bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1460: The design and biological characterization of PTP4A-targeted chemodegraders
Authors: Hart Duncan J. , Rastelli Ettore J. , Mendelson Anna J. , Letai Christopher T. , Cornelison John Robert , Sharlow Elizabeth R. , Wipf Peter , Lazo John S.
Published: July 2021

The protein tyrosine phosphatase 4A (PTP4A) family members, PTP4A1, PTP4A2 or PTP4A3, are overexpressed in many cancers, and this overexpression is associated with a distinct disadvantage to overall patient survival. Genetic knockdown or deletion of PTP4A diminishes cancer cell migration, survival and tumorigenesis. We previously demonstrated that the small molecule JMS-053 or KVX-053 (7-iminothieno[3,2-c]pyridine-4,6(5H,7H)-dione) functions as a selective, allosteric, and reversible pan-PTP4A, inhibitor. KVX-053 decreased ovarian cancer cell line spheroid viability, migration and in vivo tumor growth. We now report a conjugate of KVX-053 to an adamantyl moiety in an effort to design intracellular PTP4A chemodegraders. In addition to the linker length, we also modified the functional group attachments to the KVX-053 pharmacophore and the adamantly moiety. Significantly, several of these analogs retained potent in vitroinhibition of PTP4A3 enzymatic activity (IC50=100-750 nM), suggesting they could be dual-purpose PTP4A3 inhibitors, targeting both the catalytic activity and reducing intracellular protein concentration. Some of these KVX-053 analogs, including EJR-876-35 and EJR-887-35, exhibited enhanced cytotoxicity against human ovarian cancer, triple negative breast cancer or leukemia cell lines compared to the parent chemotype, KVX-053. Human ovarian cancer cells incubated with 1 micromolar EJR-887-35 exhibited a pronounced loss of PTP4A protein within 6 hours and the loss was sustained for at least 18 hours. These results support the further investigation of chemodegraders as specific first-in-class bifunctional PTP4A inhibitors directed against human cancers.

Citation Format: Duncan J. Hart, Ettore J. Rastelli, Anna J. Mendelson, Christopher T. Letai, John Robert Cornelison, Elizabeth R. Sharlow, Peter Wipf, John S. Lazo. The design and biological characterization of PTP4A-targeted chemodegraders [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 1460.
Abstract 1472: Novel EGFR WT sparing, HER2 selective inhibitors for the treatment of HER2 exon 20 insertion driven tumors address a clear unmet medical need
Authors: Neumüller Ralph A. , Wilding Birgit , Scharn Dirk , Böse Dietrich , Santoro Valeria , Gerlach Daniel , Ettmayer Peter , Gerstberger Thomas , Fuchs Julian , Treu Matthias , Zahn Stephan , Baum Anke , Chetta Paolo , Pearson Mark , McConnell Darryl B. , Kraut Norbert , Solca Flavio
Published: July 2021

Activating mutations in ERBB2 receptors are tractable oncogenes in 2-3% of NSCLC patients for whom no approved targeted therapies are available. In this indication, oncogenic mutations in HER2 predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. We initiated a drug discovery program aiming at discovering novel HER2 selective inhibitors sparing EGFR WT activity. Focus was set on the most frequent HER2 mutation (ERBB2 A775 insYVMA), which is least sensitive to current compounds tested in clinical trials. Here, we report the identification and pharmacological characterization of novel selective HER2 exon 20 mutation TKIs that differ from currently tested TKIs such as poziotinib, TAK-788 or BDTX-189. We could demonstrate that selective inhibition of oncogenic HER2 signaling abrogates oncogenic signaling in in vitro models. Cell survival and proliferation was reduced, which translated into tumor regressions in preclinical CRISPR engineered xenotransplantation models of HER2 exon 20 mutants. The in vivo efficacy was confirmed in patient-derived tumor models. Our results suggest that HER2 exon 20 insertions can be effectively treated by a potent and highly selective HER2 inhibitor that spares EGFR wild type. These findings warrant the upcoming clinical testing in HER2 mutant NSCLC patients in order to effectively treat this aggressive type of cancer.

Citation Format: Ralph A. Neumüller, Birgit Wilding, Dirk Scharn, Dietrich Böse, Valeria Santoro, Daniel Gerlach, Peter Ettmayer, Thomas Gerstberger, Julian Fuchs, Matthias Treu, Stephan Zahn, Anke Baum, Paolo Chetta, Mark Pearson, Darryl B. McConnell, Norbert Kraut, Flavio Solca. Novel EGFR WT sparing, HER2 selective inhibitors for the treatment of HER2 exon 20 insertion driven tumors address a clear unmet medical need [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 1472.
Abstract 1477: Co-targeting c-Met and c-Src pathways to inhibit metastatic cancer cell migration and invasion
Authors: Dai Yao , Siemann Dietmar W.
Published: July 2021

Overexpression of tyrosine kinases c-Src and c-Met has been shown to be highly associated with disease progression and poor clinical outcomes in many types of cancers. Although small molecule Src or Met inhibitors are being individually evaluated in clinical trials, patient data suggest that single treatment with any of these inhibitors only yield limited effects. One possibility is that redundant signaling pathways in tumor cells may allow tumor cell functions less interrupted upon inhibition of only one pathway. We therefore propose that c-Src and c-Met are two compensatory pathways that may cooperate to promote cancer metastasis. In order to delineate this hypothesis, we studied treatment effects of combining inhibitors targeting c-Src (dasatinib) and c-Met (ASLAN002) on multiple functional behaviors associated with tumor metastasis in c-Src and c-Met expressing metastatic cancer cells. Combination of Src- and Met-inhibitors was found to strongly inhibit cell colony formation by clonogenic survival assay. In a wound-healing assay, compared to either treatment alone, the combinatorial treatment exhibited greater suppression on cell motility. In addition, using a Matrigel-based Transwell assay, dasatinib plus ASLAN002 resulted in significant reduction of cell invasion in the presence or absence of HGF. Western blot analysis demonstrated that co-targeting Met and Src kinases lead to not only inhibition on c-Met and c-Src autophosphorylation but also multiple downstream kinases such as focal adhesion kinase (FAK), Akt and extracellular signal-regulated kinase (ERK). These data indicate that combined application of small molecule Src and Met inhibitors cause significant impairment on functional behaviors associated with cancer metastasis in vitro including survival, migration and invasion, suggesting that this combination intervention modality may serve as a potential anti-metastatic strategy that could be further tested in metastatic models in vivo.

Citation Format: Yao Dai, Dietmar W. Siemann. Co-targeting c-Met and c-Src pathways to inhibit metastatic cancer cell migration and invasion [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 1477.
Abstract 1483: Examining the role of genome editing enzymes in anti tumor immunity
Authors: Borzooee Faezeh
Published: July 2021

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

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

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

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 > MuTect > MuTect2/Strelka/LoFreq > Muse > 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

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

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

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

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.
Abstract 1523: Cell Squeeze® delivery of antigen-encoding mRNA enables human PBMCs to drive antigen-specific CD8+ T cell responses for diverse clinical applications
Authors: Maloney Michael F. , Ozay Emrah Ilker , Yee Christian , Merino Amy , Dunbar Paul R. , Mosaheb Mubeen , Volk Kelly , Smith Carolyne , Seidl Katherine J. , Bernstein Howard , Loughhead Scott M.
Published: July 2021

Antigen-specific CD8+ T cell priming is critical for mounting an effective immune response against altered self or foreign antigens that are found in most tumors and virally infected cells. T cell priming events require antigen presenting cells (APCs) to process and load antigenic peptides onto MHC molecules. One recently established method for providing APCs with foreign antigen is through delivery of mRNA. An advantage of using antigen-encoding mRNA, compared to protein or peptides, is the inherent protein amplification of mRNA translation; a single mRNA can be translated multiple times. In addition, when antigen-encoding mRNA is delivered directly to the cell, the antigens will have the appropriate post-translational modifications, potentially increasing the immunogenicity. Whereas typical mRNA vaccine platforms require modified nucleotides and lipid nanoparticle encapsulation, Cell Squeeze® technology allows for delivery of uncomplexed and unmodified mRNA directly into the cytosol.

Here, we use ex vivo microfluidic Cell Squeeze® technology to deliver uncomplexed and unmodified mRNA encoding for disease related antigens into human peripheral blood mononuclear cells (PBMCs) to generate PBMC APCs. We demonstrate that squeezing PBMCs with mRNA results in effective delivery and translation of mRNA in major cell subsets including T cells, B cells, NK cells, and monocytes. Delivery of antigen-encoding mRNA to human PBMCs enables them to function as APCs, capable of presenting antigenic peptides on MHC molecules for activation of antigen-specific T cells. Specifically, we demonstrate squeezing antigen-encoding mRNA (e.g. HPV16 E7) into human PBMCs results in PBMC APCs that can elicit robust in vitro activation of antigen-specific CD8+ T cells. This approach to APC engineering demonstrates Cell Squeeze® technology's potential to leverage the modularity, cost-effectiveness, and streamlined manufacturing of multiple mRNAs to create cellular vaccines for treating various tumor and infectious disease targets.

Citation Format: Michael F. Maloney, Emrah Ilker Ozay, Christian Yee, Amy Merino, Paul R. Dunbar, Mubeen Mosaheb, Kelly Volk, Carolyne Smith, Katherine J. Seidl, Howard Bernstein, Scott M. Loughhead. Cell Squeeze® delivery of antigen-encoding mRNA enables human PBMCs to drive antigen-specific CD8+ T cell responses for diverse clinical applications [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 1523.
Abstract 1540: CARs and TCRs: Myth and reality
Authors: Wang Xueyin , Martin Aaron D. , Negri Kathleen , McElvain Michele , Oh Julyun , Wu Ming-Lun , Lee Wen-Hua , Hamburger Agnes , Xu Han , Kamb Alexander
Published: July 2021

Here we address questions of comparative TCR and CAR function: How different are CARs from TCRs with regard to antigen sensitivity, selectivity, co-stimulation dependence, checkpoint control, cytokine sensitivity, and antigen-dependent exhaustion (defined as loss of cytotoxicity and proliferative capacity over time). We used optimized CARs and TCRs matched to the same target pMHCs, allowing direct comparison of these important parameters. We describe a large-scale, systematic comparison of 17 CARs and 5 TCRs directed at 5 different pMHC targets in both acute and chronic settings in vitro. Surprisingly, we find little fundamental difference between CARs and TCRs with respect to their relative sensitivity to known regulators of T cell activation, including co-stimulation, checkpoint, and cytokine control. Though TCRs often emerge from human blood directly as potent, selective receptors, CARs must be heavily optimized to attain these properties for pMHC targets. Nonetheless, when iteratively improved, CARs can achieve levels of sensitivity and selectivity similar to their vaunted counterparts. Thus, other than signaling sensitivity (and perhaps selectivity), there may be little to distinguish CARs from TCRs in the context of engineered-cell therapy. CARs appear similar to TCRs with regard to immune control, undermining the notion that CARs can, of themselves, overcome regulators that limit T cell activity. Nevertheless, CARs tolerate much more sequence variability within their structural components and this, coupled with their capability to bind different antigen classes, smaller size and subunit simplicity, confers advantages that may foster a leading role for CARs in the next generation of cell therapeutics. We believe that the major conclusions reached here should instruct thinking about the boundaries around therapeutic designs for CARs, which appear to be very wide, and those around differentiation from TCRs, which appear to be much narrower.

Citation Format: Xueyin Wang, Aaron D. Martin, Kathleen Negri, Michele McElvain, Julyun Oh, Ming-Lun Wu, Wen-Hua Lee, Agnes Hamburger, Han Xu, Alexander Kamb. CARs and TCRs: Myth and reality [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 1540.
Abstract 1541: Incorporation of an IgG4-derived spacer improves the activity of nanobody-based CAR T cells targeting a membrane-distal epitope of glypican 1 in pancreatic cancer
Authors: Li Nan , Li Dan , Pan Jiajia , Ho Mitchell
Published: July 2021

Background & Aims: Epitope recognition and antigen density have emerged as major parameters affecting the anti-tumor activity of chimeric antigen receptor (CAR) T cells. Glypican 1 (GPC1) is a cell surface proteoglycan that is expressed in pancreatic cancer. Here, we investigate how the choice of an epitope and an extracellular spacer in CAR design can determine T cell activation in high- or low- GPC1-expressing tumors.

Methods: We use mouse hybridoma and phage display technologies to isolate GPC1-specific antibodies or nanobodies with distinct epitopes, and analyze GPC1 protein expression in pancreatic cancer. We construct CARs with different extracellular spacers and test them in GPC1-positive cell and mouse models. We use droplet digital PCR and genomic sequencing methods to analyze persistent CAR T cells in mice.

Results: We isolate a camel nanobody D4 and a mouse monoclonal antibody HM2 recognizing the membrane-distal and membrane-proximal epitopes of GPC1, respectively, with similar high affinity. GPC1 protein was frequently detected in pancreatic tumor tissues and normal pancreas adjacent to tumors. Both D4 and HM2 CAR T cells with the hinge and transmembrane domain (TM) from CD8 regressed high GPC1-expressing xenograft tumors. Interestingly, D4 CAR containing a short IgG4 hinge spacer and CD28 TM outperformed the initial D4 CAR against low GPC1-expressing tumors in vitro and in vivo.

Conclusion: By optimizing spacer and TM designs, the activity of nanobody D4-based CAR T cell targeting a membrane-distal epitope of GPC1 is significantly enhanced against low GPC1-expressing tumors, suggesting it might improve the efficacy of GPC1-targeted CAR T cell therapy in pancreatic cancer.

Citation Format: Nan Li, Dan Li, Jiajia Pan, Mitchell Ho. Incorporation of an IgG4-derived spacer improves the activity of nanobody-based CAR T cells targeting a membrane-distal epitope of glypican 1 in pancreatic cancer [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 1541.
Abstract 1567: IL-15 and anti-PD-1 augment the abscopal efficacy of agonistic intratumoral anti-CD40 in the TRAMP-C2 murine tumor model
Authors: Chen Wei
Published: July 2021

IL-15 promotes activation and maintenance of natural killer (NK) and CD8+ T-effector memory (TEM) cells making it a potential immunotherapeutic agent for the treatment of cancer. However, high doses of γc cytokines lead to the paralysis/depression of naïve CD4 but not CD8 T-cells that is mediated through transient expression of suppressor of cytokine signaling 3 (SOCS3). Thus, the out-of-normal order addition of γc cytokines is associated with generation of “helpless” tumor antigen nonspecific CD8 T cells. The role of CD4 helper T cells can be alternatively mediated by CD40 signaling through the addition of agonistic anti-CD40 antibodies. However, parenteral administration of an agonistic anti-CD40 antibody is associated with unacceptable toxicity that can be avoided by intratumoral administration. Intratumoral immunotherapy that aims at generating a potent low toxic priming of antitumor immunity uses the tumor as its own vaccine. We investigated the combination of IL-15 with an intratumoral anti-CD40 monoclonal antibody using a dosing schedule based on our previous study in the TRAMP-C2 murine prostate cancer model. Here we demonstrated that given intratumorally anti-CD40 had abscopal efficacy, and that the combination of IL-15 with the intratumoral anti-CD40 showed an augmented immune response with an increase in the number of tumor specific tetramer positive CD8+ T-cells. Furthermore, anti-PD-1 antibody was additive to the anti-CD40 IL-15 combination. These studies support the initiation of a clinical trial in patients with cancer involving IL-15 in association with intratumoral optimized anti-CD40.

Citation Format: Wei Chen. IL-15 and anti-PD-1 augment the abscopal efficacy of agonistic intratumoral anti-CD40 in the TRAMP-C2 murine tumor model [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 1567.
Abstract 1582: T-SIGn cancer gene therapy and anti-EGFR CAR-T cells synergize in combination therapy to clear A549 lung tumor xenografts and lung metastases in NSG mice
Authors: Sonzogni Olmo , Zak Daniel , West Katy , Lear Rochelle , Muntzer Alice , Champion Brian R. , Rottman James B.
Published: July 2021

Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for hematologic malignancies but has demonstrated limited efficacy in solid tumors. This is largely attributed to the immunosuppressive tumor microenvironment (TME) that impairs the ability of CAR-T cells to infiltrate, become activated, and kill tumor cells. Virotherapy is another emerging cancer treatment that has so far shown only limited clinical efficacy as a monotherapy for solid tumors. Importantly, in addition to their ability to directly kill tumor cells, viral vectors can also be leveraged to deliver transgenes to “reprogram” the TME from an immunosuppressive to a pro-inflammatory environment. We hypothesized that IV-deliverable T-SIGn cancer gene therapy vectors based on the oncolytic adenovirus enadenotucirev, which carry a transgene cassette encoding human immunomodulatory molecules to aid recruitment and activation of T-cells, could enhance the anti-tumor activity of human anti-EGFR CAR T cells in vivo. To test this hypothesis, NSG mice implanted subcutaneously with human lung cancer A549 xenografts, were treated intravenously with the T-SIGn virus followed by treatment with human anti-EGFR CAR-T cells. This treatment regimen demonstrated clearance of the xenograft and pulmonary metastases, whereas either therapy alone showed either no efficacy or delayed disease progression. When xenograft transcript analysis was performed, robust expression of the virus-encoded transgenes, as well as an upregulation of transcripts involved in type I IFN responses (e.g. IRF1, SOCS1), antigen presentation (e.g. HLA.DRA, TAP1), T cell activation (IFNγ, GrB), and inflammation (e.g. CXCL9, TNFSF10) was measured in the xenografts of animals treated with combination therapy, indicating a reprogramming of the TME to more effectively recruit and/or enhance T-cell responses. In addition, the combination treatment also dramatically reduced the number of A549 tumor cell micrometastases detectable in the lungs. Since both agents were delivered systemically, this observation may reflect direct therapeutic activity at the metastatic sites. Taken together, these results indicate that combination therapy with a T-SIGn vector and CAR T cells holds great potential to be efficacious in humans with solid tumors.

Citation Format: Olmo Sonzogni, Daniel Zak, Katy West, Rochelle Lear, Alice Muntzer, Brian R. Champion, James B. Rottman. T-SIGn cancer gene therapy and anti-EGFR CAR-T cells synergize in combination therapy to clear A549 lung tumor xenografts and lung metastases in NSG mice [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 1582.
Abstract 1584: Efficacy and pharmacodynamic effect of anti-CD73/PD-L1 monoclonal antibodies in combination with chemotherapy: Observations from mouse tumor models
Authors: Eyles Jim , Watkins Amanda , Ilieva Kristina , Mullins Stef , Anderton Jude , Galvani Elena , Garcon Fabien , Ryan Kelli , Kaistha Brajesh P. , Dannhorn Andreas , Ling Stephanie , Slidel Tim , Kar Gozde , Schuller Alwin , Cooper Zachary A. , Sachsenmeier Kris , Luheshi Nadia , Kumar Rakesh , Wilkinson Robert W.
Published: July 2021

Intratumoral adenosine is a key immunosuppressive factor linked to poor prognosis and reduced efficacy of T cell checkpoint inhibitors. CD73 is an ectoenzyme and key node in the catabolic pathway responsible for sequential hydrolysis of extracellular ATP to adenosine. ATP is released from necrotic and damaged tumor cells; a phenomenon enhanced as consequence of cytotoxic chemotherapy or radiotherapy. A CD73 inhibiting human monoclonal IgG1-TM antibody, Oleclumab, is currently in phase 2 clinical development for treatment of patients with various solid tumors. The combination of CD73 inhibition with chemotherapy and T cell checkpoint inhibition was tested using two murine cancer models, CT26 (colorectal) or MCA205 (fibrosarcoma) implanted subcutaneously in BALB/c mice or C57BL/6 mice, respectively. Tumor bearing mice were treated with combinations of oxaliplatin and 5-fluorouracil and murine surrogate monoclonal antibodies for Oleclumab and Durvalumab (anti-PD-L1). CT26 implanted mice were also treated with the murine surrogate antibodies in the presence and absence of Docetaxel. In an attempt to define contribution of components, comparator groups received monotherapies and other iterations of the combination. Treatment with anti-CD73 and anti-PD-L1 antibodies, concomitantly with chemotherapy, resulted in improved tumor growth inhibition, plus increased proportions of complete tumor regression (P<0.05). FACS analysis of CT26 tumors highlighted elevated frequencies of intratumoral IFN-gamma secreting T and NK cells in the chemotherapy plus double IO combination group (P<0.05). Consistent with this, selective depletion of CD8 T cells significantly diminished tumor control and long term survival (P<0.05). RNAseq and Mass spectrometry based imaging techniques were utilized as tools to explore biomarker changes and mechanism.

Citation Format: Jim Eyles, Amanda Watkins, Kristina Ilieva, Stef Mullins, Jude Anderton, Elena Galvani, Fabien Garcon, Kelli Ryan, Brajesh P. Kaistha, Andreas Dannhorn, Stephanie Ling, Tim Slidel, Gozde Kar, Alwin Schuller, Zachary A. Cooper, Kris Sachsenmeier, Nadia Luheshi, Rakesh Kumar, Robert W. Wilkinson. Efficacy and pharmacodynamic effect of anti-CD73/PD-L1 monoclonal antibodies in combination with chemotherapy: Observations from mouse tumor models [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 1584.
Abstract 1604: Anticancer nanocage platforms for combined immunotherapy designed to harness immune checkpoints and deliver anticancer drugs
Authors: Kim Soyoun , Kim Minseong
Published: July 2021

The interaction of programmed cell death 1 ligand 1 (PD-L1) with its receptor, programmed cell death 1 (PD-1), inhibits T-cell responses. Monoclonal antibodies that block this interaction have been shown effective as immunotherapy. However, only a subset of cancers exhibits a durable response to PD-1/PD-L1 blockade. Moreover, antibody-based immune checkpoint blockade is costly and is occasionally accompanied by systemic side effects. To overcome these limitations of antibody-based immune checkpoint blockade, an immune checkpoint-blocking ferritin nanocage displaying 24 PD-L1 binding peptides (PD-L1pep1) on its surface was designed and constructed. These ferritin nanocages displaying PD-L1pep1 (PpNF) specifically bind to PD-L1 expressed on cancer cells or to purified PD-L1 with a ~30 nM binding affinity. The addition of PpNF to cocultures of T cells and cancer cells inhibited PD-1/PD-L1 interactions and restored T-cell activities. In a mouse model of syngeneic colon cancer, PpNF specifically targeted tumors and showed antitumor activity. Moreover, PpNF nanocages encapsulating the chemotherapeutic drug doxorubicin had more potent antitumor activity than a monoclonal antibody against PD-L1. These results demonstrate that ferritin nanocages displaying surface PD-L1pep1 can be efficiently applied for immunotherapy, especially when encapsulating small chemotherapeutic drugs. These nanocages may have promise as an immunotherapeutic nanomedicine against various solid tumors.

Citation Format: Soyoun Kim, Minseong Kim. Anticancer nanocage platforms for combined immunotherapy designed to harness immune checkpoints and deliver anticancer drugs [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 1604.