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Mutation-associated neoantigens (MANAs) are exquisitely cancer-specific therapeutic targets. However, MANAs are present at ultra-low densities on the cancer cell surface (as few as 1-2 copies per cell), leading to the challenge of eliciting a sufficiently robust therapeutic effect. We combined components of both T cell receptors (TCRs) and chimeric antigen receptors (CARs) to create a new receptor with improved potency against an ultra-low-density MANA. From CARs, we utilized the antibody-based antigen recognition domain (i.e. the single chain variable fragment, scFv) and the integrated co-stimulation that amplifies T cell activation. From TCRs, we utilized the multi-chain signaling platform that facilitates high antigen sensitivity. This new receptor, termed a TCR Embedded ScFv for Long-term Activation (TESLA), showed promising characteristics when tested with the H2-scFv which targets the p53 R175H mutation presented on HLA-A*02:01 (R175H/A2). Using CRISPR-based homology directed repair in primary human T cells, we tested 15 configurations of appending the H2-scFv to subunits of the TCR complex to identify a design that maximized T cell cytotoxicity and interferon gamma release in co-cultures with cancer cells expressing endogenous levels of the R175H/A2 antigen. In this system, we showed that the optimal TCR-embedded configuration of the H2-scFv produced similar levels of cytotoxicity and interferon gamma secretion as patient-derived TCRs targeting the same R175H/A2 MANA, while conventional H2-CARs were unable to produce any T-cell activation. We then used a multiple stimulation co-culture system to identify a co-stimulation domain combination (MyD88 and CD40) that improved serial cytotoxicity and proliferation of H2-TESLAs when incorporated on the intracellular side of the TCRbeta chain. Finally, we compared the H2-TESLA receptor to patient-derived TCRs modified with the same MyD88 and CD40 co-stimulation domains. In vivo, H2-TESLAs cured all mice in a tumor model, while co-stimulation-modified TCRs produced only temporary tumor control. Moreover, in vivo, H2-TESLAs elicited 100-fold greater T cell expansion than co-stimulation-modified TCRs. In conclusion, we demonstrated that by combining aspects of both CARs and TCRs, the TESLA receptor improved T cell reactivity against an ultra-low-density neoantigen compared to conventional CARs and patient-derived TCRs. Citation Format: Brian J. Mog, Sarah R. DiNapoli, Michael S. Hwang, Tushar D. Nichakawade, Jacqueline Douglass, Emily Han-Chung Hsiue, Katharine M. Wright, Alexander H. Pearlman, Maximilian F. Konig, Suman Paul, Nicolas Wyhs, Nikita Marcou, Stephanie Glavaris, Jiaxin Ge, Michelle S. Miller, P. Aitana Azurmendi, Evangeline Watson, Drew M. Pardoll, Sandra B. Gabelli, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. Hybrid TCR-CAR design surpasses conventional CARs and patient-derived TCRs in targeting an ultra-low-density neoantigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB095.

Single cell RNA sequencing (scRNA-seq) has gained increased popularity in recent years and has revolutionized the study of cell populations; however, this technology presents several caveats regarding specific gene expression measurement. Here we examine the expression levels of several immune checkpoint genes, which are currently assessed in clinical studies. We find that unlike in most bulk sequencing studies, PVRIG and murine TIGIT suffers from poor detection in 10x Chromium scRNA-seq and other types of assays that utilize the GENCODE gene model. We show that the default GENCODE gene model, typically used in the analysis of such data, is incorrect in the PVRIG genomic region and contains also a predicted read-through transcript to which PVRIG reads co-align, causing these to be discarded and hence hindering its proper detection. Moreover, we find that the murine TIGIT 3’ UTR is mis-annotated, leading to the loss of legitimate reads. We thus generated a corrected reference genome, by removing the faulty read-through in the case of PVRIG and by extending the TIGIT 3’ UTR and demonstrate that by employing these changes we can correctly capture genuine expression levels of these checkpoints, and which align with our findings at the protein level using FACS and CITEseq. Furthermore, we show that specialized read multimap algorithms such as RSEM and STARsolo can also partially improve the detection of PVRIG. Our study provides means to better interrogate the expression of PVRIG and murine TIGIT in scRNA-seq and emphasize the importance of optimizing gene models and alignment algorithms to enable accurate gene expression measurement in scRNA-seq and bulk sequencing. Moreover, our results support detailed study of the expression of immune checkpoints in clinical and pre-clinical studies towards the development of cancer immunotherapy treatments. Citation Format: Sergey Nemzer, Niv Sabath, Assaf Wool, Zoya Altber, Hirofumi Ando, Drew M. Pardoll, Sudipto Ganguly, Yaron Turpaz, Zurit Levine, Roy Z. Granit. Gene model correction for PVRIG and TIGIT in single cell sequencing data enables accurate detection and study of its functional relevance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4292.

INTRODUCTION: Tumor mutation burden (TMB) is a commonly used biomarker for cancer immunotherapy however TMB only partially captures tumor foreignness. We hypothesized that mutations in single-copy regions of the genome or mutations present in multiple copies (hereafter referred to as persistent mutations) are retained during cancer evolution and immunoediting, may render the tumor continuously visible to the immune system and promote sustained tumor control during immune checkpoint blockade (ICB). METHODS: We performed pan-cancer analyses of whole exome sequencing data across 31 tumor types in TCGA to quantify the landscape of persistent mutations (n=9,242). We then evaluated the association between persistent tumor mutation burden (pTMB) and ICB response compared to TMB in eight ICB-treated cohorts of patients with NSCLC, melanoma, mesothelioma, and head and neck cancer (n=524). To investigate the clonal evolution of persistent mutations we serially analyzed whole exome sequence data from NSCLCs prior to and at emergence of acquired resistance to ICB. Finally, we evaluated the composition of the tumor microenvironment (TME) in baseline and on-ICB melanomas by RNA sequencing differential enrichment analyses and deconvolution. RESULTS: Integration of sequence alterations in only-copy and multi-copy states for 9,242 tumors across 31 tumor types revealed a cancer lineage-dependent distribution of persistent mutations that was largely independent of the overall TMB. In evaluating differential classification based on pTMB- vs TMB-high, we found re-classification rates as high as 53% in individual tumor types, with a median reclassification rate of 33% across all tumor types (range 15% - 53%). We then evaluated the clonal composition of persistent mutations and found a wide range of correlations between pTMB and fraction of clonal mutations (Spearman ρ range: -0.11 - 0.59). In ICB-treated cohorts, pTMB better distinguished responding tumors compared to TMB, and a number of mutation and copy-number related features including tumor aneuploidy (melanoma: Mann-Whitney p=2.3e-06, NSLC: p CONCLUSIONS: Persistent mutations represent a biologically distinct subset within the overall TMB that is unlikely to be lost under selective pressure of ICB and may function as an intrinsic driver of sustained immunologic tumor control. Citation Format: Noushin Niknafs, Archana Balan, Christopher Cherry, Karlijn Hummelink, Kim Monkhorst, Xiaoshan M. Shao, Zineb Belcaid, Kristen A. Marrone, Joseph Murray, Kellie N. Smith, Benjamin Levy, Josephine Feliciano, Christine L. Hann, Vincent Lam, Drew M. Pardoll, Rachel Karchin, Tanguy Y. Seiwert, Julie R. Brahmer, Patrick M. Forde, Victor E. Velculescu, Valsamo K. Anagnostou. Persistent mutation burden drives sustained anti-tumor immune responses in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1393.

Despite the initial successes of immunotherapy, there is an urgent clinical need for molecular assays that identify patients more likely to respond. Here, we report that ultrasensitive measures of circulating tumor DNA (ctDNA) and T-cell expansion can be used to assess responses to immune checkpoint blockade in metastatic lung cancer patients (N = 24). Patients with clinical response to therapy had a complete reduction in ctDNA levels after initiation of therapy, whereas nonresponders had no significant changes or an increase in ctDNA levels. Patients with initial response followed by acquired resistance to therapy had an initial drop followed by recrudescence in ctDNA levels. Patients without a molecular response had shorter progression-free and overall survival compared with molecular responders [5.2 vs. 14.5 and 8.4 vs. 18.7 months; HR 5.36; 95% confidence interval (CI), 1.57–18.35; P = 0.007 and HR 6.91; 95% CI, 1.37–34.97; P = 0.02, respectively], which was detected on average 8.7 weeks earlier and was more predictive of clinical benefit than CT imaging. Expansion of T cells, measured through increases of T-cell receptor productive frequencies, mirrored ctDNA reduction in response to therapy. We validated this approach in an independent cohort of patients with early-stage non–small cell lung cancer (N = 14), where the therapeutic effect was measured by pathologic assessment of residual tumor after anti-PD1 therapy. Consistent with our initial findings, early ctDNA dynamics predicted pathologic response to immune checkpoint blockade. These analyses provide an approach for rapid determination of therapeutic outcomes for patients treated with immune checkpoint inhibitors and have important implications for the development of personalized immune targeted strategies. Significance: Rapid and sensitive detection of circulating tumor DNA dynamic changes and T-cell expansion can be used to guide immune targeted therapy for patients with lung cancer. See related commentary by Zou and Meyerson, p. 1038

Introduction: Metastatic prostate cancer is currently incurable and kills more than 30,000 men in the United States each year, making it the second leading cancer related cause of death and an area of unmet need. Previously, we reported increased infiltration of immunosuppressive CD206-positive tumor associated macrophages (TAMs) with disease progression in prostate adenocarcinoma.[1] Glutamine metabolism has been implicated in immunosuppressive TAMs as well as metastatic castration resistant prostate cancer (mCRPC)[2] and radioresistant urothelial carcinoma.[3] As such, we hypothesize that rational metabolic interventions could be a promising strategy to simultaneously target both TAMs and cancer cells, resulting in anti-tumor immunity. Methods: We utilized a novel pro-drug (JHU083) derived from 6-Diazo-5-oxo-L-norleucine (DON) which has significantly lowered toxicity to treat two urological syngeneic immunogenic mouse tumor models in vivo; B6CaP (prostate cancer) and MB49 (bladder cancer). We studied the direct effect on tumor cells, as well as the required immune compartment for drug efficacy in vivo using antibody targeted depletions or adoptively transferred TAMs. Moreover, we used RNA-sequencing and multi-parameter flow cytometry to characterize the effect of JHU083 on TAMs transcriptional programming and proteomic expression profiles in vivo. Lastly, we assessed the phagocytotic capacity of macrophages after treatment with JHU083 with flow cytometry and immunofluorescence (IF) microscopy. Results: JHU083 treatment showed significant tumor regression in both urologic cancer models. Using in vivo depletion of CD4 or CD8 T cells, or adoptively transferring previously in-vivo JHU083 treated TAMs we established a direct anti-tumor role of TAMs. These TAMs were more inflammatory as shown by the increased TNF-positivity. Strikingly, in the TME we also observed an increase of Glut1 and Hexokinase II indicating metabolic reprogramming of these TAMs towards glycolytic phenotype. Importantly, JHU083-treated TAMs showed significantly increased phagocytic activity of cancer cells, providing direct evidence of functional reprogramming. Conclusion: We found that JHU083 has two distinct functions in vivo; first, it directly impairs cancer cells which are glutamine dependent and second, it reprograms TAMs from an immunosuppressive to an inflammatory state via antagonism of glutamine metabolism in vivo. These macrophages convert to a highly glycolytic state, have increased TNF production, and have improved phagocytic activity against tumor cells. As urologic cancers are heavily infiltrated with immunosuppressive TAMs, JHU083 is an excellent preclinical candidate for these diseases. It remains to be seen if JHU083 treatment can be effectively combined with checkpoint therapy to elicit durable anti-tumor immunity. Citation Format: Monali Praharaj, Fan Shen, Liang Zhao, Thomas R. Nirschl, Laura Sena, Alok K. Singh, Debebe Theodros, Xiaoxu Wang, Raekwon A. Williams, Elizabeth Thompson, Ada Tam, Srinivasan Yegnasubramanian, Robert D. Leone, Jesse Al, Rana Rais, Barbara S. Slusher, Drew M. Pardoll, Jonathan D. Powell, Jelani C. Zarif. Glutamine blockade via prodrug JHU083 reprograms immunosuppressive tumor associated macrophages (TAMs) and drives tumor immunity in urologic cancers [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 2115.

Background: Multidimensional, spatially resolved analyses of immune and tumor cells within the TME of patients treated with checkpoint inhibitors will provide clinically translatable mechanistic insights and potentiate biomarker discovery. To achieve this goal, information from pathology specimens needs to be captured at a single cell level with high fidelity and in meaningfully sized cohorts. To date, efforts have been limited by inadequate tissue sampling and previously unrecognized errors in staining, imaging and data analysis. Here we describe the ‘AstroPath' platform, where strategies from the field of astronomy were adapted to study pathology specimens and generate large high quality mIF data. Methods: Potential error was identified and addressed at each stage of 6-plex (PD-1, PD-L1, FoxP3, CD163, CD8, tumor marker) mIF assay development. Whole slides from formalin-fixed paraffin embedded tissue specimens were stained with the optimized assay and imaged using a multispectral microscope (Vectra 3.0) with 20% overlap of high power fields (HPFs). The overlaps were used to quantify and correct optical lens distortion, HPF alignment, and illumination variation. Errors from cell segmentation algorithms, batch-to-batch staining variation, and HPF sampling were also addressed. The resultant mIF data were organized and analyzed using a large, relational database. Results: The optimized mIF assay captured equivalent signal compared to gold standard chromogenic immunohistochemistry and 2x more signal for PD-1, PD-L1 and FoxP3 compared to the manufacturer's recommended protocol. Errors and corrections for imaging included: pixel alignment error reduced from ~10 to Conclusion: Here we present an end-to-end pathology workflow with rigorous quality control for creating quantitative, spatially resolved mIF datasets using lessons derived from the field of astronomy. Such approaches will vastly improve standardization and scalability of mIF technologies, enabling cross-site comparisons and eventual clinical translation as biomarker discovery platforms or standard diagnostic tests. Citation Format: Sneha Berry, Nicolas Giraldo, Benjamin Green, Elizabeth Engle, Haiying Xu, Aleksandra Ogurtsova, Daphne Wang, Julie E. Stein, Peter Nguyen, Suzanne Topalian, Angelo DeMarzo, Drew M. Pardoll, Robert A. Anders, Tricia R. Cottrell, Alexander S. Szalay, Janis M. Taube. The ‘AstroPath' platform for spatially resolved, single cell analysis of the tumor microenvironment (TME) using multispectral immunofluorescence (mIF) [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 6584.

Castration resistant prostate cancer (CRPC) first manifests as a sustained rise in the androgen-responsive gene, PSA, consistent with reactivation of a functioning androgen receptor (AR) axis. This observation led to the development of “second-line” therapies aimed at further blocking androgen/AR signaling. Unfortunately, resistance to these agents can also develop quickly. Paradoxically, several studies have suggested that the growth of AR-positive human CRPC cell lines may be inhibited by supraphysiologic levels of testosterone (SupT). These studies suggested that the adaptive reliance on AR signaling by CRPC cells becomes a therapeutic liability that can be exploited through the administration of SupT, which we termed as bipolar androgen therapy (BAT). Understanding how BAT works at the molecular and cellular levels might help in rationally combining BAT with other agents to achieve increased efficacy and tumor responses. Our data indicates that SupT induces autophagy mediated degradation of ferritin in prostate cancer (PCa) cells. Degradation of ferritin results in increase in labile iron pool increasing lipid peroxidation. Our data further indicates that SupT distinctly induces ferroptosis, a nonapoptotic regulated form of cell death induced by the accumulation of labile iron. Ferroptosis is thought to have tumor suppressing capabilities by clearing tumor cells via immune system activation. BAT has been discussed as a potential therapy for prostate cancer, but further research is needed to understand its full potential. Future combination of BAT with existing immunotherapeutics including immune checkpoint blockade may prove beneficial for treatment of CRPC. Citation Format: Rajendra Kumar, Kavya Boyapati, Naiju Thomas, Deven Topiwala, Suthicha Kanacharoen, Olutosin Owoyemi, Max Coffey, Michael Carducci, Mark C. Markowski, Emmanuel S. Antonarakis, Drew Pardoll, Samuel Denmeade, Sushant K. Kachhap. Supraphysiological androgens induce ferroptotic cell death in prostate cancer cells [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 2410.

While immune checkpoint inhibitors (ICIs) have revolutionized the treatment of many cancers by producing durable antitumor responses, only 10-30% of treated patients respond and the ability to predict response to treatment remains elusive. Preliminary studies suggest the gut microbiome may be a novel biomarker for tumor response rates, including high alpha-diversity and a few specific bacterial species that associate with improved tumor responses to ICIs in melanoma, renal cell cancer (RCC), and non-small cell lung cancer (NSCLC). Despite these reports, the specific bacteria or bacterial communities helpful or harmful to ICI responses have been inconsistent across study populations and various malignancies, and further correlation with immune and mutational biomarkers is limited or lacking. We hypothesized that, by use of a larger sample size and a consistent computational approach, we would derive a clearer microbial profile that correlated with immunotherapeutic outcomes in more than one cancer type and in response to ICIs that target different immune checkpoints. To test this hypothesis, we have reanalyzed the available raw 16S rRNA amplicon and metagenomic sequencing data across five recently published studies (n=303) using Resphera Insight v2.2 and MetaPhlAn2, respectively, for taxonomic assignment. We used pathway prediction algorithms (PICRUSt) to examine functional characteristics enriched among responders and nonresponders, as well as effect of antibiotic usage and virulence factors using multiple reference databases. Our results confirm signals reported in each study, though some bacteria reported initially were not statistically significant after correction for false discovery rate. Likely, in part, because our analysis allows for comparison of individual species across cohorts, we were able to identify new bacterial signatures associated with clinical response or nonresponse. Further, these new results enabled us to re-evaluate and develop response and nonresponse indicator indexes. When our composite index was compared to an index assembled from the literature, some improvement occurred in a sensitivity and specificity analysis. Moreover, while lower alpha-diversity has been associated with disease states and higher alpha-diversity with healthy states, we found that alpha-diversity was not consistently predictive of response or nonresponse to ICIs. In summary, this bioinformatics platform improves on existing pipelines by standardizing critical preprocessing and downstream analysis tools, enabling comprehensive evaluations of taxonomic and functional signals across sequencing datasets. These analyses allow for identification of novel bacterial signatures associated with clinical responses that could potentially be used as biomarkers in patients undergoing treatment with checkpoint inhibitors. Results from these analyses will be validated in subsequent analyses of ICI therapy and clinical outcomes in our ongoing prospective cohorts. Citation Format: Fyza T. Shaikh, James R. White, Joell G. Gills, Jarushka Naidoo, Evan Lipson, Drew M. Pardoll, Cynthia L. Sears. Meta-analysis improves identification of microbiome associations with antitumor response in melanoma, lung, and kidney cancer patients treated with checkpoint inhibitors [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A14.

Although immune checkpoint inhibitors (ICIs) have shown promise in treating various cancers, fewer than half of patients with most tumor types experience a durable response. Thus, there is a need for biomarkers to better predict outcomes. Recent studies suggest that the presence of a handful of microbial species and greater alpha-diversity in the gut may serve as a biomarker for and might facilitate ICI responses. However, the specific bacteria, or bacterial communities, that associate with improved ICI responses vary across study populations, and the factors that contribute to these discrepant findings remain elusive. Thus, a standardized method by which biospecimens may be collected, transported, and stored for gut microbiome studies in the context of ICI therapy is needed. In this study, we evaluated a method for shipping fecal samples using a low-cost (1 year of durable tumor response after ICI therapy. Patients were provided with stool collection kits and either asked to collect fecal samples at home within 48 hours of their clinic visit and store at 4°C (fresh) or asked to place stool in preservative and ship at ambient temperature to our laboratory (fixed). For fresh samples, a portion of each sample was frozen and another portion placed into preservative as a paired control. DNA was extracted using Zymo Quick-DNA™ Fecal/Soil Microbe kit. For fixed samples, methanol was removed by evaporation prior to DNA extraction. Microbial composition was analyzed with 16S rRNA amplicon sequencing with V1-V2 primers with 150bp paired-end sequencing using an Illumina platform. Among n=10 samples collected in clinic, fixed portions demonstrated decreased alpha-diversity and a uniform shift in beta-diversity compared to the paired frozen portions. In all fixed samples, Faecalibacterium and Roseburia relative abundance decreased with a corresponding increase in Bacteroides. Among a second set of n=11 samples that were placed in preservative by patients and then shipped to our laboratory, we analyzed the effects of shipping by comparing fresh stool samples and shipped fixed samples collected by the same patient within one month. The data revealed similar trends, suggesting that fixation, rather than shipping, drives the overall effect. We are currently verifying the relative abundance of specific bacterial species in both sets of samples using quantitative RT-PCR. Our data show that methanol-based preservation must be optimized prior to clinical utilization for accurate assessment. If optimized, we have identified a low-cost method to collect fecal samples that could be adopted in community practices and low-income areas. Ongoing work from our group includes optimization of processing procedures, ratio of fecal matter to preservative, and storage conditions. Citation Format: Kimberly E. Peloza, Joell J. Gills, Fyza Y. Shaikh, James R. White, Sara Glass, Carisse Lansiquot, Courtney Stevens, William Assan, William H. Sharfman, Dung T. Le, Jarushka Naidoo, Evan J. Lipson, Drew M. Pardoll, Cynthia L. Sears. Development of a low-cost method for collecting fecal samples in clinical trials [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B13.

The tumor immune microenvironment (TiME) of mismatch-repair deficient (MMRd) colorectal cancer (CRC) is characterized by a cytotoxic T cell immune signature and counter-expression of IFNγ -inducible T cell checkpoints, features underlining intratumoral adaptive immunosuppression and sensitivity to immune checkpoint blockade. Since single-agent checkpoint inhibitors have not demonstrated, so far, similar meaningful clinical activity for MMR proficient (MMRp) CRC, we compared the tumor immune microenvironment (TiME) in CRC patients that responded to anti-PD1 therapy with the TiME of patients that did not (NCT01876511) to identify immune signatures and biomarkers that could help deciding on combinatorial immunotherapies to treat patients with MMRp CRC. With this approach, we detected immunoreactive MMRp (irMMRp) tumors characterized by a dense infiltration with cytotoxic T cells (CTL) as well as high expression of IFNγ and CD274 (PD-L1), despite their low tumor mutation burden. We observed that these irMMRp colon tumors are characterized by a negative association between Indolamine 2,3 dioxygenase 1 (IDO1) expression and Th17 infiltration. Despite the high expression of CD8 and PD-L1 expression, only irMMRp CRC with high expression of IDO1 on tumor cells and low infiltration with Th17 cells have a TiME resembling the TiME of CRC responding to anti-PD1. Since IDO1-derived tryptophan metabolites, kynurenines, are known to repress Th17 differentiation, these results suggested that the use of IDO1 small molecule inhibitors may derepress Th17 infiltration in CRC and blunt the effect of anti-PD1. Combining therapy with IDO1/PD1 inhibitors with drugs inhibiting IL-17 signaling pathway may help to treat irMMRp CRC patients. Citation Format: Nicolas Llosa, Anas H. Awan, Ada J. Tam, Hongni Fan, Kellie N. Smith, Cynthia L. Sears, Hao Wang, Drew M. Pardoll, Robert A. Anders, Franck Housseau. Mismatch repair proficient colorectal cancer and adaptive immunosuppression of endogenous anti-tumor immune response: Implications for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2793.

Artificial intelligence is poised to revolutionize every aspect of human life, finding applications in everything from self-driving cars to diagnosing cancer. In fact, almost any task that involves pattern recognition can be formulated in a way that modern AI algorithms can be used to achieve super-human performance. The immune synapse is a highly complex interaction between several proteins and peptides that allows for a constant surveillance of foreign invaders. However, modeling these interactions is extremely difficult as the combinations of interactions is simply intractable. In immuno-oncology, the study of this interaction is crucial as anti-tumor responses rely on sensitive and specific recognition of tumor-specific antigens. Implications of accurately predicting and modeling these interactions in immune-oncology range from improved and potent vaccine design to biomarkers for predicting response to immunotherapy. Our group has developed a variety of deep learning models to model the signal transmission within the immune synapse. At the core of all architectures designed, convolutional layers, similar to ones used to learn features in images, are used to learn motifs within the sequencing data for a predictive or descriptive purpose.We first present AI-MHC, an applied deep convolutional neural network for class-specific MHC binding algorithm that achieves state-of-the-art performance in both Class and Class II predictions. By incorporating ‘meaning’ of the allele within the network, we are able to model the interaction of allele and peptide within the context of a neural network. We take these concepts further in the development of DeepMANA, a deep learning framework which combines sequence-specific information about an allele/peptide pairing to not only predict binding affinity for any allele with a known protein sequence but also provide an antigen ‘quality’ score, based on the “non-self/foreign-ness” of a neoantigen. We observe in three previously published immunotherapy clinical trials, these quality neoantigens are enriched in long-term survivors or responders. Finally, we present DeepTCR, a collection of unsupervised and supervised deep learning algorithms capable of revealing structure in T-cell receptor repertoire. We demonstrate that DeepTCR achieves state-of-the-art performance in clustering antigen-specific TCR’s and is capable of learning a predictive signature in TIL repertoire of mice treated with various immunotherapies.These types of AI technologies could yield an entire new area of biomarker discovery as well as improve our understanding of the complex interaction occurring at the immune synapse that is ultimately required for a successful anti-tumor response. Citation Format: John-William Sidhom, Drew M. Pardoll, Alexander S. Baras. Deep learning of the immune synapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4444.

Background: Patients with metastatic osteosarcoma (OS) have a 5 year overall survival of Methods: Immunohistochemistry (IHC) slides from 66 formalin-fixed paraffin-embedded OS tissue blocks were analyzed. Laser capture microdissection and RNA extraction was performed on 13 OS specimens, and gene expression profiles of the tumor interior vs. tumor interface region were compared utilizing RNA seq. Tumor infiltrating lymphocytes (TILs) were isolated from 25 freshly obtained OS specimens and analyzed by multiparameter flow cytometry (MFC). Results: Digital image analysis of IHC specimens revealed significantly higher immune cell infiltration (CD3+ and CD8+ cells) in the pulmonary metastases compared to primary bone tumors, particularly at the interface between the OS lesion and normal lung tissue. There is increased expression at the interface of the immune checkpoint molecules programmed cell death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3) and lymphocyte activation gene 3 (LAG-3). Gene expression profiling showed increased CD8 T cell and resting CD4 memory T cell signature at the interface compared to the tumor interior, and a strong M2 and M0 macrophage signature in both regions. TILs isolated from pulmonary metastases and analyzed by MFC had higher expression of PD-1 and other immune checkpoint molecules, and these TILs were more capable of producing the effector molecules IFN-gamma and granzyme B. Conclusions: Pulmonary metastatic OS lesions are more highly infiltrated with immune cells than primary bone lesions, particularly at the interface. The fact that these immune cells are shown via MFC to be capable of producing cytotoxic cytokines and proteases suggests that these TILs may be tumor-specific lymphocytes capable of acting against the tumor if they were not being inhibited by multiple immune checkpoint molecules. The inability of these immune cells to penetrate further into the tumor interior may represent an adaptive immune response by the tumor through a combination of repellant cytokines and inhibition of TILs via upregulation of PD-1, TIM-3 and LAG-3. Treatment with a combination of anti-PD-1, anti-TIM-3 or anti-LAG-3 directed treatments may unleash these immune cells and allow them to destroy OS tumor cells. Citation Format: John A. Ligon, Teniola F. Oke, Woonyoung Choi, Megan H. Fong, Adam Levin, Daniel S. Rhee, Carol D. Morris, Nicholas Siegel, Emily H. Hsieu, Christian F. Meyer, David J. McConkey, Robert A. Anders, Drew M. Pardoll, Nicolas J. Llosa. The immunosuppressive tumor microenvironment of metastatic osteosarcoma inhibits the cytotoxic effect of tumor-infiltrating lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4973.

Multiple classes of pharmacologic agents have the potential to induce the expression and release of proinflammatory factors from dying tumor cells. As a result, these cells can in theory elicit an immune response through various defined mechanisms to permanently eradicate disseminated cancer. However, the impact of chemotherapy on the tumor-specific immune response in the context of the tumor microenvironment is largely unknown. Within the tumor microenvironment, the immune response promoted by chemotherapy is antagonized by an immune-suppressive milieu, and the balance of these opposing forces dictates the clinical course of disease. Here, we report that high antigen exposure within the tumor microenvironment following chemotherapy is sufficient to skew this balance in favor of a productive immune response. In elevating antigen exposure, chemotherapy can achieve long-term control of tumor progression without the need of an additional adjuvant. We found that chemotherapy initiated this phenomenon in the tumor microenvironment through an accumulation of dendritic cells, which stimulated CD8+ T cells and the type I IFN pathway. From this conceptual base, we developed a simple approach to cancer therapy combining chemotherapy and vaccination that may be widely applicable. Cancer Res; 73(8); 2493–504. ©2013 AACR.

Both STAT3 and NF-κB are persistently activated in diverse cancers and promote tumor cell proliferation, survival, angiogenesis, and metastasis through transcriptional activation of multiple common genes. Paradoxically, STAT3 also suppresses many NF-κB–inducible genes involved in innate and adaptive antitumor immunity in spite of elevated levels of NF-κB in tumors. In this study, we show that expression of many NF-κB downstream target genes in tumors depends on STAT3 DNA binding. When STAT3 is elevated in tumor cells and tumor-infiltrating immune cells, persistently activated NF-κB interacts with STAT3 and preferentially binds to genes with STAT3-binding site(s) in promoters. A large number of NF-κB downstream genes associated with oncogenesis and chronic inflammation contain STAT3 DNA-binding site(s). However, in contrast, many genes frequently associated with antitumor immunity lack STAT3 DNA-binding site(s) and can only be activated by NF-κB when STAT3 is inhibited in tumors. The introduction of STAT3 DNA-binding sequences by site-specific mutagenesis in an immunostimulatory gene promoter allows its transcriptional activation by NF-κB in tumor cells. Furthermore, STAT3 facilitates NF-κB binding to genes that are important for tumor growth while inhibiting its binding to Th-1 immunostimulatory genes in growing tumors, including in tumor-infiltrating immune cells. The results of this study provide insight into how some of the oncogenic/inflammatory and Th-1 immunostimulatory genes are differentially regulated in cancer. Cancer Res; 71(11); 3772–80. ©2011 AACR.

Inflammatory conditions elicited by extrinsic environmental factors promote malignant cell transformation, tumor growth, and metastasis. Although most attention has been focused on innate immune mechanisms of inflammatory carcinogenesis, more recently the role of T cells in cancer promotion has been examined. Although IFN-dependent Th1 responses that promote Stat1 signaling inhibit tumor growth, the role of T helper type 17 responses, and interleukin-17 (IL-17) in particular, has been controversial. Indeed, IL-17 has been reported to either enhance or inhibit the growth of transplantable tumors, depending on the system. Little is known about the role of IL-17 in de novo carcinogenesis. Using IL-17 knockout mice, we examined the role of IL-17 in the classic DMBA/TPA-induced skin carcinogenesis model. Disruption of IL-17 dramatically reduced tumorigenesis in this model in a manner correlated with diminished Stat3 activation in the tumor microenvironment. IL-17 loss reduced Stat3-associated proliferative and antiapoptotic gene expression along with epidermal cell proliferation and hyperplasia. In addition, IL-17 loss was associated with reduced expression of Stat3-regulated chemokines that attract myeloid cells and a decreased infiltration of myeloid cells into the local tumor microenvironment. Together, our findings point to a critical role of the IL-17–Stat3 pathway in supporting cancer-associated inflammation in the tumor microenvironment. Therapeutic approaches that target this pathway may therefore be effective to inhibit carcinogenesis. Cancer Res; 70(24); 10112–20. ©2010 AACR.

Paracrine cross-talk between tumor cells and immune cells within the tumor microenvironment underlies local mechanisms of immune evasion. Signal transducer and activator of transcription 3 (STAT3), which is constitutively activated in diverse cancer types, is a key regulator of cytokine and chemokine expression in murine tumors, resulting in suppression of both innate and adaptive antitumor immunity. However, the immunologic effects of STAT3 activation in human cancers have not been studied in detail. To investigate how STAT3 activity in human head and neck squamous cell carcinoma (HNSCC) might alter the tumor microenvironment to enable immune escape, we used small interfering RNA and small-molecule inhibitors to suppress STAT3 activity. STAT3 inhibition in multiple primary and established human squamous carcinoma lines resulted in enhanced expression and secretion of both proinflammatory cytokines and chemokines. Although conditioned medium containing supernatants from human HNSCC inhibited lipopolysaccharide-induced dendritic cell activation in vitro, supernatants from STAT3-silenced tumor cells reversed this immune evasion mechanism. Moreover, supernatants from STAT3-silenced tumor cells were able to stimulate the migratory behavior of lymphocytes from human peripheral blood in vitro. These results show the importance of STAT3 activation in regulating the immunomodulatory mediators by human tumors and further validate STAT3 as a promising target for therapeutic intervention. Cancer Res; 70(16); 6467–76. ©2010 AACR.

Introduction: Angiogenesis plays an important role in the malignancy of glioblastoma (GBM) as well as in cancer immunotherapy. In addition, PD-1 checkpoint receptors are upregulated in GBM. We investigate the treatment effects of combination immunotherapy with checkpoint inhibitor anti-PD-1 and anti-CXCR4, an antagonist of a chemokine receptor involved in immune cell homing as well as vasculature development, in a murine glioma model. Methods: C57Bl/6 mice were implanted with GL261-Luc+ glioma cells and randomized into 4 treatment arms: 1) control, 2) anti-PD-1 monotherapy, 3) anti-CXCR4 monotherapy, and 4) combination anti-PD-1 and anti-CXCR4 therapy. Overall survival and median survival were assessed. Brain samples from untreated mice were stained for CXCR4 expression on plasma membrane, and immunohistochemistry studies were conducted on human glioblastoma specimens as well. Immune cell activation and cell population changes were assessed through flow cytometry analysis. Results: Both murine and human glioma specimens demonstrated robust positive expression of CXCR4 on tumor-infiltrating immune cells and endothelial cells of vasculature specific to the tumor bed. Combination therapy with anti-PD-1 and anti-CXCR4 conferred survival benefit compared to control and both monotherapy arms. Long-term survivors that had received combination therapy demonstrate immune memory and decreased populations of immunosuppressive, tumor-promoting immune cells. For instance, the monocytic myeloid-derived suppressor cell population was decreased in the brain in mice treated with combination therapy. The pattern of change in microglia was similar in the brain compartment for the combination treatment group as well. Conclusion: Anti-CXCR4 and anti-PD-1 synergistic immunotherapy not only modulates the immune cell make-up of the glioma microenvironment but also affects vasculature within the tumor region. Dual therapy targeting both checkpoint and chemokine receptors results in improved survival rates. Citation Format: Adela Wu, Pina Cardarelli, Miho Oyasu, Daniel Menezes, Paul Ponath, John Cogswell, Russell Maxwell, Andrew Luksik, Alice Hung, Eileen Kim, Zineb Belcaid, Henry Brem, Drew Pardoll, Michael Lim. The combination of CXCR4 and checkpoint receptor inhibition improves survival in an orthotopic murine glioma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1736.

Nasopharyngeal carcinoma (NPC) is an EBV-driven tumor that shows variable expression of PD-L1 and ~20% objective response rate to anti-PD-1 monotherapy. As novel immune checkpoint inhibitors are being developed, combination therapies may allow for more effective treatment of both newly diagnosed and relapsed NPC. We characterized the TME in 13 cases of EBV+ NPC from the Johns Hopkins Pathology archives (7 primary tumors, 6 metastases). EBV status was confirmed with EBER ISH. Immunohistochemistry (IHC) was conducted on all cases for CD3, CD4, CD8, CD20, FoxP3, PD-1, PD-L1, LAG-3, TIM-3, GITR, IDO, COX2, and pSTAT3. Gene expression profiling (GEP) was performed on 7 cases with sufficient material (4 primary lesions, 3 metastases), using multiplex qRT-PCR for a panel of 61 candidate immune-related genes (Duffield, Blood Advances 2017). The immunosuppressive ligand PD-L1 was expressed on tumor cells in 11/13 cases (mean 22% tumor cells+, range 0-57%), as well as on infiltrating macrophages. The NPC inflammatory infiltrate was diverse, including CD4+, CD8+, CD20+ and CD68+ cells, and showed variable expression of immune-regulatory molecules. In all 13 cases, lymphocytes expressing PD-1 (mean 36% positive, range 8-70%), LAG-3 (7%; 1-30%) and GITR (12%; 2-27%) were found. FoxP3+ and TIM-3+ lymphocytes were infrequent. IDO+ macrophages were also infrequent; however, 7/13 NPCs showed expression of the immunosuppressive metabolic enzyme IDO by a proportion of tumor cells. Compared to 12 EBV+ Hodgkin lymphomas (Duffield, Blood Advances 2017), EBV+ NPCs demonstrated a Th17 cytokine profile with overexpression of IL1A, IL17RC, IL23A, and IL23R. The generation of pathogenic Th17 responses requires phosphorylation of the STAT3 transcription factor, and IHC confirmed that a subset of inflammatory cells in all NPC cases expressed pSTAT3 (mean 10%; range 1-40%). Additionally, upregulated gene expression characterizing activated macrophages was found (IDO, IL1A, IL12A, LYZ, TLR3). Of note, several molecules upregulated in the NPC TME are capable of inducing PD-L1 expression on human monocytes in vitro, including IL-1A and IL-32-gamma (Taube, Clin Cancer Res 2015; Duffield, Blood Advances 2017). Importantly PTGS2 (COX2), with known proinflammatory and immunosuppressive properties, was over-expressed in NPCs along with the downstream modulator CXCL8 (IL-8); IHC revealed COX2 expression in tumor cells but not infiltrating immune cells. In summary, NPC is characterized by markers of an immunosuppressive TME, including immune checkpoints and metabolic modulators. While these findings should be explored in a larger cohort, they have potential implications for designing combination NPC treatment regimens with anti-PD-1, which might include inhibitors of LAG-3, IDO, IL-17/-23, COX2, and/or IL-8. Funded by the Bristol-Myers Squibb International Immuno-Oncology Network and NCI R01 CA142779 Citation Format: Amy S. Duffield, Maria Libera Ascierto, Robert A. Anders, Janis M. Taube, Tracee L. McMiller, Elizabeth L. Engle, Alan K. Meeker, Alan E. Berger, Drew M. Pardoll, Richard F. Ambinder, Suzanne L. Topalian. The immunosuppressive tumor microenvironment (TME) in nasopharyngeal carcinoma: implications for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4750.

In tumor immunotherapy, regulatory T lymphocytes (Tregs) play an important role in the suppression of beneficial anti-tumor responses. In the non-immunogenic B16-F10 mouse melanoma model, tumors are found to be highly infiltrated by Tregs. DAB389IL-2 (Ontak, denileukin diftitox) is an IL-2 diphtheria toxin-related fusion protein that has been showed to deplete Tregs in both humans and mice. We treated mice with established B16F10 tumors with s-DAB389IL-2 (produced as a secreted protein from C. diphtheria) and observed a significant decrease in tumor growth rate in C57BL/6 mice. Also, s-DAB389IL-2 treatment increased the frequency of CD8+IFNγ+ T cells (TILs) in tumors and spleens. S-DAB389IL-2 treatment followed by PD-1 blockade resulted in greater tumor growth inhibition than either monotherapy alone. These data suggest that s-DAB389IL-2 plus anti-PD1 treatment can improve anti-tumor responses in non-immunogenic B16F10 melanoma and inhibit tumor growth. Additionally, we mutated a vascular leak inducing motif to construct s-DAB389IL-2 (V6A), which cause less vascular leak in vitro. S-DAB389IL-2 (V6A) inhibited tumor growth with similar efficacy to the original fusion toxin. As vascular leak is the major adverse effect caused by Ontak, DAB389IL-2 (V6A) shows promise as a better tolerated drug for further studies as a cancer immunotherapy. Citation Format: Juan Fu, Drew Pardoll, Laurene Cheung. IL-2 diphtheria toxin fusion protein with anti-PD1: in mouse B16-F10 melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-293.

There is great interest in using PD-(L)1 blockading drugs as neoadjuvant therapy for patients with resectable NSCLC. Early results demonstrated a 45% (9/20) major pathologic response (MPR) rate in patients with Stage I-III NSCLC after receiving nivolumab (NCT02259621). Major pathologic response (MPR) criteria were developed in the context of cytotoxic chemotherapy, defined as ≤10% residual viable tumor cells (RVT). The features of immune-mediated tumor regression following anti-PD-1 have yet to be described. We reviewed H&E-stained slides from resection specimens in 19 patients treated with neoadjuvant nivolumab [n=9 MPR, n=3 partial responders, n=7 non-responders (>70% RVT)] to identify histopathologic features of immune-mediated tumor regression. Specimens were assessed for immune characteristics (tumor infiltrating lymphocyte (TIL) and macrophage density, and presence/absence of, lymphoid aggregates, tertiary lymphoid structures (TLS), dense plasma cell infiltrates, neutrophils, giant cells, etc.) and non-immune features (necrosis, hemosiderin, hyalinized and proliferative fibrosis). We found that immune-mediated tumor regression is characterized by a fibroinflammatory stroma with features of (1) immune activation, including dense TIL and macrophages, TLS, and granulomas; (2) massive [tumor] cell death, including cholesterol clefts and giant cells; and (3) tissue repair, including neovascularization and proliferative fibrosis (each enriched in MPR vs. non-responders, Fisher's exact test p Citation Format: Tricia R. Cottrell, Julie E. Stein, Jamie E. Chaft, Elizabeth D. Thompson, Natasha Rekhtman, Valsamo Anagnostou, Kellie N. Smith, Amy S. Duffield, Robert A. Anders, James M. Isbell, David R. Jones, Jonathan D. Cuda, Richard Battafarano, Stephen C. Yang, Peter B. Illei, Edward Gabrielson, Frederic Askin, Moises Velez, Matthew D. Hellmann, Jennifer L. Sauter, Ludmila Danilova, Victor E. Velculescu, Jedd D. Wolchok, Suzanne L. Topalian, Julie R. Brahmer, Drew M. Pardoll, Ashley Cimino-Mathews, Patrick M. Forde, Janis M. Taube. Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small cell lung carcinoma (NSCLC): A proposal for quantitative immune-related pathologic response criteria (irPRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-154.

IFN-producing killer dendritic cells (IKDC) represent a recently discovered cell type in the immune system that possesses a number of functions contributing to innate and adaptive immunity, including production of type 1and 2 IFNs, interleukin (IL)-12, natural killing, and ultimately antigen presentation to naïve T cells. Here, we compared in vitro and in vivo responses of mouse IKDC, conventional dendritic cells (DC), and natural killer (NK) cells to murine cytomegalovirus infection and found distinct functions among these cell subsets. Upon recognition of infected fibroblasts, IKDC, as well as NK, produced high level of IFN-γ, but unlike NK, IKDC simultaneously produced IL-12p40 and up-regulated MHC class II (MHC-II) and costimulatory molecules. Using MHC-II molecule expression as a phenotypic marker to distinguish activated IKDC from activated NK, we further showed that highly purified MHC-II+ IKDC but not NK cross-present MHC class I-restricted antigens derived from MCMV-infected targets to CD8+ T cells in vitro and in vivo. Our findings emphasize the unique nature of IKDC as a killer antigen-presenting cell directly linking innate and adaptive immunity. ©2009 American Association for Cancer Research., link_to_subscribed_fulltext

The liver represents a major and frequently sole site of metastases for many types of cancer, particularly gastrointestinal cancers. We showed previously that coadministration of an engineered hepatic-targeting Listeria monocytogenes (LM) with a cancer vaccine enhanced the antitumor effect of vaccine-induced T cells selectively against hepatic metastases. Here, we show that administration of multiple doses of LM, in the absence of vaccine, generates therapeutic responses against hepatic metastases. LM treatment of mice bearing hepatic metastases induced tumor-specific CD8+ T-cell responses that were enhanced by depletion of regulatory T (Treg) cells by either anti-CD25 or cyclophosphamide treatment. Antitumor activity of LM further depended on natural killer (NK) cell activation but was inhibited by presence of a subset of NK T cells. These results show the utility of LM in the treatment of hepatic metastases even in the absence of vaccine administration and further suggest that blockade of Treg cells and NK T cells will enhance antitumor activity. [Cancer Res 2007;67(20):10058–66]

Immune escape is an important reason why the immune system cannot control tumor growth, but how escape variants emerge during immunotherapy remains poorly understood. Here, we identify a new mechanism of tumor immune escape using an in vivo selection strategy. We generated a highly immune-resistant cancer cell line (P3) by subjecting a susceptible cancer cell line (P0/TC-1) to multiple rounds of in vivo immune selection. Microarray analysis of P0 and P3 revealed that vascular cell adhesion molecule-1 (VCAM-1) is up-regulated in the P3-resistant variant. Retroviral transfer of VCAM-1 into P0 significantly increased its resistance against a vaccine-induced immune response. Analysis of tumors showed a dramatic decrease in the number of tumor-infiltrating cluster of differentiation 8+ (CD8+) T cells in the tumors expressing VCAM-1. In vitro transwell migration assays showed that VCAM-1 can promote the migration of CD8+ T cells through its interaction with the α4β1 integrin. Site-directed mutagenesis of VCAM-1 at amino acid residues required for interaction with α4β1 integrin completely abolished the immune resistance conferred by VCAM-1 in vivo. Surface staining showed that most renal cell carcinomas (RCC) express VCAM-1, whereas an RCC that responded to vaccination was VCAM-1 negative. These data provide evidence that tumor expression of VCAM-1 represents a new mechanism of immune evasion and has important implications for the development of immunotherapy for human RCC. [Cancer Res 2007;67(4):1832–41]

Background: While blockade of the CTLA4 and PD1 pathways has emerged as an effective treatment of cancer, the majority of patients do not derive long term benefit. This provides a rationale for identifying and targeting additional checkpoints. Employing our unique computational algorithms, we identified PVRIG, a new member of the B7/CD28 family. We report here the expression pattern, functional characterization, and anti-tumor activity of blocking antibodies targeting PVRIG as well as characterization of PVRIG KO mice. Materials and Methods: PVRIG is expressed by T and NK cells within the tumor microenvironment. We identified PVRL2 as its counterpart and characterized the PVRIG-PVRL2 interaction. Antibody discovery was carried out with phage display and hybridoma platforms and antibodies against the human protein were screened for their ability to enhance T-cell activity in vitro, while surrogate antibodies targeting the mouse protein were assessed in syngeneic models for effects on tumor growth. PVRIG -/- KO mice were generated and characterized including phenotyping and anti-tumor immune response. Results: PVRIG is expressed on different T cell subsets and on NK, NKT and γδ T-cells. Within T cells, memory subsets possess the highest level of PVRIG and its expression is induced upon long term activation with different stimuli. Within tumor microenvironment, PVRIG was found to be expressed on NK and CD8+ T cells in multiple cancers. A high affinity lead Ab was selected, COM701, for further clinical development and demonstrated blockade of the interaction of PVRIG with PVRL2 as well as enhancement of activation of both primary and tumor-derived effector immune cells through a PVRL2-dependent mechanism. Moreover, COM-701 showed notable enhancement of T cell function in-vitro when combined with PD1 or TIGIT Ab blockade. The lead antibody, COM-701, is currently in preclinical development. A surrogate antibody, that blocks PVRIG-PVRL2 interaction, was shown to inhibit growth of colon carcinoma and melanoma in syngeneic models upon combined treatment with anti-PDL1 antibody. Comparative analysis of PVRIG KO versus WT derived T cells revealed enhanced reactivity of PVRIG null T cells upon polyclonal activation in presence of PVRL2-Ig. Accordingly, MC38 tumors grew slower in PVRIG KO than in WT mice and ex vivo analysis pointed to the quantitative and functional differences in anticancer immunity developed in these mice. Conclusion: We describe the identification of PVRIG as a novel T cell immune checkpoint. We further demonstrate that antibody blockade of the PVRIG-PVRL2 interaction has the potential to be efficiently combined with PD1 or TIGIT blockade for enhancing anti-tumor immunity. COM-701 is a high affinity antagonistic antibody that is currently in preclinical development. Taken together, these data demonstrate the utility of targeting PVRIG in addition to other B7 family checkpoints for the treatment of cancer. Citation Format: Ofer Levy, Chris Chan, Gady Cojocaru, Spencer Liang, Eran Ophir, Sudipto Ganguly, Maya Kotturi, Tal Friedman, Benjamin Murter, Liat Dassa, Ling Leung, Shirley Greenwald, Meir Azulay, Sandeep Kumar, Zoya Alteber, Xiaoyu Pan, Andy Drake, Ran Salomon, Arthur Machlenkin, John Hunter, Zurit Levine, Drew Pardoll, Mark White. Discovery and development of COM701, a therapeutic antibody targeting the novel immune checkpoint PVRIG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 581. doi:10.1158/1538-7445.AM2017-581

Correlative studies from checkpoint inhibitor trials have indcated that better understanding of human leukocytic trafficking into the human tumor microenvironment can expedite the translation of future immune-oncologic agents. In order to directly characterize signaling pathways that can regulate human leukocytic trafficking into the tumor, we have developed a completely autologous xenotransplanation method to reconstitute the human tumor immune microenvironment in vivo. When we analyzed the TCGA database of human head and neck squamous cell carcinoma(HNSCC), we found that STAT3 signaling was associated with worse prognostic mesenchymal subtye. We silenced STAT3 signaling in the tumor compartment in these autologously reconstituted humanized mice, and we noted increased tumor infiltrating lymphocytes and slower tumor growth rate. We also used this novel agents that can alter endogenous leukocytic infiltration into the tumor. Taken together, we present a valuable method to study individialized human tumor microenvironments in vivo without confound allgeneic responses. Citation Format: Juan Fu, Rupashree Sen, David L Masica, Rachel Karchin, Drew Pardoll, Young Kim. Autologous resconstitution of human cancer and immune system in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3843. doi:10.1158/1538-7445.AM2017-3843

Tumor cells contain nonsynonymous somatic mutations that alter the amino acid sequences of the proteins encoded by the affected genes. Those alterations are foreign to the immune system and may therefore represent tumor-specific neoantigens capable of inducing antitumor immune responses. Somatic mutational and neoantigen density has recently been shown to correlate with long-term benefit from immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma, suggesting that a high density of neoepitopes stemming from somatic mutations may enhance clinical benefit from blockade of immune checkpoints that unleash endogenous responses to these mutation-associated neoantigens (MANAs). Expression of the programmed cell death ligand 1 (PD-L1) in tumors or tumor-infiltrating immune cells has been associated with responses to PD-1 blockade; however, PD-L1 expression or other immune biomarkers have not been sufficient to fully explain therapeutic outcomes. Among the patients that initially respond to PD-1 blockade, some become resistant to the therapy. Up-regulation of alternate immune checkpoints, loss of HLA haplotypes, or somatic mutations in HLA or JAK1/JAK2 genes have been proposed as mechanisms of evasion to immune recognition in some patients, but the mechanisms underlying response and acquired resistance to immune checkpoint blockade have remained elusive. To examine mechanisms of resistance to immunotherapy, we performed genome-wide sequence analysis of protein coding genes and T-cell receptor (TCR) clonotype analysis, followed by functional assays of autologous T-cell activation of patients who demonstrated initial response to immune checkpoint blockade but ultimately developed progressive disease.Of a cohort of 42 NSCLC patients treated with single-agent PD-1 or combined PD-1 and CTLA4 blockade, we identified all consecutive cases that at the time of the analysis developed acquired resistance (two subjects treated with nivolumab and two with ipilimumab and nivolumab) and where paired tumor specimens were available both before and after therapy. To examine the landscape of genomic alterations and associated neoantigens, we performed whole exome sequencing of tumors from these patients. Pretreatment and postprogression specimens were obtained from the same anatomic location or from sites in close anatomic proximity. We examined multiple immune-related parameters of peptides stemming from somatic alterations using a computational multidimensional neoantigen prediction platform. This approach allowed for identification of peptides within mutated genes that were predicted to be processed and presented by MHC class I proteins and therefore had the potential to elicit an immune response. The algorithm evaluated the binding of mutant peptides (8-11mers) to patient-specific HLA class I alleles and ranked the neoantigens according to MHC binding affinity, antigen processing, and self-similarity. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. While algorithm-based predictions of antigenicity are valuable in narrowing down the large number of peptides capable of being generated by a mutation to a set potential antigenic peptides presented by self-MHC alleles, functional T cell recognition is critical to evaluate immune responsiveness. To this end, we developed a sensitive approach for assessing T-cell response to candidate MANAs (cMANAs) that utilized next-generation sequencing of TCR-Vb CDR3 regions as a measure of T-cell clonality. To evaluate T-cell recognition of eliminated neoantigens, purified peripheral blood T cells from the patients described above were stimulated with autologous peripheral blood mononuclear cells (PBMCs) loaded with cMANA peptides in a ten-day culture system. We subsequently used TCR next-generation sequencing to assess the differential abundance of neoantigen-specific T cell clonotypes in these expanded T cell populations. In order to further investigate the importance of eliminated MANAs, we generated peptides from retained and gained MANAs and assessed their potential to elicit a MANA-specific T-cell expansion. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. These findings indicate that patient-derived T cells recognized the eliminated neoantigens and suggest that these neoantigens were relevant targets for the achievement of initial therapeutic response to checkpoint blockade. Conceptually, neoantigen loss occurs through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations. To evaluate the contribution of these mechanisms to the loss of neoantigens, we analyzed the tumors both before and after therapy using the SCHISM pipeline and incorporating mutation frequency, tumor purity, and copy number variation to infer the fraction of cells containing a specific mutation (mutation cellularity). Consistent with our hypothesis, we observed both mechanisms of neoantigen elimination: loss of truncal changes through genetic events involving chromosomal deletions and loss of heterozygosity (LOH) and loss of subclonal neoantigens either by LOH or through elimination of tumor subclones. Both truncal and subclonal changes were among the eliminated neoantigens that were functionally validated. To evaluate the impact of changes in neoantigen landscape on cytotoxic T-cell receptor repertoire, we analyzed serially collected PBMCs, prior to immunotherapy initiation, at clinical response, and at resistance. We hypothesized that loss of neoantigens would lead to a decrease in clonality of cytotoxic TCR clonotypes, thus reflecting tumor immune evasion at the time of emergence of resistance. We observed peripheral T-cell expansion of a subset of the top 100 most frequent intratumoral clones, with the most frequent clones reaching up to a 44-fold increase in abundance in the blood at the time of response, followed by a decrease to pretreatment levels at the time of resistance. As a comparison, such decreases in TCR frequencies were not observed in a NSCLC patient with durable response to PD-1 blockade and no change in intratumoral TCR frequencies was seen in a NSCLC patient with primary resistance to PD-1 blockade. Taken together, these observations suggest that TCR expansion may be both a useful measure of response to checkpoint blockade and an indicator of acquired therapeutic resistance through neoantigen loss. As immune checkpoint therapy has become standard of care for many cancer types, the development of acquired resistance is being recognized more commonly. Through our comprehensive genomic analyses, we have identified changes in the genomic landscape of tumors during immune checkpoint blockade. These analyses show that emergence of acquired resistance is associated with loss of mutations encoding for putative tumor-specific neoantigens, through both elimination of tumor subclones and chromosomal loss of truncal alterations. Using a new approach to assess neoantigen reactivity by T cells, we found that some of these eliminated mutations indeed encoded peptides recognized by T cells in the peripheral circulation of the respective patients. Given that the antitumor efficacy of checkpoint blockade likely involves release of endogenous T-cell responses to tumor antigens generated by coding mutations, our findings are consistent with a mechanism of acquired resistance to immune checkpoint blockade that involves therapy-induced immune editing of MANAs. Acquisition of somatic resistance mutations is a common mechanism of therapeutic resistance to targeted therapies. However, elimination of genomic alterations and more specifically loss of somatic mutations through subsequent genetic events is uncommon in the context of natural tumor evolution or therapeutic resistance. Elimination of mutation associated antigens by a T-cell-dependent immunoselection process has been proposed as a mechanism of cancer immunoediting in melanoma after adoptive T cell transfer; however, the evolution of neoantigen loss as an escape mechanism under the selective pressure of immune checkpoint blockade in lung cancer has not been previously studied. In addition, we examined a variety of other mechanisms that have been proposed in the development of resistance to immunotherapies. We did not observe any differences in PD-L1 expression in tumor cells between responsive and resistant tumor samples. Likewise, we evaluated known potential genomic mechanisms of immunotherapy resistance; however, there were no new genomic alterations in the CD274 gene encoding for PD-L1, PDCD1 encoding for PD-1, CTLA4, JAK1 or JAK2 genes, in HLA genes, beta 2 microglobulin, or other antigen presentation-associated genes. This work demonstrates for the first time that acquired resistance to anti-PD-1 or anti-PD1/anti-CTLA4 therapy in lung cancer can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of tumor neoantigen reactivity may mitigate the development of acquired resistance. Citation Format: Valsamo Anagnostou, Kellie N. Smith, Patrick M. Forde, Noushin Niknafs, Rohit Bhattacharya, James White, Vilmos Adleff, Jillian Phallen, Neha Wali, Carolyn Hruban, Violeta B. Guthrie, Kristen Rodgers, Jarushka Naidoo, Hyunseok Kang, William Sharfman, Christos Georgiades, Franco Verde, Peter Illei, Qing Ka Li, Edward Gabrielson, Malcolm Brock, Cynthia Zahnow, Stephen B. Baylin, Rob Scharpf, Julie R. Brahmer, Rachel Karchin, Drew M. Pardoll, Victor E. Velculescu. Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr NG01. doi:10.1158/1538-7445.AM2017-NG01

Biomarker studies have shown that expression of the T-cell coregulatory ligand PDL1 on tumor cells correlates with clinical responsiveness to the PD1 antibody nivolumab. Here, we report the findings of a preclinical cancer vaccine study demonstrating vaccine-dependent PDL1 upregulation in the tumor microenvironment. We formulated an IFNγ-inducing cancer vaccine called TEGVAX that combined GM-CSF and multiple Toll-like receptor agonists to increase the number of activated dendritic cells. Treatment of established tumors with TEGVAX retarded tumor growth in a manner associated with enhanced systemic antitumor immunity. Unexpectedly, TEGVAX also upregulated PDL1 expression in the tumor microenvironment, possibly explaining why tumors were not eliminated completely. In support of this likelihood, PDL1 upregulation in this setting relied upon IFNγ-expressing tumor-infiltrating CD4+ and CD8+ T cells and administration of a PD1-blocking antibody with TEGVAX elicited complete regression of established tumors. Taken together, our findings provide a mechanistic rationale to combine IFNγ-inducing cancer vaccines with immune checkpoint blockade. Cancer Res; 74(15); 4042–52. ©2014 AACR.

Human papillomavirus-associated head and neck squamous cell carcinomas (HPV-HNSCC) originate in the tonsils, the major lymphoid organ that orchestrates immunity to oral infections. Despite its location, the virus escapes immune elimination during malignant transformation and progression. Here, we provide evidence for the role of the PD-1:PD-L1 pathway in HPV-HNSCC immune resistance. We show membranous expression of PD-L1 in the tonsillar crypts, the site of initial HPV infection. In HPV-HNSCCs that are highly infiltrated with lymphocytes, PD-L1 expression on both tumor cells and CD68+ tumor-associated macrophages is geographically localized to sites of lymphocyte fronts, whereas the majority of CD8+ tumor-infiltrating lymphocytes express high levels of PD-1, the inhibitory PD-L1 receptor. Significant levels of mRNA for IFN-γ, a major cytokine inducer of PD-L1 expression, were found in HPV+ PD-L1(+) tumors. Our findings support the role of the PD-1:PD-L1 interaction in creating an “immune-privileged” site for initial viral infection and subsequent adaptive immune resistance once tumors are established and suggest a rationale for therapeutic blockade of this pathway in patients with HPV-HNSCC. Cancer Res; 73(6); 1733–41. ©2012 AACR.

Myeloid-derived-suppression cells (MDSC) are functionally defined to suppress T-cells in their primary means of immune evasion mechanism in cancer. However, we have discovered that MDSC can have direct, T-cell independent pro-carcinogenic effect on tumor cells. Sorted monocytic CD14+/CD11b+/HLA-DRlow MDSCs from head and neck squamous cell carcinoma (HNSCC) patients were found to increase the proliferation index of HNSCC cells. This induction of tumor proliferation was not dependent on MDSC-tumor cell contact. Exome analysis showed that these human MDSC from cancer patients expressed high levels of inflammasome complex, and supernatant from the MDSCs were found to secrete IL-1b and IL-18. When we probed the functional significance of these inflammasome complex, we found that MDSC's promotion of tumor proliferative index of the tumor was found to be Caspase-1 dependent. To test this in vivo, T-cell depleted caspase-1 null mice showed significant decrease in tumor growth rate. To confirm the importance of myeloid inflammasome signaling in carcinogenesis, we suppressed MyD88 gene in tumor cell line Cal27 and found that the ability of MDSC to promoting tumor proliferation is diminished. Taken together, our findings demonstrate that tumor infiltrating MDSCs may play a prominent role in chronic inflammation associated carcinogenesis. Citation Format: Qi Zeng, Jesse R. Qualliotine, Lee Blosser, Drew Pardoll, Young J. Kim. Caspase-1 from MDSC promote MyD88 dependent carcinogenesis that is T-cell independent. [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 3250.

Background: In previously treated MEL patients (pts), the results of an early phase I trial with NIVO monotherapy (CA209-003) demonstrated tumor responses that were durable even after treatment discontinuation (Topalian et al. J Clin Oncol 2014;32:1020). We report extended follow-up with 5-year overall survival (OS) data from this study. Methods: IPI-naïve pts (N = 107) who had received 1-5 prior systemic therapies for MEL were treated with NIVO (0.1, 0.3, 1, 3, or 10 mg/kg) every 2 weeks for ?96 weeks. Pts were followed for OS, progression-free survival (PFS), long-term safety, and response duration after discontinuing NIVO treatment. Pts began treatment in October 2008, and current data were analyzed in October 2015 with a minimum follow-up of 45 months (time from when the last pt received his or her first dose of NIVO). Results: Median age of the pts was 61 years, 67% were male, 97% had an ECOG performance status of 0 or 1, 62% had received ?2 prior systemic therapies, and 36% had elevated lactate dehydrogenase levels at baseline. In all 107 pts, the 60-month OS rate was 34% (95% confidence interval [CI]: 25-43) and median OS was 17.3 months (95% CI: 12.5-37.8) (Table). OS rates appeared to plateau at ∼48 months, although further follow-up is needed. Similar results were observed with NIVO at 3 mg/kg, the currently approved monotherapy dose (Table). At the last timepoint for tumor assessment, PFS rates at 30 months were 18.6% and 25.7% for all pts and those who received NIVO at 3 mg/kg, respectively. Conclusions: This analysis represents the longest survival follow-up of pts who received anti-PD-1 therapy in a clinical study. In this heavily pretreated population of MEL pts, these results suggest durable, long-term survival following NIVO monotherapy, with 34% of pts alive at 5 years. Characteristics of long-term survivors and updated safety data will also be presented. OS ratesNIVO 3 mg/kg (n=17)All Patients (N=107)OS rate, % (95% CI)*12-month64.7 (37.7-82.3)62.7 (52.6-71.2)24-month47.1 (23.0-68.0)48.0 (38.1-57.2)36-month41.2 (18.6-62.6)42.1 (32.4-51.4)48-month35.3 (14.5-57.0)34.8 (25.7-44.1)60-month35.3 (14.5-57.0)33.6 (24.6-42.9)Median OS, months (95% CI)20.3 (7.2-NR)17.3 (12.5-37.8)*Based on Kaplan-Meier estimates.NR, not reached. Citation Format: F. Stephen Hodi, Harriet Kluger, Mario Sznol, Richard Carvajal, Donald Lawrence, Michael Atkins, John Powderly, William Sharfman, Igor Puzanov, David Smith, Philip Leming, Evan Lipson, Janis Taube, Robert Anders, Christine Horak, Joel Jiang, David McDermott, Jeffrey Sosman, Julie Brahmer, Drew Pardoll, Suzanne Topalian. Durable, long-term survival in previously treated patients with advanced melanoma (MEL) who received nivolumab (NIVO) monotherapy in a phase I trial. [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 CT001.

Epigenetic treatment and immune checkpoint blockade are novel therapeutic approaches being investigated in NSCLC. Our previous studies showed that DNA methyltransferase inhibitor azacitidine (AZA) treatment in NSCLC cell lines led to up-regulation of genes related to the response to interferon and adaptive immune attack. We hypothesize that Interferon-γ(IFN-γ), an immune effecter made primarily by activated T cells and NK cells, has a direct tumor suppression effect in NSCLC cells, which can be enhanced by the DNA hypomethylation agent. To test this hypothesis, NSCLC cells (H838 and/or H1299) were first treated with or without 500nM AZA for 3 days, and then received IFN-γ. The samples were subsequently tested for cell viability, apoptosis, cell cycle, and gene expression assays. This preclinical study is part of the collective efforts to investigate the efficacy of combined epigenetic and immune therapy in NSCLC. IFN-γ causes moderate direct growth inhibition in H838 and H1299 cells, which was enhanced, using cell viability assays as a readout, by pretreatment with AZA. The AZA sensitizing effect lasted at least 3 weeks and was not detectable 8 weeks after stopping treatment. This timing is consistent with initial reprogramming of the cells and eventual waning of this effect. Flow cytometry studies showed that AZA enhances lung cell apoptosis induced by IFN-γ. However, no significant change in cell cycle was observed. To examine the mechanisms behind these effects on cell growth, we further determined how AZA altered IFN-γ induced gene expression in H1299 cells, first using Agilent expression arrays, and then validating specific changes in key IFN and cell death pathway genes with RT-PCR based methods. IFN-γ significantly up-regulated a group of known IFN targeted genes, including IFI27, IFITM1, ISG20, ICAM1, CCL5, CXCL10, MX1 and others, and this upregulation was further enhanced by AZA pretreatment. AZA also enhanced IFN-γ induced expression of CASP1, CASP4, TNFSF 10, and BCL2A. These AZA enhanced IFN-γ induced genes are important in tumor immune response and evasion, cell death, cytokine response and other critical cellular process. Our studies demonstrated that AZA enhanced lung cancer cell response to the immune effecter IFN-γ. This supports the clinical exploration of epigenetic “priming” and its combination with subsequent immune therapy in NSCLC treatment. It may also provide mechanistically derived biomarkers that can be used to monitor and predict epigenetic and immune response in NSCLC. Citation Format: Kai He, Xin Zhang, Ludmila Danilova, Xiaoyu Pan, Julie Brahmer, Drew Pardoll, Malcolm Brock, Stephen Baylin, James Herman, John Wrangle. Azacitidine pretreatment sensitizes NSCLC cells to interferon-γ. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5033. doi:10.1158/1538-7445.AM2015-5033

VEGFR3 plays a key role in the regulation of lymphangiogenesis in adults, and is also important for tumor angiogenesis and metastasis. In order to characterize human cancer tissues for imunohistochemical (IHC) localization and prevalence of VEGFR3 protein, we developed a robust IHC assay in our lab using a mouse monoclonal primary antibody (Millipore). To optimize the various assay parameters, we utilized high-quality VEGFR3 positive (and internal negative) control tissues (Kaposi's sarcoma, angiosarcoma, lymph node). Reagent negative controls were satisfactory. Specificity of the primary anti-VEGFR3-antibody was supported by a discrete band at the expected molecular weight on western blots using a VEGFR3 transfected cell lysate and negative control cell lines. We also demonstrated lack of specific vascular VEGFR3 staining in pre-absorption experiments with VEGFR3 (but not VEGFR1 or 2) recombinant protein, supporting selectivity of the primary antibody for VEGFR3. Using the fully optimized assay, we stained a human multi-tumor screening tissue microarray (TMA) to demonstrate full range of specific vascular VEGFR3 staining in glioblastoma and carcinomas of the colon, breast, ovary, pancreas, lung, larynx, kidney, cervix, bladder, extrahepatic cholangiocarcinoma and malignant melanoma. Specific, unequivocal VEGFR3 immunoreactivity was interpreted qualitatively (VEGFR3 positive/negative) in tumor blood and lymphatic vessels. No tumor cell staining was seen. Some colon cancer tissues in multi-tumor TMA were VEGFR3+, while others were VEGFR3-. The observed variation in VEGFR3 expression and vascular distribution on screening TMA were further evaluated in two independent cohorts of well-characterized human colorectal cancer tissues (organ-specific TMAs) by a Board-certified, subspecialty GI pathologist (AN), with overall VEGFR3 prevalence rates of 62% (36 of 58 cases) and 56% (55 of 98 cases). Using CD34 and D2-40 IHC assays from a CLIA-certified lab, we confirmed IHC localization of VEGFR3 protein both in the stromal blood vessels and lymphatics within the invasive colorectal cancer stroma. In conclusion, following well-established IHC assay development and standardization protocols and efficient workflow paradigm, we have used a technically robust IHC assay to characterize routinely processed archival human cancer tissues and have demonstrated specific VEGFR3 expression patterns, tissue localization and prevalence in human colorectal cancer tissues. Based on its performance in our hands, this assay can be used to further evaluate patterns of VEGFR3 expression in areas of tumor angiogenesis in other human cancer tissues. Data-driven hypotheses generated from such investigations will be relevant to corroborate VEGF receptor biology and emerging clinical experience with anti-VEGF/anti-VEGFR therapies. Citation Format: Timothy R. Holzer, Drew M. Nedderman, Aejaz Nasir. Robust immunohistochemical assay to characterize human cancer tissues for prevalence of vascular endothelial growth factor receptor 3 (VEGFR3). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2015-4175

Background: The PD-1/PD-L1 immune checkpoint pathway limits host immune responses to cancer in the local tumor microenvironment. Monoclonal antibodies blocking PD-1 or PD-L1 have shown promising clinical results in a variety of advanced human cancers including renal cell carcinoma (RCC). We previously reported that response to anti-PD-1 therapy correlates with PD-L1 expression by tumor cells in pre-treatment biopsies. Although 20-30% of patients with metastatic RCC respond to anti-PD-1 therapy, many patients with PD-L1+ tumors still do not respond. The current study was undertaken to understand mechanisms underlying the failure of anti-PD-1 targeted therapies in patients with PD-L1+ RCC. Methods: The specimen cohort included formalin-fixed, paraffin-embedded (FFPE) pre-treatment tumor biopsies expressing PD-L1, derived from 13 RCC patients treated with nivolumab (anti-PD-1) at a single institution [4 responders (R), 9 non-responders (NR); RECIST]. PD-L1+ specimens were defined as those having ≥5% of tumor cells with cell surface PD-L1 expression by immunohistochemistry (IHC). RNA was isolated from PD-L1+ regions on FFPE slides. Whole genome microarray profiling with cDNA-mediated Annealing, Selection, extension and Ligation (DASL) was performed. Global gene expression analysis was profiled using BRBArrayTools. Multiplex quantitative (q)RT-PCR was used to validate differential expression of genes of interest, and IHC was used to validate protein expression from select genes, in R vs. NR. Results: Whole genome analysis revealed 234 transcripts that were differentially expressed in R vs. NR (p value ≤ 0.01, fold change ≥1.5). Ingenuity Pathway Analysis (IPA) of these transcripts showed the involvement of metabolic and immune pathways as well as genes encoding oxidation stress response molecules. Multiplex qRT-PCR for a subset of 60 differentially expressed genes validated significant over-expression of genes with metabolic functions, such as drug glucuronidation (UGT1A6/A1/A3), glucose transport (SLC23A1), and mitochondrial oxidation (AKR1C3) in NR vs. R. Conversely, R were found to overexpress immune markers such as BMP1, which has been shown to positively regulate PD-L1 expression, and CCL3 involved in leukocyte migration. Conclusions: Although tumor PD-L1 expression is associated with an increased likelihood of response to anti-PD-1/PD-L1 therapy, tumor cell-intrinsic metabolism may contribute to treatment resistance in PD-L1+ patients. Our data suggest that overexpression of certain metabolic factors may contribute to the failure of PD-L1+ RCC to respond to PD-1 pathway blockade, while immune factors in the tumor immune microenvironment may contribute to success. Treatment strategies that co-target these factors may be needed to enhance responses to anti-PD-1 immunotherapy in RCC. Supported by grants from Bristol-Myers Squibb and Stand Up to Cancer Citation Format: Maria Libera Ascierto, Tracee McMiller, Alan Berger, Robert A. Anders, Chris Cheadle, Haiying Hu, Charles Drake, Drew Pardoll, Janis Taube, Suzanne L. Topalian. Transcriptional signatures associated with lack of response to anti-PD-1 therapy in patients with renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1312. doi:10.1158/1538-7445.AM2015-1312

Stimulator of Interferon Genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides, activating TBK-1/IRF-3, NF-κB, and STAT6 signaling pathways to induce robust type I interferons and proinflammatory cytokines. Cyclic dinucleotide ligands were formulated with GM-CSF-producing cellular cancer vaccines - termed STINGVAX - that demonstrated potent in vivo anti-tumor efficacy in multiple therapeutic models of established cancer. We found that rationally designed synthetic CDN derivative molecules, including one with an Rp,Rp dithio diastereomer and non-canonical c[A(2′,5′)pA(3′,5′)p] phosphate bridge structure conferred significantly enhanced therapeutic anti-tumor efficacy in multiple aggressive therapeutic models of established cancer in mice. Anti-tumor activity was STING-dependent and correlated with increased activation of dendritic cells and tumor antigen-specific CD8+ T cells. Tumors from STINGVAX treated mice demonstrated dramatic PD-L1 upregulation, which was associated with tumor infiltrating CD8+IFNγ+ T-cells. When combined with PD-1 blockade, STINGVAX induced regression of palpable, poorly immunogenic tumors that did not respond to PD-1 blockade alone. We found that the STINGVAX's anti-tumor efficacy is NK cell dependent, and demonstrated that CDN are potent activators of IFNg in NK cells. Citation Format: Young Kim, Drew Pardoll, Juan Fu, Tom Dubensky. STING agonists formulated into cancer vaccines (STINGVAX) can cure established tumor resistant to immune checkpoint blockade by activating NK cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2495. doi:10.1158/1538-7445.AM2015-2495

We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+ CTL as well as activated Th1 cells characterized by IFN-γ production and the Th1 transcription factor Tbet. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly up-regulated expression of multiple immune checkpoints, including five - PD-1, PD-L1, CTLA-4, LAG-3 and IDO - currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of CRC. Significance. The findings reported in this article are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair defective subset of CRC selectively up-regulates at least 5 checkpoint molecules that are targets of inhibitors currently being clinically tested. Citation Format: Nicolas Jose Llosa, Michael Cruise, Ada Tam, Elizabeth Wick, Elizabeth Hechenbleikner, Janis Taube, Lee Blosser, Hongni Fan, Hao Wang, Ming Zhang, Brandon Luber, Nickolas Papadopoulos, Kenneth Kinzler, Bert Vogelstein, Cynthia Sears, Robert Anders, Drew Pardoll, Franck Housseau. The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 451. doi:10.1158/1538-7445.AM2015-451

The Vascular Endothelial Growth Factor (VEGF) pathway plays a prominent role in the growth and progression of human cancers, including non-small cell lung carcinoma (NSCLC). The key mediators of VEGF signaling are a family of related receptor tyrosine kinases that include VEGFR1, VEGFR2, and VEGFR3. The relative expression levels, activity and cross-talk among these receptors may contribute to clinical response of NSCLC patients to anti-angiogenic therapies. Using a well-annotated tissue microarray (TMA) and robust immunohistochemical (IHC) assays developed, standardized and implemented in our laboratory, we comparatively evaluated expression of the three VEGFRs in archival primary NSCLC tissues (n = 97). VEGFR1 and VEGFR2 were localized both in tumor vessels and cells, while VEGFR3 was only localized in tumor vessels. VEGFR1 immunoreactivity was reported as negative/low, medium, or high, based on intensity and proportion of stained tumor cell by an experienced solid tumor immunopathologist (AN), who was blinded to clinico-pathologic details. VEGFR2 and VEGFR3 positive vessels were counted by manual assessment of each core by the same immunopathologist. For systematic comparative analysis of VEGFR data, IHC expression thresholds were selected based on the 25% and 75% quartiles around the median of the range of counts for VEGFR2 and VEGFR3: 0-2, 3-10 and >10 (VEGFR2+ vascular count) respectively; and 0-1, 2-9 and >9 (VEGFR3+ vascular count) respectively. Based on VEGFR (1,2 and3) expression levels defined above, a set of eight VEGFR staining profiles were identified: Triple VEGFR positive (n = 11, 11.3%), VEGFR1 predominant (22, 22.7%), VEGFR2 predominant (9, 9.3%), VEGFR3 predominant (3, 3.1%), VEGFR1/2 predominant (13, 13.4%), VEGFR1/3 predominant (2, 2.1%), VEGFR2/3 predominant (9, 9.3%), and triple VEGFR negative (28, 28.9%). These new data provide original insights on the tissue distribution, subcellular localization and heterogeneity of expression of VEGFRs in human NSCLC cells and stromal vessels. The proposed human NSCLC sub-classification, based on the observed differential VEGFR 1, 2, 3 expression profiles, has identified various subsets of human NSCLC, especially triple VEGFR +, triple VEGFR -, VEGFR1 predominant and VEGFR 1 / 2 predominant. These profiles are distinct from the VEGF receptor profiles that we reported in a prior study on colorectal carcinomas (Holzer, Nasir et al., AACR 2014), of which VEGFR1/2 predominant subset was 50% and triple VEGFR-negative subset was only 4%. This work suggests distinct patterns of heterogeneity of VEGF receptor profiles in human NSCLC and CRCs. These data also support further evaluation of whether the reported VEGFR profiles correlate with differential sensitivity to therapeutic VEGF/VEGFR pathway inhibition. Citation Format: Timothy R. Holzer, Angie D. Fulford, Leslie O'Neill Reising, Drew M. Nedderman, Laura E. Benjamin, Andrew E. Schade, Aejaz Nasir. Heterogeneity of vascular endothelial growth factor receptors 1, 2, and 3 in human non-small cell lung carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4157. doi:10.1158/1538-7445.AM2015-4157

The ability of cancers to evade immune surveillance and resist immunotherapy raises a fundamental question of how tumor cells survive in the presence of a competent immune system. Studies to address this question have primarily focused on mechanisms by which tumor cells avoid recognition by or induce tolerance in the immune system. However, little is known about whether cancer cells also acquire an intrinsic ability to resist killing by immune effectors. We find that cancer cells enhance their ability to withstand an attack by cytotoxic immune effector cells via acquisition of specific genetic alterations that interfere with the shared mitochondrial death signaling pathway entrained by granzyme B, IFN-γ, and Apo2 ligand/tumor necrosis factor–related apoptosis inducing ligand (Apo2L/TRAIL), three key mediators of immunologic cell–mediated cytotoxicity. We show that the coexistence of specific mitochondrial signaling defects (either deletion of Bax, overexpression of Bcl-xL, or deletion of Smac) with expression of X-linked inhibitor of apoptosis protein decreases the sensitivity of cancer cells to IFN-γ/Apo2L/TRAIL– or granzyme B–induced apoptosis, lymphocyte-mediated cytotoxicity in vitro, and adoptive cellular immunotherapy in vivo. Conversely, negating X-linked inhibitor of apoptosis protein expression or function in tumor cells with defective mitochondrial signaling enables direct activation of caspase-3/-7 by granzyme B or Apo2L/TRAIL, and restores their susceptibility to immunologic cytotoxicity. These findings identify an important mechanism by which cancers evade elimination by immune effector cells and suggest that cancer immunotherapy might be improved by concurrent strategies to alleviate or circumvent the intrinsic mitochondrial death signaling defects that help cancer cells resist immunologic cytotoxicity. (Cancer Res 2006; 66(3): 1730-9)

A phase I clinical trial with granulocyte-macrophage colony-stimulating factor tumor cell vaccines in patients with metastatic renal cell carcinoma (RCC) showed immune cell infiltration at vaccine sites and delayed-type hypersensitivity (DTH) responses to autologous tumor cells indicative of T-cell immunity. To further characterize RCC T-cell responses and identify relevant RCC-associated antigens, we did a detailed analysis of CD8+ T-cell responses in two vaccinated RCC patients who generated the greatest magnitude of DTH response and also displayed a strong clinical response to vaccination (>90% reduction in metastatic tumor volume). Three separate CD8+ T-cell lines (and subsequent derived clones) derived from patient 24 recognized distinct RCC-associated antigens. One recognized a shared HLA-A*0201-restricted antigen expressed by both renal cancer cells and normal kidney cells. This recognition pattern correlated with a positive DTH test to normal kidney cells despite no evidence of impairment of renal function by the patient's remaining kidney after vaccination. A second line recognized a shared HLA-C7-restricted antigen that was IFN-γ inducible. A third line recognized a unique HLA-A*0101-restricted RCC antigen derived from a mutated KIAA1440 gene specific to the tumor. In addition, two independent CTL lines and three clones were also generated from patient 26 and they recognized autologous tumor cells restricted through HLA-A*0205, HLA-A/B/C, and HLA-B/C. These results show that paracrine granulocyte-macrophage colony-stimulating factor tumor vaccines may generate a diverse repertoire of tumor-reactive CD8+ T-cell responses and emphasize the importance of polyvalency in the design of cancer immunotherapies.

The majority of colorectal cancers have lost/inactivated the p53 tumor suppressor gene. Using isogenic human colon cancer cells that differ only in their p53 status, we demonstrate that loss of p53 renders tumor cells relatively resistant to the topoisomerase I inhibitor, irinotecan. Whereas irinotecan-induced up-regulation of the proapoptotic proteins PUMA and Noxa requires p53, we find that irinotecan inhibits Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 and 5 (STAT3/5) signaling in both p53-proficient and p53-deficient tumor cells. We show that irinotecan inhibits JAK2-STAT3/5-dependent expression of survival proteins (Bcl-xL and XIAP) and cooperates with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) to facilitate p53-independent apoptosis of colon cancer cells. Whereas xenografts of p53-deficient colon cancer cells are relatively resistant to irinotecan compared with their p53-proficient counterparts, combined treatment with irinotecan and Apo2L/TRAIL eliminates hepatic metastases of both p53-proficient and p53-deficient cancer cells in vivo and significantly improves the survival of animals relative to treatment with either agent alone. Although the synergy between chemotherapy and Apo2L/TRAIL has been ascribed to p53, our data demonstrate that irinotecan enhances Apo2L/TRAIL-induced apoptosis of tumor cells via a distinct p53-independent mechanism involving inhibition of JAK2-STAT3/5 signaling. These findings identify a novel p53-independent channel of cross-talk between topoisomerase I inhibitors and Apo2L/TRAIL and suggest that the addition of Apo2L/TRAIL can improve the therapeutic index of irinotecan against both p53-proficient and p53-deficient colorectal cancers, including those that have metastasized to the liver.