GD2-targeting CAR T-cell therapy for patients with GD2+ medulloblastoma.

PURPOSE: Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation and chemotherapy, is often responsible for cognitive, neurologic and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T-cells directed towards the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. EXPERIMENTAL DESIGN: GD2 expression was evaluated on primary tumor biopsies of MB children by flow-cytometry. GD2 expression in MB cells was evaluated also in response to an EZH2 inhibitor (Tazemetostat). In in vitro, as well as in in vivo models, GD2+MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible-caspase-9. RESULTS: GD2 was expressed in 73.17% of MB tumors. The SHH and G4 subtypes expressed the highest levels of GD2, while the WNT subtype the lowest. In in-vitro co-culture assays, CAR.GD2 T-cells were able to kill GD2+MB cells. Pre-treatment with Tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T-cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T-cells significantly controlled tumor growth, prolonging overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood brain barrier and to eliminate both blood circulating and tumor infiltrating CAR.GD2 T-cells. CONCLUSIONS: Our experimental data indicate the feasibility of CAR.GD2 T-cell therapy. A phase I/II clinical trial will be conducted to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

Neutralizing IFNγ improves safety without compromising efficacy of CAR-T cell therapy in B-cell malignancies.

Chimeric antigen receptor T (CAR-T) cell therapy may achieve long-lasting remission in patients with B-cell malignancies not responding to conventional therapies. However, potentially severe and hard-to-manage side effects, including cytokine release syndrome (CRS), neurotoxicity and macrophage activation syndrome, and the lack of pathophysiological experimental models limit the applicability and development of this form of therapy. Here we present a comprehensive humanized mouse model, by which we show that IFNγ neutralization by the clinically approved monoclonal antibody, emapalumab, mitigates severe toxicity related to CAR-T cell therapy. We demonstrate that emapalumab reduces the pro-inflammatory environment in the model, thus allowing control of severe CRS and preventing brain damage, characterized by multifocal hemorrhages. Importantly, our in vitro and in vivo experiments show that IFNγ inhibition does not affect the ability of CD19-targeting CAR-T (CAR.CD19-T) cells to eradicate CD19+ lymphoma cells. Thus, our study provides evidence that anti-IFNγ treatment might reduce immune related adverse effect without compromising therapeutic success and provides rationale for an emapalumab-CAR.CD19-T cell combination therapy in humans.

Dimethylmyricacene: An In Vitro and In Silico Study of a Semisynthetic Non-Camptothecin Derivative Compound, Targeting Human DNA Topoisomerase 1B.

Human topoisomerase 1B regulates the topological state of supercoiled DNA enabling all fundamental cell processes. This enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by nicking one DNA strand and forming a transient protein-DNA covalent complex. The interaction of human topoisomerase 1B and dimethylmyricacene, a compound prepared semisynthetically from myricanol extracted from Myrica cerifera root bark, was investigated using enzymatic activity assays and molecular docking procedures. Dimethylmyricacene was shown to inhibit both the cleavage and the religation steps of the enzymatic reaction, and cell viability of A-253, FaDu, MCF-7, HeLa and HCT-116 tumor cell lines.

Direct CD32 T-cell cytotoxicity: implications for breast cancer prognosis and treatment.

The FcγRII (CD32) ligands are IgFc fragments and pentraxins. The existence of additional ligands is unknown. We engineered T cells with human chimeric receptors resulting from the fusion between CD32 extracellular portion and transmembrane CD8α linked to CD28/ζ chain intracellular moiety (CD32-CR). Transduced T cells recognized three breast cancer (BC) and one colon cancer cell line among 15 tested in the absence of targeting antibodies. Sensitive BC cell conjugation with CD32-CR T cells induced CD32 polarization and down-regulation, CD107a release, mutual elimination, and proinflammatory cytokine production unaffected by human IgGs but enhanced by cetuximab. CD32-CR T cells protected immunodeficient mice from subcutaneous growth of MDA-MB-468 BC cells. RNAseq analysis identified a 42 gene fingerprint predicting BC cell sensitivity and favorable outcomes in advanced BC. ICAM1 was a major regulator of CD32-CR T cell-mediated cytotoxicity. CD32-CR T cells may help identify cell surface CD32 ligand(s) and novel prognostically relevant transcriptomic signatures and develop innovative BC treatments.

From Antarctica to cancer research: a novel human DNA topoisomerase 1B inhibitor from Antarctic sponge Dendrilla antarctica.

Nature has been always a great source of possible lead compounds to develop new drugs against several diseases. Here we report the identification of a natural compound, membranoid G, derived from the Antarctic sponge Dendrilla antarctica displaying an in vitro inhibitory activity against human DNA topoisomerase 1B. The experiments indicate that membranoid G, when pre-incubated with the enzyme, strongly and irreversibly inhibits the relaxation of supercoiled DNA. This compound completely inhibits the cleavage step of the enzyme catalytic mechanism by preventing protein binding to the DNA. Membranoid G displays also a cytotoxic effect on tumour cell lines, suggesting its use as a possible lead compound to develop new anticancer drugs.

Cow Milk Extracellular Vesicle Effects on an In Vitro Model of Intestinal Inflammation.

Extracellular vesicles (EVs) are lipid bilayer nano-dimensional spherical structures and act mainly as signaling mediators between cells, in particular modulating immunity and inflammation. Milk-derived EVs (mEVs) can have immunomodulatory and anti-inflammatory effects, and milk is one of the most promising food sources of EVs. In this context, this study aimed to evaluate bovine mEVs anti-inflammatory and immunomodulating effects on an in vitro co-culture (Caco-2 and THP-1) model of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release through ELISA. After establishing a pro-inflammatory environment due to IFN-γ and LPS stimuli, CXCL8, IL1B, TNFA, IL12A, IL23A, TGFB1, NOS2, and MMP9 were significantly up-regulated in inflamed Caco-2 compared to the basal co-culture. Moreover, IL-17, IL-1ß, IL-6, TNF-α release was increased in supernatants of THP-1. The mEV administration partially restored initial conditions with an effective anti-inflammatory activity. Indeed, a decrease in gene expression and protein production of most of the tested cytokines was detected, together with a significant gene expression decrease in MMP9 and the up-regulation of MUC2 and TJP1. These results showed a fundamental capability of mEVs to modulate inflammation and their potential beneficial effect on the intestinal mucosa.

In Vitro and In Silico Characterization of an Antimalarial Compound with Antitumor Activity Targeting Human DNA Topoisomerase IB.

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.

Natural Compounds as Therapeutic Agents: The Case of Human Topoisomerase IB.

Natural products are widely used as source for drugs development. An interesting example is represented by natural drugs developed against human topoisomerase IB, a ubiquitous enzyme involved in many cellular processes where several topological problems occur due the formation of supercoiled DNA. Human topoisomerase IB, involved in the solution of such problems relaxing the DNA cleaving and religating a single DNA strand, represents an important target in anticancer therapy. Several natural compounds inhibiting or poisoning this enzyme are under investigation as possible new drugs. This review summarizes the natural products that target human topoisomerase IB that may be used as the lead compounds to develop new anticancer drugs. Moreover, the natural compounds and their derivatives that are in clinical trial are also commented on.

Swapping of The N-Terminal Domain of Human Topoisomerase 1B with the Corresponding Plasmodium Falciparum Counterpart Strongly Impairs Enzyme Activity.

BACKGROUND: DNA topoisomerases 1B are a class of ubiquitous enzyme that solves the topological problems associated with biological processes such as replication, transcription and recombination. Numerous sequence alignment of topoisomerase 1B from different species shows that the lengths of different domains as well as their amino acids sequences are quite different. In the present study a hybrid enzyme, generated by swapping the N-terminal of Plasmodium falciparum into the corresponding domain of the human, has been characterized. METHODS: The chimeric enzyme was generated using different sets of PCR. The in vitro characterization was carried out using different DNA substrate including radio-labelled oligonucleotides. RESULTS: The chimeric enzyme displayed slower relaxation activity, cleavage and re-ligation kinetics strongly perturbed when compared to the human enzyme. CONCLUSION: These results indicate that the N-terminal domain has a crucial role in modulating topoisomerase activity in different species.

Topoisomerase IB: a relaxing enzyme for stressed DNA.

DNA topoisomerase I enzymes relieve the torsional strain in DNA; they are essential for fundamental molecular processes such as DNA replication, transcription, recombination, and chromosome condensation; and act by cleaving and then religating DNA strands. Over the past few decades, scientists have focused on the DNA topoisomerases biological functions and established a unique role of Type I DNA topoisomerases in regulating gene expression and DNA chromosome condensation. Moreover, the human enzyme is being investigated as a target for cancer chemotherapy. The active site tyrosine is responsible for initiating two transesterification reactions to cleave and then religate the DNA backbone, allowing the release of superhelical tension. The different steps of the catalytic mechanism are affected by various inhibitors; some of them prevent the interaction between the enzyme and the DNA while others act as poisons, leading to TopI-DNA lesions, breakage of DNA, and eventually cellular death. In this review, our goal is to provide an overview of mechanism of human topoisomerase IB action together with the different types of inhibitors and their effect on the enzyme functionality.

CD16-158-valine chimeric receptor T cells overcome the resistance of KRAS-mutated colorectal carcinoma cells to cetuximab.

KRAS mutations hinder therapeutic efficacy of epidermal growth factor receptor (EGFR)-specific monoclonal antibodies cetuximab and panitumumab-based immunotherapy of EGFR+ cancers. Although cetuximab inhibits KRAS-mutated cancer cell growth in vitro by natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), KRAS-mutated colorectal carcinoma (CRC) cells escape NK cell immunosurveillance in vivo. To overcome this limitation, we used cetuximab and panitumumab to redirect Fcγ chimeric receptor (CR) T cells against KRAS-mutated HCT116 colorectal cancer (CRC) cells. We compared four polymorphic Fcγ-CR constructs including CD16158F -CR, CD16158V -CR, CD32131H -CR, and CD32131R -CR transduced into T cells by retroviral vectors. Percentages of transduced T cells expressing CD32131H -CR (83.5 ± 9.5) and CD32131R -CR (77.7 ± 13.2) were significantly higher than those expressing with CD16158F -CR (30.3 ± 10.2) and CD16158V -CR (51.7 ± 13.7) (p < 0.003). CD32131R -CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16158V -CR T cells released high levels of interferon gamma (IFNγ = 1,145.5 pg/ml ±16.5 pg/ml, p < 0.001) and tumor necrosis factor alpha (TNFα = 614 pg/ml ± 21 pg/ml, p < 0.001) upon incubation with cetuximab-opsonized HCT116 cells. Moreover, only CD16158V -CR T cells combined with cetuximab killed HCT116 cells and A549 KRAS-mutated cells in vitro. CD16158V -CR T cells also effectively controlled subcutaneous growth of HCT116 cells in CB17-SCID mice in vivo. Thus, CD16158V -CR T cells combined with cetuximab represent useful reagents to develop innovative EGFR+KRAS-mutated CRC immunotherapies.

In vitro elimination of epidermal growth factor receptor-overexpressing cancer cells by CD32A-chimeric receptor T cells in combination with cetuximab or panitumumab.

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). Although the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects the poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on natural killer (NK) cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32A) to a similar extent. Our study compares the ability of T cells, engineered with a novel low-affinity CD32A131R -chimeric receptor (CR), and those engineered with the low-affinity CD16158F -CR T cells, in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. After T-cell transduction, the percentage of CD32A131R -CR T cells was 74 ± 10%, whereas the percentage of CD16158F -CR T cells was 46 ± 15%. Only CD32A131R -CR T cells bound panitumumab. CD32A131R -CR T cells combined with the mAb 8.26 (anti-CD32) and CD16158F -CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116FcγR+ cells, in a reverse ADCC assay in vitro. Crosslinking of CD32A131R -CR on T cells by cetuximab or panitumumab and CD16158F -CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and the secretion of interferon gamma and tumor necrosis factor alpha. Neither cetuximab nor panitumumab induced Fcγ-CR T antitumor activity against Kirsten rat sarcoma (KRAS)-mutated HCT116, nonsmall-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. The ADCC of Fcγ-CR T cells was associated with the overexpression of EGFR on ECCs. In conclusion, CD32A131R -CR T cells are efficiently redirected by cetuximab or panitumumab against breast cancer cells overexpressing EGFR.

Recent perspective on CAR and Fcγ-CR T cell immunotherapy for cancers: Preclinical evidence versus clinical outcomes.

The chimeric antigen receptor T cell (CAR-T cell) immunotherapy currently represents a hot research trend and it is expected to revolutionize the field of cancer therapy. Promising outcomes have been achieved using CAR-T cell therapy for haematological malignancies. Despite encouraging results, several challenges still pose eminent hurdles before being fully recognized. Directing CAR-T cells to target a single tumour associated antigen (TAA) as the case in haematological malignancies might be much simpler than targeting the extensive inhibitory microenvironments associated with solid tumours. This review focuses on the basic principles involved in development of CAR-T cells, emphasizing the differences between humoral IgG, T-cell receptors, CAR and Fcγ-CR constructs. It also highlights the complex inhibitory network that is usually associated with solid tumours, and tackles recent advances in the clinical studies that have provided great hope for the future use of CAR-T cell immunotherapy. While current Fcγ-CR T cell immunotherapy is in pre-clinical stage, is expected to provide a sound therapeutic approach to add to existing classical chemo- and radio-therapeutic modalities.

The human DNA topoisomerase I mutant Gly717Asp: Higher religation rate is not always associated with camptothecin resistance.

DNA topoisomerases are key enzyme responsible for modulating the topological state of the DNA by breaking and rejoining of DNA strand. Characterization of a Gly717Asp mutation in the human topoisomerase was performed using several catalytic assays. The mutant enzyme was shown to have comparable cleavage and fast religation rate as compared to the wild-type protein. Addition of the anticancer drug camptothecin significantly reduced the religation step. The simulative approaches and analysis of the cleavage/religation equilibrium indicate that the mutation is able to modify the architecture of the drug binding site, increasing the persistence of the drug for the enzyme-DNA covalent complex. Taken together these results indicate that the structure modification of the drug binding site is the key reason for the increasing CPT persistence and furthermore provide the possibility for new anti-cancer drug discovery.

Engineering a responsive DNA triple helix into an octahedral DNA nanostructure for a reversible opening/closing switching mechanism: a computational and experimental integrated study.

We propose an experimental and simulative approach to study the effect of integrating a DNA functional device into a large-sized DNA nanostructure. We selected, as a test bed, a well-known and characterized pH-dependent clamp-switch, based on a parallel DNA triple helix, to be integrated into a truncated octahedral scaffold. We designed, simulated and experimentally characterized two different functionalized DNA nanostructures, with and without the presence of a spacer between the scaffold and the functional elements. The experimental and simulative data agree in validating the need of a spacer for the occurrence of the pH dependent switching mechanism. The system is fully reversible and the switching can be monitored several times without any perturbation, maintaining the same properties of the isolated clamp switch in solution.

Entry, fate and degradation of DNA nanocages in mammalian cells: a matter of receptors.

DNA has been used to build nanostructures with potential biomedical applications. However, their use is limited by the lack of information on the mechanism of entry, intracellular fate and degradation rate of nanostructures inside cells. We generated octahedral DNA nanocages functionalized with folic acid and investigated the cellular uptake mediated by two distinctive internalization pathways, using two cellular systems expressing the oxidized low-density lipoprotein receptor-1 (LOX-1) and the α isoform of the folate receptor (αFR), respectively. Here, we report that DNA nanocages are very efficiently and selectively internalized by both receptors with an efficiency at least 30 times higher than that observed in cells not expressing the receptors. When internalized by LOX-1, nanocages traffic to lysosomes within 4 hours and are rapidly degraded. When the uptake is mediated by αFR, DNA nanocages are highly stable (>48 hours) and accumulate inside cells in a time-dependent way. These data demonstrate that the selection of the cellular receptor is crucial for targeting specific sub-cellular compartments and for modulating the DNA nanocage intracellular half-life, indicating that vitamin-mediated uptake may constitute a protected pathway for intracellular drug delivery.

Real-time analysis of cleavage and religation activity of human topoisomerase 1 based on ternary fluorescence resonance energy transfer DNA substrate.

Human topoisomerase 1B is a ubiquitous and essential enzyme involved in relaxing the topological state of supercoiled DNA to allow the progression of fundamental DNA metabolism. Its enzymatic catalytic cycle consists of cleavage and religation reaction. A ternary fluorescence resonance energy transfer biosensor based on a suicide DNA substrate conjugated with three fluorophores has been developed to monitor both cleavage and religation Topoisomerase I catalytic function. The presence of fluorophores does not alter the specificity of the enzyme catalysis on the DNA substrate. The enzyme-mediated reaction can be tracked in real-time by simple fluorescence measurement, avoiding the use of risky radioactive substrate labeling and time-consuming denaturing gel electrophoresis. The method is applied to monitor the perturbation brought by single mutation on the cleavage or religation reaction and to screen the effect of the camptothecin anticancer drug monitoring the energy transfer decrease during religation reaction. Pathological mutations usually affect only the cleavage or the religation reaction and the proposed approach represent a fast protocol for assessing chemotherapeutic drug efficacy and analyzing mutant's properties.

Selective targeting and degradation of doxorubicin-loaded folate-functionalized DNA nanocages.

Selective targeting is a crucial property of nanocarriers used for drug delivery in cancer therapy. We generated biotinylated octahedral DNA nanocages functionalized with folic acid through bio-orthogonal conjugation chemistry. Molecular modelling indicated that a distance of about 2.5 nm between folic acid and DNA nanocage avoids steric hindrance with the folate receptor. HeLa cells, a folate receptor positive tumour cell line, internalize folate-DNA nanocages with efficiency greater than 40 times compared to cells not expressing the folate receptors. Functionalized DNA nanocages are highly stable, not cytotoxic and can be efficiently loaded with the chemotherapeutic agent doxorubicin. After entry into cells, doxorubicin-loaded nanoparticles are confined in vesicular structures, indicating that DNA nanocages traffic through the endocytic pathway. Doxorubicin release from loaded DNA cages, facilitated by low pH of endocytic vesicles, induces toxic pathways that, besides selectively killing folate receptor-positive cancer cells, leads to cage degradation avoiding nanoparticles accumulation inside cells.

Simulative and Experimental Characterization of a pH-Dependent Clamp-like DNA Triple-Helix Nanoswitch.

Here we couple experimental and simulative techniques to characterize the structural/dynamical behavior of a pH-triggered switching mechanism based on the formation of a parallel DNA triple helix. Fluorescent data demonstrate the ability of this structure to reversibly switch between two states upon pH changes. Two accelerated, half microsecond, MD simulations of the system having protonated or unprotonated cytosines, mimicking the pH 5.0 and 8.0 conditions, highlight the importance of the Hoogsteen interactions in stabilizing the system, finely depicting the time-dependent disruption of the hydrogen bond network. Urea-unfolding experiments and MM/GBSA calculations converge in indicating a stabilization energy at pH 5.0, 2-fold higher than that observed at pH 8.0. These results validate the pH-controlled behavior of the designed structure and suggest that simulative approaches can be successfully coupled with experimental data to characterize responsive DNA-based nanodevices.

DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family.

In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands.