Faculty Bibliography

Background Mesothelin (MSLN) is expressed on a variety of solid tumors, including mesothelioma and ovarian, uterine, gastric, pancreatic, and lung cancers.1 However, efforts to target MSLN using cellular therapies have been hampered by severe on-target, off-tumor toxicities associated with damage to normal tissues expressing MSLN.2 To avoid these toxicities, we have developed a logic-gated engineered cell therapy, TmodTM, which is composed of two chimeric antigen receptors (CARs): an activator that targets a tumor-associated antigen and an inhibitory receptor (blocker) gated by an antigen expressed on normal tissue but lost in tumor cells due to loss of heterozygosity (LOH). A2B694 is an MSLN-specific Tmod construct combining a third-generation MSLN CAR with an LIR-1-based inhibitory receptor specific for human leukocyte antigen A*02 (HLA-A*02). Methods Lentivirus encoding i) the CAR, ii) the blocker, and iii) an shRNA targeting b2M was used to transduce T cells from HLA-A*02 donors and generate MSLN Tmod cells. In vitro cytotoxicity measurements were performed using fluorescence-based imaging and luciferase readouts. In vivo assessments were performed in NSG mice subcutaneously implanted with normal cells (MSLN[+]A*02[+]), or tumor cells (MSLN[+]A*02[-]), in the left and right flanks, respectively. Following engraftment, mice were randomized and treated intravenously with MSLN Tmod cells or controls. Grafts were measured via caliper. Results MSLN Tmod cells preferentially killed tumor cells (MSLN[+]A*02[-]) over normal cells (MSLN[+]A*02[+]) in vitro, unlike clinically active comparator M5 CAR T cells, which indiscriminately killed both target cell types (figure 1A). Soluble MSLN, tested across a 0-2 mg/mL range, did not impact MSLN Tmod function. Additionally, in mixed cell cultures where T cells and tumor and normal cells were simultaneously cultured (1:1:1 ratio), MSLN Tmod cells selectively killed tumor targets while sparing normal cells. Further, MSLN Tmod cells cycled between activated and blocked states in vitro when repeatedly challenged with tumor or normal target cells. Finally, while MSLN CAR T cells killed both normal and tumor grafts in vivo, MSLN Tmod cells selectively killed tumor grafts while sparing normal grafts (figure 1B, C). Conclusions A2B694 is an autologous MSLN Tmod cell product that leverages LOH at the HLA locus in cancer cells, providing a mechanism to discriminate between normal and tumor cells. BASECAMP-1 (NCT04981119), an observational study that will identify patients with somatic HLA LOH, is currently recruiting. Eligible patients with metastatic colorectal, pancreatic, or non-small cell lung cancer will be apheresed for a future A2B694 interventional study (EVEREST-2)

Background: Metastatic colorectal (CRC), pancreatic (PANC), and gastroesophageal (GE) cancers are the leading causes of GI cancer- related mortality (5-yr survival rate, 14%, 3% and 5-6%, respectively). T-cell immunotherapy targeting GI-associated tumor antigens has been attempted, but efficacy has been constrained by on-target off-tumor toxicity, limiting the therapeutic window. The Tmod (TM) platform is an AND-NOT logic-gated CAR T modular system, versions of which have a CEA-or MSLN-targeting CAR activator and a separate HLA-A*02-targeting blocker receptor to protect normal cells. Tmod CAR T exploits HLA LOH, common in GI malignancies (10-33% in primary solid tumors [TCGA]) and can kill tumor cells without harming healthy cells in vitro and in vivo. However, the prevalence of HLA LOH across GI tumors is unknown in the real-world setting. We utilized the Tempus xT oncology NGS database of patients with multiple GI tumors. From a standard-of-care NGS assay, GI cancer patients can be readily identified for HLA LOH and future treatment with Tmod CAR T therapy.
Method(s): The occurrence of HLA LOH in GI tumors of 1439 patients was assessed using paired germline and somatic DNA sequencing using a research assay [6]. CRC, PANC and GE patients with >= stage 3 were then extracted, and rates of HLA LOH were identified (ie, whether loss occurred across high-frequency HLA-A alleles). In addition, mutations in KRAS and BRAF, as well as MSI status were stratified to determine any association with HLA-A LOH.
Result(s): HLA-A LOH was detected in 830 (17.3%) of all solid tumor records, and a similar proportion when all GI cancer records were analyzed (17.0%). For GI subtypes, these values ranged from 13.5% to 23.1% (Table). No high-frequency HLA-A allele (A*01, A*02, A*03, A*11) was more likely to be lost. Clinical biomarkers (KRAS, BRAF and MSI status) were not associated with HLA-LOH.
Conclusion(s): The frequency of HLA LOH among advanced solid tumor cancers in this dataset is 17.3%, with a range of 13.5-23% between CRC, PANC and GE. The HLA LOH frequency observed in these GI tumors is consistent with that in primary tumors from TCGA, which also used germline-matched and tumor samples. Clinical biomarkers were not associated with HLA LOH. Tempus NGS was able to identify HLA LOH, which can be used for Tmod CAR T therapy to an enhanced therapeutic window. Identification of these patients in BASECAMP-1 (NCT04981119) will enable novel Tmod CAR T therapy. (Table Presented)

Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.

PURPOSE/OBJECTIVE:To investigate the safety of yttrium-90 radioembolization in combination with checkpoint inhibitor immunotherapy for the treatment of hepatocellular carcinoma (HCC). MATERIALS AND METHODS/METHODS:This single-center retrospective study included 26 consecutive patients with HCC who received checkpoint inhibitor immunotherapy within 90 days of radioembolization from April 2015 to May 2018. Patients had preserved liver function (Child-Pugh scores A-B7) and either advanced HCC due to macrovascular invasion or limited extrahepatic disease (21 patients) or aggressive intermediate stage HCC that resulted in earlier incorporation of systemic immunotherapy (5 patients). Clinical documentation, laboratory results, and imaging results at 1- and 3-month follow-up intervals were reviewed to assess treatment-related adverse events and treatment responses. RESULTS:The median follow-up period after radioembolization was 7.8 months (95% confidence interval [CI], 5.6-11.8). There were no early (30-day) mortality or grades 3/4 hepatobiliary or immunotherapy-related toxicities. Delayed grades 3/4 hepatobiliary toxicities (1-3 months) occurred in 2 patients in the setting of HCC disease progression. One patient developed pneumonitis. The median overall survival from first immunotherapy was 17.2 months (95% CI, 10.9-23.4). The median overall survival from first radioembolization was 16.5 months (95% CI, 6.6-26.4). From first radioembolization, time to tumor progression was 5.7 months (95% CI, 4.2-7.2), and progression-free survival was 5.7 months (95% CI, 4.3-7.1). CONCLUSIONS:Radioembolization combined with checkpoint inhibitor immunotherapy in cases of HCC appears to be safe and causes limited treatment-related toxicity. Future prospective studies are needed to identify the optimal combination treatment protocols and evaluate the efficacy of combination therapy.

Background: Tyrosine kinase inhibitors can prolong survival in advanced HCC patients, but response rates have been minimal. Recently, immune checkpoint inhibition with nivolumab (nivo) demonstrated objective response rates (ORR) of 15% (escalation phase) and 20% (expansion phase) in the Checkmate 040 study. Pre-clinical and translational studies have demonstrated that IL-8 and tumor associated macrophages (TAMs) contribute to HCC progression and recurrence following treatment. Therefore, rationale exists to evaluate combinatorial approaches to target TAM function combined with checkpoint inhibitory therapy. This phase II, randomized study will evaluate the safety and efficacy of combined anti-CSF1R (Cabiralizumab) or anti-IL-8 (BMS-986253) in combination with Nivo in advanced HCC. Method(s): Advanced HCC patients without prior systemic treatment and disease measurable by RECISTv1.1 with Childs A liver function are eligible. Patients will be enrolled (n=25 per arm) to Nivo 240 mg IV Q2 weeks monotherapy, Nivo 240 mg IV + BMS-986253 1200 mg IV Q2 weeks, or Nivo 240 mg IV + Cabiralizumab 4 mg/kg IV Q2 weeks. Primary endpoints include safety and ORR determined by RECISTv1.1. Secondary endpoints include time to response, duration of response, progression free survival, and overall survival. Exploratory endpoints include analysis of tumor microenvironment immune and tumor cell profiling of pre- and on-treatment tumor tissue

Hepatocellular Carcinoma (HCC) is one of the most lethal cancers with a high mortality and recurrence rate. Circulating tumor cell (CTC) detection offers various opportunities to advance early detection and monitoring of HCC tumors which is crucial for improving patient outcome. We developed and optimized a novel Labyrinth microfluidic device to efficiently isolate CTCs from peripheral blood of HCC patients. CTCs were identified in 88.1% of the HCC patients over different tumor stages. The CTC positivity rate was significantly higher in patients with more advanced HCC stages. In addition, 71.4% of the HCC patients demonstrated CTCs positive for cancer stem cell marker, CD44, suggesting that the major population of CTCs could possess stemness properties to facilitate tumor cell survival and dissemination. Furthermore, 55% of the patients had the presence of circulating tumor microemboli (CTM) which also correlated with advanced HCC stage, indicating the association of CTM with tumor progression. Our results show effective CTC capture from HCC patients, presenting a new method for future noninvasive screening and surveillance strategies. Importantly, the detection of CTCs with stemness markers and CTM provides unique insights into the biology of CTCs and their mechanisms influencing metastasis, recurrence and therapeutic resistance.

CD73, a cell-surface N-linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N-linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N-linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity (P < 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity in vitro, similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N-linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC.

Background and aims: Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is a leading cause of [Figure Presented] cancer-related deaths globally. Adenosine-3 receptor (A3R) activation blocks HCC progression in rodents, and small molecule A3R agonists are undergoing clinical development for HCC patients. Here, we examined the regulation of ecto-5'-nucleotidase (CD73), a novel target of cancer immunotherapy that produces extracellular adenosine, in human HCC.
Method(s): We analyzed CD73 expression and correlation with tumor characteristics and patient outcomes using The Cancer Genome Atlas (TCGA). In addition, we extracted CD73 from paired surgical specimens of HCC tumor and non-tumor adjacent liver tissues and normal control liver tissues collected under approved protocols, and performed glycopeptide mass spectrometry analysis. RNAseq analysis was performed to compare the expression of glycoprotein-encoding genes in addition to molecular, biochemical, and cell biological assays using multiple human HCC cell lines.
Result(s): High expression of the CD73-encoding gene (NT5E) correlated with poor survival in HCC patients (N = 365; p = 0.012) but did not correlate with a specific HCC immune subtype, based on TCGA data. We show using analysis of surgical specimens from HCC and adjacent liver tissues similar CD73 protein levels between normal and tumor hepatocytes. However, tumor-associated CD73 was primarily intracellular and exhibited altered N-linked glycosylation, independently of HCC etiology or fibrosis presence. The altered CD73 glycosylation correlated with a 2.7-fold decrease in tumor associated 5'-nucleotidase activity (p < 0.0001), which was otherwise necessary to induce cell death in HCC cultured cells. Mass spectrometry profiling of CD73 from tumors demonstrated a significant increase in high-mannose CD73 glycans on residues N311/N333. Blocking CD73 N311/N333 glycosylation caused intracellular CD73 accumulation and decreased 5'-nucleotidase activity in vitro, similar to the phenotypes observed in the primary human HCC tumors. Aberrant Golgi function was implicated as a potential mechanism, based on altered tumor expression and organization of the structural protein GM130 and genes encoding other N-linked glycoproteins, as determined by RNAseq analysis.
Conclusion(s): Our results reveal a novel glycosylation mechanism for regulating endogenous purinergic signaling in HCC tumors.
Copyright

Purpose: As Y90 radioembolization has been shown to activate an immune response, synergistic effects with check-point inhibitor immunotherapy have been proposed. However, the safety of concurrent Y90 and immunotherapy has not been reported. This study retrospectively evaluated the safety of Y90 with concurrent immunotherapy in patients with primary or metastatic liver cancer. Materials: The retrospective study was conducted with IRB approval. Patients who received Y90 treatment within 30 days of immunotherapy were considered to have concurrent therapy. Baseline laboratory values obtained within one month prior to Y90 and at 1 and 3 months after Y90 were evaluated. Hepatobiliary and immunotherapy-related adverse events were characterized according to NCI CTCAE v5.0. Patient survival was estimated using Kaplan-Meier analysis.
Result(s): Between June 2015 and March 2018, 18 patients received concurrent therapy. 14 patients had hepatocellular carcinoma (3 BCLC B, and 11 BCLC C), and 4 had metastatic disease to the liver (3 melanoma, 1 gastric cancer). The median interval between Y90 and immunotherapy was 7 days. Grade >=3 hepatobiliary toxicity occurred in 1 patient at 1 month (6%) and in 3 patients at 3 months (17%) after Y90. Grade >=3 toxicities occurred only in patients with advanced HCC (BCLC C). No grade >=3 immune-associated toxicities occurred at 1 or 3 months in any patients. Median overall survival from first Y90 was 27.4 months for patients with HCC and 13.7 months for patients with metastatic disease to the liver.
Conclusion(s): Concurrent Y90 radioembolization and checkpoint-inhibitor immunotherapy appears to be safe with a low incidence of toxicity. Toxicities were limited to patients with advanced HCC and may be confounded by disease progression.
Copyright