Engineering CD3/CD137 dual specificity into a DLL3-targeted T-cell engager enhances T-cell infiltration and efficacy against small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICIs) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all SCLC patients are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared to a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.

Engineering CD3/CD137 Dual Specificity into a DLL3-Targeted T-Cell Engager Enhances T-Cell Infiltration and Efficacy against Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.

Combination of T cell-redirecting bispecific antibody ERY974 and chemotherapy reciprocally enhances efficacy against non-inflamed tumours.

Identifying a strategy with strong efficacy against non-inflamed tumours is vital in cancer immune therapy. ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody that recognizes glypican-3 and CD3. Here we examine the combination effect of ERY974 and chemotherapy (paclitaxel, cisplatin, and capecitabine) in the treatment of non-inflamed tumours in a xenograft model. ERY974 monotherapy shows a minor antitumour effect on non-inflamed NCI-H446 xenografted tumours, as infiltration of ERY974-redirected T cells is limited to the tumour-stromal boundary. However, combination therapy improves efficacy by promoting T cell infiltration into the tumour centre, and increasing ERY974 distribution in the tumour. ERY974 increases capecitabine-induced cytotoxicity by promoting capecitabine conversion to its active form by inducing thymidine phosphorylase expression in non-inflamed MKN45 tumour through ERY974-induced IFNγ and TNFα in T cells. We show that ERY974 with chemotherapy synergistically and reciprocally increases antitumour efficacy, eradicating non-inflamed tumours.

A case of spontaneous autoimmune skin disease in a cynomolgus monkey (Macaca fascicularis).

Pemphigus is an autoimmune blistering disease characterized by lesions on the skin and mucous membranes. To date, no spontaneous cases of this disease have been reported in cynomolgus monkeys. This report describes the histopathological characteristics of spontaneous pemphigus in a cynomolgus monkey. Macroscopically, redness and scaling with pruritus were observed on the skin of the entire body. Histopathologically, the epidermis showed intercellular edema, and eosinophils and mononuclear cells infiltrated the epidermis. There was no obvious acantholysis in the epidermis. The perivascular area showed edema, and eosinophils and mononuclear cells infiltrated the vessels in the dermis. Immunohistochemically, the intercellular area in the epidermis was positive for Immunoglobulin G and Complement component 3. Serologically, anti-desmoglein 1 and desmoglein 3 antibodies in the serum were negative. From these findings, this case was diagnosed as an autoimmune skin disease, suspected to be pemphigus, and concluded as lesions being similar to those in human "pemphigus herpetiformis".

Antibody to CD137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.

Diet modification and its influence on metabolic and related pathological alterations in the SHR/NDmcr-cp rat, an animal model of the metabolic syndrome.

SHR/NDmcr-cp (SHR/NDcp) rats, which carry a nonsense mutation of the leptin receptor gene, are known to spontaneously develop hypertension, obesity and hyperlipidemia, and have therefore found use as an animal model of the metabolic syndrome and type 2 diabetes. However, some recent studies on SHR/NDcp rats revealed only mild elevation of blood glucose levels. To investigate whether metabolic factors including blood glucose and histopathological alterations of SHR/NDcp rats deteriorate with a diabetogenic diet, biochemical and histopathological examinations were conducted with animals fed normal or diabetogenic diets for 20 weeks. SHR/NDcp rats receiving the normal diet displayed obesity, hypertension, hyperlipidemia, and mild elevation of blood glucose and HbA1c levels. Urinary glucose excretion was noted in only 1 out of 6 animals. Histologically, macro- and micro-vesicular steatosis in the liver, glomerular and tubular damages in the kidney and islet hyperplasia mainly of beta cells in the pancreas were characteristically noted. In SHR/NDcp rats fed the diabetogenic diet, obesity was more severe, with higher blood glucose and HbA1c levels, increased numbers of animals with urinary glucose excretion, and more pronounced hepatic steatosis and renal tubular changes. However, elevation of blood glucose levels and urinary glucose excretion proved transient. These observations indicate that the diabetic state and associated histopathological alterations in SHR/NDcp rats are exacerbated by feeding a diabetogenic diet, but the effects are limited. Elevated islet function with compensative insulin secretion might be related to amelioration of the hyperglycemic state. Further diet modification could be needed to induce a more prominent and persistent diabetic state in SHR/NDcp rats.

A Hepatocellular Adenoma in a Diet-induced Obese Mouse.

A hepatic nodule was noted in a C57BL/6J mouse with diet-induced obesity at 53 weeks of age. Macroscopically, a protruding yellowish white nodule was observed on the visceral surface of the left lateral lobe. Light microscopy demonstrated clear demarcation from the compressed adjacent parenchyma, with loss of the distinct lobular pattern. The proliferating cells of the lesion varied in shape and showed cellular atypia and prominent nucleoli along with vacuoles of various sizes. Some of the cells contained various-sized eosinophilic inclusion bodies in their cytoplasm, and electron microscopy revealed the presence of lipid droplets in the rough endoplasmic reticulum. Eosinophilic inclusions were observed as electron dense granular material in the rough endoplasmic reticulum, with one or a few low density central cores. A diagnosis of hepatocellular adenoma was made based on these findings.

Induction of suppressors of cytokine signaling by the trichothecene deoxynivalenol in the mouse.

Deoxynivalenol (DON), a trichothecene mycotoxin found in grains and cereal-based foods worldwide, impairs weight gain in experimental animals but the underlying mechanisms remain undetermined. Oral exposure to DON induces rapid and transient upregulation of proinflammatory cytokine expression in the mouse. The latter are known to induce several suppressors of cytokine signaling (SOCS), some of which impair growth hormone (GH) signaling. We hypothesized that oral exposure to DON will induce SOCS expression in the mouse. Real-time PCR and cytokine bead array revealed that oral gavage with DON rapidly (1 h) induced tumor necrosis factor-alpha and interleukin-6 mRNA and protein expression in several organs and plasma, respectively. Upregulation of mRNAs for four well-characterized SOCS (CIS [cytokine-inducible SH2 domain protein], SOCS1, SOCS2, and SOCS3) was either concurrent with (1 h) or subsequent to cytokine upregulation (2 h). Notably, DON-induced SOCS3 mRNAs in muscle, spleen and liver, with CIS1, SOCS1, and SOCS2 occurring to a lesser extent. Hepatic SOCS3 mRNA was a very sensitive indicator of DON exposure with SOCS3 protein being detectable in the liver well after the onset of cytokine decline (5 h). Furthermore, hepatic SOCS upregulation was associated with about 75% suppression of GH-inducible insulin-like growth factor acid labile subunit. Taken together, DON-induced cytokine upregulation corresponded to increased expression of several SOCS, and was associated with suppression of GH-inducible gene expression in the liver.

Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage.

Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultant fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.

Purification and comparative neurotoxicity of the trichothecenes satratoxin G and roridin L2 from Stachybotrys chartarum.

Satratoxin G (SG), a macrocyclic trichothecene produced by Stachybotrys chartarum, induces apoptosis in cultured neuronal cells as well as nasal olfactory sensory neurons (OSN) in the nose and brain of mice exposed intranasally to this toxin. The purposes of this study were to (1) develop a facile method for production and purification of both SG and its putative biosynthetic precursor, roridin L2 (RL2), from S. chartarum cultures and (2) compare their relative neurotoxicity in vitro and in vivo. Stachybotrys chartarum 29-58-17 was cultured in Fernbach flasks on rice (5 x 10(5) spores/250 g rice) for 4 to 6 wk. Following extraction with acetonitrile, the extract was dried, dissolved in dichloromethane, and subjected to Michel-Miller silica-gel chromatography using a stepwise acetonitrile-dichloromethane gradient with SG and RL2 eluting in the 30 and 40% acetonitrile fractions, respectively. Purification of the two compounds was completed by C18 semipreparative reverse-phase liquid chromatography using an acetonitrile-water gradient, and purity was confirmed by electrospray ionization/collision-induced dissociation (ESI-CID) tandem mass spectroscopy. Although viability significantly decreased in PC-12 neuronal cells treated with 10 to 25 ng/ml of SG, RL2 at concentrations up to 1000 ng/ml was not toxic. Flow cytometry and agarose DNA fragmentation assays revealed that SG at 10 to 25 ng/ml induced apoptotic death in the PC-12 cells, while RL2 at concentrations up to 1000 ng/ml was without effect. In a similar fashion, intranasal exposure of mice (female B6C3F1) to SG at 100 microg/kg body weight (bw) induced marked OSN apoptosis and atrophy of the olfactory epithelium, whereas RL2 at the equivalent dose did not exhibit toxicity. Taken together, an optimized protocol for production and isolation of trichothecenes from S. chartarum cultures is described and further demonstrates that while the macrocyclic SG was neurotoxic in vitro and in vivo, its biosynthetic precursor, RL2, was nontoxic.