Structure-Activity Relationship of Antibody-Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody-Phosphorodiamidate Morpholino Oligomer Conjugates for Drug Development.

Antibody-oligonucleotide conjugates (AOCs) are promising treatments for Duchenne muscular dystrophy (DMD). They work via induction of exon skipping and restoration of dystrophin protein in skeletal and heart muscles. The structure-activity relationships (SARs) of AOCs comprising antibody-phosphorodiamidate morpholino oligomers (PMOs) depend on several aspects of their component parts. We evaluate the SAR of antimouse transferrin receptor 1 antibody (αmTfR1)-PMO conjugates: cleavable and noncleavable linkers, linker location on the PMO, and the impact of drug-to-antibody ratios (DARs) on plasma pharmacokinetics (PK), oligonucleotide delivery to tissues, and exon skipping. AOCs containing a stable linker with a DAR9.7 were the most effective PMO delivery vehicles in preclinical studies. We demonstrate that αmTfR1-PMO conjugates induce dystrophin protein restoration in the skeletal and heart muscles of mdx mice. Our results show that αmTfR1-PMO conjugates are a potentially effective approach for the treatment of DMD.

Tobacco mosaic virus for the targeted delivery of drugs to cells expressing prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a membrane-bound protein that is preferentially expressed in the prostate gland and induced in many prostate cancers, making it an important target for new diagnostics and therapeutics. To improve the efficacy of nanoparticle formulations for the imaging and/or eradication of prostate cancer, we synthesized the PSMA-binding glutamic acid derivative DUPA and conjugated it to the external surface of tobacco mosaic virus (TMV) particles. DUPA-targeted TMV was subsequently loaded with the antineoplastic agent mitoxantrone (MTO) or conjugated internally with the fluorescent dye cyanine 5 (Cy5). We found that TMV particles could be efficiently decorated with DUPA and loaded with MTO or Cy5 while maintaining structural integrity. DUPA-targeted TMV particles were able to bind more efficiently to the surface of PSMA+ LNCaP cells compared to non-targeted TMV; but there was little difference in binding efficiency between targeted and untargeted TMV when we tested PSMA- PC3 cells (both cell lines are prostate cancer cell lines). DUPA-targeted TMV particles were internalized by LNCaP cells enabling drug delivery. Finally, we loaded the DUPA-targeted TMV particles and untargeted control particles with MTO to test their cytotoxicity against LNCaP cells in vitro. The cytotoxicity of the TMV-MTO particles (IC50 = 10.2 nM) did not differ significantly from that of soluble MTO at an equivalent dose (IC50 = 12.5 nM) but the targeted particles (TMV-DUPA-MTO) were much more potent (IC50 = 2.80 nM). The threefold increase in cytotoxicity conferred by the DUPA ligand suggests that MTO-loaded, DUPA-coated TMV particles are promising as a therapeutic strategy for PSMA+ prostate cancer and should be advanced to preclinical testing in mouse models of prostate cancer.

Use of a Single CAR T Cell and Several Bispecific Adapters Facilitates Eradication of Multiple Antigenically Different Solid Tumors.

Most solid tumors are comprised of multiple clones that express orthogonal antigens, suggesting that novel strategies must be developed in order to adapt chimeric antigen receptor (CAR) T-cell therapies to treat heterogeneous solid tumors. Here, we utilized a cocktail of low-molecular-weight bispecific adapters, each comprised of fluorescein linked to a different tumor-specific ligand, to bridge between an antifluorescein CAR on the engineered T cell and a unique antigen on the cancer cell. This formation of an immunologic synapse between the CAR T cell and cancer cell enabled use of a single antifluorescein CAR T cell to eradicate a diversity of antigenically different solid tumors implanted concurrently in NSG mice. Based on these data, we suggest that a carefully designed cocktail of bispecific adapters in combination with antifluorescein CAR T cells can overcome tumor antigen escape mechanisms that lead to disease recurrence following many CAR T-cell therapies. SIGNIFICANCE: A cocktail of tumor-targeted bispecific adapters greatly augments CAR T-cell therapies against heterogeneous tumors, highlighting its potential for broader applicability against cancers where standard CAR T-cell therapy has failed.

Methodological Recommendations for Trials of Psychological Interventions.

Recent years have seen major developments in psychotherapy research that suggest the need to address critical methodological issues. These recommendations, developed by an international group of researchers, do not replace those for randomized controlled trials, but rather supplement strategies that need to be taken into account when considering psychological treatments. The limitations of traditional taxonomy and assessment methods are outlined, with suggestions for consideration of staging methods. Active psychotherapy control groups are recommended, and adaptive and dismantling study designs offer important opportunities. The treatments that are used, and particularly their specific ingredients, need to be described in detail for both the experimental and the control groups. Assessment should be performed blind before and after treatment and at long-term follow-up. A combination of observer- and self-rated measures is recommended. Side effects of psychotherapy should be evaluated using appropriate methods. Finally, the number of participants who deteriorate after treatment should be noted according to the methods that were used to define response or remission.

Development of a Small Molecule Tubulysin B Conjugate for Treatment of Carbonic Anhydrase IX Receptor Expressing Cancers.

Carbonic anhydrase IX (CAIX) is a membrane-spanning zinc metalloenzyme that catalyzes the reversible consumption of CO2 and water to form H+ + HCO3-. Many human cancers upregulate CAIX to help control the pH in their hypoxic microenvironments. The consequent overexpression of CAIX on malignant cells and low expression on normal tissues render CAIX a particularly attractive target for small molecule inhibitors, antibody-drug conjugates, and ligand-targeted drugs. In this study, CAIX-targeted fluorescent reporter molecules were initially exploited to investigate CAIX-specific binding to multiple cancer cell lines, where they were shown to display potent and selective binding to CAIX positive cells. A small molecule CAIX-targeted tubulysin B conjugate was then synthesized and examined for its ability to kill CAIX-expressing tumor cells in vitro. Potent therapeutic conjugates were subsequently tested in vivo and demonstrated to eliminate solid human tumor xenografts in murine tumor models without exhibiting overt signs of toxicity. Because most solid tumors contain hypoxic regions where CAIX is overexpressed, development of a method to selectively deliver drugs to these hypoxic regions could aid in the therapy of otherwise difficult to treat tumors.

Single-item screening for agoraphobic symptoms: validation of a web-based audiovisual screening instrument.

The advent of web-based treatments for anxiety disorders creates a need for quick and valid online screening instruments, suitable for a range of social groups. This study validates a single-item multimedia screening instrument for agoraphobia, part of the Visual Screener for Common Mental Disorders (VS-CMD), and compares it with the text-based agoraphobia items of the PDSS-SR. The study concerned 85 subjects in an RCT of the effects of web-based therapy for panic symptoms. The VS-CMD item and items 4 and 5 of the PDSS-SR were validated by comparing scores to the outcomes of the CIDI diagnostic interview. Screening for agoraphobia was found moderately valid for both the multimedia item (sensitivity.81, specificity.66, AUC.734) and the text-based items (AUC.607-.697). Single-item multimedia screening for anxiety disorders should be further developed and tested in the general population and in patient, illiterate and immigrant samples.

The effectiveness of problem solving therapy in deprived South African communities: results from a pilot study.

BACKGROUND: The majority of South Africans with a DSM-IV diagnosis receive no treatment for their mental health problems. There is a move to simplify treatment for common mental disorders (CMDs) in order to ease access. Brief problem solving therapy (PST) might fill the treatment gap for CMD's in deprived communities in South Africa. This pilot study evaluates the feasibility, acceptability and effectiveness of this PST program for CMD's in deprived communities around Cape Town. METHODS: A Dutch problem solving program was adapted and translated into English, Xhosa and Afrikaans and thereafter implemented in townships around Cape Town. An initial attempt to recruit participants for online PST proved difficult, and so the program was adapted to a booklet format. Volunteers experiencing psychological distress were invited to participate in the either individually or group delivered 5-week during self-help program. To evaluate the effectiveness, psychological distress was administered through self-report questionnaires. After completion of the intervention participants also rated the program on various acceptability aspects. RESULTS: Of 103 participants, 73 completed 5 weeks of brief PST in a booklet/workshop format. There were significantly more dropouts in those who used the booklet individually than in the group. Psychological distress measured on the K-10 and SRQ fell significantly and the program was evaluated positively. CONCLUSIONS: The results suggest that brief problem solving in a booklet/workshop format may be an effective, feasible and acceptable short-term treatment for people with CMD's in deprived communities. In this setting, group delivery of PST had lower drop-out rates than individual delivery, and was more feasible and acceptable. Randomized controlled trials are needed to evaluate the effect of brief self-help PST more rigorously.

Strain-promoted "click" chemistry for terminal labeling of DNA.

1,3-Dipolar [3 + 2] cycloaddition between azides and alkynes--an archetypal "click" chemistry--has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here, we describe the solid-phase synthesis and characterization of 5'-dibenzocyclooctyne (DIBO)-modified oligonucleotides, using a new DIBO phosphoramidite, which react with azides via copper-free, strain-promoted alkyne-azide cycloaddition (SPAAC). We found that the DIBO group not only survived the standard acidic and oxidative reactions of solid-phase oligonucleotide synthesis (SPOS), but that it also survived the thermal cycling and standard conditions of the polymerase chain reaction (PCR). As a result, PCR with DIBO-modified primers yielded "clickable" amplicons that could be tagged with azide-modified fluorophores or immobilized on azide-modified surfaces. Given its simplicity, SPAAC on DNA could streamline the bioconjugate chemistry of nucleic acids in a number of modern biotechnologies.

Quick and easy self-rating of Generalized Anxiety Disorder: validity of the Dutch web-based GAD-7, GAD-2 and GAD-SI.

Screening of Generalized Anxiety Disorder (GAD), which is often undetected and untreated, could be improved by quick and easy web-based data collection. This paper aims to validate the web-based self-rated 7-item GAD-7, its shortened 2-item GAD-2 and a single item from the GAD-7 scale (GAD-SI) to screen for GAD. Of a total of 502 subjects aged 18-80 who rated the web-based GAD-7, Center for Epidemiological Studies Depression scale (CES-D) and Hospital Anxiety and Depression Scale (HADS), 157 (31%) subjects had a WHO Composite International Diagnostic Interview for DSM-IV-disorders by telephone. The GAD-7 had good reliability. Subjects with a GAD-diagnosis had significantly higher means on GAD-2, GAD-SI and GAD-7 than subjects without a GAD-diagnosis. The AUC (Area Under the Curve) of the GAD-SI and GAD-2 was accurate and not significantly different to the GAD-7 AUC. The web-based GAD-SI, GAD-2 and GAD-7 are reliable, valid tools to quickly screen for GAD in busy mental health settings and clinical research. More research is needed to validate the GAD-SI 'Do you have trouble relaxing' to see if its screening properties approach those of the GAD-7.

Social support moderates outcome in a randomized controlled trial of exposure therapy and (or) cognitive restructuring for chronic posttraumatic stress disorder.

OBJECTIVE: To understand what predicts good outcome in psychiatric treatments, thus creating a pathway to improving efficacy. METHOD: Our study investigated relations between predictor variables and outcome (on the Clinician Administered Posttraumatic Stress Disorder [PTSD] Scale [CAPS]) at posttreatment for 77 treatment completers in a randomized controlled trial of exposure therapy and (or) cognitive restructuring, compared with relaxation, for chronic PTSD in adults. RESULTS: More social support on the Significant Others Scale significantly predicted better outcome on the CAPS, even after controlling for the effects of treatment group and of pretreatment severity. Importantly, social support was only a significant predictor of outcome for participants receiving cognitive restructuring and (or) exposure therapy and not for participants in the relaxation condition. CONCLUSIONS: Better social support is associated with significantly greater gain following cognitive restructuring and (or) exposure therapy for PTSD. Future interventions should consider augmenting social support as an adjunct to treatment.

Brief self-rated screening for depression on the Internet.

BACKGROUND: The Internet offers promising possibilities for the quick screening of depression for treatment and research purposes. This paper aims to validate three self-rated measures to screen for depression on the Internet: SID (single-item depression scale), CES-D (Center for Epidemiological Studies Depression scale) and K10 (Kessler psychological distress scale). METHODS: Of the 502 subjects aged 18-80 who rated the SID, CES-D and K10 measures on the Internet, 157 (31%) subjects were also interviewed by telephone using the WHO Composite International Diagnostic Interview (C)IDI) for DSM-IV-disorders. RESULTS: Cronbach's alpha for both web self-rated measures CES-D and K10 was 0.90. The SID correlated 0.68 (P<0.001) with the CES-D and with the K10. The CES-D correlated 0.84 with the K10 (P<0.001). Subjects with a DSM-IV diagnosis for any depressive disorder had significantly higher means (P<0.001) on the three self-rated measures for depressive symptoms than subjects without a diagnosis of any depressive disorder. Using any depressive disorder as the gold standard, the area under the curve (AUC) of the SID was 0.71 (95% CI: 0.63-0.79), which was significantly lower than the AUC of the CES-D (AUC: 0.84; 95% CI: 0.77-0.90, P=0.003) and of the K10 (AUC: 0.81; 95% CI: 0.73-0.88, P=0.0024). The AUCs for the K10 and CES-D did not differ significantly from each other. LIMITATIONS: The CIDI interviews were not recorded, so inter-rater reliability could not be calculated. CONCLUSIONS: The CES-D and K10 are reliable, valid tools for care providers to quickly screen depressive patients on the Internet and for researchers to collect data.

Implementation of Internet-based preventive interventions for depression and anxiety: role of support? The design of a randomized controlled trial.

BACKGROUND: Internet-based self-help is an effective preventive intervention for highly prevalent disorders, such as depression and anxiety. It is not clear, however, whether it is necessary to offer these interventions with professional support or if they work without any guidance. In case support is necessary, it is not clear which level of support is needed. This study examines whether an internet-based self-help intervention with a coach is more effective than the same intervention without a coach in terms of clinical outcomes, drop-out and economic costs. Moreover, we will investigate which level of support by a coach is more effective compared to other levels of support. METHODS: In this randomized controlled trial, a total of 500 subjects (18 year and older) from the general population with mild to moderate depression and/or anxiety will be assigned to one of five conditions: (1) web-based problem solving through the internet (self-examination therapy) without a coach; (2) the same as 1, but with the possibility to ask help from a coach on the initiative of the respondent (on demand, by email); (3) the same as 1, but with weekly scheduled contacts initiated by a coach (once per week, by email); (4) weekly scheduled contacts initiated by a coach, but no web-based intervention; (5) information only (through the internet). The interventions will consist of five weekly lessons. Primary outcome measures are symptoms of depression and anxiety. Secondary outcome measures are drop-out from the intervention, quality of life, and economic costs. Other secondary outcome measures that may predict outcome are also studied, e.g. client satisfaction and problem-solving skills. Measures are taken at baseline (pre-test), directly after the intervention (post-test, five weeks after baseline), 3 months later, and 12 months later. Analysis will be conducted on the intention-to-treat sample. DISCUSSION: This study aims to provide more insight into the clinical effectiveness, differences in drop-out rate and costs between interventions with and without support, and in particular different levels of support. This is important to know in relation to the dissemination of internet-based self-help interventions. TRIAL REGISTRATION: Nederlands Trial Register (NTR): TC1355.

A brief Web-based screening questionnaire for common mental disorders: development and validation.

BACKGROUND: The advent of Internet-based self-help systems for common mental disorders has generated a need for quick ways to triage would-be users to systems appropriate for their disorders. This need can be met by using brief online screening questionnaires, which can also be quickly used to screen patients prior to consultation with a GP. OBJECTIVE: To test and enhance the validity of the Web Screening Questionnaire (WSQ) to screen for: depressive disorder, alcohol abuse/dependence, GAD, PTSD, social phobia, panic disorder, agoraphobia, specific phobia, and OCD. METHODS: A total of 502 subjects (aged 18 - 80) answered the WSQ and 9 other questionnaires on the Internet. Of these 502, 157 were assessed for DSM-IV-disorders by phone in a WHO Composite International Diagnostic Interview with a CIDI-trained interviewer. RESULTS: Positive WSQ "diagnosis" had significantly (P < .001) higher means on the corresponding validating questionnaire than negative WSQ "diagnosis". WSQ sensitivity was 0.72 - 1.00 and specificity was 0.44 - 0.77 after replacing three items (GAD, OCD, and panic) and adding one question for specific phobia. The Areas Under the Curve (AUCs) of the WSQ's items with scaled responses were comparable to AUCs of longer questionnaires. CONCLUSIONS: The WSQ screens appropriately for common mental disorders. While the WSQ screens out negatives well, it also yields a high number of false positives.

Computer-aided self-exposure therapy for phobia/panic disorder: a pilot economic evaluation.

Phobia/panic disorder is common. It improves with exposure therapy, even when guided mainly by a computer self-help system such as FearFighter (FF), but such therapy must also demonstrate cost-effectiveness. This study compares the cost-effectiveness of FF with computed-aided relaxation and clinician-led exposure. Data were obtained on patients from a randomised controlled trial of FF. Economic analyses used pretreatment and 1-month follow-up self-ratings of the main problem and global phobia. Clinician costs were calculated using the number of therapist hours and the cost of FF. Incremental cost-effectiveness ratios were calculated and cost-effectiveness acceptability curves were produced. Data were available on 62 patients with main problem ratings and 60 with global phobia ratings. FF and clinician-led exposure were more effective than relaxation but more expensive. Compared with relaxation, producing an extra unit of improvement on the main problem scale cost 64 UK pounds with FF and 100 UK pounds with clinician-led exposure. FF appeared to be more cost-effective using the global phobia rating (112 UK pounds per extra unit of improvement vs. 128 UK pounds for clinician-led exposure). The cost-effectiveness of FF could be enhanced if users had less highly trained supporters. FF would be less cost-effective if face-to-face therapy was delivered by less qualified professionals. Caution is urged regarding these indicative findings given that these were secondary analyses.

Computer-aided psychological treatments: evolving issues.

Evidence is growing that several computer-aided psychotherapy (CP) systems can effectively improve a range of common mental health problems. Most clients find CP acceptable because of its convenience, confidentiality, and reduction of stigma. CP can be accessed in a clinic, but recently clients have used CP especially on the Internet at home, with brief support on a telephone helpline and/or by email. Brief and efficient screening and support greatly reduce attrition. CP's efficacy, and encouragement of its dissemination and implementation by some national funding bodies and governmental agencies, has led to its spread as a regular care option and is increasing access to psychological therapies in some countries. Transfer of this new approach from use in tight research studies to use as an integrated part of everyday care under widely varying conditions generates teething problems that are being managed in diverse ways across different centers. Anonymized Internet audit of CP outcomes facilitates effective care and clinical governance. This review examines the current state of the art as well as the science and broad applications of CP.

Computer-aided psychotherapy for anxiety disorders: a meta-analytic review.

Computer-aided psychotherapy (CP) is said to (1) be as effective as face-to-face psychotherapy, while requiring less therapist time, for anxiety disorder sufferers, (2) speed access to care, and (3) save traveling time. CP may be delivered on stand-alone or Internet-linked computers, palmtop computers, phone-interactive voice response, DVDs, and cell phones. The authors performed a meta-analysis of 23 randomised controlled studies (RCTs) that compared CP with non-CP in anxiety disorders: phobias, n = 10; panic disorder/agoraphobia, n = 9; PTSD, n = 3; obsessive-compulsive disorder, n = 1. Overall mean effect size of CP compared with non-CP was 1.08 (95% confidence interval: 0.84-1.32). CP and face-to-face psychotherapy did not differ significantly from each other (13 comparisons, d = -0.06). Much caution is needed when interpreting the findings indicating that outcome was unrelated to type of disorder, type of comparison group, mode of CP delivery (Internet, stand-alone PC, palmtop), and recency of the CP system and that effect size decreased when more therapist time was replaced by the computer. Because CP as a whole was as effective as face-to-face psychotherapy, certain forms of CP deserve to be integrated into routine practice.

Meta-analysis of computer-aided psychotherapy: problems and partial solutions.

Meta-analysis can be valuable if it heeds its originators' caution that intimate communing with the data is essential. A critique of the authors' own meta-analysis shows that the danger of overly broad conclusions could be reduced by attention to specificities and awareness of potentially hidden sources of variance. Conclusions from even good meta-analyses are best placed in perspective, along with naturalistic reviews, open studies, and even anecdotes to yield a fair picture of what computer-aided psychotherapy or any other treatment can achieve under varying conditions. The most realistic picture comes from zooming in and out and melding meta-analyses with further types of evidence.