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Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody–dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro. A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

Background IL-27 is a heterodimeric cytokine consisting of IL 27p28 and Epstein-Barr virus-induced gene 3 (EBI3) that binds the IL-27 receptor subunit alpha and glycoprotein 130. IL-27 is produced by activated macrophages and dendritic cells and limits the intensity and duration of immune responses in the tumor microenvironment by inducing the expression of immunoregulatory receptors (PD-L1, TIM3, LAG-3, TIGIT) and inhibiting production of proinflammatory cytokines (IFNγ, IL-17, TNFα). The IL-27 subunit EBI3 is elevated in plasma from patients with certain cancers including renal cell carcinoma, where it correlates with poor outcome. Based on high expression of IL-27 transcript in tumors from patients with hepatocellular carcinoma (HCC), the role of IL-27 was further explored in patient samples and a mouse model of HCC. Methods Gene expression profiles from the Cancer Genome Atlas (TCGA) were analyzed to identify tumors with elevated IL-27 transcripts. Serum from patients with HCC was analyzed for levels of the IL-27 subunit EBI3. The ability of SRF388, a first-in-class IL-27-blocking antibody that binds to IL-27p28, to reverse IL-27-induced inhibition of cytokine production in human immune cell cultures from patients with HCC was assessed in vitro. Finally, the anti-tumor activity of SRF388 was assessed in an orthotopic murine model of HCC. Results TCGA expression data revealed that IL-27p28 transcripts were elevated in tumors from patients with HCC relative to other indications. Serum levels of EBI3 were: 1) elevated in a subset of HCC patients; 2) inversely correlated with survival; 3) independent of serum alpha-fetoprotein levels; and 4) elevated in both hepatitis B/C virus positive and negative patients. Treatment with SRF388 stimulated increased cytokine production in activated peripheral blood mononuclear cells from patients with HCC that was further enhanced when combined with PD-1 blockade. Furthermore, SRF388 inhibited the growth of orthotopic Hepa1-6 liver tumors. mRNA transcriptional profiling of treated tumors revealed that SRF388 profoundly altered the transcriptional landscape in this model. In particular, treatment with SRF388 inhibited expression of immunoregulatory receptors PD-L1 and TIGIT, repressed transcripts associated with TGF-β signaling, and altered myeloid and natural killer cell transcripts. Conclusions These data indicate that elevated IL-27 subunit EBI3 is a hallmark of HCC and is associated with poor outcomes in these patients. Blockade of IL-27 with SRF388, currently being evaluated in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04374877), may represent a promising therapy for patients with HCC where it can potentiate anti-tumor immune responses.

BackgroundInterleukin (IL)-27 is a heterodimeric immunoregulatory cytokine that signals through the JAK/STAT pathway to increase the expression of coinhibitory receptors on immune cells (e.g. PD-L1, TIM-3, LAG-3) and dampen inflammatory cytokine production. Blockade of IL-27 leads to antitumor activity in preclinical mouse models of lung metastases. A Phase 1 trial of SRF388 (NCT04374877), a first-in-class anti–IL-27 antibody, has demonstrated monotherapy antitumor activity in a patient with non-small cell lung cancer (NSCLC).1 The current study aimed to characterize the immunoregulatory impact of IL-27 signaling by gene expression profiling.MethodsGene expression changes induced by IL-27 were examined in activated human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs), and the IL-27RA–expressing lung cancer cell line NCI-H2228 by microarray or single cell RNA-sequencing. The resulting IL-27 signature genes were interrogated by Gene Set Enrichment Analysis (GSEA) using publicly available datasets, including single cell RNA-seq analysis of the tumor microenvironment, from patients with NSCLC.2ResultsIL-27 induced a robust gene expression program in human immune cells that included several inhibitory receptors and canonical interferon regulated genes such as guanylate-binding proteins and interferon regulatory factors. GSEA and interferon signature analysis showed a striking overlap with those genes regulated by interferon-beta, a cytokine known to drive immune suppression associated with chronic viral infection and that is used therapeutically for controlling inflammation associated with the autoimmune disease multiple sclerosis. Moreover, interferon regulated pathways have recently emerged as a mechanism of resistance to immune checkpoint blockade in cancer. Exploration of the IL-27 gene signature in published datasets showed enrichment in macrophage populations associated with progressive disease in patients with NSCLC. While many of the properties of IL-27–mediated immune regulation have focused on hematopoietic cells, IL-27RA is also expressed on tumor cells from NSCLC patients with progressive disease as well as lung cancer cell lines in which IL-27 can upregulate PD-L1, IDO1, and other canonical interferon regulated genes.ConclusionsThese studies elucidate the transcriptional networks that are engaged after IL-27 signaling in immune and cancer cells and highlight the parallels with interferon-associated immune regulation. Blockade of IL-27 provides a novel therapeutic strategy to alleviate a gene transcriptional program implicated in immune suppression and checkpoint resistance.ReferencesPatnaik A, Morgensztern D, Mantia C, et al. Results of a phase 1 study of SRF388, a first-in-human, first-in-class, high-affinity anti-IL-27 antibody in advanced solid tumors. J Clin Oncol 2021;39:2551–2551.Maynard A, McCoach CE, Rotow JK, et al. Therapy-induced evolution of human lung cancer revealed by single-cell RNA sequencing. Cell 2020;182:1232–1251.

CD39 (ENTPD1) is a key enzyme responsible for the degradation of extracellular adenosine triphosphate (ATP) and is upregulated in the tumor microenvironment (TME). High levels of extracellular ATP, often generated in the TME as a result of tissue damage and immunogenic cell death, can initiate proinflammatory responses that are blocked by the enzymatic activity of CD39. In addition, extracellular adenosine accumulates in cancerous tissues through the degradation of ATP by CD39 and other ectonucleotidases (eg, CD73), and constitutes an important mechanism of tumor immune escape, induction of angiogenesis, and metastasis. Thus, inhibition of CD39 can convert a suppressive TME to a proinflammatory environment. SRF617 is an investigational fully human anti-CD39 antibody that binds to human CD39 with nanomolar affinity and potently inhibits its enzymatic function. Results of the current studies show that CD39 is predominantly expressed in tumor stroma and on tumor-infiltrating immune cells in samples from patients with cancer. Similar expression patterns are observed in various murine tumor models. In vivo, SRF617 has significant single-agent anti-tumor activity in a variety of cell line-derived xenograft models that express CD39. Cancer therapies, such as immunogenic cell death agents, can increase inflammation and ATP levels in the TME; combination studies of SRF617 with these agents showed improved preclinical efficacy and led to significant improvement in survival. In addition, a mouse-specific anti-CD39 surrogate antibody demonstrated potent binding and enzymatic inhibition of murine CD39 in vitro and significantly decreased tumor growth in a syngeneic murine tumor model. Pharmacodynamic studies demonstrated mechanistic changes in the tumor-infiltrating leukocytes and plasma chemokine levels. Combination treatment of a murine anti-CD39 surrogate and an anti-mouse PD-1 displayed improved activity and an increase in overall survival in the CT-26 mouse model. In addition, administration of SRF617 in combination with other immunotherapies also demonstrated a similar improvement in activity and survival. In summary, these studies demonstrate that SRF617 is a potent inhibitor of CD39 enzymatic activity both in vitro and in vivo. Inhibition of CD39 potentiates the activity of chemotherapy and immunotherapy agents to improve tumor growth inhibition and survival in mice. These findings support future clinical studies of SRF617 as monotherapy and in combination with other therapeutic agents in treating patients with cancer. Citation Format: Sonia G. Das, Austin Dulak, Gege Tan, Marc Johnson, Tauqeer H. Zaidi, Michael C. Warren, Secil Koseoglu, Erik Devereaux, Marisa O. Peluso, Alison M. Paterson, Benjamin H. Lee, Vito J. Palombella, Pamela M. Holland, Andrew C. Lake. SRF617, a potent enzymatic inhibitor of CD39, demonstrates single-agent activity and cooperates with various cancer therapies in both solid tumor and hematologic malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6639.

Platelet exocytosis is regulated partially by the granular/cellular membrane lipids and proteins. Some platelets contain a membrane-bound tube, called an open canalicular system (OCS), which assists in granular release events and increases the membrane surface area for greater spreading. The OCS is not found in all species, and variations in membrane composition can cause changes in platelet secretion. Since platelet studies use various animal models, it is important to understand how platelets differ in both their composition and granular release to draw conclusions among various models. The relative phospholipid composition of the platelets with (mouse, rabbit) and without (cow) an OCS was quantified using UPLC-MS/MS. Cholesterol and protein composition was measured using an Amplex Red Assay and BCA Assay. TEM and dark field platelet images were gathered and analyzed with Image J. Granular release was monitored with single cell carbon fiber microelectrode amperometry. Cow platelets contained greater amounts of cholesterol and sphingomyelin. In addition, they yield greater serotonin release and longer δ granule secretion times. Finally, they showed greater spreading area with a greater range of spread. Platelets containing an OCS had more similarities in their membrane composition and secretion kinetics compared to cow platelets. However, cow platelets showed greater fusion pore stability which could be due to extra sphingomyelin and cholesterol, the primary components of lipid rafts. In addition, their greater stability may lead to many granules assisting in spreading. This study highlights fundamental membrane differences and their effects on platelet secretion.

The cellular phospholipid membrane plays an important role in cell function and cell-cell communication, but its biocomplexity and dynamic nature presents a challenge for examining cellular uptake of phospholipids and the resultant effects on cell function. Platelets, small anuclear circulating cell bodies that influence a wide variety of physiological functions through their dynamic secretory and adhesion behavior, present an ideal platform for exploring the effects of exogenous phospholipids on membrane phospholipid content and cell function. In this work, a broad range of platelet functions are quantitatively assessed by leveraging a variety of analytical chemistry techniques, including ultraperformance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-MS/MS), vasculature-mimicking microfluidic analysis, and single cell carbon-fiber microelectrode amperometry (CFMA). The relative enrichments of phosphatidylserine (PS) and phosphatidylethanolamine (PE) were characterized with UPLC-MS/MS, and the effects of the enrichment of these two phospholipids on both platelet secretory behavior and adhesion were examined. Results show that, in fact, both PS and PE influence platelet adhesion and secretion. PS was enriched dramatically and decreased platelet adhesion as well as secretion from δ-, α-, and lysosomal granules. PE enrichment was moderate and increased secretion from platelet lysosomes. These insights illuminate the critical connection between membrane phospholipid character and platelet behavior, and both the methods and results presented herein are likely translatable to other mammalian cell systems.

While ion-selective electrodes (ISEs) based on anion ionophores with guanidine groups were reported in the past, their response mechanism was poorly understood. In this study, an ionophore with one guanidine group was prepared as a model compound, and the chloride and sulfate responses of ISE membranes containing this ionophore were investigated. The electrodes exhibit Nernstian responses resulting from a neutral or charged ionophore mode when added cationic or anionic sites are used, respectively. Based on this observation, it might be expected that membranes without added ionic sites exhibit Nernstian responses according to a charged carrier mechanism. However, experimental results show an apparently ‘two–thirds Nernstian’ equilibrium response, which can be explained on the basis of the phase boundary model.

TAK-164 is an antibody-drug conjugate comprising a full-length, fully human IgG1 monoclonal antibody (mAb) directed towards the extracellular domain of guanylyl cyclase C (GCC). The mAb is conjugated using the peptide-linked indolino-benzodiazepine DNA alkylator DGN549 (also known as IGN-P1). TAK-164 demonstrates efficient binding to antigen-expressing cells in vitro, and GCC-dependent uptake that results in cytotoxicity. Following a single administration of TAK-164 in female SCID mice bearing GCC-positive xenografts, the pharmacokinetics of the conjugated antibody component of TAK-164 displayed low plasma clearance. Exposure to the ADC resulted in a dose- and time-dependent increase in DNA damage as measured by the pharmacodynamic marker pH2A.X in GCC-positive tumor-bearing xenografts. To further these observations, antitumor activity of TAK-164 was evaluated in multiple GCC-positive patient-derived xenografts. In these studies, a single intravenous administration of TAK-164 resulted in durable antitumor activity. Furthermore, the use of preclinical imaging demonstrated that TAK-164 preferentially accumulates in GCC-positive tumors, and that treatment response can be monitored using FDG-PET. These promising preclinical data warrant advancement of this ADC to clinical evaluation. Citation Format: Adnan O. Abu-Yousif, Bret M. Bannerman, Donna Cvet, Melissa Gallery, Michelle L. Ganno, Michael D. Smith, Katharine C. Lai, Thomas A. Keating, Jayaprakasam Bolleddula, Bradley Stringer, Mark G. Qian, Afrand Kamali, Kurt Eng, Secil Koseoglu, Cindy Q. Xia, O. Petter Veiby. TAK-164, a GCC-targeted antibody-drug conjugate (ADC) for the treatment of colorectal cancers and other GI malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B120.

Secreted bioactive lipids play critical roles in cell-to-cell communication and have been implicated in inflammatory immune responses such as anaphylaxis, vasodilation, and bronchoconstriction. Analysis of secreted bioactive lipids can be challenging due to their relatively short lifetimes and structural diversity. Herein, a method has been developed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to quantify five cell-secreted, structurally and functionally diverse bioactive lipids (PGD2, LTC4, LTD4, LTE4, PAF) that play roles in inflammation. Sample analysis time is 5 min, and isotopically labeled internal standards are used for quantification. This method was applied to an immortal secretory cell line (RBL-2H3), a heterogeneous primary cell culture containing peritoneal mast cells, and murine platelets. In RBL cell supernatant samples, intrasample precisions ranged from 7.32-21.6%, averaging 17.0%, and spike recoveries in cell supernatant matrices ranged from 88.0-107%, averaging 97.0%. Calibration curves were linear from 10 ng mL(-1) to 250 ng mL(-1), and limits of detection ranged from 0.0348 ng mL(-1) to 0.803 ng mL(-1). This method was applied to the determination of lipid secretion from mast cells and platelets, demonstrating broad applicability for lipid measurement in primary culture biological systems.

To inhibit pathological thrombus formation without impairing hemostasis is the holy grail of anticoagulant therapy. Recent data from animal models have indicated that factor XII (FXII) may be a promising new antithrombotic target that is particularly intriguing due to the longstanding clinical observation that severe congenital FXII deficiency is not associated with a bleeding diathesis in humans. FXII is thought to participate in thrombus formation after being activated in high shear arterial environments. FXIIa then initiates downstream activation of the contact pathway, culminating in thrombin generation. However, the relevant cell surface for FXII activation remains unclear. Here we compare the role of platelets versus endothelial cells in FXII activation and study the function of surface phospholipids in this process. To explore the effect of FXII inhibition on thrombus formation in vivo, we used antibody X210-C01, a novel human IgG1 developed using phage-display technology that blocks both mouse and human FXIIa. Using a mouse laser injury model of arterial thrombosis, we showed that X210-C01 inhibited both fibrin formation and platelet accumulation at sites of vascular injury. Plasma removed from animals after completion of these experiments was used to quantify the concentrations of X210-C01 achieved in vivo at a given dose. FXII inhibition was somewhat more potent in preventing platelet accumulation (IC50 dose = 27 mg/kg, R2=0.93) than fibrin formation (IC50 dose = 43 mg/kg, R2=0.95). Importantly, treatment with X210-C01 at 100 mg/kg did not prolong bleeding times or increase total blood loss in a tail bleeding assay. To evaluate the mechanism underlying our in vivo observations, we studied the differential role of FXII in thrombin generation by stimulated platelets and endothelium. X210-C01 did not globally inhibit SFLLRN-induced platelet aggregation or granule release. We next performed a fluorogenic thrombin generation assay (TGA) using human platelets treated with the peptide agonist SFLLRN. X210-C01 inhibited platelet-based thrombin generation in a dose-dependent fashion, whereas anti-tissue factor (TF) and anti-factor VIIa (FVIIa) antibodies did not. By contrast, in a similar TGA using SFLLRN-stimulated endothelial cells, X210-C01 had no effect, while anti-TF antibodies abrogated thrombin generation. These results indicate that stimulated endothelium generates thrombin by a mechanism distinct from that of platelets. FXII is known to be activated in vitro by anionic surfaces. Because phosphatidylserine (PS) is a negatively-charged phospholipid expressed on the surface of stimulated platelets, we reasoned that PS may serve as the platelet-based activator of FXII. To test this hypothesis, we used lactadherin, a potent and specific inhibitor of PS, in the platelet-based TGA and showed that PS blockade inhibited platelet-based thrombin generation at concentrations as low as 10 nM. We then used a chromogenic FXIIa activity assay to test the ability of PS-containing liposomes to activate FXII. Liposomes containing 80% phosphatidylcholine (PC) and 20% PS (PC-PS 80/20) failed to activate FXII at concentrations as high as 100 µM. In this assay, SFLLRN-stimulated platelets led to significantly greater FXII activation than either resting platelets or the PC-PS 80/20 liposomes tested. In summary, we have made the unexpected finding that thrombin generation on the surface of stimulated platelets proceeds by a FXIIa-dependent pathway and does not require FVIIa or TF. By contrast, thrombin generation on endothelium requires TF but not FXIIa. Additionally, surface PS is necessary but not sufficient for platelet-based FXII activation and thrombin generation, pointing to the involvement of a second platelet component. Further studies will be directed towards investigating the in vivo role of platelet-based FXII activation in arterial thrombus formation. Disclosures Mason: Shire Pharmaceuticals: Employment. Kenniston:Shire Pharmaceuticals: Employment.

Platelets are traditionally recognized as critical cells in hemostasis and thrombosis but have also recently been identified to play a significant role in many diseases, including bacterial infections, cancer, and allergic asthma. Platelets store and release important messenger molecules via exocytosis from two populations of granules (α- granules with adhesive protein species and δ- granules with small molecule/ion species). However, very little known about how chemical messengers are stored in these granules and the driving forces for secretion into the blood stream, thus limiting the development of new therapeutic approaches to manage the role of platelets in many physiological events. Carbon-fiber microelectrode amperometry (CFMA) enables both quantal and kinetic analysis of the exocytotic event of single cell with sub-ms time resolution but is limited to measuring electroactive species from δ-granules. Localized surface plasmon resonance (LSPR) spectroscopy is a complementary tool that offers an outstanding sensing ability by measuring the extremely small changes in the sensing media. This work aims to overcome the gap in fundamental knowledge about platelet granular storage and secretion by detecting serotonin secretion from δ- granules using CFMA and Platelet factor 4 (PF4) secretion from α- granules using LSPR. These measurements are performed while exposing platelets to different stimulants, including ionomycin, adenosine diphosphate (ADP), and thrombin. Significant differences are seen in both α- and δ-granule secretion with varied stimulant exposure, and these combined measurements yield biophysical insight into the mechanism behind these secretory behaviors.

Cell–cell communication is often achieved via granular exocytosis, as in neurons during synaptic transmission or neuroendocrine cells during blood hormone control. Owing to its critical role in membrane properties and SNARE function, cholesterol is expected to play an important role in the highly conserved process of exocytosis. In this work, membrane cholesterol concentration is systematically varied in primary culture mouse chromaffin cells, and the change in secretion behavior of distinct vesicle pools as well as pool recovery following stimulation is measured using carbon-fiber microelectrode amperometry. Amperometric traces obtained from activation of the younger readily releasable and slowly releasable pool (RRP/SRP) vesicles at depleted cholesterol levels showed fewer sustained fusion pore features (6.1 ± 1.1% of spikes compared with 11.2 ± 1.0% for control), revealing that cholesterol content influences fusion pore formation and stability during exocytosis. Moreover, subsequent stimulation of RRP/SRP vesicles showed that cellular cholesterol level influences both the quantal recovery and kinetics of the later release events. Finally, diverging effects of cholesterol on RRP and the older reserve pool vesicle release suggest two different mechanisms for the release of these two vesicular pools.