Diverse immunotherapies can effectively treat syngeneic brainstem tumors in the absence of overt toxicity

Background: Immunotherapy has shown remarkable clinical promise in the treatment of various types of cancers. However, clinical benefits derive from a highly inflammatory mechanism of action. This presents unique challenges for use in pediatric brainstem tumors including diffuse intrinsic pontine glioma (DIPG), since treatment-related inflammation could cause catastrophic toxicity. Therefore, the goal of this study was to investigate whether inflammatory, immune-based therapies are likely to be too dangerous to pursue for the treatment of pediatric brainstem tumors. Methods: To complement previous immunotherapy studies using patient-derived xenografts in immunodeficient mice, we developed fully immunocompetent models of immunotherapy using transplantable, syngeneic tumors. These four models – HSVtk/GCV suicide gene immunotherapy, oncolytic viroimmunotherapy, adoptive T cell transfer, and CAR T cell therapy – have been optimized to treat tumors outside of the CNS and induce a broad spectrum of inflammatory profiles, maximizing the chances of observing brainstem toxicity. Results: All four models achieved anti-tumor efficacy in the absence of toxicity, with the exception of recombinant vaccinia virus expressing GMCSF, which demonstrated inflammatory toxicity. Histology, imaging, and flow cytometry confirmed the presence of brainstem inflammation in all models. Where used, the addition of immune checkpoint blockade did not introduce toxicity. Conclusions: It remains imperative to regard the brainstem with caution for immunotherapeutic intervention. Nonetheless, we show that further careful development of immunotherapies for pediatric brainstem tumors is warranted to harness the potential potency of anti-tumor immune responses, despite their possible toxicity within this anatomically sensitive location

Combination therapy with reovirus and anti-PD-1 blockade controls tumor growth through innate and adaptive immune responses.

Oncolytic reovirus can be delivered both systemically and intratumorally, in both pre-clinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against a variety of tumor types and anti-tumor activity is directly associated with immune activation by virus replication in tumors. Immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, and the generation of adaptive anti-tumor immune responses as a result of in vivo priming against tumor-associated antigens. We tested the combination of local oncolytic reovirus therapy with systemic immune checkpoint inhibition. We show that treatment of subcutaneous B16 melanomas with a combination of intravenous (i.v.) anti-PD-1 antibody and intratumoral (i.t.) reovirus significantly enhanced survival of mice compared to i.t. reovirus (p<0.01) or anti-PD-1 therapy alone. In vitro immune analysis demonstrated that checkpoint inhibition improved the ability of NK cells to kill reovirus-infected tumor cells, reduced Treg activity, and increased the adaptive CD8(+) T cell dependent anti-tumor T cell response. PD-1 blockade also enhanced the anti-viral immune response but through effector mechanisms which overlapped with, but also differed from those affecting the antitumor response. Therefore, combination with checkpoint inhibition represents a readily translatable next step in the clinical development of reovirus

APOBEC3B-mediated Corruption of the Tumor Cell Immunopeptidome Induces Heteroclitic Neoepitopes for Cancer Immunotherapy

APOBEC3B, an anti-viral cytidine deaminase which induces DNA mutations, has been implicated as a mediator of cancer evolution and therapeutic resistance. Mutational plasticity also drives generation of neoepitopes, which prime anti-tumor T cells. Here, we show that overexpression of APOBEC3B in tumors increases resistance to chemotherapy, but simultaneously heightens sensitivity to immune checkpoint blockade in a murine model of melanoma. However, in the vaccine setting, APOBEC3B-mediated mutations reproducibly generate heteroclitic neoepitopes in vaccine cells which activate de novo T cell responses. These cross react against parental, unmodified tumors and lead to a high rate of cures in both subcutaneous and intra-cranial tumor models. Heteroclitic Epitope Activated Therapy (HEAT) dispenses with the need to identify patient specific neoepitopes and tumor reactive T cells ex vivo. Thus, actively driving a high mutational load in tumor cell vaccines increases their immunogenicity to drive anti-tumor therapy in combination with immune checkpoint blockade

Subversion of NK-cell and TNFα Immune Surveillance Drives Tumor Recurrence

Understanding how incompletely cleared primary tumors transition from minimal residual disease (MRD) into treatment resistant, immune-invisible recurrences has major clinical significance. We show here that this transition is mediated through the subversion of two key elements of innate immune surveillance. In the first, the role of TNFalpha changes from an antitumor effector against primary tumors into a growth promoter for MRD. Second, whereas primary tumors induced a natural killer (NK)-mediated cytokine response characterized by low IL6 and elevated IFNgamma, PD-L1hi MRD cells promoted the secretion of IL6 but minimal IFNgamma, inhibiting both NK cell and T-cell surveillance. Tumor recurrence was promoted by trauma- or infection-like stimuli inducing VEGF and TNFalpha which stimulated the growth of MRD tumors. Finally, therapies which blocked PD1, TNFalpha or NK cells delayed or prevented recurrence. These data show how innate immune surveillance mechanisms, which control infection and growth of primary tumors, are exploited by recurrent, competent tumors and identifies therapeutic targets in patients with MRD known to be at high risk of relapse

Angiopoietin-1 inhibits tumour growth and ascites formation in a murine model of peritoneal carcinomatosis

Angiopoietin-1 is an important regulator of endothelial cell survival. Angiopoietin-1 also reduces vascular permeability mediated by vascular endothelial growth factor. The effects of angiopoietin-1 on tumour growth and angiogenesis are controversial. We hypothesised that angiopoietin-1 would decrease tumour growth and ascites formation in peritoneal carcinomatosis. Human colon cancer cells (KM12L4) were transfected with vector (pcDNA) alone (control) or vector containing angiopoietin-1 and injected into the peritoneal cavities of mice. After 30 days, the following parameters were measured: number of peritoneal nodules, ascites volume, and diameter of the largest tumour. Effects of angiopoietin-1 on vascular permeability were investigated using an intradermal Miles assay with conditioned media from transfected cells. Seven of the nine mice in the pcDNA group developed ascites (1.3±0.5 ml (mean±s.e.m.)), whereas no ascites was detectable in the angiopoietin-1 group (0 out of 10) (P<0.01). Number of peritoneal metastases (P<0.05), tumour volume, (P<0.05), vessel counts (P<0.01), and tumour cell proliferation (P<0.01) were significantly reduced in angiopoietin-1-expressing tumours. Conditioned medium from angiopoietin-1-transfected cells decreased vascular permeability more than did conditioned medium from control cells (P<0.05). Our results suggest that angiopoietin-1 is an important mediator of angiogenesis and vascular permeability and thus could theoretically serve as an anti-neoplastic agent for patients with carcinomatosis from colorectal cancer

Data mining of high density genomic variant data for prediction of Alzheimer's disease risk

<p>Abstract</p> <p>Background</p> <p>The discovery of genetic associations is an important factor in the understanding of human illness to derive disease pathways. Identifying multiple interacting genetic mutations associated with disease remains challenging in studying the etiology of complex diseases. And although recently new single nucleotide polymorphisms (SNPs) at genes implicated in immune response, cholesterol/lipid metabolism, and cell membrane processes have been confirmed by genome-wide association studies (GWAS) to be associated with late-onset Alzheimer's disease (LOAD), a percentage of AD heritability continues to be unexplained. We try to find other genetic variants that may influence LOAD risk utilizing data mining methods.</p> <p>Methods</p> <p>Two different approaches were devised to select SNPs associated with LOAD in a publicly available GWAS data set consisting of three cohorts. In both approaches, single-locus analysis (logistic regression) was conducted to filter the data with a less conservative p-value than the Bonferroni threshold; this resulted in a subset of SNPs used next in multi-locus analysis (random forest (RF)). In the second approach, we took into account prior biological knowledge, and performed sample stratification and linkage disequilibrium (LD) in addition to logistic regression analysis to preselect loci to input into the RF classifier construction step.</p> <p>Results</p> <p>The first approach gave 199 SNPs mostly associated with genes in calcium signaling, cell adhesion, endocytosis, immune response, and synaptic function. These SNPs together with <it>APOE and GAB2 </it>SNPs formed a predictive subset for LOAD status with an average error of 9.8% using 10-fold cross validation (CV) in RF modeling. Nineteen variants in LD with <it>ST5, TRPC1, ATG10, ANO3, NDUFA12, and NISCH </it>respectively, genes linked directly or indirectly with neurobiology, were identified with the second approach. These variants were part of a model that included <it>APOE </it>and <it>GAB2 </it>SNPs to predict LOAD risk which produced a 10-fold CV average error of 17.5% in the classification modeling.</p> <p>Conclusions</p> <p>With the two proposed approaches, we identified a large subset of SNPs in genes mostly clustered around specific pathways/functions and a smaller set of SNPs, within or in proximity to five genes not previously reported, that may be relevant for the prediction/understanding of AD.</p

The evolution of cyclodextrin glucanotransferase product specificity

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, β- and γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity

Contrasted Patterns of Selection on MHC-Linked Microsatellites in Natural Populations of the Malagasy Plague Reservoir

Plague (Yersinia pestis infection) is a highly virulent rodent disease that persists in many natural ecosystems. The black rat (Rattus rattus) is the main host involved in the plague focus of the central highlands of Madagascar. Black rat populations from this area are highly resistant to plague, whereas those from areas in which the disease is absent (low altitude zones of Madagascar) are susceptible. Various lines of evidence suggest a role for the Major Histocompatibility Complex (MHC) in plague resistance. We therefore used the MHC region as a candidate for detecting signatures of plague-mediated selection in Malagasy black rats, by comparing population genetic structures for five MHC-linked microsatellites and neutral markers in two sampling designs. We first compared four pairs of populations, each pair including one population from the plague focus and one from the disease-free zone. Plague-mediated selection was expected to result in greater genetic differentiation between the two zones than expected under neutrality and this was observed for one MHC-class I-linked locus (D20Img2). For this marker as well as for four other MHC-linked loci, a geographic pattern of genetic structure was found at local scale within the plague focus. This pattern would be expected if plague selection pressures were spatially variable. Finally, another MHC-class I-linked locus (D20Rat21) showed evidences of balancing selection, but it seems more likely that this selection would be related to unknown pathogens more widely distributed in Madagascar than plague