In vivo biodistribution study of TAT-L-Sco2 fusion protein, developed as protein therapeutic for mitochondrial disorders attributed to SCO2 mutations.

The rapid progress achieved in the development of many biopharmaceuticals had a tremendous impact on the therapy of many metabolic/genetic disorders. This type of fruitful approach, called protein replacement therapy (PRT), aimed to either replace the deficient or malfunctional protein in human tissues that act either in plasma membrane or via a specific cell surface receptor. However, there are also many metabolic/genetic disorders attributed to either deficient or malfunctional proteins acting intracellularly. The recent developments of Protein Transduction Domain (PTD) technology offer new opportunities by allowing the intracellular delivery of recombinant proteins of a given therapeutic interest into different subcellular sites and organelles, such as mitochondria and other entities. Towards this pathway, we applied successfully PTD Technology as a protein therapeutic approach, in vitro, in SCO2 deficient primary fibroblasts, derived from patient with mutations in human SCO2 gene, responsible for fatal, infantile cardioencephalomyopathy and cytochrome c oxidase deficiency. In this work, we radiolabeled the recombinant TAT-L-Sco2 fusion protein with technetium-99 m to assess its in vivo biodistribution and fate, by increasing the sensitivity of detection of even low levels of the transduced recombinant protein. The biodistribution pattern of [99mTc]Tc-TAT-L-Sco2 in mice demonstrated fast blood clearance, significant hepatobiliary and renal clearance. In addition, western blot analysis detected the recombinant TAT-L-Sco2 protein in the isolated mitochondria of several mouse tissues, including heart, muscle and brain. These results pave the way to further consider this PTD-mediated Protein Therapy Approach as a potentially alternative treatment of genetic/metabolic disorders.

Ubiquitin is a versatile scaffold protein for the generation of molecules with de novo binding and advantageous drug-like properties.

In the search for effective therapeutic strategies, protein-based biologicals are under intense development. While monoclonal antibodies represent the majority of these drugs, other innovative approaches are exploring the use of scaffold proteins for the creation of binding molecules with tailor-made properties. Ubiquitin is especially suited for this strategy due to several key characteristics. Ubiquitin is a natural serum protein, 100% conserved across the mammalian class and possesses high thermal, structural and proteolytic stability. Because of its small size and lack of posttranslational modifications, it can be easily produced in Escherichia coli. In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo. In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4. Cellular assays based on different signaling pathways of the receptor were conducted with the natural agonist SDF-1 as a benchmark. In none of the assays could a response to ubiquitin treatment be elicited. Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.