Imaging of cell surface biomarkers is emerging as an increasingly important approach in cancer detection and monitoring therapeutic efficacy. We have previously shown that engineered antibody fragments (i.e. diabodies and minibodies) retain excellent tumor targeting in mouse models in vivo
, coupled with rapid clearance from the circulation. Since the pharmacokinetic of diabodies match very well with the half-life of F-18,6
F-labeled versions represent a novel class of PET tracers with broad potential utility for imaging cell surface phenotype in vivo
. Therefore there is a great need for developing efficient procedures to produce 18
F-labeled diabody-based tracers in high yield sufficient for routine PET imaging.
Because the amounts of proteins produced in the R&D environment are often restrained, the extent to which trial-and-error optimizations of the [18
F]SFB labeling reactions can be conducted is rather limited. Alternatively, brute force approaches using entire batches of proteins and high activity (>1 Ci) were often used to ensure sufficient activity produced for microPET studies.6
Therefore, a practical means to perform optimization using only small amount of proteins and radiolabeling tags is critical to the progress of this field. Since the minute masses required for radiotracer production are well matched to the small volumes required in microfluidics, we have designed and fabricated a DMDG chip which can “digitally” generate composition-specific and size-controlled droplets on demand. This microfluidic-based optimization method provides a simple and effective means to perform screening pH and concentration droplet-by-droplet. The unique features of this device allow: (1) the independent control of volume and composition for every droplet, enabling reaction condition screening with minimal reagent consumption (Supplemental Material, Movie S1
); (2) the ability to pause, modify, and restart the droplet generation process, e.g. for replacement/change of reagents (Supplemental Material, Movie S2
); and (3) the use of nitrogen gas rather than oil to separate droplets, eliminating the need for oil removal steps later. Furthermore, the current device, distinct from other droplet-based microfluidics,14, 28
is unique in its ability to operate with very low total volumes of samples and reagents with little to no loss. As each droplet can be driven out slowly in a well-controlled matter using the on-chip peristaltic pump, one single droplet can be easily used for determining the corresponding RLY. The volume and radioactivity of a single droplet is around 120 nL and can represent 0.5–2 µCi, respectively. For example, 5 µL of diabody solution (2 mg/mL) could be used in screening for more than 125 different conditions in single micro-droplets.
To probe specific biological process in vivo without exerting mass effects within the same living systems under study, for example, measuring receptor-ligand interactions, PET tracers with high SA are desired. Our strategy to obtain 18F-labeled diabody of high SA (in our case, ESA) is to first, identify the pH range leading to the highest RLY, and subsequently optimize the protein concentration to generate the highest ESA at the previously-determined optimal pH. In the [18F]SFB labeling reactions, RLYs generally increase at higher protein concentrations at the optimal pH. However, continuing to increase the amount of protein used in the labeling reaction will lower the final ESA since the RLY eventually plateaus (). Using our DMDG chip, a series of experiments with different pHs and concentrations can be rapidly performed, allowing mapping the area of optimal conditions. This is especially important since the optimal parameters for [18F]SFB labeling vary depending on the sequence and structure of the individual protein being radiolabeled. Furthermore, batch-to-batch variations can occur for the protein and [18F]SFB as well, making rapid optimization using small aliquots of reactants even more critical. Our method utilizing the DMDG chip can address requirements above by identification of optimal reaction parameters in a rapid, reagent-economical fashion. Once the optimal reaction parameters are identified, larger-scale reactions can be performed under the same chip-derived conditions to produce sufficient amount of 18F-labeled biomolecules.
Compared to the previously described 124
I-labeled minibody currently in clinical development,29
the new [18
F]FB-A2 Db opens the possibility of same-day PET imaging with superior image contrast in a clinical setting. The key advantage of using diabodies, the smallest bivalent fragments based on antibody combining sites, is the potential to obtain clear tumor visualization just a few hours (4–6 h) after injection 30
. Earlier microPET imaging results using 124
I-labeled anti-PSCA diabodies suggested that the best image contrast required a 12 h delay and the tumor-to-blood (ROI) ratio was around 331
. In contrast, the tumor-to-soft tissue ratio using [18
F]FB-A2 Db in this study was 13.6 at 4 h p.i., which represents a significant improvement. There are several possible explanations for this improvement. First, the half-life and positron yield of F-18 is better suited for PET detection within 4–6 h p.i. Second, due to the optimization of microfludic method, the resulting [18
F]FB-A2 Db was obtained with high immunoreactivity to PSCA, radiochemical purity and ESA. Third, the radioiodination method used to label anti-PSCA diabodies 27
was not optimal and could result in deiodination that can affect the tumor uptake and reduce image contrast. It is difficult to isolate the degree of influence among those individual parameters from in vivo
imaging results, and it is likely that all of the factors listed above are important. Regardless, in order to obtain an effective protein-based immunoPET tracer, it is necessary to optimize individual 18
F-labeling condition under which a sufficient amount of tracer with high immunoreactivity, ESA and radiochemical purity can be generated.
To show the versatility and generality of our method, we also applied this microfluidic-based method to optimize the [18
F]SFB labeling of an additional diabody (anti-HER2 Db) specific to Human Epidermal Growth Factor Receptor 2 (HER2) and, under optimized conditions (Supplemental Material, Fig. S12
F]FB-anti-HER2 Db was obtained for microPET studies in a breast cancer xenograft-bearing mouse (MCF-7/HER2) (Supplemental Material, Fig. S13
). These successful examples utilizing a microfluidic-based approach for optimization of labeling conditions provide a practical means to produce [18
F]fluorobenzoylated diabodies ([18
F]FB-labeled Dbs) or other scarce biomolecules. Furthermore, our microfluidic-based method is a de novo
approach and can be applied in the beginning of each labeling experiment to access the quality of biomolecule and determine of best labeling condition for the day.