This study may further inspire development of new 44Sc/47Sc-based radiopharmaceuticals for immunoPET imaging and personalized cancer management

This study may further inspire development of new 44Sc/47Sc-based radiopharmaceuticals for immunoPET imaging and personalized cancer management. Experimental Section Chemicals Erbitux (Cetuximab) was obtained from ImClone LLC, NJ. with 111In.15,16 However, it took several hours to obtain satisfactory contrast SR9238 between the tumor and normal tissues after administration of the radiolabeled agent.16 This is especially disadvantageous when repeated imaging is required within short time intervals, for example, when studying the dynamics of EGFR expression during treatment. Therefore, we aimed to further improve EGFR targeting kinetics by using monovalent (Fab) fragments RGS20 of Cetuximab for PET imaging. Cetuximab-Fab fragments, comprising both VH and VL domains, are expected to retain the specificity and antigen-binding affinity of the parent antibody while demonstrating improved pharmacokinetics for tissue penetration.12 The decay half-life of 44Sc matches the biological half-life of Fab fragments, which is another desirable feature for successful immunoPET imaging.12 Herein, we report the generation of a Cetuximab-Fab fragment and its radiolabeling with 44Sc at room temperature using CHX-A-DTPA (and characteristics of 44ScCCHX-A-DTPACCetuximab-Fab were investigated for PET imaging of EGFR expression in a human glioblastoma (U87MG) tumor model. The present study is the first report, to the best SR9238 of our knowledge, of the radiolabeling and preclinical evaluation of 44Sc-labeled protein molecules. This strategy can be extended for SR9238 radiolabeling other temperature-sensitive biomolecules with 44Sc for PET imaging. Results Generation of Cetuximab-Fab and Its Characterization Cetuximab-Fab was generated from the intact antibody upon papain digestion for 4 h (Figure ?(Figure1A).1A). Protein A columns were used for separation of Cetuximab-Fab from the intact antibody and Fc fragments. The Protein A resin binds specifically to the Fc region of immunoglobulin molecules, especially IgGs,17?19 allowing intact antibody and the Fc fragments generated during papain digestion to be trapped in the column and purified Cetuximab-Fab to pass through. Purified Cetuximab-Fab solution was further concentrated and buffer exchanged into PBS by ultrafiltration. SDS-PAGE showed the disappearance of the intact Cetuximab band (150 kDa) and the appearance of a band corresponding to Cetuximab-Fab (50 kDa), indicating complete digestion of Cetuximab by papain to yield a high-quality Fab fragment (Figure ?(Figure1B).1B). The molecular weight of Cetuximab-Fab, as determined by mass spectrometry, was 49.9 kDa (Figure ?(Figure1C).1C). The purified Cetuximab-Fab was further used for bioconjugation and preclinical investigation in targeted, blocking, and negative control groups. For non-targeted groups, the purified Fab fragments were denatured by high-energy ultrasonication for over 1 h Open in a separate window Figure 1 Generation of Cetuximab-Fab and its characterization. (A) Schematic diagram for Cetuximab-Fab generation from intact antibody, conjugation, and radiolabeling. The figures are not drawn to scale. (B) SDS-PAGE to confirm the purity of Cetuximab-Fab (lane 1, ladder; lane 2, intact Cetuximab; lane 3, unpurified Cetuximab-Fab after papain digestion; and lane 4, purified Cetuximab-Fab after passing through Protein A column). (C) Mass spectrometry of Cetuximab-Fab (49.9 kDa). Flow Cytometry To confirm that the generated Cetuximab-Fab retained the EGFR-binding characteristics of the intact antibody, targeting experiments were carried out using U87MG (high EGFR expression) and Caco-2 (low EGFR expression) cells for flow cytometry. Fluorescein isothiocynate (FITC; excitation = 494 nm/emission = 521 nm) conjugated Cetuximab-Fab (50 nM) significantly enhanced the mean fluorescence intensity of U87MG cells (20-fold higher than that of unstained cells), whereas treatment with a blocking dose of Cetuximab (1 M) reduced the cell fluorescence by about 10-fold (Figure ?(Figure2A).2A). These results demonstrate that FITCCCetuximab-Fab specifically binds to EGFR on the U87MG cells. Meanwhile, the fluorescence.