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The promise of IL12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL12, a novel, engineered exosome therapeutic that displays functional IL12 on the surface of an exosome. IL12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN resulting in equivalent potency in vitro to recombinant IL12 (rIL12) as demonstrated by IFNγ production. Following intratumoral injection, exoIL12 exhibited prolonged tumor retention and greater antitumor activity than rIL12. Moreover, exoIL12 was significantly more potent than rIL12 in tumor growth inhibition. In the MC38 model, complete responses were observed in 63% of mice treated with exoIL12; in contrast, rIL12 resulted in 0% complete responses at an equivalent IL12 dose. This correlated with dose-dependent increases in tumor antigen–specific CD8+ T cells. Rechallenge studies of exoIL12 complete responder mice showed no tumor regrowth, and depletion of CD8+ T cells completely abrogated antitumor activity of exoIL12. Following intratumoral administration, exoIL12 exhibited 10-fold higher intratumoral exposure than rIL12 and prolonged IFNγ production up to 48 hours. Retained local pharmacology of exoIL12 was further confirmed using subcutaneous injections in nonhuman primates. This work demonstrates that tumor-restricted pharmacology of exoIL12 results in superior in vivo efficacy and immune memory without systemic IL12 exposure and related toxicity. ExoIL12 is a novel cancer therapeutic candidate that overcomes key limitations of rIL12 and thereby creates a therapeutic window for this potent cytokine.

Mean Cytokine Values in Cynomolgus Plasma After Subcutaneous Dosing

Fold-change Cytokine Values in Cynomolgus Plasma After Subcutaneous Dosing

Figures S1 through S6

20201229pdtedSupplementaryFigures.pdf from Exosome Surface Display of IL12 Results in Tumor-Retained Pharmacology with Superior Potency and Limited Systemic Exposure Compared with Recombinant IL12

Although biologiocal ancillay materials (AMs) have specific risk associated with their derivations, it plays key role to manufature cell and gene therapy (CGT) products. It is important to understand the regulation relevant to AMs for developers.The authors investigated the guidelines and pharmacopeia (hereinafter referred to as "guidelines") for biological AMs used for the manufacture of CGT products in Asia (China, India, Japan, Korea and Taiwan). In addition, the authors benchmarked the relevant guidelines in the United States (US) and European Union (EU).The guidelines could be classified into two types based on whether specific AMs are scoped: (i) general guidelines for risk assessment of AMs and (ii) guidelines for specific AMs. The authors compared the risk categories for each type of AM provided in the general guidelines between the US and China and the specific requirements for bovine serum and trypsin in the guidelines of China, Japan, Taiwan, US and EU. The authors further compiled in-depth descriptions of the respective regulations in China, India, Japan, Korea and Taiwan. There is limited availability of some guidelines for specific AMs. Moreover, there are no common requirements established across the surveyed countries and regions. Therefore, the authors suggest a risk assessment approach for AMs with consideration of their biological origin and traceability, production steps applied and ability to control or remove AMs from the final medicinal product over the CGT manufacturing process.

CD19-directed chimerical antigen receptor T-cell (CAR-T) products have gained US Food and Drug Administration approval for systemic large B-cell lymphoma. Because of concerns about potential immune cell-associated neurotoxicity syndrome (ICANS), patients with primary central nervous system (CNS) lymphoma (PCNSL) were excluded from all pivotal CAR-T studies. We conducted a phase 1/2 clinical trial of tisagenlecleucel in a highly refractory patients with PCNSL and significant unmet medical need. Here, we present results of 12 relapsed patients with PCNSL who were treated with tisagenlecleucel and followed for a median time of 12.2 months (range, 3.64-23.5). Grade 1 cytokine release syndrome was observed in 7/12 patients (58.3%), low-grade ICANS in 5/12 (41.6%) patients, and only 1 patient experienced grade 3 ICANS. Seven of 12 patients (58.3%) demonstrated response, including a complete response in 6/12 patients (50%). There were no treatment-related deaths. Three patients had ongoing complete remission at data cutoff. Tisagenlecleucel expanded in the peripheral blood and trafficked to the CNS. Exploratory analysis identified T-cell, CAR T, and macrophage gene signatures in cerebrospinal fluid following infusion when compared with baseline. Overall, tisagenlecleucel was well tolerated and resulted in a sustained remission in 3/7 (42.9%) of initial responders. These data suggest that tisagenlecleucel is safe and effective in this highly refractory patient population. This trial was registered at www.clinicaltrials.gov as #NCT02445248.

Solid stress, distinct from fluid pressure, is a physical force contained in and transmitted by solid components of the brain tumor, including cells and the matrix they produce. Solid stress has been shown to promote tumor progression, and decrease anticancer therapy efficacy. This is especially relevant in brain tumors, as the rigid skull results in these trapped forces, increasing intracranial pressure, and potentially leading to other complications, including neuronal cell death. Here we present a novel method of quantifying these physical stresses in situ in both mice (glioblastoma [U87], brain metastasis [BT474], and ependymoma models) and patients. Briefly, following a craniotomy, mechanical forces that include solid stress are released, which causes the tissue to deform in peaks (areas under compression) and valleys (areas originally under tension). This tissue deformation is imaged via high-resolution ultrasound and analysed via custom MATLAB code to produce an accurate 3D model of the entire mouse brain, including the tumour region. For human samples, a pre-operative MRI is used to generate a detailed 3D model of the human brain. During surgery, the trapped physical stresses results in a bulge of the dura post craniotomy. We use BrainLab to measure the craniotomy induced brain deformation, which is then registered to the pre-operation MRI before being analysed in an identical fashion to the murine models using SolidWorks and Abaqus. We further show that in the brain metastases model, chemotherapy reduces compression stresses by 51%. Further, our technique results in fast processing time (~ 15 minutes), and has the potential to prevent the need for intraoperative MRI based on position simulations. As such, solid stress measurements provide a new class of mechanical biomarkers that can be correlated to clinical outcomes for predictive and prognostic value.

Purpose Although maximal resection of wild-type GBM tumors is associated with longer survival, the association of genomic alterations with the ability to resect disease has not been elucidated. This study aimed to determine whether certain molecular alterations are less amenable to surgical resection and whether achieving a surgical margin devoid of tumor cell burden provides a clinical benefit. Methods Molecular profiling of 148 consecutive tumor samples from newly diagnosed patients with IDH-WT GBM SNaPshot genotyping, and methylation-specific PCR for MGMT promoter methylation. The association of alterations with residual volume and progression-free survival was evaluated in regression analyses. We prospectively investigated the impact of TERTp status at the surgical margin on progression-free survival (PFS) in 47 patients. PFS and overall survival (OS) were evaluated with the log-rank test and Cox regression models.Results Overall, 148 patients (91 men [61.5%], median age, 62.5 years [range 56–70 years] had maximally safe surgery. The median follow-up duration was 18 months (IQR, 4-32 months). The rate of PFS was significantly lower in MGMT promoter unmethylated IDH-WT GBM amenable to resection with residual volume >4.9cc (hazard ratio [HR], 2.35; 95% CI, 1.44-3.84; P=.005). Gene amplification (PDGFRA, KIT, KDR) on 4q12 and TP53 and RB1 mutations were associated with greater RV. Prospective trial revealed median PFS for tumors with TERTp mutations in the surgical margin was 10.1 months while tumors with undetectable TERTp mutations in the margin did not progress (median follow-up 8.1 months, IQR 1.6-14.6). Surgical tumor margins devoid of TERTp mutations were associated with significantly different probability of PFS (0.23; 95%CI 0.15-0.43; P< 0.01)Conclusion In conclusion, 4q12 amplification (KDR/PDGFRA/KIT) and tumor suppressor gene alterations (RB1, TP53) were associated with greater residual volume. Tumor-free margins defined spatially by intraoperative navigation as assessed by quantifying TERTp mutation allelic fraction was correlated with prolonged PFS.

Targeting solid tumors with CAR T cells has proven to be more difficult due to their heterogenous target antigen expression, antigen escape, and due to their hostile tumor microenvironment (TME). Mesothelin represents a promising surface tumor antigen, since it has been associated with tumor invasion and is highly expressed on various cancer types, including pancreatic adenocarcinoma. As clinical trials of mesothelin targeting CAR have yet to show efficacy, we hypothesized that tumor stromal cells such as cancer-associated fibroblasts (CAFs) may play a role in this resistance. To provide a more favorable TME for CAR T cells, we generated a bicistronic lentiviral vector encoding a mesothelin CAR along with a secreted T cell engaging molecule (TEAM) that targets fibroblast activation protein (FAP), which is expressed by CAFs. We termed our constructs CARTEAM, and in this case, mesoFAP. We have assessed the activation and proliferative capacity of these CARTEAM through in vitro assays. We showed that TEAMs secreted by CAR T cells bind their appropriate target antigen by adding supernatant from CARTEAM to target cells expressing FAP. In a co-culture assay using a transwell system we demonstrated the cytotoxic effect of secreted TEAM interacting and recruiting bystander T cells against CAFs. In a real-time cell analysis (RTCA) co-culture assay with a pancreatic cancer cell line (AsPC1) and FAP expressing CAFs, we showed cell death of AsPC1 upon CAR recognition and cell death of CAFs through TEAM-mediated recruitment of bystander T cells and CART cells. We show in these co-culture systems, mimicking tumor and TME, that our CARTEAM construct is superior in the elimination of both cancer cell line and CAF, in comparison to control constructs, including mesothelin targeting CAR T cells (meso CAR) and meso CAR T cells secreting an unspecific CD19 TEAM (mesoCD19). We also used acoustic force microscopy to evaluate the additive effect of the TEAM molecule secreted to binding to tumor cells by mesothelin CAR T cells. In vivo experiments of subcutaneously injected tumor cells admixed with CAFs, show superior tumor control when treated with CARTEAM in comparison to control constructs. Based on these data, we demonstrate both the effective in vitro elimination of CAFs and pancreatic cancer cells through the application of CARTEAM and control of pancreatic tumor growth in vivo. Our studies provide a deeper insight into a dual targeting strategy using a novel CAR T cell secreting a TEAM against pancreatic cancer and its tumor microenvironment. Citation Format: Marc Wehrli, Adam Kuo, Rebecca Larson, Irene Scarfò, Amanda Bouffard, Korneel Grauwet, Mark Leick, Andrea Schmidts, Stefanie Bailey, Tamina Kienka, Michael Kann, Sonika Vatsa, Harrison Silva, Kathleen Gallagher, Max Jan, Bryan Choi, David Ting, Marcela Maus. Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME) [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 569.

Background and Purpose— A potential way to improve prehospital stroke care and patient handoff is hospital-directed feedback for emergency medical service (EMS) providers. We evaluated whether a hospital-directed EMS stroke follow-up tool improved documentation of adherence to the Rhode Island state prehospital stroke protocol for EMS providers. Methods— A standardized, 10-item feedback tool was developed in 2012 and sent to EMS directors for every transported patient with a discharge diagnosis of ischemic stroke. We reviewed patient charts meeting these criteria between January 2008 and December 2013. Performance on the tool was compared between the preintervention (January 2008 through January 2012) and postintervention (February 2012 through December 2013) periods. Results— We identified 1176 patients with ischemic stroke who arrived by EMS in the study period: 668 in the preintervention period and 508 in the postintervention period. The overall score for the preintervention group was 5.31 and for the postintervention group 6.42 ( P Conclusions— Hospital-directed feedback to EMS was associated with improved overall compliance with state protocols and documentation of 9 out of 10 individual items. Future confirmatory studies in different locales and studies on the impact of this intervention on actual tissue-type plasminogen activator administration rates and EMS personnel knowledge and behavior are needed.

Exosomes are natural and abundant nanoscale vesicles for intercellular communication, capable of transferring biological instructions between neighboring and distant cell types. Translational research efforts have focused on exploiting this communication mechanism to deliver exogenous pharmacologic payloads to treat a variety of diseases including cancer. Functionalization of the exosome surface with proteins and peptides is an important strategy to maximize the potential of exosomes as therapeutics. Comparative proteomic analysis (LC/MS) of stringently purified exosomes led to the identification of several highly enriched and unique proteins, including a transmembrane glycoprotein (Protein X, PrX), belonging to the immunoglobulin superfamily. Stable expression of PrX in a producer cell line resulted in a 200-fold increase of PrX on the secreted exosomes. Protein X was extensively characterized and the minimum structural requirements for exosome enrichment were determined. With our engExTM platform, we developed precision engineered exosome therapeutics using PrX as a scaffold to enable high-density exosome surface display of an array of structurally and biologically diverse proteins, including enzymes, antibodies, type I cytokines, and TNF superfamily members. These proteins were genetically fused to PrX and overexpressed in a producer cell. Significantly higher transgene expression on secreted exosomes was achieved compared to conventional scaffolds, including the tetraspanins CD9/CD63/CD81 and LAMP2B. Oligomerization of PrX coupled with avidity effects inherent in exosome surface display resulted in a clear activity advantage compared to free protein. Protein X-mediated display of CD40L on exosomes resulted in a 20-fold potency increase in B cell activation over recombinant CD40L. Furthermore, expression of CD40L redirected exosome uptake from phagocytic antigen presenting cells (APCs) to B cells, demonstrating exosome surface modifications can alter cellular tropism. We also evaluated the functionality of IL-12 tethered to the exosome surface and demonstrated superior tumor retention compared to free cytokine, resulting in robust anti-tumor activity in anti-PD-1 refractory B16F10 tumor models. These results demonstrate the potential of the engExTM platform to generate novel exosome therapeutics. Citation Format: Kevin Dooley, Ke Xu, Sonya Haupt, Nuruddeen Lewis, Rane Harrison, Shelly Martin, Christine McCoy, Chang Ling Sia, Su Chul Jang, Katherine Kirwin, Russell McConnell, Bryan Choi, Adam T. Boutin, Damian Houde, Jorge Sanchez-Salazar, Agata Villiger-Oberbek, Kyriakos D. Economides, John D. Kulman, Sriram Sathyanarayanan. engEx: A novel exosome engineering platform enabling targeted transfer of pharmacological molecules [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 2150.

To assess immune-based gene therapy in a murine floor of mouth (FOM) squamous cell carcinoma (SCC) model.In vitro and in vivo testing of immune therapy for SCC.Multiple SCC lines were infected by using advRSV-interleukin-12 (IL-12) and advCMV-interleukin-12/granulocyte macrophage colony-stimulating factor (IL-12/GM-CSF) and monitored for production of IL-12 and GM-CSF. Intratumoral injections of viral vectors were administered with systemic Ig-4-1BB ligand in an orthotopic murine FOM SCC model and followed for tumor size and survival.In vitro, all cell lines produced substantial levels of IL-12 and GM-CSF. In vivo, tumors treated with advCMV-IL-12/GM-CSF and Ig-4-1BBL showed a striking reduction in tumor volume (vs control P0.0001) and improved median survival (38 days vs 19 days for control, P0.0001).Combination immune-based therapies effectively improve survival in mice bearing FOM SCC over single-modality therapy.

Quality Improvement (QI) is required in all aspects of the healthcare field. Emergency Medical Services (EMS) poses unique QI challenges. This article explores some of these challenges and provides some points to consider when performing QI in EMS services.

Objectives: Vesicular stomatitis virus (VSV) is a negative-strand ribonucleic acid (RNA) virus that replicates specifically in tumor cells and has oncolytic effects in a variety of malignant tumors. We previously demonstrated recombinant VSV vectors incorporating viral fusion protein (rVSV-F) and interleukin 12 (rVSV-IL12) to have significant antitumor effects against squamous cell carcinoma (SCC) in a murine model. Here we evaluate the potential to combine a potent chemotherapeutic agent for SCC (cisplatin) with rVSV-F and rVSV-IL12 to improve efficacy. Study Design: In vitro, three SCC cell lines were tested using rVSV-F and rVSV-IL12 with cisplatin, monitoring viral replication and cell survival. In an orthotopic floor of mouth murine SCC model, intratumoral injections of virus combined with systemic cisplatin were tested for tumor control and animal survival. Results: In vitro, virus and cisplatin combination demonstrated rapid replication and enhanced tumor cell kill. Human keratinocytes were unaffected by virus and cisplatin. In vivo, combined rVSV-F with cisplatin reduced tumor burden and improved survival (P = .2 for both), while rVSV-IL12 monotherapy had better tumor control (P = .06) and survival (P = .024) than combination therapy. Conclusions: Addition of cisplatin did not affect the ability of either virus to replicate in or kill murine SCC cells in vitro. In vivo, combination therapy enhancedrVSV-F antitumor activity, but diminished rVSV-IL12 antitumor activity. Combination therapy may provide useful treatment for SCC with the development of more efficient viral vectors in combination with different chemotherapy agents or immunostimulatory agents.

Invasive fungal infections are increasingly common in the nosocomial setting. Furthermore, because risk factors for these infections continue to increase in frequency, it is likely that nosocomial fungal infections will continue to increase in frequency in the coming decades. The predominant nosocomial fungal pathogens include Candida spp., Aspergillus spp., Mucorales, Fusarium spp., and other molds, including Scedosporium spp. These infections are difficult to diagnose and cause high morbidity and mortality despite antifungal therapy. Early initiation of effective antifungal therapy and reversal of underlying host defects remain the cornerstones of treatment for nosocomial fungal infections. In recent years, new antifungal agents have become available, resulting in a change in standard of care for many of these infections. Nevertheless, the mortality of nosocomial fungal infections remains high, and new therapeutic and preventative strategies are needed.

Objectives: Replication-competent, vesicular stomatitis virus (VSV) has been demonstrated to be an effective oncolytic agent in a variety of malignant tumors. Cytokine gene transfer has also been used as immunomodulatory therapy for cancer. To test the use of combining these two approaches, an oncolytic VSV vector (rVSV-IL12) was designed to express the murine interleukin 12 (IL12) gene. This cytokine-carrying oncolytic virus was compared with an analogous noncytokine-carrying fusogenic virus (rVSV-F) in the treatment of murine SCC VII squamous cell carcinoma (SCC). Study Design and Setting: The authors performed in vitro testing of recombinant VSV-F and recombinant VSV-IL12 in SCC cell lines. In vivo testing of multiple direct intratumoral injections of rVSV-F or rVSV-IL12 in an orthotopic floor of mouth murine model was performed. Each cell line was tested using rVSV-F or rVSV-IL12 at multiplicity of infection of 0.01. The ability of each virus to replicate was tested by real-time reverse transcriptase-polymerase chain reaction over 48 hours to determine viral copies of RNA. Cell survival was determined by MTT assay over 72 hours. IL12 expression by rVSV-IL12-treated cells was determined by enzyme-linked immunosorbent assay. Results: Both viruses demonstrated similar infection efficiency, viral replication, and cytotoxicity in vitro. In an SCC VII orthotopic floor of mouth model in immunocompetent C3H/HeJ mice, multiple intratumoral injections with each virus caused a significant reduction in tumor volume when compared with saline injections alone. The rVSV-IL12-treated tumors showed a striking reduction in tumor volume when compared with rVSV-F and saline-treated tumors (P

This study investigates the efficacy of recombinant fusogenic VSV [rVSV-NDV/F(L289A) or rVSV-F] in the treatment of head and neck squamous cell carcinoma (HNSCC).The in vitro replication and cytotoxicity of rVSV-F were studied in two human SCC cell lines, in one murine SCC cell line, and in human keratinocytes. The effects on tumor size and animal survival were investigated following in vivo rVSV-F treatment of floor-of-mouth tumor model C3H/HeJ mice.Recombinant VSV-F preferentially induced rapid syncytia formation, and replicated in (P0.04) and killed (P1 x 10(-13)) all three SCC lines tested. The virus had no observable effect on human keratinocytes. Tumor size was smaller (P0.03) and overall survival was better (P0.001) for treated animals than for control animals.Recombinant VSV-F confers a modest survival benefit for HNSCC in this orthotopic murine model. This oncolytic virus holds promise as a novel cancer treatment for recurrent HNSCC.

Incisional hernias commonly occur following abdominal wall surgery. Human acellular dermal matrices (HADM) are widely used in abdominal wall defect repair. Xenograft acellular dermal matrices, particularly those made from porcine tissues (PADM), have recently experienced increased usage. The purpose of this study was to compare the effectiveness of HADM and PADM in the repair of incisional abdominal wall hernias in a rabbit model. A review from earlier work of differences between human allograft acellular dermal matrices (HADM) and porcine xenograft acellular dermal matrices (PADM) demonstrated significant differences (P 0.05) in mechanical properties: Tensile strength 15.7 MPa vs. 7.7 MPa for HADM and PADM, respectively. Cellular (fibroblast) infiltration was significantly greater for HADM vs. PADM (Armour). The HADM exhibited a more natural, less degraded collagen by electrophoresis as compared to PADM. The rabbit model surgically established an incisional hernia, which was repaired with one of the two acellular dermal matrices 3 weeks after the creation of the abdominal hernia. The animals were euthanized at 4 and 20 weeks and the wounds evaluated. Tissue ingrowth into the implant was significantly faster for the HADM as compared to PADM, 54 vs. 16% at 4 weeks, and 58 vs. 20% for HADM and PADM, respectively at 20 weeks. The original, induced hernia defect (6 cm(2)) was healed to a greater extent for HADM vs. PADM: 2.7 cm(2) unremodeled area for PADM vs. 1.0 cm² for HADM at 20 weeks. The inherent uniformity of tissue ingrowth and remodeling over time was very different for the HADM relative to the PADM. No differences were observed at the 4-week end point. However, the 20-week data exhibited a statistically different level of variability in the remodeling rate with the mean standard deviation of 0.96 for HADM as contrasted to a mean standard deviation of 2.69 for PADM. This was significant with P 0.05 using a one tail F test for the inherent variability of the standard deviation. No significant differences between the PADM and HADM for adhesion, inflammation, fibrous tissue or neovascularization were noted.