The study of tumour-specific antigens (TSAs) as targets for antitumour therapies has accelerated within the past decade. The most commonly studied class of TSAs are those derived from non-synonymous ...single-nucleotide variants (SNVs), or SNV neoantigens. However, to increase the repertoire of available therapeutic TSA targets, 'alternative TSAs', defined here as high-specificity tumour antigens arising from non-SNV genomic sources, have recently been evaluated. Among these alternative TSAs are antigens derived from mutational frameshifts, splice variants, gene fusions, endogenous retroelements and other processes. Unlike the patient-specific nature of SNV neoantigens, some alternative TSAs may have the advantage of being widely shared by multiple tumours, allowing for universal, off-the-shelf therapies. In this Opinion article, we will outline the biology, available computational tools, preclinical and/or clinical studies and relevant cancers for each alternative TSA class, as well as discuss both current challenges preventing the therapeutic application of alternative TSAs and potential solutions to aid in their clinical translation.
The ability to non-invasively monitor tumor-infiltrating T cells in vivo could provide a powerful tool to visualize and quantify tumor immune infiltrates. For non-invasive evaluations in vivo, an ...anti-CD3 mAb was modified with desferrioxamine (DFO) and radiolabeled with zirconium-89 (Zr-89 or 89Zr). Radiolabeled 89Zr-DFO-anti-CD3 was tested for T cell detection using positron emission tomography (PET) in both healthy mice and mice bearing syngeneic bladder cancer BBN975. In vivo PET/CT and ex vivo biodistribution demonstrated preferential accumulation and visualization of tracer in the spleen, thymus, lymph nodes, and bone marrow. In tumor bearing mice, 89Zr-DFO-anti-CD3 demonstrated an 11.5-fold increase in tumor-to-blood signal compared to isotype control. Immunological profiling demonstrated no significant change to total T cell count, but observed CD4+ T cell depletion and CD8+ T cell expansion to the central and effector memory. This was very encouraging since a high CD8+ to CD4+ T cell ratio has already been associated with better patient prognosis. Ultimately, this anti-CD3 mAb allowed for in vivo imaging of homeostatic T cell distribution, and more specifically tumor-infiltrating T cells. Future applications of this radiolabeled mAb against CD3 could include prediction and monitoring of patient response to immunotherapy.
Combination immunotherapy has recently emerged as a powerful cancer treatment strategy. A promising treatment approach utilizes coadministration of antagonistic antibodies to block checkpoint ...inhibitor receptors, such as antiprogrammed cell death‐1 (aPD1), alongside agonistic antibodies to activate costimulatory receptors, such as antitumor necrosis factor receptor superfamily member 4 (aOX40). Optimal T‐cell activation is achieved when both immunomodulatory agents simultaneously engage T‐cells and promote synergistic proactivation signaling. However, standard administration of these therapeutics as free antibodies results in suboptimal T‐cell binding events, with only a subset of the T‐cells binding to both aPD1 and aOX40. Here, it is shown that precise spatiotemporal codelivery of aPD1 and aOX40 using nanoparticles (NP) (dual immunotherapy nanoparticles, DINP) results in improved T‐cell activation, enhanced therapeutic efficacy, and increased immunological memory. It is demonstrated that DINP elicits higher rates of T‐cell activation in vitro than free antibodies. Importantly, it is demonstrated in two tumor models that combination immunotherapy administered in the form of DINP is more effective than the same regimen administered as free antibodies. This work demonstrates a novel strategy to improve combination immunotherapy using nanotechnology.
Combination immunotherapy has recently emerged as a powerful cancer‐treatment strategy. However, standard administration of these therapeutics as free antibodies results in suboptimal T‐cell binding events. Precise spatiotemporal codelivery of aPD1 and aOX40 using nanoparticles (dual‐immunotherapy nanoparticles) is shown to result in improved T‐cell activation, enhanced therapeutic efficacy, and increased immunological memory.
High-grade urothelial cancer contains intrinsic molecular subtypes that exhibit differences in underlying tumor biology and can be divided into luminal-like and basal-like subtypes. We describe here ...the first subtype-specific murine models of bladder cancer and show that Upk3a-Cre
; Trp53
; Pten
; Rosa26
(UPPL, luminal-like) and BBN (basal-like) tumors are more faithful to human bladder cancer than the widely used MB49 cells. Following engraftment into immunocompetent C57BL/6 mice, BBN tumors were more responsive to PD-1 inhibition than UPPL tumors. Responding tumors within the BBN model showed differences in immune microenvironment composition, including increased ratios of CD8
:CD4
and memory:regulatory T cells. Finally, we predicted and confirmed immunogenicity of tumor neoantigens in each model. These UPPL and BBN models will be a valuable resource for future studies examining bladder cancer biology and immunotherapy.
This work establishes human-relevant mouse models of bladder cancer.
.
Measures of the adaptive immune response have prognostic and predictive associations in melanoma and other cancer types. Specifically, intratumoral T cell density and function have considerable ...prognostic and predictive value in skin cutaneous melanoma (SKCM). Less is known about the significance of tumor-infiltrating B cells in SKCM. Our goal was to understand the prognostic and predictive value of B cell phenotypic subsets in SKCM using RNA sequencing.
We used our previously published algorithm, V'DJer, to assemble B cell receptor (BCR) repertoires and estimate diversity from short-read RNA sequencing (RNA-seq). We applied machine learning-based cellular phenotype classifiers to measure relative similarity of bulk tumor sample gene expression profiles and different B cell phenotypes. We assessed these aspects of B cell biology in 473 SKCM from the Cancer Genome Atlas Project (TCGA) as well as in RNA-seq data corresponding to tumor samples procured from patients who received CTLA-4 and PD-1 inhibitors for metastatic SKCM.
We found that the BCR repertoire was associated with different clinical factors, such as tumor tissue site and sex. However, increased clonality of the BCR repertoire was favorably prognostic in SKCM and was prognostic even after first conditioning on various clinical factors. Mutation burden was not correlated with any BCR measurement, and no specific mutation had an altered BCR repertoire. Lack of an assembled BCR in pre-treatment tumor tissues was associated with a lack of anti-tumor response to a CTLA-4 inhibitor in metastatic SKCM.
These findings suggest an important prognostic and predictive role for B cell characteristics in SKCM. This has implications for melanoma immunobiology and potential development of immunogenomics features to predict survival and response to immunotherapy.
Human endogenous retroviruses (hERVs) are remnants of exogenous retroviruses that have integrated into the genome throughout evolution. We developed a computational workflow, hervQuant, which ...identified more than 3,000 transcriptionally active hERVs within The Cancer Genome Atlas (TCGA) pan-cancer RNA-Seq database. hERV expression was associated with clinical prognosis in several tumor types, most significantly clear cell renal cell carcinoma (ccRCC). We explored two mechanisms by which hERV expression may influence the tumor immune microenvironment in ccRCC: (i) RIG-I-like signaling and (ii) retroviral antigen activation of adaptive immunity. We demonstrated the ability of hERV signatures associated with these immune mechanisms to predict patient survival in ccRCC, independent of clinical staging and molecular subtyping. We identified potential tumor-specific hERV epitopes with evidence of translational activity through the use of a ccRCC ribosome profiling (Ribo-Seq) dataset, validated their ability to bind HLA in vitro, and identified the presence of MHC tetramer-positive T cells against predicted epitopes. hERV sequences identified through this screening approach were significantly more highly expressed in ccRCC tumors responsive to treatment with programmed death receptor 1 (PD-1) inhibition. hervQuant provides insights into the role of hERVs within the tumor immune microenvironment, as well as evidence that hERV expression could serve as a biomarker for patient prognosis and response to immunotherapy.
Although immune-checkpoint inhibitors (ICIs) have been a remarkable advancement in bladder cancer treatment, the response rate to single-agent ICIs remains suboptimal. There has been substantial ...interest in the use of epigenetic agents to enhance ICI efficacy, although precisely how these agents potentiate ICI response has not been fully elucidated. We identified entinostat, a selective HDAC1/3 inhibitor, as a potent antitumor agent in our immune-competent bladder cancer mouse models (BBN963 and BBN966). We demonstrate that entinostat selectively promoted immune editing of tumor neoantigens, effectively remodeling the tumor immune microenvironment, resulting in a robust antitumor response that was cell autonomous, dependent upon antigen presentation, and associated with increased numbers of neoantigen-specific T cells. Finally, combination treatment with anti-PD-1 and entinostat led to complete responses and conferred long-term immunologic memory. Our work defines a tumor cell-autonomous mechanism of action for entinostat and a strong preclinical rationale for the combined use of entinostat and PD-1 blockade in bladder cancer.
Abstract
Motivation
Elimination of cancer cells by T cells is a critical mechanism of anti-tumor immunity and cancer immunotherapy response. T cells recognize cancer cells by engagement of T cell ...receptors with peptide epitopes presented by major histocompatibility complex molecules on the cancer cell surface. Peptide epitopes can be derived from antigen proteins coded for by multiple genomic sources. Bioinformatics tools used to identify tumor-specific epitopes via analysis of DNA and RNA-sequencing data have largely focused on epitopes derived from somatic variants, though a smaller number have evaluated potential antigens from other genomic sources.
Results
We report here an open-source workflow utilizing the Nextflow DSL2 workflow manager, Landscape of Effective Neoantigens Software (LENS), which predicts tumor-specific and tumor-associated antigens from single nucleotide variants, insertions and deletions, fusion events, splice variants, cancer-testis antigens, overexpressed self-antigens, viruses, and endogenous retroviruses. The primary advantage of LENS is that it expands the breadth of genomic sources of discoverable tumor antigens using genomics data. Other advantages include modularity, extensibility, ease of use, and harmonization of relative expression level and immunogenicity prediction across multiple genomic sources. We present an analysis of 115 acute myeloid leukemia samples to demonstrate the utility of LENS. We expect LENS will be a valuable platform and resource for T cell epitope discovery bioinformatics, especially in cancers with few somatic variants where tumor-specific epitopes from alternative genomic sources are an elevated priority.
Availability and implementation
More information about LENS, including code, workflow documentation, and instructions, can be found at (https://gitlab.com/landscape-of-effective-neoantigens-software).
BP180, also termed collagen XVII, is a hemidesmosomal transmembrane glycoprotein expressed in basal keratinocytes, and functions as a cell-matrix adhesion molecule in the dermal-epidermal junction of ...the skin. Its function, other than cell-matrix adhesion, remains unclear. We generated a mouse strain with BP180 dysfunction (termed ∆NC16A), which develops spontaneous skin inflammation accompanied by an influx of myeloid derived suppressor cells (MDSCs). We used the B16 mouse melanoma model to demonstrate that BP180 dysfunction in either skin or basal keratinocytes promotes MDSC influx into skin and tumor progression. MDSC depletion reduced tumor progression in ∆NC16A mice, demonstrating a critical role for BP180 dysfunction-driven MDSCs in melanoma progression. This study provides the first direct evidence that BP180, a cell-cell matrix adhesion molecule, possesses antitumor function through modulating infiltration of MDSCs. Basal keratinocytes actively participate in skin microenvironment changes caused by BP180 dysfunction. ∆NC16A mice could be a new animal model to study the melanoma microenvironment.
Early in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4
T cell, and CD8
T cell stimulation. The rationale for this design was ...to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE).
We explored the set of computationally predicted SARS-CoV-2 HLA-I and HLA-II ligands, examining protein source, concurrent human/murine coverage, and population coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, sequence conservation, source protein abundance, and coverage of high frequency HLA alleles. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering for surface accessibility, sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites.
From 58 initial candidates, three B cell epitope regions were identified. From 3730 (MHC-I) and 5045 (MHC-II) candidate ligands, 292 CD8
and 284 CD4
T cell epitopes were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we proposed a set of 22 SARS-CoV-2 vaccine peptides for use in subsequent murine studies. We curated a dataset of ~ 1000 observed T cell epitopes from convalescent COVID-19 patients across eight studies, showing 8/15 recurrent epitope regions to overlap with at least one of our candidate peptides. Of the 22 candidate vaccine peptides, 16 (n = 10 T cell epitope optimized; n = 6 B cell epitope optimized) were manually selected to decrease their degree of sequence overlap and then synthesized. The immunogenicity of the synthesized vaccine peptides was validated using ELISpot and ELISA following murine vaccination. Strong T cell responses were observed in 7/10 T cell epitope optimized peptides following vaccination. Humoral responses were deficient, likely due to the unrestricted conformational space inhabited by linear vaccine peptides.
Overall, we find our selection process and vaccine formulation to be appropriate for identifying T cell epitopes and eliciting T cell responses against those epitopes. Further studies are needed to optimize prediction and induction of B cell responses, as well as study the protective capacity of predicted T and B cell epitopes.
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