218 Autologous glioblastoma tumor cells and an antisense oligonucleotide against insulin-like growth factor type 1 receptor protect against tumor challenge and generate T cell anti-tumor responses

BackgroundIGV-001 is a novel immunotherapy that combines irradiated, patient-derived glioblastoma tumor cells and an antisense oligonucleotide against insulin-like growth factor type 1 receptor (IMV-001) in biodiffusion chambers (0.1-micron pore size). We recently evaluated IGV-001 in patients with newly diagnosed glioblastoma.1 In a subgroup of IGV-001-treated, Stupp-eligible patients2 with methylated O6-methylguanine–DNA methyl-transferase (MGMT) promoter, median progression free survival was 38.4 months1 compared with 8.3 months in historical standard-of-care-treated patients (p=0.0008).2 We utilized the GL261-Luciferase (-Luc) glioblastoma orthotopic murine model and conducted in vitro immunological assays using patient-derived GBM tumor cells and matched peripheral blood mononuclear cells (PBMC) to unravel the potential mechanisms associated with the activity of IGV-001.MethodsBiodiffusion chambers containing phosphate-buffered saline (PBS) alone or IGV-001 prepared with 1x106 GL261-Luc cells were implanted in the flanks of C57BL/6 albino mice and explanted 48 hours later, as per the clinical protocol. GL261-Luc intracranial tumor challenge was conducted 28 days after chamber implantation. Mice were monitored for survival and tumor growth, as determine by bioluminescence intensity (BLI). For in vitro experiments, IGV-001 prepared with patient tumor cells were co-cultured with patient-derived PBMC to evaluate activated and memory T cell subsets and responses. To elucidate the immunostimulatory underpinnings of IGV-001, ATP release assay was conducted as a surrogate measure of immunogenic cell death.Results59% of IGV-001 treated mice were alive and continued to gain weight at the termination of the study, 58 days post–intracranial tumor challenge. In comparison, there were no survivors in the PBS group by day 24 (p<0.001). Fluorospot assays demonstrated enhanced T cell IFN-gamma responses to tumor cell antigens. In IGV-001 treated mice, serum IL-6 was positively correlated with BLI, meaning that treated mice with lower BLI signal had less circulating IL-6 (p<0.01). Fluorospot assays demonstrated enhanced T cell IFN-gamma responses to tumor cell antigens. Tumor co-culture studies showed elevated percentage of activated CD4 and CD8 T cells as well as increased central and effector memory phenotypes in both T cell subsets compared to IMV-001-treated PBMC controls. Lastly, tumor cells treated with IMV-001 released significantly more (p<0.01) ATP than untreated or sense oligonucleotide-treated controls.ConclusionsThese data support the antitumor activity of IGV-001 in newly diagnosed glioblastoma, as evidenced in the phase 1 study. Th1 anti-tumor T cell activity was demonstrated. The ATP results suggest a possible immunogenic conversion by which IGV-001 stimulates the immune system and suppresses tumor growth, which can be quantified via circulating IL-6.ReferencesAndrews DW, Judy KD, Scott CB, Garcia S, Harshyne LA, Kenyon L, Talekar K, Flanders A, Atsina KB, Kim L, Martinez N, Shi W, Werner-Wasik M, Liu H, Prosniak M, Curtis M, Kean R, Ye DY, Bongiorno E, Sauma S, Exley MA, Pigott K, Hooper DC. Phase Ib clinical trial of IGV-001 for patients with newly diagnosed glioblastoma. Clin Cancer Res 2021 April 1;27(7):1912–1922. doi: 10.1158/1078-0432.CCR-20-3805. Epub 2021 Jan 26. PMID: 33500356.Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO, European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005 March 10;352(10):987–96. doi: 10.1056/NEJMoa043330. PMID: 15758009.Ethics ApprovalEthical consent was obtained for all human biospecimens with the appropriate IRB approval.ConsentInformed consent was obtained for all human biospecimens with the appropriate IRB approval.

Irreversible electroporation augments checkpoint immunotherapy in prostate cancer and promotes tumor antigen-specific tissue-resident memory CD8+ T cells

Memory CD8+ T cells populate non-lymphoid tissues (NLTs) following pathogen infection, but little is known about the establishment of endogenous tumor-specific tissue-resident memory T cells (TRM) during cancer immunotherapy. Using a transplantable mouse model of prostate carcinoma, here we report that tumor challenge leads to expansion of naïve neoantigen-specific CD8+ T cells and formation of a small population of non-recirculating TRM in several NLTs. Primary tumor destruction by irreversible electroporation (IRE), followed by anti-CTLA-4 immune checkpoint inhibitor (ICI), promotes robust expansion of tumor-specific CD8+ T cells in blood, tumor, and NLTs. Parabiosis studies confirm that TRM establishment following dual therapy is associated with tumor remission in a subset of cases and protection from subsequent tumor challenge. Addition of anti-PD-1 following dual IRE + anti-CTLA-4 treatment blocks tumor growth in non-responsive cases. This work indicates that focal tumor destruction using IRE combined with ICI is a potent in situ tumor vaccination strategy that generates protective tumor-specific TRM.

LILRB4 suppresses immunity in solid tumors and is a potential target for immunotherapy

Immune receptors expressed on TAMs are intriguing targets for tumor immunotherapy. In this study, we found inhibitory receptor LILRB4 on a variety of intratumoral immune cell types in murine tumor models and human cancers, most prominently on TAMs. LILRB4, known as gp49B in mice, is a LILRB family receptor. Human and murine LILRB4 have two extracellular domains but differ in the number of intracellular ITIMs (three versus two). We observed a high correlation in LILRB4 expression with other immune inhibitory receptors. After tumor challenge, LILRB4−/− mice and mice treated with anti-LILRB4 antibody showed reduced tumor burden and increased survival. LILRB4−/− genotype or LILRB4 blockade increased tumor immune infiltrates and the effector (Teff) to regulatory (Treg) T cell ratio and modulated phenotypes of TAMs toward less suppressive, CD4+ T cells to Th1 effector, and CD8+ T cells to less exhausted. These findings reveal that LILRB4 strongly suppresses tumor immunity in TME and that alleviating that suppression provides antitumor efficacy.

Leukemia vaccine overcomes limitations of checkpoint blockade by evoking clonal T cell responses in a murine acute myeloid leukemia model

We have developed a personalized vaccine whereby patient derived leukemia cells are fused to autologous dendritic cells, evoking a polyclonal T cell response against shared and neo-antigens. We postulated that the dendritic cell (DC)/AML fusion vaccine would demonstrate synergy with checkpoint blockade by expanding tumor antigen specific lymphocytes that would provide a critical substrate for checkpoint blockade mediated activation. Using an immunocompetent murine leukemia model, we examined the immunologic response and therapeutic efficacy of vaccination in conjunction with checkpoint blockade with respect to leukemia engraftment, disease burden, survival and the induction of tumor specific immunity. Mice treated with checkpoint blockade alone had rapid leukemia progression and demonstrated only a modest extension of survival. Vaccination with DC/AML fusions resulted in the expansion of tumor specific lymphocytes and disease eradication in a subset of animals, while the combination of vaccination and checkpoint blockade induced a fully protective tumor specific immune response in all treated animals. Vaccination followed by checkpoint blockade resulted in upregulation of genes regulating activation and proliferation in memory and effector T cells. Long term survivors exhibited increased T cell clonal diversity and were resistant to subsequent tumor challenge. The combined DC/AML fusion vaccine and checkpoint blockade treatment offers unique synergy inducing the durable activation of leukemia specific immunity, protection from lethal tumor challenge and the selective expansion of tumor reactive clones.

93 Targeting sirt2 rescues the metabolic fitness and effector functions of tumor-reactive T cells within the metabolically restricted tumor microenvironment

BackgroundThe majority of cancer patients remain refractory to existing cancer immunotherapies. Despite the growing evidence that dysregulated metabolism contributes to the exhaustion of tumor-infiltrating T lymphocytes (TILs) and the loss of their effector functions within the metabolically restricted tumor microenvironment (TME), actionable targets to rescue metabolic fitness and anti-tumor activity of TILs remain elusive.Memory T (TM) cells and TILs rely on fatty acid catabolism to preserve their effector functions due to nutrient competition for glucose with tumor cells. Therefore, enhancing fatty acid catabolism of TILs represents an attractive strategy to increase the efficacy of immunotherapies.Sirt2 is an NAD+ dependent histone deacetylase. We previously showed that upregulation of Sirt2 in human TILs negatively correlates with response to TIL therapy in advanced non-small cell lung cancer (NSCLC) and Sirt2 deficiency leads to hyper-reactive T cells with superior antitumor activity.MethodsSirt2 expression was analyzed by flow cytometry and Western blot. The role of Sirt2 in tumor immunity was studied using in vivo B16F10 tumor challenge models as well as ex vivo analysis including RNA-sequencing, CFSE proliferation assay, DAPI/AnnexinV staining, IFN-γ ELISpot assay, intracellular staining of effector molecules and LDH cytotoxicity assay on WT versus Sirt2KO T cells. Molecular partners of Sirt2 were identified using mass spectrometry (MS) and Co-immunoprecipitation analyses. The role of Sirt2 in T cell metabolism was investigated using seahorse bioanalyzer and LC-MS/MS Metabolomic profiling. AGK2, a Sirt2 selective inhibitor, was used for Sirt2 blockade in human T cells.ResultsSirt2 expression is upregulated during T cell activation, TM stage, and within the TME. Our molecular studies revealed that Sirt2 negatively impacts the acetylation status and the activity of the trifunctional protein, the key enzyme of fatty acid oxidation (FAO). Accordingly, Sirt2 deficiency enhanced FAO and metabolic fitness of activated T cells and mouse TILs isolated from B16F10 tumor nodules. As a consequence of enhanced FAO, Sirt2 deficient mice displayed increased accumulation of TM cells, which was associated with decreased apoptosis and increased survival after tumor challenge leading to superior tumor rejection. Most importantly, pharmacologic inhibition of Sirt2 in human TILs isolated from NSCLC patients enhanced their metabolic fitness and cytotoxic activity against their autologous tumor cells.ConclusionsOur findings indicate Sirt2 as a suppressor of T cell metabolism amenable to therapeutic targeting, and Sirt2 inhibition reprograms T cell metabolic fitness to optimally sustain their effector function within the hypoglycemic TME, thus, leading to an effective anti-tumor immune response.AcknowledgementsThis work was supported in part by K08 CA194273, ACS IRG-17-173-22, NCI Cancer Center Support Grant (P30-CA076292) and the Moffitt Foundation.

Near-infrared absorbing Ru(ii) complexes act as immunoprotective photodynamic therapy (PDT) agents against aggressive melanoma

Ru(ii) photosensitizers (PSs) destroy aggressive melanoma cells, triggering an immune response that leads to protection against tumor challenge and mouse survival.

Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.