bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A145: The prognostic significance of programmed cell death-ligand 1 expression in pulmonary squamous cell carcinoma
Authors: Hung Jung-Jyh , Wu Yu-Chung , Hsu Wen-Hu
Published: November 2016

Introduction: The programmed cell death-ligand 1 (PD-L1) pathway plays an important role in maintaining immune homeostasis. PD-L1 pathway may also protect tumor from attack of cytotoxic T cells. The prognostic significance of PD-L1 expression in pulmonary squamous cell carcinoma has not been well demonstrated.

Methods: A total of 98 patients with resected pulmonary squamous cell carcinoma were included in the study. PD-L1 expression was determined by immunohistochemistry in tumor specimens. Relationship between PD-L1 expression and clinicopathological variables was investigated. The prognostic value of PD-L1 expression in overall survival (OS) and probability of freedom from recurrence [FFR] was also demonstrated in the study.

Results: PD-L1 expression was identified in 72 (73.5%) of the 98 lung tumor samples. PD-L1 expression was significantly associated with advanced stage (P = 0.045). PD-L1 expression (hazard ratio, 0.347; 95% confidence interval, 0.148 to 0.814; P = 0.015) was a significant prognostic factor for better OS in multivariate analysis. For patients with stage I/II lung squamous cell carcinoma, PD-L1 expression was a significant prognostic factor for better OS (HR, 0.299; 95% CI, 0.114 to 0.786; P = 0.014) and probability of FFR (HR, 0.346; 95% CI, 0.143 to 0.836; P = 0.025) in multivariate analysis.

Conclusions: PD-L1 expression was significantly associated with advanced stage in pulmonary squamous cell carcinoma. PD-L1 expression was a significant prognostic factor for better OS in patients with pulmonary squamous cell carcinoma, and also a significant prognostic factor for better probability of FFR in those with early-stage pulmonary squamous cell carcinoma.

Citation Format: Jung-Jyh Hung, Yu-Chung Wu, Wen-Hu Hsu. The prognostic significance of programmed cell death-ligand 1 expression in pulmonary squamous cell carcinoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A145.
Abstract A148: 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: November 2016

Modulating regulatory T cell (Treg) function to promote anti-tumor immunity is a desirable strategy for cancer immunotherapy. Blockade of checkpoint molecules, CTLA-4 and PD-1, has shown clinical efficacy in advanced metastatic melanoma and other malignancies. We recently reported that Treg stimulation induced the recruitment of protein kinase C-eta (PKCη), together with the GIT-PAK-PIX signaling complex that mediates focal adhesion disassembly, to CTLA-4, an inhibitory receptor highly expressed on Foxp3+ Tregs, which is important for Treg suppression. This resulted in PKCη localization in the Treg immunological synapse. These events promoted and were obligatory for Treg-mediated contact-dependent suppression, including suppression of tumor immunity via a mechanism likely involving depletion of costimulatory CD80/86 ligands from APCs (Nat Immunol. 2014;15:465-72). PKCη-deficient (PKCη−/−) Tregs were defective in the suppression of anti-tumor immunity against B16 melanoma and the slow growing prostate adenocarcinoma TRAMP-C1 but, surprisingly, retained the ability to inhibit experimental autoimmune colitis, a disease that often occurs in cancer patients treated with checkpoint inhibitors. Moreover, PKCη loss in Treg cells increased the number of intratumoral CD4+ and CD8+ effector T cells without affecting Treg numbers, resulting in a significant increase of the intratumoral Teff:Treg cell ratio, a parameter considered to predict a better clinical outcome. Impaired activation of the GIT-PAK-PIX complex and the resulting defect in CD80/86 depletion are likely responsible for the defective suppressive activity of PKCη−/- Tregs. These findings establish the novel CTLA-4-PKCη signaling pathway as critical for suppressing tumor immunity, and implicate it as an attractive cancer immunotherapy target.

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 Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A148.
Abstract B011: Human IL-2 mutant exhibits a higher antitumor efficacy and lower toxicity than wildtype IL-2 in different preclinical contexts
Authors: Carmenate Tania , Montalvo Magela , Rojas Gertrudis , Fuentes Dasha , Leon Kalet
Published: November 2016

High dose IL-2 is registered for the treatment of melanoma and renal cell carcinoma, however this therapy has limited efficacy and severe toxicity. It has been shown that in patients HD IL-2 drives preferential expansion of regulatory T cells, which dampen the antitumor immunity. This results explain at least partially the limited efficacy of this therapy. In this work, we characterize a human IL-2 mutant which differs from wildtype IL-2 by four mutations at the interface with the a-subunit of IL-2R. In vitro, IL-2 mutant induces proliferation of CD8+CD44hi and NK1.1 cells as efficiently as wtIL-2 does, but it shows a reduced capacity to induce proliferation of CD4+Foxp3+ regulatory T cells. In vivo, IL-2 mutant clearly induces preferential proliferation of CD8+CD44hi witha quite reduced proliferation of CD4+Foxp3+ regulatory T cells. The IL-2 mutant shows higher antimetastatic effect than wtIL-2 in several transplantable tumor models. i.e. the experimental metastasis model of MB16F0; the experimental and spontaneous metastasis models for the mouse pulmonary carcinoma 3LL-D1222 and the metastasis mammary carcinoma 4T1. Moreover initial data also support a higher potency of this IL2 mutant, when used in combination with other immune-therapies. Relevantly, the IL-2 mutant also exhibits lower lung and liver toxicity than does wtIL-2 when used at high doses in mice. The IL-2 mutant described could be a good candidate for improving cancer therapy based on IL-2.

Citation Format: Tania Carmenate, Magela Montalvo, Gertrudis Rojas, Dasha Fuentes, Kalet Leon. Human IL-2 mutant exhibits a higher antitumor efficacy and lower toxicity than wildtype IL-2 in different preclinical contexts [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B011.
Abstract B049: Immunotherapy of EBV associated gastric cancer using EBV specific T cells
Authors: Moon Jae Seung , Kim Jung Ho , Lee Sang Kyou
Published: November 2016

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is mediated by EBV-infected malignant cells in gastric intestinal tract. About 10% of gastric carcinoma are EBVaGC worldwide, and over 85,000 patients are developed EBVaGC annually. EBVaGC shows several different aspects with EBV-negative gastric cancer such as clinicopathology, CpG island methylation status and expression of EBV-related genes. Therefore, immunotherapy to treat EBVaGC would be a good approach because expression pattern of EBVaGC is evidently different with that of EBV-negative cells in stomach. Adoptive T cell transfer has been effective for treatment of EBV-positive nasopharyngeal carcinoma (NPC) and lymphomas, which express similar EBV antigens to those of EBVaGC cells. Thus, we wanted to use system of NPC clinical trials to EBVaGC. Targeted EBV antigens expressed both in NPC and EBVaGC were latent membrane proteins (LMPs)-1,2 and EBV-determined nuclear antigen (EBNA) 1. To generate EBV-specific cytotoxic T lymphocytes (EBV-CTLs), we firstly isolated peripheral blood mononuclear cells (PBMCs) from healthy human donor blood by using Ficoll and sorted dendritic cells (DCs) to deliver LMP2A and EBNA1 expressing-vectors by nucleofection into DCs. Nucleofected DCs were cocultured with PBMCs in order to cross-present EBV antigens toward CD4+, CD8+ T cells in DC maturation condition. Generated EBV-CTLs were incubated with IL-2, 7, 15-supplemented CTL media for inducing memory-like cells and expanded for 14 days to apply to adoptive transfer of EBVaGC model. Cytotoxicity of EBV-CTLs were specific for EBV-expressing gastric cancer cell line (SNU 719) in vitro, not EBV-negative gastric cancer cell line (MKN45). To confirm efficacy of EBV-CTLs in mouse model, we developed EBVaGC mouse model using SNU719 and injected 1 × 107 cells/mouse once a week for three times. EBV-CTLs had an effect on the reduction of tumor size of EBVaGC and effectively migrated into tumor site. Also, administered EBV-CTLs stably existed in blood and tumor tissue even over 3 days analyzed by FACS and immunohistochemistry. In conclusion, EBV-CTLs represented improved anti-tumor activity to treat EBVaGC and specific lysis to EBV-expressing cells. Also, any side effects or autoimmune diseases were not detected. Therefore, EBV-CTLs would be likely to play important roles in targeting many EBV-associated cancers and be applied to clinical trials.

Note: This abstract was not presented at the conference.

Citation Format: Jae Seung Moon, Jung Ho Kim, Sang Kyou Lee. Immunotherapy of EBV associated gastric cancer using EBV specific T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B049.
Abstract B064: Induction of protective antitumor immunity through attenuation of endoplasmic reticulum aminopeptidase 1 function
Authors: Sugiyarto Gessa , Bailey Ian , Elliott Tim , James Edward
Published: November 2016

Background: The ability to generate potent anti-tumor CTL responses is fundamental to the rejection of many tumors. However many potent anti-tumor responses are suppressed by regulatory T cells (Treg) preventing this. Recent clinical trial studies, using checkpoint inhibitors have shown great promise in inducing protective anti-tumor responses albeit in a minority of patients. In the mouse CT26 tumor model, removal of Treg suppression uncovers potent cryptic anti-GSW11 CTL, which are immunodominant and protective. The generation of GSW11 peptide in the ER is susceptible to destruction by endoplasmic reticulum aminopeptidase 1 (Erap1) significantly reducing the amount presented at the cell surface to T cells. Here we investigate the ability to overcome Treg suppression by modulating the function of Erap1.

Methods: GSW11-specific T cells were characterised from CT26 challenged Treg replete and depleted mice by flow cytometry. Attenuation of Erap1 function was achieved using either the inhibitor Leucine-thiol or siRNA and its effect on GSW11 generation determined by T cell activation assays and reverse phase HPLC.

Results: We identified that Treg suppressed GSW11-specific T cells exhibit a clonally exhausted phenotype in CT26 challenged mice. Examination of GSW11 peptide generation showed that attenuation of Erap1 function by inhibition or siRNA significantly increased the generation of GSW11 (∼75-fold increase). Strikingly, Treg replete mice challenged with Erap1 siRNA CT26 were able overcome Treg suppression and induce GSW11-specific T cell responses resulting in tumor protection. In addition, in vivo intra-tumoral administration of Erap1 inhibitor in established CT26 tumors resulted in the attenuation or regression of tumor growth in the majority of mice.

Conclusions: These results reveal the therapeutic potential of modulating Erap1 function to alter tumor-specific antigen presentation to overcome clonal exhaustion and generate potent anti-tumor T cell immunity.

Citation Format: Gessa Sugiyarto, Ian Bailey, Tim Elliott, Edward James. Induction of protective antitumor immunity through attenuation of endoplasmic reticulum aminopeptidase 1 function [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B064.
Abstract B069: The best is not enough—when it is alone: Current clinical doses of humanized anti-Her2 antibodies should be applied in combination to delay development of trastuzumab resistance by additively enhancing ADCC
Authors: Tóth Gábor , Szöőr Árpád , Simon László , Yarden Yosef , Szöllősi János , Vereb György
Published: November 2016

Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab')2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies.Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be.The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition.

Citation Format: Gábor Tóth, Árpád Szöőr, László Simon, Yosef Yarden, János Szöllősi, György Vereb. The best is not enough—when it is alone: Current clinical doses of humanized anti-Her2 antibodies should be applied in combination to delay development of trastuzumab resistance by additively enhancing ADCC [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B069.
Abstract B073: Protein nanoparticles for controllable T cell activation
Authors: Lajoie Marc J. , Butterfield Gabriel , Gray Elizabeth , DaPron Kate , Stetson Daniel , Baker David , King Neil
Published: November 2016

The immunosuppressive nature of cancer prevents a sustained response to T-cell-inducing vaccines. We hypothesize that co-delivering antigens and adjuvants directly to sub-compartments of antigen presenting cells could elicit specific CTL and Th1 activation, providing more control over the immune response. To accomplish this goal, we are developing protein nanoparticles capable of delivering payloads consisting of RNA, protein, and small molecules to target cells. One class of protein nanoparticles is produced in E. coli and is capable of packaging its own RNA to enable evolution of improved functions such as increased RNA packaging, improved serum stability, and pH-mediated disassembly for endosomal payload delivery. Another class of protein nanoparticles can bud from HEK293F cells containing small molecule adjuvants and mRNA-encoded antigens. By pseudotyping these enveloped protein nanoparticles with viral fusion proteins, we can deliver nucleic acids to the cytosol of target cells where they are translated to produce the desired protein antigen. These non-viral delivery devices could improve our ability to control immune responses against cancer.

Citation Format: Marc J. Lajoie, Gabriel Butterfield, Elizabeth Gray, Kate DaPron, Daniel Stetson, David Baker, Neil King. Protein nanoparticles for controllable T cell activation [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B073.
Abstract B083: Novel mRNA manufacturing techniques using modification of nucleosides and purification improves the efficacy of mRNA chimeric antigen receptor T cells
Authors: Foster Jessica , Choudhari Namrata , Barrett David , Resnick Adam
Published: November 2016

Chimeric Antigen Receptor (CAR) T-cells made with mRNA offer a transient and safe alternative to viral CARs, mitigating the concern for persistent unwanted side effects from constitutively active T-cells. Previous studies have shown that mRNA CARs are transiently effective, but lack CAR persistence and have struggled to show success across tumor types. We hypothesized the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology including the use of modified nucleosides and novel purification methods of the RNA to prevent double stranding that induces toxicity. Using the established CD19 CAR model in B-cell leukemia, we created mRNA CARs using previously described methods and compared them to mRNA CARs created using the modifications of pseudo-uridine or N-methyl pseudo-uridine instead of uridine, and purification to remove any aberrant double stranded mRNA. Comparing the modified and purified mRNA CAR T cells to those created using prior methods of making mRNA, the novel mRNA CAR T cells showed a two-fold increase in expression of the CAR on their surface initially, as well as a two-fold improvement in cytotoxic killing of leukemia cells that persisted for up to five days. The modified and purified mRNA CAR T cells also showed reduced expression of negative checkpoint regulators compared to original RNA CAR T cells. In vivo studies using a leukemia mouse model with a single dose of CAR T cells also showed marked improvement using modified and purified mRNA CAR T cells, with purified RNA showing the most robust 2-log enhanced suppression of leukemic burden. Our results provide a novel technique that can be easily incorporated into RNA production for use in clinical trials, and poise RNA CARs for increased efficacy as new CAR targets emerge and are being tested.

Citation Format: Jessica Foster, Namrata Choudhari, David Barrett, Adam Resnick. Novel mRNA manufacturing techniques using modification of nucleosides and purification improves the efficacy of mRNA chimeric antigen receptor T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B083.
Abstract B090: Evaluation of a novel impedance-based assay for label free and dynamic assessment of CAR-T constructs efficacy against B-cell leukemia and lymphoma
Authors: Xue Chelsea , Cerignoli Fabio , Senyukov Vladimir , Pierog Piotr , Miller Thomas , Shaw Amy , Xi Biao , Muir Lincoln , Abassi Yama , Kassim Sadik
Published: November 2016

Immunotherapy is emerging as a promising approach in cancer treatment, allowing specific targeting of cancer cells based on surface marker expression. In vitro characterization of the effectiveness of the treatment is a necessary step before moving into clinical trials, but currently technologies rely on endpoint assays that are unable to capture the complexity and dynamics of the in vivo immune response. Here we present the adaptation of an impedance-based technology to measure immune-mediated cell killing of B lymphoid and lymphoma tumor cells. Impedance technology is able to detect alterations in cell adhesion, morphology and cell number of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. The variation is reported through an impedance-related parameter termed Cell Index (CI). The platform enables label-free and continuous monitoring of cell viability, leaving the same cells still available for secondary end point assays. To adapt such adhesion-based technology to monitor non-adherent B cells, we developed a strategy where the bottom of the wells is coated with an anti-CD40 antibody. The coating allows specific adhesion and retention of B cells and measurement of changes in impedance that are proportional to cell number. Using primary T lymphocytes expressing Chimeric Antigen Receptors (CAR) against CD19 or unrelated targets, we were able to demonstrate specific killing of tumor B cell lines and primary cells also at very low effector:target ratios. The results were also confirmed by flow cytometry. Taken together our results demonstrate the high sensitivity of this high throughput approach when compared to end point assays like 51Cr release or flow cytometry, improving the workflow for therapy validation and optimization in the context of personalized medicine.

Citation Format: Chelsea Xue, Fabio Cerignoli, Vladimir Senyukov, Piotr Pierog, Thomas Miller, Amy Shaw, Biao Xi, Lincoln Muir, Yama Abassi, Sadik Kassim. Evaluation of a novel impedance-based assay for label free and dynamic assessment of CAR-T constructs efficacy against B-cell leukemia and lymphoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B090.
Abstract B097: Analyze and recover functionally defined single immune cell clones through Opto-Electro-Positioning technology
Authors: Wang Xiaohua , Bronevetsky Yelena , Stadler Guido , Beaumont Kristin , Bao Xiaoyan Robert , Shimizu Saki , Smith Duane , Chapman Kevin
Published: November 2016

Cancer patients mount protective innate and adaptive immune responses against tumor cells. Since such responses are able to control or even cure eliminate cancer if harnessed properly, identifying clonal adaptive immune cells targeting tumor cells with designated function is extremely valuable but remains challenging. We provide evidence here that the combination of the Opto-Electro-Positioning (OEP) technology and the nanofluidic culture enables high throughput screening and harvesting functionally defined immune cells through single cell based analysis. Immune cells can be maneuvered deterministically through OEP into defined nanofluidic culture space which supports efficient antigen specific T/B cell clonal expansion and functional profiling ex vivo. The viable T/B cell colonies with designated functions can subsequently be harvested after analysis for downstream applications including rapid expansion, antigen receptor sequencing, and epigenetic analysis etc. Integration of these two basic technologies with computer controlled automation leads to our Beacon platform that allows researchers to screen tens of thousands of live single immune cells simultaneously. The system is also exceptionally efficient in investigating immune cell population from the tumor biopsies that are typically of low cellularity. In summary, the OEP and nanofluidic device based Beacon solution provides researchers an “all-purpose” single cell based discovery platform for both basic and clinical oncology investigations.

Citation Format: Xiaohua Wang, Yelena Bronevetsky, Guido Stadler, Kristin Beaumont, Xiaoyan Robert Bao, Saki Shimizu, Duane Smith, Kevin Chapman. Analyze and recover functionally defined single immune cell clones through Opto-Electro-Positioning technology [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B097.
Abstract B101: Mechanism of T cell activation by phase separation
Authors: Su Xiaolei , Ditlev Jonathon , Hui Enfu , Rosen Michael , Vale Ronald
Published: November 2016

Activation of many cell surface receptors triggers the reorganization of downstream signaling molecules into micron- or submicron-sized clusters. However, the functional consequences of such clustering have been unclear. We biochemically reconstituted a 12-component signaling pathway on model membranes, beginning with T cell receptor (TCR) activation and ending with actin assembly. When TCR phosphorylation was triggered, downstream signaling proteins spontaneously separated into liquid-like clusters that promoted signaling outputs both in vitro and in human Jurkat T cells. Reconstituted clusters were enriched in kinases but excluded phosphatases and enhanced actin filament assembly by recruiting and organizing actin regulators. These results demonstrate that protein phase separation can create a distinct physical and biochemical compartment that facilitates signaling.

Note: This abstract was not presented at the conference.

Citation Format: Xiaolei Su, Jonathon Ditlev, Enfu Hui, Michael Rosen, Ronald Vale. Mechanism of T cell activation by phase separation [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B101.
Abstract B102: A microengineered human lymph node-on-a-chip to study cancer immunotherapy
Authors: Goyal Girija , Ingber Donald
Published: November 2016

Preclinical studies in cancer immunotherapy, such as those involving checkpoint blockade are usually conducted and validated in mice; however, rodent immune responses do not faithfully mimic those observed in humans. The overall goal of this project is to leverage recent advances in human organ-on-chip technology to create a human lymph-node-on-chip (hLN-on-chip) that exhibits complex human immune functionality. For maximum applicability, we have started with human peripheral blood mononuclear cells (PBMC) such that these devices may be widely used as non-invasive surrogates for patient testing. We have optimized matrix, cellular composition, chip design and microfluidic perfusion to induce B cell clusters reminiscent of follicles found in the lymph node. T and B cell function is maintained in the hLN-on-chip as evidenced by IL-2 and immunoglobulin M production in response to heat killed formalin fixed Staph aureus Cowan I (SAC), commonly used to induce a T cell dependent antibody response. Current studies are focused on defining the alterations in sensitivity and activity of the T cells. For instance, lymph node resident T and B cells are known to be polarized. Thus, we have tested if coinhibitory receptors such as CTLA-4, known to be important in anti-tumor immunity are also polarized under tissue like conditions. We also present preliminary data on optimizing the perfusion of the lymphocyte parenchyma with microengineered channels lined with lymphatic and vascular endothelial cells to model antigen delivery and immune cell trafficking. These devices will enable near real-time assessment of secreted factors, cell trafficking and high-resolution imaging of dynamic cellular interactions that occur in the human lymph node, and hence, they should prove invaluable in studying human cancer response to anti-cancer immunotherapies in the future.

Citation Format: Girija Goyal, Donald Ingber. A microengineered human lymph node-on-a-chip to study cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B102.
Abstract B121: Oncogenic MYC contributes to tumor super-enhancers via eRNA amplification
Authors: Guo Yang Eric , Seyffert Michael , Abraham Brian , Ji Xiong , Young Richard
Published: November 2016

Super-enhancers (SEs) are large clusters of enhancers that control transcription of genes that define cell identity, and they are frequently acquired by cancer cells to drive key oncogenes. Indeed, the prominent oncogene MYC recurrently acquires SEs in many types of cancer, which may explain its overexpression in a broad array of cancers. Elevated levels of MYC promote transcriptional elongation at promoters of all active genes, thereby amplifying RNA output of the cellular gene expression program. High levels of MYC also lead to increased binding of MYC at enhancers, but the significance of enhancer invasion by MYC is unclear. Here, we propose a model that at tumor cell SEs MYC amplifies the production of enhancer RNAs (eRNAs), which in turn facilitate maintenance of SEs by stabilizing transcription factor and co-factor occupancy. We found that in Burkitt's lymphoma cells SEs depend on high levels of MYC and eRNAs, and that MYC is required for elevated levels of eRNA transcription. We demonstrate that eRNAs directly interact with RNA-binding transcription factors at SEs, and that perturbation of eRNA affects SE signal. Elevated MYC thus contributes to the dynamic formation and maintenance of SEs through amplification of eRNAs. Our results may identify novel RNA targets for new therapies for Burkitt's lymphoma, as well as other cancers. New ways to modulate the immune response may also be revealed by our studies.

Citation Format: Yang Eric Guo, Michael Seyffert, Brian Abraham, Xiong Ji, Richard Young. Oncogenic MYC contributes to tumor super-enhancers via eRNA amplification [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B121.
Abstract B122: Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures
Authors: Ru Heng , Chambers Melissa G. , Fu Tian-Min , Tong Alexander B. , Liao Maofu , Wu Hao
Published: November 2016

A hallmark of vertebrate immunity is the diverse repertoire of antigen-receptor genes that results from combinatorial splicing of gene coding segments by V(D)J recombination. The dimeric (RAG1-RAG2)2 recombinase (RAG) recognizes specific recombination signal sequences (RSSs), each containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS). RAG only combines one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the fidelity of V(D)J recombination. RAG introduces precise breaks at RSS-coding segment junctions to generate cleaved RSSs and hairpin coding segments. Here we report cryo-electron microscopy structures of multiple RAG complexes reconstituted with 12-RSS and 23-RSS intermediates and HMGB1 at up to 3.4 angstrom resolution. These structures reveal a closed conformation of RAG upon RSS synapsis, with base flipping and base-specific recognition of RSSs. Distortion at the RSS-coding segment junction and base flipping in the coding segment uncovers the geometry for a two-metal-ion catalytic mechanism. Both 12-RSS and 23-RSS are exceedingly bent by the bound HMGB1. The nonamer-binding domain dimer of RAG1 is flexibly attached to the active site dimer of RAG and is tilted towards the bent 12-RSS but away from the bent 23-RSS in the synaptic complexes, which provides an induced fit mechanism for the 12/23 rule. Collectively, our structures illustrate the elegant mechanisms of RAG-catalyzed V(D)J recombination at the molecular level.

Citation Format: Heng Ru, Melissa G. Chambers, Tian-Min Fu, Alexander B. Tong, Maofu Liao, Hao Wu. Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B122.
Abstract B127: Free DNA and tolerance
Authors: Filaci Gilberto , Ferrera Francesca , Tardito Samuele , Altosole Tiziana , Bernardi Cinzia , Parodi Alessia , Fenoglio Daniela
Published: November 2016

Soluble DNA circulates in the peripheral blood of healthy subjects, and, at increased concentrations, of pregnant women and patients affected by tumors and vasculitis. Since the discovery of intracellular DNA sensors, inducing inflammatory responses after binding to DNA, DNA has been included among danger signals. However, this view does not explain the presence of abundant circulating DNA in conditions characterized by tolerance (pregnancy) or immunodeficiency (tumor). In order to verify whether DNA mediates also an immunoregulating activity, a 20 base-pair long oligonucleotide (poly-CG) was synthesized and tested for its capacity to: 1) bind MHC class II molecules; 2) exert immunoregulatory activity. The oligo binding to PMJ2-PC macrophages was inhibited by anti-MHC monoclonal antibodies (mAb). The specific binding of DNA to MHC class II molecules was confirmed by western blot analysis. Proliferation of OVA-specific splenocytes was inhibited by incubation with the oligo as well as by circulating DNA from cancer patients; moreover, fluorescent oligo, injected intravenously to NZB/W F1 mice, bound only on MHC class II positive cells (macrophages and B lymphocytes). Finally, nephritic NZB/W F1 mice, administered weekly with the oligo, showed delayed onset of protenuria and improved survival with respect to untreated animals, a phenomenon associated with increased frequencies of Treg. Interestingly, in vitro incubation of PMJ2-PC macrophages with the oligo induced increased CCL22 secretion. These data demonstrate for the first time that free DNA may mediate immunoregulatory activities.

Citation Format: Gilberto Filaci, Francesca Ferrera, Samuele Tardito, Tiziana Altosole, Cinzia Bernardi, Alessia Parodi, Daniela Fenoglio. Free DNA and tolerance [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B127.
Abstract B128: Clinical translation of oncolytic measles virus for T cell activation
Authors: Engeland Christine E. , Veinalde Rūta , Kalle Christof von , Jaeger Dirk , Ungerechts Guy
Published: November 2016

The oncolytic measles virus (MeV) vaccine strain provides a flexible vector platform which allows for targeted, tumor-restricted immunomodulation. We have developed MeV vectors to support different phases of the cancer immunity cycle. This enables therapy directly tailored to the specific immune micromilieu of individual tumors.

We have validated MeV vectors for T cell activation in preclinical models. MeV encoding bispecific T cell engagers (BiTEs) recruit cytotoxic T cells to the tumor site. Tumor antigen-specific T cell activation is achieved with MeV oncolytic vaccines. MeV encoding IL-12 directs T cell responses towards a Th1 phenotype. Combining MeV with immune checkpoint inhibition increases the ratio of effector to regulatory T cells. Furthermore, our preclinical data support the notion that MeV-mediated oncolysis can break resistance to checkpoint antibodies.

A clinical Phase Ib/II trial combining MeV with an anti-PD1 antibody in patients with metastatic pancreatic adenocarcinoma is currently in preparation. Aside from safety and anti-tumor efficacy, a main focus of the trial is the accompanying translational research program. Sequential biopsies for analysis of tumor-infiltrating immune cells, cytokine and chemokine profiling, transcriptome and immunoreceptor sequencing are performed to identify biomarkers predictive of response.

Citation Format: Christine E. Engeland, Rūta Veinalde, Christof von Kalle, Dirk Jaeger, Guy Ungerechts. Clinical translation of oncolytic measles virus for T cell activation [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B128.
Abstract B132: Importance and mechanism of poly(A) tail length-mediated translational control in different immune cells
Authors: Xiang Kehui , Bartel David
Published: November 2016

Translational control is an essential regulatory mechanism in immune cells. Although it has been suggested that poly(A) tail length-mediated translational control plays important roles in regulating select cytokine production in memory T cells and macrophages, whether this is a transcriptome-wide regulatory principle in all immune cells remains unknown. We set out to perform RNA-seq, ribosome-footprint profiling and poly(A) tail length profiling across major types of immune cells to examine the relationship between poly(A) tail length and translational efficiency in these systems. We also plan to delineate the transcriptome-wide landscape of poly(A) tail length before and after immune activation, and investigate if the coupling of poly(A) tail length and translational efficiency plays a regulatory role in this process.

Meanwhile, to understand the general mechanistic details of how poly(A) tail length affects translational efficiency in a cell context-dependent manner, we carried out RNA pull-down experiments and isolated proteins from known coupled (Xenopus oocytes) and uncoupled (rabbit reticulocyte lysates) systems. By quantitative mass spectrometry analysis, we identified key factors that are responsible for coupling translational efficiency to poly(A) tail length. Among these candidates, poly(A) binding protein (PABPC1) plays an essential role. Overexpression of PABPC1 in Xenopus oocytes renders the translation of reporter RNAs with either a short poly(A) tail or a long poly(A) tail equally well, turning a coupled system into an uncoupled system. We have also generated an effective degron-induced PABPC1-knockdown cell line and are performing analysis to see if the coupling can be established in a normally uncoupled system. Our results suggest that cells can modify the expression of PABPC1 to help determine if poly(A) tail length regulates translational efficiency.

Citation Format: Kehui Xiang, David Bartel. Importance and mechanism of poly(A) tail length-mediated translational control in different immune cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B132.
Abstract B138: Immunotherapy effect on survival of acute myeloid leukemia patients
Authors: Shi Runhua , Li Teena , Shi Lawrence , Mills Glenn
Published: November 2016

Background: Immunotherapy has been used to treat many cancers for years. It has been combined with other treatments such as chemotherapy and stem cell transplant. The purpose of the current study is to assess the impact of immunotherapy and payer status on Acute Myeloid Leukemia (AML) patient survival.

Patients and Methods: Patients in the National Cancer Data Base who were registered between 2004 and 2012 and followed up to the end of 2013 were used in this research. We evaluated a cohort of 50272 AML patients between the ages of 18 to 65 years old who did not undergo radiation or hormonal therapy. The effect of treatments (immunotherapy, stem cell transplant, and adjuvant chemotherapy) on the survival of AML was investigated. Additional variables adjusted included sex, age, race, Charlson Comorbidity Index, education, income, payer status, distance traveled, facility type, diagnosing/treating facility, and treatment delay. Multivariate Cox regression was used to investigate the effect of immunotherapy on the survival of AML patients while adjusting for other factors.

Results: The mean age of the patients was 49 years (SD = 12) old. The proportion of patients that received treatments were immunotherapy (1.47%), stem cell transplant (6.11%), and chemotherapy (74.43%). The median overall survival for all patients was 8.76 years. While using multivariate analysis to adjust for other factors, effects of treatment on survival were significant for chemotherapy (hazard ratio (HR) = 1.7, 95% CI: 1.49-2.0), immunotherapy (HR = 0.87, 95% CI: 0.76-0.99), and stem cell transplant (HR = 0.946, 95% CI: 0.88-1.01). While adjusting for other factors, patients who received immunotherapy had a reduced risk of death by 13% compared to patients without immunotherapy whereas patients who received chemotherapy had an increased risk of death by 70% compared to patients without chemotherapy. In addition, compared to patients with private insurance, the HR were 1.38, 1.43, and 1.2 for Medicaid, Medicare and uninsured patients, respectively and were all statistically significant. Compared to patients with a Comorbidity Index score of 0, the HR were 1.35 and 1.71 for Comorbidity Index score of 1 and 2+, respectively.

Conclusion: Immunotherapy was found to be a significant benefactor in the survival of AML patients. In addition, payer status and Comorbidity index score were also significant predictors for the survival of AML patients.

Citation Format: Runhua Shi, Teena Li, Lawrence Shi, Glenn Mills. Immunotherapy effect on survival of acute myeloid leukemia patients [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B138.
Abstract B141: Targeting the complement system to improve radiation treatment
Authors: Olcina Monica M. , Giaccia Amato J.
Published: November 2016

The standard of care for a variety of cancers consists of therapies that are associated with significant toxicity, however, relatively little is known regarding what strategies could be undertaken to reduce treatment toxicity without compromising tumor control. We aim to establish whether targeting the complement system could be used to minimize treatment toxicity while improving tumor sensitivity to radiation. Complement is an important pathway in immunity; however, its inappropriate activation may result in tissue injury. Our data suggests that in response to radiotherapy, expression of complement regulators is decreased significantly in normal colon. Importantly, these expression changes correlate with an increase in the C5b-9 complex (thought to be responsible for cell lysis) in irradiated colon in vivo. Furthermore, our results suggest that targeting the complement system (either genetically or pharmacologically) can result in increased survival of mice following radiotherapy, through protection of the gastrointestinal tract from radiation-induced toxicity.

Citation Format: Monica M. Olcina, Amato J. Giaccia. Targeting the complement system to improve radiation treatment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B141.
Abstract IA04: T cell recognition and tumor resistance in human cancer
Authors: Schumacher Ton
Published: November 2016

The T cell-based immune system has evolved to recognize and destroy cells that differ from “self” on the basis of the antigens they express. Many human tumors contain large numbers of mutations, of which many hundreds can be present within expressed genes. As the resulting altered protein sequences are foreign to the immune system, immune recognition of such “neo-antigens” has been proposed as a major factor in the activity of clinically used immunotherapeutics such as anti-CTLA-4 and anti-PD-1.

In support of this hypothesis, using in-house developed technologies we have demonstrated that both CD4 and CD8 T cell recognition of the consequences of DNA damage is a common feature in human melanoma and can be boosted by T cell checkpoint blockade. Furthermore, to extend these data to other tumor types, we have benchmarked the foreign antigenic space in a variety of human cancers against a series of human pathogens. Collectively, these data, and data from other groups, establish “tumor foreignness” as one of the critical determinants in effective cancer immunotherapy.

Clearly, the sensitivity of tumor cells to the tumor-specific T cell pool that can be present in intrinsically foreign tumors forms an additional requirement for tumor control. To address factors that control such sensitivity, we a currently determining mechanisms of immune escape upon T cell targeting of human tumors. Results from this analysis are expected to contribute to the further definition of biomarkers that predict the outcome of the interaction between human tumors and the T cell-based immune system.

Citation Format: Ton Schumacher. T cell recognition and tumor resistance in human cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA04.
Abstract IA05: Targeting regulatory T cells in tumors
Authors: Vignali Dario A.
Published: November 2016

Regulatory T cells (Tregs) inhibit beneficial antitumor responses. Treg depletion enhances tumor rejection in animal models and the clinic but also leads to substantial adverse events. Thus approaches have been sought to target Tregs in tumors while limiting systemic autoimmune and inflammatory manifestations. Two approaches will be discussed. First, the signals that maintain Treg stability and potentiate their function remain obscure. We have shown that the immune cell surface ligand semaphorin-4a (Sema4a) on conventional T cells and DCs, and the Treg-restricted receptor neuropilin-1 (Nrp1) interact to potentiate Treg function. Mice with a Treg-restricted deletion of Nrp1 exhibit limited tumor-induced tolerance and thus substantial resistance to tumors, yet do not develop any autoimmune manifestations. Thus, Nrp1 ligation maintains Treg stability and function in highly inflammatory sites but is dispensable for the maintenance of immune homeostasis, highlighting Nrp1 as a potential immunotherapeutic target in cancer. Second, the relative importance of different Treg suppressive mechanisms remains contentious. Interleukin-35 (IL35) is a Treg-secreted cytokine known to inhibit effector T cell proliferation and mediate infectious tolerance via induction of suppressive IL35-producing induced Tregs, iTr35. Using antibody-mediated neutralization, Treg-restricted deletion of Ebi3 and novel reporter mice, we have shown that IL35 facilitated tumor growth by limiting antitumor immunity in transplantable and genetically-induced murine models of melanoma and lung carcinoma. These findings reveal the previously unappreciated importance of IL35 in limiting antitumor immunity and present IL35 as a potential therapeutic target in cancer.

Citation Format: Dario A. Vignali. Targeting regulatory T cells in tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA05.