PET radiometals for antibody labeling

Abstract

Recent advances in molecular characterization of tumors have made possible the emergence of new types of cancer therapies where traditional cytotoxic drugs and nonspecific chemotherapy can be complemented with targeted molecular therapies. One of the main revolutionary treatments is the use of monoclonal antibodies (mAbs) that selectively target the disseminated tumor cells while sparing normal tissues. mAbs and related therapeutics can be efficiently radiolabeled with a wide range of radionuclides to facilitate preclinical and clinical studies. Non-invasive molecular imaging techniques, such as Positron Emission Tomography (PET), using radiolabeled mAbs provide useful information on the whole-body distribution of the biomolecules, which may enable patient stratification, diagnosis, selection of targeted therapies, evaluation of treatment response, and prediction of dose limiting tissue and adverse effects. In addition, when mAbs are labeled with therapeutic radionuclides, the combination of immunological and radiobiological cytotoxicity may result in enhanced treatment efficacy. The pharmacokinetic profile of antibodies demands the use of long half-life isotopes for longitudinal scrutiny of mAb biodistribution and precludes the use of well-stablished short half-life isotopes. Herein, we review the most promising PET radiometals with chemical and physical characteristics that make the appealing for mAb labeling, highlighting those with theranostic radioisotopes.

1 | INTRODUCTION

Monoclonal antibodies (mAbs) have become indispensable tools for the modern clinical management of cancer. Currently, approximately 76 mAbs or antibody-related therapeutics have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of several primary and metastatic cancer types. Some of the advantages of mAbs as therapeutic agents include an exquisite affinity and specificity for their cognate antigen, relatively long circulation half-lives, and the ability to elicit mAb-mediated cell killing.^1,2 Additionally, the process of generating cancer-specific mAbs is relatively straightforward compared with their small molecule counterparts. In contrast to conventional chemotherapy drugs, which are non-specific and incur serious toxicities, mAb-targeted antigens over-express in cancer cells compared with normal tissues.³ This broadens the therapeutic window of these agents while reducing the incidence of severe side effects. However, the effectiveness of mAb therapies depends on the careful selection of likely responders based on expression of the target of interest. Therefore, the parallel development of noninvasive, reliable methods to scrutinize the expression of a given molecular target is vital to the efficacious implementation of mAb regimes.

Positron emission tomography (PET) imaging is a versatile nuclear medicine technique to investigate the expression of molecular targets noninvasively. PET imaging tracks the spatial distribution of a positron-emitting radionuclide that is typically conjugated to a targeting molecule. Due to the high sensitivity of PET, concentrations of radiotracers as low as 10⁻¹² M can be detected, facilitating noninvasive functional imaging with minimal pharmacological effects.⁴ A plethora of positron-emitting radionuclides with diverse chemistries and decay properties are available for conjugation to biologically active molecules ranging from simple molecules like glucose to more complex macromolecules such as proteins and polymers.

The radiolabeling of mAbs with positron emitters for PET imaging (immunoPET) may provide valuable information about the in vivo biodistribution of these molecules and their related therapeutics.⁵ ImmunoPET imaging can elucidate drug target expression via quantification of tracer uptake in the tumor, describe tumor saturation and heterogeneity, and provide data to support drug development, particularly regarding patient selection, stratification, and monitoring of treatment response.¹ In fact, extensive preclinical and clinical studies highlight the increasing importance of immunoPET as a diagnostic tool in oncology.^6,7 In addition, mAbs can also be labeled with therapeutic radionuclides (eg, ¹⁷⁷Lu, ⁶⁷Cu, and ⁹⁰Y) to combine immunological and radiobiological cytotoxicity.^5,8 Within this context, the use of diagnostic surrogate radioisotopes will facilitate quantification of the therapeutic agents’ biodistribution and dosimetry.

For each application, the selection of the optimal radioisotope is crucial. It starts by matching the half-life of the radionuclide with the pharmacokinetic profile of the mAb in vivo. This step is essential to radiotracers’ “smart” design and ensures that the time course of the radioactivity matches that of the mAb.⁷ Typically, due to prolonged circulation half-lives, antibodies’ accumulation in tumors tends to peak days after injection, which makes necessary the use of long half-life isotopes (eg, ⁸⁹Zr, ⁶⁴Cu, and ⁸⁶Y) instead of more traditional choices such as ¹¹C, ¹⁸F, or ⁶⁸Ga. In instances where the conventional isotopes do not suit the desired application, other interesting radionuclides have been investigated which offer more appropriate chemical or decay properties. Notable examples of such attractive radionuclides include ⁵²Mn, ⁵⁵Co, ¹⁵²Tb, ⁹⁰Nb, ⁶⁶Ga, ⁷²As, and ⁶⁹Ge. The utilization of these relatively long-lived PET isotopes often requires the leveraging of inorganic metal complexation chemistry with bifunctional chelators (BFCs) containing both a polydentate radiometal ligand and a bioconjugation functional group. The conjugation of many such BFC moieties to the free -COOH, -NH2, or -SH groups in mAb amino acid side chains allows efficient labeling of mAbs with a wide range of radionuclides. In this review, we present an overview of the most promising radiometals for immunoPET focusing on those that also possess an isotopic pair that could be used for theranostic applications.

1.1 | Zr-89

Currently, ⁸⁹Zr (t_1/2 = 78.4 hours, 22.7% β⁺, E_{β + ave} = 396 keV; Figure 1A and Table 1) is utilized in clinical trials much more extensively than any other PET-radiometal.⁹ Due to its relatively long half-life, ⁸⁹Zr is particularly well suited for centralized production and national and international transport. It decays via positron emission and electron capture to ^89mY (t_1/2 = 15.7 s), which decays via γ ray emission (909 keV, I_γ = 99.1%) to stable ⁸⁹Y. The 3-day half-life of ⁸⁹Zr matches the typical pharmacokinetic time scales of mAbs as mentioned previously. ⁸⁹Zr’s relatively soft positron emissions are attractive in terms of achievable PET resolution. Additionally, ⁸⁹Zr is a residualizing radionuclide: upon internalization and degradation, “free” ⁸⁹Zr is trapped inside tumor cells, which results in improved tumor retention and enhanced tumor-to-normal tissue compared with non-residualizing radionuclides such as ¹²⁴I.^10–12

FIGURE 1.

A, Simplified decay scheme of ⁸⁹Zr (data taken from the National Nuclear Data Center: www.nndc.bnl.gov), B, schematic overview of ⁸⁹Zr-labeled antibody using DFO as chelator, and C, biodistribution of ⁸⁹Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer (reprinted with permission from Dijkers EC, et al Clinical Pharmacology & Therapeutics 2010; 87: 586–592)

TABLE 1.

Decay properties, production methods, chelators, and theranostic pair for potential PET-radiometals. The gamma spectrum for each radiometal listed below is provided as Supporting Information (Figure S1)

Radionuclide	t1/2	Average β+Energy/ Iβ+ (%)	Principle γ Emissions / Iγ (%)	Optimal Production Method	Typical Chelators	Theranostic Partner Isotope
89Zr	78.4 h	396 keV/ 23%	909 keV/ 99%	Cyclotron	DFO HOPO DOTA	-
64Cu	12.7 h	278 keV/ 18%	-	Cyclotron	NOTA DOTA TETA SAR family	67Cu
86Y	14.7 h	660 keV/ 32%	1076 keV/83% 628 keV/33% 1153 keV/31% 777 keV/22% 1921 keV/21% 1854 keV/17%	Cyclotron	DOTA DTPA	90Y
52Mn	5.6d	242 keV/ 29%	1434 keV/ 100% 935 keV/95% 744 keV/90%	Cyclotron	DOTA	-
55Co	17.5 h	570 keV/76%	931 keV/75% 1409 keV/17%	Cyclotron	DOTA HBED TETA NOTA	58mCo
152Tb	17.5 h	1140 keV/20%	-	Proton-induced spallation	DOTA	161Tb
90Nb	14.6 h	620 keV/51%	1129 keV/93% 2319 keV/82% 141 keV/67% 2186 keV/18%	High energy cyclotron	DFOA	-
66Ga	9.3 h	1750 keV/57%	1039 keV/37% 2752 keV/23% 4295 keV/4%	Cyclotron	DOTA NOTA	71Ga
72As	26.0 h	1170 keV/88%	834 keV/81% 630 keV/8%	Cyclotron	Trithiol / lipoic acid	77As
69Ge	39.1 h	490 keV/24%	1107 keV/36% 574 keV/13% 872 keV/12%	Cyclotron	Metal oxide nanoparticles	-

⁸⁹Zr is mainly produced in small biomedical cyclotrons via the transmutation reaction ^natY(p,n)⁸⁹Zr and proton irradiation of natural-abundance yttrium foils with yields of ~50 MBq/μAh.¹³ Following irradiation ⁸⁹Zr is separated from the target material using a commercially available hydroxamic acid functionalized resin (hydroxamate resin), whose development built upon previously reported methodologies.^{14,15 89}Zr is conveniently eluted in small volumes as ⁸⁹Zr-oxalate; however, it can be reprocessed as ⁸⁹ZrCl₄ if desired.^14,16 Our recent results have shown the possibility of obtaining ⁸⁹ZrCl₄ using a direct chloride-based ⁸⁹Zr/^natY separation strategy which might simplify radiochemical automation and improve the chemical purity and the ⁸⁹Zr recovery.^{17 89}Zr is typically obtained with high radiochemical yields (~90%) and effective molar (specific) activities of around 60 GBq/μmol, as measured by desferrioxamine (DFO) titration.¹⁴

Currently, DFO is the most commonly employed chelator for ⁸⁹Zr-radiolabeling (Figure 1B).^9,18 However, it is now generally accepted that DFO is not optimal for ⁸⁹Zr coordination, as bone uptake-evidence of ⁸⁹Zr transchelation-has been observed in preclinical in vivo experiments regardless of the DFO-based radio-tracer.^9,19,20 Emerging reports have shown that extended DFO (DFO* or DFO-Sq) are superior because they allow for higher coordination numbers with the metal ion despite showing low radiolabeling efficiency and poor water solubility.⁹ Other promising alternatives are chelators featuring hydroxypyridinone moieties including HOPO and 2,3-HOPO which display higher in vitro and in vivo stabilities compared with DFO. Due to the well-known macrocyclic effect,²¹ macrocyclic chelators have been proposed as a viable alternative to less stable open chelators. However, the synthesis of chelators incorporating 4 hydroxamate or hydroxypyridinone moieties into a macrocyclic ring has proven challenging. Recent work has shown an excellent stability of ⁸⁹Zr- DOTA complexes compared with linear chelators, but the synthesis of such Zr-chelates requires high temperatures and the use of ⁸⁹Zr in the form of ⁸⁹ZrCl₄.¹⁸ The necessity for a heating step precludes the direct ⁸⁹Zr labeling of the mAb using DOTA as chelator. Nonetheless, the long half-life of ⁸⁹Zr is conducive to the implementation of a post-radiolabeling conjugation strategy, where ⁸⁹Zr chelation is carried out prior to the BFC-mAb bioconjugation step, potentially overcoming the limitations imposed by the elevated temperatures needed for radiosynthesis.

The first ⁸⁹Zr-labeled mAb (⁸⁹Zr-labeled anti-EpCam antibody 323/A3) was successfully used to visualize human OVCAR-3 xenografts in immunodeficient mice in 1997.²² Less than a decade later, the first clinical ⁸⁹Zr- labeled mAbs showed that primary head and neck squamous cell carcinomas might be detected by PET imaging using ⁸⁹Zr-labeled chimeric anti-CD44v6 antibody U36.¹⁰ Since then, numerous ⁸⁹Zr-labeled mAbs have been developed targeting several tumor-associated antigens, i.e., EGFR, HER2, PSMA, CD44v6, CD20, and VEGF-A.^7,9,23 For example, ⁸⁹Zr-trastuzumab has been used for the detection of lung, liver, bone, and brain metastatic lesions in patients with HER2-positive breast cancer (Figure 1C).¹¹ Within this context, immunoPET was able to unequivocally diagnose patients with suspected liver and mediastinal metastases, which could not be properly confirmed by biopsies.²⁴ Additionally, ⁸⁹Zr-trastuzumab has also been employed for the prediction and monitoring of therapy response.^25,26 Despite recent advancements in the field indicating a need for the optimization of the chelators and bioconjugation approaches used for clinical immunoPET with ⁸⁹Zr immunoconjugates, the increasing number of successful preclinical studies using ⁸⁹Zr- labeled mAbs for cancer imaging has established this radiometal as one of the most promising for antibody labeling.^7,9

1.2 | Cu-64

⁶⁴Cu (t_1/2 = 12.7 hours, 17.4% β⁺, E_{β + ave} = 278 keV; Figure 2A and Table 1) represents another convenient alternative for antibody and protein labeling as it has an intermediate half-life and negligible contaminating gamma emissions. Due to the favorable excitation function of the ⁶⁴Ni (p,n)⁶⁴Cu reaction at low energies (11 MeV), ⁶⁴Cu can be economically produced by proton irradiation of isotopically enriched ⁶⁴Ni targets with yields of 0.37 GBq/μAh.²⁷ Our group and others have developed efficient methods for radiochemical isolation of produced ⁶⁴Cu from Ni and Co targets and the subsequent recovery of the enriched ⁶⁴Ni.²⁷

FIGURE 2.

A, Simplified decay scheme of ⁶⁴Cu (data taken from the National Nuclear Data Center: www.nndc.bnl.gov), B, schematic overview of ⁶⁴Cu-labeled antibody using DOTA as chelator, and C, ⁶⁴Cu-DOTA-trastuzumab PET images of HER2-positive metastatic brain lesions (arrows) (This research was originally published in JNM. Tamura K et al. 64Cu-DOTA-Trastuzumab PET Imaging in Patients with HER2-Positive Breast Cancer. J Nucl Med 2013; 54:1869–1875. © by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

The chemistry of the intermediate-hard metal ion Cu²⁺ is well understood, particularly its coordination with chelators with amino and carboxylic acid functional groups.²⁸ Many macrocyclic chelators have been used for the coordination of Cu²⁺ isotopes with excellent kinetic and thermodynamic stabilities including DOTA (Figure 2B), DTPA, TETA, NOTA, and its derivatives.^28,29 Another family of chelators, the hexaazamacrobicyclic cage-type ligands (namely SAR ligands) have shown excellent conjugation efficiencies at room temperature within minutes and much faster chelation kinetics compared with those with TETA derivatives (CB-TE1A1P and CB-TE2P).³⁰ Despite the number of emerging Cu²⁺chelators, most conventional ligands have acceptable in vivo stabilities within a 48-hour time-frame post- injection (PI), and NOTA remains the gold standard for the radiolabeling of Cu isotopes.^29,31

The intermediate half-life of ⁶⁴Cu makes it an appropriate radioactive tag for a wide variety of molecules with a range of molecular weights and pharmacokinetic profiles.^{5 64}Cu has been used for immunoPET imaging to scrutinize the expression of several cancer-specific receptors including the epidermal growth-factor receptor (EGFR; cetuximab),³² CD105 (TRC105),³¹ human α_vβ₃ integrin (etaracizumab),³³ epithelial cell adhesion molecule (EpCAM; mAb7),³⁴ CD20 (rituxi-mab),³⁵ human epidermal growth factor receptor 2 (HER2; trastuzumab),^5,36 and CD146 (YY146).³⁷ An example of ⁶⁴Cu-DOTA-Trastuzumab PET images of HER2-positive metastatic brain lesions is shown in Figure 2C. These studies have demonstrated the feasibility of immunoPET using ⁶⁴Cu-labeled mAb for quantitative, noninvasive evaluation of the expression of cancer- specific molecules and pointed to the advantages of ⁶⁴Cu over ⁸⁹Zr in terms of procedural radiation exposure.³⁸ Despite being appropriate for most preclinical immunoPET applications, the intermediate half-life of ⁶⁴Cu limits the implementation of longitudinal or delayed imaging studies that require imaging time points beyond 48 hours PI.

The availability of theranostic isotope pairs is of great interest because image-guided, targeted radionuclide therapy (TRT) can be seamlessly implemented. ⁶⁴Cu not only possesses intrinsic theranostic properties due to its mixed β⁺ and β⁻ (38.5%, E_{β − ave} = 191 keV) decay modes, but can also serve as surrogate for treatment planning and dose estimation of TRT using ⁶⁷Cu (t_1/2 = 2.58 days, 100% β⁻ , E_{β − ave} = 141 keV). Owing to a half-life similar to the biological half-life of many mAbs, a lower whole-body dose to the patient, and simple radiolabeling procedures, ⁶⁷Cu is well-suited to radioimmunotherapy (RIT).³⁶ Several mAbs have been labeled with ⁶⁷Cu, including chCE7, an anti-L1-cell adhesion molecule mAb for the treatment of neuroblastoma, ovarian cancer, and renal carcinoma; Lym-1 for non- Hodgkin’s lymphoma; C595 an anti-MUC1 for bladder cancer treatment³⁹; and trastuzumab for the treatment of HER2 positive tumors.⁴⁰ Currently, the requirement for high energy charged particle bombardments, or high intensity neutral particle irradiations limits the availability of ⁶⁷Cu. Notably, the development of the Facility for Rare Isotope Beams (FRIB) at the Michigan State University (MSU) will allow the harvesting of ⁶⁷Cu in GBq-scale quantities from the aqueous beam dump produced at this projectile fragmentation facility.⁴¹ With further development of novel facilities and methods for the production of ⁶⁷Cu, ⁶⁴Cu will receive additional consideration for mAb-based theranostic applications.

1.3 | Y-86

Another isotope with promising characteristics for immunoPET is ⁸⁶Y (t_1/2 = 14.7 hours, 31.9% β⁺, E_{β + ave} = 660 keV; Figure 3 and Table 1). This radionuclide can be produced in a small biomedical cyclotron by the proton-induced transmutation reaction ⁸⁶Sr(p,n)⁸⁶Y at less than 18 MeV.⁸ Solid targets of isotopically enriched ⁸⁶SrCO₃ are required to produce ⁸⁶Y with a radionuclidic purity higher than 98%. This constitutes a challenge in the production of ⁸⁶Y because the elevated market price of ⁸⁶SrCO₃ ($9/mg) mandates the recycling of the target material, which involves a series of precipitation/reconstitution steps.⁴² The target material is problematic due to its low thermal conductivity and difficulty coupling it to backing materials in geometries that can dissipate more than a few tens of watts of deposited beam power, limiting production yields to a few hundred MBq. Several methods have been reported for the separation of ⁸⁶Y from the irradiated ⁸⁶SrCO₃ targets, including co-precipitation^42,43 and ion exchange,^44–46 electrolysis,^47,48 single column chromatography,⁴⁹ multiple column chromatography,^50,51 and solvent extraction.⁴⁵ Although each method has its own advantages and disadvantages, in general, all have achieved the efficient separation of the radionuclide. Unfortunately, ⁸⁶Y emits a large number of gamma rays with nontrivial intensities and energies greater than a MeV, limiting injectable quantities to approximately 100 MBq.

FIGURE 3.

Simplified decay scheme of ⁸⁶Y (data taken from the National Nuclear Data Center: www.nndc.bnl.gov)

The pseudo-lanthanide Y³⁺ has hard-acid character and tends to form complexes with ligands containing hard donor atoms, displaying high coordination numbers, usually 8 or 9. Consequently, the labeling of antibodies and proteins with ⁸⁶Y has been performed using mainly polyaminocarboxylic ligands, for example DTPA, DOTA, or its derivatives. DOTA and DOTA-derivatives show particularly high thermodynamic stability and enhanced kinetic inertness because the cavity size of DOTA is well matched to the ionic radii of the yttrium trivalent metal ion.^5,28

⁸⁶Y has been used for longitudinal immunoPET recordings of the biodistribution of mAbs such as cetuximab.⁵² Compared with ⁶⁴Cu and ⁸⁹Zr, ⁸⁶Y production rates are typically lower, and its decay properties, namely half-life and a cluttered gamma emission spectrum, have affected the quantity of literature studies using ⁸⁶Y as a PET isotope.⁵³ The main advantage of ⁸⁶Y is the potential of its well-established therapeutic radioisotope: ⁹⁰Y (t_1/2 = 64.0 hours, 100% β⁻ , E_{β − ave} = 934 keV). In fact, the theranostic concept in nuclear medicine was first coined for the use of the radionuclide pair ⁸⁶Y/⁹⁰Y at the Research Center Jülich, Germany in 1992,⁸ which allowed a combination of PET and TRT.

⁹⁰Y has been extensively used as a therapeutic radio-nuclide in the treatment of various malignancies, including lymphoma, ovarian, colorectal, leukemia, pancreatic, and bone cancers.⁸ In fact, one of the most efficacious TRT agents reported to date is ⁹⁰Y-labeled mAb, ⁹⁰Y- ibritumomab tiuxetan (Zevalin®, Spectrum Pharmaceuticals, Henderson, NV, USA), which was approved in 2002 by the US Food and Drug Administration for the targeting of CD20 in Non-Hodgkin’s lymphoma patients and remains a part of the standard of care today.⁵ Following the success of Zevalin, several proof-of-concept studies exploited the potential of the ⁸⁶Y/⁹⁰Y theranostic pair. For example, ⁹⁰Y-labeled cetuximab combined with External Beam Radiotherapy (EBRT) was tracked by ⁸⁶Y-cetuximab immunoPET.⁵² Due to a high positron branching ratio, convenient physical half-life, and the success of therapies employing ⁹⁰Y, ⁸⁶Y represents a valuable choice for PET imaging that is currently limited primarily by the scalability of its production. For additional discussion, the reader is referred to the recent review by the Jülich group.⁸

2 | EMERGING RADIOISOTOPES

2.1 | Mn-52

⁵²Mn (t_1/2 = 5.59 days, 29.4% β⁺, E_{β + ave} = 242 keV; Figure 4 and Table 1) may offer advantages over conventional ⁸⁹Zr or ⁶⁴Cu in situations where treatment response monitoring at late time-points (2–3 weeks) is desired.⁵⁴ In cases where RIT is initiated with long-lived nuclides, multi-week treatment time-courses can be monitored by ⁵²Mn PET. Additionally, due to the abundance of coincident high energy gamma emission, ⁵²Mn is one of the relatively few nuclides that may be used in third-gamma coincidence PET for either dual nuclide event tagging or combined Compton telescope PET tomography.⁵⁵ Despite this, the clinical translation must be carried out with caution due to the preponderance of the coincident high energy gammas: 744 keV (90%), 935 keV (95%), and 1434 keV (100%). Together with the countless biological roles of manganese, which may lead to prolonged retention in critical organs, the biodistribution and dosimetry of ⁵²Mn-labeled agents require evaluation prior to clinical translation.

FIGURE 4.

Simplified decay scheme of ⁵²Mn (data taken from the National Nuclear Data Center: www.nndc.bnl.gov)

⁵²Mn can be produced in small biomedical cyclotrons at low proton energies using pressed natural chromium targets.⁵⁶ For incident proton energies of 16 MeV and “thick” targets, average production yields of 6.2 MBq/μAh are typical. Importantly, due to the high natural abundance of ⁵²Cr (83.8%) and low coproduction yields of Mn radioisotopic impurities, ^natCr targets are a viable, inexpensive alternative to the enriched target material for preclinical studies.⁵⁴ The main radionuclidic impurity from the irradiation of ^natCr is ⁵⁴Mn (t_1/2 = 312 days), representing 0.1% to 0.4% of the ⁵²Mn activity at the end of a short bombardment at 16 MeV.⁵⁴ Adjustment of incident energy affords some reduction in the relative contamination of ⁵⁴Mn but cannot eliminate its contamination. The most recently reported separations of ⁵²Mn from the target material use solid-phase anion exchange in ethanolic HCl and recover >90% of the ⁵²Mn with 10^5–6 decontamination factors from chromium, copper, iron, cobalt, and zinc.⁵⁶

Similar to other hard transition metals, Mn²⁺ forms highly stable complexes with polyaminocarboxylic acid chelators. From those, DOTA has been the most common choice of chelator for the manganese chelation.^54,56 DOTA can be rapidly and quantitatively radiolabeled with ⁵²Mn reaction times less than 1 minute, and the resulting complex shows excellent in vitro stability.⁵⁶ These results have motivated the use of ⁵²Mn in several preclinical investigations.^57–59

Numerous applications of ⁵²Mn have been reported, including myocardial perfusion tracer,⁶⁰ neural tractography,⁶¹ stem cell tracking,⁶² and biological toxicity assays.^54,63 However, the extension to immunoPET requires high-specific-activity ⁵²Mn in a chemical state suitable for macromolecule labeling. Thanks to the contributions of our group and the group at the Hevesy Laboratory, ⁵²Mn has been produced with sufficient specific activity for the labeling of a mAb for immunoPET.^54,56 Using a DOTA-based BFC, TRC105, an anti-CD105 mAb was radiolabeled (⁵²Mn-DOTA-TRC105) for the imaging of angiogenesis in a syngeneic 4T1 xenograft model of breast adenocarcinoma. The elevated stability of ⁵²Mn- DOTA-TRC105 over the course of several days proves the usefulness of ⁵²Mn as a radiotracer for immunoPET.⁵⁴

2.2 | Co-55

⁵⁵Co (t_1/2 = 17.5 hours, 76% β⁺, E_{β + ave} = 570 keV, 24% EC; Figure 5 and Table 1) offers a high positron yield and moderate abundance of co-emitted gamma-rays.^{64 55}Co can be produced via either the ⁵⁸Ni(p,α)⁵⁵Co or the ⁵⁴Fe(d,n)⁵⁵Co nuclear reactions at low energy biomedical cyclotrons and possesses a half-life permissive of centralized production and distribution.^64,65 After irradiation, the produced ⁵⁵Co can be separated from the target material using an extraction resin functionalized with N,N,N′, N′-tetrakis-2-ethylhexyldiglycolamide, or branched DGA (Eichrom, Illinois).⁶⁴ As with many of the radiometals discussed herein, radiocobalt can be efficiently coordinated using common hard donor chelators such as such as DOTA, HBED, TETA, and NOTA,^66–68 but to date, strategies for the labeling of mAbs or proteins with ⁵⁵Co have not been well established.

FIGURE 5.

Simplified decay scheme of ⁵⁵Co (data taken from the National Nuclear Data Center: www.nndc.bnl.gov)

Nevertheless, ⁵⁵Co has been used as a PET imaging agent in diverse applications such as ischemic stroke,⁶⁹ imaging of renal function,⁷⁰ and multiple sclerosis.⁷¹ Recent reports show that high-resolution PET images can be obtained when labeling small peptides such as bombesin with radiocobalt.^67,72 More recently, this radio-nuclide has shown promise for labeling affibodies. The DOTA-Z2395-C affibody was efficiently and stably labeled with ⁵⁷Co.⁶⁸ The intercellular retention of radiocobalt- labeled DOTA-Z2395-C was comparable to the retention of its ¹¹¹In-labeled counterpart in vitro. In vivo, the radiocobalt label provided better tumor-to-organ ratios than the radionuclide ¹¹¹In. We anticipate future studies with DOTA-coupled affibody molecules will show efficient ⁵⁵Co radiolabeling and be used for in vivo PET imaging applications.

The recent realization of the therapeutic potential of the Auger electron emitter ^58mCo (t_1/2 = 9.10 hours, 100% IC) has revived interest in ⁵⁵Co as an imaging surrogate of ^58mCo for theranostic applications.^64,73,74 Thisgaard et al were the first to demonstrate the possibility of producing therapeutic quantities of ^58mCo using a small biomedical cyclotron.⁷³ In a more recent study, Valdovinos and colleagues showed the viability of obtaining high specific activity ^58mCo conducive to the quantitative radiolabeling of mAb.⁶⁴ However, so far, the therapeutic potential of ^58mCo has not been demonstrated in vivo. Nonetheless, the availability of this pair of radionuclides is compelling to the implementation of TRT with Auger electrons.

2.3 | Tb-152

Terbium offers 4 clinically interesting radioisotopes with complementary physical decay characteristics: ¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tb, and ¹⁶¹Tb.⁷⁵ The identical chemical characteristics of these radioisotopes allow the preparation of radiopharmaceutical isosteres for PET (¹⁵²Tb) and SPECT (¹⁵⁵Tb) imaging, and for α (¹⁴⁹Tb) and β⁻ (¹⁶¹Tb) therapy. In addition, the half-life of ¹⁵²Tb (t_1/2 = 17.5 hours, 20.3% β⁺, E_{β + ave} = 1140 keV; Figure 6 and Table 1) makes this radionuclide in conjunction with ¹⁶¹Tb (t_1/2 = 6.7 days, E_β − _ave = 134 keV) a potential isotopic pair for mAb-based theranostic applications. ¹⁵²Tb can be produced by proton-induced spallation of tantalum targets, and carrier-free terbium radioisotopes can be obtained after purification in mass separators, with activities around 600 MBq.⁷⁵ Being a lanthanide, Tb³⁺ has chemical properties that resemble those of group III elements (Sc and Y), including the formation of stable coordination complexes with DOTA and DOTA-like chelators, which facilitates the radiolabeling of well-established targeting agents.^75–78

FIGURE 6.

Simplified decay scheme of ¹⁵²Tb (data taken from the National Nuclear Data Center: www.nndc.bnl.gov)

Because of its unique production facility requirements, only a limited number of studies using ¹⁵²Tb have been reported. The first reported study using ¹⁵²Tb as PET radionuclide radiolabeled a peptide (DOTANOC) for in vivo PET imaging of somatostatin receptor expression in AR42J tumor-bearing mice.⁷⁷ The results of this study paved the way for a more recent first-in-human multi-disciplinary PET/CT study using this promising radionuclide.⁷⁸ To this end, ¹⁵²Tb-DOTATOC longitudinal PET scans were acquired over a period of 24 hours, allowing the visualization of even small metastases with excellent tumor-to-background contrast ratios. However, to the best of our knowledge, the use of this radionuclide for the labeling of mAbs has not yet been reported.

Few studies have also reported the use of the γ-rays co- emission of ¹⁶¹Tb (48.9 keV-17%, 57.2 keV-1.8%, 74.6–10%) for monitoring the in-vivo behavior of radio-labeled bio-molecules and their effects on malignant tissue.⁷⁵ It can be produced by neutron capture and subsequent decay via the reaction chain ¹⁶⁰Gd(n, γ)¹⁶¹Gd→β-decay→¹⁶¹Tb in nuclear reactors with activities in the range of 15 GBq.⁷⁵ Preliminary results using this radionuclide suggested that ¹⁶¹Tb-labeled compounds may elicit more robust therapeutic effects than other therapeutic radionuclides like ¹⁷⁷Lu when applied at similar activity levels.^75,79 Based on these preliminary results, ¹⁵²Tb/ ¹⁶¹Tb seems to be a compelling pair for mAb-based theranostic applications. However, in our opinion, the complexity and high cost of the production of ¹⁵²Tb constitute major limitations to the widespread implementation of this radioisotopes.

2.4 | Nb-90

Another interesting radionuclide with suitable decay properties as a radiometal for immunoPET is ⁹⁰Nb (t_1/2 = 14.6 hours, 51.2% β⁺, E_{β + ave} = 620 keV, 24% EC; Figure 7 and Table 1), which may allow the noninvasive, high-resolution visualization and quantification of the whole-body distribution of macromolecules including mAb, mAb fragments, polymers, and nanoparticles.^{80 90}Nb can be produced via ⁹⁰Zr(p,n)⁹⁰Nb nuclear reaction using low-energy proton (20 MeV) irradiation of natural zirconium targets.⁸¹ No-carrier-added ⁹⁰Nb can be isolated by a multi-step separation procedure involving liquid extraction and ion-exchange chromatography.⁸¹ Recent advancements in optimization of the separation process of ⁹⁰Nb from irradiated targets have allowed efficient (>90%) and fast (less than 1 hour) recovery of ⁹⁰Nb with a radionuclidic purity higher than 97% (decay corrected to EoB) and molar (specific) activity suitable for the radiolabeling of mAbs at 4.5 TBq/mmol.⁸¹ However, the co-production of other long half-life Nb radioisotopes such as ^92mNb (t_1/2 = 10.2 days), ^95mNb (t_1/2 = 3.6 days), ⁹⁵Nb (t_1/2 = 35 days), or ⁹⁶Nb (t_1/2 = 23.4 hours) may make enriched ⁹⁰Zr targets necessary for clinical applications.

FIGURE 7.

Simplified decay scheme of ⁹⁰Nb (data taken from the National Nuclear Data Center: www.nndc.bnl.gov)

The coordination of ⁹⁰Nb with different chelators has been evaluated in terms of radiolabeling efficiency and stability of the radiolabeled Nb⁵⁺ complex.⁸² Results indicate that DFO shows the best properties as it is able to form quantitative complexes at a wide range of pHs (4–7) at room temperature. In addition, it was verified that bi-functionalization does not affect the complex formation parameters of the DFO and that the complex remains stable in vivo.⁸² In follow-up studies, the mAb rituximab was radiolabeled with ⁹⁰Nb, and stability measurements revealed that the complex was more than 99% stable over a prolonged period of 18 days.⁸³ Recently, as a proof of concept, ⁹⁰Nb was used to label the mAb bevacizumab (Avastin®), and in vitro and in vivo stability was evaluated in normal swiss mice and tumor-bearing SCID mice.⁸¹ In- vivo PET imaging in tumor-bearing SCID mice after the injection of ⁹⁰Nb-bevacizumab showed avid localization of the radiotracer in the tumor, while uptake in the liver, spleen, kidneys, or bones remained low. Overall, these results indicate the feasibility of ⁹⁰Nb-labeled antibodies for immunoPET. Nevertheless, the need for enriched targets for ⁹⁰Nb production may be a significant drawback for future clinical implementation.

2.5 | Ga-66

The radionuclide ⁶⁶Ga (t_1/2 = 9.3 hours, 56.5% β⁺, E_{β + ave} = 1750 keV, 43.5% EC; Figure 8 and Table 1) constitutes an intriguing alternative to ⁶⁷Ga (t_1/2 = 78.3 hours) and ⁶⁸Ga (t_1/2 = 68.3 minutes), which are used for SPECT and PET imaging, respectively.⁸⁴ Particularly, the relatively long half-life of ⁶⁶Ga makes it a more practical radiolabel for antibodies and proteins, whose slower in- vivo kinetics are poorly matched by the much shorter half-life of ⁶⁸Ga.

FIGURE 8.

Simplified decay scheme of ⁶⁶Ga (data taken from the National Nuclear Data Center: www.nndc.bnl.gov)

Lewis et al described the irradiation of natural Zn and enriched ⁶⁶Zn targets to produce ⁶⁶Ga, which was then purified by cation exchange chromatography and solvent extraction.⁸⁵ This separation method has been used in most ⁶⁶Ga-based studies to date; however, modest effective specific activities of ⁶⁶Ga were obtained (4.6 GBq/ μmol). More recently, our own laboratory in collaboration with researchers at the Autonomous University of Mexico (UNAM) reported the production of high specific activity ⁶⁶Ga (>70 GBq/μmol) from the ^natZn(p,n)⁶⁶Ga and ⁶⁶Zn(p,n)⁶⁶Ga nuclear reactions, using a small biomedical cyclotron and energies of 16 → 7 MeV.⁸⁶ The subsequent separation of ⁶⁶Ga was accomplished using anion exchange chromatography.⁸⁷ This result increases the potential of this non-conventional radionuclide for cancer imaging applications.

The coordination chemistry of radiogallium has been extensively studied due to the availability of ⁶⁸Ge/⁶⁸Ga generator-produced ⁶⁸Ga. The choice of chelator for the radiolabeling of ⁶⁶Ga has mirrored that of the ⁶⁸Ga, with quantitative labeling being achieved with the macrocyclic chelators DOTA and NOTA.^84,88 Although, limited studies have been reported on ⁶⁶Ga-based immunoPET imaging including the work describing the radiolabeling of the mAb TRC105 with ⁶⁶Ga for the in-vivo annotation of CD105 expression in a murine model of breast cancer.⁸⁴ TRC105, a chimeric IgG1 mAb which binds to both human and murine CD105, was conjugated with NOTA and efficiently radiolabeled with ⁶⁶Ga. PET imaging revealed fast, prominent, and CD105- specific tumor targeting in mice bearing 4T1 tumor xenografts. Such successful immunoPET imaging studies demonstrate the feasibility of using ⁶⁶Ga to expand the spectrum of accessible radiogallium-based imaging agents to include macromolecules with slower pharmacokinetic profiles. Among the main disadvantages of ⁶⁶Ga are the high energy of the positron (E_{β + ave} = 1.75 MeV), which decreases the achievable quality of acquired PET images-especially in the preclinical setting-and the abundance of high-energy gamma rays: 1039 keV (37%), 2752 (23%), and 4295 keV (4%) that impose a challenge from the dosimetric point of view. These facts, together with the relatively low effective molar activities, achieved using the commonly reported methods are the main reasons for the limited number of studies using ⁶⁶Ga. However, it remains the only option for long time-point PET studies of the accumulation of Ga-labeled vectors.

2.6 | As-72

The radioisotopes of arsenic have played a role in nuclear medicine because the use of ⁷⁴As (t_1/2 = 17.77 days, 29% β⁺, E_{β + ave} = 440 keV; Figure 9 and Table 1) in Sweet and Brownell’s pioneering work imaging intracranial lesions through positroencephalography.⁸⁹ More recently, efforts have focused on utilizing the theranostic pair of ⁷²As (t_1/2 = 26.0 hours, 88% β⁺, E_{β + ave} = 1170 keV) and ⁷⁷As (t_1/2 = 38.8 hours, 100% β⁻ , E_{β − ave} = 225 keV). Additionally, ⁷¹As (t_1/2 = 65.3 hours, 28% β⁺, E_{β + ave} = 350 keV) and ⁷⁰As (t_1/2 = 50.6 m, 91% β⁺, E_{β + ave} = 980 keV) are alternative PET diagnostic isotopes for targeting vectors with longer or shorter biological half-lives and ⁷³As (t_1/2 = 80.3 days) has potential in Auger- emission TRT. This broad palette of radioisotopes, along with the metalloid element’s unique soft-Lewis-acid chemical properties, makes these radioisotopes compelling for use in theranostic applications with mAbs.

FIGURE 9.

Simplified decay scheme of ⁷²As (data taken from the National Nuclear Data Center: www.nndc.bnl.gov)

Radioarsenic can be produced through the irradiation of ^natGe_(m) or ^natGeO₂ using a small biomedical cyclotron with solid target capabilities.^90,91 However, proton irradiation of natural enrichment germanium produces ⁷²As with low radionuclidic purity, with significant end-of- bombardment contamination from ⁷⁰As (600% of ⁷²As), ⁷⁶As (8% of ⁷²As), and ⁷⁴As (6% of ⁷²As).⁹¹ More recently, isotopically enriched ⁷²Ge_(m) targets have been used to produce ⁷²As with high yield (90 MBq/μAh) and radionuclidic purity (99.4%)⁹² with a biomedical cyclotron. The isolation of radioarsenic from germanium target material has been accomplished through a variety of methods, including precipitation,^91,93,94 dry distillation,⁹⁵ HCl distillation,^90,92 solvent extraction,^90,94 polystyrene- based solid phase extraction,^92,96 and chromatography with anion exchange resins,^90–92 silica,^97,98 titania,⁹⁹ and zirconia.¹⁰⁰ Additionally, ⁷²As can be produced as the radioactive decay product of ⁷²Se (t_1/2 = 8.5 days), allowing for the production of a ⁷²Se/⁷²As generator system, broadening the accessibility of ⁷²As to sites without access to a solid-target-capable biomedical cyclotron.^101,102

It is well known that arsenic is a very biologically reactive element with significant binding to proteins, as evidenced by its acute systemic toxicity estimated at approximately 0.6 mg/kg daily.¹⁰³ This biological activity and protein binding capacity are largely due to the metalloid’s significant soft-Lewis-acid character that allows it to bind especially strongly to soft ligands such as the sulfhydryl groups on cysteine amino acid sidechains.¹⁰⁴ Jennewein et al aimed to take advantage of this binding mechanism to label the anti-phosphatidylserine mAb, bavituximab, with ⁷⁴As.¹⁰⁵ Bavituximab was first modified using N- succinimidyl S-acetylthioacetate (SATA) to exhibit additional sulfhydryl functionality and then labeled using a trivalent ⁷⁴AsI₃ compound. The ⁷⁴As-S-bavituximab radioimmunoconjugate appeared to exhibit good in-vitro stability and showed promising tumor-to-liver and tumor- to-muscle ratios in R3327-AT1 tumor-bearing rats.¹⁰⁵ Despite this, it exhibited significant biological clearance with only 0.25% injected dose (ID)/g remaining in tumor and 0.125%ID/g in liver at 48 hours post injection. This clearance was dramatically faster than that of ⁶⁴Cu-labeled bavituximab, which showed 3.2%ID/g in tumor and 20.6% ID/g in liver at 48 hours post injection in LNCaP-tumor bearing mice.¹⁰⁶ This discrepancy is most likely explained by significant dearsenylation of the ⁷⁴As-S-bavituximab. More recent work labeling the anti-CD105 mAb, TRC105, through similar direct sulfhydryl radioarsenic labeling methods also yielded rapid dearsenylation of the radioimmunoconjugate with the resulting radiotracer biodistribution indistinguishable from unlabeled radioarsenic.⁹² These studies demonstrate the need for the development of next generation methods for stable incorporation radioarsenic isotopes into radioimmunoconjugates. Several methods are currently under investigation for this, including the development of a trithiol containing chelators¹⁰⁷ and the utilization of the dithiol containing mitochondrial enzyme cofactor, lipoic acid.⁹²

2.7 | Ge-69

A much less common but potentially useful PET radioisotope is ⁶⁹Ge (t_1/2 = 39.05 hours, 21% β⁺, E_{β + ave} = 490 keV;Figure 10 and Table 1). Only a handful of studies have been reported on the production, separation, and radiochemistry of ⁶⁹Ge and its potential therapeutic counter-part, the Auger-emitter ⁷¹Ge (t_1/2 = 11.4 days). However, they constitute an intriguing isotopic pair whose application niche is not yet established.¹⁰⁸

FIGURE 10.

Simplified decay scheme of ⁶⁹Ge (data taken from the National Nuclear Data Center: www.nndc.bnl.gov)

The production of this radionuclide can be accomplished in a simple and cost-effective manner via the bombardment of a Ga/Ni alloys, which are also proposed for the production of ⁶⁸Ge with 11-MeV protons.¹⁰⁸ Separation of ⁶⁹Ge from target material is carried out by column chromatography using a DGA extraction resin in HNO₃ media. One limitation is ⁶⁹Ge’s complex chemical speciation in aqueous media, which has led to a lack of suitable radiolabeling techniques for the preparation of ⁶⁹Ge- based agents. The distribution of germanium species depends on the total concentration and the pH of the medium with the 4 major hydrolysis products identified as Ge(OH)₄, [GeO(OH)₃]⁻ , [GeO₂(OH)₂]²⁻ , and[[Ge(OH)₄]₈(OH)₃]^3-,109 making it a major challenge for radiolabeling with ⁶⁹Ge using traditional chelator-based methods.

In a recent effort to develop conjugation techniques for ⁶⁹Ge, superparamagnetic iron oxide nanoparticles (SPION) were recently used as a matrix for chelator free radiolabeling of this nanoconstruct with ⁶⁹Ge.¹⁰⁸ The incorporation of Ge into metal oxide matrix (such as TiO₂, ZrO₂, CeO₂, SnO₂, Fe₂O₃, Fe₃O₄, Al₂O₃) has been extensively exploited for the preparation of the widely used ⁶⁸Ge/⁶⁸Ga generators.^110,111 Therefore, SPION nanoparticles were employed as a platform to radiolabel ⁶⁹Ge without the need for functionalized chelators. As an added benefit, given the magnetic properties of SPIONs simultaneous PET and magnetic resonance imaging (MRI) data were acquired using these constructs.¹¹² Despite having nuclear properties favorable for mAb work, ⁶⁹Ge remains underutilized as a PET radiolabel due to the lack of traditional BFC-based methods for incorporating it into targeting moieties of interest.

3 | CONCLUSIONS

The field of positron emission tomography imaging using labeled mAbs is rapidly progressing toward widespread clinical adoption. Radiolabeled immunoconjugates play an essential role in drug development and aid in patient stratification and monitoring of the treatment response. Moreover, the availability of theranostic isotopic pairs facilitates the implementation of TRT using therapeutic radionuclides that allow the combination of immunological and radiobiological cytotoxic effects for higher anti-tumor efficacy. Within this context, a parallel development of isotope production, separation, and radiochemistry methods is vital to nurture a steady development of both preclinical and clinical studies using different PET radiometals, which will ultimately result in the realization of radiolabeled mAbs as promising tools in the management of cancer. Nevertheless, especially for those emerging radiometals, the production, isolation, and labeling need to be optimized to efficiently fulfill the clinical requirements for human uses.

Biographies

Eduardo Aluicio-Sarduy received his undergraduate education at the Higher Institute of Applied Sciences and Technology (InSTEC) in Havana, Cuba, where he completed with honors his BSc (2007) and MSc in Radiochemistry (2011). In 2016, he earned his PhD in Materials Engineering at Politecnico di Milano, and currently, he is a postdoctoral researcher at the University of Wisconsin-Madison working on the production and radiochemical isolation of long-lived radionuclides for theranostics applications.

Jonathan Ward Engle is an assistant professor and cyclotron jockey in the departments of Medical Physics and Radiology at the University of Wisconsin. He has experience in radionuclide production, accelerator targetry, analytical and preparative radiochemistry for clinical and pre-clinical positron emission tomography (PET) scanning, and automation. He has degrees in Religion, Education, and Physics.