bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A141: The vigorous immune microenvironment of microsatellite instable colon cancer isbalanced by multiple counter-inhibitory checkpoints
Authors: Llosa Nicolas J. , Housseau Franck , Wick Elizabeth , Lizzy Hechenbleikner , Cruise Michael , Anders Robert , Sears Cynthia , Pardoll Drew M. , Fan Hongni , Siegel Nicholas , Blosser Lee , Tam Ada , Wang Hao
Published: May 2016

We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+ CTL as well as activated Th1 cells characterized by IFN-gamma production and the Th1 transcription factor Tbet. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly up-regulated expression of multiple immune checkpoints, including five - PD-1, PD-L1, CTLA-4, LAG-3 and IDO - currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of CRC. Our findings are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair defective subset of CRC selectively up-regulates at least 5 checkpoint molecules that are targets of inhibitors currently being clinically tested. Furthermore, our results were clinically validated in a phase 2 study at Hopkins which showed mismatch-repair status as a predictor of clinical benefit to immune checkpoint blockade with pembrolizumab.

Citation Format: Nicolas J. Llosa, Franck Housseau, Elizabeth Wick, Hechenbleikner Lizzy, Michael Cruise, Robert Anders, Cynthia Sears, Drew M. Pardoll, Hongni Fan, Nicholas Siegel, Lee Blosser, Ada Tam, Hao Wang. The vigorous immune microenvironment of microsatellite instable colon cancer isbalanced by multiple counter-inhibitory checkpoints. [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 A141.
Abstract A155: Unmasking cancer to innate sensing by anti-CD47 antibody triggers adaptive immune-mediated destruction of immunogenic tumors
Authors: Xu Meng
Published: May 2016

Blocking macrophage phagocytosis of tumor cells by anti-CD47 antibody (Ab) has demonstrated therapeutic efficacy in xenograft models. We have observed that in an immunocompetent host, a CTL adaptive response is essential for immungenic tumor regression after anti-CD47 Ab treatment. Furthermore, anti-CD47 Ab treatment promotes DC but not macrophage-mediated cross priming of CTLs in a type I IFN dependent fashion. In addition, we found that anti-CD47 Ab-mediated immunity requires the functional adaptor protein STING, but not MyD88, suggesting that cytosolic sensing of DNA from tumor cells is enhanced by anti-CD47 Ab treatment futher bridging the innate and adaptive responses. Importantly, we observed that the timing of standard chemotherapeutics impacts anti-CD47 Ab-initiated anti-tumor immunity. Together, our study reveals that anti-CD47 antibody can trigger DNA sensing pathway for adaptive immune-mediated destruction of immungenic tumors.

Citation Format: Meng Xu. Unmasking cancer to innate sensing by anti-CD47 antibody triggers adaptive immune-mediated destruction of immunogenic tumors. [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 A155.
Abstract A158: MAGED4B specific peptides: Short or long or combination?
Authors: Lim Kue Peng , Chun Ai Leng , Ponniah Sathibalan , Cheong Sok Ching
Published: May 2016

Immunotherapy has emerged as one of the most efficacious form of cancer therapy. We have previously demonstrated MAGED4B short peptides are efficacious in inducing patients' immune responses in vitro. However, effective vaccination against tumours requires generation of both CD8+ and CD4+ T-cell responses. A critical role of CD4+ helper T cells in the development of CD8+ tumour-specific effector function has been shown by numerous studies. In this pilot study, we aim to compare the efficacy of MAGED4B specific-short peptides (SP), long peptide (LP) and the combination of both short and long peptides in inducing patients' immune responses in vitro. Briefly, peripheral blood mononuclear cells (PBMC) from HLA-A2 positive healthy volunteers were incubated with 10 μg/ml of peptide in the presence of IL-2 and IL-7 for 7 days; peptide exposed PBMC were then incubated with cancer cells expressing MAGED4B overnight. Capability of T cells to secrete IFN-γ was accessed using ELISPOT assay and CEF peptides pool was used as positive control. Our data demonstrated that all MAGED4B peptides (short and long) are capable to induce similar and/or higher cytokine secretion compared to CEF peptides pool suggesting that these peptides are highly immunogenic. MAGED4B specific SP are better in inducing cytolytic activity compared to MAGED4B specific LP; however when both LP and SP were mixed as a cocktail, we observed a reduction in cytokine secreting T cells when the immune cells were co-incubated with MAGED4B expressing cell line, advocating an inhibition instead of synergizing reaction. In summary, our data has corroborated our previous finding that HLA-A2 restricted MAGED4B specific SP are immunogenic. However, we did not observed a synergistic event when SP and LP were used in a cocktail, questioning the combinatorial effect of CD4+ and CD8+ T cells. These preliminary findings would need to be validated in a bigger cohort of patients.

Citation Format: Kue Peng Lim, Ai Leng Chun, Sathibalan Ponniah, Sok Ching Cheong. MAGED4B specific peptides: Short or long or combination? [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 A158.
Abstract A169: NY-ESO-1 protein cancer vaccine with TLR 3 and 4 agonists
Authors: Takeoka Tomohira , Nagase Hirotsugu , Miyazaki Yasuhiro , Takahashi Tsuyoshi , Kurokawa Yukinori , Makino Tomoki , Yamasaki Makoto , Takiguchi Shuji , Mori Masaki , Doki Yuichiro , Wada Hisashi
Published: May 2016

NY-ESO-1 is expressed in wide range of human malignancies expect testis in normal tissues and induces spontaneous antibody in patients with cancers expressing NY-ESO-1. Because of its unique expression and high immunogenicity, NY-ESO-1 is promising as a target of cancer vaccine. We have conducted serial clinical trials with the NY-ESO-1 protein since 2004 and antigen-specific immune responses were observed in most of patients immunized. Some patients showed feasible clinical responses however they were limited and novel immune modulators which could make NY-ESO-1 protein vaccination effective are needed. Recently, stimulation through the Toll-like receptors (TLRs) enhance the induction of antigen-specific immune responses upon cancer vaccine has become apparent. In this study, we conducted a phase I cancer vaccine clinical trial using NY-ESO-1 protein combined with Poly ICLC and Picibanil OK-432 as TLR 3 and 4 agonist, respectively.

This trial was designed to evaluate the safety, induction of immune response and clinical response. Patients with advanced cancers expressing NY-ESO-1 were eligible. 200μg NY-ESO-1 protein mixed with Montanide in combination of Poly ICLC and/or Picibanil OK-432 was administered subcutaneously once every 2 weeks in six times. Seven patients with esophageal cancer, three with gastric cancer, two with lung cancer, two with ovarian cancer and one with malignant melanoma were enrolled in four groups, i) NY-ESO-1 protein alone (n=3), ii) NY-ESO-1 protein with OK-432 (n=3), iii) NY-ESO-1 protein with Poly ICLC (n=3), iv) NY-ESO-1 protein with OK-432 and Poly ICLC (n=6). All patients developed an injection-site reaction and fever (grade1 or 2) but no severe adverse events related to the drug were observed. An increase in the NY-ESO-1 antibody response was observed in all patients after vaccination. Earlier induction and higher frequency of NY-ESO-1 responsible T cells were observed in patients immunized with Poly ICLC than those without Poly ICLC. NY-ESO-1 protein vaccine with Poly ICLC and OK432 was safe and antigen-specific immune responses were induced in all patients. TLR agonists would be useful and effective immune modulators for cancer vaccines.

Citation Format: Tomohira Takeoka, Hirotsugu Nagase, Yasuhiro Miyazaki, Tsuyoshi Takahashi, Yukinori Kurokawa, Tomoki Makino, Makoto Yamasaki, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Hisashi Wada. NY-ESO-1 protein cancer vaccine with TLR 3 and 4 agonists. [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 A169.
Abstract A179: Ex vivo generation of clinical grade T cells by using activating and propagating feeder cells to cross-link T cell receptor
Authors: Jena Bipulendu , Taguchi Hiroaki , Nowicka Aleksandra , Korngold Ana B. , Huls Helen , Cooper Laurence JN
Published: May 2016

Success of T-cell based immunotherapy depends largely on exogenous modification and propagation of patient or donor derived T cells. The conventional paradigm is to stimulate primary T cells cross-linking endogenous CD3-complex with a monoclonal antibody (mAb) such as CD3ε targeting OKT3 alone or in combination with super-agonist mAb anti-CD28. The crosslinking via agonist mAb(s) lead to downstream activation signaling, phosphorylation of immunotyrosine activation motif and associated Ca2+ influx resulting in T-cell division and proliferation in vitro. We report an alternate approach to targeting T-cell signaling complex by cross-linking αβ-TCR of T cells through a novel TCR-specific single chain antibody. Towards this objective, first we generated several TCR targeted monoclonal antibodies. Next, we designed a panel of high affinity scFvs derived from a mAb clone A79 from which a specific mutant named SAYON-15 (Single chain AffinitY Optimized Novel mutant 15) possessing highest affinity towards TCR α-chain was selected. Affinity enhancement was achieved in silico via homology modeling and scanning mutations in the original scFv (A79). SAYON-15 in combination with either truncated human CD8 transmembrane or truncated human IgG1-Fc chain scaffold was expressed on K562 (HLA C-neg) cells. As activating and propagating feeder cells K562 HLA C-neg expressing SAYON-15 could drive expansion of αβ TCR+ T cells from donor derived peripheral or umbilical cord blood mononuclear cells in comparable fashion to gene modified K562 cells loaded with CD3ε specific mAb (OKT3). Ex vivo propagated T cells growth kinetics, phenotypic composition, TCR repertoire were studied and compared between two separate T-cell activation strategies described here. We analyzed ex vivo cultured T-cells immune repertoire with respect to TCRV gene rearrangement, clonal diversity, CDR3 composition by next generation sequencing. SAYON-15 expressing AaPc when compared with OKT3 expressing AaPC showed greater clonality, equivalent level of functional V-gene rearrangement, CDR3 chain length and V-J combinations. In summary, we report a novel TCR-specific scFv and harnessed its ability to numerically expand T cells ex vivo. This could potentially be used for clinical applications in the context of adoptive cell therapy.

Citation Format: Bipulendu Jena, Hiroaki Taguchi, Aleksandra Nowicka, Ana B. Korngold, Helen Huls, Laurence JN Cooper. Ex vivo generation of clinical grade T cells by using activating and propagating feeder cells to cross-link T cell receptor. [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 A179.
Abstract A181: Dissecting the spatiotemporal functions of WAVE complex during chemotaxis using CRISPR and optogenetics
Authors: Graziano Brian R. , Weiner Orion D.
Published: May 2016

Neutrophils comprise a crucial component of the innate immune system and rely on chemotaxis to track down, engulf, and destroy pathogens. However, neutrophils also migrate to sites of developing tumors where they may promote angiogenesis and set the stage for metastasis to occur. The WAVE regulatory complex (WRC) serves a central role in mediating chemotaxis, as it links the actin dynamics powering cell movement to the signaling cascade which responds to environmental stimuli. Actively migrating neutrophils must respond quickly (within seconds) to changes in their environment; unfortunately, traditional methods one may use to alter WRC activity (e.g, RNAi and gene deletion) require hours/days to take effect. Consequently, while we know that WRC is generally required for efficient chemotaxis, its specific functions during each temporal phase, such as establishment and maintenance of a chemotactic response, remain unknown. Furthermore, the roles of WRC in this process are convoluted by additional actin assembly and assembly-promoting factors that act alongside WRC to drive neutrophil movement: Cells may compensate for prolonged loss of WRC through altering the activities of one or more of these factors.

Here, I employed CRISPR to knockout the HEM1 subunit of WRC in neutrophil-like HL60 cells. Lacking functional WRC, these cells exhibit impaired chemotaxis, similar to cells where HEM1 expression is knocked-down. Unexpectedly however, the knockout neutrophils display additional defects in morphology and actin organization that are never observed in knockdown cells, despite knockdown cells containing less than 10% of normal Hem1 levels. I have also, in parallel, generated and validated optogenetic tools that can be used to rapidly and reversibly recruit WRC to different subcellular locations. When expressed in neutrophils lacking HEM1, these tools will open up the possibility of acutely ‘activating’ or ‘inactivating’ WRC during different temporal stages of chemotaxis (e.g., polarity establishment or maintenance). Ultimately, a more nuanced understanding of neutrophil chemotaxis should lead to the identification of new drug targets that may be used to block the accumulation of neutrophils at sites of tumor growth.

Citation Format: Brian R. Graziano, Orion D. Weiner. Dissecting the spatiotemporal functions of WAVE complex during chemotaxis using CRISPR and optogenetics. [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 A181.
Abstract A193: Bioengineered Dectin-1 CAR+ T cells to control invasive fungal infection
Authors: Kumaresan Pappanaicken R. , Albert Nathaniel , Singh Harjeet , Olivares Simon , Maiti Sourindra N. , Mi Tiejuan , Huls Helen , Champlin Richard E. , Kontoyiannis Dimitrios P. , Cooper Laurence J.N.
Published: May 2016

Myelosuppressive and ablative regimens are used to deplete lymphocytes in some patients before adoptive cell therapy. CD19-specific engineered T-cells have been used successfully to eliminate B-cells in patients with B-cell leukemias and lymphomas. Prolonged immunosuppression, including B-cell aplasia is associated with increased risk of invasive fungal infection (IFI). Thus, patients will benefit if bio-engineered T cells can control IFI as well as kill tumor cells. The Sleeping Beauty gene transfer system was used to render T cells to express a chimeric antigen receptor (CAR) on the cell surface to redirect specificity via Dectin-1. Upon binding with the β-1,3-glucan sugar moiety found in IFI, Dectin-1 CAR+ T cells signal via chimeric CD28 and CD3-ζ signaling domain. The activated T cells secrete perforin, granzyme and granulysin to damage hyphae and thus inhibit the hyphal growth of Aspergillus and Candida. The T cells also secrete IFN-γ to activate other immune cells to target IFI. To ready the Dectin-1 CAR-modified T cells for the human application, we have modified the Fc domain of the extracellular stalk to alleviate deleterious uptake by Fcγ receptors and thus in vivo loss of infused T cells. We have shown that the Dectin-1 CAR+ T cells do not kill the yeast form of Candida so normal commensals in the gut microbiota should not be affected. We have also demonstrated that Dectin-1 CAR+ T cells can control Aspergillus infection in the presence of immunosuppressive drugs at physiological concentrations. Finally, we have generated dual CAR+ T cells by co-expressing a CD19-specific CAR with Dectin-1 CAR rendering the resultant genetically modified T cells able to target both B-cell malignant cells as well as fungal hyphae. These dual CAR-T cells can thus control both leukemia and invasive fungal infections. These data provide a path forward for clinical trials testing Dectin-1 CAR+ T cells.

Citation Format: Pappanaicken R. Kumaresan, Nathaniel Albert, Harjeet Singh, Simon Olivares, Sourindra N. Maiti, Tiejuan Mi, Helen Huls, Richard E. Champlin, Dimitrios P. Kontoyiannis, Laurence J.N. Cooper. Bioengineered Dectin-1 CAR+ T cells to control invasive fungal infection . [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 A193.
Abstract B009: Design and development of HDAC6-selective inhibitors for hematological cancer treatment and solid tumor immunotherapy
Authors: Shuttleworth Stephen J.
Published: May 2016

Despite its name, HDAC6 is primarily a tubulin deacetylase, not a histone deacetylase, and it has emerged as a particularly interesting – and unique – cancer target as it plays an essential role in stress response and in aggresomal protein degradation: specifically, HDAC6 binds polyubiquitinated misfolded proteins and recruits misfolded protein cargo to dynein motors for transport to aggresomes. Cells deficient in HDAC6 fail to clear these misfolded protein aggregates from the cytoplasm, cannot form aggresomes properly, and are hypersensitive to the accumulation of misfolded proteins, which leads to activation of apoptosis. Further, recent evidence has emerged of HDAC6 modulating immune-related pathways in solid tumors, notably regulating PD-L1 expression through STAT3 modulation. In order to explore the clinical potential of HDAC6 in both hematological and solid tumor immunotherapy, we have de novo-designed a class of highly-potent, ultra-selective and orally-active HDAC6 inhibitors with potential in the treatment of multiple tumor types, including through immunotherapeutic mechanisms. Additionally, owing to the selectivity we have achieved for HDAC6 with our lead compounds, much greater opportunities for broader combination modalities can be realized, and we have established potentiation of our leads with other tumor immunotherapeutics in in vivo syngeneic models. The profile of our preclinical development candidate will be presented.

Citation Format: Stephen J. Shuttleworth. Design and development of HDAC6-selective inhibitors for hematological cancer treatment and solid tumor 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 B009.
Abstract B022: Functional bifurcation in T cells expressing chimeric antigen receptors
Authors: Chang ZeNan L. , Silver Pamela A. , Chen Yvonne Y.
Published: May 2016

Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) can induce durable regressions in cancer patients, particularly in the treatment of B-cell malignancies. The production and expansion of CAR-T cells relies on in vitro T-cell stimulation and more work is needed to explore the phenotypic changes that occur in this process to elucidate features that are predictive of anti-tumor functionality. Here, we present a systematic study of T cells expressing a second-generation, anti-CD19 CAR and use flow cytometry to interrogate the dynamic phenotypic and functional changes exhibited by CAR-T cells undergoing varied antigen stimulation courses. By tracking CAR–T-cell viability, proliferation, cytokine production, lytic abilities, and expression of various T-cell activation markers and inhibitory receptors across time, we find that CAR-T cells consistently bifurcate into two distinct subpopulations with dramatically different functional capacities and surface marker expression profiles. Furthermore, the proportion of CAR-T cells that demonstrate the superior functions is dependent on the particular degree of antigen stimulation and the composition of the T cell population (CD4 vs CD8, T-cell memory compartment breakdown, % transduced). Altogether, the systematic characterization methods and results reported here can guide future efforts to improve CAR–T-cell therapies.

Citation Format: ZeNan L. Chang, Pamela A. Silver, Yvonne Y. Chen. Functional bifurcation in T cells expressing chimeric antigen receptors. [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 B022.
Abstract B035: Self-tumor-antigen-specific CD8 T cells were suppressed by regulatory T cells
Authors: Maeda Yuka , Nishikawa Hiroyoshi , Sakaguchi Shimon
Published: May 2016

Background: Naturally occurring regulatory T (Treg) cells play an essential role for immunological unresponsiveness to self-tumor-antigens. Yet, how they stably suppress self-tumor-antigen-specific T cells remains unclear. In particular, it is unknown whether Treg cell-mediated suppression for a limited period has a critical long-lasting effect on cell fate and antigen reactivity of self-tumor-antigen-specific T cells.

Experimental procedures: To demonstrate Treg-cell effect on proliferation, cytokine production, and cell fate of self-tumor-antigen-specific CD8+ T cells, CD8+ T cells from peripheral blood mononuclear cells (PBMCs) of healthy individuals were stimulated with a melanocyte differentiation antigen Melan-A/ MART-1, expressed by normal melanocytes and some melanoma cells, with/without FoxP3+CD25+CD4+ Treg cells. Melan-A/ MART-1-specific CD8+ T-cell induction and their phenotypes were investigated.

Results: Melan-A/ MART-1-specific CD8+ T-cell induction was reduced by the presence of Treg cells. Self-tumor-antigen-specific CD8+ T cells co-cultured with Treg cells underwent one cell division and stopped further proliferation due to the lack of co-stimulation from antigen-presenting cells through Treg-cell suppression. They harbored low-affinity T-cell receptors and an anergic phenotype (i.e., hypo-proliferative and cytokine hypo-producing upon antigen re-stimulation) defined by expression of CCR7 and co-inhibitory molecules such as CTLA-4. Melan-A/ MART-1-specific CD8+ T cells with this anergic phenotype were detected ex vivo in the periphery and contributed to the tolerance of self-tumor-antigens.

Conclusions: Treg-cell-mediated induction of anergy in self- antigen tumor-specific CD8+ T cells inhibits their activation in cancer patients after immunotherapy, and neo-antigens associated with tumor-specific gene mutations may become a main target of CD8+ T cells inducing actual tumor regression.

Citation Format: Yuka Maeda, Hiroyoshi Nishikawa, Shimon Sakaguchi. Self-tumor-antigen-specific CD8 T cells were suppressed by regulatory T cells. [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 B035.
Abstract B037: Improved CD8+ T-cell recognition of neuroblastoma after innate immune attack by natural killer cells
Authors: Spel Lotte , Boelens Jaap J. , Til Niek van , Steen Dirk M. van der , Blokland Nina J.G. , Noesel Max M. van , Molenaar Jan J. , Heemskerk Mirjam H.M. , Boes Marianne , Nierkens Stefan
Published: May 2016

Neuroblastoma is the most common solid tumor in children, with mortality rates after conventional treatments still being 80% for high-risk (stage 4) disease. There is a clear medical need for development of new treatment strategies. Recently, immunotherapy has gained traction. However, restrained MHC I expression evades recognition of neuroblastoma by immune cells, for a suppressed immunogenic status of neuroblastoma tumors. We here describe a preclinical study aimed to improve neuroblastoma immunogenicity. We found that neuroblastoma cells significantly increase surface expression of MHC I upon exposure to active NK cells and thereby readily sensitize neuroblastoma cells for recognition by cytotoxic T lymphocytes (CTLs). We show that oncoprotein PRAME serves as a T-cell antigen in neuroblastoma as NK-modulated neuroblastoma cells are recognized by PRAMESLLQHLIGL/A2-specific CTL clones. Furthermore, NK cell induced MHC I upregulation in neuroblastoma cells requires IFNγ production by NK cells that is triggered by neuroblastoma contact. Our results demonstrate remarkable plasticity in the immunogenic properties of neuroblastoma cells, which are exposed when neuroblastoma cells attempt to escape innate immune attack. These findings support the exploration of NK cells in combined immunotherapy to enforce tumor-specific CTL responses, and thereby strengthen immune-mediated tumor reactivity.

Citation Format: Lotte Spel, Jaap J. Boelens, Niek van Til, Dirk M. van der Steen, Nina J.G. Blokland, Max M. van Noesel, Jan J. Molenaar, Mirjam H.M. Heemskerk, Marianne Boes, Stefan Nierkens. Improved CD8+ T-cell recognition of neuroblastoma after innate immune attack by natural killer cells. [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 B037.
Abstract B045: Immunosuppressive plasmocytes impede T-cell-dependent immunogenic chemotherapy
Authors: Shalapour Shabnam , Karin Michael
Published: May 2016

Cancer-associated genetic alterations induce expression of tumor antigens that can activate CD8+ cytotoxic T cells (CTLs), but the microenvironment of established tumors promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumor-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic that is effective in aggressive prostate cancer. Mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumor-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFβ receptor signaling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumors.

Citation Format: Shabnam Shalapour, Michael Karin. Immunosuppressive plasmocytes impede T-cell-dependent immunogenic 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 B045.
Abstract B049: Evaluation of attenuated HSV1716 in combination with chimeric antigen receptor T cells for solid tumors
Authors: Haile Samuel , Conner Joe , Mackall Crystal
Published: May 2016

Neuroblastoma, osteosarcoma, and rhabdomyosarcoma are among the most prevalent childhood solid tumors. Each of these tumor types as well as melanomas exhibit increased levels of the tumor associated carbohydrate, GD2 on their cell surface making them ideal targets for chimeric antigen receptor (CAR) T cell-directed therapies. Despite the ability of GD2 CAR T cells to target GD2-expressing tumor cells in vitro, there is great interest in improving tumor clearance in vivo, especially for solid tumors where current outcomes remain poor. We hypothesize that the immunosuppressive milieu present within the solid tumor microenvironment serves as a major factor limiting the effectiveness of GD2 CAR T cells and propose that administration of oncolytic viruses could induce inflammation within the tumor microenvironment that may enhance, rather than inhibit, the effectiveness of immune based therapies. GD2 CAR T cells composed of the 14G2a single chain variable fragment linked to the cytoplasmic signaling domains of CD28 and CD3 zeta (GD2-28z) were expressed in murine lymphocytes and evaluated for the ability to target and lyse GD2-expressing tumor cells. Additionally GD2-28z T cells were co-cultured with tumor cells to access their ability to secrete proinflammatory cytokines IFNγ and IL-2. In order to determine the oncolytic ability of attenuated HSV1716, tumor cells were cultured in the presence or absence of HSV1716 and relative cell survival was measured. We observed specific lysis of GD2-expressing tumor cells when co-cultured with GD2-28z, but not mock T cells. Furthermore, GD2-28z T cells secrete IFNγ and IL-2 following co-culture with GD2-expressing tumor cells. Interestingly, melanoma cell lines were not susceptible to oncolytic lysis while rhabdomycosarcoma (RMS) cell lines were susceptible. Using a melanoma model, GD2-28z T cells displayed anti-tumor activity. The combination of GD2-28z and HSV1716 enhanced CAR persistence in a melanoma model. Given that the melanoma cells in our model are not susceptible to oncolytic lysis yet we observe an increased T cell persistence when used in combination with HSV1716, this supports our hypothesis that HSV1716 in inducing inflammation, which is then triggering T cell expansion.

Citation Format: Samuel Haile, Joe Conner, Crystal Mackall. Evaluation of attenuated HSV1716 in combination with chimeric antigen receptor T cells for solid tumors. [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 B049.
Abstract B057: Optimizing the high throughput in vitro priming of tumor specific T cells
Authors: Prokop Lea , Souczek Stefanie , Peper Janet , Rammensee Hans-Georg , Stevanović Stefan
Published: May 2016

Peptide-based vaccines are a promising strategy for immunotherapy against cancer. Our group has characterized large numbers of HLA ligands from tumor cells; among them many that might be used for in vivo induction of tumor-specific immune responses. For the development of peptide-based immunotherapies such peptides should be able to induce in vivo tumor-directed immune responses, especially by CD8+ T cells that have a key role in inducing death of tumor cells. The aim of our work is to establish a fast and efficient workflow for immunogenicity testing of tumor-extracted HLA ligands. T cells usually depend on professional antigen presenting cells such as autologous dendritic cells (DCs) for specific induction and expansion. Since the generation of dendritic cells is expensive and time consuming with varying quality and amount of differentiated DCs, we established an artificial system to replace cell-based in vitro priming of T cells, streptavidin-coated artificial antigen presenting cells loaded with defined amounts of recombinant HLA molecules and costimulatory antibodies such as anti-CD28. Using the standard protocol, CD8+ T cells are stimulated three times with artificial antigen presenting cells in weekly intervals in the presence of IL-2 and IL-12. Peptide-specific CD8+ T cells are identified by tetramer staining. Our group started to vary different parameters of this protocol to increase the frequency and number of antigen specific T cells. Alternate costimulatory antibodies and cytokines proved successful to reach that goal. Employing artificial APCs for in vitro priming of tumor-reactive T cells will foster immunogenicity analysis of novel peptides for immunotherapy.

Citation Format: Lea Prokop, Stefanie Souczek, Janet Peper, Hans-Georg Rammensee, Stefan Stevanović. Optimizing the high throughput in vitro priming of tumor specific T cells. [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 B057.
Abstract B060: The canonical NF-κB pathway is crucial for Treg homeostasis during the antitumor response
Authors: Grinberg-Bleyer Yenkel , Ho Hyunju , Schmid Roland , Klein Ulf , Ghosh Sankar
Published: May 2016

Cancers use a wide variety of mechanisms to dampen tumor immune response. Among them, CD4+Foxp3+ regulatory T cells (Tregs) are largely described to inhibit the function of effector cells. In several murine models of cancer, Treg depletion suppresses tumor growth. Interestingly, it has been proposed that anti-CTLA-4 therapies exert their anti-tumor effects by targeting Tregs. Therefore, precisely understanding the mechanisms governing Treg homeostasis may be a valuable strategy to enhance immune responses against cancer. We and other have previously shown that transcription factor NF-κB plays a major role in Treg development; we are now analyzing the role of different NF-κB subunits in Tregs by using conditional knock-out mice.

We found that both p65 (RelA) and c-Rel subunits of NF-κB in Tregs, were required for both development of thymic Treg progenitors and subsequent differentiation into Foxp3-expressing mature Tregs. Ablation of NF-κB also prevented in vitro differentiation of inducible Tregs. Furthermore, specific deletion of either p65 or c-Rel subunits in natural Tregs using FoxP3-cre led to the development of a lethal autoimmune syndrome by 8 to 15 weeks of age. Ablation of both subunits further shortened the lifespan of animals to around 3 weeks of age. This was associated with profound changes in the molecular signature of NF-κB deficient Tregs, which down-regulated Treg-associated genes while acquiring an effector-like phenotype. Also, tamoxifen-induced acute deletion of NF-κB in mature Tregs reduced their survival and expression of Treg-associated markers. These data highlight a crucial role for NF-κB in maintaining Treg identity in vivo.

We next explored the role of NF-κB in Treg homeostasis during tumor growth. Strikingly, melanoma growth was drastically reduced in mice lacking c-Rel, but not p65, in Tregs. This was associated with an increased polyclonal and antigen-specific effector T cell activation. Besides, CD8 T cell depletion abolished the protection against tumor growth. We are now proceeding to test the effect of specific c-Rel inhibitors on tumor growth. We believe our work reveals a central molecular mechanism in the maintenance of Treg identity and thus highlights novel therapeutic target for the treatment of cancer.

Citation Format: Yenkel Grinberg-Bleyer, Hyunju Ho, Roland Schmid, Ulf Klein, Sankar Ghosh. The canonical NF-κB pathway is crucial for Treg homeostasis during the antitumor response. [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 B060.
Abstract B064: The effects of tumor-associated calreticulin mutants on antigen presentation
Authors: Arshad Najla , Cresswell Peter
Published: May 2016

The phenomenon of antigen processing and presentation aids the immune system in distinguishing between normal and malignant cells. In cancer cells, Major Histocompatibility Complex (MHC) proteins present tumor-associated antigens (TAAs) that may serve as targets of immunotherapy. Antigen presentation by MHC class I-β2m heterodimers (MHC-I) requires peptide association to occur in in the endoplasmic reticulum (ER). Binding is facilitated by the Peptide Loading Complex (PLC), which consists of the TAP transporter, tapasin, the thiol-oxidoreductase ERp57, and a lectin chaperone, calreticulin (CRT). CRT associates with MHC-I via the single, conserved, glycan present on MHC-I, drawing it into the PLC to facilitate peptide loading.

CRT is comprised of an N-terminal lectin domain, a proline-rich central P domain, and a negatively charged C-terminal domain (CTD) that contains an ER retention motif. Two recent studies report a mutant CRT associated with myeloproliferative neoplasms (MPNs). These MPNs carry a somatic frame-shift CRT mutation (CRTFS) that generates a novel CTD that lacks the ER-retention signal and renders it positively charged. Previous studies have shown that ablation of CRT expression in mouse embryonic fibroblast cells result in decreased surface expression of MHC-I, which is reversed on reconstitution of full-length CRT. Interestingly, reconstitution of CRT lacking the CTD did not restore MHC-I expression, indicating a role for the CTD in MHC-I expression. We therefore suspect that antigen presentation in MPNs carrying the CRTFS is altered.

To address the role of CRTFS in antigen presentation in a human cell line, we have generated a CRT knockout of HeLaM cells using CRISPR/Cas9 technology. The expression of MHC-I in HeLaM cells dramatically increases on exposure to interferon-γ and we are currently assessing the alterations in MHC-I expression in these CRT knockout cells using various biochemical techniques. Future experiments include analysis of the peptide repertoire displayed on the surface of these cells as well as changes in the molecular interactions of the mutant CRT with other members of the PLC. After a preliminary study in HeLaM cells, we will study the effect of CRTFS in antigen presentation in MPN cell lines to identify unique TAAs to facilitate the specific targeting of these cells by immunotherapy.

Citation Format: Najla Arshad, Peter Cresswell. The effects of tumor-associated calreticulin mutants on antigen presentation. [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 B064.
Abstract B075: CD103 integrin promotes T-cell recruitment and activation within epithelial tumors
Authors: Boutet Marie , Mami-Chouaib Fathia
Published: May 2016

Cancer immunotherapies specifically target tumors by optimizing the capacity of host cytotoxic T lymphocytes (CTL) to recognize and kill malignant cells. To destroy their target, CTL must migrate to the tumor, infiltrate the stroma and then interact with cancer cells. Recruitment of T lymphocytes within the tumor involves several sequential steps regulated by molecular interactions with a variety of stromal cell types and coordinated by chemotactic agents, extracellular matrix proteins and cell surface adhesion molecules. Integrins are one of the major families of adhesion molecules that contribute to lymphocyte development, migration, extravasation and activation. CD103 integrin and its ligand, the epithelial cell marker E-cadherin, play a crucial role in promoting both tumor cell cytotoxicity and cytokine secretion. Accumulating evidence also indicates that CD103 on CD8+ tumor-infiltrating lymphocytes (TIL) is associated with increased intraepithelial T-cell infiltration and that an enhanced CD103+ TIL subset correlates with improved patient survival in several cancers. However, the contribution of CD103 to promoting T-cell recruitment within epithelial tumor regions, and the influence of TGF-beta, abundant within the tumor microenvironment, on integrin functions and signaling remain poorly understood. In the present study we provide evidence that CD103 is directly involved in T-cell recruitment within epithelial tumor islets and in local early T-cell signaling. Moreover, TGF-beta triggers integrin-mediated functional activities by promoting phosphorylation of integrin-linked kinase (ILK) and its subsequent binding to CD103, thus initiating an “inside-out” signaling leading to activation and clustering of the integrin and thereby initiating an “outside-in” signal that contributes to T-cell stimulation, spreading and cytotoxicity. We conclude that TGF-beta, while known for its protumoral effects, promotes an antitumor CTL response by activating CD103 integrin.

Citation Format: Marie Boutet, Jr., Fathia Mami-Chouaib, Sr. CD103 integrin promotes T-cell recruitment and activation within epithelial tumors. [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 B075.
Abstract B077: Evaluating oncolytic vaccines targeting mutated epitopes in a murine fibrosarcoma model CMS-5
Authors: Stephenson Kyle , Atherton Matthew , Bell John , Lichty Brian
Published: May 2016

Oncolytic viruses have traditionally been used as stand alone therapy delivered to animals and patients to lytically destroy tumor tissue. However, it has recently become clear that involvement of the immune system is an extremely important aspect of these therapies. To further enhance anti-tumor immune responses we have utilized an oncolytic vaccine approach. This approach involves priming the immune system to a specific tumor associated antigen (TAA) with an Adenovirus and then subsequently delivering an oncolytic Rhabdovirus expressing the same tumor antigen leading to robust anti-tumor immune responses and improved efficacy.

For the studies presented herein we have developed oncolytic vaccines targeting mutated epitopes expressed in CMS-5. Immune responses generated towards these epitopes through oncolytic vaccination will be characterized and their ability to control tumor growth will be evaluated. We hypothesize that the foreign nature of these mutated epitopes will allow for the generation of substantial immune responses following oncolytic vaccination in tumor bearing mice.

In preliminary studies, oncolytic vaccination has been able to generate robust immune responses leading to as many as 60% of circulating CD8+ T cells responding to a single mutated epitope in tumor free animals. Therapeutic oncolytic vaccination of CMS-5 tumor bearing animals was still able to generate large anti-tumor immune responses, however these responses were cut in half compared to tumor free animals. The resultant anti-tumor immune response was able to significantly control tumor growth, and the magnitude of these responses correlated with survival. Even though there was a delay in tumor growth following therapy, the responses were not durable and all mice ultimately failed the therapy.

We are currently engaged in studies to further characterize the immune responses generated and determine what, if any, immunosuppression led to failure of our therapy; even in the presence of anti-tumor immune responses exceeding 20% of peripheral CD8+ T cells. Nanostring analysis of the tumor microenvironment are being conducted focusing on gene signatures for immune cells and inflammation and these data will be confirmed by histology and flow cytometry. These studies will help shed light on potential mechanisms of immune evasion/suppression following oncolytic vaccination and point to potential targets for modulation of the oncolytic vaccine induced immune response.

Citation Format: Kyle Stephenson, Matthew Atherton, John Bell, Brian Lichty. Evaluating oncolytic vaccines targeting mutated epitopes in a murine fibrosarcoma model CMS-5. [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 B077.