Efficacy In Tumor-bearing Mice Research Articles

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

A Personalized Cancer Nanovaccine that Enhances T-Cell Responses and Efficacy through Dual Interactions with Dendritic Cells and T Cells.

Conventional approaches to developing therapeutic cancer vaccines that primarily activate tumor-specific T cells via dendritic cells (DCs) often demonstrate limited efficacy due to the suboptimal activation of these T cells. To address this limitation, here we develop a therapeutic cancer nanovaccine that enhances T cell responses by interacting with both DCs and T cells. The nanovaccine is based on a cancer cell membrane nanoparticle (CCM-MPLA) that utilizes monophosphoryl lipid A (MPLA) as an adjuvant. To allow direct interaction between the nanovaccine and tumor-specific T cells, anti-CD28 antibodies (aCD28) are conjugated onto CCM-MPLA, resulting in CCM-MPLA-aCD28. This nanovaccine activates tumor-specific CD8+ T cells in both presence and absence of DCs. Compared with nanovaccines that interact with either DCs (CCM-MPLA) or T cells (CCM-aCD28), CCM-MPLA-aCD28 induces more potent responses of tumor-specific CD8+ T cells and exhibits a higher antitumor efficacy in tumor-bearing mice. No differences in T cell activation efficiency and therapeutic efficacy are observed between CCM-MPLA and CCM-aCD28. This approach may lead to the development of effective personalized therapeutic cancer vaccines prepared from autologous cancer cells. This article is protected by copyright. All rights reserved.

Modulating the Chemical Reactivity of Gold Complexes in Living Systems: From Concept to Biomedical Applications.

ConspectusOver the past few decades, research on the chemistry of gold has progressed rapidly, encompassing topics like catalysis, supramolecular chemistry, molecular recognition, etc. These chemical properties are of great value in developing therapeutics or orthogonal catalysts in biology. However, the presence of concentrated nucleophiles and reductants, particularly thiol-containing serum albumin in blood and glutathione (GSH) inside cells that can strongly bind and quench the active gold species, makes it difficult to translate the chemistry of gold from test tubes into living systems. In this regard, modulating the chemical reactivity of gold complexes to conquer nonspecific interactions with thiols and meanwhile to controllably activate their reactivity in a spatiotemporal manner is of pivotal importance to develop gold complexes for biomedical applications. In this account, we aim to highlight the concept of developing stimuli-activatable gold complexes with masked chemical properties, the bioactivity of which can be spatiotemporally activated at the target site by leveraging approaches from classic structure design to recently emerged photo- and bioorthogonal-activation.A straightforward approach to tuning the reactivity of gold complexes is based on structure modification. This is achieved by introducing strong carbon donor ligands, such as N-heterocyclic carbene, alkynyl, and diphosphine, to improve the stability of gold(I) complexes against off-target thiols. Likewise, GSH-responsive gold(III) prodrug and supramolecular Au(I)-Au(I) interaction have been harnessed to keep a reasonable stability against serum albumin and confer tumor-targeted cytotoxicity by inhibiting thiol- and selenol-containing thioredoxin reductase (TrxR) for potent cancer treatment in vivo. To achieve better spatiotemporal controllability, photoactivatable prodrugs are developed. These complexes are equipped with cyclometalated pincer-type ligands and carbanion or hydride as ancillary ligands, rendering high thiol-stability in the dark, but upon photoirradiation, the complexes can undergo unprecedented photoinduced ligand substitution, β-hydride elimination, and/or reduction to release active gold species for TrxR inhibition at the diseased tissue. To further improve the therapeutic activity, an oxygen-dependent conditional photoreactivity of gold(III) complexes by evolving from photodynamic into photoactivated chemotherapy has been achieved, resulting in highly potent antitumor efficacy in tumor-bearing mice. Of equal importance is harnessing the bioorthogonal activation approach by chemical inducers, as exemplified by a palladium-triggered transmetalation reaction to selectively activate the chemical reactivities of gold including its TrxR inhibition and catalytic activity in living cells and zebrafish. Collectively, strategies to modulate gold chemistry in vitro and in vivo are emerging, and it is hoped that this Account will spur the creation of better approaches to advance gold complexes closer to clinical application.

Novel MR imaging nanoprobe for hepatocellular carcinoma detection based on manganese-zinc ferrite nanoparticles: in vitro and in vivo assessments.

Achieving new contrast enhancer agents that can produce high-resolution images in magnetic resonance imaging (MRI) with a minimum dose and side effects has always been important. Herein, the pegylated curcumin-coated manganese-zinc ferrite nanoparticles (MZF@CA-PEG-CUR NPs) have been reported as an MR imaging nanoprobe in hepatocellular carcinoma detection in the murine model for the first time.In vitrostudies were done on HEPA 1-6 cancer cells and L929 as normal cells, andin vivostudies were done on hepatocellular carcinoma (HCC) using xenograft models of HCC. The prepared NP had a diameter of 105nm with narrow size distribution and was superparamagnetic with a saturated magnetization (Ms) of 39emu/g. The NP was biocompatible without any significant hemolysis and cytotoxicity. Prussian blue staining showed more cellular uptake of HEPA 1-6 compared to L929 control cells after incubation (P < 0.05). The concentration of Fe in mice blood confirmed the plasma half-life of about 3h; it seems the PEGylation increased the circulation time. ICP-OES of Fe showed the highest tumor localization for MZF@CA-CUR-PEG NPs, due to passive accumulation, compared to the other mice studied organs. The r2 relaxivity of NPs was 134.89mM-1s-1, andin vitroMRI demonstrated better effects in HEPA 1-6 cells than in L929 (P < 0.05). Also,in vivoMR images showed signal enhancement efficacy in tumor-bearing mice. This study demonstrated that the MZF@CA-CUR-PEG nanoprobe could be a promising candidate as an MR imaging agent in hepatocellular carcinoma early detection.

Integrin α2β1 Targeting DGEA-Modified Liposomal Doxorubicin Enhances Antitumor Efficacy against Breast Cancer.

Breast cancer was the leading cause of newly diagnosed cases of tumors in 2020, ranking as the second highest cause of female death. Chemotherapy remains the conventional treatment of choice for breast tumors in most clinical cases. However, it is often accompanied by a poor prognosis and severe side effects, resulting from an insufficient accumulation of the drug at tumor sites and an unsystematic distribution of the drug across the body. Inspired by the fact that breast tumor cells overexpress integrin α2β1 on the surface, we designed and constructed an integrin α2β1 targeting DGEA-modified liposomal doxorubicin (DGEA-Lipo-DOX) platform for application in breast cancer therapy. The DGEA-Lipo-DOX was stable with a uniform particle size of 121.1 ± 3.8 nm and satisfactory drug encapsulation. Demonstrated in vitro and in vivo, the constructed platform exhibited improved antitumor ability. The DGEA-Lipo-DOX showed 4-fold enhanced blood circulation and 6-fold increased accumulation of DOX at the tumor sites compared to those of free DOX, resulting in a significantly enhanced antitumor efficacy in tumor-bearing mice. A preliminary safety evaluation suggested that the systemic toxicity of DOX was relieved by DGEA-Lipo delivery. Collectively, binding integrin α2β1 by DGEA may represent an alternative therapeutic strategy for potentially safer breast cancer treatment.

Polyethyleneimine- -Tocopherol Hydrogen Succinate/Hyaluronic Acid-Quercetin (PEI-TOS/HA-QU) Core-Shell Micelles Delivering Paclitaxel for Combinatorial Treatment of MDR Breast Cancer.

Multidrug resistance (MDR) remains a significant impediment to chemotherapy during cancer therapy. In this study, the amphiphilic biomaterials PEI-TOS and HA-QU were synthesized to self-assemble into PEI-TOS/HA-QU core-shell micelles for the targeted codelivery of paclitaxel (PTX) and quercetin (QU) to alleviate multidrug drug resistance and enhance therapeutic efficacy. The PTX-loaded micelles possessed a uniform particle size (167.60 ± 8.185 nm), stable negative charge (-19.13 ± 0.321 mV), and pH-responsive drug release with good compatibility. The drug-loaded micelles increased the chemosensitivity of MDR tumor cells (MDA-MB-231/MDR1) to PTX and activated mitochondria-dependent apoptotic pathways (the IC50 was 2.22-fold lower than that of PTX alone). Moreover, PEI-TOS/HA-QU micelles increased the cellular uptake of lipophilic antitumor drugs by downregulating P-gp expression in MDA-MB-231/MDR1 cells. Compared with Taxol, PTX-loaded PEI-TOS/HA-QU micelles presented excellent antitumor efficacy in tumor-bearing mice, with an average tumor size that was 3.7-fold lower than that of the control group. The drug-loaded formulation showed low in vitro / in vivo toxicity and better tumor accumulation than the free drug, which led to a high tumor inhibition rate of 80.56% and considerable biocompatibility. This work describes a new platform for the codelivery of lipophilic anticancer drugs and natural active ingredients such as PTX and QU for the treatment of MDR cancer cells.

Pectin supplement significantly enhanced the anti-PD-1 efficacy in tumor-bearing mice humanized with gut microbiota from patients with colorectal cancer.

Background: Anti-PD-1-based immunotherapy has emerged as a promising therapy for several cancers. However, it only benefits a small subset of colorectal cancer (CRC) patients. Mounting data supports the pivotal role of gut microbiota in shaping immune system. Pectin, a widely consumed soluble fiber, has been reported to ameliorate the imbalance of gut microbiota. Therefore, we aimed to explore the effect and the underlying mechanisms of pectin in improving anti-PD-1 mAb efficacy.Methods: The C57BL/6 mice were treated with a broad-spectrum antibiotic (ATB) cocktail to depleted endogenous gut microbiota and subsequently humanized with feces from healthy controls or newly diagnosed CRC patients. The antitumor efficacies of anti-PD-1 mAb combined with or without pectin were assessed using these mice. Flow cytometry and immunohistochemistry (IHC) were conducted to investigate the tumor immune microenvironment after treatment. The gut microbiota profiles and short-chain fatty acids (SCFAs) levels were determined by 16S ribosomal RNA (16S rRNA) gene sequencing and gas chromatography-mass spectrometry (GC-MS), respectively. The effect of gut microbiota on anti-PD-1 mAb efficacy after pectin supplement was further tested by fecal microbiota transplantation (FMT).Results: The anti-PD-1 mAb efficacy was largely impaired in the mice humanized with feces from newly diagnosed CRC patients compared to those from healthy controls. However, pectin significantly enhanced the anti-PD-1 mAb efficacy in the tumor-bearing mice humanized with CRC patient gut microbiota. Flow cytometry and IHC analysis revealed increased T cell infiltration and activation in the tumor microenvironment of mice treated with anti-PD-1 mAb plus pectin. In vivo depletion of CD8+ T cells diminished the anti-tumor effect of anti-PD-1 mAb combined with pectin. 16S rRNA gene sequencing showed that pectin significantly increased gut microbial diversity and beneficially regulated microbial composition. In addition, we identified unique bacterial modules that were significantly enriched in the anti-PD-1 mAb + pectin group, which composed of butyrate-producing bacteria indicative of good response to immunotherapy. Meanwhile, GC-MS showed that pectin altered the level of SCFA butyrate. Furthermore, butyrate, a main product of dietary fiber in gut microbial fermentation, was found to be sufficient to promote T cells infiltration and thus enhance the efficacy of anti-PD-1 mAb. In addition, FMT demonstrated the effects of pectin were dependent on gut microbiota. Importantly, the beneficial effects of pectin were confirmed in the mice humanized with gut microbiota from patient with resistance to anti-PD-1 mAb.Conclusion: Pectin facilitated the anti-PD-1 mAb efficacy in CRC via regulating the T cell infiltration in the tumor microenvironment, which was potentially mediated by the metabolite butyrate.

Antitumor activity of the third generation EphA2 CAR-T cells against glioblastoma is associated with interferon gamma induced PD-L1

ABSTRACT Glioblastoma (GBM) is the most common and aggressive brain malignancy in adults and is currently incurable with conventional therapies. The use of chimeric antigen receptor (CAR) modified T cells has been successful in clinical treatment of blood cancers, except solid tumors such as GBM. This study generated two third-generation CARs targeting different epitopes of ephrin type-A receptor 2 (EphA2) and examined their anti-GBM efficacy in vitro and in tumor-bearing mice. We observed that these two types of T cells expressing CAR (CAR-T) targeting EphA2 could be activated and expanded by EphA2 positive tumor cells in vitro. The survival of tumor-bearing mice after EphA2 CAR-T cell treatment was significantly improved. T cells transduced with one of the two EphA2 CARs exhibited better anti-tumor activity, which is related to the upregulation of CXCR-1/2 and appropriate interferon-γ (IFN-γ) production. CAR-T cells expressed excessively high level of IFN-γ exhibited poor anti-tumor activity resulting from inducing the upregulation of PD-L1 in GBM cells. The combination of CAR-T cells with poor anti-tumor activity and PD1 blockade improved the efficacy in tumor-bearing mice. In conclusion, both types of EphA2 CAR-T cells eliminated 20%-50% of GBM in xenograft mouse models. The appropriate combination of IFN-γ and CXCR-1/2 levels is a key factor for evaluating the antitumor efficiency of CAR-T cells.

Effect of local activation via SQ3370 on the safety of chemotherapy with concomitant anti-tumor immune response.

e15525 Background: With systemic chemotherapy, only 1-2% of the dose given reaches a local tumor, while the remaining leads to off-target toxicities. Hence, there is a critical need to locally deliver cytotoxics directly to the tumor. Our approach (SQ3370) consists of: 1. SQL70 - drug-activating biomaterial carrying 2. SQP33 - chemically-modified prodrug of doxorubicin (Dox) with attenuated activity. Methods: SQL70 is injected at the tumor site followed by SQP33 given systemically. The prodrug is captured by the biomaterial due to their complementary chemical reactivities. The active drug is then spontaneously released, providing local delivery directly to the tumor region while reducing systemic side effects. Results: We have shown that the greater safety of SQ3370 allows significantly higher doses to be administered compared to conventional Dox. PK studies in rodents and dogs show highly efficient capture and activation of the prodrug, likely due to the rapid reaction of SQP33 with SQL70. Without SQL70, SQP33 shows minimal spontaneous conversion to active Dox. A single SQL70 injection selectively activated multiple doses of SQP33 at the target site, maximizing the local therapeutic index. Additionally, SQP33 was capable of being activated by SQL70 injected in various regions in the body, including peri/intratumoral, intraperitoneal, subcutaneous and intramuscular sites. SQ3370 treatment also enhances efficacy in tumor-bearing mice: In a syngeneic MC38 colorectal cancer model, SQ3370 significantly improved anti-tumor response and overall survival compared to conventional Dox. In mice bearing two tumors, SQL70 injection at one tumor site followed by systemic SQP33 treatment induced an anti-tumor response in both the injected and non-injected tumors. This technology also enabled a sustained anti-tumor response upon rechallenge with MC38 tumor cells, without additional treatment. Furthermore, SQ3370 increased CD4 and CD8 TILs and decreased regulatory T-cells in both injected and non-injected tumors, suggesting that SQ3370 can induce an immune-mediated anti-tumor effect, thus expanding potential treatment options in the clinic and highlighting the advantages of immune-sparing cytotoxic therapy. Conclusions: SQ3370 enables delivery of cytotoxic drugs to multiple target sites while limiting off-target exposure, leading to improved safety and efficacy. A multicenter, first-in-human, Phase 1 dose-escalation clinical trial (NCT04106492) in patients with advanced solid tumor malignancies is planned to commence enrollment in April, 2020.

Tumor Antigen Mediated Conformational Changes of Nanoplatform for Activated Photodynamic Therapy.

Photodynamic therapy (PDT) is a noninvasive powerful tool for tumor treatment. However, phototoxicity seriously limits the clinical application of PDT, and activated PDT specifically response to tumor cell antigen is rarely reported. Herein, a tumor cell specific "switch-on" PDT nanoplatform, which employs a well-designed hairpin structure mucl protein (MUC1) aptamer (Apt) as specific linker to conjugate gold nanorod and Chlorin e6 (Ce6) (GNR/Apt-Ce6) is prepared, and "switch on" via conformational changes of aptamer-induced fluorescence resonance energy transfer missing between GNR and Ce6 for selective tumor therapy. In the absence of tumor cells, MUC1 Apt keeps a hairpin structure, leading to Ce6 closely adhered to the surface of GNR, PDT is in an "off" state even under the irradiations. On the contrary, in the presence of tumor cells with overexpressed MUC1, Apt specifically recognizes and binds to MUC1, resulting in conformational changes of Apt from regular hairpin to extended chain structure. Thus with an enlarged distance between Ce6 and GNR, PDT is switched-on. GNR/Apt-Ce6 shows excellent PDT efficacy in tumor-bearing mice (55.1% vs 1.3%, tumor apoptosis rate of GNR/Apt-Ce6 vs GNR/random sequence-Ce6) due to its high tumor-targeting and "switch-on" properties. The strategy of tumor antigen activated PDT is expected to provide a new perspective for clinical application.

Abstract 4961: Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells and IL-12

Abstract Adoptive T cell therapy (ACT) is a promising new modality for malignancies. Here, we report that adoptive T cell efficacy in tumor-bearing mice is significantly affected by differences in the native composition of the gut microbiome or treatment with antibiotics, or by heterologous fecal transfer. Depletion of bacteria with vancomycin decreased the rate of tumor growth in mice from The Jackson Laboratory receiving ACT, whereas treatment with neomycin and metronidazole had no effect, indicating the role of specific bacteria in host response. Vancomycin treatment induced an increase in systemic CD8α+ DCs, which sustained systemic adoptively transferred antitumor T cells in an IL-12-dependent manner. In subjects undergoing allogeneic hematopoietic cell transplantation, we found that oral vancomycin also increased IL-12 levels. Collectively, our findings demonstrate an important role played by the gut microbiota in the antitumor effectiveness of ACT and suggest potentially new avenues to improve response to ACT by altering the gut microbiota. Citation Format: Mireia Uribe-Herranz, Kyle Bittinger, Stavros Rafail, Stefano Pierini, Sonia Guedan, Frederic Bushman, Carl June, Andrea Facciabene. Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells and IL-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4961.

Abstract 3798: Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells

Abstract Adoptive T-Cell therapy (ACT) is a promising new modality for malignancies. Here, we report that adoptive T-cell efficacy in tumor-bearing mice is significantly affected by differences in the native composition of the gut microbiome, treatment with antibiotics or by heterologous fecal transfer. Depletion of bacteria with vancomycin, decreased the rate of tumor growth in mice receiving ACT, whereas treatment with neomycin and metronidazole had no effect, indicating the role of specific bacteria in host response. Vancomycin treatment induced an increase in systemic CD8α+ dendritic cells, which mediated a sustained systemic expansion of adoptively transferred antitumor T cells in an IL-12-dependent manner. In subjects undergoing allogeneic hematopoietic cell transplantation, we found that oral vancomycin also increased IL-12 levels. Collectively, our findings demonstrate an important role played by the gut microbiota in the antitumor effectiveness of ACT and suggest new avenues to improve response to ACT by altering the gut microbiota. Citation Format: Mireia Uribe-Herranz, Kyle Bittinger, Stavros Rafail, Sonia Guedan, Stefano Pierini, Ceylan Tanes, Saar A. Gill, Janos Tany, Mark A. Morgan, Frederic D. Bushman, Carl H. June, Andrea Facciabene. Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3798.

Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells and IL-12.

Adoptive T cell therapy (ACT) is a promising new modality for malignancies. Here, we report that adoptive T cell efficacy in tumor-bearing mice is significantly affected by differences in the native composition of the gut microbiome or treatment with antibiotics, or by heterologous fecal transfer. Depletion of bacteria with vancomycin decreased the rate of tumor growth in mice from The Jackson Laboratory receiving ACT, whereas treatment with neomycin and metronidazole had no effect, indicating the role of specific bacteria in host response. Vancomycin treatment induced an increase in systemic CD8α+ DCs, which sustained systemic adoptively transferred antitumor T cells in an IL-12-dependent manner. In subjects undergoing allogeneic hematopoietic cell transplantation, we found that oral vancomycin also increased IL-12 levels. Collectively, our findings demonstrate an important role played by the gut microbiota in the antitumor effectiveness of ACT and suggest potentially new avenues to improve response to ACT by altering the gut microbiota.

Inhibition of intrinsic coagulation improves safety and tumor-targeted drug delivery of cationic solid lipid nanoparticles

Cationic solid lipid nanoparticles (cSLNs) are promising nanoparticles for controlled drug delivery. Increasing surface charge and/or reducing PEG density enhance cellular uptake of cSLNs in vitro, but for unknown reasons fail to improve drug delivery in vivo. Herein, we show that cSLNs present a risk for systemic platelet activation and aggregation in vivo, and this toxic effect can be significantly augmented by increasing the surface charge and reducing the PEG density. Furthermore, thrombotic toxicity significantly reduces blood circulation time and in vivo cellular uptake of cSLNs. Mechanistic studies revealed that the intrinsic coagulation pathway is responsible for cSLN-induced platelet activation. Importantly, pretreatment of the recipient mice with heparin, a clinically-approved intrinsic coagulation inhibitor, was highly effective in preventing toxicity, prolonging the circulation time of cSLNs, and improving cSLN-based antitumor drug delivery and therapeutic efficacy in tumor-bearing mice. This study offers a useful strategy for improving both the safety and efficacy of cSLN-based anticancer therapies.

Specific Inhibition of Bacterial β-Glucuronidase by Pyrazolo[4,3-c]quinoline Derivatives via a pH-Dependent Manner To Suppress Chemotherapy-Induced Intestinal Toxicity.

The direct inhibition of bacterial β-glucuronidase (βG) activity is expected to reduce the reactivation of glucuronide-conjugated drugs in the intestine, thereby reducing drug toxicity. In this study, we report on the effects of pyrazolo[4,3-c]quinolines acting as a new class of bacterial βG-specific inhibitors in a pH-dependent manner. Refinement of this chemotype for establishing structure-activity relationship resulted in the identification of potential leads. Notably, the oral administration of 3-amino-4-(4-fluorophenylamino)-1H-pyrazolo[4,3-c]quinoline (42) combined with chemotherapeutic CPT-11 treatment prevented CPT-11-induced serious diarrhea while maintaining the antitumor efficacy in tumor-bearing mice. Importantly, the inhibitory effects of 42 to E. coli βG was reduced as the pH decreased due to the various surface charges of the active pocket of the enzyme, which may make their combination more favorable at neutral pH. These results demonstrate novel insights into the potent bacterial βG-specific inhibitor that would allow this inhibitor to be used for the purpose of reducing drug toxicity.

Targeted Cancer Therapy Using Fusion Protein of TNFα and Tumor-Associated Fibronectin-Specific Aptide.

Tumor necrosis factor-α has shown potent antitumor effects in preclinical and clinical studies. However, severe side effects at less than therapeutic doses have limited its systemic delivery, prompting the need for a new strategy for targeted delivery of the protein to tumors. Here, we report a fusion protein of mouse tumor necrosis factor (TNF)-α (mTNFα) and a cancer-targeting, high-affinity aptide and investigate its therapeutic efficacy in tumor-bearing mice. A fusion protein consisting of mTNFα, a linker, and an aptide specific to extra domain B (EDB) of fibronectin (APTEDB), designated mTNFα-APTEDB, was successfully produced by expression in Escherichia coli. mTNFα-APTEDB retained specificity and affinity for its target, EDB. In mice bearing EDB-overexpressing fibrosarcomas, mTNFα-APTEDB showed greater efficacy in inhibiting tumor growth than mTNFα alone or mTNFα linked to a nonrelevant aptide, without causing an appreciable loss in body weight. Moreover, in vivo antitumor efficacy was further significantly increased by combination treatment with the chemotherapeutic drug, melphalan, suggesting a synergistic effect attributable to enhanced drug uptake into the tumor as a result of TNFα-mediated enhanced vascular permeability. These results suggest that a fusion protein of mTNFα with a cancer-targeting peptide could be a new anticancer therapeutic option for ensuring potent antitumor efficacy after systemic delivery.

Subcellular-Scale Drug Transport via Ultrasound-Degradable Mesoporous Nanosilicon to Bypass Cancer Drug Resistance.

Delivering and releasing anticancer agents directly to their subcellular targets of action in a controlled manner are almost the ultimate goal of pharmacology, but it is challenging. In recent decades, plenty of efforts have been made to send drugs to tumor tissue or even specifically to cancer cells; however, at the subcellular scale, cancer cells have multiple cunning ways to hinder drugs from reaching their final action targets. Here, we demonstrate a strategy to bypass the last defense of cancer drug resistance by contolling the drug transportation and release at subcellular scale. We developed a platform based on ultrasound-degradable mesoporous nanosilicon, which allows drug delivery towards, ultrasound controlled drug release into the cell nucleus. This strategy altered the drug distribution within cells and remarkably enhanced the drug accumulation ratio at the action target, i.e. nucleus. In vitro and in vivo studies proved that this strategy reduced the drug dosage by an order of magnitude, prolonged drug retention and amplified therapeutic efficacy in tumor-bearing mice. These results offer new insights into bypassing cancer drug resistance through transport and release drugs directly to their action targets in a controlled manner.

IL-10 expression defines an immunosuppressive dendritic cell population induced by antitumor therapeutic vaccination.

Vaccination induces immunostimulatory signals that are often accompanied by regulatory mechanisms such as IL-10, which control T-cell activation and inhibit vaccine-dependent antitumor therapeutic effect. Here we characterized IL-10-producing cells in different tumor models treated with therapeutic vaccines. Although several cell subsets produced IL-10 irrespective of treatment, an early vaccine-dependent induction of IL-10 was detected in dendritic cells (DC). IL-10 production defined a DC population characterized by a poorly mature phenotype, lower expression of T-cell stimulating molecules and upregulation of PD-L1. These IL-10+ DC showed impaired in vitro T-cell stimulatory capacity, which was rescued by incubation with IL-10R and PD-L1-inhibiting antibodies. In vivo IL-10 blockade during vaccination decreased the proportion of IL-10+ DC and improved their maturation, without modifying PD-L1 expression. Similarly, PD-L1 blockade did not affect IL- 10 expression. Interestingly, vaccination combined with simultaneous blockade of IL-10 and PD-L1 induced stronger immune responses, resulting in a higher therapeutic efficacy in tumor-bearing mice. These results show that vaccine-induced immunoregulatory IL- 10+ DC impair priming of antitumor immunity, suggesting that therapeutic vaccination protocols may benefit from combined targeting of inhibitory molecules expressed by this DC subset.

Abstract 2348: Nanovaccine : A novel immunotherapeutic strategy to treat bladder cancer

Abstract Introduction Bladder cancer is a significant healthcare problem. According to the National Cancer Institute, bladder cancer is the 6th most common type of cancer and represents 4.5% of total cancer patients in the U.S. Intravesical therapy with Bacillus Calmette-Guerin (BCG) vaccine is the gold standard for treating non-muscle invasive bladder cancer. However, BCG has several drawbacks. First, BCG is a tuberculosis vaccine, which often fails to trigger immune response against bladder cancer. This leads to low therapeutic efficacy and high recurrence rates. Secondly, intravesical injection is painful and causes urinary side effects, including burning sensation and increased frequency of urination. Therefore, a therapeutic regimen that can specifically treat bladder cancer and can be administered through other routes is highly needed. Immunotherapeutic strategy using nanoparticle can fulfill these needs. Polymeric nanoparticles encapsulating an immune adjuvant and co-injected with a tumor-associated antigen can trigger robust immune response against bladder cancer. Moreover, nanoparticles formulated using the FDA approved polymer poly (lactide-co-glycolide) (PLGA) is approved for human use and can be injected subcutaneously. Methods Nanoparticles encapsulating a novel imidazoquinoline derivative that activates both Toll-Like Receptor (TLR) 7 and 8 was used as vaccine adjuvants. These novel nanoparticle-encapsulated TLR agonists were used in combination with a model antigen, ovalbumin, to create the vaccine. This is referred to as nanovaccine in this study. C57BL/6 mice were vaccinated subcutaneously with 1-5 doses of the nanovaccine or various control treatments. Two days after the final vaccination dose, mice were sacrificed and organs were harvested. Splenocytes and lymphocytes were analyzed using flow cytometry. Results Nanovaccine improved dendritic cell activation in the lymph node. Frequency of co-stimulatory molecule CD80+ dendritic cells was two fold higher in the lymph node of vaccinated group. Greater activation of T cells and increased frequency of antigen-specific T cells were found in the spleen of vaccinated group. Antigen-specific CD44+ CD8 Tcell frequency was two fold higher in the vaccinated group. Nanovaccine elevated the frequency of both antigen-specific CD4 T cells and antigen-specific CD44+ CD4 T cells by ten fold. Conclusion Nanovaccine generated antigen-specific immune response that can maximize the efficacy of immunotherapy. Moreover, nanoparticles were physically stable and remained active after subcutaneous injection, which can overcome the administration difficulties of current BCG therapy. Next step is to generate nanovaccine with bladder tumor-specific antigen or using whole cell lysate and test the therapeutic efficacy in tumor bearing mice. Generation of a bladder cancer-specific immune response will allow stronger and durable inhibition of tumor growth and diminished risk of progression. Citation Format: Hyunjoon Kim, Lin Niu, Peter Larson, Katherine Murphy, Tamara Kucaba, David Ferguson, Thomas Griffith, Jayanth Panyam. Nanovaccine : A novel immunotherapeutic strategy to treat bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2348.

Incorporation of lapatinib into human serum albumin nanoparticles with enhanced anti-tumor effects in HER2-positive breast cancer

Lapatinib, a selective small-molecule dual-tyrosine kinase inhibitor of HER2 and EGFR, is effective in HER2-positive patients with advanced metastatic breast cancer. However, its low and variable oral absorption, large required daily dose and serious gastrointestinal side effects all limit its clinical use. Intravenous administration offers a good option to overcome these disadvantages. However, the poor solubility of lapatinib in water and organic solvents causes lapatinib to fail in a common injectable preparation. Considering lapatinib's high albumin binding ability (>99%), in this study, we developed human serum albumin nanoparticles loaded with lapatinib (LHNPs) by Nab technology for intravenous administration and investigated its efficacy against HER2-positive breast cancer. Raman shift, X-ray diffraction and X-ray photoelectron spectroscopy studies demonstrated that lapatinib was successfully incorporated into nanoparticles, and LHNPs exhibited good stability and sustained-release effect in vitro. LHNPs could be effectively taken up by SKBr3 cells in a concentration- and time-dependent manner, and the uptake was mediated by energy-dependent endocytosis, which involved clathrin-dependent pinocytosis. Furthermore, in vitro and in vivo data indicated that LHNPs presented the strong ability to induce apoptosis and superior anti-tumor efficacy in tumor-bearing mice to the commercial tablet Tykerb through the inhibition of HER2 phosphorylation. Subchronic toxicity assays indicated that LHNPs had no hepatic or kidney toxicity. With mature technology for industrial production and enhanced therapeutic effects, LHNPs are likely to have great potential as a safe therapeutic candidate against HER2-positive breast cancer in the clinic.