The specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacologic half-lives necessitate the use of radioisotopes with long physical halsives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodology for pretargeted PET imaging based on the bioorthogonal Diels–Alder click reaction between tetrazine and transcyclooctene. Methods A proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and 64Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with 64Cu. Pretargeted in vivo biodistribution and PET imaging experiments were performed with athymic nude mice bearing A33 antigen–expressing, SW1222 colorectal cancer xenografts. Results The huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the 64Cu-NOTA–labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9–10 MBq/μg. For in vivo experiments, mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with 64Cu-NOTA–labeled tetrazine for PET imaging and biodistribution experiments. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per gram), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution experiments performed using A33 directly labeled with either 64Cu or 89Zr revealed that although absolute tumor uptake was higher with the directly radiolabeled antibodies, the pre-targeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calculations revealed that the 64Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with 89Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, respectively). Conclusion The high quality of the images produced by this pretargeting approach, combined with the ability of the methodology to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clinical translation.

The ability to generate heat under an alternating magnetic field (AMF) makes magnetic iron oxide nanoparticles (MIONs) an ideal heat source for biomedical applications including cancer thermoablative therapy, tissue preservation, and remote control of cell function. However, there is a lack of quantitative understanding of the mechanisms governing heat generation of MIONs, and the optimal nanoparticle size for magnetic fluid heating (MFH) applications. Here, we show that MIONs with large sizes (>20 nm) have a specific absorption rate (SAR) significantly higher than that predicted by the widely used linear theory of MFH. The heating efficiency of MIONs in both the superparamagnetic and ferromagnetic regimes increased with size, which can be accurately characterized with a modified dynamic hysteresis model. In particular, the 40 nm ferromagnetic nanoparticles have an SAR value approaching the theoretical limit under a clinically relevant AMF. An in vivo study further demonstrated that the 40 nm MIONs could effectively heat tumor tissues at a minimal dose. Our experimental results and theoretical analysis on nanoparticle heating offer important insight into the rationale design of MION-based MFH for therapeutic applications.

A modular system for the construction of radiometalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4,5-tetrazine and the strained dienophile norbornene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes 64Cu and 89Zr were employed as a model system. The antibody was first covalently coupled to norbornene, and this stock of norbornene-modified antibody was then reacted with tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with 64Cu and 89Zr, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific activity (2.7–5.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the 64Cu-DOTA- and 89Zr-DFO-based probes were shown to be nearly identical in terms of stability, the number of chelates per antibody, and immunoreactivity (>93% in all cases). In vivo PET imaging and acute biodistribution experiments revealed significant, specific uptake of the 64Cu- and 89Zr-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-MB-468 xenografts or other tissues. This modular system—one in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelators—will allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals.

Superparamagnetic iron oxide nanoparticles (SPIOs) have the potential for use as a multimodal cancer therapy agent due to their ability to carry anticancer drugs and generate localized heat when exposed to an alternating magnetic field, resulting in combined chemotherapy and hyperthermia. To explore this potential, we synthesized SPIOs with a phospholipid-polyethylene glycol (PEG) coating, and loaded Doxorubicin (DOX) with 30.8% w/w loading capacity when the PEG length is optimized. We found that DOX-loaded SPIOs exhibited a sustained DOX release over 72 hours where the release kinetics could be altered by PEG length. In contrast, the heating efficiency of the SPIOs showed minimal change with PEG length. With a core size of 14 nm, the SPIOs could generate sufficient heat to raise the local temperature to 43°C, enough to trigger apoptosis in cancer cells. Further, we found that DOX-loaded SPIOs resulted in cell death comparable to free DOX, and that the combined effect of DOX and SPIO-induced hyperthermia enhanced cancer cell death in vitro. This study demonstrates the potential of using phospholipid-PEG coated SPIOs for chemotherapy-hyperthermia combinatorial cancer treatment with increased efficacy.