bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract B003: Developing an analytical method for second messenger cyclic GMP-AMP
Authors: Li Tuo , Chen Zhijian J. 
Published: May 2016

Abstract
The presence of double-stranded DNA (dsDNA) in the cytosol is a danger signal associated with microbial infections and tumor microenvironment. The cytosolic DNA surveillance pathway (CDSP) is critical for sensing such signal to elicit type I interferon response, which facilitates infection clearance and tumor rejection. Uncontrolled engagement of CDSP is also linked to autoimmune disease. The key components of CDSP are the DNA sensor—cyclic GMP-AMP synthase (cGAS), the second messenger—cyclic GMP-AMP (cGAMP), and the endoplasmic reticulum localized adaptor—stimulator of interferon inducible genes (STING). While numerous efforts, including ours, are focusing on developing STING agonists formulated cancer vaccine, an analytical method that is sensitive and reliable for cGAMP and other cyclic dinucleotides is also in high demand to enable quantitative investigations. In this work, we developed and optimized a rapid quantification workflow that integrates solid phase extraction, hydrophilic liquid chromatography and multiple reaction monitoring mass spectrometry. This workflow enables detection of cGAMP in cells at sub-femtomole levels and is amenable for scaling up for high throughput analysis. A biochemically synthesized nonradioactive isotope labeled cGAMP standard enables absolute quantification. As a proof of principle, this method is first applied to probe cGAMP in macrophages infected with Mycobacterium tuberculosis, and to help establish cGAS as the innate immune sensor for the pandemic pathogen. Second, constitutive levels of cGAMP in organs of autoimmune mouse models, of Trex1 or Dnase2 deficiencies, are quantified. The result indicates a direct engagement of the cGAS-cGAMP-STING pathway in the autoimmune pathology. Finally, we show that cGAMP is produced when the apoptosis program is initiated in conjunction with pro-apoptotic caspase inhibition. This shows that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response and to inactivate this response in a caspase-dependent manner. Altogether, these studies demonstrated the usefulness of this analytical method for quantifying cGAMP in a high dynamic range under different biological contexts. We are currently applying this method to measure the activation of the cGAS pathway in tumor immunity and immunotherapy.
Citation Format: Tuo Li, Zhijian J. Chen. Developing an analytical method for second messenger cyclic GMP-AMP. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B003.
Abstract B005: An immunohistochemical PD-L1 companion diagnostic assay for treatment with durvalumab (MEDI4736) in NSCLC patients
Authors: Rebelatto Marlon C. , Mistry Amita , Sabalos Constantine , Schechter Nicole , Walker Jill , Midha Anita , Steele Keith E. , Robbins Paul B. , Li Xia , Shi Li , Blake-Haskins John A. , Ibrahim Ramy A. , Richman Laura 
Published: May 2016

Abstract
Background: A high quality PD-L1 companion diagnostic may help predict which patients are more likely to respond to PD-1/PD-L1 antibody-based therapy. Here we describe a PD-L1 immunohistochemical (IHC) diagnostic test developed by Ventana Medical Systems for use with durvalumab.
Methods: An anti-human PD-L1 rabbit monoclonal antibody (SP263) was optimized for use with Ventana OptiView DAB IHC Detection Kit on the automated BenchMark ULTRA platform. The PD-L1 IHC assay was validated for use in formalin-fixed, paraffin-embedded samples of NSCLC in a series of studies addressing sensitivity, specificity, robustness, and precision. Durvalumab is a human IgG1 mAb that blocks PD-L1 binding to PD-1 and CD80 with high affinity and selectivity. A subset of clinical trial samples from a Phase 1/2 study of durvalumab (NCT01693562) was analyzed to determine optimal cut-off for enriching response to durvalumab. Inter-reader precision was established by 3 pathologists who evaluated 81 NSCLC samples across the range of expression levels.
Results: The Ventana PD-L1 IHC (SP263) assay met all pre-defined acceptance criteria. The scoring algorithm was defined using statistical analysis of clinical response data and PD-L1 staining parameters observed in a set of NCT01693562 clinical trial samples. Samples of both cancer types are considered test positive when the membrane of ≥ 25% of tumor cells stain for PD-L1 at any intensity. Inter-reader precision in determining PD-L1 status resulted in an overall percentage agreement of 97% for NSCLC. PD-L1+ patients identified by the scoring algorithm had a higher response rate than PD-L1- patients. Interlaboratory testing was performed at 3 external laboratories and demonstrated an overall agreement rate of 86.4%.
Conclusions: These results highlight the robustness and reproducibility of the PD-L1 IHC (SP263) assay in a clinical setting. In NSCLC patients treated with durvalumab, PD-L1+ patients identified by the scoring algorithm had a higher response rate than PD-L1- patients. The clinical utility of the PD-L1 diagnostic assay will be further validated in a prospective manner using additional patients in this study and in other durvalumab studies.
Citation Format: Marlon C. Rebelatto, Amita Mistry, Constantine Sabalos, Nicole Schechter, Jill Walker, Anita Midha, Keith E. Steele, Paul B. Robbins, Xia Li, Li Shi, John A. Blake-Haskins, Ramy A. Ibrahim, Laura Richman. An immunohistochemical PD-L1 companion diagnostic assay for treatment with durvalumab (MEDI4736) in NSCLC patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B005.
Abstract B018: Developmental and functional characterization of immunoglobulin kappa chain-bridged single chain bi-specific antibody for targeted antitumor therapy
Authors: Lo Yu Hsun , Yu Jei-Hwa , Hsui Yu-Hsen , Yuan Ta-Tung 
Published: May 2016

Abstract
The immune system has routinely been the focus of cancer treatments. One of the strategies that targets the immune system is by use of monoclonal antibodies to specifically target tumor antigens leading to immune activation and tumor destruction. Therefore, many biopharmaceutical companies are interested/invested in antibody development, including the development of next-generation antibodies such as bispecific antibodies. The Development Center for Biotechnology (DCB) in Taiwan has developed a new platform for the generation of immunoglobulin kappa chain-bridged single chain bispecific antibody. Using this platform, DCB constructed a kappa domain-bridged HER2/CD3 bispecific single chain antibody known as the LBscbHER2-CD3. Utilizing a mammalian expression system, the antibody can be easily purified by affinity chromatography via kappa fragment capture. Binding affinity studies have revealed that the purified LBscbHER2-CD3 antibody is able to independently as well as simultaneously bind to HER2+ tumor cells and CD3+ T cells at low nanomolar concentrations. In co-culture experiments, the addition of purified LBscbHER2-CD3 antibody resulted in a significant increase in IL-2 production which is a strong indicator of human T cell activation. In addition, the co-engagement of either IL-2 primed or un-primed human T cells and HER2+ tumor cells by the LBscbHER2-CD3 antibody facilitated the killing of tumor cells from breast, colon and pancreatic cancers. Lastly, when administered in vivo, LBscbHER2-CD3 showed strong tumor growth inhibition in SCID mice xenograft models with implanted HER2+ SW480 cancer cells, HT29 human colon carcinoma cells, and Capan-1 human pancreatic cancer cells. In conclusion, the LBscbHER2-CD3 bispecific antibody has strong affinity for both the cancer cell-expressing receptor and the T cell antigen which leads to cancer cell elimination in vitro and in vivo. These results suggest that LBscbHER2-CD3 may have therapeutic potential for drug development in the treatment of human cancers.
Citation Format: Yu Hsun Lo, Jei-Hwa Yu, Yu-Hsen Hsui, Ta-Tung Yuan. Developmental and functional characterization of immunoglobulin kappa chain-bridged single chain bi-specific antibody for targeted antitumor therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B018.
Abstract B038: GMP production of cord blood-derived dendritic cell-based vaccine to prevent relapses after hematopoietic cell transplantation in children with AML
Authors: Haar Colin de , Dunnebach Ester , Til Niek van , Plantinga Maud C. , Blokland Nina JG , Nierkens Stefan , Boelens Jaap Jan 
Published: May 2016

Abstract
Introduction: Pediatric patients with refractory/relapsed acute myeloid leukemia (AML) have only one treatment option: hematopoietic cell transplantation (HCT). Using cord blood (CB)-derived stem cells, instead of cells from bone marrow cells or peripheral blood, results in less relapses (increased anti-tumor reactivity) and less graft-versus-host disease (increased safety). Although this treatment is potentially curative, still more than half of the children die from relapses. We therefore aim to develop additional powerful and safe CB-derived immunotherapies for pediatric AML.
Methods: We have successfully translated our pre-clinical protocol for generations of a dendritic cell (DC) vaccine from CD34+ CB stem cells into GMP production. This production process enables us to generate sufficient CBDC for at least 4 vaccinations when using the 20% fraction of the CB unit. After CliniMACS isolation the CD34+ cells are expanded in culture bags using medium containing FLT3L, SCF, IL-3 and IL-6. When sufficient expansion is reached the cells are differentiated into DCs using medium containing FLT3L, SCF, GM-CSF and IL-4. The CBDCs are then matured using proinflammatory cytokines and electroporated with Wilms' Tumor 1 (WT1) mRNA as tumor antigen. After 4 hours recovery, cells are cryopreserved until time of validation or intradermal vaccination
Results: CD34+ CB stem cells were expanded and differentiated into DCs. CBDCs upregulated co-stimulatory molecules after maturation and showed enhanced CCR7-dependent migration towards CCL19 in a trans-well migrations assay. In addition, CBDCs expressed the tumor antigen Wilms' Tumor 1 (WT1) protein after electroporation with WT1-mRNA. These WT1 expressing CBDCs were not only able to stimulate T cells in a mixed lymphocyte reaction but in an antigen-specific setting as well.
Conclusions: We are able to produce a GMP CBDC vaccine with the goal to stimulate the anti-tumor reactivity of the newly developing immune system in AML patients after CB-HCT in a Phase 2 clinical trial starting early 2016.
Citation Format: Colin de Haar, Ester Dunnebach, Niek van Til, Maud C. Plantinga, Nina JG Blokland, Stefan Nierkens, Jaap Jan Boelens. GMP production of cord blood-derived dendritic cell-based vaccine to prevent relapses after hematopoietic cell transplantation in children with AML. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B038.
Abstract B051: Control of regulatory T (Treg) cell function by protein kinase C-eta (PKCη): A novel target for cancer immunotherapy
Authors: Pedros Christophe , Kong Kok-Fai , Altman Amnon 
Published: May 2016

Abstract
Modulating regulatory T (Treg) cell functions to specifically promote anti-tumor immune responses is a desirable strategy for cancer immunotherapy. Blockade of checkpoint molecules, CTLA-4 and PD-1, has proven clinical efficacy in advanced metastatic melanoma and other malignancies. We recently found that protein kinase C-eta (PKCη), a member of the PKC enzyme family, physically associated with CTLA-4, an inhibitory receptor highly expressed on Treg cells, which is important for Treg cell-mediated suppression. PKCη is recruited to the Treg cell immunological synapse, and mediates contact-dependent suppression by Treg cells. PKCη-deficient (Prkch-/-) Treg cells are defective in the suppression of anti-tumor immunity against B16 melanoma but retain the ability to control the development of autoimmune colitis. Here, we extended our findings regarding the importance of the CTLA-4-PKCη complex in contact-dependent Treg cell suppressive activity to an additional tumor model, i.e., the slow growing prostate adenocarcinoma TRAMP-C1, indicating that the novel CTLA-4-PKCη signaling pathway may be relevant to a broad spectrum of solid cancers. We further found that in the tumor microenvironment, the loss of PKCη in Treg cells substantially promoted the infiltration of CD4+ and CD8+ effector T cells without affecting the Treg cell numbers. As a result, there was a significant increase in the intratumoral CD8+ to Treg cell ratio, a parameter considered to be predictive of a better clinical outcome. Thus, our data reveal that the novel CTLA-4-PKCη signaling pathway, which we identified, is critical for suppressing anti-tumor immunity and, hence, implicate this pathway as an attractive cancer immunotherapy target for a broad spectrum of clinically debilitating malignancies.
Citation Format: Christophe Pedros, Kok-Fai Kong, Amnon Altman. Control of regulatory T (Treg) cell function by protein kinase C-eta (PKCη): A novel target for cancer immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B051.
Abstract B063: Unsaturated fatty acids cause CD8+ T cell exhaustion in melanoma
Authors: Cui Guoliang , Kaech Susan 
Published: May 2016

Abstract
Remarkable progress in cancer immunotherapy has revealed the power of harnessing immunity to combat cancer. However, the immunosuppressive nature of the tumor microenvironment remains a fundamental challenge to improving immune-based anti-cancer therapies. We aim to develop new immunotherapies by studying the nutrient utilization of immune cells in the tumors. Using an inducible Braf/Pten melanoma model, we found that tumors contain higher amounts of available free fatty acids (FFAs) and lower amounts of glucose than the blood. Exposure to high concentration of FFAs induced an exhaustion-like phenotype of CD8+ T cells. Furthermore, mice deficient of FFA transporter CD36 developed smaller tumors compared to wild-type control mice. Tumor-infiltrating Cd36-/- CD8+ T cells expressed lower level of inhibitory receptors, such as PD-1 and LAG-3. This study highlights an important role of lipid metabolism in regulating T cell exhaustion in the tumor microenvironment and reveals CD36 as a potential drug candidate for immunotherapy.
Citation Format: Guoliang Cui, Susan Kaech. Unsaturated fatty acids cause CD8+ T cell exhaustion in melanoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B063.
Abstract B067: Successful immunotherapy of cancer stem cells requires memory and the adaptive immune system of the host
Authors: Schmidt Patrick , Desbaillets Nicolas , Dessen Pierre , Thompson Nancy , Huelsken Joerg , Jäger Dirk 
Published: May 2016

Abstract
The Cancer Stem Cell (CSC) hypothesis puts a, not necessarily rare, subpopulation of cells on the top of a tumor forming hierarchy, pointing to the importance of their destruction when aiming on a final cure. However, current studies targeting CSCs rather focus on the tumor initiating capacities of these cells than on their maintaining capacity in an established lesion. In this presentation we describe an immunotherapeutic approach of targeting the minor CSC subfraction by exploiting the stem cell related wnt-pathway. Within a fully syngeneic mouse tumor model, we labelled 4T1 breast tumor cells by lentiviral transduction to express a foreign antigen in wnt-active cells. When transferred into the host, these cells recapitulated CSC behavior and were concomitantly targetable by chimeric antigen receptor engineered T cells. All mice receiving a CSC specific CAR treatment showed long term metastases free survival, although a minority of cells served as actual aim of treatment. We investigated the consequences of CSC eradication at different time points after treatment and found that a memory effect, provided by engineered T cells, is essential to keep the re-formation of CSCs from bulk tumor cells under control. Furthermore, our treatment setting is able to re-activate the host's non engineered T cells against the majority of tumor cells, which leads to a long lasting tumor-free survival in our mouse model system. These findings implicate a leading role for immunotherapy when targeting the tumor stem cell fraction.
Citation Format: Patrick Schmidt, Nicolas Desbaillets, Pierre Dessen, Nancy Thompson, Joerg Huelsken, Dirk Jäger. Successful immunotherapy of cancer stem cells requires memory and the adaptive immune system of the host. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B067.
Abstract B074: Tumor-reactive CD4+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy
Authors: Pelletier Sandy , Turcotte Simon 
Published: May 2016

Abstract
Purpose: To evaluate whether patients with metastatic gastrointestinal adenocarcinomas refractory to chemotherapy harbor tumor-reactive CD4+ T cells.
Experimental Design: Expansion of CD4+ tumor-infiltrating lymphocytes (TIL) and cancer cell lines was performed from gastrointestinal cancer metastases in 5 patients for the study of antitumor immune recognition. Retroviral transduction of genes encoding the class II, major histocompatibility complex, transactivator (CIITA) was used to induce the expression of major histocompatibility complex (MHC) class II in tumor cell lines. Recognition of autologous tumor cell lines by TIL was evaluated by up-regulation of 4-1BB and OX40 by flow cytometry, and/or secretion of IFNg by ELISA.
Results: TIL were expanded from metastases, and new tumor cell lines were generated in 5 patients. Retroviral transduction of CIITA in tumor cell lines effectively induced expression of MHC class II in &gt;80% of cells. Autologous tumor recognition was found in CD4+ TIL from 2 of these 5 patients. In a patient with gastric cancer liver metastases, tumor-reactive CD4+OX40+ TIL were cell-sorted from a TIL cell line. These cells up-regulated OX40 in the presence of all 4 autologous cancer cell lines albeit at different levels, but they did not produce IFNg. This recognition was specifically abolished by pan-MHC class II blocking antibodies. CD4+ TIL clones have been isolated and are being further characterised. Interestingly, tumor-reactive CD8+ TIL were previously identified and characterized in this patient. In a second patient with colon cancer abdominal wall metastases, tumor-reactive CD4+OX40+ TIL were cell-sorted from a TIL cell line. These cells were reactive to all 4 autologous cancer cell lines as they up-regulated OX40 and secreted IFNg. Recognition was blocked by anti-HLA-DR blocking antibodies. CD4+ TIL clones have been isolated and are being further characterized. In the near future, we expect to identify the TCR and HLA restriction element for both patients. We will also determine if CD4+ TIL clones have cross-reactivity against allogeneic HLA-matched gastrointestinal tumor cell lines.
Conclusions: This study provides a basis for the development of immunotherapy for patients with advanced gastrointestinal malignancies by first establishing the presence of naturally occurring tumor-reactive CD4+ TIL at the molecular level.
Citation Format: Sandy Pelletier, Simon Turcotte. Tumor-reactive CD4+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B074.
Abstract B102: Releasing oxygen-restricted CTL function: Insights from live intratumoral imaging
Authors: Feferman Tali , Manaster Yoav , Gruper Yael , Shipony Zohar , Shakhar Guy 
Published: May 2016

Abstract
Poor tumor vascularization is an obstacle to immunotherapy by CTLs. It impairs tumor infiltration but also introduces hypoxia, known to interfere with T cell migration. It is yet unknown how suboptimal vascularization affects CTL migration and function within tumors. To study this question, we combined immunohistochemistry of human melanoma samples with two-photon imaging in live mice. Orthotopically implanted B16-OVA tumors were studied after adoptive transfer of in-vitro matured antigen-specific OT-I CTLs. In patients, CD8 T cells concentrated around peripheral vessels in the tumor and sparsely infiltrated avascular areas. This pattern was clearest in poorly-infiltrated tumors. In mice, OT-I CTLs, as well as polyclonal CTLs, entered tumors through inflamed intratumoral vessels, and crawled only in oxygenated areas within 50 µm of flowing blood vessels. Gradually, CTLs formed dense perivascular swarms, which eliminated nearby target cells leading to patchy tumor clearance around blood vessels. Occluding intratumoral blood vessels triggered immediate arrest of CTL motility, which was quickly reversed when flow was resumed. Immunohistology indicated that CTLs avoided hypoxic tumor areas. Live CTL imaging in vitro showed deceleration under hypoxic conditions and when oxidative phosphorylation was blocked. To circumvent intratumoral CTL dysfunction we attempted to increase vascular density by implanting tumors in matrices containing bFGF. bFGF-laced tumors were more easily rejected after transfer of CTLs and displayed delayed growth in untreated mice but were not affected in mice deficient in CD8 T-cells. CTLs infiltrated such tumors in normal numbers, but displayed enhanced motility in highly-vascular tumors, suggesting that enhanced rejection resulted from improved intratumoral CTL migration. Taken together, the results suggest that hypoxia limits CTL function away from blood vessels, and that alleviating it may synergize with immunotherapy.
Citation Format: Tali Feferman, Yoav Manaster, Yael Gruper, Zohar Shipony, Guy Shakhar. Releasing oxygen-restricted CTL function: Insights from live intratumoral imaging. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B102.