In Vivo Evaluation and Dosimetry Estimate for a High Affinity Affibody PET Tracer Targeting PD-L1.

PURPOSE: In vivo imaging of programmed death ligand 1 (PD-L1) during immunotherapy could potentially monitor changing PD-L1 expression and PD-L1 expression heterogeneity within and across tumors. Some protein constructs can be used for same-day positron emission tomography (PET) imaging. Previously, we evaluated the PD-L1-targeting Affibody molecule [18F]AlF-NOTA-ZPD-L1_1 as a PET tracer in a mouse tumor model of human PD-L1 expression. In this study, we evaluated the affinity-matured Affibody molecule ZPD-L1_4, to determine if improved affinity for PD-L1 resulted in increased in vivo targeting of PD-L1. PROCEDURES: ZPD-L1_4 was conjugated with NOTA and radiolabeled with either [18F]AlF or 68Ga. [18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 were evaluated in immunocompromised mice with LOX (PD-L1+) and SUDHL6 (PD-L1-) tumors with PET and ex vivo biodistribution measurements. In addition, whole-body PET studies were performed in rhesus monkeys to predict human biodistribution in a model with tracer binding to endogenous PD-L1, and to calculate absorbed radiation doses. RESULTS: Ex vivo biodistribution measurements showed that both tracers had > 25 fold higher accumulation in LOX tumors than SUDHL6 ([18F]AlF-NOTA-ZPD-L1_4: LOX: 8.7 ± 0.7 %ID/g (N = 4) SUDHL6: 0.2 ± 0.01 %ID/g (N = 6), [68Ga]NOTA-ZPD-L1_4: LOX: 15.8 ± 1.0 %ID/g (N = 6) SUDHL6: 0.6 ± 0.1 %ID/g (N = 6)), considerably higher than ZPD-L1_1. In rhesus monkeys, both PET tracers showed fast clearance through kidneys and low background signal in the liver ([18F]AlF-NOTA-ZPD-L1_4: 1.26 ± 0.13 SUV, [68Ga]NOTA-ZPD-L1_4: 1.11 ± 0.06 SUV). PD-L1-expressing lymph nodes were visible in PET images, indicating in vivo PD-L1 targeting. Dosimetry estimates suggest that both PET tracers can be used for repeated clinical studies, although high kidney accumulation may limit allowable radioactive doses. CONCLUSIONS: [18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 are promising candidates for same-day clinical PD-L1 PET imaging, warranting clinical evaluation. The ability to use either [18F] or [68Ga] may expand access to clinical sites.

Automated multi-dimensional purification of tagged proteins.

The capacity for high throughput purification (HTP) is essential in fields such as structural genomics where large numbers of protein samples are routinely characterized in, for example, studies of structural determination, functionality and drug development. Proteins required for such analysis must be pure and homogenous and available in relatively large amounts. AKTA 3D system is a powerful automated protein purification system, which minimizes preparation, run-time and repetitive manual tasks. It has the capacity to purify up to 6 different His6- or GST-tagged proteins per day and can produce 1-50 mg protein per run at >90% purity. The success of automated protein purification increases with careful experimental planning. Protocol, columns and buffers need to be chosen with the final application area for the purified protein in mind.

Purification and partial characterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the recombinant transposase, TniA.

A recombinant transposase, TniA, a basic DNA binding protein, was chromatographically purified and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) methods. Escherichia coli cells, overexpressing native TniA, were ultrasonically disrupted and the clarified supernatant was used as starting material for anion-exchange chromatography on SOURCE1 15Q 4.6/100 PE (Tricorn), at pH 7.5. This initial step was proven to be a fast and simple way of removing acidic proteins like proteases. TniA was collected from the flow-through fraction and applied onto HiTrap heparin HP 5 ml in order to capture the basic TniA. This was followed by cation-exchange chromatography through Mono S 5/50 GL (Tricorn), at pH 6.5 which resulted in a purity of TniA of about 95%. The molecular mass of TniA was determined to 62 869 rel. mol. mass units with MALDI-TOF-MS and the identity of the protein was confirmed by peptide mass fingerprinting of trypsin-digested TniA. Partial amino acid sequencing was achieved after derivatization of tryptic peptides using Ettan CAF MALDI Sequencing Kit and post source decay. The fact that transposases are DNA-binding and that many of them possess basic isoelectric point values suggest that the outlined purification protocol may serve as a general method for the purification of recombinant nontagged transposases and other basic DNA-binding proteins.

Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases.

The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions.