Anti-4-1BB×PD-L1 Bispecific Antibody Reinvigorates Tumor-Specific Exhausted CD8+ T Cells and Enhances the Efficacy of Anti-PD-1 Blockade.

PURPOSE: To overcome the limited efficacy of immune checkpoint blockade, there is a need to find novel cancer immunotherapeutic strategies for the optimal treatment of cancer. The novel anti-4-1BB×PD-L1 bispecific antibody-ABL503 (also known as TJ-L14B)-was designed to simultaneously target PD-L1 and 4-1BB, and demonstrated strong antitumor T-cell responses without considerable toxicity. Here, we investigated how the combination of ABL503 and anti-PD-1 blockade affected the reinvigoration of exhausted tumor-infiltrating CD8+ T cells (CD8+ TILs) and anti-tumor efficacy. EXPERIMENTAL DESIGN: Single cell suspensions of hepatocellular carcinoma and ovarian cancer from treatment-naive patients were used for immunophenotyping of CD8+ TILs and in vitro functional assays. Humanized hPD-1/hPD-L1/h4-1BB triple knock-in mice were used to evaluate the effects of ABL503 and anti-PD-1 blockade in vivo. RESULTS: We observed that ABL503 successfully restored the functions of 4-1BB+ exhausted CD8+ TILs, which were enriched for tumor-specific T cells but unresponsive to anti-PD-1 blockade. Importantly, compared to anti-PD-1 blockade alone, the combination of ABL503 and anti-PD-1 blockade further enhanced the functional restoration of human CD8+ TILs in vitro. Consistently, the combination of ABL503 with anti-PD-1 in vivo significantly alleviated tumor growth, and induced enhanced infiltration and activation of CD8+ TILs. CONCLUSIONS: ABL503-a PD-L1 and 4-1BB dual-targeting bispecific antibody-elicits pronounced additive tumor growth inhibition, with increased infiltration and functionality of exhausted CD8+ T cells, which in turn enhances the anti-cancer effects of anti-PD-1 blockade. These promising findings suggest that ABL503 (TJ-L14B) in combination with PD-1 inhibitors will likely further enhance therapeutic benefit in clinical trials.

Toxicology and safety study of L-tryptophan and its impurities for use in swine.

L-tryptophan, an essential amino acid for physiological processes, metabolism, development, and growth of organisms, is widely utilized in animal nutrition and human health as a feed additive and nutritional supplement, respectively. Despite its known benefits, safety concerns have arisen due to an eosinophilia-myalgia syndrome (EMS) outbreak linked to L-tryptophan consumed by humans. Extensive research has established that the EMS outbreak was caused by an L-tryptophan product that contained certain impurities. Therefore, safety validations are imperative to endorse the use of L-tryptophan as a supplement or a feed additive. This study was conducted in tertiary hybrid [(Landrace × Yorkshire) × Duroc] pigs to assess general toxicity and potential risks for EMS-related symptoms associated with L-tryptophan used as a feed additive. Our investigation elucidated the relationship between L-tryptophan and EMS in swine. No mortalities or clinical signs were observed in any animals during the administration period, and the test substance did not induce toxic effects. Hematological analysis and histopathological examination revealed no changes in EMS-related parameters, such as eosinophil counts, lung lesions, skin lesions, or muscle atrophy. Furthermore, no test substance-related changes occurred in other general toxicological parameters. Through analyzing the tissues and organs of swine, most of the L-tryptophan impurities that may cause EMS were not retained. Based on these findings, we concluded that incorporating L-tryptophan and its impurities into the diet does not induce EMS in swine. Consequently, L-tryptophan may be used as a feed additive throughout all growth stages of swine without safety concerns.

The homeoprotein HOXB2 limits triple-negative breast carcinogenesis via extracellular matrix remodeling.

Homeobox genes and their encoded DNA-binding homeoproteins are master regulators of development. Consequently, these homeotic elements may regulate key steps in cancer pathogenesis. Here, using a combination of in silico analyses of large-scale patient datasets, in vitro RNAi phenotyping, and in vivo validation studies, we investigated the role of HOXB2 in different molecular subtypes of human breast cancer (BC). The gene expression signatures of HOXB2 are different across distinct BC subtypes due to various genetic alterations, but HOXB2 was specifically downregulated in the aggressive triple-negative subtype (TNBC). We found that the reduced expression of HOXB2 was correlated with the metastatic abilities (epithelial-to-mesenchymal transition) of TNBC cells. Further, we revealed that HOXB2 restrained TNBC aggressiveness by ECM organization. HOXB2 bound to the promoter regions of MATN3 and ECM2 and regulated their transcription levels. Forced expression of HOXB2 effectively prevented TNBC progression and metastasis in a mouse xenograft model. Reduction of HOXB2 and the HOXB2/MATN3/ECM2 transcriptional axis correlated with poor survival in patients with various cancers. Further, we found the long non-coding RNA HOXB-AS1 in complex with SMYD3, a lysine methyltransferase, as an epigenetic switch controlling HOXB2 expression. Overall, our results indicate a tumor-suppressive role of HOXB2 by maintaining ECM organization and delineate potential clinical utility of HOXB2 as a marker for TNBC patients.

Toxicology and safety study of L-tryptophan and its impurities for use in broiler feed.

L-tryptophan has been utilized as a feed additive in animal nutrition to improve growth performance, as well as a dietary supplement to alleviate various emotional symptoms in humans. Despite its benefits, concerns regarding its safety arose following the outbreak of eosinophilia-myalgia syndrome (EMS) among individuals who consumed L-tryptophan. The causative material of EMS was determined to be not L-tryptophan itself, but rather L-tryptophan impurities resulting from a specific manufacturing process. To investigate the effect of L-tryptophan and its impurities on humans who consume meat products derived from animals that were fed L-tryptophan and its impurities, an animal study involving broiler chickens was conducted. The animals in test groups were fed diet containing 0.065%-0.073% of L-tryptophan for 27 days. This study aimed to observe the occurrence of toxicological or EMS-related symptoms and analyze the residues of L-tryptophan impurities in meat products. The results indicated that there was no evidence of adverse effects associated with the test substance in the investigated parameters. Furthermore, most of the consumed EMS-causing L-tryptophan impurities did not remain in the meat of broiler chickens. Thus, this study demonstrated the safety of L-tryptophan and some of its impurities as a feed additive.

Regulation of sexually dimorphic placental adaptation in LPS exposure-induced intrauterine growth restriction.

BACKGROUND: Sexual dimorphism in placental physiology affects the functionality of placental adaptation during adverse pregnancy. Defects of placental function compromise fetal programming, affecting the offspring's adult life. However, studies focusing on the relationship between sex-specific placental adaptation and consequent fetal maldevelopment under sub-optimal uterus milieu are still elusive. METHODS: Here, we investigated the effects of maternal lipopolysaccharide (LPS) exposure between placental sex. Pregnant ICR mice received intraperitoneal injection of phosphate-buffered saline or 100, 200, and 400 µg/kg LPS on the gestational day (GD) 15.5. To determine whether prenatal maternal LPS exposure resulted in complicated pregnancy outcomes, survival rate of embryos was calculated and the growth of embryos and placentas was examined. To elucidate global transcriptomic changes occurring in the placenta, total RNA-sequencing (RNA-seq) was performed in female and male placentas. RESULTS: LPS administration induced placental inflammation in both sexes at GD 17.5. Prenatal infection resulted in growth retardation in both sexes of embryos, and especially more prevalently in male. Impaired placental development was observed in a sex-specific manner. LPS 400 µg/kg reduced the percentage area of the labyrinth in females and junctional zone in males, respectively. RNA-sequencing revealed widespread sexually dimorphic transcriptional changes in placenta. In particular, representative changes were involved in biological processes such as trophoblast differentiation, nutrient/ion transporter, pregnancy, and immune system. CONCLUSIONS: Our results present the sexually dimorphic responses of placental physiology in intrauterine growth restriction model and provide tentative relationship further to be elucidated between sex-biased placental functional change and long-term effects on the offspring's later life.

Evaluation of the 52-week chronic toxicity of a novel phthalate-free plasticizer, Eco-DEHCH (bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate), in Han Wistar rats.

Phthalate esters (PEs) are the most widely used class of plasticizers. Several PEs, however, were found to have adverse effects on the health of animals. A new phthalate-free plasticizer, Eco-DEHCH (bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate), was recently developed as an ecofriendly replacement for phthalate plasticizers and to be less harmful to organisms. The present study evaluated the long-term toxicity of Eco-DEHCH in Wistar Han rats to explore adverse effects and predict hazardous potential to humans. Forty male and forty female Wistar Han rats were exposed to Eco-DEHCH in dietary feed for 52 weeks, and their hematologic, coagulation, and serum biochemical parameters were monitored. The rats were subjected to close clinical, ophthalmic, and histopathologic examinations and urinalysis throughout the consumption of Eco-DEHCH. The effects of this plasticizer on food consumption and organ weight were also determined. Chronic exposure to Eco-DEHCH was generally safe, although it also resulted in α2u-globulin accumulation, a parameter with no human relevance. In conclusion, Eco-DEHCH can serve as a safe and promising alternative plasticizer.

Akt1 Decreases Gcn5 Protein Stability through Regulating The Ubiquitin-Proteasome Pathway in Mouse Embryonic Fibroblasts.

General control non-derepressible 5 (Gcn5) is a member of histone acetyltransferase (HAT) that plays key roles during embryogenesis as well as in the development of various human cancers. Gcn5, an epigenetic regulator of Hoxc11, has been reported to be negatively regulated by Akt1 in the mouse embryonic fibroblasts (MEFs). However, the exact mechanism by which Akt1 regulates Gcn5 is not well understood. Using protein stability chase assay, we observed that Gcn5 is negatively regulated by Akt1 at the post-translational level in MEFs. The stability of Gcn5 protein is determined by the competitive binding with the protein partner that interacts with Gcn5. The interaction of Gcn5 and Cul4a-Ddb1 complex predominates and promotes ubiquitination of Gcn5 in the wild-type MEFs. On the other hand, in the Akt1-null MEFs, the interaction of Gcn5 and And-1 inhibits binding of Gcn5 and Cul4a-Dbd1 E3 ubiquitin ligase complex, thereby increasing the stability of the Gcn5 protein. Taken together, our study indicates that Akt1 negatively controls Gcn5 via the proteasomal degradation pathway, suggesting a potential mechanism that regulates the expression of Hox genes.

Comparative Analyses of Inflammatory Response and Tissue Integration of 14 Hyaluronic Acid-Based Fillers in Mini Pigs.

PURPOSE: Hyaluronic acid (HA)-based dermal fillers have been approved for various clinical indications, both cosmetic and medical. Previous studies that have assessed the performance of HA dermal fillers have primarily focused on evaluating filler durability, and only a few have studied their distribution within the tissues. The present study aimed to compare tissue integration of various types of HA dermal fillers having different clinical indications and varying injection depths. METHODS: To examine the local inflammatory response and distribution pattern of 14 HA dermal fillers (six Neuramis [NEU], one Belotero [BEL], three Juvéderm [JUV], and four Restylane [RES]), each product was injected intradermally and subcutaneously at the backs of two male miniature pigs. Histopathological evaluation and visual examination of the tissue sections were conducted 1 and 4 weeks after injection. RESULTS: Mean inflammatory cell infiltration scores tended to be lower in response to fillers from the NEU and BEL series than to those from the JUV and RES series after intradermal and subcutaneous injection. Furthermore, the inflammatory response to fillers with higher physicochemical properties specifically designed for injection into deeper layers of the skin tended to be slightly higher than those designated for injection into more superficial layers. There was no significant difference in tissue integration according to clinical indication and injection depth, although fillers from the NEU and BEL series exhibited better tissue integration than those from the JUV and RES series. CONCLUSION: Our findings not only suggest that the local inflammatory response and tissue integration differ across HA dermal filler products, but also that these parameters could vary according to the recommended clinical indication and injection depth of the products.

Depletion of CTCF disrupts PSG gene expression in the human trophoblast cell line Swan 71.

Pregnancy-specific glycoproteins (PSGs) are fetal proteins secreted by the placenta during pregnancy. The PSG level in maternal serum is an indicator of risk for pregnancy complications. However, little is known about the molecular mechanisms underlying PSG gene expression. Recently, the importance of epigenetic regulation of placental genes has been emphasized in the study of developmental defects and placental disease. In this study, the role of the CCCTC-binding factor (CTCF) in regulation of PSG expression was investigated to better understand the epigenetic regulatory mechanisms of the PSG genes. Inhibition of CTCF expression disturbed transcription of several PSG genes: PSG1, PSG2, PSG4, PSG5, PSG8, and PSG9 were upregulated and PSG6 and PSG11 were downregulated. These transcriptional changes were correlated with decreased CTCF binding and changes in histone modification at the PSG promoters. Our data demonstrate that CTCF is a potential mediator in the regulation of PSG gene expression.

Elevated GCN5 expression confers tamoxifen resistance by upregulating AIB1 expression in ER-positive breast cancer.

Approximately 70% of breast cancers are estrogen receptor (ER)-positive and treated with endocrine therapy. A commonly used treatment agent, tamoxifen, shows high efficacy for improving prognosis. However, approximately one-third of patients treated with tamoxifen develop resistance to this drug. Here, we investigated the function of general control non-derepressible 5 (GCN5) and its downstream effectors in tamoxifen-resistant (TamR) breast cancer. TamR-MCF7 breast cancer cells maintained high GCN5 levels due to its attenuated proteasomal degradation. GCN5 overexpression upregulated amplified in breast cancer 1 (AIB1) expression, resulting in decreased p53 stability and tamoxifen resistance. Conversely, the sensitivity of GCN5-AIB1-overexpressing MCF7 cells to tamoxifen was restored by forced p53 expression. An in vivo study demonstrated a positive correlation between GCN5 and AIB1 and their contribution to tamoxifen resistance. We concluded that GCN5 promotes AIB1 expression and tamoxifen resistance in breast cancer by reducing p53 levels, suggesting the utility of GCN5 and its downstream effectors as therapeutic targets to either prevent or overcome tamoxifen resistance in breast cancer.

Transcriptional activation of EGFR by HOXB5 and its role in breast cancer cell invasion.

HOX genes are transcription factors that play important roles in body patterning and many cellular processes during embryonic, fetal, and adult development. Given their important function in normal tissues, it is reasonable to assume that abnormal expression of HOX genes in adults could lead to serious diseases such as cancer. Our previous study reported HOXB5 to be significantly up-regulated in breast cancer, and its expression was found to be associated with tumor cell proliferation and invasion. Furthermore, the epidermal growth factor receptor (EGFR), a cellular tyrosine kinase that plays an important role in breast cancer progression, was found significantly up-regulated by HOXB5 in ER-positive breast cancer cells. In the present study, we demonstrated that HOXB5 regulates EGFR expression at the transcriptional level by directly binding to its promoter region and promotes phosphorylation of EGFR as well as its downstream effectors. Patients with ER-positive breast cancer, having high co-expression of HOXB5 and EGFR, had poor prognosis than those with low expression. Knockdown studies validated a key role played by EGFR in the HOXB5-induced invasion of breast cancer cells. These results suggest that targeting EGFR could be an effective strategy to treat breast cancer in patients with high HOXB5 expression.