ImmunoPET: Antibody-Based PET Imaging in Solid Tumors

Immuno-positron emission tomography (immunoPET) is a molecular imaging modality combining the high sensitivity of PET with the specific targeting ability of monoclonal antibodies. Various radioimmunotracers have been successfully developed to target a broad spectrum of molecules expressed by malignant cells or tumor microenvironments. Only a few are translated into clinical studies and barely into clinical practices. Some drawbacks include slow radioimmunotracer kinetics, high physiologic uptake in lymphoid organs, and heterogeneous activity in tumoral lesions. Measures are taken to overcome the disadvantages, and new tracers are being developed. In this review, we aim to mention the fundamental components of immunoPET imaging, explore the groundbreaking success achieved using this new technique, and review different radioimmunotracers employed in various solid tumors to elaborate on this relatively new imaging modality.


INTRODUCTION
There is an expanding insight into the role of different molecules and pathways in the development and progression of cancer. The growing knowledge about the involved molecules and processes has resulted in the modification in cancer management; therefore, targeted therapies and immunotherapies are increasingly utilized to treat different malignancies (1)(2)(3). This process was accelerated by the production of monoclonal antibodies (mAbs), following advances in DNA technology and Ab engineering (4). The targets can be membrane receptors, enzymes, or various molecules in signaling pathways, which are overexpressed or specifically present in a particular tumor or its microenvironment (5). Targeting molecules include Abs and Ab fragments, small molecule inhibitors, selective high-affinity ligands, some peptides, and aptamers (5,6).
The first human radioimmunoimaging was conducted in 1978 using 131 l-labeled whole immunoglobin G (lgG) targeting carcinoembryonic antigen (CEA) (7) with inherent drawbacks. Since then, significant efforts have been implemented to develop ideal radioimmunoimaging tracers and radiopharmaceuticals for different cancers. The ideal tracer should be target-specific, biologically inert, highly stable in serum, minimally immunogenic, with rapid biodistribution and background clearance. Physiochemical characteristics to facilitate radiolabeling are also crucial (6, 8). For instance, manufactured small-sized Ab fragments (Fab) show higher specificity and rapid biodistribution and provide superior imaging characteristics over whole Abs (9, 10).
In the era of ever-growing targeted therapy, there is a requirement for accurate targeted imaging. Although immunohistochemistry (IHC) is the integral modality for detecting biomarkers (11), the non-invasive evaluation of the whole-body remains a compelling field of research, especially for patient selection and response evaluation. Medical imaging has a fundamental role in managing solid tumors, among which positron emission tomography (PET) is of particular importance (12). PET-based imaging demonstrates different functional and biochemical procedures occurring in normal tissues and malignant tumors at the cellular and molecular levels (12, 13).
The recent advances in PET acquisition systems, providing highly sensitive imaging (13), coupled with developments in labeling methods (14) and the specific targeting offered by mAbs, build the foundation of immunoPET. ImmunoPET is molecular imaging used for (1) the evaluation of biodistribution of Abs or their fragments in normal and malignant tissues, (2) the non-invasive detection of expression of target molecules and their heterogeneity in whole-body, and 3) prediction of response to targeted therapies (15). Although the concept of immunoPET is simple, it is an umbrella term covering almost all aspects of medical imaging, including oncology, infection/inflammation, neurological diseases, and drug development.
In this review, we aimed to provide a simplified summary of the current state of immunoPET in oncology. First, we briefly present the principles of immunoPET. Afterward, we focus on the Ab-based immunoPET in solid tumors and discuss the various developed probes in preclinical and clinical studies for each cancer.

THE CONCEPT OF IMMUNOPET
PET is a non-invasive and powerful imaging procedure with a wide range of clinical and research applications. PET provides the three-dimensional mapping of organs and lesions using a radioactive tracer. Radionuclides are incorporated either into compounds normally used by the organs, such as glucose, or into molecules that bind to receptors, peptides, cytokines, or other components of cellular pathways (16,17). Recent advances in the development of PET systems and sophisticated software enable rapid, highly sensitive imaging (18). The combination of the superior targeting specificity of immune system-associated molecules and the inherent high sensitivity of PET technique establishes the principle of ImmunoPET (19). These tracers can specifically target various molecular pathways involved in the tumor biology (4).
Generally, the successful development of immunoPET in oncology is highly dependent on knowledge about the processes involved in tumor biology, choice of tumor-targeting vectors, radionuclides and chelators, and conjugation strategies. A vast number of molecules and processes are involved in tumor development and progression (20), the details of which are beyond the scope of this review. Some of the studied targets for imaging are discussed in more detail in the next section.
A variety of tumor-targeting vectors have been investigated for immunoPET. Full-length Abs are among the most used forms (21). Abs and associated amino acid-based macromolecules have been developed to display high specificity and binding affinity toward molecular targets overexpressed by cancer cells and tumor microenvironment (4). Their development dates back to the beginning of the twentieth century when Paul Ehrlich brought up the "magic bullet" idea to seek out and eradicate the spirochete of syphilis without affecting normal tissues (22).
MAbs are Abs with specificity for one particular epitope on an antigen (23). Despite the clinical success, these Abs come with a number of limitations including slow blood clearance, serum sickness, low target-to-background ratio (TBR), and the necessity of repetitive imaging (24). Moreover, high costs of production limit their use in developing countries. Thus, smaller Ab constructs have been engineered to overcome these limitations (25). Engineered Abs have a faster clearance rate, higher TBR, and are suggested to penetrate solid tumors more effectively (26).
Immunoglobulin G (IgG) is the most common type of Abs. IgG is composed of two main parts, crystallizable fragment (Fc) and antigen-binding Fragment (Fab), which contain polypeptides of heavy and light chains forming the constant and variable fragments of the IgG. The smaller fragments used for radiolabeling contain the variable domain. Fab and (Fab ′ )2 fragments are made by omitting the Fc region from Abs. They can have more rapid renal clearance and improved tumor penetration. However, their production is difficult and cannot be obtained from all subclasses of Abs (27). The smaller molecule, single-chain fragment variable (scFv), consists only of light and heavy variable chains. scFvs are produced more easily and are one of the most popular used fragments (28). The chains are attached with a non-covalent association, making scFv normally unstable. A disulfide bond between chains is used to increase stability, forming disulfide fragment variable (dsFv), and singlechain disulfide fragment variable (scdsFv). Proper tumor uptake and retention could be achieved by increasing the valency of scFvs molecules. Multivalent scFvs such as diabodies, tribodies and tetrabodies are favorable agents for radioimmunoimaging. In comparison with monovalent scFvs, tumor the retention time is augmented in multivalent scFvs. Also, their clearance time is shorter than whole Abs but equivalent to monovalent scFvs (25). scFvs are cleared through the urinary system, and there is significant retention in the kidneys (29).
Minibody is another engineered Ab fragment, produced by combining scFv molecule with human IgG1 constant heavy chain-3 (CH3) (30). Application of minibody is a mean to surmount slow renal clearance, which is the major problem of scFvs (29). Nanobodies or single-domain antibodies (sdAbs) are the smallest fragments of Abs obtained from the camelid heavychain-only Abs (31). Nanobodies are easier to produce and are more stable than scFvs (31). Finally, there is a smaller engineered targeting protein, affibody, which is derived from the IgG binding region. Affibodies seem to be a suitable protein for imaging but suffer from rapid clearance and decreased avidity to the targets (Figure 1) (27). FIGURE 1 | The schematic illustration of an antibody and its fragments. The schematic illustration of an antibody and its fragments. IgG has two main parts, crystallizable Fragment (Fc) and antigen-binding Fragment (Fab)

IMMUNOPET IN DIFFERENT MALIGNANCIES
ImmunoPET is known to provide excellent specificity and sensitivity in detecting some tumors (32, 33). However, some drawbacks include suboptimal imaging properties (feasibility, long imaging protocols), low expression of the targets in tumoral lesions, and high background activity in some organs.
Several molecules are involved in the development of different malignancies ( Table 1). Receptor tyrosine kinases (RTKs) have been among the most explored targets for developing anticancer therapeutic and imaging agents. Substantial efforts have been made to establish immunoPET probes for revealing the heterogeneous expression of RTKs. The human epidermal growth factor (HER) family is one of the most evaluated RTKs, including four members, HER1-4 (101). A number of mAbs and Ab fragments have been developed for preclinical and clinical studies targeting anti-epidermal growth factor receptor (EGFR, known as HER1) in various malignancies (101), HER2 mostly in breast cancer (102), and HER3 in different solid tumors (103)(104)(105)(106). Other RTKs, such as vascular endothelialderived growth factor/receptor (VEGF/VEGFR) (107) and platelet-derived growth factor/receptor (PDGF/PDGFR) (82), and insulin-like growth factor-1 receptor (IGF-1R) (74) have also been targeted for immunoPET imaging.
Processes other than only high Ag-Ab affinity are required for appropriate targeting. The heterogeneous tracer uptake and high physiologic activity, especially in the liver and lymphoid tissue, can make imaging with these probes challenging (15, 106,108). Preinjection of the cold tracer might decrease the hepatic uptake (109). Also, long radioimmunotracer clearance time for whole Abs can limit the tumor visualization (67,110,111). On the other hand, the renal retention of the smaller fragments is higher (112)(113)(114)(115). Moreover, the poor vascular permeability of the tumors rather than the unfavorable characteristics of the probe can affect the outcome (17, 116).
The binding of programmed cell death protein ligand-1 (PD-L1) on the tumor cells to PD-1 on T-cells suppresses Tcell function and helps tumors escape the immune system. Immune checkpoint inhibitors (ICIs) are Abs that block PD-L1 and show therapeutic effect in various malignancies (117). Several radiolabeled ICIs have been developed for immunoPET, showing high physiologic uptake in lymphoid tissues (118,119). Preinjection of unlabeled anti-PD-L1 may be useful for the PD-L1 expression evaluation (120). Also, T-cells in the tumor microenvironment impact response to therapies (121,122) that have been evaluated in different malignancies (123)(124)(125)(126), which can be used as a biomarker for response evaluation.
Many other proteins and molecules are overexpressed in different tumors, making them potential targets for immunoPET imaging. Some molecules and pathways are engaged in the development of various malignancies, and some are particularly found in distinct tumors. Below, we review the relevant evaluated probes in different malignancies.     89 Zr-labeled pertuzumab and trastuzumab in the preclinical (132,133) and phase I clinical studies (102,131,134,135).
Although new lesions were found (102), some HER2-positive lesions showed no significant uptake (102,134), and the detection rate of 79-89% was reported (102,134). Interestingly, HER2-targeting tracer uptake, suggesting HER2-positivity, was demonstrated in metastases from HER2-negative primary tumors (130,131). The heterogenicity was also illustrated in metabolism on [ 18 89 Zr]Zr-trastuzumab PET/CT increased physicians' confidence or altered management in a substantial ratio of patients (136). Also, early changes in [ 89 Zr]Zr-trastuzumab uptake correlated with response to therapy with a new experimental agent (137). The liver uptake is substantial with whole Abs, which may be decreased by the cold pharmaceutical preinjection (109).
Regulation of the immune system by ICIs have shown therapeutic efficacy in triple-negative breast cancer (143). Evaluating PD-L1 expression, Bensch et al. reported a heterogeneous tracer uptake in a few cancer types, including triple-negative breast cancer (144). The intensity of [ 89 Zr]Zratezolizumab (anti-PD-L1) uptake best correlated with outcome compared to IHC in their study (144). Currently, the role of [ 89 Zr]Zr-atezolizumab PET/CT is being investigated for patient selection for ICIs therapy (NCT02478099 and NCT02453984). Moreover, [ 89 Zr]Zr-Avelumab, another anti-PD-L1 ligand, is developed for the imaging of breast cancer in preclinical studies (145,146).
In addition, CEA is a cell surface adhesion molecule (53), correlating with poor survival in breast cancer (54). Imaging with anti-CEA immunoPET seems promising. TF2 is a bispecific trivalent mAb comprising two humanized anti-CEA and an antihistamine-succinyl-glycine (HSG) hapten Fab fragments. This unique structure enables TF2 to be used in pretargeted imaging of malignancies with CEA expression (147). It showed more bone metastases than conventional imaging (148,149) and helped delineate the clinical target volume for stereotactic body radiotherapy in CEA-positive metastatic breast cancer (148).
There are also other prognostic biomarkers which have been evaluated for the imaging of triple-negative breast cancer in preclinical studies, such as syndecan-1 (CD138) (83) and IGF-1 (74).

Lung Cancer
Non-small cell lung cancer (NSCLC) is also a heterogeneous malignancy (156). Radioimmunoimaging in lung cancer has been performed in preclinical and clinical studies for various purposes.
The presence of tumor-infiltrating lymphocytes (TILs) correlates with survival in different malignancies (96). Also, the amount of T-cell infiltration after immunotherapy in the tumor microenvironment impacts the response (121,122). The non-invasive evaluation of these cells may predict response to immunomodulatory therapies. Probes have been developed to monitor TILs dynamics after therapy (123)(124)(125)(126). In this regard, an anti-CD8 minibody, [ 89 Zr]Zr-IAB22M2C, showed a favorable biodistribution (174), and currently, it is under investigation in phase II clinical trial for different solid tumors (NCT03802123). Also, the presence of tumor-associated macrophages (TAMs), indicating poorer survival (92), CD30, a marker of T-cell activation (175), and inducible T-cell costimulatory receptor (ICOS), a costimulatory signaling molecule (73) have been assessed using immunoPET.
MET is an RTK, a receptor of hepatocyte growth factor (HGF), which plays a role in the development, progression, and therapy resistance (70). ImmunoPET shows potential in the non-invasive evaluation of MET expression (176,177). Another biomarker, CD146, interacts with VEGFR (17). It is overexpressed in about 50% of lung cancers and correlates with poor survival (178,179

Colorectal Cancer
Tumor biomarkers are being investigated in metastatic colorectal cancer and are used to guide patient selection due to variable responses to available targeted therapies (181,182).
Anti-EGFR therapy is used for Kirsten rat sarcoma virus (KRAS) wild-type tumors; however, not all patients respond to this therapy (183,184). Anti-EGFR immunoPET using cetuximab and panitumumab showed specific but heterogeneous uptake in EGFR-expressing preclinical models (67,160,185,186) and colorectal cancer patients (184). Evaluating the clinical impact in patient selection for therapy, van-Helden et al. failed to demonstrate a relation between [ 89 Zr]Zr-cetuximab-positivity and treatment response or outcome (183). Also, the high liver sequestration of [ 89 Zr]Zr-cetuximab in normal liver tissue is vital in colorectal cancer, limiting its diagnostic ability in liver metastasis (108).
A33 is a glycoprotein responsible for cell adhesion, overexpressed in 95% of colorectal cancers (34). [ 124 I]I-huA33 PET/CT localized tumoral lesions in colorectal cancer patients (34,199); however, the tracer uptake was also seen in normal bowel (34). To improve pharmacokinetics, pretargeted imaging was also evaluated, producing high-quality images in the preclinical studies (200,201). The expression of another prognostic cell adhesion glycoprotein, CD44, was also demonstrated in colorectal cancer models (202,203). Tumorassociated glycoprotein-72 (TAG-72) was also targeted (204), which is a prognostic cell membrane molecule overexpressed in colorectal cancer with mucin (MUC) properties.
Moreover, the cancer stem cell hypothesis suggests the possible role of cancer stem cells in the progression of malignancies. In this regard, a few molecules have successfully targeted stem cells in preclinical studies, such as CD133 (205) and LGR5 (leucine-rich repeat-containing, G protein-coupled receptor 5) (77,206). Additionally, probes monitoring TILs in response to immunotherapy have been evaluated in colorectal cancer models (124,125).
There are other factors involved in tumor development, progression, and poor prognosis in colorectal cancer, assessed as targets for immunoPET in preclinical studies for the detection of colorectal cancer cell lines, including HER3 (69)

Prostate Cancer
Ongoing studies are striving to improve imaging in prostate cancer. The prostate-specific membrane antigen (PSMA) is known as a suitable target for prostate cancer imaging and therapy (208,209). Agents targeting PSMA are classified into three groups: Abs, aptamers, and PSMA inhibitors (210). Here, we discuss Abs against the PSMA molecule and other targets for immunoPET imaging of prostate cancer. ProstaScint ([ 111 In]In-capromab-pentitide) was the first FDAapproved mAb-based imaging, employing a mAb against PSMA (7E11) to detect occult pelvic lymph node metastases and recurrence of prostate cancer (211,212). However, the binding of 7E11 to the intracellular epitope of PSMA resulted in low sensitivity for detecting metastases (213). To overcome this limitation, extracellular-binding mAbs were developed, such as J591 (88), labeled with imaging and therapeutic radionuclides (214)(215)(216). In a clinical study, the humanized [ 89 Zr]Zr-J591 (huJ591) showed accuracies of 60% and 95% for the detection of soft-tissue and bone metastases (217). However, mAbs have a prolonged circulation time due to their large size (218). In this regard, the third generation mAbs (minibodies/diabodies) were developed (219,220). [ 89  One of the challenges in prostate cancer imaging is tumors with no or negligible PSMA uptake. Therefore, tracers targeting biomarkers of poorer prognosis may help detect or treat this subset of patients.
Various other Abs or Ab fragments have been evaluated in preclinical studies. Prostate stem cell antigen (PSCA) is a cell membrane protein that plays a part in signal transduction. Because of the slow kinetics of whole mAbs, different Ab fragments have been produced to target PSCA in preclinical studies (87,(226)(227)(228). It may become helpful in response evaluation to anti-androgen therapy (87). Low non-specific uptake has been reported with [ 124 I]I-A11 minibody (226) and slightly better with its smaller fragment, [ 89 Zr]Zr-A2cDb diabody (227,229). Furthermore, TROP-2, known as tumor-associated calcium signal transducer 2, is a glycoprotein possessing stemcell like qualities, overexpressed in some malignancies, (99). Pretargeted imaging with bsAbs, anti-TROP-2 × anti-HSG, may improve TBR and provide a fast, sensitive, and specific tool for prostate cancer imaging (218,230). Also, a scFv against CD133, [ 89 Zr]Zr-HA10, localized in aggressive prostate cancer models (231,232).
Others are anti-EpCAM Ab against epithelial cell adhesion molecule (233) (208,209), have cast a shadow on other targets in prostate cancer. However, immunoPET may further help evaluate the aggressive variants (231,232) or androgen receptor status or guide androgen receptor deprivation therapy (71). Also, it can have a role in radioimmunotherapy of patients who are not candidates or not responding to the currently available therapies.

Gastric Cancer
ImmunoPET has been investigated in gastric cancer only in limited studies. Targeted therapy against HER2 is recommended to treat gastric cancer with HER2-overexpression (168). ImmunoPET has shown promising results in the non-invasive evaluation of HER-positive lesions. Radiolabeled anti-HER2 (236, 237) and anti-HER3 (mAb3481) (238) successfully detected gastric cancer cells in mice models. Also, to assess therapy response after HER2-targeted therapy, a decrease in [ 89 Zr]Zr-trastuzumab uptake was demonstrated in the gastric cancer xenografts (239). Additionally, the dynamics of HER2 expression after pretreatment with lovastatin was evaluated using HER2 targeted immunoPET (240,241). In initial clinical studies, [ 89 Zr]Zr-trastuzumab has shown a wide range of no to intense uptake in HER2-positive gastroesophageal cancers (242,243). The uniformly high [ 89 Zr]Zr-trastuzumab uptake in all lesions was associated with a good response to therapy (243).
A few studies evaluated the MET/HGF pathway in gastric cancer. Radiolabeled rilotumumab (anti-HGF mAb) and onartuzumab (anti-MET mAb) non-invasively detected HGF and MET expression, respectively (244,245). Additionally, MG7, a gastric cancer-specific antigen, is a prognostic biomarker (78). PET imaging of anti-MG7 Abs in gastric cancer xenografts showed a favorable binding affinity (246), but its application may be limited due to the overexpression of MG7 in helicobacterrelated gastric disease (247). Also, radiolabeled anti-cadherin17 Ab (CDH17, an adhesion protein) was introduced as a potential probe for CDH17-positive gastric cancer (248).

Esophageal Cancer
ImmunoPET of esophageal cancer using multiple targets can provide useful information to detect primary tumors and metastases and identify tumor phenotype to facilitate patient selection for targeted therapies (57, 84,256,257).
Anti-EGFR imaging has been evaluated in a few preclinical studies, showing potential for [ 64 Cu]Cu-cetuximab PET/CT for the EGFR-expression detection and patient selection for cetuximab treatment (257,258). Moreover, the detection of HER2 expression was evaluated in a small number of patients with gastroesophageal cancer (242). Similar to other malignancies, there was a wide range of uptake intensity from no uptake to SUV of 22.7 (242). Additionally, the decrease in vascular density after anti-VGEF treatment was depicted by [ 89 Zr]Zrbevacizumab immunoPET (259). Finally, the overexpression of some biomarkers, such as periostin, an extracellular matrix (ECM) protein (84), and atypical chemokine receptor 3 (ACKR3), a cell adhesion transmembrane protein (35), was noninvasively depicted in esophageal cell lines.

Head and Neck Squamous Cell Carcinoma (HNSCC)
A few targets such as EGFR and CD44v6 have been evaluated in HNSCC (260,261). Therapeutic Abs targeting EGFR showed promising outcomes in HNSCC treatment (262). Hence, ImmunoPET can be a valuable approach for diagnosis, treatment response prediction, and RIT planning in this group of cancers (46, [263][264][265][266]. Several studies assessed the correlation of radiolabeled cetuximab uptake with EGFR expression to predict response to anti-EGFR therapies in HNSCC (67,(266)(267)(268), showing a clear tumor visualization in almost all patients (8/9) in phase I clinical trial (111). Similar to other malignancies, a mismatch of [ 89 Zr]Zr-cetuximab (67,111,268) and [ 64 Cu]Cupanitumumab (116) uptake and EGFR expression was reported. However, an anti-EGFR affibody strongly correlated with EGFR expression alteration in response to cetuximab (269). Heat shock protein 90 (HSP90) is significantly associated with many oncoproteins and an interesting target for therapy (260). After HSP90 inhibitor therapy in a preclinical model, the amount of [ 124 I]I-cetuximab uptake was decreased in xenografts, but the uptake of [ 124 I]I-CD4v6 was unaffected (260). CD44v6 is a specific isomer of CD44, expressed in cells with squamous differentiation and overexpressed in squamous cell carcinomas in different organs (270). Preclinical studies on engineered anti-CD44v6 Abs, Ab fragments, and minibodies reported specific tumor uptake and more favorable kinetics in HNSCC animal models (46,271,272). In one of the first clinical studies, 89 Zrlabeled anti-CD44v6, U36, detected primary tumors in all subjects and had equal performance to CT and MRI for the metastatic lymph node detection (273).

Thyroid Cancer
Thyroid cancer is a common malignancy, effectively managed with radioactive iodine. However, management of less common thyroid cancer subtypes is challenging. Recently, various molecules were evaluated for targeted imaging and therapy in these cancers (274,275).
Expression of PDGFRα has been correlated with treatment resistance and risk of recurrence in papillary thyroid carcinoma (PTC). [ 64 Cu]Cu-D13C6, a PDGFRα targeting mAb, had specific uptake in PTC models, which can be of significant potential given that PTC is the most prevalent type of thyroid cancer (276). Moreover, β-galactoside-binding protein galectin-3 (Gal-3) is a biomarker, expressed only in thyroid cancer cells (63). 89 Zrlabeled targeting IgG and Fab fragments successfully detected thyroid cancer lesions and can potentially be used for identifying recurrence and metastasis (277)(278)(279)(280)(281).
Some anaplastic thyroid carcinomas (ATC) overexpress HER2. In this regard, dual-tracer imaging by 89 Zr-and IRDye 800CW-labeled pertuzumab depicted HER2-expressing ATC cell lines (282). Similar results were observed for an intercellular adhesion molecule-1 (ICAM-1), and tissue factor (TF or CD142, a mediator of hemostasis and inflammation) targeting probes in ATC models (72,283). These findings can be of significant value in the diagnosis and management of this aggressive subtype. [ 124 I]I-U36, an anti-CD44v6, also demonstrated high ATC tumor accumulation independent of the iodine uptake (284).

Pancreatic Cancer
Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic malignancy (52). There is a need to improve the imaging modalities for the diagnosis and staging of PDAC (287,288). Various Ab-based targets have been developed and are majorly evaluated only in preclinical studies, which may promote the role of immunoPET in PDAC (288).
Carbohydrate antigen 19-9 (CA 19-9), the known serum marker for PDAC, is detected on tumor cells in the vast majority of patients (289). Among agents targeting CA 19-9, a mAb, 5B1, demonstrated very high affinity and specificity (290), and immunoPET with [ 89 Zr]Zr-5B1 showed remarkable radiotracer uptake in the CA 19-9-positive models (291). Also, a duallabeled tracer ( 89 Zr-and NIRF) successfully delineated tumoral lesions (292). It showed negligible non-specific uptake in CA 19-9-negative tumoral cell lines (292). However, the circulating CA 19-9 and long mAb half-life are problematic, increasing the background activity. In this regard, preinjection of cold 5B1 improved the image quality (293). Additionally, other pretargeting methods were assessed to overcome the drawbacks, including the development of a new-generation tracer (which exploits the advantages of reagents) (294) and an Ab-gold nanoparticle conjugate (295).
In the first clinical trial, [ 89 Zr]Zr-HuMab-5B1 PET/CT successfully detected the primary sites of PDACs, metastases, and small lymph nodes, overlooked by other imaging techniques (296). The tumor uptake was high, showing an average SUV of 19.7 ± 2.9, and the pretargeting increased TBR (296). Interestingly, serum CA 19-9 levels did not affect the physiologic distribution (296). Another ongoing phase I trial is assessing the performance of [ 89 Zr]Zr-HuMab-5B1 PET/CT in CA 19-9positive tumors (NCT02687230).
Imaging with mAb was first conducted in PDAC using a murine Ab targeting MUC (297). Mucins are transmembrane glycoproteins highly expressed on several epithelial cancers, usually with an altered glycosylation pattern (37, 298). MUC1 or cancer antigen 15-3 (CA 15-3) may have a role in the management of PDAC (299). Also, carbohydrate antigen 125 (CA125), an extracellular domain of MUC16, is a well-known ovarian cancer biomarker (38). The newer generation of Abs targeted the unshed domain of MUC16 rather than CA125 and showed specific tracer uptake in PDAC xenografts with superior imaging characteristics (38).
Integrin αvβ6 is a cell surface protein, overexpression of which correlates with tumor progression (300) and seems as a potential theranostic agent in PDAC (301). However, the αvβ6targeting peptides (302)(303)(304) are superior to Abs (300) and are more investigated.
Tissue factor (TF) is a transmembrane protein that plays a role in initiating coagulation and regulation of inflammation. The radiolabeled anti-TF, ALT-836, localized PDAC cell lines (93,305). Additionally, a bsAb consisting of anti-TF × anti-endoglin improved binding affinity and localization of the xenografts (306,307).
Mesothelin (MSLN) is a membrane-bound glycoprotein overexpressed in more than 80-100% of pancreatic and ovarian cancer with an unknown specific role in tumor progression (108). MSLN-based immunoPET was previously used to evaluate the efficacy of MSLN-targeted antibodydrug conjugate (ADC) in preclinical models (308). In a clinical study on a small group of ovarian and pancreatic cancer patients, an 89 Zr-labeled anti-MSLN Ab detected the majority of malignant lesions with minimal non-specific uptake. Tumor tracer uptake also correlated with MSLN expression on IHC, but no correlation was observed with progression-free survival (309).

Melanoma
The first trials of Ab-based imaging in melanoma date back to more than 25 years ago when Abs against the melanin-associated antigen (335) and a mouse monoclonal anti-melanoma Ab (336) were used for imaging in patients with metastatic malignant melanoma.
Currently, targeted therapies are the main systemic treatments for advanced melanoma (337,338). It is crucial to document the presence of targets before the initiation of therapy to achieve the best response and avoid side effects (339,340). Several preclinical studies showed favorable binding affinity and imaging of melanoma cell lines using Abs and Ab fragments targeting immune checkpoints, PD-1/PD-L1 (9, 119, 340-345) and CTLA-4 (346). However, the uptake in the lymphoid tissue was also high (119,347). Moreover, radiotherapy-induced PD-L1 upregulation was demonstrated in melanoma mouse models, using radiolabeled anti-PD-L1 Abs (348,349). Another immune checkpoint, T-cell immunoglobulin and mucin domaincontaining-3 (TIM-3), was a successful target for murine melanoma models (97). One clinical study evaluated [ 89 Zr]Zrpembrolizumab in melanoma, reporting relations between tracer uptake and response to therapy and survival in patients with advanced or metastatic disease (347). Also, an ongoing phase II clinical trial is investigating the biodistribution and tumor uptake of [ 89 Zr]Zr-ipilimumab (anti-CTLA-4) in advanced melanoma (NCT03313323).
In addition to immune checkpoints, the number of CD4-and CD8-positive T-cells (121,122) and the presence of costimulatory signaling molecules (64), namely OX40 (member 4 of tumor necrosis factor [TNF] family) and GITR (glucocorticoidinduced TNF Receptor), in the tumor microenvironment impact the response to immunotherapy. Preclinical studies using immunoPET probes successfully detected CD4-and CD8positive rich tissues and upregulation of costimulatory substances (64,122,174,350,351). These probes have the potential to monitor immune response following different therapies. Moreover, immunoPET targeting ICAM-1 (72), CD146 (352), VEGF (353), and integrin-αvβ6 (75) was also successful in the detection of melanoma cells in preclinical studies.

Ovarian Cancer
Ovarian cancer comprises multiple subtypes and a diverse profile of overexpressed molecules (354). Moreover, the lack of effective specific treatment makes such molecules valuable for targeted therapies guided by immunoPET (108).
With the wide use of anti-HER2 mAbs, several studies evaluated 89 Zr/ 64 Cu-labeled anti-HER2 mAbs and their engineered fragments in preclinical models and compared different chelators and labeling strategies, or their optimal performance in vivo (138,140,(355)(356)(357)(358)(359)(360)(361). Radiolabeledtrastuzumab has been used to monitor response not only to anti-HER2 mAbs but also to HSP90 inhibitors, which downregulate HER2 (361,362). Using dual labeled mAbs combines pre-operative PET imaging and intra-operative optical imaging data. It can improve precise tumor and metastasis excision, especially in metastasis-prone tumors, such as ovarian cancer. Dual-labeled pertuzumab with 89 Zr and NIRF was used successfully for image-guided tumor resection in ovarian cancer xenografts (363).
CA125, an extracellular domain of MUC16, is a serological biomarker for treatment monitoring and recurrence of ovarian cancer (298). Preclinical studies on the 89 Zr/ 64 Cu-labeled oregovomab, an anti-CA125 mAb, reported high uptake in ovarian cancer xenografts (367,368). Furthermore, [ 89 Zr]Zroregovomab detected histologically-confirmed lymph node involvement (368). The newer generation of Abs targeting MUC16 showed superior imaging characteristics (38, 369). Additionally, an 89 Zr-labeled anti-MUC1 or CA 15-3 also showed proper performance in vivo (37). The first clinical study on a Fab targeting carbonic anhydrase 6 (CA6) epitope of MUC1 reported the safety of this probe in an ovarian cancer patient, and the probe correctly reflected the low tumor expression of CA6 observed in IHC (370).
REGN4018, a T-cell engaging bsAb targeting MUC16 × CD3, is currently being investigated in a clinical trial for ovarian cancer (NCT03564340). It had high specific uptake in tumors and lymphoid organs in preclinical studies (371).
[ 89 Zr]Zr-ERY974, a bsAb targeting CD3ε on T-cells × GPC3, showed high specific uptake in GPC3-positive ovarian cancer xenografts ingrafted with immune cells compared to xenografts in immunodeficient mice, highlighting the potential value of such probes in immunotherapy response monitoring (372).
Finally, as mentioned in the pancreatic cancer section, MSLN expression may be non-invasively evaluated in ovarian cancer, using 89 Zr-labeled anti-MSLN Ab (309), which may help patient selection for therapy.

Central Nervous System (CNS)
Precision imaging and targeted therapy in the central nervous system (CNS) is challenging due to the limited distribution of radiopharmaceuticals beyond the blood-brain barrier (BBB), especially for high molecular weight compounds such as Abs. However, disruption of BBB by tumors can lead to the appropriate probe uptake (373). Nanobodies are attractive probes for CNS malignancies given their small size, easy BBB penetration, and faster blood clearance (374). ImmunoPET has also been helpful to monitor brain drug delivery alteration in response to different methods of increasing BBB permeability (375)(376)(377)(378) and predict response to intrathecal radioimmunotherapy (379).
Several studies, mainly preclinical, investigated radioimmunotracers targeting molecules involved in angiogenesis, such as VEGF (380,381), CD146 (382,383), and PDGFRβ (384) in the setting of brain tumors. On clinical studies of the mentioned targets, [ 89 Zr]Zr-bevacizumab imaging was feasible and safe in children with diffuse intrinsic pontine glioma; however, significant uptake heterogeneity was observed (385). Interestingly, high expression of PSMA by neovascular endothelium has been reported in a number of highly vascularized tumors, including gliomas and some brain metastases (386). This phenomenon makes PSMA a potential target to monitor response to anti-angiogenesis treatments in brain malignancies. Matsuda et al. showed high PSMA expression in histological specimens for a wide range of brain malignancies, including gliomas and metastatic brain lesions. Next, they successfully imaged three patients with recurrent glioma or brain metastasis with [ 89 Zr]Zr-IAB2M (387).

Others
ImmunoPET studies on other malignancies are still in the preclinical setting. For instance, early murine studies targeting EGFR and CA 19-9 were promising in bladder cancer models (404,405). An anti-CD3 probe also detected T-cell infiltration in bladder cancer-bearing mice, reflecting the potential for future studies on immunotherapy response prediction in urothelial cancers (406).

CONCLUSION
In the era of precision medicine and molecular targeted therapy, the need for accurate targeted imaging is inevitable. Given the inherent favorable characteristics, immunoPET seems very promising in this regard. A broad spectrum of both tumorspecific and common general molecules in different malignancies has already become targets for radioimmunoimaging of cancer. However, only a few have been introduced in clinical studies (Supplementary Table 1). Some common obstacles to the wide implementation of immunoPET include the high costs, advanced technology to commercially produce pure mAbs, difficulties in conjugation and in vivo tracer stability, as well as high circulation time and physiologic uptake after administration. Recent developments are answering these needs (97,411) and will continue to evolve. Although immunoPET can become a diagnostic tool in specific conditions, its primary role seems to be a complementary imaging tool for therapy guidance.
Almost all studies mention the heterogeneous uptake of probes in different tumors. That is where the immunoPET strength lies, i.e., non-invasively depicting the heterogeneity of tumoral lesions. ImmunoPET tracks target expression and pharmacokinetics of mAbs in vivo before and after certain treatments, pointing out its potential value for patient recruiting and response monitoring of targeted therapies. Noteworthy, smaller Ab fragments provide more favorable imaging properties that can help increase the detection rate and accuracy of the imaging. However, the implementation of whole Abs is crucial since these structures are used for treatment, and the goal is to demonstrate their in vivo distribution.
Another advantage of developing new radioimmunoimaging probes is the theranostic application. The probes can be labeled with positron-emitting imaging and beta/alpha-emitting therapeutic radiometals, providing another possible treatment option for different malignancies.
Besides developing new targeting probes, future studies should also focus on the predictive and prognostic value of the radioimmunotracers after targeted therapies to further elaborate on their impact on treatment selection.

AUTHOR CONTRIBUTIONS
RM-F, CP, and MB came up with the main study topic and designed the outline. RM-F and BA performed the initial search and screened the literature to finalize the topics to cover. RM-F, BA, SR, ZA, and MM performed the literature search and extracted and summarized the data. All authors contributed to writing the initial draft of the manuscript under the supervision of RM-F, CP, and MB. ZA, BA, and RM-F drafted the tables. RM-F and SR prepared the figure. BA proofread and edited the final version of the manuscript. All authors read and approved the final version prior to submission.