Retroviral Antigens Research Articles

Radiation Therapy for Bridging and Improving CAR-T Cell Therapy

<h3>Purpose/Objective(s)</h3> CD19-targeted chimeric antigen receptor (CAR) T cells have transformed the treatment of patients with relapsed or refractory CD19-positive hematologic malignancies. However, a significant subset of these patients either fails to respond or eventually relapses. Moreover, patients who are candidates for CART-19 therapy often have symptomatic disease that requires some form of treatment to support them during the manufacture period. An ideal bridging therapy would simultaneously both (1) sensitize the tumor to CART-19 attack to increase antitumor control and (2) debulk the disease during manufacture. Radiotherapy (RT) is an established curative and palliative cancer treatment regimen, with approximately half of cancer patients with solid tumors receiving RT sometime during their disease. Several reports have underscored the existence of threshold doses (and regimens) that are able to switch on different damage programs that profoundly affect responses to therapy by eliciting potent immune modulatory effects, prompting immunologically mediated tumor cell death. These effects have been attributed to multiple pathways, including the activation of the cGMP-cAMP Synthetase (cGAS), Stimulator of Interferon Genes (STING) pathway with Type I Interferons expression, and tumor associated antigen (TAA) cross priming with anti-tumor CD8⁺ T cell elicitation, ultimately inducing "abscopal" effects. By virtue of the immunomodulatory effects of RT, we hypothesize that RT could serve as a successful bridging strategy for CAR T-cell therapy. <h3>Materials/Methods</h3> To investigate this hypothesis, we established the A20 Lymphoma CART-19 therapy mouse model in our lab and used it to perform preliminaries experiments. A20 cells were implanted into both flanks of the animals. Mimicking human immune adjuvant doses used in our institute for RT bridging, 20 days after tumor cell implantation, we radiated one of the two tumors using a dose of 8Gy divided in two fractions of 4Gy each. Twenty-four hours later 1 × 10ep6 CART-19 cells were infused intravenously. <h3>Results</h3> While the effects of RT in combination with CART-19 have similar impact on the tumor growth of the irradiated mass, a significant increase of the antitumor effects of the single therapy was observed on the non-irradiated tumor. FACS studies demonstrated heavy leukocyte infiltration with increased presence of infused CART-19 as well indigenous T cells. To investigate possible molecular mechanisms involved in the increased effects on the non-irradiated tumor, we performed a qPCR array and observed increased expression of genes linked to cross priming, including IFN type I and Batf3. A20 cells express the ectopic retroviral antigen gp70 with well characterized AH1 immune-dominant epitope. AH1 tetramer staining on infiltrating T cells demonstrated increased staining in tumor from mice treated with the combination. <h3>Conclusion</h3> Overall these results suggest RT may serve as optimal debulking and bridging therapy for CAR-T cell therapy.

An Immunogenic Model of KRAS-Mutant Lung Cancer Enables Evaluation of Targeted Therapy and Immunotherapy Combinations.

This study develops a mouse model of immunogenic KRAS-mutant lung cancer to facilitate the investigation of optimal combinations of targeted therapies with immunotherapies.

Abstract 3449: Radiation therapy for bridging and improving CAR-T cells therapy

Abstract CD19-targeted chimeric antigen receptor (CAR) T cells have transformed the treatment of patients with relapsed or refractory CD19-positive hematologic malignancies. However, a significant subset of these patients either fails to respond or eventually relapses. Moreover, patients who are candidates for CART-19 therapy often have symptomatic disease that requires some form of treatment to support them during the manufacture period. An ideal bridging therapy would simultaneously both (1) sensitize the tumor to CART-19 attack to increase antitumor control and (2) debulk the disease during manufacture. Radiotherapy (RT) is an established curative and palliative cancer treatment regimen, with approximately half of cancer patients with solid tumors receiving RT some time during their disease. Several reports have underscored the existence of threshold doses (and regimens) that are able to switch on different damage programs that profoundly affect responses to therapy by eliciting potent immune modulatory effects, prompting immunologically mediated tumor cell death. These effects have been attributed to multiple pathways, including the activation of the cGMP-cAMP Synthetase (cGAS), Stimulator of Interferon Genes (STING) pathway with Type I Interferons expression, and tumor associated antigen (TAA) cross priming with anti-tumor CD8+ T cell elicitation, ultimately inducing “abscopal” effects. By virtue of the immunomodulatory effects of RT, we hypothesize that RT could serve as a successful bridging strategy for CAR T-cell therapy. To investigate this hypothesis we established the A20 Lymphoma CART-19 therapy mouse model in our lab and use it to perform preliminaries experiments. A20 cells were implanted into both flanks of the animals. Mimicking human immune adjuvant dose used in our institute for RT bridging, 20 days after the A20 challenge we RT treated one of the two tumors using a dose of 8Gy divided in two fractions of 4Gy each. Twenty-four hours later 1x10ep6 CART-19 cells were infused intravenously. While the effects of RT in combination with CART-19 have similar impact on the tumor growth of the irradiated mass a significant increase of the antitumor effects of the single therapy was observed on the non-irradiated tumor. FACS studies demonstrated heavy leukocytes infiltration with increased presence of infused CART-19 as well indigenous T cells. To investigate possible molecular mechanisms involved in the increased effects on the non-irradiated tumor, we perform a qPCR array and observed increased expression of genes linked to cross priming, including IFN type I and Batf3. A20 cells express the ectopic retroviral antigen gp70 with well characterized AH1 immune-dominant epitope. AH1 tetramer staining on infiltrating T cells demonstrated increase staining in tumor from mice treated with the combination. Overall these results suggest RT may serve as optimal debulking and adjuvant bridging therapy for CAR-T cells therapy. Citation Format: Nektarios Kostopoulos, Costanitnos Koumeins, John P. Plastaras, Stephen J. Schuster, Andrea Facciabene. Radiation therapy for bridging and improving CAR-T cells therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3449.

Abstract 1390: Immunotherapy boosts B cell responses in lung adenocarcinoma

Abstract Introduction: B cells constitute a major part of the lung tumour microenvironment where they are organised in tertiary lymphoid structures (TLS) that resemble antibody-producing germinal centres. The presence of TLS has been associated with improved patient outcome and response to immunotherapy, though the mechanisms remain unclear. Similarly, the contribution of B cells and TLS to anti-tumour immunity in non-small cell lung cancer (NSCLC) remains largely unexplored. Methods: We evaluated TRACERx and TCGA NSCLC cohorts to compare B cell and TLS signatures as well as BCR sequences from human adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Using a novel immunogenic mouse model of LUAD, we applied lineage tracing, 3D IF, and single-cell sequencing to characterise the evolution of lung germinal centre and antibody responses over tumour progression and immunotherapy treatment. We validated immunotherapy observations in a cohort of melanoma patients with pre- and post-treatment samples. Results: We found that B cell and TLS gene set scores were significantly higher in LUAD compared to LUSC. We examined B cell signals in specific mutational backgrounds, and observed an increase in BCR CDR3 diversity and IgG class switch in the context of clonal TP53 mutation. We therefore investigated the mechanistic basis of B cells in LUAD using an immunogenic mouse model on a KRAS/TP53 background. We found that TLS organisation was triggered by CXCL13 and that exogenous CXCL13 treatment increased B cell infiltration and overall survival. Using serum transfer experiments, we demonstrated that tumour-binding class-switched antibodies played an anti-tumour role and increased survival. We found that immunotherapy increased germinal centre B cell responses and antibody quantity and quality. Using single-cell BCR sequencing of immunotherapy-treated mice, we showed clonal expansion of B cells reactive to re-expressed endogenous retroviral antigens. Furthermore, we found that KRAS G12C but not MEK inhibition boosted B cell and antibody responses. Finally, combined treatment with CXCL13 and checkpoint blockade improved survival, suggesting that boosting local B cell responses can improve immunotherapy outcome. In checkpoint blockade-treated melanoma patients, we found that BCR diversity and the number of unique antigen specificities was elevated in responders post-therapy. Non-responders showed a loss of expanded clones, suggesting that both maintenance of expanded clones and recruitment of new clones associate with successful response. Conclusions: Our work describes a novel role for B cells in the lung tumour microenvironment and establish that TLS directly contribute to anti-tumour immunity by producing class-switched protective antibodies. We demonstrate that selective boosting of anti-tumour B cells shows therapeutic potential in combination with targeted and immunotherapy. Citation Format: Katey Enfield, Kevin Ng, Jesse Boumelha, Kevin Litchfield, Oriol Pich, TRACERx Consortium, Charles Swanton, George Kassiotis, Julian Downward. Immunotherapy boosts B cell responses in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1390.

Targeting the Atf7ip-Setdb1 Complex Augments Antitumor Immunity by Boosting Tumor Immunogenicity.

Substantial progress has been made in understanding how tumors escape immune surveillance. However, few measures to counteract tumor immune evasion have been developed. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR-Cas9 suppressor screen for genes encoding epigenetic modifiers. We found that disruption of the genes encoding either of the chromatin modifiers activating transcription factor 7-interacting protein (Atf7ip) or its interacting partner SET domain bifurcated histone lysine methyltransferase 1 (Setdb1) in tumor cells restored tumor antigen expression. This resulted in augmented tumor immunogenicity concomitant with elevated endogenous retroviral (ERV) antigens and mRNA intron retention. ERV disinhibition was associated with a robust type I interferon response and increased T-cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1. ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T-cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy.

Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.

Endogenous retroviruses expressed in human tumours cannot be used as targets for anti-tumour vaccines

Many tumour cells express on their surface proteins of endogenous retroviruses (ERVs) and there are suggestions to use these retroviral antigens as target for anti-tumour vaccines. However, until now there is no convincing data showing that this strategy works, in contrast, there are considerations suggesting that this strategy may be harmful if applied.

Genome-wide analysis of CCHC-type zinc finger (ZCCHC) proteins in yeast, Arabidopsis, and humans.

The diverse eukaryotic proteins that contain zinc fingers participate in many aspects of nucleic acid metabolism, from DNA transcription to RNA degradation, post-transcriptional gene silencing, and small RNA biogenesis. These proteins can be classified into at least 30 types based on structure. In this review, we focus on the CCHC-type zinc fingers (ZCCHC), which contain an 18-residue domain with the CX2CX4HX4C sequence, where C is cysteine, H is histidine, and X is any amino acid. This motif, also named the "zinc knuckle", is characteristic of the retroviral Group Antigen protein and occurs alone or with other motifs. Many proteins containing zinc knuckles have been identified in eukaryotes, but only a few have been studied. Here, we review the available information on ZCCHC-containing factors from three evolutionarily distant eukaryotes-Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens-representing fungi, plants, and metazoans, respectively. We performed systematic searches for proteins containing the CX2CX4HX4C sequence in organism-specific and generalist databases. Next, we analyzed the structural and functional information for all such proteins stored in UniProtKB. Excluding retrotransposon-encoded proteins and proteins harboring uncertain ZCCHC motifs, we found seven ZCCHC-containing proteins in yeast, 69 in Arabidopsis, and 34 in humans. ZCCHC-containing proteins mainly localize to the nucleus, but some are nuclear and cytoplasmic, or exclusively cytoplasmic, and one localizes to the chloroplast. Most of these factors participate in RNA metabolism, including transcriptional elongation, polyadenylation, translation, pre-messenger RNA splicing, RNA export, RNA degradation, microRNA and ribosomal RNA biogenesis, and post-transcriptional gene silencing. Several human ZCCHC-containing factors are derived from neofunctionalized retrotransposons and act as proto-oncogenes in diverse neoplastic processes. The conservation of ZCCHCs in orthologs of these three phylogenetically distant eukaryotes suggests that these domains have biologically relevant functions that are not well known at present.

Endogenous retroviral proteins provide an immunodominant but not requisite antigen in a murine immunotherapy tumor model

ABSTRACT Clinical observations suggest that responses to cancer immunotherapy are correlated with intra-tumoral T cell receptor (TCR) clonality, tumor mutation burden (TMB) and host HLA genotype, highlighting the importance of host T cell recognition of tumor antigens. However, the dynamic interplay between T cell activation state and changes in TCR repertoire in driving the identification of potential immunodominant antigen(s) remains largely unexplored. Here, we performed single-cell RNA-sequencing on CD8+ tumor-infiltrating T cells (TILs) using the murine colorectal tumor model MC38 to identify unique TCR sequences and validate their tumor reactivity. We found that the majority of clonally expanded TILs are tumor-reactive and their TCR repertoire is unique amongst individual MC38 tumor-bearing mice. Our query identified that multiple expanded TCR clones recognized the retroviral epitope p15E as an immunodominant antigen. In addition, we found that the endogenous retroviral genome encoding for p15E is highly expressed in MC38 tumors, but not in normal tissues, due to epigenetic derepression. Further, we demonstrated that the p15E-specific TILs exhibit an activated phenotype and an increase in frequency upon treatment with anti-41BB and anti-PD-1 combination immunotherapy. Importantly, we showed that although p15E-specific TILs are not required to mount a primary anti-tumor response, they contributed to the development of strong immune memory. Overall our results revealed that endogenous retroviral antigens expressed by tumor cells may represent an important and underappreciated category of tumor antigens that could be readily targeted in the clinic.

Antibody-Based Delivery of Cytokine Payloads to Carbonic Anhydrase IX Leads to Cancer Cures in Immunocompetent Tumor-Bearing Mice.

Antibody-cytokine fusion proteins can have the potential to increase the density and activity of subsets of leukocytes within the tumor mass. Here, we describe the design, production, and characterization of four novel antibody-cytokine fusion proteins directed against human carbonic anhydrase IX, a highly validated marker of hypoxia that is overexpressed in clear cell renal cell carcinoma and other malignancies. As immunomodulatory payloads we used TNF, IL2, IFNα2 (corresponding to products that are in clinical use), and IL12 (as this cytokine potently activates T cells and NK cells). Therapy experiments were performed in BALB/c mice, bearing CT26 tumors transfected with human carbonic anhydrase IX, in order to assess the performance of the fusion proteins in an immunocompetent setting. The biopharmaceuticals featuring TNF, IL2, or IL12 as payloads cured all mice in their therapy groups, whereas only a subset of mice was cured by the antibody-based delivery of IFNα2. Although the antibody fusion with TNF mediated a rapid hemorrhagic necrosis of the tumor mass, a slower regression of the neoplastic lesions (which continued after the last injection) was observed with the other fusion proteins, and treated mice acquired protective anticancer immunity. A high proportion of tumor-infiltrating CD8+ T cells was specific to the retroviral antigen AH1; however, the LGPGREYRAL peptide derived from human carbonic anhydrase IX was also present on tumor cells. The results described herein provide a rationale for the clinical use of fully human antibody-cytokine fusions specific to carbonic anhydrase IX.

The antibody-based delivery of interleukin-12 to solid tumors boosts NK and CD8+ T cell activity and synergizes with immune checkpoint inhibitors.

We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 antibody in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively spliced EDB domain of fibronectin), retained the activity of the parental cytokine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent antitumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with checkpoint blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case, cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anticancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.

Retrovirus insertion site analysis of LGL leukemia patient genomes

BackgroundLarge granular lymphocyte (LGL) leukemia is an uncommon cancer characterized by sustained clonal proliferation of LGL cells. Antibodies reactive to retroviruses have been documented in the serum of patients with LGL leukemia. Culture or molecular approaches have to date not been successful in identifying a retrovirus.MethodsBecause a retrovirus must integrate into the genome of an infected cell, we focused our efforts on detecting a novel retrovirus integration site in the clonally expanded LGL cells. We present a new computational tool that uses long-insert mate pair sequence data to search the genome of LGL leukemia cells for retrovirus integration sites. We also utilize recently published methods to interrogate the status of polymorphic human endogenous retrovirus type K (HERV-K) provirus in patient genomes.ResultsOur data show that there are no new retrovirus insertions in LGL genomes of LGL leukemia patients. However, our insertion call tool did detect four HERV-K provirus integration sites that are polymorphic in the human population but absent from the human reference genome, hg19. To determine if the prevalence of these or other polymorphic proviral HERV-Ks differed between LGL leukemia patients and the general population, we used a recently developed tool that reports sites in the human genome occupied by a known proviral HERV-K. We report that there are significant differences in the number of polymorphic HERV-Ks in the genomes of LGL leukemia patients of European origin compared to individuals with European ancestry in the 1000 genomes (KGP) data.ConclusionsOur study confirms that the clonal expansion of LGL cells in LGL leukemia is not driven by the integration of a new infectious or endogenous retrovirus, although we do not rule out that these cells are responding to retroviral antigens produced in other cell types. However, our computational analyses revealed that the genomes of LGL leukemia patients carry a higher burden of polymorphic HERV-K proviruses compare to individuals from KGP of European ancestry. Our research emphasizes the merits of comprehensive genomic assessment of HERV-K in cancer samples and suggests that further analyses to determine contributions of HERV-K to LGL leukemia are warranted.

Friend retrovirus infection induces the development of memory-like natural killer cells

Traditionally, NK cells belong to the innate immune system and eliminate virus-infected cells through their germline-encoded receptors. However, NK cells were recently reported to possess memory-like functions that were predominantly provided by hepatic NK cells. Memory properties were mainly documented in contact hypersensitivity models or during cytomegalovirus infections. However, the precise role and the physiologic importance of memory-like NK cells during retroviral infections are still under investigation. Here, we show that Friend retrovirus (FV) infection of mice induced a population of phenotypically memory-like NK cells at 28 days post infection. Upon secondary antigen encounter, these NK cells showed an increased production of the pro-inflammatory cytokines IFNγ and TNFα as well as the death ligand FasL in comparison to naïve NK cells. Furthermore, we found an augmented elimination of antigen-matched but not antigen-mismatched target cells by these memory-like NK cells. In adoptive cell transfer experiments, equal antiviral activities of splenic and hepatic memory-like NK cells during the late phase of acute FV infection were found. Our results strongly imply the existence and antiviral activity of spleen and liver memory-like NK cells in FV infection, which efficiently respond upon secondary exposure to retroviral antigens.

Endogenous retroviral signatures predict immunotherapy response in clear cell renal cell carcinoma.

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.

Correlates of Follicular Helper Bias in the CD4 T Cell Response to a Retroviral Antigen.

CD4+ T cell differentiation is influenced by a plethora of intrinsic and extrinsic factors, providing the immune system with the ability to tailor its response according to specific stimuli. Indeed, different classes of pathogens may induce a distinct balance of CD4+ T cell differentiation programmes. Here, we report an uncommonly strong bias toward follicular helper (Tfh) differentiation of CD4+ T cells reactive with a retroviral envelope glycoprotein model antigen, presented in its natural context during retroviral infection. Conversely, the response to the same antigen, presented in different immunization regimens, elicited a response typically balanced between Tfh and T helper 1 cells. Comprehensive quantitation of variables known to influence Tfh differentiation revealed the closest correlation with the strength of T cell receptor (TCR) signaling, leading to PD-1 expression, but not with surface TCR downregulation, irrespective of TCR clonotypic avidity. In contrast, strong TCR signaling leading to TCR downregulation and induction of LAG3 expression in high TCR avidity clonotypes restrained CD4+ T cell commitment and further differentiation. Finally, stunted Th1 differentiation, correlating with limited IL-2 availability in retroviral infection, provided permissive conditions for Tfh development, suggesting that Tfh differentiation is the default program of envelope-reactive CD4+ T cells.

Abstract IA21: Immunocompetent models of head and neck cancer

Abstract Background: Syngeneic cell line based cancer model systems have advanced our understanding of host tumor interactions in developing cancers and in immunotherapeutic approaches. Our group initiated development of murine head and neck squamous cell carcinomas (HNSCC) model systems in 2007-2008 as no organ specific models had been described. The best described animal model systems for HNSCC utilized the hamster cheek pouch model where the carcinogen 7,12-Dimethylbenz(a)anthracene was the initiating and tumor promoting agent. We adapted this model to murine systems and generated primary oral squamous cell carcinomas that we used to make transplantable cell line models. These mouse oral carcinoma cell lines (MOC model) have a conserved carcinogen origin and retain pathologic, genomic and biologic features of the human disease and thus represent a robust preclinical surrogate. Here we describe considerations in development of immunocompetent models of HNSCC, validation approaches used for the MOC model and highlight recent work on checkpoint responses in the MOC model. Methods: To define the genomic landscape and assess the fidelity of the MOC model to human disease, whole exome sequencing (WES) and expression arrays were performed. In order to evaluate checkpoint-based immunotherapeutic approaches in murine OSCC, independent cohorts of mice were treated with blocking monoclonal antibodies to PD-1. Tumor growth was monitored by twice-weekly measurement. Whole exome sequencing data were processed via MHC Class I binding algorithms including NetMHC to predict high-affinity neoantigen candidates. Mutant peptides were synthesized and used in Enzyme-Linked ImmunoSpot (ELISPOT) assays using tumor-infiltrating lymphocytes generated from mice bearing MOC1 and MOC22 tumors and ELISPOT data was validated using H-2Kb dual-color tetramer assays. Results: Next-generation sequencing of the MOC cell lines demonstrated significant homology to human head and neck squamous cell carcinoma (HNSCC), including identification of driver pathway mutations in Trp53, mitogen-activated protein kinase, phosphoinositide 3-kinase, NOTCH, JAK/STAT, and Fat1-4. Furthermore, the most aggressive MOC cell line, MOC2, demonstrated an expression signature consistent with more aggressive human HNSCC tumors, including upregulation of pERK and CD44. This finding led to a biomarker window-of-opportunity clinical trial of a MEK inhibitor, trametinib, for patients with aggressive OSCC. Next, looking at checkpoint inhibitors, we identified three distinct phenotypes in response to anti-PD1 in the three murine OSCC tumors. Anti-PD1 had no impact on the growth kinetics or frequency of metastases in MOC2. MOC1, a moderately immunogenic tumor, exhibited significant tumor regression followed by outgrowth in a subset of treated mice. When this “escape” tumor was harvested, expanded in vitro and retransplanted into mice, it was more aggressive than the parental and no longer exhibited any growth delay with anti-PD1. MOC22 demonstrated complete response, consistently rejecting 20-30 days following treatment. We then applied a neoantigen discovery pipeline to MOC22 as these tumors demonstrated the most robust immune response with checkpoint blockade. Predicted high-affinity neoepitopes were screened using tumor-infiltrating lymphocytes assessing for interferon-gamma (IFNg) response with ELISPOT. TIL from MOC22 demonstrated reactivity to a mutated ICAM-1 (mICAM1) protein with a proline to leucine (P315L) substitution which induced a 145-fold increase in H-2Kb affinity (2.45 nM for mutant versus 357.21 nM for wild type). In addition to screening for mutation-derived neoepitopes, we also identified that MOC22 expressed an endogenous retroviral derived H2-Kb restricted antigen from the p15e protein, previously described in several C57BL/6 derived tumor models. Likewise, in ELISPOT assays, we found strong reactivity to the p15e antigen as well. To validate the ELISPOT data, we utilized dual-color H-2Kb restricted tetramers with our two candidate antigens, miCAM1 (TVYNFSAL) and p15e (KSPWFTTL) and identified a subset of T cells with specificity for each antigen. Ongoing work is focused on defining the time course of TIL infiltration in these tumors and how to optimize use of these tumor antigens in a vaccine setting. Conclusions: We have developed and characterized three syngeneic mouse models of OSCC that demonstrate conservation of mutations and transcriptional pathway activation with human HNSCC. Findings in this model have demonstrated translational relevancy leading to demonstration of MEK targeting as an option in aggressive OSCC. Furthermore, we have identified a genomically-derived tumor neoantigen and an endogenous retroviral antigen that are candidates for checkpoint inhibitor-induced tumor rejection in a murine OSCC model. These data represent the translational relevance of the MOC model and provide a preclinical foundation for development of personalized and targeted cancer immunotherapeutics in OSCC. Citation Format: Ravindra Uppaluri. Immunocompetent models of head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA21.

Autoimmune Responses to Exosomes and Candidate Antigens Contribute to Type 1 Diabetes in Non-Obese Diabetic Mice.

The initial autoimmune trigger of type 1 diabetes (T1D) remains unclear. In non-obese diabetic (NOD) mice, islet inflammation starts early in life, suggesting the presence of an endogenous trigger for the spontaneous autoimmune response in this T1D mouse model. In this review, we argue that abnormal release of exosomes might be the trigger of the early inflammatory and autoimmune responses in the islets. Exosomes are nano-sized membrane complexes that are secreted by cells following fusion of late endosomes and/or multivesicular bodies with the plasma membrane. They are known extracellular messengers, communicating among neighboring cells via transporting large molecules from parent cells to recipient cells. Recent evidence demonstrates that these extracellular vesicles can modulate immune responses. It has been shown that insulinoma and islet mesenchymal stem cell-released exosomes are potent immune stimuli that can induce autoreactive B and T cells. Searching for candidate antigens in the exosomes identified endogenous retrovirus (ERV) Env and Gag antigens, which are homologous to an endogenous murine leukemia retrovirus. Autoantibodies and autoreactive T cells spontaneously developed in NOD mice can react to these retroviral antigens. More importantly, expression of the retroviral antigens in the islet mesenchymal stem cells is associated with disease susceptibility, and the expression is restricted to T1D-susceptible but not resistant mouse strains. Exosomes are novel autoimmune targets, carrying autoantigens that can stimulate innate and adaptive immune responses. An abnormal or excess release of exosomes, particularly those ones containing endogenous retroviral antigens might be responsible for triggering tissue-specific inflammatory and autoimmune responses.

Sarcoma Eradication by Doxorubicin and Targeted TNF Relies upon CD8+ T-cell Recognition of a Retroviral Antigen.

Antibody-cytokine complexes may offer new tools to treat cancer. Here, we show how TNF-linked antibodies, which recognize tumor-selective splice isoforms of fibronectin (F8-TNF), can be exploited to eradicate sarcomas in immunocompetent mice. We treated mice bearing WEHI-164 fibrosarcoma with a combination of F8-TNF and doxorubicin, curing the majority of treated animals (29/37). Notably, cured mice were resistant to rechallenge not only by WEHI-164 cells but also heterologous C51 or CT26 colorectal tumor cells in a CD8+ T-cell-dependent process. Mechanistic analyses revealed that each tumor cell line presented AH1, a common endogenous retroviral peptide. Numbers of AH1-specific CD8+ T cells exhibiting cytotoxic capacity were increased by F8-TNF plus doxorubicin treatment, arguing that cognate CD8+ T cells contributed to tumor eradication. Sequence analysis of T-cell receptors of CD8+ T cells revealed the presence of H-2Ld/AH1-specific T cells and an expansion of sequence diversity in treated mice. Overall, our findings provide evidence that retroviral genes contribute to tumoral immunosurveillance in a process that can be generally boosted by F8-TNF and doxorubicin treatment. Cancer Res; 77(13); 3644-54. ©2017 AACR.

Type 1 diabetes pathogenesis is modulated by spontaneous autoimmune responses to endogenous retrovirus antigens in NOD mice

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and Tcells, causing Type 1 Diabetes in non-obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well-characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus-like particles produced by co-expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes-resistant mice developed anti-Env autoantibodies that increase in titer as disease progresses. A lentiviral-based RNA interference knockdown of Gag revealed that Gag contributes to the MV-induced T-cell response, whose diabetogenic function can be demonstrated via cell-transfer into immune-deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet-derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes-resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti-retroviral responses to trigger autoimmunity.

Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies.