On Demand Bioorthogonal Switching of an Antibody-Conjugated SPECT Probe to a Cytotoxic Payload: from Imaging to Therapy.

Antibody-drug conjugates (ADCs) for the treatment of cancer aim to achieve selective delivery of a cytotoxic payload to tumor cells while sparing normal tissue. In vivo, multiple tumor-dependent and -independent processes act on ADCs and their released payloads to impact tumor-versus-normal delivery, often resulting in a poor therapeutic window. An ADC with a labeled payload would make synchronous correlations between distribution and tissue-specific pharmacological effects possible, empowering preclinical and clinical efforts to improve tumor-selective delivery; however, few methods to label small molecules without destroying their pharmacological activity exist. Herein, we present a bioorthogonal switch approach that allows a radiolabel attached to an ADC payload to be removed tracelessly at will. We exemplify this approach with a potent DNA-damaging agent, the pyrrolobenzodiazepine (PBD) dimer, delivered as an antibody conjugate targeted to lung tumor cells. The radiometal chelating group, DOTA, was attached via a novel trans-cyclooctene (TCO)-caged self-immolative para-aminobenzyl (PAB) linker to the PBD, stably attenuating payload activity and allowing tracking of biodistribution in tumor-bearing mice via SPECT-CT imaging (live) or gamma counting (post-mortem). Following TCO-PAB-DOTA reaction with tetrazines optimized for extra- and intracellular reactivity, the label was removed to reveal the unmodified PBD dimer capable of inducing potent tumor cell killing in vitro and in mouse xenografts. The switchable antibody radio-drug conjugate (ArDC) we describe integrates, but decouples, the two functions of a theranostic given that it can serve as a diagnostic for payload delivery in the labeled state, but can be switched on demand to a therapeutic agent (an ADC).

Preclinical development of ZED8, an 89Zr immuno-PET reagent for monitoring tumor CD8 status in patients undergoing cancer immunotherapy.

BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.

Effect of molecular size on interstitial pharmacokinetics and tissue catabolism of antibodies.

Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for biologic therapies and expanding the need to understand how various structural aspects affect their distribution properties. To assess the effect of antibody size on systemic pharmacokinetics (PK) and tissue distribution with or without neonatal Fc receptor (FcRn) binding, we evaluated a series of non-mouse-binding anti-glycoprotein D monoclonal antibody formats, including IgG [~150 kDa], one-armed IgG [~100 kDa], IgG-HAHQ (attenuated FcRn binding) [~150 kDa], F(ab')2 [~100 kDa], and F(ab) [~50 kDa]. Tissue-specific concentration-time profiles were corrected for blood content based on vascular volumes and normalized based on interstitial volumes to allow estimation of interstitial concentrations and interstitial:serum concentration ratios. Blood correction demonstrated that the contribution of circulating antibody on total uptake was greatest at early time points and for highly vascularized tissues. Tissue interstitial PK largely mirrored serum exposure profiles. Similar interstitial:serum ratios were obtained for the two FcRn-binding molecules, IgG and one-armed IgG, which reached pseudo-steady-state kinetics in most tissues. For non-FcRn-binding molecules, interstitial:serum ratios changed over time, suggesting that these molecules did not reach steady-state kinetics during the study. Furthermore, concentration-time profiles of both intact and catabolized molecule were measured by a dual tracer approach, enabling quantification of tissue catabolism and demonstrating that catabolism levels were highest for IgG-HAHQ. Overall, these data sets provide insight into factors affecting preclinical distribution and may be useful in estimating interstitial concentrations and/or catabolism in human tissues.

Assessment of university policies for service animals and emotional support animals.

OBJECTIVE: Assess administrative responsibilities and experiential effects of emotional support animal (ESA) and service animal (SA) policies on college campuses. PARTICIPANTS: Students at two four-year universities participated in an emotional support animals and service animals survey. Selected students and professional personnel participated in interviews and focus groups. METHODS: This mixed-methods study included quantitative survey data from 1,363 students, qualitative individual interviews (3) and a focus group (1) regarding emotional support animal (ESA) and service animal (SA). RESULTS: Seventy-one students reported having ESAs, 18 had SAs. Barriers for ESAs on campus included no ESAs outside of dorms, while SA-owners reported fewer barriers. University administrators followed federal guidelines for SAs but lacked clear guidelines for ESAs. Qualitative themes included lack of awareness, education, support for SA, and ESA accommodations. CONCLUSIONS: ESA and SA accommodations continue to rise, on university campuses. Clear guidelines and implementation processes are imperative for future improvements.

Imaging Reveals Importance of Shape and Flexibility for Glomerular Filtration of Biologics.

Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure-activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv)2Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell-dependent bispecific molecules, many of which have nonstandard antibody structures.

Valency of HER2 Targeting Antibodies Influences Tumor Cell Internalization and Penetration.

T-cell-dependent bispecific antibodies (TDB) have been a major advancement in the treatment of cancer, allowing for improved targeting and efficacy for large molecule therapeutics. TDBs are comprised of one arm targeting a surface antigen on a cancer cell and another targeting an engaging surface antigen on a cytotoxic T cell. To impart this function, the antibody must be in a bispecific format as opposed to the more conventional bivalent format. Through in vitro and in vivo studies, we sought to determine the impact of changing antibody valency on solid tumor distribution and catabolism. A bivalent anti-HER2 antibody exhibited higher catabolism than its full-length monovalent binding counterpart in vivo by both invasive tissue harvesting and noninvasive single photon emission computed tomography/X-ray computed tomography imaging despite similar systemic exposures for the two molecules. To determine what molecular factors drove in vivo distribution and uptake, we developed a mechanistic model for binding and catabolism of monovalent and bivalent HER2 antibodies in KPL4 cells. This model suggests that observed differences in cellular uptake of monovalent and bivalent antibodies are caused by the change in apparent affinity conferred by avidity as well as differences in internalization and degradation rates of receptor bound antibodies. To our knowledge, this is the first study to directly compare the targeting abilities of monovalent and bivalent full-length antibodies. These findings may inform diverse antibody therapeutic modalities, including T-cell-redirecting therapies and drug delivery strategies relying upon receptor internalization.

Antibody Format and Serum Disposition Govern Ocular Pharmacokinetics of Intravenously Administered Protein Therapeutics.

Protein therapeutics have witnessed tremendous use and application in recent years in treatment of various diseases. Predicting efficacy and safety during drug discovery and translational development is a key factor for successful clinical development of these therapies. In general, drug related toxicities are predominantly driven by pharmacokinetic (PK) exposure at off-target sites. This work explores the ocular PK of intravenously administered protein therapeutics to understand impact of antibody format on off-site exposure. Species matched non-binding rabbit antibody proteins (rabFab and rabIgG) were intravenously administered to male New Zealand White rabbits at a single 1 mg bolus dose and exposure was measured up to 3 weeks. As anticipated based on absence of FcRn recycling, rabFab has relatively fast systemic PK (CL-943 mL/day and t1/2-1.93 days) compared to rabIgG (CL-18.5 mL/day and t1/2-8.93 days). Similarly, rabFab has lower absolute ocular exposure in ocular compartments (e.g., vitreous and aqueous humor) compared to rabIgG, despite higher relative exposures (measured as percent tissue partition in ocular tissues relative to serum, based on Cmax and AUC). In general, percent tissue partition based on AUC (in aqueous and vitreous humor) relative to serum exposure were 10.4 and 8.62 for rabFab respectively and 1.11 and 0.64 for rabIgG respectively. This work emphasizes size and format based ocular exposure of intravenously administered protein therapeutics. Findings from this work enable prediction of format based ocular exposure for systemically administered antibody based therapeutics and aid in selection of molecule format for clinical candidate to minimize ocular exposure.

Anti-LYPD1/CD3 T-Cell-Dependent Bispecific Antibody for the Treatment of Ovarian Cancer.

Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient in vivo antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.

Preclinical optimization of Ly6E-targeted ADCs for increased durability and efficacy of anti-tumor response.

Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.

Balancing the Affinity and Pharmacokinetics of Antibodies by Modulating the Size of Charge Patches on Complementarity-Determining Regions.

A localized positive charge on IgG (referred to as a "charge patch") shows an adverse effect on pharmacokinetics (PK), so it would seem to be best practice to avoid charge patches during the discovery stage and closely monitor charge interactions during the development process. In certain circumstances, however, charge patches are required for target binding, in which case completely removing charge patches is not feasible. Therefore, quantitative measurement of a charge patch and its impact on PK is critical to the success of therapeutic antibody development. In this article, we generated mutations of a recombinant human antibody (referred to as mAb1) with disrupted charge patches to investigate how charge patches on IgG antibodies impact both target-binding affinity and PK-related factors. We conclude that it is important to modulate the size of the charge patch in order to balance target-binding affinity and PK.

Effect of Modulating FcRn Binding on Direct and Pretargeted Tumor Uptake of Full-length Antibodies.

Full-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted trans-cyclooctene (TCO)-modified antibodies. Noninvasive SPECT imaging of mice bearing HER2-expressing xenografts was performed both directly (111In-labeled antibody) and indirectly (pretargeted TCO-modified antibody followed by 111In-labeled tetrazine). Pharmacokinetic modulation of antibodies was achieved by two distinct methods: Fc engineering to reduce binding affinity to FcRn, and delayed administration of an antibody that competes with binding to FcRn. Tumor imaging with directly labeled antibodies was feasible in the absence of FcRn binding, rapidly attaining high tumor-to-blood ratios, but accompanied by moderate liver and spleen uptake. Pretargeted imaging of tumors with non-FcRn-binding antibody was also feasible, but systemic click reaction still occurred, albeit at lower levels than with parental antibody. Our findings demonstrate that FcRn binding impairment of full-length IgG antibodies moderately lowers tumor accumulation of radioactivity, and shifts background activity from blood pool to liver and spleen. Furthermore, reduction of FcRn binding did not eliminate systemic click reaction, but yielded greater improvements in tumor-to-blood ratio when imaging with directly labeled antibodies than with pretargeting.

Biodistribution and efficacy of an anti-TENB2 antibody-drug conjugate in a patient-derived model of prostate cancer.

TENB2, a transmembrane proteoglycan protein, is a promising target for antibody drug conjugate (ADC) therapy due to overexpression in human prostate tumors and rapid internalization. We previously characterized how predosing with parental anti-TENB2 monoclonal antibody (mAb) at 1 mg/kg in a patient-derived LuCap77 explant model with high (3+) TENB2 expression could (i) block target-mediated intestinal uptake of tracer (& 0.1 mg/kg) levels of radiolabeled anti-TENB2-monomethyl auristatin E ADC while preserving tumor uptake, and (ii) maintain efficacy relative to ADC alone. Here, we systematically revisit this strategy to evaluate the effects of predosing on tumor uptake and efficacy in LuCap96.1, a low TENB2-expressing (1+) patient-derived model that is more responsive to ADC therapy than LuCap77. Importantly, rather than using tracer (& 0.1 mg/kg) levels, radiolabeled ADC tumor uptake was assessed at 1 mg/kg - one of the doses evaluated in the tumor growth inhibition study - in an effort to bridge tissue distribution (PK) with efficacy (PD). Predosing with mAb up to 1 mg/kg had no effect on efficacy. These findings warrant further investigations to determine whether predosing prior to ADC therapy might improve therapeutic index by preventing ADC disposition and possible toxicological liabilities in antigen-expressing healthy tissues.

Hyaluronic Acid-Antibody Fragment Bioconjugates for Extended Ocular Pharmacokinetics.

Treatment of ocular diseases associated with neovascularization currently requires frequent intravitreal injections of antivascular endothelial growth factor (anti-VEGF) therapies. Reducing the required frequency of anti-VEGF injections and associated clinical visits may improve patient adherence to the prescribed treatment regimen and improve outcomes. Herein, we explore conjugation of rabbit and fragment antibodies (Fab) to the biopolymer hyaluronic acid (HA) as a half-life modifying strategy, and assess the impact on Fab biophysical properties and vitreal pharmacokinetics. HA-Fab conjugates of three distinct molecular weights and hydrodynamic radii (RH) were assessed for in vivo pharmacokinetic performance relative to unconjugated Fab after intravitreal injection in rabbits. Covalent conjugation to HA did not significantly alter the thermal stability or secondary or tertiary structure, or diminish the potency of the Fab, thereby preserving its pharmacological properties. Conjugation to HA did significantly slow the in vivo clearance of Fab from the rabbit vitreous in an RH-dependent manner. Compared to free Fab (observed vitreal half-life of 2.8 days), HA-Fab conjugates cleared with observed half-lives of 7.6, 10.2, and 18.3 days for 40 kDa, 200 kDa, and 600 kDa HA conjugates, respectively. This work elucidates a possible strategy for long-acting delivery of proteins intended for the treatment of chronic posterior ocular diseases.

Production, characterization, and in vivo half-life extension of polymeric IgA molecules in mice.

IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.

Neuroinflammatory signals drive spinal curve formation in zebrafish models of idiopathic scoliosis.

The etiopathogenesis of idiopathic scoliosis (IS), a highly prevalent spinal deformity that occurs in the absence of obvious congenital or physiological abnormalities, is poorly understood. Although recent zebrafish genetic studies have linked cilia motility and cerebrospinal fluid (CSF) flow defects with scoliosis progression, underlying mechanisms were not identified. Here, we use next-generation sequencing and conditional genetic methodologies to define the spatial and biological origins of spinal curve formation in ptk7 mutant zebrafish, a faithful IS model. We demonstrate that focal activation of proinflammatory signals within the spinal cord is associated with, and sufficient for, induction of spinal curvatures. Furthermore, administration of acetylsalicylic acid (aspirin) or N-acetylcysteine (NAC) to juvenile ptk7 mutants significantly reduces the incidence and/or severity of scoliosis phenotypes. Together, our results implicate neuroinflammation, downstream of CSF defects, in spinal curve formation and provide intriguing evidence that simple immunomodulating therapies might prove effective in managing idiopathic-like spinal deformities.

Tissue Physiology of Cynomolgus Monkeys: Cross-Species Comparison and Implications for Translational Pharmacology.

We previously performed a comparative assessment of tissue-level vascular physiological parameters in mice and rats, two of the most commonly utilized species in translational drug development. The present work extends this effort to non-human primates by measuring tissue- and organ-level vascular volumes (Vv), interstitial volumes (Vi), and blood flow rates (Q) in cynomolgus monkeys. These measurements were accomplished by red blood cell labeling, extracellular marker infusion, and rubidium chloride bolus distribution, respectively, the same methods used in previous rodent measurements. In addition, whole-body blood volumes (BV) were determined across species. The results demonstrate that Vv, Vi, and Q, measured using our methods scale approximately by body weight across mouse, rat, and monkey in the tissues considered here, where allometric analysis allowed extrapolation to human parameters. Significant differences were observed between the values determined in this study and those reported in the literature, including Vv in muscle, brain, and skin and Q in muscle, adipose, heart, thymus, and spleen. The impact of these differences for selected tissues was evaluated via sensitivity analysis using a physiologically based pharmacokinetic model. The blood-brain barrier in monkeys was shown to be more impervious to an infused radioactive tracer, indium-111-pentetate, than in mice or rats. The body weight-normalized total BV measured in monkey agreed well with previously measured value in rats but was lower than that in mice. These findings have important implications for the common practice of scaling physiological parameters from rodents to primates in translational pharmacology.

A Placebo-controlled Study of PF-06473871 (Anti-Connective Tissue Growth Factor Antisense Oligonucleotide) in Reducing Hypertrophic Skin Scarring.

BACKGROUND: Connective tissue growth factor (CTGF) is a matricellular protein that plays a key role in wound healing and scar formation. Inhibition of CTGF by a specific antisense oligonucleotide significantly reduced scarring and fibrosis in animal models. This study examined whether an antisense oligonucleotide that inhibits human CTGF expression could reduce the severity of hypertrophic scar formation in patients following surgical revision of preexisting breast scars. METHODS: This study was a 24-week multicenter, randomized, double-blind, within-subject, placebo-controlled phase 2b study evaluating the efficacy and safety of PF-06473871 in 2 regimens of either 3 or 4 intradermal injections (postsurgery weeks 2, 5, 8, and 11) of 5 mg/cm adjacent to the new surgical incision. One hundred subjects with bilateral hypertrophic scars resulting from prior breast surgery were randomized. Efficacy was determined by the Patient and Observer Scar Assessment Scale (POSAS). RESULTS: The Physician/Observer POSAS overall opinion score at (week 24) for the 4-injection regimen demonstrated a statistically significant (P = 0.022) treatment difference from placebo of 0.68, and the treatment difference for the 3-injection regimen was nonsignificant (P = 0.4). Physician evaluation of scar severity at (week 24) with the photo-guide in the 4-injection regimen had a significant reduction (point estimate of treatment difference of 0.43 favoring PF-06473871). The surgical effect was approximately 2.0 at week 24 and was nearly 3 times greater than the treatment effect. Patient evaluations using the POSAS and photo-guide were not significantly improved with either dose regimen. PF-06473871 was generally well tolerated systemically and locally. CONCLUSION: The 4-dose regimen of PF-06473871 provided statistically significant improvement, inhibiting severity of hypertrophic scar formation based on physician assessment. However, the effect of revision surgery alone is significant and may dominate the treatment effect of PF-06473871.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging.

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder 'click' reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

Engagement, empowerment, and job satisfaction before implementing an academic model of shared governance.

AIM: Baseline information was obtained from a School of Nursing faculty and staff about perceptions of job satisfaction, empowerment, and engagement in the workplace before the introduction of an integrated faculty and staff shared governance system. BACKGROUND: Governance structure in schools of nursing has the potential to enhance or impose constraints on the work environment for faculty, staff, and stakeholders. RESEARCH METHODS: RESULTS: Faculty and staff perceptions of job satisfaction and engagement in the workplace before the introduction of a new model of shared governance are presented. Statistical differences were found between faculty and staff responses on the overall or total scales and select subscales, and group patterns of relationships differed. CONCLUSIONS: We provided a description of the first shared governance structure derived from the perspective of shared governance as defined and operationalized in Magnet Hospital health care systems and includes administrators, faculty, and staff in decision-making councils. As academia embarks on this change in governance structure from hierarchical to a more flattened approach findings support examining levels of work engagement, structural and psychological empowerment, and job satisfaction as key monitors of the work environment.