Cancer neoantigens are antigens that result from somatic mutations present in individual cancers. Neoantigens are considered important targets for cancer immunotherapy because of their immunogenicity ...and lack of expression in normal tissues. Next-generation sequencing technologies and computational analysis have recently made neoantigen discovery possible. Although neoantigens are important targets of checkpoint blockade therapy, neoantigen vaccines are currently being investigated in preclinical models and early-phase human clinical trials. Preliminary results from these clinical trials demonstrate that dendritic cell, synthetic long peptide, and RNA-based neoantigen vaccines are safe, and capable of inducing both CD8+ and CD4+ neoantigen-specific T-cell responses. We and others are testing neoantigen vaccines in melanoma, breast cancer, non-small-cell lung cancer and other cancer types. Since cancers have evolved mechanisms to escape immune control, it is particularly important to study the efficacy of neoantigen vaccines in combination with other immunotherapies including checkpoint blockade therapy, and immune therapies targeting the immunosuppressive tumor microenvironment.
Next generation sequencing of human cancer mutations has identified novel therapeutic targets. Activating Ras oncogene mutations play a central role in oncogenesis, and Ras-driven tumorigenesis ...upregulates an array of genes and signaling cascades that can transform normal cells into tumor cells. In this study, we investigated the role of altered localization of epithelial cell adhesion molecule (EpCAM) in Ras-expressing cells. Analysis of microarray data demonstrated that Ras expression induced EpCAM expression in normal breast epithelial cells. Fluorescent and confocal microscopy showed that H-Ras mediated transformation also promoted epithelial-to-mesenchymal transition (EMT) together with EpCAM. To consistently localize EpCAM in the cytosol, we generated a cancer-associated EpCAM mutant (EpCAM-L240A) that is retained in the cytosol compartment. Normal MCF-10A cells were transduced with H-Ras together with EpCAM wild-type (WT) or EpCAM-L240A. WT-EpCAM marginally effected invasion, proliferation, and soft agar growth. EpCAM-L240A, however, markedly altered cells and transformed to mesenchymal phenotype. Ras-EpCAM-L240A expression also promoted expression of EMT factors FRA1, ZEB1 with inflammatory cytokines IL-6, IL-8, and IL1. This altered morphology was reversed using MEK-specific inhibitors and to some extent JNK inhibition. Furthermore, these transformed cells were sensitized to apoptosis using paclitaxel and quercetin, but not other therapies. For the first time, we have demonstrated that EpCAM mutations can cooperate with H-Ras and promote EMT. Collectively, our results highlight future therapeutic opportunities in EpCAM and Ras mutated cancers.
During the process of cross-presentation, viral or tumor-derived antigens are presented to CD8
T cells by
dependent CD8α
/XCR1
classical dendritic cells (cDC1s). We designed a functional CRISPR ...screen for previously unknown regulators of cross-presentation, and identified the BEACH domain-containing protein WDFY4 as essential for cross-presentation of cell-associated antigens by cDC1s in mice. However, WDFY4 was not required for major histocompatibility complex class II presentation, nor for cross-presentation by monocyte-derived dendritic cells. In contrast to
mice,
mice displayed normal lymphoid and nonlymphoid cDC1 populations that produce interleukin-12 and protect against
infection. However, similar to
mice,
mice failed to prime virus-specific CD8
T cells in vivo or induce tumor rejection, revealing a critical role for cross-presentation in antiviral and antitumor immunity.
Neoantigens are newly formed peptides created from somatic mutations that are capable of inducing tumor-specific T cell recognition. Recently, researchers and clinicians have leveraged next ...generation sequencing technologies to identify neoantigens and to create personalized immunotherapies for cancer treatment. To create a personalized cancer vaccine, neoantigens must be computationally predicted from matched tumor-normal sequencing data, and then ranked according to their predicted capability in stimulating a T cell response. This candidate neoantigen prediction process involves multiple steps, including somatic mutation identification, HLA typing, peptide processing, and peptide-MHC binding prediction. The general workflow has been utilized for many preclinical and clinical trials, but there is no current consensus approach and few established best practices. In this article, we review recent discoveries, summarize the available computational tools, and provide analysis considerations for each step, including neoantigen prediction, prioritization, delivery, and validation methods. In addition to reviewing the current state of neoantigen analysis, we provide practical guidance, specific recommendations, and extensive discussion of critical concepts and points of confusion in the practice of neoantigen characterization for clinical use. Finally, we outline necessary areas of development, including the need to improve HLA class II typing accuracy, to expand software support for diverse neoantigen sources, and to incorporate clinical response data to improve neoantigen prediction algorithms. The ultimate goal of neoantigen characterization workflows is to create personalized vaccines that improve patient outcomes in diverse cancer types.
Identification of neoantigens is a critical step in predicting response to checkpoint blockade therapy and design of personalized cancer vaccines. This is a cross-disciplinary challenge, involving ...genomics, proteomics, immunology, and computational approaches. We have built a computational framework called pVACtools that, when paired with a well-established genomics pipeline, produces an end-to-end solution for neoantigen characterization. pVACtools supports identification of altered peptides from different mechanisms, including point mutations, in-frame and frameshift insertions and deletions, and gene fusions. Prediction of peptide:MHC binding is accomplished by supporting an ensemble of MHC Class I and II binding algorithms within a framework designed to facilitate the incorporation of additional algorithms. Prioritization of predicted peptides occurs by integrating diverse data, including mutant allele expression, peptide binding affinities, and determination whether a mutation is clonal or subclonal. Interactive visualization via a Web interface allows clinical users to efficiently generate, review, and interpret results, selecting candidate peptides for individual patient vaccine designs. Additional modules support design choices needed for competing vaccine delivery approaches. One such module optimizes peptide ordering to minimize junctional epitopes in DNA vector vaccines. Downstream analysis commands for synthetic long peptide vaccines are available to assess candidates for factors that influence peptide synthesis. All of the aforementioned steps are executed via a modular workflow consisting of tools for neoantigen prediction from somatic alterations (pVACseq and pVACfuse), prioritization, and selection using a graphical Web-based interface (pVACviz), and design of DNA vector-based vaccines (pVACvector) and synthetic long peptide vaccines. pVACtools is available at http://www.pvactools.org.
ObjectiveChemokine pathways are co-opted by pancreatic adenocarcinoma (PDAC) to facilitate myeloid cell recruitment from the bone marrow to establish an immunosuppressive tumour microenvironment ...(TME). Targeting tumour-associated CXCR2+neutrophils (TAN) or tumour-associated CCR2+ macrophages (TAM) alone improves antitumour immunity in preclinical models. However, a compensatory influx of an alternative myeloid subset may result in a persistent immunosuppressive TME and promote therapeutic resistance. Here, we show CCR2 and CXCR2 combined blockade reduces total tumour-infiltrating myeloids, promoting a more robust antitumour immune response in PDAC compared with either strategy alone.MethodsBlood, bone marrow and tumours were analysed from PDAC patients and controls. Treatment response and correlative studies were performed in mice with established orthotopic PDAC tumours treated with a small molecule CCR2 inhibitor (CCR2i) and CXCR2 inhibitor (CXCR2i), alone and in combination with chemotherapy.ResultsA systemic increase in CXCR2+ TAN correlates with poor prognosis in PDAC, and patients receiving CCR2i showed increased tumour-infiltrating CXCR2+ TAN following treatment. In an orthotopic PDAC model, CXCR2 blockade prevented neutrophil mobilisation from the circulation and augmented chemotherapeutic efficacy. However, depletion of either CXCR2+ TAN or CCR2+ TAM resulted in a compensatory response of the alternative myeloid subset, recapitulating human disease. This was overcome by combined CCR2i and CXCR2i, which augmented antitumour immunity and improved response to FOLFIRINOX chemotherapy.ConclusionDual targeting of CCR2+ TAM and CXCR2+ TAN improves antitumour immunity and chemotherapeutic response in PDAC compared with either strategy alone.
Derived from genetic alterations, cancer neoantigens are proteins with novel amino acid sequences that can be recognized by the immune system. Recent evidence demonstrates that cancer neoantigens ...represent important targets of cancer immunotherapy. The goal of cancer neoantigen vaccines is to induce neoantigen-specific immune responses and antitumor immunity, while minimizing the potential for autoimmune toxicity. Advances in sequencing technologies, neoantigen prediction ?algorithms,? and other technologies have dramatically improved the ability to identify and prioritize cancer neoantigens. These advances have generated considerable enthusiasm for ?the ?development of neoantigen vaccines. Several neoantigen vaccine platforms are currently being evaluated in early phase clinical trials including the synthetic long peptide (SLP), RNA, dendritic cell (DC), and DNA vaccine platforms.
In this review, we describe, evaluate the mechanism(s) of action, compare the advantages and disadvantages, and summarize early clinical experience with each vaccine platform. We provide perspectives on the future directions of the neoantigen vaccine field. All data are derived from PubMed and ClinicalTrials search updated in October 2020.
Although the initial clinical experience is promising, significant challenges to the success of neoantigen vaccines include limitations in neoantigen identification and the need to successfully target the immunosuppressive tumor microenvironment.
Conventional type 1 dendritic cells (cDC1)
are thought to perform antigen cross-presentation, which is required to prime CD8
T cells
, whereas cDC2 are specialized for priming CD4
T cells
. CD4
T ...cells are also considered to help CD8
T cell responses through a variety of mechanisms
, including a process whereby CD4
T cells 'license' cDC1 for CD8
T cell priming
. However, this model has not been directly tested in vivo or in the setting of help-dependent tumour rejection. Here we generated an Xcr1
mouse strain to evaluate the cellular interactions that mediate tumour rejection in a model requiring CD4
and CD8
T cells. As expected, tumour rejection required cDC1 and CD8
T cell priming required the expression of major histocompatibility class I molecules by cDC1. Unexpectedly, early priming of CD4
T cells against tumour-derived antigens also required cDC1, and this was not simply because they transport antigens to lymph nodes for processing by cDC2, as selective deletion of major histocompatibility class II molecules in cDC1 also prevented early CD4
T cell priming. Furthermore, deletion of either major histocompatibility class II or CD40 in cDC1 impaired tumour rejection, consistent with a role for cognate CD4
T cell interactions and CD40 signalling in cDC1 licensing. Finally, CD40 signalling in cDC1 was critical not only for CD8
T cell priming, but also for initial CD4
T cell activation. Thus, in the setting of tumour-derived antigens, cDC1 function as an autonomous platform capable of antigen processing and priming for both CD4
and CD8
T cells and of the direct orchestration of their cross-talk that is required for optimal anti-tumour immunity.
Dendritic cells (DCs) play a central role in the orchestration of effective T cell responses against tumors. However, their functional behavior is context-dependent. DC type, transcriptional program, ...location, intratumoral factors, and inflammatory milieu all impact DCs with regard to promoting or inhibiting tumor immunity. The following review introduces important facets of DC function, and how subset and phenotype can affect the interplay of DCs with other factors in the tumor microenvironment. It will also discuss how current cancer treatment relies on DC function, and survey the myriad ways with which immune therapy can more directly harness DCs to enact antitumor cytotoxicity.
Purpose
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells that are upregulated in cancer. Little is known about the prevalence and importance of MDSC ...in pancreas adenocarcinoma (PA).
Experimental design
Peripheral blood, bone marrow, and tumor samples were collected from pancreatic cancer patients, analyzed for MDSC (CD15
+
CD11b
+
) by flow cytometry and compared to cancer-free controls. The suppressive capacity of MDSC (CD11b
+
Gr-1
+
) and the effectiveness of MDSC depletion were assessed in C57BL/6 mice inoculated with Pan02, a murine PA, and treated with placebo or zoledronic acid, a potent aminobisphosphonate previously shown to target MDSC. The tumor microenvironment was analyzed for MDSC (Gr1
+
CD11b
+
), effector T cells, and tumor cytokine levels.
Results
Patients with PA demonstrated increased frequency of MDSC in the bone marrow and peripheral circulation which correlated with disease stage. Normal pancreas tissue showed no MDSC infiltrate, while human tumors avidly recruited MDSC. Murine tumors similarly recruited MDSC that suppressed CD8
+
T cells in vitro and accelerated tumor growth in vivo. Treatment with zoledronic acid impaired intratumoral MDSC accumulation resulting in delayed tumor growth rate, prolonged median survival, and increased recruitment of T cells to the tumor. This was associated with a more robust type 1 response with increased levels of IFN-γ and decreased levels of IL-10.
Conclusions
MDSC are important mediators of tumor-induced immunosuppression in pancreatic cancer. Inhibiting MDSC accumulation with zoledronic acid improves the host anti-tumor response in animal studies suggesting that efforts to block MDSC may represent a novel treatment strategy for pancreatic cancer.
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