bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract IA15: “Next-gen” genomics-based clinical trials
Authors: Siu Lillian L.
Published: May 2016

Next generation sequencing (NGS) technologies have gained increasing clinical applications as a strategy to screen and identify patients for genomic based clinical trials in oncology. Molecular characterization programs using NGS have been established in many cancer centers worldwide, including some that comprise part of nation-based efforts. With a growing number of patients whose tumors have undergone molecular profiling, there is an urgent need to justify these activities and translate genomic findings into clinically relevant outcome. Molecular profiling offers information beyond the histopathological classification of tumors, subdividing them into smaller genomic subsets ranging from those harboring common recurrent aberrations to others with rare variants of uncertain significance. The current era of genomic based clinical trials is populated by “umbrella” and “basket” trials which allocate patients with druggable genomic signatures to specific genotype-drug matched groups that remain histology-based (“umbrella” trials) or are histology-agnostic (“basket” trials). The pros and cons of these clinical trial designs, and their ability to bring the value of precision medicine to bear, will be discussed. Looking beyond the present clinical trials framework, it is unlikely that genomics is sufficient as a standalone strategy to fully characterize the complex molecular landscape of cancer. “Next-gen” clinical trials need to target the dynamic status of tumors and take into consideration intratumoral heterogeneity in their design. The extension of profiling algorithms to include transcriptomics and epigenetics is attractive to encompass other alterations that can lead to unchecked tumor growth. A systems biology approach should be considered to understand signaling pathway interactions and decipher mechanisms of oncogenic dependence, especially in tumors which lack readily actionable profiles. There are close interactions between the genome and the immunome such that NGS may inform the selection of patients most likely to benefit from immune-based therapies. “Next-gen” clinical trials must be capable of interrogating cancer in a multifaceted and dynamic manner in order to achieve substantial incremental benefits in therapeutic outcome.

Citation Format: Lillian L. Siu. “Next-gen” genomics-based clinical trials. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA15.
Abstract PR06: Precision medicine for newly diagnosed and refractory/recurrent pediatric cancer patients: Lessons learned from “N=1” studies
Authors: McEachron Troy A. , Zabokrtsky Keri B. , Sassoon Aaron F. , Nasser Sara , Izatt Tyler , Garner Chad P. , Craig David W. , Carpten John D. , Sender Leonard S.
Published: May 2016

Whole genome sequencing has rapidly emerged as the preferred method for unbiased genomic interrogation. Leveraging this technology, we have developed a precision medicine clinical research protocol for pediatric, adolescent, and young adult cancer patients. The primary objective of this study is to arrive at genome informed treatment recommendations for individual patients suffering from aggressive and/or worsening disease in a clinically relevant time frame. The second objective is to survey the genomic landscape of newly diagnosed and recurrent/refractory patients. To accomplish this, we have employed an interdisciplinary workflow including a customized bioinformatic analysis pipeline, contextual biological interpretation of the data, and case reviews during weekly molecular tumor board meetings. Here we report the results of having sequenced over 50 patients diagnosed with both common and rare malignancies. Factors affecting the successful identification of therapeutically actionable targets and subsequent intervention are varied. The most consistent findings are the increased mutation burden in patients who have received chemotherapy and/or radiation prior to tissue collection. Lastly, we highlight the realistic opportunities, challenges, and insight gained from an ongoing single institution experience.

This abstract is also presented as Poster 27.

Citation Format: Troy A. McEachron, Keri B. Zabokrtsky, Aaron F. Sassoon, Sara Nasser, Tyler Izatt, Chad P. Garner, David W. Craig, John D. Carpten, Leonard S. Sender. Precision medicine for newly diagnosed and refractory/recurrent pediatric cancer patients: Lessons learned from “N=1” studies. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr PR06.
Abstract A006: Analysis of immune checkpoint control protein co-expression in breast and ovarian cancer using novel rabbit monoclonal antibodies and multiplex IHC
Authors: Silver Matthew R. , Ziello Jennifer E. , Haack Herbert
Published: May 2016

With an increasing number of biomarkers, and often limited availability of biopsy material, there is a growing need for multiplexed assays for both research and clinical purposes. IHC based solutions are particularly attractive in the field of immuno-oncology, as maintaining special context within the tumor microenvironment provides meaningful, and potentially actionable, information. Immuno-assays with high specificity and sensitivity are a powerful tool; however, there are challenges associated with antibody based multiplexing when more than 2-3 markers are required. A multiplex IHC solution that bypasses antibody species/isotype concerns, while providing signal amplification, was used to examine the co-expression of immune checkpoint control proteins, such as PD-L1, B7-H3, B7-H4, VISTA, in breast and ovarian cancer.

Citation Format: Matthew R. Silver, Jennifer E. Ziello, Herbert Haack. Analysis of immune checkpoint control protein co-expression in breast and ovarian cancer using novel rabbit monoclonal antibodies and multiplex IHC. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A006.
Abstract A033: Phase Ia study of FoxP3+ CD4 Treg depletion by infusion of a humanized anti-CCR4 antibody, KW-0761, in cancer patients
Authors: Kurose Koji , Ohue Yoshihiro , Wada Hisashi , Doi Toshihiko , Nishikawa Hiroyoshi , Oka Mikio , Ueda Ryuzo
Published: May 2016

Purpose: FoxP3+ Tregs inhibit immune responses against tumors. KW-0761 is a humanized anti-human CCR4 monoclonal antibody (mAb) that has ADCC activity. Depletion of CCR4-expressing FoxP3+ CD4 Tregs by KW-0761 infusion was investigated in solid cancer patients.

Experimental Design: We conducted a phase Ia clinical trial of KW-0761 infusion in 7 lung and 3 esophageal cancer patients. Toxicity, clinical efficacy, changes in lymphocyte subpopulations, including Tregs, and induction of immune responses were analyzed.

Results: The results showed that KW-0761 infusion at dose range between 0.1 mg/kg to 1.0 mg/kg was safe and well tolerated. No DLT was observed. Four of 10 patients showed stable disease during treatment and were long survivors. The monitoring of FoxP3+ Tregs in the PBMCs during treatment indicated efficient depletion of those cells even at the lowest dose of 0.1 mg/kg used. The reduction in Th 1 CD4 T-cells and CD8 T-cells was limited, while a significant reduction was observed with Th 2 and Th 17 CD4 T-cells. Immune responses to cancer/testis (CT) antigens and an autoantibody response to thyroid peroxidase were observed in some patients.

Conclusions: The findings showed Tregs depletion and the possible occurrence of an immune response following KW-0761 infusion. Combined use of KW-0761 to deplete FoxP3+ Tregs with other immunotherapies, such as cancer vaccines or checkpoint inhibitors, is promising to augment immune responses.

Citation Format: Koji Kurose, Yoshihiro Ohue, Hisashi Wada, Toshihiko Doi, Hiroyoshi Nishikawa, Mikio Oka, Ryuzo Ueda. Phase Ia study of FoxP3+ CD4 Treg depletion by infusion of a humanized anti-CCR4 antibody, KW-0761, in cancer patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A033.
Abstract A059: Efficacy of anti-ICOS agonist monoclonal antibodies in preclinical tumor models provides a rationale for clinical development as cancer immunotherapeutics
Authors: Elpek Kutlu , Harvey Christopher , Duong Ellen , Simpson Tyler , Shu Jenny , Shallberg Lindsey , Wallace Matt , Sathy Sriram , Mabry Robert , Michaelson Jennifer , Briskin Michael
Published: May 2016

ICOS (inducible co-stimulator molecule) is a T cell encoded member of the extended B7/CD28 superfamily that is up-regulated upon early T cell activation. Previous studies have shown that higher ICOS expression on circulating T cells, post treatment with ipilimumab, is associated with better clinical outcome. In complementary pre-clinical studies, efficacy observed with a whole cell vaccine consisting of ICOS-L expressing B16 melanoma (IVAX) suggests that agonism of this pathway could provide therapeutic benefit in the cancer setting.

In accordance with these observations, we generated a panel of anti-ICOS monoclonal antibodies with in vitro agonist properties. A lead series of mAbs were shown to be efficacious as monotherapies in syngeneic tumors models with enhanced tumor inhibition observed in combination with anti-PD1. Mechanistic studies demonstrate that tumor regression is associated with enhanced ratios of cytotoxic CD8/regulatory T (Tregs) cells and that this change is related to preferential reduction in ICOS high Tregs in the tumor microenvironment.

These data provided the rationale for development of a high affinity humanized agonist monoclonal antibody to be tested in both mono therapy and in combination with other T cell checkpoints. Our lead anti-ICOS antibody is currently in IND-enabling studies and will be tested for activity in solid tumor indications.

Citation Format: Kutlu Elpek, Christopher Harvey, Ellen Duong, Tyler Simpson, Jenny Shu, Lindsey Shallberg, Matt Wallace, Sriram Sathy, Robert Mabry, Jennifer Michaelson, Michael Briskin. Efficacy of anti-ICOS agonist monoclonal antibodies in preclinical tumor models provides a rationale for clinical development as cancer immunotherapeutics. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A059.
Abstract A064: Modeling checkpoint blockade inhibitor resistant immunoregulation induced by squamous epithelial cancers
Authors: Mattarollo Stephen , Leggatt Graham , Wells James , Lambert Paul , Frazer Ian H.
Published: May 2016

Aim: Squamous epithelial cancers are immunogenic, as demonstrated by an increased risk of development in immunosuppressed patients. Some however evade host immunity, and establishing the mechanism of immune evasion could assist with squamous cancer immunotherapy.

Method: We have used a model of squamous epithelial pre malignancy, in which HPV16 E7 transgenic and hyperproliferative mouse skin is transplanted to naïve mice, and have explored local regulation of effector T cell function by E7 transgenic epithelial cells through manipulation of the graft donor immune system.

Results: E7 transgenic skin is not rejected by naïve recipient mice, or by mice immunised with E7 protein and expressing E7 specific cytotoxic T cells. E7 expressing hyper proliferative epithelium demonstrates caspase-1 activation, production of IL-1β, IL-18, and CCL2, and attraction and activation of NKT cells and mast cells. Rejection of E7 transgenic skin grafts can be observed following depletion from the donor skin of bone marrow derived cells, of NKT cells or of mast cells, whereas adjacent E7 transgenic grafts, otherwise unmanipulated, are not rejected. E7 graft rejection can also be induced by suppression in the donor skin or recipient animal of NKT cell secreted IL-17 or IFN-γ, or by depletion of the soluble IL-1 receptor IL-1R1 or blockade of Arginase activity in the recipient. Inhibition of graft rejection is not overcome by antibody to PD-1, PD-L1, or PD-L2. Hyperproliferative cervix epithelium in patients with CIN3 expresses the factors identified as inhibitory to E7 transgenic skin graft rejection, whereas skin from E7 transgenic mice with a mutated Rb gene preventing E7 induced epithelial proliferation lacks the inhibitory factors.

Conclusion: Epithelial hyper proliferation induces immunosuppressive mechanisms that locally prevent cytotoxic T cell function through mechanisms independent of checkpoint blockade. We hypothesise from our data that the local balance of production of IL-1β and Il-18 locally determines cytotoxic T cell effector function targeted at epithelial antigens.

Citation Format: Stephen Mattarollo, Graham Leggatt, James Wells, Paul Lambert, Ian H. Frazer. Modeling checkpoint blockade inhibitor resistant immunoregulation induced by squamous epithelial cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A064.
Abstract A098: Helicobacter-specific T cell responses in gut homeostasis and disease
Authors: Xu Mo , Littman Dan R.
Published: May 2016

The gastrointestinal tract is the major site for constant immunological challenge to the host immune system. The host must be able to mount protective immune responses against pathogenic micro-organisms while maintaining tolerance to most microbes under homeostatic conditions. A number of Helicobacter species, including Helicobacter hepaticus, have been isolated as the etiological agents of gastric ulcer disease or colitis in their respective hosts. H. hepaticus infection induces severe colitis in a number of immunocompromised mouse mutants, but interestingly does not usually cause disease in most WT mouse strains. Although Th17/Th1 responses are known to play an important role in H. hepaticus-mediated inflammation, how the immune response is contained such that it is harmless under homeostatic conditions and how perturbations lead to pathogenicity remain elusive questions. To answer these, it will be important to track H. hepaticus-specific T helper cells, which form a small proportion of total gut T helper cells, under homeostatic conditions. We have characterized TCRs specific for H. hepaticus antigens, which has allowed for development of TCR transgenic mice and MHCII tetramers to interrogate the specific T cell response that can contribute to inflammatory bowel disease in mouse models.

Citation Format: Mo Xu, Dan R. Littman. Helicobacter-specific T cell responses in gut homeostasis and disease. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A098.
Abstract A104: Eradication of large solid tumors in immunocompetent mice using dual specific CAR T cells and vaccination
Authors: Slaney Clare Y. , Westwood Jennifer A. , Beavis Paul A. , Darcy Phillip K. , Kershaw Michael H.
Published: May 2016

While adoptive cell transfer (ACT) using chimeric antigen receptor-expressing (CAR) T cells has led to remarkable complete responses in leukemia, complete responses of solid tumors in patients are few. Similarly, previous investigations in mice have demonstrated clearance of large burdens of disseminated cancer using CAR T cells, but large solid tumors (>1 cm2) have not responded fully. Here we report the generation of transgenic mice containing dual specific CAR T cells with specificity for Her2 and the melanoma-associated antigen pMEL (gp100), designated CARaMEL mice. In response to Her2-positive cancer cells, T cells from CARaMEL mice could secrete cytokines and engage in cytolysis mediated by an anti-Her2 CAR. Through their endogenous TCR, CARaMEL T cells could proliferate extensively in response to gp100. We demonstrate the ability of CARaMEL T cells in ACT incorporating vaccination (ACTIV) therapy to induce eradication of large tumors of various histologies, including orthotopic breast tumors, subcutaneous sarcoma and colon carcinoma liver tumors. Tumor models were performed in immunocompetent mice expressing human Her2 in normal brain and breast tissue. Effective ACTIV therapy included a lymphodepleting preparative regimen, ACT, high-dose IL-2 and recombinant vaccinia virus expressing gp100. ACTIV therapy was associated with expansion of dual-specific T cells and extensive tumor infiltration by CARaMEL T cells. Infiltration of CARaMEL T cells was also observed in normal tissues including brain. Long term surviving mice were totally resistant to rechallenge with Her2-expressing tumor cells, and partially resistant to Her2-negative tumor cells. We anticipate studies currently underway to provide mechanistic insight into the antitumor activity of ACTIV therapy.

Citation Format: Clare Y. Slaney, Jennifer A. Westwood, Paul A. Beavis, Phillip K. Darcy, Michael H. Kershaw. Eradication of large solid tumors in immunocompetent mice using dual specific CAR T cells and vaccination. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A104.
Abstract A145: Radiation therapy induces tumor-promoting immune suppression in the microenvironment of pancreatic carcinoma
Authors: Tomkötter Lena , Werba Gregor , Nguy Susanna , Alothman Sara , Alqunaibit Dalia , Tiwari Shaun , Ly Nancy Ngoc Giao , Daley Donnele , Ochi Atsuo , Barilla Rocky , Torres-Hernandez Alejandro , Pellicciotta Ilenia , Du Kevin , Miller George
Published: May 2016

Radiation therapy (RT) has shown marginal efficacy in patients with pancreatic ductal adenocarcinoma (PDA). Two of the past three randomized controlled trials investigating the efficacy of RT for patients with locally advanced unresectable PDA have shown a statistically significant worse survival by 40% or more for patients receiving RT. We postulated that RT reprograms inflammatory cells within the tumor microenvironment to an immune suppressive phenotype limiting the efficacy of RT in invasive PDA and accelerating disease progression in surrounding pre-invasive foci. We found that RT markedly accelerates the progression of pre-invasive PDA in a dose-dependent manner and reduces animal survival by more than 6 months. In both invasive and pre-invasive PDA, RT reprograms immunogenic macrophages towards an immune-suppressive M2 phenotype resulting in CD8 T cell scarcity and Th2 and Treg differentiation of CD4 T cells. Moreover, adoptive transfer of T cells harvested from RT-treated tumors accelerates tumor growth in recipient hosts. We show that M-CSF blockade concurrent with RT prevents immune-suppressive macrophage and T cell reprogramming and markedly enhances the efficacy of RT in PDA. These data suggest that targeting macrophage reprogramming can unleash the utility of RT for PDA.

Citation Format: Lena Tomkötter, Gregor Werba, Susanna Nguy, Sara Alothman, Dalia Alqunaibit, Shaun Tiwari, Nancy Ngoc Giao Ly, Donnele Daley, Atsuo Ochi, Rocky Barilla, Alejandro Torres-Hernandez, Ilenia Pellicciotta, Kevin Du, George Miller. Radiation therapy induces tumor-promoting immune suppression in the microenvironment of pancreatic carcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A145.
Abstract A161: Angiotensin II type 1 receptor antagonism suppresses tumor metastasis through inhibition of CCL2-CCR2 mediated monocyte recruitment
Authors: Regan Daniel P. , Guth Amanda M. , Hansen Ryan J. , Gustafson Daniel L. , Dow Steven W.
Published: May 2016

Purpose: Inflammatory monocytes have been shown to play key roles in cancer metastasis through promotion of tumor cell extravasation, seeding, growth, and angiogenesis, as well as suppression of anti-tumor immunity. Migration of inflammatory monocytes to sites of inflammation or tumor metastasis is mediated primarily via the action of the CCL2-CCR2 chemotactic axis. Thus, disruption of this axis through receptor or ligand blockade represents an attractive therapeutic target for the treatment of metastatic disease.

Methods: Losartan, an angiotensin II type 1 receptor (AT1R) antagonist, has been previously reported to suppress tumor growth through mechanisms associated with tumor angiogenesis and inhibition of TGF-β signaling. However, our studies also suggest an important contribution to the anti-tumor effects of losartan from monocyte migration inhibition. Therefore, we conducted studies in murine breast (4T1) and colon (CT26) carcinoma experimental pulmonary metastasis models, to determine the degree to which losartan's anti-metastatic effect was predominantly dependent on disruption of CCL2-CCR2 mediated monocyte recruitment to tumor metastases.

Results: We found that daily treatment with losartan prolonged survival, and significantly reduced metastatic burden, percentages of CD11b+/Ly6C+ lung monocytes, and tumor microvessel density in mice with 4T1 and CT26 pulmonary metastases. However, compared to pure CCR2 antagonists, our studies show that losartan does not act as a direct competitive antagonist to CCR2. Rather, in vitro assays and studies in CCR2 -\- mice indicated that losartan's anti-metastatic effect was primarily mediated via suppression of monocyte recruitment through indirect interruption of the CCL2-CCR2 axis. Importantly, losartan proved more effective at reducing monocyte recruitment and slowing CT26 pulmonary metastatic tumor growth compared to a specific CCR2 antagonist.

Conclusions: We conclude therefore that losartan exerts significant anti-metastatic activity in aggressive murine metastasis models, and that losartan's major anti-metastatic effect is mediated by blockade of CCL2-CCR2 dependent monocyte recruitment. Thus, by virtue of its myeloid cell activity, losartan, in combination with conventional cancer therapeutics, has potential for use as an anti-metastatic agent in animals and humans at high risk for tumor metastasis.

Citation Format: Daniel P. Regan, Amanda M. Guth, Ryan J. Hansen, Daniel L. Gustafson, Steven W. Dow. Angiotensin II type 1 receptor antagonism suppresses tumor metastasis through inhibition of CCL2-CCR2 mediated monocyte recruitment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A161.
Abstract A162: Cancer-specific T cell receptor isolation for cancer immunotherapy
Authors: Ryan Ruth , Hibbert Linda , Weigand Luise , Paston Samantha , Baker Debbie , Donnellan Zoe , Clark Vanessa , Hale Kathy , Conlon Louise , Dukes Joseph , Boudousquie Caroline , Bossi Giovanna , Hickman Emma , Powlesland Alex , Vuidepot Annelise , Hassan Namir , Jakobsen Bent
Published: May 2016

Malignant cells may be recognised by T cells binding cell surface Class I HLA (Human Leukocyte Antigen)-peptide complexes presenting disease-associated epitopes. Many cancer patients have been shown to generate CD8 cytotoxic T cell responses to tumour-associated antigens. However, this is often insufficient for the immune system to clear tumours, resulting in progression of cancer. This is in part due to the low avidity of these T cells, as well as the ability of cancer cells to develop escape mechanisms to avoid destruction by T cells. To overcome these issues, we have engineered novel, bi-functional protein therapeutics termed ImmTACs (Immune mobilising monoclonal TCR Against Cancer) which re-direct the immune system to target and destroy tumour cells with a high degree of potency and specificity. An ImmTAC comprises a high affinity ‘monoclonal’ T cell receptor (mTCR) targeting a cancer-associated HLA-peptide complex, fused to an anti-CD3 scFv domain which activates an anti-tumour T cell response.

In order to produce ImmTACs, we have developed an integrated in-house process leading to the isolation of TCRs specific for validated cancer epitopes. The critical steps in this process are: antigen selection, epitope identification, T cell cloning, TCR isolation and binding to soluble peptide:MHC on Biacore. High affinity ImmTACs are then generated through a process of affinity maturation. We describe the steps leading to cloning of wild type TCRs and present data to illustrate the successful isolation of TCRs as a result of this procedure.

Citation Format: Ruth Ryan, Linda Hibbert, Luise Weigand, Samantha Paston, Debbie Baker, Zoe Donnellan, Vanessa Clark, Kathy Hale, Louise Conlon, Joseph Dukes, Caroline Boudousquie, Giovanna Bossi, Emma Hickman, Alex Powlesland, Annelise Vuidepot, Namir Hassan, Bent Jakobsen. Cancer-specific T cell receptor isolation for cancer immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A162.
Abstract B003: Developing an analytical method for second messenger cyclic GMP-AMP
Authors: Li Tuo , Chen Zhijian J.
Published: May 2016

The presence of double-stranded DNA (dsDNA) in the cytosol is a danger signal associated with microbial infections and tumor microenvironment. The cytosolic DNA surveillance pathway (CDSP) is critical for sensing such signal to elicit type I interferon response, which facilitates infection clearance and tumor rejection. Uncontrolled engagement of CDSP is also linked to autoimmune disease. The key components of CDSP are the DNA sensor—cyclic GMP-AMP synthase (cGAS), the second messenger—cyclic GMP-AMP (cGAMP), and the endoplasmic reticulum localized adaptor—stimulator of interferon inducible genes (STING). While numerous efforts, including ours, are focusing on developing STING agonists formulated cancer vaccine, an analytical method that is sensitive and reliable for cGAMP and other cyclic dinucleotides is also in high demand to enable quantitative investigations. In this work, we developed and optimized a rapid quantification workflow that integrates solid phase extraction, hydrophilic liquid chromatography and multiple reaction monitoring mass spectrometry. This workflow enables detection of cGAMP in cells at sub-femtomole levels and is amenable for scaling up for high throughput analysis. A biochemically synthesized nonradioactive isotope labeled cGAMP standard enables absolute quantification. As a proof of principle, this method is first applied to probe cGAMP in macrophages infected with Mycobacterium tuberculosis, and to help establish cGAS as the innate immune sensor for the pandemic pathogen. Second, constitutive levels of cGAMP in organs of autoimmune mouse models, of Trex1 or Dnase2 deficiencies, are quantified. The result indicates a direct engagement of the cGAS-cGAMP-STING pathway in the autoimmune pathology. Finally, we show that cGAMP is produced when the apoptosis program is initiated in conjunction with pro-apoptotic caspase inhibition. This shows that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response and to inactivate this response in a caspase-dependent manner. Altogether, these studies demonstrated the usefulness of this analytical method for quantifying cGAMP in a high dynamic range under different biological contexts. We are currently applying this method to measure the activation of the cGAS pathway in tumor immunity and immunotherapy.

Citation Format: Tuo Li, Zhijian J. Chen. Developing an analytical method for second messenger cyclic GMP-AMP. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B003.
Abstract B005: An immunohistochemical PD-L1 companion diagnostic assay for treatment with durvalumab (MEDI4736) in NSCLC patients
Authors: Rebelatto Marlon C. , Mistry Amita , Sabalos Constantine , Schechter Nicole , Walker Jill , Midha Anita , Steele Keith E. , Robbins Paul B. , Li Xia , Shi Li , Blake-Haskins John A. , Ibrahim Ramy A. , Richman Laura
Published: May 2016

Background: A high quality PD-L1 companion diagnostic may help predict which patients are more likely to respond to PD-1/PD-L1 antibody-based therapy. Here we describe a PD-L1 immunohistochemical (IHC) diagnostic test developed by Ventana Medical Systems for use with durvalumab.

Methods: An anti-human PD-L1 rabbit monoclonal antibody (SP263) was optimized for use with Ventana OptiView DAB IHC Detection Kit on the automated BenchMark ULTRA platform. The PD-L1 IHC assay was validated for use in formalin-fixed, paraffin-embedded samples of NSCLC in a series of studies addressing sensitivity, specificity, robustness, and precision. Durvalumab is a human IgG1 mAb that blocks PD-L1 binding to PD-1 and CD80 with high affinity and selectivity. A subset of clinical trial samples from a Phase 1/2 study of durvalumab (NCT01693562) was analyzed to determine optimal cut-off for enriching response to durvalumab. Inter-reader precision was established by 3 pathologists who evaluated 81 NSCLC samples across the range of expression levels.

Results: The Ventana PD-L1 IHC (SP263) assay met all pre-defined acceptance criteria. The scoring algorithm was defined using statistical analysis of clinical response data and PD-L1 staining parameters observed in a set of NCT01693562 clinical trial samples. Samples of both cancer types are considered test positive when the membrane of ≥ 25% of tumor cells stain for PD-L1 at any intensity. Inter-reader precision in determining PD-L1 status resulted in an overall percentage agreement of 97% for NSCLC. PD-L1+ patients identified by the scoring algorithm had a higher response rate than PD-L1- patients. Interlaboratory testing was performed at 3 external laboratories and demonstrated an overall agreement rate of 86.4%.

Conclusions: These results highlight the robustness and reproducibility of the PD-L1 IHC (SP263) assay in a clinical setting. In NSCLC patients treated with durvalumab, PD-L1+ patients identified by the scoring algorithm had a higher response rate than PD-L1- patients. The clinical utility of the PD-L1 diagnostic assay will be further validated in a prospective manner using additional patients in this study and in other durvalumab studies.

Citation Format: Marlon C. Rebelatto, Amita Mistry, Constantine Sabalos, Nicole Schechter, Jill Walker, Anita Midha, Keith E. Steele, Paul B. Robbins, Xia Li, Li Shi, John A. Blake-Haskins, Ramy A. Ibrahim, Laura Richman. An immunohistochemical PD-L1 companion diagnostic assay for treatment with durvalumab (MEDI4736) in NSCLC patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B005.
Abstract B018: Developmental and functional characterization of immunoglobulin kappa chain-bridged single chain bi-specific antibody for targeted antitumor therapy
Authors: Lo Yu Hsun , Yu Jei-Hwa , Hsui Yu-Hsen , Yuan Ta-Tung
Published: May 2016

The immune system has routinely been the focus of cancer treatments. One of the strategies that targets the immune system is by use of monoclonal antibodies to specifically target tumor antigens leading to immune activation and tumor destruction. Therefore, many biopharmaceutical companies are interested/invested in antibody development, including the development of next-generation antibodies such as bispecific antibodies. The Development Center for Biotechnology (DCB) in Taiwan has developed a new platform for the generation of immunoglobulin kappa chain-bridged single chain bispecific antibody. Using this platform, DCB constructed a kappa domain-bridged HER2/CD3 bispecific single chain antibody known as the LBscbHER2-CD3. Utilizing a mammalian expression system, the antibody can be easily purified by affinity chromatography via kappa fragment capture. Binding affinity studies have revealed that the purified LBscbHER2-CD3 antibody is able to independently as well as simultaneously bind to HER2+ tumor cells and CD3+ T cells at low nanomolar concentrations. In co-culture experiments, the addition of purified LBscbHER2-CD3 antibody resulted in a significant increase in IL-2 production which is a strong indicator of human T cell activation. In addition, the co-engagement of either IL-2 primed or un-primed human T cells and HER2+ tumor cells by the LBscbHER2-CD3 antibody facilitated the killing of tumor cells from breast, colon and pancreatic cancers. Lastly, when administered in vivo, LBscbHER2-CD3 showed strong tumor growth inhibition in SCID mice xenograft models with implanted HER2+ SW480 cancer cells, HT29 human colon carcinoma cells, and Capan-1 human pancreatic cancer cells. In conclusion, the LBscbHER2-CD3 bispecific antibody has strong affinity for both the cancer cell-expressing receptor and the T cell antigen which leads to cancer cell elimination in vitro and in vivo. These results suggest that LBscbHER2-CD3 may have therapeutic potential for drug development in the treatment of human cancers.

Citation Format: Yu Hsun Lo, Jei-Hwa Yu, Yu-Hsen Hsui, Ta-Tung Yuan. Developmental and functional characterization of immunoglobulin kappa chain-bridged single chain bi-specific antibody for targeted antitumor therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B018.
Abstract B038: GMP production of cord blood-derived dendritic cell-based vaccine to prevent relapses after hematopoietic cell transplantation in children with AML
Authors: Haar Colin de , Dunnebach Ester , Til Niek van , Plantinga Maud C. , Blokland Nina JG , Nierkens Stefan , Boelens Jaap Jan
Published: May 2016

Introduction: Pediatric patients with refractory/relapsed acute myeloid leukemia (AML) have only one treatment option: hematopoietic cell transplantation (HCT). Using cord blood (CB)-derived stem cells, instead of cells from bone marrow cells or peripheral blood, results in less relapses (increased anti-tumor reactivity) and less graft-versus-host disease (increased safety). Although this treatment is potentially curative, still more than half of the children die from relapses. We therefore aim to develop additional powerful and safe CB-derived immunotherapies for pediatric AML.

Methods: We have successfully translated our pre-clinical protocol for generations of a dendritic cell (DC) vaccine from CD34+ CB stem cells into GMP production. This production process enables us to generate sufficient CBDC for at least 4 vaccinations when using the 20% fraction of the CB unit. After CliniMACS isolation the CD34+ cells are expanded in culture bags using medium containing FLT3L, SCF, IL-3 and IL-6. When sufficient expansion is reached the cells are differentiated into DCs using medium containing FLT3L, SCF, GM-CSF and IL-4. The CBDCs are then matured using proinflammatory cytokines and electroporated with Wilms' Tumor 1 (WT1) mRNA as tumor antigen. After 4 hours recovery, cells are cryopreserved until time of validation or intradermal vaccination

Results: CD34+ CB stem cells were expanded and differentiated into DCs. CBDCs upregulated co-stimulatory molecules after maturation and showed enhanced CCR7-dependent migration towards CCL19 in a trans-well migrations assay. In addition, CBDCs expressed the tumor antigen Wilms' Tumor 1 (WT1) protein after electroporation with WT1-mRNA. These WT1 expressing CBDCs were not only able to stimulate T cells in a mixed lymphocyte reaction but in an antigen-specific setting as well.

Conclusions: We are able to produce a GMP CBDC vaccine with the goal to stimulate the anti-tumor reactivity of the newly developing immune system in AML patients after CB-HCT in a Phase 2 clinical trial starting early 2016.

Citation Format: Colin de Haar, Ester Dunnebach, Niek van Til, Maud C. Plantinga, Nina JG Blokland, Stefan Nierkens, Jaap Jan Boelens. GMP production of cord blood-derived dendritic cell-based vaccine to prevent relapses after hematopoietic cell transplantation in children with AML. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B038.
Abstract B051: Control of regulatory T (Treg) cell function by protein kinase C-eta (PKCη): A novel target for cancer immunotherapy
Authors: Pedros Christophe , Kong Kok-Fai , Altman Amnon
Published: May 2016

Modulating regulatory T (Treg) cell functions to specifically promote anti-tumor immune responses is a desirable strategy for cancer immunotherapy. Blockade of checkpoint molecules, CTLA-4 and PD-1, has proven clinical efficacy in advanced metastatic melanoma and other malignancies. We recently found that protein kinase C-eta (PKCη), a member of the PKC enzyme family, physically associated with CTLA-4, an inhibitory receptor highly expressed on Treg cells, which is important for Treg cell-mediated suppression. PKCη is recruited to the Treg cell immunological synapse, and mediates contact-dependent suppression by Treg cells. PKCη-deficient (Prkch-/-) Treg cells are defective in the suppression of anti-tumor immunity against B16 melanoma but retain the ability to control the development of autoimmune colitis. Here, we extended our findings regarding the importance of the CTLA-4-PKCη complex in contact-dependent Treg cell suppressive activity to an additional tumor model, i.e., the slow growing prostate adenocarcinoma TRAMP-C1, indicating that the novel CTLA-4-PKCη signaling pathway may be relevant to a broad spectrum of solid cancers. We further found that in the tumor microenvironment, the loss of PKCη in Treg cells substantially promoted the infiltration of CD4+ and CD8+ effector T cells without affecting the Treg cell numbers. As a result, there was a significant increase in the intratumoral CD8+ to Treg cell ratio, a parameter considered to be predictive of a better clinical outcome. Thus, our data reveal that the novel CTLA-4-PKCη signaling pathway, which we identified, is critical for suppressing anti-tumor immunity and, hence, implicate this pathway as an attractive cancer immunotherapy target for a broad spectrum of clinically debilitating malignancies.

Citation Format: Christophe Pedros, Kok-Fai Kong, Amnon Altman. Control of regulatory T (Treg) cell function by protein kinase C-eta (PKCη): A novel target for cancer immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B051.
Abstract B063: Unsaturated fatty acids cause CD8+ T cell exhaustion in melanoma
Authors: Cui Guoliang , Kaech Susan
Published: May 2016

Remarkable progress in cancer immunotherapy has revealed the power of harnessing immunity to combat cancer. However, the immunosuppressive nature of the tumor microenvironment remains a fundamental challenge to improving immune-based anti-cancer therapies. We aim to develop new immunotherapies by studying the nutrient utilization of immune cells in the tumors. Using an inducible Braf/Pten melanoma model, we found that tumors contain higher amounts of available free fatty acids (FFAs) and lower amounts of glucose than the blood. Exposure to high concentration of FFAs induced an exhaustion-like phenotype of CD8+ T cells. Furthermore, mice deficient of FFA transporter CD36 developed smaller tumors compared to wild-type control mice. Tumor-infiltrating Cd36-/- CD8+ T cells expressed lower level of inhibitory receptors, such as PD-1 and LAG-3. This study highlights an important role of lipid metabolism in regulating T cell exhaustion in the tumor microenvironment and reveals CD36 as a potential drug candidate for immunotherapy.

Citation Format: Guoliang Cui, Susan Kaech. Unsaturated fatty acids cause CD8+ T cell exhaustion in melanoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B063.
Abstract B067: Successful immunotherapy of cancer stem cells requires memory and the adaptive immune system of the host
Authors: Schmidt Patrick , Desbaillets Nicolas , Dessen Pierre , Thompson Nancy , Huelsken Joerg , Jäger Dirk
Published: May 2016

The Cancer Stem Cell (CSC) hypothesis puts a, not necessarily rare, subpopulation of cells on the top of a tumor forming hierarchy, pointing to the importance of their destruction when aiming on a final cure. However, current studies targeting CSCs rather focus on the tumor initiating capacities of these cells than on their maintaining capacity in an established lesion. In this presentation we describe an immunotherapeutic approach of targeting the minor CSC subfraction by exploiting the stem cell related wnt-pathway. Within a fully syngeneic mouse tumor model, we labelled 4T1 breast tumor cells by lentiviral transduction to express a foreign antigen in wnt-active cells. When transferred into the host, these cells recapitulated CSC behavior and were concomitantly targetable by chimeric antigen receptor engineered T cells. All mice receiving a CSC specific CAR treatment showed long term metastases free survival, although a minority of cells served as actual aim of treatment. We investigated the consequences of CSC eradication at different time points after treatment and found that a memory effect, provided by engineered T cells, is essential to keep the re-formation of CSCs from bulk tumor cells under control. Furthermore, our treatment setting is able to re-activate the host's non engineered T cells against the majority of tumor cells, which leads to a long lasting tumor-free survival in our mouse model system. These findings implicate a leading role for immunotherapy when targeting the tumor stem cell fraction.

Citation Format: Patrick Schmidt, Nicolas Desbaillets, Pierre Dessen, Nancy Thompson, Joerg Huelsken, Dirk Jäger. Successful immunotherapy of cancer stem cells requires memory and the adaptive immune system of the host. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B067.
Abstract B074: Tumor-reactive CD4+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy
Authors: Pelletier Sandy , Turcotte Simon
Published: May 2016

Purpose: To evaluate whether patients with metastatic gastrointestinal adenocarcinomas refractory to chemotherapy harbor tumor-reactive CD4+ T cells.

Experimental Design: Expansion of CD4+ tumor-infiltrating lymphocytes (TIL) and cancer cell lines was performed from gastrointestinal cancer metastases in 5 patients for the study of antitumor immune recognition. Retroviral transduction of genes encoding the class II, major histocompatibility complex, transactivator (CIITA) was used to induce the expression of major histocompatibility complex (MHC) class II in tumor cell lines. Recognition of autologous tumor cell lines by TIL was evaluated by up-regulation of 4-1BB and OX40 by flow cytometry, and/or secretion of IFNg by ELISA.

Results: TIL were expanded from metastases, and new tumor cell lines were generated in 5 patients. Retroviral transduction of CIITA in tumor cell lines effectively induced expression of MHC class II in >80% of cells. Autologous tumor recognition was found in CD4+ TIL from 2 of these 5 patients. In a patient with gastric cancer liver metastases, tumor-reactive CD4+OX40+ TIL were cell-sorted from a TIL cell line. These cells up-regulated OX40 in the presence of all 4 autologous cancer cell lines albeit at different levels, but they did not produce IFNg. This recognition was specifically abolished by pan-MHC class II blocking antibodies. CD4+ TIL clones have been isolated and are being further characterised. Interestingly, tumor-reactive CD8+ TIL were previously identified and characterized in this patient. In a second patient with colon cancer abdominal wall metastases, tumor-reactive CD4+OX40+ TIL were cell-sorted from a TIL cell line. These cells were reactive to all 4 autologous cancer cell lines as they up-regulated OX40 and secreted IFNg. Recognition was blocked by anti-HLA-DR blocking antibodies. CD4+ TIL clones have been isolated and are being further characterized. In the near future, we expect to identify the TCR and HLA restriction element for both patients. We will also determine if CD4+ TIL clones have cross-reactivity against allogeneic HLA-matched gastrointestinal tumor cell lines.

Conclusions: This study provides a basis for the development of immunotherapy for patients with advanced gastrointestinal malignancies by first establishing the presence of naturally occurring tumor-reactive CD4+ TIL at the molecular level.

Citation Format: Sandy Pelletier, Simon Turcotte. Tumor-reactive CD4+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B074.