bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B2: Coexpression of effector T cell-associated chemokine receptors defines T-cell subsets with different roles in antitumor response and toxicity of dual checkpoint blockade
Authors: Gonzales Rodney J.M. Macedo , Harle David , Wang Ximi K. , Matschiner Alexandra , Reshef Ran
Published: March 2020

Dual checkpoint blockade has yielded impressive clinical responses in cancer treatment, but only a subset of patients responds and immune-related adverse events (irAE) are common, frequently leading to treatment discontinuation. Both CCR5 and CXCR3 expression are associated with effector T-cell trafficking and favorable outcomes in various tumors, but only CXCR3 was shown to exhibit a nonredundant role for cytotoxic T-cell trafficking across tumor vessels, while CCR5 has been implicated in recruitment of regulatory T cells to the tumor microenvironment. Here, we explored the coexpression of T cell-associated chemokine receptors in human melanoma and studied the effect of genetic and pharmacologic manipulation of CCR5 on PD-1/CTLA-4 (P1C4) blockade efficacy and toxicity observed in B16 tumor-bearing mice. We found that in human melanoma CCR5 and CXCR3 expression characterizes different T-cell subsets based on their coexpression with other chemokine receptors at the single-cell level. These subsets exhibit distinct phenotypic and functional characteristics in blood and tumor; CXCR3 indicated less differentiated T cells with greater proliferative potential while CCR5 expression was more abundant on terminal effector cells. In addition, subsets expressing only CXCR3 were increased in patients who received checkpoint blockade while subsets that express CCR5 were unaffected by the treatment. In murine melanoma, dual checkpoint inhibition using PD-1/CTLA-4 blockade significantly increased CXCR3, but not CCR5 expression on T cells. Moreover, a murine model of PD-1/CTLA-4 blockade-induced irAE showed high levels of CCR5+ T cells in toxicity target organs. When PD-1/CTLA-4 inhibitors were administered to either CCR5-KO mice or WT mice treated with the CCR5 antagonist maraviroc, there was a significant decrease in tumor growth and improved survival compared to WT mice receiving P1C4 alone. Together these results reveal distinct, potentially opposite roles for CCR5 and CXCR3 subsets specifically in the setting of dual checkpoint blockade, that can be redirected to improve efficacy and reduce toxicity observed in melanoma.

Citation Format: Rodney J.M. Macedo Gonzales, David Harle, Ximi K. Wang, Alexandra Matschiner, Ran Reshef. Coexpression of effector T cell-associated chemokine receptors defines T-cell subsets with different roles in antitumor response and toxicity of dual checkpoint blockade [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 B2.
Abstract B21: Dual immunotherapy targeting macrophages and PD-1 immune checkpoint for treatment of melanoma
Authors: Shudde Claire E. , Patel Jalpa , Rajan Robin , Shmeeda Hilary , Nguyen Dung , Gabizon Alberto A. , La-Beck Ninh M.
Published: March 2020

Blockading antibodies against PD-1/PD-L1 immune checkpoints are approved for melanoma treatment; however, only a minority of patients experience durable remission. Our previous work suggested that pegylated liposomal alendronate (PLA) targets tumor-associated macrophages, another mediator of immunosuppression, and polarized them towards an antitumor phenotype. We hypothesized that dual immunotherapy with PLA and PD-1 inhibitor would result in enhanced efficacy by targeting nonoverlapping immune escape mechanisms. In vitro cytotoxicity studies were performed in B16-OVA melanoma and murine bone marrow-derived dendritic cells. In vivo efficacy/toxicity studies were conducted in seven-week old C57BL/6 mice with 100,000 B16-OVA cells implanted on the rear flank. Once tumors were palpable, mice (n=4-5/group) were randomized to receive PLA (alendronate 4 mg/kg) + α-PD-1 mAb (10 mg/kg), vehicle + IgG-isotype, PLA + IgG-isotype, or vehicle + α-PD-1. Tumor growth was monitored, and at endpoint, tissues (tumors, lymph nodes, livers, kidneys, spleen, intestines, and lungs) were harvested for analysis by flow cytometry and histopathology. Neither PLA nor α-PD-1 were cytotoxic to melanoma or dendritic cells in vitro when compared to doxorubicin (cytotoxic chemotherapy). Mice treated with PLA + α-PD-1 had significantly smaller tumors compared to placebo controls (p=0.0015) and α-PD-1 monotherapy (p=0.0168).There were no significant pathologic changes in tissues. Assessment of immune responses in tumors and sentinel lymph nodes is ongoing. Dual immunotherapy targeting tumor-associated macrophages and the PD-1 checkpoint is safe and more efficacious than monotherapies solely targeting the PD-1 checkpoint in the treatment of murine melanoma. This is rapidly translatable to clinical trials, as the separate components are already approved for use in humans.

Citation Format: Claire E. Shudde, Jalpa Patel, Robin Rajan, Hilary Shmeeda, Dung Nguyen, Alberto A. Gabizon, Ninh M. La-Beck. Dual immunotherapy targeting macrophages and PD-1 immune checkpoint for treatment of melanoma [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 B21.
Abstract B24: Using L-fucose to render melanomas immune hot: Roles of melanoma HLA-DRB1 and CD4+T cell-mediated antitumor immunity
Authors: Lester Daniel K. , Mercurio Matt , Innamorato Pasquale , Krithika Kodumudi , Danial Williamson , Gregory Watson , Shari Pilon-Thomas , Jane Messina , Thomas Kerri L. , McCarthy Susan , Markowitz Joseph , Haltiwanger Robert , Lau Eric
Published: March 2020

Responsiveness to immunotherapies is limited in melanoma to subsets of patients. Despite attempts to improve responsiveness to immunotherapies by testing combination therapies, patients often experience significant adverse events that require the termination of treatment. Although causes of nonresponsiveness remain unclear and the subject of active investigation, a commonality among refractory tumors is low pretreatment levels of tumor-infiltrating lymphocytes (TILs). Thus, studies aiming to elucidate the interactions between melanoma and immune cells and to increase TIL levels are anticipated to improve therapies. We previously reported that fucosylation (the post-translational modification of proteins with the dietary plant sugar L-fucose), is altered in melanoma. Increasing fucosylation levels in melanoma—genetically or with oral L-fucose—suppresses tumor growth, metastasis, and increases TILs in immune competent mouse models of melanoma (Lau et al., 2015). The mechanisms underlying how melanoma fucosylation appears to trigger antitumor immunity are unclear. Here, we identified TIL subpopulations triggered by fucosylation to mediate tumor suppression and a key molecular mechanism. In NRAS- and BRAF-mutant melanoma models, dietary L-fucose suppressed tumor growth by ~50-60% and increased TILs by ~10-50-fold. Of all TIL subpopulations examined, CD3+ T cells were most increased by L-fucose, doubling or increasing by 15-fold, in NRAS- or BRAF-mutant models, respectively. Depletion of CD4+ T cells abrogated L-fucose-triggered TIL increases and tumor suppression, indicating that CD4+ T cells are central for antitumor immune effects of L-fucose. We identified the Class II MHC protein HLA-DRB1 as expressed and fucosylated in melanoma cells and confirmed its requirement for L-fucose-triggered, CD4+ T cell-dependent tumor suppression in vivo. Roles of HLA-DRB1 fucosylation in melanoma, effects on CD4+ T cell biology, and how L-fucose can enhance immunotherapies will be discussed. Our studies highlight how L-fucose supplementation can render tumors “immune hot” and represents a potential therapeutic strategy for improving immunotherapies.

Citation Format: Daniel K. Lester, Matt Mercurio, Pasquale Innamorato, Kodumudi Krithika, Williamson Danial, Watson Gregory, Pilon-Thomas Shari, Messina Jane, Kerri L. Thomas, Susan McCarthy, Joseph Markowitz, Robert Haltiwanger, Eric Lau. Using L-fucose to render melanomas immune hot: Roles of melanoma HLA-DRB1 and CD4+T cell-mediated 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 B24.
Abstract B28: ERAP2 overexpression is a marker for reduced anti-PD-1 response in nasopharyngeal carcinoma
Authors: Tay Joshua K. , Ma Brigette B.Y. , Varma Sushama , Lim Yaw Chyn , Tan Chee Seng , Lo Kwok Wai , Sunwoo John B. , Cullen Kevin J. , Goh Boon Cher , West Robert B.
Published: March 2020

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial cancer common in the Southern Chinese population. Patients with recurrent or metastatic NPC treated with PD-1 blockade showed a response rate of 20.5%, with no correlation observed between the expression of PD-L1 and treatment response. In an effort to identify biomarkers for anti-PD-1 treatment response, we evaluated a cohort of 10 NPC patients with recurrent or metastatic NPC treated with nivolumab or pembrolizumab. Laser capture microdissection was performed on pretreatment biopsies, with tumor and microenvironment compartments separately microdissected. Nasopharyngeal epithelium from normal nasopharyngeal biopsies were also microdissected as controls. Smart-3SEQ, a novel 3´ end RNA-Seq technique that allows for the accurate quantification of transcript abundance in FFPE samples, was performed to obtain gene expression libraries. ERAP2, a zinc metalloaminopeptidase that performs N-terminal trimming of antigenic epitopes for presentation by MHC class I, was among the most highly upregulated genes in the tumor compartment of nonresponders (adjusted p = 0.033). ERAP2 overexpression was also observed in NPC tumors compared to normal controls. Immunohistochemistry validated the overexpression of ERAP2 in nonresponders, while responders had similar expression to normal controls. In conclusion, ERAP2 is overexpressed in NPC tumors and is a promising biomarker for reduced anti-PD-1 response in NPC.

Citation Format: Joshua K. Tay, Brigette B.Y. Ma, Sushama Varma, Yaw Chyn Lim, Chee Seng Tan, Kwok Wai Lo, John B. Sunwoo, Kevin J. Cullen, Boon Cher Goh, Robert B. West. ERAP2 overexpression is a marker for reduced anti-PD-1 response in nasopharyngeal carcinoma [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 B28.
Abstract B3: Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer immunotherapy
Authors: Zhang Yan , Du Xuexiang , Liu Mingyue , Tang Fei , Zhang Peng , Ai Chunxia , Fields James K. , Sundberg Eric J. , Latinovic Olga S. , Devenport Martin , Zheng Pan , Liu Yang
Published: March 2020

It remains unclear why the clinically used anti-CTLA-4 antibodies, popularly called checkpoint inhibitors, have severe immunotherapy-related adverse effects (irAEs) and yet suboptimal cancer immunotherapeutic effects (CITE). Here we report that while irAE-prone Ipilimumab and TremeIgG1 rapidly direct cell surface CTLA-4 for lysosomal degradation, the non-irAE-prone antibodies we generated, HL12 or HL32, dissociate from CTLA-4 after endocytosis and allow CTLA-4 recycling to cell surface by the LRBA-dependent mechanism. Disrupting CTLA-4 recycling results in robust CTLA-4 downregulation by all anti-CTLA-4 antibodies and confers toxicity to a non-irAE-prone anti-CTLA-4 mAb. Conversely, increasing the pH sensitivity of TremeIgG1 by introducing designed tyrosine-to-histidine mutations prevents antibody-triggered lysosomal CTLA-4 downregulation and dramatically attenuates irAE. Surprisingly, by avoiding CTLA-4 downregulation and due to their increased bioavailability, pH-sensitive anti-CTLA-4 antibodies are more effective in intratumor regulatory T-cell depletion and rejection of large established tumors. Our data establish a new paradigm for cancer research that allows for abrogating irAE while increasing CITE of anti-CTLA-4 antibodies.

Citation Format: Yan Zhang, Xuexiang Du, Mingyue Liu, Fei Tang, Peng Zhang, Chunxia Ai, James K. Fields, Eric J. Sundberg, Olga S. Latinovic, Martin Devenport, Pan Zheng, Yang Liu. Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer 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 B3.
Abstract B32: Metformin reduces PD-L1 expression in the tumor and enhances the efficacy of vaccine-generated CD8 T cells in a murine model of triple-negative breast cancer
Authors: Munoz Luis E. , Bommireddy Ramireddy , Huang Haley L. , Pack Christopher D. , Ramachandiran Sampath , Selvaraj Periasamy
Published: March 2020

Immunotherapy has emerged as a promising treatment of breast cancer; however, response rates to therapy remain far from ideal. We have developed a new platform for personalized vaccine immunotherapy that utilizes tumor tissue to generate tumor membrane vesicles (TMVs). These TMVs can be incorporated with immunostimulatory molecules such as IL-12 and B7-1 and used for immunization. In this study, we investigated the combinatorial effect of TMV-based vaccine immunotherapy, to induce an antitumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert antitumor effects by promoting CD8 T-cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while the combination with metformin resulted in greater inhibition of primary growth. TMV vaccination reduced the metastatic burden in the lungs while the combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T-cell infiltration to the tumor, and combination with metformin caused a decrease in the accumulation of T cells, despite having better control of tumor growth. Further, we show that metformin can reduce PD-L1 expression within the tumor microenvironment and result in more IFN-γ-producing CD8 T cells within the tumor, providing a mechanistic insight for the improvement of TMV-based vaccine immunotherapy.

Citation Format: Luis E. Munoz, Ramireddy Bommireddy, Haley L. Huang, Christopher D. Pack, Sampath Ramachandiran, Periasamy Selvaraj. Metformin reduces PD-L1 expression in the tumor and enhances the efficacy of vaccine-generated CD8 T cells in a murine model of triple-negative breast 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 B32.
Abstract B35: Epitope-binding promiscuity of HLA class I molecules shapes the efficacy of checkpoint blockade immunotherapy
Authors: Manczinger Máté , Balogh Gergő , Koncz Balázs , Kemény Lajos , Papp Balázs , Pál Csaba
Published: March 2020

Immune checkpoint inhibitors (ICI), which target inhibitory receptors on T cells and thereby promote antitumor immune responses, have achieved considerable success in clinical care against a range of cancer types. However, only a subset of patients responds adequately to ICI-based immunotherapies. Therefore, it is of utmost importance to understand the immunologic mechanisms underlying resistance and adverse effects. It has been previously shown that certain MHC class I molecules are capable of promiscuous peptide binding, as they are associated with a much wider range of epitopes than others. The functional implications of such an elevated epitope-binding repertoire on the efficacy of cancer immunotherapy are unknown. In this work, we examine HLA genotypes of individuals receiving checkpoint blockade immunotherapy using two published cohorts. We demonstrate that cancer patients carrying HLA class I alleles with promiscuous peptide binding display significantly worse prognosis after ICI immunotherapy. This association remains significant after controlling for tumor types, mutational burden, and HLA genetic diversity. We also found that cancer samples of patients carrying highly promiscuous HLA class I alleles display significantly reduced expression of PD1 and CTLA-4 receptors, important markers of T-cell activation in the tumor microenvironment. Moreover, such promiscuous HLA alleles bind neoepitopes with reduced affinity. These findings indicate that high promiscuity is suboptimal due to a reduced specificity of epitope binding, yielding inadequate antitumor immune response.

Citation Format: Máté Manczinger, Gergő Balogh, Balázs Koncz, Lajos Kemény, Balázs Papp, Csaba Pál. Epitope-binding promiscuity of HLA class I molecules shapes the efficacy of checkpoint blockade 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 B35.
Abstract B39: The T-cell repertoire as a biomarker for response to anti PD-1 immunotherapy
Authors: Philip Hagit , Snir Tom , Gordin Miri , Wigelman Yoav , Zilberberg Alona , Efroni Sol
Published: March 2020

The prediction of patients’ response to checkpoint immunotherapy is currently imprecise. Here, we show that a measurement of the T-cell repertoire, from peripheral blood of mice, is enough to predict which mice would or would not respond to anti-PD1 treatment. To show this, we used a syngeneic orthotopically implanted CL261 glioma-bearing mouse model. We followed the mice over the timeline of tumor implantation and checkpoint immunotherapy, with blood samples on days 0, 7, 21, 35, 49, and 63. Since the (syngeneic) implanted tumor is bioluminescent, we were able to monitor tumor size using bioluminescence imaging (up to 9 measures, 1 measure per week), up to day 63 post-tumor inoculation. We then produced more than 130 T-cell receptor libraries for the alpha and beta chains and sequenced these libraries to produce a data set of the dynamics of the T-cell repertoire during tumor progression and during response to treatment. In this model, only 30% of animals respond to treatment, which allowed us to look at the repertoires of two distinct groups: responders and nonresponders. Using machine learning, we were able to build a model that allows us to tag, in a validation set, responders and nonresponders, thereby predicting response.

Citation Format: Hagit Philip, Tom Snir, Miri Gordin, Yoav Wigelman, Alona Zilberberg, Sol Efroni. The T-cell repertoire as a biomarker for response to anti PD-1 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 B39.
Abstract B4: High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model
Authors: Luchtel Rebecca , Bhagat Tushar , Pradhan Kith , Jacobs William , Levine Mark , Verma Amit , Shenoy Niraj
Published: March 2020

Major efforts are under way to identify agents that can potentiate effects of immune checkpoint inhibition. Ascorbic acid (AA) has been previously shown to cause DNA demethylation and corresponding increase in hydroxymethylation in multiple malignancies by enhancing the activity of the Ten-Eleven Translocation (TET) enzymes (Shenoy et al., BCJ 2017; Shenoy et al., JCI 2019; Cimmino et al., Cancer Cell 2017; Agathocleous et al., Nature 2017). Furthermore, AA has re-emerged as a promising anticancer agent based on recent knowledge of pharmacokinetics, discovery of unexpected mechanisms of action, and early phase trials with IV AA (Shenoy et al., Cancer Cell 2018). Here, we report that AA treatment caused genome-wide demethylation and enhanced expression of endogenous retroviral elements in lymphoma cells. AA also increased 5-hydroxymethylcytosine (5hmC) levels of CD8+ T cells and enhanced their cytotoxic activity in a lymphoma coculture system. High-dose AA treatment synergized with anti-PD1 therapy in a syngeneic lymphoma mouse model, resulting in marked inhibition of tumor growth compared with either agent alone. Analysis of the intratumoral epigenome revealed increased 5hmC with AA treatment, consistent with in vitro findings. Analysis of the tumor immune microenvironment revealed that AA strikingly increased intratumoral infiltration of CD8+ T cells and macrophages, suggesting enhanced tumor immune recognition. The combination treatment markedly enhanced intratumoral infiltration of macrophages and CD8+ T lymphocytes, granzyme B production by cytotoxic cells (cytotoxic T cells and natural killer cells), and IL-12 production by antigen-presenting cells compared with single-agent anti-PD1. These data strongly indicate that AA potentiates anti-PD1 checkpoint inhibition through synergistic mechanisms. The study provides a compelling rationale for testing combinations of AA and anti-PD1 agents in lymphoma patients as well as in preclinical models of other malignancies.

Citation Format: Rebecca Luchtel, Tushar Bhagat, Kith Pradhan, William Jacobs, Mark Levine, Amit Verma, Niraj Shenoy. High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse 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 B4.
Abstract B40: The phosphorylation by activated-AMPK induces ERAD of PD-L1 through abnormal glycosylation
Authors: Cha Jong-ho , Yang Wen-Hao , Hong Soon-Sun , Hung Mien-Chie
Published: March 2020

Programmed death ligand-1 (PD-L1) is a major immune checkpoint molecule that enables cancer immune evasion in various cancer types, and therefore the PD-L1/PD-1 axis has been a major target for current cancer immune checkpoint blockade (ICB). However, the regulatory mechanisms of PD-L1 expression are largely undefined. Here, we demonstrate that AMP-activated protein kinase (AMPK) phosphorylates Serine 195 of PD-L1, which induces the ER-associated degradation (ERAD) of PD-L1 via abnormal glycosylation. Further, we identified HRD1 as an ERAD E3 ligase of PD-L1. Ser195-p recruits HRD1 into PD-L1-ERAD complex and finally induces proteasomal degradation of PD-L1 through poly-ubiquitination. This study broadens our understanding of the regulation of PD-L1 expression and has the potential to improve the efficacy of ICB targeting PD-L1/PD-1 and overcome resistance to the ICB.

Citation Format: Jong-ho Cha, Wen-Hao Yang, Soon-Sun Hong, Mien-Chie Hung. The phosphorylation by activated-AMPK induces ERAD of PD-L1 through abnormal glycosylation [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 B40.
Abstract B43: Simultaneous inhibition of CXCR1/CXCR2, TGF-β, and PD-L1 overcomes immune resistance driven by tumor plasticity
Authors: Horn Lucas A. , Riskin Jeffrey , Hempel Heidi A. , Lind Hanne , Fousek Kristen , Hamilton Duane H. , McCampbell Kristen K. , Schlom Jeffrey , Palena Claudia
Published: March 2020

The occurrence of phenotypic plasticity in carcinomas, which results in the acquisition of mesenchymal features by epithelial cancer cells, associates with a poor prognosis and has been described as a mechanism of primary resistance and failure to treatment with checkpoint blockade immunotherapy targeting the PD-1/PD-L1 pathway. IL-8 and TGF-β are two of the factors that mediate phenotypic plasticity in epithelial tumors to promote an immune-suppressive microenvironment. Here we report on an immunotherapy approach aimed at overcoming escape mechanisms mediated by IL-8 and TGF-β accumulation in the tumor microenvironment via the use of two clinical-stage agents: SX-682, a dual CXCR1/2 small-molecule inhibitor, with the bifunctional anti-PD-L1/TGFβRII agent, bintrafusp alfa (M7824) that simultaneously blocks PD-L1 while trapping soluble TGF-β. We demonstrate that the concurrent inhibitions of CXCR1/CXCR2, TGF-β, and PD-L1 signaling synergize to reduce mesenchymal tumor features in murine models of breast and lung cancer, and to markedly increase expression of tumor epithelial E-cadherin while reducing infiltration with suppressive granulocytic myeloid-derived suppressor cells (G-MDSC), significantly enhancing CD4+ and CD8+ T-cell infiltration and activation in tumors, and leading to improved antitumor activity. This combination approach was effective in tumor models with varying immune cell infiltrates, degrees of plasticity, and responses to immunotherapy. The results of these studies provide the rationale for the future evaluation of this multimodal therapy in the clinic.

Citation Format: Lucas A. Horn, Jeffrey Riskin, Heidi A. Hempel, Hanne Lind, Kristen Fousek, Duane H. Hamilton, Kristen K. McCampbell, Jeffrey Schlom, Claudia Palena. Simultaneous inhibition of CXCR1/CXCR2, TGF-β, and PD-L1 overcomes immune resistance driven by tumor plasticity [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 B43.
Abstract B46: The long noncoding RNA INCA1 is a novel regulator of PD-L1 expression in tumors
Authors: Mineo Marco , Lyons Shawn M. , Ferrer-Luna Ruben , Ito Hirotaka , von Spreckelsen Niklas , Alayo Quazim A. , Khalsa Jasneet K. , Shah Khalid , Ligon Keith L. , Beroukhim Rameen , Ivanov Pavel , Anderson Paul J. , Nakashima Hiroshi , Lawler Sean E. , Chiocca E. Antonio
Published: March 2020

Long noncoding RNAs (lncRNAs) are key regulators of gene expression both in physiologic and pathologic conditions. Although the role of lncRNAs in tumorigenesis is now emerging, their function in cancer-mediated immunosuppression remains unexplored. We have identified the interferon-γ (IFNγ)-stimulated noncoding RNA 1 (INCA1) as a novel lncRNA expressed from the PD-L1 locus and showed that INCA1 is a key regulator of PD-L1 expression. Silencing INCA1 decreased PD-L1 expression both at basal level and in response to IFNγ stimulation in multiple tumor types. INCA1 knockdown sensitized tumor cells to cytotoxic T cell-mediated killing. Moreover, tumors with silenced INCA1 showed increased CAR T trafficking and activity in vivo. These findings provide novel insights into the biology of PD-L1 regulation and tumor immune evasion. INCA1 is thus a new target for cancer immunotherapy.

Citation Format: Marco Mineo, Shawn M. Lyons, Ruben Ferrer-Luna, Hirotaka Ito, Niklas von Spreckelsen, Quazim A. Alayo, Jasneet K. Khalsa, Khalid Shah, Keith L. Ligon, Rameen Beroukhim, Pavel Ivanov, Paul J. Anderson, Hiroshi Nakashima, Sean E. Lawler, E. Antonio Chiocca. The long noncoding RNA INCA1 is a novel regulator of PD-L1 expression in 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 B46.
Abstract B63: Calcium electroporation offers an effective local treatment with immunogenic cell death and the potential for systemic remission in melanoma
Authors: Tremble Liam Friel , Heffron Cynthia , Forde Patrick
Published: March 2020

Electrochemotherapy is an established treatment modality in which chemotherapeutics are delivered systemically or locally in combination with local electrical pulses. Pulsing parameters are optimized to induce the reversible formation of pores in the cell membrane, leaving cells intact and healthy. Osmotic gradients during poration result in the rapid uptake of extracellular components, which results in enhanced chemotherapeutic uptake, between 5 to 2,000-fold depending on the hydrophobicity of the drug used. Complete local response rates for dermal lesions exceed 80%. Treatment is suitable for use in proximity to vasculature and offers superior cosmetic results in comparison to surgery when used in areas such as the eyelid or mouth. Recent advances have, however, been able to replace cytotoxic chemotherapeutic agents with calcium with no decrease in efficacy. The nature of cell death induced is believed to be acute, but to date has not been clearly characterized. Using Flou-4 to track the duration of elevated intracellular calcium concentrations, we have shown that elevated calcium levels persist for hours following treatment. Cell death occurs in 60-80% of cells treated in vitro within 8 hours of treatment. Cells surviving this initial insult show no detectable loss in survival or growth potential and appear otherwise unaffected. Necrotic cell death provides a significant source of damage-associated molecular patterns and may aid the generation of anticancer immune responses. Using cocultured macrophages, we have been able to show that treatment can indirectly boost cytotoxic T-cell responses via tumor-associated macrophages. The elucidation of cell death during treatment is critical for the effective optimization of clinical treatment parameters, which has hitherto been determined empirically. Other advances, such as the use of high-frequency electroporation, are transforming the clinical delivery. It is foreseeable that the requirement of surgical theaters may be avoided with the development of a pain-free treatment in the absence of cytotoxic drugs.

Note:This abstract was not presented at the conference.

Citation Format: Liam Friel Tremble, Cynthia Heffron, Patrick Forde. Calcium electroporation offers an effective local treatment with immunogenic cell death and the potential for systemic remission in melanoma [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 B63.
Abstract B68: NF-kB p50-deficient immature myeloid cell (p50-IMC) adoptive transfer slows the growth of murine prostate and pancreatic ductal carcinoma
Authors: Suresh Rahul R. , Barakat David J. , Barberi Theresa , Zheng Lei , Jaffee Elizabeth M. , Pienta Kenneth J. , Friedman Alan D.
Published: March 2020

Macrophages and dendritic cells lacking NF-kB p50 having an increased proinflammatory phenotype. Melanoma, fibrosarcoma, colon carcinoma, and glioblastoma grow slower in p50-/- mice, with tumor-associated macrophage M2-to-M1 reprogramming. We therefore evaluated the efficacy of NF-kB-deficient immature myeloid cell (p50-IMC) adoptive transfer. Immature myeloid cells (IMCs) were utilized to favor tumor and lymph node localization. WT-IMC or p50-IMC were generated by culturing lineage-negative marrow cells from WT or p50-/- mice in media containing TPO, SCF, and FL for six days followed by M-CSF for one day. Mice inoculated with Hi-Myc prostate cancer (PCa) or K-RasG12D pancreatic ductal carcinoma (PDC) transfected with luciferase received three doses of 1E7 IMCs every 3-4 days. IMC infusions were preceded by a single dose of 5-fluorouracil (5FU) to reduce marrow production of myeloid cells, to reduce tumor myeloid cell numbers, and to potentially release tumor neoantigens. PCa grew slower in p50-/- mice, and absence of host p50 led to prolonged survival of mice inoculated orthotopically with PDC. 5FU followed by p50-IMC slowed PCa or PDC tumor growth ~3-fold in contrast to 5FU followed by WT-IMC, 5FU alone, or p50-IMC alone. Slowed tumor growth was evident for 12 of 14 PCa tumors but only 8 of 15 PDC tumors, leading us to focus on PCa for additional analyses. p50-IMC predominantly generated PCa and draining lymph node F4/80+ macrophages, but also CD11b+F4/80-CD11c+ conventional dendritic cells, with tumor and nodal macrophages having increased Ly6C and MHCII and reduced MR compared to host macrophages, indicative of a proinflammatory phenotype. p50-IMC also led to 5-fold increased activated tumor CD8 T cells, and antibody-mediated CD8 T-cell depletion obviated slower tumor growth induced by 5FU followed by p50-IMC. Adoptive transfer of patient-derived NF-kB p50-deficient p50-IMC, potentially generated by expansion and gene editing of marrow CD34+ cells, may contribute to the therapy of these and additional cancers.

Citation Format: Rahul R. Suresh, David J. Barakat, Theresa Barberi, Lei Zheng, Elizabeth M. Jaffee, Kenneth J. Pienta, Alan D. Friedman. NF-kB p50-deficient immature myeloid cell (p50-IMC) adoptive transfer slows the growth of murine prostate and pancreatic ductal carcinoma [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 B68.
Abstract B73: A novel candidate for immunologic target in treatment of multiple myeloma
Authors: Matsushita Maiko , Saito Saku , Mori Kanae , Yokoe Shinya , Ichikawa Daiju , Hattori Yutaka
Published: March 2020

Background: Despite the development of novel drugs, including immune modulatory drugs (IMiDs) and proteasome inhibitors, multiple myeloma is still an uncurable disease. In particular, patients with high-risk chromosomal abnormalities, such as t (4;14) or del17, have poor prognosis. Recently, antibodies or CAR-T cells have been shown to be effective, suggesting that myeloma cells are sensitive to immune reactions. We have previously identified an HLA-A*24:02-restiricted epitope from a novel cancer-testis antigen CXorf48. In this study, we aim to characterize cytotoxic T lymphocyte (CTL) against this antigen and investigate the possibility of the CTLs for adoptive T-cell therapy in myeloma treatments.

Methods: Expression of CXorf48 gene was examined by RT-PCR of myeloma cell lines. We then treated myeloma cells with low CXorf48 expressions by demethylating agents, 5-azacytidine (5-aza) or 5-aza-2’-deoxycytidine (DAC), and checked the expression level of this gene by quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors and their antigen recognition was examined by ELISpot assay and 51Cr cytotoxicity assay. We also analyzed T-cell receptor (TCR) repertoire using dextramer-sorted CTLs and transduced the TCR genes into Jurkat cells.

Results: Expression of CXorf48 gene was found in most myeloma cell lines including those with t (4;14) or del17. In addition, the gene expression was significantly increased by DAC in CXorf48-low myeloma cells, which caused enhanced recognition by anti-CXorf48 CTLs. We also identified CXorf48-specidfic TCR genes of CTLs from two donors, and generated TCR gene-transduced T cells.

Conclusions: CXorf48 might be a useful target for immunotherapy in combination with demethylating agents for high-risk myeloma. Aiming at CXorf48-specific adoptive therapy, we are analyzing CXorf48-specific TCR-transduced human T cells.

Citation Format: Maiko Matsushita, Saku Saito, Kanae Mori, Shinya Yokoe, Daiju Ichikawa, Yutaka Hattori. A novel candidate for immunologic target in treatment of multiple myeloma [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 B73.
Abstract B8: Multiplex quantification of immunomodulatory proteins in tissue and plasma using next-gen targeted MRM mass spectrometry
Authors: Whiteaker Jeffrey R. , Zhao Lei , Ivey Richard G. , Voytovich Julia , Schoenherr Regine M. , Huang Dongqing , Kennedy Jacob J. , Paulovich Amanda G.
Published: March 2020

Quantifying the many immunomodulatory proteins in the “cancer-immunity cycle” is critical for both patient selection and development of novel immunotherapies. Conventional technologies to measure proteins (IHC, ELISA, Western blot, flow cytometry) are often semiquantitative, not multiplexable, feature poor specificity, and are difficult to develop. Due to these limitations, investigators have turned to mRNA-based measurements to infer protein expression of immunomodulatory factors. However, as has been unequivocally demonstrated for human breast, colorectal, and ovarian cancers (Pubmed IDs: 27251275, 25043054, 27372738), RNA levels can be poor predictors of protein levels or activity. Thus, direct quantification of proteins is desirable. As part of the National Cancer Institute’s Cancer Moonshot, we are developing >300 multiplex assays for quantifying immunomodulatory proteins in tissue and plasma using a next-gen technology for protein quantification, based on targeted multiple reaction monitoring mass spectrometry (MRM-MS). MRM-MS complements traditional immunoassays by enabling highly specific, precise, harmonizable quantification of proteins in biospecimens, even at high multiplex levels. As proof of concept, we will present a novel multiplex assay based on next-gen MRM-MS for quantification of 47 immunomodulatory proteins in plasma and/or tumor tissue. The technology uses immunoaffinity enrichment coupled with MRM-MS (immuno-MRM assay). Assay bioanalytical validation was conducted in accordance with industry standards for use in FFPE tissue and plasma matrices and included determination of limits of detection, linear range of response, intraday and interday repeatability, stability of analytes in sample processing, and characterization of reproducibility of endogenous analyte measurement. The mean linear range was over three orders of magnitude with median intra- and interday CVs less than 10%. The characterized assays are being applied to a panel of tumor specimens from a variety of cancer types to document detection and determine minimum sample requirements. Additional assay panels are under development to provide measurements of up to 350 immunomodulatory proteins.

Citation Format: Jeffrey R. Whiteaker, Lei Zhao, Richard G. Ivey, Julia Voytovich, Regine M. Schoenherr, Dongqing Huang, Jacob J. Kennedy, Amanda G. Paulovich. Multiplex quantification of immunomodulatory proteins in tissue and plasma using next-gen targeted MRM mass spectrometry [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 B8.
Abstract B81: Prognostic significance of matrix metalloproteinase-3 expression in patients with resected lung adenocarcinoma
Authors: Hung Jung-Jyh , Hsu Wen-Hu
Published: March 2020

Background: Epithelial-mesenchymal transition (EMT) is one of the major molecular mechanisms inducing tumor invasion and metastasis. Many EMT regulators, such as Snail, TWIST1, and Slug, induce EMT through inhibiting E-cadherin. The process of EMT generates cells with stem-like properties. Bmi1, Oct4, and Nanog are molecules related to cancer stem cell (CSC) properties. The expression of matrix metalloproteinase-3 (MMP-3) in lung tissue leads to activation of Rac1b, and activation of EMT by Rac1b/MMP-3 pathway stimulates bypass of oncogene-induced senescence and progression to lung adenocarcinoma. However, the prognostic significance of MMP-3 expression in patients of lung cancer remains unknown.

Methods: The clinicopathologic characteristics of 92 patients with lung adenocarcinoma at Taipei Veterans General Hospital were retrospectively reviewed. Expression of MMP-3 was investigated using immunohistochemistry. Their prognostic significance in probability of overall survival was analyzed.

Results: With a median follow-up time of 27.7 months (27.4 ± 9.3), the 2-year probability of overall survival rate was 77.3%. By immunohistochemistry, overexpression of MMP-3 was identified in 12 (13.0%) of the 92 patients. Univariate analysis showed that MMP-3 overexpression was a significant prognostic factor for worse probability of overall survival (P = 0.019). Order age was also a significant prognostic factor for worse probability of overall survival (P = 0.027). Multivariate analysis showed that order age (P = 0.027) and MMP-3 overexpression (P = 0.015) were both significant prognostic factors for worse probability of overall survival.

Conclusions: Our study showed that order age and MMP-3 overexpression were both significant prognostic factors for worse probability of overall survival in resected lung adenocarcinoma.

Citation Format: Jung-Jyh Hung, Wen-Hu Hsu. Prognostic significance of matrix metalloproteinase-3 expression in patients with resected lung adenocarcinoma [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 B81.
Abstract B83: A role of a transcription factor TOX in promoting CD8+ T-cell exhaustion in cancer via single-cell transcriptome analysis
Authors: Kim Kyungsoo , Park Seyeon , Ha Sang-Jun , Lee Insuk
Published: March 2020

Recently to treat cancer, several cancer therapies have been developed such as chemotherapy, radiation therapy, immunotherapy, targeted therapy, and hormone therapy. Among several cancer therapies, immunotherapies have recently been developed and shown clinical impacts on a significant number of patients. However, even in the case of immunotherapy, every cancer therapy does not have a 100% response rate. Since the difference in the response rate is mainly due to tumor microenvironment, we need to understand the tumor microenvironment to overcome the low response rate of therapies. Previously, tumor microenvironment was analyzed using cell sorting techniques such as FACS, but it is hard to observe every cell subtype. However, recently, a new technique called single-cell RNA sequencing has been developed. Single-cell RNA sequencing sequences RNAs from every single cell from the sample. Using single-cell RNA sequencing, we can get transcriptome information from whole tumor sample. Therefore, we can observe interactions between the immune cells and the tumors cells using single-cell RNA sequencing. First of all, to focus on the microenvironment of tumor tissue, we analyzed public single-cell RNA sequencing data and unraveled the phenotype of exhausted T cells. By analyzing single-cell RNA sequencing data from the public, we found TOX, a T cell exhaustion-associated transcriptional regulator important for maintaining exhausted T cell. We confirmed that TOX is expressed in the context of exhausted T cell by FACS analysis. Also, using trajectory analysis, we observed expression pattern of TOX through T-cell exhaustion process. Additionally, we analyzed TCGA data to observe the effect of TOX on survival rate. Through analyzing several data such as expression data, binding assay, and open chromatin region of exhausted T cells, we found a potential role of the TOX gene. We also confirmed that the TOX gene can regulate the expression of other checkpoint molecules by knockdown experiment. Here, we found a novel regulator in the tumor microenvironment using single-cell transcriptomics.

Citation Format: Kyungsoo Kim, Seyeon Park, Sang-Jun Ha, Insuk Lee. A role of a transcription factor TOX in promoting CD8+ T-cell exhaustion in cancer via single-cell transcriptome analysis [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 B83.
Abstract B9: Screening the membrane proteome to determine antibody specificity and discover new immunomodulatory targets
Authors: Sullivan Tabb , Huston-Paterson Duncan , Azuelos Charles , Wilf Rona , Doranz Benjamin
Published: March 2020

Integral Molecular specializes in characterizing monoclonal antibodies (MAbs) against structurally complex targets, including GPCRs, ion channels, and immuno-oncology (I-O) targets. The Membrane Proteome Array (MPA) enables off-target analysis by screening antibodies against an expression array of >5,300 human membrane proteins. Each membrane protein is presented in live cells in its structurally intact and native conformation with appropriate post-translational modifications. Binding interactions are tested and validated by high-throughput flow cytometry, providing comprehensive assessment of off-target antibody interactions. Membrane proteins in the MPA are fully functional and can be used for phenotypic screening to identify new therapeutic targets, as demonstrated by our recent discovery of novel costimulatory molecules against cytotoxic T lymphocytes (CTLs).

Citation Format: Tabb Sullivan, Duncan Huston-Paterson, Charles Azuelos, Rona Wilf, Benjamin Doranz. Screening the membrane proteome to determine antibody specificity and discover new immunomodulatory targets [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 B9.
Abstract B91: BRCA1 mutation in breast cancer cells and generation of metastasis associated fibroblasts
Authors: Latha Neetha Rajan , Varghese Geetu Rose , Jaikumar Vishnu Sunil , Rajan Arathi , Nadhan Revathy , Warrier Arathy , Srinivas Priya
Published: March 2020

Tumor microenvironment plays an indispensable role in cancer development as it can be described as the territory where the interaction takes place between tumor and microenvironment elements such as the endothelial cells, epithelial cells, fibroblasts, immune cells, and ECM proteins. Among these structural stromal components, specifically cancer-associated fibroblasts (CAFs) have attracted substantial attention over recent decades. CAFs in the tumor microenvironment assist the accelerated growth demands of the cancer cells and enhance cancer progression through their paracrine activity by the enhanced secretion of growth factors and cytokines, which also help in remodeling the extracellular matrix (ECM). Studies reveal that CAFs have extended their role in cancer metastasis by accompanying the cancer cells to distant metastatic sites. From our in vitro studies, we have identified that coculturing the CAFs with the conditioned medium (CM) from BRCA1 mutated but not wild-type cancer cells results in the transformation of CAFs to an altered phenotype. Since these transformed CAFs showed increased migrative, invasive, and proliferative abilities, we named them “Metastasis Associated Fibroblasts” (MAF) (Sreelatha et al., 2018). The MAF thus generated showed a significant increase in the expression of the Ezrin, Radixin, and Moesin (ERM) protein complex, which may be attributed to the association of BRCA1 C-terminal (BRCT) domains of BRCA1 with the ERM protein superfamily. This association of BRCA1 with the key regulators of ERM complex can influence cell adhesion and motility, suggesting that BRCA1 may act upon their signaling pathways. To study the effect of BRCA1 mutation and ERM protein expression in CAFs in vivo, BRCA1 mutated breast cancer cells were orthotopically implanted to NOD-SCID mice. CAFs isolated from the tumor were analyzed for the expression of ERM. It was found that there is a significant increase in the expression of ERM protein in the tumors isolated from mice that were implanted with BRCA1 mutated breast cancer cells compared to the mouse group that received BRCA1 wild-type breast cancer cells. Inhibitors to ezrin and CCL5 could effectively reduce the metastatic potential of BRCA1 mutant cancer models. A combination of ezrin inhibitor (NSC 668394) and plumbagin, a plant-derived naphthoquinone that effectively reduces BRCA1 mutant breast cancer cell proliferation, is under investigation.

Citation Format: Neetha Rajan Latha, Geetu Rose Varghese, Vishnu Sunil Jaikumar, Arathi Rajan, Revathy Nadhan, Arathy Warrier, Priya Srinivas. BRCA1 mutation in breast cancer cells and generation of metastasis associated fibroblasts [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 B91.