bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3612: Combination FGFR4 and ER-targeted therapy for invasive lobular carcinoma
Authors: Levine Kevin , Chen Jian , Sikora Matthew , Tasdemir Nilgun , Tseng George , Puhalla Shannon , Jankowitz Rachel , Dabbs David , McAuliffe Priscilla , Lee Adrian , Oesterreich Steffi
Published: July 2017

Background Invasive Lobular Carcinoma (ILC) is an understudied subtype of breast cancer. Distinctive properties of ILC include growth patterns, metastatic behavior, receptor status (almost universally estrogen receptor (ER) positive), and survival outcomes (Long-term survival is lower in patients with ER+ ILC compared to the other main histological subtype, invasive ductal carcinoma). Our lab recently generated six long-term estrogen deprivation (LTED) models of ILC cells. We performed RNA-Sequencing on these six LTED cell lines and identified differentially expressed genes compared to their parental cells cultured with estrogen. We overlapped these results with a previously published analysis of a tamoxifen-resistant ILC cell line and found that FGFR4 is the most consistently overexpressed gene in the setting of acquired resistance to endocrine therapy in ILC cells.

Hypothesis FGFR4 is an important mediator of resistance to endocrine therapy in ILC.

Methods To study the role of FGFR4 in acquired resistance to endocrine therapy, we used siRNA, multiple shRNAs, and specific small molecule inhibition for growth assays of ILC cells. To study the role of FGFR4 in de novo resistance to endocrine therapy, we collected 129 well curated ER+ ILC tumor specimens. We performed gene expression analysis on the pre-treatment samples using a custom NanoString panel.

Results FGFR4 inhibition decreases parental and LTED ILC cell growth in classic 2D growth conditions, in the setting of ultra-low attachment, and in colony formation assays. Mechanistically, FGFR4 and estrogen signaling are antagonistic in parental ILC cells. In our clinical samples, increased expression of FGFR4 is predictive of shorter time to distant recurrence.

Conclusion FGFR4 may play an important role in both acquired and de novo resistance to endocrine therapy in ILC. Future studies will assess the efficacy of combining FGFR4 inhibitors with ER-targeted therapy for patients with ILC.

Citation Format: Kevin Levine, Jian Chen, Matthew Sikora, Nilgun Tasdemir, George Tseng, Shannon Puhalla, Rachel Jankowitz, David Dabbs, Priscilla McAuliffe, Adrian Lee, Steffi Oesterreich. Combination FGFR4 and ER-targeted therapy for invasive lobular carcinoma [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 3612. doi:10.1158/1538-7445.AM2017-3612
Abstract 3613: Development of chemically modified peptide inhibitor ERAP targeting BIG3-PHB2 complex on hormone-resistant breast tumor
Authors: Katagiri Toyomasa , Yoshimaru Tetsuro
Published: July 2017

Approximately 70% of breast cancer cells express estrogen receptor alpha (ERα), and depend on estrogen (E2) for proliferative and metastatic activity. The current endocrine therapies for breast cancer are mainly based on targeting ERα signaling using selective ERα modulators, ERα downregulators, and aromatase inhibitor. However, up to 50% of patients with ERα-positive tumors either initially do not respond or become resistant to these drugs. The precise molecular mechanisms for the endocrine resistance contributes to be an active area of research. Therefore, identifying the factors and pathways responsible for resistance and defining ways to overcome it lead to develop novel molecular-target therapies to endocrine resistance. Recent findings support that the Brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signaling modulation in these cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using ERα activity-regulator synthetic peptide (ERAP: 165-177 amino acids) derive from a helical BIG3 sequence, a dominant-negative peptide inhibitor leads to the significant anti-tumor effect. However, duration of its effect is very short for clinical use. Here, we report the development of the chemically modified ERAP using stapling methods (stapled ERAP) to improve duration of its antitumor effects. The stapled ERAP specifically inhibited the BIG3-PHB2 interaction, thereby exhibiting the long-lasting suppressive activity and their intracellular localization without membrane-permeable polyarginine sequence supposedly through the formation of stable α-helix structure induced by the stapling. Importantly, a combination of stapled ERAP and tamoxifen caused a synergistic inhibitory effect in breast cancer cell growth. Tumor bearing mice treated with every 7 days with stapled ERAP treatment effectively prevented the BIG3-PHB2 interaction as well as daily treatment, leading to the complete regression of E2-dependent tumor in vivo. Our findings suggest that the stapled ERAP may be a promising anti-tumor drug to suppress the growth of luminal-type breast cancer in clinical use.

Citation Format: Toyomasa Katagiri, Tetsuro Yoshimaru. Development of chemically modified peptide inhibitor ERAP targeting BIG3-PHB2 complex on hormone-resistant breast tumor [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 3613. doi:10.1158/1538-7445.AM2017-3613
Abstract 3620: RARalpha interactome in breast cancer, the S100 calcium binding protein A3 binds tothe retinoid receptor
Authors: Terao Mineko
Published: July 2017

All-trans-retinoic acid (ATRA) is used in the treatment of acute promyelocytic leukemia. ATRA is endowed with promising anti-tumor activity in other tumor types, including breast cancer. In breast cancer, the anti-tumor activity of ATRA is mediated by the retinoid nuclear receptor, RARα (EMBO Mol Med. 2015 7:950-972). RARα is a ligand-dependent transcriptional factor, which is part of nuclear complexes regulating the expression of target genes. To define the RARα interactome in breast cancer cells we took a proteomic approach. We performed mass-spectrometric analysis of RARα immuno-precipitates from nuclear extracts of an ATRA-sensitive breast cancer cell line over-expressing the receptor. We identified 30 proteins differentially interacting with RARα in the absence/presence of ATRA. Three of these proteins FABP5, HSP27 and S100A3 interact with RARα in the absence of ATRA and their binding to the receptor is dramatically reduced upon addition of the retinoid ligand. Binding of the three proteins to RARα was validated by different types of immuno-precipitation experiments. We focused our attention on S100A3, a calcium binding protein whose function is largely unknown. Pull-down and Far-Western experiment indicated that S100A3 binds directly to RARα. Binding is specific to this nuclear receptor isotype as similar interactions with S100A3 are not observed with RARβ, RARγ or RXRα. S100A3 over-expression inhibited RARα ligand-dependent transcriptional activity, suggesting that the calcium-binding protein is a co-repressor. We designed specific shRNAs targeting S100A3 and we are in the process of evaluating their functional effects on ATRA-dependent anti-tumor activity in retinoid sensitive breast cancer cell lines.

Citation Format: Mineko Terao. RARalpha interactome in breast cancer, the S100 calcium binding protein A3 binds tothe retinoid receptor [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 3620. doi:10.1158/1538-7445.AM2017-3620
Abstract 3631: Delivery of a CD20 transferrin receptor VNAR bispecific antibody to the brain for CNS lymphoma
Authors: Walsh Frank S. , Wicher Krzysztof , Szary Jaroslaw , Stocki Pawel , Demydchuk Michael , Rutkowski Lynn
Published: July 2017

Penetration of the blood brain barrier (BBB) remains a significant hurdle in the development of biologic therapeutics for CNS-related cancers. Antibody-based molecules typically do not cross the BBB in amounts required for therapeutic efficacy. Receptor-mediated transporters in the luminal membrane of brain capillary endothelium, which normally transport circulating endogenous peptides and proteins, offer a non-invasive approach for the delivery of therapeutic molecules to the brain.Using a combination of in vitro and in vivo phage display technology, we isolated a panel of cross species (rodent, primate and human) binders to the transferrin receptor 1 (TfR1) from synthetic libraries of shark variable antigen receptors (VNARs). Initially, the anti-TfR1 VNARs were fused to an immunoglobulin Fc backbone for testing in mice. At therapeutic (2 mg/kg) doses delivered by tail vein injection, high levels of the VNAR-Fc antibodies were found in the brain after 18 hours, which exceeded 5% of the plasma levels. Brain fractionation studies showed that the VNAR-Fc antibodies to TfR1 translocated to the brain parenchyma and were not retained in the capillaries. The lead anti-TfR1 VNAR was then used to target aberrant B cells in the brain for conditions such as cerebral lymphoma where existing CD20 therapies to not penetrate the CNS at therapeutic doses. A family of anti-CD20/TfR1 bispecific molecules were produced by linking the small VNAR to either the N- and C- terminus of heavy and light chains to create both monovalent and bivalent fusions . We used rituxan as the CD20 agent. The bispecifics were assessed for binding to the two cognate antigens and then tested for brain penetration after tail vein injection. At least 4 different formats gave excellent brain penetration 18 hours after dosing at 3.75mg/kg. Unmodified rituxan had a brain plasma ratio of 0.25 in line with published data while the best anti-TfR1-rituximab bispecific formats were between 2 to 5%. The bispecific formats also retain antibody-dependent cell-mediated cytotoxicity and will be further tested for brain penetration in non-human primates and in rodent models of CNS lymphoma. If these results successfully translate from rodents to larger animals, the peripheral delivery of a brain penetrant CD20/TfR1 bispecific antibody would provide a safer and more effective alternative to CD20 antibody delivery by the intrathecal route and could have broad utility in B cell driven brain cancer.

Citation Format: Frank S. Walsh, Krzysztof Wicher, Jaroslaw Szary, Pawel Stocki, Michael Demydchuk, Lynn Rutkowski. Delivery of a CD20 transferrin receptor VNAR bispecific antibody to the brain for CNS lymphoma [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 3631. doi:10.1158/1538-7445.AM2017-3631
Abstract 3635: Improved T Cell activation bioassays to advance the development of bispecific antibodies and engineered T cell immunotherapies
Authors: Somberg Richard L. , Stecha Pete , Garvin Denise , Hartnett Jim , Fan Frank , Cong Mei , Cheng Jey
Published: July 2017

T cells play a central role in cell-mediated immunity and can mediate long-term, antigen-specific, effector and memory responses. In recent years, a variety of immunotherapy strategies aimed at inducing, strengthening or engineering T cell responses have emerged as promising approaches for the treatment of diseases such as cancer and autoimmunity. Current methods used to measure TCR-mediated T cell proliferation and cytokine production rely on primary PBMCs as a source of T cells, which must be stimulated via co-culture with APCs or anti-TCR/CD3 antibodies. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development settings. To overcome this barrier, we developed two reporter-based bioluminescent T cell activation bioassays that can be used for the development of bispecific antibodies and engineered T cell immunotherapies. The assays consist of Jurkat T cells genetically engineered to express luciferase downstream of either NFAT or IL-2 response elements. The T cell activation bioassays reflect the mechanisms of action of biologics designed to induce TCR and/or CD28-mediated T cell activation, as demonstrated using anti-CD3 and/or anti-CD28 antibodies as well as blinatumomab, a bispecific antibody that simultaneously binds CD3 expressed on T cells and CD19 expressed on malignant B cells. The bioassays are pre-qualified according to ICH guidelines and show assay specificity, precision, accuracy and linearity required for routine use in potency and stability studies. Finally, our data illustrate the use of reporter-based T cell activation bioassays for characterizing and measuring the activity of engineered chimeric antigen receptor T cells.

Citation Format: Richard L. Somberg, Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Jey Cheng. Improved T Cell activation bioassays to advance the development of bispecific antibodies and engineered T cell immunotherapies [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 3635. doi:10.1158/1538-7445.AM2017-3635
Abstract 3648: Targeting glypican-2 in neuroblastoma via single domain antibody-based immunotoxins and chimeric antigen receptor T cells
Authors: Li Nan , Fu Haiying , Hewitt Stephen , Dimitrov Dimiter , Khan Javed , Ho Mitchell
Published: July 2017

Neuroblastoma is the most common extracranial solid tumor in children. Approximately 45% of patients receiving standard therapy relapse and ultimately succumb to metastatic disease. One of the major challenges in the treatment of neuroblastoma and other pediatric cancers is the lack of effective targets. As such, there is an urgent need for a new therapeutic target. Here we demonstrate that glypican-2 (GPC2) protein is highly expressed in nearly half of neuroblastoma cases. High GPC2 expression has been correlated with poor overall survival when compared to patients with low GPC2 expression. The reduction of GPC2 expression inhibits neuroblastoma cell growth and induces tumor cell apoptosis through downregulation of Wnt/β-catenin signaling. We have discovered a group of human single domain antibodies specific for GPC2 and have used them to make two forms of antibody therapeutics, antibody-toxin conjugates (immunotoxins) and chimeric antigen receptor (CAR) T cells. Treatment with the immunotoxin inhibits proliferation of GPC2-positive neuroblastoma cells in vitro and mouse models. The CAR T cells targeting GPC2 suppress the growth of neuroblastoma xenograft tumors and eradicate disseminated neuroblastomas in mice. Our study establishes GPC2 as a new target of antibody-based cancer therapy and demonstrates that single domain-based antibody therapeutics can be used in the treatment of neuroblastoma.

Citation Format: Nan Li, Haiying Fu, Stephen Hewitt, Dimiter Dimitrov, Javed Khan, Mitchell Ho. Targeting glypican-2 in neuroblastoma via single domain antibody-based immunotoxins and chimeric antigen receptor 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 3648. doi:10.1158/1538-7445.AM2017-3648
Abstract 3655: Biomarker strategy to guide the clinical development of ImmTACTM molecules, a novel class of bispecific T cell engaging biologic drugs
Authors: McAlpine Cheryl , Krige David , Herrero-Gonzalez Sandra , Franklin Sarah , Hurst Jacob , Shingler William , Patel Sanjay , Johnson Andy , Parker Debbie , Coughlin Christina M. , Hassan Namir J. , Jakobsen Bent K.
Published: July 2017

IMCgp100, a first in class immunotherapy, is a T cell redirecting bispecific biologic comprising an affinity enhanced T-cell receptor specific for gp100 and an anti-CD3 scFV. Phase I/IIa data has provided evidence of a favourable safety profile, and durable responses in both cutaneous and uveal melanoma have been observed. To complement the clinical studies we have developed a comprehensive biomarker strategy to aid our understanding of pharmacodynamics, patient response and potential mechanisms of resistance which includes analysis of markers in both the tumour and periphery. The data obtained to date provide evidence of the pharmacodynamic effects of the molecule including chemokine/cytokine release, in both the tumor and periphery, and lymphocyte infiltration into tumors. In addition, changes in the levels of certain chemokines following the first dose of IMCgp100 were associated with tumor shrinkage. The biomarker strategy we have developed forms the basis for the support of the on-going Ph II development of IMCgp100 in both cutaneous and uveal melanoma and for other ImmTAC molecules, as single agents and in combination, for the treatment of solid tumours.

Citation Format: Cheryl McAlpine, David Krige, Sandra Herrero-Gonzalez, Sarah Franklin, Jacob Hurst, William Shingler, Sanjay Patel, Andy Johnson, Debbie Parker, Christina M. Coughlin, Namir J. Hassan, Bent K. Jakobsen. Biomarker strategy to guide the clinical development of ImmTACTM molecules, a novel class of bispecific T cell engaging biologic drugs [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 3655. doi:10.1158/1538-7445.AM2017-3655
Abstract 3657: Phosphatidylserine-targeting antibodies enhance anti-tumor activity of a tumor vaccine in a HPV-induced tumor model
Authors: Weir Genevieve , Quinton Tara , Hutchins Jeff T. , Freimark Bruce D. , Stanford Marianne
Published: July 2017

Antibodies targeting phosphatidylserine (PS) have been shown to induce anti-tumor responses by induction of tumor-specific T cells. Based on this observation, we evaluated the responses of PS and PD-1 targeting antibody therapy to enhance anti-tumor responses of a HPV16 peptide vaccine formulated in DepoVax™ (DPX) in mice bearing HPV-transformed C3 mouse tumors. The addition of PS-targeting antibody (mch1N11) to DPX/metronomic cyclophosphamide (mCPA) immunotherapy prolonged survival in comparison to mice receiving an isotype control in combination with DPX/mCPA. When anti-PD-1 was added to mch1N11 + mCPA, there was no increase in survival. The addition of mch1N11 to DPX/mCPA immunotherapy had no effect on tumor growth or survival in the aggressive B16-F10 model. TIL analysis revealed an increase in CD8+ T cells, antigen specific CD8+ T cells and PD-1+ T cells in the tumor with mch1N11 treatment. The expression of surface markers for macrophages (CD68high, F4/80) and dendritic cells (CD11c) were also increased in the tumors of mice treated with mch1N11. RT-qPCR analysis of the tumor confirmed higher mRNA expression of T cells markers (CD8, Granzyme B, PD-1) and antigen presenting cell markers (F4/80, CD74). In the spleen, expression of cell surface markers for monocytes (CD11b) and PD-1+ T cells (CD8) were elevated in groups treated with mch1N11 in combination with anti-PD-1. Combined, these findings indicate that in this model, PS-targeting antibodies can enhance the activity of phagocytic cells involved in antigen presentation. We have found that PD-1 expression increases as anti-tumor activity increases, therefore these results also provide an indication that antibodies targeting PS enhance the anti-tumor immune response induced by DPX/mCPA therapy. The observations suggest that PS-targeting antibodies may enhance therapeutic vaccines for the treatment of cancer.

Citation Format: Genevieve Weir, Tara Quinton, Jeff T. Hutchins, Bruce D. Freimark, Marianne Stanford. Phosphatidylserine-targeting antibodies enhance anti-tumor activity of a tumor vaccine in a HPV-induced tumor 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 3657. doi:10.1158/1538-7445.AM2017-3657
Abstract 3658: Dendritic cells dictate the responsiveness of PD-L1 blockade in cancer
Authors: Mayoux Maud , Fransen Marieke F. , Roller Andreas , Matos Ines , Pulko Vesna , Karanikas Vaios , Umana Pablo , Klein Christian , Ossendorp Ferry A. , Xu Wei
Published: July 2017

Recent advances in cancer immunotherapies with PD-1/PD-L1 pathway blockade have transformed the way that cancer is being treated, leading to durable responses and prolonged overall survival. The general thinking is that PD-1/PD-L1 blockade reinvigorates tumor-infiltrating PD-1+ T cells with exhausted phenotypes. However, a mechanistic understanding on why only a subset of patients (10-30%) responds to checkpoint inhibition remains largely unknown, as does the exact immune mechanism of PD-1/PD-L1 blockade. Here, we discovered that PD-L1 blockade mediates anti-tumor immunity via dendritic cells. In human blood, in vitro and ex vivo dendritic cells (DCs) express both PD-1 and PD-L1, and an activation signal via TLR agonists triggers downregulation of PD-1 to empower the T cell stimulatory capability of DCs. In contrast, tolerogenic DCs remain PD-1 positive, correlating to T cell-unresponsiveness. Anti-PD-L1 Ab directly induces maturation of DCs and renders them capable of stimulating T cell proliferation. Similarly, anti-PD-L1 Ab treatment in tumor-bearing mice induces massive infiltration and activation of CD11c+ DCs in the spleen and draining lymph node, indicating that PD-1 is a negative regulator in DCs. Furthermore, ablation of DCs prior to anti-PD-L1 Ab treatment in an established MC38 tumor model in CD11c-DTR mice suggests a crucial role of DCs in mediating response to PD-L1 treatment. In support of the preclinical evidence that DCs are the primary target of anti-PD-L1 Ab, we analyzed RNA-seq data from tumor biopsies at baseline in patients with renal cell carcinoma prior to treatment with atezolizumab and found that the abundance of genes related to cross-presenting DC subsets (such as XCR1) correlates with a survival advantage in response to atezolizumab (HR=0.13, median OS is 16.2 months in patients with DC gene score <50% versus NR (not reached) in those >50%). In conclusion, we discovered abundance of tumor-infiltrating DCs as a novel biomarker that can predict the clinical response of PD-L1 blockade. We postulate that checkpoint inhibition directly on tumor-infiltrating DCs likely contributes to amplification of Ag-specific priming of tumor-specific T cells.

Citation Format: Maud Mayoux, Marieke F. Fransen, Andreas Roller, Ines Matos, Vesna Pulko, Vaios Karanikas, Pablo Umana, Christian Klein, Ferry A. Ossendorp, Wei Xu. Dendritic cells dictate the responsiveness of PD-L1 blockade in 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 3658. doi:10.1158/1538-7445.AM2017-3658
Abstract 3662: Activation of CD40 while inhibiting IL6/STAT3 using oncolytic viruses induces mature DCs with high cytokine production but blocks PDL1 expression
Authors: Eriksson Emma , Milenova Ioanna , Wenthe Jessica , Dimberg Anna , Moreno Rafael , Ullenhag Gustav , Alemany Ramon , Loskog Angelica
Published: July 2017

The tumor microenvironment (TME) consists of tumor cells and stroma, including fibroblasts, blood vessels, immune cells and extracellular matrix. The TME supports tumor progression, metastasis as well as resistance to cancer therapeutics. In pancreatic cancer, the TME is dense due to overproduction of collagen and the tumor is infiltrated with suppressive myeloid cells such as M2 macrophages and myeloid suppressor cells. One key regulator of myeloid cells is CD40, a receptor expressed on a variety of cell types. CD40/CD40L signaling results in production of cytokines and chemokines by myeloid cells but also endothelial and epithelial cells to alert the immune system of immediate danger. 4-1BB is expressed by lymphocytes and dendritic cells (DCs). Stimulation via 4-1BBL drives lymphocyte expansion and regulates memory formation. IL6 signaling leads to STAT3 phosphorylation in myeloid cells and tumor cells leading to suppressive phenotypes, tumor proliferation, and angiogenesis. Further, STAT3 signaling enhances production of TGFb, which in turn leads to overexpression of collagen. We have constructed a family of oncolytic adenoviruses (LOAd) activating the CD40, 4-1BB and/or inhibiting IL6 signaling. The LOAd viruses (-, 700, 703, 713) were investigated for their capacity to activate human monocyte-derived DCs as well as their effect on pancreatic tumor cells and stroma (fibroblastic stellate cells, endothelial cells) using flow cytometry, MTS assay and ProSeek Proteomics. The LOAd viruses expressing a trimerized CD40L, 4-1BBL and/or a scFv IL6R showed efficient oncolysis of tumor cells but primary stellate cells were unaffected. However, stellate cells reduced tumor-promoting factors such as FGF5, PlGF, amphiregulin, Gal3, TGFb and collagen type I. Dendritic cells increased costimulators, cytokines as well as chemokines but PDL1 was not expressed when IL6/STAT3 was blocked. Infected endothelial cells upregulated receptors important for lymphocyte transmigration (ICAM, VCAM and E-Selectin). Taken together, our data demonstrates that it is possible to utilize oncolytic adenoviruses to spark immune activation at the same time changing biological processes via STAT3 blockade and/or CD40/4-1BB pathway activation to reduce factors that promotes tumor progression.

Citation Format: Emma Eriksson, Ioanna Milenova, Jessica Wenthe, Anna Dimberg, Rafael Moreno, Gustav Ullenhag, Ramon Alemany, Angelica Loskog. Activation of CD40 while inhibiting IL6/STAT3 using oncolytic viruses induces mature DCs with high cytokine production but blocks PDL1 expression [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 3662. doi:10.1158/1538-7445.AM2017-3662
Abstract 3663: An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+ T cell responses in tumor-bearing mice
Authors: Ueha Satoshi , Ogiwara Haru , Yokochi Shoji , Ishiwata Yoshiro , Shand Francis , Hori Shohei , Kakimi Kazuhiro , Ito Satoru , Matsushima Kouji
Published: July 2017

Administration of an anti-CD4 depleting antibody induces tumor-specific CD8+ T cell responses in tumor-bearing mice, although the mechanisms underlying this phenomenon remain unclear. CD4+ Foxp3+ regulatory T cells (Treg) impair antigen presentation by DCs through CTLA4-mediated down-regulation of CD80/CD86. In the present study, we investigated the effects of an anti-CD4 depleting monoclonal antibody (mAb) on DC responses in the B16F10 and LLC subcutaneous tumor models, and compared these effects with those of Treg targeting therapy. Administration of anti-CD4 mAb up-regulated CD80/CD86 in tumor-infiltrating DCs, and in migrating-type but not resident-type DC subsets in the tumor-draining lymph node. The upregulation of CD80/CD86 on these DC populations was correlated with increased proliferation of tumor-specific CD8+ T cells in the tumor-draining lymph node. Anti-CD4 mAb-induced CD80/CD86 up-regulation and tumor-specific CD8+ T cell responses were remarkably higher than that induced by an anti-CD25 depleting mAb or an anti-CTLA4 blocking mAb, and were equivalent to that induced by diphtheria-toxin-induced selective depletion of Tregs in Foxp3-DTR mice. Importantly, the activation of CD4+ conventional T cells and B cells in the peripheral lymph nodes that was observed following Treg depletion was not observed following anti-CD4 mAb administration. In conclusion, anti-CD4 depleting antibody therapy is an effective way to evoke anti-tumor CD8+ T cell responses by reversing regulatory T cell-induced suppression of dendritic cells, and to reduce the risk of severe autoimmunity in cancer patients.

Citation Format: Satoshi Ueha, Haru Ogiwara, Shoji Yokochi, Yoshiro Ishiwata, Francis Shand, Shohei Hori, Kazuhiro Kakimi, Satoru Ito, Kouji Matsushima. An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+ T cell responses in tumor-bearing mice [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 3663. doi:10.1158/1538-7445.AM2017-3663
Abstract 3670: Treatment of epithelial ovarian cancer with folate receptor (α/β) targeted chemotherapy is enhanced by CTLA-4 blockade: Learning from animal models
Authors: Lu Yingjuan , Johnson Theresa P. , Wheeler Leroy W. , Lloyd Alex M. , Cross Vicky A. , Westrick Elaine M. , Parker Nikki L. , Leamon Christopher P.
Published: July 2017

Epithelial ovarian cancer (EOC) remains to be the most lethal form of gynecologic malignancies despite aggressive cytoreductive surgery (i.e. debulking) followed by standard chemotherapy. The alpha folate receptor (FRα) has long been recognized as a tumor target for EOC due to its frequently intense overexpression (>80%) in the most common serous histotype. Recently, the beta folate receptor (FRβ) has been discovered on tumor-associated macrophages (TAMs) which are present at various densities in solid tumors but are also found abundant in malignant ascites of EOC patients. The ascites TAMs are strongly immunosuppressive, and high TAM frequency is correlated with poor patient prognosis and treatment resistance. Respectively, tumor- and TAM-associated FRα and FRβ are both functionally active which allows for folate-targeted delivery of small-molecule drug payloads via receptor-mediated endocytosis.

Using flow cytometry, we characterized total ascites cells harvested from human IGROV1 xenograft (hFRα) and murine ID8-CL15 syngeneic (mFRα) models at the time of euthanasia. Approximately 70% of IGROV1 and 25% of ID8-Cl15 ascites TAMs (F4/80+ CD11b+) were found to express a functional mFRβ. While ascites fluids from both models caused a dramatic reduction of FRα functionality on tumor cells in-vivo and ex-vivo, FRβ expression and functionality on ascites TAMs were largely unaffected. Interestingly, in the ID8-CL15 model, the ratio of ascites CD8+ T cells to CD4+ T cells decreased as total FR functionality on TAMs increased. Two small-molecule folate-drug conjugates with different mechanisms of action showed activity in extending the survival of tumor-bearing animals. While CTLA-4 blockage alone was completely inactive in the ID8-CL15 model, a combination regimen significantly enhanced the activity of a folate conjugate carrying a potent DNA cross-linking agent. Our data suggest that EOC treatment may require a multi-pronged approach by targeting both FRα-expressing tumor cells and FRβ-expressing TAMs. In addition, immune checkpoint inhibitors may further enhance the FR-targeted therapeutics by modulating the ascites tumor microenvironment.

Citation Format: Yingjuan Lu, Theresa P. Johnson, Leroy W. Wheeler, Alex M. Lloyd, Vicky A. Cross, Elaine M. Westrick, Nikki L. Parker, Christopher P. Leamon. Treatment of epithelial ovarian cancer with folate receptor (α/β) targeted chemotherapy is enhanced by CTLA-4 blockade: Learning from animal models [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 3670. doi:10.1158/1538-7445.AM2017-3670
Abstract 3683: Melphalan stimulates dendritic cell and CD8+ T cell expansion by inducing immunogenic cell death in melanoma cells
Authors: Johansson Junko , Kiffin Roberta , Lindnér Per , Naredi Peter , Bagge Roger Olofsson , Martner Anna
Published: July 2017

Background: Regional hyperthermic perfusion with the alkylating agent melphalan is a treatment option for patients with metastatic melanoma confined to the limbs or the liver. Following a single perfusion, tumors often decrease gradually in size during several months, suggesting an immune-mediated mechanism of action, in addition to the direct cytotoxic effects of melphalan. This study was designed to characterize the immunogenic effects of melphalan.

Materials and methods: We have established an in vitro model of regional hyperthermic perfusion where human melanoma cell lines are exposed to melphalan at 40oC for 1 h, thus mimicking the currently employed clinical protocol. The melphalan-exposed melanoma cells were analyzed for markers of immunogenic cell death and were co-cultured with peripheral blood mononuclear cells (PBMCs) in the presence or absence of IL-2. The number and activation status of various immune populations were analyzed by flow cytometry.

Results: Melphalan exposure triggered the expression of several immune-related markers on melanoma cells, including calreticulin, MHC class I, Hsp70 and PD-L1. Melphalan-treated, but not untreated melanoma cells, triggered an increase in dendritic cell (DC) numbers along with a dramatic expansion of CD8+ T cells in co-cultured PBMCs. The expanded CD8+ T cells showed an activated phenotype with the majority of cells belonging to the effector memory subtype.

Conclusions: Melanoma cells exposed to melphalan undergo immunogenic cell death and trigger DC expansion with subsequent expansion and activation of CD8+ T cells. We propose that these events may contribute to the anti-tumor efficacy of regional hyperthermic perfusion with melphalan in metastatic melanoma.

Citation Format: Junko Johansson, Roberta Kiffin, Per Lindnér, Peter Naredi, Roger Olofsson Bagge, Anna Martner. Melphalan stimulates dendritic cell and CD8+ T cell expansion by inducing immunogenic cell death in melanoma 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 3683. doi:10.1158/1538-7445.AM2017-3683
Abstract 3695: The interplay between neutrophils &It CDThe interplay between neutrophils and CD8+ cell improves survival in human colorectal cancer
Authors: Governa Valeria , Trella Emanuele , Mele Valentina , Droeser Raul , Eppenberger-Castori Serenella , Terracciano Luigi Maria , Iezzi Giandomenica , Spagnoli Giulio Cesare
Published: July 2017

Purpose: Tumor infiltration by different T lymphocyte subsets is known to be associated with favorable prognosis in colorectal cancer (CRC). Still debated is the role of innate immune system. We investigated clinical relevance, phenotypes &it functional features of CRC infiltrating CD66b+ neutrophils &it their crosstalk with CD8+ T cells.

Experimental design: CD66b+ &it CD8+ cell infiltration was analyzed by immunohistochemistry on a tissue microarray including >650 evaluable CRC samples. Phenotypic profiles of tissue infiltrating and peripheral blood CD66b+ cells were evaluated by flow cytometry. CD66b+/ CD8+ cells crosstalk was investigated by in vitro experiments.

Results: CD66b+ cell infiltration in CRC is significantly associated with increased survival. Interestingly, neutrophils frequently co-localize with CD8+ T cells in CRC. Functional studies indicate that although neutrophils are devoid of direct antitumor potential, co-culture with peripheral blood or tumor associated neutrophils (TANs) enhances CD8+ T cell activation, proliferation and cytokine release induced by suboptimal concentrations of anti-CD3 monoclonal antibody (mAb). Moreover, under optimal activation conditions, CD8+ cells initially stimulated in the presence of CD66b+ cells show decreased expression of PD-1 “exhaustion” marker and are significantly less susceptible to apoptosis induced by T- cell receptor triggered re-stimulation. Importantly, combined tumor infiltration by CD66b+ and CD8+ T lymphocytes is associated with significantly better prognosis, as compared to CD8+ T cell infiltration alone.

Conclusions: Neutrophils enhance the responsiveness of CD8+ T cells to TCR triggering. Accordingly, infiltration by neutrophils enhances the prognostic significance of CRC infiltration by CD8+ T cells, suggesting that they might effectively promote antitumor immunity.

Note: This abstract was not presented at the meeting.

Citation Format: Valeria Governa, Emanuele Trella, Valentina Mele, Raul Droeser, Serenella Eppenberger-Castori, Luigi Maria Terracciano, Giandomenica Iezzi, Giulio Cesare Spagnoli. The interplay between neutrophils &It CDThe interplay between neutrophils and CD8+ cell improves survival in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3695. doi:10.1158/1538-7445.AM2017-3695
Abstract 3697: The impact of HER3 signaling mediated PD-L1 regulation in triple negative breast cancer
Authors: Yamaguchi Ayane , Suzuki Eiji , Kawaguchi Kosuke , Nishie Mariko , Tsuda Moe , Kotake Takeshi , Toi Masakazu
Published: July 2017

Triple negative breast cancer (TNBC) is still difficult to treat partly because of lacking specific target. Although 50-70% of TNBC expresses EGFR, it is less sensitive to the treatment of EGFR inhibition for TNBC as compared to the efficacy of HER2 inhibition for HER2-positive breast cancer. Several phase II study on EGFR blockade treatment has been reported, however it has not applied in a clinical setting yet. It was reported that residual tumors after treatment with EGFR-targeted antibodies showed increased HER3 abundance leading to EGFR/HER3 receptor dimerization. The signals of HER3/EGFR dimerization to PI3K/AKT/mTOR pathways are thought to be involved in cancer survival, proliferation and also up-regulation of PD-L1 expression. Thus, we hypothesized that up-regulation of HER3 signal caused by anti-cancer treatment might induce PD-L1 expression and inhibit host anti-tumor immunity. In this study, we tested the relationships between HER3 signal and PD-L1 expression by using three basal-like breast cancer cell line; MDA-MB-231, HCC70, and MDA-MB-468. MDA-MB-231 is HER3-negative, and HCC70 and MDA-MB-468 are HER3-positive cell lines. We added neureglin 1 (NRG1: HER3 ligand) to those three cell lines and analyzed PD-L1 expression of protein by flowcytometry and mRNA by qRT-PCR. Both protein and mRNA level of PD-L1 on HCC70 and MDA-MB-468 treated with NRG1 are increased as compared with those without NRG1 while there was no change of PD-L1 expression of MDA-MB-231 either with or without NRG1. In order to confirm the significance of potential treatment target of HER3, we evaluated HER3 expression in biopsy samples by immunohistochemistry before neoadjuvant chemotherapy (NAC) including all phenotypes. Thirteen pathological complete response (pCR) cases after NAC and 6 non pCR cases were included. We scored the HER3 stainability from 0 to 3 and found that non pCR cases showed significantly higher HER3 score than pCR cases (84.6% and 33% respectively, p=0.0149).

Although further study is needed, these results suggest that HER3 signal possibly regulates PD-L1 expression in HER3-positive basal-like breast cancer and treatment with anti-HER3 targeting therapy combination with an immune checkpoint inhibition therapy for HER3 positive NAC resistant patients might be warranted.

Note: This abstract was not presented at the meeting.

Citation Format: Ayane Yamaguchi, Eiji Suzuki, Kosuke Kawaguchi, Mariko Nishie, Moe Tsuda, Takeshi Kotake, Masakazu Toi. The impact of HER3 signaling mediated PD-L1 regulation in 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 3697. doi:10.1158/1538-7445.AM2017-3697
Abstract 3701: Mutant p53 cancers reprogram tumor-associated macrophages via exosomal miR-1246
Authors: Cooks Tomer , Pateras Ioannis S. , Jenkins Lisa M. , Patel Keval M. , Robles Ana I. , Morris James , Forshew Tim , Appella Ettore , Gorgoulis Vassilis G. , Harris Curtis C.
Published: July 2017

TP53 mutants (mutp53) are involved in the pathogenesis of most human cancers. Specific mutp53 proteins gain oncogenic functions (GOF) distinct from the tumor suppressor activity of the wild-type protein. Tumor-associated-macrophages, a hallmark of solid tumors, are typically correlated with poor prognosis. Here we report a non-cell-autonomous mechanism whereby mutp53 cancer cells reprogram TAM to a tumor supportive and anti-inflammatory state. The colon cancer cells harboring GOF mutp53 selectively shed miR-1246-enriched exosomes. Uptake of these exosomes by neighboring macrophages triggers their miR-1246 dependent reprogramming into a cancer-promoting state. Mutp53-reprogammed TAM favor anti-inflammatory immunosuppression with increased activity of TGF-β. These findings, observed also in colon cancer patients, strongly support a microenvironmental GOF role for mutp53 in actively engaging the immune system to promote cancer progression and metastasis.

Citation Format: Tomer Cooks, Ioannis S. Pateras, Lisa M. Jenkins, Keval M. Patel, Ana I. Robles, James Morris, Tim Forshew, Ettore Appella, Vassilis G. Gorgoulis, Curtis C. Harris. Mutant p53 cancers reprogram tumor-associated macrophages via exosomal miR-1246 [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 3701. doi:10.1158/1538-7445.AM2017-3701
Abstract 3702: Quantitative imaging of metabolic changes in macrophage subsets
Authors: Heaster Tiffany M. , Elion David L. , Cook Rebecca S. , Skala Melissa C.
Published: July 2017

Macrophages play an important role in tumor therapeutic response. However, current methods cannot image heterogeneities in macrophage activity on a single-cell level over time within intact, living samples. Macrophage metabolism is closely linked to macrophage function, as M1-like macrophages rely on aerobic glycolysis and M2-like macrophages rely on fatty acid oxidation and oxidative phosphorylation. Thus, we propose to develop optical metabolic imaging (OMI) using two-photon microscopy to monitor cell-level changes in macrophage polarization and functional activity. OMI exploits the auto-fluorescence intensities and lifetimes of the metabolic co-enzymes NAD(P)H and FAD, and has been previously used to dynamically image in vivo tumors with cell-level resolution. The optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD) provides a global measure of redox balance within individual cells. The fluorescence lifetimes of NAD(P)H and FAD provide information in protein-binding activities in metabolic reactions. OMI was used to monitor metabolic changes in macrophages on a single-cell level during polarization and after metabolic perturbations. RAW 264.7 macrophages were stimulated towards an M1- or M2-like phenotype, and imaged with OMI at 24, 48, and 72 hours post-polarization. M1- and M2-like macrophages exhibited significant differences (p<0.05) in redox ratio and FAD fluorescence lifetime values at 48 and 72 hours post-polarization. The M1-like and M2-like character of the macrophages were validated with quantitative polymerase chain reaction (qPCR) analysis. Population density modeling of single-cell metabolism established differences in intra-sample heterogeneity across subsets over time. M1- and M2-like macrophages were also treated with 2-deoxy-glucose (glycolysis inhibitor), etomoxir (fatty acid oxidation inhibitor), and cyanide (oxidative phosphorylation inhibitor. Treatment with etomoxir caused a significant decrease in redox ratio in M2-like macrophages (p<0.05) with no significant effect on M1-like macrophages. In contrast, the redox ratio decreased in M1-like macrophages upon inhibition of glycolysis (p<0.05). Comparable to previously published data, inhibition of oxidative phosphorylation in both groups yielded a decreased NAD(P)H mean lifetimes (p<0.05) and increased FAD mean lifetimes (p<0.05). These results establish OMI as a method to image heterogeneity in macrophage populations within intact, living samples. This method could be used to understand macrophage interactions in the microenvironment of intact in vivo tumors.

Citation Format: Tiffany M. Heaster, David L. Elion, Rebecca S. Cook, Melissa C. Skala. Quantitative imaging of metabolic changes in macrophage subsets [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 3702. doi:10.1158/1538-7445.AM2017-3702
Abstract 3705: Targeting interleukin-1 to increase cetuximab efficacy in head and neck cancer
Authors: Espinosa-Cotton Madelyn , McLaren Ayana J. , Koch Adam T. , Simons Andrean L
Published: July 2017

Treatment for head and neck squamous cell carcinoma (HNSCC) patients includes the epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab. However, response to cetuximab is low and resistance remains a critical issue. Therefore, it is imperative to develop strategies to enhance tumor response to this drug. Inflammation plays an important role in tumor progression by increasing the release of cytokines that promote tumor cell survival. Our prior work has shown that a variety of pro-inflammatory cytokines involved in tumor survival are upregulated upon treatment with EGFR inhibitors in HNSCC cells, suggesting that poor tumor response to EGFR inhibitors may be due to increased tumor-associated inflammation. Our lab has shown that the increased proinflammatory tumor response due to EGFR inhibition may be activated by interleukin-1 (IL-1) signaling, suggesting that the IL-1 pathway may be an important target to enhance the efficacy of anti-EGFR therapy. Here we investigated the effect of IL-1 blockade on HNSCC tumor response to cetuximab treatment. We found that cetuximab induced IL-6 secretion from SQ20B HNSCC cells and knockdown of the adapter protein MyD88, which is essential for IL-1 signaling, suppressed IL-6 secretion at baseline and in response to cetuximab treatment. Similarly, knock down of the IL-1 receptor (IL-1R) decreased baseline and cetuximab-induced IL-6 secretion, although IL-1R knockdown did not improve response to cetuximab in SQ20B xenografts in vivo. We additionally blocked IL-1 signaling using a recombinant IL-1R antagonist (IL-1RA). IL-1RA pre-treatment reduced baseline and cetuximab-induced IL-6 secretion in vitro. Although IL-1RA did not significantly enhance SQ20B tumor response to cetuximab, IL-1RA as a single agent significantly suppressed tumor growth in female mice but not in male mice. Finally, treatment with a human-specific neutralizing IL-1 alpha (IL-1α) antibody (but not IL-1 beta (IL-1β)) reduced baseline and cetuximab-induced IL-6 secretion in vitro, and significantly increased SQ20B tumor response to cetuximab in vivo. Altogether we propose that targeting IL-1α specifically may be a viable strategy to improve HNSCC tumor response to cetuximab.

Citation Format: Madelyn Espinosa-Cotton, Ayana J. McLaren, Adam T. Koch, Andrean L, Simons. Targeting interleukin-1 to increase cetuximab efficacy in head and neck 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 3705. doi:10.1158/1538-7445.AM2017-3705