Presence Of Neoantigens Research Articles

A pan-cancer characterization of immune-related NFIL3 identifies potential predictive biomarker.

Background: Nuclear factor interleukin 3 (NFIL3) mainly focuses on the regulation of the circadian rhythm and immune system. However, the potential role of NFIL3 in human cancers has not been studied extensively. Methods: We retrieved original data from the TCGA, TARGET, and GTEx datasets via the UCSC Xena browser (http://genome.ucsc.edu/) and integrated them using R version 3.6.4. NFIL3 expression was assessed using resources such as UCSC, GEPIA (http://gepia.cancer-pku.cn/), Kaplan-Meier Plotter (KM Plotter; https://kmplot.com/), and the Human Protein Atlas (HPA; https://www.proteinatlas.org/) databases. To investigate the prognostic implications of NFIL3, we utilized GEPIA, Kaplan-Meier Plotter, and PrognoScan (http://www.abren.net/PrognoScan/) datasets. For a comprehensive analysis across multiple cancer types, we employed pan-cancer data from UCSC, examining associations between NFIL3 expression and genomic heterogeneity, tumor mutational burden (TMB), microsatellite instability (MSI), tumor purity, and neoantigens. Furthermore, we explored the relationships between NFIL3 expression and the infiltration of immune cells and the expression of immune checkpoint genes. In the context of ovarian cancer, we validated the expression and functional relevance of NFIL3. Cell Counting Kit 8 (CCK8) assays were conducted to assess cell proliferation, while scratch and transwell assays were employed to evaluate cell migration capabilities. We further examined the interaction between NFIL3 and the p53 signaling pathway through quantitative real-time polymerase chain reaction (qRT-PCR), Western blot analysis, immunofluorescence confocal, and Coimmunoprecipitation (Co-IP) assays. Results: In general, NFIL3 expression in cancerous tissues exhibited diminished levels when compared to normal tissue samples. Notably, NFIL3 expression demonstrated a robust correlation with several pivotal aspects, including prognosis, immune cell infiltration, immune checkpoint-related genes, TMB, MSI, tumor purity, and the presence of neoantigens. Experimental investigations involving scratch assays, transwell assays, and assessments of cell proliferation in ovarian cancer cells have provided indications that NFIL3 may exert influence over cell migration and proliferation processes. Moreover, a substantial association between NFIL3 and the p53 signaling pathway was discerned through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, with subsequent validation through qRT-PCR, Western blot analysis, immunofluorescence confocal, and co-immunoprecipitation (Co-IP) assays. Conclusions: Therefore, we concluded NFIL3 may serve as a possible prognostic and immunological pan-cancer biomarker.

Abstract IA13: Predicting clonal evolution in pancreatic cancer survivors

Abstract Immune editing of neoantigens is crucial for the success of immunotherapies, but it is still unknown to what extent the immune system naturally edits evolving tumors and what is the fitness cost associated with the presence of neoantigens. Here we develop a biophysically grounded neoantigen quality model, which quantifies the immunogenicity of tumor neoantigens. We use the model to define the fitness of tumor clones as a combination of negative selection due to immune recognition and positive selection due to oncogenic mutations. We investigate how pancreatic cancers – a lowly mutated, poorly immunogenic cancer, largely presumed to not be subject to immunoediting – evolve over 10 years. Our patient cohort includes a set of long-term survivors, who are characterized by high levels of immune infiltration of their primary tumors. With the fitness model, we show that long-term survivors evolve new clones of markedly lower immune fitness cost, to indicate clones with high-quality neoantigens are negatively selected. Importantly, the fitness model predicts the clonal composition of recurrent tumors of the patients. Thus, we submit longitudinal evidence that the human immune system naturally edits neoantigens. Furthermore, we present a model that describes how tumor cell populations evolve under immune pressure over time, with implications for cancer biology and therapy. Citation Format: Marta Luksza. Predicting clonal evolution in pancreatic cancer survivors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr IA13.

Demystifying emerging bulk RNA-Seq applications: the application and utility of bioinformatic methodology

Significant innovations in next-generation sequencing techniques and bioinformatics tools have impacted our appreciation and understanding of RNA. Practical RNA sequencing (RNA-Seq) applications have evolved in conjunction with sequence technology and bioinformatic tools advances. In most projects, bulk RNA-Seq data is used to measure gene expression patterns, isoform expression, alternative splicing and single-nucleotide polymorphisms. However, RNA-Seq holds far more hidden biological information including details of copy number alteration, microbial contamination, transposable elements, cell type (deconvolution) and the presence of neoantigens. Recent novel and advanced bioinformatic algorithms developed the capacity to retrieve this information from bulk RNA-Seq data, thus broadening its scope. The focus of this review is to comprehend the emerging bulk RNA-Seq-based analyses, emphasizing less familiar and underused applications. In doing so, we highlight the power of bulk RNA-Seq in providing biological insights.

Neoantigens in MDS Are Associated with Two Novel CD4+ T Cell Subsets and Improved Overall Survival

BackgroundThe immune system has been shown to be important in the pathophysiology of MDS. Adaptive immunity relies on T cells ability to discriminate between self and non-self antigens in the context of a surface bound MHC protein structure. Genomic changes occurring in acquired somatic mutations can encode novel amino acid sequences, which if translated and presented by the MHC are termed a neoantigen.To establish the impact of neoantigens in MDS we have combined a predictive algorithm to screen cases for the presence of neoantigens together with mass cytometry by time of flight (CyTOF) to identify neoantigen-related immune signatures.ResultsMutation screening was performed on 204 MDS patients using our established gene panel targeting mutations commonly occurring in MDS (Mohemdali, Leukaemia 2015). Neoantigen-MHC affinity was predicted using NetMHCpan3.0 with neoantigen affinities ranked against 400,000 naturally occurring peptides. Those in the top 2% of peptides were classed as predicted neoantigens.In a multi-variate analysis of overall survival, higher numbers of mutations were associated with a poorer outcome (HR 2.71 95%CI 1.28-5.73, p <0.01). In a subset of 72 low risk MDS cases (by IPSS) the presence of neoantigen was protective (HR 0.21 95% CI 0.05-0.84, p <0.05) (fig 1). There was no survival benefit in the presence of neoantigens in high risk MDS.To look for an immune signature in MDS, we performed deep immunophenotyping on 44 MDS cases and 8 age-matched healthy donors (HDs). Peripheral blood mononuclear cells (PBMCs) were stained with 35 metal-tagged antibodies and analysed using CyTOF. Automated clustering methods (t-distributed stochastic neighbour embedding (t-SNE), spanning-tree progression analysis of density-normalized events (SPADE) and in-house clustering) were used to identify T cell subsets. As expected, T regulatory cells were increased in MDS cases when compared to HDs (7.2% vs 4.9% p <0.00001). However, within MDS there was an increase in the recently described Treg B (Kordasti, Blood 2016) in RAEB (4.9% vs 3.6%, p <0.05). These Tregs have been shown to be activated, more suppressive, quicker in responding to IL-2 by phosphorylating STAT5 and Treg specific demethylated region (TSDR) is unmethylated. Of note, there was no difference in abundance of Tregs or other T cell subsets based on presence or absence of mutation or neoantigen.To assess other features of immune suppression we analysed expression of immune checkpoints. While there was no difference in percentage of PD1 positive cells as a proportion of T cells between MDS and HD, neoantigen positive patients had higher PD1+ T cells compared to negative cases (0.77 vs 1.69% p <0.005). To explore the phenotype of PD1 positive PBMCs further we used unsupervised clustering and identified 23 phenotypically distinct clusters of PD1 high cells (fig 2). Neoantigen positive cases had significantly lower numbers of two distinct PD1hi CD4+ T cell subsets (p<0.05). Using Marker Enrichment Modelling (MEM) to better characterise these clusters, we have shown one cluster to be part of the Treg B subpopulation and the other within T effectors (fig 3) (Diggins, Nature Methods 2017). No significant differences in numbers of these cells was seen between other subtypes of MDS, including presence or absence of mutations, or between MDS and HD.DiscussionUsing multi-dimensional analysis we have shown that increased blast counts are associated with an increase in more suppressive and proliferating Tregs (Treg B). The findings of increased numbers of PD1hi T-cells and also two specific CD4+ subsets implies a more immunosenescent phenotype in neoantigen negative MDS. The lack of this association when comparing mutation positive and negative cases suggests that this immune signature is dependent on the patient-specific interaction of the mutation with HLA type rather than driven by the presence of a mutation per se.This work demonstrates clearly for the first time the protective role of neoantigens in low risk MDS. By combining knowledge of host immune factors and tumour characteristics we have identified two novel subsets of PD1 upregulated CD4+ T cells that may play an important role in tumour surveillance and identifies cells likely to be susceptible to PD1 blockade. Further work to understand the mechanistic effects of PD1 blockade on these subsets will be crucial to identifying patients most likely to respond to checkpoint inhibition. [Display omitted] DisclosuresKulasekararaj:Akari Therapeutics Plc: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Honoraria; Ra Pharmaceuticals: Consultancy, Honoraria; Achillion pharmaceuticals: Consultancy, Honoraria. Irish:Cytobank Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Incyte: Research Funding. Kordasti:Celgene: Research Funding.

Genomic and neoantigen evolution from primary tumor to first metastases in head and neck squamous cell carcinoma.

Head and neck cell squamous-cell carcinomas (HNSCC) are a group of common cancers typically associated with tobacco use and human papilloma virus infection. Up to half of all cases will suffer a recurrence after primary treatment. As such, new therapies are needed, including therapies which promote the anti-tumor immune response. Prior work has characterized changes in the mutation burden between primary and recurrent tumors; however, little work has characterized the changes in neoantigen evolution. We characterized genomic and neoantigen changes between 23 paired primary and recurrent HNSCC tumors. Twenty-three biopsies from patients originally diagnosed with locally advanced disease were identified from the Washington University tumor bank. Whole exosome sequencing, RNA-seq, and immunohistochemistry was performed on the primary and recurrent tumors. Within these tumors, we identified 6 genes which have predicted neoantigens in 4 or more patients. Interestingly, patients with neoantigens in these shared genes had increased CD3+ CD8+ T cell infiltration and duration of survival with disease. Within HNSCC tumors examined here, there are neoantigens in shared genes by a subset of patients. The presence of neoantigens in these shared genes may promote an anti-tumor immune response which controls tumor progression.

Abstract 1137: Deep targeted and whole exome sequencing identifies driver mutations in the airway epithelium of individuals at high risk for lung cancer

Abstract Insights into tumor mutational burden of the airway epithelium may play important role in stratifying individuals at different levels of risk for lung cancer. Comparison of the genetic profiles of normal airway epithelial cells to tumor mutations can yield valuable information regarding shared mutations as well as presence of neoantigens. We hypothesized that cytologically normal bronchial epithelium of patients with lung cancer and those at high risk for lung cancer harbor driver mutations that can be identified by deep targeted sequencing of selected genes implicated in cancer and whole exome sequencing. We performed next generation sequence-based CAPP-Seq (CAncer Personalized Profiling by deep Sequencing) and whole exome sequencing (WES) of DNA from the bronchial epithelial cells from the bronchial brushings of individuals at-risk (low and high) for lung cancer and patients with lung cancer. Bronchial brushings were collected from consented individuals at the time of bronchoscopy at Vanderbilt University Medical Center and the Nashville Veterans Affairs Medical Center. We used Tammemagi risk model to calculate the risk of developing lung cancer. Lung tumor tissues from matched individuals were also sequenced. Using CAPPSeq, a total of 129 mutations were identified in bronchial brushings. Among individuals without cancer, driver mutations included TP53, PIK3CA, NOTCH1, KRAS, MET, CUL3, NF1, KEAP1 and EGFR mutations in 5 out of 13 high risk and 2 out of 7 low risk individuals. Among individuals with cancer, 13 out of 22 brushings harbored driver mutations in TP53, PIK3CA, ARID1A, PTEN, NOTCH1, KRAS, MET, NFE2L2, CUL3, NF1, and KEAP1 of which mutations in TP53 and PTEN were identified in both tumor and bronchial brushings. At least one mutation was called in 18 brushings of individuals with lung cancer. Mutations detected in both individuals with and without cancer were TP53, PIK3CA, NOTCH1, KRAS, MET, CUL3, and NF1. Using WES on the DNA from the same bronchial brushings (low risk-9; high risk-8 and lung cancer-17), we identified 90 mutations that were also identified by CAPPSeq. Most of the mutations identified by CAPPSeq with &amp;gt;5% variant allelic frequencies (VAF) were also identified by WES. However, WES was not sensitive enough to identify mutations with less than 0.5% VAF. Out of 9 mutations in TP53 identified in the brushings by CAPPSeq, 4 were identified by WES. Mutation in KMTD2, a histone methylation enzyme and believed to be a tumor suppressor, was identified by WES in the brushings and the tumor tissues of patients with lung cancer as well as in one high risk brushings. We identified mutations in cancer driver genes in the bronchial brushings of individuals with and without lung cancer. This field of cancerization effect may provide a tool for risk assessment for lung cancer. This research was funded by U01CA196405 to PPM. Citation Format: SM Jamshedur Rahman, Shih-kai Chu, Shilin Zhao, Yong Zou, Angela Hui, Thomas Stricker, Maximilian Diehn, Pierre P. Massion. Deep targeted and whole exome sequencing identifies driver mutations in the airway epithelium of individuals at high risk for lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1137.

PD-L1 regulation in colorectal cancer and role of DNA damage induced by chemotherapies

For patients with metastatic colorectal cancer (CRC), first-line therapy is based on chemotherapeutic agents, such as oxaliplatin, 5-fluorouracil and irinotecan. These drugs increase the overall survival, but resistance to therapy appears in almost 90% of patients, and the 5-year survival rate for patients with metastatic CRC is only about 12%. During the last few years, immune checkpoints blockade therapies have been developed and show good response in different cancers, including CRC with microsatellite instability (MSI). In this CRC subtype, the response rate to anti-PD-(L)1 antibodies is high thanks to the presence of neoantigens and tumor-infiltrating lymphocytes that are associated with the anti-tumor immune response. Nivolumab and pembrolizumab, two anti-PD-1 antibodies, have been approved for CRC MSI treatment. Moreover, it has been shown that the combination of chemotherapy and anti-PD-(L)1 molecules may convert cold tumors into hot tumors in which the immune system and T-cell infiltration are activated. In addition, recent studies found that DNA damage induces PD-L1 expression. ATM, ATR, DNA-PKcs and Chk1 are key sensors of the DNA damage response that regulate PD-L1 expression. This review summarizes the current knowledge on PD-L1 regulation at the genetic, epigenetic, transcriptional and translational levels. It also describes PD-L1 activation in response to chemotherapy and DNA damage. Then, it summarizes the current clinical trials that assess anti-PD-(L)1 therapies in combination with kinase inhibitors or chemotherapeutic agents in CRC.

Abstract P4-06-11: Cryoablation of murine mammary tumors induce robust immune response

Abstract Background: Breast cancer is traditionally not considered as a highly immunogenic disease. Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with reported high genomic instability and high mutation rate, indicating the possible presence of neoantigens. Cryoablation, the destruction of cells by ultra-low temperatures, can release these neoantigens and induce a tumor specific immune response. We hypothesized these neoantigens might be sufficient to trigger a robust immune response to prevent and/or reduce spread and relapse of TNBC. In this pilot study we cryoablated orthotopical 4T1 tumors in immune competent Balb/c mice and compared the results to surgery to evaluate 1) possible induction of immune responses and 2) effects on metastases formation. Methods: We used 4T1 mammary carcinoma cells to initiate tumor growth in the mammary fatpad. Tumors were treated by cryoablation, cryoablation followed by surgery (cryo-surgery), or surgery alone. Tumor growth was followed and allowed to reach 3-4mm in largest dimension. Animals were euthanized 7 days post-treatment and tissues were collected to assess cytokine levels and presence of dissociated 4T1 cells. Single-cell suspensions of tumor, tumor-draining lymph node [TDLN], and spleen were tested for secretion of mouse Th1/Th2 cytokines using a bead array and measured by flow cytometery. Possible metastatic spread was assessed by a clonogenic assay using cells from venous blood, lung, and brain. Cell suspensions were seeded in growth medium supplemented with the selection agent 6-thioguanine, allowing only resistant 4T1 cells to form colonies. Results: Cryoablation transformed tumors into a gelatinous mass surrounded by a fibrotic capsule, as typically seen in the clinic. Frozen sections of tumors revealed a necrotic core and infiltrating lymphocytes in the microenvironment. These animals displayed robust increases of Th1 and Th2 cytokines in both spleen and TDLN compared to animals with cryo-surgery treatment. TDLN of animals with surgically excised tumors secreted only IL-2. Circulating tumor cells were found in animals prior to treatment, while no 4T1 colonies formed from cell suspensions of lung and brain tissue [N=8]. At end-point, the surgery alone group had more 4T1 foci formed from lung and brain [mean foci /animal = 6.25 and 0.75, respectively; N=6] than the other two groups. Two animals in this group progressed and were euthanized early due to numerous lung metastases. The cryoablated group had the lowest number of foci formed in the lung and brain [2.25 and 0 respectively; N=8], and all animals were healthy at the predetermined end-point. Mean foci formation in the cryo-surgery group [N=7] was in-between the two other groups and one animal was euthanized early due to metastatic burden 5 days after surgery. Conclusion: Cryoablation of TNBC can induce stimulatory immune responses in vivo. These immune responses might explain why animals treated with cryoablation, though having circulating tumor cells at the time of treatment, exhibited fewer micro metastatic growths compared to surgery alone and the cryo-surgery combination. On-going experiments aim to identify long-term effects of cryoablation on the formation of metastatic foci and growth. Citation Format: Klein JD, Aukers Z, Kennedy J, Ciocca RM, Sabol JL, Carp NZ, Wallon UM. Cryoablation of murine mammary tumors induce robust immune response [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-11.

Abstract B086: Exploiting large-scale HLA peptidomics to generate novel immunotherapies: A data-driven approach to true neoantigen prioritization

Abstract Purpose of Study: In this study we demonstrate accurate prediction of the impact of somatic mutations on the HLA presentation landscape achieved by interrogating a large scale database of 1.4 million unique HLA peptide sequences that have been directly identified by mass spectrometry. Background: Peptides presented to the immune system on HLA complexes are valuable targets for immunotherapeutic treatments. Identifying the full complement of peptides derived from a particular protein that are presented on major class I HLA restrictions will provide a vital step toward increasing the speed and viability of many immunotherapeutic strategies. Advances in next-generation sequencing (NGS) and single-cell technologies have enabled the accurate capture of somatic mutations accumulated by a tumor, yet a significant hurdle remains how this information can be utilized for immunotherapeutic benefit. In particular, identifying which somatic mutations produce neoantigens (peptides that contain a somatic mutation and are presented to the immune system in complex with HLA) is crucial to linking genetic changes with immunologic impact. Materials and Methods: Our approach to understanding the targetable human HLA peptidome is based on three key principles: achieving full proteome coverage, maximising individual protein coverage, and focusing on dominant HLA restrictions. By integrating novel cell biology, mass spectrometry, and bioinformatic technologies across over 1,000 individual experiments we have dramatically increased the depth of the HLA ligandome captured and achieved near total coverage of the protein-coding genome. Over 90% of the proteome has been captured for the restriction HLA-A*02:01, dominant in Caucasian populations. Our comprehensive genome coverage has enabled us to probe both directly and indirectly for the presence of neoantigens. Known somatic mutations within immortalized lines were used to generate bespoke reference databases that has led to direct identification of many hundreds of neoantigens. Results: Proteins that were found to contain neoantigens appeared to follow the same pattern of antigen processing and presentation as their unmutated equivalents. We have therefore found our HLA peptide dataset is able to offer significant value in predicting the likelihood of a somatic mutation creating a neoantigen. To test this, somatic mutations reported in 980 cell lines were probed against the database of HLA peptides. On average we find one peptide containing the mutated amino acid for every five somatic mutations reported. By incorporating the HLA background of the cell carrying the mutation, we narrow this prediction to one high-affinity HLA peptide for every fourteen somatic mutations reported. Comparing the peptides predicted in this analysis with those directly identified by mass spectrometry, we are able to show that we can prioritize mutation data by accurately predicting the presence and relative abundance of neoantigens. Our neoantigen prediction process is fully incorporated into a large scale database system, enabling us to seamlessly integrate NGS data from individual tissue and use peptidomic data to rapidly define the targetable landscape of an individual. Conclusions: An integrative approach to HLA peptidomics has delivered a powerful reference database for developing novel immunotherapies. Citation Format: Alex S. Powlesland, Geert P.M. Mommen, Ricardo J. Carreira, Jacob Hurst, Michael J. Cundell, David Lowne, Floriana Capuano, Bent K. Jakobsen. Exploiting large-scale HLA peptidomics to generate novel immunotherapies: A data-driven approach to true neoantigen prioritization [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B086.

Abstract P1-04-02: Improving immunotherapy response by epigenetic modulation

Abstract The presence of predicted neo-antigens in any given tumor is highly correlated with total somatic mutational burden of the same tumor genome. However, many potential neo-antigens are never transcribed, translated, or presented as antigens, partially because they lie in regions of the genome that are transcriptionally-repressed by cytosine methylation of promoter-CpG islands. Treatment with DNA methyltransferase inhibitors (DNMTi) can hypothetically lead to re-expression of many potential neo-antigens. Furthermore, the presence of neo-antigens have been linked to immunotherapy outcomes in patients. Our hypothesis is that tumors with reduced mutational burden can be maximized for neo-antigen presentation by activating transcription and translation of these sequences through DNMTi treatment. Our preliminary results showed that the DNMTi guadecitabine (SGI-110), a second-generation hypomethylating agent, treatment decreases methyl-cytosine in genomic DNA both in vitro and in vivo. In addition, SGI-110 treatment enhances MHC-II expression on murine mammary carcinoma MMTV-Neu cells upon IFN-γ stimulation in vitro and increases T-cell infiltration in vivo. Currently, we are performing whole-exome-sequencing and RNA-sequencing to track somatic mutations from DNA to RNA. We will further track somatic mutations from RNA→ MHC-presented peptide sequences using MHC immunoprecipitation followed by mass spectroscopy analysis. In addition, we will explore the role of guadecitabine therapeutic priming on response to αPD-L1 immunotherapy. These studies will provide a pre-clinical data to evaluate the potential for combined epigenetic and immune-therapy in a clinical trial for breast cancer. Citation Format: Luo N, Estrada VM, Sanders ME, Balko JM. Improving immunotherapy response by epigenetic modulation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-02.

Abstract DDT01-05: First-in-class T cell-redirecting bispecific antibody targeting glypican-3: a highly tumor-selective antigen

Abstract Immune checkpoint inhibitors such as anti-PD1 antibodies have shown promising clinical responses in several solid tumors, however there remain patients who do not show an adequate response. Recent biomarker studies have revealed that the presence of neoantigens in the tumor can determine the level of response, and thus the next challenge will be how to target tumors with a neoantigen level that is too low to be recognized by endogenous cytotoxic T cells. Hope in this area is offered by a T cell-redirecting antibody (TRAB), which bispecifically engages CD3 and a tumor antigen, even at very low expression levels, to activate the inherent cytolytic potential of T cells against target tumor cells. A TRAB is highly potent because T cells are activated only in the presence of the targeted antigens and are not restricted by the specificity of the T cell receptor. Given this very potent cytotoxicity, the key to successfully achieving strong antitumor efficacy while avoiding on-target off-tumor toxicity is to select a highly tumor-selective antigen. Our fully humanized IgG TRAB recognizes CD3 and a highly tumor-selective antigen, glypican-3 (GPC3), which is a fetal protein expressed in a wide variety of tissues during development but suppressed in most adult tissues. On the other hand, an inct101e in GPC3 expression has been reported in hepatocellular carcinoma, gastric cancer, lung squamous cell carcinoma, and other cancers. In nonclinical in vitro pharmacology studies, the anti-GPC3 TRAB elicited activation and proliferation of T cells and T cell-dependent cellular cytotoxicity against a wide variety of GPC3-expressing tumor cells, and showed long-lasting in vivo efficacy against tumor expressing very low levels of GPC3 at a few thousand molecules per cell. Furthermore, in an immunocompetent mouse model using human CD3 transgenic mice, anti-GPC3 TRAB showed strong antitumor efficacy against poorly immunogenic tumors, whereas both the immune checkpoint inhibitors and a conventional ADCC-inducing antibody recognizing GPC3 did not show significant efficacy. Pharmacokinetics and toxicology studies in nonhuman primates showed a plasma half-life comparable to a standard IgG drug, allowing a QW or Q2W regimen in humans, with toxicity which was manageable and reversible; the main observations of transient cytokine elevation and associated clinical symptoms were markedly reduced by steroid premedication. Our anti-GPC3 TRAB, which is supported by proprietary antibody engineering technology (ART-Ig) that enables large-scale GMP manufacturing, has promise as a new approach in cancer immunotherapy. Citation Format: Takahiro Ishiguro, Yasuko Kinoshita, Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Natsuki Ono, Yoko Kayukawa, Mika Kamata-Sakurai, Hirotake Shiraiwa, Akihisa Kaneko, Werner Frings, Shunichiro Komatsu, Junichi Nezu, Mika Endo. First-in-class T cell-redirecting bispecific antibody targeting glypican-3: a highly tumor-selective antigen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr DDT01-05.

Epitope Spreading Is Required for Long-Term Protection Against Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) has historically been a challenging target for immunotherapy due in part to its tolerogenic effect on immune effector cells; however, CD19-targeted chimeric antigen receptor-modified T cells (CARTs) and bi-specific T cell engagers (BiTEs) show that immune tolerance to ALL can be circumvented. Unfortunately, both CD19-negative and positive relapses occur after treatment with either therapy, suggesting an approach targeting a single antigen may not be sufficient to eliminate ALL in all cases. Generating immune responses to multiple tumor antigens may be expected to provide superior relapse-free survival.We previously reported an immune-mediated spontaneous remission model of B cell precursor ALL using leukemia cells derived from Eμ-ret transgenic mice. In this model, Eμ-ret leukemia cell lines expressing green fluorescent protein (GFP) and firefly luciferase (luc) in tandem initially engraft and expand in wild type (wt) mice but are subsequently eliminated by an immune response targeted to the neoantigens. This T cell-dependent remission provides long-term immune memory against ALL. In contrast, mice transgenic for GFP/luc were not able to reject the GFP/luc ALL, suggesting pre-existing immune tolerance plays a role in undermining immune responses to ALL.In this study, we investigate the contribution of multiple antigens and the impact of antigen-specific tolerance on protection from leukemia outgrowth. Strikingly, the protective memory response against ALL is not dependent on the presence of neoantigens; mice given GFP/luc ALL cells who go into spontaneous remission do not develop disease when rechallenged with 106 wt ALL cells, while all naïve mice die of wt ALL by day 35 (p<0.0001). Furthermore, T cells from mice who have previously cleared GFP/luc ALL produce interferon gamma robustly when co-cultured with either GFP/luc ALL or wt ALL; whereas, naïve T cells do not. The neoantigen-induced immune recognition of wt ALL antigens appears to occur early, as all mice receiving wt ALL succumb to disease by day 35, while mice receiving GFP/luc ALL cells or a 1:1 mixture of GFP/luc and wt ALL fail to develop leukemia (p=0.0096).In contrast to wt mice, Eμ-ret transgenic mice show only delayed outgrowth of GFP/luc ALL compared to wt ALL (median survival 46 versus 26 days; p=0.0027) and are unable to reject the leukemia, leading to lethal disease burden in the majority of mice (p=0.014 versus wt mice with 100% survival at day 117). Given that Eμ-ret mice are likely tolerant to the transgene-related antigens, this result indicates that epitope spreading is essential to long-term protection. Finally, we evaluated the impact of non-antigen-specific immune stimulation on leukemia progression in a tolerized background. While the effect size decreased with increasing number of tolerizing antigens present in the mouse strain, treatment of GFP/luc ALL-bearing mice with the TLR9-agonist CpG oligodeoxynucleotides (CpG ODN) significantly reduced disease burden in all recipients, even GFP/luc/Eμ-ret transgenics tolerized to both transgene antigens and neoantigens (p=0.0013; Figure 1).In the present study, we show that epitope spreading, a phenomenon with important implications for targeted immunotherapies, is essential for durable long-term protection from leukemia outgrowth. While generation of a robust multi-antigen anti-leukemia response may combat immune escape in the highly successful single antigen-targeted therapies, our results suggest that tolerance to leukemia-associated antigens will be a significant hurdle that may need additional therapeutic interventions to overcome. Our results show that non-antigen-specific immune stimulation with a TLR9-agonist achieves significant reduction in disease burden even in a fully immune tolerant model; importantly, the degree of tolerance in the transgenic models may be a better representation of the high immunologic bar for autologous ALL control in the clinical setting. This result provides further pre-clinical evidence that CpG ODN may be capable of generating clinically beneficial immune activity. Augmenting anti-leukemia activity by stimulating the innate immune system may protect against CD19 escape variant relapses, an important clinical problem in targeted immunotherapies. [Display omitted] DisclosuresBassiri:GlaxoSmithKline: Equity Ownership. Barrett:Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding.

Let’s stick together

If there is one defining characteristic of a melanocyte it is the melanosome, the organelle dedicated to the synthesis and containment of melanin. Once a melanosome is made, the question is what happens next. For neural crest-derived melanocytes, as opposed to the retinal pigment epithelium, the ultimate destination of melanosomes is likely to be surrounding keratinocytes where they will function to prevent UV-induced DNA damage. Over the years, a lot has been discovered about the process of melanosome genesis and the enzymes involved in the manufacture of pigment. More recently the molecular mechanisms that direct melansomes to the cell periphery and along their dendritic processes have been deciphered, though there is still much to learn. Yet remarkably, the final step in the journey, how melanosomes are transferred to keratinocytes and what happens once transfer has taken place remains a mystery. Despite the efforts of several laboratories, no clear underlying mechanism has been described. What is clear though is that in the natural context in the skin, intimate contact between the melanocyte and surrounding keratinocytes is likely to be critical. In this issue the paper from Joshi et al., reveals a feature of the consequences of melanosome transfer to keratinocytes; the release of calcium from an intracellular source within the keratinocyte. The results further suggest that there are specific melanocyte membrane-associated proteins that are necessary for the Ca2+ signal. The paper is interesting in that it raises several interesting questions. First, what is the source of the intracellular calcium released on uptake of the melanosome? Does it arise from Ca2+ stores within the keratinocyte, or, given the ability of melanin to bind calcium as described in a recent paper in this journal from the John Simon lab, is it released from within the transferred melansome? And second, what is the role of the released Ca2+? One possibility is that it primes the keratinocyte to mobilise its intra-cellular transport machinery to correctly position the incoming melansome. Alternatively, the calcium release detected by Joshi et al could potentially be a component of the hypothesized feedback mechanism by which keratinocytes would tell melanocytes that a melanosome has been received. Whatever the answers to these questions, monitoring Ca2+ release within keratinocytes will represent a useful tool to those wishing to identify the mechanism underlying melanosome transfer. The close contact between melanocytes and keratinocytes underpins melanosome transfer, but the ability of melanocytes and keratinocytes to stick together has become a focus of research on vitiligo, a complex disease characterised by loss of epidermal and follicular melanocytes. Several models have been proposed to explain the various features of melanocytes, many of which are covered in the reviews on vitiligo genetics by Rich Spritz in the last issue of Pigment Cell Research and in the review from Westerhof and d’Ischia in this issue. It is clear that one feature of melanocyte loss in vitiligo is their destruction by an auto-immune response. Precisely what triggers the auto-immunity isn’t well defined, but both Westerhof and d’Ischia, as well as Namazi, provide a number of suggestions based on the possibility that modification of melanocyte proteins would lead to the production of neo-antigens not normally encountered by the immune system. The presence of neo-antigens would then lead to a bystander effect and the potential for an auto-immune based reaction against melanocytes. However, for neo-antigens to be seen by the immune system would require their release from within melanocytes. Here, accumulating evidence suggest that the melanocytes from vitiligo prone individuals may be more sensitive to stress. In other words, the threshold for stress-induced damage to melanocytes in vitiligo patients would be substantially lower than in normal individuals. One way in which stress might affect melanocytes is in their ability to interact with surrounding keratinocytes, and indeed stress signalling is known to down-regulate the key cell–cell adhesion molecule E-cadherin. In the paper from Cario-André et al., the response of melanocytes in skin reconstructs to various stressors is examined. In agreement with the stress-sensitivity hypothesis, the melanocytes derived from non-lesional skin from individuals with vitiligo behaved differently from melanocytes from non-affected individuals. The defects were enhanced if the melanocytes from vitiligo individuals were combined with keratinocytes from non-lesional skin. The results presented, suggest that there may well be a degree of systemic stress-hypersensitivity in the epidermal melanocyte–keratinocyte interactions in individuals with vitiligo.

Development and clinical application of a new ELISA assay to determine plasmin-alpha2-antiplasmin complexes in plasma.

Plasmin-alpha2-antiplasmin complexes (PAP) are considered good markers of fibrinolytic activation in vivo. The presence of neoantigens in these complexes offers the possibility to develop specific immunoassays to determine PAP levels. We have developed a sensitive PAP purification method in vitro by adding urokinase to fresh plasma followed by affinity chromatography to lysine-sepharose and elution with epsilon-aminocaproic acid. This material, characterized by SDS-PAGE and Western blotting, was used to raise monoclonal antibodies (MoAbs). We describe a new enzyme linked immunosorbent assay (ELISA) to quantify PAP complexes in plasma. The assay follows the sandwich principle and is based on two MoAbs, CPL12 and CPL15, that bind to the modified alpha2-antiplasmin moiety and the plasmin moiety of the complex respectively. The calibration curve was constructed with definite concentrations of purified PAP. The lower limit of the assay is 75 ng/ml and the variation coefficients are 3.5% (intra-assay) and 10-6% (interassay). A mean value of 573.5+/-131.4 ng/ml was obtained from PAP concentration in a healthy population (n = 30). Significantly higher PAP levels were observed under diverse clinical conditions in which fibrinolysis is activated: clinical sepsis, acute myocardial infarction (AMI), malignancy, diabetes, pregnancy, elderly people and thrombolytic therapy. From our results we conclude that this ELISA is suitable to measure in vivo plasma PAP levels.

Antithrombin antigen of high molecular weight associated with neoantigen in hemophilic plasma after factor IX concentrate therapy

These studies were performed to investigate the cause(s) of the cathodal shift of mobility seen in crossed immunoelectrophoresis of antithrombin antigen in plasma of hemophilic patients after factor IX concentrate therapy. These plasmas were shown to contain antithrombin neoantigen with apparent identity to the neoantigen present in normal serum but not present in normal plasma. Sephacryl S-200 gel chromatography of serum demonstrated that the neoantigen eluted with the first two, early eluting protein peaks; thus the neoantigen had a higher molecular weight than native antithrombin. When the chromatographic fractions containing the neoantigen were studied by crossed immunoelectrophoresis, they were found to contain antithrombin antigen of more cathodal mobility than normal. Sephacryl S-200 chromatography of factor IX concentrate-treated hemophilic plasma also showed an early eluting peak of antithrombin antigen of more cathodal mobility than normal in crossed immunoelectrophoresis. The mobility of this peak was identical to the cathodal peak found in normal serum and in early eluting fractions from chromatography of normal serum. These results support the conclusion that factor IX concentrate-treated hemophilic plasma contained a non-functional, high molecular weight form of antithrombin, associated with the presence of neoantigen, which may represent complexed and/or modified antithrombin produced by the action of the concentrates in vivo.

Abortive Infection and Transformation of Human Embryonic Fibroblasts by Molluscum contagiosum Virus

SUMMARY Abortive transformation of human embryonic fibroblasts was induced by Molluscum contagiosum virus. The parameters studied in order to test the establishment of a persistent infection and transformation of cells were: (1) virus interference and interferon production; (2) alterations of cell morphology and growth; (3) presence of neoantigens; (4) karyotype. The possible mechanisms leading to an abortive transformation are discussed.