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.

Pathogenic Stress Induces Human Monocyte to Express an Extracellular Web of Tunneling Nanotubes.

Actin-based tunneling nanotubes are a means of intercellular communication between remote cells. In the last decade, this type of nanotube was described in a wide variety of cell types and it became widely accepted that communication through these nanotubes is related to response to environmental changes. Few reports, however, are available regarding the expression of similar nanotubes in vivo or in primary cells. Moreover, the functional significance of this intercellular communication for health and disease is largely unknown. In this context, and as a first step in unraveling these questions, we examined the formation of similar nanotubes in primary peripheral human monocytes. To that end, we combined the use of a live cell imaging system along with advanced methods of fluorescent and scanning electron microscopy. This experimental approach reveals for the first time that the bacterial lipopolysaccharide endotoxin induces a transient expression of an unexpected abundance of actin-based tunneling nanotubes associated with vesicles. In addition, it was found that a similar response can be achieved by treating human monocytes with various bacterial and yeast membrane components, as well as with a viral component analog. In all these cases, this response is mediated by distinct complexes of toll-like receptors. Therefore, we suggest that the observed phenomena are related to a broad type of monocyte pathogen response, and raise the possibility that the phenomena described above may be involved in many clinical situations related to inflammation as a new topic of study.

Calcium-sensing receptor sequencing in 21 patients with idiopathic or familial parathyroid disorder: pitfalls and characterization of a novel I32 V loss-of-function mutation.

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor with a crucial role in calcium homeostasis. Mutations in the CaSR gene may lead to specific parathyroid disorders due to either gain-of-function (autosomal dominant hypercalciuric hypocalcemia; ADHH) or loss-of-function (familial hypocalciuric hypercalcemia; FHH). Our aim was to evaluate CaSR mutations as a cause of disease in selected patients. We identified and recruited patients with phenotypes suggestive of CaSR-related parathyroid disorders. DNA was extracted, and CaSR gene was sequenced. Live-ratiometric measurements of intracellular [Ca(2+)] and Western blot assays for evaluation of MAPK phosphorylation in response to changes in extracellular [Ca(2+)] were performed in transiently transfected HEK-293T cells to functionally characterize mutants. A total of 21 patients were evaluated, seven of them with idiopathic hypoparathyroidism (suspected ADHH) and 14 with hyperparathyroidism (suspected FHH). In the latter group two patients were found to harbor missense mutations: a novel heterozygous I32 V mutation in a female index case and a sporadic known R185Q mutation in a 1-year-old girl. In-vitro functional studies showed that I32 V is an inactivating mutation. In our study, most patients had normal CaSR sequencing. This suggests that phenotypic pitfalls may occur at time of patients' selection for CaSR sequencing. In one patient with strong positive pre-test probability based on both familial history and appropriate phenotype, a novel I32 V mutation leading to FHH was identified and characterized. In cases of familial parathyroid disorders, CaSR sequencing should be performed, but if negative, one should consider involvement of alternative genes or mechanisms.

The Na(+)/Ca(2+)-exchanger: an essential component in the mechanism governing cardiac steroid-induced slow Ca(2+) oscillations.

Plasma membrane (PM) Na(+), K(+)-ATPase, plays crucial roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically bind to the Na(+), K(+)-ATPase and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds, synthesized in and released from the adrenal gland, are considered a new family of steroid hormones. Previous studies showed that ouabain induces slow Ca(2+) oscillations in COS-7 cells by enhancing the interactions between Na(+), K(+)-ATPase, inositol 1,4,5-trisphosphate receptor (IP(3)R) and Ankyrin B (Ank-B) to form a Ca(2+) signaling micro-domain. The activation of this micro-domain, however, is independent of InsP3 generation. Thus, the mechanism underlying the induction of these slow Ca(2+) oscillations remained largely unclear. We now show that other CS, such as bufalin, can also induce Ca(2+) oscillations. These oscillations depend on extracellular Ca(2+) concentrations [Ca(2+)](out) and are inhibited by Ni(2+). Furthermore, we found that these slow oscillations are Na(+)(out) dependent, abolished by Na(+)/Ca(2+) exchanger1 (NCX1)-specific inhibitors and markedly attenuated by NCX1 siRNA knockdown. Based on these results, a model is presented for the CS-induced slow Ca(2+) oscillations in COS-7 cells.

A novel lytic peptide composed of DL-amino acids selectively kills cancer cells in culture and in mice.

The high toxicity of most chemotherapeutic drugs and their inactivation by multidrug resistance phenotypes motivated extensive search for drugs with new modes of action. We designed a short cationic diastereomeric peptide composed of d- and l-leucines, lysines, and arginines that has selective toxicity toward cancer cells and significantly inhibits lung metastasis formation in mice (86%) with no detectable side effects. Its ability to depolarize the transmembrane potential of cancer cells at the same rate (within minutes) and concentration (3 micro m), at which it shows biological activity, suggests a killing mechanism that involves plasma membrane perturbation. Confocal microscopy experiments verified that the cells died as a result of acute injury, swelling, and bursting, suggesting necrosis. Biosensor binding experiments and attenuated total reflectance-Fourier transform infrared spectroscopy using model membranes have substantiated its high selectivity toward cancer cells. Although this is an initial study that looked at tumor formation rather than the ability of the peptides to reduce established tumors, the simple sequence of the peptide, its high solubility, substantial resistance to degradation, and inactivation by serum components might make it a good candidate for future anticancer treatment.