Date of Degree
Amino Acids, Peptides, and Proteins | Biochemistry | Biotechnology | Enzymes and Coenzymes | Medicinal Chemistry and Pharmaceutics | Nanotechnology | Pharmaceutical Preparations | Therapeutics | Translational Medical Research
peptides, enzyme-responsive materials, radiotherapy, matrix metalloproteinase, dual-variable domain antibody, site-specific bioconjugation
A variety of molecules can be radiolabeled and delivered to a cancer site for the purposes of diagnostics and therapy. Among the most promising of tumor targeting molecules are peptides and antibodies. These bio-inspired molecules can be designed and synthesized to target and respond to cancer cells based on the properties of those cells. Matrix metalloproteinase (MMP) enzymes are over-expressed by some metastatic cancers, in which they are responsible for the degradation and remodeling of the extracellular matrix. In recent years, MMPs have emerged as promising targets for enzyme-responsive diagnostic probes because oligopeptides can be designed to be selectively hydrolyzed by exposure to these enzymes. With the ultimate goal of developing radio-iodinated peptides as supramolecular building blocks for MMP-sensitive tools for nuclear imaging and therapy, we designed three MMP-9-responsive peptides containing either tyrosine or iodotyrosine, in order to assess the impact of iodotyrosine introduction to peptide structure and cleavage kinetics. We found that the peptides containing iodotyrosine underwent more rapid and more complete hydrolysis by MMP-9. While the peptides under investigation were predominantly disordered, it was found that iodination increased the degree of aromatic residue-driven aggregation of the peptides. We determined that these iodination-related trends stem from improved overall intramolecular order through H- and halogen bonding, in addition to intermolecular organization of the self-assembled peptides due to steric and electrostatic effects introduced by the halogenated tyrosine.
In addition to the influence of iodination on peptide enzyme response, amino acid substitutions can have a profound effect on how peptide sequences interact with one another and are enzymatically hydrolyzed. In conducting an analysis of promising sequences for translation into MMP-activated imaging probes, we identified a three-residue modification of an 11-amino acid long fiber-forming sequence that inverted the chiral orientation of the nanofiber. Modifying and screening one amino acid variation at a time, we evaluated a series of four sequences via CD, AFM and FTIR in order to identify the amino acid responsible for the chiral switch. The peptides primarily formed β-sheets, along with some random coil, β-turn and 3(10)-helix contributions. The sequence that contained a positively charged arginine (R) one position away from a negatively charged aspartic acid (D) residue was determined to reverse the chiral orientation of the assembled nanofibers. Of the four peptides evaluated for hydrolysis by MMP-9, only one sequence was significantly hydrolyzed (approximately 30 %, compared to ~10-15 % hydrolysis observed in the three other sequences). The sequence that underwent the most hydrolysis formed nanofibers and also had the most random coil contribution as determined by FTIR compared to the dominant β-sheet secondary structure formed by the other sequences. These results indicate that the peptides within the tightly-packed anti-parallel β-sheets were likely inaccessible to the enzyme due to the strong interpeptide associations. These fundamental observations of peptide hydrolysis in response to secondary structure of assembled peptides provide insights for the development of tools for enzyme-responsive radioiodine-based tumor imaging.
Antibodies also provide a widely used and very effective platform for the delivery of a radioisotope to cancer cells. Site-specifically modified radioimmunoconjugates exhibit superior in vitro and in vivo behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, in vitro evaluation, and in vivo validation of a 177Lu-labeled radioimmunoconjugate based on HER2DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified HER2DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-A′′-DTPA (PODS-CHX-A′′-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [177Lu]Lu3+ to produce the completed radioimmunoconjugate ¾ [177Lu]Lu-CHX-A′′-DTPAPODS-HER2DVD ¾ in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/mmol. [177Lu]Lu-CHX-A′′-DTPAPODS-HER2DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetallation over a 7 d incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [177Lu]Lu-CHX-A′′-DTPAPODS-HER2DVD to that of a 177Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [177Lu]Lu-CHX-A′′-DTPAPODS-HER2DVD matched that of 177Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8 respectively at the same timepoint. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs.148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.
MacPherson, Douglas S., "Leveraging Bio-Inspired Molecules for Cancer Theranostics" (2023). CUNY Academic Works.
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