A new MVA ancestor-derived oncolytic vaccinia virus induces immunogenic tumor cell death and robust antitumor immune responses.

Vaccinia viruses (VACVs) are versatile therapeutic agents and different features of various VACV strains allow for a broad range of therapeutic applications. Modified VACV Ankara (MVA) is a particularly altered VACV strain that is highly immunogenic, incapable of replicating in mammalian hosts, and broadly used as a safe vector for vaccination. Alternatively, Western Reserve (WR) or Copenhagen (Cop) are VACV strains that efficiently replicate in cancer cells and, therefore, are used to develop oncolytic viruses. However, the immune evasion capacity of WR or Cop hinders their ability to elicit antitumor immune responses, which is crucial for efficacy in the clinic. Here, we describe a new VACV strain named Immune-Oncolytic VACV Ankara (IOVA), which combines efficient replication in cancer cells with induction of immunogenic tumor cell death (ICD). IOVA was engineered from an MVA ancestor and shows superior cytotoxicity in tumor cells. In addition, the IOVA genome incorporates mutations that lead to massive fusogenesis of tumor cells, which contributes to improved antitumor effects. In syngeneic mouse tumor models, the induction of ICD results in robust antitumor immunity directed against tumor neo-epitopes and eradication of large established tumors. These data present IOVA as an improved immunotherapeutic oncolytic vector.

4-Membered Ring Carbocations: A Positive Development in the Synthesis of 3,3-Disubstituted Oxetanes and Azetidines.

4-Membered heterocycles are low molecular weight polar scaffolds with intriguing potential for drug discovery. Despite their unquestionable value, methods to access such heterocycles remain scant. Here, we describe the generation of oxetane- and azetidine- benzylic carbocations as a general strategy to access valuable 3,3-disubstituted derivatives.

Oxetanes in Drug Discovery Campaigns.

The oxetane ring is an emergent, underexplored motif in drug discovery that shows attractive properties such as low molecular weight, high polarity, and marked three-dimensionality. Oxetanes have garnered further interest as isosteres of carbonyl groups and as molecular tools to fine-tune physicochemical properties of drug compounds such as pKa, LogD, aqueous solubility, and metabolic clearance. This perspective highlights recent applications of oxetane motifs in drug discovery campaigns (2017-2022), with emphasis on the effect of the oxetane on medicinally relevant properties and on the building blocks used to incorporate the oxetane ring. Based on this analysis, we provide an overview of the potential benefits of appending an oxetane to a drug compound, as well as potential pitfalls, challenges, and future directions.

Newly Designed Poxviral Promoters to Improve Immunogenicity and Efficacy of MVA-NP Candidate Vaccines against Lethal Influenza Virus Infection in Mice.

Influenza, a respiratory disease mainly caused by influenza A and B, viruses of the Orthomyxoviridae, is still a burden on our society's health and economic system. Influenza A viruses (IAV) circulate in mammalian and avian populations, causing seasonal outbreaks with high numbers of cases. Due to the high variability in seasonal IAV triggered by antigenic drift, annual vaccination is necessary, highlighting the need for a more broadly protective vaccine against IAV. The safety tested Modified Vaccinia virus Ankara (MVA) is licensed as a third-generation vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against different pathogens. Here, we generated and characterized recombinant MVA candidate vaccines that deliver the highly conserved internal nucleoprotein (NP) of IAV under the transcriptional control of five newly designed chimeric poxviral promoters to further increase the immunogenic properties of the recombinant viruses (MVA-NP). Infections of avian cell cultures with the recombinant MVA-NPs demonstrated efficient synthesis of the IAV-NP which was expressed under the control of the five new promoters. Prime-boost or single shot immunizations in C57BL/6 mice readily induced circulating serum antibodies' binding to recombinant IAV-NP and the robust activation of IAV-NP-specific CD8+ T cell responses. Moreover, the MVA-NP candidate vaccines protected C57BL/6 mice against lethal respiratory infection with mouse-adapted IAV (A/Puerto Rico/8/1934/H1N1). Thus, further studies are warranted to evaluate the immunogenicity and efficacy of these recombinant MVA-NP vaccines in other IAV challenge models in more detail.

Synthesis of oxetane and azetidine ethers as ester isosteres by Brønsted acid catalysed alkylation of alcohols with 3-aryl-oxetanols and 3-aryl-azetidinols.

Oxetanes and azetidines continue to draw significant interest in medicinal chemistry, as small, polar and non-planar motifs. Oxetanes also represent interesting surrogates for carbonyl-containing functional groups. Here we report a synthesis of 3,3-disubstituted oxetane- and azetidine-ethers, with comparisons made to the ester functional group. The tertiary benzylic alcohols of the 4-membered rings are selectively activated using Brønsted acid catalysis and reacted with simple alcohols to form the ethers and maintain the oxetane ring intact. This approach avoids the use of strong bases and halide alkylating agents and allows alcohol libraries to be leveraged. Oxetane ethers demonstrate excellent chemical stability across a range of conditions and an improved stability vis-à-vis analogous esters under basic and reducing conditions.

Metastatic clear-cell renal carcinoma: an exceptional cause of ileocolonic intussusception in adults

Introduction: Intussusceptions in adults are rare, representing 1% to 5% of intestinal obstructions in this age group. This condition can be caused by benign and malignant lesions acting as lead points, the latter being the most frequent. Furthermore, the diagnosis is challenging due to the non-specific symptoms with variable duration. Case Presentation: A 43-year-old man, with a history of localized clear-cell renal carcinoma (ccRCC) treated 9 years earlier with a right radical nephrectomy, presented with bowel obstruction symptoms. An abdominal computed tomography scan showed an ileocolonic intussusception. Hence, the patient required a right hemicolectomy with ileotransverse anastomosis. The histopathological analysis showed a metastatic ccRC to the terminal ileum causing the intussusception. Discussion: Adult intussusceptions are rare. However, they should be considered in the differential diagnosis of patients with abdominal pain and symptoms of bowel obstruction. Metastases of renal cancer to the small bowel are uncommon and even more so in the form of intussusception. Definitive treatment must be tailored to the patient's condition and underlying cause. (AU)

Improving poxvirus-mediated antitumor immune responses by deleting viral cGAMP-specific nuclease.

cGAMP-specific nucleases (poxins) are a recently described family of proteins dedicated to obstructing cyclic GMP-AMP synthase signaling (cGAS), an important sensor triggered by cytoplasmic viral replication that activates type I interferon (IFN) production. The B2R gene of vaccinia viruses (VACV) codes for one of these nucleases. Here, we evaluated the effects of inactivating the VACV B2 nuclease in the context of an oncolytic VACV. VACV are widely used as anti-cancer vectors due to their capacity to activate immune responses directed against tumor antigens. We aimed to elicit robust antitumor immunity by preventing viral inactivation of the cGAS/STING/IRF3 pathway after infection of cancer cells. Activation of such a pathway is associated with a dominant T helper 1 (Th1) cell differentiation of the response, which benefits antitumor outcomes. Deletion of the B2R gene resulted in enhanced IRF3 phosphorylation and type I IFN expression after infection of tumor cells, while effective VACV replication remained unimpaired, both in vitro and in vivo. In syngeneic mouse tumor models, the absence of the VACV cGAMP-specific nuclease translated into improved antitumor activity, which was associated with antitumor immunity directed against tumor epitopes.

Visible Light Photoredox-Catalyzed Decarboxylative Alkylation of 3-Aryl-Oxetanes and Azetidines via Benzylic Tertiary Radicals and Implications of Benzylic Radical Stability.

Four-membered heterocycles offer exciting potential as small polar motifs in medicinal chemistry but require further methods for incorporation. Photoredox catalysis is a powerful method for the mild generation of alkyl radicals for C-C bond formation. The effect of ring strain on radical reactivity is not well understood, with no studies that address this question systematically. Examples of reactions that involve benzylic radicals are rare, and their reactivity is challenging to harness. This work develops a radical functionalization of benzylic oxetanes and azetidines using visible light photoredox catalysis to prepare 3-aryl-3-alkyl substituted derivatives and assesses the influence of ring strain and heterosubstitution on the reactivity of small-ring radicals. 3-Aryl-3-carboxylic acid oxetanes and azetidines are suitable precursors to tertiary benzylic oxetane/azetidine radicals which undergo conjugate addition into activated alkenes. We compare the reactivity of oxetane radicals to other benzylic systems. Computational studies indicate that Giese additions of unstrained benzylic radicals into acrylates are reversible and result in low yields and radical dimerization. Benzylic radicals as part of a strained ring, however, are less stable and more π-delocalized, decreasing dimer and increasing Giese product formation. Oxetanes show high product yields due to ring strain and Bent's rule rendering the Giese addition irreversible.

Oxetan-3-ols as 1,2-bis-Electrophiles in a Brønsted-Acid-Catalyzed Synthesis of 1,4-Dioxanes.

Annulations that combine diacceptors with bis-nucleophiles are uncommon. Here, we report the synthesis of 1,4-dioxanes from 3-aryloxetan-3-ols, as 1,2-bis-electrophiles and 1,2-diols. Brønsted acid Tf2NH catalyzes both the selective activation of the oxetanol, to form an oxetane carbocation that reacts with the diol, and intramolecular ring opening of the oxetane. High regio- and diastereoselectivity are achieved with unsymmetrical diols. The substituted dioxanes and fused bicyclic products present interesting motifs for drug discovery and can be further functionalized.

Amino-oxetanes as amide isosteres by an alternative defluorosulfonylative coupling of sulfonyl fluorides.

Bioisosteres provide valuable design elements that medicinal chemists can use to adjust the structural and pharmacokinetic characteristics of bioactive compounds towards viable drug candidates. Aryl oxetane amines offer exciting potential as bioisosteres for benzamides-extremely common pharmacophores-but are rarely examined due to the lack of available synthetic methods. Here we describe a class of reactions for sulfonyl fluorides to form amino-oxetanes by an alternative pathway to the established SuFEx (sulfonyl-fluoride exchange) click reactivity. A defluorosulfonylation forms planar oxetane carbocations simply on warming. This disconnection, comparable to a typical amidation, will allow the application of vast existing amine libraries. The reaction is tolerant to a wide range of polar functionalities and is suitable for array formats. Ten oxetane analogues of bioactive benzamides and marketed drugs are prepared. Kinetic and computational studies support the formation of an oxetane carbocation as the rate-determining step, followed by a chemoselective nucleophile coupling step.

Activation of interferon regulatory factor 3 by replication-competent vaccinia viruses improves antitumor efficacy mediated by T cell responses.

Recently, oncolytic vaccinia viruses (VACVs) have shown their potential to provide for clinically effective cancer treatments. The reason for this clinical usefulness is not only the direct destruction of infected cancer cells but also activation of immune responses directed against tumor antigens. For eliciting a robust antitumor immunity, a dominant T helper 1 (Th1) cell differentiation of the response is preferred, and such polarization can be achieved by activating the Toll-like receptor 3 (TLR3)-interferon regulatory factor 3 (IRF3) signaling pathway. However, current VACVs used as oncolytic viruses to date still encode several immune evasion proteins involved in the inhibition of this signaling pathway. By inactivating genes of selected regulatory virus proteins, we aimed for a candidate virus with increased potency to activate cellular antitumor immunity but at the same time with a fully maintained replicative capacity in cancer cells. The removal of up to three key genes (C10L, N2L, and C6L) from VACV did not reduce the strength of viral replication, both in vitro and in vivo, but resulted in the rescue of IRF3 phosphorylation upon infection of cancer cells. In syngeneic mouse tumor models, this activation translated to enhanced cytotoxic T lymphocyte (CTL) responses directed against tumor-associated antigens and neo-epitopes and improved antitumor activity.

Predictors of Respiratory Dysfunction at Diagnosis of Robin Sequence.

OBJECTIVES/HYPOTHESIS: Robin sequence (RS) consists of associated micrognathia, glossoptosis, and respiratory dysfunction, with or without cleft palate. Studies on how different patient characteristics impact the severity of respiratory dysfunction are scarce and contradictory; this study investigates how different features affect respiratory obstruction severity at diagnosis of RS in controlled analysis. STUDY DESIGN: Retrospective cohort study that enrolled 71 RS patients under 90 days old who received care in our institution from 2009 to 2020. METHODS: The primary outcome, respiratory dysfunction, was categorized into four severity groups and analyzed using a multinomial logistic regression model that considered age, sex, mandible length, cleft palate, syndromic diagnosis, other airway anomalies, and degree of glossoptosis. RESULTS: Mandible length, syndromic diagnosis, and Yellon grade 3 glossoptosis were related to poorer respiratory outcomes (need for respiratory support). In univariate analysis, for each additional 1 mm of mandible length at diagnosis, a mean reduction of 28% in the risk of needing respiratory support was observed (OR = 0.72; 0.58-0.89); syndromic diagnosis and grade 3 glossoptosis also raised the risk (OR = 6.50; 1.59-26.51 and OR = 12.75; 1.03-157.14, respectively). In multivariate analysis, only mandible length significantly maintained its effects (OR = 0.73; 0.56-0.96), a 27% reduction. CONCLUSIONS: Mandible length was an independent predictor for more severe respiratory dysfunction in RS patients, with larger mandibles showing protective effects. Syndromic diagnosis and Yellon grade 3 glossoptosis are also likely to be associated with poorer respiratory outcomes, although this was not demonstrated in multivariate analysis. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:2811-2816, 2021.

Oncolytic Virus-Mediated Targeting of PGE2 in the Tumor Alters the Immune Status and Sensitizes Established and Resistant Tumors to Immunotherapy.

Immunotherapies are highly promising cancer treatments, but understanding the factors mediating their resistance remains critical. Successes in randomized clinical testing have supported the growing appreciation that oncolytic virotherapies primarily act as immunotherapies. Here we identified prostaglandin E2 (PGE2) in the tumor as a key mediator of resistance to immunotherapies, including oncolytic vaccinia virotherapy. Elevated levels of PGE2 coupled to suppressive chemokine profiles and high levels of granulocytic myeloid-derived suppressor cells resulted in loss of immunotherapeutic potential. Viral vectors engineered to target PGE2 were capable of overcoming localized immunosuppression leading to profound changes in the tumor's immune status. This allowed the viral vectors to raise robust anti-tumor adaptive immune responses and sensitized established and previously resistant tumors to immunotherapies.

Manipulating TLR Signaling Increases the Anti-tumor T Cell Response Induced by Viral Cancer Therapies.

The immune response plays a key role in enhancing the therapeutic activity of oncolytic virotherapies. However, to date, investigators have relied on inherent interactions between the virus and the immune system, often coupled to the expression of a single cytokine transgene. Recently, the importance of TLR activation in mediating adaptive immunity has been demonstrated. We therefore sought to influence the type and level of immune response raised after oncolytic vaccinia therapy through manipulation of TLR signaling. Vaccinia naturally activates TLR2, associated with an antibody response, whereas a CTL response is associated with TLR3-TRIF-signaling pathways. We manipulated TLR signaling by vaccinia through deglycosylation of the viral particle to block TLR2 activation and expression of a TRIF transgene. The resulting vector displayed greatly reduced production of anti-viral neutralizing antibody as well as an increased anti-tumor CTL response. Delivery in both naive and pre-treated mice was enhanced and immunotherapeutic activity dramatically improved.

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging.

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule-based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter-tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.

Defining Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy.

PURPOSE: Recent data from randomized clinical trials with oncolytic viral therapies and with cancer immunotherapies have finally recapitulated the promise these platforms demonstrated in preclinical models. Perhaps the greatest advance with oncolytic virotherapy has been the appreciation of the importance of activation of the immune response in therapeutic activity. Meanwhile, the understanding that blockade of immune checkpoints (with antibodies that block the binding of PD1 to PDL1 or CTLA4 to B7-2) is critical for an effective antitumor immune response has revitalized the field of immunotherapy. The combination of immune activation using an oncolytic virus and blockade of immune checkpoints is therefore a logical next step. EXPERIMENTAL DESIGN: Here, we explore such combinations and demonstrate their potential to produce enhanced responses in mouse tumor models. Different combinations and regimens were explored in immunocompetent mouse models of renal and colorectal cancer. Bioluminescence imaging and immune assays were used to determine the mechanisms mediating synergistic or antagonistic combinations. RESULTS: Interaction between immune checkpoint inhibitors and oncolytic virotherapy was found to be complex, with correct selection of viral strain, antibody, and timing of the combination being critical for synergistic effects. Indeed, some combinations produced antagonistic effects and loss of therapeutic activity. A period of oncolytic viral replication and directed targeting of the immune response against the tumor were required for the most beneficial effects, with CD8(+) and NK, but not CD4(+) cells mediating the effects. CONCLUSIONS: These considerations will be critical in the design of the inevitable clinical translation of these combination approaches. Clin Cancer Res; 21(24); 5543-51. ©2015 AACR.See related commentary by Slaney and Darcy, p. 5417.

Safety and efficacy of VCN-01, an oncolytic adenovirus combining fiber HSG-binding domain replacement with RGD and hyaluronidase expression.

PURPOSE: Tumor targeting upon intravenous administration and subsequent intratumoral virus dissemination are key features to improve oncolytic adenovirus therapy. VCN-01 is a novel oncolytic adenovirus that combines selective replication conditional to pRB pathway deregulation, replacement of the heparan sulfate glycosaminoglycan putative-binding site KKTK of the fiber shaft with an integrin-binding motif RGDK for tumor targeting, and expression of hyaluronidase to degrade the extracellular matrix. In this study, we evaluate the safety and efficacy profile of this novel oncolytic adenovirus. EXPERIMENTAL DESIGN: VCN-01 replication and potency were assessed in a panel of tumor cell lines. VCN-01 tumor-selective replication was evaluated in human fibroblasts and pancreatic islets. Preclinical toxicity, biodistribution, and efficacy studies were conducted in mice and Syrian hamsters. RESULTS: Toxicity and biodistribution preclinical studies support the selectivity and safety of VCN-01. Antitumor activity after intravenous or intratumoral administration of the virus was observed in all tumor models tested, including melanoma and pancreatic adenocarcinoma, both in immunodeficient mice and immunocompetent hamsters. CONCLUSIONS: Oncolytic adenovirus VCN-01 characterized by the expression of hyaluronidase and the RGD shaft retargeting ligand shows an efficacy-toxicity prolife in mice and hamsters by intravenous and intratumoral administration that warrants clinical testing.

Transposon tagging in diploid strawberry.

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T1 progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T0 launch pads, putative transposants in the T1 generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T1 plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T1 plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T0 generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T2 transposon-tagged plants. The mutant collection has been catalogued in an on-line database.

Theranostic potential of oncolytic vaccinia virus.

Biological cancer therapies, such as oncolytic, or replication-selective viruses have advantages over traditional therapeutics as they can employ multiple different mechanisms to target and destroy cancers (including direct cell lysis, immune activation and vascular collapse). This has led to their rapid recent clinical development. However this also makes their pre-clinical and clinical study complex, as many parameters may affect their therapeutic potential and so defining reason for treatment failure or approaches that might enhance their therapeutic activity can be complicated. The ability to non-invasively image viral gene expression in vivo both in pre-clinical models and during clinical testing will considerably enhance the speed of oncolytic virus development as well as increasing the level and type of useful data produced from these studies. Further, subsequent to future clinical approval, imaging of reporter gene expression might be used to evaluate the likelihood of response to oncolytic viral therapy prior to changes in tumor burden. Here different reporter genes used in conjunction with oncolytic viral therapy are described, along with the imaging modalities used to measure their expression, while their applications both in pre-clinical and clinical testing are discussed. Possible future applications for reporter gene expression from oncolytic viruses in the phenotyping of tumors and the personalizing of treatment regimens are also discussed.

Minimal RB-responsive E1A promoter modification to attain potency, selectivity, and transgene-arming capacity in oncolytic adenoviruses.

Oncolytic adenoviruses are promising anticancer agents due to their ability to self-amplify at the tumor mass. However, tumor stroma imposes barriers difficult to overcome by these agents. Transgene expression is a valuable strategy to counteract these limitations and to enhance antitumor activity. For this purpose, the genetic backbone in which the transgene is inserted should be optimized to render transgene expression compatible with the adenovirus replication cycle and to keep genome size within the encapsidation size limit. In order to design a potent and selective oncolytic adenovirus that keeps intact all the viral functions with minimal increase in genome size, we inserted palindromic E2F-binding sites into the endogenous E1A promoter. The insertion of these sites controlling E1A-Δ24 results in a low systemic toxicity profile in mice. Importantly, the E2F-binding sites also increased the cytotoxicity and the systemic antitumor activity relative to wild-type adenovirus in all cancer models tested. The low toxicity and the increased potency results in improved antitumor efficacy after systemic injection and increased survival of mice carrying tumors. Furthermore, the constrained genome size of this backbone allows an efficient and potent expression of transgenes, indicating that this virus holds promise for overcoming the limitations of oncolytic adenoviral therapy.