bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5549: Antitumor efficacy of the oncolytic peptide LTX-315 in genetic mouse models that resist conventional chemo- and immunotherapeutic treatments
Authors: Pittet Mikael J. , Liao Hsin-Wei , Sveinbjørnsson Baldur , Rekdal Øystein
Published: August 2018

LTX-315 is an oncolytic peptide that induces immunogenic cancer cell death and is currently evaluated in phase 1 clinical trials for its ability to trigger recruitment of tumor-specific T cells to tumors. Previous experimental studies identified LTX-315's ability to control cancer in mice grafted with various tumor cell lines. Here we extend our analyses to conditional genetic mouse models of both melanoma (driven by Braf and Pten alterations) and soft tissue sarcoma (driven by Kras and P53 alterations). In these genetic models, cancer cells are derived from somatic cells that are transformed in their normal tissue microenvironment and progress to high-grade tumors that are poorly infiltrated by T cells and typically resist prescribed chemo- and immunotherapeutic treatments. We report that LTX-315 not only delays tumor progression substantially in both models, but also profoundly alters the tumor microenvironment, most notably characterized with CD8+ T cell, NK cell and dendritic cell infiltration. Transition from a ‘cold' to a ‘hot' tumor microenvironment was also observed in patients with melanoma, sarcoma and breast cancer. Furthermore, CD8+ T cell depletion in mice abrogated long-term antitumor efficacy of LTX-315, indicating that these cells are required for drug-induced tumor control. Importantly, the ability to convert non-T-cell-infiltrated tumors into ones that display antitumor T cell immunity opens the possibility to prime and make unresponsive tumors sensitive for systemic immune treatments.

Citation Format: Mikael J. Pittet, Hsin-Wei Liao, Baldur Sveinbjørnsson, Øystein Rekdal. Antitumor efficacy of the oncolytic peptide LTX-315 in genetic mouse models that resist conventional chemo- and immunotherapeutic treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5549.
Abstract 5560: PD-1/PD-L1 blockade enhances MDM2 inhibitor activity in p53 wild-type cancers
Authors: Wang Hui Qin , Liang Jinsheng , Mulford Iain , Sharp Fiona , Gaulis Swann , Chen Yan , Trabucco Gina , Quinn David , Growney Joseph D. , Meyer Matthew J. , Williams Juliet , Hammerman Peter , Hofmann Francesco , Dranoff Glenn , Engelman Jeffrey , Mataraza Jennifer , Halilovic Ensar
Published: August 2018

p53 is a transcription factor that plays a central role in guarding genomic stability of the cell through cell cycle arrest or induction of apoptosis. It has also been reported that p53 participates in the regulation of tumor immunity and in homeostatic regulation of immune responses. However, the immunomodulatory effect of p53 in tumor and the tumor microenvironment is not well understood. From gene expression and immunohistochemical analysis of pre- and post-treatment biopsies from patients treated with the MDM2 inhibitor NVP-CGM097, we observed upregulation of immune checkpoint transcripts (PD1 and PDL1), and an increase in the number of CD8+ tumor infiltrating lymphocytes. To investigate the immunomodulatory effect of p53 in more detail, we studied murine syngeneic models treated with NVP-HDM201, a potent and selective second generation MDM2 inhibitor. We performed multi-color FACS analysis on tumors and tumor draining lymph nodes. With NVP-HDM201 treatment, we observed increase in numbers of CD103+ DC cells, capable of antigen cross-presentation. Furthermore, NVP-HDM201 increased the percentage of Tbet+EOMES-T cells in tumors as well as tumor draining lymph nodes and also resulted in increased ratios of CD8+ Tcells/Treg in tumor. In addition, levels of both PD-L1 on CD45- cells and PD-1 on CD4+ T cells were increased. Importantly, PD-1 and PD-L1 blockade enhanced HDM201 activity in p53 WT syngeneic mouse models but not in p53 mutated models. The rate of complete tumor regressions (CR) was significantly increased with combination treatment as compared to either treatment alone. The animals that

achieved complete regressions also developed long lasting anti-tumor memory against the specific tumor cells as evidenced by re-challenge experiments. Taken together, these results demonstrate that MDM2 inhibition triggered adaptive immunity which was further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in cancer patients with wildtype p53.

Citation Format: Hui Qin Wang, Jinsheng Liang, Iain Mulford, Fiona Sharp, Swann Gaulis, Yan Chen, Gina Trabucco, David Quinn, Joseph D. Growney, Matthew J. Meyer, Juliet Williams, Peter Hammerman, Francesco Hofmann, Glenn Dranoff, Jeffrey Engelman, Jennifer Mataraza, Ensar Halilovic. PD-1/PD-L1 blockade enhances MDM2 inhibitor activity in p53 wild-type cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5560.
Abstract 5562: A 3D image-based quantification of T cell-mediated killing of CRC organoids in the presence of immuno-modulators
Authors: Goverse Gera , Daszkiewicz Lidia , Yan Kuan , Tleis Mohamed , Madej Mariusz , Salinaro Lucia , Price Leo
Published: August 2018

BACKGROUND

Cancer immunotherapy has already yielded promising clinical results but most patients still do not respond. The mechanisms of action of these treatment modalities are also not fully understood and the progress in this direction is hampered by a lack of appropriate pre-clinical testing models. To investigate the role of novel therapeutics targeting the immune cell compartment to kill tumor cells, we developed an in vitro assay based on 3D cultures and image based analysis in a 384-wells plate format. Immune cells are added to recapitulate the tumor micro-environment and its complex interactions between different cellular players. Specifically, infiltration of immune cells into the tumoroids and their killing are visualized and measured, enabling a better understanding of the immune-modulatory profile of different treatments.

METHODS

Autologous patient derived colon organoids from normal and tumor tissue from several patients were cultured in a 3D environment. HLA-matched PBMCs with and without activation were added and immune-cell infiltration and killing of the CRC organoids was visualized in 3D using automated microscopy. Quantification of immune cell effects was achieved with morphometric analysis with OMinerTM software.

RESULTS

3D image data analysis enabled the discrimination of immune-tumor cell interactions and revealed a higher immune cell infiltration and tumoroid killing upon activation. In addition, we were able to compare both normal and tumor colon tissue from the same patient and compare between patients, demonstrating patient related differences and elucidating the effect of immune cell targeting on normal colon tissue.

CONCLUSIONS

The 3D assay presented here allows the analysis of different cell types that engage in a more realistic setting than when culturing them in traditional 2D cultures. Using image-based analysis, immune-tumor interactions can be visualized and quantified. The 3D environment, both for the cell culture and image analysis, allows for measurement of spatially resolved information, not accessible by monolayer cultures or biochemical assays. This new and innovative platform can empower immunotherapy drug developers to select the most promising candidates and better understand their mechanism of action.

Citation Format: Gera Goverse, Lidia Daszkiewicz, Kuan Yan, Mohamed Tleis, Mariusz Madej, Lucia Salinaro, Leo Price. A 3D image-based quantification of T cell-mediated killing of CRC organoids in the presence of immuno-modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5562.
Abstract 557A: The clinicopathoogical significance of FBXW7 expression in GIST
Authors: Koga Yuki , Iwatsuki Masaaki , Yamashita Kouhei , Kiyozumi Yuki , Sawayama Hiroshi , Hiyoshi Yukiharu , Baba Yoshihfumi , Miyamoto Yuji , Yoshida Naoya , Baba Hideo
Published: August 2018

Objective: Gastrointestinal stromal tumor (GIST) is a rare mesenchymal tumor originated from the interstitial cells of Cajal in the gastrointestinal tract. A significant breakthrough was achieved with the identification of the expression of the c-kit (CD117) antigen in GISTs. However, alternate oncogenic pathways have not sufficiently been understood. FBXW7 is a ubiquitin ligase that mediates ubiquitylation of oncoproteins, which is known as tumor-suppressor gene in various malignancies. Previous reports showed that FBXW7 is associated with tumor progression by regulating some oncoprotein such as c-myc and c-jun. The aim of this study is to elucidate the clinicopathological significance of FBXW7 in GIST.

Methodology: (i) We examined the FBXW7 expression in GIST cells by immunostaining and correlation clinicopathological characteristics. (ii) We examined cell proliferation by Cytotoxicity assays in GIST-T1 cells suppressed by FBXW7-specific siRNA.

Results: (i) In 90 cases of GIST samples with surgical resection, high FBXW7 and observed are 47 cases and low expression are 43 cases. There are significantly more large size, located small intestine, high mitotic index in low FBXW7 cases (P=0.05, P=0.01, P=0.04). There are significantly more high risk cases in modified-Fletcher classification in low FBXW7 expression cases (P=0.0008). There are significantly higher value of SUV max in high risk cases of modified-Fletcher

classification and low FBXW7 expression cases (P=0.007, P=0.02). In GIST cases with distant metastasis, there are significantly more low FBXW7 expression cases than GIST cases without distant metastasis (P=0.009). FBXW7 expression is significantly inversely correlated with C-MYC expression (P=0.0004). (ii) The GIST cells suppressed FBXW7 expression by siRNA have higher proliferation rate compared to control GIST cells (P=0.02).

Conclusion: We demonstrated that FBXW7 expression is correlated with modified-Fletcher classification, SUV max value, c-myc expression and cell proliferation in GIST. These results are suggesting that FBXW7 may be associated with tumor progression. These findings suggest that it is possible that FBXW7 can be a promising biomarker for disease progression of GIST.

Citation Format: Yuki Koga, Masaaki Iwatsuki, Kouhei Yamashita, Yuki Kiyozumi, Hiroshi Sawayama, Yukiharu Hiyoshi, Yoshihfumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. The clinicopathoogical significance of FBXW7 expression in GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 557A.
Abstract 5584: Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung cancer patients
Authors: Andree Kiki C. , Tamminga Menno , Mentink Anouk , Hilterman T Jeroen , Groen Harry J. , Terstappen Leon W.
Published: August 2018

Introduction The number of circulating tumor cells (CTC) isolated by the FDA approved CellSearch (CS) system in 7.5 mL of blood of metastatic and non-metastatic non-small cell lung cancer (NSCLC) patients is low or absent. Processing a larger blood volume could yield a greater amount of CTC from these patients. An approach to probe larger volumes is using diagnostic leukapheresis (DLA) where the blood can be split into its components after which selective harvesting of mononuclear cells (MNC) including CTC can take place.

Methods Patients with histologically proven NSCLC who either have been recently diagnosed or are eligible for new therapy can participate in this ongoing study. Patients had DLA for MNC collection derived from one total body blood volume. Before and after DLA, whole blood was drawn in a CellSave blood collection tube and analyzed by CS. The obtained DLA product was divided for analysis. 2x108 cells (on average 2mL of DLA product, representing ~100mL of blood) was diluted in 7.5 mL buffer, and analyzed by CS. 9 mL of the DLA product (on average 1x109 cells) was fixed and depleted of leukocytes using the RosetteSep CD45 depletion kit (Stemcell Technologies), allowing processing of a larger volume, followed by CS analysis. Results of molecular characterization of the detected CTC is pending.

Results So far, a total of 8 DLA's were performed in 7 stage 4 NSCLC patients. Sample collection took place before (t=0) and/or after (t=1) treatment. Patients underwent DLA in about 110 minutes, at this time an average of 4860 mL of blood was processed. No complications occurred, no post-procedure complaints were recorded. Similar DLA products with an average of 95 mL were obtained. CTC counts for these patient samples are shown in the table below.

CTC counts blood and DLAPatientt=#CTC pre blood#CTC post blood#CTC DLA (~2mL, 200x106 cells)#CTC depleted DLA (~9mL, 900x106 cells)101n/a114113020203514602031000040n/a04495000166000029020114

Conclusion DLA is a novel method to collect CTC from the blood. DLA can result in the detection of CTC where the use of 7.5mL of blood is not sufficient. Additionally, DLA shows an increase of CTC yield in patients with CTC detected in 7.5 mL of blood.

Citation Format: Kiki C. Andree, Menno Tamminga, Anouk Mentink, T Jeroen Hilterman, Harry J. Groen, Leon W. Terstappen. Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5584.
Abstract 559: Development of a novel approach to identify cancer drivers for hepatitis B virus-associated hepatocellular carcinoma
Authors: SHEN SHIH-CHUN , Lu Amy Jiaen , Shieh John Caleb , Su Yih-Ping , Nasir Bilal , Ling Ning , Lau Daryl T. , Steffen Jamin D. , Shieh Fwu-Shan , Song Wei , Su Ying-Hsiu
Published: August 2018

Infection with Hepatitis B virus (HBV) is a major etiology of hepatocellular carcinoma (HCC), the third leading cause of cancer-related deaths worldwide. Studies have shown that some cells infected with HBV contain integrated viral DNA, and integrated viral DNA has been observed more frequently in HCC than in non-HCC infected liver. Although integration occurs randomly throughout host chromosomes, recurrent integration sites have been reported. Interestingly, these integration sites are located in genes known to be involved in carcinogenesis. During HCC carcinogenesis, tumor cells undergo uncontrolled clonal expansion in which a particular integration junction will become dominant (or a major junction). We have adapted a method known as primer extension capture (PEC) to enrich integrated HBV DNA for next-generation sequencing (NGS) to detect these integration junctions that occur at low frequency. We next developed a novel program, “HccDriverFinder,” to identify major junctions, incorporate PubMed data mining for driver identification, detect HBV mutations (revealing a striking 98.8% HCC-linked mutations identified in cancer), and visualize integration events. By analyzing NGS data generated from 13 matched HCC and adjacent non-HCC tissue samples, we found specific integrations in known targets such as TERT and CCNE1, and uncovered new recurrent HBV integration sites in genes including CSAD and ABCC13 (both of which are reportedly linked with tumorigenesis). We have further optimized the PEC approach to enrich for the whole HBV genome and have applied it to urine DNA from 31 hepatitis, 27 cirrhosis, and 29 HCC patients for the potential of identifying HBV junctions in circulation. Our approach holds the promise of building a driver identification kit for HCC detection, drug development and precision medicine.

Citation Format: SHIH-CHUN SHEN, Amy Jiaen Lu, John Caleb Shieh, Yih-Ping Su, Bilal Nasir, Ning Ling, Daryl T. Lau, Jamin D. Steffen, Fwu-Shan Shieh, Wei Song, Ying-Hsiu Su. Development of a novel approach to identify cancer drivers for hepatitis B virus-associated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 559.
Abstract 5591: Analytical and clinical validation of blood-based NGS panel for detection of genomic alterations in DNA repair pathways
Authors: Zhao Zhixin
Published: August 2018

Circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA) in cancer patients are valuable resource of biomarkers for early detection, diagnosis, treatment and monitoring of disease progression. Compared to cfDNA, cfRNA is more labile to degrade and fluctuate, and thus more challenging to be detected in blood. In the present study, we reported PrediSeq, a validated NGS-based liquid biopsy assay to detect fusions, splicing and mutation events using cfRNA. By using Predicine proprietary ultra-sensitive DeepSeaTM (Deterministic, Efficient, Precise, Sensitive Algorithm) variant detection algorithm, pre-analytical study demonstrated RNA-based detection of fusion, RNA splicing, and mutations using reference materials. In a LOD study, PrediSeq NGS assay is more sensitive than the digital PCR based assay in terms of mutation detection. In clinical validation study, tissue and plasma samples from various types of cancer patients (lung, breast, prostate etc) were used, where concordant fusion events with tissue were observed and RNA mutations confirmed variants detected at DNA level. In conclusion, a highly sensitive and accurate RNA-based liquid biopsy test has been developed to complement the use of cfDNA for detection of fusion, splicing and mutations.

Citation Format: Zhixin Zhao. Analytical and clinical validation of blood-based NGS panel for detection of genomic alterations in DNA repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5591.
Abstract 5607: Tissue factor is a novel oncotarget for immunotherapy of triple-negative breast cancer using a second generation ICON in orthotopic mouse models of human and murine TNBC cell line- and patient-derived xenografts
Authors: Hu Zhiwei , Shen Rulong , Campbell Amanda , McMichael Elizabeth , Yu Lianbo , Ramaswamy Bhuvaneswari , London Cheryl A. , Xu Tian , Carson William E.
Published: August 2018

Triple-negative breast cancer (TNBC) is often associated with BRCA1 and BRCA2 mutation and is a leading cause of breast cancer death. Due to the lack of validated target molecules, there is currently no approved targeted therapy for TNBC. Tissue factor (TF) is a common yet specific surface target receptor for cancer cells, tumor vascular endothelial cells and cancer stem cells in several types of solid cancers. Here we report evidence supporting the idea that TF is a novel surface target for TNBC patients and cancer lines with BRCA1 and BRCA2 mutations. We showed that TF is over-expressed on TNBC cells and tumor neovasculature in greater than 85% of TNBC patients (n=14) when using standard paraffin-embedded tumor tissues and in nearly 60% of TNBC patients (n=157) when employing tissue microarray slides, but was not detected in adjacent normal breast tissue. We also describe the development of a second-generation TF-targeting immunoconjugate with improved efficacy and higher safety threshold compared to the original immunoconjugate. We showed that L-ICON1 is effective in killing TNBC cells via antibody-dependent cell-mediated cytotoxicity in vitro and in treating human and murine TNBC cell line- and patient-derived xenografts in vivo in preclinical orthotopic mouse models. These results suggest that TF is a novel oncotarget in TNBC and targeting TF may constitute a novel targeted immunotherapy for TNBC patients. The findings in this preclinical study warrant further clinical investigation.

Citation Format: Zhiwei Hu, Rulong Shen, Amanda Campbell, Elizabeth McMichael, Lianbo Yu, Bhuvaneswari Ramaswamy, Cheryl A. London, Tian Xu, William E. Carson. Tissue factor is a novel oncotarget for immunotherapy of triple-negative breast cancer using a second generation ICON in orthotopic mouse models of human and murine TNBC cell line- and patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5607.
Abstract 5608: Evaluation of retargeted T cell cytotoxicity of a bispecific antibody targeting CDH17 and CD3 in 2D- and 3D-colon cancer models
Authors: Irigoyen Macarena , Dai Eric , Luk John , Castillo Gonzalo , Staunton Don
Published: August 2018

Cadherin-17 (CDH17) is an adhesion molecule that binds to self and integrin a2b1, its expression in the adult is limited to gastrointestinal tissue and is involved in cancer progression. CDH17 is expressed de novo or overexpressed in several gastro-intestinal cancers including colorectal, gastric and pancreatic, making it an ideal target for immunotherapies. A humanized bifunctional antibody was developed, ARB-201, that binds to both CDH17 and the CD3/TCR complex (CD3) with high affinity. ARB-201 induced retargeted T cell cytotoxicity was determined in a 2D and 3D model. Relative to 2D, cytotoxicity of 3D tumors may be considered more predictive of efficacy for solid tumors. DLD-1, a colorectal adenocarcinoma cell line, was targeted in the 2D and 3D T cell cytotoxicity assays. Pre-labeled DLD-1 were grown in standard 384-well microplate (2D) or Kuraray's 384-well Elplasia microplates (3D). Effector PBMCs, freshly isolated from healthy donors were added to the target DLD-1 cells containing a concentration response curve of ARB-201. Samples were evaluated for the specific cellular cytotoxicity using cell imaging. The results show at 48 hours an EC50 of 12 pM for 2D and 47 pM for the 3D model. ARB-201 induced potent T cell cytotoxicity of DLD-1. Although cytotoxicity in the 3D tumor model was relatively less, we observed an increase over time, demonstrating that ARB-201 progressively kills 3D tumor cells and possesses cytotoxic activity that may translate into clinical efficacy for solid tumors.

Citation Format: Macarena Irigoyen, Eric Dai, John Luk, Gonzalo Castillo, Don Staunton. Evaluation of retargeted T cell cytotoxicity of a bispecific antibody targeting CDH17 and CD3 in 2D- and 3D-colon cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5608.
Abstract 5615: Antibodies targeting TIP1 enhance the efficacy of radiotherapy in lung cancer and glioblastoma
Authors: Kapoor Vaishali , Singh Abhay K. , Dadey David , Hoye Kelly , Collins Andrea , Thotala Dinesh , Hallahan Dennis
Published: August 2018

Antigens that are over-expressed in cancer in response to radiation are being used as novel targets. We showed tax interacting protein 1 (TIP-1) to be radiation-inducible that translocated to the surface of the cancer cell following irradiation. TIP-1, which consists of a single PDZ domain plays an important role in cell signaling, cancer development, and progression. TIP-1's involvement in various survival pathways makes it an attractive target for anticancer therapeutics. We used antibodies specific to this PDZ domain to determine its role in cancer cell survival. We monitored the proliferation of lung cancer (A549 and H460) and glioblastoma (D54 and U251) cells after 24, 48, 72 and 96h treatment with the anti-PDZ antibody. We observed a time-dependent proliferation arrest with anti-PDZ antibody treatment which was associated with increased apoptosis. The anti-PDZ antibody, when combined with radiation (3Gy), led to reduced proliferation and colony formation. Anti-PDZ antibody had no effect on the proliferation of normal lung (MRC-5) and endothelial (HUVEC) cells. Cells treated with anti-PDZ antibody showed decreased levels of the phosphorylated forms of AKT, mTOR, and a downstream substrate of mTOR, 4EBP1. Anti-PDZ antibody treatment also led to an overall reduction in basal levels of AKT, mTOR, and 4EBP1. Further, we evaluated the effect of the anti-PDZ antibody on tumor growth in heterotopic mouse models of lung cancer (A549) and glioma (U251). We observed significant growth delay in mice treated with anti-PDZ antibody treatment when compared to mice treated with the isotype control. The combination of the anti-PDZ antibody with radiation showed an additive effect. Immunoblot analysis of tumor tissues also showed downregulation of phosphorylated and total levels of AKT, mTOR and 4EBP1 in the tumors treated with anti-PDZ antibody. Overall, our results suggest that TIP-1 is a promising therapeutic target for treatment of lung cancer and glioblastoma. Antibodies specific to the PDZ domain of TIP-1 enhance the efficacy of radiotherapy. The anti-PDZ antibodies need to be optimized further before translating it into the clinic.

Citation Format: Vaishali Kapoor, Abhay K. Singh, David Dadey, Kelly Hoye, Andrea Collins, Dinesh Thotala, Dennis Hallahan. Antibodies targeting TIP1 enhance the efficacy of radiotherapy in lung cancer and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5615.
Abstract 5625: Myeloid derived suppressor cells (MDSCs) express Sialyl Tn (STn) and are a therapeutic target for anti-STn antibody drug conjugates
Authors: Eavarone David A. , Rao Patricia E. , Prendergast Jillian M. , Curtis Adam D. , Zhang Rong , Shopland Lindsay S. , Stein Jenna N. , Behrens Jeff , Dransfield Daniel T.
Published: August 2018

Introduction: Myeloid derived suppressor cells (MDSCs) are functionally defined by their capacity to suppress T cell immunity; therefore inhibiting these cells is of great interest for immuno-oncology applications. STn, the sialylated version of the carbohydrate Tn antigen, is broadly expressed in human cancers such as breast, ovarian, bladder, cervical, colon, and lung and is rarely expressed in normal adult human tissues. The presence of STn in tumors is associated with metastatic disease, poor prognosis, and reduced overall survival. Tumor STn expression is well established and can be leveraged for targeted cancer therapy, however its expression on immuno-suppressive tumor infiltrating lymphocytes such as MDSCs has not been established. Methods: We generated humanized anti-STn mAbs having no cross-reactivity to the asialylated form of STn (Tn) or other glycan antigens. The epitope targeted by these mAbs is the STn glycan itself, not a particular glycopeptide or carrier protein, which offers the broadest potential to bind to multiple STn-glycosylated proteins on cell surfaces. We used these mAbs to quantify specifically the expression of tumor and MDSC STn expression across a set of patient tumor samples. We further treated mice with a humanized anti-STn-MMAE antibody-drug conjugate to evaluate targeted depletion of STn+ MDSCs. In order to evaluate the therapeutic potential of targeting this MDSC phenotype, we further evaluated the immunosuppressive environment of tumors containing STn+ and STn- MDSCs. Results: For the first time, we report the detection of STn on the surface of MDSCs in a growing body of patient tumor samples. Our data in patients as well as xenograft models additionally links STn expression on MDSCs to tumor cell STn expression. We have also demonstrated depletion of STn+ MDSCs after treatment with an anti-STn antibody-drug conjugate in an ovarian carcinoma xenograft model. Conclusions: Our data demonstrated that STn provides a uniquely glycan-specific and potent target for treatment of solid tumors. Anti-STn therapeutics offer the potential to go beyond tumor targeting to also directly target and deplete immune-suppressive MDSCs, thus fostering immune re-engagement and increasing the potential for better patient outcomes.

Citation Format: David A. Eavarone, Patricia E. Rao, Jillian M. Prendergast, Adam D. Curtis, Rong Zhang, Lindsay S. Shopland, Jenna N. Stein, Jeff Behrens, Daniel T. Dransfield. Myeloid derived suppressor cells (MDSCs) express Sialyl Tn (STn) and are a therapeutic target for anti-STn antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5625.
Abstract 5632: Targeted immunotherapy with SV-BR-1-GM: Mechanism of action and companion diagnostic development
Authors: Lacher Markus D. , Graeve Sanne , Wiseman Charles L. , Kong Ying Y. , Kwok William W. , Williams William V.
Published: August 2018

SV-BR-1-GM is a GM-CSF-engineered whole-cell targeted immunotherapy derived from a breast cancer cell line (SV-BR-1). We are currently assessing SV-BR-1-GM in a phase I/IIa clinical trial in metastatic and locally recurrent breast cancer (ClinicalTrials.gov identifier NCT03066947). Subjects who fail to respond with tumor regression are offered participation in a “roll-over” study in which an immune checkpoint inhibitor (pembrolizumab or ipilimumab) is added to the SV-BR-1-GM regimen (ClinicalTrials.gov identifier NCT03328026). To identify patients likely to respond to SV-BR-1-GM we are co-developing a companion diagnostic.

In a pilot phase I study with four evaluable patients, one subject (A002), with metastatic breast cancer, experienced systemic tumor regression including in soft tissue, lung, and brain lesions. In contrast to the three nonresponders, A002 matched with SV-BR-1-GM at both a class I (HLA-A) and a class II (HLA-DRB3) HLA allele. Strikingly, SV-BR-1-GM cells also express HLA class II “accessory” factors such as HLA-DMA and -DMB, HLA-DRA, and CD74 (encoding invariant chain and CLIP), and immunostimulators such as IL6, Il18, and KITLG, and the damage-associated molecular pattern (DAMP) protein HMGB1. SV-BR-1-GM cells do not express the T cell co-stimulatory ligands CD80 or CD86. These findings are consistent with a potential to activate previously primed but not naïve CD4+ T cells. In agreement, peptide-pulsed SV-BR-1-GM cells selectively activated peptide-HLA specific, pre-primed T cells.

Given this functional data and the expression of the “immune signature”, comprised of factors including those mentioned above, we hypothesized that SV-BR-1-GM cells can act as antigen-presenting cells (APCs) and present tumor-associated antigens (TAAs) directly to HLA-matched T cells, which in turn would induce an immune response directed to the patients' tumors. To evaluate this hypothesis, we have begun developing bioassays for assessing antibody and T cell responses to SV-BR-1-GM.

Here, we present data on SV-BR-1-GM's molecular makeup and development of anti-SV-BR-1-GM humoral and cellular immune responses. Our findings support SV-BR-1-GM acting as antigen-presenting cells for HLA class I and class II restricted immune responses.

Citation Format: Markus D. Lacher, Sanne Graeve, Charles L. Wiseman, Ying Y. Kong, William W. Kwok, William V. Williams. Targeted immunotherapy with SV-BR-1-GM: Mechanism of action and companion diagnostic development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5632.
Abstract 5633: Development of glycoconjugate-based anticancer vaccines
Authors: Huang Xuefei
Published: August 2018

The development of an effective vaccine construct against tumor associated carbohydrate antigens (TACAs) is an attractive approach towards cancer treatment and prevention. However, a significant challenge is that TACAs are only weakly immunogenic and direct administration of TACAs cannot elicit a powerful antibody response to protect the host from cancer development. In this presentation, we will present our strategies to significantly enhance the immunogenicity of TACAs as potential anti-cancer vaccines using mucin-1 (MUC1) as a representative antigen. Mucin-1 (MUC1) is one of the top ranked tumor associated antigens. MUC1 peptides and glycopeptides have been covalently conjugated to an immunogenic carrier. Immunization of mice with these constructs led to super strong antibody responses with IgG titers over one million, which are one of the highest IgG titers reported to date. Furthermore, the constructs elicited MUC1 specific cytotoxic T cells, which can selectively kill MUC1 positive tumor cells. The abilities of MUC1 conjugates to induce both antibody and cytotoxic T cell immunity targeting tumor cells highlight the potential of these constructs as anti-tumor vaccines.

Citation Format: Xuefei Huang. Development of glycoconjugate-based anticancer vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5633.
Abstract 5634: Eradication of breast carcinoma with bone metastasis by autologous formalin-fixed tumor vaccine (AFTV) combined with radiation and chemotherapy
Authors: Kuranishi Fumito , Imaoka Yuki , Iwako Hiroshi , Uemae Yoji , Ishihara Takeshi , Miyazaki Tsubasa , Ohno Tadao
Published: August 2018

No standardized treatment has been established for skeletal metastasis of breast carcinoma which is refractory to intensive chemo-radiation therapy and therefore is assumed impossible to cure. Here we show, however, the 3rd successful case concomitantly treated with the tumor vaccine, AFTV, and chemo-radiation therapy (the 1st and 2nd cases have been presented in AACR2013 and AACR2015, respectively). A 50-year old woman presented 2.5-cm mass of tubular carcinoma in her left breast in a MRI image. The tumor accompanied with no lymph node metastasis was resected in March 2009. We found the tumor was ER(+), PgR(-), and HER2 (-). Four months after the resection, she selected one course of AFTV as the first-line therapy. Response to delayed-type hypersensitivity (DTH) test became strongly positive. We then treated her with the aromatase inhibitor, anastrozole. Six years later, in March 2015, a PET image revealed bone-metastasis to sternum which was confirmed by needle-biopsy and ABC immunostaining as class V metastatic ductal carcinoma. We treated her again with second course of AFTV and palliative radiation therapy (36Gy/12fractions/19days) together with letrozole, bisphosphonate, additional 2 shots of nivolmab (40mg/48kg body) in October and November 2015. She was diagnosed to be complete response by PET imaging in February 2016, August 2016, and August 2017. Alteration of her blood CEA level suggests that the combined treatments eradicated the metastatic breast carcinoma. In conclusion, the present case suggests that AFTV in combination with palliative X-ray irradiation and adjuvant aromatase inhibitors eradicates skeletal metastasis of breast carcinoma with enhancement of a specific anti-tumor immune reactivity.

Citation Format: Fumito Kuranishi, Yuki Imaoka, Hiroshi Iwako, Yoji Uemae, Takeshi Ishihara, Tsubasa Miyazaki, Tadao Ohno. Eradication of breast carcinoma with bone metastasis by autologous formalin-fixed tumor vaccine (AFTV) combined with radiation and chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5634.
Abstract 5635: The development to peptide vaccine targeting heat shock protein 90 to overcome resistance to HER-2 target drugs in breast cancer
Authors: Kang Jinho
Published: August 2018

HER-2 overexpression is observed in approximately 25% of all breast cancers and is associated with poorer overall survival and decreased time to relapse. Target therapies directed against HER-2 have been developed and used clinically, but many patients continue to develop resistance. Breast cancer is immunogenic and well suited to treatment via immunomodulation. HSP90 is a chaperon for most of the important signal transduction system in HER-2 type breast cancer; HER-2, ER / PR and Akt, the downstream protein of PI3K. Therefore, HSP90 is considered as a potential therapeutic target in order to improved treatment of HER-2 positive resistant breast cancer. Immunotherapy targeting HSP90 has been tried before, however, no clinically available vaccine due to limitation of autologous cell-based approach. We questioned whether HSP90 was an immune target in HER-2 positive breast cancer. First, HSP90-specific IgG antibody immunity was detected in the sera from HER-2 positive breast cancer patients. Next, potential MHC class II epitopes derived from HSP90 with high binding affinity across multiple human HLA genotypes were identified using in silico algorithms. Top 11 peptides were synthesized and Th1 vs Th2 immunity were assessed in human PBMCs. After IFN-γ and IL-10 ELISPOT assays, epitopes inducing Th1-directed immunity were selected for in vivo experiments. Immunogenecity of HSP90 peptides vaccine was evaluated in FVB mice model and showed strong antigen-specific T cell responses in IFN-γ ELISPOT assay. Then, we assessed the anti-tumor effect of immunization with HSP90 peptides vaccine in MMTV-neu-transgenic mouse model after autologous cancer cell implantation. Peptides vaccines derived from HSP90 significantly inhibited tumor growth compared with control group (mean ± SD; 1896 ± 1211 mm3 vs. mean ± SD; 774 ± 136 mm3, respectively; p < 0.05). The tumor-inhibitory effect was associated with HSP90 peptide-specific IFN-γ-secreting T cell responses in immunization group. Also, immunohistochemical staining of tumors derived from immunized mice was performed to examine immune microenvironment and the protein expression of HER-2 pathways. Taken together, multi-epitope peptides vaccine derived from HSP90 is immunogenic in breast cancer patients and is a promising tumor antigen. Based on efficacies in tumor-rejection in vivo, further research in combination with immune modulating antibodies will be performed in the lapatinib-resistant tumor model.

Citation Format: Jinho Kang. The development to peptide vaccine targeting heat shock protein 90 to overcome resistance to HER-2 target drugs in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5635.
Abstract 5640: Development of a personalized pancreatic cancer vaccine
Authors: Cullinan Darren R. , McLellan Michael , Zhang Xiuli , Nywening Timothy M. , Miller Chris A. , Goedegebuure S. Peter , Hawkins William G. , Gillanders William E.
Published: August 2018

Introduction: Cancer neoantigens result from mutations accumulated in an individual tumor. These mutations are usually private and yield high-affinity T-cells as they are not limited by mechanisms of self-tolerance. Neoantigens presented in the context of MHC class I can be recognized by anti-tumor CD8+ T-cells.

Methods: Three consecutive patients with confirmed pancreatic ductal adenocarcinoma (PDAC) had DNA and RNA isolated from areas of high tumor cellularity. Comparative normal DNA was isolated from peripheral blood mononuclear cells (PBMC). Nonsynonymous mutations were identified by tumor/normal whole exome sequencing and expression in the tumor was verified by cDNA capture. HLA typing was inferred from exome sequencing. Candidate neoantigens were identified using multiple epitope prediction algorithms for MHC class I. Matched patient PBMC were cultured in serum-free media for 48 hours with synthetic mutated short peptides to determine the baseline immune response by interferon-gamma (IFNγ) enzyme-linked immunospot assay.

Results: A KRAS driver mutation was identified in all three patients. Using KRAS variant allele frequency (VAF) as a surrogate for tumor purity, we achieved greater than 40% tumor purity for all three samples. This provides an adequate level of purity to proceed with neoantigen identification. In these samples, the mean number of nonsynonymous mutations identified was 27 (range: 18-32; DNA VAF cutoff 10%). Of these nonsynonymous mutations, a mean of 8.6 mutations (range: 7-10) were expressed (RNA VAF cutoff >10% and FPKM >1). Following confirmation of mutation expression, a mean of 5 mutations (range: 4-6) were predicted to bind to MHC class I using multiple prediction algorithms (binding affinity <500nM). IFNγ immunospot assay revealed a baseline immune response to one neoantigen in each patient (Patient A - SDHA: 75 spot forming units per millions cells (SFU/M) vs Negative: 12.5 SFU/M, p=0.01; Patient B - ZRANB1: 588 SFU/M vs Negative: 17.5 SFU/M, p<0.001; Patient C - ATAD3C: 340 SFU/M vs Negative: 75 SFU/M, p=0.002).

Conclusion: Utilizing advanced genomic and bioinformatics tools we have successfully characterized the cancer neoantigens in a series of three consecutive patients with PDAC. From this data, we plan to open a phase I clinical trial to assess the safety and immunogenicity of a neoantigen DNA vaccine.

Citation Format: Darren R. Cullinan, Michael McLellan, Xiuli Zhang, Timothy M. Nywening, Chris A. Miller, S. Peter Goedegebuure, William G. Hawkins, William E. Gillanders. Development of a personalized pancreatic cancer vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5640.
Abstract 5645: Critical variables to reduce Western blot variability
Authors: Yukhananov Russ , Margulis Alex
Published: August 2018

Reliable immunodetection of proteins is a critical step in the discovery of new biomarkers and diagnostic. The immunodetection protocol consists of multiple steps including blocking of nonspecific binding sites, incubation using primary and secondary antibodies and extensive washing between steps that easily introduce bias and errors. The quality and reproducibility of results depends on multiple subjective and objective factors such as the qualification and technical skills of the personnel performing the assay and the accuracy of temporal and temperature control, especially during the immunodetection step. BlotCycler™, automated western blot processor, use fluidic control system that allows to eliminate the variability associated with immunodetection and to achieve higher sensitivity by optimized washing procedure. We used BlotCycler to analyze critical source of errors during immunodetection. Relatively small changes in duration and temperature of incubation significantly affect not only the intensity of signal but also the specificity of antibody interaction with antigen. Automated immunodetection at 4ºC significantly improve the specificity of detection and sensitivity that allows reproducible detection of low expressing proteins. Automated processing using BlotCycler allows optimization and control of critical variables leading to improved reproducibility and specificity of immunodetection and should be routinely used for new biomarker discovery and diagnostics, reliable protein quantification, primary antibody specificity testing, and optimization of primary and secondary antibodies concentrations.

Citation Format: Russ Yukhananov, Alex Margulis. Critical variables to reduce Western blot variability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5645.
Abstract 5646: Serum functional biomarker discovery with PEP technology
Authors: Wang Xing , Davies Michael
Published: August 2018

Proteins play essential roles in numerous biological processes and being able to pinpoint functional differences between cell or tissue samples can greatly aid in understanding disease processes and metabolic changes. The PEP technology allows systematic analysis of protein functions within a proteome. Hundreds of functional proteins can be separated and functionally assayed to generate a comprehensive three-dimensional landscape of protein families such as protein kinases, phosphatases, proteinases and oxido-reductases. This information can then be integrated into other genomic and proteomic knowledge bases to provide further insight of important biological processes such as cancer development and aging. Using the PEP technology, functional biomarker candidates have been identified from liver, lung and breast cancer patient serum. Both qualitative and quantitative differences of metabolic enzymes and proteases were observed when comparing the cancer patient serum and normal serum. Some of the active enzymes were identified by mass spectrometry and validated in selected bioassays. Deep mass spectrometry analysis revealed that some of the functional biomarkers are protein variants of important biological function. It is believed that this functional proteomics technology will provide a unique approach in the discovery of potential cancer biomarkers for diagnostic and prognostic applications. Further efforts are ongoing to develop a prototype of diagnosis kit for cancer detection based on the current discoveries.

Citation Format: Xing Wang, Michael Davies. Serum functional biomarker discovery with PEP technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5646.
Abstract 5653: VMax™: A new platform for recombinant protein expression
Authors: Hesek Eric , Wilson Chris , Wolf Lindsey , Vasu Sanjay , Sturges Michael , Gibson Daniel , Weinstock Matthew
Published: August 2018

Here we describe Vmax™, a next-generation protein expression platform based on the organism V. natriegens, an extremely rapidly growing gram-negative prokaryote capable of reaching high cell density. Vmax™ contains an IPTG inducible T7 expression system, enabling high levels of recombinant protein expression. By combining a robust expression system with a highly productive organism, Vmax allows researchers to perform experiments faster, shorten workflow times, and generate large amounts of recombinant protein.

Citation Format: Eric Hesek, Chris Wilson, Lindsey Wolf, Sanjay Vasu, Michael Sturges, Daniel Gibson, Matthew Weinstock. VMax™: A new platform for recombinant protein expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5653.