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Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses (1). However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated (2). We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous selfantigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA (3,4), RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy., DCs initiate immune responses in lymphoid tissues upon early sensing of infectious pathogens (5). Previous work aimed at gene delivery to DCs largely resorted to functionalization of nanoparticles with molecular [...]

Human papilloma virus (HPV) infection is a causative agent for several cancers types (genital, anal and head and neck region). The HPV E6 and E7 proteins are oncogenic drivers and thus are ideal candidates for therapeutic vaccination. We recently reported that a novel ribonucleic acid lipoplex (RNA-LPX)-based HPV16 vaccine, E7 RNA-LPX, mediates regression of mouse HPV16+ tumors and establishes protective T cell memory. An HPV16 E6/E7 RNA-LPX vaccine is currently being investigated in two phase I and II clinical trials in various HPV-driven cancer types; however, it remains a high unmet medical need for treatments for patients with radiosensitive HPV16+ tumors. Therefore, we set out to investigate the therapeutic efficacy of E7 RNA-LPX vaccine combined with standard-of-care local radiotherapy (LRT). We demonstrate that E7 RNA-LPX synergizes with LRT in HPV16+ mouse tumors, with potent therapeutic effects exceeding those of either monotherapy. Mode of action studies revealed that the E7 RNA-LPX vaccine induced high numbers of intratumoral-E7-specific CD8+T cells, rendering cold tumors immunologically hot, whereas LRT primarily acted as a cytotoxic therapy, reducing tumor mass and intratumor hypoxia by predisposing tumor cells to antigen-specific T cell-mediated killing. Overall, LRT enhanced the effector function of E7 RNA-LPX-primed T cell responses. The therapeutic synergy was dependent on total radiation dose, rather than radiation dose-fractionation. Together, these results show that LRT synergizes with E7 RNA-LPX and enhances its anti-tumor activity against HPV16+ cancer models. This work paves into a new translational therapy for HPV16+ cancer patients. [ABSTRACT FROM AUTHOR]

Cancer therapy is an emergent application for mRNA therapeutics. While in tumor immunotherapy, mRNA encoding for tumor-associated antigens is delivered to antigen-presenting cells in spleen and lymph nodes, other therapeutic options benefit from immediate delivery of mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNA therapeutics is still a challenge, since, in addition to delivery of the cargo to the tumor, specifics of the targeted cell type as well as its interplay with the tumor microenvironment are crucial for successful intervention. This study investigated lipoplex nanoparticle-mediated mRNA delivery to spheroid cell culture models of melanoma. Insights into cell-type specific targeting, non-cell-autonomous effects, and penetration capacity in tumor and stroma cells of the mRNA lipoplex nanoparticles were obtained. It was shown that both coculture of different cell types as well as three-dimensional cell growth characteristics can modulate distribution and transfection efficiency of mRNA lipoplex formulations. The results demonstrate that three-dimensional coculture spheroids can provide a valuable surplus of information in comparison to adherent cells. Thus, they may represent in vitro models with enhanced predictivity for the in vivo activity of cancer nanotherapeutics. [ABSTRACT FROM AUTHOR]

Research shows that the non-histone chromatin-associated protein encoding Hmga2 promoter region mediates transcription activity. Data further indicate that the polypyrimidine tract binding protein binds to both polypyrimidinic strand of Hmga2 promoter and to ppyr-containing sequences of mouse and human promoters.

Objectives: We aimed to identify clinical, anamnestic, and sociodemographic characteristics associated with a positive swab for SARS-CoV2, and to provide a predictive score to identify at risk population in children aged 2–14 years attending school and tested for clinical symptoms of COVID-19. Design: Cross sectional study. Setting: Outpatient clinic of the IRCCS Burlo Garofolo, a maternal and child health tertiary care hospital and research centre in Italy. Data collection and analysis: Data were collected through a predefined form, filled out by parents, and gathered information on sociodemographic characteristics, and specific symptoms, which were analysed to determine their association with a positive SARS-CoV-2 swab. The regression coefficients of the variables included in the multivariate analysis were further used in the calculation of a predictive score of the positive or negative test. Results: Between September 20th and December 23rd 2020, from 1484 children included in the study, 127 (8.6%) tested positive. In the multivariate analysis, the variables retained by the model were the presence of contact with a cohabiting, non-cohabiting or unspecified symptomatic case (respectively OR 37.2, 95% CI 20.1–68.7; 5.1, 95% CI 2.7–9.6; 15.6, 95% CI 7.3–33.2); female sex (OR 1.49, 95% CI 1.0–2.3); age (6–10 years old: OR 3.2, 95% CI 1.7–6.1 p<0.001; >10 years old: OR 4.8, 95% CI 2.7–8.8 p<0.001); fever (OR 3.9, 95% CI 2.3–6.4); chills (OR 1.9, 95% CI 1.1–3.3); headache (OR 1.45, 95% CI 0.9–2.4); ageusia (OR 1.3, 95% CI 0.5–4.0); sore throat (OR 0.48, 95% CI 0.3–0.8); earache (OR 0.4, 95% CI 0.1–1.3); rhinorrhoea (OR 0.8, 95% CI 0.5–1.3); and diarrhoea (OR 0.52, 95% CI 0.2–1.1). The predictive score based on these variables generated 93% sensitivity and 99% negative predictive value. Conclusions: The timely identification of SARS-CoV2 cases among children is useful to reduce the dissemination of the disease and its related burden. The predictive score may be adopted in a public health perspective to rapidly identify at risk children. [ABSTRACT FROM AUTHOR]

Antigen-encoding, lipoplex-formulated RNA (RNA-LPX) enables systemic delivery to lymphoid compartments and selective expression in resident antigen-presenting cells. We report here that the rejection of CT26 tumors, mediated by local radiotherapy (LRT), is further augmented in a CD8+ T cell-dependent manner by an RNA-LPX vaccine that encodes CD4+ T cell-recognized neoantigens (CD4 neoantigen vaccine). Whereas CD8+ T cells induced by LRT alone were primarily directed against the immunodominant gp70 antigen, mice treated with LRT plus the CD4 neoantigen vaccine rejected gp70-negative tumors and were protected from rechallenge with these tumors, indicating a potent poly-antigenic CD8+ T cell response and T cell memory. In the spleens of CD4 neoantigen-vaccinated mice, we found a high number of activated, poly-functional, Th1-like CD4+ T cells against ME1, the immunodominant CD4 neoantigen within the poly-neoantigen vaccine. LRT itself strongly increased CD8+ T cell numbers and clonal expansion. However, tumor infiltrates of mice treated with CD4 neoantigen vaccine/LRT, as compared to LRT alone, displayed a higher fraction of activated gp70-specific CD8+ T cells, lower PD-1/LAG-3 expression and contained ME1-specific IFNγ+ CD4+ T cells capable of providing cognate help. CD4 neoantigen vaccine/LRT treatment followed by anti-CTLA-4 antibody therapy further enhanced the efficacy with complete remission of gp70-negative CT26 tumors and survival of all mice. Our data highlight the power of combining synergistic modes of action and warrants further exploration of the presented treatment schema. [ABSTRACT FROM AUTHOR]

HPV16 infections are associated with a variety of cancers and there is compelling evidence that the transforming activity of HPV16 critically depends on the expression of the viral oncoproteins E6 and E7. Therapeutic cancer vaccines capable of generating durable and specific immunity against these HPV16 antigens hold great promise to achieve long-term disease control. Here we show in mice that HPV16 E7 RNA-LPX, an intravenously administered cancer vaccine based on immuno-pharmacologically optimized antigen-encoding mRNA, efficiently primes and expands antigen-specific effector and memory CD8+ T cells. HPV-positive TC-1 and C3 tumors of immunized mice are heavily infiltrated with activated immune cells and HPV16-specific T cells and are polarized towards a proinflammatory, cytotoxic and less immune-suppressive contexture. E7 RNA-LPX immunization mediates complete and durable remission of progressing tumors. Circulating memory T cells are highly cytotoxic and protect from tumor rechallenge. Moreover, E7 RNA-LPX immunization sensitizes anti-PD-L1 refractory tumors to checkpoint blockade. In conclusion, our data highlight the potential of HPV16 RNA-LPX for the treatment of HPV-driven cancers. [ABSTRACT FROM AUTHOR]

Background: Working in health care carries the risk of transmission of infected blood to patients by hospital workers and to other health personnel in the form of occupational infections. Conscientious application of the standard precautions is the main method used to avoid needle stick injuries, contamination of skin and mucous membranes, cuts with sharp tools, and inadequate disposal and recapping of needles. The aim of this work was to investigate in Friuli Venezia Giulia, a region in north-east Italy, the enhancement carried out to prevent situations of biologic risk for health care workers, and to verify the related laboratory analyses. Methods: Biological accidents occurring during the years 2012-2013 in the departments of oncology and pediatric-obstetric surgery, and in the intensive care unit at Burlo Garofolo Children's Hospital in Trieste (a large town in Friuli Venezia Giulia) were reviewed, and a new panel of tests was introduced for patients and health care workers, to also detect human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), and aspartate transaminase and immunoglobulin G. All tests were submitted for external quality assessment. Results: In total, 230 nosocomial events were reported by health care workers in the abovementioned hospital departments in 2012-2013. There were 158 accidents in 2012, including 55 accidental needle stick injuries (34.81%), 59 blood splashes (37.34%), and 44 cuts with infected instruments (27.84%). The risk of sustaining a cut was related to movement error during surgery when the appropriate procedure was not followed or when devices were being assembled and passed between doctors and nurses. Most accidents happened among physicians compared to nurses; the high percentage of needle stick injuries (34.81%) versus nurses (25.94%) were due to incorrect recapping of needles after use. No cases of health care workers being infected with HCV, HBV, or HIV were identified. In 2013, the number of biological accidents decreased to 61, comprising two needle stick injuries (3.27%), 35 blood splashes (57.37%), and 25 cuts with contaminated instruments (40.98%). The number of subcutaneous abscesses with scarring resulting from cuts with sharp instruments decreased from three in 2012 (one of which was the subject of medicolegal proceedings) to none in 2013. Although our study population was relatively small, we did detect a statistically significant decrease in the number of needle stick injuries (P,0.05, X2 test). Conclusion: In this early experience at a maternal and child health institution in the Friuli Venezia Giulia region, application of a safety protocol, centralized organization of HIV tests, improved external quality assessment, and introduction of internal quality control for immunoglobulin G contributed to a decrease in the number of work-related biological accidents and their complications, which have the potential to result in medicolegal problems. [ABSTRACT FROM AUTHOR]

Involvement of health personnel in a medical audit can reduce the number of errors in laboratory medicine. The checked control of point of care testing (POCT) could be an answer to developing a better medical service in the emergency department and decreasing the time taken to report tests. The performance of sanitary personnel from different disciplines was studied over an 18-month period in a children's hospital. Clinical errors in the emergency and laboratory departments were monitored by: nursing instruction using specific courses, POCT, and external quality control; improvement of test results and procedural accuracy; and reduction of hemolyzed and nonprotocol-conforming samples sent to the laboratory department. In January 2012, point of care testing (POCT) was instituted in three medical units (neonatology, resuscitation, delivery room) at the Children's Hospital in Trieste, northeast Italy, for analysis of hematochemical samples. In the same period, during the months of January 2012 and June 2013, 1,600 samples sent to central laboratory and their related preanalytical errors were examined for accuracy. External quality control for POCT was also monitored in the emergency department; three meetings were held with physicians, nurses, and laboratory technicians to highlight problems, ie, preanalytical errors and analytical methodologies associated with POCT. During the study, there was an improvement in external quality control for POCT from -3 or -2 standard deviations or more to one standard deviation for all parameters. Of 800 samples examined in the laboratory in January 2012, we identified 64 preanalytical errors (8.0%); in June 2013, there were 17 preanalytical errors (2.1%), representing a significant decrease (P<0.05, χ2 test). Multidisciplinary management and clinical audit can be used as tools to detect errors caused by organizational problems outside the laboratory and improve clinical and economic outcomes. [ABSTRACT FROM AUTHOR]

Background: In northeastern Italy, according to Italian legislation, authorized public facilities can accept the donation and preservation of cord blood stem cells (CB-SC). Attitudes and knowledge in pregnant women differs between the local and immigrant (non-European Union [EU]) population. In this study we assessed the choices that pregnant women have with respect to the public and private harvesting system and the main reasons driving their decisions. We examined the ethnic origin of the families and compared tests for syphilis screening and leukocyte (WBC) counts in the CB-SC bags that are required for validation of the collection. Methods: Out of a population of 3450 pregnant patients at the Institute for Maternal and Child Health of Trieste, northeast Italy, 772 women agreed to cord blood harvesting and the associated lab tests. Of these, 221 women (28.6%) were from immigrant families of non-EU countries. Their ethnic affiliation was recorded, and tests were performed for syphilis screening and for nucleated red blood cell (NRBC) interference with the WBC count in CB-SC bags to assess cellularity and to determine if storage was appropriate. Results: Of the 772 pregnant women, 648 (84.0%) accessed the public collection system, which is free of charge, and 124 (15.0%) accessed the private fee-based system. One woman from the non-EU group opted for the private fee-based system. Of the 3450 pregnant women screened for syphilis at the Institute for Maternal and Child Health, the Treponema pallidum hemagglutination (TPHA) and Venereal Disease Research Laboratory (VDRL) tests were the main tests performed (66.0% of total cases) because many gynecologists in the public harvesting system apply the Italian regulations of the 1988 Decree, while the private system requires tests on syphilis and leaves the option to the lab physicians to select the best determination method. We found that the chemiluminescence method was more specific (97.0%) than the TPHA (83.0%) and nontreponemal rapid plasma reagin VDRL (75.0%) tests (P , 0.05, χ2 test). The specificity link between the two automatic methods versus microscopes for WBC dosing and NRBC interference was r2 = 0.08 (ADVIA 120) and r2 = 0.94 (χE-2100). The public system does not include human T-cell lymphotropic virus testing; this is reserved for the population from endemic zones. Conclusion: In northeastern Italy current legislation prevents the establishment of private fee-based banks for storage of CB-SC. The cryopreservation, for future autologous personal or family use, is possible only by sending to foreign private banks, with a further fee of €300. These regulations confirm that Italian legislation tries to increase the anonymous allogenic donations and the number of CB-CS bags stored in the free-cost public system, that are available to anyone with therapeutic needs. Private banking is used almost exclusively by the wealthier local population. In the public system, many physicians continue to use older Italian laws regarding syphilis diagnosis, and NRBC interference on WBC count may have an impact on cord blood harvesting. QOur findings suggest that in the EU there is no consensus policy on donor management. The value of storage for potential use within the family is useful only with collaboration between the public and the private systems. [ABSTRACT FROM AUTHOR]

Background: The Friuli Venezia Giulia (FVG), a region of North Eastern Italy, has passed legislation (Decree No 2324/2010) to regulate the banking of umbilical cord blood samples for personal, autologous, or family-directed use, and to implement the Agreement of the State-Regions Permanent Conference (Decree No 62/CSR/2010). This paper aims to identify the formalities and the reasons why families collect and bank their cord blood in foreign banks for both personal and private use. Methods: To this end, at the Institute for Maternal and Child Health of Trieste (the regional capital city of the FVG), Italy, which assists about 1800 pregnant women a year, 129 questionnaires, drafted from January 2010 to December 2011 and concerning the granting of authorization to export samples, were examined. Results: The collected data showed that 75% of involved families had resorted to anonymous public collection, which is available to anyone with therapeutic needs, and provided compatibility and hematologic protocols recognized by the scientific and international community (main indications: leukemia, hemoglobinopaties, and inherited hematologic and immunologic disorders). Conversely, 25.0% requested private storage at a foreign bank for personal or family-dedicated use. The principal motivation by disease was for treatment for diabetes (22.4%) and celiac disease (19.7%) (a multiorgan disease for which the FVG region has provided safeguards by approving a specific law granting support to families; Decree No 561/2007). For these two types of disease we found that information was received from the internet and not from general medical physicians, with a significant difference found using the χ2 test (P < 0.01). Conclusion: The indication of treating these diseases with cord blood stem cell transplantation appears to be well grounded and encouraging, and has recently been corroborated by the international literature; however, the economic and social motivations promoting cord blood storage, for a fee, in the event of diseases that are still under study, require accurate information through general medical physicians on the actual possibilities of treatment. [ABSTRACT FROM AUTHOR]

Background: The immature (or reticulated) platelet fraction (IPF) is rich in nucleic acids, especially RNA, and can be used as a predictive factor for platelet recovery in platelet immunomediated consumption or in postchemotherapy myelosuppression. Our aim was to determine if transfusions with IPF-rich solutions, during autologous peripheral blood stem cell transplantation, reduce the occurrence of bleeding and hemorrhagic complications. Patients and methods: Transfusions were administered to 40 children, affected with hematological pathologies, who underwent autologous peripheral hematopoietic progenitor cell transplantation. There were two groups of 20 patients, one group treated with IPF-poor and the other with IPF-rich solutions. In the two groups, the conditioning regimen was the same for the same pathology (hematological pathologies: 14 acute lymphoblastic leukemia; twelve acute myelocytic leukemia; four non-Hodgkin's lymphoma; two Hodgkin's lymphoma; eight solid tumors). A new automated analyzer was used to quantify the IPF: the XE2100 (Sysmex, Kobe, Japan) blood cell counter with upgraded software. Results: The 20 patients who received solutions with a high percentage of IPF (3%-9% of total number of infused platelets) required fewer transfusions than the 20 patients who received transfusions with a low percentage of IPF (0%-1% of total number of infused platelets): 83 versus 129 (mean of number of transfusions 4.15 versus 6.45) and a significant difference was found between the two groups by using the Mann-Whitney test (P , 0.001). The prophylactic transfusions decreased from three to two per week. There was only one case of massive hemorrhage. Conclusion: The use of IPF solutions reduces the number of transfusions and bleedings after peripheral blood stem cell transplantation in pediatric patients. [ABSTRACT FROM AUTHOR]

Many investigators consider children's drawings to be an important test in the evaluation of stress and anxiety, but few studies have examined the reliability and validity of indicators of emotional distress in children's projective drawings. In this report, we describe screening tests in children coming to the Friuli Venezia Giulia region in Northeastern Italy from non-European Union regions and suspected to have celiac disease, the problems involved in diagnosis of the disease, and the "drawn double nostalgia" of Romanian children for both Italian food and traditional Romanian foods. Of 3150 Western European cases, we found 712 with positive antibodies for IgA/IgG antitransglutaminase, 174 with a positive antiendomysium antibody confirmation test, and 20 with an IgA deficit. Of the children examined, 93% were children native to Western Europe, 4% were immigrants from Eastern Europe, and 1.6% originated from Africa. Among these, four Romanian children with celiac disease brought in their drawings, as requested in a hospital questionnaire. The prevalence of celiac disease is destined to increase among immigrants. Economic problems are common, and the twin nostalgia of immigrant children for foods and tastes that are "cozonac" (from the native country) and "panettone" (Italian cake flavor) represents a problem that will be difficult to resolve. Only some children's hospitals in Italy, ie, Burlo Garofolo and Gaslini, public and private foundations, or volunteer associations would be able to deal with this problem. [ABSTRACT FROM AUTHOR]

Abstract: Intracellular antibodies or intrabodies have great potential in protein knockout strategies for intracellular antigens. We applied the Intracellular Antibody Capture Technology for the direct selection in yeast of a mouse scFv library (VL–VH format) constructed from animals immunised with recombinant non-structural protein NSP5 of Rotavirus. We selected five different intracellular antibodies (ICAbs), which specifically recognize Δ2, an NSP5 deletion mutant used as bait. The anti-NSP5 ICAbs were well expressed both in yeast and mammalian cells as cytoplasmic or nuclear-tagged forms. By immunofluorescence and co-immunoprecipitation assays we characterised the intracellular interaction of the five anti-NSP5 ICAbs with the co-expressed antigens. [Copyright &y& Elsevier]

Synchronous primary malignancies occur in a small proportion of head and neck squamous cell carcinoma (HNSCC) patients. Here, we analysed three synchronous primaries and a recurrence from one patient by comparing the genomic and transcriptomic profiles among the tumour samples and determining the recurrence origin. We found remarkable levels of heterogeneity among the primary tumours, and through the patterns of shared mutations, we traced the origin of the recurrence. Interestingly, the patient carried germline variants that might have predisposed him to carcinogenesis, together with a history of alcohol and tobacco consumption. The mutational signature analysis confirmed the impact of alcohol exposure, with Signature 16 present in all tumour samples. Characterisation of immune cell infiltration highlighted an immunosuppressive environment in all samples, which exceeded the potential activity of T cells. Studies such as the one described here have important clinical value and contribute to personalised treatment decisions for patients with synchronous primaries and matched recurrences. [ABSTRACT FROM AUTHOR]

TLR7 agonists are considered promising drugs for cancer therapy. The currently available compounds are not well tolerated when administered intravenously and therefore are restricted to disease settings amenable for topical application. Here we present the preclinical characterization of SC1, a novel synthetic agonist with exquisite specificity for TLR7. We found that intravenously administered SC1 mediates systemic release of type I interferon, but not of proinflammatory cytokines such as TNFα and IL6, and results in activation of circulating immune cells. Tumors of SC1-treated mice have brisk immune cell infiltrates and are polarized towards a Th1 type signature. Intratumoral CD8+ T cells and CD11b+ conventional dendritic cells (cDCs) are significantly increased, plasmacytoid dendritic cells (pDCs) are strongly activated and macrophages are M1 phenotype polarized, whereas myeloid-derived suppressor cells (MDSCs) are decreased. We further show that treatment of mice with SC1 profoundly inhibits the growth of established syngeneic tumors and results in significantly prolonged survival. Mice, which are tumor-free after SC1 treatment are protected from subsequent tumor rechallenge. The antitumor effect of SC1 depends on antigen-specific CD8+ T cells, which we found to be strongly enriched in the tumors of SC1-treated mice. In conclusion, this study shows that systemically administered SC1 mobilizes innate and adaptive immunity and is highly potent as single agent in mice and thereby provides a rationale for clinical testing of this compound. [ABSTRACT FROM AUTHOR]

Cancer-associated mutations, mostly single nucleotide variations, can act as neoepitopes and prime targets for effective anti-cancer T-cell immunity. T cells recognizing cancer mutations are critical for the clinical activity of immune checkpoint blockade (ICB) and they are potent vaccine antigens. High frequencies of mutation-specific T cells are rarely spontaneously induced. Hence, therapies that broaden the tumor specific T-cell response are of interest. Here, we analyzed neoepitope-specific CD8+ T-cell responses mounted either spontaneously or after immunotherapy regimens, which induce local tumor inflammation and cell death, in mice bearing tumors of the widely used colon carcinoma cell line CT26. A comprehensive immune reactivity screening of 2474 peptides covering 628 transcribed CT26 point mutations was conducted. All tested treatment regimens were found to induce a single significant CD8+ T-cell response against a non-synonymous D733A point mutation in the Smc3 gene. Surprisingly, even though Smc3 D733A turned out to be the immune-dominant neoepitope in CT26 tumor bearing mice, neither T cells specific for this neoepitope nor their T cell receptors (TCRs) were able to recognize or lyse tumor cells. Moreover, vaccination with the D733A neoepitope did not result in anti-tumoral activity despite induction of specific T cells. This is to our knowledge the first report that neoepitope specific CD8+ T cells primed by tumor-released antigen exposure in vivo can be functionally irrelevant. [ABSTRACT FROM AUTHOR]

Objective: The human body is colonized by bacteria, fungi and viruses. Resident commensal bacteria are a fundamental line of resistance to colonization by exogenous microbes. They actively regulate the production of nutrients by the host through a negative feedback mechanism, in order to prevent the availability of nutrients for potential pathogens. While only a small fraction of these microorganism may be pathogenic, the relationship between host and commensal microbiome is now studied as a whole, impacting several aspects of the host biology. Some studies have made clear the progresses in examining the role of microbiome on transplants and graft versus host disease (GVHD) severity and its pathogenesis: the risk of complications from allogenic hematopoietic stem cells transplantation (HSCT) is greater with the highest mortality if a patient has a lower bacterial diversity in the gut prior to the transplantation process beginning. Microbiota-associated molecular patterns are directly recognized by pathogen recognition receptors. The development of molecular methods has greatly expanded our knowledge of the composition and function of the microbiome in health and disease, shortening the response times vs. microbiological culture tests. The gut flora can make the difference when it comes to allo-HSCT. The aim of the study was to monitor microbiome of 10 children during allo-HSCT.Methods: Oral specimens and gut faecal microbiome (100 grams) samples were collected at 2, 16, 24 days. The samples were analysed by polymerase chain reaction and primary sequencing was done. To calculate the biodiversity of microbiome the Shannon index and the Observed species index were chosen.Results: Our study suggests some differences in the diversity indices (DIs) in 5 children affected by GVHD vs. not affected. The DIs in oral and faecal specimens show in all patients a diminution in the post-transplant phase with an improvement in species diversity after 16 days from the transplant. The Observed species index in faeces specimens after 16 days was higher in patients which had not GVHD; moreover, patients with GVHD showed a deterioration at 24 days. Oral specimens after 24 days showed a parallel trend in the two groups. The Shannon index shows a downward trend in faeces specimens of the children with GVHD at 24 days; the children without GVHD recover a good trend of entropy. Oral specimens at 24 days show low entropy in the two groups. Very aggressive bacterial species as Cronobacter and Routella in the faeces specimens of a child had not serious consequences for disease status: Cronobacter were not present 24 days after transplantation.Conclusions: The data show the microbial metabolome could have an impact on patients with GVHD vs. no GVHD. A better understanding of the role of the oral and gut microbiome in GVHD can give directions to move towards the development of innovative approaches for preventing GVHD following allo-HCT, reducing also antibiotic therapy. [ABSTRACT FROM AUTHOR]

A correction to the article "Mutant MHC class II epitopes drive therapeutic immune responses to cancer" which was published in issue #520 is presented.

Antibody-dependent cellular phagocytosis (ADCP) is a process through which myeloid cells are able to exert their phagocytic function after recognition of opsonized bacteria, viruses, infected cells or any cells targeted by a specific antibody. ADCP of tumor cells represents a potent effector mechanism of monoclonal antibody therapy mediated by tumor associated macrophages (TAM) and other phagocytic cells as an in situ anti-tumor activity. Here we described a protocol based on flow cytometry and immunofluorescence assays enabling extensive comparative studies to address whether a monoclonal antibody engaging Fcγ receptors on macrophages can mediate in vitro ADCP of tumor cells., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Despite the preclinical promise of CD40 and 4-1BB as immuno-oncology targets, clinical efforts evaluating CD40 and 4-1BB agonists as monotherapy have found limited success. DuoBody-CD40×4-1BB (GEN1042/BNT312) is a novel investigational Fc-inert bispecific antibody for dual targeting and conditional stimulation of CD40 and 4-1BB to enhance priming and reactivation of tumor-specific immunity in patients with cancer., Methods: Characterization of DuoBody-CD40×4-1BB in vitro was performed in a broad range of functional immune cell assays, including cell-based reporter assays, T-cell proliferation assays, mixed-lymphocyte reactions and tumor-infiltrating lymphocyte assays, as well as live-cell imaging. The in vivo activity of DuoBody-CD40×4-1BB was assessed in blood samples from patients with advanced solid tumors that were treated with DuoBody-CD40×4-1BB in the dose-escalation phase of the first-in-human clinical trial (NCT04083599)., Results: DuoBody-CD40×4-1BB exhibited conditional CD40 and 4-1BB agonist activity that was strictly dependent on crosslinking of both targets. Thereby, DuoBody-CD40×4-1BB strengthened the dendritic cell (DC)/T-cell immunological synapse, induced DC maturation, enhanced T-cell proliferation and effector functions in vitro and enhanced expansion of patient-derived tumor-infiltrating lymphocytes ex vivo. The addition of PD-1 blocking antibodies resulted in potentiation of T-cell activation and effector functions in vitro compared with either monotherapy, providing combination rationale. Furthermore, in a first-in-human clinical trial, DuoBody-CD40×4-1BB mediated clear immune modulation of peripheral antigen presenting cells and T cells in patients with advanced solid tumors., Conclusion: DuoBody-CD40×4-1BB is capable of enhancing antitumor immunity by modulating DC and T-cell functions and shows biological activity in patients with advanced solid tumors. These findings demonstrate that targeting of these two pathways with an Fc-inert bispecific antibody may be an efficacious approach to (re)activate tumor-specific immunity and support the clinical investigation of DuoBody-CD40×4-1BB for the treatment of cancer., Competing Interests: Competing interests: US and ÖT are management board members and employees at BioNTech (Mainz, Germany). AM, FG, KS, AT, BS, JQ, MD and SK are employees at BioNTech. Some of the authors have securities from BioNTech. ES and FV are employees at TRON. HCA III, IA, JMB, SMB, DV, VMS, MF, DPES, SK, TA, ECWB and MJ-K are employees at Genmab and own stock and/or stock options. HCA III, IA, DPES, US, AM and FG are inventors on patents and patent applications related to CD40×4-1BB bispecific antibodies., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell-mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell-mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy., Significance: DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs. See related commentary by Li et al., p. 1184. This article is highlighted in the In This Issue feature, p. 1171., (©2022 The Authors; Published by the American Association for Cancer Research.)

Antigen-encoding, lipoplex-formulated RNA (RNA-LPX) enables systemic delivery to lymphoid compartments and selective expression in resident antigen-presenting cells. We report here that the rejection of CT26 tumors, mediated by local radiotherapy (LRT), is further augmented in a CD8 + T cell-dependent manner by an RNA-LPX vaccine that encodes CD4 + T cell-recognized neoantigens (CD4 neoantigen vaccine). Whereas CD8 + T cells induced by LRT alone were primarily directed against the immunodominant gp70 antigen, mice treated with LRT plus the CD4 neoantigen vaccine rejected gp70-negative tumors and were protected from rechallenge with these tumors, indicating a potent poly-antigenic CD8 + T cell response and T cell memory. In the spleens of CD4 neoantigen-vaccinated mice, we found a high number of activated, poly-functional, Th1-like CD4 + T cells against ME1, the immunodominant CD4 neoantigen within the poly-neoantigen vaccine. LRT itself strongly increased CD8 + T cell numbers and clonal expansion. However, tumor infiltrates of mice treated with CD4 neoantigen vaccine/LRT, as compared to LRT alone, displayed a higher fraction of activated gp70-specific CD8 + T cells, lower PD-1/LAG-3 expression and contained ME1-specific IFNγ + CD4 + T cells capable of providing cognate help. CD4 neoantigen vaccine/LRT treatment followed by anti-CTLA-4 antibody therapy further enhanced the efficacy with complete remission of gp70-negative CT26 tumors and survival of all mice. Our data highlight the power of combining synergistic modes of action and warrants further exploration of the presented treatment schema., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Cancer-associated mutations, mostly single nucleotide variations, can act as neoepitopes and prime targets for effective anti-cancer T-cell immunity. T cells recognizing cancer mutations are critical for the clinical activity of immune checkpoint blockade (ICB) and they are potent vaccine antigens. High frequencies of mutation-specific T cells are rarely spontaneously induced. Hence, therapies that broaden the tumor specific T-cell response are of interest. Here, we analyzed neoepitope-specific CD8 + T-cell responses mounted either spontaneously or after immunotherapy regimens, which induce local tumor inflammation and cell death, in mice bearing tumors of the widely used colon carcinoma cell line CT26. A comprehensive immune reactivity screening of 2474 peptides covering 628 transcribed CT26 point mutations was conducted. All tested treatment regimens were found to induce a single significant CD8 + T-cell response against a non-synonymous D733A point mutation in the Smc3 gene. Surprisingly, even though Smc3 D733A turned out to be the immune-dominant neoepitope in CT26 tumor bearing mice, neither T cells specific for this neoepitope nor their T cell receptors (TCRs) were able to recognize or lyse tumor cells. Moreover, vaccination with the D733A neoepitope did not result in anti-tumoral activity despite induction of specific T cells. This is to our knowledge the first report that neoepitope specific CD8 + T cells primed by tumor-released antigen exposure in vivo can be functionally irrelevant.

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.