COVID-eVax, an electroporated DNA vaccine candidate encoding the SARS-CoV-2 RBD, elicits protective responses in animal models.

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.

MicroRNA-128-3p-mediated depletion of Drosha promotes lung cancer cell migration.

Alteration in microRNAs (miRNAs) expression is a frequent finding in human cancers. In particular, widespread miRNAs down-regulation is a hallmark of malignant transformation. In the present report, we showed that the miR-128-3p, which is up-regulated in lung cancer tissues, has Drosha and Dicer, two key enzymes of miRNAs processing, as the main modulation targets leading to the widespread down-regulation of miRNA expression. We observed that the miRNAs downregulation induced by miR-128-3p contributed to the tumorigenic properties of lung cancer cells. In particular, miR-128-3p-mediated miRNAs down-regulation contributed to aberrant SNAIL and ZEB1 expression thereby promoting the epithelial-to-mesenchymal transition (EMT) program. Drosha also resulted to be implicated in the control of migratory phenotype as its expression counteracted miR-128-3p functional effects. Our study provides mechanistic insights into the function of miR-128-3p as a key regulator of the malignant phenotype of lung cancer cells. This also enforces the remarkable impact of Drosha and Dicer alteration in cancer, and in particular it highlights a role for Drosha in non-small-cell lung cancer cells migration.

Human lung adenocarcinoma cell cultures derived from malignant pleural effusions as model system to predict patients chemosensitivity.

BACKGROUND: Lung cancer is the leading cause of cancer related deaths and Malignant Pleural Effusion (MPE) is a frequent complication. Current therapies suffer from lack of efficacy in a great percentage of cases, especially when cancer is diagnosed at a late stage. Moreover patients' responses vary and the outcome is unpredictable. Therefore, the identification of patients who will benefit most of chemotherapy treatment is important for accurate prognostication and better outcome. In this study, using malignant pleural effusions (MPE) from non-small cell lung cancer (NSCLC) patients, we established a collection of patient-derived Adenocarcinoma cultures which were characterized for their sensitivity to chemotherapeutic drugs used in the clinical practice. METHODS: Tumor cells present in MPEs of patients with NSCLC were isolated by density gradient centrifugation, placed in culture and genotyped by next generation sequencing. In a subset of cases patient derived xenografts (PDX) were obtained upon tumor cell inoculation in rag2/IL2 knock-out mice. Isolated primary cultures were characterized and tested for drug sensitivity by in vitro proliferation assays. Additivity, antagonism or synergy for combinatorial treatments were determined by analysis with the Calcusyn software. RESULTS: We have optimized isolation procedures and culture conditions to expand in vitro primary cultures from Malignant Pleural Effusions (MPEs) of patients affected by lung adenocarcinomas, the most frequent form of non small cell lung cancer. Using this approach we have been able to establish 16 primary cultures from MPEs. Cells were banked at low passages and were characterized for their mutational pattern by next generation sequencing for most common driver mutations in lung cancer. Moreover, amplified cultures were shown to engraft with high efficiency when injected in immunocompromised mice. Cancer cell sensitivity to drugs used in standard chemotherapy regimens was assessed either individually or in combination. Differential chemosensitivity and different mutation profiles were observed which suggests that this isolation method could provide a platform for predicting the efficacy of chemotherapy in the clinical setting. Most importantly for six patients it was possible to establish a correlation between drug response in vitro and response to therapy in the clinic. CONCLUSIONS: Results obtained using primary cultured cells from MPEs underscore the heterogeneity of NSCLC in advanced stage as indicated by drug response and mutation profile. Comparison of data obtained from in vitro assays with patients' responses to therapy leads to the conclusion that this strategy may provide a potentially useful approach for evaluating individual chemosensitivity profile and tailor the therapy accordingly. Furthermore, combining MPE-derived primary cultures with their genomic testing allows to identify patients eligible to trials with novel targeted agents.

Isolation and Characterization of Neutralizing Monoclonal Antibodies from a Large Panel of Murine Antibodies against RBD of the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.

Preclinical profile of antitumor activity of a novel hydrophilic camptothecin, ST1968.

ST1968 is a novel hydrophilic camptothecin (CPT) derivative of the 7-oxyiminomethyl series. Because ST1968 retained ability to form remarkably stable cleavable complexes, this study was done to investigate its preclinical profile of antitumor activity in a large panel of human tumor models, including irinotecan-resistant tumors. Although less potent than SN38 in vitro, i.v. administered ST1968 caused a marked tumor inhibition, superior to that of irinotecan, in most tested models. ST1968 exhibited an impressive activity against several tumors including models of ovarian and colon carcinoma in which a high rate of cures was observed. In the most responsive tumors, complete and persistent tumor regressions were achieved even with low suboptimal doses. Even tumors derived from intrinsically resistant cells exhibited a significant responsiveness. Histologic analysis of treated tumors supports a contribution of both proapoptotic and antiangiogenic effects to ST1968 antitumor efficacy. A study done in yeast cells transformed with CPT-resistant mutant forms of topoisomerase I documented that, in contrast to other tested CPT, ST1968 was active against yeasts expressing the mutant K720E enzyme. Based on its outstanding efficacy superior to that of irinotecan and of its good therapeutic index, ST1968 has been selected for clinical development.

Novel Symmetrical Benzazolyl Derivatives Endowed with Potent Anti-Heparanase Activity.

Heparanase is the only mammalian endo-ß-d-glucuronidase involved in a variety of major diseases. The up-regulation of heparanase expression increases tumor size, angiogenesis, and metastasis, representing a validated target in the anti-cancer field. To date, only a few small-molecule inhibitors have been described, but none have gotten through pre-clinical development. Previously, we explored 2-(4-(4-(bromo-methoxybenzamido)benzylamino)phenyl) benzazole derivatives as anti-heparanase agents, proposing this scaffold for development of broadly effective heparanase inhibitors. Herein, we report an extended investigation of new symmetrical 2-aminophenyl-benzazolyl-5-acetate derivatives, proving that symmetrical compounds are more effective than asymmetrical analogues, with the most-potent compound, 7g, being active at nanomolar concentration against heparanase. Molecular docking studies were performed on the best-acting compounds 5c and 7g to rationalize their interaction with the enzyme. Moreover, invasion assay confirmed the anti-metastatic potential of compounds 5c, 7a, and 7g, proving the inhibition of the expression of proangiogenic factors in tumor cells.

Novel Benzazole Derivatives Endowed with Potent Antiheparanase Activity.

Heparanase is the sole mammalian enzyme capable of cleaving glycosaminoglycan heparan sulfate side chains of heparan sulfate proteoglycans. Its altered activity is intimately associated with tumor growth, angiogenesis, and metastasis. Thus, its implication in cancer progression makes it an attractive target in anticancer therapy. Herein, we describe the design, synthesis, and biological evaluation of new benzazoles as heparanase inhibitors. Most of the designed derivatives were active at micromolar or submicromolar concentration, and the most promising compounds are fluorinated and/or amino acids derivatives 13a, 14d, and 15 that showed IC50 0.16-0.82 µM. Molecular docking studies were performed to rationalize their interaction with the enzyme catalytic site. Importantly, invasion assay confirmed the antimetastatic potential of compounds 14d and 15. Consistently with its ability to inhibit heparanase, compound 15 proved to decrease expression of genes encoding for proangiogenic factors such as MMP-9, VEGF, and FGFs in tumor cells.

[Corrigendum] Trastuzumab and docetaxel in a preclinical organotypic breast cancer model using tissue slices from mammary fat pad: Translational relevance.

Oncol Rep 34: [Related article:] 1146­1152, 2015; DOI: 10.3892/or.2015.4074 After the publication of the article, the authors decided to add an Acknowledgements section: Acknowledgments Research activity leading to the results shown in the paper is the continuation of the IMI Predect program that received support from the Innovative Medicines initiative Joint Undertaking under grant agreement no. 115188, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007­2013) and EFPIA companies in kind contribution. Present results and conclusions are not endorsed by the Predect Consortium.

Trastuzumab and docetaxel in a preclinical organotypic breast cancer model using tissue slices from mammary fat pad: Translational relevance.

With the ever-increasing number of drugs approved to treat cancers, selection of the optimal treatment regimen for an individual patient is challenging. Breast cancer complexity requires novel predictive methods and tools. In the present study, we set up experimental conditions to obtain an 'ex vivo' organotypic culture from xenotransplanted mice aiming at recapitulating the human clinical condition. The effect of trastuzumab (large biological molecule) and docetaxel (small chemical entity) was subsequently investigated on this organotypic model and compared with in vivo and in vitro activity on tumor cells. Tissue slices of 200 µm were obtained from mammary fat pad of SCID mice xenotransplanted with human MCF-7 breast cancer cells. Viability and proliferation were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay and Ki-67 immunohistochemistry,and apoptosis by cleaved caspase-3 immunohistochemistry. In vivo antitumor activity of trastuzumab and docetaxel was determined by caliper measurement of tumor volume and Ki-67 expression on explanted masses by immunohistochemistry. A Teflon support and normoxia were necessary experimental conditions to obtain high viability of excised breast cancer infiltrated mammary fat pad slices upon 48 h cultivation, as shown by MTT proliferation assay, and Ki-67 expression. Breast cancer tissue slices treated for 48 h with trastuzumab or docetaxel showed a significant dose­dependent reduction of viability by MTT assay. Consistently, both drugs down-modulated Ki-67 and increased cleaved caspase-3. Tumor masses collected from docetaxel- or trastuzumab­treated mice showed a similar reduction of proliferation markers. By contrast, MCF-7 cell cultures were significantly inhibited by docetaxel but not by trastuzumab. Tumor tissue slices represent a more predictive experimental cancer model compared to cell cultures for both small and large molecule antitumor efficacy. This observation supports the relevance of microenvironment in the overall tumor biology and response to therapeutics.

Synthesis and structure-activity relationships of new antiproliferative and proapoptotic retinoid-related biphenyl-4-yl-acrylic acids.

Atypical retinoids, or retinoid-related molecules (RRMs), represent a class of proapoptotic agents with a promising potential in the treatment of neoplastic diseases. In the present work, the synthesis and structure-activity relationship of a series of 3'-adamantan-1-yl-biphenyl-4-yl-acrylic acids substituted in ring A were studied. The synthesized compounds were evaluated for their antiproliferative activity in a human promyelocitic leukemia cell line (NB4), and in an ovarian carcinoma cell system including IGROV-1, carrying a functional wild-type p53, and a cisplatin-resistant subline, IGROV-1/Pt-1. The presence of at least one oxygenated substituent in positions 4' or 5' appears determinant for the antiproliferative activity. With two substituents of this kind the activity increases, particularly in the case of alkylenedioxy compounds. The activation of DNA damage response as indicated by phosphorylation of H2AX histone, RPA-2 protein, and p53 at serine 15 by the most apoptotic compounds provides additional support to the hypothesis that the genotoxic stress is a critical event mediating apoptosis induction by compounds of this group.

ST1926, a novel and orally active retinoid-related molecule inducing apoptosis in myeloid leukemia cells: modulation of intracellular calcium homeostasis.

Retinoid-related molecules (RRMs) are derivatives of retinoic acid and promising antileukemic agents with a mechanism of action different from that of other common chemotherapeutics. Here, we describe a novel chemical series designed against the RRM prototype, CD437. This includes molecules with apoptotic effects in acute promyelocytic leukemia and other myelogenous leukemia cell lines, as well as ST2065, an RRM with antagonistic properties. The most interesting apoptotic agent is ST1926, a compound more powerful than CD437 in vitro and orally active in vivo on severe combined immunodeficiency (SCID) mice that received transplants of NB4 cells. ST1926 has the same mechanism of action of CD437, as indicated by the ability to trans-activate retinoic acid receptor gamma, to induce the phosphorylation of p38 and JNK, and to down-regulate the expression of many genes negatively modulated by CD437. ST1926 causes an immediate increase in the cytosolic levels of calcium that are directly related to the apoptotic potential of the RRMs considered. The intracellular calcium elevation is predominantly the result of an inhibition of the mitochondrial calcium uptake. The phenomenon is blocked by the ST2065 antagonist, the intracellular calcium chelator BAPTA (1,2 bis (2-aminophenoxy) ethane-N, N, N',N'-tetraacetic acid tetrakis (acetoxymethyl ester), and by high concentrations of calcium blockers of the dihydropyridine type, compounds that suppress ST1926-induced apoptosis.

Bis-indols: a novel class of molecules enhancing the cytodifferentiating properties of retinoids in myeloid leukemia cells.

Enhancing the pharmacologic activity of all-trans retinoic acid (ATRA) is potentially useful in the management of acute promyelocytic leukemia (APL) and other types of myeloid leukemia. In this report, we identify a novel class of experimental agents selectively potentiating the cytodifferentiating activity of ATRA and synthetic retinoic acid receptor alpha agonists in APL and other myeloid leukemia cell lines. These agents have a bis-indolic structure (BISINDS), and ST1346 is the prototypical compound of the series. Gene-profiling experiments and determination of the level of expression of myeloid-associated markers indicate that ST1346 stimulates many aspects of the granulocytic maturation process set in motion by ATRA. Stimulation of the cytodifferentiating activity of ATRA by ST1346 enhances the efficacy of the retinoid in vivo, as demonstrated in the APL model of the severe combined immunodeficiency (SCID) mouse receiving transplants of NB4 cells. Although the molecular mechanisms underlying the ATRA-potentiating action of ST1346 and congeners have not been completely clarified, bis-indols are not ligands and do not exert any direct effect on the ATRA-dependent transactivation of nuclear receptors. However, ST1346 inhibits the down-regulation of cyclic adenosine monophosphate (cAMP)-dependent CREB transcriptional complexes and enhances the level of expression of signal transducers and activators of transcription-1 (STAT1), 2 putative molecular determinants of the differentiation process activated by ATRA in APL cells. More importantly, ST1346 relieves the down-regulation of Jun N-terminal kinases (JNK) afforded by ATRA. In addition, a specific JNK inhibitor blocks the enhancing effect of ST1346 on ATRA-induced maturation of NB4 cells. This demonstrates an important role for the mitogen-activated protein kinase in the molecular mechanisms underlying the pharmacologic activity of the bis-indol.