Tumor-targeted superantigens produce curative tumor immunity with induction of memory and demonstrated antigen spreading.

BACKGROUND: Despite remarkable progress, the immunotherapies currently used in the clinic, such as immune checkpoint blockade (ICB) therapy, still have limited efficacy against many types of solid tumors. One major barrier to effective treatment is the lack of a durable long-term response. Tumor-targeted superantigen (TTS) therapy may overcome this barrier to enhance therapeutic efficacy. TTS proteins, such as the clinical-stage molecule naptumomab estafenatox (NAP), increase tumor recognition and killing by both coating tumor cells with bacterial-derived superantigens (SAgs) and selectively expanding T-cell lineages that can recognize them. The present study investigated the efficacy and mechanism of action of repeated TTS (C215Fab-SEA) treatments leading to a long-term antitumor immune response as monotherapy or in combination with PD-1/PD-L1 inhibitors in murine tumor models. METHODS: We used syngeneic murine tumor models expressing the human EpCAM target (C215 antigen) to assess the efficacy and mechanism of action of repeated treatment with TTS C215Fab-SEA alone or with anti-PD-1/PD-L1 monoclonal antibodies. Tumor draining lymph nodes (TDLNs) and tumor tissues were processed and analyzed by immunophenotyping and immunohistochemistry. Isolated RNA from tumors was used to analyze gene expression and the TCR repertoire. Tumor rechallenge and T-cell transfer studies were conducted to test the long-term antitumor memory response. RESULTS: TTS therapy inhibited tumor growth and achieved complete tumor rejection, leading to a T-cell-dependent long-term memory response against the tumor. The antitumor effect was derived from inflammatory responses converting the immunosuppressive TME into a proinflammatory state with an increase in T-cell infiltration, activation and high T-cell diversity. The combination of TTS with ICB therapy was significantly more effective than the monotherapies and resulted in higher tumor-free rates. CONCLUSIONS: These new results indicate that TTSs not only can turn a "cold" tumor into a "hot" tumor but also can enable epitope spreading and memory response, which makes TTSs ideal candidates for combination with ICB agents and other anticancer agents.

Gne depletion during zebrafish development impairs skeletal muscle structure and function.

GNE Myopathy is a rare recessively inherited neuromuscular disorder caused by mutations in the GNE gene, which codes for the key enzyme in the metabolic pathway of sialic acid synthesis. The process by which GNE mutations lead to myopathy is not well understood. By in situ hybridization and gne promoter-driven fluorescent transgenic fish generation, we have characterized the spatiotemporal expression pattern of the zebrafish gne gene and have shown that it is highly conserved compared with the human ortholog. We also show the deposition of maternal gne mRNA and maternal GNE protein at the earliest embryonic stage, emphasizing the critical role of gne in embryonic development. Injection of morpholino (MO)-modified antisense oligonucleotides specifically designed to knockdown gne, into one-cell embryos lead to a variety of phenotypic severity. Characterization of the gne knockdown morphants showed a significantly reduced locomotor activity as well as distorted muscle integrity, including a reduction in the number of muscle myofibers, even in mild or intermediate phenotype morphants. These findings were further confirmed by electron microscopy studies, where large gaps between sarcolemmas were visualized, although normal sarcomeric structures were maintained. These results demonstrate a critical novel role for gne in embryonic development and particularly in myofiber development, muscle integrity and activity.

Variation in repeat length and heteroplasmy of the mitochondrial DNA control region along a core-edge gradient in the eastern spadefoot toad (Pelobates syriacus).

Peripheral populations are those situated at the distribution margins of a species and are often subjected to more extreme abiotic and biotic conditions than those at the core. Here, we hypothesized that shorter repeat length and fewer heteroplasmic mitochondrial DNA (mtDNA) copies, which are associated with more efficient mitochondrial function, may be related to improved survival under extreme environmental conditions. We sampled eastern spadefoot toads (mostly as tadpoles) from 43 rain pools distributed along a 300-km gradient from core to edge of the species' distribution. We show that mean pool tandem repeat length and heteroplasmy increase from edge to core, even after controlling for body size. We evaluate several alternative hypotheses and propose the Fisher hypothesis as the most likely explanation. However, additional sequential sampling and experimental studies are required to determine whether selection under extreme conditions, or alternative mechanisms, could account for the gradient in heteroplasmy and repeat length in the mtDNA control region.

The change in genetic diversity down the core-edge gradient in the eastern spadefoot toad (Pelobates syriacus).

Several hypotheses are available to predict change in genetic diversity when approaching peripheral populations. We used the eastern spadefoot toad in Israel as a model system to examine these hypotheses using population genetics analyses and network theory. Our results contradicted most of the predictions from the 'abundant centre' model, that edge populations should have lower density and lower genetic diversity than core populations. Furthermore, dispersal rate between core and peripheral populations is expected to be asymmetric, mostly directed outwards from the core population, but we did not detect such a trend. Our data did not support the hypothesis of no change or a non-linear change in genetic diversity towards the range edge. However, our results did fit the Fisher (The Genetical Theory of Natural Selection, Clarendon Press, Oxford, 1930) hypothesis, which predicts increase in genetic variability from core to edge of distribution. We attributed this finding to the much harsher climatic and abiotic conditions at the edge, which must be tolerated over generations by both tadpoles and post-metamorphic individuals in this region. Finally, our results have significant conservation implications for the survival of this species in Israel, where it is critically endangered. We identified two distinct communities, which are genetically linked through two specific rain pools in the Upper Galilee. Details on the spatial subdivision of this species are cardinal for future management and restoration of temporary wetlands in Israel.

The topical isoflavonoid NV-07alpha reduces solar-simulated UV-induced suppression of Mantoux reactions in humans.

UV radiation suppresses delayed-type hypersensitivity responses to intradermally injected tuberculin purified protein derivative in Mantoux-positive individuals. The effect of the topically administered isoflavonoid NV-07alpha, a synthetic derivative of the isoflavonoid equol, on UV-induced suppression of Mantoux reactions was assessed in 18 healthy Mantoux-positive volunteers. A single, fixed dose of solar-simulated UV radiation was delivered to the volunteers' lower backs. Different concentrations of NV-07alpha or its vehicle were applied to different sites within the irradiated field immediately after UV exposure and again 24 h later. Forty-eight hours after irradiation, Mantoux testing was performed at both the irradiated sites and adjacent, unirradiated sites. The intensity of Mantoux reactions was measured 72 h later with a reflectance erythema meter and by measuring the diameter of each reaction. Although lower concentrations of NV-07alpha (0.5 and 2 mM) did not prevent UV immunosuppression, 4 mM NV-07alpha partially but significantly attenuated UV-induced suppression of Mantoux-induced erythema. Minimal erythema doses were also determined for sites treated with NV-07alpha or its vehicle immediately after UV exposure. NV-07alpha had no significant effects on UV erythema. We conclude that 4 mM NV-07alpha prevented the suppressive effects of UV radiation on Mantoux responses in humans but did not affect UV-induced erythema at the concentrations used.