Antibody Drug Clearance: An Underexplored Marker of Outcomes with Checkpoint Inhibitors.

Immune-checkpoint inhibitor (ICI) therapy has dramatically changed the clinical landscape for several cancers, and ICI use continues to expand across many cancer types. Low baseline clearance (CL) and/or a large reduction of CL during treatment correlates with better clinical response and longer survival. Similar phenomena have also been reported with other monoclonal antibodies (mAb) in cancer and other diseases, highlighting a characteristic of mAb clinical pharmacology that is potentially shared among various mAbs and diseases. Though tempting to attribute poor outcomes to low drug exposure and arguably low target engagement due to high CL, such speculation is not supported by the relatively flat exposure-response relationship of most ICIs, where a higher dose or exposure is not likely to provide additional benefit. Instead, an elevated and/or increasing CL could be a surrogate marker of the inherent resistant phenotype that cannot be reversed by maximizing drug exposure. The mechanisms connecting ICI clearance, therapeutic efficacy, and resistance are unclear and likely to be multifactorial. Therefore, to explore the potential of ICI CL as an early marker for efficacy, this review highlights the similarities and differences of CL characteristics and CL-response relationships for all FDA-approved ICIs, and we compare and contrast these to selected non-ICI mAbs. We also discuss underlying mechanisms that potentially link mAb CL with efficacy and highlight existing knowledge gaps and future directions where more clinical and preclinical investigations are warranted to clearly understand the value of baseline and/or time-varying CL in predicting response to ICI-based therapeutics.

Decoupling FcRn and tumor contributions to elevated immune checkpoint inhibitor clearance in cancer cachexia.

High baseline clearance of immune checkpoint inhibitors (ICIs), independent of dose or systemic exposure, is associated with cachexia and poor outcomes in cancer patients. Mechanisms linking ICI clearance, cachexia and ICI therapy failure are unknown. Here, we evaluate in four murine models and across multiple antibodies whether altered baseline catabolic clearance of administered antibody requires a tumor and/or cachexia and whether medical reversal of cachexia phenotype can alleviate altered clearance. Key findings include mild cachexia phenotype and lack of elevated pembrolizumab clearance in the MC38 tumor-bearing model. We also observed severe cachexia and decreased, instead of increased, baseline pembrolizumab clearance in the tumor-free cisplatin-induced cachexia model. Liver Fcgrt expression correlated with altered baseline catabolic clearance, though elevated clearance was still observed with antibodies having no (human IgA) or reduced (human H310Q IgG1) FcRn binding. We conclude cachexia phenotype coincides with altered antibody clearance, though tumor presence is neither sufficient nor necessary for altered clearance in immunocompetent mice. Magnitude and direction of clearance alteration correlated with hepatic Fcgrt, suggesting changes in FcRn expression and/or recycling function may be partially responsible, though factors beyond FcRn also contribute to altered clearance in cachexia.

Exploration of Verticillins in High-Grade Serous Ovarian Cancer and Evaluation of Multiple Formulations in Preclinical In Vitro and In Vivo Models.

Verticillins are epipolythiodioxopiperazine alkaloids isolated from a fungus with nanomolar anti-tumor activity in high-grade serous ovarian cancer (HGSOC). HGSOC is the fifth leading cause of death in women, and natural products continue to be an inspiration for new drug entities to help tackle chemoresistance. Verticillin D was recently found in a new fungal strain and compared to verticillin A. Both compounds exhibited nanomolar cytotoxic activity against OVCAR4 and OVCAR8 HGSOC cell lines, significantly reduced 2D foci and 3D spheroids, and induced apoptosis. In addition, verticillin A and verticillin D reduced tumor burden in vivo using OVCAR8 xenografts in the peritoneal space as a model. Unfortunately, mice treated with verticillin D displayed signs of liver toxicity. Tolerability studies to optimize verticillin A formulation for in vivo delivery were performed and compared to a semi-synthetic succinate version of verticillin A to monitor bioavailability in athymic nude females. Formulation of verticillins achieved tolerable drug delivery. Thus, formulation studies are effective at improving tolerability and demonstrating efficacy for verticillins.

The Neonatal Fc Receptor Is Elevated in Monocyte-Derived Immune Cells in Pancreatic Cancer.

The neonatal Fc receptor (FcRn) is responsible for recycling of IgG antibodies and albumin throughout the body. This mechanism has been exploited for pharmaceutic delivery across an array of diseases to either enhance or diminish this function. Monoclonal antibodies and albumin-bound nanoparticles are examples of FcRn-dependent anti-cancer therapeutics. Despite its importance in drug delivery, little is known about FcRn expression in circulating immune cells. Through time-of-flight mass cytometry (CyTOF) we were able to characterize FcRn expression in peripheral blood mononuclear cell (PBMC) populations of pancreatic ductal adenocarcinoma (PDAC) patients and non-cancer donors. Furthermore, we were able to replicate these findings in an orthotopic murine model of PDAC. Altogether, we found that in both patients and mice with PDAC, FcRn was elevated in migratory and resident classical dendritic cell type 2 (cDC2) as well as monocytic and granulocytic myeloid-derived suppressor cell (MDSC) populations compared to tumor-free controls. Furthermore, PBMCs from PDAC patients had elevated monocyte, dendritic cells and MDSCs relative to non-cancer donor PBMCs. Future investigations into FcRn activity may further elucidate possible mechanisms of poor efficacy of antibody immunotherapies in patients with PDAC.

Serum Albumin: Early Prognostic Marker of Benefit for Immune Checkpoint Inhibitor Monotherapy But Not Chemoimmunotherapy.

BACKGROUND: Cancer cachexia exhibits decreased albumin and associates with short overall survival (OS) in patients with non-small cell lung cancer (NSCLC), but whether on-treatment albumin changes associate with OS in NSCLC patients treated with immune checkpoint inhibitors (ICIs) and combination chemoimmunotherapy has not been thoroughly evaluated. PATIENTS AND METHODS: We conducted a single-center retrospective study of patients with advanced NSCLC who received first-line ICI with or without chemotherapy between 2013 and 2020. The association of pretreatment albumin and early albumin changes with OS was evaluated using Kaplan-Meier method and Cox regression models. RESULTS: A total of 210 patients were included: 109 in ICI cohort and 101 in ICI + Chemo cohort. Within a median of 21 days from treatment initiation, patients with ≥ 10% of albumin decrease had significantly shorter OS compared to patients without albumin decrease in ICI cohort. Pretreatment albumin and albumin decrease within the first or second cycle of treatment were significantly and independently associated with OS in ICI cohort, but not in ICI + Chemo cohort. The lack of association between albumin and OS with the addition of chemotherapy was more pronounced among patients with ≥ 1% PD-L1 expression in subgroup analysis. CONCLUSION: Pretreatment serum albumin and early albumin decrease in ICI monotherapy was significantly associated with OS in advanced NSCLC. Early albumin change, as a routine lab value tested in clinic, may be combined with established biomarkers to improve outcome predictions of ICI monotherapy. The underlying mechanism of the observed association between decreased albumin and ICI resistance warrants further investigation.

Murine cancer cachexia models replicate elevated catabolic pembrolizumab clearance in humans.

BACKGROUND: Monoclonal antibody (mAb) immune checkpoint inhibitor (ICI) therapies have dramatically impacted oncology this past decade. However, only about one-third of patients respond to treatment, and biomarkers to predict responders are lacking. Recent ICI clinical pharmacology data demonstrate high baseline drug clearance (CL0) significantly associates with shorter overall survival, independent of ICI exposure, in patients receiving ICI mAb therapies. This suggests CL0 may predict outcomes from ICI therapy, and cachectic signalling may link elevated CL0 and poor response. Our aim was to determine if mouse models of cancer cachexia will be useful for studying these phenomena and their underlying mechanisms. METHODS: We evaluated pembrolizumab CL in the C26 and Lewis lung carcinoma mouse models of cancer cachexia. A single treatment of vehicle or pembrolizumab, at a dose of 2 or 10 mg/kg, was administered intravenously by tail vein injection. Pembrolizumab was quantified by an ELISA in serial plasma samples, and FcRn gene (Fcgrt) expression was assessed in liver using real-time quantitative reverse transcription PCR. Non-compartmental and mixed-effects pharmacokinetics analyses were performed. RESULTS: We observed higher pembrolizumab CL0 and decreased Fcgrt expression in whole liver tissue from tumour-bearing vs. tumour-free mice. In multivariate analysis, presence of tumour, total murine IgG, muscle weight and Fcgrt expression were significant covariates on CL, and total murine IgG was a significant covariate on V1 and Q. CONCLUSIONS: These data demonstrate increases in catabolic clearance of monoclonal antibodies observed in humans can be replicated in cachectic mice, in which Fcgrt expression is also reduced. Notably, FcRn activity is essential for proper antigen presentation and antitumour immunity, which may permit the study of cachexia's impact on FcRn-mediated clearance and efficacy of ICI therapies.

Targeting DNA Damage Repair Functions of Two Histone Deacetylases, HDAC8 and SIRT6, Sensitizes Acute Myeloid Leukemia to NAMPT Inhibition.

PURPOSE: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274. EXPERIMENTAL DESIGN: Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model. RESULTS: We identified two histone deacetylases (HDAC), HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells. CONCLUSIONS: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel-novel combination-based treatment for AML.

Inhibition of androgen/AR signaling inhibits diethylnitrosamine (DEN) induced tumour initiation and remodels liver immune cell networks.

A promotional role for androgen receptor (AR) signaling in hepatocellular carcinogenesis is emerging. In pre-clinical models, including diethylnitrosamine- (DEN-) induced hepatocellular carcinoma (HCC), anti-androgen therapies delay hepatocarcinogenesis. However, pharmacologic anti-androgen therapy in advanced HCC patients fails, suggesting that AR plays a role in HCC onset. This study aims to characterize AR expression and function throughout DEN-induced liver inflammation and carcinogenesis and evaluate the efficacy of prophylactic AR antagonism to prevent hepatocarcinogenesis. We demonstrate that pharmacologic AR antagonism with enzalutamide inhibits hepatocellular carcinogenesis. With enzalutamide treatment, we observe decreased CYP2E1 expression, reducing DEN-induced hepatocyte death and DNA ethyl-adducts. AR protein expression analyses show that DEN causes an initial upregulation of AR in portal fibroblasts and leukocytes, but not hepatocytes, suggesting that hepatocyte-autonomous AR signaling is not essential for DEN-induced carcinogenesis. Ablating androgen signaling by surgical castration reduced pre-carcinogen Kupffer cell populations but did not alter DEN-mediated immune cell recruitment nor AR expression. In this study, we identified that anti-androgen interventions modulate mutagenic DNA adducts, tumour initiation, and immune cell composition. Additionally, we find that AR expression in hepatocytes is not present during nor required for early DEN-mediated carcinogenesis.

Soy-tomato enriched diet reduces inflammation and disease severity in a pre-clinical model of chronic pancreatitis.

Chronic pancreatitis (CP) is a fibro-inflammatory syndrome in individuals who develop persistent pathological responses to parenchymal injury or stress. Novel therapeutic or dietary interventions that could lessen inflammation in this disease could significantly improve quality of life in patients with CP. Complex dietary foods like soy and tomatoes are composed of active metabolites with anti-inflammatory effects. Data from our group reports that bioactive agents in soy and tomatoes can reduce pro-inflammatory cytokines and suppressive immune populations. Additionally, our team has developed a novel soy-tomato juice currently being studied in healthy individuals with no toxicities, and good compliance and bioavailability. Thus, we hypothesize that administration of a soy-tomato enriched diet can reduce inflammation and severity of CP. C57BL/6 mice were injected intraperitoneally with 50 µg/kg caeurlein (7 hourly injections, twice weekly) for 6 weeks to induce CP. After 4 weeks of caerulein injections, mice were administered a control or a soy-tomato enriched diet for 2 weeks. Disease severity was measured via immunohistochemical analysis of pancreata measuring loss of acini, fibrosis, inflammation, and necrosis. Serum lipase and amylase levels were analyzed at the end of the study. Inflammatory factors in the serum and pancreas, and immune populations in the spleen of mice were analyzed by cytokine multiplex detection, qRT-PCR, and flow cytometry respectively. Infra-red (IR) sensing of mice was used to monitor spontaneous activity and distress of mice. Mice fed a soy-tomato enriched diet had a significantly reduced level of inflammation and severity of CP (p = 0.032) compared to mice administered a control diet with restored serum lipase and amylase levels (p < 0.05). Mice with CP fed a soy-tomato diet had a reduction in inflammatory factors (TNF-α, IL-1ß, IL-5) and suppressive immune populations (myeloid-derived suppressor cells; MDSC) compared to control diet fed mice (p < 0.05). Infra-red sensing to monitor spontaneous activity of mice showed that soy-tomato enriched diet improved total activity and overall health of mice with CP (p = 0.055) and CP mice on a control diet were determined to spend more time at rest (p = 0.053). These pre-clinical results indicate that a soy-tomato enriched diet may be a novel treatment approach to reduce inflammation and pain in patients with CP.

Pharmacokinetics and Tolerability of the Novel Non-immunosuppressive Fingolimod Derivative, OSU-2S, in Dogs and Comparisons with Data in Mice and Rats.

In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 µM, and Caco-2 data suggested low permeability with active efflux at 2 µM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5-20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUClast (26.1 µM*h), and Cmax (0.899 µM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.

Retraction: Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

[This retracts the article DOI: 10.18632/oncotarget.6427.].

Overcoming resistance to anabolic SARM therapy in experimental cancer cachexia with an HDAC inhibitor.

No approved therapy exists for cancer-associated cachexia. The colon-26 mouse model of cancer cachexia mimics recent late-stage clinical failures of anabolic anti-cachexia therapy and was unresponsive to anabolic doses of diverse androgens, including the selective androgen receptor modulator (SARM) GTx-024. The histone deacetylase inhibitor (HDACi) AR-42 exhibited anti-cachectic activity in this model. We explored combined SARM/AR-42 therapy as an improved anti-cachectic treatment paradigm. A reduced dose of AR-42 provided limited anti-cachectic benefits, but, in combination with GTx-024, significantly improved body weight, hindlimb muscle mass, and grip strength versus controls. AR-42 suppressed the IL-6/GP130/STAT3 signaling axis in muscle without impacting circulating cytokines. GTx-024-mediated ß-catenin target gene regulation was apparent in cachectic mice only when combined with AR-42. Our data suggest cachectic signaling in this model involves catabolic signaling insensitive to anabolic GTx-024 therapy and a blockade of GTx-024-mediated anabolic signaling. AR-42 mitigates catabolic gene activation and restores anabolic responsiveness to GTx-024. Combining GTx-024, a clinically established anabolic therapy, with AR-42, a clinically evaluated HDACi, represents a promising approach to improve anabolic response in cachectic patients.

SUV39H1 Represses the Expression of Cytotoxic T-Lymphocyte Effector Genes to Promote Colon Tumor Immune Evasion.

Despite the presence of CTLs in the tumor microenvironment, the majority of immunogenic human colon cancer does not respond to immune checkpoint inhibitor immunotherapy, and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs were present in both MSI and microsatellite stable colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 was significantly elevated in human colon carcinoma compared with normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment have high expression of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened on the basis of the SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain structure of the human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. On the basis of the structure of this small molecule, we modified it and chemically synthesized a small molecule, termed F5446, which has an EC50 of 0.496 µmol/L for SUV39H1 enzymatic activity. H3K9me3 was enriched in the promoters of GZMB, PRF1, FASLG, and IFNG in quiescent T cells. F5446 inhibited H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8+ CTL-dependent manner in vivo Our data indicate that SUV39H1 represses CTL effector gene expression and, in doing so, confers colon cancer immune escape.