Tofacitinib to prevent anti-drug antibody formation against LMB-100 immunotoxin in patients with advanced mesothelin-expressing cancers.

Background: LMB-100 is a mesothelin (MSLN)-targeting recombinant immunotoxin (iTox) carrying a Pseudomonas exotoxin A payload that has shown promise against solid tumors, however, efficacy is limited by the development of neutralizing anti-drug antibodies (ADAs). Tofacitinib is an oral Janus Kinase (JAK) inhibitor that prevented ADA formation against iTox in preclinical studies. Methods: A phase 1 trial testing LMB-100 and tofacitinib in patients with MSLN-expressing cancers (pancreatic adenocarcinoma, n=13; cholangiocarcinoma, n=1; appendiceal carcinoma, n=1; cystadenocarcinoma, n=1) was performed to assess safety and to determine if tofacitinib impacted ADA formation. Participants were treated for up to 3 cycles with LMB-100 as a 30-minute infusion on days 4, 6, and 8 at two dose levels (100 and 140 µg/kg) while oral tofacitinib was administered for the first 10 days of the cycle (10 mg BID). Peripheral blood was collected for analysis of ADA levels, serum cytokines and circulating immune subsets. Results: The study was closed early due to occurrence of drug-induced pericarditis in 2 patients. Pericarditis with the combination was not reproducible in a transgenic murine model containing human MSLN. Two of 4 patients receiving all 3 cycles of treatment maintained effective LMB-100 levels, an unusual occurrence. Sustained increases in systemic IL-10 and TNF-α were seen, a phenomenon not observed in prior LMB-100 studies. A decrease in activated T cell subsets and an increase in circulating immunosuppressive myeloid populations occurred. No radiologic decreases in tumor volume were observed. Discussion: Further testing of tofacitinib to prevent ADA formation is recommended in applicable non-malignant disease settings. Clinical trial registration: https://www.clinicaltrials.gov/study/NCT04034238.

Macrophages Under the Influence of Tumor Mesothelin Weaken Host Defenses against Pancreatic Cancer Metastasis.

Although pancreatic cancer is a systemic disease that metastasizes early in its course, the signaling systems that promote this behavior remain incompletely understood. In this issue of Cancer Research, Luckett and colleagues identify a paracrine signaling pathway between cancer cells and macrophages that promotes pancreatic cancer metastasis. The authors used immunocompetent murine pancreatic cancer models with high versus low metastatic potential, genetic knockout and complementation strategies, and The Cancer Genome Atlas human data to demonstrate that tumor-secreted mesothelin repolarizes tumor and lung macrophages to a tumor-supportive phenotype. The repolarized macrophages increase secretion of VEGF and S100A9, raising local concentrations. In turn, VEGF enhances colony formation of cancer cells, while S100A9 promotes the recruitment of neutrophils to the lungs and the formation of neutrophil extracellular traps that support tumor metastasis. Together, these findings reveal a systemic signaling pathway that promotes pancreatic cancer metastasis by co-opting macrophages typically protective against cancer to instead promote its spread. See related article by Luckett et al., p. 527.

Two rare cancers of the exocrine pancreas: to treat or not to treat like ductal adenocarcinoma?

Pancreatic cancer is an aggressive malignancy with increasing incidence. Pancreatic ductal adenocarcinoma (PDAC) accounts for > 90% of pancreatic cancer diagnoses, while other exocrine tumors are much rarer. In this review, we have focused on two rare cancers of the exocrine pancreas: adenosquamous carcinoma of the pancreas (ASCP) and pancreatic acinar cell carcinoma (PACC). The latest findings regarding their cellular and molecular pathology, clinical characteristics, prognosis, and clinical management are discussed. New genetic and transcriptomic data suggest that ASCP is related to or overlaps with the basal transcriptomic subtype of PDAC. These tumors are highly aggressive and driven by activated KRAS and MYC expression. Clinical outcomes remain poor and effective treatments are limited. PACC has no morphologic or genetic resemblance to PDAC and more favorable outcomes. Early stage PACC patients have improved survival with surgical resection and patients with advanced disease benefit most from platinum- or fluoropyrimidine-containing chemotherapy. Frequency of actionable genetic mutations is high in this disease and case reports suggest good outcomes when matched therapy is given. Dedicated clinical studies examining ASCP and PACC are limited and difficult to accrue. Further research is needed to define optimal clinical management for these rare diseases.

Systemic immune changes accompany combination treatment with immunotoxin LMB-100 and nab-paclitaxel.

LMB-100 is a novel immune-conjugate (immunotoxin) that targets mesothelin. A phase 1/2 clinical trial was conducted (NCT02810418) with primary objectives assessing the safety and efficacy of LMB-100 ± nab-paclitaxel. Participant blood samples were analyzed for changes in serum cytokines and circulating immune cell subsets associated with response or toxicity. On Arm A, participants (n = 20) received standard 30-minute LMB-100 infusion with nab-paclitaxel. Although clinical efficacy was observed, the combination caused intolerable capillary leak syndrome (CLS), a major toxicity of unclear etiology that affects many immunotoxin drugs. Participants developing CLS experienced rapid elevations in IFNγ and IL-8 compared to those without significant CLS, along with midcycle increases in Ki-67- CD4 T cells that were CD38, HLA-DR, or TIM3 positive. Additionally, a strong increase in activated CD4 and CD8 T cells and a concurrent decrease in Tregs were seen in the single Arm A patient achieving a partial response. In Arm B, administration of single agent LMB-100 to participants (n = 20) as a long infusion given over 24-48 h was investigated based on pre-clinical data that this format could reduce CLS. An optimal dose and schedule of long infusion LMB-100 were identified, but no clinical efficacy was observed even in patients receiving LMB-100 in combination with nab-paclitaxel. Despite this, both Arm A and B participants experienced increases in specific subsets of proliferating CD4 and CD8 T cells following Cycle 1 treatment. In summary, LMB-100 treatment causes systemic immune activation. Inflammatory and immune changes that accompany drug associated CLS were characterized for the first time.

Clinical Strategies Targeting the Tumor Microenvironment of Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer has a complex tumor microenvironment which engages in extensive crosstalk between cancer cells, cancer-associated fibroblasts, and immune cells. Many of these interactions contribute to tumor resistance to anti-cancer therapies. Here, new therapeutic strategies designed to modulate the cancer-associated fibroblast and immune compartments of pancreatic ductal adenocarcinomas are described and clinical trials of novel therapeutics are discussed. Continued advances in our understanding of the pancreatic cancer tumor microenvironment are generating stromal and immune-modulating therapeutics that may improve patient responses to anti-tumor treatment.

A phase II trial of the super-enhancer inhibitor Minnelide™ in advanced refractory adenosquamous carcinoma of the pancreas.

Adenosquamous carcinoma of the pancreas (ASCP) is a very rare and highly aggressive variant of pancreatic ductal adenocarcinoma, accounting for 0.5-4% of all pancreatic cancer cases in the USA. Current data indicate that epigenetic changes and MYC overexpression lead to squamous transdifferentiation of pancreatic tumor cells and development of ASCP. Minnelide™, an oral anti-super-enhancer drug that inhibits MYC expression in preclinical models of ASCP, has demonstrated safety in a phase I study. We describe the design for a phase II, open-label, single-arm trial of Minnelide in patients with advanced refractory ASCP.

Adenosquamous carcinoma of the pancreas (ASCP) is a rare and highly aggressive variant of pancreatic cancer, with limited treatment options. Changes in activation of DNA elements called super-enhancers drive the growth of ASCP. Minnelide™ is an oral drug that blocks the super-enhancer network and is safe to give to patients with advanced cancer. This trial is designed to determine whether Minnelide can shrink tumors in patients with ASCP who have already received at least one previous treatment for their cancer.  Clinical Trial Registration: NCT04896073 (ClinicalTrials.gov).

Low serum mesothelin in pancreatic cancer patients results from retention of shed mesothelin in the tumor microenvironment.

Mesothelin (MSLN) is overexpressed by many cancers, including pancreatic ductal adenocarcinoma (PDAC) and has consequently become a target for anti-cancer therapeutics. Mature, membrane bound MSLN is cleaved by proteases, releasing a shed form that transits to the circulation. Many patients with mesothelioma and ovarian cancer have abnormally high serum MSLN concentration. However, serum MSLN concentration in PDAC patients rarely exceeds levels of healthy controls. Here, serum MSLN concentration in advanced PDAC patients was examined pre- and post-treatment. Serum MSLN did not correlate with tumor MSLN expression, nor with changes in tumor burden as assessed by PDAC serum tumor marker CA19-9. Subsequently, tumor-bearing mouse models were used to investigate the fate of shed MSLN in PDAC versus a control cervical cancer model. Efficiency of MSLN secretion into the serum was cell-line dependent. Tumors from some PDAC lines had poor MSLN secretion efficiency although these lines had similar or higher MSLN shedding rate, total and surface MSLN expression. Measurements of compartment-specific MSLN concentration taken at equilibrium suggested that tumors with poor MSLN secretion efficiency trapped shed MSLN in the tumor microenvironment (TME), a finding confirmed by dynamic experiments using a doxycycline-inducible MSLN expression system. Tumors with the poorest MSLN secretion efficiency had higher collagen density and increased abundance of MSLN binding partner MUC16. The tumor with the worst secretion efficiency could rebind shed MSLN to the cancer cell surface. Altogether, these data suggest that PDAC can trap shed MSLN within the TME. This finding has potential significance for design of MSLN-targeted therapeutics.

Novel Humanized Mesothelin-Expressing Genetically Engineered Mouse Models Underscore Challenges in Delivery of Complex Therapeutics to Pancreatic Cancers.

Antibody-based therapies designed for human use frequently fail to cross-react with the murine isoform of their target. Because of this problem, preclinical studies of antibody-based mesothelin (Msl)-targeted therapeutics in immunocompetent systems have been limited by the lack of suitable mouse models. Here, we describe two immunocompetent humanized mesothelin transgenic mouse lines that can act as tolerant hosts for C57Bl/6-syngeneic cell lines expressing the human isoform of mesothelin. Thyroid peroxidase (TPO) mice have thyroid-restricted human mesothelin expression. Mesothelin (Msl) mice express human mesothelin in the typical serosal membrane distribution and can additionally be utilized to assess on-target, off-tumor toxicity of human mesothelin-targeted therapeutics. Both transgenic strains shed human mesothelin into the serum like human mesothelioma and patients with ovarian cancer, and serum human mesothelin can be used as a blood-based surrogate of tumor burden. Using these models, we examined the on-target toxicity and antitumor activity of human mesothelin-targeted recombinant immunotoxins. We found that immunotoxin treatment causes acute and chronic histologic changes to serosal membranes in Msl mice, while human mesothelin-expressing thyroid follicular cells in TPO mice are resistant to immunotoxin despite excellent drug delivery. Furthermore, poor delivery of immunotoxin to syngeneic orthotopic human mesothelin-expressing pancreatic adenocarcinoma limits antitumor activity both alone and in combination with immune checkpoint inhibition. In summary, we have developed two high-fidelity, immunocompetent murine models for human cancer that allow for rigorous preclinical evaluation of human mesothelin-targeted therapeutics.

Phase 1 study of the immunotoxin LMB-100 in patients with mesothelioma and other solid tumors expressing mesothelin.

BACKGROUND: LMB-100 is an antibody-toxin conjugate with an antimesothelin Fab linked to a 24-kilodalton portion of Pseudomonas exotoxin A with mutations that decrease immunogenicity. The objective of the current first-in-human phase 1 study was to determine the maximum tolerated dose (MTD) and safety in patients with advanced solid tumors expressing mesothelin. METHODS: Cohorts of 1 to 7 patients received intravenous LMB-100 at 7 dose levels from 40 µg/kg to 250 µg/kg intravenously on days 1, 3, and 5 of a 21-day cycle. RESULTS: Of the 25 patients accrued, 17 had mesothelioma, 3 each had ovarian or pancreatic cancer, and 2 patients had gastric cancer. Dose-limiting toxicities occurred in 2 of 4 patients treated at a dose of 250 µg/kg (capillary leak syndrome) and in 3 of 7 patients treated at a dose of 170 µg/kg (creatinine increase). The MTD of LMB-100 was 140 µg/kg. Of the 10 patients with mesothelioma who were treated at doses of 170 µg/kg or 140 µg/kg, 8 had stable disease and 2 developed progressive disease. Peak LMB-100 plasma concentrations were dose-dependent during cycle 1. The development of antidrug antibodies decreased LMB-100 blood levels in 8 of 21 patients (38%) who received cycle 2 and 9 of 11 patients (81.8%) who received cycle 3. CONCLUSIONS: The MTD for single-agent LMB-100 was found to be 140 µg/kg given on a schedule of every other day for 3 doses every 3 weeks. Although less immunogenic than the first-generation antimesothelin immunotoxin SS1P, the majority of patients developed antidrug antibodies after 2 cycles, indicating that LMB-100 has limited antitumor efficacy as a single agent. Phase 2 studies of LMB-100 plus pembrolizumab currently are ongoing for patients with mesothelioma and lung cancer. LAY SUMMARY: Mesothelin, a cell surface antigen, is an attractive target for cancer therapy given its limited expression in normal human tissues and high expression in many human cancers. LMB-100 is a recombinant antimesothelin immunotoxin consisting of a humanized antimesothelin antibody fragment fused to a truncated Pseudomonas exotoxin A. In the current study, the authors determined the safety, maximum tolerated dose, and pharmacokinetics of LMB-100, as well as the generation of antidrug antibodies. Ongoing phase 2 clinical trials are evaluating the combination of LMB-100 plus pembrolizumab in patients with treatment-refractory mesothelioma and non-small cell lung cancer.

Molecular mediators of peritoneal metastasis in pancreatic cancer.

Pancreatic cancer is the third leading cause of cancer death in the USA, and pancreatic ductal adenocarcinoma (PDA) constitutes 85% of pancreatic cancer diagnoses. PDA frequently metastasizes to the peritoneum, but effective treatment of peritoneal metastasis remains a clinical challenge. Despite this unmet need, understanding of the biological mechanisms that contribute to development and progression of PDA peritoneal metastasis is sparse. By contrast, a vast number of studies have investigated mechanisms of peritoneal metastasis in ovarian and gastric cancers. Here, we contrast similarities and differences between peritoneal metastasis in PDA as compared with those in gastric and ovarian cancer by outlining molecular mediators involved in each step of the peritoneal metastasis cascade. This review aims to provide mechanistic insights that could be translated into effective targeted therapies for patients with peritoneal metastasis from PDA.

Mesothelin-Targeted Recombinant Immunotoxins for Solid Tumors.

Mesothelin (MSLN) is a cell surface glycoprotein normally expressed only on serosal surfaces, and not found in the parenchyma of vital organs. Many solid tumors also express MSLN, including mesothelioma and pancreatic adenocarcinoma. Due to this favorable expression profile, MSLN represents a viable target for directed anti-neoplastic therapies, such as recombinant immunotoxins (iToxs). Pre-clinical testing of MSLN-targeted iTox's has yielded a strong body of evidence for activity against a number of solid tumors. This has led to multiple clinical trials, testing the safety and efficacy of the clinical leads SS1P and LMB-100. While promising clinical results have been observed, neutralizing anti-drug antibody (ADA) formation presents a major challenge to overcome in the therapeutic development process. Additionally, on-target, off-tumor toxicity from serositis and non-specific capillary leak syndrome (CLS) also limits the dose, and therefore, impact anti-tumor activity. This review summarizes existing pre-clinical and clinical data on MSLN-targeted iTox's. In addition, we address the potential future directions of research to enhance the activity of these anti-tumor agents.

Enhanced efficacy of mesothelin-targeted immunotoxin LMB-100 and anti-PD-1 antibody in patients with mesothelioma and mouse tumor models.

LMB-100 is an immunotoxin targeting the cell surface protein mesothelin, which is highly expressed in many cancers including mesothelioma. Having observed that patients receiving pembrolizumab off protocol after LMB-100 treatment had increased tumor responses; we characterized these responses and developed animal models to study whether LMB-100 made tumors more responsive to antibodies blocking programmed cell death protein 1 (PD-1). The overall objective tumor response in the 10 patients who received PD-1 inhibitor (pembrolizumab, 9; nivolumab, 1) after progression on LMB-100 was 40%, and the median overall survival was 11.9 months. Of the seven evaluable patients, four had objective tumor responses, including one complete response and three partial responses, and the overall survival for these patients was 39.0+, 27.7, 32.6+, and 13.8 months. When stratified with regard to programmed death ligand 1 (PD-L1) expression, four of five patients with tumor PD-L1 expression had objective tumor response. Patients with positive tumor PD-L1 expression also had increased progression-free survival (11.3 versus 2.1 months, P = 0.0018) compared with those lacking PD-L1 expression. There was no statistically significant difference in overall survival (27.7 versus 6.8 months, P = 0.1). LMB-100 caused a systemic inflammatory response and recruitment of CD8+ T cells in patients' tumors. The enhanced antitumor effects with LMB-100 plus anti-PD-1 antibody were also observed in a human peripheral blood mononuclear cell-engrafted mesothelioma mouse model and a human mesothelin-expressing syngeneic lung adenocarcinoma mouse model. LMB-100 plus pembrolizumab is now being evaluated in a prospective clinical trial for patients with mesothelioma.

Phase I/II Study of the Mesothelin-targeted Immunotoxin LMB-100 with Nab-Paclitaxel for Patients with Advanced Pancreatic Adenocarcinoma.

PURPOSE: LMB-100 is a recombinant immunotoxin (iTox) consisting of a mesothelin-binding Fab for targeting and a modified Pseudomonas exotoxin A payload. Preclinical studies showed that combining taxanes with iTox results in synergistic antitumor activity. The objectives of this phase I/II study were to determine the MTD of LMB-100 when administered with nanoalbumin bound (nab)-paclitaxel to patients with previously treated advanced pancreatic adenocarcinoma and to assess the objective response rate. PATIENTS AND METHODS: Patients (n = 20) received fixed-dose nab-paclitaxel (125 mg/m2 on days 1 and 8) with LMB-100 (65 or 100 µg/kg on days 1, 3, and 5) in 21-day cycles for 1-3 cycles. RESULTS: Fourteen patients were treated on the dose escalation and an additional six in the phase II expansion. MTD of 65 µg/kg was established for the combination. Dose-limiting toxicity resulting from capillary leak syndrome (CLS) was seen in two of five patients treated at 100 µg/kg and one of six evaluable phase I patients receiving the MTD. Severity of CLS was associated with increases in apoptotic circulating endothelial cells. LMB-100 exposure was unaffected by anti-LMB-100 antibody formation in five of 13 patients during cycle 2. Seven of 17 evaluable patients experienced >50% decrease in CA 19-9, including three with previous exposure to nab-paclitaxel. One patient developed an objective partial response. Patients with biomarker responses had higher tumor mesothelin expression. CONCLUSIONS: Although clinical activity was observed, the combination was not well tolerated and alternative drug combinations with LMB-100 will be pursued.

Mesothelin Enhances Tumor Vascularity in Newly Forming Pancreatic Peritoneal Metastases.

Over 90% of pancreatic ductal adenocarcinomas (PDAC) express mesothelin (MSLN). Overexpression or knockdown of MSLN has been implicated in PDAC aggressiveness. This activity has been ascribed to MSLN-induced activation of MAPK or NF-κB signaling pathways and to interaction of MSLN with its only known binding partner, MUC16. Here, we used CRISPR/Cas9 gene editing to delete MSLN from PDAC, then restored expression of wild-type (WT) or Y318A mutant MSLN by viral transduction. We found that MSLN KO cells grew in culture and as subcutaneous tumors in mouse xenografts at the same rate as WT cells but formed intraperitoneal metastases poorly. Complementation with WT MSLN restored intraperitoneal growth, whereas complementation with Y318A mutant MSLN, which does not bind MUC16, was ineffective at enhancing growth in both MUC16(+) and MUC16(-) models. Restoration of WT MSLN did enhance growth but did not affect cell-to-cell binding, cell viability in suspension or signaling pathways previously identified as contributing to the protumorigenic effect of MSLN. RNA deep sequencing of tumor cells identified no changes in transcriptional profile that could explain the observed phenotype. Furthermore, no histologic changes in tumor cell proliferation or morphology were observed in mature tumors. Examination of nascent MSLN KO tumors revealed decreased microvascular density as intraperitoneal tumors were forming, followed by decreased proliferation, which resolved by 2 weeks postimplantation. These data support a model whereby MSLN expression by tumor cells contributes to metastatic colonization. IMPLICATIONS: MSLN confers a growth advantage to tumor cells during colonization of peritoneal metastasis. Therapeutic blockade of MSLN might limit peritoneal spread.

Inherited predisposition to malignant mesothelioma and overall survival following platinum chemotherapy.

Survival from malignant mesothelioma, particularly pleural mesothelioma, is very poor. For patients with breast, ovarian, or prostate cancers, overall survival is associated with increased sensitivity to platinum chemotherapy due to loss-of-function mutations in DNA repair genes. The goal of this project was to evaluate, in patients with malignant mesothelioma, the relationship between inherited loss-of-function mutations in DNA repair and other tumor suppressor genes and overall survival following platinum chemotherapy. Patients with histologically confirmed malignant mesothelioma were evaluated for inherited mutations in tumor suppressor genes. Survival was evaluated with respect to genotype and site of mesothelioma. Among 385 patients treated with platinum chemotherapy, median overall survival was significantly longer for patients with loss-of-function mutations in any of the targeted genes compared with patients with no such mutation (P = 0.0006). The effect of genotype was highly significant for patients with pleural mesothelioma (median survival 7.9 y versus 2.4 y, P = 0.0012), but not for patients with peritoneal mesothelioma (median survival 8.2 y versus 5.4 y, P = 0.47). Effect of patient genotype on overall survival, measured at 3 y, remained independently significant after adjusting for gender and age at diagnosis, two other known prognostic factors. Patients with pleural mesothelioma with inherited mutations in DNA repair and other tumor suppressor genes appear to particularly benefit from platinum chemotherapy compared with patients without inherited mutations. These patients may also benefit from other DNA repair targeted therapies such as poly-ADP ribose polymerase (PARP) inhibitors.

Tofacitinib enhances delivery of antibody-based therapeutics to tumor cells through modulation of inflammatory cells.

The routes by which antibody-based therapeutics reach malignant cells are poorly defined. Tofacitinib, an FDA-approved JAK inhibitor, reduced tumor-associated inflammatory cells and allowed increased delivery of antibody-based agents to malignant cells. Alone, tofacitinib exhibited no antitumor activity, but combinations with immunotoxins or an antibody-drug conjugate resulted in increased antitumor responses. Quantification using flow cytometry revealed that antibody-based agents accumulated in malignant cells at higher percentages following tofacitinib treatment. Profiling of tofacitinib-treated tumor-bearing mice indicated that cytokine transcripts and various proteins involved in chemotaxis were reduced compared with vehicle-treated mice. Histological analysis revealed significant changes to the composition of the tumor microenvironment, with reductions in monocytes, macrophages, and neutrophils. Tumor-associated inflammatory cells contributed to non-target uptake of antibody-based therapeutics, with mice treated with tofacitinib showing decreased accumulation of therapeutics in intratumoral inflammatory cells and increased delivery to malignant cells. The present findings serve as a rationale for conducting trials where short-term treatments with tofacitinib could be administered in combination with antibody-based therapies.

Protein Synthesis Inhibition Activity of Mesothelin Targeting Immunotoxin LMB-100 Decreases Concentrations of Oncogenic Signaling Molecules and Secreted Growth Factors.

LMB-100 is a mesothelin-targeted recombinant immunotoxin (iTox) that carries a modified Pseuodomonas exotoxin A (PE) payload. PE kills cells by inhibiting synthesis of new proteins. We found that treatment of pancreatic cancer cells with LMB-100 for 24⁻48 h did not change total protein level despite inducing protein synthesis inhibition (PSI). Further, increased levels of ubiquitinated proteins were detected, indicating that cells may have limited ability to compensate for PSI by reducing protein degradation. Together, these data suggest that PE depletes concentrations of a minority of cellular proteins. We used reverse phase protein array and Luminex assay to characterize this subset. LMB-100 decreased the abundance of 24 of 32 cancer-related proteins (including Bcl-x, Her2, Her3 and MUC16) without compensatory increases in other analytes. Further, cancer cells failed to maintain extracellular concentrations of cancer cell secreted growth factors (CCSGFs), including Vascular Endothelial Growth Factor (VEGF) following treatment with cytostatic LMB-100 doses both in culture and in mouse tumors. Decreased VEGF concentration did not change tumor vasculature density, however, LMB-100 caused tissue-specific changes in concentrations of secreted factors made by non-cancer cells. In summary, our data indicate that PSI caused by cytostatic LMB-100 doses preferentially depletes short-lived proteins such as oncogenic signaling molecules and CCSGFs.