Cancer is a complex, multifactorial disease and is the second leading cause of death worldwide. WHO reports that 1 in 6 mortality occurs due to cancer. To change the scenario, researchers are developing newer treatment strategies. Current cancer treatment involves chemotherapy, radiotherapy, surgery, immunotherapy, and hormone-based therapy (Debela et al., 2021). Even though there are advancements in medical science, there is also a tremendous requirement for cancer diagnosis, imaging agents, and treatment regimens. The therapeutic efficacy of a treatment can only be monitored by diagnosing at regular intervals. Imaging enables constant real-time monitoring of the disease status (Seaman, Contino, Bardeesy, & Kelly, 2010). Imaging requires a contrast agent that enhances the quality of the image by creating a contrast between the tissues at the visualization site. The targeted delivery of radiometallopharmaceuticals has a dual function as an excellent contrast agent as well as targeted radiotherapy. Hence, these agents are highly encouraged in the field of cancer theranostics. In this review, we have tried to concisely describe the functionality of a theranostic reagent, i.e., as a contrast agent and a therapeutic agent (Fig. 1A). Some of the other examples of FDA-approved theranostic agents are sodium 131Iodine, HSA-131I-MIBG, 131I-Tositumomab, 131I-Metuximab, 131I-chTNT, 223Ra dichloride, 153Sm-EDTMP, 89Sr chloride, 177Lu-DOTATATE, 177Lu-PSMA-617, 90Y-microspheres, 90Y-ibritumomab tiuxetan, and 32P-colloid, 225Ac-PSMA-617 (Poot, Lam, & van Noesel, 2020) (Nindra, Lin, Becker, Roberts, & Chua, 2024).

Screening or diagnosis of cancer includes various methods that can be classified as invasive and non-invasive methods; invasive methods include organ-specific endoscopic needle aspiration, whereas non-invasive methods include liquid biopsies, Computed Tomography (CT) scan, Positron Emission Tomography/Single-photon Emission Computerized Tomography (PET/SPECT) imaging, Magnetic Resonance Imaging (MRI), ultrasound imaging, etc., (Chacko & Ankri, 2024; Folch, Costa, Wright, & VanderLaan, 2015; Yan & Wang, 2020). CT scan is one of the preliminary detection techniques. It is more cost-effective than other methods but has some limitations. The CT scan uses X-rays to obtain anatomical and functional information about the site of study (Mahersia, Zaroug, & Gabralla, 2015). Higher energy light emitted from the tissue or organ will produce better-resolution scanned images; however, this X-ray may damage the tissue. Thus, researchers have tried synthesizing theranostic agents to produce higher-resolution images, reduce tissue damage, and have therapeutic effects (Mahersia et al., 2015).

Though many imaging techniques are available to monitor various disorders, PET and SPECT are prominent techniques to detect irregularity in 3-dimensional images (Frejd & Kim, 2017). PET/SPECT scanning is based on the positron beam emitted from radiometal accumulated in the organ. Like every imaging system, PET, MRI, and SPECT require a contrast agent, where benign and malignant tumours can also be detected (Rahbar et al., 2012) (Griffeth, 2005). SPECT imaging has many advantages, including increased diagnostic accuracy, specificity, and positive predictive value (PPV) (Israel et al., 2019). PET and SPECT scans employ radiopharmaceuticals as contrast agents consisting of gamma or positron-emitting radionuclides and a localizing molecule (Coleman, 2003). These radioactive metals are known to emit gamma or positron when they undergo radioactive decay. These emitted rays can be detected by detector systems, which would be helpful for monitoring or imaging disorders (Van Dort, Rehemtulla, & Ross, 2008). Many radionuclides are employed as radiotracers, showing successful imaging and therapeutic potential in cancer (Table 1) (Velikyan, 2014). For instance, gallium has been proven to be an ideal candidate for imaging various tumours as it showed excellent resolution in PET imaging. Moreover, adding receptor-targeting moiety to gallium enhanced its tumour accumulation and showed a higher affinity to tumour cells (Al-Nahhas et al., 2007). This characteristic property of tumour accumulation often enhances the brightness of the tumours in PET imaging.

Recently, there has been a growing interest in developing radioactive metal complexes as theranostic agents for monitoring various disorders or therapeutic applications. In addition, substituting or modifying the existing chemical structures of radiometal complexes demonstrated enhanced imaging with a better pharmacokinetics profile. Some important criteria for designing an imaging agent are denoted as 4S: Stability, Sensitivity, Specificity, and Safety (Lee, Park, Hong, Choi, & Choi, 2012). Metal complexes satisfying these criteria can be considered as good candidates for imaging agents for diagnosis. The complexes containing radioactive metals such as technetium, gallium, zirconium, copper, cobalt, and indium showed high-contrast images in the detection and surveillance of cancer (Fig. 1B). In contrast, radioactive lutetium, astatine, iodine, scandium, and rhenium showed higher therapeutic efficacy (MacPherson, Fung, Cook, Francesconi, & Zeglis, 2019). Combining radionuclides with affibodies by using a chelator, sometimes utilizing metal ion chelators like DOTA, DOTAGA, NOTA, NODAGA, and DTPA as a scaffold, enables the development of radionuclide imaging probes (Fig. 1C).

Therapeutic agents selectively accumulate near the cancer tissue, which can produce α-particles against the cancer cells and thus could act as a new age of radiotherapy. For instance, Radium-233 dichloride (Xofigo) is an approved radioactive complex for metastatic prostate cancer. When these radioactive compounds reach a cancer cell, they get internalized and damage the nucleus. Due to the unstable nature of the compound, radioactive metal decays to release energy, and the amount of energy released depends on the type of radioactive metal used for the radiotherapy. Sometimes, the energy released is so intense that this radiation can penetrate and kill 20–30 cells in the surroundings (Radiopharmaceuticals Emerging as New Cancer Therapy -, 2020). This crossfire effect of radiation therapy hampering a heterogeneous population of cells has pros and cons. The advantage is that it can eliminate many cancer cells and cancer-associated cells; however, the limitation is that it could also affect the healthy neighbouring cells (Poty, Francesconi, McDevitt, Morris, & Lewis, 2018, p. 1). This radiation induces double-stranded and single-stranded breaks in the DNA of the cancer cell. This radiation affects DNA and interferes with several cellular organelles, leading to tumour cell death (Bannik et al., 2019). These radiations target mitochondria, an essential organelle that regulates cellular metabolism (Elbanna, Chowdhury, Rhome, & Fishel, 2021). It produces excessive ROS that reacts with the mitochondrial respiratory chain, reducing the tumour cell population. As a cascade of events, this leads to the activation of executionary caspases, which leads to apoptosis and/or autophagy. However, the extent of damage depends on the type of emission; for instance, if the radiation has a higher linear energy transfer (protons, neutrons, and α-rays), then the damage is about 90 %. In contrast, if the radiation is of lower linear energy transfer (β & γ rays), the damage would be 70 % (Bannik et al., 2019). The table below summarizes the list of radioactive isotopes with their half-lives and corresponding emission types (Table 1).

A target-specific imaging agent is a more exciting field of research since it provides more specific information on the location of the tumour, which ultimately assists in monitoring the tumour during the treatment regime, understanding the metastatic sites of the tumour, or locating the tumour for surgeries, etc. For targeting the receptors overexpressed on the surface of the tumour cells, ligands, such as affibodies, antibodies, peptides, nanobodies, and small molecules, are engineered to bind with high specificity to tumour-associated biomarkers, thereby enhancing the precision of diagnostic and therapeutic interventions (Altunay et al., 2021; Fu, Carroll, Yahioglu, Aboagye, & Miller, 2018; Garg, 2020; Luo, Liu, & Cheng, 2022; Yang, Liu, Huang, Liu, & Wei, 2022). Antibody-conjugated radiometallopharmaceuticals were initially employed to deliver diagnostic or therapeutic radionuclides to cancer cells with higher specificity (Sharma, Suman, & Mukherjee, 2022). The radiolabelled monoclonal antibodies enable the precise delivery of radionuclides due to their high affinity and specificity for target antigens that are overexpressed on tumours (Lin, Paolillo, Le, Macapinlac, & Ravizzini, 2021). The relatively higher molecular weight of the antibodies (~150 kDa) accounts for their slow pharmacokinetics and blood clearance, which limits their diagnostic application. However, with the availability of long-lived SPECT and PET radionuclides, several antibody-conjugated radiometallopharmaceuticals have shown promising results in clinical evaluation. The low tumour penetration and delayed clearance of the antibody-conjugated radiometallopharmaceuticals result in increased systemic toxicity and off-target effects (Leitao et al., 2020; Qi, Hoppmann, Xu, & Cheng, 2019) Dammes & Peer, 2020; Wu, 2014; Xenaki, Oliveira, & van Bergen En Henegouwen, 2017). Their prolonged half-life necessitates higher doses to achieve effective tumour targeting, potentially leading to an elevated risk of side effects (Khongorzul, Ling, Khan, Ihsan, & Zhang, 2020). These issues have led scientists to explore and develop alternatives to antibodies (Garg, 2020; Luo et al., 2022).

Affibody molecules, which are significantly smaller (~6.5 kDa), address the limitations of antibody-conjugated radiometallopharmaceuticals by offering improved tumour penetration and faster clearance from the bloodstream (Eissler et al., 2024). The small size of the affibody molecules helps to reduce the background accumulation in healthy tissues and allows for lower dosing without compromising efficacy. Additionally, affibody molecules exhibit superior stability under physiological conditions, ensuring reliable delivery of radiometals (Krasniqi et al., 2018). Their faster pharmacokinetics and reduced dosage requirements make them highly suited for clinical applications. Compared to antibodies, affibody-based radiometallopharmaceuticals offer improved tumour-to-organ ratios and have more significant potential for safe and effective cancer imaging and therapy (Zhang et al., 2025) (Table 2).

Thus, there is a requirement for synthesizing an ideal targeting agent with high binding affinity, low retention period, high penetration capability, and resistance to proteolytic cleavage (Ståhl et al., 2017). Small molecule inhibitors are toxic since they can target multiple similar receptors and some non-specific proteins inside the cell (Vallinayagam, Adil, Ahmed, Rishi, & Jamal, 2014). Peptide-based radiopharmaceutical agents also demonstrate high tumour uptake and increased specificity, like affibody-based agents. However, it is found that peptide-based agents have shown low metabolic stability in vivo conditions (Li et al., 2020). Thus, the alternative use of affibodies could overcome the issues. Affibodies are a step ahead of small molecule inhibitors in target specificity, non-target reactivity, and immunogenicity (Ståhl et al., 2017). Affibody® molecules are a class of synthetic peptides known as antibody mimics, short cysteine-free peptides of 58 amino acids (7 kDa) derived from staphylococcal protein A (SPA). As the name suggests, these molecules have similar properties to antibodies and improved properties like high selectivity, low immunogenicity, high solubility, high thermal stability, etc. (Fu et al., 2018; Ståhl et al., 2017). They possess the highest stability and can be engineered to bind with target proteins with high binding affinity. Moreover, the binding affinities of these affibodies are usually in the nM to pM range (Table 3). Affibodies originated from a bacterial antibody binding region, i.e., streptococcal protein A. The B domain of the protein has three helices that are free from cysteine residues (Fig. 2). This B domain is modified to the Z domain by a few mutations in the sequence to obtain a chemically stable molecule. The Z domain has an Fc binding specific region; altering these peptide sequences could enable affibodies to target different receptors. Thus, a combinatorial library has been created, which enables the development of a new set of affibodies. After the sequence has been identified, affibodies can be produced via the recombinant expression of the affibodies in bacteria strains, or chemical synthesis can also be done using solid-phase peptide synthesis. Both methods are relatively inexpensive compared to antibody production. Due to their small size, high binding affinities, low immunogenicity, high thermal and chemical stability, and ease of synthesis, make affibodies as an ideal ligand for targeted delivery of imaging or cytotoxic agents for therapy (Yu, Yang, Dikici, Deo, & Daunert, 2017). Their high specificity, strong binding affinity, and smaller size have made them attractive targeting molecules for theranostic applications (Tolmachev & Orlova, 2020). The Swedish firm “Affibody AB,” established in 1998, has investigated affibodies and possesses nearly 300 patents (Affibody Medical AB-The next generation biopharmaceuticals, 2025).

Tolmachev and his entire team contributed significantly to the field of affibody-based radiometal theranostics agents. This group from Uppsala University, Sweden, has contributed more than 200 research articles in two decades. They explored various affibody-conjugated radiometallopharmaceuticals and extensively studied the crucial roles of radiometals and chelators (Tolmachev & Orlova, 2020), (Eissler et al., 2024). They analyzed different novel affibodies to improve the efficacy of these theranostic agents. This review highlights affibodies conjugated radiometal-based theranostic agents that target specific tumour receptors (Fig. 2). So far, there are only a handful of reviews that have been published on affibody-conjugated radiometallopharmaceuticals; for instance, initial reviews were published by Vladimir Tolmachev and PerÅke Nygren, who are pioneers in this field, in 2007–2008 (Nygren, 2008; Orlova, Feldwisch, Abrahmsén, & Tolmachev, 2007; Tolmachev et al., 2007). A few recent review articles were reported that focused on HER2-targeted or EGFR-targeted affibody conjugates (Altunay et al., 2021; Chen, Shen, & Sun, 2019; De, Kuppusamy, & Karri, 2018; Hu et al., 2022; Zhang & Zhang, 2024). Also, articles on affibody-conjugated radiopharmaceuticals targeting breast and ovarian cancers were summarized in 2017 and 2022, respectively. So far, no attempts have been made to summarize and review the reported 200+ affibody-conjugated radiometallopharmaceuticals. Hence, this review focused on the progress of affibody-conjugated radiometallopharmaceuticals in the last 20 years; this review discusses the advantages and disadvantages of each affibody-targeted radiometal conjugate, enabling the readers to understand the concepts and assist in designing new, improved theranostic agents. We organized this article based on radiometals, 111In, 68Ga, 64Cu, 55Co, 57Co, 44Sc, 99mTc, 89Zr, 90Y, 211At, 188Re, and 177Lu. Further, we have elaborated on the choice of chelators such as DOTA, DOTAGA, NOTA, NODAGA, and DTPA and affibodies that can interact with potential cancer-associated molecular targets, such as human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor β (PDGFRβ), carbonic anhydrase IX (CAIX), human epidermal growth factor receptor 3 (HER3), insulin-like growth factor-1 receptor (IGF-1R), vascular endothelial growth factor receptor 2 (VEGFR2), Programmed Cell Death Ligand 1 (PD-L1), neonatal Fc receptor (FcRn), and B7-H3.