Subcutaneous checkpoint inhibition is equivalent to systemic delivery when combined with nelitolimod delivered via pressure-enabled drug delivery for depletion of intrahepatic myeloid-derived suppressor cells and control of liver metastases.

BACKGROUND: Toll-like receptor 9 (TLR9) agonists induce inflammatory responses that promote the killing of infectious micro-organisms, cancer cells and develop adaptive immune responses. Their ability as immunomodulators to enhance the activity of checkpoint inhibitors (CPI) in treating liver tumors is limited in part by the distinctive biology of intrahepatic myeloid-derived suppressor cells (MDSC) and challenges with tumor-specific therapeutic delivery. We have shown that the regional delivery of type C TLR9 agonist via pressure-enabled drug delivery (PEDD) system improves delivery to the tumor, enhances depletion of MDSCs and overall, stimulates the immune system in combination with or without CPI. Currently, CPIs are delivered intravenously, although there is a growing interest in its subcutaneous (SQ) administration. We compared nelitolimod formerly known as SD-101 administered using PEDD in combination with systemic (Sys) or SQ CPI in murine liver metastases (LM). METHODS: The LM model was developed by injecting MC38-Luc cells via the spleen of 8-12 week old male C57/BL6 mice followed by splenectomy. After a week, fluorescently labeled nelitolimod (10 µg/mouse) was delivered via PEDD and co-administered anti-programmed cell death-1 (α-PD-1) either via Sys or SQ. Tumor burden was monitored by in vivo imaging system. Serum cytokine levels were analyzed by Luminex. Tissues were harvested on Day 3 (D3) or Day 10 (D10) post-PEDD to enrich CD45+ cells and were analyzed via NanoString targeted transcriptomics (D3) or flow cytometry (FC, D10) to interrogate immune cell populations (D10). For NanoString analysis, the innate immune panels were selected, and for FC, MDSCs (CD11b+Gr1+), B cells (B220+), dendritic cells (DC, CD11c+), T (CD3+) cells, and M1-like macrophages (F4/80+CD38+Egr2-) were quantified. RESULTS: Nelitolimod delivered via PEDD resulted in changes in innate and adaptive immune cells within LM, including depletion of liver MDSC and increased M1-like macrophages in the liver, which are supportive of antitumor immunity. While CPI monotherapy failed to control tumor progression, nelitolimod and CPI combination improved LM control, survival and antitumor immunity beyond the nelitolimod monotherapy effect, irrespective of CPI delivery route. CONCLUSION: The SQ route of CPI delivery was equivalent to Sys in combination with nelitolimod, suggesting SQ-CPI may be a rational choice in combination with PEDD of nelitolimod for liver tumor treatment.

Pressure-Enabled Drug Delivery (PEDD) of a class C TLR9 agonist in combination with checkpoint inhibitor therapy in a murine pancreatic cancer model.

BACKGROUND: Systemic immunotherapy has had limited clinical benefit in pancreatic ductal adenocarcinoma. This is thought to be due to its desmoplastic immunosuppressive tumor microenvironment in addition to high intratumoral pressures that limit drug delivery. Recent preclinical cancer models and early-phase clinical trials have demonstrated the potential of toll-like receptor 9 agonists, including the synthetic CpG oligonucleotide SD-101, to stimulate a wide range of immune cells and eliminate suppressive myeloid cells. We hypothesized that Pressure-Enabled Drug Delivery via Pancreatic Retrograde Venous Infusion of toll-like receptor 9 agonist would improve responsiveness to systemic anti-programmed death receptor-1 checkpoint inhibitor therapy in a murine orthotopic pancreatic ductal adenocarcinoma model. METHODS: Murine pancreatic ductal adenocarcinoma (KPC4580P) tumors were implanted into the pancreatic tails of C57BL/6J mice and treated 8 days after implantation. Mice were assigned to one of the following treatment groups: Pancreatic Retrograde Venous Infusion delivery of saline, Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist, systemic anti-programmed death receptor-1, systemic toll-like receptor 9 agonist, or the combination of Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist and systemic anti-programmed death receptor-1 (Combo). Fluorescently labeled toll-like receptor 9 agonist (radiant efficiency) was used to measure uptake of the drug on day 1. Changes in tumor burden were evaluated by necropsy at 2 different time points, 7 and 10 days after toll-like receptor 9 agonist treatment. Blood and tumors were collected at necropsy 10 days after toll-like receptor 9 agonist treatment for flow cytometric analysis of tumor-infiltrating leukocytes and plasma cytokines. RESULTS: All mice analyzed survived to necropsy. Site of tumor fluorescence measurements revealed 3-fold higher intensity fluorescence in Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist compared to systemic toll-like receptor 9 agonist mice. Tumor weights were significantly lower in the Combo group compared to Pancreatic Retrograde Venous Infusion delivery of saline. Flow cytometry of the Combo group demonstrated significantly increased overall T-cell number, specifically CD4+ T-cells, and a trend toward increased CD8+ T-cells. Cytokine analysis showed significantly decreased IL-6 and CXCL1. CONCLUSION: Pressure-Enabled Drug Delivery of toll-like receptor 9 agonist by Pancreatic Retrograde Venous Infusion with systemic anti-programmed death receptor-1 demonstrated improved pancreatic ductal adenocarcinoma tumor control in a murine pancreatic ductal adenocarcinoma model. These results support study of this combination therapy in pancreatic ductal adenocarcinoma patients and expansion of ongoing Pressure-Enabled Drug Delivery clinical trials.

Draft Genome Sequence of Vibrio parahaemolyticus PSU5579, Isolated during an Outbreak of Acute Hepatopancreatic Necrosis Disease in Thailand.

Here, we announce the draft genome sequence of Vibrio parahaemolyticus strain PSU5579, isolated from a shrimp hatchery in southern Thailand during an outbreak of acute hepatopancreatic necrosis disease (AHPND). The genome contains 44 contigs with a sequence length of 5,229,426 bp, 4,861 coding sequences, and a G+C content of 45.3%.

Regional infusion of a class C TLR9 agonist enhances liver tumor microenvironment reprogramming and MDSC reduction to improve responsiveness to systemic checkpoint inhibition.

Myeloid-derived suppressor cells (MDSCs) expand in response to malignancy and suppress responsiveness to immunotherapy, including checkpoint inhibitors (CPIs). Within the liver, MDSCs have unique immunosuppressive features. While TLR9 agonists have shown promising activities in enhancing CPI responsiveness in superficial tumors amenable to direct needle injection, clinical success for liver tumors with TLR9 agonists has been limited by delivery challenges. Here, we report that regional intravascular infusion of ODN2395 into mice with liver metastasis (LM) partially eliminated liver MDSCs and reprogrammed residual MDSC. TLR9 agonist regional infusion also induced an increase in the M1/M2 macrophage ratio. Enhanced TLR9 signaling was demonstrated by an increased activation of in NFκB (pP65) and production of IL6 compared with systemic infusion. Further, PBMC-derived human MDSCs express TLR9, and treatment with class C TLR9 agonists (ODN2395 and SD101) reduced the expansion of MDSC population. TLR9 stimulation induced MDSC apoptosis and increased the M1/M2 macrophage ratio. Regional TLR9 agonist infusion along with systemic anti-PD-1 therapy improved control of LM. With effective delivery, TLR9 agonists have the potential to favorably reprogram the liver TME through reduction of MDSCs and favorable macrophage polarization, which may improve responsiveness to systemic CPI therapy.

Antigen Receptor T Cells (CAR-T) Effectively Control Tumor Growth in a Colorectal Liver Metastasis Model.

BACKGROUND: Effective treatment of solid tumors requires multi-modality approaches. In many patients with stage IV liver disease, current treatments are not curative. Chimeric antigen receptor T cells (CAR-T) are an intriguing option following success in hematological malignancies, but this has not been translated to solid tumors. Limitations include sub-optimal delivery and elevated interstitial fluid pressures. We developed a murine model to test the impact of high-pressure regional delivery (HPRD) on trafficking to liver metastases (LM) and tumor response. MATERIALS AND METHODS: CAR-T were generated from CD45.1 mice and adoptively transferred into LM-bearing CD45.2 mice via regional or systemic delivery (RD, SD). Trafficking, tumor growth, and toxicity were evaluated with flow cytometry, tumor bioluminescence (TB, photons/sec log2-foldover baseline), and liver function tests (LFTs). RESULTS: RD of CAR-T was more effective at controlling tumor growth versus SD from post-treatment days (PTD) 2-7 (P = 0.002). HPRD resulted in increased CAR-T penetration versus low-pressure RD (LPRD, P = 0.004), suppression of tumor proliferation (P = 0.03), and trended toward improved long-term control at PTD17 (TB=3.7 versus 6.1, P = 0.47). No LFT increase was noted utilizing HPRD versus LPRD (AST/ALT P = 0.65/0.84) while improved LFTs in RD versus SD groups suggested better tumor control (HPRD AST/ALT P = 0.04/0.04, LPRD AST/ALT P = 0.02/0.02). CONCLUSIONS: Cellular immunotherapy is an emerging option for solid tumors. Our model suggests RD and HPRD improved CAR-T penetration into solid tumors with improved short-term tumor control. Barriers associated with SD can be overcome using RD techniques to maximize therapeutic delivery and HPRD may further augment efficacy without increased toxicity.

Tocilizumab for severe COVID-19 related illness - A community academic medical center experience.

The SARS-CoV-2 virus responsible for the COVID-19 pandemic can result in severe or fatal disease in a subset of infected patients. While the pathogenesis of severe COVID-19 disease has yet to be fully elucidated, an overexuberant and harmful immune response to the SARS-CoV-2 virus may be a pivotal aspect of critical illness in this patient population. The inflammatory cytokine, IL-6, has been found to be consistently elevated in severely ill COVID-19 patients, prompting speculation that IL-6 is an important driver of the pathologic process. The inappropriately elevated levels of inflammatory cytokines in COVID-19 patients is similar to cytokine release syndrome (CRS) observed in cell therapy patients. We sought to describe outcomes in a series of severely ill patients with COVID-19 CRS following treatment with anti-IL-6/IL-6-Receptor (anti-IL-6/IL-6-R) therapy, including tocilizumab or siltuximab. At our academic community medical center, we formed a multi-disciplinary committee for selecting severely ill COVID-19 patients for therapy with anti-IL-6 or IL-6-R agents. Key selection criteria included evidence of hyperinflammation, most notably elevated levels of C-reactive protein (CRP) and ferritin, and an increasing oxygen requirement. By the data cutoff point, we treated 31 patients with anti-IL-6/IL-6-R agents including 12 who had already been intubated. Overall, 27 (87%) patients are alive and 24 (77%) have been discharged from the hospital. Clinical responses to anti-IL-6/IL-6-R therapy were accompanied by significant decreases in temperature, oxygen requirement, CRP, IL-6, and IL-10 levels. Based on these data, we believe anti-IL-6/IL-6-R therapy can be effective in managing early CRS related to COVID-19 disease. Further study of anti-IL-6/IL-6-R therapy alone and in combination with other classes of therapeutics is warranted and trials are underway.

HITM-SURE: Hepatic immunotherapy for metastases phase Ib anti-CEA CAR-T study utilizing pressure enabled drug delivery.

In recent years, cell therapy technologies have resulted in impressive results in hematologic malignancies. Treatment of solid tumors with chimeric antigen receptor T-cells (CAR-T) has been less successful. Solid tumors present challenges not encountered with hematologic cancers, including high intra-tumoral pressure and ineffective CAR-T trafficking to the site of disease. Novel delivery methods may enable CAR-T therapies for solid tumor malignancies. A patient with liver metastases secondary to pancreatic adenocarcinoma received CAR-T targeting carcinoembryonic antigen (CEA). Previously we reported that Pressure-Enabled Drug Delivery (PEDD) enhanced CAR-T delivery to liver metastases 5.2-fold. Three doses of anti-CEA CAR-T were regionally delivered via hepatic artery infusion (HAI) using PEDD technology to optimize the therapeutic index. Interleukin-2 was systemically delivered by continuous intravenous infusion to support CAR-T in vivo. HAI of anti-CEA CAR-T was not associated with any serious adverse events (SAEs) above grade 3 and there were no on-target/off-tumor SAEs. Following CAR-T treatment, positron emission tomography-CT demonstrated a complete metabolic response within the liver, which was durable and sustained for 13 months. The response was accompanied by normalization of serum tumor markers and an abundance of CAR+ cells found within post-treatment tumor specimens. The findings from this report exhibit biologic activity and safety of regionally infused CAR-T for an indication with limited immune-oncology success to date. Further studies will determine how HAI of CAR-T may be included in multidisciplinary treatment plans for patients with liver metastases. ClinicalTrials.gov number, NCT02850536.

Draft Genome Sequence of the Putative Marine Pathogen Thalassobius sp. I31.1.

Thalassobius sp. I31.1 is a putative pathogen involved in epizootic shell disease in the American lobster (Homarus americanus). We report here the draft genome sequence for Thalassobius sp. I31.1 and provide insight into its metabolism and links to environmental pollutant degradation.

Draft Genome Sequence of Loktanella maritima Strain YPC211, a Commensal Bacterium of the American Lobster (Homarus americanus).

Loktanella maritima strain YPC211 was isolated from the American lobster (Homarus americanus). We report here the draft genome sequence for L. maritima YPC211 and identify genes of potential importance to its role within the microbial community.

Draft Genome Sequence of the Putative Marine Pathogen Aquimarina sp. Strain I32.4.

Aquimarina sp. strain I32.4 (formerly Aquimarina sp. 'homaria') is a putative pathogen involved in epizootic shell disease in the American lobster (Homarus americanus). We report here the draft genome sequence for Aquimarina sp. strain I32.4 and describe virulence factors that may provide insight into its mechanism of pathogenicity.

Draft Genome Sequence of Bowmanella denitrificans JL63, a Bacterium Isolated from Whiteleg Shrimp (Litopenaeus vannamei) That Can Inhibit the Growth of Vibrio parahaemolyticus.

Bowmanella denitrificans strain JL63 was isolated from a whiteleg shrimp (Litopenaeus vannamei) and was determined to have antibacterial activity against an acute hepatopancreatic necrosis disease (AHPND) strain of Vibrio parahaemolyticus Here, we report the draft genome sequence of this strain and identify genes that are potentially involved in its antibacterial activity.