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10D1P demonstrates potent in vivo tumor growth inhibition in multiple xenograft tumor models

10D1F does not show rapid internalization in HER3 expressing cancer cell lines but activates ADCC

Supplementary Methods with References, Supplementary Figure Legends, and list of Supplementary Tables

Supplemental Table 2 - Summary of protein and gene expression data of HER family molecules, and their ligands, for the cancer cell lines used in this study

10D1F demonstrates superior inhibition of cancer cell line proliferation by potently inhibiting downstream signaling through the PI3K pathway in additional cancer cell lines

10D1F parental antibody, 10D1P, binds with high affinity and specificity to a species-conserved epitope on HER3, and Class 1 and Class 2 HER3 antibodies show different HER3 binding profiles in presence and absence of NRG1

In recent years, HER3 has increasingly been implicated in the progression of a variety of tumor types and in acquired resistance to EGFR and HER2 therapies. Whereas EGFR and HER2 primarily signal through the MAPK pathway, HER3, as a heterodimer with EGFR or HER2, potently activates the PI3K pathway. Despite its critical role, previous attempts to target HER3 with neutralizing antibodies have shown disappointing efficacy in the clinic, most likely due to suboptimal and indirect mechanisms of action that fail to completely block heterodimerization; for example, tumors can escape inhibition of ligand binding by upregulating ligand-independent mechanisms of HER3 activation. We therefore developed 10D1F, a picomolar affinity, highly specific anti-HER3 neutralizing antibody that binds the HER3 heterodimerization interface, a region that was hitherto challenging to raise antibodies against. We demonstrate that 10D1F potently inhibits both EGFR:HER3 and HER2:HER3 heterodimerization to durably suppress activation of the PI3K pathway in a broad panel of tumor models. Even as a monotherapy, 10D1F shows superior inhibition of tumor growth in the same cell lines both in vitro and in mouse xenograft experiments, when compared with other classes of anti-HER3 antibodies. This includes models demonstrating ligand-independent activation of heterodimerization as well as constitutively activating mutations in the MAPK pathway. Possessing favorable pharmacokinetic and toxicologic profiles, 10D1F uniquely represents a new class of anti-HER3 neutralizing antibodies with a novel mechanism of action that offers significant potential for broad clinical benefit.10D1F is a novel anti-HER3 antibody that uniquely binds the receptor dimerization interface to block ligand-dependent and independent heterodimerization with EGFR/HER2 and thus more potently inhibits tumor growth than existing anti-HER3 antibodies.

10D1F interacts with linear peptides from the HER3 domain II dimerization interface

Supplemental Table 3 - Toxicological data showing no adverse effects of 10D1F on the indicated hematological and biochemistry parameters in multiple rodent models

Supplemental Table 1 - Summary of the published functional mechanisms of action for anti-HER3 neutralizing antibodies in development

Dysregulation of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) plays important roles in atrial fibrillation (AF). This study aimed to identify crucial lncRNAs, miRNAs, and mRNAs in AF based on whole transcriptome sequencing.Thirty Sprague Dawley rats were randomly stratified into control and chronic intermittent hypoxia (CIH) groups (n = 15 in each). Hematoxylin-eosin staining, Masson staining, immunohistochemical assay, and western blotting were used to evaluate this model. Thereafter, atrial tissues were sent for whole transcriptome sequencing. Finally, fibrosis-related competing endogenous RNA (ceRNA) regulatory networks were built, and the relative levels of lncRNAs, miRNAs, and mRNAs were validated by real-time quantitative polymerase chain reaction (RT-qPCR) or western blotting.A CIH-induced atrial fibrosis rat model was successfully constructed. After sequencing, 268 differentially expressed lncRNAs (DELs), 20 differentially expressed miRNAs (DEMs), and 436 differentially expressed genes (DEGs) were identified. Functional analyses showed that these DEGs were associated with several processes and pathways, including "cell division," "IL-17 signaling pathway," "NOD-like receptor signaling pathway," and "cell adhesion molecules." Fibrosis-related ceRNA networks were then built, comprising five lncRNAs, seven miRNAs, and 19 DEGs. RT-qPCR and western blotting results showed that the patterns of lncRNAs (NONRATT016396.2, NONRATT001596.2, NONRATT011456.2), miRNAs (miR-10b-3p, miR-29b-3p), and mRNAs (Gng7 and Wnt2b) were consistent with sequencing analyses.The DELs, DEMs, and DEGs identified in this study may participate in atrial fibrosis processes, and the occurrence and progression of AF.

Purpose: To investigate the protective effect of icariin (ICA) on myocardial cells in spontaneously hypertensive rats (SHRs), and the mechanism involved. Methods: Twenty-four SPF-grade, 12-week-old SHRs were randomly assigned to model group and ICA group, with 12 rats/group. There were 12 Wistar-Kyoto (WKY) rats (aged 12 weeks) in the control group. Rats in ICA group were given ICA suspension (40 mg/kg) through gavage, daily for 3 months, while model rats were given equivalent volume of double distilled water (in place of ICA suspension) via gavage for 12 weeks. Blood pressure, cardiac function, left ventricular (LV) mass index, myocardial morphology and apoptosis-related protein levels were determined and compared among the four groups. Results: The myocardial cells were hypertrophic and disorderly arranged, with widened intercellular spaces. Besides, there were increases in protein expression levels of p53, caspase 3, Bok, Bax, GRP78, p-PERK, ATF-4, CHOP and DR5, while Bcl-2 protein was down-regulated. In contrast, the levels of these indicators in the ICA group were significantly better than those in the model group (p < 0.05). Conclusion: ICA reduces blood pressure in rats, but improves cardiac function and cardiomyocyte morphology by decreasing apoptosis in cardiomyocytes through down-regulation of mitochondrial and endoplasmic reticulum (ER) stress-related apoptosis pathways. Thus, icariin may be suitable for the treatment of hypertension; however, clinical trials need to be undertaken first.

Obesity is a global chronic disease epidemic that is attributed to the abnormal accumulation of lipids in adipose tissue. Astaxanthin (AST) from Haematococcus pluvialis, a natural carotenoid, exhibits antioxidant, anti-lipogenic, anti-diabetic and other potent effects. Herein, we evaluated the effect of AST to illuminate its efficacy and mechanisms in high-fat diet-fed mice. AST supplementation not only significantly decreased body weight and lipid droplet accumulation in the liver but also modulated liver function and serum lipid levels. Lipidomic analysis revealed that 13 lipids might be potential biomarkers responsible for the effects of AST in lipid reduction, such as total free fatty acids (FFAs), triacylglycerols (TGs) and cholesterol esters (CEs). The gut microbiota sequencing results indicated that AST alleviated HFD-induced gut microbiota dysbiosis by optimizing the ratio of Firmicutes to Bacteroides and inhibiting the abundance of obesity-related pathogenic microbiota while promoting the abundance of probiotics related to glucose and lipid metabolism. In addition, qRT-PCR demonstrated that AST could regulate the gene expressions of the AMPK/SREBP1c pathway by downregulating lipogenesis correlated-genes and upregulating the lipid oxidant related-gene. The present study revealed the new function of AST in regulating lipid metabolism, which provided a theoretical basis for the development of high-quality AST functional food and the application of diet active substances in obesity, as demonstrated in mice.

In recent decades, targeted drug delivery systems (TDDS) have been widely used as an ideal method of improving therapeutic effects and reducing systemic side effects of chemotherapeutic agents. Historically, a handful of methods have been developed to further improve the targeting ability of delivery systems. Thus, in this study, two methods, taking advantage of tumor characteristics, were used for the creation of a multi-targeted delivery system. The first was the fabrication of pH-sensitive micelles, lending the ability to increase drug release by exploiting the acidic tumor environment. The second method was through utilization of the surface-exposed phosphatidylserine (PS) of tumors, which is normally found in the inner leaflet in healthy cells. Using PS as a target site, PS binding peptide (PSBP-6) was conjugated to pH-sensitive mixed micelles, (consisting of poly (ethylene glycol)-b-poly (D, L-lactide) (PEG-PDLLA) and poly (ethylene glycol)-b-poly (L-histidine) (PEG-PHIS)). After successful preparation of micelles, paclitaxel (PTX), a common chemotherapeutic agent, was selected to measure drug loading capacity and encapsulation efficiency, showing 7.9% and 83.5%, respectively. The in vitro release of PTX from mixed micelles at pH 5.0, 6.5, and 7.4 was 78.1, 56.8, and 51.4%, respectively, indicating acid-triggered drug release. The PSBP-6-modified, mixed micelles exhibited significantly enhanced in vitro cytotoxicity and demonstrated more efficient cellular uptake compared to unmodified mixed micelles in the HeLa cell line. Moreover, pharmacokinetic, in vivo biodistribution, and fluorescence imaging studies showed that PSBP-6-PEG-PDLLA/PEG-PHIS mixed micelles provide prolonged time in blood circulation and enhanced tumor accumulation. These results suggest that the use of PS as a novel targeting site is advantageous, and that these new multi-targeted mixed micelles show great potential for realization of broad prospects in the targeted treatment of tumors for chemotherapeutic delivery.

Obesity is a global chronic disease epidemic that is attributed to the abnormal accumulation of lipids in adipose tissue. Astaxanthin (AST) from

It is a challenge to carry G2/M-phase specific antitumor drugs into G2/M phase of cancer cells. For this purpose, we set up a pH-sensitive mixed micellar system based on poly(ethylene glycol)-b-poly( l -histidine) (PEG-PHIS) and poly(ethylene glycol)-b-poly(D, l -lactide) (PEG-PDLLA), and we used duramycin, a highly specific phosphatidylethanolamine-binding peptide, as a G2/M-phase specific targeting ligand to modify PEG-PDLLA. The mixed micelles were characterized by dynamic light scattering, electron microscopy and the pyrene fluorescence probe method. The G2/M-phase specific anti-cancer drug paclitaxel was loaded with a size of 189 ± 5 nm. And the drug-loading content and encapsulation efficiency were 14.7 ± 0.3% and 89.1 ± 2.5%, respectively. In addition, cytotoxicity assay showed that paclitaxel-loaded mixed micelles had effective antitumor effects, and the blank mixed micelles were safe. Importantly, the micelles showed pH sensitivity and G2/M-phase specific uptake. What's more, pharmacokinetic and in vivo imaging study showed that the mixed micelles displayed a prolonged circulation time and improved targeted accumulation at tumor sites. All these results suggest that the mixed micelle was a promising G2/M-phase specific drug delivery system.

A hallmark for the Leukocyte Adhesion Deficiency 1 (LAD-1) syndrome is the defect in the β2 integrins due to mutations in the ITGB2 gene. To establish the correlation between LAD-1 genotype and phenotype, as well as better understand integrin function and structure, 19 LAD-1 missense mutants that recently identified were introduced into the pcDNA3 plasmid containing the ITGB2 cDNA. These plasmids were transfected into HEK293T cells together with that of either the αL, αM, αX subunits. These transfectants were subsequently examined for their ability in supporting the expression of the integrins and adhesion functions. Fourteen of these mutants do not support heterodimer expression of integrin LFA-1, Mac-1 and p150,95. Of the remaining 5, the integrins with the β2-D77N, β2-S453N and β2-P648L mutations have normal ligand binding activities. These “mutations” are therefore rare polymorphisms of the β2 subunit. In contrast, integrins with the β2-G150D mutation have no binding activities. LFA-1 with the β2-G716A mutation is constitutively active to bind ICAM-1 and requires only one activating agents to bind to ICAM-3. (It should be noted that wild-type LFA-1 requires one activating agent to bind ICAM-1 and two to bind ICAM-3.) Immunoprecipitation experiment with the reporter mAb KIM127 suggested that the LFA-1 bearing the β2-G716A is in the bent configuration. The epitope of the mAb H52 was mapped to the C-terminal half of the β2 hybrid domain. However, the epitope was abolished by the β2-L105P mutation, which is located in the N-terminal half. Based on this new information, it is concluded that the epitope is conformational. It is established that integrin activation is regulated by the divalent cations. However, Mg2+/EGTA (5 mM Mg2+ and 1.5 mM EGTA) had been shown to promote αLβ2 adhesion to ICAM-1 but not αMβ2 adhesion to denatured BSA. In order to determine the difference between αL and αM which contributes to Mg2+/EGTA sensitivity, a series of αL/αM chimeric integrins were constructed. It was shown that LFA-1 with αM calf-1 domain is not responsive to the Mg2+/EGTA activation. In the reverse experiment with Mac-1 bearing the αL calf-1 domain, the results were not clear cut due to the modified Mac-1 was constitutively active. DOCTOR OF PHILOSOPHY (SBS)

Background: Target-homing drug delivery systems are now gaining significant attention for use as novel therapeutic approaches in antitumor targeting for cancer therapy. Numerous targeted drug delivery systems have been designed to improve the targeting effects because these systems can display a range of favorable properties, thus, providing suitable characteristics for clinical applicability of anticancer drugs, such as increasing the solubility, and improving the drug distribution at target sites. The majority of these targeting systems are designed with respect to differences between cancerous and normal tissues, for instance, the low pH of tumor tissues or overexpressed receptors on tumor cell membranes. Due to the growing number of targeting possibilities, it is important to know the tumor-specific recognition strategies for designing novel, targeted, drug delivery systems. Herein, we identify and summarize literature pertaining to various recognition sites for optimizing the design of targeted drug delivery systems to augment current chemotherapeutic approaches. Objective: This review focuses on the identification of the recognition sites for developing targeted drug delivery systems for use in cancer therapeutics. Method: We have reviewed and compiled cancer-specific recognition sites and their abnormal characteristics within tumor tissues (low pH, high glutathione, targetable receptors, etc.), tumor cells (receptor overexpression or tumor cell membrane changes) and tumor cell organelles (nuclear and endoplasmic reticular dysregulation) utilizing existing scientific literature. Moreover, we have highlighted the design of some targeted drug delivery systems that can be used as homing tools for these recognition sites. Results and Conclusion: Targeted drug delivery systems are a promising therapeutic approach for tumor chemotherapy. Additional research focused on finding novel recognition sites, and subsequent development of targeting moieties for use with drug delivery systems will aid in the evaluation and clinical application of new and improved chemotherapeutics.

Focal adhesion (FA) proteins, kindlin‐2 and integrin‐linked kinase (ILK), regulate cell adhesion and migration. ILK interacts with and promotes kindlin‐2 targeting to FAs. Leu353 and Leu357 in kindlin‐2 have been reported to be important for the interaction between kindlin‐2 and ILK. However, the binding interface between kindlin‐2 and ILK remains unclear. Using molecular modeling and molecular dynamics simulations, we show that Asp344, Asp352, and Thr356 in kindlin‐2 and Arg243 and Arg334 in ILK kinase domain (KD) are important in kindlin‐2/ILK complex formation. Mutations that disrupt these interactions abrogate kindlin‐2 and ILK colocalization in HeLa cells. The interactions are direct based on data from pull‐down assays using purified recombinant kindlin‐2 F2‐pleckstrin homology and ILK KDs. These data provide additional insights into the binding interface between kindlin‐2 and ILK. ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) Accepted version

Preclinical Research The abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the development of neointimal hyperplasia after vascular injury. Nobiletin, a citrus bioflavonoid, exhibits anti-inflammatory and anti-oxidative activities. The present study evalutaed whether nobiletin could inhibit platelet-derived growth factor (PDGF)-BB- stimulated VSMC proliferation and migration and decrease neointimal hyperplasia in a rat carotid artery injury model. Cultured VSMCs from rat thoracic aortas were treated with nobiletin before being stimulated with 20 ng/ml PDGF-BB, and rats were subjected to carotid artery injury. Nobiletin inhibited PDGF-BB-induced VSMC proliferation and migration, attenuated reactive oxygen species (ROS) production and reduced phosphorylation of ERK1/2 and the expression of nuclear NF-κB p65 in PDGF-BB-stimulated VSMCs. Nobiletin decreased the intima area and the ratio of neointima to media in balloon-injured rat carotid arteries. Serum levels of TNF-α and IL-6 in nobiletin-treated rats were decreased. These results indicated that nobiletin could be a potential protective agent for the prevention and treatment of restenosis after angioplasty.

Leukocyte adhesion deficiency 1 (LAD-1) is caused by defects in the β2 integrin subunit. We studied 18 missense mutations, 14 of which fail to support the surface expression of the β2 integrins. Integrins with the β2-G150D mutation fail to bind ligands, possibly due to the failure of the α1 segment of the βI domain to assume an α-helical structure. Integrins with the β2-G716A mutation are not maintained in their resting states, and the patient has the severe phenotype of LAD-1. The β2-S453N and β2-P648L mutants support the expression of integrins and adhesion functions. They should be re-classified as polymorphic variants. Accepted version

We have shown that Mg/EGTA (5 mM Mg(2+) and 1.5 mM EGTA) could effectively promote the adhesion of integrin αLβ2 to its ligand ICAM-1 but could not promote that of the αMβ2 to denatured BSA. In order to determine the structural differences between αL and αM that specifically contribute to Mg/EGTA sensitivity, a series of αL/αM chimeras were constructed. Our results showed that αLβ2 with αM calf-1 domain completely lost the response to Mg/EGTA activation. In the reverse experiment, αMβ2 would require the presence of both the αL calf-1 and calf-2 domain to initiate the Mg/EGTA sensitivity. Accepted version

Preclinical Research The abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the development of neointimal hyperplasia after vascular injury. Nobiletin, a citrus bioflavonoid, exhibits anti-inflammatory and anti-oxidative activities. The present study evalutaed whether nobiletin could inhibit platelet-derived growth factor (PDGF)-BB- stimulated VSMC proliferation and migration and decrease neointimal hyperplasia in a rat carotid artery injury model. Cultured VSMCs from rat thoracic aortas were treated with nobiletin before being stimulated with 20 ng/ml PDGF-BB, and rats were subjected to carotid artery injury. Nobiletin inhibited PDGF-BB-induced VSMC proliferation and migration, attenuated reactive oxygen species (ROS) production and reduced phosphorylation of ERK1/2 and the expression of nuclear NF-κB p65 in PDGF-BB-stimulated VSMCs. Nobiletin decreased the intima area and the ratio of neointima to media in balloon-injured rat carotid arteries. Serum levels of TNF-α and IL-6 in nobiletin-treated rats were decreased. These results indicated that nobiletin could be a potential protective agent for the prevention and treatment of restenosis after angioplasty.

We showed that the αLβ2 integrin with the non-functional mutation G150D cannot be induced with Mg/EGTA to express the mAb KIM127 epitope, which reports the leg-extended conformation. We extended the study to the αIIbβ3, an integrin without an αI domain. The equivalent mutation, i.e. G161D, also resulted in an expressible, but non-adhesive αIIbβ3 integrin. An NMR study of synthetic peptides spanning the α1-α1′ helix of the β3 I domain shows that both wild-type and mutant peptides are α-helical. However, whereas in the wild-type peptide this helix is continuous, the mutant presents a discontinuity, or kink, precisely at the site of mutation G161D. Our results suggest that the mutation may lock integrin heterodimers in a bent conformation that prevents integrin activation via conformational extension., Highlights • Integrin αLβ2-G150D cannot be activated to an extended conformation. • Integrin αIIbβ3-G161D can be expressed on transfectants. • Transfectants expressing the αIIbβ3-G161D integrin cannot adhere to fibrinogen. • The Asp locks the α1-α1′ helices to into a kinked structure.