Nimotuzumab Site-Specifically Labeled with 89 Zr and 225 Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

Simple Summary: Monoclonal antibodies (IgG) are excellent probes for targeting cell surface receptors for imaging and therapeutic applications. These theranostic agents are often developed by randomly conjugating radioisotopes/drugs/chelators to the primary amine of lysine or the sulfhydryl groups of cysteine on the antibody. Random conjugation often alters the properties of the antibody. We have site-specifically radiolabeled nimotuzumab an anti-epidermal growth factor receptor (EGFR) monoclonal antibody with 89Zr and 225Ac using SpyTag: increment N-SpyCatcher for positron emission tomography (PET) imaging and alpha particle radiotherapy, and evaluated these agents in a model of EGFR-positive triple negative breast cancer (TNBC). Nimotuzumab-SpyTag- increment N-SpyCatcher constructs showed improved binding in vitro compared with randomly conjugated constructs. 89Zr-nimotuzumab-SpyTag- increment N-SpyCatcher specifically delineated EGFR-positive xenograft in vivo using microPET/CT imaging. Compared with control treatment groups, 225Ac-nimotuzumab-SpyTag- increment N-SpyCatcher more than doubled the survival of mice bearing EGFR-positive MDA-MB-231 TNBC xenograft. This work highlights a facile method to site-specifically radiolabel antibodies using SpyTag: increment N-SpyCatcher. To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using increment N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, Delta N-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive Delta N-SpyCatcher-DFO. The Delta N-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag- increment N-SpyCatcher-DFO. Radiolabeling was performed with Zr-89, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, increment N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (Ac-225) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of Zr-89-nimotuzumab-SpyTag- increment N-SpyCatcher with 6.0 +/- 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of Ac-225-nimotuzumab-SpyTag- increment N-SpyCatcher and Ac-225-control-IgG-SpyTag- increment N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 +/- 3.3 Bq/mL (0.04 +/- 0.03 nM) and 18.5 +/- 4.4 Bq/mL (0.2 +/- 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, Ac-225-nimotuzumab-SpyTag- increment N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to Ac-225-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/ increment N-SpyCatcher to nimotuzumab did not significantly (p > 0. 05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. Ac-225-nimotuzumab-SpyTag- increment N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.