Development of an 18F-labeled anti-human CD8 VHH for same-day immunoPET imaging.

PURPOSE: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. METHODS: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. RESULTS: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. CONCLUSION: Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.

Nanolipoprotein Particles as a Delivery Platform for Fab Based Therapeutics.

Nanolipoprotein particles (NLPs), a lipid bilayer-based nanoparticle platform, have recently been developed for in vivo delivery of a variety of molecules of therapeutic interest, but their potential to deliver Fabs with valencies that exceed those of current multivalent formats has not yet been evaluated. Here we describe the development, optimization, and characterization of Fab-NLP conjugates. NLPs were generated with maleimide reactive lipids for conjugation to a Fab with a C-terminal cysteine. Of note, maleimide reactive lipids were shown to conjugate to the apolipoprotein when the NLPs were assembled at pH 7.4. However, this undesirable reaction was not observed when assembled at pH 6. Site-specific Fab conjugation conditions were then optimized, and conjugation of up to 30 Fab per NLP was demonstrated. Interestingly, although conjugation of higher numbers of Fabs had a significant impact on NLP molecular weight, only a minimal impact on NLP hydrodynamic radius was observed, indicating that particle size is largely dictated by the discoidal shape of the NLP. Fab-NLP viscosity and its stability upon lyophilization were also evaluated as an assessment of the manufacturability of the Fab-NLP. Significantly higher Fab concentrations were achieved with the Fab-NLP conjugates relative to another multivalent format (Fab-PEG conjugates). Fab conjugation to the NLP was also not found to have an impact on Fab activity in both an inhibitory and agonist setting. Finally, the stability of the Fab-NLP conjugates was evaluated in 50% serum and Fab-NLPs demonstrated increased stability, with >63% of Fab-NLP remaining intact after 24 h at Fab per particle ratios of 7 or greater. Our findings suggest Fab-NLPs are a promising platform for the targeted delivery of Fabs in a multivalent format and are compatible with established manufacturing processes.

The association between pollutant levels and asthma-related emergency department visits in the Bronx after the World Trade Center attacks.

Objective: To examine the potential impact of the World Trade Center (WTC) attacks on asthma-related emergency department visits (AREDV) in the New York City borough of the Bronx. Methods: We obtained daily nitrogen dioxide (NO2), sulfur dioxide (SO2) and ozone (O3) values from the National Climatic Data Center's collection station in the Bronx from 1999 and 2002, a year before and after the WTC attacks. We compared daily AREDV and pollutant levels between 1999 and 2002 using the Wilcoxon signed rank sum test. We considered each season separately due to seasonal variations of AREDV and pollutants. We then used multiple linear regression models to assess the relationships between the changes in AREDV and the changes in pollutants from 1999 to 2002 in each season. Results: There were statistically significant increases from 1999 to 2002 in the daily NO2 in the summer. Significant increases for daily SO2 and O3 values from 1999 to 2002 occurred in all seasons. Significant increases occurred in daily AREDV values in the spring and fall. Multiple linear regression analyses showed that increases in the daily O3 values were significantly associated with increases in AREDV from 1999 to 2002 in the summer season. Conclusion: We observed a possible association between the WTC attacks and significant increases in O3 and SO2 for all seasons, and NO2 for the summer. AREDV significantly increased following the WTC attacks. Increases in daily O3 values were significantly associated with increases in AREDV in the summer season.

The association of environmental, meteorological, and pollen count variables with asthma-related emergency department visits and hospitalizations in the Bronx.

Objective: To better understand how meteorological variables, air quality variables, and pollen counts collectively contribute to asthma-related emergency department visits (AREDV) and asthma-related hospitalizations (ARH) among pediatric and adult patients in the New York City borough of the Bronx. Methods: The numbers of daily adult and pediatric AREDV and ARH from 2001 to 2008 were obtained from three Bronx hospitals. After removing outliers, interpolating missing data, and standardizing variable values by scaling the data using z-scores, data were analyzed using Spearman rank tests and linear regression models for the full year and each season. Results: There were a total of 42,065 AREDV and 1,664 ARH at both Bronx hospitals. With the exception of a spring peak in AREDVs, AREDVs and ARHs follow a cyclical pattern, climbing in the fall, plateauing in the winter, dropping in the spring, and reaching a low in the summer. Among the 11 air quality, meteorological, and pollen count variables, temperature and tree pollen made the greatest contribution to AREDV with scaled coefficients of -0.337 and 0.311 respectively; equating to an additional AREDV for every 5.0-unit decrease in temperature and an additional AREDV for every 186.0-unit increase in tree pollen. These two variables were confirmed to have independent associations with AREDV prior to the data interpolation. Grass pollen was also found to have a relatively large contribution to AREDV during the summer with a scaled coefficient of 0.314, equating to an additional AREDV for every 2.3-unit increase in grass pollen. Conclusion: There are distinct peaks of increased AREDVs that are closely associated with increased tree pollen counts in the spring and decreasing temperatures in the fall. Early anticipation of these air quality, meteorological, and pollen factor changes based on ongoing surveillance could potentially guide clinical practice and minimize AREDVs in the Bronx.

Changes in antidepressant medication use in the workplace and sustainable employability: a qualitative study of experiences, strategies and solutions.

This study explores patients' experiences of how antidepressant medication transition events (ceasing, changing or reducing) affect employment and workplace functioning. An anonymous online survey was conducted with adults who had experienced antidepressant medication transition events (AMTEs). Data were analyzed using a hybrid inductive and deductive thematic analysis approach. While a majority of participants perceived many positive impacts of antidepressant medication on their workplace functioning, considerable negative effects during AMTEs were reported. Participants provided practical solutions to assist employers, policy and clinicians. Significant and detrimental impact of antidepressant medication changes occurred in the workplace. There is an urgent need to raise awareness of the vulnerability of people during AMTEs and to develop educational and supportive resources to assist clinicians and practitioners to support people during this vulnerable time.

Implementation of an Automated High-Throughput Plasmid DNA Production Pipeline.

Biologics sample management facilities are often responsible for a diversity of large-molecule reagent types, such as DNA, RNAi, and protein libraries. Historically, the management of large molecules was dispersed into multiple laboratories. As methodologies to support pathway discovery, antibody discovery, and protein production have become high throughput, the implementation of automation and centralized inventory management tools has become important. To this end, to improve sample tracking, throughput, and accuracy, we have implemented a module-based automation system integrated into inventory management software using multiple platforms (Hamilton, Hudson, Dynamic Devices, and Brooks). Here we describe the implementation of these systems with a focus on high-throughput plasmid DNA production management.