexpressing cells. Antisense reduction of EGFR in A431 and U87-MG cells resulted in decreased boron accumulation compared to control cells, indicating that cellular uptake of B-ASOs is related to EGFR-dependent internalization. The data obtained suggest that EGFR-mediated cellular uptake of B-ASO represents a novel strategy for cellular delivery of therapeutic nucleic acids (and possibly other medicines) conjugated to boron clusters.

Molecular mechanism of the uptake and toxicity of EGF-LipoAgNPs in EGFR-overexpressing cancer cells

The surface of silver nanoparticles (AgNPs) is characterized by high reactivity resulting in prooxidative and cytotoxic properties. These effects are observed both in normal and in cancer cells, which overexpress the Epidermal Growth Factor Receptor (EGFR). In our previous paper, we have demonstrated that, with the use of liposomes labeled with the Epidermal Growth Factor (EGF), it is possible to direct the toxic effect of AgNPs in EGFR-overexpressing cells. Unfortunately, the mechanism of uptake and toxicity induction by such liposomes is still unknown. Therefore, the aim of this study was to determine the impact of EGF-LipoAgNPs on certain genes related to endocytosis and toxicity induction by such liposomes in human lung (A549) and tongue (SCC-15) cancer cells. The siRNA knock-out gene method was used in this study to determine the engagement of EGFR in this process. The confocal microscopy study revealed that the number of liposomes in the cytoplasm of the A549EGFR- and SCC-15EGFR- cells was lowered by 51.99 × 103 RFU and 138.50 × 103 RFU, respectively, proving the crucial role of EGFR in the liposome uptake. Moreover, the expression of the SHH and ATM genes was significantly increased, whereas the expression of the NRF2 gene was decreased after the treatment with EGF-LipoAgNPs and native AgNPs. Furthermore, the expression of the CLTC, AP2M1, CAV1, and SH3GLB1 genes indicated that the tested liposomes are uptaken via the clathrin-dependent pathway with engagement of the AP-2 complex and endophilin in this process. Summarizing, the created targeted delivery system of AgNPs causes an increase in the prooxidative and toxic effect of such NPs and has an impact on endocytosis regulatory genes, especially those related to the clathrin-mediated endocytosis.

Meditopia: Meditope‐Enabled Bonds and their Affinity with Cetuximab

A meditope is a peptide found in the fragment antigen‐binding (Fab) region of an antibody, and can be used to bind substances, such as monoclonal antibodies (mAbs) and pharmaceuticals. We centered on four mutations that can increase the affinity between the meditope and the mAb, allowing for a stronger bond. Our focus was on the anti‐epidermal growth factor receptor mAb cetuximab, which tracks the overexpression of the epidermal growth factor receptor (EGFR) by cancer cells. Utilizing a meditope, cetuximab can be linked to chemotherapy, creating a more effective treatment that only attacks cancer cells, significantly reducing possible long‐term side effects. In this study, yeast display and deep mutational scanning tracked, enhanced, and joined four mutations within the meditope, increasing affinity, or bond strength, for cetuximab 10‐fold, from Kd 130 nm to 15 nm. Those mutations led to heightened cetuximab recruitment to EGFR‐overexpressing cancer cells, and therefore more efficient targeting of cancer. The methods used in this study can develop other effective meditope‐linked monoclonal antibodies and chemotherapy treatments. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team will use 3‐D modeling and printing technology to examine structure‐function relationships of meditope‐mAb binding within cetuximab. The visual model will be a valuable tool in developing our story.

Epidermal Growth Factor-labeled liposomes as a way to target the toxicity of silver nanoparticles into EGFR-overexpressing cancer cells in vitro

Silver nanoparticles (AgNPs) are the most toxic nanostructures for both cancer and healthy cells. Thus, their usefulness in the anticancer therapy is limited. Interestingly, the epidermal growth factor receptor (EGFR) is overexpressed in many cancer cells, e.g. in lung and tongue cancers. Therefore, the aim of this study was to develop a way to direct the cytotoxic effect of AgNPs against cancer cells, saving healthy ones by entrapping these NPs inside liposomes labeled with the epidermal growth factor (EGF-LipoAgNPs) and directing these structures into EGFR-overexpressing cancer cells. The obtained results showed spherical structures with a 107.9 nm size and − 16.60 mV zeta-potential. The UV–Vis scan and TEM images did not show free AgNPs in the solution. The obtained complexes were able to decrease the metabolic activity in the A549 and SCC-15 cells more effectively than native AgNPs. Furthermore, the ROS production, lactate dehydrogenase release, and caspase-9 and -3 activity were significantly increased after the treatment with EGF-LipoAgNPs for 24 and 48 h. The expression of genes encoding catalase, superoxide dismutase, and p53 protein increased significantly, while the KI67 gene expression decreased, especially in the A549 and SCC-15 cells. Moreover, the KI67 protein expression was lower than in the cells treated with native AgNPs, while catalase activity was decreased significantly after the treatment with the obtained complexes. In turn, SOD activity increased more efficiently in the EGFR-overexpressing cancer cells. In all tested parameters, EGF-LipoAgNPs exerted a lower toxic effect on the BJ cells than native AgNPs. Summarizing, the created liposomal system reduces the toxicity of AgNPs against normal human fibroblasts and enhances the toxic and proapoptotic effect of these NPs, which may be caused by improvement of their uptake by EGFR-overexpressing cancer cells.

Magnetic nanoparticles covalently immobilizing epidermal growth factor receptor by SNAP-Tag protein as a platform for drug discovery.

Magnetic nanoparticles (NPs) cloaked with cell membranes expressing high levels of the epidermal growth factor receptor (EGFR) have been used to screen for EGFR-targeting active compounds in traditional Chinese medicine (TCM) formulations. However, previous strategies involved physical immobilization of the biomaterials on the surface of the nanocarrier, resulting in highly unstable platforms since the biological materials could dislodge easily. Chemical bonding of biomaterials to the nanoparticles surface can improve the stability of the biomimetic platforms. In this study, membrane fragments from cells expressing SNAP-Tag-EGFR (ST-EGFR) were immobilized on the surface of magnetic NPs. The ST-EGFR magnetic cell membrane nanoparticles (ST-EGFR/MCMNs) showed greater stability, and higher binding capacity, selectivity adsorption of gefitinib after 7 days compared to the un-immobilized magnetic cell membrane nanoparticles (EGFR/MCMNs). The ST-EGFR/MCMNs were used to screen for the EGFR-targeting active compounds of Zanthoxyli Radix (ZR), and identified toddalolactone and nitidine chloride. The latter significantly inhibited the proliferation of EGFR-overexpressing cancer cells, and was more effective compared to gefitinib. This innovative technology can be used to rapidly screen for active compounds from complex extracts, and aid in drug discovery.

First Clinical Results of Fluorescence Lifetime-enhanced Tumor Imaging Using Receptor-targeted Fluorescent Probes.

Fluorescence molecular imaging, using cancer-targeted near infrared (NIR) fluorescent probes, offers the promise of accurate tumor delineation during surgeries and the detection of cancer specific molecular expression in vivo. However, nonspecific probe accumulation in normal tissue results in poor tumor fluorescence contrast, precluding widespread clinical adoption of novel imaging agents. Here we present the first clinical evidence that fluorescence lifetime (FLT) imaging can provide tumor specificity at the cellular level in patients systemically injected with panitumumab-IRDye800CW, an EGFR-targeted NIR fluorescent probe. We performed wide-field and microscopic FLT imaging of resection specimens from patients injected with panitumumab-IRDye800CW under an FDA directed clinical trial. We show that the FLT within EGFR-overexpressing cancer cells is significantly longer than the FLT of normal tissue, providing high sensitivity (>98%) and specificity (>98%) for tumor versus normal tissue classification, despite the presence of significant nonspecific probe accumulation. We further show microscopic evidence that the mean tissue FLT is spatially correlated (r > 0.85) with tumor-specific EGFR expression in tissue and is consistent across multiple patients. These tumor cell-specific FLT changes can be detected through thick biological tissue, allowing highly specific tumor detection and noninvasive monitoring of tumor EFGR expression in vivo. Our data indicate that FLT imaging is a promising approach for enhancing tumor contrast using an antibody-targeted NIR probe with a proven safety profile in humans, suggesting a strong potential for clinical applications in image guided surgery, cancer diagnostics, and staging.

Theranostic potential of self-luminescent branched polyethyleneimine-coated superparamagnetic iron oxide nanoparticles.

Polyethylenimine (PEI), which is frequently used for polyplex formation and effective gene transfection, is rarely recognized as a luminescent polymer. Therefore, it is usually tagged with an organic fluorophore to be optically tracked. Recently, we developed branched PEI (bPEI) superparamagnetic iron oxide nanoparticles (SPION@bPEI) with blue luminescence 1200 times stronger than that of bPEI without a traditional fluorophore, due to partial PEI oxidation during the synthesis. Here, we demonstrate in vitro dye-free optical imaging and successful gene transfection with luminescent SPION@bPEI, which was further modified for receptor-mediated delivery of the cargo selectively to cancer cell lines overexpressing the epidermal growth factor receptor (EGFR). Pro-apoptotic polyinosinic–polycytidylic acid sodium (PIC) was delivered to HeLa cells with SPION@bPEI and caused a dramatic reduction in the cell viability at otherwise non-toxic nanoparticle concentrations, proving that bPEI coating is still an effective component for the delivery of an anionic cargo. Besides, a strong intracellular optical signal supports the optically traceable nature of these nanoparticles. SPION@bPEI nanoparticles were further conjugated with Erbitux (Erb), which is an anti-EGFR antibody for targeting EGFR-overexpressing cancer cell lines. SPION@bPEI-Erb was used for the delivery of a GFP plasmid wherein the transfection was confirmed by the luminescence of the expressed gene within the transfected cells. Poor GFP expression in MCF7, a slightly better expression in HeLa, and a significant enhancement in the transfection of HCT116 cells proved a selective uptake and hence the targeting ability of Erb-tagged nanoparticles. Altogether, this study proves luminescent, cationic, and small SPION@bPEI nanoparticles as strong candidates for imaging and gene therapy.

Cetuximab-modified doxorubicin bovine serum albumin nanoparticles for the treatment of epidermal growth factor receptor-overexpressing colon cancer cells

In this study, we developed an active targeting nano-immunoplatform of doxorubicin (DOX)-loaded bovine serum albumin (BSA) modified with the humanized antibody cetuximab, which can target the epidermal growth factor receptor (EGFR) of EGFR-overexpressing colon cancer, and subjected it to a series ofin vitroevaluation. The obtained cetuximab-modified nanoparticles (cetuximab-DOX-immuno-NPs) had an average particle size of 218.2±2.2 nm, a polydispersity index of 0.162±0.023, a zeta potential of −23.28±0.75 mV, and a cetuximabloading efficiency of 33.6%±3.5%. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity experiments showed that the cytotoxic of cetuximab-DOX-immuno-NPs in EGFR-overexpressing RKO colon cancer cells was significantly higher than it was in EGFR-under-expressing LS174 T cells. The results of flow cytometry showed that when DOX-immuno-NPs were used, DOX was eliminated soon after entering the cells, whereas when cetuximab-DOX-immuno-NPs were used, DOX efflux was reduced. Confocal laser-scanning microscopy was used to visualize the endocytosis of nanoparticles, and revealed that the fluorescence intensity of cetuximab-DOX-immuno-NPs was significantly higher than that of non-specific IgG-DOX-immuno-NPs in RKO cells. Moreover, in EGFR-under-expressing LS174 T cells, both cetuximab-DOXimmuno-NPs and Immunoglobulin G-DOX-immuno-NPs showed weak DOX fluorescence intensity, reflecting the specificity and selectivity of cetuximab-modified nanoparticles toward EGFR-overexpressing cancer cells. This study demonstrated that cetuximab modification of DOX-loaded BSA nanoparticles can increase their selectivity and reduce their toxicity. As this would decrease the side effects of these treatments in patients, this study shows that cetuximab-modified doxorubicin-loaded bovine serum albumin nanoparticles are promising candidates for targeted colon cancer therapy.

Capsule-like molecular imprinted polymer nanoparticles for targeted and chemophotothermal synergistic cancer therapy

Selective cancer cell targeting, controlled drug release, easy construction and multiple therapeutic modalities are some of the desirable characteristics of drug delivery systems. We designed and built simple capsule-like molecular imprinted polymer (MIP)-based nanoparticles for targeted and chemo-photothermal synergistic cancer therapy. Using dopamine (DA) as functional monomer, cross-linking agent as well as photo-thermal agent, ZIF-8 (zeoliticimidazolate framework-8) as drug carrier, epitope of EGFR (epidermal growth factor receptor) as template molecules, molecular imprinted polymer (MIP) drug carrier was constructed. The ability of MIP layer to bind to EGFR epitope endowed the MD (DOX@MIP) particles to recognize EGFR-overexpressing cancer cells, while the pH-responsiveness and photothermal conversion ability of PDA (polydopamine) achieved chemo-photothermal synergistic effects upon NIR irradiation. Taken together, the MD nanoparticles integrated cancer cell targeting recognition, intelligent drug release, biocompatibility and chemo-photothermal effects, and is therefore a promising tool for targeted cancer therapy with minimal toxicity to normal cells, as well as tumor imaging.

Metformin Resensitizes Sorafenib-Resistant HCC Cells Through AMPK-Dependent Autophagy Activation

Despite the activation of autophagy may enable residual cancer cells to survive and allow tumor relapse, excessive activation of autophagy may eventually lead to cell death. However, the details of the association of autophagy with primary resistance in hepatocellular carcinoma (HCC) remain less clear. In this study, cohort analysis revealed that HCC patients receiving sorafenib with HBV had higher mortality risk. We found that high epidermal growth factor receptor (EGFR) expression and activity may be linked to HBV-induced sorafenib resistance. We further found that the resistance of EGFR-overexpressed liver cancer cells to sorafenib is associated with low activity of AMP-activated protein kinase (AMPK) and CCAAT/enhancer binding protein delta (CEBPD) as well as insufficient autophagic activation. In response to metformin, the AMPK/cAMP-response element binding protein (CREB) pathway contributes to CEBPD activation, which promotes autophagic cell death. Moreover, treatment with metformin can increase sorafenib sensitivity through AMPK activation in EGFR-overexpressed liver cancer cells. This study suggests that AMPK/CEBPD-activated autophagy could be a potent strategy for improving the efficacy of sorafenib in HCC patients.

Abstract 1723: Trispecific natural killer cell nanoengagers for targeted chemoimmunotherapy

Abstract Background: Activation of the innate immune system and natural killer (NK) cells has been a key effort in cancer immunotherapy research. Several NK-cell-targeted therapeutics are under clinical investigation. Current research is focused on bispecific antibodies that can bind to and target both tumor cells and NK cells. However, NK activation remains a key challenge. One approach is to simultaneously co-activate more than one NK cell stimulatory molecule, such as 4-1BB and CD16 (FcγRIII), to increase immune activation. To achieve this, we developed a new nanoparticle (NP)-based trispecific NK cell engager (nano-TriNKE) platform that can target epidermal growth factor receptor (EGFR) overexpressing tumors and promote the recruitment and activation of NK cells to eradicate these cancer cells. Moreover, the nanoengager platform allows the targeted delivery of chemotherapeutics such as epirubicin (EPI) to further improve the therapeutic efficacy. Methods: EGFR-targeted drug-free/EPI-encapsulated nano-TriNKEs were prepared by conjugating cetuximab (anti-EGFR), anti-4-BB, and anti-CD16 agonistic antibodies to PEG-PLGA NPs. Their binding specificities were evaluated in expanded murine NK cells, EGFR-overexpressed A431 (epidermoid carcinoma), MB468 (mammary gland adenocarcinoma), and HT29 (colorectal adenocarcinoma) cells in vitro. The in vitro NK cell anticancer activities in nano-TriNKE-pretreated A431, MB468, and HT29 cells were evaluated via viability assays. We also performed comprehensive in vivo efficacy studies in A431, MB468, and HT29 tumor model to investigate the anticancer activities of EGFR-targeted drug-free/EPI-encapsulated nano-TriNKEs. Further biodistribution and mechanistic studies were performed to verify the anticancer activities of nano-TriNKEs. Results: In vitro binding assay confirmed that the EGFR-targeted nano-TriNKEs bind selectively to EGFR-overexpressed cancer cells. In vitro anticancer activity studies demonstrated that effective NK cell activation can be achieved by spatiotemporal co-activation of CD16 and 4-1BB stimulatory molecules on NK cells with nanoengagers, and that the nanoengagers are more effective than free antibodies. Comprehensive in vivo efficacy studies in A431, MB468, and HT29 tumor models demonstrated that the EGFR-targeted nano-TriNKEs can augment both NK activating agents and chemotherapy as highly effective anticancer agents, providing robust chemoimmunotherapy against a broad range of EGFR-overexpressed cancers. Conclusions: Our data demonstrated that nano-TriNKEs are more effective in NK activation than free agonistic antibodies, and that the nanoengager platform provides more effective biological targeting, which is critical in NK-mediated cancer immunotherapy. Citation Format: Kin Man Au, Steven Park, Andrew Wang. Trispecific natural killer cell nanoengagers for targeted chemoimmunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1723.

Quantification of EGFR and EGFR-overexpressed cancer cells based on carbon dots@bimetallic CuCo Prussian blue analogue.

A new bimetallic CuCo Prussian blue analogue (CuCo PBA) loaded with carbon dots (CDs) was prepared (represented by CD@CuCoPBA) and developed as a scaffold for anchoring the epidermal growth factor receptor (EGFR) aptamer to detect EGFR and living EGFR-overexpressed cancer cells. The basic characterizations revealed CuCo PBA exhibited nanocube shape and still remained its nanostructure and physical/chemical properties after coupling with large amounts of CDs. As compared with the pristine CuCo PBA, the CD@CuCoPBA displayed good electrochemical activity, strong binding interaction toward aptamer, and high stability of aptamer-EGFR G-quadruplex in aqueous solution. As such, the results of electrochemical impedance spectroscopy measurements indicated that the CD@CuCoPBA-based aptasensor displayed an ultra-low detection limit toward EGFR (0.42 fg mL−1) and living EGFR-overexpressed MCF-7 cancer cells (80 cell per mL), as well as high selectivity, good reproducibility, high stability, repeatability, and acceptable applicability. Consequently, the constructed CD@CuCoPBA-based aptasensor can be extended to be a promising universal method for early diagnosis of cancers.

An epidermal growth factor receptor-targeted and endoplasmic reticulum-localized organic photosensitizer toward photodynamic anticancer therapy

The endoplasmic reticulum (ER), as the largest organelle in eukaryotic cells, plays complex but pivotal roles in multiple intracellular metabolic functions, including biosynthesis, sensing, and signal transduction, especially in proteins folding and post-translation modification. The ER is regarded as a promising target for anticancer therapy. Based on previous tumor-targeted photodynamic therapy (PDT), we chemically modified the phthalocyanine-based photosensitizer molecule with the small molecular anticancer-targeting drug erlotinib and the ER-targetable moiety methyl sulfonamide to develop an advanced photosensitizer EB-ER-Pc that can specifically target the subcellular organelle ER of EGFR-overexpressing cancer cells. The in vitro experiments show that the dual-target photosensitizer EB-ER-Pc can generate ROS in situ in the ER of the tumor target region to induce ER stress, upregulate Ca2+ ion level, and decrease mitochondrial membrane potential (MMP) to mediate cancer cells death and ablation. The results suggest that EB-ER-Pc is a promising candidate for effective photodynamic cancer therapy.

Sulforaphane-Conjugated Carbon Dots: A Versatile Nanosystem for Targeted Imaging and Inhibition of EGFR-Overexpressing Cancer Cells.

Because of the demand for precision medicine, the investigatation on the application of carbon-dots-based nanosystems in the field of biomedicine is attracting more and more attention. Therefore, we have built a multifunctional nanosystem based on sulforaphane-conjugated carbon dots (SFN-CDs) with thiourea skeleton and applied for EGFR-overexpressing cancer cells targeted imaging and inhibiting. The SFN-CDs are formed by grafting sulforaphane on the amino-rich yellow fluorescent carbon dots, which have excellent optical stability and can be distinguished from normal cells for targeted imaging of cancer cells. The vitro toxicity experiments demonstrated that the SFN-CDs can effectively inhibit EGFR-overexpressing cancer cell proliferation at concentrations below 100 μg mL-1. All these results validated that SFN-CDs nanoparticles with integration of diagnostic and therapeutic functions can be used as a potential nanodurg in early stage of cancer control. Moreover, this work provides useful insight into targeted nanoparticle design in the biological nanomedicine field.

Abstract 994: Combining ruthenium metallo-intercalators and targeted radionuclide therapy for EGFR-overexpressing oesophageal cancer

Abstract Targeted radionuclide therapy (TRT) combines the specificity of molecular targeting with the potent cytotoxicity of ionising radiation. An emerging question is how to most effectively integrate TRT with radiosensitizing small molecules [1]. Auger electron-emitting radionuclides such as 111In are well-suited as the therapeutic radionuclide as their short-range reduces “cross fire” damage to neighbouring cells. As a novel chemical class of radiosensitizing agent, we have found ruthenium polypyridyl complexes (RPCs) that intercalate between DNA base pairs such as Ru(phen)2(tpphz)2+ (phen = 1,10 phenanthroline, tpphz = tetrapyridophenazine) act to stall replication forks and impede mitotic progression in human oesophageal cancer cells [2,3]. This dual mechanism of action results in preferential growth inhibition of rapidly-proliferating oesophageal cancer cells with elevated mitotic indices. In contrast to cisplatin and other DNA-damaging agents, relatively low DSB levels are generated in response to RPC metallo-intercalation and reduced cytotoxicity towards non-cancer cells is observed. In addition to this, treatment is able to enhance cancer cell sensitivity to IR by increasing IR-induced DNA double-strand break (DSB) damage, decreasing cell survival in a synergistic manner. Based on these discoveries, we explore how nanoparticle-mediated delivery of Ru(phen)2(tpphz)2+ and the Auger electron emitting radionuclide 111In can achieve localised cytotoxicity enhancement specifically in EGFR-overexpressing oesophageal cancer cells, thereby demonstrating both a combinational effect alongside molecular targeting for this novel combination [4].

A novel tumor and mitochondria dual-targeted photosensitizer showing ultra-efficient photodynamic anticancer activities.

We designed and synthesized a simple, but highly effective photosensitizer (G-Mito-Pc), which can precisely target the mitochondria of epidermal growth factor receptor (EGFR)-overexpressing cancer cells, to achieve dual targeting function at both cell and organelle levels in cancer therapy. We further explored the possible molecular mechanism of the enhanced bioactivity of G-Mito-Pc compared to that of the reference photosensitizer using molecular dynamics simulations on their interactions with a physiologically relevant mitochondrial membrane model.

Bypassing pro-survival and resistance mechanisms of autophagy in EGFR-positive lung cancer cells by targeted delivery of 5FU using theranostic Ag2S quantum dots.

Targeted drug delivery systems that combine imaging and therapeutic functions in a single structure have become very popular in nanomedicine. Near-infrared (NIR) emitting Ag2S quantum dots (QDs) are excellent candidates for this task. Here, we have developed PEGylated Ag2S QDs functionalized with Cetuximab (Cet) antibody and loaded with an anticancer drug, 5-fluorouracil (5FU). These theranostic QDs were used for targeted NIR imaging and treatment of lung cancer using low (H1299) and high (A549) Epidermal Growth Factor Receptor (EGFR) overexpressing cell lines. The Cet conjugated QDs effectively and selectively delivered 5FU to A549 cells and provided significantly enhanced cell death associated with apoptosis. Interestingly, while treatment of cells with free 5FU activated autophagy, a cellular mechanism conferring resistance to cell death, these EGFR targeting multimodal QDs significantly overcame drug resistance compared to 5FU treatment alone. The improved therapeutic outcome of 5FU delivered to A549 cells by Cet conjugated Ag2S QDs is suggested as the synergistic outcome of enhanced receptor mediated uptake of nanoparticles, and hence the drug, coupled with suppressed autophagy even in the absence of addition of an autophagy suppressor.