bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A26: Towards the identification of novel tumor antigens in human lung cancer
Authors: Chiou Shin-Heng , Tseng Diane , Yang Xinbo , Reuben Alexandre , McSween Alana , Wilhelmy Julie , Wang Chunlin , Shrager Joseph , Zhang Jianjun (Jay) , Garcia K. Christopher , Mackall Crystal , Davis Mark M
Published: March 2020

Immunotherapy has changed the treatment paradigm for patients with lung cancer and other solid tumors, but little is known about the antigen specificities of the T cell residing in tumors. In order to identify novel shared tumor antigens, we have previously developed an algorithm that allows the inference of T-cell specificities based on shared motifs within the T-cell receptor (TCR) CDR3 sequences. Leveraging with a dataset of 789K CDR3 sequences from 178 HLA-typed non-small cell lung cancer (NSCLC) patients, we have found up to 40K motifs after applying stringent cutoffs. Publicly available CDR3 sequences from various tetramer databases assisted with robust assignments of unique T-cell clones from the NSCLC cohorts to known specificities. For example, we have identified and validated multiple tumor-infiltrating T-cell clones that are specific to viral antigens. Furthermore, the availability of HLA genotype results for our lung cancer cohorts facilitated prediction of motifs that are potentially “restricted” to a given HLA allele or supertype. Together with single cell-sorted T-cell clones from our Stanford NSCLC cohort (n = 5,448), we have narrowed down to 60 tumor-specific CDR3 motifs (wherein at least one Stanford sequence could be found) that are potentially restricted to the A02 allele. Within these top A02-enriched motifs, 49 candidates TCR alpha/beta pairs were identified. In an effort to find novel tumor antigens, we prioritized four top T-cell candidates and screened A02 yeast display libraries with distinct peptide length (8-11mer). Four rounds of repeated screens led to the enrichment of a few dominant mimotopes with similar sequences. Database search identified a near-perfect match to a peptide 9mer from the TMEM161A locus that is predicted to bind HLA-A02 with high affinity. Functionally, the synthetic TMEM161A peptide 9mer stimulated Jurkat cells expressing the specific TCR alpha/beta chains as well as the top mimotopes. In conclusion, our results highlight the robustness of our current pipeline for the identification of novel shared tumor antigens. The ongoing work involves further characterizing the functional state of these specific T cells within the tumor microenvironment.

Citation Format: Shin-Heng Chiou, Diane Tseng, Xinbo Yang, Alexandre Reuben, Alana McSween, Julie Wilhelmy, Chunlin Wang, Joseph Shrager, Jianjun (Jay) Zhang, K. Christopher Garcia, Crystal Mackall, Mark M Davis. Towards the identification of novel tumor antigens in human lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A26.
Abstract A27: Training an immuno-army: Exploiting immunoengineering for the treatment of glioblastoma
Authors: Ferber Shiran , Cryer Alexander M. , Gorelick Noah , Tyler Betty , Brem Henry , Langer Robert S. , Artzi Natalie
Published: March 2020

Gliomas are the most common solid tumors and the greatest cause of cancer-related deaths among children in the U.S. Treatment for this group of heterogeneous malignancies involves surgery and chemotherapy; however, tumor recurrence is inevitable. Immunotherapy is a treatment modality that can stimulate the intrinsic immune defenses of the body to eliminate tumor cells. This has been challenging for gliomas, though, due to the exclusionary anatomic barriers and the immunologically quiescent environment of the brain. This project aims to develop an injectable hydrogel patch for controlled local delivery of combination immunotherapy directly to the postsurgical formed cavity of the brain to treat residual disease and to prevent tumor recurrence. Using the hydrogel patch, we seek to overcome the delivery and immunosuppressive barriers of the brain by locally releasing, as a programmed regimen, the following immunomodulatory entities: i) C-X-C motif chemokine 10 (CXCL10; to promote T-lymphocyte recruitment), ii), a new generation of programmed death-ligand 1 (PD-L1) inhibitors (to prevent T-cell exhaustion), iii) FMS-like tyrosine kinase 3 ligand (FLT3L; to induce differentiation and expansion of dendritic cells), and iv) a stimulator of interferon genes (STING) agonist (to trigger cross-priming of CD8+ cytotoxic T cells). Our novel technology possesses the flexibility to personalize an array of antigens/adjuvants or other components of immunotherapy and enables the prevention of brain metastasis by creating an immunologically inhospitable setting for circulating tumor cells. As pediatric gliomas are notoriously resistant to treatment, our hydrogel formulation seeks to address an unmet clinical need for more effective therapeutic modalities.

Note:This abstract was not presented at the conference.

Citation Format: Shiran Ferber, Alexander M. Cryer, Noah Gorelick, Betty Tyler, Henry Brem, Robert S. Langer, Natalie Artzi. Training an immuno-army: Exploiting immunoengineering for the treatment of glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A27.
Abstract A32: Characterizing patterns of cytokine coexpression with immune checkpoint markers in CD4 and CD8 tumor-infiltrating lymphocytes
Authors: Jackson Christopher M. , Dinalankara Wikum , Choi John , Nirschl Thomas R. , Kochel Christina M. , Pant Ayush , Routkevitch Denis , Saleh Laura , Jackson Christina , Skaist Alyza M. , Gupta Anuj , Snyder Linda A. , Schaeffer Edward M. , Ross Ashley E. , Carter Ballentine , Allaf Mohamad E. , Bivalacqua Trinity J. , DeMarzo Angelo M. , Weingart Jon D. , Bettegowda Chetan , Brem Henry , Pardoll Drew M. , Marchionni Luigi , Drake Charles G. , Lim Michael
Published: March 2020

In recent years, immunotherapy has become one of the most exciting and promising avenues to cancer treatment. Treatment with immune checkpoint inhibitors has managed to produce long-term remission of solid tumors in many patients. However, patients who respond well to such treatment are often a minority; this is particularly the case with some cancers such as renal cell carcinoma, non-small cell lung cancer, and glioblastoma, where many patients either derive no benefit or only a short-term benefit. In this analysis, we examined gene expression data from RNA sequencing experiments that compared tumor-infiltrating lymphocytes (TIL) with paired circulating lymphocytes from patients with renal cell carcinoma (RCC), bladder cancer (BLCA), prostate cancer (PRAD), and glioblastoma (GBM). Our analysis helped to characterize global CD4 and CD8 TIL gene expression patterns among these four cohorts. Further, using the expression profiles for known immune checkpoint markers PD-1, TIM-3, and LAG-3 in CD8 cells, we dichotomized the patient samples into potential checkpoint inhibitor responder and nonresponder groups. This model was then used to identify other genes that are associated with CD8 TIL exhaustion, which may lead to the identification of cytokines useful in discovering specific therapeutic targets.

Citation Format: Christopher M. Jackson, Wikum Dinalankara, John Choi, Thomas R. Nirschl, Christina M. Kochel, Ayush Pant, Denis Routkevitch, Laura Saleh, Christina Jackson, Alyza M. Skaist, Anuj Gupta, Linda A. Snyder, Edward M. Schaeffer, Ashley E. Ross, Ballentine Carter, Mohamad E. Allaf, Trinity J. Bivalacqua, Angelo M. DeMarzo, Jon D. Weingart, Chetan Bettegowda, Henry Brem, Drew M. Pardoll, Luigi Marchionni, Charles G. Drake, Michael Lim. Characterizing patterns of cytokine coexpression with immune checkpoint markers in CD4 and CD8 tumor-infiltrating lymphocytes [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A32.
Abstract A37: Blockading PD1 on tumor-primed CD4 T cells instigates antitumor immunity
Authors: Hao Xingxing , Falo Louis D. , Chen Guo , Carey Cara D. , Falo Louis D. , You Zhaoyang
Published: March 2020

Despite the remarkable treatment benefits associated with interventional checkpoint PD1 blockade in cancer, most patients ultimately progress on therapy. Revealing mechanisms underlying tumors responsive to PD1 blockade is critical to identifying biomarkers for predicting responses to PD1 therapy and developing novel PD1 combined therapeutic regimens for their broad use in clinic. In a clinically reflective mouse model of spontaneous metastatic breast carcinoma, effective and durable PD1 therapy is dependent on systemic immunity including Type 1 conventional dendritic cells (cDC1: CD8Î±+DC/CD103+DC), B cells, CD4 T cells, and CD8 T cells. Increased circulating and/or tumor-infiltrating IL12+CD103+DC and IL2+CD4/CD8 T cells are associated with tumors responsive to PD1 therapy. PD1 blockade results in tumor-primed CD44+IL2+CD4 T cells in the periphery regardless of cDC1. Adoptive transfer of PD1 blockade-driven CD44+CD4 T cells ignites protective immunity against new tumors. Neutralization of IL2 post the adoptive transfer abrogates the antitumor immunity. These data suggest the prominent role of PD1 blockade on tumor-primed CD4 T cells in newly induced T cells via cDC1 in response to PD1 therapy.

Citation Format: Xingxing Hao, Louis D. Falo III, Guo Chen, Cara D. Carey, Louis D. Falo Jr., Zhaoyang You. Blockading PD1 on tumor-primed CD4 T cells instigates antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A37.
Abstract A38: Targeting the androgen receptor to increase sensitivity to checkpoint immunotherapy
Authors: Sehrawat Archana , Polesso Fanny , Hipfinger Christina , Guan Xiangnan , Xia Zheng , Graff Julie , Moran Amy
Published: March 2020

Advanced prostate cancer is the second leading cause of cancer-associated mortality in men with ~32,000 deaths each year. Castration-resistant prostate cancer has no cure and an overall survival of approximately 5 years. Strategies to block androgen receptor (AR) signaling form the backbone of prostate-cancer therapy and despite suppression of serum testosterone, intratumor concentrations of androgens can remain high. Blocking AR nuclear translocation and its transcriptional function delays disease progression, but not overall survival, in castration-resistant prostate cancer patients. In a paradigm-challenging clinical trial, anti-PD-1 blockade induced long-term durable responses in a subset of metastatic castration-resistant prostate cancer patients treated with the AR inhibitor, enzalutamide. Interestingly, T cells also express AR and their function can be directly modulated by changes in androgen availability. Given this and the success of this clinical trial, we asked if enzalutamide sensitized T cells to checkpoint immunotherapy. Using in vitro and in vivo assays, we reveal a direct effect of enzalutamide on T-cell proliferation and function. In a novel implantable mouse model of advanced prostate cancer, we highlight a requirement for androgen receptor blockade to sensitize prostate tumor-bearing animals to immunotherapy. Transcriptomic analysis of leukocytes from the tumor and draining lymph node revealed a small cohort of genes differentially regulated by enzalutamide that are consistent with increased sensitivity to checkpoint blockade. Perhaps most significant, in patients who responded to anti-PD-1 therapy, some of these same genes were upregulated prior to treatment, suggesting these could be a biomarker for immunotherapy sensitization in advanced prostate cancer patients. Taken together, our data suggest a novel mechanism targeting androgen receptor-mediated immune suppression to improve immunotherapy outcomes in advanced prostate cancer.

Note:This abstract was not presented at the conference.

Citation Format: Archana Sehrawat, Fanny Polesso, Christina Hipfinger, Xiangnan Guan, Zheng Xia, Julie Graff, Amy Moran. Targeting the androgen receptor to increase sensitivity to checkpoint immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A38.
Abstract A41: Absence of central tolerance as a sculpting mechanism of immune-checkpoint therapy
Authors: Benitez Asiel A. , Khalil-Aguero Sara , Nandakumar Anjali , Gupta Namita , Zhang Wendy , Atwal Gurinder S. , Murphy Andrew J. , Sleeman Matthew A. , Haxhinasto Sokol
Published: March 2020

Therapeutic targeting of immune checkpoint pathways, which are exploited by tumors to evade the immune system, can result in increased overall survival for some patients with specific types of cancers. The endogenous antitumor response is limited in part by the absence of tumor-reactive T cells, an inevitable consequence of thymic central tolerance mechanisms ensuring their deletion to protect against autoimmunity. In this study, we show that compared to wild-type mice, tumor rejection induced by immune checkpoint blockade is significantly enhanced in Aire-deficient mice, the epitome of central tolerance breakdown. The observed synergy in tumor rejection extended to different tumor models and was accompanied by increased numbers of activated T cells expressing high levels of genes such as Gzma, Gzmb, Perforin, and Cxcr3, and tumors contained high levels of the chemokines Cxcl9 and Cxcl10 compared to wild-type mice. Furthermore, there was an enrichment of T cells expressing markers associated with the potent antitumor tissue-resident memory T cells expressing Cd103. Consistent with Aire’s central role in T cells’ repertoire selection, single-cell TCR sequencing unveiled the expansion of several clones with high tumor reactivity. Together, these results show that a break in central tolerance in combination with immune checkpoint blockade can potently enhance antitumor immunity and serve as a model system to unmask novel antitumor therapies.

Citation Format: Asiel A. Benitez, Sara Khalil-Aguero, Anjali Nandakumar, Namita Gupta, Wendy Zhang, Gurinder S. Atwal, Andrew J. Murphy, Matthew A. Sleeman, Sokol Haxhinasto. Absence of central tolerance as a sculpting mechanism of immune-checkpoint therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A41.
Abstract A47: scRNA-seq reveals functionally distinct gd T cells in human colorectal tumors
Authors: Harmon Cathal , Zaborowski Alex , Kane Harry , Cunningham Stephen , Agudelo Leandro , Kellis Manolis , Winter Des , Lynch Lydia
Published: March 2020

Colorectal cancer is the third most common malignancy worldwide, with increasing numbers due to the spreading obesity epidemic and Western diet. Colorectal tumors are subclassed by mutational burden, caused by microsatellite instability (MSI). MSI-high tumors tend to induce robust T-cell responses and respond well to immunotherapy. However, microsatellite-stable (MSS) tumors lack high mutational burden and conventional T-cell recognition. While these tumors have therefore been considered immunologically “cold,”, MSS tumors are infiltrated by innate lymphocytes, including gd T cells, natural killer (NK) cells, and mucosal associated invariant T (MAIT) cells. We therefore focused on the innate antitumor response in these patients. Colon tumor and uninvolved tissue were sampled from 6 patients undergoing surgical resection for MSS tumors. Using the 10x genomics platform, single-cell RNAseq was performed on isolated populations of gd T cells, NK cells, MAIT cells, and conventional T cells. Single-cell analysis of these tumors revealed significant heterogeneity in all cell subsets analyzed, revealing phenotypic and functional changes not previously observed in bulk sequencing data. A subset of gdT cells adopted a wound healing phenotype in all colorectal cancer samples. These soluble factors are involved in the tissue repair response and barrier integrity, providing mitogenic signals to epithelial cells. In addition, they are associated with immune suppression and induction of regulatory T cells. In humans, this phenotype can be induced in Vd1 T cells in response to proinflammatory cytokine stimulation. In the murine gut, resident gd T cells adopt this phenotype basally and increase production in response to inflammatory cytokine production. The combination of pro-tissue growth and immune suppression, by gd T cells, likely contributes to the poor immunogenicity of MSS tumors. Depleting or converting these cells represents a novel immunotherapeutic target in colorectal cancer.

Citation Format: Cathal Harmon, Alex Zaborowski, Harry Kane, Stephen Cunningham, Leandro Agudelo, Manolis Kellis, Des Winter, Lydia Lynch. scRNA-seq reveals functionally distinct gd T cells in human colorectal tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A47.
Abstract A49: Acquired resistance of non-small cell lung cancer to EGFR-TKI: Role of AKT3
Authors: Huang Shih-Hsiang , Shih Jin-Yuan , Chen Ching-Chow
Published: March 2020

The discovery of EGFR-TKIs has had a dramatic impact on the treatment of NSCLC with EGFR activation mutation. Although they improve the overall survival, the main obstacle is the emergence of acquired resistance. Two resistant cells, HCC827/IR (IRESSA resistance) and H1975/AR (AZD9291 resistance), generated and showing “off-target resistance,” were employed to investigate the molecular and cellular characteristics of the acquired resistance to EGFR-TKIs. AKT activation (p-AKT) in both parental cells was more sensitive to EGFR-TKIs than that in resistant cells. We found that mRNA and protein of AKT3, but not AKT1 and AKT2, were upregulated in both HCC827/IR and H1975/AR cells. Immunoprecipitation of AKT3 demonstrated its contribution to AKT activation. Knockdown of AKT3 in both resistant cells decreased basal AKT activation (p-AKT). However, the insensitive inhibition of gefitinib on AKT activation (p-AKT) in HCC827/IR cells and AZD9291in H1975/AR cells remained even if AKT3 was knocked down. AKT3 protein, but not AKT1 and AKT2, was found to be overexpressed in resistant cells treated with gefitinib or AZD9291 as well as AKT3 KD, indicating the crucial role of AKT3 in acquired resistance to EGFR-TKIs. Combination of AKT inhibitors with AZD9291 showed synergistic inhibition on cell viability of H1975/AR cells. Therefore, AKT3 upregulation might be one of the mechanisms for acquired resistance to EGFR-TKIs, and it might serve as a predictive biomarker for EGFR-TKIs therapy.

Citation Format: Shih-Hsiang Huang, Jin-Yuan Shih, Ching-Chow Chen. Acquired resistance of non-small cell lung cancer to EGFR-TKI: Role of AKT3 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A49.
Abstract A71: Chitinase-3-like-1: A new immunomodulatory target in lung cancer
Authors: Akosman Bedia , Ma Bing , Kamle Suchitra , Kurtulus Dilara , Lee Chang-Min , Lee Chun , Elias Jack
Published: March 2020

One of the most exciting breakthroughs in the cancer field is the discovery of immune checkpoint molecules (ICPs) such as PD-1 and its ligands PD-L1 and PD-L2 as an immune evasion mechanism for cancer cells. Although the immunotherapeutics targeting ICPs have great success in many tumors, including lung cancer, still the response rates are very limited. Thus, understanding the underlying regulatory mechanism for these molecules is essential to develop rational and new immunotherapeutic strategies to maximize the clinical outcomes for patients. Chitinase 3-like-1, as a proinflammatory and protumorigenic molecule, has increased expression in many solid tumors including lung cancer. Circulating levels of Chi3l1 are also highly correlated with poor prognosis and decreased survival rates. However, the specific role of Chi3l1 in tumor initiation and progression and as an immunomodulator in the pulmonary tumor microenvironment has not been adequately defined. Recently we have discovered that melanoma lung metastasis was significantly reduced in Chi3l1 null mutant (Brp39−/−) mice and the mice treated with anti-Chi3l1 antibody (FRG) while it was increased in overexpressing transgenic mice (YKL-40 Tg) compared to the controls, suggesting a critical role of Chi3l1 in metastatic spread and colonization. We also showed that Chi3l1 transgene expression leads to infiltration of immune cells, particularly myeloid cells into the lung, and causes dysregulated expression of PD-L1 on the macrophages. In vitro studies by using bone marrow-derived macrophages demonstrated a blunted IFNg induced PD-L1 expressions in Brp39−/− cells compared to WT controls. Additionally, we have found that the FRG antibody has synergizing effect on anti-PD1 treatment in metastatic lung cancer model. FRG antibody treatment can also effectively inhibit tumor development and also promotes CD8+ T-cell infiltration into the tumor area compared to control treatment in primary lung cancer model (K-ras+/G12D/ P53−/−mice). Overall, our studies demonstrated that Chi3l1 plays an important role in lung cancer development or progression through regulating PD-1/PD-L1/L2 axis and T-cell infiltration. These studies also highlight a significant therapeutic potential of FRG antibody for lung cancer as a single or combinational immunotherapeutic agent.

Citation Format: Bedia Akosman, Bing Ma, Suchitra Kamle, Dilara Kurtulus, Chang-Min Lee, Chun Lee, Jack Elias. Chitinase-3-like-1: A new immunomodulatory target in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A71.
Abstract A73: Fucosylation in CD4+ T cell-mediated melanoma suppression
Authors: Lester Daniel K. , Innamarato Pasquale , Kodumudi Krithika , Watson Gregory , Mercurio Matt , Pilon-Thomas Shari , Messina Jane , McCarthy Susan , Lau Eric
Published: March 2020

While immunotherapies have had a striking efficacy in melanoma patients, significant proportions of melanoma patients exhibit poor responsiveness to immunotherapies. Further understanding of immunoregulatory mechanisms is needed to improve immunotherapies. Previously, we discovered increasing tumor fucosylation in melanomas reduces tumor growth and metastasis (Lau et al., 2015). To identify tumor-infiltrating lymphocytes (TILs) affected by L-fucose, we profiled lymphocytes from syngeneic tumors of control- or fucose-fed mice tumors. Dietary supplementation of L-fucose increased TILs by ~10-50-fold. Of total TILs, CD3+ T cells (including CD4+ and CD8+ T cells) doubled. L-fucose did not trigger tumor suppression in immune-deficient mouse melanoma models, indicating the immune system’s requirement for L-fucose-triggered suppression. Immunodepletion of CD4+ or CD8+ T cells during L-fucose treatment revealed that CD4+ T cells are crucial for tumor suppression. CD4+ T-cell depletion abrogated infiltration of NK, dendritic, and CD8+ T cells in the tumors, implicating these populations as downstream effectors of fucosylation- and CD4+ T cell-triggered tumor suppression. To identify proteins on melanomas contributing to fucosylation-triggered immune responses, we performed mass spectrometric analysis of all fucosylated proteins in melanoma and identified 2 MHC proteins, HLA-A and HLA-DRB1, as fucosylated. Knockdown of HLA- DRB1 and HLA-A in vivo revealed HLA-DRB1 is required for L-fucose-mediated tumor suppression, and notably, for recruitment of CD4+ T cells. We further investigated how fucosylation affects CD4+ T-cell biology using CD4+ T cells isolated from healthy donors, which we pharmacologically modulated fucosylation. The roles of fucosylation on tumor vs. CD4+ T cells and clinical utility/implications for melanoma patients will be discussed. Our data show how increased fucosylation drives tumor suppression through CD4+ T cells, and support the potential of dietary L-fucose to boost immunity in melanomas.

Citation Format: Daniel K. Lester, Pasquale Innamarato, Krithika Kodumudi, Gregory Watson, Matt Mercurio, Shari Pilon-Thomas, Jane Messina, Susan McCarthy, Eric Lau. Fucosylation in CD4+ T cell-mediated melanoma suppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A73.
Abstract A79: Inhibition of p53-MDM2 protein interaction reduces tumor growth in a mouse melanoma model
Authors: Ingelshed Katrine , Lianoudaki Danai , Lama Dilraj , Sohn Silke , Fritz Nicolas , Jiang Long , Wermeling Fredrik , Karlsson Mikael , Lane David P. , Sedimbi Saikiran K.
Published: March 2020

Tumor suppressor protein p53 is mutated in close to 50% of human tumors. Under steady state conditions, the two E3 ligases MDM2 and MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 has therefore been considered a therapeutic strategy. In this study, we tested p53-MDM2 protein-protein interaction inhibition using the small molecule AMG 232, which is currently being tested in phase 1b/2a clinical trials. In vitro, AMG 232 induced a significant, p53-dependent growth arrest in the mouse melanoma cell line B16-F10. Using mass spectrometry-based proteomics, we identified differential protein expression patterns following AMG 232 treatment of B16-F10 melanoma cells. In vivo, the growth of B16-F10 melanoma cells implanted in WT C57BL/6 mice was significantly reduced by AMG 232 treatment. Our data demonstrate that AMG 232 induces a p53-dependent tumor growth arrest in an immunocompetent mouse model, and we are currently testing whether AMG232 can synergize with checkpoint immunotherapy.

Citation Format: Katrine Ingelshed, Danai Lianoudaki, Dilraj Lama, Silke Sohn, Nicolas Fritz, Long Jiang, Fredrik Wermeling, Mikael Karlsson, David P. Lane, Saikiran K. Sedimbi. Inhibition of p53-MDM2 protein interaction reduces tumor growth in a mouse melanoma model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A79.
Abstract A89: Microglial galectin-3 enhances the metastatic phenotype of breast cancer cells in brain metastases
Authors: Kersch Cymon N. , Morris DreeAnna F. , Muldoon Leslie L. , Neuwelt Edward A.
Published: March 2020

Background: Breast cancer brain metastases have poor prognosis and few treatment options. When breast cancer cells invade the brain, they interact with the vasculature and resident brain cells, including microglia and astrocytes. We hypothesize that brain cells produce factors that enhance the growth and invasion of breast cancer cells in the brain.

Methods: Human breast cancer cell lines (MDA-MB231BR-HER2 and HCC1954) were inoculated intracranially in athymic rats as xenograft models of brain metastasis. Brains were analyzed for protumorigenic factor expression in the tumor microenvironment using fluorescent immunohistochemistry. In vitro assays assessed factors involved in breast cancer cell proliferation, morphology, and migration.

Results: The brain tumor xenografts showed infiltration into the perivascular space. Galectin-3 (Gal3) colocalized with reactive microglia in and around the tumors. In vitro, Gal3 increased HCC1954 cell proliferation and migration. In transwell coculture assays, BV2 microglial cells increased the migration of HCC1954 breast cancer cells >25 fold, which was prevented by cilengitide, an inhibitor of alpha(v)beta(3)-integrin cell adhesion protein. ELISA analysis demonstrated that BV2 microglia secrete Gal3 in the presence of HCC1954 cells. Gal3 is known to bind and induce clustering of alpha(v)beta(3)-integrin, which is expressed on metastatic breast cancer cells. Immunohistochemistry of clinical specimens revealed that Gal3 is expressed in/around human breast cancer brain metastases.

Conclusions: These data suggest that factors produced in the tumor microenvironment promote the growth and migration of breast cancer cells in the brain. Gal3, produced and secreted by activated microglia in vitro and expressed in and around brain metastases, increases the invasive capability of breast cancer cells. The interactions of neoplastic cells with the brain environment may provide a target to improve therapy of brain metastases.

Citation Format: Cymon N. Kersch, DreeAnna F. Morris, Leslie L. Muldoon, Edward A. Neuwelt. Microglial galectin-3 enhances the metastatic phenotype of breast cancer cells in brain metastases [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A89.
Abstract A9: Systemic immune response profiling with SYLARAS implicates a role for CD45R/B220+ CD8+ T cells in glioblastoma immunology
Authors: Baker Gregory J. , Palaniappan Sucheendra K. , Moore Jodene K. , Davis Stephanie H. , Santagata Sandro , Sorger Peter K.
Published: March 2020

Research tools for the rapid and accurate assessment of bulky single-cell datasets are needed to achieve a more integrated comprehension of immune response to disease and therapy. Here we describe SYLARAS (Systemic Lymphoid Architecture Response Assessment), a modular and extensible research platform for the acquisition, statistical analysis, and visual display of multi-organ immune response data. Leveraging our technology against a syngeneic mouse model of glioblastoma (GBM), we reveal the tumor’s influence on immune cell subsets hitherto undescribed in the disease, including CD45R/B220+ CD8+ T cells characterized by their ability to constrain immune response to autologous antigens. Ancillary studies show that these cells are transcriptionally and morphologically distinguishable from conventional CD8+ T lymphocytes and infiltrate the brain tumor microenvironment of rodents and humans. The ability of SYLARAS to provide ample content on the cell and molecular mechanisms governing systemic immune response makes it a broadly useful preclinical research tool in the era of immunotherapy.

Citation Format: Gregory J. Baker, Sucheendra K. Palaniappan, Jodene K. Moore, Stephanie H. Davis, Sandro Santagata, Peter K. Sorger. Systemic immune response profiling with SYLARAS implicates a role for CD45R/B220+ CD8+ T cells in glioblastoma immunology [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A9.
Abstract B10: Novel CRISPR/Cas9 vectors for in vivo tumor functional genomics
Authors: Dubrot Juan , Manguso Robert T. , Lane-Reticker Sarah Kate , Ayer Austin , Kessler Emily , Wolfe Clara , Mahapatra Animesh
Published: March 2020

During the last decade, there has been notable progress in cancer immunotherapy, including promising clinical success of immune checkpoint inhibitors. Moreover, there is an increasing number of new potential targets for cancer immunotherapy that are currently being developed both as monotherapy or in combination. However, the lack of durable clinical responses due in part to the resistance mechanisms that tumors exhibit in a significant proportion of patients urges novel approaches to find the right therapeutic strategies. The understanding of such tumor evasion mechanisms and the complex interactions that take place in the tumor microenvironment is essential to obtain successful treatments. Functional genomics has emerged as a powerful tool that can help to reveal some of these unknown processes, with CRISPR-Cas9 technology the most prominent example. However, in vivo genetic screens require stable expression of several components that in most cases are not derived from murine cells, which represents a major obstacle in the context of cancer immunology. The inoculation of engineered tumor cell lines in immunocompetent mice results in either tumor rejection or an aberrant response to immunotherapy, making the experiments technically impossible in the first case or providing inconsistent results in the latter. We have designed new vector strategies to overcome the altered immunogenicity of Cas9-expressing tumor cell lines using the Cre-lox system. Expression and effective removal of Cas9 and vector components are monitored using fluorescent reporter proteins. Both genome-editing capacity and normal in vivo behavior of all tested cell lines remain largely unaltered using this novel strategy. This technology represents a major advance for preclinical functional genomics, allowing studies in fully competent animals looking for better combination treatments and resistance mechanism in particular cancer models, and ultimately will help in the design of clinical trials.

Citation Format: Juan Dubrot, Robert T. Manguso, Sarah Kate Lane-Reticker, Austin Ayer, Emily Kessler, Clara Wolfe, Animesh Mahapatra. Novel CRISPR/Cas9 vectors for in vivo tumor functional genomics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B10.
Abstract B16: Identification of tumor-specific antigens shared by induced pluripotent stem cells
Authors: Apavaloaei Anca , Hardy Marie-Pierre , Larouche Jean-David , Zhao Qingchuan , Vincent Krystel , Laverdure Jean-Philippe , Durette Chantal , Lanoix Joel , Thibault Pierre , Perreault Claude
Published: March 2020

The development of cancer immunotherapeutics is limited by the tumor-specific antigen (TSA) discovery. Cancer cells and pluripotent stem cells, i.e., embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), show a significant transcriptomic overlap. Indeed, when used as prophylactic vaccines in another study, iPSCs prevented tumor growth in mouse models of lung, breast, and skin cancer, suggesting that we could mine iPSCs for the discovery of TSAs. We use a recently developed proteogenomic approach combining RNA-sequencing and mass spectrometry to characterize the immunopeptidome (MHC class I-associated peptides) of iPSCs. Analyses of hitherto one human iPS cell line allowed us to identify 9 MHC class I-associated antigens that were not expressed by normal somatic tissues and thus are likely immunogenic. 50% of these antigens were highly expressed in different cancer types from The Cancer Genome Atlas, highlighting their potential for cancer immunotherapy. Expression of these TSAs resulted from aberrant expression of noncoding genomic regions and not from mutations, making them ideal candidates for antigen-based cancer vaccines. Our study indicates which stem cell features present in cancer cells might be harnessed for cancer immunotherapy. Moreover, we show for the first time that the immunopeptidome of iPSCs reflects the state of pluripotency. Cancer stem cells are known to drive disease relapse after treatment, while cancer circulating cells possessing pluripotent stem cell features are more efficient at producing metastases. These factors indicate that treatments targeted at stemness could improve clinical outcome, and that iPSC-informed TSAs hold promise for prophylactic and therapeutic cancer vaccines.

Citation Format: Anca Apavaloaei, Marie-Pierre Hardy, Jean-David Larouche, Qingchuan Zhao, Krystel Vincent, Jean-Philippe Laverdure, Chantal Durette, Joel Lanoix, Pierre Thibault, Claude Perreault. Identification of tumor-specific antigens shared by induced pluripotent stem cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B16.
Abstract B17: Identification of drugs that synergize with immunotherapies
Authors: Sahu Avinash D. , Wang Xiaoman , Flaherty Keith , Klomp Jan , Oubrie Arthur , Liu Shirley
Published: March 2020

Introduction: Immune checkpoint blockers (ICB) revitalize the immune system against cancer cells and have led to breakthroughs in cancer treatment and unprecedented survival benefits. However, they are ineffective in the majority of advanced cancer patients. For designing new treatments combining ICB with cancer drugs for treating late-stage cancer patients, there is an unmet need for a systematic approach to identify immunomodulatory drugs. Addressing this, we developed DeepImmune, the first in silico predictor of immunomodulatory drugs.

Methods: DeepImmune engages artificial intelligence (AI) to build a novel ICB response predictor and employs drug-induced transcriptomic changes to identify drugs that synergize with ICB and prioritize them for clinical trials. The parameters of the predictor are trained on 32,000 non-ICB patients’ data and transferred to ICB setting, where the parameters are fine-tuned on data from 1,000 ICB patients.

Results: We applied DeepImmune to 1,600 investigational drugs that explicated oxidative phosphorylation (Oxphos) inhibition as immunomodulatory. DeepImmune predicted inhibition of Oxphos in tumors would enhance antigen presentation, infiltration of CD8-T and proinflammatory macrophage, and will decrease T-regs and MDSC in tumor microenvironment. We validated these predictions in patient tumor data. Furthermore, the analysis of single-cell RNA-seq revealed that a decrease in Oxphos was associated with polarization toward proinflammatory (M1) macrophage. Drug inhibition of Oxphos in immune cold murine models (4T1 and B16F10) enhanced T-cell infiltration, decreased T-regs, and MDSC in tumors. The Oxphos inhibition-induced immune response in mouse models was accompanied by tumor regression, validating DeepImmune’s predictions.

Conclusion: Our analysis suggests that Oxphos inhibition can turn immune-cold tumor hot. Importantly, AI could be engaged to identify new immunomodulatory small molecules, which will help to rationalize immuno-oncology clinical trials and accelerate therapy optimization.

Citation Format: Avinash D. Sahu, Xiaoman Wang, Keith Flaherty, Jan Klomp, Arthur Oubrie, Shirley Liu. Identification of drugs that synergize with immunotherapies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B17.
Abstract B29: The importance of epitope spreading in the generation of effective antitumor immunity in mice treated with combination immunotherapy
Authors: Lee Karin L. , Palena Claudia , Schlom Jeffrey , Hamilton Duane H.
Published: March 2020

Recent clinical successes of PD-1/L1 and CTLA-4 blockade have demonstrated the potential of immunotherapies for the treatment of cancer. Research suggests the clinical efficacy of these checkpoint blockade therapies requires the presence of tumor-reactive T cells prior to therapy. It is thought that these tumor-reactive T cells primarily recognize nonsynonymous mutations (neoepitopes) uniquely expressed by a patient’s tumor. Unfortunately, at present the majority of patients fail to respond to checkpoint blockade, and it is hypothesized that one can improve their efficacy by combining them with vaccines targeting neoepitopes. Using the MC38 murine colon cancer model, we previously showed that an effective antitumor immunity could be generated when combining either a peptide or recombinant adenoviral-based vaccine targeting neoepitopes with three rationally selected immune-oncology agents. We utilized an IL-15 superagonist (N-803) to promote the generation and expansion of T cells and a tumor-targeted IL12 (NHS-IL12) and PD-L1 blockade to facilitate T-cell expansion and function within the tumor microenvironment. This multifaceted treatment regimen was effective at inducing tumor clearance in the majority of animals treated. In the present study, we examine the immune response in animals that fail to respond to our treatment regimen. By analyzing the spread of immunity to neoepitopes expressed by the MC38 tumor, but not contained within the vaccine, we were able to correlate treatment failures to less diversity in the antitumor immune response. To examine this observation more closely, tumor-bearing mice were treated with different combinations of neoepitope vaccine, N-803, PD-L1 blockade, and NHS-IL12, to ascertain which components of the combination therapy facilitated epitope spreading. We also examined which cell populations were able to cooperate with tumor-reactive CD8+ T cells to promote and/or inhibit epitope spread. Our observations demonstrate the importance of a diverse antitumor immune response and offers insights on how one might increase the diversity of the antitumor immune response following treatment with immune-oncology agents. These studies were performed in collaboration with NantOmics, NantBio and ImmunityBio, and EMD Serono as a part of Collaborative Research and Development Agreements (CRADA) with the NCI.

Citation Format: Karin L. Lee, Claudia Palena, Jeffrey Schlom, Duane H. Hamilton. The importance of epitope spreading in the generation of effective antitumor immunity in mice treated with combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B29.
Abstract B42: Human CD141+ dendritic cells (cDC1) are impaired in advanced melanoma patients but can be targeted to increase efficacy of anti-PD-1 checkpoint inhibitor therapy
Authors: O’Brien Liam , Lee Yoke Seng , Walpole Carina , Rojas Ingrid Leal , Masterman Kelly-Anne , Atkinson Victoria , Barbour Andrew , Radford Kristen
Published: March 2020

Dendritic cells (DCs) as professional antigen-presenting cells with unique T-cell stimulatory capacity represent a potential means by which to improve response rates towards immune checkpoint inhibitor antibodies (ICIs) such as anti-PD-1 (pembrolizumab) in melanoma. The conventional type 1 DC subset (cDC1) is indispensable for the efficacy of ICIs in animal models; however, very little is known about the role of cDC1 in human cancer patients. To address this, we developed a whole-blood assay for quantifying and characterizing human DC subsets (cDC1, cDC2 and plasmacytoid DCs [pDCs]) by flow cytometry and compared these in healthy donors and stage III and IV metastatic melanoma patients. cDC1 and pDC numbers were significantly reduced in stage IV melanoma patients compared to healthy controls. Moreover, cDC1s in melanoma patients were selectively impaired in their ability to upregulate CD83 expression after stimulation with TLR3 and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-CTLA-4 ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in nonresponding patients. To examine whether harnessing cDC1 could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with CD34+ hematopoietic stem cells from umbilical cord blood followed by transplantation of a human melanoma cell line. Treatment with anti-PD-1 in this model was ineffective at controlling tumor growth, but efficacy was enhanced by indirectly expanding and activating DCs in vivo with Flt-3 ligand (Flt3L) and TLR3 agonist polyI:C. Moreover, intratumoral injection of cDC1s, but not cDC2s, resulted in reduced tumor growth when combined with anti-PD-1 treatment. Together, these data illustrate impairments in cDC1 in advanced melanoma patients and provide rationale for harnessing them to increase immunogenicity and response rates to ICIs.

Citation Format: Liam O’Brien, Yoke Seng Lee, Carina Walpole, Ingrid Leal Rojas, Kelly-Anne Masterman, Victoria Atkinson, Andrew Barbour, Kristen Radford. Human CD141+ dendritic cells (cDC1) are impaired in advanced melanoma patients but can be targeted to increase efficacy of anti-PD-1 checkpoint inhibitor therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B42.
Abstract B47: CHIME screening identifies PTPN2 as a novel regulator of antitumor immunity
Authors: LaFleur Martin , Nguyen Thao , Coxe Matthew , Miller Brian , Yates Kathleen , Gillis Jacob , Sen Debattama , Doench John , Haining Nicholas , Sharpe Arlene
Published: March 2020

Despite the significant clinical responses observed with checkpoint blockade, there is an urgent need to identify new therapeutic targets in immune cells for combination therapies. However, the use of functional genomic approaches in immune cells to discover immunotherapy targets is limited by inefficient vector delivery or perturbation of cell states. To circumvent these limitations, we developed CHIME: CHimeric IMmune Editing, a bone marrow chimeric CRISPR-Cas9 delivery system, to rapidly evaluate gene function in innate and adaptive immune cells in vivo without prior ex vivo manipulation. This approach enables efficient deletion of genes of interest in major immune lineages without altering immune development or function. To discover novel immunotherapy targets we performed an in vivo pooled genetic screen for negative regulators of CD8+ T-cell responses to LCMV Clone 13 viral infection. We found that deletion of the phosphatase Ptpn2 enhances CD8+ T-cell responses to chronic pathogens and cancer. In models of both chronic viral infection and transplantable tumors, Ptpn2 null CD8+ T cells expand more and show increased expression of effector genes compared to wild-type cells. Consistent with this, in the LCMV Clone 13 model, Ptpn2 deletion in CD8+ T cells affects the differentiation and expansion of exhausted subpopulations by increasing IFN-I signaling. Furthermore, deletion of Ptpn2 in the immune system induces a CD8+ T cell-dependent clearance of tumors and synergizes with PD-1 immune checkpoint blockade. Our results suggest that therapeutic inhibition of Ptpn2 in immune cells may enhance CD8+ T-cell effector function and mediate antitumor immunity to improve tumor control. More generally, these findings suggest that this genetic platform can enable rapid target discovery through pooled loss-of-function screening in immune cell lineages in vivo and presents a novel target for potential immune based therapies.

Citation Format: Martin LaFleur, Thao Nguyen, Matthew Coxe, Brian Miller, Kathleen Yates, Jacob Gillis, Debattama Sen, John Doench, Nicholas Haining, Arlene Sharpe. CHIME screening identifies PTPN2 as a novel regulator of antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B47.