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We present the preclinical pharmacology of BNT142, a lipid nanoparticle (LNP)–formulated RNA (RNA-LNP) encoding a T cell–engaging bispecific antibody that monovalently binds the T cell marker CD3 and bivalently binds claudin 6 (CLDN6), an oncofetal antigen that is absent from normal adult tissue but expressed on various solid tumors. Upon BNT142 RNA-LNP delivery in cell culture, mice, and cynomolgus monkeys, RNA is translated, followed by self-assembly into and secretion of the functional bispecific antibody RiboMab02.1. In vitro, RiboMab02.1 mediated CLDN6 target cell–specific activation and proliferation of T cells, and potent target cell killing. In mice and cynomolgus monkeys, intravenously administered BNT142 RNA-LNP maintained therapeutic serum concentrations of the encoded antibody. Concentrations of RNA-encoded RiboMab02.1 were maintained longer in circulation in mice than concentrations of directly injected, sequence-identical protein. Weekly injections of mice with BNT142 RNA-LNP in the 0.1- to 1-μg dose range were sufficient to eliminate CLDN6-positive subcutaneous human xenograft tumors and increase survival over controls. Tumor regression was associated with an influx of T cells and depletion of CLDN6-positive cells. BNT142 induced only transient and low cytokine production in CLDN6-positive tumor-bearing mice humanized with peripheral blood mononuclear cells (PBMCs). No signs of adverse effects from BNT142 RNA-LNP administration were observed in mice or cynomolgus monkeys. On the basis of these and other findings, a phase 1/2 first-in-human clinical trial has been initiated to assess the safety and preliminary efficacy of BNT142 RNA-LNP in patients with CLDN6-positive advanced solid tumors (NCT05262530). Editor's summary: Bispecific T cell–engaging antibodies (BsAbs) have been approved to treat hematological malignancies; however, current forms are limited because of manufacturing and short serum half-lives. To improve these BsAbs, Stadler et al. have created BNT142, a lipid nanoparticle (LNP)–formulated RNA encoding a BsAb against CD3 and the oncofetal antigen claudin 6 (CLDN6). The authors then showed inhibition of tumor growth in treated preclinical mouse models and favorable pharmacokinetic profiles and no adverse effects in cynomolgus monkeys. This has led to initiation of a phase 1/2 first-in-human clinical trial, which will be used to assess safety and efficacy in patients with advanced solid tumors (NCT05262530). —Dorothy Hallberg [ABSTRACT FROM AUTHOR]

IMAB362/Zolbetuximab, a first-in-class IgG1 antibody directed against the cancer-associated gastric-lineage marker CLDN18.2, has recently been reported to have met its primary endpoint in two phase 3 trials as a first-line treatment in combination with standard of care chemotherapy in CLDN18.2-positive Her2 negative advanced gastric cancer. Here we characterize the preclinical pharmacology of BNT141, a nucleoside-modified RNA therapeutic encoding the sequence of IMAB362/Zolbetuximab, formulated in lipid nanoparticles (LNP) for liver uptake. We show that the mRNA-encoded antibody displays a stable pharmacokinetic profile in preclinical animal models, mediates CLDN18.2-restricted cytotoxicity comparable to IMAB362 recombinant protein and inhibits human tumor xenograft growth in immunocompromised mice. BNT141 administration did not perpetrate mortality, clinical signs of toxicity, or gastric pathology in animal studies. A phase 1/2 clinical trial with BNT141 mRNA-LNP has been initiated in advanced CLDN18.2-expressing solid cancers (NCT04683939). [ABSTRACT FROM AUTHOR]

Successful outcome of immune checkpoint blockade in patients with solid cancers is in part associated with a high tumor mutational burden (TMB) and the recognition of private neoantigens by T-cells. The quality and quantity of target recognition is determined by the repertoire of 'neoepitope'-specific T-cell receptors (TCRs) in tumor-infiltrating lymphocytes (TIL), or peripheral T-cells. Interferon gamma (IFN-γ), produced by T-cells and other immune cells, is essential for controlling proliferation of transformed cells, induction of apoptosis and enhancing human leukocyte antigen (HLA) expression, thereby increasing immunogenicity of cancer cells. TCR αβ-dependent therapies should account for tumor heterogeneity and availability of the TCR repertoire capable of reacting to neoepitopes and functional HLA pathways. Immunogenic epitopes in the tumor-stroma may also be targeted to achieve tumor-containment by changing the immune-contexture in the tumor microenvironment (TME). Non protein-coding regions of the tumor-cell genome may also contain many aberrantly expressed, non-mutated tumor-associated antigens (TAAs) capable of eliciting productive anti-tumor immune responses. Whole-exome sequencing (WES) and/or RNA sequencing (RNA-Seq) of cancer tissue, combined with several layers of bioinformatic analysis is commonly used to predict possible neoepitopes present in clinical samples. At the ImmunoSurgery Unit of the Champalimaud Centre for the Unknown (CCU), a pipeline combining several tools is used for predicting private mutations from WES and RNA-Seq data followed by the construction of synthetic peptides tailored for immunological response assessment reflecting the patient's tumor mutations, guided by MHC typing. Subsequent immunoassays allow the detection of differential IFN-γ production patterns associated with (intra-tumoral) spatiotemporal differences in TIL or peripheral T-cells versus TIL. These bioinformatics tools, in addition to histopathological assessment, immunological readouts from functional bioassays and deep T-cell 'adaptome' analyses, are expected to advance discovery and development of next-generation personalized precision medicine strategies to improve clinical outcomes in cancer in the context of i) anti-tumor vaccination strategies, ii) gauging mutation-reactive T-cell responses in biological therapies and iii) expansion of tumor-reactive T-cells for the cellular treatment of patients with cancer. [ABSTRACT FROM AUTHOR]

Memory formation, guided by microbial ligands, has been reported for innate immune cells. Epigenetic imprinting plays an important role herein, involving histone modification after pathogen-/danger-associated molecular patterns (PAMPs/DAMPs) recognition by pattern recognition receptors (PRRs). Such "trained immunity" affects not only the nominal target pathogen, yet also non-related targets that may be encountered later in life. The concept of trained innate immunity warrants further exploration in cancer and how these insights can be implemented in immunotherapeutic approaches. In this review, we discuss our current understanding of innate immune memory and we reference new findings in this field, highlighting the observations of trained immunity in monocytic and natural killer cells. We also provide a brief overview of trained immunity in non-immune cells, such as stromal cells and fibroblasts. Finally, we present possible strategies based on trained innate immunity that may help to devise host-directed immunotherapies focusing on cancer, with possible extension to infectious diseases. [ABSTRACT FROM AUTHOR]

Summary: Human cytomegalovirus (CMV) is a ubiquitous, persistent beta herpesvirus. CMV infection contributes to the accumulation of functional antigen‐specific CD8+ T‐cell pools with an effector‐memory phenotype and enrichment of these immune cells in peripheral organs. We review here this 'memory T‐cell inflation' phenomenon and associated factors including age and sex. 'Collateral damage' due to CMV‐directed immune reactivity may occur in later stages of life – arising from CMV‐specific immune responses that were beneficial in earlier life. CMV may be considered an age‐dependent immunomodulator and a double‐edged sword in editing anti‐tumour immune responses. Emerging evidence suggests that CMV is highly prevalent in patients with a variety of cancers, particularly glioblastoma. A better understanding of CMV‐associated immune responses and its implications for immune senescence, especially in patients with cancer, may aid in the design of more clinically relevant and tailored, personalized treatment regimens. 'Memory T‐cell inflation' could be applied in vaccine development strategies to enrich for immune reactivity where long‐term immunological memory is needed, e.g. in long‐term immune memory formation directed against transformed cells. The immunomodulatory effect of inflationary cytomegalovirus (CMV)‐specific memory T cells stretches across immunological niches, including long‐term memory T cells. The CMV‐driven cellular immune repertoire may cause shifts in immune recognition profiles, or lead to reduced survival of 'inflationary T cells', and potentially shapes the quality and quantity of the anti‐tumour cellular immune response in patients with cancer. [ABSTRACT FROM AUTHOR]

KRAS is a driver mutation for malignant transformation. It is found in 30% of all cancers and in 90% of pancreatic cancers. The identification of small molecules selectively inhibiting KRAS mutants has been challenging, yet mutant KRAS has recently been shown to be targeted by tumor-infiltrating lymphocyte (TIL)-derived T cells that confer tumor regression upon adoptive transfer. Furthermore, a human IgG1 monoclonal antibody interfering with mutant KRAS function inside the cell has been described to inhibit growth of KRAS-mutant xenografts in tumor-bearing mice. B cells have been described to infiltrate pancreatic cancer and may be associated with tertiary lymphoid structures associated with good prognosis, or, in contrast, promote tumor growth. However, their function, nor their antigen-specificity has been clearly defined. We discuss here the presence of tumor-infiltrating B cells (TIB) in patients with pancreatic cancer that produce KRAS-mutant specific IgG, underlining that intratumoral T and B cells may exclusively target mutant KRAS. KRAS-specific IgG may, therefore, serve as a readout of the activation of both arms of the anti-tumor adaptive immune armament although some B-cell populations may promote tumor progression. [ABSTRACT FROM AUTHOR]

Virus-specific T-cell responses are crucial to control cytomegalovirus (CMV) infections/reactivation in immunocompromised individuals. Adoptive cellular therapy with CMVspecific T-cells has become a viable treatment option. High-affinity anti-viral cellular immune responses are associated with improved long-term immune protection against CMV infection. To date, the characterization of high-affinity T-cell responses against CMV has not been achieved in blood from patients after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, the purpose of this study was to describe and analyze the phenotype and clinical impact of different CMV-specific CD8+ cytotoxic T-lymphocytes (CMV-CTL) classes based on their T-cell receptor (TCR) affinity. T-cells isolated from 23 patients during the first year following HSCT were tested for the expression of memory markers, programmed cell death 1 (PD-1), as well as TCR affinity, using three different HLA-A*02:01 CMVNLVPMVATV-Pp65 tetramers (wild-type, a245v and q226a mutants). High-affinity CMV-CTL defined by q226a tetramer binding, exhibited a higher frequency in CD8+ T-cells in the first month post-HSCT and exhibited an effector memory phenotype associated with strong PD-1 expression as compared to the medium- and low-affinity CMV-CTLs. High-affinity CMV-CTL was found at higher proportion in patients with chronic graft-versus-host disease (p < 0.001). This study provides a first insight into the detailed TCR affinities of CMV-CTL. This may be useful in order to improve current immunotherapy protocols using isolation of viral-specific T-cell populations based on their TCR affinity. [ABSTRACT FROM AUTHOR]

Treatment of multidrug-resistant tuberculosis (MDR-TB) is extremely challenging due to the virulence of the etiologic strains of Mycobacterium tuberculosis (M. tb), the aberrant host immune responses and the diminishing treatment options with TB drugs. New treatment regimens incorporating therapeutics targeting both M. tb and host factors are urgently needed to improve the clinical management outcomes of MDR-TB. Host-directed therapies (HDT) could avert destructive tuberculous lung pathology, facilitate eradication of M. tb, improve survival and prevent long-term functional disability. In this review we (1) discuss the use of HDT for cancer and other infections, drawing parallels and the precedent they set for MDR-TB treatment, (2) highlight preclinical studies of pharmacological agents commonly used in clinical practice which have HDT potential, and (3) outline developments in cellular therapy to promote clinically beneficial immunomodulation to improve treatment outcomes in patients with pulmonary MDR-TB. The use of HDTs as adjuncts to MDR-TB therapy requires urgent evaluation. [ABSTRACT FROM AUTHOR]

Summary Anti-tuberculosis drug treatment is known to affect the number, phenotype, and effector functionality of antigen-specific T-cells. In order to objectively gauge Mycobacterium tuberculosis (MTB)-specific CD8+ T-cells at the single-cell level, we developed soluble major histocompatibility complex (MHC) class I multimers/peptide multimers, which allow analysis of antigen-specific T-cells without ex vivo manipulation or functional tests. We constructed 38 MHC class I multimers covering some of the most frequent MHC class I alleles (HLA-A*02:01, A*24:02, A*30:01, A*30:02, A*68:01, B*58:01, and C*07:01) pertinent to a South African or Zambian population, and presenting the following MTB - derived peptides: the early expressed secreted antigens TB10.4 (Rv0288), Ag85B (Rv1886c), and ESAT-6 (Rv3875), as well as intracellular enzymes, i.e., glycosyltransferase 1 (Rv2957), glycosyltransferase 2 (Rv2958c), and cyclopropane fatty acid synthase (Rv0447c). Anti-TB treatment appeared to impact on the frequency of multimer-positive CD8+ T-cells, with a general decrease after 6 months of therapy. Also, a reduction in the total central memory CD8+ T-cell frequencies, as well as the antigen-specific compartment in CD45RA−CCR7+ T-cells was observed. We discuss our findings on the basis of differential dynamics of MTB-specific T-cell frequencies, impact of MTB antigen load on T-cell phenotype, and antigen-specific T-cell responses in tuberculosis. [ABSTRACT FROM AUTHOR]

Summary: Tuberculosis continues to kill 1·4 million people annually. During the past 5 years, an alarming increase in the number of patients with multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis has been noted, particularly in eastern Europe, Asia, and southern Africa. Treatment outcomes with available treatment regimens for drug-resistant tuberculosis are poor. Although substantial progress in drug development for tuberculosis has been made, scientific progress towards development of interventions for prevention and improvement of drug treatment outcomes have lagged behind. Innovative interventions are therefore needed to combat the growing pandemic of multidrug-resistant and extensively drug-resistant tuberculosis. Novel adjunct treatments are needed to accomplish improved cure rates for multidrug-resistant and extensively drug-resistant tuberculosis. A novel, safe, widely applicable, and more effective vaccine against tuberculosis is also desperately sought to achieve disease control. The quest to develop a universally protective vaccine for tuberculosis continues. So far, research and development of tuberculosis vaccines has resulted in almost 20 candidates at different stages of the clinical trial pipeline. Host-directed therapies are now being developed to refocus the anti-Mycobacterium tuberculosis-directed immune responses towards the host; a strategy that could be especially beneficial for patients with multidrug-resistant tuberculosis or extensively drug-resistant tuberculosis. As we are running short of canonical tuberculosis drugs, more attention should be given to host-directed preventive and therapeutic intervention measures. [ABSTRACT FROM AUTHOR]

Bacillus Calmette–Guérin (BCG), the only approved tuberculosis vaccine, provides only limited protection. Previously, we generated a recombinant derivative (BCG ΔureC::hly), which secretes the pore-forming toxin listeriolysin O (LLO) of Listeria monocytogenes. This vaccine shows superior protection against tuberculosis in preclinical models and is safe in humans. Here we describe two new vaccine strains which express human interleukin-7 (hIL)-7 or hIL-18 in the genetic background of BCG ΔureC::hly to modulate specific T cell immunity. Both strains exhibited an uncompromised in vitro growth pattern, while inducing a proinflammatory cytokine profile in human dendritic cells (DCs). Human DCs harbouring either strain efficiently promoted secretion of IL-2 by autologous T cells in a coculture system, suggesting superior immunogenicity. BALB/c mice vaccinated with BCG ΔureC::hly, BCG ΔureC::hly_hIL7 or BCG ΔureC::hly_hIL18 developed a more robust Th1 response than after vaccination with parental BCG. Both strains provided significantly better protection than BCG in a murine Mycobacterium tuberculosis challenge model but efficacy remained comparable to that afforded by BCG ΔureC::hly. We conclude that expression of hIL-7 or hIL-18 enhanced specific T cell responses but failed to improve protection over BCG ΔureC::hly in mice. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis ( Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F420-dependent anti-oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F420-deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/ NAD+ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F420− mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin-dependent nitroreductase ( Ddn) and its two homologues Rv1261c and Rv1558 encode for an F420H2-dependent quinone reductase ( Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F420H2-specific obligate two-electron reduction of endogenous quinones, thereby competing with the one-electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F420 biosynthesis pathway or Fqr-class proteins as a mechanism to potentiate the action of bactericidal agents. [ABSTRACT FROM AUTHOR]

Immune responses to human cytomegalovirus (CMV) can be used to assess immune fitness in an individual. Further to its clinical significance in posttransplantation settings, emerging clinical and translational studies provide examples of immune correlates of protection pertaining to anti-CMV immune responses in the context of cancer or infectious diseases, e.g., tuberculosis. In this viewpoint, we provide a brief overview about CMV-directed immune reactivity and immune fitness in a clinical context and incorporate some of our own findings obtained from peripheral blood or tumour-infiltrating lymphocytes (TIL) from patients with advanced cancer. Observations in patients with solid cancers whose lesions contain both CMV and tumour antigen-specific T-cell subsets are highlighted, due to a possible CMV-associated "bystander" effect in amplifying local inflammation and subsequent tumour rejection. The role of tumour-associated antibodies recognising diverse CMV-derived epitopes is also discussed in light of anti-cancer immune responses. We discuss here the use of anti-CMV immune responses as a theranostic tool—combining immunodiagnostics with a personalised therapeutic potential—to improve treatment outcomes in oncological indications. [ABSTRACT FROM AUTHOR]

The article offers information on the development of tuberculosis vaccine for patients with human immunodeficiency virus type 1. Topics discussed include the use of BCG vaccine for tuberculosis, the Ag85A antigen when tested proved not to decrease mycobacterium tuberculosis infection, and the relevance of cytotoxic T lymphocytes clinically in tuberculosis.

Tumor-infiltrating lymphocytes (TILs) may represent a viable source of T cells for the biological treatment of patients with gliomas. Glioma tissue was obtained from 16 patients, tumor cell lines were established, and TILs were expanded in 16/16 cases using a combination of IL-2/IL-15/IL-21. Intracellular cytokine staining (ICS, IL-2, IL-17, TNFα and IFNγ production) as well as a cytotoxicity assay was used to detect TIL reactivity against autologous tumor cells or shared tumor-associated antigens (TAAs; i.e., NY-ESO-1, Survivin or EGFRvIII). TILs were analyzed by flow cytometry, including T-cell receptor (TCR) Vβ family composition, exhaustion/activation and T-cell differentiation markers (CD45RA/CCR7). IL-2/IL-15/IL-21 expanded TILs exhibited a mixture of CD4+, CD8+, as well as CD3+CD4−CD8−T cells with a predominant central memory CD45RA−CCR7+phenotype. TIL showed low frequencies of T cells testing positive for PD-1, TIM-3 and CTLA-4. LAG3 tested positive in up to 30% of CD8+TIL, with low (1.25%) frequencies in CD4+T cells. TIL cultures exhibited preferential usage of Vβ families and recognition of autologous tumor cells defined by cytokine production and cytotoxicity. IL-2/IL-15/IL-21 expanded TILs represent a viable source for the cellular therapy of patients with gliomas. [ABSTRACT FROM AUTHOR]

Despite extensive global efforts in the fight against killer infectious diseases, they still cause one in four deaths worldwide and are important causes of long-term functional disability arising from tissue damage. The continuing epidemics of tuberculosis, HIV, malaria, and influenza, and the emergence of novel zoonotic pathogens represent major clinical management challenges worldwide. Newer approaches to improving treatment outcomes are needed to reduce the high morbidity and mortality caused by infectious diseases. Recent insights into pathogen-host interactions, pathogenesis, inflammatory pathways, and the host's innate and acquired immune responses are leading to identification and development of a wide range of host-directed therapies with different mechanisms of action. Host-directed therapeutic strategies are now becoming viable adjuncts to standard antimicrobial treatment. Host-directed therapies include commonly used drugs for non-communicable diseases with good safety profiles, immunomodulatory agents, biologics (eg monoclonal antibodies), nutritional products, and cellular therapy using the patient's own immune or bone marrow mesenchymal stromal cells. We discuss clinically relevant examples of progress in identifying host-directed therapies as adjunct treatment options for bacterial, viral, and parasitic infectious diseases. [ABSTRACT FROM AUTHOR]

Summary Three years after its first discovery in Jeddah Saudi Arabia, the novel zoonotic pathogen of humans, the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) continues to be a major threat to global health security. 1 Sporadic community acquired cases of MERS continue to be reported from the Middle East. The recent nosocomial outbreaks in hospitals in Seoul, Korea and at the National Guard Hospital in Riyadh, Saudi Arabia indicate the epidemic potential of MERS-CoV. Currently there are no effective anti-MERS-CoV anti-viral agents or therapeutics and MERS is associated with a high mortality rate (40%) in hospitalised patients. A large proportion of MERS patients who die have a range of pulmonary pathology ranging from pneumonia to adult respiratory distress syndrome with multi-organ failure, compounded by co-morbidities, reflecting a precarious balance of interactions between the host-immune system and MERS-CoV. Whilst we wait for new MERS-CoV specific drugs, therapeutics and vaccines to be developed, there is a need to advance a range of Host-Directed Therapies. A range of HDTs are available, including commonly used drugs with good safety profiles, which could augment host innate and adaptive immune mechanisms to MERS-CoV, modulate excessive inflammation and reduce lung tissue destruction. We discuss the rationale and potential of using Host-Directed Therapies for improving the poor treatment outcomes associated with MERS. Carefully designed randomized controlled trials will be needed to determine whether HDTs could benefit patients with MERS. The recurrent outbreaks of MERS-CoV infections at hospitals in the Middle East present unique opportunities to conduct randomized clinical trials. The time has come for a more coordinated global response to MERS and a multidisciplinary global MERS-CoV response group is required to take forward priority research agendas. [ABSTRACT FROM AUTHOR]

The intricate interplay between the immune system and microbes is an essential part of the physiological homeostasis in health and disease. Immunological recognition of commensal microbes, such as bacterial species resident in the gut or lung as well as dormant viral species, i.e., cytomegalovirus (CMV) or Epstein-Barr virus (EBV), in combination with a balanced immune regulation, is central to achieve immune-protection. Emerging evidence suggests that immune responses primed to guard against commensal microbes may cause unexpected pathological outcomes, e.g., chronic inflammation and/or malignant transformation. Furthermore, translocation of immune cells from one anatomical compartment to another, i.e., the gut-lung axis via the lymphatics or blood has been identified as an important factor in perpetrating systemic inflammation, tissue destruction, as well as modulating host-protective immune responses. We present in this review immune response patterns to pathogenic as well as non-pathogenic microbes and how these immune-recognition profiles affect local immune responses or malignant transformation. We discuss personalized immunological therapies which, directly or indirectly, target host biological pathways modulated by antimicrobial immune responses., (Copyright © 2020 Lérias, Paraschoudi, de Sousa, Martins, Condeço, Figueiredo, Carvalho, Dodoo, Castillo-Martin, Beltrán, Ligeiro, Rao, Zumla and Maeurer.)

We review state-of-the-art in translational and clinical studies focusing on the tumor microenvironment (TME) with a focus on tumor-infiltrating B cells (TIBs). The TME is a dynamic matrix of mutations, immune-regulatory networks, and distinct cell-to-cell interactions which collectively impact on disease progress. We discuss relevant findings concerning B cells in pancreatic cancer, the concepts of "bystander" B cells, the role of antigen-specific B cells contributing to augmenting anticancer-directed immune responses, the role of B cells as prognostic markers for response to checkpoint inhibitors (ICBs), and the potential use in adoptive cell tumor-infiltrating lymphocyte (TIL) products.

The biology and clinical efficacy of immune cells from patients with infectious diseases or cancer are associated with metabolic programming. Host immune- and stromal-cell genetic and epigenetic signatures in response to the invading pathogen shape disease pathophysiology and disease outcomes. Directly linked to the immunometabolic axis is the role of the host microbiome, which is also discussed here in the context of productive immune responses to lung infections. We also present host-directed therapies (HDT) as a clinically viable strategy to refocus dysregulated immunometabolism in patients with infectious diseases, which requires validation in early phase clinical trials as adjuncts to conventional antimicrobial therapy. These efforts are expected to be continuously supported by newly generated basic and translational research data to gain a better understanding of disease pathology while devising new molecularly defined platforms and therapeutic options to improve the treatment of patients with pulmonary infections, particularly in relation to multidrug-resistant pathogens.

[This corrects the article DOI: 10.18632/oncotarget.24955.].

Purpose: We investigated serum cytokine and T-cell responses directed against tumour-associated antigens (TAAs) in association with survival of patients with glioblastoma multiforme (GBM)., Patients and Methods: Peripheral blood from 205 treatment-naïve patients with glioma (GBM = 145; non-GBM = 60) was obtained on the day of surgery to measure (i) circulating T-cells reacting to viral antigens and TAAs, in the presence or absence of cytokine conditioning with IL-2/IL-15/IL-21 or IL-2/IL-7, and (ii) serum cytokine levels (IL-4, IL-5, IL-6, TNF-α, IFN-γ and IL-17A). Patients were followed-up for at least 1000 days post-surgery. Survivin protein and gene expression in resected GBM tumour tissue were confirmed by immunohistochemistry and real-time polymerase chain reaction, respectively. Antigen-specific T-cell responses were gauged by ICS (intracellular cytokine production). Associations between patient survival and immunological reactivity patterns were analysed using univariate and multivariate statistics., Results: Approximately 2% of patients with GBM and 18% of patients with non-GBM glioma, were alive beyond 1000 days of surgery. Univariate analysis indicated that the combination of three cytokines (IL-4/IL-5/IL-6, p = .0022; IFN-γ/TNF-α/IL-17A, p = .0083) but not a 'partial' combination of these cytokines, the IFN-γ immune response to EBV-EBNA-1 (p < .0001) as well as T-cell responses to the survivin 97-111 peptide (p = .0152) correlated with longer survival among patients with GBM. Multivariate analysis identified survivin 97-111 -directed IFN-γ production with IL-2/IL-15/IL-21 conditioning (p = .024), and the combined presence of serum IFN-γ/TNF-α/IL-17a (p = .003) as independent predictors of survival., Conclusion: Serum cytokine patterns and lymphocyte reactivity to survivin 97-111 , particularly with IL-2, IL-15 and IL-21 conditioning may be instrumental in predicting survival among patients with GBM. This has implications for clinical follow-up of patients with GBM and the targeted development of immunotherapy for patients with CNS tumours., (Copyright © 2018. Published by Elsevier B.V.)

Most patients with pancreatic cancer present with extensive metastasis at diagnosis, with a 5-year survival rate of approximately 5%, despite chemotherapy and surgery. New treatment modalities are needed to improve survival. Mesothelin is a tumor-associated antigen (TAA) in patients with pancreatic cancer that could be used to gauge cellular immune responses directed against transformed cells since up to 100 percent of pancreatic ductal adenocarcinoma cells have been shown to strongly express mesothelin. A prospective, observational study was carried out in twenty-six, chemotherapy-naïve patients with resectable pancreatic ductal adenocarcinoma. Participants were between 48 and 81 years (median age: 64.5 years), 15 males and 11 females. All participants were clinically followed-up between 439 and 853 days post-surgery (n=14) or until death (n=12). Peripheral blood drawn on the day of surgery was stimulated with a mesothelin peptide pool (42 peptides, non-overlapping), individual mesothelin peptides, positive (anti-CD3 antibody, OKT3) and negative controls (medium) with or without adding IL-21. Kaplan-Meier estimators were used to gauge patients' survival pattern in relation to mesothelin-specific IFN-γ responses. A survival benefit was linked with IFN-γ responses to peptides corresponding to mature mesothelin (p=0.018) and targeted recognition of the mesothelin 601-615 epitope (MQEALSGTPCLLGPG) (p=0.006) in the presence of IL-21. Conversely, production of high levels of IFN-γ to OKT3 stimulation with IL-21 conditioning was associated with reduced survival of patients (p=0.016). Gauging anti-Mesothelin- directed immune responses will aid to identify patients i) in need of a more intensive clinical follow-up and ii) who may benefit from immunotherapeutic approaches targeting mesothelin., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Neoepitope-specific T-cell responses have been shown to induce durable clinical responses in patients with advanced cancers. We explored the recognition patterns of tumor-infiltrating T lymphocytes (TILs) from patients with glioblastoma multiforme (GBM), the most fatal form of tumors of the central nervous system. Whole-genome sequencing was used for generating DNA sequences representing the entire spectrum of 'private' somatic mutations in GBM tumors from five patients, followed by 15-mer peptide prediction and subsequent peptide synthesis. For each mutated peptide sequence, the wildtype sequence was also synthesized and individually co-cultured with autologous GBM TILs, which had been expanded in vitro with a combination of interleukin (IL)-2, IL-15 and IL-21. After seven days of culture, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and/or IL-17A production was measured by ELISA in culture supernatants, and used as an epitope-specific immune response readout. Mutated peptides that induced a strong cytokine response were considered to contain legitimate neoepitopes. TILs from 5/5 patients with GBM exhibited specific immune reactivity profiles to the nominal target peptides, defined by IFN-γ and/or TNF-α production, as well as IL-17A. Neoepitopes, defined by mutated peptides inducing IFN-γ and/or TNF-α production without or only minimal reactivity to the wildtype sequences, were found for each individual patient. CD8+ TILs dominated the patients' responses to private neoepitopes. The present study shows that neoepitope-specific TIL reactivity constitutes an important arm of anti-tumor immune responses in patients with GBM, and thus a powerful tool for developing next-generation personalized immunotherapies., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Background: Patients with advanced malignancies, e.g. lung cancer, ovarian cancer or melanoma, frequently present with brain metastases. Clinical presentation and disease progression of cancer is in part shaped by the interaction of the immune system with malignant cells. Antigen-targeted immune responses have been implicated in the prolonged survival of patients with cancer. This includes the tumor-associated antigen (TAA) mature mesothelin, a 40kDa cell surface-bound antigen that is overexpressed in several malignancies including lung ovarian and pancreatic cancer. We examined in an observational, prospective study the survival of patients with brain metastases in association with clinical parameters and cellular immune responses to molecularly defined TAAs or viral (control) target antigens., Methods: Immune cells in peripheral blood obtained from thirty-six patients with brain metastases were tested for cytokine production in response to a broad panel of defined viral and TAA target antigens, including full-length mesothelin. Incubation of immune cells with antigenic targets was carried out in i) medium alone, (ii) in a cytokine cocktail of interleukin (IL)-2/IL-15/IL-21, or (iii) IL-2/IL-7. Supernatants were tested for interferon gamma (IFN-γ) production, after which univariate and multivariate analyses (Cox stepwise regression model) were performed to identify independent clinical and immunological factors associated with patient survival. Patients were followed-up for at least 500days after surgery or until death., Findings: Univariate analysis identified age, gender, radiotherapy and mutational load as clinical parameters affecting survival of patients with brain metastases. Cox multivariate analysis showed that radiotherapy (P=0·004), age (P=0·029) and IFN-γ responses to mature mesothelin, conditioned by IL-2/IL-7 (P=0·045) were independent predictors of the survival of patients from surgery up to follow-up or death., Interpretation: This is the first evidence that immune responses to mesothelin serve as a marker of increased overall survival in patients with brain metastases, regardless of the primary tumor origin. Analyses of immunological markers could potentially serve as prognostic markers in patients with brain metastases and help to select patients in need for adjunct, immunological, treatment strategies., (Copyright © 2017. Published by Elsevier B.V.)

Glioblastoma multiforme (GBM) presents the most malignant form of glioma, with a 5-year survival rate below 3% despite standard therapy. Novel immune-based therapies in improving treatment outcomes in GBM are therefore warranted. Several molecularly defined targets have been identified mediating anti-GBM cellular immune responses. Mesothelin is a tumor-associated antigen (TAA) which is expressed in several solid tumors with different histology. Here, we report the immunological significance of mesothelin in human malignant glioma. Expression of mature, surface-bound mesothelin protein was found to bein human GBM defined by immunofluorescence microscopy, and on freshly isolated, single cell suspension of GBM tumor cells and GBM tumor cell lines, determined by based on flow cytometric analysis. Peripheral blood (PB) from patients with GBM, stimulated with mesothelin peptides and IL-2, IL-15 and IL-21, exhibited increased antigen-specific IFN-γ and TNF-α production. Anti-mesothelin directed T-cell responses could also be detected in tumor - infiltrating lymphocytes (TIL) isolated from GBM speciments. Furthermore, T cells cultured in the presence of IL-2, IL-15 and IL-21 displayed enhanced mesothelin-specific CD4+ and CD8+ subset proliferation, based on ELISA and flow cytometric readouts. Mesothelin-specific IgG antibodies as well as (shed) mature mesothelin protein were detected in plasma samples from patients with GBM by indirect ELISA. Finally yet importantly, we identified distinct immune recognition hotspots within the mature mesothelin component, defined by peptide-specific IFN-γ responses from peripheral T-cells from patients with GBM. Mesothelin may therefore qualify as a viable target for immunotherapeutic approaches for patients with GBM., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest