bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3625: Thyroid hormone, thyroxine, promotes cell proliferation and β-catenin activation in colorectal cancer
Authors: Lee Yee-Shin , Hsieh Meng-Ti , Chin Yu-Tang , Hercbergs Aleck , Davis Paul J. , Wu Han-Chung , Lin Hung-Yun
Published: July 2017

Backgrounds: Being a crucial hormone regulating growth, metabolism and various physiological processes, the status of thyroid hormone has long been implicated in cancer risks and tumor developments. In the present study, we aimed to look at the role of thyroid hormone, thyroxine (T4), in colorectal cancer cell proliferation and β-catenin activation, which is highly involved in both normal and oncogenic developments of the gut.

Materials and Methods: The effects of T4 in colorectal cancer cell lines HCT 116 (APC wild type) and HT-29 (APC mutant) as well as the primary cells derived from colorectal cancer patients were studied. Cell proliferation was evaluated according to cell counting, MTT assay and qRT-PCR. The activation of β-catenin was examined using Western blotting, qRT-PCR and immunoprecipitation.

Results: The results showed that T4 increased the cell number of both HCT 116 and HT-29 cells compared to the untreated cells. In both cell lines, T4 induced nuclear β-catenin accumulation and the protein levels of WNT/β-catenin targets Cyclin D1 and c-Myc. The mRNA expression of CTNNB1 was elevated by T4 in HCT 116 cells, but not in HT-29 cells. In APC wild type HCT 116 cells, T4 increased the WNT4 mRNA expression and decreased the association between β-catenin and the WNT-regulated β-catenin destruction complex. Moreover, the cell numbers of T4-treated primary cells were higher compared to the untreated cells while the mRNA expressions of proliferative genes PCNA, CCND1 and c-Myc were elevated by T4. In the primary cells, T4-induced nuclear β-catenin accumulation, protein levels of Cyclin D1 and c-Myc, and mRNA expressions of CTNNB1 and WNT4 were also observed.

Conclusions: T4 promoted cell proliferation and β-catenin activation in colorectal cancer. In the cells with different APC mutation status, the activation of β-catenin was regulated by different mechanisms.

Citation Format: Yee-Shin Lee, Meng-Ti Hsieh, Yu-Tang Chin, Aleck Hercbergs, Paul J. Davis, Han-Chung Wu, Hung-Yun Lin. Thyroid hormone, thyroxine, promotes cell proliferation and β-catenin activation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2017-3625
Abstract 3632: BiTE® antibody constructs for the treatment of SCLC
Authors: Giffin Michael J. , Lobenhofer Ed K. , Cooke Keegan , Raum Tobias , Stevens Jennitte , Beltran Pedro J. , Coxon Angela , Hughes Paul E.
Published: July 2017

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor with a poor prognosis and limited therapeutic options. Most patients present with extensive stage disease, where recent advances in immunotherapies, including bi-specific T cell engager (BiTE®) antibody constructs, represent a promising new therapeutic approach. BiTE® molecules have demonstrated durable complete responses in the clinic in hematological malignancies. Similar to hematological malignancies, SCLC is also a widely-disseminated malignancy that shows very high response rate to first line therapies with high rates of disease recurrence, features which may support efficacy of the BiTE® modality. Next generation sequencing (NGS) identified Delta-like Ligand 3 (DLL3) as a highly specific tumor associated antigen for SCLC, with consistent expression in tumors and very low expression in normal tissues. Tumor expression of DLL3 protein was confirmed by IHC, with 30 of 35 SCLC tumors staining positive for DLL3. DLL3 BiTE® antibody constructs showed low pM potency in vitro and also demonstrated significant inhibition of tumor growth in vivo in an orthotopic model of SCLC. A half-life extended (HLE) BiTE® targeting DLL3 demonstrated antibody-like pharmacokinetic properties in single-dose studies in non-human primates (NHP), with a half-life of 11 days. This is predicted to support every other week dosing in humans. The combination of high potency and excellent PK properties suggests that HLE BiTE® molecules may provide a useful tool for targeting residual disease in SCLC patients whose tumors express DLL3.

Citation Format: Michael J. Giffin, Ed K. Lobenhofer, Keegan Cooke, Tobias Raum, Jennitte Stevens, Pedro J. Beltran, Angela Coxon, Paul E. Hughes. BiTE® antibody constructs for the treatment of SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3632. doi:10.1158/1538-7445.AM2017-3632
Abstract 3640: Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site
Authors: Eavarone David A. , Prendergast Jillian , Rao Patricia E. , Stein Jenna , Behrens Jeff , Dransfield Daniel T.
Published: July 2017

Tumor-associated carbohydrate antigens (TACAs) historically have been challenging targets for antibody therapeutics. Sialyl-Tn (STn) is a cancer specific antigen that is expressed on the surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy. We have developed humanized bispecific antibodies targeting STn and CD3 for T-cell recruitment and activation at the tumor site. These bispecific antibodies were selected for optimal tumor targeting using our glycan microarray that enriches for candidates whose binding is protein-independent and glycan specific. STn-selective binding was demonstrated. Current lead candidates exhibited low nanomolar EC50 binding in flow cytometric assays against both STn expressing tumor cells and T cells. Quantification of T-cell activation and T-cell induced tumor killing in vitro provides a basis for the further clinical development of these bispecific antibody candidates.

Citation Format: David A. Eavarone, Jillian Prendergast, Patricia E. Rao, Jenna Stein, Jeff Behrens, Daniel T. Dransfield. Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3640. doi:10.1158/1538-7445.AM2017-3640
Abstract 3665: Tumor Treating Fields (TTFields) plus anti-PD-1 therapy induce immunogenic cell death resulting in enhanced antitumor efficacy
Authors: Voloshin Tali , Tal-Yitzhaki Orna , Kaynan Noa , Giladi Moshe , Shteingauz Anna , Munster Mijal , Blat Roni , Porat Yaara , Schneiderman Rosa S. , Cahal Shay , Itzhaki Aviran , Kirson Eilon D. , Weinberg Uri , Palti Yoram
Published: July 2017

Tumor Treating Fields (TTFields) are an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. TTFields is approved for the treatment of both newly diagnosed and recurrent glioblastoma. TTFields interrupt cancer cell mitosis by disrupting microtubules and septin filaments, which play key roles in mitosis. The mitotic effects of TTFields include abnormal chromosome segregation and ER stress, which trigger different forms of cell death.

We evaluated the in vitro and in vivo effects of TTFields combined with an immune checkpoint inhibitor (anti-PD1) on immunogenic cell death.

Murine Lewis lung carcinoma (LLC) and ovarian surface epithelial (MOSE) cells were treated with TTFields using the inovitroTM system. Levels of calreticulin (CRT) on the surface of treated cells and intracellular ATP levels were evaluated using flow cytometry. High mobility group box 1 (HMGB1) secretion was measured using an ELISA assay. Mice were implanted with LLC cells were treated with TTFields, anti-PD-1, or a combination of the two modalities. Tumor volume was monitored; flow cytometry analysis was performed for phenotypic characterization of infiltrating immune cells.

TTFields induced elevated cell surface expression of CRT, decreased intracellular ATP levels, and promoted HMGB1 secretion. In vivo, the combined treatment of lung tumor-bearing mice with TTFields plus anti-PD-1 led to a significant decrease in tumor volume compared to anti-PD-1 alone or to the control group. Significant increases in CD45+ tumor infiltrating cells were observed in the TTFields plus anti-PD-1 group. Infiltrating cells demonstrated a significant upregulation of surface PD-L1 expression. Both F4/80+CD11b+ cells and CS11c+ cells exhibited higher tumor infiltration and elevated PD-L1 expression as compared to infiltrating immune cell in the control group.

Our results demonstrate that TTFields treatment potentiates immunogenic cell death in cancer cells. Combining TTFields with specific immunotherapies such as anti-PD-1 may enhance antitumor immunity and result in increased tumor control.

Citation Format: Tali Voloshin, Orna Tal-Yitzhaki, Noa Kaynan, Moshe Giladi, Anna Shteingauz, Mijal Munster, Roni Blat, Yaara Porat, Rosa S. Schneiderman, Shay Cahal, Aviran Itzhaki, Eilon D. Kirson, Uri Weinberg, Yoram Palti. Tumor Treating Fields (TTFields) plus anti-PD-1 therapy induce immunogenic cell death resulting in enhanced antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2017-3665
Abstract 3682: Synergistic immunostimulatory effects and therapeutic benefit of combined histone deacetylase and bromodomain inhibition in non-small cell lung cancer
Authors: Adeegbe Dennis O. , Liu Yan , Lizotte Patrick , Kamihara Yusuke , Awad Mark , Barbie David , Ritz Jerome , Jones Simon , Quayle Steven , Hammerman Peter , Wong Kwok-Kin
Published: July 2017

Effective therapies for non-small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with potential to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. This study evaluated the immunoregulatory properties of two classes of drugs that modulate the epigenome, histone deacetylase (HDAC) and bromodomain inhibitors with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes associated with enhanced T-cell priming and function of antigen presenting cells. The bromodomain inhibitor JQ1 attenuated CD4+Foxp3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Collectively, our findings highlight immunomodulatory effects of two epigenetic modifiers that together promote T-cell-mediated anti-tumor immunity and demonstrate their therapeutic potential for NSCLC treatment.

Citation Format: Dennis O. Adeegbe, Yan Liu, Patrick Lizotte, Yusuke Kamihara, Mark Awad, David Barbie, Jerome Ritz, Simon Jones, Steven Quayle, Peter Hammerman, Kwok-Kin Wong. Synergistic immunostimulatory effects and therapeutic benefit of combined histone deacetylase and bromodomain inhibition in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3682. doi:10.1158/1538-7445.AM2017-3682
Abstract 3686: Generation of an off-the-shelf cell-based platform (PDC-vac) for active antitumor immunotherapy
Authors: Plumas Joel , Lenogue Kevin , Walencik Alexandre , Molens Jean-Paul , Chaperot Laurence , Pule Martin
Published: July 2017

The development of efficient vaccines for the treatment of cancers in combination or not with other agents such as checkpoint inhibitors represents a major public health issue but remains a challenge. A dozen of vaccine drug products is expected to be marketed in the next few years. In this context, off-the-shelf and potent platforms are more attractive than individualized vaccines that are tailored to each patient in many aspects. We have recently developed a new cell-based vaccine approach named PDC*vac (formerly GeniusVac). This cell-based vaccine was developed thanks to the establishment, from leukemic PDC, of unique human PDC line (PDC*line). We have previously shown the potential of peptide-loaded PDC*line to induce strong and fully functional HLA-A*02:01-restricted anti-tumor immunity in vitro and in vivo (Aspord et al, 2010, 2012). PDC*mel (GenusVac-Mel4), our first product is currently developed for the treatment of melanoma patients (NCT01863108). The high efficiency of PDC*vac renders the strategy very attractive for further clinical developments in active immunotherapy field. Our aim is to optimize PDC*vac technology by passing use peptides and render the vaccine easier to manufacture. We engineered our PDC*line using retrovirus to force the endogenous expression of one or more tumor or viral antigenic peptides or whole tumor/viral proteins (gp100, tyrosinase, Melan-A, Mage-A3, CMVpp65, flu, EBV) that broaden the panel of antigens presented. Data obtaining with different polypeptide and protein gene coding constructions and the expansion of multi-specific antigen-specific T-cells at the same time will be presented. Moreover, we will present how we succeed to obtain 100% of efficacy in antigen presentation by the PDC*line engineered to express tumor proteins or polyepitopes. We have validated the use of retrovirus constructs to create a new generation of an “off-the-shelf” cancer vaccines highly potent to stimulate antitumor-specific T-cells.

Citation Format: Joel Plumas, Kevin Lenogue, Alexandre Walencik, Jean-Paul Molens, Laurence Chaperot, Martin Pule. Generation of an off-the-shelf cell-based platform (PDC-vac) for active antitumor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3686. doi:10.1158/1538-7445.AM2017-3686
Abstract 369: Therapeutic targeting of the Wnt antagonist DKK1 with a humanized monoclonal antibody in oncology indications
Authors: Kagey Michael H. , Wu Yinyuan , Zhang Xinjun , Sirad Cynthia A. , Mulligan Shane E. , He Xi , Mirabelli Christopher K.
Published: July 2017

Wnt signaling is a fundamental pathway that is dysregulated in oncology. The Wnt antagonist DKK1 is expressed in a variety of tumor types which frequently correlates with a poor prognosis, including overall survival. DKK1 has known oncogenic activity by stimulating proliferation, metastasis, and angiogenesis, and recently been implicated in contributing to an immunosuppressive tumor microenvironment. The neutralization of DKK1 is hypothesized to have efficacy from both a direct antitumor effect and through an immune stimulated response. Here we describe the characterization of DKN-01, a humanized monoclonal therapeutic antibody to DKK1. DKN-01 binds DKK1 with high affinity and selectivity, disrupts the interaction of DKK1 with the LRP6 co-receptor, and neutralizes DKK1 activity in a cell based assay. In vivo, DKN-01 has efficacy both as a monotherapy and in combination with chemotherapies in a non-small cell lung (NSCLC) cancer A549 xenograft model. Results suggest that DKN-01 has an antiangiogenic effect and may stimulate a NK cell mediated antitumor response. Clinically, DKN-01 is being evaluated in relapsed/refractory esophageal cancer patients in combination with paclitaxel, and preliminary results demonstrate promising activity. Archival patient tumor samples are currently being analyzed genetically and by IHC for DKK1 and β-catenin staining for biomarker identification. Taken together, our results suggest that DKN-01, a novel therapeutic, has clinical efficacy by disrupting Wnt signaling, which results in a direct anti-tumor effect and stimulates a pro-inflammatory tumor response.

Citation Format: Michael H. Kagey, Yinyuan Wu, Xinjun Zhang, Cynthia A. Sirad, Shane E. Mulligan, Xi He, Christopher K. Mirabelli. Therapeutic targeting of the Wnt antagonist DKK1 with a humanized monoclonal antibody in oncology indications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 369. doi:10.1158/1538-7445.AM2017-369
Abstract 3721: DLL3 analysis of circulating tumor cells predict treatment outcome in phase 1 rova-T study in small cell lung cancer
Authors: Roy Somdutta , Martinez Kevin , Dilip Archana , Karsunky Holger , Dylla Scott
Published: July 2017

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with early dissemination and poor prognosis that accounts for 15-20% of all lung cancer cases worldwide. Most cases are inoperable and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs), which are prevalent in SCLC, present a readily accessible liquid biopsy. Here we present CTC data from aggressive, late stage SCLC patients who were enrolled on a Phase 1 trial studying Rova-T (anti-DLL3 antibody drug conjugate). CTCs were captured using a microfluidic platform (IsoFlux) that uses flow control and immunomagnetic capturing of epithelial cell surface marker (EpCAM)-positive cells. CTCs were isolated before dosing (baseline) of Rova-T and at successive time points post treatment. CTCs from each time point were distinguished and enumerated using DAPI, EpCAM and the cytoplasmic marker (pan-CK), in addition to the absence of CD45. Enriched CTCs were also collected for RT-PCR for expression analysis of genes confirmed to be absent in blood cells. The number of CTCs varied among the 58 baseline samples collected, ranging from 5-1000 CTCs per 7.5ml of blood. When the CTCs were assessed for DLL3 target expression, 37/58 baseline samples expressed DLL3 by RT-PCR. Interestingly, the expression of DLL3 on the baseline CTCs correlated with treatment outcome. When additional time points after treatment were enumerated for CTCs, patients that presented with DLL3 expression on CTC at baseline showed a significant decrease in CTCs after treatment. This suggests that Rova-T might kill DLL3-expressing CTCs. These data support continued investigation of DLL3 expression on CTC in SCLC patients with the goal of facilitating a liquid biopsy able to assess DLL3 status as a prospective companion diagnostic for Rova-T.

Citation Format: Somdutta Roy, Kevin Martinez, Archana Dilip, Holger Karsunky, Scott Dylla. DLL3 analysis of circulating tumor cells predict treatment outcome in phase 1 rova-T study in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3721. doi:10.1158/1538-7445.AM2017-3721
Abstract 3729: Tumor-specific antibody labeling of pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model using a fluorescent humanized anti-CEA antibody
Authors: Lwin Thinzar , Murakami Takashi , Hoffman Robert M. , Yazaki Paul J. , Bouvet Michael
Published: July 2017

Introduction: Delineation of tumor margins is critical in oncologic surgery, particularly in resection of pancreatic cancer. Surgeons are limited in visualization with bright light surgery. Fluorescence guided surgery (FGS) can help surgeons better visualize all potential cancer cells during the operation. In the present study, we show that the use of an anti-CEA antibody conjugated to an 800nm NIR fluorescent dye can selectively label pancreatic cancer in both pancreatic cancer cell lines and patient derived xenograft mouse models (PDOX).

Materials/Methods: BxPC3-GFP cells or pancreatic cancer tissue specimens were grafted into flanks of nude mice. Both types of tumors were allowed to grow until 1 cm. Fragments tumors (2 mm3) were grafted onto the pancreatic tail of recipient mice to create PDOX models. After tumors reached 7-10mm in size, 75 ug of Anti-CEA-800 dye was injected into the tail-vein (LI-COR 800 CW IRDye®, Lincoln, NE). Mice were imaged via the Maestro CRI imaging system (Perkin Elmer, Waltham, MA) 24 hours after injection.

Results: Images obtained after fluorescent antibody injection showed that anti-CEA-800 specifically labeled both the cell-line-derived tumors and patient-derived tumors with an adequate tumor to background contrast at 24 hours. The dye co-localized with GFP in tagged tumor cells lines. GFP fluorescence had poor penetration if covered by overlying tissue, which was not an issue with the anti-CEA-800 dye. The dye allowed clear identification of the tumor and tumor margin. In the BxPC-3 based tumor, the dye allowed identification of a small 1mm satellite lesion in the spleen that would otherwise have been missed.

Conclusions: Fluorescent humanized anti-CEA-800 antibody specifically labeled orthotopically implanted pancreatic cancer xenografts from cell lines and patient derived tissue. Tumor-specific fluorescence imaging clearly identified the primary tumor and satellite lesion. The 800nm wavelength allowed deeper tissue penetration, particularly in areas of tumor covered by normal pancreatic parenchyma. Humanized anti-CEA antibodies conjugated to a radio-labeling agent and LI-COR 800 dye is already in Phase I/II trials. Humanized anti-CEA conjugated with the IR-800 dye is a promising agent for future clinical FGS applications.

Citation Format: Thinzar Lwin, Takashi Murakami, Robert M. Hoffman, Paul J. Yazaki, Michael Bouvet. Tumor-specific antibody labeling of pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model using a fluorescent humanized anti-CEA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3729. doi:10.1158/1538-7445.AM2017-3729
Abstract 3730: Early metabolic response to tumor-treating fields in patients with recurrent glioblastoma
Authors: Mittal Sandeep , Barger Geoffrey R. , Bosnyák Edit , Shah Varun B. , Juhász Csaba
Published: July 2017

PURPOSE. Tumor-Treating Fields (TTFields) is an FDA-approved treatment approach for patients with both newly-diagnosed and recurrent glioblastoma. The main purpose of the present study was to explore if amino acid positron emission tomography (PET) is able to detect an early, objective metabolic tumor response during the first months of TTFields therapy.

METHODS. Patients with MRI signs of recurrent glioblastoma eligible for TTFields along with maintenance temozolomide were recruited for PET scanning with alpha[C-11]-methyl-L-tryptophan (AMT) at baseline (before start of TTFields application) and up to 3 months later. Serial MRIs were also performed in 1-2 months intervals. AMT accumulation in the MRI-detected tumor mass was measured by standardized uptake values (SUVs), and PET-based tumor volumes were also calculated. Interval changes of these PET variables were compared to changes in areas of MRI enhancement.

RESULTS. Out of 7 recruited patients, 5 showed appropriate compliance (>75%) with the device and underwent follow-up PET. Four of the 5 patients showed a clear metabolic response during TTF treatment on PET imaging. Two patients, who received TTFields treatment only (in addition to maintenance temozolomide), showed 25% and 100% decrease of PET-based tumor volumes, respectively. MRI was stable (patient #1) or showed increasing contrast enhancement (#2) during the same period. In this latter patient, TTFields was continued with bevacizumab treatment, leading to decreased contrast enhancement and further decrease of AMT uptake on a second follow-up PET. In two other patients (#3 and #4), TTFields were combined with bevacizumab rescue treatment. Patient #3 showed a markedly decreased tumoral AMT uptake and metabolic volume along with stable MRI enhancement. Patient #4 showed interval decrease of tryptophan PET uptake 2 months after initiation of TTFields therapy. Finally, patient #5 showed an interval expansion of the PET-based tumor volume along with MRI progression during a 2-month follow-up despite TTFields combined with bevacizumab and carboplatin.

CONCLUSIONS. This is the first study to demonstrate an objective metabolic response of recurrent glioblastoma within 1.5-3 months after initiation of TTFields. The data suggest that amino acid PET can identify early responders to TTFields therapy.

Citation Format: Sandeep Mittal, Geoffrey R. Barger, Edit Bosnyák, Varun B. Shah, Csaba Juhász. Early metabolic response to tumor-treating fields in patients with recurrent glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3730. doi:10.1158/1538-7445.AM2017-3730
Abstract 3731: A novel multidimensional cfDNA assay for real time analysis of chemotherapy response
Authors: Shah Pavan P. , Patsch Katherin , Matasci Naim , Soudararajan Anjana , Diaz Patricia , Agus David B. , Ruderman Daniel , Gross Mitchell E.
Published: July 2017

Due to its non-invasive nature and transient lifespan (~30 mins), circulating cell free DNA (cfDNA) may provide novel insights into cancer evolution and response to therapy. Here, we have developed a multidimensional cfDNA assay to measure patients’ responses to chemotherapy on a time scale that would be impossible using traditional biopsy methodologies.

We extracted cfDNA from plasma collected immediately before and after infusion of cytotoxic chemotherapy, and 24 hours after treatment. We developed assays to (1) quantify cfDNA via qPCR of a 288 base pair amplicon of Long Interspersed Nuclear Elements (LINE1), (2) determine the DNA Integrity Index based on additional analysis of 115bp and 516bp LINE1 amplicons, and (3) analyze androgen receptor exon 8 via targeted deep sequencing.

We analyzed cfDNA extracted from 17 patients with metastatic castration-resistant prostate cancer through sequential application of these 3 assays. Median PSA levels were 69 (0-2223) ng/dl, Hgb 12 (8.2-15) g/L, Alk Phos 117 (35-879) IU/mL. Deep sequencing of AR exon 8 revealed 6 of the 17 patients presented with 1-2 SNVs (H874Y, T877A, D890H or D879Y) at ≥1% frequency. Considering LINE1 quantification alone, the average change pre/post chemotherapy was 38.78 pg/mL (0.5-292.0 pg/mL). A variety of responses was noted including: 8 of the patients expressed elevated LINE1 levels post-treatment when compared to baseline, and 5 expressed depressed levels. Importantly, therapy did not affect tumor subclones equally, as demonstrated by sequencing and quantitation of mutant AR exon 8 species. Additional data on changes in DNA Integrity Index will be presented.

We conclude that cytotoxic chemotherapy produces immediately observable effects on cfDNA. The effect of docetaxel chemotherapy on distinct clones within the tumor can be measured within hours. Now that a short-time dynamics response has been demonstrated, follow up studies will be performed involving more patients to determine the prognostic value.

Citation Format: Pavan P. Shah, Katherin Patsch, Naim Matasci, Anjana Soudararajan, Patricia Diaz, David B. Agus, Daniel Ruderman, Mitchell E. Gross. A novel multidimensional cfDNA assay for real time analysis of chemotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3731. doi:10.1158/1538-7445.AM2017-3731
Abstract 3733: Adenine nucleotide translocase2 mediates 18F-FDG uptake in dedifferentiated thyroid cancer
Authors: Lee Chul-Hee , Youn Hyewon , Chung Seock-Jin , Kim Ha , Park Cho Rong , Kim Mi Jeong , Park Young Joo , Cho Sun Wook , Kang Keon Wook , Chung June-Key
Published: July 2017

Objectives: 18F-fluorodeoxyglucose (FDG), an analogue of glucose, provides valuable functional information based on the increased glucose uptake and glycolysis in malignant tumor cells. Although both glucose transporter-1 (GLUT-1) and hexokinase2 (HK2) activity have been considered to associate with FDG uptake, the molecular mechanisms that determine FDG uptake are still largely unknown. Adenine nucleotide translocase 2 (ANT2) in the mitochondria inner membrane was reported to relate with tumor malignancy. We investigated the correlation between FDG uptake and Glut-1, HK2, and ANT2 expression in thyroid cancers throughout various spectrums of differentiation status.

Methods: N-thy-ori (normal human thyroid cells), WRO (follicular cancer), BHP10-3 and TPC-1 (papillary cancer), and FRO (anaplastic cancer) were used for this research. GLUT-1, HK2, and ANT2 expressions were measured by western blot. ANT2 siRNAs and pcDNA3.1-ANT2 vectors were used to modify ANT2 expression. FDG uptakes were measured in thyroid cells and human embryonic kidney cells (293FT) with HK2 or ANT2 transfection. For patient tissue analysis, 95 thyroid tissue-array cores were evaluated, and which are classified 36 as normal, 44 as poorly differentiated (PD), and 15 as anaplastic thyroid cancer (ATC). ANT2 expression was measured by immunostaining, scored from 1 to 5.

Results: FDG uptake in thyroid cancer cells was increased in anaplastic and poorly differentiated cells (P<0.001). GLUT-1 expression was higher in both TPC-1 and FRO than other cells. Whereas, HK2 was expressed only in cancer cells. ANT2 was expressed only in FRO cells and the highest FDG accumulation was also observed in FRO. ANT2 siRNA showed decreased FDG uptake (0.55-fold) and ANT2 overexpression increased FDG uptake (1.7-fold). In 293FT cells, HK2 and ANT2 transfection increased FDG uptake (P<0.01). PD tissues (mean = 41.7, SD = 19.7) and ATC (mean = 48.0, SD = 25.6) tissues from patients showed higher ANT2 expression than normal (P<0.05).

Conclusion: We showed that ANT2 was expressed only in anaplastic thyroid cancer cells, and this was related to FDG uptake. Higher level of ANT2 expression was observed in dedifferentiated cancer, and this indicates that ANT2 can be used as a marker of malignancy in thyroid cancer.

Citation Format: Chul-Hee Lee, Hyewon Youn, Seock-Jin Chung, Ha Kim, Cho Rong Park, Mi Jeong Kim, Young Joo Park, Sun Wook Cho, Keon Wook Kang, June-Key Chung. Adenine nucleotide translocase2 mediates 18F-FDG uptake in dedifferentiated thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3733. doi:10.1158/1538-7445.AM2017-3733
Abstract 3747: msa2 biotin-binding CAR combined with biotinylated tumor-specific antibodies or DNA aptamers for universal cancer immunotherapy
Authors: Lohmueller Jason J. , Ham James D. , Finn Olivera J.
Published: July 2017

Chimeric antigen receptor T cells (CAR-Ts) are highly promising as cancer therapeutics, but the creation of CARs for new antigens remains a laborious process. As an alternative strategy researchers are designing universal “tag-CARs” that instead of directly recognizing tumor antigens, recognize tags on tumor-specific antibodies bound to tumor cells, thereby activating T cell effector functions. Here we report the creation of a novel tag-CAR that uses the high affinity monomeric streptavidin 2 (msa2) biotin binding domain to target cancer cells labeled with biotinylated tumor-specific antibodies or DNA aptamers. We constructed lentiviral vectors encoding msa2 CARs with three different signaling architectures - CD28-zeta, CD28-OX40-zeta, and 4-1BB-zeta. After confirming that each msa2 CAR was efficiently expressed on the surface of Jurkat and primary human T cells, we found that plate-immobilized biotin was capable of specifically inducing T cell activation markers and cytokine production. We then stained a variety of cancer cell lines with various tumor-specific biotinylated antibodies or DNA aptamers targeting tumor antigens such as MUC1, CD19, CD20, and EGFR and found that the stained cells specifically activated the msa2-CAR cells and led to cytokine production. We are currently investigating the ability of msa2-CAR cells to lyse antibody and aptamer-bound cancer cells and will be assessing their ability to shrink tumors in a human tumor xenograft mouse model.

Citation Format: Jason J. Lohmueller, James D. Ham, Olivera J. Finn. msa2 biotin-binding CAR combined with biotinylated tumor-specific antibodies or DNA aptamers for universal cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3747. doi:10.1158/1538-7445.AM2017-3747
Abstract 3751: Allogeneic EGFRvIII chimeric antigen receptor T cells for treatment of glioblastoma
Authors: Wong Oi Kwan , Dusseaux Mathilde , Ma Jing-Tyan , Au Melinda , Leduc Sophie , Chou Joyce , Yu Jessica M. , Bateman Marjorie , Pertel Thomas , Lindquist Kevin C. , Smith Julianne , Sasu Barbra , Liu Shu-Hui
Published: July 2017

Glioblastoma multiforme (GBM) is a highly aggressive form of brain tumors with a 5-year survival rate of less than 10%. Standard-of-care combining radiation therapy with temozolomide only yields a median survival of 14.6 months and more effective therapeutic options to extend patient lives are urgently needed. EGFR variant III (EGFRvIII) is a tumor specific mutant of EGFR found in 25-30% of GBM but not expressed in healthy tissues. EGFRvIII is formed by an in-frame deletion of exons 2-7 of the wild-type EGFR which leads to removal of 267 amino acids in the extracellular domain of the receptor. The truncated receptor loses its ability to bind ligands but acquires constitutive kinase activity. The lack of normal tissue expression makes EGFRvIII an ideal target for developing CAR T therapy. Most CAR T therapies in clinical development use the patients’ own T cells for CAR T manufacturing. Our approach is to develop an “off-the-shelf” CAR-T treatment for GBM using healthy donor T cells with the TCR (T-cell receptor) disrupted to prevent graft-versus-host disease. This allogeneic approach could circumvent challenges faced by some patients due to limited availability and quality of their own T cells and rapid progress of their diseases, and has the potential to reduce the high cost associated with autologous CART therapy. Using phage panning and hybridoma approaches, we generated a series of humanized and fully human EGFRvIII antibodies with a wide range of affinities. Recombinant EGFRvIII protein binding by Biacore and FACS assays using EGFRvIII and EGFR wild-type expressing cells demonstrated that these antibodies are highly specific for EGFRvIII. The antibodies were converted to ScFvs and cloned into a CAR vector. Subsequently, EGFRvIII CARs were transduced into primary human T cells for functional studies. We developed a series of assays to evaluate CAR expression, degranulation activity and target dependent cytotoxicity. Our goal is to select CAR candidate that is safe, persistent and has potent target dependent cytotoxicity.

Citation Format: Oi Kwan Wong, Mathilde Dusseaux, Jing-Tyan Ma, Melinda Au, Sophie Leduc, Joyce Chou, Jessica M. Yu, Marjorie Bateman, Thomas Pertel, Kevin C. Lindquist, Julianne Smith, Barbra Sasu, Shu-Hui Liu. Allogeneic EGFRvIII chimeric antigen receptor T cells for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3751. doi:10.1158/1538-7445.AM2017-3751
Abstract 3757: Ewing Sarcoma regression by Allo-MHC/Chm1 specific T cells without GVHD
Authors: Thiel Uwe , Schober Sebastian , Einspieler ingo , Kirschner Andreas , Schirmer David , Grünewald Thomas , Lüttichau Irene Teichert-von , Richter Guenther , Sorensen Poul , Burdach Stefan
Published: July 2017

Background: Chondromodulin-I (CHM1) sustains malignancy in Ewing sarcoma (ES). Refractory ES carries a dismal prognosis and patients with bone marrow (BM) metastases do not survive irrespective of therapy. We assessed HLA-A*02:01/ CHM1 specific allorestricted T cell receptor (TCR) wildtype and transgenic cytotoxic (CD8+) T cells against ES.

Patients and Methods: Three refractory HLA-A2+ ES patients were treated with HLA-A*02:01/peptide specific allorepertoire-derived (i.e. allorestricted) CD8+ T cells. Patient #1 received up to 4.8x105/kg body weight HLA-A*02:01- allorestricted donor derived wild type CD8+ T cells. Patient #2 received up to 8.2x106/kg HLA-A*02:01- donor derived and patient #3 up to 6x106/kg autologous allorestricted TCR transgenic CD8+ T cells. All patients were treated with the same TCR complementary determining region 3 allorecognition sequence for CHM1319.

Results: HLA-A*02:01/CHM1319 specific allorestricted CD8+ T cells showed specific in vitro lysis of all patient-derived ES cell lines. Therapy was well tolerated and did not cause graft versus host disease (GVHD). Patients #1 and #3 showed slow progression, whereas patient #2, while having BM involvement, showed partial metastatic regression associated with T cell homing to involved lesions. CHM1319 TCR transgenic T cells could be tracked in his BM for weeks.

Conclusions: CHM1319-TCR transgenic T cells home to affected BM and may cause partial disease regression. HLA-A*02:01/antigen specific allorestricted T cells proliferate in vivo without causing GVHD.

Citation Format: Uwe Thiel, Sebastian Schober, ingo Einspieler, Andreas Kirschner, David Schirmer, Thomas Grünewald, Irene Teichert-von Lüttichau, Guenther Richter, Poul Sorensen, Stefan Burdach. Ewing Sarcoma regression by Allo-MHC/Chm1 specific T cells without GVHD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3757. doi:10.1158/1538-7445.AM2017-3757
Abstract 3769: Stem cell-engineered invariant natural killer T cells for cancer therapy
Authors: Yang Lili , Smith Drake , Liu Siyuan , Ji Sunjong , Liu Bo
Published: July 2017

Invariant natural killer T (iNKT) cells comprise a small population of αβ T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer, infections, allergies and autoimmunity. However, the study of iNKT cell biology and the therapeutic applications of these cells are greatly limited by their small numbers in vivo (~0.01-1% in mouse and human blood). Here, we report a new method to generate large numbers of iNKT cells in mice through T cell receptor (TCR) gene engineering of hematopoietic stem cells (HSCs). We showed that iNKT TCR-engineered HSCs could generate a clonal population of iNKT cells. These HSC-engineered iNKT cells displayed the typical iNKT cell phenotype and functionality. They followed a two-stage developmental path, first in thymus and then in the periphery, resembling that of endogenous iNKT cells. When tested in a mouse melanoma lung metastasis model, the HSC-engineered iNKT cells effectively protected mice from tumor metastasis. This method provides a powerful and high-throughput tool to investigate the in vivo development and functionality of clonal iNKT cells in mice. More importantly, this method takes advantage of the self-renewal and longevity of HSCs to generate a long-term supply of engineered iNKT cells, thus opening up a new avenue for iNKT cell-based immunotherapy.

Citation Format: Lili Yang, Drake Smith, Siyuan Liu, Sunjong Ji, Bo Liu. Stem cell-engineered invariant natural killer T cells for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3769. doi:10.1158/1538-7445.AM2017-3769
Abstract 3770: CAR-T cell harboring a camelid single domain antibody as a targeting agent to kill tumors expressing VEGFR2
Authors: Chao Heman , Tian Baomin , Uger Marni , Wong Wah
Published: July 2017

Modulation of the immune system is showing tremendous promise in the treatment of malignancies. In addition to checkpoint inhibitors that re-activate T cells present in the tumor microenvironment, exogenously transduced chimeric antigen receptor (CAR) T cells are providing excellent responses in clinical trials for the treatment of leukemias. In this study, we describe CAR-T cells that target VEGFR2-expressing tumors.

Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor (VEGF), which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFR). As VEGFR2 is also expressed by a variety of tumors, we investigated the utility of anti-VEGFR2 CAR-T cells as a method to treat VEGFR2-expressing tumors.

Camelid antibodies are small (14 kD) single chain antibodies. To generate a camelid antibody targeting the extracellular domain of VEGFR2, a llama was immunized with recombinant VEGFR2/Fc. A phage display library was generated and screened to identify an antibody with high binding affinity to VEGFR2. The selected antibody was expressed in the E. coli. BL21 (DE3) pT7 system. The purified antibody was characterized by SEC, LC-MS peptide mapping and ELISA.

CAR-T cells were engineered to express the camelid anti-VEGFR2 antibody in combination with the CD28 and 4-1BB costimulatory molecules and the CD3 zeta chain. Tumor cells were screened for expression of VEGFR2, and the HL-60 acute promyelocytic leukemia, ZR-75-30 breast ductal carcinoma and NCI-H23 non-small cell lung adenocarcinoma were identified. Co-incubation of anti-VEGFR2 CAR-T cells with the VEGFR-2-expressing cell lines resulted in dose-dependent target cell toxicity as measured by LDH release. In addition, T cell activity was confirmed, as high levels of IL-2 and IFN-γ were detected in the cell culture media. These results suggest that anti-VEGFR2 CAR-T may be useful in directly targeting VEGFR2-expressing tumors.

We previously showed the utility of camelid antibodies in CAR-T constructs as anti-CEACAM6 CAR-T cells show both in vitro and in vitro efficacy against the pancreatic tumor Bx-PC3. The use of the a camelid V21 antibody to target VEGFR2-expressing tumors provides hope that camelid single domain antibodies can be developed for CAR-T therapies.

Citation Format: Heman Chao, Baomin Tian, Marni Uger, Wah Wong. CAR-T cell harboring a camelid single domain antibody as a targeting agent to kill tumors expressing VEGFR2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3770. doi:10.1158/1538-7445.AM2017-3770
Abstract 3771: Concurrent GVAX and anti-OX40 therapy in murine intracranial glioma model
Authors: Jahan Nusrat , Talat Hammad , Curry William T.
Published: July 2017

Introduction: Malignant glioma is the most aggressive brain tumor with a median survival time of less than 1.5 years post diagnosis despite standard treatments. However, recent advancements in immunotherapy hold significant promise. Irradiated whole tumor cell vaccination stimulates antitumor immunity and prolongs survival in mice with intracranial glioma tumors. We have shown that vaccination and immune checkpoint blockade is synergistic and these efforts are underway in the clinic. In contrast to immune checkpoint blockade, agonist stimulation of OX40, a member of the TNFR/TNF superfamily that is expressed on activated CD4 and CD8 T lymphocytes increases T cell proliferation, clonal memory differentiation, and cytokine release. We hypothesized that subcutaneous vaccination with irradiated GM-CSF expressing glioma cells (GVAX) would, therefore, synergize with systemic delivery of stimulating anti-OX40 antibody and provide effective immunity against intracranial gliomas.

Methods: Gl261 cells were implanted in the right frontal lobes of syngeneic c57/bl6 mice using a stereotactic implantation technique. Mice received concurrent subcutaneous injection of irradiated GM-CSF expressing glioma cells and intraperitoneal injection of OX40 antibody, either monotherapy, or control treatment on days 3, 6 and 9 following tumor implantation.

Results: Combination immunotherapy with irradiated GM-CSF expressing glioma cells and intraperitoneal delivery of agonist monoclonal antibody against OX40 was more effective than either therapy alone and prolonged survival in mice with established intracranial GL261 tumors. Elispot analyses of peripheral lymphocytes revealed that combination immunotherapy reverses the Th2 cytokine skew that is induced by the presence of intracranial glioma. OX40 signaling alone and in combination with vaccination drove high levels of interferon gamma expression by splenocytes harvested from glioma-bearing animals, while IL-10 expression was little changed. In the tumor microenvironment, vaccination improves the CD8+ /FoxP3+ T lymphocyte ratio, while OX40 ligation overcomes tumor-driven lymphocyte exhaustion, demonstrated by reduced expression of PD1 and LAG3, measured by flow cytometery 2 weeks after initiation of treatment.

Conclusions: Combination treatment with GVAX and OX-40 agonist antibody increased survival in mice bearing intracranial gliomas and are mechanistically complementary.

Citation Format: Nusrat Jahan, Hammad Talat, William T. Curry. Concurrent GVAX and anti-OX40 therapy in murine intracranial glioma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3771. doi:10.1158/1538-7445.AM2017-3771