Early Convalescent Plasma for High-Risk Outpatients with Covid-19.

BACKGROUND: Early administration of convalescent plasma obtained from blood donors who have recovered from coronavirus disease 2019 (Covid-19) may prevent disease progression in acutely ill, high-risk patients with Covid-19. METHODS: In this randomized, multicenter, single-blind trial, we assigned patients who were being treated in an emergency department for Covid-19 symptoms to receive either one unit of convalescent plasma with a high titer of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or placebo. All the patients were either 50 years of age or older or had one or more risk factors for disease progression. In addition, all the patients presented to the emergency department within 7 days after symptom onset and were in stable condition for outpatient management. The primary outcome was disease progression within 15 days after randomization, which was a composite of hospital admission for any reason, seeking emergency or urgent care, or death without hospitalization. Secondary outcomes included the worst severity of illness on an 8-category ordinal scale, hospital-free days within 30 days after randomization, and death from any cause. RESULTS: A total of 511 patients were enrolled in the trial (257 in the convalescent-plasma group and 254 in the placebo group). The median age of the patients was 54 years; the median symptom duration was 4 days. In the donor plasma samples, the median titer of SARS-CoV-2 neutralizing antibodies was 1:641. Disease progression occurred in 77 patients (30.0%) in the convalescent-plasma group and in 81 patients (31.9%) in the placebo group (risk difference, 1.9 percentage points; 95% credible interval, -6.0 to 9.8; posterior probability of superiority of convalescent plasma, 0.68). Five patients in the plasma group and 1 patient in the placebo group died. Outcomes regarding worst illness severity and hospital-free days were similar in the two groups. CONCLUSIONS: The administration of Covid-19 convalescent plasma to high-risk outpatients within 1 week after the onset of symptoms of Covid-19 did not prevent disease progression. (SIREN-C3PO ClinicalTrials.gov number, NCT04355767.).

Prediction of in-hospital deterioration in normotensive pulmonary embolism remains elusive: external validation of the calgary acute pulmonary embolism score.

Acute pulmonary embolism (PE) is a frequently diagnosed condition. Prediction of in-hospital deterioration is challenging with current risk models. The Calgary Acute Pulmonary Embolism (CAPE) score was recently derived to predict in-hospital adverse PE outcomes but has not yet been externally validated. Retrospective cohort study of normotensive acute pulmonary embolism cases diagnosed in our emergency department between 2017 and 2019. An external validation of the CAPE score was performed in this population for prediction of in-hospital adverse outcomes and a secondary outcome of 30-day all-cause mortality. Performance of the simplified Pulmonary Embolism Severity Index (sPESI) and Bova score was also evaluated. 712 patients met inclusion and exclusion criteria, with 536 patients having a sPESI score of 1 or more. Among this population, the CAPE score had a weak discriminative power to predict in-hospital adverse outcomes, with a calculated c-statistic of 0.57. In this study population, an external validation study found weak discriminative power of the CAPE score to predict in-hospital adverse outcomes among normotensive PE patients. Further efforts are needed to define risk assessment models that can identify normotensive PE patients at risk for in hospital deterioration. Identification of such patients will better guide intensive care utilization and invasive procedural management of PE.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

Targeted In Vivo Loading of Red Blood Cells Markedly Prolongs Nanocarrier Circulation.

Engineering drug delivery systems for prolonged pharmacokinetics (PK) has been an ongoing pursuit for nearly 50 years. The gold standard for PK enhancement is the coating of nanoparticles with polymers, namely polyethylene glycol (PEGylation), which has been applied in several clinically used products. In the present work, we utilize the longest circulating and most abundant component of blood─the erythrocyte─to improve the PK behavior of liposomes. Antibody-mediated coupling of liposomes to erythrocytes was tested in vitro to identify a loading dose that did not adversely impact the carrier cells. Injection of erythrocyte targeting liposomes into mice resulted in a ∼2-fold improvement in the area under the blood concentration versus time profile versus PEGylated liposomes and a redistribution from the plasma into the cellular fraction of blood. These results suggest that in vivo targeting of erythrocytes is a viable strategy to improve liposome PK relative to current, clinically viable strategies.

Molecularly Engineered Nanobodies for Tunable Pharmacokinetics and Drug Delivery.

The use of single-domain antibody fragments, or nanobodies, has gained popularity in recent years as an alternative to traditional monoclonal antibody-based approaches. Relatively little is known, however, about the utility of nanobodies as targeting agents for delivery of therapeutic cargoes, particularly to vascular epitopes or in the setting of acute inflammatory conditions. We used a nanobody (VCAMelid) directed against mouse vascular cell adhesion molecule 1 (VCAM-1) and techniques for site-specific radiolabeling and bioconjugation to measure targeting to sites of constitutive and inducible antigen expression and investigate the impact of various characteristics (affinity, valence, circulation time) on nanobody biodistribution and pharmacokinetics. Engineering of VCAMelid for bivalent binding (BiVCAMelid) increased affinity by an order of magnitude and provided 2.8- and 3.6-fold enhancements in splenic and brain targeting in naive mice, with a further 2.6-fold increase in brain uptake in the setting of focal CNS inflammation. In contrast, introduction of an albumin-binding arm (VCAM/ALB8) did not affect binding affinity, but its prolonged circulation time resulted in 3.5-fold and 17.4-fold increases in splenic and brain uptake at 20 min post-dose and remarkable 40-, 25-, and 15-fold enhancements in overall exposure of blood, spleen, and brain, respectively, relative to both VCAMelid and BiVCAMelid. Both therapeutic protein (superoxide dismutase, SOD-1) and nanocarrier (liposome) delivery were enhanced by conjugation to VCAM-1 targeted nanobodies. The bispecific VCAM/ALB8 maintained its superiority over VCAMelid in enhancing both circulation time and organ targeting of SOD-1, but its advantages were largely blunted by conjugation to liposomes.

Shoulder Pseudodislocation Associated with Calcific Tendinitis/Bursitis and Diagnosed by Point of Care Ultrasound.

BACKGROUND: Shoulder pseudodislocation, or "drooping shoulder," presents with acute pain and deformity of the joint, with radiographs demonstrating inferior subluxation of the humeral head relative to the glenoid fossa. The diagnosis must be made promptly and distinguished from true glenohumeral dislocation, both to avoid unnecessary attempts at closed reduction and to facilitate investigation of the underlying cause, which may include septic arthritis, hemarthrosis, or other emergent etiologies. Point-of-care ultrasound (POCUS) may be useful in the evaluation of emergency department (ED) patients with suspected pseudodislocation. CASE REPORT: A 50-year old female presented to the ED with an acutely painful and deformed shoulder but atypical history and physical examination. Initial radiography appeared to show a glenohumeral dislocation, but POCUS, done to guide intra-articular lidocaine injection, led to recognition of pseudodislocation and subsequent diagnosis of calcific tendinitis/bursitis, a condition not previously associated with inferior humeral subluxation. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Shoulder pseudodislocation must be considered in the evaluation of patients with suspected glenohumeral dislocation, but atypical features on history, physical examination, or initial plain radiography. POCUS may facilitate prompt diagnosis and identification of the underlying etiology.

Vascular Targeting of Radiolabeled Liposomes with Bio-Orthogonally Conjugated Ligands: Single Chain Fragments Provide Higher Specificity than Antibodies.

Liposomes are a proven, versatile, and clinically viable technology platform for vascular delivery of drugs and imaging probes. Although targeted liposomes have the potential to advance these applications, complex formulations and the need for optimal affinity ligands and conjugation strategies challenge their translation. Herein, we employed copper-free click chemistry functionalized liposomes to target platelet-endothelial cell adhesion molecule (PECAM-1) and intracellular adhesion molecule (ICAM-1) by conjugating clickable monoclonal antibodies (Ab) or their single chain variable fragments (scFv). For direct, quantitative tracing, liposomes were surface chelated with 111In to a >90% radiochemical yield and purity. Particle size and distribution, stability, ligand surface density, and specific binding to target cells were characterized in vitro. Biodistribution of liposomes after IV injection was characterized in mice using isotope detection in organs and by noninvasive imaging (single-photon emission computed tomography/computed tomography, SPECT/CT). As much as 20-25% of injected dose of liposomes carrying PECAM and ICAM ligands, but not control IgG accumulated in the pulmonary vasculature. The immunospecificity of pulmonary targeting of scFv/liposomes to PECAM-1 and ICAM-1, respectively, was 10-fold and 2.5-fold higher than of Ab/liposomes. Therefore, the combination of optimal ligands, benign conjugation, and labeling yields liposomal formulations that may be used for highly effective and specific vascular targeting.

Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging.

Genetic incorporation of biologically orthogonal functional groups into macromolecules has the potential to yield efficient, controlled, reproducible, site-specific conjugation of affinity ligands, contrast agents, or therapeutic cargoes. Here, we applied this approach to ferritin, a ubiquitous iron-storage protein that self-assembles into multimeric nanocages with remarkable stability, size uniformity (12 nm), and endogenous capacity for loading and transport of a variety of inorganic and organic cargoes. The unnatural amino acid, 4-azidophenylalanine (4-AzF), was incorporated at different sites in the human ferritin light chain (hFTL) to allow site-specific conjugation of alkyne-containing small molecules or affinity ligands to the exterior surface of the nanocage. The optimal positioning of the 4-AzF residue was evaluated by screening a library of variants for the efficiency of copper-free click conjugation. One of the engineered ferritins, hFTL-5X, was found to accommodate ∼14 small-molecule fluorophores (AlexaFluor 488) and 3-4 IgG molecules per nanocage. Intravascular injection in mice of radiolabeled hFTL-5X carrying antibody to cell adhesion molecule ICAM-1, but not control IgG, enabled specific targeting to the lung due to high basal expression of ICAM-1 (43.3 ± 6.99 vs 3.48 ± 0.14%ID/g for Ab vs IgG). Treatment of mice with endotoxin known to stimulate inflammatory ICAM-1 overexpression resulted in 2-fold enhancement of pulmonary targeting (84.4 ± 12.89 vs 43.3 ± 6.99%ID/g). Likewise, injection of fluorescent, ICAM-targeted hFTL-5X nanocages revealed the effect of endotoxin by enhancement of near-infrared signal, indicating potential utility of this approach for both vascular targeting and imaging.

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting.

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo in vitro and in vivo.

Targeting thrombomodulin to circulating red blood cells augments its protective effects in models of endotoxemia and ischemia-reperfusion injury.

Endothelial thrombomodulin (TM) regulates coagulation and inflammation via several mechanisms, including production of activated protein C (APC). Recombinant APC and soluble fragments of TM (sTM) have been tested in settings associated with insufficiency of the endogenous TM/APC pathway, such as sepsis. We previously designed a fusion protein of TM [single-chain variable fragment antibody (scFv)/TM] targeted to red blood cells (RBCs) to improve pharmacokinetics and antithrombotic effects without increasing bleeding. Here, scFv/TM was studied in mouse models of systemic inflammation and ischemia-reperfusion injury. Injected concomitantly with or before endotoxin, scFv/TM provided more potent protection against liver injury and release of pathological mediators than sTM, showing similar efficacy at up to 50-fold lower doses. scFv/TM provided protection when injected after endotoxin, whereas sTM did not, and augmented APC production by thrombin ∼50-fold more than sTM. However, scFv/TM injected after endotoxin did not reduce thrombin/antithrombin complexes; nor did antibodies that block APC anticoagulant activity suppress the prophylactic anti-inflammatory effect of scFv/TM. Therefore, similar to endogenous TM, RBC-anchored scFv/TM activates several protective pathways. Finally, scFv/TM was more effective at reducing cerebral infarct volume and alleviated neurological deficits than sTM after cerebral ischemia/reperfusion injury. These results indicate that RBC-targeted scFv/TM exerts multifaceted cytoprotective effects and may find utility in systemic and focal inflammatory and ischemic disorders.-Carnemolla, R., Villa, C. H., Greineder, C. F., Zaitseva, S., Patel, K. R., Kowalska, M. A., Atochin, D. N., Cines, D. B., Siegel, D. L., Esmon, C. T., Muzykantov, V. R. Targeting thrombomodulin to circulating red blood cells augments its protective effects in models of endotoxemia and ischemia-reperfusion injury.

Mechanisms that determine nanocarrier targeting to healthy versus inflamed lung regions.

Inflamed organs display marked spatial heterogeneity of inflammation, with patches of inflamed tissue adjacent to healthy tissue. To investigate how nanocarriers (NCs) distribute between such patches, we created a mouse model that recapitulates the spatial heterogeneity of the inflammatory lung disease ARDS. NCs targeting the epitope PECAM strongly accumulated in the lungs, but were shunted away from inflamed lung regions due to hypoxic vasoconstriction (HVC). In contrast, ICAM-targeted NCs, which had lower whole-lung uptake than PECAM/NCs in inflamed lungs, displayed markedly higher NC levels in inflamed regions than PECAM/NCs, due to increased regional ICAM. Regional HVC, epitope expression, and capillary leak were sufficient to predict intra-organ of distribution of NCs, antibodies, and drugs. Importantly, these effects were not observable with traditional spatially-uniform models of ARDS, nor when examining only whole-organ uptake. This study underscores how examining NCs' intra-organ distribution in spatially heterogeneous animal models can guide rational NC design.

Vascular Accessibility of Endothelial Targeted Ferritin Nanoparticles.

Targeting nanocarriers to the endothelium, using affinity ligands to cell adhesion molecules such as ICAM-1 and PECAM-1, holds promise to improve the pharmacotherapy of many disease conditions. This approach capitalizes on the observation that antibody-targeted carriers of 100 nm and above accumulate in the pulmonary vasculature more effectively than free antibodies. Targeting of prospective nanocarriers in the 10-50 nm range, however, has not been studied. To address this intriguing issue, we conjugated monoclonal antibodies (Ab) to ICAM-1 and PECAM-1 or their single chain antigen-binding fragments (scFv) to ferritin nanoparticles (FNPs, size 12 nm), thereby producing Ab/FNPs and scFv/FNPs. Targeted FNPs retained their typical symmetric core-shell structure with sizes of 20-25 nm and ∼4-5 Ab (or ∼7-9 scFv) per particle. Ab/FNPs and scFv/FNPs, but not control IgG/FNPs, bound specifically to cells expressing target molecules and accumulated in the lungs after intravenous injection, with pulmonary targeting an order of magnitude higher than free Ab. Most intriguing, the targeting of Ab/FNPs to ICAM-1, but not PECAM-1, surpassed that of larger Ab/carriers targeted by the same ligand. These results indicate that (i) FNPs may provide a platform for targeting endothelial adhesion molecules with carriers in the 20 nm size range, which has not been previously reported; and (ii) ICAM-1 and PECAM-1 (known to localize in different domains of endothelial plasmalemma) differ in their accessibility to circulating objects of this size, common for blood components and nanocarriers.

Dual targeting of therapeutics to endothelial cells: collaborative enhancement of delivery and effect.

Anchoring pharmacologic agents to the vascular lumen has the potential to modulate critical processes at the blood-tissue interface, avoiding many of the off-target effects of systemically circulating agents. We report a novel strategy for endothelial dual targeting of therapeutics, which both enhances drug delivery and enables targeted agents to partner enzymatically to generate enhanced biologic effect. Based on the recent discovery that paired antibodies directed to adjacent epitopes of platelet endothelial cell adhesion molecule (PECAM)-1 stimulate each other's binding, we fused single-chain fragments (scFv) of paired anti-mouse PECAM-1 antibodies to recombinant murine thrombomodulin (TM) and endothelial protein C receptor (EPCR), endothelial membrane proteins that partner in activation of protein C (PC). scFv/TM and scFv/EPCR bound to mouse endothelial PECAM-1 with high affinity (EC50 1.5 and 3.8 nM, respectively), and codelivery induced a 5-fold increase in PC activation not seen when TM and EPCR are anchored to distinct cell adhesion molecules. In a mouse model of acute lung injury, dual targeting reduces both the expression of lung inflammatory markers and trans-endothelial protein leak by as much as 40%, as compared to either agent alone. These findings provide proof of principle for endothelial dual targeting, an approach with numerous potential biomedical applications.

Advanced drug delivery systems for antithrombotic agents.

Despite continued achievements in antithrombotic pharmacotherapy, difficulties remain in managing patients at high risk for both thrombosis and hemorrhage. Utility of antithrombotic agents (ATAs) in these settings is restricted by inadequate pharmacokinetics and narrow therapeutic indices. Use of advanced drug delivery systems (ADDSs) may help to circumvent these problems. Various nanocarriers, affinity ligands, and polymer coatings provide ADDSs that have the potential to help optimize ATA pharmacokinetics, target drug delivery to sites of thrombosis, and sense pathologic changes in the vascular microenvironment, such as altered hemodynamic forces, expression of inflammatory markers, and structural differences between mature hemostatic and growing pathological clots. Delivery of ATAs using biomimetic synthetic carriers, host blood cells, and recombinant fusion proteins that are activated preferentially at sites of thrombus development has shown promising outcomes in preclinical models. Further development and translation of ADDSs that spare hemostatic fibrin clots hold promise for extending the utility of ATAs in the management of acute thrombotic disorders through rapid, transient, and targeted thromboprophylaxis. If the potential benefit of this technology is to be realized, a systematic and concerted effort is required to develop clinical trials and translate the use of ADDSs to the clinical arena.

Targeting to endothelial cells augments the protective effect of novel dual bioactive antioxidant/anti-inflammatory nanoparticles.

Oxidative stress and inflammation are intertwined contributors to numerous acute vascular pathologies. A novel dual bioactive nanoparticle with antioxidant/anti-inflammatory properties was developed based on the interactions of tocopherol phosphate and the manganese porphyrin SOD mimetic, MnTMPyP. The size and drug incorporation efficiency were shown to be dependent on the amount of MnTMPyP added as well as the choice of surfactant. MnTMPyP was shown to retain its SOD-like activity while in intact particles and to release in a slow and controlled manner. Conjugation of anti-PECAM antibody to the nanoparticles provided endothelial targeting and potentiated nanoparticle-mediated suppression of inflammatory activation of these cells manifested by expression of VCAM, E-selectin, and IL-8. This nanoparticle technology may find applicability with drug combinations relevant for other pathologies.

Bispecific antibody shuttles targeting CD98hc mediate efficient and long-lived brain delivery of IgGs.

The inability of antibodies to penetrate the blood-brain barrier (BBB) is a key limitation to their use in diverse applications. One promising strategy is to deliver IgGs using a bispecific BBB shuttle, which involves fusing an IgG to a second affinity ligand that engages a cerebrovascular endothelial target and facilitates transport across the BBB. Nearly all prior efforts have focused on shuttles that target transferrin receptor (TfR-1) despite inherent delivery and safety challenges. Here, we report bispecific antibody shuttles that engage CD98hc, the heavy chain of the large neutral amino acid transporter (LAT1), and efficiently transport IgGs into the brain. Notably, CD98hc shuttles lead to much longer-lived brain retention of IgGs than TfR-1 shuttles while enabling more specific targeting due to limited CD98hc engagement in the brain parenchyma, which we demonstrate for IgGs that either agonize a neuronal receptor (TrkB) or target other endogenous cell-surface proteins on neurons and astrocytes.

Optimizing Use of High-Sensitivity Troponin for Risk-Stratification of Acute Pulmonary Embolism.

BACKGROUND: High-sensitivity troponin T (HS-TnT) may improve risk-stratification in hemodynamically stable acute pulmonary embolism (PE), but an optimal strategy for combining this biomarker with clinical risk-stratification tools has not been determined. STUDY HYPOTHESIS: We hypothesized that different HS-TnT cutoff values may be optimal for identifying (1) low-risk patients who may be eligible for outpatient management and (2) patients at increased risk of clinical deterioration who might benefit from advanced PE therapies. METHODS: Retrospective analysis of hemodynamically stable patients in the University of Michigan acute ED-PE registry with available HS-TnT values. Primary and secondary outcomes were 30-day mortality and need for intensive care unit-level care. Receiver operating characteristic curves were used to determine optimal HS-TnT cutoffs in the entire cohort, and for those at higher risk based on the simplified Pulmonary Embolism Severity Index (PESI) or imaging findings. RESULTS: The optimal HS-TnT cutoff in the full cohort, 12 pg/mL, was significantly associated with 30-day mortality (odds ratio [OR]: 3.94, 95% confidence interval [CI]: 1.48-10.50) and remained a significant predictor after adjusting for the simplified PESI (sPESI) score and serum creatinine (adjusted OR: 3.05, 95% CI: 1.11-8.38). A HS-TnT cutoff of 87 pg/mL was associated with 30-day mortality (OR: 5.01, 95% CI: 2.08-12.06) in patients with sPESI ≥1 or right ventricular dysfunction. CONCLUSION: In this retrospective, single-center study of acute PE patients, we identified distinct optimal HS-TnT values for different clinical uses-a lower cutoff, which identified low-risk patients even in the absence of other risk-stratification methods, and a higher cutoff, which was strongly associated with adverse outcomes in patients at increased risk.

Affinity targeting of therapeutic proteins to the bone surface - local delivery of sclerostin neutralizing antibody enhances efficacy.

Currently available biotherapeutics for the treatment of osteoporosis lack explicit mechanisms for bone localization, potentially limiting efficacy and inducing unintended off-target toxicities. While various strategies have been explored for targeting the bone surface, critical aspects remain poorly understood, including the optimal affinity ligand, the role of binding avidity and circulation time, and, perhaps most importantly, whether or not this strategy can enhance the functional activity of clinically relevant protein therapeutics. To investigate, we generated fluorescent proteins (e.g., mCherry) with site-specifically attached small molecule (bisphosphonate, BP) or peptide (deca-aspartate, D10) affinity ligands. While both affinity ligands successfully anchored fluorescent protein to the bone surface, quantitative radiotracing revealed only modest femoral and vertebral accumulation and suggested a need for enhanced circulation time. To achieve this, we fused mCherry to the Fc fragment of human IgG1 and attached D10 peptides to each C-terminus. mCherry-Fc-D10 demonstrated ~80-fold increase in plasma exposure and marked increases in femoral and vertebral accumulation (13.6 ± 1.4% and 11.4 ± 1.3% of the injected dose/gram [%ID/g] at 24 hours, respectively). To determine if bone surface targeting could enhance the efficacy of a clinically relevant therapeutic, we generated a bone-targeted sclerostin neutralizing antibody, anti-sclerostin-D10. The targeted antibody demonstrated marked increases in bone accumulation and retention (20.9 ± 2.5% and 19.5 ± 2.5% ID/g in femur and vertebrae at 7 days) and enhanced effects in a murine model of ovariectomy-induced bone loss (BV/TV, connectivity density, and structure model index all increased [p < 0.001] vs. untargeted anti-sclerostin). Collectively, our results indicate the importance of both bone affinity and circulation time in achieving robust targeting of therapeutic proteins to the bone surface and suggest that this approach may enable lower doses and/or longer dosing intervals without reduction in biotherapeutic efficacy. Future studies will be needed to determine the translational potential of this strategy and its potential impact on off-site toxicities.

Several biologic therapies have been approved for osteoporosis, but they lack means of localization to bone tissue, potentially limiting their efficacy and leading to off-target toxicities. This manuscript investigates strategies for targeting biotherapeutics to the bone surface and asks the question of whether or not this approach can enhance functional activity and allow for lower or less frequent dosing. To define the key determinants of bone surface targeting, we begin by synthesizing fluorescent model proteins with different bone targeting tags. We show that even one tag is enough to make the surface of the femur and vertebrae fluorescent following systemic administration. The results are relatively modest at first, but when we combine the bone targeting tag with a second modification that makes the protein circulate in the body for a longer period of time, we observe a huge increase in bone surface delivery. We then synthesize a bone surface targeted version of a sclerostin-inhibiting antibody and show that it is more effective than the untargeted antibody and provides near complete protection of bone density despite relatively low dose. Our findings could have translational implications for existing bone therapies and help guide design of future strategies for optimized bone surface targeting.

Platelet endothelial cell adhesion molecule targeted oxidant-resistant mutant thrombomodulin fusion protein with enhanced potency in vitro and in vivo.

Thrombomodulin (TM) is a glycoprotein normally present in the membrane of endothelial cells that binds thrombin and changes its substrate specificity to produce activated protein C (APC) that has antithrombotic and anti-inflammatory features. To compensate for loss of endogenous TM in pathology, we have fused recombinant TM with single chain variable fragment (scFv) of an antibody to mouse platelet endothelial cell adhesion molecule-1 (PECAM). This fusion, anti-PECAM scFv/TM, anchors on the endothelium, stimulates APC production, and provides therapeutic benefits superior to sTM in animal models of acute thrombosis and inflammation. However, in conditions of oxidative stress typical of vascular inflammation, TM is inactivated via oxidation of the methionine 388 (M388) residue. Capitalizing on the reports that M388L mutation renders TM resistant to oxidative inactivation, in this study we designed a mutant anti-PECAM scFv/TM M388L. This mutant has the same APC-producing capacity and binding to target cells, yet, in contrast to wild-type fusion, it retains APC-producing activity in an oxidizing environment in vitro and in vivo. Therefore, oxidant resistant mutant anti-PECAM scFv/TM M388L is a preferable targeted biotherapeutic to compensate for loss of antithrombotic and anti-inflammatory TM functions in the context of vascular oxidative stress.