bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5916: Proton-sensor GPR4 potentiates intestinal inflammation in the DSS-induced colitis mouse model
Authors: Sanderlin Edward J. , Leffler Nancy R. , Lertpiriyapong Kvin , Cai Qi , Hong Heng , Bakthavatchalu Vasudevan , Fox James G. , Oswald Joani Z. , Justus Calvin R. , Krewson Elizabeth A. , O’Rourke Dorcas , Yang Li V.
Published: July 2017

Inflammation and tissue acidosis are two factors that co-exist in inflammatory bowel disease (IBD) and can contribute to increased risk of colorectal cancer (CRC) development. GPR4 is a proton-sensing G protein-coupled receptor that can be activated by extracellular acidosis through several histidine residues and subsequently signal through downstream G-protein pathways. Recently, GPR4 has been shown to be activated by acidosis and can increase the expression of numerous inflammatory and stress response genes in vascular endothelial cells (ECs) and has functionally increased EC-leukocyte adhesion. Subsequently, genetic and small molecule approaches for the inhibition of GPR4 activity have reduced endothelial cell inflammation. In this study, we examined the role of GPR4 in intestinal inflammation using a dextran sulfate sodium (DSS)-induced colitis mouse model. We observed that GPR4 mRNA expression was increased in mouse and human IBD tissues when compared to control intestinal tissues. To determine the function of GPR4 in intestinal inflammation, wild-type and GPR4-deficient mice were treated with 3% DSS for acute and chronic time points for the induction of colitis. Our results showed that the severity of colitis was decreased in GPR4-deficient DSS-treated mice in comparison to wild-type DSS-treated mice. Clinical parameters, macroscopic disease indicators, and histopathological features were less severe in the DSS-treated GPR4-deficient mice than the DSS-treated wild-type mice. Inflammatory gene expression, endothelial adhesion molecule expression, leukocyte infiltration, and isolated lymphoid follicle (ILF) formation were reduced in intestinal tissues of DSS-treated GPR4-null mice. In summary, our results suggest GPR4 potentiates intestinal inflammation as the absence of GPR4 ameliorates intestinal inflammation in the DSS-induced colitis mouse model. Use of GPR4 inhibitors could prove a valuable therapeutic in the reduction of intestinal inflammation and subsequent CRC development.

Note: This abstract was not presented at the meeting.

Citation Format: Edward J. Sanderlin, Nancy R. Leffler, Kvin Lertpiriyapong, Qi Cai, Heng Hong, Vasudevan Bakthavatchalu, James G. Fox, Joani Z. Oswald, Calvin R. Justus, Elizabeth A. Krewson, Dorcas O’Rourke, Li V. Yang. Proton-sensor GPR4 potentiates intestinal inflammation in the DSS-induced colitis mouse 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 5916. doi:10.1158/1538-7445.AM2017-5916
Abstract 5922: Cellular interaction between pancreatic cancer cells and MSC as tumor microenvironment
Authors: Kondo Eisaku , Saito Ken
Published: July 2017

Tumor microenvironment such as neoangiogenesis, stroma and tumor immunity has recently gained much attention because it is significantly involved in tumor progression. Pancreatic adenocarcinoma (PAC; pancreatic invasive ductal carcinoma) is recognized as one of the most intractable malignancies and required to disclose a key biological characteristics to provide a clue for its diagnosis and therapeutics. Here we focus on the biological role of bone marrow-derived human mesenchymal stem cells (MSCs) in PAC, which were reported to be accumulated in various cancer tissues such as colon, breast cancers.

We detected small numbers of MSCs in the stroma of PAC in human pathological tissues using double immunofluorescence in combination with several MSC markers including CD73, 105, 34, 45, SMA and so on. Then, in in vitro coculture system, we found that MSCs significantly facilitated invasion of BxPC3 cells, a human PAC cell line, in comparison with the culture of BxPC3 cell alone by transwell chamber assay. Proteome profiling of the cocultured supernatant revealed augmented production of soluble molecules including CXCL7, CCL7 as a cytokine, integrin beta1, transferrin R, ALCAM, and Amphiregulin (AREG) as a soluble receptor and ligand, metalloprotease (MMP), ADAMTS-1 as a soluble protease-related molecule, all which were significantly increased comparing to the culture sup. of BxPC3 alone or MSC alone.

Based on the result of proteome profiling, we further examined mRNA upreguttion on the cells of each culture by real-time PCR (qPCR) to identify which molecule would be more specifically upregulated by the coculture sample of PAC cells and MSCs.

Consequently, upregutaion of AREG and MMP-3, in addition to downregulation of MMP-9 was observed by qPCR, and knockdown of AREG using the specific siRNA on the cocultured cells corroborated marked decrease of PAC invasion in transwell chamber assay. Interestingly, AREG expression on the tumor cells of tissue specimens of the patient with PAC was observed especially on the cells at invasive front, which suggests stromal MSCs may interact with PAC cells and facilitate invasion of cancer cells by regulating expression of the specific genes such as AREG and MMPs.

Note: This abstract was not presented at the meeting.

Citation Format: Eisaku Kondo, Ken Saito. Cellular interaction between pancreatic cancer cells and MSC as tumor microenvironment [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 5922. doi:10.1158/1538-7445.AM2017-5922
Abstract 5931: Effect of necrosis on the migration, invasion and miRNA-mRNA association in glioblastoma cells
Authors: Ahn So-Hee , Park Hyunju , Kim Jeong Seon , Ahn Young-Ho , Kim Sewha , Cho Min-Sun , Kang Jihee Lee , Choi Youn-Hee
Published: July 2017

Grade IV astrocytoma, also called glioblastoma multiforme (GBM), is considered the most malignant glial tumor. The purpose of our study is to understand the molecular mechanisms underlying the signaling network between necrotic tissues and GBM cells by exploring the effect of necrosis on GBM migration, invasion and miRNA-mRNA association. The migration and invasion of the CRT-MG human astroglioma cells were significantly enhanced by treatment with necrotic cells, as shown by assays for scratch wound healing and spheroid invasion. Incubation with necrotic cells induced IL-8 secretion in CRT-MG in dose-dependent manner. Immunohistochemical and immunoflurorescence analysis for IL-8 in human GBM tissues showed that positively stained cells were mainly distributed in perinecrotic region. Necrotic cells induced NF-κB and AP-1 activation and their DNA binding to IL-8 promoter, leading to enhanced IL-8 production and secretion in GBM cells. To determine the effect of necrosis on the miRNA profiles and regulatory relationship, the expression profiles of miRNA and mRNA and association between mRNA and their corresponding miRNAs were analyzed by next-generation sequencing (NGS). Our results demonstrate that necrotic cells enhance the migration and invasion of GBM cells through NF-κB/AP-1/IL-8 signaling pathway and induce dysregulation of miRNA and mRNA expression profiles in GBM cells.

Citation Format: So-Hee Ahn, Hyunju Park, Jeong Seon Kim, Young-Ho Ahn, Sewha Kim, Min-Sun Cho, Jihee Lee Kang, Youn-Hee Choi. Effect of necrosis on the migration, invasion and miRNA-mRNA association in glioblastoma cells [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 5931. doi:10.1158/1538-7445.AM2017-5931
Abstract 5933: Cancer-associated fibroblasts (CAFs) release exosomal microRNAs that dictate an aggressive phenotype in breast cancer
Authors: Condorelli Gerolama , Donnarumma Elvira , Fiore Danilo , Nappa Martina , Roscigno Giuseppina , Adamo Assunta , Iaboni Margherita , Russo Valentina , Affinito Alessandra , Quintavalle Cristina , Rienzo Anna , Piscuoglio Salvatore , Thomas Renato
Published: July 2017

Cancer-associated fibroblasts (CAFs), the major components of the tumor microenvironment may drive tumor progression. Exosomes mediate horizontal transfer of miRs, mRNAs and proteins, thus affecting breast cancer progression. Differential expression profile analysis identified three miRs increased in exosomes from CAFs as compared from normal fibroblasts. Immunofluorescence indicated that exosomes may be transferred from CAFs to breast cancer cells, releasing their cargo miRs. Breast cancer cells exposed to CAF exosomes or transfected with those miRs exhibited a significant increased capacity to form mammospheres, increased stem cell and epithelial-mesenchymal transition (EMT) markers, and anchorage-independent cell growth. These effects were reverted by transfection with anti-miRs. Similarly to CAF exosomes, normal fibroblast exosomes transfected with CAFs-miRs promoted the stemness and EMT phenotype of breast cancer cells. Thus, we provided evidence of the role of CAF exosomes and their miRs in the induction of the stemness and EMT phenotype in different breast cancer cell lines.

Note: This abstract was not presented at the meeting.

Citation Format: Gerolama Condorelli, Elvira Donnarumma, Danilo Fiore, Martina Nappa, Giuseppina Roscigno, Assunta Adamo, Margherita Iaboni, Valentina Russo, Alessandra Affinito, Cristina Quintavalle, Anna Rienzo, Salvatore Piscuoglio, Renato Thomas. Cancer-associated fibroblasts (CAFs) release exosomal microRNAs that dictate an aggressive phenotype in breast 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 5933. doi:10.1158/1538-7445.AM2017-5933
Abstract 5943: Role of tissue stiffness and oxygen tension in promoting breast cancer stem cells
Authors: Pang Mei-Fong , Radisky Derek C. , Nelson Celeste M.
Published: July 2017

Breast tumors are stiff and hypoxic. Nevertheless, it remains unclear how stiff and hypoxic conditions within the tumor microenvironment promote breast cancer progression. Using an innovative engineered culture model to recapitulate these features, we found that, under stiff and hypoxic conditions, breast cancer cells have enhanced integrin-linked kinase (ILK) expression and acquire cancer stem cell (CSC)-like traits, suggesting tissue stiffness and oxygen tension can modulate ILK expression to induce breast CSC formation. Knocking down ILK impairs the mechanosensing of invasive breast cancer cells on stiff microenvironments, inhibits CSC markers and properties. In contrast, ectopic expression of ILK promotes breast CSC formation. In addition to promoting CSC-like phenotype, microarray analysis reveals that stiff and hypoxic microenvironments also regulate genes involved in mRNA processing, splicing and the spliceosome. These data suggest that the non-cellular compartment of the tumor microenvironment, namely tissue mechanics and oxygen tension, can promote breast cancer progression by controlling mechanotransduction and post-transcriptional regulation of breast cancer cells.

Note: This abstract was not presented at the meeting.

Citation Format: Mei-Fong Pang, Derek C. Radisky, Celeste M. Nelson. Role of tissue stiffness and oxygen tension in promoting breast cancer stem cells [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 5943. doi:10.1158/1538-7445.AM2017-5943
Abstract 5944: Matrilysin/MMP-7 compromises a novel border interaction between perlecan/HSPG2 and semaphorin 3A to support prostate cancer invasion
Authors: Grindel Brian J. , Harrington Daniel A. , Chung Leland , Farach-Carson Mary C.
Published: July 2017

Prostate cancer (PCa) cells interact with the stroma and extracellular matrix (ECM) during tumor expansion and invasion during metastasis. Dissecting the complex matrix-cancer interplay is essential to halting tumor progression. A vital component of the ECM is perlecan/heparan sulfate proteoglycan 2 (HSPG2). As a large extracellular proteoglycan, perlecan helps orchestrate tumor angiogenesis, proliferation, differentiation and invasion. Actively metastasizing cancer cells must proteolytically degrade several tissue borders which perlecan patrols, including the basement membrane, vasculature, reactive stromal matrix and bone marrow. Investigating the effects of perlecan on metastatic PCa cells showed full length perlecan and specifically conserved domain IV of perlecan triggers clustering of PCa cells. Perlecan cleavage by the pro-tumor protease matrilysin/matrix metalloproteinase (MMP-7) reverses this, favoring cell dispersion and tumor dyscohesion. Network analysis of a reverse phase protein array and subsequent western blots revealed clustered cells on intact perlecan/Dm IV induced a strong focal adhesion kinase (FAK) dephosphorylation/deactivation. Alternatively, FAK was highly phosphorylated/activated on MMP-7 cleaved perlecan/Dm IV consistent with an adhesive and dispersive/metastatic phenotype. Several lines of evidence indicate Dm IV interacts with the classically known axon guidance protein semaphorin 3A (Sema3A), to shut off pro-metastatic FAK activation. Notably, a C-terminus directed Sema3A antibody mimics the clustering activity of Dm IV. We showed Dm IV binds to Sema3A in ELISA and quartz crystal microbalance experiments and the interaction is abrogated by the Sema3A antibody. Interestingly, Sema3A also is sensitive to MMP-7 proteolytic activity. Finally, transient knockdown of Sema3A in PCa cells inhibits the clustering effect by a Dm IV substrate. This work shows that perlecan is an effective border molecule because it coordinates with Sema3A to repulse and cluster cells by switching off FAK activity. However, PCa cells expressing active MMPs, such as MMP-7, can destroy this Sema3A/perlecan complex to develop an invasive, metastatic phenotype associated with lethal disease.

Citation Format: Brian J. Grindel, Daniel A. Harrington, Leland Chung, Mary C. Farach-Carson. Matrilysin/MMP-7 compromises a novel border interaction between perlecan/HSPG2 and semaphorin 3A to support prostate cancer invasion [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 5944. doi:10.1158/1538-7445.AM2017-5944
Abstract 607: PDL1 expression associated with triple negative breast ductal carcinomas in African American women
Authors: Khan Farhan , Kanaan Yasmine , Ricks-Santi Luisel J. , Zafar Rabia , Aymut Hagos , Naab Tammey
Published: July 2017

Background: Tumor cells avoid host immune response through expression of inhibitory T cell regulator,

Programmed cell death ligand (PDL1).Our objective was to evaluate PDL1

expression by immunohistochemistry in the four major subtypes of breast

carcinoma (Luminal A, Luminal B, HER2, and Triple Negative) in a population of

202 African-American (AA) women with other clinicopathological factors.

Design: Tissue microarrays

(TMAs) were constructed from FFPE tumor blocks from primary ductal breast

carcinomas in 202 African-American females.

Two separate 1 mm cores represented each case. Five micrometer sections were stained with rabbit

monoclonal antibody against PDL1.

The sections were evaluated for the percentage and intensity of membrane

staining in both tumor and immune cells. Cut off was > 1%. Bivariate

analysis was done via χ2 analysis and survivability data was calculated via the

generation of Kaplan-Meier curves (SPSS v19). Statistical significance was

assumed if p < 0.05.

Results: PDL1 expression in

only tumor cells, only immune cells and in combination was associated with ER negative

(p<0.0001), PR negative (p<0.0001), Triple negative subtype (p<0.0001)

and high grade (p<0.0001) breast ductal cancers.

Conclusion: Our study finding of selective expression of PDL1 in triple negative breast ductal cancers (TNBC) in AA women suggests that inhibition of adaptive immune response is involved in the progression of these cancers. PDL1/PD1 is the inhibitory check point of immune response resulting in decreased T cell activation and cytokine production. Some studies have shown that PDL1 expression is associated with loss of PTEN and phosphatide inositol 3 kinase pathway (PI3K) activation. Our previous study showed loss of PTEN in TNBC. Combining PDL1 inhibitors with PI3K inhibitors may be useful therapy for aggressive TNBC.

Citation Format: Farhan Khan, Yasmine Kanaan, Luisel J. Ricks-Santi, Rabia Zafar, Hagos Aymut, Tammey Naab. PDL1 expression associated with triple negative breast ductal carcinomas in African American women [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 607. doi:10.1158/1538-7445.AM2017-607
Abstract 608: Augmenting and broadening T cell responses against mutated tumour neo-antigens
Authors: Robinson Bruce W. , Ma Shaokang , Chee Jonathan , Rive Craig , Miert Paula Van , Holt Rob A. , Creaney Jenette
Published: July 2017

Cytotoxic T lymphocytes (CTLs) recognize mutated tumor proteins (neo-antigens) and are important for anti-tumor immunity, especially in the context of immune checkpoint blockade immunotherapy (ICPB). Positive outcomes to ICPB are associated with high neo-antigen loads and with neo-antigen specific CTL responses. However only around 20% of patients respond to ICPB.

In order to examine ways in which the non-responders might become responders we examined several strategies to improve response rates, using anti-CTLA4 initially as the ICPB therapy in BALB/c mice in which Uqcrc2 has been defined as a DNA/RNAseq-identified neo-antigen in AB1 tumor lines induced by a relevant human carcinogen(1).

a) We first examined therapy-induced changes in T-cell responses against Uqcrc2. Anti-CTLA4 alone increased the magnitude of responses against Uqcrc2. Anti-CTLA4 combined with anti-GITR induced determinant spreading, unmasked responses against a new neo-antigen UNC45A that was undetectable during normal tumour growth. Immunogenic chemotherapy also unmasked responses against UNC45A, suggesting that subdominant neo-antigens can be unmasked by the appropriate immunotherapy.

b) We then evaluated neo-antigen vaccination strategies. Uqcrc2 vaccination only protected against tumor growth when administered in combination with partial Treg depletion (Foxp3.DTR mice), suggesting that neo-antigen vaccination will only be maximally effective when administered in combination with therapies that modulate existing immune restraints, including Tregs.

c) We determined optimal anatomical location for tracking neo-antigen CTL responses and identified the draining lymph node as an optimal, though not exclusive, location for response testing compared to blood or tumor.

We are currently examining these observations using sequencing-defined neo-antigens in our patients and will present data on changes in human neo-antigen responses to therapy. These observations have important translational implications for identification of key neo-antigens, choice of therapy and monitoring of anti-tumor responses.

1.

J. Creaney et al., Strong spontaneous tumor neoantigen responses induced by a natural human carcinogen. Oncoimmunology 4, e1011492 (2015).

Citation Format: Bruce W. Robinson, Shaokang Ma, Jonathan Chee, Craig Rive, Paula Van Miert, Rob A. Holt, Jenette Creaney. Augmenting and broadening T cell responses against mutated tumour neo-antigens [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 608. doi:10.1158/1538-7445.AM2017-608
Abstract 61: Zymelink drug conjugate platform: redefining the therapeutic window for ADCs
Authors: Barnscher Stuart , Babcook John , Rich Jamie , Winters Geoff , Garnett Graham , Hernandez Andrea , Fung Vincent , Yin Kevin , Hamblett Kevin , Davies Rupert
Published: July 2017

Antibody drug conjugates (ADCs) combine the specificity of monoclonal antibodies with potent antineoplastic small molecules, and promise efficacy without the systemic toxicity of chemotherapy. Despite this tremendous potential, most clinical ADCs have failed to provide sufficient therapeutic benefit before the onset of off-target dose-limiting platform toxicities.

Here we report the development of proprietary protease cleavable N-acyl sulfonamide linked hemiasterlin and auristatin payloads, Zymelink, that allow the generation of an efficacious ADC platform with improved tolerability. Both Zymelink drug-linkers were conjugated via maleimides to endogenous cysteines. As Zymelink drug-linkers are more polar than maleimide valine citrulline monomethyl auristatin E drug-linker, the resulting ADCs can be produced with potential advantages in PK, efficacy and safety. The resulting ADCs exhibit potent in vitro cytotoxicity. A trastuzumab-based ADC prepared with Zymelink drug-linker and demonstrated at least equivalent efficacy compared to a trastuzumab-based ADC prepared with MMAE, promoting durable complete regressions in a patient-derived xenograft model.

Zymelink hemiasterlin and auristatin ADCs were tolerated at 5-6 fold higher doses compared to a MMAE ADC in cynomolgus monkeys. The maximum tolerated dose of the MMAE ADC was just 3 mg/kg based on severe neutropenia. Zymelink hemiasterlin ADC was tolerated at 15 mg/kg with no evidence of neutropenia or elevations in transaminases. Zymelink auristatin ADC was tolerated at 18 mg/kg based on increased levels of transaminases at 24 mg/kg. Moreover, Zymelink ADCs exhibited greater serum exposure at equivalent doses. These results suggest Zymelink ADCs have a greatly expanded therapeutic window compared to MMAE conjugates.

Citation Format: Stuart Barnscher, John Babcook, Jamie Rich, Geoff Winters, Graham Garnett, Andrea Hernandez, Vincent Fung, Kevin Yin, Kevin Hamblett, Rupert Davies. Zymelink drug conjugate platform: redefining the therapeutic window for ADCs [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 61. doi:10.1158/1538-7445.AM2017-61
Abstract 610: Blockade of tumor expression of PD-1 promotes lung cancer growth in a murine model
Authors: Du Shisuo , Guan Qing , Dicker Adam P. , LU Bo
Published: July 2017

Lung cancer remains one of the most prevalent and fatal malignancies worldwide. There are substantial evidences that immunotherapies offer significant benefits among lung cancer patients. Pembrolizumab, a humanized programmed death receptor-1(anti-PD-1) antibody is now approved by FDA as a first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC). Recent data demonstrate that PD-1 is not only expressed by immune cells, but also by human melanoma cells. However, little is known about the biological significance of PD-1 pathway in cancer including NSCLC. Thus, we examined PD-1 expression in a series of established murine lung cancer cell lines. RT-PCR amplification and sequencing of the full coding sequence of the murine PD-1 (PDCD1) gene revealed the presence of PDCD1 mRNA in the M109 lung cancer cells. Flow cytometry and immunofluorescence analysis demonstrated PD-1 protein expression in these murine lung cancer cells. To determine the molecular function of tumor PD-1 in lung cancer survival, we treated M109 cells in culture with either anti- mouse PD-1(100ug/ml) or isotype control antibody and found that PD-1 inhibition significantly enhanced cancer cell viability as measured by MTT and clonogenic assay. Alternative PD1 inhibition by either knockdown of PDCD1 via siRNA or knockout of PDCD1 by CRISPR-Cas9 all lead to increased survival of M109 cells. Furthermore, PDCD1-knockout M109 cells had no survival difference in the presence of anti-PD1 compared to its PD1-expressing parental cells. Conversely, treatment with recombinant PD-L1 fusion protein exhibited significantly increased apoptotic rates (7.5±3.2% vs 2.1±1.2%) suppressing the tumor cell growth in the wild-type M109 in culture. To validate these results in in vivo models, NSG mice were used to determine the direct impact of anti-PD1 on M109 xenografts without the interplay of host immune system. M109 xenografts treated with antiPD-1 (10mg/kg i.p.) antibodies showed accelerated growth compared to those treated by IgG control (2487±473 vs 1499±292mm3 at days 21 after tumor inoculation, p=0.013). M109 subcutaneous tumors in the PD-1 knockout mice also manifested more rapid growth than the same tumors implanted in wild-type mice. Taken together, our results uncover a novel function of PD-1/PD-L1 axis in lung cancer survival and implicate possible tumor-promoting effects from anti-PD-1 therapy upon PD1-expressing lung cancer in the absence of effective immune response. We plan to interrogate bio-specimens collected through NRG trials to establish the translational potential of these findings.

Citation Format: Shisuo Du, Qing Guan, Adam P. Dicker, Bo LU. Blockade of tumor expression of PD-1 promotes lung cancer growth in a murine 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 610. doi:10.1158/1538-7445.AM2017-610
Abstract 613: Development of FPA154, a novel tetravalent anti-GITR antibody, for the treatment of solid tumors
Authors: Barbee Susannah D. , Chen Amanda , Johnson Susan , Bellovin David I. , Timmer John C. , Wondyfraw Nebiyu , Mkrtichyan Mikayel , Razai Amir S. , Jones Kyle S. , Hata Chelsie Y. , Gonzalez Denise , Deveraux Quinn , Eckelman Brendan P. , Borges Luis
Published: July 2017

Glucocorticoid-induced TNFR-related (GITR, TNFRSF18) is a member of the TNFR superfamily with pleiotropic T cell modulatory activity. We are developing a novel anti-GITR antibody with enhanced agonist activity for the treatment of solid tumors. Our candidate molecule, FPA154, is constructed with single-domain antibodies (sdAbs) in a tetravalent format, with an effector-competent IgG1 Fc domain. Both FPA154 and the mouse-reactive surrogate molecule (cmFPA154, mIgG2a isotype) bind to cell-surface GITR with high affinity. GITR is most highly expressed in vivo on activated and intratumoral Treg, and our data indicates that FPA154 and cmFPA154 potently mediate ADCC activity against Treg expressing high levels of GITR. In contrast, activated effector T cells express modest levels of cell-surface GITR, and FPA154 and cmFPA154 drive GITR-induced NF-κB activation. This activity is independent of Fc-mediated crosslinking, which is normally required for bivalent GITR antibodies to induce GITR signaling. cmFPA154 has potent antitumor activity in several syngeneic mouse tumors, both as a monotherapy treatment and in combination with anti-PD-1. In summary, FPA154 is a promising candidate with multiple mechanisms of action that contribute to generation of an antitumor immune response mediated by different T cell subsets.

Citation Format: Susannah D. Barbee, Amanda Chen, Susan Johnson, David I. Bellovin, John C. Timmer, Nebiyu Wondyfraw, Mikayel Mkrtichyan, Amir S. Razai, Kyle S. Jones, Chelsie Y. Hata, Denise Gonzalez, Quinn Deveraux, Brendan P. Eckelman, Luis Borges. Development of FPA154, a novel tetravalent anti-GITR antibody, for the treatment of solid tumors [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 613. doi:10.1158/1538-7445.AM2017-613
Abstract 633: Induction of ADCC resistance profoundly alters tumor cell phenotype and stress response
Authors: Aldeghaither Dalal S. , Murray Joseph C. , Roth Sarah M. , Fertig Elana J. , Tang Shaojun , Jablonski Sandra A. , Weiner Louis M.
Published: July 2017

Antibody dependent cellular cytotoxicity (ADCC) is one mechanism by which monoclonal antibodies (mAb) work. In addition to ADCC, mAbs act on their target by causing signal perturbation and complement activation. Cetuximab is an EGFR targeting mAb used to target EGFR overexpression and amplification seen in many types of cancer. While mechanisms of resistance to EGFR targeted therapy have been extensively studied, resistance to ADCC has not, mainly due to the lack of ADCC-resistance models for study. To address this limitation we established a model system for anti-EGFR ADCC using NK92-CD16V effector cells, cetuximab, and the high EGFR-expressing squamous cell carcinoma cell line A431. Continuous A431 exposure to ADCC yielded an ADCC resistant phenotype (ADCCr) that exhibits a stable phenotype in the absence of continued ADCC selection. We have explored: ADCCr cell gene expression profile, NK cell activation, metabolic signature and subsequent RPPA analysis to further understand the causes and properties associated with this resistance. This ADCCr cell line has a distinctive transcriptional profile highlighted by overexpression of histone- and interferon-related genes, reduced sensitivity to antimetabolites, DNA-intercalating and ABC transporter-regulated cytotoxic agents. Intense ADCC selection causes epigenetic modification and stress response characterized by the transcriptional overexpression of PCAF (KAT2B), which initiates histone hyper-acetylation and epigenetic changes, inducing DNA replication arrest, DNA damage and stress responses that activate checkpoint signaling in the cell cycle. The pharmacologic inhibition of KAT2B reverses the ADCCr phenotype. Immune checkpoints such as PD-L1 do not modulate ADCC in this model system. These results shed light on new mechanisms of ADCC resistance and inform future combinatorial treatments for mAb therapy. We are exploring the possibility that stress response mechanisms are responsible for resistance for diverse selection pressures imposed by immune synapse-mediated cytotoxic attack.

Citation Format: Dalal S. Aldeghaither, Joseph C. Murray, Sarah M. Roth, Elana J. Fertig, Shaojun Tang, Sandra A. Jablonski, Louis M. Weiner. Induction of ADCC resistance profoundly alters tumor cell phenotype and stress 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 633. doi:10.1158/1538-7445.AM2017-633
Abstract 64: Meditope SnAP-body technology facilitates enhanced internalization and in vivo efficacy of antibody therapeutics
Authors: Forster Karin , Gardiner Elisabeth , Hsieh Stephanie
Published: July 2017

Meditope Biosciences has developed a way to use SnAP technology to promote internalization of antibody receptor complexes through a novel construct called a SnAP-body. Meditope’s SnAP platform functionalizes monoclonal antibodies to bind to specific meditope peptides; this property can be used to directly facilitate receptor crosslinking when meditope-enabled antibodies are bound to cell surface receptors. SnAP-bodies are self-crosslinking meditope-enabled antibodies which permit a meditope peptide to specifically interact with a meditope-enabled antibody at the cell surface upon antigen engagement. Antigen-antibody complex formation through the specific contact of the SnAP-bodies and their cognate epitopes on the target antigen can increase clustering or co-localization of a cell surface antigen on a cell, which can promote enhanced internalization of the SnAP-body. The increased internalization can be used to clear or reduce the concentration of a cell surface antigen from the surface of a target cell or tumor. Enhancing internalization can increase the efficacy of antibody-based therapeutics, and in the case of antibody-drug conjugates, which require receptor internalization to deliver cytotoxic payloads, can reduce the amount of drug necessary to achieve a therapeutic effect. In vitro studies of SnAP-body binding to cell surface antigens demonstrate accelerated internalization of the antibody. In vivo, this translates into enhanced efficacy. The ability to see equivalent efficacy in the absence of toxin suggests a unique role for SnAP-body constructs in cancer therapeutics, especially in cases where lack of efficacy for a specific target is correlated with poor receptor internalization.

Citation Format: Karin Forster, Elisabeth Gardiner, Stephanie Hsieh. Meditope SnAP-body technology facilitates enhanced internalization and in vivo efficacy of antibody therapeutics [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 64. doi:10.1158/1538-7445.AM2017-64
Abstract 640: Subsets of HLA alleles are capable of binding neoantigens derived from mutations within cancer driving genes such as KRAS and EGFR
Authors: Nguyen Andrew , Sanborn J Zachary , Vaske Charles J. , Rabizadeh Shahrooz , Niazi Kayvan , Soon-Shiong Patrick , Benz Steve
Published: July 2017

Background: Immunoncology has shown great promise as a low toxicity tool to combat several cancers. Use of checkpoint inhibitors against PD1 or CTLA4 unlocks the immune system’s ability to recognize tumor antigens and, more specifically, neoantigens caused by random mutations within cancers. The vast majority of neoantigens consist of private mutations unique to a patient’s tumor genome, but several cancers harbor recurrent mutations. Mutations in the KRAS gene, such as p.G12V, occur in roughly 25% of colorectal cancers. Mutations in EGFR occur in 10% and 35% of patients with non-small cell lung cancer in the US and East Asia, respectively. Even more prevalent are mutations within the TP53 tumor suppressor gene, with roughly 23000 unique protein variants reported to date. If these mutations in cancer driving genes are so prevalent in cancers, why are neoantigens against these targets not more readily available?

Results: We collected recurrent mutations across a variety of cancer driving genes such as KRAS, EGFR, TP53 and MYC and performed binding analysis using netMHC 3.4 to see which HLA alleles are capable of binding specific cancer mutations such as KRAS p.G12V. Using this method, we report all possible HLA alleles capable of binding these recurrent mutations within cancer genes. We further performed 3-dimensional modeling to determine whether complexes created by the HLA alleles and cancer neoepitopes are stable.

Conclusions: Several HLA alleles are capable of binding recurrent cancer mutations. These include both MHC Class 1 and Class 2 alleles. The variation in alleles capable of binding commonly mutated genes such as EGFR may explain the difference in prevalence of these mutations between geographic populations. Determining whether a certain HLA allele confers resistance to common cancer gene mutations may lead to identification of immune cells within these populations that can recognize neoantigens from commonly mutated cancer genes.

Citation Format: Andrew Nguyen, J Zachary Sanborn, Charles J. Vaske, Shahrooz Rabizadeh, Kayvan Niazi, Patrick Soon-Shiong, Steve Benz. Subsets of HLA alleles are capable of binding neoantigens derived from mutations within cancer driving genes such as KRAS and EGFR [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 640. doi:10.1158/1538-7445.AM2017-640
Abstract 649: Emigrant pre-REP tumor infiltrating lymphocytes profoundly differ from remnant T-cells
Authors: Simpson-Abelson Michelle R. , Mosychuk Christopher , Fardis Maria , Lotze Michael T.
Published: July 2017

Adoptive T cell therapy with autologous tumor infiltrating lymphocytes (TIL) provides up to 56% objective response rates and a complete response in 24% of patients with metastatic melanoma. The process of generating TIL from resected tumor involves morcellating the tumor into 1-3 mm3 fragments and expanding TIL in the presence of Interleukin 2 (IL-2) in a pre-Rapid Expansion Protocol (pre-REP). During the ‘pre-REP’, tumor-resident immune cells emigrate (eTIL) and proliferate. The length of the pre-REP varies between 11-21 days, depending on cell growth. Residual tumor fragments (remnants) are discarded and the expanded eTIL are subjected to a Rapid Expansion Protocol (REP) with irradiated PBMC feeders, anti-CD3 and IL-2. Viable cells remaining in the tumor remnants (rTIL) following the pre-REP were investigated to assess their function and phenotype. We evaluated and compared the rTIL and eTIL in melanoma, breast, renal, pancreatic, lung and colorectal tumors (n=9). Tumor rTIL are consistently phenotypically distinct from eTIL, as determined by differential expression of various markers (Table 1). The fundamental differences in rTIL were: Increased CD69+ (7 fold MFI in CD4+) (p<.001); diminished LAG3 (2 fold MFI in CD8) (p<.05); TIM3 (3 and 2 fold MFI in CD8 and CD4 respectively) (p<.05/.01); CD154 (3 fold MFI in CD4) (p<.01); and CD56 (5%) (p<.05). Surprisingly, a REP of rTIL and eTIL resulted in comparable expansion. The phenotypic signature of TIL was sustained post-REP with fidelity of the individual expression of LAG3, Tim3, and CD28. These studies have identified significant differences in the biology of cell populations in terms of tissue-resident T cells and the signals associated with emigration and retention. These data provide additional insights on the individual TIL populations that could be utilized for adoptive T-cell therapy in patients and raise important questions about the nature of tissue-resident T cells in sites of chronic inflammation such as tumor.

Table 1:Tumor resident remnant T cells are phenotypically distinct from emigrating T cells (N=9)Marker ExpressionLAG3 (CD8/ CD4) MFITim3 (CD8/ CD4) MFIPD-1 (CD8/ CD4) %CD69 (CD8/ CD4) MFICD154 (CD8/ CD4) MFICD28 (CD8/ CD4) MFICD57 (CD8/ CD4) %CD56%eTIL507/ 1442832/ 175636.95/ 471320/ 15431498/ 37511163/ 503618.76/ 19.65.615rTIL209/ 106877/ 74242.8/ 483437/ 223.41034/ 1167458.3/ 27959.16/8.51.027*P-values(CD8/ CD4) 0.05/ 0.210.05/ 0.010.38/ 0.890.11/ 0.0010.55/ 0.010.05/ 0.110.05/ 0.060.05 _______

Citation Format: Michelle R. Simpson-Abelson, Christopher Mosychuk, Maria Fardis, Michael T. Lotze. Emigrant pre-REP tumor infiltrating lymphocytes profoundly differ from remnant T-cells [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 649. doi:10.1158/1538-7445.AM2017-649
Abstract 650: B lymphocytes promote upregulation of an IL-1-NfkB dependent signaling and increase invasiveness of triple negative breast cancer
Authors: Flynn Nicole , Somasundaram Rajasekharan , Sims-Mourtada Jennifer
Published: July 2017

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer that progresses quickly from a non-invasive carcinoma in situ to an invasive state. Chronic inflammation associated to humoral immune responses has been found to promote aggressiveness in a number of solid tumor types. In breast cancer, B lymphocytes are associated with microinvasive disease and correlate with expression of inflammatory genes. The purpose of our work is to study the impact of B lymphocytes on the tumor microenvironment and increased invasiveness of TNBC cells. Through real-time PCR, we demonstrate that co-culture of B lymphocytes and TNBC cells leads to increased mRNA levels of IL1β and its downstream target interleukin 8 (IL8) in both B lymphocytes and in TNBC cells. Western Blot analysis shows that co-culture also leads to increased phosphorylation of p65, indicating IL-1 β activation of NFκB signaling. Additionally, co-culture of B lymphocytes with TNBC cells leads to increased expression of matrix metalloproteinases (MMPs) and cellular invasion through a matrigel invasion chamber. Gelatin zymography reveals increased functional MMP2 and MMP9 in tumor cell supernatant following co-culture with B lymphocytes. To complement our in vitro studies, we examined the presence of CD20+ B cells and expression of inflammatory cytokines IL-1β and IL-8 by immunohistochemistry in serial sections of tissue microarrays from patients with estrogen receptor (ER) positive and negative ductal carcinoma in situ (DCIS) and invasive carcinoma. Large areas of densely populated B cells were observed in ER-DCIS and in invasive TNBC compared to ER+ DCIS. Furthermore, in ER- DCIS and TNBC, both B lymphocytes and tumor cells are found to express IL1β whereas IL8 is found more specifically to be expressed by tumor cells. Our findings support the hypothesis that B lymphocytes promote a chronic inflammatory environment leading to increased invasion of TNBC cells.

Citation Format: Nicole Flynn, Rajasekharan Somasundaram, Jennifer Sims-Mourtada. B lymphocytes promote upregulation of an IL-1-NfkB dependent signaling and increase invasiveness of triple negative breast 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 650. doi:10.1158/1538-7445.AM2017-650
Abstract 651: Evaluation of the expression and function of TIM-3 relative to PD-1 in human tumors
Authors: McEachern Kristen , Ghosh Srimoyee , Zhao Yonghong , Zhang Qiyao , Zhang Norman , Jenkins David W.
Published: July 2017

While immunotherapies directed against PD-1 and PD-L1 have proven effective across multiple indications, there is still a large unmet medical need for therapies for patients who do not respond or who develop acquired resistance during the course of treatment. There are several emerging hypotheses to explain the lack of response, including the overall presence and localization of immune cells, as well as the up-regulation of additional T-cell checkpoints, including TIM-3 in tumor infiltrating lymphocytes. To investigate the potential role of TIM-3, we set out to evaluate the expression and function of TIM-3 and the relationship to PD-1 in several systems. Firstly, we developed a flow cytometry methodology to enumerate T-cell, including CD4 and CD8 positive cells, as well as other immune cell populations in a panel of human tumor samples, including non-small cell lung cancer (NSCLC). In these samples, we investigated the expression profiles of TIM-3 and PD-1 in both T-cell and non-T-cell populations. In addition, we performed complementary genomic studies to explore gene expression profiles of not only the bulk tumor samples but also of isolated PD1 positive/TIM-3 negative versus PD1 and TIM-3 double positive cell populations. The profiling identified tumors with a range of tumor infiltrating lymphocyte content and also differences in PD1 and TIM-3 expression. It was found that in addition to T-cells, TIM-3 was expressed on myeloid cell populations and furthermore, that there were distinct differences in the gene expression profiles of PD1 single positive versus PD1 and TIM-3 double positive cells. Building on the expression studies, the functional role of Tim-3 in T-cells and also on myeloid-derived cells was explored. Taken together, these studies provide further evidence for TIM-3 as an important immunological checkpoint and as a relevant therapeutic target for the treatment of cancer with the potential for biological effects in both the T-cell and myeloid compartments of the tumor microenvironment.

Citation Format: Kristen McEachern, Srimoyee Ghosh, Yonghong Zhao, Qiyao Zhang, Norman Zhang, David W. Jenkins. Evaluation of the expression and function of TIM-3 relative to PD-1 in human tumors [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 651. doi:10.1158/1538-7445.AM2017-651
Abstract 66: Anthracycline-based antibody drug conjugates with potent immune-stimulatory functions
Authors: Beerli Roger R.
Published: July 2017

Antibody drug conjugates (ADCs) are highly potent and selective anti-tumor drugs, combining the specific targeting of monoclonal antibodies with the potency of small molecule toxic payloads. Here, we employed enzymatic, site-specific conjugation to generate homogenous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a non-cleavable peptide linker, using the anti-HER-2 antibody trastuzumab (part of trastuzumab emtansine) and the anti-CD30 antibody cAC10 (part of brentuximab vedotin). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as trastuzumab emtansine and brentuximab vedotin. Anti-tumor activity in an immune-competent host involved activation of the immune system, as shown by evaluation of a trastuzumab-PNU ADC in a Kadcyla-resistant HER2-positive orthotopic breast cancer model. Depletion of CD8 T cells severely reduced the anti-tumor activity of the ADC, demonstrating an important role for T cells in driving tumor regression. Furthermore, when tumor free animals were re-challenged with the same tumor, tumor growth was entirely inhibited in the absence of any further ADC administration, indicating the development of an immunological memory. In summary, we present a novel ADC format endowed not only with highly potent cytotoxicity, but also effective immune-stimulatory functions.

Citation Format: Roger R. Beerli. Anthracycline-based antibody drug conjugates with potent immune-stimulatory functions [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 66. doi:10.1158/1538-7445.AM2017-66
Abstract 667: Immunoprofiling circulating blood as a means to early detection of solid tumors
Authors: Kumar Amit , Gabrilovich Dimitry , Rauscher Frank J. , Dominguez George , Sholevar Cyrus , Roop John , Campisi Anthony , Polo Alexander
Published: July 2017

Our goal was to evaluate whether profiling Myeloid Derived Suppressor Cells (MDSCs) in circulation correlated with the existence and stage of multiple, biopsy-verified solid tumor types and to evaluate whether such analyses could enable early detection. The tumor micro-environment (TME) is replete with numerous immune cells, and the type and concentration of such cells can provide prognostic information. The link between high concentrations of MDSCs and poor prognosis is most likely due to the immuno-suppressive effects of such cells. Some fraction of these cells spill into the blood stream. We utilized flow cytometry to phenotypically quantify subsets of MDSCs and other leukocytes in the circulation of biopsy-verified cancer patients, as well as in age and sex matched healthy donors. Our results indicate a marked increase in MDSC levels in the circulation of tumor patients relative to healthy donors. We have analyzed patients presenting over a dozen solid tumor types (lung, breast, ovarian, colon, melanoma, liver, thyroid, pancreatic, uterine, osteosarcoma, appendiceal, leiomyosarcoma, liposarcoma, and vulvar) and found notable differences in the immune-profiles of circulating blood in these patients. It appears that the immune response, as measured by our flow cytometry technique, is general for most tumor types. We will present correlations and inter-relations between different cell types and evidence of tumors. While our studies to date have been performed in an unblinded manner, we will present performance data (specificity and sensitivity) for our approach.

Citation Format: Amit Kumar, Dimitry Gabrilovich, Frank J. Rauscher, George Dominguez, Cyrus Sholevar, John Roop, Anthony Campisi, Alexander Polo. Immunoprofiling circulating blood as a means to early detection of solid tumors [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 667. doi:10.1158/1538-7445.AM2017-667