408 oncolytic vaccines in combination with pd l1 blockade for the treatment of melanoma

The Immunological escape of tumors represents one of the main obstacles to the treatment of malignancies. The approval of drugs able to disrupt the immune suppressive pathways through anti-CTLA-4 monoclonal antibodies represented a milestone in the history of immunotherapy. However, treatment with these immune checkpoint inhibitors (ICIs) seems to be effective only in small cohorts of patients. It has been proposed that the efficacy of ICIs relies on the presence of an undergoing immunological response. For this reason, we hypothesized that oncolytic vaccines, able to elicit a tumor specific response, would synergize with anti-PD-L1 therapy. B16 murine melanomas were established in immunocompetent C57 mice. Then mice were treated with anti-PD-L1 monotherapy, PeptiCRAd (oncolytic vaccine) monotherapy or a combination of the two. The growth of the tumors was analyzed. At the end of the experiment, all the mice mice were euthanized and organs collected for immunological analysis. We investigated antigen-specific T-cell responses and immune suppressive background by flow cytometry and ELISPOT assays

Towards the use of an amino acid cleavable linker for solid-phase chemical synthesis of peptides and proteins

The synthesis of proteins by solid-phase chemical ligation (SPCL) suffers from the paucity of linkers that can be cleaved under mild conditions. Here, we deployed a spontaneous nickel-assisted cleavage (SNAC) tag, known to undergo spontaneous cleavage in the presence of nickel(ii), as a linker for C-to-N SPCL

Effect of trimethine cyanine dye- and folate-conjugation on the in vitro biological activity of proapoptotic peptides

Despite continuous advances, anticancer therapy still faces several technical hurdles, such as selectivity on cellular and subcellular targets of therapeutics. Toward addressing these limitations, we have combined the use of proapoptotic peptides, trimethine cyanine dye, and folate to target the mitochondria of tumor cells. A series of proapoptotic peptides and their conjugates with a cyanine dye and/or folate were synthesized in the solid phase, and their toxicity in different human cell lines was assessed. Cyanine-bearing conjugates were found to be up to 100-fold more cytotoxic than the parent peptides and to localize in mitochondria. However, the addition of a folate motif did not enhance the potency or selectivity of the resulting conjugates toward tumor cells that overexpress folate receptor α. Furthermore, while dual-labeled constructs were also found to localize within the target organelle, they were not generally selective towards folate receptor α-positive cell lines in vitro

642 oncolytic vaccines with modified tumor epitopes for cancer immunotherapy

Oncolytic adenoviruses (OAds) are capable of killing tumor cells while activating the immune system due to their immunogenicity. Hance, they are an excellent platform for oncolytic vaccine. We previously demonstrated that the injection of peptide-coated conditionally replicating adenoviruses (PeptiCRAd) is capable of reducing the growth of established aggressive melanomas (murine B16).Oncolytic vaccines, like PeptiCRAds, often rely on inducing an immune response against specific tumor antigens. However, many tumor antigens are also self-antigens, hence the peripheral tolerance might impair the activity of tumor-reactive T-cells. Therefore, mutated epitopes represent an optimal tool to break the tolerance, exploiting the cross-reactivity of T-cells. To this end we developed an Epitope Discovery and Improvement System (EDIS) framework to study native epitopes and predict, in silico, mutated forms suitable for cancer therapy. The novel aspect of EDIS is the ability to interrogate different prediction servers, integrate the different results and validate these by molecular dynamics simulations.We started by studying the model epitope SIINFEKL. According to our in silico predictions, two mutated variants were suggested to be more immunogenic than the native SIINFEKL peptide. To test whether this prediction would reflect in enhanced vaccine effect we studied the immune response against these peptides in B16-OVA bearing mice. Mice were challenged with B16-OVA and treated with three different PeptiCRAds coated with SIINFEKL and the two predicted derivates. By ELISPOT assay we assessed the anti-peptide response and demonstrated that the two mutated forms were in fact more effective in reducing the growth of established B16OVA tumors.Finally, we studied the native epitope SVYDFFVWL from the tyrosinase-related protein 2 (TRP2), a melanoma antigen in clinical evaluation. By using the EDIS framework we selected two mutated variants that show increased MHC-I binding affinity and we tested them by treating aggressive B16F10 tumors. As expected, treatment with the native TRP2 reduced the growth of the tumors compared to the controls. Suprisingly, one of the two analogues improved significantly the survival of mice and reduced the growth of their tumors compared to the group treated with the native TRP2 epitope. In conclusion, we demonstrated that the integration of different in silico methods increases the accuracy when predicting mutated epitopes for cancer immunotherapy

Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

Asparaginyl endopeptidases (AEPs) are ideal for peptide and protein labeling. However, because of the reaction reversibility, a large excess of labels or backbone modified substrates are needed. In turn, simple and cheap reagents can be used to label N-terminal cysteine, but its availability inherently limits the potential applications. Aiming to address these issues, we have created a chemo-enzymatic labeling system that exploits the substrate promiscuity of AEP with the facile chemical reaction between N-terminal cysteine and 2-formyl phenylboronic acid (FPBA). In this approach, AEP is used to ligate polypeptides with a Asn–Cys–Leu recognition sequence with counterparts possessing an N-terminal Gly–Leu. Instead of being a labeling reagent, the commercially available FPBA serves as a scavenger converting the byproduct Cys–Leu into an inert thiazolidine derivative. This consequently drives the AEP labeling reaction forward to product formation with a lower ratio of label to protein substrate. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP-ligation/FPBA-coupling system was further demonstrated by site-specifically labeling the N- or C-termini of various proteins

Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes

Organelle-specific delivery systems are of significant clinical interest. We demonstrate the use of common cyanine dyes Cy3 and Cy5 as vectors for targeting and delivering cargoes to mitochondria in cancer cells. Specifically, conjugation to the dyes can increase cytotoxicity by up to 1000-fold

Diabetic ketoacidosis at the onset of disease during a national awareness campaign: a 2-year observational study in children aged 0-18 years

After a previous survey on the incidence of diabetic ketoacidosis (DKA) at onset of type 1 diabetes in children in 2013-2014 in Italy, we aimed to verify a possible decline in the incidence of DKA at onset during a national prevention campaign

The Silent Epidemic of Diabetic Ketoacidosis at Diagnosis of Type 1 Diabetes in Children and Adolescents in Italy During the COVID-19 Pandemic in 2020

To compare the frequency of diabetic ketoacidosis (DKA) at diagnosis of type 1 diabetes in Italy during the COVID-19 pandemic in 2020 with the frequency of DKA during 2017-2019