Molecular mimicry and cancer vaccine development.

BACKGROUND: The development of cancer immunotherapeutic strategies relies on the identification and validation of optimal target tumor antigens, which should be tumor-specific as well as able to elicit a swift and potent anti-tumor immune response. The vast majority of such strategies are based on tumor associated antigens (TAAs) which are shared wild type cellular self-epitopes highly expressed on tumor cells. Indeed, TAAs can be used to develop off-the-shelf cancer vaccines appropriate to all patients affected by the same malignancy. However, given that they may be also presented by HLAs on the surface of non-malignant cells, they may be possibly affected by immunological tolerance or elicit autoimmune responses. MAIN BODY: In order to overcome such limitations, analogue peptides with improved antigenicity and immunogenicity able to elicit a cross-reactive T cell response are needed. To this aim, non-self-antigens derived from microorganisms (MoAs) may be of great benefit.

Immune profiling of SARS-CoV-2 epitopes in asymptomatic and symptomatic pediatric and adult patients.

BACKGROUND: The infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unpredictable manifestations of coronavirus disease (COVID-19) and variable clinical course with some patients being asymptomatic whereas others experiencing severe respiratory distress, or even death. We aimed to evaluate the immunoglobulin G (IgG) response towards linear peptides on a peptide array containing sequences from SARS-CoV-2, Middle East respiratory syndrome-related coronavirus (MERS) and common-cold coronaviruses 229E, OC43, NL63 and HKU1 antigens, in order to identify immunological indicators of disease outcome in SARS-CoV-2 infected patients. METHODS: We included in the study 79 subjects, comprising 19 pediatric and 30 adult SARS-CoV-2 infected patients with increasing disease severity, from mild to critical illness, and 30 uninfected subjects who were vaccinated with one dose of SARS-CoV-2 spike mRNA BNT162b2 vaccine. Serum samples were analyzed by a peptide microarray containing 5828 overlapping 15-mer synthetic peptides corresponding to the full SARS-CoV-2 proteome and selected linear epitopes of spike (S), envelope (E) and membrane (M) glycoproteins as well as nucleoprotein (N) of MERS, SARS and coronaviruses 229E, OC43, NL63 and HKU1 (isolates 1, 2 and 5). RESULTS: All patients exhibited high IgG reactivity against the central region and C-terminus peptides of both SARS-CoV-2 N and S proteins. Setting the threshold value for serum reactivity above 25,000 units, 100% and 81% of patients with severe disease, 36% and 29% of subjects with mild symptoms, and 8% and 17% of children younger than 8-years reacted against N and S proteins, respectively. Overall, the total number of peptides in the SARS-CoV-2 proteome targeted by serum samples was much higher in children compared to adults. Notably, we revealed a differential antibody response to SARS-CoV-2 peptides of M protein between adults, mainly reacting against the C-terminus epitopes, and children, who were highly responsive to the N-terminus of M protein. In addition, IgG signals against NS7B, NS8 and ORF10 peptides were found elevated mainly among adults with mild (63%) symptoms. Antibodies towards S and N proteins of other coronaviruses (MERS, 229E, OC43, NL63 and HKU1) were detected in all groups without a significant correlation with SARS-CoV-2 antibody levels. CONCLUSIONS: Overall, our results showed that antibodies elicited by specific linear epitopes of SARS-CoV-2 proteome are age dependent and related to COVID-19 clinical severity. Cross-reaction of antibodies to epitopes of other human coronaviruses was evident in all patients with distinct profiles between children and adult patients. Several SARS-CoV-2 peptides identified in this study are of particular interest for the development of vaccines and diagnostic tests to predict the clinical outcome of SARS-CoV-2 infection.

Mutations in the telomerase reverse transcriptase promoter and PIK3CA gene are common events in penile squamous cell carcinoma of Italian and Ugandan patients.

Penile carcinoma develops either through human papillomavirus (HPV) related or unrelated carcinogenic pathways. Genetic alterations and nucleotide changes in coding regions (ie, TP53, CDKN2A, PIK3CA and NOTCH1) are main cancer driver events either in HPV positive or in HPV negative tumours. We investigated the presence of hotspot nucleotide mutations in TERT promoter (TERTp) and PIK3CA exon 9 and their relationship with HPV status in 69 penile cancer cases from Italian and Ugandan patients. Genetic variations and viral sequences have been characterised by end-point polymerase chain reaction (PCR) and Sanger sequencing. The mutant allele frequencies (MAFs) of TERTp -124A/-146A and PIK3CA E545K have been determined by droplet digital PCR (ddPCR) assays. The results showed that TERTp mutations are highly prevalent in penile carcinoma (53.6%) and significantly more frequent in HPV negative (67.6%) than HPV positive (32.4%) cases (P = .0482). PIK3CA mutations were similarly distributed in virus-related and unrelated cases (25.9% and 26.7%, respectively) and coexisted with TERTp changes in 15.8% of penile carcinoma samples. Notably, MAFs of co-occurring mutations were frequently discordant indicating that PIK3CA E545K nucleotide changes are subsequent genetic events occurring in subclones of TERTp mutated cells. The frequencies of TERTp and PIK3CA mutations were higher among Italian compared to Ugandan cases and inversely correlated with the HPV status. In conclusion, TERTp mutations are very common in penile carcinoma and their coexistence with PIK3CA in a substantial number of cases may represent a novel oncogenic synergy relevant for patient stratification and use of therapeutic strategies against new actionable targets.

Molecular mimicry between tumor associated antigens and microbiota-derived epitopes.

BACKGROUND: The gut microbiota profile is unique for each individual and are composed by different bacteria species according to individual birth-to-infant transitions. In the last years, the local and systemic effects of microbiota on cancer onset, progression and response to treatments, such as immunotherapies, has been extensively described. Here we offer a new perspective, proposing a role for the microbiota based on the molecular mimicry of tumor associated antigens by microbiome-associated antigens. METHODS: In the present study we looked for homology between published TAAs and non-self microbiota-derived epitopes. Blast search for sequence homology was combined with extensive bioinformatics analyses. RESULTS: Several evidences for homology between TAAs and microbiota-derived antigens have been found. Strikingly, three cases of 100% homology between the paired sequences has been identified. The predicted average affinity to HLA molecules of microbiota-derived antigens is very high (< 100 nM). The structural conformation of the microbiota-derived epitopes is, in general, highly similar to the corresponding TAA. In some cases, it is identical and contact areas with both HLA and TCR chains are indistinguishable. Moreover, the spatial conformation of TCR-facing residues can be identical in paired TAA and microbiota-derived epitopes, with exactly the same values of planar as well as dihedral angles. CONCLUSIONS: The data reported in the present study show for the first time the high homology in the linear sequence as well as in structure and conformation between TAAs and peptides derived from microbiota species of the Firmicutes and the Bacteroidetes phyla, which together account for 90% of gut microbiota. Cross-reacting CD8+ T cell responses are very likely induced. Therefore, the anti-microbiota T cell memory may turn out to be an anti-cancer T cell memory, able to control the growth of a cancer developed during the lifetime if the expressed TAA is similar to the microbiota epitope. This may ultimately represent a relevant selective advantage for cancer patients and may lead to a novel preventive anti-cancer vaccine strategy.

Antigenic molecular mimicry in viral-mediated protection from cancer: the HIV case.

BACKGROUND: People living with HIV/AIDS (PLWHA) show a reduced incidence for three cancer types, namely breast, prostate and colon cancers. In the present study, we assessed whether a molecular mimicry between HIV epitopes and tumor associated antigens and, consequently, a T cell cross-reactivity could provide an explanation for such an epidemiological evidence. METHODS: Homology between published TAAs and non-self HIV-derived epitopes have been assessed by BLAST homology. Structural analyses have been performed by bioinformatics tools. Immunological validation of CD8+ T cell cross-reactivity has been evaluated ex vivo by tetramer staining. FINDINGS: Sequence homologies between multiple TAAs and HIV epitopes have been found. High structural similarities between the paired TAAs and HIV epitopes as well as comparable patterns of contact with HLA and TCR α and ß chains have been observed. Furthermore, cross-reacting CD8+ T cells have been identified. INTERPRETATION: This is the first study showing a molecular mimicry between HIV antigens an TAAs identified in breast, prostate and colon cancers. Therefore, it is highly reasonable that memory CD8+ T cells elicited during the HIV infection may play a key role in controlling development and progression of such cancers in the PLWHA lifetime. This represents the first demonstration ever that a viral infection may induce a natural "preventive" anti-cancer memory T cells, with highly relevant implications beyond the HIV infection.

Long-term memory T cells as preventive anticancer immunity elicited by TuA-derived heteroclitic peptides.

The host's immune system may be primed against antigens during the lifetime (e.g. microorganisms antigens-MoAs), and swiftly recalled upon growth of a tumor expressing antigens similar in sequence and structure. C57BL/6 mice were immunized in a preventive setting with tumor antigens (TuAs) or corresponding heteroclitic peptides specific for TC-1 and B16 cell lines. Immediately or 2-months after the end of the vaccination protocol, animals were implanted with cell lines. The specific anti-vaccine immune response as well as tumor growth were regularly evaluated for 2 months post-implantation. The preventive vaccination with TuA or their heteroclitic peptides (hPep) was able to delay (B16) or completely suppress (TC-1) tumor growth when cancer cells were implanted immediately after the end of the vaccination. More importantly, TC-1 tumor growth was significantly delayed, and suppressed in 6/8 animals, also when cells were implanted 2-months after the end of the vaccination. The vaccine-specific T cell response provided a strong immune correlate to the pattern of tumor growth. A preventive immunization with heteroclitic peptides resembling a TuA is able to strongly delay or even suppress tumor growth in a mouse model. More importantly, the same effect is observed also when tumor cells are implanted 2 months after the end of vaccination, which corresponds to 8 - 10 years in human life. The observed potent tumor control indicates that a memory T cell immunity elicited during the lifetime by a antigens similar to a TuA, i.e. viral antigens, may ultimately represent a great advantage for cancer patients and may lead to a novel preventive anti-cancer vaccine strategy.

PDCD1 and IFNL4 genetic variants and risk of developing hepatitis C virus-related diseases.

BACKGROUND: Genetic variants of IFNL4 and PDCD1 genes have been shown to influence the spontaneous clearance of hepatitis C virus (HCV) infection. We investigated the IFNL4 rs12979860 and the PDCD1 polymorphisms in 734 HCV-positive patients, including 461 cases with liver disease of varying severity and 273 patients with lymphoproliferative disorders to determine the association of these genes with patient's outcome. METHODS: Expression levels of PDCD1 mRNA encoded by haplotypes were investigated by quantitative PCR in hepatocellular carcinoma (HCC) tissue and peripheral blood mononuclear cells. Flow cytometry was used to detect PD-1 and its ligand PD-L1. RESULTS: The frequency of IFNL4 rs12979860 C/T or T/T genotypes was significantly higher in patients with HCV-related diseases than blood donors (P < .0001). Patients expressing the IFNλ4 variant with one amino acid change that reduces IFNλ4 secretion was found increased in frequency in HCV-related diseases compared to HCC PDCD1 mRNA levels in HCC tissue were significantly higher in cases carrying the PD-1.3 A or the PD-1.7 G allele (P = .0025 and P = .0167). Linkage disequilibrium (LD) between PD-1.3 and IFNL4 was found in patients with mixed cryoglobulinaemia (MC) only (LD = 0 in HCC; LD = 72 in MC). PBMCs of MC patients expressed low levels of PD-L1 in CD19+IgM+B cells and of PD-1 in CD4+T cells suggesting the involvement of regulatory B cell-T cell interaction to the pathogenesis of MC. CONCLUSION: Collectively, our data indicate an important contribution of IFNλ4 expression to the development of HCV-related HCC and an epistatic contribution of IFNL4 and PDCD1 in MC. LAY SUMMARY: Studies of IFNL4 and PDCD1 genes are helpful to better understand the role of host genetic factors and immune antigens influencing the outcome of HCV-related diseases. Our data support an association between the expression of IFNλ4, which prevents the expression of IFNλ3, with all the different HCV-related diseases studied, and besides, evidence that a higher IFNλ4 expression is associated with hepatocellular at a younger age. The expression pattern of low PD-L1 on B cells and high PD-1 on CD4+T-cells in patients with HCV-positive cryoglobulinaemia suggests a critical role of the PD-1/PD-L1 signaling in modulating B cell-T cell interaction in this lymphoproliferative disease.

Detection of a large spectrum of viral infections in conjunctival premalignant and malignant lesions.

To study the interaction between HIV and other carcinogenic infections in conjunctival squamous cell carcinoma (SCC), we evaluated the presence of a broad spectrum of human viruses in conjunctiva specimens. Beta Human papillomavirus (HPV; n = 46), gamma HPV (n = 52), polyomaviruses (n = 12) and herpes viruses (n = 3) was determined in DNA extracted from 67 neoplastic and 55 non-neoplastic conjunctival tissues of HIV-positive and HIV negative subjects by Luminex-based assays. Next-generation sequencing (NGS) was also used to further characterize the presence of cutaneous HPVs. Detection of beta-2 HPV infections was associated with the risk of neoplasia (adjusted odds ratio [aOR] 3.0; 95% confidence interval [CI] 1.3-6.8), regardless of HIV status (HIV positive, aOR 2.6, 95% CI 0.9-7.7; HIV negative, aOR 3.5, 95% CI 0.9-14.4). EBV was strongly associated with the risk of neoplasia (aOR 12.0, 95% CI 4.3-33.5; P < .01) mainly in HIV individuals (HIV positive, aOR 57.5; 95% CI: 10.1-327.1; HIV negative aOR 2.6; 95% CI: 0.2-34.7). NGS allowed to identify 13 putative novel HPVs in cases and controls. Our findings suggest a role of beta HPV types and EBV, in conjunctival SCC. However, additional studies of viral expression in tumor tissue are required to confirm the causal association.

SARS-CoV-2 RNA polymerase as target for antiviral therapy.

A new human coronavirus named SARS-CoV-2 was identified in several cases of acute respiratory syndrome in Wuhan, China in December 2019. On March 11 2020, WHO declared the SARS-CoV-2 infection to be a pandemic, based on the involvement of 169 nations. Specific drugs for SARS-CoV-2 are obviously not available. Currently, drugs originally developed for other viruses or parasites are currently in clinical trials based on empiric data. In the quest of an effective antiviral drug, the most specific target for an RNA virus is the RNA-dependent RNA-polymerase (RdRp) which shows significant differences between positive-sense and negative-sense RNA viruses. An accurate evaluation of RdRps from different viruses may guide the development of new drugs or the repositioning of already approved antiviral drugs as treatment of SARS-CoV-2. This can accelerate the containment of the SARS-CoV-2 pandemic and, hopefully, of future pandemics due to other emerging zoonotic RNA viruses.

Immunological effects of adjuvants in subsets of antigen presenting cells of cancer patients undergoing chemotherapy.

BACKGROUND: We have previously shown that HCC patients and healthy subjects are equally responsive to a RNAdjuvant®, a novel TLR-7/8/RIG-I agonist based on noncoding RNA developed by CureVac, by an ex vivo evaluation. However, the immunological effect of adjuvants on immune cells from cancer patients undergoing chemotherapy remains to be demonstrated. Different adjuvants currently used in cancer vaccine clinical trials were evaluated in the present study on immune cells from cancer patients before and after chemotherapy in an ex vivo setting. METHODS: PBMCs were obtained from 4 healthy volunteers and 23 patients affected by either colon (OMA) or lung cancer (OT). The effect of CpG, Poly I:C, Imiquimod and RNA-based adjuvant (RNAdjuvant®) was assessed using a multiparametric approach to analyze network dynamics of early immune responses. Evaluation of CD80, CD86 and HLA-DR expression as well as the downstream effect on CD4+ T cell phenotyping was performed by flow cytometry; cytokine and chemokine production was evaluated by Bio-Plex ProTM. RESULTS: Treatment with RNAdjuvant® induced the strongest response in cancer patients in terms of activation of innate and adoptive immunity. Indeed, CD80, CD86 and HLA-DR expression was found upregulated in circulating dendritic cells, which promoted a CD4+ T cell differentiation towards an effector phenotype. RNAdjuvant® was the only one to induce most of the cytokines/chemokines tested with a pronounced Th1 cytokine pattern. According to the different parameters evaluated in the study, no clear cut difference in immune response to adjuvants was observed between healthy subjects and cancer patients. Moreover, in the latter group, the chemotherapy treatment did not consistently correlate to a significant altered response in the different parameters. CONCLUSIONS: The present study is the first analysis of immunological effects induced by adjuvants in cancer patients who undergo chemotherapy, who are enrolled in the currently ongoing cancer vaccine clinical trials. The results show that the RNAdjuvant® is a potent and Th1 driving adjuvant, compared to those tested in the present study. Most importantly, it is demonstrated that chemotherapy does not significantly impair the immune system, implying that cancer patients are likely to respond to a cancer vaccine even after a chemotherapy treatment.

Antimicrobial Peptides as Anticancer Agents: Functional Properties and Biological Activities.

Antimicrobial peptides (AMPs), or host defense peptides, are small cationic or amphipathic molecules produced by prokaryotic and eukaryotic organisms that play a key role in the innate immune defense against viruses, bacteria and fungi. AMPs have either antimicrobial or anticancer activities. Indeed, cationic AMPs are able to disrupt microbial cell membranes by interacting with negatively charged phospholipids. Moreover, several peptides are capable to trigger cytotoxicity of human cancer cells by binding to negatively charged phosphatidylserine moieties which are selectively exposed on the outer surface of cancer cell plasma membranes. In addition, some AMPs, such as LTX-315, have shown to induce release of tumor antigens and potent damage associated molecular patterns by causing alterations in the intracellular organelles of cancer cells. Given the recognized medical need of novel anticancer drugs, AMPs could represent a potential source of effective therapeutic agents, either alone or in combination with other small molecules, in oncology. In this review we summarize and describe the properties and the mode of action of AMPs as well as the strategies to increase their selectivity toward specific cancer cells.

Distinct profiles of TERT promoter mutations and telomerase expression in head and neck cancer and cervical carcinoma.

Two recurrent mutations (-124 G > A and -146 G > A) in the core promoter region of the human telomerase reverse transcriptase (TERT) gene create consensus binding sites for ETS transcription factors and cause increased TERT expression in several tumour types. We analyzed TERT promoter mutations and TERT mRNA levels in head and neck cancer, cervical carcinoma and cervical intraepithelial neoplasia (CIN) as well as in C-4I, CaSki, HeLa and SiHa cervical cell lines. Nucleotide sequence analysis of TERT promoter region showed that 33.3% of oral squamous cell carcinoma (SCC) and 16.8% of cervical SCC harboured mutually exclusive G to A transitions at nucleotide position -124 or -146. TERT promoter was mutated at nucleotide -146 (G > A) in SiHa cell line. Other nucleotide changes creating in some cases putative ETS binding sites were more frequent in oral SCC (26.7%) than in cervical carcinoma (4.8%). The frequency of mutations was independent of human papillomavirus (HPV) tumour status in both cervical and oral cancer. Expression of TERT gene was significantly higher in TERT promoter mutated (-124G > A or -146G > A) cervical SCC compared to not mutated SCC irrespective of HPV16 E6 and E7 levels. Such hot spot changes were not detected in oropharyngeal SCC, cervical adenocarcinoma and CIN lesions. Our results suggest that TERT promoter mutations play a relevant role in oral SCC as well as in cervical SCC, besides the already known effect of HPV16 E6 protein on TERT expression.

Telomerase promoter mutations in human immunodeficiency virus-related conjunctiva neoplasia.

BACKGROUND: Squamous cell carcinoma (SCC) of the conjunctiva is a common cancer in Africa mainly associated with solar ultraviolet (UV) exposure and human immunodeficiency virus (HIV) infection. We analyzed the role of HIV on the occurrence of telomerase reverse transcriptase (TERT) promoter mutations among a cohort of conjunctiva neoplasia Ugandan patients. METHODS: Telomerase reverse transcriptase promoter mutations were searched in 72 conjunctiva neoplasia cases, comprising SCC and intraepithelial neoplasia grade 1-3 (CIN1-3), as well as in 53 conjunctiva normal tissues and in 24 HIV-related Kaposi sarcoma. RESULTS: The average prevalence of TERT promoter mutations in conjunctiva neoplasia was 31.9%. The mutation rates were significantly higher in HIV-positive (31.8% of CIN1 and CIN2, 46.2% of CIN3 and SCC,) than HIV-negative patients (22.2% of CIN1 and CIN2, 13.3% of CIN3 and SCC). Such mutations were rarely identified among HIV-positive conjunctiva controls (3.6%) and never in Kaposi sarcoma lesions. The most frequent variations were the hot spots - 124G>A and - 146G>A and tandem transitions - 124_125GG>AA and - 138_139GG>AA. CONCLUSIONS: Telomerase reverse transcriptase promoter mutations are early events in conjunctival neoplasia and could be used for timely diagnosis of conjunctiva tumours. The high frequency of UV-signatures in HIV-positive conjunctiva lesions suggests an additive effect of the virus to UV-related mutagenesis.

Unique true predicted neoantigens (TPNAs) correlates with anti-tumor immune control in HCC patients.

BACKGROUND: A novel prediction algorithm is needed for the identification of effective tumor associated mutated neoantigens. Only those with no homology to self wild type antigens are true predicted neoantigens (TPNAs) and can elicit an antitumor T cell response, not attenuated by central tolerance. To this aim, the mutational landscape was evaluated in HCV-associated hepatocellular carcinoma. METHODS: Liver tumor biopsies and adjacent non-tumor liver tissues were obtained from 9 HCV-chronically infected subjects and subjected to RNA-Seq analysis. Mutant peptides were derived from single nucleotide variations and TPNAs were predicted using two prediction servers (e.g. NetTepi and NetMHCstabpan) by comparison with corresponding wild-type sequences, non-related self and pathogen-related antigens. Immunological confirmation was obtained in preclinical as well as clinical setting. RESULTS: The development of such an improved algorithm resulted in a handful of TPNAs despite the large number of predicted neoantigens. Furthermore, TPNAs may share homology to pathogen's antigens and be targeted by a pre-existing T cell immunity. Cross-reactivity between such antigens was confirmed in an experimental pre-clinical setting. Finally, TPNAs homologous to pathogen's antigens were found in the only HCC long-term survival patient, suggesting a correlation between the pre-existing T cell immunity specific for these TPNAs and the favourable clinical outcome. CONCLUSIONS: The new algorithm allowed the identification of the very few TPNAs in cancer cells, and those targeted by a pre-existing immunity strongly correlated with long-term survival. Only such TPNAs represent the optimal candidates for immunotherapy strategies.

The Role of Circulating Free DNA and MicroRNA in Non-Invasive Diagnosis of HBV- and HCV-Related Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third and the fifth leading cause of cancer related death worldwide in men and in women, respectively. HCC generally has a poor prognosis, with a very low 5-year overall survival, due to delayed diagnosis and treatment. Early tumour detection and timely intervention are the best strategies to reduce morbidity and mortality in HCC patients. Histological evaluation of liver biopsies is the gold standard for cancer diagnosis, although it is an invasive, time-consuming and expensive procedure. Recently, the analysis of circulating free DNA (cfDNA) and RNA molecules released by tumour cells in body fluids, such as blood serum, saliva and urine, has attracted great interest for development of diagnostic assays based on circulating liver cancer molecular biomarkers. Such "liquid biopsies" have shown to be useful for the identification of specific molecular signatures in nucleic acids released by cancer cells, such as gene mutations and altered methylation of DNA as well as variations in the levels of circulating microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Body fluids analysis may represent a valuable strategy to monitor liver disease progression in subjects chronically infected with hepatitis viruses or cancer relapse in HCC treated patients. Several studies showed that qualitative and quantitative assays evaluating molecular profiles of circulating cell-free nucleic acids could be successfully employed for early diagnosis and therapeutic management of HCC patients. This review describes the state of art on the use of liquid biopsy for cancer driver gene mutations, deregulated DNA methylation as well as miRNA levels in HCC diagnosis.

Cell Penetrating Peptides as Molecular Carriers for Anti-Cancer Agents.

Cell membranes with their selective permeability play important functions in the tight control of molecular exchanges between the cytosol and the extracellular environment as the intracellular membranes do within the internal compartments. For this reason the plasma membranes often represent a challenging obstacle to the intracellular delivery of many anti-cancer molecules. The active transport of drugs through such barrier often requires specific carriers able to cross the lipid bilayer. Cell penetrating peptides (CPPs) are generally 5-30 amino acids long which, for their ability to cross cell membranes, are widely used to deliver proteins, plasmid DNA, RNA, oligonucleotides, liposomes and anti-cancer drugs inside the cells. In this review, we describe the several types of CPPs, the chemical modifications to improve their cellular uptake, the different mechanisms to cross cell membranes and their biological properties upon conjugation with specific molecules. Special emphasis has been given to those with promising application in cancer therapy.

Immunological effects of a novel RNA-based adjuvant in liver cancer patients.

Evaluation of biological effects of adjuvants on immune cells has been assessed in a limited number of studies. Moreover, no data are available on samples derived from cancer patients who may have a severe immune impairment. The effects of a novel RNA-based adjuvant (RNAdjuvant® developed by CureVac) were assessed in an ex vivo setting on PBMCs obtained from 8 healthy volunteers and 17 HCC patients, using a multiparametric approach to analyze network dynamics of early immune responses. Evaluation of CD80, CD86 and HLA-DR expression, cytokine production as well as gene expression was performed. Moreover, the downstream effect on CD4+ T cell phenotyping was evaluated. Treatment with RNAdjuvant® showed comparable effects on PBMCs of both HCC and healthy subjects. In particular, CD80, CD86 and HLA-DR expression was found up-regulated in circulating dendritic cells, which promoted a CD4+ T cell differentiation toward an effector phenotype. A mixed Th1/Th2 cytokine pattern was induced, although a more predominant production of TNFα and IFNγ was observed in HCC patients versus healthy controls. The cytokine profile was further confirmed by gene transcriptional analysis, which showed up-regulation of several genes involved in innate and adaptive immune-related pathways. The present study is the first demonstration that HCC patients and healthy subjects are equally responsive to an adjuvant. This may suggest that the same vaccine formulation including the RNAdjuvant® might have similar potency in healthy subjects and cancer patients.

MDM2 gene polymorphisms and risk of classic Kaposi's sarcoma among Iranian patients.

A single-nucleotide polymorphism (SNP) in the promoter region of MDM2 (SNP309T>G, rs2279744) has been shown to increase the expression of the MDM2 protein in various cancer types. However, only one study has analyzed the role of the MDM2 polymorphism in the development of Kaposi's sarcoma (KS). The association of MDM2 SNP309 with classic KS risk was evaluated in 79 Iranian patients with classic KS and 123 healthy controls. The MDM2 SNP309 was genotyped using PCR and restriction fragment length polymorphism methods. No significant correlation was found between the SNP309 polymorphism in MDM2 promoter and classic KS risk. There was no significant correlation between gender and disease stage. However, a significant association was found between SNP309 GG genotype and younger age (≤50 years) (odds ratio 9.5, 95% confidence intervals 1.5-60, p = 0.03). Our findings support no major role for the MDM2 SNP309 in KS development although it might influence the clinical outcome of KS in younger patients.

New Insights in the Design of Bioactive Peptides and Chelating Agents for Imaging and Therapy in Oncology.

Many synthetic peptides have been developed for diagnosis and therapy of human cancers based on their ability to target specific receptors on cancer cell surface or to penetrate the cell membrane. Chemical modifications of amino acid chains have significantly improved the biological activity, the stability and efficacy of peptide analogues currently employed as anticancer drugs or as molecular imaging tracers. The stability of somatostatin, integrins and bombesin analogues in the human body have been significantly increased by cyclization and/or insertion of non-natural amino acids in the peptide sequences. Moreover, the overall pharmacokinetic properties of such analogues and others (including cholecystokinin, vasoactive intestinal peptide and neurotensin analogues) have been improved by PEGylation and glycosylation. Furthermore, conjugation of those peptide analogues to new linkers and bifunctional chelators (such as AAZTA, TETA, TRAP, NOPO etc.), produced radiolabeled moieties with increased half life and higher binding affinity to the cognate receptors. This review describes the most important and recent chemical modifications introduced in the amino acid sequences as well as linkers and new bifunctional chelators which have significantly improved the specificity and sensitivity of peptides used in oncologic diagnosis and therapy.