bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1616: S100A4 blockage alleviates agonistic anti-CD137 antibody induced liver pathology without disruption of anti-tumor immunity
Authors: Zhang Jinhua , Song Kun , Qin Zhihai
Published: July 2017

Liver-related autoimmune toxicities triggered by anti-CD137 agonist antibodies have greatly limited their use in clinic applications. Here we found that anti-CD137 monoclonal antibody (mAb) treatment in mice induced the infiltration of a large number of S100A4+ macrophages into the liver. Depletion of these cells or deficiency of S100A4 decreased inflammatory cytokine profiles, and drastically reduced the number of liver pathogenic CD8+ T cells. Mechanistically, soluble S100A4 directly activated the Akt pathway and specifically prolonged CD8+ T cell survival. Interestingly, one S100A4 neutralizing monoclonal antibody selectively alleviated liver abnormalities but did not affect the anti-tumor immunity induced by anti-CD137 therapy. Thus, our study presents a novel molecular link to the liver pathology induced by an immune stimulatory antibody, and proposes that combinational immunotherapies targeting those pathways could potentially elicit optimal anti-tumor immunity with minimal side effects.

Note: This abstract was not presented at the meeting.

Citation Format: Jinhua Zhang, Kun Song, Zhihai Qin. S100A4 blockage alleviates agonistic anti-CD137 antibody induced liver pathology without disruption of anti-tumor immunity [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 1616. doi:10.1158/1538-7445.AM2017-1616
Abstract 1620: Oral IL-10 suppresses colon carcinogenesis via elimination of pathogenic CD4+ T-cells and induction of antitumor CD8+ T-cell activity
Authors: GU TAO , DeJesus Magdia , Burris Thomas P. , Egilmez Nejat K.
Published: July 2017

An oral sustained-release formulation of Interleukin-10 suppressed tumor growth and enhanced survival in the APCmin/+ / Bacteroides fragilis spontaneous colon cancer model. Therapeutic benefit was associated with a 5-fold reduction in CD4+RORγt+Foxp3-IL-17+ T-helper cell, CD4+RORγt+Foxp3+IL-17+ pathogenic T-regulatory cell (pgTreg) and CD4+RORγt-Foxp3+IL-17- conventional T-regulatory cell (cTreg) numbers; and a concurrent enhancement of CD8+ T-cell cytotoxicity in the colonic lamina propria. Selective subset depletion and functional blockade studies demonstrated that CD4+RORγt+IL-17+ T-cell subsets and CD4+Foxp3+ cTreg supported tumorigenesis/tumor progression whereas CD8+ cytotoxic T-lymphocytes suppressed tumor growth. These findings establish the utility of oral particulate IL-10 as a potential new therapeutic in the management of colon cancer and shed light on the cellular mechanisms that underlie its antitumor activity.

Note: This abstract was not presented at the meeting.

Citation Format: TAO GU, Magdia DeJesus, Thomas P. Burris, Nejat K. Egilmez. Oral IL-10 suppresses colon carcinogenesis via elimination of pathogenic CD4+ T-cells and induction of antitumor CD8+ T-cell activity [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 1620. doi:10.1158/1538-7445.AM2017-1620
Abstract 1621: Adoptively transferred B cells directly kill tumor cells via the CXCR4/CXCL12 & perforin pathways
Authors: Xia Leiming , Xia Yang , Chen Quanning , Wang Yi , Bao Yangyi , Lundy Steven K. Lundy , Dai Fu , Chang Alfred E , Li Qiao
Published: July 2017

Over the years, the role of B cells in the host immune response to malignancy has been overshadowed by our focus on T cells. The role played by B cells in cancer immunology is complex and controversial. The observation made by our lab that activated B cells alone can mediate tumor regression in the adoptive immunotherapy of solid tumors is innovative (JI 2009; CCR 2011; JSO 2012; EJI 2015). One novel mechanism by which activated B cells mediate tumor regression is via direct tumor cell cytotoxicity in the absence of antibodies. We previously reported that antitumor B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. In this study, we defined additional mechanisms involved in B cell antitumor immunity. Administration of IL-2 significantly augmented the therapeutic efficacy of adoptively transferred tumor-draining lymph node (TDLN) B cells which express IL-2R. Culture supernatant of purified B splenocytes harvested from the mice that received adoptive transfer of 4T1 TDLN B cells plus IL-2 administration produced larger amounts of IgG which bound to 4T1, resulting in 4T1 lysis. Furthermore, we detected CXCR4 expression on 4T1 TDLN B cells, and 4T1 tumor cells produced its ligand CXCL12. Transwell experiments demonstrated the chemotraction of CXCR4-expressing 4T1 TDLN B cells towards CXCL12-producing 4T1 cells. Blockade of CXCR4 using a CXCR4-specific inhibitor, AMD3100, significantly reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Blockade of FasL and CXCR4 concurrently inhibited B cell-mediated direct killing of tumor cells in an additive manner, indicating that both Fas/FasL and CXCL12/CXCR4 pathways are involved in the direct killing of 4T1 cells by 4T1 TDLN B cells. TDLN B cells produced perforin. Additional transwell experiments showed that effector B cells could directly kill tumor cells in cell-cell contact via the Fas/FasL and CXCR4/CXCL12 pathways as well as perforin, while without cell contact, perforin secreted by B cells led to tumor cell cytotoxicity. These findings underscore the diversity of function by which B cells can play an important role in the host immune response to tumor, and clearly indicated that transferred effector B cells can act independently of T cells in causing tumor destruction in adoptive immunotherapy.

Citation Format: Leiming Xia, Yang Xia, Quanning Chen, Yi Wang, Yangyi Bao, Steven K. Lundy Lundy, Fu Dai, Alfred E Chang, Qiao Li. Adoptively transferred B cells directly kill tumor cells via the CXCR4/CXCL12 & perforin pathways [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 1621. doi:10.1158/1538-7445.AM2017-1621
Abstract 164: Enhanced antitumor efficacy by recruiting macrophages as effector cells via bispecific antibodies mediated by CD89
Authors: Li Bingyu , Xu Lijun , Xie Kun , Tao Fei , Li Renhao , Gu Hua , Fang Jianmin
Published: July 2017

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of key mechanisms through which antibody therapeutics can lyse tumor target cells. ADCC is usually mediated by IgG through recruiting effector cells, mostly NK cells, via IgG Fc receptor. In order to enhance specific tumor lysis by ADCC, a common strategy is to design a bispecific antibody that binds a tumor cell surface antigen and T cell marker CD3, thus to recruit T cells as effector cells. An example of this type of design is the FDA-approved bispecific antibody blinatumomab. We are interested in designing novel bispecific strategies to achieve improved ADCC effect. Since tumors are often infiltrated by macrophages and neutrophils, it would be advantageous to turn these types of cells into effector cells for ADCC. We have designed several bispecific strategies that can target a tumor associated antigen (TAA) and CD89 (FcαRI). The aim is to lyse tumors through connecting tumor cells to CD89-expressing immune effector cells, such as macrophages and neutrophils. The CD89 bispecific antibodies were produced by HEK293 cells and showed binding activities to both CD89 and TAA. The CD89 bispecific antibodies can recruit CD89-expressing immune cells and induced enhanced tumor cells-killing in human whole blood ADCC assays. Since mouse does not express human CD89 homologue, we generated a transgenic mouse strain with specific expression of CD89 on macrophages and monocytes. In this transgenic mouse model, the CD89 bispecific antibodies showed significant anti-tumor activities, demonstrating that the bispecific antibodies can redirect macrophages, including M2 macrophages, to mediate additional effector function in tumor microenvironment.

Citation Format: Bingyu Li, Lijun Xu, Kun Xie, Fei Tao, Renhao Li, Hua Gu, Jianmin Fang. Enhanced antitumor efficacy by recruiting macrophages as effector cells via bispecific antibodies mediated by CD89 [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 164. doi:10.1158/1538-7445.AM2017-164
Abstract 1642: Evaluation of ENMD-2076 in combination with anti-PD1 in syngeneic cancer models
Authors: Fletcher Graham C. , Kiarash Reza , Bray Mark R. , Hu Amanda S. , Ren Ken K.
Published: July 2017

ENMD-2076 is a clinical stage compound with potent activity towards Aurora A and angiogenic kinases. ENMD-2076 has shown promising activity in multiple Phase 1 clinical trials, as well as in a Phase 2 trial in advanced ovarian cancer. ENMD-2076 is currently the subject of several ongoing Phase 2 clinical trials including fibrolamellar carcinoma, triple-negative breast cancer (TNBC), advanced/metastatic soft tissue sarcoma (STS), and advanced ovarian clear cell carcinomas (OCCC). ENMD-2076 has been developed to date as a single agent, however ENMD-2076 inhibits a spectrum of targets including Aurora A, FAK, CSF1R, c-Kit, and KDR, that are potentially involved in immune evasion mechanisms. These kinases have been shown in published studies, when inhibited, to enhance or augment the activity of immune checkpoint inhibitors such as anti-PD1. A study was thereby conducted in syngeneic models to determine the utility of ENMD-2076 combined with immune checkpoint inhibition as a rational strategy for cancer therapy. The study evaluated the efficacy of ENMD-2076 administered daily by oral gavage in the MC38 and CT26 colon cancer models, alone and in combination with an anti-PD1 antibody. Xenografts were established in the appropriate mouse strain (C57BL/6 and BALBc, respectively) by the subcutaneous inoculation of MC38 or CT26 cells into the right flank of female mice. Treatment was initiated 7 days following inoculation when tumor volumes had reached a mean volume of approximately 85 mm3. All treatments were well tolerated, with no significant body weight loss seen during either study. While CT26 tumors were relatively refractory to single agent ENMD-2076, tumor regression was observed in several MC38-bearing animals suggesting an immune activating mechanism. In both models a trend was observed for an augmentation of anti-tumor response in combination relative to single agent ENMD-2076 and anti-PD1 alone. Further studies to evaluate mechanism and an assessment of re-challenge experiments in animals exhibiting complete regression of tumors will be discussed. These studies support the further evaluation of ENMD-2076 in combination with immune checkpoint inhibition as a strategy for cancer therapy.

Citation Format: Graham C. Fletcher, Reza Kiarash, Mark R. Bray, Amanda S. Hu, Ken K. Ren. Evaluation of ENMD-2076 in combination with anti-PD1 in syngeneic cancer 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 1642. doi:10.1158/1538-7445.AM2017-1642
Abstract 1645: CEACAM1-blockade for T-cell activation and antitumor T-cell response
Authors: Eun So-Young
Published: July 2017

Immune checkpoint blockade became the most powerful way to control cancer without causing tremendous side effects, since anti-PD-1 blocking antibodies proved their superiority in clinical outcomes to those of traditional chemo- or targeted therapeutics. CEACAM1 (CCM1) is one of the several immune checkpoint receptors expressed on T cells that mediate suppression of inflammatory T cell response. CCM1 caught our attention for a cancer immune-therapeutic target, because of its inhibitory role in TCR proximal signaling complex. The anti-cancer therapeutic potential of CCM1-blockade has already been studied in mouse models, and one of the anti-human CCM1 antibody clones recently emerged in clinical Phase I studies. CCM1-targeting antibody candidates were screened out of our in-house synthetic libraries, among which a few of the clones stood out showing their significant blocking activities against CCM1-CCM1 homophilic interaction, enhancement of T cell activation, cytokine release, NFAT signaling, and tumor lysis. One of the clones (C25) showed its strongest efficacy equivalent to that of a reference Ab in CCM1-dependent T cell activation, proliferation, and IL-2 release, in parental Jurkat cells as well as in CCM1-overexpressing stable Jurkat cells. Moreover, C25 also enhanced tumor lysis by CCM1+ NK cells or CCM1+ cytotoxic T cells in a CCM1-dependent manner. In addition, C25 shows distinct characteristics in binding to CCM family from those of a reference Ab. Thus, C25 is thought to have a strong therapeutic potential on CCM1+ cancer.

Citation Format: So-Young Eun. CEACAM1-blockade for T-cell activation and antitumor T-cell 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 1645. doi:10.1158/1538-7445.AM2017-1645
Abstract 1647: Characterization of MiXenoTM humanized mouse model for immuno-oncological therapy evaluation
Authors: Zhang Juan , Shi Qian , Zhang Lan
Published: July 2017

Backgrounds: Despite the unprecedented success of immune checkpoint antibody therapy in patients with various cancer types, lack of stable experimental immunotherapy models is still a big challenge in development of effective new immune-therapeutics. Different strategies of humanized mouse model system, including human hematopoietic stem cell (HSC) or human PBMC humanization in immune-deficient mice, or chimeric models harboring human immune checkpoint targets in immune-competent mice have been reported. CrownBio has successfully established a number of human PBMC-humanized xenograft models MiXenoTM for in vivo IO therapy evaluation, which has been shown to be a rapid and simple strategy of humanization. However, several outstanding questions, e.g. the optimal PBMC injection route and cell number, the donor dependence of the efficacy result and impact of donor HLA type on the efficacy, etc., need to be further addressed.

Results: PBMC number was titrated in one of our MiXenoTM model HCC827, and PBMC injection via i.p. vs. i.v. was compared. Tumor growth curve, graft versus host disease (GVHD), and blood immunophenotyping were followed under different settings. Furthermore, the antitumor activity of PD-1 antibody pembrolizumab in a panel of PBMC donor context that bears diversified HLA types was precisely analyzed. We found that the efficacy was highly donor dependent, and there was lack of correlation between efficacy and donor/tumor HLA match.

Conclusion: The MiXenoTM model provides an alternative to the full stem cell reconstitution approach. Better understanding on the characteristics of the model may allow a rational model selection and study design in the discovery IO therapeutics.

Citation Format: Juan Zhang, Qian Shi, Lan Zhang. Characterization of MiXenoTM humanized mouse model for immuno-oncological therapy evaluation [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 1647. doi:10.1158/1538-7445.AM2017-1647
Abstract 1656: Comprehensive evaluation of human immune system reconstitution in NSGTM and NSGTM-SGM3 toward the development of a novel Onco-HuTM xenograft model
Authors: Middlebrook Aaron J. , Snowden Eileen , Porter Warren , Hahn Friedrich , Ferguson Mitchell , Soper Brian , Keck James , Malcolm Joan , Dillmore Shannon , Ghanekar Smita , Blaesius Rainer
Published: July 2017

The recent successes of immunotherapeutic approaches to the treatment of melanoma and the promise of similar treatments in a variety of other cancers underscore the importance of the immune system in cancer. Indeed, effective therapeutic design and evaluation require a comprehensive understanding of the interplay between the immune compartment and the proliferating tumor cells that comprise the tumor microenvironment. A humanized mouse strain engrafted with cancerous tissue from a patient derived xenograft (PDX) tumor provides researchers with a highly sophisticated tool, ideally suited to facilitate the design of treatment strategies that prevent tumor evasion of immune cells and that improve cytotoxic responses. Severely combined immunodeficient mice such as NOD scid gamma (NSGTM) and triple transgenic NSG mice expressing human cytokines KITLG, CSF2, and IL-3 (NSGTM-SGM3) are proven hosts for the engraftment of human tumors and establishment of human immune system components following hematopoietic stem cell (CD34+) transplantation. The endogenous expression of cytokines that support the development of myeloid lineages and regulatory T cells potentially represents a substantial improvement over standard NSG mice. Here we employ four 14-color flow cytometry panels to perform a comprehensive and detailed analysis of the entire immune system. The four panels are designed to fully characterize specific branches of the immune system: 1) T cells 2) NK cells/dendritic cells/B cells 3) myeloid lineages, and 4) immune checkpoint markers. Blood, spleen, and bone marrow tissue from both NSG and NSG-SGM3 mice were evaluated at 9, 16, 21, and 31 weeks of age using each of the four phenotyping panels. Our results indicate that the triple transgenic NSG-SGM3 mice exhibit a more completely humanized immune system compared to NSG mice, with specific improvements in the distribution of T-cell subsets and overall representation of the myeloid lineage. NSG mice engrafted with allogeneic human tumors represent a valuable preclinical testing platform for immuno-oncology.

Citation Format: Aaron J. Middlebrook, Eileen Snowden, Warren Porter, Friedrich Hahn, Mitchell Ferguson, Brian Soper, James Keck, Joan Malcolm, Shannon Dillmore, Smita Ghanekar, Rainer Blaesius. Comprehensive evaluation of human immune system reconstitution in NSGTM and NSGTM-SGM3 toward the development of a novel Onco-HuTM xenograft 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 1656. doi:10.1158/1538-7445.AM2017-1656
Abstract 1657: Evaluating anti-tumor activity of the human anti-PD-1 antibody pembrolizumab using humanized mouse models
Authors: Linn Douglas E. , Cristescu Razvan , Ray Kallol , Zhang Shuli , Dhandapani Sripriya , Kaliyaperumal Sarav , Yearley Jennifer H. , Long Brian J.
Published: July 2017

Targeting immune checkpoints has proven to be an effective strategy for reactivating the immune system to elicit potent anti-tumor activity. For example, the anti-PD-1 antibody pembrolizumab has shown robust and durable clinical activity in numerous malignancies including melanoma and non-small cell lung cancer. Unfortunately, a subset of patients do not benefit from pembrolizumab treatment. Ongoing molecular analyses of patient samples are establishing gene signatures which aim to more accurately predict which patients will benefit from immunotherapy. However, there are currently no preclinical in vivo platforms that comprehensively translate these clinical findings. Therefore, we have explored the use of mice engineered with a human immune system to examine the anti-tumor activity of pembrolizumab against a panel of human tumor xenografts derived from both cultured cell lines as well as patient tissue. Tumor models were selected based on parameters including PD-L1 expression and mutational load in order to closely mimic responder patients in the ongoing pembrolizumab clinical trials. Our data confirm that pembrolizumab can induce significant anti-tumor efficacy in humanized mice inoculated with human tumor models derived from both cancer cell lines and patient tumors. In responsive models, pembrolizumab caused significant tumor growth inhibition and, in some instances, tumor regressions. FACS analyses confirmed effective targeting of PD-1+ T cell populations. Anti-tumor efficacy correlated with increased effector T cell to regulatory T cell ratios as well as an elevated T cell infiltrate presence. Nonresponsive models lacked robust immune infiltration detectable by FACS or IHC, consistent with clinical data. Furthermore, presence of cytolytic activity at baseline and T-cell inflamed environment were evaluated using transcriptome analysis and subsequently linked to pembrolizumab response and to somatic neoantigen burden of the tumors. Together these studies validate the utility of humanized mice in the preclinical development of novel cancer immunotherapies and may also be beneficial in better defining the patient responder hypothesis and its prognostic value. Additionally, humanized mice may help identify optimal combination treatment regimens to provide more meaningful clinical benefit to patients who do not respond to single-agent therapy with pembrolizumab.

Citation Format: Douglas E. Linn, Razvan Cristescu, Kallol Ray, Shuli Zhang, Sripriya Dhandapani, Sarav Kaliyaperumal, Jennifer H. Yearley, Brian J. Long. Evaluating anti-tumor activity of the human anti-PD-1 antibody pembrolizumab using humanized mouse 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 1657. doi:10.1158/1538-7445.AM2017-1657
Abstract 1670: Identification and characterization of novel mediators of tumor-induced T-cell dysfunction
Authors: Markowitz Geoffrey J. , Philip Mary , Schietinger Andrea , Mittal Vivek
Published: July 2017

Immunotherapies targeting T-cell functionality have been shown to have efficacy in numerous cancers, including non-small cell lung cancer (NSCLC). However, treatment with currently available therapeutics, such as anti-PD-1 and anti-CTLA-4, induce responses only in a subset of patients. This raises the possibility that numerous other effectors of T-cell dysfunction remain to be elucidated. To evaluate possible novel mediators of T-cell dysfunction, we isolated CD8+ T-cells from orthotopic KrasG12D/p53-/- murine lung adenocarcinomas (HKP-1) that exhibited either stable or progressive disease as a function of tumor growth. RNAseq analysis revealed increased expression of several genes including Tim3, PD-1, Lag3, 2B4, and CISH, and decreased expression of GzmB and Eomes in CD8+ T-cells isolated from progressive tumors indicative of a dysfunctional state, as well as multiple novel genes not previously associated with T-cell dysfunction. To explore the functional role of the novel candidate genes, we have developed in vitro and in vivo assays leveraging Ova and GFP overexpression in our HKP-1 cell line (KP1-Ova-GFP). We have begun to target these unique set of candidate genes in tumor-specific T-cells associated with dysfunction through innovative RNAi/shRNA and adoptive transfer approaches to facilitate therapeutic reprogramming and enhanced T-cell-mediated immunity in lung cancer. We expect to provide novel strategies for designing rational immunotherapies targeting dysfunctional tumor-specific CD8+ T-cells to generate durable anti-tumor immune responses for the treatment of lung adenocarcinoma.

Citation Format: Geoffrey J. Markowitz, Mary Philip, Andrea Schietinger, Vivek Mittal. Identification and characterization of novel mediators of tumor-induced T-cell dysfunction [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 1670. doi:10.1158/1538-7445.AM2017-1670
Abstract 1673: Mass spectrometric characterization of peptides associated with molecules of the major histocompatibility complex
Authors: Ford Michael , Jones Richard , Allen David , Amunugama Ravi , Rizzo Paul Del , Pisano Michael , Mobley James , Domanski Paul , Ho Bill , Bochar Daniel
Published: July 2017

The major histocompatibility complex (MHC) is a region of highly polymorphic genes encoding for glycoproteins (MHC molecules) that form part of the cell-mediated branch of the acquired immune system. In the cytosol, cellular self and foreign (non-self) proteins are constantly being degraded; it is the peptides generated that are presented, non-covalently bound to MHC molecules, on the surface of cells for inspection by cytotoxic T-lymphocytes (CTLs). Non recognition of the presented peptide ultimately leads to cell destruction.

Characterizing the factors associated with Non recognition is an attractive proposition for anyone interested in generating tools for targeted cell destruction. In the field of oncology the obvious application then is the targeted destruction of cancerous cells. To enable the molecular level characterization of peptides associated with molecules of the major histocompatibility complex requires a targeted protein complex enrichment, an unbiased peptide elution and finally a peptide analysis method. Most frequently an immunoprecipitation is used to isolate the target complex. The peptide elution is performed under conditions minimizing protein contamination and finally peptide analysis is accomplished by mass spectrometry.

Here we present a case study of our recent work optimizing and performing a workflow for the analysis of peptides associated with Class I MHC molecules. The goal of the assay optimization was to minimize the amount of antibody required for the assay, to minimize the amount of biological material needed from which the complex is isolated and to achieve the optimum sensitivity towards the hitherto unknown target peptides.

Citation Format: Michael Ford, Richard Jones, David Allen, Ravi Amunugama, Paul Del Rizzo, Michael Pisano, James Mobley, Paul Domanski, Bill Ho, Daniel Bochar. Mass spectrometric characterization of peptides associated with molecules of the major histocompatibility complex [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 1673. doi:10.1158/1538-7445.AM2017-1673
Abstract 1674: Quantifying tumor-infiltrating leukocytes in hematoxylin stained NSCLC tissue samples using morphometric features
Authors: Ergon Elliott , Harney Allison S. , Martin Nathan , Paces Will , Aeffner Famke , Wilson Kristin , Patterson-Kane Janet , Ryall Karen , Rudmann Daniel G. , Hirsch Brooke , Krueger Joseph
Published: July 2017

Quantification of tumor-infiltrating lymphocytes (TILs) in non-small cell lung cancers (NSCLC) is valuable for understanding patient prognosis and survival. TILs comprise a subset of tumor-infiltrating leukocytes that modulate immune evasion and response to therapy. Understanding the composition of TIL subsets, especially relative to the total tumor leukocyte population, may provide additional context for understanding NSCLC pathogenesis and patient response to treatment. However, availability of tissues and use of chromogenic assays can limit the number of TIL and leukocyte subset markers assayed in a tissue section. Therefore, this study evaluated the identification of total leukocyte component in NSCLC using morphometric parameters and routine TIL marker monoplex immunohistochemistry (IHC) assays to further identify the composition of TIL subsets. Computational Tissue Analysis (cTA™) tools were used to determine the morphometric parameters which could identify immune cells in the absence of biomarker stain. The morphometric features which characterized immune infiltrates were used to quantify the total immune cell population frequency in the tumor nests and surrounding stroma in hematoxylin-stained tissues. The leukocyte population identified with morphometric parameters was correlated with CD45+ cell frequencies identified by cTA based on biomarker staining in CD45-stained serial sections. This morphometric ruleset was then applied to CD3- and CD8-stained tissues to evaluate the frequency of CD3+ and CD8+ TILs in the context of total infiltrating leukocytes. The relative populations of CD3+ and CD8+ TILs were consistent with available literature demonstrating that the morphometric ruleset could be utilized to enable evaluation of TIL sub-types relative to total leukocyte population without the need for additional IHC stains. The approach could, therefore, provide an added dimension of analysis for tissues stained by IHC for identifying the total immune cell infiltrating component without requiring additional biomarker staining.

Citation Format: Elliott Ergon, Allison S. Harney, Nathan Martin, Will Paces, Famke Aeffner, Kristin Wilson, Janet Patterson-Kane, Karen Ryall, Daniel G. Rudmann, Brooke Hirsch, Joseph Krueger. Quantifying tumor-infiltrating leukocytes in hematoxylin stained NSCLC tissue samples using morphometric features [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 1674. doi:10.1158/1538-7445.AM2017-1674
Abstract 1690: Epitope-minigenes for optimal induction of the immune response against tumor associated antigens and neoantigens
Authors: Bandini Silvio , Luberto Laura , Palombo Fabio , Roscilli Giuseppe , Marra Emanuele , Ciliberto Gennaro , Aurisicchio Luigi
Published: July 2017

We have recently established a workflow that allows the identification of T cell epitopes within Tumor Associated Antigens (TAAs) and the construction of genetic cancer vaccine based on the use of minigenes. The T-cell epitope in silico prediction approach is based on three criteria: 1) binding to MHC Class I alleles; 2) uniqueness to the antigen of interest; 3) increased likelihood of natural processing. The combination of in silico prediction and a biochemical binding/stability assay resulted in an accurate identification of novel TAA-derived epitopes. Predicted T cell epitopes were connected by furin sensitive linkers and linked to human tissue plasminogen activator (TPA) signal and E. Coli enterotoxin B subunit, to construct an optimal minigene scaffold used as vaccine candidate. The present study was aimed at evaluating HER2/neu and hTERT (telomerase) minigenes with the same technology platform. First of all, minigenes delivered via Electro Gene Transfer (DNA-EGT) were more immunogenic than genetic vectors encoding the full-length protein or peptides injected subcutaneously and they were able to break immune tolerance in wild type and HLA-A0201 transgenic mice. Moreover, this technology applied to epitopes selected within Mutated Tumor Antigens results in strong immunogenicity and significant antitumor effects in mouse models. In conclusion, we show that minigenes delivered via DNA-EGT and based on predicted and/or experimentally identified epitopes are powerful tools to induce immune responses and combat cancer. Combination studies of minigenes with peptide vaccination, chemotherapy and immune checkpoint blockade may define new therapeutic opportunities for cancer patients.

Citation Format: Silvio Bandini, Laura Luberto, Fabio Palombo, Giuseppe Roscilli, Emanuele Marra, Gennaro Ciliberto, Luigi Aurisicchio. Epitope-minigenes for optimal induction of the immune response against tumor associated antigens and neoantigens [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 1690. doi:10.1158/1538-7445.AM2017-1690
Abstract 1692: KRAS-mediated therapeutic resistance abrogates immunogenic cell death in colorectal cancer cells
Authors: Wang Yi-Jun , Chen Dongshi , Tong Jingshan , Yu Jian , Bardelli Alberto , Zhang Lin
Published: July 2017

A hallmark of cancer is compromised immune surveillance, which is characterized by failure of the immune system to recognize tumor-associated antigens and specifically eliminate malignant cells. A number of studies showed the tumor microenvironment in colorectal cancer (CRC) patients is highly immunosuppressive. Conventional chemotherapy and recent targeted therapy can restore anti-tumor immune response through induction of immunogenic cell death (ICD) in tumor cells. However, the mechanism and functional role of ICD in CRC treatment remains vague. In this study, we found FOLFIRI (Leucovorin/5-Fluorouracil/Irinotecan) combined with the anti-EGFR antibody cetuximab, a commonly used regimen for CRC treatment, induces death of CRC cells with characteristics of necroptosis, including ATP depletion, release of HMGB1, and phosphorylation of RIP3 and MLKL. Cells subjected to the combination treatment exhibited features of ICD including plasma membrane exposure of calreticulin and phagocytosis by dendritic cells. We identified the BH3-only Bcl-2 family protein PUMA as a key mediator of necroptosis induced by the combination treatment in vitro and in vivo. Furthermore, we found that CRC cells with acquired cetuximab resistance due to KRAS mutation or amplification are deficient in ICD. Collectively, our results suggest that oncogenic mutations cause resistance of CRC cells to anticancer therapies in part by suppressing ICD.

Citation Format: Yi-Jun Wang, Dongshi Chen, Jingshan Tong, Jian Yu, Alberto Bardelli, Lin Zhang. KRAS-mediated therapeutic resistance abrogates immunogenic cell death in colorectal cancer 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 1692. doi:10.1158/1538-7445.AM2017-1692
Abstract 1695: Exploiting the pancreatic cancer mutome for immune interception
Authors: Kinkead Heather , Lutz Eric , Dubensky Thomas W. , Jaffee Elizabeth
Published: July 2017

This study aims to translate tumor-specific mutations identified by next-generation sequencing techniques into an effective anti-tumor vaccine for a classically non-immunogenic tumor, pancreatic ductal adenocarcinoma (PDA). Pancreatic ductal adenocarcinomas develop an average of 63 mutations during tumorigenesis, collectively referred to as the “mutome.” Of these, approximately 45 are amino acid point mutations. These mutations are not present in normal cells and provide a source of tumor-specific neoepitopes for targeted immunotherapy. Next-generation sequencing technologies allow for rapid identification of mutations present in patient tumors; however, methods for rapid identification of immunogenic neoepitopes which can be effectively targeted for tumor clearance, and optimized vaccination strategies for targeting them, still need to be developed.

In this study, we are using a transplantable murine PDA model, Panc02, to develop vaccination strategies for inducing neoepitope-specific T cell antitumor responses, and to define parameters for selecting appropriate targets. Exome sequencing identified 878 nonsynonymous mutations in the Panc02 cell line. Three immunological epitope prediction servers identified 878 potentially immunogenic peptides in the Panc02 model, 29 of which were strong candidates (scores <50) and another 240 candidate epitopes within the limits of prediction (scores <1000). Panc02 neoepitope peptide vaccinations in mice confirmed 14% of the predicted epitopes to be immunogenic by IFNγ ELISPOT, which may include both CD8+ and CD4+ T cell responses. Tumor transplant experiments demonstrate that therapeutic vaccination against neoepitopes in combination with a human STING-activating cyclic dinucleotide adjuvant is capable of inducing a temporary regression of implanted tumors. After tumor escape and regrowth, flow cytometry reveals a large population of infiltrating lymphocytes, the majority of which are co-expressing the checkpoint molecules PD-1, Tim3, and Lag3, indicating that treatment of vaccinated animals with checkpoint blockade therapy may potentiate infiltrating lymphocytes and ultimately result in permanent tumor regression. Thus, a broad and unbiased approach for screening vaccine targets will allow for the evaluation of predictive algorithms and ultimately the development of more effective vaccination approaches for targeting the tumor mutome.

Citation Format: Heather Kinkead, Eric Lutz, Thomas W. Dubensky, Elizabeth Jaffee. Exploiting the pancreatic cancer mutome for immune interception [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 1695. doi:10.1158/1538-7445.AM2017-1695
Abstract 1698: Immunoprevention of KRAS-driven lung adenocarcinoma by a multi-peptide KRAS vaccine
Authors: Pan Jing , Zhang Qi , Sei Shizuko , Shoemaker Robert H. , Lubet Ronald A. , Wang Yian , You Ming
Published: July 2017

Lung cancer remains the leading cause of cancer death worldwide. Mutations in KRAS are detected in up to 30% of lung cancer cases, about 80% of which occur in codon 12, yet no effective therapies specifically targeting mutant KRAS have been developed. Boosting the host immune response to cancer cells by vaccinating against defined tumor-associated antigens (TAAs) as a means of treating established tumors or preventing the development of tumors in high risk individuals such as current or former smokers is an area of intense research. The present study evaluated both immunogenicity and anti-tumor efficacy of a newly formulated multipeptide (peptides 15-17 amino acids long) vaccine targeting multiple epitopes of the KRAS molecule in a mouse model of KRAS-driven lung tumor. Vaccination was performed in the prevention setting in a transgenic mouse model, where mutated mouse Kras (G12D) is conditionally over-expressed in the lungs of mice using a CCSP promoter. A multi-score prediction algorithm was used to identify likely immunogenic epitopes in the KRAS protein sequence. The identified peptides were synthesized and screened in naïve mice for immunogenicity. The formulated vaccine contained the top four peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. The multipeptide KRAS vaccine was then tested in an inducible CCSP-TetO-KRASG12D mouse model, where the vaccines mixed with the Freund’s adjuvant or adjuvant alone were administered prior to activating the mutant KRAS protein. Our results show the KRAS peptide vaccine exhibited striking efficacy, reducing tumor number and tumor burden by >80% when compared with adjuvant alone. Splenocytes collected from vaccinated animals at the end of the study showed a robust immunologic response to the immunizing peptides. Furthermore, in vitro stimulation of these splenocytes by the vaccine peptides resulted in the secretion of cytokines indicative of Th1 (interferon gamma) and Th17 (IL17) responses but with minimal secretion of Th2 related cytokines e.g. IL3 and IL4. In the draining lymph nodes from vaccinated mice rejecting tumors, there was a substantial increase in CD4 cells but a limited increase in CD8 cells. In summary, the multipeptide KRAS vaccine was immunogenic and efficacious in the primary prevention of KRAS-induced lung cancer in mice, indicating that the approach can potentially be used for the prevention of KRAS-driven cancers either alone or in combination with other modalities.

Citation Format: Jing Pan, Qi Zhang, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. Immunoprevention of KRAS-driven lung adenocarcinoma by a multi-peptide KRAS vaccine [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 1698. doi:10.1158/1538-7445.AM2017-1698
Abstract 1706: Neoantigen frequency as an independent prognostic factor in patients with clear cell ovarian carcinoma (CCOC)
Authors: Matsushita Hirokazu , Hasegawa Kosei , Oda Katsutoshi , Yamamoto Shogo , Nishijima Akira , Imai Yuichi , Asada Kayo , Ikeda Yuji , Karasaki Takahiro , Fujiwara Keiichi , Aburatani Hiroyuki , Kakimi Kazuhiro
Published: July 2017

Aim: Neoantigens derived from tumor-specific somatic mutations are thought to be targets for antitumor immune responses. It has been reported that mutation/neoantigen burdens correlate with prognosis of certain cancers under checkpoint blockade immunotherapy. In clear cell ovarian carcinoma (CCOC), checkpoint inhibitors provided durable responses in a subset of patients. To test whether the same may apply in CCOC patients, who are not on immunomodulatory therapy, we investigated the number of mutations or neoantigens, and immune signature of CCOC by integrated molecular analysis.

Methods: We performed exome sequencing and expression array for 74 CCOC patients. Candidate neoantigens derived from mutations were identified by MHC class I binding prediction algorism (NetMHCpan v 2.8). Immune signature was assessed by gene ontology and gene set enrichment analyses for immune cell lineage or molecule genes. We also analyzed their relationships with clinical outcomes.

Results: Neither number of missense mutations nor neoantigens correlated with clinical outcomes in CCOC. However, we found neoantigen frequency defined here as the number of neoantigens per missense mutation correlated with clinical outcomes. A cox multivariate regression analysis demonstrated that high neoantigen frequency correlated with decreased progression free survival (PFS) and was an independent prognostic factor (p=0.032). Immune-related genes including those related to effector memory CD8 T cell were dominantly expressed in tumors with low neoantigen frequency. This may suggest that CD8 T cell-mediated immunity is constantly eliminating immunogenic subclones expressing neoantigens (immunoediting). In contrast, we observed increased ratio of CTLA-4 and PD-1 to CD8A expression in tumors with high neoantigen frequency. This may imply that restricted antitumor immunity by immunoinhibitory mechanism result in the limited immunoediting and poor prognosis

Conclusions: Neoantigen frequency underlying immunoediting in CCOC is an independent prognostic factor for clinical outcome and may become a potential candidate biomarker for an immunomodulatory-based treatment.

Citation Format: Hirokazu Matsushita, Kosei Hasegawa, Katsutoshi Oda, Shogo Yamamoto, Akira Nishijima, Yuichi Imai, Kayo Asada, Yuji Ikeda, Takahiro Karasaki, Keiichi Fujiwara, Hiroyuki Aburatani, Kazuhiro Kakimi. Neoantigen frequency as an independent prognostic factor in patients with clear cell ovarian carcinoma (CCOC) [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 1706. doi:10.1158/1538-7445.AM2017-1706
Abstract 171: Targeting the tumor microenvironment: inhibition of carbonic anhydrase 9 impedes brain tumor initiating cell chemoresistance and delays glioblastoma growth in vivo
Authors: Boyd Nathaniel H. , Walker Kiera , McDonald Paul C. , Bevensee Mark O. , Gillespie Yancey G. , Nabors Burt , Dedhar Shoukat , Hjelmeland Anita B.
Published: July 2017

Brain tumor initiating cells (BTICs) are resistant to chemo- and radiotherapy, providing a reservoir for tumor recurrence and a desirable target for glioma treatments. Standard of care for glioblastoma (GBM; grade IV astrocytoma) includes the chemotherapeutic agent temozolomide, which prolongs life expectancy by months and is not curative. Prior studies suggested the efficacy of chemotherapies including temozolomide was increased by reducing expression of carbonic anhydrase 9 (CA9). CA9 is a hypoxia responsive gene elevated in tumors that is important for regulating intracellular pH and contributing to the acidic extracellular microenvironment. After confirming basal and hypoxia-induced expression of CA9 in GBM BTICs, we targeted CA9 activity with the small molecule inhibitor SLC-0111 alone or in combination with temozolomide. In multiple GBM BTIC lines, SLC-0111 reduced cell growth in vitro and showed additional benefit when used concurrently with temozolomide. Importantly, SLC-0111 inhibited the enrichment of BTICs after temozolomide treatment as determined via BTIC marker expression and neurosphere formation capacity. These data suggested the potential of SLC-0111 as a chemosensitizer, which we next evaluated in preclinical studies using a subcutaneous recurrent GBM model. GBMs treated with SLC-0111 in combination with temozolomide significantly regressed and the resulting in vivo growth delay was greater than that of temozolomide or SLC-0111 alone. Together, our data suggest that SLC-0111 can sensitize GBM BTICs to the chemotherapy temozolomide and significantly delay disease progression.

Citation Format: Nathaniel H. Boyd, Kiera Walker, Paul C. McDonald, Mark O. Bevensee, Yancey G. Gillespie, Burt Nabors, Shoukat Dedhar, Anita B. Hjelmeland. Targeting the tumor microenvironment: inhibition of carbonic anhydrase 9 impedes brain tumor initiating cell chemoresistance and delays glioblastoma growth in vivo [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 171. doi:10.1158/1538-7445.AM2017-171