Pretargeted nuclear imaging and radiotherapy have recently attracted increasing attention for diagnosis and treatment of cancer with nanomedicines. This is because it conceptually offers better imaging contrast and therapeutic efficiency while reducing the dose to radiosensitive tissues compared to conventional strategies. In conventional imaging and radiotherapy, a directly radiolabeled nano-sized vector is administered and allowed to accumulate in the tumor, typically on a timescale of several days. In contrast, pretargeting is based on a two-step approach. First, a tumor-accumulating vector carrying a tag is administered followed by injection of a fast clearing radiolabeled agent that rapidly recognizes the tag of the tumor-bound vector in vivo. Therefore, pretargeting circumvents the use of long-lived radionuclides that is a necessity for sufficient tumor accumulation and target-to-background ratios using conventional approaches. In this review, we give an overview of recent advances in pretargeted imaging strategies. We will critically reflect on the advantages and disadvantages of current state-of-the-art conventional imaging approaches and compare them to pretargeted strategies. We will discuss the pretargeted imaging concept and the involved chemistry. Finally, we will discuss the steps forward in respect to clinical translation, and how pretargeted strategies could be applied to improve state-of-the-art radiotherapeutic approaches.

In vivo radionuclide generators make complex combinations of physical and chemical properties available for medical diagnostics and therapy. Perhaps the best-known in vivo generator is²¹²Pb/²¹²Bi, which takes advantage of the extended half-life of²¹²Pb to execute a targeted delivery of the therapeutic short-livedα-emitter²¹²Bi. Often, as in the case of⁸¹Rb/⁸¹Kr, chemical changes resulting from the transmutation of the parent are relied upon for diagnostic value. In other instances such as with extended alpha decay chains, chemical changes may lead to unwanted consequences. This article reviews some common and not-so-common in vivo generators with the purpose of understanding their value in medicine and medical research. This is currently relevant in light of a recent push for alpha emitters in targeted therapies, which often come with extended decay chains.

The bioorthogonal reaction between a tetrazine and strained trans-cyclooctene (TCO) has garnered success in pretargeted imaging. This reaction was first validated in nuclear imaging using an ¹¹¹In-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-linked bispyridyl tetrazine (Tz) ([¹¹¹In]In-DOTA-PEG₁₁-Tz) and a TCO functionalized CC49 antibody. Given the initial success of this Tz, it has been paired with TCO functionalized small molecules, diabodies, and affibodies for in vivo pretargeted studies. Furthermore, the single photon emission tomography (SPECT) radionuclide, ¹¹¹In, has been replaced with the β-emitter, ¹⁷⁷Lu and α-emitter, ²¹²Pb, both yielding the opportunity for targeted radiotherapy. Despite use of the ‘universal chelator’, DOTA, there is yet to be an analogue suitable for positron emission tomography (PET) using a widely available radionuclide. Here, a ⁶⁸Ga-labeled variant ([⁶⁸Ga]Ga-DOTA-PEG₁₁-Tz) was developed and evaluated using two different in vivo pretargeting systems (Aln-TCO and TCO-CC49). Small animal imaging and ex vivo biodistribution studies were performed and revealed target specific uptake of [⁶⁸Ga]Ga-DOTA-PEG₁₁-Tz in the bone (3.7 %ID/g, knee) in mice pretreated with Aln-TCO and tumor specific uptake (5.8 %ID/g) with TCO-CC49 in mice bearing LS174 xenografts. Given the results of this study, [⁶⁸Ga]Ga-DOTA-PEG₁₁-Tz can serve as an alternative to [¹¹¹In]In-DOTA-PEG₁₁-Tz.

A small library of [2 + 1] ^99mTc(i) complexes based on phenyl-imidazole-fused phenanthroline (PIP) ligands were synthesized and evaluated as multimodal molecular imaging probes.