bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1419: Metastasis and cancer stem cell markers identify two colorectal cancer subtypes with different molecular and clinical features
Authors: Alsaihati Burair , Zhao Shaying
Published: August 2019

Introduction Colorectal cancer (CRC) falls among the top prevalent cancer types with lethal outcome both in the United States and worldwide. The heterogeneity of CRC highlights the importance of identifying molecular markers for meaningful classification and prognosis. The recently published Consensus Molecular Subtypes (CMS) represent a widely-used molecular subtyping of CRC. However, our analyses indicate that clear heterogeneity still exists in some CMSs. To better classify CRC, we performed the following study.

Methods We collected from literature 19 gene markers classified as metastasis or cancer stem cell (CSC) markers in CRC. We then examined their expression in 622 CRC samples by The Cancer Genome Atlas (TCGA), and validated the findings using another published dataset, GSE39582, with 566 CRC samples.

Results We found among the 19 gene markers examined, 9 genes cluster into two oppositely co-expressed groups (VEGFC, SMAD4, TNIK, ITGB1, CD44 and ALCAM in group 1 and VEGFB, OTUB1 and DLL4 in group 2). Principal component analysis and hierarchical sample clustering of theses 9 genes identify two subtypes among the samples of each CMS, particularly CMS2 and CMS4. We named the two subtypes S1, which corresponds to increased expression in group 1 genes, and S2, which corresponds to higher expression of group 2 genes. Analysis of cancer hallmarks indicates a strong enrichment of six pathways (androgen response, protein secretion, mitotic spindle, UV response down, PI3K and TGF-β signaling), a common and defining feature of the S1 subtype in all CMSs. Immune activation pathways and most immune cells are strongly enriched in the S1 subtype of CMS4 samples only. Conversely, S2 is the subtype that exhibits immune activation across CMS2 samples. Tumor microenvironment signatures indicate more lymph vessels and stromal invasion in S1 samples within CMS4. However, within CMS2, a higher leukocyte fraction and invasive signature feature the S2 subtype. The epithelial and proliferative signatures are associated with the S1 subtype in general, but more prominently within CMS2. Consistent with the invasive signature of S2 samples in CMS2, the analysis of TCGA clinical data indicates increased lymphatic invasion, metastasized tumors and worse overall survival for them. On the other hand, GSE39582 clinical data show that worse overall survival is associated with the S1 subtype in CMS4.

Conclusion Each of CMS2 and CMS4 can be further divided into two subtypes, S1 and S2. While S2 may define more aggressive CMS2 cancers, S1 can be linked to worse prognosis in CMS4 patients.

Citation Format: Burair Alsaihati, Shaying Zhao. Metastasis and cancer stem cell markers identify two colorectal cancer subtypes with different molecular and clinical features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1419.
Abstract 1424: Preclinical development of TAC01-CD19, a CD19 specific T-cell Antigen Coupler (TAC) therapy for the treatment of CD19-positive B cell malignancies
Authors: Helsen Christopher W. , Hayes Danielle , Hammill Joanne , Aarts Criag , Bader Andreas , Rill Donna , Bramson Jonathan
Published: August 2019

Background: The T cell antigen coupler (TAC) is a chimeric receptor co-opting the natural TCR receptor signaling complex to target tumor antigens in an MHC-independent manner (Helsen et. al., Nat. Comm. 2018). TAC engineered T cells mediate biological effects that are distinct from conventional chimeric antigen receptors (CARs) and may offer safety advantages due to greater target selectivity and no detectable off-target toxicity. Here, we present preclinical data of TAC01-CD19, an autologous CD19-specific TAC-T cell product candidate for clinical testing in patients with CD19-positive B cell malignancies

Materials and Methods: T cells from healthy donors and lymphoma patients were engineered with a CD19-specific TAC using lentivirus. Anti-tumor activity, T cell expansion and persistence were measured in the CD19-positive NALM-6 and Jeko-1 mouse tumor xenograft models. We used flow cytometry to determine surface expression of CD19-TAC, cytokine production and in vitro T cell proliferation. Cytotoxicity was evaluated in vitro using luciferase-based killing assay.

Results: Cytotoxicity was measured against multiple CD19 positive tumors cells (NALM6/Jeko-1/Raji) showing strong cytotoxicity in all cases. Treatment of established NALM-6 (acute lymphoblastic leukemia) and Jeko-1 xenografts (mantle cell lymphoma) with CD19 TAC-T cells resulted in clearance of tumors within a few weeks of T cell infusion. In vivo efficacy was tested across multiple donors, establishing consistent and reproducible anti-tumor efficacy. Monitoring of TAC T cells post-infusion revealed robust expansion that peaked in the peripheral blood 1-2 weeks post-infusion. Mice that cleared tumors following TAC-T cell treatment were resistant to subsequent challenge with fresh tumor cells demonstrating persistence of TAC-T cells. In contrast, treatment with control TAC-T had no impact on tumor growth. A histological examination of normal mouse tissues failed to detect TAC-T cell induced toxicity.

Conclusion: The pre-clinical evaluation of TAC01-CD19 has demonstrated specific anti-tumor responses without signs of toxicity. Results were reproducible across multiple donors demonstrating the robustness of the CD19 TAC system. TAC01-CD19 is expected to enter clinical testing in patients with CD19-positive hematological malignancies in Q2 2019.

Citation Format: Christopher W. Helsen, Danielle Hayes, Joanne Hammill, Criag Aarts, Andreas Bader, Donna Rill, Jonathan Bramson. Preclinical development of TAC01-CD19, a CD19 specific T-cell Antigen Coupler (TAC) therapy for the treatment of CD19-positive B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1424.
Abstract 1427: IL6 trans-signaling robustly promotes the expansion and antitumor activity of CAR T cells
Authors: Li Peng , Jiang Zhiwu , Liao Rui , Tang Zhaoyang
Published: August 2019

Chimeric antigen receptor T (CAR T) cell immunotherapy targeting CD19 positive B cells malignancies induces promising clinical remissions. However, the treatments are often accompanied with high levels of IL-6 characterized cytokine release syndrome (CRS). Previous studies had shown that sIL-6R are constitutively present in high concentrations in serum. Therefore, when the level of IL-6 is elevated (above nomogram per ml), sIL-6R combines with IL6 to form the IL-6/sIL-6R complex (trans-signaling). The IL-6 trans-signaling plays important roles in regulating immune responses, cell survival, apoptosis, and proliferation. To our knowledge, comprehensive assessment of the IL-6 signaling in biologic functions of CAR T has not been well addressed. We thus tested whether the IL-6 trans-signaling promotes expansion and anti-tumor activity of CAR T. We found that the levels of serum IL-6 and IL-6/sIL-6R complex were positively correlated with anti-CD19 CAR T cell expansion and anti-leukemia response in patients. To simulate the IL-6 trans-signaling, we next constructed a constitutively expression of Hyper IL-6 (HIL-6) with various CAR T cells. Anti-tumor efficacy of HIL-6-CAR T cells were confirmed in leukemia (anti-CD19) and solid tumors (anti-MUC1 and anit-GPC3 CAR T cells targeting lung cancer and hepatocellular carcinoma, respectively). Our results demonstrate that HIL-6-CAR T cells are more effective for suppressing tumor growth and more persistent in xenograft models. Transcriptomic profiling analysis revealed that the expression of genes that facilitate T-cell migration, early memory differentiation and the IL-6/GP130/STAT3 signaling was upregulated in HIL-6 simulated CAR T cells compared to the ones without HIL-6 overexpression. Because IL-6 trans-signaling activates downstream signaling via GP130, we also generated a novel CAR vector with constituted GP130 activation and demonstrated the CAR T cells were superior in anti-tumor activity with delayed xGVHD in xenograft models of leukemia, lung cancer and liver cancer. Taken together, these results showed that IL-6 trans-signaling can significantly enhanced the expansion anti-tumor activity of CAR T cells via GP130/STAT3 activation and GP130/CAR T may have the potential to promote anti-tumor effects with reduced toxicity in vivo.

Keywords: Chimeric antigen receptor T, cytokine release syndrome, IL-6 trans-signaling, GP130

Citation Format: Peng Li, Zhiwu Jiang, Rui Liao, Zhaoyang Tang. IL6 trans-signaling robustly promotes the expansion and antitumor activity of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1427.
Abstract 1428: Allogeneic CRISPR/Cas9 gene-edited CAR-T cells targeting CD33 show potent preclinical activity against AML cells
Authors: McEwan Brigid , Padalia Zinkal , Porras Ashley , Sagert Jason , Terrett Jonathan A. , Ho Tony , Kalaitzidis Demetrios
Published: August 2019

Acute myelogenous leukemia (AML) has a high mortality rate and remains difficult to treat, making new treatment approaches critical. Chimeric antigen receptor T (CAR-T) cell therapies have shown impressive clinical responses in B-cell neoplasia. However, comparable successes have not been reported to date in myeloid malignancies, potentially due to difficulty in manufacturing efficacious CAR-T cells from AML patients and lack of a suitable tumor antigen for AML. To address these issues, we have developed allogeneic CAR-T cells from healthy donors targeting the CD33/Siglec-3 antigen, a protein expressed on most AML cells and subpopulations in the majority of AML patients at presentation and relapse. Allogeneic anti-CD33 CAR-T cells were produced from healthy donor-derived T cells using CRISPR/Cas9 gene editing. In these cells, the TRAC locus was disrupted to reduce the risk of graft versus host disease (GvHD). At the same time, a CAR construct targeting CD33 was inserted site-specifically into the TRAC locus. In addition, the beta-2-microglobulin locus was disrupted to prevent clearance of the allogeneic CAR-T cells by the host immune system. These allogeneic anti-CD33 CAR-T cells showed potent effector activity in vitro against human AML-derived cell lines, as measured by both tumor cell lysis and effector cytokine secretion. The allogeneic anti-CD33 CAR-T cells also potently reduced AML tumors in vivo in xenograft mouse models.

Citation Format: Brigid McEwan, Zinkal Padalia, Ashley Porras, Jason Sagert, Jonathan A. Terrett, Tony Ho, Demetrios Kalaitzidis. Allogeneic CRISPR/Cas9 gene-edited CAR-T cells targeting CD33 show potent preclinical activity against AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1428.
Abstract 144: Characterization of glioma subtype-specific macrophage-tumor interactions using single-cell RNA-seq
Authors: Rao Rohit , Verma Ravinder , Wang Jincheng , Lu Qing Richard
Published: August 2019

Glioblastomas are malignant brain tumors that carry a poor prognosis. The tumor microenvironment has been identified as an important regulator of tumor growth and may represent a novel target for therapy. We have developed mouse models corresponding to two different glioma molecular subtypes, proneural and mesenchymal, to identify changes in microenvironment composition between glioma subtypes. We performed Drop-Seq on endpoint tumors from the proneural glioma model and found that tumor associated macrophages and microglia are the most prominent infiltrating microenvironmental population in proneural glioma. We profiled receptor-ligand interactions between tumor macrophages and tumor cells to identify the mechanisms by which macrophages regulate tumor growth. We identified several regulators previously published in the literature that regulate glioma growth such as pleiotrophin, Igf-1, and osteopontin, as well as some new candidate regulators prosaposin, oncostatin M and thrombospondin. Future directions include validation of tumor-microenvironment interactions using CRISPR activation in vitro, characterization of the tumor microenvironment in mesenchymal-like glioblastoma via single-cell RNA-sequencing, and changes in tumor microenvironment composition and function following treatment with chemotherapy or radiotherapy. A better understanding of tumor-microenvironment interactions in glioma will aid development of new therapies to promote tumor regression

Citation Format: Rohit Rao, Ravinder Verma, Jincheng Wang, Qing Richard Lu. Characterization of glioma subtype-specific macrophage-tumor interactions using single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 144.
Abstract 1458: Study on cervical cancer preventive effect of HPV vaccine in Japan
Authors: Nishimura Masato , Kagawa Tomohiro , Mineda Ayuka , Takiguchi Eri , Abe Akiko , Irahara Minoru
Published: August 2019

Objective: In Japan, side effects are emphasized and ingestion of HPV vaccine is not widespread. Meanwhile, the number of people suffering from cervical cancer has increased, and there is concern that deaths due to cervical cancer will increase. The objective of this study is to clarify the differences in the frequency of cytological abnormalities by presence or absence of HPV vaccination, sex hormone intake, and smoking history.

Methods: We examined each item described in medical questionnaire of a consultant who received cervical cancer screening in April 2015 - March 2016 and cytological diagnosis.

Results: There are 23,001 consultation examiners for 1 year. Of these, 3609 people aged 20-29, 6457 people aged 30-39 years old, 4433 people aged 40-49 years old were examined each item. With or without HPV vaccination, the frequency of cytological abnormalities (ASC-US and more) in 20-29 years old was 8/262 (3.1%), 121/3307 (3.7%), and there was no difference. Frequency of LSIL or more was significantly lower in vaccinates as 0/262 (0%) vs 70/3307 (2.3%) p<0.05. There was no difference in the frequency of cytological abnormalities between the groups at 30-39 years old and 40-49 years old. With regard to the presence or absence of sex hormones, there was no difference in the frequency of cytological abnormalities in all ages. With smoking history, the frequency of LSIL or more was significantly higher in a group with smoking history, at the ages of 20-29 years (3.7% vs 1.4%) and 30-39 years (2.3% vs 1.2%).

Conclusion: If there is smoking history, it is important to undergo regular cervical cancer screening. In addition, HPV vaccination reduces the frequency of cytological abnormalities, so HPV vaccine is also important for the prevention of cervical cancer.

Citation Format: Masato Nishimura, Tomohiro Kagawa, Ayuka Mineda, Eri Takiguchi, Akiko Abe, Minoru Irahara. Study on cervical cancer preventive effect of HPV vaccine in Japan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1458.
Abstract 146: Multiple modalities of NanoString GeoMx™ Digital Spatial Profiler allow for spatially-resolved, multiplexed quantification of protein and mRNA distribution and abundance
Authors: Demirkan Gokhan , Viboch Elena , Merritt Chris , Ong Giang , Zevin Kristi , Sorg Kristina , Irving Lindy , Dunaway Dwayne , Geiss Gary K. , Beechem Joseph
Published: August 2019

Characterization of the spatial distribution and abundance of proteins and mRNAs with morphological context within tissues enables a better understanding of biological systems in many research areas, including immunology and oncology. However, it has proven difficult to perform such studies in a highly multiplexed manner. To address this unmet need, we have developed a novel optical-barcode based microscope and tissue-sampling platform designed to simultaneously analyze hundreds of proteins or mRNAs on a single FFPE section from distinct tissue spatial regions (GeoMxTM Digital Spatial Profiler, DSP).

Here, we present a series of modalities and associated applications for the GeoMxTM DSP platform and its integrated software. First, geometric profiling can be utilized by drawing automated circles, squares or even manual hand-drawn polygons as regions of interest (ROI) to characterize tissue heterogeneity. Second, gridded profiling offers high resolution unbiased tumor profiling by placing a grid on tissues and separately analyzing each segment in the grid. Third, contour profiling employs concentric rings, or any custom shape at a growing distance from a site of interest. Finally, segment and rare cell profiling exploits fluorophore-conjugated antibodies to profile specific cell types. These techniques can be used to discover drug mechanism of action or immune activation status, as well as to facilitate prediction of treatment response and disease progression or investigation of specific rare cell populations’ molecular profiles.

Using these multiple modalities, we spatially resolve protein and mRNA expression over 30 immune targets on FFPE tissue sections from various organs, including colon and tonsil. We demonstrate multiplexed detection from discrete regions within a tumor (tumor center and immune invasive margin), enabling systematic interrogation of immune activity in FFPE samples. Finally, we present the utility of each modality under different scenarios.

Citation Format: Gokhan Demirkan, Elena Viboch, Chris Merritt, Giang Ong, Kristi Zevin, Kristina Sorg, Lindy Irving, Dwayne Dunaway, Gary K. Geiss, Joseph Beechem. Multiple modalities of NanoString GeoMx™ Digital Spatial Profiler allow for spatially-resolved, multiplexed quantification of protein and mRNA distribution and abundance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 146.
Abstract 1460: Generation of a novel, allogeneic cell-based, Gp96-Ig/OX40L cancer vaccine, improves anti-tumor immunity and long-term memory T-cell generation
Authors: Tahiliani Vikas , Miriyala Jayalakshmi , Dillon Patrick , Rose Jason , Giffin Louise , Hutchins Jeff , Seavey Matthew M.
Published: August 2019

Our technology is focused on developing a next generation cellular vaccine platform – referred to as ComPACT (COMbination Pan-Antigen Cytotoxic Therapy), that incorporates a tumor antigen chaperone (gp96-Ig) with T-cell costimulation (OX40L-Ig), into a single tumor cell line that secretes both. Viagenpumatucel-L (HS-110; ImPACT), a human lung adenocarcinoma cell line, stably transfected to express gp96-Ig is being tested in a phase 1/2 clinical trial (NCT#02439450) with checkpoint inhibition for NSCLC. A similar line is being generated that will complement HS-110, providing costimulation in the form of secreted OX40-Ig (HS-130). To model how the addition of human HS-130 to HS-110 may impact anti-tumor immune responses, we generated mouse surrogates of these human lines and established an analogous system, that treats tumor-bearing animals with tissue-matched irradiated cancer cell lines (B16F10) expressing gp96-Ig (mHS-110) and OX40L-Ig (mHS-130) both expressing ovalbumin, as our model tumor-associated antigen. Single dose vaccination with mHS-110 identified that 1 to 10 million cells (290 ng to 2,900 ng of secreted gp96-Ig) provided sufficient anti-tumor CD8+ T-cell expansion, in vivo (*p<0.01; 1-way ANOVA), with the greatest expansion observed on day 7, post-immunization. To identify the best ratio of mHS-110 to mHS-130; a dose ratio study was performed. Fixed numbers of mHS-110 (1 million cells, 290 ng of secreted gp96-Ig) were matched with different ratios of mHS-130. Similar to our single dose vaccination study, our results demonstrated that the peak CD8+ T-cell expansion occurred on day 7 post-immunization, and that the addition of mHS-130 further boosted anti-tumor CD8+ T-cell expansion by 3-fold when the ratios of mHS-110 to mHS-130 were at a 1-to-1 ratio (290 ng of secreted gp96-Ig to 290 ng of secreted OX40L-Ig) (*p<0.05, Mann-Whitney). These animals were subsequently boosted 14-days post-immunization, and we similarly found that the 1-to-1 ratio of mHS-110 to mHS-130 gave the maximum expansion of CD8+ T cell responses, peaking on day 21 and contracting thereafter (*p<0.05; Mann-Whitney). Importantly, these ratios led to high frequencies of IFNγ+ antigen-specific CD8+ T cells at both priming and boosting, which enhanced rejection of established B16F10 tumors and increased overall survival.

Citation Format: Vikas Tahiliani, Jayalakshmi Miriyala, Patrick Dillon, Jason Rose, Louise Giffin, Jeff Hutchins, Matthew M. Seavey. Generation of a novel, allogeneic cell-based, Gp96-Ig/OX40L cancer vaccine, improves anti-tumor immunity and long-term memory T-cell generation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1460.
Abstract 1466: Combination immunotherapy successfully control tumor growth in a transgenic mouse model
Authors: Lim Kue Peng , Zainal Syafinaz , Zulaziz Natasha , Gan Chai Phei , Chai San Jiun , Lye Bryan Kit Weng , Wang Chuan , Sutavani Ruhcha V. , Ottensmeier Christian , King Emma , Thomas Gareth , Savelyeva Natalia , Cheong Sok Ching
Published: August 2019

The use of checkpoint inhibitor anti-PD1 has achieved significant clinical benefit, in recurrent and metastatic head and neck cancer patients, where the overall response rate is between 16-20%. The use of antigen-specific cancer vaccine to increase the level of cytotoxic T cells is believed to work synergistically with anti-PD1. In this study, we evaluated the efficacy of antigen-specific DNA vaccine when it’s used in combination with anti-PD1. The B6.Cg-Tg(HLA-A/H2-D)2Enge/J mice bearing established tumours overexpressing the tumour antigen were vaccinated either with anti-PD1, DNA vaccine or anti-PD1 in combination with DNA vaccine. Tumour volume was monitored and antigen-specific immune responses were evaluated at the endpoint. Our data demonstrated DNA vaccine induced antigen-specific immune responses and animals vaccinated with DNA vaccine harboured smaller tumour volume compared to controls. Significantly, animals that received DNA vaccine expressed consistently higher levels IFN-γ and resulted in the upregulation of PD1 compared to control animals. We then demonstrated mice vaccinated with both DNA vaccine and anti-PD1 had near to complete tumour control, indicating a synergistic effect of DNA vaccine with anti-PD1. Animals treated with DNA vaccine in combination with anti-PD1 also have better overall survival. Importantly, these animals showed increased antigen-specific responses by the ELISPOT assay. In summary, our data suggest antigen-specific DNA vaccine works synergistically with anti-PD1 and conferred an excellent tumour control; this opens up a new opportunity of combinatory immunotherapy that might benefit a wider population of patients in an antigen-specific manner.

Citation Format: Kue Peng Lim, Syafinaz Zainal, Natasha Zulaziz, Chai Phei Gan, San Jiun Chai, Bryan Kit Weng Lye, Chuan Wang, Ruhcha V. Sutavani, Christian Ottensmeier, Emma King, Gareth Thomas, Natalia Savelyeva, Sok Ching Cheong. Combination immunotherapy successfully control tumor growth in a transgenic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1466.
Abstract 1468: Synergistic cancer immunotherapy combination of MVA-CD40L with tumor targeting antibodies or checkpoint blockade to achieve strong antitumor immune responses against large, established tumors
Authors: Medina-Echeverz Jose , Hinterberger Maria , Giessel Raphael , Bathke Barbara , Kassub Ronny , Fiore Giovanna , Chaplin Paul , Hochrein Hubertus , Lauterbach Henning
Published: August 2019

Virus-based vaccines and appropriate costimulation potently enhance antigen-specific T cell immunity against cancer. In the present study, we exploit both innate and adaptive immune responses triggered by recombinant modified vaccinia virus Ankara (rMVA) encoding costimulatory CD40L against large, established tumors in different combinatory settings. Therapeutic treatment with rMVA-CD40L resulted in the control or eradication of solid tumors in several unrelated tumor models. The expansion of non-exhausted, tumor-specific cytotoxic CD8+ T cells was essential for the therapeutic activity and was partially dependent on CD8α+ cross-presenting dendritic cells. Combination of rMVA-CD40L with PD-1 checkpoint blockade further enhanced the therapeutic activity of MVA virotherapy in colorectal carcinoma. In addition, rMVA-CD40L combined with antibodies targeting Tumor-Associated Antigens (TAA) resulted in increased therapeutic antitumor efficacy against two unrelated tumor models. We describe a translationally relevant therapeutic synergy between viral vaccination and CD40L costimulation. We show strengthened antitumor immune responses when both rMVA-CD40L-induced innate and adaptive immune mechanisms are exploited by combining immunotherapeutic regimes, such as checkpoint blockade and TAA targeting antibodies. This novel immunotherapeutic approach could translate into clinical cancer therapies where ADCC competent TAA targeting antibodies PD-1 checkpoint blockade are employed.

Citation Format: Jose Medina-Echeverz, Maria Hinterberger, Raphael Giessel, Barbara Bathke, Ronny Kassub, Giovanna Fiore, Paul Chaplin, Hubertus Hochrein, Henning Lauterbach. Synergistic cancer immunotherapy combination of MVA-CD40L with tumor targeting antibodies or checkpoint blockade to achieve strong antitumor immune responses against large, established tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1468.
Abstract 147: Deletion of the RNA regulator HuR in microglia/macrophages promotes an anti-tumor microenvironment in glioblastoma
Authors: Wang Jiping , Leavenworth Jianmei , Hjelmeland Anita , Borg Ben , King Peter H.
Published: August 2019

Glioblastoma is a malignant brain tumor that portends a poor prognosis. Its resilience, in part, is related to a remarkable capacity for manipulating the microenvironment to promote its growth and survival. Microglia/macrophages are a prime target, being drawn into the tumor and stimulated to produce factors that support tumor growth and immune system evasion. Here we show that the RNA regulator, HuR, plays a key role in the tumor-promoting response of microglia/macrophages. Knockout of HuR in microglia/macrophages led to reduced tumor growth and prolonged survival in a murine model of glioblastoma. Analysis of tumor composition by flow cytometry showed that tumor associated macrophages were decreased, more polarized toward an M1-like phenotype, and had attenuated PD-L1 expression. There was a concomitant reduction in tumor-associated polymorphonuclear myeloid-derived suppressor cells, but an increase in Treg cells. In vitro assays showed reduced migration of HuR-/- microglia toward secreted factors derived from glioblastoma cells, and a decrease in migration of glioblastoma cells toward secreted factors derived from HuR-/- microglia. The molecular response of HuR-/- microglia was altered including reduced CXCL1, 2, MMP2, PD-L1 and VEGF expression and an increase in CXCL10 and several MMPs. There was a mixed effect on cytokines and other factors associated with both proinflammatory and alternatively activated phenotypes. In summary, HuR is a key modulator of the tumor microenvironment in glioblastoma, promoting tumor progression through its molecular regulation of factors produced by microglia/macrophages. These findings underscore the relevance of HuR as a therapeutic target in glioblastoma.

Citation Format: Jiping Wang, Jianmei Leavenworth, Anita Hjelmeland, Ben Borg, Peter H. King. Deletion of the RNA regulator HuR in microglia/macrophages promotes an anti-tumor microenvironment in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 147.
Abstract 1473: Dual oncolytic viral immunotherapy generates large polyfunctional tumour-specific CD8 + T cell responses that infiltrate immunosuppressive tumors
Authors: Stojdl David
Published: August 2019

Generating effective T cell mediated clearance of solid tumors remains an unmet challenge in cancer therapy. Suppressive tumor microenvironments limit the generation of significant numbers of tumor specific T effector cells, their migration to tumor beds, and their subsequent functionality within tumors, thereby blocking the patient’s normally potent acquired immune response from contributing to tumor control.

In an effort to meet this challenge, we have developed novel oncolytic viruses that were bioselected and engineered to cause cancer cell death through two distinct and complementary mechanisms-of-action, direct cancer lysis and tumor-antigen specific T cell generation. Specifically, we have selected and engineered MG1 Maraba virus which both infects tumor tissue to reverse immune suppressive programs while simultaneously delivering vector encoded tumor antigens to the spleen to vaccinate against the patient’s tumor. The result is a large increase in peripheral and tumor infiltrating CD8+ T effectors cells, strong intratumoral inflammatory signatures and ultimately curative efficacy in preclinical solid tumor models. This first-in-class therapeutic strategy is currently being evaluated in Phase 1 and Phase 2 clinical trials.

In this new study, we describe the development of a novel viral immunotherapy platform based on Farmington virus that is: (1) oncolytic in solid tumor models, (2) a potent inducer of highly-functional antigen-specific T cells (expanding tumor specific CD8+ T effector pools over 1000 fold from pre-existing T central memory) and is (3) immunologically distinct from our clinical MG1 Maraba platform. We show that when used sequentially in a heterologous boosting regimen, T cell responses to encoded tumor antigens, either foreign (HPV E7), self (TRP2) or multi-neoantigen, can exceed greater than 50% of all CD8+ T cells in the periphery. The majority of these CD8+ T effectors show markers of polyfunctionality with little expression of the PD-1 exhaustion marker. We will describe our current data assessing the phenotype, localization and potency of these T cell responses in preclinical models of solid tumors and propose strategies to deploy our novel dual oncolytic viral immunotherapy boosting paradigm to the clinic.

Citation Format: David Stojdl. Dual oncolytic viral immunotherapy generates large polyfunctional tumour-specific CD8 + T cell responses that infiltrate immunosuppressive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1473.
Abstract 1476: Decoding the immunologic landscape of human clear cell renal carcinoma
Authors: Chowdhury Nivedita , Obradovic Aleksandar , Aggen David , Wang Vinson , Guo Xinzheng , DeCastro Guarionex J. , Ding Hongxu , Califano Andrea , Drake Charles G.
Published: August 2019

Immune cells in the tumor microenvironment (TME) play a critical role in regulating tumor development and response to targeted immunotherapy. While it is well-accepted that clear cell renal carcinoma (ccRCC) is characterized by a T-cell infiltrate, differential activation states within the tumor and the contribution of other immunomodulatory cells remain relatively unexplored. We sequenced approximately 30,000 immune cells at the single-cell level from tumor and matched adjacent normal tissue in ccRCC patients. With a median of 3300 Unique Molecular Identifiers (UMIs)/cell, we were able to capture the expression of 1000 genes per cell on average and 21,000 genes across all cells from each patient. We used Multidimensional Scaling (MDS) -transformation to visualize the various clusters and found that the tumor and adjacent normal tissue were distinct, with each having two major sub-clusters - lymphoid and myeloid cells. The data demonstrated a higher fraction of lymphoid infiltrate(~70-75%) than myeloid in a relatively stable ratio across patients, with tumor containing a slightly higher fraction of lymphoid cells. The normal and tumor cell subtypes were distinguished by multiple iterations of PAM clustering (Partitioning Around Mediods) with feature selection using the iterClust algorithm. Top differentially expressed genes in each cluster were identified and cross-referenced with known immune cell markers along with pathway enrichment analysis for cell typing. Among the tumor lymphoid population, genes specific for T-cells, including markers of functional exhaustion were the most highly expressed. A separate sub-cluster of cells consistent with cytotoxic T and/or NK cells was also identified. We found a distinct population of monocytes within the myeloid cluster that was present exclusively in the tumor tissue. This population expressed high levels of complement factors C1QA, C1QB, C1QC, as well as ApoE. The latter has been implicated as a driver of myeloid cell immunosuppression, indicating a possible regulatory role in the TME of ccRCC that has not previously been described. The phenotype of the immune cell populations in tumor was further validated by spectral flow cytometry. This study demonstrates relative abundance of cell types in the immune microenvironment within ccRCC tumors and also identifies a novel C1Q-high myeloid sub-population specific to the tumor, suggesting its possible role in RCC tumorigenesis or progression.

Citation Format: Nivedita Chowdhury, Aleksandar Obradovic, David Aggen, Vinson Wang, Xinzheng Guo, Guarionex J. DeCastro, Hongxu Ding, Andrea Califano, Charles G. Drake. Decoding the immunologic landscape of human clear cell renal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1476.
Abstract 1480: A novel mechanism of immunosuppression via nanotube mediated mitochondrial trafficking between cancer cell and immune cell
Authors: Saha Tanmoy , Dash Chinmayee , Khiste Sachin , Sengupta Shiladitya
Published: August 2019

Cancer cells deploy multiple mechanisms for immune evasion. Here we report a novel mechanism of cancer cell-induced immune evasion via organelle trafficking between cancer cells and immune cells through physical nanotubes. Using high resolution scanning electron microscopy to analyze a co-culture assay of breast cancer cells and NKT cells revealed that the cancer cells and NKT cells form physical nanotube connections at early time points. Immunofluorescence imaging and quantification using flow cytometry revealed that the cancer cells rapidly hijack mitochondria from immune cells through these nanotubes. The transfer of mitochondria from immune cell to cancer cells imposes a metabolic impairment and results in a reduced population of immune cells. This is not observed in a control coculture using a Boyden chamber, where the two cell types are prevented from forming nanotubes. As the nanotubes are assembled via cytoskeletal rearrangement, we next tested whether the members of Rho-family of GTPases, Cdc42, Ral and Rac are involved in the actin polymerization. Indeed, an inhibitor for Cdc42/Rac1 GTPase inhibitor (ML141) and a geranylgeranyltransferase 1 inhibitor (L-778,123) significantly decreased mitochondrial transfer, and prevented the depletion of NKT cells in co-culture. Furthermore, treating 4T1 tumor-bearing animals with a combination of L-778,123 and a PD1 immune checkpoint inhibitor increased the anti-tumor efficacy compared to PD1 inhibition alone.

Conclusion: Our study sheds insights on a novel mechanism of cancer immune suppression via nanotube-mediated intercellular organelle trafficking. The efficacy of GTPase- or geranylgeranyltransferase-inhibitors, in perturbing this process, and the increased efficacy of the combination with PD1 inhibitor, opens up the possibility of developing these as novel immunotherapy agents for cancer.

Citation Format: Tanmoy Saha, Chinmayee Dash, Sachin Khiste, Shiladitya Sengupta. A novel mechanism of immunosuppression via nanotube mediated mitochondrial trafficking between cancer cell and immune cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1480.
Abstract 1486: The immune atlas and chemokine profile of giant cell tumor of bone
Authors: Li Binghao , Wang Zhan , Ye Zhaoming
Published: August 2019

Background and purpose: Giant cell tumor of bone (GCTB) is a locally aggressive tumor with metastatic potential. Given that GCTBs are composed of three major cell populations and the bone-resorption activity is attributed to the multinucleated giant cells which are conjectured to be recruited by “real” tumor cells, it is still elusive that how and what are these immune cells recruited. The purpose of this study is to present the immune landscape of GCTB, as well as to find out responsible chemokines during immune cell recruiting.

Experimental procedures: We collected fresh GCTB tissues from surgical excisions. To identify immune cell composition within the tumor microenvironment, we performed mass cytometry (CyTOF) and conventional multicolor cytometry. Some fresh osteosarcoma and chondrosarcoma samples were also used to compare the immune atlas. For chemokine profiling, chemokine arrays were utilized. Further analysis on potential association between chemokines and immune cells were conducted.

Results: For last updated, 47 GCTB fresh samples were collected, of which 12 cases received CyTOF analysis and the rest 35 samples were analyzed by multicolor cytometry. All GCTB samples were analyzed by chemokine array In-depth interrogation of the immune landscapes showed that compared with osteosarcoma and chondrosarcoma, GCTBs had some unique immune cell subgroups. Some CD163+ CD206+ macrophage clusters which expressed various immune suppressive markers were significantly enriched in GCTBs, while CD4+ CD25+ Treg cells were fewer in GCTB. In addition, some macrophage-like mononuclear cells (CD14- CD68+ CD163- CD206+ HLA-DR+) were only seen in GCTBs.Chemokine array revealed that higher levels of CXCL12, CCL22, and CCL7 might be associated with moremacrophage-like mononuclear cells in GCTBs.

Conclusion: GCTB have very different immune features from other malignant bone tumors. Multiple chemokines may play important roles in recruiting “functional” monocytes. Such in-depth understanding has important implications in appropriate therapeutics for GCTB. Bigger sample size will further help in studying the immune and chemokine features of GCTB.

Note: This abstract was not presented at the meeting.

Citation Format: Binghao Li, Zhan Wang, Zhaoming Ye. The immune atlas and chemokine profile of giant cell tumor of bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1486.
Abstract 1496: Novel hCD3e mouse models for preclinical pharmacology studies of therapeutic bi-specific antibodies
Authors: Xiang Jie , Li Yanan , Shen Yuelei , Guo Yanan
Published: August 2019

Recently it has been demonstrated that T cell-mediated immunotherapy is an attractive treatment strategy for various diseases. For this therapeutic approach, the CD3 complex is one of the key targets to modulate T cell activation; however, in many instances we cannot directly evaluate antibody candidates in mice because the monoclonal antibodies specific to human CD3 cannot cross-react to mouse endogenous CD3. In order to overcome the current model limitation, we have generated CD3e-extracellular domain KI mice for anti-human CD3e bispecific antibody in vivo efficacy evaluation. In this model, the mouse CD3e extracellular domain was replaced by human CD3e. We used conventional mouse ES/HR technology to knock-in human CD3e genomic DNA from the 3’ end of exon 2 to the 5’ end of exon7 to replace murine exon 2-exon 6. CD3e is one of four subunits of the CD3 co-receptor, which plays an important role in the immune response, associating with the T cell receptor (TCR) to couple antigen recognition to intracellular signal transduction, leading to T cell activation. We have characterized our B-hCD3e mice with the following features:

1.Mouse CD3e+ cells were detected in the wildtype C57BL/6 mice, while human CD3e+ cells were only detected in the homozygous B-hCD3e mice.

2.T cell development was normal in B-hCD3e homozygous mice.

3.T cell subtypes in the spleen, thymus, lymph nodes, and PBMC in B-hCD3e mice were similar and comparable to that in wildtype C57BL/6 mice.

4.Normal in vitro T cell proliferation and cytokine production were found in anti-hCD3e antibody treated humanized B-hCD3e mice

5.Mouse anti-PD-1 antibody can significantly repress the growth of MC38 tumor cells engrafted on B-hCD3e mice, suggesting that T cells in B-hCD3e mice are functionally normal

6.Treatment of B-hCD3e mice with anti-human CD3e antibody OKT leads to T cell depletion, resulting in more aggressive tumor growth.

Furthermore, efficacy and toxicity evaluation of CD3e based bispecific antibodies in B-hCD3e mice are ongoing. Based on our data from extensive phenotypical analyses of B-hCD3e mice, we believe this novel humanized CD3e mouse model will be very useful for in vivo efficacy studies of CD3- mediated therapy, especially anti-CD3e bi-specific antibodies.

Citation Format: Jie Xiang, Yanan Li, Yuelei Shen, Yanan Guo. Novel hCD3e mouse models for preclinical pharmacology studies of therapeutic bi-specific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1496.
Abstract 1504: Characterization of a MC38 mouse syngeneic tumor model expressing human PD-L1 in the transgenic C57BL/6 mouse system expressing human PD-1 and PD-L1
Authors: Koratich Michael , Green Ted , Liu Jie , Hammond Charlotte , Durbin LaJuana , Zhou Jerry , Chen Anna , Stackhouse Murray
Published: August 2019

The human checkpoint targets PD-1 and PDL-1 continue to demonstrate great promise in the clinic. With much of the current focus turning toward combination regiments, an animal model using the human clinical antibodies will be beneficial for evaluating new combination strategies. Here we continue characterization of a C57BL/6 transgenic mouse model expressing both the human PD-1 and PD-L1 combined with a modified murine MC38 colon tumor cell line expressing human PD-L1. Both PD-1 and PD-L1 expression of the transgenic mice were verified in ex vivo stimulated splenocytes by flow cytometry analysis. Expression of PD-L1 on the genetically modified MC38 cells was also demonstrated by flow cytometry. For in vivo efficacy evaluation, tumor-implanted mice were treated with the clinical agents nivolumab and pembrolizumab at 100 µg and atezolizumab at 1 mg on Days 3, 7, 10, and 14 post implant. Treatment with nivolumab and pembrolizumab initiated tumor regression by Day 17. Complete tumor regressions were seen at Day 28 in nivolumab, 62.5% complete regression, and pembrolizumab, 71.4% complete regression. Growth inhibition was 83.9%, 69.7%, and 95.0% on Day 28 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. To compare the human anti-PD-1 and PD-L1 clinical agent specificity, the non-transgenic parent C57BL/6 mouse strain was implanted with the unmodified MC38 colon tumor cells. Treatment with pembrolizumab and atezolizumab was conducted as with the transgenic animals. Through 28 days no growth inhibition, tumor size 105.9% of control, was seen in the pembrolizumab treated group, demonstrating lack of cross reactivity of the human therapeutic in the standard mouse model. Atezolizumab did demonstrate a 56.9% growth inhibition compared to controls and is consistent with the known cross reactivity of atezolizumab between human and mouse. We have shown a genetically modified MC38 colon tumor expressing human PD-L1 in transgenic mice expressing both human PD-1 and PD-L1 to be a suitable model for checkpoint inhibitor evaluation of the human form of the particular checkpoint therapeutic. Further research will involve combining each of the checkpoint antibodies with various chemotherapeutic agents.

Citation Format: Michael Koratich, Ted Green, Jie Liu, Charlotte Hammond, LaJuana Durbin, Jerry Zhou, Anna Chen, Murray Stackhouse. Characterization of a MC38 mouse syngeneic tumor model expressing human PD-L1 in the transgenic C57BL/6 mouse system expressing human PD-1 and PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1504.