Generation and characterization of antagonistic anti-human CD39 nanobodies.

CD39 is the major enzyme controlling the levels of extracellular adenosine triphosphate (ATP) via the stepwise hydrolysis of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). As extracellular ATP is a strong promoter of inflammation, monoclonal antibodies (mAbs) blocking CD39 are utilized therapeutically in the field of immune-oncology. Though anti-CD39 mAbs are highly specific for their target, they lack deep penetration into the dense tissue of solid tumors, due to their large size. To overcome this limitation, we generated and characterized nanobodies that targeted and blocked human CD39. From cDNA-immunized alpacas we selected 16 clones from seven nanobody families that bind to two distinct epitopes of human CD39. Among these, clone SB24 inhibited the enzymatic activity of CD39. Of note, SB24 blocked ATP degradation by both soluble and cell surface CD39 as a 15kD monomeric nanobody. Dimerization via fusion to an immunoglobulin Fc portion further increased the blocking potency of SB24 on CD39-transfected HEK cells. Finally, we confirmed the CD39 blocking properties of SB24 on human PBMCs. In summary, SB24 provides a new small biological antagonist of human CD39 with potential application in cancer therapy.

Exploring parental risk perception regarding childhood unintentional home injuries: A cross-sectional study.

INTRODUCTION: While previous studies on childhood injury focused mainly on the relationship between parents' preventive behavior and its determinants, knowledge about parental risk perception that can help to develop, implement, and evaluate successful interventions to prevent childhood unintentional home injuries is still limited. The aim of this study was to gain a deeper understanding of parental risk perception regarding unintentional home injuries of children under six years of age. METHOD: A convenience sample of 469 parents, residing in Germany, completed a web-based questionnaire assessing factors potentially associated with parental risk perception on the child, parental, and environmental level. Descriptive statistics, p for trend, and Spearman's Rho correlation coefficients were calculated. RESULTS: Key factors influencing parental risk perception included children's sex, age, migrant status, emotionality, and injury risk as well as parents' parenting style and external locus of control. While parental risk perception was positively associated with children's injury risk, the analyses showed no associations between parental risk perception and parents' preventive behavior. CONCLUSIONS: Our findings add to a better understanding of parental risk perception, provide practical implications for injury prevention, and indicate that the relationship between parental risk perception and parents' preventive behavior is based on a complex mechanism which is possibly moderated by parents' locus of control. PRACTICAL APPLICATIONS: The identified key factors help to assess parental risk perception more accurately. Therefore, they should be considered in the development of tailored interventions to prevent unintentional home injuries of children, for instance, by targeting specific groups of parental risk perception.

Associations between older adults' physical fitness level and their engagement in different types of physical activity: cross-sectional results from the OUTDOOR ACTIVE study.

OBJECTIVES: To describe the prevalence of different types of physical activities and to explore the association between engagement in these physical activities and performance in the physical fitness dimensions among older adults living in Bremen, Germany. DESIGN: Cross-sectional study. SETTING: Twelve subdistricts in Bremen, Germany. PARTICIPANTS: 1583 non-institutionalised adults aged 65-75 years residing in one of the 12 subdistricts in Bremen, Germany (53.1% female). PRIMARY OUTCOME MEASURES: Level of physical fitness in five dimensions: handgrip strength (hand dynamometry), lower muscle strength (30 s-chair stand test), aerobic endurance (2 min-step test), lower body flexibility (sit-and-reach test), and upper body flexibility (back scratch test); classified using normative values. RESULTS: In this study population, home-based activities (housework, gardening) and activities for transport (walking, cycling) were performed by nearly all the participants, while leisure activities were less prevalent. Logistic regression revealed a positive association between being in or over norm in handgrip strength with cycling (OR: 1.56, 95% CI: 1.13 to 2.15), hiking/running (OR: 1.50, 95% CI: 1.05 to 2.16) and other sports (OR: 3.22, 95% CI: 1.37 to 7.56). Lower muscle strength was positively related to cycling (OR: 1.91, 95% CI: 1.37 to 2.65), gym training (OR: 1.62, 95% CI: 1.16 to 2.26) and dancing (OR: 2.15, 95% CI: 1.00 to 4.61). Aerobic endurance was positively associated with cycling (OR: 1.90, 95% CI: 1.37 to 2.65), gym training (OR: 1.68, 95% CI: 1.20 to 2.36), aerobics (OR: 1.64, 95% CI: 1.19 to 2.26), dancing (OR: 2.62, 95% CI: 1.10 to 6.22) and ball sports (OR: 2.07, 95% CI: 1.30 to 3.29). Apart from housework and upper body flexibility (OR: 0.39, 95% CI: 0.19 to 0.78), flexibility dimensions did not show any significant associations. CONCLUSIONS: While muscle strength dimensions and aerobic endurance were associated with several physical activities, flexibility dimensions were associated with none of the investigated activities apart from housework. Especially cycling and leisure activities (eg, hiking/ running, gym training, aerobics, dancing) showed great potential for sustaining and increasing physical fitness in older age.

Meal Patterns of Older Adults: Results from the OUTDOOR ACTIVE Study.

Eating habits have a substantial effect on health, not only because of consumed foods and nutrients, but also because of the regularity of meals. This study investigates meal patterns in older adults. Data from 1198 adults (52.8% female) aged between 65 and 75 years, who resided in Bremen, Germany, were included in this cross-sectional study. Using a self-administered questionnaire, daily meals were assessed and categorised into three meal pattern types: 'regular eaters' (eating at least three meals a day), 'meal skippers' (skipping one meal a day), and 'irregular eaters' (skipping more than one meal a day). Descriptive analyses were carried out, stratified by sex and meal pattern types. Most women and men were regular eaters (51.5% and 51.7%, respectively), 33.8% of women and 33.3% of men were meal skippers, and 14.7% of women and 15.0% of men were irregular eaters. Differences between meal patterns were seen with regard to socioeconomic status; self-rated health; body-mass index; hypertension; consumption of self-prepared meals; and consumption of whole-grain products, green vegetables, meat, and alcohol. The results provide first insights into possible associations between meal patterns and sociodemographic and health factors, and can benefit disease prevention and health promotion in older adults.

Adherence to Lifestyle Recommendations for Bone Health in Older Adults with and without Osteoporosis: Cross-Sectional Results of the OUTDOOR ACTIVE Study.

Sustaining good bone health into older age is key for preventing osteoporosis. Bone health is associated with several lifestyle factors. This study investigates older adults' adherence to bone health-promoting lifestyle recommendations dependent on osteoporosis status. Cross-sectional data of 1610 community-dwelling older adults (65-75 years) residing in Bremen, Germany (53.4% female) were included. The Osteoporosis Self-Assessment Tool and self-reported osteoporosis diagnosis were used to classify participants by osteoporosis status (low risk, high risk, diagnosis). Adherence to bone health recommendations regarding calcium and vitamin D intake, sun exposure, alcohol consumption, resistance/weight-bearing exercise, and physical activity were assessed. Descriptive statistics were applied, stratified by sex and osteoporosis status. A total of 91 women (10.6%) and 15 men (2.0%) reported an osteoporosis diagnosis, 457 women (43.2%) and 311 men (41.4%) were classified as having a high risk, and 311 women (36.2%) and 425 men (56.6%) as having a low risk. Adherence to bone health recommendations was high for calcium intake (93.3-100.0%), vitamin D intake (77.8-93.3%), and sun exposure (86.7-97.7%). Lower adherence was observed regarding resistance/weight-bearing exercise (36.3-54.4%), physical activity (14.3-57.7%), and alcohol consumption (40.0-72.4%). In conclusion, tailored prevention strategies are needed that focus on older adults with an osteoporosis diagnosis or who are at high risk.

Associations between socioeconomic status and physical activity among older adults: cross-sectional results from the OUTDOOR ACTIVE study.

BACKGROUND: Regular physical activity (PA) is an important strategy for healthy ageing. Socioeconomic status was found to be a key determinant of PA, however, evidence on associations between socioeconomic status and PA among older adults is limited. The aim of this study was to contribute to research on the associations of socioeconomic status and PA among older adults by including self-reported and objectively measured PA data. Furthermore, we examined the self-reported PA data more closely by looking at the activities separately. METHODS: Cross-sectional data of 1507 participants (52.5% female) of the OUTDOOR ACTIVE study between 65 and 75 years, residing in Bremen, Germany, were included in the analyses. Self-reported PA was assessed via questionnaire and comprised all organised and non-organised activities. For analyses, mean hours per week of total and moderate to vigorous PA, and mean metabolic equivalents per week were used. Objectively measured PA was assessed using accelerometers over seven consecutive days. Socioeconomic status was included as an additive social class index containing education, income, and occupation. To test for associations between PA and socioeconomic status, linear regressions were carried out. RESULTS: Self-reported PA showed significant negative associations with socioeconomic status for both men and women. Objectively measured PA was positively associated with socioeconomic status, which was significant in men but not in women. When examining physical activities separately, time spent on housework, gardening, biking, and walking decreased with increasing socioeconomic status. Women in the second SES quintile and men in the third quintile reported the most, and women in the first quintile and men in the fifth quintile the least hours per week spent on exercise. CONCLUSIONS: The results of this study contributed to the existing research gap on the associations of socioeconomic status and PA among older adults. Moreover, we provided information on both self-reported and objectively measured PA, and showed the discrepancies in the two methods' results. These findings can help to develop PA promotion interventions targeting specific socioeconomic status groups and to develop accurate, valid, and reliable self-reported and objective measurements of PA for older adults.

Cross-sectional survey on researchers' experience in using accelerometers in health-related studies.

Objectives: Accelerometers are widely applied in health studies, but lack of standardisation regarding device placement, sampling and data processing hampers comparability between studies. The objectives of this study were to assess how accelerometers are applied in health-related research and problems with accelerometer hardware and software encountered by researchers. Methods: Researchers applying accelerometry in a health context were invited to a cross-sectional web-based survey (August 2020-September 2020). The questionnaire included quantitative questions regarding the application of accelerometers and qualitative questions on encountered hardware and software problems. Descriptive statistics were calculated for quantitative data and content analysis was applied to qualitative data. Results: In total, 116 health researchers were included in the study (response: 13.7%). The most used brand was ActiGraph (67.2%). Independently of brand, the main reason for choosing a device was that it was the standard in the field (57.1%-83.3%). In children and adolescent populations, sampling frequency was higher (mean: 73.3 Hz ±29.9 Hz vs 47.6 Hz ±29.4 Hz) and epoch length (15.0s±15.6s vs 30.1s±25.9s) and non-wear time (42.9 min ±23.7 min vs 65.3 min ±35.4 min) were shorter compared with adult populations. Content analysis revealed eight categories of hardware problems (battery problems, compliance issues, data loss, mechanical problems, electronic problems, sensor problems, lacking waterproofness, other problems) and five categories of software problems (lack of user-friendliness, limited possibilities, bugs, high computational burden, black box character). Conclusions: The study confirms heterogeneity regarding accelerometer use in health-related research. Moreover, several hardware and software problems were documented. Both aspects must be tackled to increase validity, practicability and comparability of research.

The Role of Community-Based Men's Sheds in Health Promotion for Older Men: A Mixed-Methods Systematic Review.

Men's Sheds are a community-based health promotion concept which brings men together to engage in joint activities. Prior research reported beneficial effects on health and well-being of the participants; however, evidence is limited. The main objective of this systematic review is to provide an extensive overview of current research on the effectiveness of Men's Sheds on self-rated health, social isolation, and well-being by applying a mixed-methods approach. In addition, this review aims to identify how to successfully transfer and implement the concept. Eligible for inclusion were all studies published in English, German, or French that specifically referred to the concept of Men's Sheds. Four databases were searched for eligible studies, followed by a hand search on websites and reference lists. Methodological quality of included studies was assessed using checklists developed by the Joanna Briggs Institute. Following the convergent integrated approach, quantitative data were transformed and merged with qualitative data to conduct a thematic analysis. Overall, 35 qualitative, nine quantitative, and eight mixed-methods studies were included. We found evidence regarding benefits of shed participation on self-rated health, social isolation, and well-being. We identified three key characteristics of a successful Men's Shed, including appropriate shed facilities, sufficient funding as well as a participant-driven management and organization of the shed. This mixed-methods systematic review provides a comprehensive overview of the evidence base concerning Men's Sheds and highlights the need for longitudinal studies investigating causal relationships and gathering sufficient information on how to successfully transfer and implement the concept of Men's Sheds in other countries.

Domain-specific life satisfaction among older adults with and without children: The role of intergenerational contact.

BACKGROUND: Life satisfaction is associated with many important health outcomes among older adults and is an indicator of successful ageing. The present study aims to replicate earlier findings regarding relationships between satisfaction with various life domains and life satisfaction in older adults. The study furthermore explores how parental status is associated with satisfaction with different life domains and how two types of intergenerational contact (contact with own children; post-retirement work in childcare) relate to life satisfaction. METHODS: Participants were 1978 older adults, aged 65-75 year (51.7% female), who live in Bremen and took part in the OUTDOOR ACTIVE study. 82.6% of the participants had one or more children. All participants completed a questionnaire, which among others comprised items assessing life satisfaction as well as satisfaction with six different life domains (satisfaction with living situation, financial situation, leisure time, health, family, neighbors and friends). RESULTS: LS is significantly related to all of the investigated life domains, independent of sex and age. For the participants with children, life satisfaction had the highest association with satisfaction with family (ß: 0.202; 95%CI: 0.170-0.235), followed by satisfaction with neighbors and friends (ß: 0.151; 95%CI: 0.111-0.191), and health satisfaction (ß: 0.148; 95%CI: 0.120-0.176). In comparison to that, participants without children had the highest association between life satisfaction and satisfaction with health (ß: 0.193; 95%CI: 0.135-0.252), followed by satisfaction with family (ß: 0.175; 95%CI: 0.114-0.236) and satisfaction with neighbors and friends (ß: 0.154; 95%CI: 0.077-0.232). In participants with children, there was a non-significant negative association between life satisfaction and work in childcare (ß: -0.031; 95%CI: -0.178-0.116), while life satisfaction was statistically significantly positively associated to work in childcare in participants without own children (ß: 0.681; 95%CI: 0.075-1.288). CONCLUSIONS: The results suggest that the domain-specific approach to life satisfaction can elucidate differences in the correlates of life satisfaction and well-being between older adults with and without children. They further suggest that the benefits of working with children for life satisfaction may be more pronounced in older adults without children than older adults with children.

Men's sheds as community-based health promotion for men aged 50 plus: protocol for a mixed-methods systematic review.

BACKGROUND: Men are less likely to participate in health promotion. One approach to reach men is the concept of men's sheds. This community-based health promotion concept brings older men together to engage in joint activities. Prior research revealed various health-related effects of men's sheds, such as benefits for well-being and mental health. To strengthen the current evidence base of men's sheds, a mixed-methods systematic review analysing the association between participation and self-rated health, subjective well-being, and social isolation will be conducted. Moreover, information on how to successfully implement men's sheds will be gathered. METHODS: This mixed-methods systematic review will follow the guidelines of the Joanna Briggs Institute (JBI). The databases MEDLINE (via PubMed), Scopus, Web of Science, and OpenGrey and the websites of men's sheds associations will be searched for publications. Additionally, a hand search in the reference lists of the included publications will be conducted. Qualitative and quantitative studies published in English, German, or French will be considered for inclusion. The quality of the selected studies will be assessed using the JBI critical appraisal checklists. Following the convergent integrated approach, quantitative data will be transformed into textual descriptions, and subsequently combined with data from qualitative studies as well as from the qualitative components of mixed-methods studies in a simultaneous data synthesis. DISCUSSION: The results of this systematic review will lead to a comprehensive understanding of the current evidence base regarding the effectiveness of men's sheds. Furthermore, they will provide useful implications for the implementation of men's sheds. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020219390.

Generation and validation of ActiGraph GT3X+ accelerometer cut-points for assessing physical activity intensity in older adults. The OUTDOOR ACTIVE validation study.

The study of physical activity in older adults is becoming more and more relevant. For evaluation of physical activity recommendations, intensity-specific accelerometer cut-points are utilized. However, research on accelerometer cut-points for older adults is still scarce. The aim of the study was to generate placement-specific cut-points of ActiGraph GT3X+ activity counts and raw measures of acceleration to determine physical activity intensity in older adults. A further aim was to compare the validity of the generated cut-points for a range of different physical activities. The study was a single experimental trial using a convenience sample. Study participants were 20 adults aged 59 to 73 years. Accelerometers were worn at six different placements (one on each wrist, one on each ankle, and two at the hip) and breath-by-breath indirect calorimetry was used as the reference for energy. The experiment comprised of two parts; a) The first required participants to walk on a treadmill at incremental speeds (3.0-5.0 km·h-1), and b) Five different everyday activities (reading, cleaning, shopping, cycling, aerobics) were staged in the laboratory setting. Accelerometer cut-points (activity counts, raw data) were derived for each of the investigated placements by linear regression using the treadmill part. Performance of the cut-points was assessed by applying the cut-points to the everyday activities. We provide cut-points for six placements and two accelerometer metrics in the specific age group. However, the derived cut-points did not outperform published ones. More research and innovative approaches are needed for improving internal and external validity of research results across populations and age groups.

Sex- and age-specific normative values for handgrip strength and components of the Senior Fitness Test in community-dwelling older adults aged 65-75 years in Germany: results from the OUTDOOR ACTIVE study.

BACKGROUND: Physical fitness is a key component of independent living and healthy ageing. For the measurement of physical fitness in older adults, the Senior Fitness Test is a commonly used tool. The objective of this study is to calculate sex- and age-specific normative values for handgrip strength and components of the Senior Fitness Test for older adults (65-75 years) in Germany. METHODS: Cross-sectional data of 1657 community-dwelling older adults residing in Bremen, Germany (53% female) were included in this study. Physical fitness was assessed using the following measurements of the Senior Fitness Test battery: 30s-chair stand test, 2 min-step test, sit-and-reach test, and back scratch test. In addition, handgrip strength was measured using a Saehan DHD-3 digital hand dynamometer SH1003. Sex- and age specific normative values were calculated for the 1st, 3rd, 10th, 25th, 50th, 75th, 90th, 97th, and 99th percentile using the GAMLSS method. RESULTS: The normative values show differences dependent on sex and age. For handgrip strength, the 30s-chair stand test and the 2 min-step test, normative values were higher for men, while women reached higher values in the sit-and-reach test and the back scratch test. For both, men and women, normative values declined with age. CONCLUSIONS: This study provides sex- and age-specific normative values for handgrip strength and components of the Senior Fitness Test for older adults in Germany. They might be useful for future research and for the application in practice.

Promoting Physical Activity Among Older Adults Using Community-Based Participatory Research With an Adapted PRECEDE-PROCEED Model Approach: The AEQUIPA/OUTDOOR ACTIVE Project.

PURPOSE: The PRECEDE-PROCEED model (PPM) is a community-based participatory research (CBPR) framework for health promotion, yet the direct application of the PPM into practice is unclear. This paper describes how the PPM was adapted for the development and application of a pilot intervention study to promote outdoor physical activity (PA) in older adults (OUTDOOR ACTIVE). We illustrate the steps and adaptations we applied to put the PPM into practice and present the developed interventions. DESIGN: The PPM was adapted by incorporating a socio-ecological model. This ensured the design of any resultant intervention would explicitly address multi-level determinants of physical activity. The list of possible program components to select from for the design of an intervention was also extended. SETTING: Bremen, Germany. PARTICIPANTS: Participants in the intervention development were 924 noninstitutionalized older adults, aged 65-75 years (response: 25.2%), living in Bremen-Hemelingen, Germany. For implementation of the intervention and to ensure sustainability, several groups of stakeholders were involved throughout the process. METHODS: A mixed method design was employed (e.g., focus groups, quantitative survey) to identify determinants. A round table and participatory workshops were held to involve the target group and community stakeholders in the design of an intervention using the adapted PPM model. RESULTS: A conceptual model was developed illustrating the integration of a socio-ecological model into the PPM. The model received ecological validity, as it was affirmed by community stakeholders as an appropriate method for designing a community-level PA intervention. Target goals to address PA determinants were selected by target group members. An intervention to meet the goals was developed and implemented with target group input. CONCLUSION: The adapted PPM is a promising starting point for developing multi-level interventions. Steps should be taken to ensure all social groups are participating in the process and all levels of determinants are addressed.

Time allocation to active domains, physical activity, and health indicators in older adults: cross-sectional results from the OUTDOOR ACTIVE study.

BACKGROUND: Physical activity (PA) is one of the key determinants of healthy ageing. Research showed that time allocation plays an important role in PA. Therefore, an understanding of the time use of older adults is crucial for developing PA programs. The aim of this study was to examine the associations of time allocation and objectively measured PA, and several health indicators in older adults. METHODS: In this cross-sectional study all 915 participants of the OUTDOOR ACTIVE study were included. The participants were 65 to 75 years old and resided in a subdistrict of Bremen, Germany (50.9% female). The active domains were derived from the SLOTH model (leisure activities, occupation, active transport, home-based activities). PA was objectively measured with accelerometers over seven consecutive days. Binary logistic regressions were used to test the associations of total PA and time spent in the domains with several health indicators (self-rated health, overweight, obesity, activities of daily living (ADL)). RESULTS: Participants over the age of 70 years were significantly less physically active than those under 70 years and women were significantly more physically active than men. Regardless of age and sex, most time was spent on home-based activities (women: 118.5 ± 87.8 min/day; men: 80.2 ± 69.4 min/day). Both PA and time spent on leisure activities were associated with a lower risk of bad self-rated health (0.36; 95%-CL: 0.20, 0.65 for PA; 0.93; 95%-CL: 0.87, 0.99 for leisure activities) and less limitations in ADL. PA and active transport seemed to lower the risk of overweight (0.39; 95%-CL: 0.25, 0.62 for PA; 0.80; 95%-CL: 0.69, 0.93 for active transport) and obesity (0.36; 95%-CL: 0.21, 0.60 for PA; 0.77; 95%-CL: 0.64, 0.92 for active transport). Having an occupation was associated with a lower risk of bad self-rated health (0.60; 95%-CL: 0.40, 0.92). CONCLUSIONS: The results of this study provide insights in the time allocation to active domains and total PA of older adults, as well as the associations with health indicators. These findings have important implications for the development of PA programs and guidelines. Future research should examine the associations further in longitudinal studies.

Nanobody-based CD38-specific heavy chain antibodies induce killing of multiple myeloma and other hematological malignancies.

Rationale: CD38 is a target for the therapy of multiple myeloma (MM) with monoclonal antibodies such as daratumumab and isatuximab. Since MM patients exhibit a high rate of relapse, the development of new biologics targeting alternative CD38 epitopes is desirable. The discovery of single-domain antibodies (nanobodies) has opened the way for a new generation of antitumor therapeutics. We report the generation of nanobody-based humanized IgG1 heavy chain antibodies (hcAbs) with a high specificity and affinity that recognize three different and non-overlapping epitopes of CD38 and compare their cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. Methods: We generated three humanized hcAbs (WF211-hcAb, MU1067-hcAb, JK36-hcAb) that recognize three different non-overlapping epitopes (E1, E2, E3) of CD38 by fusion of llama-derived nanobodies to the hinge- and Fc-domains of human IgG1. WF211-hcAb shares the binding epitope E1 with daratumumab. We compared the capacity of these CD38-specific hcAbs and daratumumab to induce CDC and ADCC in CD38-expressing tumor cell lines in vitro and in patient MM cells ex vivo as well as effects on xenograft tumor growth and survival in vivo. Results: CD38-specific heavy chain antibodies (WF211-hcAb, MU1067-hcAb, JK36-hcAb) potently induced antibody-dependent cellular cytotoxicity (ADCC) in CD38-expressing tumor cell lines and in primary patient MM cells, but only little if any complement-dependent cytotoxicity (CDC). In vivo, CD38-specific heavy chain antibodies significantly reduced the growth of systemic lymphomas and prolonged survival of tumor bearing SCID mice. Conclusions: CD38-specific nanobody-based humanized IgG1 heavy chain antibodies mediate cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. These promising results of our study indicate that CD38-specific hcAbs warrant further clinical development as therapeutics for multiple myeloma and other hematological malignancies.

Targeting CD38-Expressing Multiple Myeloma and Burkitt Lymphoma Cells In Vitro with Nanobody-Based Chimeric Antigen Receptors (Nb-CARs).

The NAD-hydrolyzing ecto-enzyme CD38 is overexpressed by multiple myeloma and other hematological malignancies. We recently generated CD38-specific nanobodies, single immunoglobulin variable domains derived from heavy-chain antibodies naturally occurring in llamas. Nanobodies exhibit high solubility and stability, allowing easy reformatting into recombinant fusion proteins. Here we explore the utility of CD38-specific nanobodies as ligands for nanobody-based chimeric antigen receptors (Nb-CARs). We cloned retroviral expression vectors for CD38-specific Nb-CARs. The human natural killer cell line NK-92 was transduced to stably express these Nb-CARs. As target cells we used CD38-expressing as well as CRISPR/Cas9-generated CD38-deficient tumor cell lines (CA-46, LP-1, and Daudi) transduced with firefly luciferase. With these effector and target cells we established luminescence and flow-cytometry CAR-dependent cellular cytotoxicity assays (CARDCCs). Finally, the cytotoxic efficacy of Nb-CAR NK-92 cells was tested on primary patient-derived CD38-expressing multiple myeloma cells. NK-92 cells expressing CD38-specific Nb-CARs specifically lysed CD38-expressing but not CD38-deficient tumor cell lines. Moreover, the Nb-CAR-NK cells effectively depleted CD38-expressing multiple myeloma cells in primary human bone marrow samples. Our results demonstrate efficacy of Nb-CARs in vitro. The potential clinical efficacy of Nb-CARs in vivo remains to be evaluated.

Accelerometer-assessed outdoor physical activity is associated with meteorological conditions among older adults: Cross-sectional results from the OUTDOOR ACTIVE study.

BACKGROUND: Meteorological conditions are potential determinants of physical activity (PA). A profound understanding of the determinants of PA behaviour is required for PA promotion. This study examined the association between accelerometer-assessed PA and meteorological conditions among older adults. METHODS: This cross-sectional study included data of 577 adults aged 65-75 years living in Bremen, Germany (52% female; 3278 days). PA was measured with accelerometers for seven consecutive days (10/15-08/16). A threshold of 240 lx was used to differentiate between outdoor physical activity (OPA) and indoor physical activity (IPA). Linear mixed models estimated the association between PA (daily accelerometer counts per minute (CPM)) and meteorological factors (temperature, cloud cover, wind, and no precipitation) derived by principal component analysis. RESULTS: The analyses showed associations between PA in CPM and the meteorological factors temperature (93.7; 95%-CL: 64.9, 122.5) and no precipitation (48.4; 95%-CL: 19.8, 77.0) in women and wind (-40.3; 95%-CL: -59.7, -20.8) and no precipitation (30.1; 95%-CL: 5.6, 54.6) in men. After distinguishing in OPA and IPA for a subsample of 128 participants (473 days), the sex differences were no longer present. OPA in CPM was associated with temperature (women: 174.5; 95%-CL: 81.3, 267.6; men: 183.3; 95%-CL: 81.2, 285.4), cloud cover (women: -153.0; 95%-CL: -200.3, -105.7; men: -123.2; 95%-CL: -174.7, -71.7), and wind (women: -118.6; 95%-CL: -189.6; -47.7; men: -96.9; 95%-CL: -177.0, -16.7). No association between OPA and no precipitation was found (women: 2.9; 95%-CL: -89.0, 94.8; men: -17.1; 95%-CL: -116.7, 82.4). CONCLUSIONS: The results of this study emphasize the importance of meteorological conditions as environmental determinants of PA among older adults. Meteorological conditions should be accounted for in the unbiased assessment of habitual PA and the development of PA promotion programs. Future research should focus on the associations of OPA and IPA with meteorological conditions in different climatic regions.

Nanobody-Enhanced Targeting of AAV Gene Therapy Vectors.

A limiting factor for the use of adeno-associated viruses (AAVs) as vectors in gene therapy is the broad tropism of AAV serotypes, i.e., the parallel infection of several cell types. Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Their small size and high solubility allow easy reformatting into fusion proteins. Herein we show that a membrane protein-specific nanobody can be inserted into a surface loop of the VP1 capsid protein of AAV2. Using three structurally distinct membrane proteins-a multispan ion channel, a single-span transmembrane protein, and a glycosylphosphatidylinositol (GPI)-anchored ectoenzyme-we show that this strategy can dramatically enhance the transduction of specific target cells by recombinant AAV2. Moreover, we show that the nanobody-VP1 fusion of AAV2 can be incorporated into the capsids of AAV1, AAV8, and AAV9 and thereby effectively redirect the target specificity of other AAV serotypes. Nanobody-mediated targeting provides a highly efficient AAV targeting strategy that is likely to open up new avenues for genetic engineering of cells.

CD38-Specific Biparatopic Heavy Chain Antibodies Display Potent Complement-Dependent Cytotoxicity Against Multiple Myeloma Cells.

CD38 is overexpressed by multiple myeloma cells and has emerged as a target for therapeutic antibodies. Nanobodies are soluble single domain antibody fragments derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. We previously identified distinct llama nanobodies that recognize three non-overlapping epitopes of the extracellular domain of CD38. Here, we fused these VHH domains to the hinge, CH2, and CH3 domains of human IgG1, yielding highly soluble chimeric llama/human heavy chain antibodies (hcAbs). We analyzed the capacity of these hcAbs to mediate complement-dependent cytotoxicity (CDC) to CD38-expressing human multiple myeloma and Burkitt lymphoma cell lines. Combinations of two hcAbs that recognize distinct, non-overlapping epitopes of CD38 mediated potent CDC, in contrast to the hcAb monotherapy with only weak CDC capacity. Similarly, combining daratumumab with a hcAb that recognizes a non-overlapping epitope resulted in dramatically enhanced CDC. Further, introducing the E345R HexaBody mutation into the CH3 domain strongly enhanced the CDC potency of hcAbs to CD38-expressing cells. Exploiting their high solubility, we genetically fused two distinct nanobodies into heteromeric dimers via a flexible peptide linker and then fused these nanobody dimers to the hinge, CH2 and CH3 domains of human IgG1, yielding highly soluble, biparatopic hcAbs. These biparatopic hcAbs elicited CDC toward CD38-expressing myeloma cells more effectively than daratumumab. Our results underscore the advantage of nanobodies vs. pairs of VH and VL domains for constructing bispecific antibodies. Moreover, the CD38-specific biparatopic heavy chain antibodies described here represent potential new powerful therapeutics for treatment of multiple myeloma.

A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation.

Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic and antagonistic Nbs against several cell surface ecto-enzymes and ligand-gated ion channels.