High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers.

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Indwelling and Retrieval Complications of Denali and Celect Infrarenal Vena Cava Filters.

PURPOSE: To compare indwelling and retrieval complications of Denali and Celect filters placed in the infrarenal inferior vena cava (IVC). MATERIALS AND METHODS: A retrospective study was conducted over 2 years at a single institution in which 171 Denali and 162 Celect filters were placed in 333 patients with a mean age of 62.3 years ± 15.7 (161 men; 48.3%). Filter indications included venous thromboembolic disease (n = 320; 96.1%) and surgical prophylaxis (n = 13; 3.9%). A jugular approach was used to place 303 filters (91.0%). Computed tomography (CT) follow-up, complications, and retrieval data were obtained. RESULTS: Follow-up CT imaging was performed on 58 filters from each group with lower incidences of caval strut penetration (one vs 12) and filter tilt (one vs 15) in the Denali filter group (P = .002 and P < .001, respectively). There was no difference in incidences of breakthrough pulmonary embolism (P = .68). Retrieval attempts were performed on 43 Denali and 53 Celect filters with mean indwelling times at retrieval of 128.2 and 144.1 days, respectively (P = .40). Mean fluoroscopy time at retrieval was lower in the Denali group (3.1 min vs 6.0 min; P = .01). There were fewer cases of complex retrieval in the Denali group (n = 2 vs 10; P = .06). Tilt, fluoroscopy time, and air kerma were associated with complex retrieval (P = .04, P < .001, and P < .001, respectively). There was one Denali filter deployment complication that led to retrieval failure. CONCLUSIONS: This study suggests that Denali filters are associated with lower incidences of strut penetration and filter tilt as well as shorter fluoroscopy time at retrieval compared with Celect filters when placed in the infrarenal IVC.

Optimization and automation of an end-to-end high throughput microscale transient protein production process.

High throughput protein production from transient transfection of mammalian cells is used in multiple facets of research and development studies. Commonly used formats for these high number expressions are 12-, 24- and 96-well plates at various volumes. However there are no published examples of a 96-deep well plate microscale (1,000 µL) suspension process for mammalian transient expression. For this reason, we aimed to determine the optimal operating conditions for a high producing, microscale HEK293 transient system. We evaluated the hydrodynamic flow and measured the oxygen transfer rate (OTR) and transient protein expression for 96-deep well plates of different well geometries filled at 600-1,000 µL working volumes and agitated at various speeds and orbital diameters. Ultimately, a round well-round bottom (RR) 96-deep well plate with a working volume of 1,000 µL agitated at 1,000 RPM and a 3 mm orbital diameter yielded the highest and most consistent total transient protein production. As plate cultures are subject to evaporation, water loss from different plate seals was measured to identify an optimal plate sealing method. Finally, to enable higher capacity protein production, both expression and purification processes were automated. Functionality of this end-to-end automation workflow was demonstrated with the generation of high levels of human IgG1 antibodies (≥360 µg/mL) with reproducible productivity, product quality and ≥78% purification recovery.

Strut penetration: local complications, breakthrough pulmonary embolism, and retrieval failure in patients with Celect vena cava filters.

PURPOSE: To investigate strut penetration in patients with Celect filters, specifically local complications and association with breakthrough pulmonary embolism (PE) or retrieval failure. MATERIALS AND METHODS: A retrospective single-center study was conducted to evaluate patients who received Celect filters between January 2007 and May 2013. A total of 595 filters were placed during the study period. Primary indications included thromboembolic disease (93%) and primary surgical prophylaxis (7%). Complications and retrieval data were assessed by computed tomography (CT) and electronic medical records. RESULTS: A total of 193 patients underwent follow-up abdominal CT at a mean follow-up interval of 176.2 days (range, 0-1,739 d). The rate of strut penetration more than 3 mm outside the caval wall was 28.5% (n = 55). One patient had CT evidence of clinically major strut penetration (1.8%) with strut compression of the right ureter causing hydronephrosis. Indwelling filter time longer than 100 days was associated with strut penetration (P < .001). Age, sex, and history of thromboembolic disease were not associated with strut penetration (P = .51, P = .81, and P = .89). Sixty-three patients presented for follow-up CT pulmonary angiography at a mean of 128.1 days (range, 1-895 d). The rate of breakthrough PE was 12.7%. The overall retrieval success rate was 96.7% (n = 150). Strut penetration was not associated with breakthrough PE or retrieval failure (P = .49 and P = .22). CONCLUSIONS: Although strut penetration is a common complication with Celect filters, there is no association with breakthrough PE or retrieval failure. CT evidence of local complications associated with strut penetration is rare.

Magnetic resonance diffusion tensor imaging (MRDTI) of the optic nerve and optic radiations at 3T in children with neurofibromatosis type I (NF-1).

BACKGROUND: Optic pathway glioma (OPG) is a characteristic hallmark of neurofibromatosis type I (NF-I). OBJECTIVE: To evaluate the feasibility of magnetic resonance diffusion tensor imaging (MRDTI) at 3T to detect abnormalities of the optic nerves and optic radiations in children with NF-I. MATERIALS AND METHODS: 3-T MRDTI was prospectively performed in 9 children with NF-I (7 boys, 2 girls, average age 7.8 years, range 3-17 years) and 44 controls (25 boys, 19 girls, average age 8.1 years, range 3-17 years). Fractional anisotropy (FA) and mean diffusivity were determined by region-of-interest analysis for the optic nerves and radiations. Statistical analysis compared controls to NF-I patients. RESULTS: Two NF-I patients had bilateral optic nerve gliomas, three had chiasmatic gliomas and four had unidentified neurofibromatosis objects (UNOs) along the optic nerve pathways. All NF-I patients had statistically significant decreases in FA and elevations in mean diffusivity in the optic nerves and radiations compared to age-matched controls. CONCLUSION: MRDTI can evaluate the optic pathways in children with NF-I. Statistically significant abnormalities were detected in the diffusion tensor metrics of the optic nerves and radiations in children with NF-I compared to age-matched controls.

Complications and Retrieval Data of Vena Cava Filters Based on Specific Infrarenal Location.

PURPOSE: Although recommended placement of IVC filters is with their tips positioned at the level of the renal vein inflow, in practice, adherence is limited due to clinical situation or IVC anatomy. We seek to evaluate the indwelling and retrieval complications of IVC filters based on their specific position within the infrarenal IVC. MATERIALS AND METHODS: Retrospective, single institution study of 333 consecutive infrarenal vena cava filters placed by interventional radiologists in patients with an average age of 62.2 ± 15.7 years was performed between 2013 and 2015. Primary indication was venous thromboembolic disease (n = 320, 96.1%). Filters were classified based on location of the apex below the lowest renal vein inflow on the procedural venogram: less than 1 cm (n = 180, 54.1%), 1-2 cm (n = 96, 28.8%), and greater than 2 cm (n = 57, 17.1%). Denali (n = 171, 51.4%) and Celect (n = 162, 48.6%) filters were evaluated. CT follow-up, indwelling complications, and retrieval data were obtained. RESULTS: Follow-up CT imaging performed for symptomatic indications occurred for 38.3% of filters placed < 1 cm below the lowest renal vein, 27.1% of filters placed 1-2 cm, and 36.8% placed > 2 cm (p = .16). There was no difference in caval strut penetration, penetration of adjacent viscera, time to penetration, filter migration, or tilt (p = .15, .27, .41, .57, .93). No filter fractures occurred. There was no difference in the incidence of breakthrough PE or complex filter retrieval (p = .83, .59). Only one retrieval failure occurred. CONCLUSIONS: This study suggests filter apex location within the infrarenal IVC, including placement > 2 cm below the level of the renal vein inflow, is not associated with differences in indwelling or retrieval complications. LEVEL OF EVIDENCE: Level 3 non-randomized controlled follow-up study.

The Emprint™ Ablation System with Thermosphere™ Technology: One of the Newer Next-Generation Microwave Ablation Technologies.

Microwave ablation is a recent development in the field of tumor ablation that uses electromagnetic waves to establish a microwave near-field with direct tissue heating. Some of the limitations of the earlier generation devices had been unpredictable size and shape of the ablation zones with changes in the surrounding tissue environment as well as differences across various different tissue types. The Emprint Ablation System with Thermosphere Technology (Covidien, Boulder, CO) is the most recent generation ablation system that attempts to produce predictable large spherical zones of ablation despite varying tissue environments across different tissue types such as liver, lung, and bone to name a few. This article will discuss these recent device developments as well as review some basic microwave characteristics.

Core Lung Biopsy for Biomarker Analysis: Is There Increased Risk Compared With Conventional Biopsy?

PURPOSE: The aim of the study was to compare the rate of pneumothorax and chest tube placement in patients undergoing conventional lung biopsy with those undergoing core lung biopsy for biomarker analysis. MATERIALS AND METHODS: Twenty-three patients had biopsies performed for biomarker analysis (5 male, 18 female patients, mean age 67 y), and 173 patients underwent standard diagnostic lung biopsy (86 male, 87 female patients, mean age 68 y). All biopsies were performed under computed tomography guidance using the coaxial technique (19 G introducer needle and 20 G core biopsy needle). The number of core samples was noted for each case, and all complications were recorded in accordance with Society of Interventional Radiology guidelines. RESULTS: In the biomarker analysis group, a mean of 5.1 core samples (range, 1 to 10) was obtained. In the conventional biopsy group, a mean of 2.9 core samples (range, 1 to 6) was obtained. The pneumothorax rate was 37.6% in the conventional biopsy group and 30.4% in the biomarker analysis group (P=0.505). The rate of chest tube placement was 16.8% in the conventional biopsy group and 8.7% in the biomarker analysis group (P=0.319). Lesion size was found to be an independent predictor of pneumothorax (P=0.031), whereas biopsy tract length was found to be an independent predictor of both pneumothorax (P<0.001) and chest tube placement (P=0.005) upon multivariate analysis. CONCLUSIONS: There is no statistically significant difference in the incidence of pneumothorax or chest tube placement between patients undergoing standard diagnostic lung biopsy and those requiring increased core samples for biomarker analysis.

Dual-lumen chest port infection rates in patients with head and neck cancer.

PURPOSE: The aim of this study was to investigate dual-lumen chest port infection rates in patients with head and neck cancer (HNC) compared to those with other malignancies (non-HNC). MATERIALS AND METHODS: An IRB-approved retrospective study was performed on 1,094 consecutive chest ports placed over a 2-year period. Patients with poor follow-up (n = 53), no oncologic history (n = 13), or single-lumen ports (n = 183) were excluded yielding a study population of 845 patients. The electronic medical records were queried for demographic information, data regarding ports and infections, and imaging review. RESULTS: HNC patients experienced more infections (42 vs. 30), an increased infection rate per 1,000 catheter days (0.68 vs. 0.21), and more early infections within 30 days compared to non-HNC patients (10 vs. 6) (p < 0.001, p < 0.001, p = 0.02, respectively). An existing tracheostomy at the time of port placement was associated with infection in the HNC group (p = 0.02) but was not an independent risk factor for infection in the study population overall (p = 0.06). There was a significant difference in age, male gender, and right-sided ports between the HNC and non-HNC groups (p < 0.01, p < 0.001, and p = 0.01), although these were not found to be independent risk factors for infection (p = 0.32, p = 0.76, p = 0.16). CONCLUSION: HNC patients are at increased risk for infection of dual-lumen chest ports placed via a jugular approach compared to patients with other malignancies. Tracheostomy is associated with infection in HNC patients but is not an independent risk factor for infection in the oncologic population as a whole.

Development of a semi-automated high throughput transient transfection system.

Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.