Association of Suppressive Myeloid Cell Enrichment with Aggressive Oropharynx Squamous Cell Carcinoma

BACKGROUND: While immune-cell infiltrated tumors, such as human papillomavirus positive (HPV+) ororpharyngeal squamous cell carcinomas (OPSCC) have been associated with an improved clinical prognosis, there is evidence to suggest that OPSCCs are also subjected to increased immunoregulatory influence. The objective of this study was to assess whether patients with clinically aggressive OPSCC have a distinct immunosuppressive immune signature in the primary tumor. METHODS: This retrospective case-control study analyzed 37 pre-treatment tissue samples from HPV+ and HPV-negative OPSCC patients treated at a single institution. The cases were patients with known disease recurrence and the controls were patients without disease recurrence. An mRNA-expression immune-pathway profiling was performed, and correlated to clinical outcomes. The TCGA head and neck cancer database was utilized to make comparisons with the institutional cohort. RESULTS: In our cohort, HPV-negative and HPV+ patients with known disease recurrence both had significantly increased suppressive monoctyte/macrophage and granulocyte cell-expression-profile enrichment. Similar findings were found in the TCGA cohort when comparing HPV-negative to positive patients. CONCLUSIONS: our study demonstrates that patients with recurrent HPV+ OPSCC had suppressive monocyte/macrophage and granulocyte immune-cell enrichment, similar to those seen in the more aggressive HPV-negative OPSCC

A Novel Role for P-Selectin in the Pathogenesis of Acute Chest Syndrome

Abstract Acute chest syndrome (ACS) is a potentially fatal lung complication of sickle cell disease (SCD). There are currently no mechanistic based therapies for ACS and it continues to be a leading cause of death in SCD. Recent studies have identified independent links between extracellular heme with ACS, and with vaso-occlusive crisis (VOC) a common prodrome of ACS. Our experimental ACS model involves intravenous injection of purified hemin typically 35 µmoles/kg, which raises total plasma heme to a clinically relevant pathophysiologic level equivalent to ~0.5g/dl of hemoglobin, and causes respiratory failure exclusively in transgenic SCD mice of both the Townes and Berkeley strains (Ghosh et al., J Clin Invest, 2013). We have determined using time-lapse video microscopy and electrical cell impedance that hemin rapidly disrupts the pulmonary endothelial barrier in vitro. Hemopexin blocks hemin-induced pulmonary endothelial barrier disruption in vitro and respiratory failure in hemin-challenged SS mice. Importantly, genetic polymorphisms in the gene encoding heme oxygenase-1 the rate-limiting heme degradation enzyme are associated with ACS risk in the Cooperative Study of Sickle Cell Disease and the Silent Infarct Transfusion cohorts. These human data and our mechanistic results in SS mice support the heme hypothesis for ACS pathogenesis. P-selectin is a cell adhesion molecule involved in vascular inflammation. It is normally sequestered inside quiescent endothelium. However, recent evidence indicates extracellular heme activates the release of P-selectin unto the endothelial surface wall. Thus, in this study, we tested the hypothesis that P-selectin promotes ACS development. Infusion of a function blocking anti-P-selectin antibody protected SS mice (n=3) from hemin-induced ACS while all control SS mice (n=3) pretreated with IgG before the hemin challenge died (p=0.03) with severe hypoxemia and postmortem evidence of alveolar flooding. The apparent requirement for P-selectin in heme-induced lethal acute lung injury was confirmed by a 100% survival of P-selectin-/- (n=5) and 100% lethality of congenic P-selectin+/+ (n=5) mice challenged with hemin infusions (p=0.005). To identify the cell population involved in this disease process we generated bone marrow chimeric C57BL/6 mice lacking P-selectin in hematopoietic (P-selectinPLT-/-) and non-hematopoietic (P-selectinEC-/-) compartments. Seventy-five percent of P-selectinEC-/- mice were protected while all P-selectinPLT-/- congenic controls succumbed to hemin (n=3-4). The severity of lung injury in the P-selectinPLT-/- C57BL/6 mice was reflected by severe hypoxemia (SpO2: 82.75±2.14%) and a significantly higher lung wet/dry weight ratio compared to the ratio of the P-selectinEC-/- mouse lungs (p&lt;0.05). Next, we transplanted congenic P-selectin-/- and P-selectin+/+ mice with SS mouse bone marrow to generate chimeric SS mice lacking endothelial P-selectin or expressing endothelial P-selectin respectively. Induction of ACS resulted in 40% lethality in the SS/P-selectinEC-/- mice (n=7) and 100% lethality in SS/P-selectinEC+/+ mice (n=7; p&lt;0.01). The development of respiratory failure in the hemin modelinvolves de novo heme release from acute intravascular hemolysis. In this study, we found that SS/P-selectinEC-/- mice cleared ~70% of the hemin bolus within 30 min, which attenuated intravascular hemolysis and damped de novo heme release, while total plasma heme increased over 2-fold in the SS/P-selectinEC+/+ mice during the same time interval. Together these results demonstrate a critical intermediary role for P-selectin in the ACS triggered by acute intravascular hemolysis and elevated extracellular heme. In addition, we provide proof-of-principle in mice that antagonists of P-selectin can prevent and potentially treat ACS. Disclosures No relevant conflicts of interest to declare. </jats:sec

COVD-02. ADAPTING RNA-NANOPARTICLE VACCINES FROM GLIOBLASTOMA TO SARS-COV-2

Abstract BACKGROUND Glioblastoma (GBM) can be an effective teacher in the war on COVID-19, as an operative vaccine for either must elicit near-immediate protective responses that overcomes disease heterogeneity and immune suppression. Current prophylactic strategies against COVID-19 utilize mRNA vaccines targeting small fragments of the SARS-CoV-2 genome, but these may not induce robust T cell responses or elicit immunity quickly enough. OBJECTIVE We sought to adapt an FDA-IND approved mRNA vaccine in GBM against COVID-19 for: 1) activation of near immediate immune responses, 2) targeting of full-length SARS-CoV-2 structural proteins, and 3) induction of bidirectional (B and T cell) adaptive immunity. METHODS We utilized a novel engineering design that layers mRNA into a lipid-nanoparticle (NP) shell (much like an onion); this allows greater packaging of mRNA per particle to quickly boost innate/adaptive immune responses against full-length glioblastoma antigens or SARS-CoV-2 structural proteins. RESULTS In small and large animal models, RNA-NPs safely mimic viremia activating the quiescent immune system in only a few hours for induction of protective immunity against its mRNA payload. RNA-NPs activate dendritic cells (DCs), upregulate critical innate gene signatures, and induce antigen-specific cellular and humoral immunity. We found that mice receiving SARS-CoV-2 spike RNA-NPs had more effector T cells after vaccination with significant memory recall expansion after in vitro re-stimulation with overlapping SARS-CoV-2 spike peptide mix. We also found increased release of MIP-1-alpha (i.e. CCL3) previously shown by our group (Mitchell et al. Nature 2015) to be responsible for Th1 mediated memory recall to infectious vaccine antigens in GBM patients. CONCLUSION SARS-CoV-2 RNA-NPs elicit memory recall response after vaccination. We have obtained FDA-IND approval (BB-19304, Sayour) in GBM with SARS-CoV-2 specific amendment (BB-20871) underway to support first-in-human trials of RNA-NPs targeting both GBM and COVID-19. </jats:sec

IMMU-13. CUSTOMIZABLE MULTI-LAMELLAR RNA-NANOPARTICLES FOR PEDIATRIC GLIOMA

Abstract Background Since the preponderance of pediatric gliomas are mutationally ‘bland,’ immune checkpoint inhibitors are unlikely to mediate therapeutic benefit. Alternately, immunologic response can be induced de novo against pediatric gliomas with mRNA cancer vaccines. Messenger RNA represents a paradigm shift in vaccinology (i.e. COVID-19) given its flexibility, commercialization, and propensity to confer rapid protection with only a single vaccine. Objective We sought to develop a new mRNA platform with an optimized backbone for insertion of both personalized and/or “off the shelf’ (i.e. H3K27M) transcripts for rapid induction of anti-tumor activity against pediatric gliomas. Approach We synthesized an mRNA backbone with optimized 5’ and 3’ UTRs for delivery of gene transcripts pertinent to pediatric brain tumors using a lipid-nanoparticle (NP) delivery vehicle. This vaccine utilizes a novel engineering design that layers tumor derived mRNA into a lipid-nanoparticle (NP) “onion-like” or multi-lamellar package. Results We demonstrate immunogenicity of RNA-NPs delivering either personalized glioma mRNA or H3K27M mRNA. RNA-NPs localize to myeloid cells in murine KR158b brain tumors and activate dendritic cells that supplant regulatory intratumoral myeloid populations inducing antigen-recall response with long-term survivor benefit. Our optimized mRNA backbone yielded significantly improved anti-tumor efficacy compared with commercial backbones. We have shown this approach can be refined for co-delivery of immunomodulatory RNAs (i.e. GM-CSF) and/or delivery of siRNAs targeting immunoregulatory axes (PD-L1) in murine brain tumors (GL261). We have since established safety of RNA-NPs in acute/chronic murine GLP toxicity studies without cross-reactivity to normal-brain, and launched a large-animal canine brain tumor trial which demonstrated RNA-NPs to be feasible, safe and immunologically active. Conclusion RNA-NPs reprogram the brain tumor microenvironment while inducing a glioma-specific immune response. We have since received FDA-IND approval for first-in-human trials (IND#BB-19304) in pediatric patients with high-grade gliomas (PNOC020 study, NCT04573140). </jats:sec

IMMU-32. RNA-NANOPARTICLE VACCINES MEDIATE T CELL TRAFFICKING NECESSARY FOR BBB PASSAGE AND ANTI-GLIOMA IMMUNE RESPONSE

Abstract BACKGROUND The blood-brain barrier (BBB) remains a potent obstacle for development of new therapies against glioblastoma (GBM). While activated T cells can cross the BBB, immunotherapy has yet to be fully unlocked for malignant brain tumors due their heterogeneity and immunosuppressive microenvironments. To overcome these challenges, our group has developed a novel treatment platform, which leverages the use of a clinically translatable nanoparticles (NPs) combined with personalized tumor derived mRNA to peripherally activate T cells against a heterogenous source of tumor antigens and reprogram the intratumoral milieu into an immune activated state. OBJECTIVE We sought to assess if RNA-NPs could activate systemic/intratumoral dendritic cells (DCs) and mediate a peripheral T cell response that could penetrate the GBM microenvironment. RESULTS We uncovered that RNA-NPs elicit potent innate immunomodulating effects through release of interferon-α (IFN-α) from plasmacytoid DCs (pDCs). After only a single RNA-NP vaccine, the bulk of systemic and intratumoral DCs in mice display an activated phenotype; DCs, harvested from intracranial tumors, elicit expansion of antigen specific T cell immunity. Tumor-specific RNA-NPs elicited enhanced survival outcomes in immunocompetent animals bearing NF-1/p53 mutant gliomas with increased intratumoral memory CD8+T-cells. Unlike immune checkpoint blockade (anti-PD-L1 mAbs), we found that RNA-NPs increase LFA-1 on peripheral CD8 splenocytes, which is necessary for activated T cell passage across the BBB. RNA-NPs also increased CCR2 on peripheral CD8 cells, which was dependent on IFN-α/β, as the percentage of CCR2+CD8+ splenocytes and anti-tumor activity (mediated by RNA-NPs) was abrogated in animals receiving concomitant type I IFN receptor (IFNAR1) mAbs. CONCLUSION Since LFA-1 is important for T cell trafficking across the BBB and CCR2 may promote chemotaxis to the brain (as GBMs are known to secrete CCL2), RNA-NPs may offer a new treatment modality and immunologic mechanism for unlocking peripheral T cell immunity against malignant gliomas. </jats:sec

IMMU-47. RNA-NANOPARTICLE VACCINES ARE SAFE AND IMMUNOLOGICALLY ACTIVE IN CLIENT-OWNED CANINES WITH TERMINAL GLIOMAS

Abstract BACKGROUND The lack of appropriate preclinical murine glioblastoma models limits comprehensive toxicity/efficacy evaluation of investigational agents. To overcome this challenge, we evaluated the safety and activity of a new immunotherapeutic technology that we have pioneered (composed of tumor mRNA complexed into a custom lipid-nanoparticle formulation) in client-owned canines (pet dogs) diagnosed with malignant gliomas. OBJECTIVE/ METHODS Canine malignant gliomas were biopsied for generation of personalized tumor mRNA loaded into our custom lipid-nanoparticle (NP) vector. The patients received RNA-NPs intravenously beginning two weeks after their biopsy once weekly (x3) and no other anti-tumor therapeutic interventions. RESULTS Within a few hours after administration, tumor specific RNA-NPs elicited margination of peripheral blood mononuclear cells, which increased in the subsequent days/weeks post-treatment; suggesting that RNA-NPs mediate lymphoid honing of immune cell populations before egress. RNA-NPs also elicited increased: 1) serum interferon-α that spiked at 2 hours; 2) CD80 and MHCII on CD11c+ cells (demonstrating activation of peripheral DCs); and 3) interferon-γ + T-cells (i.e. activated T-cells). After receiving weekly RNA-NPs (×3), the canines had a steady course. Aside from low-grade fevers on the vaccination days, personalized tumor RNA-NPs (1x) were well tolerated with stable blood counts, chemistries, and renal/liver function tests. All patients assessed developed immunologic response with pseudoprogression or stable/smaller tumors by MRI. Although we have treated a small cohort, we have observed improvement in median/overall survival in all canine patients with terminal gliomas receiving RNA-NPs (compared with historical controls). CONCLUSION RNA-NPs were feasible, safe and immunologically active in client-owned canines with terminal gliomas. We have not appreciated significant toxicities in canines that would preclude investigation in humans at 1x dosing. Although these results need to be validated in larger canine data sets, these results suggest safety and activity of tumor specific RNA-NPs in canines with terminal gliomas. </jats:sec

Macrophage Hypercellularity Accompanies Erythroid Hyperplasia in Sickle Cell Mice and during Recovery from Blood Loss in Wild Type Mice

Red blood cell (RBC) homeostasis tightly balances production of new erythroid cells and clearance of damaged or senescent RBCs. Macrophages play a central role in this process in the specialized niches termed erythroblastic island. Macrophages expressing CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G play important roles in physiological and pathological erythropoiesis. Sickle cell disease (SCD) is characterized by chronic stress erythropoiesis as a compensatory mechanism for anemia. Recent publications have detected macrophage hypercellularity correlated with erythroid hyperplasia in genetic models of erythrocytosis and monocyte-derived macrophage proliferation in the spleen. Because macrophages are integral part of the erythropoietic niche, our objective was to assess the macrophage compartment in transgenic sickle cell mice (SS) at steady state compared to AA controls. Furthermore, we used acute blood loss in C57BL/6J wild type (WT) to confirm if macrophage hypercellularity accompanies erythroid hyperplasia in a mouse model without erythrocytosis or sickling. Using flow cytometry, macrophages were identified with F4/80, CD45, CD11b and CD169, while erythroid progenitor cells were identified with Ter119 and CD71 in the BM and spleen of SS mice and age-matched control (AA) mice. Each subset of macrophages is 3.8 to 6.8-fold higher in SS mice bone marrow compared to AA at steady state (p≤0.05, Fig 1a). Findings were similar for other CD169 subsets in the spleens of SS mice. This macrophage hypercellularity was accompanied by expected erythroid hyperplasia, with 12-fold higher immature erythroid progenitor cells (CD71hiTer119hi) in SS mice than AA controls (p≤0.001). To confirm the increase in macrophage numbers that accompanied erythroid hyperplasia in SS mice as a response to erythroid stress, we induce stress with acute blood loss in WT mice by phlebotomy (once). Interestingly, we found macrophage hypercellularity accompanies erythroid hyperplasia in WT mice during recovery from acute blood loss. WT mice showed about 47-61% increases in CD169 macrophages subsets in the marrow and 47-83% increase in the spleen during recovery from acute blood loss (5-7 days) compared to untreated mice (Fig. 1b). This macrophage hypercellularity was accompanied with 88% increase in immature erythroid progenitor cell. Similarly, we investigated if there was age dependence in macrophage hypercellularity that accompanies erythroid hyperplasia in SS mice. In SS mice, there was no significant differences in macrophage numbers in the BM juvenile mice (5 weeks old) compared to young adults (14 weeks old), presumably because the higher demand for erythroid production to compensate for anemia had already started in the juvenile animals. However, there was a significantly higher macrophage hypercellularity in the spleen at 14 weeks compared to juvenile animals (p≤0.001). There was also a reduction in hematocrit (p≤0.05) and hemoglobin (p≤0.01) from 5 to 14 weeks in SS mice, although this does not correlate to either macrophage hypercellularity or erythroid hyperplasia. We also quantified mRNA expression of heme oxygenase 1 (Hmox1), a critical regulator of erythroid stress response, in the BM of untreated SS mice, and untreated and stressed WT mice. As expected, BM cells from SS mice at steady state expressed significantly higher Hmox1 expression than in WT mice (p≤0.001). Acute blood loss induced-stress raised the Hmox1 expression in WT mice to comparable levels to that of SS mice at steady state. Our investigation is the first to our knowledge to detect BM macrophage hypercellularity is associated with erythroid hyperplasia in response to acute blood loss and in congenital hemolytic anemia. Our data of macrophage hypercellularity in SCD opens up further research opportunity to elucidate the role of these cells in multiorgan complication in SCD. Figure 1: Macrophage hypercellularity accompanies erythroid hyperplasia in sickle cell disease and during recovery from acute blood loss. (a) BM macrophage quantification (per femur) in untreated sickle cell mice compared to AA controls (n = 5). Student's Ttest * SS mice compared to AA mice. (b) Acute blood loss induced macrophage hypercellularity in the BM of WT mice (n = 4 - 5). Student's Ttest * Untreated compared to stressed WT mice. Figure 1 Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding. </jats:sec