bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 1483: Examining the role of genome editing enzymes in anti tumor immunity
Authors: Borzooee Faezeh 
Published: July 2021

Abstract
Background: Tumor antigens (TA) on cancer cells can trigger immune response TA are peptides that are either not present, or are present to a much lesser extent, on normal cells compared to tumor cells. CD8+ Cytotoxic T-cells (CTL) recognize TAs bound to class I major histocompatibility complex (MHC) molecules on the surface of tumor cells. We and others have shown that viruses subvert the mutational activity of the endogenous DNA-editing APOBEC enzymes in human host cells towards immune escape. Viruses do so by adapting their genomic DNA code to position APOBEC's favored mutational “hotspot” DNA sequences such that upon being mutated by the host APOBEC enzymes, the mutated viral protein is less immunogenic for CTL recognition. Like viruses, cancer cells have genomes that are highly plastic and adaptable through mutation; therefore, we hypothesize that the APOBEC DNA-editing enzymes are exploited by cancer cells for modulating TA immunogenicity.
Aims of the Study: Aim1: To elucidate the impact of A3-mediated mutations on modulating TA immunogenicity for CTL recognition. Aim 2: To examine whether genomic sequences encoding TAs in the human genome have co-evolved to either attract or avoid APOBEC-driven mutations.
Methods: Aim 1: Using the human reference proteome from EMBL/EBI, we located APOBEC-mutable hotspots in genomic DNA encoding the immunopeptidome. We simulated APOBEC-mediated mutations and translated to peptide TAs. We used NetMHCpan 4 to measure the impact of TA mutations on MHC class I binding affinity. In parallel, we are searching for already-identified APOBEC-mediated mutations in sequenced tumor genomes in cancer genome databases such as The Cancer Genome Atlas (TCGA), Catalogue of Observed Somatic Mutations in Cancer (COSMIC) and International Cancer Genome Consortium (ICGC). Aim 2: To investigate the enrichment of APOBEC-hotspots in genomic sequences encoding TAs restricted to each HLA, we are tabulating APOBEC hotspots in genomic DNA sequencing encoding TAs vs. non-TA-encoding sequences. At the sequence level, the ratio of hotspot frequency, in combination with scores of predicted impacts on immunogenicity as obtained in aim 1, will be used to glean whether and how the human genomic sequence has co-evolved with APOBECs from the standpoint of TAs.
Results and Significance: Based on the preliminary data we observe that APOBEC-mediated mutations can diminish or enhance immunogenicity. Understanding the balance between these two effects is key in informing how APOBEC expression and activity ought to be regarded in the context of cancer immunotherapy.
Citation Format: Faezeh Borzooee. Examining the role of genome editing enzymes in anti tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1483.
Abstract 1486: Inhibition of host PI3K-gamma modulates anti-tumor immunity in poorly immunogenic HNSCC
Authors: Anderson Kelvin , Ryan Nathan , Alkhimovitch Anastasia , Siddiqui Arham , Oghumu Steve 
Published: July 2021

Abstract
HNSCC is the 6th most common cancer, with around 650,000 new cases yearly. Gain of function mutations in the PI3K pathway are common in HNSCC, and inhibition of the PI3K p110γ subunit has shown promise in HNSCC treatment. However, given that PI3K p110γ plays an important role in myeloid and lymphoid immune cell function, it is essential to understand how PI3K p110γ inhibition affects the anti-tumor immune response independent of tumor cells. To elucidate PI3K p110γ function in HNSCC, we employed an orthotopic mouse model using poorly immunogenic and aggressive cell line MOC2 on Pik3cg-/- mice. We observed that wild-type and Pik3cg-/- mice displayed similar rates of HNSCC tumor growth and metastasis after 20 days post tumor injection. T cell infiltration and intrinsic T cell responses to MOC2 oral tumors were comparable between wild-type and Pik3cg-/- mice. Interestingly, the immune response of tumor-bearing Pik3cg-/- mice was marked by increased anti-tumor cytotoxic molecules (IFN-γ, IL-17, PD-1) by T cells, and immune checkpoint marker (PD-L1) expression by myeloid cells compared to tumor bearing wild type mice. Taken together, our findings demonstrate that inhibition of PI3K p110γ modulates tumor associated immune cells, which likely potentiates HNSCC treatment when used in combination with selective PD-L1 inhibitors.
Citation Format: Kelvin Anderson, Nathan Ryan, Anastasia Alkhimovitch, Arham Siddiqui, Steve Oghumu. Inhibition of host PI3K-gamma modulates anti-tumor immunity in poorly immunogenic HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1486.
Abstract 1492: Chimeric antigen receptor (CAR) T cell donor dependent dysfunction modulation with activation of autophagy and inhibition of trogocytosis
Authors: Liang Jiyong , Fang Dexing , Heimberger Amy B. 
Published: July 2021

Abstract
Introduction: Effective anti-tumor immunity requires the presence of abundant effector T cells in the tumor microenvironment with both cancer cell specificity and cytotoxic potency. Chimeric antigen receptor (CAR) T cells engineered ex vivo to fulfill such quintessence are effective in both experimental and clinical settings with curative potential. However, the efficacy of CAR T cells can be limited by trogocytosis whereby CAR T cells extract cognate antigens from target cells thereby eliciting antigen escape and then express the target antigen on the CAR triggering fratricide that reduces the CAR persistence and efficacy.
Methods: Epidermal growth factor variant III (EGFRvIII) specific CAR T cells were generated from various donors and analyzed for EGFRvIII cytotoxicity, trogocytosis, and in vivo therapeutic activity which was modulated with verteporfin, an inducer of autophagy and bafilomycin A1, an inhibitor of autophagy. A three-way co-culture system modeling the glioblastoma microenvironment of the CAR T cell interaction with targets (U87-EGFRvIII) and bystanders (U87-EGFR) was used to evaluate phagocytic and autophagy functions of the CAR T cells.
Results: Glioblastoma-specific EGFRvIII CARs induce trogocytosis of the cognate tumor antigen upon engaging target tumor cells within 2 hours. Trogocytic activity varies in a donor-dependent manner and correlates with impaired CAR performance in murine models of orthotopic gliomas expressing EGFRvIII. Trogocytosis is blocked with verteporfin which restores cytolytic capacity of the CAR. In the three-way co-culture system, therapeutically effective in vivo CARs demonstrate superior phagocytic clearance of U87-EGFRvIII released mitochondria that is blocked by autophagy inhibition. The induction of immune checkpoints such as PD-L1 during CAR-target interaction is enhanced when autophagy is blocked. Furthermore, autophagy blockade prevents phagocytic clearance of cytolytic target release of mitochondria. We further demonstrate that pharmacological facilitation of autophagy with verteporfin inhibits trogocytic expression of target tumor antigen on CARs and thereby markedly increases CAR persistence in the glioma microenvironment in mice.
Conclusions: Our data establish autophagy as an antagonizing mechanism counteracting CAR-induced trogocytosis and immune checkpoint activation, providing evidence for a new strategy to innovate high-performance CARs.
Citation Format: Jiyong Liang, Dexing Fang, Amy B. Heimberger. Chimeric antigen receptor (CAR) T cell donor dependent dysfunction modulation with activation of autophagy and inhibition of trogocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1492.
Abstract 151: An improved computational pipeline for tumor somatic alterations detection
Authors: Yan Cheng , Ye Yingjiang , Guo Weihua , Yang Junhui , Yang Yufei 
Published: July 2021

Abstract
Background: As high throughput sequencing (HTS) technique advances, it has been widely used in cancer research. More specifically, HTS enables genome-wide detection of somatic alterations which implicate the dynamic of cancer development as well as therapeutic strategy. However, a number of complications, such as sample tissue lesion, imperfect experimental procedure and nucleotide polymorphism between individual, often induce non-somatic noise and jeopardize the detection of somatic alterations. We present a validated computational pipeline that allows accurate somatic alteration identification.
Method: We created a number of cell line mixture samples and sequenced at averagely 1500x depth. Each mixture sample was composed of cell lines with pre-specified proportion in order to represent distinct tumor purity and clonality. Prior to mixture creation, variant candidates in each cell line sample were labeled as one of three categories: germline variant of high confidence, artifact or blacklist (excluded from future assessment). The categorization was based on HaplotypeCaller results followed by a series of quality control. The allele frequency distribution of germline variants from each cell line was validated in mixture samples. We used MuTect as prototype pipeline and implemented further optimization on a number of parameter settings in a supervised fashion. Lastly, the optimized pipeline was evaluated using an independent dataset and its performance was compared with a number of alternative variant callers.
Result: Our benchmark showed that default parameter setting of ‘alt_allele_in_normal' and ‘nearby_gap_events' in MuTect were over-strict. 73% of variants rejected by these parameters were actually real somatic variants. We believed that, instead of using constant as parameter settings, the optimum parameters should be trained on data with similar sequencing depth. We therefore re-configured the parameters using logistic regression. Furthermore, minimum supporting depth and minimum allele frequency were determined by AUROC. With optimized pipeline, we were able to achieve 96% sensitivity and 2/M false positive rate (FPR) compared with 90% sensitivity and 8/M FPR using default parameters. We further tested the optimized pipeline in an independent dataset and compared the performance with several well-known variant callers including MuSE, VarScan2, Strelka, LoFreq, VarDict and MuTect2. The performance of each caller was ranked as following: our optimized pipeline &gt; MuTect &gt; MuTect2/Strelka/LoFreq &gt; Muse &gt; VarScan2/VarDict.
Citation Format: Cheng Yan, Yingjiang Ye, Weihua Guo, Junhui Yang, Yufei Yang. An improved computational pipeline for tumor somatic alterations detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 151.
Abstract 1510: Effects of the lung tumor microenvironment on T cell therapy
Authors: Schmidt Leah Marie , Tran Oanh , Jenkins Cody , Oda Shannon , Inman Quintin , Leeper Haley , Tan Sasha , Greenberg Philip 
Published: July 2021

Abstract
Immune checkpoint therapy (ICT) is a front-line treatment for lung cancer; however, low mutational burden and ‘non-T cell inflamed' signatures predict poor responses to ICT in ~50% of patients. Adoptive cellular therapy (ACT) with T cells engineered to express T cell receptors (TCRs) specific for tumor-associated antigens (TAAs; proteins overexpressed by cancers) is an approach that circumvents the need for endogenous T cell responses. TCR-ACT is effective against hematologic cancers, but ACT against solid tumors is in the early stages of exploration. A deeper understanding of complex interactions between therapeutic cells and the tumor microenvironment (TME) is crucial for identifying successful strategies to enhance function and mitigate toxicity. Genetically engineered mouse models (GEMMs) achieve in situ tumor development alongside competent immune systems, recapitulating the native TME over the full spectrum of disease progression in a preclinical setting. KP GEMM lung tumors harbor few mutations, are poorly infiltrated by T cells, and are refractory to ICT, modeling intractable patient disease. We show that TAA-specific T cells recognize KP lung cancer cells and efficiently home to tumors, but rapidly lose function in the lung TME (compared to KP pancreatic tumors). Repetitive TCR-ACT can extend survival of animals with lung tumors, but animals eventually succumb to disease. These results show promise for TCR-ACT against lung cancer but highlight tissue-specific obstacles that must be overcome to enhance efficacy. Preliminary studies indicate that therapeutic T cells expressing immunomodulatory fusion proteins (IFPs) or accompanied by engineered helper T cells exhibit increased function in lung tumors, indicating that increased costimulatory signaling and T cell help can partially overcome T cell suppression in the lung TME. As we continue to leverage these models to advance our understanding of tissue-specific effects on therapeutic outcomes, these findings offer inroads to uncovering more effective therapies, with the ultimate goal of benefitting cancer patients.
Citation Format: Leah Marie Schmidt, Oanh Tran, Cody Jenkins, Shannon Oda, Quintin Inman, Haley Leeper, Sasha Tan, Philip Greenberg. Effects of the lung tumor microenvironment on T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1510.
Abstract 1514: Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells
Authors: Donaghey Julie , Kieffer-Kwon Philippe , Gomez-Rodriguez Julio , Patterson Troy , Gierut Jessica , Chan Tiffany , Milione Ryan , Daniel Joshua , Horton Holly , Tighe Robert , Hofmeister Robert , Gutierrez Dario A. 
Published: July 2021

Abstract
The natural T cell receptor (TCR) recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules and requires HLA matching of α/β TCR-engineered T cells for cancer therapy. To bypass the need for HLA matching, we have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein that comprises an antibody-derived binder tethered to the CD3ϵ signaling subunit. Upon integration of the TRuC into the TCR, it recognizes tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report the engineering of off-the-shelf TRuC-T cells directed against CD19 and mesothelin, respectively. To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprising antibody binders fused to the murine TCRα or β constant domains, or alternatively, to the TCRγ and δ constant domains. Similar to their autologous counterparts, allogeneic CD19 and MSLN-targeting TRuC-T cells upregulate activation markers, secrete cytokines, and lyse tumor cells in an antigen-specific manner. Importantly, TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft model comparable to autologous anti-CD19 TRuC-T cells, but without signs of GvHD. Likewise, the functional activity and efficacy of allogeneic TRuC-T cells directed against mesothelin is similar to that of autologous anti-MSLN TRuC-T cells. To reduce host rejection and enhance the persistence of the allogeneic TRuC-T cells, MHC class I expression on the surface of the TRuC-T cells was down-regulated by means of RNAi. In summary we have engineered allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with comparable efficacy as donor-matched autologous TRuC-T cells; moreover, these T cells have the potential to persist in an allogeneic host by diminishing the risk of rejection by the host.
Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Julio Gomez-Rodriguez, Troy Patterson, Jessica Gierut, Tiffany Chan, Ryan Milione, Joshua Daniel, Holly Horton, Robert Tighe, Robert Hofmeister, Dario A. Gutierrez. Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1514.
Abstract 1516: Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors
Authors: Barber Amorette E. 
Published: July 2021

Abstract
Adoptive transfer of T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 4-1BB, GITR, ICOS, or OX40 costimulatory domains in the chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10), chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies.
Citation Format: Amorette E. Barber. Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1516.
Abstract 1519: PD1 TurboCAR™ T cells: PD1-resistant CAR T cells with programmable cytokine signaling outputs
Authors: Lin Regina , Zhang Yi , Srinivasan Surabhi , Lay Cecilia , Lang Shanshan , Sutton Janette , Manion Candice , Van Blarcom Tom , Sasu Barbra , Panowski Siler 
Published: July 2021

Abstract
CAR T cell therapy has attained unprecedented success in the treatment of hematological malignancies. However, clinical benefit in solid tumor indications has been limited, potentially in part due to suppressive solid tumor microenvironments (TME) that inhibit T cell effector function and persistence. While the provision of cytokine support can help CAR T cells overcome suppressive TME, combining CAR T therapy with systemically-administered cytokines/cytokine mimetics can result in toxicities and locally secreted cytokines may enhance rejection of allogeneic cell products by host cells. For this reason we had previously designed and tested a novel cytokine-stimulated CAR T cell designated TurboCARs. TurboCAR T cells coexpress a CAR and a Turbo domain (i.e. a homodimeric cytokine receptor chimera) that transmits CAR T cell-intrinsic cytokine signals, resulting in enhanced potency, expansion and persistence in preclinical studies. However, TurboCAR T cells may remain sensitive to inhibition by immune barriers, such as negative signaling through the PD1 receptor. We reasoned that a two-pronged approach aimed at inhibiting immune-suppressive PD1 signaling while simultaneously transmitting immune-potentiating cytokine signaling may augment CAR T cell activity in solid tumors. To this end, we engineered PD1 TurboCAR T cells, in which the Turbo domain is fused to a PD1 ectodomain that serves as a dominant-negative receptor. The PD1 ectodomain was further modified for high-affinity binding to PD1 ligands, allowing for preferential ligand sequestration and more effective inhibition of endogenous PD1 signaling. Despite having a larger lentiviral vector cargo, PD1 TurboCAR T cell products remained manufacturable and retained a favorable memory phenotype. PD1 TurboCAR T cells directed towards PDL1-expressing solid tumor target cells showed increased functionality compared to CAR T cells combined with PD1 blockade or to the parental TurboCAR T cells alone. In conclusion, PD1 TurboCARs augmented with a PD1 dominant negative ectodomain conferred CAR T cells with resistance to PD1-mediated inhibition, while simultaneously transmitting cytokine signals in a CAR T cell-intrinsic fashion. As an all-in-one product, PD1 TurboCAR T cells may obviate the need for combination therapy with anti-PD1 antibodies, while circumventing safety risks associated with systemic cytokine administration.
Citation Format: Regina Lin, Yi Zhang, Surabhi Srinivasan, Cecilia Lay, Shanshan Lang, Janette Sutton, Candice Manion, Tom Van Blarcom, Barbra Sasu, Siler Panowski. PD1 TurboCAR™ T cells: PD1-resistant CAR T cells with programmable cytokine signaling outputs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1519.