ImmunoPET: Concept, Design, and Applications

Abstract

Immuno-positron emission tomography (immunoPET) is a paradigm-shifting molecular imaging modality combining the superior targeting specificity of monoclonal antibody (mAb) and the inherent sensitivity of PET technique. A variety of radionuclides and mAbs have been exploited to develop immunoPET probes, which has been driven by the development and optimization of radiochemistry and conjugation strategies. In addition, tumor-targeting vectors with a short circulation time (e.g., Nanobody) or with an enhanced binding affinity (e.g., bispecific antibody) are being used to design novel immunoPET probes. Accordingly, several immunoPET probes, such as ⁸⁹Zr-Df-pertuzumab and ⁸⁹Zr-atezolizumab, have been successfully translated for clinical use. By noninvasively and dynamically revealing the expression of heterogeneous tumor antigens, immunoPET imaging is gradually changing the theranostic landscape of several types of malignancies. ImmunoPET is the method of choice for imaging specific tumor markers, immune cells, immune checkpoints, and inflammatory processes. Furthermore, the integration of immunoPET imaging in antibody drug development is of substantial significance because it provides pivotal information regarding antibody targeting abilities and distribution profiles. Herein, we present the latest immunoPET imaging strategies and their preclinical and clinical applications. We also emphasize current conjugation strategies that can be leveraged to develop next-generation immunoPET probes. Lastly, we discuss practical considerations to tune the development and translation of immunoPET imaging strategies.

Graphical Abstract

1. INTRODUCTION

Molecular imaging is defined as “visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems” by using molecular imaging agents and tools.¹ Positron emission tomography (PET) imaging is the foundation of molecular imaging and has drastically improved global healthcare since its inception in the clinical practice.^2–4 With the gradual discovery of the molecular pathogenesis of cancers and contemporaneous understanding of the host immune system, molecularly targeted therapies (e.g., small-molecule inhibitors and monoclonal antibodies [mAbs]) and immunotherapies (e.g., immune checkpoint inhibitors) have been developed. The clinical use of these novel regimens is changing the therapeutic landscape for numerous cancers.^5–7 In the era of molecularly targeted therapy and cancer immunotherapy, it is clear that PET imaging with traditional radiotracers is inadequate.⁸ For instance, 18F-fluorodeoxyglucose (¹⁸F-FDG) PET/computed tomography (CT) has been integrated into several criteria in predicting and assessing responses to targeted therapies or immunotherapies.^9,10 However, several studies have reported that 18F-FDG PET/CT parameters, such as SUVmax and SUVmean, did not correlate with clinical responses for immunotherapy regimens.^11,12 Additionally, it is challenging to differentiate immune-related adverse events (e.g., sarcoidosis) and pseudoprogression on ¹⁸F-FDG PET images,^13,14 leading to misinterpretation.

To further improve the clinical management of cancers and noncancerous diseases, the integration of novel molecular imaging approaches into routine diagnostic toolbox is critically important.¹⁵ Antibody-derived molecular imaging probes have been instrumental in visualizing target expression and pharmacokinetics of therapeutic mAbs in living subjects. Although several antibody-based tracers for single-photon emission computed tomography (SPECT) imaging exist in the clinic,¹⁶ PET imaging with antibody-based tracers has distinct advantages in terms of image quality, spatial resolution, and quantification.¹⁷

2. CONCEPT OF IMMUNOPET

Immuno-positron emission tomography (immunoPET or iPET), which exquisitely fuses the extraordinary targeting specificity of mAb and the superior sensitivity and resolution of PET, is a paradigm shift for molecular imaging modalities.¹⁸ The concept of immunoPET was manifested more than two decades ago,^19,20 but its development rapidly accelerated in recent years with the increasing approval of therapeutic antibodies and the more widespread production of long half-life radionuclides. Meanwhile, the concept of immunoPET has evolved over the years with the incorporation of antibody fragments or mimetics as targeting moieties. More importantly, the clinical application of immunoPET imaging has increased our understanding of tumor heterogeneity and refined clinical disease management. For instance, the status of programmed death ligand-1 (PD-L1) assessed by ⁸⁹Zr-atezolizumab immunoPET, but not by immunohistochemistry (IHC) or RNA sequencing, predicted the therapeutic response of atezolizumab in patients with three types of tumors.²¹

Despite the existence of several reviews on immunoPET, there are none that comprehensively describe the design strategies and the application landscape of this novel imaging modality. In this review, we first elaborate on the development of immunoPET imaging strategies by introducing positron-emitting radionuclides, associated chelators, targeting vectors (e.g., mAbs and antibody fragments), as well as traditional and novel conjugation strategies. We then introduce the role of immunoPET in imaging cancers and noncancerous diseases, followed by a recapitulation of how immunoPET imaging aids in the development of antibody and antibody-based therapeutics. In the last part of the review, we discuss practical considerations for future development and translation of immunoPET imaging tracers.

3. DESIGN AND CONJUGATION STRATEGIES OF IMMUNOPET

ImmunoPET applications require simple, fast, and specific radiolabeling of antibody vectors under mild conditions. Optimal immunoPET imaging is attributed to a highly specific tumor uptake and low background retention. Toward this end, it is essential for a tracer to specifically saturate its target as fast as possible, with the unbound tracer cleared out rapidly from the blood circulation. Generally, the successful development of immunoPET probes is highly dependent on the choice of tumor-targeting vectors, radionuclides, bifunctional chelators, and conjugation strategies as discussed below.

3.1. Antibodies, Antibody Fragments, and VHHs

3.1.1. Full-Length Antibodies.

The development and use of mAbs have achieved considerable success, and various kinds of mAbs have been adapted to treat solid tumors, hematological malignancies, as well as noncancerous diseases.^5,22–24 In 2018, the Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved 13 antibody therapeutics for clinical use.²⁵ Although only five new antibody therapeutics were approved in 2019, it is anticipated that at least 13 products will be granted approval in 2020.²⁶ The therapeutic mechanisms of mAbs mainly include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), interruption of a signaling pathway, inhibition of enzymatic activity, and inhibition of immune checkpoint, which are discussed extensively in other reviews.^27,28 For therapeutic purposes, immunoglobulin G (IgG) is considered to have the most favorable balance between clearance and tumor uptake.²⁹ Over the past decade, immunoPET imaging with radiolabeled mAbs has progressed rapidly together with the development of antibody engineering and production of long-lived PET radionuclides.¹⁸ Currently, mAb-based immunoPET imaging is being actively investigated in preclinical models and has attracted considerable attention in clinical practice. The tumor-targeting and treatment efficacy of mAbs can be maximized by generating bispecific antibodies (BsAb) or trispecific antibodies.^30,31 By targeting multiple tumor antigens, these novel polyspecific antibodies are alternatives for developing immunoPET probes.³²

3.1.2. Limitations of Full-Length mAbs in Immuno-PET Imaging.

Despite the clinical success, mAb-derived immunoPET probes suffer from several disadvantages. First, the size of mAb (150 kDa; Figure 1a) exceeds the clearance cutoff value (60 kDa) of glomerular filtration. Additionally, the interaction between the Fc domain of IgG and the neonatal Fc receptor (FcRn) in endothelial cells further protects serum IgG from degradation.^33–35 Consequently, long-lived radionuclides that match the serum half-life of mAbs are required to develop the radiotracers. These factors synergistically contribute to the typical features of mAb-based immunoPET imaging, such as slow blood clearance, less optimal target-to-background [T/B] ratio, and the necessity to image repeatedly after administration of a single dose of the tracer. In addition, the pharmacology of antibody–antigen binding and the internalization of the antibody–antigen complex must also be considered when developing mAb-derived PET imaging tracers.^36–38 To maximize tumor uptake and detect liver malignancies or metastases, preloading or coadministration of unlabeled antibody is required in the course of immunoPET imaging.³⁹ However, the required blocking dose differs in a target- and antibody-dependent manner⁴⁰ and is greatly affected by the antibody treatment schedule at the time of tracer injection.⁴¹ Therefore, a feasible and reproducible imaging protocol needs to be carefully established before carrying out regular immunoPET scanning. Antibodies are produced in eukaryotic cell lines due to their complex expression and post-translational modifications.⁴² Because of this, the use of large amounts of antibodies is costly, further increased from the expenses of producing radiometals. Therefore, high costs may limit the widespread use of mAb-based immunoPET imaging tracers.

Figure 1.

Schematic of representative antibody and antibody fragments. (a) Conventional IgG is composed of two identical heavy chains and two identical light chains. While each heavy chain consists of three constant domains (i.e., C_H1, C_H2, and C_H3) and a variable domain (VH), an IgG light chain has one constant domain (CL) and one variable domain (VL). (b) Heavy-chain-only antibody (HCAb) lacks the light chains and the typical C_H1 domain. (c) The antigen-binding specificity of a HCAb is due to the single VHH domain. (d) Single-chain variable fragment (scFv) is the smallest unit of the IgG molecule that retains antigen-binding capacity. Using scFv as the building block, (e) diabody (dimers of scFv), and (f) minibody (dimers of scFv–C_H3) can be constructed.

3.1.3. VHHs.

Because mAbs tend to circulate in the blood and deposit in normal organs such as the liver, spleen, and bone marrow, smaller antibody constructs have been employed to accelerate the clearance of unbound radiotracer from systemic circulation and correspondingly achieve higher T/B ratios. Furthermore, small antibody fragments may penetrate solid tumors more efficiently and homogeneously.⁴³ Several types of smaller targeting vectors are available, including camelid heavy-chain-only antibodies (HCAbs) and shark-derived immunoglobulin new antigen receptors (IgNARs).⁴⁴ HCAbs (Figure 1b) are naturally occurring antigen-binding antibodies in Camelidae. HCAbs can be obtained by immunization of camels, llamas, or dromedaries or from näive or synthetic phage libraries.⁴⁵ HCAbs have only two constant domains, as opposed to the three constant domains of an IgG. HCAbs are generally humanized for theranostic purposes.^46,47

The variable domain of the heavy chain of a HCAb (VHH, Figure 1c), often referred to as Nanobody (a trade name of Ablynx) or single-domain antibody (sdAb), is the smallest antigen-binding derivative. With its molecular weight around 15 kDa and diameter <4 nm, VHH can penetrate deeply into tumor tissues while retaining its antigen recognition ability.⁴⁸ VHHs targeting various cellular or subcellular receptors or oncogenic proteins have also been generated.^49,50 Manifold techniques are available to achieve chemical functionalization of VHHs, which indubitably facilitates more sophisticated applications of VHHs.^51,52 Specifically, strategies like PEGylation and albumin hitchhiking may be used to prolong the circulatory half-life of VHH,^53–55 enabling more thorough and efficient targeting of the targets.

It has been suggested that VHHs are “magic bullets” for molecular cancer imaging.⁵⁶ For radiometal labeling, cysteine (Cys) or lysine (Lys) residues on VHHs are chemically modified with bifunctional chelators. Whereas for radio-iodination of VHHs, either direct electrophilic radioiodination or indirect radiolabeling methods can be used.⁵⁷ Because of the variable number of Lys or Cys residues in the complementary-determining region 3 (CDR3) of VHHs, it is challenging to radiolabel VHHs homogeneously using these standard methods. Methodologies that enable site-specific radiolabeling are readily available to prepare homogeneous VHH-based radiotracers (described in section 3.3.3.).^58,59 Despite the favorable pharmacokinetics, radiotracers based on VHHs have very high kidney accumulation due to the renal clearance of the excess material, limiting their role in detecting lesions located in the urinary system or in the vicinity of kidneys. Several factors (e.g., the sequence of the VHH, conjugation method, as well as specific receptors in the glomeruli) may all contribute to the high kidney retention of the developed radiotracers.^60,61 However, there are strategies to circumvent this phenomenon, including coinfusion of gelofusine and Lys,⁶² removal of polyhistidine tag (His-tag),⁶³ PEGylation,⁶⁴ and site-specific radiohalogen labeling.⁶⁵ Furthermore, VHHs are also being actively exploited for therapeutic purposes,⁶⁶ either in the form of radioimmunotherapy (RIT),^67–69 or in the form of photoimmunotherapy (PIT).^70,71

3.1.4. Other Engineered Antibody Fragments and Proteins.

Several other antibody fragments have been engineered for imaging purposes.⁷² In general, these antibody fragments lack the Fc region and are smaller in size. Single-chain variable fragment (scFv, Figure 1d) is one of the most popular antibody fragments with a molecular size of ~25 kDa. A scFv clears exceptionally fast from the bloodstream and creates much higher T/B ratios compared with an intact IgG. scFv is composed of variable light and variable heavy chains that are joined by a flexible peptide linker. As such, the length and amino acid composition of the peptide linker between the two domains significantly affect the binding affinity and size of the engineered scFv.⁷³ A significant drawback of scFv molecules is their monovalent antigen-binding specificity. In certain cases, engineering a monovalent scFv into multivalent constructs may enhance the avidity and optimize the tumor-targeting capability. Diabody (~60 kDa, Figure 1e) is a divalent variant of the monovalent scFv. Typically, a Cysmodified diabody (Cys-diabody) is constructed and used for site-specific radiolabeling.⁷⁴ Similar to radiolabeled VHHs, radiolabeled scFv, and diabody are rapidly cleared by the urinary system, resulting in high accumulation of the radiotracers in the kidneys. Other larger divalent forms, such as minibody (Mb, Figure 1f) and (scFv)₂-Fc constructs, have also been developed as targeting vectors.^75,76 Several multivalent scFv fragments, such as triabody (~90 kDa) and trimerbody (110 kDa), also showed potential as ligands for immunoPET imaging.^77,78

In the pursuit of proteins with enhanced or novel functions, a multitude of protein scaffolds has been generated and used in the field of molecular imaging. These low-molecular-weight proteins lack disulfide bonds and glycosylation, so they can be expressed in bacterial systems with proper conformation rapidly.⁷⁹ Currently, one of the most commonly engineered protein scaffolds for PET imaging is the Affibody (6 kDa).⁸⁰ For instance, Z_HER2:342 is an Affibody molecule targeting human epidermal growth factor receptor 2 (HER2) and has been widely studied for PET imaging. The most attractive advantages of HER2-targeting Affibodies are their unique binding sites on HER2, which are distinct from HER2-targeted therapeutics (e.g., trastuzumab).^81,82 Therefore, novel imaging approaches employing these Affibodies may help discriminate the downregulation and saturation of HER2 following HER2-targeted therapies. Other similar molecules that have already been used for molecular imaging include adnectins,⁸³ fibronectin,⁸⁴ knottins,^85,86 and anticalins.^87,88 Like VHHs, the primary benefits of these small antigen-targeting moieties are to permit same-day molecular imaging.⁸⁹ The advantages from accelerated clearance are compensated by lower tumor uptake, yielding modest imaging quality. To enhance tumor retention and decrease kidney retention, several approaches (e.g., PEGylation) can be used to modify the targeting vectors.^90–92

Of the BsAbs, bispecific T-cell engager (BiTE) antibody constructs (~55 kDa) are designed to induce context-dependent anticancer immune responses by cross-linking tumor cells with cytotoxic T cells.^93–95 One successful example is the blinatumomab, which simultaneously targets CD19-positive B cells and then recruits CD3-positive cytotoxic T cells.⁹⁶ It is much more challenging to design T-cell-dependent BsAb constructs because each arm of the antibody has a different antigen-binding affinity. Moreover, the T-cell-targeting arm substantially affects the in vivo distribution of the antibody and, therefore, the fate of the developed molecular imaging tracers.^97,98

3.2. Radionuclides and Chelators

In recent years, various antibodies targeting diverse antigens have been labeled with gamma-emitting radionuclides (e.g., ¹³¹I, ¹²³I, ¹¹¹In, or ^99mTc) and used for diagnosis by SPECT or planar imaging or for therapeutic applications. Because of their poor diagnostic performance, very few are routinely used in the clinic. With the global installation of cyclotrons, a variety of novel positron-emitting radionuclides is being produced.^99,100 High-purity radiometals, a fundamental component in immunoPET imaging probes, are increasingly being produced and used in recent years.^101,102 Traditionally, radiometals are eluted from generators or produced using solid targets with cyclotrons.¹⁰³ As a supplement to solid targets, liquid targets (solution targets) can also be used to produce radiometals upon optimization.¹⁰⁴ Several factors need to be considered before exploiting them for radiolabeling, which include physical properties (e.g., half-life [T_1/2] and decay mode), chemical properties, production efficiency, safety profiles, and price. The T_1/2 of a chosen positron emitter has to closely match the biological half-life of the targeting vector. In conjugating immunoPET imaging probes, the positron emitter is generally complexed with an inert chelator that is attached to the targeting antibody. The major principle is that the binding affinity, stability, and pharmacokinetic characteristics of the final radiopharmaceutical are in concert with the naive antibody. There are several review articles describing various radiometals and their coordination chemistry^101,105,106 and ¹⁸F radiolabeling of heat-sensitive molecules.¹⁰⁷ In this section, we will confine to the most promising radionuclides and related chelators used for immunoPET imaging (Table 1).

Table 1.

Representative Radionuclides Used in Developing ImmunoPET Imaging Probes^a

isotope	T1/2	emission profiles	production methods	ref
89Zr	78.4 h	β+: 22.8%, Eβ+max = 901 keV; EC: 77%, Eγ = 909 keV	89Y(d,2n)89Zr, 89Y(p,n)89Zr, natSr(α,xn)89Zr, etc.	111,120
64Cu	12.7 h	β+: 19%, Eβ+max = 656 keV; EC 41%, Eγ = 1346 keV; β−: 40%, Eβ−max = 579 keV	64Ni(p,n)64Cu 64Ni(d, 2n)64Cu 68Zn(p,α)64Cu	141
124I	4.18 d	β+: 22%, Eβ+max = 2.13 MeV	124Te(p,n)124I	180
86Y	14.7 h	β+: 34%, Eβ+max = 3.153 MeV; EC: 66%, Eγ = 1043 keV	86Sr(p,n)86Y 86Sr(d,2n)86Y	163,164
68Ga	1.1 h	β+: 89%, Eβ+max = 1899 keV; EC: 11%, Eγ = 1077 keV	68Ge/68Ga generator 68Zn(p,n)68Ga	225,226
44Sc	3.9 h	β+: 94%, Eβ+max = 1474 keV; EC: 6%, Eγ = 1157 keV	44Ti/44Sc generator natCa(p,n)44Sc 44Ca(p,n)44Sc	981–983
18F	109.8 min	β+: 97%, Eβ+max = 635 keV	18O(p,n)18F	196
52Mn	5.591 d	β+: 29.4%, Eβ+max = 575 keV; Eγ = 1434	52Cr(p,n)52 Mn natCr(p,x)52 Mn	246

^aAbbreviations: T_1/2, half-life; EC, electron capture (e.c.). The methods given in this Table are commonly used approaches to produce radionuclides. The readers are recommended to refer to the cited references for production details.

3.2.1. Zirconium-89.

Zirconium-89 (⁸⁹Zr, T_1/2 = 78.4 h) has been extensively used in the biomedical imaging field due to its fitting emission energy properties and long half-life, which matches the circulation half-life of mAbs.^{108,109 89}Zr can be produced via several different nuclear reaction pathways, such as the ^natSr(α,xn)⁸⁹Zr reaction, ⁸⁹Y(d,2n)⁸⁹Zr reaction, or ⁸⁹Y(p,n)⁸⁹Zr reaction.¹¹⁰ However, the production of ⁸⁹Zr using solid targets with a small medical cyclotron might still be challenging. Recent studies have reported the production of ⁸⁹Zr via solution targets, which are filled with a yttrium nitrate solution (Y(NO₃)₃·6H₂O). ^111,112 Further refinement of the irradiation procedures of liquid targets may potentially broaden the availability of ⁸⁹Zr and therefore the development of ⁸⁹Zr-based PET imaging. In developing ⁸⁹Zr-mAb conjugates, ⁸⁹Zroxalate (⁸⁹Zr–Zr(ox)₂) can be converted to 89Zr-chloride (⁸⁹Zr-ZrCl₄), which tends to undergo aquation and chelation with chelator-modified mAbs more rapidly.^113,114 Moreover, ⁸⁹Zr-chloride lacks the toxic oxalic acid of ⁸⁹Zr-oxalate.¹¹⁵

⁸⁹Zr is coupled to a mAb of interest through a bifunctional chelator, which possesses a ligand for capturing ⁸⁹Zr and a reactive group for conjugating Lys or Cys residues on the mAb surface. Desferrioxamine (Figure 2a), denoted as Df or DFO, is a clinically used chelator for complexation of ⁸⁹Zr. Traditionally, the preparation of 89Zr-mAb conjugates involves a multistep synthesis, in which a succinylated-derivative of desferrioxamine B (N-sucDf) was used to modify mAbs.^116–118 This pioneering work paved the way for subsequent preclinical and more importantly, the clinical success of ⁸⁹Zr-mAb immunoPET imaging. The development of a novel p-isothiocyanatobenzyl-derivative of desferrioxamine B (known variously as p-SCN-Bn-deferoxamine, Df-Bz-NCS or DFO-pPhe-NCS; Figure 2b) further allowed efficient and rapid preparation of ⁸⁹Zr-mAb conjugates. This process involves the first coupling of Df–Bz–NCS to the lysine-NH₂ groups of a mAb under alkaline conditions (pH 8.9–9.1) followed by radiolabeling with ⁸⁹Zr-oxalate.^119,120

Figure 2.

Chemical structures of chelators used in ⁸⁹Zr-labeling of antibody vectors.

Despite their attractive characteristics, such chelators are unable to saturate the octavalent demands of the Zr⁴⁺ cation,¹²¹ which results in less stable ⁸⁹Zr-immunoconjugates as indicated by accumulation of free 89Zr in the bone. Preclinical studies suggested that ⁸⁹Zr’s tropism for bone may introduce undesirable radiation to bone marrow and confound imaging conclusions of bone malignancies or joint inflammation.^122,123 The clinical impact of unbound ⁸⁹Zr remains to be determined. A family of novel bifunctional chelators that impart enhanced stabilities has been developed to surmount this problem. One of these, the tetrahydroxamate chelator called DFO* (Figure 2c) and its derivative DFO*-pPhe-NCS (Figure 2d) were synthesized successfully.^{124,125 89}Zr-DFO*-trastuzumab was thermodynamically more stable and had significantly lower bone uptake compared to the DFO modified mAb.¹²⁵ More recently, DFOcyclo*-pPhe-NCS (Figure 3a), a novel DFO* derivative, was developed.¹²⁶ When competed with excess DFO, this novel chelator was more stable than DFO and DFO* for chelating 89Zr. ImmunoPET imaging and biodistribution studies further demonstrated significantly lower bone uptake of 89Zr-DFOcyclo*-trastuzumab than ⁸⁹Zr-DFO-trastuzumab. Despite this, ⁸⁹Zr-DFOcyclo*-trastuzumab and ⁸⁹Zr-DFO*-trastuzumab showed comparable imaging performance (Figure 3b). Desferrichromes (DFC) and related compounds have also been explored for coordinating ⁸⁹Zr, but the DFC system did not show a dramatic advantage over DFO in in vivo imaging studies.¹²⁷

Figure 3.

Comparison DFO, DFO*, and DFOcyclo* in immunoPET imaging. (a) Chemical structure of DFOcyclo*-pPhe-NCS. (b) ImmunoPET imaging with ⁸⁹Zr-DFO-trastuzumab (left), ⁸⁹Zr-DFOcyclo*-trastuzumab (middle), and ⁸⁹Zr-DFO*-trastuzumab (right) in HER2⁺ SKOV-3 models. The results showed bone uptake in mice injected with ⁸⁹Zr-DFO-trastuzumab but not with ⁸⁹Zr-DFOcyclo*-trastuzumab or ⁸⁹Zr-DFO*-trastuzumab at 168 h after injection of the radiotracers. Reproduced with permission from ref 126. Copyright 2019 Springer Berlin Heidelberg under [CC LICENSE] [http://creativecommons.org/licenses/by/4.0/].

Other novel ⁸⁹Zr chelators include those that do not contain hydroxamate moieties, such as p-SCN-Bn-H6phospa,¹²⁸ 3,4,3-(LI-1,2-HOPO),¹²⁹ p-SCN-Bn-HOPO,^130,131 and many other novel chelators containing hydroxamate moieties.^132–137 For instance, DFO-1-hydroxy-2-pyridone ligand (DFO-HOPO) is an octadentate chelator for ⁸⁹Zr. ⁸⁹Zr-DFO-HOFO showed excellent renal clearance and significantly lower bone uptake compared with ⁸⁹Zr-DFO.¹³⁵ Meanwhile, other novel ⁸⁹Zr chelators with cyclic structures have been developed.^136,138,139 Most recently, ligands containing carboxylate or amino donors have been tested as ⁸⁹Zr chelators.¹⁴⁰ Of these, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, vide infra) has been shown to outperform other analogues because ⁸⁹Zr-DOTA demonstrated exceptional stability and more rapid systemic clearance. However, the high temperature (90 °C), longer reaction duration (45 min), and the need to use ⁸⁹Zr-ZrCl₄ may hinder its application in the immunoPET imaging field.

3.2.2. Copper-64.

Copper-64 (⁶⁴Cu, T_1/2 = 12.7 h) stands out as an immunoPET imaging radionuclide because of its ready availability and favorable properties. ⁶⁴Cu is typically produced by bombardment of an enriched nickel target via the ⁶⁴Ni(p,n)⁶⁴Cu nuclear reaction.¹⁴¹ Interestingly, a recent study reported the possibility of ⁶⁴Cu production using a liquid target.¹⁴² Because ⁶⁴Cu undergoes β⁻ emission in addition to β⁺ emission, it is a promising theranostic radionuclide. Furthermore, the combination of ⁶⁴Cu and ⁶⁷Cu (T_1/2 = 61.8 h, β⁻: 100%) results in an attractive theranostic pair.¹⁴³ To avoid nonspecific deposition of ⁶⁴Cu in healthy tissues, various macrocyclic ligands and their derivatives have been developed. DOTA and its derivatives (Figure 4a–d) are the most commonly used ones to chelate ⁶⁴Cu for PET imaging.^144,145 However, it has been shown that DOTA is not the chelator of choice to develop immunoPET tracers.^146,147 NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)-based chelators (Figure 4e,f) are the most successful for chelating both ⁶⁴Cu and ⁶⁸Ga (vide infra) and are well-suited for radiolabeling of heat-sensitive antibody vectors at room temperature (rt). A comparison of the NOTA derivative 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA, Figure 4g) and DOTA for labeling a mAb with ⁶⁴Cu demonstrated better in vivo performance of ⁶⁴Cu-NODAGA-mAb than that of ⁶⁴Cu-DOTA-mAb.¹⁴⁸ p-SCN-Bz-MANOTA (Figure 4h), another NOTA derivative, outperformed DOTA and NODAGA as ⁶⁴Cu-MANOTA-Fab showed the highest stability and the lowest background uptake in immunoPET imaging studies.¹⁴⁹

Figure 4.

Chemical structures of chelators used in ⁶⁴Cu-labeling of antibody vectors.

In addition, a series of cyclam-based macrocycles have been devised for ⁶⁴Cu-labeling of antibodies. One of such agents, CB - TE2A (4, 11 - bis (carboxymethyl) - 1, 4, 8, 11 - tetraazabicyclo[6.6.2]hexadecane, Figure 4i), has shown its merits as an effective chelator for ⁶⁴Cu.¹⁵⁰ Unfortunately, the unfriendly labeling conditions (95 °C, 60 min, pH 6–7) limit the use of CB-TE2A in applications with antibody-based agents.¹⁵¹ CB-TE1A1P [(1,4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid), Figure 4j] bearing a methanephosphonic acid and a carboxymethyl pendant arm can be radiolabeled with ⁶⁴Cu at rt.^152,153 While CB-TE2P [1,4,8,11-tetraazacyclotetradecane-1,8-di-(methanephosphonic acid), Figure 4k] can also be used for ⁶⁴Cu-labeling at mild conditions,¹⁵⁴ CB-TE1A1P is more favorable for antibody labeling because the carboxylate group allows for facile bioconjugation. Indeed, several studies have used ⁶⁴Cu-CB-TE1A1P for immunoPET imaging.^155,156 To further capitalize the better radiochemical yield (RCY) of cyclam derivatives, several other cyclam-based bis-(phosphinate)-bearing ligands for conventional or click chemistry-based ⁶⁴Cu-labeling were developed.^157,158 These novel cyclam-based bifunctional ligands are highly promising for developing immunoPET probes with ⁶⁴Cu under mild conditions (25–37 °C, 10–20 min, pH 5.5–6.2).¹⁵⁸ SarAr (Figure 4l) is a sarcophagine-based chelator used for developing immunoPET probes and can be labeled with radiometals under mild conditions (20–37 °C, 5–30 min, pH 5–5.5).^159,160

The use of different chelators may result in varied accumulation patterns of the ⁶⁴Cu-labeled mAb in the blood pool and in other healthy organs (e.g., liver) despite the similar tumor uptake.¹⁶¹ Generally, ⁶⁴Cu undergoes hepatobiliary clearance which may result in increased liver and intestine signals, limiting the detection of diseases at these sites as well as diseases at the adjacent organs or tissues (e.g., pancreas). However, this problem can be resolved with 64Cu-labeled VHHs, which precisely detected small pancreatic tumors with clarity and high T/B ratio.¹⁶²

3.2.3. Yttrium-86.

Yttrium-86 (⁸⁶Y, T_1/2 = 14.7 h) decays via electron capture (ec) and positron emission, accompanied by the emission of γ rays. Several nuclear reactions have been explored to produce ⁸⁶Y. To date, the recommended reaction is ⁸⁶Sr(p,n)⁸⁶Y reaction,^163–165 where the target material SrCO₃ or SrO is enriched for irradiation with energies from 8–15 MeV. Although a liquid target has also been used to produce ⁸⁶Y,¹⁰⁴ the yield is generally low and methods for separating radiation-induced chemical species need to be established. With the refinement of methods for separating radioyttrium,¹⁶⁶ the large scale production of ⁸⁶Y is feasible with hospital-based cyclotrons. The most appealing application of ⁸⁶Y is in tandem with yttrium-90 (⁹⁰Y, T_1/2 = 64.1 h), which is a pure beta emitter with excellent therapeutic properties.^167,168 The advantage of this theranostic pair is that quantitative PET imaging with ⁸⁶Y allows precise dosimetry of ⁹⁰Y-based radiopharmaceuticals.¹⁶⁹ Both clinical and preclinical studies have suggested that sequential use of ⁸⁶Y and ⁹⁰Y is an attractive theranostic pair if proper targeting vectors are used.^170,171 Derivatives of ethylenediaminetetraacetic acid (EDTA, Figure 5a,b), diethylenetriamine pentaacetic acid (DTPA, Figure 5c), and DOTA are the most widely used chelators for yttrium radiolabeling of mAbs.^172,173 Several studies have shown that incorporating the isothiocyanatobenzyl group (SCN-Bz) into the backbone of DTPA (Figure 5d) may sterically hinder the release of yttrium from the radiopharmaceuticals.^174,175 To further improve the coordination efficiency of DTPA derivatives, CHX-A′′-DTPA (Figure 5e) and p-SCN-Bn-CHX-A′′-DTPA (Figure 5f) bearing a cyclohexyl were developed, and these chelators possessed improved stability over DTPA in radiolabeling mAbs.^176–179

Figure 5.

Chemical structures of chelators used in ⁸⁶Y-labeling of antibody vectors.

3.2.4. Radioiodine-124.

Radioisotopes of iodine have long been used as theranostic agents in the field of thyroid cancer.¹⁸⁰ One among these, 124I (T_1/2 = 4.18 d) can be produced through the 124Te(p,n)¹²⁴I reaction.^181,182 Iodine-124 has gained interest in radiolabeling mAbs since the clinical feasibility of immunoPET imaging with a ¹²⁴I-labeled HMFGI mAb was first demonstrated in 1991.^183–186 ImmunoPET imaging with ¹²⁴I-labeled antibody agents are useful for evaluating bone metastases, another major benefit compared to those labeled with bone-seeking radiometals (e.g., ⁸⁹Zr). It is important to mention that ¹²⁴I-labeled immunoPET probes may not be appropriate for detecting primary thyroid cancers, stomach cancers, and urinary malignancies (e.g., bladder cancer and prostate cancer) because thyroid and stomach can scavenge iodide produced by deiodination and iodide is cleared via the urinary system. In addition to its role in PET imaging, ¹²⁴I is a theranostic agent because of Auger electrons produced during its decay.¹⁸⁷ ImmunoPET imaging using ¹²⁴I-mAb is fully concordant with 131I-mAb RIT, where immunoPET imaging acts as a scouting procedure prior to RIT.¹⁸⁸ Currently, the IODO-GEN method is the method of choice for radioiodination of noninternalizing mAbs.¹⁸⁹ In an attempt to trap radioiodinated mAbs inside the tumor cells for improved molecular imaging, residualizing prosthetic agents for radioiodination have been developed and used by several groups.^190–195

3.2.5. Fluorine-18.

With a high positron yield of 97%, a low mean positron range of 0.5 mm, and no simultaneous γ ray emission, fluorine-18 (¹⁸F, T_1/2 = 110 min) is an ideal radionuclide for PET imaging.¹⁹⁶ Diabodies and VHHs labeled with ¹⁸F have short plasma half-lives and can permit same-day imaging,¹⁹⁷ a practical advantage over mAb-based imaging tracers. However, 18F has not been used to develop immunoPET probes until recently due to the harsh radio-labeling conditions and low RCY.

With the development of automated chemistry stations, several prosthetic groups for radiofluorination have been reported.¹⁹⁸ [¹⁸F]Fluorobenzaldehyde ([¹⁸F]FBA, Figure 6a) is among the most popular prosthetic groups used for radio-fluorination of biomolecules.¹⁹⁹ N-Succinimidyl-4-[¹⁸F]-fluorobenzoate ([¹⁸F]SFB, Figure 6b) is another prosthetic group which can form a stable amide bond with the Lys residue on proteins or peptides.^200–202 Generally, an average RCY of 30%–35% will be obtained when [¹⁸F]SFB is used for labeling proteins or peptides.²⁰³ N-[2-(4-[¹⁸F]-Fluorobenzamido)-ethyl]maleimide ([¹⁸F]FBEM, Figure 6c) is a thiol-reactive ¹⁸F-labeling agent that can be site-specifically conjugated to Cys residues.^204–207 However, the radiosynthesis of [¹⁸F]FEBM is a multistep and time-consuming process and often results in low RCY. It has been reported that the preparation of [¹⁸F]FBEM using automated radiochemical procedures requires less time and provides higher RCY (~17%).²⁰⁸ Another prosthetic group that facilitates radio-labeling of biomolecules under mild conditions (37–40 °C, 15 min, pH 8.5–9.0) is 2,3,5,6-tetrafluorophenyl 6-[¹⁸F]-fluoronicotinate ([¹⁸F]TFPFN, Figure 6d).^209,210 Recent studies simplified the synthesis of [¹⁸F]TFPFN without drying [¹⁸F]fluoride,^211,212 but the unfavorable RCY (~5%) in labeling VHHs may limit its applications.²¹³ Direct ¹⁸F-labeling approaches include the silicon–fluoride acceptor approach (¹⁸F-SiFA),^214–217 and the organotrifluoroborate ([¹⁸F]BF₃) method,²¹⁸ but one concern is that the solvents (e.g., acetonitrile) or the acidic conditions (pH 2.0–2.5) used in these methods are detrimental for sensitive antibodies.

Figure 6.

Chemical structures of prosthetic groups and chelators in ¹⁸F-labeling of antibody vectors.

In 2009, McBride et al. reported the aluminum-fluoride (Al¹⁸F) chelation strategy where fluorine is firmly bound to Al³⁺ by forming Al¹⁸F, which is complexed to NOTA with the resultant complex conjugated to the biomolecule of interest.²¹⁹ This method has since been widely used for radiofluorination of various biomolecules.²²⁰ Although this procedure allows rapid fluorination of peptides, the high temperature (~100 °C) necessary for the complexation is unsuitable for most antibodies and some peptides. To overcome this drawback, a facile two-step procedure was described,²²¹ where [¹⁸F]AlF was first complexed to NODA-MPAEM at high temperature (105–109 °C, 15–20 min; Figure 6e) and the purified intermediate then conjugated to antibodies via the maleimide–thiol reaction at rt for 10 min. Building upon this work, a series of novel acyclic polydentate ligands permitting facile Al¹⁸F radiolabeling of antibodies have been developed.^222,223 RESCA-tetrafluorophenyl ester ((±)-H₃RESCA-TFP, Figure 6f) and RESCA-maleimide ((±)-H₃RESCA-Mal, Figure 6g) are two chelators that can be used to conjugate biomolecules via the Lys or Cys residues, respectively.²²⁴ Future studies are warranted to evaluate the diagnostic value of Al¹⁸F-RESCA-labeled antibodies.

3.2.6. Gallium-68.

Gallium-68 (⁶⁸Ga, T_1/2 = 1.1 h) is an attractive positron-emitting radionuclide because it is readily available from an affordable in-house ⁶⁸Ge/⁶⁸Ga generator. Because ⁶⁸Ge has a half-life of 270.8 days, the shelf life of the generator is about 6–12 months based on elution schedules.²²⁵ Meanwhile, other means have been explored to produce ⁶⁸Ga on a large scale.^226–228 Although extensively studied for ⁶⁷Ga/⁶⁸Ga complexation, the DOTA-Gallium complex is less stable than its counterpart NOTA analogue. Currently, NOTA and its derivatives are the “gold standard” for ⁶⁷Ga/⁶⁸Ga complexation because of their fast and efficient radiolabeling at rt and high in vivo stability.²²⁹ HBED and its derivatives (Figure 7a–c) enabled ⁶⁸Ga-labeling of heat-labile antibodies and antibody fragments at ambient temperatures.^230,231 H2dedpa and its bifunctional derivative p-SCN-Bn-H2dedpa (Figure 7d,e) were developed for labeling peptides with ⁶⁸Ga or 64Cu and tracers based on these chelators have shown promising imaging potentials.^232–234 CP256 (Figure 7f) and YM103 (Figure 7g) are two other acyclic ligands that have yielded encouraging imaging results.²³⁵ PCTA and its derivative (Figure 7h,i) were superior with respect to kinetics and RCY for radiolabeling mAbs with ⁶⁴Cu.^236,237 Similarly, ⁶⁸Ga-PCTA complex also showed improvement over ⁶⁸Ga-NOTA for conjugating peptides.²³⁸ TRAP-Pr, a derivative of NOTA bearing phosphinic acid groups, showed significantly improved specificity for Ga³⁺ in radiolabeling peptides,²³⁹ but the harsh radiolabeling conditions (95 °C, pH 3.2) prohibit its use in radiolabeling of antibody vectors. To the best of our knowledge, many of the chelators mentioned above (e.g., p-SCN-Bn-H2dedpa, YM103, and PCTA) have not been utilized in developing immunoPET probes, but it is plausible that these chelators may have a certain value for ⁶⁸Ga-based immunoPET probes.

Figure 7.

Chemical structures of chelators used in ⁶⁸Ga-labeling of antibody vectors.

3.2.7. Other Radiometals.

Scandium-44 (⁴⁴Sc, T_1/2 = 3.9h) is a positron-emitting isotope and can be produced from a generator or a cyclotron source.^240–242 Our team showed that CHX-A”-DTPA as opposed to other conventional chelators(i.e., DOTA, NOTA, DTPA), achieved ⁴⁴Sc-labeling of a Fab fragment at rt.²⁴³ We are positive that the development of novel chelation strategies will further expand applications of ⁴⁴Sc in the biomedical imaging field.^244,245

Manganese-52 (⁵²Mn, T_1/2 = 5.591 d) can be produced via several nuclear reactions including the natCr(p,x)⁵²Mn reaction.^246,247 Manganese-52 has a higher β⁺ branching ratio of 29.4% and a lower β⁺ energy of 575 keV when compared to ⁸⁹Zr (β⁺: 22.8%, E_β+max = 901 keV), making it a promising alternative to ⁸⁹Zr for immunoPET imaging. In a proof-of-concept study, we reported that the chelation of ⁵²Mn via DOTA is possible and immunoPET imaging with 52Mn-DOTA-mAb is feasible over the course of several days.²⁴⁸ Other radiometals that can be incorporated into immunoPET imaging include ¹⁵²Tb (T_1/2 = 17.5 h),^{249 76}Br (T_1/2 = 16.2h),^250,251 and ¹³²La (T_1/2 = 4.59 h).^252,253

3.3. New Conjugation Strategies

Lysine-based random conjugation is the most prevalent method used for chemical modification of antibodies, followed by nonspecific Cys-based conjugation. Indeed, many clinical-grade radiolabeled antibodies have been produced via lysine functionalization. However, modification of antibodies at undesirable sites may compromise the immunoreactivity and distribution profiles of the radiolabeled antibodies. Hence, continuous efforts have been devoted to developing site-specific conjugation strategies to produce well-defined radio-tracers for high-quality imaging.²⁵⁴

3.3.1. Conventional Site-Specific Conjugation.

For site-specific modification purposes, proteins and antibodies are produced with short peptide tags via standard protein engineering and recombinant expression protocols. Generally, tags are introduced at the C-terminal end of the mAbs or sdAbs to avoid antigen-binding interference. Cys engineering is the most frequently used method for site-specific radiolabeling of antibody vectors (Figure 8a).^255,256 The most commonly used strategy for Cys modification is via the maleimide conjugation, which is reversible and may result in the release of the maleimide scaffold in plasma (retro-Michael reaction).²⁵⁷ Recently, a novel strategy that may realize irreversible Cys bioconjugation was described.²⁵⁸ Thanks to the continuous advancement of the biomedical field, more sophisticated techniques are being exponentially developed for site-specific modification of proteins.²⁵⁹ Future studies may synthesize novel immunoPET agents taking advantage of these emerging techniques.

Figure 8.

Site-specific radiolabeling strategies. (a) The maleimide–cysteine reaction is among the most commonly used strategies for site-specific radiolabeling of antibody vectors. R = chelator of interest. (b) The strain-promoted azide–alkyne cycloaddition (SPAAC) reaction. R1 = antibody of interest, R2 = chelator of interest. (c) The inverse electron demand Diels–Alder (IEDDA) cycloaddition reaction. R1 = chelator of interest, R2 = antibody of interest. It is worth noting that radiolabeling via the click chemistry reaction goes both ways.

3.3.2. Click Chemistry-Mediated Radiolabeling.

Click chemistry has been increasingly applied to develop new molecular imaging probes.^260,261 Of various click chemistry reactions, the Cu(I)-catalyzed 1,3-dipolar cycloaddition between azides and alkynes (CuAAC) is frequently used to develop radiopharmaceuticals. 18F-labeled small-molecule probes prepared via the CuAAC reaction are actively assessed in clinical settings.^262,263 If it is necessary to avoid the use of Cu(I) catalyst, particularly when developing immunoPET probes with radiometals, the catalyst-free strain-promoted azide–alkyne cycloaddition (SPAAC) reaction is a bioorthogonal alternative (Figure 8b).^264,265 However, the synthetic complexity and hydrophobicity of the cyclooctyne precursors in the SPAAC system may potentially limit its widespread application. The inverse electron demand Diels–Alder (IEDDA) reaction between strained trans-cyclooctene (TCO) and electron-deficient tetrazine (Tz) is a giant step forward in the field of bioorthogonal chemistry in terms of reactivity and application possibilities (Figure 8c).²⁶⁶ As such, this chemistry has been applied in a myriad of uses developing molecular imaging probes.^267–269 Moreover, the axial TCO isomers were found to be more reactive than their equatorial analogues.²⁷⁰

Photoclick chemistry has been used in the field of chemical biology for years.^271,272 On the basis of the previous success, several groups have used photochemical methods to develop immunoPET probes recently.^273–275 Upon further refinement, these novel bioconjugation methods may eliminate time-consuming purification steps and maximize RCY, which is particularly needed for short-lived radionuclides such as ¹¹C (T_1/2 ≈ 20 min) and ⁶⁸Ga (T_1/2 = 1.1 h).

3.3.3. Enzyme-Mediated Radiolabeling.

Enzymatic methods are well suited to achieve site-specific labeling of antibody vectors. Prominent one among them utilizes sortase A (SrtA), an enzyme derived primarily from Gram-positive Staphylococcus aureus. Typically, SrtA recognizes substrates containing C-terminal LPXTG motifs (where X = any amino acid except proline) and cleaves the peptide between threonine and glycine (Gly), leading to loss of the downstream part of the substrates (e.g., His-tag) and formation of new peptide bonds with nucleophilic substrates containing N-terminal Gly residues.²⁷⁶ While SrtA is a well-established enzyme responsible for anchoring LPXTG-containing proteins to the growing cell wall and pili of various Gram-positive bacteria, recombinant SrtA has been developed into a valuable protein engineering tool in recent years.²⁷⁷ By employing sortase-mediated transpeptidation, it is facile to install functional moieties (e.g., chelator and dye) onto the N- or C-terminus of an antibody in a site-specific fashion (Figure 9). The use of SrtA has facilitated site-specific radiolabeling of VHHs using either ¹⁸F^55,278 or radiometals.^64,162 A unique two-step modular system is also available to conjugate immunoPET probes. In this system, SrtA is used to incorporate the strained cyclooctyne functional groups into the targeting vector of interest, followed by azide–alkyne cycloaddition reaction between the click chemistry handles.^279,280 With further improvement of the catalytic activity of SrtA and evolution of Ca²⁺ independent SrtA mutants,^281–283 SrtA will serve as a versatile platform for developing more sophisticated immuno-PET probes.

Figure 9.

Sortase-catalyzed site-specific labeling of antibody moieties. (a) For C-terminal labeling, the LPXTG motif is expressed at the C-terminus of the targeting vector (e.g., VHH and antibody fragment). (b) For N-terminal labeling, sortase recognition tag (i.e., LPXTG) is positioned at the C-terminus of the modification (e.g., chelator and dye) with the oligoglycine nucleophile inserted at the N-terminus of the targeting vector.

Butelase-1 is another transpeptidase found in Clitoria ternatea (butterfly pea) and recognizes a tripeptide motif, Asn-His-Val.^284,285 Butelase-1 efficiently cyclizes peptides and proteins with a high yield. Although it is the fastest peptide ligase, its biological applications are limited because it cannot be produced as of now using recombinant techniques. Theoretically, a combination of butelase-1 and SrtA may facilitate the labeling of proteins at two distinct sites. This was proven in a recent work by Harmand et al.,²⁸⁶ which reported that the combination of SrtA and butelase 1 allowed facile preparation of C-to-C fusion proteins and dual-labeling of an IgG1 molecule with two fluorescent dyes. A combination of butelase-1 with other transpeptidases may be possible in the future.

Recently, a chemoenzymatic strategy combining glycan engineering and click chemistry was invented. The combination of these strategies allowed site-specific attachment of molecules to the heavy chain glycans. This methodology involves the following steps: (1) removal of galactose residues on the C_H2 domain of the heavy chains of an antibody using β−1,4-galactosidase, (2) attachment of azide-modified monosaccharide to the heavy chain glycans using β-galactosyltransferase mutant to Gal-T(Y289L), (3) synthesis of chelator or dye-containing cyclic dibenzocyclooctyne (DIBO or DBCO), (4) catalyst-free click chemistry between azide-bearing antibody and DIBO-bearing payload (e.g., chelator or dye), and (5) radiolabeling of the site selectively modified antibody using radionuclides of interest (Figure 10).²⁸⁷ This method has been applied to design dual-labeled agents for PET and optical imaging of colorectal cancers.²⁸⁸ A recent study demonstrated that ⁸⁹Zr-DFO-trastuzumab developed using this chemoenzymatic strategy outperformed its counterpart developed by random conjugation method because the site-specifically modified ⁸⁹Zr-DFO-trastuzumab showed enhanced immunoreactivity and stability in immunoPET imaging studies.²⁸⁹ Although being able to yield homogeneous and well-defined products, this method suffers from a lengthy and relatively tedious protocol.

Figure 10.

Schematic of a chemoenzymatic methodology for site-specifically grafting cargoes (e.g., chelator) to the heavy-chain glycans of an antibody of interest. Reproduced with permission from ref 261. Copyright 2016 American Chemical Society.

HaloTag is a genetic construct with multifunctional and versatile capabilities. The molecular mechanism of this system is based on a mutant bacterial haloalkane dehalogenase enzyme, which is obtained from Rhodococcus rhodochrous.^290,291 Use of the HaloTag technology begins with the fusion of the HaloTag (33 kDa) to the protein of interest. A HaloTag-specific ligand is then introduced, resulting in the formation of an irreversible covalent bond between the HaloTag-modified protein and the ligand.^292,293 As we all know, His-tag is ubiquitously introduced in protein production, but its role is merely limited to the isolation and purification of proteins.^294,295 In comparison, HaloTag can be employed to rapidly purify proteins and the isolated proteins may further enable multimodal molecular imaging. Preliminary evidence has demonstrated the feasibility of HaloTag-based pretargeted imaging. This strategy involves first administering a HaloTag-modified antibody for pretargeting and then a small radiolabeled molecule with a short circulation time for imaging.²⁹⁶

Analogous to these, Schibli et al. reported that microbial transglutaminase (mTGase) can modify mAbs in a stoichio-metric manner and the site-specifically engineered mAbs are of particular interest for molecular imaging.^297,298 Another bacterial enzyme lipoic acid ligase (LplA) was also used with [¹⁸F]fluorooctanoic acid ([¹⁸F]FA) for site-specific radio-labeling of a Fab fragment.²⁹⁹ Although mTGase and LplA have shown potential in mediating site-specific radiolabeling of biomolecules, their robustness needs to be confirmed by future studies.

3.3.4. Pretargeted ImmunoPET Imaging Strategies.

The slow blood clearance of mAb is problematic because it leads to high background activity and radiation exposure, especially to the red bone marrow. Aside from reducing the molecular size and removing or blocking the Fcγ receptors (FcγRs), pretargeted immunoPET imaging holds promise to improve the imaging quality while decreasing radiation exposure.³⁰⁰ In addition, this imaging approach enables the use of short-lived PET radionuclides (e.g., the widely available ⁶⁸Ga and ¹⁸F).³⁰¹ Pretargeted imaging was initially achieved using the biotin–streptavidin interaction and BsAbs.^302,303 In the avidin–biotin pretargeting approach, a streptavidinmodified immunoconjugate, and radiolabeled biotin were used.^304,305 Streptavidin and biotin-based pretargeting systems have been investigated in several clinical studies.^306–308 However, the bacterially derived streptavidin constructs are prone to have immunogenicity, which may limit repeated imaging or therapy.³⁰⁹ A second concern for this system is that endogenous biotin in patient blood and tissues may competitively occupy and block the binding sites of streptavidin, thus preventing the binding of radiolabeled biotin.

In a BsAb-based pretargeted imaging system, a BsAb that can bind to target antigen and radiolabeled hapten is first injected, enabling the saturation of the target and washing out of the unbound antibodies. Once the unbound antibody is cleared from the blood and normal tissues, a radiolabeled chelate is injected and a portion will be captured by the BsAbs at the tumor sites with the remainder eliminated rapidly from the body. This strategy has been refined over the years and used in clinical studies for pretargeted radioimmunotherapy (pRIT),³¹⁰ as well as for pretargeted immunoSPECT and immunoPET imaging.^311–314 Traditionally, BsAbs used for pretargeted imaging and therapy were produced by chemical methods^315,316 or by recombinant expression from Escherichia coli or myeloma cell cultures.^317,318 Although several clinical studies have validated the therapeutic effect of pRIT using the antibodies produced this way,^319,320 the murine or chimeric property of such agents limit their clinical use. To further advance the clinical translation and application of pretargeted imaging and therapy, a more innovative Dock-and-Lock method is now being used to develop humanized recombinant BsAbs on a large scale.³²¹ One such example is the anti-CEA × anti-HSG TF2 BsAb, which contains a humanized antihist-amine-succinyl-glycine (HSG) Fab fragment from the anti-HSG mAb 679 and two humanized anti-CEA Fab fragments derived from the hMN14 mAb (labetuzumab).^219,321 As the hapten peptides of the pretargeted system, the radiolabeled small molecules bear two HSG groups and various chelators (Figure 11), allowing versatile labeling with radionuclides of interest (e.g., ⁶⁸Ga and [¹⁸F]AlF). Currently, several other such BsAbs (e.g., TF4 for CD20 and TF10 for a mucin antigen) produced via this approach are also being actively investigated for theranostic purposes.^322–324 Using the directed evolution and yeast surface display,³²⁵ Orcutt and coauthors at the Massachusetts Institute of Technology (MIT) constructed another pretargeted system,^326,327 which exploits a principle similar to the streptavidin–biotin system but replaces streptavidin with a C825 scFv capturing benzyl(Bn)-DOTA-radiometal complex. In this system, use of a clearing agent (e.g., dextran-(Y)-DOTA-Bn conjugate) further improved the imaging quality and therapeutic outcome.^328,329

Figure 11.

Chemical structures of DOTA-HSG and NOTA-HSG hapten peptides used in pretargeted immunoPET imaging.

In recent years, bioorthogonal chemical reactions have been developed as alternatives to biologic pretargeting interactions for recruiting radiolabeled probes to the tumor-bound mAb, as excellently described in several reviews.^330–332 Since its initial report,²⁶⁶ the IEDDA reaction has been widely used for pretargeted tumor imaging. Various TCO-conjugated antibodies and ¹¹¹In-, ¹⁸F-, or ⁶⁴Cu-labeled Tz probes have been used to achieve pretargeted imaging.^333–335 These novel strategies substantially improved T/B ratios and reduced radiation dose to the bone marrow. Ideal imaging results were achieved when the tagged biomolecules (e.g., TCO-modified mAb) were completely cleared from the circulation before injecting the radiolabeled Tz, which could be accomplished by injecting a clearing agent (e.g., Tz-galactose-albumin).^336,337 Furthermore, sequential use of the enzyme-mediated site-specific modification and click chemistry produced improved imaging results.³³⁸ On the basis of the available evidence, the added value of this strategy is confined to the production of homogeneous immunoconjugate and reduced use of the antibody because the imaging performance was comparable to that of the randomly labeled analogous construct.³³⁹ Along with this, several other Tz-modified chelators have been developed for labeling peptides with radiometals,^340,341 but their performance with antibodies remains to be determined. It is also notable that some chemical reactions are not suitable for in vivo pretargeted imaging due either to the interactions of the radioligand with serum albumin or to the slow reaction kinetics.^342,343

As discussed above, high-affinity Affibodies have been successfully applied for molecular imaging of cancers. The unfavorable part is that their rapid renal clearance, reabsorption, and internalization unavoidably lead to high accumulation of the radiotracers in the proximal tubule of the kidneys. Pretargeted imaging approaches have been harnessed to overcome this disadvantage.^344–347 One such approach is the peptide nucleic acid (PNA)-mediated hybridization system, where the primary PNA strand used for tumor targeting can selectively and rapidly hybridize with the secondary complementary PNA strand equipped with radio-nuclides. For instance, Vorobyeva et al. developed a modular system consisting of Z_HER2:342-SR-HP1 (primary targeting agent) and 68Ga-HP2 (secondary targeting agent).³⁴⁸ They reported that this Affibody-based imaging approach yielded increased tumor uptake and decreased kidney uptake in preclinical ovary cancer models.

3.3.5. Clearance-Enhanced ImmunoPET Imaging.

Other than the above-mentioned methods for increasing image contrast and improving image quality, use of urokinase and a urokinase-cleavable bifunctional chelator (CB-TE1A1PUSL-DBCO) is a promising radionuclide clearance enhancement system.³⁴⁹ This system has been used to develop ⁶⁴Cu-CB-TE1A1P-USL-trastuzumab,¹⁵⁶ where injection of urokinase triggers urokinase-responsive cleavage of the radiotracer, leading to enhanced elimination of radioactivity from the blood circulation, enhanced hepatic radioactivity clearance, and significantly increased tumor-to-blood ratio. These studies indicate that urokinase and urokinase substrate linkers can be used to induce clearance of radioactivity from the nontargeted tissues and shorten the time required to obtain optimal immunoPET imaging contrast.

4. IMMUNOPET IMAGING OF CANCERS

The major application of immunoPET imaging is to facilitate better management of cancer patients. According to several clinical reports, immunoPET imaging provided excellent specificity and sensitivity in detecting primary tumors.^184,186 ImmunoPET imaging is also an appealing option for detecting lymph node and distant metastases.^118,350 More importantly, accumulating clinical evidence suggests that immunoPET can detect previously unknown lymph node and distant meta-stases.^39,41 These impressive results indicate that immunoPET may complement IHC staining in clinical dilemmas when suspected lesions are inaccessible for biopsy. However, suboptimal imaging conditions (e.g., imaging protocol and facility performance) and low expression of the target in small tumor lesions may lead to underestimation of the tumor burden and target abundance. Following immunoPET imaging, patients with positive findings can be selected for subsequent therapies (e.g., antibody therapy and antibody-based RIT), whereas patients with negative or heterogeneous findings may need multidisciplinary treatments. ImmunoPET is a useful diagnostic tool but also a theranostic companion for radiation dosimetry prior to administering the therapeutic radiopharmaceuticals (discussed in section 8). Moreover, immunoPET imaging is useful for improved triage during early disease stages and to facilitate image-guided surgery.^351,352 The information provided by immunoPET will significantly enhance the existing diagnostic methods for better tumor characterization. One can envision that tumors may be classified not only according to their origins and mutation status but also according to the expression of specific tumor antigens in the future.

4.1. Receptor Tyrosine Kinases

Receptor tyrosine kinases (RTKs) are often overexpressed and/or mutated in a variety of cancers.³⁵³ As the best-studied oncogenic drivers, RTKs have been among the most explored targets for developing anticancer therapeutics. Indeed, mAbs and small-molecule tyrosine kinase inhibitors (TKIs) suppressing RTKs or their ligands are the most typical examples of targeted cancer therapies. Along with this success, substantial efforts have been dedicated to developing immunoPET imaging approaches for revealing the heterogeneous status of RTKs in cancers.³⁵⁴

4.1.1. Epidermal Growth Factor Receptor.

Human epidermal growth factor receptor (EGFR) is a RTK regulated by at least seven activating ligands in humans.³⁵⁵ Several mAbs (e.g., cetuximab, panitumumab, and nimotuzumab) targeting the extracellular domain of EGFR and TKIs (e.g., erlotinib) targeting the intracellular domain of EGFR have been approved for treating EGFR-positive cancers. An initial clinical study demonstrated that ⁸⁹Zr-Df-cetuximab immunoPET imaging could visualize EGFR expression and predict the treatment efficacy of cetuximab in advanced colorectal cancers.³⁵⁶ However, a follow-up study showed that ⁸⁹Zr-Dfcetuximab uptake failed to predict the efficacy of cetuximab monotherapy in patients with RAS wild-type metastatic colorectal cancer.^{357 89}Zr-Df-cetuximab was further investigated in nine patients with head and neck squamous cell carcinoma (HNSCC) or nonsmall-cell lung cancer (NSCLC). The results showed no direct relationship between EGFR expression and tumor uptake of the radiotracer in terms of the T/B ratio,³⁵⁸ which was in concert with the results reported by two other studies.^359,360 Because cetuximab irreversibly binds to EGFR expressed in liver cells, van Loon et al. reasoned that an optimal preloading of unlabeled cetuximab is needed to first saturate liver EGFRs.³⁵⁸ In addition, Pool et al. found that shed EGFR ectodomain levels in liver and plasma interfere with EGFR-targeted immunoPET imaging agents, and increased administration of radiotracer could improve tumor visualization.³⁶¹

Panitumumab is a fully human mAb targeting EGFR.³⁶² Niu et al. initially reported that ⁶⁴Cu-DOTA-panitumumab immunoPET imaging failed to quantify EGFR protein expression in three different HNSCC xenografts,³⁶³ probably due to the poor penetration of the antibody and varying tumor vasculature in the used models. However, several other studies showed that uptake of ⁸⁹Zr-panitumumab was associated with EGFR expression in other tumor models.^364–366 Additionally, a group reported the clinical safety and feasibility of ⁸⁹Zrpanitumumab immunoPET in noninvasively characterizing EGFR expression.^367,368 Scott et al. screened a variety of mouse mAbs and found that mAb 806 specifically targets the overexpressed or activated forms of EGFR.^369,370 ch806, a chimeric form of mAb 806, has been validated as an effective therapeutic antibody and ch806-based molecular imaging showed specific accumulation of the antibody at multiple tumor sites.^371,372 In an attempt to trap the radioiodinated ch806 in lysosomes, ch806 was further radiolabeled with residualizing peptides ¹²⁴I-IMP-R4 and ¹²⁴I-PEG₄-tptddYddtpt. The tumor uptake of ¹²⁴I-IMP-R4-ch806 (Figure 12) and ¹²⁴I PEG₄-tptddYddtpt-ch806 was apparent in preclinical glioma models.^373,374 More recently, two other preclinical studies have demonstrated the value of ⁸⁹Zr-Df-nimotuzumab immunoPET diagnosing epidermoid carcinomas and gliomas.^375,376

Figure 12.

ImmunoPET imaging of EGFR expression using ¹²⁴I-labeled residualizing radiotracer. (a–c) ¹²⁴I-IMP-R4-ch806 immuno-PET imaging clearly delineated EGFR-positive gliomas with negligible uptake in normal tissues. Reproduced with permission from ref 373. Copyright 2010 SNMMI.

Affibody-based PET imaging probes are also being developed to image EGFR expression.^377–381 Burley et al. developed two Affibody-based EGFR-targeting radioligands ⁸⁹Zr-DFO-Z_EGFR:03115 and ¹⁸F-AlF-NOTA-Z_EGFR:03115).³⁸² The authors found that 18F-AlF-NOTA-Z_EGFR:03115 PET imaging could correlate EGFR downregulation in response to cetuximab treatment in preclinical HNSCC models. These probes may have clinical utility because of the poor performance of ⁸⁹Zr-Df-cetuximab and the dichotomous role ⁶⁴Cu-DOTA-panitumumab in mapping EGFR levels. Although the development of next-generation EGFR-targeting mAbs may overcome cetuximab-induced resistance,^383,384 VHH-based EGFR-targeting therapeutics may also overcome cetuximab-induced resistance. One such VHH, 7D12, penetrates more deeply and homogeneously into tumors than cetuximab.^385,386 Therefore, 7D12-based nuclear medicine imaging approaches may serve as promising tools in selecting patients suitable for VHH-based therapies.^387,388

4.1.2. Human Epidermal Growth Factor Receptor 2.

Human epidermal growth factor receptor 2 (HER2/ErbB2) has attracted much interest as a molecular imaging target in the past two decades. Along with the clinical approval of HER2-targeted antibody therapeutics (e.g., trastuzumab, trastuzumab emtansine [T-DM1], and pertuzumab), several antibody-based radiotracers have been developed for imaging HER2 expression.³⁸⁹ Of them, two initial clinical studies using ¹¹¹In-DTPA-trastuzumab reported uptake of the tracer in the myocardial wall and detection of new HER2-positive breast cancer lesions.^16,390 Since ⁸⁹Zr became clinically available,¹¹⁸ successive translational studies have reported the value of ⁸⁹Zr-Df-trastuzumab immunoPET in detecting both previously known and unknown metastatic breast cancer lesions,⁴¹ detecting heterogeneous HER2 expression in breast cancer lesions before T-DM1 treatment and predicting T-DM1 treatment outcomes.^{391–393 64}Cu-DOTA-trastuzumab is an alternative that has also been tested in the clinic.^394,395 In addition to trastuzumab, pertuzumab is another FDA-approved mAb targeting HER2. ⁸⁹Zr-Df-pertuzumab has been successfully translated into the clinic and ⁸⁹Zr-Df-pertuzumab immunoPET imaging was able to detect primary breast cancers and distant breast cancer metastases including brain metastases (Figure 13a–d).³⁹⁶ However, no clinical studies have directly compared the diagnostic efficacies of ⁸⁹Zr-Dftrastuzumab and ⁶⁴Cu-DOTA-trastuzumab.

Figure 13.

ImmunoPET imaging of HER2 expression. (a) T1-weighed MR imaging of a 46-year-old woman showed brain metastases from breast cancer (red arrows). (b–d) ⁸⁹Zr-Dfpertuzumab immunoPET/CT imaging of the same patient demonstrated varying uptake of the radiotracer in brain metastases (red arrows) and minimal uptake in the superior sagittal sinus (red arrowhead). Reproduced with permission from ref 396. Copyright 2018 SNMMI. (e) Chemical structure of [¹⁸F]AlF-NOTA-Tz-TCOGK-2Rs15d. (f) ImmunoPET/CT imaging of a human ovarian cancer xenograft at 2 and 3 h after injection of [¹⁸F]AlF-NOTA-Tz-TCOGK-2Rs15d. Reproduced with permission from ref 416. Copyright 2018 American Chemical Society.

Increasing evidence supports HER2 as a broad tumor biomarker beyond its established role in breast cancers.³⁹⁷ Preliminary studies have reported the value of HER2-specific immunoPET in elucidating HER2 expression levels in gastric cancer and esophagogastric adenocarcinoma.^398–400 Because HER2 serves as a biomarker for ovarian cancer and also potentially for advanced thyroid cancers, it is rational that HER2-targeted immunoPET imaging was able to map HER2 expression in these solid tumors.^401–404

Although a combination of 18F-FDG PET and ⁸⁹Zr-Dftrastuzumab immunoPET robustly predicted the treatment efficacy of T-DM1, ⁸⁹Zr-Df-trastuzumab did not accumulate in a proportion of HER2-positive lesions.³⁹¹ Temporal modulation of HER2 expression with mucolytic treatment enhanced tumor uptake of ⁸⁹Zr-Df-trastuzumab in a preclinical breast cancer model.⁴⁰⁵ Moreover, it has been shown that caveolin-1 mediates HER2 internalization and depletion of caveolin-1 with lovastatin increased tumor uptake of ⁸⁹Zr-DFO-trastuzumab.⁴⁰⁶ This effect was further validated by a more recent study where oral administration of lovastatin enhanced tumor accumulation of ⁸⁹Zr-DFO-pertuzumab in preclinical gastric cancer models.⁴⁰⁷ More importantly, image-guided modulation of HER2 expression and internalization could improve the efficacy of trastuzumab treatment.⁴⁰⁸ These results together demonstrate that modulation of HER2 expression or internalization could increase tumor uptake of HER2-targeted immunoPET probes and also the efficacies of HER2-targeted therapeutic regimens.

Small biomolecules (e.g., antibody fragments, sdAbs, and Affibodies) are also being used as HER2-targeting vectors.^409,410 Beylergil et al. developed 68Ga-DOTA-F(ab′)_2-trastuzumab and investigated the diagnostic utility of this radiotracer in 15 patients with breast cancer. Although less optimal than radiolabeled trastuzumab, this imaging approach detected diseases in 4/8 patients with HER2-positive breast cancers.⁴¹¹ 2Rs15d is a sdAb developed against HER2 and has shown excellent targeting of HER2 in both preclinical settings,⁶³ and clinical settings.⁴¹² In both cases, 68Ga was randomly conjugated on Lys residues of 2Rs15d without compromising the targeting affinity of 2Rs15d, in part due to the absence of Lys residues in its antigen-binding domains. (Note: 2Rs15d contains six lysines that are dispersed in the framework and away from the receptor-binding regions.) A recent study revealed that SrtA-mediated site-specific radiolabeling of 2Rs15d yielded homogeneous ⁶⁸Ga-NOTA-2Rs15d, and immunoPET imaging with this radiotracer readily visualized HER2-positive breast cancers.⁴¹³ However, the renal clearance of ⁶⁸Ga-NOTA-2Rs15d and the resultant high tracer retention in bilateral kidneys is problematic, which was also observed in several other 18F-labeled VHHs.^202,414 In this setting, two HER2-specific VHHs (i.e., 2Rs15d and 5F7) were radiolabeled with [¹⁸F]TFPFN and subsequent immunoPET imaging with the synthesized probes successfully detected tumors with prominent tumor uptake and substantially lower renal uptake.⁴¹⁵ Furthermore, [¹⁸F]AlF-NOTA-Tz-TCO-GK-2Rs15d was developed by the IEDDA reaction and incorporation of a renal brush border enzyme-cleavable Gly-Lys (GK) linker in the prosthetic moiety. This strategy achieved quite high RCY (~17%) while also maintaining high T/B ratios (Figure 13e,f).⁴¹⁶ 2Rs15d has been validated useful as a vehicle for RIT after labeling with ¹⁷⁷Lu or ¹³¹I,^67,417 providing valuable therapeutic options accompanying the above-mentioned immunoPET imaging. A clinical trial evaluating the safety and distribution of ¹³¹I-SGMIB-2Rs15d in breast cancers has completed patient recruitment (NCT02683083).

Z_HER2:342 is a second-generation HER2-targeting Affibody and has been radiolabeled nonselectively with ¹²⁵I and ¹¹¹In^418,419 or site-specifically with ¹⁸F-FBEM.^205,420,421 Xu et al. further modified Z_HER2:342 with a hydrophilic linker (Cys-Gly-Gly-Gly-Arg-Asp-Asn) that is conjugated with maleimidomonoamide-NOTA and radiolabeled the derivative with Al¹⁸F.⁴²² The authors found that this modification produced excellent imaging contrast and low abdomen uptake of the developed tracer. DOTA-Z_{HER2:342‑pep2} (ABY-002) is a chemically synthesized derivative of Z_HER2:342 with a DOTA coupled to its NH₂ terminus. Therefore, this agent could be site-specifically radiolabeled with 111In or 68Ga.^423–425 Furthermore, 111In-ABY-002 and 68Ga-ABY-002 demonstrated the potential in visualizing HER2-expressing metastatic lesions in patients with breast cancer.⁴²⁶ Another group of HER2-specific molecular imaging tracers is based on the Affibody MMA-DOTA-Cys⁶¹-Z_HER2:2891-Cys (ABY-025).^427–429 Of them, ⁶⁸Ga-ABY-025 can be produced in compliance with Good Manufacturing Practice (GMP)⁴³⁰ and can be used to accurately image HER2 expression in metastatic breast cancers.^431–434 Because ¹⁸F is a radionuclide validated for clinical use and its longer half-life enables imaging over a longer time window compared to ⁶⁸Ga, Glaser et al. used three methods (i.e., ¹⁸F-SiFA, ¹⁸F-AlF-NOTA, and ¹⁸F-FBA) to radiolabel ABY-025. They found that ¹⁸F-FBA-ZHER2:2891 (GE226) emerged as a highly specific candidate for imaging HER2 expression in pre- and post-treatment settings.^435,436 Z_HER2:2395 is another variant of Z_HER2:342 and has a C-terminal Cys.⁴³⁷ This Affibody molecule was site-specifically labeled with Al¹⁸F-NOTA (90 °C, 15 min) with an acceptable RCY (21% ± 5.7%).⁴³⁸

Aiming to improve the therapeutic effect of HER2-targeted therapies, BsAbs are being increasingly produced,^439–442 which may serve as appealing immunoPET imaging components. Because of the progress in preclinical and clinical studies, we are optimistic that HER2-targeted immunoPET imaging will provide a noninvasive and dynamic visualization and quantification of HER2 expression in heterogeneous tumors, which will refine clinical management of HER2-targeted therapeutics. Apart from detecting the heterogeneous HER2 expression in tumor tissues for initial patient selection,⁴⁴³ HER2-specific immunoPET may serve as a useful tool in predicting therapeutic responses following HER2-targeted therapies.^444,445 This will be especially useful when the applied HER2-specific vector binds to epitopes different from that of the clinically approved antibodies (e.g., trastuzumab and pertuzumab).⁴⁴⁶

4.1.3. Human Epidermal Growth Factor Receptor 3.

Human epidermal growth factor receptor 3 (HER3/ErbB3) has weak intracellular kinase activity and does not form homodimers, but it is a signal amplifier after forming heterodimers with other EGFR family members (i.e., HER2 and EGFR). It has been well established that HER3 is related to the development and progression of several types of cancers.⁴⁴⁷ With the broad clinical application of HER2- and EGFR-targeting agents, increasing evidence indicates that HER3 is also implicated in the resistance of HER2- or EGFR-targeting therapeutics.⁴⁴⁸ As such, more than 13 HER3-targeting mAbs (e.g., patritumab, lumretuzumab, KTN3379, REGN1400) are under clinical investigation as either monotherapy agents or components of combination therapies.⁴⁴⁹ Additionally, duligotuzumab (MEHD7945A) is a human IgG1 mAb dually targeting HER3 and EGFR.^450,451

Patritumab is a fully human anti-HER3 mAb and has shown to have an excellent safety profile in clinical trials. Although a preclinical study showed the feasibility of ⁶⁴Cu-DOTA-patritumab immunoPET in imaging HER3 expression,⁴⁵² the clinical use of ⁶⁴Cu-DOTA-patritumab was not satisfactory because tumor uptake of 64Cu-DOTA-patritumab was not robust.⁴⁵³ The discrepancy observed in these two studies may possibly be caused by the substantial uptake of the radiotracer in human livers and less potent targeting property of the antibody. GSK2849330 is another fully human HER3-specific mAb and dose-dependent, saturable uptake of the agent was reported in a preclinical study where ⁸⁹Zr-GSK2849330 was employed.⁴⁵⁴ More recently, van Oordt et al. characterized the value of ⁸⁹Zr-GSK2849330 immunoPET in clinical settings.⁴⁵⁵ The authors validated ⁸⁹Zr-Df-GSK2849330 saturation after preloading with unlabeled GSK2849330 and also observed a significant uptake of the tracer in the liver and spleen. Lumretuzumab (RG7116) is a humanized mAb targeting the extracellular domain of HER3.⁴⁵⁶ ImmunoPET imaging with ⁸⁹Zr-lumretuzumab provided useful information on HER3 expression in multiple tumor-bearing mouse models.⁴⁵⁷ A follow-up clinical study using ⁸⁹Zr-lumretuzumab further demonstrated tumor uptake of the tracer.⁴⁵⁸ The study also revealed significant liver uptake of the tracer, which was partially caused by Kupffer cell-mediated capture and clearance of the glycoengineered antibody.

Several other HER3-targeting vectors, including HER3 specific antibody fragments and Affibody, have been developed and employed as PET and fluorescent imaging probes.^459–462 However, probes of long retention time are favored for imaging HER3 because this receptor has a low density on the tumor cells but high abundance in the normal organs and tissues, such as the small intestine. Furthermore, Affibody-based imaging probes have very fast blood clearance due to their small sizes (~7 kDa), which results in substantial kidney retention that interferes with image interpretation.^463,464 To this end, a biparatopic VHH construct MSB0010853 (39.5 kDa), which blocks two different HER3 epitopes, was developed (Figure 14a).⁴⁶⁵ Warnders et al. reported that tumor uptake of ⁸⁹Zr-MSB0010853 correlated with HER3 expression (Figure 14b), and its uptake in tissues was dose-dependent. Owing to the relatively larger size and the albumin-binding capacity of ⁸⁹Zr-MSB0010853, bloodstream circulation of this tracer was relatively longer and renal uptake is relatively lower (<15% ID/g).⁴⁶⁵

Figure 14.

ImmunoPET imaging of HER3 expression. (a) MSB0010853 is composed of two Nanobodies targeting two different epitopes of HER3 and an additional Nanobody targeting albumin. (b) ⁸⁹Zr-MSB0010853 immunoPET imaging of HER3-positive mouse xenografts (H441 and FaDu) and HER3-negative mouse xenograft (Calu-1) demonstrated the ability of this imaging approach to reveal varying HER3 expression levels. Reproduced with permission from ref 465. Copyright 2017 SNMMI.

4.1.4. Vascular Endothelial Growth Factor Receptor.

Vascular endothelial-derived growth factor (VEGF)/VEGF receptor (VEGFR) signaling pathway is the key pathway regulating vasculogenesis and angiogenesis during physiologic homeostasis and diseases.⁴⁶⁶ A number of therapeutic agents targeting VEGF (e.g., bevacizumab and ramucirumab) and VEGFR (e.g., sorafenib and sunitinib) have been approved for clinical use around the world.⁴⁶⁷ As a neutralizing mAb targeting VEGF-A, the benefits of bevacizumab have been validated for different oncological indications.⁴⁶⁸ To date, clinical immunoPET studies using ⁸⁹Zr-Df-bevacizumab were performed in a variety of tumors, including breast cancer,⁴⁶⁹ neuroendocrine tumors,⁴⁷⁰ renal cell carcinoma (RCC),⁴⁷¹ NSCLC,⁴⁷² and glioma.^{473,474 89}Zr-Df-bevacizumab immuno-PET imaging detected VEGF-A downregulation induced either by the mammalian target of rapamycin inhibitor (everolimus) in patients with neuroendocrine tumors,⁴⁷⁰ or by the HSP90 inhibitor, luminespib (NVP-AUY922), in ovarian cancer xenografts.⁴⁷⁵ However, clinical ⁸⁹Zr-Df-bevacizumab immunoPET imaging failed to monitor VEGF reduction in patients with breast cancer following NVP-AUY922 treatment.⁴⁷⁶ To enhance the penetration of bevacizumab across the blood–brain barrier (BBB), intra-arterial administration and blood–brain barrier opening (BBBO) are two emerging strategies.⁴⁷⁷ In agreement with this clinical evidence, BBBO with mannitol followed by intra-arterial administration of ⁸⁹Zr-Df-bevacizumab resulted in significantly higher accumulation of the tracer in the ipsilateral hemisphere.⁴⁷⁸

To gain a more thorough insight into tumor response following antiangiogenic treatment, ranibizumab (a humanized Fab fragment targeting all isoforms of VEGF-A) was radiolabeled with ⁸⁹Zr.⁴⁷⁹ Uptake of ⁸⁹Zr-ranibizumab in the tumor center reduced substantially following sunitinib treatment. However, immunoPET scanning performed 7 days after the termination of the treatment showed that tracer accumulation in the tumor centers increased and returned to baseline. It is worthwhile to note that uptake of ⁸⁹Zr-Dfbevacizumab in RCC and normal organs may also rebound following sunitinib treatment,⁴⁷¹ but a similar phenomenon was not observed following sorafenib or everolimus treatment of RCC.^480,481 All of the clinical evidence indicates that expression of VEGF not only varies in different patients but also among the metastases and within the tumor in a single patient. VEGF-directed immunoPET is useful for visualizing the dynamic changes of VEGF before and after VEGF-targeted therapies. It has been postulated that antiangiogenic therapies induce the apoptosis of the endothelial cells and “normalize” the hyper-permeability of the tumor vasculature.⁴⁸² Therefore, disruption of the tumor vasculature may lead to reduced tumor uptake of the radiotracer, regardless of the VEGF levels. This factor should be taken into consideration when interpreting the imaging results.

Although VEGF-A binds to both VEGFR-1 and VEGFR-2, VEGFR-2 plays a key role in regulating angiogenesis and vascular permeability. Results from several preclinical studies demonstrated that it was also feasible to image tumor vasculature by targeting VEGFR-2.^483–485

4.1.5. Other Receptor Tyrosine Kinases.

Other than the EGFR family members, there are many other RTKs implicated in the pathogenesis and progression of cancers and non-cancerous diseases.⁴⁸⁶ Of them, c-MET (also known as mesenchymal–epithelial transition factor), platelet-derived growth factor (PDGF) receptor (PDGFR), and insulin-like growth factor-1 receptor (IGF-1R) are emerging therapeutic and diagnostic targets.

c-MET is the receptor for the hepatocyte growth factor (HGF). The HGF/c-MET pathway is vital for the development and metastatic progression of gastrointestinal cancers,⁴⁸⁷ NSCLC,⁴⁸⁸ and several other malignancies.⁴⁸⁹ Furthermore, c-MET is closely related to the acquired resistance of EGFR-targeted or VEGFR2-targeted therapies in a broad range of solid tumors.⁴⁹⁰ Current efforts are directed to validate c-MET as a biomarker and HGF/c-MET pathway inhibitors as anticancer therapeutics. However, several clinical trials have failed to demonstrate the synergistic effect of c-MET or HGF inhibitors in combination therapies, such as onartuzumab plus erlotinib in NSCLC,⁴⁹¹ and rilotumumab plus epirubicin, cisplatin, and capecitabine in gastric or gastro-esophageal junction adenocarcinoma.⁴⁹² Therefore, development and clinical translation of companion diagnostic probes may underpin clinical investigation of HGF/c-MET pathway inhibition for cancer therapies. Luo et al. produced a recombinant human HGF (rh-HGF) and demonstrated that ⁶⁴Cu-NOTA-rh-HGF PET imaging indirectly visualized c-MET-positive human glioblastoma in mouse models.⁴⁹³ Using a fully human mAb rilotumumab (AMG102) which selectively targets HGF, Price et al. developed ⁸⁹Zr-DFO-AMG102 and reported that immunoPET imaging with this tracer determined HGF in the local tumor microenvironment (TME).⁴⁹⁴ To directly delineate c-MET abundance, several radiotracers have been developed and tested in preclinical mouse models.^495,496 Onartuzumab is a one-armed monovalent antibody targeting the c-MET.⁴⁹⁷ Pool et al. demonstrated that ⁸⁹Zr-Dfonartuzumab immunoPET imaging could visualize erlotinib-induced c-MET upregulation and luminespib-induced c-MET downregulation in NSCLC models.⁴⁹⁸ Escorcia et al. reported the theranostic value of ⁸⁹Zr-Df-onartuzumab and 177Lu-DTPA-onartuzumab in pancreatic ductal adenocarcinoma (PDAC) xenograft models. In this theranostic scenario, immunoPET imaging with ⁸⁹Zr-Df-onartuzumab identified c-MET-positive PDAC xenografts and targeted radioligand therapy with ¹⁷⁷Lu-DTPA-onartuzumab efficiently delayed the growth of the selected tumors.⁴⁹⁹ A more recent study by Klingler et al. reported the synthesis of ⁸⁹Zr-DFO-azepinonartuzumab within 10 min via the one-pot photoradiochemical conjugation reaction.⁵⁰⁰ When compared to the conventional ⁸⁹Zr-DFO-Bn-NCS-onartuzumab, immunoPET imaging with ⁸⁹Zr-DFO-azepin-onartuzumab resulted in comparable T/B ratios but a lower uptake in the liver. These results highlight the feasibility of immunoPET in assessing the dynamics of the HGF/c-MET signaling pathway and selecting candidates most likely to benefit from HGF/c-MET-targeted therapies.

PDGF and PDGF receptors (i.e., PDGFRα and PDGFRβ) stimulate the growth of tumor cells and regulate tumor angiogenesis as well as tumor stromal fibroblasts.^501,502 Although no PDGF-specific TKIs have been approved, olaratumab (LY3012207, IMC-3G3), a human IgG1 mAb targeting PDGFRα, was approved by the FDA in 2016 for treating soft-tissue sarcomas.⁵⁰³ To directly image stromal PDGFR, PDGFRβ-targeting Affibodies have been radiolabeled with ¹¹¹In or ⁶⁸Ga and tested in preclinical glioma models.^504–506 By using a dual cysteine disulfide bond linker, a dimeric Affibody molecule Z_PDGFRβ was produced and ⁸⁹Zr-DFO-Z_PDGFRβ immunoPET imaging visualized PDGFRβ-expressing colorectal adenocarcinomas.⁵⁰⁷ Overexpression of PDGFRa has been reported to be associated with lymph node metastases of papillary thyroid cancers.^508–511 Accordingly, Wagner et al. developed ⁶⁴Cu-NOTA-D13C6 and reported that immunoPET imaging with this tracer identified papillary thyroid cancers that have the potential to metastasize (Figure 15).⁵¹²

Figure 15.

ImmunoPET imaging of PDGFRa expression using ⁶⁴Cu-NOTA-D13C6. While lower uptake of ⁶⁴Cu-NOTA-D13C6 was seen in the PDGFRα-negative B-CPAP tumor (left flank), higher accumulation of the radiotracer was observed in the transfected PDGFRα-positive B-CPAP tumor (right flank) at late time-points. Reproduced with permission from ref 512. Copyright 2018 Elsevier Inc.

IGF-1R is universally expressed in hematologic and solid tumors.^513,514 Insulin-like growth factors and IGF-1R are attractive targets for cancer therapy and imaging.⁵¹⁵ R1507 is a mAb targeting IGF-1R and molecular imaging with R1507-based radiotracers, ¹¹¹In-R1507 and ⁸⁹Zr-Df-R1507, successfully determined IGF-1R expression in breast cancer xenografts.⁵¹⁶ We screened an IGF-1R-specific mAb 1A2G11 and developed an immunoPET probe ⁸⁹Zr-Df-1A2G11,⁵¹⁷ which specifically accumulated in IGF-1R-positive pancreatic cancers.⁵¹⁸ In accordance with the antibody-based imaging findings, an Affibody-based immunoPET tracer also specifically delineated U87MG tumors with enhanced clearance from the renal-urine system.⁵¹⁹ These preclinical results imply that IGF-1R-specific immunoPET may help identify patients that would benefit from anti-IGF-1R therapies and enable dynamic monitoring of the IGF-1R expression following the therapies.⁵²⁰

4.2. Clusters of Differentiation

Clusters of differentiation (CD) antigens have long been investigated as either diagnostic or therapeutic targets for a broad spectrum of diseases. In this review, we will describe in-depth some selected markers (i.e., CD20, CD38, CD146, and CD105). However, there are many other CD antigens that have shown potential as molecular imaging targets, including CD54 (known as intercellular adhesion molecule, ICAM-1),⁵²¹ CD44,^{118,522–524} CD47,^525–527 and CD138.^528,529

4.2.1. CD20.

Lymphoma is an umbrella term for a large group of cancers that often arise from the lymph nodes. CD20 and CD30 are two common biomarkers for molecular imaging of lymphoma.^530–532 Rituximab, a CD20-specific chimeric mAb, was approved by the FDA for the treatment of non-Hodgkin’s lymphoma (NHL) in 1997 and rheumatoid arthritis (RA) in 2006.⁵³³ Studies have reported the feasibility of ⁶⁴Cu-DOTA-rituximab and ⁸⁹Zr-rituximab immunoPET in revealing CD20 expression in NHL-bearing humanized mouse models.^534,535 Of them, ⁸⁹Zr-rituximab has been translated for clinical use. Muylle et al. have reported that ⁸⁹Zr-rituximab immunoPET/CT scanning without preloading of cold rituximab enabled clearer tumor visualization and higher tumor uptake (Figure 16).⁵³⁶

Figure 16.

ImmunoPET imaging of non-Hodgkin’s lymphomas. (a) In a patient with circulating CD20⁺ lymphocytes, significant uptake of ⁸⁹Zr-rituximab was observed in the spleen, which was blocked by preloading with unlabeled rituximab (250 mg/m²) prior to injection of ⁸⁹Zr-rituximab. The spleen is indicated with black arrows. (b) In the same patient, preloading reduced ⁸⁹Zr-rituximab uptake in the involved lymph nodes (white arrows), but enhanced uptake of the radiotracer in the visceral lesions (blue arrows). Reproduced with permission from ref 536. Copyright 2015 Springer Berlin Heidelberg.

Recently, antibody fragments targeting human CD20 have been engineered and investigated after being labeled with ¹²⁴I, ⁸⁹Zr, or ⁶⁴Cu. Olafsen et al. engineered two rituximab fragments of different sizes (a Mb of 80 kDa and a scFv-Fc fragment of 105 kDa). The authors reported that both fragments offered CD20-specific imaging, but the Mb-based radiotracer resulted in images of higher contrast.⁵³⁷ Humanized obinutuzumab (GA101) and fully human ofatumumab are two other CD20-targeting mAbs showing superior activity when compared to rituximab.⁵³⁸ Yoon et al. radiolabeled these two mAbs with ⁸⁹Zr and compared their diagnostic efficacies with ⁸⁹Zr-rituximab.⁵³⁹ The authors reported that both ⁸⁹Zrobinituzumab and 89Zr-ofatumumab localized lymphoma xenografts more clearly than 89Zr-rituximab. Zettlitz et al. further engineered antibody fragments from obinutuzumab and radiolabeled these CD20-specific vectors with ⁸⁹Zr, ¹²⁴I, and ¹⁸F.^197,540 Similarly, the authors found that obinutuzumab-based radiotracers outperformed rituximab-based radiotracers in delineating CD20 expression.⁵⁴⁰ To further conquer the poor tumor penetration and undesirable pharmacokinetics of full-length antibodies, a set of hCD20-targeting sdAbs were generated.⁶⁸ One of these sdAbs, sdAb 9079 was radiolabeled with ⁶⁸Ga and ¹⁷⁷Lu and used for immunoPET imaging and targeted therapy of lymphoma, respectively.

Both 124I-rituximab and ⁸⁹Zr-rituximab immunoPET/CT imaging was able to visualize CD20 expression in patients with RA.^541,542 In addition to imaging NHL and RA, recent studies have reported the value of ⁸⁹Zr-rituximab immunoPET imaging in diagnosing orbital inflammatory diseases and interstitial pneumonitis,^543–545 but rituximab treatment has not been approved for these diseases.

4.2.2. CD38.

CD38 is a transmembrane glycoprotein highly and uniformly expressed on multiple myeloma (MM), NHL, and several types of solid tumor cells.^546,547 Several CD38-specific mAbs (e.g., daratumumab, isatuximab, and MOR202) have been developed. Daratumumab is a fully human mAb and has been proven a clinical success for treating MM.⁵⁴⁸ Recently, both 64Cu- and 89Zr-labeled daratumumab have shown excellent imaging attributes for detecting subcutaneous and disseminated MM (Figure 17a–c).^549,550 Preclinical applications of CD38-targeted immunoPET imaging has also been extended to detecting lung cancer, hepatocellular carcinoma, and lymphoma (Figure 17d).^551–553 Additionally, CD38-specific sdAbs were generated for imaging and treating MM.^554,555 Small agents like sdAbs penetrate more effectively into the disseminated MM lesions than the conventional mAbs. Because daratumumab administration interferes with CD38 detection and some of the sdAbs binds to CD38 independently of daratumumab,^555,556 immunoPET probes derived from sdAbs will be very useful for detecting MM at early stages and evaluating the efficacy of daratumumab treatment.

Figure 17.

ImmunoPET imaging of CD38 expression. (a) ⁸⁹Zr-DFO-daratumumab immunoPET/CT imaging of a mouse bearing bilateral MM1.S tumors (T₁ and T₂). (b) ⁸⁹Zr-DFO-daratumumab immuno-PET/CT imaging of a mouse bearing a unilateral MM1.S tumor (T₃) in the presence of unlabeled daratumumab as a blocking agent. (c) Representative bioluminescence imaging of the mice in the blocking group receiving an injection of cold daratumumab. The bioluminescent signal indicates the successful establishment of the tumor on the right flank of the mouse. Reproduced with permission from ref 550. Copyright 2018 SNMMI. (d) ⁸⁹Zr-DFO-daratumumab immunoPET imaging of lymphoma (Ramos tumor) at 120 h after administration of the tracer. Reproduced with permission from ref 552. Copyright 2018 Springer Berlin Heidelberg.

Accompanying the above imaging success, substantial preclinical evidence has demonstrated that CD38-targeted RIT could achieve the eradication of disseminated MM.^557–559 Using a less immunogenic two-step pRIT strategy consisting of a novel 028-Fc-C825 bispecific protein (targeting CD38 antigen and yttrium-DOTA ligand) and a ⁹⁰Y-DOTA-biotin dramatically reduced tumor growth and increased survival in both MM and NHL models.⁵⁶⁰ These results indicate the superiority of CD38 for both immunoPET imaging and pRIT. Clinical translation of these CD38-targeted theranostic agents will likely improve the management of patients with MM (NCT03665155).⁵⁶¹

4.2.3. CD146.

CD146, also known as MUC18 or MCAM, was originally identified as a marker for melanoma and now is a novel biomarker for several cancers.⁵⁶² Recently, Jiang et al. demonstrated that CD146 interacts with VEGFR-2 in the endothelium and promotes angiogenesis. Additionally, they found that an anti-CD146 mAb (AA98) or CD146 siRNA could successfully inhibit the CD146/VEGFR-2 pathway.⁵⁶³ It has been proven that AA98 has anticancer effects in several types of cancer, including leiomyosarcoma, pancreatic cancer, hepatocellular carcinoma, breast cancer, among others.^564–567 The work is of importance because intrinsic or acquired resistance to bevacizumab occurs in clinical practice,⁵⁶⁸ and novel agents targeting CD146 may have therapeutic benefits.

Our group initially generated a murine anti-CD146 mAb (denoted as YY146) and radiolabeled it for immunoPET imaging of glioblastoma multiforme.⁵⁶⁹ In the work, we reported that ⁶⁴Cu-NOTA-YY146 immunoPET imaging could delineate U87MG tumors as small as 2 mm in diameter. Additionally, we explored the potential therapeutic effects of YY146 on U87MG cells. Another immunoPET imaging agent based on YY146 was developed by labeling the YY146 with ⁸⁹Zr.⁵⁷⁰ Follow-up studies from our group revealed that ⁶⁴Cu-NOTA-YY146 immunoPET could clearly visualize both subcutaneous and metastatic lung cancer xenografts.^571,572 Furthermore, ⁸⁹Zr-Df-YY146-ZW800, a dual-modality imaging tool, enabled immunoPET and near-infrared fluorescence (NIRF) imaging of CD146-positive hepatocellular carcinomas (Figure 18a,b).⁵⁷³ The prominent and persistent uptake of ⁸⁹Zr-Df-YY146-ZW800 also facilitated image-guided resection of the orthotopic HepG2 tumors (Figure 18c). On the basis of the previous success, a more recent study further developed a theranostic pair consisting of ⁸⁹Zr-Df-YY146 and IR700-YY146.¹²² While immunoPET imaging with ⁸⁹Zr-Df-YY146 precisely diagnosed CD146-positive melanomas, PIT with IR700-YY146 efficiently eradicated a large portion of CD146-positive small melanomas in an image-guided manner. These promising results imply that the development of humanized YY146 (huYY146) would be worthwhile. In future clinical scenarios, immunoPET imaging with ⁶⁴Cu-NOTA-huYY146 or ⁸⁹Zr-Df-huYY146 may identify patients with an increased likelihood of responding to CD146-targeted therapies. CD146-targeted immunoPET imaging will be able to monitor early treatment responses to such therapies.

Figure 18.

ImmunoPET probes targeting CD146 and CD105. (a) ImmunoPET and (b) near-infrared fluorescence (NIRF) imaging performed at different time-points after intravenous injection of ⁸⁹Zr-Df-YY146-ZW800 demonstrated prominent and persistent uptake of the tracer in HepG2 tumors but not in the YY146 blocking group. H (heart), L (liver), and T (tumor). (c) Clear delineation of orthotopic HepG2 tumors by both PET and NIRF imaging was enabled through ⁸⁹Zr-Df-YY146-ZW800, which further facilitated image-guided resection of the multiple tumors (red and yellow arrows). Reproduced with permission from ref 573. Copyright 2016 Ivyspring International Publisher. (d) Serial coronal immunoPET imaging using a tissue factor and CD105 dual-targeting ⁶⁴Cu-NOTA-heterodimer at 3, 15, 24, and 30 h postinjection of the tracer clearly detected the BxPC-3 tumor. (e) Coronal PET images of mice bearing an orthotopic BxPC-3 tumor at 3, 15, 24, and 30 h following injection of ⁶⁴Cu-NOTA-heterodimer. This imaging technique realized an easy diagnosis of the orthotopic BxPC-3 tumor with negligible radioactivity around the surrounding tissues. Reproduced with permission from ref 590. Copyright 2016 American Association for Cancer Research.

4.2.4. CD105.

Endoglin (CD105) is a transforming growth factor-β (TGF-β) coreceptor expressed on proliferating vascular endothelium in solid tumors.^574,575 A recent study elucidated that increased tumoral cytoplasmic and endothelial expression of CD105 was significantly associated with advanced stage, renal vein invasion, and microvascular invasion of RCC.⁵⁷⁶ TRC105, a therapeutic mAb that binds to human CD105 with high avidity, has been demonstrated to be safe and effective in patients with advanced solid tumors such as RCC and hepatocellular carcinoma.^577–581

The first successful immunoPET imaging of CD105 expression in murine breast tumor models was reported by our group in 2011.^{582,583 64}Cu-DOTA-TRC105 and ⁸⁹Zr-Df-TRC105 were used in these two studies. ⁶⁶Ga-NOTATRC105,⁵⁸⁴ and multimodality imaging probes targeting CD105,^585–587 have since been developed and validated in various solid tumor models. Of note, ⁸⁹Zr-Df-TRC105–800CW immunoPET imaging could noninvasively monitor lung metastases from breast cancer and facilitate straightforward image-guided surgical removal of the tumors.⁵⁸⁵ We also generated and characterized a Fab fragment from the TRC105 and then investigated its potential utility for PET imaging of tumor angiogenesis in breast cancer models.^588,589 To develop highly specific noninvasive imaging probes for pancreatic cancer, we synthesized a bispecific heterodimer by conjugating an antitissue factor (TF) Fab with an anti-CD105 Fab and then labeled the heterodimer with ⁶⁴Cu. This dual-targeting technique provided synergistic improvements in binding affinity, and immunoPET imaging with the developed ⁶⁴Cu-NOTA-heterodimer clearly delineated both subcutaneous and orthotopic pancreatic tumors (Figure 18d,e).⁵⁹⁰ After dual-labeling of the heterodimer with ⁶⁴Cu and fluorescent dye, dual-modality PET/NIRF imaging using ⁶⁴Cu-NOTA-heterodimer-ZW800 also specifically and readily detected pancreatic tumors.⁵⁹¹

These studies strongly demonstrated the feasibility of noninvasive imaging of CD105 expression using PET or PET/NIRF technologies. Broad clinical applications of TRC105-based imaging will enable noninvasive detection of both primary and small metastatic tumor nodules, intra-operative guidance for tumor removal, selective patient stratification for TRC105 therapy, and image-guided RIT.⁵⁹² In addition, CD105 expression is upregulated on tumor endothelial cells following inhibition of the VEGF signaling pathway.^593,594 As such, TRC105-based immunoPET imaging may select patients who will potentially benefit from the combinational therapy with TRC105 and VEGF inhibitors or antibodies.

4.3. Carbohydrate Antigens

4.3.1. Carbohydrate Antigen 19.9.

Carbohydrate antigen 19.9 (CA19.9) is an established biomarker for several epithelial tumors, lung cancer, breast cancer, and PDAC. 5B1 is a fully human IgG targeting CA19.9 and has been widely used for theranostic purposes.^595–597 In a first-in-human clinical trial, immunoPET imaging with ⁸⁹Zr-DFO-5B1 detected known PDACs, metastases, and small LN metastases,³⁹ which remained undetected on conventional imaging studies.

By using the chemoenzymatic methodology described above,²⁸⁷ 5B1 was also site-specifically modified with DFO and radiolabeled with 89Zr. ImmunoPET imaging with ⁸⁹Zr-^ssDFO-5B1 showed exceptional uptake of the radiotracer in the CA19.9-positive BxPC-3 models but not the CA19.9-negative MIAPaCa-2 models. Moreover, dual-modal imaging with ⁸⁹Zr-^ssdual-5B1 delineated both primary and metastatic tumors in an orthotopically implanted PDAC model.⁵⁹⁸ Because tumors shed CA19.9 into the bloodstream, it is sometimes necessary to inject cold antibody to saturate the shed antigen.⁵⁹⁹ By utilizing the IEDDA reaction with TCO-conjugated antibodies and Tz-conjugated radioligands, several pretargeted immunoPET imaging strategies have been explored. One such system with 5B1-TCO and Tz-PEG₁₁-Al[¹⁸F]-NOTA resulted in clear delineation of the CA19.9-expressing PDAC xenografts. However, radioactivity in the intestine retained over the imaging course. Another pretargeting approach consisting of 5B1-TCO and ⁶⁴Cu-NOTA-PEG₇-Tz or ⁶⁴Cu-NOTA-Tz showed improved diagnostic value, with the former combination showed better T/B contrast at the earlier imaging time-points (Figure 19).⁶⁰⁰

Figure 19.

Pretargeted immunoPET imaging of pancreatic cancer. 5B1-TCO was first administered to target CA19.9-expressing orthotopic Capan-2 xenograft followed by injection of ⁶⁴Cu-NOTAPEG₇-Tz 3 days after the previous injection. (a) Coronal and (b) maximum-intensity projection (MIP) images demonstrated that this pretargeted imaging approach clearly delineated the Capan-2 tumor.(c) Immunohistochemistry (top left), autoradiography (bottom left), and fused PET/CT image (right) from the same mouse further showed precise colocalization of CA19.9-expressing tumor cells and ⁶⁴Cu-NOTA-PEG₇-Tz. Reproduced with permission from ref 600. Copyright 2016 SNMMI.

4.3.2. Carbohydrate Antigen 125.

Mucin 16 (MUC16) is a glycoprotein highly expressed in several types of cancers (e.g., ovarian, endometrial, and fallopian tube cancers). Carbohydrate antigen 125 (CA-125) is the released extracellular region of MUC16 following proteolytic cleavage and is regularly used to screen for ovarian cancer, to monitor cancer treatment efficacy, and to check for cancer recurrence.⁶⁰¹ B43.13 (oregovomab) is a high-affinity murine mAb and has been employed as an immunotherapeutic agent in the treatment of advanced ovarian cancers. To facilitate early detection of ovarian cancers, 64Cu-NOTA-mAb-B43.13 and ⁸⁹Zr-DFO-mAb-B43.13 were developed sequentially.^602,603 ImmunoPET imaging with these two tracers clearly delineated CA125-positive OVCAR3 tumors. ⁸⁹Zr-DFO-mAb-B43.13 immunoPET imaging is particularly attractive because it also detected LN involvement with high contrast and accuracy.⁶⁰³ REGN4018 is a human BsAb that specifically binds to MUC16 and CD3.⁶⁰⁴ Although circulating CA-125 serves as an antigen sink and interferes with MUC16-targeted therapies, the potency of REGN4018 is not hampered by the circulating CA-125. It has been shown that the site-specifically labeled ⁸⁹Zr-DFO-REGN4018 localized to the spleen and lymph nodes of nontumor-bearing mice. The imaging capabilities of ⁸⁹Zr-DFO-REGN4018 was further investigated in humanized mouse models with two humanized targets (CD3 and MUC16). In these models, the bispecific ⁸⁹Zr-DFOREGN4018 specifically accumulated in the secondary lymphoid organs as well as the MUC16-expressing tumors (Figure 20). Currently, REGN4018 is undergoing a phase I clinical trial (NCT03564340) either as a monotherapy agent or in combination with cemiplimab (an anti-PD-1 antibody).

Figure 20.

ImmunoPET imaging of ovarian cancers with a bispecific radiotracer ⁸⁹Zr-DFO-REGN4018. (a) ⁸⁹Zr-DFO-REGN4018 immunoPET/CT imaging of humanized tumor-bearing mice showed the distribution of the tracer to the spleen (yellow arrow), lymph nodes (green arrow), and tumor (red arrow). (b) Blocking with a MUC16 parental antibody reduced the tumor uptake of ⁸⁹Zr-DFO-REGN4018 without influencing the spleen and lymph node uptake. (c) Blocking with an anti-CD3 antibody substantially reduced the spleen and lymph node uptake of ⁸⁹Zr-DFO-REGN4018 without influencing the tumor uptake. Reproduced with permission from ref 604. Copyright 2019 American Association for the Advancement of Science.

4.4. Prostate-Specific Membrane Antigen

Using ¹⁸F-FDG or ¹¹C-choline PET tracers to image prostate cancer (PCa) often yields false negative or false positive uptake due to the slow growth and the low glycolytic rate of most PCas. Interpretation of ¹⁸F-FDG PET images in PCa is often difficult or even impossible because of the spillover effects from the accumulation of the tracer in the bladder and the intestine.⁶⁰⁵ Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein that is highly expressed on most prostate adenocarcinomas and has gained increasing interest as a target molecule for imaging and therapy in the past five years.^606–608 A recent study has elucidated a novel oncogenic signaling role of PSMA and reported that suppression of PSMA inhibited the PI3K signaling pathway and promoted tumor regression in preclinical PCa models.⁶⁰⁹ Studies using both antibodies and small-molecule agents have been conducted to develop PSMA-targeted SPECT and PET imaging platforms. PSMA-targeted theranostic approaches have been extensively reviewed elsewhere.^610–613 Herein, we only focus on PSMA-specific immunoPET imaging probes.

A variety of mAbs specific for intracellular and extracellular epitopes of PSMA have been developed.^{605,614–616} J591, a humanized mAb which binds to an extracellular domain of PSMA, has been clinically investigated for both imaging and therapy.^617–621 Recently, Fung et al. demonstrated that ¹²⁴IJ591 and ⁸⁹Zr-J591 had comparable surface binding and internalization rates in preclinical prostate models.⁶²² These studies imply that PCa theranostics using ¹⁷⁷Lu- and ¹²⁴I-or ⁸⁹Zr- labeled J591 is feasible, safe and may have superior targeting toward bone lesions relative to conventional imaging modalities. This may refine management strategies for patients with PSMA-positive PCas. Capromab (7E11) is a murine mAb, which has been investigated clinically as a SPECT imaging agent for recurrent and metastatic PCas⁶²³ and a therapeutic agent after labeling with ⁹⁰Y.⁶²⁴ Because capromab binds to an epitope on the intracellular domain of PSMA, tracers derived from this mAb are limited to detecting dead cells and do not possess advantages in imaging soft-tissue and bone metastases from PCas when compared to tracers binding to the extracellular domain.⁶²⁵ D2B is another PSMA-specific murine mAb for developing theranostic agents.^626,627 More recently, Barinka and co-workers reported the characterization of four novel murine PSMA-specific mAbs. One of these, 5D3, demonstrated 10-fold higher affinity compared to J591.⁶²⁸ A follow-up study further revealed that 5D3 may serve as a promising surrogate for imaging PSMA expression.⁶²⁹

When compared to mAbs, VHHs and engineered antibody fragments offer faster delivery and similar tumor delineating properties.^630,631 Viola-Villegas et al. engineered a Mb and a diabody from the intact antibody J591 and reported that immunoPET imaging with these smaller antibody fragments offered rapid tumor accumulation and accelerated clearance in PSMA-expressing PCas.⁶³² One such radiotracer, ⁸⁹Zr-IAB2M, was further translated in a dose-escalation clinical trial that included 18 patients with metastatic PCas.⁶³³ In this clinical study, immunoPET imaging with ⁸⁹Zr-IAB2M delineated metastatic PCa lesions in 17 of 18 patients 48 h after the intravenous injection of the radiotracer (10 mg). Moreover, ⁸⁹Zr-IAB2M immunoPET outperformed ^99mTc-MDP and CT in detecting bone lesions and magnetic resonance imaging (MRI) and ¹⁸F-FDG PET in detecting LN/soft tissue metastases (Figure 21).⁶³³ A phase I/IIa trial further confirmed the diagnostic efficacy of ⁸⁹Zr-IAB2M.⁶³⁴ Another advantage of this probe was its negligible accumulation in lacrimal and salivary glands because a major side effect of PSMA-targeting agents is the xerostomia.⁶³⁵ Beile et al. initially generated three mAbs against cell-adherent PSMA from spleen cells of mice^636–638 and more recently developed multimeric antibody fragments from these murine antibodies.⁶³⁹ They showed that the radiolabeled antibody fragments had stable tumor uptake and faster serum clearance.

Figure 21.

ImmunoPET imaging of prostate cancers with the minibody-based ⁸⁹Zr-IAB2M. (a) ^99mTc-MDP bone scan of a PCa patient showed multiple metastatic lesions in ribs, vertebrae, and left femur. (b) An ¹⁸F-FDG PET scanning showed the lesion in the left femur but failed to clearly detect the vertebral lesions. (c) ⁸⁹Zr-IAB2M immunoPET imaging of the same patient detected more lesions than either conventional imaging modalities. Reproduced with permission from ref 633. Copyright 2016 SNMMI.

Amassing clinical studies investigating PSMA-targeted PET imaging have demonstrated uptake of the tracers in non-prostate malignancies.^640,641 It has been proposed that uptake of PSMA-targeted tracer in nonprostate malignancies is due to the significant angiogenesis in tumor tissues.⁶⁴² Indeed, PSMA is expressed by the neovasculature endothelium but not by the tumor cells or the normal vasculature endothelium in most solid tumors.^643,644 In this context, several clinical studies have suggested that PSMA is another promising surrogate for molecular imaging of tumor neovasculature.^645,646 As such, PSMA-targeted PET and immunoPET imaging will help select patients for subsequent PSMA-targeted therapies and/or antiangiogenesis therapies (e.g., bevacizumab).

4.5. Carcinoembryonic Antigen

As a key member of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), carcinoembryonic antigen (CEA) serves as a vital tumor antigen and a serum tumor marker.⁶⁴⁷ Arcitumomab (CEAScan) is a ^99mTc-labeled hapten-peptide pretargeted imaging probe approved by the FDA and EMA for detecting colonic cancer metastases.⁶⁴⁸ However, this agent was withdrawn from the market because of competition from the more cost-effective ¹⁸F-FDG. To improve the imaging quality and specificity, several CEA-specific pretargeted immunoSPECT imaging agents were investigated. These efforts are exemplified in a study by Sharkey et al.,⁶⁴⁹ in which a superior tumor-to-blood ratio (~100:1) was obtained in human colon cancer xenografts. Alternatively, CEA-directed pretargeted immunoPET imaging was designed by first pretargeting CEA with a multivalent BsAb (which also targets HSG) followed by the injection of a radiolabeled hapten peptide. In this strategy, the DOTA-containing IMP288 peptide allowed ⁶⁸Ga complexation and the NOTA-containing IMP-449 peptide allowed facile [¹⁸F]AlF chelation.^311,650 In addition to detecting subcutaneous tumors, two recent studies demonstrated that TF2/⁶⁸Ga-IMP288 pretargeted immunoPET outperformed ¹⁸F-FDG PET in detecting disseminated human colorectal cancers (Figure 22).^651,652 Moreover, this highly sensitive pretargeting technique could be used to visualize CEA-containing human colorectal cancer tissues and normal epithelial cells.⁶⁵³ The study also demonstrated that pretargeted immunoPET has superior accuracy than ¹⁸F-FDG PET in delineating CEA⁺ tumors due to the highly specific tumor uptake and low background activity. Upon clinical translation, this novel imaging method may be useful to identify tumor lesions during surgical dissection. A recent first-in-human clinical trial has reported that TF2/⁶⁸Ga-IMP288 pretargeted immunoPET imaging revealed abnormal foci in all patients with relapsed medullary thyroid cancer.⁶⁵⁴ This study also demonstrated that a 30 h time lag between the injection of TF2 and ⁶⁸Ga-IMP288 and a TF2-to-IMP288 molar ratio of 20 were the most favorable conditions for imaging. Moreover, an ongoing clinical trial is evaluating the diagnostic role of TF2/⁶⁸Ga-IMP288 pretargeted immunoPET imaging in patients with HER2-negative but CEA⁺ breast cancers (NCT01730612).

Figure 22.

Pretargeted immunoPET imaging of metastatic colorectal cancers. (a) In this approach, CEA- and HSG-targeting BsAb TF2 was given first to saturate the LS174T tumors, followed by administration of DOTA- and HSG-containing 68Ga-IMP288 16 h later. This imaging approach clearly delineated tumors, except for two small tumor lesions (T3 and T6). Bladder (BL) uptake indicates excellent excretion of the ⁶⁸Ga-IMP288 through the urinary system. (b) ¹⁸FFDG PET/CT imaging of the same mouse showed less optimal image contrast due to uptake in the intestines. Reproduced with permission from ref 651. Copyright 2012 Springer Nature.

Along with this progress, a series of antibody fragments were engineered from a murine mAb T84.66 and have been radiolabeled with radiometals such as ⁶⁴Cu and ¹²⁴I.^655–657 To improve the T/B ratio of the engineered antibody fragments, mutation of the residues in the Fc fragment essential for FcRn binding can be performed. For instance, Kenanova et al. formatted a series of anti-CEA scFv-Fc fragments^658,659 and found that PET imaging with a ¹²⁴I-labeled scFv-Fc bearing one double mutation (H310A/H435Q) showed the highest imaging quality.⁶⁵⁸ To further permit same-day immunoPET imaging, several types of labetuzumab fragments have been radiolabeled with [¹⁸F]AlF using the chelating ligand NODAMPAEM.⁶⁶⁰ Despite the facile and rapid procedure, high kidney and liver accumulation of the developed tracers may hinder the clinical translation.⁶⁶⁰

AMG 211 (MEDI-565) is a BiTE composed of a humanized anti-CEA arm and a deimmunized anti-CD3 arm. AMG 211 could efficiently activate human T cells which lysed CEA⁺ colorectal tumor cells in a preclinical model.⁶⁶¹ However, this treatment effect was not observed in patients with advanced gastrointestinal adenocarcinoma.⁶⁶² To uncover the underlying reasons for the poor efficacy, the in vivo biodistribution and tumor targeting ability of AMG 211 was investigated with ⁸⁹Zr-AMG 211 immunoPET imaging.^663,664 The results revealed the accumulation of the tracer in CD3-rich lymphoid organs (e.g., spleen and bone marrow). Tumor uptake of ⁸⁹Zr-AMG 211 was evident yet varied strikingly within and between patients, attributed to the heterogeneous expression of CEA in different tumor lesions and varying tumor vasculature and tissue permeability.⁶⁶⁴ To specifically deliver interleukin-2 (IL-2) to CEA⁺ tumors and overcome the adverse effects of IL-2 monotherapy, cergutuzumab amunaleukin (CEA-IL2v) has been designed and is actively undergoing a clinical investigation in combination with the anti-PD-L1 atezolizumab in CEA-expressing advanced solid tumors (NCT02350673).⁶⁶⁵ Recently, a pilot immunoPET imaging study with ⁸⁹Zr-CEAIL2v showed a preferential drug accumulation in CEA⁺ tumors, and two cycles of CEA-IL2v administration reduced the number of tumor lesions and tumor uptake of the radiotracer (Figure 23).⁶⁶⁶ This exploratory study also indicated nonspecific uptake of ⁸⁹Zr-CEA-IL2 in nonmalignant lymphoid organs, suggesting the in vivo distribution of CEA-IL2v is driven by the synergistic effect of CEA targeting and IL-2 binding on immune cells.

Figure 23.

ImmunoPET imaging of solid tumors using ⁸⁹Zr-CEAIL2v. ⁸⁹Zr-CEA-IL2v immunoPET imaging of a patient with CEA⁺ colorectal cancer at cycle 1, day 5 (left) showed uptake of the radiotracer in the bilateral hilar lymph nodes and the left dorsal lung metastasis (white arrows). The uptake in these malignant lesions and a nonpathological lymph node (red arrows) decreased after the fourth cycle of CEA-IL2v treatment (right). Notably, uptake in the liver (yellow arrows) increased and uptake in the spleens (orange arrows) decreased following the treatments. Reproduced with permission from ref 666. Copyright 2018 Impact Journals, LLC.

4.6. Carbonic Anhydrase IX

Carbonic anhydrase IX (CAIX) is a cytosolic and trans-membrane enzyme belonging to the zinc-containing metal-loenzyme family.⁶⁶⁷ By catalyzing the conversion of carbon dioxide and water to carbonic acid (CO₂ + H₂O ⇄ HCO₃⁻ + H⁺), CAIX contributes to the acidic extracellular environment of hypoxic tissues, predominantly hypoxic tumors and metastases.⁶⁶⁸ CAIX is homogeneously overexpressed in 95% of clear cell renal cell carcinoma (ccRCC) cases, so it is an optimal theranostic target for ccRCC. Several mAbs (e.g., cG250 or girentuximab) targeting CAIX are undergoing clinical investigations.^669,670 On the basis of preclinical studies, where 124I-cG250 and ⁸⁹Zr-cG250 immunoPET imaging clearly visualized CAIX-expressing ccRCC xenografts,^671–673 a recent clinical study reported that ⁸⁹Zr-girentuximab immunoPET imaging precisely predicted the pathology of all the immunoPET-positive primary renal lesions. This result changed the clinical decision for 36% of patients with recurrent/metastatic ccRCC (Figure 24).⁶⁷⁴ Several proof-of-concept clinical studies have also validated the safety and superior diagnostic value of ¹²⁴I-cG250 in ccRCC,^184,186,675 with an average sensitivity and specificity of 86.2% and 85.9%, respectively.¹⁸⁶ Additionally, multimodal imaging with ¹²⁴I-cG250 could realize precise intraoperative localization of ccRCC, which further guided and confirmed complete surgical resection of the diseases.⁶⁷⁶ In contrast to ¹²⁴I-cG250, which rapidly releases ¹²⁴I after being internalized into tumor cells, the residual radiometal ⁸⁹Zr from ⁸⁹Zr-cG250 will be trapped inside the tumor cells.⁶⁷²

Figure 24.

ImmunoPET imaging of clear cell renal cell carcinoma (ccRCC) with ⁸⁹Zr-girentuximab. (a) A patient with ccRCC who previously had undergone nephrectomy was subjected to a CT scan that showed neoplasms in the right kidney and the adjacent adrenal (white circle). (b) ⁸⁹Zr-girentuximab immunoPET/CT imaging of the same patient showed that both the lesions had an uptake of the tracer. Additional uptake in the proximal radius was seen (insert), which changed the management strategy of the patient from a futile radical nephrectomy to radiotherapy. Reproduced with permission from ref 674. Copyright 2018 European Association of Urology.

M75, another mAb targeting CAIX, is regularly used in IHC studies to detect CAIX.⁶⁷⁷ In recent studies this mAb has been radiolabeled with ⁶⁴Cu and ⁶¹Cu, and the developed radio-tracers revealed specific binding in CAIX-expressing colorectal cancer models.^678,679 Moreover, several other CAIX targeting mAbs have shown preliminary anticancer effects.^680–682 Up to now, they have not been used for immunoPET imaging. Lastly, it is worthwhile to note that CAIX is highly expressed in a plethora of other tumor cells, tumor-associated stromal cells, and cancer stem cells.⁶⁶⁸ Because of the above-described merits and limited presence of CAIX in normal tissues, CAIX may serve as an ideal target for developing advanced theranostic agents.

4.7. Trophoblast Cell Surface Antigen 2

Trophoblast cell surface antigen 2 (TROP-2), a 46 kDa transmembrane glycoprotein, is overexpressed in a broad range of cancers.^683,684 Sacituzumab govitecan (IMMU-132) is an antibody–drug conjugate (ADC) targeting TROP-2 and has been approved as a third-line therapy for metastatic triple-negative breast cancers.⁶⁸⁵ Moreover, its application is being extended for the treatment of several other malignancies. hRS7 is a humanized IgG1 mAb targeting TROP-2. hRS7-based immunoPET or immunoSPECT imaging clearly visualized TROP2 expression in PCa models.⁶⁸⁶ TF12 (157 kDa) is a trivalent BsAb composed of an anti-HSG Fab and two anti-TROP-2 Fabs derived from the hRS7.⁶⁸⁷ Using TF12, pretargeted immunoPET imaging, pRIT, and image-guided surgery of human PCas have all been achieved.^688–690 While TROP-2 immunoPET imaging is of clinical interest for selecting TROP-2-positive patients, its value in evaluating the therapeutic response following IMMU-132 treatment is limited because TROP-2 is not a predictive biomarker for response.⁶⁹¹

4.8. Stem Cell Antigens

Cancer stem cells (CSCs) are characterized by undifferentiated features, which may include self-renewal, long-term replication, and diverse differentiation abilities.⁶⁹² It has been proposed that CSCs are a major driving force of tumor occurrence and metastasis.⁶⁹³ Additionally, chemotherapy and radiotherapy resistances are mediated in part by CSCs.⁶⁹⁴ Therefore, noninvasive imaging of CSCs is clinically relevant and may aid the identification and eradication of CSCs. Of the various stem cell markers, leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), prostate stem cell antigen (PSCA), and CD133 are three attractive biomarkers that have been exploited for immunoPET imaging.

LGR5, a marker of adult stem cells, is highly expressed in some human cancers.⁶⁹⁵ Noninvasive LGR5 assessment was achieved with two LGR5-targeting immunoPET probes (i.e., ⁸⁹Zr-DFO-8F2 and ⁸⁹Zr-DFO-9G5). ⁸⁹Zr-DFO-8F2 showed higher specificity over ⁸⁹Zr-DFO-9G5 in visualizing LGR5 expression in colorectal tumors.⁶⁹⁶ While PSMA-targeting agents have shown great promise in patients with PCas, PSCA is an alternative target for designing theranostic agents. Using an anti-PSCA mAb, 7F5, and a newly developed chelator, L⁵-NCS, David et al. showed that ⁶⁴Cu-L⁵-7F5 immunoPET imaging clearly visualized PSCA-positive PC3 tumors with minute activity in the liver. In comparison, ⁶⁴Cu-NODAGA-7F5 immunoPET imaging showed much less tumor accumulation but significantly higher liver uptake (Figure 25).¹⁵⁸ A11 is a humanized anti-PSCA Mb and ¹²⁴I or ⁸⁹Zr-labeled A11 was able to detect PSCA-expressing PCas⁶⁹⁷ as well as to evaluate the treatment response.⁶⁹⁸ Additionally, PSCA-targeted fluorescence or dual-modal immunoPET/fluorescence imaging agents hold great promise for intraoperatively visualizing PSCA-positive PCas and pancreatic cancers.^699–701

Figure 25.

ImmunoPET imaging of cancer stem cell markers. (a) Chemical structure of a novel chelator L⁵-NCS. (b) ⁶⁴Cu-L⁵-7F5 immunoPET imaging clearly detected PSCA-expressing PC3 tumor 2 days after injection of the tracer. (c) In comparison, ⁶⁴Cu-NODAGA-7F5 showed much lower tumor uptake and higher liver uptake. Reproduced with permission from ref 158. Copyright 2018 American Chemical Society.

AC133, an epitope of the second extracellular loop of CD133, is one of the most extensively investigated markers for CSCs.⁷⁰² An initial study showed that anti-AC133 mAb-based fluorescence imaging agents visualized CD133-overexpressing tumors.⁷⁰³ More recently, immunoPET imaging with an AC133-targeted radiotracer (⁶⁴Cu-NOTA-AC133 mAb) permitted successful detection of CD133⁺ U251 tumors and glioma stem cells. More importantly, the ⁶⁴Cu-NOTA-AC133 mAb immunoPET imaging patterns correlated well with the cytoarchitecture of the orthotopically growing gliomas.⁷⁰⁴ In the efforts for imaging CSCs, one recurring concern is that CSCs are not abundant in the TME. Taking this fact into consideration, we advocate the use of PET imaging over other imaging modalities for detecting and quantifying CSCs.

4.9. Proteases

Proteases are a group of evolutionarily conserved enzymes and can be classified into six groups by their mechanisms of action: Cys, serine, threonine, aspartate, glutamic acid proteases, and metalloprotease. Proteases play essential roles in tumor angiogenesis, invasion, and metastasis.⁷⁰⁵ Therefore, proteases are attractive targets for developing molecular imaging probes. Urokinase plasminogen activator (uPA) receptor (uPAR) is one of the validated targets. AE105, a high-affinity peptide antagonist against uPAR, has been radiolabeled and investigated extensively in preclinical studies.⁷⁰⁶ Inspired by the preclinical success, ⁶⁴Cu-DOTA-AE105 and ⁶⁸Ga-NOTA-AE105 have been successfully translated into the clinic for PET imaging of uPAR in patients with solid tumors.^707,708 Using an IgG1 mAb (ATN-291) specifically targeting human uPA, Yang and co-workers developed a radiotracer ⁸⁹Zr-Df-ATN-291.⁷⁰⁹ The authors reported the utility of ⁸⁹Zr-Df-ATN-291 immunoPET in monitoring the expression of uPA/uPAR in several mouse xenografts. Matrix metalloproteinases (MMPs) have been heralded as attractive targets for cancer therapy for decades.⁷¹⁰ LEM-2/15 is a mAb specific for membrane type 1-matrix metalloproteinase (MT1-MMP, MMP14), and immunoPET imaging with ⁸⁹Zr-DFO-LEM 2/15 specifically localized the intracranial patient-derived xenograft tumors.⁷¹¹ Moreover, ⁸⁹Zr-DFO-LEM 2/15 immunoPET imaging showed a higher specificity than ⁶⁸Ga-DOTAAF7p, a MT1-MMP-specific peptide-based tracer, in identifying PDACs.⁷¹²

Prostate-specific antigen (PSA) is a target in the androgen receptor (AR) pathway. PSA level in the blood is a reliable readout of AR pathway activity. “Free” PSA (fPSA) is a catalytically active serine protease and has been exploited as an immunoPET imaging target. 5A10 is a mAb that selectively binds fPSA. ⁸⁹Zr-5A10 immunoPET detected intratumoral fPSA expression following different pharmacological interventions.⁷¹³ However, ⁸⁹Zr-5A10 immunoPET yielded less favorable imaging contrast because ⁸⁹Zr-5A10 targeted secreted PSA with only a small proportion of the tracer directed to the tumor sites. Therefore, an immunoPET probe with increased internalization into tumor cells would image the AR pathway activity more clearly. To achieve this, two mAbs (i.e., 11B6 and its humanized version hu11B6) targeting human kallikrein-related peptidase 2 (hK2), another serine protease regulated by the AR pathway, were used for immunoPET imaging.⁷¹⁴ In contrast to the transient uptake of ⁸⁹Zr-5A10 in tumors, ⁸⁹Zr-11B6 internalized into the tumor cells and showed steadily increasing tumor uptake. Moreover, ⁸⁹Zr-11B6 immunoPET imaging delineated AR-driven hK2 expression in mice bearing subcutaneous or metastatic human PCa xenografts.⁷¹⁴

4.10. Membrane-Bound Surface Glycoprotein Mesothelin

Membrane-bound surface glycoprotein mesothelin (MSLN) is overexpressed in mesothelioma and several other solid tumors such as ovarian, lung, breast, and pancreatic cancers.^715,716 It is worth mentioning that soluble mesothelin is a superior tumor marker for monitoring tumor burden and therapeutic response in patients with malignant pleural mesothelioma.⁷¹⁷ Substantial studies have reported the feasibility of MSLN imaging using several MSLN-targeting mAbs such as a murine mAb 11–25, a chimeric mAb amatuximab, and an ADC DMOT4039A (composed of MMOT0530A and MMAE).^718–720 In a clinical study evaluating the treatment efficacy of DMOT4039A in patients with pancreatic or ovarian cancer, Lamberts et al. found that uptake of ⁸⁹Zr-MMOT0530A correlated with MSLN expression from IHC staining.⁷²¹ These results indicate that this imaging technique may guide individualized antibody-based therapies. As observed in a previous study,³⁶¹ several recent studies have demonstrated that shed antigen MSLN in blood circulation negatively regulates the circulation and tumor-targeting efficacy of amatuximab.^722,723 These results indicate the role of MSLN-targeted immunoPET in assessing the performance of mesothelin-targeted therapeutic antibodies and selecting patients for mesothelin-targeted therapies.⁷²⁴

4.11. Glycoprotein A33

Glycoprotein A33 (GPA33) is a cell surface target uniformly overexpressed in over 90% of colorectal cancers and a subset of gastric and pancreatic cancers.⁷²⁵ This antigen has been extensively studied as a target for both RIT and SPECT imaging using a murine mAb A33.^726,727 Following the humanization of A33 (huA33) and preclinical evaluation of huA33-based immunoPET tracers,^728,729 recent studies have translated ¹²⁴I-huA33 to the clinic. This immunoPET imaging technique enabled the quantitative assessment of GPA33 status in colorectal cancers.^185,730 Pretargeted immunoPET imaging has been successfully employed to delineate GPA33-expressing human colorectal carcinoma xenografts. In such a work by Zeglis et al., huA33-TCO was administered 24 h prior to ⁶⁴Cu-Tz-SarAr to reach the tumors and permit blood clearance.³³⁹ Owing to the superior stability and exclusive renal clearance of ⁶⁴Cu-Tz-SarAr, the in vivo pretargeting yielded specific uptake of the radioligand in the colorectal tumors with high T/B ratios (Figure 26). This imaging strategy also outperformed a previous pretargeted imaging strategy, in which substantial uptake of ⁶⁴Cu-Tz-NOTA was observed in the gastrointestinal tract.³³⁵ In the future, the combination of GPA33-specific immunoPET imaging and pRIT may allow precise diagnosis and treatment of colorectal cancers.^329,731,732

Figure 26.

Pretargeted immunoPET imaging of colorectal cancers. (a) Schematic of the imaging strategy, in which huA33-TCO was first administered to accumulate in the tumor followed by injection of ⁶⁴Cu-Tz-SarAr 24 h later. (b) Coronal image and (c) fused PET/CT images 24 h postinjection of the radioligand showed the effective delineation of the subcutaneous SW1222 xenografts. Reproduced with permission from ref 339. Copyright 2015 American Chemical Society.

4.12. Other Promising Tumor Biomarkers

Several other cell surface antigens have shown promise for imaging cancers at the cellular and molecular levels in either preclinical or clinical settings, such as folate receptor alpha,^733,734 tumor vascular markers,^735–737 death receptor 5,^738–740 glypican-3,^741–743 cell adhesion molecules,^744,745 six-transmembrane epithelial antigen of prostate-1 (STEAP1),^746–749 hormone receptors,^750–752 TGF-β pathway,^753–755 chemokine receptors,^756,757 and extracellular matrix-like fibronectin.^162,188 Here, we also discuss some of the promising targets briefly.

Tumor-associated glycoprotein (TAG)-72 is another glyco-protein highly expressed in the majority of adenocarcinomas. 3E8 is a humanized mAb against TAG-72 and has shown theranostic potential in colorectal cancer xenografts.⁷⁵⁸ For antibody fragment-based immunoPET imaging of TAG-72, PEGylated targeting vectors may serve as favorable surrogates for efficiently delivering the radionuclide to the tumor site while lowering kidney uptake.^759–761 Delta-like 3 (DLL3), a Notch pathway ligand, has been identified as a marker for pulmonary neuroendocrine tumors (i.e., small cell lung cancer and large cell neuroendocrine carcinoma) and neuroendocrine PCas.^762–764 As such, DLL3 may also serve as a tractable immunoPET imaging target.^765,766 Similar to the GPA33-targeted theranostic scenario, GD2-specific immunoPET imaging may help assess GD2 expression in patients with neuroblastomas.^767,768 Along with the clinical use of GD2-targeted antibody therapy and RIT,^769–771 and future implementation of pRIT,⁷⁷² GD2-targeted immunoPET imaging may guide precise anti-GD2 therapies and complement other imaging modalities to refine the management of neuroblastomas.

5. IMMUNOPET IMAGING OF IMMUNE SYSTEM

Cancer immunotherapy is increasingly becoming the standard-of-care treatment for a broad spectrum of cancers. IHC, polymerase-chain-reaction (PCR)-based assays and serum or blood biomarkers are regularly used to predict the therapeutic responses of immunotherapy regimens. Despite these approaches, it is still very challenging to properly select patients suitable for immunotherapy and precisely evaluate the treatment responses. Furthermore, immune-related adverse events are increasingly being reported. Currently, there are no reliable surveillance strategies to diagnose those unexpected complications.^773,774 Accumulating evidence has suggested that immunoPET imaging may substantially improve the clinical immunotherapy by dynamically visualizing immune responses across the whole body.^775–777 To achieve this goal, radiotracers have been developed to image interleukins and specific immune cells, including B cells,⁷⁷⁸ natural killer cells,^779,780 macrophages,^781–783 myeloid cells,^278,784 and T cells. In this section, we mainly present the most recent evidence of immunoPET strategies in delineating T cells by targeting lineage-associated antigens, immune checkpoint molecules, OX40, and interferon gamma (IFNγ).

5.1. Lineage-Associated Antigens

In addition to tracking T cells via ex vivo direct labeling or reporter gene imaging,^785–787 it is advantageous to track T cells using novel immunoPET techniques by targeting general T cell markers (e.g., CD3, CD4, CD8, CD2, and CD7). ImmunoPET imaging with a ⁸⁹Zr-labeled antimouse CD3 antibody (clone 17A2; R&D Systems) revealed a correlation between high tumor uptake of the radiotracer and better therapeutic response of anticytotoxic T lymphocyte antigen 4 (CTLA-4) therapy in a preclinical colorectal cancer model.⁷⁸⁸ However, significant liver accumulation of the radiotracer was found on immunoPET images, which was explained as liver clearance of the radiolabeled antibody. Another study also labeled the same clone (clone 17A2; Bio X Cell) with ⁸⁹Zr and confirmed the uptake of the developed radiotracer in lymphoid organs (i.e., spleen, lymph nodes, and thymus) and tumor-infiltrating lymphocytes (TILs) in syngeneic bladder cancer models.⁷⁸⁹ But what was different in this study was that the tracer was largely deposited in the spleen rather than in the liver, similar to that reported in another study.⁷⁹⁰ CD2 and CD7 are pan T cell markers and immunoPET tracers targeting these two markers have been developed to track adoptively transferred T cells.⁷⁹¹ However, the safety profiles, long-term effects, and impact of these tracers on the functionality of T cells remain to be determined.

To noninvasively detect CD8⁺ T cells, two Mbs (i.e., 2.43 Mb and YTS169 Mb) were engineered from two parental rat antimouse CD8 mAbs. Both ⁶⁴Cu-NOTA-Mbs retained high antigen specificity in imaging CD8⁺ T cells.⁷⁹² IAB22M2C is a clinical-stage Mb that targets human CD8 with high affinity. A recent phase I study has demonstrated the safety profile of ⁸⁹Zr-Df-IAB22M2C immunoPET imaging in patients with solid tumors.⁷⁹³ This study also reported a specific accumulation of the radiotracer in CD8⁺ T cell-rich tissues, such as lymph nodes, spleen, and tumors. A phase II clinical trial with this immunoPET probe investigating the correlation between 89Zr-Df-IAB22M2C immunoPET signal and CD8 status assessed by IHC in patients with metastatic cancers is currently underway (NCT03802123).

Two diabody-based anti-CD4 and anti-CD8 tracers, ⁸⁹ZrmalDFO-GK1.5 cDb and ⁸⁹Zr-malDFO-2.43 cDb, also specifically targeted various lymphoid organs and allowed longitudinal monitoring of transplanted T cells.⁷⁹⁴ To thoroughly assess CD8-targeted immunoPET imaging, ⁸⁹ZrmalDFO-169 cDb binding to CD8a of all mouse strains was assessed.⁷⁹⁵ ImmunoPET imaging with this radiotracer detected intratumoral CD8⁺ T cells after anti-CD137 or anti-PD-L1 therapy in immune-competent mice bearing CT26 tumors. Afterward, ⁶⁴Cu-TETA-169 cDb was developed and used to image CD8⁺ T cells after immunotherapy using different treatment protocols.⁷⁹⁶ As reported by the work, serial ⁶⁴Cu-TETA-169 cDb immunoPET imaging mapped T cell distribution and also detected treatment-associated hypertrophy of liver and spleen following multiple cycles of immunotherapy.

Other types of antibody vectors used for imaging T cells include VHH and monovalent antibody. Rashidian et al. produced a CD8-specific mouse VHH (VHH-X118) and fabricated ⁸⁹Zr-PEGylatedVHH-X118 using sortase-catalyzed site-specific conjugation.⁶⁴ This immunoPET probe robustly detected thymus, secondary lymphoid structures as well as CD8⁺ T cells in B16 melanomas. This study also highlighted the value of CD8-targeted immunoPET in predicting the treatment response of anti-CTLA-4 immunotherapy, where the homogeneous distribution of immunoPET signal within the tumors predicted a favorable response to the anti-CTLA-4 immunotherapy. Moreover, another recent study radiolabeled a CD8-specific monovalent antibody with ⁸⁹Zr and immuno-PET imaging with the developed agent (denoted as ZED8) efficiently detected human CD8-expressing tumors.⁷⁹⁷ This tracer was developed under quality standards appropriate for regulatory approval and is currently under clinical investigation (NCT04029181).

5.2. Immune Checkpoints

Immune checkpoints are critical components of inhibitory immune signaling pathways. The first-generation immune checkpoint inhibitors are immunomodulatory mAbs that block immune checkpoints. Notably, immune checkpoint inhibitors blocking CTLA-4, programmed death receptor 1 (PD1), and PD-L1 have emerged as trailblazers in treating various kinds of cancers.⁶ Accordingly, immunoPET probes targeting CTLA-4, PD1, or PD-L1, are extensively investigated.

CTLA-4 is a negative immune regulator highly expressed on regulatory T cells (Treg) and on activated T cells. Recent studies have elucidated that CTLA-4 and PD-1 may share one pathway by inhibiting signaling through CD28, which is a costimulatory receptor that promotes T cell activation and proliferation.⁷⁹⁸ Several studies have reported the feasibility of CTLA-4-targeted immunoPET in imaging T cells in humanized mice⁷⁹⁹ and in immune-competent mice.⁸⁰⁰ H11 is a VHH targeting mouse CTLA-4 and immunoPET with ⁸⁹Zr-H11-PEG clearly delineated CTLA-4 expression within the TME.⁸⁰¹ Moreover, several studies have shown the expression of CTLA-4 on tumor cells,⁸⁰² which was corroborated by an imaging study where ⁶⁴Cu-DOTA-ipilimumab showed persistent accumulation in the CTLA-4-expressing A549 tumors.⁸⁰³

PD1 is a characteristic marker expressed on exhausted CD8⁺ T cells. Pembrolizumab and nivolumab are anti-PD-1 checkpoint inhibitors used for treating a variety of solid tumors. Several immunoPET probes using pembrolizumab/ nivolumab and ⁸⁹Zr/⁶⁴Cu have been developed and investigated in preclinical studies.^804–808 Of them, ⁸⁹Zr-pembrolizumab and 64Cu-pembrolizumab PET/CT examinations showed prominent uptake of the radiotracers in the humanized mice bearing A375 melanomas, indicating infiltration of PD-1-positive human lymphocytes into the tumors.⁸⁰⁶ Similarly, ⁸⁹Zr-Df-nivolumab immunoPET imaging delineated PD-1-positive human lymphocytes in A549 tumors and salivary glands in humanized mice.⁸⁰⁸ More recently, a seminal study by Niemeijer et al. investigated the clinical value of ⁸⁹Zr-Dfnivolumab immunoPET imaging in 13 patients with NSCLC prior to the nivolumab treatment.⁸⁰⁹ For the first time, this study reported a correlation between ⁸⁹Zr-Df-nivolumab uptake and PD-1 expression in the TILs assessed by IHC in clinical settings (Figure 27a,b). This study also indicated the value of ⁸⁹Zr-Df-nivolumab uptake in predicting the treatment efficacy of nivolumab. However, this predictive value needs to be confirmed in a larger patient cohort.

Figure 27.

ImmunoPET imaging of immune checkpoints in nonsmall-cell lung cancer (NSCLC). (a) ¹⁸F-FDG PET/CT scan of a patient with NSCLC showed lung tumors and mediastinal lymph node metastases with high glucose metabolism. (b) PD-1-specific ⁸⁹Zr-Dfnivolumab immunoPET/CT imaging demonstrated heterogeneous uptake of the radiotracer within and between the tumor lesions. (c) Similarly, heterogeneous uptake of PD-L1-specific ¹⁸F-BMS-986192, a ¹⁸F-labeled adnectin protein, was seen within and between the tumor lesions. Reproduced with permission from ref 809. Copyright 2018 Springer Nature.

Two IHC methods for determining PD-L1 expression have been approved to predict patient response before anti–PD-L1 therapies. However, sampling limitations and multifaceted expression of PD-L1 may lead to underestimation of the target. Substantial preclinical evidence has suggested that immuno-PET imaging can assess the heterogeneous status of PD-L1 throughout the whole body and thus can overcome above drawbacks.^810–813 In a recent first-in-human clinical trial (NCT02478099), 89Zr-atezolizumab was assessed in 22 patients with progressive bladder cancer, NSCLC or triple-negative breast cancer.²¹ ImmunoPET imaging with ⁸⁹Zratezolizumab showed deposition of the radiotracer in non-malignant lymph nodes, spleens, and sites of inflammation. More importantly, ⁸⁹Zr-atezolizumab immunoPET visualized all the metastatic tumor lesions by imaging heterogeneous PD-L1 expression. High tumor uptake of ⁸⁹Zr-atezolizumab correlated with better response to atezolizumab treatment, whereas PD-L1 IHC failed to predict the treatment outcome. ⁸⁹Zr-atezolizumab immunoPET imaging could also select RCC patients who may benefit from nivolumab therapy.⁸¹⁴ Moreover, there are two ongoing clinical trials evaluating the diagnostic value of PD-L1-targeted immunoPET in advanced thoracic malignancies (NCT03746704) and in locally advanced or metastatic solid tumors (NCT02453984).

Engineered small proteins (e.g., fibronectin, ~10 kDa) are alternative targeting moieties that have been used for imaging PD-L1 expression.⁸¹⁵ Following a preclinical study which discovered the high binding affinity of ¹⁸F-BMS-986192 (an ¹⁸F-labeled adnectin) to human and cynomolgus PD-L1,⁸¹⁶ a recent clinical study reported that ¹⁸F-BMS-986192 immuno-PET could noninvasively image the heterogeneous PD-L1 status in lung cancers (Figure 27c). High-affinity consensus (HAC) PD1 (14 kDa) is another high-affinity binder engineered from PD-1 protein and can be used for imaging human PD-L.⁸¹⁷ In the course of optimizing HAC-PD1 variants for PD-L1 imaging, aglycosylated HAC-PD1 showed increased tumor uptake and decreased glandular uptake.⁸¹⁸ Other targeting vectors that have been integrated into immunoPET for imaging PD-L1 include an Affibody molecule,⁸¹⁹ VHHs,^820–822 and a Fab fragment.⁸²³

Beyond imaging PD-L1 expression pre- and postimmuno-therapy, PD-L1-targeted immunoPET is an innovative approach to monitor PD-L1 upregulation following radiation therapy^824,825 or PD-L1 downregulation after molecular-targeted therapy.⁸²⁶ Although anti-PD-L1 therapy has become the first-line treatment option for patients with NSCLC, therapeutic resistance occurs in some patients. Among many potential reasons, secreted PD-L1 splicing variants may induce resistance to anti-PD-L1 therapy by acting as decoys in plasma.⁸²⁷ Similar to that observed in other conditions,^722,723 PD-L1-targeted immunoPET may predict and assess anti-PDL1 resistance because soluble PD-L1 will relocate the radiotracer to the plasma and liver but not to the tumor sites. Besides its expression on tumor cells, PD-L1 is also expressed in normal lymphoid organs and is an independent marker for brown adipose tissue (BAT) (Figure 28),^828–830 indicating that PD-L1-targeted immunoPET may as serve as a cutting-edge imaging technique to aid basic research.

Figure 28.

ImmunoPET imaging of programmed death-ligand 1 (PDL1) in brown adipose tissue (BAT). B3 is a single domain antibody specific for mouse PD-L1 and (a) ¹⁸F-B3 immunoPET/CT imaging of a 6-week-old wild-type C57BL/6 mouse showed deposition of the radiotracer in the BAT. (b) ¹⁸F-B3 immunoPET/CT imaging of an age-matched PD-L1 knockout mouse showed the absence of PD-L1 signal in the BAT, confirming the specificity of the developed radiotracer. Reproduced with permission from ref 830. Copyright 2017 Springer Nature.

5.3. Other Emerging T Cell Markers

OX40, also known as CD134, is a 50 kDa type I membrane glycoprotein belonging to the tumor necrosis factor (TNF) receptor superfamily.⁸³¹ Binding of OX40 by its ligand OX40L results in the activation of T cells, indicating OX40 is a promising candidate for monitoring clinical immunotherapies. By radiolabeling a murine mAb (clone: OX-86; Bio X Cell) with ⁶⁴Cu, Alam et al. developed an OX40-targeted radiotracer ⁶⁴Cu-DOTA-AbOX40.⁸³² The authors found that early immunoPET imaging with ⁶⁴Cu-DOTA-AbOX40 characterized the spatiotemporal expression of OX40⁺ T cells and also predicted the response of CpG vaccination in lymphoma models. These results demonstrated that OX40-targeted immunoPET could adequately visualize the heterogeneous dynamics of OX40⁺ T cells in immune responses across different subjects. For further clinical translation, this murine antibody-based immunoPET imaging strategy needs to be optimized with human or humanized antibodies.

IFNγ is a soluble immunomodulatory factor that exerts effects on both innate and adaptive immunity. IFNγ is primarily secreted by activated lymphocytes such as CD4⁺ and CD8⁺ T cells.⁸³³ Gibson et al. recently labeled an IFNγ-targeting rat mAb (AN-18) with ⁸⁹Zr and found that tumor cell uptake of ⁸⁹Zr-anti-IFNγ was IFNγ-dependent.⁷⁹⁰ After a series of CpG vaccination and mAb treatment experiments, they further demonstrated the robust ability of ⁸⁹Zr-anti-IFNγ immunoPET in detecting T cell activation and exhaustion in spontaneous tumor models. These results together support the future development of IFNγ-targeted immunoPET for clinical use. In addition to the targets discussed above, there are several other targets successfully leveraged for imaging T cells, including granzyme B^834,835 and IL-2 receptor.⁸³⁶ However, peptides and interleukins rather than antibodies were used as the targeting vectors in these platforms.

6. IMMUNOPET IMAGING OF INFLAMMATION

Although 18F-FDG PET is a clinically viable method to noninvasively quantify inflammation, it lacks specificity due to the uptake of the tracer by metabolically active tissues. Inflammation is decisive in tumor progression, and inflammation and cancer share certain signaling molecules.⁸³⁷ Therefore, some of the aforementioned immunoPET probes could reasonably be extended to detect and evaluate inflammatory diseases.⁸³⁸ In this section, we highlight the role of immunoPET in detecting several inflammatory diseases.

6.1. Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a polygenic and multifactorial joint disease characterized by autoantibodies to various molecules such as IgG and citrullinated proteins.⁸³⁹ Treatment strategies for RA mainly include disease-modifying antirheumatic drugs, which can be further categorized into small molecules (e.g., methotrexate) and biologic agents (e.g., anti-TNFα mAbs).⁸⁴⁰ Although RA is incurable, it is important to identify and treat RA patients at earlier stages to delay the joint damage.

To track autoantibody to glucose-6-phosphate isomerase (GPI), Wipke et al. initially radiolabeled an anti-GPI IgG with ⁶⁴Cu and found that the developed probe specifically localized to the diseased joints.⁸⁴¹ This highlighted the possibility that immunoPET imaging may help us understand the sophisticated autoimmunity responses involved in human RA. F8-IL10 is a novel treatment option for RA patients. F8-IL10 was developed by fusing the Fv fragment of the human antibody F8 with the anti-inflammatory cytokine IL10, resulting in selective delivery of IL10 to the fibronectin-expressing inflammatory sites.⁸⁴² A translational study investigating [¹²⁴I]I-F8-IL10 found that this radiotracer accumulated readily in the arthritic joints of RA patients. However, this immunoPET study also revealed very rapid blood clearance of the radiotracer and unexpected high uptake in the liver and spleen.⁸⁴³ Certolizumab pegol (CZP) is a PEGylated fab fragment targeting TNFα and has shown remarkable efficacy in controlling the symptoms of RA. ⁸⁹Zr-DFO-CZP was recently synthesized and immunoPET imaging with this tracer specifically located diseased joints and paws of transgenic mice expressing human TNFα.⁸⁴⁴

Fibroblast activation protein (FAP) is a type II trans-membrane glycoprotein belonging to the family of serine prolyl oligopeptidases. In addition to being a theranostic target for cancers,^845–847 FAP is closely associated with the progression of RA. A fully human noninternalizing anti-FAP antibody 28H1 that binds to both murine and human FAP has been reported.⁸⁴⁸ When labeled with ⁸⁹Zr, it specifically visualized inflamed joints of RA models (Figure 29).⁸⁴⁹ More importantly, 28H1-based molecular imaging tracers could monitor the response of RA to different treatment options at molecular levels.^850–852

Figure 29.

ImmunoPET imaging of rheumatoid arthritis (RA). (a) ⁸⁹Zr-28H1 immunoPET/CT imaging of a mouse with collagen-II-induced arthritis 72 h after injection of the radiotracer. ⁸⁹Zr-28H1 accumulated in the inflamed joints with high contrast. (b) ¹⁸F-FDG PET/CT imaging also showed uptake in the inflamed joints but the uptake was lower than that of ⁸⁹Zr-28H1. Reproduced with permission from ref 849. Copyright 2015 SNMMI.

6.2. Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder composed of two major subtypes: Crohn’s disease and ulcerative colitis.⁸⁵³ In current clinical practice, endoscopy is the most commonly applied technique to diagnose and monitor IBD. However, this invasive examination fails to provide information regarding molecular markers involved in the development and progression of IBD. By targeting integrins and immune mediators, immunoPET imaging approaches have been adapted to sensitively grade the disease severity.⁸⁵⁴ An initial study radiolabeled a β₇ integrin-specific mAb (FIB504.64) and showed specific uptake of ⁶⁴Cu-DOTAFIB504.64 in the gut of mice with dextran sulfate sodium (DSS)-induced colitis.⁸⁵⁵ To lower background signals in nontarget organs, immunoPET probes were developed using FIB504.64 fragments.⁸⁵⁶ In these studies, ⁶⁴Cu-NOTA-FIB504.64-F(ab′)₂ outperformed ⁶⁴Cu-NOTA-FIB504.64-Fab in detecting DSS-induced colitis. Moreover, ⁶⁴Cu-NOTA-FIB504.64-F(ab′)₂ immunoPET also demonstrated better imaging contrast than ⁶⁴Cu-DOTA-DATK32, a mAb-derived tracer targeting the integrin heterodimer α₄β₇.

More recently, several immunoPET tracers targeting innate immune cells, interleukins, and CD4⁺ T cells have been characterized in murine models of colitis. Dmochowska et al. reported increased IL-1β and increased infiltration of CD11b⁺ CD3⁻ innate immune cells in the inflamed colon of colitic mice.⁸⁵⁷ ImmunoPET imaging studies in this work further corroborated the above observations. While ⁸⁹Zr-α-CD11b (clone M1/70) immunoPET detected colonic inflammation with higher sensitivity than MRI, ⁸⁹Zr-α-IL-1β (clone B122) immunoPET correlated the disease severity, although less robustly than ¹⁸F-FDG (Figure 30a–c). ⁸⁹Zr-malDFO-GK1.5 cDb is an antimouse CD4 radiotracer initially used for imaging immune system reconstitution.⁷⁹⁴ Freise et al. recently assessed the diagnostic value of ⁸⁹Zr-malDFO-GK1.5 cDb in tracking CD4⁺ T cell infiltration in DSS-induced colitis mouse models.⁸⁵⁸ ImmunoPET imaging showed radiotracer uptake in mesenteric lymph nodes and colons of the colitic mice (Figure 30d,e), which correlated with increased infiltration of CD4⁺ T cells into the inflamed colons revealed by IHC staining. Taken together, immunoPET is less intrusive and could provide useful information regarding the dynamics of immune cells throughout the intestines. This may in turn help monitor the disease severity and predict the treatment responses.

Figure 30.

ImmunoPET imaging of inflammatory bowel disease (IBD). (a) ⁸⁹Zr-a-IL-1b, (b) ⁸⁹Zr-α-CD11b immunoPET imaging, and (c) conventional ¹⁸F-FDG PET imaging all detected dextran sulfate sodium (DSS)-induced colonic inflammations, which was indicated by uptake in the colons. Reproduced with permission from ref 857. Copyright 2019 SNMMI. (d) ⁸⁹Zr-malDFO-GK1.5 cDb, an immunoPET probe targeting mouse CD4, was used to image IBD by capturing CD4⁺ T cells. Ex vivo 89Zr-malDFO-GK1.5 cDb immunoPET imaging showed increased radiotracer concentration in the DSS-treated colons, ceca, and mesenteric lymph nodes (MLNs).(e) Corresponding gross specimens obtained from the normal mice and from the colitic mice. Note that colons of the DSS-treated mice were shorter than that of the control mice. Reproduced with permission from ref 858. Copyright 2018 SNMMI.

6.3. Other Inflammatory Diseases

With the development of specific mAbs for various inflammation-related antigens, immunoPET probes have been developed to image several other inflammatory diseases such as graft versus host disease,^859,860 atherosclerotic plaques,^861–863 and inflammation-induced lymphangiogenesis.^864,865 In addition, immunoPET or immunoPET/MRI hybrid imaging are being used to detect bacterial, fungal, or viral infections.^866–870 In the case of invasive pulmonary aspergillosis, a mouse mAb mJF5 and its humanized derivative hJF5 have proven to be highly specific to mannoprotein antigens produced by Aspergillus. Preclinical studies have validated the accuracy of ⁶⁴Cu-DOTA-mJF5 and ⁶⁴Cu-NODAGA-hJF5 in detecting Aspergillus lung infection.^871–873 From these preclinical reports, it is conceivable that upon continuous investigation and clinical translation, immunoPET imaging holds enormous potential to diagnose the abovementioned inflammatory diseases.

7. IMMUNOPET IMAGING OF BETA CELL MASS

Diabetes mellitus (DM) is a metabolic disease characterized by a functional loss of beta cell mass (β cell mass, BCM) or the insufficient response of beta cells to insulin. It has been estimated that over 550 million people will suffer from diabetes by 2030.⁸⁷⁴ To monitor the early and dynamic change of BCM, various molecular imaging probes have been developed, including several antibody-based probes.⁸⁷⁵ Transmembrane protein 27 (TMEM27) is a validated BCM biomarker, and TMEM27-targeted immunoPET and fluorescent imaging probes have shown promise in imaging BCM.⁸⁷⁶ Dipeptidyl peptidase 6 (DPP6) is a newly identified biomarker for pancreatic alpha and beta cells. A VHH (i.e., 4hD29) targeting DPP6 was produced, initially radiolabeled with 99mTc, and used for immunoSPECT imaging of endocrine cell mass (ECM).⁸⁷⁷ More recently, ⁶⁸Ga-NOTA-4hD29 was developed and immunoPET imaging with this agent successfully detected DPP6-positive tumors.⁸⁷⁸ Upon clinical translation, this agent holds promise for detecting ECM, transplanted human islets, and DPP6-positive tumors.

ImmunoPET imaging of BCM is advantageous compared to other imaging modalities because of its targeting sensitivity and specificity. In preclinical models, beta cell-specific immunoPET has been used to image insulinomas and grafted human islets. However, it is very challenging for immunoPET to detect islets scattered across the pancreas in preclinical models, due either to the high background signal or to the restricted species reactivity of the antibodies. It remains to be determined whether immunoPET imaging can visualize beta cell islets in humans. If so, this novel imaging modality may improve the management of diabetes by monitoring the dynamic change of BCM over time. As we mentioned above, ⁵²Mn is a relatively new radiometal used for immunoPET. Two interesting studies have elucidated that ⁵²Mn alone serves as a sensitive tracer for measuring and quantifying BCM.^879,880 Clinical trials are warranted to confirm the unique application of ⁵²Mn PET in quantifying and monitoring BCM.

8. IMMUNOPET IMAGING-GUIDED ADVANCED THERAPEUTICS

The landscape of theranostics has evolved over time. The management of thyroid diseases (e.g., differentiated thyroid cancer and hyperthyroidism) has been revolutionized since the use of theranostic radioiodine isotopes in the 1940s.^881,882 With the identification of highly specific cancer antigens and the concomitant development of radiopharmaceuticals, PSMA-targeted and somatostatin-derived theranostic agents have achieved remarkable success in diagnosing and treating advanced PCas and neuroendocrine tumors, respectively.^883,884 This trend is expected to flourish with the discovery of newer molecular targets and synchronous advance in radiochemistry.⁸⁸⁵

ImmunoPET imaging can visualize the spatial heterogeneity of target expression and thus predict the responses of therapeutic antibodies, including the immune checkpoint inhibitors.^21,809,814 Besides selecting patients for antibody therapies, immunoPET imaging is a robust approach to select patients for antibody-based therapies, particularly RIT. Most of the aforementioned biomarkers can be leveraged to develop RIT agents. Traditional RIT has proven useful for treating radiosensitive tumors and disseminated hematologic malignancies.^886–888 However, it is less effective for radioresistant or bulky tumors. It is anticipated that innovative pRIT (e.g., agents produced via the Dock-and-Lock technology) may deliver a higher therapeutic dose to the tumor while reducing hematologic toxicity. Several exemplary targets with clinical theranostic evidence are discussed below.

B7-H3 (CD276) is an immunoregulatory glycoprotein that is also overexpressed in a broad spectrum of solid tumors.⁸⁸⁹ Preclinical studies have reported that immunoPET imaging was able to delineate CD276 expression on tumor cells and blood vessels.^890,891 8H9 (Omburtamab) is a murine mAb against B7-H3,⁸⁹² and ¹³¹I-8H9 has been used to treat several types of solid tumors after intrathecal administration.^893,894 A recent study elucidated that pretherapy immunoPET imaging with ¹²⁴I-8H9 allowed for noninvasive estimation of the therapeutic index of ¹³¹I-8H9.⁸⁹⁵ Interestingly, ¹²⁴I itself may serve as a theranostic radioisotope due to the emission of high-energy positrons. To test the clinical feasibility, a phase I clinical trial was carried out to assess the theranostic value of ¹²⁴I-8H9 in patients with diffuse intrinsic pontine glioma.⁸⁹⁶ The results demonstrated that ¹²⁴I-8H9 was precisely delivered to the brainstem lesions after intratumoral infusion (Figure 31). Moreover, immunoPET/MR imaging following ¹²⁴I-8H9 infusion quantitated the absorbed dose in the tumors and other organs. These clinical studies together demonstrate that intrathecal administration of ¹²⁴I-8H9 and ¹³¹I-8H9 in tandem (or ¹²⁴I-8H9 alone) may maximize the therapeutic outcome while limiting the systemic toxicity.

Figure 31.

Representative immunoPET/MR imaging of a patient with diffuse intrinsic pontine glioma after convection-enhanced delivery of ¹²⁴I-8H9. The axial (upper sections) and sagittal (lower sections) fused PET/MR images showed predominant retention of ¹²⁴I-8H9 in the brainstem. In this case, ¹²⁴I-8H9 servers as a theranostic agent allowing for concurrent imaging, dosimetry, and therapy. Reproduced with permission from ref 896. Copyright 2018 Elsevier Inc.

CEA is another biomarker extensively studied for cancer theranostics over the years. CEA-directed pRIT was initially investigated in patients with primary colorectal cancer⁸⁹⁷ and then in patients with medullary thyroid cancer.^319,320 However, the frequent hematological toxicity and immune responses of these first-generation approaches limited their broad applications. The IMP288 peptide allows facile radiolabeling with either therapeutic (such as ¹⁷⁷Lu, ⁹⁰Y, and ²¹³Bi) or diagnostic (such as ⁶⁸Ga, ¹¹¹In, and ⁸⁶Y) radiometals, providing a perfect platform for designing theranostic agents. A preclinical study has shown the survival benefit of TF2/¹⁷⁷Lu-IMP288 pRIT in colorectal cancer models.⁸⁹⁸ A follow-up clinical trial further reported that TF2/¹⁷⁷Lu-IMP288 pRIT was feasible in patients with colorectal cancer, but the efficacy was limited because all the patients showed progressive disease eight weeks later.^899,900 Use of ²¹³Bi (T_1/2 = 45.6 min) instead of ¹⁷⁷Lu in this pretargeted system showed comparable therapeutic efficacy in colorectal cancer models, but the dosing schedule needs to be optimized to reduce nephrotoxicity before clinical translation.⁹⁰¹ With further development of humanized antibodies,⁹⁰² dual-modal imaging probes,⁹⁰³ and the pRIT system,⁹⁰⁴ CEA-targeted theranostic toolbox holds excellent prospects for improving the management of CEA-positive human malignancies.

As mentioned earlier (section 4.3.1), a recent clinical trial has demonstrated the clinical feasibility of CA19.9-targeted immunoPET in localizing PDAC.³⁹ This study also indicated that 5B1 might deliver therapeutic doses to PDAC after labeled with beta emitters. A preclinical study explored the therapeutic benefit of CA19.9-directed pRIT, in which 5B1-TCO was administered 72 h before the injection of ¹⁷⁷Lu-DOTA-PEG₇-Tz or ¹⁷⁷Lu-CHX-A′′-DTPA-PEG₇ -Tz.⁹⁰⁵ The results demonstrated that the former combination showed a dose-dependent therapeutic response in PDAC models. Along with this preclinical evidence, an ongoing clinical trial (NCT03118349) is evaluating the safety and dosimetry of ¹⁷⁷Lu-CHX-A′′-DTPA-5B1 in patients with CA19.9-positive malignancies. 5B1 as a standalone monotherapy or in combination with chemotherapy is also under clinical investigation (NCT02672917). On the basis of the reported evidence and future clinical trial results, the 5B1-based theranostic toolbox may hopefully improve the clinical management of CA19.9-positive malignancies.

RIT is an established tool in the treatment of hematologic malignancies, such as NHL.^906,907 CD20 is a classical theranostic target since two radiolabeled murine mAbs (¹³¹I-tositumomab, Bexxar; ⁹⁰Y-ibritumomab tiuxetan, Zevalin) were approved for the treatment of B-cell NHL.⁹⁰⁸ Nonetheless, the clinical use of Bexxar and Zevalin is stagnant. Bexxar has not been commercially available since 2014 for multiple reasons (mainly due to the disappointing profits). In two recent studies, researchers reported the feasibility and satisfactory treatment efficacy of ⁹⁰Y-rituximab in patients with relapsed or refractory NHL.^909,910 Furthermore, several preclinical and clinical studies have reported the superior effectiveness of pRIT or RIT in treating lymphomas by targeting several targets, such as CD20,^911,912 CD38,⁵⁶⁰ and CD45.⁹¹³ CD33 is another alternative target for RIT of hematological malignancies^914,915 and for immunoPET imaging.⁹¹⁶ Continuous innovation of the pRIT systems and incorporation of immunoPET techniques may reinvigorate the enthusiasm for managing hematologic malignancies with nuclear medicine approaches. Readers are recommended to refer to an excellent review parsing RIT for more information.¹⁶⁷

9. IMMUNOPET IMAGING-GUIDED DRUG DEVELOPMENT

In the development of antibody- and antibody-based therapeutics, iterative approaches are needed, including the identification of antigens and screening and selection of optimal antibodies. Traditionally, several analytical and structural techniques (e.g., mass spectrometry, liquid chromatography, and electrophoresis) are used to assess the developed antibody therapeutics. Apart from these procedures, it is necessary to examine the pharmacodynamic properties and safety profiles of antibodies before clinical translation for human use.⁹¹⁷ Complementary to their role as diagnostic methods in the clinic, molecular imaging approaches are increasingly being used for fundamental research and antibody drug development.^918–920 Molecular imaging can assist antibody drug discovery (e.g., target selection and antibody optimization) as well as the clinical assessment of antibody drugs (e.g., distribution, clearance, safety profile, and therapeutic efficacy). As a result, the translation of promising antibody candidates can be accelerated from preclinical prototype to bedside reality.^921,922

With the development and use of total-body PET scanners, immunoPET imaging will facilitate the thorough assessment of mAb pharmacokinetics up to 30 days at the preclinical stage.^923–925 ImmunoPET imaging can reveal both dose-dependent and dose-independent uptake of mAb in normal organs and in tumors. This is particularly useful when the target antigen is expressed in tumors as well as normal tissues.⁹²⁶ While the dose-dependent uptake is largely mediated by relevant receptors expressed on the surface of tumor cells, the dose-independent uptake is mainly caused by osmosis and retention of mAb in the TME. ImmunoPET imaging will help estimate the therapeutic effect of antibody therapeutics in phase I dose-escalation studies and calculate the amount of unlabeled antibody required for preloading or coadministration,^41,927 which will saturate target antigens in normal organs and maximize the binding of mAb to the target antigens in tumors.

In addition to mAbs, ADCs are among the most effective targeted cancer therapeutics. Third-generation ADCs with increased potency and stability are under clinical investigation.⁹²⁸ During ADC development, cytotoxic drugs are linked to mAbs via bifunctional linkers. ImmunoPET imaging has shown its value in assessing the in vivo stability of novel linkers. This was exemplified by two recent studies where a novel platinum(II) linker was developed for ADC conjugates.^929,930 More importantly, immunoPET imaging can characterize the effect of drug-to-antibody ratio on the overall blood retention and tumor-targeting efficacies of the developed ADCs (Figure 32).^930–932 As another therapeutic alternative, RIT delivers therapeutic radionuclides to the tumor site by taking advantage of antibody specificity.⁹³³ ImmunoPET imaging permits dosimetry calculations for these therapies and further predicts dose-limiting organs prior to the RIT,⁹³⁴ optimizing the development and use of RIT agents.

Figure 32.

ImmunoPET imaging guides antibody drug development. (a) Trastuzumab-Lx-AF is an antibody–drug conjugate developed by linking trastuzumab with auristatin F (AF) via the linker Lx. To evaluate the influence of drug-to-antibody ratios (DARs), ⁸⁹Zr-DFO-trastuzumab-Lx-AF immunoPET/CT imaging was carried out at 96 h postinjection of the radiotracer. The imaging results demonstrated the varying stabilities of the Lx-based ADCs. Importantly, a DAR of 2.6 did compromise the tumor targeting. (b) Biodistribution studies further confirmed the immunoPET imaging results (black bars, DAR of 0; red bars, DAR of 2.6; blue bars, DAR of 5.2; *, P < 0.05). Reproduced with permission from ref 930. Copyright 2018 SNMMI.

ImmunoPET-guided drug development provides insightful information regarding the distribution and targeting potential of new antibody drugs or antibody-based therapeutics. However, it should not be ignored that radionuclides, chelators, and radiolabeling methods may alter the inferred pharmacokinetics of the conjugated antibody tracers.

10. FUTURE PERSPECTIVES AND CONCLUSIONS

As described in this review, immunoPET is an invaluable companion diagnostic tool actively changing the management of cancers and noncancerous diseases (Table 2). However, it must be noted that most of the immunoPET probes have only been assessed in preclinical stages or in small cohorts of patients. Further studies are needed to translate some of the promising immunoPET probes and to confirm the diagnostic value of the clinically used ones. The development of antibody therapeutics will continue to reshape the therapeutic landscape of human diseases, and more sophisticated immunoPET imaging strategies will be designed accordingly. By making the right diagnoses and optimizing subsequent therapeutic decisions, immunoPET will hopefully help clinicians refine clinical practice and realize truly personalized medicine. The ultimate purpose of designing and using immunoPET is to facilitate better management of patients and lessen the financial burden for them and society.^935,936

Table 2.

Representative Clinical-Stage ImmunoPET Imaging Probes^a

probe	target	targeting moiety	cancer types	ref
89Zr-Df-cetuximab	EGFR	mAb	solid tumors	356,358
89Zr-panitumumab	EGFR	mAb	colorectal cancer	368
89Zr-Df-trastuzumab	HER2	mAb	breast cancer, EGA	41,391,399
64Cu-DOTA-trastuzumab	HER2	mAb	breast cancer	394,395
89Zr-Df-pertuzumab	HER2	mAb	breast cancer	396
124I-trastuzumab	HER2	mAb	GC, GEC	400
68Ga-HER2-Nanobody	HER2	Nanobody	breast cancer	412
68Ga-ABY-025	HER2	Affibody	breast cancer	430,433
64Cu-DOTA-patritumab	HER3	mAb	solid tumors	453
89Zr-GSK2849330	HER3	mAb	solid tumors	455
89Zr-lumretuzumab	HER3	mAb	solid tumors	458
89Zr-Df-bevacizumab	VEGF	mAb	solid tumors	469,471,474
124I-huA33	A33	mAb	colorectal cancer	185
89Zr-cmAb U36	CD44v6	mAb	HNSCC	118
89Zr-RG7356	CD44	mAb	solid tumors	523,524
89Zr-rituximab	CD20	mAb	lymphoma	536
89Zr-DFO-5B1	CA19.9	mAb	pancreatic cancer	39
89Zr-huJ591	PSMA	mAb	prostate cancer	619,620
89Zr-Df-IAB2M	PSMA	Mb	prostate cancer	633,634
68Ga-IMP288	CEA	BsAb	medullary thyroid cancer	654
89Zr-AMG 211	CEA/CD3	BiTE	gastrointestinal adenocarcinomas	664
89Zr-CEA-IL2	CEA	immunocytokine	solid tumors
89Zr-girentuximab	CAIX	mAb	renal cell carcinoma	674
124I-cG250	CAIX	mAb	renal cell carcinoma	675
89Zr-DF0-MSTP2109A	STEAP1	mAb	prostate cancer	748,749
89Zr-fresolimumab	TGF-β	mAb	glioma	754
89Zr-Df-IAB22M2C	CD8	Mb	solid tumors	793
89Zr-atezolizumab	PD-L1	mAb	NSCLC, TNBC, bladder cancer	21
18F-BMS-986192	PD-L1	adnectin	lung cancer	809
89Zr-nivolumab	PD1	mAb	lung cancer	809
[124I]I-F8-IL10	fibronectin	Fv fragment	rheumatoid arthritis	843

^aAbbreviations: mAb, monoclonal antibody; EGA, esophagogastric adenocarcinoma; GC, gastric cancer; GEC, gastroesophageal junction cancer; HNSCC, squamous cell carcinoma of the head and neck; Mb, minibody; Fv fragment, single-chain antibody variable domain (Fv) fragment; BsAb, bispecific antibody; BiTE, bispecific T-cell engager; PSMA, prostate-specific membrane antigen; CEA, carcinoembryonic antigen; CAIX, carbonic anhydrase IX; STEAP1, six-transmembrane epithelial antigen of prostate-1; TGF-β, transforming growth factor-β; NSCLC, nonsmall cell lung cancer; TNBC, triple-negative breast cancer; PD-L1, programmed death ligand-1; PD1, programmed death receptor 1.

There are several concerns about the immunoPET technique. A fundamental question that needs to be addressed is under which settings immunoPET may be integrated into the clinical diagnostic toolbox. In our view, immunoPET is a companion diagnostic tool that should be used together with clinically approved or clinical-stage therapeutic regimens. An initial immunoPET imaging is preferred to be performed before the commencement of targeted antibody or small-molecule inhibitor treatment because it will provide pivotal information on the baseline expression level of the target. The unique information obtained at the baseline will further allow adequate restaging and evaluation of the disease. Repeated immunoPET imaging after the treatments will help evaluate the therapeutic response and also the change of the target, especially for patients with multiple biopsy-inaccessible tumor lesions. Several other concerns might be gradually resolved with the progress of the field. Specifically speaking, the development of GMP-compliant production and purification processes may spur the clinical use of this imaging technique while minimizing unnecessary radiation dose to the health care staff.^937,938 ImmunoPET imaging with total-body PET scanners will further decrease radiation exposure without compromising the image quality.^939–941 Furthermore, advances in PET technology and reconstruction algorithms will lead to improved spatial and temporal resolution of immunoPET images.⁹⁴² In most instances, immunoPET imaging is performed for patients with metastatic diseases. Consequently, another misgiving is the lack of histochemical confirmation of some of the tracer-avid lesions. However, the primary purpose of the initial immunoPET imaging is to stratify patients by mapping the expression of a specific biomarker. If the uptake or sizes of the tracer-avid lesions decrease on post-treatment immunoPET images, it is rational to consider these lesions as histopathology-positive.

For developing antibody-based radiopharmaceuticals, we would like to emphasize the importance of multidisciplinary collaboration. Specifically, multiple approaches (e.g., genomic, serological, proteomic, biological, and bioinformatical approaches) should be leveraged to identify antigens highly or exclusively overexpressed on the surface of the tumor cells, tumor stromal cells, tumor vasculature endothelial cells, immune cells, or beta cells.⁹⁴³ While the abundance and specificity are key factors when selecting suitable imaging targets, the function and stability of the targets need also to be considered.⁹⁴⁴ Of the diverse targets that are currently being investigated in preclinical studies or in clinical trials, some are relatively specific for certain tumor types (for example, TROP-2 for breast cancer and PCa, and CD138 for MM). When using antibodies as targeting vectors, it is important to remember that different IgG types have varying circulation time,⁹⁴⁵ and the interactions between the Fc domain and FcγR/FcRn dynamically regulate the immunological functions of the developed antibody.^946–948 For instance, tislelizumab (BGBA317) is a humanized IgG4 that binds to PD-1 but not to FcγRI.⁹⁴⁹ This improvement results in enhanced tumor growth inhibition when compared to BGB-A317/IgG4S228P, which has a high affinity to FcγR. Thus, Fc engineering could be used to introduce mutations that may abrogate the binding of IgG with FcγR. This strategy could also be exploited to increase the cellular accumulation of an antibody–antigen complex when targeting secreted antigens⁷¹⁴ or to enhance the persistence of VHHs in circulation.⁹⁵⁰ In addition, strategies like deglycosylation of antibodies may further improve the immunoPET imaging quality.^951,952 A high-affinity antibody does not always guarantee high tumor uptake because other factors such as circulating antigens and tumor vasculature may affect the accessibility of the radiolabeled antibody. Aside from the singly targeted imaging probes, antibody-based heterodimers, or dual-targeting probes may have higher targeting efficacy and superior specificity than their monospecific peers.^30,32,953 With the advent and evolvement of click chemistry,²⁶¹ future studies may further harness this powerful method to synthesize modular immunoPET probes with improved in vivo performance. Furthermore, pretargeting strategies may also be harnessed to optimize the imaging quality.^954,955

In the characterization of antibody-based diagnostic or theranostic probes, attention should be paid whether immunodeficient strains of animals are used in the imaging studies and how this may impact the imaging performance of the investigated probes. For example, a recent study reported that immunoPET radiotracers had inefficient tumor targeting and high off-target binding to the spleen in the highly immunodeficient mouse strains.⁷⁶⁶ This was consistent with a previous study which reported that ADCs had limited antitumor activity in the ultra immunodeficient NSG mice.⁹⁵⁶ In this setting, the use of humanized mouse models or nonhuman primates is very necessary to assess the imaging performance of the immunoPET probes prior to pilot clinical investigations.⁹⁵⁷ In addition, antibodies cross-reactive with human, nonhuman primate, and mouse antigens would be beneficial in preclinical immunoPET imaging studies.

Other than their use in immunoPET, antibodies are actively investigated as either monotherapy agents or antibody conjugates for effective cancer treatment. Antibodies can be modified with therapeutic radionuclides and photosensitizers for RIT¹⁶⁷ and PIT,^958,959 respectively, as novel treatment options. Two radiolabeled therapeutic anti-CD20 antibodies, ¹³¹I-tositumomab and ⁹⁰Y-ibritumomab tiuxetan, have been approved by the FDA for the treatment of B-cell lymphoma.^531,960 To maximize the therapeutic index, pRIT strategies may be used. For detailed information on RIT, readers are recommended to refer to other excellent reviews.^302,954,961 RTKs and oncogenic proteins are ideal candidates for RIT.^237,618,962 For instance, a recent study reported that pRIT with anti-HER2-DOTA-pRIT + ¹⁷⁷Lu-DOTA-Bn inhibited HER-2 positive breast cancers and substantially improved survival without inducing toxicity in normal tissues.⁹⁶³ Therefore, radiolabeled antibodies or antibody fragments targeting some of the selected makers discussed above may provide additional therapeutic options for cancer patients in the era of precision medicine.^964,965 The use of pairs of β⁺ and β⁻ emitting radionuclides (e.g., ⁸⁶Y/⁹⁰Y) is desirable as a promising theranostic platform for sequential imaging, dosimetry, and therapy. In addition to the traditional β-emitting therapeutic isotopes, accumulating evidence is supporting the clinical application of targeted alpha therapy, where mAbs or small molecules are labeled with therapeutic α-emitters. ²²⁵Ac (T_1/2 = 10.0 d) and ²¹³Bi are both attractive therapeutic α-emitters, but the therapeutic index of ²¹³Bilabeled agents was inferior to ²²⁵Ac-labeled agents.^966,967 In translating these radiotherapeutics to the clinic, the safety profiles of the agents need to be carefully assessed. For therapeutic radionuclides without intrinsic imaging capabilities, immunoPET imaging may help estimate the pharmacokinetics of the therapeutic radiopharmaceuticals and evaluate the dose-limiting organs.

Optical fluorescence imaging using dye-labeled mAbs or sequential PET/NIR imaging with dual-labeled mAbs has the potential to improve cancer surgery outcomes.^968–970 However, the payload of fluorophores needs to be carefully determined because the ratio of the fluorophore to mAb greatly affects both the pharmacokinetics and tumor-targeting efficiency of the developed probes.^971,972 Besides, the emission of charged particles from radionuclides traveling through dielectric materials, such as in living subjects, results in the production of Cerenkov luminescence.⁹⁷³ Cerenkov luminescence has been validated effective for triggering photodynamic therapy.^974,975 More interestingly, Cerenkov luminescence imaging (CLI) with optical imaging systems after the administration of radioactive tracers is an emerging imaging paradigm and can be exploited for image-guided surgery.⁹⁷⁶ While applications involving Cerenkov luminescence are still in their infancy, they hold great potential.^977,978

While several advantages exist over other imaging modalities, the broad application of immunoPET may be restrained by a scarcity of radiometals and antibodies in less-developed countries. Therefore, immunoSPECT imaging may alternatively fill the gap. To this end, γ-emitting radionuclides like ^99mTc, ¹²³I, and ¹¹¹In may be used to develop immunoSPECT imaging probes.⁹⁷⁹ It should be noted that the high-energy γ emission from ¹³¹I makes it unfavorable for developing immunoSPECT probes. The use of novel radiolabeling methods may improve the stability, binding affinity, internalization, and intracellular track of the radioiodinated mAbs.⁹⁸⁰

In summary, the development of immunoPET imaging strategies has achieved great success in the past decade. With continuous improvement and clinical translation, immunoPET imaging holds great promise in optimizing clinical management of human diseases, especially cancers.

ABBREVIATIONS USED

ADCP

antibody-dependent cellular phagocytosis

ADCC

antibody-dependent cell-mediated cytotoxicity

ADC

antibody–drug conjugate

AR

androgen receptor

BBB

blood–brain barrier

BiTE

bispecific T-cell engager

BCM

beta cell mass

CDC

complement-dependent cytotoxicity

CB-TE2A

4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane

CA19.9

carbohydrate antigen 19.9

CA-125

carbohydrate antigen 125

CEA

carcinoembryonic antigen

CAIX

carbonic anhydrase IXcc

RCC

clear cell renal cell carcinoma

CSC

cancer stem cell

CTLA-4

cytotoxic T lymphocyte antigen 4

CLI

Cerenkov luminescence imaging

CuAAC

Cu(I)-catalyzed 1,3-dipolar cycloaddition between azides and alkynes

DSS

dextran sulfate sodium

DLL3

delta-like 3

EDTA

ethylenediaminetetraacetic acid

EGFR

human epidermal growth factor receptor

FcγR

Fcγ receptor

FcRn

neonatal Fc receptor

¹⁸F-FDG

¹⁸F-fluorodeoxyglucose

GPI

glucose-6-phosphate isomerase

GPA33

glycoprotein A33

HNSCC

head and neck squamous cell carcinoma

HCAb

heavy-chain-only antibody

HER2/ErbB2

human epidermal growth factor receptor 2

HER3/ErbB3

Human epidermal growth factor receptor 3

HGF

hepatocyte growth factor

IHC

immunohistochemistry

IGF-1R

insulin-like growth factor-1 receptor

IBD

inflammatory bowel disease

ImmunoPET

immuno-positron emission tomography

LGR5

leucine-rich repeat-containing G-protein coupled receptor 5

mAb

monoclonal antibody

MM

multiple myeloma

MSLN

membrane-bound surface glycoprotein mesothelin

NSCLC

nonsmall-cell lung cancer

NHL

non-Hodgkin’s lymphoma

NOTA

1,4,7-triazacyclononane-1,4,7-triacetic acid

NODAGA

1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid

PDGFR

platelet-derived growth factor receptor

PD-L1

programmed death ligand-1

PET

positron emission tomography

pRIT

pretargeted radioimmunotherapy

PDAC

pancreatic ductal adenocarcinoma

PSCA

prostate stem cell antigen

PSA

prostate-specific antigen

PD1

programmed death receptor 1

PIT

photoimmunotherapy

PSMA

prostate-specific membrane antigen

RCY

radiochemical yield

RTKs

receptor tyrosine kinases

RIT

radioimmunotherapy

RA

rheumatoid arthritis

SPECT

single-photon emission computed tomography

sdAb

single-domain antibody

scFv

single-chain variable fragment

SPAAC

strain-promoted azide–alkyne cycloaddition

SrtA

sortase A

STEAP1

six-transmembrane epithelial antigen of prostate-1

TKIs

tyrosine kinase inhibitors

TME

tumor microenvironment

TGF-β

transforming growth factor-β

TROP-2

trophoblast cell-surface antigen 2

TAG-72

tumor-associated glycoprotein-72

Treg

regulatory T cells

VHH

variable domain of the heavy chain of a HCAb

VEGF

vascular endothelial-derived growth factor

VEGFR

vascular endothelial-derived growth factor receptor

Biographies

Weijun Wei obtained his M.D. and Ph.D. degrees with distinction in nuclear medicine and molecular imaging from the School of Medicine, Shanghai Jiao Tong University, in 2019. Dr. Wei was a joint graduate at the University of Wisconsin–Madison under the supervision of Prof. Weibo Cai during 09/2017–09/2018. Currently, Dr. Wei serves as a nuclear medicine physician at Renji Hospital, School of Medicine, Shanghai Jiao Tong University, where he is focusing on developing novel antibody- and nanobody-based probes for imaging and treating cancers. Dr. Wei has published more than 20 articles in several world-renowned journals.

Zachary Rosenkrans is currently a Ph.D. student in the Pharmaceutical Sciences program at the University of Wisconsin–Madison under the supervision of Dr. Weibo Cai. He previously received a B.S. in Chemical Engineering from the University of Kansas in Lawrence, KS. His research is focused on developing nanomaterial-based platforms for image-guided drug delivery and theranostics.

Prof. Jianjun Liu is the chief nuclear medicine physician at Renji Hospital, School of Medicine, Shanghai Jiao Tong University. As the director of the department, Prof. Liu leads a multidisciplinary team exploring novel therapeutic approaches for cancers on one hand and developing next-generation molecular imaging probes on the other hand. One of his specific interests is to design, validate, and translate antibody- and nanobody-based immunoPET imaging tracers. Prof. Liu has authored more than 40 research papers and tutored over 10 graduates.

Prof. Gang Huang is a well-recognized professor and leader of nuclear medicine and molecular imaging in China. His research and clinical work are focused on the diagnosis and treatment of malignancies, especially theranostics using nuclear medicine and molecular imaging approaches. He is the Elected President of the Asia Oceania Federation of Nuclear Medicine and Biology, Dean of the Asia Oceania School of Nuclear Medicine, the Ninth President of the Chinese Nuclear Medicine Society, and Editor-in-Chief of the Chinese Journal of Nuclear Medicine and Molecular Imaging. Prof. Huang has authored more than 200 articles, edited more than 30 books, given over 500 talks and received over 10 awards, such as the State Scientific and Technological Progress Award (the second place) and the Shanghai Medical Science and Technology Award (the first place), etc.

Prof. Quan-Yong Luo obtained his M.D. degree in nuclear medicine and molecular imaging from Shanghai Jiao Tong University in 2006. Currently, he is the director and chief physician at the Department of Nuclear Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Besides his interest in clinical PET and SPECT imaging, he has been actively involved in radioiodine treatment of differentiated thyroid cancers for two decades. Prof. Luo has published more than 70 articles, in which his team has thoroughly investigated the diagnostic efficacies of SPECT and PET imaging, as well as the treatment efficacy of radioiodine in patients with differentiated thyroid cancer.

Prof. Weibo Cai is a Vilas Distinguished Achievement Professor of Radiology/Medical Physics/Biomedical Engineering/Materials Science & Engineering/Pharmaceutical Sciences at the University of Wisconsin–Madison, USA. He received a Ph.D. degree in Chemistry from UCSD in 2004. Prof. Cai’s research at UW–Madison (http://mi.wisc.edu/) is mainly focused on antibody-based molecular imaging and nanotechnology. He has authored more than 300 articles (H-index: 79), edited three books, and received many awards (e.g., Fellow of AIMBE in 2018 and Fellow of SNMMI in 2019). Prof. Cai’s trainees at UW–Madison have received over 100 awards.