Quantitative Control of Gene-Engineered T-Cell Activity through the Covalent Attachment of Targeting Ligands to a Universal Immune Receptor.

Universal immune receptors represent a rapidly emerging form of adoptive T-cell therapy with the potential to overcome safety and antigen escape challenges faced by conventional chimeric antigen receptor (CAR) T-cell therapy. By decoupling antigen recognition and T-cell signaling domains via bifunctional antigen-specific targeting ligands, universal immune receptors can regulate T-cell effector function and target multiple antigens with a single receptor. Here, we describe the development of the SpyCatcher immune receptor, the first universal immune receptor that allows for the post-translational covalent attachment of targeting ligands at the T-cell surface through the application of SpyCatcher-SpyTag chemistry. The SpyCatcher immune receptor redirected primary human T cells against a variety of tumor antigens via the addition of SpyTag-labeled targeting ligands, both in vitro and in vivo. SpyCatcher T-cell activity relied upon the presence of both target antigen and SpyTag-labeled targeting ligand, allowing for dose-dependent control of function. The mutational disruption of covalent bond formation between the receptor and the targeting ligand still permitted redirected T-cell function but significantly compromised antitumor function. Thus, the SpyCatcher immune receptor allows for rapid antigen-specific receptor assembly, multiantigen targeting, and controllable T-cell activity.

In Vivo Effects of Retrobulbar Bimatoprost Injection on Orbital Fat.

PURPOSE: Recent publications have reported the adverse effects of prostaglandin analogues on the periocular tissues. These medications may cause periorbital lipodystrophy, enophthalmos, and deepening of the superior sulcus deformity. While these effects may have adverse consequences for some patients, the atrophy of the periorbital fat may have a useful role in diseases that lead to orbital and periorbital fat hypertrophy such as thyroid eye disease. In this pilot study, the authors investigated the effects of retrobulbar bimatoprost injection on the intraocular pressure and orbital fat in a rat animal model. METHODS: Three rats were sedated and intraocular pressure was measured. A 0.1 ml aliquot of bimatoprost was injected into the right orbit of all rats. In the left orbit, 0.1 ml of phosphate-buffered saline was injected as a control. Three weeks later, all rats were sedated and intraocular pressure was measured before euthanizing. Routine histologic staining was performed and thin sections through the intraconal orbital fat were obtained. Density of intraconal adipocytes was measured and adipocyte heterogeneity was determined using a computer image analysis algorithm. RESULTS: The specimens injected with bimatoprost demonstrated atrophy of orbital fat with significantly increased adipocyte density (p = 0.009) and heterogeneity (p = 0.008) when compared with control. Intraocular pressure was not significantly decreased at 3 weeks after injection of retrobulbar bimatoprost. CONCLUSIONS: In this pilot study, orbital injection of bimatoprost demonstrated atrophy of intraconal adipocytes when compared with control orbits injected with saline. The orbits injected with bimatoprost were noted to have smaller, more heterogeneous adipocytes that were densely packed in the intraconal space. The study limitations include the small sample size, which limited the ability for us to make conclusions about the effect on intraocular pressure. Nevertheless, the findings presented suggest that retrobulbar bimatoprost may present a nonsurgical alternative to induce atrophy of the orbital fat without inducing inflammation or hypotony.

LASIC: Light Activated Site-Specific Conjugation of Native IgGs.

Numerous biological applications, from diagnostic assays to immunotherapies, rely on the use of antibody-conjugates. The efficacy of these conjugates can be significantly influenced by the site at which Immunoglobulin G (IgG) is modified. Current methods that provide control over the conjugation site, however, suffer from a number of shortfalls and often require large investments of time and cost. We have developed a novel adapter protein that, when activated by long wavelength UV light, can covalently and site-specifically label the Fc region of nearly any native, full-length IgG, including all human IgG subclasses. Labeling occurs with unprecedented efficiency and speed (>90% after 30 min), with no effect on IgG affinity. The adapter domain can be bacterially expressed and customized to contain a variety of moieties (e.g., biotin, azide, fluorophores), making reliable and efficient conjugation of antibodies widely accessible to researchers at large.

Optimization of photoactive protein Z for fast and efficient site-specific conjugation of native IgG.

Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody's antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community.

Volume and composition of reflux after intravitreal injection.

PURPOSE: To quantify the amount of drug loss from cadaveric human eyes, which are injected via the pars plana with a known volume of dye at variable intraocular pressures. METHODS: Eight cadaver eyes were divided into 2 intraocular pressure groups: normal (15 mmHg; 4 eyes) or high (30 mmHg; 4 eyes). Each eye was injected with 50 µL of hematoxylin dye, and the subsequent reflux was immediately collected on a Schirmer's test strip. The test strip was scanned and digitally analyzed to determine the area of saturation and total color intensity present. Using a previously established equation, total volume of reflux and the amount of dye within that reflux were calculated. RESULTS: The average total volume of refluxed fluid was 1.68 µL (median, 0.62 µL), with a range of 0 µL to 8.05 µL. The average volume of refluxed dye was 0.37 µL (median, 0.08 µL), with a range of 0 µL to 2.15 µL. On average, only 0.74% of the original 50 µL of injected dye was lost (median, 0.15%), with a range from 0% to 4.30%. CONCLUSION: Although the presence of subconjunctival bleb formation after intravitreal injection may be a concern to the clinician, data from the present study shows that only a very small amount of the injected therapeutic agent is lost in the reflux.

Simultaneous quantification of tumor uptake for targeted and nontargeted liposomes and their encapsulated contents by ICPMS.

Liposomes are intensively being developed for biomedical applications including drug and gene delivery. However, targeted liposomal delivery in cancer treatment is a very complicated multistep process. Unfavorable liposome biodistribution upon intravenous administration and membrane destabilization in blood circulation could result in only a very small fraction of cargo reaching the tumors. It would therefore be desirable to develop new quantitative strategies to track liposomal delivery systems to improve the therapeutic index and decrease systemic toxicity. Here, we developed a simple and nonradiative method to quantify the tumor uptake of targeted and nontargeted control liposomes as well as their encapsulated contents simultaneously. Specifically, four different chelated lanthanide metals were encapsulated or surface-conjugated onto tumor-targeted and nontargeted liposomes, respectively. The two liposome formulations were then injected into tumor-bearing mice simultaneously, and their tumor delivery was determined quantitatively via inductively coupled plasma mass spectroscopy (ICPMS), allowing for direct comparisons. Tumor uptake of the liposomes themselves and their encapsulated contents was consistent with targeted and nontargeted liposome formulations that were injected individually.

Practical computer vision application to detect hip fractures on pelvic X-rays: a bi-institutional study.

BACKGROUND: Pelvic X-ray (PXR) is a ubiquitous modality to diagnose hip fractures. However, not all healthcare settings employ round-the-clock radiologists and PXR sensitivity for diagnosing hip fracture may vary depending on digital display. We aimed to validate a computer vision algorithm to detect hip fractures across two institutions' heterogeneous patient populations. We hypothesized a convolutional neural network algorithm can accurately diagnose hip fractures on PXR and a web application can facilitate its bedside adoption. METHODS: The development cohort comprised 4235 PXRs from Chang Gung Memorial Hospital (CGMH). The validation cohort comprised 500 randomly sampled PXRs from CGMH and Stanford's level I trauma centers. Xception was our convolutional neural network structure. We randomly applied image augmentation methods during training to account for image variations and used gradient-weighted class activation mapping to overlay heatmaps highlighting suspected fracture locations. RESULTS: Our hip fracture detection algorithm's area under the receiver operating characteristic curves were 0.98 and 0.97 for CGMH and Stanford's validation cohorts, respectively. Besides negative predictive value (0.88 Stanford cohort), all performance metrics-sensitivity, specificity, predictive values, accuracy, and F1 score-were above 0.90 for both validation cohorts. Our web application allows users to upload PXR in multiple formats from desktops or mobile phones and displays probability of the image containing a hip fracture with heatmap localization of the suspected fracture location. DISCUSSION: We refined and validated a high-performing computer vision algorithm to detect hip fractures on PXR. A web application facilitates algorithm use at the bedside, but the benefit of using our algorithm to supplement decision-making is likely institution dependent. Further study is required to confirm clinical validity and assess clinical utility of our algorithm. LEVEL OF EVIDENCE: III, Diagnostic tests or criteria.

Diagnostic performance of lower extremity Doppler ultrasound in detecting iliocaval obstruction.

OBJECTIVE: To retrospectively evaluate the performance of two commonly used Doppler ultrasound parameters, namely, venous flow phasicity and response to Valsalva maneuver, in detecting iliocaval obstruction. METHODS: All imaging studies of patients seen by interventional radiology for lower extremity venous disease at a single institution from 1996 to 2018 were retrospectively identified. Lower extremity ultrasounds with a concurrent magnetic resonance, computed tomography, or conventional venogram performed within the next 7 days, which served as gold standard, were further identified (n = 192 examinations, including 313 limbs). Iliocaval obstruction were assessed by two ultrasound criteria: (1) nonphasic flow and/or (2) nonresponsive flow to Valsalva in the common femoral vein. The sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) for diagnosing iliocaval obstruction were calculated for each ultrasound criterion, and also for when the two criteria were assessed jointly. RESULTS: Of the 313 limbs assessed for venous flow phasicity, 133 (42.5%) had an iliocaval obstruction confirmed on subsequent venography. Nonphasic flow demonstrated a sensitivity of 69.2%, specificity of 82.8%, NPV of 78.4%, and PPV of 74.8% for diagnosing iliocaval obstruction. Of the 212 limbs assessed for Valsalva response, 88 (41.5%) had a confirmed iliocaval obstruction. Nonresponsive flow to Valsalva demonstrated a sensitivity of 13.6%, specificity of 97.6%, NPV of 61.6%, and PPV of 80.0% for diagnosing iliocaval obstruction. Joint assessment using phasicity and Valsalva criteria demonstrated a sensitivity of 68.2%, specificity of 87.2%, NPV of 79.6%, and PPV of 78.9%. CONCLUSIONS: In this tertiary care setting, Doppler ultrasound examination was not a reliable diagnostic tool for detecting iliocaval obstruction.

Biodistribution, Clearance, and Toxicology of Polymeric Micelles Loaded with 0.9 or 5 nm Gold Nanoparticles.

Long-circulating gold nanoparticles (AuNPs) have garnered a great deal of interest as both imaging and therapeutic agents. However, their protracted elimination and long-term persistence within many organ systems remains a concern for clinical translation. To improve the excretion of long-circulating nanoparticles, we prepared -80 nm biodegradable polymeric micelles with 0.9 nm or 5 nm AuNPs tightly packed within the hydrophobic core. These gold-loaded polymeric micelles (GPMs) were expected to allow for improved excretion of gold, compared with single large AuNPs, owing to the smaller size and larger surface-to-volume ratio of the individual AuNPs within the micelle. Following intravenous administration of GPMs, organs were harvested and examined for gold content using inductively coupled plasma optical emission spectrometry (ICP-OES) for up to 3 months post-injection. While both GPM formulations showed significant clearance of gold over time, micelles containing 0.9 nm AuNPs showed a 72% and 67% reduction in gold content in the liver and spleen, respectively, between 1 day and 3 months post-injection, compared with a 38% and 35% reduction in mice receiving 5 nm GPMs. Furthermore, feces and urine analysis revealed approximately 7.5 and 100 times more gold, respectively, in mice that received 0.9 nm GPMs one day after injection. These findings suggest that the excretion profile of inorganic nanomaterials may be improved if clusters of small inorganic materials are used in favor of single solid particles.

A novel method for the measurement of reflux from intravitreal injections: data from 20 porcine eyes.

BACKGROUND: Reflux following intravitreal injection is a common phenomenon, but it is unknown how much, if any, medication is lost as a result. Reflux is known to be a combination of vitreous and the injected agent, but the relative composition is unknown. This article describes a novel method for the measurement of the volume and composition of reflux and presents data from porcine eyes. METHODS: Twenty porcine eyes were injected with 0.05 ml of dye at intraocular pressures (IOPs) of 15, 20, 25 and 30 mmHg (five eyes per subgroup). Reflux was captured on filter paper and the area of saturation and color intensity of the dye were digitally analyzed. Total refluxed volume and proportion of dye versus vitreous fluid were calculated from linear regression lines created from known standards. RESULTS: Average (median) total volume of reflux from all eyes was 1.19 µl (0.93 µl), volume of injected dye refluxed was 0.47 µl (0.11 µl) and composition of reflux was 20.8% dye (15.5%). Less than 1% of the injected dye was lost to reflux. There were no differences between IOP groups in the total volume refluxed, the total amount of dye refluxed, the average composition of the reflux or the amount of injected dye refluxed (df = 3 for all comparisons; p = 0.58, p = 0.51, p = 0.55, p = 0.51, respectively). CONCLUSIONS: This novel method allows for measurement of quantity and composition of reflux following intravitreal injection in vitro. While reflux occurs frequently, it is predominantly composed of vitreous, not the injected agent. In fact, <1% of the original injection was lost to reflux.

Multifunctional nanoparticles: cost versus benefit of adding targeting and imaging capabilities.

Nanoparticle-based drug delivery systems have been developed to improve the efficacy and reduce the systemic toxicity of a wide range of drugs. Although clinically approved nanoparticles have consistently shown value in reducing drug toxicity, their use has not always translated into improved clinical outcomes. This has led to the development of "multifunctional" nanoparticles, where additional capabilities like targeting and image contrast enhancement are added to the nanoparticles. However, additional functionality means additional synthetic steps and costs, more convoluted behavior and effects in vivo, and also greater regulatory hurdles. The trade-off between additional functionality and complexity is the subject of ongoing debate and the focus of this Review.