Recent Advances in 64Cu/67Cu-Based Radiopharmaceuticals

Copper-64 (T1/2 = 12.7 h) is a positron and beta-emitting isotope, with decay characteristics suitable for both positron emission tomography (PET) imaging and radiotherapy of cancer. Copper-67 (T1/2 = 61.8 h) is a beta and gamma emitter, appropriate for radiotherapy β-energy and with a half-life suitable for single-photon emission computed tomography (SPECT) imaging. The chemical identities of 64Cu and 67Cu isotopes allow for convenient use of the same chelating molecules for sequential PET imaging and radiotherapy. A recent breakthrough in 67Cu production opened previously unavailable opportunities for a reliable source of 67Cu with high specific activity and purity. These new opportunities have reignited interest in the use of copper-containing radiopharmaceuticals for the therapy, diagnosis, and theranostics of various diseases. Herein, we summarize recent (2018–2023) advances in the use of copper-based radiopharmaceuticals for PET, SPECT imaging, radiotherapy, and radioimmunotherapy.


Introduction
Nuclear medicine is based on the use of radiochemical properties of isotopes for the therapy and diagnosis of various diseases. Radiotherapy accompanies almost 50% of the use of chemotherapy, thus being an extremely important treatment modality not only in the treatment of tumor diseases but also in the palliative care of inoperable patients [1]. Since radiation therapy affects cancer tissues via DNA damaging, targeted, and strictly localized effects of radiation, preliminary imaging of the biodistribution of radiopharmaceuticals using theranostic pairs is extremely important [2].
A classical targeted radiopharmaceutical is a tissue-affine molecule, conjugated with chelator, which is further radiolabeled with radioactive isotope. Depending on the type of radiation emitted, imaging or therapeutic capability is assigned to the radiopharmaceutical category. Positron-emitting isotopes are used for PET imaging, and gamma-emitting radioisotopes are used for SPECT visualization; for radiotherapy, α-, β-, and Auger electron emitters are considered [3,4]. However, the design of radiopharmaceuticals is based not only on the emission properties of the isotope but also on the method of its synthesis, and the ability to produce the isotope in sufficient quantity, purity, and specific activity is extremely important. Radionuclides can be produced via nuclear reactors, linear accelerators, and medical cyclotrons, and can also be conveniently eluted from portable nuclide generators. However, the production of nuclides via nuclear reactors is a rather difficult task due to the large amount of radioactive waste produced along the way; moreover, the transportation of nuclear waste is a public safety issue. The use of cyclotrons allows the production of high-quality nuclides; however, a limited number of cyclotrons poses logistical problems [
Several theranostic pairs, such as 68 Ga/ 177 Lu, 124 I/ 131 I, 64 Cu/ 67 Cu, 43 Sc/ 44 Sc/ 47 Sc, 83 Sr/ 89 Sr, 86 Y/ 90 Y, 110 In/ 111 In, 152 Tb/ 161 Tb and 90 Y/ 177 Lu have been reported [60]. However, these theranostic couples have several disadvantages. The use of the 68 Ga/ 177 Lu theranostic pair is limited by the fact that gallium-68 generators are gradually being phased out by more cost-effective accelerator-based production, due to higher amounts of specific activity and no waiting required between productions, unlike the required 3-4 h interval between isotope elutions from portative generators [61]. 124 I, widely used in medical practice, has an accompanying positron gamma radiation, which complicates PET diagnosis due to false signal emerging and distortion of PET results [62,63]. In addition, the fact that iodine is an easily removable leaving group leads to dehalogenation reactions within the body (for example, in the diagnosis of prostate cancer [64]).
Copper is a metal essential to human health, and it is a constituent of many enzymes [65]. Natural copper has two stable isotopes, 63 Cu and 65 Cu, and five radioisotopes, 60 Cu, 61 Cu, 62 Cu, 64 Cu and 67 Cu. Two of these isotopes, namely 64 Cu and 67 Cu, are considered therapeutic β-emitters for targeted radiation therapy. However, among all copper radioisotopes, 64 Cu is the "golden mean" for PET imaging due to its unique emission properties.
Copper-64 (T 1/2 = 12.7 h; β + : 18%, β − : 39%) possesses an attractive decay profile for nuclear medicine, which provides the possibility of using 64 Cu-based radiopharmaceuticals for both PET imaging and radionuclide therapy. The combination of β + and β − emission imparts a high local radiation dose at the cellular level, and electron capture decay is accompanied by the Auger electrons' emission, which also contributes to cytotoxic potency. 64 Cu is most often produced via the 64 Ni(p,n) 64 Cu reaction on biomedical cyclotrons. Typically, with a bombardment time of 4 hours of 40 mg 64 Ni target yields 18.5 GBq of 64 Cu. The specific activity of the 64 Cu ranges from 47.4 to 474 GBq/µmol (1280 to 12,800 mCi/µmol) [66]. However, this method is limited by the high cost of 64 Ni-enriched targets [67]. Additionally, several alternative routes for 64 Cu production are reported, such as 64 Zn(n,p) 64 64 Cu and 67 Zn(p,α) 64 Cu reactions [70].
In addition to theranostic properties, a longer half-life of cyclotron-produced copper-64 provides logistical advantages over clinically used cyclotron-produced fluorine-18 (T 1/2 = 109 min) due to its ability to conduct PET studies away from the cyclotron; also, a longer half-life when compared to clinically used gallium-68 (T 1/2 = 68 min) makes it possible to provide extended tumor imaging, as well as metastatic detection. The lower positron energy of 64 Cu when compared to 68 Ga provides a lower positron mean range (0.56 mm versus 3.5 mm), resulting in improved PET image quality, increased resolution, and higher diagnostic quality [71]. Additionally, a direct comparison of PET/CT image quality and spatial resolution obtained with 18 F, 68 Ga and 64 Cu revealed a similar image quality obtained with 18 F-FDG and 64 Cu-HCl, evidently due to similar energies arising from the decay of the 18 F and 64 Cu positions. In contrast, the much higher energy of positrons arising from the decay of 68 Ga degrades image quality and spatial resolution [72].
Copper-67 (T 1/2 = 61.8 h, β − : 100%, γ: 49%) is the longest-lived radioisotope of copper, with a half-life that is suitable for imaging and beta particle energy that is appropriate for therapy. The tendency of isotopes to gamma decay with appropriate energies provides the possibility for simultaneous SPECT imaging and radiotherapy, as well as monitoring of the uptake and biodistribution of the radiotherapeutic agent during therapy [73]. A slightly higher β − emission energy than that of clinically used 177 Lu, in conjunction with a shorter half-life, causes 67 Cu to be considered as an "ideal" radionuclide for radioim-munotherapy, which is commonly associated with slow pharmacokinetics [74]. However, despite the great potential for both imaging and therapy, worthwhile research of 67 Cu potential as a theranostic radionuclide has not been carried out due to the inaccessibility of the radionuclide itself.
Obtaining a sufficient amount of the 67 Cu isotope with a high specific activity using accelerator-based production has always been a difficult task [75]. The commonly used proton-based reactions 68 Zn(p,2p) 67 Cu and 70 Zn(p,α) 67 Cu are complicated by the co-production of 64 Cu. Recently, 67 Cu production via the 70 Zn(p,α) 67 Cu reaction with a compact cyclotron was reported by Brühlmann et al. [76]. In addition, deuteron beam irradiation of enriched 70 Zn target 70 Zn(d,αn) 67 Cu is a highly promising method for 67 Cu production [77,78]. The neutron-induced reaction 67 Zn(n,p) 67 Cu requires a highflux nuclear reactor with a fast-neutron flux [79]. A breakthrough of 67 Cu production was achieved in the U.S. through the Department of Energy Isotope Program (DOE-IP). The use of high-energy photon-induced reactions on isotopically enriched 68 Zn targets 68 Zn(γ,p) 67 Cu opened opportunities for a reliable source of 67 Cu with high specific activity > 1850 GBq/mg, radionuclide purity and sufficient quantity [80,81].
In addition to the above indisputable advantages of copper radionuclides, the wellestablished coordination chemistry of copper provides its reaction with different types of chelator systems [82]. The chemical identity of imaging 64 Cu and therapeutic 67 Cu copper radioisotopes allows for the convenient use of the same bifunctional chelators for both 64 Cu and 67 Cu-based radiopharmaceuticals, for sequential PET imaging and radiotherapy. Considering the opened opportunities for obtaining 67 Cu of high purity and activity, an interest in the use of 64  Copper-based radiopharmaceuticals have been widely discussed for the treatment and diagnosis of various diseases in the last 30 years.
[ 64 Cu][Cu(ATSM)] is a group of thiosemicarbazone-based drugs, effective PETtracers of tumor hypoxia, which proved its effectiveness in both preclinical and clinical Copper-based radiopharmaceuticals have been widely discussed for the treatment and diagnosis of various diseases in the last 30 years.
[ 64 Cu][Cu(ATSM)] is a group of thiosemicarbazone-based drugs, effective PET-tracers of tumor hypoxia, which proved its effectiveness in both preclinical and clinical studies. Still, one of the 64 Cu ATSM drugs is in phase II of clinical trials for rectum cancer imaging [83]. In 2020, Liu et al. summarized the use of copper radiopharmaceuticals [Cu(ATSM)] for PET imaging of hypoxic tumors [84].
In 2020, we summarized the use of different metals, including 64 Cu in PET imaging of Alzheimer's disease [85]. In 2018, Ahmedova et al. summarized theranostic applications of copper radionuclides [86]. In 2023, Capriotti et al. summarized the use of 64 CuCl 2 for both PET imaging and theragnostic [87].
Herein, we summarize recent (2018-2023) advances in the use of copper-based radiopharmaceuticals, for PET imaging, radiotherapy and the use of 64 Cu/ 67 Cu as a theranostic pair.

Summary of Copper-Based Radiopharmaceuticals, Reported in 2018-2023
We summarized the copper-based radiopharmaceuticals developed in 2018-2023 in Table 2.

Octreotate
Somatostatin (SST) is a small peptide that regulates both cell growth and hormone secretion. Somatostatin receptors (SSTRs) are a common target for the treatment of neuroendocrine tumors (NETs) [112]. Octreotate is a peptide capable of SSTRs binding, which is used worldwide for the targeted delivery of radioactive isotopes to NETs [113,114].
Detectnet (Copper 64 Cu-dotatate) is a PET-imaging agent for the localization of SSTRpositive NETs in adult patients, which was approved by the Food and Drug Administration (FDA) in 2020 [88]. One year before, in 2019, a similar 68 Ga-based radiopharmaceutical [ 68 Ga]Ga-DOTA-TOC was approved by FDA as the first 68 Ga-radiopharmaceutical for imaging of SSTR-positive gastroenteropancreatic NETs [115]. Both radiopharmaceuticals are based on tyrosine-octreotate, conjugated with DOTA (tetraxetan) as a metal chelator ( Figure 2).

PSMA
Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein that consists of 750 amino acids, that are overexpressed in tumor tissues 100-to 1000-fold higher than that in normal ones. The commonly used substrate of PSMA is a urea-based peptide with a C-terminal glutamate, capable of active site binding [117,118]. In 2022, Debnath et al. summarized PSMA-targeting and theranostic agents [119]. In 2022, Jeitner et al. also summarized advances in PSMA theranostics [120]. Herein, we provide several copperbased radiopharmaceuticals for prostate cancer therapy and imaging.
Gallium ( 68 Ga) gozetotide is a clinically used drug for PET prostate cancer imaging, which was approved in the United States in December 2020 [121] and in the European Union in December 2022 [122].
Despite FDA approval of ( 68 Ga) gozetotide for prostate cancer imaging, several benefits of 64 Cu over 68 Ga arouse interest in 64 Cu-based radiopharmaceuticals for PET imaging of prostate cancer [123]. Additionally, 64 CuCl 2 salt showed promising therapeutic efficacy on the 3D prostate cancer model [124].
In Gallium ( 68 Ga) gozetotide is a clinically used drug for PET prostate cancer imaging, which was approved in the United States in December 2020 [121] and in the European Union in December 2022 [122].
Despite FDA approval of ( 68 Ga) gozetotide for prostate cancer imaging, several benefits of 64 Cu over 68 Ga arouse interest in 64 Cu-based radiopharmaceuticals for PET imaging of prostate cancer [123]. Additionally, 64 CuCl2 salt showed promising therapeutic efficacy on the 3D prostate cancer model [124].
In  RPS-085 was designed based on previously reported ligand RPS-063 with DOTA chelator [125]. Metal-free RPS-085 showed the ability for both PSMA inhibition and  In 2019, Zia et al. reported two sarcophagine ligands with one or two PSMA-targeting moieties ( Figure 5) [93]. The cell surface binding and internalization were evaluated in LNCaP cells and [ 64 Cu]CuSarbisPSMA displayed higher cell surface binding and internalization, which is evidence that two target-binding vectors yield better results than one.
To access imaging properties, PET images of PSMA-positive LNCaP-tumor-bearing NSG mice were obtained at 0.5, 2 and 22 h p.i., and significant tumor uptake of both radioligands was evident. Expectedly, bivalent [ 64 Cu]CuSarbisPSMA showed higher tumor uptake and retention when compared to the monomer, which was confirmed in vivo biodistribution studies. However, [ 64 Cu]CuSarPSMA showed better tumor uptake than clinically used 68 Ga-PSMA (Ga-PSMA-11) at 1 h p.i.
Since 64 Cu-CuSarbisPSMA showed both an excellent uptake and retention in LNCaP tumors, the suggestion that the 67 67 Cu than of 177 Lu (61.9 h vs. 6.7 days) shortens radiotherapy while maintaining its efficiency. 64 Cu-SAR-bisPSMA and 67 Cu-SAR-bisPSMA are currently in clinical trials as drugs for identification and treatment of PSMA-expressing metastatic castrate resistant prostate cancer (NCT04868604). In 2019, Zia et al. reported two sarcophagine ligands with one or two PSMA-targeting moieties ( Figure 5) [93]. The cell surface binding and internalization were evaluated in LNCaP cells and [ 64 Cu]CuSarbisPSMA displayed higher cell surface binding and internalization, which is evidence that two target-binding vectors yield better results than one.
To access imaging properties, PET images of PSMA-positive LNCaP-tumor-bearing NSG mice were obtained at 0.5, 2 and 22 h p.i., and significant tumor uptake of both radioligands was evident. Expectedly, bivalent [ 64 Cu]CuSarbisPSMA showed higher tumor uptake and retention when compared to the monomer, which was confirmed in vivo biodistribution studies. However, [ 64 Cu]CuSarPSMA showed better tumor uptake than clinically used 68 Ga-PSMA (Ga-PSMA-11) at 1 h p.i.
Since 64 Cu-CuSarbisPSMA showed both an excellent uptake and retention in LNCaP tumors, the suggestion that the 67

Other Peptides
In

Other Peptides
In   Three 64 Cu-labeled cross-bridged chelators showed better in vivo stability compared to the two non-cross-bridged chelators and 64 Cu-labeled PCB-TE2A-Bn-NCS proved to be the most stable. PET imaging in glioma U87MG tumor-bearing mice was obtained, and two 64 Cu-labeled PCB-TE2A conjugates exhibited higher tumor uptake compared with others. 64 Cu-PCB-TE2A-Bn-NCS-c(RGDyK) also showed 4-fold lower demetallation in blood compared with the others.
In 2022, Qiao et al. demonstrated the ability of copper-based radiopharmaceuticals to visualize melanoma. Two theranostic 64 Cu-radiolabeled α-MSH peptides were designed as potential agents for melanoma PET imaging (Figure 7) [96]. the most stable. PET imaging in glioma U87MG tumor-bearing mice was obtained, and two 64 Cu-labeled PCB-TE2A conjugates exhibited higher tumor uptake compared with others. 64 Cu-PCB-TE2A-Bn-NCS-c(RGDyK) also showed 4-fold lower demetallation in blood compared with the others. The melanocortin-1 receptors (MC1Rs) are a group of G protein-coupled receptors, which are overexpressed in human melanomas. MC1Rs can bind with alpha-melanocytestimulating hormone (α-MSH) peptides [126].
In 2022, Qiao et al. demonstrated the ability of copper-based radiopharmaceuticals to visualize melanoma. Two theranostic 64 Cu-radiolabeled α-MSH peptides were designed as potential agents for melanoma PET imaging (Figure 7) [96]. Gastrin-releasing peptide receptor (GRPR) is overexpressed on the surface of different cancers. GRPR can bind with high affinity to bombesin, a 14-amino acid peptide. Since bombesin itself exhibits low stability, its analogs have been investigated as GRPRtargeted ligands for the diagnosis and therapy of GRPR-positive tumors [127]. Gastrin-releasing peptide receptor (GRPR) is overexpressed on the surface of different cancers. GRPR can bind with high affinity to bombesin, a 14-amino acid peptide. Since bombesin itself exhibits low stability, its analogs have been investigated as GRPR-targeted ligands for the diagnosis and therapy of GRPR-positive tumors [127].

Direct Conjugation of Radiolabeled Chelator and Antibody
Radioimmunotherapy (RIT) is a subtype of radiotherapy, that uses monoclonal antibodies as a delivery agent for radionuclides. Antibodies labeled with positronemitting radionuclides are used for PET imaging and dosimetry, while radioimmunoconjugates labeled with therapeutic nuclides are used for therapy [128][129][130].
PD-1/PD-L1 inhibitors are a class of anticancer drugs, capable of blocking the activity of PD-1 and PDL1 immune checkpoint proteins [131]. Thus, anti-PD-1 or anti-PD-L1 antibodies are clinically used, and non-invasive imaging of PD-L1 expression levels in malignant tumors is of interest [132].
In 2018, Xu et al. reported the successful immunotherapy of a PD-L1 positive MC38 tumor with an anti-PD-L1 antibody, which was preliminary radiolabeled with copper-64, and its tumor accumulation was confirmed using PET [98] (Figure 9).
When PET imaging of MC38 and 4T1 tumor grafts in vivo were performed, only the PD-L1 positive MC38 tumor was visualized by radiolabeled antibody [ 64 Cu]Cu-NOTA-MX001. Immunotherapy studies provided in mice bearing MC38 tumor with MX001 antibody resulted in tumor growth suppression. In contrast, low anti-tumor efficacy of MX001 on 4T1 tumor was revealed, thereby proving the effectiveness and specificity of immunotherapy. Thus, an antibody may be successfully visualized with 64 Cu before immunotherapy.

Direct Conjugation of Radiolabeled Chelator and Antibody
Radioimmunotherapy (RIT) is a subtype of radiotherapy, that uses monoclonal antibodies as a delivery agent for radionuclides. Antibodies labeled with positron-emitting radionuclides are used for PET imaging and dosimetry, while radioimmunoconjugates labeled with therapeutic nuclides are used for therapy [128][129][130].
PD-1/PD-L1 inhibitors are a class of anticancer drugs, capable of blocking the activity of PD-1 and PDL1 immune checkpoint proteins [131]. Thus, anti-PD-1 or anti-PD-L1 antibodies are clinically used, and non-invasive imaging of PD-L1 expression levels in malignant tumors is of interest [132].
In 2018, Xu et al. reported the successful immunotherapy of a PD-L1 positive MC38 tumor with an anti-PD-L1 antibody, which was preliminary radiolabeled with copper-64, and its tumor accumulation was confirmed using PET [98] (Figure 9).
When PET imaging of MC38 and 4T1 tumor grafts in vivo were performed, only the PD-L1 positive MC38 tumor was visualized by radiolabeled antibody [ 64 Cu]Cu-NOTA-MX001. Immunotherapy studies provided in mice bearing MC38 tumor with MX001 antibody resulted in tumor growth suppression. In contrast, low anti-tumor efficacy of MX001 on 4T1 tumor was revealed, thereby proving the effectiveness and specificity of immunotherapy. Thus, an antibody may be successfully visualized with 64 Cu before immunotherapy. Trastuzumab is a human epidermal growth factor receptor protein (HER-2)-affine monoclonal antibody, clinically used in the treatment of (HER-2)+ metastatic breast cancer [133].
In 2021, Lee et al. reported a visualization of a NIH3T6.7 tumor with a 64 Curadiolabeled trastuzumab antibody. In addition, a novel conjugation approach based on click reaction was proposed [99]. For the chemical binding of the antibody with a radiolabeled chelator, Tz/TCO click reaction was used. Tz/TCO is a bio-orthogonal inverse electron-demand Diels-Alder click reaction between trans-cyclooctene (TCO) and tetrazine (Tz) (Figure 10). Copper-catalyzed azide-alkyne cycloaddition is usually not used in the synthesis of copper-chelating conjugates, due to it chelating the catalyst with reagents and the subsequent failure of the reaction. However, Lee et al. succeeded in choosing the conditions for the click reaction in which the catalytic agent is not chelated and the fast and quantitative click conjugation of the chelator and linker occurs. Since a cross-bridged chelator is not prone to complexation with Cu(II) ions at a lower temperature, Cu(I)catalyzed alkyne−azide cycloaddition was used for conjugation of chelator and linker ( Figure 11). Both 64 Cu-radiolabeled trastuzumab conjugates showed in vivo stability, and tumor accumulation and proved the ability to visualize a HER-2 positive NIH3T6.7 tumor. However, the conjugate with a PEG linker demonstrated fast body clearance.
Pertuzumab is another anti-HER-2 humanized monoclonal antibody that is used in combination with Trastuzumab in the therapy of HER-2-positive breast cancers [134].
In 2021, Hao et al. reported successful radioimmunotherapy of HER-2 positive HCC1954 tumor with radiolabeled 67 Cu-Pertuzumab [100]. [ 67 Cu]Cu-NOTA-Pertuzumab was obtained by conjugation of p-SCN-Bn-NOTA to pertuzumab and further radiolabeling. The efficacy of radioimmunotherapy was assessed on mice xenografts Trastuzumab is a human epidermal growth factor receptor protein (HER-2)-affine monoclonal antibody, clinically used in the treatment of (HER-2)+ metastatic breast cancer [133].
In 2021, Lee et al. reported a visualization of a NIH3T6.7 tumor with a 64 Cu-radiolabeled trastuzumab antibody. In addition, a novel conjugation approach based on click reaction was proposed [99]. For the chemical binding of the antibody with a radiolabeled chelator, Tz/TCO click reaction was used. Tz/TCO is a bio-orthogonal inverse electron-demand Diels-Alder click reaction between trans-cyclooctene (TCO) and tetrazine (Tz) (Figure 10). Trastuzumab is a human epidermal growth factor receptor protein (HER-2)-affine monoclonal antibody, clinically used in the treatment of (HER-2)+ metastatic breast cancer [133].
In 2021, Lee et al. reported a visualization of a NIH3T6.7 tumor with a 64 Curadiolabeled trastuzumab antibody. In addition, a novel conjugation approach based on click reaction was proposed [99]. For the chemical binding of the antibody with a radiolabeled chelator, Tz/TCO click reaction was used. Tz/TCO is a bio-orthogonal inverse electron-demand Diels-Alder click reaction between trans-cyclooctene (TCO) and tetrazine (Tz) (Figure 10). Copper-catalyzed azide-alkyne cycloaddition is usually not used in the synthesis of copper-chelating conjugates, due to it chelating the catalyst with reagents and the subsequent failure of the reaction. However, Lee et al. succeeded in choosing the conditions for the click reaction in which the catalytic agent is not chelated and the fast and quantitative click conjugation of the chelator and linker occurs. Since a cross-bridged chelator is not prone to complexation with Cu(II) ions at a lower temperature, Cu(I)catalyzed alkyne−azide cycloaddition was used for conjugation of chelator and linker ( Figure 11). Both 64 Cu-radiolabeled trastuzumab conjugates showed in vivo stability, and tumor accumulation and proved the ability to visualize a HER-2 positive NIH3T6.7 tumor. However, the conjugate with a PEG linker demonstrated fast body clearance.
Pertuzumab is another anti-HER-2 humanized monoclonal antibody that is used in combination with Trastuzumab in the therapy of HER-2-positive breast cancers [134].
In  Copper-catalyzed azide-alkyne cycloaddition is usually not used in the synthesis of copper-chelating conjugates, due to it chelating the catalyst with reagents and the subsequent failure of the reaction. However, Lee et al. succeeded in choosing the conditions for the click reaction in which the catalytic agent is not chelated and the fast and quantitative click conjugation of the chelator and linker occurs. Since a cross-bridged chelator is not prone to complexation with Cu(II) ions at a lower temperature, Cu(I)-catalyzed alkyne−azide cycloaddition was used for conjugation of chelator and linker ( Figure 11). Both 64 Cu-radiolabeled trastuzumab conjugates showed in vivo stability, and tumor accumulation and proved the ability to visualize a HER-2 positive NIH3T6.7 tumor. However, the conjugate with a PEG linker demonstrated fast body clearance.
Pertuzumab is another anti-HER-2 humanized monoclonal antibody that is used in combination with Trastuzumab in the therapy of HER-2-positive breast cancers [134].
The F(ab)'2 fragments of R-anti-mouse CD4 antibodies were conjugated to NOTA and radiolabeled with 64 Cu. PET/CT images of a mouse with collagen-induced arthritis at different time points were obtained. Despite the drug accumulation in various organs, an increased accumulation of [ 64 Cu]Cu-NOTA-IgG2b in joints with pronounced arthritis was revealed. Additionally, a decrease in tracer accumulation after dexamethasone injection confirmed a correlation of [ 64 Cu]Cu-NOTA-CD4 accumulation with arthritic inflammation levels.

Pretargeting Approach in Conjugation of Radiolabeled Chelator and Antibody
One of the main disadvantages of radioimmunotherapy is the fact that it can take several days for antibodies after administration to accumulate in their therapeutic target (tumor tissue). Thus, if antibodies are used as delivery agents for therapeutic radionuclides, only long-lived radionuclides should be used, which can lead to high radiation doses to healthy tissues. To solve this problem, in vivo pretargeting approach was suggested based on injecting the two components separately. An antibody is given several hours (or days) to accumulate in the tumor and clear from the blood. Then, the radiolabeled small molecule, capable of chemical binding with the antibody, is administered [136].
In 2020, Keinänen et al. reported an in vivo pretargeting with 64 Cu/ 67 Cu theranostic pair, with both PET imaging and subsequent radioimmunotherapy of SW1222 human colorectal carcinoma [102].
The F(ab)'2 fragments of R-anti-mouse CD4 antibodies were conjugated to NOTA and radiolabeled with 64 Cu. PET/CT images of a mouse with collagen-induced arthritis at different time points were obtained. Despite the drug accumulation in various organs, an increased accumulation of [ 64 Cu]Cu-NOTA-IgG2b in joints with pronounced arthritis was revealed. Additionally, a decrease in tracer accumulation after dexamethasone injection confirmed a correlation of [ 64 Cu]Cu-NOTA-CD4 accumulation with arthritic inflammation levels.

Pretargeting Approach in Conjugation of Radiolabeled Chelator and Antibody
One of the main disadvantages of radioimmunotherapy is the fact that it can take several days for antibodies after administration to accumulate in their therapeutic target (tumor tissue). Thus, if antibodies are used as delivery agents for therapeutic radionuclides, only long-lived radionuclides should be used, which can lead to high radiation doses to healthy tissues. To solve this problem, in vivo pretargeting approach was suggested based on injecting the two components separately. An antibody is given several hours (or days) to accumulate in the tumor and clear from the blood. Then, the radiolabeled small molecule, capable of chemical binding with the antibody, is administered [136].
In 2020, Keinänen et al. reported an in vivo pretargeting with 64 Cu/ 67 Cu theranostic pair, with both PET imaging and subsequent radioimmunotherapy of SW1222 human colorectal carcinoma [102].
Two radioligands, [ 64 Cu]CuMeCOSar-Tz and [ 67 Cu]Cu-MeCOSar-Tz, as well as TCOconjugated huA33 antibody, capable of targeting the A33 antigen, which is expressed in >95% colorectal cancers, were designed ( Figure 13).  As for therapeutic efficacy, various strategies of longitudinal therapy have been tried to find the optimal dose and interval. As a result, HuA33-TCO and [ 67 Cu]CuMeCOSar-Tz were injected 72 h apart, and a dose-dependent therapeutic response was observed. Importantly, PET images registered after injection of [ 64 Cu]Cu-MeCOSar-Tz accurately predicted the efficacy of the [ 67 Cu]Cu-MeCOSar-Tz, which was injected later, which is direct evidence of the effectiveness of the theranostic couple concept.
to find the optimal dose and interval. As a result, HuA33-TCO and [ 67 Cu]CuMeCOSar were injected 72 h apart, and a dose-dependent therapeutic response was observ Importantly, PET images registered after injection of [ 64 Cu]Cu-MeCOSar-Tz accura predicted the efficacy of the [ 67 Cu]Cu-MeCOSar-Tz, which was injected later, whic direct evidence of the effectiveness of the theranostic couple concept.
In 2022, Jallinoja et al. reported another pretargeting approach, with novel ferroce based radioligands ([ 64 Cu]Cu-NOTA-PEG3-Fc and [ 64 Cu]Cu-NOTA-PEG7-Fc) (Figure [103]. To conjugate antibodies with a chelator, a host-guest chemistry between a cucu [7]uril (CB7) and a ferrocene (Fc) was used [137]. M5A, CB7-M5A antibody can bind carcinoembryonic antigen (CEA), which expressed in several cancers, such as colorectal, gastric and pancreatic cancers, and a in some breast cancer and non-small-cell lung cancer [138]. The antibody was modifi with dibenzocyclooctyne. Both radioligands showed good in vitro stability and h similar in vivo profiles in healthy mice, with relatively slow excretion through gastrointestinal tract. The pretargeting approach has been investigated with a t interval of 120 h, and radioligands showed specific tumor uptake. In addition pretargeting approach with an extended time interval of up to 9 days still showed go tumor localization.

Another Copper-Based Radiopharmaceutical
Boron neutron capture therapy (BNCT) is based on the irradiation of boron-10-ba agents with low-energy thermal neutrons to yield high yields of lithium-7 and al particles. The heavy alpha particle has a short range, which allows for the localization the radiation effect [139]. However, despite the advantages of BNCT, mapping bor based biodistribution in the patient is unobtainable [140]. For boron mapping, opt imaging and PET imaging may be applied. One of the ways for both imaging method be optimized is the use of boronated porphyrins, which can chelate copper catio resulting in 64Сu-based agents for visualization [141].
Fluorescence imaging properties of BPN were confirmed in vivo in 4T1 tum bearing mice, and tumor imaging was performed with the system. Tumors w visualized separately from surrounding tissues. M5A, CB7-M5A antibody can bind carcinoembryonic antigen (CEA), which is expressed in several cancers, such as colorectal, gastric and pancreatic cancers, and also in some breast cancer and non-small-cell lung cancer [138]. The antibody was modified with dibenzocyclooctyne. Both radioligands showed good in vitro stability and had similar in vivo profiles in healthy mice, with relatively slow excretion through the gastrointestinal tract. The pretargeting approach has been investigated with a time interval of 120 h, and radioligands showed specific tumor uptake. In addition, a pretargeting approach with an extended time interval of up to 9 days still showed good tumor localization.

Another Copper-Based Radiopharmaceutical
Boron neutron capture therapy (BNCT) is based on the irradiation of boron-10-based agents with low-energy thermal neutrons to yield high yields of lithium-7 and alpha particles. The heavy alpha particle has a short range, which allows for the localization of the radiation effect [139]. However, despite the advantages of BNCT, mapping boronbased biodistribution in the patient is unobtainable [140]. For boron mapping, optical imaging and PET imaging may be applied. One of the ways for both imaging methods to be optimized is the use of boronated porphyrins, which can chelate copper cations, resulting in 64Cu-based agents for visualization [141].
Fluorescence imaging properties of BPN were confirmed in vivo in 4T1 tumor-bearing mice, and tumor imaging was performed with the system. Tumors were visualized separately from surrounding tissues.   These data open up a novel possibility for easy and quick radiolabeling of biomolecules with 64 Cu/ 67 Cu via using a photochemistry approach, thus yielding radiopharmaceuticals for PET imaging for radiotherapy.

Nanoparticles (Nps)
Nps are a powerful tool for various biomedical applications such as targeted drug delivery, bioimaging, diagnostics, theranostics and therapy for various diseases [142][143][144][145]. Among the countless applications of nanoparticles, their use for combined MRI-PET diagnostics, achieved by labeling Nps with imaging or theranostic isotopes is of interest [146]. Thus, an introduction of copper-64 radioactive isotope makes it possible to obtain materials for both PET diagnostics and radiotherapy of tumors. Currently, there is a clinical trial phase 1 under recruiting to evaluate 64 Cu-labeled NPs to guide the surgical treatment of prostate cancer (NCT04167969).
Recently, Pijeira et al. summarized the use of radiolabeled nanomaterials for biomedical applications [147]. Recently, Carrese et al. have also summarized the use of Nps in cancer theranostics [148]. Herein, we provide several examples of radiolabeled Nps for various biomedical applications.
In 2018, Madru et al. reported a hybrid PET-MRI probe, based on 64 Cu-radiolabeled superparamagnetic iron oxide Nps. In addition to a simple radiolabeling technique that does not require a chelating agent, successful double PET-MRI imaging of the lymph nodes has been performed [106].
Chelator-free radiolabeled [ 64 Cu]CuS Nps have also been discussed as a promising agent for simultaneous micro-PET/CT imaging and photothermal ablation therapy [149]. These data open up a novel possibility for easy and quick radiolabeling of biomolecules with 64 Cu/ 67 Cu via using a photochemistry approach, thus yielding radiopharmaceuticals for PET imaging for radiotherapy.

Nanoparticles (Nps)
Nps are a powerful tool for various biomedical applications such as targeted drug delivery, bioimaging, diagnostics, theranostics and therapy for various diseases [142][143][144][145]. Among the countless applications of nanoparticles, their use for combined MRI-PET diagnostics, achieved by labeling Nps with imaging or theranostic isotopes is of interest [146]. Thus, an introduction of copper-64 radioactive isotope makes it possible to obtain materials for both PET diagnostics and radiotherapy of tumors. Currently, there is a clinical trial phase 1 under recruiting to evaluate 64 Cu-labeled NPs to guide the surgical treatment of prostate cancer (NCT04167969).
Recently, Pijeira et al. summarized the use of radiolabeled nanomaterials for biomedical applications [147]. Recently, Carrese et al. have also summarized the use of Nps in cancer theranostics [148]. Herein, we provide several examples of radiolabeled Nps for various biomedical applications.
In 2018, Madru et al. reported a hybrid PET-MRI probe, based on 64 Cu-radiolabeled superparamagnetic iron oxide Nps. In addition to a simple radiolabeling technique that does not require a chelating agent, successful double PET-MRI imaging of the lymph nodes has been performed [106].
Chelator-free radiolabeled [ 64 Cu]CuS Nps have also been discussed as a promising agent for simultaneous micro-PET/CT imaging and photothermal ablation therapy [149] In   Thakare et al. reported a trifunctional imaging probe, based on AGuIX ® Nps [150], functionalized with NODAGA copper chelator, NIR heptamethine cyanine dye and maleimide as stabilized moiety (Figure 18) [108]. compared to PEG-[ 64 Cu]CuS, which was confirmed by both micro-PET/CT and biodistribution studies.
In 2020, Zhou et al. reported a 64 Cu -labeled PEGylated melanin Nps, which was previously reported as a promising platform for multimodality imaging [109,151]. Radiolabeled melanin Nps showed a therapeutic effect on the A431 tumor.
In 2020, Paiva et al. also reported a polymeric micellar Nps (PMNPs), conjugated with EGFR-targeting GE11 peptide via diazo-tyrosin coupling [110]. Importantly, the prelabeling strategy was used in radiolabeling of Nps with 64 Cu (Figure 19).  The resulting AGuIX ® nanoparticles, functionalized with IR-783-Lys(Mal)NODAGA are appropriate for simultaneous PET-MRI and optical trimodal imaging, which was confirmed in a TSA tumor model.
In 2020, Zhou et al. reported a 64 Cu-labeled PEGylated melanin Nps, which was previously reported as a promising platform for multimodality imaging [109,151]. Radiolabeled melanin Nps showed a therapeutic effect on the A431 tumor.
In 2020, Zhou et al. reported a 64 Cu -labeled PEGylated melanin Nps, which was previously reported as a promising platform for multimodality imaging [109,151]. Radiolabeled melanin Nps showed a therapeutic effect on the A431 tumor.
In 2020, Paiva et al. also reported a polymeric micellar Nps (PMNPs), conjugated with EGFR-targeting GE11 peptide via diazo-tyrosin coupling [110]. Importantly, the prelabeling strategy was used in radiolabeling of Nps with 64 Cu (Figure 19).   64 Cu-GE11 PMNPs displayed enhanced tumor accumulation due to EGFR targeting effects, which was confirmed by both PET imaging and biodistribution studies in vivo EGFR-positive colorectal HCT116 tumor model.
He et al. have reported antigen-delivery nanoplatforms based on poly(ethylene glycol) (PEG, Mw 500) and pyropheophorbide-A (PPa) in order to deliver the melanoma antigen peptide, Trp2180−188 (SVYDFFVWL), to dendritic cells (DCs) and stimulate CD8+ T-cell immune response [111] (Figure 20). effects, which was confirmed by both PET imaging and biodistribution studies in vivo EGFR-positive colorectal HCT116 tumor model.
He et al. have reported antigen-delivery nanoplatforms based on poly(ethylene glycol) (PEG, Mw 500) and pyropheophorbide-A (PPa) in order to deliver the melanoma antigen peptide, Trp2180−188 (SVYDFFVWL), to dendritic cells (DCs) and stimulate CD8+ T-cell immune response [111] (Figure 20). DC uptake of Trp2/PPa−PEGm was confirmed using flow cytometry. Radiolabeling of NPs with 64 Cu allows real-time monitoring of the migration process of labeled DCs to draining lymph nodes (DLNs), which was demonstrated in C57BL/6 mice.
In addition, a vaccine based on DCs treated with Trp2/PPa−PEGm NPs stimulated a significant immune response in C57BL/6 mice. Finally, a significant tumor growth inhibition was detected in C57BL/6 mice with B16-F10 melanoma tumor, injected with DCs/Trp2/PPa−PEGm NPs three times at a weekly interval. This result not only demonstrates the possibility of immunotherapy with NP-modified DCs but also the possibility of biodistribution monitoring in vivo after radiolabeling with 64 Cu. With the use of the 67 Сu isotope, a combination of immunotherapy and radiotherapy would be possible, which is of interest.

Conclusions
In 2020, copper 64 Cu-dotatate was approved by the FDA as a radioactive diagnostic agent for PET-imaging agent for SSTR-positive NETs in adult patients. The mere fact of the approval of a copper-containing drug for clinical practice encourages researchers to design new effective copper-containing drugs for the therapy, diagnosis and theragnostic of various diseases. Due to the unique emission properties of copper isotopes, there is great interest in their use as both imaging and therapeutic agents. Copper-64 is a Figure 20. PEG-PPa Trp2 melanoma antigen-delivery nanoplatform, radiolabeled with 64 Cu, designed by He et al. [111]. DC uptake of Trp2/PPa−PEGm was confirmed using flow cytometry. Radiolabeling of NPs with 64 Cu allows real-time monitoring of the migration process of labeled DCs to draining lymph nodes (DLNs), which was demonstrated in C57BL/6 mice.
In addition, a vaccine based on DCs treated with Trp2/PPa−PEGm NPs stimulated a significant immune response in C57BL/6 mice. Finally, a significant tumor growth inhibition was detected in C57BL/6 mice with B16-F10 melanoma tumor, injected with DCs/Trp2/PPa−PEGm NPs three times at a weekly interval. This result not only demonstrates the possibility of immunotherapy with NP-modified DCs but also the possibility of biodistribution monitoring in vivo after radiolabeling with 64 Cu. With the use of the 67 Cu isotope, a combination of immunotherapy and radiotherapy would be possible, which is of interest.

Conclusions
In 2020, copper 64 Cu-dotatate was approved by the FDA as a radioactive diagnostic agent for PET-imaging agent for SSTR-positive NETs in adult patients. The mere fact of the approval of a copper-containing drug for clinical practice encourages researchers to design new effective copper-containing drugs for the therapy, diagnosis and theragnostic of various diseases. Due to the unique emission properties of copper isotopes, there is great interest in their use as both imaging and therapeutic agents. Copper-64 is a cyclotronproduced nuclide with excellent energy characteristics and optimum half-life, allowing for thorough PET imaging of malignant neoplasms not available with the short-lived 68 Ga and 18 F nuclides.
The 67 Cu isotope has long been regarded as an "ideal but inaccessible" radionuclide for radiotherapy and radioimmunotherapy, due to its excellent energy characteristics and long half-life. A recent breakthrough in the production of copper-67 isotopes made it possible to essay this previously inaccessible radionuclide in action, both as a nuclide for radiotherapy and for theranostics. Additionally, the long half-life of the copper-67 isotope makes it an ideal nuclide for radioimmunotherapy, and for control of the accumulation of antibodies in a therapeutic target.
The use of the theranostic pair copper 64/67 is also of great interest due to the convenient interchangeability of copper ions. Since the nuclide 67 Cu isotope was previously not available in sufficient quantities, a 64 Cu/ 67 Cu was not tested as a theranostic pair either. Now, the opportunity for sequential PET imaging, dosimetry, radiotherapy and SPECT imaging has opened up.
In this review, we have summarized a recent successful application of copper-based radiopharmaceuticals for PET, SPECT imaging, radiotherapy, and radioimmunotherapy. Thus, several successful PET-imaging of malignant neoplasms with 64 Cu-based radiopharmaceuticals were reported [91,92,95,96], as well as imaging of rheumatoid arthritis [81], and visualization of the distribution of agents for boron neutron capture therapy [83]. A principal possibility of pre-imaging with copper-64 before radiotherapy with copper-67 has been proven by Kelly et al. [92].
Several quite interesting results in PET imaging with antibody-based agents, PET visualization of antibody biodistribution, immunotherapy, and radioimmunotherapy were also summarized. A successful PET pre-imaging of antibody accumulation in tumor, followed by effective immunotherapy, was reported by Xu et al. [98], PET-visualization of HER-2 positive NIH3T6.7 tumor with 64 Cu-labeled trastuzumab antibody was reported by Lee et al. [99]. An extremely important result, namely, a successful radioimmunotherapy of HER-2 positive HCC1954 tumor with SPECT imaging, was reported by Hao et al. [100]. These data are evidence of both the radiotherapeutic properties of the copper-67 isotope and the possibility of the use of 67 Cu-based radiopharmaceuticals as theranostic agents.
A pretargeting approach based on separate injections of the antibody and radiolabeled chelator has shown its effectiveness both for imaging of and therapy for tumor diseases [102,103]. An important and elegant study was reported by Keinänen et al. [102]. demonstrated the use of 64 Cu/ 67 Cu theranostic couple in the pretargeting assay; not only successful PET imaging of the SW1222 human colorectal carcinoma with 64 Cu-based conjugate but also therapeutic efficacy of 67 Cu-based conjugate has been shown. PET images predicted the efficacy of radiotherapy, which is direct evidence of the effectiveness of the theranostic couple concept.
The development of radiolabeled nanoparticles is definitely of interest, due to the possibility of simultaneous use of several diagnostic modalities, such as PET-MRI, fluorescence imaging, SPECT-MRI, etc. Additionally, the radiolabeling of nanoparticles with copper-67 beta-emitter is of great interest for the development of theranostic platforms.
Several incredibly successful results in the therapy, diagnosis and theranostic of tumor diseases, presented in this review, show the great potential of copper-containing radiopharmaceuticals in nuclear medicine and medicinal chemistry. Given the recent breakthrough in obtaining the copper-67 isotope in sufficient quantity and purity, the field of use of both the therapeutic radionuclide 67 Cu and the theranostic pair 64 Cu/ 67 Cu is just beginning; however, the results obtained so far are quite impressive. Thus, both the effectiveness and great potential of copper-containing radiopharmaceuticals both as imaging and therapy agents are undoubted.
Funding: This review (except Table 1) was funded by Russian Science Foundation, grant number 19-74-10059-Π; Table 1 was carried out within the framework of the Implementation Program Priority 2030 (NUST MISIS).

Conflicts of Interest:
The authors declare no conflict of interest.