Regression of EGFR positive established solid tumors in mice with the conditionally active T cell engager TAK-186.

BACKGROUND: Despite clinical success with T cell engagers (TCEs) targeting hematological malignancies, achieving a safe and efficacious dose in patients with solid tumors remains challenging. Due to potency, low levels of target antigen expression on normal tissues may not be tolerated. To overcome this, we engineered a novel conditionally active TCE design called COBRA (Conditional Bispecific Redirected Activation). Administered as prodrugs, COBRAs bind to cell surface antigens on both normal and tumor tissues but are preferentially activated within the tumor microenvironment. METHODS: A COBRA was engineered to target EGFR, TAK-186. The potency of precleaved TAK-186 relative to a non-cleavable control was assessed in vitro. Mice bearing established solid tumors expressing a range of EGFR levels were administered a single bolus of human T cells, and concurrently treated with TAK-186 and associated controls intravenously. We assessed the plasma and tumor exposure of intact and cleaved TAK-186. RESULTS: TAK-186 shows potent redirected T cell killing of antigen expressing tumor cells. In vivo efficacy studies demonstrate regressions of established solid tumors, dependent on intratumoral COBRA cleavage. Pharmacokinetic studies reveal TAK-186 is stable in circulation, but once activated is rapidly cleared due to loss of its albumin-binding half-life extension domain. CONCLUSIONS: The studies shown support the advancement of TAK-186, and the pursuit of additional COBRA TCEs for the treatment of solid tumors.

Structural arrangement of the VH and VL domains in the COBRA™ T-cell engaging single-chain diabody.

BACKGROUND: COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding variable fragment heavy chain (VH) and variable fragment light chain (VL) and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi into single-chain variable fragments (Fv) is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. METHODS: We analyzed the structure of an epidermal growth factor receptor (EGFR) COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). RESULTS: We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Nonenzymatic posttranslational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. CONCLUSION: Here, we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.

COBRA™: a highly potent conditionally active T cell engager engineered for the treatment of solid tumors.

Conditionally active COBRA™ (COnditional Bispecific Redirected Activation) T cell engagers are engineered to overcome the limitations of inherently active first-generation T cell engagers, which are unable to discern between tumor and healthy tissues. Designed to be administered as prodrugs, COBRAs target cell surface antigens upon administration, but engage T cells only after they are activated within the tumor microenvironment (TME). This allows COBRAs to be preferentially turned on in tumors while safely remaining inactive in healthy tissue. Here, we describe the development of the COBRA design and the characterization of these conditionally active T cell engagers. Upon administration COBRAs are engineered to bind to tumor-associated antigens (TAAs) and serum albumin (to extend their half-life in circulation), but are inhibited from interacting with the T cell receptor complex signaling molecule CD3. In the TME, a matrix metalloproteinase (MMP)-mediated linker cleavage event occurs within the COBRA construct, which rearranges the molecule, allowing it to co-engage TAAs and CD3, thereby activating T cells against the tumor. COBRAs are conditionally activated through cleavage with MMP9, and once active are highly potent, displaying sub-pM EC50s in T cell killing assays. Studies in tumor-bearing mice demonstrate COBRA administration completely regresses established solid tumor xenografts. These results strongly support the further characterization of the novel COBRA design in preclinical development studies.

Prospective Randomized Controlled Trial on the Efficacy of Continuous Positive Airway Pressure and Adaptive Servo-Ventilation in the Treatment of Chronic Complex Insomnia.

BACKGROUND: Complex insomnia, the comorbidity of chronic insomnia and obstructive sleep apnea (OSA), is a common sleep disorder, but the OSA component, whether presenting overtly or covertly, often goes unsuspected and undiagnosed due to a low index of suspicion. Among complex insomniacs, preliminary evidence demonstrates standard CPAP decreases insomnia severity. However, CPAP causes expiratory pressure intolerance or iatrogenic central apneas that may diminish its use. An advanced PAP mode-adaptive servo-ventilation (ASV)-may alleviate CPAP side-effects and yield superior outcomes. METHODS: In a single-site protocol investigating covert complex insomnia (ClinicalTrials.gov identifier: NCT02365064), a low index of suspicion for this comorbidity was confirmed by exclusion of 455 of 660 eligible patients who presented during the study period with overt OSA signs and symptoms. Ultimately, stringent inclusion/exclusion criteria to test efficacy yielded 40 adult, covert complex insomnia patients [average Insomnia Severity Index (ISI) moderate-severe 19.30 (95% CI 18.42-20.17)] who reported no definitive OSA symptoms or risks and who failed behavioral or drug therapy for an average of one decade. All 40 were diagnosed with OSA and randomized (using block randomization) to a single-blind, prospective protocol, comparing CPAP (n = 21) and ASV (n = 19). Three successive PAP titrations fine-tuned pressure settings, facilitated greater PAP use, and collected objective sleep and breathing data. Patients received 14 weeks of treatment including intensive biweekly coaching and follow-up to foster regular PAP use in order to accurately measure efficaciousness. Primary outcomes measured insomnia severity and sleep quality. Secondary outcomes measured daytime impact: OSA-induced impairment, fatigue severity, insomnia impairment, and quality of life. Performance on these seven variables was assessed using repeated measures ANCOVA to account for the multiple biweekly time points. FINDINGS: At intake, OSA diagnosis and OSA as a cause for insomnia were denied by all 40 patients, yet PAP significantly decreased insomnia severity scores (p = 0.021 in the primary ANCOVA analysis). To quantify effect sizes, mean intake vs endpoint analysis was conducted with ASV yielding nearly twice the effects of CPAP [- 13.2 (10.7-15.7), Hedges' g = 2.50 vs - 9.3 (6.3-12.3), g = 1.39], and between mode effect size was in the medium-large range 0.65. Clinically, ASV led to remission (ISI < 8) in 68% of cases compared to 24% on CPAP [Fisher's exact p = 0.010]. Two sleep quality measures in the ANCOVA analysis again demonstrated superior significant effects for ASV compared to CPAP (both p < 0.03), and pre- and post-analysis demonstrated substantial effects for both scales [ASV (g = 1.42; g = 1.81) over CPAP (g = 1.04; g = 0.75)] with medium size effects between modes (0.54, 0.51). Measures of impairment, residual objective sleep breathing events, and normalized breathing periods consistently demonstrated larger beneficial effects for ASV over CPAP. INTERPRETATION: PAP therapy was highly efficacious in decreasing insomnia severity in chronic insomnia patients with previously undiagnosed co-morbid OSA. ASV proved superior to CPAP in this first efficacy trial to compare advanced to traditional PAP modes in complex insomnia. Future research must determine the following: pathophysiological mechanisms to explain how OSA causes chronic insomnia; general population prevalence of this comorbidity; and, cost-effectiveness of ASV therapy in complex insomnia. Last, efforts to raise awareness of complex insomnia are urgently needed as patients and providers appear to disregard both overt and covert signs and symptoms of OSA in the assessment of chronic insomnia.

Sleep disorder prevalence in at-risk adolescents and potential effects of nightmare triad syndrome.

Objective At-risk high school students, those considered to have a higher probability for academic failure or dropping out, were assessed for various sleep disorders. Effects were compared between students with and without the nightmare triad syndrome (NTS+), the sleep disorders' cluster of frequent nightmares, insomnia disorder and suspected sleep-disordered breathing (SDB). Methods Data were gathered at a charter school for at-risk youth using: computer based surveys, physical airway exams, and mental health interviews by school social worker. Ninety-two students were enrolled, and 70 completed all study components. Results Students were teenaged [17.10 (1.50) years], male (52.2%) slightly overweight [BMI 25.50 (6.41)] Hispanics (87.0%); two-thirds (65 of 92) subjectively reported a sleep problem. Frequent nightmares (39.1%), insomnia (ISI ≥ 12, 41.3%), and SDB risk (79.3%) were common. Several presumptive sleep disorders (insomnia, SDB risk, parasomnia, or nightmares) were associated with worse sleep quality and lower quality of life. Nineteen students met criteria for NTS. Compared to NTS-, NTS+ showed significantly lower quality of life (p < 0.003, g = 0.84). Regression analyses revealed higher levels of depression and anxiety symptoms in NTS+ students. NTS was associated with reduced quality of life independent of anxiety symptoms. Conclusion Prevalence of presumptive sleep disorders was high with a tendency for clusters of sleep disorders in the same individual. Students with NTS+ showed worse outcomes and reduced quality of life, mediated partially by depression and anxiety. To examine relationships between sleep disorders and mental health in at-risk adolescents, research investigations must include both subjective and objective measurements of sleep.

Method for recovery and immunoaffinity enrichment of membrane proteins illustrated with metastatic ovarian cancer tissues.

Integral membrane proteins play key biological roles in cell signaling, transport, and pathogen invasion. However, quantitative clinical assays for this critical class of proteins remain elusive and are generally limited to serum-soluble extracellular fragments. Furthermore, classic proteomic approaches to membrane protein analysis typically involve proteolytic digestion of the soluble pieces, resulting in separation of intra- and extracellular segments and significant informational loss. In this paper, we describe the development of a new method for the quantitative extraction of intact integral membrane proteins (including GPCRs) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new (ProteoSolve-TD) buffer system. This new extraction buffer is compatible with immunoaffinity methods (e.g., ELISA and immunoaffinity chromatography), as well as conventional proteomic techniques (e.g., 2D gels, western blots). We demonstrate near quantitative recovery of membrane proteins EDG2, EDG4, FASLG, KDR, and LAMP-3 by western blots. We have also adapted commercial ELISAs for serum-soluble membrane protein fragments (e.g., sVEGFR2) to measure the tissue titers of their transmembrane progenitors. Finally, we demonstrate the compatibility of the new buffers with immunoaffinity enrichment/mass spectrometric characterization of tissue proteins.

Nocturnal time monitoring behavior ("clock-watching") in patients presenting to a sleep medical center with insomnia and posttraumatic stress symptoms.

Time monitoring behavior (TMB) commonly occurs among insomnia patients, often leads to frustration about sleeplessness, and perpetuates insomnia symptoms. Few studies have explored relationships between time monitoring and insomnia, and none have studied the potential relationships between insomnia, TMB, and posttraumatic stress symptoms (PSSs). In this retrospective chart review of 1078 patients seeking care at a sleep medical center, the patients presented with one of three chief sleep complaints (poor sleep quality, 51%; sleep-disordered breathing, 26%; and insomnia, 24%), and 32% reported moderate to severe PSSs. Both insomnia and time monitoring severity were greater in the 350 patients with PSSs compared with the 728 patients with minimal or no such symptoms. Insomnia and time monitoring severity correlated significantly with total posttraumatic stress scores and most strongly with the arousal subscale. Research on interventions to treat TMB may inform relationships between insomnia and posttraumatic stress.

Sleep disturbances and suicidal ideation in sleep medical center patients.

OBJECTIVE: The purpose of this investigation was two-fold: first, we examined associations between suicidal ideation, maladaptive sleep patterns and abnormal sleep behaviors in a sleep center population, an understudied population in the domain of suicide research; and then, we explored whether significant associations remained after accounting for the possible influence of depressive symptoms. METHOD: Data were analyzed from intake information obtained from 1584 adult patients presenting at a community-based private sleep medical center. The sample was parsed into a Suicidal Ideation (SI) group (N=211) and No Suicidal Ideation (NSI) group (N=1373). Comparisons of these groups were made on measures of self-reported sleep complaints, habits, and behaviors, suicidal ideation, depressive symptoms, and associated psychopathology. RESULTS: Approximately 13% of participants reported suicidal ideation. Clinically significant suicidal ideation was present in 4.5% of the sample. Compared to the NSI group, the SI group showed a pervasive pattern of significantly greater frequency or severity of sleep problems in areas of insomnia, nightmares and other parasomnia behaviors, poor sleep quality, and sleep-related psychophysiologic conditioning as well as worse sleep-related impairment and quality of life. Several relationships were significant after controlling for depressive symptoms. DISCUSSION: Suicidal ideation was consistently associated with a broad array of sleep complaints, even when controlling for level of depressive symptoms. As these self-reported sleep disturbances are treatable sleep disorders, future research should examine the efficacy of sleep and behavioral medicine for reducing the risk of suicidal ideation.

Nasal dilator strip therapy for chronic sleep-maintenance insomnia and symptoms of sleep-disordered breathing: a randomized controlled trial.

To test the impact of nasal dilator strips (NDSs) on insomnia severity, sleep-disordered breathing (SDB) symptoms, sleep quality, and quality of life. Randomized, controlled trial of 4 weeks' duration. Community sample of nonobese, adults with a primary sleep complaint of chronic sleep-maintenance insomnia and mild to moderate SDB symptoms (treatment, n=42; control, n=38). Primary outcomes were four validated scales: Insomnia Severity Index (ISI), Pittsburgh Sleep Quality Index (PSQI), Functional Outcomes of Sleep Questionnaire (FOSQ), and Quality of Life Enjoyment and Satisfaction Questionnaire (QLESQ). Secondary outcomes were sleep indices, nonrestorative sleep ratings, and SDB symptoms, assessed retrospectively and prospectively. Both groups received nonspecific education about sleep disorders. Treatment group also received a brief SDB education and nasal strip instructions. At 4 weeks' follow-up, the treatment group demonstrated significant (p=.0001), large improvements in ISI and PSQI (mean Cohen's d=1.18) and significant (p<.02), medium-sized improvements in FOSQ and QLESQ (mean d=0.51) compared to small, nonsignificant changes in control group (Cohen's d range=0.36-0.09). Treatment group change scores among all four primary variables were significantly correlated (mean r=0.50, p=0.01). Secondary prospective and retrospective outcomes showed medium to large improvements in treatment compared to controls for sleep indices (mean d=0.52 vs 0.28), nonrestorative sleep ratings (mean d=0.69 vs 0.11), and sleep breathing symptoms (mean d=0.47 vs 0.09). Significance was obtained for prospective sleep indices (p=0.01), retrospective, and prospective nonrestorative sleep ratings (p=0.003, <0.05), and retrospective sleep breathing symptoms (p=0.03). SDB education and NDSs demonstrated therapeutic efficacy in a select sample of insomnia patients with SDB symptoms. Replication of results requires placebo controls and objectively confirmed SDB cases.

Nasal dilator strip therapy for chronic sleep maintenance insomnia: a case series.

Sleep-disordered breathing (SDB) is a salient factor in chronic sleep maintenance insomnia. However, many insomnia patients with comorbid SDB may not be interested initially in receiving treatment with continuous positive airway pressure or oral appliance therapy. As an interim pathway, we used Breathe Right nasal dilator strip (NDS) therapy to introduce these patients to the relationship between insomnia and SDB. We hypothesized that NDS-associated improvements might motivate patients to pursue comprehensive SDB therapies. In this open label trial, three men with chronic sleep maintenance insomnia were treated with an educational session about SDB coupled with nightly NDS therapy. At 4-week follow-up, global insomnia severity, difficulty staying asleep, difficulty falling back asleep when awakened, total number of awakenings, and wake time after sleep onset systematically improved. Following NDS therapy, all three insomnia patients elected to pursue comprehensive SDB treatment at local sleep centers.