Development of a nanoparticle-based immunotherapy targeting PD-L1 and PLK1 for lung cancer treatment.

Immune checkpoint inhibitors (ICIs) targeting PD-L1 and PD-1 have improved survival in a subset of patients with advanced non-small cell lung cancer (NSCLC). However, only a minority of NSCLC patients respond to ICIs, highlighting the need for superior immunotherapy. Herein, we report on a nanoparticle-based immunotherapy termed ARAC (Antigen Release Agent and Checkpoint Inhibitor) designed to enhance the efficacy of PD-L1 inhibitor. ARAC is a nanoparticle co-delivering PLK1 inhibitor (volasertib) and PD-L1 antibody. PLK1 is a key mitotic kinase that is overexpressed in various cancers including NSCLC and drives cancer growth. Inhibition of PLK1 selectively kills cancer cells and upregulates PD-L1 expression in surviving cancer cells thereby providing opportunity for ARAC targeted delivery in a feedforward manner. ARAC reduces effective doses of volasertib and PD-L1 antibody by 5-fold in a metastatic lung tumor model (LLC-JSP) and the effect is mainly mediated by CD8+ T cells. ARAC also shows efficacy in another lung tumor model (KLN-205), which does not respond to CTLA-4 and PD-1 inhibitor combination. This study highlights a rational combination strategy to augment existing therapies by utilizing our nanoparticle platform that can load multiple cargo types at once.

Targeted Nanoparticle for Co-delivery of HER2 siRNA and a Taxane to Mirror the Standard Treatment of HER2+ Breast Cancer: Efficacy in Breast Tumor and Brain Metastasis.

The first-line treatment of advanced and metastatic human epidermal growth factor receptor type 2 (HER2+) breast cancer requires two HER2-targeting antibodies (trastuzumab and pertuzumab) and a taxane (docetaxel or paclitaxel). The three-drug regimen costs over $320,000 per treatment course, requires a 4 h infusion time, and has many adverse side effects, while achieving only 18 months of progression-free survival. To replace this regimen, reduce infusion time, and enhance efficacy, a single therapeutic is developed based on trastuzumab-conjugated nanoparticles for co-delivering docetaxel and siRNA against HER2 (siHER2). The optimal nanoconstruct has a hydrodynamic size of 100 nm and specifically treats HER2+ breast cancer cells over organ-derived normal cells. In a drug-resistant orthotopic HER2+ HCC1954 tumor mouse model, the nanoconstruct inhibits tumor growth more effectively than the docetaxel and trastuzumab combination. When coupled with microbubble-assisted focused ultrasound that transiently disrupts the blood brain barrier, the nanoconstruct inhibits the growth of trastuzumab-resistant HER2+ BT474 tumors residing in the brains of mice. The nanoconstruct has a favorable safety profile in cells and in mice. Combination therapies have become the cornerstone of cancer treatment and this versatile nanoparticle platform can co-deliver multiple therapeutic types to ensure that they reach the target cells at the same time to realize their synergy.

PLK1 and EGFR targeted nanoparticle as a radiation sensitizer for non-small cell lung cancer.

Radiation sensitizers that can selectively act on cancer cells hold great promise to patients who receive radiation therapy. We developed a novel targeted therapy and radiation sensitizer for non-small cell lung cancer (NSCLC) based on cetuximab conjugated nanoparticle that targets epidermal growth factor receptor (EGFR) and delivers small interfering RNA (siRNA) against polo-like kinase 1 (PLK1). EGFR is overexpressed in 50% of lung cancer patients and a mediator of DNA repair, while PLK1 is a key mitotic regulator whose inhibition enhances radiation sensitivity. The nanoparticle construct (C-siPLK1-NP) effectively targets EGFR + NSCLC cells and reduces PLK1 expression, leading to G2/M arrest and cell death. Furthermore, we show a synergistic combination between C-siPLK1-NP and radiation, which was confirmed in vivo in A549 flank tumors. We also demonstrate the translational potential of C-siPLK1-NP as a systemic therapeutic in an orthotopic lung tumor model, where administration of C-siPLK1-NP reduced tumor growth and led to prolonged survival. Our findings demonstrate that C-siPLK1-NP is effective as a targeted therapy and as a potent radiation sensitizer for NSCLC. Potential application to other EGFR + cancer types such as colorectal and breast cancer is also demonstrated.

Selective capture of radionuclides (U, Pu, Th, Am and Co) using functional nanoporous sorbents.

This work evaluated sorbent materials created from nanoporous silica self-assembled with monolayer (SAMMS) of hydroxypyridinone derivatives (1,2-HOPO, 3,2-HOPO, 3,4-HOPO), acetamide phosphonate (Ac-Phos), glycine derivatives (IDAA, DE4A, ED3A), and thiol (SH) for capturing of actinides and transition metal cobalt. In filtered seawater doped with competing metals (Cr, Mn, Fe, Co, Cu, Zn, Se, Mo) at levels encountered in environmental or physiological samples, 3,4-HOPO-SAMMS was best at capturing uranium (U(VI)) from pH 2-8, Ac-Phos and 1,2-HOPO-SAMMS sorbents were best at pH < 2. 3,4-HOPO-SAMMS effectively captured thorium (Th(IV)) and plutonium (239Pu(IV)) from pH 2-8, and americium (241Am(III)) from pH 5-8. Capturing cobalt (Co(II)) from filtered river water doped with competing metals (Cu, As, Ag, Cd, Hg, Tl, and Pb) was most effective from pH 5-8 with binding affinity ranged from IDAA > DE4A > ED3A > Ac-Phos > SH on SAMMS. Iminodiacetic acid (IDAA)-SAMMS was also outstanding at capturing Co(II) in ground and seawater. Within 5 min, over 99% of U(VI) and Co(II) in seawater was captured by 3,4-HOPO-SAMMS and IDAA-SAMMS, respectively. These nanoporous materials outperformed the commercially available cation sorbents in binding affinity and adsorption rate. They have great potential for water treatment and recovery of actinides and cobalt from complex matrices.

CAR-T Cells Surface-Engineered with Drug-Encapsulated Nanoparticles Can Ameliorate Intratumoral T-cell Hypofunction.

One limiting factor of CAR T-cell therapy for treatment of solid cancers is the suppressive tumor microenvironment (TME), which inactivates the function of tumor-infiltrating lymphocytes (TIL) through the production of immunosuppressive molecules, such as adenosine. Adenosine inhibits the function of CD4+ and CD8+ T cells by binding to and activating the A2a adenosine receptor (A2aR) expressed on their surface. This suppression pathway can be blocked using the A2aR-specific small molecule antagonist SCH-58261 (SCH), but its applications have been limited owing to difficulties delivering this drug to immune cells within the TME. To overcome this limitation, we used CAR-engineered T cells as active chaperones to deliver SCH-loaded cross-linked, multilamellar liposomal vesicles (cMLV) to tumor-infiltrating T cells deep within the immune suppressive TME. Through in vitro and in vivo studies, we have demonstrated that this system can be used to effectively deliver SCH to the TME. This treatment may prevent or rescue the emergence of hypofunctional CAR-T cells within the TME. Cancer Immunol Res; 6(7); 812-24. ©2018 AACR.

Combination drug delivery via multilamellar vesicles enables targeting of tumor cells and tumor vasculature.

Blood vessel development is critical for the continued growth and progression of solid tumors and, therefore, makes an attractive target for improving cancer therapy. Indeed, vascular-targeted therapies have been extensively explored but they have shown minimal efficacy as monotherapies. Combretastatin A4 (CA-4) is a tubulin-binding vascular disrupting agent that selectively targets the established tumor endothelium, causing rapid vascular beak down. Despite its potent anticancer potential, the drug has dose-limiting side effects, particularly in the form of cardiovascular toxicity. Furthermore, its poor aqueous solubility and the resulting limited bioavailability hinder its antitumor activity in the clinic. To improve the therapeutic efficacy of CA-4, we investigated its application as a combination therapy with doxorubicin (Dox) in a tumor vasculature targeted delivery vehicle: peptide-modified cross-linked multilamellar liposomal vesicles (cMLVs). In vitro cell culture studies showed that a tumor vasculature-targeting peptide, RIF7, could facilitate higher cellular uptake of drug-loaded cMLVs, and consequently enhance the antitumor efficacy in both drug resistant B16 mouse melanoma and human MDA-MB-231 breast cancer cells. In vivo, upon intravenous injection, targeted cMLVs could efficiently deliver both Dox and CA-4 to significantly slow tumor growth through the specific interaction of the targeting peptide with its receptor on the surface of tumor vasculature. This study demonstrates the potential of our novel targeted combination therapy delivery vehicle to improve the outcome of cancer treatment.

Enhanced Cancer Immunotherapy by Chimeric Antigen Receptor-Modified T Cells Engineered to Secrete Checkpoint Inhibitors.

Purpose: Despite favorable responses of chimeric antigen receptor (CAR)-engineered T-cell therapy in patients with hematologic malignancies, the outcome has been far from satisfactory in the treatment of solid tumors, partially owing to the development of an immunosuppressive tumor microenvironment. To overcome this limitation, we engineered CAR T cells secreting checkpoint inhibitors (CPI) targeting PD-1 (CAR.αPD1-T) and evaluated their efficacy in a human lung carcinoma xenograft mouse model.Experimental Design: To evaluate the effector function and expansion capacity of CAR.αPD1-T cells in vitro, we measured the production of IFNγ and T-cell proliferation following antigen-specific stimulation. Furthermore, the antitumor efficacy of CAR.αPD1-T cells, CAR T cells, and CAR T cells combined with anti-PD-1 antibody was determined using a xenograft mouse model. Finally, the underlying mechanism was investigated by analyzing the expansion and functional capacity of TILs.Results: Human anti-PD-1 CPIs secreted by CAR.αPD1-T cells efficiently bound to PD-1 and reversed the inhibitory effect of PD-1/PD-L1 interaction on T-cell function. PD-1 blockade by continuously secreted anti-PD-1 attenuated the inhibitory T-cell signaling and enhanced T-cell expansion and effector function both in vitro and in vivo In the xenograft mouse model, we demonstrated that the secretion of anti-PD-1 enhanced the antitumor activity of CAR T cells and prolonged overall survival.Conclusions: With constitutive anti-PD-1 secretion, CAR.αPD1-T cells are more functional and expandable, and more efficient at tumor eradication than parental CAR T cells. Collectively, our study presents an important and novel strategy that enables CAR T cells to achieve better antitumor immunity, especially in the treatment of solid tumors. Clin Cancer Res; 23(22); 6982-92. ©2017 AACR.

Combination Cancer Therapy Using Chimeric Antigen Receptor-Engineered Natural Killer Cells as Drug Carriers.

The therapeutic limitations of conventional chemotherapeutic drugs include chemo-resistance, tumor recurrence, and metastasis. Numerous nanoparticle-based active targeting approaches have emerged to enhance the intracellular concentration of drugs in tumor cells; however, efficient delivery of these systems to the tumor site while sparing healthy tissue remains elusive. Recently, much attention has been given to human immune-cell-directed nanoparticle drug delivery, because immune cells can traffic to the tumor and inflammatory sites. Natural killer cells are a subset of cytotoxic lymphocytes that play critical roles in cancer immunosurveillance. Engineering of the human natural killer cell line, NK92, to express chimeric antigen receptors to redirect their antitumor specificity has shown significant promise. We demonstrate that the efficacy of chemotherapy can be enhanced in vitro and in vivo while reducing off-target toxicity by using chimeric antigen receptor-engineered NK92 cells as carriers to direct drug-loaded nanoparticles to the target site.

Masked Chimeric Antigen Receptor for Tumor-Specific Activation.

Adoptive cellular therapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) cells is a powerful form of cancer immunotherapy. CAR-T cells can be redirected to specifically recognize tumor-associated antigens (TAAs) and induce high levels of antitumor activity. However, they may also display "on-target off-tumor" toxicities, resulting from low-level expression of TAAs in healthy tissues. These adverse effects have raised considerable safety concerns and limited the clinical application of this otherwise promising therapeutic modality. To minimize such side effects, we have designed an epidermal growth factor receptor (EGFR)-specific masked CAR (mCAR), which consists of a masking peptide that blocks the antigen-binding site and a protease-sensitive linker. Proteases commonly active in the tumor microenvironment can cleave the linker and disengage the masking peptide, thereby enabling CAR-T cells to recognize target antigens only at the tumor site. In vitro mCAR showed dramatically reduced antigen binding and antigen-specific activation in the absence of proteases, but normal levels of binding and activity upon treatment with certain proteases. Masked CAR-T cells also showed antitumor efficacy in vivo comparable to that of unmasked CAR. Our study demonstrates the feasibility of improving the safety profile of conventional CARs and may also inspire future design of CAR molecules targeting broadly expressed TAAs.

Generation of Targeted Adeno-Associated Virus (AAV) Vectors for Human Gene Therapy.

Adeno-associated virus (AAV) vectors are promising human gene delivery vehicles due to their ability to establish long-term gene expression in a wide variety of target tissues; however, the broad native viral tropism raises concerns over the feasibility and safety of their systemic administration. To overcome this issue, much effort has been made to redirect AAVs toward specific tissues. This review presents several design strategies that have been applied to generate AAVs that target specific tissues and cells while inhibiting the transduction of non-target tissues. Multiple methods of vector capsid engineering have shown promise in vitro, including indirect targeting by adaptor systems and direct targeting by the insertion of antibodies or receptor-specific small peptide motifs. Other strategies, including creating mosaic or chimeric capsids and directed evolution, have also been used to successfully retarget AAV vectors. This research will further expand the clinical applications of AAV vectors by enhancing the control over tissue-specific gene delivery.

Specific functions of the Wnt signaling system in gene regulatory networks throughout the early sea urchin embryo.

Wnt signaling affects cell-fate specification processes throughout embryonic development. Here we take advantage of the well-studied gene regulatory networks (GRNs) that control pregastrular sea urchin embryogenesis to reveal the gene regulatory functions of the entire Wnt-signaling system. Five wnt genes, three frizzled genes, two secreted frizzled-related protein 1 genes, and two Dickkopf genes are expressed in dynamic spatial patterns in the pregastrular embryo of Strongylocentrotus purpuratus. We present a comprehensive analysis of these genes in each embryonic domain. Total functions of the Wnt-signaling system in regulatory gene expression throughout the embryo were studied by use of the Porcupine inhibitor C59, which interferes with zygotic Wnt ligand secretion. Morpholino-mediated knockdown of each expressed Wnt ligand demonstrated that individual Wnt ligands are functionally distinct, despite their partially overlapping spatial expression. They target specific embryonic domains and affect particular regulatory genes. The sum of the effects of blocking expression of individual wnt genes is shown to equal C59 effects. Remarkably, zygotic Wnt-signaling inputs are required for only three general aspects of embryonic specification: the broad activation of endodermal GRNs, the regional specification of the immediately adjacent stripe of ectoderm, and the restriction of the apical neurogenic domain. All Wnt signaling in this pregastrular embryo is short range (and/or autocrine). Furthermore, we show that the transcriptional drivers of wnt genes execute important specification functions in the embryonic domains targeted by the ligands, thus connecting the expression and function of wnt genes by encoded cross-regulatory interactions within the specific regional GRNs.

Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides.

Adeno-associated virus type 2 (AAV2) is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs). We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy.