Long-term Survival Of Mice Research Articles

Gasdermin-E-mediated pyroptosis drives immune checkpoint inhibitor-associated myocarditis via cGAS-STING activation

Immune checkpoint inhibitor (ICI)-induced myocarditis involves intensive immune/inflammation activation; however, its molecular basis is unclear. Here, we show that gasdermin-E (GSDME), a gasdermin family member, drives ICI-induced myocarditis. Pyroptosis mediated by GSDME, but not the canonical GSDMD, is activated in myocardial tissue of mice and cancer patients with ICI-induced myocarditis. Deficiency of GSDME in male mice alleviates ICI-induced cardiac infiltration of T cells, macrophages, and monocytes, as well as mitochondrial damage and inflammation. Restoration of GSDME expression specifically in cardiomyocytes, rather than myeloid cells, in GSDME-deficient mice reproduces ICI-induced myocarditis. Mechanistically, quantitative proteomics reveal that GSDME-dependent pyroptosis promotes cell death and mitochondrial DNA release, which in turn activates cGAS-STING signaling, triggering a robust interferon response and myocardial immune/inflammation activation. Pharmacological blockade of GSDME attenuates ICI-induced myocarditis and improves long-term survival in mice. Our findings may advance the understanding of ICI-induced myocarditis and suggest that targeting the GSDME-cGAS-STING-interferon axis may help prevent and manage ICI-associated myocarditis.

Enhancing brain entry and therapeutic activity of chimeric antigen receptor T cells with intra-arterial NEO100 in a mouse model of CNS lymphoma.

Malignancies of the CNS are difficult to treat because the blood-brain barrier (BBB) prevents most therapeutics from reaching the intracranial lesions at sufficiently high concentrations. This also applies to chimeric antigen receptor (CAR) T cells, for which systemic delivery is inferior to direct intratumoral or intraventricular injection of the cells. The authors previously reported on a novel approach to safely and reversibly open the BBB of mice by applying intra-arterial (IA) injections of NEO100, a pharmaceutical-grade version of the natural monoterpene perillyl alcohol. The authors hypothesized that this method would enable enhanced brain entry and therapeutic activity of intravenously delivered CAR T cells, which the authors tested in a mouse model of CNS lymphoma. Human Raji lymphoma cells were implanted into the brains of immune-deficient mice. After tumor uptake was confirmed with bioluminescent imaging, 0.3% NEO100 was injected intra-arterially, which was followed by intravenous (IV) delivery of CD19-targeted CAR T cells. After this single intervention, tumor growth was monitored with imaging, long-term survival of mice was recorded, and select mice were euthanized to analyze the distribution of CAR T cells in brain tissue. Intravenously injected CAR T cells could be readily detected in brain tumor areas after IA injection of NEO100 but not after IA injection of the vehicle (without NEO100). Although all untreated control animals died within 3 weeks, all mice that received IA NEO100 followed by IV CAR T cells survived and thrived for 200 days, when the experiment was terminated. Of the mice that received IV CAR T cells without prior IA NEO100, 3 died within 3 weeks and 2 survived long-term. BBB opening by IA NEO100 facilitates brain entry of intravenously delivered CD19 CAR T cells. The long-term survival of all mice with CNS lymphoma, along with the disappearance of the tumor as determined with imaging, suggests that this one-time therapeutic intervention was curative. BBB opening by IA NEO100 may offer a novel option to increase brain access by CAR T cells.

TMIC-70. A TRANSCRIPTIONALLY ACTIVE EPIGENETIC IMPRINTING OF THE JAK/STAT PATHWAY MEDIATED BY H3.3-G34 MUTATIONS IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG) INDUCES AN IMMUNOREACTIVE TME

Abstract Pediatric high-grade gliomas (pHGGs) stand as the primary cause of cancer-associated fatalities among children. Sixteen percent of pHGGs found in children and young adults present Gly34Arg/Val substitutions within the histone H3.3 (H3.3-G34R/V). Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we showed that this mutation leads to downregulation of the DNA repair pathways and enhanced susceptibility to DNA damage. DNA damage response (DDR) inhibitors in vivo, further increased genetic instability, and activation of the cGAS-STING pathway, which induces the release of immune-stimulatory cytokines. We carried out an epigenetic analysis using ChIP-seq in our GEMM, and our findings indicate that this mutation triggers transcriptionally active chromatin states within genes linked to immune reactivity, particularly JAK-STAT signaling pathway genes. Correspondingly, G34-mutant pHGG cells release immune-activating cytokines that reprogram the immune cells within the tumor microenvironment (TME). Myeloid derived suppressor cells from G34-mutant pHGG are phenotypically less immunosuppressive than the MDCS form histone wild type pHGG. Moreover, G34-mutant pHGG TME exhibit higher frequency of CD8 T cells. Based on these findings, we assessed the efficacy of an adenoviral-mediated immunostimulatory gene therapy (GT). This therapy mediates expression of Flt3L within pHGG TME, which aids dendritic cell recruitment. Thymidine kinase (TK), converts ganciclovir (GSV) into a cytotoxic compound, leading to pHGG cells’ death. GT resulted in 60% of long-term survival in mice harboring G34R pHGG. The animals that survived due to the GT did not develop tumors after a rechallenge with G34R pHGG cells, demonstrating that the treatment induces antitumoral immunological memory. In conclusion, our findings suggest that G34-mutant pHGG patients could benefit from combined therapies that target genetic instability and stimulate the immune system. Funded by NIH/NINDS.

Normalization of Tumor Vessels by Lenvatinib-Based Metallo-Nanodrugs Alleviates Hypoxia and Enhances Calreticulin-Mediated Immune Responses in Orthotopic HCC and Organoids.

Immunocheckpoint inhibitors combined with Lenvatinib is the first line treatment for hepatocellular carcinoma (HCC), but their potency is hampered by the low response rate and adverse events. Herein, a targeted therapeutic strategy through the coassembly of Lenvatinib, Adriamycin, Fe3+ ion, and a natural polyphenol (metallo-nanodrugs) is presented by coordination effect for potentiating tumor vascular normalization and systematic chemo-immunotherapy to effectively inhibit the progression of HCC in both orthotopic model and patients-derived organoids. In mice with orthotopic HCC, the obtained metallo-nanodrugs efficiently increase the drug accumulation in orthotopic tumors and can respond to acidic tumor environment. The promotion of tumor vascular normalization by metallo-nanodrugs is observed, which enhances the infiltrating T lymphocytes in tumor, and reinforces the calreticulin-mediated antitumor immunity through alleviating hypoxia, reducing regulatory T cells, and down-regulating PDL1 expression of tumors. The excellent therapeutic efficiency with complete remission of orthotopic tumors (3/6) and long-term survival of mice (4/6, 42 days) are also achieved. Furthermore, the excellent therapeutic effect of metallo-nanodrugs is also validated in 5 patient-derived organoids, and hence can provide a marvelous systemic chemo-immunotherapy strategy for enhancing HCC treatment.

The future treatment for type 1 diabetes: Pig islet- or stem cell-derived β cells?

Replacement of β cells is only a curative approach for type 1 diabetes (T1D) patients to avoid the threat of iatrogenic hypoglycemia. In this pursuit, islet allotransplantation under Edmonton's protocol emerged as a medical miracle to attain hypoglycemia-free insulin independence in T1D. Shortage of allo-islet donors and post-transplantation (post-tx) islet loss are still unmet hurdles for the widespread application of this therapeutic regimen. The long-term survival and effective insulin independence in preclinical studies have strongly suggested pig islets to cure overt hyperglycemia. Importantly, CRISPR-Cas9 technology is pursuing to develop "humanized" pig islets that could overcome the lifelong immunosuppression drug regimen. Lately, induced pluripotent stem cell (iPSC)-derived β cell approaches are also gaining momentum and may hold promise to yield a significant supply of insulin-producing cells. Theoretically, personalized β cells derived from a patient's iPSCs is one exciting approach, but β cell-specific immunity in T1D recipients would still be a challenge. In this context, encapsulation studies on both pig islet as well as iPSC-β cells were found promising and rendered long-term survival in mice. Oxygen tension and blood vessel growth within the capsules are a few of the hurdles that need to be addressed. In conclusion, challenges associated with both procedures, xenotransplantation (of pig-derived islets) and stem cell transplantation, are required to be cautiously resolved before their clinical application.

Abstract LB009: A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome-engineered to express an HLA-E fusion transgene to prevent immune cell rejection

Abstract The approval and commercial launch of multiple first-generation CD19- or BCMA-directed, autologous CAR-T cell products have laid the foundation and opened a path for the development of more advanced cellular therapeutics, including CAR-T cell products with next-generation capabilities. Among these newer designs, allogeneic cell therapies are positioned to unlock the broad potential of engineered immune cells as a leading therapeutic modality. However, expansion, persistence, armoring, and trafficking of allogeneic CAR-T cells are critical to achieving long-term efficacy. Caribou Biosciences is advancing a BCMA-specific allogeneic CAR-T cell product candidate, CB-011, with an immune cloaking approach that includes both the removal of the endogenous B2M protein and the insertion of a B2M-HLA-E-peptide (B2M-HLA-E) fusion protein transgene. This strategy is designed to blunt CAR-T cell rejection by both recipient T cells and NK cells, and CB-011 is in preclinical development for relapsed or refractory multiple myeloma (r/r MM). We used Cas12a chRDNA guides in the manufacture of CB-011 to make four edits, including site-specific insertion of a proprietary humanized anti-BCMA CAR into the TRAC locus with high specificity and efficiency, thus eliminating TCR expression to prevent graft-versus-host disease (GvHD). In addition, we inserted a gene encoding a B2M-HLA-E fusion protein into the native B2M gene locus. This method simultaneously prevents the expression of the native B2M protein and expresses a minor HLA class I antigen HLA-E to blunt both T- and NK-mediated rejection of the CAR-T cells by the recipient’s immune system. B2M is a protein that stabilizes all HLA class I antigens on the cell surface, therefore its knockout eliminates endogenous HLA class I presentation on the surface of the CAR-T cells. To demonstrate that the B2M-HLA-E fusion protein expression protects CB-011 from NK-mediated cell killing, we developed an in vitro competition assay in which CB-011 cells are co-incubated with NK cells. We observed that CAR-T cells expressing the B2M-HLA-E fusion have a survival advantage over cells that do not express the fusion in the presence of NK cells in vitro, indicating that they could resist killing by a recipient’s NK cells and potentially circulate longer. The BCMA-specific CAR leads to long-term survival in mice bearing established orthotopically-engrafted MM tumor cells. Furthermore, mice treated with high doses of CB-011 did not experience symptoms typical of GvHD. This strategy should enable CB-011 CAR-T cells to remain in circulation longer in recipients, providing for increased potential of antitumor activity. Citation Format: Elizabeth Garner, Emilie Degagne, Suparna Roy, Paul Donohoue, Tristan Fowler, Morena Stanaway, Vanina Vicena, Devin Mutha, Benjamin Schilling, McKay Shaw, Mara Bryan, Leslie Edwards, Stephen Smith, Bryan Kohrs, Lynda Banh, Kyle McSweeney, Justin Skoble, Steven Kanner. A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome-engineered to express an HLA-E fusion transgene to prevent immune cell rejection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB009.

Anti-PD-L1 treatment to enhance the response of glioblastoma to radiation and produce long-term survival in mice.

e14048 Background: Glioblastoma (GBM) is the most aggressive and most common primary central nervous system cancer in adults. Blocking the interaction between Programmed Cell Death Protein-1 (PD-1) and its ligand (PD-L1) has shown remarkable success in the treatment of several cancers. However, many challenges remain in improving the efficacy of using monoclonal antibodies (Ab) against the receptor PD-1 in GBM, mainly due to the “non-immunogenic” tumor characteristics of GBM. PD-L1 has been found to be overexpressed on the surface of human GBM tumor cells and tumor-associated macrophages (TAM). Radiotherapy (RT), as one of the standard therapy of GBM, could alter the tumor microenvironment and promote an antitumor immune response. We hypothesize that anti-PD-L1 therapy can enhance the RT effects and improve the outcome of treatment when combined. Methods: Using a preclinical orthotropic syngeneic CT-2A mouse GBM tumor model, we studied the efficacy of combined therapy with anti-PD-L1 and RT. Mice were stratified into four treatment groups: control, RT, anti-PD-L1 Ab, and anti-PD-L1 Ab plus RT. RT(8 Gy) was given one time simultaneously with the first dose of anti-PD-L1, followed by systemic anti-PD-L1 maintenance treatment to the mice. Overall survival and tumor growth were monitored. Immunohistochemistry on resected tumors during treatment was performed to characterize the immune response. Single-cell RNA sequencing (scRNA-seq) was also performed to further study the immunologic parameters in the mouse brain. Results: Our results showed that anti-PD-L1 Ab in combination with RT provided a remarkable antitumor immune response and improved overall survival, with 25.5, 34, and 30 days of median survival in control (no-treatment), RT, and anti-PD-L1 groups, respectively, and achieving long-term survival and complete tumor response in 80% of the mice in the anti-PD-L1+RT treatment group (median survival not reached) in GBM tumor-bearing mice. The combined therapy promoted the recruitment of tumor-infiltrating immune cells, reversed the hostile tumor immune environment with a higher M1/TAM ratio, CD8+ /CD4+ T cell ratio, and CD8+ T cell /Treg cell ratio in the tumor area comparing with those parameters in single modality treatment groups. Furthermore, scRNA-seq data demonstrated that anti-PD-L1 combined with RT resulted in robust higher CD8 effector T cells, while lower CD4 and CD8 exhausted T cells in the tumor region compared to other groups. Increased CD4 central memory T cells and CD8 central memory T cells were seen only in tumors treated with anti-PD-L1+RT providing immunologic explanations on the durable control of GBM achieved only by the combined therapy. Conclusions: The anti-PD-L1 therapy synergizes with RT by reversing the hostile tumor immune environment resulting in improved tumor control and long-term survival in the syngeneic mouse GBM model.

An effective peptide vaccine strategy circumventing clonal MHC heterogeneity of murine myeloid leukaemia

BackgroundTherapeutic cancer vaccines are an attractive approach for treating malignant tumours, and successful tumour eradication depends primarily on controlling tumour immunosuppression status as well as heterogeneity of tumour cells driven by epigenetic alterations.MethodsPeptide-loaded dendritic cell (DC) prime and non-infectious peptide booster heterologous immunisations were assessed for the immunogenicity of polo-like kinase-1 (PLK1)-derived peptides. Heterologous vaccination regimen targeting multiple shared tumour antigens simultaneously with PD-L1 blockade was assessed against murine myeloid leukaemia.ResultsA synthetic PLK1122 (DSDFVFVVL)-based heterologous vaccination generated large numbers of long-lasting antigen-specific CD8 T-cells eliciting therapeutic effects against various established tumours. The therapeutic efficacy of single antigen-targeting PLK1122-based vaccine with sufficient endurance of PD-L1 blockade toward C1498 leukaemia relied on the heterogeneous clonal levels of MHC-I and PD-L1 expression. A novel multi-peptide-based vaccination targeting PLK1 and survivin simultaneously along with PD1 blockade led to complete tumour eradication and long-term survival in mice with clonally heterologous C1498 myeloid leukaemia.ConclusionsOur findings suggest that PLK1 could be an attractive immunotherapeutic target antigen for cancer immunotherapy, and that similar strategies would be applicable for the optimisation of cancer vaccines for the treatment of numerous viral diseases and malignant tumours.

An effective peptide vaccine strategy circumventing heterogeneity of murine myeloid leukemia

Abstract Therapeutic cancer vaccines are an attractive approach for treating malignant tumors, and successful tumor eradication depends primarily on controlling tumor immunosuppression status as well as heterogeneity of tumor cells driven by epigenetic alterations. Here we describe an effective peptide vaccination approach capable of circumventing antigenic heterogeneity by targeting multiple shared tumor antigens simultaneously in murine myeloid leukemia. Peptide-loaded dendritic cell (DC) prime and noninfectious peptide booster heterologous immunizations were assessed for the immunogenicity of polo-like kinase-1 (PLK1)-derived peptides, and targeting multiple shared tumor antigens simultaneously with PD-L1 blockade was evaluated for antitumor effects against murine myeloid leukemia. A synthetic PLK1122 (DSDFVFVVL)-based heterologous vaccination generated large numbers of long-lasting antigen-specific CD8 T-cells eliciting therapeutic effects against various established tumors. The therapeutic efficacy of single antigen-targeting PLK1122-based vaccine with sufficient endurance of PD-L1 blockade toward C1498 leukemia relied on the heterogeneous clonal levels of MHC-I and PD-L1 expression. A novel multi-peptide-based vaccination targeting PLK1 and survivin simultaneously along with PD1 blockade led to complete tumor eradication and long-term survival in mice with clonally heterologous C1498 myeloid leukemia. Our findings suggest that PLK1 could be an attractive immunotherapeutic target antigen for cancer immunotherapy, and that similar strategies would be applicable for the optimization of cancer vaccines for the treatment of numerous viral diseases and malignant tumors.

EXTH-09. FIRST-IN-HUMAN DOSING CONSIDERATIONS OF A BISPECIFIC ANTIBODY FOR TREATING GLIOBLASTOMA

Abstract Current therapy for glioblastoma (GBM) is incapacitating and limited by non-specific toxicity to the surrounding brain. We have developed an immunotherapeutic approach that selectively targets GBM by redirecting the patients’ own T cells towards the tumor in an antigen-specific manner using a bispecific antibody. Our novel bispecific antibody (“BRITE”) binds GBM-specific surface marker EGFRvIII and the CD3 receptor on T cells, resulting in crosslinking and tumor-specific cell lysis. We previously showed in patient-derived and syngeneic murine glioma models, that treatment with BRITE leads to long-term survival in mice with glioblastoma. A pharmacokinetic analysis in a CD3 humanized mouse revealed that BRITE in plasma and whole blood has an initial half-life of ~8 minutes and a terminal half-life of ~2.5 hours. Given our preclinical success, we have initiated clinical trial-enabling studies, including studies of BRITE toxicology and GMP manufacturing of drug product. A crucial consideration for our proposed Phase 1 dose escalation trial is the starting dose in humans. To this end, we utilized the FDA-recommended minimum anticipated biological effect level (MABEL) of BRITE – a holistic approach that considers all available in vitro and in vivo data – to calculate the first-in-human starting dose of BRITE.

An inhalable nanoparticulate STING agonist synergizes with radiotherapy to confer long-term control of lung metastases

Mounting evidence suggests that the tumor microenvironment is profoundly immunosuppressive. Thus, mitigating tumor immunosuppression is crucial for inducing sustained antitumor immunity. Whereas previous studies involved intratumoral injection, we report here an inhalable nanoparticle-immunotherapy system targeting pulmonary antigen presenting cells (APCs) to enhance anticancer immunity against lung metastases. Inhalation of phosphatidylserine coated liposome loaded with STING agonist cyclic guanosine monophosphate–adenosine monophosphate (NP-cGAMP) in mouse models of lung metastases enables rapid distribution of NP-cGAMP to both lungs and subsequent uptake by APCs without causing immunopathology. NP-cGAMP designed for enhanced cytosolic release of cGAMP stimulates STING signaling and type I interferons production in APCs, resulting in the pro-inflammatory tumor microenvironment in multifocal lung metastases. Furthermore, fractionated radiation delivered to one tumor-bearing lung synergizes with inhaled NP-cGAMP, eliciting systemic anticancer immunity, controlling metastases in both lungs, and conferring long-term survival in mice with lung metastases and with repeated tumor challenge.

1225P - THOR-707, a novel not-alpha IL-2, promotes all key immune system anti-tumoral actions of IL-2 without eliciting vascular leak syndrome (VLS)

BackgroundTHOR-707 is a reprogrammed, site-directed, single pegylated, recombinant human IL-2 (rIL-2) variant that lacks binding affinity for the α chain of the IL-2 receptor. Because THOR-707 has near-native binding affinity for the βγ chain of the IL-2 receptor, it produces an immune-active, CD8+ T effector-driven anti-tumor effect. At the same time because it lacks α chain affinity, it limits the proliferation of immune suppressive CD4+ Tregs and other innate lymphoid cells known to mediate the life-threatening complications of VLS. These pharmacologic properties make THOR-707 an ideal immunooncology drug development candidate. MethodsWe employed in vivo and ex vivo models to study pharmacodynamic responses to THOR-707. ResultsWe demonstrate all critical actions of IL-2 for anti-tumoral responses without inducing VLS, including: (1) maximal extravasation and expansion of key CD8+ T and NK cell populations in mice and NHPs (2) CD8+ T cell infiltration of tumor and lymph nodes in mouse tumor models, critical for antigen presentation by tumor cells and trans-presentation by antigen presenting cells and (3) induction of IFNγ release ex vivo by T cell receptor (TCR)-activated human T cells that is further potentiated by a PD-1 inhibitory antibody, important for upregulation of MHC I and II and enhancement tumor antigen presentation. In mouse syngeneic tumor models, THOR-707 promoted an increase in the number of infiltrating tumor-killing NK and CD8+ T cells without expansion of suppressive CD4+ Treg cells. Expression profiling of tumors revealed promotion of remodeling of cellular populations towards a composition favoring cytolytic phenotypes. Evaluation of TCR clonality revealed that treatment with THOR-707 increased intra-tumoral T cell diversity. ConclusionsTHOR-707 induction of CD8+ T cell tumor infiltration resulted in single agent dose-dependent anti-tumor efficacy, additive efficacy in combination with a PD-1 checkpoint inhibitor, promoted long-term survival in mice and NHPs and rejection of tumor cells on re-challenge. Based on these results, first in human studies are expected to begin this year in solid tumors both as a single agent and in combination with an immune-checkpoint inhibitor. Legal entity responsible for the studySynthorx, Inc. FundingSynthorx, Inc. DisclosureM.E. Milla: Leadership role, Shareholder / Stockholder / Stock options, Licensing / Royalties, Full / Part-time employment: Synthorx, Inc.; Licensing / Royalties: Adaptive Biotechnologies, Inc. J.L. Ptacin: Full / Part-time employment: Synthorx, Inc. L. Ma: Full / Part-time employment: Synthorx, Inc. C.E. Caffaro: Full / Part-time employment: Synthorx, Inc. H.R. Aerni: Full / Part-time employment: Synthorx, Inc. K.M. San Jose: Full / Part-time employment: Synthorx, Inc. M.J. Pena: Full / Part-time employment: Synthorx, Inc. R.W. Herman: Full / Part-time employment: Synthorx, Inc. Y. Pavlova: Full / Part-time employment: Synthorx, Inc. D.B. Chen: Full / Part-time employment: Synthorx, Inc. T.K. Ismaili: Full / Part-time employment: Synthorx, Inc. S. Li: Full / Part-time employment: Synthorx, Inc. J. Nguyen: Full / Part-time employment: Synthorx, Inc. N. Singh: Full / Part-time employment: Synthorx, Inc. L.K. Shawver: Leadership role, Shareholder / Stockholder / Stock options, Licensing / Royalties, Officer / Board of Directors: Synthorx, Inc.; Shareholder / Stockholder / Stock options: Cleave Biosciences; Shareholder / Stockholder / Stock options: Relay Therapeutics. L.K. Koriazova: Full / Part-time employment: Synthorx, Inc. I.B. Joseph: Full / Part-time employment: Synthorx, Inc.

Abstract 2059: Long-term survival of mice with relapsed ALL treated by oncolytic measles virus is terminated by expansion of persistently infected virus-resistant blasts

Abstract Background: The prognosis of patients with relapsed and refractory ALL is poor. Oncolytic virotherapy is a promising treatment modality combining cancer cell lysis with immunomodulation. Aims: To determine whether clinical-grade attenuated measles virus (MV) controls relapsed and refractory ALL in vitro and in vivo, to assess resistance mechanisms and to outline targets for immunotherapy in resistant cells. Methods: The clinical-grade MV-NIS strain was used. ALL cell lines insensitive to chemotherapy and ALL xenografts derived from patients that had died from their disease were investigated. In vitro, cells were treated with MV-NIS and viability was determined. NSG mice were transplanted with a relapsed ALL PDX. Treatment was started when the leukemic load was high, i.e. when 20-30% blasts were present in the peripheral blood, and when the disease had heavily infiltrated spleen, liver, bone marrow and CNS. One dose of i.v. MV-NIS was given. Peripheral blast counts and survival of mice were determined. At time of death the presence of MV in leukemic infiltrates was determined by immunohistochemistry and qRT-PCR, MV was isolated from blasts and serum, and infectivity of isolated MV was tested on Vero indicator cells. mRNA from the blasts, including the genome of the MV they were infected with (MV is a mRNA virus), was subjected to mRNA sequencing. Gene set enrichment and mutation analyses were performed. Results: In vitro, MV-NIS effectively killed ALL cell lines insensitive to chemotherapy and cells from relapsed patient ALL. In NSG mice with a high and disseminated load of relapsed patient ALL cells, one injection of i.v. MV-NIS sufficed to rapidly decrease leukemic load leading to long-term survival of the mice. Survival was terminated by late disease exacerbation due to expansion of virus-resistant leukemic cells persistently infected with infectious MV. Genome-wide mRNA sequencing revealed strong enrichment of antiviral pathways in the resistant cells, without mutations in the viral genome that could explain the resistance of the blasts. Conclusions: MV-NIS is preclinically effective against relapsed and refractory ALL in vitro and, initially, in vivo. Late expansion of virus-resistant cancer cells persistently infected with MV is a phenomenon hitherto not described. The viral and antiviral proteins strongly expressed in these cells, which include cell surface proteins, can serve as targets for subsequent immunotherapy. Citation Format: Annika V. Goß, Carmen Dorneburg, Medhanie Mulaw, Chun Xu, Christine Landthaler, Irmela Jeremias, Jiwu Wei, Klaus-Michael Debatin, Christian Beltinger. Long-term survival of mice with relapsed ALL treated by oncolytic measles virus is terminated by expansion of persistently infected virus-resistant blasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2059.

Abstract OT2-06-02: Chemokine modulation to enhance the effectiveness of pembrolizumab in patients with metastatic triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is found in approximately 15-20% of all breast cancer, and is associated with poor prognosis, early relapse and a significantly shorter survival following recurrence. Early phase trials with immune checkpoint inhibitors have shown modest yet durable clinical responses with a tolerable safety profile. Strategies to sensitize these tumors to checkpoint inhibition will result in decreased morbidity and mortality. Tumor infiltrating immune cells and intratumoral expression of PD-1/PD-L1/PD-L2 can predict patients' benefit from Pembrolizumab. Pre-clinical ex-vivo data show that chemokine modulating regimen consisting of Rintatolimod, IFNα and COX2 blocker (CKM), selectively attracts cytotoxic T cells into tumors, and increases intratumoral expression of PD-1/PD-L1/PD-L2, without enhancing soluble suppressive mechanisms. Furthermore, mouse data has shown safety of CKM and PD-1 blockade combination and efficacy in inducing long-term survival of mice with resistant tumors. Clinical trials of CKM in Colon and ovarian cancer (NCT01545141, NCT02432378) demonstrated safety of Rintatolimod given with IFNα/COX2, and preliminary data show local efficacy in tumor microenvironment modulation. Design: This is an open-label, single center, phase IIa study to test Chemokine modulating regimen (CKM) pre-treatment followed by Pembrolizumab in patients with metastatic TNBC, regardless of PD-1 expression, who progressed on ≥1 lines of therapy. Patients are given pre-treatment CKM, which consists of Rintatolimod (200 mg IV), IFNα-2 (20 million units/m2 IV) and Celecoxib (200 mg po BID), on 3 consecutive days for a total of 2 cycles, one week apart. The patient is then treated with Pembrolizumab 200 mg IV every 3 weeks until disease progression, intolerable side effects or withdrawal from study for up to 24 months. Study includes pre- and post CKM treatment biopsies. Eligibility: Major criteria include age ≥ 18 years, ECOG ≤ 1, histologically proven metastatic TNBC, normal organ and marrow function, no active autoimmune disease or history of transplant, no prior anti-PD1/PDL1 therapy. Aims: Primary objective is to evaluate the overall response rate to the combination therapy per irRECIST criteria. Secondary objectives include safety profile of the combination therapy, determining progression free survival, overall survival and disease control rate. Other immune exploratory objectives will include baseline and CKM-induced predictive biomarkers of clinical activity of the combination treatment. Statistical Methods: The study includes a safety lead-in of 6 patients and utilizes a Simon two-stage minimax design. 18 patients are enrolled into stage 1 of the study. If < 4 responses are observed, the treatment combination will not be considered promising and enrollment will be terminated. However, if ≥ 4 responses are observed, then an additional 19 patients will be enrolled into stage 2, for a total number of 37 patients. In stage 2, if ≥ 9 responses are observed, then the treatment combination will be considered promising for future study. Citation Format: Abdou Y, Williams LE, Kalinski P, Opyrchal M. Chemokine modulation to enhance the effectiveness of pembrolizumab in patients with metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT2-06-02.

Delayed activation of PPAR-β/δ improves long-term survival in mouse sepsis: effects on organ inflammation and coagulation.

Activation of peroxisome proliferator-activated receptor (PPAR)-β/δ reduces tissue injury in murine endotoxemia. We hypothesized that the PPAR-β/δ-agonist GW0742 improves long-term outcome after sepsis caused by cecal ligation and puncture (CLP). Fifty-one CD-1 female mice underwent CLP and received either vehicle (control), GW0742 (0.03 mg/kg/injection; five post-CLP i.v. injections), GSK0660 (PPAR-β/δ-antagonist) or both and were monitored for 28 d. Another 20 CLP mice treated with GW0742 and vehicle were sacrificed 24 h post-CLP to assess coagulopathy. Compared to vehicle, survival of CLP-mice treated with GW0742 was higher by 35% at d 7 and by 50% at d 28. CLP mice treated with GW0742 had 60% higher IFN-γ but circulating monocyte chemoattractant protein-1 and chemokine ligand were lower at 48 h post-CLP. Compared to vehicle, CLP mice treated with GW0742 exhibited a 50% reduction in the circulating plasminogen activator inhibitor-1 associated with an increase in platelet number at 24 h post-CLP (but no changes occurred in anti-thrombin-III, plasminogen, fibrinogen and clotting-times). CLP mice treated with GW0742 exhibited a similar increase in most of the biochemical markers of organ injury/dysfunction (lactate dehydrogenase, alanine aminotransferase, creatine kinase, creatinine, blood urea nitrogen, and triglycerides) measured. Treatment with GW0742 consistently improved long-term survival in septic CD-1 mice by partially modulating the post-CLP systemic cytokine response and coagulation systems.

Abstract PR03: Prophylactic nodal irradiation abrogates the synergy of tumor radiotherapy and immune checkpoint blockade

Abstract Background: Prophylactic nodal irradiation (PNI) is a strategy used to treat early stage cancers with the potential to enhance local control and prevent metastatic spread. It remains unclear whether irradiation of tumor-associated draining lymph nodes (LN) influences anti-tumor T cell responses. Our aim was to investigate the in vivo effects of nodal irradiation on the phenotype and function of tumor infiltrating lymphocytes (TIL) and the impact of PNI on the anti-tumor effects of immunotherapy. Methods: The Small Animal Radiation Research Platform (SARRP) delivered imaged-guided stereotactic radiation (RT) to tumor (T-only) or tumor and LN (T+LN). Syngeneic tumors (MC38 colon, B16 melanoma) were implanted in C57BL/6 mice and irradiated (12Gy x1) and/or treated with immune checkpoint blockade with αCTLA-4 IgG2a (depleting) or αPD-1 IgG1. The composition of the tumor microenvironment (TME) was assayed by flow cytometry. To query tumor-antigen specific T cell responses, adoptive transfer experiments were performed using OVA-specific CD8+ T-cells from Rag-/- OT-1 mice and OVA-MHC class I tetramer (SIINFEKL). Tumor lysate was also collected for chemokine analysis, and tumor outgrowth was quantified over time. Results: T-only RT resulted in a significant increase (P<0.01) in the proportion and absolute number of tumor infiltrating CD8+ effector T cells in comparison with T+LN RT. Immunosuppressive subsets (CD11b+ Gr-1hi MDSCs and FoxP3+ CD4+ Tregs) were also significantly increased in the T-only group. Ultimately, T-only RT significantly enhanced the CD8 effector:Treg ratio relative to untreated and T+LN treated tumors. Using the OVA-MHC class I tetramer to identify tumor-antigen specific CD8 T-cells, we observed that T-only RT significantly increased (P<0.05) the number of tumor-specific CD8-T cells in the TME compared with T+LN RT. Interestingly, T-only RT resulted in a a significant expansion of non-OVA specific CD8 T cells which was not observed with T+LN RT, suggesting a polyclonal anti-tumor immune response. Functionally, a significant increase (P<0.01) in the absolute number of IFNγ+ and TNFα+ antigen-specific TIL were noted with T-only RT. Mechanistically, a distinct chemokine signature correlated with robust TME immune infiltration and significantly elevated levels of CCL3/4/5 and CXCL10 (P<0.05) were observed in tumor lysate collected from T-only RT tumors relative to T+LN RT samples. In an effort to understand the implications of the RT target on potential synergy with immune checkpoint blockade we performed survival experiments with T-only and T+LN RT in combination with αPD-1 or αCTLA-4. T-only RT in combination with αCTLA-4 yielded the best outcome with a 86% long-term survival (day 90 post-RT) compared with 30-36% long-term survival in mice treated with other combinations of RT + immune checkpoint blockade. Intriguingly, favorable CD8 effector:Treg ratio was able to predict treatment response and was dramatically higher (P<0.001) among mice treated with T-only RT in combination with αCTLA-4. Conclusions: We have successfully developed a SARRP-based early stage cancer model with the ability to target or spare the tumor-associated LN. Results to date demonstrate significant immunological differences that are contingent upon inclusion/exclusion of the LN. Long-term survival experiments suggest response and survival advantages with T-only RT, particularly in combination with αCTLA-4 blockade. Taken together, these data suggest that PNI may dampen anti-tumor immune responses and that Tumor-only RT might be a better strategy in combination immunotherapy regimens, although these results should be explored in carefully designed clinical trials. This abstract is also being presented as Poster B41. Citation Format: Ariel E. Marciscano, Ali Ghasemzadeh, Thomas R. Nirschl, Brian J. Francica, Debebe Theodros, Esteban Velarde, J Wong, Daniel LJ Thorek, Theodore L. DeWeese, Charles G. Drake. Prophylactic nodal irradiation abrogates the synergy of tumor radiotherapy and immune checkpoint blockade. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR03.

Novel Roles for Obscurin Proteins in Cardiac Muscle

Obscurin is a giant myofibrillar protein important for sarcomere structure, signaling, and maintenance of sarcoplasmic reticulum (SR) organization. Surprisingly, obscurin knockout mice develop normally and only show signs of a mild skeletal muscle myopathy. However, little is known about cardiac functions of obscurin. We characterized cardiac roles of obscurin and found no changes to cardiac physiology. One explanation for the lack of a cardiac phenotype in obscurin knockout mice is that other obscurin protein family members possess a functionally redundant role. We hypothesized that obscurin-like 1 (Obsl-1) is responsible for this redundancy, in part due to its shared ability to bind the sarcomeric proteins titin and myomesin. We generated double knockout (dKO) mice by crossing the obscurin-KO mice with a cardiac specific Obsl1-KO line to investigate the roles for these proteins in cardiac development and function. Although a majority of the dKO-mice died after 12 months of age, microscopic and ultrastructural analysis failed to reveal overt changes to sarcomere organization. Dramatic changes were observed, however, in SR structure and function. Specifically we observed: 1.) loss of SR structural integrity and apparent loss of SR content, and 2.) alterations to Ca2+ transients and in the levels of Ca2+ handling proteins in KO animals. The majority of these changes were more severe in dKO-mice. Furthermore, we employed serial blockface TEM technology to precisely measure the volume of the SR and mitochondria and noticed significant differences between KO and control animals. Physiologically, these mice develop a late onset cardiomyopathy characterized by increased systolic left ventricular internal dimension and alterations in hemodynamic properties at ∼12 mo. of age. Our data suggests that obscurin and Obsl1 are not critical for sarcomerogenisis, however are important for jointly organizing SR organization, cellular Ca2+ handling, and long-term survival in mice.

337 Expansion of Dendritic Cells Using FLT3 Ligand to Treat Glioblastoma

Abstract FLT3L induces expansion and recruitment of plasmacytoid dendritic cells (pDCs) into the brain. By increasing antigen presentation, FLT3L agonists may enhance an immune response against glioblastoma (GBM). Previous results suggest that the antitumor response generated by FLT3 is secondary to production of interferon gamma. We studied the effect of FLT3L treatment of mice with a syngeneic GBM model. Under ACUC approval, 32 mice underwent implantation of 130 000 GL261 cells in the left striatum using a stereotactic frame. The presence of tumor was confirmed by bioluminescence imaging at day 7. Mice were randomly assigned to 4 groups: Control, FLT3 (courtesy of Celldex) treatment, Poly IC treatment, and FLT3+Poly IC treatment. Survival was assessed using log-rank analysis and described using Kaplan-Meier curves. A survival experiment demonstrated that FLT3L treatment of mice resulted in 50% long-term survival of mice and improved survival compared with control groups (P = .001). Addition of Poly IC (TLR3 agonist), did not add survival benefit. The present work suggests that an increase in antigen presentation by enhancing and recruiting dendritic cells into the brain and brain tumor may be beneficial and a potential therapeutic strategy for patients with GBM. The mechanism of this phenomenon is currently under investigation, but includes increased secretion of interferon gamma. Additionally, increased antigen presentation may aid the immune system mount a significant antitumor immune response. Further experiments evaluating the proportion of T cells that have been exposed to antigen are currently underway.

Study on anti-tumor effects of murine dendritic cells pulsed with PEP3-KLH peptide on prostate cancers

Objective To explore the effects of anti-tumor immunity induced by dendritic cells (DCs) vaccine pulsed with PEP3-KLH (keyhole limpet hemocyanin) on induction of specific immunity against prostate cancers. Methods DCs were propagated from bone marrow of C57BL/6 mice in vitro with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). On day 5 of culture, DCs were harvested and incubated with PEP3-KLH or KLH. Then the DC vaccine was inoculated into C57BL/6 mice by subcutaneous injection for three times with an interval of two weeks. One week after the last vaccination, the levels of IL-2, IL-12, and interferon (IFN)-γ were measured with enzyme-linked immunosorbent assay (ELISA) kit. The aforementioned immunized mice with DC vaccines were challenged with transgenic adenocarcinoma of mouse prostate (TRAMP)-C2 tumor cells, the tumor growth curve was drawn, and survival rate was checked and compared. The cytotoxic T lymphocyte (CTL) activity induced by DCs was tested with lactate dehydrogenase (LDH) release method. The percentages of CD3+ , CD4+ , or CD8+ T cells in tumor-infiltrating lymphocytes (TILs) were determined with flow cytometry. Results PEP3-KLH-DC group stimulated the body and induced higher levels of secreted IL-2, IL-12, and IFN-γ compared to DCs control and KLH-DC groups (P<0.01). The tumor of mice vaccinated with PEP3-KLH-DC grew significantly slower than that injected with DCs or KLH-DC (P<0.01). Compared to the others, the survival rate in PEP3-KLH-DC group raised remarkably (P<0.01). PEP3-KLH-DC group induced more outstanding specific CTL activities of killing tumor cells than DCs control group and KLH-DC group(P<0.01), and the cytotoxicities of TILs in PEP3-KLH-DC group was significantly enhanced (P<0.01). The percentages of CD3+ , CD4+ , or CD8+ T cells in TILs (40.9%, 34.1%) in PEP3-KLH-DC group were significantly higher than those in DC-KLH (27.3%, 5.2%) or DCs (26.2%, 5.1%) group. Conclusions PEP3-KLH-DC vaccine can inhibit effectively tumor growth, enhance long-term survival in mice, intensify the local immunologic function of tumor, and elicit and promote profound specific anti-prostate cancer cellular immune responses. Key words: Receptor, epidermal growth factor/BI; Dendritic cells; Vaccines/BI/PD; Prostatic neoplasms/IM/TH

STEM-01NEURAL STEM CELL-MEDIATED ENZYME/PRODRUG THERAPY FOR NEUROBLASTOMA: TRANSLATION TO THE CLINIC

Neuroblastoma (NB), a neuroendocrine tumor, is the most common extracranial solid tumor of childhood. 45% of these patients are diagnosed with metastatic high-risk tumors, for which treatment options are limited. Neural stem cells (NSCs), engineered to secrete a modified carboxylesterase (CE) can home to metastatic neuroblastoma tumor foci in multiple organs, and convert the prodrug CPT-11 (Irinotecan; IRN) to the 1000 fold more potent topoisomerase-1 inhibitor SN-38. The goal of our current efficacy and safety/toxicity IND-enabling studies is to identify the optimal dose and schedule of IV administered NSCs adenovirally transduced to secrete a modified human CE (hCE1m6-NSCs), followed by human equivalent doses of IRN. This would ideally provide a tumor selective, more effective and less toxic treatment for children with high-risk NB. We have determined the in vitro IC50 values of 4 human derived neuroblastoma lines to SN-38, IRN only and IRN + hCE1m6-NSC conditioned media. All NB lines were 500-2000 fold more sensitive to hCE1m6-NSC conditioned media + IRN as compared to IRN alone. In vivo clearance of hCE1m6-NSCs through peripheral organs and circulation in naive non-tumor bearing Es1e/SCID mice was within 24h, as detected using PCR analysis. In subcutaneous models of human NB we have quantified NSC tumor distribution and performed pharmacokinetic studies demonstrating tumor-specific conversion of IRN to SN-38. In our metastatic NB mouse models, preliminary results showed that hCE1m6-NSCs in combination with 3 days of IRN (15 mg/kg) had a significant decrease in bioluminescent signal as an indicator of tumor burden, as compared to the IRN only group. Treatment with hCE1m6-NSCs and IRN increased long-term survival of mice bearing CHLA-136 NB tumors. If ongoing IND-enabling studies are successful, we plan to be in phase I studies in pediatric patients with refractory or relapsed high-risk NB by 2018.