No abstract

Achieving the vision of identifying and quantifying cancer-related events and targets for future personalized oncology is predicated on the existence of synthetically accessible and economically viable probe molecules fully able to report the presence of these events and targets in a rapid, and highly selective and sensitive fashion. Delineated here are the design and evaluation of a newly synthesized turn-on probe whose intense fluorescent reporter signature is revealed only through probe activation by a specific intracellular enzyme present in tumor cells of multiple origins. Quenching of molecular probe fluorescence is achieved through unique photo-induced electron transfer (PeT) between the naphthalimide dye reporter and a covalently attached, quinone-based enzyme substrate. Fluorescence of the reporter dye is turned on by rapid removal of the quinone quencher, an event that immediately occurs only after highly selective, two-electron reduction of the sterically and conformationally restricted quinone substrate by the cancer-associated human NAD(P)H:quinone oxidoreductase isozyme 1 (hNQO1). Successes of the approach include rapid differentiation of NQO1-expressing and non-expressing cancer cell lines via the unaided eye, flow cytometry, fluorescence imaging, and two-photon microscopy. The potential for use of the turn-on probe in longer-term cellular studies is indicated by its lack of influence on cell viability and its in vitro stability.

A new quinone propionic acid-cloaked rhodamine fluorophore has its fluorescence revealed (de-cloaked) upon activation by human NAD(P)H:quinone oxidoreductase 1 (hNQO1), an upregulated enzyme in cancer cells and tumors.

quinone oxido-reductase isozyme 1 (hNQO1) results in fast trigger group removal to yield a highly fluorescent green-energy-range reporter that possesses a high molar absorptivity; there is a 136-fold increase in brightness for the enzymatically produced reporter versus probe precursor, a value 4 times greater than previously reported for the hNQO1 analyte. The novel probe is taken up and activated rapidly within only hNQO1-positive human cancer cells; addition of an hNQO1 inhibitor prevents the selective activation of the probe. Comparison of cytosolic fluorescence intensity in positive cells versus background in negative cells yields a quantitative metric (positive-to-negative ratio, PNR) for judging hNQO1 activity. We show it is possible to determine hNQO1 presence in previously studied colorectal cancer cells and the unexplored ovarian cancer cell line NIH:OVCAR-3, with respective PNR values of 926 and 34 being obtained. Even with 10 min probe incubation, ready discrimination of positive cells from negative cells is achieved. Cell viability is unaffected by probe presence, thereby highlighting the practicality of probe use in live-cell imaging applications.

Copper-67 (t1/2 = 2.58 days) decays by β− ($$\text{E}_{{\upbeta^{-}\text{max}}}$$ E β - max : 562 keV) and γ-rays (93 keV and 185 keV) rendering it with potential for both radionuclide therapy and single-photon emission computed tomography (SPECT) imaging. Prompted by the recent breakthrough of 67Cu production with high specific activity, high radionuclidic purity, and sufficient quantities, the interest in the theranostic potential of 67Cu has been rekindled. This work addresses the practicability of developing 67Cu-labeled antibodies with substantially improved quality for cancer radioimmunotheranostics. Proof of concept is demonstrated with pertuzumab, a US-FDA-approved monoclonal antibody for combination therapies of HER2-positive breast cancer. With an average number of 1.9 chelators coupled to each antibody, we achieved a two-order of magnitude increase in radiolabeling efficiency compared to literature reports. In a preclinical therapeutic study, mice (n = 4–7/group) bearing HER2+ xenografts exhibited a 67Cu-dose dependent tumor-growth inhibition from 67Cu-labeled-Pertuzumab co-administered with trastuzumab. Furthermore, greater tumor size reduction was observed with 67Cu-labeled-pertuzumab formulations of higher specific activity. The potential of SPECT imaging with 67Cu radiopharmaceuticals was tested after 67Cu-labeled-Pertuzumab administration. Impressively, all tumors were clearly visualized by SPECT imaging with 67Cu-labeled-Pertuzumab even at day 5 post injection. This work demonstrates it is practical to use 67Cu radioimmunoconjugates for cancer radioimmunotheranostics.