A Bispecific METxMET Antibody-Drug Conjugate with Cleavable Linker Is Processed in Recycling and Late Endosomes.

Most antibody-drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.

Inaccuracy of brachial blood pressure and its potential impact on treatment and aortic blood pressure estimation.

OBJECTIVE: Although brachial cuff SBP is universally used to guide hypertension management, it can differ significantly from intraarterial SBP. We examine the potential impacts of cuff-to-intraarterial brachial SBP (bSBP) mismatch on hypertension treatment and accuracy towards central SBP. METHODS: In 303 individuals, cuff bSBP (CUFF-bSBP) and central SBP were measured using a Mobil-o-Graph simultaneously to intraarterial bSBP (IA-bSBP) and aortic SBP. According to the difference between CUFF-bSBP and IA-bSBP, we identified three phenotypes: Underestimation (CUFF-bSBP < IA-bSBP by >10 mmHg); No Mismatch (CUFF-bSBP within 10 mmHg of IA-bSBP); Overestimation (CUFF-bSBP > IA-bSBP by >10 mmHg) phenotypes. Risk of overtreatment and undertreatment, and accuracy (ARTERY society criteria: mean difference ≤5 ±â€Š8 mmHg) were determined. A multiple linear regression model was used to assess variables associated with the bSBP difference. RESULTS: Underestimation (n = 142), No Mismatch (n = 136) and Overestimation (n = 25) phenotypes had relatively similar characteristics and CUFF-bSBP (124 ±â€Š17, 122 ±â€Š14, 127 ±â€Š19 mmHg, P = 0.19) but different aortic SBP (133 ±â€Š21, 120 ±â€Š16, 112 ±â€Š18 mmHg, P < 0.001). In the underestimation phenotype, 59% were at risk of undertreatment (14% in No Mismatch), whereas 50% in the Overestimation phenotype were at risk of overtreatment (17% in No Mismatch). CUFF-bSBP accurately estimated aortic SBP only in the No Mismatch Group (mean difference 1.6 ±â€Š8.2 mmHg) whereas central BP never met the accuracy criteria. Male sex, higher height and active smoking were associated with lesser underestimation of bSBP difference. CONCLUSION: The brachial cuff lacks accuracy towards intraarterial BP in a significant proportion of patients, potentially leading to increased risks of BP mismanagement and inaccurate determination of central BP. This illustrates the need to improve the accuracy of cuff-based BP monitors.

A Biparatopic Antibody-Drug Conjugate to Treat MET-Expressing Cancers, Including Those that Are Unresponsive to MET Pathway Blockade.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%-5% of lung cancers), and acquired resistance limits response durability. An antibody-drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

A Biparatopic Antibody That Modulates MET Trafficking Exhibits Enhanced Efficacy Compared with Parental Antibodies in MET-Driven Tumor Models.

PURPOSE: Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy. EXPERIMENTAL DESIGN: Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors. RESULTS: In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling. CONCLUSIONS: Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.

Circular RNA in Aging and Age-Related Diseases.

Circular RNAs (circRNAs) are widely present and participate in a variety of biological regulatory activities as a novel type of endogenous noncoding RNA molecule. With advances in RNA structure and function analysis, it was found that circRNAs are present in a myriad of life processes and longevity in model organisms such as mice, flies, and worms. Accumulating evidence indicates the involvement of circRNAs in regulation of age-related pathologies such as cancer, diabetes, cardiovascular disorders, and neurodegenerative disease, suggesting that circRNAs may have great potential implications in clinical and research fields. In this chapter, we review recent advances in circRNA functions and mechanisms and discuss their roles in aging and age-related diseases. It will provide insight into the regulatory roles of circRNAs in aging and age-related diseases.

Characterization of oromotor and limb motor dysfunction in the DJ1 -/- model of Parkinson disease.

Parkinson disease (PD) is devastating to sensorimotor function that includes cranial/oromotor and limb motor deficits. However, the onset, progression, and neural correlates of PD-related dysfunctions are poorly understood. To address this gap, we used a genetic rat model of PD, DJ1 -/-, and hypothesized that motor deficits would manifest early in the disease process, be progressive in nature, and be related to pathologies in brainstem structures associated with sensorimotor function. The present study compares homozygous DJ1 -/- male rats to age-matched wild type controls. Progressive cranial sensorimotor function (ultrasonic vocalizations and tongue motor performance) and limb motor function (tapered balance beam) was analyzed at 2, 4, 6, and 8 months of age. Additionally, tyrosine hydroxylase cell counts were performed in the locus coeruleus and correlated to behavioral measures. We found that compared to wild type controls, DJ1 -/- show deficits in ultrasonic vocalizations as well as oromotor (tongue) deficits that were progressive. Overtime, DJ1 -/- rats cross a tapered balance beam with significantly decreased speed of traversal. Additionally, in the DJ1 -/-, tyrosine hydroxylase positive cells in the locus coeruleus are significantly reduced and are negatively correlated to oromotor behaviors. Characterizing the DJ1 -/- model of PD provides important foundational work necessary to define behavioral and early-onset biomarkers that parallels early-stage PD pathology in humans.

Exercise Effects on Early Vocal Ultrasonic Communication Dysfunction in a PINK1 Knockout Model of Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a complex neurodegenerative disease with vocal communication deficits that manifest early, progress, and are largely resistant to medical interventions; however, they do respond to exercise-based speech and voice therapies. OBJECTIVE AND METHODS: To study how exercise-based vocal treatment can affect the progression of communication deficits related to PD, we studied ultrasonic vocalizations (USVs) in rats with homozygous knockout (-/-) of PINK1, a gene mutation known to cause PD, under the manipulation of a behavioral vocal exercise paradigm that allows us to precisely control dose and timing of exercise in the prodromal (prior to diagnosis) stages. RESULTS: We show that intensive vocal-training rescues frequency range and intensity deficits as well as leads to an increase in call complexity and duration of calls compared to sham-training; however, over time this training regime loses significant effect as the disease progresses. We also show effects of frequent handling and conspecific (male-female) interaction in the sham-training group as they demonstrated significantly higher call rate, intensity, frequency range, and call complexity compared to rats without any form of training and consequently less handling/interaction. Further, we confirm that this model exhibits progressive gross motor deficits that indicate neurodegeneration. DISCUSSION: This study suggests that the evolving nature of vocal communication deficits requires an adjustment of therapy targets and more intensive training over the course of this progressive disease and demonstrates the importance of frequent social experiences.

ERBB3/HER2 signaling promotes resistance to EGFR blockade in head and neck and colorectal cancer models.

EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies.

A prospective evaluation of the incidence of adverse events in nurse-administered moderate sedation guided by sedation scores or Bispectral Index.

BACKGROUND: Moderate sedation is routinely performed in patients undergoing minor therapeutic and diagnostic procedures outside the operating room. The level of sedation is often monitored by sedation nurses using clinical criteria, such as sedation scores. The Bispectral Index (BIS) is derived from changes in the electroencephalograph profile that may provide an objective measure of the level of sedation. In this prospective observational study, we investigated whether using BIS values to guide sedative drug administration influences the level of sedation and the incidence of adverse events compared with using Ramsay sedation scale (RSS) only in nurse-administered moderate sedation. We hypothesized that both depth of sedation and the incidence of adverse events related to oversedation would decrease when sedation nurses used BIS values to help guide sedative drug administration. METHODS: Sedation care was provided by trained sedation nurses under the supervision of a physician performing the procedure. The sedation regimen was initiated with IV midazolam 1 to 2 mg and fentanyl 50 mcg or hydromorphone 0.2 mg. Additional small boluses of midazolam, fentanyl, or hydromorphone were administered to maintain an RSS of 2 to 3 (cooperative, oriented, and responding to verbal command). Propofol was not used. Information including patient demographics, type of procedure, medication administered, RSS, and rates of adverse events was recorded by the sedation nurses for each patient on a computer-readable form. The study was divided into 3 phases. In phase 1 (baseline, 6 months' duration), baseline data on sedation practice were prospectively collected. There was no change from standard of care for all patients except that each patient had a BIS sensor attached, but the monitor was covered and nurses were blinded to the BIS values. In phase 2 (training, 3 months), the sedation nurses received comprehensive education on the use of BIS to guide sedative drug administration, pharmacology of commonly used drugs, and methods for rescue from oversedation. The recommended BIS range for moderate sedation was 75 to 90. Adequate training of all sedation nurses on the use of BIS was documented. In phase 3 (implementation, 6 months), the BIS values were used to guide drug administration. RESULTS: Data were available on 1766 patients (999 and 767 patients in phases 1 and 3, respectively). Most of the procedures were colonoscopies, upper gastrointestinal endoscopies, examinations under anesthesia, endoscopic retrograde cholangiopancreatography, and central venous access catheter placements. No differences in the demographics between the 2 groups were observed. The RSS was inversely associated with the BIS value, r = -0.16 (95% confidence interval, -0.19 to -0.12; P < 0.00001). An RSS of 2 to 3 was maintained in 94% of patients in phase 1 and 96% of patients in phase 3 The mean (±SD) BIS values were 80.9 ± 6.8 in phase 1 and 80.4 ± 6.5 in phase 3. The number of sedation-related adverse events was lower in our sample when BIS was used, with an odds ratio of 0.41 (95% confidence interval, 0.28-0.62; P < 0.0001), and patients with restlessness had a lower BIS value than those without this symptom (P < 0.0001). No serious adverse events or deaths were reported. CONCLUSIONS: Nurse-administered moderate sedation using midazolam and fentanyl was usually associated with appropriate levels of sedation as assessed by both the RSS and BIS with an overall low incidence of adverse events. The use of BIS did not change the mean level of sedation significantly, although the number of sedation-related adverse events appears to be lower when BIS was used.

Perivascular tissue factor is down-regulated following cutaneous wounding: implications for bleeding in hemophilia.

Healing of skin wounds is delayed in hemophilia B (HB) mice. HB mice do not bleed excessively at wounding, yet rebleed hours to days later. Tissue factor (TF) expression is up-regulated by inflammatory cytokines and has been linked to angiogenesis. We hypothesized that impaired thrombin generation in HB leads to impaired TF expression following injury. Punch biopsies were placed on wild-type (WT) and HB mice. Tissues from wound sites were immunostained for TF. Blood vessels are normally surrounded by a coat of pericytes expressing TF. Surprisingly, within a day after wounding TF disappeared from around nearby vessels; returning after 8 days in WT and 10 days in HB mice. The granulation tissue filling the wound during healing also lacked TF around angiogenic vessels. Thus, perivascular TF expression is down-regulated during wound healing. This may prevent thrombosis of neovessels during angiogenesis but renders hemophiliacs vulnerable to hemorrhage during healing.