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PDF file - 182K, Table S1 shows the correlation between all immune markers expression tested; Table S2 shows the lack of correlation between the favorable immune score and clinicopathologic characteristics. Figure S1 illustrates the quantification method for MHC Class I and Beta 2-microglobulin; Figure S2 shows the association between the immune score and the Clinical Risk Score.

Tumor-infiltrating lymphocytes (TIL) in colorectal cancer liver metastases (CLM) have been associated with more favorable patient outcomes, but whether MHC class I (MHC-I) expression on cancer cells affects prognosis is uncertain. Immunohistochemistry was performed on a tissue microarray of 158 patients with CLM, who underwent partial hepatectomy with curative intent. Using the antibody HC-10, which detects HLA-B and HLA-C antigens and a minority of HLA-A antigens, MHC-I expression was correlated with β-2 microglobulin (β2m; r = 0.7; P < 0.001), but not with T-cell density (r < 0.32). The median follow-up for survivors was 9.7 years. High levels of MHC-I expression in tumors concomitant with high T-cell infiltration (CD3, CD4, or CD8) best identified patients with favorable outcomes, compared with patients with one or none of these immune features. The median overall survival (OS) of patients with MHC-IhiCD3hi tumors (n = 31) was 116 months compared with 40 months for the others (P = 0.001), and the median time to recurrence (TTR) was not reached compared with 17 months (P = 0.008). By multivariate analysis, MHChiCD3hi was associated with OS and TTR independent of the standard clinicopathologic variables. An immune score that combines MHC-I expression and TIL density may be a valuable prognostic tool in the treatment of patients with CLM. Cancer Immunol Res; 2(6); 530–7. ©2014 AACR.

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.

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.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 logRD 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).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.

Supplemental Figure 1. In vitro cytotoxicity assay

Purpose: Chimeric antigen receptor–modified T cells (CAR-T) have demonstrated encouraging results in early-phase clinical trials. Successful adaptation of CAR-T technology for CEA-expressing adenocarcinoma liver metastases, a major cause of death in patients with gastrointestinal cancers, has yet to be achieved. We sought to test intrahepatic delivery of anti-CEA CAR-T through percutaneous hepatic artery infusions (HAIs).Experimental Design: We conducted a phase I trial to test HAI of CAR-T in patients with CEA+ liver metastases. Six patients completed the protocol, and 3 received anti-CEA CAR-T HAIs alone in dose-escalation fashion (108, 109, and 1010 cells). We treated an additional 3 patients with the maximum planned CAR-T HAI dose (1010 cells × 3) along with systemic IL2 support.Results: Four patients had more than 10 liver metastases, and patients received a mean of 2.5 lines of conventional systemic therapy before enrollment. No patient suffered a grade 3 or 4 adverse event related to the CAR-T HAIs. One patient remains alive with stable disease at 23 months following CAR-T HAI, and 5 patients died of progressive disease. Among the patients in the cohort that received systemic IL2 support, CEA levels decreased 37% (range, 19%–48%) from baseline. Biopsies demonstrated an increase in liver metastasis necrosis or fibrosis in 4 of 6 patients. Elevated serum IFNγ levels correlated with IL2 administration and CEA decreases.Conclusions: We have demonstrated the safety of anti-CEA CAR-T HAIs with encouraging signals of clinical activity in a heavily pretreated population with large tumor burdens. Further clinical testing of CAR-T HAIs for liver metastases is warranted. Clin Cancer Res; 21(14); 3149–59. ©2015 AACR.

Supplemental Figure 4. Cytokine response to treatment

Supplemental Figure 3. Radiographic studies

Supplemental Figure 2. Flow cytometry gating

Myeloid-derived suppressor cells (MDSCs) promote immunosuppressive activities in the tumor microenvironment (TME), resulting in increased tumor burden and diminishing the anti-tumor response of immunotherapies. While primary and metastatic tumors are typically the focal points of therapeutic development, the immune cells of the TME are differentially programmed by the tissue of the metastatic site. In particular, MDSCs are programmed uniquely within different organs in the context of tumor progression. Given that MDSC plasticity is shaped by the surrounding environment, the proteomes of MDSCs from different metastatic sites are hypothesized to be unique. A bottom-up proteomics approach using sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to quantify the proteome of CD11b+ cells derived from murine liver metastases (LM) and lung metastases (LuM). A comparative proteomics workflow was employed to compare MDSC proteins from LuM (LuM-MDSC) and LM (LM-MDSC) while also elucidating common signaling pathways, protein function, and possible drug-protein interactions. SWATH-MS identified 2516 proteins from 200 µg of sample. Of the 2516 proteins, 2367 have matching transcriptomic data. Upregulated proteins from lung and liver-derived murine CD11b+ cells with matching mRNA transcriptomic data were categorized based on target knowledge and level of drug development. Comparative proteomic analysis demonstrates that liver and lung tumor-derived MDSCs have distinct proteomes that may be subject to pharmacologic manipulation.

Immune checkpoint inhibitors (ICI) have demonstrated limited success in patients with metastatic uveal melanoma (MUM) with liver involvement due to an immunosuppressive tumor microenvironment (TME) driven in part by myeloid-derived suppressor cells (MDSCs). Toll-like receptor-9 agonists (TLR-9A) have improved ICI response rates in cutaneous melanoma, but delivery challenges have limited their application for MUM. Hepatic arterial infusion (HAI) of TLR-9A using a pressure-enabled drug deliveryTM (PEDDTM) device has the potential to enhance responsiveness to ICI by optimizing delivery to intrahepatic tumors and reprogramming the TME, including elimination of MDSCs.PERIO-01 is an open-label first-in-human Phase 1 trial of SD-101 given by HAI using a PEDDTM in MUM (NCT04935229). The study consists of dose-escalation cohorts of single agent SD-101 alone and with ICI. SD-101 is delivered over 2 cycles, with 3 weekly doses per cycle. Research blood, tumor and normal liver biopsies are collected serially for correlative studies. At data cutoff, a total of 20 patients were enrolled, with 13 in the single agent dose escalation cohort (2, 4, and 8 mg) and 7 patients with SD-101 (2 mg) + nivolumab. The median age was 65.5 years with an equal gender distribution. Only 2 patients were treatment-naïve and the median number of liver metastases was 5.1. The average number of SD-101 infusions was 5.2. One patient in the combination cohort experienced a serious adverse event related to treatment - asymptomatic Grade 3 increase in liver enzymes. PEDDTM resulted in high drug levels in the liver (up to 2,340 ng/ml at 8mg) with only transient exposure in the periphery ( Citation Format: Sapna P. Patel, Cara Haymaker, Rahul A. Sheth, Joshua D. Kuban, Joshua Weintraub, Eric Wehrenberg-Klee, Paula Novelli, Carin Gonsalves, Robert Adamo, Virgina Honaker, Laura Timciuc, Tarin Hennegan, Juan C. Amador Molina, Dzifa Duose, Edwin R. Parras Cuenta, Anthony Lucci, Salyna Meas, Vanessa Sarli, Victor G. Prieto, Jason LaPorte, Ann-Marie Hulstine, Ashley Moody, Bryan Cox, David Geller, Diwakar Davar, Kamaneh Montazeri, Marlana Orloff, Steven C. Katz, Richard Carvajal. PERIO-01: Initial safety experience and immunologic effects of a Class C TLR9 agonist using pressure- enabled drug delivery in a phase 1 trial of hepatic arterial infusion of SD-101 +/- checkpoint inhibition in metastatic uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5881.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that proliferate in the setting of cancer and have potent immunosuppressive functions hindering anti-tumor immunity. Here we establish that the immunologic landscape and tumor microenvironments (TME) vary between different organs which discretely shape MDSC repertoires. We found that pSTAT3 signaling exerts a dominant effect on MDSC programming in liver metastasis (LM). In contrast, in lung metastasis (LuM), MDSC programming is driven mainly by pSTAT5. Adoptive transfer of LM-MDSC into LuM resulted in a shift from pSTAT3 signaling to pSTAT5, in association with an overall shift toward lung MDSC programming. A shift from more immunosuppressive M-MDSC to G-MDSC, along with enhanced differentiation of MDSCs into pro-inflammatory M1 macrophages in LuM, indicated that MDSC plasticity and differentiation patterns are environmentally dependent. Using mass spectroscopy, we confirmed that LM-MDSCs showed enhanced expression of key proliferation pathway markers. This confirmed that liver-specific MDSC programing was comprehensive but reversible, implying that therapeutic targeting of LM-MDSC could prime the TME in a favorable manner. Our data suggest that MDSC programming in response to malignancy is highly dependent on organ-specific conditions and is modifiable.

Chimeric Antigen Receptor T cell (CAR-T) therapies have demonstrated promising clinical response rates for patients with hematological malignancies. CAR-T cells are generated by bioengineering patient T cells to express CAR proteins that can specifically target tumor antigens. For CAR-T therapy to be effective in solid tumors, several factors including trafficking, activation, expansion, and persistence are important considerations in hostile tumor microenvironment (TME). Despite significant advances in CAR-T therapies, clinical success in solid tumor indications has been limited. The complex processes involved in CAR-T manufacturing and quality control (QC) contribute to high cost of these therapies, creating barriers to widespread adoption. The processes involved are not uniformly standardized and may require numerous manual handling steps. This chapter details strategies to overcome barriers to broad CAR-T application and reviews current production processes used for CAR-T manufacturing. Emphasis is placed on reproducibility, manufacturing costs, and product release testing. Implementing new processing alternatives and innovative CAR-T designs, along with further innovation and efficiencies will be key to more consistent CAR-T cell therapy success.

We describe the use of pancreatic retrograde venous infusion in an orthotopic murine model of pancreatic ductal adenocarcinoma and hypothesize that pancreatic retrograde venous infusion delivery of gemcitabine will increase concentrations of gemcitabine in the tumor and the subsequent tumor response to treatment.Murine pancreatic ductal adenocarcinoma (KPC4580P) was transplanted onto the pancreatic tail of C57BL/6J mice. Groups (n = 15) of mice were assigned to sham laparotomy and 100 mg/kg intraperitoneal infusion of gemcitabine (systemic gemcitabine), pancreatic venous isolation with pancreatic retrograde venous infusion of 100 mg/kg gemcitabine, or pancreatic retrograde venous infusion with saline infusion. Tumor pressures were recorded during pancreatic retrograde venous infusion. Mice were killed at 1 hour or 7 days after infusion.Baseline tumor pressures were 45 ± 8 mm Hg, and pancreatic retrograde venous infusion increased tumor pressures by 29 ± 6 mm Hg (P.01). Pancreatic retrograde venous infusion gemcitabine mice had greater tumor gemcitabine concentrations compared with systemic gemcitabine (127 vs 19 ng/mg; P.01) and lesser tumor volumes compared with both systemic gem and pancreatic retrograde venous infusion with saline (274 vs 857 vs 629 mmPancreatic retrograde venous infusion increased tumor pressures greater than baseline, improved gemcitabine delivery, and increased the treatment response. These findings suggest that pressurized, regional delivery overcomes the increased pressure barrier in pancreatic ductal adenocarcinoma. Additional preclinical studies with cytotoxic and immunotherapeutic agents and clinical trials using pressure-enabled drug delivery with pancreatic retrograde venous infusion devices are underway.

With the advent of immunotherapy as an integral component of multidisciplinary solid tumor treatment, we are confronted by an unfamiliar and novel pattern of radiographic responses to treatment. Enlargement of tumors or even new lesions may not represent progression, but rather reflect what will ultimately evolve into a clinically beneficial response. In addition, the kinetics of radiographic changes in response to immunotherapy treatments may be distinct from what has been observed with cytotoxic chemotherapy and radiation. The phenomenon of pseudoprogression has been documented in patients receiving immunotherapeutic agents, such as checkpoint inhibitors and cellular therapies. Currently, there are no clinical response guidelines that adequately account for pseudoprogression and solid tumor responses to immunotherapy in general. Even so, response criteria have evolved to account for the radiographic manifestations of novel therapies. The evolution of World Health Organization (WHO) criteria and Response Evaluation Criteria in Solid Tumors (RECIST), along with the emergence of immune-related response criteria (irRC) and the immune Response Evaluation Criteria in Solid Tumors (iRECIST) reflect the need for new frameworks. This review evaluates the relationship between pseudoprogression, clinical outcomes, and our current understanding of the biology of pseudoprogression. To achieve our goal, we discuss unusual response patterns in patients receiving immunotherapy. We seek to develop a deeper understanding of radiographic responses to immunotherapy such that clinical benefit is not underappreciated in individual patients and during clinical investigation.

ObjectiveProgrammed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures.DesignWe created organotypic slice cultures from human CRLM (n=38 patients’ tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy.ResultsαIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function.ConclusionNeutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.

Metastatic liver tumors have presented challenges with the use of checkpoint inhibitors (CPIs), with only limited success. We hypothesize that regional delivery (RD) of CPIs can improve activity in the liver and minimize systemic exposure, thereby reducing immune-related adverse events (irAE). Using a murine model of colorectal cancer liver metastases (LM), we confirmed high levels of PD-L1 expression on the tumor cells and liver myeloid-derived suppressor cells (L-MDSC). In vivo, we detected improved LM response at 3 mg/kg on PTD7 via portal vein (PV) regional delivery as compared to 3 mg/kg via tail vein (TV) systemic delivery (p = 0.04). The minimal effective dose at PTD7 was 5 mg/kg (p = 0.01) via TV and 0.3 mg/kg (p = 0.02) via PV. We detected 6.7-fold lower circulating CPI antibody levels in the serum using the 0.3 mg/kg PV treatment compared to the 5 mg/kg TV cohort (p &lt, 0.001) without increased liver toxicity. Additionally, 3 mg/kg PV treatment resulted in increased tumor cell apoptotic signaling compared to 5 mg/kg TV (p &lt, 0.05). Therefore, RD of an anti-PD-1 CPI therapy for CRCLM may improve the therapeutic index by reducing the total dose required and limiting the systemic exposure. These advantages could expand CPI indications for liver tumors.

Effective chimeric antigen receptor-modified T-cell (CAR-T) therapy for liver metastases (LM) will require innovative solutions to ensure efficient delivery and minimization of systemic toxicity. We previously demonstrated the safety of CAR-T hepatic artery infusions (HAI). We subsequently conducted the phase 1b HITM-SIR trial, in which six patients (pts) with CEA+ LM received anti-CEA CAR-T HAIs and selective internal radiation therapy (SIRT). The primary endpoint was safety with secondary assessments of biologic activity. Enrolled pts had a mean LM size of 6.4 cm, 4 pts had >10 LM, and pts received an average of two lines of prior systemic therapy. No grade 4 or 5 toxicities were observed, and there were no instances of severe cytokine-release syndrome (CRS) or neurotoxicity. The mean transduction efficiency was 60.4%. Following CAR-T HAI, reduced levels of GM-CSF-R, IDO, and PD-L1 were detected in LM, and serum CEA levels were stable or decreased in all subjects. Median survival time was 8 months (mean 11, range 4–31). Anti-CEA CAR-T HAI with subsequent SIRT was well tolerated, and biologic responses were demonstrated following failure of conventional therapy. HAI of CAR-T was once again confirmed not to be associated with severe CRS or neurotoxicity.

Successful remission in hematological cancers by CAR-T cell immunotherapy has yet to be replicated in solid tumors like GBM. A significant impediment of CAR-T immunotherapy in solid tumors is poor exposure of T cells to tumor antigens resulting in suboptimal CAR-T cell activation, which ultimately fails to induce a robust anti-tumor immune response. Costimulatory moieties in advanced-generation CARs, along with additional IL2 therapy has been shown to be insufficient to overcome this hurdle and have its cytotoxic limitations. GSK3 is constitutively active in naive T cells and is transiently inactivated during T cell activation resulting in rapid T cell proliferation. Pharmacologic inhibition of GSK3 in GBM-specific CAR-T cells reduced FasL expression, increased T cell proliferation and reduced exhaustion by lowering PD-1 levels resulting in the development of CAR-T effector memory phenotype. Treatment with GSK3-inhibited CAR-T cells resulted in 100% tumor elimination during the tumor-rechallenge experiment in GBM-bearing animals and increased accumulation of memory CAR-T cells in secondary lymphoid organs. These adjuvant-like effects of GSK3 inhibition on activated CAR-T cells may be a valuable adjunct to a successful implementation of CAR-T immunotherapy against GBM and other solid tumors.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that proliferate in the setting of cancer and have potent immunosuppressive functions hindering anti-tumor immunity. Here we establish that the immunologic landscape and tumor microenvironments (TME) vary between different organs which discretely shape MDSC repertoires. We found that pSTAT3 signaling exerts a dominant effect on MDSC programming in liver metastasis (LM). In contrast, in lung metastasis (LuM), MDSC programming is driven mainly by pSTAT5. Adoptive transfer of LM-MDSC into LuM resulted in a shift from pSTAT3 signaling to pSTAT5, in association with an overall shift toward lung MDSC programming. A shift from more immunosuppressive M-MDSC to G-MDSC, along with enhanced differentiation of MDSCs into pro-inflammatory M1 macrophages in LuM, indicated that MDSC plasticity and differentiation patterns are environmentally dependent. Using mass spectroscopy, we confirmed that LM-MDSCs showed enhanced expression of key proliferation pathway markers. This confirmed that liver-specific MDSC programing was comprehensive but reversible, implying that therapeutic targeting of LM-MDSC could prime the TME in a favorable manner. Our data suggest that MDSC programming in response to malignancy is highly dependent on organ-specific conditions and is modifiable.

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.

Purpose To determine the safety and feasibility of pancreatic retrograde venous infusion (PRVI) utilizing a microvalvular infusion system (MVI) to deliver ethiodized oil (lipiodol) by means of the Pressure-Enabled Drug Delivery (PEDD) approach. Methods Utilizing transhepatic access, mapping of the pancreatic body and head venous anatomy was performed in 10 swine. PEDD was performed by cannulation of veins in the head (n = 4) and body (n = 10) of the pancreas with a MVI (Surefire® Infusion System (SIS), Surefire Medical, Inc (DBA TriSalus™ Life Sciences)) followed by infusion with lipiodol. Sets of animals were killed either immediately (n = 8) or at 4 days post-PRVI (n = 2). All pancreata were harvested and studied with micro-CT and histology. We also performed three-dimensional volumetric/multiplanar imaging to assess the vascular distribution of lipiodol within the glands. Results A total of 14 pancreatic veins were successfully infused with an average of 1.7 (0.5–2.0) mL of lipiodol. No notable change in serum chemistries was seen at 4 days. The signal-to-noise ratio (SNR) of lipiodol deposition was statistically increased both within the organ in target relative to non-target pancreatic tissue and compared to extra pancreatic tissue (p Conclusions PEDD using the RVI approach for targeted pancreatic infusions is technically feasible and did not result in organ damage in this pilot animal study.

Immunosuppressive myeloid-derived suppressor cells (MDSC) subvert antitumor immunity and limit the efficacy of chimeric antigen receptor T cells (CAR-T). Previously, we reported that the GM-CSF/JAK2/STAT3 axis drives liver-associated MDSC (L-MDSC) proliferation and blockade of this axis rescued antitumor immunity. We extended these findings in our murine liver metastasis (LM) model, by treating tumor-bearing mice with STAT3 inhibitors (STATTIC or BBI608) to further our understanding of how STAT3 drives L-MDSC suppressive function. STAT3 inhibition caused significant reduction of tumor burden as well as L-MDSC frequencies due to decrease in pSTAT3 levels. L-MDSC isolated from STATTIC or BBI608-treated mice had significantly reduced suppressive function. STAT3 inhibition of L-MDSC was associated with enhanced antitumor activity of CAR-T. Further investigation demonstrated activation of apoptotic signaling pathways in L-MDSC following STAT3 inhibition as evidenced by an upregulation of the pro-apoptotic proteins Bax, cleaved caspase-3, and downregulation of the anti-apoptotic protein Bcl-2. Accordingly, there was also a decrease of pro-survival markers, pErk and pAkt, and an increase in pro-death marker, Fas, with activation of downstream JNK and p38 MAPK. These findings represent a previously unrecognized link between STAT3 inhibition and Fas-induced apoptosis of MDSCs. Our findings suggest that inhibiting STAT3 has potential clinical application for enhancing the efficacy of CAR-T cells in LM through modulation of L-MDSC.

CpG oligodeoxynucleotides (ODNs) are TLR9 agonists (TLR9As) that stimulate pDC and B cells. Yet, multiple other cell types in the liver express TLR9. The effect of TLR9A in reshaping the tumor microenvironment (TME), particularly in the myeloid-derived suppressor cells (MDSC) compartment, is yet to be determined. Clinical success of TLR9As has been limited by toxicity when given systemically and delivery limitations when administered by direct needle injection. Here, we hypothesized that regional intra-vascular infusion of TRL9As would reprogram the liver MDSC compartment to enable immune control of liver metastases (LM). In this study, we evaluated the effect of class C ODN-2395 as TLR9A in inhibiting LM progression and its impact on the liver MDSC population. C57/BL6 mice were challenged with 2.5e6 MC38-CEA-Luc cells via intra-splenic route. After a week, mice were treated with 1, 3, 10 or 30 µg ODN2395 via PV or IV (30 µg). Bioluminescence was measured at 24 and 48 hrs post-ODN administration. CD45+ cells were isolated, and FACS analysis was performed to quantify MDSCs, monocytic MDSCs (M-MDSC), and M1-macrophage subsets, along with downstream signaling. We found that 30 µg ODN2395 delivered via PV compared to IV reduced tumor burden at 24 hrs and persisted up to 48 hrs. Also, 30 µg ODN2395 infused via PV was optimal in controlling MDSC, M-MDSC cells and polarized CD11b+F4/80+ monocytic cells towards pro-inflammatory/anti-tumorigenic M1 macrophage. Using an NFκB-dependent soluble alkaline phosphatase assay (SEAP), we determined that ODN2395 dose-dependently enhanced NFκB transcription factor activity (p Table:Readout30µg IV30µg PVP valuenBioluminescence-24 h (photon/s)4e71e7 Citation Format: Chandra Ghosh, Kara Heatherton, Kyle O'Connell, Jason Laporte, Prajna Guha, David Jaroch, Bryan Cox, Steven C. Katz. Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1793.

e14517 Background: Colorectal cancer liver metastases (CRCLM) are a major source of morbidity and mortality. Historically, curative therapy has been limited to surgical resection, but only a small fraction of patients are eligible. Cellular immunotherapy has shown promise in hematologic cancers, but challenges to solid tumor therapy remain, including lymphocyte trafficking, elevated interstitial fluid pressures, and immunosuppression. Regional intravascular infusion is a non-surgical, minimally invasive procedure commonly used in liver cancer to deliver therapeutics, which can be augmented by Pressure Enabled Drug Delivery (PEDD). We hypothesized that utilizing established regional delivery strategies to administer natural killer (NK) cells engineered to express a natural killer group 2, member D (NKG2D) activating chimeric receptor and membrane bound IL-15 (CAR NKG2D cells) could increase anti-tumor activity against liver cancer. Methods: In vitro cytotoxicity of CAR NKG2D NK cells was determined in co-culture systems. CRCLM-bearing NSG mice were treated with either CAR NKG2D, non-transduced NK cells (NT-NK), or vehicle via portal vein (PV) for regional PEDD or tail vein (TV) for systemic delivery (SD). Tumor burden was measured via tumor bioluminescence. Mann-Whitney tests were performed for statistical comparisons. Correlation of NKG2D ligand expression in tissue and serum was measured by CODEX and Luminex. Results: Multiple NKG2D ligands are highly expressed in hepatocellular and colorectal carcinoma cell lines (HCC and CRC respectively). As such, these cells lines are highly susceptible to NKG2D-mediated cytotoxicity. CAR NKG2D NK cells were 3- to 4-fold more potent in vitro than NT-NK cells against multiple HCC and CRC cell lines, including those bearing Ras pathway mutations. Using a mouse model of locoregional delivery under high pressure (10 mL/minute), we show that significant tumor reduction (p < 0.05) is only achieved when CAR NKG2D NK cells, but not vehicle or NT-NK cells, were delivered via PV and not via TV. Recovery of CAR NKG2D NK cells in hepatic tissues was on average 2-fold higher after administration via PV than that observed after TV delivery (p = 0.0001). PV delivery of NT-NK cells did not result in appreciable liver engraftment or tumor growth inhibition. Conclusions: CAR NKG2D NK cells demonstrate enhanced in vitro and in vivo cytotoxicity against CRC and HCC cell lines. Significant tumor control using regional delivery in initial studies support continued clinical development. NKX101 is an investigational agent comprised of CAR NKG2D NK cells being evaluated in a phase 1 clinical study for treatment of relapsed/refractory acute myeloid leukemia or higher risk myelodysplastic syndrome. Studies are ongoing to understand NKX101 kinetics, role of delivery pressure, and activity in combination in preclinical models of CRCLM.

Background: The primary objective of our study was to assess the association of primary tumor lymph node ratio (LNR) in stage IV colorectal adenocarcinomas (CRC) with overall survival (OS) and the extent of metastatic disease in the liver. Methods: We analyzed data on 53 stage IV CRC patients who underwent surgical resection of the primary tumor. The median LNR of 0.25 was used to stratify patients into high LNR (H-LNR) and low LNR (L-LNR) groups. Statistical comparison was performed using chi square test and multiple regression models. OS was calculated using the Kaplan-Meier (KM) method while cox regression was used for multivariate analysis. Results: H-LNR status was associated with the presence of >3 liver metastases (LM) [odds ratio (OR): 2.43, P=0.047] and bilobar LM (OR: 3.94, P=0.039). The OS in H-LNR patients was significantly worse in the entire cohort compared to L-LNR (9% vs. 34% at 3 years, P=0.027). The 5-year OS in patients undergoing liver resection for LM was also significantly worse in the H-LNR group (0% vs. 37%, P=0.013). LNR was independently associated with survival on multivariate analysis [HR: 2.63; 95% confidence intervals (CI), 1.13–6.14; P=0.025]. Conclusions: In stage IV CRC, LNR is associated with the extent of hepatic tumor burden and was an independent predictor of survival in patients undergoing liver resection.

Chimeric antigen receptor expressing T cells (CAR-T) are a promising form of immunotherapy, but the influence of age-related immune changes on CAR-T production remains poorly understood. We showed that CAR-T cells from geriatric donors (gCAR-T) are functionally impaired relative to CAR-T from younger donors (yCAR-T). Higher transduction efficiencies and improved cell expansion were observed in yCAR-T cells compared with gCAR-T. yCAR-T demonstrated significantly increased levels of proliferation and signaling activation of phosphorylated (p)Erk, pAkt, pStat3, and pStat5. Furthermore, yCAR-T contained higher proportions of CD4 and CD8 effector memory (EM) cells, which are known to have enhanced cytolytic capabilities. Accordingly, yCAR-T demonstrated higher levels of tumor antigen-specific cytotoxicity compared with gCAR-T. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin and granzyme B. yCAR-T had increased α5β1 integrin expression, a known mediator of retroviral transduction. We found that treatment with M-CSF or TGF-β1 rescued the impaired transduction efficiency of the gCAR-T by increasing the α5β1 integrin expression. Neutralization of α5β1 confirmed that this integrin was indispensable for CAR expression. Our study suggests that the increase of α5β1 integrin expression levels enhances CAR expression and thereby improves tumor killing by gCAR-T.

We have recently witnessed substantial progress with immunotherapy for selected diseases. Checkpoint inhibitors and chimeric antigen receptor T (CAR-T) cells are among the most promising agents. Whereas much of the early success with CAR-T cells has been demonstrated with hematological malignancies, important barriers remain for the application of CAR-T cell therapies for the management of metastatic solid tumors. The challenges are particularly apparent when considering primary and metastatic tumors in the liver. At baseline, the intrahepatic space is immunosuppressive and this feature is exploited by malignant cells. Fortunately, our evolving understanding of liver immune cell biology is allowing the development of novel immunotherapeutic strategies for the treatment of liver tumors. Furthermore, the unique anatomic features of the liver make possible highly selective immunotherapeutic delivery approaches that may maximize antitumor efficacy while limiting off-target damage to healthy tissues. This review summarizes the immunobiology of the intrahepatic space and how this knowledge enables identification of hurdles and potential solutions to the barriers facing immunotherapy for liver tumors.

Melanoma has been a long-standing focal point for immunotherapy development. In this review, we explore the evolution of melanoma treatments with particular attention to the history and recent advances in melanoma immunotherapy. We also discuss novel combinations of these modalities and their potential to offer novel therapeutic options for patients with advanced melanoma.

Con A hepatitis is regarded as a T cell–mediated model of acute liver injury. Mincle is a C-type lectin receptor that is critical in the immune response to mycobacteria and fungi but does not have a well-defined role in preclinical models of non-pathogen–mediated inflammation. Because Mincle can ligate the cell death ligand SAP130, we postulated that Mincle signaling drives intrahepatic inflammation and liver injury in Con A hepatitis. Acute liver injury was assessed in the murine Con A hepatitis model using C57BL/6, Mincle−/−, and Dectin-1−/− mice. The role of C/EBPβ and hypoxia-inducible factor-1α (HIF-1α) signaling was assessed using selective inhibitors. We found that Mincle was highly expressed in hepatic innate inflammatory cells and endothelial cells in both mice and humans. Furthermore, sterile Mincle ligands and Mincle signaling intermediates were increased in the murine liver in Con A hepatitis. Most significantly, Mincle deletion or blockade protected against Con A hepatitis, whereas Mincle ligation exacerbated disease. Bone marrow chimeric and adoptive transfer experiments suggested that Mincle signaling in infiltrating myeloid cells dictates disease phenotype. Conversely, signaling via other C-type lectin receptors did not alter disease course. Mechanistically, we found that Mincle blockade decreased the NF-κβ–related signaling intermediates C/EBPβ and HIF-1α, both of which are necessary in macrophage-mediated inflammatory responses. Accordingly, Mincle deletion lowered production of nitrites in Con A hepatitis and inhibition of both C/EBPβ and HIF-1α reduced the severity of liver disease. Our work implicates a novel innate immune driver of Con A hepatitis and, more broadly, suggests a potential role for Mincle in diseases governed by sterile inflammation.

Dermatofibrosarcoma protuberans (DFSP) is a rare superficial soft tissue sarcoma. Its rarity precludes large prospective studies. Clinical diagnosis requires an high index of suspicion. Effective management requires an appreciation of tumor biology and the nature of the characteristic infiltrative growth pattern. DFSP tends to recur locally, with a low risk of dissemination. Aggressive surgical resection with widely negative margins is essential to management. Radiotherapy may be indicated in special circumstances. Understanding the molecular pathogenesis has resulted in use of tyrosine kinase inhibitor therapy for patients with locally advanced disease or in metastatic disease. DFSP patients require long-term follow-up.

Effective chimeric antigen receptor-modified T-cell (CAR-T) therapy for liver metastases (LM) will require innovative solutions to ensure efficient delivery and minimization of systemic toxicity. We previously demonstrated the safety of CAR-T hepatic artery infusions (HAI). We subsequently conducted the phase 1b HITM-SIR trial, in which six patients (pts) with CEA

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Surgical resection is the only curative option for patients with colorectal liver metastases (CRLM). The objective of our study was to identify factors associated with failure to refer patients with CRLM to a surgeon with oncologic and hepatobiliary expertise.Data were retrospectively reviewed on 75 patients with CRLM treated at our institution. Patients were divided into referred and nonreferred groups for comparison. Quantitative assessment of association was tabulated using the odds ratio (OR). Statistical comparison was performed using the chi-square test and multiple regression models. Overall survival (OS) was calculated using the Kaplan-Meier method. Multivariate analysis was done using Cox regression.Factors independently associated with lower surgical referral rates included age ≥ 65 y (OR 0.29, 95% confidence interval [CI] 0.09-0.89, P = 0.032), bilobar CRLM (OR 0.35, 95% CI 0.09-0.97, P = 0.048), and presence of3 CRLM (OR 0.33, 95% CI 0.11-0.94, P = 0.044). The 5-y OS for referred patients was 33% compared with only 8% in patients who were not referred (P 0.001). Factors independently associated with worse OS included age ≥ 65 y (hazard ratio [HR] 2.01, 95% CI 1.12-3.59, P = 0.019), bilobar hepatic metastases (HR 3.04, 95% CI 1.62-5.70, P 0.001), and the presence of extrahepatic metastases (HR 2.11, 95% CI 1.02-4.16, P = 0.011). Referral to a surgeon was associated with improved OS (HR 0.42, 95% CI 0.24-0.74, P = 0.003).Failure to refer CRLM patients for surgical evaluation is associated with aggressive biologic features that do not necessarily preclude resection. Determination of resectability should be made with input from appropriately trained surgical experts.

Assumptions that liver immune cells and immunosuppressive pathways are similar to their counterparts in other spaces have led to gaps in our understanding of intrahepatic neoplasm aggressiveness. Myeloid-derived suppressor cells (MDSCs) are potent inhibitors of antitumor immunity and pose a major obstacle to solid tumor treatment. Liver MDSCs (L-MDSCs) associated with liver metastases (LM) are particularly problematic by contributing to intrahepatic immunosuppression that promotes tumor progression. L-MDSCs have been reported to expand in response to granulocyte-macrophages colony-stimulating factor (GM-CSF) and suppress antitumor immunity in LM. To extend these findings, we examined mechanisms of intrahepatic immunosuppression exploited by L-MDSCs. We found that the majority of L-MDSCs co-expressed GM-CSF receptor (GM-CSF-R), indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PD-L1), while demonstrating high levels of signal transducer and activator of transcription factor 3 (STAT3) activation. GM-CSF-secreting tumor cells induced STAT3 phosphorylation in L-MDSCs in addition to expression of IDO and PD-L1. GM-CSF or GM-CSF-R blockade markedly reduced L-MDSC IDO and PD-L1 expression, implicating tumor-derived GM-CSF in supporting L-MDSC-immunoinhibitory molecule expression. Small-molecule inhibitors of Janus-activated kinase 2 (JAK2) and STAT3 also dramatically diminished IDO and PD-L1 expression in L-MDSCs. We determined that STAT3 exerts transcriptional control over L-MDSC IDO and PD-L1 expression by binding to the IDO1 and PD-L1 promoters. Our data suggest that the GM-CSF/JAK2/STAT3 axis in L-MDSCs drives immunosuppression in a model of LM and blockade of this pathway may enable rescue of intrahepatic antitumor immunity.

Metastatic spread of colorectal cancer (CRC) to the peritoneal cavity is common and difficult to treat, with many patients dying from malignant bowel obstruction. Chimeric antigen receptor T cell (CAR-T) immunotherapy has shown great promise, and we previously reported murine and phase I clinical studies on regional intrahepatic CAR-T infusion for CRC liver metastases. We are now studying intraperitoneal (IP) delivery of CAR-Ts for peritoneal carcinomatosis. Regional IP infusion of CAR-T resulted in superior protection against carcinoembryonic antigen (CEA+) peritoneal tumors, when compared with systemically infused CAR-Ts. IP CAR-Ts also provided prolonged protection against IP tumor re-challenges and demonstrated an increase in effector memory phenotype over time. IP CAR-Ts provided protection against tumor growth at distant subcutaneous (SC) sites in association with increases in serum IFNγ levels. Given the challenges posed by immunoinhibitory pathways in solid tumors, we combined IP CAR-T treatment with suppressor cell targeting. High frequencies of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) were found within the IP tumors, with MDSC expressing high levels of immunosuppressive PD-L1. Combinatorial IP CAR-T treatment with depleting antibodies against MDSC and Treg further improved efficacy against peritoneal metastases. Our data support further development of combinatorial IP CAR-T immunotherapy for peritoneal malignancies.

Highlights • COVID-19 can cause a hyperinflammatory state akin to cytokine release syndrome (CRS). • Our report shows the efficacy and safety of using anti-IL-6/IL-6-R therapy in 31 patients. • Treatment at the onset of severe CRS may be the ideal window for intervention. • Long term effects of these agents are currently lacking., 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.

e15016 Background: Colorectal cancer liver metastases (CRCLM) are a major source of morbidity and mortality. Historically, curative therapy has been limited to surgical resection, but only a small fraction of patients are eligible. Cellular immunotherapy has shown promise in hematologic cancers, but challenges related to solid tumor therapy remain with optimal cell trafficking, elevated interstitial fluid pressures (IFP), and immunosuppression. We hypothesized that engineered natural killer (NK) cells expressing a natural killer group 2, member D (NKG2D) activating chimeric receptor (ACR) and membrane bound IL-15 (NKX101) would increase anti-tumor activity in vitro and in vivo utilizing our established regional delivery strategies. Methods: In vitro cytotoxicity and cytokine release of NKX101 cells or non-transduced NK cells (NT-NK) derived from the same donor were determined by co-culture systems with HCT116 cells that endogenously express NKG2D ligands. CRCLM-bearing NSG™ mice were treated with NKX101, NT-NK, or vehicle (CTRL) via portal vein (PV) for regional delivery (RD) or tail vein (TV) for systemic delivery (SD). Tumor burden (TB) was measured via tumor bioluminescence (TBL) and histopathology (HP). Flow cytometry (FC) determined the quantity of cells delivered. Student’s t-test and Mann-Whitney tests were performed for statistical comparisons. Results: NKX101 transduction efficiencies ranged between 63.5 – 75.6% across 3 separate healthy donors. EC50 values derived from a 4-hour cytotoxicity assay for NKX101 vs. NT-NK were 3-4 fold lower with the greatest difference observed at the 1:1 effector-to-target (E:T) ratio (mean percent cytotoxicity: 72% vs. 20%, p = 0.001). In vitro cytokine assessment revealed 2.0-2.6 fold increases in IFN-γ, GM-CSF, and TNF-α levels compared to NT-NK cells (p < 0.0001 across all groups). In vivo, FC showed 2.89-fold increase in cell delivery using RD vs. SD on PTD1 (n = 3, p = 0.006). TBL was improved with 5 x 106 cells via PV vs. TV (n = 6) from post-treatment day (PTD) 1-7, with greatest difference seen on PTD7 (12.9 vs. 42.6, p = 0.07). HP analysis showed reduction of TB at PTD7 with PV treatment. Conclusions: NKX101 demonstrated improvements in in vitro cytotoxicity and pro-inflammatory cytokine release. RD techniques in vivo revealed increased cell delivery and improved tumor control. Further studies are underway to confirm our initial findings and understand NKX101 cellular kinetics and susceptibility to immunosuppression in the liver, along with planned clinical evaluation in Phase 1 trials.

Introduction Efficacy of cellular immunotherapy for liver metastases (LM) has been limited by poor trafficking and elevated intra-tumoral pressures. In a phase 1b study, we found that regional, pressure-enabled drug delivery (PEDD, TriSalus Life Sciences, Inc.) improved LM CAR-T delivery. We hypothesized that PEDD of CAR-T in a murine model can improve LM control. Methods LM bearing CD45.2 mice were treated with CD45.1 anti-CEA CAR-T via systemic delivery (SD, tail vein) or regional delivery (RD, portal vein) at low pressure (LPRD) vs. high pressure (HPRD) to model PEDD. Flow rates were calibrated to achieve LPRD or HPRD. Tumor bioluminescence (TB), flow cytometry (FC), immunofluorescence (IF), & quantitative polymerase chain reaction (qPCR) were assessed on various post-treatment days (PTD). Results FC of liver non-parenchymal cells on PTD7 showed increased CAR-T density in HPRD & LPRD (9.4% & 11.0%, p = 0.03 & 0.002) vs. SD (3.7%). qPCR of the CAR transgene supported this observation, but was not significant (HPRD 135.8, p = 0.76, LPRD 108.6, p = 0.40 vs. SD 69.8). TB was lower in RD vs. SD up to PTD7 (log2 fold-change HPRD −1.3 & LPRD −0.9 vs. SD 4.3, p = 0.005 & 0.006). Tumor proliferation by Ki67 IF was significantly reduced with RD vs. control (HPRD & LPRD, p < 0.001 & p = 0.05) with HPRD superior to LPRD (p = 0.03). Serum liver enzymes were not significantly elevated using HPRD vs. LPRD at PTD7 (AST/ALT 154.7/36.3 vs. 139.1/29.1, p = 0.84/0.67), but did rise at PTD17, correlating with tumor progression (AST/ALT 800.7/229.3 vs. 387.5/139.5, p = 0.10/0.28). Conclusions RD increased LM CAR-T delivery and HPRD showed evidence of improved CAR-T trafficking and tumor control relative to LPRD. Further clinical testing of PEDD is planned for LM cellular therapy.