The blood brain barrier (BBB) exists to protect the brain from pathogens and other toxins but makes central nervous system (CNS) diseases hard to treat with conventional drugs. For example, many standard chemotherapeutics, that work well for other cancers, cannot be used for brain cancer patients due to BBB impassibility. The BBB is selectively permeable and allows larger proteins and hormones to pass through receptor-mediated trafficking across the barrier, a highly specific form of transport. One example is the transferrin-receptor (TfR), which binds an important iron binding protein called transferrin, and allows it to move from the blood to the brain to perform its function. Other groups have tried to take advantage of TfR-mediated entry to the CNS by using TfR-binding antibodies or conjugating drugs to transferrin itself as a sort of Trojan horse strategy to get therapeutic molecules beyond the BBB. However, these strategies have not been without issues – bulky antibodies can have poor tissue permeability, lead to unwanted immune activation, and have long serum half-lives that may be problematic if toxicity were to occur. Dr. Zach Crook, staff scientist in the Olson lab (Clinical Research Division) sought to find another way to use TfR in another way to mediate BBB permeability. The results of his study were recently published in the Journal of Molecular Biology.
The Olson group has developed a pipeline to investigate cysteine dense peptides (CDPs) for their therapeutic potential. CDPs are short peptides with multiple cysteine bridges, making them compact and very stable. Dr. Crook and his co-authors sought to find a TfR-binding CDP using their pipeline. They used a library of 10,000 naturally occurring CDPs, along with mutagenized CDPs to increase the number and diversity of possible CDPs. Using a mammalian display system, they screened their library of CDPs for their ability to bind the ectodomain of human TfR. Dr. Crook explained: "Our mammalian surface display screening platform (first published in Crook et al., Nature Communications 2017) allows us to find CDP binders to diverse targets. We turned to mammalian cells, because they have demonstrated an ability to produce CDPs from a variety of species with proper folding and in high quantities, which allows us to fully explore the medical potential of this class of proteins." Cells that could bind TfR were selected by one round of magnetic sorting, followed by three rounds of sorting by flow cytometry. The authors found one TfR binder out of their library (named TfRB1-G1), a peptide of 49 amino acids with six cysteines. They subjected TfRB1-G1 to affinity maturation by making every possible non-cysteine amino acid substitution and rescreening this library with more stringent sorting criteria to produce TfRB1-G2 (generation 2). This process was repeated to make TfRB1-G3 (generation 3). Each binder was produced as a soluble protein and validated by liquid chromatography, Western blot, and mass spectrometry. Binding to TfR was confirmed, and as expected enhanced binding was seen from TfRB1-G1 to G3. The crystal structure for TFRB1-G3 was solved, both on its own and bound to TfR, confirming the predictions that the CDP is an anti-parallel two-helix bundle and demonstrating a 2:2 binding stoichiometry with TfR. Additionally, the authors determined that TfRB1s could bind murine TfR, which validated that in vivo studies could occur in a murine host.
In order to test the activity of TfRB1 in vivo, the authors used a radiolabeled version using carbon-14. Upon intravenous administration, C14-TfRB1 accumulated in the kidney, liver, and spleen – the kidney being the likely place of removal from the blood stream, while previous reports show the liver and spleen have high expression of TfR. Using whole body autoradiography, the authors determined that TfRB1 accumulated in the CNS to approximately 25% of signal seen in the blood, well above the estimate of blood located within the CNS compartment, which suggested that the TfRB1 could cross the BBB. However, the authors wanted to confirm BBB penetration, as well as activity within the brain. To do this, they used a small protein called neurotensin (NT), which causes signaling through a CRE-driven transcriptional program but is unable to cross the BBB itself. This allowed the authors to use a CRE-driven luciferase transgenic mouse to report signaling by NT in the brain. The authors made TfRB1-NT fusions, and confirmed they were still able to bind TfR and the NT could still signal normally. Mice were given either the TfRB1 or TfRB1-NT fusion and luciferase activity was measured four hours later. Only the TfRB1-NT fusion produced luciferase signal above background, indicating that the TfRB1 was able to ferry cargo across the BBB where it could signal. The significance of their findings is summed up by Dr. Crook: "There are innumerable drugs that have been found effective against brain tumor cells in culture but ineffective in patients, likely due to poor BBB penetration. We hope that combining such drugs with TfR-binding CDPs can allow us to safely and efficiently bypass the BBB."
This study was supported by the National Institutes of Health and the Washington Research Foundation.
UW/Fred Hutch Cancer Consortium members Roland Strong and Jim Olson contributed to this work.
Crook Z, Girard E, Sevilla G, Merrill M, Friend D, Rupert P, Pakiam F, Nguyen E, Yin C, Ruff R, Hopping G, Strand A, Finton K, Coxon M, Mhyre A, Strong R, Olson J. 2020. A TfR-Binding Cystine-Dense Peptide Promotes Blood-Brain Barrier Penetration of Bioactive Molecules. Journal of Molecular Biology. Jun 26;432(14):3989-4009. doi: 10.1016/j.jmb.2020.04.002.