Purpose ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor has been produced under Good Manufacturing Practices for an FDA approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. Procedures Male and female Sprague-Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 µg/kg corresponding to 10X, 100X, and 1000X an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry and hematology were performed. In addition, pharmacokinetic clearance, and biodistribution of ABY-029 were studied in sub-groups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. Results Histopathological assessment, and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR positive and negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. Conclusion Based on the successful safety profile of ABY-029, the 1000X human microdose 24.5 mg/kg was identified as the no observed adverse effect level (NOAEL) following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.

The poor efficacy of systemic cancer therapeutics in pancreatic ductal adenocarcinoma (PDAC) is partly attributed to deposition of collagen and hyaluronan, leading to interstitial hypertension collapsing blood and lymphatic vessels, limiting drug delivery. The intrinsic micro-regional interactions between hyaluronic acid (HA), collagen and the spatial origins of mechanical stresses that close off blood vessels was investigated here. Multiple localized pressure measurements were analyzed with spatially-matched histochemical images of HA, collagen and vessel perfusion. HA is known to swell, fitting a linear elastic model with total tissue pressure (TTP) increasing above interstitial fluid pressure (IFP) directly with collagen content. However, local TTP appears to originate from collagen area fraction, as well as increased its entropy and fractal dimension, and morphologically appears to be maximized when HA regions are encapsulated by collagen. TTP was inversely correlated with vascular patency and verteporfin uptake, suggesting interstitial hypertension results in vascular compression and decreased molecular delivery in PDAC. Collagenase injection led to acute decreases in total tissue pressure and increased drug perfusion. Large microscopic variations in collagen distributions within PDAC leads to microregional TPP values that vary on the hundred micron distance scale, causing micro-heterogeneous limitations in molecular perfusion, and narrows viable treatment regimes for systemically delivered therapeutics.

Background:Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) provides selective uptake and conversion of ALA into protoporphyrin IX (PpIX) in actinic keratosis and squamous cell carcinoma, yet large response variations in effect are common between individuals. The aim of this study was to compare pre-treatment strategies that increase the therapeutic effect, including fractionated light delivery during PDT (fPDT) and use of iron chelator desferrioxamine (DFO), separately and combined.Methods:Optical measurements of fluorescence were used to quantify PpIX produced, and the total amount of PpIX photobleached as an implicit measure of the photodynamic dose. In addition, measurements of white light reflectance were used to quantify changes in vascular physiology throughout the PDT treatment.Results:fPDT produced both a replenishment of PpIX and vascular re-oxygenation during a 2 h dark interval between the first and second PDT light fractions. The absolute photodynamic dose was increased 57% by fPDT, DFO and their combination, as compared with PDT group (from 0.7 to 1.1). Despite that light fractionation increased oedema and scab formation during the week after treatment, no significant difference in long-term survival has been observed between treatment groups. However, outcomes stratified on the basis of measured photodynamic dose showed a significant difference in long-term survival.Conclusions:The assessment of implicit photodynamic dose was a more significant predictor of efficacy for ALA-PDT skin cancer treatments than prescription of an enhanced treatment strategy, likely because of high individual variation in response between subjects.

PurposeFluorescence guidance in surgical oncology provides the potential to realize enhanced molecular tumor contrast with dedicated targeted tracers, potentially with a microdose injection level. For most glioma tumors, the blood brain barrier is compromised allowing some exogenous drug/molecule delivery and accumulation for imaging. The aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers, including glioblastoma, and so the use of a near-infrared (NIR) fluorescent molecule targeted to the EGFR receptor provides the potential for improving tumor contrast during surgery. Fluorescently labeled affibody molecule (ABY-029) has high EGFR affinity and high potential specificity with reasonably fast plasma clearance. In this study, ABY-29 was evaluated in glioma versus normal brain uptake from intravenous injection at a range of doses, down to a microdose injection level.ProcedureNude rats were inoculated with the U251 human glioma cell line in the brain. Tumors were allowed to grow for 3–4 weeks. ABY-029 fluorescence ex vivo imaging of brain slices was acquired at different time points (1–48 h) and varying injection doses from 25 to 122 μg/kg (from human protein microdose equivalent to five times microdose levels).ResultsThe tumor was most clearly visualized at 1-h post-injection with 8- to 16-fold average contrast relative to normal brain. However, the tumor still could be identified after 48 h. In all cases, the ABY-029 fluorescence appeared to localize preferentially in EGFR-positive regions. Increasing the injected dose from a microdose level to five times, a microdose level increased the signal by 10-fold, and the contrast was from 8 to 16, showing that there was value in doses slightly higher than the microdose restriction. Normal tissue uptake was found to be affected by the tumor size, indicating that edema was a likely factor affecting the expected tumor to normal tissue contrast.ConclusionThese results suggest that the NIR-labeled affibody molecules provide an excellent potential to increase surgical visualization of EGFR-positive tumor regions.

Purpose While extent of tumor resection is an important predictor of outcome in glioma, margin delineation remains challenging due to lack of inherent contrast between tumor and normal parenchyma. Fluorescence-guided surgery is promising for its ability to enhance contrast through exogenous fluorophores, however, the specificity and sensitivity of the underlying contrast mechanism and tumor delivery and uptake vary widely across approved and emerging agents. Experimental Design Rats with orthotopic F98 wild-type and F98 EGFR-positive gliomas received in vivo administration of IRDye680RD, 5-aminioleuvulinic acid, and ABY-029—markers of perfusion, protoporphyrin metabolism, and EGFR expression, respectively. Ex vivo imaging demonstrates the contrast mechanism-dependent spatial heterogeneity and enables within-animal comparisons of tumor-to-background ratio (TBR). Results Generally, ABY-029 outperformed PpIX in F98EGFR orthotopic tumor margins and core (50% and 60% higher TBR, respectively). PpIX outperformed ABY-029 in F98wt margins by 60% but provided equivalent contrast in the bulk tumor. IRDye680RD provided little contrast, having an average TBR of 1.7 ± 0.2. The unique spatial patterns of each agent were combined into a single metric—the multimechanistic fluorescence-contrast index (MFCI). ABY-029 performed best in EGFR+ tumors (91% accuracy), while PpIX performed best in wild-type tumors (87% accuracy). Across all groups, ABY-029 and PpIX performed similarly (80% and 84%, respectively) but MFCI was 91% accurate, supporting multi-agent imaging when tumor genotype is unknown. Conclusions Human use of ABY-029 for glioma resection should enhance excision of EGFR+ tumors and could be incorporated into current PpIX strategies to further enhance treatment in the general glioma case.